Specific binding of (3H) lysine-vasopressin to pig kidney plasma membranes. Relationship of receptor occupancy to adenylate cyclase activation.

A plasma membrane fraction was prepared from pig kidney medulla. It contained lysine-vasopressin-sensitive adenylate cyclase activity (maximum activation: 4to 6fold, apparent Km value for the hormone: about 5 X 1OV M). Lysine-vasopressin was not inactivated by the membrane preparation even at low hormonal concentrations. The specific [3H]lysine-vasopressin (vasopressin) binding sites found are probably involved in adenylate cyclase activation; binding occurred in a concentration range giving dose-dependent activation of the adenylate cyclase. Relative affinities of vasopressin, oxytocin, (0-methyl)-tyrosine2oxytocin and angiotensin (vasopressin > oxytocin > (O-Me)Tyr2-oxy > angiotensin) for binding and adenylate cyclase activation were similar. Nonspecific binding, i.e. binding which could not be inhibited by low5 M unlabeled vasopressin, represented 12.5% of the specific binding at 10es M [ 3H]vasopressin. For all hormonal concentrations, [3H]vasopressin binding increased with time up to an equilibrium value. The binding was reversible. The time course of hormone-receptor complex formation was highly temperatureand hormone concentration-dependent. Dissociation and association rate constants at 30” varied from 0.02 to 0.088 mine1 for k-r, and from 1.3 X 1O+7 to 3.0 X lo+? M-* mine1 for kl. For the same enzyme preparation, the k-1: kl ratio was very close to the value deduced from the hormonal concentration giving half the maximum binding at equilibrium. When adenylate cyclase activation was measured as a function of time after the addition of hormone, activation was found to be progressive. The time required to reach maximum activation is similar to that needed to reach an equilibrium value for [3H]vasopressin binding at the same concentration. These data suggest that adenylate cyclase activation is a function of receptor occupancy. Reversal of adenylate cyclase activation was also progressive. When adenylate cyclase activation at equilibrium was measured for different values of receptor occupancy, it was found that for the same increment in receptor occupancy,