Subtype-selective positive cooperative interactions between brucine analogs and acetylcholine at muscarinic receptors: Functional studies

In radioligand binding studies, it has been reported that brucine, N-chloro methyl brucine, and brucine N-oxide increased the affinity of acetylcholine for M-1, M-3, and M-4 muscarinic receptors, respectively, in a manner cons istent with the predictions of the ternary complex allosteric model. We now demonstrate an equivalent ability of these three allosteric agents to modu late the actions of acetylcholine in functional studies in membranes and in whole cells. The enhancing actions of brucine and brucine N-oxide on acety lcholine (ACh) potency at M-1 and M-4 receptors respectively have been conf irmed in guanosine-5'-O-(3-[S-35]thio)triphosphate, GTPase, cAMP, and intra cellular Ca2+ mobilization assays of function. In general, neither the basa l nor the maximally stimulated response to ACh is affected. The subtype-sel ective allosteric effects of N-chloromethyl brucine on M-2 and M-3 receptor s were shown to be qualitatively and quantitatively the same in guanosine-5 '-O-(3-S-35]thio)triphosphate functional assays, in terms of both its affin ity and cooperativity with ACh, as those found in binding assays. Neutral c ooperativity of N-chloromethyl brucine with ACh on M-4 receptor function wa s also observed, thereby demonstrating its "absolute subtype selectivity": a lack of action at any concentration at M-4 receptors and an action at M-2 and M-3 receptors. The enhancing action of N-chloromethyl brucine on neuro genically released ACh binding at M-3 receptors was also detected in whole tissue as an increased contraction of the isolated guinea pig ileum to subm aximal electrical stimulation. In conclusion, these functional studies conf irm that brucine analogs are allosteric enhancers of ACh affinity at certai n muscarinic receptor subtypes.