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 (RAi), is equivalent to the ratio of microscopic affinity constants of the agonists for the active state of the receptor. We also compared the RAi 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 RAi 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 RAi estimate is a useful receptor-dependent measure of agonist activity and ligand-directed signaling.
Footnotes
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This work was supported by the National Institutes of Health [Grant GM 69829].
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ABBREVIATIONS: GPCR, G protein-coupled receptor; RAi, intrinsic relative activity; 4-DAMP mustard, N-(2-chloroethyl)-4-piperidinyldiphenyl acetate; CHO, Chinese hamster ovary; McN-A-343, 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium; KO, knockout.
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↵
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received August 11, 2008.
- Accepted November 6, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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