Bcl-2 sensitive mitochondrial potassium accumulation and swelling in apoptosis

During etoposide-induced apoptosis in HL-60 cells, cytochrome c release was associated with mitochondrial swelling caused by increased mitochondrial potassium uptake. The mitochondrial permeability transition was also observed; however, it was not the primary cause of mitochondrial swelling. Potassium uptake and swelling of mitochondria were blocked by bcl-2 overexpression. As a result, cytochrome c release was reduced, and apoptosis delayed. Residual cytochrome c release in the absence of swelling in bcl-2 expressing cells could be due to observed Bax translocation into mitochondria. This study suggests several novel aspects of apoptotic signaling: (1) potassium related swelling of mitochondria; (2) inhibition of mitochondrial potassium uptake by bcl-2; (3) co-existence within one system of multiple mechanisms of cytochrome c release: mitochondrial swelling and swelling-independent permeabilization of the outer mitochondrial membrane.     

Failure of exogenous NADH and cytochrome c to support energy-dependent swelling of mitochondria

The possibility of direct oxidation of external NADH in rat liver mitochondria and of the inner membrane potential generation in this process is still not clear. In the present work, the energy-dependent swelling of mitochondria in the medium containing valinomycin and potassium acetate was measured as one of the main criteria of the proton-motive force generation by complex III, complex IV, and both complexes III and IV of the respiratory chain. Mitochondria swelling induced by external NADH oxidation was compared with that induced by succinate or ferrocyanide oxidation, or by electron transport from succinate to ferricyanide. Mitochondria swelling, nearly equal to that promoted by ferrocyanide oxidation, was observed under external NADH oxidation, but only after the outer mitochondrial membrane was ruptured as a result of the swelling-contraction cycle, caused by succinate oxidation and its subsequent inhibition. In this case, significantly accelerated intermembrane electron transport and well-detected inner membrane potential generation, in addition to mitochondria swelling, were also observed. Presented results suggest that exogenous NADH and cytochrome c do not support the inner membrane potential generation in intact rat liver mitochondria, because the external NADH-cytochrome c reductase system, oriented in the outer mitochondrial membrane toward the cytoplasm, is inaccessible for endogenous cytochrome c reduction; as well, the inner membrane cytochrome c oxidase is inaccessible for exogenous cytochrome c oxidation.     

Interaction of cytochrome c with mitochondrial proteins and cybacrone-dextran

Interaction of cytochrome c with electron carriers in intact and damaged (with destroyed outer membrane) rat liver mitochondria was studied. It was shown that the increase in ionic strength causes changes in the respiration rate of damaged mitochondria due to the reduction of the cytochrome c affinity for its binding sites in the organelles. This suggests that cytochrome c concentration in the intermembrane space of intact mitochondria is increased by salts, whereas the increase in ionic strength has a slight influence on the rates of succinate oxidase and external rotenone-insensitive NADH-oxidase of intact mitochondria. At low ionic strength values, the Michaelis constant (KM) value of external NADH-oxidase for cytochrome c exceeds by one order of magnitude that for succinate oxidase, while the maximal activity of these two systems is nearly the same. The increase in ionic strength causes an increase in the KM value for both oxidases. Interaction of cytochrome c with mitochondrial proteins was modelled by cytochrome c interaction with cibacron-dextran anions. It was concluded that the ionic strength-sensitive electrostatic interactions play a decisive role in cytochrome c binding to electron carriers in mitochondrial membranes. However, cytochrome c content and its binding parameters in intact-mitochondrial membranes prevent the latent activity of external NADH oxidase to be revealed in intact mitochondria after the increase in the ionic strength of the surrounding medium.     

Cytochrome c sorption-desorption effects on the external NADH oxidation by mitochondria: experimental and computational study

The rupture of the outer mitochondrial membrane is known to be critical for cell death, but the mechanism, specifically its redox-signaling aspects, still needs to be studied in more detail. In this work, the external NADH oxidation by rat liver mitochondria was studied under the outer membrane rupture induced by the mitochondria hypotonic treatment or the inner membrane permeability transition. The saturation of the oxidation rate was observed as a function of mitochondrial protein concentration. This effect was shown to result from cytochrome c binding to the mitochondrial membranes. At a relatively high concentration of mitochondria, the oxidation rate was strongly activated by 4 mm Mg(2+) due to cytochrome c desorption from the membranes. A minimal kinetic model was developed to explain the main phenomena of the external NADH oxidation modulated by cytochrome c and Mg(2+) in mitochondria with the ruptured outer membrane. The computational behavior of the model closely agreed with the experimental data. We suggest that the redox state of the released cytochrome c, considered by other authors to be important for apoptosis, may strongly depend on its oxidation by the fraction of mitochondria with the ruptured outer membrane and on the cytoplasmic cytochrome c reductase activity.     

