Human Ntera-2/D1 neuronal progenitor cells endogenously express a functional P2Y1 receptor

We report here that human Ntera-2/D1 (NT-2) cells, an undifferentiated committed neuronal progenitor cell line, endogenously express a functional P2Y(1) receptor, while other P2Y subtypes, except perhaps P2Y(4), are not functionally expressed. Quantitative RT-PCR analysis showed that NT-2 cells abundantly express mRNA for P2Y(1) and P2Y(11) receptors, while P2Y(2) and P2Y(4) receptors were detected at considerably lower levels. Western blot analysis also demonstrated expression of P2Y(1) receptors and Galpha(q/11) subunits. Various nucleotides induced intracellular Ca(2+) mobilisation in NT-2 cells in a concentration-dependent manner with a rank order potency of 2-MeSADP > 2-MeSATP > ADP > ATP > UTP > ATPgammaS, a profile resembling that of human P2Y(1) receptors. Furthermore, P2Y(1) receptor-specific (A3P5P) and P2Y-selective (PPADS, suramin) antagonists inhibited adenine nucleotide-induced Ca(2+) responses in a concentration-dependent manner, consistent with expression of a P2Y(1) receptor. Moreover, of seven adenine nucleotides tested, only Bz-ATP and ATPgammaS elicited small increases in cAMP formation suggesting that few, if any, functional P2Y(11) receptors were expressed. P2Y(1) receptor-selective adenine nucleotides, including 2-MeSADP and ADP, also induced concentration-dependent phosphorylation and hence, activation of the extracellular-signal regulated protein kinases (ERK1/2). NT-2 cells, therefore, provide a useful neuronal-like cellular model for studying the precise signalling pathways and physiological responses mediated by a native P2Y(1) receptor.     

P2 receptors in the murine gastrointestinal tract

The actions of adenosine, adenosine 5'-triphosphate (ATP), 2-methylthio adenosine diphosphate ADP (2-MeSADP), 2-methylthio ATP (2-MeSATP), alpha,beta-methylene ATP (alpha,beta-meATP) and uridine triphosphate (UTP) on isolated segments of mouse stomach (fundus), duodenum, ileum and colon were investigated. The localization of P2Y(1), P2Y(2), P2Y(4), P2X(1) and P2X(2) receptors and neuronal nitric oxide synthase (NOS) were examined immunohistochemically, and P2Y(1) mRNA was examined with in situ hybridization. The order of potency for relaxation of longitudinal muscle of all regions was: 2-MeSADP>/=2-MeSATP>alpha,beta-meATP>ATP=UTP=adenosine. This is suggestive of P2Y(1)-mediated relaxation and perhaps a further P2Y receptor subtype sensitive to alpha,beta-meATP. As ATP and UTP are equipotent, the presence of a P2Y(2) receptor is indicated. ATP responses were inhibited by the P2Y(1)-selective antagonist MRS 2179, and suramin. P2Y(1) receptors were visualized immunohistochemically in the smooth muscle of the ileum and in a subpopulation for myenteric neurones, which also stained for NOS. P2Y(1) mRNA was localized in neurones in both myenteric and submucosal ganglia in the ileum. Taken together, these results suggest that ATP was acting on non-adrenergic, non-cholinergic inhibitory neurons, which release both nitric oxide (NO) and ATP. Reduced relaxations to 2-MeSADP by tetrodotoxin and N(omega)-nitro-L-arginine methyl ester, are consistent with this possibility. Adenosine acts via P1 receptors to relax smooth muscle of the mouse gut. Segments of mouse colon (in contrast to the stomach and small intestine) were contracted by nucleotides with the potency order: 2-MeSATP>alpha,betameATP>ATP; the contractions showed no desensitization and were antagonized by suramin and PPADS, consistent with responses mediated by P2X(2) receptors. Immunoreactivity to P2X(2) receptors was demonstrated on both longitudinal and circular muscle of the colon, but not in the other regions of the gut, except for a small subpopulation of myenteric neurones. In summary, neuronal P2Y(1) receptors appear to mediate relaxation, largely through NO in all regions of the mouse gut, and to a lesser extent by P2Y(1), P2Y(2) and a novel P2Y receptor subtype responsive to alpha,beta-meATP in smooth muscle, while P2X(2) receptors mediate contraction of colonic smooth muscle.     

