Jiro Nakamura

GLUTATHIONE ALTERS THE MODE OF CALCIUM-MEDIATED REGULATION OF ADENYLYL CYCLASE IN MEMBRANES FROM MOUSE BRAIN

We examined the effect of sulfhydryl compounds on the regulation of adenylyl cyclase by calcium in mouse cerebrum membranes. Isoproterenol-stimulated adenylyl cyclase (IP-AC) activity in the membranes was increased by addition of the optimum concentrations of calcium/calmodulin. However, in the presence of 0.01-0.07 mM glutathione (GSH), calcium/calmodulin inhibited the activity. At high concentrations of GSH (1-10 mM), the IP-AC activity was stimulated by calcium/calmodulin to a greater extent than that in the control (no GSH). Cysteine at less than 1.7 mM induced a similar inhibition of the IP-AC activity, but dithiothreitol did not. The activity of IP-AC measured in the absence of calmodulin decreased when calcium levels were greater than 300 microM. GSH at 0.05 mM enhanced the calcium-dependent inhibition (22% inhibition by 200 microM calcium), while 10 mM GSH lowered it. Calmodulin itself had no significant effect on the IP-AC activity, irrespective of the concentrations of GSH involved. It caused a small increase in the IP-AC activity that had been reduced by the presence of calcium and GSH. These results indicate that the redox status of sulfhydryls in adenylyl cyclase plays an important role in the calcium-mediated regulation of the enzyme. The enzyme becomes much more sensitive to the calcium-dependent inhibition after partial reduction of the sulfhydryls via the particular mode of reactin.

Relationship between alcohol consumption and the activity of GTP-binding regulatory proteins in human erythrocyte membranes

Activity of stimulatory GTP-binding regulatory protein (Gs) in human erythrocyte membranes was assessed by activation of adenylate cyclase in S49 murine lymphoma variant cells to elucidate a relationship to alcohol consumption. In apparently healthy subjects, alcohol consumption < 50 g ethanol per week did not alter the Gs activity, but it was significantly higher (14.3%, P < 0.05) in moderate drinkers (50-150 g/week) than non-drinkers. Then, the Gs activity declined with a further increase in alcohol consumption (150-550 g/week). Those subjects with drinking levels of > 50 g/week also showed significant increases in other alcohol-related markers, Na+, K(+)-ATPase and gamma-glutamyltransferase. The Gs activity was significantly low in alcoholics (a 34.9% reduction). No such reduction was noted in patients with other diseases. The results indicate that the Gs activity in erythrocyte membranes is an alcohol-related marker in humans. The variation of Gs activity is distinctive from those of other alcohol-related markers.

Protein kinase CβI interacts with the β1-adrenergic signaling pathway to attenuate lipolysis in rat adipocytes

We have shown previously that insulin attenuates beta1-adrenergic receptor (beta1-AR)-mediated lipolysis via activation of protein kinase C (PKC) in rat adipocytes. This antilipolysis persists after removal of insulin and is independent of the phosphodiesterase 3B activity, and phorbol 12-myristate 13-acetate (PMA) could substitute for insulin to produce the same effect. Here, we attempted to identify the PKC isoform responsible for antilipolysis. Isolated adipocytes were treated with high and low concentrations of PMA for up to 6 h to degrade specific PKC isoforms. In the PMA-treated cells, the downregulation profiles of PKC isoforms alpha and betaI, but not betaII, delta, epsilon, or zeta, correlated well with a decrease of lipolysis-attenuating effect of PMA. After rats fasted for 24 h, adipocyte expression of PKC isoform alpha increased, while expression of PKCdelta decreased. Fasting did not change the potency of PMA to attenuate lipolysis, however. The lipolysis-attenuating effect of PMA was blocked by the PKCbetaI/betaII inhibitor LY 333531, but not by the PKCbetaII inhibitor CGP 53353 or the PKCdelta inhibitor rottlerin. These data suggest that PKCbetaI interacts with beta1-AR signaling and attenuates lipolysis in rat adipocytes.

