Studies on Receptor-mediated Activation of Adenylyl Cyclases

Abstract A series of β-blockers inhibit competitively the increase of adenylyl cyclase activity induced by catecholamines in cell-free membrane particles from kitten hearts. Apparent equilibrium dissociation constants for the following blockers, determined on membrane particles from ventricles of reserpine-treated kittens (5 mg per kg, 24 hours), are (expressed as -log molar concentrations): (-)-KL 255, 9.39; (+)-KL 255, 7.36; (-)-oxprenolol, 8.89; (+)-oxprenolol, 7.25; (±)-pindolol, 8.99; (±)-propranolol, 8.55; (±)-alprenolol, 8.38; (-)-dichloroisoproterenol, 7.77; (±)-Ko, 592, 7.59; (±)-pronethanol, 7.28; (±)-p-chloroisoproterenol, 6.71; (±)-INPEA, 6.05; (±)-practolol, 5.97; (±)-sotalol, 5.90; and racemic methoxamine, 5.06. Each of these values is within at least 0.5 order of magnitude of the corresponding values obtained in intact kitten atria studying blockade of inotropic or chronotropic effects of catecholamines. However, catecholamines are 2 to 3 orders of magnitude less potent in increasing adenylyl cyclase activity than in producing positive inotropic and chronotropic effects. The apparent Km of activation of the (+) isomer of isoproterenol was found to be 40 to 100 times greater than that of its (-) isomer. Similar isomeric affinity ratios were observed for the two β-blockers, KL 255 and oxprenolol. Both, (-)- and (+)-isoproterenol were antagonized by (±)-propranolol to the same extent, indicating that their stimulating effects of adenylyl cyclase are mediated by a common receptor. At concentrations below 10-4 m, (+)-isoproterenol did not inhibit the action of half-maximally stimulating concentrations of (-)-isoproterenol. Kinetic experiments suggest that the high affinity β-blockers (-)-KL 255 and (±)-propranolol dissociate slower from the adrenergic receptor-activating adenylyl cyclase than (+)-KL 255 and (±)-sotalol, which have lower affinity for this receptor. It was concluded that the adrenergic receptor mediating the action of catecholamines on heart adenylyl cyclase activity can be classified as a β-receptor having similar characteristics of stereoselectivity and similar affinities for β-blockers as those of the β-adrenergic receptors mediating positive inotropism and chronotropism in the intact tissue. The stereoselectivity of the β-receptor coupled to the adenylyl cyclase system for adrenergic ligands provides an essential tool for receptor isolation studies.