Synthetic and natural polyanions induce cytochrome c release from mitochondria in vitro and in situ

A synthetic polyanion composed of styrene, maleic anhydride, and methacrylic acid (molar ratio 56:37:7) significantly inhibited the respiration of isolated rat liver mitochondria in a time-dependent fashion that correlated with 1) collapse of the mitochondrial membrane potential and 2) high amplitude mitochondrial swelling. The process is apparently Ca(2+) dependent. Since it is blocked by cyclosporin A, the process is ascribed to induction of the mitochondrial permeability transition. In mitoplasts, i.e., mitochondria lacking their outer membranes, the polyanion rapidly blocked respiration. After incubation of rat liver mitochondria with the polyanion, cytochrome c was released into the incubation medium. In solution, the polyanion modified by conjugation with fluorescein formed a complex with cytochrome c. Addition of the polyanion to cytochrome c-loaded phosphatidylcholine/cardiolipin liposomes induced the release of the protein from liposomal membrane evidently due to coordinated interplay of Coulomb and hydrophobic interactions of the polymer with cytochrome c. We conclude that binding of the polyanion to cytochrome c renders it inactive in the respiratory chain due to exclusion from its native binding sites. Apparently, the polyanion interacts with cytochrome c in mitochondria and releases it to the medium through breakage of the outer membrane as a result of severe swelling. Similar properties were demonstrated for the natural polyanion, tobacco mosaic virus RNA. An electron microscopy study confirmed that both polyanions caused mitochondrial swelling. Exposure of cerebellar astroglial cells in culture to the synthetic polyanion resulted in cell death, which was associated with nuclear fragmentation.     

Endomorphins and morphine limit anoxia-reoxygenation-induced brain mitochondrial dysfunction in the mouse

The protection of brain mitochondria from oxidative stress is an important therapeutic strategy against ischemia-reperfusion injury and neurodegenerative disorders. Isolated brain mitochondria subjected to a 5 min period of anoxia followed by 5 min reoxygenation mirrored the effect of oxidative stress in the brain. The present study attempts to evaluate the protective effects of endomorphin 1 (EM1), endomorphin 2 (EM2), and morphine (Mor) in an in vitro mouse brain mitochondria anoxia-reoxygenation model. Endomorphins (EM1/2) and Mor were added to mitochondria prior to anoxia or reoxygenation. EM1/2 and Mor markedly improved mitochondrial respiratory activity with a decrease in state 4 and increases in state 3, respiratory control ratio (RCR) and the oxidative phosphorylation efficiency (ADP/O ratio), suggesting that they may play a protective role in mitochondria. These drugs inhibited alterations in mitochondrial membrane fluidity, lipoperoxidation, and cardiolipin (CL) release, which indicates protection of the mitochondrial membranes from oxidative damage. The protective effects of these drugs were concentration-dependent. Furthermore, these drugs blocked the enhanced release of cytochrome c (Cyt c), and consequently inhibited the cell apoptosis induced by the release of Cyt c. Our results suggest that EM1/2 and Mor effectively protect brain mitochondria against oxidative stresses induced by in vitro anoxia-reoxygenation and may play an important role in the prevention of deleterious effects during brain ischemia-reperfusion and neurodegenerative diseases.     

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.     

Decreased cardiolipin synthesis corresponds with cytochrome c release in palmitate-induced cardiomyocyte apoptosis

Apoptosis has been identified recently as a component of many cardiac pathologies. However, the potential triggers of programmed cell death in the heart and the involvement of specific metabolic pathway(s) are less well characterized. Detachment of cytochrome c from the mitochondrial inner membrane is a necessary first step for cytochrome c release into the cytosol and initiation of apoptosis. The saturated long chain fatty acid, palmitate, induces apoptosis in rat neonatal cardiomyocytes and diminishes content of the mitochondrial anionic phospholipid, cardiolipin. These changes are accompanied by 1) acyl chain saturation of phosphatidic acid and phosphatidylglycerol, 2) large increases in the levels of these two phospholipids, and 3) a decline in cardiolipin synthesis. Although cardiolipin synthase activity is unchanged, saturated phosphatidylglycerol is a poor substrate for this enzyme. Under these conditions, decreased cardiolipin synthesis and release of cytochrome c are directly and significantly correlated. The results suggest that phosphatidylglycerol saturation and subsequent decreases in cardiolipin affect the association of cytochrome c with the inner mitochondrial membrane, directly influencing the pathway to cytochrome c release and subsequent apoptosis.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

