John A. Tran1, Alex Chang1, Minoru Matsui2 and Frederick J. Ehlert1 A Simple Method for Estimation of Agonist Activity at GPCRs: Characterization of Responses Elicited by M2 and M3 Muscarinic Receptors 724.10 John A. Tran1, Alex Chang1, Minoru Matsui2 and Frederick J. Ehlert1 1Department of Pharmacology, School of Medicine, University of California, Irvine, Irvine, California 92697-4625 2Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire Figure 5 Figure 3 M2/M3KO ileum M2KO ileum Figure 1 Introduction Agonist activity is usually characterized by estimation of the EC50 and Emax values for eliciting a particular response. It well known that these parameters can vary for the same agonist receptor complex, depending upon the response being measured. A more invariant approach would be to estimate the observed affinity and relative efficacy of the agonist using the method of partial receptor inactivation (Furchgott, 1966; Furchgott and Bursztyn, 1967). However this technique is more tedious and rarely used. Recently, we developed a method for estimating the product of affinity and intrinsic efficacy of an agonist expressed relative to that of another agonist, simply through the analysis of the agonist concentration-response curve (Ehlert et al., 1996; Ehlert et al., 1999; Ehlert and Griffin, 2001; Griffin et al., 2007). This parameter is termed the intrinsic relative activity (RAi), and it is mainly dependent on the agonist-receptor-G protein signaling pathway and independent of other system parameters. Consequently, the RAi estimate is useful for comparing agonist activity across a variety of systems and for quantifying ligand directed signaling. In the present the present report, we have estimated the RAi values of muscarinic agonists for eliciting M2 and M3 muscarinic responses, and compared these estimates with those calculated from the product of observed affinity and intrinsic efficacy estimated by Furchgott’s method of partial receptor inactivation. We found good agreement between the two estimates, which illustrates that our simple RAi estimate is an accurate measure of the product of affinity and efficacy of the agonist. We also found evidence that part of the contractile response to the novel muscarinic agonist McN-A-343 in the mouse ileum may be mediated through the M1 muscarinic receptor. Methods Furchgott Analysis: Equiactive concentrations of agonist before (X) and after (X’) partial receptor alkylation by 4-DAMP mustard were estimated, and the following equation was fitted to the data by nonlinear regression analysis to estimate the observed dissociation constant (Kd) and proportion of residual receptors (q): 1 Knowing the Kd value of the agonist, it is possible to plot response against receptor occupancy (Fig 1d, 2d). The relative efficacy of the agonist was estimated from this plot using the following equation: % occupancy 2 The relative intrinsic efficacy estimation is made by comparing the level of occupancy of the various agonists need to elicit the same amount of response measured as compared to the standard agonist, carbachol. The product of affinity and efficacy of the agonist of interest relative to the standard can then be made in the following manner: 3 RAi analysis: The only requirements necessary for the RAi estimate are the concentration-curves of the test and standard agonists. There are two methods to calculate the RAi value based on (1) a null equation and (2) the operational model (Griffin et al., 2007). The null method involves a fitting the following null equation to equiactive concentrations of the test and standard agonist: 4 5 Bi and Ai denote the ith concentrations of the test agonist (B) and standard agonist (A), respectively. KA denotes the dissociation constant of the standard agonist, and p and q represent the ratios of KB/KA and eB/eA. The operational model was used to estimate RAi by fitting the concentration-response curves of the agonists simultaneously to following equation: 6 7 The agonist response is denoted by R, Xj denotes the concentration of agonist A or B, Msys denotes the maximal response of the system, and m denotes the transducer slope factor. Non-linear regression analysis yields estimates for tj/Kj for each agonist (tA/KA, tB/KB). When the Emax of the test agonist B is less than that of A, it is also possible to estimate KB. M2 muscarinic receptor-mediated inhibition of forskolin-stimulated cAMP in CHO cells. a: Concentration-response curves of selected muscarinic agonists for inhibition of forskolin-stimulated cAMP accumulation in CHO M2 cells. The data represent the mean values ± SEM of 3 to 8 experiments. b: Examples of the effect of 4-DAMP mustard, an irreversible antagonist, on carbachol- and oxotremorine-M-mediated inhibition of forskolin-stimulated cAMP accumulation. c: Relationship between equiactive agonist concentrations before and after 4-DAMP mustard treatment. d: The normalized response of selected agonists is plotted against receptor occupancy. The estimate of RAi from agonist concentration-response curves yields a parameter equivalent to the product of affinity and efficacy. a: Comparison between the product of affinity and efficacy and the RAi values estimated from the cAMP response in CHO-M2 cells. b: Comparison between the product of affinity and efficacy with RAi values estimated from the contraction response in the M2 KO ileum as well as RAi values for the phosphoinositide response in CHO-M3 cells previously reported by Ehlert et al., 1999. Effect of tetrodotoxin (TTX) on contraction in the M2/M3 KO (a) and M2KO (b, c) ileum by McN-A-343 (a, c) and carbachol (b). Mean values ± SEM of 3 to 9 experiments are shown. Figure 2 Figure 4 Conclusions The RAi value estimated from the agonist concentration-response curve for M2 and M3 muscarinic responses is equivalent to the product of affinity and efficacy estimated by the method of partial inactivation. The RAi estimate is mainly a function of the agonist-receptor-G protein interaction and is therefore independent of downstream elements in the signaling cascade. Consequently, the RAi estimate is useful for comparing agonist activity across different systems and for detecting ligand-directed signaling. RAi analysis can be used to identify novel agonist behavior, such as the behavior of McN-A-343 in gastrointestinal smooth muscle, which is generally assumed to be an assay for M3 function. The anomalously high RAi estimate of McN-A-343 in mouse ileum suggests a role for the M1 receptor in this response. M3 muscarinic receptor-stimulated contractions in the ileum from M2 muscarinic receptor knockout (M2 KO) mice. a: Concentration-response curves of selected muscarinic agonists for eliciting contraction in the ileum from M2 KO mice are shown. The data represent the mean values ± SEM of 6 to 13 experiments. b: Examples of the effect of 4-DAMP mustard on carbachol- and S-aceclidine-stimulated contractions. Mean values ± SEM from 5 - 8 experiments are shown. c: Relationship between equiactive agonist concentrations before and after 4-DAMP mustard treatment. d: The normalized response of selected agonists is plotted against receptor occupancy. Effect of pirenzepine (a, b) and atropine (c, d) on the contractile action of carbachol (a, c) and McN-A-343 (b, d) in the isolated ileum from the M2KO mouse. Mean values ± SEM from 3 experiments are shown. Ehlert FJ and Griffin MT (2001). Annalytical Pharmacology 2:34-47. Ehlert FJ, Griffin MT and Glidden PF (1996). J Pharmacol Exp Ther 279:1335-1344. Ehlert FJ, Griffin MT, Sawyer GW and Bailon R (1999). J Pharmacol Exp Ther 289:981-992. Furchgott RF (1966). Adv. Drug Res. 3:21-55. Furchgott RF and Bursztyn P (1967). Ann. New York Acad. Sci. 144:882-899. Griffin MT, Figueroa KW, Liller S and Ehlert FJ (2007). J Pharmacol Exp Ther 321:1193-1207.