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Sample records for receptor inhibits progastrin-dependent

  1. AMPA receptor inhibition by synaptically released zinc

    PubMed Central

    Kalappa, Bopanna I.; Anderson, Charles T.; Lippard, Stephen J.; Tzounopoulos, Thanos

    2015-01-01

    The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses. PMID:26647187

  2. Combined Angiotensin Receptor Antagonism and Neprilysin Inhibition

    PubMed Central

    Hubers, Scott A.; Brown, Nancy J.

    2016-01-01

    Heart failure affects approximately 5.7 million people in the United States alone. Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, beta-blockers, and aldosterone antagonists have improved mortality in patients with heart failure and reduced ejection fraction, but mortality remains high. In July 2015, the FDA approved the first of a new class of drugs for the treatment of heart failure; valsartan/sacubitril (formerly known as LCZ696 and currently marketed by Novartis as Entresto) combines the angiotensin receptor blocker valsartan and the neprilysin inhibitor prodrug sacubitril in a 1:1 ratio in a sodium supramolecular complex. Sacubitril is converted by esterases to LBQ657, which inhibits neprilysin, the enzyme responsible for the degradation of the natriuretic peptides and many other vasoactive peptides. Thus, this combined angiotensin receptor antagonist and neprilysin inhibitor addresses two of the pathophysiologic mechanisms of heart failure - activation of the renin-angiotensin-aldosterone system and decreased sensitivity to natriuretic peptides. In the Prospective comparison of ARNI with ACEI to Determine Impact on Global Mortality and morbidity in Heart Failure (PARADIGM-HF) trial, valsartan/sacubitril significantly reduced mortality and hospitalization for heart failure, as well as blood pressure, compared to enalapril in patients with heart failure, reduced ejection fraction, and an elevated circulating level of brain natriuretic peptide or N-terminal pro-brain natriuretic peptide. Ongoing clinical trials are evaluating the role of valsartan/sacubitril in the treatment of heart failure with preserved ejection fraction and hypertension. We review here the mechanisms of action of valsartan/sacubitril, the pharmacologic properties of the drug, and its efficacy and safety in the treatment of heart failure and hypertension. PMID:26976916

  3. Combined Angiotensin Receptor Antagonism and Neprilysin Inhibition.

    PubMed

    Hubers, Scott A; Brown, Nancy J

    2016-03-15

    Heart failure affects ≈5.7 million people in the United States alone. Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, β-blockers, and aldosterone antagonists have improved mortality in patients with heart failure and reduced ejection fraction, but mortality remains high. In July 2015, the US Food and Drug Administration approved the first of a new class of drugs for the treatment of heart failure: Valsartan/sacubitril (formerly known as LCZ696 and currently marketed by Novartis as Entresto) combines the angiotensin receptor blocker valsartan and the neprilysin inhibitor prodrug sacubitril in a 1:1 ratio in a sodium supramolecular complex. Sacubitril is converted by esterases to LBQ657, which inhibits neprilysin, the enzyme responsible for the degradation of the natriuretic peptides and many other vasoactive peptides. Thus, this combined angiotensin receptor antagonist and neprilysin inhibitor addresses 2 of the pathophysiological mechanisms of heart failure: activation of the renin-angiotensin-aldosterone system and decreased sensitivity to natriuretic peptides. In the Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure (PARADIGM-HF) trial, valsartan/sacubitril significantly reduced mortality and hospitalization for heart failure, as well as blood pressure, compared with enalapril in patients with heart failure, reduced ejection fraction, and an elevated circulating level of brain natriuretic peptide or N-terminal pro-brain natriuretic peptide. Ongoing clinical trials are evaluating the role of valsartan/sacubitril in the treatment of heart failure with preserved ejection fraction and hypertension. We review here the mechanisms of action of valsartan/sacubitril, the pharmacological properties of the drug, and its efficacy and safety in the treatment of heart failure and hypertension.

  4. pH-Dependent Inhibition of Kainate Receptors by Zinc

    PubMed Central

    Mott, David D.; Benveniste, Morris; Dingledine, Raymond J.

    2014-01-01

    Kainate receptors contribute to synaptic plasticity and rhythmic oscillatory firing of neurons in corticolimbic circuits including hippocampal area CA3. We use zinc chelators and mice deficient in zinc transporters to show that synaptically released zinc inhibits postsynaptic kainate receptors at mossy fiber synapses and limits frequency facilitation of kainate, but not AMPA EPSCs during thetapattern stimulation. Exogenous zinc also inhibits the facilitatory modulation of mossy fiber axon excitability by kainate but does not suppress the depressive effect of kainate on CA3 axons. Recombinant kainate receptors are inhibited in a subunit-dependent manner by physiologically relevant concentrations of zinc, with receptors containing the KA1 subunit being sensitive to submicromolar concentrations of zinc. Zinc inhibition does not alter receptor desensitization nor apparent agonist affinity and is only weakly voltage dependent, which points to an allosteric mechanism. Zinc inhibition is reduced at acidic pH. Thus, in the presence of zinc, a fall in pH potentiates kainate receptors by relieving zinc inhibition. Acidification of the extracellular space, as occurs during repetitive activity, may therefore serve to unmask kainate receptor neurotransmission. We conclude that zinc modulation of kainate receptors serves an important role in shaping kainate neurotransmission in the CA3 region. PMID:18272686

  5. Glycinergic inhibition in thalamus revealed by synaptic receptor blockade.

    PubMed

    Ghavanini, Ahmad A; Mathers, David A; Puil, Ernest

    2005-09-01

    Using juvenile rat brain slices, we examined the possibility that strychnine-sensitive receptors for glycine-like amino acids contributed to synaptic inhibition in ventrobasal thalamus, where gamma-aminobutyrate (GABA) is the prevalent inhibitory transmitter. Ventrobasal nuclei showed staining for antibodies against alpha1 and alpha2 subunits of the glycine receptor. Exogenously applied glycine, taurine and beta-alanine increased membrane conductance, effects antagonized by strychnine, indicative of functional glycine receptors. Using glutamate receptor antagonists, we isolated inhibitory postsynaptic potentials and currents (IPSPs and IPSCs) evoked by high-threshold stimulation of medial lemniscus. Like the responses to glycine agonists, these synaptic responses reversed near E(Cl). In comparative tests with GABA receptor antagonists, strychnine attenuated inhibition in a majority of neurons, but did not alter slow, GABA(B) inhibition. For complete blockade, the majority of fast IPSPs required co-application of strychnine with bicuculline or gabazine, GABA(A) receptor antagonists. Strychnine acting with an IC50 approximately = 33 nM, eliminated residual fast inhibition during selective GABA(A) receptor blockade with gabazine. The latency of onset for IPSPs was compatible with polysynaptic pathways or prolonged axonal propagation time. Strychnine lacked effects on monosynaptic, GABAergic IPSPs from zona incerta. The specific actions of strychnine implicated a glycine receptor contribution to fast inhibition in somatosensory thalamus.

  6. Menthol inhibits 5-HT3 receptor-mediated currents.

    PubMed

    Ashoor, Abrar; Nordman, Jacob C; Veltri, Daniel; Yang, Keun-Hang Susan; Shuba, Yaroslav; Al Kury, Lina; Sadek, Bassem; Howarth, Frank C; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

    2013-11-01

    The effects of alcohol monoterpene menthol, a major active ingredient of the peppermint plant, were tested on the function of human 5-hydroxytryptamine type 3 (5-HT3) receptors expressed in Xenopus laevis oocytes. 5-HT (1 μM)-evoked currents recorded by two-electrode voltage-clamp technique were reversibly inhibited by menthol in a concentration-dependent (IC50 = 163 μM) manner. The effects of menthol developed gradually, reaching a steady-state level within 10-15 minutes and did not involve G-proteins, since GTPγS activity remained unaltered and the effect of menthol was not sensitive to pertussis toxin pretreatment. The actions of menthol were not stereoselective as (-), (+), and racemic menthol inhibited 5-HT3 receptor-mediated currents to the same extent. Menthol inhibition was not altered by intracellular 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid injections and transmembrane potential changes. The maximum inhibition observed for menthol was not reversed by increasing concentrations of 5-HT. Furthermore, specific binding of the 5-HT3 antagonist [(3)H]GR65630 was not altered in the presence of menthol (up to 1 mM), indicating that menthol acts as a noncompetitive antagonist of the 5-HT3 receptor. Finally, 5-HT3 receptor-mediated currents in acutely dissociated nodose ganglion neurons were also inhibited by menthol (100 μM). These data demonstrate that menthol, at pharmacologically relevant concentrations, is an allosteric inhibitor of 5-HT3 receptors.

  7. Mechanism of TGFbeta receptor inhibition by FKBP12.

    PubMed Central

    Chen, Y G; Liu, F; Massague, J

    1997-01-01

    Transforming growth factor-beta (TGFbeta) signaling requires phosphorylation of the type I receptor TbetaR-I by TbetaR-II. Although TGFbeta promotes the association of TbetaR-I with TbetaR-II, these receptor components have affinity for each other which can lead to their ligand-independent activation. The immunophilin FKBP12 binds to TbetaR-I and inhibits its signaling function. We investigated the mechanism and functional significance of this effect. FKBP12 binding to TbetaR-I involves the rapamycin/Leu-Pro binding pocket of FKBP12 and a Leu-Pro sequence located next to the activating phosphorylation sites in TbetaR-I. Mutations in the binding sites of FKBP12 or TbetaR-I abolish the interaction between these proteins, leading to receptor activation in the absence of added ligand. FKBP12 does not inhibit TbetaR-I association with TbetaR-II, but inhibits TbetaR-I phosphorylation by TbetaR-II. Rapamycin, which blocks FKBP12 binding to TbetaR-I, reverses the inhibitory effect of FKBP12 on TbetaR-I phosphorylation. By impeding the activation of TGFbeta receptor complexes formed in the absence of ligand, FKBP12 may provide a safeguard against leaky signaling resulting from the innate tendency of TbetaR-I and TbetaR-II to interact with each other. PMID:9233797

  8. Gβ promotes pheromone receptor polarization and yeast chemotropism by inhibiting receptor phosphorylation.

    PubMed

    Ismael, Amber; Tian, Wei; Waszczak, Nicholas; Wang, Xin; Cao, Youfang; Suchkov, Dmitry; Bar, Eli; Metodiev, Metodi V; Liang, Jie; Arkowitz, Robert A; Stone, David E

    2016-04-12

    Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are essential to the development and life cycles of all species. Cells use surface receptors to sense the shallow chemical gradients that elicit chemotaxis and chemotropism. Slight asymmetries in receptor activation are amplified by downstream signaling systems, which ultimately induce dynamic reorganization of the cytoskeleton. During the mating response of budding yeast, a model chemotropic system, the pheromone receptors on the plasma membrane polarize to the side of the cell closest to the stimulus. Although receptor polarization occurs before and independently of actin cable-dependent delivery of vesicles to the plasma membrane (directed secretion), it requires receptor internalization. Phosphorylation of pheromone receptors by yeast casein kinase 1 or 2 (Yck1/2) stimulates their internalization. We showed that the pheromone-responsive Gβγ dimer promotes the polarization of the pheromone receptor by interacting with Yck1/2 and locally inhibiting receptor phosphorylation. We also found that receptor phosphorylation is essential for chemotropism, independently of its role in inducing receptor internalization. A mathematical model supports the idea that the interaction between Gβγ and Yck1/2 results in differential phosphorylation and internalization of the pheromone receptor and accounts for its polarization before the initiation of directed secretion.

  9. Gβ promotes pheromone receptor polarization and yeast chemotropism by inhibiting receptor phosphorylation

    PubMed Central

    Ismael, Amber; Tian, Wei; Waszczak, Nicholas; Wang, Xin; Cao, Youfang; Suchkov, Dmitry; Bar, Eli; Metodiev, Metodi V.; Liang, Jie; Arkowitz, Robert; Stone, David E.

    2016-01-01

    Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are universal processes, which are essential to the development and life cycles of all species. Cells use surface receptors to sense the shallow chemical gradients that elicit chemotaxis and chemotropism. Slight asymmetries in receptor activation are amplified by downstream signaling systems, which ultimately induce dynamic reorganization of the cytoskeleton. During the mating response of budding yeast, a model chemotropic system, the pheromone receptor on the plasma membrane polarizes to the side of the cell closest to the stimulus. Although receptor polarization occurs before and independently of actin-cable dependent vesicle delivery (directed secretion), it requires receptor internalization. Phosphorylation of pheromone receptors by yeast casein kinase 1 or 2 (Yck1/2) stimulates their internalization. We showed that the pheromone-responsive Gβγ dimer promotes the polarization of the pheromone receptor by interacting with Yck1/2 and locally inhibiting receptor phosphorylation. We also found that receptor phosphorylation is essential for chemotropism, independent of its role in inducing receptor internalization. A mathematical model supports the idea that the interaction between Gβγ and Yck1/2 results in differential phosphorylation and internalization of the pheromone receptor and accounts for its polarization before the initiation of directed secretion. PMID:27072657

  10. Ethanol inhibits neuritogenesis induced by astrocyte muscarinic receptors.

    PubMed

    Guizzetti, Marina; Moore, Nadia H; Giordano, Gennaro; VanDeMark, Kathryn L; Costa, Lucio G

    2010-09-01

    In utero alcohol exposure can lead to fetal alcohol spectrum disorders, characterized by cognitive and behavioral deficits. In vivo and in vitro studies have shown that ethanol alters neuronal development. We have recently shown that stimulation of M(3) muscarinic receptors in astrocytes increases the synthesis and release of fibronectin, laminin, and plasminogen activator inhibitor-1, causing neurite outgrowth in hippocampal neurons. As M(3) muscarinic receptor signaling in astroglial cells is strongly inhibited by ethanol, we hypothesized that ethanol may also inhibit neuritogenesis in hippocampal neurons induced by carbachol-stimulated astrocytes. In the present study, we report that the effect of carbachol-stimulated astrocytes on hippocampal neuron neurite outgrowth was inhibited in a concentration-dependent manner (25-100 mM) by ethanol. This effect was because of the inhibition of the release of fibronectin, laminin, and plasminogen activator inhibitor-1. Similar effects on neuritogenesis and on the release of astrocyte extracellular proteins were observed after the incubation of astrocytes with carbachol in the presence of 1-butanol, another short-chain alcohol, which like ethanol is a competitive substrate for phospholipase D, but not by tert-butanol, its analog that is not a substrate for this enzyme. This study identifies a potential novel mechanism involved in the developmental effects of ethanol mediated by the interaction of ethanol with cell signaling in astrocytes, leading to an impairment in neuron-astrocyte communication.

  11. Lead inhibition of NMDA channels in native and recombinant receptors.

    PubMed

    Gavazzo, P; Gazzoli, A; Mazzolini, M; Marchetti, C

    2001-10-08

    NMDA channels are key targets for lead (Pb2+) neurotoxicity and Pb2+-induced inhibition of NMDA current is age- and subunit-dependent. In rat cerebellar granule cells maintained in high KCl, glycine affinity as well as sensitivity to ifenprodil change significantly with the days in vitro, indicating a reduction of NR2B subunit expression. Pb2+ blocked NMDA current with IC50 approximately 4 microM and this effect decreased significantly during the second week in vitro. In Xenopus laevis oocytes expressing recombinant NR1-NR2A, NR1-NR2B or NR1-NR2C receptors, Pb2+ inhibited glutamate-activated currents with IC50 of 3.3, 2.5 and 4.7 microM respectively. These data indicate that Pb2+ action is dependent on subunit composition and suggest that down-regulation of the NR2B subunit is correlated to a diminished sensitivity to Pb2+ inhibition.

  12. Inhibition of oxytocin receptor function by direct binding of progesterone.

    PubMed

    Grazzini, E; Guillon, G; Mouillac, B; Zingg, H H

    1998-04-02

    The steroid hormone progesterone (P4) is essential for establishing and maintaining pregnancy in mammals. One of its functions includes maintenance of uterine quiescence by decreasing uterine sensitivity to the uterotonic peptide hormone oxytocin. Although it is generally held that steroid hormones such as P4 act at a genomic level by binding to nuclear receptors and modulating the expression of specific target genes, we show here that the effect of P4 on uterine sensitivity to oxytocin involves direct, non-genomic action of P4 on the uterine oxytocin receptor (OTR), a member of the G-protein-coupled receptor family. P4 inhibits oxytocin binding to OTR-containing membranes in vitro, binds with high affinity to recombinant rat OTR expressed in CHO cells, and suppresses oxytocin-induced inositol phosphate production and calcium mobilization. These effects are highly steroid- and receptor-specific, because binding and signalling functions of the closely related human OTR are not affected by P4 itself but by the P4 metabolite 5beta-dihydroprogesterone. Our findings provide the first evidence for a direct interaction between a steroid hormone and a G-protein-coupled receptor and define a new level of crosstalk between the peptide- and steroid-hormone signalling pathways.

  13. [MET receptor inhibition: Hope against resistance to targeted therapies?

    PubMed

    Hochart, Audrey; Leblond, Pierre; Le Bourhis, Xuefen; Meignan, Samuel; Tulasne, David

    2017-02-01

    Overcoming the drug resistance remains a crucial issue in cancer treatment. For refractory patients, the use of MET receptor tyrosine kinase inhibitors seems to be hopeful. Indeed, important mechanisms underlying drug resistance argue for association of MET inhibitors with targeted therapies, both on first-line to prevent a primary resistance and on the second line to overcoming acquired resistance. Indeed, met gene amplification is the second most common alteration involved in acquired resistance to anti-epidermal growth factor receptor (EGFR) therapies in non-small cells lung cancer (NSCLC). Hypoxia, for its part, can activate MET transcription and amplifies HGF signaling resulting in MET activation, which could be involved in vascular endothelial growth factor (VEGF) inhibitors escape. In HER2 positive breast cancers, MET amplification may also induce tumor cells a hatch escape, resulting in secondary resistance. Finally, some patients with BRAF mutated melanoma exhibit primary resistance to BRAF inhibition by stromal HGF (ligand of MET) secretion resulting in MET receptor activation. Experimental data highlight the role of MET in primary and secondary resistance and encourage combined treatments including MET inhibitors. In this context, several promising clinical trials are in progress in numerous cancers (NSCLC, melanoma, breast cancer, glioblastoma…) using combination of anti-MET and other specific therapies targeting EGFR, BRAF, VEGF or HER2. This review summarizes the potential benefits that MET inhibition should provide to patients with cancer refractory to targeted therapies.

  14. GABAA receptor inhibition triggers a nicotinic neuroprotective mechanism

    PubMed Central

    Ferchmin, P. A; Pérez, Dinely; Alvarez, William Castro; Penzo, Mario A.; Maldonado, Héctor M.; Eterovic, Vesna A.

    2014-01-01

    Nicotinic acetylcholine receptor (nAChR)-mediated neuroprotection has been implicated in the treatment of neurodegenerative disorders such as Alzheimer’s, Parkinson’s and hypoxic ischemic events, as well as other diseases hallmarked by excitotoxic and apoptotic neuronal death. Several modalities of nicotinic neuroprotection have been reported. However, although this process generally involves α4β2 and α7 subtypes, the underlying mechanisms are largely unknown. Interestingly, both activation and inhibition of α7 nAChRs have been reported to be neuroprotective. We have shown that inhibition of α7 nAChRs protects the function of acute hippocampal slices against excitotoxicity in a α4β2-dependent manner. Neuroprotection was assessed as the prevention of the NMDA-dependent loss of the area of population spikes (PSs) in the CA1 area of acute hippocampal slices. Our results support a model in which α7 AChRs control the release of GABA. Blocking either α7 or GABAA receptors reduces the inhibitory tone on cholinergic terminals, thereby promoting α4β2 activation, which in turn mediates neuroprotection. These results shed light on how α7 nAChR inhibition can be neuroprotective through a mechanism mediated by activation of α4β2 nAChRs. PMID:23280428

  15. Dopamine D1 receptor involvement in latent inhibition and overshadowing.

    PubMed

    Nelson, Andrew J D; Thur, Karen E; Cassaday, Helen J

    2012-11-01

    Latent inhibition (LI) manifests as poorer conditioning to a stimulus that has previously been experienced without consequence. There is good evidence of dopaminergic modulation of LI, as the effect is reliably disrupted by the indirect dopamine (DA) agonist amphetamine. The disruptive effects of amphetamine on LI are reversed by both typical and atypical antipsychotics, which on their own are able to facilitate LI. However, the contribution of different DA receptors to these effects is poorly understood. Amphetamine effects on another stimulus selection procedure, overshadowing, have been suggested to be D1-mediated. Thus, in the current experiments, we systematically investigated the role of D1 receptors in LI. First, we tested the ability of the full D1 agonist SKF 81297 to abolish LI and compared the effects of this drug on LI and overshadowing. Subsequently, we examined whether the D1 antagonist SCH 23390 can lead to the emergence of LI under conditions that do not produce the effect in normal animals (weak pre-exposure). Finally, we tested the ability of SCH 23390 to block amphetamine-induced disruption of LI. We found little evidence that direct stimulation of D1 receptors abolishes LI (although there was some attenuation of LI at 0.4 mg/kg SKF 81297). Similarly, SCH 23390 failed to enhance LI. However, SCH 23390 did block amphetamine-induced disruption of LI. These data indicate that, while LI may be unaffected by selective manipulation of activity at D1 receptors, the effects of amphetamine on LI are to some extent dependent on actions at D1 receptors.

  16. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    SciTech Connect

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  17. Seizure control by decanoic acid through direct AMPA receptor inhibition

    PubMed Central

    Chang, Pishan; Augustin, Katrin; Boddum, Kim; Williams, Sophie; Sun, Min; Terschak, John A.; Hardege, Jörg D.; Chen, Philip E.

    2016-01-01

    See Rogawski (doi:10.1093/awv369) for a scientific commentary on this article.  The medium chain triglyceride ketogenic diet is an established treatment for drug-resistant epilepsy that increases plasma levels of decanoic acid and ketones. Recently, decanoic acid has been shown to provide seizure control in vivo, yet its mechanism of action remains unclear. Here we show that decanoic acid, but not the ketones β-hydroxybutryate or acetone, shows antiseizure activity in two acute ex vivo rat hippocampal slice models of epileptiform activity. To search for a mechanism of decanoic acid, we show it has a strong inhibitory effect on excitatory, but not inhibitory, neurotransmission in hippocampal slices. Using heterologous expression of excitatory ionotropic glutamate receptor AMPA subunits in Xenopus oocytes, we show that this effect is through direct AMPA receptor inhibition, a target shared by a recently introduced epilepsy treatment perampanel. Decanoic acid acts as a non-competitive antagonist at therapeutically relevant concentrations, in a voltage- and subunit-dependent manner, and this is sufficient to explain its antiseizure effects. This inhibitory effect is likely to be caused by binding to sites on the M3 helix of the AMPA-GluA2 transmembrane domain; independent from the binding site of perampanel. Together our results indicate that the direct inhibition of excitatory neurotransmission by decanoic acid in the brain contributes to the anti-convulsant effect of the medium chain triglyceride ketogenic diet. PMID:26608744

  18. Discoidin domain receptor 2 inhibits fibrillogenesis of collagen type 1.

    PubMed

    Mihai, Cosmin; Iscru, Daniel F; Druhan, Lawrence J; Elton, Terry S; Agarwal, Gunjan

    2006-09-01

    Discoidin domain receptors (DDR1 and DDR2) are widely expressed cell-surface receptors, which bind to and are activated by collagens, including collagen type 1. Activation of DDRs and the resulting downstream signaling is known to regulate the extracellular matrix. However, little is known about how DDRs interact with collagen and its direct impact on collagen regulation. Here, we have established that by binding to collagen, the extracellular domain (ECD) of DDR2 inhibits collagen fibrillogenesis and alters the morphology of collagen type 1 fibers. Our in vitro assays utilized DDR2-Fc fusion proteins, which contain only the ECD of DDR2. Using surface plasmon resonance, we confirmed that further oligomerization of DDR2-Fc (by means of anti-Fc antibody) greatly enhances its binding to immobilized collagen type 1. Collagen turbidity measurements and biochemical assays indicated that DDR2 delays the formation of collagen fibrils. Atomic force microscopy of soluble collagen revealed that a predominately monomeric state of collagen was present with DDR2, while control solutions had an abundance of polymeric collagen. Transmission electron microscopy of collagen fibers, showed that the native periodic banded structure of collagen fibers was weakened and nearly absent in the presence of DDR2. Further, using a cell-based assay we demonstrate that overexpression of full length DDR2 inhibits fibrillogenesis of collagen type 1. Our results demonstrate a novel and important functional role of the DDR2 ECD that may contribute to collagen regulation via modulation of fibrillogenesis.

  19. Argos inhibits epidermal growth factor receptor signalling by ligand sequestration.

    PubMed

    Klein, Daryl E; Nappi, Valerie M; Reeves, Gregory T; Shvartsman, Stanislav Y; Lemmon, Mark A

    2004-08-26

    The epidermal growth factor receptor (EGFR) has critical functions in development and in many human cancers. During development, the spatial extent of EGFR signalling is regulated by feedback loops comprising both well-understood activators and less well-characterized inhibitors. In Drosophila melanogaster the secreted protein Argos functions as the only known extracellular inhibitor of EGFR, with clearly identified roles in multiple stages of development. Argos is only expressed when the Drosophila EGFR (DER) is activated at high levels, and downregulates further DER signalling. Although there is ample genetic evidence that Argos inhibits DER activation, the biochemical mechanism has not been established. Here we show that Argos inhibits DER signalling without interacting directly with the receptor, but instead by sequestering the DER-activating ligand Spitz. Argos binds tightly to the EGF motif of Spitz and forms a 1:1 (Spitz:Argos) complex that does not bind DER in vitro or at the cell surface. Our results provide an insight into the mechanism of Argos function, and suggest new strategies for EGFR inhibitor design.

  20. Multiple Modes of Ryanodine Receptor 2 Inhibition by Flecainide

    PubMed Central

    Mehra, D.; Imtiaz, M. S.; van Helden, D. F.; Knollmann, B. C.

    2014-01-01

    Catecholaminergic polymorphic ventricular tachycardia (CPVT) causes sudden cardiac death due to mutations in cardiac ryanodine receptors (RyR2), calsequestrin, or calmodulin. Flecainide, a class I antiarrhythmic drug, inhibits Na+ and RyR2 channels and prevents CPVT. The purpose of this study is to identify inhibitory mechanisms of flecainide on RyR2. RyR2 were isolated from sheep heart, incorporated into lipid bilayers, and investigated by single-channel recording under various activating conditions, including the presence of cytoplasmic ATP (2 mM) and a range of cytoplasmic [Ca2+], [Mg2+], pH, and [caffeine]. Flecainide applied to either the cytoplasmic or luminal sides of the membrane inhibited RyR2 by two distinct modes: 1) a fast block consisting of brief substate and closed events with a mean duration of ∼1 ms, and 2) a slow block consisting of closed events with a mean duration of ∼1 second. Both inhibition modes were alleviated by increasing cytoplasmic pH from 7.4 to 9.5 but were unaffected by luminal pH. The slow block was potentiated in RyR2 channels that had relatively low open probability, whereas the fast block was unaffected by RyR2 activation. These results show that these two modes are independent mechanisms for RyR2 inhibition, both having a cytoplasmic site of action. The slow mode is a closed-channel block, whereas the fast mode blocks RyR2 in the open state. At diastolic cytoplasmic [Ca2+] (100 nM), flecainide possesses an additional inhibitory mechanism that reduces RyR2 burst duration. Hence, multiple modes of action underlie RyR2 inhibition by flecainide. PMID:25274603

  1. Histamine H3 receptor-mediated inhibition of noradrenaline release in the human brain.

    PubMed

    Schlicker, E; Werthwein, S; Zentner, J

    1999-01-01

    Stimulation-evoked 3H-noradrenaline release in human cerebrocortical slices was inhibited by histamine (in a manner sensitive to clobenpropit) and by imetit, suggesting H3 receptor-mediated inhibition of noradrenaline release in human brain.

  2. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    SciTech Connect

    Wang, Feng; Yang, Yong

    2014-10-03

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers.

  3. Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

    PubMed

    Chaudhary, Anu; Leite, Mara; Kulasekara, Bridget R; Altura, Melissa A; Ogahara, Cassandra; Weiss, Eli; Fu, Wenqing; Blanc, Marie-Pierre; O'Keeffe, Michael; Terhorst, Cox; Akey, Joshua M; Miller, Samuel I

    2016-07-25

    Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes.

  4. Solubilized placental membrane protein inhibits insulin receptor tyrosine kinase activity

    SciTech Connect

    Strout, H.V. Jr.; Slater, E.E.

    1987-05-01

    Regulation of insulin receptor (IR) tyrosine kinase (TK) activity may be important in modulating insulin action. Utilizing an assay which measures IR phosphorylation of angiotensin II (AII), the authors investigated whether fractions of TX-100 solubilized human placental membranes inhibited IR dependent AII phosphorylation. Autophosphorylated IR was incubated with membrane fractions before the addition of AII, and kinase inhibition measured by the loss of TSP incorporated in AII. An inhibitory activity was detected which was dose, time, and temperature dependent. The inhibitor was purified 200-fold by sequential chromatography on wheat germ agglutinin, DEAE, and hydroxyapatite. This inhibitory activity was found to correlate with an 80 KD protein which was electroeluted from preparative slab gels and rabbit antiserum raised. Incubation of membrane fractions with antiserum before the IRTK assay immunoprecipitated the inhibitor. Protein immunoblots of crude or purified fractions revealed only the 80 KD protein. Since IR autophosphorylation is crucial to IRTK activity, the authors investigated the state of IR autophosphorylation after treatment with inhibitor; no change was detected by phosphoamino acid analysis.

  5. Trace amines inhibit insect odorant receptor function through antagonism of the co-receptor subunit

    PubMed Central

    Chen, Sisi; Luetje, Charles W.

    2014-01-01

    Many insect behaviors are driven by olfaction, making insect olfactory receptors (ORs) appealing targets for insect control.  Insect ORs are odorant-gated ion channels, with each receptor thought to be composed of a representative from a large, variable family of odorant binding subunits and a highly conserved co-receptor subunit (Orco), assembled in an unknown stoichiometry.  Synthetic Orco directed agonists and antagonists have recently been identified.  Several Orco antagonists have been shown to act via an allosteric mechanism to inhibit OR activation by odorants.  The high degree of conservation of Orco across insect species results in Orco antagonists having broad activity at ORs from a variety of insect species and suggests that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to insects or may be a target of exogenous compounds, such as those produced by plants.  To test this idea, we screened a series of biogenic and trace amines, identifying several as Orco antagonists.  Of particular interest were tryptamine, a plant-produced amine, and tyramine, an amine endogenous to the insect nervous system.  Tryptamine was found to be a potent antagonist of Orco, able to block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants.  Tyramine had effects similar to those of tryptamine, but was less potent.  Importantly, both tryptamine and tyramine displayed broad activity, inhibiting odorant activation of ORs of species from three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae).  Our results suggest that endogenous and exogenous natural compounds serve as Orco ligands modulating insect olfaction and that Orco can be an important target for the development of novel insect repellants. PMID:25075297

  6. A1 adenosine receptors inhibit chloride transport in the shark rectal gland. Dissociation of inhibition and cyclic AMP.

    PubMed Central

    Kelley, G G; Poeschla, E M; Barron, H V; Forrest, J N

    1990-01-01

    In the in vitro perfused rectal gland of the dogfish shark (Squalus acanthias), the adenosine analogue 2-chloroadenosine (2Clado) completely and reversibly inhibited forskolin-stimulated chloride secretion with an IC50 of 5 nM. Other A1 receptor agonists including cyclohexyladenosine (CHA), N-ethylcarboxamideadenosine (NECA) and R-phenylisopropyl-adenosine (R-PIA) also completely inhibited forskolin stimulated chloride secretion. The "S" stereoisomer of PIA (S-PIA) was a less potent inhibitor of forskolin stimulated chloride secretion, consistent with the affinity profile of PIA stereoisomers for an A1 receptor. The adenosine receptor antagonists 8-phenyltheophylline and 8-cyclopentyltheophylline completely blocked the effect of 2Clado to inhibit forskolin-stimulated chloride secretion. When chloride secretion and tissue cyclic (c)AMP content were determined simultaneously in perfused glands, 2Clado completely inhibited secretion but only inhibited forskolin stimulated cAMP accumulation by 34-40%, indicating that the mechanism of inhibition of secretion by 2Clado is at least partially cAMP independent. Consistent with these results, A1 receptor agonists only modestly inhibited (9-15%) forskolin stimulated adenylate cyclase activity and 2Clado markedly inhibited chloride secretion stimulated by a permeant cAMP analogue, 8-chlorophenylthio cAMP (8CPT cAMP). These findings provide the first evidence for a high affinity A1 adenosine receptor that inhibits hormone stimulated ion transport in a model epithelia. A major portion of this inhibition occurs by a mechanism that is independent of the cAMP messenger system. PMID:1970583

  7. Lysophosphatidic Acid (LPA) Receptor 5 Inhibits B Cell Antigen Receptor Signaling and Antibody Response1

    PubMed Central

    Shotts, Kristin; Donovan, Erin E.; Strauch, Pamela; Pujanauski, Lindsey M.; Victorino, Francisco; Al-Shami, Amin; Fujiwara, Yuko; Tigyi, Gabor; Oravecz, Tamas; Pelanda, Roberta; Torres, Raul M.

    2014-01-01

    Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically and whose levels are elevated in certain pathological settings such as cancer and infections. Here, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13 – Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-trisphosphate receptor activity. We further show that LPA5 also limits antigen-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced antibody responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity. PMID:24890721

  8. Cinnamaldehyde suppresses toll-like receptor 4 activation mediated through the inhibition of receptor oligomerization.

    PubMed

    Youn, Hyung S; Lee, Jun K; Choi, Yong J; Saitoh, Shin I; Miyake, Kensuke; Hwang, Daniel H; Lee, Joo Y

    2008-01-15

    Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens or non-microbial endogenous molecules. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NFkappaB and IRF3 and the expression of inflammatory mediators. Deregulation of TLR activation is known to be closely linked to the increased risk of many chronic diseases. Cinnamaldehyde (3-phenyl-2-propenal) has been reported to inhibit NFkappaB activation induced by pro-inflammatory stimuli and to exert anti-inflammatory and anti-bacterial effects. However, the underlying mechanism has not been clearly identified. Our results showed that cinnamaldehyde suppressed the activation of NFkappaB and IRF3 induced by LPS, a TLR4 agonist, leading to the decreased expression of target genes such as COX-2 and IFNbeta in macrophages (RAW264.7). Cinnamaldehyde did not inhibit the activation of NFkappaB or IRF3 induced by MyD88-dependent (MyD88, IKKbeta) or TRIF-dependent (TRIF, TBK1) downstream signaling components. However, oligomerization of TLR4 induced by LPS was suppressed by cinnamaldehyde resulting in the downregulation of NFkappaB activation. Further, cinnamaldehyde inhibited ligand-independent NFkappaB activation induced by constitutively active TLR4 or wild-type TLR4. Our results demonstrated that the molecular target of cinnamaldehyde in TLR4 signaling is oligomerization process of receptor, but not downstream signaling molecules suggesting a novel mechanism for anti-inflammatory activity of cinnamaldehyde.

  9. Tumor Necrosis Factor Inhibits Glucocorticoid Receptor Function in Mice

    PubMed Central

    Van Bogaert, Tom; Vandevyver, Sofie; Dejager, Lien; Van Hauwermeiren, Filip; Pinheiro, Iris; Petta, Ioanna; Engblom, David; Kleyman, Anna; Schütz, Günther; Tuckermann, Jan; Libert, Claude

    2011-01-01

    As glucocorticoid resistance (GCR) and the concomitant burden pose a worldwide problem, there is an urgent need for a more effective glucocorticoid therapy, for which insights into the molecular mechanisms of GCR are essential. In this study, we addressed the hypothesis that TNFα, a strong pro-inflammatory mediator in numerous inflammatory diseases, compromises the protective function of the glucocorticoid receptor (GR) against TNFα-induced lethal inflammation. Indeed, protection of mice by dexamethasone against TNFα lethality was completely abolished when it was administered after TNFα stimulation, indicating compromised GR function upon TNFα challenge. TNFα-induced GCR was further demonstrated by impaired GR-dependent gene expression in the liver. Furthermore, TNFα down-regulates the levels of both GR mRNA and protein. However, this down-regulation seems to occur independently of GC production, as TNFα also resulted in down-regulation of GR levels in adrenalectomized mice. These findings suggest that the decreased amount of GR determines the GR response and outcome of TNFα-induced shock, as supported by our studies with GR heterozygous mice. We propose that by inducing GCR, TNFα inhibits a major brake on inflammation and thereby amplifies the pro-inflammatory response. Our findings might prove helpful in understanding GCR in inflammatory diseases in which TNFα is intimately involved. PMID:21646349

  10. Science Signaling Podcast for 20 December 2016: Trans-inhibition by Fc receptors.

    PubMed

    Daëron, Marc; VanHook, Annalisa M

    2016-12-20

    This Podcast features an interview with Marc Daëron, author of a Research Article that appears in the 20 December 2016 issue of Science Signaling, about a mechanism by which an Fc receptor can inhibit signaling by other receptors without aggregating with those other receptors. Engagement of Fc receptors on basophils and mast cells can either activate these cells, which promotes autoimmune and allergic inflammation, or prevent these cells from being activated. Whether these cells are activated depends upon which Fc receptors are present in clusters, because some Fc receptors can inhibit signaling by other Fc receptors that are present in the same signalosome, a phenomenon known as cis-inhibition. Malbec et al. identified a mechanism whereby inhibitory Fc receptors limit signaling by activating Fc receptors without being present in the same signalosome. This mechanism of trans-inhibition also allowed inhibitory Fc receptors to limit signaling by growth factor receptors in mast cells and oncogene-induced proliferation in mastocytoma cells.Listen to Podcast.

  11. Minimal RNA aptamer sequences that can inhibit or alleviate noncompetitive inhibition of the muscle-type nicotinic acetylcholine receptor.

    PubMed

    Sivaprakasam, Kannan; Pagán, Oné R; Hess, George P

    2010-02-01

    Combinatorially synthesized nucleotide polymers have been used during the last decade to find ligands that bind to specific sites on biological molecules, including membrane-bound proteins such as the nicotinic acetylcholine receptors (nAChRs). The neurotransmitter receptors belong to a group of four structurally related proteins that regulate signal transmission between ~10(11) neurons of the mammalian nervous system. The nAChRs are inhibited by compounds such as the anticonvulsant MK-801 [(+)-dizocilpine] and abused drugs such as cocaine. Based on predictions arising from the mechanism of receptor inhibition by MK-801 and cocaine, we developed two classes of RNA aptamers: class I members, which inhibit the nAChR, and class II members, which alleviate inhibition of the receptor by MK-801 and cocaine. The systematic evolution of ligands by the exponential enrichment (SELEX) method was used to obtain these compounds. Here, we report that we have truncated RNA aptamers in each class to determine the minimal nucleic acid sequence that retains the characteristic function for which the aptamer was originally selected. We demonstrate that a truncated class I aptamer containing a sequence of seven nucleotides inhibits the nAChR and that a truncated class II aptamer containing a sequence of only four nucleotides can alleviate MK-801 inhibition.

  12. Influence of cadmium on isolated peritoneal macrophage populations: cadmium inhibits Fc receptor internalization

    SciTech Connect

    Cook, G.B.

    1985-01-01

    In vitro experiments were performed to examine the effect of cadmium on adherent phagocytic cell populations. The authors were able to demonstrate, in vitro, a phagocytic defect that was originally observed in an in vivo system. Using in vitro methodologies, cadmium was found to inhibit opsonin-dependent but not opsonin-independent phagocytosis in two different populations of macrophages. The receptors through which the opsonized /sup 51/Cr-ElgG were internalized were characterized as Fc receptors. They were able to demonstrate that cadmium could reversibly inhibit internalization of Fc receptors. This mechanism, rather than an alteration of the receptors' binding capabilities, was responsible for the observed inhibition of Fc mediated (opsonin-dependent) phagocytosis in both populations of macrophages tested. The defect was not specific for cadmium per se. Zinc treatment caused a similar inhibition of Fc receptor mediated phagocytosis.

  13. Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism.

    PubMed

    Shim, Joong Sup; Li, Ruo-Jing; Lv, Junfang; Head, Sarah A; Yang, Eun Ju; Liu, Jun O

    2015-06-28

    Selective estrogen receptor modulators (SERM) including tamoxifen are known to inhibit angiogenesis. However, the underlying mechanism, which is independent of their action on the estrogen receptor (ER), has remained largely unknown. In the present study, we found that tamoxifen and other SERM inhibited cholesterol trafficking in endothelial cells, causing a hyper-accumulation of cholesterol in late endosomes/lysosomes. Inhibition of cholesterol trafficking by tamoxifen was accompanied by abnormal subcellular distribution of vascular endothelial growth factor receptor-2 (VEGFR2) and inhibition of the terminal glycosylation of the receptor. Tamoxifen also caused perinuclear positioning of lysosomes, which in turn trapped the mammalian target of rapamycin (mTOR) in the perinuclear region of endothelial cells. Abnormal distribution of VEGFR2 and mTOR and inhibition of VEGFR2 and mTOR activities by tamoxifen were significantly reversed by addition of cholesterol-cyclodextrin complex to the culture media of endothelial cells. Moreover, high concentrations of tamoxifen inhibited endothelial and breast cancer cell proliferation in a cholesterol-dependent, but ER-independent, manner. Together, these results unraveled a previously unrecognized mechanism of angiogenesis inhibition by tamoxifen and other SERM, implicating cholesterol trafficking as an attractive therapeutic target for cancer treatment.

  14. Effect of dopamine and serotonin receptor antagonists on fencamfamine-induced abolition of latent inhibition.

    PubMed

    de Aguiar, Cilene Rejane Ramos Alves; de Aguiar, Marlison José Lima; DeLucia, Roberto; Silva, Maria Teresa Araujo

    2013-01-05

    The purpose of this investigation was to verify the role of dopamine and serotonin receptors in the effect of fencamfamine (FCF) on latent inhibition. FCF is a psychomotor stimulant with an indirect dopaminergic action. Latent inhibition is a model of attention. Latent inhibition is blocked by dopaminergic agents and facilitated by dopamine receptor agonists. FCF has been shown to abolish latent inhibition. The serotonergic system may also participate in the neurochemical mediation of latent inhibition. The selective dopamine D(1) receptor antagonist SCH 23390 (7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol), D(2) receptor antagonists pimozide (PIM) and methoclopramide (METH), and serotonin 5-HT(2A/C) receptor antagonist ritanserin (RIT) were used in the present study. Latent inhibition was evaluated using a conditioned emotional response procedure. Male Wistar rats that were water-restricted were subjected to a three-phase procedure: preexposure to a tone, tone-shock conditioning, and a test of the effect of the tone on licking frequency. All of the drugs were administered before the preexposure and conditioning phases. The results showed that FCF abolished latent inhibition, and this effect was clearly antagonized by PIM and METH and moderately attenuated by SCH 23390. At the doses used in the present study, RIT pretreatment did not affect latent inhibition and did not eliminate the effect of FCF, suggesting that the FCF-induced abolition of latent inhibition is not mediated by serotonin 5-HT(2A/C) receptors. These results suggest that the effect of FCF on latent inhibition is predominantly related to dopamine D(2) receptors and that dopamine D(2) receptors participate in attention processes.

  15. Metabotropic glutamate receptors differentially regulate GABAergic inhibition in thalamus.

    PubMed

    Govindaiah, G; Cox, Charles L

    2006-12-27

    Thalamic interneurons and thalamic reticular nucleus (TRN) neurons provide inhibitory innervation of thalamocortical cells that significantly influence thalamic gating. The local interneurons in the dorsal lateral geniculate nucleus (dLGN) give rise to two distinct synaptic outputs: classical axonal and dendrodendritic. Activation of metabotropic glutamate receptors (mGluRs) by agonists or optic tract stimulation increases the output of these presynaptic dendrites leading to increased inhibition of thalamocortical neurons. The present study was aimed to evaluate the actions of specific mGluRs on inhibitory GABA-mediated signaling. We found that the group I mGluR (mGluR(1,5)) agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) or optic tract stimulation produced a robust increase in spontaneous IPSCs (sIPSCs) in thalamocortical neurons that was attenuated by the selective mGluR(5) antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP). In contrast, the group II mGluR (mGluR(2,3)) agonists (2R, 4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC) or (2S,2'R,3'R)-2-(2'3'-dicarboxycyclopropyl)glycine (DCG-IV) suppressed the frequency of sIPSCs. In addition, mGluR(1,5) agonist DHPG produced depolarizations and mGluR(2/3) agonists APDC or L-CCG-I [(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine] produced hyperpolarizations in dLGN interneurons. Furthermore, the enhanced sIPSC activity by optic tract stimulation was reduced when paired with corticothalamic fiber stimulation. The present data indicate that activation of specific mGluR subtypes differentially regulates inhibitory activity via different synaptic pathways. Our results suggest that activation of specific mGluR subtypes can upregulate or downregulate inhibitory activity in thalamic relay neurons, and these actions likely shape excitatory synaptic integration and thus regulate information transfer through thalamocortical circuits.

  16. Pumpkin seed extract: Cell growth inhibition of hyperplastic and cancer cells, independent of steroid hormone receptors.

    PubMed

    Medjakovic, Svjetlana; Hobiger, Stefanie; Ardjomand-Woelkart, Karin; Bucar, Franz; Jungbauer, Alois

    2016-04-01

    Pumpkin seeds have been known in folk medicine as remedy for kidney, bladder and prostate disorders since centuries. Nevertheless, pumpkin research provides insufficient data to back up traditional beliefs of ethnomedical practice. The bioactivity of a hydro-ethanolic extract of pumpkin seeds from the Styrian pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca, was investigated. As pumpkin seed extracts are standardized to cucurbitin, this compound was also tested. Transactivational activity was evaluated for human androgen receptor, estrogen receptor and progesterone receptor with in vitro yeast assays. Cell viability tests with prostate cancer cells, breast cancer cells, colorectal adenocarcinoma cells and a hyperplastic cell line from benign prostate hyperplasia tissue were performed. As model for non-hyperplastic cells, effects on cell viability were tested with a human dermal fibroblast cell line (HDF-5). No transactivational activity was found for human androgen receptor, estrogen receptor and progesterone receptor, for both, extract and cucurbitin. A cell growth inhibition of ~40-50% was observed for all cell lines, with the exception of HDF-5, which showed with ~20% much lower cell growth inhibition. Given the receptor status of some cell lines, a steroid-hormone receptor independent growth inhibiting effect can be assumed. The cell growth inhibition for fast growing cells together with the cell growth inhibition of prostate-, breast- and colon cancer cells corroborates the ethnomedical use of pumpkin seeds for a treatment of benign prostate hyperplasia. Moreover, due to the lack of androgenic activity, pumpkin seed applications can be regarded as safe for the prostate.

  17. Transcriptional Corepressor SMILE Recruits SIRT1 to Inhibit Nuclear Receptor Estrogen Receptor-related Receptor γ Transactivation*

    PubMed Central

    Xie, Yuan-Bin; Park, Jeong-Hoh; Kim, Don-Kyu; Hwang, Jung Hwan; Oh, Sangmi; Park, Seung Bum; Shong, Minho; Lee, In-Kyu; Choi, Hueng-Sik

    2009-01-01

    SMILE (small heterodimer partner interacting leucine zipper protein) has been identified as a corepressor of the glucocorticoid receptor, constitutive androstane receptor, and hepatocyte nuclear factor 4α. Here we show that SMILE also represses estrogen receptor-related receptor γ (ERRγ) transactivation. Knockdown of SMILE gene expression increases ERRγ activity. SMILE directly interacts with ERRγ in vitro and in vivo. Domain mapping analysis showed that SMILE binds to the AF2 domain of ERRγ. SMILE represses ERRγ transactivation partially through competition with coactivators PGC-1α, PGC-1β, and GRIP1. Interestingly, the repression of SMILE on ERRγ is released by SIRT1 inhibitors, a catalytically inactive SIRT1 mutant, and SIRT1 small interfering RNA but not by histone protein deacetylase inhibitor. In vivo glutathione S-transferase pulldown and coimmunoprecipitation assays validated that SMILE physically interacts with SIRT1. Furthermore, the ERRγ inverse agonist GSK5182 enhances the interaction of SMILE with ERRγ and SMILE-mediated repression. Knockdown of SMILE or SIRT1 blocks the repressive effect of GSK5182. Moreover, chromatin immunoprecipitation assays revealed that GSK5182 augments the association of SMILE and SIRT1 on the promoter of the ERRγ target PDK4. GSK5182 and adenoviral overexpression of SMILE cooperate to repress ERRγ-induced PDK4 gene expression, and this repression is released by overexpression of a catalytically defective SIRT1 mutant. Finally, we demonstrated that ERRγ regulates SMILE gene expression, which in turn inhibits ERRγ. Overall, these findings implicate SMILE as a novel corepressor of ERRγ and recruitment of SIRT1 as a novel repressive mechanism for SMILE and ERRγ inverse agonist. PMID:19690166

  18. Competitive inhibition of (TH)dexamethasone binding to mammary glucocorticoid receptor by leupeptin

    SciTech Connect

    Hsieh, L.C.C.; Su, C.; Markland, F.S. Jr.

    1987-03-01

    The inhibitory effect of leupeptin on (TH)dexamethasone binding to the glucocorticoid receptor from lactating goat mammary cytosol has been studied. Leupeptin (10 mM) caused a significant (about 35%) inhibition of (TH)dexamethasone binding to glucocorticoid receptor. Binding inhibition is further increased following filtration of unlabeled cytosolic receptor through a Bio-Gel A 0.5-m column. Binding inhibition was partially reversed by monothioglycerol at 10 mM concentration. A double reciprocal plot revealed that leupeptin appears to be a competitive inhibitor of (TH)dexamethasone binding to the glucocorticoid receptor. Low salt sucrose density gradient centrifugation revealed that the leupeptin-treated sample formed a slightly larger (approximately 9 S) receptor complex (leupeptin-free complex sediments at 8 S).

  19. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

  20. Insulin action is blocked by a monoclonal antibody that inhibits insulin receptor kinase

    SciTech Connect

    Morgan, D.O.; Ho, L.; Korn, L.J.; Roth, R.A.

    1986-01-01

    Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor ..beta.. subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor ..beta.. subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.

  1. Insulin Action is Blocked by a Monoclonal Antibody That Inhibits the Insulin Receptor Kinase

    NASA Astrophysics Data System (ADS)

    Morgan, David O.; Ho, Lisa; Korn, Laurence J.; Roth, Richard A.

    1986-01-01

    Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor β subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens, and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor β subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.

  2. Prostaglandin E₂ inhibits human lung fibroblast chemotaxis through disparate actions on different E-prostanoid receptors.

    PubMed

    Li, Ying-Ji; Wang, Xing-Qi; Sato, Tadashi; Kanaji, Nobuhiro; Nakanishi, Masanori; Kim, Miok; Michalski, Joel; Nelson, Amy J; Sun, Jian-Hong; Farid, Maha; Basma, Hesham; Patil, Amol; Toews, Myron L; Liu, Xiangde; Rennard, Stephen I

    2011-01-01

    The migration of fibroblasts is believed to play a key role in both normal wound repair and abnormal tissue remodeling. Prostaglandin E (PGE)(2), a mediator that can inhibit many fibroblast functions including chemotaxis, was reported to be mediated by the E-prostanoid (EP) receptor EP2. PGE(2), however, can act on four receptors. This study was designed to determine if EP receptors, in addition to EP2, can modulate fibroblast chemotaxis. Using human fetal lung fibroblasts, the expression of all four EP receptors was demonstrated by Western blotting. EP2-selective and EP4-selective agonists inhibited both chemotaxis toward fibronectin in the blindwell assay and migration in a wound-closure assay. In contrast, EP1-selective and EP3-selective agonists stimulated cell migration in both assay systems. These results were confirmed using EP-selective antagonists. The role of both EP2 and EP4 receptors in mediating the PGE(2) inhibition of chemotaxis was also confirmed by small interfering RNA suppression. Furthermore, the role of EP receptors was confirmed by blocking the expected signaling pathways. Taken together, these results demonstrate that PGE(2) can act on multiple EP receptors in human lung fibroblasts, to exert disparate effects. Alterations in EP receptor expression may have the potential to alter PGE(2) action. Targeting specific EP receptors may offer therapeutic opportunities in conditions characterized by abnormal tissue repair and remodeling.

  3. Nongenomic signaling of the retinoid X receptor through binding and inhibiting Gq in human platelets

    PubMed Central

    Moraes, Leonardo A.; Swales, Karen E.; Wray, Jessica A.; Damazo, Amilcar; Gibbins, Jonathan M.; Warner, Timothy D.

    2007-01-01

    Retinoid X receptors (RXRs) are important transcriptional nuclear hormone receptors, acting as either homodimers or the binding partner for at least one fourth of all the known human nuclear receptors. Functional nongenomic effects of nuclear receptors are poorly understood; however, recently peroxisome proliferator-activated receptor (PPAR) γ, PPARβ, and the glucocorticoid receptor have all been found active in human platelets. Human platelets express RXRα and RXRβ. RXR ligands inhibit platelet aggregation and TXA2 release to ADP and the TXA2 receptors, but only weakly to collagen. ADP and TXA2 both signal via the G protein, Gq. RXR rapidly binds Gq but not Gi/z/o/t/gust in a ligand-dependent manner and inhibits Gq-induced Rac activation and intracellular calcium release. We propose that RXR ligands may have beneficial clinical actions through inhibition of platelet activation. Furthermore, our results demonstrate a novel nongenomic mode for nuclear receptor action and a functional cross-talk between G-protein and nuclear receptor signaling families. PMID:17213293

  4. Inhibition of opioid release in the rat spinal cord by α2C adrenergic receptors

    PubMed Central

    Chen, Wenling; Song, Bingbing; Marvizón, Juan Carlos G.

    2008-01-01

    Neurotransmitter receptors that control the release of opioid peptides in the spinal cord may play an important role in pain modulation. Norepinephrine, released by a descending pathway originating in the brainstem, is a powerful inducer of analgesia in the spinal cord. Adrenergic α2C receptors are present in opioid-containing terminals in the dorsal horn, where they could modulate opioid release. The goal of this study was to investigate this possibility. Opioid release was evoked from rat spinal cord slices by incubating them with the sodium channel opener veratridine in the presence of peptidase inhibitors (actinonin, captopril and thiorphan), and was measured in situ through the internalization of μ-opioid receptors in dorsal horn neurons. Veratridine produced internalization in 70% of these neurons. The α2 receptor agonists clonidine, guanfacine, medetomidine and UK-14304 inhibited the evoked μ-opioid receptor internalization with IC50s of 1.7 μM, 248 nM, 0.3 nM and 22 nM, respectively. However, inhibition by medetomidine was only partial, and inhibition by UK-14304 reversed itself at concentrations higher than 50 nM. None of these agonists inhibited μ-opioid receptor internalization produced by endomorphin-2, showing that they inhibited opioid release and not the internalization itself. The inhibition produced by clonidine, guanfacine or UK-14304 was completely reversed by the selective α2C antagonist JP-1203. In contrast, inhibition by guanfacine was not prevented by the α2A antagonist BRL-44408. These results show that α2C receptors inhibit the release of opioids in the dorsal horn. This action may serve to shut down the opioid system when the adrenergic system is active. PMID:18343461

  5. Inhibition of calcium channels by neurokinin receptor and signal transduction in hamster submandibular ganglion cells.

    PubMed

    Yamada, T; Endoh, T; Suzuki, T

    1999-04-16

    Both substance P (SP) and neurokinin A (NKA) are known as neurotransmitters of the submandibular ganglion (SMG) neurons. SP released from collaterals of the sensory nerves also regulates the excitability of SMG neurons. It has recently been shown that neurokinins (NK) inhibit calcium channels in various neurons. In this study, the effects of NK on voltage-dependent calcium channel current (I(Ca)) in SMG cells were investigated using the whole-cell patch-clamp recording method. NK-1 receptor agonist and SP caused inhibition of I(Ca) in SMG cells in a dose-dependent manner. NK-1 receptor agonist inhibited L-, N- and P/Q-type I(Ca) components. GDP-beta-S included in the pipette solution reduced the NK-1 receptor agonist-induced inhibition of I(Ca). In addition, NK-1 receptor agonist-induced inhibition of I(Ca) was reduced by stimulation of protein kinase C (PKC) but not cyclic AMP-dependent protein kinase (PKA). The results provided evidence for a signal transduction pathway in which calcium channel inhibition by NK receptors required activation of G-protein and PKC-affected step phosphorylation in SMG neurons.

  6. Activation of histamine H3 receptors in human nasal mucosa inhibits sympathetic vasoconstriction.

    PubMed

    Varty, LoriAnn M; Gustafson, Eric; Laverty, Maureen; Hey, John A

    2004-01-19

    The peripheral histamine H3 receptor is a presynaptic heterologous receptor located on postganglionic sympathetic nerve fibers innervating sympathetic effector systems such as blood vessels and the heart. An extensive body of evidence shows that activation of the histamine H3 receptor attenuates sympathetic tone by presynaptic inhibition of noradrenaline release. It is proposed that this sympathoinhibitory action, in vivo, leads to reduced vasoconstriction, thereby eliciting a vasodilatory effect. In humans, the peripheral histamine H3 receptor has also been shown to exert a sympathoinhibitory function on specific peripheral autonomic effector systems. For example, human saphenous vein and heart possess functional presynaptic histamine H3 receptors on the sympathetic nerve terminals that upon activation decrease the sympathetic tone to these respective organs. The present studies were conducted to define the role of histamine H3 receptors on neurogenic sympathetic vasoconstrictor responses in human nasal turbinate mucosa. Contractility studies were conducted to evaluate the effect of histamine H3 receptor activation on sympathetic vasoconstriction in surgically isolated human nasal turbinate mucosa. We found that the histamine H3 receptor agonist, (R)-alpha-methylhistamine (30 and 300 nM), inhibited electrical field stimulation-induced (neurogenic) sympathetic vasoconstriction in a concentration-dependent fashion. Pretreatment with the selective histamine H3 receptor antagonist, clobenpropit (100 nM), blocked the sympathoinhibitory effect of (R)-alpha-methylhistamine on the neurogenic sympathetic vasoconstriction. In addition, analysis of Taqman mRNA expression studies showed a specific, high level of distribution of the histamine H3 receptor localized in the human nasal mucosa. Taken together, these studies indicate that histamine H3 receptors modulate vascular contractile responses in human nasal mucosa most likely by inhibiting noradrenaline release from

  7. Cloned M1 muscarinic receptors mediate both adenylate cyclase inhibition and phosphoinositide turnover.

    PubMed Central

    Stein, R; Pinkas-Kramarski, R; Sokolovsky, M

    1988-01-01

    The rat M1 muscarinic receptor gene was cloned and expressed in a rat cell line lacking endogenous muscarinic receptors. Assignment of the cloned receptors to the M1 class was pharmacologically confirmed by their high affinity for the M1-selective muscarinic antagonist pirenzepine and low affinity for the M2-selective antagonist AF-DX-116. Guanylyl imidodiphosphate [Gpp(NH)p] converted agonist binding sites on the receptor, from high-affinity to the low-affinity state, thus indicating that the cloned receptors couple to endogenous G-proteins. The cloned receptors mediated both adenylate cyclase inhibition and phosphoinositide hydrolysis, but by different mechanisms. Pertussis toxin blocked the inhibition of adenylate cyclase (indicating coupling of the receptor to inhibitory G-protein), but did not affect phosphoinositide turnover. Furthermore, the stimulation of phosphoinositide hydrolysis was less efficient than the inhibition of adenylate cyclase. These findings demonstrate that cloned M1 receptors are capable of mediating multiple responses in the cell by coupling to different effectors, possibly to different G-proteins. Images PMID:2846274

  8. Structure and mechanism of activity-based inhibition of the EGF-Receptor by Mig6

    PubMed Central

    Ficarro, Scott B.; Zhang, Yi; Lee, Byung Il; Cho, Ahye; Kim, Kihong; Park, Angela K.J.; Park, Woong-Yang; Murray, Bradley; Meyerson, Matthew; Beroukhim, Rameen; Marto, Jarrod A.; Cho, Jeonghee; Eck, Michael J.

    2016-01-01

    Mig6 is a feedback inhibitor that directly binds, inhibits and drives internalization of ErbB-family receptors. Mig6 selectivity targets activated receptors. Here we find that the EGF receptor phosphorylates Mig6 on Tyr394, and that this phosphorylation is primed by prior phosphorylation of an adjacent residue, Tyr395, by Src. Crystal structures of human EGFR–Mig6 complexes reveal the structural basis for enhanced phosphorylation of primed Mig6 and show how Mig6 rearranges after phosphorylation by EGFR to effectively irreversibly inhibit the same receptor that catalyzed its phosphorylation. This dual phosphorylation site allows Mig6 to inactivate EGFR in a manner that requires activation of the target receptor and can be modulated by Src. Loss of Mig6 is a driving event in human cancer; analysis of 1057 gliomas reveals frequent focal deletions of ERRFI, the gene that encodes Mig6, in EGFR-amplified glioblastomas. PMID:26280531

  9. CD44 Antibody Inhibition of Macrophage Phagocytosis Targets Fcγ Receptor- and Complement Receptor 3-Dependent Mechanisms.

    PubMed

    Amash, Alaa; Wang, Lin; Wang, Yawen; Bhakta, Varsha; Fairn, Gregory D; Hou, Ming; Peng, Jun; Sheffield, William P; Lazarus, Alan H

    2016-04-15

    Targeting CD44, a major leukocyte adhesion molecule, using specific Abs has been shown beneficial in several models of autoimmune and inflammatory diseases. The mechanisms contributing to the anti-inflammatory effects of CD44 Abs, however, remain poorly understood. Phagocytosis is a key component of immune system function and can play a pivotal role in autoimmune states where CD44 Abs have shown to be effective. In this study, we show that the well-known anti-inflammatory CD44 Ab IM7 can inhibit murine macrophage phagocytosis of RBCs. We assessed three selected macrophage phagocytic receptor systems: Fcγ receptors (FcγRs), complement receptor 3 (CR3), and dectin-1. Treatment of macrophages with IM7 resulted in significant inhibition of FcγR-mediated phagocytosis of IgG-opsonized RBCs. The inhibition of FcγR-mediated phagocytosis was at an early stage in the phagocytic process involving both inhibition of the binding of the target RBC to the macrophages and postbinding events. This CD44 Ab also inhibited CR3-mediated phagocytosis of C3bi-opsonized RBCs, but it did not affect the phagocytosis of zymosan particles, known to be mediated by the C-type lectin dectin-1. Other CD44 Abs known to have less broad anti-inflammatory activity, including KM114, KM81, and KM201, did not inhibit FcγR-mediated phagocytosis of RBCs. Taken together, these findings demonstrate selective inhibition of FcγR and CR3-mediated phagocytosis by IM7 and suggest that this broadly anti-inflammatory CD44 Ab inhibits these selected macrophage phagocytic pathways. The understanding of the immune-regulatory effects of CD44 Abs is important in the development and optimization of therapeutic strategies for the potential treatment of autoimmune conditions.

  10. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation.

    PubMed

    Macho, Alberto P; Schwessinger, Benjamin; Ntoukakis, Vardis; Brutus, Alexandre; Segonzac, Cécile; Roy, Sonali; Kadota, Yasuhiro; Oh, Man-Ho; Sklenar, Jan; Derbyshire, Paul; Lozano-Durán, Rosa; Malinovsky, Frederikke Gro; Monaghan, Jacqueline; Menke, Frank L; Huber, Steven C; He, Sheng Yang; Zipfel, Cyril

    2014-03-28

    Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

  11. Two PTP receptors mediate CSPG inhibition by convergent and divergent signaling pathways in neurons

    PubMed Central

    Ohtake, Yosuke; Wong, Daniella; Abdul-Muneer, P. M.; Selzer, Michael E.; Li, Shuxin

    2016-01-01

    Receptor protein tyrosine phosphatase σ (PTPσ) and its subfamily member LAR act as transmembrane receptors that mediate growth inhibition of chondroitin sulfate proteoglycans (CSPGs). Inhibition of either receptor increases axon growth into and beyond scar tissues after CNS injury. However, it is unclear why neurons express two similar CSPG receptors, nor whether they use the same or different intracellular pathways. We have now studied the signaling pathways of these two receptors using N2A cells and primary neurons derived from knockout mice. We demonstrate that both receptors share certain signaling pathways (RhoA, Akt and Erk), but also use distinct signals to mediate CSPG actions. Activation of PTPσ by CSPGs selectively inactivated CRMP2, APC, S6 kinase and CREB. By contrast LAR activation inactivated PKCζ, cofilin and LKB1. For the first time, we propose a model of the signaling pathways downstream of these two CSPG receptors. We also demonstrate that deleting both receptors exhibits additive enhancement of axon growth in adult neuronal cultures in vitro. Our findings elucidate the novel downstream pathways of CSPGs and suggest potential synergy of blocking their two PTP receptors. PMID:27849007

  12. Allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation.

    PubMed

    Wu, Zhuang; Li, Linlang; Zheng, Long-Tai; Xu, Zhihong; Guo, Lin; Zhen, Xuechu

    2015-09-01

    Recent studies have shown that sigma-1 receptor orthodox agonists can inhibit neuroinflammation. SKF83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), an atypical dopamine receptor-1 agonist, has been recently identified as a potent allosteric modulator of sigma-1 receptor. Here, we investigated the anti-inflammatory effects of SKF83959 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results indicated that SKF83959 significantly suppressed the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and inhibited the generation of reactive oxygen species. All of these responses were blocked by selective sigma-1 receptor antagonists (BD1047 or BD1063) and by ketoconazole (an inhibitor of enzyme cytochrome c17 to inhibit the synthesis of endogenous dehydroepiandrosterone, DHEA). Additionally, we found that SKF83959 promoted the binding activity of DHEA with sigma-1 receptors, and enhanced the inhibitory effects of DHEA on LPS-induced microglia activation in a synergic manner. Furthermore, in a microglia-conditioned media system, SKF83959 inhibited the cytotoxicity of conditioned medium generated by LPS-activated microglia toward HT-22 neuroblastoma cells. Taken together, our study provides the first evidence that allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation. SKF83959 is a potent allosteric modulator of sigma-1 receptor. Our results indicated that SKF83959 enhanced the activity of endogenous dehydroepiandrosterone (DHEA) in a synergic manner, and inhibited the activation of BV2 microglia and the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS).

  13. Azadirachtin interacts with retinoic acid receptors and inhibits retinoic acid-mediated biological responses.

    PubMed

    Thoh, Maikho; Babajan, Banaganapalli; Raghavendra, Pongali B; Sureshkumar, Chitta; Manna, Sunil K

    2011-02-11

    Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies.

  14. Potent and long-lasting inhibition of human P2X2 receptors by copper

    PubMed Central

    Punthambaker, Sukanya; Hume, Richard I.

    2013-01-01

    P2X receptors are ion channels gated by ATP. In rodents these channels are modulated by zinc and copper. Zinc is co-released with neurotransmitter at some synapses and can modulate neuronal activity, but the role of copper in the brain is unclear. Rat P2X2 receptors show potentiation by 2–100 µM zinc or copper in the presence of a submaximal concentration of ATP but are inhibited by zinc or copper at concentrations above 100 µM. In contrast, human P2X2 (hP2X2) receptors show no potentiation and are strongly inhibited by zinc over the range of 2–100 µM. The effect of copper on hP2X2 is of interest because there are human brain disorders in which copper concentration is altered. We found that hP2X2 receptors are potently inhibited by copper (IC50 = 40 nM). ATP responsiveness recovered extremely slowly after copper washout, with full recovery requiring over 1 h. ATP binding facilitated copper binding but not unbinding from this inhibitory site. A mutant receptor in which the first six extracellular cysteines were deleted, C(1–6)S, showed normal copper inhibition, however reducing agents dramatically accelerated recovery from copper inhibition in wild type hP2X2 and the C(1–6)S mutant, indicating that the final two disulfide bonds are required to maintain the high affinity copper binding site. Three histidine residues required for normal zinc inhibition were also required for normal copper inhibition. Humans with untreated Wilson’s disease have excess amounts of copper in the brain. The high copper sensitivity of hP2X2 receptors suggests that they are non-functional in these patients. PMID:24067922

  15. Relationship between muscarinic receptor occupancy and adenylate cyclase inhibition in the rabbit myocardium

    SciTech Connect

    Ehlert, F.J.

    1985-11-01

    The muscarinic receptor-binding properties of a series of muscarinic drugs were compared with their effects on adenylate cyclase in membranes of the rabbit myocardium. When measured by competitive inhibition of (TH)-N-methylscopolamine binding, the competition curves of the various agonists were adequately described by the ternary complex model. This model assumes that the receptor can bind reversibly with a guanine nucleotide binding protein in the membrane and that the affinity of the agonist for the receptor-guanine nucleotide-binding protein complex is higher than that for the free receptor. A satisfactory fit of the ternary complex model to the data could only be achieved assuming that very little receptor is precoupled with the guanine nucleotide-binding protein in the absence of agonist. There was good agreement between the efficacy of each agonist as measured by inhibition of adenylate cyclase and the estimate of the positive cooperativity between the binding of the agonist receptor complex and the guanine nucleotide-binding protein. Guanosine 5'-triphosphate (0.1 mM) had no significant effect on the binding of (TH)N-methylscopolamine but caused an increase in the concentration of the various agonists required for half-maximal receptor occupancy. There was good correlation between efficacy as measured by inhibition of adenylate cyclase and the influence of guanosine 5'-triphosphate on binding properties.

  16. Manipulation of receptor oligomerization as a strategy to inhibit signaling by TNF superfamily members.

    PubMed

    Warren, Julia T; Nelson, Christopher A; Decker, Corinne E; Zou, Wei; Fremont, Daved H; Teitelbaum, Steven L

    2014-08-19

    Signaling by receptor activator of nuclear factor κB (RANK) in response to its ligand RANKL, which is a member of the tumor necrosis factor (TNF) superfamily of cytokines, stimulates osteoclast formation and bone resorption. Thus, this ligand-receptor pair is a therapeutic target for various disorders, such as osteoporosis and metastasis of cancer to bone. RANKL exists as a physiological homotrimer, with each monomer recognizing a single molecule of RANK or the decoy receptor osteoprotegerin (OPG), which inhibits osteoclastogenesis. We engineered a RANKL protein in which all three monomers of RANKL were encoded as a single polypeptide chain, which enabled us to independently control receptor binding at each binding interface. To generate an effective RANK inhibitor, we used an unbiased forward genetic approach to identify mutations in RANKL that had a 500-fold increased affinity for RANK but had decreased affinity for the decoy receptor OPG. Incorporating mutations that blocked receptor binding into this high-affinity RANKL variant generated a mutant RANKL that completely inhibited wild-type RANKL-induced osteoclastogenesis in vitro and bone resorption in mice. Our approach may be generalized to enable the inhibition of other TNF receptor signaling systems, which are implicated in a wide range of pathological conditions.

  17. Use-dependent inhibition of P2X3 receptors by nanomolar agonist.

    PubMed

    Pratt, Emily B; Brink, Thaddeus S; Bergson, Pamela; Voigt, Mark M; Cook, Sean P

    2005-08-10

    P2X3 receptors desensitize within 100 ms of channel activation, yet recovery from desensitization requires several minutes. The molecular basis for this slow rate of recovery is unknown. We designed experiments to test the hypothesis that this slow recovery is attributable to the high affinity (< 1 nM) of desensitized P2X3 receptors for agonist. We found that agonist binding to the desensitized state provided a mechanism for potent inhibition of P2X3 current. Sustained applications of 0.5 nM ATP inhibited > 50% of current to repetitive applications of P2X3 agonist. Inhibition occurred at 1000-fold lower agonist concentrations than required for channel activation and showed strong use dependence. No inhibition occurred without previous activation and desensitization. Our data are consistent with a model whereby inhibition of P2X3 by nanomolar [agonist] occurs by the rebinding of agonist to desensitized channels before recovery from desensitization. For several ATP analogs, the concentration required to inhibit P2X3 current inversely correlated with the rate of recovery from desensitization. This indicates that the affinity of the desensitized state and recovery rate primarily depend on the rate of agonist unbinding. Consistent with this hypothesis, unbinding of [32P]ATP from desensitized P2X3 receptors mirrored the rate of recovery from desensitization. As expected, disruption of agonist binding by site-directed mutagenesis increased the IC50 for inhibition and increased the rate of recovery.

  18. Cannabinoid receptor type 1- and 2-mediated increase in cyclic AMP inhibits T cell receptor-triggered signaling.

    PubMed

    Börner, Christine; Smida, Michal; Höllt, Volker; Schraven, Burkhart; Kraus, Jürgen

    2009-12-18

    The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to G(i/o) proteins, which are associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Delta9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the zeta-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.

  19. Cannabidiol inhibits human glioma cell migration through a cannabinoid receptor-independent mechanism

    PubMed Central

    Vaccani, Angelo; Massi, Paola; Colombo, Arianna; Rubino, Tiziana; Parolaro, Daniela

    2005-01-01

    We evaluated the ability of cannabidiol (CBD) to impair the migration of tumor cells stimulated by conditioned medium. CBD caused concentration-dependent inhibition of the migration of U87 glioma cells, quantified in a Boyden chamber. Since these cells express both cannabinoid CB1 and CB2 receptors in the membrane, we also evaluated their engagement in the antimigratory effect of CBD. The inhibition of cell was not antagonized either by the selective cannabinoid receptor antagonists SR141716 (CB1) and SR144528 (CB2) or by pretreatment with pertussis toxin, indicating no involvement of classical cannabinoid receptors and/or receptors coupled to Gi/o proteins. These results reinforce the evidence of antitumoral properties of CBD, demonstrating its ability to limit tumor invasion, although the mechanism of its pharmacological effects remains to be clarified. PMID:15700028

  20. Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma.

    PubMed

    Johansson, Erik; Zhai, Qiwei; Zeng, Zhao-Jun; Yoshida, Takeshi; Funa, Keiko

    2016-05-01

    TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells.

  1. Opiate receptor blockade on human granulosa cells inhibits VEGF release.

    PubMed

    Lunger, Fabian; Vehmas, Anni P; Fürnrohr, Barbara G; Sopper, Sieghart; Wildt, Ludwig; Seeber, Beata

    2016-03-01

    The objectives of this study were to determine whether the main opioid receptor (OPRM1) is present on human granulosa cells and if exogenous opiates and their antagonists can influence granulosa cell vascular endothelial growth factor (VEGF) production via OPRM1. Granulosa cells were isolated from women undergoing oocyte retrieval for IVF. Complementary to the primary cells, experiments were conducted using COV434, a well-characterized human granulosa cell line. Identification and localization of opiate receptor subtypes was carried out using Western blot and flow cytometry. The effect of opiate antagonist on granulosa cell VEGF secretion was assessed by enzyme-linked immunosorbent assay. For the first time, the presence of OPRM1 on human granulosa cells is reported. Blocking of opiate signalling using naloxone, a specific OPRM1 antagonist, significantly reduced granulosa cell-derived VEGF levels in both COV434 and granulosa-luteal cells (P < 0.01). The presence of opiate receptors and opiate signalling in granulosa cells suggest a possible role in VEGF production. Targeting this signalling pathway could prove promising as a new clinical option in the prevention and treatment of ovarian hyperstimulation syndrome.

  2. Dopamine inhibits somatolactin gene expression in tilapia pituitary cells through the dopamine D2 receptors.

    PubMed

    Jiang, Quan; Lian, Anji; He, Qi

    2016-07-01

    Dopamine (DA) is an important neurotransmitter in the central nervous system of vertebrates and possesses key hypophysiotropic functions. Early studies have shown that DA has a potent inhibitory effect on somatolactin (SL) release in fish. However, the mechanisms responsible for DA inhibition of SL gene expression are largely unknown. To this end, tilapia DA type-1 (D1) and type-2 (D2) receptor transcripts were examined in the neurointermediate lobe (NIL) of the tilapia pituitary by real-time PCR. In tilapia, DA not only was effective in inhibiting SL mRNA levels in vivo and in vitro, but also could abolish pituitary adenylate cyclase-activating polypeptide (PACAP)- and salmon gonadotropin-releasing hormone (sGnRH)-stimulated SL gene expression at the pituitary level. In parallel studies, the specific D2 receptor agonists quinpirole and bromocriptine could mimic the DA-inhibited SL gene expression. Furthermore, the D2 receptor antagonists domperidone and (-)-sulpiride could abolish the SL response to DA or the D2 agonist quinpirole, whereas D1 receptor antagonists SCH23390 and SKF83566 were not effective in this respect. In primary cultures of tilapia NIL cells, D2 agonist quinpirole-inhibited cAMP production could be blocked by co-treatment with the D2 antagonist domperidone and the ability of forskolin to increase cAMP production was also inhibited by quinpirole. Using a pharmacological approach, the AC/cAMP pathway was shown to be involved in quinpirole-inhibited SL mRNA expression. These results provide evidence that DA can directly inhibit SL gene expression at the tilapia pituitary level via D2 receptor through the AC/cAMP-dependent mechanism.

  3. Hepatocyte insulin receptor is a calmodulin binding protein and is functionally inhibited by calmidazolium

    SciTech Connect

    Arnold, T.P.; Pollet, R.J.

    1986-05-01

    Insulin-induced autophosphorylation of the insulin receptor and changes in intracellular Ca/sup + +/ have been proposed as possible mediators of insulin action in target tissues. The authors have investigated the association of the 17kD calcium binding protein calmodulin with the insulin receptor solubilized from rat liver plasma membranes. Insulin receptors solubilized in 0.1% Triton X-100 exhibited strong binding to calmodulin-agarose affinity columns in the presence of 100..mu..M calcium and could be eluded with 100..mu..M ethelene glycol-bis (amino ethel ether) Tetra Acetic Acid (EGTA) with an 80% yield in insulin binding activity. In addition, /sup 125/I-Calmodulin was shown to bind to wheat germ agglutinin purified solubilized receptors, was specifically inhibited by EGTA (100 ..mu..M) and/or calmidazolium (10 ..mu..M) and was found to be insulin-dependent (max 10/sup -10/ M insulin). SDS-polyacrylamide gel electrophoresis data suggests that /sup 125/I-calmodulin may be associated with the 92 kD beta-subunit of the insulin receptor, consistent with the cytoplasmic domain of this subunit. While they have confirmed previous reports that the addition of calcium and calmodulin to solubilized insulin receptors preparations produces no demonstrable change in receptor phosphorylation, the addition of the calmodulin inhibitor calmidazolium did show more than 50% inhibition of insulin stimulated receptor phosphorylation, suggesting that a domain of the calmodulin molecule may be very tightly associated with the insulin receptor. These results indicate that calmodulin binds tightly and specifically to the insulin receptor of the hepatocyte and is insulin dependent. The findings also suggest that this interaction may be functionally significant in mediating insulin-induced receptor phosphorylation as well as other insulin actions. Thus, calmodulin may play a major role as an intracellular contributor to insulin action.

  4. Prejunctional inhibition of sympathetically evoked pupillary dilation in cats by activation of histamine H3 receptors.

