Molecular Pharmacology, Vol 13, 304-313, Copyright © 1977 by the American Society for Pharmacology and Experimental Therapeutics

Molecular Pharmacology of Alpha Adrenergic Receptors: Utilization of [³H]Dihydroergocryptine Binding in the Study of Pharmacological Receptor Alterations

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

The potent alpha adrenergic antagonist [³H]dihydroergocryptine has been shown to bind reversibly to alpha adrenergic receptors in uterine smooth muscle membranes. [³H]Dihydroergocryptine binding is antagonized by phentolamine and phenoxybenzamine. Inhibition of [³H]dihydroergocryptine binding by phentolamine, a reversible inhibitor of adrenergic smooth muscle contraction, was reversible and resulted in a reduction in the apparent affinity of alpha adrenergic receptors with no change in the number of available receptor binding sites. By contrast, phenoxybenzamine, an irreversible inactivator of adrenergic smooth muscle contraction, irreversibly inactivated alpha adrenergic receptor binding sites as assessed by [³H]dihydroergocryptine binding. Irreversible receptor inactivation by phenoxybenzamine occurred rapidly (t_1/2 1 min), even at low drug concentrations (0.1 µM). Exposure of membranes to 0.1 µM phenoxybenzamine resulted in a 50% reduction in the number of receptor sites, with little or no change in the apparent affinity of the unreacted binding sites for [³H]dihydroergocryptine. This characteristic nonequilibrium blockade of alpha adrenergic receptors contrasts with the competitive blockade produced by phentolamine and ergotamine. The specific sulfhydryl inhibitor p-hydroxymercuribenzoic acid inhibited [³H]dihydroergocryptine binding, causing half-maximal inhibition at a concentration of 0.3 mM. This inhibition of binding was reversed or prevented by 2 mM dithiothreitol, a sulfhydryl-containing compound. Thus the alpha adrenergic receptor binding site appears to possess an essential sulfhydryl group. The presence or absence of calcium did not alter [³H]dihydroergocryptine binding and did not alter the affinity of epinephrine for the binding sites. These results indicate that, unlike competitive antagonists, haloalkylamine alpha adrenergic antagonists act directly to inactivate the alpha adrenergic receptor binding sites irreversibly by covalent bond formation. The mechanism of this inactivation may involve the alkylation of an essential sulfhydryl group at the alpha adrenergic binding site. In contrast, the effect of calcium ion on alpha adrenergic responses is not at the receptor binding site but at some distal step.