Sample records for a1 receptor activation

  1. A restricted population of CB1 cannabinoid receptors with neuroprotective activity.

    PubMed

    Chiarlone, Anna; Bellocchio, Luigi; Blázquez, Cristina; Resel, Eva; Soria-Gómez, Edgar; Cannich, Astrid; Ferrero, José J; Sagredo, Onintza; Benito, Cristina; Romero, Julián; Sánchez-Prieto, José; Lutz, Beat; Fernández-Ruiz, Javier; Galve-Roperh, Ismael; Guzmán, Manuel

    2014-06-03

    The CB1 cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. Of note, CB1 receptors are expressed at the synapses of two opposing (i.e., GABAergic/inhibitory and glutamatergic/excitatory) neuronal populations, so the activation of one and/or another receptor population may conceivably evoke different effects. Despite the widely reported neuroprotective activity of the CB1 receptor in animal models, the precise pathophysiological relevance of those two CB1 receptor pools in neurodegenerative processes is unknown. Here, we first induced excitotoxic damage in the mouse brain by (i) administering quinolinic acid to conditional mutant animals lacking CB1 receptors selectively in GABAergic or glutamatergic neurons, and (ii) manipulating corticostriatal glutamatergic projections remotely with a designer receptor exclusively activated by designer drug pharmacogenetic approach. We next examined the alterations that occur in the R6/2 mouse, a well-established model of Huntington disease, upon (i) fully knocking out CB1 receptors, and (ii) deleting CB1 receptors selectively in corticostriatal glutamatergic or striatal GABAergic neurons. The data unequivocally identify the restricted population of CB1 receptors located on glutamatergic terminals as an indispensable player in the neuroprotective activity of (endo)cannabinoids, therefore suggesting that this precise receptor pool constitutes a promising target for neuroprotective therapeutic strategies.

  2. Alpha-1A Adrenergic receptor activation increases inhibitory tone in CA1 hippocampus

    PubMed Central

    Hillman, Kristin L.; Lei, Saobo; Doze, Van A.

    2009-01-01

    The endogenous catecholamine norepinephrine (NE) exhibits anti-epileptic properties, however it is not well understood which adrenergic receptor (AR) mediates this effect. The aim of this study was to investigate α1-adrenergic receptor (AR) activation in region CA1 of the hippocampus, a subcortical structure often implicated in temporal lobe epilepsies. Using cell-attached and whole-cell recordings in rat hippocampal slices, we confirmed that selective α1-AR activation increases action potential firing in a subpopulation of CA1 interneurons. We found that this response is mediated via the α1A-AR subtype, initiated by sodium influx, and appears independent of second messenger signaling. In CA1 pyramidal cells, α1A-AR activation decreases activity due to increased pre-synaptic GABA and somatostatin release. Examination of post-synaptic receptor involvement revealed that while GABAA receptors mediate the majority of α1A-adrenergic effects on CA1 pyramidal cells, significant contributions are also made by GABAB and somatostatin receptors. Finally, to test whether α1A-AR activation could have potential therapeutic implications, we performed AR agonist challenges using two in vitro epileptiform models. When GABAA receptors were available, α1A-AR activation significantly decreased epileptiform bursting in CA1. Together, our findings directly link stimulation of the α1A-AR subtype to release of GABA and somatostatin at the single cell level and suggest that α1A-AR activation may represent one mechanism by which NE exerts anti-epileptic effects within the hippocampus. PMID:19201164

  3. Soluble TL1A is sufficient for activation of death receptor 3.

    PubMed

    Bittner, Sebastian; Knoll, Gertrud; Füllsack, Simone; Kurz, Maria; Wajant, Harald; Ehrenschwender, Martin

    2016-01-01

    Death receptor 3 (DR3) is a typical member of the tumor necrosis factor receptor family, and was initially identified as a T-cell co-stimulatory molecule. However, further studies revealed a more complex and partly dichotomous role for DR3 and its ligand TL1A under (patho)physiological conditions. TL1A and DR3 are not only a driving force in the development of autoimmune and inflammatory diseases, but also play an important role in counteracting these processes through an increase in the number of regulatory T cells. Ligands of the tumor necrosis factor family typically occur in two forms, membrane-bound and soluble, that can differ strikingly with respect to their efficacy in activating their corresponding receptor(s). Ligand-based approaches to activate the TL1A-DR3 pathway therefore require understanding of the molecular prerequisites of TL1A-based DR3 activation. To date, this has not been addressed. Here, we show that recombinant soluble trimeric TL1A is fully sufficient to strongly activate DR3-associated pro- and anti-apoptotic signaling pathways. In contrast to the TRAIL death receptors, which are much better activated by soluble TRAIL upon secondary ligand oligomerization, but similarly to the death receptor tumor necrosis factor receptor 1, DR3 is efficiently activated by soluble TL1A trimers. Additionally, we have measured the affinity of TL1A-DR3 interaction in a cell-based system, and demonstrated TL1A-induced DR3 internalization. Identification of DR3 as a tumor necrosis factor receptor that responds to soluble ligand trimers without further oligomerization provides a basis for therapeutic exploitation of the TL1A-DR3 pathway. © 2015 FEBS.

  4. Preclinical pharmacology of bilastine, a new selective histamine H1 receptor antagonist: receptor selectivity and in vitro antihistaminic activity.

    PubMed

    Corcóstegui, Reyes; Labeaga, Luis; Innerárity, Ana; Berisa, Agustin; Orjales, Aurelio

    2005-01-01

    This study aimed to establish the receptor selectivity and antihistaminic activity of bilastine, a new selective antihistamine receptor antagonist. In vitro experiments were conducted using a receptor binding screening panel and guinea-pig and rat tissues. Antihistaminic activity was determined using H1 receptor binding studies and in vitro H1 antagonism studies conducted in guinea-pig tissues and human cell lines. Receptor selectivity was established using a receptor binding screening panel and a receptor antagonism screening conducted in guinea-pig, rat and rabbit tissues. Inhibition of inflammatory mediators was determined through the Schultz-Dale reaction in sensitised guinea-pig ileum. Bilastine binds to histamine H1-receptors as indicated by its displacement of [3H]-pyrilamine from H1-receptors expressed in guinea-pig cerebellum and human embryonic kidney (HEK) cell lines. The studies conducted on guinea-pig smooth muscle demonstrated the capability of bilastine to antagonise H1-receptors. Bilastine is selective for histamine H1-receptors as shown in receptor-binding screening conducted to determine the binding capacity of bilastine to 30 different receptors. The specificity of its H1-receptor antagonistic activity was also demonstrated in a series of in vitro experiments conducted on guinea-pig and rat tissues. The results of these studies confirmed the lack of significant antagonism against serotonin, bradykinin, leukotriene D4, calcium, muscarinic M3-receptors, alpha1-adrenoceptors, beta2-adrenoceptors, and H2- and H3-receptors. The results of the in vitro Schultz-Dale reaction demonstrated that bilastine also has anti-inflammatory activity. These preclinical studies provide evidence that bilastine has H1- antihistamine activity, with high specificity for H1-receptors, and poor or no affinity for other receptors. Bilastine has also been shown to have anti-inflammatory properties.

  5. The non-biphenyl-tetrazole angiotensin AT1 receptor antagonist eprosartan is a unique and robust inverse agonist of the active state of the AT1 receptor.

    PubMed

    Takezako, Takanobu; Unal, Hamiyet; Karnik, Sadashiva S; Node, Koichi

    2018-03-23

    Conditions such as hypertension and renal allograft rejection are accompanied by chronic, agonist-independent, signalling by angiotensin II AT 1 receptors. The current treatment paradigm for these diseases entails the preferred use of inverse agonist AT 1 receptor blockers (ARBs). However, variability in the inverse agonist activities of common biphenyl-tetrazole ARBs for the active state of AT 1 receptors often leads to treatment failure. Therefore, characterization of robust inverse agonist ARBs for the active state of AT 1 receptors is necessary. To identify the robust inverse agonist for active state of AT 1 receptors and its molecular mechanism, we performed site-directed mutagenesis, competition binding assay, inositol phosphate production assay and molecular modelling for both ground-state wild-type AT 1 receptors and active-state N111G mutant AT 1 receptors. Although candesartan and telmisartan exhibited weaker inverse agonist activity for N111G- compared with WT-AT 1 receptors, only eprosartan exhibited robust inverse agonist activity for both N111G- and WT- AT 1 receptors. Specific ligand-receptor contacts for candesartan and telmisartan are altered in the active-state N111G- AT 1 receptors compared with the ground-state WT-AT 1 receptors, suggesting an explanation of their attenuated inverse agonist activity for the active state of AT 1 receptors. In contrast, interactions between eprosartan and N111G-AT 1 receptors were not significantly altered, and the inverse agonist activity of eprosartan was robust. Eprosartan may be a better therapeutic option than other ARBs. Comparative studies investigating eprosartan and other ARBs for the treatment of diseases caused by chronic, agonist-independent, AT 1 receptor activation are warranted. © 2018 The British Pharmacological Society.

  6. Functional Selectivity and Antidepressant Activity of Serotonin 1A Receptor Ligands

    PubMed Central

    Chilmonczyk, Zdzisław; Bojarski, Andrzej Jacek; Pilc, Andrzej; Sylte, Ingebrigt

    2015-01-01

    Serotonin (5-HT) is a monoamine neurotransmitter that plays an important role in physiological functions. 5-HT has been implicated in sleep, feeding, sexual behavior, temperature regulation, pain, and cognition as well as in pathological states including disorders connected to mood, anxiety, psychosis and pain. 5-HT1A receptors have for a long time been considered as an interesting target for the action of antidepressant drugs. It was postulated that postsynaptic 5-HT1A agonists could form a new class of antidepressant drugs, and mixed 5-HT1A receptor ligands/serotonin transporter (SERT) inhibitors seem to possess an interesting pharmacological profile. It should, however, be noted that 5-HT1A receptors can activate several different biochemical pathways and signal through both G protein-dependent and G protein-independent pathways. The variables that affect the multiplicity of 5-HT1A receptor signaling pathways would thus result from the summation of effects specific to the host cell milieu. Moreover, receptor trafficking appears different at pre- and postsynaptic sites. It should also be noted that the 5-HT1A receptor cooperates with other signal transduction systems (like the 5-HT1B or 5-HT2A/2B/2C receptors, the GABAergic and the glutaminergic systems), which also contribute to its antidepressant and/or anxiolytic activity. Thus identifying brain specific molecular targets for 5-HT1A receptor ligands may result in a better targeting, raising a hope for more effective medicines for various pathologies. PMID:26262615

  7. Activation of Neurotensin Receptor Type 1 Attenuates Locomotor Activity

    PubMed Central

    Vadnie, Chelsea A.; Hinton, David J.; Choi, Sun; Choi, YuBin; Ruby, Christina L.; Oliveros, Alfredo; Prieto, Miguel L.; Park, Jun Hyun; Choi, Doo-Sup

    2014-01-01

    Intracerebroventricular administration of neurotensin (NT) suppresses locomotor activity. However, the brain regions that mediate the locomotor depressant effect of NT and receptor subtype-specific mechanisms involved are unclear. Using a brain-penetrating, selective NT receptor type 1 (NTS1) agonist PD149163, we investigated the effect of systemic and brain region-specific NTS1 activation on locomotor activity. Systemic administration of PD149163 attenuated the locomotor activity of C57BL/6J mice both in a novel environment and in their homecage. However, mice developed tolerance to the hypolocomotor effect of PD149163 (0.1 mg/kg, i.p.). Since NTS1 is known to modulate dopaminergic signaling, we examined whether PD149163 blocks dopamine receptor-mediated hyperactivity. Pretreatment with PD149163 (0.1 or 0.05 mg/kg, i.p.) inhibited D2R agonist bromocriptine (8 mg/kg, i.p.)-mediated hyperactivity. D1R agonist SKF81297 (8 mg/kg, i.p.)-induced hyperlocomotion was only inhibited by 0.1 mg/kg of PD149163. Since the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) have been implicated in the behavioral effects of NT, we examined whether microinjection of PD149163 into these regions reduces locomotion. Microinjection of PD149163 (2 pmol) into the NAc, but not the mPFC suppressed locomotor activity. In summary, our results indicate that systemic and intra-NAc activation of NTS1 is sufficient to reduce locomotion and NTS1 activation inhibits D2R-mediated hyperactivity. Our study will be helpful to identify pharmacological factors and a possible therapeutic window for NTS1-targeted therapies for movement disorders. PMID:24929110

  8. Activation of neurotensin receptor type 1 attenuates locomotor activity.

    PubMed

    Vadnie, Chelsea A; Hinton, David J; Choi, Sun; Choi, YuBin; Ruby, Christina L; Oliveros, Alfredo; Prieto, Miguel L; Park, Jun Hyun; Choi, Doo-Sup

    2014-10-01

    Intracerebroventricular administration of neurotensin (NT) suppresses locomotor activity. However, the brain regions that mediate the locomotor depressant effect of NT and receptor subtype-specific mechanisms involved are unclear. Using a brain-penetrating, selective NT receptor type 1 (NTS1) agonist PD149163, we investigated the effect of systemic and brain region-specific NTS1 activation on locomotor activity. Systemic administration of PD149163 attenuated the locomotor activity of C57BL/6J mice both in a novel environment and in their homecage. However, mice developed tolerance to the hypolocomotor effect of PD149163 (0.1 mg/kg, i.p.). Since NTS1 is known to modulate dopaminergic signaling, we examined whether PD149163 blocks dopamine receptor-mediated hyperactivity. Pretreatment with PD149163 (0.1 or 0.05 mg/kg, i.p.) inhibited D2R agonist bromocriptine (8 mg/kg, i.p.)-mediated hyperactivity. D1R agonist SKF-81297 (8 mg/kg, i.p.)-induced hyperlocomotion was only inhibited by 0.1 mg/kg of PD149163. Since the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) have been implicated in the behavioral effects of NT, we examined whether microinjection of PD149163 into these regions reduces locomotion. Microinjection of PD149163 (2 pmol) into the NAc, but not the mPFC suppressed locomotor activity. In summary, our results indicate that systemic and intra-NAc activation of NTS1 is sufficient to reduce locomotion and NTS1 activation inhibits D2R-mediated hyperactivity. Our study will be helpful to identify pharmacological factors and a possible therapeutic window for NTS1-targeted therapies for movement disorders. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Antihyperalgesic activity of a novel nonpeptide bradykinin B1 receptor antagonist in transgenic mice expressing the human B1 receptor

    PubMed Central

    Fox, Alyson; Kaur, Satbir; Li, Bifang; Panesar, Moh; Saha, Uma; Davis, Clare; Dragoni, Ilaria; Colley, Sian; Ritchie, Tim; Bevan, Stuart; Burgess, Gillian; McIntyre, Peter

    2005-01-01

    We describe the properties of a novel nonpeptide kinin B1 receptor antagonist, NVP-SAA164, and demonstrate its in vivo activity in models of inflammatory pain in transgenic mice expressing the human B1 receptor. NVP-SAA164 showed high affinity for the human B1 receptor expressed in HEK293 cells (Ki 8 nM), and inhibited increases in intracellular calcium induced by desArg10kallidin (desArg10KD) (IC50 33 nM). While a similar high affinity was observed in monkey fibroblasts (Ki 7.7 nM), NVP-SAA164 showed no affinity for the rat B1 receptor expressed in Cos-7 cells. In transgenic mice in which the native B1 receptor was deleted and the gene encoding the human B1 receptor was inserted (hB1 knockin, hB1-KI), hB1 receptor mRNA was induced in tissues following LPS treatment. No mRNA encoding the mouse or human B1 receptor was detected in mouse B1 receptor knockout (mB1-KO) mice following LPS treatment. Freund's complete adjuvant-induced mechanical hyperalgesia was similar in wild-type and hB1-KI mice, but was significantly reduced in mB1-KO animals. Mechanical hyperalgesia induced by injection of the B1 agonist desArg10KD into the contralateral paw 24 h following FCA injection was similar in wild-type and hB1-KI mice, but was absent in mB1-KO animals. Oral administration of NVP-SAA164 produced a dose-related reversal of FCA-induced mechanical hyperalgesia and desArg10KD-induced hyperalgesia in hB1-KI mice, but was inactive against inflammatory pain in wild-type mice. These data demonstrate the use of transgenic technology to investigate the in vivo efficacy of species selective agents and show that NVP-SAA164 is a novel orally active B1 receptor antagonist, providing further support for the utility of B1 receptor antagonists in inflammatory pain conditions in man. PMID:15685199

  10. The prostaglandin EP1 receptor potentiates kainate receptor activation via a protein kinase C pathway and exacerbates status epilepticus

    PubMed Central

    Rojas, Asheebo; Gueorguieva, Paoula; Lelutiu, Nadia; Quan, Yi; Shaw, Renee; Dingledine, Raymond

    2014-01-01

    Prostaglandin E2 (PGE2) regulates membrane excitability, synaptic transmission, plasticity, and neuronal survival. The consequences of PGE2 release following seizures has been the subject of much study. Here we demonstrate that the prostaglandin E2 receptor 1 (EP1, or Ptger1) modulates native kainate receptors, a family of ionotropic glutamate receptors widely expressed throughout the central nervous system. Global ablation of the EP1 gene in mice (EP1-KO) had no effect on seizure threshold after kainate injection but reduced the likelihood to enter status epilepticus. EP1-KO mice that did experience typical status epilepticus had reduced hippocampal neurodegeneration and a blunted inflammatory response. Further studies with native prostanoid and kainate receptors in cultured cortical neurons, as well as with recombinant prostanoid and kainate receptors expressed in Xenopus oocytes, demonstrated that EP1 receptor activation potentiates heteromeric but not homomeric kainate receptors via a second messenger cascade involving phospholipase C, calcium and protein kinase C. Three critical GluK5 C-terminal serines underlie the potentiation of the GluK2/GluK5 receptor by EP1 activation. Taken together, these results indicate that EP1 receptor activation during seizures, through a protein kinase C pathway, increases the probability of kainic acid induced status epilepticus, and independently promotes hippocampal neurodegeneration and a broad inflammatory response. PMID:24952362

  11. Allosteric modulation of sigma-1 receptors elicits anti-seizure activities.

    PubMed

    Guo, Lin; Chen, Yanke; Zhao, Rui; Wang, Guanghui; Friedman, Eitan; Zhang, Ao; Zhen, Xuechu

    2015-08-01

    Application of orthosteric sigma-1 receptor agonists as anti-seizure drugs has been hindered by questionable efficacy and potential adverse effects. Here, we have investigated the anti-seizure effects of the novel and potent allosteric modulator of sigma-1 receptors, SKF83959 and its derivative SOMCL-668 (3-methyl-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-ol). The anti-seizure effects of SKF83959 were investigated in three mouse models, maximal electroshock seizures, pentylenetetrazole-induced convulsions and kainic acid-induced 'status epilepticus'. Also, in rats, the cortical epileptiform activity induced by topical application of picrotoxin was recorded in electrocorticograms. In rat hippocampal brain slices, effects of the drugs on the high potassium-evoked epileptiform local field potentials were studied. Anti-seizure activities of SOMCL-668, a newly developed sigma-1 receptor selective allosteric modulator, were also investigated. SKF83959 (20, 40 mg·kg(-1) ) exhibited anti -seizure actitity in the three mouse models and reduced the cortical epileptiform activity without alteration of spontaneous motor activity and motor coordination. These effects were blocked by the sigma-1 receptor antagonist BD1047, but not the dopamine D1 receptor antagonist SCH23390. SKF83959 alone did not directly inhibit the epileptiform firing of CA3 neurons induced by high potassium in hippocampal slices, but did potentiate inhibition by the orthosteric sigma-1 receptor agonist SKF10047. Lastly, a selective sigma-1 receptor allosteric modulator SOMCL-668, which does not bind to dopamine receptors, exerted similar anti-seizure activities. SKF83959 and SOMCL-668 displayed anti-seizure activities, indicating that allosteric modulation of sigma-1 receptors may provide a novel approach for discovering new anti-seizure drugs. © 2015 The British Pharmacological Society.

  12. The pharmacology of Ro 64-6198, a systemically active, nonpeptide NOP receptor (opiate receptor-like 1, ORL-1) agonist with diverse preclinical therapeutic activity.

    PubMed

    Shoblock, James R

    2007-01-01

    The NOP receptor (formerly referred to as opiate receptor-like 1, ORL-1, LC132, OP(4), or NOP(1)) is a G protein-coupled receptor that shares high homology to the classic opioid MOP, DOP, and KOP (mu, delta, and kappa, respectively) receptors and was first cloned in 1994 by several groups. The NOP receptor remained an orphan receptor until 1995, when the endogenous neuropeptide agonist, known as nociceptin or orphanin FQ (N/OFQ) was isolated. Five years later, a group at Hoffmann-La Roche reported on the selective, nonpeptide NOP agonist Ro 64-6198, which became the most extensively published nonpeptide NOP agonist and a valuable pharmacological tool in determining the potential of the NOP receptor as a therapeutic target. Ro 64-6198 is systemically active and achieves high brain penetration. It has subnanomolar affinity for the NOP receptor and is at least 100 times more selective for the NOP receptor over the classic opioid receptors. Ro 64-6198 ranges from partial to full agonist, depending on the assay. Preclinical data indicate that Ro 64-6198 may have broad clinical uses, such as in treating stress and anxiety, addiction, neuropathic pain, cough, and anorexia. This review summarizes the pharmacology and preclinical data of Ro 64-6198.

  13. Proteinase-Activated Receptor-1 and Immunomodulatory Effects of a PAR1-Activating Peptide in a Mouse Model of Prostatitis

    PubMed Central

    Stanton, M. Mark; Nelson, Lisa K.; Benediktsson, Hallgrimur; Hollenberg, Morley D.; Buret, Andre G.; Ceri, Howard

    2013-01-01

    Background. Nonbacterial prostatitis has no established etiology. We hypothesized that proteinase-activated receptor-1 (PAR1) can play a role in prostatitis. We therefore investigated the effects of PAR1 stimulation in the context of a new model of murine nonbacterial prostatitis. Methods. Using a hapten (ethanol-dinitrobenzene sulfonic acid- (DNBS-)) induced prostatitis model with both wild-type and PAR1-null mice, we examined (1) the location of PAR1 in the mouse prostate and (2) the impact of a PAR1-activating peptide (TFLLR-NH2: PAR1-TF) on ethanol-DNBS-induced inflammation. Results. Ethanol-DNBS-induced inflammation was maximal at 2 days. In the tissue, PAR1 was expressed predominantly along the apical acini of prostatic epithelium. Although PAR1-TF on its own did not cause inflammation, its coadministration with ethanol-DNBS reduced all indices of acute prostatitis. Further, PAR1-TF administration doubled the prostatic production of interleukin-10 (IL-10) compared with ethanol-DNBS treatment alone. This enhanced IL-10 was not observed in PAR1-null mice and was not caused by the reverse-sequence receptor-inactive peptide, RLLFT-NH2. Surprisingly, PAR1-TF, also diminished ethanol-DNBS-induced inflammation in PAR1-null mice. Conclusions. PAR1 is expressed in the mouse prostate and its activation by PAR1-TF elicits immunomodulatory effects during ethanol-DNBS-induced prostatitis. However, PAR1-TF also diminishes ethanol-DNBS-induced inflammation via a non-PAR1 mechanism by activating an as-yet unknown receptor. PMID:24459330

  14. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor suppressor in hepatocellular carcinoma.

    PubMed

    Liu, Rui; Zhang, Haiyang; Zhang, Yan; Li, Shuang; Wang, Xinyi; Wang, Xia; Wang, Cheng; Liu, Bin; Zen, Ke; Zhang, Chen-Yu; Zhang, Chunni; Ba, Yi

    2017-04-01

    Peroxisome proliferator-activated receptor gamma coactivator-1 alpha plays a crucial role in regulating the biosynthesis of mitochondria, which is closely linked to the energy metabolism in various tumors. This study investigated the regulatory role of peroxisome proliferator-activated receptor gamma coactivator-1 alpha in the pathogenesis of hepatocellular carcinoma. In this study, the changes of peroxisome proliferator-activated receptor gamma coactivator-1 alpha messenger RNA levels between normal human liver and hepatocellular carcinoma tissue were examined by quantitative reverse transcription polymerase chain reaction. Knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by RNA interference in the human liver cell line L02, while overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha complementary DNA in the human hepatocarcinoma cell line HepG2. Cellular morphological changes were observed via optical and electron microscopy. Cellular apoptosis was determined by Hoechst 33258 staining. In addition, the expression levels of 21,400 genes in tissues and cells were detected by microarray. It was shown that peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression was significantly downregulated in hepatocellular carcinoma compared with normal liver tissues. After knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression in L02 cells, cells reverted to immature and dedifferentiated morphology exhibiting cancerous tendency. Apoptosis occurred in the HepG2 cells after transfection by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha. Microarray analysis showed consistent results. The results suggest that peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor

  15. Discovery of potent peptide-mimetic antagonists for the human thrombin receptor, protease-activated receptor-1 (PAR-1).

    PubMed

    Maryanoff, Bruce E; Zhang, Han-Cheng; Andrade-Gordon, Patricia; Derian, Claudia K

    2003-03-01

    Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G-protein-coupled receptors, which are enzymatically cleaved to expose a new extracellular N-terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g. platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. By using a de novo design approach, we have discovered a series of potent heterocycle-based peptide-miimetic antagonists of PAR-1, exemplified by advanced leads RWJ-56110 (22) and RWJ-58259 (32). These compounds are potent, selective PAR-1 antagonists, devoid of PAR-1 agonist and thrombin inhibitory activity: they bind to PAR-1, interfere with calcium mobilization and cellular functions associated with PAR-1, and do not affect PAR-2, PAR-3, or PAR-4. RWJ-56110 was determined to be a direct inhibitor of PAR-1 activation and internalization, without affecting PAR-1 N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, but not in human platelets; whereas, at high concentrations of TRAP-6, RWJ-56110 blocked activation responses in both cell types. This result is consistent with the presence of another thrombin receptor on human platelets, namely PAR-4. RWJ-56110 and RWJ-58259 clearly interrupt the binding of a tethered ligand to its receptor. RWJ-58259 demonstrated antirestenotic activity in a rat balloon angioplasty model and antithrombotic activity in a cynomolgus monkey arterial injury model. Such PAR-1 antagonists should not only serve as useful tools to delineate the physiological and pathophysiological roles of PAR-1, but also may have therapeutic potential for treating thrombosis and restenosis in humans.

  16. Glucose-Sensing Receptor T1R3: A New Signaling Receptor Activated by Glucose in Pancreatic β-Cells.

    PubMed

    Kojima, Itaru; Nakagawa, Yuko; Hamano, Kunihisa; Medina, Johan; Li, Longfei; Nagasawa, Masahiro

    2015-01-01

    Subunits of the sweet taste receptors T1R2 and T1R3 are expressed in pancreatic β-cells. Compared with T1R3, mRNA expression of T1R2 is considerably lower. At the protein level, expression of T1R2 is undetectable in β-cells. Accordingly, a major component of the sweet taste-sensing receptor in β-cells may be a homodimer of T1R3 rather than a heterodimer of T1R2/T1R3. Inhibition of this receptor by gurmarin or deletion of the T1R3 gene attenuates glucose-induced insulin secretion from β-cells. Hence the T1R3 homodimer functions as a glucose-sensing receptor (GSR) in pancreatic β-cells. When GSR is activated by the T1R3 agonist sucralose, elevation of intracellular ATP concentration ([ATP]i) is observed. Sucralose increases [ATP]i even in the absence of ambient glucose, indicating that sucralose increases [ATP]i not simply by activating glucokinase, a rate-limiting enzyme in the glycolytic pathway. In addition, sucralose augments elevation of [ATP]i induced by methylsuccinate, suggesting that sucralose activates mitochondrial metabolism. Nonmetabolizable 3-O-methylglucose also increases [ATP]i and knockdown of T1R3 attenuates elevation of [ATP]i induced by high concentration of glucose. Collectively, these results indicate that the T1R3 homodimer functions as a GSR; this receptor is involved in glucose-induced insulin secretion by activating glucose metabolism probably in mitochondria.

  17. In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V1 and V2

    PubMed Central

    Jamil, Khurram; Pappas, Stephen Chris; Devarakonda, Krishna R

    2018-01-01

    Terlipressin, a synthetic, systemic vasoconstrictor with selective activity at vasopressin-1 (V1) receptors, is a pro-drug for the endogenous/natural porcine hormone [Lys8]-vasopressin (LVP). We investigated binding and receptor-mediated cellular activities of terlipressin, LVP, and endogenous human hormone [Arg8]-vasopressin (AVP) at V1 and vasopressin-2 (V2) receptors. Cell membrane homogenates of Chinese hamster ovary cells expressing human V1 and V2 receptors were used in competitive binding assays to measure receptor-binding activity. These cells were used in functional assays to measure receptor-mediated cellular activity of terlipressin, LVP, and AVP. Binding was measured by [3H]AVP counts, and the activity was measured by fluorometric detection of intracellular calcium mobilization (V1) and cyclic adenosine monophosphate (V2). Binding potency at V1 and V2 was AVP>LVP>>terlipressin. LVP and terlipressin had approximately sixfold higher affinity for V1 than for V2. Cellular activity potency was also AVP>LVP>>terlipressin. Terlipressin was a partial agonist at V1 and a full agonist at V2; LVP was a full agonist at both V1 and V2. The in vivo response to terlipressin is likely due to the partial V1 agonist activity of terlipressin and full V1 agonist activity of its metabolite, LVP. These results provide supportive evidence for previous findings and further establish terlipressin pharmacology for vasopressin receptors. PMID:29302194

  18. In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V1 and V2.

    PubMed

    Jamil, Khurram; Pappas, Stephen Chris; Devarakonda, Krishna R

    2018-01-01

    Terlipressin, a synthetic, systemic vasoconstrictor with selective activity at vasopressin-1 (V 1 ) receptors, is a pro-drug for the endogenous/natural porcine hormone [Lys 8 ]-vasopressin (LVP). We investigated binding and receptor-mediated cellular activities of terlipressin, LVP, and endogenous human hormone [Arg 8 ]-vasopressin (AVP) at V 1 and vasopressin-2 (V 2 ) receptors. Cell membrane homogenates of Chinese hamster ovary cells expressing human V 1 and V 2 receptors were used in competitive binding assays to measure receptor-binding activity. These cells were used in functional assays to measure receptor-mediated cellular activity of terlipressin, LVP, and AVP. Binding was measured by [ 3 H]AVP counts, and the activity was measured by fluorometric detection of intracellular calcium mobilization (V 1 ) and cyclic adenosine monophosphate (V 2 ). Binding potency at V 1 and V 2 was AVP>LVP>terlipressin. LVP and terlipressin had approximately sixfold higher affinity for V 1 than for V 2 . Cellular activity potency was also AVP>LVP>terlipressin. Terlipressin was a partial agonist at V 1 and a full agonist at V 2 ; LVP was a full agonist at both V 1 and V 2 . The in vivo response to terlipressin is likely due to the partial V 1 agonist activity of terlipressin and full V 1 agonist activity of its metabolite, LVP. These results provide supportive evidence for previous findings and further establish terlipressin pharmacology for vasopressin receptors.

  19. β1-adrenergic receptors activate two distinct signaling pathways in striatal neurons

    PubMed Central

    Meitzen, John; Luoma, Jessie I.; Stern, Christopher M.; Mermelstein, Paul G.

    2010-01-01

    Monoamine action in the dorsal striatum and nucleus accumbens plays essential roles in striatal physiology. Although research often focuses on dopamine and its receptors, norepinephrine and adrenergic receptors are also crucial in regulating striatal function. While noradrenergic neurotransmission has been identified in the striatum, little is known regarding the signaling pathways activated by β-adrenergic receptors in this brain region. Using cultured striatal neurons, we characterized a novel signaling pathway by which activation of β1-adrenergic receptors leads to the rapid phosphorylation of cAMP Response Element Binding Protein (CREB), a transcription-factor implicated as a molecular switch underlying long-term changes in brain function. Norepinephrine-mediated CREB phosphorylation requires β1-adrenergic receptor stimulation of a receptor tyrosine kinase, ultimately leading to the activation of a Ras/Raf/MEK/MAPK/MSK signaling pathway. Activation of β1-adrenergic receptors also induces CRE-dependent transcription and increased c-fos expression. In addition, stimulation of β1-adrenergic receptors produces cAMP production, but surprisingly, β1-adrenergic receptor activation of adenylyl cyclase was not functionally linked to rapid CREB phosphorylation. These findings demonstrate that activation of β1-adrenergic receptors on striatal neurons can stimulate two distinct signaling pathways. These adrenergic actions can produce long-term changes in gene expression, as well as rapidly modulate cellular physiology. By elucidating the mechanisms by which norepinephrine and β1-adrenergic receptor activation affects striatal physiology, we provide the means to more fully understand the role of monoamines in modulating striatal function, specifically how norepinephrine and β1-adrenergic receptors may affect striatal physiology. PMID:21143600

  20. Orexin-A increases the firing activity of hippocampal CA1 neurons through orexin-1 receptors.

    PubMed

    Chen, Xin-Yi; Chen, Lei; Du, Yi-Feng

    2017-07-01

    Orexins including two peptides, orexin-A and orexin-B, are produced in the posterior lateral hypothalamus. Much evidence has indicated that central orexinergic systems play numerous functions including energy metabolism, feeding behavior, sleep/wakefulness, and neuroendocrine and sympathetic activation. Morphological studies have shown that the hippocampal CA1 regions receive orexinergic innervation originating from the hypothalamus. Positive orexin-1 (OX 1 ) receptors are detected in the CA1 regions. Previous behavioral studies have shown that microinjection of OX 1 receptor antagonist into the hippocampus impairs acquisition and consolidation of spatial memory. However, up to now, little has been known about the direct electrophysiological effects of orexin-A on hippocampal CA1 neurons. Employing multibarrel single-unit extracellular recordings, the present study showed that micropressure administration of orexin-A significantly increased the spontaneous firing rate from 2.96 ± 0.85 to 8.45 ± 1.86 Hz (P < 0.001) in 15 out of the 23 hippocampal CA1 neurons in male rats. Furthermore, application of the specific OX 1 receptor antagonist SB-334867 alone significantly decreased the firing rate from 4.02 ± 1.08 to 2.11 ± 0.58 Hz in 7 out of the 17 neurons (P < 0.05), suggesting that endogenous orexinergic systems modulate the firing activity of CA1 neurons. Coapplication of SB-334867 completely blocked orexin-A-induced excitation of hippocampal CA1 neurons. The PLC pathway may be involved in activation of OX 1 receptor-induced excitation of CA1 neurons. Taken together, the present study's results suggest that orexin-A produces excitatory effects on hippocampal neurons via OX 1 receptors. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Medial prefrontal cortex TRPV1 and CB1 receptors modulate cardiac baroreflex activity by regulating the NMDA receptor/nitric oxide pathway.

    PubMed

    Lagatta, Davi C; Kuntze, Luciana B; Ferreira-Junior, Nilson C; Resstel, Leonardo B M

    2018-05-29

    The ventral medial prefrontal cortex (vMPFC) facilitates the cardiac baroreflex response through N-methyl-D-aspartate (NMDA) receptor activation and nitric oxide (NO) formation by neuronal NO synthase (nNOS) and soluble guanylate cyclase (sGC) triggering. Glutamatergic transmission is modulated by the cannabinoid receptor type 1 (CB 1 ) and transient receptor potential vanilloid type 1 (TRPV 1 ) receptors, which may inhibit or stimulate glutamate release in the brain, respectively. Interestingly, vMPFC CB 1 receptors decrease cardiac baroreflex responses, while TRPV 1 channels facilitate them. Therefore, the hypothesis of the present study is that the vMPFC NMDA/NO pathway is regulated by both CB 1 and TRPV 1 receptors in the modulation of cardiac baroreflex activity. In order to test this assumption, we used male Wistar rats that had stainless steel guide cannulae bilaterally implanted in the vMPFC. Subsequently, a catheter was inserted into the femoral artery, for cardiovascular recordings, and into the femoral vein for assessing baroreflex activation. The increase in tachycardic and bradycardic responses observed after the microinjection of a CB 1 receptors antagonist into the vMPFC was prevented by an NMDA antagonist as well as by the nNOS and sGC inhibition. NO extracellular scavenging also abolished these responses. These same pharmacological manipulations inhibited cardiac reflex enhancement induced by TRPV 1 agonist injection into the area. Based on these results, we conclude that vMPFC CB 1 and TRPV 1 receptors inhibit or facilitate the cardiac baroreflex activity by stimulating or blocking the NMDA activation and NO synthesis.

  2. SGIP1 alters internalization and modulates signaling of activated cannabinoid receptor 1 in a biased manner.

    PubMed

    Hájková, Alena; Techlovská, Šárka; Dvořáková, Michaela; Chambers, Jayne Nicole; Kumpošt, Jiří; Hubálková, Pavla; Prezeau, Laurent; Blahos, Jaroslav

    2016-08-01

    Many diseases of the nervous system are accompanied by alterations in synaptic functions. Synaptic plasticity mediated by the endogenous cannabinoid system involves the activation of the cannabinoid receptor 1 (CB1R). The principles of CB1R signaling must be understood in detail for its therapeutic exploration. We detected the Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1) as a novel CB1R partner. SGIP1 is functionally linked to clathrin-mediated endocytosis and its overexpression in animals leads to an energy regulation imbalance resulting in obesity. We report that SGIP1 prevents the endocytosis of activated CB1R and that it alters signaling via the CB1R in a biased manner. CB1R mediated G-protein activation is selectively influenced by SGIP1, β-arrestin associated signaling is changed profoundly, most likely as a consequence of the prevention of the receptor's internalization elicited by SGIP1. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors.

    PubMed

    Zvejniece, L; Vavers, E; Svalbe, B; Vilskersts, R; Domracheva, I; Vorona, M; Veinberg, G; Misane, I; Stonans, I; Kalvinsh, I; Dambrova, M

    2014-02-01

    Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. E1R was tested for sigma receptor binding activity in a [³H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca²⁺ concentration ([Ca²⁺](i)) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca²⁺](i) increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases. © 2013 The British Pharmacological Society.

  4. The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors

    PubMed Central

    Zvejniece, L; Vavers, E; Svalbe, B; Vilskersts, R; Domracheva, I; Vorona, M; Veinberg, G; Misane, I; Stonans, I; Kalvinsh, I; Dambrova, M

    2014-01-01

    Background and Purpose Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. Experimental Approach E1R was tested for sigma receptor binding activity in a [3H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca2+ concentration ([Ca2+]i) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. Key Results Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca2+]i increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. Conclusion and Implications E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases. PMID:24490863

  5. A CONSTITUTIVELY ACTIVE FORM OF NEUROKININ 1 RECEPTOR AND NEUROKININ 1 RECEPTOR-MEDIATED APOPTOSIS IN GLIOBLASTOMAS

    PubMed Central

    Akazawa, Toshimasa; Kwatra, Shawn G.; Goldsmith, Laura E.; Richardson, Mark D.; Cox, Elizabeth A.; Sampson, John H.; Kwatra, Madan M.

    2009-01-01

    Previous studies have shown that neurokinin 1 receptor (NK1R) occurs naturally in human glioblastomas and its stimulation causes cell proliferation. In the present study we show that stimulation of NK1R in human U373 glioblastoma cells by substance P (SP) increases Akt phosphorylation by 2.5-fold, with an EC50 of 57 nM. Blockade of NK1R lowers basal phosphorylation of Akt, indicating the presence of a constitutively active form of NK1R; similar results are seen in U251 MG and DBTRG-05 glioblastoma cells. Linkage of NK1R to Akt implicates NK1R in apoptosis of glioblastoma cells. Indeed, treatment of serum-starved U373 cells with SP reduces apoptosis by 53 ± 1% (P < 0.05), and treatment with NK1R antagonist L-733,060 increases apoptosis by 64 ± 16 % (P < 0.01). Further, the blockade of NK1R in human glioblastoma cells with L-733,060 causes cleavage of Caspase-3 and proteolysis of poly (ADP-ribose) polymerase (PARP). Experiments designed to elucidate the mechanism of NK1R-mediated Akt phosphorylation revealed total involvement of non-receptor tyrosine kinase Src and PI-3-kinase, a partial involvement of epidermal growth factor receptor (EGFR), and no involvement of MEK. Taken together, the results of the present study indicate a key role for NK1R in glioblastoma apoptosis. PMID:19519779

  6. Anti-HIV-1 activity of a tripodal receptor that recognizes mannose oligomers.

    PubMed

    Rivero-Buceta, Eva; Carrero, Paula; Casanova, Elena; Doyagüez, Elisa G; Madrona, Andrés; Quesada, Ernesto; Peréz-Pérez, María Jesús; Mateos, Raquel; Bravo, Laura; Mathys, Leen; Noppen, Sam; Kiselev, Evgeny; Marchand, Christophe; Pommier, Yves; Liekens, Sandra; Balzarini, Jan; Camarasa, María José; San-Félix, Ana

    2015-12-01

    The glycoprotein gp120 of the HIV-1 viral envelope has a high content in mannose residues, particularly α-1,2-mannose oligomers. Compounds that interact with these high-mannose type glycans may disturb the interaction between gp120 and its (co)receptors and are considered potential anti-HIV agents. Previously, we demonstrated that a tripodal receptor (1), with a central scaffold of 1,3,5-triethylbenzene substituted with three 2,3,4-trihydroxybenzoyl groups, selectively recognizes α-1,2-mannose polysaccharides. Here we present additional studies to determine the anti-HIV-1 activity and the mechanism of antiviral activity of this compound. Our studies indicate that 1 shows anti-HIV-1 activity in the low micromolar range and has pronounced gp120 binding and HIV-1 integrase inhibitory capacity. However, gp120 binding rather than integrase inhibition seems to be the primary mechanism of antiviral activity of 1. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  7. LOCALIZATION OF CALCITONIN RECEPTOR-LIKE RECEPTOR (CLR) AND RECEPTOR ACTIVITY-MODIFYING PROTEIN 1 (RAMP1) IN HUMAN GASTROINTESTINAL TRACT

    PubMed Central

    Cottrell, Graeme S.; Alemi, Farzad; Kirkland, Jacob G.; Grady, Eileen F.; Corvera, Carlos U.; Bhargava, Aditi

    2012-01-01

    Calcitonin gene-related peptide (CGRP) exerts its diverse effects on vasodilation, nociception, secretion, and motor function through a heterodimeric receptor comprising of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Despite the importance of CLR•RAMP1 in human disease, little is known about its distribution in the human gastrointestinal (GI) tract, where it participates in inflammation and pain. In this study, we determined that CLR and RAMP1 mRNAs are expressed in normal human stomach, ileum and colon by RT-PCR. We next characterized antibodies that we generated to rat CLR and RAMP1 in transfected HEK cells. Having characterized these antibodies in vitro, we then localized CLR-, RAMP1-, CGRP- and intermedin-immunoreactivity (IMD-IR) in various human GI segments. In the stomach, nerve bundles in the myenteric plexus and nerve fibers throughout the circular and longitudinal muscle had prominent CLR-IR. In the proximal colon and ileum, CLR was found in nerve varicosities of the myenteric plexus and surrounding submucosal neurons. Interestingly, CGRP expressing fibers did not co-localize, but were in close proximity to CLR. However, CLR and RAMP1, the two subunits of a functional CGRP receptor were clearly localized in myenteric plexus, where they may form functional cell-surface receptors. IMD, another member of calcitonin peptide family was also found in close proximity to CLR, and like CGRP, did not co-localize with either CLR or RAMP1 receptors. Thus, CGRP and IMD appear to be released locally, where they can mediate their effect on their receptors regulating diverse functions such as inflammation, pain and motility. PMID:22484227

  8. Discovery of a Series of Imidazo[4,5-b]pyridines with Dual Activity at Angiotensin II Type 1 Receptor and Peroxisome Proliferator-Activated Receptor-[gamma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Casimiro-Garcia, Agustin; Filzen, Gary F.; Flynn, Declan

    2013-03-07

    Mining of an in-house collection of angiotensin II type 1 receptor antagonists to identify compounds with activity at the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) revealed a new series of imidazo[4,5-b]pyridines 2 possessing activity at these two receptors. Early availability of the crystal structure of the lead compound 2a bound to the ligand binding domain of human PPAR{gamma} confirmed the mode of interaction of this scaffold to the nuclear receptor and assisted in the optimization of PPAR{gamma} activity. Among the new compounds, (S)-3-(5-(2-(1H-tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-2-ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-b]pyridine (2l) was identified as a potent angiotensin II type I receptor blocker (IC{sub 50} = 1.6 nM) with partialmore » PPAR{gamma} agonism (EC{sub 50} = 212 nM, 31% max) and oral bioavailability in rat. The dual pharmacology of 2l was demonstrated in animal models of hypertension (SHR) and insulin resistance (ZDF rat). In the SHR, 2l was highly efficacious in lowering blood pressure, while robust lowering of glucose and triglycerides was observed in the male ZDF rat.« less

  9. Lordosis facilitated by GPER-1 receptor activation involves GnRH-1, progestin and estrogen receptors in estrogen-primed rats.

    PubMed

    Domínguez-Ordóñez, R; Garcia-Juárez, M; Lima-Hernández, F J; Gómora-Arrati, P; Domínguez-Salazar, E; Blaustein, J D; Etgen, A M; González-Flores, O

    2018-02-01

    The present study assessed the participation of membrane G-protein coupled estrogen receptor 1 (GPER-1) and gonadotropin releasing hormone 1 (GnRH-1) receptor in the display of lordosis induced by intracerebroventricular (icv) administration of G1, a GPER-1 agonist, and by unesterified 17β-estradiol (free E 2 ). In addition, we assessed the participation of both estrogen and progestin receptors in the lordosis behavior induced by G1 in ovariectomized (OVX), E 2 -benzoate (EB)-primed rats. In Experiment 1, icv injection of G1 induced lordosis behavior at 120 and 240min. In Experiment 2, icv injection of the GPER-1 antagonist G15 significantly reduced lordosis behavior induced by either G1 or free E 2 . In addition, Antide, a GnRH-1 receptor antagonist, significantly depressed G1 facilitation of lordosis behavior in OVX, EB-primed rats. Similarly, icv injection of Antide blocked the stimulatory effect of E 2 on lordosis behavior. In Experiment 3, systemic injection of either tamoxifen or RU486 significantly reduced lordosis behavior induced by icv administration of G1 in OVX, EB-primed rats. The results suggest that GnRH release activates both estrogen and progestin receptors and that this activation is important in the chain of events leading to the display of lordosis behavior in response to activation of GPER-1 in estrogen-primed rats. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Critical Hydrogen Bond Formation for Activation of the Angiotensin II Type 1 Receptor*

    PubMed Central

    Cabana, Jérôme; Holleran, Brian; Beaulieu, Marie-Ève; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan; Lavigne, Pierre

    2013-01-01

    G protein-coupled receptors contain selectively important residues that play central roles in the conformational changes that occur during receptor activation. Asparagine 111 (N1113.35) is such a residue within the angiotensin II type 1 (AT1) receptor. Substitution of N1113.35 for glycine leads to a constitutively active receptor, whereas substitution for tryptophan leads to an inactivable receptor. Here, we analyzed the AT1 receptor and two mutants (N111G and N111W) by molecular dynamics simulations, which revealed a novel molecular switch involving the strictly conserved residue D742.50. Indeed, D742.50 forms a stable hydrogen bond (H-bond) with the residue in position 1113.35 in the wild-type and the inactivable receptor. However, in the constitutively active mutant N111G-AT1 receptor, residue D74 is reoriented to form a new H-bond with another strictly conserved residue, N461.50. When expressed in HEK293 cells, the mutant N46G-AT1 receptor was poorly activable, although it retained a high binding affinity. Interestingly, the mutant N46G/N111G-AT1 receptor was also inactivable. Molecular dynamics simulations also revealed the presence of a cluster of hydrophobic residues from transmembrane domains 2, 3, and 7 that appears to stabilize the inactive form of the receptor. Whereas this hydrophobic cluster and the H-bond between D742.50 and W1113.35 are more stable in the inactivable N111W-AT1 receptor, the mutant N111W/F77A-AT1 receptor, designed to weaken the hydrophobic core, showed significant agonist-induced signaling. These results support the potential for the formation of an H-bond between residues D742.50 and N461.50 in the activation of the AT1 receptor. PMID:23223579

  11. Pharmacology of the hypothermic response to 5-HT1A receptor activation in humans.

    PubMed

    Lesch, K P; Poten, B; Söhnle, K; Schulte, H M

    1990-01-01

    The selective 5-HT1A receptor ligand ipsapirone (IPS) caused dose-related hypothermia in humans. The response was attenuated by the nonselective 5-HT1/2 receptor antagonist metergoline and was completely antagonized by the nonselective beta-adrenoceptor antagonist pindolol, which interacts stereoselectively with the 5-HT1A receptor. The selective beta 1-adrenergic antagonist betaxolol had no effect. The findings indicate that IPS-induced hypothermia specifically involves activation of (presynaptic) 5-HT1A receptors. Therefore, the hypothermic response to IPS may provide a convenient in vivo paradigma to assess the function of the presynaptic 5-HT receptor in affective disorders and its involvement in the effects of psychotropic drugs.

  12. Extrasynaptic targeting of NMDA receptors following D1 dopamine receptor activation and cocaine self-administration

    PubMed Central

    Ortinski, Pavel I.; Turner, Jill R.; Pierce, R. Christopher

    2013-01-01

    We previously showed that after repeated exposure to cocaine, D1-like dopamine receptor (D1DR) stimulation reverses plastic changes of AMPA receptor-mediated signaling in the nucleus accumbens shell. However, there is little information on the impact of cocaine self-administration on D1-NMDA receptor interactions in this brain region. Here, we assessed whether cocaine self-administration alters the effects of D1DR stimulation on synaptic and extrasynaptic NMDA receptors (NMDARs) using whole-cell patch-clamp recordings. In slices from cocaine-naïve rats, pre-treatment with a D1DR agonist decreased synaptic NMDAR receptor-mediated currents and increased the contribution of extrasynaptic NMDARs. In contrast, neither cocaine self-administration alone nor cocaine experience followed by D1DR stimulation had an effect on synaptic or extrasynaptic NMDAR signaling. Activation of extrasynaptic NMDARs relies on the availability of extracellular glutamate, which is regulated primarily by glutamate transporters. In cocaine-experienced animals, administration of a glutamate re-uptake blocker, DL-threo-β-benzyloxyaspartic acid (TBOA), revealed increased extrasynaptic NMDAR activity and stronger baseline activity of glutamate uptake transporters relative to cocaine-naïve rats. In cocaine-naïve rats, the D1DR-mediated increase in extrasynaptic NMDAR signaling was independent of the activity of glutamate re-uptake transporters. Taken together, these results indicate that cocaine experience blunts the influence of D1DRs on synaptic and extrasynaptic NMDAR signaling. Additionally, prior cocaine self-administration limits activation of the extrasynaptic NMDAR pool by increasing glutamate re-uptake. These findings outline a pattern of adaptive interactions between D1DRs and NMDARs in the nucleus accumbens shell and demonstrate up-regulation of extrasynaptic NMDAR signaling as a novel consequence of cocaine self-administration. PMID:23719812

  13. Ozanimod (RPC1063) is a potent sphingosine-1-phosphate receptor-1 (S1P1 ) and receptor-5 (S1P5 ) agonist with autoimmune disease-modifying activity.

    PubMed

    Scott, F L; Clemons, B; Brooks, J; Brahmachary, E; Powell, R; Dedman, H; Desale, H G; Timony, G A; Martinborough, E; Rosen, H; Roberts, E; Boehm, M F; Peach, R J

    2016-06-01

    Sphingosine1-phosphate (S1P) receptors mediate multiple events including lymphocyte trafficking, cardiac function, and endothelial barrier integrity. Stimulation of S1P1 receptors sequesters lymphocyte subsets in peripheral lymphoid organs, preventing their trafficking to inflamed tissue sites, modulating immunity. Targeting S1P receptors for treating autoimmune disease has been established in clinical studies with the non-selective S1P modulator, FTY720 (fingolimod, Gilenya™). The purpose of this study was to assess RPC1063 for its therapeutic utility in autoimmune diseases. The specificity and potency of RPC1063 (ozanimod) was evaluated for all five S1P receptors, and its effect on cell surface S1P1 receptor expression, was characterized in vitro. The oral pharmacokinetic (PK) parameters and pharmacodynamic effects were established in rodents, and its activity in three models of autoimmune disease (experimental autoimmune encephalitis, 2,4,6-trinitrobenzenesulfonic acid colitis and CD4(+) CD45RB(hi) T cell adoptive transfer colitis) was assessed. RPC1063 was specific for S1P1 and S1P5 receptors, induced S1P1 receptor internalization and induced a reversible reduction in circulating B and CCR7(+) T lymphocytes in vivo. RPC1063 showed high oral bioavailability and volume of distribution, and a circulatory half-life that supports once daily dosing. Oral RPC1063 reduced inflammation and disease parameters in all three autoimmune disease models. S1P receptor selectivity, favourable PK properties and efficacy in three distinct disease models supports the clinical development of RPC1063 for the treatment of relapsing multiple sclerosis and inflammatory bowel disease, differentiates RPC1063 from other S1P receptor agonists, and could result in improved safety outcomes in the clinic. © 2016 The British Pharmacological Society.

  14. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a

    PubMed Central

    Seredynski, Aurore L.; Balthazart, Jacques; Ball, Gregory F.

    2015-01-01

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER–mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. SIGNIFICANCE STATEMENT The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  15. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a.

    PubMed

    Seredynski, Aurore L; Balthazart, Jacques; Ball, Gregory F; Cornil, Charlotte A

    2015-09-23

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER-mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. Significance statement: The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  16. Learning and memory deficits in mice lacking protease activated receptor-1

    PubMed Central

    Almonte, Antoine G.; Hamill, Cecily E.; Chhatwal, Jasmeer P.; Wingo, Thomas S.; Barber, Jeremy A.; Lyuboslavsky, Polina N.; Sweatt, J. David; Ressler, Kerry J.; White, David A.; Traynelis, Stephen F.

    2007-01-01

    The roles of serine proteases and protease activated receptors have been extensively studied in coagulation, wound healing, inflammation, and neurodegeneration. More recently, serine proteases have been suggested to influence synaptic plasticity. In this context, we examined the role of protease activated receptor 1 (PAR1), which is activated following proteolytic cleavage by thrombin and plasmin, in emotionally-motivated learning. We were particularly interested in PAR1 because its activation enhances the function of NMDA receptors, which are required for some forms of synaptic plasticity. We examined several baseline behavioral measures, including locomotor activity, expression of anxiety-like behavior, motor task acquisition, nociceptive responses, and startle responses in C57Bl/6 mice in which the PAR1 receptor has been genetically deleted. In addition, we evaluated learning and memory in these mice using two memory tasks, passive avoidance and cued fear-conditioning. Whereas locomotion, pain response, startle, and measures of baseline anxiety were largely unaffected by PAR1 removal, PAR1−/− animals showed significant deficits in a passive avoidance task and in cued fear conditioning. These data suggest that PAR1 may play an important role in emotionally-motivated learning. PMID:17544303

  17. Activation of D4 dopamine receptor decreases AT1 angiotensin II receptor expression in rat renal proximal tubule cells

    PubMed Central

    Chen, Ken; Deng, Kun; Wang, Xiaoyan; Wang, Zhen; Zheng, Shuo; Ren, Hongmei; He, Duofen; Han, Yu; Asico, Laureano D.; Jose, Pedro A.; Zeng, Chunyu

    2014-01-01

    The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D4 dopamine receptor gene in mice produces hypertension that is associated with increased renal AT1 receptor expression. We hypothesize that the D4 receptor can inhibit AT1 receptor expression and function in renal proximal tubules (RPTs) cells from Wistar-Kyoto (WKY) rats but the D4 receptor regulation of AT1 receptor is aberrant in RPT cells from spontaneously hypertensive rats (SHRs). The D4 receptor agonist, PD168077, decreased AT1 receptor protein expression in a time and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT1 receptor protein expression. The inhibitory effect of D4 receptor on AT1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D4 receptor-mediated signaling pathway. Angiotensin II increased Na+-K+ ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na+-K+ ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D4 receptor on angiotensin II-mediated stimulation of Na+-K+ ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT1 receptor on Na+-K+ ATPase activity in SHR cells. This was confirmed in vivo; pre-treatment with PD128077 for one week augmented the anti-hypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D4 and AT1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension. PMID:25368031

  18. The transient receptor potential ankyrin-1 mediates mechanical hyperalgesia induced by the activation of B1 receptor in mice.

    PubMed

    Meotti, Flavia Carla; Figueiredo, Cláudia Pinto; Manjavachi, Marianne; Calixto, João B

    2017-02-01

    The kinin receptor B 1 and the transient receptor potential ankyrin 1 (TRPA1) work as initiators and gatekeepers of nociception and inflammation. This study reports that the nociceptive transmission induced by activation of B 1 receptor is dependent on TRPA1 ion channel. The mechanical hyperalgesia was induced by intrathecal (i.t.) injection of B 1 agonist des-Arginine 9 -bradykinin (DABK) or TRPA1 agonist cinnamaldehyde and was evaluated by the withdrawal response after von Frey Hair application in the hind paw. After behavioral experiments, lumbar spinal cord and dorsal root ganglia (DRG) were harvested to assess protein expression and mRNA by immunohistochemistry and real time-PCR, respectively. The pharmacological antagonism (HC030031) or the down-regulation of TRPA1 greatly inhibited the mechanical hyperalgesia induced by DABK. Intrathecal injection of DABK up regulated the ionized calcium binding adaptor molecule (Iba-1) in lumbar spinal cord (L5-L6); TRPA1 protein and mRNA in lumbar spinal cord; and B 1 receptor mRNA in both lumbar spinal cord and DRG. The knockdown of TRPA1 prevented microglia activation induced by DABK. Furthermore, the mechanical hyperalgesia induced by either DABK or by cinnamaldehyde was significantly reduced by inhibition of cyclooxygenase (COX), protein kinase C (PKC) or phospholipase C (PLC). In summary, this study revealed that TRPA1 positively modulates the mechanical hyperalgesia induced by B 1 receptor activation in the spinal cord and that the classical GPCR downstream molecules PLC, diacylglycerol (DAG), 3,4,5-inositide phosphate (IP 3 ) and PKC are involved in the nociceptive transmission triggered by these two receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Nonpeptide vasopressin receptor antagonists: development of selective and orally active V1a, V2 and V1b receptor ligands.

    PubMed

    Serradeil-Le Gal, C; Wagnon, J; Valette, G; Garcia, G; Pascal, M; Maffrand, J P; Le Fur, G

    2002-01-01

    The involvement of vasopressin (AVP) in several pathological states has been reported recently and the selective blockade of the different AVP receptors could offer new clinical perspectives. During the past few years, various selective, orally active AVP V1a (OPC-21268, SR49059 (Relcovaptan)), V2 (OPC-31260, OPC-41061 (Tolvaptan), VPA-985 (Lixivaptan), SR121463, VP-343, FR-161282) and mixed V1a/V2 (YM-087 (Conivaptan), JTV-605, CL-385004) receptor antagonists have been intensively studied in various animal models and have reached, Phase IIb clinical trials for some of them. For many years now, our laboratory has focused on the identification of nonpeptide vasopressin antagonists with suitable oral bioavailability. Using random screening on small molecule libraries, followed by rational SAR and modelization, we identified a chemical series of 1-phenylsulfonylindolines which first yielded SR49059, a V1a receptor antagonist prototype. This compound displayed high affinity for animal and human V1a receptors and antagonized various V1a AVP-induced effects in vitro and in vivo (intracellular [Ca2+] increase, platelet aggregation, vascular smooth muscle cell proliferation, hypertension and coronary vasospasm). We and others have used this compound to study the role of AVP in various animal models. Recent findings from clinical trials show a potential interest for SR49059 in the treatment of dysmenorrhea and in Raynaud's disease. Structural modifications and simplifications performed in the SR49059 chemical series yielded highly specific V2 receptor antagonists (N-arylsulfonyl-oxindoles), amongst them SR121463 which possesses powerful oral aquaretic properties in various animal species and in man. SR121463 is well-tolerated and dose-dependently increases urine output and decreases urine osmolality. It induces free water-excretion without affecting electrolyte balance in contrast to classical diuretics (e.g. furosemide and hydrochlorothiazide). Notably, in cirrhotic rats

  20. Activation of orphan nuclear constitutive androstane receptor requires subnuclear targeting by peroxisome proliferator-activated receptor gamma coactivator-1 alpha. A possible link between xenobiotic response and nutritional state.

    PubMed

    Shiraki, Takuma; Sakai, Noriko; Kanaya, Eiko; Jingami, Hisato

    2003-03-28

    In contrast to the classical nuclear receptors, the constitutive androstane receptor (CAR) is transcriptionally active in the absence of ligand. In the course of searching for the mediator of CAR activation, we found that ligand-independent activation of CAR was achieved in cooperation with the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha). PGC-1 beta, a PGC-1 alpha homologue, also activated CAR to less of an extent than PGC-1 alpha. Coexpression of the ligand-binding domain of a heterodimerization partner, retinoid X receptor alpha, enhanced the PGC-1 alpha-mediated activation of CAR, although it had a weak effect on the basal activity of CAR in the absence of PGC-1 alpha. Both the N-terminal region, with the LXXLL motif, and the C-terminal region, with a serine/arginine-rich domain (RS domain), in PGC-1 alpha were required for full activation of CAR. Pull-down experiments using recombinant proteins revealed that CAR directly interacted with both the LXXLL motif and the RS domain. Furthermore, we demonstrated that the RS domain of PGC-1 alpha was required for CAR localization at nuclear speckles. These results indicate that PGC-1 alpha mediates the ligand-independent activation of CAR by means of subnuclear targeting through the RS domain of PGC-1 alpha.

  1. Binding of Losartan to Angiotensin AT1 Receptors Increases Dopamine D1 Receptor Activation

    PubMed Central

    Li, Dong; Scott, Lena; Crambert, Susanne; Zelenin, Sergey; Eklöf, Ann-Christine; Di Ciano, Luis; Ibarra, Fernando

    2012-01-01

    Signaling through both angiotensin AT1 receptors (AT1R) and dopamine D1 receptors (D1R) modulates renal sodium excretion and arterial BP. AT1R and D1R form heterodimers, but whether treatment with AT1R antagonists functionally modifies D1R via allosterism is unknown. In this study, the AT1R antagonist losartan strengthened the interaction between AT1R and D1R and increased expression of D1R on the plasma membrane in vitro. In rat proximal tubule cells that express endogenous AT1R and D1R, losartan increased cAMP generation. Losartan increased cAMP in HEK 293a cells transfected with both AT1R and D1R, but it did not increase cAMP in cells transfected with either receptor alone, suggesting that losartan induces D1R activation. Furthermore, losartan did not increase cAMP in HEK 293a cells expressing AT1R and mutant S397/S398A D1R, which disrupts the physical interaction between AT1R and D1R. In vivo, administration of a D1R antagonist significantly attenuated the antihypertensive effect of losartan in rats with renal hypertension. Taken together, these data imply that losartan might exert its antihypertensive effect both by inhibiting AT1R signaling and by enhancing D1R signaling. PMID:22193384

  2. Noninvasive detection of activating estrogen receptor 1 (ESR1) mutations in estrogen receptor-positive metastatic breast cancer.

    PubMed

    Guttery, David S; Page, Karen; Hills, Allison; Woodley, Laura; Marchese, Stephanie D; Rghebi, Basma; Hastings, Robert K; Luo, Jinli; Pringle, J Howard; Stebbing, Justin; Coombes, R Charles; Ali, Simak; Shaw, Jacqueline A

    2015-07-01

    Activating mutations in the estrogen receptor 1 (ESR1) gene are acquired on treatment and can drive resistance to endocrine therapy. Because of the spatial and temporal limitations of needle core biopsies, our goal was to develop a highly sensitive, less invasive method of detecting activating ESR1 mutations via circulating cell-free DNA (cfDNA) and tumor cells as a "liquid biopsy." We developed a targeted 23-amplicon next-generation sequencing (NGS) panel for detection of hot-spot mutations in ESR1, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), tumor protein p53 (TP53), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 2 (FGFR2) in 48 patients with estrogen receptor-α-positive metastatic breast cancer who were receiving systemic therapy. Selected mutations were validated using droplet digital PCR (ddPCR). Nine baseline cfDNA samples had an ESR1 mutation. NGS detected 3 activating mutations in ESR1, and 3 hot-spot mutations in PIK3CA, and 3 in TP53 in baseline cfDNA, and the ESR1 p.D538G mutation in 1 matched circulating tumor cell sample. ddPCR analysis was more sensitive than NGS and identified 6 additional baseline cfDNA samples with the ESR1 p.D538G mutation at a frequency of <1%. In serial blood samples from 11 patients, 4 showed changes in cfDNA, 2 with emergence of a mutation in ESR1. We also detected a low frequency ESR1 mutation (1.3%) in cfDNA of 1 primary patient who was thought to have metastatic disease but was clear by scans. Early identification of ESR1 mutations by liquid biopsy might allow for cessation of ineffective endocrine therapies and switching to other treatments, without the need for tissue biopsy and before the emergence of metastatic disease. © 2015 American Association for Clinical Chemistry.

  3. Mechanisms of dopamine D1 receptor-mediated ERK1/2 activation in the parkinsonian striatum and their modulation by metabotropic glutamate receptor type 5.

    PubMed

    Fieblinger, Tim; Sebastianutto, Irene; Alcacer, Cristina; Bimpisidis, Zisis; Maslava, Natallia; Sandberg, Sabina; Engblom, David; Cenci, M Angela

    2014-03-26

    In animal models of Parkinson's disease, striatal overactivation of ERK1/2 via dopamine (DA) D1 receptors is the hallmark of a supersensitive molecular response associated with dyskinetic behaviors. Here we investigate the pathways involved in D1 receptor-dependent ERK1/2 activation using acute striatal slices from rodents with unilateral 6-hydroxydopamine (6-OHDA) lesions. Application of the dopamine D1-like receptor agonist SKF38393 induced ERK1/2 phosphorylation and downstream signaling in the DA-denervated but not the intact striatum. This response was mediated through a canonical D1R/PKA/MEK1/2 pathway and independent of ionotropic glutamate receptors but blocked by antagonists of L-type calcium channels. Coapplication of an antagonist of metabotropic glutamate receptor type 5 (mGluR5) or its downstream signaling molecules (PLC, PKC, IP3 receptors) markedly attenuated SKF38393-induced ERK1/2 activation. The role of striatal mGluR5 in D1-dependent ERK1/2 activation was confirmed in vivo in 6-OHDA-lesioned animals treated systemically with SKF38393. In one experiment, local infusion of the mGluR5 antagonist MTEP in the DA-denervated rat striatum attenuated the activation of ERK1/2 signaling by SKF38393. In another experiment, 6-OHDA lesions were applied to transgenic mice with a cell-specific knockdown of mGluR5 in D1 receptor-expressing neurons. These mice showed a blunted striatal ERK1/2 activation in response to SFK38393 treatment. Our results reveal that D1-dependent ERK1/2 activation in the DA-denervated striatum depends on a complex interaction between PKA- and Ca(2+)-dependent signaling pathways that is critically modulated by striatal mGluR5.

  4. Ability of the new AT1 receptor blocker azilsartan to block angiotensin II-induced AT1 receptor activation after wash-out.

    PubMed

    Miura, Shin-ichiro; Matsuo, Yoshino; Nakayama, Asuka; Tomita, Sayo; Suematsu, Yasunori; Saku, Keijiro

    2014-03-01

    The recently approved angiotensin II (Ang II) type 1 (AT1) receptor blocker (ARB) azilsartan strongly reduces blood pressure (BP) in patients with hypertension. We previously reported that azilsartan showed unique binding behavior to the AT1 receptor because of its 5-oxo-1,2,4-oxadiazole moiety. However, the ability of azilsartan to block Ang II-dependent AT1 receptor activation is not yet clear. Azilsartan and a derivative of azilsartan (azilsartan-7H) that lacks a carboxyl group at the benzimidazole ring were used. Ang II-induced inositol phosphate (IP) production and extracellular signal-regulated kinase (ERK) activation were analyzed in a cell-based wash-out assay. Azilsartan, but not azilsartan-7H, completely blocked Ang II-induced IP production and ERK activation. Our previous report demonstrated that azilsartan mainly interacts with Tyr(113), Lys(199), and Gln(257) in the AT1 receptor. The interactions between azilsartan and Tyr(113) and Gln(257), but not Lys(199), were critical for blocking Ang II-induced IP production and ERK activation after wash-out. Although our findings regarding the molecule-specific effects of azilsartan are based on basic research, they may lead to an exciting insight into the mechanism of azilsartan.

  5. Hypoxia-inducible factor-1α in vascular smooth muscle regulates blood pressure homeostasis through a peroxisome proliferator-activated receptor-γ-angiotensin II receptor type 1 axis.

    PubMed

    Huang, Yan; Di Lorenzo, Annarita; Jiang, Weidong; Cantalupo, Anna; Sessa, William C; Giordano, Frank J

    2013-09-01

    Hypertension is a major worldwide health issue for which only a small proportion of cases have a known mechanistic pathogenesis. Of the defined causes, none have been directly linked to heightened vasoconstrictor responsiveness, despite the fact that vasomotor tone in resistance vessels is a fundamental determinant of blood pressure. Here, we reported a previously undescribed role for smooth muscle hypoxia-inducible factor-1α (HIF-1α) in controlling blood pressure homeostasis. The lack of HIF-1α in smooth muscle caused hypertension in vivo and hyperresponsiveness of resistance vessels to angiotensin II stimulation ex vivo. These data correlated with an increased expression of angiotensin II receptor type I in the vasculature. Specifically, we show that HIF-1α, through peroxisome proliferator-activated receptor-γ, reciprocally defined angiotensin II receptor type I levels in the vessel wall. Indeed, pharmacological blockade of angiotensin II receptor type I by telmisartan abolished the hypertensive phenotype in smooth muscle cell-HIF-1α-KO mice. These data revealed a determinant role of a smooth muscle HIF-1α/peroxisome proliferator-activated receptor-γ/angiotensin II receptor type I axis in controlling vasomotor responsiveness and highlighted an important pathway, the alterations of which may be critical in a variety of hypertensive-based clinical settings.

  6. Activation of the innate immune receptor Dectin-1 upon formation of a “phagocytic synapse”

    PubMed Central

    Goodridge, Helen S.; Reyes, Christopher N.; Becker, Courtney A.; Katsumoto, Tamiko R.; Ma, Jun; Wolf, Andrea J.; Bose, Nandita; Chan, Anissa S. H.; Magee, Andrew S.; Danielson, Michael E.; Weiss, Arthur; Vasilakos, John P.; Underhill, David M.

    2011-01-01

    Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 is a pattern recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects β-glucans in fungal cell walls and triggers direct cellular anti-microbial activity, including phagocytosis and production of reactive oxygen species1, 2. In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that despite its ability to bind both soluble and particulate β-glucan polymers, Dectin-1 signalling is only activated by particulate β-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 are excluded (Supplementary Figure 1). The “phagocytic synapse” now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular anti-microbial responses only when they are required. PMID:21525931

  7. [Effect of 5-HT1A receptors in the hippocampal DG on active avoidance learning in rats].

    PubMed

    Jiang, Feng-ze; Lv, Jing; Wang, Dan; Jiang, Hai-ying; Li, Ying-shun; Jin, Qing-hua

    2015-01-01

    To investigate the effects of serotonin (5-HTIA) receptors in the hippocampal dentate gyrus (DG) on active avoidance learning in rats. Totally 36 SD rats were randomly divided into control group, antagonist group and agonist group(n = 12). Active avoidance learning ability of rats was assessed by the shuttle box. The extracellular concentrations of 5-HT in the DG during active avoidance conditioned reflex were measured by microdialysis and high performance liquid chromatography (HPLC) techniques. Then the antagonist (WAY-100635) or agonist (8-OH-DPAT) of the 5-HT1A receptors were microinjected into the DG region, and the active avoidance learning was measured. (1) During the active avoidance learning, the concentration of 5-HT in the hippocampal DG was significantly increased in the extinction but not establishment in the conditioned reflex, which reached 164.90% ± 26.07% (P <0.05) of basal level. (2) The microinjection of WAY-100635 (an antagonist of 5-HT1A receptor) into the DG did not significantly affect the active avoidance learning. (3) The microinjection of 8-OH-DPAT(an agonist of 5-HT1A receptor) into the DG significantly facilitated the establishment process and inhibited the extinction process during active avoidance conditioned reflex. The data suggest that activation of 5-HT1A receptors in hipocampal DG may facilitate active avoidance learning and memory in rats.

  8. Activation of EGF Receptor Kinase by L1-mediated Homophilic Cell Interactions

    PubMed Central

    Islam, Rafique; Kristiansen, Lars V.; Romani, Susana; Garcia-Alonso, Luis; Hortsch, Michael

    2004-01-01

    Neural cell adhesion molecules (CAMs) are important players during neurogenesis and neurite outgrowth as well as axonal fasciculation and pathfinding. Some of these developmental processes entail the activation of cellular signaling cascades. Pharmacological and genetic evidence indicates that the neurite outgrowth-promoting activity of L1-type CAMs is at least in part mediated by the stimulation of neuronal receptor tyrosine kinases (RTKs), especially FGF and EGF receptors. It has long been suspected that neural CAMs might physically interact with RTKs, but their activation by specific cell adhesion events has not been directly demonstrated. Here we report that gain-of-function conditions of the Drosophila L1-type CAM Neuroglian result in profound sensory axon pathfinding defects in the developing Drosophila wing. This phenotype can be suppressed by decreasing the normal gene dosage of the Drosophila EGF receptor gene. Furthermore, in Drosophila S2 cells, cell adhesion mediated by human L1-CAM results in the specific activation of human EGF tyrosine kinase at cell contact sites and EGF receptors engage in a physical interaction with L1-CAM molecules. Thus L1-type CAMs are able to promote the adhesion-dependent activation of EGF receptor signaling in vitro and in vivo. PMID:14718570

  9. Activation of EGF receptor kinase by L1-mediated homophilic cell interactions.

    PubMed

    Islam, Rafique; Kristiansen, Lars V; Romani, Susana; Garcia-Alonso, Luis; Hortsch, Michael

    2004-04-01

    Neural cell adhesion molecules (CAMs) are important players during neurogenesis and neurite outgrowth as well as axonal fasciculation and pathfinding. Some of these developmental processes entail the activation of cellular signaling cascades. Pharmacological and genetic evidence indicates that the neurite outgrowth-promoting activity of L1-type CAMs is at least in part mediated by the stimulation of neuronal receptor tyrosine kinases (RTKs), especially FGF and EGF receptors. It has long been suspected that neural CAMs might physically interact with RTKs, but their activation by specific cell adhesion events has not been directly demonstrated. Here we report that gain-of-function conditions of the Drosophila L1-type CAM Neuroglian result in profound sensory axon pathfinding defects in the developing Drosophila wing. This phenotype can be suppressed by decreasing the normal gene dosage of the Drosophila EGF receptor gene. Furthermore, in Drosophila S2 cells, cell adhesion mediated by human L1-CAM results in the specific activation of human EGF tyrosine kinase at cell contact sites and EGF receptors engage in a physical interaction with L1-CAM molecules. Thus L1-type CAMs are able to promote the adhesion-dependent activation of EGF receptor signaling in vitro and in vivo.

  10. A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor.

    PubMed

    Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A; Iverson, T M; Meiler, Jens; Hamm, Heidi E

    2016-09-09

    G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the α5 helix of Gαi1, especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in Gαi1 by inserting 4 alanine amino acids into the α5 helix between residues Gln(333) and Phe(334) (Ins4A). This extends the length of the α5 helix without disturbing the β6-α5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His(57), Phe(189), Phe(191), and Phe(336); it also disturbs the rotation of the α5 helix and the π-π interaction between His(57) and Phe(189) In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between α5, α1, and β2-β3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the α5 helix for G protein release from the receptor after GTP binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Orally active vasopressin V1a receptor antagonist, SRX251, selectively blocks aggressive behavior.

    PubMed

    Ferris, Craig F; Lu, Shi-Fang; Messenger, Tara; Guillon, Christophe D; Heindel, Ned; Miller, Marvin; Koppel, Gary; Robert Bruns, F; Simon, Neal G

    2006-02-01

    Arginine vasopressin functions as a neurochemical signal in the brain to affect social behavior. There is an expanding literature from animal and human studies showing that vasopressin, through the vasopressin 1A receptor (V1A), can stimulate aggressive behavior. Using a novel monocylic beta lactam platform, a series of orally active vasopressin V1a antagonists was developed with high affinity for the human receptor. SRX251 was chosen from this series of V1a antagonists to screen for effects on serenic activity in a resident-intruder model of offensive aggression. Resident, male Syrian golden hamsters were given oral doses of SRX251 or intraperitoneal Manning compound, a selective V1a receptor antagonist with reduced brain penetrance, at doses of 0.2 microg, 20 microg, 2 mg/kg or vehicle. When tested 90-120 min later, SRX251, but not Manning compound, caused a significant dose-dependent reduction in offensive aggression toward intruders as measured by latency to bite and number of bites. The reduction in aggression persisted for over 6 h and was no longer present 12 h post treatment. SRX251 did not alter the amount of time the resident investigated the intruder, olfactory communication, general motor activity, or sexual motivation. These data corroborate previous studies showing a role for vasopressin neurotransmission in aggression and suggest that V1a receptor antagonists may be used to treat interpersonal violence co-occurring with such illness as ADHD, autism, bipolar disorder, and substance abuse.

  12. Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation.

    PubMed Central

    Scheer, A; Fanelli, F; Costa, T; De Benedetti, P G; Cotecchia, S

    1996-01-01

    Site-directed mutagenesis and molecular dynamics simulations of the alpha 1B-adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B-AR to build a theoretical model which defines the essential features of R and R. The results of site-directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B-AR. (ii) The shift of R143 of the DRY sequence out of a conserved 'polar pocket' formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B-AR. Our findings might provide interesting generalities about the activation process of G protein-coupled receptors. Images PMID:8670860

  13. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

    PubMed

    Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike

    2013-01-01

    HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

  14. Cross-talk between an activator of nuclear receptors-mediated transcription and the D1 dopamine receptor signaling pathway.

    PubMed

    Schmidt, Azriel; Vogel, Robert; Rutledge, Su Jane; Opas, Evan E; Rodan, Gideon A; Friedman, Eitan

    2005-03-01

    Nuclear receptors are transcription factors that usually interact, in a ligand-dependent manner, with specific DNA sequences located within promoters of target genes. The nuclear receptors can also be controlled in a ligand-independent manner via the action of membrane receptors and cellular signaling pathways. 5-Tetradecyloxy-2-furancarboxylic acid (TOFA) was shown to stimulate transcription from the MMTV promoter via chimeric receptors that consist of the DNA binding domain of GR and the ligand binding regions of the PPARbeta or LXRbeta nuclear receptors (GR/PPARbeta and GR/LXRbeta). TOFA and hydroxycholesterols also modulate transcription from NF-kappaB- and AP-1-controlled reporter genes and induce neurite differentiation in PC12 cells. In CV-1 cells that express D(1) dopamine receptors, D(1) dopamine receptor stimulation was found to inhibit TOFA-stimulated transcription from the MMTV promoter that is under the control of chimeric GR/PPARbeta and GR/LXRbeta receptors. Treatment with the D(1) dopamine receptor antagonist, SCH23390, prevented dopamine-mediated suppression of transcription, and by itself increased transcription controlled by GR/LXRbeta. Furthermore, combined treatment of CV-1 cells with TOFA and SCH23390 increased transcription controlled by the GR/LXRbeta chimeric receptor synergistically. The significance of this in vitro synergy was demonstrated in vivo, by the observation that SCH23390 (but not haloperidol)-mediated catalepsy in rats was potentiated by TOFA, thus showing that an agent that mimics the in vitro activities of compounds that activate members of the LXR and PPAR receptor families can influence D1 dopamine receptor elicited responses.

  15. TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation.

    PubMed

    Reis, Carlos R; Chen, Ping-Hung; Bendris, Nawal; Schmid, Sandra L

    2017-01-17

    Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand-death receptor (TRAIL-DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL-DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL-DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors.

  16. TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation

    PubMed Central

    Reis, Carlos R.; Chen, Ping-Hung; Bendris, Nawal; Schmid, Sandra L.

    2017-01-01

    Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand–death receptor (TRAIL–DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL–DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL–DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors. PMID:28049841

  17. AN ANGIOTENSIN II TYPE 1 RECEPTOR ACTIVATION SWITCH PATCH REVEALED THROUGH EVOLUTIONARY TRACE ANALYSIS

    PubMed Central

    Bonde, Marie Mi; Yao, Rong; Ma, Jian-Nong; Madabushi, Srinivasan; Haunsø, Stig; Burstein, Ethan S.; Whistler, Jennifer L.; Sheikh, Søren P.; Lichtarge, Olivier; Hansen, Jakob Lerche

    2010-01-01

    Seven transmembrane (7TM) or G protein-coupled receptors constitute a large superfamily of cell surface receptors sharing a structural motif of seven transmembrane spanning alpha helices. Their activation mechanism most likely involves concerted movements of the transmembrane helices, but remains to be completely resolved. Evolutionary Trace (ET) analysis is a computational method, which identifies clusters of functionally important residues by integrating information on evolutionary important residue variations with receptor structure. Combined with known mutational data, ET predicted a patch of residues in the cytoplasmic parts of TM2, TM3, and TM6 to form an activation switch that is common to all family A 7TM receptors. We tested this hypothesis in the rat Angiotensin II (Ang II) type 1 (AT1) receptor. The receptor has important roles in the cardiovascular system, but has also frequently been applied as a model for 7TM receptor activation and signaling. Six mutations: F66A, L67R, L70R, L119R, D125A, and I245F were targeted to the putative switch and assayed for changes in activation state by their ligand binding, signaling, and trafficking properties. All but one receptor mutant (that was not expressed well) displayed phenotypes associated with changed activation state, such as increased agonist affinity or basal activity, promiscuous activation, or constitutive internalization highlighting the importance of testing different signaling pathways. We conclude that this evolutionary important patch mediates interactions important for maintaining the inactive state. More broadly, these observations in the AT1 receptor are consistent with computational predictions of a generic role for this patch in 7TM receptor activation. PMID:20227396

  18. The hallucinogen d-lysergic diethylamide (LSD) decreases dopamine firing activity through 5-HT1A, D2 and TAAR1 receptors.

    PubMed

    De Gregorio, Danilo; Posa, Luca; Ochoa-Sanchez, Rafael; McLaughlin, Ryan; Maione, Sabatino; Comai, Stefano; Gobbi, Gabriella

    2016-11-01

    d-lysergic diethylamide (LSD) is a hallucinogenic drug that interacts with the serotonin (5-HT) system binding to 5-HT 1 and 5-HT 2 receptors. Little is known about its potential interactions with the dopamine (DA) neurons of the ventral tegmental area (VTA). Using in-vivo electrophysiology in male adult rats, we evaluated the effects of cumulative doses of LSD on VTA DA neuronal activity, compared these effects to those produced on 5-HT neurons in the dorsal raphe nucleus (DRN), and attempted to identify the mechanism of action mediating the effects of LSD on VTA DA neurons. LSD, at low doses (5-20μg/kg, i.v.) induced a significant decrease of DRN 5-HT firing activity through 5-HT 2A and D 2 receptors. At these low doses, LSD did not alter VTA DA neuronal activity. On the contrary, at higher doses (30-120μg/kg, i.v.), LSD dose-dependently decreased VTA DA firing activity. The depletion of 5-HT with p-chlorophenylalanine did not modulate the effects of LSD on DA firing activity. The inhibitory effects of LSD on VTA DA firing activity were prevented by the D 2 receptor antagonist haloperidol (50μg/kg, i.v.) and by the 5-HT 1A receptor antagonist WAY-100,635 (500μg/kg, i.v.). Notably, pretreatment with the trace amine-associate receptor 1 (TAAR 1 ) antagonist EPPTB (5mg/kg, i.v.) blocked the inhibitory effect of LSD on VTA DA neurons. These results suggest that LSD at high doses strongly affects DA mesolimbic neuronal activity in a 5-HT independent manner and with a pleiotropic mechanism of action involving 5-HT 1A, D 2 and TAAR 1 receptors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor

    PubMed Central

    Cherian, Milu T; Lin, Wenwei; Wu, Jing

    2015-01-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. PMID:25762023

  20. Activation of Akt by Advanced Glycation End Products (AGEs): Involvement of IGF-1 Receptor and Caveolin-1

    PubMed Central

    Yang, Su-Jung; Chen, Chen-Yu; Chang, Geen-Dong; Wen, Hui-Chin; Chen, Ching-Yu; Chang, Shi-Chuan; Liao, Jyh-Fei; Chang, Chung-Ho

    2013-01-01

    Diabetes is characterized by chronic hyperglycemia, which in turn facilitates the formation of advanced glycation end products (AGEs). AGEs activate signaling proteins such as Src, Akt and ERK1/2. However, the mechanisms by which AGEs activate these kinases remain unclear. We examined the effect of AGEs on Akt activation in 3T3-L1 preadipocytes. Addition of AGEs to 3T3-L1 cells activated Akt in a dose- and time-dependent manner. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor DPI, suggesting the involvement of Src and NAD(P)H oxidase in the activation of PI3-kinase-Akt pathway by AGEs. AGEs-stimulated Src tyrosine phosphorylation was inhibited by NAC, suggesting that Src is downstream of NAD(P)H oxidase. The AGEs-stimulated Akt activity was sensitive to Insulin-like growth factor 1 receptor (IGF-1R) kinase inhibitor AG1024. Furthermore, AGEs induced phosphorylation of IGF-1 receptorβsubunit (IGF-1Rβ) on Tyr1135/1136, which was sensitive to PP2, indicating that AGEs stimulate Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by β-methylcyclodextrin that abolishes the structure of caveolae, and by lowering caveolin-1 (Cav-1) levels with siRNAs. Furthermore, addition of AGEs enhanced the interaction of phospho-Cav-1 with IGF-1Rβ and transfection of 3T3-L1 cells with Cav-1 Y14F mutants inhibited the activation of Akt by AGEs. These results suggest that AGEs activate NAD(P)H oxidase and Src which in turn phosphorylates IGF-1 receptor and Cav-1 leading to activation of IGF-1 receptor and the downstream Akt in 3T3-L1 cells. AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes and addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis, suggesting that IGF-1 receptor, PI3

  1. Structure and dynamics of a constitutively active neurotensin receptor

    PubMed Central

    Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo; Botos, Istvan; White, Courtney F.; Du, Haijuan; Vaidehi, Nagarajan; Grisshammer, Reinhard

    2016-01-01

    Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist. PMID:27924846

  2. Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

    PubMed Central

    Coopman, K.; Wallis, R.; Robb, G.; Brown, A. J. H.; Wilkinson, G. F.; Timms, D.

