Oxidative stress in mitochondria: its relationship to cellular Ca2+ homeostasis, cell death, proliferation, and differentiation

A variety of chemically different prooxidants causes Ca2+ release from mitochondria. This prooxidant-induced Ca2+ release occurs from intact mitochondria via a route which is physiologically relevant and may be regulated by protein monoADP-ribosylation. When the released Ca2+ is excessively 'cycled' by mitochondria (continuously taken up and released) the inner membrane is damaged. This leads to a decreased ability of mitochondria to retain Ca2+, uncoupling of mitochondria, and an impairment of ATP synthesis, which in turn deprives the cell of the energy necessary for the proper functioning of the Ca2+ ATPases of the endoplasmic (sarcoplasmic) reticulum, the nucleus and the plasma membrane. The ensuing rise of the cytosolic Ca2+ level cannot be counterbalanced by the damaged mitochondria which, under normoxic conditions, act as a safety device against an increase of the cytosolic Ca2+ concentration. The impaired ability of mitochondria to retain Ca2+ may lead to cell death. However, there is also evidence emerging that release of Ca2+ from mitochondria may be physiologically important for cell proliferation and differentiation.     

Metabolic Changes Induced by Cold Stress in Rat Liver Mitochondria

The mechanisms involved in the metabolic changes induced by cold stress in isolated rat liver mitochondria were studied. Respiration, ATP synthesis, and membrane potential as well as the contents of several metabolites were determined in liver mitochondria from cold-exposed rats. At different times of cold exposure, the force-flux relationships showed net variation in flux (enhanced respiration, diminished ATP synthesis) with no associated variation in force (H+ gradient); this suggested that decoupling rather than classical uncoupling was involved in the effects of cold stress. The flux control coefficient of the H+ leak on basal respiration was slightly increased by 380 h of cold exposure. Cold stress also induced a diminution in total membrane fatty acids, Zn2+, Fe3+, ATP, and ADP/O ratios; the content of cytochromes c + c1 and b oscillated. The contents of Ca2+, Na+, Pi, and cytochromes a + a3 were not affected, whereas matrix ADP, AMP, K+, and Mg2+ were markedly increased. Basal and oleic acid-stimulated respiration of mitochondria from cold-stressed rats was inhibited by GDP, carboxyatractyloside, or albumin. These agents did not affect basal respiration in control mitochondria. Western blot analysis showed enhanced expression of a protein of about 35 kDa, presumably the uncoupling protein 2, induced by long-term cold exposure. The overall data suggest that cold stress promoted decoupling of oxidative phosphorylation, and hence, changes in several matrix metabolites, by increasing free fatty acids and the UCP2 content.     

Uncoupling proteins in mitochondria of plants and some microorganisms.

Uncoupling proteins, members of the mitochondrial carrier family, are present in mitochondrial inner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+ re-uptake. Since 1995, it has been shown that the uncoupling protein is present in many higher plants and some microorganisms like non-photosynthetic amoeboid protozoon, Acanthamoeba castellanii and non-fermentative yeast Candida parapsilosis. In mitochondria of these organisms, uncoupling protein activity is revealed not only by stimulation of state 4 respiration by free fatty acids accompanied by decrease in membrane potential (these effects being partially released by ATP and GTP) but mainly by lowering ADP/O ratio during state 3 respiration. Plant and microorganism uncoupling proteins are able to divert very efficiently energy from oxidative phosphorylation, competing for deltamicroH+ with ATP synthase. Functional connection and physiological role of uncoupling protein and alternative oxidase, two main energy-dissipating systems in plant-type mitochondria, are discussed.     

