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

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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.

P2Y purinoceptor and nucleotide receptor-induced relaxation of precontracted bovine aortic collateral artery rings: differential sensitivity to suramin and indomethacin.

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Wilkinson, G F; McKechnie, K; Dainty, I A; Boarder, M R

1994-02-01

We have examined a series of adenine nucleotides and UTP for their ability to cause relaxation of precontracted bovine aortic collateral artery rings. The overall rank order of agonist potency for relaxation was 2-methylthioadenosine 5'-triphosphate (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) > UTP > ADP > ATP. These responses were endothelium-dependent. Interaction studies showed that responses to the selective P2Y purinoceptor agonist 2MeSATP, and to ADP, were mediated by different receptors from those mediating responses to UTP. Suramin, a P2 purinoceptor antagonist that binds to diverse sites for ATP, produced a concentration-dependent shift in the agonist concentration-effect curve to 2MeSATP, with a pKB of 5.45 +/- 0.15 and a slope of 0.94 +/- 0.09. Suramin produced a small, nonsignificant shift in the UTP response curve and had little effect on responses to ATP. Indomethacin (2.8 x 10(-6) M) had no effect on concentration-effect curves to UTP but almost abolished the relaxations produced by 2MeSATP and ADP. The concentration-effect curves to ATP and ATP gamma S showed a significant (P effects of indomethacin show that these receptors differentially modulate the release of cyclooxygenase-derived mediators of relaxation.

Medicinal chemistry of adenosine, P2Y and P2X receptors.

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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

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.

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

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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...

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

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

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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.

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.

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

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

1997-11-01

1. The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca2+ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK). 2. Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca2+, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca2+ had little effect on the peak Ca2+-response, but resulted in a more rapid decline to basal. There was no response to alpha,beta-MethylATP (alpha,beta MeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage. 3. ATP (log EC50 -5.1+/-0.2) also caused an increase in the total [3H]-inositol (poly)phosphates ([3H]-InsPx) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and alpha,beta MeATP giving no detectable response. 4. Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P3. 5. None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPgammaS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 microM forskolin. 6. Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration-dependency consistent with

The regulation of aortic endothelial cells by purines and pyrimidines involves co-existing P2y-purinoceptors and nucleotide receptors linked to phospholipase C.

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Wilkinson, G F; Purkiss, J R; Boarder, M R

1993-03-01

1. We have examined the phospholipase C responses in bovine aortic endothelial cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. The cells responded to purines in a manner consistent with the presence of P2y purinoceptors; both 2-methylthioadenosine 5'-triphosphate (2MeSATP) and adenosine 5'-0-(2-thiodiphosphate) (ADP beta S) were potent agonists (EC50 0.41 microM and 0.85 microM respectively) while beta, gamma-methylene ATP at 300 microM was not. 3. The cells also responded to UTP. The maximal response to UTP was less than that for either 2MeSATP and ADP beta S while adenosine 5'-0-(3-thiotriphosphate) (ATP gamma S) gave the largest maximal response. 4. The concentration-effect curve to UTP was additive in the presence of either 2MeSATP or ADP beta S. However, the concentration-effect curves to ATP gamma S reached the same maximum in the presence or absence of UTP. 5. Suramin, at concentrations between 10 microM and 100 microM was a competitive antagonist for the response to ADP beta S and 2MeSATP but not the response to UTP. 6. The results show that there are two separate, co-existing, receptor populations: P2y-purinoceptors (responding to purines) and nucleotide receptors (responding to both purines and pyrimidines). We conclude that purines such as ATP/ADP may regulate aortic endothelial cells by interacting with two phospholipase C-linked receptors.

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.

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.

GPR17: Molecular modeling and dynamics studies of the 3-D structure and purinergic ligand binding features in comparison with P2Y receptors

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Ranghino Graziella

2008-06-01

Full Text Available Abstract Background GPR17 is a G-protein-coupled receptor located at intermediate phylogenetic position between two distinct receptor families: the P2Y and CysLT receptors for extracellular nucleotides and cysteinyl-LTs, respectively. We previously showed that GPR17 can indeed respond to both classes of endogenous ligands and to synthetic compounds active at the above receptor families, thus representing the first fully characterized non-peptide "hybrid" GPCR. In a rat brain focal ischemia model, the selective in vivo knock down of GPR17 by anti-sense technology or P2Y/CysLT antagonists reduced progression of ischemic damage, thus highlighting GPR17 as a novel therapeutic target for stroke. Elucidation of the structure of GPR17 and of ligand binding mechanisms are the necessary steps to obtain selective and potent drugs for this new potential target. On this basis, a 3-D molecular model of GPR17 embedded in a solvated phospholipid bilayer and refined by molecular dynamics simulations has been the first aim of this study. To explore the binding mode of the "purinergic" component of the receptor, the endogenous agonist UDP and two P2Y receptor antagonists demonstrated to be active on GPR17 (MRS2179 and cangrelor were then modeled on the receptor. Results Molecular dynamics simulations suggest that GPR17 nucleotide binding pocket is similar to that described for the other P2Y receptors, although only one of the three basic residues that have been typically involved in ligand recognition is conserved (Arg255. The binding pocket is enclosed between the helical bundle and covered at the top by EL2. Driving interactions are H-bonds and salt bridges between the 6.55 and 6.52 residues and the phosphate moieties of the ligands. An "accessory" binding site in a region formed by the EL2, EL3 and the Nt was also found. Conclusion Nucleotide binding to GPR17 occurs on the same receptor regions identified for already known P2Y receptors. Agonist

Regulation of P2Y1 receptor traffic by sorting Nexin 1 is retromer independent.

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Nisar, Shaista; Kelly, Eamonn; Cullen, Pete J; Mundell, Stuart J

2010-04-01

The activity and traffic of G-protein coupled receptors (GPCRs) is tightly controlled. Recent work from our laboratory has shown that P2Y(1) and P2Y(12) responsiveness is rapidly and reversibly modulated in human platelets and that the underlying mechanism requires receptor trafficking as an essential part of this process. However, little is known about the molecular mechanisms underlying P2Y receptor traffic. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. These functions may in part be due to interactions of SNX1 with the mammalian retromer complex. In this study, we investigated the role of SNX1 in P2Y receptor trafficking. We show that P2Y(1) receptors recycle via a slow recycling pathway that is regulated by SNX1, whereas P2Y(12) receptors return to the cell surface via a rapid route that is SNX1 independent. SNX1 inhibition caused a dramatic increase in the rate of P2Y(1) receptor recycling, whereas inhibition of Vps26 and Vps35 known to be present in retromer had no effect, indicating that SNX1 regulation of P2Y(1) receptor recycling is retromer independent. In addition, inhibition of SNX4, 6 and 17 proteins did not affect P2Y(1) receptor recycling. SNX1 has also been implicated in GPCR degradation; however, we provide evidence that P2Y receptor degradation is SNX1 independent. These data describe a novel function of SNX1 in the regulation of P2Y(1) receptor recycling and suggest that SNX1 plays multiple roles in endocytic trafficking of GPCRs.

A critical look at the function of the P2Y11 receptor

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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...

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

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

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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.

Effects of luminal flow and nucleotides on [Ca(2+)](i) in rabbit cortical collecting duct.

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Woda, Craig B; Leite, Maurilo; Rohatgi, Rajeev; Satlin, Lisa M

2002-09-01

Nucleotide binding to purinergic P2 receptors contributes to the regulation of a variety of physiological functions in renal epithelial cells. Whereas P2 receptors have been functionally identified at the basolateral membrane of the cortical collecting duct (CCD), a final regulatory site of urinary Na(+), K(+), and acid-base excretion, controversy exists as to whether apical purinoceptors exist in this segment. Nor has the distribution of receptor subtypes present on the unique cell populations that constitute Ca(2+) the CCD been established. To examine this, we measured nucleotide-induced changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded rabbit CCDs microperfused in vitro. Resting [Ca(2+)](i) did not differ between principal and intercalated cells, averaging approximately 120 nM. An acute increase in tubular fluid flow rate, associated with a 20% increase in tubular diameter, led to increases in [Ca(2+)](i) in both cell types. Luminal perfusion of 100 microM UTP or ATP-gamma-S, in the absence of change in flow rate, caused a rapid and transient approximately fourfold increase in [Ca(2+)](i) in both cell types (P < 0.05). Luminal suramin, a nonspecific P2 receptor antagonist, blocked the nucleotide- but not flow-induced [Ca(2+)](i) transients. Luminal perfusion with a P2X (alpha,beta-methylene-ATP), P2X(7) (benzoyl-benzoyl-ATP), P2Y(1) (2-methylthio-ATP), or P2Y(4)/P2Y(6) (UDP) receptor agonist had no effect on [Ca(2+)](i). The nucleotide-induced [Ca(2+)](i) transients were inhibited by the inositol-1,4,5-triphosphate receptor blocker 2-aminoethoxydiphenyl borate, thapsigargin, which depletes internal Ca(2+) stores, luminal perfusion with a Ca(2+)-free perfusate, or the L-type Ca(2+) channel blocker nifedipine. These results suggest that luminal nucleotides activate apical P2Y(2) receptors in the CCD via pathways that require both internal Ca(2+) mobilization and extracellular Ca(2+) entry. The flow-induced rise in [Ca(2+)](i) is

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.

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

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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.

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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.

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.

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, 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

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.

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.

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.

P2Y6 Receptor Activation Promotes Inflammation and Tissue Remodeling in Pulmonary Fibrosis

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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.

Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

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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.

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

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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.

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

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

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.

Identification of endogenous surrogate ligands for human P2Y12 receptors by in silico and in vitro methods

International Nuclear Information System (INIS)

Nonaka, Yosuke; Hiramoto, Takeshi; Fujita, Norihisa

2005-01-01

Endogenous ligands acting on a human P2Y 12 receptor, one of the G-protein coupled receptors, were searched by in silico screening against our own database, which contains more than 500 animal metabolites. The in silico screening using the docking software AutoDock resulted in selection of cysteinylleukotrienes (CysLTs) and 5-phosphoribosyl 1-pyrophosphate (PRPP), with high free energy changes, in addition to the known P2Y 12 ligands such as 2MeSADP and ADP. These candidates were subjected to an in vitro Ca 2+ assay using the CHO cells stably expressing P2Y 12 -G 16 α fusion proteins. We found that CysLTE4 and PRPP acted on the P2Y 12 receptor as agonists with the EC 50 values of 1.3 and 7.8 nM, respectively. Furthermore, we analyzed the phylogenetic relationship of the P2Y, P2Y-like, and CysLT receptors based on sequence alignment followed by evolutionary analyses. The analyses showed that the P2Y 12 , P2Y 13 , P2Y 14 , GPR87, CysLT-1, and CysLT-2 receptors formed a P2Y-related receptor subfamily with common sequence motifs in the transmembrane regions

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

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.

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.

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

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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...

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.

Polyamidoamine (PAMAM) Dendrimer Conjugates of Clickable Agonists of the A3 Adenosine Receptor and Coactivation of the P2Y14 Receptor by a Tethered Nucleotide

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Tosh, Dilip, K. [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health; Yoo, Lena S. [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health; Chinn, Moshe [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health; Hong, Kunlun [ORNL; Kilbey, II, S Michael [ORNL; Barrett, Matthew O. [University of North Carolina School of Medicine; Fricks, Ingrid P. [University of North Carolina School of Medicine; Harden, T. Kendall [University of North Carolina School of Medicine; Jacobson, Kenneth A. [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health

2010-01-01

We previously synthesized a series of potent and selective A{sub 3} adenosine receptor (AR) agonists (North-methanocarba nucleoside 5{prime}-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed 'click' chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A{sub 3}AR activation was preserved in these multivalent conjugates, which bound with apparent Ki of 0.1-0.3 nM. They were substituted with nucleoside moieties, solely or in combination with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amide-linked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A{sub 3}AR affinity. We prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation of multiple ligands. A bifunctional conjugate activated both A{sub 3} and P2Y{sub 14} receptors (via amide-linked uridine-5{prime}-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate might be useful in disease treatment.

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

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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.

P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx

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Amstrup, Jan; Novak, Ivana

2003-01-01

P2X7 nucleotide receptors modulate a spectrum of cellular events in various cells including epithelia, such as exocrine pancreas. Although the pharmacology and channel properties of the P2X7 receptors have been studied intensively, signal transduction pathways are relatively unknown. In this study...... we applied a heterologous expression system of rat P2X7 receptors in HEK-293 cells. We followed the receptor expression and function using the enhanced green fluorescent protein (EGFP) tag, activation of intracellular proteins and increases in cellular Ca2+. EGFP-P2X7 receptors localized...... to the plasma membrane, clusters within the membrane and intracellularly. Stimulation of P2X7 receptors in HEK-293 cells led to an activation of extracellular signal-regulated kinases ERK1 and ERK2 and this activation was seen after just 1 min of stimulation with ATP. Using C- and N-terminal P2X7-receptor...

Impaired P2X1 Receptor-Mediated Adhesion in Eosinophils from Asthmatic Patients.

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Wright, Adam; Mahaut-Smith, Martyn; Symon, Fiona; Sylvius, Nicolas; Ran, Shaun; Bafadhel, Mona; Muessel, Michelle; Bradding, Peter; Wardlaw, Andrew; Vial, Catherine

2016-06-15

Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,β-methylene ATP (α,β-meATP; 10 μM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at -60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 μM) (3 ± 2 pA/pF). α,β-meATP-evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,β-meATP (10 μM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,β-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMβ2 integrin-dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration. Copyright © 2016 by The American Association of Immunologists, Inc.

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.

Convergent Synthesis of the Potent P2Y Receptor Antagonist MG 50-3-1 Based on a Regioselective Ullmann Coupling Reaction

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Christa E. Müller

2012-03-01

Full Text Available MG 50-3-1 (3, trisodium 1-amino-4-{4-[4-chloro-6-(2-sulfophenylamino-1,3,5-triazin-2-ylamino]-2-sulfophenylamino}-9,10-dioxo-9,10-dihydroanthracene 2-sulfonate is the most potent and selective antagonist (IC50 4.6 nM for “P2Y1-like” nucleotide-activated membrane receptors in guinea-pig taenia coli responsible for smooth muscle relaxation. Full characterization of the compound, however, e.g., at the human P2Y1 receptor, which is a novel potential target for antithrombotic drugs, as well as other P2 receptor subtypes, has been hampered due to difficulties in synthesizing the compound in sufficient quantity. MG 50-3-1 would be highly useful as a biological tool for detailed investigation of signal transduction in the gut. We have now developed a convenient, fast, mild, and efficient convergent synthesis of 3 based on retrosynthetic analysis. A new, regioselective Ullmann coupling reaction under microwave irradiation was successfully developed to obtain 1-amino-4-(4-amino-2-sulfophenylamino-9,10-dioxo-9,10-dihydro­anthracene 2-sulfonate (8. Four different copper catalysts (Cu, CuCl, CuCl2, and CuSO4 were investigated at different pH values of sodium phosphate buffer, and in water in the absence or presence of base. Results showed that CuSO4 in water in the presence of triethylamine provided the best conditions for the regioselective Ullmann coupling reaction yielding the key intermediate compound 8. A new synthon (sodium 2-(4,6-dichloro-1,3,5-triazin-2-ylaminobenzenesulfonate, 13 which can easily be obtained on a gram scale was prepared, and 13 was successfully coupled with 8 yielding the target compound 3.

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.

Attenuated purinergic receptor function in patients with type 2 diabetes

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Thaning, Pia; Bune, Laurids T.; Hellsten, Ylva

2010-01-01

Objective: Extra cellular nucleotides and nucleosides are involved in regulation of skeletal muscle blood flow. Diabetes induces cardiovascular dysregulation but the extent to which the vasodilatatory capacity of nucleotides and nucleosides are affected in type 2 diabetes is unknown. The present...... study investigated: 1) the vasodilatatory effect of ATP, UTP, and adenosine (ADO) and 2) the expression and distribution of P2Y(2) and P2X(1) receptors in skeletal muscles of diabetic subjects. Research Design and Methods: In 10 diabetic patients and 10 age-matched controls, leg blood flow (LBF......-DM (1.5). The distribution and mRNA-expression of receptors were similar in the two groups. Conclusions: The vasodilatatory effect of the purinergic system is severely reduced in type 2 diabetic patients. The potency of nucleotides varies with the following rank order: UTP>ATP>>>ADO. This is not due...

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.

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.

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.

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.

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.

Role of a guanine nucleotide-binding protein in α1-adrenergic receptor-mediated Ca2+ mobilization in DDT1 MF-2 cells

International Nuclear Information System (INIS)

Cornett, L.E.; Norris, J.S.

1987-01-01

In this study the mechanisms involved in α 1 -adrenergic receptor-mediated Ca 2+ mobilization at the level of the plasma membrane were investigated. Stimulation of 45 Ca 2+ efflux from saponin-permeabilized DDT 1 MF-2 cells was observed with the addition of either the α 1 -adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of [ 32 P] NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT 1 , MF-2 cells, possibly the α-subunit of N/sub i/. However, stimulation of unidirectional 45 Ca 2+ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the α 1 -adrenergic receptor to Ca 2+ mobilization in DDT 1 MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family

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.

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

Science.gov (United States)

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.

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.

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.

Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

International Nuclear Information System (INIS)

Senogles, S.E.; Caron, M.G.

1986-01-01

The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

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...

Basolateral P2X receptors mediate inhibition of NaCl transport in mouse medullary thick ascending limb (mTAL)

DEFF Research Database (Denmark)

Marques, Rita D; de Bruijn, Pauline I.A.; Sørensen, Mads Vaarby

2012-01-01

Extracellular nucleotides regulate epithelial transport via luminal and basolateral P2 receptors. Renal epithelia express multiple P2 receptors, which mediate significant inhibition of solute absorption. Recently, we identified several P2 receptors in the medullary thick ascending limb (m...