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.     

Synergistic inhibition of mitochondrial respiration by anticancer agent erucylphosphohomocholine and cyclosporin A

Alkylphosphocholines are a new class of anticancer agents. The mechanisms by which these drugs display their antitumor activities are not known. In this work, we show that erucylphosphohomocholine, a new antineoplastic compound, significantly decreased ATP synthesis in isolated rat liver mitochondria at a concentration of 50 microm or higher via permeabilization of the inner membrane. At a concentration of 25 microm, it induced a moderate swelling of mitochondria, a slight decrease of the inner membrane potential, and an increase in state 4 respiration without an essential influence on state 3 respiration or the outer membrane permeability to cytochrome c. We found that cyclosporin A did not prevent mitochondrial swelling induced by 25-100 microm erucylphosphohomocholine. Moreover, cyclosporin A induced a fast drop of the inner membrane potential in the presence of 25-50 microm erucylphosphohomocholine that seems to be due to a strong synergistic inhibition of the respiratory activity. The ratio of uncoupled to state 3 respiration rates increased from 1.3 +/- 0.1 with 25 microm erucylphosphohomocholine and from 1.5 +/- 0.1 with 1 microm cyclosporin A to 4.5 +/- 0.3 in the presence of both drugs. On the other hand, oligomycin or cyclosporin A protected certain cancer cell lines against erucylphosphohomocholine-induced apoptosis. This protection might be related to a prevention of cellular ATP hydrolysis by permeabilized mitochondria and to the inhibition of the classical permeability transition pore, respectively. Our findings provide new insight into the mechanisms by which these unusual alterations of mitochondria might be involved in anticancer activity of alkylphosphocholines.     

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.     

Polyethylenimine-mediated impairment of mitochondrial membrane potential, respiration and membrane integrity: implications for nucleic acid delivery and gene therapy

The 25 kDa branched polyethylenimine (PEI) is a highly efficient synthetic polycation used in transfection protocols, but also triggers mitochondrial-mediated apoptotic cell death processes where the mechanistic issues are poorly understood. We now demonstrate that PEI in a concentration- and time-dependent manner can affect functions (membrane potential, swelling and respiration) and ultrastructural integrity of freshly isolated rat liver mitochondria. The threshold concentration for detection of PEI-mediated impairment of rat liver mitochondrial functions is 3 μg/mL, however, lower PEI levels still exert some effects on mitochondrial morphology and respiration, and these may be related to the inherent membrane perturbing properties of this polycation. The PEI-mediated mitochondrial swelling phase is biphasic, with a fast decaying initial period (most prominent from 4 μg/mL PEI) followed by a slower, linear swelling response. The slow phase is presumably the result of a time-dependent transition permeability opening in mitochondria initially resistant to swelling/depolarization, but may further be related to PEI-induced nanoscale structural defects and/or formation of pores in the outer membrane. Respiration assessments further suggested that PEI in the presence of exogenous ADP behaves in a similar fashion to a slow-acting inhibitory compound. PEI further shows an uncoupling property that is detectable at low respiration rates. The relevance of these findings to PEI-mediated initiation of intrinsic apoptotic pathway is discussed.     

The maturation of the inner membrane of foetal rat liver mitochondria.

A new method was devised for the isolation of foetal and neonatal rat lvier mitochondria, giving higher yields than conventional methods. 2. During development from the perinatal period to the mature adult, the ratio of cytochrome oxidase/succinate-cytochrome c reductase changes. 3. The inner mitochondrial membrane of foetal liver mitochondria possesses virtually no osmotic activity; the permeability to sucrose decreases with increasing developmental age. 4. Foetal rat liver mitochondria possess only marginal respiratory control and do not maintain Ca2+-induced respiration; they also swell in respiratory-control medium in the absence of substrate. ATP enhances respiratory control and prevents swelling, adenylyl imidodiphosphate, ATP+atractyloside enhance the R.C.I. (respiratory control index), Ca2+-induced respiratory control and prevent swelling, whereas GTP and low concentrations of ADP have none of these actions. It is concluded that the effect of ATP depends on steric interaction with the inner mitochondrial membrane. 5. When 1-day pre-partum foetuses are obtained by Caesarean section and maintained in a Humidicrib for 90 min, mitochondrial maturation is "triggered", so that their R.C.I. is enhanced and no ATP is required to support Ca2+-dependent respiratory control or to inhibit mitochondrial swelling. 6. It is concluded that foetal rat liver mitochondria in utero do not respire, although they are capable of oxidative phosphorylation in spite of their low R.C.I. The different environmental conditions which the neonatal rat encounters ex utero enable the hepatic mitochondria to produce ATP, which interacts with the inner mitochondrial membrane to enhance oxidative phosphorylation by an autocatalytic mechanism.     

