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Estimation of Relative Microscopic Affinity Constants of Agonists for the Active State of the Receptor in Functional Studies on M₂ and M₃ Muscarinic Receptors

Department of Pharmacology, College of Medicine, University of California, Irvine, California (J.A.T., A.C., F.J.E.); and Department of Pharmacy, Chiba Institute of Science, Chiba, Japan (M.M.)

In prior work, we have shown that it is possible to estimate the product of observed affinity and intrinsic efficacy of an agonist expressed relative to that of a standard agonist simply through the analysis of their respective concentration-response curves. In this report, we show analytically and through mathematical modeling that this product, termed intrinsic relative activity (RA_i), is equivalent to the ratio of microscopic affinity constants of the agonists for the active state of the receptor. We also compared the RA_i estimates of selected muscarinic agonists with a relative estimate of the product of observed affinity and intrinsic efficacy determined independently through the method of partial receptor inactivation. There was good agreement between these two estimates when agonist-mediated inhibition of forskolin-stimulated cAMP accumulation was measured in Chinese hamster ovary cells stably expressing the human M₂ muscarinic receptor. Likewise, there was good agreement between the two estimates when agonist activity was measured on the ileum from M₂ muscarinic receptor knockout mice, a convenient assay for M₃ receptor activity. The RA_i estimates of agonists in the mouse ileum were similar to those estimated at the human M₃ receptor with the exception of 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium (McN-A-343), which is known to be an M₁- and M₄-selective muscarinic agonist. Additional experiments showed that the response to McN-A-343 in the mouse ileum included a non-M₃ muscarinic receptor component. Our results show that the RA_i estimate is a useful receptor-dependent measure of agonist activity and ligand-directed signaling.

Received for publication August 11, 2008.

Accepted for publication November 6, 2008.

Address correspondence to: Dr. Frederick J. Ehlert, Department of Pharmacology, University of California, Irvine, Irvine, California 92698-4625. E-mail: fjehlert{at}uci.edu