Two mechanisms by which ATP depletion potentiates induction of the mitochondrial permeability transition.

The present and a previous study [J. W. Snyder, J. G. Pastorino, A. M. Attie, and J. L. Farber, Am. J. Physiol. 264 (Cell Physiol. 33): C709-C714, 1993] define two mechanisms whereby ATP depletion promotes liver cell death. ATP depletion and cell death are linked by the mitochondrial permeability transition (MPT). Mitochondrial deenergization promotes the MPT, and ATP maintains a membrane potential by reversal of ATP synthase. With an increased influx of Ca2+ induced by the ionophore A-23187, oligomycin depleted the cells of ATP without loss of the mitochondrial membrane potential and further elevated the intracellular Ca2+ concentration. Cyclosporin A (CyA) prevented the accompanying cell killing. Fructose also preserved the viability of the cells. With the increased cytosolic Ca2+ imposed by A-23187, viability is maintained by ATP-dependent processes. Upon depletion of ATP, Ca2+ homeostasis cannot be maintained, and the MPT is induced. Rotenone also depleted the cells of ATP, and A-23187 accelerated the loss of the mitochondrial membrane potential occurring with rotenone alone. CyA and fructose prevented the cell killing with rotenone and A-23187. Oligomycin did not prevent this action of fructose. We conclude that ATP is needed to maintain Ca2+ homeostasis to prevent the MPT and the resultant liver cell death. ATP is also needed to maintain mitochondrial energization when electron transport is inhibited.

The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition.

Complete prevention of the killing of L929 fibroblasts by tumor necrosis factor alpha (TNF) in the presence of 0.5 microg/ml actinomycin D (ActD) was obtained with cyclosporin A (CyA), an inhibitor of the mitochondrial permeability transition (MPT), and aristolochic acid (ArA), a phospholipase A2 inhibitor. Peripheral benzodiazepine receptor (PBzR) agonists (PK11195, FGIN 1-27, or chlorodiazepam), agents known to potentiate induction of the MPT, potentiated the cytotoxicity of TNF in the absence of ActD, an effect prevented by CyA plus ArA. The MPT was demonstrated independently of its effect on viability as the CyA-sensitive loss of rhodamine 123 fluorescence from cells preloaded with the dye. Treatment with TNF and ActD resulted in the loss of 80% of rhodamine fluorescence within 6 h, a time prior to any loss of viability. CyA plus ArA completely prevented this effect of TNF. Potentiation of the cytotoxicity of TNF by PBzR agonists was associated with induction of the MPT, as assessed by the loss of rhodamine fluorescence. CyA plus ArA completely prevented the loss of rhodamine 123. Ceramide replaced TNF in killing L929 fibroblasts, an effect also prevented by CyA plus ArA. Ceramide in the presence of ActD resulted in the loss of rhodamine fluorescence, an effect that was again prevented by CyA plus ArA. In addition, CyA plus ArA prevented the ability of PBzR agonists to potentiate the cytotoxicity of ceramide. In the presence of each PBzR agonist, ceramide caused the loss of rhodamine fluorescence, an effect completely prevented by CyA plus ArA. D609, an inhibitor of phosphatidylcholine-specific phospholipase C, completely prevented the killing by TNF, but not by ceramide, in the presence of ActD. D609 prevented induction of the MPT occurring with TNF, but not with ceramide. Inhibitors of endocytosis, as well as lysosomotropic amines, prevented the cytotoxicity of TNF, but not that of ceramide. It is concluded that the MPT is causally linked to the genesis of irreversible cell injury with TNF. In the face of an inhibition of protein synthesis, the MPT occurs as a consequence of the formation of ceramide.

The antioxidant function of the physiological content of vitamin C.

The synthesis of vitamin C is substantially reduced in Osteogenic Disorder Shionogi (ODS) rats. Hepatocytes prepared from these rats contained approximately 12% of the wild-type content of this vitamin. In culture, the ascorbate content remained low in the absence of supplementation of the medium. Independent of their vitamin C status, cultured hepatocytes become depleted of vitamin E. Supplementation of the culture medium with 10C microM ascorbate and 1.2 microM alpha-tocopherol phosphate maintained the physiological content of both vitamins C and E in ODS hepatocytes. Thus, the antioxidant function of vitamins C and E could be evaluated in the presence of both or either vitamin or in the absence of both vitamins. Hepatocytes deficient in both vitamins were the most susceptible to lipid peroxidation (as measured by thiobarbituric acid) and to cell kllling within a 90-min exposure to 125-500 microM tert-butyl hydroperoxide (TBHP). Supplementation to achieve a physiological content of both vitamins C and E reduced the evidence of lipid peroxidation and abolished the cell killing. Supplementation with either vitamin alone resulted in an intermediate degree of both lipid peroxidation and cell killing. In ODS hepatocytes treated with TBHP, the decline in vitamin E preceded the decline in vitamin C. In ODS hepatocytes depleted of vitamin C, the loss of vitamin E after exposure to TBHP was greater than that in the presence of physiological levels of ascorbate. This greater loss of vitamin E in the face of a depletion of vitamin C was readily attributable to the increased peroxidation of lipids. Thus, the physiological level of vitamin C in cells does not seem to regenerate vitamin E. In contrast, the rate and extent of the depletion of vitamin C correlate with the degree of cell killing. These data document the antioxidant function of the physiological level of cellular vitamin C and relate this function to protection against peroxidative cell injury.

