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.     

Evidence for a P2-purinoceptor mediating vasoconstriction by UTP,ATP and related nucleotides in the isolated pulmonary vascular bed of the rat.

1. The vasoconstrictor effects of uridine 5''-triphosphate (UTP), uridine 5''-diphosphate (UDP), uridine 5''-monophosphate (UMP) and uridine were tested in the isolated pulmonary vascular bed of the rat. Comparison was made with the effects of adenine nucleotides, adenosine 5''-triphosphate (ATP), adenosine 5''-diphosphate (ADP), adenosine 5''-monophosphate (AMP) and with adenosine. The effect of P2x-purinoceptor desensitization and blockade was compared on the vascular responses to uracil and adenine nucleotides. 2. At doses ranging from 10(-8) to 10(-5) mol, UTP elicited dose-dependent vasoconstriction. UDP was equiactive to UTP, while UMP and uridine did not show vasomotor activity. Similarly, ATP showed dose-related vasoconstrictor activity. ADP was less potent than ATP in eliciting vasoconstriction, while AMP was active only at the higher doses tested and adenosine was ineffective. 3. Vasoconstriction was produced by ATP analogues with the following order of potency: alpha, beta-methylene ATP > ATP gamma S > beta, gamma-methylene ATP > 2-methylthio ATP > or = ATP. 4. Desensitization of P2x-purinoceptors by the selective agonist alpha, beta-methylene ATP did not modify the vasoconstrictor activity of UTP and UDP and only partially reduced vasoconstrictor responses to ATP, while it abolished vascular responses to alpha, beta-methylene ATP itself. 5. The antagonists of P2-purinoceptors, suramin and pyridoxalphosphate-6-azophenyl-2'', 4''-disulphonic acid (PPADS), did not affect vascular responses to UTP and UDP, but reduced vasoconstriction evoked by beta, gamma-methylene ATP and ATP by about 70 and 30%, respectively. 6. This study demonstrates that uracil nucleotides, UTP and UDP, elicit vasoconstriction in the rat pulmonary vascular bed. In addition to confirming the presence of classical P2x-purinoceptors, these results also suggest the presence of a distinct purinoceptor subtype which mediates UTP- and ATP- evoked vasoconstriction in the rat pulmonary circulation.     

Characterization of P2 receptors for purine and pyrimidine nucleotides in human placental cotyledons

1. The aim of this study was to characterize P2 receptors in the arterial vascular bed of human perfused placental cotyledons. Vasoconstrictor responses to bolus injections of purine and pyrimidine nucleotides were tested at basal tone, and vasodilator responses in preparations with tone raised by perfusion with prostaglandin F_2α (PGF_2α; 10–50 nM).
2. At basal tone, bolus injections of the P2X-selective agonist α,β-methylene ATP (α,β-meATP; 0.5–500 nmol) elicited dose-dependent vasoconstriction. ATP (0.005–5 μmol) also elicited dose-dependent vasoconstriction, but was less potent than α,β-meATP. Vasoconstriction was also elicited by other nucleotides, but only at the highest dose tested (5 μmol): UTP > CTP=ITP (n=6). GTP and TTP did not cause vasoconstriction.
3. Constrictor responses to bolus injections of α,β-meATP were resistant to desensitization and were not significantly affected when carried out in the presence of 1 μM α,β-meATP added to the perfusate. However, responses to bolus injections of α,β-meATP were partially blocked by perfusion with 10 μM α,β-meATP. In contrast, responses to ATP and UTP were unaffected by 10 μM α,β-meATP. The P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 10 and 100 μM) had no significant effect on vasoconstriction mediated by α,β-meATP and ATP.
4. Removal of the endothelium had no significant effect on constrictor responses to α,β-meATP, ATP and UTP. Inhibition of nitric oxide (NO) synthesis with N^G-nitro-L-arginine methyl ester (L-NAME; 100 μM) had no significant effect on vasoconstriction to ATP and α,β-meATP.
5. In preparations with tone raised with PGF_2α (10–50 nM) vasodilatation was elicited by nucleotides with the following order of potency: 2MeSATP=ADP >> ATP > UTP > CTP=GTP=ITP=TTP. pD₂ values were: 2MeSATP, 10.03±0.26 (n=7); ADP, 9.97±0.40 (n=5); ATP, 8.89±0.18 (n=7); UTP, 7.79±0.35 (n=7). Maximal responses to 2MeSATP and ADP were similar and were approximately 40% greater than maximal responses to ATP and UTP.
6. Vasodilator responses to nucleotides were abolished by L-NAME (100 μM) and by removal of the endothelium.
7. In conclusion, contractile responses mediated by α,β-meATP and ATP in human placental smooth muscle are resistant to desensitization and insensitive to PPADS and, thus, show a dissimilar pharmacological profile to the classic smooth muscle P2X₁ receptor. There may be two subtypes of smooth muscle P2 receptor based on differential antagonism of α,β-meATP and ATP with α,β-meATP. A smooth muscle P2 receptor mediates vasoconstriction to UTP, and may indicate a further subtype. Endothelium-dependent, NO-dependent, vasodilatation to 2MeSATP and ADP may be mediated by P2Y₁ receptors, while endothelial P2Y₂ receptors are likely to mediate NO-dependent relaxation to ATP and UTP.

