Interaction of agonists and selective antagonists with gastric smooth muscle muscarinic receptors

Summary The interaction of cholinergic agonists and antagonists with smooth muscle muscarinic receptors has been investigated by measurement of displacement of the muscarinic antagonist ³H]QNB (quinuclidinyl benzilate) in membranes prepared from toad stomach. The binding of ³H]QNB was saturable, reversible and of high affinity (K _D = 423 pM). The muscarinic receptor subtypes present in gastric smooth muscle were classified by determining the relative affinities for the selective antagonists pirenzepine (M₁), AF-DX 116 (M₂) and 4-DAMP (M₃). The results from these studies indicate the presence of a heterogeneous population of muscarinic receptor subtypes, with a majority (88%) exhibiting characteristics of M₃ receptors and a much smaller population (12%) exhibiting characteristics of M₂ receptors. The binding curve for the displacement of ³H]QNB binding by the agonist oxotremorine was complex and was consistent with presence of two affinity states: 24% of the receptors had a high affinity (K _D = 4.7 nM) for oxotremorine and 76% displayed nearly a 1,000-fold lower affinity (K _D = 4.4 M). When oxotremorine displacement of ³H]QNB binding was determined in the presence GTPS, high affinity binding was abolished, indicating that high affinity agonist binding may represent receptors coupled to G proteins. Moreover, pertussis toxin pretreatment of membranes also abolished high affinity agonist binding, indicating that the muscarinic receptors are coupled to pertussis toxin-sensitive G proteins. Reaction of smooth muscle membranes with pertussis toxin in the presence ³²P]NAD caused the ³²P]-labelling of a 40 kD protein that may represent the subunit(s) of G proteins that are known to be NAD-ribosylated by the toxin. We conclude that both M₃ and M₂ receptors may be coupled to G proteins in a pertussis-sensitive manner. Send offprint requests to T. W. Honeyman at the above address