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

Science.gov (United States)

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.

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

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

DEFF Research Database (Denmark)

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...

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

Science.gov (United States)

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.

Interaction of GABAA receptors with purinergic P2X2 receptors

International Nuclear Information System (INIS)

Shrivastava, A.

2010-01-01

GABA A Rs in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X 2 Rs are also expressed in spinal cord neurons and are known to cross-talk with GABA A Rs. Here we investigated a possible 'dynamic' interaction between GABA A Rs and P2X 2 Rs using co-immunoprecipitation and FRET studies in HEK cells along with co-localization and single particle tracking studies in spinal cord neurons. Our results suggest that a significant proportion of P2X 2 Rs forms a transient complex with GABA A Rs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface. P2X 2 Rs and GABA A Rs are then co-inserted into the cell membrane and are primarily located extra-synaptically. Furthermore, agonist induced activation of P2X 2 Rs results in disassembly of the receptor complex and destabilization of GABA A Rs whereas P2X 2 Rs are stabilized and form larger clusters. Antagonist-induced blocking of P2XRs results in co-stabilization of this receptor complex at the cell surface. These results suggest a novel mechanism where association of P2XRs with other receptors could be used for specific targeting to the neuronal membrane, thus providing an extrasynaptic receptor reserve that could regulate the excitability of neurons. We further conclude that blocking the excitatory activity of excessively released ATP under diseased state by P2XR antagonists could simultaneously enhance synaptic inhibition mediated by GABA A Rs.(author) (author) [de

Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP- and thrombin-induced human platelet activation

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Nylander, Sven; Mattsson, Christer; Ramström, Sofia; Lindahl, Tomas L

2004-01-01

The objective of this study was to investigate if there is a synergistic effect of a combination of P2Y12 and P2Y1 inhibition and P2Y12 and thrombin inhibition, on ADP- and thrombin-induced platelet activation, respectively. The rationale being that these combinations will cause a concurrent inhibition of both Gαq and Gαi signalling.Blood from healthy volunteers was preincubated with AR-C69931MX, a reversible P2Y12 antagonist; MRS2179, a reversible P2Y1 antagonist; or melagatran, a direct reversible thrombin inhibitor; alone or in various combinations prior to activation with ADP or thrombin. Platelet function in whole blood was assessed by flow cytometry using the antibody PAC-1 to estimate the expression of active αIIbβ3 (the fibrinogen receptor GPIIb/IIIa). A synergistic effect was evaluated by comparing the concentrations in the different combinations with those of corresponding equipotent concentrations of each single inhibitor alone. The equipotent single concentrations were experimentally obtained from concentration response curves performed in parallel.A synergistic effect regarding inhibition of ADP-induced platelet activation (10 μM) was obtained with different combinations of AR-C69931MX and MRS2179.Inhibition of thrombin-induced platelet activation (2 nM) with combinations of AR-C69931MX and the thrombin inhibitor melagatran did also result in a strong synergistic effect.To our knowledge, this is the first time that data supporting a synergistic effect has been published for the inhibitor combinations described.Whether this synergistic effect in vitro also results in an improved antithrombotic effect in vivo with or without an increased risk of bleeding remains to be studied in well-conducted clinical studies. PMID:15265806

Bone turnover is altered in transgenic rats overexpressing the P2Y2 purinergic receptor

DEFF Research Database (Denmark)

Ellegaard, Maria; Agca, Cansu; Petersen, Solveig

2017-01-01

overexpression on bone status and bone cell function using a transgenic rat. Three-month-old female transgenic Sprague Dawley rats overexpressing P2Y2R (P2Y2R-Tg) showed higher bone strength of the femoral neck. Histomorphometry showed increase in resorptive surfaces and reduction in mineralizing surfaces. Both...

Activation of a P2Y4-like purinoceptor triggers an increase in cytosolic [Ca2+] in the red blood cells of the lizard Ameiva ameiva (Squamata, Teiidae

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Sartorello R.

2005-01-01

Full Text Available An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential. Erythrocytes are an important source of purines, which can be released under physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells. Few studies have been conducted on lizards, and have been limited to ATP agonist itself. We have previously shown that the red blood cells (RBCs of the lizard Ameiva ameiva store Ca2+ in the endoplasmic reticulum (ER and that the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c by mobilization of the cation from internal stores. We also reported the ability of the second messenger IP3 to discharge the ER calcium pool of the ER. Here we characterize the purinoceptor present in the cytoplasmic membrane of the RBCs of the lizard Ameiva ameiva by the selective use of ATP analogues and pyrimidine nucleotides. The nucleotides UTP, UDP, GTP, and ATPgammaS triggered a dose-dependent response, while interestingly 2MeSATP, 2ClATP, alpha, ß-ATP, and ADP failed to do so in a 1- to 200-µm con- centration. The EC50 obtained for the compounds tested was 41.77 µM for UTP, 48.11 µM for GTP, 53.11 µM for UDP, and 30.78 µM for ATPgammaS. The present data indicate that the receptor within the RBCs of Ameiva ameiva is a P2Y4-like receptor due to its pharmacological similarity to the mammalian P2Y4 receptor.

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

Science.gov (United States)

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.

Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia.

Science.gov (United States)

Sen, Sanjukta; Agarwal, Rupesh; Ambulkar, Prafulla; Hinduja, Indira; Zaveri, Kusum; Gokral, Jyotsna; Pal, Asoke; Modi, Deepak

2016-02-01

The AZFc locus on the human Y chromosome harbours several multicopy genes, some of which are required for spermatogenesis. It is believed that deletion of one or more copies of these genes is a cause of infertility in some men. GOLGA2LY is one of the genes in the AZFc locus and it exists in two copies, GOLGA2P2Y and GOLGA2P3Y. The involvement of GOLGA2LY gene copy deletions in male infertility, however, is unknown. This study aimed to investigate the association of deletions of GOLGA2P2Y and GOLGA2P3Y gene copies with male infertility and with sperm concentration and motility. The frequency of GOLGA2P3Y deletion was significantly higher in oligozoospermic men compared with normozoospermic men (7.7% versus 1.2%; P = 0.0001), whereas the frequency of GOLGA2P2Y deletion was comparable between oligozoospermic and normozoospermic men (10.3% versus 11.3%). The deletion of GOLGA2P3Y but not GOLGA2P2Y was significantly higher (P = 0.03) in men with gr/gr rearrangements, indicating that GOLGA2P3Y deletions increase the susceptibility of men with gr/gr rearrangements to oligozoospermia. Furthermore, men with GOLGA2P3Y deletion had reduced sperm concentration and motility compared with men without deletion or with deletion of GOLGA2P2Y. These findings indicate GOLGA2P3Y gene copy may be candidate AZFc gene for male infertility. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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.

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.

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

Science.gov (United States)

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.

Intercellular calcium signaling occurs between human osteoblasts and osteoclasts and requires activation of osteoclast P2X7 receptors

DEFF Research Database (Denmark)

Jørgensen, Niklas R; Henriksen, Zanne; Sørensen, Ole

2002-01-01

that human osteoclasts expressed functional P2Y1 receptors, but, unexpectedly, desensitization of P2Y1 did not block calcium signaling to osteoclasts. We also found that osteoclasts expressed functional P2X7 receptors and showed that pharmacological inhibition of these receptors blocked calcium signaling...

Acidic pH facilitates peripheral αβmeATP-mediated nociception in rats: differential roles of P2X, P2Y, ASIC and TRPV1 receptors in ATP-induced mechanical allodynia and thermal hyperalgesia.

Science.gov (United States)

Seo, Hyoung-Sig; Roh, Dae-Hyun; Kwon, Soon-Gu; Yoon, Seo-Yeon; Kang, Suk-Yun; Moon, Ji-Young; Choi, Sheu-Ran; Beitz, Alvin J; Lee, Jang-Hern

2011-03-01

Peripheral ischemia is commonly associated with an increase in tissue ATP concentration and a decrease in tissue pH. Although in vitro data suggest that low tissue pH can affect ATP-binding affinities to P2 receptors, the mechanistic relationship between ATP and low pH on peripheral nociception has not been fully examined. This study was designed to investigate the potential role of an acidified environment on intraplantar αβmeATP-induced peripheral pain responses in rats. The mechanical allodynia (MA) produced by injection of αβmeATP was significantly increased in animals that received the drug diluted in pH 4.0 saline compared to those that received the drug diluted in pH 7.0 saline. Moreover, animals injected with αβmeATP (100 nmol) in pH 4.0 saline developed thermal hyperalgesia (TH), which did not occur in animals treated with αβmeATP diluted in pH 7.0 saline. To elucidate which receptors were involved in this pH-related facilitation of αβmeATP-induced MA and TH, rats were pretreated with PPADS (P2 antagonist), TNP-ATP (P2X antagonist), MRS2179 (P2Y1 antagonist), AMG9810 (TRPV1 antagonist) or amiloride (ASIC blocker). Both PPADS and TNP-ATP dose-dependently blocked pH-facilitated MA, while TH was significantly reduced by pre-treatment with MRS2179 or AMG9810. Moreover, amiloride injection significantly reduced low pH-induced facilitation of αβmeATP-mediated MA, but not TH. These results demonstrate that low tissue pH facilitates ATP-mediated MA via the activation of P2X receptors and ASICs, whereas TH induced by ATP under low pH conditions is mediated by the P2Y1 receptor and TRPV1, but not ASIC. Thus distinct mechanisms are responsible for the development of MA and TH under conditions of tissue acidosis and increased ATP. Copyright © 2010 Elsevier Ltd. All rights reserved.

Slow spontaneous [Ca2+]i oscillations reflect nucleotide release from renal epithelia

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Geyti, Christine Stride; Odgaard, Elvin V. P.; Overgaard, Morten Thaarup

2008-01-01

Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca(2+) concentration [Ca(2+)](i) through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca(2+)](i) oscillations, a feature suggested to involve nucl...

Palindromic nucleotide analysis in human T cell receptor rearrangements.

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Santosh K Srivastava

Full Text Available Diversity of T cell receptor (TCR genes is primarily generated by nucleotide insertions upon rearrangement from their germ line-encoded V, D and J segments. Nucleotide insertions at V-D and D-J junctions are random, but some small subsets of these insertions are exceptional, in that one to three base pairs inversely repeat the sequence of the germline DNA. These short complementary palindromic sequences are called P nucleotides. We apply the ImmunoSeq deep-sequencing assay to the third complementarity determining region (CDR3 of the β chain of T cell receptors, and use the resulting data to study P nucleotides in the repertoire of naïve and memory CD8(+ and CD4(+ T cells. We estimate P nucleotide distributions in a cross section of healthy adults and different T cell subtypes. We show that P nucleotide frequency in all T cell subtypes ranges from 1% to 2%, and that the distribution is highly biased with respect to the coding end of the gene segment. Classification of observed palindromic sequences into P nucleotides using a maximum conditional probability model shows that single base P nucleotides are very rare in VDJ recombination; P nucleotides are primarily two bases long. To explore the role of P nucleotides in thymic selection, we compare P nucleotides in productive and non-productive sequences of CD8(+ naïve T cells. The naïve CD8(+ T cell clones with P nucleotides are more highly expanded.

P2X receptors in epithelia

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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...

Inhibitory Effect of Flavonolignans on the P2Y12 Pathway in Blood Platelets.

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Bijak, Michal; Szelenberger, Rafal; Dziedzic, Angela; Saluk-Bijak, Joanna

2018-02-10

Adenosine diphosphate (ADP) is the major platelet agonist, which is important in the shape changes, stability, and growth of the thrombus. Platelet activation by ADP is associated with the G protein-coupled receptors P2Y1 and P2Y12. The pharmacologic blockade of the P2Y12 receptor significantly reduces the risk of peripheral artery disease, myocardial infarction, ischemic stroke, and vascular death. Recent studies demonstrated the inhibition of ADP-induced blood platelet activation by three major compounds of the flavonolignans group: silybin, silychristin, and silydianin. For this reason, the aim of the current work was to verify the effects of silybin, silychristin, and silydianin on ADP-induced physiological platelets responses, as well as mechanisms of P2Y12-dependent intracellular signal transduction. We evaluated the effect of tested flavonolignans on ADP-induced blood platelets' aggregation in platelet-rich plasma (PRP) (using light transmission aggregometry), adhesion to fibrinogen (using the static method), and the secretion of PF-4 (using the ELISA method). Additionally, using the double labeled flow cytometry method, we estimated platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. We demonstrated a dose-dependent reduction of blood platelets' ability to perform ADP-induced aggregation, adhere to fibrinogen, and secrete PF-4 in samples treated with flavonolignans. Additionally, we observed that all of the tested flavonolignans were able to increase VASP phosphorylation in blood platelets samples, which is correlated with P2Y12 receptor inhibition. All of these analyses show that silychristin and silybin have the strongest inhibitory effect on blood platelet activation by ADP, while silydianin also inhibits the ADP pathway, but to a lesser extent. The results obtained in this study clearly demonstrate that silybin, silychristin, and silydianin have inhibitory properties against the P2Y12 receptor and block ADP-induced blood platelet

LPS-induced systemic inflammation is more severe in P2Y12 null mice.

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Liverani, Elisabetta; Rico, Mario C; Yaratha, Laxmikausthubha; Tsygankov, Alexander Y; Kilpatrick, Laurie E; Kunapuli, Satya P

2014-02-01

Thienopyridines are a class of antiplatelet drugs that are metabolized in the liver to several metabolites, of which only one active metabolite can irreversibly antagonize the platelet P2Y12 receptor. Possible effects of these drugs and the role of activated platelets in inflammatory responses have also been investigated in a variety of animal models, demonstrating that thienopyridines could alter inflammation. However, it is not clear whether it is caused only by the P2Y12 antagonism or whether off-target effects of other metabolites also intervene. To address this question, we investigated P2Y12 KO mice during a LPS-induced model of systemic inflammation, and we treated these KO mice with a thienopyridine drug (clopidogrel). Contrary to the reported effects of clopidogrel, numbers of circulating WBCs and plasma levels of cytokines were increased in LPS-exposed KO mice compared with WT in this inflammation model. Moreover, both spleen and bone marrow show an increase in cell content, suggesting a role for P2Y12 in regulation of bone marrow and spleen cellular composition. Finally, the injury was more severe in the lungs of KO mice compared with WT. Interestingly, clopidogrel treatments also exerted protective effects in KO mice, suggesting off-target effects for this drug. In conclusion, the P2Y12 receptor plays an important role during LPS-induced inflammation, and this signaling pathway may be involved in regulating cell content in spleen and bone marrow during LPS systemic inflammation. Furthermore, clopidogrel may have effects that are independent of P2Y12 receptor blockade.

A2BR Adenosine Receptor Modulates Sweet Taste in Circumvallate Taste Buds

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Yang, Dan; Shultz, Nicole; Vandenbeuch, Aurelie; Ravid, Katya; Kinnamon, Sue C.; Finger, Thomas E.

2012-01-01

In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields. PMID:22253866

A2BR adenosine receptor modulates sweet taste in circumvallate taste buds.

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Kataoka, Shinji; Baquero, Arian; Yang, Dan; Shultz, Nicole; Vandenbeuch, Aurelie; Ravid, Katya; Kinnamon, Sue C; Finger, Thomas E

2012-01-01

In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields.

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...

Modulation of the TGF-β1-induced epithelial to mesenchymal transition (EMT) mediated by P1 and P2 purine receptors in MDCK cells.

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Zuccarini, Mariachiara; Giuliani, Patricia; Buccella, Silvana; Di Liberto, Valentina; Mudò, Giuseppa; Belluardo, Natale; Carluccio, Marzia; Rossini, Margherita; Condorelli, Daniele Filippo; Rathbone, Michel Piers; Caciagli, Francesco; Ciccarelli, Renata; Di Iorio, Patrizia

2017-12-01

Epithelial to mesenchymal transition (EMT) occurs during embryogenesis or under pathological conditions such as hypoxia, injury, chronic inflammation, or tissue fibrosis. In renal tubular epithelial cells (MDCK), TGF-β1 induces EMT by reducing or increasing epithelial or mesenchymal marker expression, respectively. In this study, we confirmed that the cAMP analogues, 8-CPT-cAMP or N6-Ph-cAMP, inhibited the TGF-β1-driven overexpression of the mesenchymal markers ZEB-1, Slug, Fibronectin, and α-SMA. Furthermore, we showed that A1, A2A, P2Y1, P2Y11, and P2X7 purine receptor agonists modulated the TGF-β1-induced EMT through the involvement of PKA and/or MAPK/ERK signaling. The stimulation of A2A receptor reduced the overexpression of the EMT-related markers, mainly through the cAMP-dependent PKA pathway, as confirmed by cell pre-treatment with Myr-PKI. Both A1 and P2Y1 receptor stimulation exacerbated the TGF-β1-driven effects, which were reduced by cell pre-treatment with the MAPK inhibitor PD98059, according to the increased ERK1/2 phosphorylation upon receptor activation. The effects induced by P2Y11 receptor activation were oppositely modulated by PKA or MAPK inhibition, in line with the dual nature of the Gs- and Gq-coupled receptor. Differently, P2X7 receptor induced, per se, similar and not additive effects compared to TGF-β1, after prolonged cell exposure to BzATP. These results suggest a putative role of purine receptors as target for anti-fibrotic agents.