Regional variation in P2 receptor expression in the rat pulmonary arterial circulation

The P2 receptors that mediate contraction of the rat isolated small (SPA, 200-500 micro m i.d.) and large (LPA, 1-1.5 mM i.d.) intrapulmonary arteries were characterized. 2 In endothelium-denuded vessels the contractile order of potency was alpha,beta-methyleneATP (alpha,beta-meATP)>UDP=UTP=ATP=2-methylthioATP>ADP in the SPA and alpha,beta-meATP=UTP>or=UDP>2-methylthioATP, ATP>ADP in the LPA. alpha,beta-meATP, 2-methylthioATP and ATP had significantly greater effects in the SPA than the LPA (P<0.001), but there was no difference in the potency of UTP or UDP between the vessels. 3 In the SPA, P2X1 receptor desensitisation by alpha,beta-meATP (100 microM) inhibited contractions to alpha,beta-meATP (10 nM-300 microM), but not those to UTP or UDP (100 nM-300 microM). In the LPA, prolonged exposure to alpha,beta-meATP (100 microM) did not desensitize P2X receptors. 4 Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin and reactive blue 2 (RB2) (30-300 microM) inhibited contractions evoked by alpha,beta-meATP. UTP and UDP were potentiated by PPADS, unaffected by RB2 and inhibited, but not abolished by suramin. 1 and 3 mM suramin produced no further inhibition, indicating suramin-resistant components in the responses to UTP and UDP. 5 Thus, both P2X and P2Y receptors mediate contraction of rat large and small intrapulmonary arteries. P2Y agonist potency and sensitivity to antagonists were similar in small and large vessels, but P2X agonists were more potent in small arteries. This indicates differential expression of P2X, but not P2Y receptors along the pulmonary arterial tree.     

P2Y(1) and P2Y(2) receptors are coupled to the NO/cGMP pathway to vasodilate the rat arterial mesenteric bed

1. To assess the role of nucleotide receptors in endothelial-smooth muscle signalling, changes in perfusion pressure of the rat arterial mesenteric bed, the luminal output of nitric oxide (NO) and guanosine 3',5' cyclic monophosphate (cGMP) accumulation were measured after the perfusion of nucleotides. 2. The rank order of potency of ATP and analogues in causing relaxation of precontracted mesenteries was: 2-MeSADP=2-MeSATP>ADP>ATP=UDP=UTP>adenosine. The vasodilatation was coupled to a concentration-dependent rise in NO and cGMP production. MRS 2179 selectively blocked the 2-MeSATP-induced vasodilatation, the NO surge and the cGMP accumulation, but not the UTP or ATP vasorelaxation. 3. mRNA encoding for P2Y(1), P2Y(2) and P2Y(6) receptors, but not the P2Y(4) receptor, was detected in intact mesenteries by RT-PCR. After endothelium removal, only P2Y(6) mRNA was found. 4. Endothelium removal or blockade of NO synthase obliterated the nucleotides-induced dilatation, the NO rise and cGMP accumulation. Furthermore, 2-MeSATP, ATP, UTP and UDP contracted endothelium-denuded mesenteries, revealing additional muscular P2Y and P2X receptors. 5. Blockade of soluble guanylyl cyclase reduced the 2-MeSATP and UTP-induced vasodilatation and the accumulation of cGMP without interfering with NO production. 6. Blockade of phosphodiesterases with IBMX increased 15-20 fold the 2-MeSATP and UTP-induced rise in cGMP; sildenafil only doubled the cGMP accumulation. A linear correlation between the rise in NO and cGMP was found. 7. Endothelial P2Y(1) and P2Y(2) receptors coupled to the NO/cGMP cascade suggest that extracellular nucleotides are involved in endothelial-smooth muscle signalling. Additional muscular P2Y and P2X receptors highlight the physiology of nucleotides in vascular regulation.     

P2-purinoceptors mediating spasm of the isolated uterus of the non-pregnant guinea-pig.

