Studies on Receptor-mediated Activation of Adenylyl Cyclases: I. PREPARATION AND DESCRIPTION OF GENERAL PROPERTIES OF AN ADENYLYL CYCLASE SYSTEM IN BEEF RENAL MEDULLARY MEMBRANES SENSITIVE TO NEUROHYPOPHYSEAL HORMONES

Abstract

Abstract With use of a discontinuous sucrose density gradient centrifugation in the presence of 1 mm EDTA and 10 mm Tris-HCl, pH 7.5, membrane particles were prepared from beef renal medulla that are 4- to 6-fold enriched in fluoride- sensitive and 8- to 12-fold enriched in neurohypophyseal hormone (NHH)-sensitive adenylyl cyclase activity. Average yield of the NHH-sensitive adenylyl cyclase activity was 30 to 35%; average yield of membrane protein, starting with 80 g of beef renal medulla, was 200 to 250 mg. These membranes also contain a prostaglandin-sensitive adenylyl cyclase system, the activity of which is additive to that sensitive to NHH. Arginine-vasopressin, lysine-vasopressin, and oxytocin stimulate enzyme activity half-maximally at 10-9, 10-9, and 10-7 m, respectively. The response of the renal medullary adenylyl cyclase activity to NHH is extremely dependent on the concentrations of magnesium and ATP used in the assay. Response is maximal at low concentrations of magnesium and at concentrations of ATP varying from 0.07 to 0.10 mm. Increasing the concentration of magnesium results in selective stimulation of basal activity with concomitant loss of the relative stimulation by NHH. Increasing or decreasing the concentration of ATP results in selective loss of activity due to NHH stimulation, also leading to reduced relative stimulation by NHH. GTP at 10-5 m inhibits stimulation of the beef renal medullary adenylyl cyclase by NHH. 5'-Adenylyl-imidodiphosphate (AMP-PNP), an analogue of ATP that is not hydrolyzed by membrane ATPases, was evaluated as substrate for renal adenylyl cyclase. Depending on the concentration of the analogue and magnesium used, AMP-PNP yields activities that are between 10 and 25% of those obtained with ATP. Maximal hormonal stimulation requires higher concentrations of AMP-PNP than ATP; excess AMP-PNP does not result in inhibition of hormonal stimulation. Activities with the analogue exhibit higher pH optima than with ATP. The possibility that these differences in activity are due to a different exit rate of inorganic imidodiphosphate as compared to PPi or to the different divalent cation binding properties of AMP-PNP and ATP, or both, is discussed. The extreme sensitivity of hormonal stimulation to variations of nucleotide concentration, be it ATP or GTP, coupled to the relatively high yield of activity from beef renal medulla, make this preparation useful for the study of modes of regulation of hormonal stimulation of adenylyl cyclase systems. In addition, several minor modifications of the procedure of Krishna et al. (Krishna, G., Weiss, B., and Brodie, B. B. (1968) J. Exp. Ther. Pharmacol. 163, 379–385) for isolation and determination of cyclic adenosine 3':5'-monophosphate (cAMP) formed during incubations for adenylyl cyclase are described. They result in higher yields of cAMP (60 to 65%) and in lower reaction blanks (0.0005 to 0.0010% of initially added ATP).