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Sample records for p2 receptor-mediated signaling

  1. P2 receptor-mediated signaling in mast cell biology.

    PubMed

    Bulanova, Elena; Bulfone-Paus, Silvia

    2010-03-01

    Mast cells are widely recognized as effector cells of allergic inflammatory reactions. They contribute to the pathogenesis of different chronic inflammatory diseases, wound healing, fibrosis, thrombosis/fibrinolysis, and anti-tumor immune responses. In this paper, we summarized the role of P2X and P2Y receptors in mast cell activation and effector functions. Mast cells are an abundant source of ATP which is stored in their granules and secreted upon activation. We discuss the contribution of mast cells to the extracellular ATP release and to the maintenance of extracellular nucleotides pool. Recent publications highlight the importance of purinergic signaling for the pathogenesis of chronic airway inflammation. Therefore, the role of ATP and P2 receptors in allergic inflammation with focus on mast cells was analyzed. Finally, ATP functions as mast cell autocrine/paracrine factor and as messenger in intercellular communication between mast cells, nerves, and glia in the central nervous system.

  2. P2Y6 Receptor-Mediated Proinflammatory Signaling in Human Bronchial Epithelia

    PubMed Central

    Hao, Yuan; Liang, Jocelyn F.; Chow, Alison W.; Cheung, Wing-tai; Ko, Wing-hung

    2014-01-01

    P2Y receptors are expressed in virtually all epithelia and are responsible for the control of fluid and electrolyte transport. In asthmatic inflammation, the bronchial epithelia are damaged by eosinophil-derived, highly toxic cationic proteins, such as major basic protein (MBP). Consequently, extracellular nucleotides are released into the extracellular space from airway epithelial cells, and act in an autocrine or paracrine fashion to regulate immune functions. Our data show damage to the human bronchial epithelial cell line, 16HBE14o-, by poly-L-arginine-induced UDP release into the extracellular medium. Activation of P2Y6 receptor by its natural ligand, UDP, or its specific agonist, MRS 2693, led to the production of two proinflammatory cytokines, interleukin (IL)-6 and IL-8. This may have resulted from increased IL-6 and IL-8 mRNA expression, and activation of p38 and ERK1/2 MAPK, and NF-κB pathways. Our previous study demonstrated that UDP stimulated transepithelial Cl− secretion via both Ca2+- and cAMP-dependent pathways in 16HBE14o- epithelia. This was further confirmed in this study by simultaneous imaging of Ca2+ and cAMP levels in single cells using the Fura-2 fluorescence technique and a FRET-based approach, respectively. Moreover, the P2Y6 receptor-mediated production of IL-6 and IL-8 was found to be dependent on Ca2+, but not the cAMP/PKA pathway. Together, these studies show that nucleotides released during the airway inflammatory processes will activate P2Y6 receptors, which will lead to further release of inflammatory cytokines. The secretion of cytokines and the formation of such “cytokine networks” play an important role in sustaining the airway inflammatory disease. PMID:25243587

  3. Receptor-mediated signaling in Aspergillus fumigatus

    PubMed Central

    Grice, C. M.; Bertuzzi, M.; Bignell, E. M.

    2013-01-01

    Aspergillus fumigatus is the most pathogenic species among the Aspergilli, and the major fungal agent of human pulmonary infection. To prosper in diverse ecological niches, Aspergilli have evolved numerous mechanisms for adaptive gene regulation, some of which are also crucial for mammalian infection. Among the molecules which govern such responses, integral membrane receptors are thought to be the most amenable to therapeutic modulation. This is due to the localization of these molecular sensors at the periphery of the fungal cell, and to the prevalence of small molecules and licensed drugs which target receptor-mediated signaling in higher eukaryotic cells. In this review we highlight the progress made in characterizing receptor-mediated environmental adaptation in A. fumigatus and its relevance for pathogenicity in mammals. By presenting a first genomic survey of integral membrane proteins in this organism, we highlight an abundance of putative seven transmembrane domain (7TMD) receptors, the majority of which remain uncharacterized. Given the dependency of A. fumigatus upon stress adaptation for colonization and infection of mammalian hosts, and the merits of targeting receptor-mediated signaling as an antifungal strategy, a closer scrutiny of sensory perception and signal transduction in this organism is warranted. PMID:23430083

  4. Contribution of the P2Y12 receptor-mediated pathway to platelet hyperreactivity in hypercholesterolemia

    PubMed Central

    Nagy, Béla; Jin, Jianguo; Ashby, Barrie; Reilly, Michael P.; Kunapuli, Satya P.

    2011-01-01

    Summary Background In hypercholesterolemia, platelets demonstrate increased reactivity and promote the development of cardiovascular disease. Objective This study was carried out to investigate the contribution of the ADP receptor P2Y12-mediated pathway in platelet hyperreactivity due to hypercholesterolemia. Methods Low-density lipoprotein receptor deficient mice and C57Bl/6 wild type mice were fed on normal chow and high-fat (Western or Paigen) diets for 8 weeks to generate differently elevated cholesterol levels. P2Y12 receptor induced functional responses via Gi signaling were studied ex vivo when washed murine platelets were activated by 2MeSADP and PAR4 agonist AYPGKF in the presence and absence of indomethacin. Platelet aggregation, secretion, αIIbβ3 receptor activation and the phosphorylation of extracellular signal-regulated protein kinase (ERK) and Akt were analyzed. Results Plasma cholesterol levels ranged from 69±10 to 1011±185 mg/dl depending on diet in mice with different genotypes. Agonist-dependent aggregation, dense and α-granule secretion and JON/A binding were gradually and significantly (P < 0.05) augmented at low agonist concentration in correlation with the increasing plasma cholesterol levels even if elevated thromboxane generation was blocked. These functional responses were induced via increased level of Gi mediated ERK and Akt phosphorylation in hypercholesterolemic mice versus normocholesterolemic animals. In addition, blocking of the P2Y12 receptor by AR-C69931MX (Cangrelor) resulted in strongly reduced platelet aggregation in mice with elevated cholesterol levels compared to normocholesterolemic controls. Conclusions These data revealed that the P2Y12 receptor pathway was substantially involved in platelet hyperreactivity associated with mild and severe hypercholesterolemia. PMID:21261805

  5. 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.

  6. Rho/ROCK acts downstream of lysophosphatidic acid receptor 1 in modulating P2X3 receptor-mediated bone cancer pain in rats

    PubMed Central

    Wu, Jing-xiang; Yuan, Xiao-min; Wang, Qiong; Wei, Wang

    2016-01-01

    Background Lysophosphatidic acid receptor 1 and Rho/ROCK signaling is implicated in bone cancer pain development. However, it remains unknown whether the two signaling pathways function together in P2X3 receptor-mediated bone cancer pain. Results In this study, using a rat model of bone cancer, we examined the expression of P2X3 and lysophosphatidic acid receptor 1 in rat dorsal root ganglion neurons and further dissected whether lysophosphatidic acid receptor 1 and Rho/ROCK-mediated pathways interacted in modulating rat pain behavior. Bone cancer was established by inoculating Walker 256 cells into the left tibia of female Wistar rats. We observed a gradual and yet significant decline in mean paw withdrawal threshold in rats with bone cancer, but not in control rats. Our immunohistochemical staining revealed that the number of P2X3- and lysophosphatidic acid receptor 1-positive dorsal root ganglion neurons was significantly greater in rats with bone cancer than control rats. Lysophosphatidic acid receptor 1 blockade with VPC32183 significantly attenuated decline in mean paw withdrawal threshold. Flinching behavior test further showed that lysophosphatidic acid receptor 1 inhibition with VPC32183 transiently but significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Rho inhibition by intrathecal BoTXC3 caused a rapid reversal in decline in mean paw withdrawal threshold of rats with bone cancer. Flinching behavior test showed that BoTXC3 transiently and significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Similar findings were observed with ROCK inhibition by intrathecal Y27632. Furthermore, VPC32183 and BoTXC3 effectively aborted the appearance of lysophosphatidic acid-induced calcium influx peak. Conclusions Lysophosphatidic acid and its receptor LPAR1, acting through the Rho-ROCK pathway, regulate P2X3 receptor in the development of both mechanical and spontaneous pain in bone cancer. PMID:27094551

  7. P2X7 receptor-mediated calcium dynamics in HEK293 cells: experimental characterization and modelling approach

    NASA Astrophysics Data System (ADS)

    Di Garbo, A.; Alloisio, S.; Nobile, M.

    2012-04-01

    The P2X7 receptor (P2X7R) induces ionotropic Ca2 + signalling in different cell types. It plays an important role in the immune response and in the nervous system. Here, the mechanisms underlying intracellular Ca2 + variations evoked by 3‧-O-(4-benzoyl)benzoyl-ATP (BzATP), a potent agonist of the P2X7R, in transfected HEK293 cells, are investigated both experimentally and theoretically. We propose a minimal model of P2X7R that is capable of reproducing, qualitatively and quantitatively, the experimental data. This approach was also adopted for the P2X7R variant, which lacks the entire C-terminus tail (trP2X7R). Then we introduce a biophysical model describing the Ca2 + dynamics in HEK293. Our model gives an account of the ionotropic Ca2 + influx evoked by BzATP on the basis of the kinetics model of P2X7R. To explain the complex Ca2 + responses evoked by BzATP, the model predicted that an impairment in Ca2 + extrusion flux through the plasma membrane is a key factor for Ca2 + homeostasis in HEK293 cells.

  8. P2X1 Receptor-Mediated Ca(2+) Influx Triggered by DA-9801 Potentiates Nerve Growth Factor-Induced Neurite Outgrowth.

    PubMed

    Back, Moon Jung; Lee, Hae Kyung; Lee, Joo Hyun; Fu, Zhicheng; Son, Mi Won; Choi, Sang Zin; Go, Hyo Sang; Yoo, Sungjae; Hwang, Sun Wook; Kim, Dae Kyong

    2016-11-16

    Nerve growth factor (NGF)-induced neuronal regeneration has emerged as a strategy to treat neuronal degeneration-associated disorders. However, direct NGF administration is limited by the occurrence of adverse effects at high doses of NGF. Therefore, development of a therapeutic strategy to promote the NGF trophic effect is required. In view of the lack of understanding of the mechanism for potentiating the NGF effect, this study investigated molecular targets of DA-9801, a well-standardized Dioscorea rhizome extract, which has a promoting effect on NGF. An increase in intracellular calcium ion level was induced by DA-9801, and chelation of extracellular calcium ions with ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) suppressed the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. In addition, EGTA treatment reduced the DA-9801-induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), the major mediators of neurite outgrowth. To find which calcium ion-permeable channel contributes to the calcium ion influx induced by DA-9801, we treated PC12 cells with various inhibitors of calcium ion-permeable channels. NF449, a P2X1 receptor selective antagonist, significantly abolished the potentiating effect of DA-9801 on NGF-induced neurite outgrowth and abrogated the DA-9801-induced ERK1/2 phosphorylation. In addition, transfection with siRNA of P2X1 receptor significantly reduced the DA-9801-enhanced neurite outgrowth. In conclusion, calcium ion influx through P2X1 receptor mediated the promoting effect of DA-9801 on NGF-induced neurite outgrowth via ERK1/2 phosphorylation.

  9. Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells

    SciTech Connect

    Okajima, F.; Sho, K.; Kondo, Y.

    1988-08-01

    Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrations below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.

  10. Site of action of the general anesthetic propofol in muscarinic M1 receptor-mediated signal transduction.

    PubMed

    Murasaki, Osamu; Kaibara, Muneshige; Nagase, Yoshihisa; Mitarai, Sayaka; Doi, Yoshiyuki; Sumikawa, Koji; Taniyama, Kohtaro

    2003-12-01

    Although a potential target site of general anesthetics is primarily the GABA A receptor, a chloride ion channel, a previous study suggested that the intravenous general anesthetic propofol attenuates the M1 muscarinic acetylcholine receptor (M1 receptor)-mediated signal transduction. In the present study, we examined the target site of propofol in M1 receptor-mediated signal transduction. Two-electrode voltage-clamp method was used in Xenopus oocytes expressing both M1 receptors and associated G protein alpha subunits (Gqalpha). Propofol inhibited M1 receptor-mediated signal transduction in a dose-dependent manner (IC50 = 50 nM). Injection of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) into oocytes overexpressing Gqalpha was used to investigate direct effects of propofol on G protein coupled with the M1 receptor. Propofol did not affect activation of Gqalpha-mediated signal transduction with the intracellular injection of GTPgammaS. We also studied effects of propofol on l-[N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding and M1 receptor-mediated signal transduction in mammalian cells expressing M1 receptor. Propofol inhibited the M1 receptor-mediated signal transduction but did not inhibit binding of [3H]NMS. Effects of propofol on Gs- and Gi/o-coupled signal transduction were investigated, using oocytes expressing the beta2 adrenoceptor (beta2 receptor)/cystic fibrosis transmembrane conductance regulator or oocytes expressing the M2 muscarinic acetylcholine receptor (M2 receptor)/Kir3.1 (a member of G protein-gated inwardly rectifying K(+) channels). Neither beta2 receptor-mediated nor M2 receptor-mediated signal transduction was inhibited by a relatively high concentration of propofol (50 microM). These results indicate that propofol inhibits M1 receptor-mediated signal transduction by selectively disrupting interaction between the receptor and associated G protein.

  11. P2X7 receptor-mediated PARP1 activity regulates astroglial death in the rat hippocampus following status epilepticus

    PubMed Central

    Kim, Ji Yang; Ko, Ah-Reum; Kim, Ji-Eun

    2015-01-01

    Poly(ADP-ribose) polymerase-1 (PARP1) plays a regulatory role in apoptosis, necrosis, and other cellular processes after injury. Recently, we revealed that PARP1 regulates the differential neuronal/astroglial responses to pilocarpine-induced status epilepticus (SE) in the distinct brain regions. In addition, P2X7 receptor (P2X7R), an ATP-gated ion channel, activation accelerates astroglial apoptosis, while it attenuates clasmatodendrosis (lysosome-derived autophagic astroglial death). Therefore, we investigated whether P2X7R regulates regional specific astroglial PARP1 expression/activation in response to SE. In the present study, P2X7R activation exacerbates SE-induced astroglial apoptosis, while P2X7R inhibition attenuates it accompanied by increasing PARP1 activity in the molecular layer of the dentate gyrus following SE. In the CA1 region, however, P2X7R inhibition deteriorates SE-induced clasmatodendrosis via PARP1 activation following SE. Taken together, our findings suggest that P2X7R function may affect SE-induced astroglial death by regulating PARP1 activation/expression in regional-specific manner. Therefore, the selective modulation of P2X7R-mediated PARP1 functions may be a considerable strategy for controls in various types of cell deaths. PMID:26388738

  12. P2X7 receptor-mediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages1

    PubMed Central

    Lees, Michael P.; Fuller, Stephen J.; McLeod, Rima; Boulter, Nicola R.; Miller, Catherine M.; Zakrzewski, Alana M.; Mui, Ernest J.; Witola, William H.; Coyne, Jessica J.; Hargrave, Aubrey C.; Jamieson, Sarra E.; Blackwell, Jenefer M.; Wiley, James S.; Smith, Nicholas C.

    2010-01-01

    The P2X7 receptor (P2X7R)4 is highly expressed on the macrophage cell surface and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms (SNPs) that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this paper we show that macrophages from people with the 1513C (rs3751143) loss-of-function P2X7R SNP are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knock-out mice (P2X7R−/−) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production. PMID:20488797

  13. The C-Kit Receptor-Mediated Signal Transduction and Tumor-Related Diseases

    PubMed Central

    Liang, Jing; Wu, Yan-Ling; Chen, Bing-Jia; Zhang, Wen; Tanaka, Yoshimasa; Sugiyama, Hiroshi

    2013-01-01

    As an important member of tyrosine kinase family, c-kit receptor causes specific expression of certain genes, regulates cell differentiation and proliferation, resists cell apoptosis, and plays a key role in tumor occurrence, development, migration and recurrence through activating the downstream signaling molecules following interaction with stem cell factor (SCF). The abnormality of SCF/c-kit signaling pathway is closely related to some certain tumors. The discovery of c-kit receptor-targeted drugs has promoted clinical-related cancer's diagnosis and treatment. In this paper, we review recent research progress on c-kit receptor-mediated signal transduction and its potential therapeutic application as a target in tumor-related diseases. PMID:23678293

  14. MAGI-1 acts as a scaffolding molecule for NGF receptor-mediated signaling pathway.

    PubMed

    Ito, Hidenori; Morishita, Rika; Iwamoto, Ikuko; Mizuno, Makoto; Nagata, Koh-ichi

    2013-10-01

    We have recently found that the membrane-associated guanylate kinase with inverted organization-1 (MAGI-1) was enriched in rat nervous tissues such as the glomeruli in olfactory bulb of adult rats and dorsal root entry zone in spinal cord of embryonic rats. In addition, we revealed the localization of MAGI-1 in the growth cone of the primary cultured rat dorsal root ganglion cells. These results point out the possibility that MAGI-1 is involved in the regulation of neurite extension or guidance. In this study, we attempted to reveal the physiological role(s) of MAGI-1 in neurite extension. We found that RNA interference (RNAi)-mediated knockdown of MAGI-1 caused inhibition of nerve growth factor (NGF)-induced neurite outgrowth in PC12 rat pheochromocytoma cells. To clarify the involvement of MAGI-1 in NGF-mediated signal pathway, we tried to identify binding partners for MAGI-1 and identified p75 neurotrophin receptor (p75NTR), a low affinity NGF receptor, and Shc, a phosphotyrosine-binding adaptor. These three proteins formed an immunocomplex in PC12 cells. Knockdown as well as overexpression of MAGI-1 caused suppression of NGF-stimulated activation of the Shc-ERK pathway, which is supposed to play important roles in neurite outgrowth of PC12 cells. These results indicate that MAGI-1 may act as a scaffolding molecule for NGF receptor-mediated signaling pathway.

  15. Cellular mechanisms of the 5-HT7 receptor-mediated signaling

    PubMed Central

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes. PMID:25324743

  16. Cellular mechanisms of the 5-HT7 receptor-mediated signaling.

    PubMed

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes.

  17. Optimization of stress response through the nuclear receptor-mediated cortisol signalling network

    PubMed Central

    Kolodkin, Alexey; Sahin, Nilgun; Phillips, Anna; Hood, Steve R.; Bruggeman, Frank J.; Westerhoff, Hans V.; Plant, Nick

    2013-01-01

    It is an accepted paradigm that extended stress predisposes an individual to pathophysiology. However, the biological adaptations to minimize this risk are poorly understood. Using a computational model based upon realistic kinetic parameters we are able to reproduce the interaction of the stress hormone cortisol with its two nuclear receptors, the high-affinity glucocorticoid receptor and the low-affinity pregnane X-receptor. We demonstrate that regulatory signals between these two nuclear receptors are necessary to optimize the body’s response to stress episodes, attenuating both the magnitude and duration of the biological response. In addition, we predict that the activation of pregnane X-receptor by multiple, low-affinity endobiotic ligands is necessary for the significant pregnane X-receptor-mediated transcriptional response observed following stress episodes. This integration allows responses mediated through both the high and low-affinity nuclear receptors, which we predict is an important strategy to minimize the risk of disease from chronic stress. PMID:23653204

  18. P2X(7) receptor-mediated release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases.

    PubMed

    Lopez-Castejon, Gloria; Theaker, Jill; Pelegrin, Pablo; Clifton, Andrew D; Braddock, Martin; Surprenant, Annmarie

    2010-08-15

    The ATP-gated P2X(7) receptor (P2X(7)R) is a promising therapeutic target in chronic inflammatory diseases with highly specific antagonists currently under clinical trials for rheumatoid arthritis. Anti-inflammatory actions of P2X(7)R antagonists are considered to result from inhibition of P2X(7)R-induced release of proinflammatory cytokines from activated macrophages. However, P2X(7)Rs are also expressed in resting macrophages, suggesting that P2X(7)R may also signal via cytokine-independent mechanisms involved in joint disease. In this study, we examined P2X(7)R function in resting human lung macrophages and mouse bone marrow-derived macrophages and found that ATP induced rapid release of the lysosomal cysteine proteases cathepsin B, K, L, and S and that was independent of the presence of the proinflammatory cytokines IL-1beta and IL-18. Cathepsins released into the medium were effective to degrade collagen extracellular matrix. ATP-induced cathepsin release was abolished by P2X(7)R antagonists, absent from P2X(7)R(-/-) mouse macrophages, and not associated with cell death. Our results suggest P2X(7)R activation may play a novel and direct role in tissue damage through release of cathepsins independently of its proinflammatory actions via IL-1 cytokines.

  19. P2X and P2Y receptor signaling in red blood cells.

    PubMed

    Sluyter, Ronald

    2015-01-01

    Purinergic signaling involves the activation of cell surface P1 and P2 receptors by extracellular nucleosides and nucleotides such as adenosine and adenosine triphosphate (ATP), respectively. P2 receptors comprise P2X and P2Y receptors, and have well-established roles in leukocyte and platelet biology. Emerging evidence indicates important roles for these receptors in red blood cells. P2 receptor activation stimulates a number of signaling pathways in progenitor red blood cells resulting in microparticle release, reactive oxygen species formation, and apoptosis. Likewise, activation of P2 receptors in mature red blood cells stimulates signaling pathways mediating volume regulation, eicosanoid release, phosphatidylserine exposure, hemolysis, impaired ATP release, and susceptibility or resistance to infection. This review summarizes the distribution of P2 receptors in red blood cells, and outlines the functions of P2 receptor signaling in these cells and its implications in red blood cell biology.

  20. Wnt3a mitigates acute lung injury by reducing P2X7 receptor-mediated alveolar epithelial type I cell death

    PubMed Central

    Guo, Y; Mishra, A; Weng, T; Chintagari, N R; Wang, Y; Zhao, C; Huang, C; Liu, L

    2014-01-01

    Acute lung injury (ALI) is characterized by pulmonary endothelial and epithelial cell damage, and loss of the alveolar–capillary barrier. We have previously shown that P2X7 receptor (P2X7R), a cell death receptor, is specifically expressed in alveolar epithelial type I cells (AEC I). In this study, we hypothesized that P2X7R-mediated purinergic signaling and its interaction with Wnt/β-catenin signaling contributes to AEC I death. We examined the effect of P2X7R agonist 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP) and Wnt agonist Wnt3a on AEC I death in vitro and in vivo. We also assessed the therapeutic potential of Wnt3a in a clinically relevant ALI model of intratracheal lipopolysaccharide (LPS) exposure in ventilated mice. We found that the activation of P2X7R by BzATP caused the death of AEC I by suppressing Wnt/β-catenin signaling through stimulating glycogen synthase kinase-3β (GSK-3β) and proteasome. On the other hand, the activation of Wnt/β-catenin signaling by Wnt3a, GSK-3β inhibitor, or proteasome inhibitor blocked the P2X7R-mediated cell death. More importantly, Wnt3a attenuated the AEC I damage caused by intratracheal instillation of BzATP in rats or LPS in ventilated mice. Our results suggest that Wnt3a overrides the effect of P2X7R on the Wnt/β-catenin signaling to prevent the AEC I death and restrict the severity of ALI. PMID:24922070

  1. PKC-dependent ERK phosphorylation is essential for P2X7 receptor-mediated neuronal differentiation of neural progenitor cells

    PubMed Central

    Tsao, H-K; Chiu, P-H; Sun, S H

    2013-01-01

    Purinergic receptors have been shown to be involved in neuronal development, but the functions of specific subtypes of P2 receptors during neuronal development remain elusive. In this study we investigate the distribution of P2X7 receptors (P2X7Rs) in the embryonic rat brain using in situ hybridization. At E15.5, P2X7R mRNA was observed in the ventricular zone and subventricular zone, and colocalized with nestin, indicating that P2X7R might be expressed in neural progenitor cells (NPCs). P2X7R mRNA was also detected in the subgranular zone and dentate gyrus of the E18.5 and P4 brain. To investigate the roles of P2X7R and elucidate its mechanism, we established NPC cultures from the E15.5 rat brain. Stimulation of P2X7Rs induced Ca2+ influx, inhibited proliferation, altered cell cycle progression and enhanced the expression of neuronal markers, such as TUJ1 and MAP2. Similarly, knockdown of P2X7R by shRNA nearly abolished the agonist-stimulated increases in intracellular Ca2+ concentration and the expression of TUJ1 and NeuN. Furthermore, stimulation of P2X7R induced activation of ERK1/2, which was inhibited by the removal of extracellular Ca2+ and treatment with blockers for P2X7R and PKC activity. Stimulation of P2X7R also induced translocation of PKCα and PKCγ, but not of PKCβ, whereas knockdown of either PKCα or PKCγ inhibited ERK1/2 activation. Inhibition of PKC or p-ERK1/2 also caused a decrease in the number of TUJ1-positive cells and a concomitant increase in the number of GFAP-positive cells. Taken together, the activation of P2X7R in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway. PMID:23907465

  2. 5-HT7 receptor-mediated fear conditioning and possible involvement of extracellular signal-regulated kinase.

    PubMed

    Takeda, Kotaro; Tsuji, Minoru; Miyagawa, Kazuya; Takeda, Hiroshi

    2017-01-18

    Fear conditioning is a valuable behavioral paradigm for studying the neural basis of emotional learning and memory. The present study examined the involvement of extracellular signal-regulated kinase 1/2 (ERK) signaling on the serotonin (5-HT)7 receptor-mediated fear conditioning. Conditioning was performed in a trial in which a tone was followed by an electrical foot-shock. Context- and tone-dependent fear were examined in tests conducted 24 and 48h after conditioning, respectively. The selective 5-HT7 receptor antagonist 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,-tetrahydrobenzo(c,d)indol-2-(1H)-one (DR4004) (5mg/kg), when administered intraperitoneally (i.p.) immediately after conditioning, caused a significant decrease in both context- and tone-dependent fear responses (freezing behavior). A significant increase in ERK activity was observed in the amygdala of mice that displayed context- or tone-dependent fear responses, and these changes were also inhibited by the administration of DR4004 (5mg/kg, i.p.) immediately after conditioning. In contrast, the increase in hippocampal ERK activity in mice that displayed context-dependent fear responses was further enhanced by the administration of DR4004 (5mg/kg, i.p.). These results suggest that 5-HT7 receptor-mediated ERK signaling may play a significant role in the processes of emotional learning and memory.

  3. ATP sensitivity of preBötzinger complex neurones in neonatal rat in vitro: mechanism underlying a P2 receptor-mediated increase in inspiratory frequency

    PubMed Central

    Lorier, A R; Lipski, J; Housley, G D; Greer, J J; Funk, G D

    2008-01-01

    P2 receptor (R) signalling plays an important role in the central ventilatory response to hypoxia. The frequency increase that results from activation of P2Y1Rs in the preBötzinger complex (preBötC; putative site of inspiratory rhythm generation) may contribute, but neither the cellular nor ionic mechanism(s) underlying these effects are known. We applied whole-cell recording to rhythmically-active medullary slices from neonatal rat to define, in preBötC neurones, the candidate cellular and ionic mechanisms through which ATP influences rhythm, and tested the hypothesis that putative rhythmogenic preBötC neurones are uniquely sensitive to ATP. ATP (1 mm) evoked inward currents in all non-respiratory neurones and the majority of respiratory neurons, which included inspiratory, expiratory and putative rhythmogenic inspiratory neurones identified by sensitivity to substance P (1 μm) and DAMGO (50 μm) or by voltage-dependent pacemaker-like activity. ATP current densities were similar in all classes of preBötC respiratory neurone. Reversal potentials and input resistance changes for ATP currents in respiratory neurones suggested they resulted from either inhibition of a K+ channel or activation of a mixed cationic conductance. The P2YR agonist 2MeSADP (1 mm) evoked only the latter type of current in inspiratory and pacemaker-like neurones. In summary, putative rhythmogenic preBötC neurones were sensitive to ATP. However, this sensitivity was not unique; ATP evoked similar currents in all types of preBötC respiratory neurone. The P2Y1R-mediated frequency increase is therefore more likely to reflect activation of a mixed cationic conductance in multiple types of preBötC neurone than excitation of one, highly sensitive group. PMID:18174215

  4. P2Y receptor mediated inhibitory modulation of noradrenaline release in response to electrical field stimulation and ischemic conditions in superfused rat hippocampus slices.

    PubMed

    Csölle, Cecília; Heinrich, Attila; Kittel, Agnes; Sperlágh, Beáta

    2008-07-01

    In this study, the inhibitory regulation of the release of noradrenaline (NA) by P2 receptors was investigated in hippocampus slices pre-incubated with [(3)H]NA. Electrical field stimulation (EFS; 2 Hz, 240 shocks, and 1 ms) released NA in an outside [Ca(2+)]-dependent manner, and agonists of P2Y receptors inhibited the EFS-evoked [(3)H]NA release with pharmacological profile similar to that of the P2Y(1) and P2Y(13) receptor subtypes. This inhibitory modulation was counteracted by bicuculline and 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline + 2-amino-5-phosphonovalerate and 2-amino-4-phosphonobutyrate. In contrast, the excess release in response to 30 min combined oxygen and glucose deprivation was outside [Ca(2+)] independent, but still sensitive to the inhibition of both facilitatory P2X(1) and inhibitory P2Y(1) receptors. Whereas mRNA encoding P2Y(12) and P2Y(13) receptor subunits were expressed in the brainstem, P2Y(1) receptor immunoreactivity was localized to neuronal somata and dendrites innervated by the mossy fiber terminals in the CA3 region of the hippocampus, as well as somata of granule cells and interneurons in the dentate gyrus. In summary, in addition to the known facilitatory modulation via P2X receptors, EFS-evoked [(3)H]NA outflow in the hippocampus is subject to inhibitory modulation by P2Y(1)/P2Y(13) receptors. Furthermore, endogenous activation of both facilitatory and inhibitory P2 receptors may participate in the modulation of pathological NA release under ischemic-like conditions.

  5. Homocysteine-NMDA receptor mediated activation of extracellular-signal regulated kinase leads to neuronal cell death

    PubMed Central

    Poddar, Ranjana; Paul, Surojit

    2009-01-01

    Hyper-homocysteinemia is an independent risk factor for stroke and neurological abnormalities. However the underlying cellular mechanisms by which elevated homocysteine can promote neuronal death is not clear. In the present study we have examined the role of NMDA receptor mediated activation of the extracellular-signal regulated mitogen activated protein (ERK MAP) kinase pathway in homocysteine-dependent neurotoxicity. The study demonstrates that in neurons L-homocysteine-induced cell death is mediated through activation of NMDA receptors. The study also shows that homocysteine-dependent NMDA receptor stimulation and resultant Ca2+ influx leads to rapid and sustained phosphorylation of ERK MAP kinase. Inhibition of ERK phosphorylation attenuates homocysteine mediated neuronal cell death thereby demonstrating that activation of ERK MAP kinase signaling pathway is an intermediate step that couples homocysteine mediated NMDA receptor stimulation to neuronal death. The findings also show that cAMP response-element binding protein (CREB), a pro-survival transcription factor and a downstream target of ERK, is only transiently activated following homocysteine exposure. The sustained activation of ERK but a transient activation of CREB together suggest that exposure to homocysteine initiates a feedback loop that shuts off CREB signaling without affecting ERK phosphorylation and thereby facilitates homocysteine mediated neurotoxicity. PMID:19508427

  6. P2Y12 receptor-mediated activation of spinal microglia and p38MAPK pathway contribute to cancer-induced bone pain

    PubMed Central

    Liu, Mingjuan; Yao, Ming; Wang, Hanqi; Xu, Longsheng; Zheng, Ying; Huang, Bing; Ni, Huadong; Xu, Shijie; Zhou, Xuyan; Lian, Qingquan

    2017-01-01

    Background Cancer-induced bone pain (CIBP) is one of the most challenging clinical problems due to a lack of understanding the mechanisms. Recent evidence has demonstrated that activation of microglial G-protein-coupled P2Y12 receptor (P2Y12R) and proinflammatory cytokine production play an important role in neuropathic pain generation and maintenance. However, whether P2Y12R is involved in CIBP remains unknown. Methods The purpose of this study was to investigate the role of P2Y12R in CIBP and its molecular mechanisms. Using the bone cancer model inoculated with Walker 256 tumor cells into the left tibia of Sprague Dawley rat, we blocked spinal P2Y12R through intrathecal administration of its selective antagonist MRS2395 (400 pmol/µL, 15 µL). Results We found that not only the ionized calcium-binding adapter molecule 1 (Iba-1)-positive microglia in the ipsilateral spinal cord but also mechanical allodynia was significantly inhibited. Furthermore, it decreased the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and the production of proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6), whereas it increased tumor necrosis factor-α (TNF-α). Conclusion Taken together, our present results suggest that microglial P2Y12R in the spinal cord may contribute to CIBP by the activation of spinal microglia and p38MAPK pathway, thus identifying a potential therapeutic target for the treatment of CIBP. PMID:28243146

  7. Mediator mechanisms involved in TRPV1, TRPA1 and P2X receptor-mediated sensory transduction of pulmonary ROS by vagal lung C-fibers in rats.

    PubMed

    Lin, Yu-Jung; Hsu, Hsao-Hsun; Ruan, Ting; Kou, Yu Ru

    2013-10-01

    We investigated the mediator mechanisms involved in the sensory transduction of pulmonary reactive oxygen species (ROS) by vagal lung C-fibers in anesthetized rats. Airway challenge of aerosolized H₂O₂ (0.4%) stimulated these afferent fibers. The H₂O₂-induced responses were reduced by a cyclooxygenase inhibitor or ATP scavengers and also attenuated by an antagonist of TRPV1, TRPA1 or P2X receptors. The suppressive effect of the cyclooxygenase inhibitor was not affected by a combined treatment with the TRPV1 or TRPA1 antagonist, but was amplified by a combined treatment with the P2X antagonists. The suppressive effect of ATP scavengers was not affected by a combined treatment with the P2X antagonist, but was amplified by a combined treatment with the TRPV1 or TRPA1 antagonist. Thus, the actions of cyclooxygenase metabolites are mediated through the functioning of the TRPV1 and TRPA1 receptors, whereas the action of ATP is mediated through the functioning of P2X receptors.

  8. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    SciTech Connect

    Magno, Aaron L.; Ingley, Evan; Brown, Suzanne J.; Conigrave, Arthur D.; Ratajczak, Thomas; Ward, Bryan K.

    2011-09-09

    Highlights: {yields} A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. {yields} The second zinc finger of LIM domain 1 of testin is critical for interaction. {yields} Testin bound to a region of the receptor tail important for cell signalling. {yields} Testin and receptor interaction was confirmed in mammalian (HEK293) cells. {yields} Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  9. Ectodysplasin receptor-mediated signaling is essential for embryonic submandibular salivary gland development.

    PubMed

    Jaskoll, Tina; Zhou, Yan-Min; Trump, Gary; Melnick, Michael

    2003-04-01

    Hypohidrotic (anhidrotic) ectodermal dysplasia (HED), the most common of the approximately 150 described ectodermal dysplasias, is a disorder characterized by abnormal hair, teeth, sweat glands, and salivary glands. Mutations in the EDA (ectodysplasin-A) and EDAR (ectodysplasin-A receptor) genes are responsible for X-linked and autosomal HED, respectively. Abnormal phenotypes similar to HED are seen in Tabby (Eda(Ta)) and downless (Edar(dl)) mutant mice. Although recent studies have focused on the role of Eda/Edar signaling during hair and tooth development, very little is known about its role during embryonic submandibular salivary gland (SMG) development. To this end, we analyzed the SMG phenotypes in Tabby (Ta) and downless (dl) mutant mice and determined that Ta SMGs are hypoplastic, whereas dl SMGs are severely dysplastic. The absence of SMG ducts and acini in dl SMGs suggests that Eda/Edar signaling is essential for lumina formation and glandular histodifferentiation. Our localization of Eda and Edar proteins at sites of lumen and acini formation supports this conclusion. Moreover, the presence of SMGs in both Ta and dl mutant mice, as well as the absence of immunodetectable Eda and Edar protein in Initial Bud and Early Pseudoglandular stage SMGs, indicate that Eda/Edar-mediated signaling is important for branching morphogenesis and histodifferentiation, but not for initial gland formation. To initially delineate the morphoregulatory role of Eda/Edar-mediated signaling during embryonic SMG development, we cultured embryonic day 14 SMGs with enhanced or abrogated Eda/Edar signaling. Eda supplementation induced a significant increase in SMG branching, and enhanced activation of NF-kappaB. Abrogating Eda/Edar signaling by adding the soluble form of Edar to bind endogenous ligand in embryonic SMGs results in a significant dose-dependent decrease in branching morphogenesis. Taken together, our results suggest that the Eda/Edar/NF-kappaB pathway exerts its effect

  10. Metabotropic glutamate receptor-mediated signaling dampens the HPA axis response to restraint stress.

    PubMed

    Evanson, Nathan K; Herman, James P

    2015-10-15

    Glutamate is an important neurotransmitter in the regulation of the neural portion of hypothalamus-pituitary-adrenal (HPA) axis activity, and signals through ionotropic and metabotropic receptors. In the current studies we investigated the role of hypothalamic paraventricular group I metabotropic glutamate receptors in the regulation of the HPA axis response to restraint stress in rats. Direct injection of the group I metabotropic glutamate receptor agonist 3,5-dihydroxyphenylglycine (DHPG) into the PVN prior to restraint leads to blunting of the HPA axis response in awake animals. Consistent with this result, infusion of the group I receptor antagonist hexyl-homoibotenic acid (HIBO) potentiates the HPA axis response to restraint. The excitatory effect of blocking paraventricular group I metabotropic glutamate signaling is blocked by co-administration of dexamethasone into the PVN. However, the inhibitory effect of DHPG is not affected by co-administration of the cannabinoid CB1 receptor antagonist AM-251 into the PVN. Together, these results suggest that paraventricular group I metabotropic glutamate receptor signaling acts to dampen HPA axis reactivity. This effect appears to be similar to the rapid inhibitory effect of glucocorticoids at the PVN, but is not mediated by endocannabinoid signaling.

  11. Activation of oncogenic tyrosine kinase signaling promotes insulin receptor-mediated cone photoreceptor survival

    PubMed Central

    Rajala, Ammaji; Wang, Yuhong; Rajala, Raju V.S.

    2016-01-01

    In humans, daylight vision is primarily mediated by cone photoreceptors. These cells die in age-related retinal degenerations. Prolonging the life of cones for even one decade would have an enormous beneficial effect on usable vision in an aging population. Photoreceptors are postmitotic, but shed 10% of their outer segments daily, and must synthesize the membrane and protein equivalent of a proliferating cell each day. Although activation of oncogenic tyrosine kinase and inhibition of tyrosine phosphatase signaling is known to be essential for tumor progression, the cellular regulation of this signaling in postmitotic photoreceptor cells has not been studied. In the present study, we report that a novel G-protein coupled receptor–mediated insulin receptor (IR) signaling pathway is regulated by non-receptor tyrosine kinase Src through the inhibition of protein tyrosine phosphatase IB (PTP1B). We demonstrated the functional significance of this pathway through conditional deletion of IR and PTP1B in cones, in addition to delaying the death of cones in a mouse model of cone degeneration by activating the Src. This is the first study demonstrating the molecular mechanism of a novel signaling pathway in photoreceptor cells, which provides a window of opportunity to save the dying cones in retinal degenerative diseases. PMID:27391439

  12. 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

  13. Differential T cell receptor-mediated signaling in naive and memory CD4 T cells.

    PubMed

    Farber, D L; Acuto, O; Bottomly, K

    1997-08-01

    Naive and memory CD4 T cells differ in cell surface phenotype, function, activation requirements, and modes of regulation. To investigate the molecular bases for the dichotomies between naive and memory CD4 T cells and to understand how the T cell receptor (TCR) directs diverse functional outcomes, we investigated proximal signaling events triggered through the TCR/CD3 complex in naive and memory CD4 T cell subsets isolated on the basis of CD45 isoform expression. Naive CD4 T cells signal through TCR/CD3 similar to unseparated CD4 T cells, producing multiple tyrosine-phosphorylated protein species overall and phosphorylating the T cell-specific ZAP-70 tyrosine kinase which is recruited to the CD3zeta subunit of the TCR. Memory CD4 T cells, however, exhibit a unique pattern of signaling through TCR/CD3. Following stimulation through TCR/CD3, memory CD4 T cells produce fewer species of tyrosine-phosphorylated substrates and fail to phosphorylate ZAP-70, yet unphosphorylated ZAP-70 can associate with the TCR/CD3 complex. Moreover, a 26/28-kDa phosphorylated doublet is associated with CD3zeta in resting and activated memory but not in naive CD4 T cells. Despite these differences in the phosphorylation of ZAP-70 and CD3-associated proteins, the ZAP-70-related kinase, p72syk, exhibits similar phosphorylation in naive and memory T cell subsets, suggesting that this kinase could function in place of ZAP-70 in memory CD4 T cells. These results indicate that proximal signals are differentially coupled to the TCR in naive versus memory CD4 T cells, potentially leading to distinct downstream signaling events and ultimately to the diverse functions elicited by these two CD4 T cell subsets.

  14. Caveolin-3 regulates compartmentation of cardiomyocyte beta2-adrenergic receptor-mediated cAMP signaling.

    PubMed

    Wright, Peter T; Nikolaev, Viacheslav O; O'Hara, Thomas; Diakonov, Ivan; Bhargava, Anamika; Tokar, Sergiy; Schobesberger, Sophie; Shevchuk, Andrew I; Sikkel, Markus B; Wilkinson, Ross; Trayanova, Natalia A; Lyon, Alexander R; Harding, Sian E; Gorelik, Julia

    2014-02-01

    The purpose of this study was to investigate whether caveolin-3 (Cav3) regulates localization of β2-adrenergic receptor (β2AR) and its cAMP signaling in healthy or failing cardiomyocytes. We co-expressed wildtype Cav3 or its dominant-negative mutant (Cav3DN) together with the Förster resonance energy transfer (FRET)-based cAMP sensor Epac2-camps in adult rat ventricular myocytes (ARVMs). FRET and scanning ion conductance microscopy were used to locally stimulate β2AR and to measure cytosolic cAMP. Cav3 overexpression increased the number of caveolae and decreased the magnitude of β2AR-cAMP signal. Conversely, Cav3DN expression resulted in an increased β2AR-cAMP response without altering the whole-cell L-type calcium current. Following local stimulation of Cav3DN-expressing ARVMs, β2AR response could only be generated in T-tubules. However, the normally compartmentalized β2AR-cAMP signal became diffuse, similar to the situation observed in heart failure. Finally, overexpression of Cav3 in failing myocytes led to partial β2AR redistribution back into the T-tubules. In conclusion, Cav3 plays a crucial role for the localization of β2AR and compartmentation of β2AR-cAMP signaling to the T-tubules of healthy ARVMs, and overexpression of Cav3 in failing myocytes can partially restore the disrupted localization of these receptors.

  15. Neurobiology of microglial action in CNS injuries: receptor-mediated signaling mechanisms and functional roles

    PubMed Central

    Hu, Xiaoming; Liou, Anthony K.F.; Leak, Rehana K.; Xu, Mingyue; An, Chengrui; Suenaga, Jun; Shi, Yejie; Gao, Yanqin; Zheng, Ping; Chen, Jun

    2014-01-01

    Microglia are the first line of immune defense against central nervous system (CNS) injuries and disorders. These highly plastic cells play dualistic roles in neuronal injury and recovery and are known for their ability to assume diverse phenotypes. A broad range of surface receptors are expressed on microglia and mediate microglial ‘On’ or ‘Off’ responses to signals from other host cells as well as invading microorganisms. The integrated actions of these receptors result in tightly regulated biological functions, including cell mobility, phagocytosis, the induction of acquired immunity, and trophic factor/inflammatory mediator release. Over the last few years, significant advances have been made towards deciphering the signaling mechanisms related to these receptors and their specific cellular functions. In this review, we describe the current state of knowledge of the surface receptors involved in microglial activation, with an emphasis on their engagement of distinct functional programs and their roles in CNS injuries. It will become evident from this review that microglial homeostasis is carefully maintained by multiple counterbalanced strategies, including, but not limited to, ‘On’ and ‘Off’ receptor signaling. Specific regulation of theses microglial receptors may be a promising therapeutic strategy against CNS injuries. PMID:24923657

  16. 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

  17. 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

  18. 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

  19. 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.

  20. Calmodulin Regulates Ca2+-sensing Receptor-mediated Ca2+ Signaling and Its Cell Surface Expression*

    PubMed Central

    Huang, Yun; Zhou, Yubin; Wong, Hing-Cheung; Castiblanco, Adriana; Chen, Yanyi; Brown, Edward M.; Yang, Jenny J.

    2010-01-01

    The Ca2+-sensing receptor (CaSR) is a member of family C of the GPCRs responsible for sensing extracellular Ca2+ ([Ca2+]o) levels, maintaining extracellular Ca2+ homeostasis, and transducing Ca2+ signaling from the extracellular milieu to the intracellular environment. In the present study, we have demonstrated a Ca2+-dependent, stoichiometric interaction between CaM and a CaM-binding domain (CaMBD) located within the C terminus of CaSR (residues 871–898). Our studies suggest a wrapping around 1–14-like mode of interaction that involves global conformational changes in both lobes of CaM with concomitant formation of a helical structure in the CaMBD. More importantly, the Ca2+-dependent association between CaM and the C terminus of CaSR is critical for maintaining proper responsiveness of intracellular Ca2+ responses to changes in extracellular Ca2+ and regulating cell surface expression of the receptor. PMID:20826781

  1. Human Fc gamma RII, in the absence of other Fc gamma receptors, mediates a phagocytic signal.

    PubMed Central

    Indik, Z; Kelly, C; Chien, P; Levinson, A I; Schreiber, A D

    1991-01-01

    Fc gamma receptors are important components in the binding and phagocytosis of IgG-sensitized cells. Studies on the role of these receptors have been limited by the fact that most hematopoietic cells express more than one Fc gamma receptor. We studied the role of Fc gamma RIIA in isolation on a human erythroleukemia cell line (HEL) which expresses Fc gamma RIIA as its only Fc gamma receptor. HEL cells were observed to bind and phagocytose IgG-sensitized red blood cells (RBCs) in a dose-dependent manner. We then examined the role of Fc gamma RI and Fc gamma RII in isolation and in combination, in transfected COS-1 cells. Fc gamma RIIA-transfected COS cells also mediated both the binding and phagocytosis of IgG-sensitized RBCs. In contrast, phagocytosis was not observed in Fc gamma RI-transfected cells, although these cells avidly bound IgG-sensitized RBCs. Furthermore, coexpression of both receptors by doubly transfected cells did not affect the phagocytic efficiency of Fc gamma RIIA. These studies establish that Fc gamma RIIA can mediate phagocytosis and suggest that transfected COS-1 cells provide a model for examining this process. Since HEL cells exhibit characteristics of cells of the megakaryocyte-platelet lineage, including expression of Fc gamma RII as the only Fc gamma receptor, Fc gamma RIIA on megakaryocytes and platelets may be involved in the ingestion of IgG-containing immune complexes. Furthermore, these studies indicate that Fc gamma RI and Fc gamma RIIA differ in their requirements for transduction of a phagocytic signal. Images PMID:1834702

  2. Ror2 Receptor Mediates Wnt11 Ligand Signaling and Affects Convergence and Extension Movements in Zebrafish*

    PubMed Central

    Bai, Yan; Tan, Xungang; Zhang, Haifeng; Liu, Chengdong; Zhao, Beibei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng

    2014-01-01

    The receptor-tyrosine kinase Ror2 acts as an alternative receptor or co-receptor for Wnt5a and mediates Wnt5a-induced convergent extension movements during embryogenesis in mice and Xenopus as well as the polarity and migration of several cell types during development. However, little is known about whether Ror2 function is conserved in other vertebrates or is involved in other non-canonical Wnt ligands in vivo. In this study we demonstrated that overexpression of dominant-negative ror2 (ror2-TM) mRNA in zebrafish embryos resulted in convergence and extension defects and incompletely separated eyes, which is consistent with observations from slb/wnt11 mutants or wnt11 knockdown morphants. Moreover, the co-injection of ror2-TM mRNA and a wnt11 morpholino or the coexpression of ror2 and wnt11 in zebrafish embryos synergetically induced more severe convergence and extension defects. Transplantation studies further demonstrated that the Ror2 receptor responded to the Wnt11 ligand and regulated cell migration and cell morphology during gastrulation. DnRor2 inhibited the action of Wnt11, which was revealed by a decreased percentage of Wnt11-induced convergence and extension defects. Ror2 physically interacts with Wnt11. The intracellular Tyr-647 and Ser-863 sites of Ror2 are essential for mediating the action of Wnt11. Dishevelled and RhoA act downstream of Wnt11-Ror2 to regulate convergence and extension movements. Overall, our data suggest an important role of Ror2 in mediating Wnt11 signaling and in regulating convergence and extension movements in zebrafish. PMID:24928507

  3. Gastrin promotes intestinal polyposis through cholecystokinin-B receptor-mediated proliferative signaling and fostering tumor microenvironment.

    PubMed

    Han, Y-M; Park, J-M; Park, S-H; Hahm, K B; Hong, S P; Kim, E-H

    2013-08-01

    Increased serum gastrin concentrations in patients with colorectal cancer suggested the tumorigenic trophic effect of gastrin. Detailed and global molecular mechanisms explaining trophic effect of gastrin had not been revealed. In the current study, intestinal polyposis of APC(Min/⁺) mice was compared between phosphate buffered saline (PBS) injected and gastrin (10 μg/kg, thrice per week) injected group. Total number of intestinal polyposis was counted and immunohistochemical staining with F4/80 and CD3 was done. MTT assay, cell cycle analysis, and Western blot for cyclin D1, CDK4, and β-catenin were performed in Raw 264.7 and HCT116 cells before and after gastrin administration. Experiments were repeated with YM022 or transfection with si-cholecystokinin-B receptor (CCK-B-R). Intraperitoneal gastrin significantly increased intestinal polyposis in APC(Min/⁺) mice (P<0.005), in which significant increases in macrophage were noted on F4/80 immunohistochemical staining (Plt;0.05) as well as Ki-67 staining (Plt;0.05) after gastrin. On comparative cytokine array, gastrin increased interleukin-1β (IL-1β), interleukin 3Rβ (IL-3Rβ), stromal cell-derived factor-1α (SDF-1α), thymus and activation-regulated chemokine (TARC), and thymus-derived chemotactic agent 3 (TCA-3) in macrophage cells, which was further confirmed with real time polymerase chain reaction (RT-PCR) analysis (P<0.05). In addition to increased inflammatory cytokines, gastrin increased macrophage proliferation accompanied with increased cyclin D1 and CDK4. Targeted for HCT116 cells, gastrin significantly increased proliferation as well as increases in synthetic phase of cell cycle. YM022 as gastrin antagonist significantly abolished the trophic actions of gastrin (P<0.05). HCT116 cells transfected with siCCK-B-R, gastrin did not increase either cell cycle or β-catenin in spite of gastrin administration. Conclusively, gastrin promoted intestinal polyposis through either direct gastrin receptor-mediated

  4. Epac–protein kinase C alpha signaling in purinergic P2X3R-mediated hyperalgesia after inflammation

    PubMed Central

    Gu, Yanping; Li, Guangwen; Chen, Yong; Huang, Li-Yen Mae

    2016-01-01

    Abstract Sensitization of purinergic P2X3 receptors (P2X3Rs) is a major mechanism contributing to injury-induced exaggerated pain responses. We showed in a previous study that cyclic adenosine monophosphate (cAMP)–dependent guanine nucleotide exchange factor 1 (Epac1) in rat sensory dorsal root ganglia (DRGs) is upregulated after inflammatory injury, and it plays a critical role in P2X3R sensitization by activating protein kinase C epsilon (PKCε) inside the cells. protein kinase C epsilon has been established as the major PKC isoform mediating injury-induced hyperalgesic responses. On the other hand, the role of PKCα in receptor sensitization was seldom considered. Here, we studied the participation of PKCα in Epac signaling in P2X3R-mediated hyperalgesia. The expression of both Epac1 and Epac2 and the level of cAMP in DRGs are greatly enhanced after complete Freund adjuvant (CFA)–induced inflammation. The expression of phosphorylated PKCα is also upregulated. Complete Freund adjuvant (CFA)–induced P2X3R-mediated hyperalgesia is not only blocked by Epac antagonists but also by the classical PKC isoform inhibitors, Go6976, and PKCα-siRNA. These CFA effects are mimicked by the application of the Epac agonist, 8-(4-chlorophenylthio)-2 -O-methyl-cAMP (CPT), in control rats, further confirming the involvement of Epacs. Because the application of Go6976 prior to CPT still reduces CPT-induced hyperalgesia, PKCα is downstream of Epacs to mediate the enhancement of P2X3R responses in DRGs. The pattern of translocation of PKCα inside DRG neurons in response to CPT or CFA stimulation is distinct from that of PKCε. Thus, in contrast to prevalent view, PKCα also plays an essential role in producing complex inflammation-induced receptor-mediated hyperalgesia. PMID:26963850

  5. Signal transduction of receptor-mediated antiproliferative action of melatonin on human prostate epithelial cells involves dual activation of Gα(s) and Gα(q) proteins.

    PubMed

    Shiu, Stephen Y W; Pang, Bo; Tam, Chun W; Yao, Kwok-Ming

    2010-10-01

    Melatonin has been shown to inhibit the proliferation of malignant and transformed human prostate epithelial cells by transcriptional up-regulation of p27(Kip1) expression via MTNR1A receptor-mediated activation of protein kinase A (PKA) and protein kinase C (PKC) in parallel. Given that melatonin MTNR1A receptor is a G protein-coupled receptor, this study was conducted to identify the specific G proteins that mediate the antiproliferative action of melatonin on human prostate epithelial cells. In 22Rv1 and RWPE-1 cells, knockdown of either Gα(s) or Gα(q) , but not Gα(i2) expression by RNA interference, abrogated the effects of melatonin on p27(Kip1) and cell proliferation. Conversely, cellular overexpression of activated mutants of Gα(s) and Gα(q) in 22Rv1 and RWPE-1 cells mimicked the effects of melatonin on prostate epithelial cell antiproliferation by increasing p27(Kip1) expression through downstream activation of PKA and PKC in parallel. Moreover, melatonin or 2-iodomelatonin induced elevation of adenosine-3',5'-cyclic monophosphate (cAMP) in 22Rv1 and RWPE-1 cells. The effects of 2-iodomelatonin on cAMP were blocked by the nonselective MTNR1A/MTNR1B receptor antagonist luzindole but were not affected by the selective MTNR1B receptor antagonist 4-phenyl-2-propionamidotetraline (4-P-PDOT). Furthermore, knockdown of Gα(s) mitigated the stimulatory effects of 2-iodomelatonin on cAMP. Collectively, the data demonstrated, for the first time, functional coupling of MTNR1A receptor to Gα(s) in cancerous or transformed human cells expressing endogenous melatonin receptors. Our results also showed that dual activation of Gα(s) and Gα(q) proteins is involved in the signal transduction of MTNR1A receptor-mediated antiproliferative action of melatonin on human prostate epithelial cells.

  6. Dynamics of receptor-operated Ca(2+) currents through TRPC channels controlled via the PI(4,5)P2-PLC signaling pathway.

    PubMed

    Mori, Masayuki X; Itsuki, Kyohei; Hase, Hideharu; Sawamura, Seishiro; Kurokawa, Tatsuki; Mori, Yasuo; Inoue, Ryuji

    2015-01-01

    Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that carry receptor-operated Ca(2+) currents (ROCs) triggered by receptor-induced, phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Within the vasculature, TRPC channel ROCs contribute to smooth muscle cell depolarization, vasoconstriction, and vascular remodeling. However, TRPC channel ROCs exhibit a variable response to receptor-stimulation, and the regulatory mechanisms governing TRPC channel activity remain obscure. The variability of ROCs may be explained by their complex regulation by PI(4,5)P2 and its metabolites, which differentially affect TRPC channel activity. To resolve the complex regulation of ROCs, the use of voltage-sensing phosphoinositide phosphatases and model simulation have helped to reveal the time-dependent contribution of PI(4,5)P2 and the possible role of PI(4,5)P2 in the regulation of ROCs. These approaches may provide unprecedented insight into the dynamics of PI(4,5)P2 regulation of TRPC channels and the fundamental mechanisms underlying transmembrane ion flow. Within that context, we summarize the regulation of TRPC channels and their coupling to receptor-mediated signaling, as well as the application of voltage-sensing phosphoinositide phosphatases to this research. We also discuss the controversial bidirectional effects of PI(4,5)P2 using a model simulation that could explain the complicated effects of PI(4,5)P2 on different ROCs.

  7. Histamine H4 Receptor mediates interleukin-8 and TNF-α release in human mast cells via multiple signaling pathways.

    PubMed

    Chen, X-F; Zhang, Z; Dou, X; Li, J-J; Zhang, W; Yu, Y-Y; Yu, B; Yu, B

    2016-01-27

    Histamine, mainly produced by mast cells, is an important inflammatory mediator in immune response. Recently Histamine H4 Receptor (H4R) was also identified in mast cells, from which pro-inflammatory cytokines and chemokines are released. However, the mechanism of how H4R mediates these cytokines and chemokines release in mast cells was still unclear. To further explore the role of H4R in the immune inflammatory response in mast cells, we tested the release of inflammatory cytokine tumor necrosis factor-α (TNF-α), chemokine interleukin-8 (IL-8) and the relevant signaling pathways activated by H4R on LAD2 cells (a human mast cell line). We found that the release of IL-8 and TNF-α were blocked by inhibitors of PI3K, ERK and Ca2+-Calcineurin-NFAT signaling pathways, while the release of these cytokines and chemokines were enhanced by the inhibitor of P38 signaling pathway. However, inhibitors of the JNK and NF-κB signaling pathways had little effect on the expression of the pro-inflammatory mediators. Moreover, activation of the H4R could induce phosphorylation of ERK, p38 and AKT in mast cells. In conclusion, we found that H4R mediates the release of inflammatory cytokine TNF-α and chemokine IL-8 in human mast cells via PI3K, Ca2+-Calcineurin-NFAT and MAPKs signaling pathways.

  8. Ligation of human Fc receptor like-2 by monoclonal antibodies down-regulates B-cell receptor-mediated signalling

    PubMed Central

    Shabani, Mahdi; Bayat, Ali Ahmad; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Hojjat-Farsangi, Mohammad; Ulivieri, Cristina; Amirghofran, Zahra; Baldari, Cosima Tatiana; Shokri, Fazel

    2014-01-01

    B-cell antigen receptor (BCR) signalling and its regulation through negative and positive regulators are critical for balancing B-cell response and function. Human Fc receptor like-2 (FCRL2), a member of the newly identified FCRL family, could influence B-cell signalling due to possession of both immunoreceptor tyrosine-based activation and inhibitory motifs (ITAM and ITIM). Since the natural ligand of FCRL2 has not been identified, we generated FCRL2-specific monoclonal antibodies (mAbs) and employed them to investigate the influence of FCRL2 stimulation on BCR signalling in an FCRL2-expressing B-cell line. Two anti-FCRL2 mAb-producing hybridoma clones (5A7-E7 and 3D8-G8) were selected. None of the mAbs displayed any cross-reactivity with the other members of the FCRL family including recombinant FCRL1, -3, -4 and -5, as tested by FACS and ELISA techniques. Engagement of the FCRL2 by these mAbs resulted in significant inhibition of BCR signalling mediators such as calcium mobilization and phosphorylation of the mitogen-activated protein kinases Erk, p38 and Jnk. These findings indicate that the FCRL2 ITIM motifs are functional and the anti-FCRL2 mAbs may mimic the natural ligand of FCRL2 by induction of inhibitory signals in B cells. PMID:24797767

  9. Chronic Valproate Treatment Blocks D2-like Receptor-Mediated Brain Signaling via Arachidonic Acid in Rats

    PubMed Central

    Ramadan, Epolia; Basselin, Mireille; Taha, Ameer Y.; Cheon, Yewon; Chang, Lisa; Chen, Mei; Rapoport, Stanley I.

    2011-01-01

    Background and Objective Hyperdopaminergic signaling and an upregulated brain arachidonic acid (AA) cascade may contribute to bipolar disorder (BD). Lithium and carbamazepine, FDA-approved for the treatment of BD, attenuate brain dopaminergic D2-like (D2, D3, and D4) receptor signaling involving AA when given chronically to awake rats. We hypothesized that valproate (VPA), with mood-stabilizing properties, would also reduce the D2-like-mediated signaling via AA. Methods An acute dose of quinpirole (1 mg/kg) or saline was administered to unanesthetized rats that had been treated for 30 days with a therapeutically relevant dose of VPA (200 mg/kg/day) or vehicle. Regional brain AA incorporation coefficients, k*, and incorporation rates, Jin, markers of AA signaling and metabolism, were measured by quantitative autoradiography after intravenous [1-14C]AA infusion. Whole brain concentrations of prostaglandin (PG)E2 and thromboxane (TX)B2 also were measured. Results Quinpirole compared to saline significantly increased k* in 40 of 83 brain regions, and increased brain concentrations of PGE2 in chronic vehicle-treated rats. VPA treatment by itself reduced concentrations of plasma unesterified AA and whole brain PGE2 and TXB2, and blocked the quinpirole-induced increments in k* and PGE2. Conclusion These results further support our hypothesis that similar to lithium and carbamazepine, VPA downregulates brain dopaminergic D2-like receptor-signaling involving AA. PMID:21839100

  10. The role of G protein coupled receptor-mediated signaling in the biological properties of Acanthamoeba castellanii of the T4 genotype.

    PubMed

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Manan, Zainab; Khan, Naveed Ahmed

    2015-04-01

    Despite advances in antimicrobial chemotherapy and supportive care, the prognosis of Acanthamoeba infections remains poor, suggesting that new targets are needed that can affect parasite survival and host-pathogen interactions. G proteins and their coupled receptors are well known regulators of a variety of cellular functions. The overall aim of the present study was to study the role of G-protein coupled receptor, β adrenergic receptor on the biology and pathogenesis of keratitis isolate of Acanthamoeba castellanii of the T4 genotype. Inhibition of β adrenergic receptor using antagonist, propranolol had detrimental effects on the extracellular proteolytic activities A. castellanii as determined using zymographic assays. Conversely, β adrenergic receptor agonist, isoprenaline showed increased proteases. Interestingly, β adrenergic receptor inhibition affected A. castellanii growth (using amoebistatic assays), viability (using amoebicidal assays by measuring uptake of Trypan blue) and encystation as determined by trophozoite transformation into the cyst form. Pre-treatment of parasites with propranolol hampered A. castellanii-mediated human brain microvascular endothelial cell cytotoxicity, as measured by the lacatate dehydrogenase release. The aforementioned findings suggest that G-protein coupled receptor, β adrenergic receptor-mediated signaling in A. castellanii biology and pathogenesis may offer new pharmacological targets.

  11. Cathepsin Inhibition Prevents Autophagic Protein Turnover and Downregulates Insulin Growth Factor-1 Receptor-Mediated Signaling in Neuroblastoma.

    PubMed

    Soori, Mehrnoosh; Lu, Guizhen; Mason, Robert W

    2016-02-01

    Inhibition of the major lysosomal proteases, cathepsins B, D, and L, impairs growth of several cell types but leads to apoptosis in neuroblastoma. The goal of this study was to examine the mechanisms by which enzyme inhibition could cause cell death. Cathepsin inhibition caused cellular accumulation of fragments of the insulin growth factor 1 (IGF-1) receptor. The fragments were located in dense organelles that were characterized as autophagosomes. This novel discovery provides the first clear link between lysosomal function, autophagy, and IGF-1- mediated cell proliferation. A more in-depth analysis of the IGF1 signaling pathway revealed that the mitogen-activated protein kinase (MAPK) cell-proliferation pathway was impaired in inhibitor treated cells, whereas the Akt cell survival pathway remained functional. Shc, an adapter protein that transmits IGF-1 signaling through the MAPK pathway, was sequestered in autophagosomes; whereas IRS-2, an adapter protein that transmits IGF-1 signaling through the Akt pathway, was unaffected by cathepsin inhibition. Furthermore, Shc was sequestered in autophagosomes as its active form, indicating that autophagy is a key mechanism for downregulating IGF-1-induced cell proliferation. Cathepsin inhibition had a greater effect on autophagic sequestration of the neuronal specific adapter protein, Shc-C, than ubiquitously expressed Shc-A, providing mechanistic support for the enhanced sensitivity of neuronally derived tumor cells. We also observed impaired activation of MAPK by epidermal growth factor treatment in inhibitor-treated cells. The Shc adapter proteins are central to transducing proliferation signaling by a range of receptor tyrosine kinases; consequently, cathepsin inhibition may become an important therapeutic approach for treating neuroblastoma and other tumors of neuronal origin.

  12. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling.

    PubMed

    Magno, Aaron L; Ingley, Evan; Brown, Suzanne J; Conigrave, Arthur D; Ratajczak, Thomas; Ward, Bryan K

    2011-09-09

    The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  13. Evidence of the receptor-mediated influence of melatonin on pancreatic glucagon secretion via the Gαq protein-coupled and PI3K signaling pathways.

    PubMed

    Bähr, Ina; Mühlbauer, Eckhard; Albrecht, Elke; Peschke, Elmar

    2012-11-01

    Melatonin has been shown to modulate glucose metabolism by influencing insulin secretion. Recent investigations have also indicated a regulatory function of melatonin on the pancreatic α-cells. The present in vitro and in vivo studies evaluated whether melatonin mediates its effects via melatonin receptors and which signaling cascade is involved. Incubation experiments using the glucagon-producing mouse pancreatic α-cell line αTC1 clone 9 (αTC1.9) as well as isolated pancreatic islets of rats and mice revealed that melatonin increases glucagon secretion. Preincubation of αTC1.9 cells with the melatonin receptor antagonists luzindole and 4P-PDOT abolished the glucagon-stimulatory effect of melatonin. In addition, glucagon secretion was lower in the pancreatic islets of melatonin receptor knockout mice than in the islets of the wild-type (WT) control animals. Investigations of melatonin receptor knockout mice revealed decreased plasma glucagon concentrations and elevated mRNA expression levels of the hepatic glucagon receptor when compared to WT mice. Furthermore, studies using pertussis toxin, as well as measurements of cAMP concentrations, ruled out the involvement of Gαi- and Gαs-coupled signaling cascades in mediating the glucagon increase induced by melatonin. In contrast, inhibition of phospholipase C in αTC1.9 cells prevented the melatonin-induced effect, indicating the physiological relevance of the Gαq-coupled pathway. Our data point to the involvement of the phosphatidylinositol 3-kinase signaling cascade in mediating melatonin effects in pancreatic α-cells. In conclusion, these findings provide evidence that the glucagon-stimulatory effect of melatonin in pancreatic α-cells is melatonin receptor mediated, thus supporting the concept of melatonin-modulated and diurnal glucagon release.

  14. Angiotensin receptor-mediated oxidative stress is associated with impaired cardiac redox signaling and mitochondrial function in insulin-resistant rats

    PubMed Central

    Vázquez-Medina, José Pablo; Popovich, Irina; Thorwald, Max A.; Viscarra, Jose A.; Rodriguez, Ruben; Sonanez-Organis, Jose G.; Lam, Lisa; Peti-Peterdi, Janos; Nakano, Daisuke; Nishiyama, Akira

    2013-01-01

    Activation of angiotensin receptor type 1 (AT1) contributes to NADPH oxidase (Nox)-derived oxidative stress during metabolic syndrome. However, the specific role of AT1 in modulating redox signaling, mitochondrial function, and oxidative stress in the heart remains more elusive. To test the hypothesis that AT1 activation increases oxidative stress while impairing redox signaling and mitochondrial function in the heart during diet-induced insulin resistance in obese animals, Otsuka Long Evans Tokushima Fatty (OLETF) rats (n = 8/group) were treated with the AT1 blocker (ARB) olmesartan for 6 wk. Cardiac Nox2 protein expression increased 40% in OLETF compared with age-matched, lean, strain-control Long Evans Tokushima Otsuka (LETO) rats, while mRNA and protein expression of the H2O2-producing Nox4 increased 40–100%. ARB treatment prevented the increase in Nox2 without altering Nox4. ARB treatment also normalized the increased levels of protein and lipid oxidation (nitrotyrosine, 4-hydroxynonenal) and increased the redox-sensitive transcription factor Nrf2 by 30% and the activity of antioxidant enzymes (SOD, catalase, GPx) by 50–70%. Citrate synthase (CS) and succinate dehydrogenase (SDH) activities decreased 60–70%, whereas cardiac succinate levels decreased 35% in OLETF compared with LETO, suggesting that mitochondrial function in the heart is impaired during obesity-induced insulin resistance. ARB treatment normalized CS and SDH activities, as well as succinate levels, while increasing AMPK and normalizing Akt, suggesting that AT1 activation also impairs cellular metabolism in the diabetic heart. These data suggest that the cardiovascular complications associated with metabolic syndrome may result from AT1 receptor-mediated Nox2 activation leading to impaired redox signaling, mitochondrial activity, and dysregulation of cellular metabolism in the heart. PMID:23771688

  15. Angiotensin receptor-mediated oxidative stress is associated with impaired cardiac redox signaling and mitochondrial function in insulin-resistant rats.

