Binding of 3H-catecholamines to alpha-noradrenergic receptor sites in calf brain.

The binding of (+ )-L3Hlepinephrine and (-PHlnorepinephrine to calf cortical membranes in the presence of 1.0 mM pyrocatechol has properties indicating an association with a-noradrenergic receptors. Binding of the “H-catecholamines associated with /3-noradrenergic receptors has not been detected, even at low concentrations of pyrocatechol. Chromatographic studies reveal that radioactivity bound to membranes is authentic catecholamine. Binding of both “Hcatecholamines is saturable with dissociation constants at 37” of 18 nM for (?)-[3Hlepinephrine and 26 nM for c-j[“Hlnorepinephrine. The Hill coefficients for binding of both “H-ligands are 1.0. At 37, both “H-catecholamines associate rapidly to equilibrium, and binding is completely reversible. Kinetically determined dissociation constants correspond well with values obtained from equilibrium studies. Binding is stereoselective for both ligands, with (-)-norepinephrine displaying about 40 times greater affinity than (+)-norepinephrine. The structure-activity relationship of a series of phenylethanolamine derivatives in competing for “H-catecholamine binding sites parallels the known relative potencies of these compounds at peripheral a-receptors. (YAntagonists and partial agonists are potent competitors, whereas /&antagonists and catechol derivatives are extremely weak. The structurally dissimilar a-agonist, [“Hlclonidine, seems to bind to the same calf cortical membrane sites as the “H-catecholamines, whereas the a-antagonist, “H-labeled WB-4101, binds to sites with the same LYreceptor characteristics, but which have greater affinity for antagonists and lesser affinity for agonists. At 25” and 4”, the “H-catecholamines and agonist competitors have 2 to 4 times greater affinity for the binding sites than at 37”, whereas antagonists are 2 to 10 times weaker, and partial agonists have approximately the same affinity. At 4” and 25”, “H-catecholamines dissociate in a biphasic manner which is time-dependent.