1. The isolated uterus of the non-pregnant guinea-pig has been suggested to contain P1-, and possibly P2-purinoceptors mediating spasm. The presence of P1-purinoceptors has been confirmed and these receptors have been further characterized. 2. In the presence of the adenosine uptake inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 300 nM) and a pA100 concentration of the P1-purinoceptor antagonist 8-sulphophenyltheophylline (140 microM), the potency order of agonists as spasmogens was: 2 methylthio ATP >> alpha,beta methylene ATP = UTP = ATP >> beta,gamma methylene ATP. This order is not consistent with any single recognised P2-purinoceptor subtype. 3. Indomethacin (1 microM) treatment abolished responses to 2 methylthio ATP, alpha,beta methylene ATP and UTP, while spasm to ATP was significantly inhibited. When the endometrial and circular smooth muscle cell layers were removed, spasmogenic responses to ATP, 2 methylthio ATP, alpha,beta methylene ATP and UTP were significantly reduced. 4. 2-methylthio ATP was able to cause desensitization to itself, but not to UTP, indicating that these agonists act at different receptor sites. 5. The P2-purinoceptor antagonist, suramin antagonized 2 methylthio ATP with a PA2 of 5.9 +/- 0.3. Suramin was also an antagonist of ATP and UTP. In the case of ATP, the antagonism was not dependent on suramin concentration, while for UTP the interaction appeared to be non-equilibrium. Pyridoxalphosphate-6-azophenyl-2'',4''-disulphonic acid (PPADS, 10 microM) had no effect on spasm to ATP, UTP or 2 methythio ATP. 6. In the presence of indomethacin, responses to ATP were unaffected by 8-sulphophenyltheophylline (140 microM) or by suramin (100 microM), but PPADS (10 microM) antagonized ATP. 7. These results suggest that the isolated uterus of the non-pregnant guinea-pig contains a mixture of P2-purinoceptors. P2U- (or UTP-selective pyrimidinoceptors) and P2Y-purinoceptors appear to be present, probably mainly located on the endometrial or circular smooth muscle layer. Activation of these receptors leads to spasm via increases in prostanoid generation. There appears also to be a third class of non-P2X-, non p2Y-purinoceptor present, at which ATP is an agonist and PPADS is an antagonist, located on the longitudinal smooth muscle, activation of which causes spasm independent of changes in prostanoids.     

The effects of purine compounds on the isolated aorta of the frog Rana temporaria.

1. In the isolated aorta of the frog, Rana temporaria, adenosine concentration-dependently, endothelium-independently relaxed adrenaline pre-constricted vessels. None of the adenosine analogues including D-5''-(N-ethylcarboxamide) adenosine (NECA), R- and S-N6-(2-phenylisopropyl) adenosine (R-and S-PIA) and 2-chloroadenosine (2-CA), or the more selective A1, A2 and A3 agonists cyclopentyladenosine (CPA), CGS 21680 and N6-(3-iodobenzyl) adenosine-5''-N-methylcarboxamide (IB-MECA) respectively, had any effect. 2. The non-selective adenosine antagonist, 8-p-sulphophenyl-theophylline (8-pSPT; 30 microM) failed to inhibit adenosine relaxations, as did NG-nitro-L-arginine methyl ester (L-NAME; 0.1 mM) and indomethacin (30 microM). 3. Adenosine 5''-triphosphate (ATP), alpha, beta-methylene ATP (alpha, beta-MeATP), beta, gamma-methylene ATP (beta, gamma-MeATP), 2-methylthio ATP (2-MeSATP) and uridine 5''-triphosphate (UTP) all concentration-dependently contracted the frog aorta. ATP and alpha, beta-MeATP were equipotent and more potent than UTP and beta, gamma-MeATP; 2-MeSATP had little activity. 4. The P2-purinoceptor antagonist, suramin (0.1 mM) inhibited contractions to alpha, beta-MeATP but not to ATP. Pyridoxalphosphate-6-azophenyl-2'',4''-disulphonic acid (PPADS; 30 microM) also inhibited contractions to alpha, beta-MeATP but not to ATP. Contractions to ATP were, however, inhibited by indomethacin (30 microM). 5. In conclusion, in the frog aorta there appears to be a novel subclass of P1-purinoceptor mediating vasodilatation, although like the A3 subclass it is not blocked by methylxanthines; a P2-purinoceptor mediates vasconstriction which resembles a P2x subtype, based on the agonist potency of alpha, beta-MeATP being more potent than 2-MeSATP (UTP has moderate activity) and PPADS is an effective antagonist. There is no evidence for the presence of a P2y-purinoceptor, mediating vasodilatation, in this preparation.     

