Molecular mechanisms of platelet P2Y(12) receptor regulation.

Science.gov (United States)

Cunningham, Margaret R; Nisar, Shaista P; Mundell, Stuart J

2013-02-01

Platelets are critical for haemostasis, however inappropriate activation can lead to the development of arterial thrombosis, which can result in heart attack and stroke. ADP is a key platelet agonist that exerts its actions via stimulation of two surface GPCRs (G-protein-coupled receptors), P2Y(1) and P2Y(12). Similar to most GPCRs, P2Y receptor activity is tightly regulated by a number of complex mechanisms including receptor desensitization, internalization and recycling. In the present article, we review the molecular mechanisms that underlie P2Y(1) and P2Y(12) receptor regulation, with particular emphasis on the structural motifs within the P2Y(12) receptor, which are required to maintain regulatory protein interaction. The implications of these findings for platelet responsiveness are also discussed.

17β estradiol regulation of connexin 43-based gap junction and mechanosensitivity through classical estrogen receptor pathway in osteocyte-like MLO-Y4 cells.

KAUST Repository

Ren, Jian

2013-04-01

Connexin 43 (Cx43) plays an essential role in osteocyte mechanotransduction. Although estrogen involves in the adaptive responses of bone cells to mechanical loadings, its effects on osteocytic Cx43-based gap junction intercellular communication (GJIC) remain obscure. We found that 17β estradiol (E2) up-regulated Cx43, and enhanced GJIC in osteocyte-like MLO-Y4 cells in fluorescence recovery after photobleaching (FRAP) assay. Combination of E2 pre-treatment and oscillating fluid flow (OFF) further enhanced Cx43 expression and mitogen-activated protein kinase (MAPK) phosphorylation, comparing to E2 or OFF treatment alone. Both blocking of classical estrogen receptors (ERα/β) by fulvestrant and ERα knockdown by small interfering RNA inhibited E2-mediated Cx43 increase, while a GPR30-specific agonist G-1 failed to promote Cx43 expression. Our results suggest that the presence of E2 enhanced Cx43-based GJIC mainly via ERα/β pathway, and sensitized osteocytes to mechanical loading. © 2012 Elsevier Inc. All rights reserved.

Chemerin Elicits Potent Constrictor Actions via Chemokine-Like Receptor 1 (CMKLR1), not G-Protein-Coupled Receptor 1 (GPR1), in Human and Rat Vasculature.

Science.gov (United States)

Kennedy, Amanda J; Yang, Peiran; Read, Cai; Kuc, Rhoda E; Yang, Lucy; Taylor, Emily J A; Taylor, Colin W; Maguire, Janet J; Davenport, Anthony P

2016-10-14

Circulating levels of chemerin are significantly higher in hypertensive patients and positively correlate with blood pressure. Chemerin activates chemokine-like receptor 1 (CMKLR1 or ChemR23) and is proposed to activate the "orphan" G-protein-coupled receptor 1 (GPR1), which has been linked with hypertension. Our aim was to localize chemerin, CMKLR1, and GPR1 in the human vasculature and determine whether 1 or both of these receptors mediate vasoconstriction. Using immunohistochemistry and molecular biology in conduit arteries and veins and resistance vessels, we localized chemerin to endothelium, smooth muscle, and adventitia and found that CMKLR1 and GPR1 were widely expressed in smooth muscle. C9 (chemerin149-157) contracted human saphenous vein (pD 2 =7.30±0.31) and resistance arteries (pD 2 =7.05±0.54) and increased blood pressure in rats by 9.1±1.0 mm Hg at 200 nmol. Crucially, these in vitro and in vivo vascular actions were blocked by CCX832, which we confirmed to be highly selective for CMKLR1 over GPR1. C9 inhibited cAMP accumulation in human aortic smooth muscle cells and preconstricted rat aorta, consistent with the observed vasoconstrictor action. Downstream signaling was explored further and, compared to chemerin, C9 showed a bias factor=≈5000 for the G i protein pathway, suggesting that CMKLR1 exhibits biased agonism. Our data suggest that chemerin acts at CMKLR1, but not GPR1, to increase blood pressure. Chemerin has an established detrimental role in metabolic syndrome, and these direct vascular actions may contribute to hypertension, an additional risk factor for cardiovascular disease. This study provides proof of principle for the therapeutic potential of selective CMKLR1 antagonists. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Targeting CB2-GPR55 Receptor Heteromers Modulates Cancer Cell Signaling*

Science.gov (United States)

Moreno, Estefanía; Andradas, Clara; Medrano, Mireia; Caffarel, María M.; Pérez-Gómez, Eduardo; Blasco-Benito, Sandra; Gómez-Cañas, María; Pazos, M. Ruth; Irving, Andrew J.; Lluís, Carme; Canela, Enric I.; Fernández-Ruiz, Javier; Guzmán, Manuel; McCormick, Peter J.; Sánchez, Cristina

2014-01-01

The G protein-coupled receptors CB2 (CB2R) and GPR55 are overexpressed in cancer cells and human tumors. Because a modulation of GPR55 activity by cannabinoids has been suggested, we analyzed whether this receptor participates in cannabinoid effects on cancer cells. Here we show that CB2R and GPR55 form heteromers in cancer cells, that these structures possess unique signaling properties, and that modulation of these heteromers can modify the antitumoral activity of cannabinoids in vivo. These findings unveil the existence of previously unknown signaling platforms that help explain the complex behavior of cannabinoids and may constitute new targets for therapeutic intervention in oncology. PMID:24942731

Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2.

Science.gov (United States)

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

2016-01-01

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

Orphan receptor GPR110, an oncogene overexpressed in lung and prostate cancer

International Nuclear Information System (INIS)

Lum, Amy M; Wang, Bruce B; Beck-Engeser, Gabriele B; Li, Lauri; Channa, Namitha; Wabl, Matthias

2010-01-01

GPR110 is an orphan G protein-coupled receptor--a receptor without a known ligand, a known signaling pathway, or a known function. Despite the lack of information, one can assume that orphan receptors have important biological roles. In a retroviral insertion mutagenesis screen in the mouse, we identified GPR110 as an oncogene. This prompted us to study the potential isoforms that can be gleaned from known GPR110 transcripts, and the expression of these isoforms in normal and transformed human tissues. Various epitope-tagged isoforms of GPR110 were expressed in cell lines and assayed by western blotting to determine cleavage, surface localization, and secretion patterns. GPR110 transcript and protein levels were measured in lung and prostate cancer cell lines and clinical samples, respectively, by quantitative PCR and immunohistochemistry. We found four potential splice variants of GPR110. Of these variants, we confirmed three as being expressed as proteins on the cell surface. Isoform 1 is the canonical form, with a molecular mass of about 100 kD. Isoforms 2 and 3 are truncated products of isoform 1, and are 25 and 23 kD, respectively. These truncated isoforms lack the seven-span transmembrane domain characteristic of GPR proteins and thus are not likely to be membrane anchored; indeed, isoform 2 can be secreted. Compared with the median gene expression of ~200 selected genes, GPR110 expression was low in most tissues. However, it had higher than average gene expression in normal kidney tissue and in prostate tissues originating from older donors. Although identified as an oncogene in murine T lymphomas, GPR110 is greatly overexpressed in human lung and prostate cancers. As detected by immunohistochemistry, GPR110 was overexpressed in 20 of 27 (74%) lung adenocarcinoma tissue cores and in 17 of 29 (59%) prostate adenocarcinoma tissue cores. Additionally, staining with a GPR110 antibody enabled us to differentiate between benign prostate hyperplasia and potential

Short-chain free fatty acid receptors FFA2/GPR43 and FFA3/GPR41 as new potential therapeutic targets.

Science.gov (United States)

Ulven, Trond

2012-01-01

The deorphanization of the free fatty acid (FFA) receptors FFA1 (GPR40), FFA2 (GPR43), FFA3 (GPR41), GPR84, and GPR120 has made clear that the body is capable of recognizing and responding directly to nonesterified fatty acid of virtually any chain length. Colonic fermentation of dietary fiber produces high concentrations of the short-chain fatty acids (SCFAs) acetate, propionate and butyrate, a process which is important to health. The phylogenetically related 7-transmembrane (7TM) receptors free fatty acid receptor 2 (FFA2) and FFA3 are activated by these SCFAs, and several lines of evidence indicate that FFA2 and FFA3 mediate beneficial effects associated with a fiber-rich diet, and that they may be of interest as targets for treatment of inflammatory and metabolic diseases. FFA2 is highly expressed on immune cells, in particular neutrophils, and several studies suggest that the receptor plays a role in diseases involving a dysfunctional neutrophil response, such as inflammatory bowel disease (IBD). Both FFA2 and FFA3 have been implicated in metabolic diseases such as type 2 diabetes and in regulation of appetite. More research is however required to clarify the potential of the receptors as drug targets and establish if activation or inhibition would be the preferred mode of action. The availability of potent and selective receptor modulators is a prerequisite for these studies. The few modulators of FFA2 or FFA3 that have been published hitherto in the peer-reviewed literature in general have properties that make them less than ideal as such tools, but published patent applications indicate that better tool compounds might soon become available which should enable studies critical to validate the receptors as new drug targets.

Short-chain free fatty acid receptors FFA2/GPR43 and FFA3/GPR41 as new potential therapeutic targets

Directory of Open Access Journals (Sweden)

Trond eUlven

2012-10-01

Full Text Available The deorphanization of the free fatty acid (FFA receptors FFA1 (GPR40, FFA2 (GPR43, FFA3 (GPR41, GPR84 and GPR120 made clear that the body is capable of recognizing and responding directly to nonesterified fatty acid of virtually any chain length. Colonic fermentation of dietary fiber produces high concentrations of the short-chain fatty acids (SCFAs acetate, propionate and butyrate, a process which is important to health. The phylogenetically related 7-transmembrane receptors free fatty acid receptor 2 (FFA2 and FFA3 are activated by these SCFAs, and several lines of evidence indicate that FFA2 and FFA3 mediate beneficial effects associated with a fiber-rich diet, and that they may be of interest as targets for treatment of inflammatory and metabolic diseases. FFA2 is highly expressed on immune cells, in particular neutrophils, and several studies suggest that the receptor plays a role in diseases involving a dysfunctional neutrophil response, such as inflammatory bowel disease (IBD. Both FFA2 and FFA3 have been implicated in metabolic diseases such as type 2 diabetes and regulation of appetite. More research is however required to clarify potential of the receptors as drug targets and establish if activation or inhibition would be the preferred mode of action. The availability of potent and selective receptor modulators is a prerequisite for these studies. The few modulators of FFA2 or FFA3 that have been published hitherto in the peer-reviewed literature in general have properties that make them less than ideal as such tools, but published patent applications indicate that the situation may soon improve, and that proper tool compounds will enable studies critical to validate the receptors as new drug targets.

Arrestin scaffolds NHERF1 to the P2Y12 receptor to regulate receptor internalization.

Science.gov (United States)

Nisar, Shaista P; Cunningham, Margaret; Saxena, Kunal; Pope, Robert J; Kelly, Eamonn; Mundell, Stuart J

2012-07-13

We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y(12) receptor (P2Y(12)R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y(12)R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y(12)R internalization. In vitro and prior to agonist stimulation P2Y(12)R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization.

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

Science.gov (United States)

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

2012-01-01

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

Embelin and its derivatives unravel the signaling, proinflammatory and antiatherogenic properties of GPR84 receptor.

Science.gov (United States)

Gaidarov, Ibragim; Anthony, Todd; Gatlin, Joel; Chen, Xiaohua; Mills, David; Solomon, Michelle; Han, Sangdon; Semple, Graeme; Unett, David J

2018-05-01

GPR84 is an orphan G-protein coupled receptor, expressed on monocytes, macrophages and neutrophils and is significantly upregulated by inflammatory stimuli. The physiological role of GPR84 remains largely unknown. Medium chain fatty acids (MCFA) activate the receptor and have been proposed to be its endogenous ligands, although the high concentrations of MCFAs required for receptor activation generally exceed normal physiological levels. We identified the natural product embelin as a highly potent and selective surrogate GPR84 agonist (originally disclosed in patent application WO2007027661A2, 2007) and synthesized close structural analogs with widely varying receptor activities. These tools were used to perform a comprehensive study of GPR84 signaling and function in recombinant cells and in primary human macrophages and neutrophils. Activation of recombinant GPR84 by embelin in HEK293 cells results in G i/o as well as G12/13-Rho signaling. In human macrophages, GPR84 initiates PTX sensitive Erk1/2 and Akt phosphorylation, PI-3 kinase activation, calcium flux, and release of prostaglandin E2. In addition, GPR84 signaling in macrophages elicits G i Gβγ-mediated augmentation of intracellular cAMP, rather than the decrease expected from G iα engagement. GPR84 activation drives human neutrophil chemotaxis and primes them for amplification of oxidative burst induced by FMLP and C5A. Loss of GPR84 is associated with attenuated LPS-induced release of proinflammatory mediators IL-6, KC-GROα, VEGF, MIP-2 and NGAL from peritoneal exudates. While initiating numerous proinflammatory activities in macrophages and neutrophils, GPR84 also possesses GPR109A-like antiatherosclerotic properties in macrophages. Macrophage receptor activation leads to upregulation of cholesterol transporters ABCA1 and ABCG1 and stimulates reverse cholesterol transport. These data suggest that GPR84 may be a target of therapeutic value and that distinct modes of receptor modulation (inhibition vs

The orphan G protein-coupled receptor 25 (GPR25) is activated by Apelin and Apela in non-mammalian vertebrates.

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Zhang, Jiannan; Wan, Yiping; Fang, Chao; Chen, Junan; Ouyang, Wangan; Li, Juan; Wang, Yajun

2018-06-22

G protein-coupled receptor 25 (GPR25) is an orphan G protein-coupled receptor in vertebrates, that has been implicated to be associated with autoimmune diseases and regulate blood pressure in humans. However, the endogenous ligand of GPR25 remains unknown in vertebrates. Here, we reported that in non-mammalian vertebrates (zebrafish, spotted gars, and pigeons), GPR25 could be activated by Apelin and Apela peptides, which are also the two endogenous ligands of vertebrate Apelin receptor (APLNR). Using the pGL3-CRE-luciferase reporter assay and confocal microscopy, we first demonstrated that like APLNR, zebrafish GPR25 expressing in HEK293 cells could be effectively activated by zebrafish Apelin and Apela peptides, leading to the inhibition of forskolin-stimulated cAMP production and receptor internalization. Like zebrafish GPR25, pigeon and spotted gar GPR25 could also be activated by Apelin and Apela, and their activation could inhibit forskolin-induced cAMP accumulation. Interestingly, unlike zebrafish (/spotted gar/pigeon) GPR25, human GPR25 could not be activated by Apelin and Apela under the same experimental conditions. RNA-seq analysis further revealed that GPR25 is expressed in a variety of tissues, including the testes and intestine of zebrafish/spotted gars/humans, implying the potential roles of GPR25 signaling in many physiological processes in vertebrates. Taken together, our data not only provides the first proof that the orphan receptor GPR25 possesses two potential ligands 'Apelin and Apela' and its activation decreases intracellular cAMP levels in non-mammalian vertebrates, but also facilitates to unravel the physiological roles of GPR25 signaling in vertebrates. Copyright © 2018 Elsevier Inc. All rights reserved.

Deletion of the pH sensor GPR4 decreases renal acid excretion.

Science.gov (United States)

Sun, Xuming; Yang, Li V; Tiegs, Brian C; Arend, Lois J; McGraw, Dennis W; Penn, Raymond B; Petrovic, Snezana

2010-10-01

Proton receptors are G protein-coupled receptors that accept protons as ligands and function as pH sensors. One of the proton receptors, GPR4, is relatively abundant in the kidney, but its potential role in acid-base homeostasis is unknown. In this study, we examined the distribution of GPR4 in the kidney, its function in kidney epithelial cells, and the effects of its deletion on acid-base homeostasis. We observed GPR4 expression in the kidney cortex, in the outer and inner medulla, in isolated kidney collecting ducts, and in cultured outer and inner medullary collecting duct cells (mOMCD1 and mIMCD3). Cultured mOMCD1 cells exhibited pH-dependent accumulation of intracellular cAMP, characteristic of GPR4 activation; GPR4 knockdown attenuated this accumulation. In vivo, deletion of GPR4 decreased net acid secretion by the kidney and resulted in a nongap metabolic acidosis, indicating that GPR4 is required to maintain acid-base homeostasis. Collectively, these findings suggest that GPR4 is a pH sensor with an important role in regulating acid secretion in the kidney collecting duct.

Genetic deletion of GPR52 enhances the locomotor-stimulating effect of an adenosine A2A receptor antagonist in mice: A potential role of GPR52 in the function of striatopallidal neurons.

Science.gov (United States)

Nishiyama, Keiji; Suzuki, Hirobumi; Maruyama, Minoru; Yoshihara, Tomoki; Ohta, Hiroyuki

2017-09-01

G protein-coupled receptor 52 (GPR52) is largely co-expressed with dopamine D 2 receptor (DRD2) in the striatum and nucleus accumbens, and this expression pattern is similar to that of adenosine A 2A receptor (ADORA2A). GPR52 has been proposed as a therapeutic target for positive symptoms of schizophrenia, based on observations from pharmacological and transgenic mouse studies. However, the physiological role of GPR52 in dopaminergic functions in the basal ganglia remains unclear. Here, we used GPR52 knockout (KO) mice to examine the role of GPR52 in dopamine receptor-mediated and ADORA2A-mediated locomotor activity and dopamine receptor signaling. High expression of GPR52 protein in the striatum, nucleus accumbens, and lateral globus pallidus of wild type (WT) littermates was confirmed by immunohistochemical analysis. GPR52 KO and WT mice exhibited almost identical locomotor responses to the dopamine releaser methamphetamine and the N-methyl-d-aspartate antagonist MK-801. In contrast, the locomotor response to the ADORA2A antagonist istradefylline was significantly augmented in GPR52 KO mice compared to WT mice. Gene expression analysis revealed that striatal expression of DRD2, but not of dopamine D 1 receptor and ADORA2A, was significantly decreased in GPR52 KO mice. Moreover, a significant reduction in the mRNA expression of enkephalin, a marker of the activity of striatopallidal neurons, was observed in the striatum of GPR52 KO mice, suggesting that GPR52 deletion could enhance DRD2 signaling. Taken together, these results imply the physiological relevance of GPR52 in modulating the function of striatopallidal neurons, possibly by interaction of GPR52 with ADORA2A and DRD2. Copyright © 2017 Elsevier B.V. All rights reserved.

Mutations in the pH-Sensing G-protein-Coupled Receptor GPR68 Cause Amelogenesis Imperfecta.

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Parry, David A; Smith, Claire E L; El-Sayed, Walid; Poulter, James A; Shore, Roger C; Logan, Clare V; Mogi, Chihiro; Sato, Koichi; Okajima, Fumikazu; Harada, Akihiro; Zhang, Hong; Koruyucu, Mine; Seymen, Figen; Hu, Jan C-C; Simmer, James P; Ahmed, Mushtaq; Jafri, Hussain; Johnson, Colin A; Inglehearn, Chris F; Mighell, Alan J

2016-10-06

Amelogenesis is the process of dental enamel formation, leading to the deposition of the hardest tissue in the human body. This process requires the intricate regulation of ion transport and controlled changes to the pH of the developing enamel matrix. The means by which the enamel organ regulates pH during amelogenesis is largely unknown. We identified rare homozygous variants in GPR68 in three families with amelogenesis imperfecta, a genetically and phenotypically heterogeneous group of inherited conditions associated with abnormal enamel formation. Each of these homozygous variants (a large in-frame deletion, a frameshift deletion, and a missense variant) were predicted to result in loss of function. GPR68 encodes a proton-sensing G-protein-coupled receptor with sensitivity in the pH range that occurs in the developing enamel matrix during amelogenesis. Immunohistochemistry of rat mandibles confirmed localization of GPR68 in the enamel organ at all stages of amelogenesis. Our data identify a role for GPR68 as a proton sensor that is required for proper enamel formation. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

A critical look at the function of the P2Y11 receptor

DEFF Research Database (Denmark)

Dreisig, Karin; Kornum, Birgitte Rahbek

2016-01-01

The P2Y11 receptor is a member of the purinergic receptor family. It has been overlooked, somewhat due to the lack of a P2ry11 gene orthologue in the murine genome, which prevents the generation of knockout mice, which have been so helpful for defining the roles of other P2Y receptors. Furthermor...

Medium-chain fatty acid-sensing receptor, GPR84, is a proinflammatory receptor.

Science.gov (United States)

Suzuki, Masakatsu; Takaishi, Sachiko; Nagasaki, Miyuki; Onozawa, Yoshiko; Iino, Ikue; Maeda, Hiroaki; Komai, Tomoaki; Oda, Tomiichiro

2013-04-12

G protein-coupled receptor 84 (GPR84) is a putative receptor for medium-chain fatty acids (MCFAs), whose pathophysiological roles have not yet been clarified. Here, we show that GPR84 was activated by MCFAs with the hydroxyl group at the 2- or 3-position more effectively than nonhydroxylated MCFAs. We also identified a surrogate agonist, 6-n-octylaminouracil (6-OAU), for GPR84. These potential ligands and the surrogate agonist, 6-OAU, stimulated [(35)S]GTP binding and accumulated phosphoinositides in a GPR84-dependent manner. The surrogate agonist, 6-OAU, internalized GPR84-EGFP from the cell surface. Both the potential ligands and 6-OAU elicited chemotaxis of human polymorphonuclear leukocytes (PMNs) and macrophages and amplified LPS-stimulated production of the proinflammatory cytokine IL-8 from PMNs and TNFα from macrophages. Furthermore, the intravenous injection of 6-OAU raised the blood CXCL1 level in rats, and the inoculation of 6-OAU into the rat air pouch accumulated PMNs and macrophages in the site. Our results indicate a proinflammatory role of GPR84, suggesting that the receptor may be a novel target to treat chronic low grade inflammation associated-disease.

Medicinal chemistry of adenosine, P2Y and P2X receptors.

Science.gov (United States)

Jacobson, Kenneth A; Müller, Christa E

2016-05-01

Pharmacological tool compounds are now available to define action at the adenosine (ARs), P2Y and P2X receptors. We present a selection of the most commonly used agents to study purines in the nervous system. Some of these compounds, including A1 and A3 AR agonists, P2Y1R and P2Y12R antagonists, and P2X3, P2X4 and P2X7 antagonists, are potentially of clinical use in treatment of disorders of the nervous system, such as chronic pain, neurodegeneration and brain injury. Agonists of the A2AAR and P2Y2R are already used clinically, P2Y12R antagonists are widely used antithrombotics and an antagonist of the A2AAR is approved in Japan for treating Parkinson's disease. The selectivity defined for some of the previously introduced compounds has been revised with updated pharmacological characterization, for example, various AR agonists and antagonists were deemed A1AR or A3AR selective based on human data, but species differences indicated a reduction in selectivity ratios in other species. Also, many of the P2R ligands still lack bioavailability due to charged groups or hydrolytic (either enzymatic or chemical) instability. X-ray crystallographic structures of AR and P2YRs have shifted the mode of ligand discovery to structure-based approaches rather than previous empirical approaches. The X-ray structures can be utilized either for in silico screening of chemically diverse libraries for the discovery of novel ligands or for enhancement of the properties of known ligands by chemical modification. Although X-ray structures of the zebrafish P2X4R have been reported, there is scant structural information about ligand recognition in these trimeric ion channels. In summary, there are definitive, selective agonists and antagonists for all of the ARs and some of the P2YRs; while the pharmacochemistry of P2XRs is still in nascent stages. The therapeutic potential of selectively modulating these receptors is continuing to gain interest in such fields as cancer, inflammation, pain

Neuropharmacology of purinergic receptors in human submucous plexus: Involvement of P2X₁, P2X₂, P2X₃ channels, P2Y and A₃ metabotropic receptors in neurotransmission.

Science.gov (United States)

Liñán-Rico, A; Wunderlich, J E; Enneking, J T; Tso, D R; Grants, I; Williams, K C; Otey, A; Michel, K; Schemann, M; Needleman, B; Harzman, A; Christofi, F L

2015-08-01

The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 > P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R. Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are

P2Y2 Receptor and EGFR Cooperate to Promote Prostate Cancer Cell Invasion via ERK1/2 Pathway.

Science.gov (United States)

Li, Wei-Hua; Qiu, Ying; Zhang, Hong-Quan; Tian, Xin-Xia; Fang, Wei-Gang

2015-01-01

As one member of G protein-coupled P2Y receptors, P2Y2 receptor can be equally activated by extracellular ATP and UTP. Our previous studies have proved that activation of P2Y2 receptor by extracellular ATP could promote prostate cancer cell invasion and metastasis in vitro and in vivo via regulating the expressions of some epithelial-mesenchymal transition/invasion-related genes (including IL-8, E-cadherin, Snail and Claudin-1), and the most significant change in expression of IL-8 was observed after P2Y2 receptor activation. However, the signaling pathway downstream of P2Y2 receptor and the role of IL-8 in P2Y2-mediated prostate cancer cell invasion remain unclear. Here, we found that extracellular ATP/UTP induced activation of EGFR and ERK1/2. After knockdown of P2Y2 receptor, the ATP -stimulated phosphorylation of EGFR and ERK1/2 was significantly suppressed. Further experiments showed that inactivation of EGFR and ERK1/2 attenuated ATP-induced invasion and migration, and suppressed ATP-mediated IL-8 production. In addition, knockdown of IL-8 inhibited ATP-mediated invasion and migration of prostate cancer cells. These findings suggest that P2Y2 receptor and EGFR cooperate to upregulate IL-8 production via ERK1/2 pathway, thereby promoting prostate cancer cell invasion and migration. Thus blocking of the P2Y2-EGFR-ERK1/2 pathway may provide effective therapeutic interventions for prostate cancer.

Purine receptor P2Y_6 mediates cellular response to γ-ray-induced DNA damage

International Nuclear Information System (INIS)

Ide, Shunta; Nishimaki, Naoko; Tsukimoto, Mitsutoshi; Kojima, Shuji

2014-01-01

We previously showed that nucleotide P2 receptor agonists such as ATP and UTP amplify γ-ray-induced focus formation of phosphorylated histone H2A variant H2AX (γH2AX), which is considered to be an indicator of DNA damage so far, by activating purine P2Y_6 and P2Y_1_2 receptors. Therefore, we hypothesized that these P2 receptors play a role in inducing the repair response to γ-ray-induced DNA damage. In the present study, we tested this idea by using human lung cancer A549 cells. First, reverse-transcription polymerase chain reaction (RT-PCR) showed that P2Y_6 receptor is highly expressed in A549 cells, but P2Y_1_2 receptor is only weakly expressed. Next, colony formation assay revealed that P2Y_6 receptor antagonist MRS2578 markedly reduced the survival rate of γ-ray-exposed A549 cells. The survival rate was also significantly reduced in P2Y_6-knock-down cells, compared with scramble siRNA-transfected cells. Since it has reported that phosphorylation of ERK1/2 after activation of EGFR via P2Y_6 and P2Y_1_2 receptors is involved in the repair response to γ-ray-induced DNA damage, we next examined whether γ-ray-induced phosphorylation of ERK1/2 was also inhibited by MRS2578 in A549 cells. We found that it was. Taken together, these findings indicate that purinergic signaling through P2Y_6 receptor, followed by ERK1/2 activation, promotes the cellular repair response to γ-ray-induced DNA damage. (author)

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

Science.gov (United States)

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

2016-05-10

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.

Tools and drugs for uracil nucleotide-activated P2Y receptors.

Science.gov (United States)

Rafehi, Muhammad; Müller, Christa E

2018-04-13

P2Y receptors (P2YRs) are a family of G protein-coupled receptors activated by extracellular nucleotides. Physiological P2YR agonists include purine and pyrimidine nucleoside di- and triphosphates, such as ATP, ADP, UTP, UDP, nucleotide sugars, and dinucleotides. Eight subtypes exist, P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 , which represent current or potential future drug targets. Here we provide a comprehensive overview of ligands for the subgroup of the P2YR family that is activated by uracil nucleotides: P2Y 2 (UTP, also ATP and dinucleotides), P2Y 4 (UTP), P2Y 6 (UDP), and P2Y 14 (UDP, UDP-glucose, UDP-galactose). The physiological agonists are metabolically unstable due to their fast hydrolysis by ectonucleotidases. A number of agonists with increased potency, subtype-selectivity and/or enzymatic stability have been developed in recent years. Useful P2Y 2 R agonists include MRS2698 (6-01, highly selective) and PSB-1114 (6-05, increased metabolic stability). A potent and selective P2Y 2 R antagonist is AR-C118925 (10-01). For studies of the P2Y 4 R, MRS4062 (3-15) may be used as a selective agonist, while PSB-16133 (10-06) represents a selective antagonist. Several potent P2Y 6 R agonists have been developed including 5-methoxyuridine 5'-O-((R p )α-boranodiphosphate) (6-12), PSB-0474 (3-11), and MRS2693 (3-26). The isocyanate MRS2578 (10-08) is used as a selective P2Y 6 R antagonist, although its reactivity and low water-solubility are limiting. With MRS2905 (6-08), a potent and metabolically stable P2Y 14 R agonist is available, while PPTN (10-14) represents a potent and selective P2Y 14 R antagonist. The radioligand [ 3 H]UDP can be used to label P2Y 14 Rs. In addition, several fluorescent probes have been developed. Uracil nucleotide-activated P2YRs show great potential as drug targets, especially in inflammation, cancer, cardiovascular and neurodegenerative diseases. Copyright © 2018. Published by Elsevier Inc.

Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40

Energy Technology Data Exchange (ETDEWEB)

Lu, Jun; Byrne, Noel; Wang, John; Bricogne, Gerard; Brown, Frank K.; Chobanian, Harry R.; Colletti, Steven L.; Di Salvo, Jerry; Thomas-Fowlkes, Brande; Guo, Yan; Hall, Dawn L.; Hadix, Jennifer; Hastings, Nicholas B.; Hermes, Jeffrey D.; Ho, Thu; Howard, Andrew D.; Josien, Hubert; Kornienko, Maria; Lumb, Kevin J.; Miller, Michael W.; Patel, Sangita B.; Pio, Barbara; Plummer, Christopher W.; Sherborne, Bradley S.; Sheth, Payal; Souza, Sarah; Tummala, Srivanya; Vonrhein, Clemens; Webb, Maria; Allen, Samantha J.; Johnston, Jennifer M.; Weinglass, Adam B.; Sharma, Sujata; Soisson, Stephen M. (Merck); (Globel Phasing)

2017-06-05

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

Expression of the Fatty Acid Receptors GPR84 and GPR120 and Cytodifferentiation of Epithelial Cells in the Gastric Mucosa of Mouse Pups in the Course of Dietary Transition.

Science.gov (United States)

Widmayer, Patricia; Kusumakshi, Soumya; Hägele, Franziska A; Boehm, Ulrich; Breer, Heinz

2017-01-01

During weaning, the ingested food of mouse pups changes from exclusively milk to solid food. In contrast to the protein- and carbohydrate-rich solid food, high fat milk is characterized primarily by fatty acids of medium chain length particularly important for the suckling pups. Therefore, it seems conceivable that the stomach mucosa may be specialized for detecting these important nutrients during the suckling phase. Here, we analyzed the expression of the G protein coupled receptors GPR84 and GPR120 (FFAR4), which are considered to be receptors for medium and long chain fatty acids (LCFAs), respectively. We found that the mRNA levels for GPR84 and GPR120 were high during the suckling period and progressively decreased in the course of weaning. Visualization of the receptor-expressing cells in 2-week-old mice revealed a high number of labeled cells, which reside in the apical as well as in the basal region of the gastric glands. At the base of the gastric glands, all GPR84-immunoreactive cells and some of the GPR120-positive cells also expressed chromogranin A (CgA), suggesting that they are enteroendocrine cells. We demonstrate that the majority of the CgA/GPR84 cells are X/A-like ghrelin cells. The high degree of overlap between ghrelin and GPR84 decreased post-weaning, whereas the overlap between ghrelin and GPR120 increased. At the apical region of the glands the fatty acid receptors were mainly expressed in unique cell types. These contain lipid-filled vacuole- and vesicle-like structures and may have absorptive functions. We detected decreased immunoreactivity for GPR84 and no lipid droplets in surface cells post-weaning. In conclusion, expression of GPR84 in ghrelin cells as well as in surface cells suggests an important role of medium chain fatty acids (MCFAs) in the developing gastric mucosa of suckling mice.

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

OpenAIRE

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 dig...

ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

Science.gov (United States)

Kanamarlapudi, Venkateswarlu; Owens, Sian E; Saha, Keya; Pope, Robert J; Mundell, Stuart J

2012-01-01

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

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Venkateswarlu Kanamarlapudi

Full Text Available Adenosine diphosphate (ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1 and P2Y(12 purinoceptors. Recently, we demonstrated that P2Y(1 and P2Y(12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6 in the internalization and function of P2Y(1 and P2Y(12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1 or P2Y(12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

Transformation of Astrocytes to a Neuroprotective Phenotype by Microglia via P2Y1 Receptor Downregulation

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Youichi Shinozaki

2017-05-01

Full Text Available Microglia and astrocytes become reactive following traumatic brain injury (TBI. However, the coordination of this reactivity and its relation to pathophysiology are unclear. Here, we show that microglia transform astrocytes into a neuroprotective phenotype via downregulation of the P2Y1 purinergic receptor. TBI initially caused microglial activation in the injury core, followed by reactive astrogliosis in the peri-injured region and formation of a neuroprotective astrocyte scar. Equivalent changes to astrocytes were observed in vitro after injury. This change in astrocyte phenotype resulted from P2Y1 receptor downregulation, mediated by microglia-derived cytokines. In mice, astrocyte-specific P2Y1 receptor overexpression (Astro-P2Y1OE counteracted scar formation, while astrocyte-specific P2Y1 receptor knockdown (Astro-P2Y1KD facilitated scar formation, suggesting critical roles of P2Y1 receptors in the transformation. Astro-P2Y1OE and Astro-P2Y1KD mice showed increased and reduced neuronal damage, respectively. Altogether, our findings indicate that microglia-astrocyte interaction, involving a purinergic signal, is essential for the formation of neuroprotective astrocytes.

Pathophysiological consequences of receptor mistraffic: Tales from the platelet P2Y12 receptor.

Science.gov (United States)

Cunningham, Margaret R; Aungraheeta, Riyaad; Mundell, Stuart J

2017-07-05

Genetic variations in G protein-coupled receptor (GPCR) genes can disrupt receptor function in a wide variety of human genetic diseases, including platelet bleeding disorders. Platelets are critical for haemostasis with inappropriate platelet activation leading to the development of arterial thrombosis, which can result in heart attack and stroke whilst decreased platelet activity is associated with an increased risk of bleeding. GPCRs expressed on the surface of platelets play key roles in regulating platelet activity and therefore function. Receptors include purinergic receptors (P2Y 1 and P2Y 12 ), proteinase-activated receptor (PAR1 and PAR4) and thromboxane receptors (TPα), among others. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis. With the advance of genomic technologies, there has been a substantial increase in the identification of naturally occurring rare and common GPCR variants. These variants include single-nucleotide polymorphisms (SNPs) and insertion or deletions that have the potential to alter GPCR expression or function. A number of defects in platelet GPCRs that disrupt receptor function have now been characterized in patients with mild bleeding disorders. This review will focus on rare, function-disrupting variants of platelet GPCRs with particular emphasis upon mutations in the P2Y 12 receptor gene that affect receptor traffic to modulate platelet function. Further this review will outline how the identification and characterization of function-disrupting GPCR mutations provides an essential link in translating our detailed understanding of receptor traffic and function in cell line studies into relevant human biological systems. Copyright © 2017. Published by Elsevier B.V.

UTP reduces infarct size and improves mice heart function after myocardial infarct via P2Y2 receptor

DEFF Research Database (Denmark)

Cohen, A; Shainberg, Asher; Hochhauser, E

2011-01-01

Pyrimidine nucleotides are signaling molecules, which activate G protein-coupled membrane receptors of the P2Y family. P2Y(2) and P2Y(4) receptors are part of the P2Y family, which is composed of 8 subtypes that have been cloned and functionally defined. We have previously found that uridine-5......'-triphosphate (UTP) reduces infarct size and improves cardiac function following myocardial infarct (MI). The aim of the present study was to determine the role of P2Y(2) receptor in cardiac protection following MI using knockout (KO) mice, in vivo and wild type (WT) for controls. In both experimental groups...... used (WT and P2Y(2)(-/-) receptor KO mice) there were 3 subgroups: sham, MI, and MI+UTP. 24h post MI we performed echocardiography and measured infarct size using triphenyl tetrazolium chloride (TTC) staining on all mice. Fractional shortening (FS) was higher in WT UTP-treated mice than the MI group...

Proton receptor GPR68 expression in dendritic-cell-like S100β-positive cells of rat anterior pituitary gland: GPR68 induces interleukin-6 gene expression in extracellular acidification.

Science.gov (United States)

Horiguchi, Kotaro; Higuchi, Masashi; Yoshida, Saishu; Nakakura, Takashi; Tateno, Kozue; Hasegawa, Rumi; Takigami, Shu; Ohsako, Shunji; Kato, Takako; Kato, Yukio

2014-11-01

S100β-positive cells, which do not express the classical pituitary hormones, appear to possess multifunctional properties and are assumed to be heterogeneous in the anterior pituitary gland. The presence of several protein markers has shown that S100β-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100β-positive cells into round-cell (dendritic-cell-like) and process-cell types. We also found the characteristic expression of anti-inflammatory factors (interleukin-6, Il-6) and membrane receptors (integrin β-6) in the round type. Here, we further investigate the function of the subpopulation of S100β-positive cells. Since IL-6 is also a paracrine factor that regulates hormone producing-cells, we examine whether a correlation exists among extracellular acid stress, IL-6 and hormone production by using primary cultures of anterior pituitary cells. Dendritic-cell-like S100β-positive cells notably expressed Gpr68 (proton receptor) and Il-6. Furthermore, the expression of Il-6 and proopiomelanocortin (Pomc) was up-regulated by extracellular acidification. The functional role of IL-6 and GPR68 in the gene expression of Pomc during extracellular acidification was also examined. Small interfering RNA for Il-6 up-regulated Pomc expression and that for Gpr68 reversed the down-regulation of Il-6 and up-regulated Pomc expression by extracellular acidification. Thus, S100β-positive dendritic-like cells can sense an increase in extracellular protons via GPR68 and respond by the production of IL-6 in order to suppress the up-regulation of Pomc expression.

Expression of the Fatty Acid Receptors GPR84 and GPR120 and Cytodifferentiation of Epithelial Cells in the Gastric Mucosa of Mouse Pups in the Course of Dietary Transition

Directory of Open Access Journals (Sweden)

Patricia Widmayer

2017-08-01

Full Text Available During weaning, the ingested food of mouse pups changes from exclusively milk to solid food. In contrast to the protein- and carbohydrate-rich solid food, high fat milk is characterized primarily by fatty acids of medium chain length particularly important for the suckling pups. Therefore, it seems conceivable that the stomach mucosa may be specialized for detecting these important nutrients during the suckling phase. Here, we analyzed the expression of the G protein coupled receptors GPR84 and GPR120 (FFAR4, which are considered to be receptors for medium and long chain fatty acids (LCFAs, respectively. We found that the mRNA levels for GPR84 and GPR120 were high during the suckling period and progressively decreased in the course of weaning. Visualization of the receptor-expressing cells in 2-week-old mice revealed a high number of labeled cells, which reside in the apical as well as in the basal region of the gastric glands. At the base of the gastric glands, all GPR84-immunoreactive cells and some of the GPR120-positive cells also expressed chromogranin A (CgA, suggesting that they are enteroendocrine cells. We demonstrate that the majority of the CgA/GPR84 cells are X/A-like ghrelin cells. The high degree of overlap between ghrelin and GPR84 decreased post-weaning, whereas the overlap between ghrelin and GPR120 increased. At the apical region of the glands the fatty acid receptors were mainly expressed in unique cell types. These contain lipid-filled vacuole- and vesicle-like structures and may have absorptive functions. We detected decreased immunoreactivity for GPR84 and no lipid droplets in surface cells post-weaning. In conclusion, expression of GPR84 in ghrelin cells as well as in surface cells suggests an important role of medium chain fatty acids (MCFAs in the developing gastric mucosa of suckling mice.

Apical membrane P2Y4 purinergic receptor controls K+ secretion by strial marginal cell epithelium

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Scofield Margaret A

2005-11-01

Full Text Available Abstract Background It was previously shown that K+ secretion by strial marginal cell epithelium is under the control of G-protein coupled receptors of the P2Y family in the apical membrane. Receptor activation by uracil nucleotides (P2Y2, P2Y4 or P2Y6 leads to a decrease in the electrogenic K+ secretion. The present study was conducted to determine the subtype of the functional purinergic receptor in gerbil stria vascularis, to test if receptor activation leads to elevation of intracellular [Ca2+] and to test if the response to these receptors undergoes desensitization. Results The transepithelial short circuit current (Isc represents electrogenic K+ secretion and was found to be decreased by uridine 5'-triphosphate (UTP, adenosine 5'-triphosphate (ATP and diadenosine tetraphosphate (Ap4A but not uridine 5'-diphosphate (UDP at the apical membrane of marginal cells of the gerbil stria vascularis. The potencies of these agonists were consistent with rodent P2Y4 and P2Y2 but not P2Y6 receptors. Activation caused a biphasic increase in intracellular [Ca2+] that could be partially blocked by 2-aminoethoxy-diphenyl borate (2-APB, an inhibitor of the IP3 receptor and store-operated channels. Suramin (100 μM did not inhibit the effect of UTP (1 μM. The ineffectiveness of suramin at the concentration used was consistent with P2Y4 but not P2Y2. Transcripts for both P2Y2 and P2Y4 were found in the stria vascularis. Sustained exposure to ATP or UTP for 15 min caused a depression of Isc that appeared to have two components but with apparently no chronic desensitization. Conclusion The results support the conclusion that regulation of K+ secretion across strial marginal cell epithelium occurs by P2Y4 receptors at the apical membrane. The apparent lack of desensitization of the response is consistent with two processes: a rapid-onset phosphorylation of KCNE1 channel subunit and a slower-onset of regulation by depletion of plasma membrane PIP2.

P2Y2 receptor knock-out mice display normal NaCl absorption in medullary thick ascending limb

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Rita Delgado Marques

2013-10-01

Full Text Available Local purinergic signals modulate renal tubular transport. Acute activation of renal epithelial P2 receptors causes inhibition of epithelial transport and thus, should favor increased water and salt excretion by the kidney. So far only a few studies have addressed the effects of extracellular nucleotides on ion transport in the thick ascending limb. In the medullary thick ascending limb (mTAL, basolateral P2X receptors markedly (~25% inhibit NaCl absorption. Although this segment does express both apical and basolateral P2Y2 receptors, acute activation of the basolateral P2Y2 receptors had no apparent effect on transepithelial ion transport. Here we studied, if the absence of the P2Y2 receptor causes chronic alterations in mTAL NaCl absorption by comparing basal and AVP-stimulated transepithelial transport rates. We used perfused mouse mTALs to electrically measure NaCl absorption in juvenile (35 days male mice. Using microelectrodes, we determined the transepithelial voltage (Vte and the transepithelial resistance (Rte and thus, transepithelial NaCl absorption (equivalent short circuit current, I’sc.We find that mTALs from adult wild type (WT mice have significantly lower NaCl absorption rates when compared to mTALs from juvenile WT mice. This could be attributed to significantly higher Rte values in mTALs from adult WT mice. This pattern was not observed in mTALs from P2Y2 receptor knockout (KO mice. In addition, adult P2Y2 receptor KO mTALs have significantly lower Vte values compared to the juvenile. No difference in absolute I´sc was observed when comparing mTALs from WT and KO mice. AVP stimulated the mTALs to similar increases of NaCl absorption irrespective of the absence of the P2Y2 receptor. No difference was observed in the medullary expression level of NKCC2 in between the genotypes.These data indicate that the lack of P2Y2 receptors does not cause substantial differences in resting and AVP-stimulated NaCl absorption in

G Protein-Coupled Receptor 87 (GPR87 Promotes Cell Proliferation in Human Bladder Cancer Cells

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Xia Zhang

2015-10-01

Full Text Available G protein-coupled receptor 87 (GPR87 is a newly deorphanized member of the cell surface molecule G protein-coupled receptor family. GPR signaling was shown to play a role in promotion of cell growth and survival, metastasis, and drug resistance. The overexpression of GPR87 has also been reported in many malignant tumors including bladder cancer. The aim of the present study is to examine the effect of silencing GPR87 expression with a replication-deficient recombinant adenoviral vector expressing short hairpin RNA targeting GPR87 (Ad-shGPR87 and to explore the underlying molecular mechanisms in bladder cancer cells. Six GPR87-expressing human bladder cancer cells, HT1197, HT1376, J82, RT112, TCCSUP and UMUC3, were used. Infection with Ad-shGPR87 effectively downregulated the GPR87 expression, and significantly reduced the percentage of viable cells in 4 of 6 cell lines as detected by an MTT assay. Significant inhibition on cell proliferation with Ad-shGPR87 was observed in the wild-type p53 bladder cancer cell lines (HT1197, RT112, TCCSUP and UMUC3, but not in the mutant p53 cells (HT1376 and J82. As represented by a wild-type p53 RT112 cell, Ad-shGPR87 infection significantly enhanced p53 and p21 expression and caused caspase-dependent apoptosis. Furthermore, the treatment with Ad-shGPR87 exerted a significant antitumor effect against the GPR87-expressing RT112 xenografts. GPR87 appeared to be a promising target for gene therapy, and Ad-shGPR87 had strong antitumor effects, specifically anti-proliferative and pro-apoptotic effects, against GPR87-expressing human bladder cancer cells.

P2Y6 Receptor Activation Promotes Inflammation and Tissue Remodeling in Pulmonary Fibrosis

Science.gov (United States)

Müller, Tobias; Fay, Susanne; Vieira, Rodolfo Paula; Karmouty-Quintana, Harry; Cicko, Sanja; Ayata, Cemil Korcan; Zissel, Gernot; Goldmann, Torsten; Lungarella, Giuseppe; Ferrari, Davide; Di Virgilio, Francesco; Robaye, Bernard; Boeynaems, Jean-Marie; Lazarowski, Eduardo R.; Blackburn, Michael R.; Idzko, Marco

2017-01-01

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and very few available treatment options. The involvement of the purinergic receptor subtypes P2Y2 and P2X7 in fibrotic lung disease has been demonstrated recently. In this study, we investigated the role of P2Y6 receptors in the pathogenesis of IPF in humans and in the animal model of bleomycin-induced lung injury. P2Y6R expression was upregulated in lung structural cells but not in bronchoalveolar lavage (BAL) cells derived from IPF patients as well as in animals following bleomycin administration. Furthermore, BAL fluid levels of the P2Y6R agonist uridine-5′-diphosphate were elevated in animals with bleomycin-induced pulmonary fibrosis. Inflammation and fibrosis following bleomycin administration were reduced in P2Y6R-deficient compared to wild-type animals confirming the pathophysiological relevance of P2Y6R subtypes for fibrotic lung diseases. Experiments with bone marrow chimeras revealed the importance of P2Y6R expression on lung structural cells for pulmonary inflammation and fibrosis. Similar effects were obtained when animals were treated with the P2Y6R antagonist MRS2578. In vitro studies demonstrated that proliferation and secretion of the pro-inflammatory/pro-fibrotic cytokine IL-6 by lung fibroblasts are P2Y6R-mediated processes. In summary, our results clearly demonstrate the involvement of P2Y6R subtypes in the pathogenesis of pulmonary fibrosis. Thus, blocking pulmonary P2Y6 receptors might be a new target for the treatment of IPF. PMID:28878780

P2Y6 Receptor Activation Promotes Inflammation and Tissue Remodeling in Pulmonary Fibrosis

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Tobias Müller

2017-08-01

Full Text Available Idiopathic pulmonary fibrosis (IPF is a disease with a poor prognosis and very few available treatment options. The involvement of the purinergic receptor subtypes P2Y2 and P2X7 in fibrotic lung disease has been demonstrated recently. In this study, we investigated the role of P2Y6 receptors in the pathogenesis of IPF in humans and in the animal model of bleomycin-induced lung injury. P2Y6R expression was upregulated in lung structural cells but not in bronchoalveolar lavage (BAL cells derived from IPF patients as well as in animals following bleomycin administration. Furthermore, BAL fluid levels of the P2Y6R agonist uridine-5′-diphosphate were elevated in animals with bleomycin-induced pulmonary fibrosis. Inflammation and fibrosis following bleomycin administration were reduced in P2Y6R-deficient compared to wild-type animals confirming the pathophysiological relevance of P2Y6R subtypes for fibrotic lung diseases. Experiments with bone marrow chimeras revealed the importance of P2Y6R expression on lung structural cells for pulmonary inflammation and fibrosis. Similar effects were obtained when animals were treated with the P2Y6R antagonist MRS2578. In vitro studies demonstrated that proliferation and secretion of the pro-inflammatory/pro-fibrotic cytokine IL-6 by lung fibroblasts are P2Y6R-mediated processes. In summary, our results clearly demonstrate the involvement of P2Y6R subtypes in the pathogenesis of pulmonary fibrosis. Thus, blocking pulmonary P2Y6 receptors might be a new target for the treatment of IPF.

A novel human gene encoding a G-protein-coupled receptor (GPR15) is located on chromosome 3

Energy Technology Data Exchange (ETDEWEB)

Heiber, M.; Marchese, A.; O`Dowd, B.F. [Univ. of Toronto, Ontario (Canada)] [and others

1996-03-05

We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor encoded by GPR15 with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1. GPR15 was mapped to human chromosome 3q11.2-q13.1. 12 refs., 2 figs.

Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

Science.gov (United States)

Hu, Liang; Chang, Lin; Zhang, Yan; Zhai, Lili; Zhang, Shenghui; Qi, Zhiyong; Yan, Hongmei; Yan, Yan; Luo, Xinping; Zhang, Si; Wang, Yiping; Kunapuli, Satya P; Ye, Hongying; Ding, Zhongren

2017-08-29

Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood. We measured P2Y 12 expression on platelets from patients with type 2 diabetes mellitus and on platelets from rats with diabetes mellitus. We also assayed platelet P2Y 12 activation by measuring cAMP and VASP phosphorylation. The antiplatelet and antithrombotic effects of AR-C78511 and cangrelor were compared in rats. Finally, we explored the role of the nuclear factor-κB pathway in regulating P2Y 12 receptor expression in megakaryocytes. Platelet P2Y 12 levels are 4-fold higher in patients with type 2 diabetes mellitus compared with healthy subjects. P2Y 12 expression correlates with ADP-induced platelet aggregation (r=0.89, P diabetes mellitus is constitutively activated. Although both AR-C78511, a potent P2Y 12 inverse agonist, and cangrelor have similar antiplatelet efficacy on platelets from healthy subjects, AR-C78511 exhibits more powerful antiplatelet effects on diabetic platelets than cangrelor (aggregation ratio 36±3% versus 49±5%, respectively, P diabetes mellitus than cangrelor (thrombus weight 4.9±0.3 mg versus 8.3±0.4 mg, respectively, P diabetes mellitus. Platelet P2Y 12 receptor expression is significantly increased and the receptor is constitutively activated in patients with type 2 diabetes mellitus, which contributes to platelet hyperactivity and limits antiplatelet drug efficacy in type 2 diabetes mellitus. © 2017 American Heart Association, Inc.

Neuropharmacology of Purinergic Receptors in Human Submucous Plexus: Involvement of P2X1, P2X2, P2X3 Channels, P2Y and A3 Metabotropic Receptors in Neurotransmission

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Liñán-Rico, A.; Wunderlich, JE.; Enneking, JT.; Tso, DR.; Grants, I.; Williams, KC.; Otey, A.; Michel, K.; Schemann, M.; Needleman, B.; Harzman, A.; Christofi, FL.

2015-01-01

Rationale The role of purinergic signaling in the human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. Experimental Approach LSCM-Fluo-4-(Ca2+)-imaging of postsynaptic Ca2+ transients (PSCaTs) was used as a reporter of neural activity. Synaptic transmission was evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons from 234 separate ganglia 107 patients; immunochemical labeling for P2XRs of neurons in ganglia from 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging was used to test effects of adenosine on fast excitatory synaptic potentials (fEPSPs). Results Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines. Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50=25μM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50=111nM) in neurons without stimulatory ADPβS responses (EC50=960nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca2+ transients or Ca2+ oscillations (EC50=400μM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20nM) or high (5μM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-ir follow the order P2X2>P2X1≫P2X3; P2X1+ P2X2 and P2X3+P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7,P2Y1,2,12-14R. Responsive neurons were also identified by HuC/D-ir. Conclusions Purines are critical regulators of neurotransmission in the human enteric nervous system. Purinergic signaling involves

Increased Retinal Expression of the Pro-Angiogenic Receptor GPR91 via BMP6 in a Mouse Model of Juvenile Hemochromatosis.

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Arjunan, Pachiappan; Gnanaprakasam, Jaya P; Ananth, Sudha; Romej, Michelle A; Rajalakshmi, Veeranan-Karmegam; Prasad, Puttur D; Martin, Pamela M; Gurusamy, Mariappan; Thangaraju, Muthusamy; Bhutia, Yangzom D; Ganapathy, Vadivel

2016-04-01

Hemochromatosis, an iron-overload disease, occurs as adult and juvenile types. Mutations in hemojuvelin (HJV), an iron-regulatory protein and a bone morphogenetic protein (BMP) coreceptor, underlie most of the juvenile type. Hjv(-/-) mice accumulate excess iron in retina and exhibit aberrant vascularization and angiomas. A succinate receptor, GPR91, is pro-angiogenic in retina. We hypothesized that Hjv(-/-) retinas have increased BMP signaling and increased GPR91 expression as the basis of angiomas. Expression of GPR91 was examined by qPCR, immunofluorescence, and Western blot in wild-type and Hjv(-/-) mouse retinas and pRPE cells. Influence of excess iron and BMP6 on GPR91 expression was investigated in ARPE-19 cells, and wild-type and Hjv(-/-) pRPE cells. Succinate was used to activate GPR91 and determine the effects of GPR91 signaling on VEGF expression. Signaling of BMP6 was studied by the expression of Smad1/5/8 and pSmad4, and the BMP-target gene Id1. The interaction of pSmad4 with GPR91 promoter was studied by ChIP. Expression of GPR91 was higher in Hjv(-/-) retinas and RPE than in wild-type counterparts. Unexpectedly, BMP signaling was increased, not decreased, in Hjv(-/-) retinas and RPE. Bone morphogenetic protein 6 induced GPR91 in RPE, suggesting that increased BMP signaling in Hjv(-/-) retinas was likely responsible for GPR91 upregulation. Exposure of RPE to excess iron and succinate as well as BMP6 and succinate increased VEGF expression. Bone morphogenetic protein 6 promoted the interaction of pSmad4 with GPR91 promoter in RPE. G-protein-coupled receptor 91 is a BMP6 target and Hjv deletion enhances BMP signaling in retina, thus underscoring a role for excess iron and hemochromatosis in abnormal retinal vascularization.

Multiple P2Y receptors couple to calcium-dependent, chloride channels in smooth muscle cells of the rat pulmonary artery

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Gurney Alison M

2005-10-01

does not correspond to any of the known P2Y subtypes. These receptors likely play a significant role in nucleotide-induced vasoconstriction.

A Newly Discovered Antifibrotic Pathway Regulated by Two Fatty Acid Receptors: GPR40 and GPR84.

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Gagnon, Lyne; Leduc, Martin; Thibodeau, Jean-Francois; Zhang, Ming-Zhi; Grouix, Brigitte; Sarra-Bournet, Francois; Gagnon, William; Hince, Kathy; Tremblay, Mikaël; Geerts, Lilianne; Kennedy, Christopher R J; Hébert, Richard L; Gutsol, Alex; Holterman, Chet E; Kamto, Eldjonai; Gervais, Liette; Ouboudinar, Jugurtha; Richard, Jonathan; Felton, Alexandra; Laverdure, Alexandre; Simard, Jean-Christophe; Létourneau, Sylvie; Cloutier, Marie-Pier; Leblond, Francois A; Abbott, Shaun D; Penney, Christopher; Duceppe, Jean-Simon; Zacharie, Boulos; Dupuis, Jocelyn; Calderone, Angelino; Nguyen, Quang T; Harris, Raymond C; Laurin, Pierre

2018-05-01

Numerous clinical conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiological pathways involved in fibrosis. GPR40 and GPR84 are G protein-coupled receptors with free fatty acid ligands and are associated with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiological processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, respectively, we uncovered an antifibrotic pathway involving these receptors. In experiments using Gpr40- and Gpr84-knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity observed in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising molecular targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compound that may be effective for managing inflammatory and fibrosis-related diseases. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

IL-1β and TNFα inhibit GPR120 (FFAR4) and stimulate GPR84 (EX33) and GPR41 (FFAR3) fatty acid receptor expression in human adipocytes: implications for the anti-inflammatory action of n-3 fatty acids.

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Muredda, Laura; Kępczyńska, Małgorzata A; Zaibi, Mohamed S; Alomar, Suliman Y; Trayhurn, Paul

2018-05-01

Regulation of the expression of GPCR fatty acid receptor genes has been examined in human adipocytes differentiated in culture. TNFα and IL-1β induced a marked reduction in GPR120 expression, mRNA level falling 17-fold at 24 h in adipocytes incubated with TNFα. In contrast, GPR84 mRNA was dramatically increased by these cytokines (>500-fold for IL-1β at 4 h); GPR41 expression was also stimulated. Rosiglitazone did not affect GPR84 expression, but GPR120 and GPR41 expression increased. Dexamethasone, insulin, linoleic and docosahexaenoic acids (DHA), and TUG891 (GPR120 agonist) had little effect on GPR120 and GPR84 expression. TUG891 did not attenuate the pro-inflammatory actions of TNFα and IL-1β. DHA slightly countered the actions of IL-1β on CCL2, IL6 and ADIPOQ expression, though not on secretion of these adipokines. GPR120 and GP84 gene expression in human adipocytes is highly sensitive to pro-inflammatory mediators; the inflammation-induced inhibition of GPR120 expression may compromise the anti-inflammatory action of GPR120 agonists.

UDP/P2Y6 receptor signaling regulates IgE-dependent degranulation in human basophils

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Manabu Nakano

2017-10-01

Conclusions: This study showed that UDP/P2Y6 receptor signaling is involved in the regulation of IgE-dependent degranulation in basophils, which might stimulate the P2Y6 receptor via the autocrine secretion of UTP. Thus, this receptor represents a potential target to regulate IgE-dependent degranulation in basophils during allergic diseases.

Bone phenotypes of P2 receptor knockout mice

DEFF Research Database (Denmark)

Orriss, Isabel; Syberg, Susanne; Wang, Ning

2011-01-01

The action of extracellular nucleotides is mediated by ionotropic P2X receptors and G-protein coupled P2Y receptors. The human genome contains 7 P2X and 8 P2Y receptor genes. Knockout mice strains are available for most of them. As their phenotypic analysis is progressing, bone abnormalities have...... been observed in an impressive number of these mice: distinct abnormalities in P2X7-/- mice, depending on the gene targeting construct and the genetic background, decreased bone mass in P2Y1-/- mice, increased bone mass in P2Y2-/- mice, decreased bone resorption in P2Y6-/- mice, decreased bone...... formation and bone resorption in P2Y13-/- mice. These findings demonstrate the unexpected importance of extracellular nucleotide signalling in the regulation of bone metabolism via multiple P2 receptors and distinct mechanisms involving both osteoblasts and osteoclasts....