Induction of Ca2+-dependent cyclosporin a-insensitive nonspecific permeability of the inner membrane of liver mitochondria and cytochrome c release by α,ω-hexadecanedioic acid in media of varying ionic strength

In liver mitochondria loaded with Ca²⁺ or Sr²⁺, α,ω-hexadecanedioic acid (HDA) can induce nonspecific permeability of the inner membrane (mitochondrial pore) by the mechanism insensitive to cyclosporin A (CsA). In this work we studied the effect of ionic strength of the incubation medium on the kinetics of the processes that accompany Ca²⁺-dependent induction of the mitochondrial pore by fatty acid: organelle swelling, Ca²⁺ release from the matrix, changes in transmembrane potential (Δψ) and rate of oxygen consumption, and the release of cytochrome c from the intermembrane space. Two basic incubation media were used: sucrose medium and isotonic ionic medium containing KCl without sucrose. We found that 200 μM Ca²⁺ and 20 μM HDA in the presence of CsA effectively induce high-amplitude swelling of mitochondria both in the case of sucrose and in the ionic incubation medium. In the presence of CsA, mitochondria can rapidly absorb Ca²⁺ and retain it in the matrix for a while without reducing Δψ. Upon incubation in the ionic medium, mitochondria retain most of the added Ca²⁺ in the matrix for a short time without reducing the Δψ. In both cases the addition of HDA to the mitochondria 2 min after the introduction of Ca²⁺ leads to the rapid release of these ions from the matrix and total drop in Δψ. The mitochondrial swelling induced by Ca²⁺ and HDA in non-ionic medium is accompanied by almost maximal stimulation of respiration. Under the same conditions, but during incubation of mitochondria in the ionic medium, it is necessary to add cytochrome c for significant stimulation of respiration. The mitochondrial swelling induced by Ca²⁺ and HDA leads to the release of cytochrome c in a larger amount in the case of ionic medium than for the sucrose medium. We conclude that high ionic strength of the incubation medium determines the massive release of cytochrome c from mitochondria and liberates it from the respiratory chain, which leads to blockade of electron transport along the respiratory chain and consequently to disruption of the energy functions of the organelles.     

BH3 death domain peptide induces cell type-selective mitochondrial outer membrane permeability

The BH3 domain is essential for the release of cytochrome c from mitochondria by pro-apoptotic Bcl-2 family proteins during apoptosis. This study tested the hypothesis that a Bax peptide that includes the BH3 domain can permeabilize the mitochondrial outer membrane and release cytochrome c in the absence of a permeability transition at the mitochondrial inner membrane. BH3 peptide (0.1-60 microm) released cytochrome c from mitochondria in the presence of physiological concentrations of ions in a cell type-selective manner, whereas a BH3 peptide with a single amino acid substitution was ineffective. The release of cytochrome c by BH3 peptide correlated with the presence of endogenous Bax at the mitochondria and its integral membrane insertion. Cytochrome c release was accompanied by adenylate kinase release, was not associated with mitochondrial swelling or substantial loss of electrical potential across the inner membrane, and was unaffected by inhibitors of the permeability transition pore. Cytochrome c release was, however, inhibited by Bcl-2. Although energy-coupled respiration was inhibited after the release of cytochrome c, mitochondria maintained membrane potential in the presence of ATP due to the reversal of the ATP synthase. Overall, results support the hypothesis that BH3 peptide releases cytochrome c by a Bax-dependent process that is independent of the mitochondrial permeability transition pore but regulated by Bcl-2.     

Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the alpha and beta subunits of the F1-ATPase were used. In addition, antibodies against a primary dehydrogenase, beta-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F1-ATPase and F0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, beta-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.     

