Calcium-mediated Interactions between the Antidiuretic Hormone and Renal Plasma Membranes

Abstract

Abstract The antidiuretic hormone-stimulated adenylate cyclase system has been characterized in a purified preparation of porcine renal plasma membranes. Adenylate cyclase activity was linear over a plasma membrane protein concentration from 30 to 300 µg, and maximal enzyme activity occurred at approximately 7 x 10-4 m ATP. Activation of the system by sodium fluoride was about 10-fold and was produced by 10 to 12 mm sodium fluoride. The adenylate cyclase was stimulated approximately 3-fold by the antidiuretic hormone. One-half maximal stimulation by hormone was measured at 2.2 x 10-8 m antidiuretic hormone. Calcium exhibited an inhibitory effect upon antidiuretic hormone-stimulated adenylate cyclase at concentrations of the metal ion above 10-6 m, but it produced little or no inhibition in the sodium fluoride-stimulated system. The chelating agent, ethylene glycol-bis(β-aminoethyl ether)-N,N'tetraacetic acid, at a concentration of 1 mm, completely inhibited antidiuretic hormone-stimulated adenyl cyclase. The activity of the chelator-inhibited enzyme was completely restored by increasing concentrations of calcium. Binding of tritiated antidiuretic hormone to the renal plasma membranes was measured by centrifugation techniques and by filtration through Millipore filters. Both methods demonstrated that binding of the hormone to the membranes occurred at a level of approximately 0.5 pmole of antidiuretic hormone per mg of protein when the hormone was added at a concentration that produced half-maximal stimulation of adenylate cyclase. The rates of association and dissociation for the interaction between the antidiuretic hormone and renal plasma membranes were measured. Binding equilibrium was reached after approximately 10 min at 37°. Dissociation of tritiated bound hormone was shown to be 100% complete after approximately 30 min. Graphical analysis of the time course of dissociation demonstrated that the kinetics of the reaction follows a simple first order rate equation. Neither adrenocorticotropic hormone nor calcitonin exchanged with antidiuretic hormone at its binding site, indicating that the receptor sites exhibit a specificity for the antidiuretic hormone. Calcium ion concentrations in the millimolar range inhibited the rate and level of binding of ADH to renal plasma membranes with complete inhibition reached at 10 mm calcium. However, ethylene glycol-bis(β-aminoethyl ether)-N,N'-tetraacetic acid at the same concentration at which it completely inhibited adenylate cyclase activity had no effect upon the binding of antidiuretic hormone to the membranes. These results suggest that calcium plays multiple regulatory roles with millimolar concentrations inhibiting binding to membrane receptor sites and lower calcium concentrations mediating at a locus within the membrane matrix between the receptor site and the site occupied by adenylate cyclase.