P2X1 receptors and the endothelium

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LS Harrington

2005-03-01

Full Text Available Adenosine triphosphate (ATP is now established as a principle vaso-active mediator in the vasculature. Its actions on arteries are complex, and are mediated by the P2X and P2Y receptor families. It is generally accepted that ATP induces a bi-phasic response in arteries, inducing contraction via the P2X and P2Y receptors on the smooth muscle cells, and vasodilation via the actions of P2Y receptors located on the endothelium. However, a number of recent studies have placed P2X1 receptors on the endothelium of some arteries. The use of a specific P2X1 receptor ligand, a, b methylene ATP has demonstrated that P2X1 receptors also have a bi-functional role. The actions of ATP on P2X1 receptors is therefore dependant on its location, inducing contraction when located on the smooth muscle cells, and dilation when expressed on the endothelium, comparable to that of P2Y receptors.

Differential roles for neuropeptide Y Y1 and Y5 receptors in anxiety and sedation

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Sørensen, Gunnar; Lindberg, Camilla; Wörtwein, Gitta

2004-01-01

PP(1-7),NPY(19-23),Ala(31),Aib(32),Gln(34)]hPP) in the elevated plus maze and open field tests. As with NPY, the Y1 agonist had a dose-dependent anxiolytic-like effect in both behavioral tests. In contrast to NPY, which caused significant sedation in the open field test, the Y1 agonist was without...... sedative effect. The Y2 agonist showed neither anxiolytic-like nor sedative effects. The Y5 agonist showed anxiolytic-like activity in both behavioral tests and caused sedation in the same dose range as NPY in the open field test. These results indicate that anxiolytic-like effects of i...... NPY receptors in anxiety and sedation remains a possibility. In the present study, we addressed this issue by testing the effects of intracerebroventricular (i.c.v.) injection of NPY as well as specific receptor agonists for the Y1 receptor ([D-His(26)]NPY), Y2 receptor (C2-NPY), and Y5 receptor ([c...

DA-6034-induced mucin secretion via Ca2+-dependent pathways through P2Y receptor stimulation.

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Lee, Hun; Kim, Eung Kweon; Kim, Ji Yeon; Yang, Yu-Mi; Shin, Dong Min; Kang, Kyung Koo; Kim, Tae-im

2014-09-11

We evaluated whether DA-6034 is involved in mucin secretion via P2Y receptor activation and/or intracellular Ca2+ concentration ([Ca2+]i) change. Also, we investigated the effect of P2Y receptor inhibitors or Ca2+ chelators on the DA-6034-induced mucin secretion and [Ca2+]i increases. Effects of DA-6034 on mucin expression in primary, cultured, conjunctival epithelial cells was studied using RT-PCR, Western blot analysis, and periodic acid-schiff (PAS) staining. To evaluate thin film layer thickness generated by mucin and fluid secretion, cells were incubated in DA-6034 with/without P2Y antagonists or extracellular/intracellular Ca2+ chelators, and were imaged with confocal microscope using Texas Red-dextran dye. In addition, DA-6034-induced Ca2+-dependent Cl- channels opening was evaluated using perforated patch clamp. Fluo-4/AM was used to measure changes in [Ca2+]i induced by DA-6034 in Ca2+-free or Ca2+-containing buffered condition, as well as P2Y antagonists. DA-6034 induced the expression of mucin genes, production of mucin protein, and increase of number of mucin-secreting cells. P2Y antagonists inhibited DA-6034-induced mucin and fluid secretion, which was also affected by extracellular/intracellular Ca2+ chelators. DA-6034 stimulated Cl- channel opening and [Ca2+]i elevation. Further, [Ca2+]i increases induced by DA-6034 were lacking in either P2Y antagonists or Ca2+-free buffered condition, and diminished when endoplasmic reticulum Ca2+ was depleted by cyclopiazonic acid in Ca2+-free buffered condition. This study demonstrated that DA-6034 has a potential to induce mucin secretion via Ca2+-dependent pathways through P2Y receptors in multilayer, cultured, human conjunctival epithelial cells. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Protein kinase C-mediated ATP stimulation of Na(+)-ATPase activity in LLC-PK1 cells involves a P2Y2 and/or P2Y4 receptor.

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Wengert, M; Ribeiro, M C; Abreu, T P; Coutinho-Silva, R; Leão-Ferreira, L R; Pinheiro, A A S; Caruso-Neves, C

2013-07-15

ATP-activated P2Y receptors play an important role in renal sodium excretion. The aim of this study was to evaluate the modulation of ATPase-driven sodium reabsorption in the proximal tubule by ATP or adenosine (Ado). LLC-PK1 cells, a model of porcine proximal tubule cells, were used. ATP (10(-6)M) or Ado (10(-6)M) specifically stimulated Na(+)-ATPase activity without any changes in (Na(+)+K(+))-ATPase activity. Our results show that the Ado effect is mediated by its conversion to ATP. Furthermore, it was observed that the effect of ATP was mimicked by UTP, ATPγS and 2-thio-UTP, an agonist of P2Y2 and P2Y4 receptors. In addition, ATP-stimulated Na(+)-ATPase activity involves protein kinase C (PKC). Our results indicate that ATP-induced stimulation of proximal tubule Na(+)-ATPase activity is mediated by a PKC-dependent P2Y2 and/or P2Y4 pathway. These findings provide new perspectives on the role of the effect of P2Y-mediated extracellular ATP on renal sodium handling. Copyright © 2013 Elsevier Inc. All rights reserved.

Novel Zn2+ Modulated GPR39 Receptor Agonists Do Not Drive Acute Insulin Secretion in Rodents.

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Ola Fjellström

Full Text Available Type 2 diabetes (T2D occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired β-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic β-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve β-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion in vivo. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn2+ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO mice and Zucker fatty rats. It is concluded that Zn2+ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.

Adenine Nucleotide Analogues Locked in a Northern Methanocarba Conformation: Enhanced Stability and Potency as P2Y1 Receptor Agonists

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Ravi, R. Gnana; Kim, Hak Sung; Servos, Jörg; Zimmermann, Herbert; Lee, Kyeong; Maddileti, Savitri; Boyer, José L.; Harden, T. Kendall; Jacobson, Kenneth A.

2016-01-01

Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5′-triphosphate agonists at P2Y1, P2Y2, P2Y4, and P2Y11 receptors, but not P2Y6 receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208–218.). We have now combined the ring-constrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y1 or human P2Y1 and P2Y2 receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC50 at the hP2Y1 receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5′-diphosphate. 2-Cl–(N)-methanocarba-ATP and its N6-Me analogue were also highly selective, full agonists at P2Y1 receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y1 receptors, while the corresponding ribosides were inactive. Although β,γ-methylene-ATP was inactive at P2Y receptors, β,γ-methylene-(N)-methanocarba-ATP was a potent hP2Y1 receptor agonist with an EC50 of 160 nM and was selective versus hP2Y2 and hP2Y4 receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5′-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and selective nucleotide agonists may be designed on the basis of

Identification of 6H1 as a P2Y purinoceptor: P2Y5.

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Webb, T E; Kaplan, M G; Barnard, E A

1996-02-06

We have determined the identity of the orphan G-protein coupled receptor cDNA, 6H1, present in activated chicken T cells, as a subtype of P2Y purinoceptor. This identification is based on first on the degree of sequence identity shared with recently cloned members of the P2Y receptor family and second on the pharmacological profile. Upon transient expression in COS-7 cells the 6H1 receptor bound the radiolabel [35S]dATP alpha S specifically and with high affinity (Kd, 10 nM). This specific binding could be competitively displaced by a range of ligands active at P2 purinoceptors, with ATP being the most active (K (i)), 116 nM). Such competition studies have established the following rank order of activity: ATP ADP 2-methylthioATP alpha, beta-methylene ATP, UTP, thus confirming 6H1 as a member of the growing family of P2Y purinoceptors. As the fifth receptor of this type to be identified we suggest that it be named P2Y5.

Genetic variants of the unsaturated fatty acid receptor GPR120 relating to obesity in dogs.

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Miyabe, Masahiro; Gin, Azusa; Onozawa, Eri; Daimon, Mana; Yamada, Hana; Oda, Hitomi; Mori, Akihiro; Momota, Yutaka; Azakami, Daigo; Yamamoto, Ichiro; Mochizuki, Mariko; Sako, Toshinori; Tamura, Katsutoshi; Ishioka, Katsumi

2015-10-01

G protein-coupled receptor (GPR) 120 is an unsaturated fatty acid receptor, which is associated with various physiological functions. It is reported that the genetic variant of GPR120, p.Arg270His, is detected more in obese people, and this genetic variation functionally relates to obesity in humans. Obesity is a common nutritional disorder also in dogs, but the genetic factors have not ever been identified in dogs. In this study, we investigated the molecular structure of canine GPR120 and searched for candidate genetic variants which may relate to obesity in dogs. Canine GPR120 was highly homologous to those of other species, and seven transmembrane domains and two N-glycosylation sites were conserved. GPR120 mRNA was expressed in lung, jejunum, ileum, colon, hypothalamus, hippocampus, spinal cord, bone marrow, dermis and white adipose tissues in dogs, as those in mice and humans. Genetic variants of GPR120 were explored in client-owned 141 dogs, resulting in that 5 synonymous and 4 non-synonymous variants were found. The variant c.595C>A (p.Pro199Thr) was found in 40 dogs, and the gene frequency was significantly higher in dogs with higher body condition scores, i.e. 0.320 in BCS4-5 dogs, 0.175 in BCS3 dogs and 0.000 in BCS2 dogs. We conclude that c.595C>A (p.Pro199Thr) is a candidate variant relating to obesity, which may be helpful for nutritional management of dogs.

Reciprocal regulation of platelet responses to P2Y and thromboxane receptor activation.

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Barton, J F; Hardy, A R; Poole, A W; Mundell, S J

2008-03-01

Thromboxane A(2) and ADP are two major platelet agonists that stimulate two sets of G protein-coupled receptors to activate platelets. Although aggregation responses to ADP and thromboxane desensitize, there are no reports currently addressing whether activation by one agonist may heterologously desensitize responses to the other. To demonstrate whether responses to ADP or U46619 may be modulated by prior treatment of platelets with the alternate agonist, revealing a level of cross-desensitization between receptor systems. Here we show that pretreatment of platelets with either agonist substantially desensitizes aggregation responses to the other agonist. Calcium responses to thromboxane receptor activation are desensitized by preactivation of P2Y(1) but not P2Y(12) receptors. This heterologous desensitization is mediated by a protein kinase C (PKC)-independent mechanism. Reciprocally, calcium responses to ADP are desensitized by pretreatment of platelets with the thromboxane analogue, U46619, and P2Y(12)-mediated inhibition of adenylate cyclase is also desensitized by pretreatment with U46619. In this direction, desensitization is comprised of two components, a true heterologous component that is PKC-independent, and a homologous component that is mediated through stimulated release of dense granule ADP. This study reveals cross-desensitization between ADP and thromboxane receptor signaling in human platelets. Cross-desensitization is mediated by protein kinases, involving PKC-dependent and independent pathways, and indicates that alterations in the activation state of one receptor may have effects upon the sensitivity of the other receptor system.

The SCFA receptor GPR43 and energy metabolism

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Ikuo eKimura

2014-06-01

Full Text Available Free fatty acids (FFAs are essential nutrients and act as signaling molecules in various cellular processes via binding with FFA receptors. Of these receptors, GPR43 is activated by short chain fatty acids (SCFAs; e.g., acetate, propionate, and butyrate. During feeding, SCFAs are produced by microbial fermentation of dietary fiber in the gut, and these SCFAs become important energy sources for the host. The gut microbiota affects nutrient acquisition and energy regulation of the host and can influence the development of obesity, insulin resistance, and diabetes. Recently, GPR43 has been reported to regulate host energy homeostasis in the gastrointestinal tract and adipose tissues. Hence, GPR43 is also thought to be a potential drug target for metabolic disorders, such as obesity and diabetes. In this review, we summarize the identification, structure, and activities of GPR43, with a focus on host energy regulation, and present an essential overview of our current understanding of its physiological roles in host energy regulation that is mediated by gut microbiota. We also discuss the potential for GPR43 as a therapeutic target.

Autocrine Regulation of UVA-Induced IL-6 Production via Release of ATP and Activation of P2Y Receptors

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Kawano, Ayumi; Kadomatsu, Remi; Ono, Miyu; Kojima, Shuji; Tsukimoto, Mitsutoshi; Sakamoto, Hikaru

2015-01-01

Extracellular nucleotides, such as ATP, are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. Exposure to the ultraviolet radiation A (UVA) component of sunlight causes molecular and cellular damage, and in this study, we investigated the involvement of extracellular nucleotides and P2 receptors in the UVA-induced cellular response. Human keratinocyte-derived HaCaT cells were irradiated with a single dose of UVA (2.5 J/cm2), and ATP release and interleukin (IL)-6 production were measured. ATP was released from cells in response to UVA irradiation, and the release was blocked by pretreatment with inhibitors of gap junction hemichannels or P2X7 receptor antagonist. IL-6 production was increased after UVA irradiation, and this increase was inhibited by ecto-nucleotidase or by antagonists of P2Y11 or P2Y13 receptor. These results suggest that UVA-induced IL-6 production is mediated by release of ATP through hemichannels and P2X7 receptor, followed by activation of P2Y11 and P2Y13 receptors. Interestingly, P2Y11 and P2Y13 were associated with the same pattern of IL-6 production, though they trigger different intracellular signaling cascades: Ca2+-dependent and PI3K-dependent, respectively. Thus, IL-6 production in response to UVA-induced ATP release involves at least two distinct pathways, mediated by activation of P2Y11 and P2Y13 receptors. PMID:26030257

Regulation of rat hepatocyte function by P2Y receptors: focus on control of glycogen phosphorylase and cyclic AMP by 2-methylthioadenosine 5'-diphosphate.

Science.gov (United States)

Dixon, C Jane; Hall, John F; Webb, Tania E; Boarder, Michael R

2004-10-01

Hepatocyte function is regulated by several P2Y receptor subtypes. Here we report that 2-methylthioadenosine 5'-diphosphate (2-MeSADP), an agonist at P2Y(1), P2Y(12), and P2Y(13) receptors, potently (threshold 30 nM) stimulates glycogen phosphorylase in freshly isolated rat hepatocytes. Antagonism by N(6)-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS 2179) confirms that this response is mediated by P2Y(1) receptors. In addition, in these cells, both 2-MeSADP and UTP inhibited glucagon-stimulated cyclic AMP accumulation. This inhibitory effect of 2-MeSADP was not reversed by the P2Y(1) antagonists, adenosine-3'-phosphate-5'-phosphate (A3P5P) or MRS 2179, both in the range 1 to 300 microM, indicating that it was not mediated by P2Y(1) receptors. This contrasts with the increase in cytosolic free Ca(2+) concentration ([Ca(2+)](c)) induced by 2-MeSADP, which has shown to be inhibited by A3P5P. Pertussis toxin abolished the inhibitory effect of both UTP and 2-MeSADP. After culture of cells for 48 h, the ability of 2-MeSADP to inhibit cyclic AMP accumulation was greatly diminished. Reverse transcriptase-polymerase chain reaction analysis revealed that during this culture period, there was a decline in the ability to detect transcripts for P2Y(12) and P2Y(13) receptors, both of which are activated by 2-MeSADP and negatively coupled to adenylyl cyclase. However, in freshly isolated cells, the P2Y(12) and P2Y(13) receptor antagonist, 2-propylthio-beta,gamma-dichloromethylene-d-ATP (AR-C67085) (10 nM to 300 microM) did not alter the ability of 2-MeSADP to inhibit glucagon-stimulated cyclic AMP accumulation. We conclude that 2-MeSADP regulates rat hepatocyte glycogen phosphorylase by acting on P2Y(1) receptors coupled to raised [Ca(2+)](c), and by inhibiting cyclic AMP levels by an unknown G(i)-coupled receptor subtype, distinct from P2Y(1), P2Y(12), or P2Y(13) receptors.

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

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Mochimaru, Yuta [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Azuma, Morio [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Oshima, Natsuki; Ichijo, Yuta; Satou, Kazuhiro [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Matsuda, Kouhei [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Asaoka, Yoichi; Nishina, Hiroshi [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Nakakura, Takashi [Department of Anatomy, Graduate School of Medicine, Teikyo University, 2-11-1 Kaga Itabashi-Ku, Tokyo 173-8605 (Japan); Mogi, Chihiro; Sato, Koichi; Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tomura, Hideaki, E-mail: tomurah@meiji.ac.jp [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan)

2015-02-20

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

A novel nonsense mutation of the GPR143 gene identified in a Chinese pedigree with ocular albinism.

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Naihong Yan

Full Text Available BACKGROUND: The purpose of this study was to elucidate the molecular basis of ocular albinism type I in a Chinese pedigree. METHODOLOGY/PRINCIPAL FINDINGS: Complete ophthalmologic examinations were performed on 4 patients, 7 carriers and 17 unaffected individuals in this five-generation family. All coding exons of four-point-one (4.1, ezrin, radixin, moesin (FERM domain-containing 7 (FRMD7 and G protein-coupled receptor 143 (GPR143 genes were amplified by polymerase chain reaction (PCR, sequenced and compared with a reference database. Ocular albinism and nystagmus were found in all patients of this family. Macular hypoplasia was present in the patients including the proband. A novel nonsense hemizygous mutation c.807T>A in the GPR143 gene was identified in four patients and the heterozygous mutation was found in seven asymptomatic individuals. This mutation is a substitution of tyrosine for adenine which leads to a premature stop codon at position 269 (p.Y269X of GPR143. CONCLUSIONS/SIGNIFICANCE: This is the first report that p.Y269X mutation of GPR143 gene is responsible for the pathogenesis of familial ocular albinism. These results expand the mutation spectrum of GPR143, and demonstrate the clinical characteristics of ocular albinism type I in Chinese population.

Astrocytes protect neurons against methylmercury via ATP/P2Y(1) receptor-mediated pathways in astrocytes.

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Noguchi, Yusuke; Shinozaki, Youichi; Fujishita, Kayoko; Shibata, Keisuke; Imura, Yoshio; Morizawa, Yosuke; Gachet, Christian; Koizumi, Schuichi

2013-01-01

Methylmercury (MeHg) is a well known environmental pollutant that induces serious neuronal damage. Although MeHg readily crosses the blood-brain barrier, and should affect both neurons and glial cells, how it affects glia or neuron-to-glia interactions has received only limited attention. Here, we report that MeHg triggers ATP/P2Y1 receptor signals in astrocytes, thereby protecting neurons against MeHg via interleukin-6 (IL-6)-mediated pathways. MeHg increased several mRNAs in astrocytes, among which IL-6 was the highest. For this, ATP/P2Y1 receptor-mediated mechanisms were required because the IL-6 production was (i) inhibited by a P2Y1 receptor antagonist, MRS2179, (ii) abolished in astrocytes obtained from P2Y1 receptor-knockout mice, and (iii) mimicked by exogenously applied ATP. In addition, (iv) MeHg released ATP by exocytosis from astrocytes. As for the intracellular mechanisms responsible for IL-6 production, p38 MAP kinase was involved. MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6. As for the mechanism of neuro-protection by IL-6, an adenosine A1 receptor-mediated pathway in neurons seems to be involved. Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y1 receptors to upregulate IL-6, thereby leading to A1 receptor-mediated neuro-protection against MeHg.

Medium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84.

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Wang, Jinghong; Wu, Xiaosu; Simonavicius, Nicole; Tian, Hui; Ling, Lei

2006-11-10

Free fatty acids (FFAs) play important physiological roles in many tissues as an energy source and as signaling molecules in various cellular processes. Elevated levels of circulating FFAs are associated with obesity, dyslipidemia, and diabetes. Here we show that GPR84, a previously orphan G protein-coupled receptor, functions as a receptor for medium-chain FFAs with carbon chain lengths of 9-14. Medium-chain FFAs elicit calcium mobilization, inhibit 3',5'-cyclic AMP production, and stimulate [35S]guanosine 5'-O-(3-thiotriphosphate) binding in a GPR84-dependent manner. The activation of GPR84 by medium-chain FFAs couples primarily to a pertussis toxin-sensitive G(i/o) pathway. In addition, we show that GPR84 is selectively expressed in leukocytes and markedly induced in monocytes/macrophages upon activation by lipopolysaccharide. Furthermore, we demonstrate that medium-chain FFAs amplify lipopolysaccharide-stimulated production of the proinflammatory cytokine interleukin-12 p40 through GPR84. Our results indicate a role for GPR84 in directly linking fatty acid metabolism to immunological regulation.

Gene-by-environment effect of house dust mite on purinergic receptor P2Y12 (P2RY12) and lung function in children with asthma.

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Bunyavanich, S; Boyce, J A; Raby, B A; Weiss, S T

2012-02-01

Distinct receptors likely exist for leukotriene (LT)E(4), a potent mediator of airway inflammation. Purinergic receptor P2Y12 is needed for LTE(4)-induced airways inflammation, and P2Y12 antagonism attenuates house dust mite-induced pulmonary eosinophilia in mice. Although experimental data support a role for P2Y12 in airway inflammation, its role in human asthma has never been studied. To test for association between variants in the P2Y12 gene (P2RY12) and lung function in human subjects with asthma, and to examine for gene-by-environment interaction with house dust mite exposure. Nineteen single nucleotide polymorphisms (SNPs) in P2RY12 were genotyped in 422 children with asthma and their parents (n = 1266). Using family based methods, we tested for associations between these SNPs and five lung function measures. We performed haplotype association analyses and tested for gene-by-environment interactions using house dust mite exposure. We used the false discovery rate to account for multiple comparisons. Five SNPs in P2RY12 were associated with multiple lung function measures (P-values 0.006–0.025). Haplotypes in P2RY12 were also associated with lung function (P-values 0.0055–0.046). House dust mite exposure modulated associations between P2RY12 and lung function, with minor allele homozygotes exposed to house dust mite demonstrating worse lung function than those unexposed (significant interaction P-values 0.0028–0.040). The P2RY12 variants were associated with lung function in a large family-based asthma cohort. House dust mite exposure caused significant gene-by-environment effects. Our findings add the first human evidence to experimental data supporting a role for P2Y12 in lung function. P2Y12 could represent a novel target for asthma treatment.

Circadian ATP Release in Organotypic Cultures of the Rat Suprachiasmatic Nucleus Is Dependent on P2X7 and P2Y Receptors

Czech Academy of Sciences Publication Activity Database

Svobodová, Irena; Bhattacharya, Anirban; Ivetic, Milorad; Bendová, Z.; Zemková, Hana

2018-01-01

Roč. 9, Mar 6 (2018), č. článku 192. ISSN 1663-9812 R&D Projects: GA ČR(CZ) GA16-12695S; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : suprachiasmatic nucleus * organotypic cultures * astrocytes * P2X7 receptor * P2Y1 receptor * P2Y2 receptor * pannexin-1 hemichannel * ATP release Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 4.400, year: 2016

Differential local tissue permissiveness influences the final fate of GPR17-expressing oligodendrocyte precursors in two distinct models of demyelination.

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Coppolino, Giusy T; Marangon, Davide; Negri, Camilla; Menichetti, Gianluca; Fumagalli, Marta; Gelosa, Paolo; Dimou, Leda; Furlan, Roberto; Lecca, Davide; Abbracchio, Maria P

2018-05-01

Promoting remyelination is recognized as a novel strategy to foster repair in neurodegenerative demyelinating diseases, such as multiple sclerosis. In this respect, the receptor GPR17, recently emerged as a new target for remyelination, is expressed by early oligodendrocyte precursors (OPCs) and after a certain differentiation stage it has to be downregulated to allow progression to mature myelinating oligodendrocytes. Here, we took advantage of the first inducible GPR17 reporter mouse line (GPR17-iCreER T2 xCAG-eGFP mice) allowing to follow the final fate of GPR17 + cells by tamoxifen-induced GFP-labeling to unveil the destiny of these cells in two demyelination models: experimental autoimmune encephalomyelitis (EAE), characterized by marked immune cell activation and inflammation, and cuprizone induced demyelination, where myelin dysfunction is achieved by a toxic insult. In both models, demyelination induced a strong increase of fluorescent GFP + cells at damaged areas. However, only in the cuprizone model reacting GFP + cells terminally differentiated to mature oligodendrocytes, thus contributing to remyelination. In EAE, GFP + cells were blocked at immature stages and never became myelinating oligodendrocytes. We suggest these strikingly distinct fates be due to different permissiveness of the local CNS environment. Based on previously reported GPR17 activation by emergency signals (e.g., Stromal Derived Factor-1), we propose that a marked inflammatory milieu, such as that reproduced in EAE, induces GPR17 overactivation resulting in impaired downregulation, untimely and prolonged permanence in OPCs, leading, in turn, to differentiation blockade. Combined treatments with remyelinating agents and anti-inflammatory drugs may represent new potential adequate strategies to halt neurodegeneration and foster recovery. © 2018 The Authors GLIA Published by Wiley Periodicals, Inc.

The platelet P2Y(12) receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [(3)H]PSB-0413.

Science.gov (United States)

Ohlmann, Philippe; Lecchi, Anna; El-Tayeb, Ali; Müller, Christa E; Cattaneo, Marco; Gachet, Christian

2013-03-01

Various radioligands have been used to characterize and quantify the platelet P2Y(12) receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y(1) and P2Y(12). We used the [(3)H]PSB-0413 selective P2Y(12) receptor antagonist radioligand to reevaluate the number of P2Y(12) receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [(3)H]PSB-0413 bound to 425 ± 50 sites/platelet (K (D) = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [(3)H]PSB-0413 bound to 1 mg protein of platelet membranes (K (D) = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y(12), with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y(1) ligand MRS2179 and the P2X(1) ligand α,β-Met-ATP did not displace [(3)H]PSB-0413 binding. Patients with severe P2Y(12) deficiency displayed virtually no binding of [(3)H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y(12) receptor had normal binding. Studies in mice showed that: (1) [(3)H]PSB-0413 bound to 634 ± 87 sites/platelet (K (D) = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [(3)H]PSB-0413 bound to 1 mg protein of platelet membranes (K (D) = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [(3)H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y(12) receptors, to identify patients with P2Y(12) deficiencies or quantify the effect of P2Y(12) targeting drugs.

Validation of antibodies for neuroanatomical localization of the P2Y receptor in macaque brain

DEFF Research Database (Denmark)

Dreisig, Karin; Degn, Matilda; Sund, Louise

2016-01-01

Focus on the purinergic receptor P2Y11 has increased following the finding of an association between the sleep disorder narcolepsy and a genetic variant in P2RY11 causing decreased gene expression. Narcolepsy is believed to arise from an autoimmune destruction of the hypothalamic neurons that pro......Focus on the purinergic receptor P2Y11 has increased following the finding of an association between the sleep disorder narcolepsy and a genetic variant in P2RY11 causing decreased gene expression. Narcolepsy is believed to arise from an autoimmune destruction of the hypothalamic neurons...

Development and Characterization of a Fluorescent Tracer for the Free Fatty Acid Receptor 2 (FFA2/GPR43)

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Hansen, Anders Højgaard; Sergeev, Eugenia; Pandey, Sunil K.

2017-01-01

The free fatty acid receptor 2 (FFA2/GPR43) is considered a potential target for treatment of metabolic and inflammatory diseases. Here we describe the development of the first fluorescent tracer for FFA2 intended as a tool for assessment of thermodynamic and kinetic binding parameters of unlabel...

P2Y6 receptor potentiates pro-inflammatory responses in macrophages and exhibits differential roles in atherosclerotic lesion development.

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Ricardo A Garcia

Full Text Available BACKGROUND: P2Y(6, a purinergic receptor for UDP, is enriched in atherosclerotic lesions and is implicated in pro-inflammatory responses of key vascular cell types and macrophages. Evidence for its involvement in atherogenesis, however, has been lacking. Here we use cell-based studies and three murine models of atherogenesis to evaluate the impact of P2Y(6 deficiency on atherosclerosis. METHODOLOGY/PRINCIPAL FINDINGS: Cell-based studies in 1321N1 astrocytoma cells, which lack functional P2Y(6 receptors, showed that exogenous expression of P2Y(6 induces a robust, receptor- and agonist-dependent secretion of inflammatory mediators IL-8, IL-6, MCP-1 and GRO1. P2Y(6-mediated inflammatory responses were also observed, albeit to a lesser extent, in macrophages endogenously expressing P2Y(6 and in acute peritonitis models of inflammation. To evaluate the role of P2Y(6 in atherosclerotic lesion development, we used P2Y(6-deficient mice in three mouse models of atherosclerosis. A 43% reduction in aortic arch plaque was observed in high fat-fed LDLR knockout mice lacking P2Y(6 receptors in bone marrow-derived cells. In contrast, no effect on lesion development was observed in fat-fed whole body P2Y(6xLDLR double knockout mice. Interestingly, in a model of enhanced vascular inflammation using angiotensin II, P2Y(6 deficiency enhanced formation of aneurysms and exhibited a trend towards increased atherosclerosis in the aorta of LDLR knockout mice. CONCLUSIONS: P2Y(6 receptor augments pro-inflammatory responses in macrophages and exhibits a pro-atherogenic role in hematopoietic cells. However, the overall impact of whole body P2Y(6 deficiency on atherosclerosis appears to be modest and could reflect additional roles of P2Y(6 in vascular disease pathophysiologies, such as aneurysm formation.

P2Y receptor-mediated transient relaxation of rat longitudinal ileum preparations involves phospholipase C activation, intracellular Ca(2+) release and SK channel activation.

