Molecular Pharmacology, Vol 12, 409-419, Copyright © 1976 by the American Society for Pharmacology and Experimental Therapeutics

Regulation of Adenylate Cyclase-Coupled Beta Adrenergic Receptor Binding Sites by Beta Adrenergic Catecholamines in Vitro

¹ Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Tolerance to catecholamines was studied using a frog erythrocyte model system in vitro. Incubation of cells with (-)-isoproterenol resulted in up to a 58% decline (p < 0.001) in maximum catecholamine-stimulated adenylate cyclase activity in membrane preparations. Desensitization was characterized by a decrease in V_max with no change in hormone affinity. Enzyme activity in the unstimulated state and after stimulation with NaF and prostaglandin E₁ (PGE₁) was unaltered. The number of functional beta adrenergic receptors, measured by binding of the beta adrenergic antagonist (-)-[³H]dihydroalprenolol, showed a parallel decline of up to 47% (p < 0.001) with no change in receptor affinity. The dose-response curve for desensitization of enzyme activity and decreased receptor binding was the same as for (-)-isoproterenol stimulation of adenylate cyclase activity. The time course for the desensitization of adenylate cyclase and decrease in receptor number was much longer than that required for receptor binding or adenosine cyclic 3',5'-monophosphate (cAMP) production, requiring 2-3 hr for completion. The ability of beta adrenergic agonists to cause a decrease in enzyme activity and in receptor number was directly related to their affinity for the beta adrenergic receptor. The potency series was isoproterenol > epinephrine > norepinephrine. The beta antagonist propranolol, while having no effect of its own, blocked the desensitization caused by isoproterenol. In contrast, the alpha adrenergic antagonist phentolamine did not interfere with the phenomenon. The desensitized state was shown to be reversible. If isoproterenol was removed from desensitized cells by washing, and cells were further incubated with propranolol, receptor binding and enzyme activity returned to nearly normal levels. Incubation of cells with PGE₁ produced desensitization to subsequent prostaglandin stimulation but had no effect on catecholamine sensitivity or beta receptor binding. Dibutyryl cAMP did not cause a decrease in binding or enzyme activity. It appears that catecholamines, through beta adrenergic receptor interactions, play a role in regulation of the number of functioning receptors at the cell surface. This may be a major mechanism for the induction of catecholamine tolerance.