cAMP regulation of phenobarbital-mediated induction of δ-aminolevulinate synthase mRNA in hepatocytes from normal and experimental diabetic rats

1994 ◽  
Vol 72 (9-10) ◽  
pp. 381-390 ◽  
Author(s):  
Cecilia L. Varone ◽  
Eduardo T. Canépa ◽  
Elena B. C. Llambías ◽  
Moisés Grinstein
Keyword(s):  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Diabetic Rats ◽  
Diabetic Rat ◽  
Dibutyryl Camp ◽  
Dependent Protein Kinase ◽  
Normal Cells ◽  
Aminolevulinate Synthase ◽  
Maximum Activation ◽  
Dependent Protein

We examined the mechanism underlying the effect of cAMP on δ-aminolevulinate synthase mRNA biosynthesis in isolated hepatocytes from normal and experimental diabetic rats. We have demonstrated that the potentiation by dibutyryl cAMP of the phenobarbital-mediated induction of δ-aminolevulinate synthase enzyme activity, observed in our previously reported studies, reflects an increased amount of its mRNA. The inducing effect exerted by phenobarbital on the biosynthesis of δ-aminolevulinate synthase mRNA in diabetic hepatocytes is greater than that observed in normal cells. This enhanced response to the increased level of endogenous cAMP in diabetic hepatocytes is apparently sufficient for a maximum activation of the cAMP-dependent protein kinase. The present results suggest that in rat liver dibutyryl cAMP modulates δ-aminolevulinate synthase mRNA biosynthesis by acting predominantly, if not exclusively, at the level of gene transcription.Key words: δ-aminolevulinate synthase mRNA, phenobarbital, cAMP, diabetic rat hepatocytes.

Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from normal and experimental-diabetic rats. Role of insulin

1990 ◽  
Vol 68 (6) ◽  
pp. 914-921 ◽  
Author(s):  
Eduardo T. Cánepa ◽  
Elena B. C. Llambías ◽  
Moisés Grinstein
Keyword(s):  
Aminolevulinic Acid ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Inhibitory Effect ◽  
Diabetic Rats ◽  
Significant Inhibition ◽  
Diabetic Rat ◽  
Experimental Conditions ◽  
Cytochrome P 450

In the present work we demonstrate that insulin decreases the phenobarbital-induced activities of δ-aminolevulinic acid synthase and ferrochelatase in isolated hepatocytes from normal and experimental-diabetic rats. Insulin concentrations required to produce significant inhibition in diabetic hepatocytes were higher than in normal cells. Under similar experimental conditions, insulin decreased the basal activities of δ-aminolevulinic acid synthase and ferrochelatase in hepatocytes from normal rats; no inhibitory effect was observed on the basal activity of δ-aminolevulinic acid synthase in hepatocytes from diabetic rats. Cytochrome P-450 content of both normal and diabetic cells was not affected by insulin in absence or presence of phenobarbital. The inhibitory action of insulin was exerted even when effective concentrations of glucagon, dexamethasone, or 8-(p-chlorophenylthio)-cAMP were present.Key words: δ-aminolevulinic acid synthase, ferrochelatase, cAMP, insulin, diabetic rat hepatocytes.

Regulation of phenobarbital-induced ferrochelatase mRNA activity by dibutyryl cAMP and glucose in normal and diabetic rat hepatocytes

1992 ◽  
Vol 70 (1) ◽  
pp. 26-33 ◽  
Author(s):  
Eduardo T. Cánepa ◽  
Marcelo Páez Pereda ◽  
Elena B. C. Llambías ◽  
Moïses Grinstein
Keyword(s):  
Experimental Evidence ◽  
Rat Hepatocytes ◽  
Diabetic Rat ◽  
Dibutyryl Camp ◽  
Glucose Effect ◽  
Normal Cells ◽  
Camp Content ◽  
Normal Rat ◽  
Increased Activity

The induction of ferrochelatase activity by phenobarbital and its potentiation by dibutyryl cAMP assayed in normal rat hepatocytes are associated with increased activity of ferrochelatase mRNA. Glucose inhibits this stimulatory effect. This inhibition can be reversed with increasing concentrations of dibutyryl cAMP. The inducing effect exerted by phenobarbital on the activity of ferrochelatase mRNA in diabetic hepatocytes is greater than that observed in normal cells. This enhanced response in diabetic rat hepatocytes is neither potentiated by adding dibutyryl cAMP nor repressed by glucose. The absence of a glucose effect persists even when the endogenous cAMP content is lowered to normal levels. The results obtained in this study are consistent with those reported in other published studies of ferrochelatase activity. This adds more experimental evidence to support the concept that ferrochelatase is inducible. The results obtained suggest that ferrochelatase is more susceptible to induction with phenobarbital in diabetic rat hepatocytes than in normal rat hepatocytes.Key words: ferrochelatase mRNA activity, phenobarbital, cAMP, glucose, diabetic rat hepatocytes.