    PubMed

    Vázquez-Medina, José Pablo; Popovich, Irina; Thorwald, Max A; Viscarra, Jose A; Rodriguez, Ruben; Sonanez-Organis, Jose G; Lam, Lisa; Peti-Peterdi, Janos; Nakano, Daisuke; Nishiyama, Akira; Ortiz, Rudy M

    2013-08-15

    Activation of angiotensin receptor type 1 (AT1) contributes to NADPH oxidase (Nox)-derived oxidative stress during metabolic syndrome. However, the specific role of AT1 in modulating redox signaling, mitochondrial function, and oxidative stress in the heart remains more elusive. To test the hypothesis that AT1 activation increases oxidative stress while impairing redox signaling and mitochondrial function in the heart during diet-induced insulin resistance in obese animals, Otsuka Long Evans Tokushima Fatty (OLETF) rats (n = 8/group) were treated with the AT1 blocker (ARB) olmesartan for 6 wk. Cardiac Nox2 protein expression increased 40% in OLETF compared with age-matched, lean, strain-control Long Evans Tokushima Otsuka (LETO) rats, while mRNA and protein expression of the H₂O₂-producing Nox4 increased 40-100%. ARB treatment prevented the increase in Nox2 without altering Nox4. ARB treatment also normalized the increased levels of protein and lipid oxidation (nitrotyrosine, 4-hydroxynonenal) and increased the redox-sensitive transcription factor Nrf2 by 30% and the activity of antioxidant enzymes (SOD, catalase, GPx) by 50-70%. Citrate synthase (CS) and succinate dehydrogenase (SDH) activities decreased 60-70%, whereas cardiac succinate levels decreased 35% in OLETF compared with LETO, suggesting that mitochondrial function in the heart is impaired during obesity-induced insulin resistance. ARB treatment normalized CS and SDH activities, as well as succinate levels, while increasing AMPK and normalizing Akt, suggesting that AT1 activation also impairs cellular metabolism in the diabetic heart. These data suggest that the cardiovascular complications associated with metabolic syndrome may result from AT1 receptor-mediated Nox2 activation leading to impaired redox signaling, mitochondrial activity, and dysregulation of cellular metabolism in the heart.

  16. Caveolin-1 regulates P2X7 receptor signaling in osteoblasts.

    PubMed

    Gangadharan, Vimal; Nohe, Anja; Caplan, Jeffrey; Czymmek, Kirk; Duncan, Randall L

    2015-01-01

    The synthesis of new bone in response to a novel applied mechanical load requires a complex series of cellular signaling events in osteoblasts and osteocytes. The activation of the purinergic receptor P2X(7)R is central to this mechanotransduction signaling cascade. Recently, P2X(7)R have been found to be associated with caveolae, a subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1), the primary protein constituent of caveolae in osteoblasts, results in increased bone mass, leading us to hypothesize that the P2X(7)R is scaffolded to caveolae in osteoblasts. Thus, upon activation of the P2X(7)R, we postulate that caveolae are endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation of MC3T3-E1 preosteoblasts showed that CAV1 was translocated to the denser cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X(7)R agonist 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7R antagonist A-839977. The P2X7R cofractionated with CAV1, but, using superresolution structured illumination microscopy, we found only a subpopulation of P2X(7)R in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular Ca(2+) response to BzATP, suggesting that caveolae regulate P2X(7)R signaling. This proposed mechanism is supported by increased mineralization in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X(7)R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatiotemporal signaling of P2X(7)R in osteoblasts.

  17. Lymphotropic Virions Affect Chemokine Receptor-Mediated Neural Signaling and Apoptosis: Implications for Human Immunodeficiency Virus Type 1-Associated Dementia

    PubMed Central

    Zheng, Jialin; Ghorpade, Anuja; Niemann, Douglas; Cotter, Robin L.; Thylin, Michael R.; Epstein, Leon; Swartz, Jennifer M.; Shepard, Robin B.; Liu, Xiaojuan; Nukuna, Adeline; Gendelman, Howard E.

    1999-01-01

    Chemokine receptors pivotal for human immunodeficiency virus type 1 (HIV-1) infection in lymphocytes and macrophages (CCR3, CCR5, and CXCR4) are expressed on neural cells (microglia, astrocytes, and/or neurons). It is these cells which are damaged during progressive HIV-1 infection of the central nervous system. We theorize that viral coreceptors could effect neural cell damage during HIV-1-associated dementia (HAD) without simultaneously affecting viral replication. To these ends, we studied the ability of diverse viral strains to affect intracellular signaling and apoptosis of neurons, astrocytes, and monocyte-derived macrophages. Inhibition of cyclic AMP, activation of inositol 1,4,5-trisphosphate, and apoptosis were induced by diverse HIV-1 strains, principally in neurons. Virions from T-cell-tropic (T-tropic) strains (MN, IIIB, and Lai) produced the most significant alterations in signaling of neurons and astrocytes. The HIV-1 envelope glycoprotein, gp120, induced markedly less neural damage than purified virions. Macrophage-tropic (M-tropic) strains (ADA, JR-FL, Bal, MS-CSF, and DJV) produced the least neural damage, while 89.6, a dual-tropic HIV-1 strain, elicited intermediate neural cell damage. All T-tropic strain-mediated neuronal impairments were blocked by the CXCR4 antibody, 12G5. In contrast, the M-tropic strains were only partially blocked by 12G5. CXCR4-mediated neuronal apoptosis was confirmed in pure populations of rat cerebellar granule neurons and was blocked by HA1004, an inhibitor of calcium/calmodulin-dependent protein kinase II, protein kinase A, and protein kinase C. Taken together, these results suggest that progeny HIV-1 virions can influence neuronal signal transduction and apoptosis. This process occurs, in part, through CXCR4 and is independent of CD4 binding. T-tropic viruses that traffic in and out of the brain during progressive HIV-1 disease may play an important role in HAD neuropathogenesis. PMID:10482576

  18. Interaction of structure-specific and promiscuous G-protein-coupled receptors mediates small-molecule signaling in Caenorhabditis elegans.

    PubMed

    Park, Donha; O'Doherty, Inish; Somvanshi, Rishi K; Bethke, Axel; Schroeder, Frank C; Kumar, Ujendra; Riddle, Donald L

    2012-06-19

    A chemically diverse family of small-molecule signals, the ascarosides, control developmental diapause (dauer), olfactory learning, and social behaviors of the nematode model organism, Caenorhabditis elegans. The ascarosides act upstream of conserved signaling pathways, including the insulin, TGF-β, serotonin, and guanylyl cyclase pathways; however, the sensory processes underlying ascaroside function are poorly understood. Because ascarosides often are multifunctional and show strongly synergistic effects, characterization of their receptors will be essential for understanding ascaroside biology and may provide insight into molecular mechanisms that produce synergistic outcomes in small-molecule sensing. Based on DAF-8 immunoprecipitation, we here identify two G-protein-coupled receptors, DAF-37 and DAF-38, which cooperatively mediate ascaroside perception. daf-37 mutants are defective in all responses to ascr#2, one of the most potent dauer-inducing ascarosides, although this mutant responds normally to other ascarosides. In contrast, daf-38 mutants are partially defective in responses to several different ascarosides. Through cell-specific overexpression, we show that DAF-37 regulates dauer when expressed in ASI neurons and adult behavior when expressed in ASK neurons. Using a photoaffinity-labeled ascr#2 probe and amplified luminescence assays (AlphaScreen), we demonstrate that ascr#2 binds to DAF-37. Photobleaching fluorescent energy transfer assays revealed that DAF-37 and DAF-38 form heterodimers, and we show that heterodimerization strongly increases cAMP inhibition in response to ascr#2. These results suggest that that the ascarosides' intricate signaling properties result in part from the interaction of highly structure-specific G-protein-coupled receptors such as DAF-37 with more promiscuous G-protein-coupled receptors such as DAF-38.

  19. Interaction of structure-specific and promiscuous G-protein–coupled receptors mediates small-molecule signaling in Caenorhabditis elegans

    PubMed Central

    Park, Donha; O'Doherty, Inish; Somvanshi, Rishi K.; Bethke, Axel; Schroeder, Frank C.; Kumar, Ujendra; Riddle, Donald L.

    2012-01-01

    A chemically diverse family of small-molecule signals, the ascarosides, control developmental diapause (dauer), olfactory learning, and social behaviors of the nematode model organism, Caenorhabditis elegans. The ascarosides act upstream of conserved signaling pathways, including the insulin, TGF-β, serotonin, and guanylyl cyclase pathways; however, the sensory processes underlying ascaroside function are poorly understood. Because ascarosides often are multifunctional and show strongly synergistic effects, characterization of their receptors will be essential for understanding ascaroside biology and may provide insight into molecular mechanisms that produce synergistic outcomes in small-molecule sensing. Based on DAF-8 immunoprecipitation, we here identify two G-protein–coupled receptors, DAF-37 and DAF-38, which cooperatively mediate ascaroside perception. daf-37 mutants are defective in all responses to ascr#2, one of the most potent dauer-inducing ascarosides, although this mutant responds normally to other ascarosides. In contrast, daf-38 mutants are partially defective in responses to several different ascarosides. Through cell-specific overexpression, we show that DAF-37 regulates dauer when expressed in ASI neurons and adult behavior when expressed in ASK neurons. Using a photoaffinity-labeled ascr#2 probe and amplified luminescence assays (AlphaScreen), we demonstrate that ascr#2 binds to DAF-37. Photobleaching fluorescent energy transfer assays revealed that DAF-37 and DAF-38 form heterodimers, and we show that heterodimerization strongly increases cAMP inhibition in response to ascr#2. These results suggest that that the ascarosides' intricate signaling properties result in part from the interaction of highly structure-specific G-protein–coupled receptors such as DAF-37 with more promiscuous G-protein–coupled receptors such as DAF-38. PMID:22665789

  20. Effects of Vanadium-Containing Compounds on Membrane Lipids and on Microdomains Used in Receptor-Mediated Signaling

    PubMed Central

    Roess, Deborah A.; Smith, Steven M. L.; Winter, Peter; Zhou, Jun; Dou, Ping; Baruah, Bharat; Trujillo, Alejandro M.; Levinger, Nancy E.; Yang, Xioda; Barisas, B. George; Crans, Debbie C.

    2011-01-01

    There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium compounds which have been shown in animal models to enhance insulin responsiveness. One possible mechanism for insulin-enhancing effects might involve direct effects of vanadium compounds on membrane lipid organization. These changes in lipid organization promote the partitioning of insulin receptors and other receptors into membrane microdomains where receptors are optimally functional. To explore this possibility, we have used several strategies involving vanadium complexes such as [VO2dipic]− (pyridin-2,6-dicarboxylatodioxovanadium(V)), decavanadate (V10O286−, V10), BMOV (bis(maltolato)oxovanadium(IV)) and [VO(saltris)]2 (2-salicylideniminato-2-(hydroxymethyl)-1,3-dihydroxypropane-oxovanadium(V)). Our strategies include an evaluation of interactions between vanadium-containing compounds and model lipid systems, an evaluation of the effects of vanadium compounds on lipid fluidity in erythrocyte membranes, and studies of the effects of vanadium-containing compounds on signaling events initiated by receptors known to use membrane microdomains as signaling platforms. PMID:18729092

  1. Effects of vanadium-containing compounds on membrane lipids and on microdomains used in receptor-mediated signaling.

    PubMed

    Roess, Deborah A; Smith, Steven M L; Winter, Peter; Zhou, Jun; Dou, Ping; Baruah, Bharat; Trujillo, Alejandro M; Levinger, Nancy E; Yang, Xioda; Barisas, B George; Crans, Debbie C

    2008-08-01

    There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium (V) compounds which have been shown in animal models to enhance insulin responsiveness. One possible mechanism for insulin-enhancing effects might involve direct effects of V compounds on membrane lipid organization. These changes in lipid organization promote the partitioning of insulin receptors and other receptors into membrane microdomains where receptors are optimally functional. To explore this possibility, we have used several strategies involving V complexes such as [VO(2)(dipic)](-) (pyridin-2,6-dicarboxylatodioxovanadium(V)), decavanadate (V(10)O(28)(6-), V(10)), BMOV (bis(maltolato)oxovanadium(IV)), and [VO(saltris)](2) (2-salicylideniminato-2-(hydroxymethyl)-1,3-dihydroxypropane-oxovanadium(V)). Our strategies include an evaluation of interactions between V-containing compounds and model lipid systems, an evaluation of the effects of V compounds on lipid fluidity in erythrocyte membranes, and studies of the effects of V-containing compounds on signaling events initiated by receptors known to use membrane microdomains as signaling platforms.

  2. Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

    PubMed Central

    Wen, Xi; Jin, Tian; Xu, Xuehua

    2016-01-01

    Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells. PMID:27684322

  3. 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.

  4. Chronic exposure to bisphenol a impairs progesterone receptor-mediated signaling in the uterus during early pregnancy

    PubMed Central

    Li, Quanxi; Davila, Juanmahel; Bagchi, Milan K.; Bagchi, Indrani C.

    2016-01-01

    Environmental and occupational exposure to endocrine disrupting chemicals (EDCs) is a major threat to female reproductive health. Bisphenol A (BPA), an environmental toxicant that is commonly found in polycarbonate plastics and epoxy resins, has received much attention due to its estrogenic activity and high risk of chronic exposure in human. Whereas BPA has been linked to infertility and recurrent miscarriage in women, the impact of its exposure on uterine function during early pregnancy remains unclear. In a recent publication in Endocrinology, we demonstrated that prolonged exposure to an environmental relevant dose of BPA disrupts progesterone receptor-regulated uterine functions, thus affecting uterine receptivity for embryo implantation and decidua morphogenesis, two critical events for establishment and maintenance of early pregnancy. In particular we reported a marked impairment of progesterone receptor (PGR) expression and its downstream effector HAND2 in the uterine stromal cells in response to chronic BPA exposure. In an earlier study we have shown that HAND2 controls embryo implantation by repressing fibroblast growth factor (FGF) expression and the MAP kinase signaling pathway, thus inhibiting epithelial proliferation. Interestingly we observed that downregulation of PGR and HAND2 expression in uterine stroma upon BPA exposure was associated with an enhanced activation of FGFR and MAPK signaling, aberrant proliferation, and lack of uterine receptivity in the epithelium. In addition, the proliferation and differentiation of endometrial stromal cells to decidual cells, an event critical for the maintenance of early pregnancy, was severely compromised in response to BPA. This research highlight will provide an overview of our findings and discuss the potential mechanisms by which chronic BPA impairs PGR-HAND2 pathway and adversely affects implantation and the establishment of pregnancy. PMID:28239613

  5. Role of Endothelin-1/Endothelin-A receptor-mediated signaling pathway in the aortic arch patterning in mice.

    PubMed Central

    Yanagisawa, H.; Hammer, R. E.; Richardson, J. A.; Williams, S. C.; Clouthier, D. E.; Yanagisawa, M.

    1998-01-01

    The intercellular signaling mediated by endothelins and their G protein-coupled receptors has recently been shown to be essential for the normal embryonic development of subsets of neural crest cell derivatives. Endothelin-1 (ET-1) is proteolytically generated from its inactive precursor by endothelin-converting enzyme-1 (ECE-1) and acts on the endothelin-A (ETA) receptor. Genetic disruption of this ET-1/ECE-1/ETA pathway results in defects in branchial arch- derived craniofacial tissues, as well as defects in cardiac outflow and great vessel structures, which are derived from cephalic (cardiac) neural crest. In this study, in situ hybridization of ETA-/- and ECE-1(-)/- embryos with a cardiac neural crest marker, cellular retinoic acid-binding protein-1, shows that the migration of neural crest cells from the neural tube to cardiac outflow tract is not affected in these embryos. Immunostaining of an endothelial marker, platelet endothelial cell adhesion molecule CD-31, shows that the initial formation of the branchial arch arteries is not disturbed in ETA-/- or ECE-1(-)/- embryos. To visualize the subsequent patterning of arch vessels in detail, we generated ETA-/- or ECE-1(-)/- embryos that expressed an SM22alpha-lacZ marker transgene in arterial smooth muscle cells. Wholemount X-gal staining of these mutant embryos reveals that the abnormal regression and persistence of specific arch arteries results in disturbance of asymmetrical remodeling of the arch arteries. These defects include abnormal regression of arch arteries 4 and 6, enlargement of arch artery 3, and abnormal persistence of the bilateral ductus caroticus and right dorsal aorta. These abnormalities eventually lead to various types of great vessel malformations highly similar to those seen in neural crest-ablated chick embryos and human congenital cardiac defects. This study demonstrates that ET-1/ETA-mediated signaling plays an essential role in a complex process of aortic arch patterning by affecting

  6. Differential action of chlorinated polycyclic aromatic hydrocarbons on aryl hydrocarbon receptor-mediated signaling in breast cancer cells.

    PubMed

    Ohura, Takeshi; Morita, Maki; Kuruto-Niwa, Ryoko; Amagai, Takashi; Sakakibara, Hiroyuki; Shimoi, Kayoko

    2010-04-01

    Chlorinated polycyclic aromatic hydrocarbons (ClPAHs), which are a series of halogenated aromatic hydrocarbons, have been found in the environment. The primary step in their metabolic activation seems to be associated with aryl hydrocarbon receptor (AhR)-mediated induction of the cytochrome P450 (CYP) 1 family, although the evidence remains unclear. In this study, we first investigated the effects of five ClPAHs with three to five rings and the corresponding parent PAHs on the expression of CYP1A1 and 1B1 in human breast cancer MCF-7 cells. For the targeted ClPAHs, Western blot analysis of ClPAH-induced CYP1A1 and 1B1 showed an enhancement in activities in comparison with induction by the corresponding parent PAHs, and the effects of chlorination were especially prominent in phenanthrene. In a further study, using 6-chlorobenzo[a]pyrene (6-ClBaP), cotreatment with 17beta-estradiol showed an increase in the expression of CYP1B1 mRNA but not CYP1A1 mRNA. Since the AhR ligand has been reported to induce formation of an AhR-estrogen receptor (ER) complex, which stimulates transcription of ER target genes, the effects of ClPAHs in MCF-7 cells transfected with estrogen response elements-regulated green fluorescent protein (GFP) reporter genes were also investigated in this study. 6-ClBaP induced a dose-dependent increase in GFP expression related to ER signaling through AhR activation in the cells, but 3,9,10-trichlorophenanthrene (3,9,10-Cl(3)ClPhe) did not, despite its ability to activate AhR. Furthermore, we investigated the effect of ClPAHs on the expression of the endogenous ER-responsive genes, cathepsin D, in MCF-7 cells. 6-ClBaP stimulated expression of the ER-responsive genes but 3,9,10-Cl(3)ClPhe did not, as in the GFP expression system. These results suggest that estrogenic action mediated ER signaling through AhR activation does not necessarily occur for every ligand that can activate AhR.

  7. β-Adrenergic Receptor-Mediated Cardiac Contractility is Inhibited via Vasopressin Type 1A-Receptor-Dependent Signaling

    PubMed Central

    Tilley, Douglas G.; Zhu, Weizhong; Myers, Valerie D.; Barr, Larry A.; Gao, Erhe; Li, Xue; Song, Jianliang; Carter, Rhonda L.; Makarewich, Catherine A.; Yu, Daohai; Troupes, Constantine D.; Grisanti, Laurel A.; Coleman, Ryan C.; Koch, Walter J.; Houser, Steven R.; Cheung, Joseph Y.; Feldman, Arthur M.

    2014-01-01

    Background Enhanced arginine vasopressin (AVP) levels are associated with increased mortality during end-stage human heart failure (HF), and cardiac AVP type 1A receptor (V1AR) expression becomes increased. Additionally, mice with cardiac-restricted V1AR overexpression develop cardiomyopathy and decreased β-adrenergic receptor (βAR) responsiveness. This led us to hypothesize that V1AR signaling regulated βAR responsiveness and in doing so contributes to HF development. Methods and Results Transaortic constriction resulted in decreased cardiac function and βAR density and increased cardiac V1AR expression, effects reversed by a V1AR-selective antagonist. Molecularly, V1AR stimulation led to decreased βAR ligand affinity, as well as βAR-induced Ca2+ mobilization and cAMP generation in isolated adult cardiomyocytes, effects recapitulated via ex vivo Langendorff analysis. V1AR-mediated regulation of βAR responsiveness was demonstrated to occur in a previously unrecognized Gq protein-independent/GRK-dependent manner. Conclusions This newly discovered relationship between cardiac V1AR and βAR may be informative for the treatment of patients with acute decompensated HF and elevated AVP. PMID:25205804

  8. Identification of ecdysteroid receptor-mediated signaling pathways in the hepatopancreas of the red swamp crayfish, Procambarus clarkii.

    PubMed

    Zhu, Baojian; Tang, Lin; Yu, Yingying; Yu, Huimin; Wang, Lei; Qian, Cen; Wei, Guoqing; Liu, Chaoliang

    2017-01-06

    The hepatopancreas of crustaceans plays an important role in lipid and carbohydrate metabolism, digestion of food, and biogenesis. In this study, the hepatopancreas transcriptome from the red crayfish Procambarus clarkii was characterized for the first time using high-throughput sequencing, producing approximately 41.4 million reads were obtained. After de novo assembly, 57,363 unigenes with an average length of 725bp were identified, Gene Ontology analysis categorized 22,580 as being involved in biological processes, among which metabolic process and cellular process groups were the most highly enriched. A total of 8034 unigenes were assigned to 223 metabolic pathways following mapping against the Kyoto encyclopedia of genes and genomes (KEGG) database. Ecdysteroid receptor (EcR)-mediated signaling pathways were investigated using digital gene expression (DGE) analysis following RNA interference targeting the EcR. A total of 529 differentially expressed genes (DEGs) were identified, including 322 downregulated and 207 upregulated unigenes. Of these, 445 (84.12%) were annotated successfully by alignment with known sequences, many of which were related to catalytic activity and binding functional categories. Using KEGG enrichment analysis, 183 DEGs were clustered into 78 pathways, and six significantly enriched pathways were predicted. The expression patterns of candidate genes identified by real-time PCR were consistent with the DGE results.

  9. Cyproheptadine enhances the I(K) of mouse cortical neurons through sigma-1 receptor-mediated intracellular signal pathway.

    PubMed

    He, Yan-Lin; Zhang, Chun-Lei; Gao, Xiao-Fei; Yao, Jin-Jing; Hu, Chang-Long; Mei, Yan-Ai

    2012-01-01

    Cyproheptadine (CPH) is a histamine- and serotonin-receptor antagonist, and its effects are observed recently in the modulation of multiple intracellular signals. In this study, we used cortical neurons and HEK-293 cells transfected with Kv2.1 α-subunit to address whether CPH modify neural voltage-gated K(+) channels by a mechanism independent of its serotonergic and histaminergic properties. Our results demonstrate that intracellularly delivered CPH increased the I(K) by reducing the activity of protein kinas A (PKA). Inhibition of G(i) eliminated the CPH-induced effect on both the I(K) and PKA. Blocking of 5-HT-, M-, D(2)-, H(1)- or H(2)-type GPCR receptors with relevant antagonists did not eliminate the CPH-induced effect on the I(K). Antagonists of the sigma-1 receptor, however, blocked the effect of CPH. Moreover, the inhibition of sigma-1 by siRNA knockdown significantly reduced the CPH-induced effect on the I(K). On the contrary, sigma-1 receptor agonist mimicked the effects of CPH on the induction of I(K). A ligand-receptor binding assay indicated that CPH bound to the sigma-1 receptor. Similar effect of CPH were obtained from HEK-293 cells transfected with the α-subunit of Kv2.1. In overall, we reveal for the first time that CPH enhances the I(K) by modulating activity of PKA, and that the associated activation of the sigma-1 receptor/G(i)-protein pathway might be involved. Our findings illustrate an uncharacterized effect of CPH on neuron excitability through the I(K), which is independent of histamine H(1) and serotonin receptors.

  10. Modulatory effect of insulin on T cell receptor mediated calcium signaling is blunted in long lasting type 1 diabetes mellitus.

    PubMed

    Demkow, Urszula; Winklewski, Paweł; Ciepiela, Olga; Popko, Katarzyna; Lipińska, Anna; Kucharska, Anna; Michalska, Beata; Wąsik, Maria

    2012-01-01

    Insulin significantly influences Ca(2+) signals evoked by various stimulants. In type 1 recent onset diabetes mellitus the proliferative response of T cells is significantly decreased. The number of clinical trials exploring the role of anti-CD3 monoclonal antibodies (mAb) as a therapeutic agent in recent onset diabetes mellitus type 1 is increasing last years. Therefore, a better understanding of the interplay between T cell receptor (TCR) dependent Ca(2+) increase, and insulin is of vital clinical significance. The aim of the study was to assess the effect of insulin on TCR evoked Ca(2+) responses in T lymphocytes obtained from healthy volunteers and patients suffering from long lasting diabetes mellitus type 1. Analysis was performed with use of the flow cytometer. We demonstrated that T cells ability to mobilize Ca(2+) was significantly reduced in long lasting diabetes mellitus type 1. Ca(2+) decrease achieved by the long term incubation with anti-CD3 mAb in T cells from healthy volunteers was restored by insulin. Strong interrelationship between baseline Ca(2+) level and plateau phase response to TCR stimulation was observed in the cytoplasm of cells pre-incubated with insulin from both healthy subjects and diabetic patients (r = 0.95, p < 0.0001 and r = 0.94, p < 0.0001, respectively). We postulate the existence of the interplay between TCR mediated activation and insulin. The TCR-insulin interplay is blunted in long lasting diabetes mellitus type 1. These observations may have an important implication for future therapeutic options in diabetes.

  11. Cyproheptadine Enhances the IK of Mouse Cortical Neurons through Sigma-1 Receptor-Mediated Intracellular Signal Pathway

    PubMed Central

    He, Yan-Lin; Zhang, Chun-Lei; Gao, Xiao-Fei; Yao, Jin-Jing; Hu, Chang-Long; Mei, Yan-Ai

    2012-01-01

    Cyproheptadine (CPH) is a histamine- and serotonin-receptor antagonist, and its effects are observed recently in the modulation of multiple intracellular signals. In this study, we used cortical neurons and HEK-293 cells transfected with Kv2.1 α-subunit to address whether CPH modify neural voltage-gated K+ channels by a mechanism independent of its serotonergic and histaminergic properties. Our results demonstrate that intracellularly delivered CPH increased the IK by reducing the activity of protein kinas A (PKA). Inhibition of Gi eliminated the CPH-induced effect on both the IK and PKA. Blocking of 5-HT-, M-, D2-, H1- or H2- type GPCR receptors with relevant antagonists did not eliminate the CPH-induced effect on the IK. Antagonists of the sigma-1 receptor, however, blocked the effect of CPH. Moreover, the inhibition of sigma-1 by siRNA knockdown significantly reduced the CPH-induced effect on the IK. On the contrary, sigma-1 receptor agonist mimicked the effects of CPH on the induction of IK. A ligand-receptor binding assay indicated that CPH bound to the sigma-1 receptor. Similar effect of CPH were obtained from HEK-293 cells transfected with the α-subunit of Kv2.1. In overall, we reveal for the first time that CPH enhances the IK by modulating activity of PKA, and that the associated activation of the sigma-1 receptor/Gi-protein pathway might be involved. Our findings illustrate an uncharacterized effect of CPH on neuron excitability through the IK, which is independent of histamine H1 and serotonin receptors. PMID:22844454

  12. CB1 cannabinoid receptor-mediated anandamide signaling mechanisms of the inferior colliculus modulate the haloperidol-induced catalepsy.

    PubMed

    Medeiros, P; de Freitas, R L; Silva, M O; Coimbra, N C; Melo-Thomas, L

    2016-11-19

    , neither the intracollicular microinjection of AM251 at the lowest (50pmol/0.2μl) nor at the highest (200pmol/0.2μl) concentration was able to block the systemic haloperidol-induced catalepsy. Furthermore, the intracollicular administration of AM251 at 100pmol/0.2μl was able to decrease the duration of catalepsy as compared to AM251 at 50pmol/0.2μl- and AM251 at 200pmol/0.2-μl-treated group. The latency for stepping down from the horizontal bar - induced by haloperidol administration - was decreased when microinjection of AEA at 50pmol/0.2μl was preceded with blockade of CB1 receptor with AM251 (100pmol/0.2μl). Our results strengthen the involvement of CB1-signaled endocannabinoid mechanisms of the IC in the neuromodulation of catalepsy induced by systemic administration of the dopaminergic receptors non-selective antagonist haloperidol.

  13. γ-Aminobutyric Acid B Receptor Mediated Inhibition of Gonadotropin-Releasing Hormone Neurons Is Suppressed by Kisspeptin-G Protein-Coupled Receptor 54 Signaling

    PubMed Central

    Zhang, Chunguang; Bosch, Martha A.; Rønnekleiv, Oline K.; Kelly, Martin J.

    2009-01-01

    γ-Aminobutyric acid (GABA) is one of the most important neurotransmitters that regulate the excitability of GnRH neurons. Numerous studies have shown that GABA activates Cl− currents in GnRH neurons, and these effects are antagonized by GABAA receptor antagonists. The GABAB receptor is a heterodimer composed of GABAB R1 and R2, and although both subunits have been localized in GnRH neurons, nothing is known about the cellular signaling of this Gαi,o-coupled receptor in GnRH neurons. Using whole-cell recordings from mouse enhanced green fluorescent protein-GnRH neurons, we found that the GABAB receptor agonist baclofen hyperpolarized GnRH neurons through activation of an inwardly rectifying K+ current in a concentration-dependent manner. The effects of baclofen were antagonized by the selective GABAB receptor antagonist CGP 52432 with a Ki (inhibitory constant) of 85 nm. Furthermore, in the presence of the GABAA receptor antagonist picrotoxin, GABA hyperpolarized GnRH neurons in a similar manner. Treatment with 17β-estradiol as compared with oil vehicle did not significantly alter either the EC50 for the baclofen-induced response (0.8 ± 0.1 vs. 1.0 ± 0.1 μm, respectively) or the maximal outward current (10.8 ± 1.7 pA vs. 11.4 ± 0.6 pA, respectively) in GnRH neurons. However, the outward current (and membrane hyperpolarization) was abrogated by submaximal concentrations of the G protein-coupled receptor 54 (GPR54) agonist kisspeptin-10 in both groups, indicating that Gαq-coupled (GPR54) can desensitize the GABAB receptor-mediated response. Therefore, the activation of GABAB receptors in GnRH neurons may provide increased inhibitory tone during estrogen-negative feedback states that is attenuated by kisspeptin during positive feedback. PMID:19164470

  14. Purine receptor mediated actin cytoskeleton remodeling of human fibroblasts

    PubMed Central

    Goldman, Nanna; Chandler-Militello, Devin; Langevin, Helene; Nedergaard, Maiken; Takano, Takahiro

    2013-01-01

    Earlier studies have shown that activation of adenosine A1 receptors on peripheral pain fibers contributes to acupuncture-induced suppression of painful input. In addition to adenosine, acupuncture triggers the release of other purines, including ATP and ADP that may bind to purine receptors on nearby fibroblasts. We here show that purine agonists trigger increase in cytosolic Ca 2+ signaling in a cultured human fibroblasts cell line. The profile of agonist-induced Ca2+ increases indicates that the cells express functional P2yR2 and P2yR4 receptors, as well as P2yR1 and P2xR7 receptors. Unexpectedly, purine-induced Ca2+ signaling was associated with a remodeling of the actin cytoskeleton. ATP induced a transient loss in F-actin stress fiber. The changes of actin cytoskeleton occurred slowly and peaked at 10 min after agonist exposure. Inhibition of ATP-induced increases in Ca2+ by cyclopiazonic acid blocked receptor-mediated cytoskeleton remodeling. The Ca2+ ionophore failed to induce cytoskeletal remodeling despite triggering robust increases in cytosolic Ca2+. These observations indicate that purine signaling induces transient changes in fibroblast cytoarchitecture that could be related to the beneficial effects of acupuncture. PMID:23462235

  15. Astrocyte-neuron interaction in the substantia gelatinosa of the spinal cord dorsal horn via P2X7 receptor-mediated release of glutamate and reactive oxygen species.

    PubMed

    Ficker, Christoph; Rozmer, Katalin; Kató, Erzsébet; Andó, Rómeó D; Schumann, Luisa; Krügel, Ute; Franke, Heike; Sperlágh, Beáta; Riedel, Thomas; Illes, Peter

    2014-10-01

    The substantia gelatinosa (SG) of the spinal cord processes incoming painful information to ascending projection neurons. Whole-cell patch clamp recordings from SG spinal cord slices documented that in a low Ca(2+) /no Mg(2+) (low X(2+) ) external medium adenosine triphosphate (ATP)/dibenzoyl-ATP, Bz-ATP) caused inward current responses, much larger in amplitude than those recorded in a normal X(2+) -containing bath medium. The effect of Bz-ATP was antagonized by the selective P2X7 receptor antagonist A-438079. Neuronal, but not astrocytic Bz-ATP currents were strongly inhibited by a combination of the ionotropic glutamate receptor antagonists AP-5 and CNQX. In fact, all neurons and some astrocytes responded to NMDA, AMPA, and muscimol with inward current, demonstrating the presence of the respective receptors. The reactive oxygen species H2 O2 potentiated the effect of Bz-ATP at neurons but not at astrocytes. Hippocampal CA1 neurons exhibited a behavior similar to, but not identical with SG neurons. Although a combination of AP-5 and CNQX almost abolished the effect of Bz-ATP, H2 O2 was inactive. A Bz-ATP-dependent and A-438079-antagonizable reactive oxygen species production in SG slices was proven by a microelectrode biosensor. Immunohistochemical investigations showed the colocalization of P2X7-immunoreactivity with microglial (Iba1), but not astrocytic (GFAP, S100β) or neuronal (MAP2) markers in the SG. It is concluded that SG astrocytes possess P2X7 receptors; their activation leads to the release of glutamate, which via NMDA- and AMPA receptor stimulation induces cationic current in the neighboring neurons. P2X7 receptors have a very low density under resting conditions but become functionally upregulated under pathological conditions.

  16. Purinergic signaling in inflammatory cells: P2 receptor expression, functional effects, and modulation of inflammatory responses.

    PubMed

    Jacob, Fenila; Pérez Novo, Claudina; Bachert, Claus; Van Crombruggen, Koen

    2013-09-01

    Extracellular ATP and related nucleotides promote a wide range of pathophysiological responses via activation of cell surface purinergic P2 receptors. Almost every cell type expresses P2 receptors and/or exhibit regulated release of ATP. In this review, we focus on the purinergic receptor distribution in inflammatory cells and their implication in diverse immune responses by providing an overview of the current knowledge in the literature related to purinergic signaling in neutrophils, macrophages, dendritic cells, lymphocytes, eosinophils, and mast cells. The pathophysiological role of purinergic signaling in these cells include among others calcium mobilization, actin polymerization, chemotaxis, release of mediators, cell maturation, cytotoxicity, and cell death. We finally discuss the therapeutic potential of P2 receptor subtype selective drugs in inflammatory conditions.

  17. P2X7 Receptor-mediated Scavenger Activity of Mononuclear Phagocytes toward Non-opsonized Particles and Apoptotic Cells Is Inhibited by Serum Glycoproteins but Remains Active in Cerebrospinal Fluid*

    PubMed Central

    Gu, Ben J.; Duce, James A.; Valova, Valentina A.; Wong, Bruce; Bush, Ashley I.; Petrou, Steven; Wiley, James S.

    2012-01-01

    Rapid phagocytosis of non-opsonized particles including apoptotic cells is an important process that involves direct recognition of the target by multiple scavenger receptors including P2X7 on the phagocyte surface. Using a real-time phagocytosis assay, we studied the effect of serum proteins on this phagocytic process. Inclusion of 1–5% serum completely abolished phagocytosis of non-opsonized YG beads by human monocytes. Inhibition was reversed by pretreatment of serum with 1–10 mm tetraethylenepentamine, a copper/zinc chelator. Inhibitory proteins from the serum were determined as negatively charged glycoproteins (pI < 6) with molecular masses between 100 and 300 kDa. A glycoprotein-rich inhibitory fraction of serum not only abolished YG bead uptake but also inhibited phagocytosis of apoptotic lymphocytes or neuronal cells by human monocyte-derived macrophages. Three copper- and/or zinc-containing serum glycoproteins, ceruloplasmin, serum amyloid P-component, and amyloid precursor protein, were identified, and the purified proteins were shown to inhibit the phagocytosis of beads by monocytes as well as phagocytosis of apoptotic neuronal cells by macrophages. Human adult cerebrospinal fluid, which contains very little glycoprotein, had no inhibitory effect on phagocytosis of either beads or apoptotic cells. These data suggest for the first time that metal-interacting glycoproteins present within serum are able to inhibit the scavenger activity of mononuclear phagocytes toward insoluble debris and apoptotic cells. PMID:22461619

  18. Desensitization properties of P2X3 receptors shaping pain signaling

    PubMed Central

    Giniatullin, Rashid; Nistri, Andrea

    2013-01-01

    ATP-gated P2X3 receptors are mostly expressed by nociceptive sensory neurons and participate in transduction of pain signals. P2X3 receptors show a combination of fast desensitization onset and slow recovery. Moreover, even low nanomolar agonist concentrations unable to evoke a response, can induce desensitization via a phenomenon called “high affinity desensitization.” We have also observed that recovery from desensitization is agonist-specific and can range from seconds to minutes. The recovery process displays unusually high temperature dependence. Likewise, recycling of P2X3 receptors in peri-membrane regions shows unexpectedly large temperature sensitivity. By applying kinetic modeling, we have previously shown that desensitization characteristics of P2X3 receptor are best explained with a cyclic model of receptor operation involving three agonist molecules binding a single receptor and that desensitization is primarily developing from the open receptor state. Mutagenesis experiments suggested that desensitization depends on a certain conformation of the ATP binding pocket and on the structure of the transmembrane domains forming the ion pore. Further molecular determinants of desensitization have been identified by mutating the intracellular N- and C-termini of P2X3 receptor. Unlike other P2X receptors, the P2X3 subtype is facilitated by extracellular calcium that acts via specific sites in the ectodomain neighboring the ATP binding pocket. Thus, substitution of serine275 in this region (called “left flipper”) converts the natural facilitation induced by extracellular calcium to receptor inhibition. Given their strategic location in nociceptive neurons and unique desensitization properties, P2X3 receptors represent an attractive target for development of new analgesic drugs via promotion of desensitization aimed at suppressing chronic pain. PMID:24367291

  19. 5-HT1A receptor-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) is modulated by regulator of G protein signaling protein 19.

    PubMed

    Wang, Qin; Terauchi, Akiko; Yee, Christopher H; Umemori, Hisashi; Traynor, John R

    2014-09-01

    The 5-HT1A receptor is a G protein coupled receptor (GPCR) that activates G proteins of the Gαi/o family. 5-HT1A receptors expressed in the raphe, hippocampus and prefrontal cortex are implicated in the control of mood and are targets for anti-depressant drugs. Regulators of G protein signaling (RGS) proteins are members of a large family that play important roles in signal transduction downstream of G protein coupled receptors (GPCRs). The main role of RGS proteins is to act as GTPase accelerating proteins (GAPs) to dampen or negatively regulate GPCR-mediated signaling. We have shown that a mouse expressing Gαi2 that is insensitive to all RGS protein GAP activity has an anti-depressant-like phenotype due to increased signaling of postsynaptic 5-HT1A receptors, thus implicating the 5-HT1A receptor-Gαi2 complex as an important target. Here we confirm that RGS proteins act as GAPs to regulate signaling to adenylate cyclase and the mitogen-activated protein kinase (MAPK) pathway downstream of the 5-HT1A receptor, using RGS-insensitive Gαi2 protein expressed in C6 cells. We go on to use short hairpin RNA (shRNA) to show that RGS19 is responsible for the GAP activity in C6 cells and also that RGS19 acts as a GAP for 5-HT1A receptor signaling in human neuroblastoma SH-SY5Y cells and primary hippocampal neurons. In addition, in both cell types the synergy between 5-HT1A receptor and the fibroblast growth factor receptor 1 in stimulating the MAPK pathway is enhanced following shRNA reduction of RGS19 expression. Thus RGS19 may be a viable new target for anti-depressant medications.

  20. Dopaminergic enhancement of excitatory synaptic transmission in layer II entorhinal neurons is dependent on D₁-like receptor-mediated signaling.

    PubMed

    Glovaci, I; Caruana, D A; Chapman, C A

    2014-01-31

    The modulatory neurotransmitter dopamine induces concentration-dependent changes in synaptic transmission in the entorhinal cortex, in which high concentrations of dopamine suppress evoked excitatory postsynaptic potentials (EPSPs) and lower concentrations induce an acute synaptic facilitation. Whole-cell current-clamp recordings were used to investigate the dopaminergic facilitation of synaptic responses in layer II neurons of the rat lateral entorhinal cortex. A constant bath application of 1 μM dopamine resulted in a consistent facilitation of EPSPs evoked in layer II fan cells by layer I stimulation; the size of the facilitation was more variable in pyramidal neurons, and synaptic responses in a small group of multiform neurons were not modulated by dopamine. Isolated inhibitory synaptic responses were not affected by dopamine, and the facilitation of EPSPs was not associated with a change in paired-pulse facilitation ratio. Voltage-clamp recordings of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptor-mediated excitatory postsynaptic currents (EPSCs) were facilitated by dopamine, but N-methyl-D-aspartate receptor-mediated currents were not. Bath application of the dopamine D₁-like receptor blocker SCH23390 (50 μM), but not the D₂-like receptor blocker sulpiride (50 μM), prevented the facilitation, indicating that it is dependent upon D₁-like receptor activation. Dopamine D₁ receptors lead to activation of protein kinase A (PKA), and including the PKA inhibitor H-89 or KT 5720 in the recording pipette solution prevented the facilitation of EPSCs. PKA-dependent phosphorylation of inhibitor 1 or the dopamine- and cAMP-regulated protein phosphatase (DARPP-32) can lead to a facilitation of AMPA receptor responses by inhibiting the activity of protein phosphatase 1 (PP1) that reduces dephosphorylation of AMPA receptors, and we found here that inhibition of PP1 occluded the facilitatory effect of dopamine. The dopamine

  1. Adenine attenuates the Ca(2+) contraction-signaling pathway via adenine receptor-mediated signaling in rat vascular smooth muscle cells.

    PubMed

    Fukuda, Toshihiko; Kuroda, Takahiro; Kono, Miki; Hyoguchi, Mai; Tajiri, Satoshi; Tanaka, Mitsuru; Mine, Yoshinori; Matsui, Toshiro

    2016-09-01

    Our previous study demonstrated that adenine (6-amino-6H-purine) relaxed contracted rat aorta rings in an endothelial-independent manner. Although adenine receptors (AdeRs) are expressed in diverse tissues, aortic AdeR expression has not been ascertained. Thus, the aims of this study were to clarify the expression of AdeR in rat vascular smooth muscle cells (VSMCs) and to investigate the adenine-induced vasorelaxation mechanism(s). VSMCs were isolated from 8-week-old male Wistar-Kyoto rats and used in this study. Phosphorylation of myosin light chain (p-MLC) was measured by western blot. AdeR mRNA was detected by RT-PCR. Intracellular Ca(2+) concentration ([Ca(2+)]i) was measured by using Fura-2/AM. Vasorelaxant adenine (10-100 μM) significantly reduced p-MLC by angiotensin II (Ang II, 10 μM) in VSMCs (P < 0.05). We confirmed the expression of aortic AdeR mRNA and the activation of PKA in VSMCs through stimulation of AdeR by adenine by ELISA. Intracellular Ca(2+) concentration ([Ca(2+)]i) measurement demonstrated that adenine inhibits Ang II- and m-3M3FBS (PLC agonist)-induced [Ca(2+)]i elevation. In AdeR-knockdown VSMCs, PKA activation and p-MLC reduction by adenine were completely abolished. These results firstly demonstrated that vasorelaxant adenine can suppress Ca(2+) contraction signaling pathways via aortic AdeR/PKA activation in VSMCs.

  2. Receptor-Mediated Tobacco Toxicity

    PubMed Central

    Arredondo, Juan; Chernyavsky, Alexander I.; Marubio, Lisa M.; Beaudet, Arthur L.; Jolkovsky, David L.; Pinkerton, Kent E.; Grando, Sergei A.

    2005-01-01

    Tobacco is a known cause of oral disease but the mechanism remains elusive. Nicotine (Nic) is a likely culprit of pathobiological effects because it displaces the local cytotransmitter acetylcholine from the nicotinic receptors (nAChRs) expressed by oral keratinocytes (KCs). To gain a mechanistic insight into tobacco-induced morbidity in the oral cavity, we studied effects of exposures to environmental tobacco smoke (ETS) versus equivalent concentration of pure Nic on human and murine KCs. Both ETS and Nic up-regulated expression of cell cycle and apoptosis regulators, differentiation marker filaggrin, and signal transduction factors at both the mRNA and protein levels. These changes could be abolished in cultured human oral KCs transfected with anti-α3 small interfering RNA or treated with the α3β2-preferring antagonist α-conotoxin MII. Functional inactivation of α3-mediated signaling in α3−/− mutant KCs prevented most of the ETS/Nic-dependent changes in gene expression. To determine relevance of the in vitro findings to the in vivo situation, we studied gene expression in oral mucosa of neonatal α3+/+ and α3−/− littermates delivered by heterozygous mice soon after their exposures to ETS or equivalent concentration of pure Nic in drinking water. In addition to reverse transcriptase-polymerase chain reaction and Western blot, the ETS/Nic-dependent alterations in gene expression were also detected by semiquantitative immunofluorescence assay directly in KCs comprising murine oral mucosa. Only wild-type mice consistently developed significant (P < 0.05) changes in the gene expression. These results identified α3β2 nAChR as a major receptor mediating effects of tobacco products on KC gene expression. Real-time polymerase chain reaction demonstrated that in all three model systems the common genes targeted by α3β2-mediated ETS/Nic toxicity were p21, Bcl-2, NF-κB, and STAT-1. The expression of the nAChR subunits α5 and β2 and the muscarinic

  3. P2Y1 Receptor Activation of the TRPV4 Ion Channel Enhances Purinergic Signaling in Satellite Glial Cells*

    PubMed Central

    Rajasekhar, Pradeep; Poole, Daniel P.; Liedtke, Wolfgang; Bunnett, Nigel W.; Veldhuis, Nicholas A.

    2015-01-01

    Transient receptor potential (TRP) ion channels of peripheral sensory pathways are important mediators of pain, itch, and neurogenic inflammation. They are expressed by primary sensory neurons and by glial cells in the central nervous system, but their expression and function in satellite glial cells (SGCs) of sensory ganglia have not been explored. SGCs tightly ensheath neurons of sensory ganglia and can regulate neuronal excitability in pain and inflammatory states. Using a modified dissociation protocol, we isolated neurons with attached SGCs from dorsal root ganglia of mice. SGCs, which were identified by expression of immunoreactive Kir4.1 and glutamine synthetase, were closely associated with neurons, identified using the pan-neuronal marker NeuN. A subpopulation of SGCs expressed immunoreactive TRP vanilloid 4 (TRPV4) and responded to the TRPV4-selective agonist GSK1016790A by an influx of Ca2+ ions. SGCs did not express functional TRPV1, TRPV3, or TRP ankyrin 1 channels. Responses to GSK1016790A were abolished by the TRPV4 antagonist HC067047 and were absent in SGCs from Trpv4−/− mice. The P2Y1-selective agonist 2-methylthio-ADP increased [Ca2+]i in SGCs, and responses were prevented by the P2Y1-selective antagonist MRS2500. P2Y1 receptor-mediated responses were enhanced in TRPV4-expressing SGCs and HEK293 cells, suggesting that P2Y1 couples to and activates TRPV4. PKC inhibitors prevented P2Y1 receptor activation of TRPV4. Our results provide the first evidence for expression of TRPV4 in SGCs and demonstrate that TRPV4 is a purinergic receptor-operated channel in SGCs of sensory ganglia. PMID:26475857

  4. Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling

    PubMed Central

    Avanzato, D.; Genova, T.; Fiorio Pla, A.; Bernardini, M.; Bianco, S.; Bussolati, B.; Mancardi, D.; Giraudo, E.; Maione, F.; Cassoni, P.; Castellano, I.; Munaron, L.

    2016-01-01

    Purinergic signaling is involved in inflammation and cancer. Extracellular ATP accumulates in tumor interstitium, reaching hundreds micromolar concentrations, but its functional role on tumor vasculature and endothelium is unknown. Here we show that high ATP doses (>20 μM) strongly inhibit migration of endothelial cells from human breast carcinoma (BTEC), but not of normal human microvascular EC. Lower doses (1–10 mm result ineffective. The anti-migratory activity is associated with cytoskeleton remodeling and is significantly prevented by hypoxia. Pharmacological and molecular evidences suggest a major role for P2X7R and P2Y11R in ATP-mediated inhibition of TEC migration: selective activation of these purinergic receptors by BzATP mimics the anti-migratory effect of ATP, which is in turn impaired by their pharmacological or molecular silencing. Downstream pathway includes calcium-dependent Adenilyl Cyclase 10 (AC10) recruitment, cAMP release and EPAC-1 activation. Notably, high ATP enhances TEC-mediated attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization. Finally, we provide the first evidence of in vivo P2X7R expression in blood vessels of murine and human breast carcinoma. In conclusion, we have identified a purinergic pathway selectively acting as an antiangiogenic and normalizing signal for human tumor-derived vascular endothelium. PMID:27586846

  5. Deletion of GIRK2 Subunit of GIRK Channels Alters the 5-HT1A Receptor-Mediated Signaling and Results in a Depression-Resistant Behavior

    PubMed Central

    Llamosas, Nerea; Bruzos-Cidón, Cristina; Rodríguez, José Julio; Ugedo, Luisa

    2015-01-01

    Background: Targeting dorsal raphe 5-HT1A receptors, which are coupled to G-protein inwardly rectifying potassium (GIRK) channels, has revealed their contribution not only to behavioral and functional aspects of depression but also to the clinical response to its treatment. Although GIRK channels containing GIRK2 subunits play an important role controlling excitability of several brain areas, their impact on the dorsal raphe activity is still unknown. Thus, the goal of the present study was to investigate the involvement of GIRK2 subunit-containing GIRK channels in depression-related behaviors and physiology of serotonergic neurotransmission. Methods: Behavioral, functional, including in vivo extracellular recordings of dorsal raphe neurons, and neurogenesis studies were carried out in wild-type and GIRK2 mutant mice. Results: Deletion of the GIRK2 subunit promoted a depression-resistant phenotype and determined the behavioral response to the antidepressant citalopram without altering hippocampal neurogenesis. In dorsal raphe neurons of GIRK2 knockout mice, and also using GIRK channel blocker tertiapin-Q, the basal firing rate was higher than that obtained in wild-type animals, although no differences were observed in other firing parameters. 5-HT1A receptors were desensitized in GIRK2 knockout mice, as demonstrated by a lower sensitivity of dorsal raphe neurons to the inhibitory effect of the 5-HT1A receptor agonist, 8-OH-DPAT, and the antidepressant citalopram. Conclusions: Our results indicate that GIRK channels formed by GIRK2 subunits determine depression-related behaviors as well as basal and 5-HT1A receptor-mediated dorsal raphe neuronal activity, becoming alternative therapeutic targets for psychiatric diseases underlying dysfunctional serotonin transmission. PMID:25956878

  6. RdgBα reciprocally transfers PA and PI at ER-PM contact sites to maintain PI(4,5)P2 homoeostasis during phospholipase C signalling in Drosophila photoreceptors.

    PubMed

    Cockcroft, Shamshad; Garner, Kathryn; Yadav, Shweta; Gomez-Espinoza, Evelyn; Raghu, Padinjat

    2016-02-01

    Phosphatidylinositol (PI) is the precursor lipid for the synthesis of PI 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane (PM) and is sequentially phosphorylated by the lipid kinases, PI 4-kinase and phosphatidylinositol 4-phosphate (PI4P)-5-kinase. Receptor-mediated hydrolysis of PI(4,5)P2 takes place at the PM but PI resynthesis occurs at the endoplasmic reticulum (ER). Thus PI(4,5)P2 resynthesis requires the reciprocal transport of two key intermediates, phosphatidic acid (PA) and PI between the ER and the PM. PI transfer proteins (PITPs), defined by the presence of the PITP domain, can facilitate lipid transfer between membranes; the PITP domain comprises a hydrophobic cavity with dual specificity but accommodates a single phospholipid molecule. The class II PITP, retinal degeneration type B (RdgB)α is a multi-domain protein and its PITP domain can bind and transfer PI and PA. In Drosophila photoreceptors, a well-defined G-protein-coupled phospholipase Cβ (PLCβ) signalling pathway, phototransduction defects resulting from loss of RdgBα can be rescued by expression of the PITP domain provided it is competent for both PI and PA transfer. We propose that RdgBα proteins maintain PI(4,5)P2 homoeostasis after PLC activation by facilitating the reciprocal transport of PA and PI at ER-PM membrane contact sites.

  7. Homeostatic regulation of the PI(4,5)P2-Ca(2+) signaling system at ER-PM junctions.

    PubMed

    Chang, Chi-Lun; Liou, Jen

    2016-08-01

    The phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-Ca(2+) signaling system is important for cell activation in response to various extracellular stimuli. This signaling system is initiated by receptor-induced hydrolysis of PI(4,5)P2 in the plasma membrane (PM) to generate the soluble second messenger inositol 1,4,5-trisphosphate (IP3). IP3 subsequently triggers the release of Ca(2+) from the endoplasmic reticulum (ER) store to the cytosol to activate Ca(2+)-mediated responses, such as secretion and proliferation. The consumed PM PI(4,5)P2 and ER Ca(2+) must be quickly restored to sustain signaling responses, and to maintain the homeostasis of PI(4,5)P2 and Ca(2+). Since phosphatidylinositol (PI), the precursor lipid for PM PI(4,5)P2, is synthesized in the ER membrane, and a Ca(2+) influx across the PM is required to refill the ER Ca(2+) store, efficient communications between the ER and the PM are critical for the homeostatic regulation of the PI(4,5)P2-Ca(2+) signaling system. This review describes the major findings that established the framework of the PI(4,5)P2-Ca(2+) signaling system, and recent discoveries on feedback control mechanisms at ER-PM junctions that sustain the PI(4,5)P2-Ca(2+) signaling system. Particular emphasis is placed on the characterization of ER-PM junctions where efficient communications between the ER and the PM occur, and the activation mechanisms of proteins that dynamically localize to ER-PM junctions to provide the feedback control during PI(4,5)P2-Ca(2+) signaling, including the ER Ca(2+) sensor STIM1, the extended synaptotagmin E-Syt1, and the PI transfer protein Nir2. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

  8. The Novel Functions of the PLC/PKC/PKD Signaling Axis in G Protein-Coupled Receptor-Mediated Chemotaxis of Neutrophils

    PubMed Central

    Xu, Xuehua; Jin, Tian

    2015-01-01

    Chemotaxis, a directional cell migration guided by extracellular chemoattractant gradients, plays an essential role in the recruitment of neutrophils to sites of inflammation. Chemotaxis is mediated by the G protein-coupled receptor (GPCR) signaling pathway. Extracellular stimuli trigger activation of the PLC/PKC/PKD signaling axis, which controls several signaling pathways. Here, we concentrate on the novel functions of PLC/PKC/PKD signaling in GPCR-mediated chemotaxis of neutrophils. PMID:26605346

  9. Ionotropic P2X ATP Receptor Channels Mediate Purinergic Signaling in Mouse Odontoblasts

    PubMed Central

    Shiozaki, Yuta; Sato, Masaki; Kimura, Maki; Sato, Toru; Tazaki, Masakazu; Shibukawa, Yoshiyuki

    2017-01-01

    ATP modulates various functions in the dental pulp cells, such as intercellular communication and neurotransmission between odontoblasts and neurons, proliferation of dental pulp cells, and odontoblast differentiation. However, functional expression patterns and their biophysical properties of ionotropic ATP (P2X) receptors (P2X1–P2X7) in odontoblasts were still unclear. We examined these properties of P2X receptors in mouse odontoblasts by patch-clamp recordings. K+-ATP, nonselective P2X receptor agonist, induced inward currents in odontoblasts in a concentration-dependent manner. K+-ATP-induced currents were inhibited by P2X4 and P2X7 selective inhibitors (5-BDBD and KN62, respectively), while P2X1 and P2X3 inhibitors had no effects. P2X7 selective agonist (BzATP) induced inward currents dose-dependently. We could not observe P2X1, 2/3, 3 selective agonist (αβ-MeATP) induced currents. Amplitudes of K+-ATP-induced current were increased in solution without extracellular Ca2+, but decreased in Na+-free extracellular solution. In the absence of both of extracellular Na+ and Ca2+, K+-ATP-induced currents were completely abolished. K+-ATP-induced Na+ currents were inhibited by P2X7 inhibitor, while the Ca2+ currents were sensitive to P2X4 inhibitor. These results indicated that odontoblasts functionally expressed P2X4 and P2X7 receptors, which might play an important role in detecting extracellular ATP following local dental pulp injury. PMID:28163685

  10. SRC Homology 2 Domain Binding Sites in Insulin, IGF-1 and FGF receptor mediated signaling networks reveal an extensive potential interactome

    PubMed Central

    2012-01-01

    Specific peptide ligand recognition by modular interaction domains is essential for the fidelity of information flow through the signal transduction networks that control cell behavior in response to extrinsic and intrinsic stimuli. Src homology 2 (SH2) domains recognize distinct phosphotyrosine peptide motifs, but the specific sites that are phosphorylated and the complement of available SH2 domains varies considerably in individual cell types. Such differences are the basis for a wide range of available protein interaction microstates from which signaling can evolve in highly divergent ways. This underlying complexity suggests the need to broadly map the signaling potential of systems as a prerequisite for understanding signaling in specific cell types as well as various pathologies that involve signal transduction such as cancer, developmental defects and metabolic disorders. This report describes interactions between SH2 domains and potential binding partners that comprise initial signaling downstream of activated fibroblast growth factor (FGF), insulin (Ins), and insulin-like growth factor-1 (IGF-1) receptors. A panel of 50 SH2 domains screened against a set of 192 phosphotyrosine peptides defines an extensive potential interactome while demonstrating the selectivity of individual SH2 domains. The interactions described confirm virtually all previously reported associations while describing a large set of potential novel interactions that imply additional complexity in the signaling networks initiated from activated receptors. This study of pTyr ligand binding by SH2 domains provides valuable insight into the selectivity that underpins complex signaling networks that are assembled using modular protein interaction domains. PMID:22974441

  11. Role of altered growth factor receptor-mediated JAK2 signaling in growth and maintenance of human acute myeloid leukemia stem cells

    PubMed Central

    Cook, Amy M.; Li, Liang; Ho, Yinwei; Lin, Allen; Li, Ling; Stein, Anthony; Forman, Stephen; Perrotti, Danilo; Jove, Richard

    2014-01-01

    Acute myeloid leukemia (AML) is sustained by small populations of leukemia stem cells (LSCs) that can resist available treatments and represent important barriers to cure. Although previous studies have shown increased signal transducer and activator of transcription (STAT)3 and STAT5 phosphorylation in AML leukemic blasts, the role of Janus kinase (JAK) signaling in primary AML compared with normal stem cells has not been directly evaluated. We show here that JAK/STAT signaling is increased in LSCs, particularly from high-risk AML. JAK2 inhibition using small molecule inhibitors or interference RNA reduced growth of AML LSCs while sparing normal stem cells both in vitro and in vivo. Increased JAK/STAT activity was associated with increased expression and altered signaling through growth factor receptors in AML LSCs, including receptor tyrosine kinase c-KIT and FMS-related tyrosine kinase 3 (FLT3). Inhibition of c-KIT and FLT3 expression significantly inhibited JAK/STAT signaling in AML LSCs, and JAK inhibitors effectively inhibited FLT3-mutated AML LSCs. Our results indicate that JAK/STAT signaling represents an important signaling mechanism supporting AML LSC growth and survival. These studies support continued evaluation of strategies for JAK/STAT inhibition for therapeutic targeting of AML LSCs. PMID:24668492

  12. Class A scavenger receptor-mediated dsRNA internalization is independent of innate antiviral signaling and does not require PI3K activity1

    PubMed Central

    Nellimarla, Srinivas; Baid, Kaushal; Loo, Yueh-Ming; Gale, Michael; Bowdish, Dawn M.; Mossman, Karen L.

    2016-01-01

    Double-stranded RNA is a potent trigger of innate immune signaling, eliciting effects within virally infected cells and following release from dying cells. Given its inherent stability, extracellular dsRNA induces both local and systemic effects. Although the class A scavenger receptors (SR-As)3 mediate dsRNA entry, it is unknown if they contribute to signaling beyond ligand internalization. Here, we investigated if SR-As contribute to innate immune signaling independent of the classic TLR and RLR pathways. We generated a stable A549 human epithelial cell line with inducible expression of the Hepatitis C virus protease NS3/4A, which efficiently cleaves TRIF and IPS-1, adaptors for TLR3 and the RLRs respectively. Cells expressing NS3/4A as well as TLR3/MDA5/IPS-1−/− mouse embryonic fibroblasts completely lacked antiviral activity to extracellular dsRNA relative to control cells, suggesting that SR-As do not possess signaling capacity independent of TLR3 or the RLRs. Previous studies implicated PI3K signaling in SR-A-mediated activities and in downstream production of type I interferon. We found that SR-A-mediated dsRNA internalization occurs independent of PI3K activation, while downstream signaling leading to interferon production was partially dependent on PI3K activity. Overall, these findings suggest that SR-A-mediated dsRNA internalization is independent of innate antiviral signaling. PMID:26363049

  13. Spinal 5-HT3 receptors mediate descending facilitation and contribute to behavioral hypersensitivity via a reciprocal neuron-glial signaling cascade

    PubMed Central

    2014-01-01

    Background It has been recently recognized that the descending serotonin (5-HT) system from the rostral ventromedial medulla (RVM) in the brainstem and the 5-HT3 receptor subtype in the spinal dorsal horn are involved in enhanced descending pain facilitation after tissue and nerve injury. However, the mechanisms underlying the activation of the 5-HT3 receptor and its contribution to facilitation of pain remain unclear. Results In the present study, activation of spinal 5-HT3 receptors by intrathecal injection of a selective 5-HT3 receptor agonist SR 57227 induced spinal glial hyperactivity, neuronal hyperexcitability and pain hypersensitivity in rats. We found that there was neuron-to-microglia signaling via the chemokine fractalkine, microglia to astrocyte signaling via cytokine IL-18, astrocyte to neuronal signaling by IL-1β, and enhanced activation of NMDA receptors in the spinal dorsal horn. Glial hyperactivation in spinal dorsal horn after hindpaw inflammation was also attenuated by molecular depletion of the descending 5-HT system by intra-RVM Tph-2 shRNA interference. Conclusions These findings offer new insights into the cellular and molecular mechanisms at the spinal level responsible for descending 5-HT-mediated pain facilitation during the development of persistent pain after tissue and nerve injury. New pain therapies should focus on prime targets of descending facilitation-induced glial involvement, and in particular the blocking of intercellular signaling transduction between neurons and glia. PMID:24913307

  14. Signal Transduction Mechanism for Serotonin 5-HT2B Receptor-Mediated DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes.

    PubMed

    Naito, Kota; Tanaka, Chizuru; Mitsuhashi, Manami; Moteki, Hajime; Kimura, Mitsutoshi; Natsume, Hideshi; Ogihara, Masahiko

    2016-01-01

    The involvement of serotonin (5-hydroxytryptamine; 5-HT) and the 5-HT2 receptor subtypes in the induction of DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes to elucidate the intracellular signal transduction mechanisms. Hepatocyte parenchymal cells maintained in a serum-free, defined medium, synthesized DNA and proliferated in the presence of 5-HT or a selective 5-HT2B receptor agonist, BW723C86, but not in the presence of 5-HT2A, or 5-HT2C receptor agonists (TCB-2 and CP809101, respectively), in a time- and dose-dependent manner. A selective 5-HT2B receptor antagonist, LY272015 (10(-7) M), and a specific phospholipase C (PLC) inhibitor, U-73122 (10(-6) M), as well as specific inhibitors of growth-related signal transducers-including AG1478, LY294002, PD98059, and rapamycin-completely inhibited 5-HT (10(-6) M)- or BW723C86 (10(-6) M)-induced hepatocyte DNA synthesis and proliferation. Both 5-HT and BW723C86 were shown to significantly stimulate the phosphorylation of epidermal growth factor (EGF)/transforming growth factor (TGF)-α receptor tyrosine kinase (p175 kDa) and extracellular signal-regulated kinase (ERK) 2 on Western blot analysis. These results suggest that the proliferative mechanism of activating 5-HT is mediated mainly through 5-HT2B receptor-stimulated Gq/PLC and EGF/TGF-α-receptor/phosphatidylinositol 3-kinase (PI3K)/ERK2/mammalian target of rapamycin (mTOR) signaling pathways in primary cultured hepatocytes.

  15. Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis

    PubMed Central

    2017-01-01

    ABSTRACT The Gram-positive bacterial cell wall (CW) peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2) ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr)-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K) signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling. PMID:28049146

  16. Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling

    SciTech Connect

    Kaowinn, Sirichat; Cho, Il-Rae; Moon, Jeong; Jun, Seung Won; Kim, Chang Seok; Kang, Ho Young; Kim, Manbok; Koh, Sang Seok; Chung, Young-Hwa

    2015-04-03

    Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling. - Highlights: • PAUF confers resistance against oncolytic parvovirus H-1 infection. • PAUF enhances the expression of IFNAR in Panc-1 cells. • Increased activation of Tyk2 or Stat1 by PAUF provides resistance to parvovirus H-1-mediated apoptosis. • Constitutive inhibition of PAUF enhances parvovirus H-1-mediated oncolysis of Bxpc3 pancreatic cancer cells.

  17. Activity-dependent bidirectional regulation of GABAA receptor channels by the 5-HT4 receptor-mediated signalling in rat prefrontal cortical pyramidal neurons

    PubMed Central

    Cai, Xiang; Flores-Hernandez, Jorge; Feng, Jian; Yan, Zhen

    2002-01-01

    Emerging evidence has implicated a potential role for 5-HT4 receptors in cognition and anxiolysis. One of the main target structures of 5-HT4 receptors on ‘cognitive and emotional’ pathways is the prefrontal cortex (PFC). As GABAergic signalling plays a key role in regulating PFC functions, we examined the effect of 5-HT4 receptors on GABAA receptor channels in PFC pyramidal neurons. Application of 5-HT4 receptor agonists produced either an enhancement or a reduction of GABA-evoked currents in PFC neurons, which are both mediated by anchored protein kinase A (PKA). Although PKA phosphorylation of GABAA receptor β3 or β1 subunits leads to current enhancement or reduction respectively in heterologous expression systems, we found that β3 and β1 subunits are co-expressed in PFC pyramidal neurons. Interestingly, altering PKA activation levels can change the direction of the dual effect, switching enhancement to reduction and vice versa. In addition, increased neuronal activity in PFC slices elevated the PKA activation level, changing the enhancing effect of 5-HT4 receptors on the amplitude of GABAergic inhibitory postsynaptic currents (IPSCs) to a reduction. These results suggest that 5-HT4 receptors can modulate GABAergic signalling bidirectionally, depending on the basal PKA activation levels that are determined by neuronal activity. This modulation provides a unique and flexible mechanism for 5-HT4 receptors to dynamically regulate synaptic transmission and neuronal excitability in the PFC network. PMID:11986365

  18. A Carma1/MALT1-dependent, Bcl10-independent, pathway regulates antigen receptor-mediated mTOR signaling in T cells

    PubMed Central

    Hamilton, Kristia S.; Phong, Binh; Corey, Catherine; Cheng, Jing; Gorentla, Balachandra; Zhong, Xiaoping; Shiva, Sruti; Kane, Lawrence P.

    2015-01-01

    Signaling to the mechanistic target of rapamycin (mTOR) regulates diverse cellular processes, including protein translation, cellular proliferation, metabolism, and autophagy. These effects are mediated in part by the mTOR targets S6 kinase (S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). Most models place Akt upstream of the best-studied mTOR complex, mTORC1; however, studies have called into question whether Akt is necessary for this pathway, at least in T cells. We found that the adaptor protein Carma1 [caspase recruitment domain (CARD)-containing membrane-associated protein 1 (Carma1)] and at least one of its associated proteins, the paracaspase MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), were required for optimal activation of mTOR in T cells in response to stimulation of the T cell receptor (TCR) and the coreceptor CD28. However, another common binding partner of Carma1 and MALT1, Bcl10, was not required for TCR-dependent activation of the mTOR pathway. Consistent with these findings, MALT1 activity was required for the proliferation of CD4+ T cells, but not early TCR-dependent activation events. Also consistent with an effect on mTOR, MALT1 activity was required for the increased metabolic flux in activated CD4+ T cells. Together, our data suggest that Carma1 and MALT1 play previously unappreciated roles in the activation of mTOR signaling in T cells after engagement of the TCR. PMID:24917592

  19. Piracy of prostaglandin E2/EP receptor-mediated signaling by Kaposi's sarcoma-associated herpes virus (HHV-8) for latency gene expression: strategy of a successful pathogen.