Interaction of peroxidized cardiolipin with rat-heart mitochondrial membranes: induction of permeability transition and cytochrome c release

Cardiolipin peroxidation plays a critical role in mitochondrial cytochrome c release and subsequent apoptotic process. Mitochondrial pore transition (MPT) is considered as an important step in this process. In this work, the effect of peroxidized cardiolipin on MPT induction and cytochrome c release in rat heart mitochondria was investigated. Treatment of mitochondria with micromolar concentrations of cardiolipin hydroperoxide (CLOOH) resulted in a dose-dependent matrix swelling, DeltaPsi collapse, release of preaccumulated Ca2+ and release of cytochrome c. All these events were inhibited by cyclosporin A and bongkrekic acid, indicating that peroxidized cardiolipin behaves as an inducer of MPT. Ca2+ accumulation by mitochondria was required for this effect. ANT (ADP/ATP translocator) appears to be involved in the CLOOH-dependent MPT induction, as suggested by the modulation by ligands and inhibitors of adenine nucleotide translocator (ANT). Together, these results indicate that peroxidized cardiolipin lowers the threshold of Ca2+ for MPT induction and cytochrome c release. This synergistic effect of Ca2+ and peroxidized cardiolipin on MPT induction and cytochrome c release in mitochondria, might be important in regulating the initial phase of apoptosis and also may have important implications in those physiopathological situations, characterized by both Ca2+ and peroxidized cardiolipin accumulation in mitochondria, such as aging, ischemia/reperfusion and other degenerative diseases.     

Direct effect of Taxol on free radical formation and mitochondrial permeability transition

To elucidate the potential role of mitochondria in Taxol-induced cytotoxicity, we studied its direct mitochondrial effects. In Percoll-gradient purified liver mitochondria, Taxol induced large amplitude swelling in a concentration-dependent manner in the microM range. Opening of the permeability pore was also confirmed by the access of mitochondrial matrix enzymes for membrane impermeable substrates in Taxol-treated mitochondria. Taxol induced the dissipation of mitochondrial membrane potential (DeltaPsi) determined by Rhodamine123 release and induced the release of cytochrome c from the intermembrane space. All these effects were inhibited by 2.5 microM cyclosporine A. Taxol significantly increased the formation of reactive oxygen species (ROS) in both the aqueous and the lipid phase as determined by dihydrorhodamine123 and resorufin derivative. Cytochrome oxidase inhibitor CN(-), azide, and NO abrogated the Taxol-induced mitochondrial ROS formation while inhibitors of the other respiratory complexes and cyclosporine A had no effect. We confirmed that the Taxol-induced collapse of DeltaPsi and the induction of ROS production occurs in BRL-3A cells. In conclusion, Taxol-induced adenine nucleotide translocase-cyclophilin complex mediated permeability transition, and cytochrome oxidase mediated ROS production. Because both cytochrome c release and mitochondrial ROS production can induce suicide pathways, the direct mitochondrial effects of Taxol may contribute to its cytotoxicity.     

Reactive oxygen species generated from the mitochondrial electron transport chain induce cytochrome c dissociation from beef-heart submitochondrial particles via cardiolipin peroxidation. Possible role in the apoptosis.

Cytochrome c release from mitochondria is a critical event in the apoptosis induction. Dissociation of cytochrome c from the mitochondrial inner membrane (IMM) is a necessary first step for cytochrome c release. In the present study, the effect of reactive oxygen species (ROS) on the dissociation of cytochrome c from beef-heart submitochondrial particles (SMP) and on the cardiolipin content was investigated. Exposure of SMP to mitochondrial-mediated ROS generation resulted in a large dissociation of cytochrome c from SMP and in a parallel loss of cardiolipin. Both these effects were directly and significantly correlated and also abolished by superoxide dismutase+catalase. These results demonstrate that ROS generation induces the dissociation of cytochrome c from IMM via cardiolipin peroxidation. The data may prove useful in clarifying the molecular mechanism underlying the release of cytochrome c from the mitochondria to the cytosol.     

