Pharmacological characterization of acetylcholine‐stimulated [35S]‐GTPγS binding mediated by human muscarinic m1–m4 receptors: antagonist studies

Abstract

1 We have used dose-ratio analysis to estimate functionally the affinity constants (pK_b) and Schild slope factors of a range of selective or atypical antagonists at human muscarinic m1–m4 receptors. 2 The functional response was the stimulation by acetylcholine of [³⁵S]-GTPγS binding to membranes from Chinese hamster ovary (CHO) cells stably expressing individual receptor subtypes. 3 A novel experimental design and analysis was used which allowed the estimation of affinity and Schild slope factor from a single antagonist inhibition curve, and the results were compared with other methods of analysis, both theoretically valid and invalid. 4 In general, the affinity estimates were very similar to previously reported values obtained in binding studies with animal tissues and cloned human receptors and the Schild slope factors were close to unity. 5 These results demonstrate the validity of the assay and provide no evidence for species differences in antagonist affinity for muscarinic receptor subtypes. 6 The results confirm both the utility of himbacine in distinguishing between m1 and m4 receptors and a previously reported modest m4-selectivity for tropicamide and secoverine. 7 The cholinesterase inhibitor, tacrine (THA), had a potency profile similar to that of gallamine but with less selectivity. Its affinity could not be determined since it had Schild slope factors of about 2 at all subtypes. 8 o-Methoxy-sila-hexocyclium had only a modest selectivity for the ml subtype.