Hormone action at the membrane level VI. Binding of (—)-[ 3H]dihydroalprenolol and (±)-[ 3H]isoproterenol to turkey erythrocytes and correlation with adenylate cyclase activity

Abstract

The binding of (±)-[ 3H]isoproterenol and (—)-[ 3H]dihydroalprenolol to intact turkey erythrocytes was studied using a rapid centrifugation technique. The binding of both ligands is rapid, dissociable, stereospecific and inhibited by (—)-propranolol. The total number of isoproterenol binding sites is 2800 sites/ cell. This consists of a low and high affinity site both of which show stereospecific binding. The high affinity isoproterenol site has a K d of 15.5—19.5 nM and has 600 sites/cell. The low affinity isoproterenol site has a K d of 195 nM and has 2200 sites/cell. The binding of (—)-[ 3H]dihydroalprenolol shows one type of site with a K d of 7.8 nM and has 2500 sites/cell. The agonists epinephrine, norepinephrine, soterenol and p-hydroxyphenylisoproterenol which were tested by competition for binding showed a 6—25-fold greater affinity for the high affinity site determined by (±)-[ 3H]isoproterenol as compared to the (—)-[ 3H]dihydroalprenolol binding site. However, the antagonists propranolol, practolol and metrapolol showed similar affinities for the binding sites as determined by competition of binding of either labeled isoproterenol or dihydroalprenolol. These studies indicate that isoproterenol binding can recognize two independent stereospecific β-adrenergic receptors or can recognize two different conformational states of a single receptor. Provisional calculations are made on the turnover number of adenylate cyclase under physiological conditions using intact erythrocytes. The turnover number is 4000 molecules of cyclic AMP/10 min per high affinity receptor.