Vol. 53, Issue 2, 304-312, February 1998

M₂ Receptor Binding of the Selective Antagonist AF-DX 384: Possible Involvement of the Common Allosteric Site

C. Tränkle, I. Andresen, G. Lambrecht, and K. Mohr

Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, 53121 Bonn, Germany (C.T., K.M.), Department of Pharmacology, University of Kiel, Hospitalstraße 4, 24105 Kiel, Germany (I.A.), and Department of Pharmacology, Biocenter Niederursel, University of Frankfurt, 60439 Frankfurt/Main, Germany (G.L.)

The hypothesis was tested that M₂-selective antagonists partially utilize the allosteric site of muscarinic M₂ receptors. 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  = 311 and  = 495, respectively). The antagonistic potency of W84 in this M₂ receptor model (W84 binding constant K_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 ( = 18) but less-than-additive effects with the M₂-preferring AF-DX 384 ( = 444). In guinea pig heart homogenates, the equilibrium binding of [³H]NMS was only partially inhibited by W84 ( = 2.4), whereas [³H]AF-DX 384 binding could be suppressed completely ( = 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.