Characterization of the β-adrenergic receptor and adenylate cyclase in skeletal muscle plasma membranes

Subcellular fractions of rat skeletal muscle enriched in plasma membrane (sarcolemma) were used to characterize properties of the β-adrenergic receptor and adenylate cyclase. The equilibrium binding of (-)-[³H]dihydroalprenolol to the sarcolemmal membrane was characterized by a high affinity, K=3.6×10⁹ liters/mol and 1.2×10^-9 mol/g of membrane protein of homogenous noninteracting sites. The binding was reversible, saturable, and stereospecific. Displacement of labeled dihydroalprenolol by adrenergic ligands revealed an order of potency corresponding to the β₂-type response: (-)-isoproterenol>(-)-epinephrine >norepinephrine. The (+)-stereoisomers and the α-adrenergic antagonist phentolamine showed little ability to compete with (-)-[³H]dihydroalprenolol for occupancy of the binding sites. In the membrane fractions, a substantial basal activity of adenylate cyclase activity was demonstrated (35-50 pmol of cyclic AMP/mg/5 min). This activity was stimulated 40- to 50-fold by sodium fluoride, 7-to 8-fold by isoproterenol, and 5- to 6-fold by guanylyl 5′-imidodiphosphate [Gpp(NH)p]. Maximum activation depended upon optimal Mg²⁺ ion concentration (10 mm). In the presence of 0.1 mm Gpp (NH)p., the isoproterenol activation of adenylate cyclase was increased twofold. In addition, the concentration of hormone required for half-maximal stimulation of enzyme activity was shifted from 2.2×10^-7m in the absence of Gpp(NH)p to 2.5×10^-8 m in the presence of Gpp(NH)p. Gpp(NH)p also stimulated epinephrine and norepinephrine activation without altering the β₂-type potency series for the enzyme. Gpp(NH)p had no effect on either the number or the affinity of (-)-[³H]dihydroalprenolol binding sites, but it did increase the dissociation constant for isoproterenol by approximately fivefold.