Estimation of relative microscopic affinity constants of agonists for the active state of the receptor in functional studies on M2 and M3 muscarinic receptors.

ABSTRACT

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 M(2) muscarinic receptor. Likewise, there was good agreement between the two estimates when agonist activity was measured on the ileum from M(2) muscarinic receptor knockout mice, a convenient assay for M(3) receptor activity. The RA(i) estimates of agonists in the mouse ileum were similar to those estimated at the human M(3) receptor with the exception of 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium (McN-A-343), which is known to be an M(1)- and M(4)-selective muscarinic agonist. Additional experiments showed that the response to McN-A-343 in the mouse ileum included a non-M(3) muscarinic receptor component. Our results show that the RA(i) estimate is a useful receptor-dependent measure of agonist activity and ligand-directed signaling.