    2011-01-01

    The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues. PMID:21868452

  3. Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-Containing NMDA Receptors

    PubMed Central

    Li, Li-Jun; Hu, Rong; Lujan, Brendan; Chen, Juan; Zhang, Jian-Jian; Nakano, Yasuko; Cui, Tian-Yuan; Liao, Ming-Xia; Chen, Jin-Cao; Man, Heng-Ye; Feng, Hua; Wan, Qi

    2016-01-01

    NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs), but not GluN2B-containing NMDA receptors (GluN2BRs), to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation. PMID:27807405

  4. An intrinsic agonist mechanism for activation of glucagon-like peptide-1 receptor by its extracellular domain

    PubMed Central

    Yin, Yanting; Zhou, X Edward; Hou, Li; Zhao, Li-Hua; Liu, Bo; Wang, Gaihong; Jiang, Yi; Melcher, Karsten; Xu, H Eric

    2016-01-01

    The glucagon-like peptide-1 receptor is a class B G protein coupled receptor (GPCR) that plays key roles in glucose metabolism and is a major therapeutic target for diabetes. The classic two-domain model for class B GPCR activation proposes that the apo-state receptor is auto-inhibited by its extracellular domain, which physically interacts with the transmembrane domain. The binding of the C-terminus of the peptide hormone to the extracellular domain allows the N-terminus of the hormone to insert into the transmembrane domain to induce receptor activation. In contrast to this model, here we demonstrate that glucagon-like peptide-1 receptor can be activated by N-terminally truncated glucagon-like peptide-1 or exendin-4 when fused to the receptor, raising the question regarding the role of N-terminal residues of peptide hormone in glucagon-like peptide-1 receptor activation. Mutations of cysteine 347 to lysine or arginine in intracellular loop 3 transform the receptor into a G protein-biased receptor and allow it to be activated by a nonspecific five-residue linker that is completely devoid of exendin-4 or glucagon-like peptide-1 sequence but still requires the presence of an intact extracellular domain. Moreover, the extracellular domain can activate the receptor in trans in the presence of an intact peptide hormone, and specific mutations in three extracellular loops abolished this extracellular domain trans-activation. Together, our data reveal a dominant role of the extracellular domain in glucagon-like peptide-1 receptor activation and support an intrinsic agonist model of the extracellular domain, in which peptide binding switches the receptor from the auto-inhibited state to the auto-activated state by releasing the intrinsic agonist activity of the extracellular domain. PMID:27917297

  5. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

    PubMed

    Zhao, Lan-Xue; Ge, Yan-Hui; Xiong, Cai-Hong; Tang, Ling; Yan, Ying-Hui; Law, Ping-Yee; Qiu, Yu; Chen, Hong-Zhuan

    2018-03-06

    M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

  6. Trigeminal Medullary Dorsal Horn Neurons Activated by Nasal Stimulation Coexpress AMPA, NMDA, and NK1 Receptors

    PubMed Central

    McCulloch, P. F.; DiNovo, K. M.; Westerhaus, D. J.; Vizinas, T. A.; Peevey, J. F.; Lach, M. A.; Czarnocki, P.

    2013-01-01

    Afferent information initiating the cardiorespiratory responses during nasal stimulation projects from the nasal passages to neurons within the trigeminal medullary dorsal horn (MDH) via the anterior ethmoidal nerve (AEN). Central AEN terminals are thought to release glutamate to activate the MDH neurons. This study was designed to determine which neurotransmitter receptors (AMPA, kainate, or NMDA glutamate receptor subtypes or the Substance P receptor NK1) are expressed by these activated MDH neurons. Fos was used as a neuronal marker of activated neurons, and immunohistochemistry combined with epifluorescent microscopy was used to determine which neurotransmitter receptor subunits were coexpressed by activated MDH neurons. Results indicate that, during nasal stimulation with ammonia vapors in urethane-anesthetized Sprague-Dawley rats, activated neurons within the superficial MDH coexpress the AMPA glutamate receptor subunits GluA1 (95.8%) and GluA2/3 (88.2%), the NMDA glutamate receptor subunits GluN1 (89.1%) and GluN2A (41.4%), and NK1 receptors (64.0%). It is therefore likely that during nasal stimulation the central terminals of the AEN release glutamate and substance P that then produces activation of these MDH neurons. The involvement of AMPA and NMDA receptors may mediate fast and slow neurotransmission, respectively, while NK1 receptor involvement may indicate activation of a nociceptive pathway. PMID:24967301

  7. Structure and dynamics of a constitutively active neurotensin receptor

    DOE PAGES

    Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo; ...

    2016-12-07

    Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecularmore » dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. In conclusion, the loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.« less

  8. Structure and dynamics of a constitutively active neurotensin receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo

    Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecularmore » dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. In conclusion, the loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.« less

  9. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sato, Chieri; Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp; Kitano, Sachie

    Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murinemore » satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

  10. AMP is an adenosine A1 receptor agonist.

    PubMed

    Rittiner, Joseph E; Korboukh, Ilia; Hull-Ryde, Emily A; Jin, Jian; Janzen, William P; Frye, Stephen V; Zylka, Mark J

    2012-02-17

    Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.

  11. Isothiocyanates from Wasabia japonica activate transient receptor potential ankyrin 1 channel.

    PubMed

    Uchida, Kunitoshi; Miura, Yosuke; Nagai, Masashi; Tominaga, Makoto

    2012-11-01

    6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) and 6-(methylthio)hexyl isothiocyanate (6-MTITC) have low pungency and are responsible for the fresh flavor of wasabi (Wasabia japonica [Miq] Matsumura). In this study, we found that these two isothiocyanates activate transient receptor potential ankyrin 1 (TRPA1), and 6-MSITC activates transient receptor potential vanilloid 1 (TRPV1), but not other transient receptor potential channels expressed in sensory neurons. Both 6-MSITC and 6-MTITCinduced intracellular Ca(2+) increases in human embryonic kidney-derived 293 cells expressing mouse TRPA1 (mTRPA1) as measured by Ca(2+) imaging. In whole-cell patch-clamp recordings, 6-MSITC and 6-MTITC dose-dependently activated both mTRPA1 (EC(50) = 147±26 µM for 6-MSITC and 30±3 µM for 6-MTITC) and human TRPA1 (hTRPA1; EC(50) = 39±4 µM for 6-MSITC and 34±3 µM for 6-MTITC). In addition, TRPA1 N-terminal cysteines, which are reported to be important for channel activation by electrophilic ligands, were involved in 6-MSITC- and 6-MTITC-evoked TRPA1 activation. These isothiocyanates also activated endogenous TRPA1 expressed in mouse dorsal root ganglion neurons and intraplantar injection of 10-30 mM 6-MSITC-evoked pain-related behaviors in mice. These results indicate the following: 1) 6-MSITC and 6-MTITC activate both mTRPA1 and hTRPA1; 2) 6-MSITC activates mTRPV1; and 3) the pharmacological functions of these isothiocyanates could be derived from TRPA1 activation.

  12. Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors.

    PubMed

    Mills, Ian G; Gaughan, Luke; Robson, Craig; Ross, Theodora; McCracken, Stuart; Kelly, John; Neal, David E

    2005-07-18

    Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription.

  13. Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors

    PubMed Central

    Mills, Ian G.; Gaughan, Luke; Robson, Craig; Ross, Theodora; McCracken, Stuart; Kelly, John; Neal, David E.

    2005-01-01

    Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription. PMID:16027218

  14. Activation of Peroxisome Proliferator–Activated Receptor β/δ Induces Lung Cancer Growth via Peroxisome Proliferator–Activated Receptor Coactivator γ-1α

    PubMed Central

    Han, ShouWei; Ritzenthaler, Jeffrey D.; Sun, XiaoJuan; Zheng, Ying; Roman, Jesse

    2009-01-01

    We previously demonstrated that a selective agonist of peroxisome proliferator–activated receptor β/δ (PPARβ/δ), GW501516, stimulated human non–small cell lung carcinoma (NSCLC) growth, partly through inhibition of phosphatase and tensin homolog deleted on chromosome 10 expression. Here, we show that GW501516 also decreases the phosphorylation of AMP-activated protein kinase α (AMPKα), a major regulator of energy metabolism. This was mediated through specific activation of PPARβ/δ, as a PPARβ/δ small interfering RNA inhibited the effect. However, AMPKα did not mediate the growth-promoting effects of GW501516, as silencing of AMPKα did not inhibit GW501516-induced cell proliferation. Instead, we found that GW501516 stimulated peroxisome proliferator–activated receptor coactivator γ (PGC)-1α, which activated the phosphatidylinositol 3 kinase (PI3-K)/Akt mitogenic pathway. An inhibitor of PI3-K, LY294002, had no effect on PGC-1α, consistent with PGC-1α being upstream of PI3-K/Akt. Of note, an activator of AMPKα, 5-amino-4-imidazole carboxamide riboside, inhibited the growth-promoting effects of GW501516, suggesting that although AMPKα is not responsible for the mitogenic effects of GW501516, its activation can oppose these events. This study unveils a novel mechanism by which GW501516 and activation of PPARβ/δ stimulate human lung carcinoma cell proliferation, and suggests that activation of AMPKα may oppose this effect. PMID:18776129

  15. Activation of peroxisome proliferator-activated receptor beta/delta induces lung cancer growth via peroxisome proliferator-activated receptor coactivator gamma-1alpha.

    PubMed

    Han, Shouwei; Ritzenthaler, Jeffrey D; Sun, Xiaojuan; Zheng, Ying; Roman, Jesse

    2009-03-01

    We previously demonstrated that a selective agonist of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta), GW501516, stimulated human non-small cell lung carcinoma (NSCLC) growth, partly through inhibition of phosphatase and tensin homolog deleted on chromosome 10 expression. Here, we show that GW501516 also decreases the phosphorylation of AMP-activated protein kinase alpha (AMPKalpha), a major regulator of energy metabolism. This was mediated through specific activation of PPARbeta/delta, as a PPARbeta/delta small interfering RNA inhibited the effect. However, AMPKalpha did not mediate the growth-promoting effects of GW501516, as silencing of AMPKalpha did not inhibit GW501516-induced cell proliferation. Instead, we found that GW501516 stimulated peroxisome proliferator-activated receptor coactivator gamma (PGC)-1alpha, which activated the phosphatidylinositol 3 kinase (PI3-K)/Akt mitogenic pathway. An inhibitor of PI3-K, LY294002, had no effect on PGC-1alpha, consistent with PGC-1alpha being upstream of PI3-K/Akt. Of note, an activator of AMPKalpha, 5-amino-4-imidazole carboxamide riboside, inhibited the growth-promoting effects of GW501516, suggesting that although AMPKalpha is not responsible for the mitogenic effects of GW501516, its activation can oppose these events. This study unveils a novel mechanism by which GW501516 and activation of PPARbeta/delta stimulate human lung carcinoma cell proliferation, and suggests that activation of AMPKalpha may oppose this effect.

  16. Dihydroartemisinin Exerts Its Anticancer Activity through Depleting Cellular Iron via Transferrin Receptor-1

    PubMed Central

    Ba, Qian; Zhou, Naiyuan; Duan, Juan; Chen, Tao; Hao, Miao; Yang, Xinying; Li, Junyang; Yin, Jun; Chu, Ruiai; Wang, Hui

    2012-01-01

    Artemisinin and its main active metabolite dihydroartemisinin, clinically used antimalarial agents with low host toxicity, have recently shown potent anticancer activities in a variety of human cancer models. Although iron mediated oxidative damage is involved, the mechanisms underlying these activities remain unclear. In the current study, we found that dihydroartemisinin caused cellular iron depletion in time- and concentration-dependent manners. It decreased iron uptake and disturbed iron homeostasis in cancer cells, which were independent of oxidative damage. Moreover, dihydroartemisinin reduced the level of transferrin receptor-1 associated with cell membrane. The regulation of dihydroartemisinin to transferrin receptor-1 could be reversed by nystatin, a cholesterol-sequestering agent but not the inhibitor of clathrin-dependent endocytosis. Dihydroartemisinin also induced transferrin receptor-1 palmitoylation and colocalization with caveolin-1, suggesting a lipid rafts mediated internalization pathway was involved in the process. Also, nystatin reversed the influences of dihydroartemisinin on cell cycle and apoptosis related genes and the siRNA induced downregulation of transferrin receptor-1 decreased the sensitivity to dihydroartemisinin efficiently in the cells. These results indicate that dihydroartemisinin can counteract cancer through regulating cell-surface transferrin receptor-1 in a non-classical endocytic pathway, which may be a new action mechanism of DHA independently of oxidative damage. PMID:22900042

  17. A novel mutation in the P2Y12 receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding.

    PubMed

    Patel, Y M; Lordkipanidzé, M; Lowe, G C; Nisar, S P; Garner, K; Stockley, J; Daly, M E; Mitchell, M; Watson, S P; Austin, S K; Mundell, S J

    2014-05-01

    The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic bleeding disorder expressing a homozygous P2RY(12) mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y(12) receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states. To determine the functional consequences of the R122C substitution for P2Y(12) function. We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y(12) stably expressed in cells was also performed. ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y(12) activity in the patient and family members. Cell surface R122C P2Y(12) expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor 1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity. Our study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait. © 2014 International Society on Thrombosis and Haemostasis.

  18. Selective Activation of Sphingosine 1-Phosphate Receptors 1 and 3 Promotes Local Microvascular Network Growth

    PubMed Central

    Sefcik, Lauren S.; Petrie Aronin, Caren E.; Awojoodu, Anthony O.; Shin, Soo J.; Mac Gabhann, Feilim; MacDonald, Timothy L.; Wamhoff, Brian R.; Lynch, Kevin R.; Peirce, Shayn M.

    2011-01-01

    Proper spatial and temporal regulation of microvascular remodeling is critical to the formation of functional vascular networks, spanning the various arterial, venous, capillary, and collateral vessel systems. Recently, our group has demonstrated that sustained release of sphingosine 1-phosphate (S1P) from biodegradable polymers promotes microvascular network growth and arteriolar expansion. In this study, we employed S1P receptor-specific compounds to activate and antagonize different combinations of S1P receptors to elucidate those receptors most critical for promotion of pharmacologically induced microvascular network growth. We show that S1P1 and S1P3 receptors act synergistically to enhance functional network formation via increased functional length density, arteriolar diameter expansion, and increased vascular branching in the dorsal skinfold window chamber model. FTY720, a potent activator of S1P1 and S1P3, promoted a 107% and 153% increase in length density 3 and 7 days after implantation, respectively. It also increased arteriolar diameters by 60% and 85% 3 and 7 days after implantation. FTY720-stimulated branching in venules significantly more than unloaded poly(D, L-lactic-co-glycolic acid). When implanted on the mouse spinotrapezius muscle, FTY720 stimulated an arteriogenic response characterized by increased tortuosity and collateralization of branching microvascular networks. Our results demonstrate the effectiveness of S1P1 and S1P3 receptor-selective agonists (such as FTY720) in promoting microvascular growth for tissue engineering applications. PMID:20874260

  19. AMP Is an Adenosine A1 Receptor Agonist*

    PubMed Central

    Rittiner, Joseph E.; Korboukh, Ilia; Hull-Ryde, Emily A.; Jin, Jian; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.

    2012-01-01

    Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5′-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5′-monophosphonate, ACP) directly activated the adenosine A1 receptor (A1R). In contrast, AMP only activated the adenosine A2B receptor (A2BR) after hydrolysis to adenosine by ecto-5′-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A1R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A1R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A1R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A1R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine. PMID:22215671

  20. OptoGluNAM4.1, a Photoswitchable Allosteric Antagonist for Real-Time Control of mGlu4 Receptor Activity.

    PubMed

    Rovira, Xavier; Trapero, Ana; Pittolo, Silvia; Zussy, Charleine; Faucherre, Adèle; Jopling, Chris; Giraldo, Jesús; Pin, Jean-Philippe; Gorostiza, Pau; Goudet, Cyril; Llebaria, Amadeu

    2016-08-18

    OptoGluNAM4.1, a negative allosteric modulator (NAM) of metabotropic glutamate receptor 4 (mGlu4) contains a reactive group that covalently binds to the receptor and a blue-light-activated, fast-relaxing azobenzene group that allows reversible receptor activity photocontrol in vitro and in vivo. OptoGluNAM4.1 induces light-dependent behavior in zebrafish and reverses the activity of the mGlu4 agonist LSP4-2022 in a mice model of chronic pain, defining a photopharmacological tool to better elucidate the physiological roles of the mGlu4 receptor in the nervous system. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Small-Molecule Inhibition and Activation-Loop Trans-Phosphorylation of the IGF1 Receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wu,J.; Li, W.; Craddock, B.

    2008-01-01

    The insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase (RTK) that has a critical role in mitogenic signalling during embryogenesis and an antiapoptotic role in the survival and progression of many human tumours. Here, we present the crystal structure of the tyrosine kinase domain of IGF1R (IGF1RK), in its unphosphorylated state, in complex with a novel compound, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1, 5-a]pyrazin-8-ylamine (PQIP), which we show is a potent inhibitor of both the unphosphorylated (basal) and phosphorylated (activated) states of the kinase. PQIP interacts with residues in the ATP-binding pocket and in the activation loop, which confers specificity for IGF1RK andmore » the highly related insulin receptor (IR) kinase. In this crystal structure, the IGF1RK active site is occupied by Tyr1135 from the activation loop of an symmetry (two-fold)-related molecule. This dimeric arrangement affords, for the first time, a visualization of the initial trans-phosphorylation event in the activation loop of an RTK, and provides a molecular rationale for a naturally occurring mutation in the activation loop of the IR that causes type II diabetes mellitus.« less

  2. Pituitary adenylate cyclase activating polypeptide and PAC1 receptor signaling increase Homer 1a expression in central and peripheral neurons.

    PubMed

    Girard, Beatrice M; Keller, Emily T; Schutz, Kristin C; May, Victor; Braas, Karen M

    2004-12-15

    Pituitary adenylate cyclase activating polypeptides (PACAP) and PAC1 receptor signaling have diverse roles in central and peripheral nervous system development and function. In recent microarray analyses for PACAP and PAC1 receptor modulation of neuronal transcripts, the mRNA of Homer 1a (H1a), which encodes the noncrosslinking and immediate early gene product isoform of Homer, was identified to be strongly upregulated in superior cervical ganglion (SCG) sympathetic neurons. Given the prominent roles of Homer in synaptogenesis, synaptic protein complex assembly and receptor/channel signaling, we have examined the ability for PACAP to induce H1a expression in sympathetic, cortical and hippocampal neurons to evaluate more comprehensively the roles of PACAP in synaptic function. In both central and peripheral neuronal cultures, PACAP peptides increased transiently H1a transcript levels approximately 3.5- to 6-fold. From real-time quantitative PCR measurements, the temporal patterns of PACAP-mediated H1a mRNA induction among the different neuronal cultures appeared similar although the onset of sympathetic H1a transcript expression appeared protracted. The increase in H1a transcripts was accompanied by increases in H1a protein levels. Comparative studies with VIP and PACAP(6-38) antagonist demonstrated that the PACAP effects reflected PAC1 receptor activation and signaling. The PAC1 receptor isoforms expressed in central and peripheral neurons can engage diverse intracellular second messenger systems, and studies using selective signaling pathway inhibitors demonstrated that the cyclic AMP/PKA and MEK/ERK cascades are principal mediators of the PACAP-mediated H1a induction response. In modulating H1a transcript and protein expression, these studies may implicate broad roles for PACAP and PAC1 receptor signaling in synaptic development and plasticity.

  3. Prefrontal cortical network activity: Opposite effects of psychedelic hallucinogens and D1/D5 dopamine receptor activation

    PubMed Central

    Lambe, Evelyn K.; Aghajanian, George K.

    2007-01-01

    The fine-tuning of network activity provides a modulating influence on how information is processed and interpreted in the brain. Here, we use brain slices of rat prefrontal cortex to study how recurrent network activity is affected by neuromodulators known to alter normal cortical function. We previously determined that glutamate spillover and stimulation of extrasynaptic NMDA receptors are required to support hallucinogen-induced cortical network activity. Since microdialysis studies suggest that psychedelic hallucinogens and dopamine D1/D5 receptor agonists have opposite effects on extracellular glutamate in prefrontal cortex, we hypothesized that these two families of psychoactive drugs would have opposite effects on cortical network activity. We found that network activity can be enhanced by DOI (a psychedelic hallucinogen that is a partial agonist of serotonin 5-HT2A/2C receptors) and suppressed by the selective D1/D5 agonist SKF 38393. This suppression could be mimicked by direct activation of adenylyl cyclase with forskolin or by addition of a cAMP analog. These findings are consistent with previous work showing that activation of adenylyl cyclase can upregulate neuronal glutamate transporters, thereby decreasing synaptic spillover of glutamate. Consistent with this hypothesis, a low concentration of the glutamate transporter inhibitor TBOA restored electrically-evoked recurrent activity in the presence of a selective D1/D5 agonist, whereas recurrent activity in the presence of a low level of the GABAA antagonist bicuculline was not resistant to suppression by the D1/D5 agonist. The tempering of network UP states by D1/D5 receptor activation may have implications for the proposed use of D1/D5 agonists in the treatment of schizophrenia. PMID:17293055

  4. Genetic variation in the peroxisome proliferator-activated receptor (PPAR) and peroxisome proliferator-activated receptor gamma co-activator 1 (PGC1) gene families and type 2 diabetes

    PubMed Central

    Villegas, Raquel; Williams, Scott M.; Gao, Yu-Tang; Long, Jirong; Shi, Jiajun; Cai, Hui; Li, Honglan; Chen, Ching-Chu; Tai, E. Shyong; Hu, Frank; Cai, Qiuyin; Zheng, Wei; Shu, Xiao-Ou

    2014-01-01

    Summary We used a two-stage study design to evaluate whether variations in the peroxisome proliferator-activated receptors (PPAR) and the peroxisome proliferator-activated receptor gamma co-activator 1 (PGC1) gene families (PPARA, PPARG, PPARD, PPARGC1A, and PPARGC1B) are associated with T2D risk. Stage I used data from a genome-wide association study (GWAS) from Shanghai, China (1,019 T2D cases and 1,709 controls) and from a meta-analysis of data from the Asian Genetic Epidemiology Network for T2D (AGEN-T2D). Criteria for selection of SNPs for stage II were: 1) P<0.05 in single marker analysis in Shanghai GWAS and P<0.05 in the meta-analysis or 2) P<10−3 in the meta-analysis alone and 3) minor allele frequency ≥0.10. Nine SNPs from the PGC1 family were assessed in stage II (an independent set of middle-aged men and women from Shanghai with 1,700 T2D cases and 1,647 controls). One SNP in PPARGC1B, rs251464, was replicated in stage II (OR=0.87; 95% CI: 0.77–0.99). Gene-body mass index (BMI) and gene-exercise interactions and T2D risk were evaluated in a combined dataset (Shanghai GWAS and stage II data: 2,719 cases and 3,356 controls). One SNP in PPARGC1A, rs12640088, had a significant interaction with BMI. No interactions between the PPARGC1B gene and BMI or exercise were observed. PMID:24359475

  5. Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

    PubMed Central

    Meng, Jingru; Ma, Xue; Wang, Ning; Jia, Min; Bi, Long; Wang, Yunying; Li, Mingkai; Zhang, Huinan; Xue, Xiaoyan; Hou, Zheng; Zhou, Ying; Yu, Zhibin; He, Gonghao; Luo, Xiaoxing

    2016-01-01

    Summary Glucagon-like peptide 1 (GLP-1) plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4) promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs), but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis. PMID:26947974

  6. Dopamine D1 receptor activation contributes to light-adapted changes in retinal inhibition to rod bipolar cells.

    PubMed

    Flood, Michael Daniel; Moore-Dotson, Johnnie M; Eggers, Erika D

    2018-05-30

    Dopamine modulation of retinal signaling has been shown to be an important part of retinal adaptation to increased background light levels but the role of dopamine modulation of retinal inhibition is not clear. We previously showed that light adaptation causes a large reduction in inhibition to rod bipolar cells, potentially to match the decrease in excitation after rod saturation. In this study we determined how dopamine D1 receptors in the inner retina contribute to this modulation. We found that D1 receptor activation significantly decreased the magnitude of inhibitory light responses from rod bipolar cells, while D1 receptor blockade during light adaptation partially prevented this decline. To determine what mechanisms were involved in the modulation of inhibitory light responses, we measured the effect of D1 receptor activation on spontaneous currents and currents evoked from electrically stimulating amacrine cell inputs to rod bipolar cells. D1 receptor activation decreased the frequency of spontaneous inhibition with no change in event amplitudes, suggesting a presynaptic change in amacrine cell activity in agreement with previous reports that rod bipolar cells lack D1 receptors. Additionally, we found that D1 receptor activation reduced the amplitude of electrically-evoked responses, showing that D1 receptors can modulate amacrine cells directly. Our results suggest that D1 receptor activation can replicate a large portion, but not all of the effects of light adaptation, likely by modulating release from amacrine cells onto rod bipolar cells.

  7. GPER-1 agonist G1 induces vasorelaxation through activation of epidermal growth factor receptor-dependent signalling pathway.

    PubMed

    Jang, Eun Jin; Seok, Young Mi; Arterburn, Jeffrey B; Olatunji, Lawrence A; Kim, In Kyeom

    2013-10-01

    The G protein-coupled oestrogen receptor-1 (GPER-1) agonist G1 induces endothelium-dependent relaxation. Activation of the epidermal growth factor (EGF) receptor leads to transduction of signals from the plasma membrane for the release of nitric oxide. We tested the hypothesis that G1 induces endothelium-dependent vasorelaxation through activation of the EGF receptor. Rat aortic rings were mounted in organ baths. After pretreatment with various inhibitors, aortic rings contracted with 11,9-epoxymethano-prostaglandin F2α or KCl were subjected to relaxation by G1. G1 induced endothelium-dependent vasorelaxation, which was attenuated by pretreatment with either L -N(ω) -nitroarginine methyl ester (L -NAME), an inhibitor of nitric oxide synthase, or (3aS,4R,9bR)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline HB-EGF, heparin-binding EGF-like growth factor, a GPER-1 antagonist. Neither a general oestrogen receptor antagonist, ICI 182 780, nor a selective oestrogen receptor-α antagonist, methyl-piperidino-pyrazole dihydrochloride (MPP), had an effect on G1-induced vasorelaxation. However, pretreatment with EGF receptor blockers, AG1478 or DAPH, resulted in attenuated G1-induced vasorelaxation. In addition, pretreatment with Src inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or Akt inhibitor VIII also resulted in attenuated vascular relaxation induced by the cumulative addition of G1. However, neither phosphatidylinositol-3 kinase inhibitors LY294002 and wortmannin nor an extracellular signal-regulated kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene monoethanolate had effect on vascular relaxation induced by the cumulative addition of G1. G1 induces endothelium-dependent vasorelaxation through Src-mediated activation of the EGF receptor and the Akt pathway in rat aorta. © 2013 Royal Pharmaceutical Society.

  8. Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.

    PubMed

    Fang, Xiao-Qian; Qiao, Haifa; Groveman, Bradley R; Feng, Shuang; Pflueger, Melissa; Xin, Wen-Kuan; Ali, Mohammad K; Lin, Shuang-Xiu; Xu, Jindong; Duclot, Florian; Kabbaj, Mohamed; Wang, Wei; Ding, Xin-Sheng; Santiago-Sim, Teresa; Jiang, Xing-Hong; Salter, Michael W; Yu, Xian-Min

    2015-11-19

    Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor

  9. Adenosine A1 receptors modulate high voltage-activated Ca2+ currents and motor pattern generation in the Xenopus embryo

    PubMed Central

    Brown, Paul; Dale, Nicholas

    2000-01-01

    Adenosine causes voltage- and non-voltage-dependent inhibition of high voltage-activated (HVA) Ca2+ currents in Xenopus laevis embryo spinal neurons. As this inhibition can be blocked by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and mimicked by N6-cyclopentyladenosine (CPA) it appears to be mediated by A1 receptors. Agents active at A2 receptors either were without effect or could be blocked by DPCPX. AMP had no agonist action on these receptors. By using ω-conotoxin GVIA we found that adenosine inhibited an N-type Ca2+ current as well as a further unidentified HVA current that was insensitive to dihydropyridines, ω-agatoxin TK and ω-conotoxin MVIIC. Both types of current were subject to voltage- and non-voltage-dependent inhibition. We used CPA and DPCPX to test whether A1 receptors regulated spinal motor pattern generation in spinalized Xenopus embryos. DPCPX caused a near doubling of, while CPA greatly shortened, the length of swimming episodes. In addition, DPCPX slowed, while CPA greatly speeded up, the rate of run-down of motor activity. Our results demonstrate a novel action of A1 receptors in modulating spinal motor activity. Furthermore they confirm that adenosine is produced continually throughout swimming episodes and acts to cause the eventual termination of activity. PMID:10856119

  10. Cannabinoid CB1 Receptor Activation Mediates the Opposing Effects of Amphetamine on Impulsive Action and Impulsive Choice

    PubMed Central

    Wiskerke, Joost; Stoop, Nicky; Schetters, Dustin; Schoffelmeer, Anton N. M.; Pattij, Tommy

    2011-01-01

    It is well known that acute challenges with psychostimulants such as amphetamine affect impulsive behavior. We here studied the pharmacology underlying the effects of amphetamine in two rat models of impulsivity, the 5-choice serial reaction time task (5-CSRTT) and the delayed reward task (DRT), providing measures of inhibitory control, an aspect of impulsive action, and impulsive choice, respectively. We focused on the role of cannabinoid CB1 receptor activation in amphetamine-induced impulsivity as there is evidence that acute challenges with psychostimulants activate the endogenous cannabinoid system, and CB1 receptor activity modulates impulsivity in both rodents and humans. Results showed that pretreatment with either the CB1 receptor antagonist/inverse agonist SR141716A or the neutral CB1 receptor antagonist O-2050 dose-dependently improved baseline inhibitory control in the 5-CSRTT. Moreover, both compounds similarly attenuated amphetamine-induced inhibitory control deficits, suggesting that CB1 receptor activation by endogenously released cannabinoids mediates this aspect of impulsive action. Direct CB1 receptor activation by Δ9-Tetrahydrocannabinol (Δ9-THC) did, however, not affect inhibitory control. Although neither SR141716A nor O-2050 affected baseline impulsive choice in the DRT, both ligands completely prevented amphetamine-induced reductions in impulsive decision making, indicating that CB1 receptor activity may decrease this form of impulsivity. Indeed, acute Δ9-THC was found to reduce impulsive choice in a CB1 receptor-dependent way. Together, these results indicate an important, though complex role for cannabinoid CB1 receptor activity in the regulation of impulsive action and impulsive choice as well as the opposite effects amphetamine has on both forms of impulsive behavior. PMID:22016780

  11. Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms.

    PubMed

    Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; El-Fakahany, Esam E; Jakubík, Jan

    2015-07-01

    We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant xanomeline. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Dopamine modulation of transient receptor potential vanilloid type 1 (TRPV1) receptor in dorsal root ganglia neurons.

    PubMed

    Chakraborty, Saikat; Rebecchi, Mario; Kaczocha, Martin; Puopolo, Michelino

    2016-03-15

    The transient receptor potential vanilloid type 1 (TRPV1) receptor plays a key role in the modulation of nociceptor excitability. To address whether dopamine can modulate the activity of TRPV1 channels in nociceptive neurons, the effects of dopamine and dopamine receptor agonists were tested on the capsaicin-activated current recorded from acutely dissociated small diameter (<27 μm) dorsal root ganglia (DRG) neurons. Dopamine or SKF 81297 (an agonist at D1/D5 receptors), caused inhibition of both inward and outward currents by ∼60% and ∼48%, respectively. The effect of SKF 81297 was reversed by SCH 23390 (an antagonist at D1/D5 receptors), confirming that it was mediated by activation of D1/D5 dopamine receptors. In contrast, quinpirole (an agonist at D2 receptors) had no significant effect on the capsaicin-activated current. Inhibition of the capsaicin-activated current by SKF 81297 was mediated by G protein coupled receptors (GPCRs), and highly dependent on external calcium. The inhibitory effect of SKF 81297 on the capsaicin-activated current was not affected when the protein kinase A (PKA) activity was blocked with H89, or when the protein kinase C (PKC) activity was blocked with bisindolylmaleimide II (BIM). In contrast, when the calcium-calmodulin-dependent protein kinase II (CaMKII) was blocked with KN-93, the inhibitory effect of SKF 81297 on the capsaicin-activated current was greatly reduced, suggesting that activation of D1/D5 dopamine receptors may be preferentially linked to CaMKII activity. We suggest that modulation of TRPV1 channels by dopamine in nociceptive neurons may represent a way for dopamine to modulate incoming noxious stimuli. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  13. Anticonvulsant activity of artificial sweeteners: a structural link between sweet-taste receptor T1R3 and brain glutamate receptors.

    PubMed

    Talevi, Alan; Enrique, Andrea V; Bruno-Blanch, Luis E

    2012-06-15

    A virtual screening campaign based on application of a topological discriminant function capable of identifying novel anticonvulsant agents indicated several widely-used artificial sweeteners as potential anticonvulsant candidates. Acesulfame potassium, cyclamate and saccharin were tested in the Maximal Electroshock Seizure model (mice, ip), showing moderate anticonvulsant activity. We hypothesized a probable structural link between the receptor responsible of sweet taste and anticonvulsant molecular targets. Bioinformatic tools confirmed a highly significant sequence-similarity between taste-related protein T1R3 and several metabotropic glutamate receptors from different species, including glutamate receptors upregulated in epileptogenesis and certain types of epilepsy. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. A second trigeminal CGRP receptor: function and expression of the AMY1 receptor

    PubMed Central

    Walker, Christopher S; Eftekhari, Sajedeh; Bower, Rebekah L; Wilderman, Andrea; Insel, Paul A; Edvinsson, Lars; Waldvogel, Henry J; Jamaluddin, Muhammad A; Russo, Andrew F; Hay, Debbie L

    2015-01-01

    Objective The trigeminovascular system plays a central role in migraine, a condition in need of new treatments. The neuropeptide, calcitonin gene-related peptide (CGRP), is proposed as causative in migraine and is the subject of intensive drug discovery efforts. This study explores the expression and functionality of two CGRP receptor candidates in the sensory trigeminal system. Methods Receptor expression was determined using Taqman G protein-coupled receptor arrays and immunohistochemistry in trigeminal ganglia (TG) and the spinal trigeminal complex of the brainstem in rat and human. Receptor pharmacology was quantified using sensitive signaling assays in primary rat TG neurons. Results mRNA and histological expression analysis in rat and human samples revealed the presence of two CGRP-responsive receptors (AMY1: calcitonin receptor/receptor activity-modifying protein 1 [RAMP1]) and the CGRP receptor (calcitonin receptor-like receptor/RAMP1). In support of this finding, quantification of agonist and antagonist potencies revealed a dual population of functional CGRP-responsive receptors in primary rat TG neurons. Interpretation The unexpected presence of a functional non-canonical CGRP receptor (AMY1) at neural sites important for craniofacial pain has important implications for targeting the CGRP axis in migraine. PMID:26125036

  15. Cross-Desensitization of CCR1, But Not CCR2, Following Activation of the Formyl Peptide Receptor (FPR1)1

    PubMed Central

    Bednar, Filip; Song, Changcheng; Bardi, Giuseppe; Cornwell, William; Rogers, Thomas J.

    2014-01-01

    The cross-regulation of G protein-coupled receptors (GPCRs) plays an important role in the immune response. Studies from several laboratories have suggested that a hierarchy of sensitivities to cross-desensitization exists for the chemoattractant GPCRs. We carried out experiments to study the capacity of the formyl peptide receptor-1 (FPR1) to desensitize chemokine receptors CCR1 and CCR2. Our results show that activation of FPR1 resulted in the desensitization and partial internalization of CCR1, but not CCR2, in both primary human monocytes and HEK293 cells co-expressing CCR1, CCR2, and FPR1 (HR1R2F cells). The desensitization of CCR1 by FPR1 stimulation was not due to the simple depletion of the Ca2+ stores, but was dependent on activation of PKC. Furthermore, we found that the cross-desensitization of CCR1 by FPR1 was associated with CCR1 phosphorylation, and moderate reduction of CCR1 cell surface expression. In contrast, CCR2 was not phosphorylated or internalized following FPR1 activation. Additional studies showed that optimal cross-talk between FPR1 and CCR1 were dependent on the functional activity of PKCβ. These results provide a mechanistic basis for the capacity of certain GPCR ligands to exert rapid and selective cross-inactivation of other chemoattractant receptors, and suggest that FPR1 is able to exert “traffic control” in the migration of inflammatory cells by rapidly inhibiting the cell responses to potentially “low priority” chemoattractants such as CCR1 agonists without inhibiting the response to “higher priority” CCR2 chemoattractants. PMID:24778447

  16. The Chemokine Receptor CCR1 Is Constitutively Active, Which Leads to G Protein-independent, β-Arrestin-mediated Internalization*

    PubMed Central

    Gilliland, C. Taylor; Salanga, Catherina L.; Kawamura, Tetsuya; Trejo, JoAnn; Handel, Tracy M.

    2013-01-01

    Activation of G protein-coupled receptors by their associated ligands has been extensively studied, and increasing structural information about the molecular mechanisms underlying ligand-dependent receptor activation is beginning to emerge with the recent expansion in GPCR crystal structures. However, some GPCRs are also able to adopt active conformations in the absence of agonist binding that result in the initiation of signal transduction and receptor down-modulation. In this report, we show that the CC-type chemokine receptor 1 (CCR1) exhibits significant constitutive activity leading to a variety of cellular responses. CCR1 expression is sufficient to induce inhibition of cAMP formation, increased F-actin content, and basal migration of human and murine leukocytes. The constitutive activity leads to basal phosphorylation of the receptor, recruitment of β-arrestin-2, and subsequent receptor internalization. CCR1 concurrently engages Gαi and β-arrestin-2 in a multiprotein complex, which may be accommodated by homo-oligomerization or receptor clustering. The data suggest the presence of two functional states for CCR1; whereas receptor coupled to Gαi functions as a canonical GPCR, albeit with high constitutive activity, the CCR1·β-arrestin-2 complex is required for G protein-independent constitutive receptor internalization. The pertussis toxin-insensitive uptake of chemokine by the receptor suggests that the CCR1·β-arrestin-2 complex may be related to a potential scavenging function of the receptor, which may be important for maintenance of chemokine gradients and receptor responsiveness in complex fields of chemokines during inflammation. PMID:24056371

  17. The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation*

    PubMed Central

    Domazet, Ivana; Martin, Stéphane S.; Holleran, Brian J.; Morin, Marie-Ève; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

    2009-01-01

    The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT1, N200C-AT1, I201C-AT1, G203C-AT1, and F204C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant I201C-N111G-AT1 became more sensitive to MTSEA, whereas mutant G203C-N111G-AT1 lost some sensitivity. Our results suggest that constitutive activation of AT1 receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side. PMID:19773549

  18. The fifth transmembrane domain of angiotensin II Type 1 receptor participates in the formation of the ligand-binding pocket and undergoes a counterclockwise rotation upon receptor activation.

    PubMed

    Domazet, Ivana; Martin, Stéphane S; Holleran, Brian J; Morin, Marie-Eve; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

    2009-11-13

    The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

  19. Emamectin is a non-selective allosteric activator of nicotinic acetylcholine receptors and GABAA/C receptors

    PubMed Central

    Xu, Xiaojun; Sepich, Caraline; Lukas, Ronald J; Zhu, Guonian; Chang, Yongchang

    2016-01-01

    Avermectins are a group of compounds isolated from a soil-dwelling bacterium. They have been widely used as parasiticides and insecticides, acting by relatively irreversible activation of invertebrate chloride channels. Emamectin is a soluble derivative of an avermectin. It is an insecticide, which persistently activates glutamate-gated chloride channels. However, its effects on mammalian ligand-gated ion channels are unknown. To this end, we tested the effect of emamectin on two cation selective nicotinic receptors and two GABA-gated chloride channels expressed in Xenopus oocytes using two-electrode voltage clamp. Our results demonstrate that emamectin could directly activate α7 nAChR, α4β2 nAChR, α1β2γ2 GABAA receptor and ρ1 GABAC receptor concentration dependently, with similar potencies for each channel. However, the potencies for it to activate these channels were at least two orders of magnitude lower than its potency of activating invertebrate glutamate-gated chloride channel. In contrast, ivermectin only activated the α1β2γ2 GABAA receptor. PMID:27049309

  20. Emamectin is a non-selective allosteric activator of nicotinic acetylcholine receptors and GABAA/C receptors.

    PubMed

    Xu, Xiaojun; Sepich, Caraline; Lukas, Ronald J; Zhu, Guonian; Chang, Yongchang

    2016-05-13

    Avermectins are a group of compounds isolated from a soil-dwelling bacterium. They have been widely used as parasiticides and insecticides, acting by relatively irreversible activation of invertebrate chloride channels. Emamectin is a soluble derivative of an avermectin. It is an insecticide, which persistently activates glutamate-gated chloride channels. However, its effects on mammalian ligand-gated ion channels are unknown. To this end, we tested the effect of emamectin on two cation selective nicotinic receptors and two GABA-gated chloride channels expressed in Xenopus oocytes using two-electrode voltage clamp. Our results demonstrate that emamectin could directly activate α7 nAChR, α4β2 nAChR, α1β2γ2 GABAA receptor and ρ1 GABAC receptor concentration dependently, with similar potencies for each channel. However, the potencies for it to activate these channels were at least two orders of magnitude lower than its potency of activating invertebrate glutamate-gated chloride channel. In contrast, ivermectin only activated the α1β2γ2 GABAA receptor. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Niessen, Markus; Jaschinski, Frank; Item, Flurin

    2007-02-15

    Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the {beta}-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH andmore » PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission.« less

  2. Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library

    PubMed Central

    Jensen, Jan K.; Malmendal, Anders; Schiøtt, Birgit; Skeldal, Sune; Pedersen, Katrine E.; Celik, Leyla; Nielsen, Niels Chr.; Andreasen, Peter A.; Wind, Troels

    2006-01-01

    The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA–PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA–PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction. PMID:16813566

  3. Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle*♦

    PubMed Central

    Tjondrokoesoemo, Andoria; Li, Na; Lin, Pei-Hui; Pan, Zui; Ferrante, Christopher J.; Shirokova, Natalia; Brotto, Marco; Weisleder, Noah; Ma, Jianjie

    2013-01-01

    Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca2+ signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca2+ sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca2+ sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca2+ sparks involves interaction between activation of IP3R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP3 production or inhibition of IP3R channel activity abolishes stress-induced Ca2+ sparks in skeletal muscle. Although genetic ablation of the type 2 IP3R does not appear to affect Ca2+ sparks in skeletal muscle, specific silencing of the type 1 IP3R leads to ablation of stress-induced Ca2+ sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP3R1 and RyR1 contributes to Ca2+ spark activation in skeletal muscle. PMID:23223241

  4. Cholecystokinin 1 Receptor - A Unique G Protein-Coupled Receptor Activated by Singlet Oxygen (GPCR-ABSO).

    PubMed

    Jiang, Hong Ning; Li, Yuan; Jiang, Wen Yi; Cui, Zong Jie

    2018-01-01

    Plasma membrane-delimited generation of singlet oxygen by photodynamic action with photosensitizer sulfonated aluminum phthalocyanine (SALPC) activates cholecystokinin 1 receptor (CCK1R) in pancreatic acini. Whether CCK1R retains such photooxidative singlet oxygen activation properties in other environments is not known. Genetically encoded protein photosensitizers KillerRed or mini singlet oxygen generator (miniSOG) were expressed in pancreatic acinar tumor cell line AR4-2J, CCK1R, KillerRed or miniSOG were expressed in HEK293 or CHO-K1 cells. Cold light irradiation (87 mW⋅cm -2 ) was applied to photosensitizer-expressing cells to examine photodynamic activation of CCK1R by Fura-2 fluorescent calcium imaging. When CCK1R was transduced into HEK293 cells which lack endogenous CCK1R, photodynamic action with SALPC was found to activate CCK1R in CCK1R-HEK293 cells. When KillerRed or miniSOG were transduced into AR4-2J which expresses endogenous CCK1R, KillerRed or miniSOG photodynamic action at the plasma membrane also activated CCK1R. When fused KillerRed-CCK1R was transduced into CHO-K1 cells, light irradiation activated the fused CCK1R leading to calcium oscillations. Therefore KillerRed either expressed independently, or fused with CCK1R can both activate CCK1R photodynamically. It is concluded that photodynamic singlet oxygen activation is an intrinsic property of CCK1R, independent of photosensitizer used, or CCK1R-expressing cell types. Photodynamic singlet oxygen CCK1R activation after transduction of genetically encoded photosensitizer in situ may provide a convenient way to verify intrinsic physiological functions of CCK1R in multiple CCK1R-expressing cells and tissues, or to actuate CCK1R function in CCK1R-expressing and non-expressing cell types after transduction with fused KillerRed-CCK1R.

  5. Caged vanilloid ligands for activation of TRPV1 receptors by 1- and 2-photon excitation†

    PubMed Central

    Zhao, Jun; Gover, Tony D.; Muralidharan, Sukumaran; Auston, Darryl A.; Weinreich, Daniel; Kao, Joseph P. Y.

    2008-01-01

    Nociceptive neurons in the peripheral nervous system detect noxious stimuli and report the information to the central nervous system. Most nociceptive neurons express the vanilloid receptor, TRPV1, a non-selective cation channel gated by vanilloid ligands such as capsaicin, the pungent essence of chili peppers. Here, we report the synthesis and biological application of two caged vanilloids—biologically inert precursors that, when photolyzed, release bioactive vanilloid ligands. The two caged vanilloids, Nb-VNA and Nv-VNA, are photoreleased with quantum efficiency of 0.13 and 0.041, respectively. Under flash photolysis conditions, photorelease of Nb-VNA and Nv-VNA is 95% complete in ∼40 μs and ∼125 μs, respectively. Through 1-photon excitation with ultraviolet light (360 nm), or 2-photon excitation with red light (720 nm), the caged vanilloids can be photoreleased in situ to activate TRPV1 receptors on nociceptive neurons. The consequent increase in intracellular free Ca2+ concentration ([Ca2+]i) can be visualized by laser-scanning confocal imaging of neurons loaded with the fluorescent Ca2+ indicator, fluo-3. Stimulation results from TRPV1 receptor activation, because the response is blocked by capsazepine, a selective TRPV1 antagonist. In Ca2+-free extracellular medium, photoreleased vanilloid can still elevate [Ca2+]i, which suggests that TRPV1 receptors also reside on endomembranes in neurons and can mediate Ca2+ release from intracellular stores. Notably, whole-cell voltage clamp measurements showed that flash photorelease of vanilloid can activate TRPV1 channels in < 4 msec at 22°C. In combination with 1- or 2-photon excitation, caged vanilloids are a powerful tool for probing morphologically distinct structures of nociceptive sensory neurons with high spatial and temporal precision. PMID:16605259

  6. SCH 206272: a potent, orally active tachykinin NK(1), NK(2), and NK(3) receptor antagonist.

    PubMed

    Anthes, John C; Chapman, Richard W; Richard, Christian; Eckel, Stephen; Corboz, Michel; Hey, John A; Fernandez, Xiomara; Greenfeder, Scott; McLeod, Robbie; Sehring, Susan; Rizzo, Charles; Crawley, Yvette; Shih, Neng-Yang; Piwinski, John; Reichard, Greg; Ting, Pauline; Carruthers, Nick; Cuss, Francis M; Billah, Motasim; Kreutner, William; Egan, Robert W

    2002-08-23

    Experiments were performed to characterize the pharmacology of SCH 206272 [(R,R)-1'[5-[(3,5-dichlorobenzoyl)methylamino]-3-(3,4-dichlorophenyl)-4(Z)-(methoxyimino)pentyl]-N-methyl-2-oxo-[1,4'bipiperidine]-3-acetamide] as a potent and selective antagonist of tachykinin (NK) NK(1), NK(2), and NK(3) receptors. SCH 206272 inhibited binding at human tachykinin NK(1), NK(2), and NK(3) receptors (K(i) = 1.3, 0.4, and 0.3 nM, respectively) and antagonized [Ca(2+)](i) mobilization in Chinese hamster ovary (CHO) cells expressing the cloned human tachykinin NK(1), NK(2), or NK(3) receptors. SCH 206272 inhibited relaxation of the human pulmonary artery (pK(b) = 7.7 +/- 0.3) induced by the tachykinin NK(1) receptor agonist, [Met-O-Me] substance P and contraction of the human bronchus (pK(b = 8.2 +/- 0.3) induced by the tachykinin NK(2) receptor agonist, neurokinin A. In isolated guinea pig tissues, SCH 206272 inhibited substance P-induced enhancement of electrical field stimulated contractions of the vas deferens, (pK(b = 7.6 +/- 0.2), NKA-induced contraction of the bronchus (pK(b) = 7.7 +/- 0.2), and senktide-induced contraction of the ileum. In vivo, oral SCH 206272 (0.1-10 mg/kg, p.o.) inhibited substance P-induced airway microvascular leakage and neurokinin A-induced bronchospasm in the guinea pig. In a canine in vivo model, SCH 206272 (0.1-3 mg/kg, p.o.) inhibited NK(1) and NK(2) activities induced by exogenous substance P and neurokinin A. Furthermore, in guinea pig models involving endogenously released tachykinins, SCH 206272 inhibited hyperventilation-induced bronchospasm, capsaicin-induced cough, and airway microvascular leakage induced by nebulized hypertonic saline. These data demonstrate that SCH 206272 is a potent, orally active tachykinin NK(1), NK(2), and NK(3) receptor antagonist. This compound may have beneficial effects in diseases thought to be mediated by tachykinins, such as cough, asthma, and chronic obstructive pulmonary disease. Copyright 2002 Elsevier

  7. A Sphingosine 1-phosphate receptor 2 selective allosteric agonist

    PubMed Central

    Satsu, Hideo; Schaeffer, Marie-Therese; Guerrero, Miguel; Saldana, Adrian; Eberhart, Christina; Hodder, Peter; Cayanan, Charmagne; Schürer, Stephan; Bhhatarai, Barun; Roberts, Ed; Rosen, Hugh; Brown, Steven J.

    2013-01-01

    Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound. PMID:23849205

  8. Valerian extract Ze 911 inhibits postsynaptic potentials by activation of adenosine A1 receptors in rat cortical neurons.

    PubMed

    Vissiennon, Z; Sichardt, K; Koetter, U; Brattström, A; Nieber, K

    2006-06-01

    In this study we evaluated the adenosine A1 receptor-mediated effect of valerian extract (Ze 911) on postsynaptic potentials (PSPs) in pyramidal cells of the rat cingulate cortex in a slice preparation. We first observed that N6-cyclopentyladenosine (CPA, 0.01 - 10 microM), an adenosine A1 receptor agonist, inhibited PSPs in a concentration-dependent manner. The CPA (10 microM)-induced inhibition was antagonized by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 microM), an adenosine A1 receptor antagonist. Ze 911 concentration dependently (0.1 - 15 mg/mL) inhibited PSPs in the presence of the adenosine A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC, 0.2 microM) and adenosine deaminase (1 U/mL). The maximal inhibition induced by 10 mg/mL was completely antagonised by DPCPX (0.1 microM), an A1 receptor blocker. The data suggest that activation of adenosine A1 receptors is involved in the pharmacological effects of the valerian extract Ze 911.

  9. G protein-coupled estrogen receptor 1 (GPER1)/GPR30 increases ERK1/2 activity through PDZ motif-dependent and -independent mechanisms.

    PubMed

    Gonzalez de Valdivia, Ernesto; Broselid, Stefan; Kahn, Robin; Olde, Björn; Leeb-Lundberg, L M Fredrik

    2017-06-16

    G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of G i/o Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin as well as by wortmannin but not by AG1478, indicating that G i/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two G i/o -mediated mechanisms, a PDZ-dependent, apparently constitutive mechanism and a PDZ-independent G-1-stimulated mechanism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Beneficial Effects of the Activation of the Angiotensin-(1–7) Mas Receptor in a Murine Model of Adriamycin-Induced Nephropathy

    PubMed Central

    Silveira, Kátia Daniela; Barroso, Lívia Corrêa; Vieira, Angélica Thomáz; Cisalpino, Daniel; Lima, Cristiano Xavier; Bader, Michael; Arantes, Rosa Maria Esteves; dos Santos, Robson Augusto Souza

    2013-01-01

    Angiotensin-(1–7) [Ang-(1–7)] is a biologically active heptapeptide that may counterbalance the physiological actions of angiotensin II (Ang II) within the renin-angiotensin system (RAS). Here, we evaluated whether activation of the Mas receptor with the oral agonist, AVE 0991, would have renoprotective effects in a model of adriamycin (ADR)-induced nephropathy. We also evaluated whether the Mas receptor contributed for the protective effects of treatment with AT1 receptor blockers. ADR (10 mg/kg) induced significant renal injury and dysfunction that was maximal at day 14 after injection. Treatment with the Mas receptor agonist AVE 0991 improved renal function parameters, reduced urinary protein loss and attenuated histological changes. Renoprotection was associated with reduction in urinary levels of TGF-β. Similar renoprotection was observed after treatment with the AT1 receptor antagonist, Losartan. AT1 and Mas receptor mRNA levels dropped after ADR administration and treatment with losartan reestablished the expression of Mas receptor and increased the expression of ACE2. ADR-induced nephropathy was similar in wild type (Mas+/+) and Mas knockout (Mas −/−) mice, suggesting there was no endogenous role for Mas receptor activation. However, treatment with Losartan was able to reduce renal injury only in Mas+/+, but not in Mas −/− mice. Therefore, these findings suggest that exogenous activation of the Mas receptor protects from ADR-induced nephropathy and contributes to the beneficial effects of AT1 receptor blockade. Medications which target specifically the ACE2/Ang-(1–7)/Mas axis may offer new therapeutic opportunities to treat human nephropathies. PMID:23762470

  11. Prohibitin is involved in the activated internalization and degradation of protease-activated receptor 1.

    PubMed

    Wang, Yan-Jie; Guo, Xiao-Long; Li, Sheng-An; Zhao, Yu-Qi; Liu, Zi-Chao; Lee, Wen-Hui; Xiang, Yang; Zhang, Yun

    2014-07-01

    The protease-activated receptor 1 (PAR1) is a G-protein-coupled receptor that is irreversibly activated by either thrombin or metalloprotease 1. Due this irrevocable activation, activated internalization and degradation are critical for PAR1 signaling termination. Prohibitin (PHB) is an evolutionarily conserved, ubiquitously expressed, pleiotropic protein and belongs to the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain family. In a previous study, we found that PHB localized on the platelet membrane and participated in PAR1-mediated human platelet aggregation, suggesting that PHB likely regulates the signaling of PAR1. Unfortunately, PHB's exact function in PAR1 internalization and degradation is unclear. In the current study, flow cytometry revealed that PHB expressed on the surface of endothelial cells (HUVECs) but not cancer cells (MDA-MB-231). Further confocal microscopy revealed that PHB dynamically associates with PAR1 in a time-dependent manner following induction with PAR1-activated peptide (PAR1-AP), though differently between HUVECs and MDA-MB-231 cells. Depletion of PHB by RNA interference significantly inhibited PAR1 activated internalization and led to sustained Erk1/2 phosphorylation in the HUVECs; however, a similar effect was not observed in MDA-MB-231 cells. For both the endothelial and cancel cells, PHB repressed PAR1 degradation, while knockdown of PHB led to increased PAR1 degradation, and PHB overexpression inhibited PAR1 degradation. These results suggest that persistent PAR1 signaling due to the absence of membrane PHB and decreased PAR1 degradation caused by the upregulation of intracellular PHB in cancer cells (such as MDA-MB-231 cells) may render cells highly invasive. As such, PHB may be a novel target in future anti-cancer therapeutics, or in more refined cancer malignancy diagnostics. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Cocaine Disrupts Histamine H3 Receptor Modulation of Dopamine D1 Receptor Signaling: σ1-D1-H3 Receptor Complexes as Key Targets for Reducing Cocaine's Effects

    PubMed Central

    Moreno, Estefanía; Moreno-Delgado, David; Navarro, Gemma; Hoffmann, Hanne M.; Fuentes, Silvia; Rosell-Vilar, Santi; Gasperini, Paola; Rodríguez-Ruiz, Mar; Medrano, Mireia; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Lluís, Carme; Ferré, Sergi; Ortiz, Jordi; Canela, Enric

    2014-01-01

    The general effects of cocaine are not well understood at the molecular level. What is known is that the dopamine D1 receptor plays an important role. Here we show that a key mechanism may be cocaine's blockade of the histamine H3 receptor-mediated inhibition of D1 receptor function. This blockade requires the σ1 receptor and occurs upon cocaine binding to σ1-D1-H3 receptor complexes. The cocaine-mediated disruption leaves an uninhibited D1 receptor that activates Gs, freely recruits β-arrestin, increases p-ERK 1/2 levels, and induces cell death when over activated. Using in vitro assays with transfected cells and in ex vivo experiments using both rats acutely treated or self-administered with cocaine along with mice depleted of σ1 receptor, we show that blockade of σ1 receptor by an antagonist restores the protective H3 receptor-mediated brake on D1 receptor signaling and prevents the cell death from elevated D1 receptor signaling. These findings suggest that a combination therapy of σ1R antagonists with H3 receptor agonists could serve to reduce some effects of cocaine. PMID:24599455

  13. Role for NK(1) and NK(2) receptors in the motor activity in mouse colon.

    PubMed

    Mulè, Flavia; Amato, Antonella; Serio, Rosa

    2007-09-10

    The present study examined the effects induced by endogenous and exogenous activation of NK(1) and NK(2) receptors on the mechanical activity of mouse proximal colon. Experiments were performed in vitro recording the changes in intraluminal pressure from isolated colonic segments. Electrical field stimulation in the presence of atropine and guanethidine produced a small relaxation, followed by nonadrenergic noncholinergic (NANC) contraction. SR140333, NK(1) receptor antagonist, or SR48968, NK(2) receptor antagonist, significantly reduced the contraction, although SR48968 appeared more efficacious. The co-administration of SR140333 and SR48968 virtually abolished the NANC contraction. [Sar(9), Met(O(2))(11)]-substance P, selective NK(1) receptor agonist, induced a concentration-dependent biphasic effect, contraction followed by reduction of the mechanical spontaneous activity. Both effects were antagonized by SR140333, but not by SR48968. [beta-Ala(8)]-neurokinin A (4-10), selective NK(2) receptor agonist, evoked concentration-dependent contraction, which was antagonized by SR48968, but not by SR140333. The contraction induced by [Sar(9), Met(O(2))(11)]-substance P, but not by [beta-Ala(8)]-neurokinin A (4-10), was reduced by tetrodotoxin or atropine, and increased by N(omega)-nitro-L-arginine methyl ester (L-NAME), inhibitor of nitric oxide synthase. The inhibitory effects induced by [Sar(9), Met(O(2))(11)]-substance P were abolished by tetrodotoxin or L-NAME. The results of the present study suggest that in mouse colon both NK(1) and NK(2) receptors are junctionally activated by endogenous tachykinins to cause an additive response. NK(1) receptors appear to be located on cholinergic and on nitrergic neurons as well as on smooth muscle cells, whereas NK(2) receptors seem to be present exclusively on smooth muscle cells.

  14. Insulin growth factor-1 (IGF-1) enhances hippocampal excitatory and seizure activity through IGF-1 receptor-mediated mechanisms in the epileptic brain.

    PubMed

    Jiang, Guohui; Wang, Wei; Cao, Qingqing; Gu, Juan; Mi, Xiujuan; Wang, Kewei; Chen, Guojun; Wang, Xuefeng

    2015-12-01

    Insulin-like growth factor-1 (IGF-1) is known to promote neurogenesis and survival. However, recent studies have suggested that IGF-1 regulates neuronal firing and excitatory neurotransmission. In the present study, focusing on temporal lobe epilepsy, we found that IGF-1 levels and IGF-1 receptor activation are increased in human epileptogenic tissues, and pilocarpine- and pentylenetetrazole-treated rat models. Using an acute model of seizures, we showed that lateral cerebroventricular infusion of IGF-1 elevates IGF-1 receptor (IGF-1R) signalling before pilocarpine application had proconvulsant effects. In vivo electroencephalogram recordings and power spectrogram analysis of local field potential revealed that IGF-1 promotes epileptiform activities. This effect is diminished by co-application of an IGF-1R inhibitor. In an in vitro electrophysiological study, we demonstrated that IGF-1 enhancement of excitatory neurotransmission and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor- and N-methyl-D-aspartate receptor-mediated currents is inhibited by IGF-1R inhibitor. Finally, activation of extracellular signal-related kinase (ERK)-1/2 and protein kinase B (Akt) in seizures in rats is increased by exogenous IGF-1 and diminished by picropodophyllin. A behavioural study reveals that the ERK1/2 or Akt inhibitor attenuates seizure activity. These results indicate that increased IGF-1 levels after recurrent hippocampal neuronal firings might, in turn, promote seizure activity via IGF-1R-dependent mechanisms. The present study presents a previously unappreciated role of IGF-1R in the development of seizure activity. © 2015 Authors; published by Portland Press Limited.

  15. Attenuation of Cocaine-Induced Conditioned Place Preference and Motor Activity via Cannabinoid CB2 Receptor Agonism and CB1 Receptor Antagonism in Rats

    PubMed Central

    Delis, Foteini; Polissidis, Alexia; Poulia, Nafsika; Justinova, Zuzana; Nomikos, George G.; Goldberg, Steven R.

    2017-01-01

    Abstract Background: Studies have shown the involvement of cannabinoid (CB) receptors in the behavioral and neurobiological effects of psychostimulants. Most of these studies have focused on the role of CB1 receptors in the psychostimulant effects of cocaine, while very few have investigated the respective role of CB2 receptors. Further studies are warranted to elucidate the extent of CB receptor involvement in the expression of cocaine-induced effects. Methods: The role of CB1 and CB2 receptors in the rewarding and motor properties of cocaine was assessed in conditioned place preference, conditioned motor activity, and open field activity in rats. Results: The CB1 receptor antagonist rimonabant (3 mg/kg) decreased the acquisition and the expression of conditioned place preference induced by cocaine (20 mg/kg). Rimonabant inhibited cocaine-elicited conditioned motor activity when administered during the expression of cocaine-induced conditioned place preference. Rimonabant decreased ambulatory and vertical activity induced by cocaine. The CB2 receptor agonist JWH-133 (10 mg/kg) decreased the acquisition and the expression of cocaine-induced conditioned place preference. JWH-133 inhibited cocaine-elicited conditioned motor activity when administered during the acquisition and the expression of cocaine-induced conditioned place preference. JWH-133 decreased ambulatory activity and abolished vertical activity induced by cocaine. The effects of JWH-133 on cocaine conditioned and stimulated responses were abolished when the CB2 receptor antagonist/inverse agonist AM630 (5 mg/kg) was preadministered. Conclusions: Cannabinoid CB1 and CB2 receptors modulate cocaine-induced rewarding behavior and appear to have opposite roles in the regulation of cocaine’s reinforcing and psychomotor effects. PMID:27994006

  16. Orphan nuclear receptor oestrogen-related receptor γ (ERRγ) plays a key role in hepatic cannabinoid receptor type 1-mediated induction of CYP7A1 gene expression

    PubMed Central

    Zhang, Yaochen; Kim, Don-Kyu; Lee, Ji-Min; Park, Seung Bum; Jeong, Won-IL; Kim, Seong Heon; Lee, In-Kyu; Lee, Chul-Ho; Chiang, John Y.L.; Choi, Hueng-Sik

    2017-01-01

    Bile acids are primarily synthesized from cholesterol in the liver and have important roles in dietary lipid absorption and cholesterol homoeostasis. Detailed roles of the orphan nuclear receptors regulating cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis, have not yet been fully elucidated. In the present study, we report that oestrogen-related receptor γ (ERRγ) is a novel transcriptional regulator of CYP7A1 expression. Activation of cannabinoid receptor type 1 (CB1 receptor) signalling induced ERRγ-mediated transcription of the CYP7A1 gene. Overexpression of ERRγ increased CYP7A1 expression in vitro and in vivo, whereas knockdown of ERRγ attenuated CYP7A1 expression. Deletion analysis of the CYP7A1 gene promoter and a ChIP assay revealed an ERRγ -binding site on the CYP7A1 gene promoter. Small heterodimer partner (SHP) inhibited the transcriptional activity of ERRγ and thus regulated CYP7A1 expression. Overexpression of ERRγ led to increased bile acid levels, whereas an inverse agonist of ERRγ, GSK5182, reduced CYP7A1 expression and bile acid synthesis. Finally, GSK5182 significantly reduced hepatic CB1 receptor-mediated induction of CYP7A1 expression and bile acid synthesis in alcohol-treated mice. These results provide the molecular mechanism linking ERRγ and bile acid metabolism. PMID:26348907

  17. Pharmacological activation of CB1 receptor modulates long term potentiation by interfering with protein synthesis.

    PubMed

    Navakkode, Sheeja; Korte, Martin

    2014-04-01

    Cognitive impairment is one of the most important side effects associated with cannabis drug abuse, as well as the serious issue concerning the therapeutic use of cannabinoids. Cognitive impairments and neuropsychiatric symptoms are caused by early synaptic dysfunctions, such as loss of synaptic connections in different brain structures including the hippocampus, a region that is believed to play an important role in certain forms of learning and memory. We report here that metaplastic priming of synapses with a cannabinoid type 1 receptor (CB1 receptor) agonist, WIN55,212-2 (WIN55), significantly impaired long-term potentiation in the apical dendrites of CA1 pyramidal neurons. Interestingly, the CB1 receptor exerts its effect by altering the balance of protein synthesis machinery towards higher protein production. Therefore the activation of CB1 receptor, prior to strong tetanization, increased the propensity to produce new proteins. In addition, WIN55 priming resulted in the expression of late-LTP in a synaptic input that would have normally expressed early-LTP, thus confirming that WIN55 priming of LTP induces new synthesis of plasticity-related proteins. Furthermore, in addition to the effects on protein translation, WIN55 also induced synaptic deficits due to the ability of CB1 receptors to inhibit the release of acetylcholine, mediated by both muscarinic and nicotinic acetylcholine receptors. Taken together this supports the notion that the modulation of cholinergic activity by CB1 receptor activation is one mechanism that regulates the synthesis of plasticity-related proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Activation of neurokinin-1 receptors during ozone inhalation contributes to epithelial injury and repair.

    PubMed

    Oslund, Karen L; Hyde, Dallas M; Putney, Leialoha F; Alfaro, Mario F; Walby, William F; Tyler, Nancy K; Schelegle, Edward S

    2008-09-01

    We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2'-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress-positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

  19. Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mochimaru, Yuta; Azuma, Morio; Oshima, Natsuki

    2015-02-20

    Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1more » overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors. - Highlights: • Zebra fish OGR1 and GPR4 homologs (zOGR1, zGPR4) are proton-sensing receptors. • The signaling pathways activated by zOGR1 and zGPR4 are different. • Histidine residues critical for sensing protons are conserved.« less

  20. Stimulating the GPR30 estrogen receptor with a novel tamoxifen analogue activates SF-1 and promotes endometrial cell proliferation.

    PubMed

    Lin, Benjamin C; Suzawa, Miyuki; Blind, Raymond D; Tobias, Sandra C; Bulun, Serdar E; Scanlan, Thomas S; Ingraham, Holly A

    2009-07-01

    Estrogens and selective estrogen receptor (ER) modulators such as tamoxifen are known to increase uterine cell proliferation. Mounting evidence suggests that estrogen signaling is mediated not only by ERalpha and ERbeta nuclear receptors, but also by GPR30 (GPER), a seven transmembrane (7TM) receptor. Here, we report that primary human endometriotic H-38 cells express high levels of GPR30 with no detectable ERalpha or ERbeta. Using a novel tamoxifen analogue, STX, which activates GPR30 but not ERs, significant stimulation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways was observed in H-38 cells and in Ishikawa endometrial cancer cells expressing GPR30; a similar effect was observed in JEG3 choriocarcinoma cells. STX treatment also increased cellular pools of phosphatidylinositol (3,4,5) triphosphate, a proposed ligand for the nuclear hormone receptor SF-1 (NR5A1). Consistent with these findings, STX, tamoxifen, and the phytoestrogen genistein were able to increase SF-1 transcription, promote Ishikawa cell proliferation, and induce the SF-1 target gene aromatase in a GPR30-dependent manner. Our findings suggest a novel signaling paradigm that is initiated by estrogen activation of the 7TM receptor GPR30, with signal transduction cascades (PI3K and MAPK) converging on nuclear hormone receptors (SF-1/LRH-1) to modulate their transcriptional output. We propose that this novel GPR30/SF-1 pathway increases local concentrations of estrogen, and together with classic ER signaling, mediate the proliferative effects of synthetic estrogens such as tamoxifen, in promoting endometriosis and endometrial cancers.

  1. Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C.

    PubMed

    Disatnik, M H; Hernandez-Sotomayor, S M; Jones, G; Carpenter, G; Mochly-Rosen, D

    1994-01-18

    Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.

  2. A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.

    PubMed

    Vyas, Falguni S; Hargreaves, Alan J; Bonner, Philip L R; Boocock, David J; Coveney, Clare; Dickenson, John M

    2016-05-01

    The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Resveratrol inhibits proteinase-activated receptor-2-induced release of soluble vascular endothelial growth factor receptor-1 from human endothelial cells

    PubMed Central

    Al-Ani, Bahjat

    2013-01-01

    We recently reported that (i) activation of the proinflammatory receptor, proteinase-activated receptor-2 (PAR-2) caused the release of an important biomarker in preeclampsia, soluble vascular endothelial growth factor receptor-1 (sVEGFR-1, also known as sFlt-1) from human umbilical vein endothelial cells (HUVECs), and (ii) that the anti-oxidant and anti-inflammatory agent, resveratrol, is capable of inhibiting the proinflammatory cytokine-induced sVEGFR-1 release from human placenta. Based on these findings and because PAR-2 is upregulated by proinflammatory cytokines, we sought to determine whether resveratrol can inhibit PAR-2-induced sVEGFR-1 release. PAR-2 expressing cells, HUVECs and human embryonic kidney cells (HEK-293) transfected with a human VEGFR-1 promoter-luciferase reporter construct were incubated with PAR-2-activating peptide and/or resveratrol. Cell supernatants were assayed for sVEGFR-1 by enzyme-linked immunosorbent assay (ELISA), and VEGFR-1 promoter-luciferase assay was performed on the harvested cell lysates. Preincubation of HEK-293 cells with resveratrol significantly inhibited PAR-2-induced VEGFR-1 promoter activity without affecting cell viability as assessed by MTT assay. The addition of resveratrol also blocked PAR-2-mediated sVEGFR-1 release from HUVECs. The present study demonstrates that resveratrol suppressed both VEGFR-1 promoter activity and sVEGFR-1 protein release induced by PAR-2 activation, which further endorses our recent findings of a potential therapeutic role for resveratrol in preeclampsia. PMID:26933402

  4. Retinoids activate the irritant receptor TRPV1 and produce sensory hypersensitivity

    PubMed Central

    Yin, Shijin; Luo, Jialie; Qian, Aihua; Du, Junhui; Yang, Qing; Zhou, Shentai; Yu, Weihua; Du, Guangwei; Clark, Richard B.; Walters, Edgar T.; Carlton, Susan M.; Hu, Hongzhen

    2013-01-01

    Retinoids are structurally related derivatives of vitamin A and are required for normal vision as well as cell proliferation and differentiation. Clinically, retinoids are effective in treating many skin disorders and cancers. Application of retinoids evokes substantial irritating side effects, including pain and inflammation; however, the precise mechanisms accounting for the sensory hypersensitivity are not understood. Here we show that both naturally occurring and synthetic retinoids activate recombinant or native transient receptor potential channel vanilloid subtype 1 (TRPV1), an irritant receptor for capsaicin, the pungent ingredient of chili peppers. In vivo, retinoids produced pain-related behaviors that were either eliminated or significantly reduced by genetic or pharmacological inhibition of TRPV1 function. These findings identify TRPV1 as an ionotropic receptor for retinoids and provide cellular and molecular insights into retinoid-evoked hypersensitivity. These findings also suggest that selective TRPV1 antagonists are potential therapeutic drugs for treating retinoid-induced sensory hypersensitivity. PMID:23925292

  5. Neurotensin receptor 1 gene activation by the Tcf/beta-catenin pathway is an early event in human colonic adenomas.

    PubMed

    Souazé, Frédérique; Viardot-Foucault, Véronique; Roullet, Nicolas; Toy-Miou-Leong, Mireille; Gompel, Anne; Bruyneel, Erik; Comperat, Eva; Faux, Maree C; Mareel, Marc; Rostène, William; Fléjou, Jean-François; Gespach, Christian; Forgez, Patricia

    2006-04-01

    Alterations in the Wnt/APC (adenomatous polyposis coli) signalling pathway, resulting in beta-catenin/T cell factor (Tcf)-dependent transcriptional gene activation, are frequently detected in familial and sporadic colon cancers. The neuropeptide neurotensin (NT) is widely distributed in the gastrointestinal tract. Its proliferative and survival effects are mediated by a G-protein coupled receptor, the NT1 receptor. NT1 receptor is not expressed in normal colon epithelial cells, but is over expressed in a number of cancer cells and tissues suggesting a link to the outgrowth of human colon cancer. Our results demonstrate that the upregulation of NT1 receptor occurring in colon cancer is the result of Wnt/APC signalling pathway activation. We first established the functionality of the Tcf response element within the NT1 receptor promoter. Consequently, we observed the activation of NT1 receptor gene by agents causing beta-catenin cytosolic accumulation, as well as a strong decline of endogenous receptor when wt-APC was restored. At the cellular level, the re-establishment of wt-APC phenotype resulted in the impaired functionality of NT1 receptor, like the breakdown in NT-induced intracellular calcium mobilization and the loss of NT pro-invasive effect. We corroborated the Wnt/APC signalling pathway on the NT1 receptor promoter activation with human colon carcinogenesis, and showed that NT1 receptor gene activation was perfectly correlated with nuclear or cytoplasmic beta-catenin localization while NT1 receptor was absent when beta-catenin was localized at the cell-cell junction in early adenomas of patients with familial adenomatous polyposis, hereditary non-polyposis colorectal cancer and loss of heterozygosity tumours. In this report we establish a novel link in vitro between the Tcf/beta-catenin pathway and NT1 receptor promoter activation.

  6. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling

    PubMed Central

    Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

    2017-01-01

    Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr52, which then promoted the dephosphorylation of CAR at Thr38 by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR. PMID:23652203

  7. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

    PubMed

    Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

    2013-05-07

    Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

  8. Dual effects of 5-HT(1a) receptor activation on breathing in neonatal mice.

    PubMed

    Corcoran, Andrea E; Commons, Kathryn G; Wu, Yuanming; Smith, Jeffrey C; Harris, Michael B; Richerson, George B

    2014-01-01

    Inhibitory 5-HT(1a) receptors are located on serotonin (5-HT) neurons (autoreceptors) as well as neurons of the respiratory network (heteroreceptors). Thus, effects on breathing of 5-HT(1a) agonists, such as (R)-(+)-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT), could either be due to decreased firing of 5-HT neurons or direct effects on the respiratory network. Mice in which the transcription factor LMX1B is genetically deleted selectively in Pet1-1-expressing cells (Lmx1b(f/f/p)) essentially have complete absence of central 5-HT neurons, providing a unique opportunity to separate the effect of activation of downstream 5-HT(1a) heteroreceptors from that of autoreceptors. We used rhythmically active medullary slices from wild-type (WT) and Lmx1b(f/f/p) neonatal mice to differentiate autoreceptor versus heteroreceptor effects of 8-OH-DPAT on hypoglossal nerve respiratory output. 8-OH-DPAT transiently increased respiratory burst frequency in Lmx1b(f/f/p) preparations, but not in WT slices. This excitation was abolished when synaptic inhibition was blocked by GABAergic/glycinergic receptor antagonists. Conversely, after 10 min of application, frequency in Lmx1b(f/f/p) slices was not different from baseline, whereas it was significantly depressed in WT slices. In WT mice in vivo, subcutaneous injection of 8-OH-DPAT produced similar biphasic respiratory effects as in Lmx1b(f/f/p) mice. We conclude that 5-HT1a receptor agonists have two competing effects: rapid stimulation of breathing due to excitation of the respiratory network, and delayed inhibition of breathing due to autoreceptor inhibition of 5-HT neurons. The former effect is presumably due to inhibition of inhibitory interneurons embedded in the respiratory network.

  9. Dual Effects of 5-HT1a Receptor Activation on Breathing in Neonatal Mice

    PubMed Central

    Commons, Kathryn G.; Wu, Yuanming; Smith, Jeffrey C.; Harris, Michael B.; Richerson, George B.

    2014-01-01

    Inhibitory 5-HT1a receptors are located on serotonin (5-HT) neurons (autoreceptors) as well as neurons of the respiratory network (heteroreceptors). Thus, effects on breathing of 5-HT1a agonists, such as (R)-(+)-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT), could either be due to decreased firing of 5-HT neurons or direct effects on the respiratory network. Mice in which the transcription factor LMX1B is genetically deleted selectively in Pet1-1-expressing cells (Lmx1bf/f/p) essentially have complete absence of central 5-HT neurons, providing a unique opportunity to separate the effect of activation of downstream 5-HT1a heteroreceptors from that of autoreceptors. We used rhythmically active medullary slices from wild-type (WT) and Lmx1bf/f/p neonatal mice to differentiate autoreceptor versus heteroreceptor effects of 8-OH-DPAT on hypoglossal nerve respiratory output. 8-OH-DPAT transiently increased respiratory burst frequency in Lmx1bf/f/p preparations, but not in WT slices. This excitation was abolished when synaptic inhibition was blocked by GABAergic/glycinergic receptor antagonists. Conversely, after 10 min of application, frequency in Lmx1bf/f/p slices was not different from baseline, whereas it was significantly depressed in WT slices. In WT mice in vivo, subcutaneous injection of 8-OH-DPAT produced similar biphasic respiratory effects as in Lmx1bf/f/p mice. We conclude that 5-HT1a receptor agonists have two competing effects: rapid stimulation of breathing due to excitation of the respiratory network, and delayed inhibition of breathing due to autoreceptor inhibition of 5-HT neurons. The former effect is presumably due to inhibition of inhibitory interneurons embedded in the respiratory network. PMID:24381267

  10. Activation of spinal and supraspinal cannabinoid-1 receptors lead to antinociception in a rat model of neuropathic spinal cord injury pain

    PubMed Central

    Hama, Aldric; Sagen, Jacqueline

    2011-01-01

    Activation of CNS cannabinoid subtype-1 (CB1) receptors has been shown to mediate the antinociceptive and other effects of systemically administered CB receptor agonists. The endogenous peptide CB receptor ligand hemopressin (HE) has previously demonstrated an antinociceptive effect in rats with a hind paw inflammation, without exhibiting characteristic CB1 receptor-mediated side-effects. The current study evaluated the effect of intrathecal (i.t.) and intracerebroventricular (i.c.v.) injection of HE in a rat model of neuropathic spinal cord injury (SCI) pain. The non-subtype selective CB receptor agonist WIN 55,212-2 was also centrally administered in SCI rats as a comparator. Four weeks following an acute compression of the mid-thoracic spinal cord, rats displayed markedly decreased hind paw withdrawal thresholds, indicative of below-level neuropathic pain. Central administration of WIN 55,212-2 significantly increased withdrawal thresholds, whereas HE did not. Hemopressin has been reported to block CB1 receptors in vitro, similar to the CB1 receptor antagonist rimonabant. Pretreatment with rimonabant completely blocked the antinociceptive effect of centrally administered WIN 55,212-2, but pretreatment with HE did not. While the data confirm that activation of either supraspinal or spinal CB1 receptors leads to significant antinociception in SCI rats, the current data do not support an antinociceptive effect from an acute blockade of central CB1 receptors, HE’s putative antinociceptive mechanism, in neuropathic SCI rats. Although such a mechanism could be useful in other models of pain with a significant inflammatory component, the current data indicate that activation of CB1 receptors is needed to ameliorate neuropathic SCI pain. PMID:21813113

  11. The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong

    2015-11-30

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibitsmore » constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.« less

  12. Novel antipsychotics activate recombinant human and native rat serotonin 5-HT1A receptors: affinity, efficacy and potential implications for treatment of schizophrenia.

    PubMed

    Newman-Tancredi, Adrian; Assié, Marie-Bernadette; Leduc, Nathalie; Ormière, Anne-Marie; Danty, Nathalie; Cosi, Cristina

    2005-09-01

    Serotonin 5-HT1A receptors are promising targets in the management of schizophrenia but little information exists about affinity and efficacy of novel antipsychotics at these sites. We addressed this issue by comparing binding affinity at 5-HT1A receptors with dopamine rD2 receptors, which are important targets for antipsychotic drug action. Agonist efficacy at 5-HT1A receptors was determined for G-protein activation and adenylyl cyclase activity. Whereas haloperidol, thioridazine, risperidone and olanzapine did not interact with 5-HT1A receptors, other antipsychotic agents exhibited agonist properties at these sites. E(max) values (% effect induced by 10 microM of 5-HT) for G-protein activation at rat brain 5-HT1A receptors: sarizotan (66.5), bifeprunox (35.9), SSR181507 (25.8), nemonapride (25.7), ziprasidone (20.6), SLV313 (19), aripiprazole (15), tiospirone (8.9). These data were highly correlated with results obtained at recombinant human 5-HT1A receptors in determinations of G-protein activation and inhibition of forskolin-stimulated adenylyl cyclase. In binding-affinity determinations, the antipsychotics exhibited diverse properties at r5-HT1A receptors: sarizotan (pK(i)=8.65), SLV313 (8.64), SSR181507 (8.53), nemonapride (8.35), ziprasidone (8.30), tiospirone (8.22), aripiprazole (7.42), bifeprunox (7.19) and clozapine (6.31). The affinity ratios of the ligands at 5-HT1A vs. D2 receptors also varied widely: ziprasidone, SSR181507 and SLV313 had similar affinities whereas aripiprazole, nemonapride and bifeprunox were more potent at D2 than 5-HT1A receptors. Taken together, these data indicate that aripiprazole has low efficacy and modest affinity at 5-HT1A receptors, whereas bifeprunox has low affinity but high efficacy. In contrast, SSR181507 has intermediate efficacy but high affinity, and is likely to have more prominent 5-HT1A receptor agonist properties. Thus, the contribution of 5-HT1A receptor activation to the pharmacological profile of action of the

  13. Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

    PubMed

    Shen, Kaitlin; Murphy, Ciara M; Chan, Ben; Kolind, Mille; Cheng, Tegan L; Mikulec, Kathy; Peacock, Lauren; Xue, Meilang; Park, Sang-Youel; Little, David G; Jackson, Chris J; Schindeler, Aaron

    2014-12-01

    Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (p<0.01) compared to rhBMP-2 alone. This was associated with a significant increase in CD31+ and TRAP+ cells in tissue sections of ectopic bone, consistent with enhanced vascularity and bone turnover. The actions of APC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  14. Constitutively active 5-HT2/α1 receptors facilitate muscle spasms after human spinal cord injury

    PubMed Central

    D'Amico, Jessica M.; Murray, Katherine C.; Li, Yaqing; Chan, K. Ming; Finlay, Mark G.; Bennett, David J.