     

A contraction-mediating receptor for UTP, presumably P2Y2, in rat vas deferens

The possible existence of a contraction-mediating P2-receptor for uracil nucleotides was investigated in the rat vas deferens. In order to minimize breakdown of nucleotides, Evans blue was used as an inhibitor of ectonucleotidases. UTP was degraded by rat vas deferens tissue, and the degradation was inhibited by Evans blue (100 microM). In the absence of other drugs, UTP and UDP elicited marginal contractions. Evans blue (100 microM) greatly enhanced contractions elicited by the uracil nucleotides. When the medium contained alpha,beta-MeATP (100 microM) in addition to Evans blue in order to desensitize contraction-mediating P2X1-receptors, responses to UTP and UDP were not changed; in contrast, responses to alpha,beta-MeATP were virtually abolished and contractions elicited by ATP and ADP were greatly reduced; EC50 values were 122 microM for UTP and 58 microM for ATP under these conditions. The P2-receptor antagonist suramin attenuated contractions elicited by UTP (320 microM) and alpha,beta-MeATP (32 microM) in the presence of Evans blue; pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS) also reduced responses to alpha,beta-MeATP but, at up to 100 microM, did not alter contractions elicited by UTP. Incubation of vasa deferentia in nominally calcium-free medium almost abolished the response to alpha,beta-MeATP (32 microM), while a major part of the contraction elicited by UTP (320 microM) was preserved. In the presence of Evans blue and alpha,beta-MeATP, prior addition of UDP (3200 microM) or ATP (320 microM), without washout, markedly reduced the response to UTP (320 microM); UTP and ATP also reduced the response to UDP; in contrast, prior addition of UTP or UDP did not alter the contraction to ATP. The results demonstrate the existence of a contraction-mediating, uracil nucleotide-sensitive P2Y-receptor in rat vas deferens, distinct from the P2X1-receptor. Pharmacological analysis indicates that it is P2Y2.     

Vasoconstrictor and vasodilator responses to various agonists in the rat perfused mesenteric arterial bed: selective inhibition by PPADS of contractions mediated via P2x-purinoceptors.

1. The effect of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on vasoconstrictor and/or vasodilator responses to various agonists and electrical field stimulation was investigated in the rat mesenteric arterial bed at basal tone and at tone raised by methoxamine (15-50 microM). 2. At basal tone, nucleotides produced vasoconstriction with the following rank order of potency: alpha,beta-methylene ATP >> 2-methylthio ATP > or = ATP = UTP. PPADS (0.3-10 microM) concentration-dependently antagonized alpha, beta-methylene ATP-, 2-methylthio ATP- and ATP-induced responses. UTP-, noradrenaline- and nerve-mediated (4-32 Hz) increases in perfusion pressure remained unaffected by 10 microM PPADS. 3. In raised tone preparations, nucleotides produced vasodilations, their rank order of potency being 2-methylthio ATP > ATP > UTP. Responses to 2-methylthio ATP were slightly antagonized, whereas ATP- and UTP-induced responses remained unaffected by 10 microM PPADS. In addition, acetylcholine- and adenosine-elicited relaxations were not influenced by 10 microM PPADS. 4. The present results confirm the previously described selective P2x antagonism by PPADS, this compound being ineffective at muscarinic M3- and adenosine P1-receptors as well as at alpha 1-adrenoceptors. There was some inhibition of P2y-purinoceptors but at a much higher concentration than required for inhibition of P2x-purinoceptors. 5. In addition, this study provides evidence for the ineffectiveness of PPADS at both vasoconstriction- and vasodilatation-mediating P2u-purinoceptors.     

