Search

Treatment of rat reticulocytes with isoproterenol caused about 50, 25, and 25% decreases in beta-adrenergic agonist-, fluoride-, and guanine nucleotide-stimulated adenylate cyclase activities, respectively. The desensitization was also induced by dibutyryl adenosine 3',5'-cyclic monophosphate (cyclic AMP) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) prevented the isoproterenol-induced desensitization, suggesting the involvement of cyclic AMP in the desensitization. Time course studies revealed that the desensitization to NaF-AlCl3 occurred faster than that to isoproterenol. Furthermore, the rate of the resensitization to NaF-AlCl3 by removal of isoproterenol was also faster than that to isoproterenol. Thus, it is likely that both guanine nucleotide-binding stimulatory regulatory protein, Ns, and beta-adrenergic receptor are sequentially involved in both desensitization and resensitization of the adenylate cyclase system in rat reticulocytes to isoproterenol.

Involvement of divalent cations was examined in the activation of catecholamine sensitive adenylate cyclase in Ehrlich ascites tumor cells. In the presence of various concentrations of either Mg2+ or Mn2+, both basal and norepinephrine-stimulated, adenylate cyclase activities were measured. Both activities were gradually increased with the increase in Mg2+ concentration. On the other hand, with Mn2+, both activities were maximum at the low (2.5 mM) concentration, and decreased at the highest concentrations, the decrease being marked in the norepinephrine-stimulated activity. Similar difference between the two divalent cations was also observed in the effect on the binding of the beta-adrenergic ligand to the membrane receptor.

The effect of barbiturates on an adenylate cyclase system in rat brain was examined. The activity of the catalytic unit of this system isolated from the synaptic membrane was inhibited by phenobarbital in dose- and time-dependent manners. The mode of the inhibition was non-competitive with respect to Mg-adenosine triphosphate (ATP). The activity of the synaptic membrane-bound adenylate cyclase was also inhibited by phenobarbital in a similar manner. The inhibitory effect of phenobarbital was more potent on the activation of the enzyme by 5'-guanylylimidodiphosphate (GppNHp) than on the basal enzyme activity. The inhibitory effect, however, was not observed in the synaptic membrane preparation in which the guanine nucleotide-binding stimulatory regulatory protein (Ns) and the catalytic unit of adenylate cyclase system had been functionally coupled by pretreatment with GppNHp. Similar results were obtained with other pharmacologically active barbiturates. These findings indicate that barbiturates primarily affect the activation of the catalytic unit by an interaction with Ns resulting in the inhibition of the enzyme activity.

Forskolin-activated adenylate cyclase activity in rat erythrocyte membranes was markedly inhibited by methanol in a concentration dependent manner. On the contrary, no change was observed in the basal cyclase activity in the presence of methanol. The methanol inhibition forskolin-activated cyclase activity was protected by the stimulation of Gs in the presence of NaF in a concentration dependent manner. These data indicate that low- and high-affinity binding sites of forskolin in rat erythrocyte membranes have different sensitivities to methanol.

Rat brain adenylate cyclase was solubilized with a combination of 0.7% sodium cholate and 0.6 M ammonium sulfate, and fractionated by addition of solid ammonium sulfate. The precipitate at 35% ammonium sulfate saturation contained neither guanine nucleotide-binding regulatory protein (G protein) nor calmodulin, and was used as the catalytic unit of the enzyme system. This catalytic unit was activated synergistically by forskolin and Mn2+. An apparent Km value for Mg-adenosine triphosphate (ATP) of the catalytic unit was about 80 microM in the basal state, while it increased in concurrence with the increase in the enzyme activity when forskolin was added to the assay system. The increase in the Km value depended on the forskolin concentration up to 1 microM, above which the value converged on ca. 200 microM. Furthermore, activation of the catalytic unit by forskolin was more marked at higher concentration of Mg-ATP. On the other hand, Mn2+ suppressed the increase in the Km value for Mg-ATP by forskolin, though the value in the basal state was not changed by Mn2+ alone. These findings indicate that the activation of the catalytic unit by forskolin is accompanied by the change in the affinity for Mg-ATP and Mn2+ modifies the change.