Pharmacological evidence for the existence of multiple P2 receptors in the circular muscle of guinea-pig colon.

1. By using the sucrose gap technique, we have investigated the effect of the metabolically stable P2Y receptor agonist, adenosine 5'-O-2-thiodiphosphate (ADPbetaS), on the membrane potential and tension in the circular muscle of the guinea-pig proximal colon. All experiments were performed in the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), nifedipine (1 microM), L-nitroarginine (L-NOARG, 100 microM) and of the tachykinin NK1 and NK2 receptor antagonists, SR 140333 (0.1 microM) and GR 94800 (0.1 microM), respectively. 2. ADPbetaS (100 microM for 15 s) evoked a tetrodotoxin- (1 microM) resistant hyperpolarization and contraction of the smooth muscle. In the presence of apamin (0.1 microM), the ADPbetaS-induced hyperpolarization was converted to depolarization and the contraction was potentiated while tetraethylammonium (TEA, 10 mM) did not affect significantly the response to ADPbetaS. The combined application of apamin and TEA reproduced the effect observed with apamin alone. 3. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acids (PPADS, 30 microM) slightly but significantly increased the ADPbetaS-induced hyperpolarization, while the contraction evoked by ADPbetaS was reduced by about 80%. Suramin (100 microM) did not affect the ADPbetaS-induced hyperpolarization but totally blocked the ADPbetaS-induced contraction. In the presence of suramin (100 microM), a small relaxation of the circular muscle was observed upon application of ADPbetaS. 4. The contraction and hyperpolarization evoked by ADPbetaS were abolished in Ca2+-free Krebs solution. The blocker of sarcoplasmic reticulum Ca2+ pump, cyclopiazonic acid (10 microM) reduced contraction and hyperpolarization induced by ADPbetaS by about 60 and 50%, respectively. 5. A comparison of our present and previous findings enables to conclude that at least 3 types of P2 receptors are present on the smooth muscle of the guinea-pig colon, as follows: (1) inhibitory P2 receptors, producing an apamin-sensitive hyperpolarization, which are activated by alpha,beta-methylene ATP (alpha,beta-meATP) and by endogenously released purines, sensitive to suramin and PPADS; (2) inhibitory P2 receptors, producing an apamin-sensitive hyperpolarization, which are activated by ADPbetaS and are resistant to suramin and PPADS; (3) excitatory P2 receptors, producing contraction, which are activated by ADPbetaS and are sensitive to suramin and PPADS. The data also support the idea of the existence of a restricted pool of specialized junctional P2 receptors producing the apamin-sensitive NANC inhibitory junction potential in response to endogenous ligand(s).