    PubMed

    Koss, M C; Hey, J A

    1993-08-01

    Frequency-dependent pupillary dilations were evoked by electrical stimulation of the pre- or post-ganglionic cervical sympathetic nerve (sympatho-excitation) or the hypothalamus (parasympatho-inhibition) in sympathectomized anesthetized cats. Systemic administration of the selective histamine H3 receptor agonist (R)-alpha-methylhistamine (R alpha MeHA) produced a dose-dependent depression of mydriasis due to direct neural sympathetic activation but had no effect on responses elicited by parasympathetic withdrawal. The histamine H2 receptor agonist, dimaprit, was inactive. R alpha MeHA was much more effective in depressing sympathetic responses obtained at lower frequencies when compared to higher frequencies of stimulation. Responses evoked both pre- and postganglionically were inhibited by R alpha MeHA. This peripheral sympatho-inhibitory action of R alpha MeHA was antagonized by the histamine H3 receptor blocker thioperamide but not by intravenous pretreatment with the histamine H1 receptor antagonist chlorpheniramine. Histamine H2 receptor blockers cimetidine and ranitidine were also without effect. R alpha MeHA did not depress pupillary responses elicited by i.v. (-)-adrenaline. The results demonstrate that histamine H3 receptors modulate sympathetic activation of the iris at a site proximal to the iris dilator muscle. The predominant mechanism of action appears to the prejunctional inhibition of noradrenaline release from postganglionic sympathetic nerve endings. However, a concomitant ganglionic inhibitory action cannot be excluded.

  5. Lamotrigine, an antiepileptic drug, inhibits 5-HT3 receptor currents in NCB-20 neuroblastoma cells

    PubMed Central

    Kim, Ki Jung; Jeun, Seung Hyun

    2017-01-01

    Lamotrigine is an antiepileptic drug widely used to treat epileptic seizures. Using whole-cell voltage clamp recordings in combination with a fast drug application approach, we investigated the effects of lamotrigine on 5-hydroxytryptamine (5-HT)3 receptors in NCB-20 neuroblastoma cells. Co-application of lamotrigine (1~300 µM) resulted in a concentration-dependent reduction in peak amplitude of currents induced by 3 µM of 5-HT for an IC50 value of 28.2±3.6 µM with a Hill coefficient of 1.2±0.1. These peak amplitude decreases were accompanied by the rise slope reduction. In addition, 5-HT3-mediated currents evoked by 1 mM dopamine, a partial 5-HT3 receptor agonist, were inhibited by lamotrigine co-application. The EC50 of 5-HT for 5-HT3 receptor currents were shifted to the right by co-application of lamotrigine without a significant change of maximal effect. Currents activated by 5-HT and lamotrigine co-application in the presence of 1 min pretreatment of lamotrigine were similar to those activated by 5-HT and lamotrigine co-application alone. Moreover, subsequent application of lamotrigine in the presence of 5-HT and 5-hydroxyindole, known to attenuate 5-HT3 receptor desensitization, inhibited 5-HT3 receptor currents in a concentration-dependent manner. The deactivation of 5-HT3 receptor was delayed by washing with an external solution containing lamotrigine. Lamotrigine accelerated the desensitization process of 5-HT3 receptors. There was no voltage-dependency in the inhibitory effects of lamotrigine on the 5-HT3 receptor currents. These results indicate that lamotrigine inhibits 5-HT3-activated currents in a competitive manner by binding to the open state of the channels and blocking channel activation or accelerating receptor desensitization. PMID:28280410

  6. Lamotrigine, an antiepileptic drug, inhibits 5-HT3 receptor currents in NCB-20 neuroblastoma cells.

    PubMed

    Kim, Ki Jung; Jeun, Seung Hyun; Sung, Ki-Wug

    2017-03-01

    Lamotrigine is an antiepileptic drug widely used to treat epileptic seizures. Using whole-cell voltage clamp recordings in combination with a fast drug application approach, we investigated the effects of lamotrigine on 5-hydroxytryptamine (5-HT)3 receptors in NCB-20 neuroblastoma cells. Co-application of lamotrigine (1~300 µM) resulted in a concentration-dependent reduction in peak amplitude of currents induced by 3 µM of 5-HT for an IC50 value of 28.2±3.6 µM with a Hill coefficient of 1.2±0.1. These peak amplitude decreases were accompanied by the rise slope reduction. In addition, 5-HT3-mediated currents evoked by 1 mM dopamine, a partial 5-HT3 receptor agonist, were inhibited by lamotrigine co-application. The EC50 of 5-HT for 5-HT3 receptor currents were shifted to the right by co-application of lamotrigine without a significant change of maximal effect. Currents activated by 5-HT and lamotrigine co-application in the presence of 1 min pretreatment of lamotrigine were similar to those activated by 5-HT and lamotrigine co-application alone. Moreover, subsequent application of lamotrigine in the presence of 5-HT and 5-hydroxyindole, known to attenuate 5-HT3 receptor desensitization, inhibited 5-HT3 receptor currents in a concentration-dependent manner. The deactivation of 5-HT3 receptor was delayed by washing with an external solution containing lamotrigine. Lamotrigine accelerated the desensitization process of 5-HT3 receptors. There was no voltage-dependency in the inhibitory effects of lamotrigine on the 5-HT3 receptor currents. These results indicate that lamotrigine inhibits 5-HT3-activated currents in a competitive manner by binding to the open state of the channels and blocking channel activation or accelerating receptor desensitization.

  7. Inhibition of radiation-induced polyuria by histamine receptor antagonists

    SciTech Connect

    Donlon, M.A.; Melia, J.A.; Helgeson, E.A.; Wolfe, W.W.

    1986-03-01

    In previous studies the authors have demonstrated that gamma radiation results in polyuria, which is preceded by polydypsia. This suggests that the increased thirst elicited by radiation causes increased urinary volume (UV). Histamine, which is released following radiation exposure, also elicits drinking by nonirradiated rats when administered exogenously. In this study the authors have investigated both the role of water deprivation and the effect of histamine receptor antagonists (HRA) on radiation-induced polyuria. Sprague-Dawley rats were housed individually in metabolic cages. Water was allowed ad libitum except in deprivation experiments where water was removed for 24 hr immediately following radiation. Cimetidine (CIM), an H2 HRA, and dexbromopheniramine (DXB), an H1 HRA, were administered i.p. (16 and 1 mg/kg, respectively) 30 min prior to irradiation (950 rads from a cobalt source). UV was determined at 24-hr intervals for 3 days preceding irradiation and 24 hr postirradiation. UV in DXB treated rats was significantly reduced 24 hr postirradiation (CON = 427 +/- 54%; DXB = 247 +/- 39% of preirradiated CON) compared to postirradiation control values. CIM did not affect postirradiation UV. These data suggest that radiation-induced polyuria is caused by polydypsia which is, in part, mediated by histamine induced by an H1 receptor.

  8. M2muscarinic receptors inhibit cell proliferation and migration in urothelial bladder cancer cells

    PubMed Central

    Pacini, Luca; De Falco, Elena; Di Bari, Maria; Coccia, Andrea; Siciliano, Camilla; Ponti, Donatella; Pastore, Antonio Luigi; Petrozza, Vincenzo; Carbone, Antonio; Tata, Ada Maria; Calogero, Antonella

    2014-01-01

    The role of muscarinic receptors in several diseases including cancer has recently emerged. To evaluate the hypothesis that muscarinic acetylcholine receptors may play a role in bladder cancer as well as in other tumor types, we investigated their expression in bladder tumor specimens. All examined samples expressed the M1, M2 and M3 receptor subtypes. We also found that the level of M2 transcripts, but not those of M1 or M3, significantly increased with the tumor histologic grade. In view of these results, we proceeded to investigate whether the M2 agonist Arecaidine had any effect on in vitro cell growth and migration of T24 cells, a bladder tumor cell line expressing the muscarinic receptors, including the M2 subtype. We observed that Arecaidine significantly reduced T24 and 5637 cell proliferation and migration in a concentration dependent manner. The silencing of M2 receptor by siRNA in T24 and 5637 cell lines showed the inability of Arecaidine (100 μM) to inhibit cell proliferation after 48 hours, whereas the use of M1 and M3 antagonists in T24 appeared not to counteract the Arecaidine effect, suggesting that the inhibition of cell proliferation was directly dependent on M2 receptor activation. These data suggest that M2 muscarinic receptors may play a relevant role in bladder cancer and represent a new attractive therapeutic target. PMID:25482946

  9. A novel role for calmodulin: Ca2+-independent inhibition of type-1 inositol trisphosphate receptors.

    PubMed Central

    Cardy, T J; Taylor, C W

    1998-01-01

    Calmodulin inhibits both inositol 1,4,5-trisphosphate (IP3) binding to, and IP3-evoked Ca2+ release by, cerebellar IP3 receptors [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U. S.A. 94, 11627-11632]. In the present study, full-length rat type-1 and -3 IP3 receptors were expressed at high levels in insect Spodoptera frugiperda 9 cells and the effects of calmodulin were examined. In the absence of Ca2+, calmodulin caused a concentration-dependent and reversible inhibition of [3H]IP3 binding to type-1 IP3 receptors by decreasing their apparent affinity for IP3. The effect was not reproduced by high concentrations of troponin C, parvalbumin or S-100. Increasing the medium free [Ca2+] ([Ca2+]m) inhibited [3H]IP3 binding to type-1 receptors, but the further inhibition caused by a submaximal concentration of calmodulin was similar at each [Ca2+]m. In the absence of Ca2+, 125I-calmodulin bound to a single site on each type-1 receptor subunit and to an additional site in the presence of Ca2+. There was no detectable binding of 125I-calmodulin to type-3 receptors and binding of [3H]IP3 was insensitive to calmodulin at all [Ca2+]m. Both peptide and conventional Ca2+-calmodulin antagonists affected neither [3H]IP3 binding directly nor the inhibitory effect of calmodulin in the absence of Ca2+, but each caused a [Ca2+]m-dependent reversal of the inhibition of [3H]IP3 binding caused by calmodulin. Camstatin, a peptide that binds to calmodulin equally well in the presence or absence of Ca2+, reversed the inhibitory effects of calmodulin on [3H]IP3 binding at all [Ca2+]m. We conclude that calmodulin specifically inhibits [3H]IP3 binding to type-1 IP3 receptors: the first example of a protein regulated by calmodulin in an entirely Ca2+-independent manner. Inhibition of type-1 IP3 receptors by calmodulin may dynamically regulate their sensitivity to IP3 in response to the changes in cytosolic free calmodulin concentration thought to accompany stimulation

  10. AMPA-Kainate Receptor Inhibition Promotes Neurologic Recovery in Premature Rabbits with Intraventricular Hemorrhage

    PubMed Central

    Dohare, Preeti; Zia, Muhammad T.; Ahmed, Ehsan; Ahmed, Asad; Yadala, Vivek; Schober, Alexandra L.; Ortega, Juan Alberto; Kayton, Robert; Ungvari, Zoltan; Mongin, Alexander A.

    2016-01-01

    Intraventricular hemorrhage (IVH) in preterm infants leads to cerebral inflammation, reduced myelination of the white matter, and neurological deficits. No therapeutic strategy exists against the IVH-induced white matter injury. AMPA-kainate receptor induced excitotoxicity contributes to oligodendrocyte precursor cell (OPC) damage and hypomyelination in both neonatal and adult models of brain injury. Here, we hypothesized that IVH damages white matter via AMPA receptor activation, and that AMPA-kainate receptor inhibition suppresses inflammation and restores OPC maturation, myelination, and neurologic recovery in preterm newborns with IVH. We tested these hypotheses in a rabbit model of glycerol-induced IVH and evaluated the expression of AMPA receptors in autopsy samples from human preterm infants. GluR1-GluR4 expressions were comparable between preterm humans and rabbits with and without IVH. However, GluR1 and GluR2 levels were significantly lower in the embryonic white matter and germinal matrix relative to the neocortex in both infants with and without IVH. Pharmacological blockade of AMPA-kainate receptors with systemic NBQX, or selective AMPA receptor inhibition by intramuscular perampanel restored myelination and neurologic recovery in rabbits with IVH. NBQX administration also reduced the population of apoptotic OPCs, levels of several cytokines (TNFα, IL-β, IL-6, LIF), and the density of Iba1+ microglia in pups with IVH. Additionally, NBQX treatment inhibited STAT-3 phosphorylation, but not astrogliosis or transcription factors regulating gliosis. Our data suggest that AMPA-kainate receptor inhibition alleviates OPC loss and IVH-induced inflammation and restores myelination and neurologic recovery in preterm rabbits with IVH. Therapeutic use of FDA-approved perampanel treatment might enhance neurologic outcome in premature infants with IVH. SIGNIFICANCE STATEMENT Intraventricular hemorrhage (IVH) is a major complication of prematurity and a large number

  11. Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin.

    PubMed

    Haga, K; Tsuga, H; Haga, T

    1997-02-11

    Agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) or rhodopsin by G protein-coupled receptor kinase 2 (GRK2) was found to be inhibited by calmodulin in a Ca2+-dependent manner. The phosphorylation was fully inhibited in the absence of G protein betagamma subunits and partially inhibited in the presence of betagamma subunits. The dose-response curve for stimulation by betagamma subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of betagamma subunits by addition of Ca2+-calmodulin. The phosphorylation by GRK2 of a glutathione S-transferase fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptors (I3-GST) was not affected by Ca2+-calmodulin in the presence or absence of betagamma subunits, but the agonist-dependent stimulation of I3-GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of betagamma subunits was suppressed by Ca2+-calmodulin. These results indicate that Ca2+-calmodulin does not directly interact with the catalytic site of GRK2 but inhibits the kinase activity of GRK2 by interfering with the activation of GRK2 by agonist-bound m2 receptors and G protein betagamma subunits. In agreement with the assumption that GRK2 activity is suppressed by the increase in intracellular Ca2+, the sequestration of m2 receptors expressed in Chinese hamster ovary cells was found to be attenuated by the treatment with a Ca2+ ionophore, A23187.

  12. Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission.

    PubMed

    Martire, Alberto; Tebano, Maria Teresa; Chiodi, Valentina; Ferreira, Samira G; Cunha, Rodrigo A; Köfalvi, Attila; Popoli, Patrizia

    2011-01-01

    An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.

  13. Voltage-dependent inhibition of recombinant NMDA receptor-mediated currents by 5-hydroxytryptamine

    PubMed Central

    Kloda, Anna; Adams, David J

    2005-01-01

    The effect of 5-HT and related indolealkylamines on heteromeric recombinant NMDA receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp recording technique. In the absence of external Mg2+ ions, 5-HT inhibited NMDA receptor-mediated currents in a concentration-dependent manner. The inhibitory effect of 5-HT was independent of the NR1a and NR2 subunit combination. The inhibition of glutamate-evoked currents by 5-HT was use- and voltage-dependent. The voltage sensitivity of inhibition for NR1a+NR2 subunit combinations by 5-HT was similar, exhibiting an e-fold change per ∼20 mV, indicating that 5-HT binds to a site deep within the membrane electric field. The inhibition of the open NMDA receptor by external Mg2+ and 5-HT was not additive, suggesting competition between Mg2+ and 5-HT for a binding site in the NMDA receptor channel. The concentration-dependence curves for 5-HT and 5-methoxytryptamine (5-MeOT) inhibition of NMDA receptor-mediated currents are shifted to the right in the presence of external Mg2+. The related indolealkylamines inhibited glutamate-evoked currents with the following order of inhibitory potency: 5-MeOT=5-methyltryptamine>tryptamine>7-methyltryptamine>5-HT≫tryptophan=melatonin. Taken together, these data suggest that 5-HT and related compounds can attenuate glutamate-mediated excitatory synaptic responses and may provide a basis for drug treatment of excitoxic neurodegeneration. PMID:15655527

  14. Cannabinoids Inhibit Insulin Receptor Signaling in Pancreatic β-Cells

    PubMed Central

    Kim, Wook; Doyle, Máire E.; Liu, Zhuo; Lao, Qizong; Shin, Yu-Kyong; Carlson, Olga D.; Kim, Hee Seung; Thomas, Sam; Napora, Joshua K.; Lee, Eun Kyung; Moaddel, Ruin; Wang, Yan; Maudsley, Stuart; Martin, Bronwen; Kulkarni, Rohit N.; Egan, Josephine M.

    2011-01-01

    OBJECTIVE Optimal glucose homeostasis requires exquisitely precise adaptation of the number of insulin-secreting β-cells in the islets of Langerhans. Insulin itself positively regulates β-cell proliferation in an autocrine manner through the insulin receptor (IR) signaling pathway. It is now coming to light that cannabinoid 1 receptor (CB1R) agonism/antagonism influences insulin action in insulin-sensitive tissues. However, the cells on which the CB1Rs are expressed and their function in islets have not been firmly established. We undertook the current study to investigate if intraislet endogenous cannabinoids (ECs) regulate β-cell proliferation and if they influence insulin action. RESEARCH DESIGN AND METHODS We measured EC production in isolated human and mouse islets and β-cell line in response to glucose and KCl. We evaluated human and mouse islets, several β-cell lines, and CB1R-null (CB1R−/−) mice for the presence of a fully functioning EC system. We investigated if ECs influence β-cell physiology through regulating insulin action and demonstrated the therapeutic potential of manipulation of the EC system in diabetic (db/db) mice. RESULTS ECs are generated within β-cells, which also express CB1Rs that are fully functioning when activated by ligands. Genetic and pharmacologic blockade of CB1R results in enhanced IR signaling through the insulin receptor substrate 2-AKT pathway in β-cells and leads to increased β-cell proliferation and mass. CB1R antagonism in db/db mice results in reduced blood glucose and increased β-cell proliferation and mass, coupled with enhanced IR signaling in β-cells. Furthermore, CB1R activation impedes insulin-stimulated IR autophosphorylation on β-cells in a Gαi-dependent manner. CONCLUSIONS These findings provide direct evidence for a functional interaction between CB1R and IR signaling involved in the regulation of β-cell proliferation and will serve as a basis for developing new therapeutic interventions to

  15. 68Ga-PSMA-11 PET Imaging of Response to Androgen Receptor Inhibition: First Human Experience.

    PubMed

    Hope, Thomas A; Truillet, Charles; Ehman, Eric C; Afshar-Oromieh, Ali; Aggarwal, Rahul; Ryan, Charles J; Carroll, Peter R; Small, Eric J; Evans, Michael J

    2017-01-01

    The purpose of this work was to evaluate the effect of androgen receptor (AR) inhibition on prostate-specific membrane antigen (PSMA) uptake imaged using (68)Ga-PSMA-11 PET in a mouse xenograft model and in a patient with castration-sensitive prostate cancer.

  16. Garlic (Allium sativum) Extracts Inhibits Lipopolysaccharide-Induced Toll-Like Receptor 4 Dimerization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Garlic has been used as a folk medicine for a long history. Numerous studies demonstrated that garlic extracts and its sulfur-containing compounds inhibit nuclear factor-kappa B (NF-kB) activation induced by various receptor agonist including lipopolysaccharide (LPS). These effects suggest that garl...

  17. Atypical effect of dopamine in modulating the functional inhibition of NMDA receptors of cultured retina cells.

    PubMed

    Do Nascimento, J L; Kubrusly, R C; Reis, R A; De Mello, M C; De Mello, F G

    1998-02-05

    Cultured retina cells released accumulated [3H]GABA (gamma-aminobutyric acid) when stimulated by L-glutamate, N-methyl-D-aspartate (NMDA) and kainate. In the absence of Mg2+, dopamine at 200 microM (IC50 60 microM), inhibited in more than 50% the release of [3H]GABA induced by L-glutamate and NMDA, but not by kainate. This effect was not blocked by the D1-like dopamine receptor antagonist, R-(+)-7-chloro-8-hydroxy-3-methyl- -phenyl-2,3,4,5-tetrahydro- H-3-benzazepine hydrochloride (SCH 23390), neither by haloperidol nor spiroperidol (dopamine D2-like receptor antagonists). The dopamine D1-like receptor agonist R(+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,diol hydrochloride (SKF 38393) at 50 microM, but not its enantiomer, also inhibited the release of [3H]GABA induced by NMDA, but not by kainate; an effect that was not prevented by the antagonists mentioned above. (+/-)-6-Chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin e hydrobromide (SKF 812497) had no effect. Neither 8BrcAMP (5 mM) nor forskolin (10 microM) inhibited the release of [3H]GABA. Our results suggest that dopamine and (+)-SKF 38393 inhibit the glutamate and NMDA-evoked [3H]GABA release through mechanisms that seem not to involve known dopaminergic receptor systems.

  18. Activation and inhibition of erythropoietin receptor function: role of receptor dimerization.

    PubMed Central

    Watowich, S S; Hilton, D J; Lodish, H F

    1994-01-01

    Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization. Images PMID:8196600

  19. Activation of GABA(A) receptors in subthalamic neurons in vitro: properties of native receptors and inhibition mechanisms.

    PubMed

    Baufreton, J; Garret, M; Dovero, S; Dufy, B; Bioulac, B; Taupignon, A

    2001-07-01

    all intact neurons. In neurons held in whole cell configuration, membrane potential hyperpolarized by -10 mV whilst input resistance decreased by 50%, indicating powerful membrane shunting. Muscimol never induced burst firing, even in neurons that exhibited the capacity of switching from regular- to burst-firing mode. These molecular and functional data indicate that native subthalamic GABA(A) receptors do not contain the epsilon protein and activation of GABA(A) receptors induces membrane shunting, which is essential for firing inhibition but prevents switching to burst-firing. They suggest that the STN, like many other parts of the brain, has the physiological and structural features of the widely expressed GABA(A) receptors consisting of alphabetagamma subunits.

  20. Helicobacter pylori lipopolysaccharide inhibition of gastric mucosal laminin receptor: effect of sulglycotide.

    PubMed

    Piotrowski, J; Czajkowski, A; Yotsumoto, F; Slomiany, A; Slomiany, B L

    1993-11-01

    1. The effect of cell-wall lipopolysaccharide from Helicobacter pylori, a bacterium implicated in the etiology of gastric disease, on the gastric mucosal laminin-receptor was investigated. 2. The receptor, isolated from gastric epithelial cell membrane by affinity chromatography on laminin-coupled Sepharose, was radioiodinated and incorporated into liposomes which exhibited specific affinity towards laminin-coated surface. 3. The binding of liposomal receptor to laminin-coated surface was inhibited by H. pylori lipopolysaccharide, which at 50 micrograms/ml caused a nearly complete (97%) inhibition in binding. 4. The inhibitory effect of the lipopolysaccharide was prevented by a cytoprotective agent, sulglycotide, that evoked a 92% restoration in binding at 40 micrograms/ml. 5. The results demonstrate that through its lipopolysaccharide H. pylori is capable of disrupting the gastric mucosal integrity and that this detrimental effect could be successfully countered by sulglycotide.

  1. Tumor necrosis factor-alpha inhibits pre-osteoblast differentiation through its type-1 receptor.

    PubMed

    Abbas, Sabiha; Zhang, Yan-Hong; Clohisy, John C; Abu-Amer, Yousef

    2003-04-01

    Tumor necrosis factor-alpha (TNF) is a pro-inflammatory cytokine with a profound role in many skeletal diseases. The cytokine has been described as a mediator of bone loss in osteolysis and other inflammatory bone diseases. In addition to its known bone resorptive action, TNF reduces bone formation by inhibiting osteoblast differentiation. Using primary and transformed osteoblastic cells, we first document that TNF inhibits expression of alkaline phosphatase and matrix deposition, both considered markers of osteoblast differentiation. The effects are dose- and time-dependent. Core-binding factor A1 (cbfa1) is a transcription factor critical for osteoblast differentiation, and we show here that it is activated by the osteoblast differentiation agent, beta-glycerophosphate. Therefore, we investigated whether the inhibitory effects of TNF were associated with altered activity of this transcription factor. Using retardation assays, we show that TNF significantly inhibits cbfal activation by beta-glycerophosphate, manifested by reduced DNA-binding activity. Next, we turned to determine the signaling pathway by which TNF inhibits osteoblast differentiation. Utilizing animals lacking individual TNF receptors, we document that TNFr1 is required for transmitting the cytokine's inhibitory effect. In the absence of this receptor, TNF failed to impact all osteoblast differentiation markers tested. In summary, TNF blocks expression of osteoblast differentiation markers and inhibits beta-glycerophosphate-induced activation of the osteoblast differentiation factor cbfa1. Importantly, these effects are mediated via a mechanism requiring the TNF type-1 receptor.

  2. Angiotensin II AT1 receptor antagonists inhibit platelet adhesion and aggregation by nitric oxide release.

    PubMed

    Kalinowski, Leszek; Matys, Tomasz; Chabielska, Ewa; Buczko, Włodzimierz; Malinski, Tadeusz

    2002-10-01

    This study investigated the process of nitric oxide (NO) release from platelets after stimulation with different angiotensin II type 1 (AT1)-receptor antagonists and its effect on platelet adhesion and aggregation. Angiotensin II AT1-receptor antagonist-stimulated NO release in platelets was compared with that in human umbilical vein endothelial cells by using a highly sensitive porphyrinic microsensor. In vitro and ex vivo effects of angiotensin II AT1-receptor antagonists on platelet adhesion to collagen and thromboxane A2 analog U46619-induced aggregation were evaluated. Losartan, EXP3174, and valsartan alone caused NO release from platelets and endothelial cells in a dose-dependent manner in the range of 0.01 to 100 micro mol/L, which was attenuated by NO synthase inhibitor N(G)-nitro-L-arginine methyl ester. The angiotensin II AT1-receptor antagonists had more than 70% greater potency in NO release in platelets than in endothelial cells. The degree of inhibition of platelet adhesion (collagen-stimulated) and aggregation (U46619-stimulated) elicited by losartan, EXP3174, and valsartan, either in vitro or ex vivo, closely correlated with the NO levels produced by each of these drugs alone. The inhibiting effects of angiotensin II AT1-receptor antagonists on collagen-stimulated adhesion and U46619-stimulated aggregation of platelets were significantly reduced by pretreatment with N(G)-nitro-L-arginine methyl ester. Neither the AT2 receptor antagonist PD123319, the cyclooxygenase synthase inhibitor indomethacin, nor the selective thromboxane A2/prostaglandin H2 receptor antagonist SQ29,548 had any effect on angiotensin II AT1-receptor antagonist-stimulated NO release in platelets and endothelial cells. The presented studies clearly indicate a crucial role of NO in the arterial antithrombotic effects of angiotensin II AT1-receptor antagonists.

  3. Histamine H3A receptor-mediated inhibition of noradrenaline release in the mouse brain cortex.

    PubMed

    Schlicker, E; Behling, A; Lümmen, G; Göthert, M

    1992-04-01

    Mouse brain cortex slices preincubated with 3H-noradrenaline were superfused with physiological salt solution containing desipramine plus a drug with alpha 2-adrenoceptor antagonist properties, and the effects of histamine receptor ligands on the electrically (0.3 Hz) evoked tritium overflow were studied. The evoked overflow (from slices superfused with phentolamine) was inhibited by histamine (pIC35 6.53), the H3 receptor agonist R-(-)-alpha-methylhistamine (7.47) and its S-(+)-enantiomer (5.82) but not influenced by the H1 receptor agonist 2-(2-thiazolyl)-ethylamine 3.2 mumol/l and the H2 receptor agonist dimaprit 10 mumol/l. The inhibitory effect of histamine was not affected by the H1 receptor antagonist dimetindene 1 mumol/l and the H2 receptor antagonist ranitidine 10 mumol/l. The concentration-response curve of histamine (determined in the presence of rauwolscine) was shifted to the right by the H3 receptor antagonists thioperamide (apparent pA2 8.67), impromidine (7.30) and burimamide (6.82) as well as by dimaprit (6.16). The pA2 values of the four drugs were compared with their affinities for H3A and H3B binding sites in rat brain membranes (West et al. 1990 Mol Pharmacol 38:610); a significant correlation was obtained for the H3A, but not for the H3B sites. The results suggest that noradrenaline release in the mouse brain cortex is inhibited by histamine via H3A receptors and that dimaprit is an H3 receptor antagonist of moderate potency.

  4. Heterologous desensitization of opioid receptors by chemokines inhibits chemotaxis and enhances the perception of pain.

    PubMed

    Szabo, Imre; Chen, Xiao-Hong; Xin, Li; Adler, Martin W; Howard, O M Z; Oppenheim, Joost J; Rogers, Thomas J

    2002-08-06

    The chemokines use G protein-coupled receptors to regulate the migratory and proadhesive responses of leukocytes. Based on observations that G protein-coupled receptors undergo heterologous desensitization, we have examined the ability of chemokines to also influence the perception of pain by cross-desensitizing opioid G protein-coupled receptors function in vitro and in vivo. We find that the chemotactic activities of both mu- and delta-opioid receptors are desensitized following activation of the chemokine receptors CCR5, CCR2, CCR7, and CXCR4 but not of the CXCR1 or CXCR2 receptors. Furthermore, we also find that pretreatment with RANTES/CCL5, the ligand for CCR1, and CCR5 or SDF-1alpha/CXCL12, the ligand for CXCR4, followed by opioid administration into the periaqueductal gray matter of the brain results in an increased rat tail flick response to a painful stimulus. Because chemokine administration into the periaqueductal gray matter inhibits opioid-induced analgesia, we propose that the activation of proinflammatory chemokine receptors down-regulates the analgesic functions of opioid receptors, and this enhances the perception of pain at inflammatory sites.

  5. Pre-synaptic GABA receptors inhibit glutamate release through GIRK channels in rat cerebral cortex.

    PubMed

    Ladera, Carolina; del Carmen Godino, María; José Cabañero, María; Torres, Magdalena; Watanabe, Masahiko; Luján, Rafael; Sánchez-Prieto, José

    2008-12-01

    Neuronal G protein-gated inwardly rectifying potassium (GIRK) channels mediate the slow inhibitory effects of many neurotransmitters post-synaptically. However, no evidence exists that supports that GIRK channels play any role in the inhibition of glutamate release by GABA(B) receptors. In this study, we show for the first time that GABA(B) receptors operate through two mechanisms in nerve terminals from the cerebral cortex. As shown previously, GABA(B) receptors reduces glutamate release and the Ca(2+) influx mediated by N-type Ca(2+) channels in a mode insensitive to the GIRK channel blocker tertiapin-Q and consistent with direct inhibition of this voltage-gated Ca(2+) channel. However, by means of weak stimulation protocols, we reveal that GABA(B) receptors also reduce glutamate release mediated by P/Q-type Ca(2+) channels, and that these responses are reversed by the GIRK channel blocker tertiapin-Q. Consistent with the functional interaction between GABA(B) receptors and GIRK channels at nerve terminals we demonstrate by immunogold electron immunohistochemistry that pre-synaptic boutons of asymmetric synapses co-express GABA(B) receptors and GIRK channels, thus suggesting that the functional interaction of these two proteins, found at the post-synaptic level, also occurs at glutamatergic nerve terminals.

  6. Histamine H3 receptor activation inhibits neurogenic sympathetic vasoconstriction in porcine nasal mucosa.

    PubMed

    Varty, LoriAnn M; Hey, John A

    2002-10-11

    Histamine release from mast cells is a primary mediator of rhinorrhea, nasal mucosal swelling, increased secretion, sneezing, pruritus and congestion that occur in allergic rhinitis. It is well known that histamine H(1) receptor antagonists inhibit the itch and rhinorhea, but do not block the allergic nasal congestion. A growing body of evidence shows that in addition to histamine H(1) receptors, activation of H(3) receptors may contribute to the procongestant nasal actions of histamine. Activation of the prejunctional histamine H(3) receptor modulates sympathetic control of nasal vascular tone and resistance. The present study was conducted to further characterize the role of histamine H(3) receptors on neurogenic sympathetic vascular contractile responses in isolated porcine nasal turbinate mucosa. We presently found that the histamine H(3) receptor agonist, (R)-alpha-methylhistamine (10-1000 nM), inhibited electrical field stimulation-induced sympathetic vasomotor contractions in a concentration-dependent fashion. Pretreatment with either of the selective histamine H(3) receptor antagonists, thioperamide and clobenpropit, blocked the sympathoinhibitory effect of (R)-alpha-methylhistamine in porcine turbinate mucosa. The effect of compound 48/80, an agent that elicits the release of endogenous histamine from mast cells on nasal sympathetic contractile responses, was also tested. The action of compound 48/80 to release mast cell-derived histamine in the nose mimics many of the nasal responses associated with allergic rhinitis, extravascular leakage and decreased nasal patency. We presently found that compound 48/80 also inhibited the electrical field stimulation-induced sympathetic response. Pretreatment with the H(3) receptor antagonist clobenpropit blocked the sympathoinhibitory action of compound 48/80 on sympathetic contractile responses in nasal mucosa. Taken together, these studies indicate that histamine H(3) receptors modulate vascular contractile

  7. Manganese inhibits NMDA receptor channel function: implications to psychiatric and cognitive effects.

    PubMed

    Guilarte, Tomás R; Chen, Ming-Kai

    2007-11-01

    Humans exposed to excess levels of manganese (Mn(2+)) express psychiatric problems and deficits in attention and learning and memory. However, there is a paucity of knowledge on molecular mechanisms by which Mn(2+) produces such effects. We now report that Mn(2+) is a potent inhibitor of [(3)H]-MK-801 binding to the NMDA receptor channel in rat neuronal membrane preparations. The inhibition of [(3)H]-MK-801 to the NMDA receptor channel by Mn(2+) was activity-dependent since Mn(2+) was a more potent inhibitor in the presence of the NMDA receptor co-agonists glutamate and glycine (K(i)=35.9+/-3.1 microM) than in their absence (K(i)=157.1+/-6.5 microM). We also show that Mn(2+) is a NMDA receptor channel blocker since its inhibition of [(3)H]-MK-801 binding to the NMDA receptor channel is competitive in nature. That is, Mn(2+) significantly increased the affinity constant (K(d)) with no significant effect on the maximal number of [(3)H]-MK-801 binding sites (B(max)). Under stimulating conditions, Mn(2+) was equipotent in inhibiting [(3)H]-MK-801 binding to NMDA receptors expressed in neuronal membrane preparations from different brain regions. However, under basal, non-stimulated conditions, Mn(2+) was more potent in inhibiting NMDA receptors in the cerebellum than other brain regions. We have previously shown that chronic Mn(2+) exposure in non-human primates increases Cu(2+), but not zinc or iron concentrations in the basal ganglia [Guilarte TR, Chen M-K, McGlothan JL, Verina T, Wong DF, Zhou Y, Alexander M, Rohde CA, Syversen T, Decamp E, Koser AJ, Fritz S, Gonczi H, Anderson DW, Schneider JS. Nigrostriatal dopamine system dysfunction and subtle motor deficits in manganese-exposed non-human primates. Exp Neurol 2006a;202:381-90]. Therefore, we also tested the inhibitory effects of Cu(2+) on [(3)H]-MK-801 binding to the NMDA receptor channel. The data shows that Cu(2+) in the presence of glutamate and glycine is a more potent inhibitor of the NMDA receptor than Mn(2

  8. Feed-forward inhibition of androgen receptor activity by glucocorticoid action in human adipocytes.

    PubMed

    Hartig, Sean M; He, Bin; Newberg, Justin Y; Ochsner, Scott A; Loose, David S; Lanz, Rainer B; McKenna, Neil J; Buehrer, Benjamin M; McGuire, Sean E; Marcelli, Marco; Mancini, Michael A

    2012-09-21

    We compared transcriptomes of terminally differentiated mouse 3T3-L1 and human adipocytes to identify cell-specific differences. Gene expression and high content analysis (HCA) data identified the androgen receptor (AR) as both expressed and functional, exclusively during early human adipocyte differentiation. The AR agonist dihydrotestosterone (DHT) inhibited human adipocyte maturation by downregulation of adipocyte marker genes, but not in 3T3-L1. It is interesting that AR induction corresponded with dexamethasone activation of the glucocorticoid receptor (GR); however, when exposed to the differentiation cocktail required for adipocyte maturation, AR adopted an antagonist conformation and was transcriptionally repressed. To further explore effectors within the cocktail, we applied an image-based support vector machine (SVM) classification scheme to show that adipocyte differentiation components inhibit AR action. The results demonstrate human adipocyte differentiation, via GR activation, upregulates AR but also inhibits AR transcriptional activity.

  9. Orphan Nuclear Receptor DAX-1 Acts as a Novel Corepressor of Liver X Receptor α and Inhibits Hepatic Lipogenesis*

    PubMed Central

    Nedumaran, Balachandar; Kim, Gwang Sik; Hong, Sungpyo; Yoon, Young-Sil; Kim, Yong-Hoon; Lee, Chul-Ho; Lee, Young Chul; Koo, Seung-Hoi; Choi, Hueng-Sik

    2010-01-01

    DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is a member of the nuclear receptor superfamily that can repress diverse nuclear receptors and has a key role in adreno-gonadal development. Our previous report has demonstrated that DAX-1 can inhibit hepatocyte nuclear factor 4α transactivity and negatively regulate gluconeogenic gene expression (Nedumaran, B., Hong, S., Xie, Y. B., Kim, Y. H., Seo, W. Y., Lee, M. W., Lee, C. H., Koo, S. H., and Choi, H. S. (2009) J. Biol. Chem. 284, 27511–27523). Here, we further expand the role of DAX-1 in hepatic energy metabolism. Transfection assays have demonstrated that DAX-1 can inhibit the transcriptional activity of nuclear receptor liver X receptor α (LXRα). Physical interaction between DAX-1 and LXRα was confirmed Immunofluorescent staining in mouse liver shows that LXRα and DAX-1 are colocalized in the nucleus. Domain mapping analysis shows that the entire region of DAX-1 is involved in the interaction with the ligand binding domain region of LXRα. Competition analyses demonstrate that DAX-1 competes with the coactivator SRC-1 for repressing LXRα transactivity. Chromatin immunoprecipitation assay showed that endogenous DAX-1 recruitment on the SREBP-1c gene promoter was decreased in the presence of LXRα agonist. Overexpression of DAX-1 inhibits T7-induced LXRα target gene expression, whereas knockdown of endogenous DAX-1 significantly increases T7-induced LXRα target gene expression in HepG2 cells. Finally, overexpression of DAX-1 in mouse liver decreases T7-induced LXRα target gene expression, liver triglyceride level, and lipid accumulation. Overall, this study suggests that DAX-1, a novel corepressor of LXRα, functions as a negative regulator of lipogenic enzyme gene expression in liver. PMID:20080977

  10. Sigma receptor ligand N,N'-di-(ortho-tolyl)guanidine inhibits release of acetylcholine in the guinea pig ileum.

    PubMed

    Cambell, B G; Keana, J F; Weber, E

    1991-11-26

    The inhibition of stimulated contractions of the guinea pig ileum longitudinal muscle/myenteric plexus preparation by sigma receptor ligands has been previously described. In this study, the stimulated release of [3H]acetylcholine from cholinergic nerve terminals in this same preparation was monitored in the presence and absence of sigma receptor ligands. N,N'-Di-(orthotolyl)guanidine (DTG) and other compounds selective for the sigma receptor inhibited stimulated [3H]acetylcholine release. These results suggest that their inhibition of stimulated contractions in this preparation was mediated by inhibition of acetylcholine release.

  11. Nitric oxide reversibly inhibits the epidermal growth factor receptor tyrosine kinase.

    PubMed Central

    Estrada, C; Gómez, C; Martín-Nieto, J; De Frutos, T; Jiménez, A; Villalobo, A

    1997-01-01

    Although it has been demonstrated that NO inhibits the proliferation of different cell types, the mechanisms of its anti-mitotic action are not well understood. In this work we have studied the possible interaction of NO with the epidermal growth factor receptor (EGFR), using transfected fibroblasts which overexpress the human EGFR. The NO donors S-nitroso-N-acetylpenicillamine (SNAP), 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NO) and N-¿4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl¿propane -1, 3-diamine (DETA-NO) inhibited DNA synthesis of fibroblasts growing in the presence of fetal calf serum, epidermal growth factor (EGF) or EGF plus insulin, as assessed by [methyl-3H]thymidine incorporation. Neither 8-bromo-cGMP nor the cGMP-phosphodiesterase inhibitor zaprinast mimicked this effect, suggesting that NO is unlikely to inhibit cell proliferation via a cGMP-dependent pathway. SNAP, DEA-NO and DETA-NO also inhibited the transphosphorylation of the EGFR and its tyrosine kinase activity toward the exogenous substrate poly-l-(Glu-Tyr), as measured in permeabilized cells using [gamma-32P]ATP as phosphate donor. In contrast, 3-[morpholinosydnonimine hydrochloride] (SIN-1), a peroxynitrite-forming compound, did not significantly inhibit either DNA synthesis or the EGFR tyrosine kinase activity. The inhibitory action of DEA-NO on the EGFR tyrosine kinase was prevented by haemoglobin, an NO scavenger, but not by superoxide dismutase, and was reversed by dithiothreitol. The binding of EGF to its receptor was unaffected by DEA-NO. The inhibitory action of DEA-NO on the EGF-dependent transphosphorylation of the receptor was also demonstrated in intact cells by immunoblot analysis using an anti-phosphotyrosine antibody. Taken together, these results suggest that NO, but not peroxynitrite, inhibits in a reversible manner the EGFR tyrosine kinase activity by S-nitrosylation of the receptor. PMID:9291107

  12. Ca2+ channel inhibition by endomorphins via the cloned mu-opioid receptor expressed in NG108-15 cells.

    PubMed

    Mima, H; Morikawa, H; Fukuda, K; Kato, S; Shoda, T; Mori, K

    1997-12-11

    Endomorphin-1 and -2, recently isolated endogenous peptides specific for the mu-opioid receptor, inhibited Ca2+ channel currents with EC50 of 6 and 9 nM, respectively, in NG108-15 cells transformed to express the cloned rat mu-opioid receptor. On the other hand, they elicited no response in nontransfected NG108-15 cells. It is concluded that endomorphin-1 and -2 induce Ca2+ channel inhibition by selectively activating the mu-opioid receptor.

  13. Ethanol-Induced Motor Impairment Mediated by Inhibition of α7 Nicotinic Receptors

    PubMed Central

    McDaid, John; Abburi, Chandrika; Wolfman, Shannon L.; Gallagher, Keith

    2016-01-01

    Nicotine and ethanol (EtOH) are among the most widely co-abused substances, and nicotinic acetylcholine receptors (nAChRs) contribute to the behavioral effects of both drugs. Along with their role in addiction, nAChRs also contribute to motor control circuitry. The α7 nAChR subtype is highly expressed in the laterodorsal tegmental nucleus (LDTg), a brainstem cholinergic center that contributes to motor performance through its projections to thalamic motor relay centers, including the mediodorsal thalamus. We demonstrate that EtOH concentrations just above the legal limits for intoxication in humans can inhibit α7 nAChRs in LDTg neurons from rats. This EtOH-induced inhibition is mediated by a decrease in cAMP/PKA signaling. The α7 nAChR-positive allosteric modulator PNU120596 [N-(5-chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea], which interferes with receptor desensitization, completely eliminated EtOH modulation of these receptors. These data suggest that EtOH inhibits α7 responses through a PKA-dependent enhancement of receptor desensitization. EtOH also inhibited the effects of nicotine at presynaptic α7 nAChRs on glutamate terminals in the mediodorsal thalamus. In vivo administration of PNU120596 either into the cerebral ventricles or directly into the mediodorsal thalamus attenuated EtOH-induced motor impairment. Thus, α7 nAChRs are likely important mediators of the motor impairing effects of moderate EtOH consumption. SIGNIFICANCE STATEMENT The motor-impairing effects of ethanol contribute to intoxication-related injury and death. Here we explore the cellular and neural circuit mechanisms underlying ethanol-induced motor impairment. Physiologically relevant concentrations of ethanol inhibit activity of a nicotinic receptor subtype that is expressed in brain areas associated with motor control. That receptor inhibition is mediated by decreased receptor phosphorylation, suggesting an indirect modulation of cell signaling pathways to achieve

  14. Prostacyclin receptor-independent inhibition of phospholipase C activity by non-prostanoid prostacyclin mimetics

    PubMed Central

    Chow, Kevin B S; Wong, Yung H; Wise, Helen

    2001-01-01

    Chinese hamster ovary (CHO) cells were transiently transfected with the mouse prostacyclin (mIP) receptor to examine IP agonist-mediated stimulation of [3H]-cyclic AMP and [3H]-inositol phosphate production.The prostacyclin analogues, cicaprost, iloprost, carbacyclin and prostaglandin E1, stimulated adenylyl cyclase activity with EC50 values of 5, 6, 25 and 95 nM, respectively. These IP agonists also stimulated the phospholipase C pathway with 10 – 40 fold lower potency than stimulation of adenylyl cyclase.The non-prostanoid prostacyclin mimetics, octimibate, BMY 42393 and BMY 45778, also stimulated adenylyl cyclase activity, with EC50 values of 219, 166 and 398 nM, respectively, but failed to stimulate [3H]-inositol phosphate production.Octimibate, BMY 42393 and BMY 45778 inhibited iloprost-stimulated [3H]-inositol phosphate production in a non-competitive manner.Activation of the endogenously-expressed P2 purinergic receptor by ATP led to an increase in [3H]-inositol phosphate production which was inhibited by the non-prostanoid prostacyclin mimetics in non-transfected CHO cells. Prostacyclin analogues and other prostanoid receptor ligands failed to inhibit ATP-stimulated [3H]-inositol phosphate production.A comparison between the IP receptor-specific non-prostanoid ONO-1310 and the structurally-related EP3 receptor-specific agonist ONO-AP-324, indicated that the inhibitory effect of non-prostanoids was specific for those compounds known to activate IP receptors.The non-prostanoid prostacyclin mimetics also inhibited phospholipase C activity when stimulated by constitutively-active mutant GαqRC, Gα14RC and Gα16QL transiently expressed in CHO cells. These drugs did not inhibit adenylyl cyclase activity when stimulated by the constitutively-active mutant GαsQL.These results suggest that non-prostanoid prostacyclin mimetics can specifically inhibit [3H]-inositol phosphate production by targeting Gq/11 and/or phospholipase C in CHO cells, and

  15. The endogenous cannabinoid anandamide inhibits alpha7 nicotinic acetylcholine receptor-mediated responses in Xenopus oocytes.

    PubMed

    Oz, Murat; Ravindran, Arippa; Diaz-Ruiz, Oscar; Zhang, Li; Morales, Marisela

    2003-09-01

    The effect of the endogenous cannabinoid ligand anandamide on the function of the cloned alpha7 subunit of the nicotinic acetylcholine (ACh) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Anandamide reversibly inhibited nicotine (10 microM) induced-currents in a concentration-dependent manner (10 nM to 30 microM), with an IC50 value of 229.7 +/- 20.4 nM. The effect of anandamide was neither dependent on the membrane potential nor meditated by endogenous Ca2+ dependent Cl- channels since it was unaffected by intracellularly injected BAPTA and perfusion with Ca2+-free bathing solution containing 2 mM Ba2+. Anandamide decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on nicotinic acetylcholine (nACh) alpha7 receptors. This effect was not mediated by CB1 or CB2 receptors, as neither the selective CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) nor CB2 receptor antagonist N-((1S)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528) reduced the inhibition by anandamide. In addition, inhibition of nicotinic responses by anandamide was not sensitive to either pertussis toxin treatment or to the membrane permeable cAMP analog 8-Br-cAMP (0.2 mM). Inhibitors of enzymes involved in anandamide metabolism including phenylmethylsulfonyl fluoride, superoxide dismutase, and indomethacin, or the anandamide transport inhibitor AM404 did not prevent anandamide inhibition of nicotinic responses, suggesting that anandamide itself acted on nicotinic receptors. In conclusion, these results demonstrate that the endogenous cannabinoid anandamide inhibits the function of nACh alpha7 receptors expressed in Xenopus oocytes in a cannabinoid receptor-independent and

  16. Potentiation of morphine-induced mechanical antinociception by σ₁ receptor inhibition: role of peripheral σ₁ receptors.

    PubMed

    Sánchez-Fernández, Cristina; Nieto, Francisco Rafael; González-Cano, Rafael; Artacho-Cordón, Antonia; Romero, Lucía; Montilla-García, Ángeles; Zamanillo, Daniel; Baeyens, José Manuel; Entrena, José Manuel; Cobos, Enrique José

    2013-07-01

    We studied the modulation of morphine-induced mechanical antinociception and side effects by σ₁ receptor inhibition. Both wild-type (WT) and σ₁ receptor knockout (σ₁-KO) mice showed similar responses to paw pressure (100-600 g). The systemic (subcutaneous) or local (intraplantar) administration of σ₁ antagonists (BD-1063, BD-1047, NE-100 and S1RA) was devoid of antinociceptive effects in WT mice. However, σ₁-KO mice exhibited an enhanced mechanical antinociception in response to systemic morphine (1-16 mg/kg). Similarly, systemic treatment of WT mice with σ₁ antagonists markedly potentiated morphine-induced antinociception, and its effects were reversed by the selective σ₁ agonist PRE-084. Although the local administration of morphine (50-200 μg) was devoid of antinociceptive effects in WT mice, it induced dose-dependent antinociception in σ₁-KO mice. This effect was limited to the injected paw. Enhancement of peripheral morphine antinociception was replicated in WT mice locally co-administered with σ₁ antagonists and the opioid. None of the σ₁ antagonists tested enhanced morphine-antinociception in σ₁-KO mice, confirming a σ₁-mediated action. Morphine-induced side-effects (hyperlocomotion and inhibition of gastrointestinal transit) were unaltered in σ₁-KO mice. These results cannot be explained by a direct interaction of σ₁ ligands with μ-opioid receptors or adaptive changes of μ-receptors in σ₁-KO mice, given that [(3)H]DAMGO binding in forebrain, spinal cord, and hind-paw skin membranes was unaltered in mutant mice, and none of the σ₁ drugs tested bound to μ-opioid receptors. These results show that σ₁ receptor inhibition potentiates morphine-induced mechanical analgesia but not its acute side effects, and that this enhanced analgesia can be induced at peripheral level.

  17. Epidermal growth factor receptor endocytic traffic perturbation by phosphatidate phosphohydrolase inhibition: new strategy against cancer.

    PubMed

    Shaughnessy, Ronan; Retamal, Claudio; Oyanadel, Claudia; Norambuena, Andrés; López, Alejandro; Bravo-Zehnder, Marcela; Montecino, Fabian J; Metz, Claudia; Soza, Andrea; González, Alfonso

    2014-05-01

    Epidermal growth factor receptor (EGFR) exaggerated (oncogenic) function is currently targeted in cancer treatment with drugs that block receptor ligand binding or tyrosine kinase activity. Because endocytic trafficking is a crucial regulator of EGFR function, its pharmacological perturbation might provide a new anti-tumoral strategy. Inhibition of phosphatidic acid (PA) phosphohydrolase (PAP) activity has been shown to trigger PA signaling towards type 4 phosphodiesterase (PDE4) activation and protein kinase A inhibition, leading to internalization of empty/inactive EGFR. Here, we used propranolol, its l- and d- isomers and desipramine as PAP inhibitors to further explore the effects of PAP inhibition on EGFR endocytic trafficking and its consequences on EGFR-dependent cancer cell line models. PAP inhibition not only made EGFR inaccessible to stimuli but also prolonged the signaling lifetime of ligand-activated EGFR in recycling endosomes. Strikingly, such endocytic perturbations applied in acute/intermittent PAP inhibitor treatments selectively impaired cell proliferation/viability sustained by an exaggerated EGFR function. Phospholipase D inhibition with FIPI (5-fluoro-2-indolyl des-chlorohalopemide) and PDE4 inhibition with rolipram abrogated both the anti-tumoral and endocytic effects of PAP inhibition. Prolonged treatments with a low concentration of PAP inhibitors, although without detectable endocytic effects, still counteracted cell proliferation, induced apoptosis and decreased anchorage-independent growth of cells bearing EGFR oncogenic influences. Overall, our results show that PAP inhibitors can counteract EGFR oncogenic traits, including receptor overexpression or activating mutations resistant to current tyrosine kinase inhibitors, perturbing EGFR endocytic trafficking and perhaps other as yet unknown processes, depending on treatment conditions. This puts PAP activity forward as a new suitable target against EGFR-driven malignancy.

  18. Inhibition of lysophosphatidic acid receptors 1 and 3 attenuates atherosclerosis development in LDL-receptor deficient mice

    PubMed Central

    Kritikou, Eva; van Puijvelde, Gijs H. M.; van der Heijden, Thomas; van Santbrink, Peter J.; Swart, Maarten; Schaftenaar, Frank H.; Kröner, Mara J.; Kuiper, Johan; Bot, Ilze

    2016-01-01

    Lysophosphatidic acid (LPA) is a natural lysophospholipid present at high concentrations within lipid-rich atherosclerotic plaques. Upon local accumulation in the damaged vessels, LPA can act as a potent activator for various types of immune cells through its specific membrane receptors LPA1/3. LPA elicits chemotactic, pro-inflammatory and apoptotic effects that lead to atherosclerotic plaque progression. In this study we aimed to inhibit LPA signaling by means of LPA1/3 antagonism using the small molecule Ki16425. We show that LPA1/3 inhibition significantly impaired atherosclerosis progression. Treatment with Ki16425 also resulted in reduced CCL2 production and secretion, which led to less monocyte and neutrophil infiltration. Furthermore, we provide evidence that LPA1/3 blockade enhanced the percentage of non-inflammatory, Ly6Clow monocytes and CD4+ CD25+ FoxP3+ T-regulatory cells. Finally, we demonstrate that LPA1/3 antagonism mildly reduced plasma LDL cholesterol levels. Therefore, pharmacological inhibition of LPA1/3 receptors may prove a promising approach to diminish atherosclerosis development. PMID:27883026

  19. Hispolon inhibits the growth of estrogen receptor positive human breast cancer cells through modulation of estrogen receptor alpha

    SciTech Connect

    Jang, Eun Hyang; Jang, Soon Young; Cho, In-Hye; Hong, Darong; Jung, Bom; Park, Min-Ju; Kim, Jong-Ho

    2015-08-07

    Human estrogen receptor α (ERα) is a nuclear transcription factor that is a major therapeutic target in breast cancer. The transcriptional activity of ERα is regulated by certain estrogen-receptor modulators. Hispolon, isolated from Phellinus linteus, a traditional medicinal mushroom called Sanghwang in Korea, has been used to treat various pathologies, such as inflammation, gastroenteric disorders, lymphatic diseases, and cancers. In this latter context, Hispolon has been reported to exhibit therapeutic efficacy against various cancer cells, including melanoma, leukemia, hepatocarcinoma, bladder cancer, and gastric cancer cells. However, ERα regulation by Hispolon has not been reported. In this study, we investigated the effects of Hispolon on the growth of breast cancer cells. We found that Hispolon decreased expression of ERα at both mRNA and the protein levels in MCF7 and T47D human breast cancer cells. Luciferase reporter assays showed that Hispolon decreased the transcriptional activity of ERα. Hispolon treatment also inhibited expression of the ERα target gene pS2. We propose that Hispolon, an anticancer drug extracted from natural sources, inhibits cell growth through modulation of ERα in estrogen-positive breast cancer cells and is a candidate for use in human breast cancer chemotherapy. - Highlights: • Hispolon decreased ERα expression at both mRNA and protein levels. • Hispolon decreased ERα transcriptional activity. • Hispolon treatment inhibited expression of ERα target gene pS2. • Shikonin is a candidate chemotherapeutic target in the treatment of human breast cancer.

  20. Inhibition of lysophosphatidic acid receptors 1 and 3 attenuates atherosclerosis development in LDL-receptor deficient mice.

    PubMed

    Kritikou, Eva; van Puijvelde, Gijs H M; van der Heijden, Thomas; van Santbrink, Peter J; Swart, Maarten; Schaftenaar, Frank H; Kröner, Mara J; Kuiper, Johan; Bot, Ilze

    2016-11-24

    Lysophosphatidic acid (LPA) is a natural lysophospholipid present at high concentrations within lipid-rich atherosclerotic plaques. Upon local accumulation in the damaged vessels, LPA can act as a potent activator for various types of immune cells through its specific membrane receptors LPA1/3. LPA elicits chemotactic, pro-inflammatory and apoptotic effects that lead to atherosclerotic plaque progression. In this study we aimed to inhibit LPA signaling by means of LPA1/3 antagonism using the small molecule Ki16425. We show that LPA1/3 inhibition significantly impaired atherosclerosis progression. Treatment with Ki16425 also resulted in reduced CCL2 production and secretion, which led to less monocyte and neutrophil infiltration. Furthermore, we provide evidence that LPA1/3 blockade enhanced the percentage of non-inflammatory, Ly6C(low) monocytes and CD4(+) CD25(+) FoxP3(+) T-regulatory cells. Finally, we demonstrate that LPA1/3 antagonism mildly reduced plasma LDL cholesterol levels. Therefore, pharmacological inhibition of LPA1/3 receptors may prove a promising approach to diminish atherosclerosis development.

  1. Reversal of gastric somatostatin receptor inhibition by Helicobacter pylori lipopolysaccharide with ebrotidine and sulglycotide.

    PubMed

    Piotrowski, J; Skrodzka, D; Slomiany, A; Slomiany, B L

    1997-05-01

    1. Among the consequences of H. pylori infection is an increase in gastric acid secretion due to the impairement in feedback inhibition by somatostatin. Here, we show that lipopolysaccharide from H. pylori inhibits the binding of somatostatin to gastric mucosal receptor, and that antiulcer agents, ebrotidine and sulglycotide, are capable of countering this effect. 2. The somatostatin receptor was prepared from the solubilized gastric mucosal epithelial cell membranes by affinity chromatography on Affi-Gel-bound [D-Tryp8] SRIF-14 and used in the binding assays for 125I-labeled somatostatin in the presence of H. pylori lipopolysaccharide and antiulcer agents. 3. The assays revealed a dose-dependent inhibition in the receptor-somatostatin binding by the lipopolysaccharide which reached a maximum of 94.1%. The effect of H. pylori lipopolysaccharide was countered by ebrotidine and sulglycotide, which at their optimal doses produced 94.9% and 84% restoration in somatostatin-receptor binding, respectively. 4. The results demonstrate that the antiulcer agents, ebrotidine and sulglycotide, possess the ability to counteract the H. pylori interference with somatostatin regulatory effect on gastric acid secretion.

  2. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

    PubMed Central

    Robinson, Samuel D.; Lee, Tet Woo; Christie, David L.; Birch, Nigel P.

    2015-01-01

    NMDA receptors (NMDARs) play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM) but not high (50 μM) concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-aminopyridine (4-AP). Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs. PMID:26500501

  3. Antipsychotic clozapine inhibits the function of alpha7-nicotinic acetylcholine receptors.

    PubMed

    Singhal, Sachin K; Zhang, Li; Morales, Marisela; Oz, Murat

    2007-02-01

    The effects of the antipsychotic clozapine on the function of the cloned alpha(7) subunit of the nicotinic acetylcholine (nACh) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Clozapine reversibly inhibited nicotine (10 microM)-induced currents in a concentration-dependent manner (300 nM to 90 microM), with an IC(50) value of 3.2+/-0.4 microM. The effect of clozapine was not dependent on the membrane potential. Clozapine did not affect the activity of endogenous Ca(2+)-dependent Cl(-) channels since the inhibition by clozapine was unaltered by the intracellularly injected Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free bathing solution containing 2mM Ba(2+). Clozapine decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on alpha(7)-nACh receptors. In hippocampal slices, the whole-cell recordings from CA1 pyramidal neurons indicated that the increases in the frequency and amplitudes of the GABA-mediated spontaneous inhibitory postsynaptic currents induced by bath application of 2 mM choline, a specific agonist for alpha(7)-nACh receptors, were abolished after 10 min application of 5 microM clozapine. In conclusion, these results demonstrate that clozapine inhibits the function of alpha(7)-nACh receptors expressed in Xenopus oocytes and in hippocampal neurons.

  4. Cell-permeable iron inhibits vascular endothelial growth factor receptor-2 signaling and tumor angiogenesis

    PubMed Central

    Kir, Devika; Saluja, Manju; Modi, Shrey; Venkatachalam, Annapoorna; Schnettler, Erica; Roy, Sabita; Ramakrishnan, Sundaram

    2016-01-01

    Angiogenesis is important for tumor growth and metastasis. Hypoxia in tumors drives this angiogenic response by stabilizing Hypoxia Inducible Factors (HIF) and target genes like Vascular Endothelial Growth Factor (VEGF). HIF stability is regulated by Prolylhydroxylases (PHD)-mediated modification. Iron is an important cofactor in regulating the enzymatic activity of PHDs. Reducing intracellular iron, for instance, mimics hypoxia and induces a pro-angiogenic response. It is hypothesized that increasing the intracellular iron levels will have an opposite, anti-angiogenic effect. We tested this hypothesis by perturbing iron homeostasis in endothelial cells using a unique form of iron, Ferric Ammonium Citrate (FAC). FAC is a cell-permeable form of iron, which can passively enter into cells bypassing the transferrin receptor mediated uptake of transferrin-bound iron. Our studies show that FAC does not decrease the levels of HIF-1α and HIF-2α in endothelial cells but inhibits the autocrine stimulation of VEGF-Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) system by blocking receptor tyrosine kinase phosphorylation. FAC inhibits VEGF-induced endothelial cell proliferation, migration, tube formation and sprouting. Finally, systemic administration of FAC inhibits VEGF and tumor cell-induced angiogenesis in vivo. In conclusion, our studies show that cell-permeable iron attenuates VEGFR-2 mediated signaling and inhibits tumor angiogenesis. PMID:27589831

  5. The Selective Estrogen Receptor Modulator Raloxifene Inhibits Neutrophil Extracellular Trap Formation

    PubMed Central

    Flores, Roxana; Döhrmann, Simon; Schaal, Christina; Hakkim, Abdul; Nizet, Victor; Corriden, Ross

    2016-01-01

    Raloxifene is a selective estrogen receptor modulator typically prescribed for the prevention/treatment of osteoporosis in postmenopausal women. Although raloxifene is known to have anti-inflammatory properties, its effects on human neutrophils, the primary phagocytic leukocytes of the immune system, remain poorly understood. Here, through a screen of pharmacologically active small molecules, we find that raloxifene prevents neutrophil cell death in response to the classical activator phorbol 12-myristate 13-acetate (PMA), a compound known to induce formation of DNA-based neutrophil extracellular traps (NETs). Inhibition of PMA-induced NET production by raloxifene was confirmed using quantitative and imaging-based assays. Human neutrophils from both male and female donors express the nuclear estrogen receptors ERα and ERβ, known targets of raloxifene. Similar to raloxifene, selective antagonists of these receptors inhibit PMA-induced NET production. Furthermore, raloxifene inhibited PMA-induced ERK phosphorylation, but not reactive oxygen species production, pathways known to be key modulators of NET production. Finally, we found that raloxifene inhibited PMA-induced, NET-based killing of the leading human bacterial pathogen, methicillin-resistant Staphylococcus aureus. Our results reveal that raloxifene is a potent modulator of neutrophil function and NET production. PMID:28003814

  6. Modafinil inhibits rat midbrain dopaminergic neurons through D2-like receptors.

    PubMed

    Korotkova, T M; Klyuch, B P; Ponomarenko, A A; Lin, J S; Haas, H L; Sergeeva, O A

    2007-02-01

    Modafinil is a well-tolerated medication for excessive sleepiness, attention-deficit disorder, cocaine dependence and as an adjunct to antidepressants with low propensity for abuse. We investigated the modafinil action on identified dopaminergic and GABAergic neurons in the ventral tegmental area (VTA) and substantia nigra (SN) of rat brain slices. Modafinil (20 microM) inhibited the firing of dopaminergic, but not GABAergic neurons. This inhibition was maintained in the presence of tetrodotoxin and was accompanied by hyperpolarization. Sulpiride (10 microM), a D2-receptor antagonist, but not prazosine (20 microM, an alpha1-adrenoreceptor blocker) abolished the modafinil action. Inhibition of dopamine reuptake with a low dose of nomifensine (1 microM) reduced the firing of DA neurons in a sulpiride-dependent manner and blunted the effect of modafinil. On acutely isolated neurons, modafinil evoked D2-receptor-mediated outward currents in tyrosine-hydroxylase positive cells, identified by single-cell RT-PCR, which reversed polarity near the K(+) equilibrium potential and were unchanged in the presence of nomifensine. Thus modafinil directly inhibits DA neurons through D2 receptors.

  7. Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels.

    PubMed

    Murbartián, Janet; Lei, Qiubo; Sando, Julianne J; Bayliss, Douglas A

    2005-08-26

    Background potassium channels determine membrane potential and input resistance and serve as prominent effectors for modulatory regulation of cellular excitability. TREK-1 is a two-pore domain background K+ channel (KCNK2, K2P2.1) that is sensitive to a variety of physicochemical and humoral factors. In this work, we used a recombinant expression system to show that activation of G alpha(q)-coupled receptors leads to inhibition of TREK-1 channels via protein kinase C (PKC), and we identified a critical phosphorylation site in a key regulatory domain that mediates inhibition of the channel. In HEK 293 cells co-expressing TREK-1 and either the thyrotropin-releasing hormone receptor (TRHR1) or the Orexin receptor (Orx1R), agonist stimulation induced robust channel inhibition that was suppressed by a bisindolylmaleimide PKC inhibitor but not by a protein kinase A blocker ((R(p))-cAMP-S). Channel inhibition by agonists or by direct activators of PKC (phorbol dibutyrate) and PKA (forskolin) was disrupted not only by alanine or aspartate mutations at an identified PKA site (Ser-333) in the C terminus, but also at a more proximal regulatory site in the cytoplasmic C terminus (Ser-300); S333A and S300A mutations enhanced basal TREK-1 current, whereas S333D and S300D substitutions mimicked phosphorylation and strongly diminished currents. When studied in combination, TREK-1 current density was enhanced in S300A/S333D but reduced in S300D/S333A mutant channels. Channel mutants were expressed and appropriately targeted to cell membranes. Together, these data support a sequential phosphorylation model in which receptor-induced kinase activation drives modification at Ser-333 that enables subsequent phosphorylation at Ser-300 to inhibit TREK-1 channel activity.

  8. Aggregation of macrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor

    SciTech Connect

    Green, S.J.; Underhill, C.B. ); Tarone, G. )

    1988-10-01

    To examine the role of the hyaluronate receptor in cell to cell adhesion, the authors have employed the K-3 monoclonal antibody (MAb) which specifically binds to the hyaluronate receptor and blocks its ability to interact with hyaluronate. In the first set of experiments, they investigated the spontaneous aggregation of SV-3T3 cells, which involves two distinct mechanisms, one of which is dependent upon the presence of divalent cation and the other is independent. The divalent cation-independent aggregation was found to be completely inhibited by both intact and Fab fragments of the K-3 MAb. In contrast, the K-3 MAb had no effect on the divalent cation-dependent aggregation of cells. In a second set of experiments, we examined alveolar macrophages. The presence of hyaluronate receptors on alveolar macrophages was demonstrated by the fact that detergent extracts of these cells could bind ({sup 3})hyaluronate, and this binding was blocked by the K-3 MAb. Immunoblot analysis of alveolar macrophages showed that the hyaluronate receptor had a M{sub r} of 99,500, which is considerably larger than the 85,000 M{sub r} for that on BHK cells. When hyaluronate was added to suspensions of alveolar macrophages, the cells were induced to aggregate. This effect was inhibited by the K-3 MAb, suggesting that the hyaluronate-induced aggregation was mediated by the receptor.

  9. Buprenorphine is a weak partial agonist that inhibits opioid receptor desensitization

    PubMed Central

    Virk, Michael S.; Arttamangkul, Seksiri; Birdsong, William T.; Williams, John T.

    2009-01-01

    Buprenorphine is a weak partial agonist at mu-opioid receptors that is used for treatment of pain and addiction. Intracellular and whole cell recordings were made from locus coeruleus (LC) neurons in rat brain slices to characterize the actions of buprenorphine. Acute application of buprenorphine caused a hyperpolarization that was prevented by previous treatment of slices with the irreversible opioid antagonist, β-chlornaltrexamine (β-CNA), but was not reversed by a saturating concentration of naloxone. As expected for a partial agonist, sub-saturating concentrations of buprenorphine decreased the [Met]5 enkephalin (ME) induced hyperpolarization or outward current. When the ME induced current was decreased below a critical value, desensitization and internalization of μ-opioid receptors (MOR) was eliminated. The inhibition of desensitization by buprenorphine was not the result of prior desensitization, slow dissociation from the receptor, or elimination of receptor reserve. Treatment of slices with sub-saturating concentrations of etorphine, methadone, oxymorphone or β-CNA also reduced the current induced by ME but did not block ME-induced desensitization. Treatment of animals with buprenorphine for a week resulted in the inhibition of the current induced by ME and a block of desensitization that was not different from the acute application of buprenorphine to brain slices. These observations show the unique characteristics of buprenorphine and further demonstrate the range of agonist selective actions that are possible through G-protein coupled receptors. PMID:19494155

  10. Oral administration of protease inhibits enterotoxigenic Escherichia coli receptor activity in piglet small intestine.

    PubMed Central

    Mynott, T L; Luke, R K; Chandler, D S

    1996-01-01

    The virulence of enterotoxigenic Escherichia coli (ETEC) is attributed to their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. A novel approach to preventing ETEC induced diarrhoea would be to prevent attachment of ETEC to intestine by proteolytically modifying the receptor attachment sites. This study aimed to examine the effect of bromelain, a proteolytic extract obtained from pineapple stems, on ETEC receptor activity in porcine small intestine. Bromelain was administered orally to piglets and K88+ ETEC attachment to small intestine was measured at 50 cm intervals using an enzyme immunoassay. K88+ ETEC attachment to intestinal sections that were not treated with bromelain varied appreciably between sampling sites. Variability in receptor activity along the intestinal surface is though to be caused by the localised effects of endogenous proteases. Oral administration of exogenous protease inhibited K88+ ETEC attachment to pig small intestine in a dose dependent manner (p < 0.05). Attachment of K88+ ETEC was negligible after treatment, resembling the levels of attachment of K88 to piglets of the genetically determined non-adhesive phenotype, which are resistant to K88+ ETEC infection. Serum biochemical analysis and histopathological examination of treated piglets showed no adverse effects of the bromelain treatment. It is concluded that administration of bromelain can inhibit ETEC receptor activity in vivo and may therefore be useful for prevention of K88+ ETEC induced diarrhoea. PMID:8566855

  11. N alpha-methylhistamine inhibits intestinal transit in mice by central histamine H1 receptor activation.

    PubMed

    Oishi, R; Adachi, N; Saeki, K

    1993-06-24

    The effects of (R)alpha-methylhistamine and N alpha-methylhistamine on intestinal transit were examined in mice. The passage of a charcoal meal in the gastrointestinal tract was dose dependently inhibited by N alpha-methylhistamine (1-20 mg/kg i.p.), but not by a selective H3 receptor agonist (R)alpha-methyl-histamine (1-50 mg/kg i.p.). The inhibitory effect of N alpha-methylhistamine (20 mg/kg) was attenuated by pretreatment with H1 receptor antagonists (mepyramine 5 mg/kg i.p. or 5 micrograms i.c.v. and triprolidine 5 mg/kg i.p.), but not by cimetidine (10 mg/kg i.p.), zolantidine (5 mg/kg i.p.), a brain-penetrating H2 receptor antagonist, or thioperamide (5 mg/kg i.p.), a selective H3 receptor antagonist. The effect of N alpha-methylhistamine was also attenuated by combined treatment with phentolamine and propranolol (5 and 15 mg/kg s.c., respectively) and by pretreatment with 6-hydroxydopamine (20 mg/kg i.p., 2 days before). N alpha-Methylhistamine markedly decreased histamine turnover in the mouse brain. These findings suggest that intestinal transit is inhibited by N alpha-methylhistamine via stimulation of central H1 but not H3 receptors and that stimulation of the sympathetic system is involved in this effect.

  12. Inhibition of Adult Rat Retinal Ganglion Cells by D1-type Dopamine Receptor Activation

    PubMed Central

    Hayashida, Yuki; Rodríguez, Carolina Varela; Ogata, Genki; Partida, Gloria J.; Oi, Hanako; Stradleigh, Tyler W.; Lee, Sherwin C.; Colado, Anselmo Felipe; Ishida, Andrew T.

    2011-01-01

    The spike output of neural pathways can be regulated by modulating output neuron excitability and/or their synaptic inputs. Dopaminergic interneurons synapse onto cells that route signals to mammalian retinal ganglion cells, but it is unknown whether dopamine can activate receptors in these ganglion cells and, if it does, how this affects their excitability. Here, we show D1a-receptor-like immunoreactivity in ganglion cells identified in adult rats by retrogradely transported dextran, and that dopamine, D1-type receptor agonists, and cAMP analogs inhibit spiking in ganglion cells dissociated from adult rats. These ligands curtailed repetitive spiking during constant current injections, and reduced the number and rate of rise of spikes elicited by fluctuating current injections without significantly altering the timing of the remaining spikes. Consistent with mediation by D1-type receptors, SCH-23390 reversed the effects of dopamine on spikes. Contrary to a recent report, spike inhibition by dopamine was not precluded by blocking Ih. Consistent with the reduced rate of spike rise, dopamine reduced voltage-gated Na+ current (INa) amplitude and tetrodotoxin, at doses that reduced INa as moderately as dopamine, also inhibited spiking. These results provide the first direct evidence that D1-type dopamine receptor activation can alter mammalian retinal ganglion cell excitability, and demonstrate that dopamine can modulate spikes in these cells by a mechanism different from the pre- and postsynaptic means proposed by previous studies. To our knowledge, our results also provide the first evidence that dopamine receptor activation can reduce excitability without altering the temporal precision of spike firing. PMID:19940196

  13. Electroacupuncture Inhibition of Hyperalgesia in Rats with Adjuvant Arthritis: Involvement of Cannabinoid Receptor 1 and Dopamine Receptor Subtypes in Striatum

    PubMed Central

    Shou, Yin; Yang, Yang; Xu, Ming-Shu; Zhao, Ying-Qian; Ge, Lin-Bao; Zhang, Bi-Meng

    2013-01-01

    Electroacupuncture (EA) has been regarded as an alternative treatment for inflammatory pain for several decades. However, the molecular mechanisms underlying the antinociceptive effect of EA have not been thoroughly clarified. Previous studies have shown that cannabinoid CB1 receptors are related to pain relief. Accumulating evidence has shown that the CB1 and dopamine systems sometimes interact and may operate synergistically in rat striatum. To our knowledge, dopamine D1/D2 receptors are involved in EA analgesia. In this study, we found that repeated EA at Zusanli (ST36) and Kunlun (BL60) acupoints resulted in marked improvements in thermal hyperalgesia. Both western blot assays and FQ-PCR analysis results showed that the levels of CB1 expression in the repeated-EA group were much higher than those in any other group (P = 0.001). The CB1-selective antagonist AM251 inhibited the effects of repeated EA by attenuating the increases in CB1 expression. The two kinds of dopamine receptors imparted different actions on the EA-induced CB1 upregulation in AA rat model. These results suggested that the strong activation of the CB1 receptor after repeated EA resulted in the concomitant phenomenon of the upregulation of D1 and D2 levels of gene expression. PMID:23762129

  14. Deletion of striatal adenosine A(2A) receptor spares latent inhibition and prepulse inhibition but impairs active avoidance learning.

    PubMed

    Singer, Philipp; Wei, Catherine J; Chen, Jiang-Fan; Boison, Detlev; Yee, Benjamin K

    2013-04-01

    Following early clinical leads, the adenosine A(2A)R receptor (A(2A)R) has continued to attract attention as a potential novel target for treating schizophrenia, especially against the negative and cognitive symptoms of the disease because of A(2A)R's unique modulatory action over glutamatergic in addition to dopaminergic signaling. Through (i) the antagonistic interaction with the dopamine D(2) receptor, and (ii) the regulation of glutamate release and N-methyl-d-aspartate receptor function, striatal A(2A)R is ideally positioned to fine-tune the dopamine-glutamate balance, the disturbance of which is implicated in the pathophysiology of schizophrenia. However, the precise function of striatal A(2A)Rs in the regulation of schizophrenia-relevant behavior is poorly understood. Here, we tested the impact of conditional striatum-specific A(2A)R knockout (st-A(2A)R-KO) on latent inhibition (LI) and prepulse inhibition (PPI) - behavior that is tightly regulated by striatal dopamine and glutamate. These are two common cross-species translational tests for the assessment of selective attention and sensorimotor gating deficits reported in schizophrenia patients; and enhanced performance in these tests is associated with antipsychotic drug action. We found that neither LI nor PPI was significantly affected in st-A(2A)R-KO mice, although a deficit in active avoidance learning was identified in these animals. The latter phenotype, however, was not replicated in another form of aversive conditioning - namely, conditioned taste aversion. Hence, the present study shows that neither learned inattention (as measured by LI) nor sensory gating (as indexed by PPI) requires the integrity of striatal A(2A)Rs - a finding that may undermine the hypothesized importance of A(2A)R in the genesis and/or treatment of schizophrenia.

  15. Inhibition of the receptor-mediated virion attachment to a lipid membrane

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2012-10-01

    The forefront of the anti-viral defence is sometimes aimed at virion attachment to a host membrane. This step or, more specifically, virion contacts with cellular membrane receptors (or, e.g., glycolipids) can be inhibited by antibodies (or specially chosen or designed compounds) via their association with virions. In this case, the full-scale attachment of virions to a host membrane occurs via a subtle interplay of the formation and rupture of multiple virion-inhibitor and virion-receptor bonds. We present a kinetic model describing this interplay and illustrating general trends in the process under consideration.

  16. Novel triple neurokinin receptor antagonist CS-003 inhibits respiratory disease models in guinea pigs.

    PubMed

    Tsuchida, Hiroshi; Takahashi, Sakiko; Nosaka, Emi; Kuraya, Takako; Yamashita, Makoto; Morimoto, Kiyoshi

    2008-10-31

    Neurokinins are known to induce neurogenic inflammation related to respiratory diseases. The effects of CS-003 ([1-{2-[(2R)-(3,4-dichlorophenyl)-4-(3,4,5-trimethoxybenzoyl)morpholin-2-yl]ethyl}spiro[benzo[c]thiophene-1(3H),4'-piperidine]-(2S)-oxide hydrochloride]), a novel triple neurokinin receptor antagonist, on several respiratory disease models were evaluated in guinea pigs. As we have already shown that CS-003 is intravenously effective, we first determined if CS-003 was orally effective. CS-003 dose-dependently inhibited substance P-induced tracheal vascular hyperpermeability, neurokinin A- and neurokinin B-induced bronchoconstriction with ID(50) values of 3.6, 1.3 and 0.89 mg/kg (p.o.), respectively. CS-003 (10 mg/kg, p.o.) inhibited the number of coughs induced by capsaicin aerosol (P<0.01) and the antitussive effect was comparable to that of codeine. CS-003 (10 mg/kg, p.o.) also inhibited airway hyperresponsiveness to methacholine chloride in ovalbumin-induced asthma models (P<0.01), a milder one and a severer one. On the other hand, montelukast (10 mg/kg, p.o.), a leukotriene receptor antagonist, significantly inhibited the hyperresponsiveness only in the milder model (P<0.05). In an ovalbumin-induced rhinitis model, oral administration of CS-003 inhibited nasal blockade in a dose-dependent manner and the inhibitory effect was comparable to that of dexamethasone (10 mg/kg, p.o.). CS-003 (i.v.) also dose-dependently inhibited cigarette smoke-induced bronchoconstriction, tracheal vascular hyperpermeability and mucus secretion. These data show that CS-003, a potent orally active triple neurokinin receptor antagonist, may be useful for the treatment of respiratory diseases associated with neurokinins, such as allergic asthma, allergic rhinitis, chronic obstructive pulmonary disease and cough.

