Sequential activation of the triple excitatory transmission to the circular muscle of guinea-pig colon

Abstract

The aim of this study was to resolve the temporal relationships of the triple excitation of the circular muscle of guinea-pig colon that occurs in response to activation of the intrinsic excitatory nerves by using atropine and tachykinin NK 1 and NK 2 receptor selective antagonists to define the relative contribution of the transmitters involved. In organ bath experiments, performed in the presence of blockers of inhibitory innervation, a train of electrical pulses at 5 Hz for 300 s produced a sustained contraction of the circular muscle of guinea-pig colon: the sequential addition of atropine (1 μM), of the tachykinin NK 1 receptor antagonist, SR 140333 (0.3 μM) and of the tachykinin NK 2 receptor antagonist, MEN 11420 (1 μM) produced a cumulative inhibitory effect and progressively delayed the onset of the contractile response to nerve stimulation. In the presence of peptidase inhibitors, atropine was less effective in inhibiting the contractile response for prolonged periods of stimulation: however, the pattern of inhibition of the evoked response produced by the sequential addition of blocker drugs was not substantially affected. The selective tachykinin NK 3 receptor agonist, senktide, produced a concentration-dependent contraction of guinea-pig colon. The sequential addition of atropine (1 μM), SR 140333 (0.3 μM) and MEN 11420 (1 μM) reproduced the effect of the same drugs on the response to electrical nerve stimulation. The peptide blocker of N-type voltage-dependent calcium channels, ω-conotoxin (0.1 μM) produced a partial inhibitory effect of the response to senktide. The ω-conotoxin-resistant response to 1 μM senktide was inhibited and delayed by the progressive application of atropine, SR 140333 and MEN 11420, similar to the effect observed in the absence of ω-conotoxin. In sucrose gap, single-pulse electrical field stimulation produced a fast excitatory junction potential which was largely (>90%) inhibited by atropine; application of a low concentration of the potassium channel blocker, 4-aminopyridine (30 μM), markedly enhanced the atropine-resistant excitatory junction potential which was abolished by the NK 1 receptor antagonist, GR 82334. We conclude that, during prolonged electrical or chemical stimulation of excitatory motorneurons, there is a sequential, time-dependent activation of the three excitatory mechanisms in the circular muscle of guinea-pig colon: the pattern of activation is relatively independent of the intensity of stimulation and/or the mechanisms of secretion of released transmitters. Postjunctional factors predominate in determining the relative contribution of the three transmitters, acetylcholine, substance P and neurokinin A, in producing excitation of the circular muscle.