Identification of cu-Adrenergic Receptors in Uterine Smooth Muscle Membranes by [3H]Dihydroergocryptine Binding*

[3H]Dihydroergocryptine, a potent a-adrenergic antagonist, was used to label smooth muscle membrane binding sites which have the characteristics expected of a-adrenergic receptors. Binding of [3H]dihydroergocryptine to rabbit uterine membranes was rapid and reversible with rate constants of 1.26 x lo1 M-I min ~’ and 0.034 min’ for the forward and reverse reactions, respectively. [3H]Dihydroergocryptine binding was of high affinity, with an equilibrium dissociation constant (K,) of 8 to 10 nM. Binding was saturable with 0.14 to 0.17 pmol of [3H]dihydroergocryptine bound/mg of protein at maximal occupancy of the sites. No cooperative interactions among the sites were detected. The specificity of the binding sites for a large number of adrenergic agonists and antagonists was identical with the specificity of cu-adrenergic responses to these agents. The cr-adrenergic agonist (-)-epinephrine competed for binding with a K,, of 0.23 PM. The order of potencies for several adrenergic agonists in competing for the binding sites was (~ )-epinephrine > (-)-norepinephrine > (-)-phenylephrine > (-)-isoproterenol in agreement with their cu-adrenergic potencies. A series of 19 phenylethylamine adrenergic agonists competed for binding in a manner paralleling their potencies as a-adrenergic agonists. a-Adrenergic antagonists such as phentolamine (K a = 15 nM) and phenoxybenzamine (Ka = 18 nM) potently competed for the binding sites. In contrast, P-adrenergic antagonists such as propranolol (K, = 27,000 nM) and practolol (K, > lo6 nM) did not have high affinity for the binding sites. A series of ergot alkaloids competed for [3H]dihydroergocryptine binding in a manner which paralleled their potencies as cu-adrenergic agents. Competition for binding sites by cu-adrenergic agonists and antagonists was a stereospecific process. The (-)-stereoisomers of epinephrine, norepinephrine, and ergotamine were at least 20to 50.fold more potent than the corresponding (+)-stereoisomers. Compounds devoid of significant a-adrenergic activity, such as pyrocatechol, 3,4-dihydroxymandelic acid, normetanephrine, and n-lysergic acid, did not effectively compete for [3H]dihydroergocryptine binding sites. These rabbit uterine binding sites for [3H]dihydroergocryptine appear to have characteristics indistinguishable from those of the physiologically active oc-adrenergic receptors.