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PubMed1999

Depolarization of in situ mitochondria due to hydrogen peroxide-induced oxidative stress in nerve terminals: inhibition of alpha-ketoglutarate dehydrogenase.

Mitochondrial membrane potential (delta psi(m)) was determined in intact isolated nerve terminals using the membrane potential-sensitive probe JC-1. Oxidative stress induced by H2O2 (0.1-1 mM) caused only a minor decrease in delta psi(m). When complex I of the respiratory chain was inhibited by rotenone (2 microM), delta psi(m) was unaltered, but on subsequent addition of H2O2, delta psi(m) started to decrease and collapsed during incubation with 0.5 mM H2O2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H2O2. H2O2 also induced a marked reduction in delta psi(m) when added after oligomycin (10 microM), an inhibitor of F0F1-ATPase. H2O2 (0.1 or 0.5 mM) inhibited alpha-ketoglutarate dehydrogenase and decreased the steady-state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine delta psi(m) in the presence of H2O2: (a) The NADH level reduced owing to inhibition of alpha-ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of delta psi(m) when the F0F1-ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H2O2 prevents maintenance of delta psi(m) by F0F1-ATPase. The results indicate that to maintain delta psi(m) in the nerve terminal during H2O2-induced oxidative stress, both complex I and F0F1-ATPase must be functional. Collapse of delta psi(m) could be a critical event in neuronal injury in ischemia or Parkinson's disease when H2O2 is generated in excess and complex I of the respiratory chain is simultaneously impaired.

Animals↗
PubMed2003

Dysfunction of mitochondrial complex I and the proteasome: interactions between two biochemical deficits in a cellular model of Parkinson's disease.

Two biochemical deficits have been described in the substantia nigra in Parkinson's disease, decreased activity of mitochondrial complex I and reduced proteasomal activity. We analysed interactions between these deficits in primary mesencephalic cultures. Proteasome inhibitors (epoxomicin, MG132) exacerbated the toxicity of complex I inhibitors [rotenone, 1-methyl-4-phenylpyridinium (MPP+)] and of the toxic dopamine analogue 6-hydroxydopamine, but not of inhibitors of mitochondrial complex II-V or excitotoxins [N-methyl-d-aspartate (NMDA), kainate]. Rotenone and MPP+ increased free radicals and reduced proteasomal activity via adenosine triphosphate (ATP) depletion. 6-hydroxydopamine also increased free radicals, but did not affect ATP levels and increased proteasomal activity, presumably in response to oxidative damage. Proteasome inhibition potentiated the toxicity of rotenone, MPP+ and 6-hydroxydopamine at concentrations at which they increased free radical levels >/= 40% above baseline, exceeding the cellular capacity to detoxify oxidized proteins reduced by proteasome inhibition, and also exacerbated ATP depletion caused by complex I inhibition. Consistently, both free radical scavenging and stimulation of ATP production by glucose supplementation protected against the synergistic toxicity. In summary, proteasome inhibition increases neuronal vulnerability to normally subtoxic levels of free radicals and amplifies energy depletion following complex I inhibition.

1-Methyl-4-phenylpyridinium↗
PubMed1999

Titrating the effects of mitochondrial complex I impairment in the cell physiology.

The mitochondrial oxidative phosphorylation system consists of five multimeric enzymes (complexes I-V). NADH dehydrogenase or complex I (CI) is affected in most of the mitochondrial diseases and in some neurodegenerative disorders. We have studied the physiological consequences of a partial CI inhibition at the cellular level. We used a genetic model (40% CI-inhibited human-ape xenomitochondrial cybrids) and a drug-induced model (0-100% CI-inhibited cells using different concentrations of rotenone). We observed a quantitative correlation between the level of CI impairment and cell respiration, cell growth, free radical production, lipid peroxidation, mitochondrial membrane potential, and apoptosis. We showed that cell death was quantitatively associated with free radical production rather than with a decrease in respiratory chain function. The results obtained with human xenomitochondrial cybrid cells were compatible with those observed in rotenone-induced 40% CI-inhibited cells. At high concentrations (5-6-fold higher than the concentration necessary for 100% CI inhibition), rotenone showed a second toxic effect at the level of microtubule assembly, which also led to apoptosis. The correlation found among all the parameters studied helped clarify the physiological consequences of partial CI inhibitions at the cellular level.

Animals↗
PubMed2005

Selective vulnerability of dopaminergic neurons to microtubule depolymerization.

