Nuclear Accumulation of G-Actin in Isolated Rat Hepatocytes by Adenine Nucleotides

Incubation of hepatocytes from 24 h-starved rats in the presence of 0.5 mM-adenosine decreased gluconeogenesis from lactate, but not from alanine. The inhibition of gluconeogenesis was associated with a stimulation of ketone-body production and an inhibition of pyruvate oxidation. These metabolic changes were suppressed in the presence of iodotubercidin (an inhibitor of adenosine kinase), but were reinforced in the presence of deoxycoformycin (an inhibitor of adenosine deaminase); 2-chloroadenosine induced no change in gluconeogenesis from lactate. These data indicate that the inhibition of gluconeogenesis by adenosine probably results from its conversion into adenine nucleotides. In the presence of lactate or pyruvate, but not with alanine or asparagine, this conversion resulted in a decrease in the [ATP]/[ADP] ratio in both mitochondrial and cytosolic compartments. Adenosine decreased the Pi concentration with all gluconeogenic substrates.

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Homocysteine enhances the inhibitory effect of extracellular adenosine on the synthesis of proteins in isolated rat hepatocytes

Biochemical Journal ◽

10.1042/bj3100893 ◽

1995 ◽

Vol 310 (3) ◽

pp. 893-896 ◽

Cited By ~ 12

Author(s):

S Tinton ◽

P Buc-Calderon

Keyword(s):

Protein Synthesis ◽

Kinase Inhibitor ◽

Adenine Nucleotides ◽

Rat Hepatocytes ◽

Isolated Hepatocytes ◽

Adenosine Kinase ◽

Isolated Rat Hepatocytes ◽

Extracellular Adenosine ◽

Inhibition Of Protein Synthesis ◽

Isolated Rat

Previous work has shown that extracellular adenosine inhibits the incorporation of radiolabelled leucine into proteins in isolated rat hepatocytes [Tinton, Lefebvre, Cousin and Buc Calderon (1993) Biochim. Biophys. Acta 1176, 1-6]. In this study, we investigated whether its metabolism into adenine nucleotides, inosine or S-adenosylhomocysteine (AdoHcy) is required to induce such an impairment. Incubation of isolated hepatocytes in the presence of adenosine at 0.5 or 1 mM reduces the synthesis of proteins by about 45% after 120 min of incubation. Such an inhibition occurred without cell lysis and was not modified by adding the adenosine kinase inhibitor 5-iodotubercidin (15 microM) or the adenosine deaminase inhibitor coformycin (0.1 microM). It is therefore unlikely that the anabolic and catabolic pathways of adenosine are involved in the inhibition of protein synthesis. Adenosine (1 mM) increased the level of AdoHcy and S-adenosylmethionine by 20- and 5-fold respectively after 60 min of incubation and reduced the methylation index. These events as well as the inhibition of protein synthesis were strongly enhanced in the presence of L-homocysteine (2 mM). It is therefore concluded that the metabolism of adenosine into AdoHcy, which is known to be a potent inhibitor of cellular methylation reactions, may play an important role in the control of translation.

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Purine catabolism in isolated rat hepatocytes. Influence of coformycin

Biochemical Journal ◽

10.1042/bj1880913 ◽

1980 ◽

Vol 188 (3) ◽

pp. 913-920 ◽

Cited By ~ 67

Author(s):

Georges Van Den Berghe ◽

Françoise Bontemps ◽

Henri-Géry Hers

Keyword(s):