Exogenous phospholipids specifically affect transmembrane potential of brain mitochondria and cytochrome C release

Release of cytochrome c, a decrease of membrane potential (Deltapsi(m)), and a reduction of cardiolipin (CL) of rat brain mitochondria occurred upon incubation in the absence of respiratory substrates. Since CL is critical for mitochondrial functioning, CL enrichment of mitochondria was achieved by fusion with CL liposomes. Fusion was triggered by potassium phosphate at concentrations producing mitochondrial permeability transition pore opening but not cytochrome c release, which was observed only at >10 mm. Cyclosporin A inhibited phosphate-induced CL fusion, whereas Pronase pretreatment of mitochondria abolished it, suggesting that mitochondrial permeability transition pore and protein(s) are involved in the fusion process. Phosphate-dependent fusion was enhanced in respiratory state 3 and influenced by phospholipid classes in the order CL > phosphatidylglycerol (PG) > phosphatidylserine. The probe 10-nonylacridine orange indicated that fused CL had migrated to the inner mitochondrial membrane. In state 3, CL enrichment of mitochondria resulted in a pH decrease in the intermembrane space. Cytofluorimetric analysis of mitochondria stained with 3,3'-diexyloxacarbocyanine iodide and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzymidazolylcarbocyanine iodide showed Deltapsi(m) increase upon fusion with CL or PG. In contrast, phosphatidylserine fusion required Deltapsi(m) consumption, suggesting that Deltapsi(m) is the driving force in mitochondrial phospholipid importation. Moreover, enrichment with CL and PG brought the low energy mitochondrial population to high Deltapsi(m) values and prevented phosphate-dependent cytochrome c release.     

Tributyltin causes cytochrome C release from isolated mitochondria by two discrete mechanisms

Using isolated liver mitochondria we show that low concentrations of TBT (0.5 microM) cause the release of mitochondrial cytochrome c, in the presence of Ca(2+). This is reflected in a rapid loss of membrane potential (DeltaPsi(m)), and a large-amplitude swelling characteristic of mitochondrial permeability transition (MPT). Despite this, the inclusion of cyclosporin A could not prevent the release of cytochrome c. Further, in the absence of Ca(2+), low concentrations of TBT (0.5 microM) resulted in a slow sub-maximal shift of DeltaPsi(m), not characteristic of MPT, which was still paralleled by a release of cytochrome c. Further experiments showed that the loss of DeltaPsi(m) in the absence of Ca(2+) was due to a combination of inhibition of respiration and a direct uncoupling effect on the respiratory chain. Under these conditions, rapid swelling of mitochondria could be demonstrated, due to chloride exchange over the inner mitochondrial membrane. Taken together these data suggest that TBT can induce the release of cytochrome c in intact cells by at least two mechanisms. The first and critical mechanism is initiated immediately the mitochondria sense the presence of TBT and involves a slow loss of DeltaPsi(m) and induction of swelling, which allows release of cytochrome c in a relatively non-specific manner and independently from a rise in [Ca(2+)](i). The second mechanism involves the induction of formal MPT as intracellular [Ca(2+)](i) increases. These data help to explain previous observations in intact lymphocytes demonstrating TBT-induced release of mitochondrial cytochrome c in the absence of a rise in [Ca(2+)](i) (Stridh, H., Gigliotti, D., Orrenius, S., and Cotgreave, I. A. (1999) Biochem. Biophys. Res. Commun. 266, 460-465).     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.     

Estrogens prevent calcium-induced release of cytochrome c from heart mitochondria

We investigated the effect of estrogens on heart mitochondrial functions and whether estrogens can prevent calcium-induced release of cytochrome c from mitochondria. 10 nM-10 microM 17beta-estradiol or 4-hydroxytamoxifen did not affect mitochondrial respiration rate and membrane potential in state 3 and state 4. Higher concentrations of both agents decreased state 3 respiration rate and membrane potential. 100 nM 17beta-estradiol and 4-hydroxytamoxifen blocked high calcium-induced cytochrome c release from mitochondria but not mitochondrial swelling. Thus, at physiological concentrations estrogens do not affect mitochondrial respiratory functions but protect heart mitochondria from high calcium-induced release of cytochrome c.     

The cardiolipin-binding domain of Bid affects mitochondrial respiration and enhances cytochrome c release

Bid is cleaved by caspase 8 during apoptosis and the truncated Bid (tBid) translocates to mitochondria by targeting cardiolipin. Amino acids 103-162 of Bid were reported as the cardiolipin-binding domain (CBD). The EGFP-CBD fusion protein targets to mitochondria and induces apoptosis. Using [(3)H]cardiolipin, we proved that recombinant CBD binds cardiolipin similar to tBid and tBid(G94E), a mutant with a defective BH3 domain. CBD could induce cytochrome c release from isolated mitochondria, but much less potent than tBid. Free cardiolipin inhibited the CBD-induced cytochrome c release, suggesting that it may be mediated by interfering with mitochondrial cardiolipin, especially with the interaction between cytochrome c and cardiolipin. This is consistent with the findings that CBD induced cytochrome c release in Bax-deficient cells, and that CBD suppressed mitochondrial respiration through directly interfering with cardiolipin, a critical lipid involved in oxidative phosphorylation. These results indicate the functional importance of CBD in tBid-induced apoptosis.     

