The P2Y14 receptor activity in glioma C6 cells

In this study, we demonstrated the presence and the activity of the P2Y(14) receptor in glioma C6 cells. We found that P2Y(14) could exist in two forms, highly predominating glycosylated and non-glycosylated. Binding of UDP-glucose evoked two responses: calcium signal and adenylate cyclase inhibition, both pertussis toxin-sensitive. Separate glycosylation pattern and functional profile of these two receptor forms were observed in non-starved and serum-starved cells. During long-term serum deprivation (96 h), the level of glycosylated form strongly decreased, while non-glycosylated increased, what was correlated with the decrease of calcium signaling activity and stronger adenylate cyclase inhibition, suggesting that receptor N-glycosylation may modulate its functional activity.     

新型血小板P2Y12受体拮抗药

抑制ADP诱导的血小板聚集的药物(如氯吡格雷)已成为目前心血管领域内最重要的抗血小板药物。但氯吡格雷起效较慢,抗血小板作用相对较弱,尚有一定的低反应者和无反应者。普拉格雷是一个较新的血小板P2Y12受体拮抗药。TRITON-TIMI 38研究表明普拉格雷可使主要终点发生率(心血管死亡、非致死性心肌梗死、非致死性卒中)降低19%,但使出血并发症增加。坎格雷洛,替格雷洛,依诺格雷是更新的血小板P2Y12受体拮抗药。     

Biology and pharmacology of the platelet P2Y12 receptor

Platelets possess two receptors for ADP, P2Y(1) and P2Y(12). ADP is released from platelet dense granules upon platelet activation by numerous agonists and thereby amplifies platelet responses regardless of the initial stimulus. The P2Y(1) receptor is one of many platelet receptors coupled to Gq and initiates ADP-induced activation. The P2Y(12) receptor on the other hand is linked to Gi and plays a special role in the amplification of platelet activation initiated by numerous other pathways. Platelet activation leads to a range of responses that play a critical role in arterial thrombosis and the inflammatory responses associated with this, including platelet aggregation, dense and alpha granule secretion and procoagulant activity. P2Y(12) receptor activation yields powerful amplification of these processes such that P2Y(12) receptor antagonists may have dramatic inhibitory effects on platelet function regardless of the activating stimuli. This phenomenon, coupled with the restricted distribution of the P2Y(12) receptor in humans, makes the receptor an ideal target for pharmaceutical therapy. This has already been established by the therapeutic success of clopidogrel, which acts, via an active metabolite, on this receptor. However, current therapeutic regimens of clopidogrel yield variable and incomplete P2Y(12) receptor blockade and more effective strategies to block P2Y(12) receptor activation offer the potential of greater clinical efficacy.     

抗血小板膜P2Y12受体拮抗剂的研究进展

抗血小板药物是治疗急性冠脉综合征的必备用药,其中抗血小板膜P2Y12受体拮抗剂已成为急性冠脉综合征药物治疗研究中最活跃的一个领域,从基础到临床研究,不断地开发出系列新药.本文就对P2Y12受体拮抗剂作用机制、药代动力学、急性冠脉综合征临床研究、临床指南等进行分析,为临床合理用药提供参考.     

Molecular cloning of the platelet P2T(AC) ADP receptor: pharmacological comparison with another ADP receptor, the P2Y(1) receptor

Platelet activation plays an essential role in thrombosis. ADP-induced platelet aggregation is mediated by two distinct G protein-coupled ADP receptors, Gq-linked P2Y(1), and Gi-linked P2T(AC), which has not been cloned. The cDNA encoding a novel G protein-coupled receptor, termed HORK3, was isolated. The HORK3 gene and P2Y(1) gene were mapped to chromosome 3q21-q25. HORK3, when transfected in the rat glioma cell subline (C6-15), responded to 2-methylthio-ADP (2MeSADP) (EC(50) = 0.08 nM) and ADP (EC(50) = 42 nM) with inhibition of forskolin-stimulated cAMP accumulation. 2MeSADP (EC(50) = 1.3 nM) and ADP (EC(50) = 18 nM) also induced intracellular calcium mobilization in P2Y(1)-expressing cells. These results show that HORK3 is a Gi/o-coupled receptor and that its natural ligand is ADP. AR-C69931 MX and 2MeSAMP, P2T(AC) antagonists, selectively inhibited 2MeSADP-induced adenylyl cyclase inhibition in HORK3-expressing cells. On the other hand, A3P5PS, a P2Y(1) antagonist, blocked only 2MeSADP-induced calcium mobilization in P2Y(1)-expressing cells. HORK3 mRNA was detected in human platelets and the expression level of HORK3 was equivalent to that of P2Y(1). These observations indicate that HORK3 has the characteristics of the proposed P2T(AC) receptor. We have also determined that [(3)H]2MeSADP binds to cloned HORK3 and P2Y(1). Competition binding experiments revealed a similarity in the rank orders of potency of agonists and the selectivity of antagonists as obtained in the functional assay. These results support the view that P2Y(1) functions as a high-affinity ADP receptor and P2T(AC) as a low-affinity ADP receptor in platelets.     

