Butylated hydroxytoluene and inorganic phosphate plus Ca2+ increase mitochondrial permeability via mutually exclusive mechanisms

Mitochondria undergo a permeability transition (PT), i.e., become nonselectively permeable to small solutes, in response to a wide range of conditions/compounds. In general, opening of the permeability transition pore (PTP) is Ca²⁺- and P_i-dependent and is blocked by cyclosporin A (CsA), trifluoperazine (TFP), ADP, and butylated hydroxytoluene (BHT). Gudz and coworkers have reported [7th European Bioenergetics Conference, EBEC Short Reports (1992)7, 125], however, that, under some conditions, BHT increases mitochondrial permeability via a process that may not share all of these characteristics. Specifically, they determined that the BHT-induced permeability transition was independent of Ca²⁺ and was insensitive to CsA. We have used mitochondrial swelling to compare in greater detail the changes in permeability induced by BHT and by Ca²⁺ plus P_i with the following results. (1) The dependence of permeability on BHT concentration is triphasic: there is a threshold BHT concentration (ca. 60 nmol BHT/ mg mitochondrial protein) below which no increase occurs; BHT enhances permeability in an intermediate concentration range; and at high BHT concentrations (> 120 nmol/mg) permeability is again reduced. (2) The effects of BHT depend on the ratio of BHT to mitochondrial protein. (3) Concentrations of BHT too low to induce swelling block the PT induced by Ca²⁺ and P_i. (4) The dependence of the Ca²⁺-triggered PT on P_i concentration is biphasic. Below a threshold of 50–100 M, no swelling occurs. Above this threshold swelling increases rapidly. (5) P_i levels too low to support the Ca²⁺-induced PT inhibit BHT-induced swelling. (6) Swelling induced by BHT can bestimulated by agents and treatments that block the PT induced by Ca²⁺ plus P_i. These data suggest that BHT and Ca²⁺ plus P_i, increase mitochondrial permeability via two mutually exclusive mechanisms.     

Ca2+ Induces a Cyclosporin A-Insensitive Permeability Transition Pore in Isolated Potato Tuber Mitochondria Mediated by Reactive Oxygen Species

Oxidative damage of mammalian mitochondria induced by Ca²⁺ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca²⁺ uniport, undergo inner membrane permeabilization when treated with Ca²⁺ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg²⁺ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis–trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca²⁺ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.     

Mitochondrial ATP-Sensitive K<Superscript>+</Superscript> Channels Prevent Oxidative Stress,Permeability Transition and Cell Death

Ischemia followed by reperfusion results in impairment of cellular and mitochondrial functionality due to opening of mitochondrial permeability transition pores. On the other hand, activation of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) protects the heart against ischemic damage. This study examined the effects of mitoK_ATP and mitochondrial permeability transition on isolated rat heart mitochondria and cardiac cells submitted to simulated ischemia and reperfusion (cyanide/aglycemia). Both mitoK_ATP opening, using diazoxide, and the prevention of mitochondrial permeability transition, using cyclosporin A, protected against cellular damage, without additive effects. MitoK_ATP opening in isolated rat heart mitochondria slightly decreased Ca²⁺ uptake and prevented mitochondrial reactive oxygen species production, most notably in the presence of added Ca²⁺. In ischemic cells, diazoxide decreased ROS generation during cyanide/aglycemia while cyclosporin A prevented oxidative stress only during simulated reperfusion. Collectively, these studies indicate that opening mitoK_ATP prevents cellular death under conditions of ischemia/reperfusion by decreasing mitochondrial reactive oxygen species release secondary to Ca²⁺ uptake, inhibiting mitochondrial permeability transition.     

The permeability transition pore as a mitochondrial calcium release channel: A critical appraisal

Mitochondria from a variety of sources possess an inner membrane channel, the permeability transition pore. The pore is a voltage-dependent channel, activated by matrix Ca²⁺ and inhibited by matrix H⁺, which can be blocked by cyclosporin A, presumably after binding to mitochondrial cyclophilin. The physiological function of the permeability transition pore remains unknown. Here we evaluate its potential role as a fast Ca²⁺ release channel involved in mitochondrial and cellular Ca²⁺ homeostasis. We (i) discuss the theoretical and experimental reasons why mitochondria need a fast, inducible Ca²⁺ release channel; (ii) analyze the striking analogies between the mitochondrial permeability transition pore and the sarcoplasmic reticulum ryanodine receptor-Ca²⁺ release channel; (iii) argue that the permeability transition pore can act as a selective release channel for Ca²⁺ despite its apparent lack of selectivity for the transported speciesin vitro; and (iv) discuss the importance of mitochondria in cellular Ca²⁺ homeostasis, and how disruption of this function could impinge upon cell viability, particularly under conditions of oxidative stress.     

Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations.

