Activation Of P2Y Purinoceptors Research Articles

Hemodynamic Responses Elicited by Systemic Injections of Flavin Adenine Dinucleotide in Anesthetized Rats

Flavin adenine dinucleotide (FAD) elicits an endothelium-dependent vasodilation in isolated rat mesenteric beds via activation of P2Y-purinoceptors. The aims of this study were to characterize the hemodynamic responses elicited by systemic injections of FAD and flavin mononucleotide (FMN) in anesthetized rats and to determine the role of nitric oxide synthase (NOS), cyclooxygenase, P2Y/P2X-purinoceptors, and muscarinic receptor in these responses. FAD (0.05-1.0 micromol/kg, iv) elicited dose-dependent decreases in heart rate (HR), mean arterial blood pressure (MAP), and hindquarter vascular resistance (HQR), whereas it elicited an initial increase and then a decrease in mesenteric (MR) vascular resistance. The FAD-induced responses were not affected by the P2Y/P2X-purinoceptor antagonist suramin, the muscarinic receptor antagonist methyl-atropine, or the cyclooxygenase inhibitor indomethacin. The vasodilator actions of FAD were unaffected by the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME), whereas the bradycardia elicited by higher doses of FAD were diminished by L-NAME. FMN did not elicit hemodynamic responses in the absence or presence of L-NAME. In summary, FAD-induced bradycardia depends, in part, on the activation of NOS, whereas the vasodilator actions of FAD are not obviously due to newly synthesized nitrosyl factors. These findings and those in our companion manuscript support the concepts that the adenine moiety confers biological activity to FAD, which releases preformed pools of nitrosyl factors.

Enhanced IPC by activation of pertussis toxin-sensitive and -insensitive G protein-coupled purinoceptors.

Extracellular ATP plays an important role in ischemic preconditioning (IPC) through the activation of P(2y) purinoceptors. This study examined whether ATP-stimulated P(2y) purinoceptors are coupled to pertussis toxin (PTX)-insensitive G protein and whether activation of this pathway enhances myocardial protection afforded by IPC. The rat was treated with PTX for 48 h, and the heart was then isolated and buffer perfused. The heart underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. Isovolumic left ventricular function was measured, and functional recovery at 30 min after reperfusion was taken as an end point of myocardial protection. PTX pretreatment partially inhibited functional protection by IPC. Treatment with 100 microM 8-(p-sulfophenyl) theophylline (SPT) during IPC had no further effect on PTX-induced inhibition of functional protection by IPC, whereas suramin (300 microM) or reactive blue (RB) (10 microM) completely abolished myocardial protection in the preconditioned heart pretreated with PTX. Supplementation with adenosine (30 microM), ATP (30 microM), or UTP (50 microM) significantly enhanced IPC-induced functional protection, although preconditioning with these nucleotides without IPC had no protective effect. Adenosine-enhanced IPC was inhibited by pretreatment with PTX and SPT but not by suramin or RB, whereas ATP-enhanced IPC was inhibited by suramin or RB in combination with PTX pretreatment. On the other hand, UTP-enhanced IPC was not affected by PTX pretreatment but was inhibited by suramin or RB. Adenosine supplemented IPC without PTX pretreatment and ATP supplemented IPC with PTX pretreatment were not affected by nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (100 microM). Although the protein kinase C inhibitor Ro318425 (0.3 microM) or tyrosine kinase inhibitor genistein (50 microM) had no significant effect on the functional protection afforded by adenosine-supplemented IPC without PTX pretreatment and ATP-supplemented IPC with PTX pretreatment, the combination of Ro318425 and genistein attenuated functional protection afforded by both the purinoceptor agonist-supplemented IPC. These results suggest the crucial involvement of PTX-sensitive and -insensitive G protein coupled purinoceptors in enhanced IPC by supplementation with adenosine, ATP, and UTP.

Participation of ATP in cell volume regulation in the endothelium after hypotonic stress.