  17. Rapid Glucocorticoid Feedback Inhibition of ACTH Secretion Involves Ligand-Dependent Membrane Association of Glucocorticoid Receptors

    PubMed Central

    Deng, Qiong; Riquelme, Denise; Trinh, Loc; Low, Malcolm J.; Tomić, Melanija; Stojilkovic, Stanko

    2015-01-01

    The hypothesis that rapid glucocorticoid inhibition of pituitary ACTH secretion mediates a feedforward/feedback mechanism responsible for the hourly glucocorticoid pulsatility was tested in cultured pituitary cells. Perifusion with 30 pM CRH caused sustained the elevation of ACTH secretion. Superimposed corticosterone pulses inhibited CRH-stimulated ACTH release, depending on prior glucocorticoid clearance. When CRH perifusion started after 2 hours of glucocorticoid-free medium, corticosterone levels in the stress range (1 μM) caused a delayed (25 min) and prolonged inhibition of CRH-stimulated ACTH secretion, up to 60 minutes after corticosterone withdrawal. In contrast, after 6 hours of glucocorticoid-free medium, basal corticosterone levels inhibited CRH-stimulated ACTH within 5 minutes, after rapid recovery 5 minutes after corticosterone withdrawal. The latter effect was insensitive to actinomycin D but was prevented by the glucocorticoid receptor antagonist, RU486, suggesting nongenomic effects of the classical glucocorticoid receptor. In hypothalamic-derived 4B cells, 10 nM corticosterone increased immunoreactive glucocorticoid receptor content in membrane fractions, with association and clearance rates paralleling the effects on ACTH secretion from corticotrophs. Corticosterone did not affect CRH-stimulated calcium influx, but in AtT-20 cells, it had biphasic effects on CRH-stimulated Src phosphorylation, with early inhibition and late stimulation, suggesting a role for Src phosphorylation on the rapid glucocorticoid feedback. The data suggest that the nongenomic/membrane effects of classical GR mediate rapid and reversible glucocorticoid feedback inhibition at the pituitary corticotrophs downstream of calcium influx. The sensitivity and kinetics of these effects is consistent with the hypothesis that pituitary glucocorticoid feedback is part of the mechanism for adrenocortical ultradian pulse generation. PMID:26121342

  18. Inhibition of noradrenaline release in the rat brain cortex via presynaptic H3 receptors.

    PubMed

    Schlicker, E; Fink, K; Hinterthaner, M; Göthert, M

    1989-12-01

    The effects of histamine and related drugs on the evoked tritium overflow from superfused rat brain cortex slices preincubated with 3H-noradrenaline were determined. Tritium overflow was stimulated electrically (3 Hz; slices superfused with normal physiological salt solution) or by introduction of CaCl2 1.3 mmol/l (slices superfused with Ca2(+)-free medium containing K+ 20 mmol/l). Histamine slightly decreased the electrically evoked 3H overflow in slices superfused in the presence of desipramine. The degree of inhibition obtained with histamine was doubled when both desipramine and phentolamine were present in the superfusion medium (pIC15 6.46). Under the latter condition, the evoked overflow was inhibited by the H3 receptor agonist R-(-)-alpha-methylhistamine and its S-(+) enantiomer (pIC15 7.36 and 5.09, respectively), but was not affected by the H2 receptor agonist dimaprit and the H1 receptor agonist 2-thiazolylethylamine (both at up to 32 mumols/l). The concentration-response curve of histamine was shifted to the right by the H3 receptor antagonists thioperamide, impromidine and burimamide (apparent pA2 8.37, 6.86 and 7.05, respectively), by the H2 receptor antagonist ranitidine (apparent pA2 4.27) and was not affected by the H1 receptor antagonist dimetindene (32 mumols/l). The inhibitory effect of R-(-)-alpha-methylhistamine on the evoked overflow was also counteracted by thioperamide. Given alone, none of the five histamine receptor antagonists affected the evoked overflow. In the absence of desipramine plus phentolamine, impromidine and burimamide facilitated the electrically evoked 3H overflow whereas thioperamide had no effect. The facilitatory effects of impromidine and burimamide were abolished by phentolamine, but not affected by desipramine.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Extracellular nucleotides inhibit growth of human oesophageal cancer cells via P2Y2-receptors

    PubMed Central

    Maaser, K; Höpfner, M; Kap, H; Sutter, A P; Barthel, B; von Lampe, B; Zeitz, M; Scherübl, H

    2002-01-01

    Extracellular ATP is known to inhibit growth of various tumours by activating specific purinergic receptors (P2-receptors). Since the therapy of advanced oesophageal cancer is unsatisfying, new therapeutic approaches are mandatory. Here, we investigated the functional expression and potential antiproliferative effects of P2-purinergic receptors in human oesophageal cancer cells. Prolonged incubation of primary cell cultures of human oesophageal cancers as well as of the squamous oesophageal cancer cell line Kyse-140 with ATP or its stable analogue ATPγS dose-dependently inhibited cell proliferation. This was due to both an induction of apoptosis and cell cycle arrest. The expression of P2-receptors was examined by RT-PCR, immunocytochemistry, and [Ca2+]i-imaging. Application of various extracellular nucleotides dose-dependently increased [Ca2+]i. The rank order of potency was ATP=UTP>ATPγS>ADP=UDP. 2-methylthio-ATP and α,β-methylene-ATP had no effects on [Ca2+]i. Complete cross-desensitization between ATP and UTP was observed. Moreover, the phospholipase C inhibitor U73122 dose-dependently reduced the ATP triggered [Ca2+]i signal. The pharmacological features strongly suggest the functional expression of G-protein coupled P2Y2-receptors in oesophageal squamous cancer cells. P2Y2-receptors are involved in the antiproliferative actions of extracellular nucleotides. Thus, P2Y2-receptors are promising target proteins for innovative approaches in oesophageal cancer therapy. British Journal of Cancer (2002) 86, 636–644. DOI: 10.1038/sj/bjc/6600100 www.bjcancer.com © 2002 Cancer Research UK PMID:11870549

  20. Evidence for inhibition mediated by coassembly of GABAA and GABAC receptor subunits in native central neurons.

    PubMed

    Milligan, Carol J; Buckley, Noel J; Garret, Maurice; Deuchars, Jim; Deuchars, Susan A

    2004-08-18

    Fast inhibition in the nervous system is commonly mediated by GABA(A) receptors comprised of 2alpha/2beta/1gamma subunits. In contrast, GABA(C) receptors containing only rho subunits (rho1-rho3) have been predominantly detected in the retina. However, here using reverse transcription-PCR and in situ hybridization we show that mRNA encoding the rho1 subunit is highly expressed in brainstem neurons. Immunohistochemistry localized the rho1 subunit to neurons at light and electron microscopic levels, where it was detected at synaptic junctions. Application of the GABA(C) receptor agonist cis-4-aminocrotonic acid (100-800 microM) requires the rho1 subunit to elicit responses, which surprisingly are blocked independently by antagonists to GABA(A) (bicuculline, 10 microM) and GABA(C) [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA); 40-160 microM] receptors. Responses to GABA(C) agonists were also enhanced by the GABA(A) receptor modulator pentobarbitone (300 microM). Spontaneous and evoked IPSPs were reduced in amplitude but never abolished by TPMPA, but were completely blocked by bicuculline. We therefore tested the hypothesis that GABA(A) and GABA(C) subunits formed a heteromeric receptor. Immunohistochemistry indicated that rho1 and alpha1 subunits were colocalized at light and electron microscopic levels. Electrophysiology revealed that responses to GABA(C) receptor agonists were enhanced by the GABA(A) receptor modulator zolpidem (500 nm), which acts on the alpha1 subunit when the gamma2 subunit is also present. Finally, coimmunoprecipitation indicated that the rho1 subunit formed complexes that also containedalpha1 and gamma2 subunits. Taken together these separate lines of evidence suggest that the effects of GABA in central neurons can be mediated by heteromeric complexes of GABA(A) and GABA(C) receptor subunits.

  1. CH5137291, an androgen receptor nuclear translocation-inhibiting compound, inhibits the growth of castration-resistant prostate cancer cells.

    PubMed

    Ishikura, Nobuyuki; Kawata, Hiromitsu; Nishimoto, Ayako; Nakamura, Ryo; Tsunenari, Toshiaki; Watanabe, Miho; Tachibana, Kazutaka; Shiraishi, Takuya; Yoshino, Hitoshi; Honma, Akie; Emura, Takashi; Ohta, Masateru; Nakagawa, Toshito; Houjo, Takao; Corey, Eva; Vessella, Robert L; Aoki, Yuko; Sato, Haruhiko

    2015-04-01

    Resistance of prostate cancer to castration is currently an unavoidable problem. The major mechanisms underlying such resistance are androgen receptor (AR) overexpression, androgen-independent activation of AR, and AR mutation. To address this problem, we developed an AR pure antagonist, CH5137291, with AR nuclear translocation-inhibiting activity, and compared its activity and characteristics with that of bicalutamide. Cell lines corresponding to the mechanisms of castration resistance were used: LNCaP-BC2 having AR overexpression and LNCaP-CS10 having androgen-independent AR activation. VCaP and LNCaP were used as hormone-sensitive prostate cancer cells. In vitro functional assay clearly showed that CH5137291 inhibited the nuclear translocation of wild-type ARs as well as W741C- and T877A-mutant ARs. In addition, it acted as a pure antagonist on the transcriptional activity of these types of ARs. In contrast, bicalutamide did not inhibit the nuclear translocation of these ARs, and showed a partial/full agonistic effect on the transcriptional activity. CH5137291 inhibited cell growth more strongly than bicalutamide in VCaP and LNCaP cells as well as in LNCaP-BC2 and LNCaP-CS10 cells in vitro. In xenograft models, CH5137291 strongly inhibited the tumor growth of LNCaP, LNCaP-BC2, and LNCaP-CS10, whereas bicalutamide showed a weaker effect in LNCaP and almost no effect in LNCaP-BC2 and LNCaP-CS10 xenografts. Levels of prostate-specific antigen (PSA) in plasma correlated well with the antitumor effect of both agents. CH5137291 inhibited the growth of LNCaP tumors that had become resistant to bicalutamide treatment. A docking model suggested that CH5137291 intensively collided with the M895 residue of helix 12, and therefore strongly inhibited the folding of helix 12, a cause of AR agonist activity, in wild-type and W741C-mutant ARs. In cynomolgus monkeys, the serum concentration of CH5137291 increased dose-dependently and PSA level decreased 80% at 100 mg/kg. CH

  2. Highly chlorinated PCBs inhibit the human xenobiotic response mediated by the steroid and xenobiotic receptor (SXR).

    PubMed

    Tabb, Michelle M; Kholodovych, Vladyslav; Grün, Felix; Zhou, Changcheng; Welsh, William J; Blumberg, Bruce

    2004-02-01

    Polychlorinated biphenyls (PCBs) are a family of persistent organic contaminants suspected to cause adverse effects in wildlife and humans. In rodents, PCBs bind to the aryl hydrocarbon (AhR) and pregnane X receptors (PXR) inducing the expression of catabolic cytochrome p450 enzymes of the CYP1A and 3A families. We found that certain highly chlorinated PCBs are potent activators of rodent PXR but antagonize its human ortholog, the steroid and xenobiotic receptor (SXR), inhibiting target gene induction. Thus, exposure to PCBs may blunt the human xenobiotic response, inhibiting the detoxification of steroids, bioactive dietary compounds, and xenobiotics normally mediated by SXR. The antagonistic PCBs are among the most stable and abundant in human tissues. These findings have important implications for understanding the biologic effects of PCB exposure and the use of animal models to predict the attendant risk.

  3. Highly chlorinated PCBs inhibit the human xenobiotic response mediated by the steroid and xenobiotic receptor (SXR).

    PubMed Central

    Tabb, Michelle M; Kholodovych, Vladyslav; Grün, Felix; Zhou, Changcheng; Welsh, William J; Blumberg, Bruce

    2004-01-01

    Polychlorinated biphenyls (PCBs) are a family of persistent organic contaminants suspected to cause adverse effects in wildlife and humans. In rodents, PCBs bind to the aryl hydrocarbon (AhR) and pregnane X receptors (PXR) inducing the expression of catabolic cytochrome p450 enzymes of the CYP1A and 3A families. We found that certain highly chlorinated PCBs are potent activators of rodent PXR but antagonize its human ortholog, the steroid and xenobiotic receptor (SXR), inhibiting target gene induction. Thus, exposure to PCBs may blunt the human xenobiotic response, inhibiting the detoxification of steroids, bioactive dietary compounds, and xenobiotics normally mediated by SXR. The antagonistic PCBs are among the most stable and abundant in human tissues. These findings have important implications for understanding the biologic effects of PCB exposure and the use of animal models to predict the attendant risk. PMID:14754570

  4. Glycine Transporter-1 Inhibition Promotes Striatal Axon Sprouting via NMDA Receptors in Dopamine Neurons

    PubMed Central

    Castagna, Candace; Mrejeru, Ana; Lizardi-Ortiz, José E.; Klein, Zoe; Lindsley, Craig W.

    2013-01-01

    NMDA receptor activity is involved in shaping synaptic connections throughout development and adulthood. We recently reported that brief activation of NMDA receptors on cultured ventral midbrain dopamine neurons enhanced their axon growth rate and induced axonal branching. To test whether this mechanism was relevant to axon regrowth in adult animals, we examined the reinnervation of dorsal striatum following nigral dopamine neuron loss induced by unilateral intrastriatal injections of the toxin 6-hydroxydopamine. We used a pharmacological approach to enhance NMDA receptor-dependent signaling by treatment with an inhibitor of glycine transporter-1 that elevates levels of extracellular glycine, a coagonist required for NMDA receptor activation. All mice displayed sprouting of dopaminergic axons from spared fibers in the ventral striatum to the denervated dorsal striatum at 7 weeks post-lesion, but the reinnervation in mice treated for 4 weeks with glycine uptake inhibitor was approximately twice as dense as in untreated mice. The treated mice also displayed higher levels of striatal dopamine and a complete recovery from lateralization in a test of sensorimotor behavior. We confirmed that the actions of glycine uptake inhibition on reinnervation and behavioral recovery required NMDA receptors in dopamine neurons using targeted deletion of the NR1 NMDA receptor subunit in dopamine neurons. Glycine transport inhibitors promote functionally relevant sprouting of surviving dopamine axons and could provide clinical treatment for disorders such as Parkinson's disease. PMID:24133278

  5. Inhibition of gastric mucosal mucin receptor by Helicobacter pylori lipopolysaccharide: effect of sulglycotide.

    PubMed

    Piotrowski, J; Majka, J; Murty, V L; Czajkowski, A; Slomiany, A; Slomiany, B L

    1994-09-01

    1. A receptor for mucin was isolated from the solubilized gastric epithelial cell membrane by affinity chromatography on Sepharose-bound wheat germ agglutinin. 2. The receptor protein displayed a molecular weight of 97 kDa and exhibited specific affinity towards mucin-coated surfaces. The optimum for mucin binding occurred at 60-100 micrograms/ml, while the values for the receptor were 2.0-3.1 micrograms/ml. 3. The mucin binding to the receptor was susceptible to Helicobacter pylori lipopolysaccharide which caused maximum inhibition of 91% at 30 mu/ml. This inhibitory effect of the lipopolysaccharide was abolished by a gastroprotective agent, sulglycotide. 4. The effect of sulglycotide was dose dependent and at 50 micrograms/ml produced a 94% restoration in receptor-mucin binding. Furthermore, sulglycotide was also capable of enhancing (97%) the mucin binding to its receptor in the absence of the lipopolysaccharide. 5. The results demonstrate that H. pylori through its lipopolysaccharide interferes in the interaction of mucin with gastric epithelial surfaces and that a gastroprotective agent, sulglycotide, counteracts this effect, and hence is capable of preventing the loss of mucin coat continuity occurring with H. pylori infection.

  6. Molecular Determinants for Competitive Inhibition of α4β2 Nicotinic Acetylcholine Receptors

    PubMed Central

    Carbone, Annalisa; García-Beltrán, Olimpo; Livingstone, Phil D.; Biggin, Philip C.; Cassels, Bruce K.; Wonnacott, Susan; Zapata-Torres, Gerald; Bermudez, Isabel

    2010-01-01

    The Erythrina alkaloids erysodine and dihydro-β-erythroidine (DHβE) are potent and selective competitive inhibitors of α4β2 nicotinic acetylcholine receptors (nAChRs), but little is known about the molecular determinants of the sensitivity of this receptor subtype to inhibition by this class of antagonists. We addressed this issue by examining the effects of DHβE and a range of aromatic Erythrina alkaloids on [3H]cytisine binding and receptor function in conjunction with homology models of the α4β2 nAChR, mutagenesis, and functional assays. The lactone group of DHβE and a hydroxyl group at position C-16 in aromatic Erythrina alkaloids were identified as major determinants of potency, which was decreased when the conserved residue Tyr126 in loop A of the α4 subunit was substituted by alanine. Sensitivity to inhibition was also decreased by substituting the conserved aromatic residues α4Trp182 (loop B), α4Tyr230 (loop C), and β2Trp82 (loop D) and the nonconserved β2Thr84; however, only α4Trp182 was predicted to contact bound antagonist, suggesting α4Tyr230, β2Trp82, and β2Thr84 contribute allosterically to the closed state elicited by bound antagonist. In addition, homology modeling predicted strong ionic interactions between the ammonium center of the Erythrina alkaloids and β2Asp196, leading to the uncapping of loop C. Consistent with this, β2D196A abolished sensitivity to inhibition by DHβE or erysodine but not by epierythratidine, which is not predicted to form ionic bonds with β2Asp196. This residue is not conserved in subunits that comprise nAChRs with low sensitivity to inhibition by DHβE or erysodine, which highlights β2Asp196 as a major determinant of the receptor selectivity of Erythrina alkaloids. PMID:20547737

  7. Ligand-independent and tissue-selective androgen receptor inhibition by pyrvinium

    PubMed Central

    Lim, Minyoung; Otto-Duessel, Maya; He, Miaoling; Su, Leila; Nguyen, Dan; Chin, Emily; Alliston, Tamara; Jones, Jeremy O.

    2014-01-01

    Pyrvinium pamoate (PP) is a potent non-competitive inhibitor of the androgen receptor (AR). Using a novel method of target identification, we demonstrate that AR is a direct target of PP in prostate cancer cells. We demonstrate that PP inhibits AR activity via the highly conserved DNA binding domain (DBD), the only AR inhibitor that functions via this domain. Furthermore, computational modeling predicts that pyrvinium binds at the interface of the DBD dimer and the minor groove of the AR response element. Because PP acts through the DBD, PP is able to inhibit the constitutive activity of AR splice variants, which are thought to contribute to the growth of castration resistant prostate cancer (CRPC). PP also inhibits androgen-independent AR activation by HER2 kinase. The anti-androgen activity of pyrvinium manifests in the ability to inhibit the in vivo growth of CRPC xenografts that express AR splice variants. Interestingly, PP was most potent in cells with endogenous AR expression derived from prostate or bone. PP was able to inhibit several other hormone nuclear receptors (NRs), but not structurally unrelated transcription factors. PP inhibition of other NRs was similarly cell-type selective. Using dual-energy X-ray absorptiometry, we demonstrate that the cell-type specificity of PP manifests in tissue-selective inhibition of AR activity in mice, as PP decreases prostate weight and bone mineral density, but does not affect lean body mass. Our results suggest that the non-competitive AR inhibitor pyrvinium has significant potential to treat CRPC, including cancers driven by ligand-independent AR signaling. PMID:24354286

  8. 5-HT2 presynaptic receptors mediate inhibition of glutamate release from cerebellar mossy fibre terminals.

    PubMed

    Maura, G; Carbone, R; Guido, M; Pestarino, M; Raiteri, M

    1991-09-17

    'Giant' synaptosomes originating from mossy fibre terminals and having sedimentation properties different from those of standard synaptosomes were obtained from rat cerebellum. Exposure of superfused giant synaptosomes to 15 mM KCl caused the release of endogenous glutamate in a largely (about 80%) calcium-dependent manner. The K(+)-evoked overflow of glutamate was inhibited in a concentration-dependent manner by 5-hydroxytryptamine (5-HT) and by the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI), but not by the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The effects of 5-HT and DOI were quite potent, already reaching significant inhibition (about 25%) at 10 nM. The 5-HT2 receptor antagonist ketanserin counteracted the inhibitory effect of 5-HT. In cerebellar slices, ketanserin increased on its own the calcium-dependent K(+)-evoked release of glutamate and this effect was not prevented by tetrodotoxin (TTX). The results support the idea that cerebellar mossy fibres use glutamate as a transmitter and show that the release of glutamate can be inhibited via presynaptic heteroreceptors of the 5-HT2 type probably localized on the mossy fibre terminals.

  9. The Cellular Prion Protein Prevents Copper-Induced Inhibition of P2X4 Receptors

    PubMed Central

    Lorca, Ramón A.; Varela-Nallar, Lorena; Inestrosa, Nibaldo C.; Huidobro-Toro, J. Pablo

    2011-01-01

    Although the physiological function of the cellular prion protein (PrPC) remains unknown, several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ through a domain composed by four to five repeats of eight amino acids. Previously, we have shown that the perfusion of this domain prevents and reverses the inhibition by Cu2+ of the adenosine triphosphate (ATP)-evoked currents in the P2X4 receptor subtype, highlighting a modulatory role for PrPC in synaptic transmission through regulation of Cu2+ levels. Here, we study the effect of full-length PrPC in Cu2+ inhibition of P2X4 receptor when both are coexpressed. PrPC expression does not significantly change the ATP concentration-response curve in oocytes expressing P2X4 receptors. However, the presence of PrPC reduces the inhibition by Cu2+ of the ATP-elicited currents in these oocytes, confirming our previous observations with the Cu2+ binding domain. Thus, our observations suggest a role for PrPC in modulating synaptic activity through binding of extracellular Cu2+. PMID:22114745

  10. Inhibition of human α7 nicotinic acetylcholine receptors by cyclic monoterpene carveol.

    PubMed

    Lozon, Yosra; Sultan, Ahmed; Lansdell, Stuart J; Prytkova, Tatiana; Sadek, Bassem; Yang, Keun-Hang Susan; Howarth, Frank Christopher; Millar, Neil S; Oz, Murat

    2016-04-05

    Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order carveol>thymoquinone>carvacrol>menthone>thymol>limonene>eugenole>pulegone≥carvone≥vanilin. Among the monoterpenes, carveol showed the highest potency on acetylcholine-induced responses, with IC50 of 8.3µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies.

  11. A non-peptide NK1-receptor antagonist, RP 67580, inhibits neurogenic inflammation postsynaptically.

    PubMed Central

    Moussaoui, S. M.; Montier, F.; Carruette, A.; Blanchard, J. C.; Laduron, P. M.; Garret, C.

    1993-01-01

    1. The non-peptide neurokinin NK1-receptor antagonist, RP 67580 (3aR, 7aR), a perhydroisoindolone derivative, powerfully reduced plasma extravasation in rat hind paw skin induced by local application of xylene (ID50 = 0.03 mg kg-1, i.v.) or capsaicin (ID50 = 0.06 mg kg-1, i.v.), or by i.v. injection of exogenous substance P (SP) or septide ([pGlu6,Pro9]SP(6-11)) (ID50 = 0.04-0.05 mg kg-1, i.v.). RP 67580 (1 mg kg-1, i.v.) also abolished capsaicin-induced nasal fluid hypersecretion (by 82 +/- 5%). These effects were found to be stereospecific, the enantiomer, RP 68651 (3aS, 7aS), being inactive at 1 mg kg-1, i.v. 2. In rats neonatally treated with capsaicin (50 mg kg-1, s.c.), plasma extravasation induced by SP was significantly increased (by 43 +/- 7%). RP 67580 (1 mg kg-1, i.v.) completely inhibited the SP-induced plasma extravasation in capsaicin neonatally treated-animals, as it did in control animals. This result suggests that RP 67580 acts at the postsynaptic level for the inhibition of plasma extravasation. 3. Opioid receptor agonists, mu-(morphine) and kappa-(PD-117302) at 10 mg kg-1, s.c., in contrast to NK1-receptor antagonists, did not inhibit plasma extravasation induced by exogenous SP. They were, however, partially effective against plasma extravasation induced by electrical nerve stimulation (74 +/- 4% and 48 +/- 9% inhibition at 10 mg kg-1, s.c. of morphine and PD-117302, respectively, compared to 90 +/- 3% inhibition obtained with RP 67580, 3 mg kg-1, s.c.).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7684305

  12. Anandamide inhibits nuclear factor-kappaB activation through a cannabinoid receptor-independent pathway.

    PubMed

    Sancho, Rocío; Calzado, Marco A; Di Marzo, Vincenzo; Appendino, Giovanni; Muñoz, Eduardo

    2003-02-01

    Anandamide (arachidonoylethanolamine, AEA), an endogenous agonist for both the cannabinoid CB(1) receptor and the vanilloid VR1 receptor, elicits neurobehavioral, anti-inflammatory, immunomodulatory, and proapoptotic effects. Because of the central role of nuclear factor-kappaB (NF-kappaB) in the inflammatory process and the immune response, we postulated that AEA might owe some of its effects to the suppression of NF-kappaB. This study shows that AEA inhibits tumor necrosis factor-alpha (TNFalpha)-induced NF-kappaB activation by direct inhibition of the IkappaB kinase (IKK)beta and, to a lesser extent, the IKKalpha subunits of kappaB inhibitor (IkappaB) kinase complex, and that IKKs inhibition by AEA correlates with inhibition of IkappaBalpha degradation, NF-kappaB binding to DNA, and NF-kappaB-dependent transcription in TNFalpha-stimulated cells. AEA also prevents NF-kappaB-dependent reporter gene expression induced by mitogen-activated protein kinase kinase kinase and NF-kappaB-inducing kinase. The NF-kappaB inhibitory activity of AEA was independent of CB(1) and CB(2) activation in TNFalpha-stimulated 5.1 and A549 cell lines, which do not express vanilloid receptor 1, and was not mediated by hydrolytic products formed through the activity of the enzyme fatty acid amide hydrolase. Chemical modification markedly affected AEA inhibitory activity on NF-kappaB, suggesting rather narrow structure-activity relationships and the specific interaction with a molecular target. Substitution of the alkyl moiety with less saturated fatty acids generally reduced or abolished activity. However, replacement of the ethanolamine "head" with a vanillyl group led to potent inhibition of TNFalpha-induced NF-kappaB-dependent transcription. These findings provide new mechanistic insights into the anti-inflammatory and proapoptotic activities of AEA, and should foster the synthesis of improved analogs amenable to pharmaceutical development as anti-inflammatory agents.

  13. Estrogen receptor alpha inhibits RLR-mediated immune response via ubiquitinating TRAF3.

    PubMed

    Wang, Changxing; Huang, Yue; Sheng, Jianzhong; Huang, Hefeng; Zhou, Jun

    2015-10-01

    RIG-I-like receptors (RLRs) function as key sentinel receptor for invading viruses. Moderate activation of RLR signaling is critical for efficient viral clearance without harmful immunopathology. Estrogen receptor alpha (ERα) is a member of the nuclear receptor superfamily of ligand-activated transcription factors and is involved in the regulation of innate immune responses. However, the effects of ERα on RLR signaling and the molecular mechanisms are poorly understood. In this study, we identify ERα as a negative regulator of RLR-triggered antiviral immune responses. The expression level of ERα is upregulated following RLR activation in macrophages. In the absence of ligand, VSV infection phosphorylates ERα at serine 167. ERα inhibits VSV-induced IRF3 activation. We further demonstrate that ERα directly interacts with TRAF3 and promotes K48-linked proteasomal degradation of TRAF3. Consistently, ERα inhibits VSV-triggered IFN-β production in macrophages in a ligand independent mechanism. Thus, ERα functions as a negative feedback regulator of RLR-triggered antiviral immune responses. These findings also provide the insights that separate the immune effects of ERα from its ligand-induced hormonal effects.

  14. Inhibition of RNA synthesis by bradykinin involves both the B1 and B2 receptor subtypes.

    PubMed

    Yau, L; Pinsk, M; Zahradka, P

    1996-04-01

    The efficacy of angiotensin converting enzyme inhibitors in the treatment of heart disease is due in part to the accumulation of bradykinin (BK). Since BK can exert its effect by influencing cell proliferation, we chose to study the effect of BK on the growth of A10 vascular smooth muscle cells. Ligand binding studies to determine which BK receptor subtypes are present on A10 cells showed that both B1 and B2 receptors were present in approximately equal numbers. Examination of RNA synthesis demonstrated that BK inhibits uridine incorporation in a dose-dependent manner. This decrease in RNA synthesis was blocked by both B1 and B2 receptor antagonists, as well as by addition of indomethacin, a cyclooxygenase inhibitor. The latter result suggested that prostaglandins mediate the biological actions of BK. Consequently, we examined the direct effect of two prostaglandins, PGE2 and PGI2 (prostacyclin), on A10 cells. PGE2 caused a decrease in RNA synthesis, thus mimicking the effect of BK, while PGI2 did not. Therefore, the inhibition of RNA synthesis in A10 vascular smooth muscle cells by BK requires both B1 and B2 receptor subtypes and this action of BK is apparently mediated by de novo synthesis of prostaglandins.

  15. Inhibition of Sphingosine-1-phosphate receptors in ischemia reperfusion injured autoimmunity-prone mice.

    PubMed

    Edison, Jess; Frattalone, Sharon; Tracy, Christopher; Woodard, Geoffrey E; Butts, Melissa; Moratz, C M

    2017-01-01

    B6.MRL/lpr mice, an autoimmune strain, have an accelerated injury time course, increased intensity of tissue damage, and increased CD4+ T cell infiltration in the mesenteric ischemia/reperfusion injury model. In this study, the mechanism by which CD4+ T cells were recruited into injured tissue was addressed. Fingolimod (FTY720) was utilized to assess the role of infiltrating CD4+ T cells. FTY720 treatment was more effective in attenuating injury in B6.MRL/lpr mice then in control mice. Reduced CD4+ cell infiltration and tissue injury correlated with decreased neutrophil infiltration and pro-inflammatory cytokine generation. Inhibiting downstream Sphingosine-1-phosphate (S1P) receptor signaling, specifically GαI mediated signaling, did not inhibit injury, suggesting differential utilization of the S1P receptors between control and MRL/lpr strains. Analysis of S1P receptor expression exposed a predominance of S1P2 in the B6.MRL/lpr strain. Reliance on alternate S1P receptors in the autoimmune strain will alter the progress of inflammation and tissue injury.

  16. The Wedelolactone Derivative Inhibits Estrogen Receptor-Mediated Breast, Endometrial, and Ovarian Cancer Cells Growth

    PubMed Central

    Xu, Defeng; Lin, Tzu-Hua; Cheng, Max A.; Chen, Lu-Min; Chang, Chawnshang; Yeh, Shuyuan

    2014-01-01

    Estrogen and estrogen receptor (ER)-mediated signaling pathways play important roles in the etiology and progression of human breast, endometrial, and ovarian cancers. Attenuating ER activities by natural products and their derivatives is a relatively practical strategy to control and reduce breast, endometrial, and ovarian cancer risk. Here, we found 3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one (BTB), a new derivative of wedelolactone, could effectively inhibit the 17-estradiol (E2)-induced ER transactivation and suppress the growth of breast cancer as well as endometrial and ovarian cancer cells. Our results indicate that 2.5 μM BTB effectively suppresses ER-positive, but not ER-negative, breast, endometrial, and ovarian cancer cells. Furthermore, our data indicate that BTB can modulate ER transactivation and suppress the expression of E2-mediated ER target genes (Cyclin D1, E2F1, and TERT) in the ER-positive MCF-7, Ishikawa, and SKOV-3 cells. Importantly, this BTB mediated inhibition of ER activity is selective since BTB does not suppress the activities of other nuclear receptors, including glucocorticoid receptor and progesterone receptor, suggesting that BTB functions as a selective ER signaling inhibitor with the potential to treat breast, endometrial, and ovarian cancers. PMID:25221777

  17. GABA-B receptor activation inhibits the in vitro migration of malignant hepatocytes.

    PubMed

    Lodewyks, Carly; Rodriguez, Jose; Yan, Jing; Lerner, Betty; Lipschitz, Jeremy; Nfon, Charles; Rempel, Julia Darlene; Uhanova, Julia; Minuk, Gerald Yosel

    2011-06-01

    There are conflicting data regarding whether activation of γ-aminobutyric acid-B (GABA-B) receptors results in inhibition of tumor growth and invasion. The objectives of this study were to document the effects of the GABA-B receptor agonist baclofen on malignant hepatocyte proliferation and migration. We also sought to determine whether any effects on cell migration were mediated by changes in cyclic adenosine monophosphate (cAMP) signaling or matrix metalloproteinase (MMP) expression. Finally, GABA-B(1) and -B(2) receptor expression was documented in 2 malignant hepatocyte cell lines (PLC/PRF/5 and Huh-7) and 12 sets of human hepatocellular carcinoma and adjacent nontumor tissues. Cell proliferative activity was documented by WST-1 absorbance, migration by wound healing assays, cAMP levels by enzyme-linked immunoassay (ELISA), MMP by immunohistochemistry and ELISA, and GABA-B receptor expression by flow cytometry and reverse transcriptase - polymerase chain reaction. Although baclofen had no effect on cell proliferation, wound healing was delayed, an effect that was reversed by the GABA-B receptor antagonist CGP. cAMP levels were decreased in Huh-7 but not PLC cells exposed to baclofen. MMP expression remained unaltered in both cell lines. Finally, GABA-B(1) receptor expression was present and consistently expressed, but GABA-B(2) expression was limited and varied with the number of cell passages and (or) duration of culture. In conclusion, activation of GABA-B receptors has no effect on malignant hepatocyte proliferation but does decrease cell migration. This inhibitory effect may involve cAMP signaling but not MMP expression. GABA-B(2) receptor expression is limited and variable, which may help to explain discrepancies with previously published results.

  18. Neuropeptide Y inhibits the trigeminovascular pathway through NPY Y1 receptor: implications for migraine

    PubMed Central

    Oliveira, Margarida-Martins; Akerman, Simon; Tavares, Isaura; Goadsby, Peter J.

    2016-01-01

    Abstract Migraine is a painful neurologic disorder with premonitory symptomatology that can include disturbed appetite. Migraine pathophysiology involves abnormal activation of trigeminocervical complex (TCC) neurons. Neuropeptide Y (NPY) is synthesized in the brain and is involved in pain modulation. NPY receptors are present in trigeminal ganglia and trigeminal nucleus caudalis suggesting a role in migraine pathophysiology. The present study aimed to determine the effect of systemic administration of NPY on TCC neuronal activity in response to dural nociceptive trigeminovascular activation. We performed in vivo electrophysiology in anesthetized rats, administered NPY (10, 30, and 100 µg·kg−1), and investigated the receptors involved by studying NPY Y1 (30 µg·kg−1), Y2 (30 µg·kg−1), and Y5 receptor agonists (100·µg·kg−1), and NPY Y1 receptor antagonist (30 µg·kg−1). NPY (30 and 100 µg·kg−1) significantly reduced TCC neuronal firing in response to dural-evoked trigeminovascular activation, but only NPY (30 µg·kg−1) significantly reduced spontaneous trigeminal firing. NPY Y1 receptor agonist also significantly reduced dural-evoked and spontaneous TCC neuronal firing. NPY (10 µg·kg−1), NPY Y2, and Y5 receptor agonists, and the NPY Y1 receptor antagonist had no significant effects on nociceptive dural-evoked neuronal firing in the TCC or spontaneous trigeminal firing. This study demonstrates that NPY dose dependently inhibits dural-evoked trigeminal activity, through NPY Y1 receptor activation, indicating antinociceptive actions of NPY in a migraine animal model. Based on the role of NPY in appetite regulation, it is possible that disruption of the NPY system might explain changes of appetite in migraineurs. PMID:27023421

  19. Lenalidomide Stabilizes the Erythropoietin Receptor by Inhibiting the E3 Ubiquitin Ligase RNF41.

    PubMed

    Basiorka, Ashley A; McGraw, Kathy L; De Ceuninck, Leentje; Griner, Lori N; Zhang, Ling; Clark, Justine A; Caceres, Gisela; Sokol, Lubomir; Komrokji, Rami S; Reuther, Gary W; Wei, Sheng; Tavernier, Jan; List, Alan F

    2016-06-15

    In a subset of patients with non-del(5q) myelodysplastic syndrome (MDS), lenalidomide promotes erythroid lineage competence and effective erythropoiesis. To determine the mechanism by which lenalidomide promotes erythropoiesis, we investigated its action on erythropoietin receptor (EpoR) cellular dynamics. Lenalidomide upregulated expression and stability of JAK2-associated EpoR in UT7 erythroid cells and primary CD71+ erythroid progenitors. The effects of lenalidomide on receptor turnover were Type I cytokine receptor specific, as evidenced by coregulation of the IL3-Rα receptor but not c-Kit. To elucidate this mechanism, we investigated the effects of lenalidomide on the E3 ubiquitin ligase RNF41. Lenalidomide promoted EpoR/RNF41 association and inhibited RNF41 auto-ubiquitination, accompanied by a reduction in EpoR ubiquitination. To confirm that RNF41 is the principal target responsible for EpoR stabilization, HEK293T cells were transfected with EpoR and/or RNF41 gene expression vectors. Steady-state EpoR expression was reduced in EpoR/RNF41 cells, whereas EpoR upregulation by lenalidomide was abrogated, indicating that cellular RNF41 is a critical determinant of drug-induced receptor modulation. Notably, shRNA suppression of CRBN gene expression failed to alter EpoR upregulation, indicating that drug-induced receptor modulation is independent of cereblon. Immunohistochemical staining showed that RNF41 expression decreased in primary erythroid cells of lenalidomide-responding patients, suggesting that cellular RNF41 expression merits investigation as a biomarker for lenalidomide response. Our findings indicate that lenalidomide has E3 ubiquitin ligase inhibitory effects that extend to RNF41 and that inhibition of RNF41 auto-ubiquitination promotes membrane accumulation of signaling competent JAK2/EpoR complexes that augment Epo responsiveness. Cancer Res; 76(12); 3531-40. ©2016 AACR.

  20. Thiocolchicoside inhibits the activity of various subtypes of recombinant GABA(A) receptors expressed in Xenopus laevis oocytes.

    PubMed

    Mascia, Maria Paola; Bachis, Elisabetta; Obili, Nicola; Maciocco, Elisabetta; Cocco, Giovanni Antonio; Sechi, Gian Pietro; Biggio, Giovanni

    2007-03-08

    Thiocolchicoside is a myorelaxant drug with anti-inflammatory and analgesic properties as well as pronounced convulsant activity. To characterize the mechanisms of action of this drug at the molecular level, we examined its effects on the function of various recombinant neurotransmitter receptors expressed in Xenopus oocytes. Electrophysiological recordings from recombinant human gamma-aminobutyric acid type A (GABA(A)) receptors consisting of alpha1beta1gamma2L, alpha1beta2gamma2L, or alpha2beta2gamma2L subunit combinations revealed that thiocolchicoside inhibited GABA-evoked Cl(-) currents with similar potencies (median inhibitory concentrations of 0.13 to 0.2 microM) and in a competitive manner. Consistent with previous observations, thiocolchicoside also inhibited the binding of GABA to rat cerebral cortical membranes. Thiocolchicoside inhibited the function of recombinant human strychnine-sensitive glycine receptors composed of the alpha1 subunit with a potency (median inhibitory concentration of 47 microM) lower than that apparent with recombinant GABA(A) receptors. It also inhibited the function of human nicotinic acetylcholine receptors composed of the alpha4 and beta2 subunits, but this effect was only partial and apparent at high concentrations. In contrast, thiocolchicoside had no effect on the function of 5-HT(3A) serotonin receptors. Our results thus provide molecular evidence that the epileptogenic activity of thiocolchicoside might be due to inhibition of the function of inhibitory receptors in the central nervous system, especially that of GABA(A) receptors.

  1. Tumor necrosis factor inhibits ligand-stimulated EGF receptor activation through a TNF receptor 1-dependent mechanism

    PubMed Central

    McElroy, Steven J.; Frey, Mark R.; Yan, Fang; Edelblum, Karen L.; Goettel, Jeremy A.; John, Sutha; Polk, D. Brent

    2008-01-01

    Tumor necrosis factor (TNF) and epidermal growth factor (EGF) are key regulators in the intricate balance maintaining intestinal homeostasis. Previous work from our laboratory shows that TNF attenuates ligand-driven EGF receptor (EGFR) phosphorylation in intestinal epithelial cells. To identify the mechanisms underlying this effect, we examined EGFR phosphorylation in cells lacking individual TNF receptors. TNF attenuated EGF-stimulated EGFR phosphorylation in wild-type and TNFR2−/−, but not TNFR1−/−, mouse colon epithelial (MCE) cells. Reexpression of wild-type TNFR1 in TNFR1−/− MCE cells rescued TNF-induced EGFR inhibition, but expression of TNFR1 deletion mutant constructs lacking the death domain (DD) of TNFR1 did not, implicating this domain in EGFR downregulation. Blockade of p38 MAPK, but not MEK, activation of ERK rescued EGF-stimulated phosphorylation in the presence of TNF, consistent with the ability of TNFR1 to stimulate p38 phosphorylation. TNF promoted p38-dependent EGFR internalization in MCE cells, suggesting that desensitization is achieved by reducing receptor accessible to ligand. Taken together, these data indicate that TNF activates TNFR1 by DD- and p38-dependent mechanisms to promote EGFR internalization, with potential impact on EGF-induced proliferation and migration key processes that promote healing in inflammatory intestinal diseases. PMID:18467504

  2. Phorbol ester stimulates secretory activity while inhibiting receptor-activated aminopyrine uptake by gastric glands

    SciTech Connect

    Brown, M.R.; Chew, C.S.

    1986-03-05

    Both cyclic AMP-dependent and -independent secretagogues stimulate pepsinogen release, respiration and H/sup +/ secretory activity (AP uptake) in rabbit gastric glands. 12-O-tetradecanoylphorbol-13-acetate (T), a diacyglycerol analog, activates protein kinase C (PKC) and stimulates secretion in many systems. T stimulated respiration and pepsinogen release by glands and increased AP uptake by both glands and purified parietal cells. However, T reduced AP uptake by glands stimulated with carbachol (C) or histamine (H) with an apparent IC/sub 50/ of 1 nM. Preincubation with T for 30 min produced maximum inhibition which was not reversed by removal of T. T accelerated the decline of the transient C peak while the late steady state response to H was most inhibited. H-stimulated AP uptake was also inhibited by 50 ..mu..g/ml 1-oleoyl-2-acetyl-glycerol, a reported PKC activator, but not by the inactive phorbol, 4..cap alpha..-phorbol-12,13-didecanoate. In contrast, T potentiated AP uptake by glands stimulated with submaximal doses of dibutyryl cyclic AMP. These results suggest inhibition by T is a specific effect of PKC activators. The differing effects of T on secretion indicators may result from a dual action of T on receptor and post-receptor intracellular events.

  3. Bispecific designed ankyrin repeat proteins (DARPins) targeting epidermal growth factor receptor inhibit A431 cell proliferation and receptor recycling.

    PubMed

    Boersma, Ykelien L; Chao, Ginger; Steiner, Daniel; Wittrup, K Dane; Plückthun, Andreas

    2011-12-02

    The EGF receptor (EGFR) has been implicated in the development and progression of many tumors. Although monoclonal antibodies directed against EGFR have been approved for the treatment of cancer in combination with chemotherapy, there are limitations in their clinical efficacy, necessitating the search for robust targeting molecules that can be equipped with new effector functions or show a new mechanism of action. Designed ankyrin repeat proteins (DARPins) may provide the targeting component for such novel reagents. Previously, four DARPins were selected against EGFR with (sub)nanomolar affinity. As any targeting module should preferably be able to inhibit EGFR-mediated signaling, their effect on A431 cells overexpressing EGFR was examined: three of them were shown to inhibit proliferation by inducing G(1) arrest, as seen for the Food and Drug Administration-approved antibody cetuximab. To understand this inhibitory mechanism, we mapped the epitopes of the DARPins using yeast surface display. The epitopes for the biologically active DARPins overlapped with the EGF-binding site, whereas the fourth DARPin bound to a different domain, explaining the lack of a biological effect. To optimize the biological activity of the DARPins, we combined two DARPins binding to different epitopes with a flexible linker or with a leucine zipper, leading to a homodimer. The latter DARPin was able to reduce surface EGFR by inhibiting receptor recycling, leading to a dramatic decrease in cell viability. These results indicate that multispecific EGFR-specific DARPins are superior to cetuximab and may form the basis of new opportunities in tumor targeting and tumor therapy.

  4. Role of Nicotinic Acetylcholine Receptor on Efferent Inhibition in Cochlear Hair Cell

    PubMed Central

    2012-01-01

    The α9α10 nicotinic acetylcholine receptors (nAChRs) mediates efferent inhibition of hair cell function within the auditory sensory organ. Gating of the nAChRs leads to activation of calcium-dependent potassium channels to hyperpolarize the hair cell. In efferent system, main calcium providers to SK channel are nAChR and synaptic cistern, which contribution to efferent inhibition is different between avian and mammalian species. Calcium permeation is more effective in nAChRs of mammalian cochlea than avian cochlea, and mammalian calcium permeability of nAChRs is about 3 times more than avian hair cell. Thus, mammalian nAChRs is a main component of efferent inhibition in cochlear hair cell system. PMID:24653883

  5. Activation and inhibition of rat neuronal nicotinic receptors by ABT-418

    PubMed Central

    Papke, Roger L; Thinschmidt, Jeffrey S; Moulton, Becky A; Meyer, Edwin M; Poirier, Amy

    1997-01-01

    ABT-418 appeared to function as a relatively broad spectrum activator of neuronal nicotinic receptors, expressed in Xenopus oocytes, with little cross reactivity to the mammalian muscle receptor subtype. However, the relative potencies of ABT-418 at the various subtypes differed from those acetylcholine (ACh). For example, ACh was most potent at α3β2 (EC50≈30 μM) and least potent at α2β2 (EC50≈500 μM). ABT-418 was most potent at α4β2 and α2β2 (EC50≈6 μM and 11 μM, respectively) and least potent at α3β4 (EC50≈188 μM).In addition to activating neuronal receptors, ABT-418 exhibited complex properties, including the inhibition of ACh responses.The current responses elicited by relatively high concentrations of ABT-418 on the α4β2 receptor subtype were protracted beyond the application interval. The coapplication of ABT-418 with either of the use-dependent inhibitors bis(1,2,2,6,6-tetramethyl-4-pipendimyl)sebacate (BTMPS) or tetramethyl-pipenidine (TMP) eliminated the late protracted phase of the currents with only small effects on the initial activation phase. When the reversible inhibitor TMP was washed from the bath, the previously inhibited late current reappeared, suggesting that the observed mixed agonist-antagonist effects of ABT-418 and (±)-epibatidine on α4β2 were due to a concentration-dependent noncompetitive inhibition, an effect similar to that obtained for (−)-nicotine.The inhibition of α4β2 receptors by ABT-418 was voltage-dependent. When high concentrations of ABT-418 were applied under depolarizing conditions, additional late currents could be observed under conditions which suggested that a build up of ABT-418 in an unstirred layer over the surface of the oocyte was occurring. This may have been due to the dissociation of the drug from channel blocking sites on the receptors themselves, or alternatively, from the plasma membrane of the cells. PMID:9031746

  6. Mechanisms of anabolic androgenic steroid inhibition of mammalian ɛ-subunit-containing GABAA receptors

    PubMed Central

    Jones, Brian L; Whiting, Paul J; Henderson, Leslie P

    2006-01-01

    GABAergic transmission regulates the activity of gonadotrophin-releasing hormone (GnRH) neurons in the preoptic area/hypothalamus that control the onset of puberty and the expression of reproductive behaviours. One of the hallmarks of illicit use of anabolic androgenic steroids (AAS) is disruption of behaviours under neuroendocrine control. GnRH neurons are among a limited population of cells that express high levels of the ɛ-subunit of the GABAA receptor. To better understand the actions of AAS on neuroendocrine mechanisms, we have characterized modulation of GABAA receptor-mediated currents in mouse native GnRH neurons and in heterologous cells expressing recombinant α2β3ɛ-receptors. GnRH neurons exhibited robust currents in response to millimolar concentrations of GABA and a picrotoxin (PTX)-sensitive, bicuculline-insensitive current that probably arises from spontaneous openings of GABAA receptors. The AAS 17α-methyltestosterone (17α-MeT) inhibited spontaneous and GABA-evoked currents in GnRH neurons. For recombinant α2β3ɛ-receptors, 17α-MeT inhibited phasic and tonic GABA-elicited responses, accelerated desensitization and slowed paired pulse response recovery. Single channel analysis indicated that GABA-evoked events could be described by three open dwell components and that 17α-MeT enhanced residence in the intermediate dwell state. This AAS also inhibited a PTX-sensitive, spontaneous current (open probability, ∼0.15–0.2) in a concentration-dependent fashion (IC50 ≈ 9 μm). Kinetic modelling indicated that the inhibition induced by 17α-MeT occurs by an allosteric block in which the AAS interacts preferentially with a closed state and promotes accumulation in that state. Finally, studies with a G302S mutant ɛ-subunit suggest that this residue within the transmembrane domain TM2 plays a role in mediating AAS binding and modulation. In sum, our results indicate that inclusion of the ɛ-subunit significantly alters the profile of AAS

  7. The activation of liver X receptors inhibits toll-like receptor-9-induced foam cell formation.

    PubMed

    Sorrentino, Rosalinda; Morello, Silvana; Chen, Shuang; Bonavita, Eduardo; Pinto, Aldo

    2010-04-01

    Toll-like receptors (TLRs) are related to foam cell formation (FCF), key event in the establishment/progression of atherosclerosis. The activation of TLR2 and TLR4 can increase FCF. The aim of this study was to evaluate the role of TLR9 in FCF. Murine macrophages were treated with CpG-ODN, TLR9 agonist, and oxidized particles of LDL (Paz-PC) and FCF was analyzed by means of Oil Red O staining. The administration of CpG-ODN plus Paz-PC onto macrophages increased the amount of lipid droplets, correlated to increased levels of tumor necrosis factor (TNF)-alpha, IFNbeta, and IP-10. The underlying mechanism by which TLR9 ligation influenced Paz-PC in the FCF was NF-kappaB- and IRF7-dependent, as observed by higher levels of phosphorylated IkappaBalpha, increased nuclear translocation of the p65 subunit, lower levels of the total IKKalpha protein and higher release of interferon-dependent cytokines, such as IP-10. Liver X receptors (LXRs) regulate lipid cellular transport and negatively modulate TLR-dependent signaling pathways. Indeed, the addition of GW3965, synthetic LXRs agonist, significantly reduced FCF after CpG-ODN plus Paz-PC stimulation. In this condition, we observed decreased levels of the nuclear translocation of the p65 subunit, related to the higher presence of LXRalpha into the nucleus. TNF-alpha, IP-10, and IFNbeta levels were reduced by the administration of GW3965 following CpG-ODN and Paz-PC treatment. In conclusion, the activation of TLR9 facilitates the formation of foam cells in an NF-kappaB- and IRF7-dependent manner, countered by the activation of LXRs. This study further support LXRs as potential anti-atherosclerotic target.

  8. GABA(A) receptor activation is involved in noncontingent shock inhibition of instrumental conditioning in spinal rats.

    PubMed

    Ferguson, Adam R; Washburn, Stephanie N; Crown, Eric D; Grau, James W

    2003-08-01

    Previous work has demonstrated that the spinal cord, isolated from higher neural structures, can support a simple form of instrumental learning. Furthermore, preexposure to uncontrollable (noncontingent) shock to the leg or tail inhibits this form of learning. The present study explores the role of GABA(A) receptor modulation on this inhibitory effect in spinal cord-transected rats. Intrathecal administration of the GABA(A) receptor antagonist bicuculline blocked induction and expression of the inhibition. The GABA(A) receptor agonist muscimol inhibited learning in a dose-dependent manner. However, this effect was transient and showed no additivity with shock. The findings suggest that GABA(A) receptor activation may work like a pharmacological switch that is activated by noncontingent shock to inhibit instrumental conditioning within the spinal cord.

  9. Ethanol inhibits excitatory neurotransmission in the nucleus accumbens of adolescent mice through GABAA and GABAB receptors.

    PubMed

    Mishra, Devesh; Chergui, Karima

    2013-07-01

    Age-related differences in various acute physiological and behavioral effects of alcohol have been demonstrated in humans and in other species. Adolescents are more sensitive to positive reinforcing properties of alcohol than adults, but the cellular mechanisms that underlie such a difference are not clearly established. We, therefore, assessed age differences in the ability of ethanol to modulate glutamatergic synaptic transmission in the mouse nucleus accumbens (NAc), a brain region importantly involved in reward mechanisms. We measured field excitatory postsynaptic potentials/population spikes (fEPSP/PS) in NAc slices from adolescent (22-30 days old) and adult (5-8 months old) male mice. We found that 50mM ethanol applied in the perfusion solution inhibits glutamatergic neurotransmission in the NAc of adolescent, but not adult, mice. This effect is blocked by the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline and by the GABAB receptor antagonist CGP 55845. Furthermore, bicuculline applied alone produces a stronger increase in the fEPSP/PS amplitude in adult mice than in adolescent mice. Activation of GABAA receptors with muscimol produces a stronger and longer lasting depression of neurotransmission in adolescent mice as compared with adult mice. Activation of GABAB receptors with SKF 97541 also depresses neurotransmission more strongly in adolescent than in adult mice. These results demonstrate that an increased GABA receptor function associated with a reduced inhibitory tone underlies the depressant action of ethanol on glutamatergic neurotransmission in the NAc of adolescent mice.

  10. Inhibition of kinin B1 receptors attenuates pulmonary hypertension and vascular remodeling.

    PubMed

    Murugesan, Priya; Hildebrandt, Tobias; Bernlöhr, Christian; Lee, Dongwon; Khang, Gilson; Doods, Henri; Wu, Dongmei

    2015-10-01

    This study examined whether the kinin B1 receptor is involved in the pathogenesis of pulmonary hypertension, and whether its inhibition could reduce inflammation, pulmonary hypertension, vascular remodeling, and right heart dysfunction. Male Wistar rats underwent left pneumonectomy. Seven days later, the rats were injected subcutaneously with monocrotaline (60 mg/kg). The rats were then randomly assigned to receive treatment with vehicle or with BI113823 (a selective B1 receptor antagonist, 30 mg/kg, twice per day) via oral gavage from the day of monocrotaline injection to day 28. By day 28, BI113823-treated rats had significantly lower mean pulmonary artery pressure, less right ventricular hypertrophy, and pulmonary arterial neointimal formation than that of the vehicle-treated rats. Real-time polymerase chain reaction revealed that there was a significant increase in mRNA expression of B1 receptors in the lungs of monocrotaline-challenged pneumonectomized rats. Treatment with BI113823 significantly reduced macrophage recruitment, as measured via bronchoalveolar lavage. It also markedly reduced CD-68 positive macrophages and proliferating cell nuclear antigen positive cells in the perivascular areas, reduced expression of inducible nitric oxide synthase, matrix metalloproteinase 2 and 9, and B1 receptors compared with measurements in vehicle-treated rats. These findings demonstrate that kinin B1 receptors represent a novel therapeutic target for pulmonary arterial hypertension.

  11. 5-HT2B Receptor Antagonists Inhibit Fibrosis and Protect from RV Heart Failure

    PubMed Central

    Janssen, Wiebke; Schymura, Yves; Novoyatleva, Tatyana; Luitel, Himal; Tretyn, Aleksandra; Pullamsetti, Soni Savai; Weissmann, Norbert; Seeger, Werner; Ghofrani, Hossein Ardeschir; Schermuly, Ralph Theo

    2015-01-01

    Objective. The serotonin (5-HT) pathway was shown to play a role in pulmonary hypertension (PH), but its functions in right ventricular failure (RVF) remain poorly understood. The aim of the current study was to investigate the effects of Terguride (5-HT2A and 2B receptor antagonist) or SB204741 (5-HT2B receptor antagonist) on right heart function and structure upon pulmonary artery banding (PAB) in mice. Methods. Seven days after PAB, mice were treated for 14 days with Terguride (0.2 mg/kg bid) or SB204741 (5 mg/kg day). Right heart function and remodeling were assessed by right heart catheterization, magnetic resonance imaging (MRI), and histomorphometric methods. Total secreted collagen content was determined in mouse cardiac fibroblasts isolated from RV tissues. Results. Chronic treatment with Terguride or SB204741 reduced right ventricular fibrosis and showed improved heart function in mice after PAB. Moreover, 5-HT2B receptor antagonists diminished TGF-beta1 induced collagen synthesis of RV cardiac fibroblasts in vitro. Conclusion. 5-HT2B receptor antagonists reduce collagen deposition, thereby inhibiting right ventricular fibrosis. Chronic treatment prevented the development and progression of pressure overload-induced RVF in mice. Thus, 5-HT2B receptor antagonists represent a valuable novel therapeutic approach for RVF. PMID:25667920

  12. MiR-34a inhibits colon cancer proliferation and metastasis by inhibiting platelet-derived growth factor receptor α.

    PubMed

    Li, Chunyan; Wang, Yulin; Lu, Shuming; Zhang, Zhuqing; Meng, Hua; Liang, Lina; Zhang, Yan; Song, Bo

    2015-11-01

    The microRNA (miRNA), miR‑34a is significant in colon cancer progression. In the present study, the role of miR‑34a in colon cancer cell proliferation and metastasis was investigated. It was found that the expression of miR‑34a in colon cancer tissues and cell lines was lower when compared with that of normal tissues and cells. Further research demonstrated that miR‑34a inhibited cell proliferation, induced G1 phase arrest, and suppressed metastasis and epithelial mesenchymal transition in colon cancer cells. Bioinformatic prediction indicated that platelet‑derived growth factor receptor α (PDGFRA) was a potential target gene of miR‑34a and a luciferase assay identified that PDGFRA was a novel direct target gene of miR‑34a. In addition, assays of western blot analyses and quantitative reverse‑transcription polymerase chain reaction confirmed that miR‑34a decreased PDGFRA mRNA expression and protein levels in colon cancer cells. Assessment of cellular function indicated that miR‑34a inhibited colon cancer progression via PDGFRA. These findings demonstrate that miR‑34a may act as a negative regulator in colon cancer by targeting PDGFRA.

  13. Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines

    PubMed Central

    Ring, Avi; Strom, Bjorn Oddvar; Turner, Simon R.; Timperley, Christopher M.; Bird, Michael; Green, A. Christopher; Chad, John E.; Worek, Franz; Tattersall, John E. H.

    2015-01-01

    Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase), but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21) and neuronal (SH-SY5Y) cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning. PMID:26274808

  14. The immunosuppressant FTY720 inhibits tumor angiogenesis via the sphingosine 1-phosphate receptor 1.

    PubMed

    Schmid, Gerald; Guba, Markus; Ischenko, Ivan; Papyan, Armine; Joka, Mareile; Schrepfer, Sabine; Bruns, Christiane J; Jauch, Karl-Walter; Heeschen, Christopher; Graeb, Christian

    2007-05-01

    FTY720, a sphingosine 1-phosphate (S1P) analog, acts as an immunosuppressant through trapping of T cells in secondary lymphoid tissues. FTY720 was also shown to prevent tumor growth and to inhibit vascular permeability. The MTT proliferation assay illustrated that endothelial cells are more susceptible to the anti-proliferative effect of FTY720 than Lewis lung carcinoma (LLC1) cells. In a spheroid angiogenesis model, FTY720 potently inhibited the sprouting activity of VEGF-A-stimulated endothelial cells even at concentrations that apparently had no anti-proliferative effect. Mechanistically, the anti-angiogenic effect of the general S1P receptor agonist FTY720 was mimicked by the specific S1P1 receptor agonist SEW2871. Moreover, the anti-angiogenic effect of FTY720 was abrogated in the presence of CXCR4-neutralizing antibodies. This indicates that the effect was at least in part mediated by the S1P1 receptor and involved transactivation of the CXCR4 chemokine receptor. Additionally, we could illustrate in a coculture spheroid model, employing endothelial and smooth muscle cells (SMCs), that the latter confer a strong protective effect regarding the action of FTY720 upon the endothelial cells. In a subcutaneous LLC1 tumor model, the anti-angiogenic capacity translated into a reduced tumor size in syngeneic C57BL/6 mice. Consistently, in the Matrigel plug in vivo assay, 10 mg/kg/d FTY720 resulted in a strong inhibition of angiogenesis as demonstrated by a reduced capillary density. Thus, in organ transplant patients, FTY720 may prove efficacious in preventing graft rejection as well as tumor development.

  15. Direct inhibition of the N-methyl-D-aspartate receptor channel by dopamine and (+)-SKF38393.

    PubMed

    Castro, N G; de Mello, M C; de Mello, F G; Aracava, Y

    1999-04-01

    1. Dopamine is known to modulate glutamatergic synaptic transmission in the retina and in several brain regions by activating specific G-protein-coupled receptors. We have examined the possibility of a different type of mechanism for this modulation, one involving direct interaction of dopamine with ionotropic glutamate receptors. 2. Ionic currents induced by fast application of N-methyl-D-aspartate (NMDA) were recorded under whole-cell patch-clamp in cultured striatal, thalamic and hippocampal neurons of the rat and in retinal neurons of the chick. Dopamine at concentrations above 100 microM inhibited the NMDA response in all four neuron types, exhibiting an IC50 of 1.2 mM in hippocampal neurons. The time course of this inhibition was fast, developing in less than 100 ms. 3. The D1 receptor agonist (+)-SKF38393 mimicked the effect of dopamine, with an IC50 of 58.9 microM on the NMDA response, while the enantiomer (-)-SKF38393 was ineffective at 50 microM. However, the D1 antagonist R(+)-SCH23390 did not prevent the inhibitory effect of (+)-SKF38393. 4. The degree of inhibition by dopamine and (+)-SKF38393 depended on transmembrane voltage, increasing 2.7 times with a hyperpolarization of about 80 mV. The voltage-dependent block by dopamine was also observed in the presence of MgCl2 1 mM. 5. Single-channel recordings showed that the open times of NMDA-gated channels were shortened by (+)-SKF38393. 6. These data suggested that the site to which the drugs bound to produce the inhibitory effect was distinct from the classical D1-type dopamine receptor sites, possibly being located inside the NMDA channel pore. It is concluded that dopamine and (+)-SKF38393 are NMDA channel ligands.

  16. Direct inhibition of the N-methyl-D-aspartate receptor channel by dopamine and (+)-SKF38393

    PubMed Central

    Castro, Newton G; de Mello, Maria Christina F; de Mello, Fernando G; Aracava, Yasco

    1999-01-01

    Dopamine is known to modulate glutamatergic synaptic transmission in the retina and in several brain regions by activating specific G-protein-coupled receptors. We have examined the possibility of a different type of mechanism for this modulation, one involving direct interaction of dopamine with ionotropic glutamate receptors.Ionic currents induced by fast application of N-methyl-D-aspartate (NMDA) were recorded under whole-cell patch-clamp in cultured striatal, thalamic and hippocampal neurons of the rat and in retinal neurons of the chick. Dopamine at concentrations above 100 μM inhibited the NMDA response in all four neuron types, exhibiting an IC50 of 1.2 mM in hippocampal neurons. The time course of this inhibition was fast, developing in less than 100 ms.The D1 receptor agonist (+)-SKF38393 mimicked the effect of dopamine, with an IC50 of 58.9 μM on the NMDA response, while the enantiomer (−)-SKF38393 was ineffective at 50 μM. However, the D1 antagonist R(+)-SCH23390 did not prevent the inhibitory effect of (+)-SKF38393.The degree of inhibition by dopamine and (+)-SKF38393 depended on transmembrane voltage, increasing 2.7 times with a hyperpolarization of about 80 mV. The voltage-dependent block by dopamine was also observed in the presence of MgCl2 1 mM.Single-channel recordings showed that the open times of NMDA-gated channels were shortened by (+)-SKF38393.These data suggested that the site to which the drugs bound to produce the inhibitory effect was distinct from the classical D1-type dopamine receptor sites, possibly being located inside the NMDA channel pore. It is concluded that dopamine and (+)-SKF38393 are NMDA channel ligands. PMID:10372829

  17. Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain.

    PubMed

    Strick, Christine A; Li, Cheryl; Scott, Liam; Harvey, Brian; Hajós, Mihály; Steyn, Stefanus J; Piotrowski, Mary A; James, Larry C; Downs, James T; Rago, Brian; Becker, Stacey L; El-Kattan, Ayman; Xu, Youfen; Ganong, Alan H; Tingley, F David; Ramirez, Andres D; Seymour, Patricia A; Guanowsky, Victor; Majchrzak, Mark J; Fox, Carol B; Schmidt, Christopher J; Duplantier, Allen J

    2011-01-01

    Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents.

  18. Development of a New Class of Drugs to Inhibit All Forms of Androgen Receptor in Castration-Resistant Prostate Cancers

    DTIC Science & Technology

    2015-10-01

    AWARD NUMBERS: W81XWH-14-1-0519 TITLE: Development of a New Class of Drugs to Inhibit All Forms of Androgen Receptor in Castration-Resistant...of Drugs to Inhibit All Forms of Androgen Receptor in Castration-Resistant Prostate Cancers 5b. GRANT NUMBER W81XWH-14- 1-0519 5c. PROGRAM...castration. However, despite a growing armamentarium of drugs targeting the androgen/AR signaling axis, progression of castration-resistant prostate

  19. Presynaptic Inhibition of Glutamate Transmission by Alpha-2 Receptors in the VTA

    PubMed Central

    Jiménez-Rivera, Carlos A.; Figueroa, Johnny; Vázquez, Rafael; Vélez, María; Schwarz, David; Velásquez-Martinez, María C.; Arencibia-Albite, Francisco

    2013-01-01

    The ventral tegmental area (VTA) forms part of the mesocorticolimbic system and plays a pivotal role in reward and reinforcing actions of drugs of abuse. Glutamate transmission within the VTA controls important aspects of goal-directed behavior and motivation. Noradrenergic receptors also present in the VTA have important functions in the modulation of neuronal activity. Here we studied the effects of alpha-2 noradrenergic receptor activation in the alteration of glutamate neurotransmission in VTA dopaminergic neurons from male Sprague-Dawley rats. We used whole cell patch clamp recordings from putative VTA dopaminergic neurons and measured excitatory postsynaptic currents. Clonidine (40 μM) and UK 13,408 (40 μM), both alpha-2 receptor agonists, reduced (~ 40%) the amplitude of glutamate-induced excitatory postsynaptic currents. After clonidine administration, there was a dose-dependent reduction over the concentration range of 15–40 μM. Using yohimbine (20μM) and two other alpha-2 adrenergic receptor antagonists, idaxozan (40 μM) and atipemazole (20μM), we demonstrated that the inhibitory action is specifically mediated by alpha-2 receptors. Moreover, by inhibiting protein kinases with H-7 (75 μM), Rp-adenosine 3′,5′-cyclic (11 μM) and chelerythrine (1 μM) it was shown that the clonidine-induced inhibition seems to involve a selective activation of the protein kinase C intracellular pathway. An increased paired-pulse ratios and changes in spontaneous and miniature excitatory postsynaptic currents frequencies but not amplitudes indicated that the alpha-2 agonist’s effect was presynaptically mediated. It is suggested that the suppression of glutamate excitatory inputs onto VTA dopaminergic neurons might be relevant in the regulation of reward and drug seeking behaviors. PMID:22564071

  20. Uridine Triphosphate Thio Analogues Inhibit Platelet P2Y12 Receptor and Aggregation

    PubMed Central

    Gündüz, Dursun; Tanislav, Christian; Sedding, Daniel; Parahuleva, Mariana; Santoso, Sentot; Troidl, Christian; Hamm, Christian W.; Aslam, Muhammad

    2017-01-01

    Platelet P2Y12 is an important adenosine diphosphate (ADP) receptor that is involved in agonist-induced platelet aggregation and is a valuable target for the development of anti-platelet drugs. Here we characterise the effects of thio analogues of uridine triphosphate (UTP) on ADP-induced platelet aggregation. Using human platelet-rich plasma, we demonstrate that UTP inhibits P2Y12 but not P2Y1 receptors and antagonises 10 µM ADP-induced platelet aggregation in a concentration-dependent manner with an IC50 value of ~250 µM. An eight-fold higher platelet inhibitory activity was observed with a 2-thio analogue of UTP (2S-UTP), with an IC50 of 30 µM. The 4-thio analogue (4S-UTP) with an IC50 of 7.5 µM was 33-fold more effective. A three-fold decrease in inhibitory activity, however, was observed by introducing an isobutyl group at the 4S- position. A complete loss of inhibition was observed with thio-modification of the γ phosphate of the sugar moiety, which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y12 receptor was verified by P2Y12 receptor binding and cyclic AMP (cAMP) assays. These novel data demonstrate for the first time that 2- and 4-thio analogues of UTP are potent P2Y12 receptor antagonists that may be useful for therapeutic intervention. PMID:28146050

  1. Selective inhibition of beta(2)-adrenergic receptor-mediated cAMP generation by activation of the P2Y(2) receptor in mouse pineal gland tumor cells.

    PubMed

    Suh, B C; Kim, J S; Namgung, U; Han, S; Kim, K T

    2001-06-01

    Rhythmic noradrenergic signaling from the hypothalamic clock in the suprachiasmatic nucleus to the pineal gland causes an increase in intracellular cAMP which regulates the circadian fluctuation of melatonin synthesis. The activation of phospholipase C (PLC)-coupled P2Y(2) receptors upon treatment with ATP and UTP exclusively inhibited the isoproterenol-stimulated cAMP production in mouse pineal gland tumor cells. However, the activation of other PLC-coupled receptors including P2Y(1) and bombesin receptors had little or no effect on the isoproterenol-stimulated cAMP production. Also, ATP did not inhibit cAMP production caused by forskolin, prostaglandin E(2), or the adenosine analog NECA. These results suggest a selective coupling between signalings of P2Y(2) and beta(2)-adrenergic receptors. The binding of [(3)H]CGP12177 to beta(2)-adrenergic receptors was not effected by the presence of ATP or UTP. Ionomycin decreased the isoproterenol-stimulated cAMP production, whereas phorbol 12-myristate 13-acetate slightly potentiated the isoproterenol response. Chelation of intracellular Ca(2+), however, had little effect on the ATP-induced inhibition of cAMP production, while it completely reversed the ionomycin-induced inhibition. Treatment of cells with pertussis toxin almost completely blocked the inhibitory effect of nucleotides. Pertussis toxin also inhibited the nucleotide-induced increase in intracellular Ca(2+) and inositol 1,4,5-trisphosphate production by 30-40%, suggesting that the ATP-mediated inhibition of the cAMP generation and the partial activation of PLC are mediated by pertussis toxin-sensitive G(i)-protein. We conclude that one of the functions of P2Y(2) receptors on the pineal gland is the selective inhibition of beta-adrenergic receptor-mediated signaling pathways via the inhibitory G-proteins.

  2. Activation of histamine H3-receptors inhibits carrier-mediated norepinephrine release during protracted myocardial ischemia. Comparison with adenosine A1-receptors and alpha2-adrenoceptors.

    PubMed

    Imamura, M; Lander, H M; Levi, R

    1996-03-01

    We previously showed that prejunctional histamine H3-receptors downregulate norepinephrine exocytosis, which is markedly enhanced in early myocardial ischemia. In the present study, we investigated whether H3-receptors modulate nonexocytotic norepinephrine release during protracted myocardial ischemia. In this setting, decreased pH(i) in sympathetic nerve endings sequentially leads to a compensatory activation of the Na+-H+ antiporter (NHE), accumulation of intracellular Na+, reversal of the neuronal uptake of norepinephrine, and thus carrier-mediated release of norepinephrine. Accordingly, norepinephrine overflow from isolated guinea pig hearts undergoing 20-minute global ischemia and 45-minute reperfusion was attenuated approximately 80% by desipramine (10 nmol/L) and 70% by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 10 micromol/L), inhibitors of norepinephrine uptake and NHE, respectively. The H3-receptor agonist imetit (0.1 micromol/L) decreased carrier-mediated norepinephrine release by approximately 50%. This effect was blocked by the H3-receptor antagonist thioperamide (0.3 micromol/L), indicating that H-receptor activation inhibits carrier-mediated norepinephrine release. At lower concentrations, imetit (10 nmol/L) or EIPA (3 micromol/L) did not inhibit carrier-mediated norepinephrine release. However, a 25% inhibition occurred with imetit (10 nmol/L) and EIPA (3 micromol/L) combined. This synergism suggests an association between H-receptors and NHE. Conceivably, activation of H-receptors may lead to inhibition of NHE. In fact, alpha2-adrenoceptor activation, which is known to stimulate NHE, enhanced norepinephrine release, whereas alpha2-adrenoceptor blockade attenuated it. Furthermore, activation of adenosine A1-receptors markedly attenuated norepinephrine release, whereas their inhibition potentiated it. Because norepinephrine directly correlated with the severity of reperfusion arrhythmia and imetit reduced the incidence of ventricular fibrillation by 50

  3. Intrathecal alpha2 adrenoceptor agonist clonidine inhibits mechanical transmission in mouse spinal cord via activation of muscarinic M1 receptors.

    PubMed

    Honda, Kenji; Koga, Kohei; Moriyama, Tomoko; Koguchi, Masako; Takano, Yukio; Kamiya, Hiro-o

    2002-04-12

    We examined the role of the spinal muscarinic receptor subtype in the anti-nociceptive effect of intrathecal (i.t.) alpha2 adrenoceptor agonist clonidine in mice. I.t. injection of the muscarinic receptor antagonist atropine completely inhibited i.t. clonidine-induced increase in the mechanical threshold, but did not affect the increase in tail-flick latency induced by i.t. clonidine. The clonidine-induced increase in mechanical threshold was inhibited by i.t. injection of the M1 receptor antagonist pirenzepine in a dose-dependent manner, and by the M3 receptor antagonist 4-DAMP, but not by the M2 receptor antagonist methoctramine. The potency of pirenzepine was greater than that of 4-DAMP. These results suggest that the clonidine-induced increase in mechanical threshold is mediated via the activation of M1 receptors in the spinal cord.

  4. Structure-Activity Relationship of Indole-Tethered Pyrimidine Derivatives that Concurrently Inhibit Epidermal Growth Factor Receptor and Other Angiokinases

    PubMed Central

    Song, Jiho; Yoo, Jakyung; Kwon, Ara; Kim, Doran; Nguyen, Hong Khanh; Lee, Bong-Yong; Suh, Wonhee; Min, Kyung Hoon

    2015-01-01

    Antiangiogenic agents have been widely investigated in combination with standard chemotherapy or targeted cancer agents for better management of advanced cancers. Therapeutic agents that concurrently inhibit epidermal growth factor receptor and other angiokinases could be useful alternatives to combination therapies for epidermal growth factor receptor-dependent cancers. Here, we report the synthesis of an indole derivative of pazopanib using a bioisosteric replacement strategy, which was designated MKP101. MKP101 inhibited not only the epidermal growth factor receptor with an IC50 value of 43 nM but also inhibited angiokinases as potently as pazopanib. In addition, MKP101 effectively inhibited vascular endothelial growth factor-induced endothelial proliferation, tube formation, migration of human umbilical vein endothelial cells and proliferation of HCC827, an epidermal growth factor receptor-addicted cancer cell line. A docking model of MKP101 and the kinase domain of the epidermal growth factor receptor was generated to predict its binding mode, and validated by synthesizing and evaluating MKP101 derivatives. Additionally, a study of structure-activity relationships of indolylamino or indolyloxy pyrimidine analogues derived from MKP101 demonstrated that selectivity for epidermal growth factor receptor and other angiokinases, especially vascular endothelial growth factor receptor 2 depends on the position of substituents on pyrimidine and the type of link between pyrimidine and the indole moiety. We believe that this study could provide a basis for developing angiokinase inhibitors having high affinity for the epidermal growth factor receptor, from the pyrimidine scaffold. PMID:26401847

  5. Error correction in latent inhibition and its disruption by opioid receptor blockade with naloxone.

    PubMed

    Leung, Hiu T; Killcross, A S; Westbrook, R Frederick

    2013-11-01

    Latent inhibition refers to the retardation in the development of conditioned responding when a pre-exposed stimulus is used to signal an unconditioned stimulus. This effect is described by error-correction models as an attentional deficit and is commonly used as an animal model of schizophrenia. A series of experiments studied the role of error-correction mechanism in latent inhibition and its interaction with the endogenous opioid system. Systemic administration of the competitive opioid receptor antagonist naloxone before rats were pre-exposed to a target stimulus prevented latent inhibition of its subsequent fear conditioning; it was without effect on a non-pre-exposed stimulus and did not produce state-dependent learning (Experiments 1a and 1b). Naloxone did not reverse the latent inhibitory effect already accrued to a pre-exposed target. However, it did prevent the enhancement of latent inhibition by a long retention interval interpolated between its initial exposure and re-exposure (Experiment 2) or by a novel stimulus compounded with the pre-exposed target during re-exposure (Experiment 3). These results provide evidence that attentional loss in latent inhibition is instructed by an opioid-mediated error signal which diminishes with repeated stimulus exposures but recovers with the passage of time or reintroduction of novelty.

  6. Soluble human complement receptor type 1 inhibits complement-mediated host defense.

    PubMed

    Swift, A J; Collins, T S; Bugelski, P; Winkelstein, J A

    1994-09-01

    Soluble complement receptor type 1 (sCR1) is a powerful inhibitor of complement activation. Because of this ability, sCR1 may prove to be an important therapeutic agent that can be used to block the immunopathologic effects of uncontrolled complement activation in a variety of clinically significant disorders. Although several previous studies have examined the ability of sCR1 to inhibit complemented-mediated immunopathologic damage, there is no information on its ability to interfere with the host's defense against infection. In the current experiments sCR1 exerted a concentration-dependent inhibitory effect on the phagocytosis of Streptococcus pneumoniae by human polymorphonuclear leukocytes in vitro. Not only di sCR1 inhibit complement-dependent opsonization of the pneumococcus but at higher concentrations it also inhibited the ingestion of bacteria which had been previously opsonized. Furthermore, when rats were injected with sCR1, it inhibited both their serum hemolytic activity and serum opsonic activity in a dose-dependent fashion. Finally, for rats treated with sCR1, the 50% lethal dose was S. pneumoniae and Pseudomonas aeruginosa. These data demonstrate that sCR1 significantly inhibits complement-mediated host against bacterial infection.

  7. Inhibition of prolyl hydroxylase domain-containing protein downregulates vascular angiotensin II type 1 receptor.

    PubMed

    Matsuura, Hirohide; Ichiki, Toshihiro; Ikeda, Jiro; Takeda, Kotaro; Miyazaki, Ryohei; Hashimoto, Toru; Narabayashi, Eriko; Kitamoto, Shiro; Tokunou, Tomotake; Sunagawa, Kenji

    2011-09-01

    Inhibition of prolyl hydroxylase domain-containing protein (PHD) by hypoxia stabilizes hypoxia-inducible factor 1 and increases the expression of target genes, such as vascular endothelial growth factor. Although the systemic renin-angiotensin system is activated by hypoxia, the role of PHD in the regulation of the renin-angiotensin system remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin II type 1 receptor (AT(1)R). Hypoxia, cobalt chloride, and dimethyloxalylglycine, all known to inhibit PHD, reduced AT(1)R expression in vascular smooth muscle cells. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT(1)R expression. Cobalt chloride diminished angiotensin II-induced extracellular signal-regulated kinase phosphorylation. Cobalt chloride decreased AT(1)R mRNA through transcriptional and posttranscriptional mechanisms. Oral administration of cobalt chloride (14 mg/kg per day) to C57BL/6J mice receiving angiotensin II infusion (490 ng/kg per minute) for 4 weeks significantly attenuated perivascular fibrosis of the coronary arteries without affecting blood pressure level. These data suggest that PHD inhibition may be beneficial for the treatment of cardiovascular diseases by inhibiting renin-angiotensin system via AT(1)R downregulation.