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 oxidation of exogenous cytochrome c by mitochondria. Resolution of a long-standing controversy

Several reports in the past have dealt with the oxidation of cytochrome c added to suspensions of rat liver mitochondria. Yet, it is generally believed that the cytochrome cannot penetrate the outer membrane. Probably it has been assumed that the permeability of the outer membrane to cytochrome c is very low but finite, and that fast oxidation may be observed if time is allowed for sufficient penetration before initiation of electron flow. Here we show that this view is false. The main fraction of rat liver mitochondria, as isolated by conventional procedures, does not catalyse any significant oxidation of added cytochrome c, even after prolonged incubation. The observed appreciable oxidation of added cytochrome c is catalysed by a very small fraction (5-12%) of the mitochondria that apparently has a damaged outer membrane. Consequently, the turnover of cytochrome oxidase is very high in this fraction during oxidation of added cytochrome c. This finding readily explains why Moyle and Mitchell (e.g., FEBS Lett. 88 (1978) 268-272; 90 (1978) 361-365) have failed to observe proton translocation by cytochrome oxidase during oxidation of ferrocytochrome c added to rat liver mitochondria, which has been their main reason for rejecting the proton-pumping function of cytochrome oxidase.     

Resonance Raman investigations of cytochrome c conformational change upon interaction with the membranes of intact and Ca2+-exposed mitochondria

The conformational states of cytochrome c inside intact and Ca(2+)-exposed mitochondria have been investigated using resonance Raman spectroscopy. Intact and swelling bovine heart and rat liver mitochondria were examined with an excitation wavelength (413.1 nm) in resonance with the Soret transition of ferrous cytochrome c. The different b- to c-type cytochrome concentration ratio in mitochondria from two different tissues was used to help assign the Raman spectral components. Resonance Raman spectra were also recorded for mitochondria fractions (supernatants and pellets) obtained from swollen (Ca(2+)-exposed) mitochondria after differential centrifugation. The results illustrate that cytochrome c has an altered vibrational spectrum in solution, in intact, and in swollen mitochondria. When cytochrome c is released from mitochondria, its Raman spectrum becomes identical to that of ferrous cytochrome c in solution. The spectra of mitochondrial pellets indicate that a small amount of structurally modified cytochrome c remains associated with the heavy membrane fraction. Indeed, spectroscopic shifts in the low-frequency fingerprint and the high-frequency marker-band regions suggest that membrane binding leads to a partial opening of the heme pocket and an alteration of the heme thioether bonds. The results support the conclusion that most cytochrome c molecules in mitochondria are membrane-bound and that the cytochrome c structure changes upon binding. Furthermore, changes in the resonance Raman active mode located at 675 cm(-)(1) in the spectra of intact, swollen, and fractionated mitochondria indicate that b-type cytochromes may also undergo structural alterations during mitochondrial swelling and disruption.     

A folding variant of human alpha-lactalbumin induces mitochondrial permeability transition in isolated mitochondria.

A human milk fraction containing multimeric alpha-lactalbumin (MAL) is able to kill cells via apoptosis. MAL is a protein complex of a folding variant of alpha-lactalbumin and lipids. Previous results have shown that upon treatment of transformed cells, MAL localizes to the mitochondria and cytochrome c is released into the cytosol. This is followed by activation of the caspase cascade. In this study, we further investigated the involvement of mitochondria in apoptosis induced by the folding variant of alpha-lactalbumin. Addition of MAL to isolated rat liver mitochondria induced a loss of the mitochondrial membrane potential (Delta Psi(m)), mitochondrial swelling and the release of cytochrome c. These changes were Ca(2+)-dependent and were prevented by cyclosporin A, an inhibitor of mitochondrial permeability transition. MAL also increased the rate of state 4 respiration in isolated mitochondria by exerting an uncoupling effect. This effect was due to the presence of fatty acids in the MAL complex because it was abolished completely by BSA. BSA delayed, but failed to prevent, mitochondrial swelling as well as dissipation of Delta Psi(m), indicating that the fatty acid content of MAL facilitated, rather than caused, these effects. Similar results were obtained with HAMLET (human alpha-lactalbumin made lethal to tumour cells), which is native alpha-lactalbumin converted in vitro to the apoptosis-inducing folding variant of the protein in complex with oleic acid. Our findings demonstrate that a folding variant of alpha-lactalbumin induces mitochondrial permeability transition with subsequent cytochrome c release, which in transformed cells may lead to activation of the caspase cascade and apoptotic death.     