2-Alkylthio-substituted platelet P2Y12 receptor antagonists reveal pharmacological identity between the rat brain Gi-linked ADP receptors and P2Y12

The Gi-linked platelet ADP receptor, now designated as P2Y12, accounts for ADP-induced inhibition of adenylyl cyclase in platelets and certain clonal rat cell lines. The pharmacology of this receptor is well characterized. Based on the functional approach of [35S]GTPgammaS autoradiography, we recently disclosed the widespread presence of Gi-linked ADP receptors in the rat nervous system. Based on initial pharmacological analysis, these receptors were strikingly similar with P2Y12. Here, we extend this analysis by comparing the potencies of six 2-alkylthio-substituted ATP analogues, including the adenosine-aspartate conjugate 2-hexylthio-AdoOC(O)Asp2 and five AR-C compounds (AR-C67085, AR-C69931, AR-C78511, AR-C69581, AR-C70300) with wide range of affinities towards P2Y12, in reversing 2-methylthio-ADP stimulated G protein activity in rat brain sections and human platelet membranes. Closely matching pIC50 values (r2=0.99) revealed pharmacological similarity between the two receptors with one exception: AR-C67085 more avidly recognized the platelet P2Y12. Further analysis of the rat brain pIC50 data against those available for three of the AR-C compounds in reversing P2Y12-mediated adenylyl cyclase inhibition in rat platelets (r2=0.96) and rat C6 glioma cells (r2=1.00) demonstrated that the three P2Y receptors are pharmacologically indistinguishable. We conclude that the rat brain Gi-linked ADP receptors, as revealed using [35S]GTPgammaS autoradiography, correspond to P2Y12.     

Two subtypes of G protein-coupled nucleotide receptors, P2Y(1) and P2Y(2) are involved in calcium signalling in glioma C6 cells

1. In glioma C6 cells, the stimulation of P2Y receptors by ADP, ATP and UTP initiated an increase in the intracellular Ca2+ concentration, in a process that involved the release of Ca2+ from InsP(3)-sensitive store and the capacitative, extracellular Ca2+ entry. The presence of external Ca2+ was not necessary to elevate Ca(2+). 2. The rank order of potencies of nucleotide analogues in stimulating [Ca2+](i) was: 2MeSADP > ADP > 2MeSATP = 2ClATP > ATP > UTP. alpha,beta-Methylene ATP, adenosine and AMP were ineffective. 3. ADP and UTP effects were additive, while actions of ATP and UTP were not additive on [Ca2+](i) increase. Similarly, cross-desensitization between ATP and UTP but not between ADP and UTP occurred. 4. Suramin, a non-specific nucleotide receptors inhibitor, antagonized ATP-, UTP- and ADP-evoked Ca2+ responses. PPADS, a selective antagonist of the P2Y(1) receptor-generated InsP(3) accumulation, decreased ADP-initiated Ca2+ response with no effect on ATP and UTP. 5. Pertussis toxin (PTX) reduced ADP- and ATP-induced Ca2+ increases. Short-term treatment with TPA, inhibited both ATP and ADP stimulatory effects on [Ca2+](i). 6. ADP inhibited isoproterenol-induced cyclic AMP accumulation. PTX blocked this effect, but PPADS did not. 7. RT - PCR analysis revealed the molecular identity of P2Y receptors expressed by glioma C6 cells to be both P2Y(1) and P2Y(2). 8. It is concluded that both P2Y(1) and P2Y(2) receptors co-exist in glioma C6 cells. ADP acts as agonist of the first, and ATP and UTP of the second one. Both receptors are linked to phospholipase C (PLC).     

