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

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.

Download Full-text

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

AJP Cell Physiology ◽

10.1152/ajpcell.1989.257.2.c347 ◽

1989 ◽

Vol 257 (2) ◽

pp. C347-C354 ◽

Cited By ~ 176

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.

Download Full-text

pH-dependent nonlysosomal proteolysis contributes to lethal anoxic injury of rat hepatocytes

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1993.264.4.g744 ◽

1993 ◽

Vol 264 (4) ◽

pp. G744-G751 ◽

Cited By ~ 5

Author(s):

S. F. Bronk ◽

G. J. Gores

Keyword(s):

Cell Death ◽

Protease Inhibitor ◽

Cell Survival ◽

Protease Activity ◽

Cell Injury ◽

Rat Hepatocytes ◽

Extracellular Acidosis ◽

Anoxic Injury ◽

Lysosomal Proteolysis ◽

Ph Dependent

Our aim was to test the hypothesis that pH-dependent nonlysosomal proteolysis is a key mechanism culminating in lethal anoxic injury of rat hepatocytes. Although lysosomal proteolysis was suppressed during anoxia, total nonlysosomal proteolysis was increased twofold compared with aerobic controls. Extracellular acidosis inhibited total nonlysosomal proteolysis and improved cell survival during anoxia. Indeed, we found a direct highly significant linear relationship between cell death and total nonlysosomal proteolysis as modulated by changes in the extracellular pH (r = 0.99, P < 0.01). Glycolytic generation of ATP from fructose during anoxia suppressed total nonlysosomal proteolysis and improved cell survival. An increase in a pH-dependent calpain-like protease activity was also identified during anoxia, but calpain-like protease activity only accounted for 16% of total nonlysosomal protease activity. In addition, the calpain protease inhibitor Cbz-Leu-Leu-Tyr-CHN2 only partially protected against cell killing despite complete inhibition of calpain-like protease activity. These data suggest that pH-dependent total nonlysosomal proteolysis contributes to lethal cell injury during anoxia. However, calpain protease activity only partially contributes to total nonlysosomal protease activity and cell death.

Download Full-text

Effects of chloride channel inhibitors on H2O2-induced renal epithelial cell injury

AJP Renal Physiology ◽

10.1152/ajprenal.2000.278.1.f83 ◽

2000 ◽

Vol 278 (1) ◽

pp. F83-F90 ◽

Cited By ~ 13

Author(s):

Xianmin Meng ◽

W. Brian Reeves

Keyword(s):

Lipid Peroxidation ◽

Dna Damage ◽

Cell Death ◽

Epithelial Cell ◽

Small Molecules ◽

Cell Injury ◽

Membrane Integrity ◽

Atp Depletion ◽

Renal Epithelial Cell ◽

Epithelial Cell Injury

Oxidative stress contributes to renal epithelial cell injury in certain settings. Chloride influx has also been proposed as an important component of acute renal epithelial cell injury. The present studies examined the role of Cl− in H2O2-induced injury to LLC-PK1 renal epithelial cells. Exposure of LLC-PK1 cells to 1 mM H2O2 resulted in the following: depletion of intracellular ATP content; DNA damage; lipid peroxidation; and a loss of membrane integrity to both small molecules, e.g., trypan blue, and macromolecules, e.g., lactate dehydrogenase (LDH), and cell death. Substitution of Cl− by isethionate or the inclusion of certain Cl− channel blockers, e.g., diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino)· benzoate (NPPB), and niflumic acid, prevented the H2O2-induced loss of membrane integrity to LDH. In addition, the H2O2-induced loss of membrane integrity was prevented by raising the osmolality of the extracellular solutions, by depletion of cell ATP, and by inhibitors of volume-sensitive Cl− channels. However, these maneuvers did not prevent the H2O2-induced permeability to small molecules or H2O2-induced ATP depletion, DNA damage, lipid peroxidation, or cell death. These results support the view that volume-sensitive Cl− channels play a role in the progressive loss of cell membrane integrity during injury.

