Effect of ethanol on adenosine triphosphate, cytosolic free calcium, and cell injury in rat hepatocytes (original) (raw)
Related papers
Archives of Biochemistry and Biophysics, 1998
The effect of chronic ethanol consumption on hepatic Previous studies (Ivester et al., Arch. Biochem. Bio-energy state is complex. Recent studies have demonphys. 322, 14-21, 1995) have established that periportal strated that ATP concentrations, phosphorylation poand perivenous hepatocytes isolated from ethanol-fed tential, and energy charge in liver or hepatocytes from rats demonstrate lower ATP concentrations than ethanol-fed rats are normal if the preparations are adethose in control preparations when the cells are mainquately oxygenated (1, 2). In contrast, if the tissue or tained at very low oxygen tension. In the present inveshepatocytes are allowed to become anoxic, the energy tigation, experiments were implemented with periporstate decreases to significantly lower values in preparatal and perivenous hepatocytes to determine the eftions from ethanol-fed rats as compared with control fects of chronic ethanol consumption on cellular animals (1, 2). This latter observation is consistent respiratory and glycolytic activities, since both conwith earlier reports of ethanol-related decreases in hetribute to maintenance of the energy state of the liver patic energy state measured in tissues allowed to go cell. Both periportal and perivenous hepatocytes from anaerobic before being freeze-clamped for adenine nuethanol-fed rats demonstrated significantly increased, cleotide analyses (3-6). This ethanol-related effect on rather than decreased, respiratory activity when monhepatic energy state has also been observed in intact itored with oxygen concentrations ranging from 16 to animals. French and co-workers (7), using in vivo NMR 140 mM. Whole liver hepatocytes from control and ethspectroscopy, demonstrated more dramatic decreases anol-fed animals demonstrated equivalent oxygen utiin hepatic ATP in ethanol-fed rats than in controls lization, however. Glycolytic activity, monitored by when the animals were subjected to a hypoxic episode. lactate / pyruvate concentrations obtained after both These studies emphasize the increased sensitivity of anaerobic and aerobic incubation protocols, was decreased in both cell types from ethanol-fed animals. liver energy state to oxygen tension in animals sub-The glycogen concentrations in freshly isolated perijected to chronic ethanol administration. portal and perivenous hepatocytes were also de-1 This project was supported by Grant 02887 from the National Since chronic consumption damages hepatic mito-Institute on Alcohol Abuse and Alcoholism. C.G.V.H. was supported chondria such that there are depressions in the rates by NIAAA Training Grant 07565. of electron transport activity (8, 9) and of ATP synthe-2 To whom correspondence should be addressed at Wake Forest sis (10), measurements of oxygen utilization in soni
Ethanol-induced increases in [Ca2+]i and inositol (1,4,5) triphosphate in rat hepatocytes
Biochemical and Biophysical Research Communications, 1990
Rat hepatocytes were studied for [Ca2+]i with Fura-2 at the single cell level using a microfluorometer-imaging system which showed that both the number of cells elevating [Ca2+]i and the magnitude of [Ca2+]i increase were directly dependent upon ethanol concentration between 50 mM and 1 M. Peak [Ca2+]i increases ranged from 27 nM with 50 mM ethanol to 57 nM after 1 M ethanol. Ethanol appeared to initiate calcium release from intracellular stores and caused a dose dependent production of inositol(1,4,5) triphosphate (Ins(1,4,5)P3) in hepatocytes. Low concentrations of ethanol (50-100 mM) did not significantly raise Ins(1,4,5)P3 although 300 mM-1 M increased Ins(1,4,5)P3 comparable to that found with vasopressin (5 nM). In summary, physiologic amounts of ethanol raise [Ca2+]i in rat hepatocytes, although at lower levels (50-100 mM) the changes may or may not be related to an Ins(1,4,5)P3 pathway.
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2011
Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD + ratio, and the concentrations of SAM and SAH in the liver tissue were determined by highperformance liquid chromatography. The increase in the NADH/NAD + ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD +-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD + ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADHdependent enzyme lactate dehydrogenase, restored the NADH/NAD + ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD + redox couple and the effects of ethanol on methionine metabolism in the liver.
Effects of adenosine on ethanol-induced modifications of liver metabolism
Biochemical Pharmacology, 1980
total prevention of ethanol-induced fatty liver by the simultaneous administration of adenosine and allopurinol was observed. Under these conditions, adenosine ameliorated the reduction in the cytoplasmic NAD+/NADH ratio produced by ethanol metabolism, increased the rate of ethanol oxidation, and decreased the blood ketone bodies, reflecting an inhibition of hepatic fatty acid oxidation. Moreover, in rats treated with 4-methylpyrazole, the effect of the nucleoside on the increased rate of ethanol oxidation was not observed. The effect of adenosine on the modifications induced by the administration of ethanol in the mitochondrial redox potential was tested. When the nucleoside was administered to ethanol-treated animals, a reversal of the ethanol-induced diminution in the mitochondrial NAD+/NADH ratio and in the redox potential was observed after 2-4 hr of treatment. These data lend further support to the suggestion that adenosine promotes the capacity of the cell to handle the reducing equivalents generated during ethanol metabolism. Moreover, these experiments suggest that hydrogen peroxide, generated through purine metabolism, plays a minimal role in the action of adenosine on ethanol metabolism. Finally, a second mechanism by which the nucleoside prevents fatty liver in the presence of allopurinol was evident and this was related to an inhibition of fatty acid metabolism.
