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).     

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

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.     

Endothelium-dependent relaxation evoked by ATP and UTP in the aorta of P2Y2-deficient mice

Based on pharmacological criteria, we previously suggested that in the mouse aorta, endothelium-dependent relaxation by nucleotides is mediated by P2Y1 (adenosine diphosphate (ADP)), P2Y2 (adenosine triphosphate (ATP)) and P2Y6 (uridine diphosphate (UDP)) receptors. For UTP, it was unclear whether P2Y2, P2Y6 or yet another subtype was involved. Therefore, in view of the lack of selective purinergic agonists and antagonists, we used P2Y2-deficient mice to clarify the action of UTP. Thoracic aorta segments (width 2 mm) of P2Y2-deficient and wild-type (WT) mice were mounted in organ baths to measure isometric force development and intracellular calcium signalling.Relaxations evoked by ADP, UDP and acetylcholine were identical in knockout and WT mice, indicating that the receptors for these agonists function normally. P2Y2-deficient mice showed impaired ATP- and adenosine 5'[gamma-thio] triphosphate (ATPgammaS)-evoked relaxation, suggesting that in WT mice, ATP and ATPgammaS activate predominantly the P2Y2 subtype. The ATP/ATPgammaS-evoked relaxation and calcium signals in the knockout mice were partially rescued by P2Y1, as they were sensitive to 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS2179), a P2Y1-selective antagonist.In contrast to ATP, the UTP-evoked relaxation was not different between knockout and WT mice. Moreover, the action of UTP was not sensitive to MRS2179. Therefore, the action of UTP is probably mediated mainly by a P2Y6(like) receptor subtype.In conclusion, we demonstrated that ATP-evoked relaxation of the murine aorta is mainly mediated by P2Y2. But this P2Y2 receptor has apparently no major role in UTP-evoked relaxation. The vasodilator effect of UTP is probably mediated mainly by a P2Y6(like) receptor.     

Purinergic and pyrimidinergic receptors as potential drug targets

In the last decade, the field of purinergic pharmacology has continued to grow as the complexity of the receptor families and the various enzymes involved in purine metabolism have been defined in molecular terms. A major theme that has emerged from these studies is the functional complexity of the interactions between P1 and P2 receptors, based upon the dynamic interrelationship between ATP and adenosine as extracellular signaling molecules. It is now clear that ATP and its degradation products (particularly ADP and adenosine) form a complex cascade for the regulation of cell-to-cell communication that can function to attenuate the consequences of tissue trauma (e.g. ischemia) that involve alterations in cellular energy charge and depletion of ATP stores. In addition to the P2 receptor family, alterations in cellular ATP stores can also affect the function of other receptors, e.g. K(ATP) channels, and mitochondrial function. The discovery of pyrimidine-preferring (UTP/UDP) P2Y receptors has also raised the possibility that the corresponding nucleoside, uracil, may function as a signaling molecule.     

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

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.     

Characterisation of P2Y(1)-like receptor in cultured rat pineal glands

The rat pineal gland possesses P2 receptors which potentiate the effect of noradrenaline-induced N'-acetyl-5-hydroxytryptamine (N'-acetyl-5-HT) production. In the current study, this receptor was characterised according to agonist selectivity and signal transduction mechanisms. 2-MethylthioATP (2MeSATP), 2-chloroATP (2-ClATP), adenosine 5'-O-2-thiodiphosphate, (ADPbetaS), ATP and ADP, but not UTP, potentiated noradrenaline-induced N'-acetyl-5-HT production in a concentration-dependent manner. 2MeSATP neither induced the production of adenosine 3':5'-cyclic monophosphate (cyclic AMP), nor inhibited its formation when the glands were stimulated by forskolin. The phospholipase C inhibitor 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), but not the inactive analogue, 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione (U73343), blocked the 2MeSATP effect. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-dissulphonic acid (PPADS), which inhibits phospholipase C-coupled P2Y(1) receptors, blocked the 2MeSATP effect. In conclusion, our data strongly suggest that the P2-like receptor that is present in rat pinealocytes and which is responsible for the potentiation of noradrenaline-induced N'-acetyl-5-HT production is a P2Y(1)-like receptor, coupled to a G protein which stimulates phospholipase C.     

Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) as a tool for differentiation of P2Y2-receptor-mediated vasorelaxation in guinea pig aorta

The action of the putative P2Y1-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) was studied in guinea pig aorta for potential use in the differentiation between P2Y1 and P2Y2 purine receptors. Concentration-effect curves to 2-methylthioadenosine triphosphate (2MeSATP) and uridine triphosphate (UTP), agonists at P2Y1- and P2Y2-receptor sites, respectively, and the common agonist adenosine triphosphate (ATP) were constructed in guinea pig aortic ring preparations with the tone raised by 5 microM of noradrenaline. A ranked order of agonist potency of 2MeSATP > ATP > UTP resulted from the construction of concentration response curves to vasorelaxation of the agonists. Deendothelialization virtually abolished vasorelaxation to UTP but made no significant difference to 2MeSATP-induced responses. PPADS exhibited noncompetitive inhibition at P2Y1-receptor sites by reducing the maximal response to 2MeSATP, although a trend towards a right shift of the concentration-effect curves was observed. In total contrast, PPADS enhanced P2Y2-mediated vasorelaxation to UTP by shifting the concentration-effect curves to the left and increasing maximal responses. Thus, PPADS is a noncompetitive antagonist at P2Y1 receptors but enhances responses, at least to UTP, at P2Y2 receptors in guinea pig aorta via a hitherto unknown mechanism. Thus, PPADS is a potentially useful substance that may be used for differentiation between P2Y1 and P2Y2 receptors in the guinea pig aorta.     

Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta

Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. Nucleotides evoked complete (adenosine 5' triphosphate (ATP), uridine 5' triphosphate (UTP), uridine 5' diphosphate (UDP); >90%) or partial (adenosine 5' diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. The rank order of potency was: UDP approximately UTP approximately ADP>adenosine 5'-[gamma-thio] triphosphate (ATPgammaS)>ATP, with respective pD2 values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y1 (ADP>ATP), P2Y2 or P2Y4 (ATP and UTP) and P2Y6 (UDP) receptors. P2Y4 receptors were not involved, since P2Y4-deficient mice displayed unaltered responses to ATP and UTP.The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pKb for ATP (4.53+/-0.07) was compatible with literature, but the pKb for UTP (5.19+/-0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non-P2Y2 receptor subtype(s). Further, pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). Finally, 2'-deoxy-N6-methyladenosine3',5'-bisphosphate (MRS2179) inhibited ADP-evoked relaxation only. Taken together, these results point to the presence of functional P2Y1 (ADP), P2Y2 (ATP, UTP) and P2Y6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP-evoked vasodilatation.     

Relative contribution of P2U- and P2Y-purinoceptors to endothelium-dependent vasodilatation in the golden hamster isolated mesenteric arterial bed.

1. P2-purinoceptors were characterized pharmacologically in the constantly perfused isolated mesenteric arterial vascular bed of the golden hamster. Vasoconstrictor and vasodilator responses to the nucleotides ATP, ADP, 2 methylthio ATP (2MeSATP), alpha,beta-methylene ATP (alpha,beta-meATP) and uridine 5'-triphosphate (UTP) and a role for ATP in sympathetic constriction were examined. 2. At basal tone nucleotides elicited dose-dependent vasoconstriction with an observed rank order of potency of alpha,beta-meATP >> 2MeSATP > ATP = ADP > UTP (based on the doses required to elicit constrictor responses of 25 mmHg). Adenosine had no vasoconstrictor action at doses up to 5 mumol. After application of a single dose (0.5 mumol) of alpha,beta-meATP preparations were desensitized to constriction by subsequent application of nucleotides. 3. Electrical field stimulation (4-64 Hz, 90 V, 1 ms, 30 s) elicited frequency-dependent constrictions which were abolished by guanethidine (5 microM) and by prazosin (1 microM). 4. The non-selective P2-purinoceptor antagonist suramin (100 microM) did not significantly affect vasoconstrictor responses to ATP. The P2X-selective purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 3 microM), virtually abolished responses to ATP. When the endothelium was removed vasoconstrictor responses to ATP and noradrenaline were augmented. 5. In preparations with tone raised with methoxamine (10-80 microM) nucleotides elicited vasodilatation with an observed potency order of ATP = UTP > ADP >> adenosine. 2MeSATP had relatively minor vasodilator effects and at the highest dose tested (50 nmol) elicited only vasoconstriction. alpha,beta-meATP did not elicit vasodilatation but produced further constriction of the raised tone preparation. At the highest doses of ATP and ADP (0.5 microM) responses were biphasic with vasoconstriction preceding vasodilatation. After removal of the endothelium, with the exception of adenosine, vasodilator responses to purines and to UTP were abolished; vasoconstriction to ATP, ADP, UTP and 2MeSATP was evident at the highest doses. 6. Suramin (100 microM) inhibited vasodilatation to both ATP and UTP and abolished responses to 2MeSATP. PPADS (3 microM) inhibited relaxation to 2MeSATP but did not affect relaxation to ATP, UTP, adenosine and acetylcholine and ADP. 7. Reactive blue 2 (30 microM) blocked vasodilator responses to ATP, UTP, 2MeSATP and acetylcholine; it was without effect when used at 3 microM. 8. The results of this study show that ATP elicits vasoconstriction of mesenteric arteries of the golden hamster via P2X-purinoceptors located on the smooth muscle, and vasodilatation via P2U-receptors which are located on the endothelium. 2MeSATP has marginal vasodilator activity, suggesting that P2Y-purinoceptors contribute minimally to relaxation to ATP in hamster mesenteric arteries.     