The pharmacology of nucleotide receptors on primary rat brain endothelial cells grown on a biological extracellular matrix: effects on intracellular calcium concentration

1. Brain capillary endothelial cells express a variety of nucleotide receptors, but differences have been reported between culture models. This study reports examination of nucleotide receptors on primary cultured rat brain capillary endothelial cells (RBCEC) grown on a biological extracellular matrix (ECM) to produce a more differentiated phenotype. 2. Fura-2 fluorescence ratio imaging was used to monitor intracellular free calcium concentration [Ca(2+)](i). ATP, UTP, and 2-methylthioATP (2-MeSATP) increased [Ca(2+)](i) to similar levels, while 2-MeSADP, ADP and adenosine gave smaller responses. 3. Removal of extracellular calcium caused no significant change in the [Ca(2+)](i) response to 2-MeSATP, evidence that the response was mediated by a metabotropic (P2Y) receptor. 4. All cells tested responded to ATP, UTP, 2-MeSATP and ADP, while 63% responded to adenosine and 50% to 2-MeSADP. No cells responded to alpha, beta-methyleneATP. Cells grown on rat tail collagen instead of ECM gave smaller and less uniform [Ca(2+)](i) responses, suggesting that the differentiating effect of the ECM contributed to a more uniform receptor profile. 5. The [Ca(2+)](i) response to the P2Y(1)-selective agonist 2-MeSADP was abolished in the presence of the subtype-selective antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS). 6. The P2Y(2) antagonist suramin completely blocked the response to ATP and inhibited the response to UTP by 66%. 7. The A(1) subtype-selective adenosine receptor agonist N(6)-Cyclopentyladenosine (CPA) gave a small but characteristic [Ca(2+)](i) response, while A(2A) and A(2B) subtype-selective agonists failed to generate [Ca(2+)](i) changes. 8. The results are consistent with the presence on RBCEC of a P2Y(2)-like receptor coupled to phospholipase C, and a P2Y(1)-like receptor mobilizing intracellular Ca(2+). The role of multiple nucleotide receptors in the function of the brain endothelium is discussed.     

Evidence that 2-methylthioATP and 2-methylthioADP are both agonists at the rat hepatocyte P2Y(1) receptor

In the absence of selective antagonists, pharmacological characterization of P2Y receptor subtypes has relied heavily upon their distinct agonist profiles. 2-methylthioADP (2-MeSADP) is a selective agonist for the P2Y(1) receptor. The agonist action of 2-MeSATP at the P2Y(1) receptor has recently been questioned. The effects of both 2-MeSADP and 2-MeSATP have been studied on rat hepatocytes injected with the bioluminescent Ca(2+) indicator, aequorin. Single hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca(2+)](i)) when stimulated with agonists acting through the phosphoinositide signalling pathway. The transients induced by 2-MeSADP and 2-MeSATP in the same cell were indistinguishable, indicating that they act at a common receptor. In contrast the transients evoked by ATP and UTP had very different profiles. Treatment of 2-MeSATP with an ATP-regenerating system to remove contaminating 2-MeSADP did not abolish its agonist activity. Application of the P2Y(1) antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P) inhibited the transients induced by both 2-MeSADP and 2-MeSATP. In contrast the transients induced by ATP and UTP were enhanced by the addition of A3P5P. These results indicate that both 2-MeSADP and 2-MeSATP are agonists at the rat hepatocyte P2Y(1) receptor.     

Responses of the longitudinal muscle and the muscularis mucosae of the rat duodenum to adenine and uracil nucleotides.