Deletion of P2X2 and P2X3 receptor subunits does not alter motility of the mouse colon

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Matthew DeVries

2010-03-01

Full Text Available Purinergic P2X receptors contribute to neurotransmission in the gut. P2X receptors are ligand-gated cation channels that mediate synaptic excitation in subsets of enteric neurons. The present study evaluated colonic motility in vitro and in vivo in wild type (WT and P2X2 and P2X3 subunit knockout (KO mice. The muscarinic receptor agonist, bethanechol (0.3-3 micromolar, caused similar contractions of the longitudinal muscle in colon segments from WT, P2X2 and P2X3 subunit KO mice. Nicotine (1-300 micromolar, acting at neuronal nicotinic receptors, caused similar longitudinal muscle relaxations in colonic segments from WT and P2X2 and P2X3 subunit KO mice. Nicotine-induced relaxations were inhibited by nitro-L-arginine (NLA, 100 micromolar and apamin (0.1 micromolar which block inhibitory neuromuscular transmission. ATP (1-1000 micromolar caused contractions only in the presence of NLA and apamin. ATP-induced contractions were similar in colon segments from WT, P2X2 and P2X3 KO mice. The mouse colon generates spontaneous migrating motor complexes (MMCs in vitro. The MMC frequency was higher in P2X2 KO compared to WT tissues; other parameters of the MMC were similar in colon segments from WT, P2X2 and P2X3 KO mice. 5-Hydroxytryptophan-induced fecal output was similar in WT, P2X2 and P2X3 KO mice. These data indicate that nicotinic receptors are located predominately on inhibitory motor neurons supplying the longitudinal muscle in the mouse colon. P2X2 or P2X3 subunit containing receptors are not localized to motorneurons supplying the longitudinal muscle. Synaptic transmission mediated by P2X2 or P2X3 subunit containing receptors is not required for propulsive motility in the mouse colon.

Transcription factor FOXO1 promotes cell migration toward exogenous ATP via controlling P2Y1 receptor expression in lymphatic endothelial cells.

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Niimi, Kenta; Ueda, Mizuha; Fukumoto, Moe; Kohara, Misaki; Sawano, Toshinori; Tsuchihashi, Ryo; Shibata, Satoshi; Inagaki, Shinobu; Furuyama, Tatsuo

2017-08-05

Sprouting migration of lymphatic endothelial cell (LEC) is a pivotal step in lymphangiogenic process. However, its molecular mechanism remains unclear including effective migratory attractants. Meanwhile, forkhead transcription factor FOXO1 highly expresses in LEC nuclei, but its significance in LEC migratory activity has not been researched. In this study, we investigated function of FOXO1 transcription factor associated with LEC migration toward exogenous ATP which has recently gathered attentions as a cell migratory attractant. The transwell membrane assay indicated that LECs migrated toward exogenous ATP, which was impaired by FOXO1 knockdown. RT-PCR analysis showed that P2Y1, a purinergic receptor, expression was markedly reduced by FOXO1 knockdown in LECs. Moreover, P2Y1 blockage impaired LEC migration toward exogenous ATP. Western blot analysis revealed that Akt phosphorylation contributed to FOXO1-dependent LEC migration toward exogenous ATP and its blockage affected LEC migratory activity. Furthermore, luciferase reporter assay and ChIP assay suggested that FOXO1 directly bound to a conserved binding site in P2RY1 promoter and regulated its activity. These results indicated that FOXO1 serves a pivotal role in LEC migration toward exogenous ATP via direct transcriptional regulation of P2Y1 receptor. Copyright © 2017 Elsevier Inc. All rights reserved.

Purinergic receptors and calcium signalling in human pancreatic duct cell lines

DEFF Research Database (Denmark)

Hansen, Mette R; Krabbe, Simon; Novak, Ivana

2008-01-01

pancreatic duct cell lines PANC-1 and CFPAC-1. Expression of P2 receptors was examined using RT-PCR and immunocytochemistry. Both cell lines, and also Capan-1 cells, express RNA transcripts for the following receptors: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11-14 and P2X1, P2X2, P2X4, P2X5, P2X6 and P2X7. Using Fura-2...... and single-cell imaging we tested effects of various nucleotide analogues on intracellular Ca(2+) signals in PANC-1 and CFPAC-1 cells. The cell lines responded to all nucleotides with the following efficiency: UTP >or= ATP = ATPgammaS > BzATP. ATP, UTP and ATPgammaS elicited oscillatory responses. Bz...

A2BR adenosine receptor modulates sweet taste in circumvallate taste buds.

Directory of Open Access Journals (Sweden)

Shinji Kataoka

Full Text Available In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3 on taste nerves as well as metabotropic (P2Y purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate, but not anterior (fungiform, palate taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields.

P2X receptors, sensory neurons and pain.

Science.gov (United States)

Bele, Tanja; Fabbretti, Elsa

2015-01-01

Pain represents a very large social and clinical problem since the current treatment provides insufficient pain relief. Plasticity of pain receptors together with sensitisation of sensory neurons, and the role of soluble mediators released from non-neuronal cells render difficult to understand the spatial and temporal scale of pain development, neuronal responses and disease progression. In pathological conditions, ATP is one of the most powerful mediators that activates P2X receptors that behave as sensitive ATP-detectors, such as neuronal P2X3 receptor subtypes and P2X4 and P2X7 receptors expressed on non-neuronal cells. Dissecting the molecular mechanisms occurring in sensory neurons and in accessory cells allows to design appropriate tissue- and cell- targeted approaches to treat chronic pain.

THE ROLE OF CLOPIDOGREL IN PATIENTS WITH ACUTE CORONARY SYNDROME AFTER THE EMERGENCE OF MORE POWERFUL INHIBITORS OF P2Y12 RECEPTOR

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S. R. Gilyarevsky

2014-07-01

Full Text Available The role of P2Y12 receptor blocker clopidogrel after the introduction into clinical practice of new, more powerful and stable operating drugs belonging to this class of antiplatelet agents is discussed. The advantages and disadvantages of each of the currently used antiplatelet drugs that block the receptor P2Y12 are reviewed. On the basis of the analysis concludes that, despite the emergence of new antiplatelet agents clopidogrel, appears to be widely used for a long time in the treatment of patients with acute coronary syndrome and / or after coronary stenting. This is primarily due to the presence of large evidence base, and confirmation of safety of long-term therapy clopidogrel.

Uridine adenosine tetraphosphate (Up4A) is a strong inductor of smooth muscle cell migration via activation of the P2Y2 receptor and cross-communication to the PDGF receptor

International Nuclear Information System (INIS)

Wiedon, Annette; Tölle, Markus; Bastine, Joschika; Schuchardt, Mirjam; Huang, Tao; Jankowski, Vera; Jankowski, Joachim; Zidek, Walter; Giet, Markus van der

2012-01-01

Highlights: ► Up 4 A induces VSMC migration. ► VSMC migration towards Up 4 A involves P2Y 2 activation. ► Up 4 A-induced VSMC migration is OPN-dependent. ► Activation of ERK1/2 pathway is necessary for VSMC migration towards Up 4 A. ► Up 4 A-directed VSMC migration cross-communicates with the PDGFR. -- Abstract: The recently discovered dinucleotide uridine adenosine tetraphosphate (Up 4 A) was found in human plasma and characterized as endothelium-derived vasoconstrictive factor (EDCF). A further study revealed a positive correlation between Up 4 A and vascular smooth muscle cell (VSMC) proliferation. Due to the dominant role of migration in the formation of atherosclerotic lesions our aim was to investigate the migration stimulating potential of Up 4 A. Indeed, we found a strong chemoattractant effect of Up 4 A on VSMC by using a modified Boyden chamber. This migration dramatically depends on osteopontin secretion (OPN) revealed by the reduction of the migration signal down to 23% during simultaneous incubation with an OPN-blocking antibody. Due to inhibitory patterns using specific and unspecific purinoreceptor inhibitors, Up 4 A mediates it’s migratory signal mainly via the P2Y 2 . The signaling behind the receptor was investigated with luminex technique and revealed an activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway. By use of the specific PDGF receptor (PDGFR) inhibitor AG1296 and siRNA technique against PDGFR-β we found a strongly reduced migration signal after Up 4 A stimulation in the PDGFR-β knockdown cells compared to control cells. In this study, we present substantiate data that Up 4 A exhibits migration stimulating potential probably involving the signaling cascade of MEK1 and ERK1/2 as well as the matrix protein OPN. We further suggest that the initiation of the migration process occurs predominant through direct activation of the P2Y 2 by Up 4 A and via transactivation of the PDGFR.

Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding.

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Mundell, S J; Rabbolini, D; Gabrielli, S; Chen, Q; Aungraheeta, R; Hutchinson, J L; Kilo, T; Mackay, J; Ward, C M; Stevenson, W; Morel-Kopp, M-C

2018-01-01

Essentials Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. To date, there has been no phenotype/genotype correlation explaining their dominant transmission. Proline plays an important role in P2Y12R ligand binding and signaling defects. P2Y12R homodimer formation is critical for the receptor function and signaling. Background Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is

ATP mediates NADPH oxidase/ROS generation and COX-2/PGE2 expression in A549 cells: role of P2 receptor-dependent STAT3 activation.

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Shin-Ei Cheng

Full Text Available BACKGROUND: Up-regulation of cyclooxygenase (COX-2 and its metabolite prostaglandin E(2 (PGE(2 are frequently implicated in lung inflammation. Extracellular nucleotides, such as ATP have been shown to act via activation of P2 purinoceptors, leading to COX-2 expression in various inflammatory diseases, such as lung inflammation. However, the mechanisms underlying ATP-induced COX-2 expression and PGE(2 release remain unclear. PRINCIPAL FINDINGS: Here, we showed that ATPγS induced COX-2 expression in A549 cells revealed by western blot and real-time PCR. Pretreatment with the inhibitors of P2 receptor (PPADS and suramin, PKC (Gö6983, Gö6976, Ro318220, and Rottlerin, ROS (Edaravone, NADPH oxidase [diphenyleneiodonium chloride (DPI and apocynin], Jak2 (AG490, and STAT3 [cucurbitacin E (CBE] and transfection with siRNAs of PKCα, PKCι, PKCμ, p47(phox, Jak2, STAT3, and cPLA(2 markedly reduced ATPγS-induced COX-2 expression and PGE(2 production. In addition, pretreatment with the inhibitors of P2 receptor attenuated PKCs translocation from the cytosol to the membrane in response to ATPγS. Moreover, ATPγS-induced ROS generation and p47(phox translocation was also reduced by pretreatment with the inhibitors of P2 receptor, PKC, and NADPH oxidase. On the other hand, ATPγS stimulated Jak2 and STAT3 activation which were inhibited by pretreatment with PPADS, suramin, Gö6983, Gö6976, Ro318220, GF109203X, Rottlerin, Edaravone, DPI, and apocynin in A549 cells. SIGNIFICANCE: Taken together, these results showed that ATPγS induced COX-2 expression and PGE(2 production via a P2 receptor/PKC/NADPH oxidase/ROS/Jak2/STAT3/cPLA(2 signaling pathway in A549 cells. Increased understanding of signal transduction mechanisms underlying COX-2 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies.

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

NPY2-receptor variation modulates iconic memory processes.

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Arning, Larissa; Stock, Ann-Kathrin; Kloster, Eugen; Epplen, Jörg T; Beste, Christian

2014-08-01

Sensory memory systems are modality-specific buffers that comprise information about external stimuli, which represent the earliest stage of information processing. While these systems have been the subject of cognitive neuroscience research for decades, little is known about the neurobiological basis of sensory memory. However, accumulating evidence suggests that the glutamatergic system and systems influencing glutamatergic neural transmission are important. In the current study we examine if functional promoter variations in neuropeptide Y (NPY) and its receptor gene NPY2R affect iconic memory processes using a partial report paradigm. We found that iconic memory decayed much faster in individuals carrying the rare promoter NPY2R G allele which is associated with increased expression of the Y2 receptor. Possibly this effect is due to altered presynaptic inhibition of glutamate release, known to be modulated by Y2 receptors. Altogether, our results provide evidence that the functionally relevant single nucleotide polymorphism (SNP) in the NPY2R promoter gene affect circumscribed processes of early sensory processing, i.e. only the stability of information in sensory memory buffers. This leads us to suggest that especially the stability of information in sensory memory buffers depends on glutamatergic neural transmission and factors modulating glutamatergic turnover. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

Multiple roles of the extracellular vestibule amino acid residues in the function of the rat P2X4 receptor.

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Milos B Rokic

Full Text Available The binding of ATP to trimeric P2X receptors (P2XR causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.

Rationale and design of the Affordability and Real-world Antiplatelet Treatment Effectiveness after Myocardial Infarction Study (ARTEMIS): A multicenter, cluster-randomized trial of P2Y12 receptor inhibitor copayment reduction after myocardial infarction.

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Doll, Jacob A; Wang, Tracy Y; Choudhry, Niteesh K; Cannon, Christopher P; Cohen, David J; Fonarow, Gregg C; Henry, Timothy D; Bhandary, Durgesh D; Khan, Naeem; Davidson-Ray, Linda D; Anstrom, Kevin; Peterson, Eric D

2016-07-01

The use of oral P2Y12 receptor inhibitors after acute myocardial infarction (MI) can reduce risks of subsequent major adverse cardiovascular events (composite of all-cause death, recurrent MI, and stroke), yet medication persistence is suboptimal. Although copayment cost has been implicated as a factor influencing medication persistence, it remains unclear whether reducing or eliminating these costs can improve medication persistence and/or downstream clinical outcomes. ARTEMIS is a multicenter, cluster-randomized clinical trial designed to examine whether eliminating patient copayment for P2Y12 receptor inhibitor therapy affects medication persistence and clinical outcomes. We will enroll approximately 11,000 patients hospitalized for acute ST-elevation and non-ST-elevation MI at 300 hospitals. Choice and duration of treatment with a P2Y12 receptor inhibitor will be determined by the treating physician. Hospitals randomized to the copayment intervention will provide vouchers to cover patients' copayments for their P2Y12 receptor inhibitor for up to 1 year after discharge. The coprimary end points are 1-year P2Y12 receptor inhibitor persistence and major adverse cardiovascular events. Secondary end points include choice of P2Y12 receptor inhibitor, patient-reported outcomes, and postdischarge cost of care. ARTEMIS will be the largest randomized assessment of a medication copayment reduction intervention on medication persistence, clinical outcomes, treatment selection, and cost of care after acute MI. Copyright © 2016 Elsevier Inc. All rights reserved.

Risk of estrogen receptor-positive and -negative breast cancer and single-nucleotide polymorphism 2q35-rs13387042

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Milne, Roger L; Benítez, Javier; Nevanlinna, Heli

2009-01-01

BACKGROUND: A recent genome-wide association study identified single-nucleotide polymorphism (SNP) 2q35-rs13387042 as a marker of susceptibility to estrogen receptor (ER)-positive breast cancer. We attempted to confirm this association using the Breast Cancer Association Consortium. METHODS: 2q35...

P2Y12 Receptor Localizes in the Renal Collecting Duct and Its Blockade Augments Arginine Vasopressin Action and Alleviates Nephrogenic Diabetes Insipidus.

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Zhang, Yue; Peti-Peterdi, Janos; Müller, Christa E; Carlson, Noel G; Baqi, Younis; Strasburg, David L; Heiney, Kristina M; Villanueva, Karie; Kohan, Donald E; Kishore, Bellamkonda K

2015-12-01

P2Y12 receptor (P2Y12-R) signaling is mediated through Gi, ultimately reducing cellular cAMP levels. Because cAMP is a central modulator of arginine vasopressin (AVP)-induced water transport in the renal collecting duct (CD), we hypothesized that if expressed in the CD, P2Y12-R may play a role in renal handling of water in health and in nephrogenic diabetes insipidus. We found P2Y12-R mRNA expression in rat kidney, and immunolocalized its protein and aquaporin-2 (AQP2) in CD principal cells. Administration of clopidogrel bisulfate, an irreversible inhibitor of P2Y12-R, significantly increased urine concentration and AQP2 protein in the kidneys of Sprague-Dawley rats. Notably, clopidogrel did not alter urine concentration in Brattleboro rats that lack AVP. Clopidogrel administration also significantly ameliorated lithium-induced polyuria, improved urine concentrating ability and AQP2 protein abundance, and reversed the lithium-induced increase in free-water excretion, without decreasing blood or kidney tissue lithium levels. Clopidogrel administration also augmented the lithium-induced increase in urinary AVP excretion and suppressed the lithium-induced increase in urinary nitrates/nitrites (nitric oxide production) and 8-isoprostane (oxidative stress). Furthermore, selective blockade of P2Y12-R by the reversible antagonist PSB-0739 in primary cultures of rat inner medullary CD cells potentiated the expression of AQP2 and AQP3 mRNA, and cAMP production induced by dDAVP (desmopressin). In conclusion, pharmacologic blockade of renal P2Y12-R increases urinary concentrating ability by augmenting the effect of AVP on the kidney and ameliorates lithium-induced NDI by potentiating the action of AVP on the CD. This strategy may offer a novel and effective therapy for lithium-induced NDI. Copyright © 2015 by the American Society of Nephrology.

[Replication of Streptomyces plasmids: the DNA nucleotide sequence of plasmid pSB 24.2].

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Bolotin, A P; Sorokin, A V; Aleksandrov, N N; Danilenko, V N; Kozlov, Iu I

1985-11-01

The nucleotide sequence of DNA in plasmid pSB 24.2, a natural deletion derivative of plasmid pSB 24.1 isolated from S. cyanogenus was studied. The plasmid amounted by its size to 3706 nucleotide pairs. The G-C composition was equal to 73 per cent. The analysis of the DNA structure in plasmid pSB 24.2 revealed the protein-encoding sequence of DNA, the continuity of which was significant for replication of the plasmid containing more than 1300 nucleotide pairs. The analysis also revealed two A-T-rich areas of DNA, the G-C composition of which was less than 55 per cent and a DNA area with a branched pin structure. The results may be of value in investigation of plasmid replication in actinomycetes and experimental cloning of DNA with this plasmid as a vector.

