The possible role of ATP and PACAP as mediators of apamin‐sensitive NANC inhibitory junction potentials in circular muscle of guinea‐pig colon

Abstract

1 In the presence of atropine (1 μm), guanethidine (3 μm), indomethacin (3 μm), nifedipine (1 μm), L-nitroarginine (L-NOARG, 100 μm), and the selective tachykinin NK₁ and NK₂ receptor antagonists, SR 140,333 and GR 94,800, respectively (0.1 μm each), a single pulse of electrical field stimulation (EFS) produced a monophasic non-adrenergic non-cholinergic (NANC) inhibitory junction potential (i.j.p., about 10 mV in amplitude) in the circular muscle of guinea-pig proximal colon, recorded by the modified single sucrose gap technique. 2 The P ₂ purinoceptor agonist, α, β methylene ATP (α, β mATP, 100 μm) and the pituitary adenylyl cyclase activating peptide (PACAP, 1 μm) 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 ± 0.2mN, n = 19, respectively) of the circular muscle. 3 Apamin (0.1 μm) nearly abolished (about 90% inhibition) the NANC i.j.p. and the α, β mATP-induced hyperpolarization, markedly reduced the α, β mATP-induced relaxation (73% inhibition) and the PACAP-induced hyperpolarization (65% inhibition), while the PACAP-induced relaxation 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, α, β mATP induced a biphasic response: transient depolarization and contraction followed by hyperpolarization 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 μm) and TEA (10 mM) abolished the i.j.p. and single pulse EFS evoked a pure e.j.p. with latency three times longer than that of the i.j.p. In the majority of strips tested, α, β mATP and PACAP elicited a biphasic response: depolarization and small contraction followed by hyperpolarization and relaxation. 6 The P₂ purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) inhibited the NANC i.j.p. in concentration-dependent manner and inhibited the α, β mATP-induced hyperpolarization and relaxation, without affecting the hyperpolarization and relaxation induced by PACAP. On the other hand, the P₂ purinoceptor antagonist, suramin (100 μm) inhibited to a similar extent (60–80%) the NANC i.j.p. and the hyperpolarization and relaxation induced by α, β mATP or PACAP. 7 PPADS and suramin reduced the NANC e.j.p. evoked by a single pulse EFS in the presence of apamin and TEA (100 μm of PPADS and 300 μm of suramin inhibited the e.j.p. by about 40%). 8 We conclude 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 excitatory P₂ purinoceptors. The subtypes of P₂ purinoceptor involved in the inhibitory and excitatory responses remain to be established. The data suggest that excitatory P₂ purinoceptors may be located extrajunctionally.