Ca2+ release from mitochondria induced by prooxidants

A variety of chemically different prooxidants causes Ca2+ release from mitochondria. The prooxidant-induced Ca2+ release occurs from intact mitochondria via a route which is physiologically relevant and may be regulated by protein ADP-ribosylation. When the released Ca2+ is excessively cycled by mitochondria they are damaged. This leads to uncoupling, a decreased ATP supply, and a decreased ability of mitochondria to retain Ca2+. Excessive Ca2+ cycling by mitochondria will deprive cells of ATP. As a result, Ca2+ ATPases of the endoplasmic (sarcoplasmic) reticulum and the plasma membrane are stopped. The rising cytosolic Ca2+ level cannot be counterbalanced due to damage of mitochondria which, under normoxic conditions, act as safety device against increased cytosolic Ca2+. It is proposed that prooxidants are toxic because they impair the ability of mitochondria to retain Ca2+.     

The anti-cancer agent nemorosone is a new potent protonophoric mitochondrial uncoupler

Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1-25 μM) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50-500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca(2+) from Ca(2+)-loaded mitochondria, decreased Ca(2+) uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells.     

Intramitochondrial K+ as activator of carboxyatractyloside-induced Ca2+ release.

The role of intramitochondrial K+ content on the increase in membrane permeability to Ca2+, as induced by carboxyatractyloside was studied. In mitochondria containing a high K+ concentration (83 nmol/mg), carboxyatractyloside induced a fast and extensive mitochondrial Ca2+ release, membrane de-energization, and swelling. Conversely, in K(+)-depleted mitochondria (11 nmol/mg), carboxyatractyloside was ineffective. The addition of 40 mM K+ to K(+)-depleted mitochondria restored the capability of atractyloside to induce an increase in membrane permeability to Ca2+ release. The determination of matrix free Ca2+ concentration showed that, at an external free-Ca2+ concentration of 0.8 microM, control mitochondria contained 3.9 microM of free Ca2+ whereas K(+)-depleted mitochondria contained 0.9 microM free Ca2+. It is proposed that intramitochondrial K+ affects the matrix free Ca2+ concentration required to induce a state of high membrane permeability.     

Regulation of steady-state free Ca2+ levels by the ATP/ADP ratio and orthophosphate in permeabilized RINm5F insulinoma cells

Stimulation of insulin secretion in the pancreatic beta-cell by a fuel such as glucose requires the metabolism of the fuel and is accompanied by increases in oxygen consumption and intracellular free Ca2+. A very early signal for these events could be a decrease in the cytosolic ATP/ADP ratio due to fuel phosphorylation. To test this hypothesis the regulation of free Ca2+ was evaluated in permeabilized RINm5F insulinoma cells that sequester Ca2+ and maintain a low medium free Ca2+ concentration (set point), between 100 and 200 nM, in the presence of Mg2+ and ATP. ATP, creatine, creatine phosphate, and creatine phosphokinase were added to the media to achieve various constant ratios of ATP/ADP. Free Ca2 was monitored using fura-2. The results demonstrated that the steady-state free Ca2+ concentration varied inversely with the ATP/ADP ratio and orthophosphate (Pi) levels. In contrast, no correlation between free Ca2+ and the phosphorylation potential (ATP/ADP.Pi) was found. Regulation of the Ca2+ set point by the ATP/ADP ratio was observed at ratios between 5 and 50 and at Pi concentrations between 1 and 7 mM, irrespective of whether mitochondria were participating in Ca2+ sequestration or were inhibited. Increasing the ATP/ADP ratio stimulated Ca2+ uptake by the nonmitochondrial pool but did not modify Ca2+ efflux. Glucose 6-phosphate (1 mM) had no effect on the Ca2+ set point. The data suggest that variations in the cytosolic ATP/ADP ratio induced by fuel stimuli may regulate Ca2+ cycling across nonmitochondrial compartments and the plasma membrane by modulating the activity of Ca2+ -ATPases. A mechanism linking fuel metabolism and cytosolic ATP/ADP ratio to activation of the Ca2+ messenger system in pancreatic beta-cells is proposed.     

