Swelling, reductive stress, and cell death during chemical hypoxia in hepatocytes

1989 ◽  
Vol 257 (2) ◽  
pp. C347-C354 ◽  
Author(s):  
G. J. Gores ◽  
C. E. Flarsheim ◽  
T. L. Dawson ◽  
A. L. Nieminen ◽  
B. Herman ◽  
...  
Keyword(s):  
Cell Injury ◽  
Rat Hepatocytes ◽  
Iodoacetic Acid ◽  
Cell Killing ◽  
Cell Swelling ◽  
Reductive Stress ◽  
Chemical Hypoxia ◽  
Hydroperoxide Formation ◽  
Bleb Formation ◽  
The Relationship

In rat hepatocytes, we examined the relationship between cell volume, bleb formation, and loss of cell viability during chemical hypoxia with KCN plus iodoacetic acid. In hypotonic media (150-200 mosmol/kgH2O), cells swelled to a greater extent during chemical hypoxia than in isotonic media, but rates of cell killing were identical. Sucrose (300 mM) added to isotonic media prevented early cell swelling but actually accelerated cell killing. In contrast, mannitol (300 mM) improved cell survival but did not prevent cell swelling. Bleb formation occurred regardless of buffer tonicity. The antioxidants desferrioxamine and cyanidanol but not superoxide dismutase +/- catalase delayed lethal cell injury. Cell killing was greater during aerobic compared with anaerobic chemical hypoxia. Hydroperoxide formation was measured using a dichlorofluorescin assay and was accelerated during aerobic but not anaerobic chemical hypoxia. The results indicate that cell swelling is not the driving force for bleb formation or lethal cell injury. We conclude that “reductive stress” caused by respiratory inhibition favors formation of toxic oxygen species and may contribute to lethal cell injury during intermittent or incomplete oxygen deprivation.

Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes

1993 ◽  
Vol 264 (4) ◽  
pp. C961-C967 ◽  
Author(s):  
T. L. Dawson ◽  
G. J. Gores ◽  
A. L. Nieminen ◽  
B. Herman ◽  
J. J. Lemasters
Keyword(s):  
Reactive Oxygen Species ◽  
Rat Hepatocytes ◽  
Oxygen Radical ◽  
Cell Killing ◽  
Radical Formation ◽  
Complex Iii ◽  
Oxygen Species ◽  
Reductive Stress ◽  
Chemical Hypoxia ◽  
Hydroperoxide Formation

Cell killing, oxygen consumption, and hydroperoxide formation were determined in rat hepatocytes after glycolytic and respiratory inhibition. These conditions model the ATP depletion and reductive stress of anoxia (“chemical hypoxia”). Glycolysis was inhibited with iodoacetate, and mitochondrial electron transfer was blocked with sodium azide, cyanide, or myxothiazol. Cell killing, hydroperoxide formation, and inhibitor-insensitive oxygen consumption were greater after azide than after myxothiazol or cyanide. Desferrioxamine, an inhibitor of iron-catalyzed hydroxyl radical formation, delayed cell killing after each of the respiratory inhibitors. Anoxia also delayed cell killing during chemical hypoxia. However, during anoxic incubations, desferrioxamine did not delay the onset of cell death. These findings indicate that reactive oxygen species participate in lethal cell injury during chemical hypoxia. In isolated mitochondria, previous studies have shown that myxothiazol inhibits Q cycle-mediated ubisemiquinone formation in complex III (ubiquinol-cytochrome c oxidoreductase) and that ubisemiquinone can react with molecular oxygen to form superoxide. Decreased killing of hepatocytes with myxothiazol compared with azide suggests, therefore, that mitochondrial oxygen radical formation by complex III is involved in cell killing during reductive stress. In support of this hypothesis, myxothiazol reduced rates of cell killing and hydroperoxide formation in hepatocytes incubated with azide or cyanide. This mitochondrial mechanism for oxygen radical formation may be important in relative but not absolute hypoxia.

ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition

1994 ◽  
Vol 267 (1) ◽  
pp. C67-C74 ◽  
Author(s):  
A. L. Nieminen ◽  
A. K. Saylor ◽  
B. Herman ◽  
J. J. Lemasters
Keyword(s):  
Cell Death ◽  
Cell Injury ◽  
Rat Hepatocytes ◽  
Cell Killing ◽  
Atp Depletion ◽  
Mitochondrial Depolarization ◽  
Minimum Concentration ◽  
Atp Formation ◽  
Mitochondrial Atp Synthase ◽  
Dependent Cell

