THE POSSIBLE ROLE OF ATP AND PACAP AS MEDIATORS OF APAMIN-SENSITIVE NANC INHIBITORY JUNCTION POTENTIALS IN CIRCULAR MUSCLE OF GUINEA-PIG COLON

1 In the presence of atropine (1 mu M), guanethidine (3 mu M), indomet hacin (3 mu M), nifedipine (1 mu M), L-nitroarginine (L-NOARG, 100 mu M), and the selective tachykinin NK1 and NK2 receptor antagonists, SR 140,333 and GR 94,800, respectively (0.1 mu M each), a single pulse of electrical field stimulation (EFS) produced a monophasic non-adrenerg ic non-cholinergic (NANC) inhibitory junction potential (i.j.p., about 10 mV in amplitude) in the circular muscle of guinea-pig proximal col on, recorded by the modified single sucrose gap technique. 2 The P-2 p urinoceptor agonist, alpha,beta methylene ATP (alpha,beta mATP, 100 mu M) and the pituitary adenylyl cyclase activating peptide (PACAP, 1 mu M) both produced hyperpolarization (11 +/- 0.8 mV, n = 14 and 10.2 +/ - 0.8 mV, n = 19, respectively) and relaxation (1.1 +/- 0.2 mV, n = 14 and 1.5 +/- 0.2 mN, n = 19, respectively) of the circular muscle. 3 A pamin (0.1 mu M) nearly abolished (about 90% inhibition) the NANC i.j. p. and the alpha,beta mATP-induced hyperpolarization, markedly reduced the alpha,beta mATP-induced relaxation (73% inhibition) and the PACAP -induced hyperpolarization (65% inhibition), while the PACAP-induced r elaxation was unaffected. 4 Tetraethylammonium (TEA, 10 mM) increased the EFS-evoked i.j.p. and revealed an excitatory junction potential (e .j.p.). In the presence of TEA, alpha,beta mATP induced a biphasic res ponse: transient depolarization and contraction followed by hyperpolar ization and relaxation. The hyperpolarization to PACAP was reduced by TEA (45% inhibition) but the relaxation was unaffected. 5 The combined application of apamin (0.1 mu M) and TEA (10 mM) abolished the i.j.p. and single pulse EFS evoked a pure e.j.p. with latency three times lo nger than that of the i.j.p. In the majority of strips tested, alpha,b eta mATP and PACAP elicited a biphasic response : depolarization and s mall contraction followed by hyperpolarization and relaxation. 6 The P -2 purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disul phonic acid (PPADS) inhibited the NANC i.j.p. in concentration-depende nt manner and inhibited the alpha,beta mATP-induced hyperpolarization and relaxation, without affecting the hyperpolarization and relaxation induced by PACAP. On the other hand, the P-2 purinoceptor antagonist, suramin (100 mu M) inhibited to a similar extent (60-80%) the NANC i. j.p. and the hyperpolarization and relaxation induced by alpha,beta mA TP or PACAP. 7 PPADS and suramin reduced the NANC e.j.p. evoked by a s ingle pulse EFS in the presence of apamin and TEA (100 mu M of PPADS a nd 300 mu M of suramin inhibited the e.j.p. by about 40%). 8 We conclu de that ATP, but not PACAP, mediates the apamin-sensitive NANC i.j.p. in the circular muscle of the guinea-pig colon. After blockade of the NANC i.j.p., ATP may act as an excitatory transmitter by activating ex citatory P-2 purinoceptors. The subtypes of P-2 purinoceptor involved in the inhibitory and excitatory responses remain to be established. T he data suggest that excitatory P-2 purinoceptors may be located extra junctionally.