1. Previous studies have shown that the rat duodenum contains P1 and P2Y purinoceptors via which it relaxes to adenosine and adenosine 5'-triphosphate (ATP) respectively. It has also been shown to contract to uridine 5'-triphosphate (UTP) and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), and based on their differential inhibition by the P2 antagonist suramin it has been suggested that they act via two separate receptors. In addition, the rat duodenum has been shown to dephosphorylate ATP rapidly via ectonucleotidases and adenosine deaminase. In this study the responses of two preparations from the rat duodenum, the longitudinal muscle and the muscularis mucosae, were investigated using a series of nucleotides and suramin. 2. 2-Methylthioadenosine 5'-triphosphate (2-MeSATP), ATP, ATP-gamma-S and adenosine 5'-alpha,beta-methylene-triphosphonate (AMPCPP) each relaxed the longitudinal muscle, with an agonist potency order of 2-MeSATP > ATP = ATP-gamma-S > AMPCPP, while UTP and uridine 5'-diphosphate (UDP) were not observed to elicit relaxation. This indicates the presence of a relaxant P2Y-purinoceptor on the longitudinal muscle. The longitudinal muscle did not contract to any of the agonists at concentrations of 300 microM, apart from ATP-gamma-S which caused very weak contractions. 3. ATP-gamma-S, adenosine 5'-methylenediphosphonate (AMPCP), AMPCPP, ATP, UTP, adenosine 5'-diphosphate (ADP), UDP and 2-MeSATP each contracted the muscularis mucosae with an agonist potency order of ATP-gamma-S > or = AMPCP > or = AMPCPP = ATP = UTP = ADP = UDP >> 2-MeSATP, although maximal responses were not obtained at concentrations of 300 microM. The muscularis mucosae did not relax to any of the agonists at concentrations of 300 microM. 4. Suramin (1 mM) inhibited relaxations induced by ATP on the longitudinal muscle, shifting the relaxation concentration-response curve to the right. This further supports the presence of a P2Y-purinoceptor on this muscle layer. Suramin (1 mM) inhibited contractions induced by AMPCPP, but not those induced by ATP, UTP or ATP-gamma-S, in the muscularis mucosae. Desensitization of the muscularis mucosae was seen with AMPCPP, but not with UTP or ATP-gamma-S, and no cross-desensitization between AMPCPP and UTP or ATP-gamma-S was observed. This suggests there are two receptors which mediate contraction on the rat duodenum muscularis mucosae, one suramin-sensitive and the other suramin-insensitive. 5. ATP was rapidly degraded by the muscularis mucosae to ADP, adenosine 5'-monophosphate (AMP) and inosine, with no adenosine being detected. A similar rate of degradation was seen for UTP with UDP, uridine 5'-monophosphate (UMP) and uridine being formed and for 2-MeSATP with 2-methylthioadenosine 5'-diphosphate (2-MeSADP), 2-methylthioadenosine 5'-monophosphate (2-MeSAMP) and 2-methylthioadenosine being formed. AMPCPP and ATP-gamma-S were both degraded more slowly, AMPCPP being degraded to AMPCP, and ATP-gamma-S to ADP, AMP and inosine. Suramin (1 mM), did not significantly affect the rate and pattern of degradation of these nucleotides, apart from AMPCPP which was degraded slightly more slowly in the presence of suramin. 6. These results show that there is a P2Y-purinoceptor which mediates relaxation in the rat duodenum longitudinal muscle. They also show that there is a contraction-mediating suramin-sensitive receptor on the rat duodenum muscularis mucosae which is desensitized by AMPCPP, and thus is probably of the P2X subtype. In addition, there is a contraction-mediating suramin-insensitive receptor on the rat duodenum muscularis mucosae which is not desensitized by UTP or ATP-gamma-S, and at which ATP and UTP show equal potency, and is thus probably of the P2U subtype. In addition, the rat duodenum muscularis mucosae contains ectonucleotidases and adenosine deaminase, which rapidly degrade nucleotides, although the inhibition by suramin of this deg     

The P2Y purinoceptor in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase.

1. B10 cells, a clonal line of rat brain capillary endothelial cells, exhibit a single P2 purinoceptor, activation of which leads to increases in free intracellular calcium. In the current study the identity of this P2Y receptor was determined by its binding parameters for a range of purinoceptor ligands and by its complementary DNA (cDNA) sequence. The signal transduction mechanism activated by this receptor was also investigated. 2. The radioligand [35S]-dATP alpha S bound with high affinity (Kd = 9.8 nM) to the P2Y purinoceptor expressed on B10 cells, which was found to be extremely abundant (Bmax = 22.5 pmol mg-1 protein). The calculated Ki values of a range of P2 purinoceptor agonists which competitively displaced binding of [35S]-dATP alpha S led to the rank order of affinity: dATP alpha S (Ki 3.4 nM) > 2-chloroATP (2-ClATP) (13 nM), ATP (22 nM) > ATP gamma S (43 nM) > 2-methylthioATP (2-MeSATP) (88 nM) > ADP (368 nM) > > UTP, L-beta,gamma-methyleneATP (both > 10,000 nM). The P2 purinoceptor antagonists, Reactive blue 2 and suramin, were also able to displace binding, with Ki values of 833 and 1358 nM respectively. In contrast pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) was able to displace only 20% of [35S]-dATP alpha S binding at a concentration of 100 microM. 3. 2-ClATP (EC50 = 0.22 microM), 2-MeSATP (0.54 microM), ADP (7.9 microM) and ATP (a partial agonist), but not UTP, inhibited the cyclic AMP formation stimulated by cholera toxin, in a manner that was prevented by pertussis toxin. The purinoceptor antagonist, PPADS, was found to be inactive at a concentration of 100 microM. 4. A P2Y receptor cDNA was derived from mRNA from B10 cells and from C6-2B, a rat glioma cell line known to possess a P2Y receptor that is coupled to the inhibition of adenylate cyclase. Sequence analysis of the entire coding region revealed that both were 100% identical to the rat P2Y1 purinoceptor cDNA. No other P2Y-type receptor mRNA could be detected in B10 cells. Exactly the same sequence was isolated from rat brain cortical astrocytes, where 2-MeSATP has been shown to increase phospholipase C activity. 5. Since the receptor responsible for the transduction shares with the aforementioned binding site significant pharmacological features, including a strong activity of 2-MeSATP (characteristic of P2Y1 receptors alone among all known P2Y purinoceptors) and an unusual insensitivity to PPADS, and since abundant mRNA is present of the P2Y1 receptor but not of any other type resembling the known P2Y receptors, it is concluded that a P2Y1 receptor on rat brain microvascular endothelial cells can account for all of the observations. This single P2Y1 receptor, therefore, appears to couple in different native cell types to either adenylate cyclase inhibition or to phospholipase C activation.     