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Mader, Felix; Krause, Ludwig; Tokay, Tursonjan; Hakenberg, Oliver W; Köhling, Rüdiger; Kirschstein, Timo

2016-05-01

Purinergic signaling plays a major role in the enteric nervous system, where it governs gut motility through a number of P2X and P2Y receptors. The aim of this study was to investigate the P2Y receptor-mediated motility in rat longitudinal ileum preparations. Ileum smooth muscle strips were prepared from rats, and fixed in an organ bath. Isometric contraction and relaxation responses of the muscle strips were measured with force transducers. Drugs were applied by adding of stock solutions to the organ bath to yield the individual final concentrations. Application of the non-hydrolyzable P2 receptor agonists α,β-Me-ATP or 2-Me-S-ADP (10, 100 μmol/L) dose-dependently elicited a transient relaxation response followed by a sustained contraction. The relaxation response was largely blocked by SK channel blockers apamin (500 nmol/L) and UCL1684 (10 μmol/L), PLC inhibitor U73122 (100 μmol/L), IP3 receptor blocker 2-APB (100 μmol/L) or sarcoendoplasmic Ca(2+) ATPase inhibitor thapsigargin (1 μmol/L), but not affected by atropine, NO synthase blocker L-NAME or tetrodotoxin. Furthermore, α,β-Me-ATP-induced relaxation was suppressed by P2Y1 receptor antagonist MRS2179 (50 μmol/L) or P2Y13 receptor antagonist MRS2211 (100 μmol/L), and was abolished by co-application of the two antagonists, whereas 2-Me-S-ADP-induced relaxation was abolished by P2Y6 receptor antagonist MRS2578 (50 μmol/L). In addition, P2Y1 receptor antagonist MRS2500 (1 μmol/L) not only abolished α,β-Me-ATP-induced relaxation, but also suppressed 2-Me-S-ADP-induced relaxation. P2Y receptor agonist-induced transient relaxation of rat ileum smooth muscle strips is mediated predominantly by P2Y1 receptor, but also by P2Y6 and P2Y13 receptors, and involves PLC, IP3, Ca(2+) release and SK channel activation, but is independent of acetylcholine and NO release.

Differential local tissue permissiveness influences the final fate of GPR17‐expressing oligodendrocyte precursors in two distinct models of demyelination

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Coppolino, Giusy T.; Marangon, Davide; Negri, Camilla; Menichetti, Gianluca; Fumagalli, Marta; Gelosa, Paolo; Dimou, Leda; Furlan, Roberto; Lecca, Davide

2018-01-01

Abstract Promoting remyelination is recognized as a novel strategy to foster repair in neurodegenerative demyelinating diseases, such as multiple sclerosis. In this respect, the receptor GPR17, recently emerged as a new target for remyelination, is expressed by early oligodendrocyte precursors (OPCs) and after a certain differentiation stage it has to be downregulated to allow progression to mature myelinating oligodendrocytes. Here, we took advantage of the first inducible GPR17 reporter mouse line (GPR17‐iCreERT2xCAG‐eGFP mice) allowing to follow the final fate of GPR17+ cells by tamoxifen‐induced GFP‐labeling to unveil the destiny of these cells in two demyelination models: experimental autoimmune encephalomyelitis (EAE), characterized by marked immune cell activation and inflammation, and cuprizone induced demyelination, where myelin dysfunction is achieved by a toxic insult. In both models, demyelination induced a strong increase of fluorescent GFP+ cells at damaged areas. However, only in the cuprizone model reacting GFP+ cells terminally differentiated to mature oligodendrocytes, thus contributing to remyelination. In EAE, GFP+ cells were blocked at immature stages and never became myelinating oligodendrocytes. We suggest these strikingly distinct fates be due to different permissiveness of the local CNS environment. Based on previously reported GPR17 activation by emergency signals (e.g., Stromal Derived Factor‐1), we propose that a marked inflammatory milieu, such as that reproduced in EAE, induces GPR17 overactivation resulting in impaired downregulation, untimely and prolonged permanence in OPCs, leading, in turn, to differentiation blockade. Combined treatments with remyelinating agents and anti‐inflammatory drugs may represent new potential adequate strategies to halt neurodegeneration and foster recovery. PMID:29424466

The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis.

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Perry, Kimberly J; Johnson, Verity R; Malloch, Erica L; Fukui, Lisa; Wever, Jason; Thomas, Alvin G; Hamilton, Paul W; Henry, Jonathan J

2010-11-01

G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina, and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84's importance in lens, cornea, and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino-injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea. © 2010 Wiley-Liss, Inc.

The Zinc Sensing Receptor, ZnR/GPR39, in Health and Disease

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Michal Hershfinkel

2018-02-01

Full Text Available A distinct G-protein coupled receptor that senses changes in extracellular Zn2+, ZnR/GPR39, was found in cells from tissues in which Zn2+ plays a physiological role. Most prominently, ZnR/GPR39 activity was described in prostate cancer, skin keratinocytes, and colon epithelial cells, where zinc is essential for cell growth, wound closure, and barrier formation. ZnR/GPR39 activity was also described in neurons that are postsynaptic to vesicular Zn2+ release. Activation of ZnR/GPR39 triggers Gαq-dependent signaling and subsequent cellular pathways associated with cell growth and survival. Furthermore, ZnR/GPR39 was shown to regulate the activity of ion transport mechanisms that are essential for the physiological function of epithelial and neuronal cells. Thus, ZnR/GPR39 provides a unique target for therapeutically modifying the actions of zinc in a specific and selective manner.

Investigation of the GPR39 zinc receptor following inhibition of monoaminergic neurotransmission and potentialization of glutamatergic neurotransmission

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Młyniec, Katarzyna; Gaweł, Magdalena; Librowski, Tadeusz

2015-01-01

Zinc can regulate neural function in the brain via the GPR39 receptor. In the present study we investigated whether inhibition of serotonin, noradrenaline and dopamine synthesis and potentialization of glutamate, via administration of p-chlorophenylalanine (pCPA), α-methyl-p-tyrosine (αMT) and N......-methyl-d-aspartatic acid (NMDA), respectively, would cause changes in GPR39 levels. Western blot analysis showed GPR39 up-regulation following 3-day administration of αMT and NMDA in the frontal cortex, and GPR39 down-regulation following 10-day administration of pCPA, αMT, and NMDA in the hippocampus of CD-1 mice....... There were no changes in serum zinc levels. Additionally, we investigated tryptophan, tyrosine and glutamate concentrations in the hippocampus and frontal cortex of GPR39 knockout (GPR39 KO) mice. Liquid chromatography-mass spectrometry (LC-MS) showed a significant decrease in tryptophan and tyrosine...

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

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

2017-06-16

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

Polyunsaturated fatty acid receptors, GPR40 and GPR120, are expressed in the hypothalamus and control energy homeostasis and inflammation

DEFF Research Database (Denmark)

Dragano, Nathalia R V; Solon, Carina; Ramalho, Albina F

2017-01-01

BACKGROUND: The consumption of large amounts of dietary fats is one of the most important environmental factors contributing to the development of obesity and metabolic disorders. GPR120 and GPR40 are polyunsaturated fatty acid receptors that exert a number of systemic effects that are beneficial...... for metabolic and inflammatory diseases. Here, we evaluate the expression and potential role of hypothalamic GPR120 and GPR40 as targets for the treatment of obesity. METHODS: Male Swiss (6-weeks old), were fed with a high fat diet (HFD, 60% of kcal from fat) for 4 weeks. Next, mice underwent stereotaxic...... the treatment period. At the end of the experiment, the hypothalamus was collected for real-time PCR analysis. RESULTS: We show that both receptors are expressed in the hypothalamus; GPR120 is primarily present in microglia, whereas GPR40 is expressed in neurons. Upon intracerebroventricular treatment, GW9508...

Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

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Julie Carnesecchi

Full Text Available The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2 addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

Science.gov (United States)

Carnesecchi, Julie; Malbouyres, Marilyne; de Mets, Richard; Balland, Martial; Beauchef, Gallic; Vié, Katell; Chamot, Christophe; Lionnet, Claire; Ruggiero, Florence; Vanacker, Jean-Marc

2015-01-01

The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

Pilocarpine-Induced Status Epilepticus Increases the Sensitivity of P2X7 and P2Y1 Receptors to Nucleotides at Neural Progenitor Cells of the Juvenile Rodent Hippocampus.

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Rozmer, Katalin; Gao, Po; Araújo, Michelle G L; Khan, Muhammad Tahir; Liu, Juan; Rong, Weifang; Tang, Yong; Franke, Heike; Krügel, Ute; Fernandes, Maria José S; Illes, Peter

2017-07-01

Patch-clamp recordings indicated the presence of P2X7 receptors at neural progenitor cells (NPCs) in the subgranular zone of the dentate gyrus in hippocampal brain slices prepared from transgenic nestin reporter mice. The activation of these receptors caused inward current near the resting membrane potential of the NPCs, while P2Y1 receptor activation initiated outward current near the reversal potential of the P2X7 receptor current. Both receptors were identified by biophysical/pharmacological methods. When the brain slices were prepared from mice which underwent a pilocarpine-induced status epilepticus or when brain slices were incubated in pilocarpine-containing external medium, the sensitivity of P2X7 and P2Y1 receptors was invariably increased. Confocal microscopy confirmed the localization of P2X7 and P2Y1 receptor-immunopositivity at nestin-positive NPCs. A one-time status epilepticus in rats caused after a latency of about 5 days recurrent epileptic fits. The blockade of central P2X7 receptors increased the number of seizures and their severity. It is hypothesized that P2Y1 receptors after a status epilepticus may increase the ATP-induced proliferation/ectopic migration of NPCs; the P2X7 receptor-mediated necrosis/apoptosis might counteract these effects, which would otherwise lead to a chronic manifestation of recurrent epileptic fits. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cloning of human genes encoding novel G protein-coupled receptors

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Marchese, A.; Docherty, J.M.; Heiber, M. [Univ. of Toronto, (Canada)] [and others

1994-10-01

We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

The Proton-Activated Receptor GPR4 Modulates Glucose Homeostasis by Increasing Insulin Sensitivity

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Luca Giudici

2013-11-01

Full Text Available Background: The proton-activated G protein-coupled receptor GPR4 is expressed in many tissues including white adipose tissue. GPR4 is activated by extracellular protons in the physiological pH range (i.e. pH 7.7 - 6.8 and is coupled to the production of cAMP. Methods: We examined mice lacking GPR4 and examined glucose tolerance and insulin sensitivity in young and aged mice as well as in mice fed with a high fat diet. Expression profiles of pro- and anti-inflammatory cytokines in white adipose tissue, liver and skeletal muscle was assessed. Results: Here we show that mice lacking GPR4 have an improved intraperitoneal glucose tolerance test and increased insulin sensitivity. Insulin levels were comparable but leptin levels were increased in GPR4 KO mice. Gpr4-/- showed altered expression of PPARα, IL-6, IL-10, TNFα, and TGF-1β in skeletal muscle, white adipose tissue, and liver. High fat diet abolished the differences in glucose tolerance and insulin sensitivity between Gpr4+/+ and Gpr4-/- mice. In contrast, in aged mice (12 months old, the positive effect of GPR4 deficiency on glucose tolerance and insulin sensitivity was maintained. Liver and adipose tissue showed no major differences in the mRNA expression of pro- and anti-inflammatory factors between aged mice of both genotypes. Conclusion: Thus, GPR4 deficiency improves glucose tolerance and insulin sensitivity. The effect may involve an altered balance between pro- and anti-inflammatory factors in insulin target tissues.

Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R.

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Sundqvist, Martina; Christenson, Karin; Holdfeldt, André; Gabl, Michael; Mårtensson, Jonas; Björkman, Lena; Dieckmann, Regis; Dahlgren, Claes; Forsman, Huamei

2018-05-01

GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo. In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo. Copyright © 2018 Elsevier B.V. All rights reserved.

P2Y12 receptor upregulation in satellite glial cells is involved in neuropathic pain induced by HIV glycoprotein 120 and 2',3'-dideoxycytidine.

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Yi, Zhihua; Xie, Lihui; Zhou, Congfa; Yuan, Huilong; Ouyang, Shuai; Fang, Zhi; Zhao, Shanhong; Jia, Tianyu; Zou, Lifang; Wang, Shouyu; Xue, Yun; Wu, Bing; Gao, Yun; Li, Guilin; Liu, Shuangmei; Xu, Hong; Xu, Changshui; Zhang, Chunping; Liang, Shangdong

2018-03-01

The direct neurotoxicity of HIV and neurotoxicity of combination antiretroviral therapy medications both contribute to the development of neuropathic pain. Activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) plays a crucial role in mechanical and thermal hyperalgesia. The P2Y 12 receptor expressed in SGCs of the DRG is involved in pain transmission. In this study, we explored the role of the P2Y 12 receptor in neuropathic pain induced by HIV envelope glycoprotein 120 (gp120) combined with ddC (2',3'-dideoxycytidine). A rat model of gp120+ddC-induced neuropathic pain was used. Peripheral nerve exposure to HIV-gp120+ddC increased mechanical and thermal hyperalgesia in gp120+ddC-treated model rats. The gp120+ddC treatment increased expression of P2Y 12 receptor mRNA and protein in DRG SGCs. In primary cultured DRG SGCs treated with gp120+ddC, the levels of [Ca 2+ ] i activated by the P2Y 12 receptor agonist 2-(Methylthio) adenosine 5'-diphosphate trisodium salt (2-MeSADP) were significantly increased. P2Y 12 receptor shRNA treatment inhibited 2-MeSADP-induced [Ca 2+ ] i in primary cultured DRG SGCs treated with gp120+ddC. Intrathecal treatment with a shRNA against P2Y 12 receptor in DRG SGCs reduced the release of pro-inflammatory cytokines, decreased phosphorylation of p38 MAPK in the DRG of gp120+ddC-treated rats. Thus, downregulating the P2Y 12 receptor relieved mechanical and thermal hyperalgesia in gp120+ddC-treated rats.

Functional analysis of free fatty acid receptor GPR120 in human eosinophils: implications in metabolic homeostasis.

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Konno, Yasunori; Ueki, Shigeharu; Takeda, Masahide; Kobayashi, Yoshiki; Tamaki, Mami; Moritoki, Yuki; Oyamada, Hajime; Itoga, Masamichi; Kayaba, Hiroyuki; Omokawa, Ayumi; Hirokawa, Makoto

2015-01-01

Recent evidence has shown that eosinophils play an important role in metabolic homeostasis through Th2 cytokine production. GPR120 (FFA4) is a G protein-coupled receptor (GPCR) for long-chain fatty acids that functions as a regulator of physiological energy metabolism. In the present study, we aimed to investigate whether human eosinophils express GPR120 and, if present, whether it possesses a functional capacity on eosinophils. Eosinophils isolated from peripheral venous blood expressed GPR120 at both the mRNA and protein levels. Stimulation with a synthetic GPR120 agonist, GW9508, induced rapid down-regulation of cell surface expression of GPR120, suggesting ligand-dependent receptor internalization. Although GPR120 activation did not induce eosinophil chemotactic response and degranulation, we found that GW9508 inhibited eosinophil spontaneous apoptosis and Fas receptor expression. The anti-apoptotic effect was attenuated by phosphoinositide 3-kinase (PI3K) inhibitors and was associated with inhibition of caspase-3 activity. Eosinophil response investigated using ELISpot assay indicated that stimulation with a GPR120 agonist induced IL-4 secretion. These findings demonstrate the novel functional properties of fatty acid sensor GPR120 on human eosinophils and indicate the previously unrecognized link between nutrient metabolism and the immune system.

GPR119 agonists: a promising approach for T2DM treatment? A SWOT analysis of GPR119.

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Kang, Sang-Uk

2013-12-01

Ever since its advent as a promising therapeutic target for type 2 diabetes mellitus (T2DM), G-protein-coupled receptor 119 (GPR119) has received much interest from the pharmaceutical industry. This interest peaked in June 2010, when Sanofi-Aventis agreed to pay Metabolex (Cymabay Therapeutics) US$375 million for MBX-2982, which was a representative orally active GPR119 agonist. However, Sanofi-Aventis opted to terminate the deal in May 2011 and another leading GPR119 agonist, GSK1292263, had a loss of efficacy during its clinical trial. In this review, I discuss the pros and cons of GPR119 through a strengths, weaknesses, opportunities, and threats (SWOT) analysis and propose development strategies for the eventual success of a GPR119 agonist development program. Copyright © 2013 Elsevier Ltd. All rights reserved.

Toll-like receptor 3 signalling up-regulates expression of the HIV co-receptor G-protein coupled receptor 15 on human CD4+ T cells.

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Miriam Kiene

Full Text Available BACKGROUND: Many HIV-2 and SIV isolates, as well as some HIV-1 strains, can use the orphan 7-transmembrane receptor GPR15 as co-receptor for efficient entry into host cells. GPR15 is expressed on central memory and effector memory CD4(+ T cells in healthy individuals and a subset of these cells is susceptible to HIV-1 and SIV infection. However, it has not been determined whether GPR15 expression is altered in the context of HIV-1 infection. RESULTS: Here, we show that GPR15 expression in CD4(+ T cells is markedly up-regulated in some HIV-1 infected individuals compared to the rest of the infected patients and to healthy controls. Infection of the PM1 T cell line with primary HIV-1 isolates was found to up-regulate GPR15 expression on the infected cells, indicating that viral components can induce GPR15 expression. Up-regulation of GPR15 expression on CD4(+ T cells was induced by activation of Toll-like receptor 3 signalling via TIR-domain-containing adapter-inducing interferon-β (TRIF and was more prominent on gut-homing compared to lymph node-homing CD4(+ T cells. CONCLUSION: These results suggest that infection-induced up-regulation of GPR15 expression could increase susceptibility of CD4(+ T cells to HIV infection and target cell availability in the gut in some infected individuals.

Association of P2Y(2) receptor SNPs with bone mineral density and osteoporosis risk in a cohort of Dutch fracture patients

DEFF Research Database (Denmark)

Wesselius, Anke; Bours, Martijn J L; Henriksen, Zanne

2013-01-01

The P2Y(2) receptor is a G-protein-coupled receptor with adenosine 5'-triphosphate (and UTP) as natural ligands. It is thought to be involved in bone physiology in an anti-osteogenic manner. As several non-synonymous single nucleotide polymorphisms (SNPs) have been identified within the P2Y(2) re...

Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase.

Science.gov (United States)

Albert, J L; Boyle, J P; Roberts, J A; Challiss, R A; Gubby, S E; Boarder, M R

1997-11-01

activation at P2Y2 receptors. 2MeSATP gave a much smaller response with a lower potency than UTP. 7. These results are consistent with brain endothelial regulation by P2Y2 receptors coupled to phospholipase C, Ca2+ and MAPK; and by P2Y1-like (2MeSATP-sensitive) receptors which are linked to Ca2+ mobilization by a mechanism apparently independent of agonist stimulated Ins(1,4,5)P3 levels. A further response to ATP, acting at an undefined receptor, caused an increase in cyclic AMP levels in the presence of forskolin. The differential MAPK coupling of these receptors suggests that they exert fundamentally distinct influences over brain endothelial function.

Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y12/13 receptors among BV-2 microglia.

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Pengchong Jiang

Full Text Available Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as 'find/eat-me' signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme, suramin (broad-spectrum P2 receptor antagonist, 2-APB (IP3 receptor blocker and thapsigargin (endoplasmic reticulum calcium pump inhibitor potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 μM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.

The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells

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Gin-Wen Chang

2016-05-01

Full Text Available Natural killer (NK cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells.

G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

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Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

2014-08-08

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A common haplotype in the G-protein-coupled receptor gene GPR74 is associated with leanness and increased lipolysis

DEFF Research Database (Denmark)

Dahlman, Ingrid; Dicker, Andrea; Jiao, Hong

2007-01-01

0.36; P=.036) among those selected for obese or lean phenotypes. The ATAG haplotype was associated with increased adipocyte lipid mobilization (lipolysis) in vivo and in vitro. In human fat cells, GPR74 receptor stimulation and inhibition caused a significant and marked decrease and increase......, respectively, of lipolysis, which could be linked to catecholamine stimulation of adipocytes through beta -adrenergic receptors. These findings suggest that a common haplotype in the GPR74 gene protects against obesity, which, at least in part, is caused by a relief of inhibition of lipid mobilization from......The G-protein-coupled receptor GPR74 is a novel candidate gene for body weight regulation. In humans, it is predominantly expressed in brain, heart, and adipose tissue. We report a haplotype in the GPR74 gene, ATAG, with allele frequency ~4% in Scandinavian cohorts, which was associated...

Diindolylmethane Derivatives: Potent Agonists of the Immunostimulatory Orphan G Protein-Coupled Receptor GPR84.

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Pillaiyar, Thanigaimalai; Köse, Meryem; Sylvester, Katharina; Weighardt, Heike; Thimm, Dominik; Borges, Gleice; Förster, Irmgard; von Kügelgen, Ivar; Müller, Christa E

2017-05-11

The G i protein-coupled receptor GPR84, which is activated by (hydroxy)fatty acids, is highly expressed on immune cells. Recently, 3,3'-diindolylmethane was identified as a heterocyclic, nonlipid-like GPR84 agonist. We synthesized a broad range of diindolylmethane derivatives by condensation of indoles with formaldehyde in water under microwave irradiation. The products were evaluated at the human GPR84 in cAMP and β-arrestin assays. Structure-activity relationships (SARs) were steep. 3,3'-Diindolylmethanes bearing small lipophilic residues at the 5- and/or 7-position of the indole rings displayed the highest activity in cAMP assays, the most potent agonists being di(5-fluoro-1H-indole-3-yl)methane (38, PSB-15160, EC 50 80.0 nM) and di(5,7-difluoro-1H-indole-3-yl)methane (57, PSB-16671, EC 50 41.3 nM). In β-arrestin assays, SARs were different, indicating biased agonism. The new compounds were selective versus related fatty acid receptors and the arylhydrocarbon receptor. Selected compounds were further investigated and found to display an ago-allosteric mechanism of action and increased stability in comparison to the lead structure.

GPR120

DEFF Research Database (Denmark)

Gao, B.; Huang, Q.; Jie, Q.

2015-01-01

/2 cascade, while low concentrations elevated P38 and increased adipogenesis. The fine molecular regulation of GPR120 was implemented by up-regulating different integrin subunits (alpha 1, alpha 2 and beta 1; alpha 5 and beta 3). The administration of high doses of TUG-891 rescued oestrogen-deficient bone...... was involved in the cytoplasmic regulation of a seesaw-like balance between ERK and p38 phosphorylation. These findings provide new hope for developing novel remedies to treat osteoporosis by adjusting the GPR120-mediated differentiation balance of BMMSCs....

ATP and UTP responses of cultured rat aortic smooth muscle cells revisited: dominance of P2Y2 receptors

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Kumari, Rajendra; Goh, Gareth; Ng, Leong L; Boarder, Michael R

2003-01-01

It has previously been shown that ATP and UTP stimulate P2Y receptors in vascular smooth muscle cells (VSMCs), but the nature of these receptors, in particular the contribution of P2Y2 and P2Y4 subtypes, has not been firmly established. Here we undertake a further pharmacological analysis of [3H]inositol polyphosphate responses to nucleotides in cultured rat VSMCs. ATP generated a response that was partial compared to UTP, as reported earlier. In the presence of a creatine phosphokinase (CPK) system for regenerating nucleoside triphosphates, the response to ATP was increased, the response to UTP was unchanged, and the difference between UTP and ATP concentration–response curves disappeared. Chromatographic analysis showed that ATP was degraded slightly faster than UTP. The response to UDP was always smaller than that to UTP, but with a shallow slope and a high potency component. In the presence of hexokinase (which prevents the accumulation of ATP/UTP from ADP/UDP), the maximum response to UDP was reduced and the high-potency component of the curve was retained. By contrast, the response to ADP was weaker throughout in the presence of hexokinase. ATPγS was an effective agonist with a similar EC50 to UTP, but with a lower maximum. ITP was a weak agonist compared with UTP. Suramin was an effective antagonist of the response to UTP (pA2=4.48), but not when ATP was the agonist. However, suramin was an effective antagonist (pA2=4.45) when stimulation with ATP was in the presence of the CPK regenerating system. Taken together with the results of others, these findings indicate that the response of cultured rat VSMCs to UTP and to ATP is predominantly at the P2Y2 receptor, and that there is also a response to UDP at the P2Y6 receptor. PMID:14597595

The Proton-Sensing G-Protein Coupled Receptor GPR4 Promotes Angiogenesis in Head and Neck Cancer.

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Zhibin Jing

Full Text Available Squamous cell carcinoma of the head and neck (SCCHN is an aggressive disease with poor survival and is the sixth most common cancer worldwide. Gastroesophageal reflux is a common event in SCCHN patients. GPR4 is a proton-sensing G-protein coupled receptor, which can be activated by acidosis. The objective of this study was to explore the role of GPR4 in acid exposure and tumor angiogenesis in SCCHN. In this study, we confirmed that overexpressing GPR4 in SCCHN cells could increase the expression and secretion of IL6, IL8 and VEGFA at pH 5.9. This effect could be inhibited by SB203580 (a p38 inhibitor. Western blot analysis indicated that phosphorylation of p38 increased in GPR4 infected cells at pH 5.9, which could be inhibited by SB203580. In tube formation assay, HMEC-1 cells were incubated with conditioned medium (CM, pH 5.9, 6.5, 7.4 derived from control and GPR4 infected SCCHN cells. Tube length was significantly increased in HMEC-1 cells incubated with CM from GPR4 infected cells compared with control cells at pH5.9, which indicated the pro-angiogenic effect of GPR4 in acidic pH. The neutralizing antibodies of IL6, IL8 and VEGFA could inhibit tube formation of HMEC-1 cells. In vivo, the effect of GPR4 on angiogenesis was investigated with the chick chorioallantoic membrane (CAM model. Control and GPR4 infected SCCHN cells were seeded onto the upper CAM surface (n = 5 in each group and 5 μL DMEM/F12 (pH 5.9, 6.5, 7.4 was added to the surface of the cell every 24 h. Four days later, the upper CAM were harvested and the ratio of the vascular area to the CAM area was quantified using Image-Pro Plus 6.0 software. GPR4 infected cells could recruit more vascular than control cells at pH5.9. In conclusion, we suggested that GPR4 induces angiogenesis via GPR4-induced p38-mediated IL6, IL8 and VEGFA secretion at acidic extracellular pH in SCCHN.

An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors.

Science.gov (United States)

Wittenberger, T; Schaller, H C; Hellebrand, S

2001-03-30

We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families. Copyright 2001 Academic Press.

The Cell Surface Estrogen Receptor, G Protein- Coupled Receptor 30 (GPR30, is Markedly Down Regulated During Breast Tumorigenesis

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Indira Poola

2008-01-01

Full Text Available Background: GPR30 is a cell surface estrogen receptor that has been shown to mediate a number of non-genomic rapid effects of estrogen and appear to balance the signaling of estrogen and growth factors. In addition, progestins appear to use GPR30 for their actions. Therefore, GPR30 could play a critical role in hormonal regulation of breast epithelial cell integrity. Deregulation of the events mediated by GPR30 could contribute to tumorigenesis.Methods: To understand the role of GPR30 in the deregulation of estrogen signaling processes during breast carcinogenesis, we have undertaken this study to investigate its expression at mRNA levels in tumor tissues and their matched normal tissues. We compared its expression at mRNA levels by RT quantitative real-time PCR relative to GAPDH in ERα”—positive (n = 54 and ERα”—negative (n = 45 breast cancer tissues to their matched normal tissues.Results: We report here, for the first time, that GPR30 mRNA levels were significantly down-regulated in cancer tissues in comparison with their matched normal tissues (p 0.0001 by two sided paired t-test. The GPR30 expression levels were significantly lower in tumor tissues from patients (n = 29 who had lymph node metastasis in comparison with tumors from patients (n = 53 who were negative for lymph node metastasis (two sample t-test, p 0.02, but no association was found with ERα, PR and other tumor characteristics.Conclusions: Down-regulation of GPR30 could contribute to breast tumorigenesis and lymph node metastasis.

Association analysis of the chromosome 4p-located G protein-coupled receptor 78 (GPR78) gene in bipolar affective disorder and schizophrenia.

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Underwood, S L; Christoforou, A; Thomson, P A; Wray, N R; Tenesa, A; Whittaker, J; Adams, R A; Le Hellard, S; Morris, S W; Blackwood, D H R; Muir, W J; Porteous, D J; Evans, K L

2006-04-01

The orphan G protein-coupled receptor 78 (GPR78) gene lies within a region of chromosome 4p where we have previously shown linkage to bipolar affective disorder (BPAD) in a large Scottish family. GPR78 was screened for single-nucleotide polymorphisms (SNPs) and a linkage disequilibrium map was constructed. Six tagging SNPs were selected and tested for association on a sample of 377 BPAD, 392 schizophrenia (SCZ) and 470 control individuals. Using standard chi(2) statistics and a backwards logistic regression approach to adjust for the effect of sex, SNP rs1282, located approximately 3 kb upstream of the coding region, was identified as a potentially important variant in SCZ (chi(2) P=0.044; LRT P=0.065). When the analysis was restricted to females, the strength of association increased to an uncorrected allele P-value of 0.015 (odds ratios (OR)=1.688, 95% confidence intervals (CI): 1.104-2.581) and uncorrected genotype P-value of 0.015 (OR=5.991, 95% CI: 1.545-23.232). Under the recessive model, the genotype P-value improved further to 0.005 (OR=5.618, 95% CI: 1.460-21.617) and remained significant after correcting for multiple testing (P=0.017). No single-marker association was detected in the SCZ males, in the BPAD individuals or with any other SNP. Haplotype analysis of the case-control samples revealed several global and individual haplotypes, with P-values <0.05, all but one of which contained SNP rs1282. After correcting for multiple testing, two haplotypes remained significant in both the female BPAD individuals (P=0.038 and 0.032) and in the full sample of affected female individuals (P=0.044 and 0.033). Our results provide preliminary evidence for the involvement of GPR78 in susceptibility to BPAD and SCZ in the Scottish population. Molecular Psychiatry (2006) 11, 384-394. doi:10.1038/sj.mp.4001786; published online 3 January 2006.

