Régulation du métabolisme du glycogène dans des hépatocytes isolés de rat

dissertn: Diss. Doct

Substrate cycling between 5-amino-4-imidazolecarboxamide riboside and its monophosphate in isolated rat hepatocytes.

AICA (5-amino-4-imidazolecarboxamide)-riboside is taken up by isolated rat hepatocytes and converted by adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) into AICAR (ZMP), an intermediate of the de novo synthesis of purine nucleotides. We investigated if, in these cells, a cycle analogous to the adenosine-AMP substrate cycle operates between AICAriboside and ZMP. When 50 microM ITu, an inhibitor of adenosine kinase, was added to hepatocytes that had metabolized AICAriboside for 30 min, the concentration of ZMP decreased immediately. This was mirrored by a reincrease of AICAriboside. Rates of the ITu-induced decrease of ZMP and the increase of AICAriboside, calculated at different concentrations of ZMP, were first order, up to the highest concentration of ZMP (approx. 5 mumol/g of cells). Dephosphorylation of ZMP added to crude cytosolic extracts of rat liver displayed hyperbolic kinetics, with a Vmax of 0.65 mumol/min per g protein and an apparent Km of 5 mM, and was markedly inhibited by Pi, an inhibitor of IMP-GMP 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5). We conclude that hepatocyte ZMP is continuously dephosphorylated, most likely by IMP-GMP 5'-nucleotidase, into AICAriboside, which is rephosphorylated into ZMP by adenosine kinase. Substrate cycling was also shown to occur between other nucleoside analogs and their phosphorylated counterparts

Substrate cycling between 5-amino-4-imidazolecarboxamide riboside and its monophosphate in isolated rat hepatocytes.

AICA (5-amino-4-imidazolecarboxamide)-riboside is taken up by isolated rat hepatocytes and converted by adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) into AICAR (ZMP), an intermediate of the de novo synthesis of purine nucleotides. We investigated if, in these cells, a cycle analogous to the adenosine-AMP substrate cycle operates between AICAriboside and ZMP. When 50 microM ITu, an inhibitor of adenosine kinase, was added to hepatocytes that had metabolized AICAriboside for 30 min, the concentration of ZMP decreased immediately. This was mirrored by a reincrease of AICAriboside. Rates of the ITu-induced decrease of ZMP and the increase of AICAriboside, calculated at different concentrations of ZMP, were first order, up to the highest concentration of ZMP (approx. 5 mumol/g of cells). Dephosphorylation of ZMP added to crude cytosolic extracts of rat liver displayed hyperbolic kinetics, with a Vmax of 0.65 mumol/min per g protein and an apparent Km of 5 mM, and was markedly inhibited by Pi, an inhibitor of IMP-GMP 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5). We conclude that hepatocyte ZMP is continuously dephosphorylated, most likely by IMP-GMP 5'-nucleotidase, into AICAriboside, which is rephosphorylated into ZMP by adenosine kinase. Substrate cycling was also shown to occur between other nucleoside analogs and their phosphorylated counterparts

Pathways and control of adenine nucleotide catabolism in anoxic rat hepatocytes.

Studies are reviewed that show that in isolated rat hepatocytes subjected to anoxia, the catabolism of AMP, leading to uric acid instead of to allantoin in normoxia, proceeds almost exclusively by deamination of AMP followed by dephosphorylation of IMP. Adenosine, which is nearly undetectable in normoxic cell suspensions, accumulates to a slight extent in anoxia. The regulatory properties of liver AMP deaminase and cytosolic IMP-GMP 5'-nucleotidase were found to provide protective mechanisms for the hepatic adenine nucleotide pool in hypoxia

Mechanism of Adenosine-triphosphate Catabolism Induced By Deoxyadenosine and By Nucleoside Analogs in Adenosine Deaminase-inhibited Human-erythrocytes

The mechanism of the depletion of ATP, recorded in the erythrocytes of adenosine deaminase-deficient children and of leukemia patients treated with deoxycoformycin, was investigated in normal human erythrocytes treated with this inhibitor of adenosine deaminase. Deoxyadenosine, which accumulates in both clinical conditions, provoked a dose-dependent accumulation of dATP, depletion of ATP, and increases in the production of inosine plus hypoxanthine. Concomitantly, there was an increase of AMP and IMP, but not of adenosine, indicating that catabolism proceeded by way of AMP deaminase. A series of nucleoside analogues (9-beta-D-arabinofuranosyladenine, N6-methyladenosine, 6-methylmercaptopurine ribonucleoside, tubercidin, ribavirin, and N-1-ribosyl-5-aminoimidazole-4-carboxamide riboside) also stimulated adenine nucleotide catabolism and increased AMP and IMP to various extents. The effects of deoxyadenosine and of the nucleoside analogues were prevented by 5'-iodotubercidin, an inhibitor of adenosine kinase. Strikingly, they were reversed if the inhibitor was added after the accumulation of nucleotide analogues and initiation of adenine nucleotide catabolism. Further analyses revealed linear relationships between the rate of phosphorylation of deoxyadenosine and nucleoside analogues and the increase in AMP and between the elevation of the latter above a threshold concentration of 10 microM and the rate of adenine nucleotide catabolism. Kinetic studies with purified erythrocytic AMP deaminase, at physiological concentrations of its effectors, showed that the enzyme is nearly inactive up to 10 microM AMP and increases in activity above this threshold. We conclude that the main mechanism whereby deoxyadenosine and nucleoside analogues stimulate catabolism of adenine nucleotides by way of AMP deaminase in erythrocytes is elevation of AMP, secondary to the phosphorylation of the nucleosides

Adenine nucleotide catabolism in human erythrocytes: pathways and regulation.

Studies are reviewed which show that in human erythrocytes the catabolism of AMP, leading to hypoxanthine, proceeds by way of AMP deaminase under physiological conditions as well as upon alkalinization and addition of deoxyadenosine. In contrast, the catabolism induced by glucose deprivation, proceeds for 75% via dephosphorylation of AMP. These findings can be explained by the kinetic properties of erythrocytic AMP deaminase, which were investigated at concentrations of substrate and effectors in the physiological range