Parkinson disease (PD) is characterized by the specific degeneration of dopaminergic (DA) neurons in substantia nigra and has been linked to a variety of environmental and genetic factors. Rotenone, an environmental PD toxin, exhibited much greater toxicity to DA neurons in midbrain neuronal cultures than to non-DA neurons. The effect was significantly decreased by the microtubule-stabilizing drug taxol and mimicked by microtubule-depolymerizing agents such as colchicine or nocodazole. Microtubule depolymerization disrupted vesicular transport along microtubules and caused the accumulation of dopamine vesicles in the soma. This led to increased oxidative stress due to oxidation of cytosolic dopamine leaked from vesicles. Inhibition of dopamine metabolism significantly reduced rotenone toxicity. Thus, our results suggest that microtubule depolymerization induced by PD toxins such as rotenone plays a key role in the selective death of dopaminergic neurons.

Cell Death↗
PubMed2001

Enhancement of oxidative damage to cultured cells and Caenorhabditis elegans by mitochondrial electron transport inhibitors.

The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in primary and cultured cells as well as in the nematode Caenorhabditis elegans (C. elegans) treated simultaneously with electron transport inhibitors and oxygen gas. Oxygen loading enhanced the damage of PC 12 cells by thenoyltrifluoroacetone (TTFA, a complex II inhibitor), but did not by rotenone (a complex I inhibitor), antimycin (a complex III inhibitor), and sodium azide (a complex IV inhibitor). In primary hepatocytes, the enhancement was observed with the addition of sodium azide and rotenone, but not by TTFA or antimycin. In the nematode, only rotenone and TTFA enhanced the sensitivity under hyperoxia. These results demonstrate that highly specific inhibitors of electron transport can induce oxygen hypersensitivity in cell levels such as PC 12 cells and primary hepatocytes, and animal level of C. elegans. In addition the cell damage is different dependent on cell type and organism.

Animals↗
PubMed2005

Oxygen-sensing pathway for SK channels in the ovine adrenal medulla.

1. The intracellular pathways that modulate the opening of oxygen-sensitive ion channels during periods of hypoxia are poorly understood. Different tissues appear to use either NADPH oxidase or a rotenone-sensitive mechanism as an oxygen sensor. The aim of the present study was to identify the oxygen-sensing pathway in the oxygen-sensitive sheep adrenal medullary chromaffin cell (AMCC). 2. The whole-cell patch-clamp technique was used to measure K+ currents in dissociated adult ovine chromaffin cells as well as SK channel currents expressed in the H4IIE cell line. 3. Diphenyliodonium, an inhibitor of NADPH oxidase, had no effect on the hypoxia-evoked closure of K+ channels in primary AMCC, whereas the mitochondrial inhibitor rotenone abolished the hypoxia-evoked response. Both these compounds significantly reduced K+ current amplitude under normoxic conditions. 4. One possible mechanism through which the oxygen sensor may modulate K+ channel activity is by altering the redox state of the cell. In sheep AMCC, altering the redox state by the addition of H2O2 to the extracellular solution increased K+ conductance. 5. The oxygen-sensitive K+ (Ko2) channels in sheep chromaffin cells are from the SK family and the whole-cell conductance of cells expressing mouse SK2 or SK3, but not human SK1, was increased by H2O2 and decreased by the reducing agent dithiothreitol. 6. These studies show that, in sheep AMCC, Ko2 channels are modulated via a rotenone-sensitive mechanism and that alteration of the cellular redox state mimics the change produced by alterations in Po2. In a heterologous expression system, SK2 and SK3 channels, the channels that initiate hypoxia-evoked changes in AMCC function, are modulated appropriately by changes in cellular redox state.

Adrenal Medulla↗
PubMed1989

Effects of diltiazem on bioenergetics, K+ gradients, and free cytosolic Ca2+ levels in rat brain synaptosomes submitted to energy metabolism inhibition and depolarization.