Effects of photodynamic action on energy coupling of Ca2+ uptake in liver mitochondria

In mitochondria isolated from rat liver, incubated in the presence of 6 X 10(-3) mM hematoporphyrin and irradiated with UV light at 365 nm, respiration, oxidative phosphorylation and Ca2+ uptake were measured in order to determine the respective photosensitivity of these functions. Irradiation with increasing doses produces uncoupling of oxidative phosphorylation followed by inhibition of Ca2+ uptake and finally arrest of respiration. Ca2+ uptake stimulated by the addition of ATP was also studied in mitochondria uncoupled by irradiation which were still able to concentrate Ca2+ aerobically. Anaerobic Ca2+ uptake driven by ATP hydrolysis was found to be similar in control and in irradiated mitochondria, suggesting a different photosensitivity for the ATPase as compared to the ATP-synthase activity.     

The regulation of brain mitochondrial calcium-ion transport. The role of ATP in the discrimination between kinetic and membrane-potential-dependent calcium-ion efflux mechanisms.

Mitochondria from guinea-pig cerebral cortex incubated in the presence of Pi or acetate are unable to regulate the extramitochondrial free Ca2+ at a steady-state which is independent of the Ca2+ accumulated in the matrix. This is due to the superimposition on kinetically regulated Ca2+ cycling of a membrane-potential-dependent reversal of the Ca2+ uniporter. The latter efflux is a consequence of a low membrane potential, which correlates with a loss of adenine nucleotide loss from the matrix, enable the mitochondria to maintain a high membrane potential and allow the mitochondria to buffer the extramitochondrial free Ca2+ precisely when up to 200 nmol of Ca2+/mg of protein is accumulated in the matrix. The steady-state extramitochondrial free Ca2+ is maintained as low as 0.3 microM. The Na+-activated efflux pathway is functional in the presence of ATP and oligomycin and accounts precisely for the change in steady-state free Ca2+ induced by Na+ addition. The need to distinguish carefully between kinetic and membrane-potential-dependent efflux pathways is emphasized and the competence of brain mitochondria to regulate cytosolic free Ca2+ concentrations in vivo is discussed.     

Induction of the non-selective mitochondrial pore in lymphoid cells. 1. Permeabilized rat thymocytes.

The opening of the cyclosporin-sensitive pore in the inner membrane of mitochondria in rat thymocytes was studied. In thymocytes with digitonin-permeabilized plasma membrane, the mitochondrial pore was induced by Ca2+ overload, by uncoupling, by oxidation or cross-linking of membrane dithiols, and by atractyloside, a specific inhibitor of the adenine nucleotide transporter. Pore opening was prevented by cyclosporin A (CsA) and by its non-immunosuppressive analog MeVal-CsA. The sensitivity of the pore to CsA was decreased by atractyloside and practically disappeared when it was added in combination with uncoupler. The main properties of the pore in mitochondria from thymocytes and from hepatocytes are the same. Release of Ca2+ from thymocyte mitochondria induced by uncoupling is mediated by a specific uniporter and by the pore with similar rates.     

Mechanism of calcium potentiation of oxygen free radical injury to renal mitochondria. A model for post-ischemic and toxic mitochondrial damage

With a variety of forms of ischemic and toxic tissue injury, cellular accumulation of Ca2+ and generation of oxygen free radicals may have adverse effects upon cellular and, in particular, mitochondrial membranes. Damage to mitochondria, resulting in impaired ATP synthesis and diminished activity of cellular energy-dependent processes, could contribute to cell death. In order to model, in vitro, conditions present post-ischemia or during toxin exposure, the interactions between Ca2+ and oxygen free radicals on isolated renal mitochondria were characterized. The oxygen free radicals were generated by hypoxanthine and xanthine oxidase to simulate in vitro one of the sources of oxygen free radicals in the early post-ischemic period in vivo. With site I substrates, pyruvate and malate, Ca2+ pretreatment, followed by exposure to oxygen free radicals, resulted in an inhibition of electron transport chain function and complete uncoupling of oxidative phosphorylation. These effects were partially mitigated by dibucaine, a phospholipase A2 inhibitor. With the site II substrate, succinate, the electron transport chain defect was not manifest and respiration remained partially coupled. The electron transport chain defect produced by Ca2+ and oxygen free radicals was localized to NADH CoQ reductase. Calcium and oxygen free radicals reduced mitochondrial ATPase activity by 55% and adenine nucleotide translocase activity by 65%. By contrast oxygen free radicals alone reduced ATPase activity by 32% and had no deleterious effects on translocase activity. Dibucaine partially prevented the Ca2+-dependent reduction in ATPase activity and totally prevented the Ca2+-dependent translocase damage observed in the presence of oxygen free radicals. These findings indicate that calcium potentiates oxygen free radical injury to mitochondria. The Ca2+-induced potentiation of oxygen free radical injury likely is due in part to activation of phospholipase A2. This detrimental interaction associated with Ca2+ uptake by mitochondria and exposure of the mitochondria to oxygen free radicals may explain the enhanced cellular injury observed during post-ischemic reperfusion.     