    2013-01-01

    In animals, the recovery of motoneuron excitability in the months following a complete spinal cord injury is mediated, in part, by increases in constitutive serotonin (5-HT2) and norepinephrine (α1) receptor activity, which facilitates the reactivation of calcium-mediated persistent inward currents (CaPICs) without the ligands serotonin and norepinephrine below the injury. In this study we sought evidence for a similar role of constitutive monoamine receptor activity in the development of spasticity in human spinal cord injury. In chronically injured participants with partially preserved sensory and motor function, the serotonin reuptake inhibitor citalopram facilitated long-lasting reflex responses (spasms) previously shown to be mediated by CaPICs, suggesting that in incomplete spinal cord injury, functional descending sources of monoamines are present to activate monoamine receptors below the lesion. However, in participants with motor or motor/sensory complete injuries, the inverse agonist cyproheptadine, which blocks both ligand and constitutive 5-HT2/α1 receptor activity, decreased long-lasting reflexes, whereas the neutral antagonist chlorpromazine, which only blocks ligand activation of these receptors, had no effect. When tested in noninjured control participants having functional descending sources of monoamines, chlorpromazine was effective in reducing CaPIC-mediated motor unit activity. On the basis of these combined results, it appears that in severe spinal cord injury, facilitation of persistent inward currents and muscle spasms is mainly mediated by the activation of constitutive 5-HT2 and α1 receptor activity. Drugs that more selectively block these constitutively active monoamine receptors may provide better oral control of spasticity, especially in motor complete spinal cord injury where reducing motoneuron excitability is the primary goal. PMID:23221402

  15. Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals*

    PubMed Central

    Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn

    2011-01-01

    Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661

  16. Transcriptional activity of TGFβ1 and its receptors genes in thyroid gland.

    PubMed

    Kajdaniuk, Dariusz; Marek, Anna; Marek, Bogdan; Mazurek, Urszula; Fila-Daniłow, Anna; Foltyn, Wanda; Morawiec-Szymonik, Elżbieta; Siemińśka, Lucyna; Nowak, Mariusz; Głogowska-Szeląg, Joanna; Niedziołka-Zielonka, Danuta; Seemann, Michał; Kos-Kudła, Beata

    2016-01-01

    Determination of gene-candidates' profile expression responsible for fibrosis, immunosuppression, angiogenesis, and neoplasia processes in the pathogenesis of thyroid gland disease. Sixty-three patients underwent thyroidectomy: 27 with non-toxic nodular goitre (NG), 22 with toxic nodular goitre (TNG), six with papillary cancer (PTC), and eight with Graves' disease (GD). In thyroid tissues, transcriptional activity of TGFbeta1 and its receptors TGFbetaRI, TGFbetaRII, and TGFbetaRIII genes were assessed using RT-qPCR (Reverse Transcriptase Quantitative Polymerase Chain Reaction). Molecular analysis was performed in tissues derived from GD and from the tumour centre (PTC, NG, TNG) and from peripheral parts of the removed lobe without histopathological lesions (tissue control). Control tissue for analysis performed in GD was an unchanged tissue derived from peripheral parts of the removed lobe of patients surgically treated for a single benign tumour. Strict regulation observed among transcriptional activity of TGFb1 and their receptor TGFbetaRI-III genes in control tissues is disturbed in all pathological tissues - it is completely disturbed in PTC and GD, and partially in NG and TNG. Additionally, higher transcriptional activity of TGFb1 gene in PTC in comparison with benign tissues (NG, GD) and lower expression of mRNA TGFbRII (than in TNG, GD) and mRNA TGFbetaRIII than in all studied benign tissues (NG, TNG, GD) suggests a pathogenetic importance of this cytokine and its receptors in PTC development. In GD tissue, higher transcriptional activity of TGFbetaRII and TGFbetaRIII genes as compared to other pathological tissues was observed, indicating a participation of the receptors in the pathomechanism of autoimmune thyroid disease (AITD). TGFbeta1 blood concentrations do not reflect pathological processes taking place in thyroid gland. (Endokrynol Pol 2016; 67 (4): 375-382).

  17. MmTX1 and MmTX2 from coral snake venom potently modulate GABAA receptor activity.

    PubMed

    Rosso, Jean-Pierre; Schwarz, Jürgen R; Diaz-Bustamante, Marcelo; Céard, Brigitte; Gutiérrez, José M; Kneussel, Matthias; Pongs, Olaf; Bosmans, Frank; Bougis, Pierre E

    2015-02-24

    GABAA receptors shape synaptic transmission by modulating Cl(-) conductance across the cell membrane. Remarkably, animal toxins that specifically target GABAA receptors have not been identified. Here, we report the discovery of micrurotoxin1 (MmTX1) and MmTX2, two toxins present in Costa Rican coral snake venom that tightly bind to GABAA receptors at subnanomolar concentrations. Studies with recombinant and synthetic toxin variants on hippocampal neurons and cells expressing common receptor compositions suggest that MmTX1 and MmTX2 allosterically increase GABAA receptor susceptibility to agonist, thereby potentiating receptor opening as well as desensitization, possibly by interacting with the α(+)/β(-) interface. Moreover, hippocampal neuron excitability measurements reveal toxin-induced transitory network inhibition, followed by an increase in spontaneous activity. In concert, toxin injections into mouse brain result in reduced basal activity between intense seizures. Altogether, we characterized two animal toxins that enhance GABAA receptor sensitivity to agonist, thereby establishing a previously unidentified class of tools to study this receptor family.

  18. The Drosophila FTZ-F1 Nuclear Receptor Mediates Juvenile Hormone Activation of E75A Gene Expression through an Intracellular Pathway*

    PubMed Central

    Dubrovsky, Edward B.; Dubrovskaya, Veronica A.; Bernardo, Travis; Otte, Valerie; DiFilippo, Robert; Bryan, Heather

    2011-01-01

    Juvenile hormone (JH) regulates a wide variety of biological activities in holometabolous insects, ranging from vitellogenesis and caste determination in adults to the timing of metamorphosis in larvae. The mechanism of JH signaling in such a diverse array of processes remains either unknown or contentious. We previously found that the nuclear receptor gene E75A is activated in S2 cells as a primary response to JH. Here, by expressing an intracellular form of JH esterase, we demonstrate that JH must enter the cell in order to activate E75A. To find intracellular receptors involved in the JH response, we performed an RNAi screen against nuclear receptor genes expressed in this cell line and identified the orphan receptor FTZ-F1. Removal of FTZ-F1 prevents JH activation of E75A, whereas overexpression enhances activation, implicating FTZ-F1 as a critical component of the JH response. FTZ-F1 is bound in vivo to multiple enhancers upstream of E75A, suggesting that it participates in direct JH-mediated gene activation. To better define the role of FTZ-F1 in JH signaling, we investigated interactions with candidate JH receptors and found that the bHLH-PAS proteins MET and GCE both interact with FTZ-F1 and can activate transcription through the FTZ-F1 response element. Removal of endogenous GCE, but not MET, prevents JH activation of E75A. We propose that FTZ-F1 functions as a competence factor by loading JH signaling components to the promoter, thus facilitating the direct regulation of E75A gene expression by JH. PMID:21832074

  19. Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor.

    PubMed Central

    Reedijk, M; Liu, X Q; Pawson, T

    1990-01-01

    The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages. Images PMID:2172781

  20. Heterodimerization with beta2-adrenergic receptors promotes surface expression and functional activity of alpha1D-adrenergic receptors.

    PubMed

    Uberti, Michelle A; Hague, Chris; Oller, Heide; Minneman, Kenneth P; Hall, Randy A

    2005-04-01

    The alpha1D-adrenergic receptor (alpha1D-AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote alpha1D-AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of alpha1D-AR at the cell surface: alpha1B-AR and beta2-AR. Confocal imaging confirmed that coexpression with beta2-AR resulted in translocation of alpha1D-AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that alpha1D-AR and beta2-AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total alpha1D-AR binding sites upon coexpression with beta2-AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with beta2-AR significantly enhanced the coupling of alpha1D-AR to norepinephrine-stimulated Ca2+ mobilization. Heterodimerization of beta2-AR with alpha1D-AR also conferred the ability of alpha1D-AR to cointernalize upon beta2-AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other's activity. These findings demonstrate that the selective association of alpha1D-AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between alpha1- and beta2-ARs that is mediated through heterodimerization and cross-internalization.

  1. Activation of Neurokinin-1 Receptors during Ozone Inhalation Contributes to Epithelial Injury and Repair

    PubMed Central

    Oslund, Karen L.; Hyde, Dallas M.; Putney, Leialoha F.; Alfaro, Mario F.; Walby, William F.; Tyler, Nancy K.; Schelegle, Edward S.

    2008-01-01

    We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2′-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2′-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress–positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways. PMID:18390473

  2. Neurokinin-1 receptor activation in Botzinger complex evokes bradypnoea.

    PubMed

    Fong, Angelina Y; Potts, Jeffrey T

    2006-09-15

    In the present study, we examined the role of the neurokinin-1 receptor (NK1R) in the modulation of respiratory rhythm in a functionally identified bradypnoeic region of the ventral respiratory group (VRG) in the in situ arterially perfused juvenile rat preparation. In electrophysiologically and functionally identified bradypnoeic sites corresponding to the Bötzinger complex (BötC), microinjection of the selective NK1R agonist [Sar(9)-Met(O(2))(11)]-substance P (SSP) produced a significant reduction in phrenic frequency mediated exclusively by an increase in expiratory duration (T(E)). The reduction was characterized by a significant increase in postinspiratory (post-I) duration with no effect on either late-expiratory duration (E2) or inspiratory duration (T(I)). In contrast, in a functionally identified tachypnoeic region, corresponding to the preBötzinger complex (Pre-BötC), control microinjection of SSP elicited tachypnoea. Pretreatment with the NK1R antagonist CP99994 in the BötC significantly attenuated the bradypnoeic response to SSP injection and blunted the increase in T(E) duration. This effect of SSP mimicked the extension of T(E) produced by activation of the Hering-Breuer reflex. Therefore, we hypothesized that activation of NK1Rs in the BötC is requisite for the expiratory-lengthening effect of the Hering-Breuer reflex. Unilateral electrical stimulation of the cervical vagus nerve produced bradypnoea by exclusively extending T(E). Ipsilateral blockade of NK1Rs by CP99994 following blockade of the contralateral BötC by the GABA(A) receptor agonist muscimol significantly reduced the extension of T(E) produced by vagal stimulation. Results from the present study demonstrate that selective activation of NK1Rs in a functionally identified bradypnoeic region of the VRG can depress respiratory frequency by selectively lengthening post-I duration and provide evidence that endogenous activation of NK1Rs in the BötC appears to be involved in the

  3. The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

    NASA Astrophysics Data System (ADS)

    Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

    2015-03-01

    The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

  4. Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts.

    PubMed Central

    Burch, R M; Connor, J R; Axelrod, J

    1988-01-01

    Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s). PMID:2901097

  5. Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase.

    PubMed

    Stein, E; Huynh-Do, U; Lane, A A; Cerretti, D P; Daniel, T O

    1998-01-16

    Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and its Drosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK.

  6. G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway.

    PubMed

    Law, Nathan C; White, Morris F; Hunzicker-Dunn, Mary E

    2016-12-30

    G protein-coupled receptors (GPCRs) activate PI3K/v-AKT thymoma viral oncoprotein (AKT) to regulate many cellular functions that promote cell survival, proliferation, and growth. However, the mechanism by which GPCRs activate PI3K/AKT remains poorly understood. We used ovarian preantral granulosa cells (GCs) to elucidate the mechanism by which the GPCR agonist FSH via PKA activates the PI3K/AKT cascade. Insulin-like growth factor 1 (IGF1) is secreted in an autocrine/paracrine manner by GCs and activates the IGF1 receptor (IGF1R) but, in the absence of FSH, fails to stimulate YXXM phosphorylation of IRS1 (insulin receptor substrate 1) required for PI3K/AKT activation. We show that PKA directly phosphorylates the protein phosphatase 1 (PP1) regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) to activate PP1 associated with the IGF1R-IRS1 complex. Activated PP1 is sufficient to dephosphorylate at least four IRS1 Ser residues, Ser 318 , Ser 346 , Ser 612 , and Ser 789 , and promotes IRS1 YXXM phosphorylation by the IGF1R to activate the PI3K/AKT cascade. Additional experiments indicate that this mechanism also occurs in breast cancer, thyroid, and preovulatory granulosa cells, suggesting that the PKA-dependent dephosphorylation of IRS1 Ser/Thr residues is a conserved mechanism by which GPCRs signal to activate the PI3K/AKT pathway downstream of the IGF1R. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Activation of D1 dopamine receptors induces emergence from isoflurane general anesthesia

    PubMed Central

    Taylor, Norman E.; Chemali, Jessica J.; Brown, Emery N.; Solt, Ken

    2012-01-01

    BACKGROUND A recent study showed that methylphenidate induces emergence from isoflurane anesthesia. Methylphenidate inhibits dopamine and norepinephrine reuptake transporters. The objective of this study was to test the hypothesis that selective dopamine receptor activation induces emergence from isoflurane anesthesia. METHODS In adult rats, we tested the effects of chloro-APB (D1 agonist) and quinpirole (D2 agonist) on time to emergence from isoflurane general anesthesia. We then performed a dose–response study to test for chloro-APB-induced restoration of righting during continuous isoflurane anesthesia. SCH-23390 (D1 antagonist) was used to confirm that the effects induced by chloro-APB are specifically mediated by D1 receptors. In a separate group of animals, spectral analysis was performed on surface electroencephalogram recordings to assess neurophysiological changes induced by chloro-APB and quinpirole during isoflurane general anesthesia. RESULTS Chloro-APB decreased median time to emergence from 330s to 50s. The median difference in time to emergence between the saline control group (n=6) and the chloro-APB group (n = 6) was 222s (95% CI: 77–534s, Mann-Whitney test). This difference was statistically significant (p = 0.0082). During continuous isoflurane anesthesia, chloro-APB dose-dependently restored righting (n = 6) and decreased electroencephalogram delta power (n = 4). These effects were inhibited by pretreatment with SCH-23390. Quinpirole did not restore righting (n = 6) and had no significant effect on the electroencephalogram (n = 4) during continuous isoflurane anesthesia. CONCLUSIONS Activation of D1 receptors by chloro-APB decreases time to emergence from isoflurane anesthesia, and produces behavioral and neurophysiological evidence of arousal during continuous isoflurane anesthesia. These findings suggest that selective activation of a D1 receptor-mediated arousal mechanism is sufficient to induce emergence from isoflurane general

  8. Structural Determinants of the Transient Receptor Potential 1 (TRPV1) Channel Activation by Phospholipid Analogs*

    PubMed Central

    Morales-Lázaro, Sara L.; Serrano-Flores, Barbara; Llorente, Itzel; Hernández-García, Enrique; González-Ramírez, Ricardo; Banerjee, Souvik; Miller, Duane; Gududuru, Veeresh; Fells, James; Norman, Derek; Tigyi, Gabor; Escalante-Alcalde, Diana; Rosenbaum, Tamara

    2014-01-01

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1. PMID:25035428

  9. Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid-Activated Receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kruse, Schoen W; Suino-Powell, Kelly; Zhou, X Edward

    2010-01-12

    The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 {angstrom} crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix {alpha}10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site,more » thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.« less

  10. Sarizotan, a serotonin 5-HT1A receptor agonist and dopamine receptor ligand. 1. Neurochemical profile.

    PubMed

    Bartoszyk, G D; Van Amsterdam, C; Greiner, H E; Rautenberg, W; Russ, H; Seyfried, C A

    2004-02-01

    Sarizotan exhibited high affinities only to serotonin 5-HT1A receptors and dopamine DA D4>D3>D2 receptors with the profile of a 5-HT1A agonist and DA antagonist demonstrated by the inhibition of cAMP-stimulation and guinea pig ileum contraction, decreased accumulation of the 5-HT precursor 5-hydroxytryptophan and increased levels of 5-HT metabolites, increased accumulation of DA precursor dihydroxyphenylalanine (DOPA) and the reduced levels of DA metabolites in intact rats. However, sarizotan at higher doses decreased DA precursor accumulation in reserpinized rats and induced contralateral rotational behavior in unilaterally substantia nigra lesioned rats, indicating some intrinsic dopaminergic activity; at D2 receptors sarizotan may act as a partial agonist, depending on the dopaminergic impulse flow. Sarizotan represents a new approach for the treatment of extrapyramidal motor complications such as l-DOPA-induced dyskinesia in Parkinson's disease.

  11. Single-channel activations and concentration jumps: comparison of recombinant NR1a/NR2A and NR1a/NR2D NMDA receptors

    PubMed Central

    Wyllie, David J A; Béhé, Philippe; Colquhoun, David

    1998-01-01

    We have expressed recombinant NR1a/NR2A and NR1a/NR2D N-methyl-D-aspartate (NMDA) receptor channels in Xenopus oocytes and made recordings of single-channel and macroscopic currents in outside-out membrane patches. For each receptor type we measured (a) the individual single-channel activations evoked by low glutamate concentrations in steady-state recordings, and (b) the macroscopic responses elicited by brief concentration jumps with high agonist concentrations, and we explore the relationship between these two sorts of observation. Low concentration (5–100 nM) steady-state recordings of NR1a/NR2A and NR1a/NR2D single-channel activity generated shut-time distributions that were best fitted with a mixture of five and six exponential components, respectively. Individual activations of either receptor type were resolved as bursts of openings, which we refer to as ‘super-clusters’. During a single activation, NR1a/NR2A receptors were open for 36 % of the time, but NR1a/NR2D receptors were open for only 4 % of the time. For both, distributions of super-cluster durations were best fitted with a mixture of six exponential components. Their overall mean durations were 35.8 and 1602 ms, respectively. Steady-state super-clusters were aligned on their first openings and averaged. The average was well fitted by a sum of exponentials with time constants taken from fits to super-cluster length distributions. It is shown that this is what would be expected for a channel that shows simple Markovian behaviour. The current through NR1a/NR2A channels following a concentration jump from zero to 1 mM glutamate for 1 ms was well fitted by three exponential components with time constants of 13 ms (rising phase), 70 ms and 350 ms (decaying phase). Similar concentration jumps on NR1a/NR2D channels were well fitted by two exponentials with means of 45 ms (rising phase) and 4408 ms (decaying phase) components. During prolonged exposure to glutamate, NR1a/NR2A channels desensitized

  12. Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor.

    PubMed Central

    Göttlicher, M; Widmark, E; Li, Q; Gustafsson, J A

    1992-01-01

    Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. Testing of compounds related to lipid metabolism or peroxisomal proliferation revealed that 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activate the receptor chimera. In addition, saturated fatty acids induce the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. In conclusion, the present results indicate that fatty acids can regulate gene expression mediated by a member of the steroid nuclear receptor superfamily. Images PMID:1316614

  13. Dimerization with Cannabinoid Receptors Allosterically Modulates Delta Opioid Receptor Activity during Neuropathic Pain

    PubMed Central

    Stockton, Steven D.; Miller, Lydia K.; Devi, Lakshmi A.

    2012-01-01

    The diversity of receptor signaling is increased by receptor heteromerization leading to dynamic regulation of receptor function. While a number of studies have demonstrated that family A G-protein-coupled receptors are capable of forming heteromers in vitro, the role of these heteromers in normal physiology and disease has been poorly explored. In this study, direct interactions between CB1 cannabinoid and delta opioid receptors in the brain were examined. Additionally, regulation of heteromer levels and signaling in a rodent model of neuropathic pain was explored. First we examined changes in the expression, function and interaction of these receptors in the cerebral cortex of rats with a peripheral nerve lesion that resulted in neuropathic pain. We found that, following the peripheral nerve lesion, the expression of both cannabinoid type 1 receptor (CB1R) and the delta opioid receptor (DOR) are increased in select brain regions. Concomitantly, an increase in CB1R activity and decrease in DOR activity was observed. We hypothesize that this decrease in DOR activity could be due to heteromeric interactions between these two receptors. Using a CB1R-DOR heteromer-specific antibody, we found increased levels of CB1R-DOR heteromer protein in the cortex of neuropathic animals. We subsequently examined the functionality of these heteromers by testing whether low, non-signaling doses of CB1R ligands influenced DOR signaling in the cortex. We found that, in cortical membranes from animals that experienced neuropathic pain, non-signaling doses of CB1R ligands significantly enhanced DOR activity. Moreover, this activity is selectively blocked by a heteromer-specific antibody. Together, these results demonstrate an important role for CB1R-DOR heteromers in altered cortical function of DOR during neuropathic pain. Moreover, they suggest the possibility that a novel heteromer-directed therapeutic strategy for enhancing DOR activity, could potentially be employed to reduce

  14. Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1 Alpha as a Novel Target for Bipolar Disorder and Other Neuropsychiatric Disorders.

    PubMed

    Nierenberg, Andrew A; Ghaznavi, Sharmin A; Sande Mathias, Isadora; Ellard, Kristen K; Janos, Jessica A; Sylvia, Louisa G

    2018-05-01

    Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) is a protein that regulates metabolism and inflammation by activating nuclear receptors, especially the family of peroxisome proliferator-activated receptors (PPARs). PGC-1 alpha and PPARs also regulate mitochondrial biogenesis, cellular energy production, thermogenesis, and lipid metabolism. Brain energy metabolism may also be regulated in part by the interaction between PGC-1 alpha and PPARs. Because neurodegenerative diseases (Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis) and bipolar disorder have been associated with dysregulated mitochondrial and brain energy metabolism, PGC-1 alpha may represent a potential drug target for these conditions. The purpose of this article is to review the physiology of PGC-1 alpha, PPARs, and the role of PPAR agonists to target PGC-1 alpha to treat neurodegenerative diseases and bipolar disorder. We also review clinical trials of repurposed antidiabetic thiazolidines and anti-triglyceride fibrates (PPAR agonists) for neurodegenerative diseases and bipolar disorder. PGC-1 alpha and PPARs are innovative potential targets for bipolar disorder and warrant future clinical trials. Copyright © 2018. Published by Elsevier Inc.

  15. Endothelins activate Ca(2+)-gated K(+) channels via endothelin B receptors in CD-1 mouse erythrocytes.

    PubMed

    Rivera, A; Rotter, M A; Brugnara, C

    1999-10-01

    Cell dehydration mediated by Ca(2+)-activated K(+) channels plays an important role in the pathogenesis of sickle cell disease. CD-1 mouse erythrocytes possess a Ca(2+)-activated K(+) channel (Gardos channel) with maximal velocity (V(max)) of 0.154 +/- 0.02 mmol. l cells(-1). min(-1) and an affinity constant (K(0.5)) for Ca(2+) of 286 +/- 83 nM in the presence of A-23187. Cells pretreated with 500 nM endothelin-1 (ET-1) increased their V(max) by 88 +/- 9% (n = 8) and decreased their K(0.5) for Ca(2+) to 139 +/- 63 nM (P < 0.05; n = 4). Activation of the Gardos channel resulted in an EC(50) of 75 +/- 20 nM for ET-1 and 374 +/- 97 nM for ET-3. Analysis of the affinity of unlabeled ET-1 for its receptor showed two classes of binding sites with apparent dissociation constants of 167 +/- 51 and 785 +/- 143 nM and with capacity of binding sites of 298 +/- 38 and 1,568 +/- 211 sites/cell, respectively. The Gardos channel was activated by the endothelin B (ET(B)) receptor agonist IRL 1620 and inhibited by BQ-788, demonstrating the involvement of ET(B) receptors. Calphostin C inhibited 73% of ET-1-induced Gardos activation and 84% of the ET-1-induced membrane protein kinase C activity. Thus endothelins regulate erythrocyte Gardos channels via ET(B) receptors and a calphostin-sensitive mechanism.

  16. Activation of the sympathetic nervous system mediates hypophagic and anxiety-like effects of CB1 receptor blockade

    PubMed Central

    Bellocchio, Luigi; Soria-Gómez, Edgar; Quarta, Carmelo; Metna-Laurent, Mathilde; Cardinal, Pierre; Binder, Elke; Cannich, Astrid; Delamarre, Anna; Häring, Martin; Martín-Fontecha, Mar; Vega, David; Leste-Lasserre, Thierry; Bartsch, Dusan; Monory, Krisztina; Lutz, Beat; Chaouloff, Francis; Pagotto, Uberto; Guzman, Manuel; Cota, Daniela; Marsicano, Giovanni

    2013-01-01

    Complex interactions between periphery and the brain regulate food intake in mammals. Cannabinoid type-1 (CB1) receptor antagonists are potent hypophagic agents, but the sites where this acute action is exerted and the underlying mechanisms are not fully elucidated. To dissect the mechanisms underlying the hypophagic effect of CB1 receptor blockade, we combined the acute injection of the CB1 receptor antagonist rimonabant with the use of conditional CB1-knockout mice, as well as with pharmacological modulation of different central and peripheral circuits. Fasting/refeeding experiments revealed that CB1 receptor signaling in many specific brain neurons is dispensable for the acute hypophagic effects of rimonabant. CB1 receptor antagonist-induced hypophagia was fully abolished by peripheral blockade of β-adrenergic transmission, suggesting that this effect is mediated by increased activity of the sympathetic nervous system. Consistently, we found that rimonabant increases gastrointestinal metabolism via increased peripheral β-adrenergic receptor signaling in peripheral organs, including the gastrointestinal tract. Blockade of both visceral afferents and glutamatergic transmission in the nucleus tractus solitarii abolished rimonabant-induced hypophagia. Importantly, these mechanisms were specifically triggered by lipid-deprivation, revealing a nutrient-specific component acutely regulated by CB1 receptor blockade. Finally, peripheral blockade of sympathetic neurotransmission also blunted central effects of CB1 receptor blockade, such as fear responses and anxiety-like behaviors. These data demonstrate that, independently of their site of origin, important effects of CB1 receptor blockade are expressed via activation of peripheral sympathetic activity. Thus, CB1 receptors modulate bidirectional circuits between the periphery and the brain to regulate feeding and other behaviors. PMID:23487769

  17. Progesterone Directly and Rapidly Inhibits GnRH Neuronal Activity via Progesterone Receptor Membrane Component 1

    PubMed Central

    Bashour, Nicholas Michael

    2012-01-01

    GnRH neurons are essential for reproduction, being an integral component of the hypothalamic-pituitary-gonadal axis. Progesterone (P4), a steroid hormone, modulates reproductive behavior and is associated with rapid changes in GnRH secretion. However, a direct action of P4 on GnRH neurons has not been previously described. Receptors in the progestin/adipoQ receptor family (PAQR), as well as progesterone receptor membrane component 1 (PgRMC1) and its partner serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1) mRNA binding protein 1 (SERBP1), have been shown to mediate rapid progestin actions in various tissues, including the brain. This study shows that PgRMC1 and SERBP1, but not PAQR, are expressed in prenatal GnRH neurons. Expression of PgRMC1 and SERBP1 was verified in adult mouse GnRH neurons. To investigate the effect of P4 on GnRH neuronal activity, calcium imaging was used on primary GnRH neurons maintained in explants. Application of P4 significantly decreased the activity of GnRH neurons, independent of secretion of gamma-aminobutyric acidergic and glutamatergic input, suggesting a direct action of P4 on GnRH neurons. Inhibition was not blocked by RU486, an antagonist of the classic nuclear P4 receptor. Inhibition was also maintained after uncoupling of the inhibitory regulative G protein (Gi/o), the signal transduction pathway used by PAQR. However, AG-205, a PgRMC1 ligand and inhibitor, blocked the rapid P4-mediated inhibition, and inhibition of protein kinase G, thought to be activated downstream of PgRMC1, also blocked the inhibitory activity of P4. These data show for the first time that P4 can act directly on GnRH neurons through PgRMC1 to inhibit neuronal activity. PMID:22822163

  18. High-expression β(1) adrenergic receptor/cell membrane chromatography method based on a target receptor to screen active ingredients from traditional Chinese medicines.

    PubMed

    Yue, Yuan; Xue, Hui; Wang, Xin; Yang, Qian; Song, Yanhong; Li, Xiaoni

    2014-02-01

    β-Adrenergic receptors are important targets for drug discovery. We have developed a new β1 -adrenergic receptor cell membrane chromatography (β1 AR-CMC) with offline ultra-performance LC (UPLC) and MS method for screening active ingredients from traditional Chinese medicines. In this study, Chinese hamster ovary-S cells with high β1 AR expression levels were established and used to prepare a cell membrane stationary phase in a β1 AR-CMC model. The retention fractions were separated and identified by the UPLC-MS system. The screening results found that isoimperatorin from Rhizoma et Radix Notopterygii was the targeted component that could act on β1 AR in similar manner of metoprolol as a control drug. In addition, the biological effects of active component were also investigated in order to search for a new type of β1 AR antagonist. It will be a useful method for drug discovery as a leading compound resource. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Pindolol antagonises G-protein activation at both pre- and postsynaptic serotonin 5-HT1A receptors: a.

    PubMed

    Newman-Tancredi, A; Chaput, C; Touzard, M; Millan, M J

    2001-04-01

    The arylalkylamine, pindolol, may potentiate the clinical actions of antidepressant agents. Although it is thought to act via blockade of 5-HT1A autoreceptors, its efficacy at these sites remains controversial. Herein, we evaluated the actions of pindolol at 5-HT1A autoreceptors and specific populations of postsynaptic 5-HT1A receptors employing [35S]GTPgammaS autoradiography, a measure of receptor-mediated G-protein activation. Both 8-OH-DPAT (1 microM) and 5-HT (10 microM) elicited a pronounced increase in [35S]GTPyS binding in the dorsal raphe nucleus, which contains serotonergic cell bodies bearing 5-HT1A autoreceptors. Pindolol abolished their actions. In the dentate gyrus, lateral septum and entorhinal cortex, structures enriched in postsynaptic 5-HT1A receptors, 8-OH-DPAT (1 microM) and 5-HT (10 microM) also elicited a marked increase in [35S]GTPgammaS binding which was likewise blocked by pindolol. The antagonism of 5-HT-induced [35S]GTPgammaS labelling in the dentate gyrus was shown to be concentration-dependent, yielding a pIC50 of 5.82. Pindolol did not, itself, affect [35S]GTPgammaS binding in any brain region examined. In conclusion, these data suggest that, as characterised by [35S]GTPgammaS autoradiography, and compared with 5-HT and 8-OH-DPAT, pindolol possesses low efficacy at both pre- and postsynaptic 5-HT1A receptors.

  20. Lysophosphatidic Acid Receptor Type 1 (LPA1) Plays a Functional Role in Osteoclast Differentiation and Bone Resorption Activity*

    PubMed Central

    David, Marion; Machuca-Gayet, Irma; Kikuta, Junichi; Ottewell, Penelope; Mima, Fuka; Leblanc, Raphael; Bonnelye, Edith; Ribeiro, Johnny; Holen, Ingunn; Vales, Rùben Lopez; Jurdic, Pierre; Chun, Jerold; Clézardin, Philippe; Ishii, Masaru; Peyruchaud, Olivier

    2014-01-01

    Lysophosphatidic acid (LPA) is a natural bioactive lipid that acts through six different G protein-coupled receptors (LPA1–6) with pleiotropic activities on multiple cell types. We have previously demonstrated that LPA is necessary for successful in vitro osteoclastogenesis of bone marrow cells. Bone cells controlling bone remodeling (i.e. osteoblasts, osteoclasts, and osteocytes) express LPA1, but delineating the role of this receptor in bone remodeling is still pending. Despite Lpar1−/− mice displaying a low bone mass phenotype, we demonstrated that bone marrow cell-induced osteoclastogenesis was reduced in Lpar1−/− mice but not in Lpar2−/− and Lpar3−/− animals. Expression of LPA1 was up-regulated during osteoclastogenesis, and LPA1 antagonists (Ki16425, Debio0719, and VPC12249) inhibited osteoclast differentiation. Blocking LPA1 activity with Ki16425 inhibited expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and dendritic cell-specific transmembrane protein and interfered with the fusion but not the proliferation of osteoclast precursors. Similar to wild type osteoclasts treated with Ki16425, mature Lpar1−/− osteoclasts had reduced podosome belt and sealing zone resulting in reduced mineralized matrix resorption. Additionally, LPA1 expression markedly increased in the bone of ovariectomized mice, which was blocked by bisphosphonate treatment. Conversely, systemic treatment with Debio0719 prevented ovariectomy-induced cancellous bone loss. Moreover, intravital multiphoton microscopy revealed that Debio0719 reduced the retention of CX3CR1-EGFP+ osteoclast precursors in bone by increasing their mobility in the bone marrow cavity. Overall, our results demonstrate that LPA1 is essential for in vitro and in vivo osteoclast activities. Therefore, LPA1 emerges as a new target for the treatment of diseases associated with excess bone loss. PMID:24429286

  1. Application of Quantitative Structure–Activity Relationship Models of 5-HT1A Receptor Binding to Virtual Screening Identifies Novel and Potent 5-HT1A Ligands

    PubMed Central

    2015-01-01

    The 5-hydroxytryptamine 1A (5-HT1A) serotonin receptor has been an attractive target for treating mood and anxiety disorders such as schizophrenia. We have developed binary classification quantitative structure–activity relationship (QSAR) models of 5-HT1A receptor binding activity using data retrieved from the PDSP Ki database. The prediction accuracy of these models was estimated by external 5-fold cross-validation as well as using an additional validation set comprising 66 structurally distinct compounds from the World of Molecular Bioactivity database. These validated models were then used to mine three major types of chemical screening libraries, i.e., drug-like libraries, GPCR targeted libraries, and diversity libraries, to identify novel computational hits. The five best hits from each class of libraries were chosen for further experimental testing in radioligand binding assays, and nine of the 15 hits were confirmed to be active experimentally with binding affinity better than 10 μM. The most active compound, Lysergol, from the diversity library showed very high binding affinity (Ki) of 2.3 nM against 5-HT1A receptor. The novel 5-HT1A actives identified with the QSAR-based virtual screening approach could be potentially developed as novel anxiolytics or potential antischizophrenic drugs. PMID:24410373

  2. Proteinase-activated receptors (PARs) – focus on receptor-receptor-interactions and their physiological and pathophysiological impact

    PubMed Central

    2013-01-01

    Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects. In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease. PMID:24215724

  3. Differential Mechanisms of Activation of the Ang Peptide Receptors AT1, AT2, and MAS: Using In Silico Techniques to Differentiate the Three Receptors

    PubMed Central

    Prokop, Jeremy W.; Santos, Robson A. S.; Milsted, Amy

    2013-01-01

    The renin-angiotensin system is involved in multiple conditions ranging from cardiovascular disorders to cancer. Components of the pathway, including ACE, renin and angiotensin receptors are targets for disease treatment. This study addresses three receptors of the pathway: AT1, AT2, and MAS and how the receptors are similar and differ in activation by angiotensin peptides. Combining biochemical and amino acid variation data with multiple species sequence alignments, structural models, and docking site predictions allows for visualization of how angiotensin peptides may bind and activate the receptors; allowing identification of conserved and variant mechanisms in the receptors. MAS differs from AT1 favoring Ang-(1–7) and not Ang II binding, while AT2 recently has been suggested to preferentially bind Ang III. A new model of Ang peptide binding to AT1 and AT2 is proposed that correlates data from site directed mutagenesis and photolabled experiments that were previously considered conflicting. Ang II binds AT1 and AT2 through a conserved initial binding mode involving amino acids 111 (consensus 325) of AT1 (Asn) interacting with Tyr (4) of Ang II and 199 and 256 (consensus 512 and 621, a Lys and His respectively) interacting with Phe (8) of Ang II. In MAS these sites are not conserved, leading to differential binding and activation by Ang-(1–7). In both AT1 and AT2, the Ang II peptide may internalize through Phe (8) of Ang II propagating through the receptors’ conserved aromatic amino acids to the final photolabled positioning relative to either AT1 (amino acid 294, Asn, consensus 725) or AT2 (138, Leu, consensus 336). Understanding receptor activation provides valuable information for drug design and identification of other receptors that can potentially bind Ang peptides. PMID:23755216

  4. Anticonvulsant activity of a mGlu(4alpha) receptor selective agonist, (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid.

    PubMed

    Chapman, A G; Talebi, A; Yip, P K; Meldrum, B S

    2001-07-20

    The metabotropic Group III agonist, (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid (ACPT-1), selective for the mGlu(4alpha) receptor, suppresses sound-induced seizures in DBA/2 mice following its intracerebroventricular (i.c.v.) administration (ED(50) 5.6 [2.9-10.7], nmol i.c.v., 15 min, clonic phase) and in genetically epilepsy-prone (GEP) rats following focal administration into the inferior colliculus (ED(50) 0.08 [0.01-0.50], nmol, 60 min, clonic phase). ACPT-1 also protects against clonic seizures induced in DBA/2 mice by the Group I agonist, (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG) (ED(50) 0.60 [0.29-1.2], nmol i.c.v.) and by the Group III antagonist, (RS)-alpha-methylserine-O-phosphate (MSOP) (ED(50) 49.3 [37.9-64.1], nmol i.c.v.). Another Group III agonist, (RS)-4-phosphonophenyl-glycine (PPG), preferentially activating the mGlu(8) receptor, previously shown to protect against sound-induced seizures in DBA/2 mice and GEP rats, also protects against seizures induced in DBA/2 by 3,5-DHPG (ED(50) 3.7 [2.4-5.7], nmol i.c.v.) and by the Group III antagonist, MSOP (ED(50) 40.2 [21.0-77.0], nmol i.c.v.). At very high doses (500 nmol i.c.v. and above), Group III antagonists have pro-convulsant and convulsant activity. The anticonvulsant protection against sound-induced seizures in DBA/2 mice provided by a fully protective dose (20 nmol, i.c.v.) of the mGlu(4) receptor agonist ACPT-1, is partially reversed by the co-administration of the Group III antagonists, MSOP, (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG) or (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4), in the 20-50 nmol dose range. At doses of 50-200 nmol, MPPG and MAP4 cause further reversal of the ACPT-1 anticonvulsant protection, while the MSOP effect on ACPT-1 protection is abolished at higher doses. In contrast, the anticonvulsant protection against sound-induced seizures in DBA/2 mice provided by a fully protective dose (20 nmol, i.c.v.) of the mGlu(8) receptor agonist PPG, is not

  5. Activation of G protein-coupled estrogen receptor 1 (GPER-1) decreases fluid intake in female rats

    PubMed Central

    Santollo, Jessica; Daniels, Derek

    2015-01-01

    Estradiol (E2) decreases fluid intake in the female rat and recent studies from our lab demonstrate that the effect is at least in part mediated by membrane-associated estrogen receptors. Because multiple estrogen receptor subtypes can localize to the cell membrane, it is unclear which receptor(s) is generating the anti-dipsogenic effect of E2. The G protein-coupled estrogen receptor 1 (GPER-1) is a particularly interesting possibility because it has been shown to regulate blood pressure; many drinking-regulatory systems play overlapping roles in the control of blood pressure. Accordingly, we tested the hypothesis that activation of GPER-1 is sufficient to decrease fluid intake in female rats. In support of this hypothesis we found that treatment with the selective GPER-1 agonist G1 reduced AngII-stimulated fluid intake in OVX rats. Given the close association between food and fluid intakes in rats, and previous reports suggesting GPER-1 plays a role in energy homeostasis, we tested the hypothesis that the effect of GPER-1 on fluid intake was caused by a more direct effect on food intake. We found, however, that G1-treatment did not influence short-term or overnight food intake in OVX rats. Together these results reveal a novel effect of GPER-1 in the control of drinking behavior and provide an example of the divergence in the controls of fluid and food intakes in female rats. PMID:26093261

  6. Activation of G protein-coupled estrogen receptor 1 (GPER-1) decreases fluid intake in female rats.

    PubMed

    Santollo, Jessica; Daniels, Derek

    2015-07-01

    Estradiol (E2) decreases fluid intake in the female rat and recent studies from our lab demonstrate that the effect is at least in part mediated by membrane-associated estrogen receptors. Because multiple estrogen receptor subtypes can localize to the cell membrane, it is unclear which receptor(s) is generating the anti-dipsogenic effect of E2. The G protein-coupled estrogen receptor 1 (GPER-1) is a particularly interesting possibility because it has been shown to regulate blood pressure; many drinking-regulatory systems play overlapping roles in the control of blood pressure. Accordingly, we tested the hypothesis that activation of GPER-1 is sufficient to decrease fluid intake in female rats. In support of this hypothesis we found that treatment with the selective GPER-1 agonist G1 reduced AngII-stimulated fluid intake in OVX rats. Given the close association between food and fluid intakes in rats, and previous reports suggesting GPER-1 plays a role in energy homeostasis, we tested the hypothesis that the effect of GPER-1 on fluid intake was caused by a more direct effect on food intake. We found, however, that G1-treatment did not influence short-term or overnight food intake in OVX rats. Together these results reveal a novel effect of GPER-1 in the control of drinking behavior and provide an example of the divergence in the controls of fluid and food intakes in female rats. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Structural determinants of the transient receptor potential 1 (TRPV1) channel activation by phospholipid analogs.

    PubMed

    Morales-Lázaro, Sara L; Serrano-Flores, Barbara; Llorente, Itzel; Hernández-García, Enrique; González-Ramírez, Ricardo; Banerjee, Souvik; Miller, Duane; Gududuru, Veeresh; Fells, James; Norman, Derek; Tigyi, Gabor; Escalante-Alcalde, Diana; Rosenbaum, Tamara

    2014-08-29

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. 3,4-Dihydro-2H-benzoxazinones are 5-HT(1A) receptor antagonists with potent 5-HT reuptake inhibitory activity.

    PubMed

    Atkinson, Peter J; Bromidge, Steven M; Duxon, Mark S; Gaster, Laramie M; Hadley, Michael S; Hammond, Beverley; Johnson, Christopher N; Middlemiss, Derek N; North, Stephanie E; Price, Gary W; Rami, Harshad K; Riley, Graham J; Scott, Claire M; Shaw, Tracey E; Starr, Kathryn R; Stemp, Geoffrey; Thewlis, Kevin M; Thomas, David R; Thompson, Mervyn; Vong, Antonio K K; Watson, Jeannette M

    2005-02-01

    Starting from a high throughput screening hit, a series of 3,4-dihydro-2H-benzoxazinones has been identified with both high affinity for the 5-HT(1A) receptor and potent 5-HT reuptake inhibitory activity. The 5-(2-methyl)quinolinyloxy derivative combined high 5-HT(1A/1B/1D) receptor affinities with low intrinsic activity and potent inhibition of the 5-HT reuptake site (pK(i)8.2). This compound also had good oral bioavailability and brain penetration in the rat.

  9. Antidepressant-like activity of VN2222, a serotonin reuptake inhibitor with high affinity at 5-HT1A receptors.

    PubMed

    Tordera, Rosa M; Monge, Antonio; Del Río, Joaquín; Lasheras, Berta

    2002-05-03

    It has been suggested that drugs combining serotonin (5-hydroxytryptamine, 5-HT) transporter blockade and 5-HT1A autoreceptor antagonism could be a novel strategy for a shorter onset of action and higher therapeutic efficacy of antidepressants. The present study was aimed at characterizing the pharmacology of 1-(3-benzo[b]tiophenyl)-3-[4-(2-methoxyphenyl)-1-piperazinyl]-1-propanol (VN2222) a new synthetic compound with high affinity at both the 5-HT transporter and 5-HT1A receptors and devoid of high affinity at other receptors studied, with the only exception of alpha1-adrenoceptors. In keeping with the binding affinity at the 5-HT transporter, VN2222 inhibited 5-HT uptake in vitro both in rat cortical synaptosomes and in mesencephalic cultures and also in vivo when administered locally into the rat ventral hippocampus. After systemic administration, VN2222 exhibited an inverted U-shape effect so the inhibition of [3H]5-HT uptake ex vivo and the increase in 5-HT extracellular levels in microdialysis experiments was observed at low doses of 0.01-0.1 mg/kg whereas higher doses were ineffective. In studies related to 5-HT1A receptor function, 0.01-0.1 microM VN2222 produced a partial inhibition of forskolin-stimulated cAMP formation behaving as a weak agonist of 5-HT1A receptors. In body temperature studies, 5 mg/kg VN2222 produced a mild hypothermic effect in mice, suggesting a weak agonist activity at presynaptic 5-HT1A receptors; much lower doses (0.01-0.5 mg/kg) partially antagonized the hypothermia induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) possibly through 5-HT transporter blockade. In the learned helplessness test in rats, an animal model for antidepressants, 1-5 mg/kg VN2222 reduced significantly the number of escape failures. Consequently, VN2222 is a new compound with a dual effect on the serotonergic system, as 5-HT uptake blocker and 5-HT1A receptor partial agonist, and with a remarkable activity in an animal model of depression with

  10. Localization of angiotensin-II type 1(AT1) receptors on buffalo spermatozoa: AT1 receptor activation during capacitation triggers rise in cyclic AMP and calcium.

    PubMed

    Vedantam, Sivaram; Rani, Rita; Garg, Monica; Atreja, Suresh K

    2014-01-01

    The purpose of this study was to determine the role of Ang-II in buffalo spermatozoa; localize angiotensin type 1 (AT1) receptors on the sperm surface and understand the signaling mechanisms involved therein. Immunoblotting and immunocytochemistry using polyclonal Rabbit anti-AT1 (N-10) IgG were performed to confirm the presence of AT1 receptors. Intracellular levels of cyclic adenosine monophosphate (cAMP) were determined by non-radioactive enzyme immunoassay, while that of Calcium [Ca(2+)] were estimated by fluorimetry using Fura2AM dye. The results obtained showed that AT1 receptors were found on the post-acrosomal region, neck and tail regions. Immunoblotting revealed a single protein band with molecular weight of 40 kDa. Ang-II treated cells produced significantly higher level of cAMP compared to untreated cells (22.66 ± 2.4 vs. 10.8 ± 0.98 pmol/10(8) cells, p < 0.01). The mean levels of Ca(2+) were also higher in Ang-II treated cells compared to control (117.4 ± 6.1 vs. 61.15 ± 4.2 nmol/10(8) cells; p < 0.01). The stimulatory effect of Ang-II in both the cases was significantly inhibited in the presence of Losartan (AT1 antagonist; p < 0.05) indicating the involvement of AT1 receptors. Further, presence of neomycin (protein kinase C inhibitor) inhibited significantly the Ang-II mediated rise in Ca(2+) indicating the involvement of PKC pathway. These findings confirm the presence of AT1 receptors in buffalo spermatozoa and that Ang-II mediates its actions via the activation of these receptors. Ang-II stimulates the rise in intracellular levels of cAMP and Ca(2+) during capacitation.

  11. Agonists of proteinase-activated receptor 1 induce plasma extravasation by a neurogenic mechanism.

    PubMed

    de Garavilla, L; Vergnolle, N; Young, S H; Ennes, H; Steinhoff, M; Ossovskaya, V S; D'Andrea, M R; Mayer, E A; Wallace, J L; Hollenberg, M D; Andrade-Gordon, P; Bunnett, N W

    2001-08-01

    Thrombin, generated in the circulation during injury, cleaves proteinase-activated receptor 1 (PAR1) to stimulate plasma extravasation and granulocyte infiltration. However, the mechanism of thrombin-induced inflammation in intact tissues is unknown. We hypothesized that thrombin cleaves PAR1 on sensory nerves to release substance P (SP), which interacts with the neurokinin 1 receptor (NK1R) on endothelial cells to cause plasma extravasation. PAR1 was detected in small diameter neurons known to contain SP in rat dorsal root ganglia by immunohistochemistry and in situ hybridization. Thrombin and the PAR1 agonist TFLLR-NH(2) (TF-NH(2)) increased [Ca(2+)](i) >50% of cultured neurons (EC(50)s 24 mu ml(-1) and 1.9 microM, respectively), assessed using Fura-2 AM. The PAR1 agonist completely desensitized responses to thrombin, indicating that thrombin stimulates neurons through PAR1. Injection of TF-NH(2) into the rat paw stimulated a marked and sustained oedema. An NK1R antagonist and ablation of sensory nerves with capsaicin inhibited oedema by 44% at 1 h and completely by 5 h. In wild-type but not PAR1(-/-) mice, TF-NH(2) stimulated Evans blue extravasation in the bladder, oesophagus, stomach, intestine and pancreas by 2 - 8 fold. Extravasation in the bladder, oesophagus and stomach was abolished by an NK1R antagonist. Thus, thrombin cleaves PAR1 on primary spinal afferent neurons to release SP, which activates the NK1R on endothelial cells to stimulate gap formation, extravasation of plasma proteins, and oedema. In intact tissues, neurogenic mechanisms are predominantly responsible for PAR1-induced oedema.

  12. Characterization of Transient Receptor Potential Vanilloid-1 (TRPV1) Variant Activation by Coal Fly Ash Particles and Associations with Altered Transient Receptor Potential Ankyrin-1 (TRPA1) Expression and Asthma*

    PubMed Central

    Stockmann, Chris; Romero, Erin G.; Lu, Zhenyu; Shapiro, Darien; Stone, Bryan L.; Fassl, Bernhard; Nkoy, Flory; Uchida, Derek A.; Ward, Robert M.; Veranth, John M.; Reilly, Christopher A.

    2016-01-01

    Transient receptor potential (TRP) channels are activated by environmental particulate materials. We hypothesized that polymorphic variants of transient receptor potential vanilloid-1 (TRPV1) would be uniquely responsive to insoluble coal fly ash compared with the prototypical soluble agonist capsaicin. Furthermore, these changes would manifest as differences in lung cell responses to these agonists and perhaps correlate with changes in asthma symptom control. The TRPV1-I315M and -T469I variants were more responsive to capsaicin and coal fly ash. The I585V variant was less responsive to coal fly ash particles due to reduced translation of protein and an apparent role for Ile-585 in activation by particles. In HEK-293 cells, I585V had an inhibitory effect on wild-type TRPV1 expression, activation, and internalization/agonist-induced desensitization. In normal human bronchial epithelial cells, IL-8 secretion in response to coal fly ash treatment was reduced for cells heterozygous for TRPV1-I585V. Finally, both the I315M and I585V variants were associated with worse asthma symptom control with the effects of I315M manifesting in mild asthma and those of the I585V variant manifesting in severe, steroid-insensitive individuals. This effect may be due in part to increased transient receptor potential ankyrin-1 (TRPA1) expression by lung epithelial cells expressing the TRPV1-I585V variant. These findings suggest that specific molecular interactions control TRPV1 activation by particles, differential activation, and desensitization of TRPV1 by particles and/or other agonists, and cellular changes in the expression of TRPA1 as a result of I585V expression could contribute to variations in asthma symptom control. PMID:27758864

  13. Characterization of Transient Receptor Potential Vanilloid-1 (TRPV1) Variant Activation by Coal Fly Ash Particles and Associations with Altered Transient Receptor Potential Ankyrin-1 (TRPA1) Expression and Asthma.

    PubMed

    Deering-Rice, Cassandra E; Stockmann, Chris; Romero, Erin G; Lu, Zhenyu; Shapiro, Darien; Stone, Bryan L; Fassl, Bernhard; Nkoy, Flory; Uchida, Derek A; Ward, Robert M; Veranth, John M; Reilly, Christopher A

    2016-11-25

    Transient receptor potential (TRP) channels are activated by environmental particulate materials. We hypothesized that polymorphic variants of transient receptor potential vanilloid-1 (TRPV1) would be uniquely responsive to insoluble coal fly ash compared with the prototypical soluble agonist capsaicin. Furthermore, these changes would manifest as differences in lung cell responses to these agonists and perhaps correlate with changes in asthma symptom control. The TRPV1-I315M and -T469I variants were more responsive to capsaicin and coal fly ash. The I585V variant was less responsive to coal fly ash particles due to reduced translation of protein and an apparent role for Ile-585 in activation by particles. In HEK-293 cells, I585V had an inhibitory effect on wild-type TRPV1 expression, activation, and internalization/agonist-induced desensitization. In normal human bronchial epithelial cells, IL-8 secretion in response to coal fly ash treatment was reduced for cells heterozygous for TRPV1-I585V. Finally, both the I315M and I585V variants were associated with worse asthma symptom control with the effects of I315M manifesting in mild asthma and those of the I585V variant manifesting in severe, steroid-insensitive individuals. This effect may be due in part to increased transient receptor potential ankyrin-1 (TRPA1) expression by lung epithelial cells expressing the TRPV1-I585V variant. These findings suggest that specific molecular interactions control TRPV1 activation by particles, differential activation, and desensitization of TRPV1 by particles and/or other agonists, and cellular changes in the expression of TRPA1 as a result of I585V expression could contribute to variations in asthma symptom control. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Ionotropic and metabotropic receptor mediated airway sensory nerve activation.

    PubMed

    Lee, Min-Goo; Kollarik, Marian; Chuaychoo, Benjamas; Undem, Bradley J

    2004-01-01

    There are several receptors capable of inducing activating generator potentials in cough-associated afferent terminals in the airways. The chemical receptors leading to generator potentials can be subclassified into ionotropic and metabotropic types. An ionotropic receptor has an agonist-binding domain, and also serves directly as an ion channel that is opened upon binding of the agonist. Examples of ionotropic receptors found in airway sensory nerve terminals include receptors for serotonin (5-HT3 receptors), ATP (P2X receptors), acetylcholine (nicotinic receptors), receptors for capsaicin and related vanilloids (TRPV1 receptors), and acid receptors (acid sensing ion channels). Afferent nerve terminals can also be depolarized via activation of metabotropic or G-protein coupled receptors (GPCRs). Among the GPCRs that can lead to activation of airway afferent fibers include bradykinin B2 and adenosine A1 receptors. The signaling events leading to GPCR-mediated membrane depolarization are more complex than that seen with ionotropic receptors. The GPCR-mediated effects are thought to occur through classical second messenger systems such as activation of phospholipase C. This may lead to membrane depolarization through interaction with specific ionotropic receptors (such as TRPV1) and/or various types of calcium activated channels.

  15. 1,4-Naphthoquinones potently inhibiting P2X7 receptor activity.

    PubMed

    Faria, R X; Oliveira, F H; Salles, J P; Oliveira, A S; von Ranke, N L; Bello, M L; Rodrigues, C R; Castro, H C; Louvis, A R; Martins, D L; Ferreira, V F

    2018-01-01

    P2X7 receptor (P2X7R) is an ATP-gated ion-channel with potential therapeutic applications. In this study, we prepared and searched a series of 1,4-naphthoquinones derivatives to evaluate their antagonistic effect on both human and murine P2X7 receptors. We explored the structure-activity relationship and binding mode of the most active compounds using a molecular modeling approach. Biological analysis of this series (eight analogues and two compounds) revealed significant in vitro inhibition against both human and murine P2X7R. Further characterization revealed that AN-03 and AN-04 had greater potency than BBG and A740003 in inhibiting dye uptake, IL-1β release, and carrageenan-induced paw edema in vivo. Moreover, we used electrophysiology and molecular docking analysis for characterizing AN-03 and AN-04 action mechanism. These results suggest 1,4-napthoquinones, mainly AN-04, as potential leads to design new P2X7R blockers and anti-inflammatory drugs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  16. Virally mediated increased neurotensin 1 receptor in the nucleus accumbens decreases behavioral effects of mesolimbic system activation.

    PubMed

    Cáceda, Ricardo; Kinkead, Becky; Owens, Michael J; Nemeroff, Charles B

    2005-12-14

    Dopamine receptor agonist and NMDA receptor antagonist activation of the mesolimbic dopamine system increases locomotion and disrupts prepulse inhibition of the acoustic startle response (PPI), paradigms frequently used to study both the pharmacology of antipsychotic drugs and drugs of abuse. In rats, virally mediated overexpression of the neurotensin 1 (NT1) receptor in the nucleus accumbens antagonized d-amphetamine- and dizocilpine-induced PPI disruption, hyperlocomotion, and D-amphetamine-induced rearing. The NT receptor antagonist SR 142948A [2-[[5-(2,6-dimethoxyphenyl)-1-(4-N-(3-dimethylaminopropyl)-N-methylcarbamoyl)-2-isopropylphenyl)-1H-pyrazole-3-carbonyl]amino] adamantane-2-carboxylic acid, hydrochloride] blocked inhibition of dizocilpine-induced hyperlocomotion mediated by overexpression of the NT1 receptor. Together, these results suggest that increased nucleus accumbens NT neurotransmission, via the NT1 receptor, can decrease the effects of activation of the mesolimbic dopamine system and disruption of the glutamatergic input from limbic cortices, resembling the action of the atypical antipsychotic drug clozapine. In contrast to clozapine, virally mediated overexpression of the NT1 receptor in the nucleus accumbens had prolonged protective effects (up to 4 weeks after viral injection) without perturbing baseline PPI and locomotor behaviors. These data further confirm the NT1 receptor as the receptor mediating the antistimulant- and antipsychotic-like properties of NT and provide rationale for the development of NT1 receptor agonists as novel antipsychotic drugs. In addition, the NT1 receptor vector might be a valuable tool for understanding the mechanism of action of antipsychotic drugs and drugs of abuse and may have potential therapeutic applications.

  17. Gingerols: a novel class of vanilloid receptor (VR1) agonists

    PubMed Central

    Dedov, Vadim N; Tran, Van H; Duke, Colin C; Connor, Mark; Christie, MacDonald J; Mandadi, Sravan; Roufogalis, Basil D

    2002-01-01

    Gingerols, the pungent constituents of ginger, were synthesized and assessed as agonists of the capsaicin-activated VR1 (vanilloid) receptor. [6]-Gingerol and [8]-gingerol evoked capsaicin-like intracellular Ca2+ transients and ion currents in cultured DRG neurones. These effects of gingerols were blocked by capsazepine, the VR1 receptor antagonist. The potency of gingerols increased with increasing size of the side chain and with the overall hydrophobicity in the series. We conclude that gingerols represent a novel class of naturally occurring VR1 receptor agonists that may contribute to the medicinal properties of ginger, which have been known for centuries. The gingerol structure may be used as a template for the development of drugs acting as moderately potent activators of the VR1 receptor. PMID:12411409

  18. Receptor binding properties and antinociceptive effects of chimeric peptides consisting of a micro-opioid receptor agonist and an ORL1 receptor antagonist.

    PubMed

    Kawano, Susumu; Ito, Risa; Nishiyama, Miharu; Kubo, Mai; Matsushima, Tomoko; Minamisawa, Motoko; Ambo, Akihiro; Sasaki, Yusuke

    2007-07-01

    Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100< pmol/mouse) than when administered i.t. (ED(50): 1.34-4.51 pmol/mouse). These results suggest the involvement of nociceptin-like agonistic effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.

  19. The alpha1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family.

    PubMed

    Galarneau, L; Paré, J F; Allard, D; Hamel, D; Levesque, L; Tugwood, J D; Green, S; Bélanger, L

    1996-07-01

    The alpha1-fetoprotein (AFP) gene is located between the albumin and alpha-albumin genes and is activated by transcription factor FTF (fetoprotein transcription factor), presumed to transduce early developmental signals to the albumin gene cluster. We have identified FTF as an orphan nuclear receptor of the Drosophila FTZ-F1 family. FTF recognizes the DNA sequence 5'-TCAAGGTCA-3', the canonical recognition motif for FTZ-F1 receptors. cDNA sequence homologies indicate that rat FTF is the ortholog of mouse LRH-1 and Xenopus xFF1rA. Rodent FTF is encoded by a single-copy gene, related to the gene encoding steroidogenic factor 1 (SF-1). The 5.2-kb FTF transcript is translated from several in-frame initiator codons into FTF isoforms (54 to 64 kDa) which appear to bind DNA as monomers, with no need for a specific ligand, similar KdS (approximately equal 3 x 10(-10) M), and similar transcriptional effects. FTF activates the AFP promoter without the use of an amino-terminal activation domain; carboxy-terminus-truncated FTF exerts strong dominant negative effects. In the AFP promoter, FTF recruits an accessory trans-activator which imparts glucocorticoid reactivity upon the AFP gene. FTF binding sites are found in the promoters of other liver-expressed genes, some encoding liver transcription factors; FTF, liver alpha1-antitrypsin promoter factor LFB2, and HNF-3beta promoter factor UF2-H3beta are probably the same factor. FTF is also abundantly expressed in the pancreas and may exert differentiation functions in endodermal sublineages, similar to SF-1 in steroidogenic tissues. HepG2 hepatoma cells seem to express a mutated form of FTF.

  20. PeaTAR1B: Characterization of a Second Type 1 Tyramine Receptor of the American Cockroach, Periplaneta americana

    PubMed Central

    Balfanz, Sabine

    2017-01-01

    The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP]i) whereas type 2 tyramine receptors can mediate Ca2+ signals or both Ca2+ signals and effects on [cAMP]i. Here; we report that the American cockroach (Periplaneta americana) expresses a second type 1 tyramine receptor (PeaTAR1B) in addition to PeaTAR1A (previously called PeaTYR1). When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP]i. Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine. PMID:29084141

  1. PeaTAR1B: Characterization of a Second Type 1 Tyramine Receptor of the American Cockroach, Periplaneta americana.

    PubMed

    Blenau, Wolfgang; Balfanz, Sabine; Baumann, Arnd

    2017-10-30

    The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP] i ) whereas type 2 tyramine receptors can mediate Ca 2+ signals or both Ca 2+ signals and effects on [cAMP] i . Here; we report that the American cockroach ( Periplaneta americana ) expresses a second type 1 tyramine receptor (PeaTAR1B) in addition to PeaTAR1A (previously called PeaTYR1). When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP] i . Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana ; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine.

  2. Cardiac lipin 1 expression is regulated by the peroxisome proliferator activated receptor γ coactivator 1α/estrogen related receptor axis

    PubMed Central

    Mitra, Mayurranjan S.; Schilling, Joel D.; Wang, Xiaowei; Jay, Patrick Y.; Huss, Janice M.; Su, Xiong; Finck, Brian N.

    2011-01-01

    Lipin family proteins (lipin 1, 2, and 3) are bifunctional intracellular proteins that regulate metabolism by acting as coregulators of DNA-bound transcription factors and also dephosphorylate phosphatidate to form diacylglycerol [phosphatidate phosphohydrolase activity] in the triglyceride synthesis pathway. Herein, we report that lipin 1 is enriched in heart and that hearts of mice lacking lipin 1 (fld mice) exhibit accumulation of phosphatidate. We also demonstrate that the expression of the gene encoding lipin 1 (Lpin1) is under the control of the estrogen-related receptors (ERRs) and their coactivator the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). PGC-1α, ERRα, or ERRγ overexpression increased Lpin1 transcription in cultured ventricular myocytes and the ERRs were associated with response elements in the first intron of the Lpin1 gene. Concomitant RNAi-mediated knockdown of ERRα and ERRγ abrogated the induction of lipin 1 expression by PGC-1α overexpression. Consistent with these data, 3-fold overexpression of PGC-1α in intact myocardium of transgenic mice increased cardiac lipin 1 and ERRα/γ expression. Similarly, injection of the β2-adrenergic agonist clenbuterol induced PGC-1α and lipin 1 expression, and the induction in lipin 1 after clenbuterol occurred in a PGC-1α-dependent manner. In contrast, expression of PGC-1α, ERRα, ERRγ, and lipin 1 was down-regulated in failing heart. Cardiac phosphatidic acid phosphohydrolase activity was also diminished, while cardiac phosphatidate content was increased, in failing heart. Collectively, these data suggest that lipin 1 is the principal lipin protein in the myocardium and is regulated in response to physiologic and pathologic stimuli that impact cardiac metabolism. PMID:21549711

  3. The GIP receptor displays higher basal activity than the GLP-1 receptor but does not recruit GRK2 or arrestin3 effectively.

    PubMed

    Al-Sabah, Suleiman; Al-Fulaij, Munya; Shaaban, Ghina; Ahmed, Hanadi A; Mann, Rosalind J; Donnelly, Dan; Bünemann, Moritz; Krasel, Cornelius

    2014-01-01

    Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of insulin secretion, and their functional loss is an early characteristic of type 2 diabetes mellitus (T2DM). Pharmacological levels of GLP-1, but not GIP, can overcome this loss. GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic β-cells. Both the GLP-1 receptor (GLP-1R) and the GIP receptor (GIPR) are members of the secretin family of G protein-coupled receptors (GPCRs) and couple positively to adenylate cyclase. We compared the signalling properties of these two receptors to gain further insight into why GLP-1, but not GIP, remains insulinotropic in T2DM patients. GLP-1R and GIPR were transiently expressed in HEK-293 cells, and basal and ligand-induced cAMP production were investigated using a cAMP-responsive luciferase reporter gene assay. Arrestin3 (Arr3) recruitment to the two receptors was investigated using enzyme fragment complementation, confocal microscopy and fluorescence resonance energy transfer (FRET). GIPR displayed significantly higher (P<0.05) ligand-independent activity than GLP-1R. Arr3 displayed a robust translocation to agonist-stimulated GLP-1R but not to GIPR. These observations were confirmed in FRET experiments, in which GLP-1 stimulated the recruitment of both GPCR kinase 2 (GRK2) and Arr3 to GLP-1R. These interactions were not reversed upon agonist washout. In contrast, GIP did not stimulate recruitment of either GRK2 or Arr3 to its receptor. Interestingly, arrestin remained at the plasma membrane even after prolonged (30 min) stimulation with GLP-1. Although the GLP-1R/arrestin interaction could not be reversed by agonist washout, GLP-1R and arrestin did not co-internalise, suggesting that GLP-1R is a class A receptor with regard to arrestin binding. GIPR displays higher basal activity than GLP-1R but does not effectively recruit GRK2 or Arr3.

  4. Altered Circadian Food Anticipatory Activity Rhythms in PACAP Receptor 1 (PAC1) Deficient Mice

    PubMed Central

    Hannibal, Jens; Georg, Birgitte; Fahrenkrug, Jan

    2016-01-01

    Light signals from intrinsically photosensitive retinal ganglion cells (ipRGCs) entrain the circadian clock and regulate negative masking. Two neurotransmitters, glutamate and Pituitary Adenylate Cyclase Activating Polypeptide (PACAP), found in the ipRGCs transmit light signals to the brain via glutamate receptors and the specific PACAP type 1 (PAC1) receptor. Light entrainment occurs during the twilight zones and has little effect on clock phase during daytime. When nocturnal animals have access to food only for a few hours during the resting phase at daytime, they adapt behavior to the restricted feeding (RF) paradigm and show food anticipatory activity (FAA). A recent study in mice and rats demonstrating that light regulates FAA prompted us to investigate the role of PACAP/PAC1 signaling in the light mediated regulation of FAA. PAC1 receptor knock out (PAC1-/-) and wild type (PAC1+/+) mice placed in running wheels were examined in a full photoperiod (FPP) of 12:12 h light/dark (LD) and a skeleton photoperiod (SPP) 1:11:1:11 h L:DD:L:DD at 300 and 10 lux light intensity. Both PAC1-/- mice and PAC1+/+ littermates entrained to FPP and SPP at both light intensities. However, when placed in RF with access to food for 4–5 h during the subjective day, a significant change in behavior was observed in PAC1-/- mice compared to PAC1+/+ mice. While PAC1-/- mice showed similar FAA as PAC1+/+ animals in FPP at 300 lux, PAC1-/- mice demonstrated an advanced onset of FAA with a nearly 3-fold increase in amplitude compared to PAC1+/+ mice when placed in SPP at 300 lux. The same pattern of FAA was observed at 10 lux during both FPP and SPP. The present study indicates a role of PACAP/PAC1 signaling during light regulated FAA. Most likely, PACAP found in ipRGCs mediating non-image forming light information to the brain is involved. PMID:26757053

  5. Altered Circadian Food Anticipatory Activity Rhythms in PACAP Receptor 1 (PAC1) Deficient Mice.

    PubMed

    Hannibal, Jens; Georg, Birgitte; Fahrenkrug, Jan

    2016-01-01

    Light signals from intrinsically photosensitive retinal ganglion cells (ipRGCs) entrain the circadian clock and regulate negative masking. Two neurotransmitters, glutamate and Pituitary Adenylate Cyclase Activating Polypeptide (PACAP), found in the ipRGCs transmit light signals to the brain via glutamate receptors and the specific PACAP type 1 (PAC1) receptor. Light entrainment occurs during the twilight zones and has little effect on clock phase during daytime. When nocturnal animals have access to food only for a few hours during the resting phase at daytime, they adapt behavior to the restricted feeding (RF) paradigm and show food anticipatory activity (FAA). A recent study in mice and rats demonstrating that light regulates FAA prompted us to investigate the role of PACAP/PAC1 signaling in the light mediated regulation of FAA. PAC1 receptor knock out (PAC1-/-) and wild type (PAC1+/+) mice placed in running wheels were examined in a full photoperiod (FPP) of 12:12 h light/dark (LD) and a skeleton photoperiod (SPP) 1:11:1:11 h L:DD:L:DD at 300 and 10 lux light intensity. Both PAC1-/- mice and PAC1+/+ littermates entrained to FPP and SPP at both light intensities. However, when placed in RF with access to food for 4-5 h during the subjective day, a significant change in behavior was observed in PAC1-/- mice compared to PAC1+/+ mice. While PAC1-/- mice showed similar FAA as PAC1+/+ animals in FPP at 300 lux, PAC1-/- mice demonstrated an advanced onset of FAA with a nearly 3-fold increase in amplitude compared to PAC1+/+ mice when placed in SPP at 300 lux. The same pattern of FAA was observed at 10 lux during both FPP and SPP. The present study indicates a role of PACAP/PAC1 signaling during light regulated FAA. Most likely, PACAP found in ipRGCs mediating non-image forming light information to the brain is involved.

  6. Ligand-independent activation of the oestrogen receptor by mutation of a conserved tyrosine.

    PubMed Central

    White, R; Sjöberg, M; Kalkhoven, E; Parker, M G

    1997-01-01

    The oestrogen receptor is a member of the nuclear receptor family of transcription factors which, on binding the steroid hormone 17beta-oestradiol, interacts with co-activator proteins and stimulates gene expression. Replacement of a single tyrosine in the hormone-binding domain generated activated forms of the receptor which stimulated transcription in the absence of hormone. This increased activation is related to a decrease in hydrophobicity and a reduction in size of the side chain of the amino acid with which the tyrosine is replaced. Ligand-independent, in common with ligand-dependent transcriptional activation, requires an amphipathic alpha-helix at the C-terminus of the ligand-binding domain which is essential for the interaction of the receptor with a number of potential co-activator proteins. In contrast to the wild-type protein, constitutively active receptors were able to bind both the receptor-interacting protein RIP-140 and the steroid receptor co-activator SRC-1 in a ligand-independent manner, although in the case of SRC-1 this was only evident when the receptors were prebound to DNA. We propose, therefore, that this tyrosine is required to maintain the receptor in a transcriptionally inactive state in the absence of hormone. Modification of this residue may generate a conformational change in the ligand-binding domain of the receptor to form an interacting surface which allows the recruitment of co-activators independent of hormone binding. This suggests that this tyrosine may be a target for a different signalling pathway which forms an alternative mechanism of activating oestrogen receptor-mediated transcription. PMID:9135157

  7. Activation of 5-HT7 serotonin receptors reverses metabotropic glutamate receptor-mediated synaptic plasticity in wild-type and Fmr1 knockout mice, a model of Fragile X syndrome.

    PubMed

    Costa, Lara; Spatuzza, Michela; D'Antoni, Simona; Bonaccorso, Carmela M; Trovato, Chiara; Musumeci, Sebastiano A; Leopoldo, Marcello; Lacivita, Enza; Catania, Maria V; Ciranna, Lucia

    2012-12-01

    Fragile X syndrome (FXS) is a genetic cause of intellectual disability and autism. Fmr1 knockout (Fmr1 KO) mice, an animal model of FXS, exhibit spatial memory impairment and synapse malfunctioning in the hippocampus, with abnormal enhancement of long-term depression mediated by metabotropic glutamate receptors (mGluR-LTD). The neurotransmitter serotonin (5-HT) modulates hippocampal-dependent learning through serotonin 1A (5-HT1A) and serotonin 7 (5-HT7) receptors; the underlying mechanisms are unknown. We used electrophysiology to test the effects of 5-HT on mGluR-LTD in wild-type and Fmr1 KO mice and immunocytochemistry and biotinylation assay to study related changes of 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) glutamate receptor surface expression. Application of 5-HT or 8-OH-DPAT (a mixed 5-HT1A/5-HT7 agonist) reversed mGluR-LTD in hippocampal slices. Reversal of mGluR-LTD by 8-OH-DPAT persisted in the presence of the 5-HT1A receptor antagonist WAY-100635, was abolished by SB-269970 (5-HT7 receptor antagonist), and was mimicked by LP-211, a novel selective 5-HT7 receptor agonist. Consistently, 8-OH-DPAT decreased mGluR-mediated reduction of AMPA glutamate receptor 2 (GluR2) subunit surface expression in hippocampal slices and cultured hippocampal neurons, an effect mimicked by LP-211 and blocked by SB-269970. In Fmr1 KO mice, mGluR-LTD was abnormally enhanced; similarly to wild-type, 8-OH-DPAT reversed mGluR-LTD and decreased mGluR-induced reduction of surface AMPA receptors, an effect antagonized by SB-269970. Serotonin 7 receptor activation reverses metabotropic glutamate receptor-induced AMPA receptor internalization and LTD both in wild-type and in Fmr1 KO mice, correcting excessive mGluR-LTD. Therefore, selective activation of 5-HT7 receptors may represent a novel strategy in the therapy of FXS. Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  8. The cannabinoid receptor CB1 modulates the signaling properties of the lysophosphatidylinositol receptor GPR55.

    PubMed

    Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P; Brown, Andrew J; Heinemann, Akos; Waldhoer, Maria

    2012-12-28

    The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors.

  9. The Cannabinoid Receptor CB1 Modulates the Signaling Properties of the Lysophosphatidylinositol Receptor GPR55*

    PubMed Central

    Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P.; Brown, Andrew J.; Heinemann, Akos; Waldhoer, Maria

    2012-01-01

    The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors. PMID:23161546

  10. Effects of an orally active vasopressin V1 receptor antagonist.

    PubMed

    Burrell, L M; Phillips, P A; Stephenson, J; Risvanis, J; Hutchins, A M; Johnston, C I

    1993-05-01

    1. This paper reports on the in vitro and in vivo characteristics of a non-peptide vasopressin V1 receptor antagonist 1-(1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl)-3,4-dihydro-2( 1H)- quinolinone (OPC-21268). 2. OPC-21268 caused a concentration-dependent displacement of the selective V1 receptor antagonist radioligand, [125I]-[d(CH2)5, sarcosine7]AVP from vasopressin V1 receptors in rat liver and kidney membranes, inhibitory concentration of 50% (IC50) 4 x 10(-8), 0.3 mol/L liver and 1.5 x 10(-8), 0.2 mol/L kidney. OPC-21268 had little effect on the selective V2 antagonist radioligand [3H]desGly-NH2(9)-d(CH2)5[D-Ileu2, Ileu4]AVP binding to V2 receptors in renal membranes (IC50 > 10(-4) mol/L). 3. After oral administration to rats, OPC-21268 was an effective V1 antagonist to both liver and kidney V1 receptors, in a dose-dependent manner. 4. These studies confirm that OPC-21268 is a potent non-peptide, orally effective V1 vasopressin receptor antagonist.

  11. Angiotensin II stimulates calcineurin activity in proximal tubule epithelia through AT-1 receptor-mediated tyrosine phosphorylation of the PLC-gamma1 isoform.

    PubMed

    Lea, Janice P; Jin, Shao G; Roberts, Brian R; Shuler, Michael S; Marrero, Mario B; Tumlin, James A

    2002-07-01

    Angiotensin II (AngII) contributes to the maintenance of extracellular fluid volume by regulating sodium transport in the nephron. In nonepithelial cells, activation of phospholipase C (PLC) by AT-1 receptors stimulates the generation of 1,4,5-trisphosphate (IP(3)) and the release of intracellular calcium. Calcineurin, a serine-threonine phosphatase, is activated by calcium and calmodulin, and both PLC and calcineurin have been linked to sodium transport in the proximal tubule. An examination of whether AngII activates calcineurin in a model of proximal tubule epithelia (LLC-PK1 cells) was performed; AngII increased calcineurin activity within 30 s. An examination of whether AngII activates PLC in proximal tubule epithelia was also performed after first showing that all three families of PLC isoforms are present in LLC-PK1 cells. Application of AngII increased IP(3) generation by 60% within 15 s, which coincided with AngII-induced tyrosine phosphorylation of the PLC-gamma1 isoform also observed at 15 s. AngII-induced tyrosine phosphorylation was blocked by the AT-1 receptor antagonist, Losartan. Subsequently, an inhibitor of tyrosine phosphorylation blocked the AngII-induced activation of calcineurin, as did coincubation with an inhibitor of PLC activity and with an antagonist of the AT-1 receptor. It is therefore concluded that AngII stimulates calcineurin phosphatase activity in proximal tubule epithelial cells through a mechanism involving AT-1 receptor-mediated tyrosine phosphorylation of the PLC isoform.

  12. Improper activation of D1 and D2 receptors leads to excess noise in prefrontal cortex

    PubMed Central

    Avery, Michael C.; Krichmar, Jeffrey L.

    2015-01-01

    The dopaminergic system has been shown to control the amount of noise in the prefrontal cortex (PFC) and likely plays an important role in working memory and the pathophysiology of schizophrenia. We developed a model that takes into account the known receptor distributions of D1 and D2 receptors, the changes these receptors have on neuron response properties, as well as identified circuitry involved in working memory. Our model suggests that D1 receptor under-stimulation in supragranular layers gates internal noise into the PFC leading to cognitive symptoms as has been proposed in attention disorders, while D2 over-stimulation gates noise into the PFC by over-activation of cortico-striatal projecting neurons in infragranular layers. We apply this model in the context of a memory-guided saccade paradigm and show deficits similar to those observed in schizophrenic patients. We also show set-shifting impairments similar to those observed in rodents with D1 and D2 receptor manipulations. We discuss how the introduction of noise through changes in D1 and D2 receptor activation may account for many of the symptoms of schizophrenia depending on where this dysfunction occurs in the PFC. PMID:25814948

  13. Cannabidiolic acid prevents vomiting in Suncus murinus and nausea-induced behaviour in rats by enhancing 5-HT1A receptor activation

    PubMed Central

    Bolognini, D; Rock, EM; Cluny, NL; Cascio, MG; Limebeer, CL; Duncan, M; Stott, CG; Javid, FA; Parker, LA; Pertwee, RG

    2013-01-01

    Background and Purpose To evaluate the ability of cannabidiolic acid (CBDA) to reduce nausea and vomiting and enhance 5-HT1A receptor activation in animal models. Experimental Approach We investigated the effect of CBDA on (i) lithium chloride (LiCl)-induced conditioned gaping to a flavour (nausea-induced behaviour) or a context (model of anticipatory nausea) in rats; (ii) saccharin palatability in rats; (iii) motion-, LiCl- or cisplatin-induced vomiting in house musk shrews (Suncus murinus); and (iv) rat brainstem 5-HT1A receptor activation by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and mouse whole brain CB1 receptor activation by CP55940, using [35S]GTPγS-binding assays. Key Results In shrews, CBDA (0.1 and/or 0.5 mg·kg−1 i.p.) reduced toxin- and motion-induced vomiting, and increased the onset latency of the first motion-induced emetic episode. In rats, CBDA (0.01 and 0.1 mg·kg−1 i.p.) suppressed LiCl- and context-induced conditioned gaping, effects that were blocked by the 5-HT1A receptor antagonist, WAY100635 (0.1 mg·kg−1 i.p.), and, at 0.01 mg·kg−1 i.p., enhanced saccharin palatability. CBDA-induced suppression of LiCl-induced conditioned gaping was unaffected by the CB1 receptor antagonist, SR141716A (1 mg·kg−1 i.p.). In vitro, CBDA (0.1–100 nM) increased the Emax of 8-OH-DPAT. Conclusions and Implications Compared with cannabidiol, CBDA displays significantly greater potency at inhibiting vomiting in shrews and nausea in rats, and at enhancing 5-HT1A receptor activation, an action that accounts for its ability to attenuate conditioned gaping in rats. Consequently, CBDA shows promise as a treatment for nausea and vomiting, including anticipatory nausea for which no specific therapy is currently available. PMID:23121618

  14. Differences in 5-HT1A receptor-mediated hypothermia in rats with low or high exploratory activity.

    PubMed

    Kõiv, Kadri; Harro, Jaanus

    2010-12-01

    Alterations in the serotonin (5-HT) system and the 5-HT1A receptor function have a significant role in anxiety-related and depression-related states. This study investigated the stress-induced hyperthermia (SIH) response and sensitivity to the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetraline in rats with persistently low or high levels of exploratory activity (LE and HE, respectively), of which the LE rats show more anxiety-like and depressive-like phenotypes. No differences in the SIH in response to novel cage or injection stress were found using rectal temperature measurements. However, the LE rats had significantly less pronounced decreases in SIH in response to the 0.3 mg/kg dose of 8-hydroxy-2-(di-n-propylamino)-tetraline. Exploratory behaviour correlated significantly and positively with the magnitude of change in body temperature in response to the 5-HT1A receptor agonist. This finding suggests a less effective 5-HT1A function in the LE rats and implicates the 5-HT1A receptor in the anxiety component of passive behaviour in novel surroundings.

  15. Somatostatin, acting at receptor subtype 1, inhibits Rho activity, the assembly of actin stress fibers, and cell migration.

    PubMed

    Buchan, Alison M J; Lin, Chin-Yu; Choi, Jimmy; Barber, Diane L

    2002-08-09

    Somatostatin regulates multiple biological functions by acting through a family of five G protein-coupled receptors, somatostatin receptors (SSTRs) 1-5. Although all five receptor subtypes inhibit adenylate cyclase activity and decrease intracellular cAMP levels, specific receptor subtypes also couple to additional signaling pathways. In CCL39 fibroblasts expressing either human SSTR1 or SSTR2, we demonstrate that activation of SSTR1 (but not SSTR2) attenuated both thrombin- and integrin-stimulated Rho-GTP complex formation. The reduction in Rho-GTP formation in the presence of somatostatin was associated with decreased translocation of Rho and LIM kinase to the plasma membrane and fewer focal contacts. Activation of Rho resulted in the formation of intracellular actin stress fibers and cell migration. In CCL39-R1 cells, somatostatin treatment prevented actin stress fiber assembly and attenuated thrombin-stimulated cell migration through Transwell membranes to basal levels. To show that native SSTR1 shares the ability to inhibit Rho activation, we demonstrated that somatostatin treatment of human umbilical vein endothelial cells attenuated thrombin-stimulated Rho-GTP accumulation. These data show for the first time that a G protein-coupled receptor, SSTR1, inhibits the activation of Rho, the assembly of focal adhesions and actin stress fibers, and cell migration.

  16. A1 adenosine receptor attenuates intracerebral hemorrhage-induced secondary brain injury in rats by activating the P38-MAPKAP2-Hsp27 pathway.

    PubMed

    Zhai, Weiwei; Chen, Dongdong; Shen, Haitao; Chen, Zhouqing; Li, Haiying; Yu, Zhengquan; Chen, Gang

    2016-06-14

    This study was designed to determine the role of the A1 adenosine receptors in intracerebral hemorrhage (ICH)-induced secondary brain injury and the underlying mechanisms. A collagenase-induced ICH model was established in Sprague-Dawley rats, and cultured primary rat cortical neurons were exposed to oxyhemoglobin at a concentration of 10 μM to mimic ICH in vitro. The A1 adenosine receptor agonist N(6)-cyclohexyladenosine and antagonist 8-phenyl-1,3-dipropylxanthine were used to study the role of A1 adenosine receptor in ICH-induced secondary brain injury, and antagonists of P38 and Hsp27 were used to study the underlying mechanisms of A1 adenosine receptor actions. The protein level of A1 adenosine receptor was significantly increased by ICH, while there was no significant change in protein levels of the other 3 adenosine receptors. In addition, the A1 adenosine receptor expression could be increased by N(6)-cyclohexyladenosine and decreased by 8-phenyl-1,3-dipropylxanthine under ICH conditions. Activation of the A1 adenosine receptor attenuated neuronal apoptosis in the subcortex, which was associated with increased phosphorylation of P38, MAPK, MAPKAP2, and Hsp27. Inhibition of the A1 adenosine receptor resulted in opposite effects. Finally, the neuroprotective effect of the A1 adenosine receptor agonist N(6)-cyclohexyladenosine was inhibited by antagonists of P38 and Hsp27. This study demonstrates that activation of the A1 adenosine receptor by N(6)-cyclohexyladenosine could prevent ICH-induced secondary brain injury via the P38-MAPKAP2-Hsp27 pathway.

  17. A tachykinin NK1 receptor antagonist, CP-122,721-1, attenuates kainic acid-induced seizure activity.

    PubMed

    Zachrisson, O; Lindefors, N; Brené, S

    1998-10-01

    Substance P (SP) can play an important role in neuronal survival. To analyze the role of SP in excitotoxicity, kainic acid (KA) was administered to rats and in situ hybridization was used to analyze the levels of the SP encoding preprotachykinin-A (PPT-A) mRNA in striatal and hippocampal subregions 1, 4, and 24 h and 7 days after KA. In striatum and piriform cortex, PPT-A mRNA peaked 4 h after KA while in hippocampus, levels peaked after 24 h. KA caused seizures and neuronal toxicity as indicated by a reduction of the number of neurons in the hippocampal CA1 subregion after 7 days. KA was later administered alone or following pretreatment with the tachykinin NK1 receptor antagonist CP-122,721-1 (0.3 mg/kg). The pretreatment decreased seizure activity and a negative correlation was found between seizure activity and survival of CA1 neurons. Conclusively, treatment with CP-122,721-1 has a seizure inhibiting property and may possibly counteract KA-induced nerve cell death in CA1. Copyright 1998 Elsevier Science B.V.

  18. Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells.

    PubMed

    Montrose-Rafizadeh, C; Avdonin, P; Garant, M J; Rodgers, B D; Kole, S; Yang, H; Levine, M A; Schwindinger, W; Bernier, M

    1999-03-01

    Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.

  19. Modulation of hypoglossal motoneuron excitability by NK1 receptor activation in neonatal mice in vitro

    PubMed Central

    Yasuda, Kouichi; Robinson, Dean M; Selvaratnam, Subramaniam R; Walsh, Carmen W; McMorland, Angus J C; Funk, Gregory D

    2001-01-01

    The effects of substance P (SP), acting at NK1 receptors, on the excitability and inspiratory activity of hypoglossal (XII) motoneurons (MNs) were investigated using rhythmically active medullary-slice preparations from neonatal mice (postnatal day 0–3). Local application of the NK1 agonist [SAR9,Met (O2)11]-SP (SPNK1) produced a dose-dependent, spantide- (a non-specific NK receptor antagonist) and GR82334-(an NK1 antagonist) sensitive increase in inspiratory burst amplitude recorded from XII nerves. Under current clamp, SPNK1 significantly depolarized XII MNs, potentiated repetitive firing responses to injected currents and produced a leftward shift in the firing frequency-current relationships without affecting slope. Under voltage clamp, SPNK1 evoked an inward current and increased input resistance, but had no effect on inspiratory synaptic currents. SPNK1 currents persisted in the presence of TTX, were GR82334 sensitive, were reduced with hyperpolarization and reversed near the expected EK. Effects of the α1-noradrenergic receptor agonist phenylephrine (PE) on repetitive firing behaviour were virtually identical to those of SPNK1. Moreover, SPNK1 currents were completely occluded by PE, suggesting that common intracellular pathways mediate the actions of NK1 and α1-noradrenergic receptors. In spite of the similar actions of SPNK1 and PE on XII MN responses to somally injected current, α1-noradrenergic receptor activation potentiated inspiratory synaptic currents and was more than twice as effective in potentiating XII nerve inspiratory burst amplitude. GR82334 reduced XII nerve inspiratory burst amplitude and generated a small outward current in XII MNs. These observations, together with the first immunohistochemical evidence in the newborn for SP immunopositive terminals in the vicinity of SPNK1-sensitive inspiratory XII MNs, support the endogenous modulation of XII MN excitability by SP. In contrast to phrenic MNs (Ptak et al. 2000), blocking NMDA

  20. Activation of 5-hydroxytryptamine1B/1D/1F receptors as a mechanism of action of antimigraine drugs.

    PubMed

    Ramírez Rosas, Martha B; Labruijere, Sieneke; Villalón, Carlos M; Maassen Vandenbrink, Antoinette

    2013-08-01

    The introduction of the triptans (5-hydroxytryptamine (5-HT)1B/1D receptor agonists) was a great improvement in the acute treatment of migraine. However, shortcomings of the triptans have prompted research on novel serotonergic targets for the treatment of migraine. In this review the different types of antimigraine drugs acting at 5-HT receptors, their discovery and development are discussed. The first specific antimigraine drugs were the ergot alkaloids, consisting of ergotamine, dihydroergotamine and methysergide, which are agonists at 5-HT receptors, but can also bind α-adrenoceptors and dopamine receptors. In the 1990s, the triptans became available on the market. They are 5-HT1B/1D receptor agonists, showing fewer side effects due to their receptor specificity. In the last years, compounds that bind specifically to 5-HT1D, 5-HT1F and 5-HT7 receptors have been explored for their antimigraine potential. Furthermore, the serotonergic system seems to act in tight connection with the glutamatergic as well as the CGRP-ergic systems, which may open novel therapeutic avenues. Although the triptans are very effective in treating migraine attacks, their shortcomings have stimulated the search for novel drugs. Currently, the focus is on 5-HT1F receptor agonists, which seem devoid of vascular side effects. Moreover, novel compounds that affect multiple transmitter and/or neuropeptide systems that are involved in migraine could be of therapeutic relevance.