Diltiazem was able to decrease the oxygen consumption rate and lactate production in synaptosomes isolated from rat forebrains, both under control and depolarized (40 microM veratridine) conditions, starting from a concentration of 250 microM. This effect was particularly evident when synaptosomes were depolarized by veratridine. This depolarization-counteracting action was evident also when transplasma membrane K+ diffusion potentials were measured after depolarization induced by veratridine and by rotenone with a glucose shortage. The concentrations of ATP, phosphocreatine, and creatine were less sensitive to diltiazem action. The concentration/response relationships were the same as those found for the oxygen consumption were the same as those found for the oxygen consumption rate, lactate production, and K+ diffusion potentials. The effects of 0.5 mM diltiazem in counteracting inhibition of energy metabolism induced by rotenone without glucose were no longer detectable when either Ca2+ or Na+ was absent from the incubation medium of synaptosomes. Diltiazem at the same concentrations (starting from 250 microM) was able to inhibit both the veratridine-induced and the rotenone-without-glucose-induced increase in intrasynaptosomal free Ca2+ levels evaluated with the fluorescent probe quin2. The results are discussed in view of a possible effect of diltiazem on voltage-dependent Na+ channels and the possibility of utilizing this approach for counteracting neuronal failure due to derangement of energy metabolism or hyperexcitation.

Animals↗
PubMed1991

Interaction of 1-methyl-4-phenylpyridinium ion (MPP+) and its analogs with the rotenone/piericidin binding site of NADH dehydrogenase.

Nigrostriatal cell death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease results from the inhibition of mitochondrial respiration by 1-methyl-4-phenylpyridinium (MPP+). MPP+ blocks electron flow from NADH dehydrogenase to coenzyme Q at or near the same site as do rotenone and piericidin and protects against binding of and loss of activity due to these inhibitors. The 4'-analogs of MPP+ showed increasing affinity for the site with increasing length of alkyl chain, with the lowest Ki, for 4'-heptyl-MPP+, being 6 microM. The 4'-analogs compete with rotenone for the binding site in a concentration-dependent manner. They protect the activity of the enzyme from inhibition by piericidin in parallel to preventing its binding, indicating that the analogs and piericidin bind at the same inhibitory site(s). The optimum protection, however, was afforded by 4'-propyl-MPP+. The lesser protection by the more lipophilic MPP+ analogs with longer alkyl chains may involve a different orientation in the hydrophobic cleft, allowing rotenone and piericidin to still bind even when the pyridinium cation is in a position to interrupt electron flow from NADH to coenzyme Q.

1-Methyl-4-phenylpyridinium↗
PubMed2004

Pramipexole protects against apoptotic cell death by non-dopaminergic mechanisms.

We have investigated the ability of pramipexole, a dopamine agonist used in the symptomatic treatment of Parkinson's disease (PD), to protect against cell death induced by 1-methyl-4-phenylpyridinium (MPP+) and rotenone in dopaminergic and non-dopaminergic cells. Pre-incubation with either the active (-)- or inactive (+)-enantiomer forms of pramipexole (10 microm) decreased cell death in response to MPP+ and rotenone in dopaminergic SHSY-5Y cells and in non-dopaminergic JK cells. The protective effect was not prevented by dopamine receptor blockade using sulpiride or clozapine. Protection occurred at concentrations at which pramipexole did not demonstrate antioxidant activity, as shown by the failure to maintain aconitase activity. However, pramipexole reduced caspase-3 activation, decreased the release of cytochrome c and prevented the fall in the mitochondrial membrane potential induced by MPP+ and rotenone. This suggests that pramipexole has anti-apoptotic actions. The results extend the evidence for the neuroprotective effects of pramipexole and indicate that this is not dependent on dopamine receptor occupation or antioxidant activity. Further evaluation is required to determine whether the neuroprotective action of pramipexole is translated to a disease-modifying effect in PD patients.

1-Methyl-4-phenylpyridinium↗
PubMed2005

Ca2+-induced permeabilization promotes free radical release from rat brain mitochondria with partially inhibited complex I.

Mitochondrial complex I dysfunction has been implicated in a number of brain pathologies, putatively owing to an increased rate of reactive oxygen species (ROS) release. However, the mechanisms regulating the ROS burden are poorly understood. In this study we investigated the effect of Ca2+ loads on ROS release from rat brain mitochondria with complex I partially inhibited by rotenone. The addition of 20 nm rotenone to brain mitochondria increased ROS release. Ca2+ (100 microm) alone had no effect on ROS release, but greatly potentiated the effects of rotenone. The effect of Ca2+ was decreased by ruthenium red. Ca2+-challenged mitochondria lose about 88% of their glutathione and 46% of their cytochrome c under these conditions, although this depends only on Ca2+ loading and not complex I inhibition. ADP in combination with oligomycin decreased the loss of glutathione and cytochrome c and free radical generation. Cyclosporin A alone was ineffective in preventing these effects, but augmented the protection provided by ADP and oligomycin. Non-specific permeabilization of mitochondria with alamethicin also increased the ROS signal, but only when combined with partial inhibition of complex I. These results demonstrate that Ca2+ can greatly increase ROS release by brain mitochondria when complex I is impaired.