  8. The novel VEGF receptor 2 inhibitor YLL545 inhibits angiogenesis and growth in breast cancer

    PubMed Central

    Zhang, Jianbo; Liu, Chen; Shi, Wen; Yang, Lingling; Zhang, Quansheng; Cui, Jianlin; Fang, Yangwu; Li, Yuhao; Ren, Guosheng; Yang, Shuang; Xiang, Rong

    2016-01-01

    Their antiangiogenic effects make vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors useful for cancer treatment. However, most of these drugs have unexpected adverse side effects. Here, we show that the novel VEGFR2 inhibitor YLL545 suppressed tumor angiogenesis and growth in triple-negative breast cancer without adverse effects. YLL545 treatment also markedly inhibited proliferation, migration, invasion, and tube formation by human umbilical vascular endothelial cells (HUVECs) in vitro. These effects of YLL545 were equal to or greater than those seen with sorafenib. In addition, YLL545 inhibited VEGF-induced phosphorylation of VEGFR2 and activation of downstream signaling regulators, such as phospho-STAT3 and phospho-ERK1/2, in HUVECs. Embryonic angiogenesis assays in zebrafish and Matrigel plug assays in mice demonstrated that YLL545 inhibits angiogenesis in vivo. YLL545 also inhibited proliferation and induced apoptosis in MDA-MB-231 breast cancer cells both in vitro and in vivo, and 50 mg/kg/d YLL545 inhibited human tumor xenograft growth by more than 50% in BALB/c nude mice. These observations suggest YLL545 is a potentially useful anticancer drug candidate. PMID:27203384

  9. Characterization of prejunctional 5-HT receptors mediating inhibition of sympathetic vasopressor responses in the pithed rat.

    PubMed

    Villalón, C M; Contreras, J; Ramírez-San Juan, E; Castillo, C; Perusquía, M; Terrón, J A

    1995-12-01

    1. It has recently been shown that continuous infusions of 5-hydroxytryptamine (5-HT) are able to inhibit, in a dose-dependent manner, the pressor responses induced by preganglionic (T7-T9) sympathetic stimulation in pithed rats pretreated with desipramine (50 micrograms kg-1, i.v.). This inhibitory effect, besides being significantly more pronounced at lower frequencies of stimulation (0.03-I Hz) and devoid of tachyphylaxis, is reversible after interrupting the infusions of 5-HT (up to 5.6 micrograms kg-1 min-1). In the present study we have characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-HT. 2. The inhibition induced by 5.6 micrograms kg-1 min-1 of 5-HT on sympathetically-induced pressor responses was not blocked after i.v. treatment with physiological saline (1 ml kg-1), ritanserin (0.1 mg kg-1), MDL 72222 (0.15 mg kg-1) or tropisetron (3 mg kg-1), which did not modify the sympathetically-induced pressor responses per se, but was significantly antagonized by the 5-HT1-like and 5-HT2 receptor antagonist, methysergide (0.3 mg kg-1), which also produced a slight attenuation of the pressor responses to 0.03 and 0.1 Hz per se. 3. Unexpectedly and contrasting with methysergide, the 5-HT1-like and 5-HT2 receptor antagonists, methiothepin (0.01, 0.03 and 0.1 mg kg-1) and metergoline (1 and 3 mg kg-1), apparently failed to block the above 5-HT-induced inhibition. Nevertheless, it is noteworthy that these antagonists also blocked the electrically-induced pressor responses per se, presumably by blockade of vascular alpha 1-adrenoceptors and, indeed, this property might have masked their potential antagonism at the inhibitory 5-HT1-like receptors. 4. Consistent with the above findings, 5-carboxamidotryptamine (5-CT, a potent 5-HT1-like receptor agonist), metergoline and methysergide mimicked the inhibitory action of 5-HT with the following rank order of agonist potency: 5CT > > 5-HT > metergoline > or = methysergide. 5

  10. Characterization of prejunctional 5-HT receptors mediating inhibition of sympathetic vasopressor responses in the pithed rat.

    PubMed Central

    Villalón, C. M.; Contreras, J.; Ramírez-San Juan, E.; Castillo, C.; Perusquía, M.; Terrón, J. A.

    1995-01-01

    1. It has recently been shown that continuous infusions of 5-hydroxytryptamine (5-HT) are able to inhibit, in a dose-dependent manner, the pressor responses induced by preganglionic (T7-T9) sympathetic stimulation in pithed rats pretreated with desipramine (50 micrograms kg-1, i.v.). This inhibitory effect, besides being significantly more pronounced at lower frequencies of stimulation (0.03-I Hz) and devoid of tachyphylaxis, is reversible after interrupting the infusions of 5-HT (up to 5.6 micrograms kg-1 min-1). In the present study we have characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-HT. 2. The inhibition induced by 5.6 micrograms kg-1 min-1 of 5-HT on sympathetically-induced pressor responses was not blocked after i.v. treatment with physiological saline (1 ml kg-1), ritanserin (0.1 mg kg-1), MDL 72222 (0.15 mg kg-1) or tropisetron (3 mg kg-1), which did not modify the sympathetically-induced pressor responses per se, but was significantly antagonized by the 5-HT1-like and 5-HT2 receptor antagonist, methysergide (0.3 mg kg-1), which also produced a slight attenuation of the pressor responses to 0.03 and 0.1 Hz per se. 3. Unexpectedly and contrasting with methysergide, the 5-HT1-like and 5-HT2 receptor antagonists, methiothepin (0.01, 0.03 and 0.1 mg kg-1) and metergoline (1 and 3 mg kg-1), apparently failed to block the above 5-HT-induced inhibition. Nevertheless, it is noteworthy that these antagonists also blocked the electrically-induced pressor responses per se, presumably by blockade of vascular alpha 1-adrenoceptors and, indeed, this property might have masked their potential antagonism at the inhibitory 5-HT1-like receptors. 4. Consistent with the above findings, 5-carboxamidotryptamine (5-CT, a potent 5-HT1-like receptor agonist), metergoline and methysergide mimicked the inhibitory action of 5-HT with the following rank order of agonist potency: 5CT > > 5-HT > metergoline > or = methysergide. 5

  11. A3 adenosine receptor inhibition improves the efficacy of hypertonic saline resuscitation

    PubMed Central

    Inoue, Yoshiaki; Tanaka, Hiroshi; Sumi, Yuka; Woehrle, Tobias; Chen, Yu; Hirsh, Mark I.; Junger, Wolfgang G.

    2011-01-01

    We reported previously that hypertonic saline (HS) treatment can prevent or upregulate the function of polymorphonuclear neutrophils (PMN) via A2a adenosine receptors (A2aR) or A3 adenosine receptors (A3R), respectively. A3R translocate to the cell surface upon PMN stimulation and thus HS promotes PMN responses under conditions of delayed HS treatment. Here we investigated if inhibition of A3R improves the protective effects of HS resuscitation in a mouse sepsis model. We found that HS nearly triples extracellular adenosine concentrations in whole blood and that inhibition of A3R with the selective antagonist MRS-1191 dose-dependently improves the inhibitory effect of HS. MRS-1191 at a concentration of 1 nM enhanced the inhibitory effect of HS and reduced stimulatory effects of delayed HS treatment. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis, we found that MRS-1191 reduces acute lung injury and PMN accumulation in lung tissue. While delayed HS treatment (4 ml/kg of 7.5 % NaCl) of mice 1 h after CLP aggravated PMN accumulation, lung tissue damage, and mortality 24 h after CLP, infusion of MRS-1191 (2 ng/kg body weight) combined with HS reduced these detrimental effects of delayed HS treatment. Our data thus show that A3 receptor antagonists can strengthen the beneficial effects of HS resuscitation by avoiding stimulatory side effects that result from delayed HS administration. PMID:20661181

  12. Melanocortin 4 receptor activation inhibits presynaptic N-type calcium channels in amygdaloid complex neurons.

    PubMed

    Agosti, Francina; López Soto, Eduardo J; Cabral, Agustina; Castrogiovanni, Daniel; Schioth, Helgi B; Perelló, Mario; Raingo, Jesica

    2014-09-01

    The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor involved in food intake and energy expenditure regulation. MC4R activation modifies neuronal activity but the molecular mechanisms by which this regulation occurs remain unclear. Here, we tested the hypothesis that MC4R activation regulates the activity of voltage-gated calcium channels and, as a consequence, synaptic activity. We also tested whether the proposed effect occurs in the amygdala, a brain area known to mediate the anorexigenic actions of MC4R signaling. Using the patch-clamp technique, we found that the activation of MC4R with its agonist melanotan II specifically inhibited 34.5 ± 1.5% of N-type calcium currents in transiently transfected HEK293 cells. This inhibition was concentration-dependent, voltage-independent and occluded by the Gαs pathway inhibitor cholera toxin. Moreover, we found that melanotan II specifically inhibited 25.9 ± 2.0% of native N-type calcium currents and 55.4 ± 14.4% of evoked inhibitory postsynaptic currents in mouse cultured amygdala neurons. In vivo, we found that the MC4R agonist RO27-3225 increased the marker of cellular activity c-Fos in several components of the amygdala, whereas the N-type channel blocker ω conotoxin GVIA increased c-Fos expression exclusively in the central subdivision of the amygdala. Thus, MC4R specifically inhibited the presynaptic N-type channel subtype, and this inhibition may be important for the effects of melanocortin in the central subdivision of the amygdala.

  13. MiR-125a TNF receptor-associated factor 6 to inhibit osteoclastogenesis

    SciTech Connect

    Guo, Li-Juan; Liao, Lan; Yang, Li; Li, Yu; Jiang, Tie-Jian

    2014-02-15

    MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. In the present study, we found that miR-125a was dramatically down-regulated during macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclastogenesis of circulating CD14+ peripheral blood mononuclear cells (PBMCs). Overexpression of miR-125a in CD14+ PBMCs inhibited osteoclastogenesis, while inhibition of miR-125a promoted osteoclastogenesis. TNF receptor-associated factor 6 (TRAF6), a transduction factor for RANKL/RANK/NFATc1 signal, was confirmed to be a target of miR-125a. EMSA and ChIP assays confirmed that NFATc1 bound to the promoter of the miR-125a. Overexpression of NFATc1 inhibited miR-125a transcription, and block of NFATc1 expression attenuated RANKL-regulated miR-125a transcription. Here, we reported that miR-125a played a biological function in osteoclastogenesis through a novel TRAF6/ NFATc1/miR-125a regulatory feedback loop. It suggests that regulation of miR-125a expression may be a potential strategy for ameliorating metabolic disease. - Highlights: • MiR-125a was significantly down-regulated in osteoclastogenesis of CD14+ PBMCs. • MiR-125a inhibited osteoclast differentiation by targeting TRAF6. • NFATc1 inhibited miR-125a transciption by binding to the promoter of miR-125a. • TRAF6/NFATc1 and miR-125a form a regulatory feedback loop in osteoclastogenesis.

  14. The interaction between the Drosophila secreted protein argos and the epidermal growth factor receptor inhibits dimerization of the receptor and binding of secreted spitz to the receptor.

    PubMed

    Jin, M H; Sawamoto, K; Ito, M; Okano, H

    2000-03-01

    Drosophila Argos (Aos), a secreted protein with an epidermal growth factor (EGF)-like domain, has been shown to inhibit the activation of the Drosophila EGF receptor (DER). However, it has not been determined whether Aos binds directly to DER or whether regulation of the DER activation occurs through some other mechanism. Using DER-expressing cells (DER/S2) and a recombinant DER extracellular domain-Fc fusion protein (DER-Fc), we have shown that Aos binds directly to the extracellular domain of DER with its carboxyl-terminal region, including the EGF-like domain. Furthermore, Aos can block the binding of secreted Spitz (sSpi), a transforming growth factor alpha-like ligand of DER, to the extracellular domain of DER. We observed that sSpi stimulates the dimerization of both the soluble DER extracellular domain (sDER) and the intact DER in the DER/S2 cells and that Aos can block the sSpi-induced dimerization of both sDER and intact DER. Moreover, we have shown that, by directly interacting with DER, Aos and SpiAos (a chimeric protein that is composed of the N-terminal region of Spi and the C-terminal region of Aos) inhibit the dimerization and phosphorylation of DER that are induced by DER's overexpression in the absence of sSpi. These results indicate that Aos exerts its inhibitory function through dual molecular mechanisms: by blocking both the receptor dimerization and the binding of activating ligand to the receptor. This is the first description of this novel inhibitory mechanism for receptor tyrosine kinases.

  15. Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1.

    PubMed

    Steeland, Sophie; Puimège, Leen; Vandenbroucke, Roosmarijn E; Van Hauwermeiren, Filip; Haustraete, Jurgen; Devoogdt, Nick; Hulpiau, Paco; Leroux-Roels, Geert; Laukens, Debby; Meuleman, Philip; De Vos, Martine; Libert, Claude

    2015-02-13

    The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named "TNF Receptor-One Silencer" (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.

  16. Tributyltin and triphenyltin inhibit osteoclast differentiation through a retinoic acid receptor-dependent signaling pathway

    SciTech Connect

    Yonezawa, Takayuki; Hasegawa, Shin-ichi; Ahn, Jae-Yong; Cha, Byung-Yoon; Teruya, Toshiaki; Hagiwara, Hiromi; Nagai, Kazuo; Woo, Je-Tae; E-mail: jwoo@isc.chubu.ac.jp

    2007-03-30

    Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), have been widely used in agriculture and industry. Although these compounds are known to have many toxic effects, including endocrine-disrupting effects, their effects on bone resorption are unknown. In this study, we investigated the effects of organotin compounds, such as monobutyltin (MBT), dibutyltin (DBT), TBT, and TPT, on osteoclast differentiation using mouse monocytic RAW264.7 cells. MBT and DBT had no effects, whereas TBT and TPT dose-dependently inhibited osteoclast differentiation at concentrations of 3-30 nM. Treatment with a retinoic acid receptor (RAR)-specific antagonist, Ro41-5253, restored the inhibition of osteoclastogenesis by TBT and TPT. TBT and TPT reduced receptor activator of nuclear factor-{kappa}B ligand (RANKL) induced nuclear factor of activated T cells (NFAT) c1 expression, and the reduction in NFATc1 expression was recovered by Ro41-5253. Our results suggest that TBT and TPT suppress osteoclastogenesis by inhibiting RANKL-induced NFATc1 expression via an RAR-dependent signaling pathway.

  17. Tricyclic analogs cyclobenzaprine, amitriptyline and cyproheptadine inhibit the spinal reflex transmission through 5-HT(2) receptors.

    PubMed

    Honda, Motoko; Nishida, Takashi; Ono, Hideki

    2003-01-01

    The centrally acting muscle relaxant cyclobenzaprine decreases the amplitude of monosynaptic reflex potentials by inhibiting the facilitatory descending serotonergic influences in the spinal cord. Interestingly, the structure of cyclobenzaprine is much similar to those of amitriptyline and cyproheptadine. In the present study, we attempted to elucidate the relationship between 5-HT(2) receptor antagonistic and inhibitory effects of cyclobenzaprine, amitriptyline, cyproheptadine and ketanserin on the spinal reflexes. Cyclobenzaprine, amitriptyline, cyproheptadine, and ketanserin significantly inhibited facilitatory effects of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on flexor reflexes and mono- and polysynaptic spinal reflex potentials in spinalized rats. In intact rats, these drugs significantly reduced the mono- and polysynaptic reflex potentials. 5-HT depletion significantly prevented the depression of the spinal reflex potentials induced by these drugs. These results suggest that the inhibitory effects of cyclobenzaprine, amitriptyline, and cyproheptadine on mono- and polysynaptic reflex potentials are due to the inhibition of descending serotonergic systems through 5-HT(2) receptors in the spinal cord.

  18. A Gsα mutant designed to inhibit receptor signaling through Gs

    PubMed Central

    Iiri, Taroh; Bell, Sean M.; Baranski, Thomas J.; Fujita, Toshiro; Bourne, Henry R.

    1999-01-01

    Hormonal signals activate trimeric G proteins by substituting GTP for GDP bound to the G protein α subunit (Gα), thereby generating two potential signaling molecules, Gα–GTP and free Gβγ. The usefulness of dominant negative mutations for investigating Ras and other monomeric G proteins inspired us to create a functionally analogous dominant negative Gα mutation. Here we describe a mutant α subunit designed to inhibit receptor-mediated hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase. To construct this mutant, we introduced into the α subunit (αs) of Gs three separate mutations chosen because they impair αs function in complementary ways: the A366S mutant reduces affinity of αs for binding GDP, whereas the G226A and E268A mutations impair the protein’s ability to bind GTP and to assume an active conformation. The triple mutant robustly inhibits (by up to 80%) Gs-dependent hormonal stimulation of adenylyl cyclase in cultured cells. Inhibition is selective in that it does not affect cellular responses to expression of a constitutively active αs mutant (αs–R201C) or to agonists for receptors that activate Gq or Gi. This αs triple mutant and cognate Gα mutants should provide specific tools for dissection of G protein-mediated signals in cultured cells and transgenic animals. PMID:9892662

  19. JMV641: a potent bombesin receptor antagonist that inhibits Swiss 3T3 cell proliferation.

    PubMed

    Azay, J; Gagne, D; Devin, C; Llinares, M; Fehrentz, J A; Martinez, J

    1996-08-27

    The peptides of the bombesin family are involved in stimulation of mitogenesis in various cell lines, including cancerous cell lines. Bombesin receptor antagonists are of great interest to inhibit this proliferation. We have synthesized a potent bombesin receptor antagonist, e.g., compound JMV641 [H-DPhe-Gln-Trp-Ala-Val-Gly-His-NH-*CH[CH2-CH(CH3)2]-**CHOH- (CH2)3-CH3 [*(S); **92% of (S) isomer], in which a pseudopeptide bond mimicking the transition state analogue replaced the peptide bond between the two C-terminal residues. This compound was highly potent to dose-dependently inhibit binding of 125I-GRP to Swiss 3T3 cells (IC50 = 0.85 +/- 0.15 nM) and bombesin-stimulated Swiss 3T3 proliferation (pA2 = 8.78). However, compound JMV641 can inhibit bombesin-induced AP-1 regulated genes that are nuclear messengers mediating the actions of signal transduction pathways stimulated by growth factors.

  20. Activation of peroxisome proliferator-activated receptor γ inhibits vascular calcification by upregulating Klotho

    PubMed Central

    Cheng, Lijuan; Zhang, Lei; Yang, Jun; Hao, Lirong

    2017-01-01

    Cardiovascular diseases are common in patients with chronic kidney disease. One of the key symptoms is the calcification of the vascular smooth muscle cells (VSMCs), which is induced by dysregulated mineral metabolism with high circulating levels of inorganic phosphate (Pi) and calcium. Klotho, which was originally identified as an aging suppressor gene, has been shown to be associated with vascular calcification. Since Klotho was recently identified as a target for nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ, the present study aimed to determine whether PPARγ regulates VSMC calcification through modulating the expression levels of Klotho. It was demonstrated that the expression of PPARγ was downregulated during Pi-induced VSMC calcification. In addition, treatment with PPARγ agonists inhibited the calcification and enhanced the expression of Klotho in VSMCs in a PPARγ-dependent manner. Of note, loss of Klotho expression by RNA interference abolished the ability of PPARγ activation to inhibit VSMC calcification. Furthermore, activation of Klotho as well as PPARγ inhibited the expression of Pi transporter 1/2 and reduced Pi influx into VSMCs. To the best of our knowledge, the present study was the first to demonstrate that PPARγ regulates VSMC calcification through activating Klotho.

  1. Effects of macromolecular crowding on the inhibition of virus assembly and virus-cell receptor recognition.

    PubMed

    Rincón, Verónica; Bocanegra, Rebeca; Rodríguez-Huete, Alicia; Rivas, Germán; Mateu, Mauricio G

    2011-02-02

    Biological fluids contain a very high total concentration of macromolecules that leads to volume exclusion by one molecule to another. Theory and experiment have shown that this condition, termed macromolecular crowding, can have significant effects on molecular recognition. However, the influence of molecular crowding on recognition events involving virus particles, and their inhibition by antiviral compounds, is virtually unexplored. Among these processes, capsid self-assembly during viral morphogenesis and capsid-cell receptor recognition during virus entry into cells are receiving increasing attention as targets for the development of new antiviral drugs. In this study, we have analyzed the effect of macromolecular crowding on the inhibition of these two processes by peptides. Macromolecular crowding led to a significant reduction in the inhibitory activity of: 1), a capsid-binding peptide and a small capsid protein domain that interfere with assembly of the human immunodeficiency virus capsid, and 2), a RGD-containing peptide able to block the interaction between foot-and-mouth disease virus and receptor molecules on the host cell membrane (in this case, the effect was dependent on the conditions used). The results, discussed in the light of macromolecular crowding theory, are relevant for a quantitative understanding of molecular recognition processes during virus infection and its inhibition.

  2. Cyclin D1 represses peroxisome proliferator-activated receptor alpha and inhibits fatty acid oxidation

    PubMed Central

    Hanse, Eric A.; Mashek, Douglas G.; Mashek, Mara T.; Hendrickson, Anna M.; Mullany, Lisa K.; Albrecht, Jeffrey H.

    2016-01-01

    Cyclin D1 is a cell cycle protein that promotes proliferation by mediating progression through key checkpoints in G1 phase. It is also a proto-oncogene that is commonly overexpressed in human cancers. In addition to its canonical role in controlling cell cycle progression, cyclin D1 affects other aspects of cell physiology, in part through transcriptional regulation. In this study, we find that cyclin D1 inhibits the activity of a key metabolic transcription factor, peroxisome proliferator-activated receptor α (PPARα), a member of nuclear receptor family that induces fatty acid oxidation and may play an anti-neoplastic role. In primary hepatocytes, cyclin D1 inhibits PPARα transcriptional activity and target gene expression in a cdk4-independent manner. In liver and breast cancer cells, knockdown of cyclin D1 leads to increased PPARα transcriptional activity, expression of PPARα target genes, and fatty acid oxidation. Similarly, cyclin D1 depletion enhances binding of PPARα to target sequences by chromatin immunoprecipitation. In proliferating hepatocytes and regenerating liver in vivo, induction of endogenous cyclin D1 is associated with diminished PPARα activity. Cyclin D1 expression is both necessary and sufficient for growth factor-mediated repression of fatty acid oxidation in proliferating hepatocytes. These studies indicate that in addition to playing a pivotal role in cell cycle progression, cyclin D1 represses PPARα activity and inhibits fatty acid oxidation. Our findings establish a new link between cyclin D1 and metabolism in both tumor cells and physiologic hepatocyte proliferation. PMID:27351284

  3. Cyclin D1 represses peroxisome proliferator-activated receptor alpha and inhibits fatty acid oxidation.

    PubMed

    Kamarajugadda, Sushama; Becker, Jennifer R; Hanse, Eric A; Mashek, Douglas G; Mashek, Mara T; Hendrickson, Anna M; Mullany, Lisa K; Albrecht, Jeffrey H

    2016-07-26

    Cyclin D1 is a cell cycle protein that promotes proliferation by mediating progression through key checkpoints in G1 phase. It is also a proto-oncogene that is commonly overexpressed in human cancers. In addition to its canonical role in controlling cell cycle progression, cyclin D1 affects other aspects of cell physiology, in part through transcriptional regulation. In this study, we find that cyclin D1 inhibits the activity of a key metabolic transcription factor, peroxisome proliferator-activated receptor α (PPARα), a member of nuclear receptor family that induces fatty acid oxidation and may play an anti-neoplastic role. In primary hepatocytes, cyclin D1 inhibits PPARα transcriptional activity and target gene expression in a cdk4-independent manner. In liver and breast cancer cells, knockdown of cyclin D1 leads to increased PPARα transcriptional activity, expression of PPARα target genes, and fatty acid oxidation. Similarly, cyclin D1 depletion enhances binding of PPARα to target sequences by chromatin immunoprecipitation. In proliferating hepatocytes and regenerating liver in vivo, induction of endogenous cyclin D1 is associated with diminished PPARα activity. Cyclin D1 expression is both necessary and sufficient for growth factor-mediated repression of fatty acid oxidation in proliferating hepatocytes. These studies indicate that in addition to playing a pivotal role in cell cycle progression, cyclin D1 represses PPARα activity and inhibits fatty acid oxidation. Our findings establish a new link between cyclin D1 and metabolism in both tumor cells and physiologic hepatocyte proliferation.

  4. Inhibition of peroxisome-proliferator-activated receptor (PPAR)alpha by MK886.

    PubMed Central

    Kehrer, J P; Biswal, S S; La, E; Thuillier, P; Datta, K; Fischer, S M; Vanden Heuvel, J P

    2001-01-01

    Although MK886 was originally identified as an inhibitor of 5-lipoxygenase activating protein (FLAP), recent data demonstrate that this activity does not underlie its ability to induce apoptosis [Datta, Biswal and Kehrer (1999) Biochem. J. 340, 371--375]. Since FLAP is a fatty-acid binding protein, it is conceivable that MK886 may affect other such proteins. A family of nuclear receptors that are activated by fatty acids and their metabolites, the peroxisome-proliferator-activated receptors (PPARs), have been implicated in apoptosis and may represent a target for MK886. The ability of MK886 to inhibit PPAR-alpha, -beta and -gamma activity was assessed using reporter assay systems (peroxisome-proliferator response element--luciferase). Using a transient transfection system in monkey kidney fibroblast CV-1 cells, mouse keratinocyte 308 cells and human lung adenocarcinoma A549 cells, 10--20 microM MK886 inhibited Wy14,643 activation of PPAR alpha by approximately 80%. Similar inhibition of PPAR alpha by MK886 was observed with a stable transfection reporter system in CV-1 cells. Only minimal inhibitory effects were seen on PPAR beta and PPAR gamma. MK886 inhibited PPAR alpha by a non-competitive mechanism as shown by its effects on the binding of arachidonic acid to PPAR alpha protein, and a dose-response study using a transient transfection reporter assay in COS-1 cells. An assay assessing PPAR ligand-receptor interactions showed that MK886 prevents the conformational change necessary for active-complex formation. The expression of keratin-1, a protein encoded by a PPAR alpha-responsive gene, was reduced by MK886 in a culture of mouse primary keratinocytes, suggesting that PPAR inhibition has functional consequences in normal cells. Although Jurkat cells express all PPAR isoforms, various PPAR alpha and PPAR gamma agonists were unable to prevent MK886-induced apoptosis. This is consistent with MK886 functioning as a non-competitive inhibitor of PPAR alpha, but may

  5. Sigma-1 receptor activation inhibits osmotic swelling of rat retinal glial (Müller) cells by transactivation of glutamatergic and purinergic receptors.

    PubMed

    Vogler, Stefanie; Winters, Helge; Pannicke, Thomas; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2016-01-01

    Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Sigma (σ) receptor activation is known to have neuroprotective effects in the retina. Here, we show that the nonselective σ receptor agonist ditolylguanidine, and the selective σ1 receptor agonist PRE-084, inhibit the osmotic swelling of Müller cell somata induced by superfusion of rat retinal slices with a hypoosmotic solution containing barium ions. In contrast, PRE-084 did not inhibit the osmotic swelling of bipolar cell somata. The effects of σ receptor agonists on the Müller cell swelling were abrogated in the presence of blockers of metabotropic glutamate and purinergic P2Y1 receptors, respectively, suggesting that σ receptor activation triggers activation of a glutamatergic-purinergic signaling cascade which is known to prevent the osmotic Müller cell swelling. The swelling-inhibitory effect of 17β-estradiol was prevented by the σ1 receptor antagonist BD1047, suggesting that the effect is mediated by σ1 receptor activation. The data may suggest that the neuroprotective effect of σ receptor activation in the retina is in part mediated by prevention of the cytotoxic swelling of retinal glial cells.

  6. NR2B receptor blockade inhibits pain-related sensitization of amygdala neurons.

    PubMed

    Ji, Guangchen; Horváth, Csilla; Neugebauer, Volker

    2009-04-28

    Pain-related sensitization and synaptic plasticity in the central nucleus of the amygdala (CeA) depend on the endogenous activation of NMDA receptors and phosphorylation of the NR1 subunit through a PKA-dependent mechanism. Functional NMDA receptors are heteromeric assemblies of NR1 with NR2A-D or NR3A, B subunits. NMDA receptors composed of NR1 and NR2B subunits have been implicated in neuroplasticity and are present in the CeA. Here we used a selective NR2B antagonist (Ro-256981) to determine the contribution of NR2B-containing NMDA receptors to pain-related sensitization of CeA neurons. Extracellular single-unit recordings were made from CeA neurons in anesthetized adult male rats before and during the development of an acute arthritis. Arthritis was induced in one knee joint by intraarticular injections of kaolin and carrageenan. Brief (15 s) mechanical stimuli of innocuous (100-500 g/30 mm2) and noxious (1000-2000 g/30 mm2) intensity were applied to the knee and other parts of the body. In agreement with our previous studies, all CeA neurons developed increased background and evoked activity after arthritis induction. Ro-256981 (1, 10 and 100 muM; 15 min each) was administered into the CeA by microdialysis 5-6 h postinduction of arthritis. Ro-256981 concentration-dependently decreased evoked responses, but not background activity. This pattern of effect is different from that of an NMDA receptor antagonist (AP5) in our previous studies. AP5 (100 microM - 5 mM) inhibited background activity and evoked responses. The differential effects of AP5 and Ro-256981 may suggest that NMDA receptors containing the NR2B subunit are important but not sole contributors to pain-related changes of CeA neurons.

  7. Exposure to D2-like dopamine receptor agonists inhibits swimming in Daphnia magna.

    PubMed

    Barrozo, Enrico R; Fowler, David A; Beckman, Matthew L

    2015-10-01

    Daphnia are freshwater crustaceans that have been used for decades in ecotoxicology research. Despite the important role that Daphnia have played in environmental toxicology studies, very little is known about the neurobiology of Daphnia. Although many studies have investigated the swimming movements of these "water fleas", few studies have examined the underlying neurochemical basis for these movements. To characterize the locomotor effect of drugs in Daphnia, a two-dimensional video imaging tool was developed and animal tracking was performed with freely available software, CTRAX. Due to the central role that dopamine plays in the movement of animals, we sought to determine the role of dopamine receptor signaling in Daphnia movement by characterizing the effect of ten drugs that are agonists or antagonists of dopamine receptors. At 1, 2, and 6h of treatment with a 10μM drug, several dopamine receptor agonists with documented effects on the D2-like class of receptors decreased the movement. Further, we determined behavioral inhibition values (IC50) at 1h of treatment for (1R,3S)-1-(aminomethyl)-3-phenyl-3,4-dihydro-1H-isochromene-5,6-diol (A68930) to be 1.4μM and for bromocriptine to be 6.6μM. This study describes a new method to study Daphnia swimming and establishes this organism as a useful model for studies of dopaminergic signaling. Specifically, this study shows that a dopamine receptor signaling pathway, mediated by putative D2-like receptors, is involved in the control of Daphnia swimming behavior. Due to its ease of use and its rich motor program we propose that Daphnia should be considered for future studies of dopamine neuron toxicity and protection.

  8. Muscarinic Acetylcholine Receptor M3 Modulates Odorant Receptor Activity via Inhibition of β-Arrestin-2 Recruitment

    PubMed Central

    Jiang, Yue; Li, Yun Rose; Tian, Huikai; Ma, Minghong; Matsunami, Hiroaki

    2015-01-01

    The olfactory system in rodents serves a critical function in social, reproductive, and survival behaviors. Processing of chemosensory signals in the brain is dynamically regulated in part by an animal's physiological state. We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odor-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odors. Here, we show that an M3-R antagonist attenuates odor-induced responses in OSNs from wild-type, but not M3-R-null mice. Using a novel molecular assay, we demonstrate that the activation of M3-Rs inhibits the recruitment of β-arrestin-2 to ORs, resulting in a potentiation of odor-induced response in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system. PMID:25800153

  9. The MYND domain-containing protein BRAM1 inhibits lymphotoxin beta receptor-mediated signaling through affecting receptor oligomerization.

    PubMed

    Liu, Hao-Ping; Chung, Pei-Jung; Liang, Chih-Lung; Chang, Yu-Sun

    2011-01-01

    MYND (myeloid-Nervy-DEAF-1) domains exist in a large number of proteins that are functionally important in development or associated with cancers. We have previously demonstrated that a MYND domain-containing protein, the bone morphogenesis protein receptor-associated molecule 1 (BRAM1), is able to interact with Epstein-Barr virus-encoded latent membrane protein 1 (LMP1), which acts as a constitutively activated tumor necrosis factor receptor (TNFR). Herein we further demonstrated that BRAM1 additionally associates with the TNFR-superfamily member, the lymphotoxin beta receptor (LTβR), and hence inhibits LTβR-mediated function. Using the yeast two-hybrid assay, we demonstrated that BRAM1 interacts with LTβR mainly through the self-association domain of LTβR (aa 336-398). The co-immunoprecipitation experiment further revealed that BRAM1 as well as MYND domain-containing proteins, MTG8 and DEAF-1, interacts with LTβR via their MYND domains. The BRAM1-LTβR interaction impedes the self-association of LTβR and the recruitment of TNFR-associated factors 2 and 3 (TRAF2 and TRAF3), leading to abolishment of LTβR-induced NF-κB signaling, JNK activation, and caspase-dependent cell death. In sum, our data demonstrate that the MYND-containing protein BRAM1 abrogates LTβR function through a protein-protein interaction. These findings may provide a direction for the treatment of dysregulation of LTβR-mediated signaling.

  10. Adenosine A1 receptors mediate inhibition of cAMP formation in vitro in the pontine, REM sleep induction zone.

    PubMed

    Marks, Gerald A; Birabil, Christian G; Speciale, Samuel G

    2005-11-09

    Microinjection of adenosine A1 receptor agonist or an inhibitor of adenylyl cyclase into the caudal, oral pontine reticular formation (PnOc) of the rat induces a long-lasting increase in REM sleep. Here, we report significant inhibition of forskolin-stimulated cAMP in dissected pontine tissue slices containing the PnOc incubated with the A1 receptor agonist, cyclohexaladenosine (10(-8) M). These data are consistent with adenosine A1 receptor agonist actions on REM sleep mediated through inhibition of cAMP.

  11. Cranberries inhibit LDL oxidation and induce LDL receptor expression in hepatocytes.

    PubMed

    Chu, Yi-Fang; Liu, Rui Hai

    2005-08-26

    Cardiovascular disease (CVD) is the leading cause of death in most industrialized countries. Cranberries were evaluated for their potential roles in dietary prevention of CVD. Cranberry extracts were found to have potent antioxidant capacity preventing in vitro LDL oxidation with increasing delay and suppression of LDL oxidation in a dose-dependent manner. The antioxidant activity of 100 g cranberries against LDL oxidation was equivalent to 1000 mg vitamin C or 3700 mg vitamin E. Cranberry extracts also significantly induced expression of hepatic LDL receptors and increased intracellular uptake of cholesterol in HepG2 cells in vitro in a dose-dependent manner. This suggests that cranberries could enhance clearance of excessive plasma cholesterol in circulation. We propose that additive or synergistic effects of phytochemicals in cranberries are responsible for the inhibition of LDL oxidation, the induced expression of LDL receptors, and the increased uptake of cholesterol in hepatocytes.

  12. Inhibiting androgen receptor nuclear entry in castration-resistant prostate cancer

    PubMed Central

    Pollock, Julie A.; Wardell, Suzanne E.; Parent, Alexander A.; Stagg, David B.; Ellison, Stephanie J.; Alley, Holly M.; Chao, Christina A.; Lawrence, Scott A.; Stice, James P.; Spasojevic, Ivan; Baker, Jennifer G.; Kim, Sung Hoon; McDonnell, Donald P.; Katzenellenbogen, John A.; Norris, John D.

    2016-01-01

    Clinical resistance to the second-generation antiandrogen enzalutamide in castration resistant prostate cancer (CRPC), despite persistent androgen receptor (AR) activity in tumors, highlights the unmet medical need for next generation antagonists. We have identified and characterized tetra-aryl cyclobutanes (CBs) as a new class of competitive AR antagonists that exhibit a unique mechanism of action. These CBs are structurally distinct from current antiandrogens (hydroxyflutamide, bicalutamide, and enzalutamide), and inhibit AR-mediated gene expression, cell proliferation, and tumor growth in several models of CRPC. Conformational profiling revealed that CBs stabilize an AR conformation resembling an unliganded receptor. Using a variety of techniques, it was determined that the AR:CB complex was not recruited to AR-regulated promoters and, like apo AR, remains sequestered in the cytoplasm bound to heat shock proteins. Thus, we have identified third generation AR antagonists whose unique mechanism of action suggests that they may have therapeutic potential in CRPC. PMID:27501397

  13. Glucocorticoid Receptor Activation Inhibits Chemotherapy-induced Cell Death in High-grade Serous Ovarian Carcinoma

    PubMed Central

    Stringer-Reasor, Erica M.; Baker, Gabrielle M.; Skor, Maxwell N.; Kocherginsky, Masha; Lengyel, Ernst; Fleming, Gini F.; Conzen, Suzanne D.

    2015-01-01

    Objectives To test the hypothesis that glucocorticoid receptor (GR) activation increases resistance to chemotherapy in high-grade serous ovarian cancer (HGS-OvCa) and that treatment with a GR antagonist will improve sensitivity to chemotherapy. Methods GR expression was assessed in OvCa cell lines by qRT-PCR and Western blot analysis and in xenografts and primary human tumors using immunohistochemistry (IHC). We also examined the effect of GR activation versus inhibition on chemotherapy-induced cytotoxicity in OvCa cell lines and in a xenograft model. Results With the exception of IGROV-1 cells, all OvCa cell lines tested had detectable GR expression by Western blot and qRT-PCR analysis. Twenty-five out of the 27 human primary HGS-OvCas examined expressed GR by IHC. No cell line expressed detectable progesterone receptor (PR) or androgen receptor (AR) by Western blot analysis. In vitro assays showed that in GR-positive HeyA8 and SKOV3 cells, dexamethasone (100 nM) treatment upregulated the pro-survival genes SGK1 and MKP1/DUSP1 and inhibited carboplatin/gemcitabine-induced cell death. Concurrent treatment with two GR antagonists, either mifepristone (100 nM) or CORT125134 (100 nM), partially reversed these effects. There was no anti-apoptotic effect of dexamethasone on chemotherapy-induced cell death in IGROV-1 cells, which did not have detectable GR protein. Mifepristone treatment alone was not cytotoxic in any cell line. HeyA8 OvCa xenograft studies demonstrated that adding mifepristone to carboplatin/gemcitabine increased tumor shrinkage by 48% compared to carboplatin/gemcitabine treatment alone (P=0.0004). Conclusions These results suggest that GR antagonism sensitizes GR+ OvCa to chemotherapy-induced cell death through inhibition of GR-mediated cell survival pathways. PMID:26115975

  14. Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

    PubMed Central

    Abraham, R T; Ho, S N; Barna, T J; Rusovick, K M; McKean, D J

    1988-01-01

    The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells. Images PMID:2977423

  15. Targeting receptor for advanced glycation end products (RAGE) expression induces apoptosis and inhibits prostate tumor growth

    SciTech Connect

    Elangovan, Indira; Thirugnanam, Sivasakthivel; Chen, Aoshuang; Zheng, Guoxing; Bosland, Maarten C.; Kajdacsy-Balla, Andre; Gnanasekar, Munirathinam

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer Targeting RAGE by RNAi induces apoptosis in prostate cancer cells. Black-Right-Pointing-Pointer Silencing RAGE expression abrogates rHMGB1 mediated cell proliferation. Black-Right-Pointing-Pointer Down regulation of RAGE by RNAi inhibits PSA secretion of prostate cancer cells. Black-Right-Pointing-Pointer Knock down of RAGE abrogates prostate tumor growth in vivo. Black-Right-Pointing-Pointer Disruption of RAGE expression in prostate tumor activates death receptors. -- Abstract: Expression of receptor for advanced glycation end products (RAGE) plays a key role in the progression of prostate cancer. However, the therapeutic potential of targeting RAGE expression in prostate cancer is not yet evaluated. Therefore in this study, we have investigated the effects of silencing the expression of RAGE by RNAi approach both in vitro and in vivo. The results of this study showed that down regulation of RAGE expression by RNAi inhibited the cell proliferation of androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells. Furthermore, targeting RAGE expression resulted in apoptotic elimination of these prostate cancer cells by activation of caspase-8 and caspase-3 death signaling. Of note, the levels of prostate specific antigen (PSA) were also reduced in LNCaP cells transfected with RAGE RNAi constructs. Importantly, the RAGE RNAi constructs when administered in nude mice bearing prostate tumors, inhibited the tumor growth by targeting the expression of RAGE, and its physiological ligand, HMGB1 and by up regulating death receptors DR4 and DR5 expression. Collectively, the results of this study for the first time show that targeting RAGE by RNAi may be a promising alternative therapeutic strategy for treating prostate cancer.

  16. Targeting colorectal cancer via its microenvironment by inhibiting IGF-1 Receptor-insulin receptor substrate and STAT3 signaling

    PubMed Central

    Sanchez-Lopez, Elsa; Flashner-Abramson, Efrat; Shalapour, Shabnam; Zhong, Zhenyu; Taniguchi, Koji; Levitzki, Alexander; Karin, Michael

    2015-01-01

    The tumor microenvironment (TME) exerts critical pro-tumorigenic effects through cytokines and growth factors that support cancer cell proliferation, survival, motility and invasion. Insulin-like growth factor-1 (IGF-1) and Signal transducer and activator of transcription 3 (STAT3) stimulate colorectal cancer (CRC) development and progression via cell autonomous and microenvironmental effects. Using a unique inhibitor, NT157, which targets both IGF-1 receptor (IGF-1R) and STAT3, we show that these pathways regulate many TME functions associated with sporadic colonic tumorigenesis in CPC-APC mice, in which cancer development is driven by loss of the Apc tumor suppressor gene. NT157 causes a substantial reduction in tumor burden by affecting cancer cells, cancer-associated fibroblasts (CAF) and myeloid cells. Decreased cancer cell proliferation and increased apoptosis were accompanied by inhibition of CAF activation and decreased inflammation. Furthermore, NT157 inhibited expression of pro-tumorigenic cytokines, chemokines and growth factors, including IL-6, IL-11 and IL-23 as well as CCL2, CCL5, CXCL7, CXCL5, ICAM1 and TGFβ; decreased cancer cell migratory activity and reduced their proliferation in the liver. NT157 represents a new class of anti-cancer drugs that affect both the malignant cell and its supportive microenvironment. PMID:26364612

  17. 5-HT(1A)-like receptor activation inhibits abstinence-induced methamphetamine withdrawal in planarians.

    PubMed

    Rawls, Scott M; Shah, Hardik; Ayoub, George; Raffa, Robert B

    2010-10-29

    No pharmacological therapy is approved to treat methamphetamine physical dependence, but it has been hypothesized that serotonin (5-HT)-enhancing drugs might limit the severity of withdrawal symptoms. To test this hypothesis, we used a planarian model of physical dependence that quantifies withdrawal as a reduction in planarian movement. Planarians exposed to methamphetamine (10 μM) for 60 min, and then placed (tested) into drug-free water for 5 min, displayed less movement (i.e., withdrawal) than either methamphetamine-naïve planarians tested in water or methamphetamine-exposed planarians tested in methamphetamine. A concentration-related inhibition of withdrawal was observed when methamphetamine-exposed planarians were placed into a solution containing either methamphetamine and 5-HT (0.1-100 μM) or methamphetamine and the 5-HT(1A) receptor agonist 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) (10, 20 μM). Planarians with prior methamphetamine exposure displayed enhanced withdrawal when tested in a solution of the 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide (WAY 100635) (1 μM). Methamphetamine-induced withdrawal was not affected by the 5-HT(2B/2C) receptor agonist meta-chlorophenylpiperazine (m-CPZ) (0.1-20 μM). These results provide pharmacological evidence that serotonin-enhancing drugs inhibit expression of methamphetamine physical dependence in an invertebrate model of withdrawal, possibly through a 5-HT(1A)-like receptor-dependent mechanism.

  18. Adenosine A1 receptors mediate inhibition of tachykinin release from perifused enteric nerve endings.

    PubMed

    Broad, R M; McDonald, T J; Brodin, E; Cook, M A

    1992-03-01

    A perifused preparation of guinea pig myenteric nerve varicosities (synaptosomes) was used to determine the characteristics of evoked tachykinin release and the inhibition of such release by adenosine analogues. Release of substance P-like immunoreactivity (SP-LI) and neurokinin A-like immunoreactivity (NKA-LI) was evoked by elevated extracellular [K+] in a reversible and repeatable manner. This release was completely abolished in the absence of extracellular Ca2+. Perifusion in the presence of 5'-N-ethylcarboxamidoadenosine (NECA), a nonselective A1/A2 adenosine receptor agonist, decreased K(+)-evoked release of SP-LI and NKA-LI compared with that in the absence of the nucleoside. Similar decrements in peptide release were obtained with N6-cyclopentyl adenosine (CPA), a selective A1 agonist, and 2-[p-(2-carboxyethyl)]phenethylamino-5'-N-ethyl-carboxamidoadenosi ne (CGS 21680), a selective A2 agonist. Response to all nucleosides was graded. Potency order of adenosine analogues was CPA greater than NECA much greater than CGS 21680. Inhibition due to the nucleosides was diminished in the presence of the highly selective A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) while perifusion in the presence of DPCPX alone did not alter evoked release of either peptide. These findings provide direct measurements of inhibitory effects of adenine nucleosides on the release, from enteric nerve endings, of endogenous neuromediators SP and NKA. The findings also directly demonstrate the presence of functional adenosine receptors of the A1 subtype on enteric nerve endings coupled negatively to release of tachykinins. The presence of A2 receptors on enteric nerve endings is neither supported nor excluded.

  19. Bipartite inhibition of Drosophila epidermal growth factor receptor by the extracellular and transmembrane domains of Kekkon1.

    PubMed Central

    Alvarado, Diego; Rice, Amy H; Duffy, Joseph B

    2004-01-01

    In Drosophila, signaling by the epidermal growth factor receptor (EGFR) is required for a diverse array of developmental decisions. Essential to these decisions is the precise regulation of the receptor's activity by both stimulatory and inhibitory molecules. To better understand the regulation of EGFR activity we investigated inhibition of EGFR by the transmembrane protein Kekkon1 (Kek1). Kek1 encodes a molecule containing leucine-rich repeats (LRR) and an immunoglobulin (Ig) domain and is the founding member of the Drosophila Kekkon family. Here we demonstrate with a series of Kek1-Kek2 chimeras that while the LRRs suffice for EGFR binding, inhibition in vivo requires the Kek1 juxta/transmembrane region. We demonstrate directly, and using a series of Kek1-EGFR chimeras, that Kek1 is not a phosphorylation substrate for the receptor in vivo. In addition, we show that EGFR inhibition is unique to Kek1 among Kek family members and that this function is not ligand or tissue specific. Finally, we have identified a unique class of EGFR alleles that specifically disrupt Kek1 binding and inhibition, but preserve receptor activation. Interestingly, these alleles map to domain V of the Drosophila EGFR, a region absent from the vertebrate receptors. Together, our results support a model in which the LRRs of Kek1 in conjunction with its juxta/transmembrane region direct association and inhibition of the Drosophila EGFR through interactions with receptor domain V. PMID:15166146

  20. Morphinans and isoquinolines: acetylcholinesterase inhibition, pharmacophore modeling, and interaction with opioid receptors.

    PubMed

    Schuster, Daniela; Spetea, Mariana; Music, Melisa; Rief, Silvia; Fink, Monika; Kirchmair, Johannes; Schütz, Johannes; Wolber, Gerhard; Langer, Thierry; Stuppner, Hermann; Schmidhammer, Helmut; Rollinger, Judith M

    2010-07-15

    Following indications from pharmacophore-based virtual screening of natural product databases, morphinan and isoquinoline compounds were tested in vitro for acetylcholinesterase (AChE) inhibition. After the first screen, active and inactive compounds were used to build a ligand-based pharmacophore model in order to prioritize compounds for biological testing. Among the virtual hits tested, the enrichment of actives was significantly higher than in a random selection of test compounds. The most active compounds were biochemically tested for their activity on mu, delta, and kappa opioid receptors.

  1. Phenoxy herbicides and fibrates potently inhibit the human chemosensory receptor subunit T1R3

    PubMed Central

    Maillet, Emeline L.; Margolskee, Robert F.; Mosinger, Bedrich

    2009-01-01

    We show that phenoxy-auxin herbicides and lipid-lowering fibrates inhibit human but not rodent T1R3. T1R3 as a co-receptor in taste cells responds to sweet compounds and amino-acids; in endocrine cells of gut and pancreas T1R3 contributes to glucose sensing. Thus, certain effects of fibrates in treating hyperlipidemia and type II diabetes may be via actions on T1R3. Likewise, phenoxy-herbicides may have adverse metabolic effects in humans that would have gone undetected in studies on rodents. PMID:19817384

  2. H3 receptor-mediated inhibition of the neurogenic vasopressor response in pithed rats.

    PubMed

    Malinowska, B; Schlicker, E

    1991-12-03

    In pithed rats, the H3 agonist R-(-)-alpha-methylhistamine (R alpha MeHA) inhibited the electrically induced increase in blood pressure without affecting the vasopressor response to exogenous noradrenaline. The effect of R alpha MeHA was not affected by the H1 and H2 antagonists dimetindene and ranitidine, but attenuated by the H3 antagonist thioperamide. At higher doses, R alpha MeHA itself increased basal blood pressure; this effect was not affected by the H1, H2 and H3 antagonists. In conclusion, the neurogenic vasopressor response can be modulated via H3 receptors, probably located presynaptically on postganglionic sympathetic nerve fibres.

  3. Methods for recording and measuring tonic GABAA receptor-mediated inhibition.

    PubMed

    Bright, Damian P; Smart, Trevor G

    2013-12-05

    Tonic inhibitory conductances mediated by GABAA receptors have now been identified and characterized in many different brain regions. Most experimental studies of tonic GABAergic inhibition have been carried out using acute brain slice preparations but tonic currents have been recorded under a variety of different conditions. This diversity of recording conditions is likely to impact upon many of the factors responsible for controlling tonic inhibition and can make comparison between different studies difficult. In this review, we will firstly consider how various experimental conditions, including age of animal, recording temperature and solution composition, are likely to influence tonic GABAA conductances. We will then consider some technical considerations related to how the tonic conductance is measured and subsequently analyzed, including how the use of current noise may provide a complementary and reliable method for quantifying changes in tonic current.

  4. A review of PCSK9 inhibition and its effects beyond LDL receptors.

    PubMed

    Dixon, Dave L; Trankle, Cory; Buckley, Leo; Parod, Eric; Carbone, Salvatore; Van Tassell, Benjamin W; Abbate, Antonio

    2016-01-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an integral role in the degradation of low-density lipoprotein receptors (LDL-R), making it an intriguing target for emerging pharmacotherapy. Two PCSK9 inhibitors, alirocumab and evolocumab, have been approved and are available in the United States and European Union. However, much of the PCSK9 story remains to be told. The pipeline for additional pharmacotherapy options is rich with several compounds under development, using alternative strategies for inhibiting PCSK9. Perhaps, more intriguing is the interaction between PCSK9 and non-LDL-R targets, including mediators of inflammation and immunological processes, which remain under intense investigation. This review will discuss the currently available PCSK9 inhibitors, the development of novel approaches to PCSK9 modulation, and the potential non-LDL-R-mediated effects of PCSK9 inhibition.

  5. Inhibition of T cell receptor signaling by cholesterol sulfate, a naturally occurring derivative of membrane cholesterol

    PubMed Central

    Wang, Feng; Beck-García, Katharina; Zorzin, Carina; Schamel, Wolfgang W. A.; Davis, Mark M.

    2016-01-01

    Most adaptive immune responses require the activation of specific T cells through the T cell antigen receptor–CD3 complex (TCR). Here we show that cholesterol sulfate (CS), a naturally occurring analog of cholesterol, inhibits CD3 ITAM phosphorylation, a crucial first step in T cell activation. Biochemical studies show that CS disrupted TCR multimers, apparently by displacing cholesterol, known to bind TCRβ. Moreover, CS-deficient mice displayed a heightened sensitivity to a self-antigen, whereas increasing CS content by intrathymic injection inhibited thymic selection, indicating that this molecule is an intrinsic regulator of thymocyte development. These results reveal a regulatory role for CS in TCR signaling and thymic selection, highlighting the importance of the membrane microenvironment in modulating cell surface receptor activation. PMID:27213689

  6. Methods for recording and measuring tonic GABAA receptor-mediated inhibition

    PubMed Central

    Bright, Damian P.; Smart, Trevor G.

    2013-01-01

    Tonic inhibitory conductances mediated by GABAA receptors have now been identified and characterized in many different brain regions. Most experimental studies of tonic GABAergic inhibition have been carried out using acute brain slice preparations but tonic currents have been recorded under a variety of different conditions. This diversity of recording conditions is likely to impact upon many of the factors responsible for controlling tonic inhibition and can make comparison between different studies difficult. In this review, we will firstly consider how various experimental conditions, including age of animal, recording temperature and solution composition, are likely to influence tonic GABAA conductances. We will then consider some technical considerations related to how the tonic conductance is measured and subsequently analyzed, including how the use of current noise may provide a complementary and reliable method for quantifying changes in tonic current. PMID:24367296

  7. Blocking the tropomyosin receptor kinase A (TrkA) receptor inhibits pain behaviour in two rat models of osteoarthritis

    PubMed Central

    Nwosu, Lilian N; Mapp, Paul I; Chapman, Victoria; Walsh, David A

    2016-01-01

    Objectives Tropomyosin receptor kinase A (TrkA) mediates nociceptor sensitisation by nerve growth factor (NGF), but it is unknown whether selective TrkA inhibition will be an effective strategy for treating osteoarthritis (OA) pain. We determined the effects of a TrkA inhibitor (AR786) on pain behaviour, synovitis and joint pathology in two rat OA models. Methods Knee OA was induced in rats by intra-articular monosodium-iodoacetate (MIA) injection or meniscal transection (MNX) and compared with saline-injected or sham-operated controls. Pain behaviour was assessed as weight-bearing asymmetry and paw withdrawal threshold to punctate stimulation. Oral doses (30 mg/kg) of AR786 or vehicle were administered twice daily in either preventive (day −1 to –27) or treatment (day 14–28) protocols. Effect maintenance was evaluated for 2 weeks after treatment discontinuation. Alterations in knee structure (cartilage, subchondral bone and synovium) were examined by macroscopic visualisation of articular surfaces and histopathology. Results Preventive AR786 treatment inhibited pain behaviour development and therapeutic treatment attenuated established pain behaviour. Weight-bearing asymmetry increased 1 week after treatment discontinuation, but remained less than in vehicle-treated arthritic rats, whereas paw withdrawal thresholds returned to levels of untreated rats within 5 days of treatment discontinuation. AR786 treatment reduced MIA-induced synovitis and did not significantly affect osteochondral pathology in either model. Conclusions Blocking NGF activity by inhibiting TrkA reduced pain behaviour in two rat models of OA. Analgesia was observed both using preventive and treatment protocols, and was sustained after treatment discontinuation. Selective inhibitors of TrkA therefore hold potential for OA pain relief. PMID:26286016

  8. Antipsychotic drugs inhibit prolactin release from rat anterior pituitary cells in culture by a mechanism not involving the dopamine receptor.

    PubMed

    West, B; Dannies, P S

    1979-04-01

    Bromocriptine, a dopamine agonist, inhibited secretion of PRL and did not affect GH release from rat anterior pituitary cells in culture. The reversal of this inhibition of PRL release by butaclamol, a dopamine antagonist, was stereospecific; 10 nM d-butaclamol completely reversed the inhibition caused by 10 nM bromocriptine, while l-butaclamol had no effect at concentrations up to 10 microM. However, both enantiomers at 10 microM inhibited PRL release to 30% and GH release to 91% of control values. Two other dopamine antagonists also inhibited hormone release. Haloperidol (10 microM) inhibited PRL release to 23% of control values and did not affect GH release; 3.3 microM pimozide inhibited PRL and GH release to 18% and 38% of control values, respectively. These data indicate that, the inhibition of PRL by antipsychotic drugs is not mediated through the dopamine receptor.

  9. Melanocortin 4 receptor constitutive activity inhibits L-type voltage-gated calcium channels in neurons.

    PubMed

    Agosti, F; Cordisco Gonzalez, S; Martinez Damonte, V; Tolosa, M J; Di Siervi, N; Schioth, H B; Davio, C; Perello, M; Raingo, J

    2017-03-27

    The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that is expressed in several brain nuclei playing a crucial role in the regulation of energy balance controlling the homeostasis of the organism. It displays both agonist-evoked and constitutive activity, and moreover, it can couple to different G proteins. Most of the research on MC4R has been focused on agonist-induced activity, while the molecular and cellular basis of MC4R constitutive activity remains scarcely studied. We have previously shown that neuronal N-type voltage-gated calcium channels (CaV2.2) are inhibited by MC4R agonist-dependent activation, while the CaV subtypes that carry L- and P/Q-type current are not. Here, we tested the hypothesis that MC4R constitutive activity can affect CaV, with focus on the channel subtypes that can control transcriptional activity coupled to depolarization (L-type, CaV1.2/1.3) and neurotransmitter release (N- and P/Q-type, CaV2.2 and CaV2.1). We found that MC4R constitutive activity inhibits specifically CaV1.2/1.3 and CaV2.1 subtypes of CaV. We also explored the signaling pathways mediating this inhibition, and thus propose that agonist-dependent and basal MC4R activation modes signal differentially through Gs and Gi/o pathways to impact on different CaV subtypes. In addition, we found that chronic incubation with MC4R endogenous inverse agonist, agouti and agouti-related peptide (AgRP), occludes CaV inhibition in a cell line and in amygdaloid complex cultured neurons as well. Thus, we define new mechanisms of control of the main mediators of depolarization-induced calcium entry into neurons by a GPCR that displays constitutive activity.

  10. The TM2 6′ Position of GABAA Receptors Mediates Alcohol Inhibition

    PubMed Central

    Howard, Rebecca J.; Trudell, James R.; Harris, R. Adron

    2012-01-01

    Ionotropic GABAA receptors (GABAARs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABAARs. We used Xenopus laevis oocytes to investigate the 6′ position in the second transmembrane region of GABAARs as a site influencing alcohol inhibition. We asked whether modification of the 6′ position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6′ position in either α2 or β2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6′ position. We verified a role for the 6′ position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABAARs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABAAR homolog, revealing that the 6′ position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6′ positions in both α2 and β2 GABAAR subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels. PMID:22072732

  11. Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells.

    PubMed

    Khan, Shaheen; Liu, Shengxi; Stoner, Matthew; Safe, Stephen

    2007-08-15

    The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ERalpha crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1alpha or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NFkappaB which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide.

  12. COBALTOUS CHLORIDE AND HYPOXIA INHIBIT ARYL HYDROCARBON RECEPTOR-MEDIATED RESPONSES IN BREAST CANCER CELLS

    PubMed Central

    Khan, Shaheen; Liu, Shengxi; Stoner, Matthew; Safe, Stephen

    2007-01-01

    The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-Erα crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1α or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NFκB which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 hr, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 hr, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 hr enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide. PMID:17599377

  13. The TM2 6' position of GABA(A) receptors mediates alcohol inhibition.

    PubMed

    Johnson, W David; Howard, Rebecca J; Trudell, James R; Harris, R Adron

    2012-02-01

    Ionotropic GABA(A) receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABA(A)Rs. We used Xenopus laevis oocytes to investigate the 6' position in the second transmembrane region of GABA(A)Rs as a site influencing alcohol inhibition. We asked whether modification of the 6' position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6' position in either α2 or β2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6' position. We verified a role for the 6' position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABA(A)Rs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABA(A)R homolog, revealing that the 6' position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6' positions in both α2 and β2 GABA(A)R subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels.

  14. Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells

    SciTech Connect

    Khan, Shaheen; Liu Shengxi; Stoner, Matthew; Safe, Stephen

    2007-08-15

    The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ER{alpha} crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1{alpha} or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NF{kappa}B which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide.

  15. Presynaptic inhibition of synaptic transmission in the rat hippocampus by activation of muscarinic receptors: involvement of presynaptic calcium influx

    PubMed Central

    Qian, Jing; Saggau, Peter

    1997-01-01

    Modulation of presynaptic voltage-dependent calcium channels (VDCCs) by muscarinic receptors at the CA3–CA1 synapse of rat hippocampal slices was investigated by using the calcium indicator fura-2. Stimulation-evoked presynaptic calcium transients ([Capre]t) and field excitatory postsynaptic potentials (fe.p.s.ps) were simultaneously recorded. The relationship between presynaptic calcium influx and synaptic transmission was studied. Activation of muscarinic receptors inhibited [Capre]t, thereby reducing synaptic transmission. Carbachol (CCh, 10 μM) inhibited [Capre]t by 35% and reduced fe.p.s.p. by 85%. The inhibition was completely antagonized by 1 μM atropine. An approximate 4th power relationship was found between presynaptic calcium influx and postsynaptic responses. Application of the N-type VDCC-blocking peptide toxin ω-conotoxin GVIA (ω-CTx GVIA, 1 μM) inhibited [Capre]t and fe.p.s.ps by 21% and 49%, respectively, while the P/Q-type VDCC blocker ω-agatoxin IVA (ω-Aga IVA, 1 μM) reduced [Capre]t and fe.p.s.ps by 35% and 85%, respectively. Muscarinic receptor activation differentially inhibited distinct presynaptic VDCCs. ω-CTx GVIA-sensitive calcium channels were inhibited by muscarinic receptors, while ω-Aga IVA-sensitive channels were not. The percentage inhibition of ω-CTx GVIA-sensitive [Capre]t was about 63%. Muscarinic receptors inhibited presynaptic VDCCs in a way similar to adenosine (Ad) receptors. The percentage inhibition of ω-CTx GVIA-sensitive [Capre]t by Ad (100 μM) was about 59%. There was no significant inhibition of ω-Aga IVA-sensitive channels by Ad. The inhibitions of [Capre]t by CCh and Ad were mutually occlusive. These results indicate that inhibition of synaptic transmission by muscarinic receptors is mainly the consequence of a reduction of the [Capre]t due to inhibition of presynaptic VDCCs. PMID:9351508

  16. δ-Tocopherol inhibits receptor tyrosine kinase-induced AKT activation in prostate cancer cells.

    PubMed

    Wang, Hong; Hong, Jungil; Yang, Chung S

    2016-11-01

    The cancer preventive activity of vitamin E is suggested by epidemiological studies and supported by animal studies with vitamin E forms, γ-tocopherol and δ-tocopherol (δ-T). Several recent large-scale cancer prevention trials with high dose of α-tocopherol, however, yielded disappointing results. Whether vitamin E prevents or promotes cancer is a serious concern. A better understanding of the molecular mechanisms of action of the different forms of tocopherols would enhance our understanding of this topic. In this study, we demonstrated that δ-T was the most effective tocopherol form in inhibiting prostate cancer cell growth, by inducing cell cycle arrest and apoptosis. By profiling the effects of δ-T on the cell signaling using the phospho-kinase array, we found that the most inhibited target was the phosphorylation of AKT on T308. Further study on the activation of AKT by EGFR and IGFR revealed that δ-T attenuated the EGF/IGF-induced activation of AKT (via the phosphorylation of AKT on T308 induced by the activation of PIK3). Expression of dominant active PIK3 and AKT in prostate cancer cell line DU145 in which PIK3, AKT, and PTEN are wild type caused the cells to be reflectory to the inhibition of δ-T, supporting that δ-T inhibits the PIK3-mediated activation of AKT. Our data also suggest that δ-T interferes with the EGF-induced EGFR internalization, which leads to the inhibition of the receptor tyrosine kinase-dependent activation of AKT. In summary, our results revealed a novel mechanism of δ-T in inhibiting prostate cancer cell growth, supporting the cancer preventive activity δ-T. © 2015 Wiley Periodicals, Inc.

  17. Anti-MUC1 antibody inhibits EGF receptor signaling in cancer cells

    SciTech Connect

    Hisatsune, Akinori; Nakayama, Hideki; Kawasaki, Mitsuru; Horie, Ichiro; Miyata, Takeshi; Isohama, Yoichiro; Kim, Kwang Chul; Katsuki, Hiroshi

    2011-02-18

    Research highlights: {yields} We identified changes in the expression and function of EGFR by anti-MUC1 antibody. {yields} An anti-MUC1 antibody GP1.4 decreased EGFR from cell surface by internalization. {yields} GP1.4 specifically inhibited ERK signaling triggered EGF-EGFR signaling pathway. {yields} Internalization of EGFR was dependent on the presence of MUC1 on cell surface. {yields} GP1.4 significantly inhibited EGF-dependent cancer cell proliferation and migration. -- Abstract: MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. We previously reported that MUC1 is internalized by the binding of the anti-MUC1 antibody, from the cell surface to the intracellular region via the macropinocytotic pathway. Since MUC1 is closely associated with ErbBs, such as EGF receptor (EGFR) in cancer cells, we examined the effect of the anti-MUC1 antibody on EGFR trafficking. Our results show that: (1) anti-MUC1 antibody GP1.4, but not another anti-MUC1 antibody C595, triggered the internalization of EGFR in pancreatic cancer cells; (2) internalization of EGFR by GP1.4 resulted in the inhibition of ERK phosphorylation by EGF stimulation, in a MUC1 dependent manner; (3) inhibition of ERK phosphorylation by GP1.4 resulted in the suppression of proliferation and migration of pancreatic cancer cells. We conclude that the internalization of EGFR by anti-MUC1 antibody GP1.4 inhibits the progression of cancer cells via the inhibition of EGFR signaling.

  18. Blockage of transient receptor potential vanilloid 4 inhibits brain edema in middle cerebral artery occlusion mice.

    PubMed

    Jie, Pinghui; Tian, Yujing; Hong, Zhiwen; Li, Lin; Zhou, Libin; Chen, Lei; Chen, Ling

    2015-01-01

    Brain edema is an important pathological process during stroke. Activation of transient receptor potential vanilloid 4 (TRPV4) causes an up-regulation of matrix metalloproteinases (MMPs) in lung tissue. MMP can digest the endothelial basal lamina to destroy blood brain barrier, leading to vasogenic brain edema. Herein, we tested whether TRPV4-blockage could inhibit brain edema through inhibiting MMPs in middle cerebral artery occlusion (MCAO) mice. We found that the brain water content and Evans blue extravasation at 48 h post-MCAO were reduced by a TRPV4 antagonist HC-067047. The increased MMP-2/9 protein expression in hippocampi of MCAO mice was attenuated by HC-067046, but only the increased MMP-9 activity was blocked by HC-067047. The loss of zonula occludens-1 (ZO-1) and occludin protein in MCAO mice was also attenuated by HC-067047. Moreover, MMP-2/9 protein expression increased in mice treated with a TRPV4 agonist GSK1016790A, but only MMP-9 activity was increased by GSK1016790A. Finally, ZO-1 and occludin protein expression was decreased by GSK1016790A, which was reversed by an MMP-9 inhibitor. We conclude that blockage of TRPV4 may inhibit brain edema in cerebral ischemia through inhibiting MMP-9 activation and the loss of tight junction protein.

  19. Retinoic-acid-orphan-receptor-C inhibition suppresses Th17 cells and induces thymic aberrations

    PubMed Central

    Guntermann, Christine; Piaia, Alessandro; Hamel, Marie-Laure; Theil, Diethilde; Rubic-Schneider, Tina; del Rio-Espinola, Alberto; Dong, Linda; Billich, Andreas; Kaupmann, Klemens; Dawson, Janet; Hoegenauer, Klemens; Orain, David; Hintermann, Samuel; Stringer, Rowan; Patel, Dhavalkumar D.; Doelemeyer, Arno; Deurinck, Mark

    2017-01-01

    Retinoic-acid-orphan-receptor-C (RORC) is a master regulator of Th17 cells, which are pathogenic in several autoimmune diseases. Genetic Rorc deficiency in mice, while preventing autoimmunity, causes early lethality due to metastatic thymic T cell lymphomas. We sought to determine whether pharmacological RORC inhibition could be an effective and safe therapy for autoimmune diseases by evaluating its effects on Th17 cell functions and intrathymic T cell development. RORC inhibitors effectively inhibited Th17 differentiation and IL-17A production, and delayed-type hypersensitivity reactions. In vitro, RORC inhibitors induced apoptosis, as well as Bcl2l1 and BCL2L1 mRNA downregulation, in mouse and nonhuman primate thymocytes, respectively. Chronic, 13-week RORC inhibitor treatment in rats caused progressive thymic alterations in all analyzed rats similar to those in Rorc-deficient mice prior to T cell lymphoma development. One rat developed thymic cortical hyperplasia with neoplastic features, including increased mitosis and reduced IKAROS expression, albeit without skewed T cell clonality. In summary, pharmacological inhibition of RORC not only blocks Th17 cell development and related cytokine production, but also recapitulates thymic aberrations seen in Rorc-deficient mice. While RORC inhibition may offer an effective therapeutic principle for Th17-mediated diseases, T cell lymphoma with chronic therapy remains an apparent risk. PMID:28289717

  20. Losartan, a selective inhibitor of subtype AT1 receptors for angiotensin II, inhibits the binding of N-formylmethionyl-leucyl-phenylalanine to neutrophil receptors.

    PubMed

    Raiden, S; Giordano, M; Andonegui, G; Trevani, A S; López, D H; Nahmod, V; Geffner, J R

    1997-05-01

    Losartan, a selective antagonist of AT1 receptors for angiotensin II, is widely used clinically to manage hypertension. We report here that losartan markedly inhibits neutrophil shape change, adherence and chemiluminescence responses triggered by N-formylmethionyl-leucyl-phenylalanine (fMLP), without affecting responses induced by immune complexes, zymosan or concanavalin A. Neither saralasin, another antagonist of angiotensin II receptors, nor captopril, an angiotensin-converting enzyme inhibitor, reproduced the effects of losartan. It was also observed that neutrophil responses triggered by fMLP were not affected by exogenously added angiotensin II. The effect of losartan on the binding of fMLP was measured using [3H]fMLP. It was found that losartan inhibits the binding of [3H]fMLP to neutrophil receptors. As observed for neutrophils, studies performed with monocytes showed that losartan inhibits chemiluminescence emission triggered by fMLP, without affecting chemiluminescence responses triggered by immune complexes, zymosan or concanavalin A.

  1. Histamine H3 receptor-mediated inhibition of excitatory synaptic transmission in the rat dentate gyrus in vivo.

    PubMed

    Chang, M; Saito, H; Abe, K

    1998-07-01

    We investigated the effects of histamine H3-receptor ligands on hippocampal synaptic transmission by using anesthetized rats in vivo. The medial perforant path was stimulated, and the population excitatory postsynaptic potential (pEPSP) and population spike were recorded from the granule cell layer of the dentate gyrus. Intracerebroventricular injection of the H3-receptor agonist (R)-alpha-methylhistamine decreased both the pEPSP and population spike, while H3-receptor antagonists, clobenpropit and thioperamide, increased both the pEPSP and population spike. These results suggest that the histaminergic system plays a role in inhibition of hippocampal synaptic excitation via the H3 receptor.

  2. Halothane inhibits the cholinergic-receptor-mediated influx of calcium in primary culture of bovine adrenal medulla cells

    SciTech Connect

    Yashima, N.; Wada, A.; Izumi, F.

    1986-04-01

    Adrenal medulla cells are cholinoceptive cells. Stimulation of the acetylcholine receptor causes the influx of Ca to the cells, and Ca acts as the coupler of the stimulus-secretion coupling. In this study, the authors investigated the effects of halothane on the receptor-mediated influx of /sup 45/Ca using cultured bovine adrenal medulla cells. Halothane at clinical concentrations (0.5-2%) inhibited the influx of /sup 45/Ca caused by carbachol, with simultaneous inhibition of catecholamine secretion. The influx of /sup 45/Ca and the secretion of catecholamines caused by K depolarization were inhibited by a large concentration of Mg, which competes with Ca at Ca channels, but not inhibited by halothane. Inhibition of the /sup 45/Ca influx by halothane was not overcome by increase in the carbachol concentration. Inhibition of the /sup 45/Ca influx by halothane was examined in comparison with that caused by a large concentration of Mg by the application of Scatchard analysis as the function of the external Ca concentration. Halothane decreased the maximal influx of /sup 45/Ca without altering the apparent kinetic constant of Ca to Ca channels. On the contrary, a large concentration of Mg increased the apparent kinetic constant without altering the maximal influx of /sup 45/Ca. Based on these findings, the authors suggest that inhibition of the /sup 45/Ca influx by halothane was not due to the direct competitive inhibition of Ca channels, nor to the competitive antagonism of agonist-receptor interaction. As a possibility, halothane seems to inhibit the receptor-mediated activation of Ca channels through the interference of coupling between the receptor and Ca channels.

  3. Non-competitive androgen receptor inhibition in vitro and in vivo.

    PubMed

    Jones, Jeremy O; Bolton, Eric C; Huang, Yong; Feau, Clementine; Guy, R Kiplin; Yamamoto, Keith R; Hann, Byron; Diamond, Marc I

    2009-04-28

    Androgen receptor (AR) inhibitors are used to treat multiple human diseases, including hirsutism, benign prostatic hypertrophy, and prostate cancer, but all available anti-androgens target only ligand binding, either by reduction of available hormone or by competitive antagonism. New strategies are needed, and could have an important impact on therapy. One approach could be to target other cellular mechanisms required for receptor activation. In prior work, we used a cell-based assay of AR conformation change to identify non-ligand inhibitors of AR activity. Here, we characterize 2 compounds identified in this screen: pyrvinium pamoate, a Food and Drug Administration-approved drug, and harmol hydrochloride, a natural product. Each compound functions by a unique, non-competitive mechanism and synergizes with competitive antagonists to disrupt AR activity. Harmol blocks DNA occupancy by AR, whereas pyrvinium does not. Pyrvinium inhibits AR-dependent gene expression in the prostate gland in vivo, and induces prostate atrophy. These results highlight new therapeutic strategies to inhibit AR activity.

  4. Lipopolysaccharide Inhibits the Channel Activity of the P2X7 Receptor

    PubMed Central

    Leiva-Salcedo, Elias; Coddou, Claudio; Rodríguez, Felipe E.; Penna, Antonello; Lopez, Ximena; Neira, Tanya; Fernández, Ricardo; Imarai, Mónica; Rios, Miguel; Escobar, Jorge; Montoya, Margarita; Huidobro-Toro, J. Pablo; Escobar, Alejandro; Acuña-Castillo, Claudio

    2011-01-01

    The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance. PMID:21941410

  5. Phosphorylation of Jak2 on Ser523 Inhibits Jak2-Dependent Leptin Receptor Signaling†

    PubMed Central

    Ishida-Takahashi, Ryoko; Rosario, Felicia; Gong, Yusong; Kopp, Keely; Stancheva, Zlatina; Chen, Xiaohong; Feener, Edward P.; Myers, Martin G.

    2006-01-01

    The leptin receptor, LRb, and other cytokine receptors are devoid of intrinsic enzymatic activity and rely upon the activity of constitutively associated Jak family tyrosine kinases to mediate intracellular signaling. In order to clarify mechanisms by which Jak2, the cognate LRb-associated Jak kinase, is regulated and mediates downstream signaling, we employed tandem mass spectroscopic analysis to identify phosphorylation sites on Jak2. We identified Ser523 as the first-described site of Jak2 serine phosphorylation and demonstrated that this site is phosphorylated on Jak2 from intact cells and mouse spleen. Ser523 was highly phosphorylated in HEK293 cells independently of LRb-Jak2 activation, suggesting a potential role for the phosphorylation of Ser523 in the regulation of LRb by other pathways. Indeed, mutation of Ser523 sensitized and prolonged signaling by Jak2 following activation by the intracellular domain of LRb. The effect of Ser523 on Jak2 function was independent of Tyr570-mediated inhibition. Thus, the phosphorylation of Jak2 on Ser523 inhibits Jak2 activity and represents a novel mechanism for the regulation of Jak2-dependent cytokine signaling. PMID:16705160

  6. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    PubMed Central

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes. PMID:23530060

  7. Inhibition of androgen receptor by decoy molecules delays progression to castration-recurrent prostate cancer

    PubMed Central

    Myung, Jae-Kyung; Wang, Gang; Chiu, Helen H. L.; Wang, Jun; Mawji, Nasrin R.; Sadar, Marianne D.

    2017-01-01

    Androgen receptor (AR) is a member of the steroid receptor family and a therapeutic target for all stages of prostate cancer. AR is activated by ligand binding within its C-terminus ligand-binding domain (LBD). Here we show that overexpression of the AR NTD to generate decoy molecules inhibited both the growth and progression of prostate cancer in castrated hosts. Specifically, it was shown that lentivirus delivery of decoys delayed hormonal progression in castrated hosts as indicated by increased doubling time of tumor volume, prolonged time to achieve pre-castrate levels of serum prostate-specific antigen (PSA) and PSA nadir. These clinical parameters are indicative of delayed hormonal progression and improved therapeutic response and prognosis. Decoys reduced the expression of androgen-regulated genes that correlated with reduced in situ interaction of the AR with androgen response elements. Decoys did not reduce levels of AR protein or prevent nuclear localization of the AR. Nor did decoys interact directly with the AR. Thus decoys did not inhibit AR transactivation by a dominant negative mechanism. This work provides evidence that the AR NTD plays an important role in the hormonal progression of prostate cancer and supports the development of AR antagonists that target the AR NTD. PMID:28306720

  8. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    PubMed Central

    Yin, Yongjun; Ren, Xiaodi; Smith, Craig; Guo, Qianxu; Malabunga, Maria; Guernah, Ilhem; Zhang, Yiwei; Shen, Juqun; Sun, Haijun; Chehab, Nabil; Loizos, Nick; Ludwig, Dale L.; Ornitz, David M.

    2016-01-01

    ABSTRACT Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. PMID:27056048

  9. The vascular endothelial growth factor receptor inhibitor PTK787/ZK222584 inhibits aromatase.

    PubMed

    Banerjee, Susana; Zvelebil, Marketa; Furet, Pascal; Mueller-Vieira, Ursula; Evans, Dean B; Dowsett, Mitch; Martin, Lesley-Ann

    2009-06-01

    Endocrine therapy is well established for the treatment of breast cancer, and antiangiogenic agents are showing considerable promise. Targeting the vascular endothelial growth factor (VEGF) and estrogen receptor (ER) signaling pathways concomitantly may provide enhanced therapeutic benefit in ER-positive breast cancer. Therefore, the effects of the VEGF receptor (VEGFR) tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) were investigated using human breast cancer cell lines engineered to express aromatase. As expected in this system, estrogen (E2) or androstenedione induced a proliferative response and increased ER-mediated transcription in ER-positive cell lines expressing aromatase. However, surprisingly, in the presence of androstenedione, PTK/ZK suppressed both the androstenedione-stimulated proliferation and ER-mediated transcription. PTK/ZK alone and in the presence of E2 had no observable effect on proliferation or ER-mediated transcription. These effects result from PTK/ZK having previously unrecognized antiaromatase activity and PTK/ZK being a competitive aromatase inhibitor. Computer-assisted molecular modeling showed that PTK/ZK could potentially bind directly to aromatase. The demonstration that PTK/ZK inhibits aromatase and VEGFR indicates that agents cross-inhibiting two important classes of targets in breast cancer could be developed.

  10. The inhibition of release by mGlu7 receptors is independent of the Ca2+ channel type but associated to GABAB and adenosine A1 receptors.