Cloned and transfected P2Y4 receptors: characterization of a suramin and PPADS-insensitive response to UTP.

The P2Y family of receptors are G protein-coupled receptors for ATP, ADP, UTP and UDP. Recently several members of this family have been cloned, including the P2Y4, which is activated by UTP but not by ATP. In the present report, using receptors stably transfected into 1321N1 cells, we show that suramin acts as an antagonist at cloned P2Y1 and (less potently) P2Y2 receptors, but not at the cloned P2Y4 receptor. Furthermore, PPADS (pyridoxal-phosphate-6-azophenyl-2'',4''-disulphonic acid), a potent antagonist at the P2Y1 receptor, is a relatively inneffective antagonist at the cloned P2Y4 receptor. This work moves us closer to the goal of classifying the native P2Y receptors on the basis of agonist and antagonist profiles.     

Discrimination by PPADS between endothelial P2Y- and P2U-purinoceptors in the rat isolated mesenteric arterial bed.

1. The main aim of this study was to characterize the antagonistic effects of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) at coexisting endothelial P2Y- and P2U-purinoceptors. Studies were conducted in Krebs-perfused mesenteric arterial preparations isolated from the rat, with tone raised by methoxamine (5-50 microM). 2. Purine and pyrimidine compounds elicited vasodilatation with a rank order of potency of 2-methylthio ATP (2-MeSATP) = ADP > ATP = UTP > P1, P3-diadenosine triphosphate (Ap3A) > P1, P2-diadenosine pyrophosphate (Ap2A) > NADP > adenosine. 8-para-Sulphophenyltheophylline (8-PSPT; 3 microM) had no effect on vasodilator responses to 2MeSATP, ADP, ATP, UTP, Ap3A or NADP, but blocked responses to adenosine and the maximal response to Ap2A. 3. PPADS (3-100 microM) attenuated vasodilator responses to the P2Y-selective agonists 2MeSATP and ADP, shifting the dose-response curves to the right. The pA2 values for PPADS at 2MeSATP and ADP were 5.97 +/- 0.69 and 5.98 +/- 0.86 respectively. In contrast, PPADS had no effect on vasodilator responses mediated by the P2U-selective agonist, UTP, or on vasodilator responses mediated by ATP. 4. PPADS (10 microM) was used to characterize responses mediated by the adenine dinucleotides; dose-response curves for vasodilator responses to Ap3A and NADP, but not those to Ap2A, were shifted to the right by PPADS. The estimated pA2 values for the effect of PPADS on Ap3A and NADP were 6.38 and 6.26 respectively. 5. Indomethacin (10 microM) had no effect on vasodilator responses to 2MeSATP, ADP, ATP or UTP. 6. In conclusion, these results show that PPADS is an antagonist at endothelial P2Y- but not P2U-purinoceptors in rat mesenteric arteries. These receptors cannot be discriminated by inhibition of prostaglandin synthesis; P2Y-purinoceptors are, however, sensitive to ADP. Selective antagonism by use of PPADS showed that ATP acts at P2U- and not P2Y-purinoceptors. Ap3A and NADP mediate vasodilatation via P2Y-purinoceptors, whereas vasodilatation to Ap2A is mediated partly via P1- and possibly via P2U-purinoceptors.     

Stimulation of P2 purinergic receptors induces the release of eosinophil cationic protein and interleukin-8 from human eosinophils