Nanoparticle-Encapsulated Curcumin Inhibits Diabetic Neuropathic Pain Involving the P2Y12 Receptor in the Dorsal Root Ganglia

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Tianyu Jia

2018-01-01

Full Text Available Diabetic peripheral neuropathy results in diabetic neuropathic pain (DNP. Satellite glial cells (SGCs enwrap the neuronal soma in the dorsal root ganglia (DRG. The purinergic 2 (P2 Y12 receptor is expressed on SGCs in the DRG. SGC activation plays an important role in the pathogenesis of DNP. Curcumin has anti-inflammatory and antioxidant properties. Because curcumin has poor metabolic stability in vivo and low bioavailability, nanoparticle-encapsulated curcumin was used to improve its targeting and bioavailability. In the present study, our aim was to investigate the effects of nanoparticle-encapsulated curcumin on DNP mediated by the P2Y12 receptor on SGCs in the rat DRG. Diabetic peripheral neuropathy increased the expression levels of the P2Y12 receptor on SGCs in the DRG and enhanced mechanical and thermal hyperalgesia in rats with diabetes mellitus (DM. Up-regulation of the P2Y12 receptor in SGCs in the DRG increased the production of pro-inflammatory cytokines. Up-regulation of interleukin-1β (IL-1β and connexin43 (Cx43 resulted in mechanical and thermal hyperalgesia in rats with DM. The nanoparticle-encapsulated curcumin decreased up-regulated IL-1β and Cx43 expression and reduced levels of phosphorylated-Akt (p-Akt in the DRG of rats with DM. The up-regulation of P2Y12 on SGCs and the up-regulation of the IL-1β and Cx43 in the DRG indicated the activation of SGCs in the DRG. The nano-curcumin treatment inhibited the activation of SGCs accompanied by its anti-inflammatory effect to decrease the up-regulated CGRP expression in the DRG neurons. Therefore, the nanoparticle-encapsulated curcumin treatment decreased the up-regulation of the P2Y12 receptor on SGCs in the DRG and decreased mechanical and thermal hyperalgesia in rats with DM.

Characterising pharmacological ligands to study the long chain fatty acid receptors GPR40/FFA1 and GPR120/FFA4

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Milligan, G; Alvarez-Curto, E; Watterson, K R

2015-01-01

The G protein-coupled receptors FFA1 (previously designated GPR40) and FFA4 (previously GPR120) are both activated by saturated and unsaturated longer-chain free fatty acids. With expression patterns and functions anticipated to directly or indirectly promote insulin secretion, provide homeostati...

PI3K/Akt Activated by GPR30 and Src Regulates 17β-Estradiol-Induced Cultured Immature Boar Sertoli Cells Proliferation.

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Yang, Wei-Rong; Zhu, Feng-Wei; Zhang, Jiao-Jiao; Wang, Yi; Zhang, Jia-Hua; Lu, Cheng; Wang, Xian-Zhong

2016-05-24

Sertoli cell (SC) is a key element in the process of spermatogenesis. Accumulating research show that estrogen plays an important role in regulating boar SC proliferation. However, it is unclear whether phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/Akt) is involved in this process. In the present study, the role of PI3K/Akt on the 17β-estradiol-induced piglet SC proliferation was explored. In addition, we also explained the roles of G-protein-coupled estrogen receptor (GPR30) and Sarcoma protein (Src) in this process. Our study demonstrated that, 17β-estradiol induced activation of PI3K in a time-dependent manner. Both G-15 (an antagonist of GPR30, 0.1 μmol/L) and PP2 (an inhibitor of Src, 2.0 μmol/L) inhibited 17β-estradiol-induced activation of PI3K, reduced SC proliferation, and decreased messenger RNA (mRNA) and protein expression of S-phase kinase-associated protein 2 (Skp2). We also found that 17β-estradiol induced activation of Akt in a time-dependent manner. Both LY294002 (an inhibitor of PI3K) and 10-DEBC (an inhibitor of Akt) significantly reduced 17β-estradiol-induced SC proliferation and reduced mRNA and protein expression of Skp2. In addition, LY294002 inhibited 17β-estradiol-induced activation of Akt. The results indicated that 17β-estradiol regulates SC proliferation by activating PI3K/Akt. Both GPR30 and Src are involved in 17β-estradiol-induced phosphorylation of PI3K/Akt. Activation of PI3K/Akt enhances the expression of Skp2, which promotes SC proliferation. © The Author(s) 2016.

P2X4: A fast and sensitive purinergic receptor

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Jaanus Suurväli

2017-10-01

Full Text Available Extracellular nucleotides have been recognized as important mediators of activation, triggering multiple responses via plasma membrane receptors known as P2 receptors. P2 receptors comprise P2X ionotropic receptors and G protein-coupled P2Y receptors. P2X receptors are expressed in many tissues, where they are involved in a number of functions including synaptic transmission, muscle contraction, platelet aggregation, inflammation, macrophage activation, differentiation and proliferation, neuropathic and inflammatory pain. P2X4 is one of the most sensitive purinergic receptors (at nanomolar ATP concentrations, about one thousand times more than the archetypal P2X7. P2X4 is widely expressed in central and peripheral neurons, in microglia, and also found in various epithelial tissues and endothelial cells. It localizes on the plasma membrane, but also in intracellular compartments. P2X4 is preferentially localized in lysosomes, where it is protected from proteolysis by its glycosylation. High ATP concentration in the lysosomes does not activate P2X4 at low pH; P2X4 gets activated by intra-lysosomal ATP only in its fully dissociated tetra-anionic form, when the pH increases to 7.4. Thus, P2X4 is functioning as a Ca2+-channel after the fusion of late endosomes and lysosomes. P2X4 modulates major neurotransmitter systems and regulates alcohol-induced responses in microglia. P2X4 is one of the key receptors mediating neuropathic pain. However, injury-induced upregulation of P2X4 expression is gender dependent and plays a key role in pain difference between males and females. P2X4 is also involved in inflammation. Extracellular ATP being a pro-inflammatory molecule, P2X4 can trigger inflammation in response to high ATP release. It is therefore involved in multiple pathologies, like post-ischemic inflammation, rheumatoid arthritis, airways inflammation in asthma, neurodegenerative diseases and even metabolic syndrome. Although P2X4 remains poorly

Purinergic P2Y12 Receptor Activation in Eosinophils and the Schistosomal Host Response.

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Muniz, Valdirene S; Baptista-Dos-Reis, Renata; Benjamim, Claudia F; Mata-Santos, Hilton A; Pyrrho, Alexandre S; Strauch, Marcelo A; Melo, Paulo A; Vicentino, Amanda R R; Silva-Paiva, Juliana; Bandeira-Melo, Christianne; Weller, Peter F; Figueiredo, Rodrigo T; Neves, Josiane S

2015-01-01

Identifying new target molecules through which eosinophils secrete their stored proteins may reveal new therapeutic approaches for the control of eosinophilic disorders such as host immune responses to parasites. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils; however, its functional role in this cell type and its involvement in eosinophilic inflammation remain unknown. Here, we investigated functional roles of P2Y12R in isolated human eosinophils and in a murine model of eosinophilic inflammation induced by Schistosoma mansoni (S. mansoni) infection. We found that adenosine 5'-diphosphate (ADP) induced human eosinophils to secrete eosinophil peroxidase (EPO) in a P2Y12R dependent manner. However, ADP did not interfere with human eosinophil apoptosis or chemotaxis in vitro. In vivo, C57Bl/6 mice were infected with cercariae of the Belo Horizonte strain of S. mansoni. Analyses performed 55 days post infection revealed that P2Y12R blockade reduced the granulomatous hepatic area and the eosinophilic infiltrate, collagen deposition and IL-13/IL-4 production in the liver without affecting the parasite oviposition. As found for humans, murine eosinophils also express the P2Y12R. P2Y12R inhibition increased blood eosinophilia, whereas it decreased the bone marrow eosinophil count. Our results suggest that P2Y12R has an important role in eosinophil EPO secretion and in establishing the inflammatory response in the course of a S. mansoni infection.

Characterization of a Ca2+ response to both UTP and ATP at human P2Y11 receptors: evidence for agonist-specific signaling.

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White, Pamela J; Webb, Tania E; Boarder, Michael R

2003-06-01

Previous reports on heterologously-expressed human P2Y11 receptors have indicated that ATP, but not UTP, is an agonist stimulating both phosphoinositidase C and adenylyl cyclase. Consistent with these findings, we report that in 1321N1 cells expressing human P2Y11 receptors, UTP stimulation did not lead to accumulation of inositol(poly)phosphates under conditions in which ATP gave a robust, concentration-dependent effect. Unexpectedly, however, both UTP and ATP stimulated increases in cytosolic Ca2+ concentration ([Ca2+]c), with both nucleotides achieving similar EC50 and maximal responses. The responses to maximally effective concentrations of ATP and UTP were not additive. The [Ca2+]c increase in response to UTP was less dependent on extracellular Ca2+ than was the response to ATP. AR-C67085 (2-propylthio-beta,gamma-difluoromethylene-d-ATP, a P2Y11-selective agonist), adenosine 5'-O-(3-thiotriphosphate), and benzoyl ATP were all full agonists with potencies similar to those of ATP and UTP. In desensitization experiments, exposure to ATP resulted in loss of the UTP response; this response was more sensitive to desensitization than that of ATP. Pertussis toxin pretreatment attenuated the response to UTP but left the ATP response unaffected. The presence of 2-aminoethyl diphenylborate differentially affected the responses of ATP and UTP. No mRNA transcripts for P2Y2 or P2Y4 were detectable in the P2Y11-expressing cells. We conclude that UTP is a Ca2+-mobilizing agonist at P2Y11 receptors and that ATP and UTP acting at the same receptor recruit distinct signaling pathways. This example of agonist-specific signaling is discussed in terms of agonist trafficking and differential signal strength.

Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells.

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Katie M Lowther

Full Text Available G protein-coupled receptor 3 (GPR3 is a constitutively active receptor that maintains high 3'-5'-cyclic adenosine monophosphate (cAMP levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2 and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third

Novel G Protein-Coupled Oestrogen Receptor GPR30 Shows Changes in mRNA Expression in the Rat Brain over the Oestrous Cycle

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Emma J. Spary

2012-02-01

Full Text Available Oestrogen influences autonomic function via actions at classical nuclear oestrogen receptors α and β in the brain, and recent evidence suggests the orphan G protein-coupled receptor GPR30 may also function as a cytoplasmic oestrogen receptor. We investigated the expression of GPR30 in female rat brains throughout the oestrous cycle and after ovariectomy to determine whether GPR30 expression in central autonomic nuclei is correlated with circulating oestrogen levels. In the nucleus of the solitary tract (NTS, ventrolateral medulla (VLM and periaqueductal gray (PAG GPR30 mRNA, quantified by real-time PCR, was increased in proestrus and oestrus. In ovariectomised (OVX rats, expression in NTS and VLM appeared increased compared to metoestrus, but in the hypothalamic paraventricular nucleus and PAG lower mRNA levels were seen in OVX. GPR30-like immunoreactivity (GPR30-LI colocalised with Golgi in neurones in many brain areas associated with autonomic pathways, and analysis of numbers of immunoreactive neurones showed differences consistent with the PCR data. GPR30-LI was found in a variety of transmitter phenotypes, including cholinergic, serotonergic, catecholaminergic and nitrergic neurones in different neuronal groups. These observations support the view that GPR30 could act as a rapid transducer responding to oestrogen levels and thus modulate the activity of central autonomic pathways.

Differential endosomal sorting of a novel P2Y12 purinoreceptor mutant.

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Cunningham, Margaret R; Nisar, Shaista P; Cooke, Alexandra E; Emery, Elizabeth D; Mundell, Stuart J

2013-05-01

P2Y12 receptor internalization and recycling play an essential role in ADP-induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y12 (P341A) whose P2Y12 receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild-type (WT) P2Y12 recycling and investigated P2Y12 -P341A receptor traffic. Treatment with ADP resulted in delayed Rab5-dependent internalization of P341A when compared with WT P2Y12 . While WT P2Y12 rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7-positive endosomes with considerable agonist-dependent accumulation in the trans-Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT-P2Y12 . Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y12 receptor traffic and explain the compromised receptor function in the platelets of the P2Y12 -P341A-expressing patient. © 2013 John Wiley & Sons A/S.

A medium-chain fatty acid receptor Gpr84 in zebrafish: expression pattern and roles in immune regulation.

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Huang, Qiaoyan; Feng, Dong; Liu, Kai; Wang, Peng; Xiao, Hongyan; Wang, Ying; Zhang, Shicui; Liu, Zhenhui

2014-08-01

Gpr84 was recently identified as a receptor for medium-chain fatty acids, but its functions remain to be clarified. We reported the identification of a zebrafish Gpr84 homologue (zGpr84), which has a higher gene expression in the tissues of intestine, heart and liver. During embryogenesis, zGpr84 is maternally expressed and a significant increase is observed at segmentation period, and it is mainly restricted to the head region, pectoral fins, branchial arches, intestine and lateral line neuromast. Fasting or treatment with lipopolysaccharide (LPS) can induce significant up-regulation of zGpr84. We further demonstrated that zGpr84 is involved in the accumulation of lipid droplets in cells. Moreover, undecanoic acid (UA) can amplify LPS induced production of the proinflammatory cytokine IL-12 p40 through zGpr84, supporting the proposal that Gpr84 may play a role in directly linking fatty acid metabolism to immunological regulation. The resulting data in fish lay a foundation for a comprehensive exploration of the functions and evolution of Gpr84. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of P2Y receptors mediating ATP induced relaxation in guinea pig airway smooth muscle: involvement of prostaglandins and K+ channels.

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Montaño, Luis M; Cruz-Valderrama, José E; Figueroa, Alejandra; Flores-Soto, Edgar; García-Hernández, Luz M; Carbajal, Verónica; Segura, Patricia; Méndez, Carmen; Díaz, Verónica; Barajas-López, Carlos

2011-10-01

In airway smooth muscle (ASM), adenosine 5'-triphosphate (ATP) induces a relaxation associated with prostaglandin production. We explored the role of K(+) currents (I (K)) in this relaxation. ATP relaxed the ASM, and this effect was abolished by indomethacin. Removal of airway epithelium slightly diminished the ATP-induced relaxation at lower concentration without modifying the responses to ATP at higher concentrations. ATPγS and UTP induced a concentration-dependent relaxation similar to ATP; α,β-methylene-ATP was inactive from 1 to 100 μM. Suramin or reactive blue 2 (RB2), P2Y receptor antagonists, did not modify the relaxation, but their combination significantly reduced this effect of ATP. The relaxation was also inhibited by N-ethylmaleimide (NEM; which uncouples G proteins). In myocytes, the ATP-induced I (K) increment was not modified by suramin or RB2 but the combination of both drugs abolished it. This increment in the I (K) was also completely nullified by NEM and SQ 22,536. 4-Amynopyridine or iberiotoxin diminished the ATP-induced I (K) increment, and the combination of both substances diminished ATP-induced relaxation. The presence of P2Y(2) and P2Y(4) receptors in smooth muscle was corroborated by Western blot and confocal images. In conclusion, ATP: (1) produces relaxation by inducing the production of bronchodilator prostaglandins in airway smooth muscle, most likely by acting on P2Y(4) and P2Y(2) receptors; (2) induces I (K) increment through activation of the delayed rectifier K(+) channels and the high-conductance Ca(2+)-dependent K(+) channels, therefore both channels are implicated in the ATP-induced relaxation; and (3) this I (K) increment is mediated by prostaglandin production which in turns increase cAMP signaling pathway.

Essential Regulation of Lung Surfactant Homeostasis by the Orphan G Protein-Coupled Receptor GPR116

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Mi Young Yang

2013-05-01

Full Text Available GPR116 is an orphan seven-pass transmembrane receptor whose function has been unclear. Global disruption of the Gpr116 gene in mice revealed an unexpected, critical role for this receptor in lung surfactant homeostasis, resulting in progressive accumulation of surfactant lipids and proteins in the alveolar space, labored breathing, and a reduced lifespan. GPR116 expression analysis, bone marrow transplantation studies, and characterization of conditional knockout mice revealed that GPR116 expression in ATII cells is required for maintaining normal surfactant levels. Aberrant packaging of surfactant proteins with lipids in the Gpr116 mutant mice resulted in compromised surfactant structure, function, uptake, and processing. Thus, GPR116 plays an indispensable role in lung surfactant homeostasis with important ramifications for the understanding and treatment of lung surfactant disorders.

The orphan G protein-coupled receptor GPR139 is activated by the peptides

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Jensen, Anne Cathrine Nøhr; Shehata, Mohamed A; Hauser, Alexander S

2017-01-01

GPR139 is an orphan G protein-coupled receptor that is expressed primarily in the brain. Not much is known regarding the function of GPR139. Recently we have shown that GPR139 is activated by the amino acids l-tryptophan and l-phenylalanine (EC50 values of 220 μM and 320 μM, respectively), as well...

Regulating the effects of GPR21, a novel target for type 2 diabetes

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Leonard, Siobhán; Kinsella, Gemma K.; Benetti, Elisa; Findlay, John B. C.

2016-05-01

Type 2 diabetes is a chronic metabolic disorder primarily caused by insulin resistance to which obesity is a major contributor. Expression levels of an orphan G protein-coupled receptor (GPCR), GPR21, demonstrated a trend towards a significant increase in the epididymal fat pads of wild type high fat high sugar (HFHS)-fed mice. To gain further insight into the potential role this novel target may play in the development of obesity-associated type 2 diabetes, the signalling capabilities of the receptor were investigated. Overexpression studies in HEK293T cells revealed GPR21 to be a constitutively active receptor, which couples to Gαq type G proteins leading to the activation of mitogen activated protein kinases (MAPKs). Overexpression of GPR21 in vitro also markedly attenuated insulin signalling. Interestingly, the effect of GPR21 on the MAPKs and insulin signalling was reduced in the presence of serum, inferring the possibility of a native inhibitory ligand. Homology modelling and ligand docking studies led to the identification of a novel compound that inhibited GPR21 activity. Its effects offer potential as an anti-diabetic pharmacological strategy as it was found to counteract the influence of GPR21 on the insulin signalling pathway.

Possible involvement of neuropeptide Y Y1 receptors in antidepressant like effect of agmatine in rats.

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Kotagale, Nandkishor R; Paliwal, Nikhilesh P; Aglawe, Manish M; Umekar, Milind J; Taksande, Brijesh G

2013-09-01

Agmatine and neuropeptide Y (NPY) are widely distributed in central nervous system and critically involved in modulation of depressive behavior in experimental animals. However their mutual interaction, if any, in regulation of depression remain largely unexplored. In the present study we explored the possible interaction between agmatine and neuropeptide Y in regulation of depression like behavior in forced swim test. We found that acute intracerebroventricular (i.c.v.) administration of agmatine (20-40μg/rat), NPY (5 and 10μg/rat) and NPY Y1 receptor agonist, [Leu(31), Pro(34)]-NPY (0.4 and 0.8ng/rat) dose dependently decreased immobility time in forced swim test indicating their antidepressant like effects. In combination studies, the antidepressant like effect of agmatine (10μg/rat) was significantly potentiated by NPY (1 and 5μg/rat, icv) or [Leu(31), Pro(34)]-NPY (0.2 and 0.4ng/rat, icv) pretreatment. Conversely, pretreatment of animals with NPY Y1 receptor antagonist, BIBP3226 (0.1ng/rat, i.c.v.) completely blocked the antidepressant like effect of agmatine (20-40μg/rat) and its synergistic effect with NPY (1μg/rat, icv) or [Leu(31), Pro(34)]-NPY (0.2ng/rat, icv). The results of the present study showed that, agmatine exerts antidepressant like effects via NPYergic system possibly mediated by the NPY Y1 receptor subtypes and suggest that interaction between agmatine and neuropeptide Y may be relevant to generate the therapeutic strategies for the treatment of depression. Copyright © 2013 Elsevier Inc. All rights reserved.

Getting from A to B-exploring the activation motifs of the class B adhesion G protein-coupled receptor subfamily G member 4/GPR112

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Cornelia Peeters, Miriam; Mos, Iris; Lenselink, Eelke B

2016-01-01

The adhesion G protein-coupled receptors (ADGRs/class B2 G protein-coupled receptors) constitute an ancient family of G protein-coupled receptors that have recently been demonstrated to play important roles in cellular and developmental processes. Here, we describe a first insight...... into the structure-function relationship of ADGRs using the family member ADGR subfamily G member 4 (ADGRG4)/GPR112 as a model receptor. In a bioinformatics approach, we compared conserved, functional elements of the well-characterized class A and class B1 secretin-like G protein-coupled receptors with the ADGRs. We...... identified several potential equivalent motifs and subjected those to mutational analysis. The importance of the mutated residues was evaluated by examining their effect on the high constitutive activity of the N-terminally truncated ADGRG4/GPR112 in a 1-receptor-1-G protein Saccharomyces cerevisiae...

Microglia P2Y6 receptor is related to Parkinson’s disease through neuroinflammatory process

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Xiaodong Yang

2017-02-01

Full Text Available Abstract Background Microglia in the central nervous system (CNS were reported to play crucial role in neurodegeneration. Previous studies showed that P2Y6 receptor (P2Y6R mainly contributed to microglia activation and phagocytosis in CNS. However, the level of P2Y6R in Parkinson’s disease (PD patients is unclear. Therefore, we measured the level of P2Y6R in PD patients and speculated whether it could be a potential biomarker for PD. Given on the basis that P2Y6R was higher in PD patients, we further explored the mechanisms underlying P2Y6R in the pathogenesis of PD. Methods We tested the expression level of P2Y6R in the peripheral blood mononuclear cells (PBMCs among 145 PD patients, 170 healthy controls, and 30 multiple system atrophy (MSA patients. We also used a lipopolysaccharide (LPS-stimulated microglial cell culture model to investigate (i the effects of LPS on P2Y6R expression with western blot and RT-PCR, (ii the effects of LPS on UDP expression using HPLC, (iii the effects of UDP/P2Y6R signaling on cytokine expression using western blot, RT-PCR, and ELISA, and (iv the signaling pathways activated by the P2Y6R involved in the neuroinflammation. Results Expression levels of P2Y6R in PD patients were higher than healthy controls and MSA patients. P2Y6R could be a good biomarker of PD. P2Y6R was also upregulated in LPS-treated BV-2 cells and involved in proinflammatory cytokine release through an autocrine loop based on LPS-triggered UDP secretion and accelerated neuroinflammatory responses through the ERK1/2 pathway. Importantly, blocking UDP/P2Y6R signaling could reverse these pathological processes. Conclusions P2Y6R may be a potential clinical biomarker of PD. Blocking P2Y6R may be a potential therapeutic approach to the treatment of PD patients through inhibition of microglia-activated neuroinflammation.

The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis

OpenAIRE

Perry, Kimberly J.; Johnson, Verity R.; Malloch, Erica L.; Fukui, Lisa; Wever, Jason; Thomas, Alvin G.; Hamilton, Paul W.; Henry, Jonathan J.

2010-01-01

G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84’s importance in lens, cornea and retinal development. Ex...

The G protein-coupled receptor GPR30 mediates the proliferative and invasive effects induced by hydroxytamoxifen in endometrial cancer cells

Energy Technology Data Exchange (ETDEWEB)

Du, Gui-Qiang; Zhou, Long; Chen, Xiao-Yue [Department of Obstetrics and Gynecology, The International Peace Maternity and Child Health Hospital of the China Welfare Institute Affiliated to Shanghai Jiao Tong University, 910, Hengshan Road, Shanghai (China); Wan, Xiao-Ping, E-mail: wanxiaoping61@126.com [Department of Obstetrics and Gynecology, The International Peace Maternity and Child Health Hospital of the China Welfare Institute Affiliated to Shanghai Jiao Tong University, 910, Hengshan Road, Shanghai (China); He, Yin-Yan [Department of Obstetrics and Gynecology, Shanghai First People' s Hospital, Shanghai Jiao Tong University, Shanghai (China)

2012-04-06

Highlights: Black-Right-Pointing-Pointer We assessed hydroxytamoxifen (OHT) effects in two endometrial cancer cell lines. Black-Right-Pointing-Pointer GPR30 mediates the proliferative effects induced by OHT. Black-Right-Pointing-Pointer GPR30 mediates the invasive effects induced by OHT. Black-Right-Pointing-Pointer GPR30 expression was up-regulated by OHT in endometrial cancer cell line. -- Abstract: The selective ER modulator tamoxifen (TAM) is the most widely used ER antagonist for treatment of women with hormone-dependent breast tumor. However, long-term treatment is associated with an increased risk of endometrial cancer. The aim of the present study was to demonstrate new insight into the role of G-protein coupled receptor 30 (GPR30) in the activity of TAM, which promoted endometrial cancer. In endometrial cancer cell lines ISHIKAWA and KLE, the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, 17{beta}-estradiol (E2) and G1, a non-steroidal GPR30-specific agonist to promote cell proliferation and invasion was evaluated. All agents above induced high proliferative and invasive effects, while the down-regulation of GPR30 or the interruption of MAPK signal pathway partly or completely prevented the action of the regent. Moreover, the RNA and protein expression of GPR30 was up-regulated by G1, E2 or OHT in both cell lines. The present study provided a new insight into the mechanism involved in the agonistic activity exerted by TAM in the uterus.

The G protein-coupled receptor GPR30 mediates the proliferative and invasive effects induced by hydroxytamoxifen in endometrial cancer cells

International Nuclear Information System (INIS)

Du, Gui-Qiang; Zhou, Long; Chen, Xiao-Yue; Wan, Xiao-Ping; He, Yin-Yan

2012-01-01

Highlights: ► We assessed hydroxytamoxifen (OHT) effects in two endometrial cancer cell lines. ► GPR30 mediates the proliferative effects induced by OHT. ► GPR30 mediates the invasive effects induced by OHT. ► GPR30 expression was up-regulated by OHT in endometrial cancer cell line. -- Abstract: The selective ER modulator tamoxifen (TAM) is the most widely used ER antagonist for treatment of women with hormone-dependent breast tumor. However, long-term treatment is associated with an increased risk of endometrial cancer. The aim of the present study was to demonstrate new insight into the role of G-protein coupled receptor 30 (GPR30) in the activity of TAM, which promoted endometrial cancer. In endometrial cancer cell lines ISHIKAWA and KLE, the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, 17β-estradiol (E2) and G1, a non-steroidal GPR30-specific agonist to promote cell proliferation and invasion was evaluated. All agents above induced high proliferative and invasive effects, while the down-regulation of GPR30 or the interruption of MAPK signal pathway partly or completely prevented the action of the regent. Moreover, the RNA and protein expression of GPR30 was up-regulated by G1, E2 or OHT in both cell lines. The present study provided a new insight into the mechanism involved in the agonistic activity exerted by TAM in the uterus.

GPR119 - a major Enteroendocrine Sensor of Dietary Triglyceride Metabolites Co-acting in Synergy with FFA1 (GPR40)

DEFF Research Database (Denmark)

Ekberg, Jeppe H; Pedersen, Maria Hauge; Kristensen, Line V

2016-01-01

the FFA1 agonist was administered on top of appropriately low doses of the GPR119 agonist a clear synergistic, i.e. more than additive effect was observed. It is concluded that the 2-MAG receptor GPR119 is at least as important as the LCFA receptor FFA1 in mediating the triglyceride-induced secretion......Triglycerides are among the most efficacious stimulators of incretin secretion; however the relative importance of FFA1 (GPR40), FFA4 (GPR120) and GPR119 which all recognize triglyceride metabolites, i.e. LCFA and 2-MAG respectively, is still unclear. Here, we find all three receptors to be highly...... the synthetic FFA1 agonists, TAK-875 stimulated GLP-1 secretion to a similar extent as the prototype GLP-1 secretagogue neuromedin C, this however only corresponded to approx. half the maximal efficiency of the GPR119 agonist AR231453 whereas the GPR120 agonist Metabolix-209 had no effect. Importantly, when...

Cell adhesion controlled by adhesion G protein-coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo-Dock.

Science.gov (United States)

Hernández-Vásquez, Magda Nohemí; Adame-García, Sendi Rafael; Hamoud, Noumeira; Chidiac, Rony; Reyes-Cruz, Guadalupe; Gratton, Jean Philippe; Côté, Jean-François; Vázquez-Prado, José

2017-07-21

Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gβγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses. © 2017 by The American Society for

P2Y1 receptor antagonists mitigate oxygen and glucose deprivation‑induced astrocyte injury.

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Guo, Hui; Liu, Zhong-Qiang; Zhou, Hui; Wang, Zhi-Ling; Tao, Yu-Hong; Tong, Yu

2018-01-01

The aim of the present study was to elucidate the effects of blocking the calcium signaling pathway of astrocytes (ASs) on oxygen and glucose deprivation (OGD)‑induced AS injury. The association between the changes in the concentrations of AS‑derived transmitter ATP and glutamic acid, and the changes in calcium signaling under the challenge of OGD were investigated. The cortical ASs of Sprague Dawley rats were cultured to establish the OGD models of ASs. The extracellular concentrations of ATP and glutamic acid in the normal group and the OGD group were detected, and the intracellular concentration of calcium ions (Ca2+) was detected. The effects of 2'‑deoxy‑N6‑methyl adenosine 3', 5'‑diphosphate diammonium salt (MRS2179), a P2Y1 receptor antagonist, on the release of calcium and glutamic acid of ASs under the condition of OGD were observed. The OGD challenge induced the release of glutamic acid and ATP by ASs in a time‑dependent manner, whereas elevation in the concentration of glutamic acid lagged behind that of the ATP and Ca2+. The concentration of Ca2+ inside ASs peaked 16 h after OGD, following which the concentration of Ca2+ was decreased. The effects of elevated release of glutamic acid by ASs when challenged by OGD may be blocked by MRS2179, a P2Y1 receptor antagonist. Furthermore, MRS2179 may significantly mitigate OGD‑induced AS injury and increase cell survival. The ASs of rats cultured in vitro expressed P2Y1 receptors, which may inhibit excessive elevation in the concentration of intracellular Ca2+. Avoidance of intracellular calcium overload and the excessive release of glutamic acid may be an important reason why MRS2179 mitigates OGD‑induced AS injury.

In vitro and in vivo effects of kisspeptin antagonists p234, p271, p354, and p356 on GPR54 activation.

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C H J Albers-Wolthers

Full Text Available Kisspeptins (KPs and their receptor (GPR54 or KiSS1R play a key-role in regulation of the hypothalamic-pituitary-gonadal axis and are therefore interesting targets for therapeutic interventions in the field of reproductive endocrinology. As dogs show a rapid and robust LH response after the administration of KP10, they can serve as a good animal model for research concerning KP signaling. The aims of the present study were to test the antagonistic properties of KP analogs p234, p271, p354, and p356 in vitro, by determining the intracellular Ca2+ response of CHEM1 cells that stably express human GPR54, and to study the in vivo effects of these peptides on basal plasma LH concentration and the KP10-induced LH response in female dogs. Exposure of the CHEM1 cells to KP-10 resulted in a clear Ca2+ response. P234, p271, p354, and p356 did not prevent or lower the KP10-induced Ca2+ response. Moreover, the in vivo studies in the dogs showed that none of these supposed antagonists lowered the basal plasma LH concentration and none of the peptides lowered the KP10-induced LH response. In conclusion, p234, p271, p354, and p356 had no antagonistic effects in vitro nor any effect on basal and kisspeptin-stimulated plasma LH concentration in female dogs.