1. The role of ATP in the regulatory volume decrease (RVD) after hypotonic cell swelling was examined in cultured endothelial cells isolated from the rat caudal artery. 2. Hypotonic stress increased [Ca2+]i in addition to increasing the overflow of ATP and cell volume. The hypotonicity induced increase in [Ca2+]i was prevented by pyridoxalphosphate-6-azophenyl-1-2',4'-disulphonic acid (PPADS; a P2 purinoceptor antagonist), U-73122 (a phospholipase C inhibitor) and thapsigargin (a Ca2+ pump inhibitor). However, the hypotonicity induced increase in cell volume was potentiated by PPADS, U-73122 and thapsigargin. 3. Similar changes were observed in cells treated with 2-methylthioATP, a P2Y purinoceptor agonist, but not by alpha,beta-methylene ATP, a P2X purinoceptor agonist. Thus, it appears that the responses observed following hypotonic stress are mediated by activation of P2Y purinoceptors. 4. On the basis of these findings, it is suggested that ATP, which is released by hypotonicity, may participate in the RVD as a substantial regulator or initiator via P2 purinoceptor-induced increases in [Ca2+]i.

Potassium efflux triggered by P2Y purinoceptor activation in cultured pituicytes.

We have previously investigated the effects of extracellular ATP on the concentration of free cytosolic calcium ([Ca2+]i) from rat cultured neurohypophysial astrocytes (pituicytes). We demonstrated that ATP acts via a P2Y receptor to increase [Ca2+]i. In the present study, we examine the effect of ATP on K+ efflux using 86Rb+ as an isotopic tracer, in order to characterize the possible presence of a Ca2+-activated K+ conductance and to establish the implications of pituicytes in the regulation of stimulus-secretion coupling. ATP evoked an increase in 86Rb+ efflux from cultured pituicytes. This effect was Ca2+ dependent, as indicated by the unresponsiveness of cells loaded with BAPTA/AM (20 microM). Furthermore, the effect of ATP was mimicked by 2-methylthio-adenosine-5'-triphosphate (2MeSATP), a P2 purinoceptor agonist, and abolished by Reactive Blue 2 (RB-2), a selective P2Y antagonist, implying a role for the P2Y purinoreceptor. A pharmacological study revealed that Ba2+ and tetraethylammonium (TEA), two inhibitors of K+ channels, both strongly reduced the ATP-stimulated 86Rb+ efflux. In addition, the effect of ATP was modulated by different peptidic toxins. Apamin (100 nM), an inhibitor of the small-conductance Ca2+-activated K+ channels, partly blocked ATP-induced 86Rb+ efflux. Leiurus quinquestriatus hebraeus (LQH) scorpion venom (20 microg/ml) and Buthus tamulus (BT) scorpion venom (20-200 microg/ml) inhibited ATP-induced 86Rb+ efflux. The specificity of the effects of the crude venoms was checked using charybdotoxin (100 nM) and iberiotoxin (1 pM), which are the active toxins extracted from the LQH and BT venoms, respectively. These data indicate the involvement of several types of Ca2+-activated K+ channels in the ATP-dependent K+ efflux, and lead to the proposal that, in the neurohypophysis, extracellular ATP released by nerve terminals may act directly on the pituicytes and induce a K+ efflux via a P2Y purinoreceptor.

ATP-activated cation currents in single guinea-pig hepatocytes.

1. Responses of single guinea-pig liver cells to the application of external ATP were studied using the whole-cell voltage clamp technique. 2. When the cells were loaded with 5 mM EGTA in the absence of K+ and Cl- in both internal and external solutions, application of ATP (0.03-100 microM) elicited a large cation-selective inward current at negative holding potentials. The current densities at the peak of the response to 100 microM ATP were 4.5 +/- 0.5 pA pF-1 (mean +/- s.e.m., n = 18) in the presence of Na+ and Ca2+ in the external medium and 3.3 +/- 0.7 pA pF-1 (n = 6) with Ca2+ as the major permeant ion. 3. Divalent cations, when added during the response to ATP in the presence of Na+ and Ca2+, exerted different effects: CdSO4 (2 mM) totally and NiSO4 (2 mM) partially blocked the inward current whereas MnSO4 (2 mM) did not block it. The ATP-activated conductance was permeable to all the divalent cations tested in this study, i.e. Ca2+, Cd2+, Ni2+, Mn2+ and Mg2+. No response to ATP was observed in the absence of external cations. 4. The activation of the inward current was not maintained in the continuous presence of ATP. The effect of Ca2+ ions on the desensitization of the response was studied in different external solutions. The decline in the amplitude of the inward current after the peak was fitted with a single exponential with a time constant of about 2 s for pure Ca2+, Cd2+ or Ni2+ currents, 3 s for Mg2+ or Mn2+ and 4 s in the presence of both Na+ and Ca2+. 5. Under more physiological conditions, the entry of Ca2+ evoked after the stimulation of P2X purinoceptors was associated with an increase in fluo-3 fluorescence and a marked reduction in the delay before the mobilization of internal Ca2+ stores following the activation of P2Y purinoceptors.