  1. Solubilization of active opiate receptors.

    PubMed Central

    Simonds, W F; Koski, G; Streaty, R A; Hjelmeland, L M; Klee, W A

    1980-01-01

    Receptors that reversibly bind opiates and opioid peptides have been solubilized from brain and neuroblastoma-glioma hybrid cell NG108-15 membranes. Active receptors are specifically solubilized with a new type of detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, which is a zwitterionic derivative of cholic acid. The solubilized receptor complexes behave as large molecules with a Stokes radius of 70 A and contain protein as an essential constituent. PMID:6254034

  2. An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.

    PubMed

    Butcher, Adrian J; Bradley, Sophie J; Prihandoko, Rudi; Brooke, Simon M; Mogg, Adrian; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; Edwards, Jennifer M; Bottrill, Andrew R; Challiss, R A John; Broad, Lisa M; Felder, Christian C; Tobin, Andrew B

    2016-04-22

    Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228) These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Roles of steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF) 2 in androgen receptor activity in mice

    PubMed Central

    Ye, Xiangcang; Han, Sang Jun; Tsai, Sophia Y.; DeMayo, Francesco J.; Xu, Jianming; Tsai, Ming-Jer; O'Malley, Bert W.

    2005-01-01

    Genetic disruption of the steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF)2/SRC-2 in mouse resulted in distinctive mutant phenotypes. To quantify their roles in the function of androgen receptor (AR) transcriptional activity in vivo, we generated a unique transgenic AR-reporter mouse and analyzed the cell-specific contributions of SRC-1 and TIF2 to the activity of AR in mouse testis. Transgenic AR-luciferase and transgenic AR-lacZ mice harbor a recombinant mouse AR gene, ARGAL4DBD, which is functionally coupled with a upstream activation sequence-mediated reporter gene (AR activity indicator). After characterization of these mice in terms of AR function, we further derived bigenic mice by crossing AR activity indicator mice with the SRC-1-/- or TIF2+/- mutant mice. Analyses of the resultant bigenic mice by in vivo imaging and luciferase assays showed that testicular AR activity was decreased significantly in those with the TIF2+/- mutation but not in the SRC-1+/- background, suggesting that TIF2 serves as the preferential coactivator for AR in testis. Immunohistological analysis confirmed that AR and TIF2 coexist in mouse testicular Sertoli cell nuclei under normal conditions. Although SRC-1 concentrates in Sertoli cell nuclei in the absence of TIF2, nuclear SRC-1 is not able to rescue AR activity in the TIF2 mutant background. Interestingly, SRC-1 appears to negatively influence AR activity, thereby counterbalancing the TIF2-stimulated AR activity. Our results provide unique in vivo insights to the multidimensional cell-type-specific interactions between AR and coregulators. PMID:15983373

  4. Roles of steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF) 2 in androgen receptor activity in mice.

    PubMed

    Ye, Xiangcang; Han, Sang Jun; Tsai, Sophia Y; DeMayo, Francesco J; Xu, Jianming; Tsai, Ming-Jer; O'Malley, Bert W

    2005-07-05

    Genetic disruption of the steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF)2/SRC-2 in mouse resulted in distinctive mutant phenotypes. To quantify their roles in the function of androgen receptor (AR) transcriptional activity in vivo, we generated a unique transgenic AR-reporter mouse and analyzed the cell-specific contributions of SRC-1 and TIF2 to the activity of AR in mouse testis. Transgenic AR-luciferase and transgenic AR-lacZ mice harbor a recombinant mouse AR gene, AR(GAL4DBD), which is functionally coupled with a upstream activation sequence-mediated reporter gene (AR activity indicator). After characterization of these mice in terms of AR function, we further derived bigenic mice by crossing AR activity indicator mice with the SRC-1-/- or TIF2+/- mutant mice. Analyses of the resultant bigenic mice by in vivo imaging and luciferase assays showed that testicular AR activity was decreased significantly in those with the TIF2+/- mutation but not in the SRC-1+/- background, suggesting that TIF2 serves as the preferential coactivator for AR in testis. Immunohistological analysis confirmed that AR and TIF2 coexist in mouse testicular Sertoli cell nuclei under normal conditions. Although SRC-1 concentrates in Sertoli cell nuclei in the absence of TIF2, nuclear SRC-1 is not able to rescue AR activity in the TIF2 mutant background. Interestingly, SRC-1 appears to negatively influence AR activity, thereby counterbalancing the TIF2-stimulated AR activity. Our results provide unique in vivo insights to the multidimensional cell-type-specific interactions between AR and coregulators.

  5. Receptor-heteromer mediated regulation of endocannabinoid signaling in activated microglia. Role of CB1 and CB2 receptors and relevance for Alzheimer's disease and levodopa-induced dyskinesia.

    PubMed

    Navarro, Gemma; Borroto-Escuela, Dasiel; Angelats, Edgar; Etayo, Íñigo; Reyes-Resina, Irene; Pulido-Salgado, Marta; Rodríguez-Pérez, Ana I; Canela, Enric I; Saura, Josep; Lanciego, José Luis; Labandeira-García, José Luis; Saura, Carlos A; Fuxe, Kjell; Franco, Rafael

    2018-01-01

    Endocannabinoids are important regulators of neurotransmission and, acting on activated microglia, they are postulated as neuroprotective agents. Endocannabinoid action is mediated by CB 1 and CB 2 receptors, which may form heteromeric complexes (CB 1 -CB 2 Hets) with unknown function in microglia. We aimed at establishing the expression and signaling properties of cannabinoid receptors in resting and LPS/IFN-γ-activated microglia. In activated microglia mRNA transcripts increased (2 fold for CB 1 and circa 20 fold for CB 2 ), whereas receptor levels were similar for CB 1 and markedly upregulated for CB 2 ; CB 1 -CB 2 Hets were also upregulated. Unlike in resting cells, CB 2 receptors became robustly coupled to G i in activated cells, in which CB 1 -CB 2 Hets mediated a potentiation effect. Hence, resting cells were refractory while activated cells were highly responsive to cannabinoids. Interestingly, similar results were obtained in cultures treated with ß-amyloid (Aß 1-42 ). Microglial activation markers were detected in the striatum of a Parkinson's disease (PD) model and, remarkably, in primary microglia cultures from the hippocampus of mutant β-amyloid precursor protein (APP Sw,Ind ) mice, a transgenic Alzheimer's disease (AD) model. Also of note was the similar cannabinoid receptor signaling found in primary cultures of microglia from APP Sw,Ind and in cells from control animals activated using LPS plus IFN-γ. Expression of CB 1 -CB 2 Hets was increased in the striatum from rats rendered dyskinetic by chronic levodopa treatment. In summary, our results showed sensitivity of activated microglial cells to cannabinoids, increased CB 1 -CB 2 Het expression in activated microglia and in microglia from the hippocampus of an AD model, and a correlation between levodopa-induced dyskinesia and striatal microglial activation in a PD model. Cannabinoid receptors and the CB 1 -CB 2 heteroreceptor complex in activated microglia have potential as targets in the

  6. Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.

    PubMed

    Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J

    1993-06-15

    alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first

  7. Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.

    PubMed Central

    Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J

    1993-01-01

    alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first

  8. Characterization of the intrinsic activity for a novel class of cannabinoid receptor ligands: Indole Quinuclidine analogues

    PubMed Central

    Franks, Lirit N.; Ford, Benjamin M.; Madadi, Nikhil R.; Penthala, Narsimha R.; Crooks, Peter A.; Prather, Paul L.

    2014-01-01

    Our laboratory recently reported that a group of novel indole quinuclidine analogues bind with nanomolar affinity to cannabinoid type-1 and type-2 receptors. This study characterized the intrinsic activity of these compounds by determining whether they exhibit agonist, antagonist, or inverse agonist activity at cannabinoid type-1 and/or type-2 receptors. Cannabinoid receptors activate Gi/Go-proteins that then proceed to inhibit activity of the downstream intracellular effector adenylyl cyclase. Therefore, intrinsic activity was quantified by measuring the ability of compounds to modulate levels of intracellular cAMP in intact cells. Concerning cannabinoid type-1 receptors endogenously expressed in Neuro2A cells, a single analogue exhibited agonist activity, while eight acted as neutral antagonists and two possessed inverse agonist activity. For cannabinoid type-2 receptors stably expressed in CHO cells, all but two analogues acted as agonists; these two exceptions exhibited inverse agonist activity. Confirming specificity at cannabinoid type-1 receptors, modulation of adenylyl cyclase activity by all proposed agonists and inverse agonists was blocked by co-incubation with the neutral cannabinoid type-1 antagonist O-2050. All proposed cannabinoid type-1 receptor antagonists attenuated adenylyl cyclase modulation by cannabinoid agonist CP-55,940. Specificity at cannabinoid type-2 receptors was confirmed by failure of all compounds to modulate adenylyl cyclase activity in CHO cells devoid of cannabinoid type-2 receptors. Further characterization of select analogues demonstrated concentration-dependent modulation of adenylyl cyclase activity with potencies similar to their respective affinities for cannabinoid receptors. Therefore, indole quinuclidines are a novel structural class of compounds exhibiting high affinity and a range of intrinsic activity at cannabinoid type-1 and type-2 receptors. PMID:24858620

  9. Angiotensin AT1A receptors on leptin receptor-expressing cells control resting metabolism.

    PubMed

    Claflin, Kristin E; Sandgren, Jeremy A; Lambertz, Allyn M; Weidemann, Benjamin J; Littlejohn, Nicole K; Burnett, Colin M L; Pearson, Nicole A; Morgan, Donald A; Gibson-Corley, Katherine N; Rahmouni, Kamal; Grobe, Justin L

    2017-04-03

    Leptin contributes to the control of resting metabolic rate (RMR) and blood pressure (BP) through its actions in the arcuate nucleus (ARC). The renin-angiotensin system (RAS) and angiotensin AT1 receptors within the brain are also involved in the control of RMR and BP, but whether this regulation overlaps with leptin's actions is unclear. Here, we have demonstrated the selective requirement of the AT1A receptor in leptin-mediated control of RMR. We observed that AT1A receptors colocalized with leptin receptors (LEPRs) in the ARC. Cellular coexpression of AT1A and LEPR was almost exclusive to the ARC and occurred primarily within neurons expressing agouti-related peptide (AgRP). Mice lacking the AT1A receptor specifically in LEPR-expressing cells failed to show an increase in RMR in response to a high-fat diet and deoxycorticosterone acetate-salt (DOCA-salt) treatments, but BP control remained intact. Accordingly, loss of RMR control was recapitulated in mice lacking AT1A in AgRP-expressing cells. We conclude that angiotensin activates divergent mechanisms to control BP and RMR and that the brain RAS functions as a major integrator for RMR control through its actions at leptin-sensitive AgRP cells of the ARC.

  10. Scavenger receptor B1 facilitates macrophage uptake of silver nanoparticles and cellular activation

    NASA Astrophysics Data System (ADS)

    Aldossari, Abdullah A.; Shannahan, Jonathan H.; Podila, Ramakrishna; Brown, Jared M.

    2015-07-01

    Due to increased use of silver nanoparticles (AgNPs) for their antimicrobial activity, concerns have risen regarding potential adverse human health effects. Scavenger receptor B1 (SR-B1), a major receptor for high-density lipoprotein (HDL), is expressed by macrophages and has also been reported to play a role in recognition of negatively charged particles. We, therefore, hypothesized that SR-B1 mediates macrophage uptake of AgNPs and inflammatory activation. To test this hypothesis, we exposed a mouse macrophage cell line RAW264.7 (RAW) and bone marrow-derived macrophages (BMDM) to 20 nm citrate-suspended AgNPs. To verify the role of the SR-B1 receptor, we utilized a SR-B1 inhibitor (Blt2). In vitro studies demonstrated uptake of AgNPs and HDL-coated AgNPs by macrophages which were significantly reduced following pretreatment with Blt2. Inflammatory cytokine arrays revealed that macrophages exposed to AgNPs up-regulated expression of Tnf- α, Oncostatin m (OSM), Ccl4, Il17f, Ccl7, and Ccl2, whereas Il16 was found to be down-regulated. Macrophage activation was observed following AgNP and HDL-coated AgNP exposure as measured by OSM protein production and increased surface expression of CD86. These markers of activation were reduced with Blt2 pretreatment. The in vitro findings were confirmed in vivo through pulmonary instillation of AgNPs in mice. Pulmonary instillation of AgNPs resulted in a recruitment of inflammatory cells that were reduced in SR-B1-deficient mice or following Blt2 pretreatment. This study suggests that SR-B1 plays a major role in cellular recognition of AgNPs and the induction of cell responses that could contribute to inflammation caused by AgNP exposure.

  11. Transient Receptor Potential Ankyrin 1 Receptor Activation In Vitro and In Vivo by Pro-tussive Agents: GRC 17536 as a Promising Anti-Tussive Therapeutic

    PubMed Central

    Mukhopadhyay, Indranil; Kulkarni, Abhay; Aranake, Sarika; Karnik, Pallavi; Shetty, Mahesh; Thorat, Sandeep; Ghosh, Indraneel; Wale, Dinesh; Bhosale, Vikram; Khairatkar-Joshi, Neelima

    2014-01-01

    Cough is a protective reflex action that helps clear the respiratory tract which is continuously exposed to airborne environmental irritants. However, chronic cough presents itself as a disease in its own right and despite its global occurrence; the molecular mechanisms responsible for cough are not completely understood. Transient receptor potential ankyrin1 (TRPA1) is robustly expressed in the neuronal as well as non-neuronal cells of the respiratory tract and is a sensor of a wide range of environmental irritants. It is fast getting acceptance as a key biological sensor of a variety of pro-tussive agents often implicated in miscellaneous chronic cough conditions. In the present study, we demonstrate in vitro direct functional activation of TRPA1 receptor by citric acid which is routinely used to evoke cough in preclinical and clinical studies. We also show for the first time that a potent and selective TRPA1 antagonist GRC 17536 inhibits citric acid induced cellular Ca+2 influx in TRPA1 expressing cells and the citric acid induced cough response in guinea pigs. Hence our data provides a mechanistic link between TRPA1 receptor activation in vitro and cough response induced in vivo by citric acid. Furthermore, we also show evidence for TRPA1 activation in vitro by the TLR4, TLR7 and TLR8 ligands which are implicated in bacterial/respiratory virus pathogenesis often resulting in chronic cough. In conclusion, this study highlights the potential utility of TRPA1 antagonist such as GRC 17536 in the treatment of miscellaneous chronic cough conditions arising due to diverse causes but commonly driven via TRPA1. PMID:24819048

  12. Involvement of neuropeptide Y Y1 receptors in the regulation of neuroendocrine corticotropin-releasing hormone neuronal activity.

    PubMed

    Dimitrov, Eugene L; DeJoseph, M Regina; Brownfield, Mark S; Urban, Janice H

    2007-08-01

    The neuroendocrine parvocellular CRH neurons in the paraventricular nucleus (PVN) of the hypothalamus are the main integrators of neural inputs that initiate hypothalamic-pituitary-adrenal (HPA) axis activation. Neuropeptide Y (NPY) expression is prominent within the PVN, and previous reports indicated that NPY stimulates CRH mRNA levels. The purpose of these studies was to examine the participation of NPY receptors in HPA axis activation and determine whether neuroendocrine CRH neurons express NPY receptor immunoreactivity. Infusion of 0.5 nmol NPY into the third ventricle increased plasma corticosterone levels in conscious rats, with the peak of hormone levels occurring 30 min after injection. This increase was prevented by pretreatment with the Y1 receptor antagonist BIBP3226. Immunohistochemistry showed that CRH-immunoreactive neurons coexpressed Y1 receptor immunoreactivity (Y1r-ir) in the PVN, and a majority of these neurons (88.8%) were neuroendocrine as determined by ip injections of FluoroGold. Bilateral infusion of the Y1/Y5 agonist, [leu(31)pro(34)]NPY (110 pmol), into the PVN increased c-Fos and phosphorylated cAMP response element-binding protein expression and elevated plasma corticosterone levels. Increased expression of c-Fos and phosphorylated cAMP response element-binding protein was observed in populations of CRH/Y1r-ir cells. The current findings present a comprehensive study of NPY Y1 receptor distribution and activation with respect to CRH neurons in the PVN. The expression of NPY Y1r-ir by neuroendocrine CRH cells suggests that alterations in NPY release and subsequent activation of NPY Y1 receptors plays an important role in the regulation of the HPA.

  13. IGF-1 receptor cleavage in hypertension.

    PubMed

    Cirrik, Selma; Schmid-Schönbein, Geert W

    2018-06-01

    Increased protease activity causes receptor dysfunction due to extracellular cleavage of different membrane receptors in hypertension. The vasodilatory effects of insulin-like growth factor-1 (IGF-1) are decreased in hypertension. Therefore, in the present study the association of an enhanced protease activity and IGF-1 receptor cleavage was investigated using the spontaneously hypertensive rats (SHRs) and their normotensive Wistar Kyoto (WKY) controls (n = 4). Matrix metalloproteinase (MMP) activities were determined using gelatin zymography on plasma and different tissue samples. WKY aorta rings were incubated in WKY or SHR plasma with or without MMP inhibitors, and immunohistochemistry was used to quantify the densities of the alpha and beta IGF-1 receptor (IGF-1R) subunits and to determine receptor cleavage. The pAkt and peNOS levels in the aorta were investigated using immunoblotting as a measure of IGF-IR function. Increased MMP-2 and MMP-9 activities were detected in plasma and peripheral tissues of SHRs. IGF-1R beta labeling was similar in both groups without plasma incubation, but the fraction of immunolabeled area for IGF-1R alpha was lower in the endothelial layer of the SHR aorta (p < 0.05). A 24-h incubation of WKY aorta with SHR plasma did not affect the IGF-1R beta labeling density, but reduced the IGF-1R alpha labeling density in the endothelium (p < 0.05). MMP inhibitors prevented this decrease (p < 0.01). Western blot analyses revealed that the pAkt and peNOS levels under IGF-1-stimulated and -unstimulated conditions were lower in SHRs (p < 0.05). A reduced IGF-1 cellular response in the aorta was associated with the decrease in the IGF-1R alpha subunit in the SHR hypertension model. Our results indicate that MMP-dependent receptor cleavage contributed to the reduced IGF-1 response in SHRs.

  14. Glucagon-like peptide-1 receptor expression on human eosinophils and its regulation of eosinophil activation.

    PubMed

    Mitchell, P D; Salter, B M; Oliveria, J P; El-Gammal, A; Tworek, D; Smith, S G; Sehmi, R; Gauvreau, G M; Butler, M; O'Byrne, P M

    2017-03-01

    Glucagon-like peptide-1 (GLP-1) and its receptor are part of the incretin family of hormones that regulate glucose metabolism. GLP-1 also has immune modulatory roles. To measure the expression of the GLP-1 receptor (GLP-1R) on eosinophils and neutrophils in normal and asthmatic subjects and evaluate effects of a GLP-1 analog on eosinophil function. Peripheral blood samples were taken from 10 normal and 10 allergic asthmatic subjects. GLP-1R expression was measured on eosinophils and neutrophils. Subsequently, the asthmatic subjects underwent allergen and diluent inhalation challenges, and GLP-1R expression was measured. Purified eosinophils, collected from mild asthmatic subjects, were stimulated with lipopolysaccharide (LPS) and a GLP-1 analog to evaluate eosinophil cell activation markers CD11b and CD69 and cytokine (IL-4, IL-5, IL-8 and IL-13) production. Glucagon-like peptide-1 receptor is expressed on human eosinophils and neutrophils. Eosinophil, but not neutrophil, expression of GLP-1R is significantly higher in normal controls compared to allergic asthmatics. The expression of GLP-1R did not change on either eosinophils or neutrophils following allergen challenge. A GLP-1 analog significantly decreased the expression of eosinophil-surface activation markers following LPS stimulation and decreased eosinophil production of IL-4, IL-8 and IL-13, but not IL-5. Glucagon-like peptide-1 receptor is expressed on human eosinophils and neutrophils. A GLP-1 analog attenuates LPS-stimulated eosinophil activation. GLP-1 agonists may have additional adjunctive indications in treating persons with concomitant type 2 diabetes mellitus and asthma. © 2016 John Wiley & Sons Ltd.

  15. Interaction of valerian extracts of different polarity with adenosine receptors: identification of isovaltrate as an inverse agonist at A1 receptors.

    PubMed

    Lacher, Svenja K; Mayer, Ralf; Sichardt, Kathrin; Nieber, Karen; Müller, Christa E

    2007-01-15

    A series of extracts of valerian roots (Valeriana officinalis L.) was prepared with solvents of different polarity. Polar as well as nonpolar extracts were found to interact with adenosine A(1) receptors. While polar extracts activated A(1) receptors (partial agonistic activity), nonpolar extracts showed antagonistic or inverse agonistic activity at A(1) receptors, as demonstrated by GTPgammaS binding assays at human recombinant A(1) receptors stably expressed in Chinese hamster ovary (CHO) cells. Guided by radioligand binding assays, fractionation of a lipophilic petroleum ether:diethyl ether (1:1) extract led to the isolation of isovaltrate, which was characterized as a potent, highly efficacious inverse agonist at adenosine A(1) receptors (K(i) rat A(1): 2.05 microM). In experiments at rat brain slices measuring post-synaptic potentials (PSPs) in cortical neurons, isovaltrate at least partly reversed the reduction in the PSPs induced by the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA). Isovaltrate may serve as a new lead structure for the development of inverse agonists at adenosine A(1) receptors. The common use of hydrophilic, but not lipophilic valerian extracts as mild sleep-inducing agents is consistent with the opposite actions of hydrophilic and lipophilic extracts on adenosine receptors.

  16. A role of the SAM domain in EphA2 receptor activation.

    PubMed

    Shi, Xiaojun; Hapiak, Vera; Zheng, Ji; Muller-Greven, Jeannine; Bowman, Deanna; Lingerak, Ryan; Buck, Matthias; Wang, Bing-Cheng; Smith, Adam W

    2017-03-24

    Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization.

  17. Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation.

    PubMed

    Law, Nathan C; Hunzicker-Dunn, Mary E

    2016-02-26

    The ubiquitous phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)-dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, granulosa cells secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser(789). Knockdown of PP1β blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through protein kinase B (AKT) and FOXO1 (forkhead box protein O1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Thrombin Receptors and Protease-Activated Receptor-2 in Human Placentation

    PubMed Central

    O’Brien, Peter J.; Koi, Hideki; Parry, Samuel; Brass, Lawrence F.; Strauss, Jerome F.; Wang, Li-Peng; Tomaszewski, John E.; Christenson, Lane K.

    2003-01-01

    Proteolysis of the thrombin receptor, protease activated receptor-1 (PAR1), may enhance normal and pathological cellular invasion, and indirect evidence suggests that activation of PAR1 expressed by invasive extravillous trophoblasts (EVTs) influences human placentation. Here we describe PAR1, PAR2, and PAR3 protein distribution in the developing human placenta and implicate PAR1 and PAR2 activation in functions central to EVT invasion. PAR1, PAR2, and PAR3 are expressed in cultured 8- to 13-week-old EVTs, and in situ in 18- to 20-week-old placental syncytiotrophoblasts and invasive trophoblasts. Thrombin, but not the PAR2 agonist peptide SLIGKV, inhibited proliferation in cultured EVTs, although both agonists stimulated phosphoinositide hydrolysis and EVT invasion through Matrigel barriers. Thrombin-induced phosphoinositide hydrolysis was completely inhibited and the thrombin effect on proliferation was prevented when PAR1 cleavage was first blocked with specific monoclonal antibodies, indicating that PAR1 is the predominant thrombin receptor on EVTs. Together these results support a role for PAR1, and potentially PAR2 and PAR3 in the invasive phase of human placentation. PMID:14507634

  19. Transactivation Assays that Identify Indirect and Direct Activators of Human Pregnane X Receptor (PXR, NR1I2) and Constitutive Androstane Receptor (CAR, NR1I3).

    PubMed

    Pinne, Marija; Ponce, Elsa; Raucy, Judy L

    2017-01-01

    Nuclear Receptors (NRs), including PXR and CAR, are presumed to be ligand-dependent transcription factors, but ligand binding is not an absolute requirement for activation. Indeed, many compounds activate PXR and CAR by indirect mechanisms. Detecting these indirect activators of specific nuclear receptors in vitro has been difficult. As NR activation of either or both PXR and CAR can lead to drug-drug interactions and adverse drug effects, false negatives obtained with screening tools incapable of detecting indirect activators could present liabilities. The aim of this study was to establish assays that identify indirect activators of human PXR and CAR. Commercially available human PXR and CAR transactivation assays were used for analyses. We show that transactivation assays containing full-length nuclear receptors with native promoters can identify indirect activators of human CAR and PXRwhen compared to those of commercially available assays containing only the LBD of PXR and CAR. Of these two assay systems, only human PXR and CAR1 assays with full-length receptors and native promoters are capable of detecting indirect and ligand activators. With this capability, several kinase inhibitors were identified that activate PXR and CAR by indirect mechanisms. Furthermore by using both the LBD and full-length receptors, phenobarbital and midostaurin were found to be direct and indirect activators of PXR while human CAR activation by phenobarbital occurs by indirect mechanisms only. Cell based transactivation assays employing the full-length receptors and native promoters identify both direct and indirect activators of either or both human PXR and CAR. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Type-7 metabotropic glutamate receptors negatively regulate α1-adrenergic receptor signalling.

    PubMed

    Iacovelli, Luisa; Di Menna, Luisa; Peterlik, Daniel; Stangl, Christina; Orlando, Rosamaria; Molinaro, Gemma; De Blasi, Antonio; Bruno, Valeria; Battaglia, Giuseppe; Flor, Peter J; Uschold-Schmidt, Nicole; Nicoletti, Ferdinando

    2017-02-01

    We studied the interaction between mGlu7 and α 1 -adrenergic receptors in heterologous expression systems, brain slices, and living animals. L-2-Amino-4-phosphonobutanoate (L-AP4), and l-serine-O-phosphate (L-SOP), which activate group III mGlu receptors, restrained the stimulation of polyphosphoinositide (PI) hydrolysis induced by the α 1 -adrenergic receptor agonist, phenylephrine, in HEK 293 cells co-expressing α 1 -adrenergic and mGlu7 receptors. The inibitory action of L-AP4 was abrogated by (i) the mGlu7 receptor antagonist, XAP044; (ii) the C-terminal portion of type-2 G protein coupled receptor kinase; and (iii) the MAP kinase inhibitors, UO126 and PD98059. This suggests that the functional interaction between mGlu7 and α 1 -adrenergic receptors was mediated by the βγ-subunits of the G i protein and required the activation of the MAP kinase pathway. Remarkably, activation of neither mGlu2 nor mGlu4 receptors reduced α 1 -adrenergic receptor-mediated PI hydrolysis. In mouse cortical slices, both L-AP4 and L-SOP were able to attenuate norepinephrine- and phenylephrine-stimulated PI hydrolysis at concentrations consistent with the activation of mGlu7 receptors. L-AP4 failed to affect norepinephrine-stimulated PI hydrolysis in cortical slices from mGlu7 -/- mice, but retained its inhibitory activity in slices from mGlu4 -/- mice. At behavioural level, i.c.v. injection of phenylephrine produced antidepressant-like effects in the forced swim test. The action of phenylephrine was attenuated by L-SOP, which was inactive per se. Finally, both phenylephrine and L-SOP increased corticosterone levels in mice, but the increase was halved when the two drugs were administered in combination. Our data demonstrate that α 1 -adrenergic and mGlu7 receptors functionally interact and suggest that this interaction might be targeted in the treatment of stress-related disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Anticonvulsant effect of AMP by direct activation of adenosine A1 receptor.

    PubMed

    Muzzi, Mirko; Coppi, Elisabetta; Pugliese, Anna Maria; Chiarugi, Alberto

    2013-12-01

    Purinergic neurotransmission mediated by adenosine (Ado) type 1 receptors (A1Rs) plays pivotal roles in negative modulation of epileptic seizures, and Ado is thought to be a key endogenous anticonvulsant. Recent evidence, however, indicates that AMP, the metabolic precursor of Ado, also activate A1Rs. Here, we evaluated the antiepileptic effects of AMP adopting in vitro and in vivo models of epilepsy. We report that AMP reversed the increase in population spike (PS) amplitude and the decrease in PS latency induced by a Mg(2+)-free extracellular solution in CA1 neurons of mouse hippocampal slices. The AMP effects were inhibited by the A1R antagonist DPCPX, but not prevented by inhibiting conversion of AMP into Ado, indicating that AMP inhibited per se sustained hippocampal excitatory neurotransmission by directly activating A1Rs. AMP also reduced seizure severity and mortality in a model of audiogenic convulsion. Of note, the anticonvulsant effects of AMP were potentiated by preventing its conversion into Ado and inhibited by DPCPX. When tested in a model of kainate-induced seizure, AMP prolonged latency of convulsions but had no effects on seizure severity and mortality. Data provide the first evidence that AMP is an endogenous anticonvulsant acting at A1Rs. © 2013.

  2. Shifting Topographic Activation and 5-HT1A-Receptor Mediated Inhibition of Dorsal Raphe Serotonin Neurons Produced by Nicotine Exposure and Withdrawal

    PubMed Central

    Sperling, Robin; Commons, Kathryn G.

    2011-01-01

    Nicotine activates serotonin (5-HT) neurons innervating the forebrain and this is thought to reduce anxiety. Nicotine withdrawal has also been associated with an activation of 5-HT neurotransmission, although withdrawal increases anxiety. In each case, 5-HT1A receptors have been implicated in the response. To determine if there are different subgroups of 5-HT cells activated during nicotine administration and withdrawal, we mapped the appearance of Fos, a marker of neuronal activation, in 5-HT cells of the dorsal and median raphe nuclei (DR and MR). To understand the role 5-HT1A receptor feedback inhibitory pathways on 5-HT cell activity during these conditions, we administered a selective 5-HT1A-receptor antagonist and measured novel disinhibited Fos expression within 5-HT cells. Using these approaches, we found evidence that acute nicotine activates 5-HT neurons rostrally and in the lateral wings of the DR while there is 5-HT1A dependent inhibition of cells located ventrally both at rostral and mid levels. Previous chronic nicotine exposure did not modify the pattern of Fos activation produced by acute nicotine, but increased 5-HT1A-dependent inhibition of 5-HT cells in the caudal DR. This pattern was nearly reversed during nicotine withdrawal when there was evidence for caudal activation and mid- and rostral-5-HT1A-dependent inhibition. These results suggest that the distinct behavioral states produced by nicotine exposure and withdrawal correlate with reciprocal rostral-caudal patterns of activation and 5-HT1A-mediated inhibition of DR 5-HT neurons. The complimentary patterns of activation and inhibition suggest that 5-HT1A receptors may help shape distinct topographic patterns of activation within the DR. PMID:21501256

  3. The 5HT(1A) receptor ligand, S15535, antagonises G-protein activation: a [35S]GTPgammaS and [3H]S15535 autoradiography study.

    PubMed

    Newman-Tancredi, A; Rivet, J; Chaput, C; Touzard, M; Verrièle, L; Millan, M J

    1999-11-19

    4-(Benzodioxan-5-yl)1-(indan-2-yl)piperazine (S15535) is a highly selective ligand at 5-HT(1A) receptors. The present study compared its autoradiographic labelling of rat brain sections with its functional actions, visualised by guanylyl-5'-[gamma-thio]-triphosphate ([35S]GTPgammaS) autoradiography, which affords a measure of G-protein activation. [3H]S15535 binding was highest in hippocampus, frontal cortex, entorhinal cortex, lateral septum, interpeduncular nucleus and dorsal raphe, consistent with specific labelling of 5-HT(1A) receptors. In functional studies, S15535 (10 microM) did not markedly stimulate G-protein activation in any brain region, but abolished the activation induced by the selective 5-HT(1A) agonist, (+)-8-hydroxy-dipropyl-aminotetralin ((+)-8-OH-DPAT, 1 microM), in structures enriched in [3H]S15535 labelling. S15535 did not block 5-HT-stimulated activation in caudate nucleus or substantia nigra, regions where (+)-8-OH-DPAT was ineffective and [3H]S15535 binding was absent. Interestingly, S15535 attenuated (+)-8-OH-DPAT and 5-HT-stimulated G-protein activation in dorsal raphe, a region in which S15535 is known to exhibit agonist properties in vivo [Lejeune, F., Millan, M.J., 1998. Induction of burst firing in ventral tegmental area dopaminergic neurons by activation of serotonin (5-HT)(1A) receptors: WAY100,635-reversible actions of the highly selective ligands, flesinoxan and S15535. Synapse 30, 172-180.]. The present data show that (i) [3H]S15535 labels pre- and post-synaptic populations of 5-HT(1A) sites in rat brain sections, (ii) S15535 exhibits antagonist properties at post-synaptic 5-HT(1A) receptors in corticolimbic regions, and (iii) S15535 also attenuates agonist-stimulated G-protein activation at raphe-localised 5-HT(1A) receptors.

  4. Activation of the ζ receptor 1 suppresses NMDA responses in rat retinal ganglion cells.

    PubMed

    Zhang, X-J; Liu, L-L; Jiang, S-X; Zhong, Y-M; Yang, X-L

    2011-03-17

    The sigma receptor 1 (σR1) has been shown to modulate the activity of several voltage- and ligand-gated channels. Using patch-clamp techniques in rat retinal slice preparations, we demonstrated that activation of σR1 by SKF10047 (SKF) or PRE-084 suppressed N-methyl-D-aspartate (NMDA) receptor-mediated current responses from both ON and OFF type ganglion cells (GCs), dose-dependently, and the effect could be blocked by the σR1 antagonist BD1047 or the σR antagonist haloperidol. The suppression by SKF of NMDA currents was abolished with pre-incubation of the G protein inhibitor GDP-β-S or the Gi/o activator mastoparan. We further explored the intracellular signaling pathway responsible for the SKF-induced suppression of NMDA responses. Application of either cAMP/the PKA inhibitor Rp-cAMP or cGMP/the PKG inhibitor KT5823 did not change the SKF-induced effect, suggesting the involvement of neither cAMP/PKA nor cGMP/PKG pathway. In contrast, suppression of NMDA responses by SKF was abolished by internal infusion of the phosphatidylinostiol-specific phospholipase C (PLC) inhibitor U73122, but not by the phosphatidylcholine-PLC inhibitor D609. SKF-induced suppression of NMDA responses was dependent on intracellular Ca2+ concentration ([Ca2+]i), as evidenced by the fact that the effect was abolished when [Ca2+]i was buffered with 10 mM BAPTA. The SKF effect was blocked by xestospongin-C/heparin, IP3 receptor antagonists, but unchanged by ryanodine/caffeine, ryanodine receptor modulators. Furthermore, application of protein kinase C inhibitors Bis IV and Gö6976 eliminated the SKF effect. These results suggest that the suppression of NMDA responses of rat retinal GCs caused by the activation of σR1 may be mediated by a distinct [Ca2+]i-dependent PLC-PKC pathway. This effect of SKF could help ameliorate malfunction of GCs caused by excessive stimulation of NMDA receptors under pathological conditions. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights

  5. AmTAR2: Functional characterization of a honeybee tyramine receptor stimulating adenylyl cyclase activity.

    PubMed

    Reim, Tina; Balfanz, Sabine; Baumann, Arnd; Blenau, Wolfgang; Thamm, Markus; Scheiner, Ricarda

    2017-01-01

    The biogenic monoamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they employ octopamine and tyramine for comparable physiological functions. These biogenic amines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Based on pharmacological data obtained on heterologously expressed receptors, α- and β-adrenergic-like octopamine receptors are better activated by octopamine than by tyramine. Conversely, GPCRs forming the type 1 tyramine receptor clade (synonymous to octopamine/tyramine receptors) are better activated by tyramine than by octopamine. More recently, receptors were characterized which are almost exclusively activated by tyramine, thus forming an independent type 2 tyramine receptor clade. Functionally, type 1 tyramine receptors inhibit adenylyl cyclase activity, leading to a decrease in intracellular cAMP concentration ([cAMP] i ). Type 2 tyramine receptors can mediate Ca 2+ signals or both Ca 2+ signals and effects on [cAMP] i . We here provide evidence that the honeybee tyramine receptor 2 (AmTAR2), when heterologously expressed in flpTM cells, exclusively causes an increase in [cAMP] i . The receptor displays a pronounced preference for tyramine over octopamine. Its activity can be blocked by a series of established antagonists, of which mianserin and yohimbine are most efficient. The functional characterization of two tyramine receptors from the honeybee, AmTAR1 (previously named AmTYR1) and AmTAR2, which respond to tyramine by changing cAMP levels in opposite direction, is an important step towards understanding the actions of tyramine in honeybee behavior and physiology, particularly in comparison to the effects of octopamine. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Solid-phase synthesis and structure-activity relationships of novel biarylethers as melanin-concentrating hormone receptor-1 antagonists.

    PubMed

    Ma, Vu; Bannon, Anthony W; Baumgartner, Jamie; Hale, Clarence; Hsieh, Faye; Hulme, Christopher; Rorrer, Kirk; Salon, John; van Staden, Carlo; Tempest, Paul

    2006-10-01

    Melanin-concentrating hormone (MCH) is a cyclic 19 amino acid orexigenic neuropeptide. The action of MCH on feeding is thought to involve the activation of its respective G protein-coupled receptor MCH-R1. Consequently, antagonists that block MCH regulated MCH-R1 activity may provide a viable approach to the treatment of diet-induced obesity. This communication reports the discovery of a novel MCH-R1 receptor antagonist, the biarylether 7, identified through high throughput screening. The solid-phase synthesis and structure-activity relationship of related analogs is described.

  7. The human luteinizing hormone receptor gene promoter: activation by Sp1 and Sp3 and inhibitory regulation.

    PubMed

    Geng, Y; Tsai-Morris, C H; Zhang, Y; Dufau, M L

    1999-09-24

    To understand the transcriptional mechanism(s) of human LH receptor (LHR) gene expression, we have identified the dominant functional cis-elements that regulate the activity of the promoter domain (-1 to -176 bp from ATG). Mutagenesis demonstrated that the promoter activity was dependent on two Sp1 domains (-79 bp, -120 bp) in a transformed normal placental cell (PLC) and the choriocarcinoma JAR cell. Both elements interacted with endogenous Sp1 and Sp3 factors but not with Sp2 or Sp4. In Drosophila SL2 cells, the promoter was activated by either Sp1 or Sp3. An ERE half-site (EREhs) at -174 bp was inhibitory (by 100%), but was unresponsive to estradiol and did not bind the estrogen receptor or orphan receptors ERR1 and SF-1. The 5' upstream sequence (-177 to -2056 bp) inhibited promoter activity in PLC by 60%, but only minimally in JAR cells. Activation of the human LHR promoter through Sp1/3 factors is negatively regulated through EREhs and upstream sequences to exert control of gene expression. Copyright 1999 Academic Press.

  8. EphA2 receptor activation with ephrin-A1 ligand restores cetuximab efficacy in NRAS-mutant colorectal cancer cells.

    PubMed

    Cuyàs, Elisabet; Queralt, Bernardo; Martin-Castillo, Begoña; Bosch-Barrera, Joaquim; Menendez, Javier A

    2017-07-01

    Patients with wild-type KRAS metastatic colorectal cancer (mCRC) that harbors NRAS activating mutations do not benefit from anti-EGFR therapies. Very little is known about oncogenic NRAS signaling driving mCRC unresponsiveness to the EGFR-directed antibody cetuximab. Using a system of paired NRAS-mutant and wild-type isogenic mCRC cell lines to explore signaling pathways engaged by the common oncogenic NRAS Q61K variant upon challenge with cetuximab, we uncovered an unexpected mechanism of resistance to cetuximab involving dysregulation of the ephrin-A1/EphA2 signaling axis. Parental NRAS+/+ cells, but not NRASQ61K/+ cells, activated the ephrin receptor ephA1 in response to cetuximab treatment. Moreover, whereas cetuximab treatment significantly downregulated EPHA2 gene expression in NRAS+/+ cells, EPHA2 expression in NRASQ61K/+ cells was refractory to cetuximab. Remarkably, pharmacologically mimicked ephrin-A1 engagement to ephA2 converted NRAS-mutant into RAS wild-type mCRC cells in terms of cetuximab efficacy. Accordingly, activation of the ephA2 receptor by bioactive recombinant human ephrin-A1/Fc-fusion protein suppressed the cetuximab-unresponsive hyperactivation of MAPK and AKT and fully restored cetuximab activity in NRAS-mutant colorectal cells. Collectively, these findings reveal that the clinical benefit of cetuximab in mCRC might necessarily involve the suppression of the ligandless oncogenic signaling of the ephA2 receptor. Hence, ligand-dependent tumor suppressor signaling using therapeutic ephA2 agonists might offer new therapeutic opportunities to clinically widen the use of cetuximab in NRAS-mutated and/or ephA2-dependent mCRC tumors.

  9. MAS1 Receptor Trafficking Involves ERK1/2 Activation Through a β-Arrestin2-Dependent Pathway.

    PubMed

    Cerniello, Flavia M; Carretero, Oscar A; Longo Carbajosa, Nadia A; Cerrato, Bruno D; Santos, Robson A; Grecco, Hernán E; Gironacci, Mariela M

    2017-11-01

    The MAS1 receptor (R) exerts protective effects in the brain, heart, vessels, and kidney. R trafficking plays a critical function in signal termination and propagation and in R resensitization. We examined MAS1R internalization and trafficking on agonist stimulation and the role of β-arrestin2 in the activation of ERK1/2 (extracellular signal-regulated kinase 1/2) and Akt after MAS1R stimulation. Human embryonic kidney 293T cells were transfected with the coding sequence for MAS1R-YFP (MAS1R fused to yellow fluorescent protein). MAS1R internalization was evaluated by measuring the MAS1R present in the plasma membrane after agonist stimulation using a ligand-binding assay. MAS1R trafficking was evaluated by its colocalization with trafficking markers. MAS1R internalization was blocked in the presence of shRNAcaveolin-1 and with dominant negatives for Eps15 (a protein involved in endocytosed Rs by clathrin-coated pits) and for dynamin. After stimulation, MAS1R colocalized with Rab11-a slow recycling vesicle marker-and not with Rab4-a fast recycling vesicle marker-or LysoTracker-a lysosome marker. Cells transfected with MAS1R showed an increase in Akt and ERK1/2 activation on angiotensin-(1-7) stimulation, which was blocked when the clathrin-coated pits pathway was blocked. Suppression of β-arrestin2 by shRNA reduced the angiotensin-(1-7)-induced ERK1/2 activation, whereas Akt activation was not modified. We conclude that on agonist stimulation, MAS1R is internalized through clathrin-coated pits and caveolae in a dynamin-dependent manner and is then slowly recycled back to the plasma membrane. MAS1R induced Akt and ERK1/2 activation from early endosomes, and the activation of ERK1/2 was mediated by β-arrestin2. Thus, MAS1R activity and density may be tightly controlled by the cell. © 2017 American Heart Association, Inc.

  10. Activation of temperature-sensitive TRPV1-like receptors in ARC POMC neurons reduces food intake

    PubMed Central

    Jeong, Jae Hoon; Lee, Dong Kun; Liu, Shun-Mei; Chua, Streamson C.; Schwartz, Gary J.

    2018-01-01

    Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) respond to numerous hormonal and neural signals, resulting in changes in food intake. Here, we demonstrate that ARC POMC neurons express capsaicin-sensitive transient receptor potential vanilloid 1 receptor (TRPV1)-like receptors. To show expression of TRPV1-like receptors in ARC POMC neurons, we use single-cell reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiology, TRPV1 knock-out (KO), and TRPV1-Cre knock-in mice. A small elevation of temperature in the physiological range is enough to depolarize ARC POMC neurons. This depolarization is blocked by the TRPV1 receptor antagonist and by Trpv1 gene knockdown. Capsaicin-induced activation reduces food intake that is abolished by a melanocortin receptor antagonist. To selectively stimulate TRPV1-like receptor-expressing ARC POMC neurons in the ARC, we generate an adeno-associated virus serotype 5 (AAV5) carrying a Cre-dependent channelrhodopsin-2 (ChR2)–enhanced yellow fluorescent protein (eYFP) expression cassette under the control of the two neuronal POMC enhancers (nPEs). Optogenetic stimulation of TRPV1-like receptor-expressing POMC neurons decreases food intake. Hypothalamic temperature is rapidly elevated and reaches to approximately 39 °C during treadmill running. This elevation is associated with a reduction in food intake. Knockdown of the Trpv1 gene exclusively in ARC POMC neurons blocks the feeding inhibition produced by increased hypothalamic temperature. Taken together, our findings identify a melanocortinergic circuit that links acute elevations in hypothalamic temperature with acute reductions in food intake. PMID:29689050

  11. Activation of temperature-sensitive TRPV1-like receptors in ARC POMC neurons reduces food intake.

    PubMed

    Jeong, Jae Hoon; Lee, Dong Kun; Liu, Shun-Mei; Chua, Streamson C; Schwartz, Gary J; Jo, Young-Hwan

    2018-04-01

    Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) respond to numerous hormonal and neural signals, resulting in changes in food intake. Here, we demonstrate that ARC POMC neurons express capsaicin-sensitive transient receptor potential vanilloid 1 receptor (TRPV1)-like receptors. To show expression of TRPV1-like receptors in ARC POMC neurons, we use single-cell reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiology, TRPV1 knock-out (KO), and TRPV1-Cre knock-in mice. A small elevation of temperature in the physiological range is enough to depolarize ARC POMC neurons. This depolarization is blocked by the TRPV1 receptor antagonist and by Trpv1 gene knockdown. Capsaicin-induced activation reduces food intake that is abolished by a melanocortin receptor antagonist. To selectively stimulate TRPV1-like receptor-expressing ARC POMC neurons in the ARC, we generate an adeno-associated virus serotype 5 (AAV5) carrying a Cre-dependent channelrhodopsin-2 (ChR2)-enhanced yellow fluorescent protein (eYFP) expression cassette under the control of the two neuronal POMC enhancers (nPEs). Optogenetic stimulation of TRPV1-like receptor-expressing POMC neurons decreases food intake. Hypothalamic temperature is rapidly elevated and reaches to approximately 39 °C during treadmill running. This elevation is associated with a reduction in food intake. Knockdown of the Trpv1 gene exclusively in ARC POMC neurons blocks the feeding inhibition produced by increased hypothalamic temperature. Taken together, our findings identify a melanocortinergic circuit that links acute elevations in hypothalamic temperature with acute reductions in food intake.

  12. Chewing rescues stress-suppressed hippocampal long-term potentiation via activation of histamine H1 receptor.

    PubMed

    Ono, Yumie; Kataoka, Tsuyoshi; Miyake, Shinjiro; Sasaguri, Kenichi; Sato, Sadao; Onozuka, Minoru

    2009-08-01

    We have previously found in rats that chewing, an active behavioral strategy to cope with a stressful situation, rescues long-term potentiation (LTP) in the hippocampus through activating stress-suppressed N-methyl-D-aspartate (NMDA) receptor function. To further examine the mechanisms underlying this ameliorative effect of chewing, we studied the involvement of the histaminergic system, which has been shown to be activated by mastication, in the LTP of hippocampal slices of rats that were allowed to chew a wooden stick during exposure to immobilization stress. Chewing failed to rescue stress-suppressed LTP in the rats treated with histamine H1 receptor (H1R) antagonist pyrilamine (5 mg/kg, i.p.) before exposure to stress, although administration of pyrilamine did not affect LTP in naive rats and in stressed rats that did not chew. However, when pyrilamine was administrated immediately after exposure to stress, chewing rescued LTP whose magnitude was statistically comparable to that in the rats that chewed without drug treatment. These results suggest that chewing-induced histamine release in the hippocampus and the subsequent H1 receptor activation may be essential to rescue stress-suppressed synaptic plasticity.

  13. Endothelial nitric-oxide synthase (eNOS) is activated through G-protein-coupled receptor kinase-interacting protein 1 (GIT1) tyrosine phosphorylation and Src protein.

    PubMed

    Liu, Songling; Premont, Richard T; Rockey, Don C

    2014-06-27

    Nitric oxide (NO) is a critical regulator of vascular tone and plays an especially prominent role in liver by controlling portal blood flow and pressure within liver sinusoids. Synthesis of NO in sinusoidal endothelial cells by endothelial nitric-oxide synthase (eNOS) is regulated in response to activation of endothelial cells by vasoactive signals such as endothelins. The endothelin B (ETB) receptor is a G-protein-coupled receptor, but the mechanisms by which it regulates eNOS activity in sinusoidal endothelial cells are not well understood. In this study, we built on two previous strands of work, the first showing that G-protein βγ subunits mediated activation of phosphatidylinositol 3-kinase and Akt to regulate eNOS and the second showing that eNOS directly bound to the G-protein-coupled receptor kinase-interacting protein 1 (GIT1) scaffold protein, and this association stimulated NO production. Here we investigated the mechanisms by which the GIT1-eNOS complex is formed and regulated. GIT1 was phosphorylated on tyrosine by Src, and Y293F and Y554F mutations reduced GIT1 phosphorylation as well as the ability of GIT1 to bind to and activate eNOS. Akt phosphorylation activated eNOS (at Ser(1177)), and Akt also regulated the ability of Src to phosphorylate GIT1 as well as GIT1-eNOS association. These pathways were activated by endothelin-1 through the ETB receptor; inhibiting receptor-activated G-protein βγ subunits blocked activation of Akt, GIT1 tyrosine phosphorylation, and ET-1-stimulated GIT1-eNOS association but did not affect Src activation. These data suggest a model in which Src and Akt cooperate to regulate association of eNOS with the GIT1 scaffold to facilitate NO production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Activation of the umami taste receptor (T1R1/T1R3) initiates the peristaltic reflex and pellet propulsion in the distal colon.

    PubMed

    Kendig, Derek M; Hurst, Norman R; Bradley, Zachary L; Mahavadi, Sunila; Kuemmerle, John F; Lyall, Vijay; DeSimone, John; Murthy, Karnam S; Grider, John R

    2014-12-01

    Intraluminal nutrients in the gut affect the peristaltic reflex, although the mechanism is not well defined. Recent evidence supports the presence of taste receptors and their signaling components in enteroendocrine cells, although their function is unclear. This study aimed to determine if nutrients modify colonic motility through activation of taste receptors. Colonic sections were immunostained for the umami taste receptor T1R1/T1R3, which mediates the response to umami ligands, such as monosodium glutamate (MSG), in taste cells. Ascending contraction, descending relaxation, and calcitonin gene-related peptide release were measured in three-chamber flat-sheet preparations of rat colon in response to MSG alone or with inosine 5'-monophosphate (IMP). Velocity of artificial fecal pellet propulsion was measured by video recording in guinea pig distal colon. T1R1/T1R3 receptors were present in enteroendocrine cells of colonic sections from human, rat, mouse, and guinea pig. MSG initiated ascending contraction and descending relaxation components of the peristaltic reflex and calcitonin gene-related peptide release in flat-sheet preparations. IMP augmented the MSG-induced effects, suggesting activation of T1R1/T1R3 receptors. In T1R1(-/-) mice, mucosal stroking, but not MSG, elicited a peristaltic reflex. Intraluminal perfusion of MSG enhanced the velocity of artificial fecal pellet propulsion, which was also augmented by IMP. Propulsion was also increased by l-cysteine, but not l-tryptophan, supporting a role of T1R1/T1R3 receptors. We conclude that T1R1/T1R3 activation by luminal MSG or l-cysteine elicits a peristaltic reflex and CGRP release and increases the velocity of pellet propulsion in distal colon. This mechanism may explain how nutrients regulate colonic propulsion. Copyright © 2014 the American Physiological Society.

  15. Activation of the umami taste receptor (T1R1/T1R3) initiates the peristaltic reflex and pellet propulsion in the distal colon

    PubMed Central

    Kendig, Derek M.; Hurst, Norman R.; Bradley, Zachary L.; Mahavadi, Sunila; Kuemmerle, John F.; Lyall, Vijay; DeSimone, John; Murthy, Karnam S.

    2014-01-01

    Intraluminal nutrients in the gut affect the peristaltic reflex, although the mechanism is not well defined. Recent evidence supports the presence of taste receptors and their signaling components in enteroendocrine cells, although their function is unclear. This study aimed to determine if nutrients modify colonic motility through activation of taste receptors. Colonic sections were immunostained for the umami taste receptor T1R1/T1R3, which mediates the response to umami ligands, such as monosodium glutamate (MSG), in taste cells. Ascending contraction, descending relaxation, and calcitonin gene-related peptide release were measured in three-chamber flat-sheet preparations of rat colon in response to MSG alone or with inosine 5′-monophosphate (IMP). Velocity of artificial fecal pellet propulsion was measured by video recording in guinea pig distal colon. T1R1/T1R3 receptors were present in enteroendocrine cells of colonic sections from human, rat, mouse, and guinea pig. MSG initiated ascending contraction and descending relaxation components of the peristaltic reflex and calcitonin gene-related peptide release in flat-sheet preparations. IMP augmented the MSG-induced effects, suggesting activation of T1R1/T1R3 receptors. In T1R1−/− mice, mucosal stroking, but not MSG, elicited a peristaltic reflex. Intraluminal perfusion of MSG enhanced the velocity of artificial fecal pellet propulsion, which was also augmented by IMP. Propulsion was also increased by l-cysteine, but not l-tryptophan, supporting a role of T1R1/T1R3 receptors. We conclude that T1R1/T1R3 activation by luminal MSG or l-cysteine elicits a peristaltic reflex and CGRP release and increases the velocity of pellet propulsion in distal colon. This mechanism may explain how nutrients regulate colonic propulsion. PMID:25324508

  16. Discovery of Tetrahydropyrazolopyridine as Sphingosine 1-Phosphate Receptor 3 (S1P3)-Sparing S1P1 Agonists Active at Low Oral Doses.

    PubMed

    Demont, Emmanuel H; Bailey, James M; Bit, Rino A; Brown, Jack A; Campbell, Colin A; Deeks, Nigel; Dowell, Simon J; Eldred, Colin; Gaskin, Pam; Gray, James R J; Haynes, Andrea; Hirst, David J; Holmes, Duncan S; Kumar, Umesh; Morse, Mary A; Osborne, Greg J; Renaux, Jessica F; Seal, Gail A L; Smethurst, Chris A; Taylor, Simon; Watson, Robert; Willis, Robert; Witherington, Jason

    2016-02-11

    FTY720 is the first oral small molecule approved for the treatment of people suffering from relapsing-remitting multiple sclerosis. It is a potent agonist of the S1P1 receptor, but its lack of selectivity against the S1P3 receptor has been linked to most of the cardiovascular side effects observed in the clinic. These findings have triggered intensive efforts toward the identification of a second generation of S1P3-sparing S1P1 agonists. We have recently disclosed a series of orally active tetrahydroisoquinoline (THIQ) compounds matching these criteria. In this paper we describe how we defined and implemented a strategy aiming at the discovery of selective structurally distinct follow-up agonists. This effort culminated with the identification of a series of orally active tetrahydropyrazolopyridines.

  17. Estrogen receptor ESR1 mediates activation of ERK1/2, CREB, and ELK1 in the corpus of the epididymis.

    PubMed

    Cavalcanti, Fernanda N; Lucas, Thais F G; Lazari, Maria Fatima M; Porto, Catarina S

    2015-06-01

    Expression of the estrogen receptor ESR1 is higher in the corpus than it is in the initial segment/caput and cauda of the epididymis. ESR1 immunostaining in the corpus has been localized not only in the nuclei but also in the cytoplasm and apical membrane, which indicates that ESR1 plays a role in membrane-initiated signaling. The present study investigated whether ESR1 mediates the activation of rapid signaling pathways by estradiol (E2) in the epididymis. We investigated the effect of E2 and the ESR1-selective agonist (4,4',4''-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT) on the activation of extracellular signal-regulated protein kinases (ERK1/2), CREB protein, and ETS oncogene-related protein (ELK1). Treatment with PPT did not affect ERK1/2 phosphorylation in the cauda, but it rapidly increased ERK1/2 phosphorylation in the initial segment/caput and corpus of the epididymis. PPT also activated CREB and ELK1 in the corpus of the epididymis. The PPT-induced phosphorylation of ERK1/2, CREB, and ELK1 was blocked by the ESR1-selective antagonist MPP and by pretreatment with a non-receptor tyrosine kinase SRC inhibitor, an EGFR kinase inhibitor, an MEK1/2 inhibitor, and a phosphatidylinositol-3-kinase inhibitor. In conclusion, these results indicate that the corpus, which is a region with high expression of the estrogen receptor ESR1, is a major target in the epididymis for the activation of rapid signaling by E2. The sequence of events that follow E2 interaction with ESR1 includes the SRC-mediated transactivation of EGFR and the phosphorylation of ERK1/2, CREB, and ELK1. This rapid estrogen signaling may modulate gene expression in the corpus of the epididymis, and it may play a role in the dynamic microenvironment of the epididymal lumen. © 2015 Society for Endocrinology.

  18. Activation of mas-related G-protein-coupled receptors by the house dust mite cysteine protease Der p1 provides a new mechanism linking allergy and inflammation.

    PubMed

    Reddy, Vemuri B; Lerner, Ethan A

    2017-10-20

    Cysteine and serine proteases function via protease-activated and mas-related G-protein-coupled receptors (Mrgprs) to contribute to allergy and inflammation. Der p1 is a cysteine protease and major allergen from the house dust mite and is associated with allergic rhinitis and allergic asthma. Der p1 activates protease-activated receptor 2 and induces the release of the pro-inflammatory cytokine IL-6 from cells. However, the possibility that Der p1 acts on Mrgprs has not been considered. We report here that ratiometric calcium imaging reveals that Der p1 activates the human receptor MRGPRX1 and the mouse homolog MrgprC11, implicated previously in itch. Der p1 cleavage of N-terminal receptor peptides followed by site-directed mutagenesis of the cleavage sites links receptor activation to specific amino acid residues. Der p1 also induced the release of IL-6 from heterologous cells expressing MRGPRX1. In summary, activation of Mrgprs by the allergen Der p1 may contribute to inflammation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor {alpha}/retinoid X receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Belanger, Adam J.; Luo Zhengyu; Vincent, Karen A.

    2007-12-21

    In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid {beta}-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid {beta}-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1{alpha}/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferasemore » I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for {beta}-oxidation. Furthermore, adenovirus-mediated expression of HIF-1{alpha}/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})/retinoid X receptor (RXR), in the presence or absence of a PPAR{alpha} ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPAR{alpha}/RXR.« less

  20. Mixed-ligand copper(II) complexes activate aryl hydrocarbon receptor AhR and induce CYP1A genes expression in human hepatocytes and human cell lines.

    PubMed

    Kubešová, Kateřina; Dořičáková, Aneta; Trávníček, Zdeněk; Dvořák, Zdeněk

    2016-07-25

    The effects of four copper(II) mixed-ligand complexes [Cu(qui1)(L)]NO3·H2O (1-3) and [Cu(qui2)(phen)]NO3 (4), where qui1=2-phenyl-3-hydroxy-4(1H)-quinolinone, Hqui2=2-(4-amino-3,5-dichlorophenyl)-N-propyl-3-hydroxy-4(1H)-quinolinone-7-carboxamide, L=1,10-phenanthroline (phen) (1), 5-methyl-1,10-phenanthroline (mphen) (2), bathophenanthroline (bphen) (3), on transcriptional activities of steroid receptors, nuclear receptors and xenoreceptors have been studied. The complexes (1-4) did not influence basal or ligand-inducible activities of glucocorticoid receptor, androgen receptor, thyroid receptor, pregnane X receptor and vitamin D receptor, as revealed by gene reporter assays. The complexes 1 and 2 dose-dependently induced luciferase activity in stable gene reporter AZ-AhR cell line, and this induction was reverted by resveratrol, indicating involvement of aryl hydrocarbon receptor (AhR) in the process. The complexes 1, 2 and 3 induced CYP1A1 mRNA in LS180 cells and CYP1A1/CYP1A2 in human hepatocytes through AhR. Electrophoretic mobility shift assay EMSA showed that the complexes 1 and 2 transformed AhR in its DNA-binding form. Collectively, we demonstrate that the complexes 1 and 2 activate AhR and induce AhR-dependent genes in human hepatocytes and cancer cell lines. In conclusion, the data presented here might be of toxicological importance, regarding the multiple roles of AhR in human physiology and pathophysiology. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Activation of the chemosensing transient receptor potential channel A1 (TRPA1) by alkylating agents.

    PubMed

    Stenger, Bernhard; Zehfuss, Franziska; Mückter, Harald; Schmidt, Annette; Balszuweit, Frank; Schäfer, Eva; Büch, Thomas; Gudermann, Thomas; Thiermann, Horst; Steinritz, Dirk

    2015-09-01

    The transient receptor potential ankyrin 1 (TRPA1) cation channel is expressed in different tissues including skin, lung and neuronal tissue. Recent reports identified TRPA1 as a sensor for noxious substances, implicating a functional role in the molecular toxicology. TRPA1 is activated by various potentially harmful electrophilic substances. The chemical warfare agent sulfur mustard (SM) is a highly reactive alkylating agent that binds to numerous biological targets. Although SM is known for almost 200 years, detailed knowledge about the pathophysiology resulting from exposure is lacking. A specific therapy is not available. In this study, we investigated whether the alkylating agent 2-chloroethyl-ethylsulfide (CEES, a model substance for SM-promoted effects) and SM are able to activate TRPA1 channels. CEES induced a marked increase in the intracellular calcium concentration ([Ca(2+)]i) in TRPA1-expressing but not in TRPA1-negative cells. The TRP-channel blocker AP18 diminished the CEES-induced calcium influx. HEK293 cells permanently expressing TRPA1 were more sensitive toward cytotoxic effects of CEES compared with wild-type cells. At low CEES concentrations, CEES-induced cytotoxicity was prevented by AP18. Proof-of-concept experiments using SM resulted in a pronounced increase in [Ca(2+)]i in HEK293-A1-E cells. Human A549 lung epithelial cells, which express TRPA1 endogenously, reacted with a transient calcium influx in response to CEES exposure. The CEES-dependent calcium response was diminished by AP18. In summary, our results demonstrate that alkylating agents are able to activate TRPA1. Inhibition of TRPA1 counteracted cellular toxicity and could thus represent a feasible approach to mitigate SM-induced cell damage.

  2. Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    He, Rui-Qing; Tang, Xiao-Feng; Zhang, Bao-Li

    Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-β and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediatedmore » Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes. - Highlights: • Direct activation of PAR1 and PAR2 led to adventitial fibroblast (AF) activation. • PAR1 and PAR2 antagonists attenuated Ang II-induced AF activation. • Ang II induced the upregulation and transactivation of PAR1/PAR2 in AFs.« less

  3. Novel mechanisms of G-protein-coupled receptors functions: AT1 angiotensin receptor acts as a signaling hub and focal point of receptor cross-talk.

    PubMed

    Tóth, András D; Turu, Gábor; Hunyady, László; Balla, András

    2018-04-01

    AT 1 angiotensin receptor (AT 1 R), a prototypical G protein-coupled receptor (GPCR), is the main receptor, which mediates the effects of the renin-angiotensin system (RAS). AT 1 R plays a crucial role in the regulation of blood pressure and salt-water homeostasis, and in the development of pathological conditions, such as hypertension, heart failure, cardiovascular remodeling, renal fibrosis, inflammation, and metabolic disorders. Stimulation of AT 1 R leads to pleiotropic signal transduction pathways generating arrays of complex cellular responses. Growing amount of evidence shows that AT 1 R is a versatile GPCR, which has multiple unique faces with distinct conformations and signaling properties providing new opportunities for functionally selective pharmacological targeting of the receptor. Biased ligands of AT 1 R have been developed to selectively activate the β-arrestin pathway, which may have therapeutic benefits compared to the conventional angiotensin converting enzyme inhibitors and angiotensin receptor blockers. In this review, we provide a summary about the most recent findings and novel aspects of the AT 1 R function, signaling, regulation, dimerization or oligomerization and its cross-talk with other receptors, including epidermal growth factor (EGF) receptor, adrenergic receptors and CB 1 cannabinoid receptor. Better understanding of the mechanisms and structural aspects of AT 1 R activation and cross-talk can lead to the development of novel type of drugs for the treatment of cardiovascular and other diseases. Copyright © 2018. Published by Elsevier Ltd.

  4. CRF1 receptor-deficiency increases cocaine reward.

    PubMed

    Contarino, Angelo; Kitchener, Pierre; Vallée, Monique; Papaleo, Francesco; Piazza, Pier-Vincenzo

    2017-05-01

    Stimulant drugs produce reward but also activate stress-responsive systems. The corticotropin-releasing factor (CRF) and the related hypothalamus-pituitary-adrenal (HPA) axis stress-responsive systems are activated by stimulant drugs. However, their role in stimulant drug-induced reward remains poorly understood. Herein, we report that CRF 1 receptor-deficient (CRF 1 -/-), but not wild-type, mice show conditioned place preference (CPP) responses to a relatively low cocaine dose (5 mg/kg, i.p.). Conversely, wild-type, but not CRF 1 -/-, mice display CPP responses to a relatively high cocaine dose (20 mg/kg, i.p.), indicating that CRF 1 receptor-deficiency alters the rewarding effects of cocaine. Acute pharmacological antagonism of the CRF 1 receptor by antalarmin also eliminates cocaine reward. Nevertheless, CRF 1 -/- mice display higher stereotypy responses to cocaine than wild-type mice. Despite the very low plasma corticosterone concentration, CRF 1 -/- mice show higher nuclear glucocorticoid receptor (GR) levels in the brain region of the hippocampus than wild-type mice. Full rescue of wild-type-like corticosterone and GR circadian rhythm and level in CRF 1 -/- mice by exogenous corticosterone does not affect CRF 1 receptor-dependent cocaine reward but induces stereotypy responses to cocaine. These results indicate a critical role for the CRF 1 receptor in cocaine reward, independently of the closely related HPA axis activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. NK1 receptor activation in rat rostral ventrolateral medulla selectively attenuates somato-sympathetic reflex while antagonism attenuates sympathetic chemoreflex.

    PubMed

    Makeham, John M; Goodchild, Ann K; Pilowsky, Paul M

    2005-06-01

    The effects of activation and blockade of the neurokinin 1 (NK1) receptor in the rostral ventrolateral medulla (RVLM) on arterial blood pressure (ABP), splanchnic sympathetic nerve activity (sSNA), phrenic nerve activity, the somato-sympathetic reflex, baroreflex, and chemoreflex were studied in urethane-anesthetized and artificially ventilated Sprague-Dawley rats. Bilateral microinjection of either the stable substance P analog (pGlu5, MePhe8, Sar9)SP(5-11) (DiMe-SP) or the highly selective NK1 agonist [Sar9, Met (O(2))11]SP into the RVLM resulted in an increase in ABP, sSNA, and heart rate and an abolition of phrenic nerve activity. The effects of [Sar9, Met (O(2))11]SP were blocked by the selective nonpeptide NK1 receptor antagonist WIN 51708. NK1 receptor activation also dramatically attenuated the somato-sympathetic reflex elicited by tibial nerve stimulation, while leaving the baroreflex and chemoreflex unaffected. This effect was again blocked by WIN 51708. NK1 receptor antagonism in the RVLM, with WIN 51708 significantly attenuated the sympathoexcitatory response to hypoxia but had no effect on baseline respiratory function. Our findings suggest that substance P and the NK1 receptor play a significant role in the cardiorespiratory reflexes integrated within the RVLM.

  6. Interaction between Calpain 5, Peroxisome proliferator-activated receptor-gamma and Peroxisome proliferator-activated receptor-delta genes: a polygenic approach to obesity

    PubMed Central

    Sáez, María E; Grilo, Antonio; Morón, Francisco J; Manzano, Luis; Martínez-Larrad, María T; González-Pérez, Antonio; Serrano-Hernando, Javier; Ruiz, Agustín; Ramírez-Lorca, Reposo; Serrano-Ríos, Manuel

    2008-01-01

    Context Obesity is a multifactorial disorder, that is, a disease determined by the combined effect of genes and environment. In this context, polygenic approaches are needed. Objective To investigate the possibility of the existence of a crosstalk between the CALPAIN 10 homologue CALPAIN 5 and nuclear receptors of the peroxisome proliferator-activated receptors family. Design Cross-sectional, genetic association study and gene-gene interaction analysis. Subjects The study sample comprise 1953 individuals, 725 obese (defined as body mass index ≥ 30) and 1228 non obese subjects. Results In the monogenic analysis, only the peroxisome proliferator-activated receptor delta (PPARD) gene was associated with obesity (OR = 1.43 [1.04–1.97], p = 0.027). In addition, we have found a significant interaction between CAPN5 and PPARD genes (p = 0.038) that reduces the risk for obesity in a 55%. Conclusion Our results suggest that CAPN5 and PPARD gene products may also interact in vivo. PMID:18657264

  7. Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

    PubMed

    Nuñez, S B; Medin, J A; Braissant, O; Kemp, L; Wahli, W; Ozato, K; Segars, J H

    1997-03-14

    Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

  8. Excessive D1 Dopamine Receptor Activation in the Dorsal Striatum Promotes Autistic-Like Behaviors.

    PubMed

    Lee, Yunjin; Kim, Hannah; Kim, Ji-Eun; Park, Jin-Young; Choi, Juli; Lee, Jung-Eun; Lee, Eun-Hwa; Han, Pyung-Lim

    2018-07-01

    The dopamine system has been characterized in motor function, goal-directed behaviors, and rewards. Recent studies recognize various dopamine system genes as being associated with autism spectrum disorder (ASD). However, how dopamine system dysfunction induces ASD pathophysiology remains unknown. In the present study, we demonstrated that mice with increased dopamine functions in the dorsal striatum via the suppression of dopamine transporter expression in substantia nigra neurons or the optogenetic stimulation of the nigro-striatal circuitry exhibited sociability deficits and repetitive behaviors relevant to ASD pathology in animal models, while these behavioral changes were blocked by a D1 receptor antagonist. Pharmacological activation of D1 dopamine receptors in normal mice or the genetic knockout (KO) of D2 dopamine receptors also produced typical autistic-like behaviors. Moreover, the siRNA-mediated inhibition of D2 dopamine receptors in the dorsal striatum was sufficient to replicate autistic-like phenotypes in D2 KO mice. Intervention of D1 dopamine receptor functions or the signaling pathways-related D1 receptors in D2 KO mice produced anti-autistic effects. Together, our results indicate that increased dopamine function in the dorsal striatum promotes autistic-like behaviors and that the dorsal striatum is the neural correlate of ASD core symptoms.

  9. Cyclin D1 Repression of Peroxisome Proliferator-Activated Receptor γ Expression and Transactivation

    PubMed Central

    Wang, Chenguang; Pattabiraman, Nagarajan; Zhou, Jian Nian; Fu, Maofu; Sakamaki, Toshiyuki; Albanese, Chris; Li, Zhiping; Wu, Kongming; Hulit, James; Neumeister, Peter; Novikoff, Phyllis M.; Brownlee, Michael; Scherer, Philipp E.; Jones, Joan G.; Whitney, Kathleen D.; Donehower, Lawrence A.; Harris, Emily L.; Rohan, Thomas; Johns, David C.; Pestell, Richard G.

    2003-01-01

    The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPARγ induces hepatic steatosis, and liganded PPARγ promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPARγ function, transactivation, expression, and promoter activity. PPARγ transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB- and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix (HLH) structure. The cyclin D1 HLH region was also required for repression of the PPARγ ligand-binding domain linked to a heterologous DNA binding domain. Adipocyte differentiation by PPARγ-specific ligands (BRL49653, troglitazone) was enhanced in cyclin D1−/− fibroblasts and reversed by retroviral expression of cyclin D1. Homozygous deletion of the cyclin D1 gene, enhanced expression by PPARγ ligands of PPARγ and PPARγ-responsive genes, and cyclin D1−/− mice exhibit hepatic steatosis. Finally, reduction of cyclin D1 abundance in vivo using ponasterone-inducible cyclin D1 antisense transgenic mice, increased expression of PPARγ in vivo. The inhibition of PPARγ function by cyclin D1 is a new mechanism of signal transduction cross talk between PPARγ ligands and mitogenic signals that induce cyclin D1. PMID:12917338

  10. Direct involvement of sigma-1 receptors in the dopamine D1 receptor-mediated effects of cocaine.

    PubMed

    Navarro, Gemma; Moreno, Estefanía; Aymerich, Marisol; Marcellino, Daniel; McCormick, Peter J; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Canela, Enric I; Ortiz, Jordi; Fuxe, Kjell; Lluís, Carmen; Ferré, Sergi; Franco, Rafael

    2010-10-26

    It is well known that cocaine blocks the dopamine transporter. This mechanism should lead to a general increase in dopaminergic neurotransmission, and yet dopamine D(1) receptors (D(1)Rs) play a more significant role in the behavioral effects of cocaine than the other dopamine receptor subtypes. Cocaine also binds to σ-1 receptors, the physiological role of which is largely unknown. In the present study, D(1)R and σ(1)R were found to heteromerize in transfected cells, where cocaine robustly potentiated D(1)R-mediated adenylyl cyclase activation, induced MAPK activation per se and counteracted MAPK activation induced by D(1)R stimulation in a dopamine transporter-independent and σ(1)R-dependent manner. Some of these effects were also demonstrated in murine striatal slices and were absent in σ(1)R KO mice, providing evidence for the existence of σ(1)R-D(1)R heteromers in the brain. Therefore, these results provide a molecular explanation for which D(1)R plays a more significant role in the behavioral effects of cocaine, through σ(1)R-D(1)R heteromerization, and provide a unique perspective toward understanding the molecular basis of cocaine addiction.

  11. Interaction of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and opioid receptors in spinal cord nociceptive reflexes.

    PubMed

    Ramos-Zepeda, Guillermo; Herrero, Juan F

    2013-08-14

    We previously observed that the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) is a very effective antinociceptive agent on intact but not on spinalized adult rats with inflammation. Since a close connection between opioid and adenosine A1 receptors has been described, we studied a possible relationship between these systems in the spinal cord. CPA-mediated antinociception was challenged by the selective adenosine A1 receptor antagonist 8-cyclopentyl-1, 3-dimethylxanthine (CPT) and by the opioid receptor antagonist naloxone on male adult Wistar rats with carrageenan-induced inflammation. Withdrawal reflexes activated by noxious mechanical and electrical stimulation were recorded using the single motor technique in intact and sham-spinalized animals. CPA was very effective in intact and sham spinalized rats but not in spinalized animals. Full reversal of CPA antinociception was observed with i.v. 1mg/kg of naloxone but not with 20mg/kg of CPT i.v. in responses to noxious mechanical and electrical stimulation. CPT fully prevented CPA from any antinociceptive action whereas naloxone did not modify CPA activity. These results suggest a centrally-mediated action, since CPA depressed the wind-up phenomenon which is derived of the activity of spinal cord neurons. The present study provides strong in vivo evidence of an antinociceptive activity mediated by the adenosine A1 receptor system in the spinal cord, linked to an activation of opioid receptors in adult animals with inflammation. © 2013.

  12. An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory*♦

    PubMed Central

    Butcher, Adrian J.; Bradley, Sophie J.; Prihandoko, Rudi; Brooke, Simon M.; Mogg, Adrian; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; Edwards, Jennifer M.; Bottrill, Andrew R.; Challiss, R. A. John; Broad, Lisa M.; Felder, Christian C.; Tobin, Andrew B.

    2016-01-01

    Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo. Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser228) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser228. These data supported the hypothesis that phosphorylation at Ser228 was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser228 on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser228 phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser228 not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning. PMID:26826123

  13. Mechanisms of transient receptor potential vanilloid 1 activation and sensitization by allyl isothiocyanate.

    PubMed

    Gees, Maarten; Alpizar, Yeranddy A; Boonen, Brett; Sanchez, Alicia; Everaerts, Wouter; Segal, Andrei; Xue, Fenqin; Janssens, Annelies; Owsianik, Grzegorz; Nilius, Bernd; Voets, Thomas; Talavera, Karel

    2013-09-01

    Allyl isothiocyanate (AITC; aka, mustard oil) is a powerful irritant produced by Brassica plants as a defensive trait against herbivores and confers pungency to mustard and wasabi. AITC is widely used experimentally as an inducer of acute pain and neurogenic inflammation, which are largely mediated by the activation of nociceptive cation channels transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 (TRPV1). Although it is generally accepted that electrophilic agents activate these channels through covalent modification of cytosolic cysteine residues, the mechanism underlying TRPV1 activation by AITC remains unknown. Here we show that, surprisingly, AITC-induced activation of TRPV1 does not require interaction with cysteine residues, but is largely dependent on S513, a residue that is involved in capsaicin binding. Furthermore, AITC acts in a membrane-delimited manner and induces a shift of the voltage dependence of activation toward negative voltages, which is reminiscent of capsaicin effects. These data indicate that AITC acts through reversible interactions with the capsaicin binding site. In addition, we show that TRPV1 is a locus for cross-sensitization between AITC and acidosis in nociceptive neurons. Furthermore, we show that residue F660, which is known to determine the stimulation by low pH in human TRPV1, is also essential for the cross-sensitization of the effects of AITC and low pH. Taken together, these findings demonstrate that not all reactive electrophiles stimulate TRPV1 via cysteine modification and help understanding the molecular bases underlying the surprisingly large role of this channel as mediator of the algesic properties of AITC.

  14. Screening of phytochemicals against protease activated receptor 1 (PAR1), a promising target for cancer.

    PubMed

    Kakarala, Kavita Kumari; Jamil, Kaiser

    2015-02-01

    Drug resistance and drug-associated toxicity are the primary causes for withdrawal of many drugs, although patient recovery is satisfactory in many instances. Interestingly, the use of phytochemicals in the treatment of cancer as an alternative to synthetic drugs comes with a host of advantages; minimum side effects, good human absorption and low toxicity to normal cells. Protease activated receptor 1 (PAR1) has been established as a promising target in many diseases including various cancers. Strong evidences suggest its role in metastasis also. There are no natural compounds known to inhibit its activity, so we aimed to identify phytochemicals with antagonist activity against PAR1. We screened phytochemicals from Naturally Occurring Plant-based Anticancer Compound-Activity-Target database (NPACT, http://crdd.osdd.net/raghava/npact/ ) against PAR1 using virtual screening workflow of Schrödinger software. It analyzes pharmaceutically relevant properties using Qikprop and calculates binding energy using Glide at three accuracy levels (high-throughput virtual screening, standard precision and extra precision). Our study led to the identification of phytochemicals, which showed interaction with at least one experimentally determined active site residue of PAR1, showed no violations to Lipinski's rule of five along with predicted high human absorption. Furthermore, structural interaction fingerprint analysis indicated that the residues H255, D256, E260, S344, V257, L258, L262, Y337 and S344 may play an important role in the hydrogen bond interactions of the phytochemicals screened. Of these residues, H255 and L258 residues were experimentally proved to be important for antagonist binding. The residues Y183, L237, L258, L262, F271, L332, L333, Y337, L340, A349, Y350, A352, and Y353 showed maximum hydrophobic interactions with the phytochemicals screened. The results of this work suggest that phytochemicals Reissantins D, 24,25-dihydro-27-desoxywithaferin A, Isoguaiacin

  15. Synthesis, structure-activity relationships, and in vivo evaluation of N3-phenylpyrazinones as novel corticotropin-releasing factor-1 (CRF1) receptor antagonists.

    PubMed

    Hartz, Richard A; Ahuja, Vijay T; Arvanitis, Argyrios G; Rafalski, Maria; Yue, Eddy W; Denhart, Derek J; Schmitz, William D; Ditta, Jonathan L; Deskus, Jeffrey A; Brenner, Allison B; Hobbs, Frank W; Payne, Joseph; Lelas, Snjezana; Li, Yu-Wen; Molski, Thaddeus F; Mattson, Gail K; Peng, Yong; Wong, Harvey; Grace, James E; Lentz, Kimberley A; Qian-Cutrone, Jingfang; Zhuo, Xiaoliang; Shu, Yue-Zhong; Lodge, Nicholas J; Zaczek, Robert; Combs, Andrew P; Olson, Richard E; Bronson, Joanne J; Mattson, Ronald J; Macor, John E

    2009-07-23

    Evidence suggests that corticotropin-releasing factor-1 (CRF(1)) receptor antagonists may offer therapeutic potential for the treatment of diseases associated with elevated levels of CRF such as anxiety and depression. A pyrazinone-based chemotype of CRF(1) receptor antagonists was discovered. Structure-activity relationship studies led to the identification of numerous potent analogues including 12p, a highly potent and selective CRF(1) receptor antagonist with an IC(50) value of 0.26 nM. The pharmacokinetic properties of 12p were assessed in rats and Cynomolgus monkeys. Compound 12p was efficacious in the defensive withdrawal test (an animal model of anxiety) in rats. The synthesis, structure-activity relationships and in vivo properties of compounds within the pyrazinone chemotype are described.

  16. Exercise reduces adipose tissue via cannabinoid receptor type 1 which is regulated by peroxisome proliferator-activated receptor-delta.

    PubMed

    Yan, Zhen Cheng; Liu, Dao Yan; Zhang, Li Li; Shen, Chen Yi; Ma, Qun Li; Cao, Ting Bing; Wang, Li Juan; Nie, Hai; Zidek, Walter; Tepel, Martin; Zhu, Zhi Ming

    2007-03-09

    Obesity is one major cardiovascular risk factor. We tested effects of endurance exercise on cannabinoid receptor type 1 (CB1) and peroxisome proliferator-activated receptor-delta (PPAR-delta)-dependent pathways in adipose tissue. Male Wistar rats were randomly assigned to standard laboratory chow or a high-fat diet without and with regular endurance exercise. Exercise in rats on high-fat diet significantly reduced visceral fat mass, blood pressure, and adipocyte size (each p<0.05). Adipocyte hypertrophy induced by high-fat diet was accompanied by increased CB1 expression in adipose tissue, whereas exercise significantly reduced CB1 expression (each p<0.05). CB1 receptor expression and adipocyte differentiation were directly regulated by PPAR-delta. Adipocyte hypertrophy induced by high-fat diet was accompanied by reduced PPAR-delta. Furthermore, selective silencing of PPAR-delta by RNA interference in 3T3-L1-preadipocyte cells significantly increased CB1 expression from 1.00+/-0.06 (n=3) to 1.91+/-0.06 (n=3; p<0.01) and increased adipocyte differentiation, whereas adenovirus-mediated overexpression of PPAR-delta significantly reduced CB1 expression to 0.39+/-0.03 (n=3; p<0.01) and reduced adipocyte differentiation. In the presence of the CB1 antagonist rimonabant adipocyte differentiation in stimulated 3T3 L1 preadipocyte cells was significantly reduced. The study indicates that high-fat diet-induced hypertrophy of adipocytes is associated with increased CB1 receptor expression which is directly regulated by PPAR-delta. Both CB1 and PPAR-delta are intimately involved in therapeutic interventions against a most important cardiovascular risk factor.

  17. Activation of D1/5 Dopamine Receptors: A Common Mechanism for Enhancing Extinction of Fear and Reward-Seeking Behaviors.

    PubMed

    Abraham, Antony D; Neve, Kim A; Lattal, K Matthew

    2016-07-01

    Dopamine is critical for many processes that drive learning and memory, including motivation, prediction error, incentive salience, memory consolidation, and response output. Theories of dopamine's function in these processes have, for the most part, been developed from behavioral approaches that examine learning mechanisms in appetitive tasks. A parallel and growing literature indicates that dopamine signaling is involved in consolidation of memories into stable representations in aversive tasks such as fear conditioning. Relatively little is known about how dopamine may modulate memories that form during extinction, when organisms learn that the relation between previously associated events is severed. We investigated whether fear and reward extinction share common mechanisms that could be enhanced with dopamine D1/5 receptor activation. Pharmacological activation of dopamine D1/5 receptors (with SKF 81297) enhanced extinction of both cued and contextual fear. These effects also occurred in the extinction of cocaine-induced conditioned place preference, suggesting that the observed effects on extinction were not specific to a particular type of procedure (aversive or appetitive). A cAMP/PKA biased D1 agonist (SKF 83959) did not affect fear extinction, whereas a broadly efficacious D1 agonist (SKF 83822) promoted fear extinction. Together, these findings show that dopamine D1/5 receptor activation is a target for the enhancement of fear or reward extinction.

  18. The kinase activity of fibroblast growth factor receptor 3 with activation loop mutations affects receptor trafficking and signaling.