Peripherally Administered Y2-Receptor Antagonist BIIE0246 Prevents Diet-Induced Obesity in Mice With Excess Neuropeptide Y, but Enhances Obesity in Control Mice.

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Ailanen, Liisa; Vähätalo, Laura H; Salomäki-Myftari, Henriikka; Mäkelä, Satu; Orpana, Wendy; Ruohonen, Suvi T; Savontaus, Eriika

2018-01-01

Neuropeptide Y (NPY) plays an important role in the regulation of energy homeostasis in the level of central and sympathetic nervous systems (SNSs). Genetic silencing of peripheral Y 2 -receptors have anti-obesity effects, but it is not known whether pharmacological blocking of peripheral Y 2 -receptors would similarly benefit energy homeostasis. The effects of a peripherally administered Y 2 -receptor antagonist were studied in healthy and energy-rich conditions with or without excess NPY. Genetically obese mice overexpressing NPY in brain noradrenergic nerves and SNS (OE-NPY DβH ) represented the situation of elevated NPY levels, while wildtype (WT) mice represented the normal NPY levels. Specific Y 2 -receptor antagonist, BIIE0246, was administered (1.3 mg/kg/day, i.p.) for 2 or 4.5 weeks to OE-NPY DβH and WT mice feeding on chow or Western diet. Treatment with Y 2 -receptor antagonist increased body weight gain in both genotypes on chow diet and caused metabolic disturbances (e.g., hyperinsulinemia and hypercholesterolemia), especially in WT mice. During energy surplus (i.e., on Western diet), blocking of Y 2 -receptors induced obesity in WT mice, whereas OE-NPY DβH mice showed reduced fat mass gain, hepatic glycogen and serum cholesterol levels relative to body adiposity. Thus, it can be concluded that with normal NPY levels, peripheral Y 2 -receptor antagonist has no potential for treating obesity, but oppositely may even induce metabolic disorders. However, when energy-rich diet is combined with elevated NPY levels, e.g., stress combined with an unhealthy diet, Y 2 -receptor antagonism has beneficial effects on metabolic status.

Peripheral-specific y2 receptor knockdown protects mice from high-fat diet-induced obesity.

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Shi, Yan-Chuan; Lin, Shu; Castillo, Lesley; Aljanova, Aygul; Enriquez, Ronaldo F; Nguyen, Amy D; Baldock, Paul A; Zhang, Lei; Bijker, Martijn S; Macia, Laurence; Yulyaningsih, Ernie; Zhang, Hui; Lau, Jackie; Sainsbury, Amanda; Herzog, Herbert

2011-11-01

Y2 receptors, particularly those in the brain, have been implicated in neuropeptide Y (NPY)-mediated effects on energy homeostasis and bone mass. Recent evidence also indicates a role for Y2 receptors in peripheral tissues in this process by promoting adipose tissue accretion; however their effects on energy balance remain unclear. Here, we show that adult-onset conditional knockdown of Y2 receptors predominantly in peripheral tissues results in protection against diet-induced obesity accompanied by significantly reduced weight gain, marked reduction in adiposity and improvements in glucose tolerance without any adverse effect on lean mass or bone. These changes occur in association with significant increases in energy expenditure, respiratory exchange ratio, and physical activity and despite concurrent hyperphagia. On a chow diet, knockdown of peripheral Y2 receptors results in increased respiratory exchange ratio and physical activity with no effect on lean or bone mass, but decreases energy expenditure without effecting body weight or food intake. These results suggest that peripheral Y2 receptor signaling is critical in the regulation of oxidative fuel selection and physical activity and protects against the diet-induced obesity. The lack of effects on bone mass seen in this model further indicates that bone mass is primarily controlled by non-peripheral Y2 receptors. This study provides evidence that novel drugs that target peripheral rather than central Y2 receptors could provide benefits for the treatment of obesity and glucose intolerance without adverse effects on lean and bone mass, with the additional benefit of avoiding side effects often associated with pharmaceuticals that act on the central nervous system.

The regulation of aortic endothelial cells by purines and pyrimidines involves co-existing P2y-purinoceptors and nucleotide receptors linked to phospholipase C.

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Wilkinson, G. F.; Purkiss, J. R.; Boarder, M. R.

1993-01-01

1. We have examined the phospholipase C responses in bovine aortic endothelial cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. The cells responded to purines in a manner consistent with the presence of P2y purinoceptors; both 2-methylthioadenosine 5'-triphosphate (2MeSATP) and adenosine 5'-0-(2-thiodiphosphate) (ADP beta S) were potent agonists (EC50 0.41 microM and 0.85 microM respectively) while beta, gamma-methylene ATP at 300 microM was not. 3....

Enhancement of the response to purinergic agonists in P2Y1 transfected 1321N1 cells by antagonists suramin and PPADS.

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Brown, C A; Charlton, S J; Boarder, M R

1997-03-01

1. We have previously shown that both suramin and pyridoxal-phosphate-6-azophenyl-2',4' disulphonic acid (PPADS) act as antagonists at transfected P2Y1 receptors. Here we show that under certain experimental conditions these two P2 antagonists can enhance the response to agonists acting at these receptors. 2. The expression of either P2Y1 or P2Y2 receptors in 1321N1 human astrocytoma cells results, on a change of medium, in an elevation of basal (no added agonist) accumulation of [3H]-inositol(poly)phosphates([3H]-InsPx) compared to cells not expressing these receptors. This elevation is much greater in P2Y1 transfectants than in P2Y, transfectants. 3. Both PPADS and suramin reduced this basal level of [3H]-InsPx accumulation in the P2Y1 expressing cells. 4. When a protocol was used which required changing the culture medium, antagonists were added at a concentration which reduced the basal accumulation by about 50%, there was a significant stimulation in response to increasing concentrations of 2-methylthioadenosine 5'-triphosphate (2MeSATP), in the absence of antagonists there was no significant effect of the agonist. 5. However, when 2MeSATP was added in the absence of a change of medium and with no antagonist present, there was a several fold increase in [3H]-InsPx accumulation. These results show that a release of endogenous agonist activity (possibly ATP/ADP) from the P2Y1 expressing cells can create conditions in which a response to an agonist such as 2MeSATP can only be seen in the presence of a competitive antagonist.

The role of P2X receptors in bone biology.

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Jørgensen, N R; Syberg, S; Ellegaard, M

2015-01-01

Bone is a highly dynamic organ, being constantly modeled and remodeled in order to adapt to the changing need throughout life. Bone turnover involves the coordinated actions of bone formation and bone degradation. Over the past decade great effort has been put into the examination of how P2X receptors regulate bone metabolism and especially for the P2X7 receptor an impressive amount of evidence has now documented its expression in osteoblasts, osteoclasts, and osteocytes as well as important functional roles in proliferation, differentiation, and function of the cells of bone. Key evidence has come from studies on murine knockout models and from pharmacologic studies on cells and animals. More recently, the role of P2X receptors in human bone diseases has been documented. Loss-of-functions polymorphisms in the P2X7 receptorare associated with bone loss and increased fracture risk. Very recently a report from a genetic study in multiple myeloma demonstrated that decreased P2X7 receptor function was associated with increased risk of developing multiple myeloma. In contrast, the risk of developing myeloma bone disease and subsequent vertebral fractures was increased in subjects carrying P2X7 receptor gain-of-function alleles as compared to subjects only carrying loss-of-function or normal functioning alleles. It is evident that P2X receptors are important in regulating bone turnover and maintaining bone mass, and thereby holding great potential as novel drug targets for treatment of bone diseases. However, further research is needed before we fully understand the roles and effects of P2X receptors in bone.

Communication between corneal epithelial cells and trigeminal neurons is facilitated by purinergic (P2) and glutamatergic receptors.

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Oswald, Duane J; Lee, Albert; Trinidad, Monique; Chi, Cheryl; Ren, Ruiyi; Rich, Celeste B; Trinkaus-Randall, Vickery

2012-01-01

Previously, we demonstrated that nucleotides released upon mechanical injury to corneal epithelium activate purinergic (P2) receptors resulting in mobilization of a Ca(2+) wave. However, the tissue is extensively innervated and communication between epithelium and neurons is critical and not well understood. Therefore, we developed a co-culture of primary trigeminal neurons and human corneal limbal epithelial cells. We demonstrated that trigeminal neurons expressed a repertoire of P2Yand P2X receptor transcripts and responded to P2 agonists in a concentration-dependent manner. Mechanical injuries to epithelia in the co-cultures elicited a Ca(2+) wave that mobilized to neurons and was attenuated by Apyrase, an ectonucleotidase. To elucidate the role of factors released from each cell type, epithelial and neuronal cells were cultured, injured, and the wound media from one cell type was collected and added to the other cell type. Epithelial wound media generated a rapid Ca(2+) mobilization in neuronal cells that was abrogated in the presence of Apyrase, while neuronal wound media elicited a complex response in epithelial cells. The rapid Ca(2+) mobilization was detected, which was abrogated with Apyrase, but it was followed by Ca(2+) waves that occurred in cell clusters. When neuronal wound media was preincubated with a cocktail of N-methyl-D-aspartate (NMDA) receptor inhibitors, the secondary response in epithelia was diminished. Glutamate was detected in the neuronal wound media and epithelial expression of NMDA receptor subunit transcripts was demonstrated. Our results indicate that corneal epithelia and neurons communicate via purinergic and NMDA receptors that mediate the wound response in a highly orchestrated manner.

Communication between corneal epithelial cells and trigeminal neurons is facilitated by purinergic (P2 and glutamatergic receptors.

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Duane J Oswald

Full Text Available Previously, we demonstrated that nucleotides released upon mechanical injury to corneal epithelium activate purinergic (P2 receptors resulting in mobilization of a Ca(2+ wave. However, the tissue is extensively innervated and communication between epithelium and neurons is critical and not well understood. Therefore, we developed a co-culture of primary trigeminal neurons and human corneal limbal epithelial cells. We demonstrated that trigeminal neurons expressed a repertoire of P2Yand P2X receptor transcripts and responded to P2 agonists in a concentration-dependent manner. Mechanical injuries to epithelia in the co-cultures elicited a Ca(2+ wave that mobilized to neurons and was attenuated by Apyrase, an ectonucleotidase. To elucidate the role of factors released from each cell type, epithelial and neuronal cells were cultured, injured, and the wound media from one cell type was collected and added to the other cell type. Epithelial wound media generated a rapid Ca(2+ mobilization in neuronal cells that was abrogated in the presence of Apyrase, while neuronal wound media elicited a complex response in epithelial cells. The rapid Ca(2+ mobilization was detected, which was abrogated with Apyrase, but it was followed by Ca(2+ waves that occurred in cell clusters. When neuronal wound media was preincubated with a cocktail of N-methyl-D-aspartate (NMDA receptor inhibitors, the secondary response in epithelia was diminished. Glutamate was detected in the neuronal wound media and epithelial expression of NMDA receptor subunit transcripts was demonstrated. Our results indicate that corneal epithelia and neurons communicate via purinergic and NMDA receptors that mediate the wound response in a highly orchestrated manner.

Neuropeptide Y2 receptors in anteroventral BNST control remote fear memory depending on extinction training.

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Verma, Dilip; Tasan, Ramon; Sperk, Guenther; Pape, Hans-Christian

2018-03-01

The anterior bed nucleus of stria terminalis (BNST) is involved in reinstatement of extinguished fear, and neuropeptide Y2 receptors influence local synaptic signaling. Therefore, we hypothesized that Y2 receptors in anteroventral BNST (BNSTav) interfere with remote fear memory and that previous fear extinction is an important variable. C57BL/6NCrl mice were fear-conditioned, and a Y2 receptor-specific agonist (NPY 3-36 ) or antagonist (JNJ-5207787) was applied in BNSTav before fear retrieval at the following day. Remote fear memory was tested on day 16 in two groups of mice, which had (experiment 1) or had not (experiment 2) undergone extinction training after conditioning. In the group with extinction training, tests of remote fear memory revealed partial retrieval of extinction, which was prevented after blockade of Y2 receptors in BNSTav. No such effect was observed in the group with no extinction training, but stimulation of Y2 receptors in BNSTav mimicked the influence of extinction during tests of remote fear memory. Pharmacological manipulation of Y2 receptors in BNSTav before fear acquisition (experiment 3) had no effect on fear memory retrieval, extinction or remote fear memory. Furthermore, partial retrieval of extinction during tests of remote fear memory was associated with changes in number of c-Fos expressing neurons in BNSTav, which was prevented or mimicked upon Y2 blockade or stimulation in BNSTav. These results indicate that Y2 receptor manipulation in BNSTav interferes with fear memory and extinction retrieval at remote stages, likely through controlling neuronal activity in BNSTav during extinction training. Copyright © 2018 Elsevier Inc. All rights reserved.

Purinergic receptors in the endocrine and exocrine pancreas.

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Novak, I

2008-09-01

The pancreas is a complex gland performing both endocrine and exocrine functions. In recent years there has been increasing evidence that both endocrine and exocrine cells possess purinergic receptors, which influence processes such as insulin secretion and epithelial ion transport. Most commonly, these processes have been viewed separately. In beta cells, stimulation of P2Y(1) receptors amplifies secretion of insulin in the presence of glucose. Nucleotides released from secretory granules could also contribute to autocrine/paracrine regulation in pancreatic islets. In addition to P2Y(1) receptors, there is also evidence for other P2 and adenosine receptors in beta cells (P2Y(2), P2Y(4), P2Y(6), P2X subtypes and A(1) receptors) and in glucagon-secreting alpha cells (P2X(7), A(2) receptors). In the exocrine pancreas, acini release ATP and ATP-hydrolysing and ATP-generating enzymes. P2 receptors are prominent in pancreatic ducts, and several studies indicate that P2Y(2), P2Y(4), P2Y(11), P2X(4) and P2X(7) receptors could regulate secretion, primarily by affecting Cl(-) and K(+) channels and intracellular Ca(2+) signalling. In order to understand the physiology of the whole organ, it is necessary to consider the full complement of purinergic receptors on different cells as well as the structural and functional relation between various cells within the whole organ. In addition to the possible physiological function of purinergic receptors, this review analyses whether the receptors could be potential therapeutic targets for drug design aimed at treatment of pancreatic diseases.

Chronic administration of the selective P2X3, P2X2/3 receptor antagonist, A-317491, transiently attenuates cancer-induced bone pain in mice

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Hansen, Rikke Rie; Nasser, Arafat; Falk, Sarah

2012-01-01

The purinergic P2X3 and P2X2/3 receptors are in the peripheral nervous system almost exclusively confined to afferent sensory neurons, where they are found both at peripheral and central synapses. The P2X3 receptor is implicated in both neuropathic and inflammatory pain. However, the role of the ......X3 receptor in chronic cancer-induced bone pain is less known. Here we investigated the effect of systemic acute and chronic administration of the selective P2X3, P2X2/3 receptor antagonist (5-[[[(3-Phenoxyphenyl)methyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl]-1...

DESENSITIZATION PROPERTIES OF P2X3 RECEPTORS SHAPING PAIN SIGNALLING

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Rashid eGiniatullin

2013-12-01

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

Adeno-associated viral vector-induced overexpression of neuropeptide Y Y2 receptors in the hippocampus suppresses seizures

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Woldbye, David Paul Drucker; Ängehagen, Mikael; Gøtzsche, Casper René

2010-01-01

Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure...... recombinant adeno-associated viral vectors. In two temporal lobe epilepsy models, electrical kindling and kainate-induced seizures, vector-based transduction of Y2 receptor complementary DNA in the hippocampus of adult rats exerted seizure-suppressant effects. Simultaneous overexpression of Y2...... and neuropeptide Y had a more pronounced seizure-suppressant effect. These results demonstrate that overexpression of Y2 receptors (alone or in combination with neuropeptide Y) could be an alternative strategy for epilepsy treatment....

Comparison of P2 purinergic receptors of aortic endothelial cells with those of adrenal medulla: evidence for heterogeneity of receptor subtype and of inositol phosphate response.

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Allsup, D J; Boarder, M R

1990-07-01

Vascular endothelial cells from different parts of the circulation are known to show different functional responses, presumably corresponding to physiological roles. Previous studies have shown that ATP acts on P2 purinergic receptors of endothelial cells of major blood vessels, stimulating the formation of inositol phosphates. Here we have compared the action of ATP and congeners acting on endothelial cells of bovine thoracic aorta with cells derived from the microvasculature of bovine adrenal medulla. With measurement of total inositol phosphates, cells from the aorta showed a rank order of agonist potency of 2-methylthio-ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) greater than ADP greater than ATP greater than beta, gamma-imido-ATP greater than beta, gamma-methylene-ATP, consistent with action at receptors of the P2Y subtype. However, with adrenal cells the rank order of potency was ATP gamma S greater than ATP greater than beta, gamma-imido-ATP greater than ADP greater than beta, gamma-methylene-ATP = 2-methylthio-ATP. This profile is not consistent with either P2X or P2Y receptors. When the nature of this inositol phosphate response was analyzed with anion exchange chromatography, it was found that the aortic cells showed an inositol trisphosphate stimulation that peaked within a few seconds and rapidly declined, whereas the response of the adrenal medulla cells continued to rise through 5 min. Analysis of isomers of inositol phosphates revealed a different pattern of metabolism between the two cell types, which may account for the different time course of response. With adrenal cells, ATP at low micromolar concentrations caused a dose-dependent increase in levels of cyclic AMP and had a greater than additive effect on cyclic AMP levels when combined with submaximal stimulation by prostaglandin E2. These results suggest the presence of a P2Y receptor on aortic endothelial cells, with an 'atypical' purinocepter, i.e., neither P2X nor P2Y

Early versus delayed invasive strategy for intermediate- and high-risk acute coronary syndromes managed without P2Y12 receptor inhibitor pretreatment: Design and rationale of the EARLY randomized trial.

