Identification of alpha-adrenergic receptors in human platelets by [3H]dihydroergocryptine binding.
Binding of [(3)H]dihydroergocryptine to platelet lysates appears to have all the characteristics of binding to alpha-adrenergic receptors. At 25 degrees C binding reaches equilibrium within 20 min and is reversible upon addition of excess phentolamine. Binding is saturable with 183+/-22 fmol of [(3)H]dihydroergocryptine bound per mg of protein at saturation, corresponding to 220+/-26 sites per platelet. Kinetic and equilibrium studies indicate the dissociation constant of [(3)H]dihydroergocryptine for the receptors is 1-3 nM. The specificity of the binding sites is typical of an alpha-adrenergic receptor. Catecholamine agonists compete for occupancy of the [(3)H]dihydroergocryptine binding sites with an order of potency (-)epinephrine> (-)norepinephrine>> (-)isoproterenol. Stereospecificity was demonstrated inasmuch as the (+)isomers of epinephrine and norepinephrine were 10-20-fold less potent than the (-)isomers. The potent alpha-adrenergic antagonists phentolamine, phenoxybenzamine, and yohimbine competed potently for the sites, whereas beta-antagonists such as propranolol and dichlorisoproterenol were quite weak. Dopamine and serotonin competed only at high concentrations (0.1 mM). The [(3)H]dihydroergocryptine binding sites could also be demonstrated in intact platelets where they displayed comparable specificity, stereospecificity, and saturability. Saturation binding studies with the intact platelets indicated 220+/-45 receptors per platelet, in good agreement with the value derived from studies with platelet lysates. Ability of alpha-adrenergic agonists to inhibit adenylate cyclase and of alpha-adrenergic antagonists to antagonize this inhibitory effect directly paralleled ability to interact with the [(3)H]dihydroergocryptine binding sites. These data demonstrate the feasibility of directly studying alpha-adrenergic receptor binding sites in human platelets with [(3)H]dihydroergocryptine.
D000262:Adenylyl Cyclases / Q000097:blood; D000316:Adrenergic alpha-Agonists / Q000097:blood / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000097:blood / Q000494:pharmacology; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D004088:Dihydroergotoxine / Q000097:blood* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D010974:Platelet Aggregation / Q000187:drug effects; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*
D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D011941:Receptors, Adrenergic; D011942:Receptors, Adrenergic, alpha; D004088:Dihydroergotoxine; D000262:Adenylyl Cyclases