    PubMed

    Lievens, Patricia M-J; Mutinelli, Chiara; Baynes, Darcie; Liboi, Elio

    2004-10-08

    Amino acid substitutions at the Lys-650 codon within the activation loop kinase domain of fibroblast growth factor receptor 3 (FGFR3) result in graded constitutive phosphorylation of the receptor. Accordingly, the Lys-650 mutants are associated with dwarfisms with graded clinical severity. To assess the importance of the phosphorylation level on FGFR3 maturation along the secretory pathway, hemagglutinin A-tagged derivatives were studied. The highly activated SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) mutant accumulates in its immature and phosphorylated form in the endoplasmic reticulum (ER), which fails to be degraded. Furthermore, the Janus kinase (Jak)/STAT pathway is activated from the ER by direct recruitment of Jak1. Abolishing the autocatalytic property of the mutated FGFR3 by replacing the critical Tyr-718 reestablishes the receptor full maturation and inhibits signaling. Differently, the low activated hypochondroplasia mutant is present as a mature phosphorylated form on the plasma membrane, although with a delayed transition in the ER, and is completely processed. Signaling does not occur in the presence of brefeldin A; instead, STAT1 is activated when protein secretion is blocked with monensin, suggesting that the hypochondroplasia receptor signals at the exit from the ER. Our results suggest that kinase activity affects FGFR3 trafficking and determines the spatial segregation of signaling pathways. Consequently, the defect in down-regulation of the highly activated receptors results in the increased signaling capacity from the intracellular compartments, and this may determine the severity of the diseases.

  19. Activation of Muscarinic M1 Acetylcholine Receptors Induces Long-Term Potentiation in the Hippocampus

    PubMed Central

    Dennis, Siobhan H.; Pasqui, Francesca; Colvin, Ellen M.; Sanger, Helen; Mogg, Adrian J.; Felder, Christian C.; Broad, Lisa M.; Fitzjohn, Steve M.; Isaac, John T.R.; Mellor, Jack R.

    2016-01-01

    Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires NMDA receptor activity and bi-directionally occludes with synaptically induced LTP. Thus, we describe synergistic mechanisms by which acetylcholine acting through M1Rs excites CA1 pyramidal neurons and induces LTP, to profoundly increase activation of CA1 pyramidal neurons. These features are predicted to make a major contribution to the pro-cognitive effects of cholinergic transmission in rodents and humans. PMID:26472558

  20. Effects of 5-HT1A Receptor Stimulation on D1 Receptor Agonist-Induced Striatonigral Activity and Dyskinesia in Hemiparkinsonian Rats

    PubMed Central

    2013-01-01

    Accumulating evidence supports the value of 5-HT1A receptor (5-HT1AR) agonists for dyskinesias that arise with long-term L-DOPA therapy in Parkinson’s disease (PD). Yet, how 5-HT1AR stimulation directly influences the dyskinetogenic D1 receptor (D1R)-expressing striatonigral pathway remains largely unknown. To directly examine this, one cohort of hemiparkinsonian rats received systemic injections of Vehicle + Vehicle, Vehicle + the D1R agonist SKF81297 (0.8 mg/kg), or the 5-HT1AR agonist ±8-OH-DPAT (1.0 mg/kg) + SKF81297. Rats were examined for changes in abnormal involuntary movements (AIMs), rotations, striatal preprodynorphin (PPD), and glutamic acid decarboxylase (GAD; 65 and 67) mRNA via RT-PCR. In the second experiment, hemiparkinsonian rats received intrastriatal pretreatments of Vehicle (aCSF), ±8-OH-DPAT (7.5 mM), or ±8-OH-DPAT + the 5-HT1AR antagonist WAY100635 (4.6 mM), followed by systemic Vehicle or SKF81297 after which AIMs, rotations, and extracellular striatal glutamate and nigral GABA efflux were measured by in vivo microdialysis. Results revealed D1R agonist-induced AIMs were reduced by systemic and intrastriatal 5-HT1AR stimulation while rotations were enhanced. Although ±8-OH-DPAT did not modify D1R agonist-induced increases in striatal PPD mRNA, the D1R/5-HT1AR agonist combination enhanced GAD65 and GAD67 mRNA. When applied locally, ±8-OH-DPAT alone diminished striatal glutamate levels while the agonist combination increased nigral GABA efflux. Thus, presynaptic 5-HT1AR stimulation may attenuate striatal glutamate levels, resulting in diminished D1R-mediated dyskinetic behaviors, but maintain or enhance striatal postsynaptic factors ultimately increasing nigral GABA levels and rotational activity. The current findings offer a novel mechanistic explanation for previous results concerning 5-HT1AR agonists for the treatment of dyskinesia. PMID:23496922

  1. CRIP1a inhibits endocytosis of G-protein coupled receptors activated by endocannabinoids and glutamate by a common molecular mechanism.

    PubMed

    Mascia, Fabrizio; Klotz, Lisa; Lerch, Judith; Ahmed, Mostafa H; Zhang, Yan; Enz, Ralf

    2017-05-01

    The excitability of the central nervous system depends largely on the surface density of neurotransmitter receptors. The endocannabinoid receptor 1 (CB 1 R) and the metabotropic glutamate receptor mGlu 8 R are expressed pre-synaptically where they reduce glutamate release into the synaptic cleft. Recently, the CB 1 R interacting protein cannabinoid receptor interacting protein 1a (CRIP1a) was identified and characterized to regulate CB 1 R activity in neurons. However, underlying molecular mechanisms are largely unknown. Here, we identified a common mechanism used by CRIP1a to regulate the cell surface density of two different types of G-protein coupled receptors, CB 1 R and mGlu 8a R. Five amino acids within the CB 1 R C-terminus were required and sufficient to reduce constitutive CB 1 R endocytosis by about 72% in the presence of CRIP1a. Interestingly, a similar sequence is present in mGlu 8a R and consistently, endocytosis of mGlu 8a R depended on CRIP1a, as well. Docking analysis and molecular dynamics simulations identified a conserved serine in CB 1 R (S468) and mGlu 8a R (S894) that forms a hydrogen bond with the peptide backbone of CRIP1a at position R82. In contrast to mGlu 8a R, the closely related mGlu 8b R splice-variant carries a lysine (K894) at this position, and indeed, mGlu 8b R endocytosis was not affected by CRIP1a. Chimeric constructs between CB 1 R, mGlu 8a R, and mGlu 8b R underline the role of the identified five CRIP1a sensitive amino acids. In summary, we suggest that CRIP1a negatively regulates endocytosis of two different G-protein coupled receptor types, CB 1 R and mGlu 8a R. © 2017 International Society for Neurochemistry.

  2. Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

    PubMed Central

    De Henau, Olivier; Degroot, Gaetan-Nagim; Imbault, Virginie; Robert, Virginie; De Poorter, Cédric; Mcheik, Saria; Galés, Céline; Parmentier, Marc; Springael, Jean-Yves

    2016-01-01

    Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties. PMID:27716822

  3. Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2.

    PubMed

    De Henau, Olivier; Degroot, Gaetan-Nagim; Imbault, Virginie; Robert, Virginie; De Poorter, Cédric; Mcheik, Saria; Galés, Céline; Parmentier, Marc; Springael, Jean-Yves

    2016-01-01

    Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties.

  4. Selective and non-selective OT receptor agonists induce different locomotor behaviors in male rats via central OT receptors and peripheral V1a receptors.

    PubMed

    Wolfe, Monica; Wisniewska, Halina; Tariga, Hiroe; Ibanez, Gerardo; Collins, James C; Wisniewski, Kazimierz; Qi, Steve; Srinivasan, Karthik; Hargrove, Diane; Lindstrom, Beatriz Fioravanti

    2018-05-21

    Oxytocin (OT) continues to inspire much research due to its diverse physiological effects. While the best-understood actions of OT are uterine contraction and milk ejection, OT is also implicated in maternal and bonding behaviors, and potentially in CNS disorders such as autism, schizophrenia, and pain. The dissection of the mechanism of action of OT is complicated by the fact that this peptide activates not only its cognate receptor but also vasopressin type 1a (V1a) receptors. In this study, we evaluated OT and a selective OT receptor (OTR) agonist, FE 204409, in an automated assay that measures rat locomotor activity. The results showed: 1) Subcutaneous (sc) administration of OT decreased locomotor behavior (distance traveled, stereotypy, and rearing). This effect was reversed by a V1a receptor (V1aR) antagonist ([Pmp1,Tyr(ME)2]AVP, sc), suggesting that OT acts through peripheral V1aR to inhibit locomotor activity. 2) A selective OTR agonist (FE 204409, sc) increased stereotypy. This effect was reversed by an OTR antagonist dosed icv, suggesting a central OTR site of action. Our findings identify distinct behavioral effects for OT and the selective agonist FE 204409, adding to the growing body of evidence that the V1aR mediates many effects attributed to OT and that peptides administered systemically at supra-physiological doses may activate receptors in the brain. Our studies further emphasize the importance of utilizing selective agonists and antagonists to assess therapeutic indications. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Antagonistic actions of renal dopamine and 5-hydroxytryptamine: increase in Na+, K(+)-ATPase activity in renal proximal tubules via activation of 5-HT1A receptors.

    PubMed Central

    Soares-da-Silva, P.; Pinto-do-O, P. C.; Bertorello, A. M.

    1996-01-01

    1. 5-Hydroxytryptamine (5-HT) is antinatriuretic. Since this effect of 5-HT is not accomplished by changes in glomerular haemodynamics, we have examined in this study whether 5-HT may influence sodium excretion by affecting the Na+, K(+)-ATPase activity in renal cortical tubules. 2. Na+, K(+)-ATPase activity was determined as the rate of [32P]-ATP hydrolysis in renal cortical tubules in suspension. Basal Na+, K(+)-ATPase activity in renal tubules was 4.8 +/- 0.4 mumol Pi mg-1 protein h-1 (n = 8). The 5-HT1A receptor agonist, (+/-)-8-hydroxy-2-(di-n-propylamino) tetraline (8-OH-DPAT) (10 to 3000 nM) induced a concentration-dependent increase (P < 0.05) in Na+, K(+)-ATPase activity with an EC50 value of 355 nM (95% confidence limits: 178, 708). Maximal stimulation elicited by 3000 nM of 8-OH-DPAT was antagonized by the selective 5-HT1A receptor antagonist, (+)-WAY 100135 10 to 1000 nM) with an IC50 value of 20 nM (14, 29); 0.3 microM (+)-WAY 100135 completely abolished (P < 0.01) the stimulatory effect of 8-OH-DPAT. The stimulatory effect of 8-OH-DPAT was found to be time-dependent (15 +/- 2% and 66 +/- 7% increase at 2.5 and 5.0 min, respectively). The 5-HT2 receptor agonist alpha-methyl-5-HT (100 to 3000 nM) did not induce any significant changes in Na+, K(+)-ATPase activity (5.0 +/- 1.5 mumol Pi mg-1 protein h-1; n = 4). 3. The stimulatory effect 8-OH-DPAT was absent when homogenates were used. Stimulation occurred at a Vmax concentration (70 mM) of sodium supporting the notion that stimulation occurs independently of increasing sodium permeability. 4. The inhibitory effect of dopamine (P < 0.05) on Na+, K(+)-ATPase activity was blunted by co-incubation with 8-OH-DPAT (0.5 microM). 5. It is concluded that activation of 5-HT1A receptors increases Na+, K(+)-ATPase activity in renal cortical tubules; this effect may represent an important cellular mechanism, at the tubule level, responsible for the antinatriuretic effect of 5-HT. Images Figure 4 PMID:8882616

  6. Sulphur-containing compounds of durian activate the thermogenesis-inducing receptors TRPA1 and TRPV1.

    PubMed

    Terada, Yuko; Hosono, Takashi; Seki, Taiichiro; Ariga, Toyohiko; Ito, Sohei; Narukawa, Masataka; Watanabe, Tatsuo

    2014-08-15

    Durian (Durio zibethinus Murr.) is classified as a body-warming food in Indian herbalism, and its hyperthermic effect is empirically known in Southeast Asia. To investigate the mechanism underlying this effect, we focused on the thermogenesis-inducing receptors, TRPA1 and TRPV1. Durian contains sulphides similar to the TRPA1 and TRPV1 agonists of garlic. Accordingly, we hypothesized that the thermogenic effect of durian is driven by sulphide-induced TRP channel activation. To investigate our hypothesis, we measured the TRPA1 and TRPV1 activity of the sulphur-containing components of durian and quantified their content in durian pulp. These sulphur-containing components had a stronger effect on TRPA1 than TRPV1. Furthermore, sulphide content in the durian pulp was sufficient to evoke TRP channel activation and the main agonist was diethyl disulphide. From these results, we consider that the body-warming effect of durian is elicited by TRPA1 activation with its sulphides, as can be seen in spices. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Brain reward-system activation in response to anticipation and consumption of palatable food is altered by glucagon-like peptide-1 receptor activation in humans.

    PubMed

    van Bloemendaal, L; Veltman, D J; Ten Kulve, J S; Groot, P F C; Ruhé, H G; Barkhof, F; Sloan, J H; Diamant, M; Ijzerman, R G

    2015-09-01

    To test the hypothesis that food intake reduction after glucagon-like peptide-1 (GLP-1) receptor activation is mediated through brain areas regulating anticipatory and consummatory food reward. As part of a larger study, we determined the effects of GLP-1 receptor activation on brain responses to anticipation and receipt of chocolate milk versus a tasteless solution, using functional MRI (fMRI). Obese subjects with type 2 diabetes, and obese and lean subjects with normoglycaemia (n = 48) underwent three fMRI sessions at separate visits with intravenous infusion of the GLP-1 receptor agonist exenatide, exenatide with prior GLP-1 receptor blockade by exendin-9-39 or placebo, during somatostatin pituitary-pancreatic clamps. Body mass index negatively correlated with brain responses to receipt of chocolate milk and positively correlated with anticipation of receipt of chocolate milk in brain areas regulating reward, appetite and motivation. Exenatide increased brain responses to receipt of chocolate milk and decreased anticipation of receipt of chocolate milk compared with placebo, paralleled by reductions in food intake. Exendin-9-39 largely prevented these effects. Our findings show that GLP-1 receptor activation decreases anticipatory food reward, which may reduce cravings for food and increases consummatory food reward, which may prevent overeating. © 2015 John Wiley & Sons Ltd.

  8. Amadori products promote cellular senescence activating insulin-like growth factor-1 receptor and down-regulating the antioxidant enzyme catalase.

    PubMed

    Del Nogal-Ávila, María; Troyano-Suárez, Nuria; Román-García, Pablo; Cannata-Andía, Jorge B; Rodriguez-Puyol, Manuel; Rodriguez-Puyol, Diego; Kuro-O, Makoto; Ruiz-Torres, María P

    2013-07-01

    Activation of the insulin growth factor receptor-1 signaling pathways has been largely related to the aging process. Amadori products are produced in pathological conditions such as diabetes and aging, and are potentially involved in diabetic nephropathy or age-associated decline of renal function. We hypothesize that Amadori products induce senescence in primary human mesangial cells through the activation of IGF-1 receptor and investigate, in the present work, the intracellular mechanism involved after this activation. We treated cultured human mesangial cells with glycated albumin, one of the most abundant Amadori product, and senescence was assessed by determining the senescence associated β-galactosidase activity and the expression of the cell cycle regulators p53 and p21. We demonstrated that prolonged exposition (more than 24h) to glycated albumin induced senescence and, in parallel, incremented the release of IGF-1 and the activation of the IGF-1 receptor. Inhibition of the IGF-1 activation prevented the GA induced senescence. Activation of IGF-1R, after GA addition, promoted a reduction in the catalase content through the constitutive activation of Ras and erk1/2 proteins which were, in turn, responsible of the observed GA-induced senescence. In conclusion, we propose that the Amadori product, glycated albumin, promotes premature cell senescence in mesangial cells through the activation of the IGF-1 receptor and the subsequent reduction in the antioxidant enzyme catalase. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. MT1 and MT2 Melatonin Receptors: A Therapeutic Perspective

    PubMed Central

    Liu, Jiabei; Clough, Shannon J.; Hutchinson, Anthony J.; Adamah-Biassi, Ekue B.; Popovska-Gorevski, Marina; Dubocovich, Margarita L.

    2016-01-01

    Melatonin, or 5-methoxy-N-acetyltryptamine, is synthesized and released by the pineal gland and locally in the retina following a circadian rhythm, with low levels during the day and elevated levels at night. Melatonin activates two high-affinity G protein–coupled receptors, termed MT1 and MT2, to exert beneficial actions in sleep and circadian abnormality, mood disorders, learning and memory, neuroprotection, drug abuse, and cancer. Progress in understanding the role of melatonin receptors in the modulation of sleep and circadian rhythms has led to the discovery of a novel class of melatonin agonists for treating insomnia, circadian rhythms, mood disorders, and cancer. This review describes the pharmacological properties of a slow-release melatonin preparation (i.e., Circadin®) and synthetic ligands (i.e., agomelatine, ramelteon, tasimelteon), with emphasis on identifying specific therapeutic effects mediated through MT1 and MT2 receptor activation. Discovery of selective ligands targeting the MT1 or the MT2 melatonin receptors may promote the development of novel and more efficacious therapeutic agents. PMID:26514204

  10. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

    PubMed

    Martin, Adam L; Steurer, Michael A; Aronstam, Robert S

    2015-01-01

    The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.

  11. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors

    PubMed Central

    Martin, Adam L.; Steurer, Michael A.; Aronstam, Robert S.

    2015-01-01

    The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention. PMID:26384023

  12. The Arrestin-selective Angiotensin AT1 Receptor Agonist [Sar1,Ile4,Ile8]-AngII Negatively Regulates Bradykinin B2 Receptor Signaling via AT1-B2 Receptor Heterodimers*

    PubMed Central

    Wilson, Parker C.; Lee, Mi-Hye; Appleton, Kathryn M.; El-Shewy, Hesham M.; Morinelli, Thomas A.; Peterson, Yuri K.; Luttrell, Louis M.; Jaffa, Ayad A.

    2013-01-01

    The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar1,Ile4,Ile8]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar1,Ile4, Ile8]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar1,Ile4,Ile8]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar1,Ile4,Ile8]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar1,Ile4,Ile8]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar1,Ile4,Ile8]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic actions. PMID:23661707

  13. The arrestin-selective angiotensin AT1 receptor agonist [Sar1,Ile4,Ile8]-AngII negatively regulates bradykinin B2 receptor signaling via AT1-B2 receptor heterodimers.

    PubMed

    Wilson, Parker C; Lee, Mi-Hye; Appleton, Kathryn M; El-Shewy, Hesham M; Morinelli, Thomas A; Peterson, Yuri K; Luttrell, Louis M; Jaffa, Ayad A

    2013-06-28

    The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar(1),Ile(4),Ile(8)]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar(1),Ile(4), Ile(8)]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar(1),Ile(4),Ile(8)]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar(1),Ile(4),Ile(8)]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar(1),Ile(4),Ile(8)]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar(1),Ile(4),Ile(8)]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic

  14. Effect of active immunization against angiotensin II type 1 (AT1) receptor on hypertension & arterial remodelling in spontaneously hypertensive rats (SHR).

    PubMed

    Li, Liu-Dong; Tian, Miao; Liao, Yu-Hua; Zhou, Zi-Hua; Wei, Fen; Zhu, Feng; Wang, Min; Wang, Bin; Wei, Yu-Miao

    2014-04-01

    a0 ngiotensin II receptor type 1 (AT1) is known to be involved in the pathogenesis of hypertension. t0 his study was undertaken to explore the effect of active immunization against AT1 receptor on blood pressure and small artery remodelling in spontaneously hypertensive rat (SHR). Male SHR and Wistar rats aged two months were actively immunized with different peptides (ATR12185ͱͲATR10014 and ATR12181) corresponding to particular sequences of rat AT1 receptor, while another SHR group was given losartan (10 mg/kg/day) orally once a day. Anti-AT1 receptor antibodies were detected by ELISA and blood pressure was measured. The effect of the antibodies on the artery and vascular smooth muscle cells (VSMCs) proliferation was studied. all immunized animals produced antibodies against the particular peptides. The systolic blood pressure was decreased in the SHR immunized with peptide-ATR12181 compared with the control. However, no changes were observed in the SHR immunized with other two peptides. The Wistar rats immunized with the three peptides did not show any changes in blood pressure. The media/lumen area ratio of the mesenteric artery was reduced in SHR immunized with ATR12181 and similar to that of the SHR treated with losartan. The antibody from SHR immunized with ATR12181 had no effect on the proliferation of VSMC. But it could inhibit the proliferation caused by angiotensin II and its effect at the titre of 1:40 was similar to that of 1µmol/l losartan. Our findings demonstrated that the antibody from SHR immunized with ATR12181 had the effect of reducing blood pressure and target organ protection similar to losartan. Active immunization against AT1 receptor may be a promising strategy in future for the treatment of hypertension.

  15. Action of novel antipsychotics at human dopamine D3 receptors coupled to G protein and ERK1/2 activation.

    PubMed

    Bruins Slot, Liesbeth A; Palmier, Christiane; Tardif, Stéphanie; Cussac, Didier

    2007-08-01

    The effects of new generation antipsychotic drugs (APDs) targeting dopamine D(2) and serotonin 5-HT(1A) receptors were compared with typical and atypical APDs on phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and measures of G protein activation in CHO cell lines stably expressing the human dopamine D(3) receptor. The preferential dopamine D(3) agonists (+)-7-OH-DPAT and PD128907, like dopamine and quinelorane, efficaciously stimulated ERK 1/2 phosphorylation at dopamine D(3) receptors. In contrast, in [(35)S]GTPgammaS binding experiments, (+)-7-OH-DPAT exhibited partial agonist properties, while PD128907 and quinelorane maintained full agonist properties. The preferential dopamine D(3) ligand BP 897 and the antidyskinetic sarizotan partially activated ERK 1/2 phosphorylation while exerting no agonist activity on GTPgammaS binding, suggesting signal amplification at the MAP kinase level. Antipsychotics differed in their ability to inhibit both agonist-stimulated GTPgammaS binding and ERK 1/2 phosphorylation, but all typical and atypical compounds tested acted as dopamine D(3) receptor antagonists with the exception of n-desmethylclozapine, the active metabolite of clozapine, which partially activated dopamine D(3) receptor-mediated ERK 1/2 phosphorylation. Among the new generation dopamine D(2)/serotonin 5-HT(1A) antipsychotics, only F 15063 and SLV313 acted as pure dopamine D(3) receptor antagonists, bifeprunox was highly efficacious whereas SSR181507 and aripiprazole showed marked partial agonist properties for ERK 1/2 phosphorylation. In contrast, in the GTPgammaS binding study, aripiprazole was devoid of agonist properties and bifeprunox, and to an even lesser extent SSR181507, only weakly stimulated GTPgammaS binding. In summary, these findings underline the differences of dopamine D(3) properties of new generation antipsychotics which may need to be considered in understanding their diverse therapeutic actions.

  16. Activation of G-protein coupled estrogen receptor inhibits the proliferation of cervical cancer cells via sustained activation of ERK1/2.

    PubMed

    Zhang, Qiong; Wu, Yuan-Zhe; Zhang, Yan-Mei; Ji, Xiao-Hong; Hao, Qun

    2015-04-01

    Cervical cancer is one of the most common gynaecological women cancer and suggested to be modulated by estrogenic signals. G protein-coupled receptor (GPER), a seven-transmembrane G protein-coupled receptor, has been reported to regulate the cell proliferation of various cancers. But there is no study investigating the effects of GPER on the progression of cervical cancer. In the present study, we revealed for the first time that GPER was also highly expressed in various human cervical cancer cells. Activation of GPER via its specific agonist G-1 induced G2/M cell cycle arrest and down regulation of cyclin B via a time dependent manner. Furthermore, G-1 treatment induced sustained activation of extracellular-signal-regulated kinases (ERK)1/2 via epidermal growth factor receptor (EGFR) signals. Both inhibitors of ERK1/2 and EGFR significantly abolished G-1-induced suppression of cell proliferation and down regulation of cyclin B. Generally, our study revealed that GPER is highly expressed in human cervical cancer cells and its activation inhibits cell proliferation via EGFR/ERK1/2 signals. It suggested that G-1 can be considered as a potential new pharmacological tool to reduce the growth of cervical cancer. Copyright © 2015 John Wiley & Sons, Ltd.

  17. Increased peroxisome proliferator-activated receptor γ activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells

    PubMed Central

    Wang, Jueqiong; Lu, Liu; Kok, Chung H.; Saunders, Verity A.; Goyne, Jarrad M.; Dang, Phuong; Leclercq, Tamara M.; Hughes, Timothy P.; White, Deborah L.

    2017-01-01

    Imatinib is actively transported by organic cation transporter-1 (OCT-1) influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Herein we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor γ agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor γ antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to BCR-ABL kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor γ-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor γ transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; P<0.0001), suggesting that peroxisome proliferator-activated receptor γ activation has a negative impact on the intracellular uptake of imatinib and consequent BCR-ABL kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor γ activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor γ agonist pioglitazone was reported to act synergistically with imatinib, targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor γ ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor γ activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in

  18. Phosphorylated Nuclear Receptor CAR Forms a Homodimer To Repress Its Constitutive Activity for Ligand Activation

    PubMed Central

    Shizu, Ryota; Osabe, Makoto; Perera, Lalith; Moore, Rick; Sueyoshi, Tatsuya

    2017-01-01

    ABSTRACT The nuclear receptor CAR (NR1I3) regulates hepatic drug and energy metabolism as well as cell fate. Its activation can be a critical factor in drug-induced toxicity and the development of diseases, including diabetes and tumors. CAR inactivates its constitutive activity by phosphorylation at threonine 38. Utilizing receptor for protein kinase 1 (RACK1) as the regulatory subunit, protein phosphatase 2A (PP2A) dephosphorylates threonine 38 to activate CAR. Here we demonstrate that CAR undergoes homodimer-monomer conversion to regulate this dephosphorylation. By coexpression of two differently tagged CAR proteins in Huh-7 cells, mouse primary hepatocytes, and mouse livers, coimmunoprecipitation and two-dimensional gel electrophoresis revealed that CAR can form a homodimer in a configuration in which the PP2A/RACK1 binding site is buried within its dimer interface. Epidermal growth factor (EGF) was found to stimulate CAR homodimerization, thus constraining CAR in its inactive form. The agonistic ligand CITCO binds directly to the CAR homodimer and dissociates phosphorylated CAR into its monomers, exposing the PP2A/RACK1 binding site for dephosphorylation. Phenobarbital, which is not a CAR ligand, binds the EGF receptor, reversing the EGF signal to monomerize CAR for its indirect activation. Thus, the homodimer-monomer conversion is the underlying molecular mechanism that regulates CAR activation, by placing phosphorylated threonine 38 as the common target for both direct and indirect activation of CAR. PMID:28265001

  19. Activation of Stat1 by mutant fibroblast growth-factor receptor in thanatophoric dysplasia type II dwarfism.

    PubMed

    Su, W C; Kitagawa, M; Xue, N; Xie, B; Garofalo, S; Cho, J; Deng, C; Horton, W A; Fu, X Y

    1997-03-20

    The achondroplasia class of chondrodysplasias comprises the most common genetic forms of dwarfism in humans and includes achondroplasia, hypochondroplasia and thanatophoric dysplasia types I and II (TDI and TDII), which are caused by different mutations in a fibroblast growth-factor receptor FGFR3 (ref. 1). The molecular mechanism and the mediators of these FGFR3-related growth abnormalities are not known. Here we show that mutant TDII FGFR3 has a constitutive tyrosine kinase activity which can specifically activate the transcription factor Stat1 (for signal transducer and activator of transcription). Furthermore, expression of TDII FGFR3 induced nuclear translocation of Stat1, expression of the cell-cycle inhibitor p21(WAF1/CIP1), and growth arrest of the cell. Thus, TDII FGFR3 may use Stat1 as a mediator of growth retardation in bone development. Consistent with this, Stat1 activation and increased p21(WAF1/CIP1) expression was found in the cartilage cells from the TDII fetus, but not in those from the normal fetus. Thus, abnormal STAT activation and p21(WAF1/CIP1) expression by the TDII mutant receptor may be responsible for this FGFR3-related bone disease.

  20. Novel antipsychotic agents with 5-HT(1A) agonist properties: role of 5-HT(1A) receptor activation in attenuation of catalepsy induction in rats.

    PubMed

    Kleven, Mark S; Barret-Grévoz, Catherine; Bruins Slot, Liesbeth; Newman-Tancredi, Adrian

    2005-08-01

    Compounds possessing 5-HT(1A) agonist properties attenuate catalepsy induced by D(2) receptor blockade. Here we examined the role of 5-HT(1A) receptor agonism in the reduced cataleptogenic potential of several novel antipsychotic agents in the crossed leg position (CLP) and the bar catalepsy tests in rats. When administered alone, ziprasidone produced marked catalepsy, whereas aripiprazole, bifeprunox, SLV313, SSR181507 and sarizotan did not. However, when 5-HT(1A) receptors were blocked with the selective antagonist, WAY100635 (0.63 mg/kg, SC), robust cataleptogenic properties of SLV313, bifeprunox and sarizotan were unmasked and the catalepsy induced by ziprasidone was accentuated. In contrast, only modest catalepsy was induced by aripiprazole and SSR181507, even following a higher dose of WAY100635 (2.5 mg/kg). This suggests that these compounds possess other anti-cataleptic properties, such as partial agonism at dopamine D(2) receptors. The capacity to reverse neuroleptic-induced catalepsy was investigated in interaction studies with haloperidol (2.5 mg/kg, SC). Whereas ziprasidone and aripiprazole did not markedly reduce the effects of haloperidol, SLV313 and sarizotan attenuated CLP catalepsy. In contrast, SSR181507 and bifeprunox potently inhibited both CLP and bar catalepsy. Taken together, these data show that 5-HT(1A) receptor activation reduces the cataleptogenic potential of novel antipsychotic agents but indicate marked diversity in the contribution of 5-HT(1A) and/or other mechanisms to the profiles of the drugs.

  1. Signal transduction and functional selectivity of F15599, a preferential post-synaptic 5-HT1A receptor agonist

    PubMed Central

    Newman-Tancredi, A; Martel, J-C; Assié, M-B; Buritova, J; Lauressergues, E; Cosi, C; Heusler, P; Slot, L Bruins; Colpaert, FC; Vacher, B; Cussac, D

    2009-01-01

    Background and purpose: Activation of post-synaptic 5-HT1A receptors may provide enhanced therapy against depression. We describe the signal transduction profile of F15599, a novel 5-HT1A receptor agonist. Experimental approach: F15599 was compared with a chemical congener, F13714, and with (+)8-OH-DPAT in models of signal transduction in vitro and ex vivo. Key results: F15599 was highly selective for 5-HT1A receptors in binding experiments and in [35S]-GTPγS autoradiography of rat brain, where F15599 increased labelling in regions expressing 5-HT1A receptors. In cell lines expressing h5-HT1A receptors, F15599 more potently stimulated extracellular signal-regulated kinase (ERK1/2) phosphorylation, compared with G-protein activation, internalization of h5-HT1A receptors or inhibition of cAMP accumulation. F13714, (+)8-OH-DPAT and 5-HT displayed a different rank order of potency for these responses. F15599 stimulated [35S]-GTPγS binding more potently in frontal cortex than raphe. F15599, unlike 5-HT, more potently and efficaciously stimulated Gαi than Gαo activation. In rat prefrontal cortex (a region expressing post-synaptic 5-HT1A receptors), F15599 potently activated ERK1/2 phosphorylation and strongly induced c-fos mRNA expression. In contrast, in raphe regions (expressing pre-synaptic 5-HT1A receptors) F15599 only weakly or did not induce c-fos mRNA expression. Finally, despite its more modest affinity in vitro, F15599 bound to 5-HT1A receptors in vivo almost as potently as F13714. Conclusions and implications: F15599 showed a distinctive activation profiles for 5-HT1A receptor-mediated signalling pathways, unlike those of reference agonists and consistent with functional selectivity at 5-HT1A receptors. In rat, F15599 potently activated signalling in prefrontal cortex, a feature likely to underlie its beneficial effects in models of depression and cognition. PMID:19154445

  2. Evidence against dopamine D1/D2 receptor heteromers

    PubMed Central

    Frederick, Aliya L.; Yano, Hideaki; Trifilieff, Pierre; Vishwasrao, Harshad D.; Biezonski, Dominik; Mészáros, József; Sibley, David R.; Kellendonk, Christoph; Sonntag, Kai C.; Graham, Devon L.; Colbran, Roger J.; Stanwood, Gregg D.; Javitch, Jonathan A.

    2014-01-01

    Hetero-oligomers of G-protein-coupled receptors have become the subject of intense investigation because their purported potential to manifest signaling and pharmacological properties that differ from the component receptors makes them highly attractive for the development of more selective pharmacological treatments. In particular, dopamine D1 and D2 receptors have been proposed to form hetero-oligomers that couple to Gαq proteins, and SKF83959 has been proposed to act as a biased agonist that selectively engages these receptor complexes to activate Gαq and thus phospholipase C. D1/D2 heteromers have been proposed as relevant to the pathophysiology and treatment of depression and schizophrenia. We used in vitro bioluminescence resonance energy transfer (BRET), ex vivo analyses of receptor localization and proximity in brain slices, and behavioral assays in mice to characterize signaling from these putative dimers/oligomers. We were unable to detect Gαq or Gα11 protein coupling to homomers or heteromers of D1 or D2 receptors using a variety of biosensors. SKF83959-induced locomotor and grooming behaviors were eliminated in D1 receptor knockout mice, verifying a key role for D1-like receptor activation. In contrast, SKF83959-induced motor responses were intact in D2 receptor and Gαq knockout mice, as well as in knock-in mice expressing a mutant Ala286-CaMKIIα, that cannot autophosphorylate to become active. Moreover, we found that in the shell of the nucleus accumbens, even in neurons in which D1 and D2 receptor promoters are both active, the receptor proteins are segregated and do not form complexes. These data are not compatible with SKF83959 signaling through Gαq or through a D1–D2 heteromer and challenge the existence of such a signaling complex in the adult animals that we used for our studies. PMID:25560761

  3. Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

    PubMed

    Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael

    2015-12-01

    Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

  4. Modulating Estrogen Receptor-related ReceptorActivity Inhibits Cell Proliferation*

    PubMed Central

    Bianco, Stéphanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc

    2009-01-01

    High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERRα-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21waf/cip1 at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice. PMID:19546226

  5. The role of 5-HT(1A) receptors in learning and memory.

    PubMed

    Ogren, Sven Ove; Eriksson, Therese M; Elvander-Tottie, Elin; D'Addario, Claudio; Ekström, Joanna C; Svenningsson, Per; Meister, Björn; Kehr, Jan; Stiedl, Oliver

    2008-12-16

    The ascending serotonin (5-HT) neurons innervate the cerebral cortex, hippocampus, septum and amygdala, all representing brain regions associated with various domains of cognition. The 5-HT innervation is diffuse and extensively arborized with few synaptic contacts, which indicates that 5-HT can affect a large number of neurons in a paracrine mode. Serotonin signaling is mediated by 14 receptor subtypes with different functional and transductional properties. The 5-HT(1A) subtype is of particular interest, since it is one of the main mediators of the action of 5-HT. Moreover, the 5-HT(1A) receptor regulates the activity of 5-HT neurons via autoreceptors, and it regulates the function of several neurotransmitter systems via postsynaptic receptors (heteroreceptors). This review assesses the pharmacological and genetic evidence that implicates the 5-HT(1A) receptor in learning and memory. The 5-HT(1A) receptors are in the position to influence the activity of glutamatergic, cholinergic and possibly GABAergic neurons in the cerebral cortex, hippocampus and in the septohippocampal projection, thereby affecting declarative and non-declarative memory functions. Moreover, the 5-HT(1A) receptor regulates several transduction mechanisms such as kinases and immediate early genes implicated in memory formation. Based on studies in rodents the stimulation of 5-HT(1A) receptors generally produces learning impairments by interfering with memory-encoding mechanisms. In contrast, antagonists of 5-HT(1A) receptors facilitate certain types of memory by enhancing hippocampal/cortical cholinergic and/or glutamatergic neurotransmission. Some data also support a potential role for the 5-HT(1A) receptor in memory consolidation. Available results also implicate the 5-HT(1A) receptor in the retrieval of aversive or emotional memories, supporting an involvement in reconsolidation. The contribution of 5-HT(1A) receptors in cognitive impairments in various psychiatric disorders is still

  6. Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk Is Essential for Toll-like Receptor Activation and Cellular Signaling*

    PubMed Central

    Abdulkhalek, Samar; Amith, Schammim Ray; Franchuk, Susan L.; Jayanth, Preethi; Guo, Merry; Finlay, Trisha; Gilmour, Alanna; Guzzo, Christina; Gee, Katrina; Beyaert, Rudi; Szewczuk, Myron R.

    2011-01-01

    The signaling pathways of mammalian Toll-like receptors (TLRs) are well characterized, but the precise mechanism(s) by which TLRs are activated upon ligand binding remains poorly defined. Recently, we reported a novel membrane sialidase-controlling mechanism that depends on ligand binding to its TLR to induce mammalian neuraminidase-1 (Neu1) activity, to influence receptor desialylation, and subsequently to induce TLR receptor activation and the production of nitric oxide and proinflammatory cytokines in dendritic and macrophage cells. The α-2,3-sialyl residue of TLR was identified as the specific target for hydrolysis by Neu1. Here, we report a membrane signaling paradigm initiated by endotoxin lipopolysaccharide (LPS) binding to TLR4 to potentiate G protein-coupled receptor (GPCR) signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP9) activation to induce Neu1. Central to this process is that a Neu1-MMP9 complex is bound to TLR4 on the cell surface of naive macrophage cells. Specific inhibition of MMP9 and GPCR Gαi-signaling proteins blocks LPS-induced Neu1 activity and NFκB activation. Silencing MMP9 mRNA using lentivirus MMP9 shRNA transduction or siRNA transfection of macrophage cells and MMP9 knock-out primary macrophage cells significantly reduced Neu1 activity and NFκB activation associated with LPS-treated cells. These findings uncover a molecular organizational signaling platform of a novel Neu1 and MMP9 cross-talk in alliance with TLR4 on the cell surface that is essential for ligand activation of TLRs and subsequent cellular signaling. PMID:21873432

  7. Statins Increase Plasminogen Activator Inhibitor Type 1 Gene Transcription through a Pregnane X Receptor Regulated Element

    PubMed Central

    Stanley, Frederick M.; Linder, Kathryn M.; Cardozo, Timothy J.

    2015-01-01

    Plasminogen activator inhibitor type 1 (PAI-1) is a multifunctional protein that has important roles in inflammation and wound healing. Its aberrant regulation may contribute to many disease processes such as heart disease. The PAI-1 promoter is responsive to multiple inputs including cytokines, growth factors, steroids and oxidative stress. The statin drugs, atorvastatin, mevastatin and rosuvastatin, increased basal and stimulated expression of the PAI-1 promoter 3-fold. A statin-responsive, nuclear hormone response element was previously identified in the PAI-1 promoter, but it was incompletely characterized. We characterized this direct repeat (DR) of AGGTCA with a 3-nucleotide spacer at -269/-255 using deletion and directed mutagenesis. Deletion or mutation of this element increased basal transcription from the promoter suggesting that it repressed PAI-1 transcription in the unliganded state. The half-site spacing and the ligand specificity suggested that this might be a pregnane X receptor (PXR) responsive element. Computational molecular docking showed that atorvastatin, mevastatin and rosuvastatin were structurally compatible with the PXR ligand-binding pocket in its agonist conformation. Experiments with Gal4 DNA binding domain fusion proteins showed that Gal4-PXR was activated by statins while other DR + 3 binding nuclear receptor fusions were not. Overexpression of PXR further enhanced PAI-1 transcription in response to statins. Finally, ChIP experiments using Halo-tagged PXR and RXR demonstrated that both components of the PXR-RXR heterodimer bound to this region of the PAI-1 promoter. PMID:26379245

  8. Compensatory insulin receptor (IR) activation on inhibition of insulin-like growth factor-1 receptor (IGF-1R): rationale for cotargeting IGF-1R and IR in cancer.

    PubMed

    Buck, Elizabeth; Gokhale, Prafulla C; Koujak, Susan; Brown, Eric; Eyzaguirre, Alexandra; Tao, Nianjun; Rosenfeld-Franklin, Maryland; Lerner, Lorena; Chiu, M Isabel; Wild, Robert; Epstein, David; Pachter, Jonathan A; Miglarese, Mark R

    2010-10-01

    Insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and critical activator of the phosphatidylinositol 3-kinase-AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis. These observations have spurred anticancer drug discovery and development efforts for both biological and small-molecule IGF-1R inhibitors. The ability for one RTK to compensate for another to maintain tumor cell viability is emerging as a common resistance mechanism to antitumor agents targeting individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), we asked whether IR is tumorigenic and whether IR-AKT signaling contributes to resistance to IGF-1R inhibition. Both IGF-1R and IR(A) are tumorigenic in a mouse mammary tumor model. In human tumor cells coexpressing IGF-1R and IR, bidirectional cross talk was observed following either knockdown of IR expression or treatment with a selective anti-IGF-1R antibody, MAB391. MAB391 treatment resulted in a compensatory increase in phospho-IR, which was associated with resistance to inhibition of IRS1 and AKT. In contrast, treatment with OSI-906, a small-molecule dual inhibitor of IGF-1R/IR, resulted in enhanced reduction in phospho-IRS1/phospho-AKT relative to MAB391. Insulin or IGF-2 activated the IR-AKT pathway and decreased sensitivity to MAB391 but not to OSI-906. In tumor cells with an autocrine IGF-2 loop, both OSI-906 and an anti-IGF-2 antibody reduced phospho-IR/phospho-AKT, whereas MAB391 was ineffective. Finally, OSI-906 showed superior efficacy compared with MAB391 in human tumor xenograft models in which both IGF-1R and IR were phosphorylated. Collectively, these data indicate that cotargeting IGF-1R and IR may provide superior antitumor efficacy compared with targeting IGF-1R alone.

  9. A chimeric receptor of the insulin-like growth factor receptor type 1 (IGFR1) and a single chain antibody specific to myelin oligodendrocyte glycoprotein activates the IGF1R signalling cascade in CG4 oligodendrocyte progenitors.

    PubMed

    Annenkov, Alexander; Rigby, Anne; Amor, Sandra; Zhou, Dun; Yousaf, Nasim; Hemmer, Bernhard; Chernajovsky, Yuti

    2011-08-01

    In order to generate neural stem cells with increased ability to survive after transplantation in brain parenchyma we developed a chimeric receptor (ChR) that binds to myelin oligodendrocyte glycoprotein (MOG) via its ectodomain and activates the insulin-like growth factor receptor type 1 ‎‎(IGF1R) signalling cascade. Activation of this pro-survival pathway in response to ligand broadly available in the brain might increase neuroregenerative potential of transplanted precursors. The ChR was produced by fusing a MOG-specific single ‎chain antibody with the extracellular boundary of the IGF1R transmembrane segment. The ChR is expressed on the cellular surface, predominantly as a monomer, and is not N-glycosylated. To show MOG-dependent functionality of the ChR, neuroblastoma cells B104 expressing this ChR were stimulated with monolayers of cells expressing recombinant MOG. The ChR undergoes MOG-dependent tyrosine phosphorylation and homodimerisation. It promotes insulin and IGF-independent growth of the oligodendrocyte progenitor cell line CG4. The proposed mode of the ChR activation is by MOG-induced dimerisation which promotes kinase domain transphosphorylation, by-passing the requirement of conformation changes known to be important for IGF1R activation. Another ChR, which contains a segment of the β-chain ectodomain, was produced in an attempt to recapitulate some of these conformational changes, but proved non-functional. 2011 Elsevier B.V. All rights reserved.

  10. Ethanol exposure in early adolescence inhibits intrinsic neuronal plasticity via sigma-1 receptor activation in hippocampal CA1 neurons

    PubMed Central

    Sabeti, Jilla

    2011-01-01

    Background We demonstrated previously that rats exposed to chronic intermittent ethanol (CIE) vapors in early adolescence show increased magnitudes of long-term potentiation (LTP) of excitatory transmission when recorded at dendritic synapses in hippocampus. Large amplitude LTP following CIE exposure is mediated by sigma-1 receptors; however, not yet addressed is the role of sigma-1 receptors in modulating the intrinsic properties of neurons to alter their action potential firing during LTP. Methods Activity-induced plasticity of spike firing was investigated using rat hippocampal slice recordings to measure changes in both field excitatory postsynaptic potentials (fEPSPs) and population spikes (pop. spikes) concomitantly at dendritic inputs and soma of CA1 pyramidal neurons, respectively. Results We observed unique modifications in plasticity of action potential firing in hippocampal slices from CIE exposed adolescent rats, where the induction of large amplitude LTP by 100 Hz stimulations was accompanied by reduced CA1 neuronal excitability—reflected as decreased pop. spike efficacy and impaired activity-induced fEPSP-to-spike (E-S) potentiation. By contrast, LTP induction in ethanol-naïve control slices resulted in increased spike efficacy and robust E-S potentiation. E-S potentiation impairments emerged at 24 hr after CIE treatment cessation, but not before the alcohol withdrawal period, and were restored with bath-application of the sigma-1 receptor selective antagonist BD1047, but not the NMDA receptor antagonist D-AP5. Further evidence revealed a significantly shortened somatic fEPSP time course in adolescent CIE-withdrawn hippocampal slices during LTP; however, paired-pulse data show no apparent correspondence between E-S dissociation and altered recurrent feedback inhibition. Conclusions Results here suggest that acute withdrawal from adolescent CIE exposure triggers sigma-1 receptors that act to depress the efficacy of excitatory inputs in triggering

  11. Substance P and central respiratory activity: a comparative in vitro study in NK1 receptor knockout and wild-type mice.

    PubMed

    Ptak, K; Hunt, S P; Monteau, R

    2000-07-01

    Neurokinin-1 receptors (NK1) are present within the respiratory medullary network and in the phrenic nucleus, which controls the diaphragm. We compared the efficacy of substance P (SP) at inducing changes in respiratory frequency or the amplitude of the respiratory motor output between NK1 knockout (NK1-/-) and wild-type mice, using the in vitro brainstem-spinal cord preparation. The in vitro respiratory frequency, as well as the variability of the rhythm and the amplitude of the motor output were similar in both lines. In wild-type mice, application of exogenous SP induced either an increase in respiratory frequency (superfusion of the medulla) or an increase of the inspiratory motor output, as defined by the integral of C4 cervical ventral root activity (superfusion of the spinal cord). These two effects were not apparent in NK1-/- mice. In conclusion, NK1 receptors mediate the respiratory responses to SP but the lack of NK1 receptors in newborn NK1-/- mice does not change the respiratory activity.

  12. Activation of 5-HT1A receptors in the rat dorsomedial hypothalamus inhibits stress-induced activation of the hypothalamic-pituitary-adrenal axis.

    PubMed

    Stamper, Christopher E; Hassell, James E; Kapitz, Adam J; Renner, Kenneth J; Orchinik, Miles; Lowry, Christopher A

    2017-03-01

    Acute activation of the hypothalamic-pituitary-adrenal (HPA) axis, leading to the release of corticosteroid hormones into the circulation, is an adaptive response to perceived threats. Persistent activation of the HPA axis can lead to impaired physiological or behavioral function with maladaptive consequences. Thus, efficient control and termination of stress responses is essential for well-being. However, inhibitory control mechanisms governing the HPA axis are poorly understood. Previous studies suggest that serotonergic systems, acting within the medial hypothalamus, play an important role in inhibitory control of stress-induced HPA axis activity. To test this hypothesis, we surgically implanted chronic jugular cannulae in adult male rats and conducted bilateral microinjection of vehicle or the 5-HT 1A receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT; 8 nmol, 0.2 μL, 0.1 μL/min, per side) into the dorsomedial hypothalamus (DMH) immediately prior to a 40 min period of restraint stress. Repeated blood sampling was conducted using an automated blood sampling system and plasma corticosterone concentrations were determined using enzyme-linked immunosorbent assay. Bilateral intra-DMH microinjections of 8-OH-DPAT suppressed stress-induced increases in plasma corticosterone within 10 min of the onset of handling prior to restraint and, as measured by area-under-the-curve analysis of plasma corticosterone concentrations, during the 40 min period of restraint. These data support an inhibitory role for serotonergic systems, acting within the DMH, on stress-induced activation of the HPA axis. Lay summary: Inhibitory control of the hypothalamic-pituitary-adrenal (HPA) stress hormone response is important for well-being. One neurochemical implicated in inhibitory control of the HPA axis is serotonin. In this study we show that activation of serotonin receptors, specifically inhibitory 5-HT 1A receptors in the dorsomedial

  13. Chronic administration of aripiprazole activates GSK3β-dependent signalling pathways, and up-regulates GABAA receptor expression and CREB1 activity in rats.

    PubMed

    Pan, Bo; Huang, Xu-Feng; Deng, Chao

    2016-07-20

    Aripiprazole is a D2-like receptor (D2R) partial agonist with a favourable clinical profile. Previous investigations indicated that acute and short-term administration of aripiprazole had effects on PKA activity, GSK3β-dependent pathways, GABAA receptors, NMDA receptor and CREB1 in the brain. Since antipsychotics are used chronically in clinics, the present study investigated the long-term effects of chronic oral aripiprazole treatment on these cellular signalling pathways, in comparison with haloperidol (a D2R antagonist) and bifeprunox (a potent D2R partial agonist). We found that the Akt-GSK3β pathway was activated by aripiprazole and bifeprunox in the prefrontal cortex; NMDA NR2A levels were reduced by aripiprazole and haloperidol. In the nucleus accumbens, all three drugs increased Akt-GSK3β signalling; in addition, both aripiprazole and haloperidol, but not bifeprunox, increased the expression of Dvl-3, β-catenin and GABAA receptors, NMDA receptor subunits, as well as CREB1 phosphorylation levels. The results suggest that chronic oral administration of aripiprazole affects schizophrenia-related cellular signalling pathways and markers (including Akt-GSK3β signalling, Dvl-GSK3β-β-catenin signalling, GABAA receptor, NMDA receptor and CREB1) in a brain-region-dependent manner; the selective effects of aripiprazole on these signalling pathways might be associated with its unique clinical effects.

  14. Chronic administration of aripiprazole activates GSK3β-dependent signalling pathways, and up-regulates GABAA receptor expression and CREB1 activity in rats

    PubMed Central

    Pan, Bo; Huang, Xu-Feng; Deng, Chao

    2016-01-01

    Aripiprazole is a D2-like receptor (D2R) partial agonist with a favourable clinical profile. Previous investigations indicated that acute and short-term administration of aripiprazole had effects on PKA activity, GSK3β-dependent pathways, GABAA receptors, NMDA receptor and CREB1 in the brain. Since antipsychotics are used chronically in clinics, the present study investigated the long-term effects of chronic oral aripiprazole treatment on these cellular signalling pathways, in comparison with haloperidol (a D2R antagonist) and bifeprunox (a potent D2R partial agonist). We found that the Akt-GSK3β pathway was activated by aripiprazole and bifeprunox in the prefrontal cortex; NMDA NR2A levels were reduced by aripiprazole and haloperidol. In the nucleus accumbens, all three drugs increased Akt-GSK3β signalling; in addition, both aripiprazole and haloperidol, but not bifeprunox, increased the expression of Dvl-3, β-catenin and GABAA receptors, NMDA receptor subunits, as well as CREB1 phosphorylation levels. The results suggest that chronic oral administration of aripiprazole affects schizophrenia-related cellular signalling pathways and markers (including Akt-GSK3β signalling, Dvl-GSK3β-β-catenin signalling, GABAA receptor, NMDA receptor and CREB1) in a brain-region-dependent manner; the selective effects of aripiprazole on these signalling pathways might be associated with its unique clinical effects. PMID:27435909

  15. Dopamine acting at D1-like, D2-like and α1-adrenergic receptors differentially modulates theta and gamma oscillatory activity in primary motor cortex.

    PubMed

    Özkan, Mazhar; Johnson, Nicholas W; Sehirli, Umit S; Woodhall, Gavin L; Stanford, Ian M

    2017-01-01

    The loss of dopamine (DA) in Parkinson's is accompanied by the emergence of exaggerated theta and beta frequency neuronal oscillatory activity in the primary motor cortex (M1) and basal ganglia. DA replacement therapy or deep brain stimulation reduces the power of these oscillations and this is coincident with an improvement in motor performance implying a causal relationship. Here we provide in vitro evidence for the differential modulation of theta and gamma activity in M1 by DA acting at receptors exhibiting conventional and non-conventional DA pharmacology. Recording local field potentials in deep layer V of rat M1, co-application of carbachol (CCh, 5 μM) and kainic acid (KA, 150 nM) elicited simultaneous oscillations at a frequency of 6.49 ± 0.18 Hz (theta, n = 84) and 34.97 ± 0.39 Hz (gamma, n = 84). Bath application of DA resulted in a decrease in gamma power with no change in theta power. However, application of either the D1-like receptor agonist SKF38393 or the D2-like agonist quinpirole increased the power of both theta and gamma suggesting that the DA-mediated inhibition of oscillatory power is by action at other sites other than classical DA receptors. Application of amphetamine, which promotes endogenous amine neurotransmitter release, or the adrenergic α1-selective agonist phenylephrine mimicked the action of DA and reduced gamma power, a result unaffected by prior co-application of D1 and D2 receptor antagonists SCH23390 and sulpiride. Finally, application of the α1-adrenergic receptor antagonist prazosin blocked the action of DA on gamma power suggestive of interaction between α1 and DA receptors. These results show that DA mediates complex actions acting at dopamine D1-like and D2-like receptors, α1 adrenergic receptors and possibly DA/α1 heteromultimeric receptors to differentially modulate theta and gamma activity in M1.

  16. Protein kinase D1 phosphorylates HDAC7 and induces its nuclear export after T-cell receptor activation.

    PubMed

    Parra, Maribel; Kasler, Herbert; McKinsey, Timothy A; Olson, Eric N; Verdin, Eric

    2005-04-08

    HDAC7, a class II histone deacetylase that is highly expressed in thymocytes, inhibits both transcription of the orphan steroid nuclear receptor Nur77 and induction of apoptosis in response to activation of the T-cell receptor (TCR). Here, we report that HDAC7 is exported to the cytoplasm by a calcium-independent signaling pathway after TCR activation. Protein kinase D1 (PKD1) was activated after TCR engagement, interacted with HDAC7, and phosphorylated three serines (Ser155, Ser318, and Ser448) at its N terminus, leading to its export from the nucleus. Mutation of Ser155, Ser318, and Ser448 blocked the nucleocytoplasmic shuttling of HDAC7 in response to TCR activation, as did overexpression of a kinase-inactive form of PKD1. Consistent with the regulatory role of HDAC7 in Nur77 expression, PKD1 activation led to the transcriptional activation of Nur77 via myocyte enhancer factor 2-binding sites in its promoter. In a mouse model of negative selection, PKD1 was activated during thymocyte activation. These observations indicate that PKD1 regulates the expression of Nur77 during thymocyte activation at least in part by phosphorylating HDAC7.

  17. Effects of selective activation of M1 and M4 muscarinic receptors on object recognition memory performance in rats.

    PubMed

    Galloway, Claire R; Lebois, Evan P; Shagarabi, Shezza L; Hernandez, Norma A; Manns, Joseph R

    2014-01-01

    Acetylcholine signaling through muscarinic receptors has been shown to benefit memory performance in some conditions, but pan-muscarinic activation also frequently leads to peripheral side effects. Drug therapies that selectively target M1 or M4 muscarinic receptors could potentially improve memory while minimizing side effects mediated by the other muscarinic receptor subtypes. The ability of three recently developed drugs that selectively activate M1 or M4 receptors to improve recognition memory was tested by giving Long-Evans rats subcutaneous injections of three different doses of the M1 agonist VU0364572, the M1 positive allosteric modulator BQCA or the M4 positive allosteric modulator VU0152100 before performing an object recognition memory task. VU0364572 at 0.1 mg/kg, BQCA at 1.0 mg/kg and VU0152100 at 3.0 and 30.0 mg/kg improved the memory performance of rats that performed poorly at baseline, yet the improvements in memory performance were the most statistically robust for VU0152100 at 3.0 mg/kg. The results suggested that selective M1 and M4 receptor activation each improved memory but that the likelihood of obtaining behavioral efficacy at a given dose might vary between subjects even in healthy groups depending on baseline performance. These results also highlighted the potential of drug therapies that selectively target M1 or M4 receptors to improve memory performance in individuals with impaired memory.

  18. Identification and characterization of a receptor for tissue ferritin on activated rat lipocytes.

    PubMed Central

    Ramm, G A; Britton, R S; O'Neill, R; Bacon, B R

    1994-01-01

    Hepatic iron overload causes lipocyte activation with resultant fibrogenesis. This study examines whether rat lipocytes express ferritin receptors, which could be involved in paracellular iron movement and in cellular regulation. Lipocytes from normal rat liver were cultured on plastic and incubated with 125I-labeled rat liver ferritin (RLF) +/- a 100-fold excess of either unlabeled RLF or human heart ferritin, human liver ferritin, human recombinant H-ferritin, a mutant human recombinant L-ferritin, or a variety of nonspecific proteins. Specific binding sites for ferritin were demonstrated by displacement of 125I-RLF by RLF (64.5 +/- 4.3%) and by other ferritins (55-60%), but not by recombinant L-ferritin. Scatchard analysis demonstrated a single class of binding sites with a Kd of 5.1 +/- 2.9 x 10(-10) M, maximum binding capacity of 4.7 +/- 1.3 x 10(-12) M, and 5,000-10,000 receptor sites/cell. Ferritin receptor expression was observed only in activated lipocytes. Internalization of RLF was observed within 15 min using FITC-RLF and confocal microscopy. This study demonstrates that (a) activated lipocytes express a specific high affinity ferritin receptor; (b) the binding appears to be dependent on the H-ferritin subunit; and (c) lipocytes internalize ferritin. Expression of ferritin receptors in activated lipocytes suggests that the receptor may either be involved in the activation cascade or may be a marker of activation. Images PMID:8040296

  19. TSH/IGF-1 Receptor Cross-Talk Rapidly Activates Extracellular Signal-Regulated Kinases in Multiple Cell Types.

    PubMed

    Krieger, Christine C; Perry, Joseph D; Morgan, Sarah J; Kahaly, George J; Gershengorn, Marvin C

    2017-10-01

    We previously showed that thyrotropin (TSH)/insulinlike growth factor (IGF)-1 receptor cross-talk appears to be involved in Graves' orbitopathy (GO) pathogenesis and upregulation of thyroid-specific genes in human thyrocytes. In orbital fibroblasts from GO patients, coadministration of TSH and IGF-1 induces synergistic increases in hyaluronan secretion. In human thyrocytes, TSH plus IGF-1 synergistically increased expression of the sodium-iodide symporter that appeared to involve ERK1/2 activation. However, the details of ERK1/2 activation were not known, nor was whether ERK1/2 was involved in this synergism in other cell types. Using primary cultures of GO fibroblasts (GOFs) and human thyrocytes, as well as human embryonic kidney (HEK) 293 cells overexpressing TSH receptors (HEK-TSHRs), we show that simultaneous activation of TSHRs and IGF-1 receptors (IGF-1Rs) causes rapid, synergistic phosphorylation/activation of ERK1 and ERK2 in all three cell types. This effect is partially inhibited by pertussis toxin, an inhibitor of TSHR coupling to Gi/Go proteins. In support of a role for Gi/Go proteins in ERK1/2 phosphorylation, we found that knockdown of Gi(1-3) and Go in HEK-TSHRs inhibited ERK1/2 phosphorylation stimulated by TSH and TSH plus IGF-1. These data demonstrate that the synergistic effects of TSH plus IGF-1 occur early in the TSHR signaling cascade and further support the idea that TSHR/IGF-1R cross-talk is an important mechanism for regulation of human GOFs and thyrocytes.

  20. Glutamate mediates platelet activation through the AMPA receptor

    PubMed Central

    Morrell, Craig N.; Sun, Henry; Ikeda, Masahiro; Beique, Jean-Claude; Swaim, Anne Marie; Mason, Emily; Martin, Tanika V.; Thompson, Laura E.; Gozen, Oguz; Ampagoomian, David; Sprengel, Rolf; Rothstein, Jeffrey; Faraday, Nauder; Huganir, Richard; Lowenstein, Charles J.

    2008-01-01

    Glutamate is an excitatory neurotransmitter that binds to the kainate receptor, the N-methyl-D-aspartate (NMDA) receptor, and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR). Each receptor was first characterized and cloned in the central nervous system (CNS). Glutamate is also present in the periphery, and glutamate receptors have been identified in nonneuronal tissues, including bone, heart, kidney, pancreas, and platelets. Platelets play a central role in normal thrombosis and hemostasis, as well as contributing greatly to diseases such as stroke and myocardial infarction. Despite the presence of glutamate in platelet granules, the role of glutamate during hemostasis is unknown. We now show that activated platelets release glutamate, that platelets express AMPAR subunits, and that glutamate increases agonist-induced platelet activation. Furthermore, we demonstrate that glutamate binding to the AMPAR increases intracellular sodium concentration and depolarizes platelets, which are important steps in platelet activation. In contrast, platelets treated with the AMPAR antagonist CNQX or platelets derived from GluR1 knockout mice are resistant to AMPA effects. Importantly, mice lacking GluR1 have a prolonged time to thrombosis in vivo. Our data identify glutamate as a regulator of platelet activation, and suggest that the AMPA receptor is a novel antithrombotic target. PMID:18283118

  1. Methylphenidate enhances NMDA-receptor response in medial prefrontal cortex via sigma-1 receptor: a novel mechanism for methylphenidate action.

    PubMed

    Zhang, Chun-Lei; Feng, Ze-Jun; Liu, Yue; Ji, Xiao-Hua; Peng, Ji-Yun; Zhang, Xue-Han; Zhen, Xue-Chu; Li, Bao-Ming

    2012-01-01

    Methylphenidate (MPH), commercially called Ritalin or Concerta, has been widely used as a drug for Attention Deficit Hyperactivity Disorder (ADHD). Noteworthily, growing numbers of young people using prescribed MPH improperly for pleasurable enhancement, take high risk of addiction. Thus, understanding the mechanism underlying high level of MPH action in the brain becomes an important goal nowadays. As a blocker of catecholamine transporters, its therapeutic effect is explained as being due to proper modulation of D1 and α2A receptor. Here we showed that higher dose of MPH facilitates NMDA-receptor mediated synaptic transmission via a catecholamine-independent mechanism, in layer V∼VI pyramidal cells of the rat medial prefrontal cortex (PFC). To indicate its postsynaptic action, we next found that MPH facilitates NMDA-induced current and such facilitation could be blocked by σ1 but not D1/5 and α2 receptor antagonists. And this MPH eliciting enhancement of NMDA-receptor activity involves PLC, PKC and IP3 receptor mediated intracellular Ca(2+) increase, but does not require PKA and extracellular Ca(2+) influx. Our additional pharmacological studies confirmed that higher dose of MPH increases locomotor activity via interacting with σ1 receptor. Together, the present study demonstrates for the first time that MPH facilitates NMDA-receptor mediated synaptic transmission via σ1 receptor, and such facilitation requires PLC/IP3/PKC signaling pathway. This novel mechanism possibly explains the underlying mechanism for MPH induced addictive potential and other psychiatric side effects.

  2. Methylphenidate Enhances NMDA-Receptor Response in Medial Prefrontal Cortex via Sigma-1 Receptor: A Novel Mechanism for Methylphenidate Action

    PubMed Central

    Liu, Yue; Ji, Xiao-Hua; Peng, Ji-Yun; Zhang, Xue-Han; Zhen, Xue-Chu; Li, Bao-Ming

    2012-01-01

    Methylphenidate (MPH), commercially called Ritalin or Concerta, has been widely used as a drug for Attention Deficit Hyperactivity Disorder (ADHD). Noteworthily, growing numbers of young people using prescribed MPH improperly for pleasurable enhancement, take high risk of addiction. Thus, understanding the mechanism underlying high level of MPH action in the brain becomes an important goal nowadays. As a blocker of catecholamine transporters, its therapeutic effect is explained as being due to proper modulation of D1 and α2A receptor. Here we showed that higher dose of MPH facilitates NMDA-receptor mediated synaptic transmission via a catecholamine-independent mechanism, in layer V∼VI pyramidal cells of the rat medial prefrontal cortex (PFC). To indicate its postsynaptic action, we next found that MPH facilitates NMDA-induced current and such facilitation could be blocked by σ1 but not D1/5 and α2 receptor antagonists. And this MPH eliciting enhancement of NMDA-receptor activity involves PLC, PKC and IP3 receptor mediated intracellular Ca2+ increase, but does not require PKA and extracellular Ca2+ influx. Our additional pharmacological studies confirmed that higher dose of MPH increases locomotor activity via interacting with σ1 receptor. Together, the present study demonstrates for the first time that MPH facilitates NMDA-receptor mediated synaptic transmission via σ1 receptor, and such facilitation requires PLC/IP3/PKC signaling pathway. This novel mechanism possibly explains the underlying mechanism for MPH induced addictive potential and other psychiatric side effects. PMID:23284812

  3. [Coactivators in energy metabolism: peroxisome proliferator-activated receptor-gamma coactivator 1 family].

    PubMed

    Wang, Rui; Chang, Yong-sheng; Fang, Fu-de

    2009-12-01

    Peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1) family is highly expressed in tissues with high energy metabolism. They coactivate transcription factors in regulating genes engaged in processes such as gluconeogenesis, adipose beta-oxydation, lipoprotein synthesis and secretion, mitochondrial biogenesis, and oxidative metabolism. Protein conformation studies demonstrated that they lack DNA binding domains and act as coactivators through physical interaction with transcription factors. PGC1 activity is regulated at transcription level or by multiple covalent chemical modifications such as phosphorylation, methylation and acetylation/deacetylation. Abnormal expression of PGC1 coactivators usually is closely correlated with diseases such as diabetes, obesity, hyperglycemia, hyperlipemia, and arterial and brain neuron necrosis diseases.

  4. The vanilloid receptor TRPV1 is tonically activated in vivo and involved in body temperature regulation.

    PubMed

    Gavva, Narender R; Bannon, Anthony W; Surapaneni, Sekhar; Hovland, David N; Lehto, Sonya G; Gore, Anu; Juan, Todd; Deng, Hong; Han, Bora; Klionsky, Lana; Kuang, Rongzhen; Le, April; Tamir, Rami; Wang, Jue; Youngblood, Brad; Zhu, Dawn; Norman, Mark H; Magal, Ella; Treanor, James J S; Louis, Jean-Claude

    2007-03-28

    The vanilloid receptor TRPV1 (transient receptor potential vanilloid 1) is a cation channel that serves as a polymodal detector of pain-producing stimuli such as capsaicin, protons (pH <5.7), and heat. TRPV1 antagonists block pain behaviors in rodent models of inflammatory, neuropathic, and cancer pain, suggesting their utility as analgesics. Here, we report that TRPV1 antagonists representing various chemotypes cause an increase in body temperature (hyperthermia), identifying a potential issue for their clinical development. Peripheral restriction of antagonists did not eliminate hyperthermia, suggesting that the site of action is predominantly outside of the blood-brain barrier. Antagonists that are ineffective against proton activation also caused hyperthermia, indicating that blocking capsaicin and heat activation of TRPV1 is sufficient to produce hyperthermia. All TRPV1 antagonists evaluated here caused hyperthermia, suggesting that TRPV1 is tonically activated in vivo and that TRPV1 antagonism and hyperthermia are not separable. TRPV1 antagonists caused hyperthermia in multiple species (rats, dogs, and monkeys), demonstrating that TRPV1 function in thermoregulation is conserved from rodents to primates. Together, these results indicate that tonic TRPV1 activation regulates body temperature.

  5. A Novel Chemical Inhibitor of ABA Signaling Targets All ABA Receptors1

    PubMed Central

    Ye, Yajin; Liu, Xue; Liu, Hao; Li, Deqiang; Cao, Minjie; Chen, Haifeng; Zhu, Jian-kang

    2017-01-01

    Abscisic acid (ABA), the most important stress-induced phytohormone, regulates seed dormancy, germination, plant senescence, and the abiotic stress response. ABA signaling is repressed by group A type 2C protein phosphatases (PP2Cs), and then ABA binds to its receptor of the ACTIN RESISTANCE1 (PYR1), PYR1-LIKE (PYL), and REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) family, which, in turn, inhibits PP2Cs and activates downstream ABA signaling. The agonist/antagonist of ABA receptors have the potential to reveal the ABA signaling machinery and to become lead compounds for agrochemicals; however, until now, no broad-spectrum antagonists of ABA receptors blocking all PYR/PYL-PP2C interactions have been identified. Here, using chemical genetics screenings, we identified ABA ANTAGONIST1 (AA1), the first broad-spectrum antagonist of ABA receptors in Arabidopsis (Arabidopsis thaliana). Physiological analyses revealed that AA1 is sufficiently active to block ABA signaling. AA1 interfered with all the PYR/PYL-HAB1 interactions, and the diminished PYR/PYL-HAB1 interactions, in turn, restored the activity of HAB1. AA1 binds to all 13 members. Molecular dockings, the non-AA1-bound PYL2 variant, and competitive binding assays demonstrated that AA1 enters into the ligand-binding pocket of PYL2. Using AA1, we tested the genetic relationships of ABA receptors with other core components of ABA signaling, demonstrating that AA1 is a powerful tool with which to sidestep this genetic redundancy of PYR/PYLs. In addition, the application of AA1 delays leaf senescence. Thus, our study developed an efficient broad-spectrum antagonist of ABA receptors and demonstrated that plant senescence can be chemically controlled through AA1, with a simple and easy-to-synthesize structure, allowing its availability and utility as a chemical probe synthesized in large quantities, indicating its potential application in agriculture. PMID:28193765

  6. Hippocampal 5-HT1A Receptor and Spatial Learning and Memory

    PubMed Central

    Glikmann-Johnston, Yifat; Saling, Michael M.; Reutens, David C.; Stout, Julie C.

    2015-01-01

    Spatial cognition is fundamental for survival in the topographically complex environments inhabited by humans and other animals. The hippocampus, which has a central role in spatial cognition, is characterized by high concentration of serotonin (5-hydroxytryptamine; 5-HT) receptor binding sites, particularly of the 1A receptor (5-HT1A) subtype. This review highlights converging evidence for the role of hippocampal 5-HT1A receptors in spatial learning and memory. We consider studies showing that activation or blockade of the 5-HT1A receptors using agonists or antagonists, respectively, lead to changes in spatial learning and memory. For example, pharmacological manipulation to induce 5-HT release, or to block 5-HT uptake, have indicated that increased extracellular 5-HT concentrations maintain or improve memory performance. In contrast, reduced levels of 5-HT have been shown to impair spatial memory. Furthermore, the lack of 5-HT1A receptor subtype in single gene knockout mice is specifically associated with spatial memory impairments. These findings, along with evidence from recent cognitive imaging studies using positron emission tomography (PET) with 5-HT1A receptor ligands, and studies of individual genetic variance in 5-HT1A receptor availability, strongly suggests that 5-HT, mediated by the 5-HT1A receptor subtype, plays a key role in spatial learning and memory. PMID:26696889

  7. Mild deficits in mice lacking pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) performing on memory tasks.

    PubMed

    Sauvage, M; Brabet, P; Holsboer, F; Bockaert, J; Steckler, T

    2000-12-08

    Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor subtype 1 (PAC1) have been suggested to play a role in the modulation of learning and memory. However, behavioral evidence for altered mnemonic function due to altered PAC1 activity is missing. Therefore, the role of PAC1 in learning and memory was studied in mouse mutants lacking this receptor (PAC1 knock-out mice), tested in water maze two-choice spatial discrimination, one-trial contextual and cued fear conditioning, and multiple-session contextual discrimination. Water maze spatial discrimination was unaffected in PAC1 mutants, while a mild deficit was observed in multiple session contextual discrimination in PAC1 knock-out mice. Furthermore, PAC1 knock-out mice were able to learn the association between context and shock in one-trial contextual conditioning, but showed faster return to baseline than wild-type mice. Thus, the effects of PAC1 knock-out on modulating performance in these tasks were subtle and suggest that PAC1 only plays a limited role in learning and memory.

  8. Contrasting contribution of 5-hydroxytryptamine 1A receptor activation to neurochemical profile of novel antipsychotics: frontocortical dopamine and hippocampal serotonin release in rat brain.

    PubMed

    Assié, Marie-Bernadette; Ravailhe, Véronique; Faucillon, Valérie; Newman-Tancredi, Adrian

    2005-10-01

    Several novel antipsychotics, such as aripiprazole, bifeprunox, SSR181507 [(3-exo)-8-benzoyl-N-(((2S)7-chloro-2,3-dihydro-1,4-benzodioxin-1-yl)methyl)-8-azabicyclo(3.2.1)octane-3-methanamine], and SLV313 [1-(2,3-dihydro-benzo[1,4]dioxin-5-yl)-4-[5-(4-fluorophenyl)-pyridin-3-ylmethyl]-piperazine], activate serotonin 5-hydroxytryptamine (5-HT)1A receptors. Such activity is associated with enhanced treatment of negative symptoms and cognitive deficits, which may be mediated by modulation of cerebral dopamine and serotonin levels. We employed microdialysis coupled to high pressure liquid chromatography with electrochemical detection to examine 5-HT1A receptor activation in the modulation of extracellular dopamine in medial prefrontal cortex and serotonin in hippocampus of freely moving rats. The above compounds were compared with drugs that have less interaction with 5-HT1A receptors (clozapine, nemonapride, ziprasidone, olanzapine, risperidone, and haloperidol). Hippocampal 5-HT was decreased by bifeprunox, SSR181507, SLV313, sarizotan, and nemonapride, effects similar to those seen with the 5-HT1A agonist, (+)-8-hydroxy-2-(di-n-propylamino)tetralin [(+)8-OH-DPAT], consistent with activation of 5-HT1A autoreceptors. These decreases were reversed by the selective 5-HT1A antagonist, WAY100635 [N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide]. In contrast, haloperidol, risperidone, clozapine, olanzapine, ziprasidone, and aripiprazole did not significantly modify hippocampal serotonin levels. In medial prefrontal cortex, dopamine levels were increased by SSR181507, SLV313, sarizotan, and (+)8-OH-DPAT. These effects were reversed by WAY100635, indicating mediation by 5-HT1A receptors. In contrast, the increases in dopamine levels induced by clozapine, risperidone, olanzapine, and ziprasidone were not blocked by WAY100635, consistent with predominant influence of other mechanisms in the actions of these drugs. Haloperidol, nemonapride

  9. Activation of cannabinoid CB1 receptors modulates evoked action potentials in rat retinal ganglion cells.

    PubMed

    Jiang, Shu-Xia; Li, Qian; Wang, Xiao-Han; Li, Fang; Wang, Zhong-Feng

    2013-08-25

    Activation of cannabinoid CB1 receptors (CB1Rs) regulates a variety of physiological functions in the vertebrate retina through modulating various types of ion channels. The aim of the present study was to investigate the effects of this receptor on cell excitability of rat retinal ganglion cells (RGCs) in retinal slices using whole-cell patch-clamp techniques. The results showed that under current-clamped condition perfusing WIN55212-2 (WIN, 5 μmol/L), a CB1R agonist, did not significantly change the spontaneous firing frequency and resting membrane potential of RGCs. In the presence of cocktail synaptic blockers, including excitatory postsynaptic receptor blockers CNQX and D-APV, and inhibitory receptor blockers bicuculline and strychnine, perfusion of WIN (5 μmol/L) hardly changed the frequencies of evoked action potentials by a series of positive current injection (from +10 to +100 pA). Phase-plane plot analysis showed that both average threshold voltage for triggering action potential and delay time to reach threshold voltage were not affected by WIN. However, WIN significantly decreased +dV/dtmax and -dV/dtmax of action potentials, suggestive of reduced rising and descending velocities of action potentials. The effects of WIN were reversed by co-application of SR141716, a CB1R selective antagonist. Moreover, WIN did not influence resting membrane potential of RGCs with synaptic inputs being blocked. These results suggest that activation of CB1Rs may regulate intrinsic excitability of rat RGCs through modulating evoked action potentials.

  10. The peroxisome proliferator-activated receptor: A family of nuclear receptors role in various diseases

    PubMed Central

    Tyagi, Sandeep; Gupta, Paras; Saini, Arminder Singh; Kaushal, Chaitnya; Sharma, Saurabh

    2011-01-01

    Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of nuclear hormone receptor superfamily comprising of the following three subtypes: PPARα, PPARγ, and PPARβ/δ. Activation of PPAR-α reduces triglyceride level and is involved in regulation of energy homeostasis. Activation of PPAR-γ causes insulin sensitization and enhances glucose metabolism, whereas activation of PPAR-β/δ enhances fatty acids metabolism. Thus, PPAR family of nuclear receptors plays a major regulatory role in energy homeostasis and metabolic function. The present review critically analyzes the protective and detrimental effect of PPAR agonists in dyslipidemia, diabetes, adipocyte differentiation, inflammation, cancer, lung diseases, neurodegenerative disorders, fertility or reproduction, pain, and obesity. PMID:22247890

  11. Activation of neurokinin-1 receptor by substance P inhibits melanogenesis in B16-F10 melanoma cells.

    PubMed

    Ping, Fengfeng; Shang, Jing; Zhou, Jia; Song, Jing; Zhang, Luyong

    2012-12-01

    Skin pigmentation plays a number of valuable roles and its regulation is a complex process that is controlled by different factors. Substance P (SP) regulates many biological functions, including neurogenic inflammation, pain, and stress. However, to date, the regulatory role of SP in the control of melanogenesis has not been elucidated. The present study was designed to investigate the effects of SP on melanogenesis and to elucidate its underlying mechanism(s). After treatment for 48 h in mouse B16-F10 melanoma cells, SP (1 and 10nM) significantly down-regulated tyrosinase activity and melanin content. Importantly, western blot analysis revealed the presence of neurokinin-1 receptor (NK-1 R) in B16-F10 cells and the activation of it after SP treatment. It was also found that preincubation with NK-1 receptor antagonist Spantide I could partially reversed SP-induced down-regulations of tyrosinase activity, melanin content and the expression of tyrosinase and tyrosinase-related protein 1. Furthermore, SP could remarkably inhibit the expressions of microphtalmia-associated transcription factor (MITF) and p-p38 MAPK and stimulated p-p70 S6K1. These effects could also be partially reversed by the pretreatment with Spantide I. These results collectively suggested that SP inhibited melanogenesis in B16-F10 cells, which might be attributed to the fact that SP induces the activation of NK-1 receptor, stimulates the phosphorylation of p70 S6K1 and inhibits that of p38 MAPK, decreases the tyrosinase and tyrosinase-related protein 1 expression through MITF, finally resulting in the suppression of melanogenesis. These observations in vitro indicated that the regulation of the SP/NK-1 receptor system might be a useful novel management for skin pigmentation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Activation of pregnane X receptor and induction of MDR1 by dietary phytochemicals.

    PubMed

    Satsu, Hideo; Hiura, Yuto; Mochizuki, Keiichi; Hamada, Mika; Shimizu, Makoto

    2008-07-09

    The pregnane X receptor (PXR) is understood to be the key regulator for gene expression of such drug-metabolizing enzymes and transporters as multidrug-resistant protein 1 (MDR1) and the cytochrome P450 (CYP) family. We examined the effect of dietary phytochemicals on the PXR-dependent transcriptional activity in human intestinal LS180 cells by using a reporter assay. Among approximately 40 kinds of phytochemicals, tangeretin and ginkgolides A and B markedly induced the PXR-dependent transcriptional activity and also the activity of the human MDR1 promoter. The expression levels of MDR1 mRNA as well as of CYP3A4 mRNA, another gene regulated by PXR, were significantly increased by these phytochemicals. Furthermore, an increase was observed of the MDR1 protein and its functional activity by tangeretin and by ginkgolides A and B. These findings strongly suggest that tangeretin and ginkgolides A and B activated PXR, thereby regulating detoxification enzymes and transporters in the intestines.

  13. Mechanism of Activation of Enteric Nociceptive Neurons via Interaction of TLR4 and TRPV1 Receptors.

    PubMed

    Filippova, L V; Fedorova, A V; Nozdrachev, A D

    2018-03-01

    Evidence obtained by immunohistochemical double labeling and confocal laser scanning microscopy suggests that capsaicin, a ligand of the TRPV1 nociceptive vanilloid receptor, increases the number of TLR4-positive neurons in the rat colon myenteric plexus. In colitis caused by trinitrobenzene sulfonate, an increase in TRPV1 expression was more significant in both plexuses. Specific inhibitor of the TLR4 (C34) pattern-recognition receptor reduces TRPV1 expression in enteric neurons of both intact rats and rats with induced acute colitis. Thus, stimulation of nociceptive neurons by means of direct activation of their receptors of innate immunity (TLR4) is one of the possible mechanisms underlying the visceral pain in bacterial invasion and inflammatory bowel diseases.

  14. Sigma-1 receptor ligands control a switch between passive and active threat responses

    PubMed Central

    Rennekamp, Andrew J.; Huang, Xi-Ping; Wang, You; Patel, Samir; Lorello, Paul J.; Cade, Lindsay; Gonzales, Andrew P. W.; Yeh, Jing-Ruey Joanna; Caldarone, Barbara J.; Roth, Bryan L.; Kokel, David; Peterson, Randall T.

    2016-01-01

    Humans and many animals exhibit freezing behavior in response to threatening stimuli. In humans, inappropriate threat responses are fundamental characteristics of several mental illnesses. To identify small molecules that modulate threat responses, we developed a high-throughput behavioral assay in zebrafish (Danio rerio) and characterized the effects of 10,000 compounds on freezing behavior. We found three classes of compounds that switch the threat response from freezing to escape-like behavior. We then screened these for binding activity across 45 candidate targets. Using target profile clustering we implicated the sigma-1 receptor in the mechanism of behavioral switching and confirmed that known sigma-1 ligands also disrupt freezing behavior. Furthermore, mutation of the sigma-1 gene prevented the behavioral effect of escape-inducing compounds. The compound ‘finazine’ potently bound mammalian sigma-1 and altered rodent threat response behavior. Thus, pharmacological and genetic interrogation of the freezing response revealed sigma-1 as a mediator of vertebrate threat responses. PMID:27239788

  15. Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

    PubMed

    Cunningham, Margaret R; McIntosh, Kathryn A; Pediani, John D; Robben, Joris; Cooke, Alexandra E; Nilsson, Mary; Gould, Gwyn W; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

    2012-05-11

    Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

  16. Metalloprotease cleavage of the N terminus of the orphan G protein-coupled receptor GPR37L1 reduces its constitutive activity.

    PubMed

    Coleman, James L J; Ngo, Tony; Schmidt, Johannes; Mrad, Nadine; Liew, Chu Kong; Jones, Nicole M; Graham, Robert M; Smith, Nicola J

    2016-04-12

    Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gα(s) when heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3',5'-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gα(s) or Gα(i) signaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue. Copyright © 2016, American Association for the Advancement of Science.

  17. Potentiation of transient receptor potential V1 functions by the activation of metabotropic 5-HT receptors in rat primary sensory neurons

    PubMed Central

    Ohta, Toshio; Ikemi, Yuki; Murakami, Matsuka; Imagawa, Toshiaki; Otsuguro, Ken-ichi; Ito, Shigeo

    2006-01-01

    5-Hydroxytryptamine (5-HT) is one of the major chemical mediators released in injured and inflamed tissue and is capable of inducing hyperalgesia in vivo. However, the cellular mechanisms of 5-HT-induced hyperalgesia remain unclear. Transient receptor potential V1 (TRPV1) plays a pivotal role in nociceptive receptors. In the present study, we determined whether 5-HT changes TRPV1 functions in cultured dorsal root ganglion (DRG) neurons isolated from neonatal rats, using Ca2+ imaging and whole-cell patch-clamp techniques. In more than 70% of DRG neurons, 5-HT potentiated the increases of [Ca2+]i induced by capsaicin, protons and noxious heat. Capsaicin-induced current and depolarizing responses, and proton-induced currents were also augmented by 5-HT. RT-PCR analysis revealed the expression of 5-HT2A and 5-HT7 receptors in rat DRG neurons. Agonists for 5-HT2A and 5-HT7 receptors mimicked the potentiating effect of 5-HT, and their antagonists decreased it. In DRG ipsilateral to the complete Freund's adjuvant-injected inflammation side, expression levels of 5-HT2A and 5-HT7 mRNAs increased, and the potentiating effect of 5-HT was more prominent than in the contralateral control side. These results suggest that the PKC- and PKA-mediated signalling pathways are involved in the potentiating effect of 5-HT on TRPV1 functions through the activation of 5-HT2A and 5-HT7 receptors, respectively. Under inflammatory conditions, the increases of the biosynthesis of these 5-HT receptors may lead to further potentiation of TRPV1 functions, resulting in the generation of inflammatory hyperalgesia in vivo. PMID:16901936

  18. Cannabinoids Modulate Neuronal Activity and Cancer by CB1 and CB2 Receptor-Independent Mechanisms

    PubMed Central

    Soderstrom, Ken; Soliman, Eman; Van Dross, Rukiyah

    2017-01-01

    Cannabinoids include the active constituents of Cannabis or are molecules that mimic the structure and/or function of these Cannabis-derived molecules. Cannabinoids produce many of their cellular and organ system effects by interacting with the well-characterized CB1 and CB2 receptors. However, it has become clear that not all effects of cannabinoid drugs are attributable to their interaction with CB1 and CB2 receptors. Evidence now demonstrates that cannabinoid agents produce effects by modulating activity of the entire array of cellular macromolecules targeted by other drug classes, including: other receptor types; ion channels; transporters; enzymes, and protein- and non-protein cellular structures. This review summarizes evidence for these interactions in the CNS and in cancer, and is organized according to the cellular targets involved. The CNS represents a well-studied area and cancer is emerging in terms of understanding mechanisms by which cannabinoids modulate their activity. Considering the CNS and cancer together allow identification of non-cannabinoid receptor targets that are shared and divergent in both systems. This comparative approach allows the identified targets to be compared and contrasted, suggesting potential new areas of investigation. It also provides insight into the diverse sources of efficacy employed by this interesting class of drugs. Obtaining a comprehensive understanding of the diverse mechanisms of cannabinoid action may lead to the design and development of therapeutic agents with greater efficacy and specificity for their cellular targets. PMID:29066974

  19. Activity-dependent ubiquitination of GluA1 mediates a distinct AMPA receptor endocytosis and sorting pathway.

    PubMed

    Schwarz, Lindsay A; Hall, Benjamin J; Patrick, Gentry N

    2010-12-08

    The accurate trafficking of AMPA receptors (AMPARs) to and from the synapse is a critical component of learning and memory in the brain, whereas dysfunction of AMPAR trafficking is hypothesized to be an underlying mechanism of Alzheimer's disease. Previous work has shown that ubiquitination of integral membrane proteins is a common posttranslational modification used to mediate endocytosis and endocytic sorting of surface proteins in eukaryotic cells. Here we report that mammalian AMPARs become ubiquitinated in response to their activation. Using a mutant of GluA1 that is unable to be ubiquitinated at lysines on its C-terminus, we demonstrate that ubiquitination is required for internalization of surface AMPARs and their trafficking to the lysosome in response to the AMPAR agonist AMPA but not for internalization of AMPARs in response to the NMDA receptor agonist NMDA. Through overexpression or RNA interference-mediated knockdown, we identify that a specific E3 ligase, Nedd4-1 (neural-precursor cell-expressed developmentally downregulated gene 4-1), is necessary for this process. Finally, we show that ubiquitination of GluA1 by Nedd4-1 becomes more prevalent as neurons mature. Together, these data show that ubiquitination of GluA1-containing AMPARs by Nedd4-1 mediates their endocytosis and trafficking to the lysosome. Furthermore, these results provide insight into how hippocampal neurons regulate AMPAR trafficking and degradation with high specificity in response to differing neuronal signaling cues and suggest that changes to this pathway may occur as neurons mature.