    PubMed

    Martín, Ricardo; Ladera, Carolina; Bartolomé-Martín, David; Torres, Magdalena; Sánchez-Prieto, José

    2008-09-01

    Neurotransmitter release is inhibited by G-protein coupled receptors (GPCRs) through signalling pathways that are negatively coupled to Ca2+ channels and adenylyl cyclase. Through Ca2+ imaging and immunocytochemistry, we have recently shown that adenosine A1, GABAB and the metabotropic glutamate type 7 receptors coexist in a subset of cerebrocortical nerve terminals. As these receptors inhibit glutamate release through common intracellular signalling pathways, their co-activation occluded each other responses. Here we have addressed whether the occlusion of receptor responses is restricted to the glutamate release mediated by N-type Ca2+ channels by analysing this process in nerve terminals from mice lacking the alpha1B subunit (Cav 2.2) of these channels. We found that glutamate release from cerebrocortical nerve terminals without these channels, in which release relies exclusively on P/Q type Ca2+ channels, is not modulated by mGlu7 receptors. Furthermore, there is no occlusion of the release inhibition by GABAB and adenosine A1. Hence, in the cerebrocortical preparation, these three receptors only appear to coexist in N-type channel containing nerve terminals. In contrast, in hippocampal nerve terminals lacking this subunit, where mGlu7 receptors modulate glutamate release via P/Q type channels, the occlusion of inhibitory responses by co-stimulation of adenosine A1, GABAB and mGlu7 receptors was observed. Thus, occlusion of the responses by the three GPCRs is independent of the Ca2+ channel type but rather, it is associated to functional mGlu7 receptors.

  11. 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.

  12. Inhibition of Androgen Receptor Transcriptional Activity as a Novel Mechanism of Action of Arsenic

    PubMed Central

    Rosenblatt, Adena E.; Burnstein, Kerry L.

    2009-01-01

    Environmental sodium arsenite is a toxin that is associated with male infertility due to decreased and abnormal sperm production. Arsenic trioxide (ATO), another inorganic trivalent semimetal, is an effective therapy for acute promyelocytic leukemia, and there is investigation of its possible efficacy in prostate cancer. However, the mechanism of arsenic action in male urogenital tract tissues is not clear. Because the androgen receptor (AR) plays an important role in spermatogenesis and prostate cancer, we explored the possibility that trivalent arsenic regulates AR function. We found that arsenic inhibited AR transcriptional activity in prostate cancer and Sertoli cells using reporter gene assays testing several androgen response element-containing regions and by assessing native target gene expression. Arsenic inhibition of AR activity was not due to down-regulation of AR protein levels, decreased hormone binding to AR, disruption of AR nuclear translocation, or interference with AR-DNA binding in vitro. However, chromatin immunoprecipitation studies revealed that arsenic inhibited AR recruitment to an AR target gene enhancer in vivo. Consistent with a deficiency in AR-chromatin binding, arsenic disrupted AR amino and carboxyl termini interaction. Furthermore, ATO caused a significant decrease in prostate cancer cell proliferation that was more pronounced in cells expressing AR compared with cells depleted of AR. In addition, inhibition of AR activity by ATO and by the AR antagonist, bicalutamide, was additive. Thus, arsenic-induced male infertility may be due to inhibition of AR activity. Further, because AR is an important target in prostate cancer therapy, arsenic may serve as an effective therapeutic option. PMID:19131511

  13. Inhibition of carbonic anhydrase augments GABAA receptor-mediated analgesia via a spinal mechanism of action

    PubMed Central

    Asiedu, Marina N.; Mejia, Galo L.; Hübner, Christian A.; Kaila, Kai; Price, Theodore J.

    2014-01-01

    Peripheral nerve injury negatively influences spinal GABAergic networks via a reduction in the neuron-specific K+-Cl- cotransporter KCC2. This process has been linked to the emergence of neuropathic allodynia. In vivo pharmacological and modeling studies show that a loss of KCC2 function results in a decrease in the efficacy of GABAA -mediated spinal inhibition. One potential strategy to mitigate this effect entails inhibition of carbonic anhydrase activity to reduce HCO3- -dependent depolarization via GABAA receptors when KCC2 function is compromised. We have tested this hypothesis here. Our results show that, similarly to when KCC2 is pharmacologically blocked, peripheral nerve injury causes a loss of analgesic effect for neurosteroid GABAA allosteric modulators at maximally effective doses in naïve mice in the tail flick test. Remarkably, inhibition of carbonic anhydrase activity with intrathecal acetazolamide rapidly restores an analgesic effect for these compounds suggesting an important role of carbonic anhydrase activity in regulating GABAA -mediated analgesia after peripheral nerve injury. Moreover, spinal acetazolamide administration leads to a profound reduction in the mouse formalin pain test indicating that spinal carbonic anhydrase inhibition produces analgesia when primary afferent activity is driven by chemical mediators. Finally, we demonstrate that systemic administration of acetazolamide to rats with peripheral nerve injury produces an anti-allodynia effect by itself and an enhancement of the peak analgesic effect with a change in the shape of the dose response curve of the α1-sparing benzodiazepine L-838,417. Thus, carbonic anhydrase inhibition mitigates the negative effects of loss of KCC2 function after nerve injury in multiple species and through multiple administration routes resulting in an enhancement of analgesic effects for several GABAA allosteric modulators. We suggest that carbonic anhydrase inhibitors, many of which are clinically

  14. EP3 receptors inhibit antidiuretic-hormone-dependent sodium transport across frog skin epithelium.

    PubMed

    Rytved, K A; Nielsen, R

    1999-01-01

    We examined the effect of prostaglandin E2 (PGE2) on antidiuretic hormone (ADH)-dependent Na+ transport and cAMP production in isolated frog skin epithelium. ADH caused an increase in transepithelial Na+ transport and a decrease in cellular potential, indicating an increase in apical Na+ permeability. Subsequent addition of PGE2 decreased Na+ transport and repolarised the cells. The PGE2 receptor EP1/3-selective analogue sulprostone and the PGE2 receptor EP2/3-selective analogue misoprostol were able to mimic the effect of PGE2. ADH increased cellular cAMP levels, whereas PGE2, sulprostone and misoprostol were able to reduce the ADH-dependent cAMP production. Measurements of intracellular Ca2+ concentration ([Ca2+]i) revealed that it was unaffected by both PGE2 and sulprostone. The inhibitory effect of PGE2 on ADH-dependent Na+ transport was also observed in Ca2+-depleted epithelia. We conclude that ADH stimulates transepithelial Na+ transport by increasing cellular cAMP levels, whereas PGE2 inhibits ADH-dependent Na+ transport by activating EP3-type receptors, which decrease cellular cAMP levels. We have found no evidence that [Ca2+]i is involved in the regulation of ADH-dependent Na+ transport by PGE2.

  15. Inhibition of epidermal growth factor receptor attenuates atherosclerosis via decreasing inflammation and oxidative stress.

    PubMed

    Wang, Lintao; Huang, Zhouqing; Huang, Weijian; Chen, Xuemei; Shan, Peiren; Zhong, Peng; Khan, Zia; Wang, Jingying; Fang, Qilu; Liang, Guang; Wang, Yi

    2017-04-04

    Atherosclerosis is a progressive disease leading to loss of vascular homeostasis and entails fibrosis, macrophage foam cell formation, and smooth muscle cell proliferation. Recent studies have reported that epidermal growth factor receptor (EGFR) is involved vascular pathophysiology and in the regulation of oxidative stress in macrophages. Although, oxidative stress and inflammation play a critical role in the development of atherosclerosis, the underlying mechanisms are complex and not completely understood. In the present study, we have elucidated the role of EGFR in high-fat diet-induced atherosclerosis in apolipoprotein E null mice. We show increased EGFR phosphorylation and activity in atherosclerotic lesion development. EGFR inhibition prevented oxidative stress, macrophage infiltration, induction of pro-inflammatory cytokines, and SMC proliferation within the lesions. We further show that EGFR is activated through toll-like receptor 4. Disruption of toll-like receptor 4 or the EGFR pathway led to reduced inflammatory activity and foam cell formation. These studies provide evidence that EGFR plays a key role on the pathogenesis of atherosclerosis, and suggests that EGFR may be a potential therapeutic target in the prevention of atherosclerosis development.

  16. NMDA receptor activation inhibits alpha-secretase and promotes neuronal amyloid-beta production.

    PubMed

    Lesné, Sylvain; Ali, Carine; Gabriel, Cecília; Croci, Nicole; MacKenzie, Eric T; Glabe, Charles G; Plotkine, Michel; Marchand-Verrecchia, Catherine; Vivien, Denis; Buisson, Alain

    2005-10-12

    Acute brain injuries have been identified as a risk factor for developing Alzheimer's disease (AD). Because glutamate plays a pivotal role in these pathologies, we studied the influence of glutamate receptor activation on amyloid-beta (Abeta) production in primary cultures of cortical neurons. We found that sublethal NMDA receptor activation increased the production and secretion of Abeta. This effect was preceded by an increased expression of neuronal Kunitz protease inhibitory domain (KPI) containing amyloid-beta precursor protein (KPI-APP) followed by a shift from alpha-secretase to beta-secretase-mediated APP processing. This shift is a result of the inhibition of the alpha-secretase candidate tumor necrosis factor-alpha converting enzyme (TACE) when associated with neuronal KPI-APPs. This KPI-APP/TACE interaction was also present in AD brains. Thus, our findings reveal a cellular mechanism linking NMDA receptor activation to neuronal Abeta secretion. These results suggest that even mild deregulation of the glutamatergic neurotransmission may increase Abeta production and represent a causal risk factor for developing AD.

  17. Histamine H3 receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion.

    PubMed

    Iida, Tomomitsu; Yoshikawa, Takeo; Matsuzawa, Takuro; Naganuma, Fumito; Nakamura, Tadaho; Miura, Yamato; Mohsen, Attayeb S; Harada, Ryuichi; Iwata, Ren; Yanai, Kazuhiko

    2015-07-01

    Histamine is a physiological amine which initiates a multitude of physiological responses by binding to four known G-protein coupled histamine receptor subtypes as follows: histamine H1 receptor (H1 R), H2 R, H3 R, and H4 R. Brain histamine elicits neuronal excitation and regulates a variety of physiological processes such as learning and memory, sleep-awake cycle and appetite regulation. Microglia, the resident macrophages in the brain, express histamine receptors; however, the effects of histamine on critical microglial functions such as chemotaxis, phagocytosis, and cytokine secretion have not been examined in primary cells. We demonstrated that mouse primary microglia express H2 R, H3 R, histidine decarboxylase, a histamine synthase, and histamine N-methyltransferase, a histamine metabolizing enzyme. Both forskolin-induced cAMP accumulation and ATP-induced intracellular Ca(2+) transients were reduced by the H3 R agonist imetit but not the H2 R agonist amthamine. H3 R activation on two ubiquitous second messenger signalling pathways suggests that H3 R can regulate various microglial functions. In fact, histamine and imetit dose-dependently inhibited microglial chemotaxis, phagocytosis, and lipopolysaccharide (LPS)-induced cytokine production. Furthermore, we confirmed that microglia produced histamine in the presence of LPS, suggesting that H3 R activation regulate microglial function by autocrine and/or paracrine signalling. In conclusion, we demonstrate the involvement of histamine in primary microglial functions, providing the novel insight into physiological roles of brain histamine.

  18. Dopamine and angiotensin type 2 receptors cooperatively inhibit sodium transport in human renal proximal tubule cells.

    PubMed

    Gildea, John J; Wang, Xiaoli; Shah, Neema; Tran, Hanh; Spinosa, Michael; Van Sciver, Robert; Sasaki, Midori; Yatabe, Junichi; Carey, Robert M; Jose, Pedro A; Felder, Robin A

    2012-08-01

    Little is known regarding how the kidney shifts from a sodium and water reclaiming state (antinatriuresis) to a state where sodium and water are eliminated (natriuresis). In human renal proximal tubule cells, sodium reabsorption is decreased by the dopamine D(1)-like receptors (D(1)R/D(5)R) and the angiotensin type 2 receptor (AT(2)R), whereas the angiotensin type 1 receptor increases sodium reabsorption. Aberrant control of these opposing systems is thought to lead to sodium retention and, subsequently, hypertension. We show that D(1)R/D(5)R stimulation increased plasma membrane AT(2)R 4-fold via a D(1)R-mediated, cAMP-coupled, and protein phosphatase 2A-dependent specific signaling pathway. D(1)R/D(5)R stimulation also reduced the ability of angiotensin II to stimulate phospho-extracellular signal-regulated kinase, an effect that was partially reversed by an AT(2)R antagonist. Fenoldopam did not increase AT(2)R recruitment in renal proximal tubule cells with D(1)Rs uncoupled from adenylyl cyclase, suggesting a role of cAMP in mediating these events. D(1)Rs and AT(2)Rs heterodimerized and cooperatively increased cAMP and cGMP production, protein phosphatase 2A activation, sodium-potassium-ATPase internalization, and sodium transport inhibition. These studies shed new light on the regulation of renal sodium transport by the dopaminergic and angiotensin systems and potential new therapeutic targets for selectively treating hypertension.

  19. Phosphorylation inhibits DNA-binding of alternatively spliced aryl hydrocarbon receptor nuclear translocator

    SciTech Connect

    Kewley, Robyn J. . E-mail: rkewley@csu.edu.au; Whitelaw, Murray L.

    2005-12-09

    The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer.

  20. Inhibition of the Androgen Receptor by Antiandrogens in Spinobulbar Muscle Atrophy.

    PubMed

    Baniahmad, Aria

    2016-03-01

    Spinal-bulbar muscle atrophy (SBMA) or also named Kennedy's Disease is caused by a polyglutamine expansion (PolyQ) of the coding region of the androgen receptor (AR). The AR is a ligand-controlled transcription factor and member of the nuclear hormone receptor superfamily. The central characteristics of the SBMA pathogenicity are muscle weakness, the loss of motoneurons and the occurrence of AR-containing protein aggregates that are observed in spinal cord motoneurons and skeletal muscles induced by the AR-PolyQ expansion in the presence of androgens. The PolyQ triggers a misfolding in the AR-PolyQ and leads to protein aggregation in spinal cord motoneurons and muscle cells. The AR-PolyQ toxicity is activated by the AR ligand testosterone and dihydrotestosterone that activate the receptor and triggers nuclear toxicity by inducing AR nuclear translocation. In line with this, androgen treatment of SBMA patients worsened the SBMA symptoms. SBMA has been modeled in AR-overexpressing and AR-PolyQ-knock-in animals, but precisely how the PolyQ expansion leads to neurodegeneration is unclear. The androgen-induced toxicity and androgen-dependent nuclear accumulation of AR-PolyQ protein seems to be central to the pathogenesis. Therefore, the inhibition of the androgen-activated AR-PolyQ might be a therapeutic option. Here the use of AR antagonists for treatment option of SBMA will be reviewed and discussed.

  1. FDA-Approved Selective Estrogen Receptor Modulators Inhibit Ebola Virus Infection

    PubMed Central

    Johansen, Lisa M.; Brannan, Jennifer M.; Delos, Sue E.; Shoemaker, Charles J.; Stossel, Andrea; Lear, Calli; Hoffstrom, Benjamin G.; DeWald, Lisa Evans; Schornberg, Kathryn L.; Scully, Corinne; Lehár, Joseph; Hensley, Lisa E.; White, Judith M.; Olinger, Gene G.

    2014-01-01

    Ebola viruses remain a substantial threat to both civilian and military populations as bioweapons, during sporadic outbreaks, and from the possibility of accidental importation from endemic regions by infected individuals. Currently, no approved therapeutics exist to treat or prevent infection by Ebola viruses. Therefore, we performed an in vitro screen of Food and Drug Administration (FDA)– and ex–US-approved drugs and selected molecular probes to identify drugs with antiviral activity against the type species Zaire ebolavirus (EBOV). From this screen, we identified a set of selective estrogen receptor modulators (SERMs), including clomiphene and toremifene, which act as potent inhibitors of EBOV infection. Anti-EBOV activity was confirmed for both of these SERMs in an in vivo mouse infection model. This anti-EBOV activity occurred even in the absence of detectable estrogen receptor expression, and both SERMs inhibited virus entry after internalization, suggesting that clomiphene and toremifene are not working through classical pathways associated with the estrogen receptor. Instead, the response appeared to be an off-target effect where the compounds interfere with a step late in viral entry and likely affect the triggering of fusion. These data support the screening of readily available approved drugs to identify therapeutics for the Ebola viruses and other infectious diseases. The SERM compounds described in this report are an immediately actionable class of approved drugs that can be repurposed for treatment of filovirus infections. PMID:23785035

  2. Histamine H3-receptors inhibit sympathetic neurotransmission in guinea pig myocardium.

    PubMed

    Luo, X X; Tan, Y H; Sheng, B H

    1991-11-12

    The histamine H3 agonist, (R)-alpha-methylhistamine (alpha-MeHA, 10(-10) to 10(-5) M), caused a concentration-dependent inhibition of the sympathetic contractile response to electrical field stimulation of guinea pig isolated atria, but alpha-MeHA did not alter the basal tension or the contraction induced by exogenously applied norepinephrine. Blockade of H1 and H2 histamine receptors, and alpha- and beta-adrenoceptors failed to prevent the inhibitory effect of alpha-MeHA, whereas the specific H3 receptor antagonist, thioperamide, concentration dependently reversed the inhibitory effect of alpha-MeHA. At the concentration of 10(-7) M, which was effective for antagonizing the action of alpha-MeHA, thioperamide did not modify the sympathetic responses facilitated by the beta 2-adrenoceptor agonist, clenbuterol, or attenuated by the alpha 2-adrenoceptor agonist, clonidine. Our results suggest that H3 receptors exist on the cardiac sympathetic terminals, which may modulate adrenergic neurotransmission in guinea pig myocardium.

  3. SKF-83566, a D1-dopamine receptor antagonist, inhibits the dopamine transporter.

    PubMed

    Stouffer, Melissa A; Ali, Solav; Reith, Maarten E A; Patel, Jyoti C; Sarti, Federica; Carr, Kenneth D; Rice, Margaret E

    2011-09-01

    Dopamine (DA) is an important transmitter in both motor and limbic pathways. We sought to investigate the role of D(1)-receptor activation in axonal DA release regulation in dorsal striatum using a D(1)-receptor antagonist, SKF-83566. Evoked DA release was monitored in rat striatal slices using fast-scan cyclic voltammetry. SKF-83566 caused a concentration-dependent increase in peak single-pulse evoked extracellular DA concentration, with a maximum increase of ∼ 65% in 5 μM SKF-83566. This was accompanied by a concentration-dependent increase in extracellular DA concentration clearance time. Both effects were occluded by nomifensine (1 μM), a dopamine transporter (DAT) inhibitor, suggesting that SKF-83566 acted via the DAT. We tested this by examining [(3)H]DA uptake into LLc-PK cells expressing rat DAT, and confirmed that SKF-83566 is a competitive DAT inhibitor with an IC(50) of 5.7 μM. Binding studies with [(3)H]CFT, a cocaine analog, showed even more potent action of SKF-83566 at the DAT cocaine binding site (IC(50) = 0.51 μM). Thus, data obtained using SKF-83566 as a D(1) DA-receptor antagonist may be confounded by concurrent DAT inhibition. More positively, however, SKF-83566 might be a candidate to attenuate cocaine effects in vivo because of the greater potency of this drug at the cocaine versus DA binding site of the DAT.

  4. Amphetamine decreases behavioral inhibition by stimulation of dopamine D2, but not D3, receptors.

    PubMed

    van Gaalen, Marcel M; Unger, Liliane; Jongen-Rêlo, Ana-Lucia; Schoemaker, Hans; Gross, Gerhard

    2009-09-01

    Behavioral disinhibition is a manifestation of impulsive behavior that is prominent in the psychopathology of various psychiatric disorders such as addiction, attention-deficit hyperactivity disorder, mania, and personality disorders. Impulsivity may be studied by measuring anticipatory responses made before the presentation of a food-predictive, brief light stimulus in a two-choice serial reaction time task. In such serial reaction time tasks, amphetamine has been shown to produce dose-dependent increases in premature responding in a manner dependent on dopamine D(2)-like receptor stimulation. So far, it is unknown whether it is the D(2) or D(3) receptor that is involved in this form of impulsivity. In this study, rats were trained in a two-choice serial reaction time task until baseline performance was stable. Next, effects of the dopamine D(2) preferring antagonist L-741,626 and selective D(3) antagonist SB-277011 were assessed alone and in the presence of amphetamine. Neither L-741,626 nor SB-277011 affected behavioral inhibition, although the latter significantly increased reaction time at 10 mg/kg. Amphetamine dose-dependently increased impulsivity. The effect of amphetamine was attenuated by L-741,626 (3 mg/kg), whereas SB-277011 (3 mg/kg) had no effect. Therefore, amphetamine-induced behavioral disinhibition depends on D(2), but not D(3), receptor stimulation.

  5. ApoC-III inhibits clearance of triglyceride-rich lipoproteins through LDL family receptors

    PubMed Central

    Gordts, Philip L.S.M.; Son, Ni-Huiping; Ramms, Bastian; Lew, Irene; Gonzales, Jon C.; Thacker, Bryan E.; Basu, Debapriya; Lee, Richard G.; Mullick, Adam E.; Graham, Mark J.; Goldberg, Ira J.; Crooke, Rosanne M.; Witztum, Joseph L.

    2016-01-01

    Hypertriglyceridemia is an independent risk factor for cardiovascular disease, and plasma triglycerides (TGs) correlate strongly with plasma apolipoprotein C-III (ApoC-III) levels. Antisense oligonucleotides (ASOs) for ApoC-III reduce plasma TGs in primates and mice, but the underlying mechanism of action remains controversial. We determined that a murine-specific ApoC-III–targeting ASO reduces fasting TG levels through a mechanism that is dependent on low-density lipoprotein receptors (LDLRs) and LDLR-related protein 1 (LRP1). ApoC-III ASO treatment lowered plasma TGs in mice lacking lipoprotein lipase (LPL), hepatic heparan sulfate proteoglycan (HSPG) receptors, LDLR, or LRP1 and in animals with combined deletion of the genes encoding HSPG receptors and LDLRs or LRP1. However, the ApoC-III ASO did not lower TG levels in mice lacking both LDLR and LRP1. LDLR and LRP1 were also required for ApoC-III ASO–induced reduction of plasma TGs in mice fed a high-fat diet, in postprandial clearance studies, and when ApoC-III–rich or ApoC-III–depleted lipoproteins were injected into mice. ASO reduction of ApoC-III had no effect on VLDL secretion, heparin-induced TG reduction, or uptake of lipids into heart and skeletal muscle. Our data indicate that ApoC-III inhibits turnover of TG-rich lipoproteins primarily through a hepatic clearance mechanism mediated by the LDLR/LRP1 axis. PMID:27400128

  6. Inhibition of epidermal growth factor receptor attenuates atherosclerosis via decreasing inflammation and oxidative stress

    PubMed Central

    Wang, Lintao; Huang, Zhouqing; Huang, Weijian; Chen, Xuemei; Shan, Peiren; Zhong, Peng; Khan, Zia; Wang, Jingying; Fang, Qilu; Liang, Guang; Wang, Yi

    2017-01-01

    Atherosclerosis is a progressive disease leading to loss of vascular homeostasis and entails fibrosis, macrophage foam cell formation, and smooth muscle cell proliferation. Recent studies have reported that epidermal growth factor receptor (EGFR) is involved vascular pathophysiology and in the regulation of oxidative stress in macrophages. Although, oxidative stress and inflammation play a critical role in the development of atherosclerosis, the underlying mechanisms are complex and not completely understood. In the present study, we have elucidated the role of EGFR in high-fat diet-induced atherosclerosis in apolipoprotein E null mice. We show increased EGFR phosphorylation and activity in atherosclerotic lesion development. EGFR inhibition prevented oxidative stress, macrophage infiltration, induction of pro-inflammatory cytokines, and SMC proliferation within the lesions. We further show that EGFR is activated through toll-like receptor 4. Disruption of toll-like receptor 4 or the EGFR pathway led to reduced inflammatory activity and foam cell formation. These studies provide evidence that EGFR plays a key role on the pathogenesis of atherosclerosis, and suggests that EGFR may be a potential therapeutic target in the prevention of atherosclerosis development. PMID:28374780

  7. Diurnal inhibition of NMDA-EPSCs at rat hippocampal mossy fibre synapses through orexin-2 receptors

    PubMed Central

    Perin, Martina; Longordo, Fabio; Massonnet, Christine; Welker, Egbert; Lüthi, Anita

    2014-01-01

    Diurnal release of the orexin neuropeptides orexin-A (Ox-A, hypocretin-1) and orexin-B (Ox-B, hypocretin-2) stabilises arousal, regulates energy homeostasis and contributes to cognition and learning. However, whether cellular correlates of brain plasticity are regulated through orexins, and whether they do so in a time-of-day-dependent manner, has never been assessed. Immunohistochemically we found sparse but widespread innervation of hippocampal subfields through Ox-A- and Ox-B-containing fibres in young adult rats. The actions of Ox-A were studied on NMDA receptor (NMDAR)-mediated excitatory synaptic transmission in acute hippocampal slices prepared around the trough (Zeitgeber time (ZT) 4–8, corresponding to 4–8 h into the resting phase) and peak (ZT 23) of intracerebroventricular orexin levels. At ZT 4–8, exogenous Ox-A (100 nm in bath) inhibited NMDA receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) at mossy fibre (MF)–CA3 (to 55.6 ± 6.8% of control, P = 0.0003) and at Schaffer collateral–CA1 synapses (70.8 ± 6.3%, P = 0.013), whereas it remained ineffective at non-MF excitatory synapses in CA3. Ox-A actions were mediated postsynaptically and blocked by the orexin-2 receptor (OX2R) antagonist JNJ10397049 (1 μm), but not by orexin-1 receptor inhibition (SB334867, 1 μm) or by adrenergic and cholinergic antagonists. At ZT 23, inhibitory effects of exogenous Ox-A were absent (97.6 ± 2.9%, P = 0.42), but reinstated (87.2 ± 3.3%, P = 0.002) when endogenous orexin signalling was attenuated for 5 h through i.p. injections of almorexant (100 mg kg−1), a dual orexin receptor antagonist. In conclusion, endogenous orexins modulate hippocampal NMDAR function in a time-of-day-dependent manner, suggesting that they may influence cellular plasticity and consequent variations in memory performance across the sleep–wake cycle. PMID:25085886

  8. Diurnal inhibition of NMDA-EPSCs at rat hippocampal mossy fibre synapses through orexin-2 receptors.

    PubMed

    Perin, Martina; Longordo, Fabio; Massonnet, Christine; Welker, Egbert; Lüthi, Anita

    2014-10-01

    Diurnal release of the orexin neuropeptides orexin-A (Ox-A, hypocretin-1) and orexin-B (Ox-B, hypocretin-2) stabilises arousal, regulates energy homeostasis and contributes to cognition and learning. However, whether cellular correlates of brain plasticity are regulated through orexins, and whether they do so in a time-of-day-dependent manner, has never been assessed. Immunohistochemically we found sparse but widespread innervation of hippocampal subfields through Ox-A- and Ox-B-containing fibres in young adult rats. The actions of Ox-A were studied on NMDA receptor (NMDAR)-mediated excitatory synaptic transmission in acute hippocampal slices prepared around the trough (Zeitgeber time (ZT) 4-8, corresponding to 4-8 h into the resting phase) and peak (ZT 23) of intracerebroventricular orexin levels. At ZT 4-8, exogenous Ox-A (100 nm in bath) inhibited NMDA receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) at mossy fibre (MF)-CA3 (to 55.6 ± 6.8% of control, P = 0.0003) and at Schaffer collateral-CA1 synapses (70.8 ± 6.3%, P = 0.013), whereas it remained ineffective at non-MF excitatory synapses in CA3. Ox-A actions were mediated postsynaptically and blocked by the orexin-2 receptor (OX2R) antagonist JNJ10397049 (1 μm), but not by orexin-1 receptor inhibition (SB334867, 1 μm) or by adrenergic and cholinergic antagonists. At ZT 23, inhibitory effects of exogenous Ox-A were absent (97.6 ± 2.9%, P = 0.42), but reinstated (87.2 ± 3.3%, P = 0.002) when endogenous orexin signalling was attenuated for 5 h through i.p. injections of almorexant (100 mg kg(-1)), a dual orexin receptor antagonist. In conclusion, endogenous orexins modulate hippocampal NMDAR function in a time-of-day-dependent manner, suggesting that they may influence cellular plasticity and consequent variations in memory performance across the sleep-wake cycle.

  9. Inhibition of solid tumor growth by gene transfer of VEGF receptor-1 mutants.

    PubMed

    Heidenreich, Regina; Machein, Marcia; Nicolaus, Anke; Hilbig, Andreas; Wild, Carola; Clauss, Matthias; Plate, Karl H; Breier, Georg

    2004-09-01

    Vascular endothelial growth factor (VEGF) and the high-affinity VEGF receptor Flk-1/KDR (VEGFR-2) are key regulators of tumor angiogenesis. Strategies to block VEGF/VEGFR-2 signaling were successfully used to inhibit experimental tumor growth and indicated that VEGFR-2 is the main signaling VEGF receptor in proliferating tumor endothelium. Here, we investigated the role of the VEGF receptor-1 (VEGFR-1/Flt-1) in the vascularization of 2 different experimental tumors in vivo. VEGFR-1 mutants were generated that lack the intracellular tyrosine kinase domain. Retrovirus-mediated gene transfer of the VEGFR-1 mutants led to a strong reduction of tumor growth and angiogenesis in xenografted C6 glioma and in syngeneic BFS-1 fibrosarcoma. Histological analysis of the inhibited fibrosarcoma revealed reduced vascular density, decreased tumor cell proliferation as well as increased tumor cell apoptosis and the formation of necrosis. The retroviral gene transfer of the full length VEGFR-1 also caused a significant reduction of tumor growth in both models. The inhibitory effects of the VEGFR-1 mutants and the full length VEGFR-1 in BFS-1 fibrosarcoma were mediated through host tumor endothelial cells because the BFS-1 fibrosarcoma cells were not infected by the retrovirus. The formation of heterodimers between VEGFR-2 and full length or truncated VEGFR-1 was observed in vitro and might contribute to the growth inhibitory effect by modulating distinct signal transduction pathways. The results of our study underline the central role of the VEGF/VEGFR-1 signaling system in tumor angiogenesis and demonstrate that VEGFR-1 can serve as a target for anti-angiogenic gene therapy.

  10. Purinergic receptor activation inhibits osmotic glial cell swelling in the diabetic rat retina.

    PubMed

    Wurm, Antje; Iandiev, Ianors; Hollborn, Margrit; Wiedemann, Peter; Reichenbach, Andreas; Zimmermann, Herbert; Bringmann, Andreas; Pannicke, Thomas

    2008-10-01

    The anti-inflammatory glucocorticoid, triamcinolone acetonide, is used clinically for the rapid resolution of diabetic macular edema. Osmotic swelling of glial cells may contribute to the development of retinal edema. Triamcinolone inhibits the swelling of retinal glial cells of diabetic rats. Here, we determined whether the effect of triamcinolone is mediated by a receptor-dependent mechanism. Hyperglycemia was induced in rats with streptozotocin injection. After 6-10 months, the swelling properties of glial cells in retinal slices upon hypotonic challenge were determined. Nucleotide-degrading ecto-enzymes were immunostained in retinal slices and glial cells. Hypotonic challenge did not change the size of glial cell bodies from control retinas but induced swelling of cells from diabetic animals. Triamcinolone inhibited glial cell swelling; this effect was prevented by a selective antagonist of adenosine A1 receptors, an inhibitor of nucleoside transporters, inhibitors of adenylyl cyclase and protein kinase A activation, and inhibitors of potassium and chloride channels. In diabetic (but not control) retinas, the effect of triamcinolone apparently involves extracellular nucleotide degradation. Glial cells from diabetic retinas displayed immunolabeling against nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) which was not observed in control retinas. The mRNA expression for NTPDase1 was significantly increased in the retina of diabetic rats. It is suggested that triamcinolone induces the release and formation of endogenous adenosine that subsequently activates A1 receptors resulting in ion efflux through potassium and chloride channels and prevention of osmotic swelling. Whereas adenosine is liberated via facilitated transport in control retinas, an extracellular formation of adenosine contributes to the effect of triamcinolone in diabetic retinas.

  11. LINGO-1 receptor promotes neuronal apoptosis by inhibiting WNK3 kinase activity.

    PubMed

    Zhang, Zhaohuan; Xu, Xiaohui; Xiang, Zhenghua; Yu, Zhongwang; Feng, Jifeng; He, Cheng

    2013-04-26

    LINGO-1 is a functional component of the Nogo receptor 1 · p75(NTR) · LINGO-1 and Nogo receptor 1 · TAJ (TNFRSF19/TROY)·LINGO-1 signaling complexes. It has recently been shown that LINGO-1 antagonists significantly improve neuronal survival after neural injury. However, the mechanism by which LINGO-1 signaling influences susceptibility to apoptosis remains unknown. In an effort to better understand how LINGO-1 regulates these signaling pathways, we used an established model of serum deprivation (SD) to induce neuronal apoptosis. We demonstrate that treatment either with a construct containing the intracellular domain of LINGO-1 or with Nogo66, a LINGO-1 receptor complex agonist, resulted in an enhanced rate of apoptosis in primary cultured cortical neurons under SD. Reducing the expression levels of the serine/threonine kinase WNK3 using shRNA or inhibiting its kinase activity had similar effects on the survival of serum-deprived neurons. Consistent with these observations, we found that LINGO-1 and WNK3 co-localized and co-precipitated in cultured cortical neurons and brain tissue. Significantly, this co-association was enhanced by Nogo66 treatment. Binding of WNK3 to the intracellular domain of LINGO-1 led to a reduction in WNK3 kinase activity, as did Nogo66 stimulation. Moreover, in vitro and in vivo evidence indicates that endogenous WNK3 suppresses SD-induced neuronal apoptosis in a kinase-dependent manner, as the expression of either a WNK3 RNAi construct or a kinase-dead N-terminal fragment of WNK3 led to increased apoptosis. Taken together, our results show that LINGO-1 potentiates neuronal apoptosis, likely by inhibiting WNK3 kinase activity.

  12. Fulvestrant-mediated inhibition of estrogen receptor signaling slows lung cancer progression.

    PubMed

    Tang, Hexiao; Liao, Yongde; Zhang, Chao; Chen, Guang; Xu, Liqiang; Liu, Zhaoguo; Fu, Shengling; Yu, Li; Zhou, Sheng

    2014-01-01

    Estrogens are key signaling molecules that regulate various physiological processes such as cell growth, development, and differentiation. They also play a major role in many pathological conditions, such as hormone-dependent cancer. The importance of inhibiting estrogen receptor signaling in diseases of estrogen target tissues, such as breast cancer, is well documented. However, the role of estrogen signaling in diseases of nontarget tissues, such as lung cancer, is not well characterized. The aim of the current study is to examine the expression of estrogen receptor β (ERβ) and the roles of estradiol (E2) and fulvestrant on the progression of lung cancer. Tissue microarray (TMA) and immunohistochemistry (IHC) analyses were used to detect the expression of aromatase, ERα, and ERβ in 198 patients. We performed analyses to determine if there was any correlation among these three proteins. A mouse model of urethane-induced lung adenocarcinoma was used in the study. Mice were divided into three treatment groups: blank control, E2 alone, and E2 + fulvestrant (ERβ antagonist). Western blot analysis and fluorescence quantitative PCR (FQ-PCR) were used to measure expression of ERβ protein and mRNA levels, respectively. ERβ, but not ERα, was overexpressed in NSCLC samples. Lung cancer progression in mice treated with E2 was significantly increased compared to either the control group or the E2 + fulvestrant group. Mice in the E2 treatment group had significantly increased expression of ERβ at both the mRNA and protein levels compared to mice treated with E2 + fulvestrant or control. Our data suggest that ERβ promotes lung cancer progression in mice and that this progression can be inhibited with fulvestrant. These findings may help elucidate the role of ERβ in lung cancer and suggest that estrogen receptor antagonists, such as fulvestrant, may be therapeutically beneficial for the treatment of the disease.

  13. Spinal alpha3beta2* nicotinic acetylcholine receptors tonically inhibit the transmission of nociceptive mechanical stimuli.

    PubMed

    Young, Tracey; Wittenauer, Shannon; McIntosh, J Michael; Vincler, Michelle

    2008-09-10

    The presence of non-alpha4beta2, non-alpha7 nicotinic acetylcholine receptors (nAChR) in the rat spinal cord has been suggested previously, but the identity of these nAChRs had not been shown. Intrathecal administration of the alpha3beta2*/alpha6beta2* selective alpha-conotoxin MII (alpha-CTX MII) dose- and time-dependently reduced paw withdrawal thresholds to mechanical pressure in normal rats. The pronociceptive effect of alpha-CTX MII was partially blocked by NMDA receptor antagonism and lost completely following ablation of C-fibers. The effect of spinal nerve ligation on alpha-CTX MII-induced mechanical hypersensitivity was also assessed. Sensitivity was lost in the hind paw ipsilateral to spinal nerve ligation, but maintained in the contralateral hind paw at control levels. Radioligand binding in spinal cord membranes revealed high and low affinity alpha-CTX MII binding sites. Spinal nerve ligation did not significantly alter alpha-CTX MII binding ipsilateral to ligation. Finally, no evidence for the presence of alpha6-containing nAChRs was identified. The results of these studies show the presence of 2 populations of alpha-CTX MII-sensitive nAChRs containing the alpha3 and beta2, but not the alpha6, subunits in the rat spinal cord that function to inhibit the transmission of nociceptive mechanical stimuli via inhibiting the release of glutamate from C-fibers. Spinal nerve ligation produces a unilateral loss of alpha-CTX MII-induced mechanical hypersensitivity without altering alpha-CTX MII binding sites. Our data support a peripheral injury-induced loss of a cholinergic inhibitory tone at spinal alpha3beta2* nAChRs, without the loss of the receptors themselves, which may contribute to mechanical hypersensitivity following spinal nerve ligation.

  14. Spinal α3β2* nicotinic acetylcholine receptors tonically inhibit the transmission of nociceptive mechanical stimuli

    PubMed Central

    Young, Tracey; Wittenauer, Shannon; McIntosh, J. Michael; Vincler, Michelle

    2008-01-01

    The presence of non-α4β2, non-α7 nicotinic acetylcholine receptors (nAChR) in the rat spinal cord has been suggested previously, but the identity of these nAChRs had not been shown. Intrathecal administration of the α3β2*/α6β2* selective α-conotoxin MII (α-CTX MII) dose- and time-dependently reduced paw withdrawal thresholds to mechanical pressure in normal rats. The pronociceptive effect of α-CTX MII was partially blocked by NMDA receptor antagonism and lost completely following ablation of C-fibers. The effect of spinal nerve ligation on α-CTX MII-induced mechanical hypersensitivity was also assessed. Sensitivity was lost in the hind paw ipsilateral to spinal nerve ligation, but maintained in the contralateral hind paw at control levels.. Radioligand binding in spinal cord membranes revealed high and low affinity α-CTX MII binding sites. Spinal nerve ligation did not significantly alter α-CTX MII binding ipsilateral to ligation. Finally, no evidence for the presence of α6-containing nAChRs was identified. The results of these studies show the presence of 2 populations of α-CTX MII-sensitive nAChRs containing the α3 and β2, but not the α6, subunits in the rat spinal cord that function to inhibit the transmission of nociceptive mechanical stimuli via inhibiting the release of glutamate from C-fibers. Spinal nerve ligation produces a unilateral loss of α-CTX MII-induced mechanical hypersensitivity without altering α-CTX MII binding sites. Our data support a peripheral injury-induced loss of a cholinergic inhibitory tone at spinal α3β2* nAChRs, without the loss of the receptors themselves, which may contribute to mechanical hypersensitivity following spinal nerve ligation. PMID:18634758

  15. Inhibition of the production of endothelium-derived hyperpolarizing factor by cannabinoid receptor agonists

    PubMed Central

    Fleming, I; Schermer, B; Popp, R; Busse, R

    1999-01-01

    The endogenous cannabinoid, anandamide, has been reported to induce an 'endothelium-derived hyperpolarizing factor (EDHF)-like' relaxation in vitro. We therefore investigated the effects of cannabinoid CB1 receptor agonists; HU 210, Δ9-tetrahydrocannabinol (Δ9-THC) and anandamide, and a CB1 antagonist/inverse agonist, SR 141716A, on nitric oxide (NO) and EDHF-mediated relaxation in precontracted rings of porcine coronary, rabbit carotid and mesenteric arteries. In rings of mesenteric artery HU 210 and Δ9-THC induced endothelium- and cyclo-oxygenase-independent relaxations which were sensitive to SR 141716A. Anandamide (0.03–30 μM) induced a slowly developing, endothelium-independent relaxation which was abolished by diclofenac and was therefore mediated by cyclo-oxygenase product(s). None of the CB1 agonists tested affected the tone of precontracted rings of rabbit carotid or porcine coronary artery. In endothelium-intact segments, HU 210, Δ9-THC and anandamide did not affect NO-mediated responses but under conditions of continuous NO synthase/cyclo-oxygenase blockade, significantly inhibited acetylcholine and bradykinin-induced relaxations which are attributed to the production of EDHF. The effects of HU 210 and Δ9-THC were not observed when experiments were performed in the presence of SR 141716A suggesting the involvement of the CB1 receptor. In a patch clamp bioassay of EDHF production, HU 210 decreased the EDHF-mediated hyperpolarization of detector smooth muscle cells when applied to the donor segment but was without effect on the membrane potential of detector cells. The inhibition of EDHF production was unrelated to alterations in Ca2+-signalling or cytochrome P450 activity. These results suggest that the activation of endothelial CB1 receptors appears to be negatively coupled to the production of EDHF. PMID:10193775

  16. Inhibition of Protease-activated Receptor 1 Ameliorates Intestinal Radiation Mucositis in a Preclinical Rat Model

    SciTech Connect

    Wang, Junru; Kulkarni, Ashwini; Chintala, Madhu; Fink, Louis M.; Hauer-Jensen, Martin

    2013-01-01

    Purpose: To determine, using a specific small-molecule inhibitor of protease-activated receptor 1 (PAR1) signaling, whether the beneficial effect of thrombin inhibition on radiation enteropathy development is due to inhibition of blood clotting or to cellular (PAR1-mediated) thrombin effects. Methods and Materials: Rats underwent fractionated X-irradiation (5 Gy Multiplication-Sign 9) of a 4-cm small-bowel segment. Early radiation toxicity was evaluated in rats receiving PAR1 inhibitor (SCH602539, 0, 10, or 15 mg/kg/d) from 1 day before to 2 weeks after the end of irradiation. The effect of PAR1 inhibition on development of chronic intestinal radiation fibrosis was evaluated in animals receiving SCH602539 (0, 15, or 30 mg/kg/d) until 2 weeks after irradiation, or continuously until termination of the experiment 26 weeks after irradiation. Results: Blockade of PAR1 ameliorated early intestinal toxicity, with reduced overall intestinal radiation injury (P=.002), number of myeloperoxidase-positive (P=.03) and proliferating cell nuclear antigen-positive (P=.04) cells, and collagen III accumulation (P=.005). In contrast, there was no difference in delayed radiation enteropathy in either the 2- or 26-week administration groups. Conclusion: Pharmacological blockade of PAR1 seems to reduce early radiation mucositis but does not affect the level of delayed intestinal radiation fibrosis. Early radiation enteropathy is related to activation of cellular thrombin receptors, whereas platelet activation or fibrin formation may play a greater role in the development of delayed toxicity. Because of the favorable side-effect profile, PAR1 blockade should be further explored as a method to ameliorate acute intestinal radiation toxicity in patients undergoing radiotherapy for cancer and to protect first responders and rescue personnel in radiologic/nuclear emergencies.

  17. Inhibition of Nicotinamide Phosphoribosyltransferase Induces Apoptosis in Estrogen Receptor-Positive MCF-7 Breast Cancer Cells

    PubMed Central

    Alaee, Mohammad; Khaghani, Shahnaz; Behroozfar, Kiarash; Hesari, Zahra; Ghorbanhosseini, Seyedeh Sara

    2017-01-01

    Purpose Tumor cells have increased turnover of nicotinamide adenine dinucleotide (NAD+), the main coenzyme in processes including adenosine diphosphate-ribosylation, deacetylation, and calcium mobilization. NAD+ is predominantly synthesized in human cells via the salvage pathway, with the first component being nicotinamide. Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme in this pathway, and its chemical inhibition by FK866 has elicited antitumor effects in several preclinical models of solid and hematologic cancers. However, its efficacy in estrogen receptor (ER)-positive and human epidermal growth factor receptor 2-positive breast cancer cells has not been previously investigated. In this study, we aimed to deplete the NAD+ content of MCF-7 cells, a model cell line for ER-positive breast cancer, by inhibiting NAMPT in order to evaluate downstream effects on p53 and its acetylation, p21 and Bcl-2-associated X protein (BAX) expression, and finally, apoptosis in MCF-7 breast cancer cells. Methods MCF-7 cells were cultured and treated with FK866. NAD+ levels in cells were determined colorimetrically. Levels of p53 and its acetylated form were determined by Western blotting. Expression of p21 and BAX was determined by real-time polymerase chain reaction. Finally, levels of apoptosis were assessed by flow cytometry using markers for annexin V and propidium iodide. Results FK866 treatment was able to increase p53 levels and acetylation, upregulate BAX and p21 expression, and induce apoptosis in MCF-7 cells. Addition of exogenous NAD+ to cells reversed these effects, suggesting that FK866 exerted its effects by depleting NAD+ levels. Conclusion Results showed that FK866 could effectively inhibit NAD+ biosynthesis and induce programmed cell death in MCF-7 cells, suggesting that NAMPT inhibitors may be useful for the treatment of ER-positive breast cancers. PMID:28382091

  18. 5-HT1D receptor inhibits renal sympathetic neurotransmission by nitric oxide pathway in anesthetized rats.

    PubMed

    García-Pedraza, José-Ángel; García, Mónica; Martín, María-Luisa; Morán, Asunción

    2015-09-01

    Although serotonin has been shown to inhibit peripheral sympathetic outflow, serotonin regulation on renal sympathetic outflow has not yet been elucidated. This study investigated which 5-HT receptor subtypes are involved. Wistar rats were anesthetized (sodium pentobarbital; 60mg/kg, i.p.), and prepared for in situ autoperfused rat kidney, which allows continuous measurement of systemic blood pressure (SBP), heart rate (HR) and renal perfusion pressure (PP). Electrical stimulation of renal sympathetic nerves resulted in frequency-dependent increases in PP (18.3±1.0, 43.7±2.7 and 66.7±4.0 for 2, 4 and 6Hz, respectively), without altering SBP or HR. 5-HT, 5-carboxamidotryptamine (5-HT1/7 agonist) (0.00000125-0.1μg/kg each) or l-694,247 (5-HT1D agonist; 0.0125μg/kg) i.a. bolus inhibited vasopressor responses by renal nerve electrical stimulation, unlike i.a. bolus of agonists α-methyl-5-HT (5-HT2), 1-PBG (5-HT3), cisapride (5-HT4), AS-19 (5-HT7), CGS-12066B (5-HT1B) or 8-OH-DPAT (5-HT1A) (0.0125μg/kg each). The effect of l-694,247 did not affect the exogenous norepinephrine-induced vasoconstrictions, whereas was abolished by antagonist LY310762 (5-HT1D; 1mg/kg) or l-NAME (nitric oxide; 10mg/kg), but not by indomethacin (COX1/2; 2mg/kg) or glibenclamide (ATP-dependent K(+) channel; 20mg/kg). These results suggest that 5-HT mechanism-induced inhibition of rat vasopressor renal sympathetic outflow is mainly mediated by prejunctional 5-HT1D receptors via nitric oxide release.

  19. Inhibition of androgen receptor binding by natural and synthetic steroids in cultured human genital skin fibroblasts.

    PubMed

    Breiner, M; Romalo, G; Schweikert, H U

    1986-08-15

    The ability of various natural and synthetic steroids (some of which are widely used in clinical practice) to compete with dihydrotestosterone receptor binding in human genital skin fibroblasts was studied. Binding was assessed in fibroblast monolayers after incubation for 1 h at 37 degrees C with 2 nM 3H-dihydrotestosterone in the presence or absence of increasing concentrations of the steroid to be tested. Inhibition constants (Ki) were determined as the concentration of competitor-required for 50% inhibition of 3H-dihydrotestosterone binding. In addition, relative binding activity (RBA) of each test compound was calculated. Each competitor was tested in at least two different cell strains. The concentrations of unlabeled methyltrienolone (a synthetic nonmetabolizable androgen) and dihydrotestosterone for 50% inhibition of 3H-dihydrotestosterone binding were in the same order of magnitude, namely, 2 nM (2.2 respectively, 2.4 nM), whereas the affinity of testosterone was approximately one-fifth that of dihydrotestosterone. Other potent competitors for dihydrotestosterone binding were three progestins (norgestrel, gestoden, and medroxyprogesterone acetate) which have Ki values similar to testosterone. An order of magnitude lower Ki values (around 10(-7) M) were found for the androgen 17 alpha-propylmesterolone, the antiandrogen cyproterone acetate, and the progestin norethisterone acetate. Binding affinities of all other steroids to the androgen receptor were markedly lower and showed the following order of potency: estrogens (estradiol, ethinyl estradiol, diethylstilbestrol) greater than glucocorticoids as well as aromatase inhibitors and potassium canrenoate.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. PKCɛ mediates substance P inhibition of GABAA receptors-mediated current in rat dorsal root ganglion.

    PubMed

    Li, Li; Zhao, Lei; Wang, Yang; Ma, Ke-tao; Shi, Wen-yan; Wang, Ying-zi; Si, Jun-qiang

    2015-02-01

    The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 μmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 μmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 μmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 μmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²⁺-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.

  1. Inhibition of experimental auto-immune uveitis by the A3 adenosine receptor agonist CF101.

    PubMed

    Bar-Yehuda, Sara; Luger, Dror; Ochaion, Avivit; Cohen, Shira; Patokaa, Renana; Zozulya, Galina; Silver, Phyllis B; de Morales, Jose Maria Garcia Ruiz; Caspi, Rachel R; Fishman, Pnina

    2011-11-01

    Uveitis is an inflammation of the middle layer of the eye with a high risk of blindness. The Gi protein associated A3 adenosine receptor (A3AR) is highly expressed in inflammatory cells whereas low expression is found in normal cells. CF101 is a highly specific agonist at the A3AR known to induce a robust anti-inflammatory effect in different experimental animal models. The CF101 mechanism of action entails down-regulation of the NF-κB-TNF-α signaling pathway, resulting in inhibition of pro-inflammatory cytokine production and apoptosis of inflammatory cells. In this study the effect of CF101 on the development of retinal antigen interphotoreceptor retinoid-binding protein (IRBP)-induced experimental autoimmune uveitis (EAU) was assessed. Oral treatment with CF101 (10 µg/kg, twice daily), initiated upon disease onset, improved uveitis clinical score measured by fundoscopy and ameliorated the pathological manifestations of the disease. Shortly after treatment with CF101 A3AR expression levels were down-regulated in the lymph node and spleen cells pointing towards receptor activation. Downstream events included a decrease in PI3K and STAT-1 and proliferation inhibition of IRPB auto-reactive T cells ex vivo. Inhibition of interleukin-2, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production and up-regulation of interleukin-10 was found in cultured splenocytes derived from CF101-treated animals. Overall, the present study data point towards a marked anti-inflammatory effect of CF101 in EAU and support further exploration of this small molecule drug for the treatment of uveitis.

  2. Delphinidin inhibits cell proliferation and invasion via modulation of Met receptor phosphorylation

    SciTech Connect

    Syed, Deeba N.; Afaq, Farrukh; Sarfaraz, Sami; Khan, Naghma; Kedlaya, Rajendra; Setaluri, Vijayasaradhi; Mukhtar, Hasan

    2008-08-15

    The HGF/Met signaling pathway is deregulated in majority of cancers and is associated with poor prognosis in breast cancer. Delphinidin, present in pigmented fruits and vegetables possesses potent anti-oxidant, anti-inflammatory and anti-angiogenic properties. Here, we assessed the anti-proliferative and anti-invasive effects of delphinidin on HGF-mediated responses in the immortalized MCF-10A breast cell line. Treatment of cells with delphinidin prior to exposure to exogenous HGF resulted in the inhibition of HGF-mediated (i) tyrosyl-phosphorylation and increased expression of Met receptor, (ii) phosphorylation of downstream regulators such as FAK and Src and (iii) induction of adaptor proteins including paxillin, Gab-1 and GRB-2. In addition, delphinidin treatment resulted in significant inhibition of HGF-activated (i) Ras-ERK MAPKs and (ii) PI3K/AKT/mTOR/p70S6K pathways. Delphinidin was found to repress HGF-activated NF{kappa}B transcription with a decrease in (i) phosphorylation of IKK{alpha}/{beta} and I{kappa}B{alpha}, and (ii) activation and nuclear translocation of NF{kappa}B/p65. Inhibition of HGF-mediated membrane translocation of PKC{alpha} as well as decreased phosphorylation of STAT3 was further observed in delphinidin treated cells. Finally, decreased cell viability of Met receptor expressing breast cancer cells treated with delphinidin argues for a potential role of the agent in the prevention of HGF-mediated activation of various signaling pathways implicated in breast cancer.

  3. Coantagonism of Glutamate Receptors and Nicotinic Acetylcholinergic Receptors Disrupts Fear Conditioning and Latent Inhibition of Fear Conditioning

    ERIC Educational Resources Information Center

    Gould, Thomas J.; Lewis, Michael C.

    2005-01-01

    The present study investigated the hypothesis that both nicotinic acetylcholinergic receptors (nAChRs) and glutamate receptors ([alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) and N-methyl-D-aspartate glutamate receptors (NMDARs)) are involved in fear conditioning, and may modulate similar processes. The effects of the…

  4. Light and hydrogen peroxide inhibit C. elegans feeding through gustatory receptor orthologs and pharyngeal neurons

    PubMed Central

    Bhatla, Nikhil; Horvitz, H. Robert

    2015-01-01

    SUMMARY While gustatory sensing of the five primary flavors (sweet, salty, sour, bitter, and savory) has been extensively studied, pathways that detect non-canonical taste stimuli remain relatively unexplored. In particular, while reactive oxygen species cause generalized damage to biological systems, no gustatory mechanism to prevent ingestion of such material has been identified in any organism. We observed that light inhibits C. elegans feeding and used light as a tool to uncover molecular and neural mechanisms for gustation. Light can generate hydrogen peroxide, and we discovered that hydrogen peroxide similarly inhibits feeding. The gustatory receptor family members LITE-1 and GUR-3 are required for the inhibition of feeding by light and hydrogen peroxide. The I2 pharyngeal neurons increase calcium in response to light and hydrogen peroxide, and these responses require GUR-3 and a conserved antioxidant enzyme peroxiredoxin PRDX-2. Our results demonstrate a gustatory mechanism that mediates the detection and blocks ingestion of a non-canonical taste stimulus, hydrogen peroxide. PMID:25640076

  5. A3 adenosine receptor agonist reduces brain ischemic injury and inhibits inflammatory cell migration in rats.

    PubMed

    Choi, In-Young; Lee, Jae-Chul; Ju, Chung; Hwang, Sunyoung; Cho, Geum-Sil; Lee, Hyuk Woo; Choi, Won Jun; Jeong, Lak Shin; Kim, Won-Ki

    2011-10-01

    A3 adenosine receptor (A3AR) is recognized as a novel therapeutic target for ischemic injury; however, the mechanism underlying anti-ischemic protection by the A3AR agonist remains unclear. Here, we report that 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyl-4'-thioadenosine (LJ529), a selective A3AR agonist, reduces inflammatory responses that may contribute to ischemic cerebral injury. Postischemic treatment with LJ529 markedly reduced cerebral ischemic injury caused by 1.5-hour middle cerebral artery occlusion, followed by 24-hour reperfusion in rats. This effect was abolished by the simultaneous administration of the A3AR antagonist MRS1523, but not the A2AAR antagonist SCH58261. LJ529 prevented the infiltration/migration of microglia and monocytes occurring after middle cerebral artery occlusion and reperfusion, and also after injection of lipopolysaccharides into the corpus callosum. The reduced migration of microglia by LJ529 could be related with direct inhibition of chemotaxis and down-regulation of spatiotemporal expression of Rho GTPases (including Rac, Cdc42, and Rho), rather than by biologically relevant inhibition of inflammatory cytokine/chemokine release (eg, IL-1β, TNF-α, and MCP-1) or by direct inhibition of excitotoxicity/oxidative stress (not affected by LJ529). The present findings indicate that postischemic activation of A3AR and the resultant reduction of inflammatory response should provide a promising therapeutic strategy for the treatment of ischemic stroke.

  6. Metabotropic glutamate subtype 5 receptors modulate fear-conditioning induced enhancement of prepulse inhibition in rats.

    PubMed

    Zou, Dan; Huang, Juan; Wu, Xihong; Li, Liang

    2007-02-01

    Non-startling acoustic events presented shortly before an intense startling sound can inhibit the acoustic startle reflex. This phenomenon is called prepulse inhibition (PPI), and is widely used as a model of sensorimotor gating. The present study investigated whether PPI can be modulated by fear conditioning, whose acquisition can be blocked by the specific antagonist of metabotropic glutamate receptors subtype 5 (mGluR5), 2-methyl-6-(phenylethynyl)-pyridine (MPEP). The results show that a gap embedded in otherwise continuous noise sounds, which were delivered by two spatially separated loudspeakers, could inhibit the startle reflex induced by an intense sound that was presented 50 ms after the gap. The inhibitory effect depended on the duration of the gap, and was enhanced by fear conditioning that was introduced by temporally pairing the gap with footshock. Intraperitoneal injection of MPEP (0.5 or 5mg/kg) 30 min before fear conditioning blocked the enhancing effect of fear conditioning on PPI, but did not affect either the baseline startle magnitude or PPI if no fear conditioning was introduced. These results indicate that PPI is enhanced when the prepulse signifies an aversive event after fear conditioning. Also, mGlu5Rs play a role in preserving the fear-conditioning-induced enhancement of PPI.

  7. Flavonoids Suppress Pseudomonas aeruginosa Virulence through Allosteric Inhibition of Quorum-sensing Receptors.

    PubMed

    Paczkowski, Jon E; Mukherjee, Sampriti; McCready, Amelia R; Cong, Jian-Ping; Aquino, Christopher J; Kim, Hahn; Henke, Brad R; Smith, Chari D; Bassler, Bonnie L

    2017-03-10

    Quorum sensing is a process of cell-cell communication that bacteria use to regulate collective behaviors. Quorum sensing depends on the production, detection, and group-wide response to extracellular signal molecules called autoinducers. In many bacterial species, quorum sensing controls virulence factor production. Thus, disrupting quorum sensing is considered a promising strategy to combat bacterial pathogenicity. Several members of a family of naturally produced plant metabolites called flavonoids inhibit Pseudomonas aeruginosa biofilm formation by an unknown mechanism. Here, we explore this family of molecules further, and we demonstrate that flavonoids specifically inhibit quorum sensing via antagonism of the autoinducer-binding receptors, LasR and RhlR. Structure-activity relationship analyses demonstrate that the presence of two hydroxyl moieties in the flavone A-ring backbone are essential for potent inhibition of LasR/RhlR. Biochemical analyses reveal that the flavonoids function non-competitively to prevent LasR/RhlR DNA binding. Administration of the flavonoids to P. aeruginosa alters transcription of quorum sensing-controlled target promoters and suppresses virulence factor production, confirming their potential as anti-infectives that do not function by traditional bacteriocidal or bacteriostatic mechanisms.

  8. Tumor suppressor ING4 inhibits estrogen receptor activity in breast cancer cells

    PubMed Central

    Keenen, Madeline M; Kim, Suwon

    2016-01-01

    Resistance to antiestrogen therapy remains a significant problem in breast cancer. Low expression of inhibitor of growth 4 (ING4) in primary tumors has been correlated with increased rates of recurrence in estrogen receptor-positive (ER+) breast cancer patients, suggesting a role for ING4 in ER signaling. This study provides evidence that ING4 inhibits ER activity. ING4 overexpression increased the sensitivity of T47D and MCF7 ER+ breast cancer cells to hormone deprivation. ING4 attenuated maximal estrogen-dependent cell growth without affecting the dose–response of estrogen. These results indicated that ING4 functions as a noncompetitive inhibitor of estrogen signaling and may inhibit estrogen-independent ER activity. Supportive of this, treatment with fulvestrant but not tamoxifen rendered T47D cells sensitive to hormone deprivation as did ING4 overexpression. ING4 did not affect nuclear ERα protein expression, but repressed selective ER-target gene transcription. Taken together, these results demonstrated that ING4 inhibited estrogen-independent ER activity, suggesting that ING4-low breast tumors recur faster due to estrogen-independent ER activity that renders tamoxifen less effective. This study puts forth fulvestrant as a proposed therapy choice for patients with ING4-low ER+ breast tumors. PMID:27895513

  9. Targeted Inhibition of Complement Using Complement Receptor 2-Conjugated Inhibitors Attenuates EAE

    PubMed Central

    Hu, Xianzhen; Tomlinson, Stephen; Barnum, Scott R.

    2012-01-01

    Multiple sclerosis (MS) is the most common autoimmune demyelinating disease, affecting millions of individuals worldwide. In the last two decades, many therapeutic options for the treatment of MS have become available, however they are limited in terms of effectiveness and some remain plagued by safety issues. The currently available treatment options target relapsing remitting forms of MS and are not effective against the more progressive forms of the disease. These limitations highlight a significant unmet treatment need for MS. In experimental autoimmune encephalomyelitis (EAE) studies from our laboratory, we have previously shown, using a number of complement mutant and transgenic mice, that inhibition of the alternative complement pathway and the C3 convertase confers significant protection from disease. We report here that targeted inhibition of complement activation using complement receptor 2 (CR2)-conjugated inhibitors significantly attenuates EAE. Administration of CR2-Crry (blocks all complement pathways at C3 activation) and CR2-fH (specifically blocks the alternative pathway) just prior to and during the onset of EAE blocks progression of both acute and chronic disease. These data indicate that inhibition of complement may offer an effective therapeutic approach to treating both acute and chronic forms of demyelinating disease through blocking the alternative pathway or complement convertases. PMID:23079547

  10. Targeted inhibition of complement using complement receptor 2-conjugated inhibitors attenuates EAE.

    PubMed

    Hu, Xianzhen; Tomlinson, Stephen; Barnum, Scott R

    2012-11-30

    Multiple sclerosis (MS) is the most common autoimmune demyelinating disease, affecting millions of individuals worldwide. In the last two decades, many therapeutic options for the treatment of MS have become available, however they are limited in terms of effectiveness and some remain plagued by safety issues. The currently available treatment options target relapsing remitting forms of MS and are not effective against the more progressive forms of the disease. These limitations highlight a significant unmet treatment need for MS. In experimental autoimmune encephalomyelitis (EAE) studies from our laboratory, we have previously shown, using a number of complement mutant and transgenic mice, that inhibition of the alternative complement pathway and the C3 convertase confers significant protection from disease. We report here that targeted inhibition of complement activation using complement receptor 2 (CR2)-conjugated inhibitors significantly attenuates EAE. Administration of CR2-Crry (blocks all complement pathways at C3 activation) and CR2-fH (specifically blocks the alternative pathway) just prior to and during the onset of EAE blocks progression of both acute and chronic disease. These data indicate that inhibition of complement may offer an effective therapeutic approach to treating both acute and chronic forms of demyelinating disease through blocking the alternative pathway or complement convertases.

  11. Hydrazinobenzoylcurcumin inhibits androgen receptor activity and growth of castration-resistant prostate cancer in mice

    PubMed Central

    Wu, Min; Kim, Sahn-Ho; Datta, Indrani; Levin, Albert; Dyson, Gregory; Li, Jing; Kaypee, Stephanie; Swamy, M. Mahadeva; Gupta, Nilesh; Kwon, Ho Jeong; Menon, Mani; Kundu, Tapas K.; Reddy, G. Prem-Veer

    2015-01-01

    There is a critical need for therapeutic agents that can target the amino-terminal domain (NTD) of androgen receptor (AR) for the treatment of castration-resistant prostate cancer (CRPC). Calmodulin (CaM) binds to the AR NTD and regulates AR activity. We discovered that Hydrazinobenzoylcurcumin (HBC), which binds exclusively to CaM, inhibited AR activity. HBC abrogated AR interaction with CaM, suppressed phosphorylation of AR Serine81, and blocked the binding of AR to androgen-response elements. RNA-Seq analysis identified 57 androgen-regulated genes whose expression was significantly (p ≤ 0.002) altered in HBC treated cells as compared to controls. Oncomine analysis revealed that genes repressed by HBC are those that are usually overexpressed in prostate cancer (PCa) and genes stimulated by HBC are those that are often down-regulated in PCa, suggesting a reversing effect of HBC on androgen-regulated gene expression associated with PCa. Ingenuity Pathway Analysis revealed a role of HBC affected genes in cellular functions associated with proliferation and survival. HBC was readily absorbed into the systemic circulation and inhibited the growth of xenografted CRPC tumors in nude mice. These observations demonstrate that HBC inhibits AR activity by targeting the AR NTD and suggest potential usefulness of HBC for effective treatment of CRPC. PMID:25704883

  12. Heme from Alzheimer's brain inhibits muscarinic receptor binding via thiyl radical generation.

    PubMed

    Venters, H D; Bonilla, L E; Jensen, T; Garner, H P; Bordayo, E Z; Najarian, M M; Ala, T A; Mason, R P; Frey, W H

    1997-08-01

    An endogenous inhibitor (< 3500 Da) of antagonist binding to the muscarinic acetylcholine receptor (mAChR) has been reported to be elevated 3-fold in Alzheimer's disease (AD) brain. This endogenous inhibitor was found to require the presence of reducing agents such as reduced glutathione (GSH) for optimal activity. In the presence of GSH, the inhibitor was shown to generate thiyl radicals which irreversibly inhibited the mAChR. We now report that the inhibitor contains free heme, a well-established source of oxidative stress capable of generating free radicals and causing neurotoxicity. While FeSO4, microperoxidase and hemin all inhibited antagonist binding to the mAChR, only hemin shared the inhibitor's requirement for GSH. Both the free radical scavengers Trolox and Mn2+, and the metal chelator, EDTA, blocked the activity of the endogenous AD inhibitor and of hemin. Heme oxygenase-1 (HO-1) markedly reduced the activity of both the endogenous AD inhibitor and hemin, indicating that the endogenous inhibitor contains heme. Mass spectrometric analysis confirmed the presence of free heme and heme fragments in fractions of the endogenous AD inhibitor. The antioxidants estrogen, vitamin E and vitamin C all protected the mAChR from irreversible inhibition by the endogenous inhibitor or hemin. These antioxidants may function to protect the integrity of the mAChR in vivo and may have therapeutic potential in AD where free heme could be a source of oxidative stress.

  13. Gemini vitamin D analogues inhibit estrogen receptor-positive and estrogen receptor-negative mammary tumorigenesis without hypercalcemic toxicity.

    PubMed

    Lee, Hong Jin; Paul, Shiby; Atalla, Nadi; Thomas, Paul E; Lin, Xinjie; Yang, Ill; Buckley, Brian; Lu, Gang; Zheng, Xi; Lou, You-Rong; Conney, Allan H; Maehr, Hubert; Adorini, Luciano; Uskokovic, Milan; Suh, Nanjoo

    2008-11-01

    Numerous preclinical, epidemiologic, and clinical studies have suggested the benefits of vitamin D and its analogues for the prevention and treatment of cancer. However, the hypercalcemic effects have limited the use of 1alpha,25(OH)(2)D(3), the hormonally active form of vitamin D. To identify vitamin D analogues with better efficacy and low toxicity, we have tested >60 novel Gemini vitamin D analogues with a unique structure of two side chains for growth inhibition of breast cancer cells. Our initial studies found that some Gemini analogues are 5-15 times more active than 1alpha,25(OH)(2)D(3) in growth inhibition assay. In vivo experiments were designed to study the inhibitory effect of selected Gemini vitamin D analogues against mammary carcinogenesis by using (a) an N-methyl-N-nitrosourea-induced estrogen receptor (ER)-positive mammary tumor model and (b) an MCF10DCIS.com xenograft model of ER-negative mammary tumors. Among vitamin D analogues we tested, Gemini 0072 [1alpha,25-dihydroxy-20S-21(3-trideuteromethyl-3-hydroxy-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-19-nor-cholecalciferol] and Gemini 0097 [1alpha,25-dihydroxy-20R-21(3-trideuteromethyl-3-hydroxy-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-19-nor-cholecalciferol] administration inhibited by 60% the NMU-induced mammary tumor burden compared with the NMU-treated control group, but these compounds were devoid of hypercalcemia toxicity. In an ER-negative xenograft model, Gemini 0097 significantly suppressed tumor growth without hypercalcemia toxicity. We found that the inhibitory effect of Gemini 0097 was associated with an increased level of cyclin-dependent kinase inhibitor p21 and the insulin-like growth factor binding protein 3 in both ER-positive and ER-negative mammary tumors. Our results suggest that Gemini vitamin D analogues may be potent agents for the prevention and treatment of both ER-positive and ER-negative breast cancer without hypercalcemia toxicity.

  14. Inhibition of tumor angiogenesis and growth by a small-molecule multi-FGF receptor blocker with allosteric properties.

    PubMed

    Bono, Françoise; De Smet, Frederik; Herbert, Corentin; De Bock, Katrien; Georgiadou, Maria; Fons, Pierre; Tjwa, Marc; Alcouffe, Chantal; Ny, Annelii; Bianciotto, Marc; Jonckx, Bart; Murakami, Masahiro; Lanahan, Anthony A; Michielsen, Christof; Sibrac, David; Dol-Gleizes, Frédérique; Mazzone, Massimiliano; Zacchigna, Serena; Herault, Jean-Pascal; Fischer, Christian; Rigon, Patrice; Ruiz de Almodovar, Carmen; Claes, Filip; Blanc, Isabelle; Poesen, Koen; Zhang, Jie; Segura, Inmaculada; Gueguen, Geneviève; Bordes, Marie-Françoise; Lambrechts, Diether; Broussy, Roselyne; van de Wouwer, Marlies; Michaux, Corinne; Shimada, Toru; Jean, Isabelle; Blacher, Silvia; Noel, Agnès; Motte, Patrick; Rom, Eran; Rakic, Jean-Marie; Katsuma, Susumu; Schaeffer, Paul; Yayon, Avner; Van Schepdael, Ann; Schwalbe, Harald; Gervasio, Francesco Luigi; Carmeliet, Geert; Rozensky, Jef; Dewerchin, Mieke; Simons, Michael; Christopoulos, Arthur; Herbert, Jean-Marc; Carmeliet, Peter

    2013-04-15

    Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment.

  15. Copper Inhibits NMDA Receptor-Independent LTP and Modulates the Paired-Pulse Ratio after LTP in Mouse Hippocampal Slices

    PubMed Central

    Salazar-Weber, Nina L.; Smith, Jeffrey P.

    2011-01-01

    Copper misregulation has been implicated in the pathological processes underlying deterioration of learning and memory in Alzheimer's disease and other neurodegenerative disorders. Supporting this, inhibition of long-term potentiation (LTP) by copper (II) has been well established, but the exact mechanism is poorly characterized. It is thought that an interaction between copper and postsynaptic NMDA receptors is a major part of the mechanism; however, in this study, we found that copper (II) inhibited NMDA receptor-independent LTP in the CA3 region of hippocampal slices. In addition, in the CA3 and CA1 regions, copper modulated the paired-pulse ratio (PPR) in an LTP-dependent manner. Combined, this suggests the involvement of a presynaptic mechanism in the modulation of synaptic plasticity by copper. Inhibition of the copper-dependent changes in the PPR with cyclothiazide suggested that this may involve an interaction with the presynaptic AMPA receptors that regulate neurotransmitter release. PMID:22028985

  16. Inhibition of nicotinic receptor-mediated responses in bovine chromaffin cells by diltiazem.

    PubMed

    Gandía, L; Villarroya, M; Sala, F; Reig, J A; Viniegra, S; Quintanar, J L; García, A G; Gutiérrez, L M

    1996-07-01

    1. The effects of diltiazem on various functional parameters were studied in bovine cultured adrenal chromaffin cells stimulated with the nicotinic receptor agonist dimethylphenylpiperazinium (DMPP) or with depolarizing Krebs-HEPES solutions containing high K+ concentrations. 2. The release of [3H]-noradrenaline induced by DMPP (100 microM for 5 min) was gradually and fully inhibited by increasing concentrations of diltiazem (IC50 = 1.3 microM). In contrast, the highest concentration of diltiazem used (10 microM) inhibited the response to high K+ (59 mM for 5 min) by only 25%. 3. 45Ca2+ uptake into cells stimulated with DMPP (100 microM for 1 min) was also blocked by diltiazem in a concentration-dependent manner (IC50 = 0.4 microM). Again, diltiazem blocked the K(+)-evoked 45Ca2+ uptake (70 mM K+ for 1 min) only by 20%. In contrast, the N-P-Q-type Ca2+ channel blocker omega-conotoxin MVIIC depressed the K+ signal by 70%. In the presence of this toxin, diltiazem exhibited an additional small inhibitory effect, indicating that the compound was acting on L-type Ca2+ channels. 4. Whole-cell Ba2+ currents through Ca2+ channels in voltage-clamped chromaffin cells were inhibited by 3-10 microM diltiazem by 20-25%. The inhibition was readily reversed upon washout of the drug. 5. The whole-cell currents elicited by 100 microM DMPP (IDMPP) were inhibited in a concentration-dependent and reversible manner by diltiazem. Maximal effects were found at 10 microM, which reduced the peak IDMPP by 70%. The area of each curve represented by total current (QDMPP) was reduced more than the peak current. At 10 microM, the inhibition amounted to 80%; the IC50 for QDMPP inhibition was 0.73 microM, a figure close to the IC50 for 45Ca2+ uptake (0.4 microM) and [3H]-noradrenaline release (1.3 microM). The blocking effects of diltiazem developed very quickly and did not exhibit use-dependence; thus the drug blocked the channel in its closed state. The blocking effects of 1 microM diltiazem on

  17. Suppression of prostaglandin E2 receptor subtype EP2 by PPARgamma ligands inhibits human lung carcinoma cell growth.

    PubMed

    Han, ShouWei; Roman, Jesse

    2004-02-20

    Prostaglandin E(2) (PGE(2)), a major cyclooxygenase (COX-2) metabolite, plays important roles in tumor biology and its functions are mediated through one or more of its receptors EP1, EP2, EP3, and EP4. We have shown that the matrix glycoprotein fibronectin stimulates lung carcinoma cell proliferation via induction of COX-2 expression with subsequent PGE(2) protein biosynthesis. Ligands of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibited this effect and induced cellular apoptosis. Here, we explore the role of the PGE(2) receptor EP2 in this process and whether the inhibition observed with PPARgamma ligands is related to effects on this receptor. We found that human non-small cell lung carcinoma cell lines (H1838 and H2106) express EP2 receptors, and that the inhibition of cell growth by PPARgamma ligands (GW1929, PGJ2, ciglitazone, troglitazone, and rosiglitazone [also known as BRL49653]) was associated with a significant decrease in EP2 mRNA and protein levels. The inhibitory effects of BRL49653 and ciglitazone, but not PGJ2, were reversed by a specific PPARgamma antagonist GW9662, suggesting the involvement of PPARgamma-dependent and -independent mechanisms. PPARgamma ligand treatment was associated with phosphorylation of extracellular regulated kinase (Erk), and inhibition of EP2 receptor expression by PPARgamma ligands was prevented by PD98095, an inhibitor of the MEK-1/Erk pathway. Butaprost, an EP2 agonist, like exogenous PGE(2) (dmPGE(2)), increased lung carcinoma cell growth, however, GW1929 and troglitazone blocked their effects. Our studies reveal a novel role for EP2 in mediating the proliferative effects of PGE(2) on lung carcinoma cells. PPARgamma ligands inhibit human lung carcinoma cell growth by decreasing the expression of EP2 receptors through Erk signaling and PPARgamma-dependent and -independent pathways.

  18. The activation of cannabinoid receptors in striatonigral GABAergic neurons inhibited GABA uptake.

    PubMed

    Romero, J; de Miguel, R; Ramos, J A; Fernández-Ruiz, J J

    1998-01-01

    Cannabinoid receptors (CNRs) in basal ganglia are located on striatal efferent neurons which are gamma-aminobutiric acid (GABA)-containing neurons. Recently, we have demonstrated that CN-induced motor inhibition is reversed by GABA-B, but not GABA-A, receptor antagonists, presumably indicating that the activation of CNRs in striatal outflow nuclei, mainly in the substantia nigra, should be followed by an increase of GABA concentrations into the synaptic cleft of GABA-B receptor synapses. The present study was designed to examine whether this was originated by increasing GABA synthesis and/or release or by decreasing GABA uptake. We analyzed: (i) GABA synthesis, by measuring the activity of glutamic acid decarboxylase (GAD) and GABA contents in brain regions that contain striatonigral GABAergic neurons, after in vivo administration of CNs and/or the CNR antagonist SR141716; (ii) [3H]GABA release in vitro in the presence or the absence of a synthetic CN agonist, HU-210, by using perifusion of small fragments of substantia nigra; and (iii) [3H]GABA uptake in vitro in the presence or the absence of WIN-55,212-2, by using synaptosomes obtained from either globus pallidus or substantia nigra. Results were as follows. Delta9-tetrahydrocannabinol (delta9-THC) and HU-210, did not alter neither GAD activity nor GABA contents in both the striatum and the ventral midbrain at any of the two times tested, thus suggesting that CNs apparently failed to change GABA synthesis in striatonigral GABAergic neurons. A similar lack of effect of HU-210 on in vitro [3H]GABA release, both basal and K+-evoked, was seen when this CN was added to perifused substantia nigra fragments, also suggesting no changes at the level of GABA release. However, when synaptosome preparations obtained from the substantia nigra were incubated in the presence of WIN-55,212-2, a decrease in [3H]GABA uptake could be measured. This lowering effect was specific of striatonigral GABAergic neurons since it was not

  19. Inhibition of histamine receptor 3 suppresses glioblastoma tumor growth, invasion, and epithelial-to-mesenchymal transition

    PubMed Central

    Cai, Wen-Ke; Yang, Yong-Xiang; Sun, Chao; Zhang, Zhuo; Xu, Yu-Qiao; Chang, Ting; Li, Zhu-Yi

    2015-01-01

    Histamine receptor 3 (H3R) is expressed in various tumors and correlated with malignancy and tumor proliferation. However, the role of H3R in tumor invasion and epithelial to mesenchymal transition (EMT) remains unknown. Here, we explored the H3R in the highly invasive glioblastoma (GBM) and U87MG cells. We found that H3R mRNA and protein levels were up-regulated in the GBM and glioma cell lines compared to normal brain tissue and astrocytes. In U87MG cell line, inhibition of H3R by siRNA or the antagonist ciproxifan (CPX) suppressed proliferation, invasiveness, and the expression of EMT activators (Snail, Slug and Twist). In addition, expression of epithelial markers (E-cadherin and ZO-1) was up-regulated and expression of mesenchymal markers (vimentin and N-cadherin) was down-regulated in vitro and in vivo in a xenograft model. In addition, we also showed that inhibition of H3R by siRNA or CPX inactivated the PI3K/Akt and MEK/ERK signaling pathways, while inhibition of Akt or ERK activity with antagonists or siRNAs suppressed H3R agonist (R)-(α)-(−)- methylhistamine dihydrobromide (RAMH) mediated invasion and reorganization of cadherin-household. In conclusion, overexpression of H3R is associated with glioma progression. Inhibition of H3R leads to suppressed invasion and EMT of GBM by inactivating the PI3K/Akt and MEK/ERK pathways in gliomas. PMID:25940798

  20. Protein Expression Signatures for Inhibition of Epidermal Growth Factor Receptor-mediated Signaling*

    PubMed Central

    Myers, Matthew V.; Manning, H. Charles; Coffey, Robert J.; Liebler, Daniel C.