    PubMed

    George Paul, Arun; Sharma-Walia, Neelam; Kerur, Nagaraj; White, Carl; Chandran, Bala

    2010-05-01

    Kaposi's sarcoma-associated herpes virus (KSHV) is implicated in the pathogenesis of KS, a chronic inflammation-associated malignancy. Cyclooxygenase-2 (COX-2) and its metabolite prostaglandin E2 (PGE2), two pivotal proinflammatory/oncogeneic molecules, are proposed to play roles in the expression of major KSHV latency-associated nuclear antigen-1 (LANA-1). Microsomal PGE2 synthase, PGE2, and its receptors (EP1, EP2, EP3, and EP4) were detected in KS lesions with the distinct staining of EP2/EP4 in KS lesions. In latently infected endothelial TIVE-LTC cells, EP receptor antagonists downregulated LANA-1 expression as well as Ca(2+), p-Src, p-PI3K, p-PKCzeta/lambda, and p-NF-kappaB, which are also some of the signal molecules proposed to be important in KS pathogenesis. Exogenous PGE2 and EP receptor agonists induced the LANA-1 promoter in 293 cells, and YY1, Sp1, Oct-1, Oct-6, C/EBP, and c-Jun transcription factors seem to be involved in this induction. PGE2/EP receptor-induced LANA-1 promoter activity was downregulated significantly by the inhibition of Ca(2+), p-Src, p-PI3K, p-PKCzeta/lambda, and p-NF-kappaB. These findings implicate the inflammatory PGE2/EP receptors and the associated signal molecules in herpes virus latency and uncover a novel paradigm that shows the evolution of KSHV genome plasticity to use inflammatory response for its survival advantage of maintaining latent gene expression. These data also suggest that potential use of anti-COX-2 and anti-EP receptor therapy may not only ameliorate the chronic inflammation associated with KS but could also lead to elimination of the KSHV latent infection and the associated KS lesions.

  20. Insulinotropic toxins as molecular probes for analysis of glucagon-like peptide-1 receptor-mediated signal transduction in pancreatic β-cells

    PubMed Central

    Holz, George G.; Leech, Colin A.; Habener, Joel F.

    2010-01-01

    Cholera toxin, pertussis toxin, mastoparan, maitotoxin, and α-latrotoxin are complex protein or polyether-based toxins of bacterial, insect, or phytoplankton origin that act with high potency at the endocrine pancreas to stimulate secretion of insulin from β-cells located in the islets of Langerhans. The remarkable insulinotropic properties of these toxins have attracted considerable attention by virtue of their use as selective molecular probes for analyses of β-cell stimulus-secretion coupling. Targets of the toxins include heptahelical cell surface receptors, GTP-binding proteins, ion channels, Ca2+ stores, and the exocytotic secretory apparatus. Here we review the value of insulinotropic toxins from the perspective of their established use in the study of signal transduction pathways activated by the blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1). Our analysis of one insulinotropic toxin (α-latrotoxin) leads us to conclude that there exists a process of molecular mimicry whereby the ‘lock and key’analogy inherent to hormone-receptor interactions is reproduced by a toxin related in structure to GLP-1. PMID:11086221

  1. Zbtb16 (PLZF) is stably suppressed and not inducible in non-innate T cells via T cell receptor-mediated signaling.

    PubMed

    Zhang, Sai; Laouar, Amale; Denzin, Lisa K; Sant'Angelo, Derek B

    2015-07-16

    The transcription factor PLZF (promyelocytic leukemia zinc finger; zbtb16) is essential for nearly all of the unique characteristics of NKT cells including their rapid and potent response to antigen. In the immune system, zbtb16 expression is only found in innate cells. Conventional T cells that ectopically express PLZF spontaneously acquire an activated, effector phenotype. Activation induced expression of lineage defining transcription factors such as T-bet, FoxP3, RORγt, GATA3 and others is essential for naïve T cell differentiation into effector T cells. In this study, we used sensitive genetic-based approaches to assess the induction of PLZF expression in non-innate T cells by T cell receptor (TCR)-mediated activation. Surprisingly, we found that PLZF was stably repressed in non-innate T cells and that TCR-mediated signaling was not sufficient to induce PLZF in conventional T cells. The inactivated state of PLZF was stably maintained in mature T cells, even under inflammatory conditions imposed by bacterial infection. Collectively, our data show that, in contrast to multiple recent reports, PLZF expression is highly specific to innate T cells and cannot be induced in conventional T cells via TCR-mediated activation or inflammatory challenge.

  2. Zbtb16 (PLZF) is stably suppressed and not inducible in non-innate T cells via T cell receptor-mediated signaling

    PubMed Central

    Zhang, Sai; Laouar, Amale; Denzin, Lisa K.; Sant’Angelo, Derek B.

    2015-01-01

    The transcription factor PLZF (promyelocytic leukemia zinc finger; zbtb16) is essential for nearly all of the unique characteristics of NKT cells including their rapid and potent response to antigen. In the immune system, zbtb16 expression is only found in innate cells. Conventional T cells that ectopically express PLZF spontaneously acquire an activated, effector phenotype. Activation induced expression of lineage defining transcription factors such as T-bet, FoxP3, RORγt, GATA3 and others is essential for naïve T cell differentiation into effector T cells. In this study, we used sensitive genetic-based approaches to assess the induction of PLZF expression in non-innate T cells by T cell receptor (TCR)-mediated activation. Surprisingly, we found that PLZF was stably repressed in non-innate T cells and that TCR-mediated signaling was not sufficient to induce PLZF in conventional T cells. The inactivated state of PLZF was stably maintained in mature T cells, even under inflammatory conditions imposed by bacterial infection. Collectively, our data show that, in contrast to multiple recent reports, PLZF expression is highly specific to innate T cells and cannot be induced in conventional T cells via TCR-mediated activation or inflammatory challenge. PMID:26178856

  3. Neurons and astroglia govern microglial endotoxin tolerance through macrophage colony-stimulating factor receptor-mediated ERK1/2 signals

    PubMed Central

    Chu, Chun-Hsien; Wang, Shijun; Li, Chia-Ling; Chen, Shih-Heng; Hu, Chih-Fen; Chung, Yi-Lun; Chen, Shiou-Lan; Wang, Qingshan; Lu, Ru-Band; Gao, Hui-Ming; Hong, Jau-Shyong

    2016-01-01

    Endotoxin tolerance (ET) is a reduced responsiveness of innate immune cells like macrophages/monocytes to an endotoxin challenge following a previous encounter with the endotoxin. Although ET in peripheral systems has been well studied, little is known about ET in the brain. The present study showed that brain immune cells, microglia, being different from peripheral macrophages, displayed non-cell autonomous mechanisms in ET formation. Specifically, neurons and astroglia were indispensable for microglial ET. Macrophage colony-stimulating factor (M-CSF) secreted from these non-immune cells was essential for governing microglial ET. Neutralization of M-CSF deprived the neuron-glia conditioned medium of its ability to enable microglia to form ET when microglia encountered two lipopolysaccharide (LPS) treatments. Recombinant M-CSF protein rendered enriched microglia refractory to the second LPS challenge leading to microglial ET. Activation of microglial M-CSF receptor (M-CSFR; also known as CSF1R) and the downstream ERK1/2 signals was responsible for M-CSF-mediated microglial ET. Endotoxin-tolerant microglia in neuron-glia cultures displayed M2-like polarized phenotypes, as shown by upregulation of M2 marker Arg-1, elevated production of anti-inflammatory cytokine interleukin 10, and decreased secretion of pro-inflammatory mediators (tumor necrosis factor α, nitric oxide, prostaglandin E2 and interleukin 1β). Endotoxin-tolerant microglia protected neurons against LPS-elicited inflammatory insults, as shown by reduced neuronal damages in LPS pre-treatment group compared with the group without LPS pre-treatment. Moreover, while neurons and astroglia became injured during chronic neuroinflammation, microglia failed to form ET. Thus, this study identified a distinct non-cell autonomous mechanism of microglial ET. Interactions of M-CSF secreted by neurons and astroglia with microglial M-CSFR programed microglial ET. Loss of microglial ET could be an important

  4. Regulation of ATP-Gated P2X Channels: From Redox Signaling to Interactions with Other Proteins

    PubMed Central

    Leiva-Salcedo, Elías; Rokic, Milos B.; Coddou, Claudio

    2014-01-01

    Abstract Significance: The family of purinergic P2X receptors (P2XRs) is a part of ligand-gated superfamily of channels activated by extracellular adenosine-5′-triphosphate. P2XRs are present in virtually all mammalian tissues as well as in tissues of other vertebrate and nonvertebrate species and mediate a large variety of functions, including fast transmission at central synapses, contraction of smooth muscle cells, platelet aggregation, and macrophage activation to proliferation and cell death. Recent Advances: The recent solving of crystal structure of the zebrafish P2X4.1R is a major advance in the understanding of structural correlates of channel activation and regulation. Combined with growing information obtained in the post-structure era and the reinterpretation of previous work within the context of the tridimensional structure, these data provide a better understanding of how the channel operates at the molecular levels. Critical Issues: This review focuses on the relationship between redox signaling and P2XR function. We also discuss other allosteric modulation of P2XR gating in the physiological/pathophysiological context. This includes the summary of extracellular actions of trace metals, which can be released to the synaptic cleft, pH decrease that happens during ischemia and inflammation, and calcium, an extracellular and intracellular messenger. Future Directions: Our evolving understanding of activation and regulation of P2XRs is helpful in clarifying the mechanism by which these channels trigger and modulate cellular functions. Further research is required to identify the signaling pathways contributing to the regulation of the receptor activity and to develop novel and receptor-specific allosteric modulators, which could be used in vivo with therapeutic potential. Antioxid. Redox Signal. 21, 953–970. PMID:23944253

  5. Nucleus of the solitary tract (pro)renin receptor-mediated antihypertensive effect involves nuclear factor-κB-cytokine signaling in the spontaneously hypertensive rat.

    PubMed

    Zubcevic, Jasenka; Jun, Joo Y; Lamont, Gwyneth; Murça, Tatiane M; Shi, Peng; Yuan, Wei; Lin, Fan; Carvajal, Jessica Marulanda; Li, Qiuhong; Sumners, Colin; Raizada, Mohan K; Shan, Zhiying

    2013-03-01

    The importance of the (pro)renin receptor (PRR) in the function of the central nervous system is increasingly evident because PRR seems to play a role in neuronal control of cardiovascular function. PRR expression is elevated in the nucleus of the solitary tract (NTS) of spontaneously hypertensive rats (SHR). In this study, we tested the hypothesis that altered activity of PRR in the NTS is linked to hypertension. Eight weeks of chronic knockdown of the NTS PRR, using recombinant adeno-associated virus type 2 (AAV2)-PRR-small hairpain RNA (shRNA)-mediated gene transduction, caused a significant increase in mean arterial pressure (MAP) in the SHR (shRNA, 173±5; Control, 151±6 mm Hg) but not in Wistar Kyoto rats (shRNA, 108±7; Control, 106±6 mm Hg). The MAP elevation in the SHR was associated with decreased inflammatory markers tumor necrosis factor-α, interleukin-6, C-C motif ligand 5, and their transcription factor, nuclear factor-κB. Consistent with the pressor effects of the PRR knockdown, acute bilateral NTS injection of human renin (2 pmol/side) decreased MAP and heart rate (HR) in SHR (ΔMAP, -38±4 mm Hg; Δheart rate, -40±10 bpm), with negligible responses in Wistar Kyoto rats (ΔMAP, -4±3 mm Hg; Δheart rate, -12±7 bpm). These effects in SHR were attenuated (80%) by prorenin handle region peptide but were not affected by angiotensin II type 1 or angiotensin II type 2 receptor blockers. Finally, PRR activation in SHR neuronal cultures by prorenin activated nuclear factor-κB and increased mRNA levels of interleukin-1β (250-fold), tumor necrosis factor-α (32-fold), interleukin-6 (35-fold), C-C motif ligand 5 (12-fold), and interleukin-10 (7-fold) in a nuclear factor-κB-dependent but angiotensin II type 1 receptor-independent manner. Therefore, NTS PRR mediates antihypertensive effects via an angiotensin II-independent mechanism in SHR, which involves stimulation of the nuclear factor-κB-cytokine signaling pathway.

  6. Cholesterol reduction by methyl-β-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells

    PubMed Central

    Huang, Peng; Xu, Wei; Yoon, Su-In; Chen, Chongguang; Chong, Parkson Lee-Gau; Liu-Chen, Lee-Yuan

    2008-01-01

    Opioid receptors have been shown to be located in and regulated by lipid rafts/caveolae in caveolin-rich non-neuronal cells. Here, we found that caveolin-1 level was very low in rat brain and undetectable in NG108-15 cells, which endogenously express delta opioid receptors (DOR). Rat caudate putamen (CPu) membranes, NG108-15 cells and CHO cells stably transfected with FLAG-mouse-DOR (CHO-FLAG-mDOR) were homogenized, sonicated in a detergent-free 0.5 M Na2CO3 buffer and fractionated through discontinuous or continuous sucrose density gradients. About 70% of opioid receptors in CPu and DOR in both cell lines were present in low-density (5-20% sucrose) membrane-domains enriched in cholesterol and ganglioside M1 (GM1), characteristics of lipid rafts in plasma membranes. In both cells, stimulation with permeable or non-permeable full agonists, but not with partial or inverse agonists, for 30 min shifted ∼25% of DORs out of rafts, by a naloxone-reversible and pertussis toxin-insensitive mechanism, which may undergo internalization. Methyl-β-cyclodextrin (MCD) treatment greatly reduced cholesterol and shifted DOR to higher-density fractions and decreased DPDPE affinities. MCD treatment attenuated DPDPE-induced [35S]GTPγS binding in CPu and NG108-15 cells, but enhanced it in CHO-FLAG-mDOR cells. In CHO-FLAG-mDOR cells, Gαi co-immunoprecipitated with caveolin-1, which was shown to inhibit Gαi/o, and MCD treatment dramatically reduced the association leading to disinhibition. Thus, although localization in rafts and agonist-induced shift of DOR are independent of caveolin-1, lipid rafts sustain DOR-mediated signaling in caveolin-deficient neuronal cells, but appear to inhibit it in caveolin-enriched non-neuronal cells. Cholesterol-dependent association of caveolin-1 with and the resulting inhibition of G proteins may be a contributing factor. PMID:17141202

  7. Sprouty2 Regulates PI(4,5)P2/Ca2+ Signaling and HIV-1 Gag Release

    PubMed Central

    Ehrlich, Lorna S.; Medina, Gisselle N.; Carter, Carol A.

    2011-01-01

    We recently reported that activation of the inositol 1,4,5-triphosphate receptor (IP3R) is required for efficient HIV-1 Gag trafficking and viral particle release (Ehrlich 2010). IP3R activation requires phospholipase C (PLC)-catalyzed hydrolysis of PI(4,5)P2 to IP3 and diacylglycerol. Here we show that Sprouty2 (Spry2), which binds PI(4,5)P2 and PLCγ, interfered with PI(4,5)P2 in a manner similar to U73122, an inhibitor of PI(4,5)P2 hydrolysis, suggesting that Spry2 may negatively regulate IP3R by preventing formation of its activating ligand, IP3. Mutation to Asp of R252, a critical determinant of PI(4,5)P2 binding in the C-terminal domain of Spry2, prevented the interference completely, indicating that binding to the phospholipid is required. In contrast, deletion of the PLCγ binding region or mutation of a critical Tyr residue in the region did not prevent the interference but Spry2-PI(4,5)P2 colocalization was not detected, suggesting that PLC binding is required for their stable association. Like U73122, Spry2 over-expression inhibited WT Gag release as virus-like particles. The inhibition was relieved by disrupting either binding determinant. IP3R-mediated Ca2+ signaling, in turn, was found to influence Spry2 subcellular distribution and ERK, a Spry2 regulator. Our findings suggest that Spry2 influences IP3R function through control of PI(4,5)P2 and IP3R influences Spry2 function by controlling its distribution and ERK activation. PMID:21762810

  8. Modulation of bladder afferent signals in normal and spinal cord-injured rats by purinergic P2X3 and P2X2/3 receptors

    PubMed Central

    Munoz, Alvaro; Somogyi, George T.; Boone, Timothy B.; Ford, Anthony P.; Smith, Christopher P.

    2015-01-01

    OBJECTIVE • To evaluate the role of bladder sensory purinergic P2X3 and P2X2/3 receptors on modulating the activity of lumbosacral neurones and urinary bladder contractions in vivo in normal or spinal cord-injured (SCI) rats with neurogenic bladder overactivity. MATERIALS AND METHODS • SCI was induced in female rats by complete transection at T8 – T9 and experiments were performed 4 weeks later, when bladder overactivity developed. Non-transected rats were used as controls (normal rats). • Neural activity was recorded in the dorsal horn of the spinal cord and field potentials were acquired in response to intravesical pressure steps via a suprapubic catheter. Field potentials were recorded under control conditions, after stimulation of bladder mucosal purinergic receptors with intravesical ATP (1 mm), and after intravenous injection of the P2X3/P2X2/3 antagonist AF-353 (10 mg/kg and 20 mg/kg). • Cystometry was performed in urethaneanaesthetised rats intravesically infused with saline. AF-353 (10 mg/kg) was systemically applied after baseline recordings; the rats also received a second dose of AF-353 (20 mg/kg). Changes in the frequency of voiding (VC) and non-voiding (NVC) contractions were evaluated. RESULTS • SCI rats had significantly higher frequencies for field potentials and NVC than NL rats. Intravesical ATP increased field potential frequency in control but not SCI rats, while systemic AF-353 significantly reduced this parameter in both groups. • AF-353 also reduced the inter-contractile interval in control but not in SCI rats; however, the frequency of NVC in SCI rats was significantly reduced. CONCLUSION • The P2X3/P2X2/3 receptors on bladder afferent nerves positively regulate sensory activity and NVCs in overactive bladders. PMID:22540742

  9. Extracellular ATP signaling via P2X(4) receptor and cAMP/PKA signaling mediate ATP oscillations essential for prechondrogenic condensation.

    PubMed

    Kwon, Hyuck Joon

    2012-09-01

    Prechondrogenic condensation is the most critical process in skeletal patterning. A previous study demonstrated that ATP oscillations driven by Ca(2+) oscillations play a critical role in prechondrogenic condensation by inducing oscillatory secretion. However, it remains unknown what mechanisms initiate the Ca(2+)-driven ATP oscillations, mediate the link between Ca(2+) and ATP oscillations, and then result in oscillatory secretion in chondrogenesis. This study has shown that extracellular ATP signaling was required for both ATP oscillations and prechondrogenic condensation. Among P2 receptors, the P2X(4) receptor revealed the strongest expression level and mediated ATP oscillations in chondrogenesis. Moreover, blockage of P2X(4) activity abrogated not only chondrogenic differentiation but also prechondrogenic condensation. In addition, both ATP oscillations and secretion activity depended on cAMP/PKA signaling but not on K(ATP) channel activity and PKC or PKG signaling. This study proposes that Ca(2+)-driven ATP oscillations essential for prechondrogenic condensation is initiated by extracellular ATP signaling via P2X(4) receptor and is mediated by cAMP/PKA signaling and that cAMP/PKA signaling induces oscillatory secretion to underlie prechondrogenic condensation, in cooperation with Ca(2+) and ATP oscillations.

  10. Subcellular distribution and early signalling events of P2X7 receptors from mouse cerebellar granule neurons.

    PubMed

    Sánchez-Nogueiro, Jesús; Marín-García, Patricia; Bustillo, Diego; Olivos-Oré, Luis Alcides; Miras-Portugal, María Teresa; Artalejo, Antonio R

    2014-12-05

    The subcellular distribution and early signalling events of P2X7 receptors were studied in mouse cerebellar granule neurons. Whole-cell patch-clamp recordings evidenced inwardly directed non-desensitizing currents following adenosine 5'-triphosphate (ATP; 600 µM) or 2'-3'-o-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP; 100 µM) administration to cells bathed in a medium with no-added divalent cations (Ca(2+) and Mg(2+)). Nucleotide-activated currents were inhibited by superfusion of 2.5 mM Ca(2+), 1.2 mM Mg(2+) or 100 nM Brilliant Blue G (BBG), hence indicating the expression of ionotropic P2X7 receptors. Fura-2 calcium imaging showed [Ca(2+)]i elevations in response to ATP or BzATP at the somas and at a small number of axodendritic regions of granule neurons. Differential sensitivity of these [Ca(2+)]i increases to three different P2X7 receptor antagonists (100 nM BBG, 10 μM 4-[(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester, KN-62, and 1 μM 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine hydrochloride hydrate, A-438079) revealed that P2X7 receptors are co-expressed with different P2Y receptors along the plasmalemma of granule neurons. Finally, experiments with the fluorescent dye YO-PRO-1 indicated that prolonged stimulation of P2X7 receptors does not lead to the opening of a membrane pore permeable to large cations. Altogether, our results emphasise the expression of functional P2X7 receptors at both the axodendritic and somatic levels in mouse cerebellar granule neurons, and favour the notion that P2X7 receptors might function in a subcellular localisation-specific manner: presynaptically, by controlling glutamate release, and on the cell somas, by supporting granule neuron survival against glutamate excytotoxicity.

  11. The C-terminal domain of chikungunya virus nsP2 independently governs viral RNA replication, cytopathicity, and inhibition of interferon signaling.

    PubMed

    Fros, Jelke J; van der Maten, Erika; Vlak, Just M; Pijlman, Gorben P

    2013-09-01

    Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S only in combination with KR649AA or adaptive mutation D711G allowed noncytopathic replicon RNA replication, (ii) prohibiting nsP2 nuclear localization abrogates inhibition of antiviral interferon-induced JAK-STAT signaling, and (iii) nsP2 independently affects RNA replication, cytopathicity, and JAK-STAT signaling.

  12. Receptor-mediated DNA-targeted photoimmunotherapy.

    PubMed

    Karagiannis, Tom C; Lobachevsky, Pavel N; Leung, Brenda K Y; White, Jonathan M; Martin, Roger F

    2006-11-01

    We show the efficacy of a therapeutic strategy that combines the potency of a DNA-binding photosensitizer, UV(A)Sens, with the tumor-targeting potential of receptor-mediated endocytosis. The photosensitizer is an iodinated bibenzimidazole, which, when bound in the minor groove of DNA and excited by UV(A) irradiation, induces cytotoxic lesions attributed to a radical species resulting from photodehalogenation. Although reminiscent of photochemotherapy using psoralens and UV(A) irradiation, an established treatment modality in dermatology particularly for the treatment of psoriasis and cutaneous T-cell lymphoma, a critical difference is the extreme photopotency of the iodinated bibenzimidazole, approximately 1,000-fold that of psoralens. This feature prompted consideration of combination with the specificity of receptor-mediated targeting. Using two in vitro model systems, we show the UV(A) cytotoxicity of iodo ligand/protein conjugates, implying binding of the conjugate to cell receptors, internalization, and degradation of the conjugate-receptor complex, with release and translocation of the ligand to nuclear DNA. For ligand-transferrin conjugates, phototoxicity was inhibited by coincubation with excess native transferrin. Receptor-mediated UV(A)-induced cytotoxicity was also shown with the iodo ligand conjugate of an anti-human epidermal growth factor receptor monoclonal antibody, exemplifying the potential application of the strategy to other cancer-specific targets to thus improve the specificity of phototherapy of superficial lesions and for extracorporeal treatments.

  13. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD)

    PubMed Central

    Okada, Yasunobu; Maeno, Emi; Shimizu, Takahiro; Dezaki, Katsuya; Wang, Jun; Morishima, Shigeru

    2001-01-01

    A fundamental property of animal cells is the ability to regulate their own cell volume. Even under hypotonic stress imposed by either decreased extracellular or increased intracellular osmolarity, the cells can re-adjust their volume after transient osmotic swelling by a mechanism known as regulatory volume decrease (RVD). In most cell types, RVD is accomplished mainly by KCl efflux induced by parallel activation of K+ and Cl− channels. We have studied the molecular mechanism of RVD in a human epithelial cell line (Intestine 407). Osmotic swelling results in a significant increase in the cytosolic Ca2+ concentration and thereby activates intermediate-conductance Ca2+-dependent K+ (IK) channels. Osmotic swelling also induces ATP release from the cells to the extracellular compartment. Released ATP stimulates purinergic ATP (P2Y2) receptors, thereby inducing phospholipase C-mediated Ca2+ mobilization. Thus, RVD is facilitated by stimulation of P2Y2 receptors due to augmentation of IK channels. In contrast, stimulation of another G protein-coupled Ca2+-sensing receptor (CaR) enhances the activity of volume-sensitive outwardly rectifying Cl− channels, thereby facilitating RVD. Therefore, it is possible that Ca2+ efflux stimulated by swelling-induced and P2Y2 receptor-mediated intracellular Ca2+ mobilization activates the CaR, thereby secondarily upregulating the volume-regulatory Cl− conductance. On the other hand, the initial process towards apoptotic cell death is coupled to normotonic cell shrinkage, called apoptotic volume decrease (AVD). Stimulation of death receptors, such as TNFα receptor and Fas, induces AVD and thereafter biochemical apoptotic events in human lymphoid (U937), human epithelial (HeLa), mouse neuroblastoma × rat glioma hybrid (NG108-15) and rat phaeochromocytoma (PC12) cells. In those cells exhibiting AVD, facilitation of RVD is always observed. Both AVD induction and RVD facilitation as well as succeeding apoptotic events can be

  14. Molecular determinants of P2Y2 nucleotide receptor function: implications for proliferative and inflammatory pathways in astrocytes.

    PubMed

    Weisman, Gary A; Wang, M; Kong, Q; Chorna, N E; Neary, J T; Sun, Grace Y; González, Fernando A; Seye, C I; Erb, L

    2005-01-01

    In the mammalian nervous system, P2 nucleotide receptors mediate neurotransmission, release of proinflammatory cytokines, and reactive astrogliosis. Extracellular nucleotides activate multiple P2 receptors in neurons and glial cells, including G protein-coupled P2Y receptors and P2X receptors, which are ligand-gated ion channels. In glial cells, the P2Y2 receptor subtype, distinguished by its ability to be equipotently activated by ATP and UTP, is coupled to pro-inflammatory signaling pathways. In situ hybridization studies with rodent brain slices indicate that P2Y2 receptors are expressed primarily in the hippocampus and cerebellum. Astrocytes express several P2 receptor subtypes, including P2Y2 receptors whose activation stimulates cell proliferation and migration. P2Y2 receptors, via an RGD (Arg-Gly-Asp) motif in their first extracellular loop, bind to alphavbeta3/beta5 integrins, whereupon P2Y2 receptor activation stimulates integrin signaling pathways that regulate cytoskeletal reorganization and cell motility. The C-terminus of the P2Y2 receptor contains two Src-homology-3 (SH3)-binding domains that upon receptor activation, promote association with Src and transactivation of growth factor receptors. Together, our results indicate that P2Y2 receptors complex with both integrins and growth factor receptors to activate multiple signaling pathways. Thus, P2Y2 receptors present novel targets to control reactive astrogliosis in neurodegenerative diseases.

  15. F-actin links Epac-PKC signaling to purinergic P2X3 receptor sensitization in dorsal root ganglia following inflammation

    PubMed Central

    Gu, Yanping; Wang, Congying; Li, GuangWen

    2016-01-01

    Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund’s adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund’s adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors. PMID:27385722

  16. EXPRESS: F-actin links Epac-PKC signaling to purinergic P2X3 receptors sensitization in dorsal root ganglia following inflammation.

    PubMed

    Gu, Yanping; Wang, Congying; Li, Guangwen; Huang, Li-Yen Mae

    2016-01-01

    Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund’s adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund’s adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors.

  17. Extracellular UDP and P2Y6 function as a danger signal to protect mice from vesicular stomatitis virus infection through an increase in IFN-β production.

    PubMed

    Li, Ruimei; Tan, Binghe; Yan, Yan; Ma, Xiaobin; Zhang, Na; Zhang, Zhi; Liu, Mingyao; Qian, Min; Du, Bing

    2014-11-01

    Extracellular nucleotides that constitute a "danger signal" play an important role in the regulation of immune responses. However, the function and mechanism of extracellular UDP and P2Y6 in antiviral immunity remain unknown. In this study, we demonstrated the in vitro and in vivo protection of UDP/P2Y6 signaling in vesicular stomatitis virus (VSV) infection. First, we demonstrated that VSV-infected cells secrete UDP from the cytoplasm as a danger signal to arouse surrounding cells. Meanwhile, expression of the UDP-specific receptor P2Y6 also was enhanced by VSV. Consequently, UDP protects RAW 264.7 cells, murine embryonic fibroblasts, bone marrow-derived macrophages, and L929 cells from VSV and GFP lentivirus infection. This protection can be blocked by the P2Y6 selective antagonist MRS2578 or IFN-α/β receptor-blocking Ab. VSV-induced cell death and virus replication were both enhanced significantly by knocking down and knocking out P2Y6 in different cells. Mechanistically, UDP facilitates IFN-β secretion through the p38/JNK- and ATF-2/c-Jun-signaling pathways, which are crucial in promoting antiviral immunity. Interestingly, UDP was released through a caspase-cleaved pannexin-1 channel in VSV-induced apoptotic cells and protected cells from infection through P2Y6 receptor in an autocrine or paracrine manner. Furthermore, UDP also protected mice from VSV infection through P2Y6 receptors in an acute neurotropic infection mouse model. Taken together, these results demonstrate the important role of extracellular UDP and P2Y6 as a danger signal in antiviral immune responses and suggest a potential therapeutic role for UDP/P2Y6 in preventing and controlling viral diseases.

  18. Emerging evidence of signalling roles for PI(3,4)P2 in Class I and II PI3K-regulated pathways.

    PubMed

    Hawkins, Phillip T; Stephens, Len R

    2016-02-01

    There are eight members of the phosphoinositide family of phospholipids in eukaryotes; PI, PI3P, PI4P, PI5P, PI(4,5)P2, PI(3,4)P2, PI(3,5)P2 and PI(3,4,5)P3. Receptor activation of Class I PI3Ks stimulates the phosphorylation of PI(4,5)P2 to form PI(3,4,5)P3. PI(3,4,5)P3 is an important messenger molecule that is part of a complex signalling network controlling cell growth and division. PI(3,4,5)P3 can be dephosphorylated by both 3- and 5-phosphatases, producing PI(4,5)P2 and PI(3,4)P2, respectively. There is now strong evidence that PI(3,4)P2 generated by this route does not merely represent another pathway for removal of PI(3,4,5)P3, but can act as a signalling molecule in its own right, regulating macropinocytosis, fast endophilin-mediated endocytosis (FEME), membrane ruffling, lamellipodia and invadopodia. PI(3,4)P2 can also be synthesized directly from PI4P by Class II PI3Ks and this is important for the maturation of clathrin-coated pits [clathrin-mediated endocytosis (CME)] and signalling in early endosomes. Thus PI(3,4)P2 is emerging as an important signalling molecule involved in the coordination of several specific membrane and cytoskeletal responses. Further, its inappropriate accumulation contributes to pathology caused by mutations in genes encoding enzymes responsible for its degradation, e.g. Inpp4B.

  19. Involvement of P2X7 receptor signaling on regulating the differentiation of Th17 cells and type II collagen-induced arthritis in mice

    PubMed Central

    Fan, Zhi-Dan; Zhang, Ya-Yuan; Guo, Yi-Hong; Huang, Na; Ma, Hui-Hui; Huang, Hui; Yu, Hai-Guo

    2016-01-01

    Interleukin (IL)-17 producing T helper (Th17) cells are major effector cells in the pathogenesis of rheumatoid arthritis (RA). The P2X7 receptor (P2X7R) has emerged as a potential site in the regulation of inflammation in RA but little is known of its functional role on the differentiation of Th17 cells. This study investigates the in vitro and in vivo effects of P2X7R on Th17 cell differentiation during type II collagen (CII) induced experimental arthritis model. In CII-treated dendritic cells (DCs) and DC/CD4+ T coculture system, pretreatment with pharmacological antagonists of P2X7R (Suramin and A-438079) caused strong inhibition of production of Th17-promoting cytokines (IL-1β, TGF-β1, IL-23p19 and IL-6). Exposure to CII induced the elevation of mRNAs encoding retinoic acid receptor-related orphan receptor α and γt, which were abolished by pretreatment with P2X7R antagonists. Furthermore, blocking P2X7R signaling abolished the CII-mediated increase in IL-17A. Blockade of P2X7R remarkably inhibited hind paw swelling and ameliorated pathological changes in ankle joint of the collagen-induced arthritis mice. Thus, we demonstrated a novel function for P2X7R signaling in regulating CII-induced differentiation of Th17 cells. P2X7R signaling facilitates the development of the sophisticated network of DC-derived cytokines that favors a Th17 phenotype. PMID:27775097

  20. The Ca(2+) -binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer.

    PubMed

    Kato, Mariko; Aoyama, Takashi; Maeshima, Masayoshi

    2013-05-01

    Plasma membrane-associated Ca(2+) -binding protein-2 (PCaP2) of Arabidopsis thaliana is a novel-type protein that binds to the Ca(2+) /calmodulin complex and phosphatidylinositol phosphates (PtdInsPs) as well as free Ca(2+) . Although the PCaP2 gene is predominantly expressed in root hair cells, it remains unknown how PCaP2 functions in root hair cells via binding to ligands. From biochemical analyses using purified PCaP2 and its variants, we found that the N-terminal basic domain with 23 amino acids (N23) is necessary and sufficient for binding to PtdInsPs and the Ca(2+) /calmodulin complex, and that the residual domain of PCaP2 binds to free Ca(2+) . In mutant analysis, a pcap2 knockdown line displayed longer root hairs than the wild-type. To examine the function of each domain in root hair cells, we over-expressed PCaP2 and its variants using the root hair cell-specific EXPANSIN A7 promoter. Transgenic lines over-expressing PCaP2, PCaP2(G2A) (second glycine substituted by alanine) and ∆23PCaP2 (lacking the N23 domain) exhibited abnormal branched and bulbous root hair cells, while over-expression of the N23 domain suppressed root hair emergence and elongation. The N23 domain was necessary and sufficient for the plasma membrane localization of GFP-tagged PCaP2. These results suggest that the N23 domain of PCaP2 negatively regulates root hair tip growth via processing Ca(2+) and PtdInsP signals on the plasma membrane, while the residual domain is involved in the polarization of cell expansion.

  1. Heat Shock Protein 90 Inhibitors Reduce Trafficking of ATP-gated P2X1 Receptors and Human Platelet Responsiveness*

    PubMed Central

    Lalo, Ulyana; Jones, Sarah; Roberts, Jonathan A.; Mahaut-Smith, Martyn P.; Evans, Richard J.

    2012-01-01

    We have used selective inhibitors to determine whether the molecular chaperone heat shock protein 90 (HSP90) has an effect on both recombinant and native human P2X1 receptors. P2X1 receptor currents in HEK293 cells were reduced by ∼70–85% by the selective HSP90 inhibitor geldanamycin (2 μm, 20 min). This was associated with a speeding in the time course of desensitization as well as a reduction in cell surface expression. Imaging in real time of photoactivatable GFP-tagged P2X receptors showed that they are highly mobile. Geldanamycin almost abolished this movement for P2X1 receptors but had no effect on P2X2 receptor trafficking. P2X1/2 receptor chimeras showed that the intracellular N and C termini were involved in geldanamycin sensitivity. Geldanamycin also inhibited native P2X1 receptor-mediated responses. Platelet P2X1 receptors play an important role in hemostasis, contribute to amplification of signaling to a range of stimuli including collagen, and are novel targets for antithrombotic therapies. Platelet P2X1 receptor-, but not P2Y1 receptor-, mediated increases in intracellular calcium were reduced by 40–45% following HSP90 inhibition with geldanamycin or radicicol. Collagen stimulation leads to ATP release from platelets, and calcium increases to low doses of collagen were also reduced by ∼40% by the HSP90 inhibitors consistent with an effect on P2X1 receptors. These studies suggest that HSP90 inhibitors may be as effective as selective antagonists in regulating platelet P2X1 receptors, and their potential effects on hemostasis should be considered in clinical studies. PMID:22851178

  2. PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions.

    PubMed

    Gómez-Villafuertes, Rosa; García-Huerta, Paula; Díaz-Hernández, Juan Ignacio; Miras-Portugal, M Teresa

    2015-12-21

    The expression of purinergic P2X7 receptor (P2X7R) in neuroblastoma cells is associated to accelerated growth rate, angiogenesis, metastasis and poor prognosis. Noticeably, P2X7R allows the survival of neuroblastoma cells under restrictive conditions, including serum and glucose deprivation. Previously we identified specificity protein 1 (Sp1) as the main factor involved in the transcriptional regulation of P2rx7 gene, reporting that serum withdrawal triggers the expression of P2X7R in Neuro-2a (N2a) neuroblastoma cell line. Here we demonstrate that PI3K/Akt pathway is crucial for the upregulation of P2X7R expression in serum-deprived neuroblastoma cells, circumstance that facilitates cell proliferation in the absence of trophic support. The effect exerted by PI3K/Akt is independent of both mTOR and GSK3, but requires the activation of EGF receptor (EGFR). Nuclear levels of Sp1 are strongly reduced by inhibition of PI3K/Akt pathway, and blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene expression following serum withdrawal. Furthermore, atypical PKCζ plays a key role in the regulation of P2X7R expression by preventing phosphorylation and, consequently, activation of Akt. Altogether, these data indicate that activation of EGFR enhanced the expression of P2X7R in neuroblastoma cells lacking trophic support, being PI3K/Akt/PKCζ signaling pathway and Sp1 mediating this pro-survival outcome.

  3. PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions

    PubMed Central

    Gómez-Villafuertes, Rosa; García-Huerta, Paula; Díaz-Hernández, Juan Ignacio; Miras-Portugal, Mª Teresa

    2015-01-01

    The expression of purinergic P2X7 receptor (P2X7R) in neuroblastoma cells is associated to accelerated growth rate, angiogenesis, metastasis and poor prognosis. Noticeably, P2X7R allows the survival of neuroblastoma cells under restrictive conditions, including serum and glucose deprivation. Previously we identified specificity protein 1 (Sp1) as the main factor involved in the transcriptional regulation of P2rx7 gene, reporting that serum withdrawal triggers the expression of P2X7R in Neuro-2a (N2a) neuroblastoma cell line. Here we demonstrate that PI3K/Akt pathway is crucial for the upregulation of P2X7R expression in serum-deprived neuroblastoma cells, circumstance that facilitates cell proliferation in the absence of trophic support. The effect exerted by PI3K/Akt is independent of both mTOR and GSK3, but requires the activation of EGF receptor (EGFR). Nuclear levels of Sp1 are strongly reduced by inhibition of PI3K/Akt pathway, and blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene expression following serum withdrawal. Furthermore, atypical PKCζ plays a key role in the regulation of P2X7R expression by preventing phosphorylation and, consequently, activation of Akt. Altogether, these data indicate that activation of EGFR enhanced the expression of P2X7R in neuroblastoma cells lacking trophic support, being PI3K/Akt/PKCζ signaling pathway and Sp1 mediating this pro-survival outcome. PMID:26687764

  4. Large-conductance channel formation mediated by P2X7 receptor activation is regulated through distinct intracellular signaling pathways in peritoneal macrophages and 2BH4 cells.

    PubMed

    Faria, R X; Cascabulho, C M; Reis, R A M; Alves, Luiz Anastácio

    2010-07-01

    The P2X(7) receptor (P2X7R) is a ligand-gated ATP receptor that acts as a low- and large-conductance channel (pore) and is known to be coupled to several downstream effectors. Recently, we demonstrated that the formation of a large-conductance channel associated with the P2X(7) receptor is induced by increasing the intracellular Ca(2+) concentration (Faria et al., Am J Physiol Cell Physiol 297:C28-C42, 2005). Here, we investigated the intracellular signaling pathways associated with P2X(7) large-conductance channel formation using the patch clamp technique in conjunction with fluorescent imaging and flow cytometry assays in 2BH4 cells and peritoneal macrophages. Different antagonists were applied to investigate the following pathways: Ca(2+)-calmodulin, phospholipase A, phospholipase D, phospholipase C, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), MAPK/extracellular signal-regulated kinase, phosphoinositide 3-kinase (PI3K), and cytoskeletal proteins. Macroscopic ionic currents induced by 1 mM ATP were reduced by 85% in the presence of PKC antagonists. The addition of antagonists for MAPK, PI3K, and the cytoskeleton (actin, intermediary filament, and microtubule) blocked 92%, 83%, and 95% of the ionic currents induced by 1 mM ATP, respectively. Our results show that PKC, MAPK, PI3K, and cytoskeletal components are involved in P2X(7) receptor large-channel formation in 2BH4 cells and peritoneal macrophages.

  5. Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

    PubMed Central

    2013-01-01

    Background Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Results Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2-octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Conclusions Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors. PMID

  6. NLRP3 inflammasome as a target of berberine in experimental murine liver injury: interference with P2X7 signalling.

    PubMed

    Vivoli, Elisa; Cappon, Andrea; Milani, Stefano; Piombanti, Benedetta; Provenzano, Angela; Novo, Erica; Masi, Alessio; Navari, Nadia; Narducci, Roberto; Mannaioni, Guido; Moneti, Gloriano; Oliveira, Claudia P; Parola, Maurizio; Marra, Fabio

    2016-10-01

    Berberine (BRB) is commonly used in herbal medicine, but its mechanisms of action are poorly understood. In the present study, we tested BRB in steatohepatitis induced by a methionine- and choline-deficient (MCD) diet, in acute acetaminophen intoxication and in cultured murine macrophages. BRB markedly improved parameters of liver injury and necroinflammation induced by the MCD diet, although increased mortality was observed by mechanisms independent of bacterial infections or plasma levels of BRB. The MCD diet induced up-regulation of all components of the NLRP3 (NACHT, LRR and PYD domain-containing protein 3) inflammasome, and increased hepatic levels of mature IL-1β (interleukin 1β). All of these parameters were significantly reduced in mice treated with BRB. In mice administered an acetaminophen overdose, a model dependent on inflammasome activation, BRB reduced mortality and ALT (alanine aminotransferase) elevation, and limited the expression of inflammasome components. In vitro, LPS (lipopolysaccharide)-induced activation of NLRP3 inflammasome in RAW264.7 murine macrophages was markedly decreased by pre-incubation with BRB. BRB significantly limited the activation of the purinergic receptor P2X7, involved in the late phases of inflammasome activation. Upon P2X7 knockdown, the ability of BRB to block LPS-induced secretion of IL-1β was lost. These data indicate that administration of BRB ameliorates inflammation and injury in two unrelated murine models of liver damage. We demonstrate for the first time that BRB interferes with activation of the NLRP3 inflammasome pathway in vivo and in vitro, through a mechanism based on interference with activation of P2X7, a purinergic receptor involved in inflammasome activation.

  7. N-linked glycosylation of platelet P2Y12 ADP receptor is essential for signal transduction but not for ligand binding or cell surface expression.

    PubMed

    Zhong, Xiaotian; Kriz, Ron; Seehra, Jasbir; Kumar, Ravindra

    2004-03-26

    P(2)Y(12) receptor is a G(i)-coupled adenosine diphosphate (ADP) receptor with a critical role in platelet aggregation. It contains two potential N-linked glycosylation sites at its extra cellular amino-terminus, which may modulate its activity. Studies of both tunicamycin treatment and site-directed mutagenesis have revealed a dispensable role of the N-linked glycosylation in the receptor's surface expression and ligand binding activity. However, the non-glycosylated P(2)Y(12) receptor is defective in the P(2)Y(12)-mediated inhibition of the adenylyl cyclase activity. Thus the study uncovers an unexpected vital role of N-linked glycans in receptor's signal transducing step but not in surface expression or ligand binding.

  8. LAPTM4B is a PtdIns(4,5)P2 effector that regulates EGFR signaling, lysosomal sorting, and degradation.

    PubMed

    Tan, Xiaojun; Sun, Yue; Thapa, Narendra; Liao, Yihan; Hedman, Andrew C; Anderson, Richard A

    2015-02-12

    Lysosomal degradation is essential for the termination of EGF-stimulated EGF receptor (EGFR) signaling. This requires EGFR sorting to the intraluminal vesicles (ILVs) of multi-vesicular endosomes (MVEs). Cytosolic proteins including the ESCRT machineries are key regulators of EGFR intraluminal sorting, but roles for endosomal transmembrane proteins in receptor sorting are poorly defined. Here, we show that LAPTM4B, an endosomal transmembrane oncoprotein, inhibits EGF-induced EGFR intraluminal sorting and lysosomal degradation, leading to enhanced and prolonged EGFR signaling. LAPTM4B blocks EGFR sorting by promoting ubiquitination of Hrs (an ESCRT-0 subunit), which inhibits the Hrs association with ubiquitinated EGFR. This is counteracted by the endosomal PIP kinase, PIPKIγi5, which directly binds LAPTM4B and neutralizes the inhibitory function of LAPTM4B in EGFR sorting by generating PtdIns(4,5)P2 and recruiting SNX5. PtdIns(4,5)P2 and SNX5 function together to protect Hrs from ubiquitination, thereby promoting EGFR intraluminal sorting. These results reveal an essential layer of EGFR trafficking regulated by LAPTM4B, PtdIns(4,5)P2 signaling, and the ESCRT complex and define a mechanism by which the oncoprotein LAPTM4B can transform cells and promote tumor progression.

  9. Effects of pioglitazone on platelet P2Y12-mediated signalling in clopidogrel-treated patients with type 2 diabetes mellitus.

    PubMed

    Suryadevara, Siva; Ueno, Masafumi; Tello-Montoliu, Antonio; Ferreiro, Jose Luis; Desai, Bhaloo; Rollini, Fabiana; Box, Lyndon C; Zenni, Martin; Guzman, Luis A; Bass, Theodore A; Angiolillo, Dominick J

    2012-11-01

    Patients with type 2 diabetes mellitus (T2DM) have impaired clopidogrel-induced antiplatelet effects, which may be in part attributed to their reduced sensitivity to insulin and consequently, results in upregulation of the P2Y12 signalling pathway. It has been hypothesised that insulin sensitising strategies may enhance clopidogrel-mediated P2Y12 inhibitory effects. The aim of this pilot pharmacodynamics (PD) study was to assess the impact of pioglitazone on clopidogrel-mediated P2Y12 inhibitory effects in patients with T2DM. This was a prospective, randomised, double-blind, placebo-controlled, cross-over PD study. Patients with T2DM and stable coronary artery disease on maintenance aspirin and clopidogrel were randomised to receive either pioglitazone 30 mg or matching placebo daily for 14 days. PD assessments were measured at baseline, 14 days after randomisation, at the end of the wash-out period, and 14 days after cross-over. The primary endpoint measure was maximal platelet aggregation (MPA) to 20 μM adenosine diphosphate (ADP) as assessed by light transmittance aggregometry (LTA). Flow cytometric analysis of vasodilator-stimulated phosphoprotein phosphorylation (VASP-PRI), and VerifyNow P2Y12 testing were also performed. A total of 15 randomised patients completed the study. MPA to 20 μM ADP (primary endpoint) was not significantly different with pioglitazone compared with placebo (49.53 ± 4.76 vs. 52.52 ± 3.89%; p = 0.594). Similarly, other PD measures did not differ significantly between the groups. In conclusion, in patients with T2DM on maintenance aspirin and clopidogrel therapy, the adjunctive use of pioglitazone does not result in enhanced inhibition of platelet P2Y12 mediated signalling.

  10. ATP induces NO production in hippocampal neurons by P2X(7) receptor activation independent of glutamate signaling.

    PubMed

    Codocedo, Juan Francisco; Godoy, Juan Alejandro; Poblete, Maria Ines; Inestrosa, Nibaldo C; Huidobro-Toro, Juan Pablo

    2013-01-01

    To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2'(3')-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by N(ω)-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.

  11. ATP Induces NO Production in Hippocampal Neurons by P2X7 Receptor Activation Independent of Glutamate Signaling

    PubMed Central

    Codocedo, Juan Francisco; Godoy, Juan Alejandro; Poblete, Maria Ines; Inestrosa, Nibaldo C.; Huidobro-Toro, Juan Pablo

    2013-01-01

    To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2′(3′)-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by Nω-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity. PMID:23472093

  12. CD3ζ-based chimeric antigen receptors mediate T cell activation via cis- and trans-signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy.

    PubMed

    Bridgeman, J S; Ladell, K; Sheard, V E; Miners, K; Hawkins, R E; Price, D A; Gilham, D E

    2014-02-01

    Chimeric antigen receptors (CARs) can mediate redirected lysis of tumour cells in a major histocompatibility complex (MHC)-independent manner, thereby enabling autologous adoptive T cell therapy for a variety of malignant neoplasms. Currently, most CARs incorporate the T cell receptor (TCR) CD3ζ signalling chain; however, the precise mechanisms responsible for CAR-mediated T cell activation are unclear. In this study, we used a series of immunoreceptor tyrosine-based activation motif (ITAM)-mutant and transmembrane-modified receptors to demonstrate that CARs activate T cells both directly via the antigen-ligated signalling chain and indirectly via associated chains within the TCR complex. These observations allowed us to generate new receptors capable of eliciting polyfunctional responses in primary human T cells. This work increases our understanding of CAR function and identifies new avenues for the optimization of CAR-based therapeutic interventions.

  13. Important roles of P2Y receptors in the inflammation and cancer of digestive system

    PubMed Central

    Wan, Han-Xing; Hu, Jian-Hong; Xie, Rei; Yang, Shi-Ming; Dong, Hui

    2016-01-01

    Purinergic signaling is important for many biological processes in humans. Purinoceptors P2Y are widely distributed in human digestive system and different subtypes of P2Y receptors mediate different physiological functions from metabolism, proliferation, differentiation to apoptosis etc. The P2Y receptors are essential in many gastrointestinal functions and also involve in the occurrence of some digestive diseases. Since different subtypes of P2Y receptors are present on the same cell of digestive organs, varying subtypes of P2Y receptors may have opposite or synergetic functions on the same cell. Recently, growing lines of evidence strongly suggest the involvement of P2Y receptors in the pathogenesis of several digestive diseases. In this review, we will focus on their important roles in the development of digestive inflammation and cancer. We anticipate that as the special subtypes of P2Y receptors are studied in depth, specific modulators for them will have good potentials to become promising new drugs to treat human digestive diseases in the near future. PMID:26908460

  14. Quercetin modulates toll-like receptor-mediated protein kinase signaling pathways in oxLDL-challenged human PBMCs and regulates TLR-activated atherosclerotic inflammation in hypercholesterolemic rats.

    PubMed

    Bhaskar, Shobha; Helen, A

    2016-12-01

    Toll-like receptors (TLRs) are pattern recognition receptors that have a unique and essential function in innate immunity. The effect of quercetin on TLR-mediated downstream signaling mechanism and its effect on TLR-mediated MAP kinase and Akt pathways were studied in oxLDL-stimulated hPBMCs using specific inhibitors. The pretreatment of hPBMCs with specific TLR inhibitor, CLI-095, decreased the NF-κB nuclear translocation and TNF-α release by oxLDL. When the cells treated with inhibitor and quercetin together, the inhibition was more effective. The specific inhibitor for p38 MAPK, SB203580, reduced the phosphorylated p38 level and decreased the NF-κB activation and TNF-α release by oxLDL-challenged hPBMCs. This inhibitor showed enhanced inhibition when treated with quercetin together. The inhibitors for ERK1/2, PD98059, and for JNK, SP606125, also showed inhibitory effect on NF-κB activation and TNF-α release by oxLDL-simulated hPBMCs. Quercetin supplementation enhanced the inhibition of nuclear translocation of NF-κB and the release of cytokines. TLR4 inhibition study confirmed the downstream signaling mechanism mediated by NF-κB which is involved in the oxLDL-induced inflammatory response, and quercetin suppresses the cytokine, TNF-α release by modulating TLR-NF-κB signaling pathway. In addition to NF-κB signaling pathway, inflammation induced by oxLDL was also related to the activation of p38MAPK, ERK1/2 and JNK, and Akt pathways, and the protective effect of quercetin may be also related to the inhibition of activation of these pathways. Quercetin significantly downregulated the elevated mRNA expression of TLRs and cytokine TNF-α in HCD-fed atherosclerotic rats in vivo. As quercetin possesses inhibition on both TLR-NF-κB signaling pathway and TLR-mediated MAPK pathway, it is evident that it can be used as a therapeutic agent to ameliorate atherosclerotic inflammation. Since quercetin is the major flavonoid and forms the backbone of many other

  15. RDGBα, a PtdIns-PtdOH transfer protein, regulates G-protein-coupled PtdIns(4,5)P2 signalling during Drosophila phototransduction

    PubMed Central

    Yadav, Shweta; Garner, Kathryn; Georgiev, Plamen; Li, Michelle; Gomez-Espinosa, Evelyn; Panda, Aniruddha; Mathre, Swarna; Okkenhaug, Hanneke; Cockcroft, Shamshad; Raghu, Padinjat

    2015-01-01

    ABSTRACT Many membrane receptors activate phospholipase C (PLC) during signalling, triggering changes in the levels of several plasma membrane lipids including phosphatidylinositol (PtdIns), phosphatidic acid (PtdOH) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. It is widely believed that exchange of lipids between the plasma membrane and endoplasmic reticulum (ER) is required to restore lipid homeostasis during PLC signalling, yet the mechanism remains unresolved. RDGBα (hereafter RDGB) is a multi-domain protein with a PtdIns transfer protein (PITP) domain (RDGB-PITPd). We find that, in vitro, the RDGB-PITPd binds and transfers both PtdOH and PtdIns. In Drosophila photoreceptors, which experience high rates of PLC activity, RDGB function is essential for phototransduction. We show that binding of PtdIns to RDGB-PITPd is essential for normal phototransduction; however, this property is insufficient to explain the in vivo function because another Drosophila PITP (encoded by vib) that also binds PtdIns cannot rescue the phenotypes of RDGB deletion. In RDGB mutants, PtdIns(4,5)P2 resynthesis at the plasma membrane following PLC activation is delayed and PtdOH levels elevate. Thus RDGB couples the turnover of both PtdIns and PtdOH, key lipid intermediates during G-protein-coupled PtdIns(4,5)P2 turnover. PMID:26203165

  16. 4-tert-Octylphenol stimulates the expression of cathepsins in human breast cancer cells and xenografted breast tumors of a mouse model via an estrogen receptor-mediated signaling pathway.

    PubMed

    Lee, Hye-Rim; Choi, Kyung-Chul

    2013-02-08

    Endocrine disrupting chemicals (EDCs) are defined as environmental compounds that modulate steroid hormone receptor-dependent responses an abnormal manner, resulting in adverse health problems for humans such as cancer growth and metastasis. Cathepsins are proteases that have been implicated in cancer progression. However, there have been few studies about the association between cathepsins and estrogenic chemicals during the cancer progression. In this study, we examined the effect(s) of 4-tert-octylphenol (OP), a potent EDC, on the expression of cathepsins B and D in human MCF-7 breast cancer cells and a xenograft mouse model. Treatment with OP significantly induced the proliferation MCF-7 cells in an MTT assay. In addition, the expression of cathepsins B and D was markedly enhanced in MCF-7 cells at both the transcriptional and the translational levels following treatment with E2 or OP up to 48h. These results demonstrated the ability of OP to disrupt normal transcriptional regulation of cathepsins B and D in human breast cancer cells. However, the effects of OP on cell growth or overexpression of cathepsins by inhibiting ER-mediated signaling were abolished by an ER antagonist and siRNA specific for ERα. In conclusion, our findings suggest that OP at 10(-6)M, like E2, may accelerate breast cancer cell proliferation and the expression of cathepsins through an ER-mediated signaling pathway. In addition, the breast cancer cells exposed with OP to a xenograft mouse model were more aggressive according to our histological analysis and showed markedly increased expression of cathepsin B. These effects of mouse model resulted in an increased potential for metastasis in breast cancer. Taken together, we determined that OP can adversely affect human health by promoting cancer proliferation and metastasis through the amplification of cathepsins B and D via the ER-mediated signaling pathway.

  17. Serotonin 5-HT3 receptor-mediated vomiting occurs via the activation of Ca2+/CaMKII-dependent ERK1/2 signaling in the least shrew (Cryptotis parva).

    PubMed

    Zhong, Weixia; Hutchinson, Tarun E; Chebolu, Seetha; Darmani, Nissar A

    2014-01-01

    Stimulation of 5-HT3 receptors (5-HT3Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT3 receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT3R channel has high permeability to extracellular calcium (Ca(2+)) and upon stimulation allows increased Ca(2+) influx. We examined the contribution of Ca(2+)/calmodulin-dependent protein kinase IIα (Ca(2+)/CaMKIIα), interaction of 5-HT3R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca(2+) levels in brainstem slices and that the selective 5-HT3R antagonist palonosetron, can abolish the induced Ca(2+) signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca(2+) channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca2+-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT3R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study

  18. Serotonin 5-HT3 Receptor-Mediated Vomiting Occurs via the Activation of Ca2+/CaMKII-Dependent ERK1/2 Signaling in the Least Shrew (Cryptotis parva)

    PubMed Central

    Zhong, Weixia; Hutchinson, Tarun E.; Chebolu, Seetha; Darmani, Nissar A.

    2014-01-01

    Stimulation of 5-HT3 receptors (5-HT3Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT3 receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT3R channel has high permeability to extracellular calcium (Ca2+) and upon stimulation allows increased Ca2+ influx. We examined the contribution of Ca2+/calmodulin-dependent protein kinase IIα (Ca2+/CaMKIIα), interaction of 5-HT3R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca2+ levels in brainstem slices and that the selective 5-HT3R antagonist palonosetron, can abolish the induced Ca2+ signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca2+ channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca2+-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT3R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study demonstrates

  19. Receptor-mediated stimulation of lipid signalling pathways in CHO cells elicits the rapid transient induction of the PDE1B isoform of Ca2+/calmodulin-stimulated cAMP phosphodiesterase.

    PubMed

    Spence, S; Rena, G; Sullivan, M; Erdogan, S; Houslay, M D

    1997-01-01

    Chinese hamster ovary cells (CHO cells) do not exhibit any Ca2+/calmodulin-stimulated cAMP phosphodiesterase (PDE1) activity. Challenge of CHO cells with agonists for endogenous P2-purinoceptors, lysophosphatidic acid receptors and thrombin receptors caused a similar rapid transient induction of PDE1 activity in each instance. This was also evident on noradrenaline challenge of a cloned CHO cell line transfected so as to overexpress alpha 1B-adrenoceptors. This novel PDE1 activity appeared within about 15 min of exposure to ligands, rose to a maximum value within 30 min to 1 h and then rapidly decreased. In each case, the expression of novel PDE1 activity was blocked by the transcriptional inhibitor actinomycin D. Challenge with insulin of either native CHO cells or a CHO cell line transfected so as to overexpress the human insulin receptor failed to induce PDE1 activity. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1C isoform, did not amplify any fragment from RNA preparations of CHO cells expressing PDE1 activity, although they did so from the human thyroid carcinoma FTC133 cell line. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1A and PDE1B isoforms, successfully amplified a fragment of the predicted size from RNA preparations of both CHO cells expressing PDE1 activity and human Jurkat T-cells. Sequencing of the PCR products, generated using the PDE1A/B primers, yielded a novel sequence which, by analogy with sequences reported for bovine and murine PDE1B forms, suggests that the PDE1 species induced in CHO cells through protein kinase C activation and that expressed in Jurkat T-cells are PDE1B forms.

  20. Learning and memory deficits consequent to reduction of the fragile X mental retardation protein result from metabotropic glutamate receptor-mediated inhibition of cAMP signaling in Drosophila.

    PubMed

    Kanellopoulos, Alexandros K; Semelidou, Ourania; Kotini, Andriana G; Anezaki, Maria; Skoulakis, Efthimios M C

    2012-09-19

    Loss of the RNA-binding fragile X protein [fragile X mental retardation protein (FMRP)] results in a spectrum of cognitive deficits, the fragile X syndrome (FXS), while aging individuals with decreased protein levels present with a subset of these symptoms and tremor. The broad range of behavioral deficits likely reflects the ubiquitous distribution and multiple functions of the protein. FMRP loss is expected to affect multiple neuronal proteins and intracellular signaling pathways, whose identity and interactions are essential in understanding and ameliorating FXS symptoms. We used heterozygous mutants and targeted RNA interference-mediated abrogation in Drosophila to uncover molecular pathways affected by FMRP reduction. We present evidence that FMRP loss results in excess metabotropic glutamate receptor (mGluR) activity, attributable at least in part to elevation of the protein in affected neurons. Using high-resolution behavioral, genetic, and biochemical analyses, we present evidence that excess mGluR upon FMRP attenuation is linked to the cAMP decrement reported in patients and models, and underlies olfactory associative learning and memory deficits. Furthermore, our data indicate positive transcriptional regulation of the fly fmr1 gene by cAMP, via protein kinase A, likely through the transcription factor CREB. Because the human Fmr1 gene also contains CREB binding sites, the interaction of mGluR excess and cAMP signaling defects we present suggests novel combinatorial pharmaceutical approaches to symptom amelioration upon FMRP attenuation.

  1. Myelin-associated glycoprotein modulates apoptosis of motoneurons during early postnatal development via NgR/p75NTR receptor-mediated activation of RhoA signaling pathways

    PubMed Central

    Palandri, A; Salvador, V R; Wojnacki, J; Vivinetto, A L; Schnaar, R L; Lopez, P H H

    2015-01-01

    Myelin-associated glycoprotein (MAG) is a minor constituent of nervous system myelin, selectively expressed on the periaxonal myelin wrap. By engaging multiple axonal receptors, including Nogo-receptors (NgRs), MAG exerts a nurturing and protective effect the axons it ensheaths. Pharmacological activation of NgRs has a modulatory role on p75NTR-dependent postnatal apoptosis of motoneurons (MNs). However, it is not clear whether this reflects a physiological role of NgRs in MN development. NgRs are part of a multimeric receptor complex, which includes p75NTR, Lingo-1 and gangliosides. Upon ligand binding, this multimeric complex activates RhoA/ROCK signaling in a p75NTR-dependent manner. The aim of this study was to analyze a possible modulatory role of MAG on MN apoptosis during postnatal development. A time course study showed that Mag-null mice suffer a loss of MNs during the first postnatal week. Also, these mice exhibited increased susceptibility in an animal model of p75NTR-dependent MN apoptosis induced by nerve-crush injury, which was prevented by treatment with a soluble form of MAG (MAG-Fc). The protective role of MAG was confirmed in in vitro models of p75NTR-dependent MN apoptosis using the MN1 cell line and primary cultures. Lentiviral expression of shRNA sequences targeting NgRs on these cells abolished protection by MAG-Fc. Analysis of RhoA activity using a FRET-based RhoA biosensor showed that MAG-Fc activates RhoA. Pharmacological inhibition of p75NTR/RhoA/ROCK pathway, or overexpression of a p75NTR mutant unable to activate RhoA, completely blocked MAG-Fc protection against apoptosis. The role of RhoA/ROCK signaling was further confirmed in the nerve-crush model, where pretreatment with ROCK inhibitor Y-27632 blocked the pro-survival effect of MAG-Fc. These findings identify a new protective role of MAG as a modulator of apoptosis of MNs during postnatal development by a mechanism involving the p75NTR/RhoA/ROCK signaling pathway. Also, our results

  2. Myelin-associated glycoprotein modulates apoptosis of motoneurons during early postnatal development via NgR/p75(NTR) receptor-mediated activation of RhoA signaling pathways.

    PubMed

    Palandri, A; Salvador, V R; Wojnacki, J; Vivinetto, A L; Schnaar, R L; Lopez, P H H

    2015-09-03

    Myelin-associated glycoprotein (MAG) is a minor constituent of nervous system myelin, selectively expressed on the periaxonal myelin wrap. By engaging multiple axonal receptors, including Nogo-receptors (NgRs), MAG exerts a nurturing and protective effect the axons it ensheaths. Pharmacological activation of NgRs has a modulatory role on p75(NTR)-dependent postnatal apoptosis of motoneurons (MNs). However, it is not clear whether this reflects a physiological role of NgRs in MN development. NgRs are part of a multimeric receptor complex, which includes p75(NTR), Lingo-1 and gangliosides. Upon ligand binding, this multimeric complex activates RhoA/ROCK signaling in a p75(NTR)-dependent manner. The aim of this study was to analyze a possible modulatory role of MAG on MN apoptosis during postnatal development. A time course study showed that Mag-null mice suffer a loss of MNs during the first postnatal week. Also, these mice exhibited increased susceptibility in an animal model of p75(NTR)-dependent MN apoptosis induced by nerve-crush injury, which was prevented by treatment with a soluble form of MAG (MAG-Fc). The protective role of MAG was confirmed in in vitro models of p75(NTR)-dependent MN apoptosis using the MN1 cell line and primary cultures. Lentiviral expression of shRNA sequences targeting NgRs on these cells abolished protection by MAG-Fc. Analysis of RhoA activity using a FRET-based RhoA biosensor showed that MAG-Fc activates RhoA. Pharmacological inhibition of p75(NTR)/RhoA/ROCK pathway, or overexpression of a p75(NTR) mutant unable to activate RhoA, completely blocked MAG-Fc protection against apoptosis. The role of RhoA/ROCK signaling was further confirmed in the nerve-crush model, where pretreatment with ROCK inhibitor Y-27632 blocked the pro-survival effect of MAG-Fc. These findings identify a new protective role of MAG as a modulator of apoptosis of MNs during postnatal development by a mechanism involving the p75(NTR)/RhoA/ROCK signaling pathway

  3. The effect of purinergic signaling via the P2Y11 receptor on vascular function in a rat model of acute inflammation.

    PubMed

    Dănilă, Maria D; Privistirescu, Andreea; Duicu, Oana M; Rațiu, Corina D; Angoulvant, Denis; Muntean, Danina M; Sturza, Adrian

    2017-02-17

    There is a growing body of evidence pointing to the role of purinergic signaling in the development and progression of various conditions that have inflammation as a common pathogenetic denominator. The aim of the present study was to assess the involvement of P2Y11 purinergic receptors in the regulation of vascular function in aortic segments obtained using an experimental model of acute inflammation, the lipopolysaccharide (LPS, 8 mg/kg, i.p)-treated rats. Twelve hours after LPS administration, thoracic aortas were isolated and used for studies of vascular reactivity in the organ bath and for the measurement of reactive oxygen species (ROS) generation, respectively. LPS treatment significantly increased contractility to phenylephrine and attenuated the endothelium-dependent relaxation of the vascular segments in response to acetylcholine; an increased production of hydrogen peroxide (H2O2) was also recorded. The P2Y11 activator, NF546, decreased the LPS-induced aortic H2O2 release and partially normalized the vasomotor function, namely reduced contractility and improved relaxation. The effect was abolished by co-treatment with the P2Y11 inhibitor, NF340, and also after endothelium denudation. Importantly, NF546 did not elicit an antioxidant effect by acting as a H2O2 scavenger, suggesting that the beneficial outcome of this treatment on the vasculature is the consequence of P2Y11 stimulation. In conclusion, purinergic P2Y11 receptors stimulation improves vascular function and mitigates oxidative stress in the setting of acute systemic inflammation, revealing salutary effects and therapeutic potential in pathologies associated with endothelial dysfunction.

  4. P2 receptors and immunity

    PubMed Central

    Rayah, Amel; Kanellopoulos, Jean M.; Di Virgilio, Francesco

    2012-01-01

    Immune cells express receptors for extracellular nucleotides named P2 receptors. P2 receptors transduce signals delivered by nucleotides present in the extracellular environment. Accruing evidence shows that purinergic signalling has a profound effect on multiple immune cell responses such as T lymphocyte proliferation, chemotaxis, cytokine release, phagocytosis, Ag presentation and cytotoxicity. This makes P2 receptors an attractive target for the therapy of immuno-mediated disease and cancer. PMID:22909902

  5. A Point Mutation in the Ubiquitin Ligase RNF170 That Causes Autosomal Dominant Sensory Ataxia Destabilizes the Protein and Impairs Inositol 1,4,5-Trisphosphate Receptor-mediated Ca2+ Signaling*

    PubMed Central

    Wright, Forrest A.; Lu, Justine P.; Sliter, Danielle A.; Dupré, Nicolas; Rouleau, Guy A.; Wojcikiewicz, Richard J. H.

    2015-01-01

    RNF170 is an endoplasmic reticulum membrane ubiquitin ligase that contributes to the ubiquitination of activated inositol 1,4,5-trisphosphate (IP3) receptors, and also, when point mutated (arginine to cysteine at position 199), causes autosomal dominant sensory ataxia (ADSA), a disease characterized by neurodegeneration in the posterior columns of the spinal cord. Here we demonstrate that this point mutation inhibits RNF170 expression and signaling via IP3 receptors. Inhibited expression of mutant RNF170 was seen in cells expressing exogenous RNF170 constructs and in ADSA lymphoblasts, and appears to result from enhanced RNF170 autoubiquitination and proteasomal degradation. The basis for these effects was probed via additional point mutations, revealing that ionic interactions between charged residues in the transmembrane domains of RNF170 are required for protein stability. In ADSA lymphoblasts, platelet-activating factor-induced Ca2+ mobilization was significantly impaired, whereas neither Ca2+ store content, IP3 receptor levels, nor IP3 production were altered, indicative of a functional defect at the IP3 receptor locus, which may be the cause of neurodegeneration. CRISPR/Cas9-mediated genetic deletion of RNF170 showed that RNF170 mediates the addition of all of the ubiquitin conjugates known to become attached to activated IP3 receptors (monoubiquitin and Lys48- and Lys63-linked ubiquitin chains), and that wild-type and mutant RNF170 have apparently identical ubiquitin ligase activities toward IP3 receptors. Thus, the Ca2+ mobilization defect seen in ADSA lymphoblasts is apparently not due to aberrant IP3 receptor ubiquitination. Rather, the defect likely reflects abnormal ubiquitination of other substrates, or adaptation to the chronic reduction in RNF170 levels. PMID:25882839

  6. A Point Mutation in the Ubiquitin Ligase RNF170 That Causes Autosomal Dominant Sensory Ataxia Destabilizes the Protein and Impairs Inositol 1,4,5-Trisphosphate Receptor-mediated Ca2+ Signaling.