High Speed ◽

Adenine Nucleotide ◽

Adenine Nucleotides ◽

Rat Hepatocytes ◽

Amp Deaminase ◽

Purine Nucleotides ◽

Isolated Rat Hepatocytes ◽

Adenine Nucleotide Pool ◽

Isolated Rat

1. The catabolism of purine nucleotides was investigated by both chemical and radiochemical methods in isolated rat hepatocytes, previously incubated with [14C]adenine. The production of allantoin reached 32±5nmol/min per g of cells (mean±s.e.m.) and as much as 30% of the radioactivity incorporated in the adenine nucleotides was lost after 1h. This rate of degradation is severalfold in excess over values previously reported to occur in the liver in vivo. An explanation for this enhancement of catabolism may be the decrease in the concentration of GTP. 2. In a high-speed supernatant of rat liver, adenosine deaminase was maximally inhibited by 0.1μm-coformycin. The activity of AMP deaminase, measured in the presence of its stimulator ATP in the same preparation, as well as the activity of the partially purified enzyme, measured after addition of its physiological inhibitors GTP and Pi, required 50μm-coformycin for maximal inhibition. 3. The production of allantoin by isolated hepatocytes was not influenced by the addition of 0.1μm-coformycin, but was decreased by concentrations of coformycin that were inhibitory for AMP deaminase. With 50μm-coformycin the production of allantoin was decreased by 85% and the formation of radioactive allantoin from [14C]adenine nucleotides was completely suppressed. 4. In the presence of 0.1μm-coformycin or in its absence, the addition of fructose (1mg/ml) to the incubation medium caused a rapid degradation of ATP, without equivalent increase in ADP and AMP, followed by transient increases in IMP and in the rate of production of allantoin; adenosine was not detectable. In the presence of 50μm-coformycin, the fructose-induced breakdown of ATP was not modified, but the depletion of the adenine nucleotide pool proceeded much more slowly and the rate of production of allantoin increased only slightly. No rise in IMP concentration could be detected, but AMP increased manyfold and reached values at which a participation of soluble 5′-nucleotidase in the catabolism of adenine nucleotides is most likely. 5. These results are in agreement with the hypothesis that the formation of allantoin is controlled by AMP deaminase. They constitute further evidence that 5′-nucleotidase is inactive on AMP, unless the concentration of this nucleotide rises to unphysiological values.

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Fructose-induced adenine nucleotide catabolism in isolated rat hepatocytes

Canadian Journal of Biochemistry ◽

10.1139/o77-185 ◽

1977 ◽

Vol 55 (12) ◽

pp. 1237-1240 ◽

Cited By ~ 14

Author(s):

Camilla M. Smith ◽

Liisa M. Rovamo ◽

Kari O. Raivio

Keyword(s):

Adenosine Deaminase ◽

Adenine Nucleotide ◽

Adenine Nucleotides ◽

Rat Hepatocytes ◽

Amp Deaminase ◽

Isolated Rat Hepatocytes ◽

Previous Hypothesis ◽

Adenine Nucleotide Pool ◽

Isolated Rat

The mechanism of fructose-induced nucleotide catabolism was studied using isolated rat hepatocytes in which the adenine nucleotide pool was prelabelled with [14C]adenine. Incubation of these cells with fructose caused a rapid depletion of the adenine nucleotides and a corresponding increase in allantoin. There was no accumulation of radioactivity in adenosine in the presence or absence of the adenosine deaminase inhibitor 9-erythro-(2-hydroxy-3-nonyl)adenine. This confirms the previous hypothesis that fructose-induced adenine nucleotide catabolism occurs by way of AMP deaminase (AMP amino-hydrolase, EC 3.5.4.6).

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Mechanisms of elevation of adenosine levels in anoxic hepatocytes

Biochemical Journal ◽

10.1042/bj2900671 ◽

1993 ◽

Vol 290 (3) ◽

pp. 671-677 ◽

Cited By ~ 42

Author(s):

F Bontemps ◽

M F Vincent ◽

G Van den Berghe

Keyword(s):

