M 2 Muscarinic Receptors Inhibit Forskolin-but not Isoproterenol-Mediated Relaxation in Bovine Tracheal Smooth Muscle 1

The ability of the M2 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 M3 receptors were inactivated by treatment with N-(2-chloroethyl)-4-piperidinyl diphenylacetate. In the presence of histamine (20 mM), the histamine H2 antagonist cimetidine (10 mM) and forskolin (4 mM), 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 mM) in a manner consistent with contractions mediated predominantly by M2 receptors. When similar experiments were conducted in the presence of isoproterenol (0.1 mM) instead of forskolin, contractions were antagonized in a manner consistent with an M3 receptor-mediated response. In similar experiments, we measured the relaxant potency of isoproterenol and forskolin against histamineinduced contractions in N-(2-chloroethyl)-4-piperidinyl diphenylacetate-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 M2 receptors inhibit the relaxant effects of forskolin, but not isoproterenol. In untreated tissues, the relaxant responses to isoproterenol and forskolin were 10.8and 14.2-fold more potent, respectively, against histamine than against oxotremorine-M-induced contractions 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 oxotremorine-M (80 nM) compared to that measured in the presence of histamine (20 mM). Analysis of the data indicated that isoproterenol 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 M2 receptor. Muscarinic receptor populations in mammalian smooth muscle of the gastrointestinal tract and the airways are known to consist of approximately 80% M2 and 20% M3 receptors (Candell et al., 1990; Gies et al., 1989; Haddad et al., 1991; Maeda et al., 1988). M3 receptors mediate direct contractions of smooth muscle and couple to the Gq family of G proteins causing activation of phospholipase C-b (Candell et al., 1990; Roffel et al., 1990; Yang et al., 1991). The more predominant M2 receptor is known to mediate inhibition of adenylyl cyclase through the pertussis toxin-sensitive G protein, Gi, in airway smooth muscle and in cells transfected with the M2 subtype (Kurose et al., 1983; Sankary et al., 1988). Conversely, beta adrenergic agonists stimulate adenylyl cyclase through the cholera toxin-sensitive G protein, Gs and induce relaxation of smooth muscle via cAMP-dependent activation of protein kinase A. M2 receptors have been shown to mediate contractions by inhibiting the relaxant effects of isoproterenol or the direct adenylyl cyclase activator, forskolin, 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 M3 receptors selectively. Under this condition, responses elicited by a muscarinic agonist in the presence of histamine and either isoproterenol or forskolin were attributed to M2 receptors through the use of selective antagonists. The contractile mechanism involved an M2 receptor-mediated inhibition of the relaxant effect of isoproterenol or forskolin on histamine-induced contractions. Furthermore, the ability of muscarinic agonists to inhibit the relaxant effects of isoproterenol or forskolin has been correlated with M2 receptormediated inhibition of stimulated cAMP accumulation (Ostrom 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 39, 59 cyclic monophosphate; AF-DX 116, [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6Hpyrido[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 A PE T Jornals on M ay 7, 2017 jpet.asjournals. org D ow nladed from The contractile role of the M2 receptor is less clear in airway smooth muscle. Using the method of inactivating M3 receptors with 4-DAMP mustard, Thomas and coworkers showed that the M2 receptor inhibited the relaxant effect of forskolin on histamine-induced contractions of the guinea pig trachea (Thomas and Ehlert, 1996). However, using a similar strategy, Watson et. al. (1995) were unable to detect a role for the M2 receptor when isoproterenol was used as the relaxant agent. One reason for this discrepancy may lie in the ability of forskolin to stimulate more cAMP than isoproterenol. Unfortunately, cAMP accumulation is difficult to measure in the small amount of smooth muscle of the guinea pig trachea, so this hypothesis remains untested. However, in equine trachea it was recently demonstrated that isoproterenol stimulated far less cAMP at equivalent levels of relaxation than did forskolin (Kume et al., 1994). These investigators also demonstrated that beta adrenergic receptors stimulate calcium-activated potassium channels through direct coupling to Gsa. The M2 receptor, however, appears to be capable of inhibiting this same channel directly via Gia irrespective of any effect on cAMP (Kume and Kotlikoff, 1991). Our study was undertaken to investigate the contractile role of the M2 receptor in bovine tracheal smooth muscle. The large size of the bovine trachea enabled us to measure both contraction and cAMP accumulation in a relatively homogeneous preparation of smooth muscle cells. We show that activation of M2 receptors inhibits the increase in cAMP elicited by both forskolin and isoproterenol. However, activation of M2 receptors only inhibited the relaxant effects of forskolin, not isoproterenol, on histamine-induced contraction. These results suggest that isoproterenol mediates at least part of its relaxant effects through a non-cAMP-dependent mechanism resistant to functional antagonism by the M2 receptor.