Molecular Pharmacology, Vol 16, 719-728, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

Quantitation and Characterization of Human Platelet Alpha-Adrenergic Receptors Using [³H]Phentolamine

¹ Department of Surgery, Beth Israel Hospital and Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215

The binding of [³H]phentolamine to human platelet membranes was studied, and this ligand was found to bind to a single class of noncooperative sites with a dissociation constant (K_d) of 12 ± 3 nM. At saturation, 0.165 ± 0.060 pmoles were bound per mg protein, and each platelet was noted to possess 260 ± 95 binding sites. [³H]phentolamine binding was rapid (t_1/2 < 15 sec) and reversible (t_1/2 for dissociation <30 sec) at 30°. [³H]phentolamine binding was stereospecifically inhibited by epinephrine. Inhibition of [³H]phentolamine binding by catecholamines occurred with an order of potency of epinephrine > norepinephrine > isoproterenol and propranolol (1 µM) did not inhibit [³H]phentolamine binding. These observations indicate that [³H]phentolamine binds to sites having the characteristics of -adrenergic receptors. Dihydroergocryptine was also noted to cause time and concentration-dependent inhibition of [³H]phentolamine binding. Comparison of these data to those previously reported using [³H]dihydroergocryptine as the binding probe (Newman, K. D., Williams, L. T., Bishopric, N. H. et al. J. Clin. Invest. 61: 395-402, 1978; Alexander, R. W., Cooper, B. and Handin, R. I. J. Clin. Invest. 61, 1136-1144, 1978) suggests that both [³H]phentolamine and [³H]dihydroergocryptine interact with the same binding sites. Dopamine and serotonin were found to inhibit [³H]phentolamine binding (K_d 1.9 and 15.1 mM respectively) indicating either that phentolamine can bind to receptors for dopamine and serotonin or these ligands bind to the platelet -receptor. The binding of [³H]phentolamine to platelet membranes was not altered when GTP (0.01 mM) was present, but the nucleotide caused a tenfold shift in the inhibition curve for epinephrine, indicating that epinephrine was less avidly bound in the presence of GTP.

Note:
ACKNOWLEDGMENT The authors gratefully acknowledge the assistance of Dr. W. Rein of Ciba-Geigy for help in obtaining [³H]phentolamine.