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.     

Extended pharmacological profiles of rat P2Y2 and rat P2Y4 receptors and their sensitivity to extracellular H+ and Zn2+ ions

1. Two molecularly distinct rat P2Y receptors activated equally by adenosine-5'-triphosphate (ATP) and uridine-5'-triphosphate (UTP) (rP2Y2 and rP2Y4 receptors) were expressed in Xenopus oocytes and studied extensively to find ways to pharmacologically distinguish one from the other. 2. Both P2Y subtypes were activated fully by a number of nucleotides. Tested nucleotides were equipotent at rP2Y4 (ATP=UTP=CTP=GTP=ITP), but not at rP2Y2 (ATP=UTP>CTP>GTP>ITP). For dinucleotides (ApnA, n=2-6), rP2Y4 was only fully activated by Ap4A, which was as potent as ATP. All tested dinucleotides, except for Ap2A, fully activated rP2Y2, but none were as potent as ATP. ATP gamma S and BzATP fully activated rP2Y2, whereas ATP gamma S was a weak agonist and BzATP was inactive (as an agonist) at rP2Y4 receptors. 3. Each P2Y subtype showed different sensitivities to known P2 receptor antagonists. For rP2Y2, the potency order was suramin>PPADS= RB-2>TNP-ATP and suramin was a competitive antagonist (pA2, 5.40). For rP2Y4, the order was RB-2>suramin>PPADS> TNP-ATP and RB-2 was a competitive antagonist (pA2, 6.43). Also, BzATP was an antagonist at rP2Y4 receptors. 4. Extracellular acidification (from pH 8.0 to pH 5.5) enhanced the potency of ATP and UTP by 8-10-fold at rP2Y4 but did not affect agonist responses at rP2Y2 receptors. 5. Extracellular Zn2+ ions (0.1-300 microM) coapplied with ATP inhibited agonist responses at rP2Y4 but not at rP2Y2 receptors. 6. These two P2Y receptors differ significantly in terms of agonist and antagonist profiles, and the modulatory activities of extracellular H+ and Zn2+ ions. These pharmacological differences will help to distinguish between rP2Y2 and rP2Y4 receptors, in vivo.     

Subclasses of purinoceptors in feline bladder.

Purines have been shown to inhibit and excite feline detrusor smooth muscle through P1 and P2 receptor activation. Several recent studies have demonstrated differences in agonist potency orders for subclasses of purinoceptors, including P2Y and nucleotide, or P2U receptors. The current studies were performed to determine the presence of such receptor subtypes in feline detrusor smooth muscle. Cats were surgically prepared for monitoring detrusor smooth muscle contractions as increases in intravesical pressure. Contractions were induced by pelvic nerves stimulation (PNS), ATP, and ATP analogs, such as beta, gamma-methylene ATP (APPCP), 5' adenylimido diphosphate (AMP-PNP) and 2-methylthio ATP (2-MeSATP), ATP gamma S, UTP, CTP and GTP. These agents all produced contractions and had an agonist potency order of AMP-PNP = APPCP > ATP gamma S = 2-MeSATP > ATP > UTP = CTP = GTP. The agonist potency order for inhibition of PNS nerve-evoked bladder contractions was APPCP = AMP-PNP = ATP gamma S > 2-MeSATP = ATP > UTP = CTP = GTP. Reactive Blue 2 and Coomassie's Brilliant Blue G, two putative P2Y receptor antagonists, antagonized purine-induced actions. This antagonism and the agonist potency orders suggest the possible presence of novel receptors in detrusor smooth muscle and/or the presence of multiple receptors in detrusor smooth muscle.     