Adenine Nucleotides ◽

Rat Hepatocytes ◽

Adenosine Kinase ◽

Slight Effect ◽

Isolated Rat Hepatocytes ◽

Adenosylhomocysteine Hydrolase ◽

Adenosine Concentration ◽

Isolated Rat ◽

Rate Of Accumulation ◽

In Cells

Previous work has shown that normoxic isolated rat hepatocytes continuously produce adenosine from AMP and that the nucleoside is not catabolized further but immediately rephosphorylated by adenosine kinase [Bontemps, Van den Berghe and Hers (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2829-2833]. We now report the effect of anoxia on adenosine production and on the AMP/adenosine substrate cycle. In cell suspensions incubated in O2/CO2, the adenosine concentration was about 0.4 microM. It increased 30-fold in cells incubated in N2/CO2 or with 5 mM KCN, and 20-fold in cells incubated with 2 mM amytal. Adenosine production, measured in hepatocytes in which adenosine kinase and adenosine deaminase were inhibited by 5-iodotubercidin and deoxycoformycin respectively, was about 18 nmol/min per g of cells in normoxia; it increased about 2-fold in anoxia, although AMP increased 8-16-fold in this condition. From studies with inhibitors of membrane 5′-nucleotidase and of S-adenosylhomocysteine hydrolase, it was deduced that adenosine is produced by the latter enzyme and by cytosolic 5′-nucleotidase in normoxia, and by cytosolic and membrane 5′-nucleotidases in anoxia. Unlike in normoxic hepatocytes, inhibition of adenosine kinase by 5-iodotubercidin neither elevated the adenosine concentration nor enhanced total purine release from adenine nucleotides in cells treated with N2/CO2 or KCN; it had only a slight effect in cells treated with amytal. This indicates that recycling of adenosine is suppressed or profoundly inhibited in anoxia. The rate of accumulation of adenosine in anoxia was several-fold lower than the rate of its rephosphorylation upon reoxygenation. It is concluded that the elevation of adenosine in anoxic hepatocytes is much more dependent on decreased recycling of adenosine by adenosine kinase than on increased production by dephosphorylation of AMP.

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The pathway of adenine nucleotide catabolism and its control in isolated rat hepatocytes subjected to anoxia

Biochemical Journal ◽

10.1042/bj2020117 ◽

1982 ◽

Vol 202 (1) ◽

pp. 117-123 ◽

Cited By ~ 44

Author(s):

Marie-Françoise Vincent ◽

Georges Van Den Berghe ◽

Henri-Géry Hers

Keyword(s):

Uric Acid ◽

Adenine Nucleotide ◽

Adenine Nucleotides ◽

Rat Hepatocytes ◽

Urate Oxidase ◽

Amp Deaminase ◽

Isolated Rat Hepatocytes ◽

Adenine Nucleotide Pool ◽

Glycogen Stores ◽

Isolated Rat

1. The breakdown of the adenine nucleotide pool provoked by the replacement of the O2/CO2 gas phase by N2/CO2 was studied in isolated rat hepatocytes with the purpose of defining the pathway of the catabolism of AMP in anoxic conditions. 2. Approx. 40% of the adenine nucleotide pool was lost after 40–60 min of anoxia. In hepatocytes from fed rats there was a slow disappearance of ATP. This is explained by the presence of glycogen stores, allowing the generation of ATP by anaerobic glycolysis. In hepatocytes from 24h-starved rats, ATP almost completely disappeared within 5 min, and was partly replaced by an accumulation of AMP. This indicates that another mechanism protects the adenine nucleotide pool in the starved state. In both conditions, the loss of adenine nucleotides was mainly accounted for by an accumulation of uric acid, owing to the oxygen-dependence of urate oxidase. 3. Incubation of the hepatocytes before the suppression of O2 with coformycin at concentrations known to inhibit selectively adenosine deaminase did not result in an accumulation of adenosine and did not influence the formation of uric acid. This indicates that the degradation of AMP does not proceed by way of 5′-nucleotidase under these conditions. In the presence of coformycin at concentrations which are inhibitory to AMP deaminase, however, the formation of uric acid was nearly suppressed, demonstrating that the initial degradation of AMP was catalysed by the latter enzyme. 4. The accumulation of AMP in the starved state can be explained by the pronounced decrease in ATP, the major stimulator of AMP deaminase, and the enhanced increase in Pi, one of its physiological inhibitors. The modifications of these effectors can also explain the increased inhibition of the cytoplasmic 5′-nucleotidase, shown by the accumulation of IMP in the absence of coformycin, in hepatocytes from starved rats. 5. Reoxygenation of the hepatocytes after 20 min of anoxia induced a prompt regeneration of ATP, which reached concentrations equal to the pre-existing concentration of AMP. 6. No explanation was found for the accumulation of IMP observed after preincubation of the hepatocytes with 0.1μm-coformycin, since the activities of the IMP-metabolizing enzymes were not influenced by this inosine analogue.

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