Noradrenaline and extracellular nucleotide cotransmission involves activation of vasoconstrictive P2X(1,3)- and P2Y6-like receptors in mouse perfused kidney

Nucleotides like ATP and UTP act as potent extracellular signalling molecules. Released from sympathetic nerve endings as cotransmitters of noradrenaline or paracrine from nonexcitatory cells, they activate specific receptors (ion-gated P2X(1-7) and G-protein-coupled P2Y(1,2,4,6,11-15)). Which of these subtypes, however, are able to modulate vasoconstriction in the kidney is unclear. Wild-type- and P2Y4-receptor-deficient mice kidneys were isolated and perfused with Krebs-Henseleit solution. Pressor responses to renal nerve stimulations (RNS) and added drugs were recorded. Release of endogenous noradrenaline was measured by HPLC. RNS (1-15 Hz) induced a frequency-dependent increase in the perfusion pressor (14.2+/-5.1-67.3+/-6.9 mmHg) and noradrenaline release (1.4+/-0.3-24.2+/-3.4 ng g(-1) kidney). Pressor responses to RNS were not (1-2 Hz) or only partially (5-15 Hz) blocked by the alpha-adrenoceptor antagonist phentolamine (1 microM). Combination of phentolamine and the P2-receptor blocker PPADS (5 microM) prevented RNS-induced pressor responses. The P2X(1,3)-receptor selective antagonist NF279 (10 microM) reduced RNS-induced pressor responses in a frequency-dependent manner. Perfusion of ATP, ADP, UTP, UDP and alpha,beta-meATP concentration dependently increased perfusion pressor with the following rank order of potency alpha,beta-meATP>ADP approximately ATP approximately UDP > or = UTP. NF279 (10 microM) reduced alpha,beta-meATP- (0.1 microM) (21.7+/-3.9% of control) but not UTP- (0.3 microM) (102.6+/-15.3% of control) induced pressor responses. No differences in nucleotide-induced effects were detected among wild-type and P2Y4-receptor knockout mice. Continuous perfusion of alpha,beta-meATP (0.01 microM) potentiated UTP-, UDP- and ATP-gamma S-induced pressor responses. Neuronally and paracrine-released nucleotides evoked renal vasoconstriction by activation of P2X(1,3)- and P2Y6-like receptors in mice. Pretreatment with the P2X(1,3)-receptor agonist alpha,beta-meATP potentiated P2Y6-like receptor-mediated vasoconstrictions.     

UTP induces vascular responses in the isolated and perfused canine epicardial coronary artery via UTP-preferring P2Y receptors

1. Vasoconstrictor responses of the isolated and perfused canine epicardial coronary artery to uridine 5′-triphosphate (UTP) were analysed pharmacologically.
2. At basal perfusion pressure, UTP induced vasoconstriction in a dose-related manner and the vasoconstriction was sometimes followed by a slight vasodilatation at large doses (more than 10 nmol). The rank order of potency for vasoconstriction was UTP=UDP>ATP>TTP⩾ITP>> UMP. At raised perfusion pressure by 20 mM KCl, the vasoconstriction was not changed and a small vasodilatation was induced at large doses. The rank order of potency for vasodilatation was induced at large doses. The rank order of potency for vasodilatation was ATP>>ITP⩾UDP>UTP⩾TTP. The maximal vasodilator response to UTP was much less than that to ATP. UMP did not induce vasodilatation.
3. The P2X receptor agonist and desensitizing agent α,β-methylene ATP (1 μM) and the P2 receptor antagonist suramin (100 μM) inhibited the vasoconstrictor responses to ATP but not those to UTP and UDP. The P2 receptor antagonist reactive blue 2 (30 μM) did not inhibit the vascular responses to UTP.
4. UTP (200 μM) desensitized the vasoconstrictor responses to UTP, but not either the vasodilator responses to UTP or the vasoconstrictor responses to ATP and UDP. UDP (200 μM) did not desensitize the vascular responses to UTP.
5. Preincubating the UDP stock solution and arterial preparation with hexokinase (10 and 1 uml⁻¹, respectively) did not change the vasoconstrictor responses to UDP.
6. The Ca channel blocker diltiazem (1 μM) inhibited the vasoconstrictor responses to UTP but not those to ATP and UDP. Incubation in a Ca²⁺-free solution containing 1 mM EGTA inhibited the vascular responses to ATP, UTP and UDP.
7. Removal of the endothelium by an intraluminal injection of saponin (1 mg) inhibited the vasodilator responses to UTP. Indomethacin, a cyclo-oxygenase inhibitor (1 μM), inhibited the vasodilator responses to UTP, but N^G-nitro-L-arginine, a nitric oxide synthase inhibitor (300 μM), did not have an inhibitory effect.
8. The results suggest that (1) UTP induces vasoconstriction via UTP-preferring P2Y receptors on the smooth muscle and vasodilatation via receptors different from those mediating the vasoconstriction induced by UTP and mediating the vasodilatation by ATP on the endothelium, through mainly the release of prostacyclin in the canine epicardial coronary artery; (2) UDP induces vasoconstriction via UDP-preferring P2Y receptors; and (3) L-type Ca ion channels are involved in the vasoconstriction induced by UTP, but not in that induced by UDP.