Changes in mitochondrial membrane potential during staurosporine-induced apoptosis in Jurkat cells

Cytochrome c release from mitochondria is central to apoptosis, but the events leading up to it are disputed. The mitochondrial membrane potential has been reported to decrease, increase or remain unchanged during cytochrome c release. We measured mitochondrial membrane potential in Jurkat cells undergoing apoptosis by the uptake of the radiolabelled lipophilic cation TPMP, enabling small changes in potential to be determined. The ATP/ADP ratio, mitochondrial and cell volumes, plasma membrane potential and the mitochondrial membrane potential in permeabilised cells were also measured. Before cytochrome c release the mitochondrial membrane potential increased, followed by a decrease in potential associated with mitochondrial swelling and the release of cytochrome c and DDP-1, an intermembrane space house keeping protein. Mitochondrial swelling and cytochrome c release were both blocked by bongkrekic acid, an inhibitor of the permeability transition. We conclude that during apoptosis mitochondria undergo an initial priming phase associated with hyperpolarisation which leads to an effector phase, during which mitochondria swell and release cytochrome c.     

The effect of reactive oxygen species generated from the mitochondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart submitochondrial particles

The effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of cytochrome c oxidase and on the cardiolipin content in bovine heart submitochondrial particles (SMP) was studied. ROS were produced by treatment of succinate-respiring SMP with antimycin A. This treatment resulted in a large production of superoxide anion, measured by epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial mediated ROS generation, led to a marked loss of cytochrome c oxidase activity and to a parallel loss of cardiolipin content. Both these effects were completely abolished by SOD+catalase. Added cardiolipin was able to almost completely restore the ROS-induced loss of cytochrome c oxidase activity. No restoration was obtained with peroxidized cardiolipin. These results demonstrate that mitochondrial mediated ROS generation affects the activity of cytochrome c oxidase via peroxidation of cardiolipin which is needed for the optimal functioning of this enzyme complex. These results may prove useful in probing molecular mechanism of ROS-induced peroxidative damage to mitochondria which have been proposed to contribute to aging, ischemia/reperfusion and chronic degenerative diseases.     

Brain mitochondria are primed by moderate Ca2+ rise upon hypoxia/reoxygenation for functional breakdown and morphological disintegration

In animal models, brain ischemia causes changes in respiratory capacity, mitochondrial morphology, and cytochrome c release from mitochondria as well as a rise in cytosolic Ca2+ concentration. However, the causal relationship of the cellular processes leading to mitochondrial deterioration in brain has not yet been clarified. Here, by applying various techniques, we used isolated rat brain mitochondria to investigate how hypoxia/reoxygenation and nonphysiological Ca2+ concentrations in the low micromolar range affect active (state 3) respiration, membrane permeability, swelling, and morphology of mitochondria. Either transient hypoxia or a micromolar rise in extramitochondrial Ca2+ concentration, given as a single insult alone, slightly decreased active respiration. However, the combination of both insults caused devastating effects. These implied almost complete loss of active respiration, release of both NADH and cytochrome c, and rupture of mitochondria, as shown by electron microscopy. Mitochondrial respiration deteriorated even in the presence of cyclosporin A, documenting that membrane permeabilization occurred independent of mitochondrial permeability transition pore. Ca2+ has to enter the mitochondrial matrix in order to mediate this mitochondrial injury, because blockade of the mitochondrial Ca2+-transport system by ruthenium red in combination with CGP37157 completely prevented damage. Furthermore, protection of respiration from Ca2+-mediated damage by the adenine nucleotide ADP, but not by AMP, during hypoxia/reoxygenation is consistent with the delayed susceptibility of brain mitochondria to prolonged hypoxia, which is observed in vivo.     