Download Full-text

Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria

AJP Cell Physiology ◽

10.1152/ajpcell.00083.2008 ◽

2008 ◽

Vol 295 (5) ◽

pp. C1247-C1260 ◽

Cited By ~ 33

Author(s):

Erin M. Baggaley ◽

Austin C. Elliott ◽

Jason I. E. Bruce

Keyword(s):

Oxidative Stress ◽

Plasma Membrane ◽

Cell Injury ◽

Mitochondrial Permeability Transition ◽

Acinar Cells ◽

Atp Depletion ◽

Pancreatic Acinar Cells ◽

Spatiotemporal Pattern ◽

Mitochondrial Depolarization

Impairment of the normal spatiotemporal pattern of intracellular Ca2+ ([Ca2+]i) signaling, and in particular, the transition to an irreversible “Ca2+ overload” response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca2+ overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H2O2) evokes a Ca2+ overload response and inhibition of plasma membrane Ca2+-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938–C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca2+]i clearance assay in which mitochondrial Ca2+ uptake was blocked with Ru-360, H2O2 (50 μM–1 mM) markedly inhibited the PMCA activity. This H2O2-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H2O2-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca2+ handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca2+ overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.

Download Full-text

Increased Uptake of Zinc in Malignant Cells is Associated with Enhanced Activation of MAPK Signalling and P53-Dependent Cell Injury

Acta Medica (Hradec Kralove Czech Republic) ◽

10.14712/18059694.2017.7 ◽

2008 ◽

Vol 51 (1) ◽

pp. 43-49 ◽

Cited By ~ 2

Author(s):

Emil Rudolf

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Cell Injury ◽

Cell Damage ◽

Zinc Homeostasis ◽

Malignant Cells ◽

Intracellular Zinc ◽

Free Zinc ◽

Dependent Cell ◽

Zinc Levels

Excess intracellular zinc has been demonstrated to be responsible for cell injury and cell death in various experimental as well as clinical models. While the cells possess a system of mechanisms regulating intracellular zinc homeostasis, their saturation by acutely increased zinc levels or by a sustained exposure to elevated zinc levels results in liberation of free zinc stores within the cells and ultimate cell damage and cell death. Here we report that in Hep-2 malignant cells enhanced uptake of zinc causes activation of mitogen-activated protein kinase (MAPK) signaling with resulting p53-dependent cell injury which can be significantly prevented by specific p53 inhibition and by prevention of oxidative stress. Our observations are consistent with the view that subacutely increased intracellular free zinc levels stimulate via oxidative stress p53-dependent pathways which are responsible for the final cell damage in tumor cells.

Download Full-text

Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide

Biochemical Journal ◽

10.1042/bj3070099 ◽

1995 ◽

Vol 307 (1) ◽

pp. 99-106 ◽

Cited By ~ 286

Author(s):

A L Nieminen ◽

A K Saylor ◽

S A Tesfai ◽

B Herman ◽

J J Lemasters

Keyword(s):

Cell Death ◽

Laser Scanning ◽

Mitochondrial Permeability Transition ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Atp Depletion ◽

Fluorescence Probes ◽

Mitochondrial Depolarization ◽

Intact Cells ◽

Mitochondrial Permeability

We have developed a novel method for monitoring the mitochondrial permeability transition in single intact hepatocytes during injury with t-butylhydroperoxide (t-BuOOH). Cultured hepatocytes were loaded with the fluorescence probes, calcein and tetramethylrhodamine methyl ester (TMRM). Depending on loading conditions, calcein labelled the cytosolic space exclusively and did not enter mitochondria or it stained both cytosol and mitochondria. TMRM labelled mitochondria as an indicator of mitochondrial polarization. Fluorescence of two probes was imaged simultaneously using laser-scanning confocal microscopy. During normal incubations, TMRM labelled mitochondria indefinitely (longer than 63 min), and calcein did not redistribute between cytosol and mitochondria. These findings indicate that the mitochondrial permeability transition pore (‘megachannel’) remained closed continuously. After addition of 100 microM t-BuOOH, mitochondria filled quickly with calcein, indicating the onset of mitochondrial permeability transition. This event was accompanied by mitochondrial depolarization, as shown by loss of TMRM. Subsequently, the concentration of ATP declined and cells lost viability. Trifluoperazine, a phospholipase inhibitor that inhibits the permeability transition in isolated mitochondria, prevented calcein redistribution into mitochondria, mitochondrial depolarization, ATP depletion and cell death. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, also rapidly depolarized mitochondria of intact hepatocytes but did not alone induce a permeability transition. Trifluoperazine did not prevent ATP depletion and cell death after the addition of CCCP. In conclusion, the permeability transition pore does not ‘flicker’ open during normal incubation of hepatocytes but remains continuously closed. Moreover, mitochondrial depolarization per se does not cause the permeability transition in intact cells. During oxidative stress, however, a permeability transition occurs quickly which leads to mitochondrial depolarization and cell death.

