M 2 Muscarinic Receptors Inhibit Forskolin- but not Isoproterenol-Mediated Relaxation in Bovine Tracheal Smooth Muscle 1 RENNOLDS S. OSTROM and FREDERICK J. EHLERT Department of Pharmacology, College of Medicine, University of California, Irvine, Irvine, California Accepted for publication March 16, 1998 This paper is available online at http://www.jpet.org ABSTRACT The ability of the M 2 muscarinic receptor to inhibit the relaxant effects of forskolin and isoproterenol was investigated in bovine trachea. In most experiments, we measured contractile responses to oxotremorine-M in smooth muscle isolated from bovine trachea in which a majority of M 3 receptors were inactivated by treatment with N-(2-chloroethyl)-4-piperidinyl diphenylacetate. In the pres- ence of histamine (20 M), the histamine H 2 antagonist cimetidine (10 M) and forskolin (4 M), responses to oxotremorine-M were antagonized by [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]- 5,11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one (1 M) in a manner consistent with contractions mediated predominantly by M 2 receptors. When similar experiments were conducted in the presence of isoproterenol (0.1 M) instead of forskolin, contrac- tions were antagonized in a manner consistent with an M 3 recep- tor-mediated response. In similar experiments, we measured the relaxant potency of isoproterenol and forskolin against histamine- induced contractions in N-(2-chloroethyl)-4-piperidinyl diphenyla- cetate-treated trachea. By itself, oxotremorine-M (7.5 nM) had no contractile effect; however, it caused a substantial reduction in the relaxant potency of forskolin although having little effect on that of isoproterenol. These experiments establish that M 2 receptors in- hibit the relaxant effects of forskolin, but not isoproterenol. In untreated tissues, the relaxant responses to isoproterenol and forskolin were 10.8- and 14.2-fold more potent, respectively, against histamine than against oxotremorine-M-induced contrac- tions of equal magnitude. Similarly, the maximal stimulation of cAMP accumulation elicited by isoproterenol and forskolin was inhibited 58 and 62%, respectively, in the presence of oxotremo- rine-M (80 nM) compared to that measured in the presence of histamine (20 M). Analysis of the data indicated that isoprotere- nol elicited relaxation at concentrations well beyond those that stimulated maximal levels of cAMP accumulation. Our results indicate that part of the relaxant response to isoproterenol is mediated through a non-cAMP-dependent mechanism, and that this mechanism is largely unopposed by the M 2 receptor. Muscarinic receptor populations in mammalian smooth muscle of the gastrointestinal tract and the airways are known to consist of approximately 80% M 2 and 20% M 3 receptors (Candell et al., 1990; Gies et al., 1989; Haddad et al., 1991; Maeda et al., 1988). M 3 receptors mediate direct contractions of smooth muscle and couple to the G q family of G proteins causing activation of phospholipase C- (Candell et al., 1990; Roffel et al., 1990; Yang et al., 1991). The more predominant M 2 receptor is known to mediate inhibition of adenylyl cyclase through the pertussis toxin-sensitive G pro- tein, G i , in airway smooth muscle and in cells transfected with the M 2 subtype (Kurose et al., 1983; Sankary et al., 1988). Conversely, beta adrenergic agonists stimulate adeny- lyl cyclase through the cholera toxin-sensitive G protein, G s and induce relaxation of smooth muscle via cAMP-dependent activation of protein kinase A. M 2 receptors have been shown to mediate contractions by inhibiting the relaxant effects of isoproterenol or the direct adenylyl cyclase activator, forsko- lin, in smooth muscle of the gastrointestinal tract (Reddy et al., 1995; Thomas et al., 1993). These studies used treatment of tissues with 4-DAMP mustard to inactivate a majority of the M 3 receptors selectively. Under this condition, responses elicited by a muscarinic agonist in the presence of histamine and either isoproterenol or forskolin were attributed to M 2 receptors through the use of selective antagonists. The con- tractile mechanism involved an M 2 receptor-mediated inhi- bition of the relaxant effect of isoproterenol or forskolin on histamine-induced contractions. Furthermore, the ability of muscarinic agonists to inhibit the relaxant effects of isopro- terenol or forskolin has been correlated with M 2 receptor- mediated inhibition of stimulated cAMP accumulation (Os- trom and Ehlert, 1997). Received for publication October 29, 1997. 1 This work was supported by National Institutes of Health Grant NS 30882. ABBREVIATIONS: cAMP, adenosine 3',5' cyclic monophosphate; AF-DX 116, [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3b][1,4]benzodiazepine-6-one; 4-DAMP mustard, N-(2-chloroethyl)-4-piperidinyl diphenylacetate; IBMX, isobutylmethylxanthine; KRB buffer, Krebs ringer bicarbonate buffer. 0022-3565/98/2861-0234$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 286, No. 1 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 286:234 –242, 1998 234 at ASPET Journals on June 9, 2015 jpet.aspetjournals.org Downloaded from