1. Extracellular nucleotides are the focus of increasing attention for their role as extracellular mediators since they are released into the extracellular environment in a regulated manner and/or as a consequence of cell damage. 2. Here, we show that human eosinophils stimulated with different nucleotides release eosinophil cationic protein (ECP) and the chemokine interleukin 8 (IL-8), and that release of these two proteins has a different nucleotide requirement. 3. Release of ECP was triggered in a dose-dependent manner by ATP, UTP and UDP, but not by 2'-&3'-o-(4-benzoyl-benzoyl)adenosine 5'-triphosphate (BzATP), ADP and alpha,beta-methylene adenosine 5' triphosphate (alpha,beta-meATP). Release of IL-8 was triggered by UDP, ATP, alpha,beta-meATP and BzATP, but not by UTP or ADP. Pretreatment with pertussis toxin abrogated nucleotide-stimulated ECP but not IL-8 release. 4. Release of IL-8 stimulated by BzATP was fully blocked by the P2X(7) blocker KN-62, while release triggered by ATP was only partially inhibited. IL-8 secretion due to UDP was fully insensitive to KN-62 inhibition. 5. Priming of eosinophils with GM-CSF increased IL-8 secretion irrespectively of the nucleotide used as a stimulant. 6. It is concluded that extracellular nucleotides trigger secretion of ECP by stimulating a receptor of the P2Y subfamily (possibly P2Y(2)), while, on the contrary, nucleotide-stimulated secretion of IL-8 can be due to activation of both P2Y (P2Y(6)) and P2X (P2X(1) and P2X(7)) receptors.     

P_2嘌呤受体的研究进展

P2 嘌呤受体最初被分为P2X和P2Y两种亚型 ,其后扩展到P2T、P2Z 和P2D亚型。 90年代发现 ,一些组织标本对UTP、ATP和ATPγS反应良好 ,而对α ,β MeATP和 2 MeSATP不敏感 ,此类受体被称为“P2U受体”。晚近又证明 ,存在一种对UTP敏感而对ATP不敏感的“嘧啶受体”。据此IUPHAR(Internationalunionofpharmacology)规定 ,任何被核苷酸激活的离子通道型和G蛋白偶联型受体的亚型均分别命名为P2Xn和P2Yn受体。随着分子生物学技术的发展以及上述受体的克隆和表达 ,此分类系统得到了有力的支持。     

Evidence for a discrete UTP receptor in cardiac endothelial cells.

1. We have examined the effects of various purine and pyrimidine nucleotides upon cells cultured from guinea-pig cardiac endothelium (CEC), and find the P2Y-agonist 2-methylthioadenosine triphosphate (2MeSATP) to be a potent (EC50 = 85 +/- 10.2 nM) stimulator of increase in intracellular calcium concentrations, while uridine 5'-triphosphate (UTP) and adenosine 5'-triphosphate (ATP) are less potent but equipotent with one another (EC50s = 2.1 +/- 0.3 and 1.8 +/- 0.2 microM, respectively). 2. While the P2Y receptor exhibited rapid homologous desensitization, this had no effect upon subsequent responsiveness of CEC to either ATP or UTP. Effects of maximal concentrations of ATP and UTP were not only additive, but did not cross-desensitize. Responses to UTP (but not to ATP or 2MeSATP) were blocked by treatment with pertussis toxin (PTX); all three nucleotides appeared to liberate calcium from an intracellular pool. 3. Suramin (30 microM) significantly (P < 0.05) increased the EC50 for ATP-dependent increases in intracellular calcium (5.3 +/- 2.2 microM vs. 2.0 +/- 0.9 microM in the absence of suramin), while it completely blocked the response to 2MeSATP. Suramin had no effect upon responses to UTP at concentrations of 100 microM. 4. We conclude that in addition to the P2Y and P2U subtypes of the ATP receptor, an additional receptor responsive to UTP but exhibiting no affinity for purine nucleotides is present in CEC; this "pyrimidine receptor' liberates intracellular calcium via a G-protein, and may partly mediate the contractile response to UTP in the coronary vasculature.     

Neuronal "nucleotide" receptor linked to phospholipase C and phospholipase D? Stimulation of PC12 cells by ATP analogues and UTP.

We have investigated the characteristics of the receptor for ATP on neuronal cells and the involvement of phospholipase C and phospholipase D in the effector mechanisms, using PC12 rat phaeochromocytoma cells in culture. We show that the cells respond, with generation of total inositol phosphates, to ATP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) but not to 2-methylthioadenosine5'-triphosphate (2MeSATP), beta,gamma-methylene ATP, or adenosine 5'-O-(2-thiodiphosphate) (ADP beta S). The largest response to ATP gamma S was mainly independent of extracellular calcium, had an EC50 of 7.93 +/- 0.76 microM, and was competitively inhibited by the nonspecific antagonist suramin. The pyrimidine nucleotide UTP also elicited a response in these cells. Measurement of [3H]inositol triphosphate showed a rapid rise to maximum (10-15 sec) in response to both ATP gamma S and UTP but no response to 2MeSATP. Cells prelabeled with 32Pi and stimulated in the presence of 50 mM butanol responded to ATP gamma S, ATP, and UTP with enhanced formation of [32P]phosphatidylbutanol as well as [32P]phosphatidic acid, indicating that agonist-stimulated phosphatidic acid occurs by both phospholipase D and phospholipase C activity. The stimulation of phospholipase D was inhibited by the presence of a protein kinase C inhibitor, Ro 31-8220. The dose-response curve for the stimulation by ATP gamma S of phospholipase C was shifted to the right by the presence of UTP, indicating that both compounds act on the same receptors. The data provide the first evidence for the existence of a nucleotide receptor on neuronal cells (insensitive to both purines and pyrimidines) and show that this receptor is linked to both phospholipase C and phospholipase D.     