    2012-01-01

    Analysis of cellular signaling networks typically involves targeted measurements of phosphorylated protein intermediates. However, phosphoproteomic analyses usually require affinity enrichment of phosphopeptides and can be complicated by artifactual changes in phosphorylation caused by uncontrolled preanalytical variables, particularly in the analysis of tissue specimens. We asked whether changes in protein expression, which are more stable and easily analyzed, could reflect network stimulation and inhibition. We employed this approach to analyze stimulation and inhibition of the epidermal growth factor receptor (EGFR) by EGF and selective EGFR inhibitors. Shotgun analysis of proteomes from proliferating A431 cells, EGF-stimulated cells, and cells co-treated with the EGFR inhibitors cetuximab or gefitinib identified groups of differentially expressed proteins. Comparisons of these protein groups identified 13 proteins whose EGF-induced expression changes were reversed by both EGFR inhibitors. Targeted multiple reaction monitoring analysis verified differential expression of 12 of these proteins, which comprise a candidate EGFR inhibition signature. We then tested these 12 proteins by multiple reaction monitoring analysis in three other models: 1) a comparison of DiFi (EGFR inhibitor-sensitive) and HCT116 (EGFR-insensitive) cell lines, 2) in formalin-fixed, paraffin-embedded mouse xenograft DiFi and HCT116 tumors, and 3) in tissue biopsies from a patient with the gastric hyperproliferative disorder Ménétrier's disease who was treated with cetuximab. Of the proteins in the candidate signature, a core group, including c-Jun, Jagged-1, and Claudin 4, were decreased by EGFR inhibitors in all three models. Although the goal of these studies was not to validate a clinically useful EGFR inhibition signature, the results confirm the hypothesis that clinically used EGFR inhibitors generate characteristic protein expression changes. This work further outlines a prototypical

  1. Further evidence for inhibition of episodic luteinizing hormone release in ovariectomized rats by stimulation of dopamine receptors.

    PubMed

    Drouva, S V; Gallo, R V

    1977-03-01

    Stimulation of dopamine receptors by apomorphine inhibits episodic LH release in ovariectomized rats. The present study was designed to examine further the role of dopamine in this process. Unrestrained, unanesthetized rats with indwelling right atrial cannulae were bled continuously (30 or 50 microliters of whole blood/5 min for 3-6 h) and whole blood samples analyzed for LH by radioimmunoassay. Animals were treated with various compounds reported to stimulate or block dopamine receptors. ET 495, a long acting dopamine receptor stimulating agent, caused a marked inhibition of episodic LH release (2 1/2-4 h). Control injections of distilled water had no effect. d-Butaclamol, a blocker of dopamine receptors, did not itself alter episodic LH release but prevented the inhibitory effects seen following apomorphine or ET 495. I-butaclamol, a biologically inactive form of butaclamol, had no effect. Measurement of plasma corticosterone levels in these same animals indicated increased values following apomorphine or ET 495 alone (when LH release was inhibited), as well as after apomorphine or ET 495 administration to d-butaclamol-pretreated rats (when LH levels did not change). These data support our previous hypothesis that in ovariectomized adult rats, activation of dopamine receptors is capable of inhibiting episodic LH release, but that dopamine may not play an inhibitory role under normal physiological conditions in the modulation of LH secretion. In addition, the inhibitory action of apomorphine and ET 495 does not appear to be exerted via a stress-induced release of adrenal corticosterone.

  2. Bromoenol Lactone Inhibits Voltage-Gated Ca2+ and Transient Receptor Potential Canonical ChannelsS⃞

    PubMed Central

    Chakraborty, Saikat; Berwick, Zachary C.; Bartlett, Paula J.; Kumar, Sanjay; Thomas, Andrew P.; Sturek, Michael; Tune, Johnathan D.

    2011-01-01

    Circulating hormones stimulate the phospholipase Cβ (PLC)/Ca2+ influx pathway to regulate numerous cell functions, including vascular tone. It was proposed previously that Ca2+-independent phospholipase A2 (iPLA2)-dependent store-operated Ca2+ influx channels mediate hormone-induced contractions in isolated arteries, because bromoenol lactone (BEL), a potent irreversible inhibitor of iPLA2, inhibited such contractions. However, the effects of BEL on other channels implicated in mediating hormone-induced vessel contractions, specifically voltage-gated Ca2+ (CaV1.2) and transient receptor potential canonical (TRPC) channels, have not been defined clearly. Using isometric tension measurements, we found that thapsigargin-induced contractions were ∼34% of those evoked by phenylephrine or KCl. BEL completely inhibited not only thapsigargin- but also phenylephrine- and KCl-induced ring contractions, suggesting that CaV1.2 and receptor-operated TRPC channels also may be sensitive to BEL. Therefore, we investigated the effects of BEL on heterologously expressed CaV1.2 and TRPC channels in human embryonic kidney cells, a model system that allows probing of individual protein function without interference from other signaling elements of native cells. We found that low micromolar concentrations of BEL inhibited CaV1.2, TRPC5, TRPC6, and heteromeric TRPC1–TRPC5 channels in an iPLA2-independent manner. BEL also attenuated PLC activity, suggesting that the compound may inhibit TRPC channel activity in part by interfering with an initial PLC-dependent step required for TRPC channel activation. Conversely, BEL did not affect endogenous voltage-gated K+ channels in human embryonic kidney cells. Our findings support the hypothesis that iPLA2-dependent store-operated Ca2+ influx channels and iPLA2-independent hormone-operated TRPC channels can serve as smooth muscle depolarization triggers to activate CaV1.2 channels and to regulate vascular tone. PMID:21795434

  3. Mechanical vibration inhibits osteoclast formation by reducing DC-STAMP receptor expression in osteoclast precursor cells.

    PubMed

    Kulkarni, Rishikesh N; Voglewede, Philip A; Liu, Dawei

    2013-12-01

    It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-specific transmembrane protein (DC-STAMP) and P2X7 receptor (P2X7R). RAW264.7 (a murine osteoclastic-like cell line) cells were treated with 20ng/ml receptor activator of NF-κB ligand (RANKL). For 3 consecutive days, the cells were subjected to 1h of mechanical vibration with 20μm displacement at a frequency of 4Hz and compared to the control cells that were treated under the same condition but without the vibration. After 5days of culture, osteoclast formation was determined. Gene expression of DC-STAMP and P2X7R by RAW264.7 cells was determined after 1h of mechanical vibration, while protein production of the DC-STAMP was determined after 6h of postincubation after vibration. As a result, mechanical vibration of RAW264.7 cells inhibited the formation of osteoclasts. Vibration down-regulated DC-STAMP gene expression by 1.6-fold in the presence of RANKL and by 1.4-fold in the absence of RANKL. Additionally, DC-STAMP protein production was also down-regulated by 1.4-fold in the presence of RANKL and by 1.2-fold in the absence of RANKL in RAW264.7 cells in response to mechanical vibration. However, vibration did not affect P2X7R gene expression. Mouse anti-DC-STAMP antibody inhibited osteoclast formation in the absence of vibration. Our results suggest that mechanical vibration of osteoclast precursor cells reduces DC-STAMP expression in osteoclast precursor cells leading to the inhibition of osteoclast formation.

  4. Gangliosides and Nogo receptors independently mediate myelin-associated glycoprotein inhibition of neurite outgrowth in different nerve cells.

    PubMed

    Mehta, Niraj R; Lopez, Pablo H H; Vyas, Alka A; Schnaar, Ronald L

    2007-09-21

    In the injured nervous system, myelin-associated glycoprotein (MAG) on residual myelin binds to receptors on axons, inhibits axon outgrowth, and limits functional recovery. Conflicting reports identify gangliosides (GD1a and GT1b) and glycosylphosphatidylinositol-anchored Nogo receptors (NgRs) as exclusive axonal receptors for MAG. We used enzymes and pharmacological agents to distinguish the relative roles of gangliosides and NgRs in MAG-mediated inhibition of neurite outgrowth from three nerve cell types, dorsal root ganglion neurons (DRGNs), cerebellar granule neurons (CGNs), and hippocampal neurons. Primary rat neurons were cultured on control substrata and substrata adsorbed with full-length native MAG extracted from purified myelin. The receptors responsible for MAG inhibition of neurite outgrowth varied with nerve cell type. In DRGNs, most of the MAG inhibition was via NgRs, evidenced by reversal of inhibition by phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves glycosylphosphatidylinositol anchors, or by NEP1-40, a peptide inhibitor of NgR. A smaller percentage of MAG inhibition of DRGN outgrowth was via gangliosides, evidenced by partial reversal by addition of sialidase to cleave GD1a and GT1b or by P4, an inhibitor of ganglioside biosynthesis. Combining either PI-PLC and sialidase or NEP1-40 and P4 was additive. In contrast to DRGNs, in CGNs MAG inhibition was exclusively via gangliosides, whereas inhibition of hippocampal neuron outgrowth was mostly reversed by sialidase or P4 and only modestly reversed by PI-PLC or NEP1-40 in a non-additive fashion. A soluble proteolytic fragment of native MAG, dMAG, also inhibited neurite outgrowth. In DRGNs, dMAG inhibition was exclusively NgR-dependent, whereas in CGNs it was exclusively ganglioside-dependent. An inhibitor of Rho kinase reversed MAG-mediated inhibition in all nerve cells, whereas a peptide inhibitor of the transducer p75(NTR) had cell-specific effects quantitatively similar to Ng

  5. Serine dipeptide lipids of Porphyromonas gingivalis inhibit osteoblast differentiation: Relationship to Toll-like receptor 2.

    PubMed

    Wang, Yu-Hsiung; Nemati, Reza; Anstadt, Emily; Liu, Yaling; Son, Young; Zhu, Qiang; Yao, Xudong; Clark, Robert B; Rowe, David W; Nichols, Frank C

    2015-12-01

    Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential

  6. Inhibition of insulin receptor gene expression and insulin signaling by fatty acid: interplay of PKC isoforms therein.

    PubMed

    Dey, Debleena; Mukherjee, Mohua; Basu, Dipanjan; Datta, Malabika; Roy, Sib Sankar; Bandyopadhyay, Arun; Bhattacharya, Samir

    2005-01-01

    Fatty acids are known to play a key role in promoting the loss of insulin sensitivity causing insulin resistance and type 2 diabetes. However, underlying mechanism involved here is still unclear. Incubation of rat skeletal muscle cells with palmitate followed by I(125)- insulin binding to the plasma membrane receptor preparation demonstrated a two-fold decrease in receptor occupation. In searching the cause for this reduction, we found that palmitate inhibition of insulin receptor (IR) gene expression effecting reduced amount of IR protein in skeletal muscle cells. This was followed by the inhibition of insulin-stimulated IRbeta tyrosine phosphorylation that consequently resulted inhibition of insulin receptor substrate 1 (IRS 1) and IRS 1 associated phosphatidylinositol-3 kinase (PI3 Kinase), phosphoinositide dependent kinase-1 (PDK 1) phosphorylation. PDK 1 dependent phosphorylation of PKCzeta and Akt/PKB were also inhibited by palmitate. Surprisingly, although PKCepsilon phosphorylation is PDK1 dependent, palmitate effected its constitutive phosphorylation independent of PDK1. Time kinetics study showed translocation of palmitate induced phosphorylated PKCepsilon from cell membrane to nuclear region and its possible association with the inhibition of IR gene transcription. Our study suggests one of the pathways through which fatty acid can induce insulin resistance in skeletal muscle cell.

  7. Anandamide (arachidonylethanolamide), a brain cannabinoid receptor agonist, reduces sperm fertilizing capacity in sea urchins by inhibiting the acrosome reaction.

    PubMed Central

    Schuel, H; Goldstein, E; Mechoulam, R; Zimmerman, A M; Zimmerman, S

    1994-01-01

    Anandamide (arachidonylethanolamide) is an endogenous cannabinoid receptor agonist in mammalian brain. Sea urchin sperm contain a high-affinity cannabinoid receptor similar to the cannabinoid receptor in mammalian brain. (-)-delta 9-Tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. We now report that anandamide produces effects similar to those previously obtained with THC in Strongylocentrotus purpuratus in reducing sperm fertilizing capacity and inhibiting the egg jelly-stimulated acrosome reaction. Arachidonic acid does not inhibit the acrosome reaction under similar conditions. The adverse effects of anandamide on sperm fertilizing capacity and the acrosome reaction are reversible. The receptivity of unfertilized eggs to sperm and sperm motility are not impaired by anandamide. Under conditions where anandamide completely blocks the egg jelly-stimulated acrosome reaction, it does not inhibit the acrosome reaction artificially initiated by ionomycin, which promotes Ca2+ influx, and nigericin, which activates K+ channels in sperm. These findings provide additional evidence that the cannabinoid receptor in sperm plays a role in blocking the acrosome reaction, indicate that anandamide or a related molecule may be the natural ligand for the cannabinoid receptor in sea urchin sperm, and suggest that binding of anandamide to the cannabinoid receptor modulates stimulus-secretion-coupling in sperm by affecting an event prior to ion channel opening. PMID:8052642

  8. Inhibition of noradrenaline release from the sympathetic nerves of the human saphenous vein by presynaptic histamine H3 receptors.

    PubMed

    Molderings, G J; Weissenborn, G; Schlicker, E; Likungu, J; Göthert, M

    1992-07-01

    The human saphenous vein was used to examine whether presynaptic histamine receptors can modulate noradrenaline release and, if so, to determine their pharmacological characteristics. Strips of this blood vessel were incubated with [3H]noradrenaline and subsequently superfused with physiological salt solution containing desipramine and corticosterone. Electrically (2 Hz) evoked 3H overflow was inhibited by histamine and the H3 receptor agonist R-(-)-alpha-methylhistamine. Histamine-induced inhibition of electrically evoked tritium overflow was not affected by alpha 2-adrenoceptor blockade by rauwolscine. S-(+)-alpha-methylhistamine (up to 10 mumol/l) as well as the histamine H1 and H2 receptor agonists 2-(2-thiazolyl)ethylamine (up to 3 mumol/l) and dimaprit (up to 30 mumol/l), respectively, were ineffective. The selective histamine H3 receptor antagonist thioperamide abolished the inhibitory effect of histamine. The histamine H2 and H1 receptor antagonists ranitidine and pheniramine, respectively, did not affect the histamine-induced inhibition of evoked tritium overflow. The present results are compatible with the suggestion that the sympathetic nerves of the human saphenous vein are endowed with inhibitory presynaptic histamine receptors of the H3 class.

  9. Cannabinoids inhibit insulin secretion and cytosolic Ca2+ oscillation in islet beta-cells via CB1 receptors.

    PubMed

    Nakata, Masanori; Yada, Toshihiko

    2008-01-10

    Obesity is the main risk factor for the development of metabolic syndrome. Endogenous cannabinoids act on the cannabinoid type 1 (CB1) receptor, a GPCR, and stimulate appetite via central and peripheral actions, while blockade of CB1 receptor reduces body weight in humans. In this study, we aimed to explore a role of the peripheral endocannabinoid system in insulin secretion, which could be important in the metabolic effects of the cannabinoid-CB1 system. We found that mRNA for CB1 receptor, but not CB2 receptor, was expressed in mouse pancreatic islets using RT-PCR. Immunohistochemical study revealed that CB1 receptor was expressed in beta-cells. Furthermore, anandamide and a CB1 agonist, arachidonylcyclopropylamide (ACPA), inhibited glucose-induced insulin secretion from mouse pancreatic islets. Both anandamide and ACPA inhibited glucose-induced cytosolic Ca(2+) oscillation in mouse pancreatic beta-cells. These results demonstrate a novel peripheral action of cannabinoids to inhibit insulin secretion via CB1 receptors.

  10. Low nanomolar serotonin inhibits the glutamate receptor/nitric oxide/cyclic GMP pathway in slices from adult rat cerebellum.

    PubMed

    Maura, G; Guadagnin, A; Raiteri, M

    1995-09-01

    The function of serotonin afferents to the cerebellum has been investigated by monitoring the effects of serotoninergic drugs on the production of cyclic GMP elicited in cerebellar slices by activation of ionotropic glutamate receptors. Exposure of adult rat cerebellar slices to N-methyl-D-aspartate (1 nM to 1 microM) or to (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 1 nM to 10 microM) elicited concentration-dependent and saturable rises in the levels of cyclic GMP. These responses were blocked by selective antagonists at the N-methyl-D-aspartate or AMPA receptors and by inhibiting nitric oxide synthase, but were insensitive to tetrodotoxin. When tested between 0.1 and 10 nM, serotonin, the serotonin1A receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin and the serotonin2 receptor agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane inhibited, concentration-dependently, the cyclic GMP responses evoked by near-maximal (0.1 microM) concentrations of N-methyl-D-aspartate or AMPA. The EC50 values (concentrations causing half-maximal effect) ranged between 0.7 and 2.1 nM. The actions of serotonin were totally abolished by methiothepin, a mixed-type serotonin receptor antagonist. Thus, the serotonergic cerebellar afferents may exert a potent inhibitory control on the excitatory transmission mediated by N-methyl-D-aspartate and AMPA receptors; the inhibition occurs through both serotonin1A and serotonin2 receptors. As the glutamate receptor-dependent cyclic GMP responses involve production of nitric oxide, a diffusible activator of guanylate cyclase, the above inhibitory serotonin receptors may have multiple localization.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. MiRNA-101 inhibits breast cancer growth and metastasis by targeting CX chemokine receptor 7.

    PubMed

    Li, Jun-Tang; Jia, Lin-Tao; Liu, Ning-Ning; Zhu, Xiao-Shan; Liu, Qin-Qin; Wang, Xiu-Li; Yu, Feng; Liu, Yan-Li; Yang, An-Gang; Gao, Chun-Fang

    2015-10-13

    Whereas miR-101 is involved in the development and progression of breast cancer, the underlying molecular mechanisms remain to be elucidated. Here, we report that miR-101 expression is inversely correlated with the clinical stage, lymph node metastasis and prognosis in breast cancers. Introduction of miR-101 inhibited breast cancer cell proliferation and invasion in vitro and suppressed tumor growth and lung metastasis of in vivo. CX chemokine receptor 7 (CXCR7) is a direct target of miR-101, positively correlating with the histological grade and the incidence of lymph node metastasis in breast cancer patients. The effects of miR-101 were mimicked and counteracted by CXCR7 depletion and overexpression, respectively. STAT3 signaling downstream of CXCR7 is involved in miR-101 regulation of breast cancer cell behaviors. These findings have implications for the potential application of miR-101 in breast cancer treatment.

  12. Selective inhibition of Ebola entry with selective estrogen receptor modulators by disrupting the endolysosomal calcium

    PubMed Central

    Fan, Hanlu; Du, Xiaohong; Zhang, Jingyuan; Zheng, Han; Lu, Xiaohui; Wu, Qihui; Li, Haifeng; Wang, Han; Shi, Yi; Gao, George; Zhou, Zhuan; Tan, Dun-Xian; Li, Xiangdong

    2017-01-01

    The Ebola crisis occurred in West-Africa highlights the urgency for its clinical treatments. Currently, no Food and Drug Administration (FDA)-approved therapeutics are available. Several FDA-approved drugs, including selective estrogen receptor modulators (SERMs), possess selective anti-Ebola activities. However, the inhibitory mechanisms of these drugs remain elusive. By analyzing the structures of SERMs and their incidental biological activity (cholesterol accumulation), we hypothesized that this incidental biological activity induced by SERMs could be a plausible mechanism as to their inhibitory effects on Ebola infection. Herein, we demonstrated that the same dosages of SERMs which induced cholesterol accumulation also inhibited Ebola infection. SERMs reduced the cellular sphingosine and subsequently caused endolysosomal calcium accumulation, which in turn led to blocking the Ebola entry. Our study clarified the specific anti-Ebola mechanism of SERMs, even the cationic amphiphilic drugs (CADs), this mechanism led to the endolysosomal calcium as a critical target for development of anti-Ebola drugs. PMID:28117364

  13. G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition.

    PubMed

    Zhang, Panpan; He, Qiuping; Chen, Dongbo; Liu, Weixiao; Wang, Lu; Zhang, Chunxia; Ma, Dongyuan; Li, Wei; Liu, Bing; Liu, Feng

    2015-10-01

    In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs.

  14. Myeloid mineralocorticoid receptor deficiency inhibits aortic constriction-induced cardiac hypertrophy in mice.

    PubMed

    Li, Chao; Zhang, Yu Yao; Frieler, Ryan A; Zheng, Xiao Jun; Zhang, Wu Chang; Sun, Xue Nan; Yang, Qing Zhen; Ma, Shu Min; Huang, Baozhuan; Berger, Stefan; Wang, Wang; Wu, Yong; Yu, Ying; Duan, Sheng Zhong; Mortensen, Richard M

    2014-01-01

    Mineralocorticoid receptor (MR) blockade has been shown to suppress cardiac hypertrophy and remodeling in animal models of pressure overload (POL). This study aims to determine whether MR deficiency in myeloid cells modulates aortic constriction-induced cardiovascular injuries. Myeloid MR knockout (MMRKO) mice and littermate control mice were subjected to abdominal aortic constriction (AAC) or sham operation. We found that AAC-induced cardiac hypertrophy and fibrosis were significantly attenuated in MMRKO mice. Expression of genes important in generating reactive oxygen species was decreased in MMRKO mice, while that of manganese superoxide dismutase increased. Furthermore, expression of genes important in cardiac metabolism was increased in MMRKO hearts. Macrophage infiltration in the heart was inhibited and expression of inflammatory genes was decreased in MMRKO mice. In addition, aortic fibrosis and inflammation were attenuated in MMRKO mice. Taken together, our data indicated that MR deficiency in myeloid cells effectively attenuated aortic constriction-induced cardiac hypertrophy and fibrosis, as well as aortic fibrosis and inflammation.

  15. Development of a New Class of Drugs to Inhibit All Forms of Androgen Receptor in Castration-Resistant Prostate Cancers

    DTIC Science & Technology

    2015-10-01

    AWARD NUMBERS: W81XWH-14-1-0518 TITLE: Development of a New Class of Drugs to Inhibit All Forms of Androgen Receptor in Castration-Resistant...COVERED 30 Sep 2014 - 29 Sep 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Development of a New Class of Drugs to Inhibit All Forms of Androgen...designed to block AR activity that re- emerges during castration. However, despite a growing armamentarium of drugs targeting the androgen/AR signaling

  16. Development of a New Class of Drugs to Inhibit All Forms of Androgen Receptor in Castration Resistant Prostate Cancers

    DTIC Science & Technology

    2015-10-01

    AWARD NUMBERS: W81XWH-14-1-0520 TITLE: Development of a New Class of Drugs to Inhibit All Forms of Androgen Receptor in Castration-Resistant...COVERED 30 Sep 2014 - 29 Sep 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-14-1-0520 Development of a New Class of Drugs to Inhibit All Forms...re- emerges during castration. However, despite a growing armamentarium of drugs targeting the androgen/AR signaling axis, progression of castration

  17. Influence of intramuscular heat stimulation on modulation of nociception: complex role of central opioid receptors in descending facilitation and inhibition.

    PubMed

    You, Hao-Jun; Lei, Jing; Ye, Gang; Fan, Xiao-Li; Li, Qiang

    2014-10-01

    It has been reported that the threshold to activate 'silent' or inactive descending facilitation of nociception is lower than that of descending inhibition. Thus, the development of pain therapy to effectively drive descending inhibition alone, without the confounding influences of facilitation is a challenge. To address this issue we investigated the effects of intramuscular stimulation with a heating-needle on spinal nociception, assessed by measuring nociceptive paw withdrawal reflex in rats. Additionally, involvement of the thalamic 'nociceptive discriminators' (thalamic mediodorsal (MD) and ventromedial (VM) nuclei), and opioid-mediated mechanisms were further explored. Descending facilitation and inhibition were elicited by 46°C noxious heating-needle stimulation, and were regulated by thalamic MD and VM nuclei, respectively. In contrast, innocuous heating-needle stimulation at a temperature of 43°C elicited descending inhibition modulated by the thalamic VM nucleus alone. Microinjection of μ/δ/κ-opioid receptor antagonists β-funaltrexamine hydrochloride/naltrindole/nor-binaltorphimine, into the VM nucleus attenuated the 46°C intramuscular heating-needle stimulation-evoked descending inhibition, whereas treatment of the MD nucleus with β-funaltrexamine hydrochloride significantly decreased the descending facilitation. By contrast, descending inhibition evoked by 43°C heating-needle stimulation was only depressed by naltrindole, as opposed to μ- and κ-opioid receptor antagonists, which failed to influence descending inhibition. The present study reveals distinct roles of μ-opioid receptors in the function of thalamic MD and VM nuclei,which exert facilitatory and inhibitory actions on nociception. Furthermore, innocuous, but not noxious, intramuscular heating-needle stimulation targeting δ-opioid receptors is suggested to be a promising avenue for the effective inhibition of pain.

  18. Inhibition of atrial receptor-induced renal responses by stimulation of carotid baroreceptors in anaesthetized dogs.

    PubMed Central

    Karim, F; Majid, D S

    1991-01-01

    excitation of carotid baroreceptors can completely inhibit the reflex renal haemodynamic and functional responses to atrial receptor stimulation. PMID:1886066

  19. Inhibition of T-tropic HIV Strains by Selective Antagonization of the Chemokine Receptor CXCR4

    PubMed Central

    Schols, Dominique; Struyf, Sofie; Damme, Jo Van; Esté, José A.; Henson, Geoffrey; Clercq, Erik De

    1997-01-01

    Bicyclams are a novel class of antiviral compounds that are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. Surprisingly, however, when the prototype compound AMD3100 was tested against M-tropic virus strains such as BaL, ADA, JR-CSF, and SF-162 in human peripheral blood mononuclear cells, the compound was completely inactive. Because of the specific and potent inhibitory effect of AMD3100 on T-tropic viruses, but not M-tropic viruses, it was verified that AMD3100 interacts with the CXC-chemokine receptor CXCR4, the main coreceptor used by T-tropic viruses. AMD3100 dose dependently inhibited the binding of a specific CXCR4 monoclonal antibody to SUP-T1 cells as measured by flow cytometry. It did not inhibit the binding of the biotinylated CC-chemokine macrophage inflammatory protein (MIP) 1α or MIP-1β, ligands for the chemokine receptor CCR5 (the main coreceptor for M-tropic viruses). In addition, AMD3100 completely blocked (a) the Ca2+ flux at 100 ng/ml in lymphocytic SUP-T1 and monocytic THP-1 cells, and (b) the chemotactic responses of THP-1 cells induced by stromal cell–derived factor 1α, the natural ligand for CXCR4. Finally, AMD3100 had no effect on the Ca2+ flux induced by the CC-chemokines MIP-1α, regulated on activation normal T cell expressed and secreted (RANTES; also a ligand for CCR5), or monocyte chemoattractant protein 3 (a ligand for CCR1 and CCR2b), nor was it able to induce Ca2+ fluxes by itself. The bicyclams are, to our knowledge, the first low molecular weight anti-HIV agents shown to act as potent and selective CXCR4 antagonists. PMID:9334378

  20. Inhibition of T-tropic HIV strains by selective antagonization of the chemokine receptor CXCR4.

    PubMed

    Schols, D; Struyf, S; Van Damme, J; Esté, J A; Henson, G; De Clercq, E

    1997-10-20

    Bicyclams are a novel class of antiviral compounds that are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. Surprisingly, however, when the prototype compound AMD3100 was tested against M-tropic virus strains such as BaL, ADA, JR-CSF, and SF-162 in human peripheral blood mononuclear cells, the compound was completely inactive. Because of the specific and potent inhibitory effect of AMD3100 on T-tropic viruses, but not M-tropic viruses, it was verified that AMD3100 interacts with the CXC-chemokine receptor CXCR4, the main coreceptor used by T-tropic viruses. AMD3100 dose dependently inhibited the binding of a specific CXCR4 monoclonal antibody to SUP-T1 cells as measured by flow cytometry. It did not inhibit the binding of the biotinylated CC-chemokine macrophage inflammatory protein (MIP) 1alpha or MIP-1beta, ligands for the chemokine receptor CCR5 (the main coreceptor for M-tropic viruses). In addition, AMD3100 completely blocked (a) the Ca2+ flux at 100 ng/ml in lymphocytic SUP-T1 and monocytic THP-1 cells, and (b) the chemotactic responses of THP-1 cells induced by stromal cell-derived factor 1alpha, the natural ligand for CXCR4. Finally, AMD3100 had no effect on the Ca2+ flux induced by the CC-chemokines MIP-1alpha, regulated on activation normal T cell expressed and secreted (RANTES; also a ligand for CCR5), or monocyte chemoattractant protein 3 (a ligand for CCR1 and CCR2b), nor was it able to induce Ca2+ fluxes by itself. The bicyclams are, to our knowledge, the first low molecular weight anti-HIV agents shown to act as potent and selective CXCR4 antagonists.

  1. Modulators of estrogen receptor inhibit proliferation and migration of prostate cancer cells.

    PubMed

    Piccolella, Margherita; Crippa, Valeria; Messi, Elio; Tetel, Marc J; Poletti, Angelo

    2014-01-01

    In the initial stages, human prostate cancer (PC) is an androgen-sensitive disease, which can be pharmacologically controlled by androgen blockade. This therapy often induces selection of androgen-independent PC cells with increased invasiveness. We recently demonstrated, both in cells and mice, that a testosterone metabolite locally synthetized in prostate, the 5α-androstane-3β, 17β-diol (3β-Adiol), inhibits PC cell proliferation, migration and invasion, acting as an anti-proliferative/anti-metastatic agent. 3β-Adiol is unable to bind androgen receptor (AR), but exerts its protection against PC by specifically interacting with estrogen receptor beta (ERβ). Because of its potential retro-conversion to androgenic steroids, 3β-Adiol cannot be used "in vivo", thus, the aims of this study were to investigate the capability of four ligands of ERβ (raloxifen, tamoxifen, genistein and curcumin) to counteract PC progression by mimicking the 3β-Adiol activity. Our results demonstrated that raloxifen, tamoxifen, genistein and curcumin decreased DU145 and PC3 cell proliferation in a dose-dependent manner; in addition, all four compounds significantly decreased the detachment of cells seeded on laminin or fibronectin. Moreover, raloxifen, tamoxifen, genistein and curcumin-treated DU145 and PC3 cells showed a significant decrease in cell migration. Notably, all these effects were reversed by the anti-estrogen, ICI 182,780, suggesting that their actions are mediated by the estrogenic pathway, via the ERβ, the only isoform present in these PCs. In conclusion, these data demonstrate that by selectively activating the ERβ, raloxifen, tamoxifen, genistein and curcumin inhibit human PC cells proliferation and migration favoring cell adesion. These synthetic and natural modulators of ER action may exert a potent protective activity against the progression of PC even in its androgen-independent status.

  2. Aryl hydrocarbon receptor activation inhibits in vitro differentiation of human monocytes and Langerhans dendritic cells.

    PubMed

    Platzer, Barbara; Richter, Susanne; Kneidinger, Doris; Waltenberger, Darina; Woisetschläger, Maximilian; Strobl, Herbert

    2009-07-01

    The transcription factor aryl hydrocarbon receptor (AhR) represents a promising therapeutic target in allergy and autoimmunity. AhR signaling induced by the newly described ligand VAF347 inhibits allergic lung inflammation as well as suppresses pancreatic islet allograft rejection. These effects are likely mediated via alterations in dendritic cell (DC) function. Moreover, VAF347 induces tolerogenic DCs. Langerhans cells (LCs) are immediate targets of exogenous AhR ligands at epithelial surfaces; how they respond to AhR ligands remained undefined. We studied AhR expression and function in human LCs and myelopoietic cell subsets using a lineage differentiation and gene transduction model of human CD34(+) hematopoietic progenitors. We found that AhR is highly regulated during myeloid subset differentiation. LCs expressed highest AhR levels followed by monocytes. Conversely, neutrophil granulocytes lacked AhR expression. AhR ligands including VAF347 arrested the differentiation of monocytes and LCs at an early precursor cell stage, whereas progenitor cell expansion or granulopoiesis remained unimpaired. AhR expression was coregulated with the transcription factor PU.1 during myeloid subset differentiation. VAF347 inhibited PU.1 induction during initial monocytic differentiation, and ectopic PU.1 restored monocyte and LC generation in the presence of this compound. AhR ligands failed to interfere with cytokine receptor signaling during LC differentiation and failed to impair LC activation/maturation. VAF347-mediated antiproliferative effect on precursors undergoing LC lineage differentiation occurred in a clinically applicable serum-free culture model and was not accompanied by apoptosis induction. In conclusion, AhR agonist signaling interferes with transcriptional processes leading to monocyte/DC lineage commitment of human myeloid progenitor cells.

  3. Piperlongumine inhibits atherosclerotic plaque formation and vascular smooth muscle cell proliferation by suppressing PDGF receptor signaling

    SciTech Connect

    Son, Dong Ju; Kim, Soo Yeon; Han, Seong Su; Kim, Chan Woo; Kumar, Sandeep; Park, Byeoung Soo; Lee, Sung Eun; Yun, Yeo Pyo; Jo, Hanjoong; Park, Young Hyun

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Anti-atherogenic effect of PL was examined using partial carotid ligation model in ApoE KO mice. Black-Right-Pointing-Pointer PL prevented atherosclerotic plaque development, VSMCs proliferation, and NF-{kappa}B activation. Black-Right-Pointing-Pointer Piperlongumine reduced vascular smooth muscle cell activation through PDGF-R{beta} and NF-{kappa}B-signaling. Black-Right-Pointing-Pointer PL may serve as a new therapeutic molecule for atherosclerosis treatment. -- Abstract: Piperlongumine (piplartine, PL) is an alkaloid found in the long pepper (Piper longum L.) and has well-documented anti-platelet aggregation, anti-inflammatory, and anti-cancer properties; however, the role of PL in prevention of atherosclerosis is unknown. We evaluated the anti-atherosclerotic potential of PL in an in vivo murine model of accelerated atherosclerosis and defined its mechanism of action in aortic vascular smooth muscle cells (VSMCs) in vitro. Local treatment with PL significantly reduced atherosclerotic plaque formation as well as proliferation and nuclear factor-kappa B (NF-{kappa}B) activation in an in vivo setting. PL treatment in VSMCs in vitro showed inhibition of migration and platelet-derived growth factor BB (PDGF-BB)-induced proliferation to the in vivo findings. We further identified that PL inhibited PDGF-BB-induced PDGF receptor beta activation and suppressed downstream signaling molecules such as phospholipase C{gamma}1, extracellular signal-regulated kinases 1 and 2 and Akt. Lastly, PL significantly attenuated activation of NF-{kappa}B-a downstream transcriptional regulator in PDGF receptor signaling, in response to PDGF-BB stimulation. In conclusion, our findings demonstrate a novel, therapeutic mechanism by which PL suppresses atherosclerosis plaque formation in vivo.

  4. Endogenous Inhibition of the Trigeminally Evoked Neurotransmission to Cardiac Vagal Neurons by Muscarinic Acetylcholine Receptors

    PubMed Central

    Gorini, C.; Philbin, K.; Bateman, R.

    2010-01-01

    Stimulation of the nasal mucosa by airborne irritants or water evokes a pronounced bradycardia accompanied by peripheral vasoconstriction and apnea. The dive response, which includes the trigeminocardiac reflex, is among the most powerful autonomic responses. These responses slow the heart rate and reduce myocardial oxygen consumption. Although normally cardioprotective, exaggeration of this reflex can be detrimental and has been implicated in cardiorespiratory diseases, including sudden infant death syndrome (SIDS). An essential component of the diving response and trigeminocardiac reflex is activation of the parasympathetic cardiac vagal neurons (CVNs) in the nucleus ambiguus that control heart rate. This study examined the involvement of cholinergic receptors in trigeminally evoked excitatory postsynaptic currents in CVNs in an in vitro preparation from rats. CVNs were identified using a retrograde tracer injected into the fat pads at the base of the heart. Application of the acetylcholinesterase inhibitor neostigmine significantly decreased the amplitude of glutamatergic neurotransmission to CVNs on stimulation of trigeminal fibers. Whereas nicotine did not have any effect on the glutamatergic responses, the muscarinic acetylcholine receptor (mAChR) agonist bethanechol significantly decreased the excitatory neurotransmission. Atropine, an mAChR antagonist, facilitated these responses indicating this trigeminally evoked brain stem pathway in vitro is endogenously inhibited by mAChRs. Tropicamide, an m4 mAChR antagonist, prevented the inhibitory action of the muscarinic agonist bethanechol. These results indicate that the glutamatergic synaptic neurotransmission in the trigeminally evoked pathway to CVNs is endogenously inhibited in vitro by m4 mAChRs. PMID:20719927

  5. Autoantibodies against muscarinic type 3 receptor in Sjögren's syndrome inhibit aquaporin 5 trafficking.

    PubMed

    Lee, Byung Ha; Gauna, Adrienne E; Perez, Geidys; Park, Yun-jong; Pauley, Kaleb M; Kawai, Toshihisa; Cha, Seunghee

    2013-01-01

    Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS.

  6. Due to interleukin-6 type cytokine redundancy only glycoprotein 130 receptor blockade efficiently inhibits myeloma growth

    PubMed Central

    Burger, Renate; Günther, Andreas; Klausz, Katja; Staudinger, Matthias; Peipp, Matthias; Penas, Eva Maria Murga; Rose-John, Stefan; Wijdenes, John; Gramatzki, Martin

    2017-01-01

    Interleukin-6 has an important role in the pathophysiology of multiple myeloma where it supports the growth and survival of the malignant plasma cells in the bone marrow. It belongs to a family of cytokines which use the glycoprotein 130 chain for signal transduction, such as oncostatin M or leukemia inhibitory factor. Targeting interleukin-6 in plasma cell diseases is currently evaluated in clinical trials with monoclonal antibodies. Here, efforts were made to elucidate the contribution of interleukin-6 and glycoprotein 130 signaling in malignant plasma cell growth in vivo. In the xenograft severe combined immune deficiency model employing our interleukin-6-dependent plasma cell line INA-6, the lack of human interleukin-6 induced autocrine interleukin-6 production and a proliferative response to other cytokines of the glycoprotein 130 family. Herein, mice were treated with monoclonal antibodies against human interleukin-6 (elsilimomab/B-E8), the interleukin-6 receptor (B-R6), and with an antibody blocking glycoprotein 130 (B-R3). While treatment of mice with interleukin-6 and interleukin-6 receptor antibodies resulted in a modest delay in tumor growth, the development of plasmacytomas was completely prevented with the anti-glycoprotein 130 antibody. Importantly, complete inhibition was also achieved using F(ab’)2-fragments of monoclonal antibody B-R3. Tumors harbor activated signal transducer and activator of transcription 3, and in vitro, the antibody inhibited leukemia inhibitory factor stimulated signal transducer and activator of transcription 3 phosphorylation and cell growth, while being less effective against interleukin-6. In conclusion, the growth of INA-6 plasmacytomas in vivo under interleukin-6 withdrawal remains strictly dependent on glycoprotein 130, and other glycoprotein 130 cytokines may substitute for interleukin-6. Antibodies against glycoprotein 130 are able to overcome this redundancy and should be explored for a possible therapeutic window

  7. VEGF receptor inhibition blocks liver cyst growth in pkd2(WS25/-) mice.

    PubMed

    Amura, Claudia R; Brodsky, Kelley S; Groff, Rachel; Gattone, Vincent H; Voelkel, Norbert F; Doctor, R Brian

    2007-07-01

    Proliferation of cyst-lining epithelial cells is an integral part of autosomal dominant polycystic kidney disease (ADPKD) cyst growth. Cytokines and growth factors within cyst fluids are positioned to induce cyst growth. Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor present in ADPKD liver cyst fluids (human 1,128 +/- 78, mouse 2,787 +/- 136 pg/ml) and, to a lesser extent, in ADPKD renal cyst fluids (human 294 +/- 41, mouse 191 +/- 90 pg/ml). Western blotting showed that receptors for VEGF (VEGFR1 and VEGFR2) were present in both normal mouse bile ducts and pkd2(WS25/-) liver cyst epithelial cells. Treatment of pkd2(WS25/-) liver cyst epithelial cells with VEGF (50-50,000 pg/ml) or liver cyst fluid induced a proliferative response. The effect on proliferation of liver cyst fluid was inhibited by SU-5416, a potent VEGF receptor inhibitor. Treatment of pkd2(WS25/-) mice between 4 and 8 mo of age with SU-5416 markedly reduced the cyst volume density of the liver (vehicle 9.9 +/- 4.3%, SU-5416 1.8 +/- 0.7% of liver). SU-5416 treatment between 4 and 12 mo of age markedly protected against increases in liver weight [pkd2(+/+) 4.8 +/- 0.2%, pkd2(WS25/-)-vehicle 10.8 +/- 1.9%, pkd2(WS25/-)-SU-5416 4.8 +/- 0.4% body wt]. The capacity of VEGF signaling to induce in vitro proliferation of pkd2(WS25/-) liver cyst epithelial cells and inhibition of in vivo VEGF signaling to retard liver cyst growth in pkd2(WS25/-) mice indicates that the VEGF signaling pathway is a potentially important therapeutic target in the treatment of ADPKD liver cyst disease.

  8. Farnesoid X receptor inhibits glucagon-like peptide-1 production by enteroendocrine L cells.

    PubMed

    Trabelsi, Mohamed-Sami; Daoudi, Mehdi; Prawitt, Janne; Ducastel, Sarah; Touche, Véronique; Sayin, Sama I; Perino, Alessia; Brighton, Cheryl A; Sebti, Yasmine; Kluza, Jérôme; Briand, Olivier; Dehondt, Hélène; Vallez, Emmanuelle; Dorchies, Emilie; Baud, Grégory; Spinelli, Valeria; Hennuyer, Nathalie; Caron, Sandrine; Bantubungi, Kadiombo; Caiazzo, Robert; Reimann, Frank; Marchetti, Philippe; Lefebvre, Philippe; Bäckhed, Fredrik; Gribble, Fiona M; Schoonjans, Kristina; Pattou, François; Tailleux, Anne; Staels, Bart; Lestavel, Sophie

    2015-07-02

    Bile acids are signalling molecules, which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex bile acids in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces glucagon-like peptide-1 (GLP-1) production by L cells which potentiates β-cell glucose-induced insulin secretion. Whether FXR is expressed in L cells and controls GLP-1 production is unknown. Here, we show that FXR activation in L cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycaemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes.

  9. Both neuropeptide Y knockdown and Y1 receptor inhibition modulate CART-mediated appetite control.

    PubMed

    Chu, Shu-Chen; Chen, Pei-Ni; Ho, Ying-Jui; Yu, Ching-Han; Hsieh, Yih-Shou; Kuo, Dong-Yih

    2015-01-01

    Amphetamine (AMPH)-induced appetite suppression has been attributed to its inhibition of neuropeptide Y (NPY)-containing neurons in the hypothalamus. This study examined whether hypothalamic cocaine- and amphetamine-regulated transcript (CART)-containing neurons and NPY Y1 receptor (Y1R) were involved in the action of AMPH. Rats were treated daily with AMPH for four days, and changes in feeding behavior and expression levels of NPY, CART, and POMC were assessed and compared. The results showed that both feeding behavior and NPY expression decreased during AMPH treatment, with the biggest reduction occurring on Day 2. By contrast, the expression of CART and melanocortin 3 receptor (MC3R), a member of the POMC neurotransmission, increased with the maximum response on Day 2, directly opposite to the NPY expression results. The intracerebroventricular infusion of NPY antisense or Y1R inhibitor both modulated AMPH-induced anorexia and the expression levels of MC3R and CART. The results suggest that in the hypothalamus both POMC- and CART-containing neurons participate in regulating NPY-mediated appetite control during AMPH treatment. These results may advance the knowledge of molecular mechanism of anorectic drugs.

  10. A host deficiency of discoidin domain receptor 2 (DDR2) inhibits both tumour angiogenesis and metastasis.

    PubMed

    Zhang, Shuya; Bu, Xin; Zhao, Hu; Yu, Jiangtian; Wang, Yingmei; Li, Di; Zhu, Chuchao; Zhu, Tong; Ren, Tingting; Liu, Xinping; Yao, Libo; Su, Jin

    2014-03-01

    Discoidin domain receptor 2 (DDR2) is a unique receptor tyrosine kinase (RTK) that signals in response to collagen binding and is implicated in tumour malignant phenotypes such as invasion and metastasis. Although it has been reported that DDR2 expression is up-regulated in activated endothelial cells (ECs), functional studies are lacking. Herein, we found that enforced expression of DDR2 promoted proliferation, migration and tube formation of primary human umbilical vein endothelial cells (HUVECs). The results of immunohistochemical analysis showed a strikingly high level of DDR2 in human tumour ECs. Most significantly, we discovered that a host deficiency of DDR2 inhibits subcutaneous angiogenesis induced by either VEGF or tumour cells. In addition, the remaining tumour vessels in DDR2-deficient mice exhibit some normalized properties. These vascular phenotypes are accompanied by the up-regulation of anti-angiogenic genes and down-regulation of pro-angiogenic genes, as well as by alleviated tumour hypoxia. By use of a tail vein metastasis model of melanoma, we uncovered that loss of stromal DDR2 also suppresses tumour metastasis to the lung. Hence, our current data disclose a new mechanism by which DDR2 affects tumour progression, and may strengthen the feasibility of targeting DDR2 as an anticancer strategy.

  11. Corticosterone Inhibits the Proliferation of C6 Glioma Cells via the Translocation of Unphosphorylated Glucocorticoid Receptor.

    PubMed

    Nakatani, Yoshihiko; Amano, Taku; Takeda, Hiroshi

    2016-01-01

    Astroglial cells have been considered to have passive brain function by helping to maintain neurons. However, recent studies have revealed that the dysfunction of such passive functions may be associated with various neuropathological diseases, such as schizophrenia, Alzheimer's disease, amyotrophic lateral sclerosis and major depression. Corticosterone (CORT), which is often referred to as the stress hormone, is a well-known regulator of peripheral immune responses and also shows anti-inflammatory properties in the brain. However, it is still obscure how CORT affects astroglial cell function. In this study, we investigated the effects of CORT on the proliferation and survival of astroglial cells using C6 glioma cells. Under treatment with CORT for 24h, the proliferation of C6 glioma cells decreased in a dose-dependent manner. Moreover, this inhibition was diminised by treatment with mifepristone, a glucocorticoid receptor (GR) antagonist, but not by spironolactone, a mineralocorticoid receptor (MR) antagonist, and was independent of GR phosphorylation and other GR-related intracellular signaling cascades. Furthermore, it was observed that the translocation of GR from the cytosol to the nucleus was promoted by the treatment with CORT. These results indicate that CORT decreases the proliferation of C6 glioma cells by modifying the transcription of a particular gene related to cell proliferation independent of GR phosphorylation.

  12. Structural basis for subtype-specific inhibition of the P2X7 receptor

    SciTech Connect

    Karasawa, Akira; Kawate, Toshimitsu

    2016-12-09

    The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

  13. Farnesoid X Receptor Inhibits Glucagon-Like Peptide-1 Production by Enteroendocrine L-cells

    PubMed Central

    TRABELSI, Mohamed-Sami; DAOUDI, Mehdi; PRAWITT, Janne; DUCASTEL, Sarah; TOUCHE, Véronique; SAYIN, Sama I.; PERINO, Alessia; BRIGHTON, Cheryl A.; SEBTI, Yasmine; KLUZA, Jérôme; BRIAND, Olivier; DEHONDT, Hélène; VALLEZ, Emmanuelle; DORCHIES, Emilie; BAUD, Grégory; SPINELLI, Valeria; HENNUYER, Nathalie; CARON, Sandrine; BANTUBUNGI, Kadiombo; CAIAZZO, Robert; REIMANN, Frank; MARCHETTI, Philippe; LEFEBVRE, Philippe; BÄCKHED, Fredrik; GRIBBLE, Fiona M.; SCHOONJANS, Kristina; PATTOU, François; TAILLEUX, Anne; STAELS, Bart; LESTAVEL, Sophie

    2015-01-01

    Bile acids (BA) are signalling molecules which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex BA in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces Glucagon-Like Peptide-1 (GLP-1) production by L-cells which potentiates β-cell glucose-induced insulin secretion. Whether FXR is expressed in L-cells and controls GLP-1 production is unknown. Here we show that FXR activation in L-cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR-deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes. PMID:26134028

  14. Niclosamide enhances abiraterone treatment via inhibition of androgen receptor variants in castration resistant prostate cancer

    PubMed Central

    Liu, Chengfei; Armstrong, Cameron; Zhu, Yezi; Lou, Wei; Gao, Allen C.

    2016-01-01

    Considerable evidence from both clinical and experimental studies suggests that androgen receptor variants, particularly androgen receptor variant 7 (AR-V7), are critical in the induction of resistance to enzalutamide and abiraterone. In this study, we investigated the role of AR-V7 in the cross-resistance of enzalutamide and abiraterone and examined if inhibition of AR-V7 can improve abiraterone treatment response. We found that enzalutamide-resistant cells are cross-resistant to abiraterone, and that AR-V7 confers resistance to abiraterone. Knock down of AR-V7 by siRNA in abiraterone resistant CWR22Rv1 and C4-2B MDVR cells restored their sensitivity to abiraterone, indicating that AR-V7 is involved in abiraterone resistance. Abiraterone resistant prostate cancer cells generated by chronic treatment with abiraterone showed enhanced AR-V7 protein expression. Niclosamide, an FDA-approved antihelminthic drug that has been previously identified as a potent inhibitor of AR-V7, re-sensitizes resistant cells to abiraterone treatment in vitro and in vivo. In summary, this preclinical study suggests that overexpression of AR-V7 contributes to resistance to abiraterone, and supports the development of combination of abiraterone with niclosamide as a potential treatment for advanced castration resistant prostate cancer. PMID:27049719

  15. Structural basis for subtype-specific inhibition of the P2X7 receptor

    PubMed Central

    Karasawa, Akira; Kawate, Toshimitsu

    2016-01-01

    The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases. DOI: http://dx.doi.org/10.7554/eLife.22153.001 PMID:27935479

  16. Niclosamide enhances abiraterone treatment via inhibition of androgen receptor variants in castration resistant prostate cancer.

    PubMed

    Liu, Chengfei; Armstrong, Cameron; Zhu, Yezi; Lou, Wei; Gao, Allen C

    2016-05-31

    Considerable evidence from both clinical and experimental studies suggests that androgen receptor variants, particularly androgen receptor variant 7 (AR-V7), are critical in the induction of resistance to enzalutamide and abiraterone. In this study, we investigated the role of AR-V7 in the cross-resistance of enzalutamide and abiraterone and examined if inhibition of AR-V7 can improve abiraterone treatment response. We found that enzalutamide-resistant cells are cross-resistant to abiraterone, and that AR-V7 confers resistance to abiraterone. Knock down of AR-V7 by siRNA in abiraterone resistant CWR22Rv1 and C4-2B MDVR cells restored their sensitivity to abiraterone, indicating that AR-V7 is involved in abiraterone resistance. Abiraterone resistant prostate cancer cells generated by chronic treatment with abiraterone showed enhanced AR-V7 protein expression. Niclosamide, an FDA-approved antihelminthic drug that has been previously identified as a potent inhibitor of AR-V7, re-sensitizes resistant cells to abiraterone treatment in vitro and in vivo. In summary, this preclinical study suggests that overexpression of AR-V7 contributes to resistance to abiraterone, and supports the development of combination of abiraterone with niclosamide as a potential treatment for advanced castration resistant prostate cancer.

  17. Cholera Toxin Inhibits the T-Cell Antigen Receptor-Mediated Increases in Inositol Trisphosphate and Cytoplasmic Free Calcium

    NASA Astrophysics Data System (ADS)

    Imboden, John B.; Shoback, Dolores M.; Pattison, Gregory; Stobo, John D.

    1986-08-01

    The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor.

  18. Peripheral Administration of a Long-Acting Peptide Oxytocin Receptor Agonist Inhibits Fear-Induced Freezing

    PubMed Central

    Modi, Meera E.; Majchrzak, Mark J.; Fonseca, Kari R.; Doran, Angela; Osgood, Sarah; Vanase-Frawley, Michelle; Feyfant, Eric; McInnes, Heather; Darvari, Ramin; Buhl, Derek L.

    2016-01-01

    Oxytocin (OT) modulates the expression of social and emotional behaviors and consequently has been proposed as a pharmacologic treatment of psychiatric diseases, including autism spectrum disorders and schizophrenia; however, endogenous OT has a short half-life in plasma and poor permeability across the blood-brain barrier. Recent efforts have focused on the development of novel drug delivery methods to enhance brain penetration, but few efforts have aimed at improving its half-life. To explore the behavioral efficacy of an OT analog with enhanced plasma stability, we developed PF-06655075 (PF1), a novel non–brain-penetrant OT receptor agonist with increased selectivity for the OT receptor and significantly increased pharmacokinetic stability. PF-06478939 was generated with only increased stability to disambiguate changes to selectivity versus stability. The efficacy of these compounds in evoking behavioral effects was tested in a conditioned fear paradigm. Both central and peripheral administration of PF1 inhibited freezing in response to a conditioned fear stimulus. Peripheral administration of PF1 resulted in a sustained level of plasma concentrations for greater than 20 hours but no detectable accumulation in brain tissue, suggesting that plasma or cerebrospinal fluid exposure was sufficient to evoke behavioral effects. Behavioral efficacy of peripherally administered OT receptor agonists on conditioned fear response opens the door to potential peripheral mechanisms in other behavioral paradigms, whether they are mediated by direct peripheral activation or feed-forward responses. Compound PF1 is freely available as a tool compound to further explore the role of peripheral OT in behavioral response. PMID:27217590

  19. Kainate receptor-mediated presynaptic inhibition at the mouse hippocampal mossy fibre synapse

    PubMed Central

    Kamiya, Haruyuki; Ozawa, Seiji

    2000-01-01

    The presynaptic action of kainate (KA) receptor activation at the mossy fibre-CA3 synapse was examined using fluorescence measurement of presynaptic Ca2+ influx as well as electrophysiological recordings in mouse hippocampal slices. Bath application of a low concentration (0·2 μM) of KA reversibly increased the amplitude of presynaptic volley evoked by stimulation of mossy fibres to 146 ± 6 % of control (n = 6), whereas it reduced the field excitatory postsynaptic potential (EPSPs) to 30 ± 4 %. The potentiating effect of KA on the presynaptic volleys was also observed in Ca2+-free solution, and was partly antagonized by (2S,4R)-4-methylglutamic acid (SYM 2081, 1 μM), which selectively desensitizes KA receptors. The antidromic population spike of dentate granule cells evoked by stimulation of mossy fibres was increased by application of 0·2 μM KA to 160 ± 10 % of control (n = 6). Whole-cell current-clamp recordings revealed that the stimulus threshold for generating antidromic spikes recorded from a single granule cell was lowered by KA application. Application of KA (0·2 μM) suppressed presynaptic Ca2+ influx to 78 ± 4 % of control (n = 6), whereas the amplitude of the presynaptic volley was increased. KA at 0·2 μM reversibly suppressed excitatory postsynaptic currents (EPSCs) evoked by mossy fibre simulation to 38 ± 9 % of control (n = 5). These results suggest that KA receptor activation enhances the excitability of mossy fibres, probably via axonal depolarization, and reduces action potential-induced Ca2+ influx, thereby inhibiting mossy fibre EPSCs presynaptically. This novel presynaptic inhibitory action of KA at the mossy fibre-CA3 synapse may regulate the excitability of highly interconnected CA3 networks. PMID:10718745

  20. Histamine H3 receptor-mediated inhibition of noradrenaline release in pig retina discs.

    PubMed

    Schlicker, E; Schunack, W; Göthert, M

    1990-11-01

    Discs of pig retina were preincubated with 3H-noradrenaline, 3H-dopamine or 3H-serotonin and then superfused. Electrical field stimulation increased the outflow of tritium from discs preincubated with 3H-noradrenaline or 3H-dopamine, but no from discs preincubated with 3H-serotonin. The tritium content at the end of superfusion was similar in discs preincubated with 3H-noradrenaline or 3H-dopamine but about tenfold lower in discs preincubated with 3H-serotonin. The tritium content in discs preincubated with 3H-noradrenaline was markedly reduced when desipramine was present during preincubation but was not affected by selective inhibitors of dopamine and serotonin uptake. The tritium content in discs preincubated with 3H-dopamine and 3H-serotonin, in contrast, was reduced or tended to be reduced by a selective dopamine and serotonin uptake inhibitor, respectively. The electrically evoked overflow of tritium from discs preincubated with 3H-noradrenaline was abolished by tetrodotoxin or omission of Ca2+. In discs superfused with desipramine, the electrically evoked overflow was enhanced by phentolamine but not affected by histamine. When both desipramine and phentolamine were present in the superfusion medium, histamine inhibited the evoked overflow (pIC15 6.85). This effect was mimicked by the histamine H3 receptor agonist R-(-)-alpha-methylhistamine as well as by its S-(+)-enantiomer (pIC15 7.85 and 5.30, respectively) but not by the H1 receptor agonist 2-(2-thiazolyl)ethylamine and the H2 receptor agonist dimaprit (each 10 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Endostatin inhibits androgen-independent prostate cancer growth by suppressing nuclear receptor-mediated oxidative stress.

    PubMed

    Lee, Joo Hyoung; Kang, Minsung; Wang, Hong; Naik, Gurudatta; Mobley, James A; Sonpavde, Guru; Garvey, W Timothy; Darley-Usmar, Victor M; Ponnazhagan, Selvarangan

    2017-04-01

    Androgen-deprivation therapy has been identified to induce oxidative stress in prostate cancer (PCa), leading to reactivation of androgen receptor (AR) signaling in a hormone-refractory manner. Thus, antioxidant therapies have gained attention as adjuvants for castration-resistant PCa. Here, we report for the first time that human endostatin (ES) prevents androgen-independent growth phenotype in PCa cells through its molecular targeting of AR and glucocorticoid receptor (GR) and downstream pro-oxidant signaling. This reversal after ES treatment significantly decreased PCa cell proliferation through down-regulation of GR and up-regulation of manganese superoxide dismutase and reduced glutathione levels. Proteome and biochemical analyses of ES-treated PCa cells further indicated a significant up-regulation of enzymes in the major reactive oxygen species (ROS) scavenging machinery, including catalase, glutathione synthetase, glutathione reductase, NADPH-cytochrome P450 reductase, biliverdin reductase, and thioredoxin reductase, resulting in a concomitant reduction of intracellular ROS. ES further augmented the antioxidant system through up-regulation of glucose influx, the pentose phosphate pathway, and NAD salvaging pathways. This shift in cancer cell redox homeostasis by ES significantly decreased the effect of protumorigenic oxidative machinery on androgen-independent PCa growth, suggesting that ES can suppress GR-induced resistant phenotype upon AR antagonism and that the dual targeting action of ES on AR and GR can be further translated to PCa therapy.-Lee, J. H., Kang, M., Wang, H., Naik, G., Mobley, J. A., Sonpavde, G., Garvey, W. T., Darley-Usmar, V. M., Ponnazhagan, S. Endostatin inhibits androgen-independent prostate cancer growth by suppressing nuclear receptor-mediated oxidative stress.

  2. The Ly49E Receptor Inhibits the Immune Control of Acute Trypanosoma cruzi Infection

    PubMed Central

    Filtjens, Jessica; Coltel, Nicolas; Cencig, Sabrina; Taveirne, Sylvie; Van Ammel, Els; Van Acker, Aline; Kerre, Tessa; Matthys, Patrick; Taghon, Tom; Vandekerckhove, Bart; Carlier, Yves; Truyens, Carine; Leclercq, Georges

    2016-01-01

    The protozoan parasite Trypanosoma cruzi circulates in the blood upon infection and invades various cells. Parasites intensively multiply during the acute phase of infection and persist lifelong at low levels in tissues and blood during the chronic phase. Natural killer (NK) and NKT cells play an important role in the immune control of T. cruzi infection, mainly by releasing the cytokine IFN-γ that activates the microbicidal action of macrophages and other cells and shapes a protective type 1 immune response. The mechanisms by which immune cells are regulated to produce IFN-γ during T. cruzi infection are still incompletely understood. Here, we show that urokinase plasminogen activator (uPA) is induced early upon T. cruzi infection and remains elevated until day 20 post-infection. We previously demonstrated that the inhibitory receptor Ly49E, which is expressed, among others, on NK and NKT cells, is triggered by uPA. Therefore, we compared wild type (WT) to Ly49E knockout (KO) mice for their control of experimental T. cruzi infection. Our results show that young, i.e., 4- and 6-week-old, Ly49E KO mice control the infection better than WT mice, indicated by a lower parasite load and less cachexia. The beneficial effect of Ly49E depletion is more obvious in 4-week-old male than in female mice and weakens in 8-week-old mice. In young mice, the lower T. cruzi parasitemia in Ly49E KO mice is paralleled by higher IFN-γ production compared to their WT controls. Our data indicate that Ly49E receptor expression inhibits the immune control of T. cruzi infection. This is the first demonstration that the inhibitory Ly49E receptor can interfere with the immune response to a pathogen in vivo. PMID:27891126

  3. Transactivation of ErbB Family of Receptor Tyrosine Kinases Is Inhibited by Angiotensin-(1-7) via Its Mas Receptor

    PubMed Central

    Akhtar, Saghir; Chandrasekhar, Bindu; Attur, Sreeja; Dhaunsi, Gursev S.; Yousif, Mariam H. M.; Benter, Ibrahim F.

    2015-01-01

    Transactivation of the epidermal growth factor receptor (EGFR or ErbB) family members, namely EGFR and ErbB2, appears important in the development of diabetes-induced vascular dysfunction. Angiotensin-(1–7) [Ang-(1–7)] can prevent the development of hyperglycemia-induced vascular complications partly through inhibiting EGFR transactivation. Here, we investigated whether Ang-(1–7) can inhibit transactivation of ErbB2 as well as other ErbB receptors in vivo and in vitro. Streptozotocin-induced diabetic rats were chronically treated with Ang-(1–7) or AG825, a selective ErbB2 inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in cultured vascular smooth muscle cells (VSMCs). Ang-(1–7) or AG825 treatment inhibited diabetes-induced phosphorylation of ErbB2 receptor at tyrosine residues Y1221/22, Y1248, Y877, as well as downstream signaling via ERK1/2, p38 MAPK, ROCK, eNOS and IkB-α in the mesenteric vascular bed. In VSMCs cultured in high glucose (25 mM), Ang-(1–7) inhibited src-dependent ErbB2 transactivation that was opposed by the selective Mas receptor antagonist, D-Pro7-Ang-(1–7). Ang-(1–7) via Mas receptor also inhibited both Angiotensin II- and noradrenaline/norephinephrine-induced transactivation of ErbB2 and/or EGFR receptors. Further, hyperglycemia-induced transactivation of ErbB3 and ErbB4 receptors could be attenuated by Ang-(1–7) that could be prevented by D-Pro7-Ang-(1–7) in VSMC. These data suggest that Ang-(1–7) via its Mas receptor acts as a pan-ErbB inhibitor and might represent a novel general mechanism by which Ang-(1–7) exerts its beneficial effects in many disease states including diabetes-induced vascular complications. PMID:26536590

  4. Transient Receptor Potential Vanilloid 4 Inhibits γ-Aminobutyric Acid-Activated Current in Hippocampal Pyramidal Neurons

    PubMed Central

    Hong, Zhiwen; Tian, Yujing; Qi, Mengwen; Li, Yingchun; Du, Yimei; Chen, Lei; Liu, Wentao; Chen, Ling

    2016-01-01

    The balance between excitatory and inhibitory neurotransmitter systems is crucial for the modulation of neuronal excitability in the central nervous system (CNS). The activation of transient receptor potential vanilloid 4 (TRPV4) is reported to enhance the response of hippocampal glutamate receptors, but whether the inhibitory neurotransmitter system can be regulated by TRPV4 remains unknown. γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the CNS. Here, we show that application of transient receptor potential vanilloid 4 (TRPV4) synthetic (GSK1016790A or 4α-PDD) or endogenous agonist (5,6-EET) inhibited GABA-activated current (IGABA) in hippocampal CA1 pyramidal neurons, which was blocked by specific antagonists of TRPV4 and of GABAA receptors. GSK1016790A increased the phosphorylated AMP-activated protein kinase (p-AMPK) and decreased the phosphorylated protein kinase B (p-Akt) protein levels, which was attenuated by removing extracellular calcium or by a calcium/calmodulin-dependent protein kinase kinase-β antagonist. GSK1016790A-induced decrease of p-Akt protein level was sensitive to an AMPK antagonist. GSK1016790A-inhibited IGABA was blocked by an AMPK antagonist or a phosphatidyl inositol 3 kinase (PI3K) agonist. GSK1016790A-induced inhibition of IGABA was also significantly attenuated by a protein kinase C (PKC) antagonist but was unaffected by protein kinase A or calcium/calmodulin-dependent protein kinase II antagonist. We conclude that activation of TRPV4 inhibits GABAA receptor, which may be mediated by activation of AMPK and subsequent down-regulation of PI3K/Akt signaling and activation of PKC signaling. Inhibition of GABAA receptors may account for the neuronal hyperexcitability caused by TRPV4 activation. PMID:27616980

  5. Lactisole inhibits the glucose-sensing receptor T1R3 expressed in mouse pancreatic β-cells.

    PubMed

    Hamano, Kunihisa; Nakagawa, Yuko; Ohtsu, Yoshiaki; Li, Longfei; Medina, Johan; Tanaka, Yuji; Masuda, Katsuyoshi; Komatsu, Mitsuhisa; Kojima, Itaru

    2015-07-01

    Glucose activates the glucose-sensing receptor T1R3 and facilitates its own metabolism in pancreatic β-cells. An inhibitor of this receptor would be helpful in elucidating the physiological function of the glucose-sensing receptor. The present study was conducted to examine whether or not lactisole can be used as an inhibitor of the glucose-sensing receptor. In MIN6 cells, in a dose-dependent manner, lactisole inhibited insulin secretion induced by sweeteners, acesulfame-K, sucralose and glycyrrhizin. The IC50 was ∼4 mmol/l. Lactisole attenuated the elevation of cytoplasmic Ca2+ concentration ([Ca2+]c) evoked by sucralose and acesulfame-K but did not affect the elevation of intracellular cAMP concentration ([cAMP]c) induced by these sweeteners. Lactisole also inhibited the action of glucose in MIN6 cells. Thus, lactisole significantly reduced elevations of intracellular [NADH] and intracellular [ATP] induced by glucose, and also inhibited glucose-induced insulin secretion. To further examine the effect of lactisole on T1R3, we prepared HEK293 cells stably expressing mouse T1R3. In these cells, sucralose elevated both [Ca2+]c and [cAMP]c. Lactisole attenuated the sucralose-induced increase in [Ca2+]c but did not affect the elevation of [cAMP]c. Finally, lactisole inhibited insulin secretion induced by a high concentration of glucose in mouse islets. These results indicate that the mouse glucose-sensing receptor was inhibited by lactisole. Lactisole may be useful in assessing the role of the glucose-sensing receptor in mouse pancreatic β-cells.

  6. Restraint stress-induced reduction in prepulse inhibition in Brown Norway rats: Role of the CRF2 receptor

    PubMed Central

    Sutherland, Jane E.; Conti, Lisa H.

    2011-01-01

    Stress plays a role in many psychiatric disorders that are characterized by deficits in prepulse inhibition (PPI), a form of sensorimotor gating. Corticotropin-releasing factor (CRF) is one of the most important neurotransmitters involved in behavioral components of the stress response. Central infusion of CRF reduces PPI in both rats and mice. In mice, it has been shown that CRF1 receptor activation mediates the effect of exogenous CRF on PPI. However, the roles of the two CRF receptors in a stress-induced reduction in PPI are not known. We sought to determine whether CRF1 and/or CRF2 receptor blockade attenuates a stress-induced reduction of PPI in rats. In separate experiments, we assessed PPI in Brown Norway rats after exposure to five days of 2-hour restraint, and after pretreatment with the CRF1 receptor antagonist, CP-154,526 (20.0 mg/kg), or the CRF2 receptor antagonist, antisauvagine-30 (10.0 µg). Repeated, but not acute, restraint decreased PPI and attenuated the increase in PPI caused by repeated PPI testing. Blockade of the CRF1 receptor did not attenuate the effect of repeated restraint on PPI or grooming behavior. While CRF2 receptor blockade did attenuate the effect of repeated restraint on PPI, repeated ICV infusion of the selective CRF2 receptor agonist urocortin III, did not affect PPI. These findings demonstrate the effect of stress on sensorimotor gating and suggest that the CRF2 receptor mediates this effect in rats. PMID:21185316

  7. An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

    SciTech Connect

    Basu, Niladri; Stamler, Christopher J.; Loua, Kovana Marcel; Chan, H.M. . E-mail: laurie.chan@mcgill.ca

    2005-05-15

    Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl{sub 2}) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl{sub 2} and MeHg on [{sup 3}H]-quinuclidinyl benzilate ([{sup 3}H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B {sub max}) and ligand affinity (K {sub d}). Subsequently, samples were exposed to HgCl{sub 2} or MeHg to derive IC50 values and inhibition constants (K {sub i}). Results demonstrate that HgCl{sub 2} is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [{sup 3}H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies.

  8. Methanandamide allosterically inhibits in vivo the function of peripheral nicotinic acetylcholine receptors containing the alpha 7-subunit.

    PubMed

    Baranowska, Urszula; Göthert, Manfred; Rudz, Radoslaw; Malinowska, Barbara

    2008-09-01

    Methanandamide (MAEA), the stable analog of the endocannabinoid anandamide, has been proven in Xenopus oocytes to allosterically inhibit the function of the alpha7-nicotinic acetylcholine receptors (nAChRs) in a cannabinoid (CB) receptor-independent manner. The present study aimed at demonstrating that this mechanism can be activated in vivo. In anesthetized and vagotomized pithed rats treated with atropine, we determined the tachycardic response to electrical stimulation of preganglionic sympathetic nerves via the pithing rod or to i.v. nicotine (0.7 micromol/kg) activating nAChRs on the cardiac postganglionic sympathetic neurons. MAEA (3 and 10 micromol/kg) inhibited the electrically induced tachycardia (maximally by 15-20%; abolished by the CB(1) receptor antagonist AM 251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide]; 3 micromol/kg) in pentobarbitone-anesthetized pithed rats, but not in urethane-anesthetized pithed rats, which, thus, are suitable to study the CB(1) receptor-independent inhibition of nicotine-evoked tachycardia. The subunit-nonselective nAChR antagonist hexamethonium (100 micromol/kg) and the selective alpha7-subunit antagonist methyllycaconitine (MLA; 3 and 10 micromol/kg) decreased the nicotine-induced tachycardia by 100 and 40%, respectively (maximal effects), suggesting that nAChRs containing the alpha7-subunit account for 40% of the nicotine-induced tachycardia. MAEA (3 micromol/kg) produced an AM 251-insensitive inhibition (maximum again by 40%) of the nicotine-induced tachycardia. Simultaneous or sequential coadministration of MLA and MAEA inhibited the nicotine-induced tachycardia to the same extent (maximally by 40%) as each of the drugs alone. In conclusion, according to nonadditivity of the effects, MAEA mediates in vivo inhibition by the same receptors as MLA, namely alpha7-subunit-containing nAChRs, although at an allosteric instead of the orthosteric site.

  9. Ciproxifan, a histamine H3 receptor antagonist, reversibly inhibits monoamine oxidase A and B

    PubMed Central

    Hagenow, S.; Stasiak, A.; Ramsay, R. R.; Stark, H.

    2017-01-01

    Ciproxifan is a well-investigated histamine H3 receptor (H3R) inverse agonist/antagonist, showing an exclusively high species-specific affinity at rodent compared to human H3R. It is well studied as reference compound for H3R in rodent models for neurological diseases connected with neurotransmitter dysregulation, e.g. attention deficit hyperactivity disorder or Alzheimer’s disease. In a screening for potential monoamine oxidase A and B inhibition ciproxifan showed efficacy on both enzyme isoforms. Further characterization of ciproxifan revealed IC50 values in a micromolar concentration range for human and rat monoamine oxidases with slight preference for monoamine oxidase B in both species. The inhibition by ciproxifan was reversible for both human isoforms. Regarding inhibitory potency of ciproxifan on rat brain MAO, these findings should be considered, when using high doses in rat models for neurological diseases. As the H3R and monoamine oxidases are all capable of affecting neurotransmitter modulation in brain, we consider dual targeting ligands as interesting approach for treatment of neurological disorders. Since ciproxifan shows only moderate activity at human targets, further investigations in animals are not of primary interest. On the other hand, it may serve as starting point for the development of dual targeting ligands. PMID:28084411

  10. A novel monoclonal antibody targeting coxsackie virus and adenovirus receptor inhibits tumor growth in vivo

    PubMed Central

    Kawada, Manabu; Inoue, Hiroyuki; Kajikawa, Masunori; Sugiura, Masahito; Sakamoto, Shuichi; Urano, Sakiko; Karasawa, Chigusa; Usami, Ihomi; Futakuchi, Mitsuru; Masuda, Tohru

    2017-01-01

    To create a new anti-tumor antibody, we conducted signal sequence trap by retrovirus-meditated expression method and identified coxsackie virus and adenovirus receptor (CXADR) as an appropriate target. We developed monoclonal antibodies against human CXADR and found that one antibody (6G10A) significantly inhibited the growth of subcutaneous as well as orthotopic xenografts of human prostate cancer cells in vivo. Furthermore, 6G10A also inhibited other cancer xenografts expressing CXADR, such as pancreatic and colorectal cancer cells. Knockdown and overexpression of CXADR confirmed the dependence of its anti-tumor activity on CXADR expression. Our studies of its action demonstrated that 6G10A exerted its anti-tumor activity primarily through both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Moreover, 6G10A reacted with human tumor tissues, such as prostate, lung, and brain, each of which express CXADR. Although we need further evaluation of its reactivity and safety in human tissues, our results show that a novel anti-CXADR antibody may be a feasible candidate for cancer immunotherapy. PMID:28074864

  11. Dysregulation of Ack1 inhibits down-regulation of the EGF receptor

    SciTech Connect

    Grovdal, Lene Melsaether; Johannessen, Lene E.; Rodland, Marianne Skeie; Madshus, Inger Helene; Stang, Espen

    2008-04-01

    The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of {sup 125}I-EGF internalization, whereas internalization of {sup 125}I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized {sup 125}I-EGF was increased, while degradation of {sup 125}I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.

  12. Chemical inhibition of prometastatic lysyl-tRNA synthetase–laminin receptor interaction

    PubMed Central

    Kim, Dae Gyu; Lee, Jin Young; Kwon, Nam Hoon; Fang, Pengfei; Zhang, Qian; Wang, Jing; Young, Nicolas L.; Guo, Min; Cho, Hye Young; Mushtaq, AmeeqUl; Jeon, Young Ho; Choi, Jin Woo; Han, Jung Min; Kang, Ho Woong; Joo, Jae Eun; Hur, Youn; Kang, Wonyoung; Yang, Heekyoung; Nam, Do-Hyun; Lee, Mi-Sook; Lee, Jung Weon; Kim, Eun-Sook; Moon, Aree; Kim, Kibom; Kim, Doyeun; Kang, Eun Joo; Moon, Youngji; Rhee, Kyung Hee; Han, Byung Woo; Yang, Jee Sun; Han, Gyoonhee; Yang, Won Suk; Lee, Cheolju; Wang, Ming-Wei; Kim, Sunghoon

    2014-01-01

    Lysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds to KRS, impinged on interaction of KRS with 67LR and suppressed metastasis in 3 different mouse models. The compound inhibited KRS–67LR interaction in two ways. First, it directly blocked the association between KRS and 67LR. Second, it suppressed the dynamic movement of the N-terminal extension of KRS and reduced membrane localization of KRS. However, it did not affect the catalytic activity of KRS. Our results suggest that specific modulation of a cancer-related KRS–67LR interaction may offer a way to control metastasis while avoiding the toxicities associated with inhibition of the normal functions of KRS. PMID:24212136

  13. Phasic, Nonsynaptic GABA-A Receptor-Mediated Inhibition Entrains Thalamocortical Oscillations

    PubMed Central

    Rovó, Zita; Mátyás, Ferenc; Barthó, Péter; Slézia, Andrea; Lecci, Sandro; Pellegrini, Chiara; Astori, Simone; Dávid, Csaba; Hangya, Balázs

    2014-01-01

    GABA-A receptors (GABA-ARs) are typically expressed at synaptic or nonsynaptic sites mediating phasic and tonic inhibition, respectively. These two forms of inhibition conjointly control various network oscillations. To disentangle their roles in thalamocortical rhythms, we focally deleted synaptic, γ2 subunit-containing GABA-ARs in the thalamus using viral intervention in mice. After successful removal of γ2 subunit clusters, spontaneous and evoked GABAergic synaptic currents disappeared in thalamocortical cells when the presynaptic, reticular thalamic (nRT) neurons fired in tonic mode. However, when nRT cells fired in burst mode, slow phasic GABA-AR-mediated events persisted, indicating a dynamic, burst-specific recruitment of nonsynaptic GABA-ARs. In vivo, removal of synaptic GABA-ARs reduced the firing of individual thalamocortical cells but did not abolish slow oscillations or sleep spindles. We conclude that nonsynaptic GABA-ARs are recruited in a phasic manner specifically during burst firing of nRT cells and provide sufficient GABA-AR activation to control major thalamocortical oscillations. PMID:24849349

  14. Angiotensin receptor neprilysin inhibition in heart failure: mechanistic action and clinical impact.

    PubMed

    Buggey, Jonathan; Mentz, Robert J; DeVore, Adam D; Velazquez, Eric J

    2015-09-01

    Heart failure (HF) is an increasingly common syndrome associated with high mortality and economic burden, and there has been a paucity over the past decade of new pharmacotherapies that improve outcomes. However, recent data from a large randomized controlled trial compared the novel agent LCZ696, a dual-acting angiotensin receptor blocker and neprilysin inhibitor (ARNi), with the well established angiotensin-converting enzyme (ACE) inhibitor enalapril and found significant reduction in mortality among the chronic reduced ejection fraction HF population. Preclinical and clinical data suggest that neprilysin inhibition provides beneficial outcomes in HF patients by preventing the degradation of natriuretic peptides and thereby promoting natriuresis and vasodilatation and counteracting the negative cardiorenal effects of the up-regulated renin-angiotensin-aldosterone system. Agents such as omapatrilat combined neprilysin and ACE inhibition but had increased rates of angioedema. Goals of an improved safety profile provided the rationale for the development of the ARNi LCZ696. Along with significant reductions in mortality and hospitalizations, clinical trials suggest that LCZ696 may improve surrogate markers of HF severity. In this paper, we review the preclinical and clinical data that led to the development of LCZ696, the understanding of the underlying mechanistic action, and the robust clinical impact that LCZ696 may have in the near future.

  15. Devazepide, a nonpeptide antagonist of CCK receptors, induces apoptosis and inhibits Ewing tumor growth.

    PubMed

    Carrillo, Jaime; Agra, Noelia; Fernández, Noemí; Pestaña, Angel; Alonso, Javier

    2009-08-01

    The Ewing family of tumors is a group of highly malignant tumors that mainly arise in bone and most often affect children and young adults in the first two decades of life. Despite the use of multimodal therapy, the long-term disease-free survival rate of patients with Ewing tumors is still disappointingly low, making the discovery of innovative therapeutic strategies all the more necessary. We have recently shown that cholecystokinin (CCK), a neuroendocrine peptide, involved in many biological functions, including cell growth and proliferation, is a relevant target of the EWS/FLI1 oncoprotein characteristic of Ewing tumors. CCK silencing inhibits cell proliferation and tumor growth in vivo, suggesting that CCK acts as an autocrine growth factor for Ewing cells. Here, we analyzed the impact of two CCK receptor antagonists, devazepide (a CCK1-R antagonist) and L365 260 (a CCK2-R antagonist), on the growth of Ewing tumor cells. Devazepide (10 micromol/l) inhibited cell growth of four different Ewing tumor cells in vitro (range 85-88%), whereas the effect of the CCK2-R antagonist on cell growth was negligible. In a mouse tumor xenograft model, devazepide reduced tumor growth by 40%. Flow cytometry experiments showed that devazepide, but not L365 260, induced apoptosis of Ewing tumor cells. In summary, devazepide induces cell death of Ewing tumor cells, suggesting that it could represent a new therapeutic approach in the management of Ewing's tumor patients.