  20. DYRK1A-mediated phosphorylation of GluN2A at Ser(1048) regulates the surface expression and channel activity of GluN1/GluN2A receptors.

    PubMed

    Grau, Cristina; Arató, Krisztina; Fernández-Fernández, José M; Valderrama, Aitana; Sindreu, Carlos; Fillat, Cristina; Ferrer, Isidre; de la Luna, Susana; Altafaj, Xavier

    2014-01-01

    N-methyl-D-aspartate glutamate receptors (NMDARs) play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs were modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser(1048) of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser(1048) hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser(1048) increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons.

  1. DYRK1A-mediated phosphorylation of GluN2A at Ser1048 regulates the surface expression and channel activity of GluN1/GluN2A receptors

    PubMed Central

    Grau, Cristina; Arató, Krisztina; Fernández-Fernández, José M.; Valderrama, Aitana; Sindreu, Carlos; Fillat, Cristina; Ferrer, Isidre; de la Luna, Susana; Altafaj, Xavier

    2014-01-01

    N-methyl-D-aspartate glutamate receptors (NMDARs) play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs were modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser1048 of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser1048 hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser1048 increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons. PMID:25368549

  2. Protease-Activated Receptor 1 Inhibition by SCH79797 Attenuates Left Ventricular Remodeling and Profibrotic Activities of Cardiac Fibroblasts

    PubMed Central

    Sonin, Dmitry L.; Wakatsuki, Tetsuro; Routhu, Kasi V.; Harmann, Leanne M.; Petersen, Matthew; Meyer, Jennifer; Strande, Jennifer L.

    2013-01-01

    Purpose Fibroblast activity promotes adverse left ventricular (LV) remodeling that underlies the development of ischemic cardiomyopathy. Transforming growth factor-β (TGF-β) is a potent stimulus for fibrosis, and the extracellular signal-regulated kinases(ERK) 1/2 pathway also contributes to the fibrotic response. The thrombin receptor, protease-activated receptor 1 (PAR1), has been shown to play an important role in the excessive fibrosis in different tissues. The aim of this study was to investigate the influence of a PAR1 inhibitor, SCH79797, on cardiac fibrosis, tissue stiffness and postinfarction remodeling, and effects of PAR1 inhibition on thrombin-induced TGF-β and (ERK) 1/2 activities in cardiac fibroblasts. Methods We used a rat model of myocardial ischemia–reperfusion injury, isolated cardiac fibroblasts, and 3-dimensional (3D) cardiac tissue models fabricated to ascertain the contribution of PAR1 activation on cardiac fibrosis and LV remodeling. Results The PAR1 inhibitor attenuated LV dilation and improved LV systolic function of the reperfused myocardium at 28 days. This improvement was associated with a nonsignificant decrease in scar size (%LV) from 23 ± % in the control group (n = 10) to 16% ± 5.5% in the treated group (n = 9; P = .052). In the short term, the PAR1 inhibitor did not rescue infarct size or LV systolic function after 3 days. The PAR1 inhibition abolished thrombin-mediated ERK1/2 phosphorylation, TGF-β and type I procollagen production, matrix metalloproteinase-2/9 activation, myofibroblasts transformation in vitro, and abrogated the remodeling of 3D tissues induced by chronic thrombin treatment. Conclusion These studies suggest PAR1 inhibition initiated after ischemic injury attenuates adverse LV remodeling through late-stage antifibrotic events. PMID:23598708

  3. Inhibition of Protease-Activated Receptor (PAR1) Reduces Activation of the Endothelium, Coagulation, Fibrinolysis and Inflammation during Human Endotoxemia.

    PubMed

    Schoergenhofer, Christian; Schwameis, Michael; Gelbenegger, Georg; Buchtele, Nina; Thaler, Barbara; Mussbacher, Marion; Schabbauer, Gernot; Wojta, Johann; Jilma-Stohlawetz, Petra; Jilma, Bernd

    2018-06-04

    The protease-activated receptor-1 (PAR-1) is critically involved in the co-activation of coagulation and inflammatory responses. Vorapaxar is a reversible, orally active, low molecular weight, competitive antagonist of PAR-1.We investigated the effects of PAR-1 inhibition by vorapaxar on the inflammatory response, the activation of coagulation, fibrinolysis and endothelium during experimental endotoxemia. In this randomized, double blind, crossover trial, 16 healthy volunteers received a bolus infusion of 2 ng/kg lipopolysaccharide (LPS) ± placebo/vorapaxar with a washout period of 8 weeks. Vorapaxar dosing was guided by thrombin receptor-activating peptide-6-induced whole blood aggregometry. Participants received 10 mg vorapaxar or placebo as an initial dose and, depending on the aggregometry, potentially an additional 10 mg. Goal was > 80% inhibition of aggregation compared with baseline. Vorapaxar significantly reduced the LPS-induced increase in pro-thrombin fragments F1 + 2 by a median of 27% (quartiles: 11-49%), thrombin-anti-thrombin concentrations by 22% (-3 to 46%) and plasmin-anti-plasmin levels by 38% (23-53%). PAR-1 inhibition dampened peak concentrations of tumour necrosis factor -α, interleukin-6 and consequently C-reactive protein by 66% (-11-71%), 50% (15-79%) and 23% (16-38%), respectively. Vorapaxar decreased maximum von Willebrand factor levels by 29% (26-51%) and soluble E-selectin concentrations by 30% (25-38%) after LPS infusion. PAR-1 inhibition did not affect thrombomodulin, soluble P-selectin and platelet factor-4 concentrations.PAR-1 inhibition significantly reduced the activation of coagulation, fibrinolysis, the inflammatory response and endothelial activation during experimental human endotoxemia. Schattauer GmbH Stuttgart.

  4. Effects of protease-activated receptor 1 inhibition on anxiety and fear following status epilepticus.

    PubMed

    Bogovyk, Ruslan; Lunko, Oleksii; Fedoriuk, Mihail; Isaev, Dmytro; Krishtal, Oleg; Holmes, Gregory L; Isaeva, Elena

    2017-02-01

    Protease-activated receptor 1 (PAR1) is an important contributor to the pathogenesis of a variety of brain disorders associated with a risk of epilepsy development. Using the lithium-pilocarpine model of temporal lobe epilepsy (TLE), we recently showed that inhibition of this receptor during the first ten days after pilocarpine-induced status epilepticus (SE) results in substantial anti-epileptogenic and neuroprotective effects. As PAR1 is expressed in the central nervous system regions of importance for processing emotional reactions, including amygdala and hippocampus, and TLE is frequently associated with a chronic alteration of the functions of these regions, we tested the hypothesis that PAR1 inhibition could modulate emotionally driven behavioral responses of rats experiencing SE. We showed that SE induces a chronic decrease in the animals' anxiety-related behavior and an increase of locomotor activity. PAR1 inhibition after SE abolished the alteration of the anxiety level but does not affect the increase of locomotor activity in the open field and elevated plus maze tests. Moreover, while PAR1 inhibition produces an impairment of memory recall in the context fear conditioning paradigm in the control group, it substantially improves contextual and cued fear learning in rats experiencing SE. These data suggest that PAR1-dependent signaling is involved in the mechanisms underlying emotional disorders in epilepsy. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Gi proteins regulate adenylyl cyclase activity independent of receptor activation.

    PubMed

    Melsom, Caroline Bull; Ørstavik, Øivind; Osnes, Jan-Bjørn; Skomedal, Tor; Levy, Finn Olav; Krobert, Kurt Allen

    2014-01-01

    Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to G(i), some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. We used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

  6. Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

    PubMed Central

    Melsom, Caroline Bull; Ørstavik, Øivind; Osnes, Jan-Bjørn; Skomedal, Tor; Levy, Finn Olav; Krobert, Kurt Allen

    2014-01-01

    Background and purpose Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. Experimental approach We used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. Key results PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. Conclusions and implications Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs

  7. Activation of m1 muscarinic acetylcholine receptor induces surface transport of KCNQ channels through a CRMP-2-mediated pathway.

    PubMed

    Jiang, Ling; Kosenko, Anastasia; Yu, Clinton; Huang, Lan; Li, Xuejun; Hoshi, Naoto

    2015-11-15

    Neuronal excitability is strictly regulated by various mechanisms, including modulation of ion channel activity and trafficking. Stimulation of m1 muscarinic acetylcholine receptor (also known as CHRM1) increases neuronal excitability by suppressing the M-current generated by the Kv7/KCNQ channel family. We found that m1 muscarinic acetylcholine receptor stimulation also triggers surface transport of KCNQ subunits. This receptor-induced surface transport was observed with KCNQ2 as well as KCNQ3 homomeric channels, but not with Kv3.1 channels. Deletion analyses identified that a conserved domain in a proximal region of the N-terminal tail of KCNQ protein is crucial for this surface transport--the translocation domain. Proteins that bind to this domain were identified as α- and β-tubulin and collapsin response mediator protein 2 (CRMP-2; also known as DPYSL2). An inhibitor of casein kinase 2 (CK2) reduced tubulin binding to the translocation domain, whereas an inhibitor of glycogen synthase kinase 3 (GSK3) facilitated CRMP-2 binding to the translocation domain. Consistently, treatment with the GSK3 inhibitor enhanced receptor-induced KCNQ2 surface transport. M-current recordings from neurons showed that treatment with a GSK3 inhibitor shortened the duration of muscarinic suppression and led to over-recovery of the M-current. These results suggest that m1 muscarinic acetylcholine receptor stimulates surface transport of KCNQ channels through a CRMP-2-mediated pathway. © 2015. Published by The Company of Biologists Ltd.

  8. M2 receptors exert analgesic action on DRG sensory neurons by negatively modulating VR1 activity.

    PubMed

    De Angelis, Federica; Marinelli, Sara; Fioretti, Bernard; Catacuzzeno, Luigi; Franciolini, Fabio; Pavone, Flaminia; Tata, Ada Maria

    2014-06-01

    The peripheral application of the M2 cholinergic agonist arecaidine on sensory nerve endings shows anti-nociceptive properties. In this work, we analyze in vitro, the mechanisms downstream M2 receptor activation causing the analgesic effects, and in vivo the effects produced by M2 agonist arecaidine administration on nociceptive responses in a murine model of nerve growth factor (NGF)-induced pain. Cultured DRG neurons treated with arecaidine showed a decreased level of VR1 and SP transcripts. Conversely, we found an increased expression of VR1 and SP transcripts in DRG from M2/M4(-/-) mice compared to WT and M1(-/-) mice, confirming the inhibitory effect in particular of M2 receptors on SP and VR1 expression. Patch-clamp experiments in the whole-cell configuration showed that arecaidine treatment caused a reduction of the fraction of capsaicin-responsive cells, without altering the mean capsaicin-activated current in responsive cells. We also demonstrated that arecaidine prevents PKCϵ translocation to the plasma membrane after inflammatory agent stimulation, mainly in medium-small sensory neurons. Finally, in mice, we have observed that intraperitoneal injection of arecaidine reduces VR1 expression blocking hyperalgesia and allodynia caused by NGF intraplantar administration. In conclusion, our data demonstrate that in vivo M2 receptor activation induces desensitization to mechanical and heat stimuli by a down-regulation of VR1 expression and by the inhibition of PKCϵ activity hindering its translocation to the plasma membrane, as suggested by in vitro experiments. © 2013 Wiley Periodicals, Inc.

  9. Xenopus-derived glucagon-like peptide-1 and polyethylene-glycosylated glucagon-like peptide-1 receptor agonists: long-acting hypoglycaemic and insulinotropic activities with potential therapeutic utilities.

    PubMed

    Han, Jing; Fei, Yingying; Zhou, Feng; Chen, Xinyu; Zhang, Ying; Liu, Lin; Fu, Junjie

    2018-02-01

    Incretin-based therapies based on glucagon-like peptide-1 (GLP-1) receptor agonists are effective treatments of type 2 diabetes. Abundant research has focused on the development of long-acting GLP-1 receptor agonists. However, all GLP-1 receptor agonists in clinical use or development are based on human or Gila GLP-1. We have identified a potent GLP-1 receptor agonist, xGLP-1B, based on Xenopus GLP-1. To further modify the structure of xGLP-1B, alanine scanning was performed to study the structure -activity relationship of xGLP-1B. Two strategies were then employed to improve bioactivity. First, the C-terminal tail of lixisenatide was appended to cysteine-altered xGLP-1B analogues. Second, polyethylene glycol (PEG) chains with different molecular weights were conjugated with the peptides, giving a series of PEGylated conjugates. Comprehensive bioactivity studies of these conjugates were performed in vitro and in vivo. From the in vitro receptor activation potency and in vivo acute hypoglycaemic activities of conjugates 25 -36, 33 was identified as the best candidate for further biological assessments. Conjugate 33 exhibited prominent hypoglycaemic and insulinotropic activities, as well as improved pharmacokinetic profiles in vivo. The prolonged antidiabetic duration of 33 was further confirmed by pre-oral glucose tolerance tests (OGTT) and multiple OGTT. Furthermore, chronic treatment of db/db mice with 33 ameliorated non-fasting blood glucose and insulin levels, reduced HbA1c values and normalized their impaired glucose tolerance. Importantly, no in vivo toxicity was observed in mice treated with 33. Peptide 33 is a promising long-acting type 2 diabetes therapeutic deserving further investigation. © 2017 The British Pharmacological Society.

  10. Endothelin A receptor activation on mesangial cells initiates Alport glomerular disease

    PubMed Central

    Dufek, Brianna; Meehan, Daniel; Delimont, Duane; Cheung, Linda; Gratton, Michael Anne; Phillips, Grady; Song, Wenping; Liu, Shiguang; Cosgrove, Dominic

    2016-01-01

    Recent work demonstrates that Alport glomerular disease is mediated through a biomechanical strain-sensitive activation of mesangial actin dynamics. This occurs through a Rac1/CDC42 cross-talk mechanism that results in the invasion of the sub-capillary spaces by mesangial filopodia. The filopodia deposit mesangial matrix proteins in the glomerular basement membrane, including laminin 211, which activates focal adhesion kinase in podocytes culminating in the up-regulation of pro-inflammatory cytokines and metalloproteinases. These events drive the progression of glomerulonephritis. Here we test whether endothelial cell-derived endothelin-1 is upregulated in Alport glomeruli, and further elevated by hypertension. Treatment of cultured mesangial cells with endothelin-1 activates the formation of drebrin-positive actin microspikes. These microspikes do not form when cells are treated with the endothelin A receptor antagonist sitaxentan, or under conditions of siRNA knockdown of endothelin A receptor mRNA. Treatment of Alport mice with sitaxentan results in delayed onset of proteinuria, normalized glomerular basement membrane morphology, inhibition of mesangial filopodial invasion of the glomerular capillaries, normalization of glomerular expression of metalloproteinases and pro-inflammatory cytokines, increased lifespan, and prevention of glomerulosclerosis and interstitial fibrosis. Thus endothelin A receptor activation on mesangial cells is a key event in initiation of Alport glomerular disease in this model. PMID:27165837

  11. Transient receptor potential ankyrin receptor 1 is a novel target for pro-tussive agents.

    PubMed

    Andrè, E; Gatti, R; Trevisani, M; Preti, D; Baraldi, P G; Patacchini, R; Geppetti, P

    2009-11-01

    The transient receptor potential ankyrin receptor 1 (TRPA1) is a cation channel, co-expressed with the pro-tussive transient receptor potential vanilloid type 1 (TRPV1) channel in primary sensory neurons. TRPA1 is activated by a series of irritant exogenous and endogenous alpha,beta-unsaturated aldehydes which seem to play a role in airway diseases. We investigated whether TRPA1 agonists provoke cough in guinea pigs and whether TRPA1 antagonists inhibit this response. Animals were placed in a Perspex box, and cough sounds were recorded and counted by observers unaware of the treatment used. Inhalation of two selective TRPA1 agonists, allyl isothiocyanate and cinnamaldehyde, dose-dependently caused cough in control guinea pigs, but not in those with airway sensory nerves desensitized by capsaicin. Coughs elicited by TRPA1 agonists were reduced by non-selective (camphor and gentamicin) and selective (HC-030031) TRPA1 antagonists, whereas they were unaffected by the TRPV1 antagonist, capsazepine. Acrolein and crotonaldehyde, two alpha,beta-unsaturated aldehydes recently identified as TRPA1 stimulants and contained in cigarette smoke, air pollution or produced endogenously by oxidative stress, caused a remarkable tussive effect, a response that was selectively inhibited by HC-030031. Part of the cough response induced by cigarette smoke inhalation was inhibited by HC-030031, suggesting the involvement of TRPA1. A novel pro-tussive pathway involves the TRPA1 channel, expressed by capsaicin-sensitive airway sensory nerves and is activated by a series of exogenous (cigarette smoke) and endogenous irritants. These results suggest TRPA1 may be a novel target for anti-tussive medicines.

  12. Impaired locomotor activity and exploratory behavior in mice lacking histamine H1receptors

    PubMed Central

    Inoue, Isao; Yanai, Kazuhiko; Kitamura, Daisuke; Taniuchi, Ichiro; Kobayashi, Takashi; Niimura, Kaku; Watanabe, Takehiko; Watanabe, Takeshi

    1996-01-01

    From pharmacological studies using histamine antagonists and agonists, it has been demonstrated that histamine modulates many physiological functions of the hypothalamus, such as arousal state, locomotor activity, feeding, and drinking. Three kinds of receptors (H1, H2, and H3) mediate these actions. To define the contribution of the histamine H1 receptors (H1R) to behavior, mutant mice lacking the H1R were generated by homologous recombination. In brains of homozygous mutant mice, no specific binding of [3H]pyrilamine was seen. [3H]Doxepin has two saturable binding sites with higher and lower affinities in brains of wild-type mice, but H1R-deficient mice showed only the weak labeling of [3H]doxepin that corresponds to lower-affinity binding sites. Mutant mice develop normally, but absence of H1R significantly increased the ratio of ambulation during the light period to the total ambulation for 24 hr in an accustomed environment. In addition, mutant mice significantly reduced exploratory behavior of ambulation and rearings in a new environment. These results indicate that through H1R, histamine is involved in circadian rhythm of locomotor activity and exploratory behavior as a neurotransmitter. PMID:8917588

  13. Antitumor activity of a novel anti-vascular endothelial growth factor receptor-1 monoclonal antibody that does not interfere with ligand binding

    PubMed Central

    Tentori, Lucio; Scimeca, Manuel; Dorio, Annalisa S.; Atzori, Maria Grazia; Failla, Cristina M.; Morea, Veronica; Bonanno, Elena; D'Atri, Stefania; Lacal, Pedro M.

    2016-01-01

    Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase transmembrane receptor that has also a soluble isoform containing most of the extracellular ligand binding domain (sVEGFR-1). VEGF-A binds to both VEGFR-2 and VEGFR-1, whereas placenta growth factor (PlGF) interacts exclusively with VEGFR-1. In this study we generated an anti-VEGFR-1 mAb (D16F7) by immunizing BALB/C mice with a peptide that we had previously reported to inhibit angiogenesis and endothelial cell migration induced by PlGF. D16F7 did not affect binding of VEGF-A or PlGF to VEGFR-1, thus allowing sVEGFR-1 to act as decoy receptor for these growth factors, but it hampered receptor homodimerization and activation. D16F7 inhibited both the chemotactic response of human endothelial, myelomonocytic and melanoma cells to VEGFR-1 ligands and vasculogenic mimicry by tumor cells. Moreover, D16F7 exerted in vivo antiangiogenic effects in a matrigel plug assay. Importantly, D16F7 inhibited tumor growth and was well tolerated by B6D2F1 mice injected with syngeneic B16F10 melanoma cells. The antitumor effect was associated with melanoma cell apoptosis, vascular abnormalities and decrease of both monocyte/macrophage infiltration and myeloid progenitor mobilization. For all the above, D16F7 may be exploited in the therapy of metastatic melanoma and other tumors or pathological conditions involving VEGFR-1 activation. PMID:27655684

  14. Fatty Acid-binding Proteins 1 and 2 Differentially Modulate the Activation of Peroxisome Proliferator-activated Receptor α in a Ligand-selective Manner*

    PubMed Central

    Hughes, Maria L. R.; Liu, Bonan; Halls, Michelle L.; Wagstaff, Kylie M.; Patil, Rahul; Velkov, Tony; Jans, David A.; Bunnett, Nigel W.; Scanlon, Martin J.; Porter, Christopher J. H.

    2015-01-01

    Nuclear hormone receptors (NHRs) regulate the expression of proteins that control aspects of reproduction, development and metabolism, and are major therapeutic targets. However, NHRs are ubiquitous and participate in multiple physiological processes. Drugs that act at NHRs are therefore commonly restricted by toxicity, often at nontarget organs. For endogenous NHR ligands, intracellular lipid-binding proteins, including the fatty acid-binding proteins (FABPs), can chaperone ligands to the nucleus and promote NHR activation. Drugs also bind FABPs, raising the possibility that FABPs similarly regulate drug activity at the NHRs. Here, we investigate the ability of FABP1 and FABP2 (intracellular lipid-binding proteins that are highly expressed in tissues involved in lipid metabolism, including the liver and intestine) to influence drug-mediated activation of the lipid regulator peroxisome proliferator-activated receptor (PPAR) α. We show by quantitative fluorescence imaging and gene reporter assays that drug binding to FABP1 and FABP2 promotes nuclear localization and PPARα activation in a drug- and FABP-dependent manner. We further show that nuclear accumulation of FABP1 and FABP2 is dependent on the presence of PPARα. Nuclear accumulation of FABP on drug binding is driven largely by reduced nuclear egress rather than an increased rate of nuclear entry. Importin binding assays indicate that nuclear access occurs via an importin-independent mechanism. Together, the data suggest that specific drug-FABP complexes can interact with PPARα to effect nuclear accumulation of FABP and NHR activation. Because FABPs are expressed in a regionally selective manner, this may provide a means to tailor the patterns of NHR drug activation in a tissue-specific manner. PMID:25847235

  15. A novel Arabidopsis CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) mutant with enhanced pathogen-induced cell death and altered receptor processing.

    PubMed

    Petutschnig, Elena K; Stolze, Marnie; Lipka, Ulrike; Kopischke, Michaela; Horlacher, Juliane; Valerius, Oliver; Rozhon, Wilfried; Gust, Andrea A; Kemmerling, Birgit; Poppenberger, Brigitte; Braus, Gerhard H; Nürnberger, Thorsten; Lipka, Volker

    2014-12-01

    Plants detect pathogens by sensing microbe-associated molecular patterns (MAMPs) through pattern recognition receptors. Pattern recognition receptor complexes also have roles in cell death control, but the underlying mechanisms are poorly understood. Here, we report isolation of cerk1-4, a novel mutant allele of the Arabidopsis chitin receptor CERK1 with enhanced defense responses. We identified cerk1-4 in a forward genetic screen with barley powdery mildew and consequently characterized it by pathogen assays, mutant crosses and analysis of defense pathways. CERK1 and CERK1-4 proteins were analyzed biochemically. The cerk1-4 mutation causes an amino acid exchange in the CERK1 ectodomain. Mutant plants maintain chitin signaling capacity but exhibit hyper-inducible salicylic acid concentrations and deregulated cell death upon pathogen challenge. In contrast to chitin signaling, the cerk1-4 phenotype does not require kinase activity and is conferred by the N-terminal part of the receptor. CERK1 undergoes ectodomain shedding, a well-known process in animal cell surface proteins. Wild-type plants contain the full-length CERK1 receptor protein as well as a soluble form of the CERK1 ectodomain, whereas cerk1-4 plants lack the N-terminal shedding product. Our work suggests that CERK1 may have a chitin-independent role in cell death control and is the first report of ectodomain shedding in plants. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  16. Lipin-1 Phosphatidic Phosphatase Activity Modulates Phosphatidate Levels to Promote Peroxisome Proliferator-activated Receptor γ (PPARγ) Gene Expression during Adipogenesis*

    PubMed Central

    Zhang, Peixiang; Takeuchi, Kazuharu; Csaki, Lauren S.; Reue, Karen

    2012-01-01

    Adipose tissue plays a key role in metabolic homeostasis. Disruption of the Lpin1 gene encoding lipin-1 causes impaired adipose tissue development and function in rodents. Lipin-1 functions as a phosphatidate phosphatase (PAP) enzyme in the glycerol 3-phosphate pathway for triglyceride storage and as a transcriptional coactivator/corepressor for metabolic nuclear receptors. Previous studies established that lipin-1 is required at an early step in adipocyte differentiation for induction of the adipogenic gene transcription program, including the key regulator peroxisome proliferator-activated receptor γ (PPARγ). Here, we investigate the requirement of lipin-1 PAP versus coactivator function in the establishment of Pparg expression during adipocyte differentiation. We demonstrate that PAP activity supplied by lipin-1, lipin-2, or lipin-3, but not lipin-1 coactivator activity, can rescue Pparg gene expression and lipogenesis during adipogenesis in lipin-1-deficient preadipocytes. In adipose tissue from lipin-1-deficient mice, there is an accumulation of phosphatidate species containing a range of medium chain fatty acids and an activation of the MAPK/extracellular signal-related kinase (ERK) signaling pathway. Phosphatidate inhibits differentiation of cultured adipocytes, and this can be rescued by the expression of lipin-1 PAP activity or by inhibition of ERK signaling. These results emphasize the importance of lipid intermediates as choreographers of gene regulation during adipogenesis, and the results highlight a specific role for lipins as determinants of levels of a phosphatidic acid pool that influences Pparg expression. PMID:22157014

  17. 1,2,4-Benzothiadiazine-1,1-dioxide derivatives as ionotropic glutamate receptor ligands: synthesis and structure-activity relationships.

    PubMed

    Varano, Flavia; Catarzi, Daniela; Colotta, Vittoria; Squarcialupi, Lucia; Matucci, Rosanna

    2014-11-01

    Ionotropic glutamate receptor (iGluR) modulators, specially AMPA receptor antagonists, are potential tools for numerous therapeutic applications in neurological disorders, including Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, epilepsy, chronic pain, and neuropathology ensuing from cerebral ischemia or cardiac arrest. In this work, the synthesis and binding affinities at the Gly/NMDA, AMPA, and kainic acid (KA) receptors of a new series of 1,2,4-benzothiadiazine-1,1-dioxide derivatives are reported. The results show that 1,2,4-benzothiadiazine-1,1-dioxide is a new scaffold for obtaining iGluR ligands. Moreover, this work has led us to the 7-(3-formylpyrrol-1-yl)-6-trifluoromethyl substituted compound 7, which displays the highest AMPA receptor affinity and high selectivity versus the Gly/NMDA (90-fold) and KA (46-fold) receptors. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. PKCepsilon-dependent potentiation of TTX-resistant Nav1.8 current by neurokinin-1 receptor activation in rat dorsal root ganglion neurons.

    PubMed

    Cang, Chun-Lei; Zhang, Hua; Zhang, Yu-Qiu; Zhao, Zhi-Qi

    2009-06-30

    Substance P (SP), which mainly exists in a subtype of small-diameter dorsal root ganglion (DRG) neurons, is an important signal molecule in pain processing in the spinal cord. Our previous results have proved the expression of SP receptor neurokinin-1 (NK-1) on DRG neurons and its interaction with transient receptor potential vanilloid 1 (TRPV1) receptor. In this study we investigated the effect of NK-1 receptor agonist on Na(v)1.8, a tetrodotoxin (TTX)-resistant sodium channel, in rat small-diameter DRG neurons employing whole-cell patch clamp recordings. NK-1 agonist [Sar(9), Met(O2)(11)]-substance P (Sar-SP) significantly enhanced the Na(v)1.8 currents in a subgroup of small-diameter DRG neurons under both the normal and inflammatory situation, and the enhancement was blocked by NK-1 antagonist Win51708 and protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), but not the protein kinase A (PKA) inhibitor H89. In particular, the inhibitor of PKCepsilon, a PKC isoform, completely blocked this effect. Under current clamp model, Sar-SP reduced the amount of current required to evoke action potentials and increased the firing rate in a subgroup of DRG neurons. These data suggest that activation of NK-1 receptor potentiates Na(v)1.8 sodium current via PKCepsilon-dependent signaling pathway, probably participating in the generation of inflammatory hyperalgesia.

  19. A neurokinin 1 receptor antagonist decreases postoperative peritoneal adhesion formation and increases peritoneal fibrinolytic activity.

    PubMed

    Reed, Karen L; Fruin, A Brent; Gower, Adam C; Stucchi, Arthur F; Leeman, Susan E; Becker, James M

    2004-06-15

    Fibrous adhesions remain a major sequela of abdominal surgery. The proinflammatory peptide substance P (SP), known to participate in inflammatory events, may play a key role in adhesion formation. This hypothesis was tested by using an antagonist, CJ-12,255 (Pfizer), that blocks the binding of SP to the neurokinin 1 receptor (NK-1R). Adhesion formation was surgically induced in the peritoneum of rats receiving daily doses of the NK-1R antagonist (NK-1RA; 5.0 or 10.0 mg/kg per day) or saline. On postoperative day 7, both the low and high doses of NK-1RA significantly (P < 0.05) reduced adhesion formation by 45% and 53%, respectively, compared with controls. Subsequently, the effect of NK-1RA administration on peritoneal fibrinolytic activity was investigated to determine a potential mechanism for SP action in the peritoneum. Samples were collected from nonoperated controls and from animals 24 h postsurgery that were administered either NK-1RA or saline. Fibrinolytic activity in peritoneal fluid was assayed by zymography, and expression of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1, both regulators of fibrinolytic activity, was assessed in peritoneal tissue and fluid by RT-PCR and bioassay, respectively. NK-1RA administration led to a marked (P < 0.05) increase in tPA mRNA levels in peritoneal tissue compared with nonoperated and saline-administered animals. Likewise, NK-1RA administration significantly (P < 0.05) increased tPA in the peritoneal fluid. These data suggest that activation of the NK-1R promotes peritoneal adhesion formation by limiting fibrinolytic activity in the postoperative peritoneum, thus enabling fibrinous adhesions to persist.

  20. Alkyl isothiocyanates suppress epidermal growth factor receptor kinase activity but augment tyrosine kinase activity.

    PubMed

    Nomura, Takahiro; Uehara, Yoshimasa; Kawajiri, Hiroo; Ryoyama, Kazuo; Yamori, Takao; Fuke, Yoko

    2009-10-01

    We have reported the in vitro and in vivo anticancer activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) derived from a Japanese spice, wasabi. In order to obtain some clues about the mechanism of the anticancer activity, we have studied the effect of alkyl isothiocyanates (MITCs) on protein kinase activities. The anti-autophosphorylation activity of MITCs with respect to the epidermal growth factor (EGF)-stimulated receptor kinase of A431 epidermoid carcinoma cells was examined by incorporation of radioactive ATP into an acid-insoluble fraction. Their anti-phosphorylation activity with respect to the non-receptor protein kinase was analyzed by a standard SDS-PAGE method. All the tested MITCs interfered with the EGF-stimulated receptor kinase activity in a dose-dependent manner, although their effects were less than 1/10 of that of erbstatin in microg/ml. On the other hand, the MITCs did not interfere with non-receptor kinases (kinase A, kinase C, tyrosine kinase and calmodulin dependent kinase III), but enhanced non-receptor tyrosine kinase. A possible anticancer mechanism of MITCs may involve the suppression of EGF receptor kinase activity and augmentation of non-receptor PTK.

  1. Oleamide activates peroxisome proliferator-activated receptor gamma (PPARγ) in vitro.

    PubMed

    Dionisi, Mauro; Alexander, Stephen P H; Bennett, Andrew J

    2012-05-14

    Oleamide (ODA) is a fatty acid primary amide first identified in the cerebrospinal fluid of sleep-deprived cats, which exerts effects on vascular and neuronal tissues, with a variety of molecular targets including cannabinoid receptors and gap junctions. It has recently been reported to exert a hypolipidemic effect in hamsters. Here, we have investigated the nuclear receptor family of peroxisome proliferator-activated receptors (PPARs) as potential targets for ODA action. Activation of PPARα, PPARβ and PPARγ was assessed using recombinant expression in Chinese hamster ovary cells with a luciferase reporter gene assay. Direct binding of ODA to the ligand binding domain of each of the three PPARs was monitored in a cell-free fluorescent ligand competition assay. A well-established assay of PPARγ activity, the differentiation of 3T3-L1 murine fibroblasts into adipocytes, was assessed using an Oil Red O uptake-based assay. ODA, at 10 and 50 μM, was able to transactivate PPARα, PPARβ and PPARγ receptors. ODA bound to the ligand binding domain of all three PPARs, although complete displacement of fluorescent ligand was only evident for PPARγ, at which an IC50 value of 38 μM was estimated. In 3T3-L1 cells, ODA, at 10 and 20 μM, induced adipogenesis. We have, therefore, identified a novel site of action of ODA through PPAR nuclear receptors and shown how ODA should be considered as a weak PPARγ ligand in vitro.

  2. Activation of the dopamine D1 receptor can extend long-term spatial memory persistence via PKA signaling in mice.

    PubMed

    Zhang, Jiabao; Ko, Sang-Yoon; Liao, Yulan; Kwon, Yubeen; Jeon, Se Jin; Sohn, Aeree; Cheong, Jae Hoon; Kim, Dong Hyun; Ryu, Jong Hoon

    2018-05-24

    Many works have been performed to understand the mechanisms of the formation and persistence of memory. However, it is not fully understood whether the decay of long-term memory can be modulated by the activation of dopamine D 1 receptor. A Barnes maze task was employed to measure long-term spatial memory. We observed that the spatial memory acquired through 3 trials per session for 4 days had begun to fade out by the 14th day and had completely disappeared by 21 days after the first probe test. The intraperitoneal administration of SKF 38393 (a dopamine D 1 receptor agonist) for 7 days beginning on the 14th day after the first probe test prevented natural memory forgetting, and the intraperitoneal administration of SCH 23390 (a dopamine D 1 receptor antagonist) prevented this memory persistence. In the Western blotting, the administration of SKF 38393 increased the phosphorylation levels of PKA, ERK1/2, CaMKII, and CREB in the hippocampus. In addition, such increased levels were decreased by the corresponding antagonist (SCH 23390). Moreover, the inhibition of PKA could completely reverse the preservation of spatial memory induced by dopamine D 1 receptor activation. These results suggest that the activation of the dopamine D 1 receptor plays a critical role in the persistence of long-term spatial memory through the PKA signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

    PubMed Central

    Murphy, Jane E.; Roosterman, Dirk; Cottrell, Graeme S.; Padilla, Benjamin E.; Feld, Micha; Brand, Eva; Cedron, Wendy J.; Steinhoff, Martin

    2011-01-01

    Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs. PMID:21795521

  4. Effects of γ-Aminobutyric Acid A Receptor Activation on Counterregulatory Responses to Subsequent Exercise in Individuals With Type 1 Diabetes

    PubMed Central

    Hedrington, Maka S.; Mikeladze, Maia; Tate, Donna B.; Younk, Lisa M.; Davis, Ian

    2016-01-01

    The effects of γ-aminobutyric acid (GABA) A receptor activation on physiologic responses during next-day exercise in type 1 diabetes are unknown. To test the hypothesis that GABA A activation with the benzodiazepine alprazolam would blunt counterregulatory responses during subsequent exercise, 29 (15 male, 14 female) individuals with type 1 diabetes (HbA1c 7.8 ± 1%) were studied during separate 2-day protocols. Day 1 consisted of morning and afternoon 2-h euglycemic or 2.9 mmol/L hypoglycemic clamps with or without 1 mg alprazolam given 30 min before each clamp. Day 2 consisted of a 90-min euglycemic cycling exercise at 50% VO2max. Tritiated glucose was used to measure glucose kinetics. Despite equivalent day 2 insulin (93 ± 6 pmol/L) and glucose levels (5.3 ± 0.1 mmol/L), plasma epinephrine, norepinephrine, glucagon, cortisol, and growth hormone responses were similarly reduced after alprazolam or day 1 hypoglycemia compared with euglycemic control. Endogenous glucose production, lipolysis (glycerol, nonesterified fatty acid), and glycogenolysis (lactate) were also reduced during day 2 exercise after day 1 GABA A activation. We conclude that activation of GABA A receptors with alprazolam can result in widespread neuroendocrine, autonomic nervous system, and metabolic counterregulatory failure during subsequent submaximal exercise and may increase the risk of exercise-associated hypoglycemia in individuals with type 1 diabetes. PMID:27217489

  5. Protease-activated receptor 2, a receptor involved in melanosome transfer, is upregulated in human skin by ultraviolet irradiation.

    PubMed

    Scott, G; Deng, A; Rodriguez-Burford, C; Seiberg, M; Han, R; Babiarz, L; Grizzle, W; Bell, W; Pentland, A

    2001-12-01

    Previous studies have shown that the protease-activated receptor 2 is involved in skin pigmentation through increased phagocytosis of melanosomes by keratinocytes. Ultraviolet irradiation is a potent stimulus for melanosome transfer. We show that protease-activated receptor 2 expression in human skin is upregulated by ultraviolet irradiation. Subjects with skin type I, II, or III were exposed to two or three minimal erythema doses of irradiation from a solar simulator. Biopsies were taken from nonexposed and irradiated skin 24 and 96 h after irradiation and protease-activated receptor 2 expression was detected using immunohistochemical staining. In nonirradiated skin, protease-activated receptor 2 expression was confined to keratinocytes in the lower one-third of the epidermis. After ultraviolet irradiation protease-activated receptor 2 expression was observed in keratinocytes in the upper two-thirds of the epidermis or the entire epidermis at both time points studied. Subjects with skin type I showed delayed upregulation of protease-activated receptor 2 expression, however, compared with subjects with skin types II and III. Irradiated cultured human keratinocytes showed upregulation in protease-activated receptor 2 expression as determined by immunofluorescence microscopy and Western blotting. Cell culture supernatants from irradiated keratinocytes also exhibited a dose-dependent increase in protease-activated receptor-2 cleavage activity. These results suggest an important role for protease-activated receptor-2 in pigmentation in vivo. Differences in protease-activated receptor 2 regulation in type I skin compared with skin types II and III suggest a potential mechanism for differences in tanning in subjects with different skin types.

  6. Periplakin interferes with G protein activation by the melanin-concentrating hormone receptor-1 by binding to the proximal segment of the receptor C-terminal tail.

    PubMed

    Murdoch, Hannah; Feng, Gui-Jie; Bächner, Dietmar; Ormiston, Laura; White, Julia H; Richter, Dietmar; Milligan, Graeme

    2005-03-04

    In mice genetic ablation of expression of either melanin-concentrating hormone or the melanin-concentrating hormone-1 receptor results in alterations in energy metabolism and a lean phenotype. There is thus great interest in the function and regulation of this receptor. Using the yeast two-hybrid system we identified an interaction of the actin- and intermediate filament-binding protein periplakin with the intracellular C-terminal tail of the melanin-concentrating hormone-1 receptor. Direct association of these proteins was verified in pull-down and coimmunoprecipitation experiments. Truncations and internal deletions delineated the site of interaction to a group of 11 amino acids proximal to transmembrane helix VII, which was distinct from the binding site for the melanin-concentrating hormone-1 receptor-interacting zinc finger protein. Immunohistochemistry demonstrated coexpression of periplakin with melanin-concentrating hormone-1 receptor in specific cells of the piriform cortex, amygdala, and other structures of the adult mouse brain. Coexpression of the melanin-concentrating hormone-1 receptor with periplakin in human embryonic kidney 293 cells did not prevent agonist-mediated internalization of the receptor but did interfere with binding of (35)S-labeled guanosine 5'-3-O-(thio)triphosphate ([(35)S]GTPgammaS) to the G protein Galpha(o1) and the elevation of [Ca(2+)](i). Coexpression of the receptor with the interacting zinc finger protein did not modulate receptor internalization or G protein activation. The interaction of periplakin with receptors was selective. Coexpression of periplakin with the IP prostanoid receptor did not result in coimmunoprecipitation nor interfere with agonist-mediated binding of [(35)S]GTPgammaS to the G protein Galpha(s). Periplakin is the first protein described to modify the capacity of the melanin-concentrating hormone-1 receptor to initiate signal transduction.

  7. Activation of β-adrenergic receptors is required for elevated α1A-adrenoreceptors expression and signaling in mesenchymal stromal cells

    PubMed Central

    Tyurin-Kuzmin, Pyotr A.; Fadeeva, Julia I.; Kanareikina, Margarita A.; Kalinina, Natalia I.; Sysoeva, Veronika Yu.; Dyikanov, Daniyar T.; Stambolsky, Dmitriy V.; Tkachuk, Vsevolod A.

    2016-01-01

    Sympathetic neurons are important components of mesenchymal stem cells (MSCs) niche and noradrenaline regulates biological activities of these cells. Here we examined the mechanisms of regulation of MSCs responsiveness to noradrenaline. Using flow cytometry, we demonstrated that α1A adrenergic receptors isoform was the most abundant in adipose tissue-derived MSCs. Using calcium imaging in single cells, we demonstrated that only 6.9 ± 0.8% of MSCs responded to noradrenaline by intracellular calcium release. Noradrenaline increases MSCs sensitivity to catecholamines in a transitory mode. Within 6 hrs after incubation with noradrenaline the proportion of cells responding by Ca2+ release to the fresh noradrenaline addition has doubled but declined to the baseline after 24 hrs. Increased sensitivity was due to the elevated quantities of α1A-adrenergic receptors on MSCs. Such elevation depended on the stimulation of β-adrenergic receptors and adenylate cyclase activation. The data for the first time clarify mechanisms of regulation of MSCs sensitivity to noradrenaline. PMID:27596381

  8. 5-Hydroxytryptamine1A receptor/Gibetagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of src in chinese hamster ovary fibroblasts.

    PubMed Central

    Mukhin, Y V; Garnovskaya, M N; Collinsworth, G; Grewal, J S; Pendergrass, D; Nagai, T; Pinckney, S; Greene, E L; Raymond, J R

    2000-01-01

    The hypothesis of this work is that the 'serotonin' or 5-hydroxytryptamine (5-HT)(1A) receptor, which activates the extracellular signal-regulated kinase (ERK) through a G(i)betagamma-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT(1A) receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine-reactive oxidant molecules activates ERK. (3) The 5-HT(1A) receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT(1A) receptor in Chinese hamster ovary (CHO) cells downstream of G(i)betagamma subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT(1A) receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT-mediated generation of both superoxide and hydrogen peroxide. PMID:10727402

  9. Activation of the UNC5B receptor by Netrin-1 inhibits sprouting angiogenesis.

    PubMed

    Larrivée, Bruno; Freitas, Catarina; Trombe, Marc; Lv, Xiang; Delafarge, Benjamin; Yuan, Li; Bouvrée, Karine; Bréant, Christiane; Del Toro, Raquel; Bréchot, Nicolas; Germain, Stéphane; Bono, Françoise; Dol, Frédérique; Claes, Filip; Fischer, Christian; Autiero, Monica; Thomas, Jean-Léon; Carmeliet, Peter; Tessier-Lavigne, Marc; Eichmann, Anne

    2007-10-01

    Netrins are secreted molecules with roles in axonal growth and angiogenesis. The Netrin receptor UNC5B is required during embryonic development for vascular patterning, suggesting that it may also contribute to postnatal and pathological angiogenesis. Here we show that unc5b is down-regulated in quiescent adult vasculature, but re-expressed during sprouting angiogenesis in matrigel and tumor implants. Stimulation of UNC5B-expressing neovessels with an agonist (Netrin-1) inhibits sprouting angiogenesis. Genetic loss of function of unc5b reduces Netrin-1-mediated angiogenesis inhibition. Expression of UNC5B full-length receptor also triggers endothelial cell repulsion in response to Netrin-1 in vitro, whereas a truncated UNC5B lacking the intracellular signaling domain fails to induce repulsion. These data show that UNC5B activation inhibits sprouting angiogenesis, thus identifying UNC5B as a potential anti-angiogenic target.

  10. Activation of the UNC5B receptor by Netrin-1 inhibits sprouting angiogenesis

    PubMed Central

    Larrivée, Bruno; Freitas, Catarina; Trombe, Marc; Lv, Xiang; DeLafarge, Benjamin; Yuan, Li; Bouvrée, Karine; Bréant, Christiane; Del Toro, Raquel; Bréchot, Nicolas; Germain, Stéphane; Bono, Françoise; Dol, Frédérique; Claes, Filip; Fischer, Christian; Autiero, Monica; Thomas, Jean-Léon; Carmeliet, Peter; Tessier-Lavigne, Marc; Eichmann, Anne

    2007-01-01

    Netrins are secreted molecules with roles in axonal growth and angiogenesis. The Netrin receptor UNC5B is required during embryonic development for vascular patterning, suggesting that it may also contribute to postnatal and pathological angiogenesis. Here we show that unc5b is down-regulated in quiescent adult vasculature, but re-expressed during sprouting angiogenesis in matrigel and tumor implants. Stimulation of UNC5B-expressing neovessels with an agonist (Netrin-1) inhibits sprouting angiogenesis. Genetic loss of function of unc5b reduces Netrin-1-mediated angiogenesis inhibition. Expression of UNC5B full-length receptor also triggers endothelial cell repulsion in response to Netrin-1 in vitro, whereas a truncated UNC5B lacking the intracellular signaling domain fails to induce repulsion. These data show that UNC5B activation inhibits sprouting angiogenesis, thus identifying UNC5B as a potential anti-angiogenic target. PMID:17908930

  11. Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators

    PubMed Central

    Lazennec, Gwendal; Canaple, Laurence; Saugy, Damien; Wahli, Walter

    2000-01-01

    The nuclear peroxisome proliferator-activated receptors (PPARs) α, β and γ activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. The activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas the activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase dependent induction of PPARs but also their ligand-dependent induction, suggesting that the ligands may also mobilize the PKA pathway to lead to maximal transcriptional induction by PPARs. Moreover, comparing PPARα KO with PPARα wild-type mice, we show that the expression of the ACO gene can be regulated by PKA-activated PPARα in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity and we propose a model associating this pathway in the control of fatty acid β-oxidation under conditions of fasting, stress and exercise. PMID:11117527

  12. Acetylcholine modulates gamma frequency oscillations in the hippocampus by activation of muscarinic M1 receptors.

    PubMed

    Betterton, Ruth T; Broad, Lisa M; Tsaneva-Atanasova, Krasimira; Mellor, Jack R

    2017-06-01

    Modulation of gamma oscillations is important for the processing of information and the disruption of gamma oscillations is a prominent feature of schizophrenia and Alzheimer's disease. Gamma oscillations are generated by the interaction of excitatory and inhibitory neurons where their precise frequency and amplitude are controlled by the balance of excitation and inhibition. Acetylcholine enhances the intrinsic excitability of pyramidal neurons and suppresses both excitatory and inhibitory synaptic transmission, but the net modulatory effect on gamma oscillations is not known. Here, we find that the power, but not frequency, of optogenetically induced gamma oscillations in the CA3 region of mouse hippocampal slices is enhanced by low concentrations of the broad-spectrum cholinergic agonist carbachol but reduced at higher concentrations. This bidirectional modulation of gamma oscillations is replicated within a mathematical model by neuronal depolarisation, but not by reducing synaptic conductances, mimicking the effects of muscarinic M1 receptor activation. The predicted role for M1 receptors was supported experimentally; bidirectional modulation of gamma oscillations by acetylcholine was replicated by a selective M1 receptor agonist and prevented by genetic deletion of M1 receptors. These results reveal that acetylcholine release in CA3 of the hippocampus modulates gamma oscillation power but not frequency in a bidirectional and dose-dependent manner by acting primarily through muscarinic M1 receptors. © 2017 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  13. Sigma-1 receptor: the novel intracellular target of neuropsychotherapeutic drugs.

    PubMed

    Hayashi, Teruo

    2015-01-01

    Sigma-1 receptor ligands have been long expected to serve as drugs for treatment of human diseases such as neurodegenerative disorders, depression, idiopathic pain, drug abuse, and cancer. Recent research exploring the molecular function of the sigma-1 receptor started unveiling underlying mechanisms of the therapeutic activity of those ligands. Via the molecular chaperone activity, the sigma-1 receptor regulates protein folding/degradation, ER/oxidative stress, and cell survival. The chaperone activity is activated or inhibited by synthetic sigma-1 receptor ligands in an agonist-antagonist manner. Sigma-1 receptors are localized at the endoplasmic reticulum (ER) membranes that are physically associated with the mitochondria (MAM: mitochondria-associated ER membrane). In specific types of neurons (e.g., those at the spinal cord), sigma-1 receptors are also clustered at ER membranes that juxtapose postsynaptic plasma membranes. Recent studies indicate that sigma-1 receptors, partly in sake of its unique subcellular localization, regulate the mitochondria function that involves bioenergetics and free radical generation. The sigma-1 receptor may thus provide an intracellular drug target that enables controlling ER stress and free radical generation under pathological conditions. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  14. Endothelin-1 and its receptors on haemorrhoidal tissue: a potential site for therapeutic intervention.

    PubMed

    Lohsiriwat, Varut; Scholefield, John H; Wilson, Vincent G; Dashwood, Michael R

    2017-04-01

    Haemorrhoids is a common anorectal condition affecting millions worldwide. We have studied the effect of endothelin-1 (ET-1) and the role of endothelin ET A and ET B receptors in haemorrhoid tissue. Protein expression of ET-1, ET A and ET B receptors were compared between haemorrhoids and normal rectal submucosa using Western blot analysis, with the localization of proteins determined by autoradiography and immunohistochemistry. Effects of ET-1 and sarafotoxin 6a on human colonic and rectal arteries and veins was assessed by wire myography and the involvement of receptor subtypes established by selective antagonists. Dense binding of [ 125 I]-ET-1 to haemorrhoidal sections was reduced by selective receptor antagonists. A higher density of ET B than ET A receptors was found in haemorrhoidal, than in control rectal tissue and confirmed by Western blot analysis. ET A and ET B receptors were localized to smooth muscle of haemorrhoidal arteries and veins, with ET B receptors on the endothelium. Human colonic and rectal arteries and veins were similarly sensitive to ET-1 and affected by the ET A selective antagonist, but sarafotoxin S6a-induced contractions were more pronounced in veins and antagonized by a selective ET B receptor antagonist. ET A and ET B receptors are present in human haemorrhoids with ET B receptors predominating. ET A receptors are activated by ET-1 to mediate a contraction in arteries and veins, but the latter are selectively activated by sarafotoxin S6a - a response that involves ET B receptors at low concentrations. Selective ET B agonists may have therapeutic potential to reduce congestion of the haemorrhoidal venous sinusoids. © 2017 The British Pharmacological Society.

  15. Membrane receptor-mediated apoptosis and caspase activation in the differentiated EoL-1 eosinophilic cell line.

    PubMed

    Al-Rabia, Mohammed W; Blaylock, Morgan G; Sexton, Darren W; Walsh, Garry M

    2004-06-01

    Caspases are key molecules in the control of apoptosis, but relatively little is known about their contribution to eosinophil apoptosis. We examined caspase-3, -8, and -9 activities in receptor ligation-dependent apoptosis induction in the differentiated human eosinophilic cell line EoL-1. Differentiated EoL-1 exhibited bi-lobed nuclei, eosinophil-associated membrane receptors, and basic granule proteins. Annexin-V fluorescein isothiocyanate binding to EoL-1 revealed significant (P<0.01) apoptosis induction in cells cultured for 20 h with monoclonal antibodies (mAb) specific for CD45 (71%+/-4.3), CD45RA (58%+/-2.3), CD45RB (68%+/-2.4), CD95 (47%+/-2.6), and CD69 (52%+/-2.1) compared with control (23%+/-1.6) or CD45RO mAb (27%+/-3.9). The pan-caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (fmk) and inhibitors of caspase-8 (Z-Ile-Glu-Thr-Asp-fmk) and caspase-9 (Z-Leu-Glu-His-Asp-fmk) significantly inhibited mAb-induced apoptosis of EoL-1 but had no effect on constitutive (baseline) apoptosis at 16 and 20 h. Caspase activity was analyzed using the novel CaspaTag trade mark technique and flow cytometry. EoL-1 treated with pan-CD45, CD45RA, CD45RB, and CD95 mAb exhibited caspase-3 and -9 activation at 12 h post-treatment, which increased at 16 and 20 h. Activated caspase-8 was detected 12 and 16 h after ligation with CD45, CD45RA, CD45RB, and CD95 mAb followed by a trend toward basal levels at 20 h. CD69 ligation resulted in caspase-3 activation, a modest but significant activation of caspase-8, and a loss in mitochondrial transmembrane potential but had no significant effect on activation of caspase-9. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation-mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti-inflammatory therapy for asthma.

  16. Telmisartan, an AT1 receptor blocker and a PPAR gamma activator, alleviates liver fibrosis induced experimentally by Schistosoma mansoni infection.

    PubMed

    Attia, Yasmeen M; Elalkamy, Essam F; Hammam, Olfat A; Mahmoud, Soheir S; El-Khatib, Aiman S

    2013-07-05

    Hepatic schistosomiasis is considered to be one of the most prevalent forms of chronic liver disease in the world due to its complication of liver fibrosis. The demonstration of the pro-fibrogenic role of angiotensin (Ang) II in chronic liver disease brought up the idea that anti-Ang II agents may be effective in improving hepatic fibrosis by either blocking Ang II type 1 (AT1) receptors or inhibiting the angiotensin converting enzyme. Peroxisome proliferator-activated receptors gamma (PPARγ) activation has been also shown to inhibit hepatic stellate cell activation and progression of fibrosis. The present study has aimed at testing the anti-fibrogenic effects of telmisartan; an AT1 receptor blocker and a PPARγ partial agonist, alone or combined with praziquantel (PZQ) on Schistosoma mansoni-induced liver fibrosis in mice. To achieve the aim of the study, two sets of experiments were performed in which telmisartan was initiated at the 5th (set 1) and the 10th (set 2) weeks post infection to assess drug efficacy in both acute and chronic stages of liver fibrosis, respectively. Schistosoma mansoni-infected mice were randomly divided into the following four groups: infected-control (I), telmisartan-treated (II), PZQ-treated (III), and telmisartan+PZQ-treated (IV). In addition, a normal non-infected group was used for comparison. Parasitological (hepatomesenteric worm load and oogram pattern), histopathological, morphometric, immunohistochemical (hepatic expressions of matrix metalloproteinase-2; MMP-2 and tissue inhibitor of metalloproteinase-2; TIMP-2), and biochemical (serum transforming growth factor beta 1; TGF-β1 and liver function tests) studies were performed. Telmisartan failed to improve the parasitological parameters, while it significantly (P<0.05) decreased the mean granuloma diameter, area of fibrosis, and serum TGF-β1. Additionally, telmisartan increased MMP-2 and decreased TIMP-2 hepatic expression. Combined treatment failed to show any additive

  17. AMPA receptor activation controls type I metabotropic glutamate receptor signalling via a tyrosine kinase at parallel fibre-Purkinje cell synapses.

    PubMed

    Auger, Céline; Ogden, David

    2010-08-15

    Metabotropic glutamate receptors type 1 (mGluR1s) and ionotropic AMPA receptors (AMPARs) are colocalized at parallel fibre (PF) to Purkinje cell synapses of the cerebellum. Single stimulation of PFs activates fast AMPAR excitatory postsynaptic currents, whereas the activation of mGluR1s requires burst stimulation. mGluR1s signal through several pathways in Purkinje cells and the most prominent is the activation of a slow EPSC (sEPSC). To separate the two synaptic currents, studies of the sEPSC have commonly been performed in the presence of AMPA/KA receptor antagonists. We show here in rat cerebellar slices that inhibition of the fast EPSC by AMPAR antagonists strongly and selectively potentiates the mGluR1 sEPSC, showing a negative regulation of mGluR1 by AMPAR. This effect is observed with low concentrations of NBQX (300 nM to 1 microM), with the selective AMPAR antagonist GYKI 53655 and also with gamma-DGG, a low affinity glutamate receptor antagonist. When photorelease of glutamate from MNI-glutamate was used to study the postsynaptic responses in isolation, AMPAR inhibition produced a similar potentiation of the mGluR1 sEPSC, showing that the interaction is postsynaptic. Finally, perfusion of the postsynaptic cell with PP1, an inhibitor of src-family tyrosine kinase, increased the amplitude of the mGluR1 sEPSC and occluded the effect of AMPAR inhibition. Thus, at PF to Purkinje cell synapses, AMPAR activation inhibits the mGluR1 sEPSC via activation of a src-family tyrosine kinase. Consequently mGluR1 signalling will be more sensitive to spillover of glutamate than to local synaptic release. Furthermore, it will be enhanced at silent PF synapses which are the majority in Purkinje cells.

  18. Characterization of the Sweet Taste Receptor Tas1r2 from an Old World Monkey Species Rhesus Monkey and Species-Dependent Activation of the Monomeric Receptor by an Intense Sweetener Perillartine

    PubMed Central

    Cai, Chenggu; Jiang, Hua; Li, Lei; Liu, Tianming; Song, Xuejie; Liu, Bo

    2016-01-01

    Sweet state is a basic physiological sensation of humans and other mammals which is mediated by the broadly acting sweet taste receptor-the heterodimer of Tas1r2 (taste receptor type 1 member 2) and Tas1r3 (taste receptor type 1 member 3). Various sweeteners interact with either Tas1r2 or Tas1r3 and then activate the receptor. In this study, we cloned, expressed and functionally characterized the taste receptor Tas1r2 from a species of Old World monkeys, the rhesus monkey. Paired with the human TAS1R3, it was shown that the rhesus monkey Tas1r2 could respond to natural sugars, amino acids and their derivates. Furthermore, similar to human TAS1R2, rhesus monkey Tas1r2 could respond to artificial sweeteners and sweet-tasting proteins. However, the responses induced by rhesus monkey Tas1r2 could not be inhibited by the sweet inhibitor amiloride. Moreover, we found a species-dependent activation of the Tas1r2 monomeric receptors of human, rhesus monkey and squirrel monkey but not mouse by an intense sweetener perillartine. Molecular modeling and sequence analysis indicate that the receptor has the conserved domains and ligand-specific interactive residues, which have been identified in the characterized sweet taste receptors up to now. This is the first report of the functional characterization of sweet taste receptors from an Old World monkey species. PMID:27479072

  19. Characterization of the Sweet Taste Receptor Tas1r2 from an Old World Monkey Species Rhesus Monkey and Species-Dependent Activation of the Monomeric Receptor by an Intense Sweetener Perillartine.

    PubMed

    Cai, Chenggu; Jiang, Hua; Li, Lei; Liu, Tianming; Song, Xuejie; Liu, Bo

    2016-01-01

    Sweet state is a basic physiological sensation of humans and other mammals which is mediated by the broadly acting sweet taste receptor-the heterodimer of Tas1r2 (taste receptor type 1 member 2) and Tas1r3 (taste receptor type 1 member 3). Various sweeteners interact with either Tas1r2 or Tas1r3 and then activate the receptor. In this study, we cloned, expressed and functionally characterized the taste receptor Tas1r2 from a species of Old World monkeys, the rhesus monkey. Paired with the human TAS1R3, it was shown that the rhesus monkey Tas1r2 could respond to natural sugars, amino acids and their derivates. Furthermore, similar to human TAS1R2, rhesus monkey Tas1r2 could respond to artificial sweeteners and sweet-tasting proteins. However, the responses induced by rhesus monkey Tas1r2 could not be inhibited by the sweet inhibitor amiloride. Moreover, we found a species-dependent activation of the Tas1r2 monomeric receptors of human, rhesus monkey and squirrel monkey but not mouse by an intense sweetener perillartine. Molecular modeling and sequence analysis indicate that the receptor has the conserved domains and ligand-specific interactive residues, which have been identified in the characterized sweet taste receptors up to now. This is the first report of the functional characterization of sweet taste receptors from an Old World monkey species.

  20. LeEix1 functions as a decoy receptor to attenuate LeEix2 signaling.

    PubMed

    Bar, Maya; Sharfman, Miya; Avni, Adi

    2011-03-01

    The receptors for the fungal elicitor EIX (LeEix1 and LeEix2) belong to a class of leucine-rich repeat cell-surface glycoproteins with a signal for receptor-mediated endocytosis. Both receptors are able to bind the EIX elicitor while only the LeEix2 receptor mediates defense responses. We show that LeEix1 acts as a decoy receptor and attenuates EIX induced internalization and signaling of the LeEix2 receptor. We demonstrate that BAK1 binds LeEix1 but not LeEix2. In plants where BAK1 was silenced, LeEix1 was no longer able to attenuate plant responses to EIX, indicating that BAK1 is required for this attenuation. We suggest that LeEix1 functions as a decoy receptor for LeEix2, a function which requires the kinase activity of BAK1.

  1. Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and {beta}-adrenergic receptor signaling pathways

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shin, Vivian Yvonne; Jin, H.C.; Ng, Enders K.O.

    Induction of cyclooxygenase-2 (COX-2) associates with cigarette smoke exposure in many malignancies. Nicotine and its derivative, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are the two important components in cigarette smoke that contributes to cancer development. However, the molecular mechanism(s) by which nicotine or NNK promotes gastric carcinogenesis remains largely unknown. We found that nicotine and NNK significantly enhanced cell proliferation in AGS cells that expressed both alpha7 nicotinic acetylcholine receptor ({alpha}7 nAChR) and {beta}-adrenergic receptors. Treatment of cells with {alpha}-bungarotoxin ({alpha}-BTX, {alpha}7nAChR antagonist) or propranolol ({beta}-adrenergic receptor antagonist) blocked NNK-induced COX-2/PGE{sub 2} and cell proliferation, while nicotine-mediated cell growth and COX-2/PGE{sub 2} induction canmore » only be suppressed by propranolol, but not {alpha}-BTX. Moreover, in contrast to the dependence of growth promoting effect of nicotine on Erk activation, inhibitor of p38 mitogen-activated protein kinase (MAPK) repressed NNK-induced COX-2 upregulation and resulted in suppression of cell growth. In addition, nicotine and NNK mediated COX-2 induction via different receptors to modulate several G1/S transition regulatory proteins and promote gastric cancer cell growth. Selective COX-2 inhibitor (SC-236) caused G1 arrest and abrogated nicotine/NNK-induced cell proliferation. Aberrant expression of cyclin D1 and other G1 regulatory proteins are reversed by blockade of COX-2. These results pointed to the importance of adrenergic and nicotinic receptors in gastric tumor growth through MAPK/COX-2 activation, which may perhaps provide a chemoprevention strategy for cigarette smoke-related gastric carcinogenesis.« less

  2. Endothelin‐1 and its receptors on haemorrhoidal tissue: a potential site for therapeutic intervention

    PubMed Central

    Lohsiriwat, Varut; Scholefield, John H; Wilson, Vincent G

    2017-01-01

    Background and Purpose Haemorrhoids is a common anorectal condition affecting millions worldwide. We have studied the effect of endothelin‐1 (ET‐1) and the role of endothelin ETA and ETB receptors in haemorrhoid tissue. Experimental Approach Protein expression of ET‐1, ETA and ETB receptors were compared between haemorrhoids and normal rectal submucosa using Western blot analysis, with the localization of proteins determined by autoradiography and immunohistochemistry. Effects of ET‐1 and sarafotoxin 6a on human colonic and rectal arteries and veins was assessed by wire myography and the involvement of receptor subtypes established by selective antagonists. Key Results Dense binding of [125I]‐ET‐1 to haemorrhoidal sections was reduced by selective receptor antagonists. A higher density of ETB than ETA receptors was found in haemorrhoidal, than in control rectal tissue and confirmed by Western blot analysis. ETA and ETB receptors were localized to smooth muscle of haemorrhoidal arteries and veins, with ETB receptors on the endothelium. Human colonic and rectal arteries and veins were similarly sensitive to ET‐1 and affected by the ETA selective antagonist, but sarafotoxin S6a‐induced contractions were more pronounced in veins and antagonized by a selective ETB receptor antagonist. Conclusions and Implications ETA and ETB receptors are present in human haemorrhoids with ETB receptors predominating. ETA receptors are activated by ET‐1 to mediate a contraction in arteries and veins, but the latter are selectively activated by sarafotoxin S6aa response that involves ETB receptors at low concentrations. Selective ETB agonists may have therapeutic potential to reduce congestion of the haemorrhoidal venous sinusoids. PMID:28095606

  3. LE135, a retinoid acid receptor antagonist, produces pain through direct activation of TRP channels.

    PubMed

    Yin, Shijin; Luo, Jialie; Qian, Aihua; Yu, Weihua; Hu, Hongzhen

    2014-03-01

    Retinoids, through their activation of retinoic acid receptors (RARs) and retinoid X receptors, regulate diverse cellular processes, and pharmacological intervention in their actions has been successful in the treatment of skin disorders and cancers. Despite the many beneficial effects, administration of retinoids causes irritating side effects with unknown mechanisms. Here, we demonstrate that LE135 [4-(7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid], a selective antagonist of RARβ , is a potent activator of the capsaicin (TRPV1) and wasabi (TRPA1) receptors, two critical pain-initiating cation channels. We performed to investigate the excitatory effects of LE135 on TRPV1 and TRPA1 channels expressed in HEK293T cells and in dorsal root ganglia neurons with calcium imaging and patch-clamp recordings. We also used site-directed mutagenesis of the channels to determine the structural basis of LE135-induced activation of TRPV1 and TRPA1 channels and behavioural testing to examine if pharmacological inhibition and genetic deletion of the channels affected LE135-evoked pain-related behaviours. LE135 activated both the capsaicin receptor (TRPV1) and the allyl isothiocyanate receptor (TRPA1) heterologously expressed in HEK293T cells and endogenously expressed by sensory nociceptors. Mutations disrupting the capsaicin-binding site attenuated LE135 activation of TRPV1 channels and a single mutation (K170R) eliminated TRPA1 activity evoked by LE135. Intraplantar injection of LE135 evoked pain-related behaviours. Both TRPV1 and TRPA1 channels were involved in LE135-elicited pain-related responses, as shown by pharmacological and genetic ablation studies. This blocker of retinoid acid signalling also exerted non-genomic effects through activating the pain-initiating TRPV1 and TRPA1 channels. © 2013 The British Pharmacological Society.

  4. Chronic cannabinoid receptor 2 activation reverses paclitaxel neuropathy without tolerance or cannabinoid receptor 1-dependent withdrawal.

    PubMed

    Deng, Liting; Guindon, Josée; Cornett, Benjamin L; Makriyannis, Alexandros; Mackie, Ken; Hohmann, Andrea G

    2015-03-01

    Mixed cannabinoid receptor 1 and 2 (CB1 and CB2) agonists such as Δ(9)-tetrahydrocannabinol (Δ(9)-THC) can produce tolerance, physical withdrawal, and unwanted CB1-mediated central nervous system side effects. Whether repeated systemic administration of a CB2-preferring agonist engages CB1 receptors or produces CB1-mediated side effects is unknown. We evaluated antiallodynic efficacy, possible tolerance, and cannabimimetic side effects of repeated dosing with a CB2-preferring agonist AM1710 in a model of chemotherapy-induced neuropathy produced by paclitaxel using CB1 knockout (CB1KO), CB2 knockout (CB2KO), and wild-type (WT) mice. Comparisons were made with the prototypic classic cannabinoid Δ(9)-THC. We also explored the site and possible mechanism of action of AM1710. Paclitaxel-induced mechanical and cold allodynia developed to an equivalent degree in CB1KO, CB2KO, and WT mice. Both AM1710 and Δ(9)-THC suppressed established paclitaxel-induced allodynia in WT mice. In contrast to Δ(9)-THC, chronic administration of AM1710 did not engage CB1 activity or produce antinociceptive tolerance, CB1-mediated cannabinoid withdrawal, hypothermia, or motor dysfunction. Antiallodynic efficacy of systemic administration of AM1710 was absent in CB2KO mice and WT mice receiving the CB2 antagonist AM630, administered either systemically or intrathecally. Intrathecal administration of AM1710 also attenuated paclitaxel-induced allodynia in WT mice, but not CB2KO mice, implicating a possible role for spinal CB2 receptors in AM1710 antiallodynic efficacy. Finally, both acute and chronic administration of AM1710 decreased messenger RNA levels of tumor necrosis factor-α and monocyte chemoattractant protein 1 in lumbar spinal cord of paclitaxel-treated WT mice. Our results highlight the potential of prolonged use of CB2 agonists for managing chemotherapy-induced allodynia with a favorable therapeutic ratio marked by sustained efficacy and absence of tolerance, physical

  5. Sensitization of transient receptor potential vanilloid 1 by the prokineticin receptor agonist Bv8.

    PubMed

    Vellani, Vittorio; Colucci, Mariantonella; Lattanzi, Roberta; Giannini, Elisa; Negri, Lucia; Melchiorri, Pietro; McNaughton, Peter A

    2006-05-10

    Small mammalian proteins called the prokineticins [prokineticin 1 (PK1) and PK2] and two corresponding G-protein-coupled receptors [prokineticin receptor 1 (PKR1) and PKR2] have been identified recently, but the physiological role of the PK/PKR system remains mostly unexplored. Bv8, a protein extracted from frog skin, is a convenient and potent agonist for both PKR1 and PKR2, and injection of Bv8 in vivo causes a potent and long-lasting hyperalgesia. Here, we investigate the cellular basis of hyperalgesia caused by activation of PKRs. Bv8 caused increases in [Ca]i in a population of isolated dorsal root ganglion (DRG) neurons, which we identified as nociceptors, or sensors for painful stimuli, from their responses to capsaicin, bradykinin, mustard oil, or proteases. Bv8 enhanced the inward current carried by the heat and capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) via a pathway involving activation of protein kinase Cepsilon (PKCepsilon), because Bv8 caused translocation of PKCepsilon to the neuronal membrane and because PKC antagonists reduced both the enhancement of current carried by TRPV1 and behavioral hyperalgesia in rodents. The neuronal population expressing PKRs consisted partly of small peptidergic neurons and partly of neurons expressing the N52 marker for myelinated fibers. Using single-cell reverse transcriptase-PCR, we found that mRNA for PKR1 was mainly expressed in small DRG neurons. Exposure to GDNF (glial cell line-derived neurotrophic factor) induced de novo expression of functional receptors for Bv8 in a nonpeptidergic population of neurons. These results show that prokineticin receptors are expressed in nociceptors and cause heat hyperalgesia by sensitizing TRPV1 through activation of PKCepsilon. The results suggest a role for prokineticins in physiological inflammation and hyperalgesia.

  6. Bile Acid Receptor Agonist GW4064 Regulates PPARγ Coactivator-1α Expression Through Estrogen Receptor-Related Receptor α

    PubMed Central

    Dwivedi, Shailendra Kumar Dhar; Singh, Nidhi; Kumari, Rashmi; Mishra, Jay Sharan; Tripathi, Sarita; Banerjee, Priyam; Shah, Priyanka; Kukshal, Vandana; Tyagi, Abdul Malik; Gaikwad, Anil Nilkanth; Chaturvedi, Rajnish Kumar; Mishra, Durga Prasad; Trivedi, Arun Kumar; Sanyal, Somali; Chattopadhyay, Naibedya; Ramachandran, Ravishankar; Siddiqi, Mohammad Imran; Bandyopadhyay, Arun; Arora, Ashish; Lundåsen, Thomas; Anakk, Sayee Priyadarshini; Moore, David D.

    2011-01-01

    Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology. PMID:21493670

  7. Small-molecule agonists for the glucagon-like peptide 1 receptor

    PubMed Central

    Knudsen, Lotte Bjerre; Kiel, Dan; Teng, Min; Behrens, Carsten; Bhumralkar, Dilip; Kodra, János T.; Holst, Jens J.; Jeppesen, Claus B.; Johnson, Michael D.; de Jong, Johannes Cornelis; Jorgensen, Anker Steen; Kercher, Tim; Kostrowicki, Jarek; Madsen, Peter; Olesen, Preben H.; Petersen, Jacob S.; Poulsen, Fritz; Sidelmann, Ulla G.; Sturis, Jeppe; Truesdale, Larry; May, John; Lau, Jesper

    2007-01-01

    The peptide hormone glucagon-like peptide (GLP)-1 has important actions resulting in glucose lowering along with weight loss in patients with type 2 diabetes. As a peptide hormone, GLP-1 has to be administered by injection. Only a few small-molecule agonists to peptide hormone receptors have been described and none in the B family of the G protein coupled receptors to which the GLP-1 receptor belongs. We have discovered a series of small molecules known as ago-allosteric modulators selective for the human GLP-1 receptor. These compounds act as both allosteric activators of the receptor and independent agonists. Potency of GLP-1 was not changed by the allosteric agonists, but affinity of GLP-1 for the receptor was increased. The most potent compound identified stimulates glucose-dependent insulin release from normal mouse islets but, importantly, not from GLP-1 receptor knockout mice. Also, the compound stimulates insulin release from perfused rat pancreas in a manner additive with GLP-1 itself. These compounds may lead to the identification or design of orally active GLP-1 agonists. PMID:17213325

  8. Kinetic deuterium isotope effects in glucocorticoid receptor activation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aranyi, P.

    1984-01-01

    Activation and deactivation of the chick thymus glucocorticoid receptor protein was studied in ordinary and heavy water by DNA-cellulose binding of the tritiated triamcinolone acetonide-receptor complex. Activation was significantly slower in heavy water if it was promoted by incubation at elevated temperature in buffers of low ionic strength. In the presence of 300 mM KC1 or after separation from the low molecular weight cytosol constituents, the complex was activated at the same rate in both solvents. Deactivation (time dependent loss of DNA-binding capacity) was much faster in ordinary than in heavy water regardless of gel filtration or the presence ofmore » KC1. A model of receptor activation-deactivation was constructed on the basis of these data that accounts for the observed kinetic deuterium isotope effects and reveals some submolecular details of the process.« less

  9. Structural prerequisites for G-protein activation by the neurotensin receptor

    PubMed Central

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; Grisshammer, Reinhard

    2015-01-01

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1. PMID:26205105

  10. Activation of the α7 nicotinic ACh receptor induces anxiogenic effects in rats which is blocked by a 5-HT1a receptor antagonist

    PubMed Central

    Pandya, Anshul A.; Yakel, Jerrel L.

    2013-01-01

    The α7 nicotinic acetylcholine receptor (nAChR) is highly expressed in different regions of the brain and is associated with cognitive function as well as anxiety. Agonists and positive allosteric modulators (PAMs) of the α7 subtype of nAChRs have been shown to improve cognition. Previously nicotine, which activates both α7 and non-α7 subtypes of nAChRs, has been shown to have an anxiogenic effect in behavioral tests. In this study, we compared the effects of the α7-selective agonist (PNU-282987) and PAM (PNU-120596) in a variety of behavioral tests in Sprague Dawley rats to look at their effects on learning and memory as well as anxiety. We found that neither PNU-282987 nor PNU-120596 improved spatial-learning or episodic memory by themselves. However when cognitive impairment was induced in the rats with scopolamine (1 mg/kg), both PNU-120596 and PNU-282987 were able to reverse this memory impairment and restore it back to normal levels. While PNU-120596 reversed the scopolamine-induced cognitive impairment, it did not have any adverse effect on anxiety. PNU-282987 on the other hand displayed an increase in anxiety-like behavior at a higher dose (10 mg/kg) that was significantly reduced by the serotonin 5-HT1a receptor antagonist WAY-100135. However the α7 receptor antagonist methyllycaconitine was unable to reverse these anxiety-like effects seen with PNU-282987. These results suggest that α7 nAChR PAMs are pharmacologically advantageous over agonists, and should be considered for further development as therapeutic drugs targeting the α7 receptors. PMID:23321689

  11. Characterization of SB-271046: A potent, selective and orally active 5-HT6 receptor antagonist

    PubMed Central

    Routledge, Carol; Bromidge, Steven M; Moss, Stephen F; Price, Gary W; Hirst, Warren; Newman, Helen; Riley, Graham; Gager, Tracey; Stean, Tania; Upton, Neil; Clarke, Stephen E; Brown, Anthony M; Middlemiss, Derek N

    2000-01-01

    SB-271046, potently displaced [3H]-LSD and [125I]-SB-258585 from human 5-HT6 receptors recombinantly expressed in HeLa cells in vitro (pKi 8.92 and 9.09 respectively). SB-271046 also displaced [125I]-SB-258585 from human caudate putamen and rat and pig striatum membranes (pKi 8.81, 9.02 and 8.55 respectively). SB-271046 was over 200 fold selective for the 5-HT6 receptor vs 55 other receptors, binding sites and ion channels. In functional studies on human 5-HT6 receptors SB-271046 competitively antagonized 5-HT-induced stimulation of adenylyl cyclase activity with a pA2 of 8.71. SB-271046 produced an increase in seizure threshold over a wide-dose range in the rat maximal electroshock seizure threshold (MEST) test, with a minimum effective dose of ⩽0.1 mg kg−1 p.o. and maximum effect at 4 h post-dose. The level of anticonvulsant activity achieved correlated well with the blood concentrations of SB-271046 (EC50 of 0.16 μM) and brain concentrations of 0.01–0.04 μM at Cmax. These data, together with the observed anticonvulsant activity of other selective 5-HT6 receptor antagonists, SB-258510 (10 mg kg−1, 2–6 h pre-test) and Ro 04-6790 (1–30 mg kg−1, 1 h pre-test), in the rat MEST test, suggest that the anticonvulsant properties of SB-271046 are likely to be mediated by 5-HT6 receptors. Overall, these studies demonstrate that SB-271046 is a potent and selective 5-HT6 receptor antagonist and is orally active in the rat MEST test. SB-271046 represents a valuable tool for evaluating the in vivo central function of 5-HT6 receptors. PMID:10928964

  12. Fatty Acid-binding Proteins 1 and 2 Differentially Modulate the Activation of Peroxisome Proliferator-activated Receptor α in a Ligand-selective Manner.

    PubMed

    Hughes, Maria L R; Liu, Bonan; Halls, Michelle L; Wagstaff, Kylie M; Patil, Rahul; Velkov, Tony; Jans, David A; Bunnett, Nigel W; Scanlon, Martin J; Porter, Christopher J H

    2015-05-29

    Nuclear hormone receptors (NHRs) regulate the expression of proteins that control aspects of reproduction, development and metabolism, and are major therapeutic targets. However, NHRs are ubiquitous and participate in multiple physiological processes. Drugs that act at NHRs are therefore commonly restricted by toxicity, often at nontarget organs. For endogenous NHR ligands, intracellular lipid-binding proteins, including the fatty acid-binding proteins (FABPs), can chaperone ligands to the nucleus and promote NHR activation. Drugs also bind FABPs, raising the possibility that FABPs similarly regulate drug activity at the NHRs. Here, we investigate the ability of FABP1 and FABP2 (intracellular lipid-binding proteins that are highly expressed in tissues involved in lipid metabolism, including the liver and intestine) to influence drug-mediated activation of the lipid regulator peroxisome proliferator-activated receptor (PPAR) α. We show by quantitative fluorescence imaging and gene reporter assays that drug binding to FABP1 and FABP2 promotes nuclear localization and PPARα activation in a drug- and FABP-dependent manner. We further show that nuclear accumulation of FABP1 and FABP2 is dependent on the presence of PPARα. Nuclear accumulation of FABP on drug binding is driven largely by reduced nuclear egress rather than an increased rate of nuclear entry. Importin binding assays indicate that nuclear access occurs via an importin-independent mechanism. Together, the data suggest that specific drug-FABP complexes can interact with PPARα to effect nuclear accumulation of FABP and NHR activation. Because FABPs are expressed in a regionally selective manner, this may provide a means to tailor the patterns of NHR drug activation in a tissue-specific manner. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Sigma-11) Receptor in Memory and Neurodegenerative Diseases.

    PubMed

    Maurice, Tangui; Goguadze, Nino

    2017-01-01

    The sigma-11 ) receptor has been associated with regulation of intracellular Ca 2+ homeostasis, several cellular signaling pathways, and inter-organelle communication, in part through its chaperone activity. In vivo, agonists of the σ 1 receptor enhance brain plasticity, with particularly well-described impact on learning and memory. Under pathological conditions, σ 1 receptor agonists can induce cytoprotective responses. These protective responses comprise various complementary pathways that appear to be differentially engaged according to pathological mechanism. Recent studies have highlighted the efficacy of drugs that act through the σ 1 receptor to mitigate symptoms associated with neurodegenerative disorders with distinct mechanisms of pathogenesis. Here, we will review genetic and pharmacological evidence of σ 1 receptor engagement in learning and memory disorders, cognitive impairment, and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Huntington's disease.

  14. Intrinsic A(1) adenosine receptor activation during ischemia or reperfusion improves recovery in mouse hearts.

    PubMed

    Peart, J; Headrick, J P

    2000-11-01

    We assessed the role of A(1) adenosine receptor (A(1)AR) activation by endogenous adenosine in the modulation of ischemic contracture and postischemic recovery in Langendorff-perfused mouse hearts subjected to 20 min of total ischemia and 30 min of reperfusion. In control hearts, the rate-pressure product (RPP) and first derivative of pressure development over time (+dP/dt) recovered to 57 +/- 3 and 58 +/- 3% of preischemia, respectively. Diastolic pressure remained elevated at 20 +/- 2 mmHg (compared with 3 +/- 1 mmHg preischemia). Interstitial adenosine, assessed by microdialysis, rose from approximately 0.3 to 1.9 microM during ischemia compared with approximately 15 microM in rat heart. Nonetheless, these levels will near maximally activate A(1)ARs on the basis of effects of exogenous adenosine and 2-chloroadenosine. Neither A(1)AR blockade with 200 nM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) during the ischemic period alone nor A(1)AR activation with 50 nM N(6)-cyclopentyladenosine altered rapidity or extent of ischemic contracture. However, ischemic DPCPX treatment significantly depressed postischemic recovery of RPP and +dP/dt (44 +/- 3 and 40 +/- 4% of preischemia, respectively). DPCPX treatment during the reperfusion period alone also reduced recovery of RPP and +dP/dt (to 44 +/- 2 and 47 +/- 2% of preischemia, respectively). These data indicate that 1) interstitial adenosine is lower in mouse versus rat myocardium during ischemia, 2) A(1)AR activation by endogenous adenosine or exogenous agonists does not modify ischemic contracture in murine myocardium, 3) A(1)AR activation by endogenous adenosine during ischemia attenuates postischemic stunning, and 4) A(1)AR activation by endogenous adenosine during the reperfusion period also improves postischemic contractile recovery.

  15. A specific multi-nutrient formulation enhances M1 muscarinic acetylcholine receptor responses in vitro.

    PubMed

    Savelkoul, Paul J M; Janickova, Helena; Kuipers, Almar A M; Hageman, Robert J J; Kamphuis, Patrick J; Dolezal, Vladimir; Broersen, Laus M

    2012-02-01

    Recent evidence indicates that supplementation with a specific combination of nutrients may affect cell membrane synthesis and composition. To investigate whether such nutrients may also modify the physical properties of membranes, and affect membrane-bound processes involved in signal transduction pathways, we studied the effects of nutrient supplementation on G protein-coupled receptor activation in vitro. In particular, we investigated muscarinic receptors, which are important for the progression of memory deterioration and pathology of Alzheimer's disease. Nerve growth factor differentiated pheochromocytoma cells that were supplemented with specific combinations of nutrients showed enhanced responses to muscarinic receptor agonists in a membrane potential assay. The largest effects were obtained with a combination of nutrients known as Fortasyn™ Connect, comprising docosahexaenoic acid, eicosapentaenoic acid, uridine monophosphate as a uridine source, choline, vitamin B6, vitamin B12, folic acid, phospholipids, vitamin C, vitamin E, and selenium. In subsequent experiments, it was shown that the effects of supplementation could not be attributed to single nutrients. In addition, it was shown that the agonist-induced response and the supplement-induced enhancement of the response were blocked with the muscarinic receptor antagonists atropine, telenzepine, and AF-DX 384. In order to determine whether the effects of Fortasyn™ Connect supplementation were receptor subtype specific, we investigated binding properties and activation of human muscarinic M1, M2 and M4 receptors in stably transfected Chinese hamster ovary cells after supplementation. Multi-nutrient supplementation did not change M1 receptor density in plasma membranes. However, M1 receptor-mediated G protein activation was significantly enhanced. In contrast, supplementation of M2- or M4-expressing cells did not affect receptor signaling. Taken together, these results indicate that a specific

  16. Hypotensive effects of ghrelin receptor agonists mediated through a novel receptor.

    PubMed

    Callaghan, Brid; Kosari, Samin; Pustovit, Ruslan V; Sartor, Daniela M; Ferens, Dorota; Ban, Kung; Baell, Jonathan; Nguyen, Trung V; Rivera, Leni R; Brock, James A; Furness, John B

    2014-03-01

    Some agonists of ghrelin receptors cause rapid decreases in BP. The mechanisms by which they cause hypotension and the pharmacology of the receptors are unknown. The effects of ligands of ghrelin receptors were investigated in rats in vivo, on isolated blood vessels and on cells transfected with the only molecularly defined ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR1a). Three agonists of GHSR1a receptors, ulimorelin, capromorelin and CP464709, caused a rapid decrease in BP in the anaesthetized rat. The effect was not reduced by either of two GHSR1a antagonists, JMV2959 or YIL781, at doses that blocked effects on colorectal motility, in vivo. The rapid hypotension was not mimicked by ghrelin, unacylated ghrelin or the unacylated ghrelin receptor agonist, AZP531. The early hypotension preceded a decrease in sympathetic nerve activity. Early hypotension was not reduced by hexamethonium or by baroreceptor (sino-aortic) denervation. Ulimorelin also relaxed isolated segments of rat mesenteric artery, and, less potently, relaxed aorta segments. The vascular relaxation was not reduced by JMV2959 or YIL781. Ulimorelin, capromorelin and CP464709 activated GHSR1a in transfected HEK293 cells at nanomolar concentrations. JMV2959 and YIL781 both antagonized effects in these cells, with their pA2 values at the GHSR1a receptor being 6.55 and 7.84. Our results indicate a novel vascular receptor or receptors whose activation by ulimorelin, capromorelin and CP464709 lowered BP. This receptor is activated by low MW GHSR1a agonists, but is not activated by ghrelin. © 2013 The British Pharmacological Society.