Beta-adrenergic receptor regulation of NGF-mRNA content in rat C6-2B glioma cells

In C₆-2B astrocytoma cells the Beta NGF content and secretion rate are increased by isoproterenol activation of beta-adrenergic receptors (Schwartz and Costa, 1977). Utilizing poly (A⁺) RNA hybridization analysis with a cRNA probe for mouse Beta NGF it was found that isoproterenol activation of C₆-2B cells produces also a 4 fold increase of the content of messenger RNA encoding Beta NGF. This increase is specifically antagonized by 1-propanolol, but not by phentolamine. Furthermore, addition of dibutyryl-cAMP induces an increase of Beta NGF mRNA content similar to that obtained with isoproterenol. These results are consistent with the hypothesis that regulation of Beta NGF synthesis in neuroglial cells may be modulated by beta-adrenergic receptor activation.     

P2Y12 receptor blockade synergizes strongly with nitric oxide and prostacyclin to inhibit platelet activation

AimsIn vivo platelet function is a product of intrinsic platelet reactivity, modifiable by dual antiplatelet therapy (DAPT), and the extrinsic inhibitory endothelial mediators, nitric oxide (NO) and prostacyclin (PGI₂), that are powerfully potentiated by P2Y₁₂ receptor blockade. This implies that for individual patients endothelial mediator production is an important determinant of DAPT effectiveness. Here, we have investigated this idea using platelets taken from healthy volunteers treated with anti‐platelet drugs.MethodsThree groups of male volunteers (n = 8) received either prasugrel (10 mg), aspirin (75 mg) or DAPT (prasugrel + aspirin) once daily for 7 days. Platelet reactivity in the presence of diethylammonium (Z)‐1‐(N,N‐diethylamino)diazen‐1‐ium‐1,2‐diolate (DEA/NONOate) and PGI₂ was studied before and following treatment.ResultsEx vivo, PGI₂ and/or DEA/NONOate had little inhibitory effect on TRAP‐6‐induced platelet reactivity in control conditions. However, in the presence of DAPT, combination of DEA/NONOate + PGI₂ reduced platelet aggregation (74 ± 3% to 19 ± 6%, P < 0.05). In vitro studies showed even partial (25%) P2Y₁₂ receptor blockade produced a significant (67 ± 2% to 39 ± 10%, P < 0.05) inhibition when DEA/NONOate + PGI₂ was present.ConclusionsWe have demonstrated that PGI₂ and NO synergize with P2Y₁₂ receptor antagonists to produce powerful platelet inhibition. Furthermore, even with submaximal P2Y₁₂ blockade the presence of PGI₂ and NO greatly enhances platelet inhibition. Our findings highlight the importance of endothelial mediator in vivo modulation of P2Y₁₂ inhibition and introduces the concept of refining ex vivo platelet function testing by incorporating an assessment of endothelial function to predict thrombotic outcomes better and adjust therapy to prevent adverse outcomes in individual patients.     

Purinergic P2Y12 receptor blockade inhibits shear-induced platelet phosphatidylinositol 3-kinase activation

Pathologically elevated shear stress triggers aspirin-insensitive platelet thrombosis. Signaling mechanisms involved in shear-induced platelet thrombosis are not well understood. To investigate these, we examined the hypothesis that functionally important platelet phosphatidylinositol 3-kinase (PI3-K) activity is stimulated by an in vitro shear stress of 120 dynes/cm(2) (shear rate of 6,000 sec(-1)). Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) production was examined in washed human platelets subjected to pathological shear stress in a cone-plate viscometer. PIP(3) production peaks 30 s after shear begins and is initiated by von Willebrand factor (VWF) binding to the glycoprotein (Gp) Ib-IX-V complex. Inhibiting PI3-K with wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) results in the inhibition of shear-induced platelet aggregation. In resting platelets, class IA PI3-K associates with the tyrosine kinase Syk. Within 30 s of beginning shear, PI3-K-associated Syk becomes tyrosine phosphorylated. Inhibiting Syk activation with piceatannol results in the inhibition of PIP(3) production and aggregation. Selective blockade of the P2Y(12) receptor results in the inhibition of Syk phosphorylation, PIP(3) production, and aggregation. These results indicate that shear-induced VWF binding to platelet GpIb-IX-V stimulates functionally important PI3-K activity. PI3-K activation is signaled by rapid feedback amplification that involves P2Y(12) receptor-mediated activation of Syk.     

