The Glucagon-sensitive Adenylate Cyclase System in Plasma Membranes of Rat Liver: VII. HORMONAL STIMULATION: REVERSIBILITY AND DEPENDENCE ON CONCENTRATION OF FREE HORMONE

Abstract

Abstract Stimulation of rat liver adenylate cyclase activity by saturating concentrations of glucagon was found to increase with increasing concentrations of ATP from essentially no stimulation at 2.7 x 10-7 m to a maximum of 7- to 8-fold at approximately 3 x 10-3 m. Half-maximal stimulation was found at 1 x 10-4 m ATP. Stimulation (4-fold) by fluoride ion was independent of the concentration of ATP used. At low concentrations of ATP, stimulation by glucagon could also be elicited by GTP. It was concluded that the response of the rat liver adenylate cyclase system to glucagon exhibits an absolute requirement for either GTP or ATP. Addition of des-1-histidine-glucagon (DH-glucagon), a competitive inhibitor of glucagon action resulted within 45 to 60 s in complete inhibition of the effect of glucagon on adenylate cyclase activity. This action of DH-glucagon was independent of the time elapsed between the addition of glucagon and DH-glucagon and was not influenced by the presence of adenylate cyclase assay reagents prior to addition of DH-glucagon. Readdition of excess native glucagon resulted in restimulation of enzymatic activity. Exposure of liver membranes to 8 nm glucagon followed by a 4-fold dilution of the hormone resulted in glucagon-stimulated activity identical with that given by 2 mm glucagon. Inactivation of glucagon by a liver membrane process, which causes a loss of glucagon in the medium, also showed a proportional loss of stimulation of adenylate cyclase. It is concluded that glucagon reacts rapidly and reversibly with its receptor in the presence of ATP, that this interaction results in rapid and reversible stimulation of adenylate cyclase activity, and, finally, that persistence of this stimulation depends on the continuous presence of glucagon in the assay medium.