  16. Ciproxifan, a histamine H3 receptor antagonist, reversibly inhibits monoamine oxidase A and B.

    PubMed

    Hagenow, S; Stasiak, A; Ramsay, R R; Stark, H

    2017-01-13

    Ciproxifan is a well-investigated histamine H3 receptor (H3R) inverse agonist/antagonist, showing an exclusively high species-specific affinity at rodent compared to human H3R. It is well studied as reference compound for H3R in rodent models for neurological diseases connected with neurotransmitter dysregulation, e.g. attention deficit hyperactivity disorder or Alzheimer's disease. In a screening for potential monoamine oxidase A and B inhibition ciproxifan showed efficacy on both enzyme isoforms. Further characterization of ciproxifan revealed IC50 values in a micromolar concentration range for human and rat monoamine oxidases with slight preference for monoamine oxidase B in both species. The inhibition by ciproxifan was reversible for both human isoforms. Regarding inhibitory potency of ciproxifan on rat brain MAO, these findings should be considered, when using high doses in rat models for neurological diseases. As the H3R and monoamine oxidases are all capable of affecting neurotransmitter modulation in brain, we consider dual targeting ligands as interesting approach for treatment of neurological disorders. Since ciproxifan shows only moderate activity at human targets, further investigations in animals are not of primary interest. On the other hand, it may serve as starting point for the development of dual targeting ligands.

  17. Ligand independent aryl hydrocarbon receptor inhibits lung cancer cell invasion by degradation of Smad4.

    PubMed

    Lee, Chen-Chen; Yang, Wen-Hao; Li, Ching-Hao; Cheng, Yu-Wen; Tsai, Chi-Hao; Kang, Jaw-Jou

    2016-07-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent-activated transcriptional factor that regulates the metabolism of xenobiotic and endogenous compounds. Although AhR plays a crucial role in air toxicant-induced carcinogenesis, AhR expression was shown to negatively regulate tumorigenesis. Therefore, in the present study, we investigated the effect of AhR without ligand treatment on cancer invasion in lung cancer cell lines. Lung cancer cells expressing lower levels of AhR showed higher invasion ability (H1299 cells) compared with cells expressing higher levels of AhR (A549 cells). Overexpression of AhR in H1299 cells inhibited the invasion ability. We found that vimentin expression was inhibited in AhR-overexpressing H1299 cells. Additionally, the expression of EMT-related transcriptional factors Snail and ID-1 decreased. Interestingly, we found that Smad4 degradation was induced in AhR-overexpressing H1299 cells. Our data showed that AhR could interact with Jun-activation domain binding protein (Jab1) and Smad4, which may cause degradation of Smad4 by the proteasome. Our data suggest that AhR affects the transforming growth factor-β signaling pathway by inducing Smad4 degradation by the proteasome and suppressing tumor metastasis via epithelial to mesenchymal transition reduction in lung cancer cells.

  18. Mode of action of triflumezopyrim: A novel mesoionic insecticide which inhibits the nicotinic acetylcholine receptor.

    PubMed

    Cordova, Daniel; Benner, Eric A; Schroeder, Mark E; Holyoke, Caleb W; Zhang, Wenming; Pahutski, Thomas F; Leighty, Robert M; Vincent, Daniel R; Hamm, Jason C

    2016-07-01

    Triflumezopyrim, a newly commercialized molecule from DuPont Crop Protection, belongs to the novel class of mesoionic insecticides. This study characterizes the biochemical and physiological action of this novel insecticide. Using membranes from the aphid, Myzus persicae, triflumezopyrim was found to displace (3)H-imidacloprid with a Ki value of 43 nM with competitive binding results indicating that triflumezopyrim binds to the orthosteric site of the nicotinic acetylcholine receptor (nAChR). In voltage clamp studies using dissociated Periplaneta americana neurons, triflumezopyrim inhibits nAChR currents with an IC50 of 0.6 nM. Activation of nAChR currents was minimal and required concentrations ≥100 μM. Xenopus oocytes expressing chimeric nAChRs (Drosophila α2/chick β2) showed similar inhibitory effects from triflumezopyrim. In P. americana neurons, co-application experiments with acetylcholine reveal the inhibitory action of triflumezopyrim to be rapid and prolonged in nature. Such physiological action is distinct from other insecticides in IRAC Group 4 in which the toxicological mode of action is attributed to nAChR agonism. Mesoionic insecticides act via inhibition of the orthosteric binding site of the nAChR despite previous beliefs that such action would translate to poor insect control. Triflumezopyrim is the first commercialized insecticide from this class and provides outstanding control of hoppers, including the brown planthopper, Nilaparvata lugens, which is already displaying strong resistance to neonicotinoids such as imidacloprid.

  19. Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury.

    PubMed

    Zhang, Si; Ju, Peijun; Tjandra, Editha; Yeap, Yeeshan; Owlanj, Hamed; Feng, Zhiwei

    2016-10-01

    Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of OX42 + microglial cells and glial fibrillary acidic protein + astrocytes in damaged area of spinal cords. We thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages.

  20. Discovery of a novel allosteric modulator of 5-HT3 receptors: inhibition and potentiation of Cys-loop receptor signaling through a conserved transmembrane intersubunit site.

    PubMed

    Trattnig, Sarah M; Harpsøe, Kasper; Thygesen, Sarah B; Rahr, Louise M; Ahring, Philip K; Balle, Thomas; Jensen, Anders A

    2012-07-20

    The ligand-gated ion channels in the Cys-loop receptor superfamily mediate the effects of neurotransmitters acetylcholine, serotonin, GABA, and glycine. Cys-loop receptor signaling is susceptible to modulation by ligands acting through numerous allosteric sites. Here we report the discovery of a novel class of negative allosteric modulators of the 5-HT(3) receptors (5-HT(3)Rs). PU02 (6-[(1-naphthylmethyl)thio]-9H-purine) is a potent and selective antagonist displaying IC(50) values of ~1 μM at 5-HT(3)Rs and substantially lower activities at other Cys-loop receptors. In an elaborate mutagenesis study of the 5-HT(3)A receptor guided by a homology model, PU02 is demonstrated to act through a transmembrane intersubunit site situated in the upper three helical turns of TM2 and TM3 in the (+)-subunit and TM1 and TM2 in the (-)-subunit. The Ser(248), Leu(288), Ile(290), Thr(294), and Gly(306) residues are identified as important molecular determinants of PU02 activity with minor contributions from Ser(292) and Val(310), and we propose that the naphthalene group of PU02 docks into the hydrophobic cavity formed by these. Interestingly, specific mutations of Ser(248), Thr(294), and Gly(306) convert PU02 into a complex modulator, potentiating and inhibiting 5-HT-evoked signaling through these mutants at low and high concentrations, respectively. The PU02 binding site in the 5-HT(3)R corresponds to allosteric sites in anionic Cys-loop receptors, which emphasizes the uniform nature of the molecular events underlying signaling through the receptors. Moreover, the dramatic changes in the functional properties of PU02 induced by subtle changes in its binding site bear witness to the delicate structural discrimination between allosteric inhibition and potentiation of Cys-loop receptors.

  1. In vitro inhibition of methadone and oxycodone cytochrome P450-dependent metabolism: reversible inhibition by H2-receptor agonists and proton-pump inhibitors.

    PubMed

    Moody, David E; Liu, Fenyun; Fang, Wenfang B

    2013-10-01

    In vitro inhibition of oxycodone metabolism to noroxycodone and oxymorphone and R- and S-methadone metabolism to R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was measured for four H2-receptor antagonists and five proton-pump inhibitors (PPIs) using human liver microsomes (HLM) and cDNA-expressed human cytochrome P450s (rCYPs). Inhibitors were first incubated with HLM at three concentrations with and without preincubation of inhibitor, enzyme source and reducing equivalents to also screen for time-dependent inhibition (TDI). Cimetidine and famotidine (10-1,000 µM) inhibited all the four pathways >50%. Nizatidine and ranitidine did not. All the five PPIs (1-200 µM) inhibited one or more pathways >50%. Half maximal inhibitory concentrations (IC50s) were then determined using rCYPs. Cimetidine and famotidine both inhibited CYP3A4-mediated formation of noroxycodone and CYP2D6-mediated formation of oxymorphone, and famotidine inhibited CYP3A4-mediated formation of R- and S-EDDP, but IC50s were so high that only >10× therapeutic concentrations may have potential for reversible in vivo inhibition. The PPIs were more potent inhibitors; many have the potential for reversible in vivo inhibition at therapeutic concentrations. Omeprazole, esomeprazole and pantoprazole had greater effects on CYP3A4-mediated reactions, whereas lansoprazole was selective for CYP2D6-mediated formation of oxymorphone. Preincubation enhanced cimetidine inhibition of noroxycodone formation and rabeprazole inhibition of all pathways. Future studies will explore irreversible TDI.

  2. Axl receptor tyrosine kinase is a novel target of apigenin for the inhibition of cell proliferation.

    PubMed

    Kim, Kyung-Chan; Choi, Eun-Ha; Lee, Chuhee

    2014-08-01

    The Axl receptor tyrosine kinase (RTK), along with Tyro 3 and Mer, belongs to the TAM subfamily that promotes survival, stimulates proliferation and/or inhibits apoptosis. In various types of human cancer, including breast, lung and prostate cancer, Axl expression is increased and correlates with an advanced clinical stage. In this study, we examined whether apigenin has an effect on Axl expression, which in turn can affect cell proliferation. The treatment of the non‑small cell lung cancer (NSCLC) cells, A549 and H460, with apigenin decreased Axl mRNA and protein expression in a dose‑dependent manner. Axl promoter activity was also inhibited by apigenin, indicating that apigenin suppressed Axl expression at the transcriptional level. Upon treatment with apigenin, the viability of both the A549 and H460 cells was gradually decreased and the anti-proliferative effects were further confirmed by the dose‑dependent decrease in the clonogenic ability of the apigenin‑treated cells. Subsequently, we found that the viability and clonogenic ability of the cells treated with apigenin was less or more affected by transfection of the cells with a Axl-expressing plasmid or Axl targeting siRNA, compared to transfection with the empty vector or control siRNA, respectively. In addition, apigenin increased the expression of p21, a cyclin-dependent kinase inhibitor, but reduced the expression of X-linked inhibitor of apoptosis protein (XIAP). These cell cycle arrest and pro-apoptotic effects of apigenin were also attenuated or augmented by the up- or downregulation of Axl expression, respectively, which suggests that Axl is a novel target of apigenin through which it exerts its inhibitory effects on cell proliferation. Taken together, our data indicate that apigenin downregulates Axl expression, which subsequently results in the inhibition of NSCLC cell proliferation through the increase and decrease of p21 and XIAP expression, respectively.

  3. Progestin inhibition of progesterone receptor gene expression in human breast cancer cells.

    PubMed

    Alexander, I E; Clarke, C L; Shine, J; Sutherland, R L

    1989-09-01

    The present study was designed to investigate whether inhibition of progesterone receptor (PR) gene transcription and/or regulation of PR mRNA half-life were involved in the progestin-mediated decrease of PR in T-47D human breast cancer cells. Cells were treated with the progestin ORG 2058 and PR mRNA measured by Northern blot analysis of total RNA. A major PR mRNA around 13.5 kilobases and minor species around the 28S ribosomal RNA subunit were decreased upon ORG 2058 treatment. The decrease was not detectable until 2-3 h after treatment and was the same at all ORG 2058 concentrations (1-100 nM) tested. The decrease in PR mRNA was unaffected by actinomycin D in the first 3 h but was inhibited thereafter. There was a partial recovery of PR mRNA levels 24 h after ORG 2058 exposure. Immunoblot analysis showed that immunoreactive PR decreased in parallel with PR mRNA. The rate of protein loss in the first 12 h after progestin treatment was related to the ORG 2058 concentration used. Nuclear run-on experiments showed that ORG 2058 caused a decrease of up to 70% in the transcription rate of the PR gene. The half-life of PR mRNA was shown to be 2-2.5 h by [3H]uridine incorporation, which was much shorter than estimates obtained using actinomycin D, and was unaffected by ORG 2058. In summary, these data have shown that the mechanism by which progestins decrease the concentration of PR includes inhibition of transcription of the PR gene.

  4. Glucose inhibition of epinephrine stimulation of hepatic gluconeogenesis by blockade of the alpha-receptor function.

    PubMed

    Kneer, N M; Bosch, A L; Clark, M G; Lardy, H A

    1974-11-01

    For isolated rat hepatocytes, glucagon, 3':5'-cyclic AMP, 3':5'-cyclic GMP, and epinephrine stimulate the rate of gluconeogenesis from substrates not involving pathways of mitochondrial metabolism. From estimation of the rates of glucose formation, fructose 6-phosphate phosphorylation, and lactate and pyruvate formation it is concluded that epinephrine and 3':5'-cyclic GMP stimulate gluconeogenesis from either galactose or fructose by influencing the rate of reactions involving fructose 6-phosphate in a manner similar to that already reported for glucagon and 3':5'-cyclic AMP. Each agent acts to inhibit flux through phosphofructokinase (EC 2.7.1.11) and enhance flux through fructose diphosphatase (EC 3.1.3.11), resulting in the re-direction of carbon from lactate and pyruvate formation to glucose synthesis. In addition to 3':5'-cyclic GMP, dibutyryl 3':5'-cyclic GMP, 8-bromo 3':5'-cyclic GMP, 8-benzyl-thio 3':5'-cyclic GMP and 8-(4-chlorophenyl)thio 3':5'-cyclic GMP stimulate glucose formation and inhibit lactate and pyruvate formation from galactose. Guanosine monophosphate and 2':3'-cyclic GMP are inactive. As the stimulatory effect of epinephrine is inhibited by phenoxybenzamine and not by propranolol, and is not simulated by isoproterenol, it is concluded that catecholamine activity is expressed through the alpha-receptor. Increased extracellular glucose concentration (>10 mM) decreases the stimulatory effect of epinephrine, 3':5'-cyclic GMP, and partially that of 3':5'-cyclic AMP but does not alter the efficacy of glucagon.

  5. Novel CC chemokine receptor 4 antagonist RS-1154 inhibits ovalbumin-induced ear swelling in mice.

    PubMed

    Nakagami, Yasuhiro; Kawashima, Kayo; Yonekubo, Kazuki; Etori, Maki; Jojima, Takaaki; Miyazaki, Shojiro; Sawamura, Ryoko; Hirahara, Kazuki; Nara, Futoshi; Yamashita, Makoto

    2009-12-10

    CC chemokine ligand 17 (CCL17/thymus and activation-regulated chemokine: TARC) and CCL22 (macrophage-derived chemokine: MDC) selectively bind to CC chemokine receptor 4 (CCR4). The CCR4 system is considered to be responsible for the pathology of allergic diseases such as atopic dermatitis. To find and develop potential medicines against allergic diseases, we screened an in-house library to search for compounds having a profile as a CCR4 antagonist. From among the screening hits, we focused on 3-{2-[(2R)-2-phenyl-4-(4-pyridin-4-ylbenzyl)morpholin-2-yl]ethyl}quinazoline-2,4(1H,3H)-dione (named RS-1154), which had been newly synthesized in our laboratory. This compound inhibited the binding of [(125)I]CCL17 to human CCR4-expressing CHO cells with an IC(50) value of 27.7 nM and moreover inhibited CCL17-induced migration of DO11.10 mice-derived T helper 2 cells with an IC(50) value of 1.5 nM in vitro. We then examined the effect of RS-1154 in an ovalbumin-induced ear swelling assay. The ear thickness was decreased by intravenous administration of anti-CCL17 or anti-CCL22 antibodies, suggesting that the CCR4 system is involved in the ear swelling. Though partially, the oral administration of RS-1154 also significantly ameliorated the ear swelling at the doses of 30 and 100 mg/kg. Furthermore, the serum level of interleukin-4 decreased after the administration of RS-1154. In this study, we succeeded in obtaining a newly-synthesized compound, RS-1154, which has a potential to inhibit the chemotaxis of T helper 2 cells in vitro and to ameliorate ovalbumin-induced ear swelling in vivo. These results raise the possibility that RS-1154 or one of derivatives might become a therapeutic agent for atopic dermatitis patients.

  6. Detection, inhibition and disintegration of amyloid fibrils: the role of optical probes and macrocyclic receptors.

    PubMed

    Bhasikuttan, Achikanath C; Mohanty, Jyotirmayee

    2017-02-20

    Amyloid fibrils are formed by the aberrant aggregation of proteins into highly ordered β-sheet structures and are believed to be the root cause of several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Prion diseases, etc. and have been the subject of extensive biochemical, biophysical and clinical studies. Developing methods for the early detection of fibril formation using optical spectroscopic techniques and inhibition/disintegration of amyloid fibrils/plaques by introducing small molecules have been a major challenge to establish a clinically facile therapeutic intervention to combat these neurodegenerative diseases. This feature article provides an account of the recent reports from different research groups, including ours, on the optical detection, and inhibition/disintegration of mature fibrils using fluorescent probes and macrocyclic hosts such as cucurbiturils, calixarenes and cyclodextrins. Site specific or spectrally distinct fluorescence emission from a large number of fluorophores in a broad spectral region has been used to detect the fibrillation of different proteins/peptides, mainly insulin, α-synuclein, transthyretin, barstar, lysozyme, Aβ40 peptide, etc. On the one hand, while macrocyclic receptors modify the inter-protein interactions through molecular recognition of amino acid residues leading to the inhibition of amyloid fibrillation, on the other hand, one of the cavitands, p-sulfonatocalixarenes, has been demonstrated to cause the disintegration of mature fibrils, effectively through surface charge interactions, which destabilize the extended fibrillar structure into soluble or fine particles. Beneficially, the presence of extrinsic p-sulfonatocalix[4/6]arenes did not introduce any additional toxicity to the cell viability, which advocates its potential utility as a therapeutic for amyloidosis.

  7. Pharmacologic retinoid signaling and physiologic retinoic acid receptor signaling inhibit basal cell carcinoma tumorigenesis

    PubMed Central

    So, Po-Lin; Fujimoto, Michele A.; Epstein, Ervin H.

    2015-01-01

    Basal cell carcinoma (BCC) is the most common human cancer. Patients with basal cell nevus syndrome (Gorlin syndrome) are highly susceptible to developing many BCCs as a result of a constitutive inactivating mutation in one allele of PATCHED 1, which encodes a tumor suppressor that is a major inhibitor of Hedgehog signaling. Dysregulated Hedgehog signaling is a common feature of both hereditary and sporadic BCCs. Recently, we showed remarkable anti-BCC chemopreventive efficacy of tazarotene, a retinoid with retinoic acid receptor (RAR) β/γ specificity, in Ptch1 +/− mice when treatment was commenced before carcinogenic insults. In this study, we assessed whether the effect of tazarotene against BCC carcinogenesis is sustained after its withdrawal and whether tazarotene is effective against preexisting microscopic BCC lesions. We found that BCCs did not reappear for at least 5 months after topical drug treatment was stopped and that already developed, microscopic BCCs were susceptible to tazarotene inhibition. In vitro, tazarotene inhibited a murine BCC keratinocyte cell line, ASZ001, suggesting that its effect in vivo is by direct action on the actual tumor cells. Down-regulation of Gli1, a target gene of Hedgehog signaling and up-regulation of CRABPII, a target gene of retinoid signaling, were observed with tazarotene treatment. Finally, we investigated the effects of topical applications of other retinoid-related compounds on BCC tumorigenesis in vivo. Tazarotene was the most effective of the preparations studied, and its effect most likely was mediated by RARγ activation. Furthermore, inhibition of basal RAR signaling in the skin promoted BCC carcinogenesis, suggesting that endogenous RAR signaling restrains BCC growth. PMID:18483315

  8. The novel Aryl hydrocarbon receptor inhibitor biseugenol inhibits gastric tumor growth and peritoneal dissemination

    PubMed Central

    Lai, De-Wei; Karlsson, Anna Isabella; Wang, Keh-Bin; Chen, Yi-Ching; Shen, Chin-Chang; Wu, Sheng-Mao; Liu, Chia-Yu; Tien, Hsing-Ru; Peng, Yen-Chun; Jan, Yee-Jee; Chao, Te-Hsin; Lan, Keng-Hsin; Arbiser, Jack L.; Sheu, Meei-Ling

    2014-01-01

    Biseugenol (Eug) is known to antiproliferative of cancer cells; however, to date, the antiperitoneal dissemination effects have not been studied in any mouse cancer model. In this study, Aryl hydrocarbon receptor (AhR) expression was associated with lymph node and distant metastasis in patients with gastric cancer and was correlated with clinicolpathological pattern. We evaluated the antiperitoneal dissemination potential of knockdown AhR and Biseugenol in cancer mouse model and assessed mesenchymal characteristics. Our results demonstrate that tumor growth, peritoneal dissemination and peritoneum or organ metastasis implanted MKN45 cells were significantly decreased in shAhR and Biseugenol-treated mice and that endoplasmic reticulum (ER) stress was caused. Biseugenol-exposure tumors showed acquired epithelial features such as phosphorylation of E-cadherin, cytokeratin-18 and loss mesenchymal signature Snail, but not vimentin regulation. Snail expression, through AhR activation, is an epithelial-to-mesenchymal transition (EMT) determinant. Moreover, Biseugenol enhanced Calpain-10 (Calp-10) and AhR interaction resulted in Snail downregulation. The effect of shCalpain-10 in cancer cells was associated with inactivation of AhR/Snail promoter binding activity. Inhibition of Calpain-10 in gastric cancer cells by short hairpin RNA or pharmacological inhibitor was found to effectively reduced growth ability and vessel density in vivo. Importantly, knockdown of AhR completed abrogated peritoneal dissemination. Herein, Biseugenol targeting ER stress provokes Calpain-10 activity, sequentially induces reversal of EMT and apoptosis via AhR may involve the paralleling processes. Taken together, these data suggest that Calpain-10 activation and AhR inhibition by Biseugenol impedes both gastric tumor growth and peritoneal dissemination by inducing ER stress and inhibiting EMT. PMID:25226618

  9. Inhibition of melanocortin 1 receptor slows melanoma growth, reduces tumor heterogeneity and increases survival

    PubMed Central

    Kansal, Rita G.; McCravy, Matthew S.; Basham, Jacob H.; Earl, Joshua A.; McMurray, Stacy L.; Starner, Chelsey J.

    2016-01-01

    Melanoma risk is increased in patients with mutations of melanocortin 1 receptor (MC1R) yet the basis for the increased risk remains unknown. Here we report in vivo evidence supporting a critical role for MC1R in regulating melanoma tumor growth and determining overall survival time. Inhibition of MC1R by its physiologically relevant competitive inhibitor, agouti signaling protein (ASIP), reduced melanin synthesis and morphological heterogeneity in murine B16-F10 melanoma cells. In the lungs of syngeneic C57BL/6 mice, mCherry-marked, ASIP-secreting lung tumors inhibited MC1R on neighboring tumors lacking ASIP in a dose dependent manner as evidenced by a proportional loss of pigment in tumors from mice injected with 1:1, 3:1 and 4:1 mixtures of parental B16-F10 to ASIP-expressing tumor cells. ASIP-expressing B16-F10 cells formed poorly pigmented tumors in vivo that correlated with a 20% longer median survival than those bearing parental B16-F10 tumors (p=0.0005). Mice injected with 1:1 mixtures also showed survival benefit (p=0.0054), whereas injection of a 4:1 mixture showed no significant difference in survival. The longer survival time of mice bearing ASIP-expressing tumors correlated with a significantly slower growth rate than parental B16-F10 tumors as judged by quantification of numbers of tumors and total tumor load (p=0.0325), as well as a more homogeneous size and morphology of ASIP-expressing lung tumors. We conclude that MC1R plays an important role in regulating melanoma growth and morphology. Persistent inhibition of MC1R provided a significant survival advantage resulting in part from slower tumor growth, establishing MC1R as a compelling new molecular target for metastatic melanoma. PMID:27028866

  10. Suppression of calcium-sensing receptor ameliorates cardiac hypertrophy through inhibition of autophagy

    PubMed Central

    LIU, LEI; WANG, CHAO; LIN, YAN; XI, YUHUI; LI, HONG; SHI, SA; LI, HONGZHU; ZHANG, WEIHUA; ZHAO, YAJUN; TIAN, YE; XU, CHANGQING; WANG, LINA

    2016-01-01

    The calcium-sensing receptor (CaSR) releases intracellular calcium ([Ca2+]i) by accumulating inositol phosphate. Changes in [Ca2+]i initiate myocardial hypertrophy. Furthermore, autophagy associated with [Ca2+]i. Autophagy has previously been demonstrated to participate in the hypertrophic process. The current study investigated whether suppression of CaSR affects the hypertrophic response via modulating autophagy. Isoproterenol (ISO) was used to induce cardiac hypertrophy in Wistar rats. Hypertrophic status was determined by echocardiographic assessment, hematoxylin and eosin, and Masson's staining. The protein expression levels of CaSR and autophagy level were observed. Changes of hypertrophy and autophagy indicators were observed following intravenous injection of a CaSR inhibitor. An ISO-induced cardiomyocyte hypertrophy model was established and used determine the involvement of GdCl3. [Ca2+]i was determined using Fluo-4/AM dye followed by confocal microscopy. The expression levels of various active proteins were analyzed by western blotting. The size of the heart, expression levels of CaSR and autophagy level were markedly increased in hypertrophic myocardium. In addition, the present study demonstrated that the indicators of hypertrophy and autophagy were effectively suppressed by CaSR inhibitor. Furthermore, similar effects were demonstrated in neonatal rat hypertrophic cardiomyocytes treated with ISO. It was also observed that CaSR regulates the Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ)-AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling pathway induced by ISO in cardiomyocytes. Furthermore, the AMPK inhibition significantly reduced the autophagy level following CaSR stimulation (P<0.05). The results of the present demonstrated that inhibition of CaSR may ameliorate cardiac hypertrophy induced by ISO and the effect may be associated with the inhibition of autophagy and suppression of the Ca

  11. Niclosamide inhibits androgen receptor variants expression and overcomes enzalutamide resistance in castration resistant prostate cancer

    PubMed Central

    Zhu, Yezi; Nadiminty, Nagalakshmi; Schwartz, Chad T.; Evans, Christopher P.; Gao, Allen C.

    2014-01-01

    Purpose Enzalutamide, a second-generation antiandrogen, was recently approved for the treatment of castration-resistant prostate cancer (CRPC) in patients who no longer respond to docetaxel. Despite these advances that provide temporary respite, resistance to enzalutamide occurs frequently. AR splice variants such as AR-V7 have recently been shown to drive castration resistant growth and resistance to enzalutamide. This study was designed to identify inhibitors of AR variants and test its ability to overcome resistance to enzalutamide. Experimental Design The drug screening was conducted using luciferase activity assay to determine the activity of AR-V7 after treatment with the compounds in the Prestwick Chemical Library, which contains about 1120 FDA-approved drugs. The effects of the identified inhibitors on AR-V7 activity and enzalutamide sensitivity were characterized in CRPC and enzalutamide-resistant prostate cancer cells in vitro and in vivo. Results Niclosamide, an FDA-approved anti-helminthic drug, was identified as a potent AR-V7 inhibitor in prostate cancer cells. Niclosamide significantly downregulated AR-V7 protein expression by protein degradation through a proteasome dependent pathway. Niclosamide also inhibited AR-V7 transcription activity and reduced the recruitment of AR-V7 to the PSA promoter. Niclosamide inhibited prostate cancer cell growth in vitro and tumor growth in vivo. Furthermore, the combination of niclosamide and enzalutamide resulted in significantly inhibition of enzalutamide-resistant tumor growth, suggesting that Niclosamide enhances enzalutamide therapy and overcomes enzalutamide resistance in castration resistant prostate cancer cells. Conclusions Niclosamide was identified as a novel inhibitor of AR variants. Our findings offer preclinical validation of niclosamide as a promising inhibitor of androgen receptor variants to treat, either alone or in combination with current antiandrogen therapies, advanced prostate cancer patients

  12. P2X7 receptor inhibition protects against ischemic acute kidney injury in mice.

    PubMed

    Yan, Yanli; Bai, Jianwen; Zhou, Xiaoxu; Tang, Jinhua; Jiang, Chunming; Tolbert, Evelyn; Bayliss, George; Gong, Rujun; Zhao, Ting C; Zhuang, Shougang

    2015-03-15

    Activation of the purinergic P2X7 receptor (P2X7R) has been associated with the development of experimental nephritis and diabetic and hypertensive nephropathy. However, its role in acute kidney injury (AKI) remains unknown. In this study, we examined the effects of P2X7R inhibition in a murine model of ischemia-reperfusion (I/R)-induced AKI using A438079, a selective inhibitor of P2X7R. At 24 h after I/R, mice developed renal dysfunction and renal tubular damage, which was accompanied by elevated expression of P2X7R. Early administration of A438079 immediately or 6 h after the onset of reperfusion protected against renal dysfunction and attenuated kidney damage whereas delayed administration of A438079 at 24 h after restoration of perfusion had no protective effects. The protective actions of A438079 were associated with inhibition of renal tubule injury and cell death and suppression of renal expression of monocyte chemotactic protein-1 and regulated upon expression normal T cell expressed and secreted (RANTES). Moreover, I/R injury led to an increase in phosphorylation (activation) of extracellular signal-regulated kinases 1/2 in the kidney; treatment with A438079 diminished this response. Collectively, these results indicate that early P2X7R inhibition is effective against renal tubule injury and proinflammatory response after I/R injury and suggest that targeting P2X7R may be a promising therapeutic strategy for treatment of AKI.

  13. Soluble complement receptor 1 inhibits both complement and granulocyte activation during ex vivo hemodialysis.

    PubMed

    Himmelfarb, J; McMonagle, E; Holbrook, D; Toth, C

    1995-10-01

    Hemodialysis with cellulosic membranes results in both complement and granulocyte activation. We investigated the effects of soluble complement receptor 1 (sCR1), a potent complement inhibitor, on both complement and granulocyte activation in an ex vivo model of dialysis. Measurements were made of complement activation (radioimmunoassay for C3a desArg) as well as granulocyte activation (flow cytometric measurements of reactive oxygen species production, granulocyte CD11b/CD18 (MAC-1) expression and CD62L (L-selectin) expression). sCR1 completely abolished the generation of plasma C3a desArg during ex vivo hemodialysis. Without sCR1, C3a desArg levels rose from 968 +/- 373 ng/ml to 4961 +/- 40 ng/ml by the end of the ex vivo procedure (p < 0.001). sCR1 also completely inhibited MAC-1 upregulation and L-selectin shedding from granulocytes during ex vivo hemodialysis. With sCR1 there was still a statistically significant increase in granulocyte reactive oxygen species production (from 2.42 +/- 0.1 fluorescence channels to 6.47 +/- 0.7 fluorescence channels, p < 0.01) but a 50% inhibition when compared with experiments without sCR1 (3.15 +/- 0.5 to 11.2 +/- 1.9, p < 0.01). We conclude that sCR1 completely abolishes complement activation and changes in granulocyte cell adhesion molecules during ex vivo hemodialysis with cellulosic membranes. sCR1 partially inhibits granulocyte reactive oxygen species formation.

  14. Bosentan, a mixed endothelin receptor antagonist, inhibits superoxide anion-induced pain and inflammation in mice.

    PubMed

    Serafim, Karla G G; Navarro, Suelen A; Zarpelon, Ana C; Pinho-Ribeiro, Felipe A; Fattori, Victor; Cunha, Thiago M; Alves-Filho, Jose C; Cunha, Fernando Q; Casagrande, Rubia; Verri, Waldiceu A

    2015-11-01

    Bosentan is a mixed endothelin receptor antagonist widely used to treat patients with pulmonary arterial hypertension, and the emerging literature suggests bosentan as a potent anti-inflammatory drug. Superoxide anion is produced in large amounts during inflammation, stimulates cytokine production, and thus contributes to inflammation and pain. However, it remains to be determined whether endothelin contributes to the inflammatory response triggered by the superoxide anion. The present study investigated the effects of bosentan in a mouse model of inflammation and pain induced by potassium superoxide, a superoxide anion donor. Male Swiss mice were treated with bosentan (10-100 mg/kg) by oral gavage, 1 h before potassium superoxide injection, and the inflammatory response was evaluated locally and at spinal cord (L4-L6) levels. Bosentan (100 mg/kg) inhibited superoxide anion-induced mechanical and thermal hyperalgesia, overt pain-like behavior (abdominal writhings, paw flinching, and licking), paw edema, myeloperoxidase activity (neutrophil marker) in the paw skin, and leukocyte recruitment in the peritoneal cavity. Bosentan also inhibited superoxide anion-induced interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) production, while it enhanced IL-10 production in the paw skin and spinal cord. Bosentan inhibited the reduction of antioxidant capacity (reduced glutathione, ferric reducing antioxidant power, and ABTS radical scavenging ability) induced by the superoxide anion. Finally, we demonstrated that intraplantar injection of potassium superoxide induces the mRNA expression of prepro-endothelin-1 in the paw skin and spinal cord. In conclusion, our results demonstrated that superoxide anion-induced inflammation, pain, cytokine production, and oxidative stress depend on endothelin; therefore, these responses are amenable to bosentan treatment.

  15. Raloxifene inhibits cloned Kv4.3 channels in an estrogen receptor-independent manner.

    PubMed

    Chae, Yun Ju; Kim, Dae Hun; Lee, Hong Joon; Sung, Ki-Wug; Kwon, Oh-Joo; Hahn, Sang June

    2015-08-01

    Raloxifene is widely used for the treatment and prevention of postmenopausal osteoporosis. We examined the effects of raloxifene on the Kv4.3 currents expressed in Chinese hamster ovary (CHO) cells using the whole-cell patch-clamp technique and on the long-term modulation of Kv4.3 messenger RNA (mRNA) by real-time PCR analysis. Raloxifene decreased the Kv4.3 currents with an IC50 of 2.0 μM and accelerated the inactivation and activation kinetics in a concentration-dependent manner. The inhibitory effects of raloxifene on Kv4.3 were time-dependent: the association and dissociation rate constants for raloxifene were 9.5 μM(-1) s(-1) and 23.0 s(-1), respectively. The inhibition by raloxifene was voltage-dependent (δ = 0.13). Raloxifene shifted the steady-state inactivation curves in a hyperpolarizing direction and accelerated the closed-state inactivation of Kv4.3. Raloxifene slowed the time course of recovery from inactivation, thus producing a use-dependent inhibition of Kv4.3. β-Estradiol and tamoxifen had little effect on Kv4.3. A preincubation of ICI 182,780, an estrogen receptor antagonist, for 1 h had no effect on the inhibitory effect of raloxifene on Kv4.3. The metabolites of raloxifene, raloxifene-4'-glucuronide and raloxifene-6'-glucuronide, had little or no effect on Kv4.3. Coexpression of KChIP2 subunits did not alter the drug potency and steady-state inactivation of Kv4.3 channels. Long-term exposure to raloxifene (24 h) significantly decreased the expression level of Kv4.3 mRNA. This effect was not abolished by the coincubation with ICI 182,780. Raloxifene inhibited Kv4.3 channels by interacting with their open state during depolarization and with the closed state at subthreshold potentials. This effect was not mediated via an estrogen receptor.

  16. Role of 5-HT7 receptors in the inhibition of the vasodepressor sensory CGRPergic outflow in pithed rats.

    PubMed

    Cuesta, Cristina; García-Pedraza, José Ángel; García, Mónica; Villalón, Carlos M; Morán, Asunción

    2014-10-01

    The role of calcitonin gene-related peptide (CGRP) in the modulation of vascular tone has been widely documented. Indeed, electrical stimulation of the perivascular sensory outflow in pithed rats induces vasodepressor responses by activation of CGRP receptors. This study investigated the role of 5-HT7 receptors in the inhibition of the rat vasodepressor sensory outflow. Male Wistar pithed rats were pretreated with i.v. continuous infusions of hexamethonium and methoxamine, followed by physiological saline or AS-19 (a 5-HT7 receptor agonist). Then, electrical stimulation of the spinal cord resulted in frequency-dependent decreases in DBP. The infusions of AS-19, as compared to those of saline, inhibited the vasodepressor responses induced by electrical stimulation without affecting those to i.v. bolus injections of exogenous α-CGRP. This inhibition by AS-19 was abolished by the antagonists pimozide (5-HT7) or sulfisoxazole (ETA), but not by indomethacin (COX1/2) or losartan (AT1), at doses that did not affect per se the electrically-induced vasodepressor responses. Interestingly, glibenclamide (an ATP-dependent K(+) channel blocker) attenuated these vasodepressor responses. The present results suggest that AS-19-induced inhibition of the rat vasodepressor sensory CGRPergic outflow is mainly mediated by 5-HT7 receptors via endothelin release, with the possible involvement of ATP-dependent K(+) channels.

  17. EGF receptor-ligand interaction generates extracellular hydrogen peroxide that inhibits EGFR-associated protein tyrosine phosphatases.

    PubMed

    DeYulia, Garrett J; Cárcamo, Juan M

    2005-08-19

    Hydrogen peroxide (H(2)O(2)) has been shown to be an important modulator of intracellular phosphatase activity involved in cell signaling pathways, including signaling by members of the receptor tyrosine kinase family of receptors such as the epidermal growth factor receptor (EGFR). Intracellular H(2)O(2) can be generated by mitochondria-dependent pathways, whereas we recently showed that H(2)O(2) could be generated extracellularly by receptor-ligand interaction. Here, we show that H(2)O(2) produced by EGF-EGFR interaction can modulate the activity of intracellular protein tyrosine phosphatases (PTPs). Using purified proteins, we found that EGFR-ligand interaction generates H(2)O(2) that is capable of inhibiting the activity of PTP1B in vitro. Furthermore, the addition of catalase rescued phosphatase inhibition consequent to EGF-EGFR interaction. Using cells that overexpress EGFR, we found that the addition of extracellular catalase prevented EGF-induced inhibition of EGFR-associated phosphatase activity. Our findings suggest that extracellular H(2)O(2) generated by EGFR-ligand interaction permeates the plasma membrane and inhibits EGFR-associated tyrosine phosphatase activity, thereby modulating downstream signal transduction pathways.

  18. PCSK9 binds to multiple receptors and can be functionally inhibited by an EGF-A peptide.

    PubMed

    Shan, LiXin; Pang, Ling; Zhang, Rumin; Murgolo, Nicholas J; Lan, Hong; Hedrick, Joseph A

    2008-10-10

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low density lipoprotein receptor (LDLR) and induces its internalization and degradation. PCSK9 binding to LDLR is mediated through the LDLR epidermal growth factor-like repeat A (EGF-A) domain. We show for the first time that an EGF-A peptide inhibits PCSK9-mediated degradation of LDLR in HepG2 cells. In addition to LDLR, we show that PCSK9 also binds directly to ApoER2 and mouse VLDLR. Importantly, binding of PCSK9 to either LDLR or mouse VLDLR was effectively inhibited by EGF-A while binding to ApoER2 was less affected. In contrast, LDL receptor-associated protein (RAP), which interacts with LDL receptor repeat type A (LA) domains, inhibited PCSK9 binding to ApoER2 with greater efficacy than either LDLR or mVLDLR. These data demonstrate that while PCSK9 binds several receptors via its EGF-A binding domain, additional contacts with other receptor domains are also involved.

  19. G protein-coupled receptor 30 ligand G-1 increases aryl hydrocarbon receptor signalling by inhibition of tubulin assembly and cell cycle arrest in human MCF-7 cells.

    PubMed

    Tarnow, Patrick; Tralau, Tewes; Luch, Andreas

    2016-08-01

    Regulatory crosstalk between the aryl hydrocarbon receptor (AHR) and oestrogen receptor α (ERα) is well established. Apart from the nuclear receptors ERα and ERβ, oestrogen signalling further involves an unrelated G protein-coupled receptor termed GPR30. In order to investigate potential regulatory crosstalk, this study investigated the influence of G-1 as one of the few GPR30-specific ligands on the AHR regulon in MCF-7 cells. As a well-characterised model system, these human mammary carcinoma cells co-express all three receptors (AHR, ERα and GPR30) and are thus ideally suited to study corresponding regulatory pathway interactions on transcript level. Indeed, treatment with micromolar concentrations of the GPR30-specific agonist G-1 resulted in up-regulation of AHR as well as the transcripts for cytochromes P450 1A1 and 1B1, two well-known targets of the AHR regulon. While this was partly attributable to G-1-mediated inhibition of tubulin assembly and subsequent cell cycle arrest in the G2/M phase, the effects nevertheless required functional AHR. However, G-1-induced up-regulation of CYP 1A1 was not mediated by GPR30, as G15 antagonist treatment as well as a knockdown of GPR30 and AHR failed to inhibit this effect.

  20. Activation of lysophosphatidic acid receptor by gintonin inhibits Kv1.2 channel activity: involvement of tyrosine kinase and receptor protein tyrosine phosphatase α.

    PubMed

    Lee, Jun-Ho; Choi, Sun-Hye; Lee, Byung-Hwan; Hwang, Sung-Hee; Kim, Hyeon-Joong; Rhee, Jeehae; Chung, Chihye; Nah, Seung-Yeol

    2013-08-26

    Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. The primary action of gintonin is to elicit a transient increase in [Ca(2+)]i via activation of LPA receptor subtypes. Voltage-gated potassium (Kv) channels play important roles in synaptic transmission in nervous systems. The previous reports have shown that Kv channels can be regulated by Gαq/11 protein-coupled receptor ligands. In the present study, we examined the effects of gintonin on Kv1.2 channel activity expressed in Xenopus oocytes after injection of RNA encoding the human Kv1.2 α subunit. Gintonin treatment inhibited Kv1.2 channel activity in reversible and concentration-dependent manners. The inhibitory effect of gintonin on Kv1.2 channel activity was blocked by active phospholipase C inhibitor, inositol 1,4,5-triphosphate receptor antagonist, and intracellular Ca(2+) chelator. The co-expression of active receptor protein tyrosine phosphatase α (RPTPα) with Kv1.2 channel greatly attenuated gintonin-mediated inhibition of Kv1.2 channel activity, but attenuation was not observed with catalytically inactive RPTPα. Furthermore, neither genistein, a tyrosine kinase inhibitor, nor site-directed mutation of a tyrosine residue (Y132 to Y132F), which is phosphorylated by tyrosine kinase of the N-terminal of the Kv1.2 channel α subunit, significantly attenuated gintonin-mediated inhibition of Kv1.2 channel activity. These results indicate that the gintonin-mediated Kv1.2 channel regulation involves the dual coordination of both tyrosine kinase and RPTPα coupled to this receptor. Finally, gintonin-mediated regulation of Kv1.2 channel activity might explain one of the modulations of gintonin-mediated neuronal activities in nervous systems.

  1. Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4

    PubMed Central

    Schuette, Verena; Embgenbroich, Maria; Ulas, Thomas; Welz, Meike; Schulte-Schrepping, Jonas; Draffehn, Astrid M.; Quast, Thomas; Koch, Katharina; Nehring, Melanie; König, Jessica; Zweynert, Annegret; Harms, Frederike L.; Steiner, Nancy; Limmer, Andreas; Förster, Irmgard; Berberich-Siebelt, Friederike; Knolle, Percy A.; Wohlleber, Dirk; Kolanus, Waldemar; Beyer, Marc; Schultze, Joachim L.; Burgdorf, Sven

    2016-01-01

    The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8+ T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte–associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality. PMID:27601670

  2. Transient receptor potential vanilloid-1-mediated calcium responses are inhibited by the alkylamine antihistamines dexbrompheniramine and chlorpheniramine.

    PubMed

    Sadofsky, Laura R; Campi, Barbara; Trevisani, Marcello; Compton, Steven J; Morice, Alyn H

    2008-12-01

    American guidelines, unlike European guidelines, support the use of antihistamines as a first line of treatment for some causes of chronic cough. Transient receptor potential vanilloid-1 (TRPV1) is an ion channel activated by the tussive agents capsaicin, resiniferatoxin, and protons. It is predominantly expressed by C-fiber and some Adelta -fiber sensory neurons and is thought to be a cough receptor. By measuring increases in intracellular calcium as an indicator of TRPV1 activation, the authors sought to determine whether antihistamines could antagonise TRPV1 permanently expressed in HEK and Pro5 cells and TRPV1 endogenously expressed in rat dorsal root ganglia neurons. In human TRPV1-expressing HEK cells (hTRPV1-HEK), diphenhydramine and fexofenadine failed to inhibit capsaicin-triggered calcium responses. However, both dexbrompheniramine and chlorpheniramine significantly inhibited capsaicin-evoked responses in hTRPV1-HEK. Dexbrompheniramine also inhibited activation of rat TRPV1 expressed in HEK and Pro5 cells, without interfering with TRPA1 and proteinase-activated receptor-2 (PAR(2)) activation. Finally, in rat dorsal root ganglia neuron preparations, dexbrompheniramine dose-dependently inhibited capsaicin-evoked calcium responses. Thus, the inhibition of TRPV1 activation by dexbrompheniramine may provide one potential mechanism whereby this antihistamine exerts its therapeutic effect in chronic cough.

  3. H2 receptor-mediated facilitation and H3 receptor-mediated inhibition of noradrenaline release in the guinea-pig brain.

    PubMed

    Timm, J; Marr, I; Werthwein, S; Elz, S; Schunack, W; Schlicker, E

    1998-03-01

    The effect of histamine and related drugs on the tritium overflow evoked electrically (0.3 Hz) or by introduction of Ca2+ ions into Ca2+-free K+-rich (25 mmol/l) medium containing tetrodotoxin was studied in superfused guinea-pig brain cortex, cerebellum, hippocampus or hypothalamus slices and in mouse brain cortex slices preincubated with 3H-noradrenaline. The electrically evoked tritium overflow in guinea-pig cortex slices was inhibited by histamine; the H3 receptor antagonist clobenpropit reversed the effect of histamine to a slight facilitation. The facilitatory effect of histamine (obtained in the presence of clobenpropit) was not affected by the H1 receptor antagonist mepyramine but abolished by the H2 receptor antagonist ranitidine. In the absence of clobenpropit, ranitidine augmented the inhibitory effect of histamine. In slices superfused in the presence of ranitidine, the evoked overflow was inhibited by histamine and, more potently, by the H3 receptor agonist R-alpha-methylhistamine in a concentration-dependent manner (maximum inhibitory effect obtained for both agonists 30-35%). The concentration-response curve of histamine was shifted to the right by the H3 receptor antagonist thioperamide. R-alpha-methylhistamine inhibited the electrically evoked tritium overflow also in guinea-pig cerebellar, hippocampal and hypothalamic slices. In cortex slices superfused in the presence of clobenpropit, the H2 receptor agonists impromidine and, less potently, R-sopromidine facilitated the evoked overflow in a concentration-dependent manner. S-Sopromidine only tended to increase the evoked overflow. The effect of impromidine was counteracted by the H2 receptor antagonists ranitidine and cimetidine. The extent of the maximum facilitatory effect of impromidine (by 15-20%) was about the same when (i) the Ca2+ concentration in the medium was reduced from 1.3 to 0.98 mmol/l, (ii) the time of exposure to impromidine was reduced from 28 to 8 min or (iii) cerebellar

  4. Inhibition of drinking in water-deprived rats by combined central angiotensin II and cholinergic receptor blockade.

    PubMed

    Hoffman, W E; Ganten, U; Phillips, M I; Schmid, P G; Schelling, P; Ganten, D

    1978-01-01

    The effect of blockade of central angiotensin II (AII) receptors and cholinergic receptors on thirst induced by water deprivation was studied in Sprague-Dawley rats and rats with hereditary hypothalamic diabetes insipidus (DI). Neither central AII nor cholinergic blockade alone affected drinking. Antagonism of both receptors simultaneously, however, significantly inhibited water intake of both Sprague-Dawley and DI rats. This inhibitory effect was not observed in water-deprived, nephrectomized rats. The combined antagonism on water intake was specific, since milk intake in hungry rats was not affected by simultaneous AII and cholinergic blockade. Isorenin concentrations in brain tissue were at control levels in water-deprived, nephrectomized, and non-nephrectomized Sprague-Dawley rats but were increased in water-deprived DI rats. The results suggest that angiotensin and cholinergic receptors in the brain have a physiological role in thirst. Thirst is maintained when either receptor is intact, but reduced when both receptors are inhibited by antagonists. They are independently capable of maintaining thirst.

  5. An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

    1998-04-01

    Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

  6. Monoclonal antibody-induced ErbB3 receptor internalization and degradation inhibits growth and migration of human melanoma cells.

    PubMed

    Belleudi, Francesca; Marra, Emanuele; Mazzetta, Francesca; Fattore, Luigi; Giovagnoli, Maria Rosaria; Mancini, Rita; Aurisicchio, Luigi; Torrisi, Maria Rosaria; Ciliberto, Gennaro

    2012-04-01

    Members of the ErbB receptor family are targets of a growing numbers of small molecules and monoclonal antibodies inhibitors currently under development for the treatment of cancer. Although historical efforts have been directed against ErbB1 (EGFR) and ErbB2 (HER2/neu), emerging evidences have pointed to ErbB3 as a key node in the activation of proliferation/survival pathways from the ErbB receptor family and have fueled enthusiasm toward the clinical development of anti-ErbB3 agents. In this study, we have evaluated the potential therapeutic efficacy of a set of three recently generated anti-human ErbB3 monoclonals, A2, A3 and A4, in human primary melanoma cells. We show that in melanoma cells expressing ErbB1, ErbB3 and ErbB4 but not ErbB2 receptor ligands activate the PI3K/AKT pathway, and this leads to increased cell proliferation and migration. While antibodies A3 and A4 are able to potently inhibit ligand-induced signaling, proliferation and migration, antibody A2 is unable to exert this effect. In attempt to understand the mechanism of action and the basis of this different behavior, we demonstrate, through a series of combined approaches, that antibody efficacy strongly correlates with antibody-induced receptor internalization, degradation and inhibition of receptor recycling to the cell surface. Finally, fine epitope mapping studies through a peptide array show that inhibiting vs. non-inhibiting antibodies have a dramatically different mode of binding to the to the receptor extracellular domain. Our study confirms the key role of ErbB3 and points to exploitation of novel combination therapies for treatment of malignant melanoma.

  7. μ-Opioid receptor inhibition of substance P release from primary afferents disappears in neuropathic pain but not inflammatory pain

    PubMed Central

    Chen, Wenling; McRoberts, James A.; Marvizón, Juan Carlos G.

    2014-01-01

    Opiate analgesia in the spinal cord is impaired during neuropathic pain. We hypothesized that this is caused by a decrease in μ-opioid receptor inhibition of neurotransmitter release from primary afferents. To investigate this possibility, we measured substance P release in the spinal dorsal horn as neurokinin 1 receptor (NK1R) internalization in rats with chronic constriction injury (CCI) of the sciatic nerve. Noxious stimulation of the paw with CCI produced inconsistent NK1R internalization, suggesting that transmission of nociceptive signals by the injured nerve was variably impaired after CCI. This idea was supported by the fact that CCI produced only small changes in the ability of exogenous substance P to induce NK1R internalization or in the release of substance P evoked centrally from site of nerve injury. In subsequent experiments, NK1R internalization was induced in spinal cord slices by stimulating the dorsal root ipsilateral to CCI. We observed a complete loss of the inhibition of substance P release by the μ-opioid receptor agonist [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO) in CCI rats but not in sham-operated rats. In contrast, DAMGO still inhibited substance P release after inflammation of the hind paw with complete Freund’s adjuvant and in naïve rats. This loss of inhibition was not due to μ-opioid receptor downregulation in primary afferents, because their colocalization with substance P was unchanged, both in dorsal root ganglion neurons and primary afferent fibers in the dorsal horn. In conclusion, nerve injury eliminates the inhibition of substance P release by μ-opioid receptors, probably by hindering their signaling mechanisms. PMID:24583035

  8. Inhibition of muscarinic receptor-induced proliferation of astroglial cells by ethanol: mechanisms and implications for the fetal alcohol syndrome.

    PubMed

    Costa, Lucio G; Guizzetti, Marina

    2002-12-01

    In utero exposure to ethanol is deleterious to fetal brain development. Children born with the fetal alcohol syndrome (FAS) display a number of abnormalities, the most significant of which are central nervous system (CNS) dysfunctions, such as microencephaly and mental retardation. An interaction of ethanol with glial cells, particularly astrocytes, has been suggested to contribute to the developmental neurotoxicity of this alcohol. At low concentrations (10-100 mM) ethanol inhibits the proliferation of astroglial cells in vitro, particularly when stimulated by acetycholine through muscarinic M3 receptors. Of the several signal transduction pathways activated by these receptors in astrocytes or astrocytoma cells, which are involved in mitogenic signaling, only some (e.g. protein kinase C (PKC) zeta, p70S6 kinase) appear to be targeted by ethanol at the same low concentrations which effectively inhibit proliferation. Inhibition of astroglial proliferation by ethanol may contribute to the microencephaly seen in FAS.

  9. Involvement of mu opioid receptors of periaqueductal gary (PAG) in acupuncture inhibition of noxious blood pressure response in rabbits.

    PubMed

    Gao, M; Xu, W; Chen, W; He, L

    1994-01-01

    Strong electric shock stimulation of the rabbit front paw elicited a pressor blood pressure response regarded as noxious response. Ligands of mu opioid receptors were microinjected into the PAG to observe their effects on acupunture inhibition of the pressor response. (1) Ohmefentanyl (OMF), a mu agonist, significantly attenuated the pressor response. Mu antagonist TCTAP greatly enhanced the pressor response. (2) Electroacupuncture (EA) significantly inhibited the pressor response, the inhibition being readily reversed by TCTAP. The response after TCTAP was significantly greater than that of the control before EA. The results suggest that noxious stimulation is able to activate the mu opioid receptor of the PAG to modulate the noxious response and EA is able to enhance the activation.

  10. Genetic and Pharmacologic Inhibition of the Chemokine Receptor CXCR2 Prevents Experimental Hypertension and Vascular Dysfunction

    PubMed Central

    Wang, Lei; Zhao, Xue-Chen; Cui, Wei; Ma, Yong-Qiang; Ren, Hua-Liang; Zhou, Xin; Fassett, John; Yang, Yan-Zong; Chen, Yingjie; Xia, Yun-Long; Du, Jie

    2016-01-01

    Background: The recruitment of leukocytes to the vascular wall is a key step in hypertension development. Chemokine receptor CXCR2 mediates inflammatory cell chemotaxis in several diseases. However, the role of CXCR2 in hypertension development and the underlying mechanisms remain unknown. Methods: Angiotensin II (490 ng·kg-1·min-1) or deoxycorticosterone acetate (DOCA) salt–induced mouse hypertensive models in genetic ablation, pharmacologic inhibition of CXCR2, and adoptive bone marrow transfer mice were used to determine the role of CXCR2 in hypertension (measured by radiotelemetry and tail-cuff system), inflammation (verified by flow cytometry and quantitative real-time polymerase chain reaction [PCR] analysis), vascular remodeling (studied by haematoxylin and eosin and Masson’s trichrome staining), vascular dysfunction (assessed by aortic ring), and oxidative stress (indicated by nicotinamide adenine dinucleotide phosphate [NADPH] oxidase activity, dihydroethidium staining, and quantitative real-time PCR analysis). Moreover, the blood CXCR2+ cells in normotensive controls and hypertension patients were analyzed by flow cytometry. Results: Angiotensin II significantly upregulated the expression of CXCR2 mRNA and protein and increased the number of CD45+ CXCR2+ cells in mouse aorta (n=8 per group). Selective CXCR2 knockout (CXCR2-/-) or pharmacological inhibition of CXCR2 markedly reduced angiotensin II- or DOCA-salt-induced blood pressure elevation, aortic thickness and collagen deposition, accumulation of proinflammatory cells into the vascular wall, and expression of cytokines (n=8 per group). CXCR2 inhibition also ameliorated angiotensin II-induced vascular dysfunction and reduced vascular superoxide formation, NADPH activity, and expression of NADPH oxidase subunits (n=6 per group). Bone marrow reconstitution of wild-type mice with CXCR2-/- bone marrow cells also significantly abolished angiotensin II-induced responses (n=6 per group). It is important

  11. Adenosine receptor inhibition with theophylline attenuates the skin blood flow response to local heating in humans.

    PubMed

    Fieger, Sarah M; Wong, Brett J

    2010-09-01

    Mechanisms underlying the robust cutaneous vasodilatation in response to local heating of human skin remain unresolved. Adenosine receptor activation has been shown to induce vasodilatation via nitric oxide, and a substantial portion of the plateau phase to local heating of human skin has been shown to be dependent on nitric oxide. The purpose of this study was to investigate a potential role for adenosine receptor activation in cutaneous thermal hyperaemia in humans. Six subjects were equipped with four microdialysis fibres on the ventral forearm. Sites were randomly assigned to receive one of the following four treatments: (1) lactated Ringer solution to serve as a control; (2) 4 mM theophylline, a competitive, non-selective A(1)/A(2) adenosine receptor antagonist; (3) 10 mM Nomega(-)-nitro-L-arginine methyl ester (L-NAME) to inhibit NO synthase; or (4) combined 4 mm theophylline + 10 mM L-NAME. Following baseline measurements, each site was locally heated from a baseline temperature of 33 degrees C to 42 degrees C at a rate of 1 degrees C (10 s)(-1), and skin blood flow was monitored via laser-Doppler flowmetry (LDF). Cutaneous vascular conductance (CVC) was calculated as LDF divided by mean arterial pressure and normalized to maximal values (CVC(max)) via local heating to 43 degrees C and infusion of 28 mM sodium nitroprusside. The initial peak was significantly reduced in theophylline (68 +/- 2% CVC(max)) and L-NAME sites (54 +/- 5% CVC(max)) compared with control sites (81 +/- 2% CVC(max); P < 0.01 and P < 0.001, respectively). Combined theophylline + L-NAME (52 +/- 5% CVC(max)) reduced the initial peak compared with control and theophylline sites, but was not significantly different compared with L-NAME sites. The secondary plateau was attenuated in theophylline (77 +/- 2% CVC(max)), L-NAME (60 +/- 2% CVC(max)) and theophylline + L-NAME (53 +/- 1% CVC(max)) compared with control sites (94 +/- 2% CVC(max); P < 0.001 for all conditions). The secondary plateau

  12. GABAB receptor-mediated presynaptic inhibition in guinea-pig hippocampus is caused by reduction of presynaptic Ca2+ influx.

    PubMed Central

    Wu, L G; Saggau, P

    1995-01-01

    1. The hypothesis that activation of GABAB receptors inhibits evoked synaptic transmission by reducing the presynaptic Ca2+ influx was tested using a recently developed technique for simultaneously recording the presynaptic Ca2+ transient ([Ca2+]t) and the field excitatory postsynaptic potential (fEPSP) evoked by a single electrical stimulus at CA3 to CA1 synapses of guinea-pig hippocampus. 2. The GABAB receptor agonist baclofen reversibly blocked, in a dose-dependant manner, both the fEPSP and the presynaptic [Ca2+]t with similar time courses. During application of baclofen, the fEPSP was proportional to about the fourth power of the presynaptic [Ca2+]t, and the presynaptic fibre volley and the resting Ca2+ level did not change. These results are similar to those we previously observed following application of several voltage-dependent Ca2+ channel blockers, suggesting that baclofen inhibits the fEPSP by blocking the presynaptic Ca2+ influx. 3. The inhibition by baclofen of both the fEPSP and the presynaptic [Ca2+]t was blocked by the GABAB receptor antagonist CGP 35348, consistent with the causal relationship between the GABAB receptor-mediated presynaptic inhibition of the [Ca2+]t and the fEPSP. 4. The inhibition by baclofen of the [Ca2+]t was partially occluded by application of the voltage-dependent Ca2+ channel blocker omega-conotoxin-GVIA (omega-CgTX-GVIA), but not omega-agatoxin-IVA (omega-AgaTX-IVA), suggesting that baclofen reduces the presynaptic [Ca2+]t by blocking Ca2+ channels including the omega-CgTX-GVIA-sensitive type. 5. We conclude that baclofen inhibits evoked transmitter release by reducing presynaptic Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7562607

  13. Somatostatin receptor targeted liposomes with Diacerein inhibit IL-6 for breast cancer therapy.

    PubMed

    Bharti, Rashmi; Dey, Goutam; Banerjee, Indranil; Dey, Kaushik Kumar; Parida, Sheetal; Kumar, B N Prashanth; Das, Chandan Kanta; Pal, Ipsita; Mukherjee, Manabendra; Misra, Mridula; Pradhan, Anjan K; Emdad, Luni; Das, Swadesh K; Fisher, Paul B; Mandal, Mahitosh

    2017-03-01

    Selective targeting to the tumor niche remains a major challenge in successful cancer therapy. Somatostatin receptor 2 (SSTR2) is overexpressed in breast cancer cells thus making this receptor an attractive target for selective guidance of ligand-conjugated drug liposomes to the tumor site. In this study, a synthetic somatostatin analogue (SST) was used as SSTR2 targeting agent and Diacerein was employed as therapeutic molecule. Diacerein loaded liposomes (DNL) were prepared and they were further decorated with the synthetic and stable analogue of somatostatin (SST-DNL). Fabricated liposomes were nano-size in range and biocompatible. SST-DNL displayed significantly better anti-tumor efficacy as compared to free Diacerein (DN) and DNL in breast cancer models. Enhanced apoptosis in breast cancer cells was detected in SST-DNL treated groups as monitored by cell cycle analysis and changes in expression level of apoptotic/anti-apoptotic proteins Bcl-2, Bax, cleaved Caspase 3 and PARP. SST-DNL more effectively inhibited the oncogenic IL-6/IL-6R/STAT3/MAPK/Akt signalling pathways as compared to DN or DNL in cancer cells. In addition, SST-DNL effectively suppressed angiogenesis and cancer cell invasion. In vivo tumor growth in a MDA-MB-231 mouse xenograft model was significantly suppressed following SST-DNL treatment. In xenograft model, immunohistochemistry of Ki-67 and CD-31 indicated that SST-DNL improved the anti-proliferative and anti-angiogenic impacts of Diacerein. In vivo pharmacokinetic studies in rats showed enhanced circulation time in the DNL or SST-DNL treated groups as compared to free DN. Considering all of these findings, we conclude that SST-DNL provides a novel strategy with better efficacy for breast cancer therapy.

  14. Molecular requirements for inhibition of the chemokine receptor CCR8 – probe-dependent allosteric interactions

    PubMed Central

    Rummel, PC; Arfelt, KN; Baumann, L; Jenkins, TJ; Thiele, S; Lüttichau, HR; Johnsen, A; Pease, J; Ghosh, S; Kolbeck, R; Rosenkilde, MM

    2012-01-01

    BACKGROUND AND PURPOSE Here we present a novel series of CCR8 antagonists based on a naphthalene-sulfonamide structure. This structure differs from the predominant pharmacophore for most small-molecule CC-chemokine receptor antagonists, which in fact activate CCR8, suggesting that CCR8 inhibition requires alternative structural probes. EXPERIMENTAL APPROACH The compounds were tested as inverse agonists and as antagonists against CCL1-induced activity in Gαi signalling and chemotaxis. Furthermore, they were assessed by heterologous competition binding against two radiolabelled receptor ligands: the endogenous agonist CCL1 and the virus-encoded antagonist MC148. KEY RESULTS All compounds were highly potent inverse agonists with EC50 values from 1.7 to 23 nM. Their potencies as antagonists were more widely spread (EC50 values from 5.9 to 1572 nM). Some compounds were balanced antagonists/inverse agonists whereas others were predominantly inverse agonists with >100-fold lower potency as antagonists. A correspondingly broad range of affinities, which followed the antagonist potencies, was disclosed by competition with [125I]-CCL1 (Ki 3.4–842 nM), whereas the affinities measured against [125I]-MC148 were less widely spread (Ki 0.37–27 nM), and matched the inverse agonist potencies. CONCLUSION AND IMPLICATIONS Despite highly potent and direct effects as inverse agonists, competition-binding experiments against radiolabelled agonist and tests for antagonism revealed a probe-dependent allosteric effect of these compounds. Thus, minor chemical changes affected the ability to modify chemokine binding and action, and divided the compounds into two groups: predominantly inverse agonists and balanced antagonists/inverse agonists. These studies have important implications for the design of new inverse agonists with or without antagonist properties. PMID:22708643

  15. MiR-503 inhibits adipogenesis by targeting bone morphogenetic protein receptor 1a

    PubMed Central

    Man, Xiao-Fei; Tan, Shu-Wen; Tang, Hao-Neng; Guo, Yue; Tang, Chen-Yi; Tang, Jun; Zhou, Ci-La; Zhou, Hou-De

    2016-01-01

    Adipogenesis plays a key role in the regulation of whole-body energy homeostasis and is critically related to obesity. To overcome obesity and its associated disorders, it is necessary to elucidate the molecular mechanisms involved in adipogenesis. An adipogenesis-related miRNA array analysis demonstrated that miR-503 was differentially expressed before and after adipocyte differentiation; however, the exact role of miR-503 in adipocyte differentiation is unclear. Thus, the objective of this study was to further examine miR-503 in adipocyte differentiation. We found significantly decreased expression of miR-503 during adipocyte differentiation process. Using bioinformatic analysis, miR-503 was identified as a potential regulator of Bone Morphogenetic Protein Receptor 1a (BMPR1a). We then validated BMPR1a as the target of miR-503 using a dual luciferase assay, and found decreased miR-503 and increased BMPR1a expression during adipogenesis. Overexpression of miR-503 in preadipocytes repressed expression of BMPR1a and adipogenic-related factors such as CCAAT/enhancer binding protein a (C/EBPα), proliferator-activated receptor-gamma (PPARγ), and adipocyte protein 2 (AP2). In addition, miR-503 overexpression impaired the phosphoinositol-3 kinase (PI3K)/Akt pathway. Inhibition of miR-503 had the opposite effect. Additionally, BMPR1a interference by siRNA attenuated adipocyte differentiation and the accumulation of lipid droplets via downregulating the PI3K/Akt signaling pathway. Our study provides the first evidence of the role miR-503 plays in adipocyte differentiation by regulating BMPR1a via the PI3K/Akt pathway, which may become a novel target for obesity therapy. PMID:27398155

  16. Glucocorticoid receptor blockade inhibits brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus.

    PubMed

    Dunlap, Kent D; Jashari, Denisa; Pappas, Kristina M

    2011-08-01

    When animals are under stress, glucocorticoids commonly inhibit adult neurogenesis by acting through glucocorticoid receptors (GRs). However, in some cases, conditions that elevate glucocorticoids promote adult neurogenesis, and the role of glucocorticoid receptors in these circumstances is not well understood. We examined the involvement of GRs in social enhancement of brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus. In this species, long-term social interaction simultaneously elevates plasma cortisol, enhances brain cell addition and increases production of aggressive electrocommunication signals ("chirps"). We implanted isolated and paired fish with capsules containing nothing (controls) or the GR antagonist, RU486, recorded chirp production and locomotion for 7d, and measured the density of newborn cells in the periventricular zone. Compared to isolated controls, paired controls showed elevated chirping in two phases: much higher chirp rates in the first 5h and moderately higher nocturnal rates thereafter. Treating paired fish with RU486 reduced chirp rates in both phases to those of isolated fish, demonstrating that GR activation is crucial for socially induced chirping. Neither RU486 nor social interaction affected locomotion. RU486 treatment to paired fish had a partial effect on cell addition: paired RU486 fish had less cell addition than paired control fish but more than isolated fish. This suggests that cortisol activation of GRs contributes to social enhancement of cell addition but works in parallel with another GR-independent mechanism. RU486 also reduced cell addition in isolated fish, indicating that GRs participate in the regulation of cell addition even when cortisol levels are low.

  17. Pharmacological inhibition of CXCR2 chemokine receptors modulates paraquat-induced intoxication in rats.

    PubMed

    Costa, Kesiane M; Maciel, Izaque S; Kist, Luiza W; Campos, Maria M; Bogo, Maurício R

    2014-01-01

    Paraquat (PQ) is an agrochemical agent commonly used worldwide, which is allied to potential risks of intoxication. This herbicide induces the formation of reactive oxygen species (ROS) that ends up compromising various organs, particularly the lungs and the brain. This study evaluated the deleterious effects of paraquat on the central nervous system (CNS) and peripherally, with special attempts to assess the putative protective effects of the selective CXCR2 receptor antagonist SB225002 on these parameters. PQ-toxicity was induced in male Wistar rats, in a total dose of 50 mg/kg, and control animals received saline solution at the same schedule of administration. Separate groups of animals were treated with the selective CXCR2 antagonist SB225002 (1 or 3 mg/kg), administered 30 min before each paraquat injection. The major changes found in paraquat-treated animals were: decreased body weight and hypothermia, nociception behavior, impairment of locomotor and gait capabilities, enhanced TNF-α and IL-1β expression in the striatum, and cell migration to the lungs and blood. Some of these parameters were reversed when the antagonist SB225002 was administered, including recovery of physiological parameters, decreased nociception, improvement of gait abnormalities, modulation of striatal TNF-α and IL-1β expression, and decrease of neutrophil migration to the lungs and blood. Taken together, our results demonstrate that damage to the central and peripheral systems elicited by paraquat can be prevented by the pharmacological inhibition of CXCR2 chemokine receptors. The experimental evidence presented herein extends the comprehension on the toxicodynamic aspects of paraquat, and opens new avenues to treat intoxication induced by this herbicide.

  18. Nicousamide protects kidney podocyte by inhibiting the TGFβ receptor II phosphorylation and AGE-RAGE signaling

    PubMed Central

    Zhang, Sen; Wang, Dongjie; Xue, Nina; Lai, Fangfang; Ji, Ming; Jin, Jing; Chen, Xiaoguang

    2017-01-01

    Nicousamide, a clinical phase II renal protective new drug, has been demonstrated to have renal protective effect on diabetic nephropathy (DN) by experimental animal model. Its known molecular mechanisms include AGE formation blocking and moderately decreasing the blood pressure. Nicousamide shows potential on attenuating albuminuria, thereby suggests it might have protective effect on podocytes. The aim of present study was to investigate whether nicousamide could protect integrity of podocytes, and further its protection mechanisms. Sprague-Dawley (SD) rats were induced to DN by streptozotocin, and nicousamide (20 and 40 mg/kg) was orally administrated for 20 weeks. Every five weeks, the albuminuria was measured, and renal pathology was evaluated at the end of experiment. Real-time PCR and immunofluorescence were used to test expression of podocyte marker nephrin, CD2AP and podocine in rat kidney tissues. Western blot was used to test the activation and phosphorylation of TGFβ1-smad signaling pathway. surface plasmon resonance (SPR) technology was used to analyze whether nicousamide can interact with TGFβ1 receptor II (TGFβ RII) and receptor for advanced glycation endproducts (RAGE). Results demonstrate that nicousamide significantly reduces albuminuria and ameliorate the glomerulosclerosis in DN rats. RT-PCR and immunofluorescence demonstrate that nicousamide can increase the expression of podocyte markers and keep podocyte effacement. Phosphorylation of TGFβ RII and smad2 in rat kidney was inhibited by nicousamide dose dependently. SPR demonstrate that nicousamide have strong binding capability with hRAGE with Kd approximate 6 μM. These results indicate a protective effect of nicousamide against podocyte injury, and this effect might contribute from suppression of TGFβ-involved fibrosis and AGE-RAGE signaling activation. PMID:28123638

  19. Inhibition of Estrogen Receptor-DNA Binding by the "Pure" Antiestrogen ICI 164,384 Appears to be Mediated by Impaired Receptor Dimerization

    NASA Astrophysics Data System (ADS)

    Fawell, Stephen E.; White, Roger; Hoare, Susan; Sydenham, Mark; Page, Martin; Parker, Malcolm G.

    1990-09-01

    Many estrogen-antagonist and -agonist ligands have been synthesized, some of which have proved clinically important in the treatment of hormone-dependent breast tumors and endocrine disorders. Here we show that the "pure" antiestrogen ICI 164,384 inhibits mouse estrogen receptor-DNA binding in vitro. The effects of this steroid on DNA binding can be overcome by addition of an anti-receptor antibody whose epitope lies N-terminal to the receptor DNA-binding domain. Since this antibody is also capable of restoring DNA-binding activity to receptor mutants that either lack the dimerization domain or bear deleterious mutations within it, we propose that ICI 164,384 reduces DNA binding by interfering with receptor dimerization. In contrast, when complexed with the antagonist/partial agonist tamoxifen, the estrogen receptor is capable of binding to DNA in vitro, but tamoxifen does not promote the agonist-induced conformational change obtained with estradiol. The implications of these data are discussed in relation to the in vivo properties of these drugs.

  20. Inhibition of estrogen receptor-DNA binding by the "pure" antiestrogen ICI 164,384 appears to be mediated by impaired receptor dimerization.

    PubMed

    Fawell, S E; White, R; Hoare, S; Sydenham, M; Page, M; Parker, M G

    1990-09-01

    Many estrogen-antagonist and -agonist ligands have been synthesized, some of which have proved clinically important in the treatment of hormone-dependent breast tumors and endocrine disorders. Here we show that the "pure" antiestrogen ICI 164,384 inhibits mouse estrogen receptor-DNA binding in vitro. The effects of this steroid on DNA binding can be overcome by addition of anti-receptor antibody whose epitope lies N-terminal to the receptor DNA-binding domain. Since this antibody is also capable of restoring DNA-binding activity to receptor mutants that either lack the dimerization domain or bear deleterious mutations within it, we propose that ICI 164,384 reduces DNA binding by interfering with receptor dimerization. In contrast, when complexed with the antagonist/partial agonist tamoxifen, the estrogen receptor is capable of binding to DNA in vitro, but tamoxifen does not promote the agonist-induced conformational change obtained with estradiol. The implications of these data are discussed in relation to the in vivo properties of these drugs.

  1. Inhibition of estrogen receptor-DNA binding by the "pure" antiestrogen ICI 164,384 appears to be mediated by impaired receptor dimerization.

    PubMed Central

    Fawell, S E; White, R; Hoare, S; Sydenham, M; Page, M; Parker, M G

    1990-01-01

    Many estrogen-antagonist and -agonist ligands have been synthesized, some of which have proved clinically important in the treatment of hormone-dependent breast tumors and endocrine disorders. Here we show that the "pure" antiestrogen ICI 164,384 inhibits mouse estrogen receptor-DNA binding in vitro. The effects of this steroid on DNA binding can be overcome by addition of anti-receptor antibody whose epitope lies N-terminal to the receptor DNA-binding domain. Since this antibody is also capable of restoring DNA-binding activity to receptor mutants that either lack the dimerization domain or bear deleterious mutations within it, we propose that ICI 164,384 reduces DNA binding by interfering with receptor dimerization. In contrast, when complexed with the antagonist/partial agonist tamoxifen, the estrogen receptor is capable of binding to DNA in vitro, but tamoxifen does not promote the agonist-induced conformational change obtained with estradiol. The implications of these data are discussed in relation to the in vivo properties of these drugs. Images PMID:2395882

  2. Epidermal Growth Factor Receptor Inhibition in the Management of Squamous Cell Carcinoma of the Lung

    PubMed Central

    Spaans, Johanna N.

    2016-01-01

    Molecular therapies targeting epidermal growth factor receptor (EGFR) have had a profound impact on the management of advanced non-small cell lung cancer (NSCLC). EGFR inhibition with EGFR tyrosine kinase inhibitors (EGFR-TKIs) and anti-EGFR monoclonal antibodies (mAbs) in squamous NSCLC (sqNSCLC) remains controversial in patients whose tumors are not known to harbor EGFR mutations. Recent meta-analyses of EGFR-inhibition randomized trials that are adequately powered for histological subgroup analysis and anti-EGFR trials limited to patients with squamous histology afford the opportunity to revisit EGFR treatment in sqNSCLC. In unselected patients with sqNSCLC who are not eligible for chemotherapy, EGFR-TKI therapy is a valid treatment option over placebo or best supportive care, with improved progression-free survival noted in randomized controlled trials in both the first- and second-line setting and improved overall survival (OS) in the second-line setting. In patients eligible for chemotherapy, first-line combination regimens with anti-EGFR mAbs have been shown to improve OS over chemotherapy alone in patients with squamous histology in meta-analysis and more recently in the SQUIRE sqNSCLC trial (chemotherapy with and without necitumumab). In sqNSCLC patients who respond to induction chemotherapy, maintenance therapy with erlotinib delays disease progression and may improve the survival of patients with stable disease. In the second-line setting, survival outcomes are comparable between chemotherapy and EGFR-TKIs in meta-analysis, with the latter being more tolerable as a second-line therapy. Newer-generation EGFR-TKI therapies may further benefit patients with sqNSCLC who have failed first-line chemotherapy, given the positive trial results from LUX-Lung 8 (afatinib vs. erlotinib). EGFR is a valid therapeutic target in unselected/EGFR wild-type patients with squamous cell carcinoma of the lung. With the recent approval of immune checkpoint inhibitors in the

  3. Expression of 5-HT3 receptors by extrinsic duodenal afferents contribute to intestinal inhibition of gastric emptying.

    PubMed

    Raybould, Helen E; Glatzle, Jorg; Robin, Carla; Meyer, James H; Phan, Thomas; Wong, Helen; Sternini, Catia

    2003-03-01

    Intestinal perfusion with carbohydrates inhibits gastric emptying via vagal and spinal capsaicin-sensitive afferent pathways. The aim of the present study was to determine the role of 1) 5-hydroxytryptamine (5-HT)(3) receptors (5-HT(3)R) in mediating glucose-induced inhibition of gastric emptying and 2) 5-HT(3)R expression in vagal and spinal afferents in innervating the duodenum. In awake rats fitted with gastric and duodenal cannulas, perfusion of the duodenum with glucose (50 and 100 mg) inhibited gastric emptying. Intestinal perfusion of mannitol inhibited gastric emptying only at the highest concentration (990 mosm/kgH(2)O). Pretreatment with the 5-HT(3)R antagonist tropisetron abolished both glucose- and mannitol-induced inhibition of gastric emptying. Retrograde labeling of visceral afferents by injection of dextran-conjugated Texas Red into the duodenal wall was used to identify extrinsic primary afferents. Immunoreactivity for 5-HT(3)R, visualized with an antibody directed to the COOH terminus of the rat 5-HT(3)R, was found in >80% of duodenal vagal and spinal afferents. These results show that duodenal extrinsic afferents express 5-HT(3)R and that the receptor mediates specific glucose-induced inhibition of gastric emptying. These findings support the hypothesis that enterochromaffin cells in the intestinal mucosa release 5-HT in response to glucose, which activates 5-HT(3)R on afferent nerve terminals to evoke reflex changes in gastric motility. The primary glucose sensors of the intestine may be mucosal enterochromaffin cells.