    PubMed

    Wright, Forrest A; Lu, Justine P; Sliter, Danielle A; Dupré, Nicolas; Rouleau, Guy A; Wojcikiewicz, Richard J H

    2015-05-29

    RNF170 is an endoplasmic reticulum membrane ubiquitin ligase that contributes to the ubiquitination of activated inositol 1,4,5-trisphosphate (IP3) receptors, and also, when point mutated (arginine to cysteine at position 199), causes autosomal dominant sensory ataxia (ADSA), a disease characterized by neurodegeneration in the posterior columns of the spinal cord. Here we demonstrate that this point mutation inhibits RNF170 expression and signaling via IP3 receptors. Inhibited expression of mutant RNF170 was seen in cells expressing exogenous RNF170 constructs and in ADSA lymphoblasts, and appears to result from enhanced RNF170 autoubiquitination and proteasomal degradation. The basis for these effects was probed via additional point mutations, revealing that ionic interactions between charged residues in the transmembrane domains of RNF170 are required for protein stability. In ADSA lymphoblasts, platelet-activating factor-induced Ca(2+) mobilization was significantly impaired, whereas neither Ca(2+) store content, IP3 receptor levels, nor IP3 production were altered, indicative of a functional defect at the IP3 receptor locus, which may be the cause of neurodegeneration. CRISPR/Cas9-mediated genetic deletion of RNF170 showed that RNF170 mediates the addition of all of the ubiquitin conjugates known to become attached to activated IP3 receptors (monoubiquitin and Lys(48)- and Lys(63)-linked ubiquitin chains), and that wild-type and mutant RNF170 have apparently identical ubiquitin ligase activities toward IP3 receptors. Thus, the Ca(2+) mobilization defect seen in ADSA lymphoblasts is apparently not due to aberrant IP3 receptor ubiquitination. Rather, the defect likely reflects abnormal ubiquitination of other substrates, or adaptation to the chronic reduction in RNF170 levels.

  7. Receptor-Mediated Drug Delivery Systems Targeting to Glioma

    PubMed Central

    Wang, Shanshan; Meng, Ying; Li, Chengyi; Qian, Min; Huang, Rongqin

    2015-01-01

    Glioma has been considered to be the most frequent primary tumor within the central nervous system (CNS). The complexity of glioma, especially the existence of the blood-brain barrier (BBB), makes the survival and prognosis of glioma remain poor even after a standard treatment based on surgery, radiotherapy, and chemotherapy. This provides a rationale for the development of some novel therapeutic strategies. Among them, receptor-mediated drug delivery is a specific pattern taking advantage of differential expression of receptors between tumors and normal tissues. The strategy can actively transport drugs, such as small molecular drugs, gene medicines, and therapeutic proteins to glioma while minimizing adverse reactions. This review will summarize recent progress on receptor-mediated drug delivery systems targeting to glioma, and conclude the challenges and prospects of receptor-mediated glioma-targeted therapy for future applications.

  8. Rice Dwarf Virus P2 Protein Hijacks Auxin Signaling by Directly Targeting the Rice OsIAA10 Protein, Enhancing Viral Infection and Disease Development

    PubMed Central

    Jin, Lian; Qin, Qingqing; Wang, Yu; Pu, Yingying; Liu, Lifang; Wen, Xing; Ji, Shaoyi; Wu, Jianguo; Wei, Chunhong; Li, Yi

    2016-01-01

    The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB) protein and an auxin/indole-3-acetic acid (Aux/IAA) protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV), a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant) mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis. PMID:27606959

  9. Role of puerarin in the signalling of neuropathic pain mediated by P2X3 receptor of dorsal root ganglion neurons.

    PubMed

    Xu, Changshui; Xu, Wenyuan; Xu, Hong; Xiong, Wei; Gao, Yun; Li, Guilin; Liu, Shuangmei; Xie, Jinyan; Tu, Guihua; Peng, Haiying; Qiu, Shuyi; Liang, Shangdong

    2012-01-04

    Tissue injury or inflammation of the nervous system may result in chronic neuropathic pain characterized by sensitivity to painful stimuli. P2X(3) receptors play a crucial role in facilitating pain transmission. Puerarin is an active compound of a traditional Chinese medicine Ge-gen, and Ge-gen soup has anti-inflammatory effects. The present research investigated the role of puerarin in the signalling of chronic neuropathic pain mediated by P2X(3) receptors of rat dorsal root ganglion neurons. Chronic constriction injury (CCI) rat model was adopted. Sprague-Dawley rats were randomly divided into blank control group (Ctrl), sham group (Sham), puerarin-treated control group (Ctrl+PUE), chronic constriction injury (CCI) group and puerarin-treated CCI group (CCI+PUE). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured by the von-Frey test and the Hargreaves' test respectively. The stain values of P2X(3) protein and mRNA in L4/L5 dorsal root ganglion (DRG) were detected by immunohistochemistry, western blot and in situ hybridization. At day 4-7 after the operation of CCI rats, MWT and TWL in group CCI and CCI+PUE were lower than those in group Ctrl, Sham and Ctrl+PUE, while there was no difference among group Ctrl, Sham and Ctrl+PUE. At day 7-10 after operation, MWT and TWL in group CCI+PUE was higher than those in group CCI, but there was no significant difference between group CCI+PUE and group Ctrl (p>0.05). At day 14 after operation, the stain values of P2X(3) proteins and mRNAs in L4/L5 DRG of group CCI were higher than those in group Ctrl, Sham, Ctrl+PUE and CCI+PUE, while the stain values of P2X(3) proteins and mRNAs in group CCI+PUE were significantly decreased compared with those in group CCI. Therefore, puerarin may alleviate neuropathic pain mediated by P2X(3) receptors in dorsal root ganglion neurons.

  10. ATP-gated P2X3 receptors constitute a positive autocrine signal for insulin release in the human pancreatic β cell

    PubMed Central

    Jacques-Silva, M. Caroline; Correa-Medina, Mayrin; Cabrera, Over; Rodriguez-Diaz, Rayner; Makeeva, Natalia; Fachado, Alberto; Diez, Juan; Berman, Dora M.; Kenyon, Norma S.; Ricordi, Camillo; Pileggi, Antonello; Molano, R. Damaris; Berggren, Per-Olof; Caicedo, Alejandro

    2010-01-01

    Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human β cell's sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca2+ concentration, [Ca2+]i, and hormone release in vitro, we show that human β cells express ionotropic ATP receptors of the P2X3 type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X3 receptors in the β-cell plasma membrane, resulting in increased [Ca2+]i and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the β cell's secretory machinery. This may explain how the human pancreatic β cell can respond so effectively to relatively modest changes in glucose concentration under physiological conditions in vivo. PMID:20308565

  11. Signaling through P2X7 receptor in human T cells involves p56lck, MAP kinases, and transcription factors AP-1 and NF-kappa B.

    PubMed

    Budagian, Vadim; Bulanova, Elena; Brovko, Luba; Orinska, Zane; Fayad, Raja; Paus, Ralf; Bulfone-Paus, Silvia

    2003-01-17

    ATP-gated ion channel P2X receptors are expressed on the surface of most immune cells and can trigger multiple cellular responses, such as membrane permeabilization, cytokine production, and cell proliferation or apoptosis. Despite broad distribution and pleiotropic activities, signaling pathways downstream of these ionotropic receptors are still poorly understood. Here, we describe intracellular signaling events in Jurkat cells treated with millimolar concentrations of extracellular ATP. Within minutes, ATP treatment resulted in the phosphorylation and activation of p56(lck) kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase but not p38 kinase. These effects were wholly dependent upon the presence of extracellular Ca(2+) ions in the culture medium. Nevertheless, calmodulin antagonist calmidazolium and CaM kinase inhibitor KN-93 both had no effect on the activation of p56(lck) and ERK, whereas a pretreatment of Jurkat cells with MAP kinase kinase inhibitor P098059 was able to abrogate phosphorylation of ERK. Further, expression of c-Jun and c-Fos proteins and activator protein (AP-1) DNA binding activity were enhanced in a time-dependent manner. In contrast, DNA binding activity of NF-kappa B was reduced. ATP failed to stimulate the phosphorylation of ERK and c-Jun N-terminal kinase and activation of AP-1 in the p56(lck)-deficient isogenic T cell line JCaM1, suggesting a critical role for p56(lck) kinase in downstream signaling. Regarding the biological significance of the ATP-induced signaling events we show that although extracellular ATP was able to stimulate proliferation of both Jurkat and JCaM1 cells, an increase in interleukin-2 transcription was observed only in Jurkat cells. The nucleotide selectivity and pharmacological profile data supported the evidence that the ATP-induced effects in Jurkat cells were mediated through the P2X7 receptor. Taken together, these results demonstrate the ability of extracellular ATP to activate

  12. P2Y6 Receptors Require an Intact Cysteinyl Leukotriene Synthetic and Signaling System to Induce Survival and Activation of Mast Cells1

    PubMed Central

    Jiang, Yongfeng; Borrelli, Laura; Bacskai, Brian J.; Kanaoka, Yoshihide; Boyce, Joshua A.

    2008-01-01

    Cysteinyl leukotrienes (cys-LTs) induce inflammatory responses through type 1 (CysLT1R) and type 2 (CysLT2R) cys-LT receptors, and activate mast cells (MCs) in vitro. We previously demonstrated that cys-LTs cross-desensitized interleukin (IL)-4-primed primary human MCs (hMCs) to stimulation with the nucleotide uridine diphosphate (UDP). We now report that hMCs, mouse bone marrow-derived mast cells (mBMMCs), and the human MC line LAD2 all express UDP-selective P2Y6 receptors that cooperate with CysLT1R to promote cell survival and chemokine generation by a pathway involving reciprocal ligand-mediated cross-talk. LTD4, the most potent CysLT1R ligand, and UDP both induced phosphorylation of extracellular signal regulated kinase (ERK) and prolonged the survival of cytokine-starved hMCs and mBMMCs. ERK activation and cytoprotection in response to either ligand were attenuated by treatment of the cells with a selective P2Y6 receptor antagonist (MRS2578), which did not interfere with signaling through recombinant CysLT1R. Surprisingly, both UDP and LTD4-mediated ERK activation and cytoprotection were absent in mBMMCs lacking CysLT1R and the biosynthetic enzyme LTC4 synthase (LTC4S), implying a requirement for a cys-LT-mediated autocrine loop. In IL-4-primed LAD2 cells, LTD4 induced the generation of macrophage inflammatory protein 1β (MIP-1β) by IL-4-primed LAD2 cells, a response blocked by short hairpin RNA (shRNA)-mediated knockdown of CysLT1R or of P2Y6 receptors, but not of CysLT2R. Thus, CysLT1R and P2Y6 receptors, which are co-expressed with on many cell types of innate immunity, reciprocally amplify one another’s function in MCs through endogenous ligands. PMID:19124756

  13. P2Y1 Receptor Signaling Contributes to High Salt-Induced Priming of the NLRP3 Inflammasome in Retinal Pigment Epithelial Cells

    PubMed Central

    Prager, Philipp; Hollborn, Margrit; Steffen, Anja; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2016-01-01

    Background Systemic hypertension is a risk factor of age-related macular degeneration (AMD), a chronic inflammatory disease. Acute hypertension is caused by increased extracellular osmolarity after intake of dietary salt (NaCl). We determined in cultured human retinal pigment epithelial (RPE) cells whether high extracellular NaCl alters the gene expression of inflammasome-associated proteins, and whether autocrine/paracrine purinergic (P2) receptor signaling contributes to the NaCl-induced NLRP3 gene expression. Methodology/Principal Findings Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Gene and protein expression levels were determined with real-time RT-PCR and Western blot analysis, respectively. IL-1β and IL-18 levels were evaluated with ELISA. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. High extracellular NaCl induced NLRP3 and pro-IL-1β gene expression, while the gene expression of further inflammasome-associated proteins (NLRP1, NLRP2, NLRP6, NLRP7, NLRP12, NLRC4, AIM2, ASC, procaspase-1, pro-IL-18) was not altered or below the detection threshold. The NaCl-induced NLRP3 gene expression was partially dependent on the activities of phospholipase C, IP3 receptors, protein kinase C, the serum and glucocorticoid-regulated kinase, p38 MAPK, ERK1/2, JNK, PI3K, and the transcription factors HIF-1 and NFAT5. Pannexin-dependent ATP release and P2Y1 receptor activation is required for the full induction of NLRP3 gene expression. High NaCl induced a transient increase of the NLRP3 protein level and a moderate NLRP3 inflammasome activation, as indicated by the transient increase of the cytosolic level of mature IL-1β. High NaCl also induced secretion of IL-18. Conclusion High extracellular NaCl induces priming of the NLRP3 inflammasome in RPE cells, in part via P2Y1 receptor signaling. The inflammasome priming effect of NaCl suggests that high intake of dietary salt may promote

  14. P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in neuroblastoma cells.

    PubMed

    Gutiérrez-Martín, Yolanda; Bustillo, Diego; Gómez-Villafuertes, Rosa; Sánchez-Nogueiro, Jesús; Torregrosa-Hetland, Cristina; Binz, Thomas; Gutiérrez, Luis Miguel; Miras-Portugal, María Teresa; Artalejo, Antonio R

    2011-04-01

    Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca(2+)/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca(2+)-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca(2+) concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca(2+) and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation.

  15. P2X7 Receptors Trigger ATP Exocytosis and Modify Secretory Vesicle Dynamics in Neuroblastoma Cells*

    PubMed Central

    Gutiérrez-Martín, Yolanda; Bustillo, Diego; Gómez-Villafuertes, Rosa; Sánchez-Nogueiro, Jesús; Torregrosa-Hetland, Cristina; Binz, Thomas; Gutiérrez, Luis Miguel; Miras-Portugal, María Teresa; Artalejo, Antonio R.

    2011-01-01

    Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca2+/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca2+-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca2+ concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca2+ and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation. PMID:21292765

  16. NMDA Receptors Mediate Synaptic Competition in Culture

    PubMed Central

    She, Kevin; Craig, Ann Marie

    2011-01-01

    Background Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo, where activity of inputs can be differentially regulated, but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here, we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. Methodology/Principal Findings GluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons, both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures, synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures. Conclusions/Significance The enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde ‘reward’ signal generated by WT neurons, although in this paradigm there was no ‘punishment’ signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous

  17. P2Y2 purinergic receptor activation is essential for efficient hepatocyte proliferation in response to partial hepatectomy

    PubMed Central

    Tackett, Bryan C.; Sun, Hongdan; Mei, Yu; Maynard, Janielle P.; Cheruvu, Sayuri; Mani, Arunmani; Hernandez-Garcia, Andres; Vigneswaran, Nadarajah; Karpen, Saul J.

    2014-01-01

    Extracellular nucleotides via activation of P2 purinergic receptors influence hepatocyte proliferation and liver regeneration in response to 70% partial hepatectomy (PH). Adult hepatocytes express multiple P2Y (G protein-coupled) and P2X (ligand-gated ion channels) purinergic receptor subtypes. However, the identity of key receptor subtype(s) important for efficient hepatocyte proliferation in regenerating livers remains unknown. To evaluate the impact of P2Y2 purinergic receptor-mediated signaling on hepatocyte proliferation in regenerating livers, wild-type (WT) and P2Y2 purinergic receptor knockout (P2Y2−/−) mice were subjected to 70% PH. Liver tissues were analyzed for activation of early events critical for hepatocyte priming and subsequent cell cycle progression. Our findings suggest that early activation of p42/44 ERK MAPK (5 min), early growth response-1 (Egr-1) and activator protein-1 (AP-1) DNA-binding activity (30 min), and subsequent hepatocyte proliferation (24–72 h) in response to 70% PH were impaired in P2Y2−/− mice. Interestingly, early induction of cytokines (TNF-α, IL-6) and cytokine-mediated signaling (NF-κB, STAT-3) were intact in P2Y2−/− remnant livers, uncovering the importance of cytokine-independent and nucleotide-dependent early priming events critical for subsequent hepatocyte proliferation in regenerating livers. Hepatocytes isolated from the WT and P2Y2−/− mice were treated with ATP or ATPγS for 5–120 min and 12–24 h. Extracellular ATP alone, via activation of P2Y2 purinergic receptors, was sufficient to induce ERK phosphorylation, Egr-1 protein expression, and key cyclins and cell cycle progression of hepatocytes in vitro. Collectively, these findings highlight the functional significance of P2Y2 purinergic receptor activation for efficient hepatocyte priming and proliferation in response to PH. PMID:25301185

  18. Receptors mediating toxicity and their involvement in endocrine disruption.

    PubMed

    Rüegg, Joëlle; Penttinen-Damdimopoulou, Pauliina; Mäkelä, Sari; Pongratz, Ingemar; Gustafsson, Jan-Ake

    2009-01-01

    Many toxic compounds exert their harmful effects by activating of certain receptors, which in turn leads to dysregulation of transcription. Some of these receptors are so called xenosensors. They are activated by external chemicals and evoke a cascade of events that lead to the elimination of the chemical from the system. Other receptors that are modulated by toxic substances are hormone receptors, particularly the ones of the nuclear receptor family. Some environmental chemicals resemble endogenous hormones and can falsely activate these receptors, leading to undesired activity in the cell. Furthermore, excessive activation of the xenosensors can lead to disturbances of the integrity of the system as well. In this chapter, the concepts of receptor-mediated toxicity and hormone disruption are introduced. We start by describing environmental chemicals that can bind to xenosensors and nuclear hormone receptors. We then describe the receptors most commonly targeted by environmental chemicals. Finally, the mechanisms by which receptor-mediated events can disrupt the system are depicted.

  19. Multiscale Modeling of Virus Entry via Receptor-Mediated Endocytosis

    NASA Astrophysics Data System (ADS)

    Liu, Jin

    2012-11-01

    Virus infections are ubiquitous and remain major threats to human health worldwide. Viruses are intracellular parasites and must enter host cells to initiate infection. Receptor-mediated endocytosis is the most common entry pathway taken by viruses, the whole process is highly complex and dictated by various events, such as virus motions, membrane deformations, receptor diffusion and ligand-receptor reactions, occurring at multiple length and time scales. We develop a multiscale model for virus entry through receptor-mediated endocytosis. The binding of virus to cell surface is based on a mesoscale three dimensional stochastic adhesion model, the internalization (endocytosis) of virus and cellular membrane deformation is based on the discretization of Helfrich Hamiltonian in a curvilinear space using Monte Carlo method. The multiscale model is based on the combination of these two models. We will implement this model to study the herpes simplex virus entry into B78 cells and compare the model predictions with experimental measurements.

  20. Regulation of the osteogenic and adipogenic differentiation of bone marrow-derived stromal cells by extracellular uridine triphosphate: The role of P2Y2 receptor and ERK1/2 signaling

    PubMed Central

    LI, WENKAI; WEI, SHENG; LIU, CHAOXU; SONG, MINGYU; WU, HUA; YANG, YONG

    2016-01-01

    An imbalance in the osteogenesis and adipogenesis of bone marrow-derived stromal cells (BMSCs) is a crucial pathological factor in the development of osteoporosis. Growing evidence suggests that extracellular nucleotide signaling involving the P2 receptors plays a significant role in bone metabolism. The aim of the present study was to investigate the effects of uridine triphosphate (UTP) on the osteogenic and adipogenic differentiation of BMSCs, and to elucidate the underlying mechanisms. The differentiation of the BMSCs was determined by measuring the mRNA and protein expression levels of osteogenic- and adipogenic-related markers, alkaline phosphatase (ALP) staining, alizarin red staining and Oil Red O staining. The effects of UTP on BMSC differentiation were assayed using selective P2Y receptor antagonists, small interfering RNA (siRNA) and an intracellular signaling inhibitor. The incubation of the BMSCs with UTP resulted in a dose-dependent decrease in osteogenesis and an increase in adipogenesis, without affecting cell proliferation. Significantly, siRNA targeting the P2Y2 receptor prevented the effects of UTP, whereas the P2Y6 receptor antagonist (MRS2578) and siRNA targeting the P2Y4 receptor had little effect. The activation of P2Y receptors by UTP transduced to the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. This transduction was prevented by the mitogen-activated protein kinase inhibitor (U0126) and siRNA targeting the P2Y2 receptor. U0126 prevented the effects of UTP on osteogenic- and adipogenic-related gene expression after 24 h of culture, as opposed to 3 to 7 days of culture. Thus, our data suggest that UTP suppresses the osteogenic and enhances the adipogenic differentiation of BMSCs by activating the P2Y2 receptor. The ERK1/2 signaling pathway mediates the early stages of this process. PMID:26531757

  1. Characterization of protoberberine analogs employed as novel human P2X{sub 7} receptor antagonists

    SciTech Connect

    Lee, Ga Eun; Lee, Won-Gil; Lee, Song-Yi; Lee, Cho-Rong; Park, Chul-Seung; Chang, Sunghoe; Park, Sung-Gyoo; Song, Mi-Ryoung; Kim, Yong-Chul

    2011-04-15

    The P2X{sub 7} receptor (P2X{sub 7}R), a member of the ATP-gated ion channel family, is regarded as a promising target for therapy of immune-related diseases including rheumatoid arthritis and chronic pain. A group of novel protoberberine analogs (compounds 3-5), discovered by screening of chemical libraries, was here investigated with respect to their function as P2X{sub 7}R antagonists. Compounds 3-5 non-competitively inhibited BzATP-induced ethidium ion influx into hP2X{sub 7}-expressing HEK293 cells, with IC{sub 50} values of 100-300 nM. This antagonistic action on the channel further confirmed that both BzATP-induced inward currents and Ca{sup 2+} influx were strongly inhibited by compounds 3-5 in patch-clamp and Ca{sup 2+} influx assays. The antagonists also effectively suppressed downstream signaling of P2X{sub 7} receptors including IL-1{beta} release and phosphorylation of ERK1/2 and p38 proteins in hP2X{sub 7}-expressing HEK293 cells or in differentiated human monocytes (THP-1 cells). Moreover, IL-2 secretion from CD3/CD28-stimulated Jurkat T cell was also dramatically inhibited by the antagonist. These results imply that novel protoberberine analogs may modulate P2X{sub 7} receptor-mediated immune responses by allosteric inhibition of the receptor. - Graphical abstract: Display Omitted

  2. Purinergic signaling in special senses.

    PubMed

    Housley, Gary D; Bringmann, Andreas; Reichenbach, Andreas

    2009-03-01

    We consider the impact of purinergic signaling on the physiology of the special senses of vision, smell, taste and hearing. Purines (particularly ATP and adenosine) act as neurotransmitters, gliotransmitters and paracrine factors in the sensory retina, nasal olfactory epithelium, taste buds and cochlea. The associated purinergic receptor signaling underpins the sensory transduction and information coding in these sense organs. The P2 and P1 receptors mediate fast transmission of sensory signals and have modulatory roles in the regulation of synaptic transmitter release, for example in the adaptation to sensory overstimulation. Purinergic signaling regulates bidirectional neuron-glia interactions and is involved in the control of blood supply, extracellular ion homeostasis and the turnover of sensory epithelia by modulating apoptosis and progenitor proliferation. Purinergic signaling is an important player in pathophysiological processes in sensory tissues, and has both detrimental (pro-apoptotic) and supportive (e.g. initiation of cytoprotective stress-signaling cascades) effects.

  3. P2X4 receptor–eNOS signaling pathway in cardiac myocytes as a novel protective mechanism in heart failure

    PubMed Central

    Yang, Ronghua; Beqiri, Dardan; Shen, Jian-Bing; Redden, John M.; Dodge-Kafka, Kimberly; Jacobson, Kenneth A.; Liang, Bruce T.

    2014-01-01

    We have demonstrated using immunoprecipitation and immunostaining a novel physical association of the P2X4 receptor (P2X4R), a ligand-gated ion channel, with the cardioprotective, calcium-dependent enzyme endothelial nitric oxide synthase (eNOS). Treatment of murine ventricular myocytes with the P2XR agonist 2-methylthioATP (2-meSATP) to induce a current (mainly Na+) increased the formation of nitric oxide (NO), as measured using a fluorescent probe. Possible candidates for downstream effectors mediating eNOS activity include cyclic GMP and PKG or cellular protein nitrosylation. A cardiac-specific P2X4R overexpressing mouse line was protected from heart failure (HF) with improved cardiac function and survival in post-infarct, pressure overload, and calsequestrin (CSQ) overexpression models of HF. Although the role of the P2X4R in other tissues such as the endothelium and monocytes awaits characterization in tissue-specific KO, cardiac-specific activation of eNOS may be more cardioprotective than an increased activity of global systemic eNOS. The intra-myocyte formation of NO may be more advantageous over NO derived externally from a donor. A small molecule drug stimulating this sarcolemmal pathway or gene therapy-mediated overexpression of the P2X4R in cardiac myocytes may represent a new therapy for both ischemic and pressure overloaded HF. PMID:25750695

  4. Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

    PubMed

    Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael

    2015-12-01

    Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

  5. P2×7 purinergic signaling in dilated cardiomyopathy induced by auto-immunity against muscarinic M2 receptors: autoantibody levels, heart functionality and cytokine expression

    PubMed Central

    Martinez, Camila Guerra; Zamith-Miranda, Daniel; da Silva, Marcia Gracindo; Ribeiro, Karla Consort; Brandão, Izaíra Trincani; Silva, Celio Lopes; Diaz, Bruno Lourenço; Bellio, Maria; Persechini, Pedro Muanis; Kurtenbach, Eleonora

    2015-01-01

    Autoantibodies against the M2 receptors (M2AChR) have been associated with Dilated Cardiomyopathy (DCM). In the heart, P2×7 receptors influence electrical conduction, coronary circulation and response to ischemia. They can also trigger pro-inflammatory responses and the development of neurological, cardiac and renal disorders. Here, P2×7−/− mice displayed an increased heart rate and ST segment depression, but similar exercise performance when compared to wild type (WT) animals. After immunization with plasmid containing M2AChR cDNA sequence, WT mice produced anti-M2AChR antibodies, while P2×7−/− mice showed an attenuated production. Despite this, WT and P2×7−/− showed left ventricle cavity enlargement and decreased exercise tolerance. Transfer of serum from M2AChR WT immunized mice to näive recipients led to an alteration in heart shape. P2×7−/− mice displayed a significant increase in the frequency of spleen regulatory T cells population, which is mainly composed by the FoxP3+CD25− subset. M2AChR WT immunized mice showed an increase in IL-1β, IFNγ and IL-17 levels in the heart, while P2×7−/− group produced lower amounts of IL-1β and IL-17 and higher amounts of IFNγ. These results pointed to previously unnoticed roles of P2×7 in cardiovascular and immune systems, and underscored the participation of IL-17 and IFNγ in the progress of autoimmune DCM. PMID:26592184

  6. Phenobarbital but Not Diazepam Reduces AMPA/kainate Receptor Mediated Currents and Exerts Opposite Actions on Initial Seizures in the Neonatal Rat Hippocampus

    PubMed Central

    Nardou, Romain; Yamamoto, Sumii; Bhar, Asma; Burnashev, Nail; Ben-Ari, Yehezkel; Khalilov, Ilgam

    2011-01-01

    Diazepam (DZP) and phenobarbital (PB) are extensively used as first and second line drugs to treat acute seizures in neonates and their actions are thought to be mediated by increasing the actions of GABAergic signals. Yet, their efficacy is variable with occasional failure or even aggravation of recurrent seizures questioning whether other mechanisms are not involved in their actions. We have now compared the effects of DZP and PB on ictal-like events (ILEs) in an in vitro model of mirror focus (MF). Using the three-compartment chamber with the two immature hippocampi and their commissural fibers placed in three different compartments, kainate was applied to one hippocampus and PB or DZP to the contralateral one, either after one ILE, or after many recurrent ILEs that produce an epileptogenic MF. We report that in contrast to PB, DZP aggravated propagating ILEs from the start, and did not prevent the formation of MF. PB reduced and DZP increased the network driven giant depolarizing potentials suggesting that PB may exert additional actions that are not mediated by GABA signaling. In keeping with this, PB but not DZP reduced field potentials recorded in the presence of GABA and NMDA receptor antagonists. These effects are mediated by a direct action on AMPA/kainate receptors since PB: (i) reduced AMPA/kainate receptor mediated currents induced by focal applications of glutamate; (ii) reduced the amplitude and the frequency of AMPA but not NMDA receptor mediated miniature excitatory postsynaptic currents (EPSCs); (iii) augmented the number of AMPA receptor mediated EPSCs failures evoked by minimal stimulation. These effects persisted in MF. Therefore, PB exerts its anticonvulsive actions partly by reducing AMPA/kainate receptors mediated EPSCs in addition to the pro-GABA effects. We suggest that PB may have advantage over DZP in the treatment of initial neonatal seizures since the additional reduction of glutamate receptors mediated signals may reduce the severity

  7. Bacteriophage P2.

    PubMed

    Christie, Gail E; Calendar, Richard

    2016-01-01

    P2 is the original member of a highly successful family of temperate phages that are frequently found in the genomes of gram-negative bacteria. This article focuses on the organization of the P2 genome and reviews current knowledge about the function of each open reading frame.

  8. 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.

  9. Regulation of the osteogenic and adipogenic differentiation of bone marrow-derived stromal cells by extracellular uridine triphosphate: The role of P2Y2 receptor and ERK1/2 signaling.

    PubMed

    Li, Wenkai; Wei, Sheng; Liu, Chaoxu; Song, Mingyu; Wu, Hua; Yang, Yong

    2016-01-01

    An imbalance in the osteogenesis and adipogenesis of bone marrow-derived stromal cells (BMSCs) is a crucial pathological factor in the development of osteoporosis. Growing evidence suggests that extracellular nucleotide signaling involving the P2 receptors plays a significant role in bone metabolism. The aim of the present study was to investigate the effects of uridine triphosphate (UTP) on the osteogenic and adipogenic differentiation of BMSCs, and to elucidate the underlying mechanisms. The differentiation of the BMSCs was determined by measuring the mRNA and protein expression levels of osteogenic- and adipogenic-related markers, alkaline phosphatase (ALP) staining, alizarin red staining and Oil Red O staining. The effects of UTP on BMSC differentiation were assayed using selective P2Y receptor antagonists, small interfering RNA (siRNA) and an intracellular signaling inhibitor. The incubation of the BMSCs with UTP resulted in a dose-dependent decrease in osteogenesis and an increase in adipogenesis, without affecting cell proliferation. Significantly, siRNA targeting the P2Y2 receptor prevented the effects of UTP, whereas the P2Y6 receptor antagonist (MRS2578) and siRNA targeting the P2Y4 receptor had little effect. The activation of P2Y receptors by UTP transduced to the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. This transduction was prevented by the mitogen-activated protein kinase inhibitor (U0126) and siRNA targeting the P2Y2 receptor. U0126 prevented the effects of UTP on osteogenic- and adipogenic-related gene expression after 24 h of culture, as opposed to 3 to 7 days of culture. Thus, our data suggest that UTP suppresses the osteogenic and enhances the adipogenic differentiation of BMSCs by activating the P2Y2 receptor. The ERK1/2 signaling pathway mediates the early stages of this process.

  10. P2X4 Receptor Reporter Mice: Sparse Brain Expression and Feeding-Related Presynaptic Facilitation in the Arcuate Nucleus

    PubMed Central

    Xu, Ji; Bernstein, Alexander M.; Wong, Angela; Lu, Xiao-Hong; Khoja, Sheraz; Yang, X. William; Davies, Daryl L.; Micevych, Paul; Sofroniew, Michael V.

    2016-01-01

    P2X4 receptors are ATP-gated cation channels that are widely expressed in the nervous system. To identify P2X4 receptor-expressing cells, we generated BAC transgenic mice expressing tdTomato under the control of the P2X4 receptor gene (P2rx4). We found sparse populations of tdTomato-positive neurons in most brain areas with patterns that matched P2X4 mRNA distribution. tdTomato expression within microglia was low but was increased by an experimental manipulation that triggered microglial activation. We found surprisingly high tdTomato expression in the hypothalamic arcuate nucleus (Arc) (i.e., within parts of the neural circuitry controlling feeding). Immunohistochemistry and genetic crosses of P2rx4 tdTomato mice with cell-specific GFP reporter lines showed that the tdTomato-expressing cells were mainly AgRP-NPY neurons and tanycytes. There was no electrophysiological evidence for functional expression of P2X4 receptors on AgRP-NPY neuron somata, but instead, we found clear evidence for functional presynaptic P2X4 receptor-mediated responses in terminals of AgRP-NPY neurons onto two of their postsynaptic targets (Arc POMC and paraventricular nucleus neurons), where ATP dramatically facilitated GABA release. The presynaptic responses onto POMC neurons, and the expression of tdTomato in AgRP-NPY neurons and tanycytes, were significantly decreased by food deprivation in male mice in a manner that was partially reversed by the satiety-related peptide leptin. Overall, we provide well-characterized tdTomato reporter mice to study P2X4-expressing cells in the brain, new insights on feeding-related regulation of presynaptic P2X4 receptor responses, and the rationale to explore extracellular ATP signaling in the control of feeding behaviors. SIGNIFICANCE STATEMENT Cells expressing ATP-gated P2X4 receptors have proven problematic to identify and study in brain slice preparations because P2X4 expression is sparse. To address this limitation, we generated and characterized

  11. miR-9 Mediates CALHM1-Activated ATP-P2X7R Signal in Painful Diabetic Neuropathy Rats.

    PubMed

    Liu, Wenjie; Ao, Qinying; Guo, Qulian; He, Wanyou; Peng, Liangyu; Jiang, Jun; Hu, Xiaoling

    2017-03-01

    In this study, we planned to illuminate the mechanisms of the expression and function of CALHM1 in painful diabetic neuropathy (PDN). PDN rat model was constructed. The expression of CALHM1 and miR-9 in rat spinal dorsal horn neurons was detected. The correlation between the level of CALHM1 mRNA and 50 % PWT and the relationship between the expression of CALHM1 and miR-9 in rat spinal dorsal horn neurons were statistically analyzed. The effect of miR-9 and CALHM1 on each other's expression in PDN rat spinal dorsal horn neurons were tested by qRT-PCR or Western blot. The co-culture system of neurons and glias from PDN rat spinal dorsal horn was constructed. The concentration of calcium and ATP as well as the expression of P2X7 receptor regulated by CALHM1 and miR-9 in PDN rat spinal dorsal horn neurons was measured. The results showed that the expression of CALHM1 was increased in PDN rat compared with controls, while its mRNA level was negatively correlated with 50 % PWT. miR-9, which was also upregulated in the spinal dorsal horn neurons of PDN rats, was positively correlated with the expression of CALHM1. The concentration of calcium and ATP as well as the expression of P2X7 receptor in glias was also increased in PDN rats. These increases could be reverted by inhibiting CALHM1 and/or miR-9. CALHM1 is involved in miR-9-mediated ATP-P2X7 pathway between neurons and glias in PDN rat.

  12. Receptor-mediated toxicity of pahutoxin, a marine trunkfish surfactant.

    PubMed

    Kalmanzon, Eliahu; Rahamim, Yocheved; Barenholz, Yechezkel; Carmeli, Shmuel; Zlotkin, Eliahu

    2003-07-01

    Pahutoxin (PHN, choline chloride ester of 3-acetoxypalmitic acid) is a natural fish-killing (ichthyotoxic) agent derived from the defensive secretions of trunkfish. In spite of its obvious structural resemblance to synthetic cationic long-chain quaternary ammonium detergents, we show that PHN's action does not rely on its surfactant properties and is in fact, receptor-mediated. The above conclusion is supported by the following data: 1. Ichthyotoxicity is not related to its detergency or surfactivity, as indicated by the fact that the lethal concentration is about 1.5 orders of magnitude below its critical micelle concentration value (69 microM) and its liposomal/seawater partition coefficient is low (62-85); 2. The trunkfish is tolerant to its own pahutoxin; 3. Ichthyotoxicity occurs only upon application to the surrounding water, suggesting the existence of externally located receptors; 4. The receptor hypothesis was supported by the aid of equilibrium saturation binding assays revealing the presence of specific binding sites to PHN on the fish gill membranes; 5. The PHN tolerant trunkfish was shown to be devoid of PHN-binding sites. Some chemo-ecological, and environmental implications are discussed.

  13. Biologically bounded risk assessment for receptor-mediated nongenotoxic carcinogens.

    PubMed

    Gastel, J A; Sutter, T R

    1995-12-01

    We have developed a biologically bounded marginal effect model for use in risk assessment of human exposure to receptor-mediated nongenotoxic carcinogens. Schematically this model can be reduced to four components: CI, the absence of an observable biological response; CII, observable biochemical responses but no observable pathology; CIII, observable pathology; and CIV, both observable pathology and lethality. The inflection point in the marginal response curve between CI and CII is defined as the biologically evaluated scientifically tested no observable effect level (BESTNOEL). We demonstrate the utility of this approach by applying it to the well-studied nongenotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using a well-developed mechanistic understanding of the initial interactions of TCDD with the cell, we justify the selection of the minimal effective dose for CYP1A1 mRNA induction as the BESTNOEL. With allowance for variation in human sensitivity to TCDD, the BESTNOEL is assigned a human liver tissue burden of approximately 0.25-25 ppt and an allowable daily intake level in the range of 15-1500 fg/kg/day. In the future, the BESTNOEL can help establish a lower boundary for acceptable extrapolation when using either statistical or biologically based attributable risk models.

  14. Visualization of Receptor-mediated Endocytosis in Yeast

    PubMed Central

    Mulholland, Jon; Konopka, James; Singer-Kruger, Birgit; Zerial, Marino; Botstein, David

    1999-01-01

    We studied the ligand-induced endocytosis of the yeast α-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to α-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Δ. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to α-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes. PMID:10069819

  15. Hemoglobin Uptake by Paracoccidioides spp. Is Receptor-Mediated

    PubMed Central

    Bailão, Elisa Flávia Luiz Cardoso; Parente, Juliana Alves; Pigosso, Laurine Lacerda; de Castro, Kelly Pacheco; Fonseca, Fernanda Lopes; Silva-Bailão, Mirelle Garcia; Báo, Sônia Nair; Bailão, Alexandre Melo; Rodrigues, Marcio L.; Hernandez, Orville; McEwen, Juan G.; Soares, Célia Maria de Almeida

    2014-01-01

    Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms. PMID:24831516

  16. Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes

    PubMed Central

    Matute, Carlos; Sánchez-Gómez, M. Victoria; Martínez-Millán, Luis; Miledi, Ricardo

    1997-01-01

    In cultured oligodendrocytes isolated from perinatal rat optic nerves, we have analyzed the expression of ionotropic glutamate receptor subunits as well as the effect of the activation of these receptors on oligodendrocyte viability. Reverse transcription–PCR, in combination with immunocytochemistry, demonstrated that most oligodendrocytes differentiated in vitro express the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR3 and GluR4 and the kainate receptor subunits GluR6, GluR7, KA1 and KA2. Acute and chronic exposure to kainate caused extensive oligodendrocyte death in culture. This effect was partially prevented by the AMPA receptor antagonist GYKI 52466 and was completely abolished by the non-N-methyl-d-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that both AMPA and kainate receptors mediate the observed kainate toxicity. Furthermore, chronic application of kainate to optic nerves in vivo resulted in massive oligodendrocyte death which, as in vitro, could be prevented by coinfusion of the toxin with CNQX. These findings suggest that excessive activation of the ionotropic glutamate receptors expressed by oligodendrocytes may act as a negative regulator of the size of this cell population. PMID:9238063

  17. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    PubMed

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  18. Antigen-Specific Immune Modulation Targets mTORC1 Function To Drive Chemokine Receptor-Mediated T Cell Tolerance.

    PubMed

    Chen, Weirong; Wan, Xiaoxiao; Ukah, Tobechukwu K; Miller, Mindy M; Barik, Subhasis; Cattin-Roy, Alexis N; Zaghouani, Habib

    2016-11-01

    To contain autoimmunity, pathogenic T cells must be eliminated or diverted from reaching the target organ. Recently, we defined a novel form of T cell tolerance whereby treatment with Ag downregulates expression of the chemokine receptor CXCR3 and prevents diabetogenic Th1 cells from reaching the pancreas, leading to suppression of type 1 diabetes (T1D). This report defines the signaling events underlying Ag-induced chemokine receptor-mediated tolerance. Specifically, we show that the mammalian target of rapamycin complex 1 (mTORC1) is a major target for induction of CXCR3 downregulation and crippling of Th1 cells. Indeed, Ag administration induces upregulation of programmed death-ligand 1 on dendritic cells in a T cell-dependent manner. In return, programmed death-ligand 1 interacts with the constitutively expressed programmed death-1 on the target T cells and stimulates docking of Src homology 2 domain-containing tyrosine phosphatase 2 phosphatase to the cytoplasmic tail of programmed death-1. Active Src homology 2 domain-containing tyrosine phosphatase 2 impairs the signaling function of the PI3K/protein kinase B (AKT) pathway, leading to functional defect of mTORC1, downregulation of CXCR3 expression, and suppression of T1D. Thus, mTORC1 component of the metabolic pathway serves as a target for chemokine receptor-mediated T cell tolerance and suppression of T1D.

  19. Enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses in the barrel cortex of Mecp2-null mice

    PubMed Central

    Lo, Fu-Sun; Blue, Mary E.

    2015-01-01

    Rett syndrome (RTT) is a neurodevelopmental disorder that results from mutations in the X-linked gene for methyl-CpG-binding protein 2 (MECP2). The underlying cellular mechanism for the sensory deficits in patients with RTT is largely unknown. This study used the Bird mouse model of RTT to investigate sensory thalamocortical synaptic transmission in the barrel cortex of Mecp2-null mice. Electrophysiological results showed an excitation/inhibition imbalance, biased toward inhibition, due to an increase in efficacy of postsynaptic GABAA receptors rather than alterations in inhibitory network and presynaptic release properties. Enhanced inhibition impaired the transmission of tonic sensory signals from the thalamus to the somatosensory cortex. Previous morphological studies showed an upregulation of NMDA receptors in the neocortex of both RTT patients and Mecp2-null mice at early ages [Blue ME, Naidu S, Johnston MV. Ann Neurol 45: 541–545, 1999; Blue ME, Kaufmann WE, Bressler J, Eyring C, O'Driscoll C, Naidu S, Johnston MV. Anat Rec (Hoboken) 294: 1624–1634, 2011]. Although AMPA and NMDA receptor-mediated excitatory synaptic transmission was not altered in the barrel cortex of Mecp2-null mice, extrasynaptic NMDA receptor-mediated responses increased markedly. These responses were blocked by memantine, suggesting that extrasynaptic NMDA receptors play an important role in the pathogenesis of RTT. The results suggest that enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses may underlie impaired somatosensation and that pharmacological blockade of extrasynaptic NMDA receptors may have therapeutic value for RTT. PMID:26683074

  20. Synaptic NMDA receptor-mediated currents in anterior piriform cortex are reduced in the adult fragile X mouse.

    PubMed

    Gocel, James; Larson, John

    2012-09-27

    Fragile X syndrome is a neurodevelopmental condition caused by the transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. The Fmr1 knockout (KO) mouse exhibits age-dependent deficits in long term potentiation (LTP) at association (ASSN) synapses in anterior piriform cortex (APC). To investigate the mechanisms for this, whole-cell voltage-clamp recordings of ASSN stimulation-evoked synaptic currents were made in APC of slices from adult Fmr1-KO and wild-type (WT) mice, using the competitive N-methyl-D-aspartate (NMDA) receptor antagonist, CPP, to distinguish currents mediated by NMDA and AMPA receptors. NMDA/AMPA current ratios were lower in Fmr1-KO mice than in WT mice, at ages ranging from 3-18months. Since amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were no different in Fmr1-KO and WT mice at these ages, the results suggest that NMDA receptor-mediated currents are selectively reduced in Fmr1-KO mice. Analyses of voltage-dependence and decay kinetics of NMDA receptor-mediated currents did not reveal differences between Fmr1-KO and WT mice, suggesting that reduced NMDA currents in Fmr1-KO mice are due to fewer synaptic receptors rather than differences in receptor subunit composition. Reduced NMDA receptor signaling may help to explain the LTP deficit seen at APC ASSN synapses in Fmr1-KO mice at 6-18months of age, but does not explain normal LTP at these synapses in mice 3-6months old. Evoked currents and mEPSCs were also examined in senescent Fmr1-KO and WT mice at 24-28months of age. NMDA/AMPA ratios were similar in senescent WT and Fmr1-KO mice, due to a decrease in the ratio in the WT mice, without significant change in AMPA receptor-mediated mEPSCs.

  1. Enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses in the barrel cortex of Mecp2-null mice.

    PubMed

    Lo, Fu-Sun; Blue, Mary E; Erzurumlu, Reha S

    2016-03-01

    Rett syndrome (RTT) is a neurodevelopmental disorder that results from mutations in the X-linked gene for methyl-CpG-binding protein 2 (MECP2). The underlying cellular mechanism for the sensory deficits in patients with RTT is largely unknown. This study used the Bird mouse model of RTT to investigate sensory thalamocortical synaptic transmission in the barrel cortex of Mecp2-null mice. Electrophysiological results showed an excitation/inhibition imbalance, biased toward inhibition, due to an increase in efficacy of postsynaptic GABAA receptors rather than alterations in inhibitory network and presynaptic release properties. Enhanced inhibition impaired the transmission of tonic sensory signals from the thalamus to the somatosensory cortex. Previous morphological studies showed an upregulation of NMDA receptors in the neocortex of both RTT patients and Mecp2-null mice at early ages [Blue ME, Naidu S, Johnston MV. Ann Neurol 45: 541-545, 1999; Blue ME, Kaufmann WE, Bressler J, Eyring C, O'Driscoll C, Naidu S, Johnston MV. Anat Rec (Hoboken) 294: 1624-1634, 2011]. Although AMPA and NMDA receptor-mediated excitatory synaptic transmission was not altered in the barrel cortex of Mecp2-null mice, extrasynaptic NMDA receptor-mediated responses increased markedly. These responses were blocked by memantine, suggesting that extrasynaptic NMDA receptors play an important role in the pathogenesis of RTT. The results suggest that enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses may underlie impaired somatosensation and that pharmacological blockade of extrasynaptic NMDA receptors may have therapeutic value for RTT.

  2. ESCRT machinery potentiates HIV-1 utilization of the PI(4,5)P(2)-PLC-IP3R-Ca(2+) signaling cascade.

    PubMed

    Ehrlich, Lorna S; Medina, Gisselle N; Carter, Carol A

    2011-10-21

    Human immunodeficiency virus type 1 (HIV-1) release efficiency is directed by late (L) domain motifs in the viral structural precursor polyprotein Gag, which serve as links to the ESCRT (endosomal sorting complex required for transport) machinery. Linkage is normally through binding of Tsg101, an ESCRT-1 component, to the P(7)TAP motif in the p6 region of Gag. In its absence, budding is directed by binding of Alix, an ESCRT adaptor protein, to the LY(36)PX(n)L motif in Gag. We recently showed that budding requires activation of the inositol 1,4,5-triphosphate receptor (IP3R), a protein that "gates" Ca(2+) release from intracellular stores, triggers Ca(2+) cell influx and thereby functions as a major regulator of Ca(2+) signaling. In the present study, we determined whether the L domain links Gag to Ca(2+) signaling machinery. Depletion of IP3R and inactivation of phospholipase C (PLC) inhibited budding whether or not Tsg101 was bound to Gag. PLC hydrolysis of phosphatidylinositol-(4,5)-bisphosphate generates inositol (1,4,5)-triphosphate, the ligand that activates IP3R. However, with Tsg101 bound, Gag release was independent of Gq-mediated activation of PLC, and budding was readily enhanced by pharmacological stimulation of PLC. Moreover, IP3R was redistributed to the cell periphery and cytosolic Ca(2+) was elevated, events indicative of induction of Ca(2+) signaling. The results suggest that L domain function, ESCRT machinery and Ca(2+) signaling are linked events in Gag release.

  3. 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.

  4. GABAA Receptor-Mediated Activity in a Model of Cortical Dysplasia

    DTIC Science & Technology

    2012-06-29

    a model of cortical dysplasia" Name of Candidate: Joseph Abbah Doctor of Philosophy Degree June 29, 2012 DISSERTATION AND ABSTRACT APPROVED...receptor-mediated activity in a model of cortical dysplasia’ is appropriately acknowledged and, beyond brief excerpts, is with the permission of the...certifies that the use of any copyrighted material in this dissertation manuscript entitled: “GABAA receptor-mediated activity in a model of

  5. PI(4,5)P2 5-phosphatase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma.

    PubMed

    Ye, Yan; Jin, Lei; Wilmott, James S; Hu, Wang Lai; Yosufi, Benafsha; Thorne, Rick F; Liu, Tao; Rizos, Helen; Yan, Xu Guang; Dong, Li; Tay, Kwang Hong; Tseng, Hsin-Yi; Guo, Su Tang; de Bock, Charles E; Jiang, Chen Chen; Wang, Chun Yan; Wu, Mian; Zhang, Lin Jie; Hersey, Peter; Scolyer, Richard A; Zhang, Xu Dong

    2013-01-01

    Inositol polyphosphate 5-phosphatases can terminate downstream signalling of phosphatidylinositol-3 kinase; however, their biological role in the pathogenesis of cancer is controversial. Here we report that the inositol polyphosphate 5-phosphatase, phosphatidylinositol 4,5-bisphosphate 5-phosphatase, has a tumour suppressive role in melanoma. Although it is commonly downregulated in melanoma, overexpression of phosphatidylinositol 4,5-bisphosphate 5-phosphatase blocks Akt activation, inhibits proliferation and undermines survival of melanoma cells in vitro, and retards melanoma growth in a xenograft model. In contrast, knockdown of phosphatidylinositol 4,5-bisphosphate 5-phosphatase results in increased proliferation and anchorage-independent growth of melanocytes. Although DNA copy number loss is responsible for downregulation of phosphatidylinositol 4,5-bisphosphate 5-phosphatase in a proportion of melanomas, histone hypoacetylation mediated by histone deacetylases HDAC2 and HDAC3 through binding to the transcription factor Sp1 at the PIB5PA gene promoter appears to be another commonly involved mechanism. Collectively, these results establish the tumour suppressive role of phosphatidylinositol 4,5-bisphosphate 5-phosphatase and reveal mechanisms involved in its downregulation in melanoma.

  6. Induction of aryl hydrocarbon receptor-mediated and estrogen receptor-mediated activities, and modulation of cell proliferation by dinaphthofurans.

    PubMed

    Vondrácek, Jan; Chramostová, Katerina; Plísková, Martina; Bláha, Ludek; Brack, Werner; Kozubík, Alois; Machala, Miroslav

    2004-09-01

    A group of heterocyclic aromatic compounds, dinaphthofurans (DNFs), recently have been identified as potentially significant contaminants in freshwater sediments. In the present study, a battery of in vitro assays was used for detection of toxic effects of DNFs that are potentially associated with endocrine disruption and tumor promotion. Dinaphthofurans were found to act as relatively potent inducers of aryl hydrocarbon receptor (AhR)-mediated activity in the chemical-activated luciferase reporter gene expression DR-CALUX assay. The relative AhR-inducing potencies of DNFs were similar or even higher than relative potencies of unsubstituted polycyclic aromatic hydrocarbons (PAHs), with dinaphtho[1,2-b;2'3'-d]furan being the most potent AhR agonist. Two compounds, dinaphtho[2,1-b;2'3'-d]furan and dinaphtho[1,2-b;1'2'-d]furan, induced estrogen receptor (ER)-mediated activity in the estrogen receptor-mediated CALUX (the ER-CALUX) assay. Two types of potential tumor-promoting effects of DNFs were investigated, using in vitro bioassays for detection of inhibition of gap-junctional intercellular communication and detection of a release from contact inhibition. Although the acute inhibition of gap-junctional intercellular communication was not observed, all six tested DNFs were able to release rat liver epithelial WB-F344 cells from contact inhibition at concentrations as low as 100 nM. In summary, the present study indicated that DNFs can exert multiple biological effects in vitro, including induction of the AhR-mediated activity, release of cells from contact inhibition, and induction of ER-mediated activity.

  7. Different receptors mediate the electrophysiological and growth cone responses of an identified neuron to applied dopamine.

    PubMed

    Dobson, K S; Dmetrichuk, J M; Spencer, G E

    2006-09-15

    Neurotransmitters are among the many cues that may guide developing axons toward appropriate targets in the developing nervous system. We have previously shown in the mollusk Lymnaea stagnalis that dopamine, released from an identified pre-synaptic cell, differentially affects growth cone behavior of its target and non-target cells in vitro. Here, we describe a group of non-target cells that also produce an inhibitory electrophysiological response to applied dopamine. We first determined, using pharmacological blockers, which receptors mediate this physiological response. We demonstrated that the dopaminergic electrophysiological responses of non-target cells were sensitive to a D2 receptor antagonist, as are known target cell responses. However, the non-target cell receptors were linked to different G-proteins and intracellular signaling pathways than the target cell receptors. Despite the presence of a D2-like receptor at the soma, the growth cone collapse of these non-target cells was mediated by D1-like receptors. This study shows that different dopamine receptor sub-types mediated the inhibitory physiological and growth cone responses of an identified cell type. We therefore not only provide further evidence that D2- and D1-like receptors can be present on the same neuron in invertebrates, but also show that these receptors are likely involved in very different cellular functions.

  8. USP8 suppresses death receptor-mediated apoptosis by enhancing FLIPL stability.

    PubMed

    Jeong, M; Lee, E-W; Seong, D; Seo, J; Kim, J-H; Grootjans, S; Kim, S-Y; Vandenabeele, P; Song, J

    2017-01-26

    FLICE-like inhibitory protein (FLIP) is a critical regulator of death receptor-mediated apoptosis. Here, we found ubiquitin-specific peptidase 8 (USP8) to be a novel deubiquitylase of the long isoform of FLIP (FLIPL). USP8 directly deubiquitylates and stabilizes FLIPL, but not the short isoform. USP8 depletion induces FLIPL destabilization, promoting anti-Fas-, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- and tumor necrosis factor alpha-induced extrinsic apoptosis by facilitating death-inducing signaling complex or TNFR1 complex II formation, which results in the activation of caspase-8 and caspase-3. USP8 mRNA levels are elevated in melanoma and cervical cancers, and the protein levels of USP8 and FLIPL are positively correlated in these cancer cell lines. Xenograft analyses using ME-180 cervical cancer cells showed that USP8 depletion attenuated tumor growth upon TRAIL injection. Taken together, our data indicate that USP8 functions as a novel deubiquitylase of FLIPL and inhibits extrinsic apoptosis by stabilizing FLIPL.

  9. Imaging receptor-mediated endocytosis with a polymeric nanoparticle-based coherent anti-stokes Raman scattering probe.

    PubMed

    Tong, Ling; Lu, Yanhui; Lee, Robert J; Cheng, Ji-Xin

    2007-08-23

    Coherent anti-Stokes Raman scattering (CARS) microscopy was used to visualize receptor-mediated endocytosis and intracellular trafficking with the aid of a CARS probe. The probe was made of 200-nm polystyrene particles encapsulated in folate-targeted liposomes. By tuning (omega(p) - omega(s)) to 3045 cm(-1), which corresponds to the aromatic C-H stretching vibration, the polystyrene nanoparticles with a high density of aromatic C-H bonds were detected with a high signal-to-noise ratio, while the epi-detected CARS signal from cellular organelles was cancelled by the destructive interference between the resonant contribution from the aliphatic C-H vibration and the nonresonant contribution. Without any photobleaching, the CARS probe allowed single-particle tracking analysis of intracellular endosome transport. No photodamage to cells was observed under the current experimental conditions. These results show the advantages and potential of using a CARS probe to study cellular processes.

  10. Insulin-Independent GABAA Receptor-Mediated Response in the Barrel Cortex of Mice with Impaired Met Activity

    PubMed Central

    Lo, Fu-Sun; Erzurumlu, Reha S.

    2016-01-01

    Autism spectrum disorder (ASD) is a neurodevelopmental disorder caused by genetic variants, susceptibility alleles, and environmental perturbations. The autism associated gene MET tyrosine kinase has been implicated in many behavioral domains and endophenotypes of autism, including abnormal neural signaling in human sensory cortex. We investigated somatosensory thalamocortical synaptic communication in mice deficient in Met activity in cortical excitatory neurons to gain insights into aberrant somatosensation characteristic of ASD. The ratio of excitation to inhibition is dramatically increased due to decreased postsynaptic GABAA receptor-mediated inhibition in the trigeminal thalamocortical pathway of mice lacking active Met in the cerebral cortex. Furthermore, in contrast to wild-type mice, insulin failed to increase GABAA receptor-mediated response in the barrel cortex of mice with compromised Met signaling. Thus, lacking insulin effects may be a risk factor in ASD pathogenesis. SIGNIFICANCE STATEMENT A proposed common cause of neurodevelopmental disorders is an imbalance in excitatory neural transmission, provided by the glutamatergic neurons, and the inhibitory signals from the GABAergic interneurons. Many genes associated with autism spectrum disorders impair synaptic transmission in the expected cell type. Previously, inactivation of the autism-associated Met tyrosine kinase receptor in GABAergic interneurons led to decreased inhibition. In thus report, decreased Met signaling in glutamatergic neurons had no effect on excitation, but decimated inhibition. Further experiments indicate that loss of Met activity downregulates GABAA receptors on glutamatergic neurons in an insulin independent manner. These data provide a new mechanism for the loss of inhibition and subsequent abnormal excitation/inhibition balance and potential molecular candidates for treatment or prevention. PMID:27030755

  11. Distribution of P2Y2 receptors in the guinea pig enteric nervous system and its coexistence with P2X2 and P2X3 receptors, neuropeptide Y, nitric oxide synthase and calretinin.

    PubMed

    Xiang, Zhenghua; Burnstock, Geoffrey

    2005-11-01

    The distribution of P2Y2 receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y2 with P2X2 and P2X3 receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y2-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y2-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y2-ir neurons could be Dogiel type 1. About 40-60% P2X3-ir neurons were immunoreactive for P2Y2 in the myenteric plexus and all the P2X3-ir neurons expressed the P2Y2 receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y2, especially in the myenteric plexus of the small intestine; no P2Y2-ir neurons were immunoreactive for P2X2 receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y2-ir and P2X3-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.

  12. 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.

  13. GRB2 Nucleates T Cell Receptor-Mediated LAT Clusters That Control PLC-γ1 Activation and Cytokine Production

    PubMed Central

    Bilal, Mahmood Yousif; Houtman, Jon C. D.

    2015-01-01

    GRB2 is a ubiquitously expressed adaptor protein required for signaling downstream of multiple receptors. To address the role of GRB2 in receptor-mediated signaling, the expression of GRB2 was suppressed in human CD4+ T cells and its role downstream of the T cell receptor (TCR) was examined. Interestingly, GRB2 deficient T cells had enhanced signaling from complexes containing the TCR. However, GRB2 deficient T cells had substantially reduced production of IL-2 and IFN-γ. This defect was attributed to diminished formation of linker for activation of T cells (LAT) signaling clusters, which resulted in reduced MAP kinase activation, calcium flux, and PLC-γ1 recruitment to LAT signaling clusters. Add back of wild-type GRB2, but not a novel N-terminal SH3 domain mutant, rescued LAT microcluster formation, calcium mobilization, and cytokine release, providing the first direct evidence that GRB2, and its ability to bind to SH3 domain ligands, is required for establishing LAT microclusters. Our data demonstrate that the ability of GRB2 to facilitate protein clusters is equally important in regulating TCR-mediated functions as its capacity to recruit effector proteins. This highlights that GRB2 regulates signaling downstream of adaptors and receptors by both recruiting effector proteins and regulating the formation of signaling complexes. PMID:25870599

  14. GRB2 Nucleates T Cell Receptor-Mediated LAT Clusters That Control PLC-γ1 Activation and Cytokine Production.

    PubMed

    Bilal, Mahmood Yousif; Houtman, Jon C D

    2015-01-01

    GRB2 is a ubiquitously expressed adaptor protein required for signaling downstream of multiple receptors. To address the role of GRB2 in receptor-mediated signaling, the expression of GRB2 was suppressed in human CD4+ T cells and its role downstream of the T cell receptor (TCR) was examined. Interestingly, GRB2 deficient T cells had enhanced signaling from complexes containing the TCR. However, GRB2 deficient T cells had substantially reduced production of IL-2 and IFN-γ. This defect was attributed to diminished formation of linker for activation of T cells (LAT) signaling clusters, which resulted in reduced MAP kinase activation, calcium flux, and PLC-γ1 recruitment to LAT signaling clusters. Add back of wild-type GRB2, but not a novel N-terminal SH3 domain mutant, rescued LAT microcluster formation, calcium mobilization, and cytokine release, providing the first direct evidence that GRB2, and its ability to bind to SH3 domain ligands, is required for establishing LAT microclusters. Our data demonstrate that the ability of GRB2 to facilitate protein clusters is equally important in regulating TCR-mediated functions as its capacity to recruit effector proteins. This highlights that GRB2 regulates signaling downstream of adaptors and receptors by both recruiting effector proteins and regulating the formation of signaling complexes.

  15. 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.

  16. Receptor-mediated endocytosis for drug delivery in African trypanosomes: fulfilling Paul Ehrlich's vision of chemotherapy.

    PubMed

    Alsford, Sam; Field, Mark C; Horn, David

    2013-05-01

    Bloodstream-form cells of Trypanosoma brucei exhibit massively increased endocytic activity relative to the insect midgut stage, enabling rapid recycling of variant surface glycoprotein and antibody clearance from the surface. In addition, recent advances have identified a role for receptor-mediated endocytosis in the uptake of the antitrypanosomal drug, suramin, via invariant surface glycoprotein 75, and in the uptake of trypanosome lytic factor 1 via haptoglobin-haemoglobin receptor. Here, we argue that receptor-mediated endocytosis represents both a validated drug target and a promising route for the delivery of novel therapeutics into trypanosomes.

  17. Sphingosine 1-phosphate (S1P) receptor agonists mediate pro-fibrotic responses in normal human lung fibroblasts via S1P2 and S1P3 receptors and Smad-independent signaling.

    PubMed

    Sobel, Katrin; Menyhart, Katalin; Killer, Nina; Renault, Bérengère; Bauer, Yasmina; Studer, Rolf; Steiner, Beat; Bolli, Martin H; Nayler, Oliver; Gatfield, John

    2013-05-24

    Synthetic sphingosine 1-phosphate receptor 1 modulators constitute a new class of drugs for the treatment of autoimmune diseases. Sphingosine 1-phosphate (S1P) signaling, however, is also involved in the development of fibrosis. Using normal human lung fibroblasts, we investigated the induction of fibrotic responses by the S1P receptor (S1PR) agonists S1P, FTY720-P, ponesimod, and SEW2871 and compared them with the responses induced by the known fibrotic mediator TGF-β1. In contrast to TGF-β1, S1PR agonists did not induce expression of the myofibroblast marker α-smooth muscle actin. However, TGF-β1, S1P, and FTY720-P caused robust stimulation of extracellular matrix (ECM) synthesis and increased pro-fibrotic marker gene expression including connective tissue growth factor. Ponesimod showed limited and SEW2871 showed no pro-fibrotic potential in these readouts. Analysis of pro-fibrotic signaling pathways showed that in contrast to TGF-β1, S1PR agonists did not activate Smad2/3 signaling but rather activated PI3K/Akt and ERK1/2 signaling to induce ECM synthesis. The strong induction of ECM synthesis by the nonselective agonists S1P and FTY720-P was due to the stimulation of S1P2 and S1P3 receptors, whereas the weaker induction of ECM synthesis at high concentrations of ponesimod was due to a low potency activation of S1P3 receptors. Finally, in normal human lung fibroblast-derived myofibroblasts that were generated by TGF-β1 pretreatment, S1P and FTY720-P were effective stimulators of ECM synthesis, whereas ponesimod was inactive, because of the down-regulation of S1P3R expression in myofibroblasts. These data demonstrate that S1PR agonists are pro-fibrotic via S1P2R and S1P3R stimulation using Smad-independent pathways.

  18. Ligand Receptor-Mediated Regulation of Growth in Plants.

    PubMed

    Haruta, Miyoshi; Sussman, Michael R

    2017-01-01

    Growth and development of multicellular organisms are coordinately regulated by various signaling pathways involving the communication of inter- and intracellular components. To form the appropriate body patterns, cellular growth and development are modulated by either stimulating or inhibiting these pathways. Hormones and second messengers help to mediate the initiation and/or interaction of the various signaling pathways in all complex multicellular eukaryotes. In plants, hormones include small organic molecules, as well as larger peptides and small proteins, which, as in animals, act as ligands and interact with receptor proteins to trigger rapid biochemical changes and induce the intracellular transcriptional and long-term physiological responses. During the past two decades, the availability of genetic and genomic resources in the model plant species, Arabidopsis thaliana, has greatly helped in the discovery of plant hormone receptors and the components of signal transduction pathways and mechanisms used by these immobile but highly complex organisms. Recently, it has been shown that two of the most important plant hormones, auxin and abscisic acid (ABA), act through signaling pathways that have not yet been recognized in animals. For example, auxins stimulate cell elongation by bringing negatively acting transcriptional repressor proteins to the proteasome to be degraded, thus unleashing the gene expression program required for increasing cell size. The "dormancy" inducing hormone, ABA, binds to soluble receptor proteins and inhibits a specific class of protein phosphatases (PP2C), which activates phosphorylation signaling leading to transcriptional changes needed for the desiccation of the seeds prior to entering dormancy. While these two hormone receptors have no known animal counterparts, there are also many similarities between animal and plant signaling pathways. For example, in plants, the largest single gene family in the genome is the protein kinase

  19. A2A adenosine-receptor-mediated facilitation of noradrenaline release in rat tail artery involves protein kinase C activation and betagamma subunits formed after alpha2-adrenoceptor activation.

    PubMed

    Fresco, Paula; Oliveira, Jorge M A; Kunc, Filip; Soares, Ana Sofia; Rocha-Pereira, Carolina; Gonçalves, Jorge; Diniz, Carmen

    2007-07-01

    This work aimed to investigate the molecular mechanisms involved in the interaction of alpha2-adrenoceptors and adenosine A2A-receptor-mediated facilitation of noradrenaline release in rat tail artery, namely the type of G-protein involved in this effect and the step or steps where the signalling cascades triggered by alpha2-adrenoceptors and A2A-receptors interact. The selective adenosine A2A-receptor agonist 2-p-(2-carboxy ethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 100 nM) enhanced tritium overflow evoked by trains of 100 pulses at 5 Hz. This effect was abolished by the selective adenosine A2A-receptor antagonist 5-amino-7-(2-phenyl ethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine (SCH 58261; 20 nM) and by yohimbine (1 microM). CGS 21680-mediated effects were also abolished by drugs that disrupted G(i/o)-protein coupling with receptors, PTX (2 microg/ml) or NEM (40 microM), by the anti-G(salpha) peptide (2 microg/ml) anti-G(betagamma) peptide (10 microg/ml) indicating coupling of A2A-receptors to G(salpha) and suggesting a crucial role for G(betagamma) subunits in the A(2A)-receptor-mediated enhancement of tritium overflow. Furthermore, phorbol 12-myristate 13-acetate (PMA; 1 microM) or forskolin (1 microM), direct activators of protein kinase C and of adenylyl cyclase, respectively, also enhanced tritium overflow. In addition, PMA-mediated effects were not observed in the presence of either yohimbine or PTX. Results indicate that facilitatory adenosine A2A-receptors couple to G(salpha) subunits which is essential, but not sufficient, for the release facilitation to occur, requiring the involvement of G(i/o)-protein coupling (it disappears after disruption of G(i/o)-protein coupling, PTX or NEM) and/or G(betagamma) subunits (anti-G(betagamma)). We propose a mechanism for the interaction in study suggesting group 2 AC isoforms as a plausible candidate for the interaction site, as these isoforms can integrate inputs from G

  20. STAT1 Regulates the Homeostatic Component of Visual Cortical Plasticity via an AMPA Receptor-Mediated Mechanism

    PubMed Central

    Van Wart, Audra; Petravicz, Jeremy; Tropea, Daniela

    2014-01-01

    Accumulating evidence points to a role for Janus kinase/signal transducers and activators of transcription (STAT) immune signaling in neuronal function; however, its role in experience-dependent plasticity is unknown. Here we show that one of its components, STAT1, negatively regulates the homeostatic component of ocular dominance plasticity in visual cortex. After brief monocular deprivation (MD), STAT1 knock-out (KO) mice show an accelerated increase of open-eye responses, to a level comparable with open-eye responses after a longer duration of MD in wild-type (WT) mice. Therefore, this component of plasticity is abnormally enhanced in KO mice. Conversely, increasing STAT1 signaling by IFNγ treatment in WT mice reduces the homeostatic component of plasticity by impairing open-eye responses. Enhanced plasticity in KO mice is accompanied by sustained surface levels of GluA1 AMPA receptors and increased amplitude and frequency of AMPA receptor-mediated mEPSCs, which resemble changes in WT mice after a longer duration of MD. These results demonstrate a unique role for STAT1 during visual cortical plasticity in vivo through a mechanism that includes AMPA receptors. PMID:25080587

  1. NFAT regulates calcium-sensing receptor-mediated TNF production.

    PubMed

    Abdullah, Huda Ismail; Pedraza, Paulina L; Hao, Shoujin; Rodland, Karin D; McGiff, John C; Ferreri, Nicholas R

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca(2+) (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca(2+) were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  2. NFAT regulates calcium-sensing receptor-mediated TNF production

    SciTech Connect

    abdullah, huda ismail; Pedraza, Paulina L.; Hao, Shoujin; Rodland, Karin D.; McGiff, John C.; Ferreri, Nicholas R.