Lipophilic modifications to dinucleoside polyphosphates and nucleotides that confer antagonist properties at the platelet P2Y12 receptor

Platelet P2Y12 receptors play a central role in the regulation of platelet function and inhibition of this receptor by treatment with drugs such as clopidogrel results in a reduction of atherothrombotic events. We discovered that modification of natural and synthetic dinucleoside polyphosphates and nucleotides with lipophilic substituents on the ribose and base conferred P2Y12 receptor antagonist properties to these molecules producing potent inhibitors of ADP-mediated platelet aggregation. We describe methods for the preparation of these functionalized dinucleoside polyphosphates and nucleotides and report their associated activities. By analysis of these results and by deconstruction of the necessary structural elements through selected syntheses, we prepared a series of highly functionalized nucleotides, resulting in the selection of an adenosine monophosphate derivative (62) for further clinical development.     

Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family

The cloning of a human G-protein-coupled receptor (GPCR) that specifically responds to UDP-glucose and related sugar-nucleotides has been reported recently. This receptor has important structural similarities to known members of the P2Y receptor family but also shows a distinctly different pharmacological response profile. Here, the IUPHAR Subcommittee for P2Y receptor nomenclature and classification review the current knowledge of this receptor and present their reasons for including this receptor in the P2Y receptor family as the P2Y(14) receptor.     

ATP, an agonist at the rat P2Y(4) receptor, is an antagonist at the human P2Y(4) receptor

The nucleotide selectivities of the human P2Y(4) (hP2Y(4)) and rat P2Y(4) (rP2Y(4)) receptor stably expressed in 1321N1 human astrocytoma cells were determined by measuring increases in intracellular [Ca(2+)] under conditions that minimized metabolism, bioconversion, and endogenous nucleotide release. In cells expressing the hP2Y(4) receptor, UTP, GTP, and ITP all increased intracellular [Ca(2+)] with a rank order of potency of UTP (0.55) > GTP (6.59) = ITP (7.38), (EC(50), microM). ATP, CTP, xanthine 5'-triphosphate (XTP), and diadenosine 5',5"'-P(1), P(4)-tetraphosphate (Ap(4)A), all at 100 microM, were inactive at the hP2Y(4) receptor. In cells expressing the rP2Y(4) receptor, all seven nucleotides increased intracellular [Ca(2+)] with similar maximal effects and a rank order of potency of UTP (0.20) > ATP (0. 51) > Ap(4)A (1.24) approximately ITP (1.82) approximately GTP (2. 28) > CTP (7.24) > XTP (22.9). Because ATP is inactive at the hP2Y(4) receptor, we assessed whether ATP displayed antagonist activity. When coapplied, ATP shifted the concentration-response curve to UTP rightward in a concentration-dependent manner, with no change in the maximal response. A Schild plot derived from these data gave a pA(2) value of 6.15 (K(B) = 708 nM) and a slope near unity. Additionally, CTP and Ap(4)A (each at 100 microM) inhibited the response to an EC(50) concentration of UTP by approximately 40 and approximately 50%, respectively, whereas XTP had no effect. The inhibitory effects of ATP, CTP, and Ap(4)A were reversible on washout. Thus, ATP is a potent agonist at the rP2Y(4) receptor but is a competitive antagonist with moderate potency at the hP2Y(4) receptor.     

Differential effects of P2Y1 and P2Y12 nucleotide receptors on ERK1/ERK2 and phosphatidylinositol 3-kinase signalling and cell proliferation in serum-deprived and nonstarved glioma C6 cells

We have previously shown that, in glioma C6 cells, two nucleotide ADP-sensitive receptors coexist: P2Y1, coupled to PLC and responsible for Ca2+ release, and P2Y12, negatively coupled to adenylate cyclase. In the present study, we examined the effects of the stimulation of these two receptors on ERK1/2 and PI3-K activation, and cell proliferation in either serum-deprived or nonstarved C6 cells. In response to ADP and its analogues, in serum-starved cells, both p44 ERK1 and p42 ERK2 were activated in a time-dependent manner, as monitored by Western blot analysis using an antiphospho-p42/p44 MAPK antibody. The phosphorylation was reduced both by removal of the extracellular Ca2+ and partially or almost completely by MRS2179 or AR-C69931MX, specific antagonists of the P2Y1 and P2Y12 receptors, respectively. The inhibitory effect of antagonists was additive. These data indicate the involvement of both receptors, P2Y1 and P2Y12, in the ERK1/2 activation, but the P2Y12 receptor contribution predominates. ERK1/2 activity was positively correlated with cell proliferation of cultured glioma C6 cells. In nonstarved cells, ADP markedly decreased the PI3-K activity. In contrast, in serum-starved cells, ADP evoked an increase in the PI3-K activity. Blocking of the P2Y1 receptor by MRS2179 additionally increased this ADP response. These results suggest that the P2Y1 receptor has an inhibitory and the P2Y12 receptor a stimulatory effect on PI3-K signalling pathway. RT-PCR analysis revealed different mRNA expression of both receptors in starved and nonstarved cells. In nonstarved cells, the P2Y1 receptor mRNA predominates, whereas in serum-deprived cells the expression of P2Y12 mRNA becomes more pronounced.British Journal of Pharmacology (2004) 141, 497-507. doi:10.1038/sj.bjp.0705639     