Download Full-text

Inhibition of PARP prevents oxidant-induced necrosis but not apoptosis in LLC-PK1cells

AJP Renal Physiology ◽

10.1152/ajprenal.1999.277.3.f428 ◽

1999 ◽

Vol 277 (3) ◽

pp. F428-F436 ◽

Cited By ~ 20

Author(s):

Dragana M. Filipovic ◽

Xianmin Meng ◽

W. Brian Reeves

Keyword(s):

Dna Damage ◽

Cell Death ◽

Cell Injury ◽

Parp Inhibitor ◽

Atp Depletion ◽

Parp Inhibition ◽

Target Cells ◽

Necrotic Cell Death ◽

Necrotic Cell ◽

Ldh Release

Oxidant-induced cell injury has been implicated in the pathogenesis of several forms of acute renal failure. The present studies examined whether activation of poly(ADP-ribose)polymerase (PARP) by oxidant-induced DNA damage contributes to oxidant injury of renal epithelial cells. H2O2exposure resulted in an increase in PARP activity and decreases in cell ATP and NAD content. These changes were significantly inhibited by 10 mM 3-aminobenzamide (3-ABA), a PARP inhibitor. In contrast, H2O2-induced DNA damage was not prevented by 3-ABA. Exposure of LLC-PK1 cells to 1 mM H2O2for 2 h induced necrotic cell death as measured by increased lactate dehydrogenase (LDH) release. 3-ABA completely prevented the H2O2-induced LDH release. Live/dead fluorescent staining confirmed the protection by 3-ABA. These results are consistent with the view that oxidant-induced DNA damage activates PARP and that the subsequent ATP and NAD depletion contribute to necrotic cell death. Of note, although protected from necrosis, cells treated with H2O2and 3-ABA underwent apoptosis as evidenced by DNA fragmentation and bis-benzimide staining. In conclusion, activation of PARP contributes to oxidant-induced ATP depletion and necrosis in LLC-PK1 cells. However, PARP inhibition may target cells toward an apoptotic form of cell death.

Download Full-text

Depletion of ATP and Oxidative Stress Underlie Zinc-Induced Cell Injury

Acta Medica (Hradec Kralove Czech Republic) ◽

10.14712/18059694.2017.58 ◽

2007 ◽

Vol 50 (1) ◽

pp. 43-49 ◽

Cited By ~ 9

Author(s):

Emil Rudolf

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Cell Death ◽

Nadph Oxidase ◽

Cell Injury ◽

Atp Depletion ◽

Atp Production ◽

Apoptosis And Necrosis ◽

Positive Effect ◽

And Oxidative Stress

The mechanisms of cell injury resulting in a special type of cell death combining the features of apoptosis and necrosis were examined in Hep-2 cells exposed to 300 μM zinc sulfate during 24h. Acute exposure to zinc induced a rapid rise in metallothionein levels and increased oxidative stress occurring in the absence of a significant early ATP depletion. Accentuated ATP loss and elevated levels of superoxide at later treatment intervals (12h and longer) were present along with increased DNA damage. Manipulation with ATP production and inhibition of NADPH oxidase had a positive effect on zinc-related increase in oxidative stress and influenced the observed type of cell death. These results suggest that Hep-2 cells acutely exposed to zinc increase intracellular labile zinc stores and over express metalothioneins. Elevated production of peroxides in zinc-treated cells is at later treatment intervals accompanied by an increase in superoxide levels, possibly by activation of NADPH oxidase, DNA damage and severe ATP loss. Prevention of critical ATP depletion and, in particular, inhibition of oxidative stress attenuates zinc-mediated cell injury and stimulates apoptosis-like phenotype in exposed cells.

Download Full-text