Chromium(VI) interaction with plant and animal mitochondrial bioenergetics: a comparative study

The mechanism of Cr(VI)-induced toxicity in plants and animals has been assessed for mitochondrial bioenergetics and membrane damage in turnip root and rat liver mitochondria. By using succinate as the respiratory substrate, ADP/O and respiratory control ratio (RCR) were depressed as a function of Cr(VI) concentration. State 3 and uncoupled respiration were also depressed by Cr(VI). Rat mitochondria revealed a higher sensitivity to Cr(VI), as compared to turnip mitochondria. Rat mitochondrial state 4 respiration rate triplicated in contrast to negligible stimulation of turnip state 4 respiration. Chromium(VI) inhibited the activity of the NADH-ubiquinone oxidoreductase (complex I) from rat liver mitochondria and succinate-dehydrogenases (complex II) from plant and animal mitochondria. In rat liver mitochondria, complex I was more sensitive to Cr(VI) than complex II. The activity of cytochrome c oxidase (complex IV) was not sensitive to Cr(VI). Unique for plant mitochondria, exogenous NADH uncoupled respiration was unaffected by Cr(VI), indicating that the NADH dehydrogenase of the outer leaflet of the plant inner membrane, in addition to complexes III and IV, were insensitive to Cr(VI). The ATPase activity (complex V) was stimulated in rat liver mitochondria, but inhibited in turnip root mitochondria. In both, turnip and rat mitochondria, Cr(VI) depressed mitochondrial succinate-dependent transmembrane potential (Deltapsi) and phosphorylation efficiency, but it neither affected mitochondrial membrane permeabilization to protons (H+) nor induced membrane lipid peroxidation. However, Cr(VI) induced mitochondrial membrane permeabilization to K+, an effect that was more pronounced in turnip root than in rat liver mitochondria. In conclusion, Cr(VI)-induced perturbations of mitochondrial bioenergetics compromises energy-dependent biochemical processes and, therefore, may contribute to the basal mechanism underlying its toxic effects in plant and animal cells.     

Crystal violet as an uncoupler of oxidative phosphorylation in rat liver mitochondria

Crystal violet exhibited characteristics of an uncoupler of oxidative phosphorylation, i.e. it released respiratory control, hindered ATP synthesis, enhanced ATPase activity, and produced swelling of isolated rat liver mitochondria. Maximal stimulation of respiration, ATPase activity, and swelling was observed at a concentration of 40 microM. The inhibition of State 3 respiration by oligomycin was released by crystal violet. High concentrations of crystal violet inhibited mitochondrial respiration. The uncoupling effect of crystal violet required inorganic phosphate and was abolished by N-ethylmaleimide. The adenine nucleotides ADP and ATP protected mitochondria from uncoupling by the dye. The dye taken up by mitochondria was released into the incubation medium on induction of uncoupling. In the absence of phosphate, the dye did not cause uncoupling, but its retention was much greater than in the presence of phosphate. Crystal violet is suggested to induce uncoupling by acting on the membrane, rather than by its electrophoretic transfer into the mitochondria.     

L-Carnitine suppresses oleic acid-induced membrane permeability transition of mitochondria

Membrane permeability transition (MPT) of mitochondria has an important role in apoptosis of various cells. The classic type of MPT is characterized by increased Ca(2+) transport, membrane depolarization, swelling, and sensitivity to cyclosporin A. In this study, we investigated whether L-carnitine suppresses oleic acid-induced MPT using isolated mitochondria from rat liver. Oleic acid-induced MPT in isolated mitochondria, inhibited endogenous respiration, caused membrane depolarization, and increased large amplitude swelling, and cytochrome c (Cyt. c) release from mitochondria. L-Carnitine was indispensable to beta-oxidation of oleic acid in the mitochondria, and this reaction required ATP and coenzyme A (CoA). In the presence of ATP and CoA, L-carnitine stimulated oleic acid oxidation and suppressed the oleic acid-induced depolarization, swelling, and Cyt. c release. L-Carnitine also contributed to maintaining mitochondrial function, which was decreased by the generation of free fatty acids with the passage of time after isolation. These results suggest that L-carnitine acts to maintain mitochondrial function and suppresses oleic acid-mediated MPT through acceleration of beta-oxidation.     

Oxidation and reduction of exogenous cytochrome c by the activity of the respiratory chain.

Oxidation of exogenous NADH by isolated rat liver mitochondria is generally accepted to be mediated by endogenous cytochrome c which shuttles electrons from the outer to the inner mitochondrial membrane. More recently it has been suggested that, in the presence of added cytochrome c, NADH oxidation is carried out exclusively by the cytochrome oxidase of broken or damaged mitochondria. Here we show that electrons can be transferred in and out of intact mitochondria. It is proposed that at the contact sites between the inner and the outer membrane, a "bi-trans-membrane" electron transport chain is present. The pathway, consisting of Complex III, NADH-b5 reductase, exogenous cytochrome c and cytochrome oxidase, can channel electrons from the external face of the outer membrane to the matrix face of the inner membrane and viceversa. The activity of the pathway is strictly dependent on both the activity of the respiratory chain and mitochondrion integrity.