scholarly journals The roles of different protein kinases and of calmodulin in the effects of Ca2+ mobilization on 3-hydroxy-3-methylglutaryl-CoA reductase activity in isolated rat hepatocytes

1991 ◽  
Vol 273 (2) ◽  
pp. 485-488 ◽  
Author(s):  
V A Zammit ◽  
A M Caldwell
Keyword(s):  
Protein Kinase ◽  
Reductase Activity ◽  
Rat Hepatocytes ◽  
Total Activity ◽  
Isolated Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Hmg Coa Reductase ◽  
Lysosomal Proteolysis ◽  
Dependent Protein ◽  
Coa Reductase

The roles of protein kinase C, Ca2+/calmodulin-dependent protein kinase and AMP-activated protein kinase in the phosphorylation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase induced by Ca2(+)-mobilizing conditions in isolated hepatocytes were investigated. Only partial evidence for the involvement of AMP-activated kinase was found. Antagonism of calmodulin action prolonged the decrease in expressed/total activity ratio induced by vasopressin plus glucagon. Protease inhibitors active against Ca2(+)-dependent cytosolic proteases or lysosomal proteolysis did not attenuate the loss of total HMG-CoA reductase induced by glucagon plus vasopressin, but calmodulin antagonists largely prevented this effect.

Phosphorylase activation hypersensitivity in hearts of diabetic rats

1984 ◽  
Vol 246 (2) ◽  
pp. E134-E140 ◽  
Author(s):  
T. B. Miller
Keyword(s):  
Glycogen Phosphorylase ◽  
Diabetic Rats ◽  
Phosphorylase Kinase ◽  
Insulin Deficiency ◽  
Dependent Protein Kinase ◽  
Heart Perfusion ◽  
Free Intracellular Calcium ◽  
Dependent Protein ◽  
Induced Changes ◽  
Related Increase

A hypersensitivity of glycogen phosphorylase activation by epinephrine and glucagon has been demonstrated in isolated perfused working and non-working hearts from diabetic rats. Accumulation of tissue cAMP and activation of cAMP-dependent protein kinase in response to epinephrine and glucagon were no greater and usually less in hearts of diabetic than of normal rats. Insulin deficiency was not associated with greater changes in epinephrine-induced activation of glycogen phosphorylase kinase than that observed in normal hearts. Perfusion of hearts with subphysiological concentrations of calcium (0.83 mM) partially reversed the diabetes-related hypersensitivity of phosphorylase activation by epinephrine. The phosphorylase activation hypersensitivity to epinephrine was completely reversed by adrenalectomizing diabetic rats 5 days before heart perfusion, an effect potentially caused by steroid-induced changes in cardiac calcium metabolism. These data are consistent with the hypothesis that phosphorylase activation by phosphorylase kinase is allosterically increased in the diabetic due to a diabetes-related increase in free intracellular calcium concentrations.

scholarly journals Effects of vanadate on protein kinases in rat hepatocytes

1989 ◽  
Vol 262 (2) ◽  
pp. 563-567 ◽  
Author(s):  
C Villar-Palasi ◽  
J J Guinovart ◽  
A M Gómez-Foix ◽  
J E Rodriguez-Gil ◽  
F Bosch
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Activity Ratio ◽  
Glycogen Synthase ◽  
Rat Hepatocytes ◽  
Casein Kinase ◽  
Optimal Time ◽  
Dependent Protein Kinase ◽  
Dependent Protein

In rat hepatocytes, vanadate modifies neither the intracellular concentration of cyclic AMP nor the -cyclic AMP/+cyclic AMP activity ratio for cyclic AMP-dependent protein kinase. Vanadate can, however, counteract the increase in cyclic AMP and the increase in the -cyclic AMP/+cyclic AMP activity ratio of cyclic AMP-dependent protein kinase induced by glucagon. On the other hand, vanadate treatment of hepatocytes can produce a time- and concentration-dependent increase in cyclic AMP- and Ca2+-independent casein kinase activity. Maximal activation at the optimal time with 5 mM-vanadate was about 70% over control. A clear relationship was observed between the activation of casein kinase and the inactivation of glycogen synthase after vanadate treatment. These results suggest that casein kinase activity may be involved in vanadate actions in rat hepatocytes.

scholarly journals Hormonal control of fructose 2,6-bisphosphate concentration in isolated rat hepatocytes

1983 ◽  
Vol 214 (3) ◽  
pp. 829-837 ◽  
Author(s):  
R Bartrons ◽  
L Hue ◽  
E Van Schaftingen ◽  
H G Hers
Keyword(s):  
Cyclic Amp ◽  
Pyruvate Kinase ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Hormonal Control ◽  
Isolated Rat Hepatocytes ◽  
Free System ◽  
Significant Difference ◽  
Dependent Protein