Animals↗
PubMed1991

Effects of metabolic inhibitors on endothelium-dependent and endothelium-independent vasodilatation of rat and rabbit aorta.

1. Basal release of endothelium-derived relaxing factor (EDRF) rendered endothelium-containing rings of rat aorta 4.7 fold less sensitive to the contractile actions of phenylephrine and depressed the maximum response when compared with endothelium-denuded rings. The responsiveness and maximum response to phenylephrine was, however, similar in rings of rabbit aorta with or without endothelium. 2. Rotenone (1 nM-0.1 microM), an inhibitor of oxidative phosphorylation, induced a profound, irreversible blockade of phenylephrine-induced tone in endothelium-containing and endothelium-denuded rings of rat aorta, but induced only slight inhibition of tone in rings of rabbit aorta. 3. 2-Deoxy glucose (10 mM), an inhibitor of glycolysis, had no effect on phenylephrine-induced contraction in endothelium-denuded rings of rat aorta, but inhibited reversibly the endothelium-dependent depression of contraction in endothelium containing rings. 2-Deoxy glucose had no effect on phenylephrine-induced contraction in rings of rabbit aorta with or without endothelium. 4. Rotenone (0.1 microM) inhibited acetylcholine-induced, endothelium-dependent relaxation of phenylephrine-contracted rings or rat and rabbit aorta. In endothelium-denuded rings of rat aorta, relaxation induced by glyceryl trinitrate of isoprenaline was also inhibited, but relaxation induced by 8-bromo cyclic GMP or dibutyryl cyclic AMP was not. Relaxation induced by verapamil on KCl-contracted, endothelium-denuded rings of rat aorta was also unaffected. 5. 2-Deoxy glucose (10 mM) inhibited acetylcholine-induced, endothelium-dependent relaxation of phenylephrine-contracted rings of rat and rabbit aorta. In endothelium-denuded rings of rat aorta, relaxation induced by glyceryl trinitrate and by isoprenaline was also inhibited, but relaxation induced by 8-bromo cyclic GMP or dibutyryl cyclic AMP was not. Relaxation induced by verapamil on KCIcontracted, endothelium-denuded rings of rat aorta was also unaffected. 6. These data suggest that in rabbit and in rat aorta, rotenone inhibits acetylcholine-induced relaxation by inhibiting EDRF production, and by depressing smooth muscle sensitivity to EDRF, respectively. They further suggest that 2-deoxy glucose inhibits acetylcholine-induced relaxation in both tissues by depressing the sensitivity to EDRF, probably as a result of reduced synthesis of cyclic GMP. The additional possibility that 2-deoxy glucose inhibits EDRF production warrants further investigation. 7. The blockade by 2-deoxy glucose of the endothelium-dependent depression of phenylephrine-induced tone in rat aorta probably reflects blockade of the actions of spontaneously released EDRF.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Two pathways for iron uptake by guinea pig reticulocytes.

We have demonstrated that the intracellular processing of transferrin to effect iron removal involves two pathways, one sensitive to rotenone and the other not. We have also found that the effect of the rotenone is dependent on the transferrin concentration: iron uptake was suppressed with concentrations of transferrin in the micromolar range, and was not suppressed at physiologic concentrations of transferrin. Rotenone does not disturb transferrin's interaction with its extracellular receptor, indicating that its action must be intracellular. The following model is suggested: that separate pathways are entered by transferrin in the cell. The first pathway is preferentially utilized when transferrin is in short supply. It begins with an intracellular site which has a high affinity (and low capacity) for either iron or transferrin. The second pathway begins with an intracellular site which has a high capacity (but low affinity) for either iron or transferrin and is utilized when transferrin is in physiologic concentration (and the low-capacity, high-affinity site is saturated); the pathway it initiates is dominant when transferrin is abundant. We speculate that the high-affinity low-capacity pathway may serve to direct intracellular iron to sites which would be critically injured by iron excess.

Animals↗
PubMed1998

NADH: ubiquinone oxidoreductase inhibitors block induction of ornithine decarboxylase activity in MCF-7 human breast cancer cells.

