Preparation of hormone-sensitive airway smooth muscle adenylate cyclase from dissociated canine trachealis cells.
Conventional homogenizing methods produced membrane preparations of canine trachealis airway smooth muscle which contained adenylate cyclase activity that was stimulated by fluoride but not by isoproterenol. We have devised methods using collagenase digestion of minced trachealis which destroy most of the tough connective tissues but leave dissociated canine trachealis cells in suspension. Gentle homogenization of these cells permitted preparation of a particulate fraction containing adenylate cyclase that was readily stimulated by beta-adrenergic agonist of prostaglandin E2. Isoproterenol stimulation was 2.34 +/- 0.58 (S.E.) times basal and 122 +/- 25% of the stimulation induced by NaF. The beta-adrenergic blocking agent propranolol prevented isoproterenol-induced stimulation of the cyclase but had no effect on prostaglandin E2 stimulation. Catecholamine order of potency was isoproterenol greater than epinephrine greater than norepinephrine. These methods enable demonstration of stimulatory effects of hormones in broken cell preparations of airway smooth muscle that are comparable to those when hormone-stimulated cyclic AMP formation is measured in intact muscle strips.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D015232:Dinoprostone; D004285:Dogs; D004789:Enzyme Activation; D004837:Epinephrine / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006728:Hormones / Q000494:pharmacology*; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology*; D009638:Norepinephrine / Q000494:pharmacology; D011433:Propranolol / Q000494:pharmacology; D011458:Prostaglandins E / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D014132:Trachea / Q000201:enzymology
D006728:Hormones; D011458:Prostaglandins E; D006160:Guanosine Triphosphate; D012969:Sodium Fluoride; D011433:Propranolol; D000262:Adenylyl Cyclases; D015232:Dinoprostone; D007545:Isoproterenol; D009638:Norepinephrine; D004837:Epinephrine