The ability of glucagon and of adrenaline to affect the concentration of fructose 2,6-bisphosphate in isolated hepatocytes was re-investigated because of important discrepancies existing in the literature. We were unable to detect a significant difference in the sensitivity of the hepatocytes with regard to the effect of glucagon to initiate the interconversion of phosphorylase, pyruvate kinase, 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase, and also to cause the disappearance of fructose 2,6-bisphosphate. In contrast, we have observed differences in the time-course of these various changes, since the interconversions of phosphorylase and of pyruvate kinase were at least twice as fast as those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. When measured in a cell-free system in the presence of MgATP, the cyclic AMP-dependent interconversion of pyruvate kinase was 5-10-fold more rapid than those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. These data indicate that 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase are relatively poor substrates for cyclic AMP-dependent protein kinase; they also support the hypothesis that the two catalytic activities belong to a single protein. Adrenaline had only a slight effect on the several parameters under investigation, except for the activation of phosphorylase. In the absence of Ca2+ ions from the incubation medium, however, adrenaline had an effect similar to that of glucagon.

Ca2+ and lipolysis in adipocytes from exercise-trained rats

1994 ◽  
Vol 77 (6) ◽  
pp. 2618-2624 ◽  
Author(s):  
T. Izawa ◽  
T. Komabayashi
Keyword(s):  
Protein Kinase ◽  
Adrenocorticotropic Hormone ◽  
Kinase Activity ◽  
Inhibitory Effect ◽  
Protein Kinase Activity ◽  
Dibutyryl Camp ◽  
Inhibitory Effects ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

The effects of Ca2+ on lipolysis and protein kinase activity in adipocytes from exercise-trained rats were investigated. Chronic exercise significantly increased lipolytic responses to norepinephrine and dibutyryl adenosine 3′,5′-cyclic monophosphate (cAMP). The inhibitory effects of N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a calumodulin inhibitor, on norepinephrine- and dibutyryl cAMP-stimulated lipolysis were significantly greater in trained than in sedentary rats. Training did not alter cAMP-dependent protein kinase activity. However, the inhibitory effect of W-7 on cAMP-dependent protein kinase activity was much greater in trained than in sedentary rats. The basal intracellular free Ca2+ concentration ([Ca2+]i) was significantly higher in trained than in sedentary rats. The rapid and transient increases in [Ca2+]i due to adrenocorticotropic hormone and phenylephrine from basal levels were significantly lower in trained than in sedentary rats. However, the higher basal [Ca2+]i level in trained rats led to increases in sustained [Ca2+]i levels after stimulation. We concluded that in trained rats the regulation of protein kinase activity by cAMP depends to a greater degree on Ca(2+)-calmodulin complex than it does in sedentary rats and that training alters adipocyte intracellular Ca2+ homeostasis, including [Ca2+]i responsiveness to hormones.

Reversion of an S49 cell cyclic AMP-dependent protein kinase structural gene mutant occurs primarily by functional elimination of mutant gene expression

1983 ◽  
Vol 3 (2) ◽  
pp. 250-256
Author(s):  
T van Daalen Wetters ◽  
P Coffino
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Amino Acid ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Dibutyryl Camp ◽  
Dependent Protein Kinase ◽  
Type Function ◽  
Dependent Protein

The regulatory subunits of cyclic AMP (cAMP)-dependent protein kinase from a dibutyryl cAMP-resistant S49 mouse lymphoma cell mutant, clone U200/65.1, and its revertants were visualized by two-dimensional polyacrylamide gel electrophoresis. Clone U200/65.1 co-expressed electrophoretically distinguishable mutant and wild-type subunits (Steinberg et al., Cell 10:381-391, 1977). In all 48 clones examined, reversion of the mutant to dibutyryl cAMP sensitivity was accompanied by alterations in regulatory subunit labeling patterns. Some spontaneous (3 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (2 of 11) revertants retained mutant subunits, but these were altered in charge, degree of phosphorylation, or both. The charge alterations were consistent with single amino acid substitutions, suggesting that reversion was the result of second-site mutations in the mutant regulatory subunit allele that restored wild-type function, although not wild-type structure, to the gene product. The majority of spontaneous (8 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (9 of 11) revertants and all of the revertants induced by ethyl methane sulfonate (14 of 14) and ICR191 (12 of 12) displayed only wild-type subunits. Dibutyryl cAMP-resistant mutants isolated from several of these revertants displayed new mutant but not wild-type subunits, suggesting that the revertant parent expresses only a single, functional regulatory subunit allele. The mutant regulatory subunit allele can, therefore, be modified in two general ways to produce revertant phenotypes: (i) by mutations that restore its wild-type function, and (ii) by mutations that eliminate its function.

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