Ethanol-induced reduction of the amount of stimulatory GTP-binding regulatory protein, Gs, in wild-type S49 murine lymphoma cells: effect of cycloheximide and puromycin.

The activity and the amount of Gs in wild-type S49 murine lymphoma cells decreased by 16-18% and by 29%, respectively, after 24 h treatment of the cells with 50 mM ethanol, through a transient increase of the Gs activity with no alteration in its content after 4 and 8 h treatment. No decrease of the Gs activity was noted when membranes from S49 cells were treated with 50-500 mM ethanol. To study the role of protein metabolism in the ethanol-induced reduction in the amount of Gs, S49 cells were treated with ethanol for 24 h in the presence of inhibitor of protein synthesis. Cycloheximide at concentrations of 0.05-0.15 micrograms/ml and puromycin at 0.15-0.60 micrograms/ml caused a dose-dependent inhibition of cell division in S49 cells to a similar degree. The ethanol-induced reduction of the activity and the amount of Gs was completely abolished by those concentrations of cycloheximide, but not by puromycin. Cycloheximide itself caused no alteration in the Gs activity of S49 cell membranes. These results indicate that the process of the ethanol-induced event of Gs involves cycloheximide-sensitive protein synthesis.

Phorbol 12-myristate 13-acetate inhibits the antilipolytic action of insulin, probably via the activity of protein kinase Cε

Protein kinase CβI (PKCβI) mediates insulin signaling and attenuates β1-adrenoceptor-stimulated lipolysis. In this work, the effect of the PKC activator phorbol 12-myristate 13-acetate (PMA) on the antilipolytic action of insulin was determined by analyzing lipolysis induced by a β3-adrenoceptor agonist CL 316243. PMA inhibited the insulin antilipolytic action. The pan-PKC inhibitors GF 109203X and chelerythrine inhibited the PMA effect, but the PKCα/β inhibitors Gö 6976 and CGP 53353 did not. Exposure of cells to PMA downregulated PKCs α, βI, and δ within 3 h and PKCε within 12 h. The effect of PMA on insulin action greatly diminished when PKCε was downregulated. Inhibitors of phosphatidylinositol 3-kinase (PI3-K), Akt, and phosphodiesterase 3B (PDE3B) diminished the PMA effect. PMA inhibited insulin-stimulated phosphorylation of Tyr in insulin receptor β subunit and Ser/Thr in Akt. These data suggest that PMA inhibits the antilipolytic signal mediated by the insulin receptor, PI3-K, Akt, and PDE3B. The most probable target of PMA is PKCε.

Calcium ionophore, A23187 alters the mode of cAMP formation in wild-type S49 murine lymphoma cells

We examined the change in the adenylyl cyclase activity of S49 cells occurring after exposure to calcium ionophore, A23187. MnCl2-stimulated adenylyl cyclase activity in membrane preparations increased by 67 +/- 3% (after 24 h treatment with 0.3 microM A23187), while no significant change was found in the basal activity or NaF-, isoproterenol- or forskolin-stimulated activities. An activity sensitive to CaCl2/calmodulin, which could not be detected in membranes from untreated cells, was found in membranes from A23187-treated cells. These changes took place after treatment with 0.1-0.3 microM A23187 for a period longer than 16 h. A brief treatment of S49 cells with phorbol 12-myristate 13-acetate (PMA) enhances the activity of adenylyl cyclase (Bell, J.D. et al. (1985) J. Biol. Chem. 260, 2625-2628), but exposure of cells to PMA at the end of A23187-treatment did not affect the induction of the MnCl2-or CaCl2/calmodulin-sensitive activity. The results indicate that long-term treatment of S49 cells with calcium ionophore, A23187, induces adenylyl cyclase activity of a novel type, which is probably caused by an abnormal increase in free intracellular calcium.