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 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.