  4. Inhibition of Coxsackie B Virus Infection by Soluble Forms of Its Receptors: Binding Affinities, Altered Particle Formation, and Competition with Cellular Receptors

    PubMed Central

    Goodfellow, Ian G.; Evans, David J.; Blom, Anna M.; Kerrigan, Dave; Miners, J. Scott; Morgan, B. Paul; Spiller, O. Brad

    2005-01-01

    We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus B3 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37°C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed. PMID:16140777

  5. A(1) and A(3) adenosine receptors inhibit LPS-induced hypoxia-inducible factor-1 accumulation in murine astrocytes.

    PubMed

    Gessi, Stefania; Merighi, Stefania; Stefanelli, Angela; Fazzi, Debora; Varani, Katia; Borea, Pier Andrea

    2013-10-01

    Adenosine (Ado) exerts neuroprotective and anti-inflammatory functions by acting through four receptor subtypes A1, A2A, A2B and A3. Astrocytes are one of its targets in the central nervous system. Hypoxia-inducible factor-1 (HIF-1), a master regulator of oxygen homeostasis, is induced after hypoxia, ischemia and inflammation and plays an important role in brain injury. HIF-1 is expressed by astrocytes, however the regulatory role played by Ado on HIF-1α modulation induced by inflammatory and hypoxic conditions has not been investigated. Primary murine astrocytes were activated with lipopolysaccharide (LPS) with or without Ado, Ado receptor agonists, antagonists and receptor silencing, before exposure to normoxia or hypoxia. HIF-1α accumulation and downstream genes regulation were determined. Ado inhibited LPS-increased HIF-1α accumulation under both normoxic and hypoxic conditions, through activation of A1 and A3 receptors. In cells incubated with the blockers of p44/42 MAPK and Akt, LPS-induced HIF-1α accumulation was significantly decreased in normoxia and hypoxia, suggesting the involvement of p44/42 MAPK and Akt in this effect and Ado inhibited kinases phosphorylation. A series of angiogenesis and metabolism related genes were modulated by hypoxia in an HIF-1 dependent way, but not further increased by LPS, with the exception of GLUT-1 and hexochinase II that were elevated by LPS only in normoxia and inhibited by Ado receptors. Instead, genes involved in inflammation, like inducible nitric-oxide synthase (iNOS) and A2B receptors, were increased by LPS in normoxia, strongly stimulated by LPS in concert with hypoxia and inhibited by Ado, through A1 and A3 receptor subtypes. In conclusion A1 and A3 receptors reduce the LPS-mediated HIF-1α accumulation in murine astrocytes, resulting in a downregulation of genes involved in inflammation and hypoxic injury, like iNOS and A2B receptors, in both normoxic and hypoxic conditions.

  6. Cathelicidin suppresses lipid accumulation and hepatic steatosis by inhibition of the CD36 receptor

    PubMed Central

    Tran, Deanna Hoang-Yen; Tran, Diana Hoang-Ngoc; Mattai, S. Anjani; Sallam, Tamer; Ortiz, Christina; Lee, Elaine C.; Robbins, Lori; Ho, Samantha; Lee, Jung Eun; Fisseha, Elizabeth; Shieh, Christine; Sideri, Aristea; Shih, David Q; Fleshner, Philip; McGovern, Dermot PB; Vu, Michelle; Hing, Tressia C.; Bakirtzi, Kyriaki; Cheng, Michelle; Su, Bowei; Law, Ivy; Karagiannides, Iordanes; Targan, Stephan R.; Gallo, Richard L.; Li, Zhaoping; Koon, Hon Wai

    2016-01-01

    Background and Objectives Obesity is a global epidemic which increases the risk of the metabolic syndrome. Cathelicidin (LL-37 and mCRAMP) is an antimicrobial peptide with an unknown role in obesity. We hypothesize that cathelicidin expression correlates with obesity and modulates fat mass and hepatic steatosis. Materials and Methods Male C57BL/6J mice were fed a high-fat diet. Streptozotocin was injected into mice to induce diabetes. Experimental groups were injected with cathelicidin and CD36 overexpressing lentiviruses. Human mesenteric fat adipocytes, mouse 3T3-L1 differentiated adipocytes, and human HepG2 hepatocytes were used in the in vitro experiments. Cathelicidin levels in non-diabetic, prediabetic, and Type II diabetic patients were measured by ELISA. Results Lentiviral cathelicidin overexpression reduced hepatic steatosis and decreased the fat mass of high-fat diet-treated diabetic mice. Cathelicidin overexpression reduced mesenteric fat and hepatic fatty acid translocase (CD36) expression that was reversed by lentiviral CD36 overexpression. Exposure of adipocytes and hepatocytes to cathelicidin significantly inhibited CD36 expression and reduced lipid accumulation. Serum cathelicidin protein levels were significantly increased in non-diabetic and prediabetic patients with obesity, compared to non-diabetic patients with normal body mass index (BMI) values. Prediabetic patients had lower serum cathelicidin protein levels than non-diabetic subjects. Conclusions Cathelicidin inhibits the CD36 fat receptor and lipid accumulation in adipocytes and hepatocytes, leading to a reduction of fat mass and hepatic steatosis in vivo. Circulating cathelicidin levels are associated with increased BMI. Our results demonstrate that cathelicidin modulates the development of obesity. PMID:27163748

  7. Peroxisome Proliferator-Activated ReceptorInhibition Protects Against Doxorubicin-Induced Cardiotoxicity in Mice.

    PubMed

    Rahmatollahi, Mahdieh; Baram, Somayeh Mahmoodi; Rahimian, Reza; Saeedi Saravi, Seyed Soheil; Dehpour, Ahmad Reza

    2016-07-01

    Doxorubicin is an effective chemotherapeutic drug against a considerable number of malignancies. However, its toxic effects on myocardium are confirmed as major limit of utilization. PPAR-α is highly expressed in the heart, and its activation leads to an increased cardiac fatty acid oxidation and cardiomyocyte necrosis. This study was performed to adjust the hypothesis that PPAR-α receptor inhibition protects against doxorubicin-induced cardiac dysfunction in mice. Male Balb/c mice were used in this study. Left atria were isolated, and their contractility was measured in response to electrical field stimulation in a standard organ bath. PPAR-α activity was measured using specific PPAR-α antibody in an ELISA-based system coated with double-strand DNA containing PPAR-α response element sequence. Moreover, cardiac MDA and TNF-α levels were measured by ELISA method. Following incubation with doxorubicin (35 µM), a significant reduction in atrial contractility was observed (P < 0.001). Pretreatment of animals with a selective PPAR-α antagonist, GW6471, significantly improved doxorubicin-induced atrial dysfunction (P < 0.001). Furthermore, pretreatment of the mice with a non-selective cannabinoid agonist, WIN55212-2, significantly decreased PPAR-α activity in cardiac tissue, subsequently leading to significant improvement in doxorubicin-induced atrial dysfunction (P < 0.001). Also, GW6471 and WIN significantly reduced cardiac MDA and TNF-α levels compared with animals receiving doxorubicin (P < 0.001). The study showed that inhibition of PPAR-α is associated with protection against doxorubicin-induced cardiotoxicity in mice, and cannabinoids can potentiate the protection by PPAR-α blockade. Moreover, PPAR-α may be considered as a target to prevent cardiotoxicity induced by doxorubicin in patients undergoing chemotherapy.

  8. Macelignan inhibits melanosome transfer mediated by protease-activated receptor-2 in keratinocytes.

    PubMed

    Choi, Eun-Jung; Kang, Young-Gyu; Kim, Jaekyung; Hwang, Jae-Kwan

    2011-01-01

    Skin pigmentation is the result of melanosome transfer from melanocytes to keratinocytes. Protease-activated receptor-2 (PAR-2) is a key mediator of melanosome transfer, which occurs as the melanocyte extends its dendrite toward surrounding keratinocytes that take up melanosomes by phagocytosis. We investigated the effects of macelignan isolated from Myristica fragrans HOUTT. (nutmeg) on melanosome transfer and the regulation of PAR-2 in human keratinocytes (HaCaT). HaCaT cells stimulated by the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-NH₂ (SLIGRL) were treated with macelignan; PAR-2 expression was then determined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. We evaluated the effects of macelignan on calcium mobilization and keratinocyte phagocytosis. In addition, B16F10 melanoma cells and keratinocytes were co-cultured to assess the effects of macelignan on prostaglandin E₂ (PGE₂) secretion and subsequent dendrite formation. Macelignan decreased HaCaT PAR-2 mRNA and protein levels in a dose-dependent manner. Furthermore, macelignan markedly reduced intracellular calcium mobilization and significantly downregulated keratinocyte phagocytosis, as shown by decreased ingestion of Escherichia coli bioparticles and fluorescent microspheres. In co-culture experiments, macelignan reduced keratinocyte PGE₂ secretion, thereby preventing dendrite formation in B16F10 melanoma cells compared with SLIGRL-treated controls. Macelignan inhibits melanosome transfer by downregulating PAR-2, thereby reducing keratinocyte phagocytosis and PGE₂ secretion, which in turn inhibits dendrite formation in B16F10 melanoma cells. Taken together, our findings suggest that macelignan could be used as a natural depigmenting agent to ameliorate hyperpigmentation.

  9. Sulfonylurea receptors inhibit the epithelial sodium channel (ENaC) by reducing surface expression.

    PubMed

    Konstas, A A; Bielfeld-Ackermann, A; Korbmacher, C

    2001-08-01

    In the kidney the epithelial Na+ channel (ENaC) is co-expressed with the sulfonylurea receptor (SUR), an ABC protein that shares a high degree of homology with the cystic fibrosis transmembrane conductance regulator (CFTR) and reportedly modifies ENaC in various preparations. To investigate a possible regulatory relationship between SUR and ENaC, we performed co-expression studies on Xenopus laevis oocytes, which were assayed for amiloride-sensitive currents (DeltaIami). Moreover, a chemiluminescence assay was used to investigate the surface expression of extracellular hemagglutinin-tagged SUR1 (SUR1-HA) or HA-tagged ENaC (ENaC-HA). In oocytes co-injected with SUR1/ENaC (or SUR2B/ENaC) DeltaIami was reduced by congruent with 53% (or congruent with 45%) compared to DeltaIami measured in matched control oocytes injected with ENaC alone. The inhibitory effect of SUR on DeltaIami was preserved in oocytes expressing ENaC with C-terminally truncated subunits. Co-expression of SURs did not confer sensitivity of DeltaIami to diazoxide, pinacidil, tolbutamide, or glibenclamide. ENaC does not facilitate the surface expression of SUR1-HA, which is known to be retained in the endoplasmatic reticulum (ER) by an ER-retention/retrieval signal. SUR1-HAAAA, a mutant that lacks this signal, still inhibits ENaC currents. Chemiluminescence was reduced by congruent with 49% in oocytes co-expressing ENaC-HA/SUR1 compared to that in control oocytes expressing ENaC-HA alone. We conclude that SUR does not interact with ENaC at the level of the plasma membrane but that it inhibits DeltaIami by reducing surface expression of the channel.

  10. Effects of bradykinin B2 receptor antagonism on the hypotensive effects of ACE inhibition.

    PubMed Central

    Bouaziz, H; Joulin, Y; Safar, M; Benetos, A

    1994-01-01

    1. The aim of this study was to determine the participation of endogenous bradykinin (BK) in the antihypertensive effects of the angiotensin converting enzyme inhibitor (ACEI), perindoprilat, in the spontaneously hypertensive rat (SHR) on different salt diets. 2. Conscious SHRs receiving either a low or a high NaCl diet were used in order to evaluate the respective roles of angiotensin II suppression and bradykinin stimulation in the acute hypotensive effects of perindoprilat. Two different B2 receptor antagonists (B 4146 and Hoe 140) were used after bolus administration of 7 mg kg-1 of the ACEI, perindoprilat. In separate animals, Hoe 140 was administered before the injection of perindoprilat. In other experiments, the effects of Hoe 140 on the hypotensive effects of the calcium antagonist, nicardipine, were tested. 3. The different NaCl diets had no effect on baseline blood pressure. Hoe 140 injection before ACE inhibition did not modify blood pressure. Perindoprilat caused more marked hypotension in the low salt-fed rats than in the high salt animals (P < 0.01). Administration of Hoe 140 or B4146 after perindoprilat significantly reduced the antihypertensive effects of perindoprilat in the different groups, but this effect was more pronounced in high salt-fed rats. However, in SHRs receiving Hoe 140 before perindoprilat, the antihypertensive effect of perindoprilat was completely abolished in both high or low salt diet rats. In separate experiments we confirmed that Hoe 140 did not affect the hypotensive efficacy of the calcium antagonist, nicardipine. 4. Our study shows that inhibition of endogenous bradykinin degradation participates in the acute antihypertensive effects of perindoprilat in SHRs. The role of bradykinin is more pronounced following exposure to a high salt diet i.e., when the renin-angiotensin system is suppressed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7858859

  11. Serotonin 2A Receptors, Citalopram and Tryptophan-Depletion: a Multimodal Imaging Study of their Interactions During Response Inhibition

    PubMed Central

    Macoveanu, Julian; Hornboll, Bettina; Elliott, Rebecca; Erritzoe, David; Paulson, Olaf B; Siebner, Hartwig; Knudsen, Gitte M; Rowe, James B

    2013-01-01

    Poor behavioral inhibition is a common feature of neurological and psychiatric disorders. Successful inhibition of a prepotent response in ‘NoGo' paradigms requires the integrity of both the inferior frontal gyrus (IFG) and the serotonergic system. We investigated individual differences in serotonergic regulation of response inhibition. In 24 healthy adults, we used 18F-altanserin positron emission tomography to assess cerebral 5-HT2A receptors, which have been related to impulsivity. We then investigated the impact of two acute manipulations of brain serotonin levels on behavioral and neural correlates of inhibition using intravenous citalopram and acute tryptophan depletion during functional magnetic resonance imaging. We adapted the NoGo paradigm to isolate effects on inhibition per se as opposed to other aspects of the NoGo paradigm. Successful NoGo inhibition was associated with greater activation of the right IFG compared to control trials with alternative responses, indicating that the IFG is activated with inhibition in NoGo trials rather than other aspects of invoked cognitive control. Activation of the left IFG during NoGo trials was greater with citalopram than acute tryptophan depletion. Moreover, with the NoGo-type of response inhibition, the right IFG displayed an interaction between the type of serotonergic challenge and neocortical 5-HT2A receptor binding. Specifically, acute tryptophan depletion (ATD) produced a relatively larger NoGo response in the right IFG in subjects with low 5-HT2A BPP but reduced the NoGo response in those with high 5-HT2A BPP. These links between serotonergic function and response inhibition in healthy subjects may help to interpret serotonergic abnormalities underlying impulsivity in neuropsychiatric disorders. PMID:23303045

  12. P2X Receptors Inhibit NaCl Absorption in mTAL Independently of Nitric Oxide

    PubMed Central

    Svendsen, Samuel L.; Isidor, Søren; Praetorius, Helle A.; Leipziger, Jens

    2017-01-01

    Activation of basolateral P2X receptors markedly reduces NaCl absorption in mouse medullary thick ascending limb (mTAL). Here we tested the role of nitric oxide (NO) in the ATP-mediated (P2X) transport inhibition. We used isolated, perfused mTALs from mice to electrically measure NaCl absorption. By microelectrodes we determined the transepithelial voltage (Vte) and transepithelial resistance (Rte). Via these two parameters, we calculated the equivalent short circuit current, I'sc as a measure of the transepithelial Na+ absorption. Basolateral ATP (100 μM) acutely induced reversible inhibition of Na+ absorption (24 ± 4%, n = 10). Addition of L-arginine (100 μM) had no apparent effect on the ATP-induced transport inhibition. Acute reduction of extracellular [Ca2+] to either 100 nM or 0 nM by addition of EGTA had no effect on the ATP-induced transport inhibition. In the presence of the NO synthase (NOS) inhibitor L-NAME (100 μM) and/or ODQ to inhibit the guanylyl cyclase, the ATP effect remained unaffected. Increasing the concentration and incubation time for L-NAME (1 mM) still did not reveal any effect on the ATP-mediated transport inhibition. Acute addition of the NO donors SNAP (100 μM) and Spermine NONOate (10 μM) did not alter tubular transport. High concentrations of L-NAME (1 mM) in itself, however, reduced the transepithelial transport significantly. Thus, we find no evidence for nitric oxide (NO) as second messenger for P2X receptor-dependent transport inhibition in mTAL. Moreover, Ca2+ signaling appears not involved in the ATP-mediated effect. It remains undefined how P2X receptors trigger the marked reduction of transport in the TAL. PMID:28174542

  13. Acamprosate enhances N-methyl-D-apartate receptor-mediated neurotransmission but inhibits presynaptic GABA(B) receptors in nucleus accumbens neurons.

    PubMed

    Berton, F; Francesconi, W G; Madamba, S G; Zieglgänsberger, W; Siggins, G R

    1998-02-01

    Acamprosate (calcium acetylhomotaurine) is used therapeutically in Europe to reduce relapse in weaned alcoholics. However, the mechanisms of acamprosate action in the central nervous system are still obscure, although early studies suggested an action on GABA receptors. The nucleus accumbens (NAcc) is a brain region thought to underlie ethanol reinforcement. Recent studies from our laboratory have demonstrated that ethanol inhibits both N-methyl-D-aspartate (NMDA) and non-NMDA types of glutamatergic synaptic transmission in the NAcc. In the present study, we used voltage- and current-clamp intracellular recording of NAcc core neurons in a slice preparation to examine acamprosate actions on resting membrane properties and pharmacologically isolated synaptic responses. We isolated NMDA and non-NMDA receptor-mediated excitatory postsynaptic potentials or currents (EPSP/Cs) with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonovalerate (d-APV), respectively. Bicuculline was also included to block GABA(A) receptors. Superfusion of acamprosate (5, 50, and 300 microM) did not alter the resting membrane properties of NAcc neurons. However, 300 microM acamprosate significantly increased the NMDA receptor-mediated components of EPSP/Cs (NMDA-EPSP/Cs) with recovery on washout. In contrast, 300 microM acamprosate had no significant effect on the non-NMDA receptor component of the EPSP/Cs (non-NMDA-EPSP/Cs). To test acamprosate actions on the GABA system, we superfused 60 microM d-APV and 20 microM CNQX to block glutamatergic transmission and evoked monosynaptic GABA(A) receptor-mediated synaptic responses within the NAcc. Acamprosate (300 microM) did not change these monosynaptic GABA(A)-IPSCs. We also used a paired-pulse paradigm to test whether acamprosate could act on presynaptic GABA(B) autoreceptors, in the presence of d-APV and CNQX to block glutamatergic transmission. Like 0.5 microM CGP 34358 (a GABA[B] receptor blocker), acamprosate significantly

  14. Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

    SciTech Connect

    Matsui, Takanori; Yamagishi, Sho-ichi; Takeuchi, Masayoshi; Ueda, Seiji; Fukami, Kei; Okuda, Seiya

    2010-07-23

    Research highlights: {yields} Nifedipine inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma}. {yields} GW9662 treatment alone increased RAGE mRNA levels in tubular cells. {yields} Nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-{beta} gene expression in tubular cells, all of which were blocked by GW9662. -- Abstract: There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenic reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression

  15. Inhibition of testicular embryonal carcinoma cell tumorigenicity by peroxisome proliferator-activated receptor-β/δ- and retinoic acid receptor-dependent mechanisms.

    PubMed

    Yao, Pei-Li; Chen, Li Ping; Dobrzański, Tomasz P; Phillips, Dylan A; Zhu, Bokai; Kang, Boo-Hyon; Gonzalez, Frank J; Peters, Jeffrey M

    2015-11-03

    Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) has important physiological functions in control of cell growth, lipid and glucose homeostasis, differentiation and inflammation. To investigate the role of PPARβ/δ in cancer, stable human testicular embryonal carcinoma cell lines were developed that constitutively express PPARβ/δ. Expression of PPARβ/δ caused enhanced activation of the receptor, and this significantly decreased proliferation, migration, invasion, anchorage-independent growth, and also reduced tumor mass and volume of ectopic xenografts derived from NT2/D1 cells compared to controls. The changes observed in xenografts were associated with decreased PPARβ/δ-dependent expression of proliferating cell nuclear antigen and octamer-binding transcription factor-3/4, suggesting suppressed tumor proliferation and induction of differentiation. Inhibition of migration and invasion was mediated by PPARβ/δ competing with formation of the retinoic acid receptor (RAR)/retinoid X receptor (RXR) complex, resulting in attenuation of RARα-dependent matrix metalloproteinase-2 expression and activity. These results demonstrate that PPARβ/δ mediates attenuation of human testicular embryonal carcinoma cell progression through a novel RAR-dependent mechanism and suggest that activation of PPARβ/δ inhibits RAR/RXR dimerization and represents a new therapeutic strategy.

  16. Activation of mu-opioid receptors in the ventrolateral orbital cortex inhibits the GABAergic miniature inhibitory postsynaptic currents in rats.

    PubMed

    Qu, Chao-Ling; Huo, Fu-Quan; Huang, Fen-Sheng; Tang, Jing-Shi

    2015-04-10

    Previous studies have indicated that mu-opioid receptors in the ventrolateral orbital cortex (VLO) are involved in antinociception in tail flick tests and GABAergic neurons or terminals express mu-opioid receptors in the VLO. The current study examined the effect of selective mu-opioid receptor agonist DAMGO on the GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in the VLO in rats using the whole-cell patch clamp. The results demonstrated that 5 μM DAMGO application into the rat VLO slices significantly reduced the GABAergic mIPSCs frequency, without any effect on its amplitude, and this effect of DAMGO was reversed by pretreatment with selective mu-opioid receptor antagonist 1 μM CTOP. Importantly, application of CTOP alone into the VLO slices did not produce any effect on the frequency and amplitude of GABAergic mIPSCs. These results indicate a presynaptic effect of mu-opioid receptor activation on the GABAergic neurons in the VLO. The current data suggests that a presynaptic inhibition of the GABA release may contribute to the mu-opioid receptor mediated effects in the VLO and provides novel electrophysiological evidence for the underlying mechanisms of mu-opioid receptors in the VLO.

  17. Chronic FAAH inhibition during nicotine abstinence alters habenular CB1 receptor activity and precipitates depressive-like behaviors.

    PubMed

    Simonnet, A; Zamberletti, E; Cador, M; Rubino, T; Caillé, S

    2017-02-01

    The role of the endocannabinoid system in nicotine addiction is being increasingly acknowledged. Acute inhibition of anandamide (AEA) degradation efficiently reduces nicotine withdrawal-induced affective symptoms in rats and fatty acid amide hydrolase (FAAH), the degradation enzyme of AEA, has been proposed as a possible treatment against nicotine addiction. However, it is unclear whether chronic inhibition of AEA during nicotine abstinence will have beneficial or deleterious affective side-effects. Using a rat model of nicotine addiction, we found that, during abstinence, rats injected daily with a FAAH inhibitor (URB597) developed a depressive-like phenotype. Our results show that in the nicotine abstinent rats, URB597 induced low saccharin consumption, persistent immobility in the forced swim test and increased corticosterone levels in response to stress. In addition, URB597decreased CB1 receptor binding and activity in the habenula, a key structure in the control of nicotine-related emotional states. In contrast, non-treated abstinent rats showed increased CB1 receptor activity and behaviors comparable to controls. No FAAH inhibition-induced alterations were observed in animals that had a previous history of saline self-administration. Taken together, our results suggest that chronic FAAH inhibition prevents the homeostatic adaptations of habenular CB1 receptor function that are necessary for the recovery from nicotine dependence.

  18. Neurosteroid dehydroepiandrosterone sulphate inhibits persistent sodium currents in rat medial prefrontal cortex via activation of sigma-1 receptors.

    PubMed

    Cheng, Zheng-Xiang; Lan, Dan-Mei; Wu, Pei-Ying; Zhu, Yan-Hua; Dong, Yi; Ma, Lan; Zheng, Ping

    2008-03-01

    Dehydroepiandrosterone sulphate is one of the most important neurosteroids. In the present paper, we studied the effect of dehydroepiandrosterone sulphate on persistent sodium currents and its mechanism and functional consequence with whole-cell patch clamp recording method combined with a pharmacological approach in the rat medial prefrontal cortex slices. The results showed that dehydroepiandrosterone sulphate inhibited the amplitude of persistent sodium currents and the inhibitory effect was significant at 0.1 microM, reached maximum at 1 microM and decreased with the increase in the concentrations of above 1 microM. The effect of dehydroepiandrosterone sulphate on persistent sodium currents was canceled by the Gi protein inhibitor and the protein kinase C inhibitor, but not by the protein kinase A inhibitor. The effect of dehydroepiandrosterone sulphate on persistent sodium currents was also canceled by the sigma-1 receptor blockers and the sigma-1 receptor agonist could mimic the effect of dehydroepiandrosterone sulphate. Dehydroepiandrosterone sulphate had no significant influence on neuronal excitability but could significantly inhibit chemical inhibition of mitochondria-evoked increase in persistent sodium currents. These results suggest that dehydroepiandrosterone sulphate inhibits persistent sodium currents via the activation of sigma-1 receptors-Gi protein-protein kinase C-coupled signaling pathway, and the main functional consequence of this effect of DHEAS is presumably to protect neurons under ischemia.

  19. The Thyroid Hormone Receptors Inhibit Hepatic Interleukin-6 Signaling During Endotoxemia

    PubMed Central

    Contreras-Jurado, Constanza; Alonso-Merino, Elvira; Saiz-Ladera, Cristina; Valiño, Arturo José; Regadera, Javier; Alemany, Susana; Aranda, Ana

    2016-01-01

    Decreased thyroidal hormone production is found during lipopolysaccharide (LPS)-induced endotoxic shock in animals as well as in critically ill patients. Here we studied the role of the thyroid hormone receptors (TRs) in activation of STAT3, NF-κB and ERK, which play a key role in the response to inflammatory cytokines during sepsis. TR knockout mice showed down-regulation of hepatic inflammatory mediators, including interleukin 6 (IL-6) in response to LPS. Paradoxically, STAT3 and ERK activity were higher, suggesting that TRs could act as endogenous repressors of these pathways. Furthermore, hyperthyroidism increased cytokine production and mortality in response to LPS, despite decreasing hepatic STAT3 and ERK activity. This suggested that TRs could directly repress the response of the cells to inflammatory mediators. Indeed, we found that the thyroid hormone T3 suppresses IL-6 signalling in macrophages and hepatocarcinoma cells, inhibiting STAT3 activation. Consequently, the hormone strongly antagonizes IL-6-stimulated gene transcription, reducing STAT3 recruitment and histone acetylation at IL-6 target promoters. In conclusion, TRs are potent regulators of inflammatory responses and immune homeostasis during sepsis. Reduced responses to IL-6 should serve as a negative feedback mechanism for preventing deleterious effects of excessive hormone signaling during infections. PMID:27484112

  20. Oestradiol rapidly inhibits Ca2+ signals in ciliary neurons through classical oestrogen receptors in cytoplasm.

    PubMed

    Viso-León, M Carmen; Ripoll, Cristina; Nadal, Angel

    2004-10-01

    Oestrogen plays a key role in a great variety of actions in the nervous system, either through classical or alternative pathways. The classical pathways are initiated after oestrogen binding to the oestrogen receptors ERalpha or ERbeta, which translocate from the cytoplasm to the nucleus and act there as transcription factors. Alternative pathways are initiated at the plasma membrane and cytoplasm, via binding to classical or non-classical ERs. Using isolated ciliary ganglion neurons from the chick embryo and Ca2+ imaging, we demonstrated that a 10-min exposure to 17beta-oestradiol reduces Ca2+ influx through the plasma membrane. This effect was not reproduced by oestradiol conjugated to bovine serum albumin, which does not cross the plasma membrane, indicating that 17beta-oestradiol was acting intracellularly. ERalpha was detected in the cytoplasm by immunostaining and its involvement in the regulation of Ca2+ influx by ICI182,780 inhibition. The phosphatidylinositol-3 kinase (Pi3-kinase) inhibitor wortmannin and the nitric oxide synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) both blocked the oestradiol effect. The oestradiol effect was reproduced by 8Br-cGMP and abolished in the presence of the cGMP-dependent protein kinase (PKG) inhibitor KT5823. Our study indicates that 17beta-oestradiol can regulate Ca2+ influx via PI3-kinase, NOS and PKG after activation of cytoplasmic ER.

  1. Protective effects of genetic inhibition of Discoidin Domain Receptor 1 in experimental renal disease.

    PubMed

    Kerroch, Monique; Alfieri, Carlo; Dorison, Aude; Boffa, Jean-Jacques; Chatziantoniou, Christos; Dussaule, Jean-Claude

    2016-02-16

    Chronic kidney disease is a progressive incurable pathology affecting millions of people. Intensive investigations aim to identify targets for therapy. We have previously demonstrated that abnormal expression of the Discoidin Domain Receptor 1 (DDR1) is a key factor of renal disease by promoting inflammation and fibrosis. The present study investigates whether blocking the expression of DDR1 after the initiation of renal disease can delay or arrest the progression of this pathology. Severe renal disease was induced by either injecting nephrotoxic serum (NTS) or performing unilateral ureteral obstruction in mice, and the expression of DDR1 was inhibited by administering antisense oligodeoxynucleotides either at 4 or 8 days after NTS (corresponding to early or more established phases of disease, respectively), or at day 2 after ligation. DDR1 antisense administration at day 4 stopped the increase of proteinuria and protected animals against the progression of glomeruloneprhitis, as evidenced by functional, structural and cellular indexes. Antisense administration at day 8 delayed progression -but to a smaller degree- of renal disease. Similar beneficial effects on renal structure and inflammation were observed with the antisense administration of DDR1 after ureteral ligation. Thus, targeting DDR1 can be a promising strategy in the treatment of chronic kidney disease.

  2. Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid.

    PubMed

    Zhao, Mingyue; Lu, Lihui; Lei, Song; Chai, Hua; Wu, Siyuan; Tang, Xiaoju; Bao, Qinxue; Chen, Li; Wu, Wenchao; Liu, Xiaojing

    2016-01-01

    Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA.

  3. Inhibition of peroxisome proliferator-activated receptor (PPAR)-mediated keratinocyte differentiation by lipoxygenase inhibitors.

    PubMed Central

    Thuillier, Philippe; Brash, Alan R; Kehrer, James P; Stimmel, Julie B; Leesnitzer, Lisa M; Yang, Peiying; Newman, Robert A; Fischer, Susan M

    2002-01-01

    Lipoxygenase (LOX) metabolites from arachidonic acid and linoleic acid have been implicated in atherosclerosis, inflammation, keratinocyte differentiation and tumour progression. We previously showed that peroxisome proliferator-activated receptors (PPARs) play a role in keratinocyte differentiation and that the PPARalpha ligand 8S-hydroxyeicosatetraenoic acid is important in this process. We hypothesized that blocking LOX activity would block PPAR-mediated keratinocyte differentiation. Three LOX inhibitors, nordihydroguaiaretic acid, quercetin and morin, were studied for their effects on primary keratinocyte differentiation and PPAR activity. All three LOX inhibitors blocked calcium-induced expression of the differentiation marker keratin 1. In addition, activity of a PPAR-responsive element was inhibited in the presence of all three inhibitors, and this effect was mediated primarily through PPARalpha and PPARgamma. LOX inhibitors decreased the activity of a chimaeric PPAR-Gal4-ligand-binding domain reporter system and this effect was reversed by addition of PPAR ligands. Ligand-binding studies revealed that the LOX inhibitors bind directly to PPARs and demonstrate a novel mechanism for these inhibitors in altering PPAR-mediated gene expression. PMID:12069687

  4. Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit toll-like receptor 2 and 4 signaling.

    PubMed

    Kandasamy, Pitchaimani; Numata, Mari; Berry, Karin Zemski; Fickes, Rachel; Leslie, Christina C; Murphy, Robert C; Voelker, Dennis R

    2016-06-01

    The pulmonary surfactant phospholipid, 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), potently inhibits toll-like receptor (TLR)2 and TLR4 signaling from the cell surface of macrophages. Analogs of POPG that vary in polar head group length, hydroxylation, and alkyl branching were synthesized using a phospholipase D-catalyzed transphosphatidylation reaction and a 1-palmitoyl-2-oleoyl phosphatidylcholine substrate. Lipid analogs with C3 and C4 alkyl head group length (POP-propanol and POP-butanol) are less effective than POPG as TLR2 and TLR4 antagonists. However, adding a hydroxyl group at the alkyl chain 3- or 4-position (POP-propanediols or POP-butanediols) greatly increased their inhibitory effects against TLR2 and TLR4. POP-2',2'-dimethylpropanediol is a weak inhibitor of TLR2 and TLR4 activation that results in arachidonic acid release, but an effective inhibitor of TLR4 activation that results in TNF-α production. Addition of an amino group at the alkyl-2 position (POP-2'-aminopropanediol) completely abolished the antagonism of TLRs 2 and 4. Multiple analogs strongly bind to the TLR4 coreceptors, cluster of differentiation 14 (CD14) and myeloid differentiation 2, but competition for di[3-deoxy-D-manno-octulosonyl]-lipid A binding to CD14 is the best predictor of biological activity at the cellular level. Collectively, these findings identify new compounds for antagonizing TLR2 and TLR4 activation and define structural properties of POPG analogs for discriminating between two TLR systems.

  5. Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction

    PubMed Central

    Jiang, Changtao; Xie, Cen; Lv, Ying; Li, Jing; Krausz, Kristopher W.; Shi, Jingmin; Brocker, Chad N.; Desai, Dhimant; Amin, Shantu G.; Bisson, William H.; Liu, Yulan; Gavrilova, Oksana; Patterson, Andrew D.; Gonzalez, Frank J.

    2015-01-01

    The farnesoid X receptor (FXR) regulates bile acid, lipid and glucose metabolism. Here we show that treatment of mice with glycine-β-muricholic acid (Gly-MCA) inhibits FXR signalling exclusively in intestine, and improves metabolic parameters in mouse models of obesity. Gly-MCA is a selective high-affinity FXR inhibitor that can be administered orally and prevents, or reverses, high-fat diet-induced and genetic obesity, insulin resistance and hepatic steatosis in mice. The high-affinity FXR agonist GW4064 blocks Gly-MCA action in the gut, and intestine-specific Fxr-null mice are unresponsive to the beneficial effects of Gly-MCA. Mechanistically, the metabolic improvements with Gly-MCA depend on reduced biosynthesis of intestinal-derived ceramides, which directly compromise beige fat thermogenic function. Consequently, ceramide treatment reverses the action of Gly-MCA in high-fat diet-induced obese mice. We further show that FXR signalling in ileum biopsies of humans positively correlates with body mass index. These data suggest that Gly-MCA may be a candidate for the treatment of metabolic disorders. PMID:26670557

  6. Irreversible inhibition of epidermal growth factor receptor activity by 3-aminopropanamides.

    PubMed

    Carmi, Caterina; Galvani, Elena; Vacondio, Federica; Rivara, Silvia; Lodola, Alessio; Russo, Simonetta; Aiello, Stefania; Bordi, Fabrizio; Costantino, Gabriele; Cavazzoni, Andrea; Alfieri, Roberta R; Ardizzoni, Andrea; Petronini, Pier Giorgio; Mor, Marco

    2012-03-08

    Irreversible epidermal growth factor receptor (EGFR) inhibitors contain a reactive warhead which covalently interacts with a conserved cysteine residue in the kinase domain. The acrylamide fragment, a commonly employed warhead, effectively alkylates Cys797 of EGFR, but its reactivity can cause rapid metabolic deactivation or nonspecific reactions with off-targets. We describe here a new series of irreversible inhibitors containing a 3-aminopropanamide linked in position 6 to 4-anilinoquinazoline or 4-anilinoquinoline-3-carbonitrile driving portions. Some of these compounds proved to be as efficient as their acrylamide analogues in inhibiting EGFR-TK (TK = tyrosine kinase) autophosphorylation in A549 lung cancer cells. Moreover, several 3-aminopropanamides suppressed proliferation of gefitinib-resistant H1975 cells, harboring the T790M mutation in EGFR, at significantly lower concentrations than did gefitinib. A prototypical compound, N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(dimethylamino)propanamide (5), did not show covalent binding to cell-free EGFR-TK in a fluorescence assay, while it underwent selective activation in the intracellular environment, releasing an acrylamide derivative which can react with thiol groups.

  7. USP3 inhibits type I interferon signaling by deubiquitinating RIG-I-like receptors

    PubMed Central

    Cui, Jun; Song, Yanxia; Li, Yinyin; Zhu, Qingyuan; Tan, Peng; Qin, Yunfei; Wang, Helen Y; Wang, Rong-Fu

    2014-01-01

    Lysine 63 (K63)-linked ubiquitination of RIG-I plays a critical role in the activation of type I interferon pathway, yet the molecular mechanism responsible for its deubiquitination is still poorly understood. Here we report that the deubiquitination enzyme ubiquitin-specific protease 3 (USP3) negatively regulates the activation of type I interferon signaling by targeting RIG-I. Knockdown of USP3 specifically enhanced K63-linked ubiquitination of RIG-I, upregulated the phosphorylation of IRF3 and augmented the production of type I interferon cytokines and antiviral immunity. We further show that there is no interaction between USP3 and RIG-I-like receptors (RLRs) in unstimulated or uninfected cells, but upon viral infection or ligand stimulation, USP3 binds to the caspase activation recruitment domain of RLRs and then cleaves polyubiquitin chains through cooperation of its zinc-finger Ub-binding domain and USP catalytic domains. Mutation analysis reveals that binding of USP3 to polyubiquitin chains on RIG-I is a prerequisite step for its cleavage of polyubiquitin chains. Our findings identify a previously unrecognized role of USP3 in RIG-I activation and provide insights into the mechanisms by which USP3 inhibits RIG-I signaling and antiviral immunity. PMID:24366338

  8. The Thyroid Hormone Receptors Inhibit Hepatic Interleukin-6 Signaling During Endotoxemia.

    PubMed

    Contreras-Jurado, Constanza; Alonso-Merino, Elvira; Saiz-Ladera, Cristina; Valiño, Arturo José; Regadera, Javier; Alemany, Susana; Aranda, Ana

    2016-08-03

    Decreased thyroidal hormone production is found during lipopolysaccharide (LPS)-induced endotoxic shock in animals as well as in critically ill patients. Here we studied the role of the thyroid hormone receptors (TRs) in activation of STAT3, NF-κB and ERK, which play a key role in the response to inflammatory cytokines during sepsis. TR knockout mice showed down-regulation of hepatic inflammatory mediators, including interleukin 6 (IL-6) in response to LPS. Paradoxically, STAT3 and ERK activity were higher, suggesting that TRs could act as endogenous repressors of these pathways. Furthermore, hyperthyroidism increased cytokine production and mortality in response to LPS, despite decreasing hepatic STAT3 and ERK activity. This suggested that TRs could directly repress the response of the cells to inflammatory mediators. Indeed, we found that the thyroid hormone T3 suppresses IL-6 signalling in macrophages and hepatocarcinoma cells, inhibiting STAT3 activation. Consequently, the hormone strongly antagonizes IL-6-stimulated gene transcription, reducing STAT3 recruitment and histone acetylation at IL-6 target promoters. In conclusion, TRs are potent regulators of inflammatory responses and immune homeostasis during sepsis. Reduced responses to IL-6 should serve as a negative feedback mechanism for preventing deleterious effects of excessive hormone signaling during infections.

  9. Nanoparticles Containing a Liver X Receptor Agonist Inhibit Inflammation and Atherosclerosis

    PubMed Central

    Zhang, Xue-Qing; Even-Or, Orli; Xu, Xiaoyang; van Rosmalen, Mariska; Lim, Lucas; Gadde, Suresh

    2015-01-01

    Liver X receptor (LXR) signaling pathways regulate lipid metabolism and inflammation, which has generated widespread interest in developing synthetic LXR agonists as potential therapeutics for the management of atherosclerosis. In this study, we demonstrate that nanoparticles (NPs) containing the synthetic LXR agonist GW3965 (NP-LXR) exert anti-inflammatory effects and inhibit the development of atherosclerosis without causing hepatic steatosis. These NPs were engineered through self-assembly of a biodegradable diblock poly(lactide-co-glycolide)-b-poly(ethylene glycol) (PLGA-b-PEG) copolymer. NP-LXR was significantly more effective than free GW3965 at inducing LXR target gene expression and suppressing inflammatory factors in macrophages in vitro and in vivo. Addtionally, the NPs elicited negligible lipogenic gene stimulation in the liver. Using the Ldlr−/− mouse model of atherosclerosis, we saw abundant co-localization of fluorescently labeled NPs within plaque macrophages following systemic administration. Notably, six intravenous injections of NP-LXR over two weeks markedly reduced the CD68-positive cell (macrophage) content of plaques (by 50%) without increasing total cholesterol or triglycerides in the liver and plasma. Together, these findings identify GW3965-encapsulated PLGA-b-PEG NPs as a promising nanotherapeutic approach to combat atherosclerosis, providing the benefits of LXR agonists without their adverse effects on hepatic and plasma lipid metabolism. PMID:25156796

  10. Estrogen Receptor beta mediates decreased occlusal loading induced inhibition of chondrocyte maturation in female mice

    PubMed Central

    Polur, Ilona; Kamiya, Yosuke; Xu, Manshan; Cabri, Bianca S.; Alshabeeb, Marwa; Wadhwa, Sunil; Chen, Jing

    2015-01-01

    Objective Temporomandibular joint (TMJ) disorders predominantly afflict women, suggesting that estrogen may play a role in the disease process. Defects in mechanical loading-induced TMJ remodeling are believed to be a major etiological factor in TMJ degenerative disease. Previously, we found that, decreased occlusal loading caused a significant decrease in early chondrocyte maturation markers (Sox9 and Col 2) in female, but not male, C57BL/6 wild type mice (1). The goal of this study was to examine the role of Estrogen Receptor (ER) beta in mediating these effects. Design 21-day-old male (n=24) and female (n=25) ER beta KO mice were exposed to decreased occlusal loading (soft diet administration and incisor trimming) for 4 weeks. At 49 days of age the mice were sacrificed. Proliferation, gene expression, Col 2 immunohistochemistry and micro-CT analysis were performed on the mandibular condyles. Results Decreased occlusal loading triggered similar effects in male and female ER beta KO mice; specifically, significant decreases in Col 10 expression, subchondral total volume, bone volume, and trabecular number. Conclusion Decreased occlusal loading induced inhibition of chondrocyte maturation markers (Sox9 and Col 2) did not occur in female ER beta deficient mice. PMID:25791327

  11. Tonic Inhibition of Accumbal Spiny Neurons by Extrasynaptic α4βδ GABAA Receptors Modulates the Actions of Psychostimulants

    PubMed Central

    Maguire, Edward P.; Macpherson, Tom; Swinny, Jerome D.; Dixon, Claire I.; Herd, Murray B.; Belelli, Delia; Stephens, David N.

    2014-01-01

    Within the nucleus accumbens (NAc), synaptic GABAA receptors (GABAARs) mediate phasic inhibition of medium spiny neurons (MSNs) and influence behavioral responses to cocaine. We demonstrate that both dopamine D1- and D2-receptor-expressing MSNs (D-MSNs) additionally harbor extrasynaptic GABAARs incorporating α4, β, and δ subunits that mediate tonic inhibition, thereby influencing neuronal excitability. Both the selective δ-GABAAR agonist THIP and DS2, a selective positive allosteric modulator, greatly increased the tonic current of all MSNs from wild-type (WT), but not from δ−/− or α4−/− mice. Coupling dopamine and tonic inhibition, the acute activation of D1 receptors (by a selective agonist or indirectly by amphetamine) greatly enhanced tonic inhibition in D1-MSNs but not D2-MSNs. In contrast, prolonged D2 receptor activation modestly reduced the tonic conductance of D2-MSNs. Behaviorally, WT and constitutive α4−/− mice did not differ in their expression of cocaine-conditioned place preference (CPP). Importantly, however, mice with the α4 deletion specific to D1-expressing neurons (α4D1−/−) showed increased CPP. Furthermore, THIP administered systemically or directly into the NAc of WT, but not α4−/− or α4D1−/− mice, blocked cocaine enhancement of CPP. In comparison, α4D2−/− mice exhibited normal CPP, but no cocaine enhancement. In conclusion, dopamine modulation of GABAergic tonic inhibition of D1- and D2-MSNs provides an intrinsic mechanism to differentially affect their excitability in response to psychostimulants and thereby influence their ability to potentiate conditioned reward. Therefore, α4βδ GABAARs may represent a viable target for the development of novel therapeutics to better understand and influence addictive behaviors. PMID:24431441

  12. Resveratrol Inhibits Expression and Binding Activity of the Monocyte Chemotactic Protein-1 Receptor, CCR2, on THP-1 Monocytes

    PubMed Central

    Cullen, John P.; Morrow, David; Jin, Ying; von Offenberg Sweeney, Nicholas; Sitzmann, James V.; Cahill, Paul A.; Redmond, Eileen M.

    2007-01-01

    Monocyte chemotactic protein-1 and its receptor, CCR2, play a key role in atherosclerosis. We determined the effect of the polyphenol, resveratrol, on CCR2 and the mechanisms involved. Resveratrol treatment inhibited 125I-MCP-1 binding to THP-1 cells; 31%, 56%, 84% decrease for 10, 50 and 100 µM resveratrol, in the absence of any effect on receptor affinity. The inhibitory effect of resveratrol on 125I-MCP-1 binding to THP-1 cells and on CCR2 protein expression determined by FACS analysis was attenuated by treatment with L-NAME (NOS inhibitor), PD98059 (MAPK inhibitor) and LY294002 (PI3K inhibitor), whereas neither X/XO (reactive oxygen species generator) nor ICI182780 (estrogen receptor antagonist) had any effect. Concomitant with a decrease in CCR2 protein expression, resveratrol inhibited THP-1 CCR2 mRNA levels, in the absence of any effect on its stability; 26% and 45% inhibition at 10 and 50 µM resveratrol, respectively. This effect was not altered by co-treatment with L-NAME, PD98059 or ICI182780, but was potentiated by LY294002 and X/XO. Conclusions: Resveratrol inhibits monocyte CCR2 binding activity in an NO-, MAPK- and PI3K-dependent manner, whereas it inhibits CCR2 mRNA in an NO- and MAPK-independent, PI3K-dependent manner. These inhibitory effects of resveratrol on chemokine receptor binding and expression may contribute, in part, to its cardiovascular protective activity in vivo. PMID:17499741

  13. Exercise combined with low-level GABAA receptor inhibition up-regulates the expression of neurotrophins in the motor cortex.

    PubMed

    Takahashi, Kazuma; Maejima, Hiroshi; Ikuta, Gaku; Mani, Hiroki; Asaka, Tadayoshi

    2017-01-01

    Neurotrophins play a crucial role in neuroplasticity, neurogenesis, and neuroprotection in the central nervous system. Aerobic exercise is known to increase the expression of BDNF in the cerebral cortex. Several animal studies have evaluated the tonic inhibition of GABAergic synapses to enhance hippocampal plasticity as well as learning and memory, whereas the effects of GABAergic inhibition on plasticity in the cerebral cortex related to motor learning are not well characterized. The objective of the present study was to examine the interactive effect of low-level GABAA receptor inhibition and exercise on the expression of neurotrophins including BDNF in the murine motor cortex. ICR mice were randomly distributed among 4 groups based on two factors of GABAA receptor inhibition and exercise, i.e. control group, an exercise group, a bicuculline group, and an exercise plus bicuculline group. We administered GABAA receptor antagonist, bicuculline intraperitoneally to the mice (bicuculline and exercise plus bicuculline group) at a non-epileptic dose of 0.25mg/kg, whereas the mice (exercise and exercise plus bicuculline group) were exercised on a treadmill for 1h every day. After two week intervention, the expression of mRNA and protein abundance of neurotrophins in the motor cortex was assayed using Real time PCR and ELISA. BDNF gene expression was significantly increased by approximately 3-fold in the bicuculline group relative to the control, exercise, and bicuculline plus exercise groups. Protein abundance of BDNF expression was significantly increased by approximately 3-fold in the bicuculline plus exercise group relative to other groups. Therefore, the present study revealed that combined GABAA receptor inhibition and moderate aerobic exercise up-regulated BDNF protein expression in the motor cortex without producing side effects on motor or cognitive functions. Alterations in BDNF expression could positively contribute to plasticity by regulating the balance

  14. Involvement of supraspinal imidazoline receptors and descending monoaminergic pathways in tizanidine-induced inhibition of rat spinal reflexes.

    PubMed

    Kino, Yurika; Tanabe, Mitsuo; Honda, Motoko; Ono, Hideki

    2005-09-01

    The neuronal pathways involved in the muscle relaxant effect of tizanidine were examined by measurement of spinal reflexes in rats. Tizanidine (i.v. and intra-4th ventricular injection) decreased the mono- and disynaptic (the fastest polysynaptic) reflexes (MSR and DSR, respectively) in non-spinalized rats. Depletion of central noradrenaline by 6-hydroxydopamine abolished the depressant effect of tizanidine on the MSR almost completely and attenuated the effect on the DSR. Co-depletion of serotonin by 5,6-dihydroxytryptamine and noradrenaline resulted in more prominent attenuation of tizanidine-induced inhibition of the DSR. Supraspinal receptors were then studied using yohimbine- and some imidazoline-receptor ligands containing an imidazoline moiety. Idazoxan (I1, I2, I3, and alpha2), efaroxan (I1, I3, and alpha2), and RX821002 (I3 and alpha2), but not yohimbine, an alpha2-adrenergic receptor antagonist with no affinity for I receptors, antagonized the inhibitory effects of tizanidine. Thus, supraspinal I receptors (most likely I3) and descending monoaminergic influences are necessary for tizanidine-induced inhibition of spinal segmental reflexes.

  15. The MC3 receptor binding affinity of melanocortins correlates with the nitric oxide production inhibition in mice brain inflammation model.

    PubMed

    Muceniece, Ruta; Zvejniece, Liga; Liepinsh, Edgars; Kirjanova, Olga; Baumane, Larisa; Petrovska, Ramona; Mutulis, Felikss; Mutule, Ilze; Kalvinsh, Ivars; Wikberg, Jarl E S; Dambrova, Maija

    2006-06-01

    Melanocortins possess strong anti-inflammatory effects acting in the central nervous system via inhibition of the production of nitric oxide (NO) during brain inflammation. To shed more light into the role of melanocortin (MC) receptor subtypes involved we synthesized and evaluated some novel peptides, modified in the melanocyte-stimulating hormone (MSH) core structure, natural MCs and known MC receptor selective peptides - MS05, MS06. Since the study included both selective, high affinity binders and the novel peptides, it was possible to do the correlation analysis of binding activities and the NO induction-related anti-inflammatory effect of the peptides. beta-MSH, gamma1-MSH, gamma2-MSH, alpha-MSH, MS05, Ac-MS06 and Ac-[Ser12]MS06 caused dose dependent inhibition of the lipopolysaccharide (LPS)-induced increase of NO overproduction in the mice forebrain whereas MSH core modified peptides Ac-[Asp9,Ser12]MS06, [Asp9]alpha-MSH and [Asp16]beta-MSH were devoid of this effect in doses up to 10 nmol per mouse. When the minimal effective dose required for inhibition of NO production was correlated with the in vitro binding activity to MC receptor subtypes a strong and significant correlation was found for the MC3 receptor (r = 0.90; p = 0.0008), whereas weak correlation was present for the other receptors. Our results suggest that the MC3 receptor is the major player in mediating the anti-inflammatory activity of MCs in the central nervous system.

  16. The Role of cGMP on Adenosine A1 Receptor-mediated Inhibition of Synaptic Transmission at the Hippocampus

    PubMed Central

    Pinto, Isa; Serpa, André; Sebastião, Ana M.; Cascalheira, José F.

    2016-01-01

    Both adenosine A1 receptor and cGMP inhibit synaptic transmission at the hippocampus and recently it was found that A1 receptor increased cGMP levels in hippocampus, but the role of cGMP on A1 receptor-mediated inhibition of synaptic transmission remains to be established. In the present work we investigated if blocking the NOS/sGC/cGMP/PKG pathway using nitric oxide synthase (NOS), protein kinase G (PKG), and soluble guanylyl cyclase (sGC) inhibitors modify the A1 receptor effect on synaptic transmission. Neurotransmission was evaluated by measuring the slope of field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation at hippocampal slices. N6-cyclopentyladenosine (CPA, 15 nM), a selective A1 receptor agonist, reversibly decreased the fEPSPs by 54 ± 5%. Incubation of the slices with an inhibitor of NOS (L-NAME, 200 μM) decreased the CPA effect on fEPSPs by 57 ± 9% in female rats. In males, ODQ (10 μM), an sGC inhibitor, decreased the CPA inhibitory effect on fEPSPs by 23 ± 6%, but only when adenosine deaminase (ADA,1 U/ml) was present; similar results were found in females, where ODQ decreased CPA-induced inhibition of fEPSP slope by 23 ± 7%. In male rats, the presence of the PKG inhibitor (KT5823, 1 nM) decreased the CPA effect by 45.0 ± 9%; similar results were obtained in females, where KT5823 caused a 32 ± 9% decrease on the CPA effect. In conclusion, the results suggest that the inhibitory action of adenosine A1 receptors on synaptic transmission at hippocampus is, in part, mediated by the NOS/sGC/cGMP/PKG pathway. PMID:27148059

  17. The antinociceptive triterpene β-amyrin inhibits 2-arachidonoylglycerol (2-AG) hydrolysis without directly targeting cannabinoid receptors

    PubMed Central

    Chicca, A; Marazzi, J; Gertsch, J

    2012-01-01

    BACKGROUND AND PURPOSE Pharmacological activation of cannabinoid CB1 and CB2 receptors is a therapeutic strategy to treat chronic and inflammatory pain. It was recently reported that a mixture of natural triterpenes α- and β-amyrin bound selectively to CB1 receptors with a subnanomolar Ki value (133 pM). Orally administered α/β-amyrin inhibited inflammatory and persistent neuropathic pain in mice through both CB1 and CB2 receptors. Here, we investigated effects of amyrins on the major components of the endocannabinoid system. EXPERIMENTAL APPROACH We measured CB receptor binding interactions of α- and β-amyrin in validated binding assays using hCB1 and hCB2 transfected CHO-K1 cells. Effects on endocannabinoid transport in U937 cells and breakdown using homogenates of BV2 cells and pig brain, as well as purified enzymes, were also studied. KEY RESULTS There was no binding of either α- or β-amyrin to hCB receptors in our assays (Ki > 10 µM). The triterpene β-amyrin potently inhibited 2-arachidonoyl glycerol (2-AG) hydrolysis in pig brain homogenates, but not that of anandamide. Although β-amyrin only weakly inhibited purified human monoacylglycerol lipase (MAGL), it also inhibited α,β-hydrolases and more potently inhibited 2-AG breakdown than α-amyrin and the MAGL inhibitor pristimerin in BV2 cell and pig brain homogenates. CONCLUSIONS AND IMPLICATIONS We propose that β-amyrin exerts its analgesic and anti-inflammatory pharmacological effects via indirect cannabimimetic mechanisms by inhibiting the degradation of the endocannabinoid 2-AG without interacting directly with CB receptors. Triterpenoids appear to offer a very broad and largely unexplored scaffold for inhibitors of the enzymic degradation of 2-AG. LINKED ARTICLES This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8 PMID:22646533

  18. Structural basis of the anti-inflammatory activity of quercetin: inhibition of the 5-hydroxytryptamine type 2 receptor.

    PubMed

    Rotelli, Alejandra Ester; Aguilar, Carlos Fernando; Pelzer, Lilian Eugenia

    2009-09-01

    The anti-inflammatory activity of quercetin was evaluated through serotonin-induced rat-paw edema. The experiments showed that quercetin had an important effect on acute inflammatory processes. Docking of serotonin and quercetin into the homology model of the 5-Hydroxytryptamine Type 2 Receptor allowed to analyze the structural basis of the anti-inflammatory activity. Results showed that serotonin and quercetin bind in the same region of the active site with a similar binding energy but quercetin has a much bigger inhibition constant. Therefore, it seems possible that quercetin may act as a natural inhibitor of the receptor blocking the acute inflammation generated by serotonin.

  19. The CCK(2) receptor antagonist, YF476, inhibits Mastomys ECL cell hyperplasia and gastric carcinoid tumor development.

    PubMed

    Kidd, M; Siddique, Z-L; Drozdov, I; Gustafsson, B I; Camp, R L; Black, J W; Boyce, M; Modlin, I M

    2010-06-08

    YF476 is a potent and highly selective cholecystokin 2 (CCK(2)) receptor antagonist of the benzodiazepine class. It inhibits gastric neuroendocrine enterochromaffin-like (ECL) cell secretion, proliferation and spontaneous formation of gastric neuroendocrine tumors (carcinoids) in cotton rats. The Mastomys rodent species exhibits a genetic predisposition to gastric ECL neuroendocrine tumor formation which can be accelerated by acid suppression and induction of hypergastrinemia. In this respect, it mimics the human condition of atrophic gastritis, hypergastrinemia and gastric carcinoid development. We investigated whether YF476 could inhibit acid suppression-induced ECL cell hyperplasia and neoplasia in this model. In addition, we examined whether YF476 could reverse established ECL cell hyperplasia and neoplasia. Targeting the CCK(2) receptor during Loxtidine-induced hypergastrinemia resulted in a reduction in ECL cell secretion (plasma and mucosal histamine, and histidine decarboxylase (HDC) transcripts, p<0.05) and proliferation (numbers of HDC-positive cells, connective tissue growth factor (CTGF) and cyclin D1 transcription). This was associated with a decrease in ECL cell hyperplasia and a 60% reduction in gastric ECL cell microcarcinoid (tumors <0.3mm in size) formation. YF476 inhibited ECL cell neoplasia (gastric carcinoid) in animals with hyperplasia, inhibited the formation of ECL cell tumors when co-administered with Loxtidine and reversed the growth and developement of gastric ECL cell carcinoids in long-term acid suppressed Mastomys. Variable importance analysis using a logistic multinomial regression model indicated the effects of YF476 were specific to the ECL cell and alterations in ECL cell function reflected inhibition of transcripts for HDC, Chromogranin A (CgA), CCK(2) and the autocrine growth factor, CTGF. We conclude that specifically targeting the CCK(2) receptor inhibits gastrin-mediated ECL cell secretion and ECL cell proliferation and tumor

  20. Novel selective glucocorticoid receptor agonists (SEGRAs) with a covalent warhead for long-lasting inhibition.

    PubMed

    Ryabtsova, Oksana; Joossens, Jurgen; Van Der Veken, Pieter; Vanden Berghe, Wim; Augustyns, Koen; De Winter, Hans

    2016-10-15

    The synthesis and in vitro properties of six analogues of the selective glucocorticoid receptor (GR) agonist GSK866, bearing a warhead for covalent linkage to the glucocorticoid receptor, is described.

  1. Mannose-inhibitable adhesins and T3-T7 receptors of Klebsiella pneumoniae inhibit phagocytosis and intracellular killing by human polymorphonuclear leukocytes.

    PubMed Central

    Pruzzo, C; Debbia, E; Satta, G

    1982-01-01

    It has recently been shown that Klebsiella pneumoniae strains adhere to human epithelial cells and that adherence is mediated by mannose-inhibitable adhesins which are also receptors for coliphages T3 and T7. We have now found that Klebsiella strain K59, which adheres to human epithelial cells and carries the receptors for coliphages T3 and T7, adheres to human polymorphonuclear leukocytes (PMN) at 4 degrees C. Strains KRTT1 and KRTT2, which are spontaneous mutants unable to adsorb coliphages T3 and T7 and adhere to human epithelial cells, at this temperature did not adhere to PMN. Adherence of K59 cells to PMN at 4 degrees C was inhibited by D-mannose, by UV-inactivated T7 phages, and by pepsin-digested anti-K59 antibodies absorbed with KRTT1 cells. At 37 degrees C the number of PMN with KRTT bacteria associated was fourfold higher than at 4 degrees C. On the contrary, the number of PMN with K59 bacteria associated at this temperature was fourfold lower than at 4 degrees C. Phagocytosis and intracellular killing experiments performed at 37 degrees C showed that KRTT1 and KRTT2 were phagocytized and killed at a higher rate than K59. After blocking of the mannose-inhibitable adhesins and T3-T7 receptors (MIAT) by D-mannose, UV-inactivated bacteriophage T7, or specific antibodies, K59 cells became more sensitive to phagocytosis and intracellular killing at 37 degrees C. K59 cells lysogenic for prophage AP3 were approximately as sensitive to phagocytosis and intracellular killing by human PMN as strains KRTT1 and KRTT2. Unencapsulated Klebsiella strains isolated from clinical specimens were found to carry MIAT most often. Four such strains were found much more resistant to phagocytosis and intracellular killing than their spontaneous mutants resistant to bacteriophages T3 and T7. PMID:7047402

  2. Activated niacin receptor HCA2 inhibits chemoattractant-mediated macrophage migration via Gβγ/PKC/ERK1/2 pathway and heterologous receptor desensitization

    PubMed Central

    Shi, Ying; Lai, Xiangru; Ye, Lingyan; Chen, Keqiang; Cao, Zheng; Gong, Wanghua; Jin, Lili; Wang, Chunyan; Liu, Mingyong; Liao, Yuan; Wang, Ji Ming; Zhou, Naiming

    2017-01-01

    The niacin receptor HCA2 is implicated in controlling inflammatory host responses with yet poorly understood mechanistic basis. We previously reported that HCA2 in A431 epithelial cells transduced Gβγ-protein kinase C- and Gβγ-metalloproteinase/EGFR-dependent MAPK/ERK signaling cascades. Here, we investigated the role of HCA2 in macrophage-mediated inflammation and the underlying mechanisms. We found that proinflammatory stimulants LPS, IL-6 and IL-1β up-regulated the expression of HCA2 on macrophages. Niacin significantly inhibited macrophage chemotaxis in response to chemoattractants fMLF and CCL2 by disrupting polarized distribution of F-actin and Gβ protein. Niacin showed a selected additive effect on chemoattractant-induced activation of ERK1/2, JNK and PI3K pathways, but only the MEK inhibitor UO126 reduced niacin-mediated inhibition of macrophage chemotaxis, while activation of ERK1/2 by EGF alone did not inhibit fMLF-mediated migration of HEK293T cells co-expressing HCA2 and fMLF receptor FPR1. In addition, niacin induced heterologous desensitization and internalization of FPR1. Furthermore, niacin rescued mice from septic shock by diminishing inflammatory symptoms and the effect was abrogated in HCA2−/− mice. These results suggest that Gβγ/PKC-dependent ERK1/2 activation and heterologous desensitization of chemoattractant receptors are involved in the inhibition of chemoattractant-induced migration of macrophages by niacin. Thus, HCA2 plays a critical role in host protection against pro-inflammatory insults. PMID:28186140

  3. The random-coil 'C' fragment of the dihydropyridine receptor II-III loop can activate or inhibit native skeletal ryanodine receptors.

    PubMed Central

    Haarmann, Claudia S; Green, Daniel; Casarotto, Marco G; Laver, Derek R; Dulhunty, Angela F

    2003-01-01

    The actions of peptide C, corresponding to (724)Glu-Pro(760) of the II-III loop of the skeletal dihydropyridine receptor, on ryanodine receptor (RyR) channels incorporated into lipid bilayers with the native sarcoplasmic reticulum membrane show that the peptide is a high-affinity activator of native skeletal RyRs at cytoplasmic concentrations of 100 nM-10 microM. In addition, we found that peptide C inhibits RyRs in a voltage-independent manner when added for longer times or at higher concentrations (up to 150 microM). Peptide C had a random-coil structure indicating that it briefly assumes a variety of structures, some of which might activate and others which might inhibit RyRs. The results suggest that RyR activation and inhibition by peptide C arise from independent stochastic processes. A rate constant of 7.5 x 10(5) s(-1).M(-1) was obtained for activation and a lower estimate for the rate constant for inhibition of 5.9 x 10(3) s(-1).M(-1). The combined actions of peptide C and peptide A (II-III loop sequence (671)Thr-Leu(690)) showed that peptide C prevented activation but not blockage of RyRs by peptide A. We suggest that the effects of peptide C indicate functional interactions between a part of the dihydropyridine receptor and the RyR. These interactions could reflect either dynamic changes that occur during excitation-contraction coupling or interactions between the proteins at rest. PMID:12620094

  4. 5-HT7 receptor activation inhibits mechanical hypersensitivity secondary to capsaicin sensitization in mice.

    PubMed

    Brenchat, Alex; Romero, Luz; García, Mónica; Pujol, Marta; Burgueño, Javier; Torrens, Antoni; Hamon, Michel; Baeyens, José Manuel; Buschmann, Helmut; Zamanillo, Daniel; Vela, José Miguel

    2009-02-01

    This work aimed to evaluate the potential role of the 5-HT(7) receptor in nociception secondary to a sensitizing stimulus in mice. For this purpose, the effects of relevant ligands (5-HT(7) receptor agonists: AS-19, MSD-5a, E-55888; 5-HT(7) receptor antagonists: SB-258719, SB-269970; 5-HT(1A) receptor agonist: F-13640; 5-HT(1A) receptor antagonist: WAY-100635) were assessed on capsaicin-induced mechanical hypersensitivity, a pain behavior involving hypersensitivity of dorsal horn neurons (central sensitization). For the 5-HT(7) receptor agonists used, binding profile and intrinsic efficacy to stimulate cAMP formation in HEK-293F cells expressing the human 5-HT(7) receptor were also evaluated. AS-19 and E-55888 were selective for 5-HT(7) receptors. E-55888 was a full agonist whereas AS-19 and MSD-5a behaved as partial agonists, with maximal effects corresponding to 77% and 61%, respectively, of the cAMP response evoked by the full agonist 5-HT. Our in vivo results revealed that systemic administration of 5-HT(7) receptor agonists exerted a clear-cut dose-dependent antinociceptive effect that was prevented by 5-HT(7) receptor antagonists, but not by the 5-HT(1A) receptor antagonist. The order of efficacy (E-55888>AS-19>MSD-5a) matched their in vitro efficacy as 5-HT(7) receptor agonists. Contrary to agonists, a dose-dependent promotion of mechanical hypersensitivity was observed after administration of 5-HT(7) receptor antagonists, substantiating the involvement of the 5-HT(7) receptor in the control of capsaicin-induced mechanical hypersensitivity. These findings suggest that serotonin exerts an inhibitory role in the control of nociception through activation of 5-HT(7) receptors, and point to a new potential therapeutic use of 5-HT(7) receptor agonists in the field of analgesia.

  5. Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

    SciTech Connect

    Tesmer, Valerie M.; Lennarz, Sabine; Mayer, Günter; Tesmer, John J.G.

    2012-08-31

    Cardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic {alpha}F-{alpha}G loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high-affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase.

  6. Inhibition of ErbB receptors, Hedgehog and NF-kappaB signaling by polyphenols in cancer.

    PubMed

    Benvenuto, Monica; Fantini, Massimo; Masuelli, Laura; De Smaele, Enrico; Zazzeroni, Francesca; Tresoldi, Ilaria; Calabrese, Giorgio; Galvano, Fabio; Modesti, Andrea; Bei, Roberto

    2013-06-01

    Carcinogenesis is a multi-step process triggered by cumulative genetic alterations, which drive the progressive transformation of a normal cell into a cancer cell. Among the signal transduction pathways whose cross-talk plays an important role in neoplastic transformation are those mediated by ErbB receptors, NF-kappaB and the Hedgehog (HH)/glioma-associated oncogene (GLI) cascade. Polyphenols can be employed to inhibit the growth of cancer cells due to their ability to modulate the activity of multiple targets involved in carcinogenesis through simultaneous direct interaction or modulation of gene expression. This review will describe the cross-talk between ErbB receptors, NF-kappaB and the Hedgehog (HH)/glioma-associated oncogene (GLI) signaling pathways and the potential role of polyphenols in inhibiting this dialogue and the growth of cancer cells.

  7. Inhibition of a slow synaptic response by a metabotropic glutamate receptor antagonist in hippocampal CA3 pyramidal cells.

    PubMed

    Gerber, U; Lüthi, A; Gähwiler, B H

    1993-11-22

    The effects of a novel antagonist of metabotropic glutamate receptors were investigated in CA3 pyramidal cells in hippocampal slice cultures of the rat. Earlier experiments showed that selective activation of metabotropic glutamate receptors with low concentrations of an agonist, 1S, 3R-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD), induced an inward current associated with a decrease in membrane conductance and inhibition of the slow calcium-dependent potassium current. These responses were strongly and reversibly reduced by the antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 0.5-1 mM). In the presence of antagonists of ionotropic glutamate receptors, stimulation of the afferent mossy fibres evoked postsynaptic responses in CA3 pyramidal cells which paralleled those observed with exogenously applied metabotropic glutamate receptor agonists, i.e. a slow inward current and a reduction of calcium-dependent potassium current. Both responses were greatly reduced by bath-applied MCPG (1 mM). These results show that MCPG acts as an effective antagonist at metabotropic glutamate receptors coupled to potassium conductances in the hippocampus. Furthermore, they confirm that glutamate release from presynaptic terminals can modulate postsynaptic properties by activation of metabotropic glutamate receptors.

  8. Acute ethanol administration inhibits Toll-like receptor 4 signaling pathway in rat intestinal epithelia.

    PubMed

    Zhou, Chao; Zhao, Ji; Li, Jing; Wang, Haiying; Tang, Chengwei

    2013-05-01

    Excess alcohol intake, as in binge drinking, increases susceptibility to microbial pathogens. Alcohol impairs macrophage function by suppression of the Toll-like receptor 4 (TLR4) pathway. This study investigated the effects of acute ethanol intake on the TLR4 pathway in rat intestinal epithelia, which usually encounters luminal antigens at first and participates in the development of intestinal immunity. Twenty Wistar rats were randomly assigned to an ethanol group given ethanol as a 25% (v/v) solution in water at 7.5 g/kg, or a control group given saline, by oral gavage daily for 3 days. The epithelial histology and ultrastructure, the intestinal microflora, peripheral and portal venous plasma lipopolysaccharide (LPS) levels, and somatostatin (SST) levels in the peripheral plasma and small intestine were evaluated. Somatostatin receptor 2 (SSTR2), TLR4, TANK binding kinase-1 (TBK1), activated nuclear factor-κB (NF-κB), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the intestinal mucosa were assayed. LPS responsiveness with or without SST pretreatment was assayed in vitro by quantification of TLR4, TBK1, activated NF-κB, IFN-γ and TNF-α in isolated intestinal epithelia. Mucosal damage was observed in the ethanol group by light and electron microscopy. Escherichia coli cultures were unchanged in rat intestine of the ethanol group compared with controls, but lactobacilli cultures were reduced (p < 0.05). LPS levels increased in peripheral and portal venous plasma (p < 0.05), but mucosal TLR4, TBK1, nuclear NF-κB, IFN-γ and TNF-α were unchanged in the ethanol group. LPS treatment in vitro up-regulated the level of TLR4, TBK1 and nuclear NF-κB as well as the production of IFN-γ and TNF-α in isolated intestinal epithelia in the control (p < 0.05), but not the ethanol group. The stimulatory effects of LPS on intestinal epithelia isolated from the control group were significantly inhibited by SST pretreatment (p < 0.05). The

  9. Estradiol rapidly attenuates ORL-1 receptor-mediated inhibition of proopiomelanocortin neurons via Gq-coupled, membrane-initiated signaling

    PubMed Central

    Conde, Kristie; Meza, Cecilia; Kelly, Martin J.; Sinchak, Kevin; Wagner, Edward J.

    2016-01-01

    Estradiol rapidly regulates the activity of arcuate nucleus (ARH) proopiomelanocortin (POMC) neurons that project to the medial preoptic nucleus (MPN) to regulate lordosis. Orphanin FQ/nociceptin (OFQ/N) acts via opioid receptor-like (ORL)-1 receptors to inhibit these POMC neurons. Therefore, we tested the hypothesis that estradiol excites POMC neurons by rapidly attenuating inhibitory ORL-1 signaling in these cells. Hypothalamic slices through the ARH were prepared from ovariectomized rats injected with Fluorogold into the MPN. Electrophysiologic recordings were generated in ARH neurons held at or near −60 mV, and neuronal phenotype was determined posthoc by immunohistofluorescence. OFQ/N application induced robust outward currents and hyperpolarizations via GIRK channels that were attenuated by pretreatment with either 17-β estradiol (E2) or E2 conjugated to bovine serum albumin. This was blocked by the estrogen receptor (ER) antagonist ICI 182,780, and mimicked by the Gq-coupled, membrane ER (Gq-mER) ligand STX and the ERα agonist PPT. Inhibiting phosphatidylinositol-3-kinase (PI3K) blocked the estrogenic attenuation of ORL-1/GIRK currents. Antagonizing either phospholipase C (PLC), protein kinase C (PKC), protein kinase A (PKA) or neuronal nitric oxide synthase (nNOS) also abrogated E2 inhibition of ORL-1/GIRK currents, whereas activation of PKC, PKA, protein kinase B (Akt) and nNOS substrate L-arginine all attenuated the OFQ/N response. This was observed in 92 MPN-projecting, POMC-positive ARH neurons. Thus, ORL-1 receptor-mediated inhibition of POMC neurons is rapidly and negatively modulated by E2, an effect which is stereoselective and membrane initiated via Gq-coupled mER and ERα activation that signals through PLC, PKC, PKA, PI3K and nNOS. PMID:26765570

  10. Identification of a small peptide that inhibits PCSK9 protein binding to the low density lipoprotein receptor.

    PubMed

    Zhang, Yingnan; Eigenbrot, Charles; Zhou, Lijuan; Shia, Steven; Li, Wei; Quan, Clifford; Tom, Jeffrey; Moran, Paul; Di Lello, Paola; Skelton, Nicholas J; Kong-Beltran, Monica; Peterson, Andrew; Kirchhofer, Daniel

    2014-01-10

    PCSK9 (proprotein convertase subtilisin/kexin type 9) is a negative regulator of the hepatic LDL receptor, and clinical studies with PCSK9-inhibiting antibodies have demonstrated strong LDL-c-lowering effects. Here we screened phage-displayed peptide libraries and identified the 13-amino acid linear peptide Pep2-8 as the smallest PCSK9 inhibitor with a clearly defined mechanism of inhibition that has been described. Pep2-8 bound to PCSK9 with a KD of 0.7 μm but did not bind to other proprotein convertases. It fully restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. The crystal structure of Pep2-8 bound to C-terminally truncated PCSK9 at 1.85 Å resolution showed that the peptide adopted a strand-turn-helix conformation, which is remarkably similar to its solution structure determined by NMR. Consistent with the functional binding site identified by an Ala scan of PCSK9, the structural Pep2-8 contact region of about 400 Å(2) largely overlapped with that contacted by the EGF(A) domain of the LDL receptor, suggesting a competitive inhibition mechanism. Consistent with this, Pep2-8 inhibited LDL receptor and EGF(A) domain binding to PCSK9 with IC50 values of 0.8 and 0.4 μm, respectively. Remarkably, Pep2-8 mimicked secondary structural elements of the EGF(A) domain that interact with PCSK9, notably the β-strand and a discontinuous short α-helix, and it engaged in the same β-sheet hydrogen bonds as EGF(A) does. Although Pep2-8 itself may not be amenable to therapeutic applications, this study demonstrates the feasibility of developing peptidic inhibitors to functionally relevant sites on PCSK9.

  11. Estradiol Rapidly Attenuates ORL-1 Receptor-Mediated Inhibition of Proopiomelanocortin Neurons via Gq-Coupled, Membrane-Initiated Signaling.

    PubMed

    Conde, Kristie; Meza, Cecilia; Kelly, Martin J; Sinchak, Kevin; Wagner, Edward J

    2016-01-01

    Estradiol rapidly regulates the activity of arcuate nucleus (ARH) proopiomelanocortin (POMC) neurons that project to the medial preoptic nucleus (MPN) to regulate lordosis. Orphanin FQ/nociceptin (OFQ/N) acts via opioid receptor-like (ORL)-1 receptors to inhibit these POMC neurons. Therefore, we tested the hypothesis that estradiol excites POMC neurons by rapidly attenuating inhibitory ORL-1 signaling in these cells. Hypothalamic slices through the ARH were prepared from ovariectomized rats injected with Fluorogold into the MPN. Electrophysiological recordings were generated in ARH neurons held at or near -60 mV, and neuronal phenotype was determined post hoc by immunohistofluorescence. OFQ/N application induced robust outward currents and hyperpolarizations via G protein-gated, inwardly rectifying K+ (GIRK) channels that were attenuated by pretreatment with either 17-β estradiol (E2) or E2 conjugated to bovine serum albumin. This was blocked by the estrogen receptor (ER) antagonist ICI 182,780 and mimicked by the Gq-coupled membrane ER (Gq-mER) ligand STX and the ERα agonist PPT. Inhibiting phosphatidylinositol-3-kinase (PI3K) blocked the estrogenic attenuation of ORL-1/GIRK currents. Antagonizing either phospholipase C (PLC), protein kinase C (PKC), protein kinase A (PKA) or neuronal nitric oxide synthase (nNOS) also abrogated E2 inhibition of ORL-1/GIRK currents, whereas activation of PKC, PKA, protein kinase B (Akt) and nNOS substrate L-arginine all attenuated the OFQ/N response. This was observed in 92 MPN-projecting, POMC-positive ARH neurons. Thus, ORL-1 receptor-mediated inhibition of POMC neurons is rapidly and negatively modulated by E2, an effect which is stereoselective and membrane initiated via Gq-mER and ERα activation that signals through PLC, PKC, PKA, PI3K and nNOS.

  12. Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction

    PubMed Central

    Cinalli, A R; Guarracino, J F; Fernandez, V; Roquel, L I; Losavio, A S

    2013-01-01

    Background and Purpose The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved. Experimental Approach End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques. Key Results Inosine (100 μM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects inhibited by the selective A3 receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A3 receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd2+, the removal of extracellular Ca2+ and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the Gi/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62. Conclusion and Implications Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by activating A3 receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A3 receptors appear to be coupled to Gi/o protein. PKC and calmodulin may be involved in these effects of inosine. PMID:23731236

  13. The antiemetic 5-HT3 receptor antagonist Palonosetron inhibits substance P-mediated responses in vitro and in vivo.

    PubMed

    Rojas, Camilo; Li, Ying; Zhang, Jie; Stathis, Marigo; Alt, Jesse; Thomas, Ajit G; Cantoreggi, Sergio; Sebastiani, Silvia; Pietra, Claudio; Slusher, Barbara S

    2010-11-01

    Palonosetron is the only 5-HT(3) receptor antagonist approved for the treatment of delayed chemotherapy-induced nausea and vomiting (CINV) in moderately emetogenic chemotherapy. Accumulating evidence suggests that substance P (SP), the endogenous ligand acting preferentially on neurokinin-1 (NK-1) receptors, not serotonin (5-HT), is the dominant mediator of delayed emesis. However, palonosetron does not bind to the NK-1 receptor. Recent data have revealed cross-talk between the NK-1 and 5HT(3) receptor signaling pathways; we postulated that if palonosetron differentially inhibited NK-1/5-HT(3) cross-talk, it could help explain its efficacy profile in delayed emesis. Consequently, we evaluated the effect of palonosetron, granisetron, and ondansetron on SP-induced responses in vitro and in vivo. NG108-15 cells were preincubated with palonosetron, granisetron, or ondansetron; antagonists were removed and the effect on serotonin enhancement of SP-induced calcium release was measured. In the absence of antagonist, serotonin enhanced SP-induced calcium-ion release. After preincubation with palonosetron, but not ondansetron or granisetron, the serotonin enhancement of the SP response was inhibited. Rats were treated with cisplatin and either palonosetron, granisetron, or ondansetron. At various times after dosing, single neuronal recordings from nodose ganglia were collected after stimulation with SP; nodose ganglia neuronal responses to SP were enhanced when the animals were pretreated with cisplatin. Palonosetron, but not ondansetron or granisetron, dose-dependently inhibited the cisplatin-induced SP enhancement. The results are consistent with previous data showing that palonosetron exhibits distinct pharmacology versus the older 5-HT(3) receptor antagonists and provide a rationale for the efficacy observed with palonosetron in delayed CINV in the clinic.

  14. Puerarin ameliorates experimental alcoholic liver injury by inhibition of endotoxin gut leakage, Kupffer cell activation, and endotoxin receptors expression.

    PubMed

    Peng, Jing-Hua; Cui, Tuan; Huang, Fu; Chen, Liang; Zhao, Yu; Xu, Lin; Xu, Li-Li; Feng, Qin; Hu, Yi-Yang

    2013-03-01

    Puerarin, an isoflavone component extracted from Kudzu (Pueraria lobata), has been demonstrated to alleviate alcohol-related disorders. Our study examined whet