Isolation and characterization of a plasma membrane calcium pump from Dictyostelium discoideum.

During the aggregation and differentiation of amoebae of Dictyostelium discoideum, changes in free cytosolic Ca2+ appear to regulate a number of physiological processes. To understand the mechanisms regulating free intracellular Ca2+ in this organism, we have isolated and characterized an ATP/Mg2+-dependent, high-affinity Ca2+ pump. When homogenates of 2 h starved cells were fractionated on Percoll/KCl gradients, one peak of high-affinity Ca2+-pumping activity was detected. This activity was resolved from enzyme markers of the mitochondrion and the rough endoplasmic reticulum but it cosedimented with the plasma membrane marker, alkaline phosphatase. Further studies suggested that the pump was associated with 'inside-out' plasma membrane vesicles. Like plasma membrane Ca2+-transport ATPases from other systems, this isolated Ca2+ pump: (1) was Mg2+-dependent, (2) displayed a high specificity for ATP as an energy source, (3) exhibited a high affinity for free Ca2+ with a Km of 0.3 microM, and (4) was very sensitive to inhibition by vanadate (IC50 2 microM) but was unaffected by mitochondrial inhibitors, ouabain and Ca2+-channel blockers. Unlike plasma membrane Ca2+ pumps from most other systems, this enzyme appeared not to be regulated by calmodulin. During development, non-mitochondrial, vanadate-sensitive, high-affinity Ca2+-pumping activity in crude lysates remained relatively constant for at least 15 h. These observations suggest that this plasma membrane Ca2+ pump probably functions in Dictyostelium to maintain Ca2+ homeostasis by extruding free cytosolic Ca2+ from the cells.     

Regulation of Ca2+ efflux from kidney and liver mitochondria by unsaturated fatty acids and Na+ ions.

The effects of fatty acids and monovalent cations on the Ca2+ efflux from isolated liver and kidney mitochondria were investigated by means of electrode techniques. It was shown that unsaturated fatty acids and saturated fatty acids of medium chain length (C12 and C14) induced a Ca2+ efflux from mitochondria which was not inhibited by ruthenium red, but was specifically inhibited by Na+ and Li+. The Ca2+-releasing activity of unsaturated fatty acids did not correlate with their uncoupling activity. In kidney mitochondria a spontaneous, temperature-dependent Ca2+ efflux was observed which was inhibited either by albumin or by Na+. It is suggested that the net Ca2+ accumulation by mitochondria depends on the operation of independent pump and leak pathways. The pump is driven by the membrane potential and can be inhibited by ruthenium red, the leak depends on the presence of unsaturated fatty acids and is inhibited by Na+ and Li+. It is suggested that the unsaturated fatty acids produced by mitochondrial phospholipase A2 can be essential in the regulation of the Ca2+ retention in and the Ca2+ release from the mitochondria.     

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.     