  17. Hypotensive effects of ghrelin receptor agonists mediated through a novel receptor

    PubMed Central

    Callaghan, Brid; Kosari, Samin; Pustovit, Ruslan V; Sartor, Daniela M; Ferens, Dorota; Ban, Kung; Baell, Jonathan; Nguyen, Trung V; Rivera, Leni R; Brock, James A; Furness, John B

    2014-01-01

    BACKGROUND AND PURPOSE Some agonists of ghrelin receptors cause rapid decreases in BP. The mechanisms by which they cause hypotension and the pharmacology of the receptors are unknown. EXPERIMENTAL APPROACH The effects of ligands of ghrelin receptors were investigated in rats in vivo, on isolated blood vessels and on cells transfected with the only molecularly defined ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR1a). KEY RESULTS Three agonists of GHSR1a receptors, ulimorelin, capromorelin and CP464709, caused a rapid decrease in BP in the anaesthetized rat. The effect was not reduced by either of two GHSR1a antagonists, JMV2959 or YIL781, at doses that blocked effects on colorectal motility, in vivo. The rapid hypotension was not mimicked by ghrelin, unacylated ghrelin or the unacylated ghrelin receptor agonist, AZP531. The early hypotension preceded a decrease in sympathetic nerve activity. Early hypotension was not reduced by hexamethonium or by baroreceptor (sino-aortic) denervation. Ulimorelin also relaxed isolated segments of rat mesenteric artery, and, less potently, relaxed aorta segments. The vascular relaxation was not reduced by JMV2959 or YIL781. Ulimorelin, capromorelin and CP464709 activated GHSR1a in transfected HEK293 cells at nanomolar concentrations. JMV2959 and YIL781 both antagonized effects in these cells, with their pA2 values at the GHSR1a receptor being 6.55 and 7.84. CONCLUSIONS AND IMPLICATIONS Our results indicate a novel vascular receptor or receptors whose activation by ulimorelin, capromorelin and CP464709 lowered BP. This receptor is activated by low MW GHSR1a agonists, but is not activated by ghrelin. PMID:24670149

  18. Sphingosine 1-phosphate (S1P) suppresses the collagen-induced activation of human platelets via S1P4 receptor.

    PubMed

    Onuma, Takashi; Tanabe, Kumiko; Kito, Yuko; Tsujimoto, Masanori; Uematsu, Kodai; Enomoto, Yukiko; Matsushima-Nishiwaki, Rie; Doi, Tomoaki; Nagase, Kiyoshi; Akamatsu, Shigeru; Tokuda, Haruhiko; Ogura, Shinji; Iwama, Toru; Kozawa, Osamu; Iida, Hiroki

    2017-08-01

    Sphingosine 1-phosphate (S1P) is as an extracellular factor that acts as a potent lipid mediator by binding to specific receptors, S1P receptors (S1PRs). However, the precise role of S1P in human platelets that express S1PRs has not yet been fully clarified. We previously reported that heat shock protein 27 (HSP27) is released from human platelets accompanied by its phosphorylation stimulated by collagen. In the present study, we investigated the effect of S1P on the collagen-induced platelet activation. S1P pretreatment markedly attenuated the collagen-induced aggregation. Co-stimulation with S1P and collagen suppressed collagen-induced platelet activation, but the effect was weaker than that of S1P-pretreatment. The collagen-stimulated secretion of platelet-derived growth factor (PDGF)-AB and the soluble CD40 ligand (sCD40L) release were significantly reduced by S1P. In addition, S1P suppressed the collagen-induced release of HSP27 as well as the phosphorylation of HSP27. S1P significantly suppressed the collagen-induced phosphorylation of p38 mitogen-activated protein kinase. S1P increased the levels of GTP-bound Gαi and GTP-bound Gα13 coupled to S1PPR1 and/or S1PR4. CYM50260, a selective S1PR4 agonist, but not SEW2871, a selective S1PR1 agonist, suppressed the collagen-stimulated platelet aggregation, PDGF-AB secretion and sCD40L release. In addition, CYM50260 reduced the release of phosphorylated-HSP27 by collagen as well as the phosphorylation of HSP27. The selective S1PR4 antagonist CYM50358, which failed to affect collagen-induced HSP27 phosphorylation, reversed the S1P-induced attenuation of HSP27 phosphorylation by collagen. These results strongly suggest that S1P inhibits the collagen-induced human platelet activation through S1PR4 but not S1PR1. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Angiotensin II type 1 and type 2 receptor-induced cell signaling.

    PubMed

    Akazawa, Hiroshi; Yano, Masamichi; Yabumoto, Chizuru; Kudo-Sakamoto, Yoko; Komuro, Issei

    2013-01-01

    The octapeptide angiotensin II (Ang II) plays a homeostatic role in the regulation of blood pressure and water and electrolyte balance, and also contributes to the progression of cardiovascular remodeling. Ang II activates Ang II type 1 (AT1) receptor and type 2 (AT2) receptor, both of which belong to the seven-transmembrane, G protein-coupled receptor family. Most of the actions of Ang II such as promotion of cellular prolifaration, hypertrophy, and fibrosis are mediated by AT1 receptor. However, in some pathological situations, AT2 receptor shows an increase in tissue expression and functions to antagonize the actions induced by AT1 receptor. Recent studies have advanced our understanding of the molecular mechanisms underlying receptor activation and signal transduction of AT1 and AT2 receptor in the cardiovascular system.

  20. Structure-activity relationship of 5-chloro-2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole analogues as 5-HT(6) receptor agonists.

    PubMed

    Mattsson, Cecilia; Svensson, Peder; Boettcher, Henning; Sonesson, Clas

    2013-05-01

    To further investigate the structure-activity relationship (SAR) of the 5-hydroxytryptamine type 6 (5-HT6) receptor agonist 5-chloro-2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (EMD386088, 6), a series of 2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles were synthesized, and in vitro affinity to, and functional activity at 5-HT6 receptors was tested. We focused on substituents made at the indole N(1)-, 2- and 5-positions and these were found to not only influence the affinity at 5-HT6 receptors but also the intrinsic activity leading to antagonists, partial agonists and full agonists. In order for a compound to demonstrate potent 5-HT6 receptor agonist properties, the indole N(1) should be unsubstituted, an alkyl group such as 2-methyl is needed and finally halogen substituents in the indole 5-position (fluoro, chloro or, bromo) were essential requirements. However, the introduction of a benzenesulfonyl group at N(1)-position switched the full agonist 6 to be a 5-HT6 receptor antagonist (30). A few compounds within the 2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles were also screened for off-targets and generally they displayed low affinity for other 5-HT subtypes and serotonin transporter protein (SERT). Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  1. Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors activate the aryl hydrocarbon receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Moyer, Benjamin J.

    Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in the immune system by regulating tryptophan levels and T cell differentiation. Several tumor types overexpress IDO1 to avoid immune surveillance making IDO1 of interest as a target for therapeutic intervention. As a result, several IDO1 inhibitors are currently being tested in clinical trials for cancer treatment as well as several other diseases. Many of the IDO1 inhibitors in clinical trials naturally bear structural similarities to the IDO1 substrate tryptophan, as such, they fulfill many of the structural and functional criteria as potential AHR ligands. Using mouse and human cell-based luciferase genemore » reporter assays, qPCR confirmation experiments, and CYP1A1 enzyme activity assays, we report that some of the promising clinical IDO1 inhibitors also act as agonists for the aryl hydrocarbon receptor (AHR), best known for its roles in xenobiotic metabolism and as another key regulator of the immune response. The dual role as IDO antagonist and AHR agonist for many of these IDO target drugs should be considered for full interrogation of their biological mechanisms and clinical outcomes. - Highlights: • Indoleamine-2,3-dioxygenase 1 (IDO1) inhibitors are in cancer clinical trials. • Some IDO1 inhibitors also potently activate AHR signaling. • The dual role of the IDO1 inhibitors may explain some past paradoxical findings. • AHR induction studies must be included in assessing clinical suitability.« less

  2. The Second Transmembrane Domain of the Human Type 1 Angiotensin II Receptor Participates in the Formation of the Ligand Binding Pocket and Undergoes Integral Pivoting Movement during the Process of Receptor Activation*

    PubMed Central

    Domazet, Ivana; Holleran, Brian J.; Martin, Stéphane S.; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

    2009-01-01

    The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT1 receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT1, L81C-AT1, A85C-AT1, T88C-AT1, and A89C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant D74C-N111G-AT1 became insensitive to MTSEA, whereas mutant L81C-N111G-AT1 lost some sensitivity and mutant V86C-N111G-AT1 became sensitive to MTSEA. Our results suggest that constitutive activation of the AT1 receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket. PMID:19276075

  3. The second transmembrane domain of the human type 1 angiotensin II receptor participates in the formation of the ligand binding pocket and undergoes integral pivoting movement during the process of receptor activation.

    PubMed

    Domazet, Ivana; Holleran, Brian J; Martin, Stéphane S; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

    2009-05-01

    The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT(1) receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT(1), L81C-AT(1), A85C-AT(1), T88C-AT(1), and A89C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant D74C-N111G-AT(1) became insensitive to MTSEA, whereas mutant L81C-N111G-AT(1) lost some sensitivity and mutant V86C-N111G-AT(1) became sensitive to MTSEA. Our results suggest that constitutive activation of the AT(1) receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket.

  4. Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

    PubMed

    Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

    1995-03-01

    Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context

  5. A selective peroxisome proliferator-activated receptor-gamma modulator, telmisartan, binds to the receptor in a different fashion from thiazolidinediones.

    PubMed

    Tagami, Tetsuya; Yamamoto, Hiroyuki; Moriyama, Kenji; Sawai, Kuniko; Usui, Takeshi; Shimatsu, Akira; Naruse, Mitsuhide

    2009-02-01

    Angiotensin type 1 receptor blockers are widely used for the treatment of hypertension, and one angiotensin type 1 receptor blocker, telmisartan, specifically activates the peroxisome proliferator-activated receptor (PPAR)-gamma. We studied the impact of PPARgamma mutants on transcriptional control and interaction with cofactors to elucidate differences in the molecular mechanism between telmisartan and other PPARgamma agonists, thiazolidinediones (TZDs). We created several amino acid substitutions in the ligand binding domain of PPARgamma that, based on molecular modeling, may affect the binding of these agents. In transient expression experiments, wild-type PPARgamma-mediated transcription stimulated by telmisartan was more than one third of that stimulated by TZDs. The activation stimulated by TZDs was impaired, whereas activation stimulated by telmisartan was retained, in the H323Y, S342A, and H449A mutants. In the Y473A mutant, the TZD-induced activation was further impaired and lower than that of telmisartan-induced activation. Coexpression of coactivators enhanced the activation by both telmisartan and TZDs, but activation by telmisartan always exceeded that of TZDs in the Y473A mutant. Based on a mammalian two-hybrid assay, the interaction with corepressors was retained in Y473A. Telmisartan and TZDs, but not 9cis retinoic acid, dissociated corepressors from the wild-type PPARgamma. Telmisartan most effectively dissociated corepressors from Y473A. The interaction with coactivators was enhanced by TZD activation of wild-type PPARgamma and both telmisartan and TZD activation of Y473A. Thus, the Y473A mutant is selectively stimulated by telmisartan but not TZDs, suggesting that telmisartan and TZDs have differential effects on the transcriptional control. In conclusion, these PPARgamma mutants could be powerful tools for developing novel therapeutic agents that retain the metabolic efficacy of PPARgamma activation with fewer adverse effects, such as the

  6. Presynaptic M1, M2, and A1 receptors play roles in tetanic fade induced by pancuronium or cisatracurium.

    PubMed

    Bornia, Elaine Campana Sanches; Bando, Erika; Machinski, Miguel; Pereira, Monalisa Wolski; Alves-Do-Prado, Wilson

    2009-01-01

    We investigated whether presynaptic facilitatory M1 and/or inhibitory M2 muscarinic receptors contributed to pancuronium- and cisatracurium-induced tetanic fade. Phrenic nerve-diaphragm muscle preparations of rats were indirectly stimulated with tetanic frequency (75 +/- 3.3 Hz; mean +/- SD). Doses of pancuronium, cisatracurium, hexamethonium, and d-tubocurarine for producing approximately 25% fade were determined. The effects of pirenzepine and methoctramine, blockers of presynaptic M1 and M2 receptors, respectively, on the tetanic fade were investigated. The concentrations required for approximately 25% fade were 413 microM for hexamethonium (26.8 +/- 2.4% 4% fade), 55 nM for d-tubocurarine (28.7 +/- 2.55% fade), 0.32 microM for pancuronium (25.4 +/- 2.2% fade), and 0.32 microM for cisatracurium (24.7 +/- 0.8% fade). Pirenzepine or methoctramine alone did not produce the fade. Methoctramine, 1 microM, attenuated the fade induced by hexamethonium (to 16.0 +/- 2.5% fade), d-tubocurarine (to 6.0 +/- 1.6 fade), pancuronium (to 8.0 +/- 4.0% fade), and cisatracurium (to 11.0 +/- 3.3% fade). 10 nM pirenzepine attenuated only the fades produced by pancuronium (to 5.0 +/- 0.11% fade) and cisatracurium (to 13.3 +/- 5.3% fade). Cisatracurium (0.32 microM) showed antiacetylcholinesterase activity (in plasma, 14.2 +/- 1.6%; 6%; in erythrocyt 17.2 +/- 2.66%) similar to that of pancuronium (0.32 microM). The selective A1 receptor blocker, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 2.5 nM), also attenuated the fades induced by pancuronium and cisatracurium. The tetanic fades produced by pancuronium and cisatracurium depend on the activation of presynaptic inhibitory M2 receptors; these agents also have anticholinesterase activities. The fades induced by these agents also depend on the activation of presynaptic inhibitory A1 receptors through the activation of stimulatory M1 receptors by acetylcholine.

  7. Human GH Receptor-IGF-1 Receptor Interaction: Implications for GH Signaling

    PubMed Central

    Gan, Yujun; Buckels, Ashiya; Liu, Ying; Zhang, Yue; Paterson, Andrew J.; Jiang, Jing; Zinn, Kurt R.

    2014-01-01

    GH signaling yields multiple anabolic and metabolic effects. GH binds the transmembrane GH receptor (GHR) to activate the intracellular GHR-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signals, including signal transducer and activator of transcription 5 (STAT5) activation and IGF-1 gene expression. Some GH effects are partly mediated by GH-induced IGF-1 via IGF-1 receptor (IGF-1R), a tyrosine kinase receptor. We previously demonstrated in non-human cells that GH causes formation of a GHR-JAK2-IGF-1R complex and that presence of IGF-1R (even without IGF-1 binding) augments proximal GH signaling. In this study, we use human LNCaP prostate cancer cells as a model system to further study the IGF-1R's role in GH signaling. GH promoted JAK2 and GHR tyrosine phosphorylation and STAT5 activation in LNCaP cells. By coimmunoprecipitation and a new split luciferase complementation assay, we find that GH augments GHR/IGF-1R complex formation, which is inhibited by a Fab of an antagonistic anti-GHR monoclonal antibody. Short hairpin RNA-mediated IGF-1R silencing in LNCaP cells reduced GH-induced GHR, JAK2, and STAT5 phosphorylation. Similarly, a soluble IGF-1R extracellular domain fragment (sol IGF-1R) interacts with GHR in response to GH and blunts GH signaling. Sol IGF-1R also markedly inhibits GH-induced IGF-1 gene expression in both LNCaP cells and mouse primary osteoblast cells. On the basis of these and other findings, we propose a model in which IGF-1R augments GH signaling by allowing a putative IGF-1R-associated molecule that regulates GH signaling to access the activated GHR/JAK2 complex and envision sol IGF-1R as a dominant-negative inhibitor of this IGF-1R-mediated augmentation. Physiological implications of this new model are discussed. PMID:25211187

  8. Synthetic ligands of the elastin receptor induce elastogenesis in human dermal fibroblasts via activation of their IGF-1 receptors.

    PubMed

    Qa'aty, Nour; Vincent, Matthew; Wang, Yanting; Wang, Andrew; Mitts, Thomas F; Hinek, Aleksander

    2015-12-01

    We have previously reported that a mixture of peptides obtained after chemical or enzymatic degradation of bovine elastin, induced new elastogenesis in human skin. Now, we investigated the elastogenic potential of synthetic peptides mimicking the elastin-derived, VGVAPG sequence, IGVAPG sequence that we found in the rice bran, and a similar peptide, VGVTAG that we identified in the IGF-1-binding protein-1 (IGFBP-1). We now demonstrate that treatment with each of these xGVxxG peptides (recognizable by the anti-elastin antibody), up-regulated the levels of elastin-encoding mRNA, tropoelastin protein, and the deposition of new elastic fibers in cultures of human dermal fibroblasts and in cultured explants of human skin. Importantly, we found that such induction of new elastogenesis may involve two parallel signaling pathways triggered after activation of IGF-1 receptor. In the first one, the xGVxxG peptides interact with the cell surface elastin receptor, thereby causing the downstream activation of the c-Src kinase and a consequent cross-activation of the adjacent IGF-1R, even in the absence of its principal ligand. In the second pathway their hydrophobic association with the N-terminal domain (VGVTAG) of the serum-derived IGFBP-1 induces conformational changes of this IGF-1 chaperone allowing for the release of its cargo and a consequent ligand-specific phosphorylation of IGF-1R. We present a novel, clinically relevant mechanism in which products of partial degradation of dermal elastin may stimulate production of new elastic fibers by dermal fibroblasts. Our findings particularly encourage the use of biologically safe synthetic xGVxxG peptides for regeneration of the injured or aged human skin. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. Human Freud-2/CC2D1B: a novel repressor of postsynaptic serotonin-1A receptor expression.

    PubMed

    Hadjighassem, Mahmoud R; Austin, Mark C; Szewczyk, Bernadeta; Daigle, Mireille; Stockmeier, Craig A; Albert, Paul R

    2009-08-01

    Altered expression of serotonin-1A (5-HT1A) receptors, both presynaptic in the raphe nuclei and post-synaptic in limbic and cortical target areas, has been implicated in mood disorders such as major depression and anxiety. Within the 5-HT1A receptor gene, a powerful dual repressor element (DRE) is regulated by two protein complexes: Freud-1/CC2D1A and a second, unknown repressor. Here we identify human Freud-2/CC2D1B, a Freud-1 homologue, as the second repressor. Freud-2 distribution was examined with Northern and Western blot, reverse transcriptase polymerase chain reaction, and immunohistochemistry/immunofluorescence; Freud-2 function was examined by electrophoretic mobility shift, reporter assay, and Western blot. Freud-2 RNA was widely distributed in brain and peripheral tissues. Freud-2 protein was enriched in the nuclear fraction of human prefrontal cortex and hippocampus but was weakly expressed in the dorsal raphe nucleus. Freud-2 immunostaining was co-localized with 5-HT1A receptors, neuronal and glial markers. In prefrontal cortex, Freud-2 was expressed at similar levels in control and depressed male subjects. Recombinant hFreud-2 protein bound specifically to 5' or 3' human DRE adjacent to the Freud-1 site. Human Freud-2 showed strong repressor activity at the human 5-HT1A or heterologous promoter in human HEK-293 5-HT1A-negative cells and neuronal SK-N-SH cells, a model of postsynaptic 5-HT1A receptor-positive cells. Furthermore, small interfering RNA knockdown of endogenous hFreud-2 expression de-repressed 5-HT1A promoter activity and increased levels of 5-HT1A receptor protein in SK-N-SH cells. Human Freud-2 binds to the 5-HT1A DRE and represses the human 5-HT1A receptor gene to regulate its expression in non-serotonergic cells and neurons.

  10. Shear stress reduces protease activated receptor-1 expression in human endothelial cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

  11. Enhanced effects of amphetamine but reduced effects of the hallucinogen, 5-MeO-DMT, on locomotor activity in 5-HT(1A) receptor knockout mice: implications for schizophrenia.

    PubMed

    van den Buuse, Maarten; Ruimschotel, Emma; Martin, Sally; Risbrough, Victoria B; Halberstadt, Adam L

    2011-01-01

    Serotonin-1A (5-HT(1A)) receptors may play a role in schizophrenia and the effects of certain antipsychotic drugs. However, the mechanism of interaction of 5-HT(1A) receptors with brain systems involved in schizophrenia, remains unclear. Here we show that 5-HT(1A) receptor knockout mice display enhanced locomotor hyperactivity to acute treatment with amphetamine, a widely used animal model of hyperdopaminergic mechanisms in psychosis. In contrast, the effect of MK-801 on locomotor activity, modeling NMDA receptor hypoactivity, was unchanged in the knockouts. The effect of the hallucinogen 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) was markedly reduced in 5-HT(1A) receptor knockout mice. There were no changes in apomorphine-induced disruption of PPI, a model of sensory gating deficits seen in schizophrenia. Similarly, there were no major changes in density of dopamine transporters (DAT) or dopamine D(1) or D(2) receptors which could explain the behavioural changes observed in 5-HT(1A) receptor knockout mice. These results extend our insight into the possible role of these receptors in aspects of schizophrenia. As also suggested by previous studies using agonist and antagonist drugs, 5-HT(1A) receptors may play an important role in hallucinations and to modulate dopaminergic activity in the brain. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Differential Modulation of Beta-Adrenergic Receptor Signaling by Trace Amine-Associated Receptor 1 Agonists

    PubMed Central

    Nürnberg, Daniela; Grüters, Annette; Führer-Sakel, Dagmar; Krude, Heiko; Köhrle, Josef; Schöneberg, Torsten; Biebermann, Heike

    2011-01-01

    Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes. PMID:22073124

  13. Constitutive androstane receptor upregulates Abcb1 and Abcg2 at the blood-brain barrier after CITCO activation.

    PubMed

    Lemmen, Julia; Tozakidis, Iasson E P; Bele, Prachee; Galla, Hans-Joachim

    2013-03-21

    ATP-driven efflux transporters are considered to be the major hurdle in the treatment of central nervous system (CNS) diseases. Abcb1 (P-glycoprotein) and Abcg2 (breast cancer resistance protein/brain multidrug resistance protein) belong to the best known ABC-transporters. These ABC-transporters limit the permeability of the blood-brain barrier and protect the brain against toxic compounds in the blood but on the other hand they also reduce the efficacy of CNS pharmacotherapy. Even after 40 years of extensive research, the regulatory mechanisms of these efflux transporters are still not completely understood. To unravel the efflux transporter regulation, we analyzed the effect of the nuclear receptor CAR (constitutive androstane receptor) on the expression of Abcb1 and Abcg2 in primary cultures of porcine brain capillary endothelial cells (PBCEC). CAR is a xenobiotic-activated transcription factor, which is, like the other important nuclear receptor pregnane X receptor (PXR), highly expressed in barrier tissue and known to be a positive regulator of ABC-transporters. We demonstrate that activation of porcine CAR by the human CAR (hCAR) ligand CITCO (6-(4-chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde) leads to an up-regulation of both transporters, whereas the mouse-specific CAR ligand TCPOBOP (1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene) had no effect on transporter expression. The stimulation of PBCEC with CITCO caused a significant up-regulation of both efflux-transporters on RNA-level, protein level and transport level. Furthermore the additional application of a CAR inhibitor significantly decreased the transporter expression to control niveau. In conclusion our data prove CAR activation only by the human ligand CITCO leading to an increased ABC-transporter expression and transport activity. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Proteinase-Activated Receptor 1 (PAR1) Regulates Leukemic Stem Cell Functions

    PubMed Central

    Bäumer, Nicole; Krause, Annika; Köhler, Gabriele; Lettermann, Stephanie; Evers, Georg; Hascher, Antje; Bäumer, Sebastian; Berdel, Wolfgang E.

    2014-01-01

    External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1−/− hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1−/− leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance. PMID:24740120

  15. Proteinase-Activated Receptor 1 (PAR1) regulates leukemic stem cell functions.

    PubMed

    Bäumer, Nicole; Krause, Annika; Köhler, Gabriele; Lettermann, Stephanie; Evers, Georg; Hascher, Antje; Bäumer, Sebastian; Berdel, Wolfgang E; Müller-Tidow, Carsten; Tickenbrock, Lara

    2014-01-01

    External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1-/- hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1-/- leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.

  16. Activation of type 2 cannabinoid receptors (CB2R) promotes fatty acid oxidation through the SIRT1/PGC-1α pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zheng, Xuqin; Sun, Tao; Wang, Xiaodong, E-mail: xdwang666@hotmail.com

    2013-07-05

    Highlights: •TC, a CB2R specific agonist, stimulates SIRT1 activity by PKA/CREB pathway. •TC promotes PGC-1α transcriptional activity by increasing its deacetylation. •TC increases the expression of genes linked to FAO and promotes the rate of FAO. •The effects of TC in FAO are dependent on CB2R. •Suggesting CB2R as a target to treat diseases with lipid dysregulation. -- Abstract: Abnormal fatty acid oxidation has been associated with obesity and type 2 diabetes. At the transcriptional level, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) has been reported to strongly increase the ability of hormone nuclear receptors PPARα and ERRα to drive transcriptionmore » of fatty acid oxidation enzymes. In this study, we report that a specific agonist of the type 2 cannabinoid receptor (CB2R) can lead to fatty acid oxidation through the PGC-1α pathway. We have found that CB2R is expressed in differentiated C2C12 myotubes, and that use of the specific agonist trans-caryophyllene (TC) stimulates sirtuin 1 (SIRT1) deacetylase activity by increasing the phosphorylation of cAMP response element-binding protein (CREB), thus leading to increased levels of PGC-1α deacetylation. This use of TC treatment increases the expression of genes linked to the fatty acid oxidation pathway in a SIRT1/PGC-1α-dependent mechanism and also drastically accelerates the rate of complete fatty acid oxidation in C2C12 myotubes, neither of which occur when CB2R mRNA is knocked down using siRNA. These results reveal that activation of CB2R by a selective agonist promotes lipid oxidation through a signaling/transcriptional pathway. Our findings imply that pharmacological manipulation of CB2R may provide therapeutic possibilities to treat metabolic diseases associated with lipid dysregulation.« less

  17. Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-{kappa}B ligand (RANKL) expression in rheumatoid arthritis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Takeshita, Harunori; Kitano, Masayasu, E-mail: mkitano6@hyo-med.ac.jp; Iwasaki, Tsuyoshi

    Highlights: Black-Right-Pointing-Pointer MH7A cells and CD4{sup +} T cells expressed S1P1 and RANKL. Black-Right-Pointing-Pointer S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells. Black-Right-Pointing-Pointer The effect of S1P in MH7A cells was inhibited by specific Gi/Go inhibitors. -- Abstract: Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-{kappa}B ligand (RANKL) in RA synoviocytes and CD4{sup +} T cells. We demonstrated MH7Amore » cells, a human RA synovial cell line, and CD4{sup +} T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-{alpha} in MH7A cells and CD4{sup +} T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4{sup +} T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.« less

  18. Activation of Adenosine A2A Receptors Inhibits Neutrophil Transuroepithelial Migration ▿

    PubMed Central

    Säve, Susanne; Mohlin, Camilla; Vumma, Ravi; Persson, Katarina

    2011-01-01

    Adenosine has been identified as a significant inhibitor of inflammation by acting on adenosine A2A receptors. In this study, we examined the role of adenosine and A2A receptors in the transmigration of human neutrophils across an in vitro model of the transitional bladder urothelium. Human uroepithelial cells (UROtsa) were grown on transwell inserts; uropathogenic Escherichia coli (UPEC) and neutrophils were added to the transwell system; and the number of migrating neutrophils was evaluated. Reverse transcription-PCR (RT-PCR), immunohistochemistry, and flow cytometry were used to investigate the expression of adenosine receptors, the epithelial adhesion molecule ICAM-1, and the neutrophil integrin CD11b. Levels of proinflammatory interleukin-8 (IL-8) and phosphorylated IκBα were measured by enzyme-linked immunosorbent assays (ELISA) and Luminex assays, respectively. The neutrophils expressed all four adenosine receptor subtypes (A1, A2A, A2B, and A3 receptors), but A3 receptors were not expressed by UROtsa cells. UPEC stimulated neutrophil transuroepithelial migration, which was significantly decreased in response to the specific A2A receptor agonist CGS 21680. The inhibitory effect of CGS 21680 on neutrophil migration was reversed by the A2A receptor antagonist SCH 58261. The production of chemotactic IL-8 and the expression of the adhesion molecule ICAM-1 or CD11b were not significantly affected by CGS 21680. However, a significant decrease in the level of phosporylated IκBα was revealed in response to CGS 21680. In conclusion, UPEC infection in vitro evoked neutrophil migration through a multilayered human uroepithelium. The UPEC-evoked neutrophil transmigration decreased in response to A2A receptor activation, possibly through inhibition of NF-κB signaling pathways. PMID:21646447

  19. Bile Acid-regulated Peroxisome Proliferator-activated Receptor-α (PPARα) Activity Underlies Circadian Expression of Intestinal Peptide Absorption Transporter PepT1/Slc15a1*

    PubMed Central

    Okamura, Ayako; Koyanagi, Satoru; Dilxiat, Adila; Kusunose, Naoki; Chen, Jia Jun; Matsunaga, Naoya; Shibata, Shigenobu; Ohdo, Shigehiro

    2014-01-01

    Digested proteins are mainly absorbed as small peptides composed of two or three amino acids. The intestinal absorption of small peptides is mediated via only one transport system: the proton-coupled peptide transporter-1 (PepT1) encoded from the soluble carrier protein Slc15a1. In mammals, intestinal expression of PepT1/Slc15a1 oscillates during the daily feeding cycle. Although the oscillation in the intestinal expression of PepT1/Slc15a1 is suggested to be controlled by molecular components of circadian clock, we demonstrated here that bile acids regulated the oscillation of PepT1/Slc15a1 expression through modulating the activity of peroxisome proliferator-activated receptor α (PPARα). Nocturnally active mice mainly consumed their food during the dark phase. PPARα activated the intestinal expression of Slc15a1 mRNA during the light period, and protein levels of PepT1 peaked before the start of the dark phase. After food intake, bile acids accumulated in intestinal epithelial cells. Intestinal accumulated bile acids interfered with recruitment of co-transcriptional activator CREB-binding protein/p300 on the promoter region of Slc15a1 gene, thereby suppressing PPARα-mediated transactivation of Slc15a1. The time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the intestinal expression of PepT1/Slc15a1 during the daily feeding cycle that led to circadian changes in the intestinal absorption of small peptides. These findings suggest a molecular clock-independent mechanism by which bile acid-regulated PPARα activity governs the circadian expression of intestinal peptide transporter. PMID:25016014

  20. Presynaptic PICK1 facilitates trafficking of AMPA-receptors between active zone and synaptic vesicle pool.

    PubMed

    Haglerød, C; Hussain, S; Nakamura, Y; Xia, J; Haug, F-M S; Ottersen, O P; Henley, J M; Davanger, S

    2017-03-06

    Previous studies have indicated that presynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) contribute to the regulation of neurotransmitter release. In hippocampal synapses, the presynaptic surface expression of several AMPAR subunits, including GluA2, is regulated in a ligand-dependent manner. However, the molecular mechanisms underlying the presynaptic trafficking of AMPARs are still unknown. Here, using bright-field immunocytochemistry, western blots, and quantitative immunogold electron microscopy of the hippocampal CA1 area from intact adult rat brain, we demonstrate the association of AMPA receptors with the presynaptic active zone and with small presynaptic vesicles, in Schaffer collateral synapses in CA1 of the hippocampus. Furthermore, we show that GluA2 and protein interacting with C kinase 1 (PICK1) are colocalized at presynaptic vesicles. Similar to postsynaptic mechanisms, overexpression of either PICK1 or pep2m, which inhibit the N-ethylmaleimide sensitive fusion protein (NSF)-GluA2 interaction, decreases the concentration of GluA2 in the presynaptic active zone membrane. These data suggest that the interacting proteins PICK1 and NSF act as regulators of presynaptic GluA2-containing AMPAR trafficking between the active zone and a vesicle pool that may provide the basis of presynaptic components of synaptic plasticity. Copyright © 2017 IBRO. All rights reserved.

  1. Estrogen anti-inflammatory activity on human monocytes is mediated through cross-talk between estrogen receptor ERα36 and GPR30/GPER1.

    PubMed

    Pelekanou, Vasiliki; Kampa, Marilena; Kiagiadaki, Foteini; Deli, Alexandra; Theodoropoulos, Panayiotis; Agrogiannis, George; Patsouris, Efstratios; Tsapis, Andreas; Castanas, Elias; Notas, George

    2016-02-01

    Estrogens are known modulators of monocyte/macrophage functions; however, the underlying mechanism has not been clearly defined. Recently, a number of estrogen receptor molecules and splice variants were identified that exert different and sometimes opposing actions. We assessed the expression of estrogen receptors and explored their role in mediating estrogenic anti-inflammatory effects on human primary monocytes. We report that the only estrogen receptors expressed are estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30/G-protein estrogen receptor 1, in a sex-independent manner. 17-β-Estradiol inhibits the LPS-induced IL-6 inflammatory response, resulting in inhibition of NF-κB transcriptional activity. This is achieved via a direct physical interaction of ligand-activated estrogen receptor-α 36-kDa splice variant with the p65 component of NF-κB in the nucleus. G-protein coupled receptor 30/G-protein estrogen receptor 1, which also physically interacts with estrogen receptor-α 36-kDa splice variant, acts a coregulator in this process, because its inhibition blocks the effect of estrogens on IL-6 expression. However, its activation does not mimic the effect of estrogens, on neither IL-6 nor NF-κB activity. Finally, we show that the estrogen receptor profile observed in monocytes is not modified during their differentiation to macrophages or dendritic cells in vitro and is shared in vivo by macrophages present in atherosclerotic plaques. These results position estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30 as important players and potential therapeutic targets in monocyte/macrophage-dependent inflammatory processes. © Society for Leukocyte Biology.

  2. The CB₁ cannabinoid receptor signals striatal neuroprotection via a PI3K/Akt/mTORC1/BDNF pathway.

    PubMed

    Blázquez, C; Chiarlone, A; Bellocchio, L; Resel, E; Pruunsild, P; García-Rincón, D; Sendtner, M; Timmusk, T; Lutz, B; Galve-Roperh, I; Guzmán, M

    2015-10-01

    The CB1 cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. In particular, the CB1 receptor is highly expressed in the basal ganglia, mostly on terminals of medium-sized spiny neurons, where it plays a key neuromodulatory function. The CB1 receptor also confers neuroprotection in various experimental models of striatal damage. However, the assessment of the physiological relevance and therapeutic potential of the CB1 receptor in basal ganglia-related diseases is hampered, at least in part, by the lack of knowledge of the precise mechanism of CB1 receptor neuroprotective activity. Here, by using an array of pharmacological, genetic and pharmacogenetic (designer receptor exclusively activated by designer drug) approaches, we show that (1) CB1 receptor engagement protects striatal cells from excitotoxic death via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin complex 1 pathway, which, in turn, (2) induces brain-derived neurotrophic factor (BDNF) expression through the selective activation of BDNF gene promoter IV, an effect that is mediated by multiple transcription factors. To assess the possible functional impact of the CB1/BDNF axis in a neurodegenerative-disease context in vivo, we conducted experiments in the R6/2 mouse, a well-established model of Huntington's disease, in which the CB1 receptor and BDNF are known to be severely downregulated in the dorsolateral striatum. Adeno-associated viral vector-enforced re-expression of the CB1 receptor in the dorsolateral striatum of R6/2 mice allowed the re-expression of BDNF and the concerted rescue of the neuropathological deficits in these animals. Collectively, these findings unravel a molecular link between CB1 receptor activation and BDNF expression, and support the relevance of the CB1/BDNF axis in promoting striatal neuron survival.

  3. Evidence for two distinct phosphorylation pathways activated by high affinity immunoglobulin E receptors.

    PubMed

    Adamczewski, M; Paolini, R; Kinet, J P

    1992-09-05

    The high affinity receptor for immunoglobulin (Ig) E on mast cells, along with the antigen receptors on T and B cells and Fc receptors for IgG, belongs to a class of receptors which lack intrinsic kinase activity, but activate non-receptor tyrosine and serine/threonine kinases. Receptor engagement triggers a chain of signaling events leading from protein phosphorylation to activation of phosphatidylinositol-specific phospholipase C, an increase in intracellular calcium levels, and ultimately the activation of more specialized functions. IgE receptor disengagement leads to reversal of phosphorylation by undefined phosphatases and to inhibition of activation pathways. Here we show that phenylarsine oxide, a chemical which reacts with thiol groups and has been reported to inhibit tyrosine phosphatases, uncouples the IgE receptor-mediated phosphorylation signal from activation of phosphatidyl inositol metabolism, the increase in intracellular calcium levels, and serotonin release. Phenylarsine oxide inhibits neither the kinases (tyrosine and serine/threonine) phosphorylating the receptor and various cellular substrates nor, unexpectedly, the phosphatases responsible for the dephosphorylation following receptor disengagement. By contrast, it abolishes the receptor-mediated phosphorylation of phospholipase C-gamma 1, but not phospholipase C activity in vitro. Therefore the phosphorylation and activation of phospholipase C likely requires a phenylarsine oxide-sensitive element. Receptor aggregation thus activates at least two distinct phosphorylation pathways: a phenylarsine oxide-insensitive pathway leading to phosphorylation/dephosphorylation of the receptor and of various substrates and a sensitive pathway leading to phospholipase C-gamma 1 phosphorylation.

  4. Adenosine A2A receptors are required for glutamate mGluR5- and dopamine D1 receptor-evoked ERK1/2 phosphorylation in rat hippocampus: involvement of NMDA receptor.

    PubMed

    Krania, Paraskevi; Dimou, Eleni; Bantouna, Maria; Kouvaros, Stylianos; Tsiamaki, Eirini; Papatheodoropoulos, Costas; Sarantis, Konstantinos; Angelatou, Fevronia

    2018-05-01

    Interaction between mGluR5 and NMDA receptors (NMDAR) is vital for synaptic plasticity and cognition. We recently demonstrated that stimulation of mGluR5 enhances NMDAR responses in hippocampus by phosphorylating NR2B(Tyr1472) subunit, and this reaction was enabled by adenosine A 2A receptors (A 2A R) (J Neurochem, 135, 2015, 714). In this study, by using in vitro phosphorylation and western blot analysis in hippocampal slices of male Wistar rats, we show that mGluR5 stimulation or mGluR5/NMDARs co-stimulation synergistically activate ERK1/2 signaling leading to c-Fos expression. Interestingly, both reactions are under the permissive control of endogenous adenosine acting through A 2A Rs. Moreover, mGluR5-mediated ERK1/2 phosphorylation depends on NMDAR, which however exhibits a metabotropic way of function, since no ion influx through its ion channel is required. Furthermore, our results demonstrate that mGluR5 and mGluR5/NMDAR-evoked ERK1/2 activation correlates well with the mGluR5/NMDAR-evoked NR2B(Tyr1472) phosphorylation, since both phenomena coincide temporally, are Src dependent, and are both enabled by A 2A Rs. This indicates a functional involvement of NR2B(Tyr1472) phosphorylation in the ERK1/2 activation. Our biochemical results are supported by electrophysiological data showing that in CA1 region of hippocampus, the theta burst stimulation (TBS)-induced long-term potentiation coincides temporally with an increase in ERK1/2 activation and both phenomena are dependent on the tripartite A 2A , mGlu5, and NMDARs. Furthermore, we show that the dopamine D1 receptors evoked ERK1/2 activation as well as the NR2B(Tyr1472) phosphorylation are also regulated by endogenous adenosine and A 2A Rs. In conclusion, our results highlight the A 2A Rs as a crucial regulator not only for NMDAR responses, but also for regulating ERK1/2 signaling and its downstream pathways, leading to gene expression, synaptic plasticity, and memory consolidation. © 2017 International

  5. A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9.

    PubMed

    Demoulin, J B; Uyttenhove, C; Van Roost, E; DeLestré, B; Donckers, D; Van Snick, J; Renauld, J C

    1996-09-01

    Interleukin-9 (IL-9), a T-cell-derived cytokine, interacts with a specific receptor associated with the IL-2 receptor gamma chain. In this report, we analyze the functional domains of the human IL-9 receptor transfected into mouse lymphoid cell lines. Three different functions were examined: growth stimulation in factor-dependent pro-B Ba/F3 cells, protection against dexamethasone-induced apoptosis, and Ly-6A2 induction in BW5147 lymphoma cells. The results indicated that a single tyrosine, at position 116 in the cytoplasmic domain, was required for all three activities. In addition, we observed that human IL-9 reduced the proliferation rate of transfected BW5147 cells, an effect also dependent on the same tyrosine. This amino acid was necessary for IL-9-mediated tyrosine phosphorylation of the receptor and for STAT activation but not for IRS-2/4PS activation or for JAK1 phosphorylation, which depended on a domain closer to the plasma membrane. We also showed that JAK1 was constitutively associated with the IL-9 receptor. Activated STAT complexes induced by IL-9 were found to contain STAT1, STAT3, and STAT5 transcription factors. Moreover, sequence homologies between human IL-9 receptor tyrosine 116 and tyrosines (of other receptors activating STAT3 and STAT5 were observed. Taken together, these data indicate that a single tyrosine of the IL-9 receptor, required for activation of three different STAT proteins, is necessary for distinct activities of this cytokine, including proliferative responses.

  6. Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Booe, Jason M.; Walker, Christopher S.; Barwell, James

    Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less

  7. Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

    DOE PAGES

    Booe, Jason M.; Walker, Christopher S.; Barwell, James; ...

    2015-05-14

    Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less

  8. Structural prerequisites for G-protein activation by the neurotensin receptor

    DOE PAGES

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; ...

    2015-07-24

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A 3.49, L310A 6.37, F358A 7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F358 7.42 causes the conserved W321 6.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocketmore » and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L310 6.37 side chain dictates the position of R167 3.50 of the highly conserved D/ERY motif. These residues, together with the presence of E166 3.49 provide determinants for G-protein activation by NTSR1.« less

  9. Structural prerequisites for G-protein activation by the neurotensin receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A 3.49, L310A 6.37, F358A 7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F358 7.42 causes the conserved W321 6.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocketmore » and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L310 6.37 side chain dictates the position of R167 3.50 of the highly conserved D/ERY motif. These residues, together with the presence of E166 3.49 provide determinants for G-protein activation by NTSR1.« less

  10. Altered striatal function in a mutant mouse lacking D1A dopamine receptors.

    PubMed Central

    Drago, J; Gerfen, C R; Lachowicz, J E; Steiner, H; Hollon, T R; Love, P E; Ooi, G T; Grinberg, A; Lee, E J; Huang, S P

    1994-01-01

    Of the five known dopamine receptors, D1A and D2 represent the major subtypes expressed in the striatum of the adult brain. Within the striatum, these two subtypes are differentially distributed in the two main neuronal populations that provide direct and indirect pathways between the striatum and the output nuclei of the basal ganglia. Movement disorders, including Parkinson disease and various dystonias, are thought to result from imbalanced activity in these pathways. Dopamine regulates movement through its differential effects on D1A receptors expressed by direct output neurons and D2 receptors expressed by indirect output neurons. To further examine the interaction of D1A and D2 neuronal pathways in the striatum, we used homologous recombination to generate mutant mice lacking functional D1A receptors (D1A-/-). D1A-/- mutants are growth retarded and die shortly after weaning age unless their diet is supplemented with hydrated food. With such treatment the mice gain weight and survive to adulthood. Neurologically, D1A-/- mice exhibit normal coordination and locomotion, although they display a significant decrease in rearing behavior. Examination of the striatum revealed changes associated with the altered phenotype of these mutants. D1A receptor binding was absent in striatal sections from D1A-/- mice. Striatal neurons normally expressing functional D1A receptors are formed and persist in adult homozygous mutants. Moreover, substance P mRNA, which is colocalized specifically in striatal neurons with D1A receptors, is expressed at a reduced level. In contrast, levels of enkephalin mRNA, which is expressed in striatal neurons with D2 receptors, are unaffected. These findings show that D1A-/- mice exhibit selective functional alterations in the striatal neurons giving rise to the direct striatal output pathway. Images Fig. 2 Fig. 4 PMID:7809078

  11. Stat1-Vitamin D Receptor Interactions Antagonize 1,25-Dihydroxyvitamin D Transcriptional Activity and Enhance Stat1-Mediated Transcription

    PubMed Central

    Vidal, Marcos; Ramana, Chilakamarti V.; Dusso, Adriana S.

    2002-01-01

    The cytokine gamma interferon (IFN-γ) and the calcitropic steroid hormone 1,25-dihydroxyvitamin D (1,25D) are activators of macrophage immune function. In sarcoidosis, tuberculosis, and several granulomatoses, IFN-γ induces 1,25D synthesis by macrophages and inhibits 1,25D induction of 24-hydroxylase, a key enzyme in 1,25D inactivation, causing high levels of 1,25D in serum and hypercalcemia. This study delineates IFN-γ-1,25D cross talk in human monocytes-macrophages. Nuclear accumulation of Stat1 and vitamin D receptor (VDR) by IFN-γ and 1,25D promotes protein-protein interactions between Stat1 and the DNA binding domain of the VDR. This prevents VDR-retinoid X receptor (RXR) binding to the vitamin D-responsive element, thus diverting the VDR from its normal genomic target on the 24-hydroxylase promoter and antagonizing 1,25D-VDR transactivation of this gene. In contrast, 1,25D enhances IFN-γ action. Stat1-VDR interactions, by preventing Stat1 deactivation by tyrosine dephosphorylation, cooperate with IFN-γ/Stat1-induced transcription. This novel 1,25D-IFN-γ cross talk explains the pathogenesis of abnormal 1,25D homeostasis in granulomatous processes and provides new insights into 1,25D immunomodulatory properties. PMID:11909970

  12. Diarylureas as allosteric modulators of the cannabinoid CB1 receptor: structure-activity relationship studies on 1-(4-chlorophenyl)-3-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]phenyl}urea (PSNCBAM-1).

    PubMed

    German, Nadezhda; Decker, Ann M; Gilmour, Brian P; Gay, Elaine A; Wiley, Jenny L; Thomas, Brian F; Zhang, Yanan

    2014-09-25

    The recent discovery of allosteric modulators of the CB1 receptor including PSNCBAM-1 (4) has generated significant interest in CB1 receptor allosteric modulation. Here in the first SAR study on 4, we have designed and synthesized a series of analogs focusing on modifications at two positions. Pharmacological evaluation in calcium mobilization and binding assays revealed the importance of alkyl substitution at the 2-aminopyridine moiety and electron deficient aromatic groups at the 4-chlorophenyl position for activity at the CB1 receptor, resulting in several analogs with comparable potency to 4. These compounds increased the specific binding of [(3)H]CP55,940, in agreement with previous reports. Importantly, 4 and two analogs dose-dependently reduced the Emax of the agonist curve in the CB1 calcium mobilization assays, confirming their negative allosteric modulator characteristics. Given the side effects associated with CB1 receptor orthosteric antagonists, negative allosteric modulators provide an alternative approach to modulate the pharmacologically important CB1 receptor.

  13. Sigma-1 Receptor Antagonist BD1047 Reduces Mechanical Allodynia in a Rat Model of Bone Cancer Pain through the Inhibition of Spinal NR1 Phosphorylation and Microglia Activation

    PubMed Central

    Zhu, Shanshan; Wang, Chenchen; Han, Yuan; Song, Chao; Hu, Xueming; Liu, Yannan

    2015-01-01

    Previous studies have demonstrated that sigma-1 receptor plays important roles in the induction phase of rodent neuropathic pain; however, whether it is involved in bone cancer pain (BCP) and the underlying mechanisms remain elusive. The aim of this study was to examine the potential role of the spinal sigma-1 receptor in the development of bone cancer pain. Walker 256 mammary gland carcinoma cells were implanted into the intramedullary space of the right tibia of Sprague-Dawley rats to induce ongoing bone cancer-related pain behaviors; our findings indicated that, on days 7, 10, 14, and 21 after operation, the expression of sigma-1 receptor in the spinal cord was higher in BCP rats compared to the sham rats. Furthermore, intrathecal injection of 120 nmol of sigma-1 receptor antagonist BD1047 on days 5, 6, and 7 after operation attenuated mechanical allodynia as well as the associated induction of c-Fos and activation of microglial cells, NR1, and the subsequent Ca2+-dependent signals of BCP rats. These results suggest that sigma-1 receptor is involved in the development of bone cancer pain and that targeting sigma-1 receptor may be a new strategy for the treatment of bone cancer pain. PMID:26696751

  14. Pyropheophorbide-a methyl ester-mediated photosensitization activates transcription factor NF-kappaB through the interleukin-1 receptor-dependent signaling pathway.

    PubMed

    Matroule, J Y; Bonizzi, G; Morlière, P; Paillous, N; Santus, R; Bours, V; Piette, J

    1999-01-29

    Pyropheophorbide-a methyl ester (PPME) is a second generation of photosensitizers used in photodynamic therapy. We demonstrated that PPME photosensitization activated NF-kappaB transcription factor in colon cancer cells. Unexpectedly, this activation occurred in two separate waves, i.e. a rapid and transient one and a second slower but sustained phase. The former was due to photosensitization by PPME localized in the cytoplasmic membrane which triggered interleukin-1 receptor internalization and the transduction pathways controlled by the interleukin-1 type I receptor. Indeed, TRAF6 dominant negative mutant abolished NF-kappaB activation by PPME photosensitization, and TRAF2 dominant negative mutant was without any effect, and overexpression of IkappaB kinases increased gene transcription controlled by NF-kappaB. Oxidative stress was not likely involved in the activation. On the other hand, the slower and sustained wave could be the product of the release of ceramide through activation of the acidic sphingomyelinase. PPME localization within the lysosomal membrane could explain why ceramide acted as second messenger in NF-kappaB activation by PPME photosensitization. These data will allow a better understanding of the molecular basis of tumor eradication by photodynamic therapy, in particular the importance of the host cell response in the treatment.

  15. A Role for D1 Dopamine Receptors in Striatal Methamphetamine-Induced Neurotoxicity

    PubMed Central

    Friend, Danielle M.; Keefe, Kristen A.

    2015-01-01

    Methamphetamine (METH) exposure results in long-term damage to the dopamine system in both human METH abusers and animal models. One factor that has been heavily implicated in this METH-induced damage to the dopaminergic system is the activation of D1 Dopamine (DA) receptors. However, a significant caveat to the studies investigating the role of the receptor in such toxicity is that genetic and pharmacological manipulations of the D1 DA receptor also mitigate METH-induced hyperthermia. Importantly, METH-induced hyperthermia is tightly associated with the neurotoxicity, such that simply cooling animals during METH exposure protects against the neurotoxicity. Therefore, it is difficult to determine whether D1 DA receptors per se play an important role in METH-induced neurotoxicity or whether the protection observed simply resulted from a mitigation of METH-induced hyperthermia. To answer this important question, the current study infused a D1 DA receptor antagonist into striatum during METH exposure while controlling for METH-induced hyperthermia. Here we found that even when METH-induced hyperthermia is maintained, the coadministration of a D1 DA receptor antagonist protects against METH-induced neurotoxicity, strongly suggesting that D1 DA receptors play an important role in METH-induced neurotoxicity apart from the mitigation of METH-induced hyperthermia. PMID:23994061

  16. Tamoxifen and ICI 182, 780 activate hypothalamic G protein-coupled estrogen receptor 1 to rapidly facilitate lordosis in female rats

    PubMed Central

    Long, Nathan; Long, Bertha; Mana, Asma; Le, Dream; Nguyen, Lam; Chokr, Sima; Sinchak, Kevin

    2017-01-01

    In the female rat, sexual receptivity (lordosis) can be facilitated by sequential activation of estrogen receptor (ER) α and G protein-coupled estrogen receptor 1 (GPER) by estradiol. In the estradiol benzoate (EB) primed ovariectomized (OVX) rat, EB initially binds to ERα in the plasma membrane that complexes with and transactivates metabotropic glutamate receptor 1a to activate β-endorphin neurons in the arcuate nucleus of the hypothalamus (ARH) that project to the medial preoptic nucleus (MPN). This activates MPN μ-opioid receptors (MOP), inhibiting lordosis. Infusion of non-esterified 17β-estradiol into the ARH rapidly reduces MPN MOP activation and facilitates lordosis via GPER. Tamoxifen (TAM) and ICI 182,780 (ICI) are selective estrogen receptor modulators that activate GPER. Therefore, we tested the hypothesis that TAM and ICI rapidly facilitate lordosis via activation of GPER in the ARH. Our first experiment demonstrated that injection of TAM intraperitoneal, or ICI into the lateral ventricle, deactivated MPN MOP and facilitated lordosis in EB-primed rats. We then tested whether TAM and ICI were acting rapidly through a GPER dependent pathway in the ARH. In EB-primed rats, ARH infusion of either TAM or ICI facilitated lordosis and reduced MPN MOP activation within 30 minutes compared to controls. These effects were blocked by pretreatment with the GPER antagonist, G15. Our findings demonstrate that TAM and ICI deactivate MPN MOP and facilitate lordosis in a GPER dependent manner. Thus, TAM and ICI may activate GPER in the CNS to produce estrogenic actions in neural circuits that modulate physiology and behavior. PMID:28063803

  17. Tamoxifen and ICI 182,780 activate hypothalamic G protein-coupled estrogen receptor 1 to rapidly facilitate lordosis in female rats.

    PubMed

    Long, Nathan; Long, Bertha; Mana, Asma; Le, Dream; Nguyen, Lam; Chokr, Sima; Sinchak, Kevin

    2017-03-01

    In the female rat, sexual receptivity (lordosis) can be facilitated by sequential activation of estrogen receptor (ER) α and G protein-coupled estrogen receptor 1 (GPER) by estradiol. In the estradiol benzoate (EB) primed ovariectomized (OVX) rat, EB initially binds to ERα in the plasma membrane that complexes with and transactivates metabotropic glutamate receptor 1a to activate β-endorphin neurons in the arcuate nucleus of the hypothalamus (ARH) that project to the medial preoptic nucleus (MPN). This activates MPN μ-opioid receptors (MOP), inhibiting lordosis. Infusion of non-esterified 17β-estradiol into the ARH rapidly reduces MPN MOP activation and facilitates lordosis via GPER. Tamoxifen (TAM) and ICI 182,780 (ICI) are selective estrogen receptor modulators that activate GPER. Therefore, we tested the hypothesis that TAM and ICI rapidly facilitate lordosis via activation of GPER in the ARH. Our first experiment demonstrated that injection of TAM intraperitoneal, or ICI into the lateral ventricle, deactivated MPN MOP and facilitated lordosis in EB-primed rats. We then tested whether TAM and ICI were acting rapidly through a GPER dependent pathway in the ARH. In EB-primed rats, ARH infusion of either TAM or ICI facilitated lordosis and reduced MPN MOP activation within 30min compared to controls. These effects were blocked by pretreatment with the GPER antagonist, G15. Our findings demonstrate that TAM and ICI deactivate MPN MOP and facilitate lordosis in a GPER dependent manner. Thus, TAM and ICI may activate GPER in the CNS to produce estrogenic actions in neural circuits that modulate physiology and behavior. Published by Elsevier Inc.

  18. Detection of receptor ligands by monitoring selective stabilization of a Renilla luciferase-tagged, constitutively active mutant, G-protein-coupled receptor

    PubMed Central

    Ramsay, Douglas; Bevan, Nicola; Rees, Stephen; Milligan, Graeme

    2001-01-01

    The wild-type β2-adrenoceptor and a constitutively active mutant of this receptor were C-terminally tagged with luciferase from the sea pansy Renilla reniformis. C-terminal addition of Renilla luciferase did not substantially alter the levels of expression of either form of the receptor, the elevated constitutive activity of the mutant β2-adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs. Treatment of cells expressing constitutively active mutant β2-adrenoceptor-Renilla luciferase with antagonist/inverse agonist ligands resulted in upregulation of levels of this polypeptide which could be monitored by the elevated luciferase activity. The pEC50 for ligand-induced luciferase upregulation and ligand affinity to bind the receptor were highly correlated. Similar upregulation could be observed following sustained treatment with agonist ligands. These effects were only observed at a constitutively active mutant of the β2-adrenoceptor. Co-expression of the wild-type β2-adrenoceptor C-terminally tagged with the luciferase from Photinus pyralis did not result in ligand-induced upregulation of the levels of activity of this luciferase. Co-expression of the constitutively active mutant β2-adrenoceptor-Renilla luciferase and an equivalent mutant of the α1b-adrenoceptor C-terminally tagged with green fluorescent protein allowed pharmacological selectivity of adrenoceptor antagonists to be demonstrated. This approach offers a sensitive and convenient means, which is amenable to high throughput analysis, to monitor ligand binding to a constitutively active mutant receptor. As no prior knowledge of receptor ligands is required this approach may be suitable to identify ligands at orphan G protein-coupled receptors. PMID:11350868

  19. The type I BMP receptors, Bmpr1a and Acvr1, activate multiple signaling pathways to regulate lens formation

    PubMed Central

    Rajagopal, Ramya; Huang, Jie; Dattilo, Lisa K.; Kaartinen, Vesa; Mishina, Yuji; Deng, Chu-Xia; Umans, Lieve; Zwijsen, An; Roberts, Anita B.; Beebe, David C.

    2009-01-01

    BMPs play multiple roles in development and BMP signaling is essential for lens formation. However, the mechanisms by which BMP receptors function in vertebrate development are incompletely understood. To determine the downstream effectors of BMP signaling and their functions in the ectoderm that will form the lens, we deleted the genes encoding the type I BMP receptors, Bmpr1a and Acvr1, and the canonical transducers of BMP signaling, Smad4, Smad1 and Smad5. Bmpr1a and Acvr1 regulated cell survival and proliferation, respectively. Absence of both receptors interfered with the expression of proteins involved in normal lens development and prevented lens formation, demonstrating that BMPs induce lens formation by acting directly on the prospective lens ectoderm. Remarkably, the canonical Smad signaling pathway was not needed for most of these processes. Lens formation, placode cell proliferation, the expression of FoxE3, a lens-specific transcription factor, and the lens protein, αA-crystallin were regulated by BMP receptors in a Smad-independent manner. Placode cell survival was promoted by R-Smad signaling, but in a manner that did not involve Smad4. Of the responses tested, only maintaining a high level of Sox2 protein, a transcription factor expressed early in placode formation, required the canonical Smad pathway. A key function of Smad-independent BMP receptor signaling may be reorganization of actin cytoskeleton to drive lens invagination. PMID:19733164

  20. Enhanced Functional Activity of the Cannabinoid Type-1 Receptor Mediates Adolescent Behavior.

    PubMed

    Schneider, Miriam; Kasanetz, Fernando; Lynch, Diane L; Friemel, Chris M; Lassalle, Olivier; Hurst, Dow P; Steindel, Frauke; Monory, Krisztina; Schäfer, Carola; Miederer, Isabelle; Leweke, F Markus; Schreckenberger, Mathias; Lutz, Beat; Reggio, Patricia H; Manzoni, Olivier J; Spanagel, Rainer

    2015-10-14

    Adolescence is characterized by drastic behavioral adaptations and comprises a particularly vulnerable period for the emergence of various psychiatric disorders. Growing evidence reveals that the pathophysiology of these disorders might derive from aberrations of normal neurodevelopmental changes in the adolescent brain. Understanding the molecular underpinnings of adolescent behavior is therefore critical for understanding the origin of psychopathology, but the molecular mechanisms that trigger adolescent behavior are unknown. Here, we hypothesize that the cannabinoid type-1 receptor (CB1R) may play a critical role in mediating adolescent behavior because enhanced endocannabinoid (eCB) signaling has been suggested to occur transiently during adolescence. To study enhanced CB1R signaling, we introduced a missense mutation (F238L) into the rat Cnr1 gene that encodes for the CB1R. According to our hypothesis, rats with the F238L mutation (Cnr1(F238L)) should sustain features of adolescent behavior into adulthood. Gain of function of the mutated receptor was demonstrated by in silico modeling and was verified functionally in a series of biochemical and electrophysiological experiments. Mutant rats exhibit an adolescent-like phenotype during adulthood compared with wild-type littermates, with typical high risk/novelty seeking, increased peer interaction, enhanced impulsivity, and augmented reward sensitivity for drug and nondrug reward. Partial inhibition of CB1R activity in Cnr1(F238L) mutant rats normalized behavior and led to a wild-type phenotype. We conclude that the activity state and functionality of the CB1R is critical for mediating adolescent behavior. These findings implicate the eCB system as an important research target for the neuropathology of adolescent-onset mental health disorders. We present the first rodent model with a gain-of-function mutation in the cannabinoid type-1 receptor (CB1R). Adult mutant rats exhibit an adolescent-like phenotype with

  1. Functional relevance of G-protein-coupled-receptor-associated proteins, exemplified by receptor-activity-modifying proteins (RAMPs).

    PubMed

    Fischer, J A; Muff, R; Born, W

    2002-08-01

    The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8-32) and not by CGRP-(8-37), and CRLR-RAMP1, antagonized by CGRP-(8-37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.

  2. Motor effects of the non-psychotropic phytocannabinoid cannabidiol that are mediated by 5-HT1A receptors.

    PubMed

    Espejo-Porras, Francisco; Fernández-Ruiz, Javier; Pertwee, Roger G; Mechoulam, Raphael; García, Concepción

    2013-12-01

    The broad presence of CB1 receptors in the basal ganglia, mainly in GABA- or glutamate-containing neurons, as well as the presence of TRPV1 receptors in dopaminergic neurons and the identification of CB2 receptors in some neuronal subpopulations within the basal ganglia, explain the powerful motor effects exerted by those cannabinoids that can activate/block these receptors. By contrast, cannabidiol (CBD), a phytocannabinoid with a broad therapeutic profile, is generally presented as an example of a cannabinoid compound with no motor effects due to its poor affinity for the CB1 and the CB2 receptor, despite its activity at the TRPV1 receptor. However, recent evidence suggests that CBD may interact with the serotonin 5-HT1A receptor to produce some of its beneficial effects. This may enable CBD to directly influence motor activity through the well-demonstrated role of serotonergic transmission in the basal ganglia. We have investigated this issue in rats using three different pharmacological and neurochemical approaches. First, we compared the motor effects of various i.p. doses of CBD with the selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; i.p.). Second, we investigated whether the motor effects of CBD are sensitive to 5-HT1A receptor blockade in comparison with CB1 receptor antagonism. Finally, we investigated whether CBD was able to potentiate the effect of a sub-effective dose of 8-OH-DPAT. Our results demonstrated that: (i) only high doses of CBD (>10 mg/kg) altered motor behavior measured in a computer-aided actimeter; (ii) these alterations were restricted to vertical activity (rearing) with only modest changes in other parameters; (iii) similar effects were produced by 8-OH-DPAT (1 mg/kg), although this agonist affected exclusively vertical activity, with no effects on other motor parameters, and it showed always more potency than CBD; (iv) the effects of 8-OH-DPAT (1 mg/kg) and CBD (20 mg/kg) on vertical activity

  3. Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia.

    PubMed

    Pawlinski, Rafal; Pedersen, Brian; Schabbauer, Gernot; Tencati, Michael; Holscher, Todd; Boisvert, William; Andrade-Gordon, Patricia; Frank, Rolf Dario; Mackman, Nigel

    2004-02-15

    Sepsis is associated with a systemic activation of coagulation and an excessive inflammatory response. Anticoagulants have been shown to inhibit both coagulation and inflammation in sepsis. In this study, we used both genetic and pharmacologic approaches to analyze the role of tissue factor and protease-activated receptors in coagulation and inflammation in a mouse endotoxemia model. We used mice expressing low levels of the procoagulant molecule, tissue factor (TF), to analyze the effects of TF deficiency either in all tissues or selectively in hematopoietic cells. Low TF mice had reduced coagulation, inflammation, and mortality compared with control mice. Similarly, a deficiency of TF expression by hematopoietic cells reduced lipopolysaccharide (LPS)-induced coagulation, inflammation, and mortality. Inhibition of the down-stream coagulation protease, thrombin, reduced fibrin deposition and prolonged survival without affecting inflammation. Deficiency of either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) alone did not affect inflammation or survival. However, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality. These data demonstrate that hematopoietic cells are the major pathologic site of TF expression during endotoxemia and suggest that multiple protease-activated receptors mediate crosstalk between coagulation and inflammation.

  4. LE135, a retinoid acid receptor antagonist, produces pain through direct activation of TRP channels

    PubMed Central

    Yin, Shijin; Luo, Jialie; Qian, Aihua; Yu, Weihua; Hu, Hongzhen

    2014-01-01

    Background and PurposeRetinoids, through their activation of retinoic acid receptors (RARs) and retinoid X receptors, regulate diverse cellular processes, and pharmacological intervention in their actions has been successful in the treatment of skin disorders and cancers. Despite the many beneficial effects, administration of retinoids causes irritating side effects with unknown mechanisms. Here, we demonstrate that LE135 [4-(7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid], a selective antagonist of RARβ, is a potent activator of the capsaicin (TRPV1) and wasabi (TRPA1) receptors, two critical pain-initiating cation channels. Experimental ApproachWe performed to investigate the excitatory effects of LE135 on TRPV1 and TRPA1 channels expressed in HEK293T cells and in dorsal root ganglia neurons with calcium imaging and patch-clamp recordings. We also used site-directed mutagenesis of the channels to determine the structural basis of LE135-induced activation of TRPV1 and TRPA1 channels and behavioural testing to examine if pharmacological inhibition and genetic deletion of the channels affected LE135-evoked pain-related behaviours. Key ResultsLE135 activated both the capsaicin receptor (TRPV1) and the allyl isothiocyanate receptor (TRPA1) heterologously expressed in HEK293T cells and endogenously expressed by sensory nociceptors. Mutations disrupting the capsaicin-binding site attenuated LE135 activation of TRPV1 channels and a single mutation (K170R) eliminated TRPA1 activity evoked by LE135. Intraplantar injection of LE135 evoked pain-related behaviours. Both TRPV1 and TRPA1 channels were involved in LE135-elicited pain-related responses, as shown by pharmacological and genetic ablation studies. Conclusions and ImplicationsThis blocker of retinoid acid signalling also exerted non-genomic effects through activating the pain-initiating TRPV1 and TRPA1 channels. PMID:24308840

  5. Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a [35S]GTPgammaS binding study.

    PubMed

    Newman-Tancredi, A; Gavaudan, S; Conte, C; Chaput, C; Touzard, M; Verrièle, L; Audinot, V; Millan, M J

    1998-08-21

    Recombinant human (h) 5-HT1A receptor-mediated G-protein activation was characterised in membranes of transfected Chinese hamster ovary (CHO) cells by use of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS binding). The potency and efficacy of 21 5-HT receptor agonists and antagonists was determined. The agonists, 5-CT (carboxamidotryptamine) and flesinoxan displayed high affinity (subnanomolar Ki values) and high efficacy (Emax > 90%, relative to 5-HT = 100%). In contrast, ipsapirone, zalospirone and buspirone displayed partial agonist activity. EC50s for agonist stimulation of [35S]GTPgammaS binding correlated well with Ki values from competition binding (r = +0.99). Among the compounds tested for antagonist activity, methiothepin and (+)butaclamol exhibited 'inverse agonist' behaviour, inhibiting basal [35S]GTPgammaS binding. The actions of 17 antipsychotic agents were investigated. Clozapine and several putatively 'atypical' antipsychotic agents, including ziprasidone, quetiapine and tiospirone, exhibited partial agonist activity and marked affinity at h5-HT1A receptors, similar to their affinity at hD2 dopamine receptors. In contrast, risperidone and sertindole displayed low affinity at h5-HT1A receptors and behaved as 'neutral' antagonists, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Likewise the 'typical' neuroleptics, haloperidol, pimozide, raclopride and chlorpromazine exhibited relatively low affinity and 'neutral' antagonist activity at h5-HT1A receptors with Ki values which correlated with their respective Kb values. The present data show that (i) [35S]GTPgammaS binding is an effective method to evaluate the efficacy and potency of agonists and antagonists at recombinant human 5-HT1A receptors. (ii) Like clozapine, several putatively 'atypical' antipsychotic drugs display balanced serotonin h5-HT1A/dopamine hD2 receptor affinity and partial agonist activity at h5-HT1A receptors. (iii) Several 'typical' and some putatively 'atypical

  6. Glucocorticoid acts on a putative G protein-coupled receptor to rapidly regulate the activity of NMDA receptors in hippocampal neurons.

    PubMed

    Zhang, Yanmin; Sheng, Hui; Qi, Jinshun; Ma, Bei; Sun, Jihu; Li, Shaofeng; Ni, Xin

    2012-04-01

    Glucocorticoids (GCs) have been demonstrated to act through both genomic and nongenomic mechanisms. The present study demonstrated that corticosterone rapidly suppressed the activity of N-methyl-D-aspartate (NMDA) receptors in cultured hippocampal neurons. The effect was maintained with corticosterone conjugated to bovine serum albumin and blocked by inhibition of G protein activity with intracellular GDP-β-S application. Corticosterone increased GTP-bound G(s) protein and cyclic AMP (cAMP) production, activated phospholipase Cβ(3) (PLC-β(3)), and induced inositol-1,4,5-triphosphate (IP(3)) production. Blocking PLC and the downstream cascades with PLC inhibitor, IP(3) receptor antagonist, Ca(2+) chelator, and protein kinase C (PKC) inhibitors prevented the actions of corticosterone. Blocking adenylate cyclase (AC) and protein kinase A (PKA) caused a decrease in NMDA-evoked currents. Application of corticosterone partly reversed the inhibition of NMDA currents caused by blockage of AC and PKA. Intracerebroventricular administration of corticosterone significantly suppressed long-term potentiation (LTP) in the CA1 region of the hippocampus within 30 min in vivo, implicating the possibly physiological significance of rapid effects of GC on NMDA receptors. Taken together, our results indicate that GCs act on a putative G protein-coupled receptor to activate multiple signaling pathways in hippocampal neurons, and the rapid suppression of NMDA activity by GCs is dependent on PLC and downstream signaling.

  7. Transcriptional activation of NAD+-dependent protein deacetylase SIRT1 by nuclear receptor TLX.

    PubMed

    Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Horio, Yoshiyuki

    2009-09-04

    An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD(+)-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

  8. Hit-to-lead optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as a novel class of EP1 receptor antagonists.

    PubMed

    Atobe, Masakazu; Naganuma, Kenji; Kawanishi, Masashi; Morimoto, Akifumi; Kasahara, Ken-ichi; Ohashi, Shigeki; Suzuki, Hiroko; Hayashi, Takahiko; Miyoshi, Shiro

    2013-11-15

    We describe a medicinal chemistry approach to generate a series of 2-(1H-pyrazol-1-yl)thiazole compounds that act as selective EP1 receptor antagonists. The obtained results suggest that compound 12 provides the best EP1 receptor antagonist activity and demonstrates good oral pharmacokinetics. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. 5-HT1A receptor blockade reverses GABAA receptor α3 subunit-mediated anxiolytic effects on stress-induced hyperthermia

    PubMed Central

    van Oorschot, Ruud; Korte, S. Mechiel; Olivier, Berend; Groenink, Lucianne

    2010-01-01

    Rationale Stress-related disorders are associated with dysfunction of both serotonergic and GABAergic pathways, and clinically effective anxiolytics act via both neurotransmitter systems. As there is evidence that the GABAA and the serotonin receptor system interact, a serotonergic component in the anxiolytic actions of benzodiazepines could be present. Objectives The main aim of the present study was to investigate whether the anxiolytic effects of (non-)selective α subunit GABAA receptor agonists could be reversed with 5-HT1A receptor blockade using the stress-induced hyperthermia (SIH) paradigm. Results The 5-HT1A receptor antagonist WAY-100635 (0.11 mg/kg) reversed the SIH-reducing effects of the non-α-subunit selective GABAA receptor agonist diazepam (1–4 mg/kg) and the GABAA receptor α3-subunit selective agonist TP003 (1 mg/kg), whereas WAY-100635 alone was without effect on the SIH response or basal body temperature. At the same time, co-administration of WAY-100635 with diazepam or TP003 reduced basal body temperature. WAY-100635 did not affect the SIH response when combined with the preferential α1-subunit GABAA receptor agonist zolpidem (10 mg/kg), although zolpidem markedly reduced basal body temperature. Conclusions The present study suggests an interaction between GABAA receptor α-subunits and 5-HT1A receptor activation in the SIH response. Specifically, our data indicate that benzodiazepines affect serotonergic signaling via GABAA receptor α3-subunits. Further understanding of the interactions between the GABAA and serotonin system in reaction to stress may be valuable in the search for novel anxiolytic drugs. PMID:20535452

  10. Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection.

    PubMed

    Liebrand, Thomas W H; van den Berg, Grardy C M; Zhang, Zhao; Smit, Patrick; Cordewener, Jan H G; America, Antoine H P; America, Antione H P; Sklenar, Jan; Jones, Alexandra M E; Tameling, Wladimir I L; Robatzek, Silke; Thomma, Bart P H J; Joosten, Matthieu H A J

    2013-06-11

    The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4- and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.

  11. Preclinical Testing of Novel Oxytocin Receptor Activators in Models of Autism Phenotypes

    DTIC Science & Technology

    2014-09-01

    AD_________________ Award Number: TITLE: Preclinical Testing of Novel Oxytocin Receptor Activators in Models of Autism ...AUG 2013-7 Aug 2014 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Preclinical Testing of Novel Oxytocin Receptor Activators in Models of Autism ...a genetic mouse model of autism -like phenotypes, the Grin1 knockdown mouse. The Grin1 gene encodes the NR1 subunit of the NMDA receptor . In the

  12. Antiseizure Activity of Midazolam in Mice Lacking δ-Subunit Extrasynaptic GABA(A) Receptors.

    PubMed

    Reddy, Sandesh D; Younus, Iyan; Clossen, Bryan L; Reddy, Doodipala Samba

    2015-06-01

    Midazolam is a benzodiazepine anticonvulsant with rapid onset and short duration of action. Midazolam is the current drug of choice for acute seizures and status epilepticus, including those caused by organophosphate nerve agents. The antiseizure activity of midazolam is thought to result from its allosteric potentiation of synaptic GABA(A) receptors in the brain. However, there are indications that benzodiazepines promote neurosteroid synthesis via the 18-kDa cholesterol transporter protein (TSPO). Therefore, we investigated the role of neurosteroids and their extrasynaptic GABA(A) receptor targets in the antiseizure activity of midazolam. Here, we used δ-subunit knockout (DKO) mice bearing a targeted deletion of the extrasynaptic receptors to investigate the contribution of the extrasynaptic receptors to the antiseizure activity of midazolam using the 6-Hz and hippocampus kindling seizure models. In both models, midazolam produced rapid and dose-dependent protection against seizures (ED50, 0.4 mg/kg). Moreover, the antiseizure potency of midazolam was undiminished in DKO mice compared with control mice. Pretreatment with PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide], a TSPO blocker, or finasteride, a 5α-reductase neurosteroid inhibitor, did not affect the antiseizure effect of midazolam. The antiseizure activity of midazolam was significantly reversed by pretreatment with flumazenil, a benzodiazepine antagonist. Plasma and brain levels of the neurosteroid allopregnanolone were not significantly greater in midazolam-treated animals. These studies therefore provide strong evidence that neurosteroids and extrasynaptic GABA(A) receptors are not involved in the antiseizure activity of midazolam, which mainly occurs through synaptic GABA(A) receptors via direct binding to benzodiazepine sites. This study reaffirms midazolam's use for controlling acute seizures and status epilepticus. Copyright © 2015 by The American Society for

  13. Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists.

    PubMed

    Mochizuki, Michiyo; Kori, Masakuni; Kobayashi, Katsumi; Yano, Takahiko; Sako, Yuu; Tanaka, Maiko; Kanzaki, Naoyuki; Gyorkos, Albert C; Corrette, Christopher P; Cho, Suk Young; Pratt, Scott A; Aso, Kazuyoshi

    2016-03-24

    Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 μM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 μmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research.

  14. Cytoprotective signaling by activated protein C requires protease-activated receptor-3 in podocytes

    PubMed Central

    Madhusudhan, Thati; Wang, Hongjie; Straub, Beate K.; Gröne, Elisabeth; Zhou, Qianxing; Shahzad, Khurrum; Müller-Krebs, Sandra; Schwenger, Vedat; Gerlitz, Bruce; Grinnell, Brian W.; Griffin, John H.; Reiser, Jochen; Gröne, Hermann-Josef; Esmon, Charles T.; Nawroth, Peter P.

    2012-01-01

    The cytoprotective effects of activated protein C (aPC) are well established. In contrast, the receptors and signaling mechanism through which aPC conveys cytoprotection in various cell types remain incompletely defined. Thus, within the renal glomeruli, aPC preserves endothelial cells via a protease-activated receptor-1 (PAR-1) and endothelial protein C receptor-dependent mechanism. Conversely, the signaling mechanism through which aPC protects podocytes remains unknown. While exploring the latter, we identified a novel aPC/PAR-dependent cytoprotective signaling mechanism. In podocytes, aPC inhibits apoptosis through proteolytic activation of PAR-3 independent of endothelial protein C receptor. PAR-3 is not signaling competent itself as it requires aPCinduced heterodimerization with PAR-2 (human podocytes) or PAR-1 (mouse podocytes). This cytoprotective signaling mechanism depends on caveolin-1 dephosphorylation. In vivo aPC protects against lipopolysaccharide-induced podocyte injury and proteinuria. Genetic deletion of PAR-3 impairs the nephroprotective effect of aPC, demonstrating the crucial role of PAR-3 for aPC-dependent podocyte protection. This novel, aPC-mediated interaction of PARs demonstrates the plasticity and cell-specificity of cytoprotective aPC signaling. The evidence of specific, dynamic signaling complexes underlying aPC-mediated cytoprotection may allow the design of cell type specific targeted therapies. PMID:22117049

  15. Oxidative activation of CaMKIIδ in acute myocardial ischemia/reperfusion injury: A role of angiotensin AT1 receptor-NOX2 signaling axis.

    PubMed

    Rajtik, Tomas; Carnicka, Slavka; Szobi, Adrian; Giricz, Zoltan; O-Uchi, Jin; Hassova, Veronika; Svec, Pavel; Ferdinandy, Peter; Ravingerova, Tanya; Adameova, Adriana

    2016-01-15

    During ischemia/reperfusion (IR), increased activation of angiotensin AT1 receptors recruits NADPH oxidase 2 (NOX2) which contributes to oxidative stress. It is unknown whether this stimulus can induce oxidative activation of Ca(2+)/calmodulin-dependent protein kinase IIδ (CaMKIIδ) leading into the aggravation of cardiac function and whether these effects can be prevented by angiotensin AT1 receptors blockade. Losartan, a selective AT1 blocker, was used. Its effects were compared with effects of KN-93, an inhibitor of CaMKIIδ. Global IR was induced in Langendorff-perfused rat hearts. Protein expression was evaluated by immunoblotting and lipoperoxidation was measured by TBARS assay. Losartan improved LVDP recovery by 25%; however, it did not reduce reperfusion arrhythmias. Oxidized CaMKIIδ (oxCaMKIIδ) was downregulated at the end of reperfusion compared to before ischemia and losartan did not change these levels. Phosphorylation of CaMKIIδ mirrored the pattern of changes in oxCaMKIIδ levels. Losartan did not prevent the higher lipoperoxidation due to IR and did not influence NOX2 expression. Inhibition of CaMKII ameliorated cardiac IR injury; however, this was not accompanied with changes in the levels of either active form of CaMKIIδ in comparison to the angiotensin AT1 receptor blockade. In spite of no changes of oxCaMKIIδ, increased cardiac recovery of either therapy was abolished when combined together. This study showed that oxidative activation of CaMKIIδ is not elevated at the end of R phase. NOX2-oxCAMKIIδ signaling is unlikely to be involved in cardioprotective action of angiotensin AT1 receptor blockade which is partially abolished by concomitant CaMKII inhibition. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Aldosterone rapidly activates Src kinase in M-1 cells involving the mineralocorticoid receptor and HSP84.

    PubMed

    Braun, Sabine; Lösel, Ralf; Wehling, Martin; Boldyreff, Brigitte

    2004-07-16

    We investigated the effect of aldosterone on Src kinase. In the kidney cell line, M-1 aldosterone leads to a >2-fold transient activation of Src kinase seen as early as 2 min after aldosterone administration. Maximal Src kinase activation was measured at an aldosterone concentration of 1 nM. In parallel to activation, autophosphorylation at Tyr-416 of Src kinase increased. Src kinase activation was blocked by spironolactone. Aldosterone led to increased association of Src with HSP84. Furthermore, rapamycin blocked aldosterone-induced Src activation. We conclude that Src activation by aldosterone is mediated through the mineralocorticoid receptor and HSP84.

  17. Phenotypic regulation of the sphingosine 1-phosphate receptor miles apart by G protein-coupled receptor kinase 2.

    PubMed

    Burczyk, Martina; Burkhalter, Martin D; Blätte, Tamara; Matysik, Sabrina; Caron, Marc G; Barak, Lawrence S; Philipp, Melanie

    2015-01-27

    The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and β-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m(93) (mil(m93)), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that β-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling.

  18. Phenotypic Regulation of the Sphingosine 1-Phosphate Receptor Miles Apart by G Protein-Coupled Receptor Kinase 2

    PubMed Central

    2016-01-01

    The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and β-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m93 (milm93), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that β-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling. PMID:25555130

  19. High intestinal and systemic levels of decoy receptor 3 (DcR3) and its ligand TL1A in active ulcerative colitis.

    PubMed

    Bamias, Giorgos; Kaltsa, Garyfallia; Siakavellas, Spyros I; Papaxoinis, Kostis; Zampeli, Evanthia; Michopoulos, Spyros; Zouboulis-Vafiadis, Irene; Ladas, Spiros D

    2010-11-01

    Decoy receptor-3 (DcR3) is a member of the TNF receptor superfamily of proteins, which has been implicated in anti-apoptotic and anti-inflammatory pathways, via binding to TL1A, LIGHT and Fas-L. The role of the TL1A/DcR3 ligand/receptor pair in ulcerative colitis (UC) has not been studied. We investigated the systemic (peripheral blood) and local (large intestine) expression of DcR3 and TL1A in 64 patients with UC and 56 healthy controls. DcR3 serum concentrations were highly elevated in patients with active UC (P<0.0001 vs. healthy controls). This elevation was clearly related to the presence of intestinal inflammation as it was less frequently observed in patients in remission (P=0.003 vs. active UC) whereas effective treatment resulted in disappearance or significant decrease of serum DcR3 (P=0.006 vs. pre-treatment). Furthermore, DcR3 mRNA transcripts were significantly elevated in inflamed areas of the colon (P=0.002 vs. non-affected of the same patient). In addition to DcR3 elevation, we found increased circulating levels of TL1A in patients with either active or inactive UC in comparison to healthy controls (P<0.001 for both). We conclude that elevated serum DcR3 may serve as an indicator of active colonic inflammation in patients with UC. TL1A/DcR3-mediated pathways may participate in the pathogenesis of UC. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. RasGRP3 limits Toll-like receptor-triggered inflammatory response in macrophages by activating Rap1 small GTPase.

    PubMed

    Tang, Songqing; Chen, Taoyong; Yu, Zhou; Zhu, Xuhui; Yang, Mingjin; Xie, Bin; Li, Nan; Cao, Xuetao; Wang, Jianli

    2014-08-14

    Host immune cells can detect and destruct invading pathogens via pattern-recognition receptors. Small Rap GTPases act as conserved molecular switches coupling extracellular signals to various cellular responses, but their roles as regulators in Toll-like receptor (TLR) signalling have not been fully elucidated. Here we report that Ras guanine nucleotide-releasing protein 3 (RasGRP3), a guanine nucleotide-exchange factor activating Ras and Rap1, limits production of proinflammatory cytokines (especially IL-6) in macrophages by activating Rap1 on activation by low levels of TLR agonists. We demonstrate that RasGRP3, a dominant member of RasGRPs in macrophages, impairs TLR3/4/9-induced IL-6 production and relieves dextrane sulphate sodium-induced colitis and collagen-induced arthritis. In RasGRP3-deficient RAW264.7 cells obtained by CRISPR-Cas9 genome editing, TLR3/4/9-induced activation of Rap1 was inhibited while ERK1/2 activation was enhanced. Our study suggests that RasGRP3 limits inflammatory response by activating Rap1 on low-intensity pathogen infection, setting a threshold for preventing excessive inflammatory response.

