PT  - JOURNAL ARTICLE
AU  - Nigel J. M. Birdsall
AU  - Tim Farries
AU  - Parviz Gharagozloo
AU  - Shinsaku Kobayashi
AU  - Sebastian Lazareno
AU  - Masahiko Sugimoto
TI  - Subtype-Selective Positive Cooperative Interactions Between Brucine Analogs and Acetylcholine at Muscarinic Receptors: Functional Studies
DP  - 1999 Apr 01
TA  - Molecular Pharmacology
PG  - 778--786
VI  - 55
IP  - 4
4099  - http://molpharm.aspetjournals.org/content/55/4/778.short
4100  - http://molpharm.aspetjournals.org/content/55/4/778.full
SO  - Mol Pharmacol1999 Apr 01; 55
AB  - In radioligand binding studies, it has been reported that brucine,N-chloromethyl brucine, and brucineN-oxide increased the affinity of acetylcholine for M1, M3, and M4 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 M1 and M4receptors respectively have been confirmed in guanosine-5′-O-(3-[35S]thio)triphosphate, GTPase, cAMP, and intracellular Ca2+ 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 M2 and M3 receptors were shown to be qualitatively and quantitatively the same in guanosine-5′-O-(3-[35S]thio)triphosphate functional assays, in terms of both its affinity and cooperativity with ACh, as those found in binding assays. Neutral cooperativity ofN-chloromethyl brucine with ACh on M4receptor function was also observed, thereby demonstrating its “absolute subtype selectivity”: a lack of action at any concentration at M4 receptors and an action at M2 and M3 receptors. The enhancing action ofN-chloromethyl brucine on neurogenically released ACh binding at M3 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. The American Society for Pharmacology and Experimental Therapeutics