Suppression of Ca2+ oscillations in cultured rat hepatocytes by chemical hypoxia

The model of "chemical hypoxia" with KCN plus iodoacetic acid mimics the ATP depletion and reductive stress of hypoxia. Here, we examined the effects of chemical hypoxia on cytosolic free Na+ and Ca2+ in single cultured rat hepatocytes by multiparameter digitized video microscopy and ratio imaging of sodium-binding furan indicator (SBFI) and Fura-2. Intracellular Na+ increased from about 10 mM to more than 100 mM after 20 min of chemical hypoxia, whereas cytosolic free Ca2+ remained virtually unchanged. In normoxic hepatocytes, phenylephrine (50 microM) and Arg-vasopressin (20-40 nM) induced Ca2+ oscillations in 70 and 40% of cells, respectively. These Ca2+ oscillations were suppressed after one spike following the onset of chemical hypoxia. Phenylephrine and vasopressin also increased inositol phosphate formation by 22 and 147%, respectively. This effect was suppressed by KCN plus iodoacetate. Intracellular acidosis is characteristic of chemical hypoxia. Intracellular acidosis induced by 40 mM Na-acetate suppressed Ca2+ oscillations but did not inhibit hormone-induced inositol phosphate formation. Cytosolic alkalinization also suppressed Ca2+ oscillations. However, prevention of intracellular acidosis with monensin (10 microM) did not prevent suppression of Ca2+ oscillations during chemical hypoxia. Mitochondrial depolarization with uncoupler did not change free Ca2+ levels during chemical hypoxia, indicating that mitochondria do not regulate free Ca2+ during chemical hypoxia. From these results, we conclude: 1) chemical hypoxia does not block Na+ influx across the plasma membrane; 2) Chemical hypoxia inhibits hormone-stimulated Ca2+ flux pathways across cellular membranes by two different mechanisms: (a) by ATP depletion, which disrupts hormone-myo-inositol 1,4,5-triphosphate coupling, and (b) by intracellular acidosis, which inhibits myo-inositol 1,4,5-triphosphate-stimulated Ca2+ release from intracellular stores; 3) during ATP depletion by chemical hypoxia, mitochondria do not take up Ca2+ to maintain cytosolic free Ca2+ at low concentrations.     

Alteration of plant mitochondrial proton conductance by free fatty acids. Uncoupling protein involvement

We characterized the uncoupling activity of the plant uncoupling protein from Solanum tuberosum (StUCP) using mitochondria from intact potato tubers or from yeast (Saccharomyces cerevisiae) expressing the StUCP gene. Compared with mitochondria from transfected yeast, StUCP is present at very low levels in intact potato mitochondrial membranes (at least thirty times lower) as shown by immunodetection with anti-UCP1 antibodies. Under conditions that ruled out undesirable effects of nucleotides and free fatty acids on uncoupling activity measurement in plant mitochondria, the linoleic acid-induced depolarization in potato mitochondria was insensitive to the nucleotides ATP, GTP, or GDP. In addition, sensitivity to linoleic acid was similar in potato and in control yeast mitochondria, suggesting that uncoupling occurring in potato mitochondria was because of a UCP-independent proton diffusion process. By contrast, yeast mitochondria expressing StUCP exhibited a higher sensitivity to free fatty acids than those from the control yeast and especially a marked proton conductance in the presence of low amounts of linoleic acid. However, this fatty acid-induced uncoupling was also insensitive to nucleotides. Altogether, these results suggest that uncoupling of oxidative phosphorylation and heat production cannot be the dominant feature of StUCP expressed in native potato tissues. However, it could play a role in preventing reactive oxygen species production as proposed for mammalian UCP2 and UCP3.     

Calcium induced release of mitochondrial cytochrome c by different mechanisms selective for brain versus liver.

Increased mitochondrial Ca2+ accumulation is a trigger for the release of cytochrome c from the mitochondrial intermembrane space into the cytosol where it can activate caspases and lead to apoptosis. This study tested the hypothesis that Ca2+-induced release of cytochrome c in vitro can occur by membrane permeability transition (MPT)-dependent and independent mechanisms, depending on the tissue from which mitochondria are isolated. Mitochondria were isolated from rat liver and brain and suspended at 37 degrees C in a K+-based medium containing oxidizable substrates, ATP, and Mg2+. Measurements of changes in mitochondrial volume (via light scattering and electron microscopy), membrane potential and the medium free [Ca2+] indicated that the addition of 0.3 - 3.2 micromol Ca2+ mg-1 protein induced the MPT in liver but not brain mitochondria. Under these conditions, a Ca2+ dose-dependent release of cytochrome c was observed with both types of mitochondria; however, the MPT inhibitor cyclosporin A was only capable of inhibiting this release from liver mitochondria. Therefore, the MPT is responsible for cytochrome c release from liver mitochondria, whereas an MPT-independent mechanism is responsible for release from brain mitochondria.     