  1. Nuclear receptors and nonalcoholic fatty liver disease1

    PubMed Central

    Cave, Matthew C.; Clair, Heather B.; Hardesty, Josiah E.; Falkner, K. Cameron; Feng, Wenke; Clark, Barbara J.; Sidey, Jennifer; Shi, Hongxue; Aqel, Bashar A.; McClain, Craig J.; Prough, Russell A.

    2016-01-01

    Nuclear receptors are transcription factors which sense changing environmental or hormonal signals and effect transcriptional changes to regulate core life functions including growth, development, and reproduction. To support this function, following ligand-activation by xenobiotics, members of subfamily 1 nuclear receptors (NR1s) may heterodimerize with the retinoid X receptor (RXR) to regulate transcription of genes involved in energy and xenobiotic metabolism and inflammation. Several of these receptors including the peroxisome proliferator-activated receptors (PPARs), the pregnane and xenobiotic receptor (PXR), the constitutive androstane receptor (CAR), the liver X receptor (LXR) and the farnesoid X receptor (FXR) are key regulators of the gut:liver:adipose axis and serve to coordinate metabolic responses across organ systems between the fed and fasting states. Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease and may progress to cirrhosis and even hepatocellular carcinoma. NAFLD is associated with inappropriate nuclear receptor function and perturbations along the gut:liver:adipose axis including obesity, increased intestinal permeability with systemic inflammation, abnormal hepatic lipid metabolism, and insulin resistance. Environmental chemicals may compound the problem by directly interacting with nuclear receptors leading to metabolic confusion and the inability to differentiate fed from fasting conditions. This review focuses on the impact of nuclear receptors in the pathogenesis and treatment of NAFLD. Clinical trials including PIVENS and FLINT demonstrate that nuclear receptor targeted therapies may lead to the paradoxical dissociation of steatosis, inflammation, fibrosis, insulin resistance, dyslipidemia and obesity. Novel strategies currently under development (including tissue-specific ligands and dual receptor agonists) may be required to separate the beneficial effects of nuclear receptor activation from unwanted metabolic

  2. D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus

    PubMed Central

    Arnaldo, Francis B.; Villar, Van Anthony M.; Konkalmatt, Prasad R.; Owens, Shaun A.; Asico, Laureano D.; Jones, John E.; Yang, Jian; Lovett, Donald L.; Armando, Ines; Concepcion, Gisela P.

    2014-01-01

    Dopamine-mediated regulation of Na+-K+-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na+-K+-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na+-K+-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na+-K+-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na+-K+-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na+-K+-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na+-K+-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis. PMID:25080496

  3. D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus.

    PubMed

    Arnaldo, Francis B; Villar, Van Anthony M; Konkalmatt, Prasad R; Owens, Shaun A; Asico, Laureano D; Jones, John E; Yang, Jian; Lovett, Donald L; Armando, Ines; Jose, Pedro A; Concepcion, Gisela P

    2014-09-15

    Dopamine-mediated regulation of Na(+)-K(+)-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na(+)-K(+)-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na(+)-K(+)-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na(+)-K(+)-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis. Copyright © 2014 the American Physiological Society.

  4. Steroid receptor RNA activator (SRA1): unusual bifaceted gene products with suspected relevance to breast cancer

    PubMed Central

    Leygue, Etienne

    2007-01-01

    The steroid receptor RNA activator (SRA) is a unique modulator of steroid receptor transcriptional activity, as it is able to mediate its coregulatory effects as a RNA molecule. Recent findings, however, have painted a more complex picture of the SRA gene (SRA1) products. Indeed, even though SRA was initially thought to be noncoding, several RNA isoforms have now been found to encode an endogenous protein (SRAP), which is well conserved among Chordata. Although the function of SRAP remains largely unknown, it has been proposed that, much like its corresponding RNA, the protein itself might regulate estrogen and androgen receptor signaling pathways. As such, data suggest that both SRA and SRAP might participate in the mechanisms underlying breast, as well as prostate tumorigenesis. This review summarizes the published literature dealing with these two faces of the SRA gene products and underscores the relevance of this bifaceted system to breast cancer development. PMID:17710122

  5. Pharmacological Activation of Peroxisome Proliferator-Activated Receptor {Delta} Increases Sphingomyelin Synthase Activity in THP-1 Macrophage-Derived Foam Cell.

    PubMed

    Mou, Dongsheng; Yang, Hua; Qu, Changhua; Chen, Juan; Zhang, Chaogui

    2016-08-01

    Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which mediate glucose and lipid homeostasis by regulating the expression of a large number of transcription factors. Sphingomyelin synthase (SMS) is a key enzyme in the synthesis of sphingomyelin (SM), and its expression and activity have been reported to be associated with atherosclerosis (AS). Although there have been many functional PPAR and SMS studies on atherosclerosis in recent years, few have investigated the correlation between the activation of PPARδ and the activity of SMS. In his study, macrophage-induced foam cells were utilized to model important pathological changes that occur in AS. The influence of PPARδ agonism by GW501516 on SMS and its product molecule SM were measured. Results indicated that the activation of PPARδ was correlated in a positive manner with the activity of SMS2, and the content of SM was dose dependently increased by GW501516. Together, this study represents the first to suggest that PPARδ activation may be a potential risk of AS through enhancing activity of SMS2.

  6. Immunologic analysis of human breast cancer progesterone receptors. 1. Immunonaffinity purification of transformed receptors and production of monoclonal antibodies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Estes, P.A.; Suba, E.J.; Lawler-Heavner, J.

    1987-09-22

    A monoclonal antibody (MAb), designated PR-6, produced against chick oviduct progesterone receptors cross-reacts with the M/sub r/ 120,000 human B receptors. An immunomatrix prepared with PR-6 was used to purify progesterone receptors (PR) from T47D human breast cancer cells. Single-step immunoaffinity chromatography results in enrichment of B receptors (identified by immunoblot with PR-6 and by photoaffinity labeling with (/sup 3/H)promegestone) to a specific activity of 1915 pmol/mg of protein (or 23% purity) and with 27% yield. Purity and yields as judged by gel electrophoresis and densitometric scanning of the B protein were approximately 1.7-fold higher due to partial loss inmore » hormone binding activity at the elution step. B receptors purified under these conditions are transformed and biologically active. They were maintained as undergraded 120-kDa doublets and retained both hormone and DNA binding activities. These purified B receptors were used as immunogen for production of four monoclonal antibodies against human PR. Three of the MAbs, designated as B-30 (IgG/sub 1/), B-64 (IgG/sub 1/), and B-11 (IgM), are specific for B receptors. The fourth MAb, A/B-52 (IgG/sub 1/), reacts with both A and B receptors. The IgG MAbs are monospecific for human PR since they recognize and absorb native receptor-hormone complexes, displace the sedimentation of 4S receptors on salt containing sucrose gradients, and, by immunoblot assay of crude T47D cytosol, react only with receptor polypeptides. Although mice were injected with B receptors only, production of A/B-52 which recognized both A and B receptors provides evidence that these two proteins share regions of structural homology.« less

  7. Trace amine-associated receptor 1-Family archetype or iconoclast?

    PubMed

    Grandy, David K

    2007-12-01

    Interest has recently been rekindled in receptors that are activated by low molecular weight, noncatecholic, biogenic amines that are typically found as trace constituents of various vertebrate and invertebrate tissues and fluids. The timing of this resurgent focus on receptors activated by the "trace amines" (TA) beta-phenylethylamine (PEA), tyramine (TYR), octopamine (OCT), synephrine (SYN), and tryptamine (TRYP) is the direct result of 2 publications that appeared in 2001 describing the cloning of a novel G protein-coupled receptor (GPCR) referred to by their discoverers Borowsky et al. as TA1 and Bunzow et al. as TA receptor 1 (TAR1). When heterologously expressed in Xenopus laevis oocytes and various eukaryotic cell lines, recombinant rodent and human TAR dose-dependently couple to the stimulation of adenosine 3',5'-monophosphate (cAMP) production. Structure-activity profiling based on this functional response has revealed that in addition to the TA, other biologically active compounds containing a 2-carbon aliphatic side chain linking an amino group to at least 1 benzene ring are potent and efficacious TA receptor agonists with amphetamine (AMPH), methamphetamine, 3-iodothyronamine, thyronamine, and dopamine (DA) among the most notable. Almost 100 years after the search for TAR began, numerous TA1/TAR1-related sequences, now called TA-associated receptors (TAAR), have been identified in the genome of every species of vertebrate examined to date. Consequently, even though heterologously expressed TAAR1 fits the pharmacological criteria established for a bona fide TAR, a major challenge for those working in the field is to discern the in vivo pharmacology and physiology of each purported member of this extended family of GPCR. Only then will it be possible to establish whether TAAR1 is the family archetype or an iconoclast.

  8. The beta-arrestin pathway-selective type 1A angiotensin receptor (AT1A) agonist [Sar1,Ile4,Ile8]angiotensin II regulates a robust G protein-independent signaling network.

    PubMed

    Kendall, Ryan T; Strungs, Erik G; Rachidi, Saleh M; Lee, Mi-Hye; El-Shewy, Hesham M; Luttrell, Deirdre K; Janech, Michael G; Luttrell, Louis M

    2011-06-03

    The angiotensin II peptide analog [Sar(1),Ile(4),Ile(8)]AngII (SII) is a biased AT(1A) receptor agonist that stimulates receptor phosphorylation, β-arrestin recruitment, receptor internalization, and β-arrestin-dependent ERK1/2 activation without activating heterotrimeric G-proteins. To determine the scope of G-protein-independent AT(1A) receptor signaling, we performed a gel-based phosphoproteomic analysis of AngII and SII-induced signaling in HEK cells stably expressing AT(1A) receptors. A total of 34 differentially phosphorylated proteins were detected, of which 16 were unique to SII and eight to AngII stimulation. MALDI-TOF/TOF mass fingerprinting was employed to identify 24 SII-sensitive phosphoprotein spots, of which three (two peptide inhibitors of protein phosphatase 2A (I1PP2A and I2PP2A) and prostaglandin E synthase 3 (PGES3)) were selected for validation and further study. We found that phosphorylation of I2PP2A was associated with rapid and transient inhibition of a β-arrestin 2-associated pool of protein phosphatase 2A, leading to activation of Akt and increased phosphorylation of glycogen synthase kinase 3β in an arrestin signalsome complex. SII-stimulated PGES3 phosphorylation coincided with an increase in β-arrestin 1-associated PGES3 and an arrestin-dependent increase in cyclooxygenase 1-dependent prostaglandin E(2) synthesis. These findings suggest that AT(1A) receptors regulate a robust G protein-independent signaling network that affects protein phosphorylation and autocrine/paracrine prostaglandin production and that these pathways can be selectively modulated by biased ligands that antagonize G protein activation.

  9. Activation of JAK3, but not JAK1, is critical to interleukin-4 (IL4) stimulated proliferation and requires a membrane-proximal region of IL4 receptor alpha.

    PubMed

    Malabarba, M G; Kirken, R A; Rui, H; Koettnitz, K; Kawamura, M; O'Shea, J J; Kalthoff, F S; Farrar, W L

    1995-04-21

    The tyrosine kinases JAK1 and JAK3 have been shown to undergo tyrosine phosphorylation in response to interleukin-2 (IL), IL4, IL7, and IL9, cytokines which share the common IL2 receptor gamma-chain (IL2R gamma), and evidence has been found for a preferential coupling of JAK3 to IL2R gamma and JAK1 to IL2R beta. Here we show, using human premyeloid TF-1 cells, that IL4 stimulates JAK3 to a larger extent than JAK1, based upon three different evaluation criteria. These include a more vigorous tyrosine phosphorylation of JAK3 as measured by anti-phosphotyrosine immunoblotting, a more marked activation of JAK3 as determined by in vitro tyrosine kinase assays and a more manifest presence of JAK3 in activated IL4-receptor complexes. These observations suggest that IL4 receptor signal transduction does not depend on equimolar heterodimerization of JAK1 and JAK3 following IL4-induced heterodimerization of IL4R alpha and IL2R gamma. Indeed, when human IL4R alpha was stably expressed in mouse BA/F3 cells, robust IL4-induced proliferation and JAK3 activation occurred without detectable involvement of JAK1, JAK2, or TYK2. The present study suggests that JAK1 plays a subordinate role in IL4 receptor signaling, and that in certain cells exclusive JAK3 activation may mediate IL4-induced cell growth. Moreover, mutational analysis of human IL4R alpha showed that a membrane-proximal cytoplasmic region was critical for JAK3 activation, while the I4R motif was not, which is compatible with a role of JAK3 upstream of the recruitment of the insulin receptor substrate-1/4PS signaling proteins by IL4 receptors.

  10. The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific {alpha} subunit and a {beta}c subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface andmore » functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.« less

  11. The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1: Expression and action in brain.

    PubMed

    Morland, Cecilie; Lauritzen, Knut Husø; Puchades, Maja; Holm-Hansen, Signe; Andersson, Krister; Gjedde, Albert; Attramadal, Håvard; Storm-Mathisen, Jon; Bergersen, Linda Hildegard

    2015-07-01

    We have proposed that lactate is a "volume transmitter" in the brain and underpinned this by showing that the lactate receptor, G-protein-coupled receptor 81 (GPR81, also known as HCA1 or HCAR1), which promotes lipid storage in adipocytes, is also active in the mammalian brain. This includes the cerebral neocortex and the hippocampus, where it can be stimulated by physiological concentrations of lactate and by the HCAR1 agonist 3,5-dihydroxybenzoate to reduce cAMP levels. Cerebral HCAR1 is concentrated on the postsynaptic membranes of excitatory synapses and also is enriched at the blood-brain barrier. In synaptic spines and in adipocytes, HCAR1 immunoreactivity is also located on subplasmalemmal vesicular organelles, suggesting trafficking to and from the plasma membrane. Through activation of HCAR1, lactate can act as a volume transmitter that links neuronal activity, cerebral blood flow, energy metabolism, and energy substrate availability, including a glucose- and glycogen-saving response. HCAR1 may contribute to optimizing the cAMP concentration. For instance, in the prefrontal cortex, excessively high cAMP levels are implicated in impaired cognition in old age, fatigue, stress, and schizophrenia and in the deposition of phosphorylated tau protein in Alzheimer's disease. HCAR1 could serve to ameliorate these conditions and might also act through downstream mechanisms other than cAMP. Lactate exits cells through monocarboxylate transporters in an equilibrating manner and through astrocyte anion channels activated by depolarization. In addition to locally produced lactate, lactate produced by exercising muscle as well as exogenous HCAR1 agonists, e.g., from fruits and berries, might activate the receptor on cerebral blood vessels and brain cells. © 2015 Wiley Periodicals, Inc.

  12. Colonic vascular conductance increased by Daikenchuto via calcitonin gene-related peptide and receptor-activity modifying protein 1.

    PubMed

    Kono, Toru; Koseki, Takashi; Chiba, Shinichi; Ebisawa, Yoshiaki; Chisato, Naoyuki; Iwamoto, Jun; Kasai, Shinichi

    2008-11-01

    Daikencyuto (DKT) is a traditional Japanese medicine (Kampo) and is a mixture of extract powders from dried Japanese pepper, processed ginger, ginseng radix, and maltose powder and has been used as the treatment of paralytic ileus. DKT may increase gastrointestinal motility by an up-regulation of the calcitonin gene-related peptide (CGRP). CGRP is also the most powerful vasoactive substance. In the present study, we investigated whether DKT has any effect on the colonic blood flow in rats. Experiments were performed on fasted anesthetized and artificially ventilated Wistar rats. Systemic mean arterial blood pressure and heart rate were recorded. Red blood cell flux in colonic blood flow was measured using noncontact laser tissue blood flowmetry, and colonic vascular conductance (CVC) was calculated as the ratio of flux to mean arterial blood pressure. We examined four key physiological mechanisms underlying the response using blocker drugs: CGRP1 receptor blocker (CGRP(8-37)), nitric oxide synthase inhibitor, vasoactive intestinal polypeptide (VIP) receptor blocker ([4-Cl-DPhe6, Leu17]-VIP), and substance P receptor blocker (spantide). Reverse transcription-polymerase chain reaction was used for the detection of mRNA of calcitonin receptor-like receptor, receptor-activity modifying protein 1, the component of CGRP 1 receptor and CGRP. After laparotomy, a cannula was inserted into the proximal colon to administer the DKT and to measure CVC at the distal colon. Intracolonal administration of DKT (10, 100, and 300 mg/kg) increased CVC (basal CVC, 0.10 mL/mmHg) from the first 15-min observation period (0.14, 0.17, and 0.17 mL/mmHg, respectively) and with peak response at either 45 min (0.17 mL/mmHg by 10 mg/kg), or 75 and 60 min (0.23 and 0.21 mL/mmHg by 100 and 300 mg/kg, respectively). CGRP(8-37) completely abolished the DKT-induced hyperemia, whereas nitric oxide synthase inhibitor partially attenuated the DKT-induced hyperemia. [4-Cl-DPhe6, Leu17]-VIP and spantide

  13. Modulation of nociceptive dural input to the trigeminocervical complex through GluK1 kainate receptors.

    PubMed

    Andreou, Anna P; Holland, Philip R; Lasalandra, Michele P; Goadsby, Peter J

    2015-03-01

    Migraine is a common and disabling neurologic disorder, with important psychiatric comorbidities. Its pathophysiology involves activation of neurons in the trigeminocervical complex (TCC). Kainate receptors carrying the glutamate receptor subunit 5 (GluK1) are present in key brain areas involved in migraine pathophysiology. To study the influence of kainate receptors on trigeminovascular neurotransmission, we determined the presence of GluK1 receptors within the trigeminal ganglion and TCC with immunohistochemistry. We performed in vivo electrophysiologic recordings from TCC neurons and investigated whether local or systemic application of GluK1 receptor antagonists modulated trigeminovascular transmission. Microiontophoretic application of a selective GluK1 receptor antagonist, but not of a nonspecific ionotropic glutamate receptor antagonist, markedly attenuated cell firing in a subpopulation of neurons activated in response to dural stimulation, consistent with selective inhibition of postsynaptic GluK1 receptor-evoked firing seen in all recorded neurons. In contrast, trigeminovascular activation was significantly facilitated in a different neuronal population. The clinically active kainate receptor antagonist LY466195 attenuated trigeminovascular activation in all neurons. In addition, LY466195 demonstrated an N-methyl-d-aspartate receptor-mediated effect. This study demonstrates a differential role of GluK1 receptors in the TCC, antagonism of which can inhibit trigeminovascular activation through postsynaptic mechanisms. Furthermore, the data suggest a novel, possibly presynaptic, modulatory role of trigeminocervical kainate receptors in vivo. Differential activation of kainate receptors suggests unique roles for this receptor in pro- and antinociceptive mechanisms in migraine pathophysiology.

  14. Plant cysteine proteases that evoke itch activate protease-activated receptors

    PubMed Central

    Reddy, V.B.; Lerner, E.A.

    2013-01-01

    Background Bromelain, ficin and papain are cysteine proteases from plants that produce itch upon injection into skin. Their mechanism of action has not been considered previously. Objectives To determine the mechanism by which these proteases function. Methods The ability of these proteases to activate protease-activated receptors was determined by ratiometric calcium imaging. Results We show here that bromelain, ficin and papain activate protease-activated receptors 2 and 4. Conclusions Bromelain, ficin and papain function as signalling molecules and activate protease-activated receptors. Activation of these receptors is the likely mechanism by which these proteases evoke itch. PMID:20491769

  15. Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist.

    PubMed

    Ballet, Steven; Feytens, Debby; Buysse, Koen; Chung, Nga N; Lemieux, Carole; Tumati, Suneeta; Keresztes, Attila; Van Duppen, Joost; Lai, Josephine; Varga, Eva; Porreca, Frank; Schiller, Peter W; Vanden Broeck, Jozef; Tourwé, Dirk

    2011-04-14

    A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3',5'-(CF(3))(2)-Bn], 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], and 23 [Ac-Tic-NMe-3',5'-(CF(3))(2)-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], which combines the N terminus of the established Dmt(1)-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH(2)) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, that is, Dmt-D-Arg-Aba-Gly-NH(2) (36), also proved to be an extremely potent and balanced μ and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.

  16. Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist

    PubMed Central

    Ballet, Steven; Feytens, Debby; Buysse, Koen; Chung, Nga N.; Lemieux, Carole; Tumati, Suneeta; Keresztes, Attila; Van Duppen, Joost; Lai, Josephine; Varga, Eva; Porreca, Frank; Schiller, Peter W.; Broeck, Jozef Vanden; Tourwé, Dirk

    2011-01-01

    A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3′,5′-(CF3)2-Bn], 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn] and 23 [Ac-Tic-NMe-3′,5′-(CF3)2-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], which combines the N-terminus of the established Dmt1-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH2) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, i.e. Dmt-D-Arg-Aba-Gly-NH2 36, also proved to be an extremely potent and balanced μ- and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity. PMID:21413804

  17. Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment.

    PubMed

    Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Shein, S A; Grinenko, N F; Pavlov, K A; Ryabukhin, I A; Chekhonin, V P

    2012-04-01

    cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1(2-4)) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purified (metal affinity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1(2-4) showed that biotinylated VEGF specifically binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fluorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specific receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confirmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.

  18. Toll-Like Receptor Signaling Induces Nrf2 Pathway Activation through p62-Triggered Keap1 Degradation.

    PubMed

    Yin, Shasha; Cao, Wangsen

    2015-08-01

    Toll-like receptors (TLRs) induce inflammation and tissue repair through multiple signaling pathways. The Nrf2 pathway plays a key role in defending against the tissue damage incurred by microbial infection or inflammation-associated diseases. The critical event that mediates TLR-induced Nrf2 activation is still poorly understood. In this study, we found that lipopolysaccharide (LPS) and other Toll-like receptor (TLR) agonists activate Nrf2 signaling and the activation is due to the reduction of Keap1, the key Nrf2 inhibitor. TLR signaling-induced Keap1 reduction promoted Nrf2 translocation from the cytoplasm to the nucleus, where it activated transcription of its target genes. TLR agonists modulated Keap1 at the protein posttranslation level through autophagy. TLR signaling increased the expression of autophagy protein p62 and LC3-II and induced their association with Keap1 in the autophagosome-like structures. We also characterized the interaction between p62 and Keap1 and found that p62 is indispensable for TLR-mediated Keap1 reduction: TLR signaling had no effect on Keap1 if cells lacked p62 or if cells expressed a mutant Keap1 that could not interact with p62. Our study indicates that p62-mediated Keap1 degradation through autophagy represents a critical linkage for TLR signaling regulation of the major defense network, the Nrf2 signaling pathway. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. NMDA receptor activation regulates sociability by its effect on mTOR signaling activity.

    PubMed

    Burket, Jessica A; Benson, Andrew D; Tang, Amy H; Deutsch, Stephen I

    2015-07-03

    Tuberous Sclerosis Complex is one example of a syndromic form of autism spectrum disorder associated with disinhibited activity of mTORC1 in neurons (e.g., cerebellar Purkinje cells). mTORC1 is a complex protein possessing serine/threonine kinase activity and a key downstream molecule in a signaling cascade beginning at the cell surface with the transduction of neurotransmitters (e.g., glutamate and acetylcholine) and nerve growth factors (e.g., Brain-Derived Neurotrophic Factor). Interestingly, the severity of the intellectual disability in Tuberous Sclerosis Complex may relate more to this metabolic disturbance (i.e., overactivity of mTOR signaling) than the density of cortical tubers. Several recent reports showed that rapamycin, an inhibitor of mTORC1, improved sociability and other symptoms in mouse models of Tuberous Sclerosis Complex and autism spectrum disorder, consistent with mTORC1 overactivity playing an important pathogenic role. NMDA receptor activation may also dampen mTORC1 activity by at least two possible mechanisms: regulating intraneuronal accumulation of arginine and the phosphorylation status of a specific extracellular signal regulating kinase (i.e., ERK1/2), both of which are "drivers" of mTORC1 activity. Conceivably, the prosocial effects of targeting the NMDA receptor with agonists in mouse models of autism spectrum disorders result from their ability to dampen mTORC1 activity in neurons. Strategies for dampening mTORC1 overactivity by NMDA receptor activation may be preferred to its direct inhibition in chronic neurodevelopmental disorders, such as autism spectrum disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors

    PubMed Central

    Terenzio, Marco; Golding, Matthew; Russell, Matthew R G; Wicher, Krzysztof B; Rosewell, Ian; Spencer-Dene, Bradley; Ish-Horowicz, David; Schiavo, Giampietro

    2014-01-01

    We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75NTR) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75NTR available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors. PMID:24920579

  1. Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors.

    PubMed

    Terenzio, Marco; Golding, Matthew; Russell, Matthew R G; Wicher, Krzysztof B; Rosewell, Ian; Spencer-Dene, Bradley; Ish-Horowicz, David; Schiavo, Giampietro

    2014-07-17

    We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75(NTR)) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75(NTR) available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors. © 2014 The Authors.

  2. SPAK kinase is a substrate and target of PKCθ in T-cell receptor-induced AP-1 activation pathway

    PubMed Central

    Li, Yingqiu; Hu, Junru; Vita, Randi; Sun, Binggang; Tabata, Hiroki; Altman, Amnon

    2004-01-01

    Protein kinase C-θ (PKCθ) plays an important role in T-cell activation via stimulation of AP-1 and NF-κB. Here we report the isolation of SPAK, a Ste20-related upstream mitogen-activated protein kinase (MAPK), as a PKCθ-interacting kinase. SPAK interacted with PKCθ (but not with PKCα) via its 99 COOH-terminal residues. TCR/CD28 costimulation enhanced this association and stimulated the catalytic activity of SPAK. Recombinant SPAK was phosphorylated on Ser-311 in its kinase domain by PKCθ, but not by PKCα. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKCθ-deficient T cells. Transfected SPAK synergized with constitutively active PKCθ to activate AP-1, but not NF-κB. This synergistic activity, as well as the receptor-induced SPAK activation, required the PKCθ-interacting region of SPAK, and Ser-311 mutation greatly reduced these activities of SPAK. Conversely, a SPAK-specific RNAi or a dominant-negative SPAK mutant inhibited PKCθ- and TCR/CD28-induced AP-1, but not NF-κB, activation. These results define SPAK as a substrate and target of PKCθ in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-κB) activation. PMID:14988727

  3. Activation of the sigma receptor 1 modulates AMPA receptor-mediated light-evoked excitatory postsynaptic currents in rat retinal ganglion cells.

    PubMed

    Liu, Lei-Lei; Deng, Qin-Qin; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2016-09-22

    Sigma receptor (σR), a unique receptor family, is classified into three subtypes: σR1, σR2 and σR3. It was previously shown that σR1 activation induced by 1μM SKF10047 (SKF) suppressed N-methyl-d-aspartate (NMDA) receptor-mediated responses of rat retinal ganglion cells (GCs) and the suppression was mediated by a distinct Ca(2+)-dependent phospholipase C (PLC)-protein kinase C (PKC) pathway. In the present work, using whole-cell patch-clamp techniques in rat retinal slice preparations, we further demonstrate that SKF of higher dosage (50μM) significantly suppressed AMPA receptor (AMPAR)-mediated light-evoked excitatory postsynaptic currents (L-EPSCs) of retinal ON-type GCs (ON GCs), and the effect was reversed by the σR1 antagonist BD1047, suggesting the involvement of σR1. The SKF (50μM) effect was unlikely due to a change in glutamate release from bipolar cells, as suggested by the unaltered paired-pulse ratio (PPR) of AMPAR-mediated EPSCs of ON GCs. SKF (50μM) did not change L-EPSCs of ON GCs when the G protein inhibitor GDP-β-S or the protein kinase G (PKG) inhibitor KT5823 was intracellularly infused. Calcium imaging further revealed that SKF (50μM) did not change intracellular calcium concentration in GCs and persisted to suppress L-EPSCs when intracellular calcium was chelated by BAPTA. The SKF (50μM) effect was intact when protein kinase A (PKA) and phosphatidylinostiol (PI)-PLC signaling pathways were both blocked. We conclude that the SKF (50μM) effect is Ca(2+)-independent, PKG-dependent, but not involving PKA, PI-PLC pathways. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  4. Mincle suppresses Toll-like receptor 4 activation.

    PubMed

    Greco, Stephanie H; Mahmood, Syed Kashif; Vahle, Anne-Kristin; Ochi, Atsuo; Batel, Jennifer; Deutsch, Michael; Barilla, Rocky; Seifert, Lena; Pachter, H Leon; Daley, Donnele; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu R; Miller, George

    2016-07-01

    Regulation of Toll-like receptor responses is critical for limiting tissue injury and autoimmunity in both sepsis and sterile inflammation. We found that Mincle, a C-type lectin receptor, regulates proinflammatory Toll-like receptor 4 signaling. Specifically, Mincle ligation diminishes Toll-like receptor 4-mediated inflammation, whereas Mincle deletion or knockdown results in marked hyperresponsiveness to lipopolysaccharide in vitro, as well as overwhelming lipopolysaccharide-mediated inflammation in vivo. Mechanistically, Mincle deletion does not up-regulate Toll-like receptor 4 expression or reduce interleukin 10 production after Toll-like receptor 4 ligation; however, Mincle deletion decreases production of the p38 mitogen-activated protein kinase-dependent inhibitory intermediate suppressor of cytokine signaling 1, A20, and ABIN3 and increases expression of the Toll-like receptor 4 coreceptor CD14. Blockade of CD14 mitigates the increased sensitivity of Mincle(-/-) leukocytes to Toll-like receptor 4 ligation. Collectively, we describe a major role for Mincle in suppressing Toll-like receptor 4 responses and implicate its importance in nonmycobacterial models of inflammation. © Society for Leukocyte Biology.

  5. Trace Amine-Associated Receptor 1 – Family Archetype or Iconoclast?

    PubMed Central

    Grandy, David K.

    2009-01-01

    Interest has recently been rekindled in receptors that are activated by low molecular weight, non-catecholic, biogenic amines that are typically found as trace constituents of various vertebrate and invertebrate tissues and fluids. The timing of this resurgent focus on receptors activated by the ‘trace amines’ (TAs) β-phenylethylamine (PEA), tyramine (TYR), octopamine (OCT), synephrine (SYN), and tryptamine (TRYP) is the direct result of two publications that appeared in 2001 describing the cloning of a novel G protein-coupled receptor (GPCR) referred to by their discoverers as TA1 (Borowsky et al., 2001) and TAR1 (Bunzow et al., 2001). When heterologously expressed in Xenopus laevis oocytes and various eukaryotic cell lines recombinant rodent and human TA receptors dose-dependently couple to the stimulation of cAMP production. Structure-activity profiling based on this functional response has revealed that in addition to the TAs, other biologically active compounds containing a 2 carbon aliphatic side chain linking an amino group to at least one benzene ring are potent and efficacious TA receptor agonists with amphetamine, methamphetamine, 3-iodothyronamine, thyronamine, and dopamine among the most notable. Almost 100 years after the search for TA receptors began numerous TA1/TAR1-related sequences, now called Trace Amine-Associated Receptors (TAARs), have been identified in the genome of every species of vertebrate examined to date. Consequently, even though heterologously expressed TAAR1 fits the pharmacological criteria established for a bona fide TA receptor a major challenge for those working in the field is to discern the in vivo pharmacology and physiology of each purported member of this extended family of GPCRs. Only then will it be possible to establish whether TAAR1 is the family archetype or an iconoclast. PMID:17888514

  6. A role for D1 dopamine receptors in striatal methamphetamine-induced neurotoxicity.

    PubMed

    Friend, Danielle M; Keefe, Kristen A

    2013-10-25

    Methamphetamine (METH) exposure results in long-term damage to the dopamine system in both human METH abusers and animal models. One factor that has been heavily implicated in this METH-induced damage to the dopaminergic system is the activation of D1 dopamine (DA) receptors. However, a significant caveat to the studies investigating the role of the receptor in such toxicity is that genetic and pharmacological manipulations of the D1 DA receptor also mitigate METH-induced hyperthermia. Importantly, METH-induced hyperthermia is tightly associated with the neurotoxicity, such that simply cooling animals during METH exposure protects against the neurotoxicity. Therefore, it is difficult to determine whether D1 DA receptors per se play an important role in METH-induced neurotoxicity or whether the protection observed simply resulted from a mitigation of METH-induced hyperthermia. To answer this important question, the current study infused a D1 DA receptor antagonist into striatum during METH exposure while controlling for METH-induced hyperthermia. Here we found that even when METH-induced hyperthermia is maintained, the coadministration of a D1 DA receptor antagonist protects against METH-induced neurotoxicity, strongly suggesting that D1 DA receptors play an important role in METH-induced neurotoxicity apart from the mitigation of METH-induced hyperthermia. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  7. In vitro and in vivo biological activities of SR140333, a novel potent non-peptide tachykinin NK1 receptor antagonist.

    PubMed

    Emonds-Alt, X; Doutremepuich, J D; Heaulme, M; Neliat, G; Santucci, V; Steinberg, R; Vilain, P; Bichon, D; Ducoux, J P; Proietto, V

    1993-12-21

    (S)1-(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)pip eridin-3- yl]ethyl)-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride (SR140333) is a new non-peptide antagonist of tachykinin NK1 receptors. SR140333 potently, selectively and competitively inhibited substance P binding to NK1 receptors from various animal species, including humans. In vitro, it was a potent antagonist in functional assays for NK1 receptors such as [Sar9,Met(O2)11]substance P-induced endothelium-dependent relaxation of rabbit pulmonary artery and contraction of guinea-pig ileum. Up to 1 microM, it had no effect in bioassays for NK2 ([beta Ala8]neurokinin A-induced contraction of endothelium-deprived rabbit pulmonary artery) and NK3 ([MePhe7]neurokinin B-induced contraction of rat portal vein) receptors. The antagonism exerted by SR140333 toward NK1 receptors was apparently non-competitive, with pD2' values (antagonism potency evaluated by the negative logarithm of the molar concentration of antagonist that produces a 50% reduction of the maximal response to the agonist) between 9.65 and 10.16 in the different assays. SR140333 also blocked in vitro [Sar9,Met(O2)11]substance P-induced release of acetylcholine from rat striatum. In vivo, SR140333 exerted highly potent antagonism toward [Sar9,Met(O2)11]substance P-induced hypotension in dogs (ED50 = 3 micrograms/kg i.v.), bronchoconstriction in guinea-pig (ED50 = 42 micrograms/kg i.v.) and plasma extravasation in rats (ED50 = 7 micrograms/kg i.v.). Finally, it also blocked the activation of rat thalamic neurons after nociceptive stimulation (ED50 = 0.2 micrograms/kg i.v.).

  8. Antidepressant-like responses to the combined sigma and 5-HT1A receptor agonist OPC-14523.

    PubMed

    Tottori, K; Miwa, T; Uwahodo, Y; Yamada, S; Nakai, M; Oshiro, Y; Kikuchi, T; Altar, C A

    2001-12-01

    The antidepressant-like activity of a novel compound, OPC-14523, was investigated in comparison with the conventional antidepressants, fluoxetine and imipramine. OPC-14523 bound with nanomolar affinities to sigma receptors (IC(50)=47-56 nM), the 5-HT(1A) receptor (IC(50)=2.3 nM), and the 5-HT transporter (IC(50)=80 nM). OPC-14523 inhibited the in vitro reuptake of 3H-5-HT (IC(50)=27 nM), but it showed very weak inhibitory activity on 3H-NE and 3H-DA reuptake. OPC-14523 did not inhibit MAO A or B activities or muscarinic receptors. A single oral administration of OPC-14523 produced a marked antidepressant-like effect in the forced swimming test (FST) with rats (ED(50)=27 mg/kg) and mice (ED(50)=20mg/kg) without affecting the general locomotor activity. In contrast, fluoxetine and imipramine each required at least four days of repeated dosing to show this activity. The acute activity of OPC-14523 was blocked by pretreatment with the sigma receptor antagonist NE-100 or the selective 5-HT(1A) receptor antagonist WAY-100635. The induction of flat body posture by OPC-14523 was blocked by the selective 5-HT(1A) receptor antagonist NAN-190, and forebrain 5-HT biosynthesis was attenuated by OPC-14523 at behaviorally effective doses. In contrast, OPC-14523, unlike fluoxetine, failed to inhibit 5-HT reuptake at oral doses below 100mg/kg. Thus, the acute antidepressant-like action of OPC-14523 is achieved by the combined stimulation of sigma and 5-HT(1A) receptors without inhibition of 5-HT reuptake in vivo.

  9. Protease-activated receptor (PAR)-2 is required for PAR-1 signalling in pulmonary fibrosis

    PubMed Central

    Lin, Cong; von der Thüsen, Jan; Daalhuisen, Joost; ten Brink, Marieke; Crestani, Bruno; van der Poll, Tom; Borensztajn, Keren; Spek, C Arnold

    2015-01-01

    Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced lung fibrosis is diminished in both PAR-1 and PAR-2 deficient mice. We thus have been suggested that combined inactivation of PAR-1 and PAR-2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR-1 and PAR-2 agonists in the absence or presence of specific PAR-1 or PAR-2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and PAR-2 deficient mice with or without a specific PAR-1 antagonist (P1pal-12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR-1 and/or PAR-2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR-1 and PAR-2 did not show any additive effects on these pro-fibrotic responses. Strikingly, PAR-2 deficiency as well as pharmacological PAR-1 inhibition reduced bleomycin-induced pulmonary fibrosis to a similar extent. PAR-1 inhibition in PAR-2 deficient mice did not further diminish bleomycin-induced pulmonary fibrosis. Finally, we show that the PAR-1-dependent pro-fibrotic responses are inhibited by the PAR-2 specific antagonist. Targeting PAR-1 and PAR-2 simultaneously is not superior to targeting either receptor alone in bleomycin-induced pulmonary fibrosis. We postulate that the pro-fibrotic effects of PAR-1 require the presence of PAR-2. PMID:25689283

  10. Protease-activated receptor (PAR)-2 is required for PAR-1 signalling in pulmonary fibrosis.

    PubMed

    Lin, Cong; von der Thüsen, Jan; Daalhuisen, Joost; ten Brink, Marieke; Crestani, Bruno; van der Poll, Tom; Borensztajn, Keren; Spek, C Arnold

    2015-06-01

    Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced lung fibrosis is diminished in both PAR-1 and PAR-2 deficient mice. We thus have been suggested that combined inactivation of PAR-1 and PAR-2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR-1 and PAR-2 agonists in the absence or presence of specific PAR-1 or PAR-2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and PAR-2 deficient mice with or without a specific PAR-1 antagonist (P1pal-12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR-1 and/or PAR-2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR-1 and PAR-2 did not show any additive effects on these pro-fibrotic responses. Strikingly, PAR-2 deficiency as well as pharmacological PAR-1 inhibition reduced bleomycin-induced pulmonary fibrosis to a similar extent. PAR-1 inhibition in PAR-2 deficient mice did not further diminish bleomycin-induced pulmonary fibrosis. Finally, we show that the PAR-1-dependent pro-fibrotic responses are inhibited by the PAR-2 specific antagonist. Targeting PAR-1 and PAR-2 simultaneously is not superior to targeting either receptor alone in bleomycin-induced pulmonary fibrosis. We postulate that the pro-fibrotic effects of PAR-1 require the presence of PAR-2. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  11. IGF-1-dependent subunit communication of the IGF-1 holoreceptor: Interactions between. alpha. beta. heterodimeric receptor halves

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wilden, P.A.; Treadway, J.L.; Morrison, B.D.

    1989-12-12

    Examination of {sup 125}I-IGF-1 affinity cross-linking and {beta}-subunit autophosphorylation has indicated that IGF-1 induces a covalent association of isolated {alpha}{beta} heterodimeric IGF-1 receptors into an {alpha}{sub 2}{beta}{sub 2} heterotetrameric state, in a similar manner to that observed for the insulin receptor. The formation of the {alpha}{sub 2}{beta}{sub 2} heterotetrameric IGF-1 receptor complex from the partially purified {alpha}{beta} heterodimers was time dependent with half-maximal formation in approximately 30 min at saturating IGF-1 concentrations. The IGF-1-dependent association of the partially purified {alpha}{beta} heterodimers into an {alpha}{sub 2}{beta}{sub 2} heterotetrameric state was specific for the IGF-1 receptors since IGF-1 was unable to stimulatemore » the protein kinase activity of the purified {alpha}{beta} heterodimeric insulin receptor complex. Incubation of the {alpha}{sub 2}{beta}{sub 2} heterotetrameric IGF-1 holoreceptor with the specific sulfhydryl agent iodoacetamide (IAN) did not alter {sup 125}I-IGF-1 binding or IGF-1 stimulation of protein kinase activity. However, IAN treatment of the {alpha}{beta} heterodimeric IGF-1 receptors inhibited the IGF-1 dependent covalent formation of the disulfide-linked {alpha}{sub 2}{beta}{sub 2} heterotetrameric complex. These data indicate that IGF-1 induces the covalent association of isolated {alpha}{beta} heterodimeric IGF-1 receptor complexes into a disulfide-linked {alpha}{sub 2}{beta}{sub 2} heterotetrameric state whereas Mn/MgATP induces a noncovalent association. Therefore, unlike the insulin receptor in which noncovalent association is sufficient for kinase activation, only the covalent assembly of the IGF-1 receptor {alpha}{beta} heterodimers into the {alpha}{sub 2}{beta}{sub 2} heterotetrameric holoreceptor complex is associated with ligand-stimulated protein kinase activation.« less

  12. Activation of triggering receptor expressed on myeloid cells-1 on human neutrophils by marburg and ebola viruses.

    PubMed

    Mohamadzadeh, Mansour; Coberley, Sadie S; Olinger, Gene G; Kalina, Warren V; Ruthel, Gordon; Fuller, Claudette L; Swenson, Dana L; Pratt, William D; Kuhns, Douglas B; Schmaljohn, Alan L

    2006-07-01

    Marburg virus (MARV) and Ebola virus (EBOV), members of the viral family Filoviridae, cause fatal hemorrhagic fevers in humans and nonhuman primates. High viral burden is coincident with inadequate adaptive immune responses and robust inflammatory responses, and virus-mediated dysregulation of early host defenses has been proposed. Recently, a novel class of innate receptors called the triggering receptors expressed in myeloid cells (TREM) has been discovered and shown to play an important role in innate inflammatory responses and sepsis. Here, we report that MARV and EBOV activate TREM-1 on human neutrophils, resulting in DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory cytokines, and phenotypic changes. A peptide specific to TREM-1 diminished the release of tumor necrosis factor alpha by filovirus-activated human neutrophils in vitro, and a soluble recombinant TREM-1 competitively inhibited the loss of cell surface TREM-1 that otherwise occurred on neutrophils exposed to filoviruses. These data imply direct activation of TREM-1 by filoviruses and also indicate that neutrophils may play a prominent role in the immune and inflammatory responses to filovirus infections.

  13. 5-HT2A receptor activation is necessary for CO2-induced arousal

    PubMed Central

    Smith, Haleigh R.; MacAskill, Amanda; Richerson, George B.

    2015-01-01

    Hypercapnia-induced arousal from sleep is an important protective mechanism pertinent to a number of diseases. Most notably among these are the sudden infant death syndrome, obstructive sleep apnea and sudden unexpected death in epilepsy. Serotonin (5-HT) plays a significant role in hypercapnia-induced arousal. The mechanism of 5-HT's role in this protective response is unknown. Here we sought to identify the specific 5-HT receptor subtype(s) involved in this response. Wild-type mice were pretreated with antagonists against 5-HT receptor subtypes, as well as antagonists against adrenergic, cholinergic, histaminergic, dopaminergic, and orexinergic receptors before challenge with inspired CO2 or hypoxia. Antagonists of 5-HT2A receptors dose-dependently blocked CO2-induced arousal. The 5-HT2C receptor antagonist, RS-102221, and the 5-HT1A receptor agonist, 8-OH-DPAT, attenuated but did not completely block CO2-induced arousal. Blockade of non-5-HT receptors did not affect CO2-induced arousal. None of these drugs had any effect on hypoxia-induced arousal. 5-HT2 receptor agonists were given to mice in which 5-HT neurons had been genetically eliminated during embryonic life (Lmx1bf/f/p) and which are known to lack CO2-induced arousal. Application of agonists to 5-HT2A, but not 5-HT2C, receptors, dose-dependently restored CO2-induced arousal in these mice. These data identify the 5-HT2A receptor as an important mediator of CO2-induced arousal and suggest that, while 5-HT neurons can be independently activated to drive CO2-induced arousal, in the absence of 5-HT neurons and endogenous 5-HT, 5-HT receptor activation can act in a permissive fashion to facilitate CO2-induced arousal via another as yet unidentified chemosensor system. PMID:25925320

  14. Selective killing of proinflammatory synovial fibroblasts via activation of transient receptor potential ankyrin (TRPA1).

    PubMed

    Lowin, Torsten; Bleck, Janna; Schneider, Matthias; Pongratz, Georg

    2018-05-24

    Studies in rheumatoid arthritis synovial fibroblasts (RASF) demonstrated the expression of several transient receptor potential channels (TRP) such as TRPV1, TRPV2, TRPV4, TRPA1 and TRPM8. Upon ligation, these receptors increase intracellular calcium but they have also been linked to modulation of inflammation in several cell types. TNF was shown to increase the expression of TRPA1, the receptor for mustard oil and environmental poisons in SF, but the functional consequences have not been investigated yet. TRPA1 was detected by immunocytochemistry, western blot and cell-based ELISA. Calcium measurements were conducted in a multimode reader. Cell viability was assessed by quantification of lactate dehydrogenase (LDH) in culture supernatants and "RealTime-Glo" luminescent assays. IL-6 and IL-8 production by SF was quantified by ELISA. Proliferation was determined by cell titer blue incorporation. After 72 h, mimicking proinflammatory conditions by the innate cytokine TNF up-regulated TRPA1 protein levels in RASF which was accompanied by increased sensitivity to TRPA1 agonists AITC and polygodial. Under unstimulated conditions, polygodial elicited calcium flux only in the highest concentrations used (50 µM and 25 µM). TNF preincubation substantially lowered the activation threshold for polygodial (from 25 µM to 1 µM). In the absence of TNF pre-stimulation, only polygodial in high concentrations was able to reduce viability of synovial fibroblasts as determined by a real-time viability assay. However, following TNF preincubation, stimulation of TRPA1 led to a fast (<30 min) viability loss by necrosis of synovial fibroblasts. TRPA1 activation was also associated with decreased proliferation of RASFs, an effect that was also substantially enhanced by TNF preincubation. On the functional level, IL-6 and IL-8 production was attenuated by the TRPA1 antagonist A967079 but also polygodial, although the latter mediated this effect by reducing cell viability

  15. IgA Enhances IGF-1 Mitogenic Activity Via Receptor Modulation in Glomerular Mesangial Cells: Implications for IgA-Induced Nephropathy.

    PubMed

    Al-Eisa, Amal; Dhaunsi, Gursev S

    2017-01-01

    Glomerulonephritis due to mesangial proliferation is responsible for renal dysfunction in IgA nephropathy (IgAN), however molecular mechanisms of pathogenesis are not well known. We examined the effect of IgA on Insulin-like Growth Factor-1 (IGF-1) activity, a potent mitogen with vital role in growth and development of children, and IGF-1 receptor (IGF-1R) in cultures of glomerular mesangial cells (GMC). GMC were isolated from rat kidneys using sieving and enzymatic digestion of tissue homogenates, and cultured in RPMI 1640 medium. GMC cultures were treated with IgA (0-10 µg/ml) in the presence or absence of IGF-1 and fetal bovine serum (FBS), and BrdU incorporation was measured. IGF-1 levels were assayed along with real-time PCR quantification of IGF-1R mRNA. Treatment of GMC with IgA (5 -10 µg/ml) significantly (p < 0.01) increased the BrdU incorporation in the presence or absence of FBS or IGF-1. IgA-mediated effects were more pronounced in IGF-1 treated cells that were significantly (p < 0.01) blocked by pretreatment of cells with IGF-1 receptor antibody or genistein. IgA significantly increased the levels of IGF-1 in culture supernatants and GMC homogenates. IGF-1R mRNA was significantly (p < 0.01) increased in IgA treated cells particularly by co-treatment with IGF-1. These findings show that IgA enhances the IGF-1 activity in GMC via stimulation of IGF-1R gene transcription and suggest a role for IGF-1 in pathogenesis of IgAN. © 2017 The Author(s). Published by S. Karger AG, Basel.

  16. Rho-kinase signaling controls nucleocytoplasmic shuttling of class IIa Histone Deacetylase (HDAC7) and transcriptional activation of orphan nuclear receptor NR4A1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Compagnucci, Claudia; Barresi, Sabina; Petrini, Stefania

    2015-04-03

    Rho-kinase (ROCK) has been well documented to play a key role in RhoA-induced actin remodeling. ROCK activation results in myosin light chain (MLC) phosphorylation either by direct action on MLC kinase (MLCK) or by inhibition of MLC phosphatase (MLCP), modulating actin–myosin contraction. We found that inhibition of the ROCK pathway in induced pluripotent stem cells, leads to nuclear export of HDAC7 and transcriptional activation of the orphan nuclear receptor NR4A1 while in cells with constitutive ROCK hyperactivity due to loss of function of the RhoGTPase activating protein Oligophrenin-1 (OPHN1), the orphan nuclear receptor NR4A1 is downregulated. Our study identify amore » new target of ROCK signaling via myosin phosphatase subunit (MYPT1) and Histone Deacetylase (HDAC7) at the nuclear level and provide new insights in the cellular functions of ROCK. - Highlights: • ROCK regulates nucleocytoplasmic shuttling of HDAC7 via phosphorylation of MYPT1. • Nuclear export of HDAC7 and upregulation of NR4A1 occurs with low ROCK activity. • High levels of ROCK activity due to OPHN1 loss of function downregulate NR4A1.« less

  17. Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

    PubMed

    Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

    1993-06-25

    Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

  18. Evidence That Graves' Ophthalmopathy Immunoglobulins Do Not Directly Activate IGF-1 Receptors.

    PubMed

    Marcus-Samuels, Bernice; Krieger, Christine C; Boutin, Alisa; Kahaly, George J; Neumann, Susanne; Gershengorn, Marvin C

    2018-05-01

    Graves' ophthalmopathy (GO) pathogenesis involves thyrotropin (TSH) receptor (TSHR)-stimulating autoantibodies. Whether there are autoantibodies that directly stimulate insulin-like growth factor 1 receptors (IGF-1Rs), stimulating insulin-like growth factor receptor antibodies (IGFRAbs), remains controversial. This study attempted to determine whether there are stimulating IGFRAbs in patients with GO. Immunoglobulins (Igs) were purified from normal volunteers (NV-Igs) and patients with GO (GO-Igs). The effects of TSH, IGF-1, NV-Igs, and GO-Igs on pAKT and pERK1/2, members of pathways used by IGF-1R and TSHR, were compared in orbital fibroblasts from GO patients (GOFs) and U2OS-TSHR cells overexpressing TSHRs, and U2OS cells that express TSHRs at very low endogenous levels. U2OS-TSHR and U2OS cells were used because GOFs are not easily manipulated using molecular techniques such as transfection, and U2OS cells because they express TSHRs at levels that do not measurably stimulate signaling. Thus, comparing U2OS-TSHR and U2OS cells permits specifically distinguishing signaling mediated by the TSHR and IGF-1R. In GOFs, all GO-Igs stimulated pERK1/2 formation and 69% stimulated pAKT. In U2OS-TSHR cells, 15% of NV-IGs and 83% of GO-Igs stimulated increases in pERK1/2, whereas all NV-Igs and GO-Igs stimulated increases in pAKT. In U2OS cells, 70% of GO-Igs stimulated small increases in pAKT. Knockdown of IGF-1R caused a 65 ± 6.3% decrease in IGF-1-stimulated pAKT but had no effect on GO-Igs stimulation of pAKT. Thus, GO-Igs contain factor(s) that stimulate pAKT formation. However, this factor(s) does not directly activate IGF-1R. Based on the findings analyzing these two signaling pathways, it is concluded there is no evidence of stimulating IGFRAbs in GO patients.

  19. Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-α induction

    PubMed Central

    Uematsu, Satoshi; Sato, Shintaro; Yamamoto, Masahiro; Hirotani, Tomonori; Kato, Hiroki; Takeshita, Fumihiko; Matsuda, Michiyuki; Coban, Cevayir; Ishii, Ken J.; Kawai, Taro; Takeuchi, Osamu; Akira, Shizuo

    2005-01-01

    Toll-like receptors (TLRs) recognize microbial pathogens and trigger innate immune responses. Among TLR family members, TLR7, TLR8, and TLR9 induce interferon (IFN)-α in plasmacytoid dendritic cells (pDCs). This induction requires the formation of a complex consisting of the adaptor MyD88, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and IFN regulatory factor (IRF) 7. Here we show an essential role of IL-1 receptor-associated kinase (IRAK)-1 in TLR7- and TLR9-mediated IRF7 signaling pathway. IRAK-1 directly bound and phosphorylated IRF7 in vitro. The kinase activity of IRAK-1 was necessary for transcriptional activation of IRF7. TLR7- and TLR9-mediated IFN-α production was abolished in Irak-1–deficient mice, whereas inflammatory cytokine production was not impaired. Despite normal activation of NF-κB and mitogen-activated protein kinases, IRF7 was not activated by a TLR9 ligand in Irak-1–deficient pDCs. These results indicated that IRAK-1 is a specific regulator for TLR7- and TLR9-mediated IFN-α induction in pDCs. PMID:15767370

  20. Functional selectivity induced by mGlu₄ receptor positive allosteric modulation and concomitant activation of Gq coupled receptors.

    PubMed

    Yin, Shen; Zamorano, Rocio; Conn, P Jeffrey; Niswender, Colleen M

    2013-03-01

    Metabotropic glutamate receptors (mGlus) are a group of Family C Seven Transmembrane Spanning Receptors (7TMRs) that play important roles in modulating signaling transduction, particularly within the central nervous system. mGlu(4) belongs to a subfamily of mGlus that is predominantly coupled to G(i/o) G proteins. We now report that the ubiquitous autacoid and neuromodulator, histamine, induces substantial glutamate-activated calcium mobilization in mGlu(4)-expressing cells, an effect which is observed in the absence of co-expressed chimeric G proteins. This strong induction of calcium signaling downstream of glutamate activation of mGlu(4) depends upon the presence of H(1) histamine receptors. Interestingly, the potentiating effect of histamine activation does not extend to other mGlu(4)-mediated signaling events downstream of G(i/o) G proteins, such as cAMP inhibition, suggesting that the presence of G(q) coupled receptors such as H(1) may bias normal mGlu(4)-mediated G(i/o) signaling events. When the activity induced by small molecule positive allosteric modulators of mGlu(4) is assessed, the potentiated signaling of mGlu(4) is further biased by histamine toward calcium-dependent pathways. These results suggest that G(i/o)-coupled mGlus may induce substantial, and potentially unexpected, calcium-mediated signaling events if stimulation occurs concomitantly with activation of G(q) receptors. Additionally, our results suggest that signaling induced by small molecule positive allosteric modulators may be substantially biased when G(q) receptors are co-activated. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Membrane receptor-dependent Notch1/Hes1 activation by melatonin protects against myocardial ischemia-reperfusion injury: in vivo and in vitro studies.

    PubMed

    Yu, Liming; Liang, Hongliang; Lu, Zhihong; Zhao, Guolong; Zhai, Mengen; Yang, Yang; Yang, Jian; Yi, Dinghua; Chen, Wensheng; Wang, Xiaowu; Duan, Weixun; Jin, Zhenxiao; Yu, Shiqiang

    2015-11-01

    Melatonin confers profound protective effect against myocardial ischemia-reperfusion injury (MI/RI). Activation of Notch1/Hairy and enhancer of split 1 (Hes1) signaling also ameliorates MI/RI. We hypothesize that melatonin attenuates MI/RI-induced oxidative damage by activating Notch1/Hes1 signaling pathway with phosphatase and tensin homolog deleted on chromosome 10 (Pten)/Akt acting as the downstream signaling pathway in a melatonin membrane receptor-dependent manner. Male Sprague Dawley rats were treated with melatonin (10 mg/kg/day) for 4 wk and then subjected to MI/R surgery. Melatonin significantly improved cardiac function and decreased myocardial apoptosis and oxidative damage. Furthermore, in cultured H9C2 cardiomyocytes, melatonin (100 μmol/L) attenuated simulated ischemia-reperfusion (SIR)-induced myocardial apoptosis and oxidative damage. Both in vivo and in vitro study demonstrated that melatonin treatment increased Notch1, Notch1 intracellular domain (NICD), Hes1, Bcl-2 expressions, and p-Akt/Akt ratio and decreased Pten, Bax, and caspase-3 expressions. However, these protective effects conferred by melatonin were blocked by DAPT (the specific inhibitor of Notch1 signaling), luzindole (the antagonist of melatonin membrane receptors), Notch1 siRNA, or Hes1 siRNA administration. In summary, our study demonstrates that melatonin treatment protects against MI/RI by modulating Notch1/Hes1 signaling in a receptor-dependent manner and Pten/Akt signaling pathways are key downstream mediators. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Sphingosine-1-Phosphate and the S1P3 Receptor Initiate Neuronal Retraction via RhoA/ROCK Associated with CRMP2 Phosphorylation.

    PubMed

    Quarta, Serena; Camprubí-Robles, Maria; Schweigreiter, Rüdiger; Matusica, Dusan; Haberberger, Rainer V; Proia, Richard L; Bandtlow, Christine E; Ferrer-Montiel, Antonio; Kress, Michaela

    2017-01-01

    The bioactive lipid sphingosine-1-phosphate (S1P) is an important regulator in the nervous system. Here, we explored the role of S1P and its receptors in vitro and in preclinical models of peripheral nerve regeneration. Adult sensory neurons and motor neuron-like cells were exposed to S1P in an in vitro assay, and virtually all neurons responded with a rapid retraction of neurites and growth cone collapse which were associated with RhoA and ROCK activation. The S1P 1 receptor agonist SEW2871 neither activated RhoA or neurite retraction, nor was S1P-induced neurite retraction mitigated in S1P 1 -deficient neurons. Depletion of S1P 3 receptors however resulted in a dramatic inhibition of S1P-induced neurite retraction and was on the contrary associated with a significant elongation of neuronal processes in response to S1P. Opposing responses to S1P could be observed in the same neuron population, where S1P could activate S1P 1 receptors to stimulate elongation or S1P 3 receptors and retraction. S1P was, for the first time in sensory neurons, linked to the phosphorylation of collapsin response-mediated protein-2 (CRMP2), which was inhibited by ROCK inhibition. The improved sensory recovery after crush injury further supported the relevance of a critical role for S1P and receptors in fine-tuning axonal outgrowth in peripheral neurons.

  3. Identification of a G protein coupled receptor induced in activated T cells.

    PubMed

    Kaplan, M H; Smith, D I; Sundick, R S

    1993-07-15

    Many genes are induced after T cell activation to make a cell competent for proliferation and ultimately, function. Many of these genes encode surface receptors for growth factors that signal a cell to proliferate. We have cloned a novel gene (clone 6H1) that codes for a member of the G protein-coupled receptor superfamily. This gene was isolated from a chicken activated T cell cDNA library by low level hybridization to mammalian IL-2 cDNA probes. The 308 amino acid open reading frame has seven hydrophobic, presumably transmembrane domains and a consensus site for interaction with G proteins. Tissue distribution studies suggest that gene expression is restricted to activated T cells. The message appears by 1 h after activation and is maintained for at least 45 h. Transcription of 6H1 is induced by a number of T cell stimuli and is inhibited by cyclosporin A, but not by cycloheximide. This is the first description of a member of this superfamily expressed specifically in activated T cells. The gene product may provide a link between T cell growth factors and G protein activation.

  4. An Alpha-1A Adrenergic Receptor Agonist Prevents Acute Doxorubicin Cardiomyopathy in Male Mice.

    PubMed

    Montgomery, Megan D; Chan, Trevor; Swigart, Philip M; Myagmar, Bat-Erdene; Dash, Rajesh; Simpson, Paul C

    2017-01-01

    Alpha-1 adrenergic receptors mediate adaptive effects in the heart and cardiac myocytes, and a myocyte survival pathway involving the alpha-1A receptor subtype and ERK activation exists in vitro. However, data in vivo are limited. Here we tested A61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide), a selective imidazoline agonist for the alpha-1A. A61603 was the most potent alpha-1-agonist in activating ERK in neonatal rat ventricular myocytes. A61603 activated ERK in adult mouse ventricular myocytes and protected the cells from death caused by the anthracycline doxorubicin. A low dose of A61603 (10 ng/kg/d) activated ERK in the mouse heart in vivo, but did not change blood pressure. In male mice, concurrent subcutaneous A61603 infusion at 10 ng/kg/d for 7 days after a single intraperitoneal dose of doxorubicin (25 mg/kg) increased survival, improved cardiac function, heart rate, and cardiac output by echocardiography, and reduced cardiac cell necrosis and apoptosis and myocardial fibrosis. All protective effects were lost in alpha-1A-knockout mice. In female mice, doxorubicin at doses higher than in males (35-40 mg/kg) caused less cardiac toxicity than in males. We conclude that the alpha-1A-selective agonist A61603, via the alpha-1A adrenergic receptor, prevents doxorubicin cardiomyopathy in male mice, supporting the theory that alpha-1A adrenergic receptor agonists have potential as novel heart failure therapies.

  5. Type 1 receptor tyrosine kinases are differentially phosphorylated in mammary carcinoma and differentially associated with steroid receptors.

    PubMed Central

    Bacus, S. S.; Chin, D.; Yarden, Y.; Zelnick, C. R.; Stern, D. F.

    1996-01-01

    The neu/erbB-2/HER-2 proto-oncogene is amplified and/or overexpressed in up to 30% of mammary carcinomas and has been variably correlated with poor prognosis. The signaling activity of the encoded receptor tyrosine kinase is regulated by interactions with other type 1 receptors and their ligands. We have used a novel approach, phosphorylation-sensitive anti-Neu antibodies, to quantify signaling by Neu and epidermal growth factor receptor in a panel of frozen sections of mammary carcinoma specimens. We also determined the relationship of Neu, phosphorylated Neu (and epidermal growth factor receptor), and phosphotyrosine to the expression of Neu-related receptors (epidermal growth factor receptor, HER-3, and HER-4) and to prognostic factors (estrogen and progesterone receptor). We found that tyrosine phosphorylation of Neu (and hence signaling activity) is highly variable among mammary carcinomas. Neu and HER-4 were associated with divergent correlates, suggesting that they have profoundly different biological activities. These results have implications for etiology of mammary carcinoma for clinical evaluation of mammary carcinoma patients, and for development of Neu-targeted therapeutic strategies. Images Figure 1 Figure 2 PMID:8579117

  6. Dual role of SLP-76 in mediating T cell receptor-induced activation of phospholipase C-gamma1.

    PubMed

    Beach, Dvora; Gonen, Ronnie; Bogin, Yaron; Reischl, Ilona G; Yablonski, Deborah

    2007-02-02

    Phospholipase C-gamma1 (PLC-gamma1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-gamma1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-gamma1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-gamma1 GEM recruitment, but were required for PLC-gamma1 phosphorylation at Tyr(783). Thus, GEM recruitment can be insufficient for full activation of PLC-gamma1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-gamma1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-gamma1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-gamma1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.

  7. Interleukin 1 Receptor (IL-1R1) Activation Exacerbates Toxin-Induced Acute Kidney Injury.

    PubMed

    Privratsky, Jamie R; Zhang, Jiandong; Lu, Xiaohan; Rudemiller, Nathan; Wei, Qingqing; Yu, Yen-Rei; Gunn, Michael Dee; Crowley, Steven D

    2018-05-23

    Acute kidney injury (AKI) is a leading cause of morbidity and mortality. Cisplatin is an effective chemotherapeutic agent whose administration is limited by nephrotoxicity. Therapies to prevent cisplatin-induced AKI are lacking. While tumor necrosis factor-α (TNF) plays a key role in the pathogenesis of cisplatin nephrotoxicity, the immune signaling pathways that trigger TNF generation in this context require elucidation. Sterile injury triggers the release and activation of both isoforms of interleukin(IL)-1, IL-1α and IL-1β, and stimulation of the interleukin-1 receptor (IL-1R1) by these ligands engages a pro-inflammatory signaling cascade that induces TNF induction. We therefore hypothesized that IL-1R1 activation exacerbates cisplatin-induced AKI by inducing TNF production thereby augmenting inflammatory signals between kidney parenchymal cells and infiltrating myeloid cells. IL-1R1+/+ (WT) and IL-1R1-/- (KO) mice were subjected to cisplatin-induced AKI. Compared to WT mice, IL-1R1 KO mice had attenuated AKI as measured by serum creatinine and BUN; renal NGAL mRNA levels; and blinded histological analysis of kidney pathology. In the cisplatin-injured kidney, IL-1R1 KO mice had diminished levels of whole kidney TNF and fewer Ly6G-expressing neutrophils. In addition, an unbiased machine learning analysis of intra-renal immune cells revealed a diminished number of CD11bint/CD11cint myeloid cells in IL-1R1 KO injured kidneys compared to IL-1R1 WT kidneys. Following cisplatin, IL-1R1 KO kidneys, compared to WTs, had fewer TNF-producing macrophages, CD11bint/CD11cint cells, and neutrophils, consistent with an effect of IL-1R1 to polarize intra-renal myeloid cells toward a pro-inflammatory phenotype. Interruption of IL-1-dependent signaling pathways warrants further evaluation to decrease nephrotoxicity during cisplatin therapy.

  8. Principles of antibody-mediated TNF receptor activation

    PubMed Central

    Wajant, H

    2015-01-01

    From the beginning of research on receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF), agonistic antibodies have been used to stimulate TNFRSF receptors in vitro and in vivo. Indeed, CD95, one of the first cloned TNFRSF receptors, was solely identified as the target of cell death-inducing antibodies. Early on, it became evident from in vitro studies that valency and Fcγ receptor (FcγR) binding of antibodies targeting TNFRSF receptors can be of crucial relevance for agonistic activity. TNFRSF receptor-specific antibodies of the IgM subclass and secondary cross-linked or aggregation prone dimeric antibodies typically display superior agonistic activity compared with dimeric antibodies. Likewise, anchoring of antibodies to cell surface-expressed FcγRs potentiate their ability to trigger TNFRSF receptor signaling. However, only recently has the relevance of oligomerization and FcγR binding for the in vivo activity of antibody-induced TNFRSF receptor activation been straightforwardly demonstrated in vivo. This review discusses the crucial role of oligomerization and/or FcγR binding for antibody-mediated TNFRSF receptor stimulation in light of current models of TNFRSF receptor activation and especially the overwhelming relevance of these issues for the rational development of therapeutic TNFRSF receptor-targeting antibodies. PMID:26292758

  9. (+)Lysergic acid diethylamide, but not its nonhallucinogenic congeners, is a potent serotonin 5HT1C receptor agonist

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Burris, K.D.; Breeding, M.; Sanders-Bush, E.

    Activation of central serotonin 5HT2 receptors is believed to be the primary mechanism whereby lysergic acid diethylamide (LSD) and other hallucinogens induce psychoactive effects. This hypothesis is based on extensive radioligand binding and electrophysiological and behavioral studies in laboratory animals. However, the pharmacological profiles of 5HT2 and 5HT1C receptors are similar, making it difficult to distinguish between effects due to activation of one or the other receptor. For this reason, it was of interest to investigate the interaction of LSD with 5HT1C receptors. Agonist-stimulated phosphoinositide hydrolysis in rat choroid plexus was used as a direct measure of 5HT1C receptor activation.more » (+)LSD potently stimulated phosphoinositide hydrolysis in intact choroid plexus and in cultures of choroid plexus epithelial cells, with EC50 values of 9 and 26 nM, respectively. The effect of (+)LSD in both systems was blocked by 5HT receptor antagonists with an order of activity consistent with interaction at 5HT1C receptors. Neither (+)-2-bromo-LSD nor lisuride, two nonhallucinogenic congeners of LSD, were able to stimulate 5HT1C receptors in cultured cells or intact choroid plexus. In contrast, lisuride, like (+)LSD, is a partial agonist at 5HT2 receptors in cerebral cortex slices and in NIH 3T3 cells transfected with 5HT2 receptor cDNA. The present finding that (+)LSD, but not its nonhallucinogenic congeners, is a 5HT1C receptor agonist suggests a possible role for these receptors in mediating the psychoactive effects of LSD.« less

  10. An Insulin-Like Growth Factor 1 Receptor Inhibitor Induces CYP3A4 Expression through a Pregnane X Receptor-Independent, Noncanonical Constitutive Androstane Receptor-Related Mechanism

    PubMed Central

    Li, Linhao; Sinz, Michael W.; Zimmermann, Kurt

    2012-01-01

    Inhibition of insulin-like growth factor-1 receptor (IGF-1R) signaling represents an attractive therapeutic strategy for cancer treatment. A first-generation IGF-1R inhibitor (R)-4-(3-(3-chlorophenyl)-3-hydroxypropyl)-3-(4-methyl-6-morpholino-1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-one (BMS-536924), however, was associated with potent CYP3A4 induction mediated by pregnane X receptor (PXR; NR1I2) transactivation. Structural activity-based modification led to the synthesis of 4-(1-(2-(4-((2-(4-chloro-1H-pyrazol-1-yl)ethyl)amino)-2-oxo-1,2-dihydropyridin-3-yl)-4-methyl-1H-benzo[d]imidazol-6-yl)piperidin-4-yl) piperazine-1-carboxylate (BMS-665351) with no PXR activity while maintaining its ability to inhibit IGF-1R. However, BMS-665351 significantly induces CYP3A4 expression in human primary hepatocytes (HPHs). Here, we report a novel nonclassical constitutive androstane receptor (CAR; NR1I3)-related pathway of BMS-665351-mediated CYP3A4 induction. BMS-665351 treatment resulted in the significant induction of CYP3A4 in HPHs and HepG2 cells, but failed to activate either PXR or CAR in cell-based reporter assays. Moreover, BMS-665351 at concentrations that induce CYP3A4 expression was unable to translocate human CAR from the cytoplasm to the nucleus of HPHs, which represents the initial step of CAR activation. Nevertheless, quantitative polymerase chain reaction analysis demonstrated that BMS-665351 significantly enhanced the expression of CYP3A4 in CAR- but not PXR-transfected HepG2 and Huh7 cells. It is noteworthy that BMS-665351 selectively induced the expression of CAR but not PXR in all tested hepatic cell systems. Synergistic induction of CYP3A4 was observed in HPHs cotreated with BMS-665351 and prototypical activators of CAR but not PXR. In summary, our results indicate that BMS-665351-mediated induction of CYP3A4 is CAR-dependent, but BMS-665351 itself is not a typical activator of either CAR or PXR, rather it functions as a selective inducer of CAR expression and

  11. Serotonin receptor 1A–modulated phosphorylation of glycine receptor α3 controls breathing in mice

    PubMed Central

    Manzke, Till; Niebert, Marcus; Koch, Uwe R.; Caley, Alex; Vogelgesang, Steffen; Hülsmann, Swen; Ponimaskin, Evgeni; Müller, Ulrike; Smart, Trevor G.; Harvey, Robert J.; Richter, Diethelm W.

    2010-01-01

    Rhythmic breathing movements originate from a dispersed neuronal network in the medulla and pons. Here, we demonstrate that rhythmic activity of this respiratory network is affected by the phosphorylation status of the inhibitory glycine receptor α3 subtype (GlyRα3), which controls glutamatergic and glycinergic neuronal discharges, subject to serotonergic modulation. Serotonin receptor type 1A–specific (5-HTR1A–specific) modulation directly induced dephosphorylation of GlyRα3 receptors, which augmented inhibitory glycine-activated chloride currents in HEK293 cells coexpressing 5-HTR1A and GlyRα3. The 5-HTR1A–GlyRα3 signaling pathway was distinct from opioid receptor signaling and efficiently counteracted opioid-induced depression of breathing and consequential apnea in mice. Paradoxically, this rescue of breathing originated from enhanced glycinergic synaptic inhibition of glutamatergic and glycinergic neurons and caused disinhibition of their target neurons. Together, these effects changed respiratory phase alternations and ensured rhythmic breathing in vivo. GlyRα3-deficient mice had an irregular respiratory rhythm under baseline conditions, and systemic 5-HTR1A activation failed to remedy opioid-induced respiratory depression in these mice. Delineation of this 5-HTR1A–GlyRα3 signaling pathway offers a mechanistic basis for pharmacological treatment of opioid-induced apnea and other breathing disturbances caused by disorders of inhibitory synaptic transmission, such as hyperekplexia, hypoxia/ischemia, and brainstem infarction. PMID:20978350

  12. Acutely increasing δGABA(A) receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus.

    PubMed

    Whissell, Paul D; Eng, Dave; Lecker, Irene; Martin, Loren J; Wang, Dian-Shi; Orser, Beverley A

    2013-01-01

    Extrasynaptic γ-aminobutyric acid type A (GABA(A)) receptors that contain the δ subunit (δGABA(A) receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABA(A) receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABA(A) receptor-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABA(A) receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p.) on memory performance in wild-type (WT) and δGABA(A) receptor null mutant (Gabrd(-/-)) mice. Additionally, the effects of THIP on long-term potentiation (LTP), a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd(-/-) mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd(-/-) mice, an effect that was blocked by GABA(A) receptor antagonist bicuculline. Thus, acutely increasing δGABA(A) receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABA(A) receptor activity.

  13. Stretch-activation of angiotensin II type 1a receptors contributes to the myogenic response of mouse mesenteric and renal arteries.

    PubMed

    Schleifenbaum, Johanna; Kassmann, Mario; Szijártó, István András; Hercule, Hantz C; Tano, Jean-Yves; Weinert, Stefanie; Heidenreich, Matthias; Pathan, Asif R; Anistan, Yoland-Marie; Alenina, Natalia; Rusch, Nancy J; Bader, Michael; Jentsch, Thomas J; Gollasch, Maik

    2014-07-07

    Vascular wall stretch is the major stimulus for the myogenic response of small arteries to pressure. The molecular mechanisms are elusive, but recent findings suggest that G protein-coupled receptors can elicit a stretch response. To determine whether angiotensin II type 1 receptors (AT1R) in vascular smooth muscle cells exert mechanosensitivity and identify the downstream ion channel mediators of myogenic vasoconstriction. We used mice deficient in AT1R signaling molecules and putative ion channel targets, namely AT1R, angiotensinogen, transient receptor potential channel 6 (TRPC6) channels, or several subtypes of the voltage-gated K+ (Kv7) gene family (KCNQ3, 4, or 5). We identified a mechanosensing mechanism in isolated mesenteric arteries and in the renal circulation that relies on coupling of the AT1R subtype a to a Gq/11 protein as a critical event to accomplish the myogenic response. Arterial mechanoactivation occurs after pharmacological block of AT1R and in the absence of angiotensinogen or TRPC6 channels. Activation of AT1R subtype a by osmotically induced membrane stretch suppresses an XE991-sensitive Kv channel current in patch-clamped vascular smooth muscle cells, and similar concentrations of XE991 enhance mesenteric and renal myogenic tone. Although XE991-sensitive KCNQ3, 4, and 5 channels are expressed in vascular smooth muscle cells, XE991-sensitive K+ current and myogenic contractions persist in arteries deficient in these channels. Our results provide definitive evidence that myogenic responses of mouse mesenteric and renal arteries rely on ligand-independent, mechanoactivation of AT1R subtype a. The AT1R subtype a signal relies on an ion channel distinct from TRPC6 or KCNQ3, 4, or 5 to enact vascular smooth muscle cell activation and elevated vascular resistance. © 2014 American Heart Association, Inc.

  14. β1-adrenergic receptor stimulation by agonist Compound 49b restores insulin receptor signal transduction in vivo

    PubMed Central

    Jiang, Youde; Zhang, Qiuhua; Ye, Eun-Ah

    2014-01-01

    Purpose Determine whether Compound 49b treatment ameliorates retinal changes due to the lack of β2-adrenergic receptor signaling. Methods Using retinas from 3-month-old β2-adrenergic receptor-deficient mice, we treated mice with our novel β1-/β2-adrenergic receptor agonist, Compound 49b, to assess the effects of adrenergic agonists acting only on β1-adrenergic receptors due to the absence of β2-adrenergic receptors. Western blotting or enzyme-linked immunosorbent assay (ELISA) analyses were performed for β1- and β2-adrenergic receptors, as well as key insulin resistance proteins, including TNF-α, SOCS3, IRS-1Ser307, and IRTyr960. Analyses were also performed on key anti- and proapoptotic proteins: Akt, Bcl-xL, Bax, and caspase 3. Electroretinogram analyses were conducted to assess functional changes, while histological assessment was conducted for changes in retinal thickness. Results A 2-month treatment of β2-adrenergic receptor-deficient mice with daily eye drops of 1 mM Compound 49b, a novel β1- and β2-adrenergic receptor agonist, reversed the changes in insulin resistance markers (TNF-α and SOCS3) observed in untreated β2-adrenergic receptor-deficient mice, and concomitantly increased morphological integrity (retinal thickness) and functional responses (electroretinogram amplitude). These results suggest that stimulating β1-adrenergic receptors on retinal endothelial cells or Müller cells can compensate for the loss of β2-adrenergic receptor signaling on Müller cells, restore insulin signal transduction, reduce retinal apoptosis, and enhance retinal function. Conclusions Since our previous studies with β1-adrenergic receptor knockout mice confirmed that the reverse also occurs (β2-adrenergic receptor stimulation can compensate for the loss of β1-adrenergic receptor activity), it appears that increased activity in either of these pathways alone is sufficient to block insulin resistance–based retinal cell apoptosis. PMID:24966659

  15. In brown adipocytes, adrenergically induced β{sub 1}-/β{sub 3}-(G{sub s})-, α{sub 2}-(G{sub i})- and α{sub 1}-(G{sub q})-signalling to Erk1/2 activation is not mediated via EGF receptor transactivation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Yanling; Fälting, Johanna M.; Mattsson, Charlotte L.

    2013-10-15

    Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α{sub 1}-adrenoceptor coupled via G{sub q}), clonidine (α{sub 2} via G{submore » i}) or CL316243 (β{sub 3} via G{sub s}) or via β{sub 1}-receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC{sub 50} 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades. - Highlights: • In brown adipocytes, norepinephrine regulates proliferation, apoptosis, differentiation. • EGF receptor transactivation is supposed to

  16. BmpR1A is a major type 1 BMP receptor for BMP-Smad signaling during skull development.

    PubMed

    Pan, Haichun; Zhang, Honghao; Abraham, Ponnu; Komatsu, Yoshihiro; Lyons, Karen; Kaartinen, Vesa; Mishina, Yuji

    2017-09-01

    Craniosynostosis is caused by premature fusion of one or more sutures in an infant skull, resulting in abnormal facial features. The molecular and cellular mechanisms by which genetic mutations cause craniosynostosis are incompletely characterized, and many of the causative genes for diverse types of syndromic craniosynostosis have not yet been identified. We previously demonstrated that augmentation of BMP signaling mediated by a constitutively active BMP type IA receptor (ca-BmpR1A) in neural crest cells (ca1A hereafter) causes craniosynostosis and superimposition of heterozygous null mutation of Bmpr1a rescues premature suture fusion (ca1A;1aH hereafter). In this study, we superimposed heterozygous null mutations of the other two BMP type I receptors, Bmpr1b and Acvr1 (ca1A;1bH and ca1A;AcH respectively hereafter) to further dissect involvement of BMP-Smad signaling. Unlike caA1;1aH, ca1A;1bH and ca1A;AcH did not restore the craniosynostosis phenotypes. In our in vivo study, Smad-dependent BMP signaling was decreased to normal levels in mut;1aH mice. However, BMP receptor-regulated Smads (R-Smads; pSmad1/5/9 hereafter) levels were comparable between ca1A, ca1A;1bH and ca1A;AcH mice, and elevated compared to control mice. Bmpr1a, Bmpr1b and Acvr1 null cells were used to examine potential mechanisms underlying the differences in ability of heterozygosity for Bmpr1a vs. Bmpr1b or Acvr1 to rescue the mut phenotype. pSmad1/5/9 level was undetectable in Bmpr1a homozygous null cells while pSmad1/5/9 levels did not decrease in Bmpr1b or Acvr1 homozygous null cells. Taken together, our study indicates that different levels of expression and subsequent activation of Smad signaling differentially contribute each BMP type I receptor to BMP-Smad signaling and craniofacial development. These results also suggest differential involvement of each type 1 receptor in pathogenesis of syndromic craniosynostoses. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Endothelin-1 (ET-1) induces resistance to bortezomib in human multiple myeloma cells via a pathway involving the ETB receptor and upregulation of proteasomal activity.

    PubMed

    Vaiou, Maria; Pangou, Evanthia; Liakos, Panagiotis; Sakellaridis, Nikos; Vassilopoulos, George; Dimas, Konstantinos; Papandreou, Christos

    2016-10-01

    Bortezomib (BTZ) is used for the treatment of multiple myeloma (MM). However, a significant proportion of patients may be refractory to the drug. This study aimed to investigate whether the endothelin (ET-1) axis may act as an escape mechanism to treatment with bortezomib in MM cells. NCI-H929 and RPMI-8226 (human MM cell lines) were cultured with or without ET-1, BTZ, and inhibitors of the endothelin receptors. ET-1 levels were determined by ELISA, while the protein levels of its receptors and of the PI3K and MAPK pathways' components by western blot. Effects of ET-1 on cell proliferation were studied by MTT and on the ubiquitin proteasome pathway by assessing the chymotryptic activity of the 20S proteasome in cell lysates. Endothelin receptors A and B (ETAR and ETBR, respectively) were found to be expressed in both cell lines, with the RPMI-8226 cells that are considered resistant to BTZ, expressing higher levels of ETBR and in addition secreting ET-1. Treatment of the NCI-H929 cells with ET-1 increased proliferation, while co-incubation of these cells with ET-1 and BTZ decreased BTZ efficacy with concomitant upregulation of 20S proteasomal activity. Si-RNA silencing or chemical blockade of ETBR abrogated the protective effects of ET-1. Finally, data suggest that the predominant signaling pathway involved in ET-1/ETBR-induced BTZ resistance in MM cells may be the MAPK pathway. Our data suggest a possible role of the ET-1/ETBR axis in regulating the sensitivity of MM cells to BTZ. Thus, combining bortezomib with strategies to target the ET-1 axis could prove to be a novel promising therapeutic approach in MM.

  18. Effects of OPC-14523, a combined sigma and 5-HT1a ligand, on pre- and post-synaptic 5-HT1a receptors.

    PubMed

    Bermack, Jordanna E; Debonnel, Guy

    2007-01-01

    OPC-14523 (OPC) is a novel compound with high affinity for sigma and 5-HT1A receptors that shows 'antidepressant-like' effects in animal models of depression. We have previously demonstrated that OPC produces an increase in 5-HT neurotransmission and a decreased response of 5-HT neurons to the acute administration of paroxetine in the DRN, an effect that appears to be mediated by OPC's 5-HT1A receptor affinity. The current study sets out to investigate more specifically the effects of OPC on 5-HT1A pre- and post-synaptic receptors, to assess whether it acts as an agonist or antagonist. Using an electrophysiological model of in vivo extracellular recordings in anaesthetized rats, the effects of OPC was assessed on pre-synaptic DRN 5-HT1A autoreceptors and post-synaptically on hippocampal 5-HT1A receptors of CA3 pyramidal neurons. OPC applied by microiontophoresis, produced a significant decrease in the firing activity of 5-HT neurons of the DRN and of quisqualate-activated CA3 pyramidal neurons of the dorsal hippocampus. The effects of OPC on 5-HT1A receptors were significantly reduced by the co-application of the 5-HT1A antagonist WAY-100635. In addition, the effects of OPC were not blocked by the injection of the sigma antagonists NE-100 or haloperidol. Therefore, OPC is acting as an agonist on both pre- and post-synaptic 5-HT1A receptors. The current findings combined with previous data on OPC suggest a pharmacological profile that warrants further investigation.

  19. HDL activation of endothelial sphingosine-1-phosphate receptor-1 (S1P1) promotes regeneration and suppresses fibrosis in the liver.

    PubMed

    Ding, Bi-Sen; Liu, Catherine H; Sun, Yue; Chen, Yutian; Swendeman, Steven L; Jung, Bongnam; Chavez, Deebly; Cao, Zhongwei; Christoffersen, Christina; Nielsen, Lars Bo; Schwab, Susan R; Rafii, Shahin; Hla, Timothy

    2016-12-22

    Regeneration of hepatic sinusoidal vasculature is essential for non-fibrotic liver regrowth and restoration of its metabolic capacity. However, little is known about how this specialized vascular niche is regenerated. Here we show that activation of endothelial sphingosine-1-phosphate receptor-1 (S1P 1 ) by its natural ligand bound to HDL (HDL-S1P) induces liver regeneration and curtails fibrosis. In mice lacking HDL-S1P, liver regeneration after partial hepatectomy was impeded and associated with aberrant vascular remodeling, thrombosis and peri-sinusoidal fibrosis. Notably, this "maladaptive repair" phenotype was recapitulated in mice that lack S1P 1 in the endothelium. Reciprocally, enhanced plasma levels of HDL-S1P or administration of SEW2871, a pharmacological agonist specific for S1P 1 enhanced regeneration of metabolically functional vasculature and alleviated fibrosis in mouse chronic injury and cholestasis models. This study shows that natural and pharmacological ligands modulate endothelial S1P 1 to stimulate liver regeneration and inhibit fibrosis, suggesting that activation of this pathway may be a novel therapeutic strategy for liver fibrosis.

  20. Genetic deletion of CB1 receptors improves non-associative learning.

    PubMed

    Degroot, Aldemar; Salhoff, Craig; Davis, Richard J; Nomikos, George G

    2005-07-01

    Habituation (a form of non-associative learning) was measured by assessing locomotion in novel activity monitors in CB1 receptor knockout mice and juxtaposed to habituation measured in muscarinic M2, M4, and double M2/M4 receptor knockout mice. M2 and M2/M4, but not M4, receptor knockout mice appeared to have an impaired ability to habituate, whereas CB1 receptor knockout mice showed enhanced habituation compared to wild-type animals. We conclude that CB1 receptor gene invalidation improves habituation tentatively through an increase in cholinergic neurotransmission.