Inhibition of ecto-ATPase by PPADS, suramin and reactive blue in endothelial cells, C6 glioma cells and RAW 264.7 macrophages.

1. Previous studies have shown that bovine pulmonary artery endothelium (CPAE) has P2Y and P2U purinoceptors, rat C6 glioma cells have P2U purinoceptors and mouse RAW 264.7 cells have pyrimidinoceptors, all of which are coupled to phosphoinositide-specific phospholipase C (PI-PLC). The dual actions of PPADS, suramin and reactive blue as antagonists of receptor subtypes and ecto-ATPase inhibitors were studied in these three cell types. 2. In CPAE, suramin, at 3-100 microM, competitively inhibited the PI responses induced by 2MeSATP and UTP, with pA2 values of 5.5 +/- 0.3 and 4.4 +/- 0.4, respectively. Reactive blue, at 1-3 microM, produced shifts to the right of the 2MeSATP and UTP curves, but no further right shift at 10 microM. PPADS, at 10 microM, caused a 3 fold right shift of the 2MeSATP curve, but no further shift at concentrations up to 100 microM. In contrast, a dose-dependent shift to the left of the UTP curve and a weak inhibition of the ATP response were seen with PPADS. 3. In RAW 264.7 cells, suramin and reactive blue, but not PPADS, competitively inhibited the UTP response, with pA2 values of 4.8 +/- 0.5 and 5.8 +/- 0.7, respectively. 4. In C6 glioma cells, although suramin and reactive blue inhibited the ATP response, a potentiation effect on ATP and UTP responses was seen with PPADS. 5. The ecto-ATPase inhibitory activity of these three receptor antagonists were determined. All three inhibited ecto-ATPase present in CPAE, C6 and RAW 264.7 cells, with IC50 values of 4, 4.8 and 4.7 for PPADS, 4, 4.4 and > > 4 for suramin, and 4.5, 4.7 and 4.7 for reactive blue. 6. This study indicates that PPADS, suramin and reactive blue ar ecto-ATPase inhibitors. This property, combined with their antagonistic selectivity for receptor subtypes, can result in inhibition of, potentiation of, or lack of effect on agonist-mediated PI responses. Reactive blue is a more potent antagonist than suramin on P2Y, P2U and pyrimidinoceptors, and PPADS is a weak antagonist for P2Y receptors.     

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.     

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).     

Evidence for two distinct P2-purinoceptors subserving contraction of the rat anococcygeus smooth muscle.

1. The effects of the P2-purinoceptor agonists, adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP (alpha, beta-MeATP), beta, gamma-methylene ATP (beta, gamma-MeATP), L-beta, gamma-methylene ATP (L-beta, gamma-MeATP), adenosine-5'-O-(2-thiodiphosphate) (ADP beta S), and 2-methylthio ATP (2-MeSATP) were investigated on the isometric tension of the rat anococcygeus muscle. 2. Non-cumulative additions of ATP (100-1500 microM), alpha, beta-MeATP (1-300 microM), beta, gamma-MeATP (10-300 microM), L-beta, gamma-MeATP (3-100 microM) and ADP beta S (1-100 microM) produced concentration-dependent contractions, whereas 2-MeSATP (1-100 microM) had no effect. The rank order of potency was alpha, beta-MeATP > L-beta, gamma-MeATP > or = ADP beta S > beta, gamma-MeATP > > ATP > 2-MeSATP. 3. Contractions to cumulative additions of ATP, alpha, beta-MeATP, beta, gamma-MeATP and L-beta, gamma-MeATP were subject to desensitization whilst those to ADP beta S were unaffected. 4. Contractions to ATP, alpha, beta-MeATP, beta, gamma-MeATP and ADP beta S were abolished by the non-selective P2X/. P2Y-purinoceptor antagonist, suramin (100 microM). In contrast, contractions to ATP, alpha, beta-MeATP and beta, gamma-MeATP were not affected by the non-selective P1-purinoceptor antagonist 8-(p-sulphophenyl)-theophylline (8SPT, 30 microM). Blockade of P2X-purinoceptors with the selective P2X-purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM) or desensitization with L-beta, gamma-MeATP (10 microM) abolished contractions to alpha, beta-MeATP, but enhanced those to ADP beta S. The P2Y-purinoceptor antagonist, reactive blue 2 (RB2, 100 microM) enhanced contractions to ATP and alpha, beta-MeATP but abolished those to ADP beta S. 5. Simultaneous addition of alpha, beta-MeATP and ADP beta S produced an additive contraction. 6. The findings suggest that in the rat anococcygeus, smooth muscle cells are endowed with two distinct P2-purinoceptors which subserve contractions: a P2X-purinoceptor activated by ATP and its analogues, and another type of P2-purinoceptor activated by ADP beta S.     