The influence of nanomolar calcium ions and physiological levels of thyroid hormone on oxidative phosphorylation in rat liver mitochondria. A possible signal amplification control mechanism.

Using different conditions mitochondria from hypothyroid rats can show both unchanged ADP/O ratios and lowered ADP/O ratios without evidence of uncoupling when compared with euthyroid controls. Raising the free Ca2+ concentration to around 25 nM progressively lowered the ADP/O ratio in hypothyroid but not in euthyroid mitochondria. Ruthenium Red did not alter this behaviour and further increasing the Ca2+ concentration to levels below those which stimulate State 3 respiration had no additional effect. Measurements of the free Ca2+ concentration in the mitochondrial suspending medium using a Quin 2 fluorescence assay showed that the mitochondria did not buffer the free Ca2+ at these low concentrations. At 25 nM-free Ca2+, addition of 10-13) M-T3 to hypothyroid mitochondria produced an immediate and significant increase in the ADP/O ratio without altering the free Ca2+ concentration. The hormone effect was maximal by 10(-11) M. The concentration of ATP synthetase can be estimated to lie at about 10 nM in these experiments. Hence it appears possible that a substantial amplification of the hormone signal may have taken place. Comparison with binding studies suggests that T3 may have been maximally stimulating when somewhat less than half its receptor sites had been filled. The possible mechanisms by which this receptor mediated alteration of the ADP/O ratio might be achieved are discussed.     

The interaction of flavonoids with mitochondria: effects on energetic processes

The study addressed aspects of energetics of isolated rat liver mitochondria exposed to the flavonoids quercetin, taxifolin, catechin and galangin, taking into account influences of the 2,3 double bond/3-OH group and 4-oxo function on the C-ring, and o-di-OH on the B-ring of their structures, as well as mitochondrial mechanisms potentially involved in cell necrosis and apoptosis. The major findings/hypothesis, were: The 2,3 double bond/3-OH group in conjugation with the 4-oxo function on the C-ring in the flavonoid structure seems favour the interaction of these compounds with the mitochondrial membrane, decreasing its fluidity either inhibiting the respiratory chain of mitochondria or causing uncoupling; while the o-di-OH on the B-ring seems favour the respiratory chain inhibition, the absence of this structure seems favour the uncoupling activity. The flavonoids not affecting the respiration of mitochondria, induced MPT. The ability of flavonoids to induce the release of mitochondria-accumulated Ca(2+) correlated well with their ability to affect mitochondrial respiration on the one hand, and their inability to induce MPT, on the other. The flavonoids causing substantial respiratory chain inhibition or mitochondrial uncoupling, quercetin and galangin, respectively, also decreased the mitochondrial ATP levels, thus suggesting an apparent higher potential for necrosis induction in relation to the flavonoids inducing MPT, taxifolin and cathechin, which did not decrease significantly the ATP levels, rather suggesting an apparent higher potential for apoptosis induction.     

Arachidonic acid induces specific membrane permeability increase in heart mitochondria

Micromolar concentrations of arachidonic acid cause in Ca2+ loaded heart mitochondria matrix swelling and Ca2+ release. These effects appear to be unrelated to the classical membrane permeability transition (MPT), as they are CsA insensitive, membrane potential independent and can also be activated by Sr2+. Atractyloside potentiated and ATP inhibited the arachidonic acid induced swelling. These observations suggest that the ATP/ADP translocator (ANT) may be involved in the AA induced, CsA insensitive membrane permeability increase. Under the same experimental conditions used for heart mitochondria, arachidonic acid induced the classical CsA sensitive, ADP inhibitable MPT in liver mitochondria.