Adenylate cyclase and catecholamine binding in plasma membrane-enriched preparations of cardiac and skeletal muscle

Binding of [3H] epinephrine to plasma membrane-enriched fractions from guinea pig heart and rabbit skeletal muscle was investigated using the micropore filtration technique. [3H] Epinephrine and [3H] norepinephrine were found to be degraded rapidly in aqueous buffer at pH 7.6 and 37 degrees C. Deterioration of the compounds could be prevented by low concentrations of dithiothreitol. Binding of [3H] epinephrine to both membrane preparations was a slow process requiring 60 min to approach equilibrium in the case of cardiac membranes at 37 degrees C, and 20 min for skeletal muscle membranes at 0 degrees C. Binding was antagonized by the unlabeled beta agonists, isopropylnorepinephrine, epinephrine, and nor epinephrine; each was equipotent, in contrast to their different potency is eliciting beta-adrenergic responses and in stimulating adenylate cyclase. A variety of catechol compounds were as effective as antagonists of binding as the catecholamines. Methylation of one ring hydroxyl abolished inhibitory activity. The beta-adrenergic antagonists, propranolol, pronethalol, and dichlorisoproterenol, were not effective in inhibiting binding to either membrane preparation. D-Norepinephrine and L-norepinephrine were equieffective in antagonizing binding of [3H] norepinephrine to skeletal muscle membranes, suggesting that binding was not stereospecific. These and other data led to the conclusion that binding of labeled catecholamine to isolated tissue membranes using the micropore filtration technique probably does not reflect interaction with the specific beta-adrenergic recptor, but more likely reflects a less specific binding of compounds with one or more hydroxyl groups on a ring.