Subtype-Selective Positive Cooperative Interactions Between Brucine Analogs and Acetylcholine at Muscarinic Receptors: Functional Studies

• Published in: Molecular pharmacology 1999-04, Vol.55 (4), p.778
• Main Authors: Birdsall, N J, Farries, T, Gharagozloo, P, Kobayashi, S, Lazareno, S, Sugimoto, M
• Format: Article
• Language: English
• Subjects: Acetylcholine - metabolism; Allosteric Regulation; Animals; Cell Membrane - metabolism; CHO Cells; Cricetinae; Cyclic N-Oxides - pharmacology; Guinea Pigs; Humans; Male; Receptor, Muscarinic M1; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Receptors, Muscarinic - drug effects; Receptors, Muscarinic - metabolism; Strychnine - analogs & derivatives; Strychnine - chemistry; Strychnine - metabolism; Strychnine - pharmacology
• ISSN: 0026-895X; 1521-0111

In radioligand binding studies, it has been reported that brucine, N -chloromethyl brucine, and brucine N -oxide increased the affinity of acetylcholine for M 1 , M 3 , and M 4 muscarinic receptors, respectively, in a manner consistent with the predictions of the ternary complex allosteric model. We now demonstrate an equivalent ability of these three allosteric agents to modulate the actions of acetylcholine in functional studies in membranes and in whole cells. The enhancing actions of brucine and brucine N -oxide on acetylcholine (ACh) potency at M 1 and M 4 receptors respectively have been confirmed in guanosine-5′- O -(3-[ 35 S]thio)triphosphate, GTPase, cAMP, and intracellular Ca 2+ mobilization assays of function. In general, neither the basal nor the maximally stimulated response to ACh is affected. The subtype-selective allosteric effects of N -chloromethyl brucine on M 2 and M 3 receptors were shown to be qualitatively and quantitatively the same in guanosine-5′- O -(3-[ 35 S]thio)triphosphate functional assays, in terms of both its affinity and cooperativity with ACh, as those found in binding assays. Neutral cooperativity of N -chloromethyl brucine with ACh on M 4 receptor function was 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 neurogenically released ACh binding at M 3 receptors was also detected in whole tissue as an increased contraction of the isolated guinea pig ileum to submaximal electrical stimulation. In conclusion, these functional studies confirm that brucine analogs are allosteric enhancers of ACh affinity at certain muscarinic receptor subtypes.