Decreased adenylyl cyclase protein and function in airway smooth muscle by chronic carbachol pretreatment

1 Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032; and 2 Department of Environmental Health Sciences, Division of Physiology, Johns Hopkins School of Public Health, Baltimore, Maryland 21205 Cellular levels of cAMP are an important determinant of airway smooth muscle tone. We have previously shown that chronic (18 h) but not acute (30 min or 2 h) pretreatment with the muscarinic receptor agonist carbachol resulted in decreased adenylyl cyclase activity in response to GTP, isoproterenol, or forskolin via a pathway blocked by the protein kinase C inhibitor staurosporine. The present study was designed to determine if carbachol-induced decreases in adenylyl cyclase activity were due to regulatory events at the level of either G s or adenylyl cyclase. Detergent-solubilized G s from control or carbachol-pretreated bovine airway smooth muscle had similar adenylyl cyclase activity in response to either NaF or guanosine 5'- O -(3-thiotriphosphate) (GTP S) when reconstituted into S49 cyc membranes that lack endogenous G s (carbachol pretreated: GTP S, 93 ± 13% of control; NaF/AlCl 3 , 99 ± 8.6% of control; n = 4). Exogenous G s solubilized from red blood cells failed to restore normal adenylyl cyclase activity when reconstituted into carbachol-pretreated bovine airway smooth muscle (carbachol pretreated: GTP, 36 ± 10% of control; NaF/AlCl 3 , 54 ± 11% of control; n = 4). [ 3 H]forskolin radioligand saturation binding assays revealed a decreased quantity of total adenylyl cyclase protein after carbachol pretreatment (maximal binding: 152 ± 40 and 107 ± 31 fmol/mg protein in control and carbachol-pretreated airway smooth muscle, respectively). These results suggest that chronic activation of muscarinic receptors downregulates the expression of adenylyl cyclase protein in bovine airway smooth muscle. muscarinic; [ 3 H]forskolin; reconstitution; S49 cyc ; bovine