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca2+ (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca2+ were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  3. Receptor-mediated choreography of life and death.

    PubMed

    Bhardwaj, Anjana; Aggarwal, Bharat B

    2003-09-01

    The cytokine tumor necrosis factor was originally identified as a protein that kills tumor cells. So far, 18 distinct members of this family have been identified. All of them regulate cell survival, proliferation, differentiation, and cell death, also called apoptosis. The apoptosis induced by TNF, and other members of the family, for example, FasL, VEGI, and TRAIL is mediated through death receptors. The apoptotic signals by these cytokines are transduced by eight different death domain- (DD) containing receptors (TNFR1, also called DR1; Fas, also called DR2; DR3, DR4, DR5, DR6, NGFR, and EDAR). The intracellular portion of all these receptors contains a region approximately 80 amino acids long referred to as the "death domain." Upon activation by its ligand, the DD recruits various proteins that mediate both death and proliferation of the cells. These proteins in turn recruit other proteins via their DDs or death effector domains. The actual destruction of the cell, however, is accomplished by serial activation of a family of proteases referred to as caspases. Cell death is negatively regulated by a family of proteins that includes decoy receptors, silencer of DD, sentrin, cellular FLICE inhibitory protein, cellular inhibitors of apoptosis, and survivin. This review is an attempt to describe how these negative and positive players of cell death perform a harmonious dance with each other.

  4. Fuzzy C P2 spacetimes

    NASA Astrophysics Data System (ADS)

    Chaney, A.; Stern, A.

    2017-02-01

    Four-dimensional manifolds with changing signature are obtained by taking the large N limit of fuzzy C P2 solutions to a Lorentzian matrix model. The regions of Lorentzian signature give toy models of closed universes which exhibit cosmological singularities. These singularities are resolved at finite N , as the underlying C P2 solutions are expressed in terms of finite matrix elements.

  5. Food restriction increases NMDA receptor-mediated calcium-calmodulin kinase II and NMDA receptor/extracellular signal-regulated kinase 1/2-mediated cyclic amp response element-binding protein phosphorylation in nucleus accumbens upon D-1 dopamine receptor stimulation in rats.

    PubMed

    Haberny, S L; Carr, K D

    2005-01-01

    Biological drive states exert homeostatic control in part by increasing the reinforcing effects of environmental incentive stimuli. An apparent by-product of this adaptive response is the enhanced acquisition of drug self-administration behavior in food-restricted (FR) animals. While previous research has demonstrated increased central sensitivity to rewarding effects of abused drugs and direct dopamine (DA) receptor agonists in FR subjects, the underlying neurobiology is not well understood. Recently, it was demonstrated that intracerebroventricular (i.c.v.) injection of the D-1 DA receptor agonist, SKF-82958 produces a stronger activation of striatal extracellular signal-regulated kinase (ERK) 1/2 and cyclic AMP response element-binding protein (CREB) in FR relative to ad libitum (AL) fed rats. The main purpose of the present study was to characterize the involvement and mechanisms of interaction between NMDA receptor function and the augmented cellular responses to D-1 DA receptor stimulation in nucleus accumbens (NAc) of FR rats. In experiment 1, Western immunoblotting was used to demonstrate that i.c.v. injection of SKF-82958 (20 microg) produces greater phosphorylation of the NMDA NR1 subunit and calcium-calmodulin kinase II (CaMK II) in NAc of FR as compared with AL rats. In experiment 2, pretreatment of subjects with the NMDA antagonist, MK-801 (1.0 mg/kg, i.p.) decreased SKF-82958-induced activation of CaMK II, ERK1/2 and CREB, and reversed the augmenting effect of FR on activation of all three proteins. In experiment 3, pretreatment with the mitogen-activated protein kinase/ERK kinase inhibitor SL-327 (60 mg/kg, i.p.) suppressed SKF-82958- induced activation of ERK1/2 and reversed the augmenting effect of FR on CREB activation. These results point to specific neuroadaptations in the NAc of FR rats whereby D-1 DA receptor stimulation leads to increased NMDA NR1 subunit phosphorylation and consequent increases in NMDA receptor-dependent CaMK II and ERK1

  6. Cannabinoid CB1 receptor mediates glucocorticoid effects on hormone secretion induced by volume and osmotic changes.

    PubMed

    Ruginsk, S G; Uchoa, E T; Elias, L L K; Antunes-Rodrigues, J

    2012-02-01

    The present study provides the first in vivo evidence that the cannabinoid CB(1) receptor mediates the effects of dexamethasone on hormone release induced by changes in circulating volume and osmolality. Male adult rats were administered with the CB(1) receptor antagonist rimonabant (10 mg/Kg, p.o.), followed or not in 1 hour by dexamethasone (1 mg/Kg, i.p.). Extracellular volume expansion (EVE, 2 mL/100 g of body weight, i.v.) was performed 2 hours after dexamethasone or vehicle treatment using either isotonic (I-EVE, 0.15 mol/L) or hypertonic (H-EVE, 0.30 mol/L) NaCl solution. Five minutes after EVE, animals were decapitated and trunk blood was collected for all plasma measurements. Rimonabant potentiated oxytocin (OT) secretion induced by H-EVE and completely reversed the inhibitory effects of dexamethasone in response to the same stimulus. These data suggest that glucocorticoid modulation of OT release is mediated by the CB(1) receptor. Although dexamethasone did not affect vasopressin (AVP) secretion induced by H-EVE, the administration of rimonabant potentiated AVP release in response to the same stimulus, supporting the hypothesis that the CB(1) receptor regulates AVP secretion independently of glucocorticoid-mediated signalling. Dexamethasone alone did not affect atrial natriuretic peptide (ANP) release stimulated by I-EVE or H-EVE. However, pretreatment with rimonabant potentiated ANP secretion induced by H-EVE, suggesting a possible role for the CB(1) receptor in the control of peripheral factors that modulate cardiovascular function. Rimonabant also reversed the inhibitory effects of dexamethasone on H-EVE-induced corticosterone secretion, reinforcing the hypothesis that the CB(1) receptor may be involved in the negative feedback exerted by glucocorticoids on the activity of the hypothalamic-pituitary-adrenal axis. Collectively, the results of the present study indicate that the CB(1) receptor modulates neurohypophyseal hormone secretion and

  7. Tracking Progesterone Receptor-Mediated Actions in Breast Cancer

    PubMed Central

    Knutson, Todd P.; Lange, Carol A.

    2014-01-01

    Ovarian steroid hormones contribute to breast cancer initiation and progression primarily through the actions of their nuclear transcription factors, the estrogen receptor alpha (ERα) and progesterone receptors (PRs). These receptors are important drivers of the luminal A and B subtypes of breast cancer, where estrogen-blocking drugs have been effective endocrine therapies for patients with these tumors. However, many patients do not respond, or become resistant to treatment. When endocrine therapies fail, the luminal subtypes of breast cancer are more difficult to treat because these subtypes are among the most heterogeneous in terms of mutation diversity and gene expression profiles. Recent evidence suggests that progestin and PR actions may be important drivers of luminal breast cancers. Clinical trial data has demonstrated that hormone replacement therapy with progestins drives invasive breast cancer and results in greater mortality. PR transcriptional activity is dependent upon cross-talk with growth factor signaling pathways that alter PR phosphorylation, acetylation, or SUMOylation as mechanisms for regulating PR target gene selection required for increased cell proliferation and survival. Site-specific PR phosphorylation is the primary driver of gene-selective PR transcriptional activity. However, PR phosphorylation and heightened transcriptional activity is coupled to rapid PR protein degradation; the range of active PR detected in tumors is likely to be dynamic. Thus, PR target gene signatures may provide a more accurate means of tracking PR’s contribution to tumor progression rather than standard clinical protein-based (IHC) assays. Further development of antiprogestin therapies should be considered along side antiestrogens and aromatase inhibitors. PMID:24291072

  8. Role of zinc influx via AMPA/kainate receptor activation in metabotropic glutamate receptor-mediated calcium release.

    PubMed

    Takeda, Atsushi; Fuke, Sayuri; Minami, Akira; Oku, Naoto

    2007-05-01

    The uptake of free zinc into CA3 pyramidal cells and its significance was examined in rat hippocampal slices with ZnAF-2DA, a membrane-permeable zinc indicator. Intracellular ZnAF-2 signal in the CA3 pyramidal cell layer was increased during delivery of tetanic stimuli to the dentate granule cell layer. This increase was completely blocked in the presence of CNQX, an AMPA/kainate receptor antagonist. These results suggest that free zinc is taken up into CA3 pyramidal cells via activation of AMPA/kainate receptors. The effect of free zinc levels in the CA3 pyramidal cells on the increase in intracellular calcium via Group I metabotropic glutamate receptors was examined by regional delivery of tADA, a Group I metabotropic glutamate receptor agonist, to the stratum lucidum after blockade of AMPA/kainate receptor-mediated calcium and zinc influx. Intracellular calcium orange signal in the CA3 pyramidal cell layer was increased by tADA, whereas intracellular ZnAF-2 signal was not increased even in the presence of 100 muM zinc, suggesting that tADA induces calcium release from internal stores in CA3 pyramidal cells and is not involved in zinc uptake. The increase in calcium orange signal by tADA was enhanced by perfusion with pyrithione, a zinc ionophore that decreased basal ZnAF-2 signal in the CA3 pyramidal cell layer. It was blocked by perfusion with pyrithione and zinc that increased basal ZnAF-2 signal. The present study indicates that the increase in free calcium levels via the metabotropic glutamate receptor pathway is inversely related to free zinc levels in CA3 pyramidal cells.

  9. Ethanol inhibits epileptiform activity and NMDA receptor-mediated synaptic transmission in rat amygdaloid slices

    SciTech Connect

    Gean, P.W. )

    1992-02-26

    The effect of ethanol on the epileptiform activity induced by Mg{sup ++}-free solution was studied in rat amygdalar slices using intracellular recording techniques. The spontaneous and evoked epileptiform discharges consisting of an initial burst followed by afterdischarges were observed 20-30 min after switching to Mg{sup ++}-free medium. Superfusion with ethanol reversibly reduced the duration of spontaneous and evoked bursting discharges in a concentration-dependent manner. Synaptic response mediated by N-methyl-D-aspartate (NMDA) receptor activation was isolated by application of a solution containing the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and either in Mg{sup ++}-free solution or in the presence of 50 {mu}M bicuculline. Application of ethanol reversibly suppressed the duration of NMDA receptor-mediated synaptic response. These results suggest that intoxicating concentrations of ethanol possess anticonvulsant activity through blocking the NMDA receptor-mediated synaptic excitation.

  10. Menthol enhances phasic and tonic GABAA receptor-mediated currents in midbrain periaqueductal grey neurons

    PubMed Central

    Lau, Benjamin K; Karim, Shafinaz; Goodchild, Ann K; Vaughan, Christopher W; Drew, Geoffrey M

    2014-01-01

    Background and Purpose Menthol, a naturally occurring compound in the essential oil of mint leaves, is used for its medicinal, sensory and fragrant properties. Menthol acts via transient receptor potential (TRPM8 and TRPA1) channels and as a positive allosteric modulator of recombinant GABAA receptors. Here, we examined the actions of menthol on GABAA receptor-mediated currents in intact midbrain slices. Experimental Approach Whole-cell voltage-clamp recordings were made from periaqueductal grey (PAG) neurons in midbrain slices from rats to determine the effects of menthol on GABAA receptor-mediated phasic IPSCs and tonic currents. Key Results Menthol (150–750 μM) produced a concentration-dependent prolongation of spontaneous GABAA receptor-mediated IPSCs, but not non-NMDA receptor-mediated EPSCs throughout the PAG. Menthol actions were unaffected by TRPM8 and TRPA1 antagonists, tetrodotoxin and the benzodiazepine antagonist, flumazenil. Menthol also enhanced a tonic current, which was sensitive to the GABAA receptor antagonists, picrotoxin (100 μM), bicuculline (30 μM) and Zn2+ (100 μM), but unaffected by gabazine (10 μM) and a GABAC receptor antagonist, 1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA; 50 μM). In addition, menthol potentiated currents induced by the extrasynaptic GABAA receptor agonist THIP/gaboxadol (10 μM). Conclusions and Implications These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABAA receptors in native PAG neurons. The development of agents that potentiate GABAA-mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABAA-related pharmacotherapies. PMID:24460753

  11. Nanoscale imaging and mechanical analysis of Fc receptor-mediated macrophage phagocytosis against cancer cells.

    PubMed

    Li, Mi; Liu, Lianqing; Xi, Ning; Wang, Yuechao; Xiao, Xiubin; Zhang, Weijing

    2014-02-18

    Fc receptor-mediated macrophage phagocytosis against cancer cells is an important mechanism in the immune therapy of cancers. Traditional research about macrophage phagocytosis was based on optical microscopy, which cannot reveal detailed information because of the 200-nm-resolution limit. Quantitatively investigating the macrophage phagocytosis at micro- and nanoscale levels is still scarce. The advent of atomic force microscopy (AFM) offers an excellent analytical instrument for quantitatively investigating the biological processes at single-cell and single-molecule levels under native conditions. In this work, we combined AFM and fluorescence microscopy to visualize and quantify the detailed changes in cell morphology and mechanical properties during the process of Fc receptor-mediated macrophage phagocytosis against cancer cells. Lymphoma cells were discernible by fluorescence staining. Then, the dynamic process of phagocytosis was observed by time-lapse optical microscopy. Next, AFM was applied to investigate the detailed cellular behaviors during macrophage phagocytosis under the guidance of fluorescence recognition. AFM imaging revealed the distinct features in cellular ultramicrostructures for the different steps of macrophage phagocytosis. AFM cell mechanical property measurements indicated that the binding of cancer cells to macrophages could make macrophages become stiffer. The experimental results provide novel insights in understanding the Fc-receptor-mediated macrophage phagocytosis.

  12. Mannose receptor-mediated gene delivery into antigen presenting dendritic cells.

    PubMed

    Diebold, Sandra S; Plank, Christian; Cotten, Matt; Wagner, Ernst; Zenke, Martin

    2002-11-01

    Dendritic cells are professional antigen presenting cells and are unique in their ability to prime naïve T cells. Gene modification of dendritic cells is of particular interest for immunotherapy of diseases where the immune system has failed or is aberrantly regulated, such as in cancer or autoimmune disease, respectively. Dendritic cells abundantly express mannose receptor and mannose receptor-related receptors, and receptor-mediated gene transfer via mannose receptor offers a versatile tool for targeted gene delivery into these cells. Accordingly, mannose polyethylenimine DNA transfer complexes were generated and used for gene delivery into dendritic cells. Mannose receptor belongs to the group of scavenger receptors that allow dendritic cells to take up pathogenic material, which is directed for degradation and MHC class II presentation. Therefore, a limiting step of transgene expression by mannose receptor-mediated gene delivery is endosomal degradation of DNA. Several strategies have been explored to overcome this limitation including the addition of endosomolytic components to DNA transfer complexes like adenovirus particles and influenza peptides. Here, we review the current understanding of mannose receptor-mediated gene delivery into dendritic cells and discuss strategies to identify appropriate endosomolytic agents to improve DNA transfer efficacy.

  13. Slowly developing depression of N-methyl-D-aspartate receptor mediated responses in young rat hippocampi

    PubMed Central

    Dozmorov, Mikhail; Li, Rui; Xu, Hui-Ping; Jilderos, Barbro; Wigström, Holger

    2004-01-01

    Background Activation of N-methyl-D-aspartate (NMDA) type glutamate receptors is essential in triggering various forms of synaptic plasticity. A critical issue is to what extent such plasticity involves persistent changes of glutamate receptor subtypes and many prior studies have suggested a main role for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors in mediating the effect. Our previous work in hippocampal slices revealed that, under pharmacological unblocking of NMDA receptors, both AMPA and NMDA receptor mediated responses undergo a slowly developing depression. In the present study we have further adressed this phenomenon, focusing on the contribution via NMDA receptors. Pharmacologically isolated NMDA receptor mediated excitatory postsynaptic potentials (EPSPs) were recorded for two independent synaptic pathways in CA1 area using perfusion with low Mg2+ (0.1 mM) to unblock NMDA receptors. Results Following unblocking of NMDA receptors, there was a gradual decline of NMDA receptor mediated EPSPs for 2–3 hours towards a stable level of ca. 60–70 % of the maximal size. If such an experimental session was repeated twice in the same pathway with a period of NMDA receptor blockade in between, the depression attained in the first session was still evident in the second one and no further decay occurred. The persistency of the depression was also validated by comparison between pathways. It was found that the responses of a control pathway, unstimulated in the first session of receptor unblocking, behaved as novel responses when tested in association with the depressed pathway under the second session. In similar experiments, but with AP5 present during the first session, there was no subsequent difference between NMDA EPSPs. Conclusions Our findings show that merely evoking NMDA receptor mediated responses results in a depression which is input specific, induced via NMDA receptor activation, and is maintained for several hours through

  14. Regulation of muscarinic acetylcholine receptor-mediated synaptic responses by adenosine receptors in the rat hippocampus.

    PubMed Central

    Morton, R A; Davies, C H

    1997-01-01

    1. Intracellular current clamp recordings were made from CA1 pyramidal neurones in rat hippocampal slices. Experiments were performed in the presence of ionotropic glutamate receptor antagonists and gamma-aminobutyric acid (GABA) receptor antagonists to block all fast excitatory and inhibitory synaptic transmission. A single stimulus, delivered extracellularly in the stratum oriens, caused a reduction in spike frequency adaptation in response to a depolarizing current step delivered 2 s after the stimulus. A 2- to 10-fold increase in stimulus intensity evoked a slow excitatory postsynaptic potential (EPSP) which was associated with a small increase in input resistance. The peak amplitude of the EPSP occurred approximately 2.5 s after the stimulus and its magnitude (up to 30 mV) and duration (10-50 s) increased with increasing stimulus intensity. 2. The slow EPSP was unaffected by the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG; 1000 microM) but was greatly enhanced by the acetylcholinesterase inhibitor physostigmine (1-5 microM). Both the slow EPSP and the stimulus-evoked reduction in spike frequency adaptation were inhibited by the muscarinic acetylcholine receptor (mAChR) antagonist atropine (1-5 microM). These results are consistent with these effects being mediated by mAChRs. 3. Both the mAChR-mediated EPSP (EPSPm) and the associated reduction in spike frequency adaptation were reversibly depressed (up to 97%) by either adenosine (100 microM) or its non-hydrolysable analogue 2-chloroadenosine (CADO; 0.1-5.0 microM). These effects were often accompanied by postsynaptic hyperpolarization (up to 8 mV) and a reduction in input resistance (up to 11%). The selective adenosine A1 receptor agonists 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1-0.4 microM) and R(-)N6-(2-phenylisopropyl)-adenosine (R-PIA; 1 microM) both depressed the EPSPm. In contrast, the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5

  15. 5-HT2 receptors mediate functional modulation of GABAa receptors and inhibitory synaptic transmissions in human iPS-derived neurons

    PubMed Central

    Wang, Haitao; Hu, Lingli; Liu, Chunhua; Su, Zhenghui; Wang, Lihui; Pan, Guangjin; Guo, Yiping; He, Jufang

    2016-01-01

    Neural progenitors differentiated from induced pluripotent stem cells (iPS) hold potentials for treating neurological diseases. Serotonin has potent effects on neuronal functions through multiple receptors, underlying a variety of neural disorders. Glutamate and GABA receptors have been proven functional in neurons differentiated from iPS, however, little is known about 5-HT receptor-mediated modulation in such neuronal networks. In the present study, human iPS were differentiated into cells possessing featured physiological properties of cortical neurons. Whole-cell patch-clamp recording was used to examine the involvement of 5-HT2 receptors in functional modulation of GABAergic synaptic transmission. We found that serotonin and DOI (a selective agonist of 5-HT2A/C receptor) reversibly reduced GABA-activated currents, and this 5-HT2A/C receptor mediated inhibition required G protein, PLC, PKC, and Ca2+ signaling. Serotonin increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs), which could be mimicked by α-methylserotonin, a 5-HT2 receptor agonist. In contrast, DOI reduced both frequency and amplitude of mIPSCs. These findings suggested that in iPS-derived human neurons serotonin postsynaptically reduced GABAa receptor function through 5-HT2A/C receptors, but presynaptically other 5-HT2 receptors counteracted the action of 5-HT2A/C receptors. Functional expression of serotonin receptors in human iPS-derived neurons provides a pre-requisite for their normal behaviors after grafting. PMID:26837719

  16. Role of NMDA Receptor-Mediated Glutamatergic Signaling in Chronic and Acute Neuropathologies

    PubMed Central

    2016-01-01

    N-Methyl-D-aspartate receptors (NMDARs) have two opposing roles in the brain. On the one hand, NMDARs control critical events in the formation and development of synaptic organization and synaptic plasticity. On the other hand, the overactivation of NMDARs can promote neuronal death in neuropathological conditions. Ca2+ influx acts as a primary modulator after NMDAR channel activation. An imbalance in Ca2+ homeostasis is associated with several neurological diseases including schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. These chronic conditions have a lengthy progression depending on internal and external factors. External factors such as acute episodes of brain damage are associated with an earlier onset of several of these chronic mental conditions. Here, we will review some of the current evidence of how traumatic brain injury can hasten the onset of several neurological conditions, focusing on the role of NMDAR distribution and the functional consequences in calcium homeostasis associated with synaptic dysfunction and neuronal death present in this group of chronic diseases. PMID:27630777

  17. Ran GTPase promotes cancer progression via Met receptor-mediated downstream signaling

    PubMed Central

    Yuen, Hiu-Fung; Chan, Ka-Kui; Platt-Higgins, Angela; Dakir, El-Habib; Matchett, Kyle B.; Haggag, Yusuf Ahmed; Jithesh, Puthen V.; Habib, Tanwir; Faheem, Ahmed; Dean, Fennell A.; Morgan, Richard; Rudland, Philip S.; El-Tanani, Mohamed

    2016-01-01

    It has been shown previously that cancer cells with an activated oncogenic pathway, including Met activation, require Ran for growth and survival. Here, we show that knockdown of Ran leads to a reduction of Met receptor expression in several breast and lung cancer cell lines. This, in turn suppressed HGF expression and the Met-mediated activation of the Akt pathway, as well as cell adhesion, migration, and invasion. In a cell line model where Met amplification has previously been shown to contribute to gefitinib resistance, Ran knockdown sensitized cells to gefitinib-mediated inhibition of Akt and ERK1/2 phosphorylation and consequently reduced cell proliferation. We further demonstrate that Met reduction-mediated by knockdown of Ran, occurs at the post-transcriptional level, probably via a matrix metalloproteinase. Moreover, the level of immunoreactive Ran and Met are positively associated in human breast cancer specimens, suggesting that a high level of Ran may be a pre-requisite for Met overexpression. Interestingly, a high level of immunoreactive Ran dictates the prognostic significance of Met, indicating that the co-overexpression of Met and Ran may be associated with cancer progression and could be used in combination as a prognostic indicator. PMID:27716616

  18. GABA(B) receptors mediate motility signals for migrating embryonic cortical cells.

    PubMed

    Behar, T N; Smith, S V; Kennedy, R T; McKenzie, J M; Maric, I; Barker, J L

    2001-08-01

    During development, postmitotic neurons migrate from germinal regions into the cortical plate (cp), where lamination occurs. In rats, GABA is transiently expressed in the cp, near target destinations for migrating neurons. In vitro GABA stimulates neuronal motility, suggesting cp cells release GABA, which acts as a chemoattractant during corticogenesis. Pharmacological studies indicate GABA stimulates migration via GABA(B)-receptor (GABA(B)-R) activation. Using immunohistochemistry, RT-PCR and Western blotting, we examined embryonic cortical cell expression of GABA(B)-Rs in vivo. At E17, GABA(B)-R1(+) cells were identified in the ventricular zone (vz) and cp. RT-PCR and Western blotting demonstrated the presence of GABA(B)-R1a and GABA(B)-R1b mRNA and proteins. Using immuno- cytochemistry, GABA(B)-R expression was examined in vz and cp cell dissociates before and after migration to GABA in an in vitro chemotaxis assay. GABA-induced migration resulted in an increase of GABA(B)-R(+) cells in the migrated population. While <20% of each starting dissociate was GABA(B)-R(+), >70% of migrated cells were immunopositive. We used a microchemotaxis assay to analyze cp cell release of diffusible chemotropic factor(s). In vitro, cp dissociates induced vz cell migration in a cell density-dependent manner that was blocked by micromolar saclofen (a GABA(B)-R antagonist). HPLC demonstrated cp cells release micromolar levels of GABA and taurine in several hours. Micromolar levels of both molecules stimulated cell migration that was blocked by micromolar saclofen. Thus, migratory cortical cells express GABA(B)-Rs, cp cells release GABA and taurine, and both molecules stimulate cortical cell movement. Together these findings suggest GABA and/or taurine act as chemoattractants for neurons during rat cortical histogenesis via mechanisms involving GABA(B)-Rs.

  19. A whole-genome RNAi screen identifies an 8q22 gene cluster that inhibits death receptor-mediated apoptosis.

    PubMed

    Dompe, Nicholas; Rivers, Celina Sanchez; Li, Li; Cordes, Shaun; Schwickart, Martin; Punnoose, Elizabeth A; Amler, Lukas; Seshagiri, Somasekar; Tang, Jerry; Modrusan, Zora; Davis, David P

    2011-10-25

    Deregulation of apoptosis is a common occurrence in cancer, for which emerging oncology therapeutic agents designed to engage this pathway are undergoing clinical trials. With the aim of uncovering strategies to activate apoptosis in cancer cells, we used a pooled shRNA screen to interrogate death receptor signaling. This screening approach identified 16 genes that modulate the sensitivity to ligand induced apoptosis, with several genes exhibiting frequent overexpression and/or copy number gain in cancer. Interestingly, two of the top hits, EDD1 and GRHL2, are found 50 kb apart on chromosome 8q22, a region that is frequently amplified in many cancers. By using a series of silencing and overexpression studies, we show that EDD1 and GRHL2 suppress death-receptor expression, and that EDD1 expression is elevated in breast, pancreas, and lung cancer cell lines resistant to death receptor-mediated apoptosis. Supporting the relevance of EDD1 and GRHL2 as therapeutic candidates to engage apoptosis in cancer cells, silencing the expression of either gene sensitizes 8q22-amplified breast cancer cell lines to death receptor induced apoptosis. Our findings highlight a mechanism by which cancer cells may evade apoptosis, and therefore provide insight in the search for new targets and functional biomarkers for this pathway.

  20. Functional interaction of heat shock protein 90 and Beclin 1 modulates Toll-like receptor-mediated autophagy

    PubMed Central

    Xu, Congfeng; Liu, Jin; Hsu, Li-Chung; Luo, Yunping; Xiang, Rong; Chuang, Tsung-Hsien

    2011-01-01

    Autophagy is one of the downstream effector mechanisms for elimination of intracellular microbes following activation of the Toll-like receptors (TLRs). Although the detailed molecular mechanism for this cellular process is still unclear, Beclin 1, a key molecule for autophagy, has been suggested to play a role. Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the stability of signaling proteins. Herein, we show that Hsp90 forms a complex with Beclin 1 through an evolutionarily conserved domain to maintain the stability of Beclin 1. In monocytic cells, geldanamycin (GA), an Hsp90 inhibitor, effectively promoted proteasomal degradation of Beclin 1 in a concentration-dependent (EC50 100 nM) and time-dependent (t50 2 h) manner. In contrast, KNK437/Hsp inhibitor I had no effect. Hsp90 specifically interacted with Beclin 1 but not with other adapter proteins in the TLR signalsome. Treatment of cells with GA inhibited TLR3- and TLR4-mediated autophagy. In addition, S. typhimurium infection-induced autophagy was blocked by GA treatment. This further suggested a role of the Hsp90/Beclin 1 in controlling autophagy in response to microbial infections. Taken together, our data revealed that by maintaining the homeostasis of Beclin 1, Hsp90 plays a novel role in TLR-mediated autophagy.—Xu, C., Liu, J., Hsu, L. -C., Luo, Y., Xiang, R., Chuang, T. -H. Functional interaction of Hsp90 and Beclin 1 modulates Toll-like receptor-mediated autophagy. PMID:21543763

  1. P2 receptors activated by uracil nucleotides--an update.

    PubMed

    Brunschweiger, Andreas; Müller, Christa E

    2006-01-01

    Pyrimidine nucleotides, including UTP, UDP and UDP-glucose, are important signaling molecules which activate G protein-coupled membrane receptors (GPCRs) of the P2Y family. Four distinct pyrimidine nucleotide-sensitive P2Y receptor subtypes have been cloned, P2Y2, P2Y4, P2Y6 and P2Y14. P2Y2 and P2Y4 receptors are activated by UTP (the P2Y2, and the rat but not the human P2Y4 receptor are also activated by ATP), the P2Y6 receptor is activated by UDP, and the P2Y14 receptor by UDP-glucose. Furthermore, non-P2Y GPCRs, the cysteinylleukotriene receptors (CysLT1R and CysLT2R) have been described to be activated by UDP in addition to activation by cysteinylleukotrienes. While P2Y2, P2Y4, and P2Y6 receptor activation results in stimulation of phospholipase C, the P2Y14 receptor is coupled to inhibition of adenylate cyclase. Derivatives and analogs of the physiological nucleotides UTP, UDP and ATP have been synthesized and evaluated in order to obtain enzymatically stable, subtype-selective agonists. The P2Y2 receptor agonists diuridine tetraphosphate (diquafosol) and the uracil-cytosine dinucleotide denufosol are currently undergoing clinical trials for dry eye disease, retinal detachment disease, upper respiratory tract symptoms, and cystic fibrosis, respectively. The first antagonists for P2Y2 and P2Y6 receptors that appear to be selective versus other P2Y receptor subtypes have recently been described. Selective antagonists for P2Y4 and P2Y14 receptors are still lacking. Uracil nucleotide-sensitive P2Y receptor subtypes may constitute future targets for the treatment of certain cancer types, vascular diseases, inflammatory diseases, and immunomodulatory intervention. They have also been proposed to play a role in neurodegenerative diseases. This article is an updated version of "P2-Pyrimidinergic Receptors and Their Ligands" by C. E. Müller published in Curr. Pharm. Des. 2002, 8, 2353-2369.

  2. A1 and A2a receptors mediate inhibitory effects of adenosine on the motor activity of human colon.

    PubMed

    Fornai, M; Antonioli, L; Colucci, R; Ghisu, N; Buccianti, P; Marioni, A; Chiarugi, M; Tuccori, M; Blandizzi, C; Del Tacca, M

    2009-04-01

    Experimental evidence in animal models suggests that adenosine is involved in the regulation of digestive functions. This study examines the influence of adenosine on the contractile activity of human colon. Reverse transcription-polymerase chain reaction revealed A(1) and A(2a) receptor expression in colonic neuromuscular layers. Circular muscle preparations were connected to isotonic transducers to determine the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A(1) receptor antagonist), ZM 241385 (A(2a) receptor antagonist), CCPA (A(1) receptor agonist) and 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamide-adenosine (CGS 21680; A(2a) receptor agonist) on motor responses evoked by electrical stimulation or carbachol. Electrically evoked contractions were enhanced by DPCPX and ZM 241385, and reduced by CCPA and CGS 21680. Similar effects were observed when colonic preparations were incubated with guanethidine (noradrenergic blocker), L-732,138, GR-159897 and SB-218795 (NK receptor antagonists). However, in the presence of guanethidine, NK receptor antagonists and N(omega)-propyl-L-arginine (NPA; neuronal nitric oxide synthase inhibitor), the effects of DPCPX and CCPA were still evident, while those of ZM 241385 and CGS 21680 no longer occurred. Carbachol-induced contractions were unaffected by A(2a) receptor ligands, but they were enhanced or reduced by DPCPX and CCPA, respectively. When colonic preparations were incubated with guanethidine, NK antagonists and atropine, electrically induced relaxations were partly reduced by ZM 241385 or NPA, but unaffected by DPCPX. Dipyridamole or application of exogenous adenosine reduced electrically and carbachol-evoked contractions, whereas adenosine deaminase enhanced such motor responses. In conclusion, adenosine exerts an inhibitory control on human colonic motility. A(1) receptors mediate direct modulating actions on smooth muscle, whereas A(2a) receptors operate through inhibitory nitrergic nerve pathways.

  3. Chloride transporters and receptor-mediated endocytosis in the renal proximal tubule

    PubMed Central

    Devuyst, Olivier; Luciani, Alessandro

    2015-01-01

    Abstract The epithelial cells lining the proximal tubules of the kidney reabsorb a large amount of filtered ions and solutes owing to receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Dedifferentiation of proximal tubule cells and dysfunction of receptor-mediated endocytosis characterize Dent’s disease, a rare disorder caused by inactivating mutations in the CLCN5 gene that encodes the endosomal chloride–proton exchanger, ClC-5. The disease is characterized by a massive urinary loss of solutes (renal Fanconi syndrome), with severe metabolic complications and progressive renal failure. Investigations of mutations affecting the gating of ClC-5 revealed that the proximal tubule dysfunction may occur despite normal endosomal acidification. In addition to defective endocytosis, proximal tubule cells lacking ClC-5 show a trafficking defect in apical receptors and transporters, as well as lysosomal dysfunction and typical features of dedifferentiation, proliferation and oxidative stress. A similar but milder defect is observed in mouse models with defective CFTR, a chloride channel that is also expressed in the endosomes of proximal tubule cells. These data suggest a major role for endosomal chloride transport in the maintenance of epithelial differentiation and reabsorption capacity of the renal proximal tubule. Key points The reabsorptive activity of renal proximal tubule cells is mediated by receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Loss-of-function mutations of the endosomal chloride–proton exchanger ClC-5 (Dent’s disease) cause a major trafficking defect in proximal tubule cells, associated with lysosomal dysfunction, oxidative stress and dedifferentiation/proliferation. A similar but milder defect is associated with mutations in CFTR (cystic fibrosis transmembrane conductance regulator). Vesicular chloride transport appears to be important for

  4. Investigations of receptor-mediated phagocytosis by hormone-induced (imprinted) Tetrahymena pyriformis.

    PubMed

    Kovács, P; Sundermann, C A; Csaba, G

    1996-08-15

    Receptor-mediated endocytosis by Tetrahvmena pyriformis was studied using tetramethylrhodamine isothiocyanate-labeled concanavalin A (TRITC-Con A) with fluorescence and confocal microscopy. In the presence of insulin, or 24 h after insulin pretreatment (hormonal imprinting), the binding and uptake of TRITC-Con A increased when compared to controls, owing to the binding of TRITC-Con A to sugar oligomers of insulin receptors. Mannose inhibited the binding of Con A, thus demonstrating the specificity of binding. Histamine, a phagocytosis-promoting factor in mammals and Tetrahymena, and galactose, did not influence the uptake of TRITC-Con A.

  5. Receptor-Mediated Drug Delivery to Macrophages in Chemotherapy of Leishmaniasis

    NASA Astrophysics Data System (ADS)

    Mukhopadhyay, Amitabha; Chaudhuri, Gautam; Arora, Sunil K.; Sehgal, Shobha; Basu, Sandip K.

    1989-05-01

    Methotrexate coupled to maleylated bovine serum albumin was taken up efficiently through the ``scavenger'' receptors present on macrophages and led to selective killing of intracellular Leishmania mexicana amazonensis amastigotes in cultured hamster peritoneal macrophages. The drug conjugate was nearly 100 times as effective as free methotrexate in eliminating the intracellular parasites. Furthermore, in a model of experimental cutaneous leishmaniasis in hamsters, the drug conjugate brought about more than 90% reduction in the size of footpad lesions within 11 days. In contrast, the free drug at a similar concentration did not significantly affect lesion size. These studies demonstrate the potential of receptor-mediated drug delivery in the therapy of macrophage-associated diseases.

  6. Expression, assembly and function of novel C-terminal truncated variants of the mouse P2X7 receptor: re-evaluation of P2X7 knockouts

    PubMed Central

    Masin, Marianela; Young, Christopher; Lim, KoiNi; Barnes, Sara J; Xu, Xing Jian; Marschall, Viola; Brutkowski, Wojciech; Mooney, Elizabeth R; Gorecki, Dariusz C; Murrell-Lagnado, Ruth

    2012-01-01

    BACKGROUND AND PURPOSE Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor-mediated responses. Here, we investigated expression and function of C-terminal truncated (ΔC) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7−/− mice (Pfizer KO). EXPERIMENTAL APPROACH Expression in wild-type (WT) and Pfizer KO tissue was investigated by reverse transcription (RT)-PCR and Western blot analysis. ΔC variants were also cloned and expressed in HEK293 cells to investigate their assembly, trafficking and function. KEY RESULTS RT-PCR indicates expression of a ΔC splice variant in brain, salivary gland (SG) and spleen from WT and Pfizer KO mice. An additional ΔC hybrid transcript, containing sequences of P2X7 upstream of exon 12, part of exon 13 followed in-frame by the sequence of the vector used to disrupt the P2X7 gene, was also identified in the KO mice. By blue native (BN) PAGE analysis and the use of cross linking reagents followed by SDS-PAGE, P2X7 trimers, dimers and monomers were detected in the spleen and SG of Pfizer KO mice. The molecular mass was reduced compared with P2X7 in WT mice tissue, consistent with a ΔC variant. When expressed in HEK293 cells the ΔC variants were inefficiently trafficked to the cell surface and agonist-evoked whole cell currents were small. Co-expressed with P2X7A, the ΔC splice variant acted in a dominant negative fashion to inhibit function. CONCLUSIONS AND IMPLICATIONS Pfizer KO mice are not null for P2X7 receptor expression but express ΔC variants with reduced function. PMID:21838754

  7. Maternal separation increases GABA(A) receptor-mediated modulation of norepinephrine release in the hippocampus of a rat model of ADHD, the spontaneously hypertensive rat.

    PubMed

    Sterley, Toni-Lee; Howells, Fleur M; Russell, Vivienne A

    2013-02-25

    Experiencing early life stress increases the risk of developing a psychiatric disorder later in life, possibly by altering neural networks, such as the locus-coeruleus norepinephrine (LC-NE) system. Whether early life stress affects the LC-NE system directly, or whether the effects are via changes in glutamate and GABA modulation of the LC-NE system, is unclear. Early life stress has been shown to alter glutamate and GABA transmission, and in particular, to alter GABA(A) receptor expression. The LC-NE system has been implicated in attention-deficit/hyperactivity disorder (ADHD), amongst other disorders, and is over-responsive to glutamate stimulation in a validated rat model of ADHD, the spontaneously hypertensive rat (SHR). It is plausible that the LC-NE system, or glutamate and GABA modulation thereof, in an individual already genetically predisposed to develop ADHD, or in SHR, may respond in a unique way to early life stress. To investigate this we applied a mild developmental stressor, maternal separation, onto SHR, and onto their control strain, Wistar-Kyoto rats (WKY), from post-natal day (P)2-14. On P50-52, in early adulthood, we assayed glutamate and potassium stimulated release of radio-actively labelled NE ((3)[H]NE) from hippocampal slices using an in vitro superfusion technique, in the presence or absence of a GABA(A) receptor antagonist, bicuculline. Our results show that maternal separation altered GABA(A) receptor-mediated modulation of NE release in the hippocampus of the two strains in opposite directions, increasing it in SHR and decreasing it in WKY. Our findings indicate that effects of early life stress are highly dependent on genetic predisposition, since opposite changes in GABA(A) receptor-mediated modulation of NE release were observed in the rat model of ADHD, SHR, and their control strain, WKY.

  8. Extracellular α-synuclein induces sphingosine 1-phosphate receptor subtype 1 uncoupled from inhibitory G-protein leaving β-arrestin signal intact

    PubMed Central

    Zhang, Lifang; Okada, Taro; Badawy, Shaymaa Mohamed Mohamed; Hirai, Chihoko; Kajimoto, Taketoshi; Nakamura, Shun-ichi

    2017-01-01

    Parkinson’s disease (PD) is the second most common neurodegenerative disorder. The presence of α-synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, is the cytopathological hallmark of PD. Increasing bodies of evidence suggest that cell-to-cell transmission of α-Syn plays a role in the progression of PD. Although extracellular α-Syn is known to cause abnormal cell motility, the precise mechanism remains elusive. Here we show that impairment of platelet-derived growth factor-induced cell motility caused by extracellular α-Syn is mainly attributed to selective inhibition of sphingosine 1-phosphate (S1P) signalling. Treatment of human neuroblastoma cells with recombinant α-Syn caused S1P type 1 (S1P1) receptor-selective uncoupling from inhibitory G-protein (Gi) as determined by both functional and fluorescence resonance energy transfer (FRET)-based structural analyses. By contrast, α-Syn caused little or no effect on S1P2 receptor-mediated signalling. Both wild-type and α-Syn(A53T), a mutant found in familiar PD, caused uncoupling of S1P1 receptor, although α-Syn(A53T) showed stronger potency in uncoupling. Moreover, S1P1 receptor-mediated β-arrestin signal was unaltered by α-Syn(A53T). These results suggest that exogenous α-Syn modulates S1P1 receptor-mediated signalling from both Gi and β-arrestin signals into β-arrestin-biased signal. These findings uncovered a novel function of exogenous α-Syn in the cells. PMID:28300069

  9. The miR-199-dynamin regulatory axis controls receptor-mediated endocytosis.

    PubMed

    Aranda, Juan F; Canfrán-Duque, Alberto; Goedeke, Leigh; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-09-01

    Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking.

  10. Target shape dependence in a simple model of receptor-mediated endocytosis and phagocytosis.

    PubMed

    Richards, David M; Endres, Robert G

    2016-05-31

    Phagocytosis and receptor-mediated endocytosis are vitally important particle uptake mechanisms in many cell types, ranging from single-cell organisms to immune cells. In both processes, engulfment by the cell depends critically on both particle shape and orientation. However, most previous theoretical work has focused only on spherical particles and hence disregards the wide-ranging particle shapes occurring in nature, such as those of bacteria. Here, by implementing a simple model in one and two dimensions, we compare and contrast receptor-mediated endocytosis and phagocytosis for a range of biologically relevant shapes, including spheres, ellipsoids, capped cylinders, and hourglasses. We find a whole range of different engulfment behaviors with some ellipsoids engulfing faster than spheres, and that phagocytosis is able to engulf a greater range of target shapes than other types of endocytosis. Further, the 2D model can explain why some nonspherical particles engulf fastest (not at all) when presented to the membrane tip-first (lying flat). Our work reveals how some bacteria may avoid being internalized simply because of their shape, and suggests shapes for optimal drug delivery.

  11. Understanding magnetic nanoparticle osteoblast receptor-mediated endocytosis using experiments and modeling

    NASA Astrophysics Data System (ADS)

    Tran, Nhiem; Webster, Thomas J.

    2013-05-01

    Iron oxide nanoparticles are promising candidates for controlling drug delivery through an external magnetic force to treat a wide range of diseases, including osteoporosis. Previous studies have demonstrated that in the presence of hydroxyapatite coated magnetite (Fe3O4) nanoparticles, osteoblast (or bone forming cell) proliferation and long-term functions (such as calcium deposition) were significantly enhanced. Hydroxyapatite is the major inorganic component of bone. As a further attempt to understand why, in the current study, the uptake of such nanoparticles into osteoblasts was experimentally investigated and mathematically modeled. Magnetite nanoparticles were synthesized using a co-precipitation method and were coated with hydroxyapatite. A cellular uptake experiment at low temperatures indicated that receptor-mediated endocytosis contributed to the internalization of the magnetic nanoparticles into osteoblasts. A model was further developed to explain the uptake of magnetic nanoparticles into osteoblasts using receptor-mediated endocytosis. This model may explain the internalization of hydroxyapatite into osteoblasts to elevate intracellular calcium levels necessary to promote osteoblast functions to treat a wide range of orthopedic problems, including osteoporosis.

  12. Neuregulin-1-beta1 enters brain and spinal cord by receptor-mediated transport.

    PubMed

    Kastin, Abba J; Akerstrom, Victoria; Pan, Weihong

    2004-02-01

    Proteins of the neuregulin (NRG) family play important regulatory roles in neuronal survival and synaptic activity. NRG-1-beta1 has particular potential as a therapeutic agent because it enhances myelination of neurites in spinal cord explants. In this study, we determined the permeation of NRG-1-beta1 across the blood-brain and blood-spinal cord barriers (BBB and BSCB respectively). Intact radioactively labeled NRG-1-beta1 had a saturable and relatively rapid influx rate from blood to the CNS in mice. Capillary depletion studies showed that NRG-1-beta1 entered the parenchyma of the brain and spinal cord rather than being trapped in the capillaries that compose the BBB. The possible mechanism of receptor-mediated transport was shown by the ability of antibodies to erbB3 and erbB4 receptors to inhibit the influx. Lipophilicity, less important for such saturable transport mechanisms, was measured by the octanol : buffer partition coefficient and found to be low. The results indicate that NRG-1-beta1 enters spinal cord and brain by a saturable receptor-mediated mechanism, which provides the opportunity for possible therapeutic manipulation at the BBB level.

  13. Regulation and ontogeny of subtypes of muscarinic receptors and muscarinic receptor-mediated

    SciTech Connect

    Lee, W.

    1989-01-01

    The densities of total and M1 muscarinic receptors were measured using the muscarinic receptor antagonists {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine, respectively. Thus, the difference between the density of {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine binding sites represents the density of M2 sites. In addition, there is no observable change in either acetylcholine-stimulated phosphoinositide breakdown (suggested to be an M1 receptor-mediated response) or in carbachol-mediated inhibition of cyclic AMP accumulation (suggested to be an M2 receptor-mediated response) in slices of cortex+dorsal hippocampus following chronic atropine administration. In other experiments, it has been shown that the M1 and M2 receptors in rat cortex have different ontogenetic profiles. The M2 receptor is present at adult levels at birth, while the M1 receptor develops slowly from low levels at postnatal week 1 to adult levels at postnatal week 3. The expression of acetylcholine-stimulated phosphoinositide breakdown parallels the development of M1 receptors, while the development of carbachol-mediated inhibition of cyclic AMP accumulation occurs abruptly between weeks 2 and 3 postnatally.

  14. Adaptation in sound localization: from GABA(B) receptor-mediated synaptic modulation to perception.

    PubMed

    Stange, Annette; Myoga, Michael H; Lingner, Andrea; Ford, Marc C; Alexandrova, Olga; Felmy, Felix; Pecka, Michael; Siveke, Ida; Grothe, Benedikt

    2013-12-01

    Across all sensory modalities, the effect of context-dependent neural adaptation can be observed at every level, from receptors to perception. Nonetheless, it has long been assumed that the processing of interaural time differences, which is the primary cue for sound localization, is nonadaptive, as its outputs are mapped directly onto a hard-wired representation of space. Here we present evidence derived from in vitro and in vivo experiments in gerbils indicating that the coincidence-detector neurons in the medial superior olive modulate their sensitivity to interaural time differences through a rapid, GABA(B) receptor-mediated feedback mechanism. We show that this mechanism provides a gain control in the form of output normalization, which influences the neuronal population code of auditory space. Furthermore, psychophysical tests showed that the paradigm used to evoke neuronal GABA(B) receptor-mediated adaptation causes the perceptual shift in sound localization in humans that was expected on the basis of our physiological results in gerbils.

  15. P2X3 Receptors and Sensory Transduction

    NASA Astrophysics Data System (ADS)

    Kennedy, Charles

    It has been known for many years that exogenously administered adenosine 5 -triphosphate (ATP) evokes acute pain, but the physiological and pathophysiological roles of endogenous ATP in nociceptive signalling are only now becoming clear. ATP produces its effects through P2X and P2Y receptors, and the P2X3 receptor is of notable importance. It shows a selective expression, at high levels in nociceptive sensory neurons, where it forms functional receptors on its own and in combination with the P2X2 receptor. Recent studies have used gene knockout methods, antisense oligonucleotides, small interfering RNA technologies, and a novel selective P2X3 antagonist, A-317491, to show that P2X3 receptors play a prominent role in both chronic inflammatory and neuropathic pain. Several other P2X subunits also appear to be expressed in sensory neurons and there is evidence for functional P2X1/5 or P2X2/6 heteromers in some of these. These data indicate that P2X receptors, particularly the P2X3 subtype, could be targetted in the search for new, effective analgesics.

  16. Prenatal nicotine is associated with reduced AMPA and NMDA receptor-mediated rises in calcium within the laterodorsal tegmentum: a pontine nucleus involved in addiction processes.

    PubMed

    McNair, L F; Kohlmeier, K A

    2015-06-01

    Despite huge efforts from public sectors to educate society as to the deleterious physiological consequences of smoking while pregnant, 12-25% of all babies worldwide are born to mothers who smoked during their pregnancies. Chief among the negative legacies bestowed to the exposed individual is an enhanced proclivity postnatally to addict to drugs of abuse, which suggests that the drug exposure during gestation changed the developing brain in such a way that biased it towards addiction. Glutamate signalling has been shown to be altered by prenatal nicotine exposure (PNE) and glutamate is the major excitatory neurotransmitter within the laterodorsal tegmental nucleus (LDT), which is a brainstem region importantly involved in responding to motivational stimuli and critical in development of drug addiction-associated behaviours, however, it is unknown whether PNE alters glutamate signalling within this nucleus. Accordingly, we used calcium imaging, to evaluate AMPA and NMDA receptor-mediated calcium responses in LDT brain slices from control and PNE mice. We also investigated whether the positive AMPA receptor modulator cyclothiazide (CYZ) had differential actions on calcium in the LDT following PNE. Our data indicated that PNE significantly decreased AMPA receptor-mediated calcium responses, and altered the neuronal calcium response to consecutive NMDA applications within the LDT. Furthermore, CYZ strongly potentiated AMPA-induced responses, however, this action was significantly reduced in the LDT of PNE mice when compared with enhancements in responses in control LDT cells. Immunohistochemical processing confirmed that calcium imaging recordings were obtained from the LDT nucleus as determined by presence of cholinergic neurons. Our results contribute to the body of evidence suggesting that neurobiological changes are induced if gestation is accompanied by nicotine exposure. We conclude that in light of the role played by the LDT in motivated behaviour, the

  17. The SLAC P2 Marx

    SciTech Connect

    Kemp, Mark; Benwell, Andrew; Burkhart, Craig; MacNair, David; Nguyen1, Minh; /SLAC

    2012-07-05

    A proposed high energy physics accelerator, the International Linear Collider, will require greater than five hundred rf stations. Each station is composed of a klystron driven by a modulator. Recently, the SLAC P2 Marx was designated the baseline modulator for the ILC. This paper describes some key features of this modulator and presents recent experimental results. The P2 Marx is presently being transported to another facility for lifetime testing. Here, we will gain understanding of how the Marx performs into a klystron load and gain experience operating the Marx for longer periods. Long term plans include the possibility of using this rf station for L-band technology demonstration at SLAC. While the Marx was designed with the ILC in mind, the topology can be readily applied to several different applications. We are currently evaluating the use of the topology for ESS, CLIC, and upgrades for systems at Fermi National Accelerator Laboratory. Because of the modular nature of the cell and the robustness of the control system, many different combinations of series and parallel operation are possible along with different load currents and pulse shapes.

  18. Curcumin Represses NLRP3 Inflammasome Activation via TLR4/MyD88/NF-κB and P2X7R Signaling in PMA-Induced Macrophages

    PubMed Central

    Kong, Fanqi; Ye, Bozhi; Cao, Jiatian; Cai, Xueli; Lin, Lu; Huang, Shanjun; Huang, Weijian; Huang, Zhouqing

    2016-01-01

    Aims: In the NOD-like receptor (NLR) family, the pyrin domain containing 3 (NLRP3) inflammasome is closely related to the progression of atherosclerosis. This study aimed to assess the effects of curcumin on NLRP3 inflammasome in phorbol 12-myristate 13-acetate (PMA)-induced macrophages and explore its underlying mechanism. Methods: Human monocytic THP-1 cells were pretreated with curcumin for 1 h and subsequently induced with PMA for 48 h. Total protein was collected for Western blot analysis. Cytokine interleukin (IL)-1β release and nuclear factor kappa B (NF-κB) p65 translocation were detected by ELISA assay and cellular NF-κB translocation kit, respectively. Results: Curcumin significantly reduced the expression of NLRP3 and cleavage of caspase-1 and IL-1β secretion in PMA-induced macrophages. Moreover, Bay (a NF-κB inhibitor) treatment considerably suppressed the expression of NLRP3 inflammasome in PMA-induced THP-1 cells. Curcumin also markedly inhibited the upregulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphorylation level of IκB-α, and activation of NF-κB in PMA-induced macrophages. In addition, purinergic 2X7 receptor (P2X7R) siRNA was administered, and it significantly decreased NLRP3 inflammasome expression in PMA-induced macrophages. Furthermore, curcumin reversed PMA-stimulated P2X7R activation, which further reduced the expression of NLRP3 and cleavage of caspase-1 and IL-1β secretion. Silencing of P2X7R using siRNA also suppressed the activation of NF-κB pathway in PMA-induced macrophages, but P2X7R-silenced cells did not significantly decrease the expression of TLR4 and MyD88. Conclusion: Curcumin inhibited NLRP3 inflammasome through suppressing TLR4/MyD88/NF-κB and P2X7R pathways in PMA-induced macrophages. PMID:27777559

  19. Radiosensitizing Effect of P2X7 Receptor Antagonist on Melanoma in vitro and in vivo.

    PubMed

    Tanamachi, Keisuke; Nishino, Keisuke; Mori, Natsuki; Suzuki, Toshihiro; Tanuma, Sei-Ichi; Abe, Ryo; Tsukimoto, Mitsutoshi

    2017-03-24

    Melanoma is highly malignant, and generally exhibits radioresistance, responding poorly to radiation therapy. We previously reported that activation of P2X7, P2Y6, and P2Y12 receptors is involved in the DNA damage response after γ-irradiation of human lung adenocarcinoma A549 cells. However, it is not clear whether these receptors are also involved in the case of melanoma cells, although P2X7 receptor is highly expressed in various cancers, including melanoma. Here, we show that P2X7 receptor antagonist enhances radiation-induced cytotoxicity in B16 melanoma cells in vitro and in vivo. We confirmed that these cells express P2X7 receptor mRNA and exhibit P2X7 receptor-mediated activities, such as ATP-induced pore formation and cytotoxicity. We further examined the radiosensitizing effect of P2X7 receptor antagonist Brilliant Blue G (BBG) in vitro by colony formation assay of B16 cells. γ-Irradiation dose-dependently reduced cell survival, and pretreatment with BBG enhanced the radiation-induced cytotoxicity. BBG pretreatment also decreased the number of DNA repair foci in nuclei, supporting involvement of P2X7 receptor in the DNA damage response. Finally, we investigated the radiosensitizing effect of BBG on B16 melanoma cells inoculated into the hind footpad of C57BL/6 mice. Neither 1 Gy γ-irradiation alone nor BBG alone suppressed the increase of tumor volume, but the combination of irradiation and BBG significantly suppressed tumor growth. Our results suggest that P2X7 receptor antagonist BBG has a radiosensitizing effect in melanoma in vitro and in vivo. BBG, which is used as a food coloring agent, appears to be a promising candidate as a radiosensitizer.

  20. Post-translational allosteric activation of the P2X7 receptor through glycosaminoglycan chains of CD44 proteoglycans

    PubMed Central

    Moura, GEDD; Lucena, SV; Lima, MA; Nascimento, FD; Gesteira, TF; Nader, HB; Paredes-Gamero, EJ; Tersariol, ILS

    2015-01-01

    Here, we present evidence for the positive allosteric modulation of the P2X7 receptor through glycosaminoglycans (GAGs) in CHO (cell line derived from the ovary of the Chinese hamster) cells. The marked potentiation of P2X7 activity through GAGs in the presence of non-saturating agonists concentrations was evident with the endogenous expression of the receptor in CHO cells. The presence of GAGs on the surface of CHO cells greatly increased the sensitivity to adenosine 5′-triphosphate and changed the main P2X7 receptor kinetic parameters EC50, Hill coefficient and Emax. GAGs decreased the allosteric inhibition of P2X7 receptor through Mg2+. GAGs activated P2X7 receptor-mediated cytoplasmic Ca2+ influx and pore formation. Consequently, wild-type CHO-K1 cells were 2.5-fold more sensitive to cell death induced through P2X7 agonists than mutant CHO-745 cells defective in GAGs biosynthesis. In the present study, we provide the first evidence that the P2X7 receptor interacts with CD44 on the CHO-K1 cell surface. Thus, these data demonstrated that GAGs positively modulate the P2X7 receptor, and sCD44 is a part of a regulatory positive feedback loop linking P2X7 receptor activation for the intracellular response mediated through P2X7 receptor stimulation. PMID:27551441

  1. P2Y Receptors Sensitize Mouse and Human Colonic Nociceptors

    PubMed Central

    Hockley, James R. F.; Tranter, Michael M.; McGuire, Cian; Boundouki, George; Cibert-Goton, Vincent; Thaha, Mohamed A.; Blackshaw, L. Ashley; Michael, Gregory J.; Baker, Mark D.; Knowles, Charles H.; Winchester, Wendy J.

    2016-01-01

    pain-sensing nerves located in the bowel wall and their sensitization to physiological stimuli, including bowel movements, underpins the development of such pain, and is associated with mediators released during disease. This work addresses the unstudied role of purine and pyrimidine nucleotides in modulating colonic nociceptors via P2Y receptors using a combination of electrophysiological recordings from human ex vivo samples and a detailed functional study in the mouse. This is the first report to identify colonic purinergic signaling as a function of P2Y receptor activation, in addition to established P2X receptor activity, and the results contribute to our understanding of the development of visceral pain during gastrointestinal disease. PMID:26911685

  2. Decreases in mitochondrial reactive oxygen species initiate GABAA receptor-mediated electrical suppression in anoxia-tolerant turtle neurons

    PubMed Central

    Hogg, David W; Pamenter, Matthew E; Dukoff, David J; Buck, Leslie T

    2015-01-01

    Key points Anoxia induces hyper-excitability and cell death in mammalian brain but in the western painted turtle (Chrysemys picta bellii) enhanced GABA transmission prevents injury. The mechanism responsible for increased GABA transmission is unknown; however, reactive oxygen species (ROS) generated by mitochondria may play a role because this is an oxygen-sensitive process. In this study, we show that inhibition of mitochondrial ROS production is sufficient to initiate a redox-sensitive GABA signalling cascade that suppresses pyramidal neuron action potential frequency. These results further our understanding of the turtle's unique strategy for reducing ATP consumption during anoxia and highlights a natural mechanism in which to explore therapies to protect mammalian brain from low-oxygen insults (e.g. cerebral stroke). Abstract Anoxia induces hyper-excitability and cell death in mammalian brain but in the anoxia-tolerant western painted turtle (Chrysemys picta bellii) neuronal electrical activity is suppressed (i.e. spike arrest), adenosine triphosphate (ATP) consumption is reduced, and cell death does not occur. Electrical suppression is primarily the result of enhanced γ-aminobutyric acid (GABA) transmission; however, the underlying mechanism responsible for initiating oxygen-sensitive GABAergic spike arrest is unknown. In turtle cortical pyramidal neurons there are three types of GABAA receptor-mediated currents: spontaneous inhibitory postsynaptic currents (IPSCs), giant IPSCs and tonic currents. The aim of this study was to assess the effects of reactive oxygen species (ROS) scavenging on these three currents since ROS levels naturally decrease with anoxia and may serve as a redox signal to initiate spike arrest. We found that anoxia, pharmacological ROS scavenging, or inhibition of mitochondrial ROS generation enhanced all three types of GABA currents, with tonic currents comprising ∼50% of the total current. Application of hydrogen peroxide inhibited

  3. Enzyme induction and histopathology elucidate aryl hydrocarbon receptor-mediated versus non-aryl hydrocarbon receptor-mediated effects of Aroclor 1268 in American mink (Neovison vison).

    PubMed

    Folland, William R; Newsted, John L; Fitzgerald, Scott D; Fuchsman, Phyllis C; Bradley, Patrick W; Kern, John; Kannan, Kurunthachalam; Zwiernik, Matthew J

    2016-03-01

    Polychlorinated biphenyl (PCB) concentrations reported in preferred prey and blubber of bottlenose dolphins from the Turtle-Brunswick River estuary (Georgia, USA) suggest the potential for adverse effects. However, PCBs in Turtle-Brunswick River estuary dolphins are primarily derived from Aroclor 1268, and predicting toxic effects of Aroclor 1268 is uncertain because of the mixture's unique composition and associated physiochemical characteristics. These differences suggest that toxicity benchmarks for other PCB mixtures may not be relevant to dolphins exposed to Aroclor 1268. American mink (Neovison vison) were used as a surrogate model for cetaceans to characterize mechanisms of action associated with Aroclor 1268 exposure. Mink share similarities in phylogeny and life history with cetaceans and are characteristically sensitive to PCBs, making them an attractive surrogate species for marine mammals in ecotoxicity studies. Adult female mink and a subsequent F1 generation were exposed to Aroclor 1268 through diet, and effects on enzyme induction, histopathology, thyroid hormone regulation, hematology, organ weights, and body condition index were compared to a negative control and a 3,3',4,4',5-pentachlorobiphenyl (PCB 126)-positive control. Aroclor 1268 dietary exposure concentrations ranged from 1.8 µg/g wet weight to 29 µg/g wet weight. Anemia, hypothyroidism, and hepatomegaly were observed in mink exposed to Aroclor 1268 beyond various dietary thresholds. Cytochrome P450 induction and squamous epithelial proliferation jaw lesions were low in Aroclor 1268 treatments relative to the positive control. Differences in enzyme induction and the development of squamous epithelial proliferation jaw lesions between Aroclor 1268 treatments and the positive control, coupled with effects observed in Aroclor 1268 treatments not observed in the positive control, indicate that mechanisms additional to the aryl hydrocarbon receptor-mediated pathway are associated with

  4. D2-like dopamine receptors mediate the response to amphetamine in a mouse model of ADHD

    PubMed Central

    Fan, Xueliang; Hess, Ellen J.

    2007-01-01

    The mechanisms underlying the effects of psychostimulants in attention deficit hyperactivity disorder (ADHD) are not well understood, but indirect evidence implicates D2 dopamine receptors. Here we dissect the components of dopaminergic neurotransmission in the hyperactive mouse mutant coloboma to identify pre- and postsynaptic elements essential for the effects of amphetamine in these mice. Amphetamine treatment reduced locomotor activity in coloboma mice, but induced a robust increase in dopamine overflow suggesting that abnormal regulation of dopamine efflux does not account for the behavioral effect. However, the D2-like dopamine receptor antagonists haloperidol and raclopride, but not the D1-like dopamine receptor antagonist SCH23390, blocked the amphetamine-induced reduction in locomotor activity in coloboma mice, providing direct evidence that D2-like dopamine receptors mediate the effect of amphetamine in these mice. With the precedent established that it is possible to directly antagonize this response, this strategy should prove useful for identifying novel therapeutics in ADHD. PMID:17291774

  5. Bicarbonate contributes to GABAA receptor-mediated neuronal excitation in surgically resected human hypothalamic hamartomas.

    PubMed

    Kim, Do-Young; Fenoglio, Kristina A; Kerrigan, John F; Rho, Jong M

    2009-01-01

    The role of bicarbonate (HCO(3)(-)) in GABA(A) receptor-mediated depolarization of human hypothalamic hamartoma (HH) neurons was investigated using cellular electrophysiological and calcium imaging techniques. Activation of GABA(A) receptors with muscimol (30 microM) provoked neuronal excitation in over 70% of large (18-22 microM) HH neurons in HCO(3)(-) buffer. Subsequent perfusion of HCO(3)(-)-free HEPES buffer produced partial suppression of muscimol-induced excitation. Additionally, 53% of large HH neurons under HCO(3)(-)-free conditions exhibited reduced intracellular calcium accumulation by muscimol. These results suggest that HCO(3)(-) efflux through GABA(A) receptors on a subpopulation of large HH neurons may contribute to membrane depolarization and subsequent activation of L-type calcium channels.

  6. Using GFP--ligand fusions to measure receptor-mediated endocytosis in living cells.

    PubMed

    Medina-Kauwe, Lali K; Chen, Xinhua

    2002-01-01

    Recombinant DNA technology has enabled the production of many types of chimeric proteins containing heterologous functional domains that have served a variety of useful capacities for cell biology research. Among proteins gaining wide use as a fusion partner is Aequorea victoria green fluorescent protein (GFP). GFP has been employed by numerous groups as a reporter gene for cell transfection and as an autofluorescent tag by recombinant fusion to foreign sequences. Here we describe the use of GFP as a tag for ligands, and provide examples of how purified recombinant GFP-ligand fusion proteins may be used to detect ligand-receptor interactions, including receptor-mediated endocytosis. Both its utility and limitations are discussed.

  7. Current injection and receptor-mediated excitation produce similar maximal firing rates in hypoglossal motoneurons.

    PubMed

    Wakefield, Hilary E; Fregosi, Ralph F; Fuglevand, Andrew J

    2016-03-01

    The maximum firing rates of motoneurons (MNs), activated in response to synaptic drive, appear to be much lower than that elicited by current injection. It could be that the decrease in input resistance associated with increased synaptic activity (but not current injection) might blunt overall changes in membrane depolarization and thereby limit spike-frequency output. To test this idea, we recorded, in the same cells, maximal firing responses to current injection and to synaptic activation. We prepared 300 μm medullary slices in neonatal rats that contained hypoglossal MNs and used whole-cell patch-clamp electrophysiology to record their maximum firing rates in response to triangular-ramp current injections and to glutamate receptor-mediated excitation. Brief pressure pulses of high-concentration glutamate led to significant depolarization, high firing rates, and temporary cessation of spiking due to spike inactivation. In the same cells, we applied current clamp protocols that approximated the time course of membrane potential change associated with glutamate application and with peak current levels large enough to cause spike inactivation. Means (SD) of maximum firing rates obtained in response to glutamate application were nearly identical to those obtained in response to ramp current injection [glutamate 47.1 ± 12.0 impulses (imp)/s, current injection 47.5 ± 11.2 imp/s], even though input resistance was 40% less during glutamate application compared with current injection. Therefore, these data suggest that the reduction in input resistance associated with receptor-mediated excitation does not, by itself, limit the maximal firing rate responses in MNs.

  8. 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

  9. Synthesis and mannose receptor-mediated uptake of clustered glycomimetics by human dendritic cells: effect of charge.

    PubMed

    Angyalosi, Gerhild; Grandjean, Cyrille; Lamirand, Mélanie; Auriault, Claude; Gras-Masse, Hélène; Melnyk, Oleg

    2002-10-07

    Effect of charge and shape of multivalent lysine-based cluster glycomimetics on their mannose receptor-mediated uptake by human dendritic cells has been evaluated: The capture is strongly affected by the shape of the ligands. The effect of charge is less pronounced although positive charges on the ligands seem to favor non-specific endocytosis capture.

  10. Platelet adhesion and degranulation induce pro-survival and pro-angiogenic signalling in ovarian cancer cells.

    PubMed

    Egan, Karl; Crowley, Darragh; Smyth, Paul; O'Toole, Sharon; Spillane, Cathy; Martin, Cara; Gallagher, Michael; Canney, Aoife; Norris, Lucy; Conlon, Niamh; McEvoy, Lynda; Ffrench, Brendan; Stordal, Britta; Keegan, Helen; Finn, Stephen; McEneaney, Victoria; Laios, Alex; Ducrée, Jens; Dunne, Eimear; Smith, Leila; Berndt, Michael; Sheils, Orla; Kenny, Dermot; O'Leary, John

    2011-01-01

    Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.