The importance of ATP depletion and mitochondrial depolarization in the toxicity of cyanide, oligomycin, and carbonyl cyanide m-cholorophenylhydrazone (CCCP), an uncoupler, was evaluated in rat hepatocytes. Oligomycin, an inhibitor of the reversible mitochondrial ATP synthase (F1F0-adenosinetriphosphatase), caused dose-dependent cell killing with 0.1 microgram/ml being the minimum concentration causing the maximum cell killing. Oligomycin also caused rapid ATP depletion without causing mitochondrial depolarization. Fructose (20 mM), a potent glycolytic substrate in liver, protected completely against oligomycin toxicity. CCCP (5 microM) also caused rapid killing of hepatocytes. Fructose retarded cell death caused by CCCP but failed to prevent lethal cell injury. Although oligomycin (1.0 microgram/ml) was lethally toxic by itself, in the presence of fructose it protected completely against CCCP-induced cell killing. Cyanide (2.5 mM), an inhibitor of mitochondrial respiration, caused rapid cell killing that was reversed by fructose. CCCP completely blocked fructose protection against cyanide, causing mitochondrial depolarization and rapid ATP depletion. In the presence of fructose and cyanide, oligomycin protected cells against CCCP-induced ATP depletion and cell death but did not prevent mitochondrial depolarization. In every instance, cell killing was associated with ATP depletion, whereas protection against lethal cell injury was associated with preservation of ATP. In conclusion, protection by fructose against toxicity of cyanide, oligomycin, and CCCP was mediated by glycolytic ATP formation rather than by preservation of the mitochondrial membrane potential. These findings support the hypothesis that inhibition of cellular ATP formation is a crucial event in the progression of irreversible cell injury.

Exogenous fructose 1,6-bisphosphate reduces K+ permeability in isolated rat hepatocytes

1997 ◽  
Vol 273 (2) ◽  
pp. C473-C478 ◽  
Author(s):  
T. Roig ◽  
R. Bartrons ◽  
J. Bermudez
Keyword(s):  
Protective Effect ◽  
Cell Injury ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Channel Arrest ◽  
Metabolic Fluxes ◽  
Fructose 1,6 Bisphosphate ◽  
The Relationship ◽  
K Permeability ◽  
Isolated Rat

The relationship between the protective effect of fructose 1,6-bisphosphate (F-1,6-P2) against cell injury and the modifications produced in the metabolic fluxes and in the membrane permeability to K+ was studied in isolated rat hepatocytes. Incubation of these cells in the presence of F-1,6-P2 reduced metabolic activity without affecting the ATP content, which suggests a downregulation of the ATP turnover. Using 86Rb+ as a tracer, we analyzed the relationship between these metabolic changes and alterations in K+ fluxes. In the presence of F-1,6-P2 the passive and the active K+ fluxes in hepatocytes decreased. However, the Na(+)-K+ pump from semipurified membranes was not directly affected by F-1,6-P2, which suggests a secondarily induced reduction of Na(+)-K+ pump activity. Moreover, galactosamine-treated cells showed a marked increase in permeability to K+ that was abolished by the presence of F-1,6-P2. This protective effect may be related to the prevention of K+ efflux. The results reported here strongly suggest the induction of channel arrest, and the associated metabolic downregulation, as the primary protective effect of F-1,6-P2, as has been shown in the prevention of galactosamine-induced hepatotoxicity.

Cytosolic free magnesium, ATP, and blebbing during chemical hypoxia in cultured rat hepatocytes

1990 ◽  
Vol 170 (2) ◽  
pp. 477-483 ◽  
Author(s):  
Andrew W. Harman ◽  
Anna-Liisa Nieminen ◽  
John J. Lemasters ◽  
Brian Herman
Keyword(s):  
Rat Hepatocytes ◽  
Chemical Hypoxia ◽  
Cultured Rat Hepatocytes ◽  
Free Magnesium

Effect of osmotic swelling and shrinkage on Na(+)-K+ pump activity in mammalian cardiac myocytes

1993 ◽  
Vol 265 (5) ◽  
pp. C1201-C1210 ◽  
Author(s):  
D. W. Whalley ◽  
L. C. Hool ◽  
R. E. Ten Eick ◽  
H. H. Rasmussen
Keyword(s):  
Ventricular Myocytes ◽  
Pump Current ◽  
Hill Equation ◽  
Cell Swelling ◽  
Intracellular Na Activity ◽  
Single Ventricular Myocytes ◽  
Hyperosmolar Solutions ◽  
Hill Coefficients ◽  
The Hill ◽  
The Relationship

The effect on the sarcolemmal Na(+)-K+ pump of exposure to anisosmolar solutions was examined using whole cell patch clamping and ion-selective microelectrodes. Na(+)-K+ pump currents were measured in single ventricular myocytes by using pipette Na+ concentrations ([Na]pip) of 0-70 mM. The relationship between [Na]pip and pump current was well described by the Hill equation. The [Na]pip for half-maximal pump current (K0.5) was 21.4 mM in isosmolar (310 mosM) solution. K0.5 was 12.8 mM during cell swelling in hyposmolar solution (240 mosM) and 39.0 mM during cell shrinkage in hyperosmolar solution (464 mosM). The maximal pump currents, derived from the best fit of the Hill equation, and the Hill coefficients were similar in isosmolar, hyposmolar, and hyperosmolar solutions. A sustained (> 20 min) decrease in the intracellular Na+ activity developed during exposure of intact papillary muscles to hyposmolar solutions, and a sustained increase developed during exposure to hyperosmolar solutions. We conclude that osmotic myocyte swelling stimulates the sarcolemmal Na(+)-K+ pump at near-physiological levels of intracellular Na+, whereas shrinkage inhibits the pump. These changes are due to increases and decreases, respectively, in the apparent affinity of the pump for Na+.