     

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.     

Characterization of P2-purinoceptors in the smooth muscle of the rat tail artery: a comparison between contractile and electrophysiological responses.

1. The electrophysiological actions of the P2-purinoceptor agonists, adenosine 5'-triphosphate (ATP), 2-methylthioATP (2-meSATP), alpha, beta-methyleneATP (alpha, beta-meATP) and uridine 5'-triphosphate (UTP) were studied under concentration and voltage-clamp conditions in acutely dissociated rat tail artery smooth muscle cells. For comparison, their actions as vasoconstrictors were studied in intact ring preparations. 2. Rapid application of ATP (100 nM-1 microM) via a U-tube superfusion system activated concentration-dependent inward currents with a latency to onset of less than 3 ms. The inward current decayed by more than 95% during a 2 s application of 300 nM and 1 microM ATP. 3. 2-meSATP (100 mM-1 microM) and alpha, beta-meATP (100 nM-1 microM) also evoked transient inward currents. The agonist order of potency was ATP = 2-meSATP > or = alpha, beta-meATP. UTP (300 nM-1 microM) did not produce a change in the holding current. 4. A second application of ATP (300 nM and 1 microM) 10 min after the first, evoked currents which were one third of the initial amplitude. This decline was dependent upon activation of the P2-purinoceptor. Similar results were seen with 2-meSATP and alpha, beta-meATP (both 300 nM and 1 microM). Cross-desensitization was seen between ATP and 2-meSATP or alpha, beta-meATP. 5. Inward currents evoked by ATP, 2-meSATP and alpha, beta-meATP (all 1 microM) were abolished by the P2-purinoceptor antagonist suramin (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-existence of P2Y-and PPADS-insensitive P2U-purinoceptors in endothelial cells from adrenal medulla.

1. We have studied the effects of purinoceptor stimulation on Ca2+ signals in bovine adrenomedullary endothelial cells. [Ca2+]i was determined with the fluorescent probe fura-2 both in population samples and in single, isolated, endothelial cells in primary culture and after subculturing. 2. In endothelial cells, maintained in culture for more than one passage, several purinoceptor agonists elicited clear [Ca2+]i transient peaks that remained in the absence of extracellular Ca2+. Adenosine 5''-triphosphate (ATP) and uridine 5''-triphosphate (UTP) were equipotently active, with EC50 values of 8.5 +/- 0.9 microM and 6.9 +/- 1.5 microM, respectively, whereas 2-methylthioadenosine 5''-triphosphate (2MeSATP), adenosine 5''-(alpha, beta-methylene)triphosphate (alpha, beta-MeATP) and adenosine(5'')tetraphospho(5'')adenosine (Ap4A) were basically inactive. Adenosine 5''-O-(2-thiodiphosphate) (ADP beta S) was a weak agonist. The apparent potency order was UTP = ATP > ADP beta S >> 2MeSATP > alpha, beta-MeATP. 3. Cross-desensitization experiments revealed that UTP or ATP, added sequentially at concentrations of maximal effect, could completely abolish the [Ca2+]i response to the second agonist. ADP beta S exerted only a partial desensitization of the response to maximal ATP, in accordance with its lower potency in raising [Ca2+]i. 4. The effect on [Ca2+]i of 100 microM ATP in subcultured cells was reduced by only 25% with 100 microM suramin pretreatment and was negligibly affected by exposure to 10 microM pyridoxalphosphate-6-azophenyl-2'', 4''-disulphonic acid (PPADS). The concentration-effect curve for ATP was not significantly affected by PPADS, but was displaced to the right by a factor of 6.5 by 100 microM suramin. 5. In primary cultures, clear [Ca2+]i responses were elicited by 2MeSATP. Suramin totally and selectively blocked 2MeSATP responses, whereas UTP-evoked [Ca2+]i transients were mainly unaffected by suramin or PPADS. Over 80% of cells tested showed responses to both 2MeSATP and UTP. The [Ca2+]i response to UTP was not desensitized in the presence of 2MeSATP. 6. ATP and UTP stimulated the release of preloaded [3H]-arachidonic acid ([3H]-AA), both in the presence and in the absence of extracellular Ca2+, by approximately 135% with respect to basal levels. Suramin and PPADS enhanced, rather than inhibited, the [3H]-AA releasing effect of ATP by 2.5 times. Suramin also potentiated the effect of the calcium ionophore A23187. 7. These results indicate that endothelial cells from adrenomedullary capillaries co-express both P2Y- and P2U-purinoceptors. P2Y-purinoceptors are lost in culture with the first passage of the cells. The P2U-purinoceptor subtype present in these cells is insensitive to PPADS and thus similar to that found in aortic endothelial cells.     