  11. Autocrine extracellular purinergic signaling in epithelial cells derived from polycystic kidneys.

    PubMed

    Schwiebert, Erik M; Wallace, Darren P; Braunstein, Gavin M; King, Sandi R; Peti-Peterdi, Janos; Hanaoka, Kazushige; Guggino, William B; Guay-Woodford, Lisa M; Bell, P Darwin; Sullivan, Lawrence P; Grantham, Jared J; Taylor, Amanda L

    2002-04-01

    ATP and its metabolites are potent autocrine agonists that act extracellularly within tissues to affect epithelial function. In polycystic kidneys, renal tubules become dilated and/or encapsulated as cysts, creating abnormal microenvironments for autocrine signaling. Previously, our laboratory has shown that high-nanomolar to micromolar quantities of ATP are released from cell monolayers in vitro and detectable in cyst fluids from microdissected human autosomal dominant polycystic kidney (ADPKD) cysts. Here, we show enhanced ATP release from autosomal recessive polycystic kidney (ARPKD) and ADPKD epithelial cell models. RT-PCR and immunoblotting for P2Y G protein-coupled receptors and P2X purinergic receptor channels show expression of mRNA and/or protein for multiple subtypes from both families. Assays of cytosolic Ca(2+) concentration and secretory Cl(-) transport show P2Y and P2X purinergic receptor-mediated stimulation of Cl(-) secretion via cytosolic Ca(2+)-dependent signaling. Therefore, we hypothesize that autocrine purinergic signaling may augment detrimentally cyst volume expansion in ADPKD or tubule dilation in ARPKD, accelerating disease progression.

  12. Neuroprotective effects of inhibiting N-methyl-D-aspartate receptors, P2X receptors and the mitogen-activated protein kinase cascade: a quantitative analysis in organotypical hippocampal slice cultures subjected to oxygen and glucose deprivation.

    PubMed

    Rundén-Pran, E; Tansø, R; Haug, F M; Ottersen, O P; Ring, A

    2005-01-01

    Cell death was assessed by quantitative analysis of propidium iodide uptake in rat hippocampal slice cultures transiently exposed to oxygen and glucose deprivation, an in vitro model of brain ischemia. The hippocampal subfields CA1 and CA3, and fascia dentata were analyzed at different stages from 0 to 48 h after the insult. Cell death appeared at 3 h and increased steeply toward 12 h. Only a slight additional increase in propidium iodide uptake was seen at later intervals. The mitogen-activated protein kinases extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2 were activated immediately after oxygen and glucose deprivation both in CA1 and in CA3/fascia dentata. Inhibition of the specific mitogen-activated protein kinase activator mitogen-activated protein kinase kinase by PD98059 or U0126 offered partial protection against oxygen and glucose deprivation-induced cell damage. The non-selective P2X receptor antagonist suramin gave neuroprotection of the same magnitude as the N-methyl-D-aspartate channel blocker MK-801 (approximately 70%). Neuroprotection was also observed with the P2 receptor blocker PPADS. Immunogold data indicated that hippocampal slice cultures (like intact hippocampi) express several isoforms of P2X receptors at the synaptic level, consistent with the idea that the effects of suramin and PPADS are mediated by P2X receptors. Virtually complete neuroprotection was obtained by combined blockade of N-methyl-D-aspartate receptors, P2X receptors, and mitogen-activated protein kinase kinase. Both P2X receptors and N-methyl-D-aspartate receptors mediate influx of calcium. Our results suggest that inhibition of P2X receptors has a neuroprotective potential similar to that of inhibition of N-methyl-D-aspartate receptors. In contrast, our comparative analysis shows that only partial protection can be achieved by inhibiting the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase cascade, one of the

  13. P2X7 receptors stimulate AKT phosphorylation in astrocytes

    PubMed Central

    Jacques-Silva, Maria C; Rodnight, Richard; Lenz, Guido; Liao, Zhongji; Kong, Qiongman; Tran, Minh; Kang, Yuan; Gonzalez, Fernando A; Weisman, Gary A; Neary, Joseph T

    2004-01-01

    Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X7 subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. P2Y and P2X receptor agonists ATP, uridine 5′-triphosphate (UTP) and 2′,3′-O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration- and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X7 receptor. Activation was maximal at 5 – 10 min and was sustained for 60 min; the EC50 for BzATP was approximately 50 μM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X7 receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. In conclusion, our data indicate that stimulation of astrocytic P2X7 receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism. PMID:15023862

  14. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    SciTech Connect

    Xu, Yuan Cardell, Lars-Olaf

    2014-02-15

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin- (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in

  15. Histamine H4 receptor mediates eosinophil chemotaxis with cell shape change and adhesion molecule upregulation

    PubMed Central

    Ling, Ping; Ngo, Karen; Nguyen, Steven; Thurmond, Robin L; Edwards, James P; Karlsson, Lars; Fung-Leung, Wai-Ping

    2004-01-01

    During mast cell degranulation, histamine is released in large quantities. Human eosinophils were found to express histamine H4 but not H3 receptors. The possible effects of histamine on eosinophils and the receptor mediating these effects were investigated in our studies. Histamine (0.01–30 μM) induced a rapid and transient cell shape change in human eosinophils, but had no effects on neutrophils. The maximal shape change was at 0.3 μM histamine with EC50 at 19 nM. After 60 min incubation with 1 μM histamine, eosinophils were desensitized and were refractory to shape change response upon histamine restimulation. Histamine (0.01–1 μM) also enhanced the eosinophil shape change induced by other chemokines. Histamine-induced eosinophil shape change was mediated by the H4 receptor. This effect was completely inhibited by H4 receptor-specific antagonist JNJ 7777120 (IC50 0.3 μM) and H3/H4 receptor antagonist thioperamide (IC50 1.4 μM), but not by selective H1, H2 or H3 receptor antagonists. H4 receptor agonists imetit (EC50 25 nM) and clobenpropit (EC50 72 nM) could mimic histamine effect in inducing eosinophil shape change. Histamine (0.01–100 μM) induced upregulation of adhesion molecules CD11b/CD18 (Mac-1) and CD54 (ICAM-1) on eosinophils. This effect was mediated by the H4 receptor and could be blocked by H4 receptor antagonists JNJ 7777120 and thioperamide. Histamine (0.01–10 μM) induced eosinophil chemotaxis with an EC50 of 83 nM. This effect was mediated by the H4 receptor and could be blocked by H4 receptor antagonists JNJ 7777120 (IC50 86 nM) and thioperamide (IC50 519 nM). Histamine (0.5 μM) also enhanced the eosinophil shape change induced by other chemokines. In conclusion, we have demonstrated a new mechanism of eosinophil recruitment driven by mast cells via the release of histamine. Using specific histamine receptor ligands, we have provided a definitive proof that the H4 receptor mediates eosinophil chemotaxis, cell shape change and

  16. 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

    , hippocampal or hypothalamic slices were used instead of cortical slices. The Ca2+-induced tritium overflow in guinea-pig cortex slices was inhibited by histamine (in the presence of ranitidine); this effect was abolished by clobenpropit. In slices superfused in the presence of clobenpropit, impromidine failed to facilitate the Ca2+-evoked tritium overflow. The electrically evoked tritium overflow in mouse brain cortex slices was inhibited by histamine by about 60% (both in the absence or presence of ranitidine). The inhibitory effect of histamine was abolished (but not reversed) by clobenpropit. In conclusion, noradrenaline release in the guinea-pig brain cortex is inhibited via presynaptic H3 receptors and facilitated via H2 receptors not located presynaptically. In the mouse brain cortex, only inhibitory H3 receptors occur. The extent of the H3 receptor-mediated effect is more marked in the mouse than in the guinea-pig brain cortex.

  17. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

    PubMed

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves

    2013-01-01

    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

  18. Lactate Modulates the Activity of Primary Cortical Neurons through a Receptor-Mediated Pathway

    PubMed Central

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves

    2013-01-01

    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism. PMID:23951229

  19. Substance P selectively modulates GABA(A) receptor-mediated synaptic transmission in striatal cholinergic interneurons.

    PubMed

    Govindaiah, G; Wang, Yanyan; Cox, Charles L

    2010-02-01

    Substance P (SP) is co-localized and co-released with gamma-amino butyric acid (GABA) from approximately 50% of GABAergic medium spiny neurons (MSNs) in the striatum. MSNs innervate several cellular targets including neighboring MSNs and cholinergic interneurons via collaterals. However, the functional role of SP release onto striatal interneurons is unknown. Here we examined SP-mediated actions on inhibitory synaptic transmission in cholinergic interneurons using whole-cell recordings in mouse corticostriatal slices. We found that SP selectively suppressed GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs), but not excitatory post-synaptic currents (EPSCs) in cholinergic interneurons. In contrast, SP did not alter IPSCs in fast-spiking interneurons and MSNs. SP suppressed IPSC amplitude in a concentration-dependent and reversible manner, and the NK1 receptor antagonist RP67580 attenuated the SP-mediated suppression. In addition, RP67580 alone enhanced the evoked IPSC amplitude in cholinergic interneurons, suggesting an endogenous action of SP on regulation of inhibitory synaptic transmission. SP did not alter the paired-pulse ratio, but reduced the amplitudes of GABA(A) agonist muscimol-induced outward currents and miniature IPSCs in cholinergic interneurons, suggesting SP exerts its effects primarily at the post-synaptic site. Our results indicate that the physiological effects of SP are to enhance the activity of striatal cholinergic interneurons and provide a rationale for designing potential new antiparkinsonian agents.

  20. Fluid shear stress sensitizes cancer cells to receptor-mediated apoptosis via trimeric death receptors

    NASA Astrophysics Data System (ADS)

    Mitchell, Michael J.; King, Michael R.

    2013-01-01

    Cancer metastasis, the process of cancer cell migration from a primary to distal location, typically leads to a poor patient prognosis. Hematogenous metastasis is initiated by intravasation of circulating tumor cells (CTCs) into the bloodstream, which are then believed to adhere to the luminal surface of the endothelium and extravasate into distal locations. Apoptotic agents such as tumor necrosis factor apoptosis-inducing ligand (TRAIL), whether in soluble ligand form or expressed on the surface of natural killer cells, have shown promise in treating CTCs to reduce the probability of metastasis. The role of hemodynamic shear forces in altering the cancer cell response to apoptotic agents has not been previously investigated. Here, we report that human colon cancer COLO 205 and prostate cancer PC-3 cells exposed to a uniform fluid shear stress in a cone-and-plate viscometer become sensitized to TRAIL-induced apoptosis. Shear-induced sensitization directly correlates with the application of fluid shear stress, and TRAIL-induced apoptosis increases in a fluid shear stress force- and time-dependent manner. In contrast, TRAIL-induced necrosis is not affected by the application fluid shear stress. Interestingly, fluid shear stress does not sensitize cancer cells to apoptosis when treated with doxorubicin, which also induces apoptosis in cancer cells. Caspase inhibition experiments reveal that shear stress-induced sensitization to TRAIL occurs via caspase-dependent apoptosis. These results suggest that physiological fluid shear forces can modulate receptor-mediated apoptosis of cancer cells in the presence of apoptotic agents.

  1. Hormone stimulation of androgen receptor mediates dynamic changes in DNA methylation patterns at regulatory elements

    PubMed Central

    Dhiman, Vineet K.; Attwood, Kristopher; Campbell, Moray J.; Smiraglia, Dominic J.

    2015-01-01

    DNA methylation is an epigenetic modification that contributes to stable gene silencing by interfering with the ability of transcriptional regulators to bind to DNA. Recent findings have revealed that hormone stimulation of certain nuclear receptors induces rapid, dynamic changes in DNA methylation patterns alongside transcriptional responses at a subset of target loci, over time. However, the ability of androgen receptor (AR) to dynamically regulate gene transcription is relatively under-studied and its role in the regulation of DNA methylation patterns remains to be elucidated. Here we demonstrate in normal prostate cells that hormone stimulated AR activity results in dynamic changes in the transcription rate and DNA methylation patterns at the AR target genes, TIPARP and SGK1. Time-resolved chromatin immunoprecipitation experiments on the SGK1 locus reveals dynamic recruitment of AR and RNA Polymerase II, as well as the recruitment of proteins involved in the DNA demethylation process, TET1 and TDG. Furthermore, the presence of DNA methylation at dynamic regions inhibits protein binding and transcriptional activity of SGK1. These findings establish AR activity as a contributing factor to the dynamic regulation of DNA methylation patterns at target genes in prostate biology and infer further complexity involved in nuclear receptor mediation of transcriptional regulation. PMID:26646795

  2. Dual effects of anandamide on NMDA receptor-mediated responses and neurotransmission.

    PubMed

    Hampson, A J; Bornheim, L M; Scanziani, M; Yost, C S; Gray, A T; Hansen, B M; Leonoudakis, D J; Bickler, P E

    1998-02-01

    Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as delta9-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (deltaCa2+NMDA) in rat brain slices. The presence of anandamide reduced deltaCa2+NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting deltaCa2+NMDA, they also revealed another, underlying mechanism by which anandamide influences deltaCa2+NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated deltaCa2+NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.

  3. Spinal GABA receptors mediate the suppressive effect of electroacupuncture on cold allodynia in rats.

    PubMed

    Park, Jung-Hyun; Han, Jae-Bok; Kim, Sun-Kwang; Park, Jung-Hyuk; Go, Dong-Hyun; Sun, Boram; Min, Byung-Il

    2010-03-31

    This study was performed to determine whether spinal GABAergic systems mediate the relieving effects of low frequency electroacupuncture (EA) on cold allodynia in a rat tail model of neuropathic pain. For neuropathic surgery, the right superior caudal trunk was resected at the level between the S1 and S2 spinal nerves innervating the tail. Two weeks after the nerve injury, the intrathecal catheter was implanted. Five days after the catheterization, rats were intrathecally injected with gabazine (GABA(A) receptor antagonist, 0.0003, 0.001 or 0.003mug), or saclofen (GABA(B) receptor antagonist, 3, 10 or 30mug). Ten minutes after the injection, EA (2Hz) was applied to the ST36 acupoint for 30min. The cold allodynia was assessed by the tail immersion test (i.e. immersing the tail in cold (4 degrees C) water and measuring the latency of an abrupt tail movement) before and after the EA treatment. EA stimulation at ST36 significantly inhibited the cold allodynia sign, whereas EA at non-acupoint and plain acupuncture at ST36 (without electrical stimulation) did not show antiallodynic effects. Intrathecal administration of gabazine or saclofen blocked the relieving effects of ST36 EA stimulation on cold allodynia. These results suggest that spinal GABA(A) and GABA(B) receptors mediate the suppressive effect of low frequency EA on cold allodynia in the tail neuropathic rats.

  4. 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

  5. M2-like macrophages are responsible for collagen degradation through a mannose receptor-mediated pathway.

    PubMed

    Madsen, Daniel H; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S; Brenner, David A; Burgdorf, Sven; Engelholm, Lars H; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto; Bugge, Thomas H

    2013-09-16

    Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase-dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor-associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process.

  6. Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

    SciTech Connect

    Sanborn, B.B.; Schneider, A.S. )

    1990-01-01

    Inositol trisphosphate (IP{sub 3}), a product of the phosphoinositide cycle, mobilizes intracellular Ca{sup 2+} in many cell types. New evidence suggests that inositol tetrakisphosphate (IP{sub 4}), an IP{sub 3} derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP{sub 4} are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP{sub 4} in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine stimulated an increase in ({sup 3}H)IP{sub 4} and ({sup 3}H)IP{sub 3} accumulation in chromaffin cells and this effect was completely blocked by atropine. ({sup 3}H)IP{sub 4} accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP{sub 3} and IP{sub 4} hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP{sub 4} and calcium homeostasis.

  7. Accumbens shell AMPA receptors mediate expression of extinguished reward seeking through interactions with basolateral amygdala.

    PubMed

    Millan, E Zayra; McNally, Gavan P

    2011-07-01

    Extinction is the reduction in drug seeking when the contingency between drug seeking behavior and the delivery of drug reward is broken. Here, we investigated a role for the nucleus accumbens shell (AcbSh). Rats were trained to respond for 4% (v/v) alcoholic beer in one context (Context A) followed by extinction in a second context (Context B). Rats were subsequently tested in the training context, A (ABA), or the extinction context, B (ABB). Pre-test injections of the glutamate AMPA receptor antagonist, NBQX (1 µg) into AcbSh had no effect on renewal of alcoholic beer seeking when rats were returned to the training context (ABA). However, NBQX increased responding when rats were tested in the extinction context (ABB). In a second experiment, rats received training, extinction, and test in the same context. Pre-test injections of NBQX (0, 0.3, and 1 µg) into the AcbSh dose-dependently attenuated expression of extinction. We also found that NBQX in the AcbSh had no effect on initial acquisition of extinction or the motivation to respond for reward as measured by break point on a progressive ratio schedule. Finally, we show that pharmacological disconnection of a basolateral amygdala (BLA) → AcbSh pathway via NBQX in AcbSh combined with reversible inactivation of the contralateral BLA attenuates expression of extinction. Together, these results suggest that AcbSh AMPA receptors mediate expression of extinguished reward seeking through glutamatergic inputs from the BLA.

  8. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    PubMed Central

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-01-01

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism. PMID:6896821

  9. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    PubMed

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-05-15

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism.

  10. LRP6 protein regulates low density lipoprotein (LDL) receptor-mediated LDL uptake.

    PubMed

    Ye, Zhi-jia; Go, Gwang-Woong; Singh, Rajvir; Liu, Wenzhong; Keramati, Ali Reza; Mani, Arya

    2012-01-06

    Genetic variations in LRP6 gene are associated with high serum LDL cholesterol levels. We have previously shown that LDL clearance in peripheral B-lymphocytes of the LRP6(R611C) mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6(WT)) and LRP6(R611C) in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6(WT) was overexpressed and modestly reduced when it was knocked down in LDLR-deficient CHO (ldlA7) cells. These findings implicated LRP6 in LDLR-independent cellular LDL binding and uptake. However, LRP6 knockdown in wild type CHO cells resulted in a much greater decline in LDL binding and uptake compared with CHO-ldlA7 cells, suggesting impaired function of the LDLR. LDLR internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further analysis revealed that LRP6(WT) forms a complex with LDLR, clathrin, and ARH and undergoes a clathrin-mediated internalization after stimulation with LDL. LDLR and LRP6 internalizations as well as LDL uptake were all impaired in CHO-k1 cells expressing LRP6(R611C). These studies identify LRP6 as a critical modulator of receptor-mediated LDL endocytosis and introduce a mechanism by which variation in LRP6 may contribute to high serum LDL levels.

  11. S-nitrosylated SHP-2 contributes to NMDA receptor-mediated excitotoxicity in acute ischemic stroke

    PubMed Central

    Shi, Zhong-Qing; Sunico, Carmen R.; McKercher, Scott R.; Cui, Jiankun; Feng, Gen-Sheng; Nakamura, Tomohiro; Lipton, Stuart A.

    2013-01-01

    Overproduction of nitric oxide (NO) can cause neuronal damage, contributing to the pathogenesis of several neurodegenerative diseases and stroke (i.e., focal cerebral ischemia). NO can mediate neurotoxic effects at least in part via protein S-nitrosylation, a reaction that covalently attaches NO to a cysteine thiol (or thiolate anion) to form an S-nitrosothiol. Recently, the tyrosine phosphatase Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) and its downstream pathways have emerged as important mediators of cell survival. Here we report that in neurons and brain tissue NO can S-nitrosylate SHP-2 at its active site cysteine, forming S-nitrosylated SHP-2 (SNO–SHP-2). We found that NMDA exposure in vitro and transient focal cerebral ischemia in vivo resulted in increased levels of SNO–SHP-2. S-Nitrosylation of SHP-2 inhibited its phosphatase activity, blocking downstream activation of the neuroprotective physiological ERK1/2 pathway, thus increasing susceptibility to NMDA receptor-mediated excitotoxicity. These findings suggest that formation of SNO–SHP-2 represents a key chemical reaction contributing to excitotoxic damage in stroke and potentially other neurological disorders. PMID:23382182

  12. The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells

    SciTech Connect

    Kindberg, G.M.; Gudmundsen, O.; Berg, T. )

    1990-06-05

    Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions. Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C; vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content.

  13. Receptor-mediated uptake and transport of macromolecules in the human placenta.

    PubMed

    Schneider, Henning; Miller, Richard K

    2010-01-01

    The human placenta is required to be the anchor, the conduit and the controller during pregnancy. The survival of the baby and its associated placenta is dependent upon the placenta shielding the embryo/fetus from harm, e.g., autoimmune disease - thrombophilia, antiphospholipid syndrome or infections, while simultaneously providing for the passage of critical nutrients (e.g., amino acids, vitamins) and beneficial immunoglobulins. In a number of instances, the movements of macromolecules into and through the placenta can result in the passage of the intact molecules into the fetal circulation or in the case of proteins - catabolism to amino acids which are utilized by the placenta and the fetus for continued growth and development. The transfer of two such macromolecules, immunoglobulin G (IgG) and vitamin B12 (cyanocobalamin or B12), are examined as to the unique receptor-mediated transfer capability of the human placenta, its transfer specificity as related to specific receptors and the role of endogeneous placental proteins (trancobalamins) in facilitating the recognition and transport of specifically B12. Brief comparisons will be made to other animal species and the differences in specific organ transfer capabilities.

  14. Cryptococcus neoformans is internalized by receptor-mediated or 'triggered' phagocytosis, dependent on actin recruitment.

    PubMed

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both 'zipper' (receptor-mediated) and 'trigger' (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells.

  15. Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

    SciTech Connect

    Majumdar, S.; Basu, S.K. )

    1991-01-01

    p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections.

  16. A pp32-retinoblastoma protein complex modulates androgen receptor-mediated transcription and associates with components of the splicing machinery

    SciTech Connect

    Adegbola, Onikepe; Pasternack, Gary R. . E-mail: gpastern@jhmi.edu

    2005-08-26

    We have previously shown pp32 and the retinoblastoma protein interact. pp32 and the retinoblastoma protein are nuclear receptor transcriptional coregulators: the retinoblastoma protein is a coactivator for androgen receptor, the major regulator of prostate cancer growth, while pp32, which is highly expressed in prostate cancer, is a corepressor of the estrogen receptor. We now show pp32 increases androgen receptor-mediated transcription and the retinoblastoma protein modulates this activity. Using affinity purification and mass spectrometry, we identify members of the pp32-retinoblastoma protein complex as PSF and nonO/p54nrb, proteins implicated in coordinate regulation of nuclear receptor-mediated transcription and splicing. We show that the pp32-retinoblastoma protein complex is modulated during TPA-induced K562 differentiation. Present evidence suggests that nuclear receptors assemble multiprotein complexes to coordinately regulate transcription and mRNA processing. Our results suggest that pp32 and the retinoblastoma protein may be part of a multiprotein complex that coordinately regulates nuclear receptor-mediated transcription and mRNA processing.

  17. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression

    PubMed Central

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A.; Choi, Hueng-Sik

    2016-01-01

    Background Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Results Activation of the hepatic CB1 receptor by arachidonyl-2’-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Conclusion Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion. PMID:27455076

  18. Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

    PubMed Central

    Villalón, Carlos M; Heiligers, Jan P C; Centurión, David; De Vries, Peter; Saxena, Pramod R

    1997-01-01

    , sumatriptan (30, 100 and 300 μg kg−1) and indorenate (300 and 1000 μg kg−1) or the 5-HT4 receptor (partial) agonist cisapride (300 and 1000 μg kg−1) were devoid of effects on feline heart rate per se and failed to modify significantly 5-HT-induced tachycardic responses. Based upon the above rank order of agonist potency, the failure of sumatriptan, indorenate or cisapride to produce cardioacceleration and the blockade by a series of drugs showing high affinity for the cloned 5-ht7 receptor, the present results indicate that the 5-HT receptor mediating tachycardia in the cat is operationally similar to other putative 5-HT7 receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine external carotid and coronary arteries, rat systemic vasculature and guinea-pig ileum). Since these responses represent functional correlates of the 5-ht7 gene product, the 5-HT7 receptor appellation is reinforced. Therefore, the present experimental model, which is not complicated by the presence of other 5-HT receptors, can be utilized to characterize and develop new drugs with potential agonist and antagonist properties at functional 5-HT7 receptors. PMID:9249256

  19. P2X and P2Y Receptors—Role in the Pathophysiology of the Nervous System

    PubMed Central

    Puchałowicz, Kamila; Tarnowski, Maciej; Baranowska-Bosiacka, Irena; Chlubek, Dariusz; Dziedziejko, Violetta

    2014-01-01

    Purinergic signalling plays a crucial role in proper functioning of the nervous system. Mechanisms depending on extracellular nucleotides and their P2 receptors also underlie a number of nervous system dysfunctions. This review aims to present the role of purinergic signalling, with particular focus devoted to role of P2 family receptors, in epilepsy, depression, neuropathic pain, nervous system neoplasms, such as glioma and neuroblastoma, neurodegenerative diseases like Parkinson’s disease, Alzheimer’s disease and multiple sclerosis. The above-mentioned conditions are associated with changes in expression of extracellular ectonucleotidases, P2X and P2Y receptors in neurons and glial cells, as well as releasing considerable amounts of nucleotides from activated or damaged nervous tissue cells into the extracellular space, which contributes to disturbance in purinergic signalling. The numerous studies indicate a potential possibility of using synthetic agonists/antagonists of P2 receptors in treatment of selected nervous system diseases. This is of particular significance, since numerous available agents reveal a low effectiveness and often produce side effects. PMID:25530618

  20. Bradykinin B2-receptor-mediated modulation of membrane currents in guinea-pig cardiomyocytes

    PubMed Central

    Sakamoto, Naoya; Uemura, Hiroko; Hara, Yukio; Saito, Toshihiro; Masuda, Yoshiaki; Nakaya, Haruaki

    1998-01-01

    In order to define the electrophysiological mechanism(s) responsible for bradykinin (BK)-induced positive inotropic and chronotropic responses in isolated guinea-pig atria, effects of BK on the membrane currents were examined in isolated atrial cells using patch clamp techniques.BK (0.1–1000 nM) increased the L-type Ca2+ current (ICa), which was recorded from enzymatically-dissociated atrial myocytes by the nystatin-perforated patch method, in a concentration-dependent fashion, and the calculated EC50 value for increasing ICa was 5.2 nM. In conventional ruptured patch experiments, BK inhibited the muscarinic acetylcholine receptor-operated K+ current (IK.ACh) that was activated by the muscarinic agonist carbachol (1 μM) with an EC50 value of 0.57 nM. Both the increase in ICa and the decrease in IK.ACh were blocked by HOE140, a selective bradykinin B2 receptor antagonist.The BK-induced inhibition of IK.ACh was significantly attenuated by staurosporine and calphostin C, protein kinase C inhibitors. In addition, the IK.ACh inhibition by BK was also attenuated by the tyrosine kinase inhibitor genistein or tyrphostin but not by daidzein, an inactive analogue of genistein. However, neither protein kinase C inhibitor nor tyrosine kinase inhibitor affected the BK-induced increase in ICa.In the presence and absence of muscarinic stimulation, BK prolonged the action potential recorded from the atrial cells in the current clamp mode.We conclude that BK increases ICa and decreases IK.ACh in atrial cells, resulting in positive inotropic and chronotropic responses in atrial preparations. Protein kinase C activation, and possibly tyrosine kinase activation, may be involved in the B2-receptor-mediated IK.ACh inhibition. PMID:9786500

  1. Greater Beta-Adrenergic Receptor Mediated Vasodilation in Women Using Oral Contraceptives

    PubMed Central

    Limberg, Jacqueline K.; Peltonen, Garrett L.; Johansson, Rebecca E.; Harrell, John W.; Kellawan, Jeremy M.; Eldridge, Marlowe W.; Sebranek, Joshua J.; Walker, Benjamin J.; Schrage, William G.

    2016-01-01

    Background: β-adrenergic receptors play an important role in mitigating the pressor effects of sympathetic nervous system activity in young women. Based on recent data showing oral contraceptive use in women abolishes the relationship between muscle sympathetic nervous system activity and blood pressure, we hypothesized forearm blood flow responses to a β-adrenergic receptor agonist would be greater in young women currently using oral contraceptives (OC+, n = 13) when compared to those not using oral contraceptives (OC–, n = 10). Methods: Women (18–35 years) were studied during the early follicular phase of the menstrual cycle (days 1–5) or placebo phase of oral contraceptive use. Forearm blood flow (FBF, Doppler ultrasound) and mean arterial blood pressure (MAP, brachial arterial catheter) were measured at baseline and during graded brachial artery infusion of the β-adrenergic receptor agonist, Isoproterenol (ISO), as well as Acetylcholine (ACH, endothelium-dependent vasodilation) and Nitroprusside (NTP, endothelium-independent vasodilation). Forearm vascular conductance was calculated (FVC = FBF/MAP, ml/min/100 mmHg) and the rise in FVC from baseline during infusion quantified vasodilation (ΔFVC = FVCinfusion − FVCbaseline). Results: ISO increased FVC in both groups (p < 0.01) and ISO-mediated ΔFVC was greater in OC+ compared to OC– (Main effect of group, p = 0.02). Expressing data as FVC and FBF resulted in similar conclusions. FVC responses to both ACH and NTP were also greater in OC+ compared to OC–. Conclusions: These data are the first to demonstrate greater β-adrenergic receptor-mediated vasodilation in the forearm of women currently using oral contraceptives (placebo phase) when compared to those not using oral contraceptives (early follicular phase), and suggest oral contraceptive use influences neurovascular control. PMID:27375493

  2. Characterization of Parameters Influencing Receptor-Mediated Endocytosis in Cultured Soybean Cells 1

    PubMed Central

    Horn, Mark A.; Heinstein, Peter F.; Low, Philip S.

    1992-01-01

    In a recent publication, we were able to demonstrate that biotin enters plant cells by receptor-mediated endocytosis and that impermeable macromolecules can be cotransported into cells by the same pathway if they are first covalently linked to biotin. In the present study, we have exploited the biotin endocytosis pathway to evaluate the variables in the cell wall and surrounding growth medium that influence the efficiency of endocytosis in plants. Under normal growth conditions, the major constraint limiting macromolecule endocytosis was found to be the size of the internalized macromolecule. Thus, a log-linear relationship with a negative slope exists between the molecular weight of the biotin-conjugated macromolecule and its rate of internalization by cultured soybean cells. This relationship, which extends from insulin (Mr approximately 5700) to immunoglobulin G (Mr approximately 160,000), is characterized by a slope of −1.04 × 105 molecules/cell/min per log Mr unit and an x intercept (no endocytosis detectable) of approximately log 160,000 daltons. Unfortunately, mild digestion with cell wall-degrading enzymes is unable to increase significantly the upper size limit of molecules that can be internalized, but uptake of lower molecular weight proteins can be enhanced by mild cell wall digestion. The optimal extracellular pH for endocytosis was found to be 4.6, i.e. near the normal pH of the cell culture medium. Furthermore, the osmotic strength at which endocytosis occurs most rapidly was observed to be isotonic to slightly hypotonic, suggesting that turgor pressure within the plant cell must not be a major determinant of endocytosis rates by cultured soybean (Glycine max) cells. Finally, cell age was found to impact significantly on the rate of macromolecule internalization, with maximal uptake rates occurring during early exponential growth and decreasing by a factor of 2 when the cells reach stationary growth phase. PMID:16668694

  3. PAC₁ receptors mediate positive chronotropic responses to PACAP-27 and VIP in isolated mouse atria.

    PubMed

    Hoover, Donald B; Girard, Beatrice M; Hoover, Jeffrey L; Parsons, Rodney L

    2013-08-05

    PACAP and VIP have prominent effects on cardiac function in several species, but little is known about their influence on the murine heart. Accordingly, we evaluated the expression of PACAP/VIP receptors in mouse heart and the response of isolated atria to peptide agonists. Quantitative PCR demonstrated that PAC₁, VPAC₁, and VPAC₂ receptor mRNAs are present throughout the mouse heart. Expression of all three receptor transcripts was low, PAC₁ being the lowest. No regional differences in expression were detected for individual receptor mRNAs after normalization to L32. Pharmacological effects of PACAP-27, VIP, and the selective PAC₁ agonist maxadilan were evaluated in isolated, spontaneously beating atria from C57BL/6 mice of either sex. Incremental additions of PACAP-27 at 1 min intervals caused a concentration-dependent tachycardia with a logEC₅₀=-9.08 ± 0.15 M (n=7) and a maximum of 96.3 ± 5.9% above baseline heart rate. VIP and maxadilan also caused tachycardia but their potencies were about two orders of magnitude less. Increasing the dosing interval to 5 min caused a leftward shift of the concentration-response curve to maxadilan but no changes in the curves for PACAP-27 or VIP. Under this condition, neither the potency nor the efficacy of maxadilan differed from those of PACAP-27. Neither PACAP-27 nor maxadilan caused tachyphylaxis, and maximal responses to maxadilan were maintained for at least 2 h. We conclude that all three VIP/PACAP family receptors are expressed by mouse cardiac tissue, but only PAC₁ receptors mediate positive chronotropic responses to PACAP-27 and VIP.

  4. cap alpha. /sub 2/-Adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production

    SciTech Connect

    Jones, S.B.; Toews, M.L.; Turner, J.T.; Bylund, D.B.

    1987-03-01

    Preincubation of HT29 human colonic adenocarcinoma cells with ..cap alpha../sub 2/-adrenergic agonists resulted in a 10- to 20-fold increase in forskolin-stimulated cyclic AMP production as compared to cells preincubated without agonist. Similar results were obtained using either a (/sup 3/H)adenine prelabeling assay or a cyclic AMP radioimmunoassay to measure cyclic AMP levels. This phenomenon, which is termed sensitization, is ..cap alpha../sub 2/-adrenergic receptor-mediated and rapid in onset and reversal. Yohimbine, an ..cap alpha../sub 2/-adrenergic receptor-selective antagonist, blocked norepinephrine-induced sensitization, whereas prazosin (..cap alpha../sub 1/-adrenergic) and sotalol (..beta..-adrenergic) did not. The time for half-maximal sensitization was 5 min and the half-time for reversal was 10 min. Only a 2-fold sensitization of cyclic AMP production stimulated by vasoactive intestinal peptide was observed, indicating that sensitization is relatively selective for forskolin. Sensitization reflects an increased production of cyclic AMP and not a decreased degradation of cyclic AMP, since incubation with a phosphodiesterase inhibitor and forskolin did not mimic sensitization. Increasing the levels of cyclic AMP during the preincubation had no effect on sensitization, indicating that sensitization is not caused by decreased cyclic AMP levels during the preincubation. This rapid and dramatic sensitization of forskolin-stimulated cyclic AMP production is a previously unreported effect that can be added to the growing list of ..cap alpha../sub 2/-adrenergic responses that are not mediated by a decrease in cyclic AMP.

  5. Potentiating effect of eszopiclone on GABA(A) receptor-mediated responses in pedunculopontine neurons.

    PubMed

    Ye, Meijun; Garcia-Rill, Edgar

    2009-07-01

    The pedunculopontine nucleus (PPN) is part of the cholinergic arm of the reticular activating system, which is mostly active during waking and REM sleep. GABAergic modulation of this area appears to regulate sleep-wake cycles. Eszopiclone (ESZ), a nonbenzodiazepine hypnotic agent, appears to modulate GABAergic receptors. However, the action site of ESZ in the brain is still unresolved. We tested the hypothesis that ESZ acts by potentiating GABA(A) receptors on PPN neurons. Wholecell voltage clamp recordings were performed on PPN neurons in 7-15 day rat brainstem slices, and the potentiating effects of ESZ on the responses to the GABA(A) receptor agonist isoguvacine (IGV), and on GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs), were determined. In the presence of tetrodotoxin, ESZ (1) increased the amplitude of the outward current induced by IGV, (2) increased its duration, and (3) enhanced the IGV-induced decrease in input resistance (Rin). The GABA(A) receptor antagonist gabazine (GBZ) blocked these effects. ESZ alone did not induce detectable currents or change Rin at a holding potential of -60 mV, but when held at 0 mV, ESZ induced an outward current in 13/21 PPN cells, an effect blocked by GBZ. ESZ also increased the amplitude (n = 18/21), duration (n = 17/21), and frequency (n = 13/15) of IPSCs. ESZ may potentiate GABA(A) inhibition in the PPN via pre- and post-synaptic modulation, which may underlie the hypnotic effects of ESZ. The differential effects of ESZ on both pre- and post-synaptic sites may partially explain why it has less significant side effects compared to other hypnotic agents.

  6. Direct muscarinic and nicotinic receptor-mediated excitation of rat medial vestibular nucleus neurons in vitro

    NASA Technical Reports Server (NTRS)

    Phelan, K. D.; Gallagher, J. P.

    1992-01-01

    We have utilized intracellular recording techniques to investigate the cholinoceptivity of rat medial vestibular nucleus (MVN) neurons in a submerged brain slice preparation. Exogenous application of the mixed cholinergic agonists, acetylcholine (ACh) or carbachol (CCh), produced predominantly membrane depolarization, induction of action potential firing, and decreased input resistance. Application of the selective muscarinic receptor agonist muscarine (MUSC), or the selective nicotinic receptor agonists nicotine (NIC) or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also produced membrane depolarizations. The MUSC-induced depolarization was accompanied by decreased conductance, while an increase in conductance appeared to underlie the NIC- and DMPP-induced depolarizations. The muscarinic and nicotinic receptor mediated depolarizations persisted in tetrodotoxin and/or low Ca2+/high Mg2+ containing media, suggesting direct postsynaptic receptor activation. The MUSC-induced depolarization could be reversibly blocked by the selective muscarinic-receptor antagonist, atropine, while the DMPP-induced depolarization could be reversibly suppressed by the selective ganglionic nicotinic-receptor antagonist, mecamylamine. Some neurons exhibited a transient membrane hyperpolarization during the depolarizing response to CCh or MUSC application. This transient inhibition could be reversibly blocked by the gamma-aminobutyric acid (GABA) antagonist, bicuculline, suggesting that the underlying hyperpolarization results indirectly from the endogenous release of GABA acting at GABA receptors. This study confirms the cholinoceptivity of MVN neurons and establishes that individual MVN cells possess muscarinic as well as nicotinic receptors. The data provide support for a prominent role of cholinergic mechanisms in the direct and indirect regulation of the excitability of MVN neurons.

  7. 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.

  8. N-Methyl-D-Aspartate Receptor-Mediated Axonal Injury in Adult Rat Corpus Callosum

    PubMed Central

    Zhang, Jingdong; Liu, Jianuo; Fox, Howard S.; Xiong, Huangui

    2013-01-01

    Damage to white matter such as corpus callosum (CC) is a pathological characteristic in many brain disorders. Glutamate (Glut) excitotoxicity through AMPA receptors on oligodendrocyte (OL) was previously considered as a mechanism for white matter damage. Recent studies have shown that N-methyl-D-aspartate receptors (NMDARs) are expressed on myelin sheath of neonatal rat OL processes and that activation of these receptors mediated demyelization. Whether NMDARs are expressed in the adult CC and are involved in excitotoxic axonal injury remains to be determined. In this study, we demonstrate the presence of NMDARs in the adult rat CC and their distributions in myelinated nerve fibers and OL somata by means of immunocytochemical staining and Western blot. Incubation of the CC slices with Glut or NMDA induced axonal injury as revealed by analyzing amplitude of CC fiber compound action potentials (CAPs) and input–output response. Both Glut and NMDA decreased the CAP amplitude and input–output responses, suggesting an involvement of NMDARs in Glut- and NMDA-induced axonal injury. The involvement of NMDAR in Glut-induced axonal injury was further assayed by detection of β-amyloid precursor protein (β-APP) in the CC axonal fibers. Treatment of the CC slices with Glut resulted in β-APP accumulation in the CC fibers as detected by Western blot, reflecting an impairment of axonal transport function. This injurious effect of Glut on CC axonal transport was significantly blocked by MK801. Taken together, these results show that NMDARs are expressed in the adult CC and are involved in excitotoxic activity in adult CC slices in vitro. PMID:23161705

  9. Receptor-mediated cell attachment and detachment kinetics. I. Probabilistic model and analysis.

    PubMed Central

    Cozens-Roberts, C.; Lauffenburger, D. A.; Quinn, J. A.

    1990-01-01

    The kinetics of receptor-mediated cell adhesion to a ligand-coated surface play a key role in many physiological and biotechnology-related processes. We present a probabilistic model of receptor-ligand bond formation between a cell and surface to describe the probability of adhesion in a fluid shear field. Our model extends the deterministic model of Hammer and Lauffenburger (Hammer, D.A., and D.A. Lauffenburger. 1987. Biophys. J. 52:475-487) to a probabilistic framework, in which we calculate the probability that a certain number of bonds between a cell and surface exists at any given time. The probabilistic framework is used to account for deviations from ideal, deterministic behavior, inherent in chemical reactions involving relatively small numbers of reacting molecules. Two situations are investigated: first, cell attachment in the absence of fluid stress; and, second, cell detachment in the presence of fluid stress. In the attachment case, we examine the expected variance in bond formation as a function of attachment time; this also provides an initial condition for the detachment case. Focusing then on detachment, we predict transient behavior as a function of key system parameters, such as the distractive fluid force, the receptor-ligand bond affinity and rate constants, and the receptor and ligand densities. We compare the predictions of the probabilistic model with those of a deterministic model, and show how a deterministic approach can yield some inaccurate results; e.g., it cannot account for temporally continuous cell attach mentor detachment, it can underestimate the time needed for cell attachment, it can overestimate the time required for cell detachment for a given level of force, and it can overestimate the force necessary for cell detachment. PMID:2174271

  10. Fc receptor-mediated immune responses: new tools but increased complexity in HIV prevention.

    PubMed

    Vargas-Inchaustegui, Diego A; Robert-Guroff, Marjorie

    2013-07-01

    The modest success of the RV144 HIV vaccine trial in Thailand and the ensuing suggestion that a Fc-receptormediated antibody activity might have played a role in the protection observed have intensified investigations on Fcrelated immune responses. HIV neutralizing antibodies have been and continue to be the focal point of research into humoral immune protection. However, recent knowledge that their protective efficacy can be augmented by Fc-FcR interactions has increased the complexity of identifying immune correlates of protection. If anything, continued studies of both humoral and cellular immune mechanisms point to the lack of a single protective anti-HIV immune response. Here we focus on humoral immunity, analyzing the role played by Fc receptor-related responses and discussing how new knowledge of their interactions requires further investigation, but may also spur novel vaccination approaches. We initially address classical Fc-receptor mediated anti-viral mechanisms including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell mediated viral inhibition (ADCVI), and antibody-dependent cellular phagocytosis (ADCP), as well as the effector cells that mediate these functions. Next, we summarize key aspects of FcR-Fc interactions that are important for potential control of HIV/SIV such as FcR polymorphisms and post-transcriptional modifications. Finally we discuss less commonly studied non-mechanistic anti-HIV immune functions: antibody avidity and envelopespecific B cell memory. Overall, a spectrum of immune responses, reflecting the immune system's redundancy, will likely be needed to prevent HIV infection and/or disease progression. Aside from elicitation of critical immune mechanisms, a successful vaccine will need to induce mature B cell responses and long-lasting immune memory.

  11. Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients.

    PubMed

    Gorvin, Caroline M; Wilmer, Martijn J; Piret, Sian E; Harding, Brian; van den Heuvel, Lambertus P; Wrong, Oliver; Jat, Parmjit S; Lippiat, Jonathan D; Levtchenko, Elena N; Thakker, Rajesh V

    2013-04-23

    Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis

  12. Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies.

    PubMed

    Shcherbatko, Anatoly; Foletti, Davide; Poulsen, Kris; Strop, Pavel; Zhu, Guoyun; Hasa-Moreno, Adela; Melton Witt, Jody; Loo, Carole; Krimm, Stellanie; Pios, Ariel; Yu, Jessica; Brown, Colleen; Lee, John K; Stroud, Robert; Rajpal, Arvind; Shelton, David

    2016-06-03

    Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.

  13. LSD and DOB: interaction with 5-HT2A receptors to inhibit NMDA receptor-mediated transmission in the rat prefrontal cortex.

    PubMed

    Arvanov, V L; Liang, X; Russo, A; Wang, R Y

    1999-09-01

    Both the phenethylamine hallucinogen (-)-1-2, 5-dimethoxy-4-bromophenyl-2-aminopropane (DOB), a selective serotonin 5-HT2A,2C receptor agonist, and the indoleamine hallucinogen D-lysergic acid diethylamide (LSD, which binds to 5-HT1A, 1B, 1D, 1E, 1F, 2A, 2C, 5, 6, 7, dopamine D1 and D2, and alpha1 and alpha2 adrenergic receptors), but not their non-hallucinogenic congeners, inhibited N-methyl-D-aspartate (NMDA)-induced inward current and NMDA receptor-mediated synaptic responses evoked by electrical stimulation of the forceps minor in pyramidal cells of the prefrontal cortical slices. The inhibitory effect of hallucinogens was mimicked by 5-HT in the presence of selective 5-HT1A and 5-HT3 receptor antagonists. The inhibitory action of DOB, LSD and 5-HT on the NMDA transmission was blocked by the 5-HT2A receptor antagonists R-(+)-alpha-(2, 3-dimethoxyphenil)-1-[4-fluorophenylethyl]-4-piperidineme thanol (M100907) and ketanserin. However, at low concentrations, when both LSD and DOB by themselves only partially depressed the NMDA response, they blocked the inhibitory effect of 5-HT, suggesting a partial agonist action. Whereas N-(4-aminobutyl)-5-chloro-2-naphthalenesulphonamide (W-7, a calmodulin antagonist) and N-[2-[[[3-(4'-chlorophenyl)- 2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4'-methoxy-b enzenesulphonamide phosphate (KN-93, a Ca2+/CaM-KII inhibitor), but not the negative control 2-[N-4'methoxybenzenesulphonyl]amino-N-(4'-chlorophenyl)-2-propeny l-N -methylbenzylamine phosphate (KN-92), blocked the inhibitory action of LSD and DOB, the selective protein kinase C inhibitor chelerythrine was without any effect. We conclude that phenethylamine and indoleamine hallucinogens may exert their hallucinogenic effect by interacting with 5-HT2A receptors via a Ca2+/CaM-KII-dependent signal transduction pathway as partial agonists and modulating the NMDA receptors-mediated sensory, perceptual, affective and cognitive processes.

  14. Accelerated FoxP2 Evolution in Echolocating Bats

    PubMed Central

    Li, Gang; Wang, Jinhong; Rossiter, Stephen J.; Jones, Gareth; Zhang, Shuyi

    2007-01-01

    FOXP2 is a transcription factor implicated in the development and neural control of orofacial coordination, particularly with respect to vocalisation. Observations that orthologues show almost no variation across vertebrates yet differ by two amino acids between humans and chimpanzees have led to speculation that recent evolutionary changes might relate to the emergence of language. Echolocating bats face especially challenging sensorimotor demands, using vocal signals for orientation and often for prey capture. To determine whether mutations in the FoxP2 gene could be associated with echolocation, we sequenced FoxP2 from echolocating and non-echolocating bats as well as a range of other mammal species. We found that contrary to previous reports, FoxP2 is not highly conserved across all nonhuman mammals but is extremely diverse in echolocating bats. We detected divergent selection (a change in selective pressure) at FoxP2 between bats with contrasting sonar systems, suggesting the intriguing possibility of a role for FoxP2 in the evolution and development of echolocation. We speculate that observed accelerated evolution of FoxP2 in bats supports a previously proposed function in sensorimotor coordination. PMID:17878935

  15. Potentiation of epidermal growth factor receptor-mediated oncogenesis by c-Src: implications for the etiology of multiple human cancers.

    PubMed Central

    Maa, M C; Leu, T H; McCarley, D J; Schatzman, R C; Parsons, S J

    1995-01-01

    c-Src is a nontransforming tyrosine kinase that participates in signaling events mediated by a variety of polypeptide growth factor receptors, including the epidermal growth factor receptor (EGFR). Overexpression and continual ligand stimulation of the EGFR results in morphological transformation of cells in vitro and tumor development in vivo. Elevated levels of c-Src and the EGFR are found in a variety of human malignancies, raising the question of whether c-Src can functionally cooperate with the EGFR during tumorigenesis. To address this issue, we generated c-Src/EGFR double overexpressors and compared their proliferative and biochemical characteristics to those of single overexpressors and control cells. We found that in cells expressing high levels of receptor, c-Src potentiated DNA synthesis, growth in soft agar, and tumor formation in nude mice. Growth potentiation was associated with the formation of a heterocomplex between c-Src and activated EGFR, the appearance of a distinct tyrosyl phosphorylation on the receptor, and an enhancement of receptor substrate phosphorylation. These findings indicate that c-Src is capable of potentiating receptor-mediated tumorigenesis and suggest that synergism between c-Src and the EGFR may contribute to a more aggressive phenotype in multiple human tumors. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7542783

  16. P2Y nucleotide receptors: Promise of therapeutic applications

    PubMed Central

    Jacobson, Kenneth A.; Boeynaems, Jean-Marie

    2010-01-01

    Extracellular nucleotides, such as ATP and UTP, have distinct signaling roles through a class of G protein-coupled receptors, termed P2Y. However, the receptor ligands are typically charged molecules of low bioavailability and stability in vivo. Recent progress in the development of selective agonists and antagonists for P2Y receptors and study of knockout mice have led to new drug concepts based on these receptors. The rapidly accelerating progress in this field has already resulted in drug candidates for cystic fibrosis, dry eye disease, and thrombosis. On the horizon are novel treatments of cardiovascular diseases, inflammatory diseases, and neurodegeneration. PMID:20594935

  17. GABAA and GABAB receptor-mediated effects in guinea-pig ileum.

    PubMed

    Giotti, A; Luzzi, S; Spagnesi, S; Zilletti, L

    1983-03-01

    1 The effects of gamma-aminobutyric acid (GABA) and related substances were examined in guinea-pig ileum longitudinal muscle.2 GABA at doses ranging from 10(-7) M to 3 x 10(-6) M elicited a relaxation while at higher doses (3 x 10(-6) M - 10(-4) M), as previously described, it caused a contraction followed by relaxation.3 GABA-induced relaxation was bicuculline-insensitive, was mimicked by (-)-baclofen but not by homotaurine and muscimol. The effect of baclofen was stereospecific. GABA- and (-)-baclofen-induced relaxations were dose-dependent and their ED(50) values were similar. A specific cross-desensitization occurred between GABA and (-)-baclofen.4 The bicuculline-insensitive relaxation induced by GABA and (-)-baclofen was prevented by tetrodotoxin and hyoscine but not by phentolamine plus propranolol, naloxone or theophylline.5 In preparations in which the muscle tone was raised by histamine or prostaglandin F(2alpha), GABA and (-)-baclofen induced relaxation to the same extent as before increasing the tone. If the tone was raised by DMPP, a greater bicuculline-insensitive relaxation occurred.6 Contraction caused by GABA was bicuculline-sensitive and was mimicked by homotaurine and muscimol. Contraction was dose-dependent and muscimol was about three times more potent than GABA or homotaurine. A specific cross-desensitization occurred between the contractile effects of GABA and those of homotaurine or muscimol.7 Bicuculline competitively antagonized the contractile effects of GABA, homotaurine and muscimol and gave closely similar pA(2) values. The slope of the Schild plot for the above drugs was near 1, confirming the competitive nature of the antagonism.8 The bicuculline-sensitive contraction induced by GABA, homotaurine and muscimol was abolished by tetrodotoxin and was non-competitively antagonized by hyoscine, while it was unaffected by hexamethonium, mepyramine and methysergide.9 It is concluded that two receptors mediate the GABA effects in guinea

  18. 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

  19. Adenosine A2B-receptor-mediated cyclic AMP accumulation in primary rat astrocytes.

    PubMed Central

    Peakman, M. C.; Hill, S. J.

    1994-01-01

    1. The effects of adenosine receptor agonists and antagonists on the accumulation of cyclic AMP have been investigated in primary cultures of rat astrocytes. 2. Adenosine A2-receptor stimulation caused a concentration-dependent increase in the accumulation of [3H]-cyclic AMP in cells prelabelled with [3H]-adenine. The rank order of agonist potencies was 5'-N-ethylcarboxamidoadenosine (NECA; EC50 = 1 microM) > adenosine (EC50 = 5 microM) > 2-chloroadenosine (EC50 = 20 microM) >> CGS 21680 (EC50 > 10 microM). The presence of 0.5 microM dipyridamole, an adenosine uptake blocker, had no effect on the potency of adenosine. 3. The response to 10 microM NECA was antagonized in a concentration-dependent manner by the non-selective adenosine receptor antagonists, xanthine amine congener (apparent KD = 12 nM), PD 115,199 (apparent KD = 134 nM) and 8-phenyltheophylline (apparent KD = 126 nM). However, the A1-receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, had no significant effect on the responses to NECA or 2-chloroadenosine at concentrations up to 1 microM. 4. Stimulation of A1-receptors with the selective agonist, N6-cyclopentyladenosine, did not alter the basal accumulation of [3H]-cyclic AMP but inhibited a forskolin-mediated elevation of [3H]-cyclic AMP accumulation by a maximal value of 42%. This inhibition was fully reversed in the presence of 0.1 microM, 8-cyclopentyl-1,3-dipropylxanthine. 5. The time course for NECA-mediated [3H]-cyclic AMP accumulation was investigated. The results suggest that there is a substantial efflux of cyclic AMP from the cells in addition to the rapid and sustained elevation of intracellular cyclic AMP (5 fold over basal) which was also observed. 6. These data indicate that rat astrocytes in primary culture express an A2B-adenosine receptor coupled positively to adenylyl cyclase. Furthermore, the presence of A1-receptors negatively coupled to adenylyl cyclase appears to have no significant effect on the A2B-receptor-mediated

  20. The Impact of Hyperthermia on Receptor-Mediated Interleukin-6 Regulation in Mouse Skeletal Muscle

    PubMed Central

    Welc, Steven S.; Morse, Deborah A.; Mattingly, Alex J.; Laitano, Orlando; King, Michelle A.; Clanton, Thomas L.

    2016-01-01

    In inflammatory cells, hyperthermia inhibits lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) gene expression and protein secretion. Since hyperthermia alone stimulates IL-6 in skeletal muscle, we hypothesized that it would amplify responses to other receptor-mediated stimuli. IL-6 regulation was tested in C2C12 myotubes and in soleus during treatment with epinephrine (EPI) or LPS. In EPI-treated myotubes (100 ng/ml), 1 h exposure at 40.5°C-42°C transiently increased IL-6 mRNA compared to EPI treatment alone at 37°C. In LPS-treated myotubes (1 μg/ml), exposure to 41°C-42°C also increased IL-6 mRNA. In isolated mouse soleus, similar amplifications of IL-6 gene expression were observed in 41°C, during both low (1 ng/ml) and high dose (100 ng/ml) EPI, but only in high dose LPS (1 μg/ml). In myotubes, heat increased IL-6 secretion during EPI exposure but had no effect or inhibited secretion with LPS. In soleus there were no effects of heat on IL-6 secretion during either EPI or LPS treatment. Mechanisms for the effects of heat on IL-6 mRNA were explored using a luciferase-reporter in C2C12 myotubes. Overexpression of heat shock factor-1 (HSF-1) had no impact on IL-6 promoter activity during EPI stimulation, but elevated IL-6 promoter activity during LPS stimulation. In contrast, when the activator protein-1 (AP-1) element was mutated, responses to both LPS and EPI were suppressed in heat. Using siRNA against activating transcription factor-3 (ATF-3), a heat-stress-induced inhibitor of IL-6, no ATF-3-dependent effects were observed. The results demonstrate that, unlike inflammatory cells, hyperthermia in muscle fibers amplifies IL-6 gene expression to EPI and LPS. The effect appears to reflect differential engagement of HSF-1 and AP-1 sensitive elements on the IL-6 gene, with no evidence for involvement of ATF-3. The functional significance of increased IL-6 mRNA expression during heat may serve to overcome the well-known suppression of protein synthetic

  1. Lipoprotein lipase regulates Fc receptor-mediated phagocytosis by macrophages maintained in glucose-deficient medium.

    PubMed Central

    Yin, B; Loike, J D; Kako, Y; Weinstock, P H; Breslow, J L; Silverstein, S C; Goldberg, I J

    1997-01-01

    During periods of intense activity such as phagocytosis, macrophages are thought to derive most of their energy from glucose metabolism under both aerobic and anaerobic conditions. To determine whether fatty acids released from lipoproteins by macrophage lipoprotein lipase (LPL) could substitute for glucose as a source of energy for phagocytosis, we cultured peritoneal macrophages from normal and LPL knockout (LPL-KO) mice that had been rescued from neonatal demise by expression of human LPL via the muscle creatine kinase promoter. Normal and LPL-KO macrophages were cultured in medium containing normal (5 mM) or low (1 mM) glucose, and were tested for their capacity to phagocytose IgG-opsonized sheep erythrocytes. LPL-KO macrophages maintained in 1 and 5 mM glucose phagocytosed 67 and 79% fewer IgG-opsonized erythrocytes, respectively, than macrophages from normal mice. Addition of VLDL to LPL-expressing macrophages maintained in 1 mM glucose enhanced the macrophages' phagocytosis of IgG-opsonized erythrocytes, but did not stimulate phagocytosis by LPL-KO macrophages. Inhibition of secreted LPL with a monoclonal anti-LPL antibody or with tetrahydrolipstatin blocked the ability of VLDL to enhance phagocytosis by LPL-expressing macrophages maintained in 1 mM glucose. Addition of oleic acid significantly enhanced phagocytosis by both LPL-expressing and LPL-KO macrophages maintained in 1 mM glucose. Moreover, oleic acid stimulated phagocytosis in cells cultured in non-glucose-containing medium, and increased the intracellular stores of creatine phosphate. Inhibition of oxidative phosphorylation, but not of glycolysis, blocked the capacity of oleic acid to stimulate phagocytosis. Receptor-mediated endocytosis of acetyl LDL by macrophages from LPL-expressing and LPL-KO mice was similar whether the cells were maintained in 5 or 1 mM glucose, and was not augmented by VLDL. We postulate that fatty acids derived from macrophage LPL-catalyzed hydrolysis of triglycerides and

  2. Pharmacological characterization of prostanoid receptors mediating vasoconstriction in human umbilical vein

    PubMed Central

    Daray, Federico Manuel; Minvielle, Ana Itatí; Puppo, Soledad; Rothlin, Rodolfo Pedro

    2003-01-01

    This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating contraction in human umbilical vein (HUV).HUV rings were mounted in organ baths and concentration–response curves to U-46619 (TXA2 mimetic) were constructed in the absence or presence of SQ-29548 or ICI-192,605 (TP receptor antagonists). U-46619 was a potent constrictor (pEC50: 8.03). SQ-29548 and ICI-192,605 competitively antagonized responses to U-46619 with pKB values of 7.96 and 9.07, respectively.Concentration–response curves to EP receptor agonists: PGE2, misoprostol and 17-phenyl-trinor-PGE2 gave pEC50 values of 5.06, 5.25 and 5.32, respectively. Neither pEC50 nor maximum of PGE2 and 17-phenyl-trinor-PGE2 concentration–response curves were modified by the DP/EP1/EP2 receptor antagonist AH 6809 (1 μM). However, ICI-192,605 produced a concentration-dependent antagonism of the responses to all the EP receptor agonists. The pA2 estimated for ICI-192,605 against PGE2 or misoprostol were 8.91 and 9.22, respectively.Concentration–response curves to FP receptor agonists: PGF2α and fluprostenol gave pEC50 values of 6.20 and 5.82, respectively. ICI-192,605 (100 nM) was completely ineffective against PGF2α or fluprostenol. In addition, lack of antagonistic effect of AH 6809 (1 μM) against PGF2α was observed.In conclusion, the findings obtained with TP-selective agonist and antagonists provide strong evidence of the involvement of TP receptors promoting vasoconstriction in HUV. Furthermore, the action of the natural and synthetic EP receptor agonists appears to be mediated via TP receptors. On the other hand, the results employing FP receptor agonists and antagonists of different prostanoid receptors suggest the presence of FP receptors mediating vasoconstriction in this vessel. PMID:12922927

  3. 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

  4. Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

    SciTech Connect

    Hensler, J.G.; Cotterell, D.J.; Dubocovich, M.L.

    1987-12-01

    Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent (/sup 3/H)acetylcholine release from rabbit retina labeled in vitro with (/sup 3/H)choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of (/sup 3/H)acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of (/sup 3/H)acetylcholine with the following order of potency: apomorphine less than or equal to SKF(R)82526 < SKF 85174 < SKF(R)38393 less than or equal to pergolide less than or equal to dopamine (EC50 = 4.5 microM) < SKF(S)82526 less than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of (/sup 3/H)acetylcholine: SCH 23390 (IC50 = 1 nM) < (+)-butaclamol less than or equal to cis-flupenthixol < fluphenazine < perphenazine < trans-flupenthixol < R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating (/sup 3/H)acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by (/sup 3/H)SCH 23390, or as determined by adenylate cyclase activity. (/sup 3/H)SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of (/sup 3/H)SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate (/sup 3/H)acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at (/sup 3/H)SCH 23390 binding sites (r = 0.755, P < .05, n = 8).

  5. FoxP2 in songbirds.

    PubMed

    Wohlgemuth, Sandra; Adam, Iris; Scharff, Constance

    2014-10-01

    Humans with mutations in the transcription factor FOXP2 display a severe speech disorder. Songbirds are a powerful model system to study FoxP2. Like humans, songbirds communicate via vocalizations that are imitatively learned during critical periods and this learning is influenced by social factors and relies on functionally lateralized neural circuits. During the past five years significant progress has been made moving from a descriptive to a more mechanistic understanding of how FoxP2 functions in songbirds. Current evidence from molecular and electrophysiological studies indicates that FoxP2 is important for shaping synaptic plasticity of specific neuron populations. One future goal will be to identify the transcriptional regulation orchestrated by FoxP2 and its associated molecular network that brings about these physiological effects. This will be key to further unravel how FoxP2 influences synaptic function and thereby contributes to auditory guided vocal motor behavior in the songbird model.

  6. Chronic Exposure to Anabolic Androgenic Steroids Alters Neuronal Function in the Mammalian Forebrain via Androgen Receptor- and Estrogen Receptor-Mediated Mechanisms

    PubMed Central

    Penatti, Carlos A A; Porter, Donna M; Henderson, Leslie P

    2009-01-01

    Anabolic androgenic steroids (AAS) can promote detrimental effects on social behaviors for which γ-aminobutyric acid type A (GABAA) receptor-mediated circuits in the forebrain play a critical role. While all AAS bind to androgen receptors (AR), they may also be aromatized to estrogens and thus potentially impart effects via estrogen receptors (ER). Chronic exposure of wild type male mice to a combination of chemically distinct AAS increased action potential (AP) frequency, selective GABAA receptor subunit mRNAs, and GABAergic synaptic current decay in the medial preoptic area (mPOA). Experiments performed with pharmacological agents and in AR-deficient Tfm mutant mice suggest that the AAS-dependent enhancement of GABAergic transmission in wild type mice is AR-mediated. In AR-deficient mice, the AAS elicited dramatically different effects, decreasing AP frequency, sIPSC amplitude and frequency and the expression of selective GABAA receptor subunit mRNAs. Surprisingly, in the absence of AR signaling, the data indicate that the AAS do not act as ER agonists, but rather suggest a novel in vivo action in which the AAS inhibit aromatase and impair endogenous ER signaling. These results show that the AAS have the capacity to alter neuronal function in the forebrain via multiple steroid signaling mechanisms and suggest that effects of these steroids in the brain will depend not only on the balance of AR- vs. ER-mediated regulation for different target genes, but also on the ability of these drugs to alter steroid metabolism and thus the endogenous steroid milieu. PMID:19812324

  7. Chronic exposure to anabolic androgenic steroids alters neuronal function in the mammalian forebrain via androgen receptor- and estrogen receptor-mediated mechanisms.

    PubMed

    Penatti, Carlos A A; Porter, Donna M; Henderson, Leslie P

    2009-10-07

    Anabolic androgenic steroids (AAS) can promote detrimental effects on social behaviors for which GABA type A (GABA(A)) receptor-mediated circuits in the forebrain play a critical role. While all AAS bind to androgen receptors (AR), they may also be aromatized to estrogens and thus potentially impart effects via estrogen receptors (ER). Chronic exposure of wild-type male mice to a combination of chemically distinct AAS increased action potential (AP) frequency, selective GABA(A) receptor subunit mRNAs, and GABAergic synaptic current decay in the medial preoptic area (mPOA). Experiments performed with pharmacological agents and in AR-deficient Tfm mutant mice suggest that the AAS-dependent enhancement of GABAergic transmission in wild-type mice is AR-mediated. In AR-deficient mice, the AAS elicited dramatically different effects, decreasing AP frequency, spontaneous IPSC amplitude and frequency and the expression of selective GABA(A) receptor subunit mRNAs. Surprisingly, in the absence of AR signaling, the data indicate that the AAS do not act as ER agonists, but rather suggest a novel in vivo action in which the AAS inhibit aromatase and impair endogenous ER signaling. These results show that the AAS have the capacity to alter neuronal function in the forebrain via multiple steroid signaling mechanisms and suggest that effects of these steroids in the brain will depend not only on the balance of AR- versus ER-mediated regulation for different target genes, but also on the ability of these drugs to alter steroid metabolism and thus the endogenous steroid milieu.

  8. Zonal differences in ethanol-induced impairments in receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J. )

    1991-02-01

    We have shown previously that ethanol-induced defects in receptor-mediated endocytosis of asialoorosomucoid occurred as early as 1 wk after ethanol feeding. This study was undertaken as an initial attempt to establish a possible role of defective receptor-mediated endocytosis in liver injury by investigating whether differences exist in the effects of ethanol on receptor-mediated endocytosis in hepatocytes isolated from different regions of the liver. Perivenule cells, present in the distal half of the liver, are thought to be more susceptible to ethanol-induced liver injury than are the periportal cells located in the proximal half of the liver acini. For these studies, we fed male Sprague-Dawley rats for 7 days with liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate. Perivenule and periportal hepatocytes were then isolated using a digitonin-collagenase perfusion method. In control animals, cells isolated from the perivenule region bound significantly more ligand than did cells from the periportal region. Amounts of ligand internalized and degraded were also greater in perivenule than in periportal cells in these animals. After ethanol feeding, cells isolated from both the perivenule and periportal regions bound significantly less ligand than their respective controls. This impairment in surface and total binding was more pronounced in perivenule than in periportal cells. Internalization and degradation of the ligand were also more adversely affected in the centrilobular region as shown by decreases of greater than 60% in perivenule cells and by only 20% to 30% in periportal cells of ethanol-fed animals compared with controls.

  9. Wnt5a promotes cancer cell invasion and proliferation by receptor-mediated endocytosis-dependent and -independent mechanisms, respectively

    PubMed Central

    Shojima, Kensaku; Sato, Akira; Hanaki, Hideaki; Tsujimoto, Ikuko; Nakamura, Masahiro; Hattori, Kazunari; Sato, Yuji; Dohi, Keiji; Hirata, Michinari; Yamamoto, Hideki; Kikuchi, Akira

    2015-01-01

    Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity. For this action, Wnt5a-induced receptor endocytosis with clathrin is required. Wnt5a expression was previously believed to be associated with cancer cell motility but not proliferation. Recently, it was reported that Wnt5a is also implicated in cancer cell proliferation, but the mechanism was not clear. In this study, we generated a neutralizing anti-Wnt5a monoclonal antibody (mAb5A16) to investigate the mechanism by which Wnt5a regulates cancer cell proliferation. Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells. Wnt5a activated Src family kinases (SFKs) and Wnt5a-dependent cancer cell proliferation was dependent on SFKs, yet blockade of receptor-mediated endocytosis did not affect cancer cell proliferation and SFK activity. These results suggest that Wnt5a promotes invasion and proliferation of certain types of cancer cells through receptor-mediated endocytosis-dependent and -independent mechanisms, respectively. PMID:25622531

  10. P2X Receptors as Drug Targets

    PubMed Central

    Jarvis, Michael F.

    2013-01-01

    The study of P2X receptors has long been handicapped by a poverty of small-molecule tools that serve as selective agonists and antagonists. There has been progress, particularly in the past 10 years, as cell-based high-throughput screening methods were applied, together with large chemical libraries. This has delivered some drug-like molecules in several chemical classes that selectively target P2X1, P2X3, or P2X7 receptors. Some of these are, or have been, in clinical trials for rheumatoid arthritis, pain, and cough. Current preclinical research programs are studying P2X receptor involvement in pain, inflammation, osteoporosis, multiple sclerosis, spinal cord injury, and bladder dysfunction. The determination of the atomic structure of P2X receptors in closed and open (ATP-bound) states by X-ray crystallography is now allowing new approaches by molecular modeling. This is supported by a large body of previous work using mutagenesis and functional expression, and is now being supplemented by molecular dynamic simulations and in silico ligand docking. These approaches should lead to P2X receptors soon taking their place alongside other ion channel proteins as therapeutically important drug targets. PMID:23253448

  11. P2 performance measurement tools workbook: Draft

    SciTech Connect

    1995-06-01

    The underlying purpose of the Department of Energy`s (DOE) Waste Minimization and Pollution Prevention (WMin/P2) Program is compliance with the waste management regulations set forth by the DOE, the federal government, and individual state and local agencies 1. In addition to these regulatory mandates, the increases in waste management costs and public interest in environmental issues have created other drivers to develop and demonstrate an effective WMin/P2 Program. The Waste Minimization Division (EM-334) must have adequate methods to calculate and roll up pollution prevention (P2) progress to meet the WMin/P2 requirements; these requirements support DOE and national objectives and direct funding. This document outlines a system to evaluate DOE`s P2 progress towards the waste reduction requirements. The emphasis of these pollution prevention measurements is to evaluate whether P2 activities are effective, (i.e., has the required amount of waste been reduced as a result of the P2 activities) and to evaluate the cost management of P2 projects. The performance evaluation system presented in this document encompass these aspects: (1) site requirements that apply to all DOE waste generating organizations, (2) a baseline that is not affected by short-term waste generation, and (3) key indicators that can be rolled up across DOE sites and across specific Cognizant Secretarial Officers` (CSO) sites. In a performance-based management system, requirements are the fundamental link between the planning and measurement process. The site requirements are {open_quotes}targets{close_quotes} at the process or activity level. Measuring DOE`s P2 progress toward these requirements provides the necessary feedback to (1) compare performance with the requirements/standards (i.e., whether the reduction requirement of 50% by 1999 is achievable) (2) detect departures from planned levels of performance, and (3) restore performance to the planned levels or achieve new levels of performance.