Rotenone is the classical inhibitor of NADH: ubiquinone oxidoreductase and its analogue deguelin is a potent inhibitor of 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase mRNA steady state level and enzyme activity in mouse 308 cells (Gerhäuser et al. 1995). In MCF-7 human breast cancer cells, rotenone, deguelin and two structurally-unrelated miticides (pyridaben and fenazaquin) inhibit not only NADH: ubiquinone oxidoreductase but also induced ornithine decarboxylase activity with IC50 values of < 1 to 70 nM. Rotenone inhibits ornithine decarboxylase activity equally well as induced by TPA, insulin-like growth factor I and 17 beta-oestradiol. Pyridaben is the most potent of the four inhibitors not only for NADH: ubiquinone oxidoreductase activity (bovine heart enzyme) and TPA-induced ornithine decarboxylase activity and mRNA steady state level but also for TPA-induced reactive oxygen species. It is therefore proposed that NADH: ubiquinone oxidoreductase inhibitors block multiple and possibly reactive oxygen species-modulated pathways which regulate ornithine decarboxylase activity.

Breast Neoplasms↗
PubMed1969

Antiviral and antitumor antibiotics. XIV. Effects of ascochlorin and other respiration inhibitors on multiplication of Newcastle disease virus in cultured cells.

Antiviral activity on Newcastle disease virus was examined with some respiration inhibitors including ascochlorin, rotenone, antimycin A(3), piericidin A, dicoumarol, 2, 4-dinitrophenol, pentachlorophenol, and fatty acids. Of the chemicals tested, ascochlorin and rotenone showed significant inhibitory effect on the viral growth in cultured cells as determined by the plate and tube assay methods. Dose response of ascochlorin and rotenone was observed in rate and final yield of hemagglutinin synthesis. Ratio of infectivity for hemagglutinin was nearly equal at any test concentrations of ascochlorin. Thus, the possibility of inhibition of virus maturation by the antibiotic was excluded. Ascochlorin had no activity on virus particles and on viral adsorption onto host cells.

Adsorption↗
PubMed1995

Efficient selection and characterization of mutants of a human cell line which are defective in mitochondrial DNA-encoded subunits of respiratory NADH dehydrogenase.

The mitochondrial NADH dehydrogenase (complex I) in mammalian cells is a multimeric enzyme consisting of approximately 40 subunits, 7 of which are encoded in mitochondrial DNA (mtDNA). Very little is known about the function of these mtDNA-encoded subunits. In this paper, we describe the efficient isolation from a human cell line of mutants affected in any of these subunits. In the course of analysis of eight mutants of the human cell line VA2B selected for their resistance to high concentrations of the complex I inhibitor rotenone, seven were found to be respiration deficient, and among these, six exhibited a specific defect of complex I. Transfer of mitochondria from these six mutants into human mtDNA-less cells revealed, surprisingly, in all cases a cotransfer of the complex I defect but not of the rotenone resistance. This result indicated that the rotenone resistance resulted from a nuclear mutation, while the respiration defect was produced by an mtDNA mutation. A detailed molecular analysis of the six complex I-deficient mutants revealed that two of them exhibited a frameshift mutation in the ND4 gene, in homoplasmic or in heteroplasmic form, resulting in the complete or partial loss, respectively, of the ND4 subunit; two other mutants exhibited a frameshift mutation in the ND5 gene, in near-homoplasmic or heteroplasmic form, resulting in the ND5 subunit being undetectable or strongly decreased, respectively. It was previously reported (G. Hofhaus and G. Attardi, EMBO J. 12:3043-3048, 1993) that the mutant completely lacking the ND4 subunit exhibited a total loss of NADH:Q1 oxidoreductase activity and a lack of assembly of the mtDNA-encoded subunits of complex I. By contrast, in the mutant characterized in this study in which the ND5 subunit was not detectable and which was nearly totally deficient in complex I activity, the capacity to assemble the mtDNA-encoded subunits of the enzyme was preserved, although with a decreased efficiency or a reduced stability of the assembled complex. The two remaining complex I-deficient mutants exhibited a normal rate of synthesis and assembly of the mtDNA-encoded subunits of the enzyme, and the mtDNA mutation(s) responsible for their NADH dehydrogenase defect remains to be identified. The selection scheme used in this work has proven to be very valuable for the isolation of mutants from the VA2B cell line which are affected in different mtDNA-encoded subunits of complex I and may be applicable to other cell lines.