Nucleotide-mediated relaxation in guinea-pig aorta: selective inhibition by MRS2179

The vasodilatory effects of nucleotides in the guinea-pig thoracic aorta were examined to determine the relationship between molecular expression and function of P2Y receptors. In aortic rings precontracted with norepinephrine, vasodilatory responses to purine nucleotides exhibited a rank-order of potency of 2-methylthio-ATP>ADP>ATP. Responses to UTP, but not UDP suggested a functional role for P2Y4 but not P2Y6 receptors. Aortic endothelial cells express at least four P2Y receptors; P2Y1, P2Y2, P2Y4 and P2Y6. In primary culture, these cells exhibit desensitizing transient calcium responses characteristic of P2Y1, P2Y2 and P2Y4, but not P2Y6 receptors. UDP had no effect on endothelial cell calcium. The pyrimidinergic receptor agonist UTP is capable of eliciting robust vasodilation in aortic rings and causing calcium responses in cultured guineapig aortic endothelial cells. These responses are equivalent to the maximum responses observed to ATP and ADP. Measurement of intracellular calcium release in response to ATP and 2-methylthio-ATP were similar, however only the 2-methylthio-ATP response was sensitive to the P2Y1 antagonist N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179). In aortic rings, vasodilatory responses to 2-methylthio-ATP, ATP and ADP were all blocked by pre-incubation of tissues with MRS2179. MRS2179 pretreatment had no effect of the ability of UTP to cause relaxation of norepinephrine responses in aortic rings or the ability of UTP to cause calcium release in aortic endothelial cells. We demonstrate robust effects of purine and pyrimidine nucleotides in guineapig aorta and provide functional and biochemical evidence that MRS2179 is a selective P2Y1 antagonist.     

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

1. 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. 2. ATP generated a response that was partial compared to UTP, as reported earlier. 3. 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. 4. 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. 5. ATP gamma S was an effective agonist with a similar EC50 to UTP, but with a lower maximum. ITP was a weak agonist compared with UTP. 6. 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. 7. 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.     

ADP-sensitive purinoceptors induce steroidogenesis via adenylyl cyclase activation in bovine adrenocortical fasciculata cells

1. The role of P2Y receptors in the production of cAMP and the activation of protein kinase A (PKA) was studied with respect to the regulation of the steroidogenesis in primary cultures of bovine adrenocortical fasciculata cells (BAFCs). 2. ADP and ATP stimulated cAMP production with EC(50) values of 23.7+/-6.8 microM and 40.1+/-5.5 microM, respectively. In contrast, the EC(50) of BzATP for cAMP production was 153.0+/-37.4 microM. Adenosine and AMP (0.1-1000 microM) were much less effective than ADP and ATP. 2MeSADP and UTP did not exert detectable effects. ADP (10 and 100 microM) significantly stimulated steroidogenesis; the process was blocked by an adenylyl cyclase inhibitor SQ22536 (100 microM) but not by the P2Y(1) receptor antagonist MRS2179 (100 microM). 3. Real-time imaging of the PKA activity with the dye ARII, which became less fluorescent upon phosphorylation, revealed that ADP (100 microM) immediately activated PKA. These effects could be mimicked by forskolin (100 microM) and were blocked by the PKA inhibitor H89 (50 microM). UTP (100 microM) did not activate PKA. 4. The cytoplasm harvested from morphologically and electrophysiologically identified single BAFCs contained mRNA for P2Y(2) but not for P2Y(1), P2Y(4), P2Y(11) or P2Y(12) receptors, as confirmed by single-cell RT-PCR amplification (50 cycles). 5. These results suggest an expression of an ADP-sensitive G(s)-coupled purinoceptor in BAFCs. We propose that this not yet described type of P2Y receptor might mediate the extracellular purine-activated steroidogenesis via cAMP/PKA-mediated pathways, independently from the pathways involving InsP(3) production and consequent intracellular Ca(2+) increase.