  12. Characterization of P2P Systems

    NASA Astrophysics Data System (ADS)

    Stutzbach, Daniel; Rejaie, Reza

    The combination of large scale and geographically distributed nature of P2P system has led to their significant impact on the Internet. It is essential to characterize deployed P2P system for at least three reasons: (1) Accurately assessing their impact on the Internet, (2) identifying any performance bottleneck as well as any opportunity for performance improvement, (3) understanding user-driven dynamics in P2P systems. To characterize a P2P system, one needs to accurately capture snapshots of the resulting P2P overlay. This is challenging because the overlay is often large and dynamic. While the overlay is discovered by a crawler, it is changing which leads to a distorted view of the system. Capturing unbiased view of the traffic in the overlay is equally challenging because it is difficult to show that the captured behavior represent the observed behavior by all peers. In this chapter, we describe some of the fundamental problems in empirical characterization of widely deployed P2P systems. We present several examples to illustrate the effect of ad-hoc measurement/data collection on the resulting analysis/characterization. We then present two sampling techniques as a powerful approach to capture unbiased view of peer properties in a scalable fashion.

  13. Loss of dopamine D1 receptors and diminished D1/5 receptor-mediated ERK phosphorylation in the periaqueductal gray after spinal cord lesion

    PubMed Central

    Voulalas, Pamela J.; Ji, Yadong; Jiang, Li; Asgar, Jamila; Ro, Jin Y.; Masri, Radi

    2016-01-01

    Neuropathic pain resulting from spinal cord injury is often accompanied by maladaptive plasticity of the central nervous system, including the opioid receptor-rich periaqueductal gray (PAG). Evidence suggests that sensory signaling via the PAG is robustly modulated by dopamine D1- and D2-like receptors, but the effect of damage to the spinal cord on D1 and D2 receptor protein expression and function in the PAG has not been examined. Here we show that 21 days after a T10 or C6 spinothalamic tract lesion, both mice and rats display a remarkable decline in the expression of D1 receptors in the PAG, revealed by western blot analysis. These changes were associated with a significant reduction in hindpaw withdrawal thresholds in lesioned animals compared to sham-operated controls. We investigated the consequences of diminished D1 receptor levels by quantifying D1-like receptor-mediated phosphorylation of ERK1,2 and CREB, events that have been observed in numerous brain structures. In naïve animals, western blot analysis revealed that ERK1,2, but not CREB phosphorylation was significantly increased in the PAG by the D1-like agonist SKF 81297. Using immunohistochemistry, we found that SKF 81297 increased ERK1,2 phosphorylation in the PAG of sham animals. However, in lesioned animals, basal pERK1,2 levels were elevated and did not significantly increase after exposure to SKF 81297. Our findings provide support for the hypothesis that molecular adaptions resulting in a decrease in D1 receptor expression and signaling in the PAG are a consequence of SCL. PMID:27932310

  14. 5-Carboxamidotryptamine is a selective agonist at 5-hydroxytryptamine receptors mediating vasodilatation and tachycardia in anaesthetized cats.

    PubMed Central

    Connor, H. E.; Feniuk, W.; Humphrey, P. P.; Perren, M. J.

    1986-01-01

    We have attempted to characterize the 5-hydroxytryptamine (5-HT) receptors mediating bronchoconstriction, vasodilatation, vasodepression and tachycardia in anaesthetized cats following bilateral vagosympathectomy and beta-adrenoceptor blockade with propranolol. 5-HT (1-100 micrograms/kg-1 i.v.) caused dose-related bronchoconstriction and tachycardia but variable and complex effects on diastolic blood pressure and carotid arterial vascular resistance. In contrast, 5-carboxamidotryptamine (5-CT; 0.01-1 micrograms kg-1 i.v.) caused consistent, dose-related decreases in diastolic blood pressure and carotid arterial vascular resistance and increases in heart rate. 5-CT did not cause bronchoconstriction. The 5-HT-induced bronchoconstriction was dose-dependently antagonized by methiothepin, methysergide and ketanserin (10-100 micrograms kg-1 i.v.). The highest doses used of these antagonists did not antagonize bronchoconstriction induced by prostaglandin F2 alpha. The high potency of all three antagonists indicate a 5-HT2-receptor mediated effect. The 5-HT- and 5-CT-induced tachycardia as well as the 5-CT-induced vasodepressor and carotid arterial vasodilator responses were dose-dependently antagonized by low doses of methiothepin (10-100 micrograms kg-1 i.v.) and by high doses of methysergide (100-1000 micrograms kg-1 i.v.) but were little affected by ketanserin in doses up to 1000 micrograms kg-1 i.v. These selective effects of 5-CT appear to be mediated by '5-HT1-like' receptors. PMID:2937503

  15. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed Central

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-01-01

    Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells. PMID:9593770

  16. Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system

    PubMed Central

    Limaye, Arati; Koya, Vijay; Samsam, Mohtashem; Daniell, Henry

    2012-01-01

    Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins. PMID:16603603

  17. Reboxetine enhances the olanzapine-induced antipsychotic-like effect, cortical dopamine outflow and NMDA receptor-mediated transmission.

    PubMed

    Marcus, Monica M; Jardemark, Kent; Malmerfelt, Anna; Björkholm, Carl; Svensson, Torgny H

    2010-08-01

    Preclinical data have shown that addition of the selective norepinephrine transporter (NET) inhibitor reboxetine increases the antipsychotic-like effect of the D(2/3) antagonist raclopride and, in parallel, enhances cortical dopamine output. Subsequent clinical results suggested that adding reboxetine to stable treatments with various antipsychotic drugs (APDs) may improve positive, negative and depressive symptoms in schizophrenia. In this study, we investigated in rats the effects of adding reboxetine to the second-generation APD olanzapine on: (i) antipsychotic efficacy, using the conditioned avoidance response (CAR) test, (ii) extrapyramidal side effect (EPS) liability, using a catalepsy test, (iii) dopamine efflux in the medial prefrontal cortex and the nucleus accumbens, using in vivo microdialysis in freely moving animals and (iv) cortical N-methyl-D-aspartate (NMDA) receptor-mediated transmission, using intracellular electrophysiological recording in vitro. Reboxetine (6 mg/kg) enhanced the suppression of CAR induced by a suboptimal dose (1.25 mg/kg), but not an optimal (2.5 mg/kg) dose of olanzapine without any concomitant catalepsy. Addition of reboxetine to the low dose of olanzapine also markedly increased cortical dopamine outflow and facilitated prefrontal NMDA receptor-mediated transmission. Our data suggest that adjunctive treatment with a NET inhibitor may enhance the therapeutic effect of low-dose olanzapine in schizophrenia without increasing EPS liability and add an antidepressant action, thus in principle allowing for a dose reduction of olanzapine with a concomitant reduction of dose-related side effects, such as EPS and weight gain.

  18. Maltodextrin and fat preference deficits in "taste-blind" P2X2/P2X3 knockout mice.

    PubMed

    Sclafani, Anthony; Ackroff, Karen

    2014-07-01

    Adenosine triphosphate is a critical neurotransmitter in the gustatory response to the 5 primary tastes in mice. Genetic deletion of the purinergic P2X2/P2X3 receptor greatly reduces the neural and behavioral response to prototypical primary taste stimuli. In this study, we examined the behavioral response of P2X double knockout mice to maltodextrin and fat stimuli, which appear to activate additional taste channels. P2X double knockout and wild-type mice were given 24-h choice tests (vs. water) with ascending concentrations of Polycose and Intralipid. In Experiment 1, naive double knockout mice, unlike wild-type mice, were indifferent to dilute (0.5-4%) Polycose solutions but preferred concentrated (8-32%) Polycose to water. In a retest, the Polycose-experienced double knockout mice, like wild-type mice, preferred all Polycose concentrations. In Experiment 2, naive double knockout mice, unlike wild-type mice, were indifferent to dilute (0.313-2.5%) Intralipid emulsions but preferred concentrated (5-20%) Intralipid to water. In a retest, the fat-experienced double knockout mice, like wild-type mice, strongly preferred 0.313-5% Intralipid to water. These results indicate that the inherent preferences of mice for maltodextrin and fat are dependent upon adenosine triphosphate taste cell signaling. With experience, however, P2X double knockout mice develop strong preferences for the nontaste flavor qualities of maltodextrin and fat conditioned by the postoral actions of these nutrients.

  19. Adiponectin regulate growth hormone secretion via adiponectin receptor mediated Ca(2+) signalling in rat somatotrophs in vitro.

    PubMed

    Steyn, F J; Boehme, F; Vargas, E; Wang, K; Parkington, H C; Rao, J R; Chen, C

    2009-08-01

    Obesity is associated with reduced levels of growth hormone (GH) and the disruption of pulsatile GH secretion. This results in relative GH deficiency. It is likely that a regulatory relationship between GH secretion and adipose tissue exists as the secretion of GH recovers to normal levels after a reduction in body weight. This report characterise the expression and interaction of adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) and adiponectin, respectively, in regulating the activity of GH secreting cells. Polymerase chain reaction analysis of the GH3 cell line, rat anterior pituitary gland and isolated somatotroph cells from transgenic GFP expressing mice confirmed the expression of both AdipoR1 and AdipoR2 in GH secretory cells. Because GH cells expressed both receptors, it is likely that the measured increase in GH secretion, observed in primary cultured rat pituitary cells after 30 min of incubation with full-length murine adiponectin, was mediated by a direct receptor regulated process. Adiponectin induced an increase in intracellular Ca(2+) through both the influx of extracellular Ca(2+) and the release of intracellular Ca(2+) stores resulting in the secretion of GH. Furthermore, results confirm that this increase in GH secretion depended mainly on an increase in Ca(2+) influx through L-type Ca(2+) channels. It is concluded that adiponectin directly regulates GH secretion from somatotrophs by binding to either adiponectin receptor, and that this is mediated via a similar process observed after the stimulation of GH secretion by GH-releasing hormone.

  20. Human Milk Components Modulate Toll-Like Receptor-Mediated Inflammation.

    PubMed

    He, YingYing; Lawlor, Nathan T; Newburg, David S

    2016-01-01

    Toll-like receptor (TLR) signaling is central to innate immunity. Aberrant expression of TLRs is found in neonatal inflammatory diseases. Several bioactive components of human milk modulate TLR expression and signaling pathways, including soluble toll-like receptors (sTLRs), soluble cluster of differentiation (sCD) 14, glycoproteins, small peptides, and oligosaccharides. Some milk components, such as sialyl (α2,3) lactose and lacto-N-fucopentaose III, are reported to increase TLR signaling; under some circumstances this might contribute toward immunologic balance. Human milk on the whole is strongly anti-inflammatory, and contains abundant components that depress TLR signaling pathways: sTLR2 and sCD14 inhibit TLR2 signaling; sCD14, lactadherin, lactoferrin, and 2'-fucosyllactose attenuate TLR4 signaling; 3'-galactosyllactose inhibits TLR3 signaling, and β-defensin 2 inhibits TLR7 signaling. Feeding human milk to neonates decreases their risk of sepsis and necrotizing enterocolitis. Thus, the TLR regulatory components found in human milk hold promise as benign oral prophylactic and therapeutic treatments for the many gastrointestinal inflammatory disorders mediated by abnormal TLR signaling.

  1. Double P2X2/P2X3 Purinergic Receptor Knockout Mice Do Not Taste NaCl or the Artificial Sweetener SC45647

    PubMed Central

    Eddy, Meghan C.; Eschle, Benjamin K.; Barrows, Jennell; Hallock, Robert M.; Finger, Thomas E.

    2009-01-01

    The P2X ionotropic purinergic receptors, P2X2 and P2X3, are essential for transmission of taste information from taste buds to the gustatory nerves. Mice lacking both P2X2 and P2X3 purinergic receptors (P2X2/P2X3Dbl−/−) exhibit no taste-evoked activity in the chorda tympani and glossopharyngeal nerves when stimulated with taste stimuli from any of the 5 classical taste quality groups (salt, sweet, sour, bitter, and umami) nor do the mice show taste preferences for sweet or umami, or avoidance of bitter substances (Finger et al. 2005. ATP signaling is crucial for communication from taste buds to gustatory nerves. Science. 310[5753]:1495–1499). Here, we compare the ability of P2X2/P2X3Dbl−/− mice and P2X2/P2X3Dbl+/+ wild-type (WT) mice to detect NaCl in brief-access tests and conditioned aversion paradigms. Brief-access testing with NaCl revealed that whereas WT mice decrease licking at 300 mM and above, the P2X2/P2X3Dbl−/− mice do not show any change in lick rates. In conditioned aversion tests, P2X2/P2X3Dbl−/− mice did not develop a learned aversion to NaCl or the artificial sweetener SC45647, both of which are easily avoided by conditioned WT mice. The inability of P2X2/P2X3Dbl−/− mice to show avoidance of these taste stimuli was not due to an inability to learn the task because both WT and P2X2/P2X3Dbl−/− mice learned to avoid a combination of SC45647 and amyl acetate (an odor cue). These data suggest that P2X2/P2X3Dbl−/− mice are unable to respond to NaCl or SC45647 as taste stimuli, mirroring the lack of gustatory nerve responses to these substances. PMID:19833661

  2. Anonymity in P2P Systems

    NASA Astrophysics Data System (ADS)

    Manzanares-Lopez, Pilar; Muñoz-Gea, Juan Pedro; Malgosa-Sanahuja, Josemaria; Sanchez-Aarnoutse, Juan Carlos

    In the last years, the use of peer-to-peer (P2P) applications to share and exchange knowledge among people around the world has experienced an exponential growth. Therefore, it is understandable that, like in any successful communication mechanism used by a lot of humans being, the anonymity can be a desirable characteristic in this scenario. Anonymity in P2P networks can be obtained by means of different methods, although the most significant ones are broadcast protocols, dining-cryptographer (DC) nets and multiple-hop paths. Each of these methods can be tunable in order to build a real anonymity P2P application. In addition, there is a mathematical tool called entropy that can be used in some scenarios to quantify anonymity in communication networks. In some cases, it can be calculated analytically but in others it is necessary to use simulation to obtain the network entropy.

  3. Nucleotide P2Y13-stimulated phosphorylation of CREB is required for ADP-induced proliferation of late developing retinal glial progenitors in culture.

    PubMed

    Jacques, Flavia Jesus; Silva, Thayane Martins; da Silva, Flavia Emenegilda; Ornelas, Isis Moraes; Ventura, Ana Lucia Marques

    2017-03-24

    Nucleotides stimulate phosphorylation of CREB to induce cell proliferation and survival in diverse cell types. We report here that ADP induces the phosphorylation of CREB in a time- and concentration-dependent manner in chick embryo retinal progenitors in culture. ADP-induced increase in phospho-CREB is mediated by P2 receptors as it is blocked by PPADS but not by the adenosine antagonists DPCPX or ZM241385. Incubation of the cultures with the CREB inhibitor KG-501 prevents ADP-induced incorporation of [(3)H]-thymidine, indicating that CREB is involved in retinal cell proliferation. No effect of this compound is observed on the viability of retinal progenitors. While no significant increase in CREB phosphorylation is observed with the P2Y1 receptor agonist MRS2365, ADP-induced phosphorylation of CREB is blocked by the P2Y13 receptor selective antagonist MRS2211, but not by MRS2179 or PSB0739, two antagonists of the P2Y1 and P2Y12 receptors, respectively, suggesting that ADP-induced CREB phosphorylation is mediated by P2Y13 receptors. ADP-induced increase in phospho-CREB is attenuated by the PI3K inhibitor LY241385 and completely prevented by the MEK inhibitor U0126, suggesting that at least ERK is involved in ADP-induced CREB phosphorylation. A pharmacological profile similar to the activation and inhibition of CREB phosphorylation is observed in the phosphorylation of ERK, suggesting that P2Y13 receptors mediate ADP induced ERK/CREB pathway in the cultures. While no increase in [(3)H]-thymidine incorporation is observed with the P2Y1 receptor agonist MRS2365, both MRS2179 and MRS2211 prevent ADP-mediated increase in [(3)H]-thymidine incorporation, but not progenitor's survival, suggesting that both P2Y1 and P2Y13 receptor subtypes are involved in ADP-induced cell proliferation. P2Y1 receptor-mediated increase in [Ca(2+)]i is observed in glial cells only when cultures maintained for 9days are used. In glia from cultures cultivated for only 2days, no increase in [Ca

  4. Mechanism-Based Tumor-Targeting Drug Delivery System. Validation of Efficient Vitamin Receptor-Mediated Endocytosis and Drug Release

    SciTech Connect

    Chen, S.; Wong, S.; Zhao, X.; Chen, J.; Chen, J.; Kuznetsova, L.; Ojima, I.

    2010-05-01

    An efficient mechanism-based tumor-targeting drug delivery system, based on tumor-specific vitamin-receptor mediated endocytosis, has been developed. The tumor-targeting drug delivery system is a conjugate of a tumor-targeting molecule (biotin: vitamin H or vitamin B-7), a mechanism-based self-immolative linker and a second-generation taxoid (SB-T-1214) as the cytotoxic agent. This conjugate (1) is designed to be (i) specific to the vitamin receptors overexpressed on tumor cell surface and (ii) internalized efficiently through receptor-mediated endocytosis, followed by smooth drug release via glutathione-triggered self-immolation of the linker. In order to monitor and validate the sequence of events hypothesized, i.e., receptor-mediated endocytosis of the conjugate, drug release, and drug-binding to the target protein (microtubules), three fluorescent/fluorogenic molecular probes (2, 3, and 4) were designed and synthesized. The actual occurrence of these processes was unambiguously confirmed by means of confocal fluorescence microscopy (CFM) and flow cytometry using L1210FR leukemia cells, overexpressing biotin receptors. The molecular probe 4, bearing the taxoid linked to fluorescein, was also used to examine the cell specificity (i.e., efficacy of receptor-based cell targeting) for three cell lines, L1210FR (biotin receptors overexpressed), L1210 (biotin receptors not overexpressed), and WI38 (normal human lung fibroblast, biotin receptor negative). As anticipated, the molecular probe 4 exhibited high specificity only to L1210FR. To confirm the direct correlation between the cell-specific drug delivery and anticancer activity of the probe 4, its cytotoxicity against these three cell lines was also examined. The results clearly showed a good correlation between the two methods. In the same manner, excellent cell-specific cytotoxicity of the conjugate 1 (without fluorescein attachment to the taxoid) against the same three cell lines was confirmed. This mechanism

  5. The oxidized low-density lipoprotein receptor mediates vascular effects of inhaled vehicle emissions

    EPA Science Inventory

    Rationale: To determine vascular signaling pathways involved in air pollution (vehicular engine emission) exposure -induced exacerbation of atherosclerosis, associated with onset of clinical cardiovascular events. Objective: To elucidate the role of oxidized LDL (oxLDL) and its ...

  6. Adjunctive β2-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with Pompe disease

    PubMed Central

    Farah, Benjamin L.; Madden, Lauran; Li, Songtao; Nance, Sierra; Bird, Andrew; Bursac, Nenad; Yen, Paul M.; Young, Sarah P.; Koeberl, Dwight D.

    2014-01-01

    Enzyme or gene replacement therapy with acid α-glucosidase (GAA) has achieved only partial efficacy in Pompe disease. We evaluated the effect of adjunctive clenbuterol treatment on cation-independent mannose-6-phosphate receptor (CI-MPR)-mediated uptake and intracellular trafficking of GAA during muscle-specific GAA expression with an adeno-associated virus (AAV) vector in GAA-knockout (KO) mice. Clenbuterol, which increases expression of CI-MPR in muscle, was administered with the AAV vector. This combination therapy increased latency during rotarod and wirehang testing at 12 wk, in comparison with vector alone. The mean urinary glucose tetrasaccharide (Glc4), a urinary biomarker, was lower in GAA-KO mice following combination therapy, compared with vector alone. Similarly, glycogen content was lower in cardiac and skeletal muscle following 12 wk of combination therapy in heart, quadriceps, diaphragm, and soleus, compared with vector alone. These data suggested that clenbuterol treatment enhanced trafficking of GAA to lysosomes, given that GAA was expressed within myofibers. The integral role of CI-MPR was demonstrated by the lack of effectiveness from clenbuterol in GAA-KO mice that lacked CI-MPR in muscle, where it failed to reverse the high glycogen content of the heart and diaphragm or impaired wirehang performance. However, the glycogen content of skeletal muscle was reduced by the addition of clenbuterol in the absence of CI-MPR, as was lysosomal vacuolation, which correlated with increased AKT signaling. In summary, β2-agonist treatment enhanced CI-MPR-mediated uptake and trafficking of GAA in mice with Pompe disease, and a similarly enhanced benefit might be expected in other lysosomal storage disorders.—Farah, B. L., Madden, L., Li, S., Nance, S., Bird, A., Bursac, N., Yen, P. M., Young, S. P., Koeberl, D. D. Adjunctive β2-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with

  7. Aryl hydrocarbon receptor-mediated and estrogenic activities of oxygenated polycyclic aromatic hydrocarbons and azaarenes originally identified in extracts of river sediments.

    PubMed

    Machala, M; Ciganek, M; Bláha, L; Minksová, K; Vondráck, J

    2001-12-01

    Reproductive dysfunction in wildlife populations can be a result of environmental contaminants binding to aryl hydrocarbon receptor (AhR) or estrogenic receptors. Signaling by both types of receptors can be affected by polycyclic aromatic hydrocarbons (PAHs), which are potential endocrine disruptors. However, our knowledge regarding the effects of oxygenated (oxy)-PAHs and azaarenes on AhR-mediated and estrogenic activities is incomplete. In the present study, we have identified 9-fluorenone, anthrone, anthraquinone, benzanthrone, benz[a]anthracene-7,12-dione, benz[c]acridine, and dibenz[a,h]acridine as prevalent oxy-PAHs and azaarenes found in river sediments. Their concentrations in sediment samples ranged from 2.1 to 165.2 ng g(-1) for oxy-PAHs and up to 27.3 ng g(-1) for azaarenes. Their relative AhR-inducing and estrogenic potencies were quantified in vitro using two cell lines that were stably transfected with a luciferase reporter gene system and expressed as induction equivalency factors (IEFs). The only oxy-PAHs with detectable levels of in vitro AhR-mediated activity were benzanthrone and benz[a]anthracene-7,12-dione. However, their IEFs were approximately three to four orders of magnitude lower than those of benzo[a]pyrene. On the other hand, azaarenes showed a strong AhR-mediated activity, with dibenzo[a,h]acridine being a far more potent inducer of activity than benzo[a]pyrene. Benzanthrone, benz[a]anthracene-7,12-dione, anthraquinone, and benz[a]acridine were weak inducers of in vitro estrogenic activity, with IEFs similar to that of benzo[a]pyrene. Based on concentrations and relative potencies, our results suggest that dibenzo[a,h]acridine can significantly contribute to the overall AhR-mediated activity in river sediments, whereas the remaining compounds do not. No studied compound was found to contribute significantly to estrogen receptor-mediated activity in vitro.

  8. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    SciTech Connect

    Murayama, T.; Ui, M.

    1985-06-25

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.

  9. CB(1) and CB(2) cannabinoid receptors mediate different aspects of delta-9-tetrahydrocannabinol (THC)-induced T helper cell shift following immune activation by Legionella pneumophila infection.

    PubMed

    Newton, Catherine A; Chou, Ping-Jen; Perkins, Izabella; Klein, Thomas W

    2009-03-01

    Legionella pneumophila infection of mice induces proinflammatory cytokines and Th1 immunity as well as rapid increases in serum levels of IL-12 and IFNgamma and splenic IL-12Rbeta2 expression. Delta-9-tetrahydrocannabinol (THC) treatment prior to infection causes a shift from Th1 to Th2 immunity and here we demonstrate that CB(1) and CB(2) cannabinoid receptors mediate different aspects of the shift. Using cannabinoid receptor antagonists and cannabinoid receptor gene deficient mice (CB(1) (-/-) and CB(2) (-/-)), we showed that both CB(1) and CB(2) receptors were involved in the THC-induced attenuation of serum IL-12 and IFNgamma. IFNgamma production is dependent upon signaling through IL-12Rbeta2 (beta2) and THC treatment suppressed splenic beta2 message; moreover, this effect was CB(1) but not CB(2)-dependent from studies with receptor antagonists and CB1(-/-) and CB2(-/-) mice. Furthermore, observed increases in IL-4 induced by THC, were not involved in the drug effect on beta2 from studies with IL-4 deficient mice. The GATA-3 transcription factor is necessary for IL-4 production and is selectively expressed in Th2 cells. GATA-3 message levels were elevated in spleens of THC-treated and L. pneumophila-infected mice and the effect was shown to be CB(2) but not CB(1)-dependent. Furthermore, GATA-3 regulatory factors were modulated in that Notch ligand Delta4 mRNA was decreased and Jagged1 increased by THC also in a CB2-dependent manner and splenic NFkappaB p65 was increased. Together, these results indicate that CB(1) and CB(2) mediate the THC-induced shift in T helper activity in L. pneumophila-infected mice, with CB(1) involved in suppressing IL-12Rbeta2 and CB(2) involved in enhancing GATA-3.

  10. Activation of the sigma receptor 1 modulates AMPA receptor-mediated light-evoked excitatory postsynaptic currents in rat retinal ganglion cells.

    PubMed

    Liu, Lei-Lei; Deng, Qin-Qin; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2016-09-22

    Sigma receptor (σR), a unique receptor family, is classified into three subtypes: σR1, σR2 and σR3. It was previously shown that σR1 activation induced by 1μM SKF10047 (SKF) suppressed N-methyl-d-aspartate (NMDA) receptor-mediated responses of rat retinal ganglion cells (GCs) and the suppression was mediated by a distinct Ca(2+)-dependent phospholipase C (PLC)-protein kinase C (PKC) pathway. In the present work, using whole-cell patch-clamp techniques in rat retinal slice preparations, we further demonstrate that SKF of higher dosage (50μM) significantly suppressed AMPA receptor (AMPAR)-mediated light-evoked excitatory postsynaptic currents (L-EPSCs) of retinal ON-type GCs (ON GCs), and the effect was reversed by the σR1 antagonist BD1047, suggesting the involvement of σR1. The SKF (50μM) effect was unlikely due to a change in glutamate release from bipolar cells, as suggested by the unaltered paired-pulse ratio (PPR) of AMPAR-mediated EPSCs of ON GCs. SKF (50μM) did not change L-EPSCs of ON GCs when the G protein inhibitor GDP-β-S or the protein kinase G (PKG) inhibitor KT5823 was intracellularly infused. Calcium imaging further revealed that SKF (50μM) did not change intracellular calcium concentration in GCs and persisted to suppress L-EPSCs when intracellular calcium was chelated by BAPTA. The SKF (50μM) effect was intact when protein kinase A (PKA) and phosphatidylinostiol (PI)-PLC signaling pathways were both blocked. We conclude that the SKF (50μM) effect is Ca(2+)-independent, PKG-dependent, but not involving PKA, PI-PLC pathways.

  11. P2X4R+ microglia drive neuropathic pain

    PubMed Central

    Beggs, Simon; Trang, Tuan; Salter, Michael W

    2016-01-01

    Neuropathic pain, the most debilitating of all clinical pain syndromes, may be a consequence of trauma, infection or pathology from diseases that affect peripheral nerves. Here we provide a framework for understanding the spinal mechanisms of neuropathic pain as distinct from those of acute pain or inflammatory pain. Recent work suggests that a specific microglia response phenotype characterized by de novo expression of the purinergic receptor P2X4 is critical for the pathogenesis of pain hypersensitivity caused by injury to peripheral nerves. Stimulating P2X4 receptors initiates a core pain signaling pathway mediated by release of brain-derived neurotrophic factor, which produces a disinhibitory increase in intracellular chloride in nociceptive (pain-transmitting) neurons in the spinal dorsal horn. The changes caused by signaling from P2X4R+ microglia to nociceptive transmission neurons may account for the main symptoms of neuropathic pain in humans, and they point to specific interventions to alleviate this debilitating condition. PMID:22837036

  12. Functional evidence for a 5-HT2B receptor mediating contraction of longitudinal muscle in human small intestine.

    PubMed Central

    Borman, R A; Burleigh, D E

    1995-01-01

    Application of 5-hydroxytryptamine induces contraction of longitudinal muscle strips from human terminal ileum. The response was resistant to antagonism by ketanserin, ondansetron or DAU6285, but was non-surmountably antagonized by methysergide. The selective 5-HT2B/2C receptor antagonist, SB 200646A evoked a concentration-dependent, parallel and dextral displacement of the concentration-response curve to 5-HT, yielding a pA2 estimate of 7.17. Application of yohimbine, a 5-HT1 and 5-HT2B receptor antagonist, also induced a rightward shift of the response curve to 5-HT, yielding a pA2 estimate of 8.10. In conclusion, it appears that a 5-HT2B receptor mediates the contractile response of the longitudinal muscle of human small intestine to 5-HT. PMID:7599919

  13. Progesterone reverts LPS-reduced FAAH activity in murine peripheral blood mononuclear cells by a receptor-mediated fashion.

    PubMed

    Wolfson, Manuel L; Aisemberg, Julieta; Salazar, Ana I; Domínguez Rubio, Ana P; Vercelli, Claudia A; Franchi, Ana M

    2013-12-05

    Increased anandamide concentrations are associated with pregnancy failure. Anandamide levels are regulated by the fatty acid amide hydrolase (FAAH). The aim of the study was to investigate the role of progesterone (P) on FAAH modulation in murine peripheral blood mononuclear cells (PBMC) under septic conditions. We observed that in vivo administration of LPS to non-pregnant (NP) mice decreased FAAH activity of PBMC while in pregnant mice no changes in FAAH activity were observed. NP animals administered with P had a similar response to LPS as the pregnant animals. Also, NP mice injected with P antagonist and P showed that the effect of P on LPS-reduced FAAH activity was impaired. Furthermore, LPS produced a decrease in the ratio of PR-B/PR-A in NP animals. Our results showed that, in our model the endotoxin decreased PBMC's FAAH activity and this condition was reverted by P in a receptor-mediated fashion.

  14. The influence of receptor-mediated interactions on reaction-diffusion mechanisms of cellular self-organisation.

    PubMed

    Klika, Václav; Baker, Ruth E; Headon, Denis; Gaffney, Eamonn A

    2012-04-01

    Understanding the mechanisms governing and regulating self-organisation in the developing embryo is a key challenge that has puzzled and fascinated scientists for decades. Since its conception in 1952 the Turing model has been a paradigm for pattern formation, motivating numerous theoretical and experimental studies, though its verification at the molecular level in biological systems has remained elusive. In this work, we consider the influence of receptor-mediated dynamics within the framework of Turing models, showing how non-diffusing species impact the conditions for the emergence of self-organisation. We illustrate our results within the framework of hair follicle pre-patterning, showing how receptor interaction structures can be constrained by the requirement for patterning, without the need for detailed knowledge of the network dynamics. Finally, in the light of our results, we discuss the ability of such systems to pattern outside the classical limits of the Turing model, and the inherent dangers involved in model reduction.

  15. Dose-response approaches for nuclear receptor-mediated modes of action for liver carcinogenicity: Results of a workshop.

    PubMed

    Andersen, Melvin E; Preston, R Julian; Maier, Andrew; Willis, Alison M; Patterson, Jacqueline

    2014-01-01

    A public workshop, organized by a Steering Committee of scientists from government, industry, universities and research organizations, was held at the National Institute of Environmental Health Sciences (NIEHS) in September, 2010. The workshop explored the dose-response implications of toxicant modes of action (MOA) mediated by nuclear receptors. The dominant paradigm in human health risk assessment has been linear extrapolation without a threshold for cancer, and estimation of sub-threshold doses for non-cancer and (in appropriate cases) cancer endpoints. However, recent publications question the application of dose-response modeling approaches with a threshold. The growing body of molecular toxicology information and computational toxicology tools has allowed for exploration of the presence or absence of sub-threshold doses for a number of receptor-mediated MOAs. The workshop explored the development of dose-response approaches for nuclear receptor-mediated liver cancer, within a MOA Human Relevance Framework (HRF). Case studies addressed activation of the AHR, the CAR and the PPARα. This article describes the workshop process, key issues discussed and conclusions. The value of an interactive workshop approach to apply current MOA/HRF frameworks was demonstrated. The results may help direct research on the MOA and dose-response of receptor-based toxicity, since there are commonalities for many receptors in the basic pathways involved for late steps in the MOA, and similar data gaps in early steps. Three additional papers in this series describe the results and conclusions for each case-study receptor regarding its MOA, relevance of the MOA to humans and the resulting dose-response implications.

  16. Reboxetine Enhances the Olanzapine-Induced Antipsychotic-Like Effect, Cortical Dopamine Outflow and NMDA Receptor-Mediated Transmission

    PubMed Central

    Marcus, Monica M; Jardemark, Kent; Malmerfelt, Anna; Björkholm, Carl; Svensson, Torgny H

    2010-01-01

    Preclinical data have shown that addition of the selective norepinephrine transporter (NET) inhibitor reboxetine increases the antipsychotic-like effect of the D2/3 antagonist raclopride and, in parallel, enhances cortical dopamine output. Subsequent clinical results suggested that adding reboxetine to stable treatments with various antipsychotic drugs (APDs) may improve positive, negative and depressive symptoms in schizophrenia. In this study, we investigated in rats the effects of adding reboxetine to the second-generation APD olanzapine on: (i) antipsychotic efficacy, using the conditioned avoidance response (CAR) test, (ii) extrapyramidal side effect (EPS) liability, using a catalepsy test, (iii) dopamine efflux in the medial prefrontal cortex and the nucleus accumbens, using in vivo microdialysis in freely moving animals and (iv) cortical N-methyl--aspartate (NMDA) receptor-mediated transmission, using intracellular electrophysiological recording in vitro. Reboxetine (6 mg/kg) enhanced the suppression of CAR induced by a suboptimal dose (1.25 mg/kg), but not an optimal (2.5 mg/kg) dose of olanzapine without any concomitant catalepsy. Addition of reboxetine to the low dose of olanzapine also markedly increased cortical dopamine outflow and facilitated prefrontal NMDA receptor-mediated transmission. Our data suggest that adjunctive treatment with a NET inhibitor may enhance the therapeutic effect of low-dose olanzapine in schizophrenia without increasing EPS liability and add an antidepressant action, thus in principle allowing for a dose reduction of olanzapine with a concomitant reduction of dose-related side effects, such as EPS and weight gain. PMID:20463659

  17. Characterization of GABA/sub A/ receptor-mediated /sup 36/chloride uptake in rat brain synaptoneurosomes

    SciTech Connect

    Luu, M.D.; Morrow, A.L.; Paul, S.M.; Schwartz, R.D.

    1987-09-07

    ..gamma..-Aminobutyric acid (GABA) receptor-mediated /sup 36/chloride (/sup 36/Cl/sup -/) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated /sup 36/Cl/sup -/ uptake in a concentration-dependent manner with the following order of potency: Muscimol>GABA>piperidine-4-sulfonic acid (P4S)>4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP)=3-aminopropanesulfonic acid (3APS)>>taurine. Both P4S and 3APS behaved as partial agonists, while the GABA/sub B/ agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated /sup 36/Cl/sup -/ uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated /sup 36/Cl/sup -/ uptake was also dependent on the anion present in the media. The muscinol response varied in media containing the following anions: Br/sup -/>Cl/sup -/greater than or equal toNO/sub 3//sup -/>I/sup -/greater than or equal toSCN/sup -/>>C/sub 3/H/sub 5/OO/sup -/greater than or equal toClO/sub 4//sup -/>F/sup -/, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl/sup -/ channel. 43 references, 4 figures, 3 tables.

  18. Cannabinoid Receptor Activation Modifies NMDA Receptor Mediated Release of Intracellular Calcium: Implications for Endocannabinoid Control of Hippocampal Neural Plasticity

    PubMed Central

    Hampson, Robert E.; Miller, Frances; Palchik, Guillermo; Deadwyler, Sam A.

    2011-01-01

    Chronic activation or inhibition of cannabinoid receptors (CB1) leads to continuous suppression of neuronal plasticity in hippocampus and other brain regions, suggesting that endocannabinoids may have a functional role in synaptic processes that produce state-dependent transient modulation of hippocampal cell activity. In support of this, it has previously been shown in vitro that cannabinoid CB1 receptors modulate second messenger systems in hippocampal neurons that can modulate intracellular ion channels, including channels which release calcium from intracellular stores. Here we demonstrate in hippocampal slices a similar endocannabinoid action on excitatory glutamatergic synapses via modulation of NMDA-receptor mediated intracellular calcium levels in confocal imaged neurons. Calcium entry through glutamatergic NMDA-mediated ion channels increases intracellular calcium concentrations via modulation of release from ryanodine-sensitive channels in endoplasmic reticulum. The studies reported here show that NMDA-elicited increases in Calcium Green fluorescence are enhanced by CB1 receptor antagonists (i.e. rimonabant), and inhibited by CB1 agonists (i.e. WIN 55,212-2). Suppression of endocannabinoid breakdown by either reuptake inhibition (AM404) or fatty-acid amide hydrolase inhibition (URB597) produced suppression of NMDA elicited calcium increases comparable to WIN 55,212-2, while enhancement of calcium release provoked by endocannabinoid receptor antagonists (Rimonabant) was shown to depend on the blockade of CB1 receptor mediated de-phosphorylation of Ryanodine receptors. Such CB1 receptor modulation of NMDA elicited increases in intracellular calcium may account for the respective disruption and enhancement by CB1 agents of trial-specific hippocampal neuron ensemble firing patterns during performance of a short-term memory task, reported previously from this laboratory. PMID:21288475

  19. 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.

  20. D2-like dopamine receptor-mediated modulation of activity-dependent plasticity at GABAergic synapses in the subthalamic nucleus

    PubMed Central

    Baufreton, Jérôme; Bevan, Mark D

    2008-01-01

    Reciprocally connected glutamatergic subthalamic nucleus (STN) and GABAergic external globus pallidus (GP) neurons normally exhibit weakly correlated, irregular activity but following the depletion of dopamine in Parkinson's disease they express more highly correlated, rhythmic bursting activity. Patch clamp recording was used to test the hypothesis that dopaminergic modulation reduces the capability of GABAergic inputs to pattern ‘pathological’ activity in STN neurons. Electrically evoked GABAA receptor-mediated IPSCs exhibited activity-dependent plasticity in STN neurons, i.e. IPSCs evoked at frequencies between 1 and 50 Hz exhibited depression that increased with the frequency of activity. Dopamine, the D2-like dopamine receptor agonist quinpirole and external media containing a low [Ca2+] reduced both the magnitude of IPSCs evoked at 1–50 Hz and synaptic depression at 10–50 Hz. Dopamine/quinpirole also reduced the frequency but not the amplitude of miniature IPSCs recorded in the presence of tetrodotoxin. D1-like and D4 agonists were ineffective and D2/3 but not D4 receptor antagonists reversed the effects of dopamine or quinpirole. Together these data suggest that presynaptic D2/3 dopamine receptors modulate the short-term dynamics of GABAergic transmission in the STN by lowering the initial probability of transmitter release. Simulated GABAA receptor-mediated synaptic conductances representative of control or modulated transmission were then generated in STN neurons using the dynamic clamp technique. Dopamine-modulated transmission was less effective at resetting autonomous activity or generating rebound burst firing than control transmission. The data therefore support the conclusion that dopamine acting at presynaptic D2-like receptors reduces the propensity for GABAergic transmission to generate correlated, bursting activity in STN neurons. PMID:18292127

  1. FcR-Like 2 Inhibition of B Cell Receptor-Mediated Activation of B Cells

    PubMed Central

    Jackson, Tanisha A.; Haga, Christopher L.; Ehrhardt, Götz R. A.; Davis, Randall S.; Cooper, Max D.

    2017-01-01

    FcR-like (FCRL) 2 is a transmembrane protein with immunomodulatory potential that is preferentially expressed by memory B cells in humans. It has two consensus ITIMs in addition to a putative ITAM sequence in its cytoplasmic domain. We have confirmed the cellular distribution of FCRL2 and analyzed its functional potential to show that coligation with the BCR leads to tyrosine phosphorylation of its ITIM motifs and subsequent Src homology region 2 domain-containing phosphatase-1 recruitment to facilitate inhibition of BCR signaling. Mutational analysis indicates that the tyrosine residues in both inhibitory motifs of FCRL2 are required for complete inhibition of BCR signaling, whereas tyrosines in the putative activation motif are dispensable for signal modulation. These findings suggest a negative immunomodulatory function for FCRL2 in the regulation of memory B cells. PMID:21068405

  2. NMDA receptor-mediated excitotoxicity depends on the coactivation of synaptic and extrasynaptic receptors.

    PubMed

    Zhou, X; Hollern, D; Liao, J; Andrechek, E; Wang, H

    2013-03-28

    N-methyl-D-aspartate receptors (NMDAR) overactivation is linked to neurodegeneration. The current prevailing theory suggests that synaptic and extrasynaptic NMDAR (syn- and ex-NMDAR) impose counteracting effects on cell fate, and neuronal cell death is mainly mediated by the activation of ex-NMDAR. However, several lines of evidence implicate the limitation of this theory. Here, we demonstrate that activation of NMDAR bi-directionally regulated cell fate through stimulating pro-survival or pro-death signaling. While low-dose NMDA preferentially activated syn-NMDAR and stimulated the extracellular signal-regulated kinase ½-cAMP responsive element-binding protein-brain-derived neurotrophic factor pro-survival signaling, higher doses progressively activated increasing amount of ex-NMDAR along with syn-NMDAR and triggered cell death program. Interestingly, the activation of syn- or ex-NMDAR alone did not cause measurable cell death. Consistently, activation of syn- or ex-NMDAR alone stimulated pro-survival but not pro-death signaling. Next, we found that memantine, which was previously identified as an ex-NMDAR blocker, inhibited intracellular signaling mediated by syn- or ex-NMDAR. Simultaneous blockade of syn- and ex-NMDAR by memantine dose-dependently attenuated NMDAR-mediated death. Moreover, long- but not short-term treatment with high-dose NMDA or oxygen-glucose deprivation triggered cell death and suppressed pro-survival signaling. These data implicate that activation of syn- or ex-NMDAR alone is not neurotoxic. The degree of excitotoxicity depends on the magnitude and duration of syn- and ex-NMDAR coactivation. Finally, genome-wide examination demonstrated that the activation of syn- and ex-NMDAR lead to significant overlapping rather than counteracting transcriptional responses.

  3. P2Y Purinergic Regulation of the Glycine Neurotransmitter Transporters*

    PubMed Central

    Jiménez, Esperanza; Zafra, Francisco; Pérez-Sen, Raquel; Delicado, Esmerilda G.; Miras-Portugal, Maria Teresa; Aragón, Carmen; López-Corcuera, Beatriz

    2011-01-01

    The sodium- and chloride-coupled glycine neurotransmitter transporters (GLYTs) control the availability of glycine at glycine-mediated synapses. The mainly glial GLYT1 is the key regulator of the glycine levels in glycinergic and glutamatergic pathways, whereas the neuronal GLYT2 is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft. In this study, we report that stimulation of P2Y purinergic receptors with 2-methylthioadenosine 5′-diphosphate in rat brainstem/spinal cord primary neuronal cultures and adult rat synaptosomes leads to the inhibition of GLYT2 and the stimulation of GLYT1 by a paracrine regulation. These effects are mainly mediated by the ADP-preferring subtypes P2Y1 and P2Y13 because the effects are partially reversed by the specific antagonists N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate and pyridoxal-5′-phosphate-6-azo(2-chloro-5-nitrophenyl)-2,4-disulfonate and are totally blocked by suramin. P2Y12 receptor is additionally involved in GLYT1 stimulation. Using pharmacological approaches and siRNA-mediated protein knockdown methodology, we elucidate the molecular mechanisms of GLYT regulation. Modulation takes place through a signaling cascade involving phospholipase C activation, inositol 1,4,5-trisphosphate production, intracellular Ca2+ mobilization, protein kinase C stimulation, nitric oxide formation, cyclic guanosine monophosphate production, and protein kinase G-I (PKG-I) activation. GLYT1 and GLYT2 are differentially sensitive to NO/cGMP/PKG-I both in brain-derived preparations and in heterologous systems expressing the recombinant transporters and P2Y1 receptor. Sensitivity to 2-methylthioadenosine 5′-diphosphate by GLYT1 and GLYT2 was abolished by small interfering RNA (siRNA)-mediated knockdown of nitric-oxide synthase. Our data may help define the role of GLYTs in nociception and pain sensitization. PMID:21245148

  4. Data Sharing in P2P Systems

    NASA Astrophysics Data System (ADS)

    Hayek, Rabab; Raschia, Guillaume; Valduriez, Patrick; Mouaddib, Noureddine

    In this chapter, we survey P2P data sharing systems. All along, we focus on the evolution from simple file-sharing systems, with limited functionalities, to Peer Data Management Systems (PDMS) that support advanced applications with more sophisticated data management techniques. Advanced P2P applications are dealing with semantically rich data (e.g., XML documents, relational tables), using a high-level SQL-like query language. We start our survey with an overview over the existing P2P network architectures, and the associated routing protocols. Then, we discuss data indexing techniques based on their distribution degree and the semantics they can capture from the underlying data. We also discuss schema management techniques which allow integrating heterogeneous data. We conclude by discussing the techniques proposed for processing complex queries (e.g., range and join queries). Complex query facilities are necessary for advanced applications which require a high level of search expressiveness. This last part shows the lack of querying techniques that allow for an approximate query answering.

  5. P2Y2R Deficiency Attenuates Experimental Autoimmune Uveitis Development

    PubMed Central

    Relvas, Lia Judice M.; Makhoul, Maya; Dewispelaere, Remi; Caspers, Laure; Communi, Didier; Boeynaems, Jean-Marie; Robaye, Bernard; Bruyns, Catherine; Willermain, François

    2015-01-01

    We aimed to study the role of the nucleotide receptor P2Y2R in the development of experimental autoimmune uveitis (EAU). EAU was induced in P2Y2+/+ and P2Y2-/- mice by immunization with IRBP peptide or by adoptive transfer of in vitro restimulated semi-purified IRBP-specific enriched T lymphocytes from spleens and lymph nodes isolated from native C57Bl/6 or P2Y2+/+ and P2Y2-/- immunized mice. Clinical and histological scores were used to grade disease severity. Splenocytes and lymph node cell phenotypes were analyzed using flow cytometry. Semi-purified lymphocytes and MACS-purified CD4+ T lymphocytes from P2Y2+/+ and P2Y2-/- immunized mice were tested for proliferation and cytokine secretion. Our data show that clinical and histological scores were significantly decreased in IRBP-immunized P2Y2-/- mice as in P2Y2-/- mice adoptively transfered with enriched T lymphocytes from C57Bl/6 IRBP-immunized mice. In parallel, naïve C57Bl/6 mice adoptively transferred with T lymphocytes from P2Y2-/- IRBP-immunized mice also showed significantly less disease. No differences in term of spleen and lymph node cell recruitment or phenotype appeared between P2Y2-/- and P2Y2+/+ immunized mice. However, once restimulated in vitro with IRBP, P2Y2-/- T cells proliferate less and secrete less cytokines than the P2Y2+/+ one. We further found that antigen-presenting cells of P2Y2-/- immunized mice were responsible for this proliferation defect. Together our data show that P2Y2-/- mice are less susceptible to mount an autoimmune response against IRBP. Those results are in accordance with the danger model, which makes a link between autoreactive lymphocyte activation, cell migration and the release of danger signals such as extracellular nucleotides. PMID:25692550

  6. Expression of P2Y receptors in cell lines derived from the human lung.

    PubMed

    Communi, D; Paindavoine, P; Place, G A; Parmentier, M; Boeynaems, J M

    1999-05-01

    1. Northern blotting experiments have been performed with RNA extracted from several cell lines derived from the human lung in order to detect P2Y1, P2Y2, P2Y4 and P2Y6 mRNA. We have investigated the 1HAEo- and 16HBE14o- epithelial cell lines derived from the airway epithelium, the A549 cell line displaying properties of type II alveolar epithelial cells, the CALU-3 serous cells, the 6CFSMEo- submucosal cells and the HASMSC1 airway smooth muscle cells. We have also evaluated one pancreatic epithelial cell line called CFPAC-1. These experiments revealed that P2Y2 and P2Y6 mRNA are co-expressed in the IHAEo-, 16HBE14o- and A549 epithelial cell lines. The CFPAC-1 pancreatic cell line was strongly positive for the P2Y2 receptor. No signal was obtained for the P2Y1 and P2Y4 receptors. 2. We have then performed RT-PCR experiments with specific oligonucleotides of these last two P2Y receptors with the RNA used for the Northern blotting experiments. P2Y4 mRNA was detected in five cell lines: 1HAEo-, 16HBE14o-, 6CFSMEo-, HASMSC1 and CFPAC-1. P2Y1 mRNA was only detected in the CALU-3 cell line. 3. Inositol trisphosphates assays have identified a response typical of the P2Y2 receptor in the 1HAEo- and the 16HBE14o- airway epithelial cell lines which co-express P2Y2 and P2Y6 mRNA. By contrast, the 6CFSMEo- submucosal cells expressed a UTP-specific response which displayed pharmacological characteristics compatible with the human P2Y4 receptor: in particular, there was no response to UDP or ATP and the UTP effect was totally inhibited by pertussis toxin.

  7. Systematic Characterization of Dynamic Parameters of Intracellular Calcium Signals

    PubMed Central

    Mackay, Laurent; Mikolajewicz, Nicholas; Komarova, Svetlana V.; Khadra, Anmar

    2016-01-01

    Dynamic processes, such as intracellular calcium signaling, are hallmark of cellular biology. As real-time imaging modalities become widespread, a need for analytical tools to reliably characterize time-series data without prior knowledge of the nature of the recordings becomes more pressing. The goal of this study is to develop a signal-processing algorithm for MATLAB that autonomously computes the parameters characterizing prominent single transient responses (TR) and/or multi-peaks responses (MPR). The algorithm corrects for signal contamination and decomposes experimental recordings into contributions from drift, TRs, and MPRs. It subsequently provides numerical estimates for the following parameters: time of onset after stimulus application, activation time (time for signal to increase from 10 to 90% of peak), and amplitude of response. It also provides characterization of the (i) TRs by quantifying their area under the curve (AUC), response duration (time between 1/2 amplitude on ascent and descent of the transient), and decay constant of the exponential decay region of the deactivation phase of the response, and (ii) MPRs by quantifying the number of peaks, mean peak magnitude, mean periodicity, standard deviation of periodicity, oscillatory persistence (time between first and last discernable peak), and duty cycle (fraction of period during which system is active) for all the peaks in the signal, as well as coherent oscillations (i.e., deterministic spikes). We demonstrate that the signal detection performance of this algorithm is in agreement with user-mediated detection and that parameter estimates obtained manually and algorithmically are correlated. We then apply this algorithm to study how metabolic acidosis affects purinergic (P2) receptor-mediated calcium signaling in osteoclast precursor cells. Our results reveal that acidosis significantly attenuates the amplitude and AUC calcium responses at high ATP concentrations. Collectively, our data

  8. The balance of NMDA- and AMPA/kainate receptor-mediated activity in normal adult goldfish and during optic nerve regeneration.

    PubMed

    Taylor, Andrew L; Rodger, Jennifer; Stirling, R Victoria; Beazley, Lyn D; Dunlop, Sarah A

    2005-10-01

    Retinotectal topography is established during development and relies on the sequential recruitment of glutamate receptors within postsynaptic tectal cells. NMDA receptors underpin plastic changes at early stages when retinal ganglion cell (RGC) terminal arbors are widespread and topography is coarse; AMPA/kainate receptors mediate fast secure neurotransmission characteristic of mature circuits once topography is refined. Here, we have examined the relative contributions of these receptors to visually evoked activity in normal adult goldfish, in which retinotectal topography is constantly adjusted to compensate for the continual neurogenesis and the addition of new RGC arbors. Furthermore, we examined animals at two stages of optic nerve regeneration. In the first, RGC arbors are widespread and receptive fields large resulting in coarse topography; in the second, RGC arbors are pruned to reduce receptive fields leading to refined topography. Antagonists were applied to the tectum during multiunit recording of postsynaptic responses. Normal goldfish have low levels of NMDA receptor-mediated activity and high levels of AMPA/kainate. When coarse topography has been restored, NMDA receptor-mediated activity is increased and that of AMPA/kainate decreased. Once topography has been refined, the balance of NMDA and AMPA/kainate receptor-mediated activity returns to normal. The data suggest that glutamatergic neurotransmission in normal adult goldfish is dual with NMDA receptors fine-tuning topography and AMPA receptors allowing stable synaptic function. Furthermore, the normal operation of both receptors allows a response to injury in which the balance can be transiently reversed to restore topography and vision.

  9. Toluene exposure during brain growth spurt and adolescence produces differential effects on N-methyl-D-aspartate receptor-mediated currents in rat hippocampus.

    PubMed

    Chen, Hwei-Hsien; Lin, Yi-Ruu; Chan, Ming-Huan

    2011-09-10

    Toluene, an industrial organic solvent, is voluntarily inhaled as drug of abuse. Because inhibition of N-methyl-d-aspartate (NMDA) receptors is one of the possible mechanisms underlying developmental neurotoxicity of toluene, the purpose of the present study was to examine the effects of toluene exposure during two major neurodevelopmental stages, brain growth spurt and adolescence, on NMDA receptor-mediated current. Rats were administered with toluene (500 mg/kg, i.p.) or corn oil daily over postnatal days (PN) 4-9 (brain growth spurt) or PN 21-26 (early adolescence). Intracellular electrophysiological recordings employing in CA1 pyramidal neurons in the hippocampal slices were performed during PN 30-38. Toluene exposure during brain growth spurt enhanced NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) by electrical stimulation, but impaired the paired-pulse facilitation and NMDA response by exogenous application of NMDA. Toluene exposure during adolescence resulted in an increase in NMDA receptor-mediated EPSCs and a decrease in exogenous NMDA-induced currents, while lack of any effect on paired-pulse facilitation. These findings suggest that toluene exposure during brain growth spurt and adolescence might result in an increase in synaptic NMDA receptor responsiveness and a decrease in extrasynaptic NMDA receptor responsiveness, while only toluene exposure during brain growth spurt can produce presynaptic modulation in CA1 pyramidal neurons. The functional changes in NMDA receptor-mediated transmission underlying developmental toluene exposure may lead to the neurobehavioral disturbances.

  10. Human epidermal Langerhans cells cointernalize by receptor-mediated endocytosis "nonclassical" major histocompatibility complex class I molecules (T6 antigens) and class II molecules (HLA-DR antigens).

    PubMed Central

    Hanau, D; Fabre, M; Schmitt, D A; Garaud, J C; Pauly, G; Tongio, M M; Mayer, S; Cazenave, J P

    1987-01-01

    HLA-DR and T6 surface antigens are expressed only by Langerhans cells and indeterminate cells in normal human epidermis. We have previously demonstrated that T6 antigens are internalized in Langerhans cells and indeterminate cells by receptor-mediated endocytosis. This process is induced by the binding of BL6, a monoclonal antibody directed against T6 antigens. In the present study, using a monoclonal antibody directed against HLA-DR antigens, on human epidermal cells in suspension, we show that the surface HLA-DR antigens are also internalized by receptor-mediated endocytosis in Langerhans and indeterminate cells. Moreover, using immunogold double labeling, we demonstrate that T6 and HLA-DR antigens are internalized through common coated regions of the membrane of Langerhans or indeterminate cells. The receptor-mediated endocytosis that is induced involves coated pits and vesicles, receptosomes, lysosomes, and also, in Langerhans cells, the Birbeck granules. Thus, T6 antigens, which are considered to be "unusual" or "nonclassical" major histocompatibility complex class I molecules, and the major histocompatibility complex class II molecules, HLA-DR, are internalized in Langerhans and indeterminate cells through common receptor-mediated endocytosis organelles. Images PMID:3106979

  11. Pharmacological characterisation of the adenosine receptor mediating increased ion transport in the mouse isolated trachea and the effect of allergen challenge

    PubMed Central

    Kornerup, Kristin N; Page, Clive P; Moffatt, James D

    2005-01-01

    The effect of adenosine on transepithelial ion transport was investigated in isolated preparations of murine trachea mounted in Ussing chambers. The possible regulation of adenosine receptors in an established model of allergic airway inflammation was also investigated. Mucosally applied adenosine caused increases in short-circuit current (ISC) that corresponded to approximately 50% of the response to the most efficacious secretogogue, ATP (ΔISC 69.5±6.7 μA cm2). In contrast, submucosally applied adenosine caused only small (<20%) increases in ISC, which were not investigated further. The A1-selective (N6-cyclopentyladenosine, CPA, 1 nM–10 μM), A2A-selective (2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxoamido adenosine; CGS 21680; 0.1–100 μM) and A3-selective (1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide; IB-MECA; 30 nM–100 μM) adenosine receptor agonists were either equipotent or less potent than adenosine, suggesting that these receptors do not mediate the response to adenosine. The A1 receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM–1 μM) caused a rightward shift of the adenosine concentration–effect curve only at 1 μM. The mixed A2A/A2B receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) also caused rightward shift of the adenosine concentration–effect curve, again only at micromolar concentrations, suggestive of the involvement of A2B receptors. In preparations from animals sensitised to ovalbumin and challenged over 3 days with aerosol ovalbumin, a decrease in baseline ISC was observed and responses to ATP were diminished. Similarly, the amplitude of responses to adenosine were attenuated although there was no change in potency. These results suggest that the A2B receptor mediates the ISC response to adenosine in the mouse trachea. This receptor does not appear to be

  12. Pharmacological characterisation of the adenosine receptor mediating increased ion transport in the mouse isolated trachea and the effect of allergen challenge.

    PubMed

    Kornerup, Kristin N; Page, Clive P; Moffatt, James D

    2005-04-01

    The effect of adenosine on transepithelial ion transport was investigated in isolated preparations of murine trachea mounted in Ussing chambers. The possible regulation of adenosine receptors in an established model of allergic airway inflammation was also investigated. Mucosally applied adenosine caused increases in short-circuit current (I(SC)) that corresponded to approximately 50% of the response to the most efficacious secretogogue, ATP (delta I(SC) 69.5 +/- 6.7 microA cm2). In contrast, submucosally applied adenosine caused only small (<20%) increases in I(SC), which were not investigated further. The A1-selective (N6-cyclopentyladenosine, CPA, 1 nM-10 microM), A2A-selective (2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxoamido adenosine; CGS 21680; 0.1-100 microM) and A3-selective (1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purin-9-yl]-N-methyl-beta-D-ribofuranuronamide; IB-MECA; 30 nM-100 microM) adenosine receptor agonists were either equipotent or less potent than adenosine, suggesting that these receptors do not mediate the response to adenosine. The A1 receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM-1 microM) caused a rightward shift of the adenosine concentration-effect curve only at 1 microM. The mixed A2A/A2B receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) also caused rightward shift of the adenosine concentration-effect curve, again only at micromolar concentrations, suggestive of the involvement of A2B receptors. In preparations from animals sensitised to ovalbumin and challenged over 3 days with aerosol ovalbumin, a decrease in baseline I(SC) was observed and responses to ATP were diminished. Similarly, the amplitude of responses to adenosine were attenuated although there was no change in potency. These results suggest that the A2B receptor mediates the I(SC) response to adenosine in the mouse trachea. This receptor does not appear to be

  13. Perspectives of purinergic signaling in stem cell differentiation and tissue regeneration.

    PubMed

    Glaser, Talita; Cappellari, Angélica Regina; Pillat, Micheli Mainardi; Iser, Isabele Cristiana; Wink, Márcia Rosângela; Battastini, Ana Maria Oliveira; Ulrich, Henning

    2012-09-01

    Replacement of lost or dysfunctional tissues by stem cells has recently raised many investigations on therapeutic applications. Purinergic signaling has been shown to regulate proliferation, differentiation, cell death, and successful engraftment of stem cells originated from diverse origins. Adenosine triphosphate release occurs in a controlled way by exocytosis, transporters, and lysosomes or in large amounts from damaged cells, which is then subsequently degraded into adenosine. Paracrine and autocrine mechanisms induced by immune responses present critical factors for the success of stem cell therapy. While P1 receptors generally exert beneficial effects including anti-inflammatory activity, P2 receptor-mediated actions depend on the subtype of stimulated receptors and localization of tissue repair. Pro-inflammatory actions and excitatory tissue damages mainly result from P2X7 receptor activation, while other purinergic receptor subtypes participate in proliferation and differentiation, thereby providing adequate niches for stem cell engraftment and novel mechanisms for cell therapy and endogenous tissue repair. Therapeutic applications based on regulation of purinergic signaling are foreseen for kidney and heart muscle regeneration, Clara-like cell replacement for pulmonary and bronchial epithelial cells as well as for induction of neurogenesis in case of neurodegenerative diseases.

  14. Synergy between coproduced CC and CXC chemokines in monocyte chemotaxis through receptor-mediated events.

    PubMed

    Gouwy, Mieke; Struyf, Sofie; Noppen, Samuel; Schutyser, Evemie; Springael, Jean-Yves; Parmentier, Marc; Proost, Paul; Van Damme, Jo

    2008-08-01

    CC and CXC chemokines coinduced in fibroblasts and leukocytes by cytokines and microbial agents determine the number of phagocytes infiltrating into inflamed tissues. Interleukin-8/CXCL8 and stromal cell-derived factor-1/CXCL12 significantly and dose-dependently increased the migration of monocytes, expressing the corresponding CXC chemokine receptors CXCR2 and CXCR4, toward suboptimal concentrations of the monocyte chemotactic proteins CCL2 or CCL7. These findings were confirmed using different chemotaxis assays and monocytic THP-1 cells. In contrast, the combination of two CC chemokines (CCL2 plus CCL7) or two CXC chemokines (CXCL8 plus CXCL12) did not provide synergy in monocyte chemotaxis. These data show that chemokines competing for related receptors and using similar signaling pathways do not synergize. Receptor heterodimerization is probably not essential for chemokine synergy as shown in CXCR4/CCR2 cotransfectants. It is noteworthy that CCL2 mediated extracellular signal-regulated kinase 1/2 phosphorylation and calcium mobilization was significantly enhanced by CXCL8 in monocytes, indicating cooperative downstream signaling pathways during enhanced chemotaxis. Moreover, in contrast to intact CXCL12, truncated CXCL12(3-68), which has impaired receptor signaling capacity but can still desensitize CXCR4, was unable to synergize with CCL2 in monocytic cell migration. Furthermore, AMD3100 and RS102895, specific CXCR4 and CCR2 inhibitors, respectively, reduced the synergistic effect between CCL2 and CXCL12 significantly. These data indicate that for synergistic interaction between chemokines binding and signaling of the two chemokines via their proper receptors is necessary.

  15. Functional properties of five Dictyostelium discoideum P2X receptors.

    PubMed

    Baines, Abigail; Parkinson, Katie; Sim, Joan A; Bragg, Laricia; Thompson, Christopher R L; North, R Alan

    2013-07-19

    The Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD, and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels (P2XA, P2XB, P2XD, and P2XE). At P2XA receptors, ATP was the only effective agonist of 17 structurally related putative ligands that were tested. Extracellular sodium, compared with potassium, strongly inhibited ATP responses in P2XB, P2XD, and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the nonfunctional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors indicates that there is limited functional redundancy among Dictyostelium P2X receptors.

  16. The aryl hydrocarbon receptor mediates UVB radiation-induced skin tanning.

    PubMed

    Jux, Bettina; Kadow, Stephanie; Luecke, Sandra; Rannug, Agneta; Krutmann, Jean; Esser, Charlotte

    2011-01-01

    Melanogenesis is the vital response to protect skin cells against UVB-induced DNA damage. Melanin is produced by melanocytes, which transfer it to surrounding keratinocytes. Recently, we have shown that the aryl hydrocarbon receptor (AhR) is part of the UVB-stress response in epidermal keratinocytes. UVB triggers AhR signaling by generating the AhR ligand 6-formylindolo(3,2-b)carbazole from tryptophan. We show here that normal murine melanocytes express functional AhR. Using standard UVB tanning protocols, AhR-deficient mice were shown to tan significantly weaker than wild-type mice; in these mice, tyrosinase activity in the epidermis was lower as well. Tanning responses and tyrosinase activity, however, were normal in keratinocyte-specific conditional AhR knockout mice, indicating that release of melanogenic keratinocyte factors is unaffected by the UVB-AhR signaling pathway and that the diminished tanning response in AhR(-/-) mice is confined to the level of melanocytes. Accordingly, the number of dihydroxyphenylalanin-positive melanocytes increased significantly less on UVB irradiation in AhR(-/-) mice than in wild-type mice. This difference in melanocyte number was associated with a significantly reduced expression of stem cell factor-1 and c-kit in melanocytes of AhR(-/-) mice. Thus, the environmental signal sensor AhR links solar UVB radiation to skin pigmentation.

  17. Metabotropic glutamate receptor-mediated long-term depression: molecular mechanisms.

    PubMed

    Gladding, Clare M; Fitzjohn, Stephen M; Molnár, Elek

    2009-12-01

    The ability to modify synaptic transmission between neurons is a fundamental process of the nervous system that is involved in development, learning, and disease. Thus, synaptic plasticity is the ability to bidirectionally modify transmission, where long-term potentiation and long-term depression (LTD) represent the best characterized forms of plasticity. In the hippocampus, two main forms of LTD coexist that are mediated by activation of either N-methyl-d-aspartic acid receptors (NMDARs) or metabotropic glutamate receptors (mGluRs). Compared with NMDAR-LTD, mGluR-LTD is less well understood, but recent advances have started to delineate the underlying mechanisms. mGluR-LTD at CA3:CA1 synapses in the hippocampus can be induced either by synaptic stimulation or by bath application of the group I selective agonist (R,S)-3,5-dihydroxyphenylglycine. Multiple signaling mechanisms have been implicated in mGluR-LTD, illustrating the complexity of this form of plasticity. This review provides an overview of recent studies investigating the molecular mechanisms underlying hippocampal mGluR-LTD. It highlights the role of key molecular components and signaling pathways that are involved in the induction and expression of mGluR-LTD and considers how the different signaling pathways may work together to elicit a persistent reduction in synaptic transmission.

  18. Eclogitic pyroxenes, ordered with P2 symmetry

    USGS Publications Warehouse

    Clark, J.R.; Papike, J.J.

    1966-01-01

    X-ray diffraction crystal-structure analysis of omphacite from eclogite, Tiburon Peninsula, Marin County, California, shows that this clinopyroxene has P2 symmetry with a nearly ordered distribution of the multiple cation content defined by its approximate formula: (Na0.5Ca0.5) (Mg 0.4Fe2+0.1Al0.4Fe3+0.1)Si2O6. Na+ and Ca2+ tend to assume alternate locations in the structure, and (Mg,Fe2+) octahedra alternate with Al3+ or (Al,F3+) octahedra in chains along c.

  19. Activation of cannabinoid CB2 receptor-mediated AMPK/CREB pathway reduces cerebral ischemic injury.

    PubMed

    Choi, In-Young; Ju, Chung; Anthony Jalin, Angela M A; Lee, Da In; Prather, Paul L; Kim, Won-Ki

    2013-03-01

    The type 2 cannabinoid receptor (CB2R) was recently shown to mediate neuroprotection in ischemic injury. However, the role of CB2Rs in the central nervous system, especially neuronal and glial CB2Rs in the cortex, remains unclear. We, therefore, investigated anti-ischemic mechanisms of cortical CB2R activation in various ischemic models. In rat cortical neurons/glia mixed cultures, a CB2R agonist, trans-caryophyllene (TC), decreased neuronal injury and mitochondrial depolarization caused by oxygen-glucose deprivation/re-oxygenation (OGD/R); these effects were reversed by the selective CB2R antagonist, AM630, but not by a type 1 cannabinoid receptor antagonist, AM251. Although it lacked free radical scavenging and antioxidant enzyme induction activities, TC reduced OGD/R-evoked mitochondrial dysfunction and intracellular oxidative stress. Western blot analysis demonstrated that TC enhanced phosphorylation of AMP-activated protein kinase (AMPK) and cAMP responsive element-binding protein (CREB), and increased expression of the CREB target gene product, brain-derived neurotrophic factor. However, TC failed to alter the activity of either Akt or extracellular signal-regulated kinase, two major CB2R signaling pathways. Selective AMPK and CREB inhibitors abolished the neuroprotective effects of TC. In rats, post-ischemic treatment with TC decreased cerebral infarct size and edema, and increased phosphorylated CREB and brain-derived neurotrophic factor expression in neurons. All protective effects of TC were reversed by co-administration with AM630. Collectively, these data demonstrate that cortical CB2R activation by TC ameliorates ischemic injury, potentially through modulation of AMPK/CREB signaling, and suggest that cortical CB2Rs might serve as a putative therapeutic target for cerebral ischemia.