An electrophysiological study of excitatory purinergic neuromuscular transmission in longitudinal smooth muscle of chicken anterior mesenteric artery

1. The object of the present study was to clarify the neurotransmitters controlling membrane responses to electrical field stimulation (EFS) in the longitudinal smooth muscle cells of the chicken anterior mesenteric artery. 2. EFS (5 pulses at 20 Hz) evoked a depolarization of amplitude 19.7+/-2.1 mV, total duration 29.6+/-3.1 s and latency 413.0+/-67.8 ms. This depolarization was tetrodotoxin (TTX)-sensitive and its amplitude was partially decreased by atropine (0.5 microM); however, its duration was shortened by further addition of prazosin (10 microM). 3. Atropine/prazosin-resistant component was blocked by the nonspecific purinergic antagonist, suramin, in a dose-dependent manner, indicating that this component is mediated by the neurotransmitter adenosine 5'-triphosphate (ATP). 4. Neither desensitization nor blocking of P2X receptor with its putative receptor agonist alpha,beta-methylene ATP (alpha,beta-MeATP, 1 microM) and its antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic (PPADS, up to 50 microM), had significant effect on the purinergic depolarization. In contrast, either desensitization or blocking of P2Y receptor with its putative agonist 2-methylthioATP (2-MeSATP, 1 microM) and its antagonist Cibacron blue F3GA (CBF3GA, 10 microM) abolished the purinergic depolarization, indicating that this response is mediated through P2Y but not P2X receptor. 5. The purinergic depolarization was inhibited by pertussis toxin (PTX, 600 ng ml(-1)). Furthermore, it was significantly inhibited by a phospholipase C (PLC) inhibitor, U-73122 (10 microM), indicating that the receptors involved in mediating the purinergic depolarization are linked to a PTX-sensitive G-protein, which is involved in a PLC-mediated signaling pathway. 6. Data of the present study suggest that the EFS-induced excitatory membrane response occurring in the longitudinal smooth muscle of the chicken anterior mesenteric artery is mainly purinergic in nature and is mediated via P2Y purinoceptors.     

Evidence for the expression of multiple uracil nucleotide-stimulated P2 receptors coupled to smooth muscle contraction in porcine isolated arteries

BACKGROUND AND PURPOSE: The uracil nucleotides UDP and UTP have been reported to activate P2Y2, P2Y4 and P2Y6 receptors to cause vasoconstriction. We have performed a comparative analysis of these receptors in endothelium-denuded smooth muscle from porcine isolated coronary and ear arteries, using pharmacological and molecular tools. EXPERIMENTAL APPROACH: Tissue segments were used to construct non-cumulative concentration response curves for UTP and UDP, in the absence and presence of the P2 receptor antagonists PPADS or suramin. RT-PCR and immunoblot analyses were employed to define gene expression and immunoreactivity for P2Y2, P2Y4 and P2Y6 receptors. KEY RESULTS: In the coronary artery, UTP-evoked contractile responses were reduced in the presence of suramin, but not PPADS, while the smaller responses to UDP were unaffected by either antagonist. In the ear artery, contractile responses to UDP were much smaller than those to UTP; responses to UTP were inhibited by both PPADS and suramin. RT-PCR suggested predominant expression of P2Y2 receptors in the coronary artery, while P2Y4 and P2Y6 receptor gene expression appeared equivalent in both tissues. Immunoblot analyses provided evidence for P2Y6 receptors in both tissues, with equivocal evidence of P2Y2 and P2Y4 receptor immunoreactivities. CONCLUSIONS AND IMPLICATIONS: We conclude that UTP-evoked contraction of porcine coronary artery smooth muscle appears to be predominantly P2Y2-mediated, while the ear artery appears to express a uracil nucleotide-sensitive P2 receptor(s) which fails to fit readily into the current classification.