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

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 p K 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 p K 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 p K 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 n M but 3 h at 30 n M ). 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 guinea‐pig trachea in concentrations of 0.3–100 n M , by blocking prejunctional muscarinic inhibitory autoreceptors; with higher concentrations, twitch contractions were progressively diminished, as a result of blocking postjunctional M 3 receptors (apparent p K 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 [ 3 H]‐acetylcholine from guinea‐pig trachea, under conditions where there is no auto‐feedback, was blocked by tripitramine (2 h preincubation) with a p K 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 p K 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 p K 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.British Journal of Pharmacology (1997) 122 , 133–141; doi: 10.1038/sj.bjp.0701346