Autoradiography↗
PubMed1990

Energy dependence of sodium-calcium exchange in vascular smooth muscle cells.

Three different types of mitochondrial poisons (oligomycin, antimycin A, and dinitrophenol) strongly inhibited Na(+)-Ca2+ exchange in aortic myocytes. Exchange activity was assayed as 45Ca2+ uptake that depended on inverting the Na+ gradient and was inhibited by 25 microM dimethylbenzamil. Glucose markedly decreased the inhibition of exchange activity by these three poisons. Glucose also prevented rotenone from inhibiting exchange and depleting cellular ATP. In the absence of glucose, rotenone decreased ATP and exchange activity with half-times of 0.8 and 0.9 min, respectively. Almost eliminating cellular ATP with rotenone maximally inhibited exchange by 80%. Repletion of ATP with glucose substantially restored Na(+)-Ca2+ exchange activity. Ca2+ uptake by organelles, subsequent to entry via exchange for Na+, does not appear to contribute significantly to exchange activity as assayed in intact myocytes. The specific activity of Na(+)-Ca2+ exchange was approximately 30 nmol.min-1.mg protein-1. These findings suggest that ATP modulates exchange activity and that there are approximately 150,000 Na(+)-Ca2+ exchangers per cell, assuming that the turnover number is 1,000 s-1.

Animals↗
PubMed1991

Response of isolated adult canine cardiac myocytes to prolonged hypoxia and reoxygenation.

Isolated adult canine ventricular myocytes incubated in the absence of glucose with the respiratory inhibitor rotenone retained 67% of ATP (control, 26.0 +/- 0.9 nmol/mg protein) during 3-h incubation, yet phosphocreatine fell to 23% of initial content. Lactate production proceeded at a constant rate of 5 nmol.mg-1.min-1 in rotenone-treated glucose-free myocytes. A 36% decline in rod-shaped cells and an increase in percent 22Na permeation from 37% in aerobic cells (approximately 13 mM intracellular sodium) to 68% in rotenone-treated glucose-free myocytes paralleled the loss of ATP. Total exchangeable calcium was maintained at control aerobic levels. Exposure of canine cells to 3-h hypoxia in the absence of glucose followed by 5-min reoxygenation resulted in a 73% decrease in ATP, a rise in calcium from 3.3 +/- 0.2 to 6.6 +/- 1.6 nmol/mg, and an increase in 22Na permeation to 111%. Under these conditions the number of rod-shaped myocytes declined by 77%, with corresponding increases in viable contracted and hypercontracted myocytes. The response of canine myocytes to severe hypoxia and reaeration contrasts greatly to earlier studies using adult rat cardiac myocytes [see Hohl et al. Am. J. Physiol. 242 (Heart Circ. Physiol. 11): H1022-H1030, 1982]. Species differences with respect to basal metabolism, rates of ATP production and degradation, and regulation of cation movements are most likely responsible for the observed differences.

Adenosine Diphosphate↗
PubMed1995

Ca2+ depletion prevents anoxic death of hepatocytes by inhibiting mitochondrial permeability transition.

Removal of Ca2+ from the culture medium or treatment with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) prevented the killing of rat hepatocytes by anoxia and rotenone, but not by cyanide. Neither manipulation prevented the loss of the mitochondrial membrane potential or the depletion of ATP. A mitochondrial permeability transition (MPT) was demonstrated in digitonin-permeabilized hepatocytes as an increased [3H]sucrose-accessible space sensitive to cyclosporin A (CyA). Ca2+ depletion by either means prevented the MPT measured in intact cells made anoxic or treated with rotenone. In isolated mitochondria deenergized by rotenone, BAPTA-AM prevented the MPT induced by palmitoyl CoA. By contrast, in isolated mitochondria deenergized by cyanide, BAPTA-AM alone did not prevent the MPT. Rather, BAPTA-AM plus CyA were required. Similarly, the killing of cultured hepatocytes by cyanide was prevented by BAPTA-AM plus CyA, but not by either agent alone. The MPT in intact cells treated with cyanide was also prevented by BAPTA-AM plus CyA. These data define a specific requirement for Ca2+ in the killing of hepatocytes that follows the inhibition of electron transport. A model is presented in which the MPT depends on factors that modulate the sensitivity of the permeability transition to the matrix concentration of Ca2+.

Adenosine Triphosphate↗