Binding and functional affinities of the muscarinic acetylcholine (mACh) receptor antagonists darifenacin, tolterodine, oxybutynin, and atropine were assessed in Chinese hamster ovary (CHO) cells expressing the human recombinant M 2 (CHO-m2) or M 3 (CHO-m3) receptors, and in guinea pig bladder and submandibular gland. In [N-methyl-3 H]scopolamine methyl chloride binding studies in CHO cells, darifenacin displayed selectivity (14.8-fold) for the M 3 versus M 2 mACh receptor subtype. Oxybutynin was nonselective, whereas atropine and tolterodine were weakly M 2 -selective (5.1-and 6.6-fold, respectively). Antagonist functional affinity estimates were determined by the inhibition of agonist-induced [ 3 H]inositol phosphate accumulation in CHO-m3 cells and antagonism of the agonist-induced inhibition of forskolin-stimulated cyclic AMP accumulation in CHO-m2 cells. Darifenacin was the most M 3 -selective antagonist (32.4-fold), whereas oxybutynin, atropine, and tolterodine exhibited lesser selectivity. Functional affinity estimates in guinea pig urinary bladder and submandibular salivary gland using indices of phosphoinositide turnover revealed that oxybutynin, darifenacin, and tolterodine each displayed selectivity for the response in the bladder, relative to that seen in the submandibular gland (9.3-, 7.9-, and 7.4-fold, respectively). In contrast, atropine displayed a similar affinity in both tissues. These data demonstrate that in bladder, compared with submandibular gland from a single species, the mACh receptor antagonists darifenacin, tolterodine, and oxybutynin display selectivity to inhibit agonist-mediated phosphoinositide responses. It is proposed that both responses are mediated via M 3 mACh receptor activation and that differential functional affinities displayed by some, but not all, antagonists are indicative of the influence of cell background upon the pharmacology of the M 3 mACh receptor.The cholinergic nervous system is the major pathway by which bladder contraction is initiated in humans (Andersson, 1993). Molecular techniques have identified five muscarinic acetylcholine (mACh) receptor subtypes (m1-m5), whereas pharmacological data can distinguish only four subtypes, denoted as M 1 to M 4 (Caulfield and Birdsall, 1998). Both M 2 and M 3 mACh receptors have been identified in bladder (detrusor) smooth muscle at the level of mRNA (Yamaguchi et al., 1996) and protein, with quantitative immunoprecipitation demonstrating a 75 to 90% predominance of the M 2 subtype in all species studied (Wang et al., 1995). Roles for the majority M 2 receptor population in the contraction of various smooth muscle types (including bladder detrusor) have been proposed, based upon observations in tissues treated with N-2-chloroethyl-4-piperidinyl diphenylacetate selectively to inactivate the M 3 receptor population. Under these conditions M 2 receptor activation may potentiate M 3 -stimulated contraction (via activation of nonselective cation currents or inhibition of Ca 2ϩ -dependent K ϩ efflux), in addition to ...
