Selectivity of Agonists for the Active State of M-1 to M-4 Muscarinic Receptor Subtypes

We measured the intrinsic relative activity (RA(i)) of muscarinic agonists to detect possible selectivity for receptor subtypes and signaling pathways. RA(i) is a relative measure of the microscopic affinity constant of an agonist for the active state of a GPCR expressed relative to that of a standard agonist. First, we estimated RA(i) values for a panel of agonists acting at the M-4 muscarinic receptor coupled to three distinct G-protein pathways: G(i) inhibition of cAMP accumulation, G(s) stimulation of cAMP accumulation, and G alpha(15) stimulation of phosphoinositide hydrolysis. Our results show similar RA(i) values for each agonist, suggesting that the same active state of the M 4 receptor triggers the activation of the three G proteins. We also estimated RA(i) values for agonists across M-1 to M-4 muscarinic subtypes stably transfected in Chinese hamster ovary cells. Our results show selectivity of McN-A-343 [4-I-[3-chlorophenyl]carbamoyloxy)-2- butynyltrimethylammnonium chloride] for the M-1 and M-4 subtypes and selectivity of pilocarpine for the M-1 and M-3 subtypes. The other agonists tested lacked marked selectivity among M-1 to M-4 receptors. Finally, we estimated RA(i) values from published literature on M-1, M-2, and M-3 muscarinic responses and obtained results consistent with our own studies. Our results show that the RA(i) estimate is a useful receptor-dependent measure of agonist activity.