  20. P2X and P2Y nucleotide receptors as targets in cardiovascular disease.

    PubMed

    Kennedy, Charles; Chootip, Krongkarn; Mitchell, Callum; Syed, Nawazish-i-Husain; Tengah, Asrin

    2013-03-01

    Endogenous nucleotides have widespread actions in the cardiovascular system, but it is only recently that the P2X and P2Y receptor subtypes, at which they act, have been identified and subtype-selective agonists and antagonists developed. These advances have greatly increased our understanding of the physiological and pathophysiological functions of P2X and P2Y receptors, but investigation of the clinical usefulness of selective ligands is at an early stage. Nonetheless, the evidence considered in this review demonstrates clearly that various cardiovascular disorders, including vasospasm, hypertension, congestive heart failure and cardiac damage during ischemic episodes, may be viable targets. With further development of novel, selective agonists and antagonists, our understanding will continue to improve and further therapeutic applications are likely to be discovered.

  1. 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.

  2. Low doses of cyclic AMP-phosphodiesterase inhibitors rapidly evoke opioid receptor-mediated thermal hyperalgesia in naïve mice which is converted to prominent analgesia by cotreatment with ultra-low-dose naltrexone.

    PubMed

    Crain, Stanley M; Shen, Ke-Fei

    2008-09-22

    Systemic (s.c.) injection in naïve mice of cyclic AMP-phosphodiesterase (cAMP-PDE) inhibitors, e.g. 3-isobutyl-1-methylxanthine [(IBMX) or caffeine, 10 mg/kg] or the more specific cAMP-PDE inhibitor, rolipram (1 mug/kg), rapidly evokes thermal hyperalgesia (lasting >5 h). These effects appear to be mediated by enhanced excitatory opioid receptor signaling, as occurs during withdrawal in opioid-dependent mice. Cotreatment of these mice with ultra-low-dose naltrexone (NTX, 0.1 ng/kg-1 pg/kg, s.c.) results in prominent opioid analgesia (lasting >4 h) even when the dose of rolipram is reduced to 1 pg/kg. Cotreatment of these cAMP-PDE inhibitors in naïve mice with an ultra-low-dose (0.1 ng/kg) of the kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI) or the mu-opioid receptor antagonist, beta-funaltrexamine (beta-FNA) also results in opioid analgesia. These excitatory effects of cAMP-PDE inhibitors in naïve mice may be mediated by enhanced release of small amounts of endogenous bimodally-acting (excitatory/inhibitory) opioid agonists by neurons in nociceptive networks. Ultra-low-dose NTX, nor-BNI or beta-FNA selectively antagonizes high-efficacy excitatory (hyperalgesic) Gs-coupled opioid receptor-mediated signaling in naïve mice and results in rapid conversion to inhibitory (analgesic) Gi/Go-coupled opioid receptor-mediated signaling which normally requires activation by much higher doses of opioid agonists. Cotreatment with a low subanalgesic dose of kelatorphan, an inhibitor of multiple endogenous opioid peptide-degrading enzymes, stabilizes endogenous opioid agonists released by cAMP-PDE inhibitors, resulting in conversion of the hyperalgesia to analgesia without requiring selective blockade of excitatory opioid receptor signaling. The present study provides a novel pharmacologic paradigm that may facilitate development of valuable non-narcotic clinical analgesics utilizing cotreatment with ultra-low-dose rolipram plus ultra-low-dose NTX or related

  3. NR2D-containing NMDA receptors mediate tissue plasminogen activator-promoted neuronal excitotoxicity.

    PubMed

    Baron, A; Montagne, A; Cassé, F; Launay, S; Maubert, E; Ali, C; Vivien, D

    2010-05-01

    Although the molecular bases of its actions remain debated, tissue-type plasminogen activator (tPA) is a paradoxical brain protease, as it favours some learning/memory processes, but increases excitotoxic neuronal death. Here, we show that, in cultured cortical neurons, tPA selectively promotes NR2D-containing N-methyl-D-aspartate receptor (NMDAR)-dependent activation. We show that tPA-mediated signalling and neurotoxicity through the NMDAR are blocked by co-application of an NR2D antagonist (phenanthrene derivative (2S(*), 3R(*))-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid, PPDA) or knockdown of neuronal NR2D expression. In sharp contrast with cortical neurons, hippocampal neurons do not exhibit NR2D both in vitro and in vivo and are consequently resistant to tPA-promoted NMDAR-mediated neurotoxicity. Moreover, we have shown that activation of synaptic NMDAR prevents further tPA-dependent NMDAR-mediated neurotoxicity and sensitivity to PPDA. This study shows that the earlier described pro-neurotoxic effect of tPA is mediated by NR2D-containing NMDAR-dependent extracellular signal-regulated kinase activation, a deleterious effect prevented by synaptic pre-activation.

  4. Molecular Pathways: AXL, a Membrane Receptor Mediator of Resistance to Therapy

    PubMed Central

    Scaltriti, Maurizio; Elkabets, Moshe; Baselga, José

    2016-01-01

    AXL is a tyrosine kinase membrane receptor that signals via PI3K, MAPK, and protein kinase C (PKC), among other pathways. AXL has oncogenic potential and interacts with other membrane receptors, depending on their relative abundance and availability. The increased expression of AXL in cancer is often the result of pharmacologic selective pressure to a number of chemotherapies and targeted therapies and acts as a mechanism of acquired drug resistance. This resistance phenotype, frequently accompanied by epithelial-to-mesenchymal transition, can be reversed by AXL inhibition. In tumors with high levels of EGFR, including lung, head and neck, and triple-negative breast cancer, AXL dimerizes with this receptor and initiates signaling that circumvents the antitumor effects of anti-EGFR therapies. Likewise, AXL overexpression and dimerization with EGFR can overcome PI3K inhibition by activating the phospholipase C-γ-PKC cascade that, in turn, sustains mTORC1 activity. The causative role of AXL in inducing drug resistance is underscored by the fact that the suppression of AXL restores sensitivity to these agents. Hence, these observations indicate that AXL is selectively expressed in tumor cells refractory to therapy and that cotargeting AXL in this setting would potentially overcome drug resistance. The use of AXL inhibitors should be considered in the clinic. PMID:26763248

  5. Histamine H2 Receptor-Mediated Suppression of Intestinal Inflammation by Probiotic Lactobacillus reuteri

    PubMed Central

    Gao, Chunxu; Major, Angela; Rendon, David; Lugo, Monica; Jackson, Vanessa; Shi, Zhongcheng; Mori-Akiyama, Yuko

    2015-01-01

    ABSTRACT Probiotics and commensal intestinal microbes suppress mammalian cytokine production and intestinal inflammation in various experimental model systems. Limited information exists regarding potential mechanisms of probiotic-mediated immunomodulation in vivo. In this report, we demonstrate that specific probiotic strains of Lactobacillus reuteri suppress intestinal inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse colitis model. Only strains that possess the hdc gene cluster, including the histidine decarboxylase and histidine-histamine antiporter genes, can suppress colitis and mucosal cytokine (interleukin-6 [IL-6] and IL-1β in the colon) gene expression. Suppression of acute colitis in mice was documented by diminished weight loss, colonic injury, serum amyloid A (SAA) protein concentrations, and reduced uptake of [18F]fluorodeoxyglucose ([18F]FDG) in the colon by positron emission tomography (PET). The ability of probiotic L. reuteri to suppress colitis depends on the presence of a bacterial histidine decarboxylase gene(s) in the intestinal microbiome, consumption of a histidine-containing diet, and signaling via the histamine H2 receptor (H2R). Collectively, luminal conversion of l-histidine to histamine by hdc+ L. reuteri activates H2R, and H2R signaling results in suppression of acute inflammation within the mouse colon. PMID:26670383

  6. 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

  7. Cross-talk between P2X4 and gamma-aminobutyric acid, type A receptors determines synaptic efficacy at a central synapse.

    PubMed

    Jo, Young-Hwan; Donier, Emmanuelle; Martinez, Audrey; Garret, Maurice; Toulmé, Estelle; Boué-Grabot, Eric

    2011-06-03

    The essence of neuronal function is to generate outputs in response to synaptic potentials. Synaptic integration at postsynaptic sites determines neuronal outputs in the CNS. Using immunohistochemical and electrophysiological approaches, we first reveal that steroidogenic factor 1 (SF-1) green fluorescent protein (GFP)-positive neurons in the ventromedial nucleus of the hypothalamus express P2X4 subunits that are activated by exogenous ATP. Increased membrane expression of P2X4 channels by using a peptide competing with P2X4 intracellular endocytosis motif enhances neuronal excitability of SF-1 GFP-positive neurons. This increased excitability is inhibited by a P2X receptor antagonist. Furthermore, increased surface P2X4 receptor expression significantly decreases the frequency and the amplitude of GABAergic postsynaptic currents of SF-1 GFP-positive neurons. Co-immunopurification and pulldown assays reveal that P2X4 receptors complex with aminobutyric acid, type A (GABA(A)) receptors and demonstrate that two amino acids in the carboxyl tail of the P2X4 subunit are crucial for its physical association with GABA(A) receptors. Mutation of these two residues prevents the physical association, thereby blocking cross-inhibition between P2X4 and GABA(A) receptors. Moreover, disruption of the physical coupling using competitive peptides containing the identified motif abolishes current inhibition between P2X4 and GABA(A) receptors in recombinant system and P2X4 receptor-mediated GABAergic depression in SF-1 GFP-positive neurons. Our present work thus provides evidence for cross-talk between excitatory and inhibitory receptors that appears to be crucial in determining GABAergic synaptic strength at a central synapse.

  8. Cross-talk between P2X4 and γ-Aminobutyric Acid, Type A Receptors Determines Synaptic Efficacy at a Central Synapse*

    PubMed Central

    Jo, Young-Hwan; Donier, Emmanuelle; Martinez, Audrey; Garret, Maurice; Toulmé, Estelle; Boué-Grabot, Eric

    2011-01-01

    The essence of neuronal function is to generate outputs in response to synaptic potentials. Synaptic integration at postsynaptic sites determines neuronal outputs in the CNS. Using immunohistochemical and electrophysiological approaches, we first reveal that steroidogenic factor 1 (SF-1) green fluorescent protein (GFP)-positive neurons in the ventromedial nucleus of the hypothalamus express P2X4 subunits that are activated by exogenous ATP. Increased membrane expression of P2X4 channels by using a peptide competing with P2X4 intracellular endocytosis motif enhances neuronal excitability of SF-1 GFP-positive neurons. This increased excitability is inhibited by a P2X receptor antagonist. Furthermore, increased surface P2X4 receptor expression significantly decreases the frequency and the amplitude of GABAergic postsynaptic currents of SF-1 GFP-positive neurons. Co-immunopurification and pulldown assays reveal that P2X4 receptors complex with aminobutyric acid, type A (GABAA) receptors and demonstrate that two amino acids in the carboxyl tail of the P2X4 subunit are crucial for its physical association with GABAA receptors. Mutation of these two residues prevents the physical association, thereby blocking cross-inhibition between P2X4 and GABAA receptors. Moreover, disruption of the physical coupling using competitive peptides containing the identified motif abolishes current inhibition between P2X4 and GABAA receptors in recombinant system and P2X4 receptor-mediated GABAergic depression in SF-1 GFP-positive neurons. Our present work thus provides evidence for cross-talk between excitatory and inhibitory receptors that appears to be crucial in determining GABAergic synaptic strength at a central synapse. PMID:21482824

  9. P2P Reputation Management Through Social Networking

    NASA Astrophysics Data System (ADS)

    Despotovic, Zoran

    Reputation systems offer a viable solution to the problem of risk reduction in online communities, in situation in which other mechanism such as litigation or security cannot help. Building on the assumption that its participating entities engage in repeated interactions, a reputation system can either signal what happened in the past or aggregate the past feedback in such a way as to influence the future actions of the concerned entity. In the former case, the concerned entity's behavior is seen as static, while the sent signal is expected to be indicative of the entity's future actions. In the latter case, behavior is dynamic in the sense that the entity can adjust it given the observed feedback, while the purpose of the reputation system is to induce adjustments according to the designer's needs. In this chapter, we discuss these two classes of solutions in detail. In particular, we investigate how they apply to P2P networks, what additional problems and difficulties the P2P environment introduces and what scalable solutions to these problems the current research offers.

  10. Genetically designed biomolecular capping system for mesoporous silica nanoparticles enables receptor-mediated cell uptake and controlled drug release

    NASA Astrophysics Data System (ADS)

    Datz, Stefan; Argyo, Christian; Gattner, Michael; Weiss, Veronika; Brunner, Korbinian; Bretzler, Johanna; von Schirnding, Constantin; Torrano, Adriano A.; Spada, Fabio; Vrabel, Milan; Engelke, Hanna; Bräuchle, Christoph; Carell, Thomas; Bein, Thomas

    2016-04-01

    Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the development of precisely controllable and highly modular theranostic systems.Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the

  11. Inhibitory effects of benzodiazepines on the adenosine A(2B) receptor mediated secretion of interleukin-8 in human mast cells.

    PubMed

    Hoffmann, Kristina; Xifró, Rosa Altarcheh; Hartweg, Julia Lisa; Spitzlei, Petra; Meis, Kirsten; Molderings, Gerhard J; von Kügelgen, Ivar

    2013-01-30

    The activation of adenosine A(2B) receptors in human mast cells causes pro-inflammatory responses such as the secretion of interleukin-8. There is evidence for an inhibitory effect of benzodiazepines on mast cell mediated symptoms in patients with systemic mast cell activation disease. Therefore, we investigated the effects of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast cell leukaemia (HMC1) cells by an enzyme linked immunosorbent assay. The adenosine analogue N-ethylcarboxamidoadenosine (NECA, 0.3-3 μM) increased interleukin-8 production about 5-fold above baseline. This effect was attenuated by the adenosine A(2B) receptor antagonist MRS1754 (N-(4-cyanophenyl)-2-{4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy}-acetamide) 1 μM. In addition, diazepam, 4'-chlorodiazepam and flunitrazepam (1-30 μM) markedly reduced NECA-induced interleukin-8 production in that order of potency, whereas clonazepam showed only a modest inhibition. The inhibitory effect of diazepam was not altered by flumazenil 10 μM or PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide) 10 μM. Diazepam attenuated the NECA-induced expression of mRNA encoding for interleukin-8. Moreover, diazepam and flunitrazepam reduced the increasing effects of NECA on cAMP-response element- and nuclear factor of activated t-cells-driven luciferase reporter gene activities in HMC1 cells. Neither diazepam nor flunitrazepam affected NECA-induced increases in cellular cAMP levels in CHO Flp-In cells stably expressing recombinant human adenosine A(2B) receptors, excluding a direct action of benzodiazepines on human adenosine A(2B) receptors. In conclusion, this is the first study showing an inhibitory action of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast (HMC1) cells. The rank order of potency indicates the involvement of an atypical benzodiazepine binding site.

  12. Enhanced Functional Activity of the Cannabinoid Type-1 Receptor Mediates Adolescent Behavior

    PubMed Central

    Kasanetz, Fernando; Lynch, Diane L.; Friemel, Chris M.; Lassalle, Olivier; Hurst, Dow P.; Steindel, Frauke; Monory, Krisztina; Schäfer, Carola; Miederer, Isabelle; Leweke, F. Markus; Schreckenberger, Mathias; Lutz, Beat; Reggio, Patricia H.; Manzoni, Olivier J.; Spanagel, Rainer

    2015-01-01

    Adolescence is characterized by drastic behavioral adaptations and comprises a particularly vulnerable period for the emergence of various psychiatric disorders. Growing evidence reveals that the pathophysiology of these disorders might derive from aberrations of normal neurodevelopmental changes in the adolescent brain. Understanding the molecular underpinnings of adolescent behavior is therefore critical for understanding the origin of psychopathology, but the molecular mechanisms that trigger adolescent behavior are unknown. Here, we hypothesize that the cannabinoid type-1 receptor (CB1R) may play a critical role in mediating adolescent behavior because enhanced endocannabinoid (eCB) signaling has been suggested to occur transiently during adolescence. To study enhanced CB1R signaling, we introduced a missense mutation (F238L) into the rat Cnr1 gene that encodes for the CB1R. According to our hypothesis, rats with the F238L mutation (Cnr1F238L) should sustain features of adolescent behavior into adulthood. Gain of function of the mutated receptor was demonstrated by in silico modeling and was verified functionally in a series of biochemical and electrophysiological experiments. Mutant rats exhibit an adolescent-like phenotype during adulthood compared with wild-type littermates, with typical high risk/novelty seeking, increased peer interaction, enhanced impulsivity, and augmented reward sensitivity for drug and nondrug reward. Partial inhibition of CB1R activity in Cnr1F238L mutant rats normalized behavior and led to a wild-type phenotype. We conclude that the activity state and functionality of the CB1R is critical for mediating adolescent behavior. These findings implicate the eCB system as an important research target for the neuropathology of adolescent-onset mental health disorders. SIGNIFICANCE STATEMENT We present the first rodent model with a gain-of-function mutation in the cannabinoid type-1 receptor (CB1R). Adult mutant rats exhibit an adolescent

  13. Diverse Toll-like receptors mediate cytokine production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in macrophages.

    PubMed

    Park, Se-Ra; Kim, Dong-Jae; Han, Seung-Hyun; Kang, Min-Jung; Lee, Jun-Young; Jeong, Yu-Jin; Lee, Sang-Jin; Kim, Tae-Hyoun; Ahn, Sang-Gun; Yoon, Jung-Hoon; Park, Jong-Hwan

    2014-05-01

    Toll-like receptors (TLRs) orchestrate a repertoire of immune responses in macrophages against various pathogens. Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans are two important periodontal pathogens. In the present study, we investigated TLR signaling regulating cytokine production of macrophages in response to F. nucleatum and A. actinomycetemcomitans. TLR2 and TLR4 are redundant in the production of cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) in F. nucleatum- and A. actinomycetemcomitans-infected macrophages. The production of cytokines by macrophages in response to F. nucleatum and A. actinomycetemcomitans infection was impaired in MyD88-deficient macrophages. Moreover, cytokine concentrations were lower in MyD88-deficient macrophages than in TLR2/TLR4 (TLR2/4) double-deficient cells. An endosomal TLR inhibitor, chloroquine, reduced cytokine production in TLR2/4-deficient macrophages in response to F. nucleatum and A. actinomycetemcomitans, and DNA from F. nucleatum or A. actinomycetemcomitans induced IL-6 production in bone marrow-derived macrophages (BMDMs), which was abolished by chloroquine. Western blot analysis revealed that TLR2/4 and MyD88 were required for optimal activation of NF-κB and mitogen-activated protein kinases (MAPKs) in macrophages in response to F. nucleatum and A. actinomycetemcomitans, with different kinetics. An inhibitor assay showed that NF-κB and all MAPKs (p38, extracellular signal-regulated kinase [ERK], and Jun N-terminal protein kinase [JNK]) mediate F. nucleatum-induced production of cytokines in macrophages, whereas NF-κB and p38, but not ERK and JNK, are involved in A. actinomycetemcomitans-mediated cytokine production. These findings suggest that multiple TLRs may participate in the cytokine production of macrophages against periodontal bacteria.

  14. Ionotropic Glutamate Receptors Mediate Inducible Defense in the Water Flea Daphnia pulex

    PubMed Central

    Miyakawa, Hitoshi; Sato, Masanao; Colbourne, John K.; Iguchi, Taisen

    2015-01-01

    Phenotypic plasticity is the ability held in many organisms to produce different phenotypes with a given genome in response to environmental stimuli, such as temperature, nutrition and various biological interactions. It seems likely that environmental signals induce a variety of mechanistic responses that influence ontogenetic processes. Inducible defenses, in which prey animals alter their morphology, behavior and/or other traits to help protect against direct or latent predation threats, are among the most striking examples of phenotypic plasticity. The freshwater microcrustacean Daphnia pulex forms tooth-like defensive structures, “neckteeth,” in response to chemical cues or signals, referred to as “kairomones,” in this case released from phantom midge larvae, a predator of D. pulex. To identify factors involved in the reception and/or transmission of a kairomone, we used microarray analysis to identify genes up-regulated following a short period of exposure to the midge kairomone. In addition to identifying differentially expressed genes of unknown function, we also found significant up-regulation of genes encoding ionotropic glutamate receptors, which are known to be involved in neurotransmission in many animal species. Specific antagonists of these receptors strongly inhibit the formation of neckteeth in D. pulex, although agonists did not induce neckteeth by themselves, indicating that ionotropic glutamate receptors are necessary but not sufficient for early steps of neckteeth formation in D. pulex. Moreover, using co-exposure of D. pulex to antagonists and juvenile hormone (JH), which physiologically mediates neckteeth formation, we found evidence suggesting that the inhibitory effect of antagonists is not due to direct inhibition of JH synthesis/secretion. Our findings not only provide a candidate molecule required for the inducible defense response in D. pulex, but also will contribute to the understanding of complex mechanisms underlying the

  15. Epigenetic and Glucocorticoid Receptor-Mediated Regulation of Glutathione Peroxidase 3 in Lung Cancer Cells

    PubMed Central

    An, Byung Chull; Jung, Nak-Kyun; Park, Chun Young; Oh, In-Jae; Choi, Yoo-Duk; Park, Jae-Il; Lee, Seung-won

    2016-01-01

    Glutathione peroxidase 3 (GPx3), an antioxidant enzyme, acts as a modulator of redox signaling, has immunomodulatory function, and catalyzes the detoxification of reactive oxygen species (ROS). GPx3 has been identified as a tumor suppressor in many cancers. Although hyper-methylation of the GPx3 promoter has been shown to down-regulate its expression, other mechanisms by which GPx3 expression is regulated have not been reported. The aim of this study was to further elucidate the mechanisms of GPx3 regulation. GPx3 gene analysis predicted the presence of ten glucocorticoid response elements (GREs) on the GPx3 gene. This result prompted us to investigate whether GPx3 expression is regulated by the glucocorticoid receptor (GR), which is implicated in tumor response to chemotherapy. The corticosteroid dexamethasone (Dex) was used to examine the possible relationship between GR and GPx3 expression. Dex significantly induced GPx3 expression in H1299, H1650, and H1975 cell lines, which exhibit low levels of GPx3 expression under normal conditions. The results of EMSA and ChIP-PCR suggest that GR binds directly to GRE 6 and 7, both of which are located near the GPx3 promoter. Assessment of GPx3 transcription efficiency using a luciferase reporter system showed that blocking formation of the GR-GRE complexes reduced luciferase activity by 7–8-fold. Suppression of GR expression by siRNA transfection also induced down-regulation of GPx3. These data indicate that GPx3 expression can be regulated independently via epigenetic or GR-mediated mechanisms in lung cancer cells, and suggest that GPx3 could potentiate glucocorticoid (GC)-mediated anti-inflammatory signaling in lung cancer cells. PMID:27484907

  16. Differential Regulation of Progesterone Receptor-Mediated Transcription by CDK2 and DNA-PK.

    PubMed

    Treviño, Lindsey S; Bolt, Michael J; Grimm, Sandra L; Edwards, Dean P; Mancini, Michael A; Weigel, Nancy L

    2016-02-01

    Progesterone receptor (PR) function is altered by cell signaling, but the mechanisms of kinase-specific regulation are not well defined. To examine the role of cell signaling in the regulation of PR transcriptional activity, we have utilized a previously developed mammalian-based estrogen-response element promoter array cell model and automated cell imaging and analysis platform to visualize and quantify effects of specific kinases on different mechanistic steps of PR-mediated target gene activation. For these studies, we generated stable estrogen-response element array cell lines expressing inducible chimeric PR that contains a swap of the estrogen receptor-α DNA-binding domain for the DNA-binding domain of PR. We have focused on 2 kinases important for steroid receptor activity: cyclin-dependent kinase 2 and DNA-dependent protein kinase. Treatment with either a Cdk1/2 inhibitor (NU6102) or a DNA-dependent protein kinase inhibitor (NU7441) decreased hormone-mediated chromatin decondensation and transcriptional activity. Further, we observed a quantitative reduction in the hormone-mediated recruitment of select coregulator proteins with NU6102 that is not observed with NU7441. In parallel, we determined the effect of kinase inhibition on hormone-mediated induction of primary and mature transcripts of endogenous genes in T47D breast cancer cells. Treatment with NU6102 was much more effective than NU7441, in inhibiting induction of PR target genes that exhibit a rapid increase in primary transcript expression in response to hormone. Taken together, these results indicate that the 2 kinases regulate PR transcriptional activity by distinct mechanisms.

  17. P2 Receptors in Renal Autoregulation

    PubMed Central

    Guan, Zhengrong; Fellner, Robert C.; Van Beusecum, Justin; Inscho, Edward W.

    2014-01-01

    Accomplishing autoregulation of renal blood flow and glomerular filtration rate is an essential function of the renal microcirculation. While the existence of this phenomenon has been known for many years, the exact mechanisms that underlie this unique regulatory capability remain poorly understood. The work of many investigators has provided insights into many aspects of the autoregulatory mechanism, but many critical components remain elusive. This review is intended to update the reader on the role of P2 purinoceptors as a postulated mechanism responsible for renal autoregulatory resistance adjustments. It will summarize recent advances in normal function and it will touch on more recent ideas regarding autoregulatory insufficiency in hypertension and inflammation. Current thoughts on the nature of the mechanosensor responsible for myogenic behavior will be discussed as well as current thoughts on the mechanisms involved in ATP release to the extracellular fluid space. PMID:24066935

  18. Participation of peripheral P2Y1, P2Y6 and P2Y11 receptors in formalin-induced inflammatory pain in rats.

    PubMed

    Barragán-Iglesias, Paulino; Mendoza-Garcés, Luis; Pineda-Farias, Jorge Baruch; Solano-Olivares, Verónica; Rodríguez-Silverio, Juan; Flores-Murrieta, Francisco Javier; Granados-Soto, Vinicio; Rocha-González, Héctor Isaac

    2015-01-01

    Metabotropic P2Y receptors subfamily consists of eight functional mammalian receptors. Specifically, P2Y1, P2Y6 and P2Y11 receptors have been described in the sensory nervous system, but their participation, at peripheral level, in behavioral pain models is scarcely understood. This study assessed the role of peripheral P2Y1, P2Y6 and P2Y11 receptors in formalin-induced inflammatory pain. Ipsilateral, but not contralateral peripheral pre-treatment with the endogenous P2Y1 (ADP, 100-1000nmol/paw), P2Y6 (UDP, 180-300nmol/paw) and P2Y11 (ATP, 100-1000nmol/paw), or selective P2Y1 (MRS2365, 0.1-10nmol/paw), P2Y6 (PSB0474, 0.1-0.10pmol/paw) and P2Y11 (NF546, 0.3-3nmol/paw) receptor agonists increased 0.5% formalin-induced flinching behavior. Concordantly, peripheral pre-treatment with the selective P2Y1 (MRS2500, 0.01-10pmol/paw), P2Y6 (MRS2578, 3-30nmol/paw) and P2Y11 (NF340, 1-10nmol/paw) receptor antagonists significantly decreased 1% formalin-induced flinching behavior. Furthermore, the pronociceptive effect of ADP (100nmol/paw) or MRS2365 (10nmol/paw), UDP (300nmol/paw) or PSB0474 (10pmol/paw) and ATP (1000nmol/paw) or NF546 (3nmol/paw) was blocked by the selective P2Y1 (MRS2500, 0.01nmol/paw), P2Y6 (MRS2578, 3nmol/paw), and P2Y11 (NF340, 1nmol/paw) receptor antagonists, respectively. Western blot analysis confirmed the presence of P2Y1 (66kDa), P2Y6 (36kDa) and P2Y11 (75kDa) receptors in dorsal root ganglia (DRG) and sciatic nerve. Results suggest that peripheral activation of P2Y1, P2Y6 and P2Y11 receptors plays a pronociceptive role in formalin-induced pain.

  19. Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process

    PubMed Central

    de Torre-Minguela, Carlos; Barberà-Cremades, Maria; Gómez, Ana I.; Martín-Sánchez, Fátima; Pelegrín, Pablo

    2016-01-01

    The activation of P2X7 receptor (P2X7R) on M1 polarized macrophages induces the assembly of the NLRP3 inflammasome leading to the release of pro-inflammatory cytokines and the establishment of the inflammatory response. However, P2X7R signaling to the NLRP3 inflammasome is uncoupled on M2 macrophages without changes on receptor activation. In this study, we analyzed P2X7R secretome in wild-type and P2X7R-deficient macrophages polarized either to M1 or M2 and proved that proteins released after P2X7R stimulation goes beyond caspase-1 secretome. The characterization of P2X7R-secretome reveals a new function of this receptor through a fine-tuning of protein release. We found that P2X7R stimulation in macrophages is able to release potent anti-inflammatory proteins, such as Annexin A1, independently of their polarization state suggesting for first time a potential role for P2X7R during resolution of the inflammation and not linked to the release of pro-inflammatory cytokines. These results are of prime importance for the development of therapeutics targeting P2X7R. PMID:26935289

  20. Nucleotide signalling during inflammation

    PubMed Central

    Idzko, Marco; Ferrari, Davide; Eltzschig, Holger K.

    2014-01-01

    Inflammatory conditions are associated with the extracellular release of nucleotides, particularly ATP. In the extracellular compartment, ATP predominantly functions as a signalling molecule through the activation of purinergic P2 receptors. Metabotropic P2Y receptors are G-protein-coupled, whereas ionotropic P2X receptors are ATP-gated ion channels. Here we discuss how signalling events through P2 receptors alter the outcomes of inflammatory or infectious diseases. Recent studies implicate a role for P2X/P2Ysignalling in mounting appropriate inflammatory responses critical for host defence against invading pathogens or tumours. Conversely, P2X/P2Y signalling can promote chronic inflammation during ischaemia and reperfusion injury, inflammatory bowel disease or acute and chronic diseases of the lungs. Although nucleotide signalling has been used clinically in patients before, research indicates an expanding field of opportunities for specifically targeting individual P2 receptors for the treatment of inflammatory or infectious diseases. PMID:24828189

  1. P2Y1 and P2Y2 receptor distribution varies along the human placental vascular tree: role of nucleotides in vascular tone regulation

    PubMed Central

    Buvinic, Sonja; Poblete, M Inés; Donoso, M Verónica; Delpiano, Ana María; Briones, René; Miranda, Ramiro; Huidobro-Toro, J Pablo

    2006-01-01

    The expression of purinergic P2Y receptors (P2YRs) along the cord, superficial chorionic vessels and cotyledons of the human placenta was analysed and functional assays were performed to determine their vasomotor activity. Immunoblots for the P2Y1R and P2Y2R revealed a 6- to 8-fold increase in receptor expression from the cord to the chorionic or cotyledon vessels. In the cord and chorionic vessels the receptor distribution was mainly in the smooth muscle, whereas in the cotyledon vessels these receptors were equally distributed between the endothelium and smooth muscle cells. An exception was the P2Y2R at the umbilical artery, which was distributed as in the cotyledon. mRNA coding for the P2Y1R and P2Y2R were detected by RT-PCR and the mRNA coding for the P2Y4R, P2Y6R and P2Y11R was also identified. Application of 2-MeSADP and uridine triphosphate (UTP), preferential P2Y1R and P2Y2R ligands, respectively, resulted in contraction of isolated rings from umbilical and chorionic vessels. The vasoconstriction was blocked in a concentration-dependent manner by 10–100 nm indomethacin or 10 nm GR32191, suggesting the involvement of thromboxane receptors. MRS 2179, a selective P2Y1R antagonist, reduced the 2-MeSADP- but not the UTP-evoked contractions. Perfusion of cotyledons with 2-MeSADP or UTP evoked concentration-dependent reductions in perfusion pressure mediated by the NO–cGMP pathway. Blockade of NO synthase abolished the vasodilatation and the rise in luminal NO elicited by either agonist. MRS 2179 antagonized the dilatation and rise in luminal NO evoked by 2-MeSADP but not by UTP. In summary, P2Y1R and P2Y2R are unevenly distributed along the human placental vascular tree; both receptors are coupled to different signalling pathways in the cord/chorionic vessels versus the cotyledon leading to opposing vasomotor responses. PMID:16543271

  2. Requirement of Histone Methyltransferase SMYD3 for Estrogen Receptor-mediated Transcription*

    PubMed Central

    Kim, Hyunjung; Heo, Kyu; Kim, Jeong Hoon; Kim, Kyunghwan; Choi, Jongkyu; An, Woojin

    2009-01-01

    SMYD3 is a SET domain-containing protein with histone methyltransferase activity on histone H3–K4. Recent studies showed that SMYD3 is frequently overexpressed in different types of cancer cells, but how SMYD3 regulates the development and progression of these malignancies remains unknown. Here, we report the previously unrecognized role of SMYD3 in estrogen receptor (ER)-mediated transcription via its histone methyltransferase activity. We demonstrate that SMYD3 functions as a coactivator of ERα and potentiates ERα activity in response to ligand. SMYD3 directly interacts with the ligand binding domain of ER and is recruited to the proximal promoter regions of ER target genes upon gene induction. Importantly, our chromatin immunoprecipitation analyses provide compelling evidence that SMYD3 is responsible for the accumulation of di- and trimethylation of H3–K4 at the induced ER target genes. Furthermore, RNA interference-directed down-regulation of SMYD3 reveals that SMYD3 is required for ER-regulated gene transcription in estrogen signaling pathway. Thus, our results identify SMYD3 as a new coactivator for ER-mediated transcription, providing a possible link between SMYD3 overexpression and breast cancer. PMID:19509295

  3. NMDA receptor-mediated epileptiform persistent activity requires calcium release from intracellular stores in prefrontal neurons.

    PubMed

    Gao, Wen-Jun; Goldman-Rakic, Patricia S

    2006-02-01

    Various normal and pathological forms of synchronized population activity are generated by recurrent excitation among pyramidal neurons in the neocortex. However, the intracellular signaling mechanisms underlying this activity remain poorly understood. In this study, we have examined the cellular properties of synchronized epileptiform activity in the prefrontal cortex with particular emphasis on a potential role of intracellular calcium stores. We find that the zero-magnesium-induced synchronized activity is blocked by inhibition of sarco-endoplasmic reticulum Ca(2+)-ATPases, phospholipase C (PLC), the inositol 1,4,5-trisphosphate (IP3) receptor, and the ryanodine receptor. This same activity is, however, not affected by application of metabotropic glutamatergic receptor (mGluR) agonists, nor by introduction of an mGluR antagonist. These results suggest that persistent synchronized activity in vitro is dependent upon calcium release from internal calcium stores through the activation of PLC-IP3 receptor pathway. Our findings also raise the possibility that intracellular calcium release may be involved in the generation of pathologic synchronized activity in epilepsy in vivo and in physiological forms of synchronized cortical activity.

  4. Potential insight for drug discovery from high fidelity receptor-mediated transduction mechanisms in insects

    PubMed Central

    Raffa, Robert B.; Raffa, Kenneth F.

    2011-01-01

    Introduction There is a pervasive and growing concern about the small number of new pharmaceutical agents. There are many proposed explanations for this trend that do not involve the drug-discovery process per se, but the discovery process itself has also come under scrutiny. If the current paradigms are indeed not working, where are novel ideas to come from? Perhaps it is time to look to novel sources. Areas covered The receptor-signaling and 2nd-messenger transduction processes present in insects are quite similar to those in mammals (involving G proteins, ion channels, etc.). However, a review of these systems reveals an unprecedented degree of high potency and receptor selectivity to an extent greater than that modeled in most current drug-discovery approaches. Expert opinion A better understanding of insect receptor pharmacology could stimulate novel theoretical and practical ideas in mammalian pharmacology (drug discovery) and, conversely, the application of pharmacology and medicinal chemistry principles could stimulate novel advances in entomology (safer and more targeted control of pest species). PMID:21984882

  5. Hindbrain GLP-1 receptor mediation of cisplatin-induced anorexia and nausea

    PubMed Central

    De Jonghe, Bart C.; Holland, Ruby; Olivos, Diana R.; Rupprecht, Laura E.; Kanoski, Scott E.; Hayes, Matthew R.

    2016-01-01

    While chemotherapy-induced nausea and vomiting is clinically controlled in the acute (<24h) phase following treatment, the anorexia, nausea, fatigue, and other illness-type behaviors during the delayed phase (>24h) of chemotherapy are largely uncontrolled. As the hindbrain glucagon-like peptide-1 (GLP-1) system contributes to energy balance and mediates aversive and stressful stimuli, here we examine the hypothesis that hindbrain GLP-1 signaling mediates aspects of chemotherapy-induced nausea and reductions in feeding behavior in rats. Specifically, hindbrain GLP-1 receptor (GLP-1R) blockade, via 4th intracerebroventricular (ICV) exendin-(9-39) injections, attenuates the anorexia, body weight reduction, and pica (nausea-induced ingestion of kaolin clay) elicited by cisplatin chemotherapy during the delayed phase (48hr) of chemotherapy-induced nausea. Additionally, the present data provide evidence that the central GLP-1-producing preproglucagon neurons in the nucleus tractus solitarius (NTS) of the caudal brainstem are activated by cisplatin during the delayed phase of chemotherapy-induced nausea, as cisplatin led to a significant increase in c-Fos immunoreactivity in NTS GLP-1-immunoreactive neurons. These data support a growing body of literature suggesting that the central GLP-1 system may be a potential pharmaceutical target for adjunct anti-emetics used to treat the delayed-phase of nausea and emesis, anorexia, and body weight loss that accompany chemotherapy treatments. PMID:26522737

  6. Vacuolar Sorting Receptor-Mediated Trafficking of Soluble Vacuolar Proteins in Plant Cells.

    PubMed

    Kang, Hyangju; Hwang, Inhwan

    2014-08-25

    Vacuoles are one of the most prominent organelles in plant cells, and they play various important roles, such as degradation of waste materials, storage of ions and metabolites, and maintaining turgor. During the past two decades, numerous advances have been made in understanding how proteins are specifically delivered to the vacuole. One of the most crucial steps in this process is specific sorting of soluble vacuolar proteins. Vacuolar sorting receptors (VSRs), which are type I membrane proteins, are involved in the sorting and packaging of soluble vacuolar proteins into transport vesicles with the help of various accessory proteins. To date, large amounts of data have led to the development of two different models describing VSR-mediated vacuolar trafficking that are radically different in multiple ways, particularly regarding the location of cargo binding to, and release from, the VSR and the types of carriers utilized. In this review, we summarize current literature aimed at elucidating VSR-mediated vacuolar trafficking and compare the two models with respect to the sorting signals of vacuolar proteins, as well as the molecular machinery involved in VSR-mediated vacuolar trafficking and its action mechanisms.

  7. Hindbrain GLP-1 receptor mediation of cisplatin-induced anorexia and nausea.

    PubMed

    De Jonghe, Bart C; Holland, Ruby A; Olivos, Diana R; Rupprecht, Laura E; Kanoski, Scott E; Hayes, Matthew R

    2016-01-01

    While chemotherapy-induced nausea and vomiting are clinically controlled in the acute (<24 h) phase following treatment, the anorexia, nausea, fatigue, and other illness-type behaviors during the delayed phase (>24 h) of chemotherapy are largely uncontrolled. As the hindbrain glucagon-like peptide-1 (GLP-1) system contributes to energy balance and mediates aversive and stressful stimuli, here we examine the hypothesis that hindbrain GLP-1 signaling mediates aspects of chemotherapy-induced nausea and reductions in feeding behavior in rats. Specifically, hindbrain GLP-1 receptor (GLP-1R) blockade, via 4th intracerebroventricular (ICV) exendin-(9-39) injections, attenuates the anorexia, body weight reduction, and pica (nausea-induced ingestion of kaolin clay) elicited by cisplatin chemotherapy during the delayed phase (48 h) of chemotherapy-induced nausea. Additionally, the present data provide evidence that the central GLP-1-producing preproglucagon neurons in the nucleus tractus solitarius (NTS) of the caudal brainstem are activated by cisplatin during the delayed phase of chemotherapy-induced nausea, as cisplatin led to a significant increase in c-Fos immunoreactivity in NTS GLP-1-immunoreactive neurons. These data support a growing body of literature suggesting that the central GLP-1 system may be a potential pharmaceutical target for adjunct anti-emetics used to treat the delayed-phase of nausea and emesis, anorexia, and body weight loss that accompany chemotherapy treatments.

  8. Berberine reduces Toll-like receptor-mediated macrophage migration by suppression of Src enhancement.

    PubMed

    Cheng, Wei-Erh; Ying Chang, Miao; Wei, Jyun-Yan; Chen, Yen-Jen; Maa, Ming-Chei; Leu, Tzeng-Horng

    2015-06-15

    Berberine is an isoquinoline with anti-inflammatory activity. We previously demonstrated that there was a loop of signal amplification between nuclear factor kappa B and Src for macrophage mobility triggered by the engagement of Toll-like receptors (TLRs). The simultaneous suppression of lipopolysaccharide (LPS)-mediated upregulation of inducible nitric oxide synthase, cyclooxygenase 2, and cell mobility in berberine-treated macrophages suggested Src might be a target of berberine. Indeed, th reduced migration, greatly suppressed Src induction in both protein and RNA transcript by berberine were observed in macrophages exposed to LPS, peptidoglycan, polyinosinic-polycytidylic acid, and CpG-oligodeoxynucleotides. In addition to Src induction, berberine also inhibited LPS-mediated Src activation in Src overexpressing macrophages and S-nitroso-N-acetylpenicillamine (a nitric oxide donor) could partly restore it. Moreover, berberine suppressed Src activity in fibronectin-stimulated macrophages and in v-Src transformed cells. These results implied that by effectively reducing Src expression and activity, berberine inhibited TLR-mediated cell motility in macrophages.

  9. T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels.

    PubMed

    Matza, Didi; Badou, Abdallah; Klemic, Kathryn G; Stein, Judith; Govindarajulu, Usha; Nadler, Monica J; Kinet, Jean-Pierre; Peled, Amnon; Shapira, Oz M; Kaczmarek, Leonard K; Flavell, Richard A

    2016-01-01

    The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling.

  10. Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells

    PubMed Central

    Lee, Won Sup; Jung, Ji Hyun; Panchanathan, Radha; Yun, Jeong Won; Kim, Dong Hoon; Kim, Hye Jung; Kim, Gon Sup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

    2017-01-01

    Background Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. Methods Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. Results UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. Conclusions UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer. PMID:28382282

  11. T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels

    PubMed Central

    Matza, Didi; Badou, Abdallah; Klemic, Kathryn G.; Stein, Judith; Govindarajulu, Usha; Nadler, Monica J.; Kinet, Jean-Pierre; Peled, Amnon; Shapira, Oz M.; Kaczmarek, Leonard K.; Flavell, Richard A.

    2016-01-01

    The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling. PMID:26815481

  12. β-arrestins: regulatory role and therapeutic potential in opioid and cannabinoid receptor-mediated analgesia.

    PubMed

    Raehal, Kirsten M; Bohn, Laura M

    2014-01-01

    Pain is a complex disorder with neurochemical and psychological components contributing to the severity, the persistence, and the difficulty in adequately treating the condition. Opioid and cannabinoids are two classes of analgesics that have been used to treat pain for centuries and are arguably the oldest of "pharmacological" interventions used by man. Unfortunately, they also produce several adverse side effects that can complicate pain management. Opioids and cannabinoids act at G protein-coupled receptors (GPCRs), and much of their effects are mediated by the mu-opioid receptor (MOR) and cannabinoid CB1 receptor (CB1R), respectively. These receptors couple to intracellular second messengers and regulatory proteins to impart their biological effects. In this chapter, we review the role of the intracellular regulatory proteins, β-arrestins, in modulating MOR and CB1R and how they influence the analgesic and side-effect profiles of opioid and cannabinoid drugs in vivo. This review of the literature suggests that the development of opioid and cannabinoid agonists that bias MOR and CB1R toward G protein signaling cascades and away from β-arrestin interactions may provide a novel mechanism by which to produce analgesia with less severe adverse effects.

  13. A putative octopamine/tyramine receptor mediating appetite in a hungry fly

    NASA Astrophysics Data System (ADS)

    Ishida, Yuko; Ozaki, Mamiko

    2011-07-01

    In the blowfly Phormia regina, experience of simultaneous feeding with d-limonene exposure inhibits proboscis extension reflex (PER) due to decreased tyramine (TA) titer in the brain. To elucidate the molecular mechanism of TA signaling pathway related to the associated feeding behavior, we cloned cDNA encoding the octopamine/TA receptor (PregOAR/TAR). The deduced protein is composed of 607 amino acid residues and has 7 predicted transmembrane domains. Based on homology and phylogenetic analyses, this protein belongs to the OAR/TAR family. The PregOAR/TAR was mainly expressed in head, with low levels of expression in other tissues at adult stages. Gene expression profile is in agreement with a plethora of functions ascribed to TA in various insect tissues. The immunolabeled cell bodies and processes were localized in the medial protocerebrum, outer layer of lobula, antennal lobe, and subesophageal ganglion. These results suggest that decrease of TA level in the brain likely affects neurons expressing PregOAR/TAR, causing mediation of the sensitivity in the sensillum and/or output of motor neurons for PER.

  14. The Second Extracellular Loop of the Adenosine A1 Receptor Mediates Activity of Allosteric Enhancers

    PubMed Central

    Kennedy, Dylan P.; McRobb, Fiona M.; Leonhardt, Susan A.; Purdy, Michael; Figler, Heidi; Marshall, Melissa A.; Chordia, Mahendra; Figler, Robert; Linden, Joel

    2014-01-01

    Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists. PMID:24217444

  15. Receptor mediated disruption of retinal pigment epithelium function in acute glycated-albumin exposure

    PubMed Central

    Dahrouj, Mohammad; Desjardins, Danielle M.; Liu, Yueying; Crosson, Craig E.; Ablonczy, Zsolt

    2015-01-01

    Diabetic macular edema (DME) is a major cause of visual impairment. Although DME is generally believed to be a microvascular disease, dysfunction of the retinal pigment epithelium (RPE) can also contribute to its development. Advanced glycation end-products (AGE) are thought to be one of the key factors involved in the pathogenesis of diabetes in the eye, and we have previously demonstrated a rapid breakdown of RPE function following glycated-albumin (Glyc-alb, a common AGE mimetic) administration in monolayer cultures of fetal human RPE cells. Here we present new evidence that this response is attributed to apically oriented AGE receptors (RAGE). Moreover, time-lapse optical coherence tomography in Dutch-belted rabbits 48 hours post intravitreal Glyc-alb injections demonstrated a significant decrease in RPE-mediated fluid resorption in vivo. In both the animal and tissue culture models, the response to Glyc-alb was blocked by the relatively selective RAGE antagonist, FPS-ZM1 and was also inhibited by ZM323881, a relatively selective vascular endothelial growth factor receptor 2 (VEGF-R2) antagonist. Our data establish that the Glyc-alb-induced breakdown of RPE function is mediated via specific RAGE and VEGF-R2 signaling both in vitro and in vivo. These results are consistent with the notion that the RPE is a key player in the pathogenesis of DME. PMID:26070987

  16. β-Arrestins: Regulatory Role and Therapeutic Potential in Opioid and Cannabinoid Receptor-Mediated Analgesia

    PubMed Central

    Bohn, Laura M.

    2016-01-01

    Pain is a complex disorder with neurochemical and psychological components contributing to the severity, the persistence, and the difficulty in adequately treating the condition. Opioid and cannabinoids are two classes of analgesics that have been used to treat pain for centuries and are arguably the oldest of “pharmacological” interventions used by man. Unfortunately, they also produce several adverse side effects that can complicate pain management. Opioids and cannabinoids act at G protein-coupled receptors (GPCRs), and much of their effects are mediated by the mu-opioid receptor (MOR) and cannabinoid CB1 receptor (CB1R), respectively. These receptors couple to intracellular second messengers and regulatory proteins to impart their biological effects. In this chapter, we review the role of the intracellular regulatory proteins, β-arrestins, in modulating MOR and CB1R and how they influence the analgesic and side-effect profiles of opioid and cannabinoid drugs in vivo. This review of the literature suggests that the development of opioid and cannabinoid agonists that bias MOR and CB1R toward G protein signaling cascades and away from β-arrestin interactions may provide a novel mechanism by which to produce analgesia with less severe adverse effects. PMID:24292843

  17. Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors.

    PubMed

    Sodhi, Puneet; Hartwick, Andrew T E

    2016-09-01

    Melanopsin-dependent phototransduction in intrinsically photosensitive retinal ganglion cells (ipRGCs) involves a Gq-coupled phospholipase C (PLC) signaling cascade. Acetylcholine, released in the mammalian retina by starburst amacrine cells, can also activate Gq-PLC pathways through certain muscarinic acetylcholine receptors (mAChRs). Using multielectrode array recordings of rat retinas, we demonstrate that robust spiking responses can be evoked in neonatal and adult ipRGCs after bath application of the muscarinic agonist carbachol. The stimulatory action of carbachol on ipRGCs was a direct effect, as confirmed through calcium imaging experiments on isolated ipRGCs in purified cultures. Using flickering (6 Hz) yellow light stimuli at irradiances below the threshold for melanopsin activation, spiking responses could be elicited in ipRGCs that were suppressed by mAChR antagonism. Therefore, this work identified a novel melanopsin-independent pathway for stimulating sustained spiking in ganglion cell photoreceptors. This mAChR-mediated pathway could enhance ipRGC spiking responses in conditions known to evoke retinal acetylcholine release, such as those involving flickering or moving visual stimuli. Furthermore, this work identifies a pharmacological approach for light-independent ipRGC stimulation that could be targeted by mAChR agonists.

  18. Resveratrol attenuates acute kidney injury by inhibiting death receptor-mediated apoptotic pathways in a cisplatin-induced rat model

    PubMed Central

    Hao, Qiufa; Xiao, Xiaoyan; Zhen, Junhui; Feng, Jinbo; Song, Chun; Jiang, Bei; Hu, Zhao

    2016-01-01

    Acute kidney injury is a clinical syndrome characterized by a loss of renal function and acute tubular necrosis. Resveratrol exerts a wide range of pharmacological effects based on its anti-inflammatory, antioxidant and cytoprotective properties. The present study aimed to evaluate whether resveratrol attenuates acute kidney injury in a cisplatin-induced rat model and to investigate the potential mechanisms involved. Rats were randomly divided into four treatment groups: Control, cisplatin, resveratrol, and cisplatin plus resveratrol. Rats exposed to cisplatin displayed acute kidney injury, identified by analysis of renal function and histopathological observation. Resveratrol significantly ameliorated the increased serum creatinine, blood urea nitrogen, renal index and histopathological damage induced by cisplatin. Furthermore, compared with untreated control animals, cisplatin lead to significantly increased expression of Fas ligand, tumor necrosis factor-α (TNF-α), caspase-8 and Bcl-2 associated protein X apoptosis regulator (Bax), and decreased expression of anti-apoptosis regulators, BH3 interacting domain death agonist (BID) and B cell lymphoma 2 apoptosis regulator (Bcl-2). Administration of resveratrol significantly reversed the cisplatin-induced alteration in these apoptosis-associated proteins. In conclusion, these findings suggest that resveratrol attenuates cisplatin-induced acute kidney injury through inactivation of the death receptor-mediated apoptotic pathway, and may provide a new therapeutic strategy to ameliorate the process of acute kidney injury. PMID:27600998

  19. The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

    SciTech Connect

    Watanabe, H.; Grubb, J.H.; Sly, W.S. )

    1990-10-01

    The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functional receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.

  20. Receptor-mediated membrane adhesion of lipid-polymer hybrid (LPH) nanoparticles studied by dissipative particle dynamics simulations.

    PubMed

    Li, Zhenlong; Gorfe, Alemayehu A

    2015-01-14

    Lipid-polymer hybrid (LPH) nanoparticles represent a novel class of targeted drug delivery platforms that combine the advantages of liposomes and biodegradable polymeric nanoparticles. However, the molecular details of the interaction between LPHs and their target cell membranes remain poorly understood. We have investigated the receptor-mediated membrane adhesion process of a ligand-tethered LPH nanoparticle using extensive dissipative particle dynamics (DPD) simulations. We found that the spontaneous adhesion process follows a first-order kinetics characterized by two distinct stages: a rapid nanoparticle-membrane engagement, followed by a slow growth in the number of ligand-receptor pairs coupled with structural re-organization of both the nanoparticle and the membrane. The number of ligand-receptor pairs increases with the dynamic segregation of ligands and receptors toward the adhesion zone causing an out-of-plane deformation of the membrane. Moreover, the fluidity of the lipid shell allows for strong nanoparticle-membrane interactions to occur even when the ligand density is low. The LPH-membrane avidity is enhanced by the increased stability of each receptor-ligand pair due to the geometric confinement and the cooperative effect arising from multiple binding events. Thus, our results reveal the unique advantages of LPH nanoparticles as active cell-targeting nanocarriers and provide some general principles governing nanoparticle-cell interactions that may aid future design of LPHs with improved affinity and specificity for a given target of interest.

  1. Nuclear Membranes ETB Receptors Mediate ET-1-induced Increase of Nuclear Calcium in Human Left Ventricular Endocardial Endothelial Cells.

    PubMed

    Jules, Farah; Avedanian, Levon; Al-Khoury, Johny; Keita, Ramatoulaye; Normand, Alexandre; Bkaily, Ghassan; Jacques, Danielle

    2015-07-01

    In fetal human left ventricular endocardial endothelial cells (EECLs), both plasma membrane (PM) ET(A)R and ET(B)R were reported to mediate ET-1-induced increase of intracellular calcium [Ca](i); however, this effect was mediated by ET(A)R in right EECs (EECRs). In this study, we verified whether, as for the PM, nuclear membranes (NMs) ET-1 receptors activation in EECLs and EECRs induce an increase of nuclear calcium ([Ca](n)) and if this effect is mediated through the same receptor type as in PM. Using a plasmalemma-perforated technique and 3D confocal microscopy, our results showed that, as in PM intact cells, superfusion of nuclei of both cell types with cytosolic ET-1 induced a concentration-dependent sustained increase of [Ca](n). In EECRs, the ET(A)R antagonist prevented the effect of ET-1 on [Ca](n) without affecting EECLs. However, in both cell types, the effect of cytosolic ET-1 on [Ca](n) was prevented by the ETBR antagonist. In conclusion, both NMs' ET(A)R and ET(B)R mediated the effect of cytosolic ET-1 on [Ca](n) in EECRs. In contrast, only NMs' ET(B)R activation mediated the effect of cytosolic ET-1 in EECLs. Hence, the type of NMs' receptors mediating the effect of ET-1 on [Ca](n) are different from those of PM mediating the increase in [Ca](i).

  2. Fatty acyl specificity of the receptor-mediated release of polyunsaturated fatty acids from vascular endothelial cells

    SciTech Connect

    Rosenthal, M.D.

    1987-05-01

    Histamine and bradykinin appear to exhibit the same fatty acid specificity as thrombin. Incubation of human umbilical vein endothelial cells with 10 ..mu..M histamine for 10 min in buffered saline containing 50 ..mu..M fat-free albumin stimulates the release of previously incorporated (/sup 14/C)arachidonate but not (/sup 14/C)22:4(n-6) or (/sup 14/C)20:3(n-6). Similarly calf pulmonary artery endothelial cells release (/sup 14/C)arachidonate but not (/sup 14/C)22:4(n-6) in response to either bradykinin (1 /sup +/g/ml) or histamine (10..mu..M). In both types of endothelial cells, the calcium ionophore A23187 (10 ..mu..M) exhibits the same pattern of fatty acyl specificity as the receptor-mediated agonists. By contrast, mellitin (2-4 ..mu..g/ml) stimulates the release of free 22:4(n-6) and oleate in addition to arachidonate; release of 22:4(n-6) is 30-70% that of arachidonate. These results suggest that histamine, bradykinin and thrombin stimulate a common calcium-dependent fatty acyl-specific phospholipase activity.

  3. Viral infection transiently reverses activation receptor-mediated NK cell hyporesponsiveness in an MHC class I-independent mechanism.

    PubMed

    Mazumdar, Budhaditya; Bolanos, Fred D; Tripathy, Sandeep K

    2013-05-01

    Continuous engagement of the Ly49H activating receptor with its ligand (m157) in a transgenic mouse expressing m157 (m157-Tg) results in hyporesponsiveness of Ly49H(+) NK cells. The same interaction, during murine cytomegalovirus (MCMV) infection, leads to activation of Ly49H(+) NK cells. MCMV infection results in decreased MHC class I (MHC-I) expression on the infected cell as well as inflammatory responses, both of which do not take place in the uninfected m157-Tg mouse, potentially allowing for activation of NK cells in the context of MCMV infection. In this study, we demonstrated that viral infection transiently reverses activation receptor-mediated NK cell hyporesponsiveness in an MHC-I-independent mechanism. Furthermore, Ly49H(+) NK cells in an MHC-I-deficient environment remained hyporesponsive in the context of m157 expression, even when mature WT splenocytes were transferred into m157-Tg mice in an MHC-I-deficient environment. However, the administration of cytokines TNF-α, IL-12, and IFN-β resulted in a partial recovery from activation receptor-induced hyporesponsiveness. Thus, the release of the aforementioned cytokines during MCMV infection and not the downregulation of MHC-I expression appears to be responsible for partial resolution of Ly49H receptor-induced NK cell hyporesponsiveness.

  4. Centrally administered neuromedin U elevates plasma adrenaline by brain prostanoid TP receptor-mediated mechanisms in rats.

    PubMed

    Sasaki, Tsuyoshi; Shimizu, Takahiro; Wakiguchi, Hiroshi; Yokotani, Kunihiko

    2008-09-11

    Neuromedin U is a hypothalamic peptide involved in energy homeostasis and stress responses. The peptide, when administered intracerebroventricularly (i.c.v.), decreases food intake and body weight while increasing body temperature and heat production. We examined the effect of i.c.v. administered neuromedin U on plasma catecholamines with regard to the brain prostanoid using anesthetized rats. Neuromedin U (0.1, 0.5 and 1 nmol/animal, i.c.v.) effectively elevated plasma adrenaline (a maximal response was obtained at 0.5 nmol/animal), but had little effect on plasma noradrenaline. However, intravenously administered neuromedin U (0.5 nmol/animal) had no effect on plasma catecholamines. Neuromedin U (0.5 nmol/animal, i.c.v.)-induced elevation of plasma adrenaline was effectively reduced by intracerebroventricular pretreatments with indomethacin (an inhibitor of cyclooxygenase) (0.6 and 1.2 micromol/animal), furegrelate (an inhibitor of thromboxane A2 synthase) (0.9 and 1.8 micromol/animal) and (+)-S-145 (a blocker of prostanoid TP receptors) (250 and 625 nmol/animal), respectively. The neuromedin U-induced adrenaline response was also abolished by acute bilateral adrenalectomy. These results suggest that centrally administered neuromedin U evokes the secretion of adrenaline from the adrenal medulla by brain prostanoid TP receptor-mediated mechanisms in rats.

  5. Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

    PubMed Central

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both ‘zipper’ (receptor-mediated) and ‘trigger’ (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells. PMID:24586631

  6. Leukotriene D4 receptor-mediated hydrolysis of phosphoinositide and mobilization of calcium in sheep tracheal smooth muscle cells

    SciTech Connect

    Mong, S.; Miller, J.; Wu, H.L.; Crooke, S.T.

    1988-02-01

    A sheep tracheal smooth muscle primary culture cell system was developed to characterize leukotriene D4 (LTD4) receptor-mediated biochemical and pharmacological effects. (/sup 3/H)LTD4 binding to the enriched plasma membrane receptor was specific, stereoselective and saturable. LTE4 and high affinity receptor antagonists bound to the receptors with a rank-order potency that was expected from previous smooth muscle contraction studies. In the (/sup 3/H)myoinositol labeled cells, LTD4 and LTE4 induced phosphoinositide hydrolysis. The biosynthesis of (/sup 3/H)inositol-trisphosphate was rapid and the induction of biosynthesis of (/sup 3/H)inositol-monophosphate by LTs was stereoselective and specific and was inhibited specifically by a receptor antagonist, SKF 104353. In the fura-2 loaded smooth muscle cells, LTD4 and LTE4 induced transient intracellular Ca++ mobilization. The fura-2/Ca++ transient was stereoselective and specific and was inhibited by receptor antagonist, SKF 104353. These results suggest that the cultured sheep tracheal smooth muscle cells have plasma membrane receptors for LTD4. These receptors were coupled to a phospholipase C that, when activated by agonists, induced hydrolysis of inositol containing phospholipids. The hydrolysis products, e.g. diacylglycerol and inositol-trisphosphate, may serve as intracellular messengers that trigger or contribute to the contractile effect in sheep tracheal smooth muscle.

  7. The alpha(2C)-adrenergic receptor mediates hyperactivity of coloboma mice, a model of attention deficit hyperactivity disorder.

    PubMed

    Bruno, Kristy J; Hess, Ellen J

    2006-09-01

    Drugs that modify noradrenergic transmission such as atomoxetine and clonidine are increasingly prescribed for the treatment of attention deficit hyperactivity disorder (ADHD). However, the therapeutic targets of these compounds are unknown. Norepinephrine is also implicated in the hyperactivity exhibited by coloboma mice. To identify the receptor subtypes that regulate the hyperactivity, coloboma mice were systematically challenged with adrenergic drugs. The beta-adrenergic receptor antagonist propranolol and the alpha(1)-adrenergic receptor antagonist prazosin each had little effect on the hyperactivity. Conversely, the alpha(2)-adrenergic receptor antagonist yohimbine reduced the activity of coloboma mice but not control mice. Subtype-selective blockade of alpha(2C)-, but not alpha(2A)- or alpha(2B)-adrenergic receptors, ameliorated hyperactivity of coloboma mice without affecting activity of control mice, suggesting that alpha(2C)-adrenergic receptors mediate the hyperactivity. Localized in the basal ganglia, alpha(2C)-adrenergic receptors are in a prime position to impact locomotor activity and are, therefore, potential targets of pharmacotherapy for ADHD.

  8. Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells.

    PubMed

    Sapinoro, Ramil; Volcy, Ketna; Rodrigo, W W Shanaka I; Schlesinger, Jacob J; Dewhurst, Stephen

    2008-04-10

    Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcgammaRI, but not its associated gamma-chain, and was not supported by other FcgammaR family members (FcgammaRIIA, FcgammaRIIB, and FcgammaRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcgammaRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer.

  9. Receptor-mediated cell attachment and detachment kinetics. II. Experimental model studies with the radial-flow detachment assay.

    PubMed Central

    Cozens-Roberts, C; Quinn, J A; Lauffenburger, D A

    1990-01-01

    Quantitative information regarding the kinetics of receptor-mediated cell adhesion to a ligand-coated surface are crucial for understanding the role of certain key parameters in many physiological and biotechnology-related processes. Here, we use the probabilistic attachment and detachment models developed in the preceding paper to interpret transient data from well-defined experiments. These data are obtained with a simple model cell system that consists of receptor-coated latex beads (prototype cells) and a Radial-Flow Detachment Assay (RFDA) using a ligand-coated glass disc. The receptors and ligands used in this work are complementary antibodies. The beads enable us to examine transient behavior with particles that possess fairly uniform properties that can be varied systematically, and the RFDA is designed for direct observation of adhesion to the ligand-coated glass surface over a range of shear stresses. Our experiments focus on the effects of surface shear stress, receptor density, and ligand density. These data provide a crucial test of the probabilistic framework. We show that these data can be explained with the probabilistic analyses, whereas they cannot be readily interpreted on the basis of a deterministic analysis. In addition, we examine transient data on cell adhesion reported from other assays, demonstrating the consistency of these data with the predictions of the probabilistic models. Images FIGURE 2 PMID:2174272

  10. Dual actions of (-)-stepholidine on the dopamine receptor-mediated adenylate cyclase activity in rat corpus striatum.

    PubMed

    Dong, Z J; Guo, X; Chen, L J; Han, Y F; Jin, G Z

    1997-01-01

    (-)-Stepholidine (SPD) is an antagonist of normosensitive dopamine (DA) receptors, but it exhibits D1 agonistic action on rotational behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra pars compacta (SNC). In the present study, agonistic and antagonistic effects of SPD on the DA receptor-mediated synaptosomal adenylate cyclase (AC) activity in rat striatum were investigated. After blockade of D2 receptors, SPD augmented AC activity dose-dependently. The EC50 value was 41.1 +/- 8.6 micromol/L. At the concentration of 10 micromol/L, SPD increased cAMP formation from a basal level (50.8 +/- 10.3 pmol/mg protein/min) to 133.7 +/- 31.8 pmol/mg protein/min. The SPD-induced stimulation of AC activity was almost completely reversed by 10 micromol/L Sch23390. These results indicate that SPD possesses an agonistic action on the D1 receptor. Forskolin-stimulated adenylate cyclase (FSAC) activity was used as a model to elucidate the effect of SPD on D2 receptors. The results indicate that DA inhibited FSAC activity dose-dependently, while SPD partially restored FSAC activity. Taken together, these results support the conclusion that SPD has dual actions on DA receptors that mediate AC activity, i.e., an agonistic action on D1 receptors and an antagonistic action on D2 receptors.

  11. Adrenergic β2-receptors mediates visceral hypersensitivity induced by heterotypic intermittent stress in rats.

    PubMed

    Zhang, Chunhua; Rui, Yun-Yun; Zhou, Yuan-Yuan; Ju, Zhong; Zhang, Hong-Hong; Hu, Chuang-Ying; Xiao, Ying; Xu, Guang-Yin

    2014-01-01

    Chronic visceral pain in patients with irritable bowel syndrome (IBS) has been difficult to treat effectively partially because its pathophysiology is not fully understood. Recent studies show that norepinephrine (NE) plays an important role in the development of visceral hypersensitivity. In this study, we designed to investigate the role of adrenergic signaling in visceral hypersensitivity induced by heterotypical intermittent stress (HIS). Abdominal withdrawal reflex scores (AWRs) used as visceral sensitivity were determined by measuring the visceromoter responses to colorectal distension. Colon-specific dorsal root ganglia neurons (DRGs) were labeled by injection of DiI into the colon wall and were acutely dissociated for whole-cell patch-clamp recordings. Blood plasma level of NE was measured using radioimmunoassay kits. The expression of β2-adrenoceptors was measured by western blotting. We showed that HIS-induced visceral hypersensitivity was attenuated by systemic administration of a β-adrenoceptor antagonist propranolol, in a dose-dependent manner, but not by a α-adrenoceptor antagonist phentolamine. Using specific β-adrenoceptor antagonists, HIS-induced visceral hypersensitivity was alleviated by β2 adrenoceptor antagonist but not by β1- or β3-adrenoceptor antagonist. Administration of a selective β2-adrenoceptor antagonist also normalized hyperexcitability of colon-innervating DRG neurons of HIS rats. Furthermore, administration of β-adrenoceptor antagonist suppressed sustained potassium current density (IK) without any alteration of fast-inactivating potassium current density (IA). Conversely, administration of NE enhanced the neuronal excitability and produced visceral hypersensitivity in healthy control rats, and blocked by β2-adrenoceptor antagonists. In addition, HIS significantly enhanced the NE concentration in the blood plasma but did not change the expression of β2-adrenoceptor in DRGs and the muscularis externa of the colon. The

  12. Exercise intensity-dependent contribution of beta-adrenergic receptor-mediated vasodilatation in hypoxic humans.

    PubMed

    Wilkins, Brad W; Pike, Tasha L; Martin, Elizabeth A; Curry, Timothy B; Ceridon, Maile L; Joyner, Michael J

    2008-02-15

    We previously reported that hypoxia-mediated reductions in alpha-adrenoceptor sensitivity do not explain the augmented vasodilatation during hypoxic exercise, suggesting an enhanced vasodilator signal. We hypothesized that beta-adrenoceptor activation contributes to augmented hypoxic exercise vasodilatation. Fourteen subjects (age: 29 +/- 2 years) breathed hypoxic gas to titrate arterial O(2) saturation (pulse oximetry) to 80%, while remaining normocapnic via a rebreath system. Brachial artery and antecubital vein catheters were placed in the exercising arm. Under normoxic and hypoxic conditions, baseline and incremental forearm exercise (10% and 20% of maximum) was performed during control (saline), alpha-adrenoceptor inhibition (phentolamine), and combined alpha- and beta-adrenoceptor inhibition (phentolomine/propranolol). Forearm blood flow (FBF), heart rate, blood pressure, minute ventilation, and end-tidal CO(2) were determined. Hypoxia increased heart rate (P < 0.05) and minute ventilation (P < 0.05) at rest and exercise under all drug infusions, whereas mean arterial pressure was unchanged. Arterial adrenaline (P < 0.05) and venous noradrenaline (P < 0.05) were higher with hypoxia during all drug infusions. The change (Delta) in FBF during 10% hypoxic exercise was greater with phentolamine (Delta306 +/- 43 ml min(-1)) vs. saline (Delta169 +/- 30 ml min(-1)) or combined phentolamine/propranolol (Delta213 +/- 25 ml min(-1); P < 0.05 for both). During 20% hypoxic exercise, DeltaFBF was greater with phentalomine (Delta466 +/- 57 ml min(-1); P < 0.05) vs. saline (Delta346 +/- 40 ml min(-1)) but was similar to combined phentolamine/propranolol (De