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Lemesle, Gilles; Laine, Marc; Pankert, Mathieu; Puymirat, Etienne; Cuisset, Thomas; Boueri, Ziad; Maillard, Luc; Armero, Sébastien; Cayla, Guillaume; Bali, Laurent; Motreff, Pascal; Peyre, Jean-Pascal; Paganelli, Franck; Kerbaul, François; Roch, Antoine; Michelet, Pierre; Baumstarck, Karine; Bonello, Laurent

2018-01-01

According to recent literature, pretreatment with a P2Y 12 ADP receptor antagonist before coronary angiography appears no longer suitable in non-ST-segment elevation acute coronary syndrome (NSTE-ACS) due to an unfavorable risk-benefit ratio. Optimal delay of the invasive strategy in this specific context is unknown. We hypothesize that without P2Y 12 ADP receptor antagonist pretreatment, a very early invasive strategy may be beneficial. The EARLY trial (Early or Delayed Revascularization for Intermediate- and High-Risk Non-ST-Segment Elevation Acute Coronary Syndromes?) is a prospective, multicenter, randomized, controlled, open-label, 2-parallel-group study that plans to enroll 740 patients. Patients are eligible if the diagnosis of intermediate- or high-risk NSTE-ACS is made and an invasive strategy intended. Patients are randomized in a 1:1 ratio. In the control group, a delayed strategy is adopted, with the coronary angiography taking place between 12 and 72 hours after randomization. In the experimental group, a very early invasive strategy is performed within 2 hours. A loading dose of a P2Y 12 ADP receptor antagonist is given at the time of intervention in both groups. Recruitment began in September 2016 (n = 558 patients as of October 2017). The primary endpoint is the composite of cardiovascular death and recurrent ischemic events at 1 month. The EARLY trial aims to demonstrate the superiority of a very early invasive strategy compared with a delayed strategy in intermediate- and high-risk NSTE-ACS patients managed without P2Y 12 ADP receptor antagonist pretreatment. © 2018 Wiley Periodicals, Inc.

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

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Marques, Rita D; Praetorius, Helle A; Leipziger, Jens

2013-01-01

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 nuc...

P2X receptors as targets for the treatment of status epilepticus

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Henshall, David C.; Diaz-Hernandez, Miguel; Miras-Portugal, M. Teresa; Engel, Tobias

2013-01-01

Prolonged seizures are amongst the most common neurological emergencies. Status epilepticus is a state of continuous seizures that is life-threatening and prompt termination of status epilepticus is critical to protect the brain from permanent damage. Frontline treatment comprises parenteral administration of anticonvulsants such as lorazepam that facilitate γ-amino butyric acid (GABA) transmission. Because status epilepticus can become refractory to anticonvulsants in a significant proportion of patients, drugs which act on different neurotransmitter systems may represent potential adjunctive treatments. P2X receptors are a class of ligand-gated ion channel activated by ATP that contributes to neuro- and glio-transmission. P2X receptors are expressed by both neurons and glia in various brain regions, including the hippocampus. Electrophysiology, pharmacology and genetic studies suggest certain P2X receptors are activated during pathologic brain activity. Expression of several members of the family including P2X2, P2X4, and P2X7 receptors has been reported to be altered in the hippocampus following status epilepticus. Recent studies have shown that ligands of the P2X7 receptor can have potent effects on seizure severity during status epilepticus and mice lacking this receptor display altered seizures in response to chemoconvulsants. Antagonists of the P2X7 receptor also modulate neuronal death, microglial responses and neuroinflammatory signaling. Recent work also found altered neuronal injury and inflammation after status epilepticus in mice lacking the P2X4 receptor. In summary, members of the P2X receptor family may serve important roles in the pathophysiology of status epilepticus and represent novel targets for seizure control and neuroprotection. PMID:24324404

ADP stimulation of inositol phosphates in hepatocytes: role of conversion to ATP and stimulation of P2Y2 receptors.

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Dixon, C Jane; Hall, John F; Boarder, Michael R

2003-01-01

1 Accumulation of inositol (poly)phosphates (InsP(x)) has been studied in rat hepatocytes labelled with [(3)H]inositol. Stimulation with ADP resulted in a significant increase in total [(3)H]InsP(x), whereas 2-MeSADP had only a small effect and ADPbetaS was ineffective. UTP and ITP also stimulated substantial increases in [(3)H]InsP(x). 2 The dose-response curve to ADP was largely unaltered by the presence of the P2Y(1) antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P). Similarly, inclusion of MRS 2179, a more selective P2Y(1) antagonist, had no effect on the dose-response curve to ADP. 3 The inclusion of hexokinase in the assay reduced, but did not abolish, the response to ADP. 4 HPLC analysis revealed that ADP in the medium was rapidly converted to AMP and ATP. The inclusion of hexokinase removed ATP, but exacerbated the decline in ADP concentration, leading to increased levels of AMP. 2-MeSADP was stable in the medium and ATP was largely unaffected. 5 The addition of the adenylate kinase inhibitor, diadenosine pentaphosphate (Ap(5)A) significantly reduced the ADP response. HPLC analysis conducted in parallel demonstrated that this treatment inhibited conversion of ADP to ATP and AMP. 6 Inclusion of the P1 antagonist CGS 15943 had no effect on the dose-response curve to ADP. 7 These observations indicate that hepatocytes respond to ADP with an increase in inositol (poly)phosphates following conversion to ATP. P2Y(1) activation in hepatocytes does not appear to be coupled to inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) production.

Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes

International Nuclear Information System (INIS)

Sakamoto, C.; Matozaki, T.; Nagao, M.; Baba, S.

1987-01-01

Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (NI) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced 125 I-[Tyr 1 ]somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate [Gpp(NH)p]>GTP>TDP>GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg 2+ . When pancreatic acini were treated with 1 μg/ml pertussis toxin for 4 h, subsequent 125 I-[Tyr 1 ]somatostatin binding to acinar membranes was reduced. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. The present results suggest that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

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Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

2016-01-01

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Science.gov (United States)

Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

2016-01-15

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The role of P2X receptors in bone biology

DEFF Research Database (Denmark)

Jørgensen, N R; Syberg, S; Ellegaard, M

2015-01-01

receptors regulate bone metabolism and especially for the P2X7 receptor an impressive amount of evidence has now documented its expression in osteoblasts, osteoclasts, and osteocytes as well as important functional roles in proliferation, differentiation, and function of the cells of bone. Key evidence has...... come from studies on murine knockout models and from pharmacologic studies on cells and animals. More recently, the role of P2X receptors in human bone diseases has been documented. Loss-of-functions polymorphisms in the P2X7 receptorare associated with bone loss and increased fracture risk. Very...

Prostaglandin E2 potentiation of P2X3 receptor mediated currents in dorsal root ganglion neurons

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Huang Li-Yen

2007-08-01

Full Text Available Abstract Prostaglandin E2 (PGE2 is a well-known inflammatory mediator that enhances the excitability of DRG neurons. Homomeric P2X3 and heteromeric P2X2/3 receptors are abundantly expressed in dorsal root ganglia (DRG neurons and participate in the transmission of nociceptive signals. The interaction between PGE2 and P2X3 receptors has not been well delineated. We studied the actions of PGE2 on ATP-activated currents in dissociated DRG neurons under voltage-clamp conditions. PGE2 had no effects on P2X2/3 receptor-mediated responses, but significantly potentiated fast-inactivating ATP currents mediated by homomeric P2X3 receptors. PGE2 exerted its action by activating EP3 receptors. To study the mechanism underlying the action of PGE2, we found that the adenylyl cyclase activator, forskolin and the membrane-permeable cAMP analogue, 8-Br-cAMP increased ATP currents, mimicking the effect of PGE2. In addition, forskolin occluded the enhancement produced by PGE2. The protein kinase A (PKA inhibitors, H89 and PKA-I blocked the PGE2 effect. In contrast, the PKC inhibitor, bisindolymaleimide (Bis did not change the potentiating action of PGE2. We further showed that PGE2 enhanced α,β-meATP-induced allodynia and hyperalgesia and the enhancement was blocked by H89. These observations suggest that PGE2 binds to EP3 receptors, resulting in the activation of cAMP/PKA signaling pathway and leading to an enhancement of P2X3 homomeric receptor-mediated ATP responses in DRG neurons.

P2X7 receptors in satellite glial cells mediate high functional expression of P2X3 receptors in immature dorsal root ganglion neurons

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Chen Yong

2012-02-01

Full Text Available Abstract Background The purinergic P2X3 receptor (P2X3R expressed in the dorsal root ganglion (DRG sensory neuron and the P2X7 receptor (P2X7R expressed in the surrounding satellite glial cell (SGC are two major receptors participating in neuron-SGC communication in adult DRGs. Activation of P2X7Rs was found to tonically reduce the expression of P2X3Rs in DRGs, thus inhibiting the abnormal pain behaviors in adult rats. P2X receptors are also actively involved in sensory signaling in developing rodents. However, very little is known about the developmental change of P2X7Rs in DRGs and the interaction between P2X7Rs and P2X3Rs in those animals. We therefore examined the expression of P2X3Rs and P2X7Rs in postnatal rats and determined if P2X7R-P2X3R control exists in developing rats. Findings We immunostained DRGs of immature rats and found that P2X3Rs were expressed only in neurons and P2X7Rs were expressed only in SGCs. Western blot analyses indicated that P2X3R expression decreased while P2X7R expression increased with the age of rats. Electrophysiological studies showed that the number of DRG neurons responding to the stimulation of the P2XR agonist, α,β-meATP, was higher and the amplitudes of α,β-meATP-induced depolarizations were larger in immature DRG neurons. As a result, P2X3R-mediated flinching responses were much more pronounced in immature rats than those found in adult rats. When we reduced P2X7R expression with P2X7R-siRNA in postnatal and adult rats, P2X3R-mediated flinch responses were greatly enhanced in both rat populations. Conclusions These results show that the P2X7R expression increases as rats age. In addition, P2X7Rs in SGCs exert inhibitory control on the P2X3R expression and function in sensory neurons of immature rats, just as observed in adult rats. Regulation of P2X7R expression is likely an effective way to control P2X3R activity and manage pain relief in infants.

P2X receptor-mediated ATP purinergic signaling in health and disease

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Jiang LH

2012-09-01

Full Text Available Lin-Hua JiangSchool of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, United KingdomAbstract: Purinergic P2X receptors are plasma membrane proteins present in a wide range of mammalian cells where they act as a cellular sensor, enabling cells to detect and respond to extracellular adenosine triphosphate (ATP, an important signaling molecule. P2X receptors function as ligand-gated Ca2+-permeable cationic channels that open upon ATP binding to elevate intracellular Ca2+ concentrations and cause membrane depolarization. In response to sustained activation, P2X receptors induce formation of a pore permeable to large molecules. P2X receptors also interact with distinct functional proteins and membrane lipids to form specialized signaling complexes. Studies have provided compelling evidence to show that such P2X receptor-mediated ATP-signaling mechanisms determine and regulate a growing number and diversity of important physiological processes, including neurotransmission, muscle contraction, and cytokine release. There is accumulating evidence to support strong causative relationships of altered receptor expression and function with chronic pain, inflammatory diseases, cancers, and other pathologies or diseases. Numerous high throughput screening drug discovery programs and preclinical studies have thus far demonstrated the proof of concepts that the P2X receptors are druggable targets and selective receptor antagonism is a promising therapeutics approach. This review will discuss the recent progress in understanding the mammalian P2X receptors with respect to the ATP-signaling mechanisms, physiological and pathophysiological roles, and development and preclinical studies of receptor antagonists.Keywords: extracellular ATP, ion channel, large pore, signaling complex, chronic pain, inflammatory diseases

Post-translational regulation of P2X receptor channels: modulation by phospholipids

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Louis-Philippe eBernier

2013-11-01

Full Text Available P2X receptor channels mediate fast excitatory signaling by ATP and play major roles in sensory transduction, neuro-immune communication and inflammatory response. P2X receptors constitute a gene family of calcium-permeable ATP-gated cation channels therefore the regulation of P2X signaling is critical for both membrane potential and intracellular calcium homeostasis. Phosphoinositides (PIPn are anionic signaling phospholipids that act as functional regulators of many types of ion channels. Direct PIPn binding was demonstrated for several ligand- or voltage-gated ion channels, however no generic motif emerged to accurately predict lipid-protein binding sites. This review presents what is currently known about the modulation of the different P2X subtypes by phospholipids and about critical determinants underlying their sensitivity to PIPn levels in the plasma membrane.All functional mammalian P2X subtypes tested, with the notable exception of P2X5, have been shown to be positively modulated by PIPn, i.e. homomeric P2X1, P2X2, P2X3, P2X4, and P2X7, as well as heteromeric P2X1/5 and P2X2/3 receptors. Based on various results reported on the aforementioned subtypes including mutagenesis of the prototypical PIPn-sensitive P2X4 and PIPn-insensitive P2X5 receptor subtypes, an increasing amount of functional, biochemical and structural evidence converges on the modulatory role of a short polybasic domain located in the proximal C-terminus of P2X subunits. This linear motif, semi-conserved in the P2X family, seems necessary and sufficient for encoding direct modulation of ATP-gated channels by PIPn. Furthermore, the physiological impact of the regulation of ionotropic purinergic responses by phospholipids on pain pathways was recently revealed in the context of native crosstalks between phospholipase C-linked metabotropic receptors and P2X receptor channels in DRG sensory neurons and microglia.

Blockade of human P2X7 receptor function with a monoclonal antibody.

Science.gov (United States)

Buell, G; Chessell, I P; Michel, A D; Collo, G; Salazzo, M; Herren, S; Gretener, D; Grahames, C; Kaur, R; Kosco-Vilbois, M H; Humphrey, P P

1998-11-15

A monoclonal antibody (MoAb) specific for the human P2X7 receptor was generated in mice. As assessed by flow cytometry, the MoAb labeled human blood-derived macrophage cells natively expressing P2X7 receptors and cells transfected with human P2X7 but not other P2X receptor types. The MoAb was used to immunoprecipitate the human P2X7 receptor protein, and in immunohistochemical studies on human lymphoid tissue, P2X7 receptor labeling was observed within discrete areas of the marginal zone of human tonsil sections. The antibody also acted as a selective antagonist of human P2X7 receptors in several functional studies. Thus, whole cell currents, elicited by the brief application of 2',3'-(4-benzoyl)-benzoyl-ATP in cells expressing human P2X7, were reduced in amplitude by the presence of the MoAb. Furthermore, preincubation of human monocytic THP-1 cells with the MoAb antagonized the ability of P2X7 agonists to induce the release of interleukin-1beta.

Improved in Vitro Folding of the Y2 G Protein-Coupled Receptor into Bicelles

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Peter Schmidt

2018-01-01

Full Text Available Prerequisite for structural studies on G protein-coupled receptors is the preparation of highly concentrated, stable, and biologically active receptor samples in milligram amounts of protein. Here, we present an improved protocol for Escherichia coli expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (Y2R for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS. We studied the details of an improved preparation protocol including the in vitro folding of the receptor, e.g., the native disulfide bridge formation, the exchange of the denaturating detergent SDS, and the functional reconstitution into bicelle environments of varying size. Full pharmacological functionality of the Y2R preparation was shown by a ligand affinity of 4 nM and G-protein activation. Further, simple NMR experiments are used to test sample quality in high micromolar concentration.

P2X Receptors and Synaptic Plasticity

Czech Academy of Sciences Publication Activity Database

Pankratov, Y.; Lalo, U.; Krishtal, A.; Verkhratsky, Alexei

2009-01-01

Roč. 158, č. 1 (2009), s. 137-148 ISSN 0306-4522 Institutional research plan: CEZ:AV0Z50390512 Keywords : ATP * P2X receptors * synaptic plasticity Subject RIV: FH - Neurology Impact factor: 3.292, year: 2009

Molecular characterization of a rat α2B-adrenergic receptor

International Nuclear Information System (INIS)

Zeng, D.; Harrison, J.K.; D'Angelo, D.D.; Barber, C.M.; Tucker, A.L.; Lu, Z.; Lynch, K.R.

1990-01-01

α 2 -Adrenergic receptors comprise a heterogeneous population based on pharmacologic and molecular evidence. The authors have isolated a cDNA clone (pRNGα 2 ) encoding a rat α 2 -adrenergic receptor. A rat kidney cDNA library was screened with an oligonucleotide complementary to a highly conserved region found in all biogenic amine receptors described to date. The deduced amino acid sequence displays many features of guanyl nucleotide-binding protein-coupled receptors except it does not have a consensus N-linked glycosylation site near the amino terminus. Membranes prepared from COS cells transfected with pRNGα 2 DNA display high affinity an saturable binding to [ 3 H]rauwolscine. Competition curve data analysis shows that RNGα 2 protein binds to a variety of adrenergic drugs with the following rank order of potency: yohimbine ≥ chlorpromazine > prazosin ≥ clonidine > norepinephrine ≥ oxymetazoline. RNGα 2 RNA accumulates in both rat kidney and neonatal rat lung. When a cysteine residue (Cys-169) that is conserved among all members of the seven-transmembrane-region superfamily is changed to phenylalanine, the RNGα 2 protein fails to bind [ 3 H]rauwolscine after expression in COS cells. They conclude that pRNGα 2 likely represents a cDNA for a rat α 2B -adrenergic receptor

P2X7 Receptor Function in Bone-Related Cancer

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Elena Adinolfi

2012-01-01

Full Text Available Modulation of tumor microenvironment by different mediators is central in determining neoplastic formation and progression. Among these molecules extracellular ATP is emerging as a good candidate in promoting cell growth, neovascularization, tumor-host interactions, and metastatization. This paper summarizes recent findings on expression and function of P2X7 receptor for extracellular ATP in primary and metastatic bone cancers. Search of mRNA expression microchip databases and literature analysis demonstrate a high expression of P2X7 in primary bone tumors as well as in other malignancies such as multiple myeloma, neuroblastoma, breast, and prostate cancer. Evidence that P2X7 triggers NFATc1, PI3K/Akt, ROCK, and VEGF pathways in osteoblasts promoting either primary tumor development or osteoblastic lesions is also reported. Moreover, P2X7 receptor is involved in osteoclast differentiation, RANKL expression, matrix metalloproteases and cathepsin secretion thus promoting bone resorption and osteolytic lesions. Taken together these data point to a pivotal role for the P2X7 receptor in bone cancer biology.

Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation*

Science.gov (United States)

Lalo, Ulyana; Roberts, Jonathan A.; Evans, Richard J.

2011-01-01

P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation. PMID:21757694

Functional expression of P2X family receptors in macrophages is affected by microenvironment in mouse T cell acute lymphoblastic leukemia

Energy Technology Data Exchange (ETDEWEB)

Chen, Shayan; Feng, Wenli; Yang, Xiao; Yang, Wanzhu; Ru, Yongxin; Liao, Jinfeng; Wang, Lina; Lin, Yongmin; Ren, Qian [State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020 (China); Zheng, Guoguang, E-mail: zhengggtjchn@aliyun.com [State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020 (China); Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730 (China)

2014-04-18

Highlights: • We study the impact of leukemic microenvironment on P2X family receptors in Mφs. • Bone marrow and spleen Mφs are studied in Notch1-induced mouse leukemia model. • Increased expression of P2X7R is found in Mφs during the development of leukemia. • Elevated P2X7R-mediated calcium response is found in Mφs at late stage of leukemia. • More apoptotic Mφs are found in bone marrow and spleen at late stage of leukemia. - Abstract: Nucleotides are important players in intercellular signaling communication network. P2X family receptors (P2XRs) are ATP-gated plasma membrane ion channels with diverse biological functions. Macrophages are important components in the microenvironment of hematopoiesis participating in both physiological and pathological processes. However, the role of P2XRs in macrophages in leukemia has not been established. Here we investigated expression pattern and functions of P2XRs in macrophages from bone marrow (BM) and spleen of Notch1-induced T-ALL mice. Real-time PCR showed that P2XRs except P2X5R were expressed in BM and spleen macrophages. Furthermore, with the development of leukemia, the expression of P2X7R increased in both BM and spleen macrophages whereas expression of P2X1R increased in spleen macrophages. Live cell imaging recoding the Ca{sup 2+} response demonstrated that P2X7R expressed in macrophages was functional. TUNEL and electron microscopy analysis found that apoptotic macrophages were frequently observed in BM and spleen at late stage of leukemia, which was partly contributed by the activation of overexpressed P2X7R. Our results suggested that the intercellular communication mediated by nucleotides might orchestrate in the pathological process of leukemia and could be a potential target for the treatment of leukemia.

Functional expression of P2X family receptors in macrophages is affected by microenvironment in mouse T cell acute lymphoblastic leukemia

International Nuclear Information System (INIS)

Chen, Shayan; Feng, Wenli; Yang, Xiao; Yang, Wanzhu; Ru, Yongxin; Liao, Jinfeng; Wang, Lina; Lin, Yongmin; Ren, Qian; Zheng, Guoguang

2014-01-01

Highlights: • We study the impact of leukemic microenvironment on P2X family receptors in Mφs. • Bone marrow and spleen Mφs are studied in Notch1-induced mouse leukemia model. • Increased expression of P2X7R is found in Mφs during the development of leukemia. • Elevated P2X7R-mediated calcium response is found in Mφs at late stage of leukemia. • More apoptotic Mφs are found in bone marrow and spleen at late stage of leukemia. - Abstract: Nucleotides are important players in intercellular signaling communication network. P2X family receptors (P2XRs) are ATP-gated plasma membrane ion channels with diverse biological functions. Macrophages are important components in the microenvironment of hematopoiesis participating in both physiological and pathological processes. However, the role of P2XRs in macrophages in leukemia has not been established. Here we investigated expression pattern and functions of P2XRs in macrophages from bone marrow (BM) and spleen of Notch1-induced T-ALL mice. Real-time PCR showed that P2XRs except P2X5R were expressed in BM and spleen macrophages. Furthermore, with the development of leukemia, the expression of P2X7R increased in both BM and spleen macrophages whereas expression of P2X1R increased in spleen macrophages. Live cell imaging recoding the Ca 2+ response demonstrated that P2X7R expressed in macrophages was functional. TUNEL and electron microscopy analysis found that apoptotic macrophages were frequently observed in BM and spleen at late stage of leukemia, which was partly contributed by the activation of overexpressed P2X7R. Our results suggested that the intercellular communication mediated by nucleotides might orchestrate in the pathological process of leukemia and could be a potential target for the treatment of leukemia

Knocking out P2X receptors reduces transmitter secretion in taste buds

Science.gov (United States)

Huang, Yijen A.; Stone, Leslie M.; Pereira, Elizabeth; Yang, Ruibiao; Kinnamon, John C.; Dvoryanchikov, Gennady; Chaudhari, Nirupa; Finger, Thomas E.; Kinnamon, Sue C.; Roper, Stephen D.

2011-01-01

In response to gustatory stimulation, taste bud cells release a transmitter, ATP, that activates P2X2 and P2X3 receptors on gustatory afferent fibers. Taste behavior and gustatory neural responses are largely abolished in mice lacking P2X2 and P2X3 receptors (P2X2 and P2X3 double knockout, or “DKO” mice). The assumption has been that eliminating P2X2 and P2X3 receptors only removes postsynaptic targets but that transmitter secretion in mice is normal. Using functional imaging, ATP biosensor cells, and a cell-free assay for ATP, we tested this assumption. Surprisingly, although gustatory stimulation mobilizes Ca2+ in taste Receptor (Type II) cells from DKO mice, as from wild type (WT) mice, taste cells from DKO mice fail to release ATP when stimulated with tastants. ATP release could be elicited by depolarizing DKO Receptor cells with KCl, suggesting that ATP-release machinery remains functional in DKO taste buds. To explore the difference in ATP release across genotypes, we employed reverse transcriptase (RT)-PCR, immunostaining, and histochemistry for key proteins underlying ATP secretion and degradation: Pannexin1, TRPM5, and NTPDase2 (ecto-ATPase) are indistinguishable between WT and DKO mice. The ultrastructure of contacts between taste cells and nerve fibers is also normal in the DKO mice. Finally, quantitative RT-PCR show that P2X4 and P2X7, potential modulators of ATP secretion, are similarly expressed in taste buds in WT and DKO taste buds. Importantly, we find that P2X2 is expressed in WT taste buds and appears to function as an autocrine, positive feedback signal to amplify taste-evoked ATP secretion. PMID:21940456

Knocking out P2X receptors reduces transmitter secretion in taste buds.

Science.gov (United States)

Huang, Yijen A; Stone, Leslie M; Pereira, Elizabeth; Yang, Ruibiao; Kinnamon, John C; Dvoryanchikov, Gennady; Chaudhari, Nirupa; Finger, Thomas E; Kinnamon, Sue C; Roper, Stephen D

2011-09-21

In response to gustatory stimulation, taste bud cells release a transmitter, ATP, that activates P2X2 and P2X3 receptors on gustatory afferent fibers. Taste behavior and gustatory neural responses are largely abolished in mice lacking P2X2 and P2X3 receptors [P2X2 and P2X3 double knock-out (DKO) mice]. The assumption has been that eliminating P2X2 and P2X3 receptors only removes postsynaptic targets but that transmitter secretion in mice is normal. Using functional imaging, ATP biosensor cells, and a cell-free assay for ATP, we tested this assumption. Surprisingly, although gustatory stimulation mobilizes Ca(2+) in taste Receptor (Type II) cells from DKO mice, as from wild-type (WT) mice, taste cells from DKO mice fail to release ATP when stimulated with tastants. ATP release could be elicited by depolarizing DKO Receptor cells with KCl, suggesting that ATP-release machinery remains functional in DKO taste buds. To explore the difference in ATP release across genotypes, we used reverse transcriptase (RT)-PCR, immunostaining, and histochemistry for key proteins underlying ATP secretion and degradation: Pannexin1, TRPM5, and NTPDase2 (ecto-ATPase) are indistinguishable between WT and DKO mice. The ultrastructure of contacts between taste cells and nerve fibers is also normal in the DKO mice. Finally, quantitative RT-PCR show that P2X4 and P2X7, potential modulators of ATP secretion, are similarly expressed in taste buds in WT and DKO taste buds. Importantly, we find that P2X2 is expressed in WT taste buds and appears to function as an autocrine, positive feedback signal to amplify taste-evoked ATP secretion.

P2X receptor channels in endocrine glands

Czech Academy of Sciences Publication Activity Database

Stojilkovic, S. S.; Zemková, Hana

2013-01-01

Roč. 2, č. 4 (2013), s. 173-180 ISSN 2190-460X R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : ATP * purinergic P2X receptor channels * pituitary * endocrine glands Subject RIV: ED - Physiology

Comparison of Nucleotide Sequence of P2C Region in Diabetogenic and Non-Diabetogenic Coxsackie Virus B5 Isolates

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Cheng-Chong Chou

2004-11-01

Full Text Available Enteroviruses are environmental triggers in the pathogenesis of type 1 diabetes mellitus (DM. A sequence of six identical amino acids (PEVKEK is shared by the 2C protein of Coxsackie virus B and the glutamic acid decarboxylase (GAD molecules. Between 1995 and 2002, we investigated 22 Coxsackie virus B5 (CVB5 isolates from southern Taiwan. Four of these isolates were obtained from four new-onset type 1 DM patients with diabetic ketoacidosis. We compared a 300 nucleotide sequence in the 2C protein gene (p2C in 24 CVB5 isolates (4 diabetogenic, 18 non-diabetogenic and 2 prototype. We found 0.3-10% nucleotide differences. In the four isolates from type 1 DM patients, there was only 2.4-3.4% nucleotide difference, and there was only 1.7-7.1% nucleotide difference between type 1 DM isolates and non-diabetogenic isolates. Comparison of the nucleotide sequence between prototype virus and 22 CVB5 isolates revealed 18.4-24.1% difference. Twenty-one CVB5 isolates from type 1 DM and non-type 1 DM patients contained the PEVKEK sequence, as shown by the p2C nucleotide sequence. Our data showed that the viral p2C sequence with homology with GAD is highly conserved in CVB5 isolates. There was no difference between diabetogenic and non-diabetogenic CVB5 isolates. All four type 1 DM patients had at least one of the genetic susceptibility alleles HLA-DR, DQA1, DQB1. Other genetic and autoimmune factors such as HLA genetic susceptibility and GAD may also play important roles in the pathogenesis in type 1 DM.

Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P2Y- and P2U-purinoceptor-stimulated prostacyclin release.

Science.gov (United States)

Patel, V; Brown, C; Boarder, M R

1996-05-01

1. Enhanced synthesis of prostacyclin (PGI2) and inositol polyphosphates in bovine aortic endothelial cells in response to ATP and ADP is mediated by co-existing P2Y- and P2U-purinoceptors. Here we examine the regulation of these responses by isoforms of protein kinase C (PKC). 2. Immunoblots with antisera specific for 8 different PKC isoforms revealed the presence of alpha, epsilon and zeta, while no immunoreactivity was found for beta, gamma, delta, eta and theta isoforms. PKC-alpha was largely cytosolic in unstimulated cells and almost all translocated to the membrane (Triton X-100 soluble) after a 1 min treatment with the PKC activating phorbol myristate acetate (PMA); PKC-epsilon was always in a Triton X-100 insoluble membrane fraction, while PKC-zeta was found in both soluble and membrane bound (Triton X-100 soluble) forms in the unstimulated cells and was unaffected by PMA. 3. Treatment with PMA for 6 h led to a 90% downregulation of PKC-alpha, while the immunoreactivity to the epsilon and zeta isoforms remained largely unchanged. 4. After either 10 min or 6 h exposure to PMA the PGI2 response to activation of both receptors was enhanced, while the inositol 1,4,5-trisphosphate response to P2Y-purinoceptor activation was substantially attenuated and the P2U-purinoceptor response was unchanged. Thus the PGI2 response to PMA under conditions when 90% of the PKC-alpha was lost resembles that seen on acute stimulation of PKC by PMA, and the PGI2 response does not correlate with phospholipase C response. 5. Inhibition of PKC with the isoform non-selective inhibitors, Ro 31-8220 and Go 6850 abolished the PGI2 response to both P2U- and P2Y-purinoceptor stimulation. However, Go 6976, which preferentially inhibits Ca2+ sensitive isoforms (such as PKC-alpha) and not Ca2+ insensitive isoforms (such as PKC-epsilon), had no effect on the PGI2 response. 6. The results show that there is a requirement for PKC in the stimulation of PGI2 production by endothelial P2Y- and P2U

Localization of P2X receptor subtypes 2, 3 and 7 in human urinary bladder.

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Svennersten, Karl; Hallén-Grufman, Katarina; de Verdier, Petra J; Wiklund, N Peter; Poljakovic, Mirjana

2015-08-08

Voiding dysfunctions are a common problem that has a severe negative impact on the quality of life. Today there is a need for new drug targets for these conditions. The role of ATP receptors in bladder physiology has been studied for some time, primarily in animal models. The aim of this work is to investigate the localization of the ATP receptors P2X2, P2X3 and P2X7 and their colocalization with vimentin and actin in the human urinary bladder. Immunohistochemical analysis was conducted on full-thickness bladder tissues from fundus and trigonum collected from 15 patients undergoing open radical cystectomy due to chronic cystitis, bladder cancer or locally advanced prostate cancer. Colocalization analyses were performed between the three different P2X subtypes and the structural proteins vimentin and actin. Specimens were examined using epifluorescence microscopy and correlation coefficients were calculated for each costaining as well as the mean distance from the laminin positive basal side of the urothelium to the vimentin positive cells located in the suburothelium. P2X2 was expressed in vimentin positive cells located in the suburothelium. Less distinct labelling of P2X2 was also observed in actin positive smooth muscle cells and in the urothelium. P2X3 was expressed in vimentin positive cells surrounding the smooth muscle, and in vimentin positive cells located in the suburothelium. Weaker P2X3 labelling was seen in the urothelium. P2X7 was expressed in the smooth muscle cells and the urothelium. In the suburothelium, cells double positive for P2X2 and vimentin where located closer to the urothelium while cells double positive for P2X3 and vimentin where located further from the urothelium. The results from this study demonstrate that there is a significant difference in the expression of the purinergic P2X2, P2X3 and P2X7 receptors in the different histological layers of the human urinary bladder.

Heterozygosity for the Mood Disorder-Associated Variant Gln460Arg Alters P2X7 Receptor Function and Sleep Quality.

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Metzger, Michael W; Walser, Sandra M; Dedic, Nina; Aprile-Garcia, Fernando; Jakubcakova, Vladimira; Adamczyk, Marek; Webb, Katharine J; Uhr, Manfred; Refojo, Damian; Schmidt, Mathias V; Friess, Elisabeth; Steiger, Axel; Kimura, Mayumi; Chen, Alon; Holsboer, Florian; Arzt, Eduardo; Wurst, Wolfgang; Deussing, Jan M

2017-11-29

A single nucleotide polymorphism substitution from glutamine (Gln, Q) to arginine (Arg, R) at codon 460 of the purinergic P2X7 receptor (P2X7R) has repeatedly been associated with mood disorders. The P2X7R-Gln460Arg variant per se is not compromised in its function. However, heterologous expression of P2X7R-Gln460Arg together with wild-type P2X7R has recently been demonstrated to impair receptor function. Here we show that this also applies to humanized mice coexpressing both human P2X7R variants. Primary hippocampal cells derived from heterozygous mice showed an attenuated calcium uptake upon agonist stimulation. While humanized mice were unaffected in their behavioral repertoire under basal housing conditions, mice that harbor both P2X7R variants showed alterations in their sleep quality resembling signs of a prodromal disease stage. Also healthy heterozygous human subjects showed mild changes in sleep parameters. These results indicate that heterozygosity for the wild-type P2X7R and its mood disorder-associated variant P2X7R-Gln460Arg represents a genetic risk factor, which is potentially able to convey susceptibility to mood disorders. SIGNIFICANCE STATEMENT Depression and bipolar disorder are the most common mood disorders. The P2X7 receptor (P2X7R) regulates many cellular functions. Its polymorphic variant Gln460Arg has repeatedly been associated with mood disorders. Genetically engineered mice, with human P2X7R, revealed that heterozygous mice (i.e., they coexpress the disease-associated Gln460Arg variant together with its normal version) have impaired receptor function and showed sleep disturbances. Human participants with the heterozygote genotype also had subtle alterations in their sleep profile. Our findings suggest that altered P2X7R function in heterozygote individuals disturbs sleep and might increase the risk for developing mood disorders. Copyright © 2017 the authors 0270-6474/17/3711688-13$15.00/0.

Role of purinergic receptor polymorphisms in human bone

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Wesselius, Anke; Bours, Martijn J L; Agrawal, Ankita

2011-01-01

Osteoporosis is a multifactorial disease with a strong genetic component. Variations in a number of genes have been shown to associate with bone turnover and risk of osteoporosis. P2 purinergic receptors are proteins that have ATP or other nucleotides as their natural ligands. Various P2Y and P2X...