Independent antioxidant action of vitamins E and C in cultured rat hepatocytes intoxicated with allyl alcohol.

The relationship between the metabolism of alpha-tocopherol (vitamin E) and ascorbate (vitamin C) was examined in cultured hepatocytes intoxicated with allyl alcohol. Alcohol dehydrogenase rapidly metabolizes allyl alcohol to the potent electrophile acrolein. Acrolein depletes the glutathione (GSH) content of the hepatocytes, thereby sensitizing the cells to the constitutive flux of activated oxygen species. Supplementation of the medium with 1 microM alpha-tocopherol phosphate (alpha-TP) prevents the 85% decline in cellular vitamin E seen after 16-18 hr in culture. In cells supplemented with alpha-TP, allyl alcohol produced a concentration-dependent decline in the cellular content of alpha-tocopherol, and these cells were more resistant to cell killing than hepatocytes not supplemented with alpha-TP. alpha-TP concentrations that raised the cellular alpha-tocopherol above the physiological level completely protected hepatocytes against the killing by allyl alcohol. In cells with physiological alpha-tocopherol, vitamin E declined within 30 min of exposure to allyl alcohol. This decrease paralleled the peroxidation of lipids, but preceded the decrease in cellular ascorbate. Under these conditions, a decline in ascorbate correlated with the loss of cell viability. Cells supplemented with at least 3 mM ascorbate prevented the decline in alpha-tocopherol. However, ascorbate acts as an independent antioxidant at these concentrations. In the absence of killing by allyl alcohol, the loss of cellular ascorbate did not depend on the presence or absence of cellular alpha-tocopherol. These data indicate that vitamins E and C act as separate antioxidants and that ascorbate does not regenerate the tocopheroxyl radical in cultured rat hepatocytes.

Relationship of the metabolism of vitamins C and E in cultured hepatocytes treated with tert-butyl hydroperoxide.

The relationship between the metabolism of alpha-tocopherol (alpha-T) (vitamin E) and that of ascorbic acid (vitamin C) was examined in cultured hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). Unlike vitamin E, the cellular content of vitamin C did not decline after overnight culturing of freshly prepared hepatocytes. In addition, this basal vitamin C content was not affected by the presence of alpha-T phosphate in the overnight culture medium. Supplementation of the overnight culture medium with vitamin C (10 microM to 10 mM) increased the cellular content of vitamin C by > 1 order of magnitude. Increasing the cellular content of ascorbate increased the protection against TBHP toxicity, with or without the presence of a physiological content of vitamin E. In vitamin E-supplemented cells, a loss of alpha-T occurred within 15 min of exposure to TBHP and before the decrease in cellular ascorbate content. The vitamin C content declined in parallel with the loss of cell viability. Supplementation of the overnight culture medium with increasing concentrations of ascorbate progressively spared the depletion of alpha-T while preventing the cell killing. Pretreatment with the ferric iron chelator deferoxamine or the antioxidant N,N'-diphenyl-1,4-phenylenediamine prevented the loss of ascorbate and the cell killing by TBHP in hepatocytes either sufficient or deficient in alpha-T. However, neither alpha-T nor ascorbate prevented the accumulation of DNA single-strand breaks caused by TBHP, indicating that these vitamins do not effectively scavenge the TBHP-derived radicals responsible for DNA damage. The data in the present study indicate that vitamins E and C act as independent antioxidants and that ascorbate does not regenerate alpha-T in cultured rat hepatocytes.

Dexamethasone induces resistance to the lethal consequences of electron transport inhibition in cultured hepatocytes.

Pretreatment of cultured rat hepatocytes with 1 microM dexamethasone protected against cell killing by 5 microM rotenone and 1 mM cyanide. Simultaneous treatment (no pretreatment) was ineffective, as was pretreatment with 10 microM of sex hormones or the mineralocorticoid aldosterone. Protection by dexamethasone was blocked by 10 microM of glucocorticoid receptor antagonist, RU486, and by 1 microM of the inhibitor of protein synthesis, cycloheximide. Cells pretreated with dexamethasone for 6, 12, and 18 h showed increasing degrees of protection. Pretreatment with dexamethasone had no effect on either the decline of cellular ATP or the loss of the mitochondrial membrane potential. In addition, dexamethasone did not prevent the mitochondrial permeability transition. By contrast, dexamethasone prevented the increased release of [3H]arachidonic acid from phospholipids produced by cyanide. These data describe an inductive effect of dexamethasone in protecting cultured hepatocytes against inhibition of electron transport by rotenone and cyanide. It is concluded that pretreatment with dexamethasone prevents cell killing by inhibiting a mechanism that couples the mitochondrial permeability transition to the accelerated degradation of plasma membrane phospholipids.

Roles for oxidative stress and poly(ADP-ribosyl)ation in the killing of cultured hepatocytes by methyl methanesulfonate.