Cytochrome c release from rat brain mitochondria is proportional to the mitochondrial functional deficit: implications for apoptosis and neurodegenerative disease

Apoptosis may be initiated in neurons via mitochondrial release of the respiratory protein, cytochrome c. The mechanism of cytochrome c release has been studied extensively, but little is known about its dynamics. It has been claimed that release is all-or-none, however, this is not consistent with accumulating evidence of cytosolic mechanisms for 'buffering' cytochrome c. This study has attempted to model an underlying disease pathology, rather than inducing apoptosis directly. The model adopted was diminished activity of the mitochondrial respiratory chain complex I, a recognized feature of Parkinson's disease. Titration of rat brain mitochondrial respiratory function, with the specific complex I inhibitor rotenone, caused proportional release of cytochrome c from isolated synaptic and non-synaptic mitochondria. The mechanism of release was mediated, at least in part, by the mitochondrial outer membrane component Bak and voltage-dependent anion channel rather than non-specific membrane rupture. Furthermore, preliminary data were obtained demonstrating that in primary cortical neurons, titration with rotenone induced cytochrome c release that was subthreshold for the induction of apoptosis. Implications for the therapy of neurodegenerative diseases are discussed.     

A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c

Multiple apoptotic pathways release cytochrome c from the mitochondrial intermembrane space, resulting in the activation of downstream caspases. In vivo activation of Fas (CD95) resulted in increased permeability of the mitochondrial outer membrane and depletion of cytochrome c stores. Serial measurements of oxygen consumption, NADH redox state and membrane potential revealed a loss of respiratory state transitions. This tBID-induced respiratory failure did not require any caspase activity. At early time points, re-addition of exogenous cytochrome c markedly restored respiratory functions. Over time, however, mitochondria showed increasing irreversible respiratory dysfunction as well as diminished calcium buffering. Electron microscopy and tomographic reconstruction revealed asymmetric mitochondria with blebs of herniated matrix, distended inner membrane and partial loss of cristae structure. Thus, apoptogenic redistribution of cytochrome c is responsible for a distinct program of mitochondrial respiratory dysfunction, in addition to the activation of downstream caspases.     

Ischemia, rather than reperfusion, inhibits respiration through cytochrome oxidase in the isolated, perfused rabbit heart: role of cardiolipin

Ischemia and reperfusion result in mitochondrial dysfunction, with decreases in oxidative capacity, loss of cytochrome c, and generation of reactive oxygen species. During ischemia of the isolated perfused rabbit heart, subsarcolemmal mitochondria, located beneath the plasma membrane, sustain a loss of the phospholipid cardiolipin, with decreases in oxidative metabolism through cytochrome oxidase and the loss of cytochrome c. We asked whether additional injury to the distal electron chain involving cardiolipin with loss of cytochrome c and cytochrome oxidase occurs during reperfusion. Reperfusion did not lead to additional damage in the distal electron transport chain. Oxidation through cytochrome oxidase and the content of cytochrome c did not further decrease during reperfusion. Thus injury to cardiolipin, cytochrome c, and cytochrome oxidase occurs during ischemia rather than during reperfusion. The ischemic injury leads to persistent defects in oxidative function during the early reperfusion period. The decrease in cardiolipin content accompanied by persistent decrements in the content of cytochrome c and oxidation through cytochrome oxidase is a potential mechanism of additional myocyte injury during reperfusion.     

Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells

Mitochondria play a central role in apoptosis through release of cytochrome c and activation of caspases. In the present study, we showed that, in Jurkat human T cells, camptothecin-induced apoptosis is preceded by (i) an increase in cytochrome c and subunit IV of cytochrome c oxidase (COX IV) levels in mitochondria; and (ii) an elevation of the mitochondrial membrane potential (Delta(Psi)m). These events are followed by cytochrome c release into the cytosol, cytochrome c and COX IV depletion from mitochondria, externalization of phosphatidylserine (PS), disruption of Delta(Psi)m, caspase activation, poly(ADP-ribose)polymerase cleavage and DNA fragmentation. The pan-caspase inhibitor z-VAD.fmk blocked camptothecin-induced PS externalization, disruption of Delta(Psi)m and DNA fragmentation, suggesting that these events are mediated by caspase activation. In contrast, z-VAD did not prevent cytochrome c release, despite preventing cytochrome c and COX IV depletion from mitochondria. Together, these data suggest that mitochondrial cytochrome c and COX IV enrichment are early events preceding the onset of apoptosis and that cytochrome c release is upstream of caspase activation and loss of Delta(Psi)m. Furthermore, prevention by z-VAD of cytochrome c and COX IV depletion in mitochondria suggests the possibility that a caspase-like activity in mitochondria is involved in the proteolytic depletion of respiratory chain proteins. Activation of this activity may play an important role in drug-induced apoptosis.