Molecular Pharmacology, Vol 14, 549-561, Copyright © 1978 by the American Society for Pharmacology and Experimental Therapeutics

Radioligand Binding to Beta Adrenergic Receptors of Intact Cultured S49 Cells

¹ Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, and Cardiovascular Research Institute, University of California, San Francisco, California 94143

Using the radioligand [¹²⁵I]iodohydroxybenzylpindolol ([¹²⁵I]IHYP), we have characterized the beta adrenergic receptors of intact S49 lymphoma cells. Washed cells are incubated with the radioligand in a physiological growth medium containing 1 mM ascorbic acid and 0.1% horse serum. Binding of[¹²⁵I]IHYP saturates with time, equilibrium being achieved in 45-60 min, and is linear with cell number. Scatchard analysis of binding at equilibrium indicates one class of binding sites with a K_d equal to 28 pM and 1200-1300 binding sites/cell; this number of binding sites is about 4 times higher than previously observed with disrupted S49 cells. Independent estimates of the K_d derived from the forward and reverse reaction rates of binding are in agreement with the K_d derived from equilibrium studies. Competition for [¹²⁵I]IHYP binding sites by beta adrenergic antagonists corresponds closely to estimates of potency of the same antagonists in blockade of beta adrenergic-stimulated cyclic 3',5'-AMP accumulation. In contrast, beta adrenergic agonists are much more potent in stimulating cyclic AMP accumulation than in competing with [¹²⁵I]IHYP for beta adrenergic binding sites on intact S49 cells. Furthermore, agonists appear more potent in competing for [¹²⁵I]IHYP binding sites of disrupted than of intact S49 cells. The lower potency of beta adrenergic agonists in binding studies of intact cells may be attributable to the presence of endogenous guanyl nucleotides and, perhaps, to the more efficient coupling between receptors and adenylate cyclase of the intact cells. That this lower potency of agonists is not an artifact of studies with [¹²⁵I]IHYP is indicated by similar findings with the radioligand [³H]dihydroalprenolol ([³H]DHA). [³H]DHA binds to intact S49 cells rapidly (reaching equilibrium in less than 3 min) and reversibly, with a K_d of 0.67 ± 0.08 nM. An identical number of binding sites is found on intact S49 cells whether [³H]DHA or [¹²⁵I]IHYP is used, and studies in which adrenergic agents compete for ligand binding sites yield similar results for both radioligands. These studies of binding to beta adrenergic receptors of intact cultured cells using either [¹²⁵I]IHYP or [³H]DHA offer a useful tool that should complement and extend studies of receptors on plasma membranes isolated from such cells.

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ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Michele Sanda and Mary Gleason, the support of Dr. Kenneth Melmon, and the advice of Dr. Henry Bourne.