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

Cellular levels of cAMP are an important determinant of airway smooth muscl e tone. We have previously shown that chronic (18 h) but not acute (30 min or 2 h) pretreatment with the muscarinic receptor agonist carbachol resulte d in decreased adenylyl cyclase activity in response to GTP, isoproterenol, or forskolin via a pathway blocked by the protein kinase C inhibitor staur osporine. 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)alpha or adenylyl cyclase. Detergent-solubilized G(s)a lpha from control or carbachol-pretreated bovine airway smooth muscle had s imilar adenylyl cyclase activity in response to either NaF or guanosine 5'- O-(3-thiotriphosphate) (GTP gamma S) when reconstituted into S49 cyc(-) mem branes that lack endogenous G(s)alpha (carbachol pretreated: GTP gamma S, 9 3 +/- 13% of control; NaF/AlCl3, 99 +/- 8.6% of control; n = 4). Exogenous G(s)alpha solubilized from red blood cells failed to restore normal adenyly l cyclase activity when reconstituted into carbachol-pretreated bovine airw ay smooth muscle (carbachol pretreated: GTP, 36 +/- 10% of control; NaF/AlC l3, 54 +/- 11% of control; n = 4). [H-3]forskolin radioligand saturation bi nding assays revealed a decreased quantity of total adenylyl cyclase protei n after carbachol pretreatment (maximal binding: 152 +/- 40 and 107 +/- 31 fmol/mg protein in control and carbachol-pretreated airway smooth muscle, r espectively). These results suggest that chronic activation of muscarinic r eceptors downregulates the expression of adenylyl cyclase protein in bovine airway smooth muscle.