P2Y12R-Dependent Translocation Mechanisms Gate the Changing Microglial Landscape

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Ukpong B. Eyo

2018-04-01

Full Text Available Summary: Microglia are an exquisitely tiled and self-contained population in the CNS that do not receive contributions from circulating monocytes in the periphery. While microglia are long-lived cells, the extent to which their cell bodies are fixed and the molecular mechanisms by which the microglial landscape is regulated have not been determined. Using chronic in vivo two-photon imaging to follow the microglial population in young adult mice, we document a daily rearrangement of the microglial landscape. Furthermore, we show that the microglial landscape can be modulated by severe seizures, acute injury, and sensory deprivation. Finally, we demonstrate a critical role for microglial P2Y12Rs in regulating the microglial landscape through cellular translocation independent of proliferation. These findings suggest that microglial patrol the CNS through both process motility and soma translocation. : Using a chronic in vivo imaging approach, Eyo et al. show that the physical positions of brain microglia change daily and that these changes increase following certain experimental manipulations. The mechanism underlying these changes involves cell translocation controlled by microglial-specific P2Y12 receptors. Keywords: microglia, P2Y12, seizures, epilepsy, whisker trimming, microglial landscape, two photon chronic imaging

P2X7 receptor-deficient mice are susceptible to bone cancer pain

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Hansen, Rikke Rie; Nielsen, Christian K.; Nasser, Arafat

2011-01-01

The purinergic P2X7 receptor is implicated in both neuropathic and inflammatory pain, and has been suggested as a possible target in pain treatment. However, the specific role of the P2X7 receptor in bone cancer pain is unknown. We demonstrated that BALB/cJ P2X7 receptor knockout (P2X7R KO) mice...... were susceptible to bone cancer pain and moreover had an earlier onset of pain-related behaviours compared with cancer-bearing, wild-type mice. Furthermore, acute treatment with the selective P2X7 receptor antagonist, A-438079, failed to alleviate pain-related behaviours in models of bone cancer pain...... with and without astrocyte activation (BALB/cJ or C3H mice inoculated with 4T1 mammary cancer cells or NCTC 2472 osteosarcoma cells, respectively), suggesting that astrocytic P2X7 receptors play a negligible role in bone cancer pain. The results support the hypothesis that bone cancer pain is a separate pain state...

P2X receptors in the cardiovascular system and their potential as therapeutic targets in disease.

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Ralevic, Vera

2015-01-01

This review considers the expression and roles of P2X receptors in the cardiovascular system in health and disease and their potential as therapeutic targets. P2X receptors are ligand gated ion channels which are activated by the endogenous ligand ATP. They are formed from the assembly of three P2X subunit proteins from the complement of seven (P2X1-7), which can associate to form homomeric or heteromeric P2X receptors. The P2X1 receptor is widely expressed in the cardiovascular system, being located in the heart, in the smooth muscle of the majority of blood vessels and in platelets. P2X1 receptors expressed in blood vessels can be activated by ATP coreleased with noradrenaline as a sympathetic neurotransmitter, leading to smooth muscle depolarisation and contraction. There is evidence that the purinergic component of sympathetic neurotransmission is increased in hypertension, identifying P2X1 receptors as a possible therapeutic target in this disorder. P2X3 and P2X2/3 receptors are expressed on cardiac sympathetic neurones and may, through positive feedback of neuronal ATP at this prejunctional site, amplify sympathetic neurotransmission. Activation of P2X receptors expressed in the heart increases cardiac myocyte contractility, and an important role of the P2X4 receptor in this has been identified. Deletion of P2X4 receptors in the heart depresses contractile performance in models of heart failure, while overexpression of P2X4 receptors has been shown to be cardioprotective, thus P2X4 receptors may be therapeutic targets in the treatment of heart disease. P2X receptors have been identified on endothelial cells. Although immunoreactivity for all P2X1-7 receptor proteins has been shown on the endothelium, relatively little is known about their function, with the exception of the endothelial P2X4 receptor, which has been shown to mediate endothelium-dependent vasodilatation to ATP released during shear stress. The potential of P2X receptors as therapeutic targets

Internet, salud pública 2.0 y complejidad

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Rodolfo Rodríguez

2014-12-01

Full Text Available Al día de hoy, Internet ha modificado el mundo y desde su incursión comercial a principios de la década de los noventa del siglo XX, el mundo no volvió a ser el mismo. El impacto de Internet ha sido monumental e históricamente pocos inventos en la historia de la humanidad han modificado de tal forma la vida del ser humano. Sin embargo, Internet es mucho más que unos computadores conectados y unas páginas web a las cuales acceder. Adicionalmente, algunas reglas de la naturaleza para células y especies también rigen para Internet. La World Wide Web y fenómenos que habitan en ella como la salud pública 2.0 son más que una red de sitios web conectados por hiperenlaces. La Web 2.0 pertenece a una de esas inteligencias colectivas donde así como en los enjambres, el concepto de autoorganización es fundamental. La nueva revolución científica aporta diferentes conceptos para intentar comprender las dinámicas de la World Wide Web y con ello entender desde otra perspectiva fenómenos complejos como la salud pública 2.0

In Vitro Binding of [³H]PSB-0413 to P2Y₁₂ Receptors.

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Dupuis, Arnaud; Heim, Véronique; Ohlmann, Philippe; Gachet, Christian

2015-12-08

The P2Y₁₂/ADP receptor plays a central role in platelet activation. Characterization of this receptor is mandatory for studying disorders associated with a P2Y₁₂ receptor defect and for evaluating P2Y₁₂ receptor agonists and antagonists. In the absence of suitable anti-P2Y₁₂ antibodies, radioligand binding assays are the only way to conduct such studies. While various radioligands were employed in the past for this purpose, none were found to be suitable for routine use. Described in this unit are protocols for quantitatively and qualitatively assessing P2Y₁₂ receptors with [³H]PSB-0413, a selective antagonist for this site. The saturation and competition assays described herein make possible the determination of P2Y₁₂ receptor density on cells, as well as the potencies and affinities of test agents at this site. Copyright © 2015 John Wiley & Sons, Inc.

Potential Involvement of P2 Receptors in the Pathological Processes of Hyperthyroidism: A Pilot Study.

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Hong, Wu; Li, Guodong; Nie, Yijun; Zou, Lifang; Zhang, Xi; Liu, Shuangmei; Li, Guilin; Xu, Hong; Zhang, Chun-Ping; Liang, Shangdong

2016-05-01

Symptoms of hyperthyroidism manifest mainly as changes in the nervous and metabolic systems. Whether P2X receptors (ionotropic ATP purinergic receptors, including P2X3 receptor and P2X7 receptor) are involved in the alterations of these disorders still remains unclear. Thus, this study aimed to assess the association of hyperthyroidism with the expression of P2X3 and P2X7 receptors and the concentrations of ATP in blood leukocytes and catecholamine. Twelve healthy subjects and twelve patients diagnosed with hyperthyroidism were recruited. Serum free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) levels had been detected by chemiluminescence method. Meanwhile, the catecholamine levels (including adrenaline, noradrenaline, and dopamine) in plasma, ATP level and P2X receptors (including P2X3 receptor and P2X7 receptor) in peripheral blood had been detected by high performance liquid chromatography, bioluminescence method, and reverse transcription polymerase chain reaction, respectively. Levels of epinephrine and norepinephrine were significantly higher in the hyperthyroidism group compared with the control group. The concentration of ATP in the hyperthyroidism group was significantly higher than its in the control group. The expression of P2X3 mRNA and P2X7 mRNA in hyperthyroidism group were significantly increased compared with those in control group. In a conclusion, there is a relationship between the elevated expression of P2X3 receptor and P2X7 receptor in peripheral blood leukocytes and high serum epinephrine and norepinephrine levels in hyperthyroidism patients. © 2016 by the Association of Clinical Scientists, Inc.

Clopidogrel, a P2Y12 receptor antagonist, potentiates the inflammatory response in a rat model of peptidoglycan polysaccharide-induced arthritis.

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Analia E Garcia

Full Text Available The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists, which is irreversibly antagonized by the active metabolite of clopidogrel, a widely used anti-thrombotic drug. In this study, we investigated whether reduction of platelet reactivity leads to reduced inflammatory responses using a rat model of erosive arthritis. We evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS-induced arthritis model with four groups of rats: 1 untreated, 2 clopidogrel-treated, 3 PG-PS-induced, and 4 PG-PS-induced and clopidogrel-treated. There were significant differences between the PG-PS+clopidogrel group when compared to the PG-PS group including: increased joint diameter and clinical manifestations of inflammation, elevated plasma levels of pro-inflammatory cytokines (IL-1 beta, interferon (IFN gamma, and IL-6, an elevated neutrophil blood count and an increased circulating platelet count. Plasma levels of IL-10 were significantly lower in the PG-PS+clopidogrel group compared to the PG-PS group. Plasma levels of platelet factor 4 (PF4 were elevated in both the PG-PS and the PG-PS+clopidogrel groups, however PF4 levels showed no difference upon clopidogrel treatment, suggesting that the pro- inflammatory effect of clopidogrel may be due to its action on cells other than platelets. Histology indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation, blood vessel proliferation, subsynovial fibrosis and cartilage erosion upon treatment with clopidogrel in PG-PS-induced arthritis animals. In summary, animals treated with clopidogrel showed a pro-inflammatory effect in the PG-PS-induced arthritis animal model, which might not be mediated by platelets. Elucidation of the mechanism of clopidogrel-induced cell responses is important to understand the role of the P2Y12 receptor in inflammation.

Clopidogrel, a P2Y12 receptor antagonist, potentiates the inflammatory response in a rat model of peptidoglycan polysaccharide-induced arthritis.

Science.gov (United States)

Garcia, Analia E; Mada, Sripal R; Rico, Mario C; Dela Cadena, Raul A; Kunapuli, Satya P

2011-01-01

The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists, which is irreversibly antagonized by the active metabolite of clopidogrel, a widely used anti-thrombotic drug. In this study, we investigated whether reduction of platelet reactivity leads to reduced inflammatory responses using a rat model of erosive arthritis. We evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of rats: 1) untreated, 2) clopidogrel-treated, 3) PG-PS-induced, and 4) PG-PS-induced and clopidogrel-treated. There were significant differences between the PG-PS+clopidogrel group when compared to the PG-PS group including: increased joint diameter and clinical manifestations of inflammation, elevated plasma levels of pro-inflammatory cytokines (IL-1 beta, interferon (IFN) gamma, and IL-6), an elevated neutrophil blood count and an increased circulating platelet count. Plasma levels of IL-10 were significantly lower in the PG-PS+clopidogrel group compared to the PG-PS group. Plasma levels of platelet factor 4 (PF4) were elevated in both the PG-PS and the PG-PS+clopidogrel groups, however PF4 levels showed no difference upon clopidogrel treatment, suggesting that the pro- inflammatory effect of clopidogrel may be due to its action on cells other than platelets. Histology indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation, blood vessel proliferation, subsynovial fibrosis and cartilage erosion upon treatment with clopidogrel in PG-PS-induced arthritis animals. In summary, animals treated with clopidogrel showed a pro-inflammatory effect in the PG-PS-induced arthritis animal model, which might not be mediated by platelets. Elucidation of the mechanism of clopidogrel-induced cell responses is important to understand the role of the P2Y12 receptor in inflammation.

Nociceptive transmission and modulation via P2X receptors in central pain syndrome.

Science.gov (United States)

Kuan, Yung-Hui; Shyu, Bai-Chuang

2016-05-26

Painful sensations are some of the most frequent complaints of patients who are admitted to local medical clinics. Persistent pain varies according to its causes, often resulting from local tissue damage or inflammation. Central somatosensory pathway lesions that are not adequately relieved can consequently cause central pain syndrome or central neuropathic pain. Research on the molecular mechanisms that underlie this pathogenesis is important for treating such pain. To date, evidence suggests the involvement of ion channels, including adenosine triphosphate (ATP)-gated cation channel P2X receptors, in central nervous system pain transmission and persistent modulation upon and following the occurrence of neuropathic pain. Several P2X receptor subtypes, including P2X2, P2X3, P2X4, and P2X7, have been shown to play diverse roles in the pathogenesis of central pain including the mediation of fast transmission in the peripheral nervous system and modulation of neuronal activity in the central nervous system. This review article highlights the role of the P2X family of ATP receptors in the pathogenesis of central neuropathic pain and pain transmission. We discuss basic research that may be translated to clinical application, suggesting that P2X receptors may be treatment targets for central pain syndrome.

G16R single nucleotide polymorphism but not haplotypes of the ß2-adrenergic receptor gene alters cardiac output in humans

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Rokamp, Kim Z; Staalsø, Jonatan M; Gartmann, Martin

2013-01-01

Variation in genes encoding the ß2-adrenergic receptor (ADRB2) and angiotensin-converting enzyme (ACE) may influence Q¿ (cardiac output). The 46G>A (G16R) SNP (single nucleotide polymorphism) has been associated with ß2-mediated vasodilation, but the effect of ADRB2 haplotypes on Q¿ has not been...... studied. Five SNPs within ADRB2 (46G>A, 79C>G, 491C>T, 523C>A and 1053G>C by a pairwise tagging principle) and the I/D (insertion/deletion) polymorphism in ACE were genotyped in 143 subjects. Cardiovascular variables were evaluated by the Model flow method at rest and during incremental cycling exercise...... V¿O2 (oxygen uptake) in G16G subjects, but the increase was 0.5 (0.0-0.9) l/min lower in Arg16 carriers (P=0.035). A similar effect size was observed for the Arg16 haplotypes ACCCG and ACCCC. No interaction was found between ADRB2 and ACE polymorphisms. During exercise, the increase in Q¿ was 0...

Functional polymorphisms in the P2X7 receptor gene are associated with osteoporosis

DEFF Research Database (Denmark)

Husted, L B; Harsløf, T; Stenkjær, L

2013-01-01

variant allele, which has been associated with increased receptor function in monocytes, was associated with increased total hip BMD in women. With the exception of His155Tyr for which we found conflicting results in men and women, our results are consistent with the phenotype of the knockout mouse......UNLABELLED: The P2X(7) receptor is an ATP-gated cation channel. We investigated the effect of both loss-of-function and gain-of-function polymorphisms in the P2X(7) receptor gene on BMD and risk of vertebral fractures and found that five polymorphisms and haplotypes containing three...... of these polymorphisms were associated with BMD and fracture risk. INTRODUCTION: The P2X(7) receptor is an ATP-gated cation channel. P2X(7) receptor knockout mice have reduced total bone mineral content, and because several functional polymorphisms have been identified in the human P2X(7) receptor gene, we wanted...

Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel

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Milos B. Rokic

2018-04-01

Full Text Available P2X2 receptors (P2X2R exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

Purinergic signaling triggers endfoot high-amplitude Ca2+ signals and causes inversion of neurovascular coupling after subarachnoid hemorrhage.

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Pappas, Anthony C; Koide, Masayo; Wellman, George C

2016-11-01

Neurovascular coupling supports brain metabolism by matching focal increases in neuronal activity with local arteriolar dilation. Previously, we demonstrated that an emergence of spontaneous endfoot high-amplitude Ca 2+ signals (eHACSs) caused a pathologic shift in neurovascular coupling from vasodilation to vasoconstriction in brain slices obtained from subarachnoid hemorrhage model animals. Extracellular purine nucleotides (e.g., ATP) can trigger astrocyte Ca 2+ oscillations and may be elevated following subarachnoid hemorrhage. Here, the role of purinergic signaling in subarachnoid hemorrhage-induced eHACSs and inversion of neurovascular coupling was examined by imaging parenchymal arteriolar diameter and astrocyte Ca 2+ signals in rat brain slices using two-photon fluorescent and infrared-differential interference contrast microscopy. We report that broad-spectrum inhibition of purinergic (P2) receptors using suramin blocked eHACSs and restored vasodilatory neurovascular coupling after subarachnoid hemorrhage. Importantly, eHACSs were also abolished using a cocktail of inhibitors targeting G q -coupled P2Y receptors. Further, activation of P2Y receptors in brain slices from un-operated animals triggered high-amplitude Ca 2+ events resembling eHACSs and disrupted neurovascular coupling. Neither tetrodotoxin nor bafilomycin A1 affected eHACSs suggesting that purine nucleotides are not released by ongoing neurotransmission and/or vesicular release after subarachnoid hemorrhage. These results indicate that purinergic signaling via P2Y receptors contributes to subarachnoid hemorrhage-induced eHACSs and inversion of neurovascular coupling. © The Author(s) 2016.

Purinergic control of inflammation and thrombosis: Role of P2X1 receptors

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Cécile Oury

2015-01-01

Full Text Available Inflammation shifts the hemostatic mechanisms in favor of thrombosis. Upon tissue damage or infection, a sudden increase of extracellular ATP occurs, that might contribute to the crosstalk between inflammation and thrombosis. On platelets, P2X1 receptors act to amplify platelet activation and aggregation induced by other platelet agonists. These receptors critically contribute to thrombus stability in small arteries. Besides platelets, studies by our group indicate that these receptors are expressed by neutrophils. They promote neutrophil chemotaxis, both in vitro and in vivo. In a laser-induced injury mouse model of thrombosis, it appears that neutrophils are required to initiate thrombus formation and coagulation activation on inflamed arteriolar endothelia. In this model, by using P2X1−/− mice, we recently showed that P2X1 receptors, expressed on platelets and neutrophils, play a key role in thrombus growth and fibrin generation. Intriguingly, in a model of endotoxemia, P2X1−/− mice exhibited aggravated oxidative tissue damage, along with exacerbated thrombocytopenia and increased activation of coagulation, which translated into higher susceptibility to septic shock. Thus, besides its ability to recruit neutrophils and platelets on inflamed endothelia, the P2X1 receptor also contributes to limit the activation of circulating neutrophils under systemic inflammatory conditions. Taken together, these data suggest that P2X1 receptors are involved in the interplay between platelets, neutrophils and thrombosis. We propose that activation of these receptors by ATP on neutrophils and platelets represents a new mechanism that regulates thrombo-inflammation.

Y2 receptor signalling in NPY neurons controls bone formation and fasting induced feeding but not spontaneous feeding.