Inverse agonism at the P2Y12 receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor.

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Aungraheeta, Riyaad; Conibear, Alexandra; Butler, Mark; Kelly, Eamonn; Nylander, Sven; Mumford, Andrew; Mundell, Stuart J

2016-12-08

Ticagrelor is a potent antagonist of the P2Y 12 receptor (P2Y 12 R) and consequently an inhibitor of platelet activity effective in the treatment of atherothrombosis. Here, we sought to further characterize its molecular mechanism of action. Initial studies showed that ticagrelor promoted a greater inhibition of adenosine 5'-diphosphate (ADP)-induced Ca 2+ release in washed platelets vs other P2Y 12 R antagonists. This additional effect of ticagrelor beyond P2Y 12 R antagonism was in part as a consequence of ticagrelor inhibiting the equilibrative nucleoside transporter 1 (ENT1) on platelets, leading to accumulation of extracellular adenosine and activation of G s -coupled adenosine A 2A receptors. This contributed to an increase in basal cyclic adenosine monophosphate (cAMP) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P). In addition, ticagrelor increased platelet cAMP and VASP-P in the absence of ADP in an adenosine receptor-independent manner. We hypothesized that this increase originated from a direct effect on basal agonist-independent P2Y 12 R signaling, and this was validated in 1321N1 cells stably transfected with human P2Y 12 R. In these cells, ticagrelor blocked the constitutive agonist-independent activity of the P2Y 12 R, limiting basal G i -coupled signaling and thereby increasing cAMP levels. These data suggest that ticagrelor has the pharmacological profile of an inverse agonist. Based on our results showing insurmountable inhibition of ADP-induced Ca 2+ release and forskolin-induced cAMP, the mode of antagonism of ticagrelor also appears noncompetitive, at least functionally. In summary, our studies describe 2 novel modes of action of ticagrelor, inhibition of platelet ENT1 and inverse agonism at the P2Y 12 R that contribute to its effective inhibition of platelet activation. © 2016 by The American Society of Hematology.

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

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

2014-05-01

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

Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses.

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Ayaka Tobo

Full Text Available G protein-coupled receptor 4 (GPR4, previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions.

Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses.

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Tobo, Ayaka; Tobo, Masayuki; Nakakura, Takashi; Ebara, Masashi; Tomura, Hideaki; Mogi, Chihiro; Im, Dong-Soon; Murata, Naoya; Kuwabara, Atsushi; Ito, Saki; Fukuda, Hayato; Arisawa, Mitsuhiro; Shuto, Satoshi; Nakaya, Michio; Kurose, Hitoshi; Sato, Koichi; Okajima, Fumikazu

2015-01-01

G protein-coupled receptor 4 (GPR4), previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR) coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE)-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions.

P2X receptors in epithelia

DEFF Research Database (Denmark)

Leipziger, Jens Georg

2015-01-01

P2X receptors are ubiquitously expressed in all epithelial tissues but their functional roles are less well studied. Here we review the current state of knowledge by focusing on functional effects of P2X receptor in secretory and in absorptive tissues. In glandular tissue like the parotid gland...

GPR39 is coupled to TMEM16A in intestinal fibroblast-like cells.

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Fanning Zeng

Full Text Available GPR39 is a GPCR implicated as a regulator of gastrointestinal motility, although the mechanism remains elusive. Here, we report that GPR39 is expressed by a specific cell population cultured from mouse small intestine muscle layers, which was subsequently identified as fibroblast-like cells (FLCs that have recently been shown to modulate gut motility. Application of the GPR39 agonist, Zn(2+, induced large currents and membrane depolarization in FLCs cultured from wild-type mice, but not Gpr39(-/- mice. This Zn(2+-induced current could be suppressed by application of a TMEM16A antagonist, CaCC(inh-A01, or by silencing Tmem16a expression. These data suggest that GPR39 might modulate gut motility via regulating TMEM16A function in FLCs.

GPR41/FFAR3 and GPR43/FFAR2 as Cosensors for Short-Chain Fatty Acids in Enteroendocrine Cells vs FFAR3 in Enteric Neurons and FFAR2 in Enteric Leukocytes

DEFF Research Database (Denmark)

Nøhr, Mark K; Pedersen, Maria H; Gille, Andreas

2013-01-01

The expression of short-chain fatty acid receptors GPR41/FFAR3 and GPR43/ free fatty acid receptor 2 (FFAR2) was studied in the gastrointestinal tract of transgenic monomeric red fluorescent protein (mRFP) reporter mice. In the stomach free fatty acid receptor 3 (FFAR3)-mRFP was expressed...... for the majority of enteroendocrine cells of the small and large intestine and that FFAR3 and FFAR2 both act as sensors for short-chain fatty acids in enteroendocrine cells, whereas FFAR3 apparently has this role alone in enteric neurons and FFAR2 in enteric leukocytes....

Analysis of the influence of nucleotidases on the apparent activity of exogenous ATP and ADP at P2Y1 receptors

OpenAIRE

Vigne, Paul; Philippe Breittmayer, Jean; Frelin, Christian

1998-01-01

ADP is a potent agonist of rat and human P2Y1 purinoceptors. ATP is a weak competitive antagonist. This study analyses the situation in which P2Y1 receptors are exposed to ATP in the presence of exogenous ecto-nucleotidases (apyrases) that have high or low ATPase/ADPase activity ratio.Rat brain capillary endothelial cells of the B10 clone express P2Y1 receptors that couple to intracellular Ca2+ mobilization. They have low endogenous ecto-ATPase and ecto-ADPase activities.ATP did not raise int...

Deficiency of the GPR39 receptor is associated with obesity and altered adipocyte metabolism

DEFF Research Database (Denmark)

Petersen, Pia Steen; Jin, Chunyu; Madsen, Andreas Nygaard

2011-01-01

, conceivably due to decreased energy expenditure and adipocyte lipolytic activity.-Petersen, P. S., Jin, C., Madsen, A. N., Rasmussen, M., Kuhre, R., L. Egerod, K. L., Nielsen, L. B., Schwartz. T. W., Holst, B. Deficiency of the GPR39 receptor is associated with obesity and altered adipocyte metabolism....

Mutation analysis and molecular modeling for the investigation of ligand-binding modes of GPR84.

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Nikaido, Yoshiaki; Koyama, Yuuta; Yoshikawa, Yasushi; Furuya, Toshio; Takeda, Shigeki

2015-05-01

GPR84 is a G protein-coupled receptor for medium-chain fatty acids. Capric acid and 3,3'-diindolylmethane are specific agonists for GPR84. We built a homology model of a GPR84-capric acid complex to investigate the ligand-binding mode using the crystal structure of human active-state β2-adrenergic receptor. We performed site-directed mutagenesis to subject ligand-binding sites to our model using GPR84-Giα fusion proteins and a [(35)S]GTPγS-binding assay. We compared the activity of the wild type and mutated forms of GPR84 by [(35)S]GTPγS binding to capric acid and diindolylmethane. The mutations L100D `Ballesteros-Weinstein numbering: 3.32), F101Y (3.33) and N104Q (3.36) in the transmembrane helix III and N357D (7.39) in the transmembrane helix VII resulted in reduced capric acid activity but maintained the diindolylmethane responses. Y186F (5.46) and Y186H (5.46) mutations had no characteristic effect on capric acid but with diindolylmethane they significantly affected the G protein activation efficiency. The L100D (3.32) mutant responded to decylamine, a fatty amine, instead of a natural agonist, the fatty acid capric acid, suggesting that we have identified a mutated G protein-coupled receptor-artificial ligand pairing. Our molecular model provides an explanation for these results and interactions between GPR84 and capric acid. Further, from the results of a double stimulation assay, we concluded that diindolylmethane was a positive allosteric modulator for GPR84. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

GPR81, a Cell-Surface Receptor for Lactate, Regulates Intestinal Homeostasis and Protects Mice from Experimental Colitis.

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Ranganathan, Punithavathi; Shanmugam, Arulkumaran; Swafford, Daniel; Suryawanshi, Amol; Bhattacharjee, Pushpak; Hussein, Mohamed S; Koni, Pandelakis A; Prasad, Puttur D; Kurago, Zoya B; Thangaraju, Muthusamy; Ganapathy, Vadivel; Manicassamy, Santhakumar

2018-03-01

At mucosal sites such as the intestine, the immune system launches robust immunity against invading pathogens while maintaining a state of tolerance to commensal flora and ingested food Ags. The molecular mechanisms underlying this phenomenon remain poorly understood. In this study, we report that signaling by GPR81, a receptor for lactate, in colonic dendritic cells and macrophages plays an important role in suppressing colonic inflammation and restoring colonic homeostasis. Genetic deletion of GPR81 in mice led to increased Th1/Th17 cell differentiation and reduced regulatory T cell differentiation, resulting in enhanced susceptibility to colonic inflammation. This was due to increased production of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and decreased expression of immune regulatory factors (IL-10, retinoic acid, and IDO) by intestinal APCs lacking GPR81. Consistent with these findings, pharmacological activation of GPR81 decreased inflammatory cytokine expression and ameliorated colonic inflammation. Taken together, these findings identify a new and important role for the GPR81 signaling pathway in regulating immune tolerance and colonic inflammation. Thus, manipulation of the GPR81 pathway could provide novel opportunities for enhancing regulatory responses and treating colonic inflammation. Copyright © 2018 by The American Association of Immunologists, Inc.

IFNγ Induces DNA Methylation-Silenced GPR109A Expression via pSTAT1/p300 and H3K18 Acetylation in Colon Cancer.

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Bardhan, Kankana; Paschall, Amy V; Yang, Dafeng; Chen, May R; Simon, Priscilla S; Bhutia, Yangzom D; Martin, Pamela M; Thangaraju, Muthusamy; Browning, Darren D; Ganapathy, Vadivel; Heaton, Christopher M; Gu, Keni; Lee, Jeffrey R; Liu, Kebin

2015-07-01

Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A have been the subject of extensive studies; however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wild-type mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoter to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoter to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer, and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development. ©2015 American Association for Cancer Research.

IFNγ induces DNA methylation-silenced GPR109A expression via pSTAT1/p300 and H3K18 acetylation in colon cancer

Science.gov (United States)

Bardhan, Kankana; Paschall, Amy V.; Yang, Dafeng; Chen, May R.; Simon, Priscilla S.; Bhutia, Yangzom; Martin, Pamela M.; Thangaraju, Muthusamy; Browning, Darren D.; Ganapathy, Vadivel; Heaton, Christopher M.; Gu, Keni; Lee, Jeffrey R.; Liu, Kebin

2015-01-01

Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A has been the subject of extensive studies, however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wildtype mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoters to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoters to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development. PMID:25735954

A novel G protein-coupled receptor of Schistosoma mansoni (SmGPR-3 is activated by dopamine and is widely expressed in the nervous system.

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Fouad El-Shehabi

Full Text Available Schistosomes have a well developed nervous system that coordinates virtually every activity of the parasite and therefore is considered to be a promising target for chemotherapeutic intervention. Neurotransmitter receptors, in particular those involved in neuromuscular control, are proven drug targets in other helminths but very few of these receptors have been identified in schistosomes and little is known about their roles in the biology of the worm. Here we describe a novel Schistosoma mansoni G protein-coupled receptor (named SmGPR-3 that was cloned, expressed heterologously and shown to be activated by dopamine, a well established neurotransmitter of the schistosome nervous system. SmGPR-3 belongs to a new clade of "orphan" amine-like receptors that exist in schistosomes but not the mammalian host. Further analysis of the recombinant protein showed that SmGPR-3 can also be activated by other catecholamines, including the dopamine metabolite, epinine, and it has an unusual antagonist profile when compared to mammalian receptors. Confocal immunofluorescence experiments using a specific peptide antibody showed that SmGPR-3 is abundantly expressed in the nervous system of schistosomes, particularly in the main nerve cords and the peripheral innervation of the body wall muscles. In addition, we show that dopamine, epinine and other dopaminergic agents have strong effects on the motility of larval schistosomes in culture. Together, the results suggest that SmGPR-3 is an important neuronal receptor and is probably involved in the control of motor activity in schistosomes. We have conducted a first analysis of the structure of SmGPR-3 by means of homology modeling and virtual ligand-docking simulations. This investigation has identified potentially important differences between SmGPR-3 and host dopamine receptors that could be exploited to develop new, parasite-selective anti-schistosomal drugs.

GPR 120: The Potential Target for Obesity Treatment.

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Tanagho, Peter A; Shohdy, Kyrillus S

2016-01-01

G protein coupled receptor 120 (GPR120) is a class of receptors in the gastrointestinal tract (GIT) that is implicated in nutrient sensing and body weight regulation. Functions of GPR120 are thought to be mediated by the release of a group of hormones known as incretins, such as glucagon like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). We have searched PubMed with the keywords "GPR120","GLP-1" and "obesity". Relevant studies were retrieved and included in the review. Recently, many exogenous compounds have been investigated in their role in the release of GLP-1 and in causing weight loss in obese rats. However, some results question the putative role of GPR120 in metabolic homeostasis. Herein, we evaluate the potential use of GPR120 as a target receptor in obesity and found it to be ubiquitous throughout the GIT, with various functions in each site. In order to find the optimal drug, the role of GPR120 in each site needs to be defined and selectivity of the potential drug needs to be studied to ensure the success of this growing line of obesity management.

Clopidogrel (Plavix®), a P2Y(12) receptor antagonist, inhibits bone cell function in vitro and decreases trabecular bone in vivo

DEFF Research Database (Denmark)

Syberg, Susanne; Brandao-Burch, Andrea; Patel, Jessal J

2012-01-01

Clopidogrel (Plavix®), a selective P2Y(12) receptor antagonist, is widely prescribed to reduce the risk of heart attack and stroke and acts via the inhibition of platelet aggregation. Accumulating evidence now suggests that extracellular nucleotides, signalling through P2 receptors, play...... a significant role in bone, modulating both osteoblast and osteoclast function. In this study, we investigated the effects of clopidogrel treatment on (1) bone cell formation, differentiation and activity in vitro; and, (2) trabecular and cortical bone parameters in vivo. P2Y(12) receptor expression...

PPADS: an antagonist at endothelial P2Y-purinoceptors but not P2U-purinoceptors.

Science.gov (United States)

Brown, C; Tanna, B; Boarder, M R

1995-11-01

1. Bovine aortic endothelial (BAE) cells contain two co-existing receptors for extracellular ATP, the P2Y and P2U-purinoceptors. Here we have determined whether the proposed P2X-purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS) could distinguish between these two receptor subtypes. 2. Cells labelled with myo-[2-3H]-inositol were stimulated with increasing concentrations of either the P2Y-agonist, 2MeSATP, or the P2U-agonist, UTP in the absence or presence of 30 microM PPADS. The accumulation of total [3H]-inositol (poly)phosphates mediated by 2MeSATP was markedly attenuated by PPADS, whereas the response to UTP was not significantly affected. 3. Stimulation of BAE cells with increasing concentrations of ATP showed a reduced response in the presence of 10 microM PPADS, but this effect of the antagonist was not significant. By contrast, inhibition of the response to ADP was profound and highly significant. 4. These observations show that PPADS is not a selective P2X-purinoceptor antagonist, but is able to distinguish between P2Y- and P2YU-purinoceptors in BAE cells, and indicate that this compound may provide a useful tool in the study of multiple subtypes of P2-purinoceptors. Furthermore the results are consistent with the hypothesis that ATP interacts with both receptor subtypes, but that the action of ADP is primarily at the P2Y-purinoceptor in these endothelial cells.

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

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Liu MJ

2017-02-01

Full Text Available Mingjuan Liu,1 Ming Yao,1,2 Hanqi Wang,1 Longsheng Xu,1 Ying Zheng,1 Bing Huang,1 Huadong Ni,1 Shijie Xu,1 Xuyan Zhou,1 Qingquan Lian2 1Department of Anesthesiology and Pain Medicine, The First Hospital of Jiaxing, The First Affiliated Hospital of Jiaxing University, Jiaxing, 2Department of Anesthesiology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, People’s Republic of China 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. Keywords: P2Y12 receptor, cancer-induced bone pain, p38MAPK pathway, cytokines

The expression of GPR109A, NF-kB and IL-1β in peripheral blood leukocytes from patients with type 2 diabetes.

Science.gov (United States)

Liu, Fengxiu; Fu, Yucai; Wei, Chiju; Chen, Yongru; Ma, Shuhua; Xu, Wencan

2014-01-01

This study was designed to explore the association between the G protein-coupled receptor 109A (GPR109A) expression in peripheral blood leukocytes (PBLs) and type 2 diabetes (T2DM) and to discuss the regulation of inflammatory factors by GPR109A signaling. GPR109A signaling has been confirmed to be associated with homeostasis of glucose/lipid metabolism, but the role of signaling in T2DM is still poorly understood. Peripheral blood samples and biochemical data were collected from healthy individuals (normal controls) and T2DM patients. Immunocytochemical staining was used to detect the expression of GPR109A in PBLs. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure mRNA levels of GPR109A, NF-κB, and IL-1β in PBLs. Immunocytochemical staining showed that the GPR109A protein is localized in the nucleus and cytoplasm of granulocytes, monocytes, and lymphocytes. RT-PCR showed that mRNA levels of GPR109A, NF-κB, and IL-1β were higher in the T2DM group than in the control group (P<0.05). Correlation analysis showed a positive correlation both between GPR109A/NF-κB (r=0.376, P<0.05), and GPR109A/IL-1β (r=501, P<0.05) and between GPR109A and fasting plasma glucose (FPG) (r=0.179, P<0.05) and NF-κB /FPG (r=0.358, p<0.05). Our results suggest that GPR109A signaling is associated with T2DM, playing a role in regulation of the inflammatory cytokines. © 2014 by the Association of Clinical Scientists, Inc.

Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride.

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Brunner, Kurt; Omann, Markus; Pucher, Marion E; Delic, Marizela; Lehner, Sylvia M; Domnanich, Patrick; Kratochwill, Klaus; Druzhinina, Irina; Denk, Dagmar; Zeilinger, Susanne

2008-12-01

Galpha subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

Role of P2 Receptors as Modulators of Rat Eosinophil Recruitment in Allergic Inflammation.

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Anael Viana Pinto Alberto

Full Text Available ATP and other nucleotides are released from cells through regulated pathways or following the loss of plasma membrane integrity. Once outside the cell, these compounds can activate P2 receptors: P2X ionotropic receptors and G protein-coupled P2Y receptors. Eosinophils represent major effector cells in the allergic inflammatory response and they are, in fact, associated with several physiological and pathological processes. Here we investigate the expression of P2 receptors and roles of those receptors in murine eosinophils. In this context, our first step was to investigate the expression and functionality of the P2X receptors by patch clamping, our results showed a potency ranking order of ATP>ATPγS> 2meSATP> ADP> αβmeATP> βγmeATP>BzATP> UTP> UDP>cAMP. This data suggest the presence of P2X1, P2X2 and P2X7. Next we evaluate by microfluorimetry the expression of P2Y receptors, our results based in the ranking order of potency (UTP>ATPγS> ATP > UDP> ADP >2meSATP > αβmeATP suggests the presence of P2Y2, P2Y4, P2Y6 and P2Y11. Moreover, we confirmed our findings by immunofluorescence assays. We also did chemotaxis assays to verify whether nucleotides could induce migration. After 1 or 2 hours of incubation, ATP increased migration of eosinophils, as well as ATPγS, a less hydrolysable analogue of ATP, while suramin a P2 blocker abolished migration. In keeping with this idea, we tested whether these receptors are implicated in the migration of eosinophils to an inflammation site in vivo, using a model of rat allergic pleurisy. In fact, migration of eosinophils has increased when ATP or ATPγS were applied in the pleural cavity, and once more suramin blocked this effect. We have demonstrated that rat eosinophils express P2X and P2Y receptors. In addition, the activation of P2 receptors can increase migration of eosinophils in vitro and in vivo, an effect blocked by suramin.

G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane

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Funakoshi, Takeshi; Yanai, Akie; Shinoda, Koh; Kawano, Michio M.; Mizukami, Yoichi

2006-01-01

Recently, GPR30 was reported to be a novel estrogen receptor; however, its intracellular localization has remained controversial. To investigate the intracellular localization of GPR30 in vivo, we produced four kinds of polyclonal antibodies for distinct epitopes on GPR30. Immunocytochemical observations using anti-GPR30 antibody and anti-FLAG antibody show that FLAG-GPR30 localizes to the plasma membrane 24 h after transfection. Treatment with estrogen (17β-estradiol or E2) causes an elevation in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) within 10 s in HeLa cells expressing FLAG-GPR30. In addition, E2 induces the translocation of GPR30 from the plasma membrane to the cytoplasm by 1 h after stimulation. Immunohistochemical analysis shows that GPR30 exists on the cell surface of CA2 pyramidal neuronal cells. The images on transmission electron microscopy show that GPR30 is localized to a particular region associated with the plasma membranes of the pyramidal cells. These data indicate that GPR30, a transmembrane receptor for estrogen, is localized to the plasma membrane of CA2 pyramidal neuronal cells of the hippocampus in rat brain

Immune malfunction in the GPR39 zinc receptor of knockout mice

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Młyniec, Katarzyna; Trojan, Ewa; Ślusarczyk, Joanna

2016-01-01

Depression is a serious psychiatric disorder affecting not only the monaminergic, glutamatergic, and GABAergic neurosystems, but also the immune system. Patients suffering from depression show disturbance in the immune parameters as well as increased susceptibility to infections. Zinc is well known...... as an anti-inflammatory agent, and its link with depression has been proved, zinc deficiency causing depression- and anxiety-like behavior with immune malfunction. It has been discovered that trace-element zinc acts as a neurotransmitter in the central nervous system via zinc receptor GPR39. In this study we...

Characterization of the contractile P2Y14 receptor in mouse coronary and cerebral arteries

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Haanes, Kristian Agmund; Edvinsson, Lars

2014-01-01

values and immunohistochemistry illustrated the strongest P2Y14 receptor expression in the basilar artery. In the presence of pertussis toxin, UDP-glucose inhibited contraction in coronary arteries and in the basilar artery it surprisingly caused relaxation. After organ culture of the coronary artery...

Interleukin 17A and Toll-like Receptor 4 in Patients with Arterial Hypertension.

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Simundic, Tihana; Jelakovic, Bojan; Dzumhur, Andrea; Turk, Tajana; Sahinovic, Ines; Dobrosevic, Blazenka; Takac, Boris; Barbic, Jerko

2017-01-01

Immune responses are involved in arterial hypertension. An observational cross-sectional case control study was conducted to estimate the association between Toll-like receptor 4 (TLR4) expression and interleukin (IL)-17A serum levels in patients with controlled and non-controlled hypertension. We have enrolled 105 non-complicated otherwise healthy hypertensive patients: 53 with well-controlled blood pressure and 52 non-controlled. TLR4 peripheral monocytes expression and serum IL-17A levels were determined by flow cytometry and ELISA, respectively. Non-controlled patients exhibited higher TLR4 expression than well-controlled (25.60 vs. 21.99, P=0.011). TLR4 expression was lower in well-controlled patients who were prescribed beta blockers (18.9 vs. 22.6, P=0.005) and IL-17A concentration was higher in patients using diuretics in either group (1.41 vs. 2.01 pg/ml, Phypertension duration was observed in non-controlled patients (Spearman correlation coefficient . ρ=0.566, Phypertension duration and TLR4 expression (ρ=0.322, P=0.020). Arterial hypertension stimulates the immune response regardless of blood pressure regulation status. Prolonged hypertension influences peripheral monocyte TLR4 expression and IL-17A serum levels. Anti-hypertensive drugs have different immunomodulatory effects: diuretics are associated with higher IL-17A concentration and beta-blockers with lower TLR4 expression. © 2017 The Author(s)Published by S. Karger AG, Basel.

The selective antagonism of P2X7 and P2Y1 receptors prevents synaptic failure and affects cell proliferation induced by oxygen and glucose deprivation in rat dentate gyrus.

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Giovanna Maraula

Full Text Available Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS, including dentate gyrus (DG. The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD in acute rat hippocampal slices and to investigate the contribution of P2X7 and P2Y1 receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD. Application of MRS2179 (selective antagonist of P2Y1 receptor and BBG (selective antagonist of P2X7 receptor, before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ of slices prepared from rats treated with 5-Bromo-2'-deoxyuridine (BrdU were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX. The number of BrdU+ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU+ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y1 antagonist reduced the number of BrdU+ cells at this time. The data demonstrate that P2X7 and P2Y1 activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y1 receptors might play an additional and different role in promoting cell proliferation and maturation in the DG.

Role of GPR30 in estrogen-induced prostate epithelial apoptosis and benign prostatic hyperplasia.

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Yang, Deng-Liang; Xu, Jia-Wen; Zhu, Jian-Guo; Zhang, Yi-Lin; Xu, Jian-Bang; Sun, Qing; Cao, Xiao-Nian; Zuo, Wu-Lin; Xu, Ruo-Shui; Huang, Jie-Hong; Jiang, Fu-Neng; Zhuo, Yang-Jia; Xiao, Bai-Quan; Liu, Yun-Zhong; Yuan, Dong-Bo; Sun, Zhao-Lin; He, Hui-Chan; Lun, Zhao-Rong; Zhong, Wei-De; Zhou, Wen-Liang

2017-06-03

Several studies have implicated estrogen and the estrogen receptor (ER) in the pathogenesis of benign prostatic hyperplasia (BPH); however, the mechanism underlying this effect remains elusive. In the present study, we demonstrated that estrogen (17β-estradiol, or E2)-induced activation of the G protein-coupled receptor 30 (GPR30) triggered Ca 2+ release from the endoplasmic reticulum, increased the mitochondrial Ca 2+ concentration, and thus induced prostate epithelial cell (PEC) apoptosis. Both E2 and the GPR30-specific agonist G1 induced a transient intracellular Ca 2+ release in PECs via the phospholipase C (PLC)-inositol 1, 4, 5-triphosphate (IP 3 ) pathway, and this was abolished by treatment with the GPR30 antagonist G15. The release of cytochrome c and activation of caspase-3 in response to GPR30 activation were observed. Data generated from the analysis of animal models and human clinical samples indicate that treatment with the GPR30 agonist relieves testosterone propionate (TP)-induced prostatic epithelial hyperplasia, and that the abundance of GPR30 is negatively associated with prostate volume. On the basis of these results, we propose a novel regulatory mechanism whereby estrogen induces the apoptosis of PECs via GPR30 activation. Inhibition of this activation is predicted to lead to abnormal PEC accumulation, and to thereby contribute to BPH pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural and Molecular Modeling Features of P2X Receptors

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Luiz Anastacio Alves

2014-03-01

Full Text Available Currently, adenosine 5'-triphosphate (ATP is recognized as the extracellular messenger that acts through P2 receptors. P2 receptors are divided into two subtypes: P2Y metabotropic receptors and P2X ionotropic receptors, both of which are found in virtually all mammalian cell types studied. Due to the difficulty in studying membrane protein structures by X-ray crystallography or NMR techniques, there is little information about these structures available in the literature. Two structures of the P2X4 receptor in truncated form have been solved by crystallography. Molecular modeling has proven to be an excellent tool for studying ionotropic receptors. Recently, modeling studies carried out on P2X receptors have advanced our knowledge of the P2X receptor structure-function relationships. This review presents a brief history of ion channel structural studies and shows how modeling approaches can be used to address relevant questions about P2X receptors.

Bidirectional GPR119 agonism requires peptide YY and glucose for activity in mouse and human colon mucosa

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Tough, Iain R; Forbes, Sarah; Herzog, Herbert

2018-01-01

motility in wild-type (WT), GPR119-/- and PYY-/- mice.The water-soluble GPR119 agonist, AR440006 (that cannot traverse epithelial tight-junctions) elicited responses when added apically or basolaterally in mouse and human colonic mucosas. In both species, GPR119 responses were PYY, Y1 receptor...

Activation of the omega-3 fatty acid receptor GPR120 mediates anti-inflammatory actions in immortalized hypothalamic neurons.

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Wellhauser, Leigh; Belsham, Denise D

2014-03-27

Overnutrition and the ensuing hypothalamic inflammation is a major perpetuating factor in the development of metabolic diseases, such as obesity and diabetes. Inflamed neurons of the CNS fail to properly regulate energy homeostasis leading to pathogenic changes in glucose handling, feeding, and body weight. Hypothalamic neurons are particularly sensitive to pro-inflammatory signals derived locally and peripherally, and it is these neurons that become inflamed first upon high fat feeding. Given the prevalence of metabolic disease, efforts are underway to identify therapeutic targets for this inflammatory state. At least in the periphery, omega-3 fatty acids and their receptor, G-protein coupled receptor 120 (GPR120), have emerged as putative targets. The role for GPR120 in the hypothalamus or CNS in general is poorly understood. Here we introduce a novel, immortalized cell model derived from the rat hypothalamus, rHypoE-7, to study GPR120 activation at the level of the individual neuron. Gene expression levels of pro-inflammatory cytokines were studied by quantitative reverse transcriptase-PCR (qRT-PCR) upon exposure to tumor necrosis factor α (TNFα) treatment in the presence or absence of the polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA). Signal transduction pathway involvement was also studied using phospho-specific antibodies to key proteins by western blot analysis. Importantly, rHypoE-7 cells exhibit a transcriptional and translational inflammatory response upon exposure to TNFα and express abundant levels of GPR120, which is functionally responsive to DHA. DHA pretreatment prevents the inflammatory state and this effect was inhibited by the reduction of endogenous GPR120 levels. GPR120 activates both AKT (protein kinase b) and ERK (extracellular signal-regulated kinase); however, the anti-inflammatory action of this omega-3 fatty acid (FA) receptor is AKT- and ERK-independent and likely involves the GPR120-transforming growth factor

Synthesis and preliminary evaluation of [3H]PSB-0413, a selective antagonist radioligand for platelet P2Y12 receptors.