Regulation of vascular tone by UTP and UDP in isolated rat intrapulmonary arteries.

Vasoconstrictor and vasodilator responses of isolated rat intrapulmonary arteries to the pyrimidine nucleotides UTP and UDP were evaluated and compared with vascular responses to ATP and its analogues. UTP and UDP (1-500 microM) were equipotent in inducing concentration-dependent vasoconstriction, unaffected by the P2 receptor antagonists suramin (100 microM) and Reactive blue 2 (50 microM); ATP (10-500 microM) produced weaker vasoconstriction. UTP and UDP lacked vasodilator activity, while ATP and its analogue 2-methylthio ATP evoked endothelium-dependent vasodilatation. These results indicate that UTP and UDP evoke vasoconstriction of rat intrapulmonary arteries whereas ATP is predominantly a vasodilator at the same arteries. Furthermore, the pharmacological profile of the native UTP/UDP receptor differs from that of the known P2Y2, P2Y4 and P2Y6 recombinant receptors for pyrimidine nucleotides.     

Modulation by nicotinamide adenine dinucleotide of sympathetic and sensory-motor neurotransmission via P1-purinoceptors in the rat mesenteric arterial bed.

1. The pharmacological actions of the purine nucleotides beta-nicotinamide adenine dinucleotide (NAD), beta-nicotinamide adenine dinucleotide phosphate (beta-NADP), adenosine 5'-diphosphoribose (ADP-ribose), the vitamin nicotinamide and structural analogues of NAD and NADP were tested in the isolated perfused mesenteric arterial bed of the rat. Prejunctional effects of NAD were tested against sympathetic vasoconstriction at basal tone, and against sensory-motor vasodilatation at raised tone. 2. NAD and NADP had no vasoconstrictor action but were weak vasodilators of the raised-tone mesenteric arterial bed. A rank order of vasodilator potency of ADP >> ADP-ribose >> NADP > or = NAD = adenosine was observed. The P1-purinoceptor antagonist, 8-para-sulphophenyltheophylline (8-pST; 3 microM) inhibited vasodilator responses to NAD (pKB of 6.61 +/- 0.21, n = 7) and adenosine (pKB of 5.78 +/- 0.14, n = 6), but not those elicited by NADP, ADP and ADP-ribose. Nicotinamide, and analogues of NAD and NADP, namely nicotinamide-1,N6-ethenoadenine dinucleotide phosphate, beta-nicotinamide mononucleotide, nicotinamide hypoxanthine dinucleotide phosphate, nicotinamide hypoxanthine dinucleotide, nicotinamide guanine dinucleotide, and nicotinamide-1, N6-ethenoadenine dinucleotide had no vasoconstrictor or vasodilator actions (at doses of up to 50 nmol). 3. At basal tone, electrical field stimulation (EFS) (32 Hz, 1ms, 90 V, 5 s) at 2 min intervals elicited reproducible vasoconstrictor responses due to activation of sympathetic nerves. NAD and adenosine (10-100 microM) inhibited these responses in a concentration-dependent manner with similar potencies. Nicotinamide had no effect on sympathetic vasoconstriction at concentrations of up to 0.1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of transepithelial ion transport by two different purinoceptors in the apical membrane of canine kidney (MDCK) cells.

