6533b871fe1ef96bd12d1145

RESEARCH PRODUCT

Characterization of the muscarinic receptor subtype(s) mediating contraction of the guinea-pig lung strip and inhibition of acetylcholine release in the guinea-pig trachea with the selective muscarinic receptor antagonist tripitramine.

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1 The muscarinic receptor subtypes mediating contraction of the guinea-pig lung strip and inhibition of the release of acetylcholine from cholinergic vagus nerve endings in the guinea-pig trachea in vitro have previously been characterized as M-2-like, i.e. having antagonist affinity profiles that are qualitatively similar but quantitatively dissimilar compared to cardiac M-2 receptors. The present study sought to establish definitely the identity of these receptor subtypes by using the selective muscarinic receptor antagonist, tripitramine. Guinea-pig atria and guinea-pig trachea (postjunctional contractile response) were included for reference.2 It was found that tripitramine antagonized methacholine induced contractions of the guinea-pig lung strip with a pK(B) value of 8.76 +/- 0.05. Both the parallel shifts of the concentration-response curves and the slope of the Schild plot being not significantly different from unity (when antagonist preincubation was for 2 h) indicated the involvement of a single population of receptors in the contractile response. From the pK(B) values obtained with tripitramine and a range of other selective muscarinic receptor antagonists (cf. Roffel et al., 1993), this single population of receptors can only be classified as M-2-like.3 Tripitramine antagonized methacholine-induced negative chronotropic and inotropic responses in guinea-pig right and left atria with apparent pK(B) values of 9.4-9.6. However, such values were only obtained when antagonist preincubation was relatively long and/or antagonist concentration relatively high (e.g. with 1 h at 100 or 300 nM but 3 h at 30 nM). It thus appears that low concentrations of tripitramine do not readily equilibrate with M-2 receptors in guinea-pig atria nor with M-2-like receptors in the guinea-pig lung strip.4 Tripitramine increased electrical field stimulation-induced cholinergic twitch contractions in guineapig trachea in concentrations of 0.3-100 nM, by blocking prejunctional muscarinic inhibitory autoreceptors; with higher concentrations, twitch contractions were progressively diminished, as a result of blocking postjunctional M-3 receptors (apparent pK(B) value 6.07 +/- 0.15). The pEC(20) value (-log concentration that increases twitch by 20% of maximum) was 8.29 +/- 0.08, which would suggest that M-4 receptors are involved in this response.5 Oxotremorine-induced inhibition of the release of prelabelled [H-3]-acetylcholine from guinea-pig trachea, under conditions where there is no auto-feedback, was blocked by tripitramine (2 h preincubation) with a pK(B) value of 8.56 +/- 0.06. The slope of the corresponding Schild plot was not significantly different from unity, which together with the parallel shifts of the concentration-response curves indicated the involvement of a single muscarinic receptor subtype.6 Since the pK(B) value for tripitramine at prejunctional receptors in guinea-pig trachea is in between the affinities towards M-2 and M-4 receptors, correlation plots were constructed to compare the pK(B) values obtained with tripitramine and a range of other selective muscarinic receptor antagonists (cf Kilbinger et al., 1995) to reported affinities at M-1-M-4 receptors. This showed rather similar distribution patterns of the data points around the line of equality in the case of M-2 and M-4 receptor subtypes. However,the correlation coefficient was markedly better for M-2 (0.9667) than for M-4 (0.5976). Since recent evidence suggests that M-4 receptors are not expressed in cholinergic nerves from guinea-pig trachea, it is concluded that prejunctional muscarinic autoinhibitory receptors in this tissue exhibit an atypical M-2 type character, with a pharmacological profile distinct from cardiac M-2 receptors.