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El-Tayeb, Ali; Griessmeier, Kerstin J; Müller, Christa E

2005-12-15

The selective antagonist radioligand [(3)H]2-propylthioadenosine-5'-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([(3)H]PSB-0413) was prepared by catalytic hydrogenation of its propargyl precursor with a high specific radioactivity of 74Ci/mmol. In preliminary saturation binding studies, [(3)H]PSB-0413 showed high affinity for platelet P2Y(12) receptors with a K(D) value of 4.57nM. Human platelets had a high density of P2Y(12) receptors exhibiting a B(max) value of 7.66pmol/mg of protein.

P2X receptor-ion channels in the inflammatory response in adipose tissue and pancreas-potential triggers in onset of type 2 diabetes?

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Novak, Ivana; Solini, Anna

2018-01-01

-cell and adipose tissue. In the former, P2Y and possibly some P2X receptors-ion channels regulate insulin secretion, but it is still debated whether excessive ATP can via P2X receptors impair β-cell function directly or whether cell damage is due to an excessive systemic release of cytokines. In human adipocytes......, the P2X7 receptor promotes the release of inflammatory cytokines, at least in part via inflammasome activation, likely contributing to systemic insulin resistance. This receptor-inflammasome system is also strongly activated in macrophages infiltrating both pancreas and adipose tissue, mediating...

Model-Based Discovery of Synthetic Agonists for the Zn2+-Sensing G-Protein-Coupled Receptor 39 (GPR39) Reveals Novel Biological Functions

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Frimurer, Thomas M.; Mende, Franziska; Graae, Anne-Sofie

2017-01-01

binding to other receptors with similar binding pockets to select iterative series of mini-libraries. These libraries were cherry-picked from all com. available synthetic compds. A total of only 520 compds. were tested in vitro, making this method broadly applicable for tool compd. development. The compds....... of the initial library were inactive when tested alone, but lead compds. were identified using Zn2 as an allosteric enhancer. Highly selective, highly potent Zn2-independent GPR39 agonists were found in subsequent mini-libraries. These agonists identified GPR39 as a novel regulator of gastric somatostatin...

Inflammatory changes in adipose tissue enhance expression of GPR84, a medium-chain fatty acid receptor: TNFα enhances GPR84 expression in adipocytes.

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Nagasaki, Hiroshi; Kondo, Takaaki; Fuchigami, Masahiro; Hashimoto, Hiroyuki; Sugimura, Yoshihisa; Ozaki, Nobuaki; Arima, Hiroshi; Ota, Akira; Oiso, Yutaka; Hamada, Yoji

2012-02-17

In this study we aimed to identify the physiological roles of G protein-coupled receptor 84 (GPR84) in adipose tissue, together with medium-chain fatty acids (MCFAs), the specific ligands for GPR84. In mice, high-fat diet up-regulated GPR84 expression in fat pads. In 3T3-L1 adipocytes, co-culture with a macrophage cell line, RAW264, or TNFα remarkably enhanced GPR84 expression. In the presence of TNFα, MCFAs down-regulated adiponectin mRNA expression in 3T3-L1 adipocytes. Taken together, our results suggest that GPR84 emerges in adipocytes in response to TNFα from infiltrating macrophages and exacerbates the vicious cycle between adiposity and diabesity. Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

Fluoxetine, 17-β estradiol or folic acid combined with intra-lateral septal infusions of neuropeptide Y produced antidepressant-like actions in ovariectomized rats forced to swim.

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Molina-Hernández, Miguel; Téllez-Alcántara, N Patricia

2011-12-01

Folic acid is antidepressant, either alone or combined with several antidepressant drugs. However, the antidepressant-like actions of folic acid combined with intra-lateral septal (LSN) infusions of neuropeptide Y (NPY) in the forced swimming test (FST) have not been tested before. Thus, systemic injections of fluoxetine (20.0mg/kg, Pfluoxetine (15.0 mg/kg, P<0.05; s.c.) combined with subthreshold doses of NPY (2.5 μg/rat, P<0.05; intra-LSN) and these combinations produced antidepressant-like actions; which were canceled by BIBP 3226 (a NPY-Y1 receptor antagonist). It is concluded that folic acid produced antidepressant-like effects probably through the participation of the NPY Y1 receptors found in the lateral septal nuclei. Copyright © 2011 Elsevier Inc. All rights reserved.

Involvement of the Niacin Receptor GPR109a in the LocalControl of Glucose Uptake in Small Intestine of Type 2Diabetic Mice

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Tung Po Wong

2015-09-01

Full Text Available Niacin is a popular nutritional supplement known to reduce the risk of cardiovascular diseases by enhancing high-density lipoprotein levels. Despite such health benefits, niacin impairs fasting blood glucose. In type 2 diabetes (T2DM, an increase in jejunal glucose transport has been well documented; however, this is intriguingly decreased during niacin deficient state. In this regard, the role of the niacin receptor GPR109a in T2DM jejunal glucose transport remains unknown. Therefore, the effects of diabetes and high-glucose conditions on GPR109a expression were studied using jejunal enterocytes of 10-week-old m+/db and db/db mice, as well as Caco-2 cells cultured in 5.6 or 25.2 mM glucose concentrations. Expression of the target genes and proteins were quantified using real-time polymerase chain reaction (RT-PCR and Western blotting. Glucose uptake in Caco-2 cells and everted mouse jejunum was measured using liquid scintillation counting. 10-week T2DM increased mRNA and protein expression levels of GPR109a in jejunum by 195.0% and 75.9%, respectively, as compared with the respective m+/db control; high-glucose concentrations increased mRNA and protein expression of GPR109a in Caco-2 cells by 130.2% and 69.0%, respectively, which was also confirmed by immunohistochemistry. In conclusion, the enhanced GPR109a expression in jejunal enterocytes of T2DM mice and high-glucose treated Caco-2 cells suggests that GPR109a is involved in elevating intestinal glucose transport observed in diabetes.

The P2Y12 Receptor Antagonist Ticagrelor Reduces Lysosomal pH and Autofluorescence in Retinal Pigmented Epithelial Cells From the ABCA4-/- Mouse Model of Retinal Degeneration

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Wennan Lu

2018-04-01

Full Text Available The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent lysosome-associated organelles of the retinal pigmented epithelial (RPE cells, and may be related to early stages of age-related macular degeneration. While lysosomal enzymes degrade material optimally at acidic pH levels, lysosomal pH is elevated in RPE cells from the ABCA4-/- mouse model of Stargardt’s disease, an early onset retinal degeneration. Lowering lysosomal pH through cAMP-dependent pathways decreases accumulation of autofluorescent material in RPE cells in vitro, but identification of an appropriate receptor is crucial for manipulating this pathway in vivo. As the P2Y12 receptor for ADP is coupled to the inhibitory Gi protein, we asked whether blocking the P2Y12 receptor with ticagrelor could restore lysosomal acidity and reduce autofluorescence in compromised RPE cells from ABCA4-/- mice. Oral delivery of ticagrelor giving rise to clinically relevant exposure lowered lysosomal pH in these RPE cells. Ticagrelor also partially reduced autofluorescence in the RPE cells of ABCA4-/- mice. In vitro studies in ARPE-19 cells using more specific antagonists AR-C69931 and AR-C66096 confirmed the importance of the P2Y12 receptor for lowering lysosomal pH and reducing autofluorescence. These observations identify P2Y12 receptor blockade as a potential target to lower lysosomal pH and clear lysosomal waste in RPE cells.

Sustained Release of Prostaglandin E2 in Fibroblasts Expressing Ectopically Cyclooxygenase 2 Impairs P2Y-Dependent Ca2+-Mobilization

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María Pimentel-Santillana

2014-01-01

Full Text Available The nucleotide uridine trisphosphate (UTP released to the extracellular milieu acts as a signaling molecule via activation of specific pyrimidine receptors (P2Y. P2Y receptors are G protein-coupled receptors expressed in many cell types. These receptors mediate several cell responses and they are involved in intracellular calcium mobilization. We investigated the role of the prostanoid PGE2 in P2Y signaling in mouse embryonic fibroblasts (MEFs, since these cells are involved in different ontogenic and physiopathological processes, among them is tissue repair following proinflammatory activation. Interestingly, Ca2+-mobilization induced by UTP-dependent P2Y activation was reduced by PGE2 when this prostanoid was produced by MEFs transfected with COX-2 or when PGE2 was added exogenously to the culture medium. This Ca2+-mobilization was important for the activation of different metabolic pathways in fibroblasts. Moreover, inhibition of COX-2 with selective coxibs prevented UTP-dependent P2Y activation in these cells. The inhibition of P2Y responses by PGE2 involves the activation of PKCs and PKD, a response that can be suppressed after pharmacological inhibition of these protein kinases. In addition to this, PGE2 reduces the fibroblast migration induced by P2Y-agonists such as UTP. Taken together, these data demonstrate that PGE2 is involved in the regulation of P2Y signaling in these cells.

The G-protein coupled receptor, GPR84 regulates IL-4 production by T lymphocytes in response to CD3 crosslinking.

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Venkataraman, Chandrasekar; Kuo, Frederick

2005-11-15

The orphan G-protein coupled receptor, GPR84 is highly expressed in the bone marrow, and in splenic T cells and B cells. In this study, GPR84-deficient mice were generated to understand the biological function of this orphan receptor. The proliferation of T and B cells in response to various mitogens was normal in GPR84-deficient mice. Interestingly, primary stimulation of T cells with anti-CD3 resulted in increased IL-4 but not IL-2 or IFN-gamma production in GPR84(-/-) mice compared to wild-type mice. Augmented IL-4 production in GPR84-deficient T cells was not related to increased frequency of IL-4-secreting cells in response to anti-CD3 stimulation. In fact, stimulation with anti-CD3 and anti-CD28 resulted in increased levels of IL-4 but not IFN-gamma steady-state mRNA in GPR84(-/-) T cells. In addition, Th2 effector cells generated in vitro from GPR84(-/-) mice produced higher levels of IL-4, IL-5 and IL-13 compared to wild-type mice. However, there was no detectable difference in the extent of IL-4 and IL-5 production between the two groups of mice in response to antigen stimulation of spleen cells, isolated from mice previously immunized with OVA in alum. These studies reveal a novel role for GPR84 in regulating early IL-4 gene expression in activated T cells.

Neuro-psychopharmacological perspective of Orphan receptors of Rhodopsin (class A) family of G protein-coupled receptors.

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Khan, Muhammad Zahid; He, Ling

2017-04-01

In the central nervous system (CNS), G protein-coupled receptors (GPCRs) are the most fruitful targets for neuropsychopharmacological drug development. Rhodopsin (class A) is the most studied class of GPCR and includes orphan receptors for which the endogenous ligand is not known or is unclear. Characterization of orphan GPCRs has proven to be challenging, and the production pace of GPCR-based drugs has been incredibly slow. Determination of the functions of these receptors may provide unexpected insight into physiological and neuropathological processes. Advances in various methods and techniques to investigate orphan receptors including in situ hybridization and knockdown/knockout (KD/KO) showed extensive expression of these receptors in the mammalian brain and unmasked their physiological and neuropathological roles. Due to these rapid progress and development, orphan GPCRs are rising as a new and promising class of drug targets for neurodegenerative diseases and psychiatric disorders. This review presents a neuropsychopharmacological perspective of 26 orphan receptors of rhodopsin (class A) family, namely GPR3, GPR6, GPR12, GPR17, GPR26, GPR35, GPR39, GPR48, GPR49, GPR50, GPR52, GPR55, GPR61, GPR62, GPR63, GPR68, GPR75, GPR78, GPR83, GPR84, GPR85, GPR88, GPR153, GPR162, GPR171, and TAAR6. We discussed the expression of these receptors in mammalian brain and their physiological roles. Furthermore, we have briefly highlighted their roles in neurodegenerative diseases and psychiatric disorders including Alzheimer's disease, Parkinson's disease, neuroinflammation, inflammatory pain, bipolar and schizophrenic disorders, epilepsy, anxiety, and depression.

The adhesion G protein-coupled receptor G2 (ADGRG2/GPR64) constitutively activates SRE and NFκB and is involved in cell adhesion and migration

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Cornelia Peeters, Miriam; Fokkelman, Michiel; Boogaard, Bob

2015-01-01

Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which...

PPAR-γ activation increases insulin secretion through the up-regulation of the free fatty acid receptor GPR40 in pancreatic β-cells.

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Hyo-Sup Kim

Full Text Available BACKGROUND: It has been reported that peroxisome proliferator-activated receptor (PPAR-γ and their synthetic ligands have direct effects on pancreatic β-cells. We investigated whether PPAR-γ activation stimulates insulin secretion through the up-regulation of GPR40 in pancreatic β-cells. METHODS: Rat insulinoma INS-1 cells and primary rat islets were treated with rosiglitazone (RGZ and/or adenoviral PPAR-γ overexpression. OLETF rats were treated with RGZ. RESULTS: PPAR-γ activation with RGZ and/or adenoviral PPAR-γ overexpression increased free fatty acid (FFA receptor GPR40 expression, and increased insulin secretion and intracellular calcium mobilization, and was blocked by the PLC inhibitors, GPR40 RNA interference, and GLUT2 RNA interference. As a downstream signaling pathway of intracellular calcium mobilization, the phosphorylated levels of CaMKII and CREB, and the downstream IRS-2 and phospho-Akt were significantly increased. Despite of insulin receptor RNA interference, the levels of IRS-2 and phospho-Akt was still maintained with PPAR-γ activation. In addition, the β-cell specific gene expression, including Pdx-1 and FoxA2, increased in a GPR40- and GLUT2-dependent manner. The levels of GPR40, phosphorylated CaMKII and CREB, and β-cell specific genes induced by RGZ were blocked by GW9662, a PPAR-γ antagonist. Finally, PPAR-γ activation up-regulated β-cell gene expressions through FoxO1 nuclear exclusion, independent of the insulin signaling pathway. Based on immunohistochemical staining, the GLUT2, IRS-2, Pdx-1, and GPR40 were more strongly expressed in islets from RGZ-treated OLETF rats compared to control islets. CONCLUSION: These observations suggest that PPAR-γ activation with RGZ and/or adenoviral overexpression increased intracellular calcium mobilization, insulin secretion, and β-cell gene expression through GPR40 and GLUT2 gene up-regulation.

The zinc sensing receptor, ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon.

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Cohen, L; Sekler, I; Hershfinkel, M

2014-06-26

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn(2+) are not well understood. Here, we determined a role of the colonocytic Zn(2+) sensing receptor, ZnR/GPR39, in mediating Zn(2+)-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn(2+)-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn(2+) by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting

Development of [3H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([3H]PSB-12150): A Useful Tool for Studying GPR17

Science.gov (United States)

2014-01-01

The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [3H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities. PMID:24900835

Development of [(3)H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([(3)H]PSB-12150): A Useful Tool for Studying GPR17.

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Köse, Meryem; Ritter, Kirsten; Thiemke, Katharina; Gillard, Michel; Kostenis, Evi; Müller, Christa E

2014-04-10

The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [(3)H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities.

Expression of the long-chain fatty acid receptor GPR120 in the gonadotropes of the mouse anterior pituitary gland.

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Moriyama, Ryutaro; Deura, Chikaya; Imoto, Shingo; Nose, Kazuhiro; Fukushima, Nobuyuki

2015-01-01

G-protein-coupled receptor 120 (GPR120) has been known to be a receptor of long-chain fatty acids. Here, we investigated GPR120 expression in the mouse pituitary gland via real-time PCR, in situ hybridization, and immunohistochemistry. GPR120 mRNA was abundantly expressed in the pituitary gland of ad-lib fed animals. In situ hybridization and immunohistochemistry revealed GPR120 expression in the gonadotropes of the anterior pituitary gland, but not in thyrotropes, somatotropes, lactotropes, corticotropes, melanotropes, and the posterior pituitary gland. Furthermore, 24 h of fasting induced an increase in GPR120 mRNA expression in the pituitary gland. These results demonstrate that GPR120 in mouse pituitary gonadotropes is upregulated by fasting and that it may play a role in controlling gonadotropin secretion.

Expression and functional role of orphan receptor GPR158 in prostate cancer growth and progression.

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Nitin Patel

Full Text Available Prostate cancer (PCa is the second-leading cause of cancer-related mortality, after lung cancer, in men from developed countries. In its early stages, primary tumor growth is dependent on androgens, thus generally can be controlled by androgen deprivation therapy (ADT. Eventually however, the disease progresses to castration-resistant prostate cancer (CRPC, a lethal form in need of more effective treatments. G-protein coupled receptors (GPCRs comprise a large clan of cell surface proteins that have been implicated as therapeutic targets in PCa growth and progression. The findings reported here provide intriguing evidence of a role for the newly characterized glutamate family member GPR158 in PCa growth and progression. We found that GPR158 promotes PCa cell proliferation independent of androgen receptor (AR functionality and that this requires its localization in the nucleus of the cell. This suggests that GPR158 acts by mechanisms different from other GPCRs. GPR158 expression is stimulated by androgens and GPR158 stimulates AR expression, implying a potential to sensitize tumors to low androgen conditions during ADT via a positive feedback loop. Further, we found GPR158 expression correlates with a neuroendocrine (NE differentiation phenotype and promotes anchorage-independent colony formation implying a role for GPR158 in therapeutic progression and tumor formation. GPR158 expression was increased at the invading front of prostate tumors that formed in the genetically defined conditional Pten knockout mouse model, and co-localized with elevated AR expression in the cell nucleus. Kaplan-Meier analysis on a dataset from the Memorial Sloan Kettering cancer genome portal showed that increased GPR158 expression in tumors is associated with lower disease-free survival. Our findings strongly suggest that pharmaceuticals targeting GPR158 activities could represent a novel and innovative approach to the prevention and management of CRPC.

Y5 neuropeptide Y receptor overexpression in mice neither affects anxiety- and depression-like behaviours nor seizures but confers moderate hyperactivity

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Olesen, Mikkel V; Christiansen, Søren Hofman Oliveira; Gøtzsche, Casper René

2012-01-01

Neuropeptide Y (NPY) has been implicated in anxiolytic- and antidepressant-like behaviour as well as seizure-suppressant effects in rodents. Although these effects appear to be predominantly mediated via other NPY receptors (Y1 and/or Y2), several studies have also indicated a role for Y5 recepto...

Zinc Supplementation, via GPR39, Upregulates PKCζ to Protect Intestinal Barrier Integrity in Caco-2 Cells Challenged by Salmonella enterica Serovar Typhimurium.

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Shao, Yu-Xin; Lei, Zhao; Wolf, Patricia G; Gao, Yan; Guo, Yu-Ming; Zhang, Bing-Kun

2017-07-01

Background: Zinc has been shown to improve intestinal barrier function against Salmonella enterica serovar Typhimurium ( S. typhimurium ) infection, but the mechanisms involved in this process remain undefined. Objective: We aimed to explore the roles of G protein-coupled receptor (GPR)39 and protein kinase Cζ (PKCζ) in the regulation by zinc of intestinal barrier function. Methods: A Transwell Caco-2 monolayer was pretreated with 0, 50, or 100 μM Zn and then incubated with S. typhimurium for 0-6 h. Afterward, cells silenced by the small interfering RNA for GPR39 or PKCζ were pretreated with 100 μM Zn and incubated with S. typhimurium for 3 h. Finally, transepithelial electrical resistance (TEER), permeability, tight junction (TJ) proteins, and signaling molecules GPR39 and PKCζ were measured. Results: Compared with controls, S. typhimurium decreased TEER by 62.3-96.2% at 4-6 h ( P 0.1). Silencing GPR39 decreased ( P zinc-activated PKCζ and blocked ( P zinc on epithelial integrity. Furthermore, silencing PKCζ counteracted the protective effect of zinc on epithelial integrity but did not inhibit GPR39 ( P = 0.138). Conclusion: We demonstrated that zinc upregulates PKCζ by activating GPR39 to enhance the abundance of ZO-1, thereby improving epithelial integrity in S. typhimurium- infected Caco-2 cells. © 2017 American Society for Nutrition.

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

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Adam L Martin

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

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

Science.gov (United States)

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

2015-01-01

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

Neuropeptide Y Y1 receptor hippocampal overexpression via viral vectors is associated with modest anxiolytic-like and proconvulsant effects in mice

DEFF Research Database (Denmark)

Olesen, Mikkel V; Christiansen, Søren Hofman Oliveira; Gøtzsche, Casper René

2012-01-01

overexpression was found to be associated with modest anxiolytic-like effect in the open field and elevated plus maze tests, but no effect was seen on depression-like behavior using the tail suspension and forced swim tests. However, the rAAV-Y1 vector modestly aggravated kainate-induced seizures. These data...... in the hippocampus of adult mice and tested the animals in anxiety- and depression-like behavior. Hippocampal Y1 receptors have been suggested to mediate seizure-promoting effect, so the effects of rAAV-induced Y1 receptor overexpression were also tested in kainate-induced seizures. Y1 receptor transgene...

Agonists for G-protein-coupled receptor 84 (GPR84) alter cellular morphology and motility but do not induce pro-inflammatory responses in microglia.

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Wei, Li; Tokizane, Kyohei; Konishi, Hiroyuki; Yu, Hua-Rong; Kiyama, Hiroshi

2017-10-03

Several G-protein-coupled receptors (GPCRs) have been shown to be important signaling mediators between neurons and glia. In our previous screening for identification of nerve injury-associated GPCRs, G-protein-coupled receptor 84 (GPR84) mRNA showed the highest up-regulation by microglia after nerve injury. GPR84 is a pro-inflammatory receptor of macrophages in a neuropathic pain mouse model, yet its function in resident microglia in the central nervous system is poorly understood. We used endogenous, natural, and surrogate agonists for GPR84 (capric acid, embelin, and 6-OAU, respectively) and examined their effect on mouse primary cultured microglia in vitro. 6-n-Octylaminouracil (6-OAU), embelin, and capric acid rapidly induced membrane ruffling and motility in cultured microglia obtained from C57BL/6 mice, although these agonists failed to promote microglial pro-inflammatory cytokine expression. Concomitantly, 6-OAU suppressed forskolin-induced increase of cAMP in cultured microglia. Pertussis toxin, an inhibitor of Gi-coupled signaling, completely suppressed 6-OAU-induced microglial membrane ruffling and motility. In contrast, no 6-OAU-induced microglial membrane ruffling and motility was observed in microglia from DBA/2 mice, a mouse strain that does not express functional GPR84 protein due to endogenous nonsense mutation of the GPR84 gene. GPR84 mediated signaling causes microglial motility and membrane ruffling but does not promote pro-inflammatory responses. As GPR84 is a known receptor for medium-chain fatty acids, those released from damaged brain cells may be involved in the enhancement of microglial motility through GPR84 after neuronal injury.

Baicalein suppresses 17-β-estradiol-induced migration, adhesion and invasion of breast cancer cells via the G protein-coupled receptor 30 signaling pathway.

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Shang, Dandan; Li, Zheng; Zhu, Zhuxia; Chen, Huamei; Zhao, Lujun; Wang, Xudong; Chen, Yan

2015-04-01

Flavonoids are structurally similar to steroid hormones, particularly estrogens, and therefore have been studied for their potential effects on hormone-dependent cancers. Baicalein is the primary flavonoid derived from the root of Scutellaria baicalensis Georgi. In the present study, we investigated the effects of baicalein on 17β-estradiol (E2)-induced migration, adhesion and invasion of MCF-7 and SK-BR-3 breast cancer cells. The results demonstrated that baicalein suppressed E2-stimulated wound-healing migration and cell‑Matrigel adhesion, and ameliorated E2-promoted invasion across a Matrigel-coated Transwell membrane. Furthermore, baicalein interfered with E2-induced novel G protein-coupled estrogen receptor (GPR30)-related signaling, including a decrease in tyrosine phosphorylation of epidermal growth factor receptor (EGFR) as well as phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine kinase Akt, without affecting GPR30 expression. The results also showed that baicalein suppressed the expression of GPR30 target genes, cysteine-rich 61 (CYR61) and connective tissue growth factor (CTGF) induced by E2. Furthermore, baicalein prevented GPR30-related signaling activation and upregulation of CYR61 and CTGF mRNA levels induced by G1, a specific GPR 30 agonist. The results suggest that baicalein inhibits E2-induced migration, adhesion and invasion through interfering with GPR30 signaling pathway activation, which indicates that it may act as a therapeutic candidate for the treatment of GPR30-positive breast cancer metastasis.

GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

International Nuclear Information System (INIS)

Zhao, Zhuo; Wang, Hao; Lin, Marina; Groban, Leanne

2015-01-01

Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression

GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

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Zhao, Zhuo [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Department of Cardiology, Jinan Central Hospital, Affiliated with Shandong University, 105 Jiefang Road, Jinan, 250013 (China); Wang, Hao; Lin, Marina [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Groban, Leanne, E-mail: lgroban@wakehealth.edu [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Hypertension and Vascular Disease Center, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 (United States); Office of Women in Medicine and Science, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 (United States)

2015-03-27

Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression.

Transmission to interneurons is via slow excitatory synaptic potentials mediated by P2Y(1 receptors during descending inhibition in guinea-pig ileum.

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Peter D J Thornton

Full Text Available BACKGROUND: The nature of synaptic transmission at functionally distinct synapses in intestinal reflex pathways has not been fully identified. In this study, we investigated whether transmission between interneurons in the descending inhibitory pathway is mediated by a purine acting at P2Y receptors to produce slow excitatory synaptic potentials (EPSPs. METHODOLOGY/PRINCIPAL FINDINGS: Myenteric neurons from guinea-pig ileum in vitro were impaled with intracellular microelectrodes. Responses to distension 15 mm oral to the recording site, in a separately perfused stimulation chamber and to electrical stimulation of local nerve trunks were recorded. A subset of neurons, previously identified as nitric oxide synthase immunoreactive descending interneurons, responded to both stimuli with slow EPSPs that were reversibly abolished by a high concentration of PPADS (30 μM, P2 receptor antagonist. When added to the central chamber of a three chambered organ bath, PPADS concentration-dependently depressed transmission through that chamber of descending inhibitory reflexes, measured as inhibitory junction potentials in the circular muscle of the anal chamber. Reflexes evoked by distension in the central chamber were unaffected. A similar depression of transmission was seen when the specific P2Y(1 receptor antagonist MRS 2179 (10 μM was in the central chamber. Blocking either nicotinic receptors (hexamethonium 200 μM or 5-HT(3 receptors (granisetron 1 μM together with P2 receptors had no greater effect than blocking P2 receptors alone. CONCLUSIONS/SIGNIFICANCE: Slow EPSPs mediated by P2Y(1 receptors, play a primary role in transmission between descending interneurons of the inhibitory reflexes in the guinea-pig ileum. This is the first demonstration for a primary role of excitatory metabotropic receptors in physiological transmission at a functionally identified synapse.

Satellite glial cell P2Y12 receptor in the trigeminal ganglion is involved in lingual neuropathic pain mechanisms in rats

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Katagiri Ayano

2012-03-01

Full Text Available Abstract Background It has been reported that the P2Y12 receptor (P2Y12R is involved in satellite glial cells (SGCs activation, indicating that P2Y12R expressed in SGCs may play functional roles in orofacial neuropathic pain mechanisms. However, the involvement of P2Y12R in orofacial neuropathic pain mechanisms is still unknown. We therefore studied the reflex to noxious mechanical or heat stimulation of the tongue, P2Y12R and glial fibrillary acidic protein (GFAP immunohistochemistries in the trigeminal ganglion (TG in a rat model of unilateral lingual nerve crush (LNC to evaluate role of P2Y12R in SGC in lingual neuropathic pain. Results The head-withdrawal reflex thresholds to mechanical and heat stimulation of the lateral tongue were significantly decreased in LNC-rats compared to sham-rats. These nocifensive effects were apparent on day 1 after LNC and lasted for 17 days. On days 3, 9, 15 and 21 after LNC, the mean relative number of TG neurons encircled with GFAP-immunoreactive (IR cells significantly increased in the ophthalmic, maxillary and mandibular branch regions of TG. On day 3 after LNC, P2Y12R expression occurred in GFAP-IR cells but not neuronal nuclei (NeuN-IR cells (i.e. neurons in TG. After 3 days of successive administration of the P2Y12R antagonist MRS2395 into TG in LNC-rats, the mean relative number of TG neurons encircled with GFAP-IR cells was significantly decreased coincident with a significant reversal of the lowered head-withdrawal reflex thresholds to mechanical and heat stimulation of the tongue compared to vehicle-injected rats. Furthermore, after 3 days of successive administration of the P2YR agonist 2-MeSADP into the TG in naïve rats, the mean relative number of TG neurons encircled with GFAP-IR cells was significantly increased and head-withdrawal reflex thresholds to mechanical and heat stimulation of the tongue were significantly decreased in a dose-dependent manner compared to vehicle-injected rats

Screening of TYR, OCA2, GPR143, and MC1R in patients with congenital nystagmus, macular hypoplasia, and fundus hypopigmentation indicating albinism

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Preising, Markus N.; Gonser, Miriam; Lorenz, Birgit

2011-01-01

Background A broad spectrum of pigmentation of the skin and hair is found among patients diagnosed with ocular albinism (OA) and oculocutaneous albinism (OCA). Even though complexion is variable, three ocular features, i.e., hypopigmentation of the fundus, hypoplasia of the macula, and nystagmus, are classical pathological findings in these patients. We screened 172 index patients with a clinical diagnosis of OA or OCA based on the classical findings, to evaluate the frequency of sequence variants in tyrosinase (TYR), P-gene, P-protein (OCA2), and the G-protein-coupled receptor 143 gene, OA1 (GPR143). In addition, we investigated the association of sequence variants in the melanocortin receptor 1 gene (MC1R) and OCA2. Methods Pigmentation of the hair, skin, iris, and fundus were included in the evaluation of OCA and OA. Male OA patients showing X-linked inheritance were screened for GPR143. Females showing OA without family history were regarded as representing autosomal recessive OA (OA3). Direct sequencing was applied to PCR products showing aberrant single-strand conformation polymorphism–banding patterns. Results Fifty-seven male index patients were screened for OA. We identified 16 potentially pathogenic sequence variations in GPR143 (10 novel) in 22 males. In TYR, we identified 23 (7 novel), and in OCA2 28 (11 novel) possibly pathogenic variants. Variants on both alleles were identified in TYR or OCA2 in 29/79 OCA patients and 14/71 OA patients. Sequence changes in TYR were identified almost exclusively in OCA patients, while sequence changes in OCA2 occurred in OCA and OA patients. MC1R sequencing was performed in 47 patients carrying mutations in OCA2 and revealed MC1R mutations in 42 of them. Conclusions TYR gene mutations have a more severe effect on pigmentation than mutations in OCA2 and the GPR143 gene. Nevertheless, mutations in these genes affect the development of visual function either directly or by interaction with other genes like MC1R, which

Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell–matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis

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Chen, Yan; Li, Zheng; He, Yan; Shang, Dandan; Pan, Jigang; Wang, Hongmei; Chen, Huamei; Zhu, Zhuxia [Department of Physiology/Cancer Research Group, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China); Wan, Lei [Department of Pharmacology, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China); Wang, Xudong, E-mail: xdwang@gmc.edu.cn [Department of Physiology/Cancer Research Group, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China)

2014-03-01

Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer. - Highlights: • Estrogen and ICI augment adhesion to matrigel with calpain activation in MCF-7 cells. • GPR30 mediates cell–matrigel adhesion and calpain activation via ERK1/2. • Calpain is required in the cell–matrigel adhesion induced by E2 and ICI.

Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell–matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis

International Nuclear Information System (INIS)

Chen, Yan; Li, Zheng; He, Yan; Shang, Dandan; Pan, Jigang; Wang, Hongmei; Chen, Huamei; Zhu, Zhuxia; Wan, Lei; Wang, Xudong

2014-01-01

Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer. - Highlights: • Estrogen and ICI augment adhesion to matrigel with calpain activation in MCF-7 cells. • GPR30 mediates cell–matrigel adhesion and calpain activation via ERK1/2. • Calpain is required in the cell–matrigel adhesion induced by E2 and ICI

Keratin 17 is overexpressed and predicts poor survival in estrogen receptor-negative/human epidermal growth factor receptor-2-negative breast cancer.

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Merkin, Ross D; Vanner, Elizabeth A; Romeiser, Jamie L; Shroyer, A Laurie W; Escobar-Hoyos, Luisa F; Li, Jinyu; Powers, Robert S; Burke, Stephanie; Shroyer, Kenneth R

2017-04-01

Clinicopathological features of breast cancer have limited accuracy to predict survival. By immunohistochemistry (IHC), keratin 17 (K17) expression has been correlated with triple-negative status (estrogen receptor [ER]/progesterone receptor/human epidermal growth factor receptor-2 [HER2] negative) and decreased survival, but K17 messenger RNA (mRNA) expression has not been evaluated in breast cancer. K17 is a potential prognostic cancer biomarker, targeting p27, and driving cell cycle progression. This study compared K17 protein and mRNA expression to ER/progesterone receptor/HER2 receptor status and event-free survival. K17 IHC was performed on 164 invasive breast cancers and K17 mRNA was evaluated in 1097 breast cancers. The mRNA status of other keratins (16/14/9) was evaluated in 113 ER - /HER2 - ductal carcinomas. IHC demonstrated intense cytoplasmic and membranous K17 localization in myoepithelial cells of benign ducts and lobules and tumor cells of ductal carcinoma in situ. In ductal carcinomas, K17 protein was detected in most triple-negative tumors (28/34, 82%), some non-triple-negative tumors (52/112, 46%), but never in lobular carcinomas (0/15). In ductal carcinomas, high K17 mRNA was associated with reduced 5-year event-free survival in advanced tumor stage (n = 149, hazard ratio [HR] = 3.68, P = .018), and large (n = 73, HR = 3.95, P = .047), triple-negative (n = 103, HR = 2.73, P = .073), and ER - /HER2 - (n = 113, HR = 2.99, P = .049) tumors. There were significant correlations among keratins 17, 16, 14, and 9 mRNA levels suggesting these keratins (all encoded on chromosome 17) could be coordinately expressed in breast cancer. Thus, K17 is expressed in a subset of triple-negative breast cancers, and is a marker of poor prognosis in patients with advanced stage and ER - /HER2 - breast cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Genetic background can result in a marked or minimal effect of gene knockout (GPR55 and CB2 receptor in experimental autoimmune encephalomyelitis models of multiple sclerosis.

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Sofia Sisay

Full Text Available Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1 receptor and the orphan G protein receptor fifty-five (GPR55. Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational

The ADP receptor P2Y1 is necessary for normal thermal sensitivity in cutaneous polymodal nociceptors

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Jankowski Michael P

2011-02-01

Full Text Available Abstract Background P2Y1 is a member of the P2Y family of G protein-coupled nucleotide receptors expressed in peripheral sensory neurons. Using ratiometric calcium imaging of isolated dorsal root ganglion neurons, we found that the majority of neurons responding to adenosine diphosphate, the preferred endogenous ligand, bound the lectin IB4 and expressed the ATP-gated ion channel P2X3. These neurons represent the majority of epidermal afferents in hairy skin, and are predominantly C-fiber polymodal nociceptors (CPMs, responding to mechanical stimulation, heat and in some cases cold. Results To characterize the function of P2Y1 in cutaneous afferents, intracellular recordings from sensory neuron somata were made using an ex vivo preparation in which the hindlimb skin, saphenous nerve, DRG and spinal cord were dissected in continuum, and cutaneous receptive fields characterized using digitally-controlled mechanical and thermal stimuli in male wild type mice. In P2Y1-/- mice, CPMs showed a striking increase in mean heat threshold and a decrease in mean peak firing rate during a thermal ramp from 31-52°C. A similar change in mean cold threshold was also observed. Interestingly, mechanical testing of CPMs revealed no significant differences between P2Y1-/- and WT mice. Conclusions These results strongly suggest that P2Y1 is required for normal thermal signaling in cutaneous sensory afferents. Furthermore, they suggest that nucleotides released from peripheral tissues play a critical role in the transduction of thermal stimuli in some fiber types.

PPADS and suramin as antagonists at cloned P2Y- and P2U-purinoceptors.

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Charlton, S J; Brown, C A; Weisman, G A; Turner, J T; Erb, L; Boarder, M R

1996-06-01

1. The effect of suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on the stimulation of phospholipase C in 1321N1 cells transfected with the human P2U-purinoceptor (h-P2U-1321N1 cells) or with the turkey P2Y-purinoceptor (t-P2Y-1321N1 cells) was investigated. 2-Methylthioadenosine triphosphate (2MeSATP) was used as the agonist at t-P2Y-1321N1 cells and uridine triphosphate (UTP) at h-P2U-1321N1 cells. 2. Suramin caused a parallel shift to the right of the concentration-response curves for 2MeSATP in the t-P2Y-1321N1 cells, yielding a Schild plot with a slope of 1.16 +/- 0.08 and a pA2 value of 5.77 +/- 0.11. 3. Suramin also caused a shift to the right of concentration-response curves for UTP in the h-P2U-1321N1 cells, and on Schild plots gave a slope different from unity (1.57 +/- 0.19) and an apparent pA2 value of 4.32 +/- 0.13. Suramin was therefore a less potent antagonist at the P2U-purinoceptor than the P2Y-purinoceptor. 4. In the presence of the ectonucleotidase inhibitor, ARL 67156 (6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP) there was no significant difference in the EC50 or shapes of curves with either cell type, and no difference in pA2 values for suramin. 5. PPADS caused an increase in the EC50 for 2MeSATP in the t-P2Y-1321N1 cells. The Schild plot had a slope different from unity (0.55 +/- 0.15) and an X-intercept corresponding to an apparent pA2 of 5.98 +/- 0.65. 6. PPADS up to 30 microM had no effect on the concentration-response curve for UTP with the h-P2U-1321N1 cells. 7. In conclusion, suramin and PPADS show clear differences in their action at the 2 receptor types, in each case being substantially more effective as an antagonist at the P2Y-purinoceptor than at the P2U-purinoceptor. Ectonucleotidase breakdown had little influence on the nature of the responses at the two receptor types, or in their differential sensitivity to suramin.

E2GPR - Edit your geometry, Execute GprMax2D and Plot the Results!

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Pirrone, Daniele; Pajewski, Lara

2015-04-01

In order to predict correctly the Ground Penetrating Radar (GPR) response from a particular scenario, Maxwell's equations have to be solved, subject to the physical and geometrical properties of the considered problem and to its initial conditions. Several techniques have been developed in computational electromagnetics, for the solution of Maxwell's equations. These methods can be classified into two main categories: differential and integral equation solvers, which can be implemented in the time or spectral domain. All of the different methods present compromises between computational efficiency, stability, and the ability to model complex geometries. The Finite-Difference Time-Domain (FDTD) technique has several advantages over alternative approaches: it has inherent simplicity, efficiency and conditional stability; it is suitable to treat impulsive behavior of the electromagnetic field and can provide either ultra-wideband temporal waveforms or the sinusoidal steady-state response at any frequency within the excitation spectrum; it is accurate and highly versatile; and it has become a mature and well-researched technique. Moreover, the FDTD technique is suitable to be executed on parallel-processing CPU-based computers and to exploit the modern computer visualisation capabilities. GprMax [1] is a very well-known and largely validated FDTD software tool, implemented by A. Giannopoulos and available for free public download on www.gprmax.com, together with examples and a detailled user guide. The tool includes two electromagnetic wave simulators, GprMax2D and GprMax3D, for the full-wave simulation of two-dimensional and three-dimensional GPR models. In GprMax, everything can be done with the aid of simple commands that are used to define the model parameters and results to be calculated. These commands need to be entered in a simple ASCII text file. GprMax output files can be stored in ASCII or binary format. The software is provided with MATLAB functions, which

Presynaptic inhibition of spontaneous acetylcholine release mediated by P2Y receptors at the mouse neuromuscular junction.

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De Lorenzo, S; Veggetti, M; Muchnik, S; Losavio, A

2006-09-29

At the neuromuscular junction, ATP is co-released with the neurotransmitter acetylcholine (ACh) and once in the synaptic space, it is degraded to the presynaptically active metabolite adenosine. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of extracellular ATP (100 muM) and the slowly hydrolysable ATP analog 5'-adenylylimidodiphosphate lithium (betagamma-imido ATP) (30 muM) on miniature end-plate potential (MEPP) frequency. We found that application of ATP and betagamma-imido ATP decreased spontaneous secretion by 45.3% and 55.9% respectively. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A(1) adenosine receptor antagonist and alpha,beta-methylene ADP sodium salt (alphabeta-MeADP), which is an inhibitor of ecto-5'-nucleotidase, did not prevent the inhibitory effect of ATP, demonstrating that the nucleotide is able to modulate spontaneous ACh release through a mechanism independent of the action of adenosine. Blockade of Ca(2+) channels by both, Cd(2+) or the combined application of nitrendipine and omega-conotoxin GVIA (omega-CgTx) (L-type and N-type Ca(2+) channel antagonists, respectively) prevented the effect of betagamma-imido ATP, indicating that the nucleotide modulates Ca(2+) influx through the voltage-dependent Ca(2+) channels related to spontaneous secretion. betagamma-Imido ATP-induced modulation was antagonized by the non-specific P2 receptor antagonist suramin and the P2Y receptor antagonist 1-amino-4-[[4-[[4-chloro-6-[[3(or4)-sulfophenyl] amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl] amino]-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid (reactive blue-2), but not by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS), which has a preferential antagonist effect on P2X receptors. Pertussis toxin and N-ethylmaleimide (NEM), which are blockers of G(i/o) proteins, prevented the action of the nucleotide, suggesting that the effect is mediated by P2Y receptors

Acidic tumor microenvironment and pH-sensing G protein-coupled receptors.

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Justus, Calvin R; Dong, Lixue; Yang, Li V

2013-12-05

The tumor microenvironment is acidic due to glycolytic cancer cell metabolism, hypoxia, and deficient blood perfusion. It is proposed that acidosis in the tumor microenvironment is an important stress factor and selection force for cancer cell somatic evolution. Acidic pH has pleiotropic effects on the proliferation, migration, invasion, metastasis, and therapeutic response of cancer cells and the function of immune cells, vascular cells, and other stromal cells. However, the molecular mechanisms by which cancer cells and stromal cells sense and respond to acidic pH in the tumor microenvironment are poorly understood. In this article the role of a family of pH-sensing G protein-coupled receptors (GPCRs) in tumor biology is reviewed. Recent studies show that the pH-sensing GPCRs, including GPR4, GPR65 (TDAG8), GPR68 (OGR1), and GPR132 (G2A), regulate cancer cell metastasis and proliferation, immune cell function, inflammation, and blood vessel formation. Activation of the proton-sensing GPCRs by acidosis transduces multiple downstream G protein signaling pathways. Since GPCRs are major drug targets, small molecule modulators of the pH-sensing GPCRs are being actively developed and evaluated. Research on the pH-sensing GPCRs will continue to provide important insights into the molecular interaction between tumor and its acidic microenvironment and may identify new targets for cancer therapy and chemoprevention.

An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets.

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Nisar, Shaista; Daly, Martina E; Federici, Augusto B; Artoni, Andrea; Mumford, Andrew D; Watson, Stephen P; Mundell, Stuart J

2011-11-17

The platelet P2Y(12) purinoceptor (P2Y(12)R), which plays a crucial role in hemostasis, undergoes internalization and subsequent recycling to maintain receptor responsiveness, processes that are essential for normal platelet function. Here, we observe that P2Y(12)R function is compromised after deletion or mutation of the 4 amino acids at the extreme C-terminus of this receptor (ETPM), a putative postsynaptic density 95/disc large/zonula occludens-1 (PDZ)-binding motif. In cell line models, removal of this sequence or mutation of one of its core residues (P341A), attenuates receptor internalization and receptor recycling back to the membrane, thereby blocking receptor resensitization. The physiologic significance of these findings in the regulation of platelet function is shown by identification of a patient with a heterozygous mutation in the PDZ binding sequence of their P2Y(12)R (P341A) that is associated with reduced expression of the P2Y(12)R on the cell surface. Importantly, platelets from this subject showed significantly compromised P2Y(12)R recycling, emphasizing the importance of the extreme C-terminus of this receptor to ensure correct receptor traffic.

Genetic deletion of the P2Y2 receptor offers significant resistance to development of lithium-induced polyuria accompanied by alterations in PGE2 signaling.

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Zhang, Yue; Pop, Ioana L; Carlson, Noel G; Kishore, Bellamkonda K

2012-01-01

Lithium (Li)-induced polyuria is due to resistance of the medullary collecting duct (mCD) to the action of arginine vasopressin (AVP), apparently mediated by increased production of PGE(2). We previously reported that the P2Y(2) receptor (P2Y(2)-R) antagonizes the action of AVP on the mCD and may play a role in Li-induced polyuria by enhancing the production of PGE(2) in mCD. Hence, we hypothesized that genetic deletion of P2Y(2)-R should ameliorate Li-induced polyuria. Wild-type (WT) or P2Y(2)-R knockout (KO) mice were fed normal or Li-added diets for 14 days and euthanized. Li-induced polyuria, and decreases in urine osmolality and AQP2 protein abundance in the renal medulla, were significantly less compared with WT mice despite the lack of differences in Li intake or terminal serum or inner medullary tissue Li levels. Li-induced increased urinary excretion of PGE(2) was not affected in KO mice. However, prostanoid EP(3) receptor (EP3-R) protein abundance in the renal medulla of KO mice was markedly lower vs. WT mice, irrespective of the dietary regimen. The protein abundances of other EP-Rs were not altered across the groups irrespective of the dietary regimen. Ex vivo stimulation of mCD with PGE(2) generated significantly more cAMP in Li-fed KO mice (130%) vs. Li-fed WT mice (100%). Taken together, these data suggest 1) genetic deletion of P2Y(2)-R offers significant resistance to the development of Li-induced polyuria; and 2) this resistance is apparently due to altered PGE(2) signaling mediated by a marked decrease in EP3-R protein abundance in the medulla, thus attenuating the EP3-mediated decrease in cAMP levels in mCD.

Transcription of Toll-Like Receptors 2, 3, 4 and 9, FoxP3 and Th17 Cytokines in a Susceptible Experimental Model of Canine Leishmania infantum Infection

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Hosein, Shazia; Rodríguez-Cortés, Alhelí; Blake, Damer P.; Allenspach, Karin; Alberola, Jordi; Solano-Gallego, Laia

2015-01-01

Canine leishmaniosis (CanL) due to Leishmania infantum is a chronic zoonotic systemic disease resulting from complex interactions between protozoa and the canine immune system. Toll-like receptors (TLRs) are essential components of the innate immune system and facilitate the early detection of many infections. However, the role of TLRs in CanL remains unknown and information describing TLR transcription during infection is extremely scarce. The aim of this research project was to investigate the impact of L. infantum infection on canine TLR transcription using a susceptible model. The objectives of this study were to evaluate transcription of TLRs 2, 3, 4 and 9 by means of quantitative reverse transcription polymerase chain reaction (qRT-PCR) in skin, spleen, lymph node and liver in the presence or absence of experimental L. infantum infection in Beagle dogs. These findings were compared with clinical and serological data, parasite densities in infected tissues and transcription of IL-17, IL-22 and FoxP3 in different tissues in non-infected dogs (n = 10), and at six months (n = 24) and 15 months (n = 7) post infection. Results revealed significant down regulation of transcription with disease progression in lymph node samples for TLR3, TLR4, TLR9, IL-17, IL-22 and FoxP3. In spleen samples, significant down regulation of transcription was seen in TLR4 and IL-22 when both infected groups were compared with controls. In liver samples, down regulation of transcription was evident with disease progression for IL-22. In the skin, upregulation was seen only for TLR9 and FoxP3 in the early stages of infection. Subtle changes or down regulation in TLR transcription, Th17 cytokines and FoxP3 are indicative of the silent establishment of infection that Leishmania is renowned for. These observations provide new insights about TLR transcription, Th17 cytokines and Foxp3 in the liver, spleen, lymph node and skin in CanL and highlight possible markers of disease susceptibility in

Protein kinase activity associated with Fcγ/sub 2a/ receptor of a murine macrophage like cell line, P388D1

International Nuclear Information System (INIS)

Hirata, Y.; Suzuki, T.

1987-01-01

The properties of protein kinase activity associated with Fc receptor specific for IgG/sub 2a/(Fcγ/sub 2a/R) of a murine macrophage like cell line, P388D 1 , were investigated. IgG/sub 2a/-binding protein isolated from the detergent lysate of P388D 1 cells by affinity chromatography of IgG-Sepharose was found to contain four distinct proteins of M/sub r/ 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with [γ- 32 P]ATP. The autophosphorylation of Fcγ/sub 2a/ receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Phosphorylation of casein catalyzed by Fcγ/sub 2a/ receptor complex was dependent on casein concentration, increased with time or temperature, was dependent on the concentration of ATP and Mg 2+ , and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn 2+ or KCl or by a small amount of heparin and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fcγ/sub 2a/ receptor used ATP as substrate with an apparent K/sub m/ of 2 μM as well as GTP with an apparent K/sub m/ of 10 μM. Prior heating (60 0 C for 15 min) or treatment with protease (trypsin or Pronase) of Fcγ/sub 2a/ receptor complex almost totally abolished casein kinase activity. Thin-layer chromatography of a partial acid hydrolysate of the phosphorylated casein showed that the site of phosphorylation is at a seryl residue. These results suggest that Fcγ 2 /sub a/ receptor forms a molecule complex with protein kinase, whose characteristics resemble those of type II casein kinase but are different from those of cyclic nucleotide dependent protein kinase or from those of C protein kinase

GPR30 is necessary for estradiol-induced desensitization of 5-HT1A receptor signaling in the paraventricular nucleus of the rat hypothalamus.

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McAllister, C E; Creech, R D; Kimball, P A; Muma, N A; Li, Q

2012-08-01

Estrogen therapy used in combination with selective serotonin reuptake inhibitor (SSRI) treatment improves SSRI efficacy for the treatment of mood disorders. Desensitization of serotonin 1A (5-HT(1A)) receptors, which takes one to two weeks to develop in animals, is necessary for SSRI therapeutic efficacy. Estradiol modifies 5-HT(1A) receptor signaling and induces a partial desensitization in the paraventricular nucleus (PVN) of the rat within two days, but the mechanisms underlying this effect are currently unknown. The purpose of this study was to identify the estrogen receptor necessary for estradiol-induced 5-HT(1A) receptor desensitization. We previously showed that estrogen receptor β is not necessary for 5-HT(1A) receptor desensitization and that selective activation of estrogen receptor GPR30 mimics the effects of estradiol in rat PVN. Here, we used a recombinant adenovirus containing GPR30 siRNAs to decrease GPR30 expression in the PVN. Reduction of GPR30 prevented estradiol-induced desensitization of 5-HT(1A) receptor as measured by hormonal responses to the selective 5-HT(1A) receptor agonist, (+)8-OH-DPAT. To determine the possible mechanisms underlying these effects, we investigated protein and mRNA levels of 5-HT(1A) receptor signaling components including 5-HT(1A) receptor, Gαz, and RGSz1. We found that two days of estradiol increased protein and mRNA expression of RGSz1, and decreased 5-HT(1A) receptor protein but increased 5-HT(1A) mRNA; GPR30 knockdown prevented the estradiol-induced changes in 5-HT(1A) receptor protein in the PVN. Taken together, these data demonstrate that GPR30 is necessary for estradiol-induced changes in the 5-HT(1A) receptor signaling pathway and desensitization of 5-HT(1A) receptor signaling. Copyright © 2012 Elsevier Ltd. All rights reserved.

GPR158, an orphan member of G protein-coupled receptor Family C: glucocorticoid-stimulated expression and novel nuclear role.

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Patel, Nitin; Itakura, Tatsuo; Gonzalez, Jose M; Schwartz, Stephen G; Fini, M Elizabeth

2013-01-01

Members of the large G protein-coupled receptor (GPCR) clan are implicated in many physiological and disease processes, making them important therapeutic drug targets. In the present study, we follow up on results of a pilot study suggesting a functional relationship between glucocorticoid (GC)-induced ocular hypertension and GPR158, one of three orphan members of the GPCR Family C. GC treatment increases levels of GPR158 mRNA and protein through transcriptional mechanisms, in cultured trabecular meshwork (TBM) cells derived from the eye's aqueous outflow pathway. Like treatment with GCs, transient overexpression of GPR158 stimulates cell proliferation, while siRNA knockdown of endogenous GPR158 has the opposite effect. Both endogenous and overexpressed GPR158 show an unusual subcellular localization pattern, being found almost entirely in the nucleus. However, when cells are treated with inhibitors of clathrin-mediated endocytosis, GPR158 is shifted to the plasma membrane. Mutation of a bipartite nuclear localization signal (NLS) in the 8(th) helix also shifts GPR158 out of the nucleus, but in this case the protein is found in vesicles localized in the cytoplasm. These results suggest that newly synthesized GPR158 first traffics to the plasma membrane, where it rapidly undergoes endocytosis and translocation to the nucleus. Significantly, mutation of the NLS abrogates GPR158-mediated enhancement of cell proliferation, indicating a functional requirement for nuclear localization. GPR158 overexpression upregulates levels of the cell cycle regulator cyclin D1, but mutation of the NLS reverses this. Overexpression of GPR158 enhances the barrier function of a TBM cell monolayer, which is associated with an increase in the levels of tight junction proteins ZO-1 and occludin, similar to reported studies on GC treatment. Regulated paracellular permeability controls aqueous outflow facility in vivo. Since GCs stimulate GPR158 expression, the result is consistent with a

GPR55, a G-protein coupled receptor for lysophosphatidylinositol, plays a role in motor coordination.

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Chia-Shan Wu

Full Text Available The G-protein coupled receptor 55 (GPR55 is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior.

GPR55, a G-protein coupled receptor for lysophosphatidylinositol, plays a role in motor coordination.

Science.gov (United States)

Wu, Chia-Shan; Chen, Hongmei; Sun, Hao; Zhu, Jie; Jew, Chris P; Wager-Miller, James; Straiker, Alex; Spencer, Corinne; Bradshaw, Heather; Mackie, Ken; Lu, Hui-Chen

2013-01-01

The G-protein coupled receptor 55 (GPR55) is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance) in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior.

GPR143 mutations in Chinese patients with ocular albinism type 1.

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Jia, Xiuhua; Yuan, Jin; Jia, Xiaoyun; Ling, Shiqi; Li, Shiqiang; Guo, Xiangming

2017-05-01

The aim of the present study was to evaluate mutations of the G protein-coupled receptor 143 (GPR143) gene for ocular albinism type 1 (OA1) in Chinese patients. For the current study, 8 patients with OA1 were selected from the database of ocular genetic diseases. Genomic DNA of OA1 was prepared from venous leukocytes collected from the patients. Cycle sequencing was used to analyze the exons and adjacent introns of GPR143. The variation detected was analyzed by bidirectional DNA sequencing and further evaluated in 96 controls using heteroduplex‑single strand conformational polymorphism analysis. Additionally, slit lamp photography of anterior segment, fundus photography and optical coherence tomography (OCT) were performed to identify the clinical features of OA1. In five patients with OA1, 5 GPR143 gene mutations were identified and four of them there were novel mutations. The screening rate is 62.5%, including c.333G>A (p.W111X), c.353G>A (p.G118E) (known mutation), C.658+2T>G (splice mutation), c.215_216insCGCTGC (p.71‑72insAA) and c.17T>C (p. L6P). These mutations were absent in the 96 normal controls. Only one patient with OA1 in the present study was female. Patients with OA1 often have congenital nystagmus, refractive error, severe decline of visual acuity (from 0.1 to 0.4) and foveal hypoplasia. Different degrees of pigment loss were evident in the patients' iris and retina, whereas macular structure was not identified in the OCT examination. The findings of the present study expanded the gene mutation spectrum of GPR143 and investigated the clinical phenotype of patients with OA1 in the Chinese population. Additional evidence for clinical diagnosis was provided along with differential diagnosis and genetic counseling.

Targeting GPR120 and other fatty acid sensing GPCRs ameliorates insulin resistance and inflammatory diseases

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Talukdar, Saswata; Olefsky, Jerrold M; Osborn, Olivia

2011-01-01

The last decade has seen great progress in the understanding of the molecular pharmacology, physiological function and therapeutic potential of the G protein-coupled receptors. Free Fatty acids (FFAs) have been demonstrated to act as ligands of several GPCRs including GPR40, GPR43, GPR84, GPR119 and GPR120. We have recently shown that GPR120 acts as a physiological receptor of ω3 fatty acids in macrophages and adipocytes, which mediate potent anti-inflammatory and insulin sensitizing effects. The important role GPR120 plays in the control of inflammation raises the possibility that targeting this receptor could have therapeutic potential in many inflammatory diseases including obesity and type 2 diabetes. In this review, we discuss lipid-sensing GPCRs and highlight potential outcomes of targeting such receptors in ameliorating disease. PMID:21663979

Overexpression of c-myc and loss of heterozigosity on 2p, 3p, 5q, 17p and 18q in sporadic colorectal carcinoma Sobreexpresión de c-myc y pérdida de heterozigosidad en 2p, 3p, 5q, 17p y 18q en carcinoma colorrectal esporádico

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A. Sánchez-Pernaute

2005-03-01

Full Text Available Aim: the aim of the present study is to evaluate the prognostic influence of loss of heterozygosity on 2p, 3p, 5q, 17p and 18q, and c-myc overexpression on surgically treated sporadic colorectal carcinoma. Methods: tumor and non-tumor tissue samples from 153 patients were analyzed. Fifty-one percent of patients were male, and mean age in the series was 67 years. Tumors were located in the proximal colon in 37 cases, in the distal bowel in 37, and in the rectum in 79 patients. c-myc overexpression was studied by means of Northern blot analysis, and loss of heterozigosity through microsatellite analysis. Results: c-myc overexpression was detected in 25% of cases, and loss of heterozygosity in at least one of the studied regions in 48%. There was no association between clinical and pathologic features, and genetic alterations. The disease-free interval was significantly shorter for patients with both genetic alterations; the presence of both events was an independent prognostic factor for poor outcome in the multivariate analysis (RR: 4.34, p Objetivo: el objetivo del presente trabajo es evaluar la importancia pronóstica de la pérdida de heterozigosidad en las regiones 2p, 3p, 5q, 17p y 18q y de la sobreexpresión del gen c-myc en el carcinoma colorrectal esporádico, mediante el estudio de la supervivencia libre de enfermedad tras cirugía potencialmente curativa. Métodos: se han analizado muestras tumorales y no tumorales de mucosa colónica de 153 pacientes. El 51% de los pacientes eran varones y la edad media de la serie fue 67 años. Los tumores fueron proximales en 37 casos, distales en 37 y localizados en recto en 79. Se analizó la sobreexpresión del RNA de c-myc por Northern blot, y la presencia de pérdida de heterozigosidad en las diferentes regiones consideradas por análisis de microsatélites. Resultados: se detectó sobreexpresión de c-myc en el 25% de los casos, y pérdida de heterozigosidad en alguna de las regiones estudiadas

Toll-like receptor 2 or toll-like receptor 4 deficiency does not modify lupus in MRLlpr mice.

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Simon J Freeley

Full Text Available Systemic lupus erythematosus is an autoimmune disease with a high morbidity and nephritis is a common manifestation. Previous studies in murine lupus models have suggest a role for Toll-like receptor 2 and 4. We examined the role of these molecules in MRL lpr mice which is one of the most established and robust murine models. We compared disease parameters in Toll-like receptor 2 or Toll-like receptor 4 deficient mice with their littermate controls. We found no difference in the severity of glomerulonephritis as assessed by histology, serum creatinine and albuminuria when Toll-like receptor 2 or Toll-like receptor 4 deficient MRLlpr mice were compared with Toll-like receptor sufficient controls. We also found similar levels of anti-dsDNA and anti-ssDNA antibodies. These results show that Toll-like receptor 2 and Toll-like receptor 4 do not play a significant role in MRLlpr mice, and therefore they may not be important in human lupus.