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LLC-PKIL cells, a kidney-derived cell line, had sustained growth in a defined medium. When compared to the parent cell line growing with 10% fetal bovine serum, LLC-PKIL cells had about 100-times fewer vasopressin receptors. Upon modifications of the cell culture medium, the vasopressin response of the adenylate cyclase could be increased by more than 10-fold with a parallel increase in vasopressin receptor number. Using cells with high or low receptor densities, the stimulatory and inhibitory effects of N6-L-2-phenylisopropyl-adenosine on the modulation of the adenylate cyclase responsiveness to vasopressin were investigated. When high concentrations of GTP were added, low concentrations of phenylisopropyladenosine inhibited the enzyme, while higher concentrations were found to be stimulatory. The adenylate cyclase activity stimulated by vasopressin could only be inhibited by phenylisopropyladenosine under these conditions in membranes with high receptor density; only the increase in enzyme activity due to high GTP concentration was inhibitable. The analysis of the dependency of the adenylate cyclase activity as a function of the vasopressin concentrations showed that, besides reducing the maximum velocity of the system for vasopressin, the addition of phenylisopropyladenosine generated an heterogeneity in the adenylate cyclase response to vasopressin (as judged by a curvilinear Eadie plot). A high-affinity component in the adenylate cyclase response appeared when phenylisopropyladenosine was added. The growth of the cells in a medium containing adenosine deaminase gave results identical to those obtained for control cells. However, growing the cells with both phenylisopropyladenosine and adenosine deaminase abolished the inhibitory effects of the former on the adenylate cyclase and greatly reduced its stimulatory action. Under these conditions, the vasopressin response of the adenylate cyclase was not further regulated by phenylisoprophyladenosine. These results indicate a role of adenosine on vasopressin response, especially at low physiological concentrations of the hormone where a high-affinity component of the hormonal response could be demonstrated. [ABSTRACT FROM AUTHOR]

The effects of 17 non-ionic detergents on pig kidney plasma membrane adenylate cyclase were tested (digitonin; detergents from the Triton series: X45, X100, X102, X114, X165, X305, X405, N101; the Brij series: 35, 56, 96 and the Tween series: 20, 40, 60, 80, 85). Membranes (2.66 mg protein/ml) were treated with increasing amounts of detergent for 150 min at 0 °C. The adenylate cyclase activities present in treated membranes and in their corresponding non-sedimentable fractions were measured at 30 °C. Only detergents having hydrophilic lipophilic balance values from 12 to 14 were effective solubilizing agents. For these detergents, the solubilization yield was dependent on their structure. When solubilized adenylate cyclase kept at 0 °C was incubated at 30 °C, the enzyme catalytic activity exponentially decreased towards an equilibrium value with a half-time of 5 min. This temperature-dependent adjustment of enzyme activity was reversible. Its magnitude was dependent on the detergent used. A more progressive but less pronounced effect was observed with non-solubilizing detergents. The micellar form of detergent in the incubation medium was found to be responsible for the appearance of microheterogeneity in the population of solubilized enzyme molecules differing by the substrate accessibility. This effect was suppressed when reducing the detergent concentration in the incubation medium. [ABSTRACT FROM AUTHOR]

The effect of adding various Na+, K+ and Li+ salts was tested on the adenylate cyclase activity present in pig-kidney-medulla plasma membranes. Salts were found to exert dual effects: stimulation at lowest doses and inhibition at highest doses. The importance of the stimulatory effect depended on the addition of NaF, hormone or guanylyl 5′-imidodiphosphate (GMP-P(NH)P) in the incubation medium. In all cases, maximum effect (2-3.4-fold increase in activity) was observed in the presence of saturating amounts of (8-lysine)vasopressin. Rate of adenylate cyclase activation by GMP-P(NH)P was enhanced in the presence of salt. Basal adenylate cyclase activity stimulation by salt was highly dependent on the nature of the salt used. There was a 10% increase with NaSCN and a 210% increase with NaCl. The effects of salts could not be accounted for by a fluoride-like effect. Among all the tested salts, only lithium salts inhibited hormone-stimulated enzyme. The order of potency, Li+, Na+, K+, was found when considering the inhibitory effect observed with high salt concentrations. For anions, the stimulatory effect observed with low salt concentration was I-, SCN-, Br-, Cl-, N3-, HCOO- and CH3COO- in decreasing order of potency. Stimulatory effect of salt was observed when tested at 30 °C while highly inhibitory effect occurred at low temperature. Above 20 °C the energies of activation (Ea) were identical regardless of the presence of salt. At lower temperature, Ea was doubled in the presence of salt. All the stimulatory salts did not change the characteristics of adenylate cyclase activation by (8-lysine)vasopressin or its analogues, but stimulation ratio was enhanced. The above results cannot be entirely accounted for by a chaotropic action of ions on the membrane structure. [ABSTRACT FROM AUTHOR]