Identification and characterization of beta₁-adrenergic receptors in rat parotid membranes

• Beta-adrenergic receptor identification and properties are probed in rat parotid membranes utilizing the high affinity β-adrenergic antagonist(-)-[³H]dihydroalprenolol. • The binding of (-)-[³H]dihydroalprenolol to membrane preparations of parotid is rapid, equilibrium being reached in 5 min. Strict stereospecificity is observed, (-)-propanolol being 100 times more potent than (+)-propranolol in competing with (-)-[³H]dihydroalprenolol for binding sites. Beta-adrenergic agonists compete for the binding sites with (-)-[³H]dihydroalprenolol with the same characteristics, i.e., much higher concentrations of the (+)-stereoisomers than the (-)-stereoisomers are required to produce 50% inhibition, the range varies from 14-fold for epinephrine to 300-fold for isoproterenol. Among the (-)-stereoisomers, the relative potency of inhibitory action is (-)-propranolol > (-)-isoproterenol > (-)-epinephrine ≡ (-)-norepinephrine. (-)-Isoproterenol is about 20 times as potent as norepinephrine, the least potent agonist among all the catecholamine (-)-stereoisomers. • The binding of (-)-[³H]dihydroalprenolol is saturable, with a maximum number of binding sites equalling 450 fmol/mg protein and a dissociation constant of 7.9 nM. The Scatchard plots show no significant curvilinear character. Hill plots consistently give a Hill coefficient close to unity (0.92-1.05). Both pieces of evidence suggest a single-component system with no significant cooperativity. • Dissociation kinetics study after the method of De Metys et al. (1973) Biochem. Biophys. Res. Commun. 155, 154, indicates a lack of site-to-site interactions among the binding sites. The rate of dissociation of bound (-)-[³H]dihydroalprenolol is the same in the presence and absence of 1 · 10^-5 M (±)-alprenolol. • Based on the binding of (-)-[³H]dihydroalprenolol, it is concluded that the beta-adrenergic receptors can be identified in rat parotid and that these binding sites display β₁ character. Results of the study indicate a one-component system with no observable site-to-site interactions.