1. The effect of extracellular nucleotides on the transepithelial ion transport of Madin Darby canine kidney cells (MDCK) was investigated. Cells were grown up to confluency on permeable supports and the short circuit current (ISC) was measured with an Ussing chamber-like mini-perfusion system. 2. Apical ATP stimulated a biphasic ISC increase consisting of a first rapid and transient peak followed by a broader one. 3. The first peak evoked by ATP was reversibly blocked by basilen blue (BB) in a concentration-dependent fashion, with an EC50 of 7.5 microM. 4. The P2 gamma receptor agonist, 2-methylthioATP (2-MeSATP) caused a single transient ISC increase that was completely blocked by pretreatment with BB. On the contrary, the P2x agonist, alpha, beta-methylene ATP (alpha, beta-meATP) was almost completely ineffective on ISC. UTP essentially induced a monophasic response the time-course of which resembled that of the second peak stimulated by ATP. The agonist potency order was 2-MeSATP > or = ATP >> UTP, alpha, beta-meATP for the first peak and UTP > or = ATP > 2-MeSATP > alpha, beta-meATP for the second peak. 5. Monolayer incubation with the membrane permeable calcium chelator [bis-o-aminophenoxy)-ethane-N,N,N',N',-tetraacetic acid, tetra(acetoximethyl)-ester] (BAPTA/AM) inhibited the ATP-evoked first peak. 6. The non-hydrolyzable ATP analogue, adenosine-5'-O-(3-thio)-trisphosphate (ATP-gamma-S) elicited a biphasic response similar to that of ATP. The P1 receptor agonist, 2-chloroadenosine and CGS-21680, were almost unable to induce an ISC increase.2+ increase. The second induces prostaglandin synthesis probably through a P2U receptor activation.     

A study on P2X purinoceptors mediating the electrophysiological and contractile effects of purine nucleotides in rat vas deferens.

1. We have studied both the electrophysiological and contractile effects of the purine nucleotide, adenosine-5'-triphosphate (ATP), as well as a number of its structural analogues as agonists at P2X purinoceptors in the rat vas deferens in vitro. 2. Electrophysiological effects were investigated by a whole cell voltage clamp technique (holding potential-70 mV) with fast flow concentration-clamp applications of agonists in single isolated smooth muscle cells. ATP, 2-methylthio adenosine-5'-triphosphate (2-MeSATP) and alpha,beta methylene adenosine-5'-triphosphate (alpha,beta-meATP) all evoked inward currents over a similar concentration range (0.3-10 microM), being approximately equipotent with similar concentrations for threshold effects (0.3 microM). ADP (10 microM) also evoked a rapid current of similar peak amplitude to that seen with ATP (10 microM). 3. alpha,beta-meATP was the most potent agonist in producing concentrations of the rat vas deferens whole tissue preparation, with a threshold concentration equal to that in the electrophysiological studies (0.3 microM). However, ATP and 2-MeSATP were at least ten times less potent in studies measuring contraction than in the electrophysiological studies. Furthermore, their concentration-effect curves were shallow with smaller maximal responses than could be achieved with alpha,beta-meATP. ADP, AMP and adenosine were inactive at concentrations up to 1 mM. The rank order of agonist potencies observed for contraction was alpha,beta-meATP >> ATP = 2-MeSATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasoconstriction of intrapulmonary arteries to P2-receptor nucleotides in normal and pulmonary hypertensive newborn piglets.

1. The vasoconstrictor responses of isolated intrapulmonary arteries (IPA) to P2-receptor agonists was investigated during adaptation to extrauterine life in the normal piglet and the effect of pulmonary hypertension was studied following exposure of newborn animals to chronic hypobaric hypoxia (51 kPa) for 3 days. 2. At resting tone, alpha,beta-methyleneATP (alpha,beta-meATP) (P2X-receptor agonist) contracted intrapulmonary arteries from adult, but not immature pigs, and repeated application desensitized the response. 3. Adenosine 5'-triphosphate (ATP) induced endothelium-independent relaxation at low concentrations at all ages, a variable contractile response to high concentrations developed by 3 days, becoming larger and consistent by 14 days of age. 4. Uridine 5'-triphosphate (UTP) evoked a contractile response in normal intrapulmonary arteries from foetal to adult life, the magnitude of the response increasing with age. Endothelial removal and pre-incubation with Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM) increased the contractile response of adult vessels. 5. Pre-incubation with alpha,beta-meATP (100 microM), increased the contractile response to UTP in both newborn and adult vessels. ATP-induced relaxations were reduced in newborn vessels but there was no effect on the responses of adult vessels. 6. Responses to UTP, ATP and alpha,beta-meATP of intrapulmonary arteries from newborn piglets exposed to chronic hypobaric hypoxia for 3 days were normal. 7. In summary, UTP elicited marked vasoconstriction of porcine IPA at all ages. UTP and ATP responses were consistent with activation of the P2Y4-receptor recently identified in vascular smooth muscle by others. alpha, beta-meATP induced a small vasoconstriction in the adult probably via the P2X1-receptor. Responses remained normal in neonatal pulmonary hypertension.     