Dependence on dose of the acute effects of ethanol on liver metabolism in vivo
Journal of Clinical Investigation, 1975
A B S T R A C T The dose dependence of the acute effects of ethanol upon liver intermediary metabolism in vivo has been demonstrated in rats. Ethanol was given i.p. in doses of 0.69, 1.7, and 3.0 g/kg in equal volumes (20 ml/kg). The liver was freeze-clamped 120 min after injection, and multiple metabolites were measured in the perchloric acid extract of the tissue. Each group showed a significantly different pattern of metabolites, redox states, and phosphorylation potentials although the rate of ethanol disappearance, at least between the two highest dose groups, was not significantly different. The mitochondrial free [NAD+]/[NADH] ratios and the cytoplasmic free [NADP+]/ [NADPH] ratio were paradoxically most reduced with the lowest dose of ethanol and became progressively more oxidized with increasing dose. Once established, the differences in these ratios between the groups tended to persist with time, relatively independent of the concentration of ethanol. In a somewhat different pattern, the phosphorylation potential ([ATP]/[ADP][P.]) remained at the control level in the low-dose group but was significantly elevated in the two higher-dose groups.
Modulation of liver-specific cellular response to ethanol in vitro in hep G2 cells
Toxicology in vitro : an international journal published in association with BIBRA, 1998
The aim of this study was to investigate in vitro in a human hepatoblastoma cell line, Hep G2, the effect of ethanol (EtOH) toxicity. The ultrastructural changes were assessed by performing quantitative light and transmission electron microscopy. The second objective of this study was to define further EtOH-induced biochemical changes associated with mitochondrial function. In comparison with controls, after exposure to 80 mm EtOH cells showed: a threefold increase in length of mitochondria; proliferation, vesiculation and dilatation of smooth endoplasmic reticulum, and twofold increases in the size of lipid droplets and in their number/cell. Exposure of cells to two doses of EtOH augmented the ultrastructural alterations observed after a single dose. Cytoviability, assessed by metabolism of methylxanthine dye decreased significantly by (P < 0.0001) to 68% of the control after one dose and was further reduced after the second dose of EtOH (P < 0.001). Succinate dehydrogenase a...
Cellular energy charge in the heart and liver of the rat. The effects of ethanol and acetaldehyde
1986
The effects of ethanol and acetaldehyde upon adenine nucleotide concentrations in rat heart and liver were determined. 2. Ethanol administered either acutely (8 g kg °73) or chronically (20% solution in drinking water for 21 d) significantly decreased ATP concentrations, adenylate energy charge (EC) and adenylate kinase mass action ratio (I-AK) in liver but affected FAr ` only in heart. 3. Acetaldehyde treatment elicited similar effects but of lesser magnitude.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1987
In this study, a pronounced increase of ethanol oxidation was found in hepatocytes obtained from adenosine-treated rats, or after in vitro additional of the nucleoside; this finding was accompanied by a maintenance of the normal cytoplasmic redox state. These results suggest a higher availability of cytoplasmic NAD in these cells. Therefore, the metabolic pathways which carry out the reoxidation of cytosolic reducing equivalents, namely, malate-aspartate and alpha-glycerophosphate shuttles, were examined. Isolated mitochondria from adenosine-treated rats had an increased NADH oxidation by the malate-aspartate shuttle; furthermore, in vivo and in vitro addition of adenosine to the hepatocytes induced changes in the equilibrium of the malate-aspartate shuttle, as evidenced by the subcellular distribution of the intermediates of this pathway. Acetaldehyde removal was also increased by adenosine and this fact was related to an elevated NAD/NADH ratio in the mitochondria. Thus, under these conditions, an increased ethanol uptake was accompanied by enhanced acetaldehyde removal in the animal. In conclusion, adenosine administration stimulates the transport of cytoplasmic reducing equivalents to the mitochondria, mainly through the malate-aspartate shuttle. This action, which may be located at the level of the mitochondrial membrane, is reflected by an enhancement of ethanol and acetaldehyde oxidations.
Ethanol potentiates hypoxic liver injury: role of hepatocyte Na+ overload
Biochimica Et Biophysica Acta: Molecular Basis Of Disease, 2000
Centrilobular hypoxia has been suggested to contribute to hepatic damage caused by alcohol intoxication. However, the mechanisms involved are still poorly understood. We have investigated whether alterations of Na homeostasis might account for ethanol-mediated increase in hepatocyte sensitivity to hypoxia. Addition of ethanol (100 mmol/l) to isolated rat hepatocytes incubated under nitrogen atmosphere greatly stimulated cell death. An increase in intracellular Na levels preceded cell killing and Na levels in hepatocytes exposed to the combination of ethanol and hypoxia were almost twice those in hypoxic cells without ethanol. Na increase was also observed in hepatocytes incubated with ethanol in oxygenated buffer. Ethanol addition significantly lowered hepatocyte pH. Inhibiting ethanol and acetaldehyde oxidation with, respectively, 4-methylpyrazole and cyanamide prevented this effect. 4-methylpyrazole, cyanamide as well as hepatocyte incubation in a HCO 3 3-free buffer or in the presence of Na /H exchanger blocker 5-(N,N-dimethyl)-amiloride also reduced Na influx in ethanol-treated hepatocytes. 4-methylpyrazole and cyanamide similarly prevented ethanol-stimulated Na accumulation and hepatocyte killing during hypoxia. Moreover, ethanol-induced Na influx caused cytotoxicity in hepatocytes pre-treated with Na ,K-ATPase inhibitor ouabain. Also in this condition 4-methylpyrazole and 5-(N,Ndimethyl)-amiloride decreased cell killing. These results indicate that ethanol can promotes cytotoxicity in hypoxic hepatocytes by enhancing Na accumulation.