Beneficial effect of taurine depletion on osmotic sodium and calcium loading during chemical hypoxia

2002 ◽  
Vol 282 (5) ◽  
pp. C1113-C1120 ◽  
Author(s):  
Stephen W. Schaffer ◽  
Viktoriya Solodushko ◽  
David Kakhniashvili
Keyword(s):  
Cell Injury ◽  
Control Cell ◽  
Calcium Handling ◽  
Hypoxic Injury ◽  
Treated Cell ◽  
Neonatal Cardiomyocytes ◽  
Chemical Hypoxia ◽  
Calcium Loading ◽  
Taurine Deficiency ◽  
Osmotic Load

Cellular sodium excess is cytotoxic because it increases both the intracellular osmotic load and intracellular calcium concentration ([Ca2+]i). Because sodium levels rise during hypoxia, it is thought to contribute to hypoxic injury. Thus the present study tested the hypothesis that taurine-linked reductions in [Na+]i reduce hypoxia-induced cell injury. Taurine depletion was achieved by exposing isolated neonatal cardiomyocytes to medium containing the taurine analog β-Alanine. As predicted, the β-Alanine-treated cell exhibited less hypoxia-induced necrosis and apoptosis than the control, as evidenced by less swelling, shrinkage, TdT-mediated dUTP nick end labeling staining, and accumulation of trypan blue. After 1 h of chemical hypoxia, [Na+]i was 3.5-fold greater in the control than the taurine-deficient cell. Although more taurine was lost from the control cell than from the β-Alanine-treated cell during hypoxia, the combined taurine and sodium osmotic load was lower in the β-Alanine-treated cell. Taurine deficiency also reduced the degree of hypoxia-induced calcium overload. Thus the observed resistance against hypoxia-induced necrosis and apoptosis is probably related to an improvement in sodium and calcium handling.

O.04 Cell swelling increases the expression of the α2-macroglobulingene in rat hepatocytes

1998 ◽  
Vol 17 ◽  
pp. 1-2
Author(s):  
D. Meisse ◽  
S. Renouf ◽  
A. Husson ◽  
A. Lavoinne
Keyword(s):  
Rat Hepatocytes ◽  
Cell Swelling

scholarly journals Insulin-responsive cultured foetal-rat hepatocytes. Their preparation and characterization

1984 ◽  
Vol 223 (1) ◽  
pp. 39-46 ◽  
Author(s):  
D C DeSante ◽  
L Little ◽  
D E Peavy ◽  
F Vinicor
Keyword(s):  
Monolayer Culture ◽  
Cultured Cells ◽  
Rat Hepatocytes ◽  
Dependent Manner ◽  
Deoxyribonuclease I ◽  
Haematopoietic Cells ◽  
Foetal Rat ◽  
The Relationship ◽  
Dose Dependent ◽  
Scatchard Plots

An improved non-perfusion method for the preparation of cultured foetal-rat hepatocytes is described. Digestion of the liver with collagenase and deoxyribonuclease I gave yields of 40 × 10(6) hepatocytes/g of liver. The plating efficiency of hepatocytes in medium with 10 microM-cortisol was 50%. Cell morphology and metabolism were maintained through 3 days of monolayer culture, with minimal contamination by haematopoietic cells or fibroblasts. The cultured cells bound and degraded 125I-insulin in a time- and dose-dependent manner. The estimated ED50 for competitive binding at 37 degrees C was 1.1 nM. Curvilinear Scatchard plots were observed, with estimates of 16 500 high-affinity sites (Kd = 813 pM) and 53 000 low-affinity sites (Kd = 23 nM) per cell. The cultured cells demonstrated a glycogenic response to insulin, with an estimated ED50 of 120 pM. The degree of glycogenic response to insulin varied with time in culture: 500% above basal on day 1, 200% on day 2, and only 150% on day 3. Cultured foetal cells also exhibited a time-dependent uptake of 2-aminoisobutyric acid, which, in contrast with previous reports with adult cells, was not stimulated by the presence of 10 nM-insulin. Cultured foetal hepatocytes may provide an interesting model with which to study the relationship between insulin-receptor binding and insulin action.

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