Abstract

The relationship between the concentration of β-adrenergic receptors and the activation of heart muscle by isoproterenol was investigated using the irreversible β-adrenergic receptor antagonist NHNP-NBE. The interaction of NHNP-NBE with β-receptors was characterized in isolated membranes, whole cells and intact cardiac muscle, using [¹²⁵I]-iodohydroxybenzylpindolol (IHYP). Formation of the NHNP-NBE-β-receptor complex was found to be irreversible and dependent on incubation time, temperature and ligand concentration. Occupation of β-receptors by adrenergic ligands prior to NHNP-NBE exposure protected the receptors from inactivation. These data are consistent with a covalent modification of β-receptors by NHNP-NBE at a site in or near the adrenergic ligand binding site. Incubation of NHNP-NBE with intact cardiac muscle produced a dose dependent β-receptor inactivation that survived tissue homogenization and membrane isolation. NHNP-NBE dramatically affects the concentrations at which isoproterenol produces positive inotropic responses in cat papillary muscles. The ED₅₀ for isoproterenol under control conditions averaged 9.8 nM. The ED₅₀ of isoproterenol increased to 22; 70; 500 and 5623 nM subsequent to a 10 min treatment of the muscles with 0.1; 1.0; 10 and 100 µM NHNP-NBE respectively. The same maximum inotropic response was achieved with isoproterenol following each concentration of NHNP-NBE. Ten micromolar isoproterenol subsequent to 100 µM NHNP-NBE increased the papillary muscle concentration of cyclic AMP to the same extent as 10 µM isoproterenol alone. Although the maximum cyclic AMP response was essentially identical, the time course for cyclic AMP production was substantially slower following irreversible β-receptor blockade. The ED₅₀ for isoproterenol induced increases in cyclic AMP concentrations was 15 nM in the absence of NHNP-NBE and 600 nM following 100 µM NHNP-NBE. In contrast to the cardiac data, NHNP-NBE stoichiometrically inhibited IHYP binding and isoproterenol induced cyclic AMP formation in cultured human lung (VA₂) cells. These data indicate a high efficiency coupling between the isoproterenol-β-adrenergic receptor interaction, cyclic AMP formation and increased cardiac contractility, an efficiency not apparent with isoproterenol-induced cyclic AMP formation in cultured cells.