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Qi, Yue; Fu, Melissa; Herzog, Herbert

2016-02-01

Y2 receptors have been implicated in the development of obesity and are a potential target for obesity treatment due to their known role of inhibiting neuropeptide Y (NPY) induced feeding responses. However, the precise neuronal population on which Y2 receptors act to fulfil this role is less clear. Here we utilise a novel inducible, postnatal onset NPY neurons specific deletion model to investigate the functional consequences of loss of Y2 signalling in this population of neurons on feeding and energy homeostasis regulation. While the consequences of lack of Y2 signalling in NPY neurons are confirmed in terms of the uncoupling of suppression/increasing of NPY and pro-opiomelanocortin (POMC) mRNA expression in the arcuate nuclei (Arc), respectively, this lack of Y2 signalling surprisingly does not have any significant effect on spontaneous food intake. Fasting induced food intake, however, is strongly increased but only in the first 1h after re-feeding. Consequently no significant changes in body weight are being observed although body weight gain is increased in male mice after postnatal onset Y2 deletion. Importantly, another known function of central Y2 receptor signalling, the suppression of bone formation is conserved in this conditional model with whole body bone mineral content being decreased. Taken together this model confirms the critical role of Y2 signalling to control NPY and associated POMC expression in the Arc, but also highlights the possibility that others, non-NPY neuronal Y2 receptors, are also involved in controlling feeding and energy homeostasis regulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Single-nucleotide polymorphisms in the P2X7 receptor gene are associated with post-menopausal bone loss and vertebral fractures

DEFF Research Database (Denmark)

Rye Jørgensen, Niklas; Husted, Lise Bjerre; Skarratt, Kristen K

2012-01-01

to bone mass and fracture incidence in post-menopausal women. A total of 1694 women (aged 45-58) participating in the Danish Osteoporosis Prevention Study were genotyped for 12 functional P2X7 receptor variants. Bone mineral density was determined at baseline and after 10 years. In addition, vertebral...... had increased bone loss. In contrast, the Gln460Arg polymorphism was associated with protection against bone loss. The Ala348Thr polymorphism was associated with a lower vertebral fracture incidence 10 years after menopause. Finally, we developed a risk model, which integrated P2RX7 genotypes. Using...

Actividad química de los ligandos puente difósforo y difosfenilo en los complejos binucleares (Mo2Cp2(n-PCy2) (n-K2:K2-P2)(CO)2)-y (Mo2Cp2(n-PCy2)(n-K2:K2-P2Me)(CO)2)

OpenAIRE

Lozano Rivera, Raquel

2015-01-01

El trabajo de investigación que se recoge en la presente Memoria comprende un amplio estudio de la reactividad de complejos metálicos binucleares con ligandos puente difósforo y metildifosfenilo procedentes de la activación directa del fósforo blanco. Por un lado, se ha llevado a cabo un amplio análisis del comportamiento químico de la especie aniónica [Mo2Cp2(¿-PCy2)(¿-¿2:¿2-P2)(CO)2]- frente a electrófilos de distinta naturaleza, tales como clorofosfinas y complejos metálicos generadores...

Rare missense mutations in P2RY11 in narcolepsy with cataplexy

DEFF Research Database (Denmark)

Degn, Matilda; Dauvilliers, Yves; Dreisig, Karin

2017-01-01

these are single nucleotide polymorphisms in the P2RY11-EIF3G locus. It is unknown how these genetic variants affect narcolepsy pathogenesis and whether the effect is directly related to P2Y11 signalling or EIF3G function. Exome sequencing in 18 families with at least two affected narcolepsy with cataplexy...

Functional expression of P2 purinoceptors in a primary neuroglial cell culture of the rat arcuate nucleus.

Science.gov (United States)

Pollatzek, Eric; Hitzel, Norma; Ott, Daniela; Raisl, Katrin; Reuter, Bärbel; Gerstberger, Rüdiger

2016-07-07

The arcuate nucleus (ARC) plays an important role in the hypothalamic control of energy homeostasis. Expression of various purinoceptor subtypes in the rat ARC and physiological studies suggest a modulatory function of P2 receptors within the neuroglial ARC circuitry. A differentiated mixed neuronal and glial microculture was therefore established from postnatal rat ARC, revealing neuronal expression of ARC-specific transmitters involved in food intake regulation (neuropeptide Y (NPY), proopiomelanocortin (POMC), tyrosine hydroxylase (TH)). Some NPYergic neurons cosynthesized TH, while POMC and TH expression proved to be mutually exclusive. Stimulation with the general purinoceptor agonists 2-methylthioadenosine-5'triphosphate (2-MeSATP) and ATP but not the P2X1/P2X3 receptor subtype agonist α,β-methyleneadenosine-5'triphosphate (α,β-meATP) induced intracellular calcium signals in ARC neurons and astrocytes. Some 5-10% each of 2-MeSATP responsive neurons expressed POMC, NYP or TH. Supporting the calcium imaging data, radioligand binding studies to hypothalamic membranes showed high affinity for 2-MeSATP, ATP but not α,β-meATP to displace [α-(35)S]deoxyadenosine-5'thiotriphosphate ([(35)S]dATPαS) from P2 receptors. Repetitive superfusion with equimolar 2-MeSATP allowed categorization of ARC cells into groups with a high or low (LDD) degree of purinoceptor desensitization, the latter allowing further receptor characterization. Calcium imaging experiments performed at 37°C vs. room temperature showed further reduction of desensitization. Agonist-mediated intracellular calcium signals were suppressed in all LDD neurons but only 25% of astrocytes in the absence of extracellular calcium, suggestive of metabotropic P2Y receptor expression in the majority of ARC astrocytes. The highly P2Y1-selective receptor agonists MRS2365 and 2-methylthioadenosine-5'diphosphate (2-MeSADP) activated 75-85% of all 2-MeSATP-responsive ARC astrocytes. Taking into consideration the

Impact of ticagrelor on P2Y1 and P2Y12 localization and on cholesterol levels in platelet plasma membrane.

Science.gov (United States)

Rabani, Vahideh; Montange, Damien; Meneveau, Nicolas; Davani, Siamak

2017-10-11

Ticagrelor is an antiplatelet agent that inhibits platelet activation via P2Y12 antagonism. There are several studies showing that P2Y12 needs lipid rafts to be activated, but there are few data about how ticagrelor impacts lipid raft organization. Therefore, we aimed to investigate how ticagrelor could impact the distribution of cholesterol and consequently alter the organization of lipid rafts on platelet plasma membranes. We identified cholesterol-enriched raft fractions in platelet membranes by quantification of their cholesterol levels. Modifications in cholesterol and protein profiles (Flotillin 1, Flotillin 2, CD36, P2Y1, and P2Y12) were studied in platelets stimulated by ADP, treated by ticagrelor, or both. In ADP-stimulated and ticagrelor-treated groups, we found a decreased level of cholesterol in raft fractions of platelet plasma membrane compared to the control group. In addition, the peak of cholesterol in different experimental groups changed its localization on membrane fractions. In the control group, it was situated on fraction 2, while in ADP-stimulated platelets, it was located in fractions 3 to 5, and in fraction 4 in ticagrelor-treated group. The proteins studied also showed changes in their level of expression and localization in fractions of plasma membrane. Cholesterol levels of plasma membranes have a direct role in the organization of platelet membranes and could be modified by stimulation or drug treatment. Since ticagrelor and ADP both changed lipid composition and protein profile, investigating the lipid and protein composition of platelet membranes is of considerable importance as a focus for further research in anti-platelet management.

On the simultaneous Pell equations x 2 - (4m 2 - 1)y 2 = y 2 - pz 2 ...

African Journals Online (AJOL)

Let m be a positive integer, and let p be an odd prime. By using certain properties of Pell and quartic diophantine equations with some elementary number theory methods, we prove that the system of equations x2 - (4m2 - 1)y2 = 1 and y2 - pz2 = 1 has positive integer solutions (x, y, z) if and only if p ≡ 7(mod 8) and m = 1/4 ...

GluN2C/GluN2D subunit-selective NMDA receptor potentiator CIQ reverses MK-801-induced impairment in prepulse inhibition and working memory in Y-maze test in mice

Science.gov (United States)

Suryavanshi, P S; Ugale, R R; Yilmazer-Hanke, D; Stairs, D J; Dravid, S M

2014-01-01

Background and Purpose Despite ample evidence supporting the N-methyl-d-aspartate receptor (NMDAR) hypofunction hypothesis of schizophrenia, progress in the development of effective therapeutics based on this hypothesis has been limited. Facilitation of NMDA receptor function by co-agonists (d-serine or glycine) only partially alleviates the symptoms in schizophrenia; other means to facilitate NMDA receptors are required. NMDA receptor sub-types differ in their subunit composition, with varied GluN2 subunits (GluN2A-GluN2D) imparting different physiological, biochemical and pharmacological properties. CIQ is a positive allosteric modulator that is selective for GluN2C/GluN2D-containing NMDA receptors (Mullasseril et al.). Experimental Approach The effect of systemic administration of CIQ was tested on impairment in prepulse inhibition (PPI), hyperlocomotion and stereotypy induced by i.p. administration of MK-801 and methamphetamine. The effect of CIQ was also tested on MK-801-induced impairment in working memory in Y-maze spontaneous alternation test. Key Results We found that systemic administration of CIQ (20 mg·kg−1, i.p.) in mice reversed MK-801 (0.15 mg·kg−1, i.p.)-induced, but not methamphetamine (3 mg·kg−1, i.p.)-induced, deficit in PPI. MK-801 increased the startle amplitude to pulse alone, which was not reversed by CIQ. In contrast, methamphetamine reduced the startle amplitude to pulse alone, which was reversed by CIQ. CIQ also partially attenuated MK-801- and methamphetamine-induced hyperlocomotion and stereotyped behaviours. Additionally, CIQ reversed the MK-801-induced working memory deficit in spontaneous alternation in a Y-maze. Conclusion and Implications Together, these results suggest that facilitation of GluN2C/GluN2D-containing receptors may serve as an important therapeutic strategy for treating positive and cognitive symptoms in schizophrenia. PMID:24236947

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

Selective fluorescence quenching of 2,3-diazabicyclo[2.2.2]oct-2-ene by nucleotides.

Science.gov (United States)

Marquez, Cesar; Pischel, Uwe; Nau, Werner M

2003-10-16

[reaction: see text] The fluorescence quenching of 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) by nucleotides has been studied. The quenching mechanism was analyzed on the basis of deuterium isotope effects, tendencies for exciplex formation, and the quenching efficiency in the presence of a molecular container (cucurbit[7]uril). Exciplex-induced quenching appears to prevail for adenosine, cytidine, and uridine, while hydrogen abstraction becomes competitive for thymidine and guanosine. Compared to other fluorescent probes, DBO responds very selectively to the type of nucleotide.

Receptores plaquetários P2Y12: importância na intervenção coronariana percutânea

Directory of Open Access Journals (Sweden)

Felipe Jose de Andrade Falcão

2013-09-01

Full Text Available As plaquetas estão envolvidas em vários processos biológicos, desde o combate a agentes infecciosos até a coordenação do controle da permeabilidade vascular e angiogênese. Entretanto, o seu principal foco de ação consiste na modulação da cascata de coagulação. A intervenção coronariana percutânea é um procedimento com alto risco trombogênico, que induz a ativação plaquetária e de monócitos, devido à lesão direta do endotélio e pelo contato de estruturas trombogênicas com o sangue, levando ao aumento da atividade inflamatória, tanto no local do dano vascular coronariano como de forma sistêmica. Os receptores plaquetários P2Y12 desempenham papel central na amplificação da agregação induzida por todos os agonistas plaquetários, como a adenosina difosfato, o colágeno, tromboxano A2, adrenalina e serotonina. Por esse motivo, têm sido o principal alvo das drogas antiplaquetárias. Apesar de atuarem no mesmo receptor, características farmacocinéticas e farmacodinâmicas distintas conferem peculiaridades a cada agente.

Polymorphisms in the P2X7 receptor gene are associated with low lumbar spine bone mineral density and accelerated bone loss in post-menopausal women

DEFF Research Database (Denmark)

Gartland, Alison; Skarratt, Kristen K; Hocking, Lynne J

2012-01-01

The P2X7 receptor gene (P2RX7) is highly polymorphic with five previously described loss-of-function (LOF) single-nucleotide polymorphisms (SNP; c.151+1G>T, c.946G>A, c.1096C>G, c.1513A>C and c.1729T>A) and one gain-of-function SNP (c.489C>T). The purpose of this study was to determine whether th...... publication, 11 January 2012; doi:10.1038/ejhg.2011.245....

Identification and characterization of a novel P2Y 12 variant in a patient diagnosed with type 1 von Willebrand disease in the European MCMDM-1VWD study.

Science.gov (United States)

Daly, Martina E; Dawood, Ban B; Lester, William A; Peake, Ian R; Rodeghiero, Francesco; Goodeve, Anne C; Makris, Michael; Wilde, Jonathan T; Mumford, Andrew D; Watson, Stephen P; Mundell, Stuart J

2009-04-23

We investigated whether defects in the P2Y(12) ADP receptor gene (P2RY12) contribute to the bleeding tendency in 92 index cases enrolled in the European MCMDM-1VWD study. A heterozygous mutation, predicting a lysine to glutamate (K174E) substitution in P2Y(12), was identified in one case with mild type 1 von Willebrand disease (VWD) and a VWF defect. Platelets from the index case and relatives carrying the K174E defect changed shape in response to ADP, but showed reduced and reversible aggregation in response to 10 muM ADP, unlike the maximal, sustained aggregation observed in controls. The reduced response was associated with an approximate 50% reduction in binding of [(3)H]2MeS-ADP to P2Y(12), whereas binding to the P2Y(1) receptor was normal. A hemagglutinin-tagged K174E P2Y(12) variant showed surface expression in CHO cells, markedly reduced binding to [(3)H]2MeS-ADP, and minimal ADP-mediated inhibition of forskolin-induced adenylyl cyclase activity. Our results provide further evidence for locus heterogeneity in type 1 VWD.

Guanine nucleotide regulation of α1-adrenergic receptors of muscle and kidney eptihelial cells

International Nuclear Information System (INIS)

Terman, B.I.; Hughes, R.J.; Slivka, S.R.; Insel, P.A.

1986-01-01

The authors have examined the effect of guanine nucleotides on the interaction of adrenergic agents with α 1 -adrenergic receptors of two cell lines, the Madin-Darby Canine Kidney (MDCK) and BC3H-1 muscle cells. While gaunylylimidodiphosphoate (Gpp(NH)p) had no effect on the affinity or the total number of [ -3 H]prazosin binding sites in membranes prepared from these cells, the nucleotide decreased the apparent affinity of the agonist epinephrine in competing for [ 3 H]prazosin binding sites in both cell types. The EC 50 of Gpp(NH)p was ∼100 μM, and a maximal effect was seen at 500 μM. In contrast, 100 μM Gpp(NH)p yielding maximal shifts in binding of epinephrine to β-adrenergic receptors in BC3H-1 cell membranes. Guanine nucleotides were significantly more effective than adenine nucleotides in shifting agonist affinity for the α 1 -receptor and Mg ++ was required to observe a maximal effect. α 1 -receptor agonists activated phosphatidylinositol (PI) hydrolysis in both cell types, but have no direct effect on membrane adenylate cyclase activity. In intact BC3H-1 cells, α 1 -agonists inhibited β-adrenergic cAMP production, an effect which appears in preliminary studies not to result from enhanced phosphodieterase activity. These results show that agonist binding to α 1 -adrenergic receptors in mammalian kidney and muscle cells is regulated by guanine nucleotides. This regulation and inturn transmembrane signalling (PI hydrolysis) by these receptors appear to involve a guanine nucleotide binding (G) protein, which may be different than G/sub s/ and G/sub i/

Crystal structure of the β2 adrenergic receptor-Gs protein complex

DEFF Research Database (Denmark)

Rasmussen, Søren Gøgsig Faarup; DeVree, Brian T; Zou, Yaozhong

2011-01-01

-occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs...

Corneal epithelium expresses a variant of P2X(7 receptor in health and disease.

Directory of Open Access Journals (Sweden)

Courtney Mankus

Full Text Available Improper wound repair of the corneal epithelium can alter refraction of light resulting in impaired vision. We have shown that ATP is released after injury, activates purinergic receptor signaling pathways and plays a major role in wound closure. In many cells or tissues, ATP activates P2X(7 receptors leading to cation fluxes and cytotoxicity. The corneal epithelium is an excellent model to study the expression of both the full-length P2X(7 form (defined as the canonical receptor and its truncated forms. When Ca(2+ mobilization is induced by BzATP, a P2X(7 agonist, it is attenuated in the presence of extracellular Mg(2+ or Zn(2+, negligible in the absence of extracellular Ca(2+, and inhibited by the competitive P2X7 receptor inhibitor, A438079. BzATP enhanced phosphorylation of ERK. Together these responses indicate the presence of a canonical or full-length P2X(7 receptor. In addition BzATP enhanced epithelial cell migration, and transfection with siRNA to the P2X(7 receptor reduced cell migration. Furthermore, sustained activation did not induce dye uptake indicating the presence of truncated or variant forms that lack the ability to form large pores. Reverse transcription-polymerase chain reaction and Northern blot analysis revealed a P2X(7 splice variant. Western blots identified a full-length and truncated form, and the expression pattern changed as cultures progressed from monolayer to stratified. Cross-linking gels demonstrated the presence of homo- and heterotrimers. We examined epithelium from age matched diabetic and non-diabetic corneas patients and detected a 4-fold increase in P2X(7 mRNA from diabetic corneal epithelium compared to non-diabetic controls and an increased trend in expression of P2X(7variant mRNA. Taken together, these data indicate that corneal epithelial cells express full-length and truncated forms of P2X(7, which ultimately allows P2X(7 to function as a multifaceted receptor that can mediate cell proliferation and