The mechanisms by which the methylating agent methyl methanesulfonate (MMS) kills cultured hepatocytes were studied. In an amino-acid-free Krebs-Ringer buffer (KRB), 1 mM MMS depleted the cells of glutathione (GSH) within 1-2 hr. Lipid peroxidation, as measured by the accumulation of malondialdehyde (MDA), followed, and over 70% of the cells died within 3-4 hr. The iron chelator deferoxamine and the antioxidant N,N'-diphenyl-1,4-phenylenediamine (DPPD) prevented lipid peroxidation and death of the hepatocytes without any effect on the loss of GSH. 3-Aminobenzamide (ABA), a poly(ADP-ribose) polymerase inhibitor, also prevented the cell killing by attenuating the loss of GSH. In a culture medium containing amino acids and antioxidants (Williams' E medium, WEM), 1 mM MMS killed the hepatocytes more slowly, with 70% of the cells dying 8-12 hr after treatment. Lipid peroxidation accompanied the loss of viability. Deferoxamine and DPPD inhibited lipid peroxidation, while only partially protecting against the cell killing. ABA offered more protection and reduced the decline of GSH and decreased lipid peroxidation. ABA also reduced the extent of the depletion of both NAD and ATP that accompanied the cell killing by MMS in WEM. These data indicate that MMS killed the hepatocytes by different mechanisms depending on the culture conditions. In KRB, the toxicity of MMS was a consequence of oxidative cell injury that follows the depletion of GSH. In WEM, both oxidative injury and the action of poly(ADP-ribose) polymerase in response to DNA single-strand breaks contributed to the loss of viability.

Effects of vitamin E on the killing of cultured hepatocytes by tert-butyl hydroperoxide.

The disposition of vitamin E was examined in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). Culturing of the cells overnight (18-20 hr) with approximately 60 nM alpha-tocopherol (alpha-T) equivalents [Williams' E medium, 18 nM tocopherol phosphate (alpha-TP), 9% fetal calf serum, 43 nM alpha-T] resulted in a content of alpha-T that was 16% of the concentration of vitamin E measured in freshly isolated hepatocytes. Supplementation of the medium with 1 microM alpha-TP maintained the alpha-T concentration of the cultured cells at the level of freshly isolated hepatocytes. Supplemented hepatocytes exposed to TBHP showed decreased lipid peroxidation and delayed cell killing, compared with hepatocytes not cultured overnight with alpha-TP. Killing of the supplemented cells by TBHP was accompanied by a loss of alpha-T. Pretreatment of supplemented hepatocytes with the iron chelator deferoxamine prevented much of the loss of alpha-T. At the same time, deferoxamine inhibited both the lipid peroxidation and cell killing. The antioxidant N,N'-diphenyl-1,4-phenylenediamine reduced the loss of alpha-T and significantly decreased lipid peroxidation. In the presence of N,N'-diphenyl-1,4-phenylenediamine, cell killing was delayed by 15 min and reduced in extent. Overnight supplementation of hepatocytes with nonesterified alpha-T, or vitamin E esters other than alpha-TP, similarly rendered the cells less sensitive to TBHP. The nonesterified alpha-T produced a higher cell-associated vitamin E concentration than did the esters; however, nonesterified alpha-T did not result in greater protection against TBHP. These data indicate that the mechanisms of the cell killing by TBHP are the same in cultured hepatocytes that contain low or physiological concentrations of vitamin E.

Stress fiber reformation after ATP depletion.

Fluorescently labeled heavy meromyosin, alpha-actinin, and vinculin were used to localize actin, alpha-actinin, and vinculin, respectively, in permeabilized and living cells during the process of stress fiber reassembly, which occurred when cells were removed from ATP-depleting medium (20 mM sodium azide and 10 mM 2-deoxyglucose). In 80% of the cells recovering from ATP depletion, small, scattered plaques containing actin, alpha-actinin, and vinculin were replaced by long, thin, periodic fibers within 5 minutes of removal of the inhibitors. These nascent stress fibers grew broader as recovery progressed, until they attained the thickness of stress fibers in control cells. In the other 20% of the cells, the scattered plaques aggregated within 5 minutes of reversal, and almost all the actin, alpha-actinin, and vinculin in the cells became localized in one perinuclear aggregate, with a diameter of approximately 15-25 micron. As recovery progressed, all aggregates resembled rings, with diameters that increased at about 0.5 micron/minute and grew to as large as 70 micron in some giant cells. As the size of the rings increased, fibers radiated outward from them and sometimes spanned the diameter of the rings. The shape of the cells did not change during this time. By 1 hour after reversal, the rings were no longer present and all cells had networks of stress fibers. Indirect immunofluorescence techniques used to localize tubulin and vimentin indicated that microtubules and intermediate filaments were not constituents of the rings, and the rings were not closely apposed to the substrate, judging from reflection contrast optics. The rapid rearrangement of attachment plaques into a perinuclear aggregate that spreads radially in the cytoplasm occurs at the same speed as fibroblast and chromosomal movement, but is unlike other types of intracytoplasmic motility.