Comparative effects of adenosine‐5′‐triphosphate and related analogues on insulin secretion from the rat pancreas

Adenosine tri- and diphosphate (ATP and ADP) and their structural analogues stimulate insulin secretion from the isolated perfused rat pancreas, an effect mediated by P2Y-purinoceptor activation. Concerning the base moiety of the nucleotide, it was previously shown that purine but not pyrimidine nucleoside triphosphates were active and that substitution on purine C2 with the 2-methylthio group greatly enhanced the potency. In this study, we further analyze the consequences of ribose and polyphosphate chain modifications. Modifications in 2' and 3' position on the ribose led to a decrease in insulin response when bulky substitutions were made: indeed, 2'-deoxy ATP was similar in activity to ATP, whereas arylazido-aminopropionyl ATP (ANAPP3) was weakly effective and trinitrophenyl ATP (TNP-ATP) was inactive. Substitution on the gamma phosphorus of the triphosphate chain led to a decrease (gamma-anilide ATP) or no change (gamma-azido ATP) in potency; the replacement of the bridging oxygen between beta and gamma phosphorus by a peroxide group did not significantly change the activity, whereas substitution by a methylene group completely abolished stimulation of insulin secretion. As for the phosphorothioate analogues, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) induced an insulin response similar to that produced by ATP, whereas adenosine-5'-O-(2-thiodiphosphate) (ADP beta S) was about 100-fold more potent than ATP, as previously shown. In conclusion, two structural features seem to have a strategic importance for increasing the insulin secretory activity of ATP analogues: substitution at the C2 position on the adenine ring of ATP and modifications of the polyphosphate chain at the level of the beta phosphorus.

Study of the mechanisms involved in adenosine-5'-O-(2-thiodiphosphate) induced relaxation of rat thoracic aorta and pancreatic vascular bed.

1. The endothelium-dependent relaxation of blood vessels induced by P2Y-purinoceptor activation has often been shown to involve prostacyclin and/or nitric oxide (NO) release. In this work, we have investigated the mechanisms involved in the relaxant effect of the P2Y agonist, adenosine -5'-O-(2-thiodiphosphate) (ADP beta S) using two complementary preparations: rat pancreatic vascular bed and aortic ring. 2. On the pancreatic vascular bed, ADP beta S (1.5 and 15 microM) infused for 30 min induced a concentration-dependent vasodilatation; it was progressive during the first 10 min (first period) and sustained from 10 to 30 min (second period). Indomethacin (10 microM) delayed ADP beta S-induced vasodilatation (1.5 and 15 microM) by about 6 min. N omega-nitro-L-arginine methyl ester (L-NAME) (200 microM) suppressed the relaxation for about 5 min but thereafter ADP beta S at the two concentrations progressively induced an increase in the flow rate. Even the co-administration of L-NAME and indomethacin did not abolish the ADP beta S-induced vasorelaxation. 3. On 5-hydroxy tryptamine (5-HT) precontracted rings mounted in isometric conditions in organ baths, we observed that ADP beta S induced a concentration-dependent relaxation of rings with a functional endothelium; this effect was stable for 25 min. The ADP beta S relaxant effect was strongly inhibited by Reactive Blue 2 (30 microM) and was suppressed by pretreatment of rings with saponin (0.05 mg ml-1 for 30 min), which also abolished the acetylcholine-induced relaxation. 4. ADP beta S-induced relaxation of 5-HT precontracted rings is largely inhibited by indomethacin (100 or 10 microM) or L-NAME (100 microM). 5. We conclude that: the ADP beta S-induced relaxation is endothelium-dependent, mediated by P2Y-purinoceptors, and at least in part linked to NO and prostacyclin release, depending on the preparation used. Furthermore, on the pancreatic vascular bed, (an)other mechanism(s) than prostacyclin and NO releases may be involved in ADP beta S-induced vasodilatation.