title: Decreased adenylyl cyclase protein and function in airway smooth muscle by chronic carbachol pretreatment.
abstract: 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)alpha or adenylyl cyclase. Detergent-solubilized G(s)alpha 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) (GTPgammaS) when reconstituted into S49 cyc(-) membranes that lack endogenous G(s)alpha (carbachol pretreated: GTPgammaS, 93 +/- 13% of control; NaF/AlCl(3), 99 +/- 8.6% of control; n = 4). Exogenous G(s)alpha 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.
authors.affiliation: Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA. cwe5@columbia.edu
authors.forename: C W
authors.initials: CW
authors.lastname: Emala
authors.forename: J
authors.initials: J
authors.lastname: Clancy-Keen
authors.forename: C A
authors.initials: CA
authors.lastname: Hirshman
mesh_terms: D000262:Adenylyl Cyclases / Q000032:analysis / Q000378:metabolism*; D000535:Aluminum / Q000494:pharmacology; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation / Q000187:drug effects; D004912:Erythrocytes / Q000737:chemistry; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D018721:Muscarinic Agonists / Q000494:pharmacology*; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009130:Muscle, Smooth / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology*; D011869:Radioligand Assay; D012969:Sodium Fluoride / Q000494:pharmacology; D013995:Time; D014132:Trachea / Q000201:enzymology*
chemical_list: D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D001285:Atropine; D002217:Carbachol; D012969:Sodium Fluoride; D000535:Aluminum; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases