Arachidonic acid causes cytochrome c release from heart mitochondria

Arachidonic acid interaction with heart mitochondria is known to cause uncoupling as well as inhibition of pyruvate + malate and succinate-supported respiration. Here we present experiments showing that arachidonic acid causes cytochrome c release from Ca(2+)-loaded heart mitochondria. We have also measured mitochondrial matrix swelling and found a fairly good correlation between the two processes, as revealed by the same arachidonic acid concentration dependence and by the same susceptibility toward different free fatty acid species. The effects produced by arachidonic acid are not related to its protonophoric activity since, under the experimental conditions used, saturating concentrations of FCCP did not cause any effect.     

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).     

Mechanisms of cytochrome c release from mitochondria

In healthy cells, cytochrome c (Cyt c) is located in the mitochondrial intermembrane/intercristae spaces, where it functions as an electron shuttle in the respiratory chain and interacts with cardiolipin (CL). Several proapoptotic stimuli induce the permeabilization of the outer membrane, facilitate the communication between intermembrane and intercristae spaces and promote the mobilization of Cyt c from CL, allowing for Cyt c release. In the cytosol, Cyt c mediates the allosteric activation of apoptosis-protease activating factor 1, which is required for the proteolytic maturation of caspase-9 and caspase-3. Activated caspases ultimately lead to apoptotic cell dismantling. Nevertheless, cytosolic Cyt c has been associated also to vital cell functions (i.e. differentiation), suggesting that its release not always occurs in an all-or-nothing fashion and that mitochondrial outer membrane permeabilization may not invariably lead to cell death. This review deals with the events involved in Cyt c release from mitochondria, with special attention to its regulation and final consequences.     

Ceramide induces cytochrome c release from isolated mitochondria. Importance of mitochondrial redox state

In the present study we show that N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and, to a much lesser extent, C2-dihydroceramide induce cytochrome c (cyto c) release from isolated rat liver mitochondria. Ceramide-induced cyto c release is prevented by preincubation of mitochondria with a low concentration (40 nM) of Bcl-2. The release takes place when cyto c is oxidized but not when it is reduced. Upon cyto c loss, mitochondrial oxygen consumption, mitochondrial transmembrane potential (Delta Psi), and Ca2+ retention are diminished. Incubation with Bcl-2 prevents, and addition of cyto c reverses the alteration of these mitochondrial functions. In ATP-energized mitochondria, ceramides do not alter Delta Psi, neither when cyto c is oxidized nor when it is reduced, ruling out a nonspecific disturbance by ceramides of mitochondrial membrane integrity. Furthermore, ceramides decrease the reducibility of cyto c. We conclude that the apoptogenic properties of ceramides are in part mediated via their interaction with mitochondrial cyto c followed by its release and that the redox state of cyto c influences its detachment by ceramide from the inner mitochondrial membrane.     

Rapid spectrophotometric method for quantitation of cytochrome c release from isolated mitochondria or permeabilized cells revisited

This paper recalls the earlier work by Keilin, Margoliash and others at the beginning of the 20th century and shows how their results can be used for the rapid solution of new problems of modern science. It describes a rapid and simple spectrophotometric method for quantitative determination of cytochrome c release from isolated mitochondria or permeabilized cells induced by proapoptotic proteins. For this, the Soret (gamma) peak at 414 nm in the spectrum of cytochrome c is used. The results of spectrophotometric assay of cytochrome c release are in accord with those of oxygraphic determination of cytochrome c-dependent respiration of isolated mitochondria and permeabilized cardiomyocytes.     

Quantitation of cytochrome c release from rat liver mitochondria

The apoptogenic protein cytochrome c can be quantitated by reverse-phase HPLC, but this method is not utilized by those who investigate mechanisms of cell death. Here, we extend the sensitivity of the method to exceed that available from immunogenic approaches and report specific procedures for applying the method to preparations of intact mitochondria, and to supernatants and pellets that arise from mitochondrial incubations. The detection limit corresponds to 0.6% of total cytochrome c found in 100 microg of rat liver mitochondrial protein, or to all of the cytochrome c that is expected in approximately 6000 hepatocytes. A single determination can be completed in 20 min, compared to a time scale of days for Western blotting methods, or hours for ELISA-based methods. The procedures are illustrated by experiments that determine the amount of cytochrome c released following the mitochondrial permeability transition as a function of medium ionic strength, and by long-term incubations of intact mitochondria in the presence and absence of an exogenous oxidizable substrate. Swelling and the release of adenylate kinase activity have been determined simultaneously to show how the data can be applied to evaluate the role of outer membrane disruption in mechanisms that release cytochrome c.     

Release of cytochrome c from isolated mitochondria by etoposide

The efficacy of chemotherapeutic agents on tumor cells has been shown to be modulated by tumor suppressor gene p53 and its target genes such as Bcl-2 family members (Bax, Noxa, and PUMA). However, various chemotherapeutic agents can induce cell death in tumor cells that do not express the functional p53, suggesting that some chemotherapeutic agents may induce cell death in a p53-independent pathway. Here we showed that etoposide can induce the similar degree of cell death in p53-deficient HCT 116 cells, whereas 5'-FU-mediated cell death is strongly dependent on the existence of functional p53 in HCT 116 cells. Further, we provide the evidence that etoposide can induce the cytochrome c release from isolated mitochondria, and etoposide-induced cytochrome c release is not accompanied with the large amplitude swelling of mitochondria. These data suggest that etoposide can directly induce the mitochondrial dysfunction irrespective of p53 status, and it may, at least in part, account for the p53-independent pathway in cell death induced by chemotherapeutic agents.     

Caspases induce cytochrome c release from mitochondria by activating cytosolic factors.

We investigated the ability of caspases (cysteine proteases with aspartic acid specificity) to induce cytochrome c release from mitochondria. When Jurkat cells were induced to undergo apoptosis by Fas receptor ligation, cytochrome c was released from mitochondria, an event that was prevented by the caspase inhibitor, zVAD-fmk (zVal-Ala-Asp-CH2F). Purified caspase-8 triggered rapid cytochrome c release from isolated mitochondria in vitro. The effect was indirect, as the presence of cytosol was required, suggesting that caspase-8 cleaves and activates a cytosolic substrate, which in turn is able to induce cytochrome c release from mitochondria. The cytochrome c releasing activity was not blocked by caspase inhibition, but was antagonized by Bcl-2 or Bcl-xL. Caspase-8 and caspase-3 cleaved Bid, a proapoptotic Bcl-2 family member, which gains cytochrome c releasing activity in response to caspase cleavage. However, caspase-6 and caspase-7 did not cleave Bid, although they initiated cytochrome c release from mitochondria in the presence of cytosol. Thus, effector caspases may cleave and activate another cytosolic substrate (other than Bid), which then promotes cytochrome c release from mitochondria. Mitochondria significantly amplified the caspase-8 initiated DEVD-specific cleavage activity. Our data suggest that cytochrome c release, initiated by the action of caspases on a cytosolic substrates, may act to amplify a caspase cascade during apoptosis.     

Characterization of tBid-induced cytochrome c release from mitochondria and liposomes

tBid, the cleaved form of Bid, can induce cytochrome c (Cyt. c) release from rat heart mitochondria more efficiently and reproducibly than that from liver or brain mitochondria. Unlike Bax, such release was not prevented by cyclosphorin A, an inhibitor of the opening of permeability transition pore. Carbonyl-cyanide m-chlorophenyl-hydrazone or oligomycin also have no obvious effect on the release of Cyt. c. In contrast to ceramide, tBid-mediated Cyt. c release from mitochondria is independent of the redox state of Cyt. c. Furthermore, Bid or tBid can directly trigger the efflux of encapsulated Cyt. c or trypsin within liposomes without involvement of other protein factors.     

Anoxia-reoxygenation-induced cytochrome c and cardiolipin release from rat brain mitochondria

Rat brain mitochondria were successively submitted to anoxia and reoxygenation. The main mitochondrial functions were assessed at different reoxygenation times. Although the respiratory control ratio decreased, the activity for each one of the enzymes participating in the respiratory chain was not affected. However, during reoxygenation, mitochondrial membrane lipoperoxidation quickly increased and was proportional to the decrease seen in membrane fluidity. Under the same conditions, cytochrome c and cardiolipin were released from mitochondria and their rate of release increased with reoxygenation time. The release of cytochrome c and cardiolipin was followed by the collapse of the membrane potential and it was not inhibited by cyclosporin A. Addition of the antioxidant alpha-tocopherol abolished all these reoxygenation-induced changes. These data indicate that, in this model, reoxygenation promotes the uncoupling of respiratory chain, and cytochrome c and cardiolipin releases. These events are not related to the membrane potential collapse but to an oxidative stress.     

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.     

Multiple pathways of cytochrome c release from mitochondria in apoptosis

Release of cytochrome c from mitochondria is a key initiative step in the apoptotic process, although the mechanisms regulating permeabilization of the outer mitochondrial membrane and the release of intermembrane space proteins remain controversial. Here, we discuss possible scenarios of the outer membrane permeabilization. The mechanisms by which the intermembrane space proteins are released from mitochondria depend presumably on cell type and on the nature of the apoptotic stimulus. The variety of mechanisms that can lead to outer membrane permeabilization might explain diversities in the response of mitochondria to numerous apoptotic stimuli in different types of cells.     

Mitochondrial iPLA2 activity modulates the release of cytochrome c from mitochondria and influences the permeability transition

The mitochondrial Ca(2+)-independent phospholipase A(2) is activated during energy-dependent Ca(2+) accumulation under conditions where there is a sustained depression of the membrane potential. This activation is not dependent on induction of the mitochondrial permeability transition. Bromoenol lactone, which inhibits the phospholipase, is effective as an inhibitor of the transition, and this action can be overcome by low levels of exogenous free fatty acids. Apparently, activation of the Ca(2+)-independent phospholipase is a factor in the mechanisms by which depolarization and Ca(2+) accumulation promote opening of the permeability transition pore. Sustained activity of the Ca(2+)-independent phospholipase A(2) promotes rupture of the outer mitochondrial membrane and spontaneous release of cytochrome c on a time scale similar to that of apoptosis occurring in cells. However, more swelling of the matrix space must occur to provoke release of a given cytochrome c fraction when the enzyme is active, compared with when it is inhibited. Through its effects on the permeability transition and release of intermembrane space proteins, the mitochondrial Ca(2+)-independent phospholipase A(2) may be an important factor governing cell death caused by necrosis or apoptosis.     

Cardiolipin is not required for Bax-mediated cytochrome c release from yeast mitochondria

Cardiolipin (CL) is an inner mitochondrial membrane phospholipid that contributes to optimal mitochondrial function and is gaining widespread attention in studies of mitochondria-mediated apoptosis. Divergent hypotheses describing the role of CL in cytochrome c release and apoptosis have evolved. We addressed this controversy directly by comparing the spontaneous- and Bax-mediated cytochrome c release from mitochondria isolated from two strains of Saccharomyces cerevisiae: one lacking CL-synthase and therefore CL (DeltaCRD1) and the other, its corresponding wild type (WT). We demonstrated by liquid chromatography-mass spectrometry that the main yeast CL species [(16:1)2(18:1)2] differs in fatty acid composition from mammalian CL [(18:2)4], and we verified the absence of the yeast CL species in the DeltaCRD1 strain. We also demonstrated that the mitochondrial association of Bax and the resulting cytochrome c release is not dependent on the CL content of the yeast mitochondrial membranes. Bax inserted equally into both WT and DeltaCRD1 mitochondrial membranes under conditions that lead to the release of cytochrome c from both strains of yeast mitochondria. Furthermore, using models of synthetic liposomes and isolated yeast mitochondria, we found that cytochrome c was bound more "loosely" to the CL-deficient systems compared with when CL is present. These data challenge recent studies implicating that CL is required for Bax-mediated pore formation leading to the release of proteins from the mitochondrial intermembrane space. In contrast, they support our recently proposed two-step mechanism of cytochrome c release, which suggests that CL is required for binding cytochrome c to the inner mitochondrial membrane.     

Mitochondrial precursor signal peptide induces a unique permeability transition and release of cytochrome c from liver and brain mitochondria

This study tested the hypothesis that mitochondrial precursor targeting peptides can elicit the release of cytochrome c from both liver and brain mitochondria by a mechanism distinct from that mediated by the classical, Ca2+-activated permeability transition pore. Human cytochrome oxidase subunit IV signal peptide (hCOXIV1-22) at concentrations from 15 to 100 microM induced swelling, a decrease in membrane potential, and cytochrome c release in both types of mitochondria. Although cyclosporin A and bongkrekic acid were without effect, dibucaine, propanolol, dextran, and the uncoupler FCCP were each able to inhibit signal peptide-induced swelling and cytochrome c release. Adenylate kinase was coreleased with cytochrome c, arguing against a signal peptide-induced cytochrome c-specific pathway of efflux across the outer membrane. Taken together, the data indicate that a human mitochondrial signal peptide can evoke the release of cytochrome c from both liver and brain mitochondria by a unique permeability transition that differs in several characteristics from the classical mitochondrial permeability transition.     

Biosynthesis of cytochrome c oxidase by isolated rat liver mitochondria.

When isolated mitochondria which have been labeled with [3H]leucine are solubilized and treated with anti-serum specific for cytochrome c oxidase, labeled polypeptides which correspond to the three largest polypeptides of this enzyme are immunoprecipitated. This indicates that the three largest polypeptides of cytochrome c oxidase which have Mr of 66,000, 39,000, and 23,000 are synthesized by isolated mitochondria whereas the three smallest ones which have Mr of 14,000, 12,500, and 10,000 are not. The smallest polypeptides are probably synthesized on cytoplasmic ribosomes as has been demonstrated in other systems by in vivo studies. These results are the first demonstration that isolated mammalian mitochondria are capable of synthesizing some of their own polypeptide components. The antiserum used in this study was prepared to highly purified cytochrome c oxidase (12.4 nmol of heme a + a3/mg of protein) from rat liver mitochondria. This antiserum gives a single precipitin line when tested by the Ouchterlony double diffusion technique. Its specificity has been demonstrated by the fact that it: 1) only precipitates heme a + a3, not hemes b, c, or c1, when added to solubilized mitochondria, 2) inhibits cytochrome c oxidase activity at least 85%, and 3) precipitates only those polypeptides found in purified cytochrome c oxidase when added to solubilized mitochondria labeled in vivo.     

Ganglioside GD3 and its mimetics induce cytochrome c release from mitochondria

Ganglioside GD3 induced the release of cytochrome c from isolated rat liver mitochondria. This process was completely prevented by cyclosporin A and partially prevented by a cysteine protease inhibitor, n-acetyl-leu-leu-norleucinal. Cyclosporin A is a potent inhibitor of the permeability transition pore, whereas n-acetyl-leu-leu-norleucinal has no effect on this pore. These results indicate that the release of cytochrome c from mitochondria requires both the opening of the permeability transition pore and a cysteine protease inhibitor-sensitive mechanism. Gangliosides GD1a, GD1b, GT1b, and GQ1b along with the synthetic GD3 mimetics TMS-42 and CI-22, which are glycerophospholipids carrying a disialo residue, also induced cytochrome c release. In contrast, gangliosides GM1, GM2, and GM3 did not induce cytochrome c release. These results indicate that two sialo residues must play an important role in the induction of cytochrome c release by gangliosides.     

Retinol induces permeability transition and cytochrome c release from rat liver mitochondria

Biological actions of retinoids on modulation of cellular gene expression by nuclear receptors are widely known. Recently, extra-nuclear effects of retinoids have been proposed, but remain to be better elucidated. Considering that retinoids induce apoptosis in tumor cells by an unknown mechanism, and that mitochondria play a key role in controlling apoptosis via cytochrome c (cyt c) release, we exposed rat liver mitochondria to 3-40 microM of retinol (vitamin A), and observed that retinol causes mitochondrial permeability transition (MPT) and cyt c release, in a concentration-dependent pattern. Increased superoxide anion generation and lipoperoxidation were also observed. Cyclosporin A or trolox co-administration reverted all parameters tested. In view of these findings, we conclude that retinol induces mitochondria oxidative damage, leading to MPT and cyt c release by opening of the permeability transition pore, thus suggesting a putative mechanism of apoptosis activation by retinol.     

Distinct pathways for stimulation of cytochrome c release by etoposide

Induction of apoptosis by DNA-damaging agents, such as etoposide, is known to involve the release of mitochondrial cytochrome c, although the mechanism responsible for this event is unclear. In the present study, using Jurkat T-lymphocytes, a reconstituted cell-free system, or isolated liver mitochondria, we demonstrate the ability of etoposide to induce cytochrome c release via two distinct pathways. Caspase inhibition by either benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) or benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethyl ketone (z-VDVAD-fmk) attenuates cytochrome c release triggered by a low dose of etoposide via an apparent inhibition of nuclear events involving the release of protein factor(s) that is (are) able to interact with mitochondria. In contrast, caspase inhibition has no effect on cytochrome c release induced by a higher dose of etoposide. Moreover, the higher dose of etoposide heightens the sensitivity of Ca(2+)-loaded isolated mitochondria to mitochondrial permeability transition, an effect that is completely abolished by cyclosporin A. Interestingly, cyclosporin A is ineffective at preventing similar mitochondrial damage in Jurkat cells treated with etoposide. We propose that lower doses of etoposide predominantly target the nucleus and stimulate the release of caspase-sensitive protein factor(s) that interact with mitochondria to trigger cytochrome c release, whereas higher doses of the drug impart a more direct effect on mitochondria and thus are not mitigated by caspase inhibition.