CGP-12177. A hydrophilic beta-adrenergic receptor radioligand reveals high affinity binding of agonists to intact cells

A new, hydrophilic beta-adrenergic receptor radioligand, (+/-)-[3H]CGP-12177 (4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on hydrochloride), was synthesized and radiolabeled to 40 Ci/mmol. The nonspecific binding of this compound to turkey erythrocyte ghosts and C6 glioma cell membranes was less than 5% of the total binding at five times the appropriate KD. Binding assays of intact C6 glioma cells also showed low nonspecific binding, less than 20% of the total binding at five times the appropriate KD. The affinities of the antagonists (-)- and (+)-propranolol as well as of the agonist (-)-isoproterenol were examined by their potency to displace various radioligands from intact C6 glioma cell and membrane preparations. With membrane preparations, both [3H] CGP-12177 and [3H]dihydroalprenolol (DHA) were displaced stereospecifically by the antagonists (-)- and (+)-propranolol and the agonist (-)-isoproterenol. With whole cells, [3H]CGP-12177 and [3H]DHA behaved differently. [3H]DHA and [3H]carazolol could be stereospecifically displaced by antagonists but only partially displaced by agonists, while [3H]CGP-12177 could be completely displaced by both antagonists and agonists as in membranes. In contrast to [3H]CGP-12177, the lipophilic ligand [3H]DHA is actually taken up by cells. The inability of agonists to displace lipophilic radioligands from receptors on intact cells may not be due to a low affinity of agonists for receptors on cells, but to an agonist-induced change in the receptors which renders them inaccessible to hydrophilic agonists and antagonists. This change is likely to be their internalization into the cell.