Inhibition of platelet P2Y12 and alpha2A receptor signaling by cGMP-dependent protein kinase

The important role of cGMP and cGMP-dependent protein kinase (cGPK) for the inhibition of platelet activation and aggregation is well established and due to the inhibition of fundamental platelet responses such as agonist-stimulated calcium increase, exposure of adhesion receptors and actin polymerization. The diversity of cGMP binding proteins and their synergistic interaction with cAMP signaling in inhibiting platelets indicates that a variety of cGMP targets contribute to its antiplatelet action. Since stimulation of G(i)-proteins was recently shown to be essential for complete platelet activation/aggregation, the possibility that G(i)-signaling events are cGMP/cGPK targets was investigated. Thus, the effect of elevated cGMP levels and selective cGPK activation on purinergic and adrenergic receptor-evoked decrease of platelet cAMP content was closely examined. Experiments with a selective activator of cGPK demonstrate for the first time a cGMP-caused G(i)-protein inhibition and our data suggest that this effect is mediated by cGPK. Considering the essential role of G(i)-signaling for platelet activation, we propose that inhibition of G(i)-mediated signaling by cGMP/cGPK is an important mechanism of action underlying the platelet inhibition by cGMP-elevating endothelium derived factors and drugs.     

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

The objective of this study was to investigate if there is a synergistic effect of a combination of P2Y(12) and P2Y(1) inhibition and P2Y(12) and thrombin inhibition, on ADP- and thrombin-induced platelet activation, respectively. The rationale being that these combinations will cause a concurrent inhibition of both G alpha(q) and Galpha (i) signalling. Blood from healthy volunteers was preincubated with AR-C69931MX, a reversible P2Y(12) antagonist; MRS2179, a reversible P2Y(1) 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 alpha (IIb)beta(3) (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 microM) 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.     

Differential coupling of the human P2Y(11) receptor to phospholipase C and adenylyl cyclase

1. The human P2Y(11) (hP2Y(11)) receptor was stably expressed in two cell lines, 1321N1 human astrocytoma cells (1321N1-hP2Y(11)) and Chinese hamster ovary cells (CHO-hP2Y(11)), and its coupling to phospholipase C and adenylyl cyclase was assessed. 2. In 1321N1-hP2Y(11) cells, ATP promoted inositol phosphate (IP) accumulation with low microM potency (EC(50)=8.5+/-0.1 microM), whereas it was 15 fold less potent (130+/-10 microM) in evoking cyclic AMP production. 3. In CHO-hP2Y(11) cells, ATP promoted IP accumulation with slightly higher potency (EC(50)=3.6+/-1.3 microM) than in 1321N1-hP2Y(11) cells, but it was still 15 fold less potent in promoting cyclic AMP accumulation (EC(50)=62.4+/-15.6 microM) than for IP accumulation. Comparable differences in potencies for promoting the two second messenger responses were observed with other adenosine nucleotide analogues. 4. In 1321N1-hP2Y(11) and CHO-hP2Y(11) cells, down regulation of PKC by chronic treatment with phorbol ester decreased ATP-promoted cyclic AMP accumulation by 60--80% (P<0.001) with no change in its potency. Likewise, chelation of intracellular Ca(2+) decreased ATP-promoted cyclic AMP accumulation by approximately 45% in 1321N1-hP2Y(11) cells, whereas chelation had no effect on either the efficacy or potency of ATP in CHO-hP2Y(11) cells. 5. We conclude that coupling of hP2Y(11) receptors to adenylyl cyclase in these cell lines is much weaker than coupling to phospholipase C, and that activation of PKC and intracellular Ca(2+) mobilization as consequences of inositol lipid hydrolysis potentiates the capacity of ATP to increase cyclic AMP accumulation in both 1321N1-hP2Y(11) and CHO-hP2Y(11) cells.