Antagonism of the effects of 5-hydroxytryptamine on the rabbit isolated vagus nerve by BRL 43694 and metoclopramide

Summary

Depolarization and reduction in the C fibre compound action potential (C spike) in response to 5-HT were recorded simultaneously from rabbit isolated vagus nerve. 5-HT (0.1–100 μmol/l) was applied cumulatively and EC₅₀ and IC₅₀ values measured from individual concentration-response curves. Blockade of 5-HT responses by the 3-indazole carboxamide, BRL 43694, was investigated and compared with the blocking action of metoclopramide. BRL 43694 was a selective antagonist of 5-HT responses. A concentration of 10 nmol/l BRL 43694, which nearly abolished the depolarization and reduction of the C spike evoked by 5-HT (100 μmol/l), had no effect on similar responses evoked by DMPP (100 μmol/l) or GABA (100 μmol/l). Blockade of 5-HT responses by BRL 43694 (0.3 nmol/l) was slow in onset, a plateau blockade occurring after equilibrium of tissue with antagonist for 2 to 3 h. Metoclopramide induced a blockade of rapid onset. The maximal blockade was apparent within 30 min of application. Full recovery in the responsiveness of the tissue to 5-HT was observed within 30 min of washing out metoclopramide. BRL 43694 at concentrations of 0.3, 1, 3 and 10 nmol/l caused a progressive rightward shift of the concentration-response curves to 5-HT. At the highest concentration of antagonist, there was some depression of the maximal 5-HT response. The apparent pA₂ estimated from the Schild equation was 10.03 ± 0.09 (mean ± SEM, n = 20) against 5-HT depolarization and 10.31 ± 0.1 against C spike reduction. Schild plots had slopes not significantly different from 1.0. The slopes and extrapolated pA₂ fitted by linear regression were 0.91 (0.58 – 1.24) and pA₂ 10.16 (9.74–10.58; mean and 95% confidence levels) for the depolarizations. For reduction in C spikes the slope was 0.74 (0.39–1.08) with a pA₂ of 10.86 (10.24–11.49). There was no apparent use-dependent element to the blockade by BRL 43694. Blockade of 5-HT depolarization or C spike reduction by BRL 43694 (0.3 nmol/l) was not significantly different on repeated testing in the presence of the antagonist or without testing of 5-HT until 3 h incubation had elapsed. Metoclopramide at concentrations of 0.3, 1, 3, or 10 μmmol/l progressively shifted concentration-response curves to the right. However, the response maximum, especially that for depolarization, was enhanced in the presence of the antagonist. The apparent pA₂ values from the Schilde equation were 7.04 ± 0.04 (n = 20) against 5-HT depolarization and 7.13 ± 0.06 (n = 16) against C spike reduction. Schild plots had slopes significantly less than unity. The lines fitted to the relationship gave pA₂ values of 7.41 (7.25–7.57) with a slope of 0.79 (0.69–0.9) against depolarization and 7.63 (7.28–7.97) with a slope of 0.74 (0.54–0.93) against C spike reduction. Metoclopramide at concentrations above 30 μmol/l directly reduced C spike amplitude; the IC₅₀ for the local anaesthetic action was 158 ± 40 μmol/l (mean ± SEM). It is concluded that BRL 43694 is a potent and selective antagonist of 5-HT₃ receptors on the rabbit vagus nerve. At concentrations below 10 nmol/l, BRL 43694 appeared to behave as a competitive antagonist while at 10 nmol/l the antagonism was unsurmountable, suggesting a “pseudo-irreversible” antagonism due to slow dissociation of antagonist from the receptor. Although metoclopramide behaved as a surmountable antagonist, the low slope of the Schild plots and the increase in maximal response amplitude in the presence of the antagonist are unexplained features of its blocking action.