Characterization of alpha 2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine.
[3H]Yohimbine, a potent alpha 2-adrenergic antagonist, was used to label the alpha-adrenergic receptors in membranes isolated from human platelets. Binding of [3H]yohimbine to platelet membranes appears to have all the characteristics of binding to alpha-adrenergic receptors. Binding reached a steady state in 2-3 min at 37 degrees C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10(-5) M; t1/2 = 2.37 min). [3H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [3H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (-) isomer was 11-times more potent that the (+) isomer. Catecholamine agonists competed for the occupancy of the [3H]yohimbine-binding sites with an order of potency: clonidine greater than (-)-epinephrine greater than (-)-norepinephrine much greater than (-)-isoproterenol. The potent alpha-adrenergic antagonist, phentolamine, competed for the sites whereas the beta-antagonist, (+/-)-propranolol, was very weak inhibitor. 0.1 mM GTP reduced the binding affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonin competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [3H]yohimbine binding. These results suggest that [3H]yohimbine binding to hunan platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label alpha 2-adrenergic receptors.
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000328:Adult; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D004837:Epinephrine / Q000494:pharmacology; D005260:Female; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008297:Male; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium; D015016:Yohimbine / Q000378:metabolism*
D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D011941:Receptors, Adrenergic; D011942:Receptors, Adrenergic, alpha; D014316:Tritium; D015016:Yohimbine; D006160:Guanosine Triphosphate; D004837:Epinephrine