Muscarinic regulation of cyclic AMP in bovine trachealis cells

The goal of this study was to characterize the receptors and coupling mechanisms mediating muscarinic inhibition of adenylyl cyclase activity in bovine tracheal smooth muscle. In radioligand binding experiments, methoctramine and AF-DX 116 competed for approximately 85% of the 3H-quinuclidynyl benzilate (3H-QNB) binding sites on intact cells with high affinities (-log KI of 7.73 +/- 0.16 and 6.67 +/- 0.31, respectively) characteristic of binding to M2 receptors. The antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) bound the receptors on intact cells with an affinity (-log KI = 7.76 +/- 0.21) characteristic of binding at M2 receptors. In experiments measuring 3',5'-cyclic adenosine monophosphate (cAMP) accumulation, methoctramine, AF-DX 116, and 4-DAMP antagonized the inhibitory effect of carbachol on isoproterenol-stimulated cAMP accumulation with potencies consistent with mediation by M2 muscarinic receptors (-log Kb of 8.01 +/- 0.22 to 7.58 +/- 0.25 for methoctramine; 7.43 +/- 0.36 to 7.02 +/- 0.30 for AF-DX 116; and 7.60 +/- 0.21 for 4-DAMP). In other experiments, 24 +/- 3% of the inhibitory effect of carbachol was not reversed by 60 min exposure to atropine. Moreover, pertussis toxin (10, 250, and 1,000 ng/ml) decreased only a portion of the inhibitory effect of carbachol (8 +/- 19%, 32 +/- 10%, and 33 +/- 8%, respectively) on cAMP accumulation. These findings indicated that M2 receptors were coupled to adenylyl cyclase in trachealis cells, but that coupling mechanisms in addition to those of pertussis toxin-sensitive guanine nucleotide binding proteins were involved. Since the inhibitory effect of carbachol (10(-8) M) on isoproterenol-stimulated cAMP accumulation was decreased from 20 +/- 4% to -1 +/- 5% (n = 6) by okadaic acid (1 microM), protein phosphatases may regulate the processes coupling muscarinic receptors to adenylyl cyclase.