Abstract

The hypothesis was tested that M₂-selective antagonists partially utilize the allosteric site of muscarinic M₂receptors. The interactions of the allosteric agent W84 (hexane-1,6-bis[dimethyl-3′-phthalimidopropyl-ammonium bromide]) were studied with the M₂/M₄-selective AF-DX 384 [(±)-5,11-dihydro-11-{[(2-{2-[(dipropylamino)methyl]-1-piperidinyl}ethyl)amino]carbonyl}-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one], the nonselective N-methylscopolamine (NMS), and a number of other muscarinic antagonists. In isolated paced guinea pig atria, the antagonistic effect of W84 against oxotremorine- and arecaidine propargyl ester-induced negative inotropic actions reached a limiting value at higher W84 concentrations, revealing negative cooperativity (factors of cooperativity α = 311 and α = 495, respectively). The antagonistic potency of W84 in this M₂receptor model (W84 binding constantK _A ∼ 160 nm) was higher than at M₁/M₄-like receptors of rabbit vas deferens (K _B ∼800 nm) and at M₃ receptors of guinea pig ileum (K _B ∼4,000 nm). In paced atria, combinations of W84 with muscarinic antagonists yielded more-than-additive antagonistic effects against oxotremorine in case of conventional antagonists such as NMS (α = 18) but less-than-additive effects with the M₂-preferring AF-DX 384 (α = 444). In guinea pig heart homogenates, the equilibrium binding of [³H]NMS was only partially inhibited by W84 (α = 2.4), whereas [³H]AF-DX 384 binding could be suppressed completely (α = 194). The difference in cooperativity reflects that W84 inhibits [³H]NMS dissociation with a ∼40-fold higher potency (EC_diss = 900 nm) than [³H]AF-DX 384 dissociation (EC_diss = 33,300 nm). [³H]NMS dissociation also could be retarded by AF-DX 384 (EC_diss = 22,000 nm), probably via an interaction with the site used by W84. The results suggest that the binding domain of AF-DX 384 partially overlaps with the common allosteric site of the M₂ receptor protein.