Activation by ATP of a P2U 'nucleotide' receptor in an exocrine cell.

1. We employed the perforated patch whole-cell technique to investigate the effects of ATP and other related nucleotides on membrane conductances in avian exocrine salt gland cells. 2. ATP (10 microM-1 mM) evoked an increase in maxi-K+ and Cl- conductances with a reversal potential of -35 mV. At lower concentrations of ATP (< or = 100 microM) responses were generally oscillatory with a sustained response observed at higher concentrations (> or = 200 microM). 3. Both oscillatory and sustained responses were abolished by the removal of bath Ca2+. In cells preincubated in extracellular saline containing reduced Ca2+, the application of ATP resulted in a transient increase in current. 4. As increasing concentrations of ATP (and related nucleotides) evoked a graded sequence of events with little run-down we were able to establish a rank order of potency in single cells. The order of potency of ATP analogues and agonists of the various P2-receptor subtypes was UTP > ATP = 2-methylthio-ATP > ADP. Adenosine (1 microM-1 mM), AMP (1 microM-1 mM), alpha,beta-methylene-ATP (1 microM-1 mM) and beta,gamma-methylene-ATP (1 microM-1 mM) were without effect. 5. In conclusion, although unable to preclude a role for a P2Y-receptor, our results suggest that ATP binds to a P2U-receptor increasing [Ca2+]i and subsequently activating Ca(2+)-sensitive K+ and Cl- currents.     

Characterization of a P2X-purinoceptor in cultured neurones of the rat dorsal root ganglia.

1. The electrophysiological actions of the P2-purinoceptor agonists, adenosine 5'-triphosphate (ATP), 2-methylthio ATP (2-meSATP) and alpha, beta-methyleneATP (alpha, beta-meATP) and of uridine 5'-triphosphate (UTP) were studied under concentration and voltage-clamp conditions in dissociated neurones of 1-6 day old rat dorsal root ganglia. 2. ATP (10 nM-100 microM) applied rapidly via a U-tube perfusion system (equilibration time < 10 ms) activated concentration-dependent inward currents with a latency to onset of a few ms, an EC50 of 719 nM and a Hill slope of 1.47. 3. 2-meSATP (10 nM- 100 microM) and alpha, beta-meATP (100 nM - 100 microM) also evoked transient inward currents. The EC50 and Hill slopes were 450 nM and 1.58 for 2-meSATP and 1.95 microM and 1.53 for alpha, beta-meATP respectively. There was no significant difference between the maximum currents evoked by the three agonists. 4. As the concentration of ATP increased so the rate of rise and decay of the currents also increased. At 100 and 300 nM ATP the decay of the current was best fitted by a single exponential, but at 1 microM and above two exponentials were required. Log-log plots of the rise time or time constants of decay versus concentration were linear. Currents evoked by 2-meSATP and alpha, beta-meATP showed a similar concentration-dependence in their kinetics. 5. Inward currents evoked by ATP, 2-meSATP and alpha, beta-meATP (300 nM) were abolished by the P2-purinoceptor antagonist, suramin (100 microM). 6. UTP (10 microM) evoked similar transient inward currents, which were sensitive to suramin (100 microM). ATP (10 microM), applied 2 min beforehand, reduced the response to UTP (10 microM) by 80 +/- 10%. 7. This study shows that ATP, 2-meSATP and alpha, beta-meATP act via a suramin-sensitive P2x-purinoceptor to evoke rapid, transient inward currents in dissociated neurones of rat dorsal root ganglia. The pyrimidine nucleotide, UTP, was also active. It is likely that the agonists were acting at the P2x3-subtype to produce these effects.     

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.     

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.