We have studied the induction of the mitochondrial cyclosporin A-sensitive permeability transition pore (PTP) by the bifunctional SH group reagent phenylarsine oxide (PhAsO). Addition of nanomolar concentrations of the electroneutral H(+)-K+ ionophore nigericin to nonrespiring mitochondria in sucrose medium determines a dramatic increase of the time required for PTP induction by PhAsO, while no effect of nigericin is apparent in KCl medium. Using mitochondria loaded with the internal pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, we show that the effect of nigericin is mediated by the ionophore-induced acidification of matrix pH. Indeed, experimental manipulation of pHi by a number of treatments indicates that PTP induction is directly related to matrix pH, in that the PTP induction process becomes slower as pHi decreases at constant pHo. PTP induction by PhAsO in respiration-inhibited mitochondria is stimulated by Ca2+ and inhibited by a series of divalent cations. Since PhAsO induces the PTP even in the presence of excess EGTA and in the absence of respiration (Lenartowicz, E., Bernardi, P., and Azzone, G.F. (1991) J. Bioenerg. Biomembr. 23, 679-688), we have been able to study the Ca2+ dependence of the induction process. We show that the apparent Km for Ca2+ activation is about 10(-5) M and that Ca2+, cyclosporin A, and inhibitory Me2+ ions behave as if they were competing for the same binding site(s) on the pore. Since similar results are obtained from patch-clamp experiments on the mitochondrial megachannel (Szabó, I., Bernardi, P., and Zoratti, M. (1992) J. Biol. Chem. 267, 2940-2946), we suggest that (i) the PTP and the mitochondrial megachannel are the same molecular structures and (ii) the same factors affect both the process of pore induction and its open-closed orientation.     

Intracellular Ca<Superscript>2+</Superscript> Pools and Fluxes in Cardiac Muscle-Derived H9c2 Cells

Relevant Ca²⁺ pools and fluxes in H9c2 cells have been studied using fluorescent indicators and Ca²⁺-mobilizing agents. Vasopressin produced a cytoplasmic Ca²⁺ peak with half-maximal effective concentration of 6 nM, whereas thapsigargin-induced Ca²⁺ increase showed half-maximal effect at 3 nM. Depolarization of the mitochondrial inner membrane by protonophore was also associated with an increase in cytoplasmic Ca²⁺. Ionomycin induced a small and sustained depolarization, while thapsigargin had a small but transient effect. The thapsigargin-sensitive Ca²⁺ pool was also sensitive to ionomycin, whereas the protonophore-sensitive Ca²⁺ pool was not. The vasopressin-induced cytoplasmic Ca²⁺ signal, which caused a reversible discharge of the sarco-endoplasmic reticulum Ca²⁺ pool, was sensed as a mitochondrial Ca²⁺ peak but was unaffected by the permeability transition pore inhibitor cyclosporin A. The mitochondrial Ca²⁺ peak was affected by cyclosporin A when the Ca²⁺ signal was induced by irreversible discharge of the intracellular Ca²⁺ pool, i.e., adding thapsigargin. These observations indicate that the mitochondria interpret the cytoplasmic Ca²⁺ signals generated in the reticular store.     

Spectroscopic and Microscopic Studies on the Mechanisms of Mitochondrial Toxicity Induced by Different Concentrations of Cadmium

The deleterious action of Cd²⁺ on rat liver mitochondria was investigated in this work using spectroscopic and microscopic methods. The concentration dependence of Cd²⁺ on mitochondrial swelling, membrane potential and membrane fluidity was studied. Our aim was to detect the active sites of Cd²⁺ in the mitochondrial membrane treatments with cyclosporin A (CsA) and EGTA on the mitochondrial permeability transition (MPT) induced by low and high concentrations of Cd²⁺. The protective effects of dithiothreitol, human serum albumin and monobromobimane⁺ on Cd²⁺-induced MPT were also monitored. All of these investigations indicated that Cd²⁺ can directly affect MPT at two separate localization sites at different concentrations: the classic Ca²⁺ triggering site and the thiol (–SH) groups of membrane proteins matched by MPT pore opening (defined as “S” site). At the high concentration of Cd²⁺, other free –SH groups in the mitochondrial matrix may be involved in this process. These findings were supported by transmission electron microscopy and shed light on the toxic mechanism of Cd²⁺ on mitochondria.     

Mitochondrial Ca transport and permeability transition in zebrafish (Danio rerio)

We have studied mitochondrial Ca²⁺ transport and the permeability transition (PT) in the teleost zebrafish (Danio rerio), a key model system for human diseases. Permeabilized zebrafish embryo cells displayed a mitochondrial energy-dependent Ca²⁺ uptake system that, like the Ca²⁺ uniporter of mammals, was inhibited by ruthenium red. Zebrafish mitochondria underwent a Ca²⁺-dependent PT that displayed Pi-dependent desensitization by cyclosporin A, and responded appropriately to key modulators of the mammalian PT pore (voltage, pH, ubiquinone 0, dithiol oxidants and cross linkers, ligands of the adenine nucleotide translocator, arachidonic acid). Opening of the pore was documented in intact cells, where it led to death that could largely be prevented by cyclosporin A. Our results represent a necessary step toward the use of zebrafish for the screening and validation of PTP inhibitors of potential use in human diseases, as recently shown for collagen VI muscular dystrophy [Telfer et al., 2010].     

Effect of PK11195, a peripheral benzodiazepine receptor agonist,on insulinoma cell death and insulin secretion

Functional role of peripheral benzodiazepine receptor on mitochondrial membrane in apoptosis and insulin secretion from insulinoma cells was studied. A prototypic peripheral benzodiazepine receptor agonist PK11195 induced insulinoma cell apoptosis, while a central benzodiazepine receptor agonist did not. Death of insulinoma cells by PK11195 was inhibited by cyclosporin A,{ a blocker of mitochondrial permeability transition pore}. Caspase inhibitors further inhibited MIN6N8 cell death. PK11195 induced dissipation of mitochondrial potential and cytochrome c translocation to cytoplasm. PK11195 induced an increase in cytoplasmic [Ca^{2 +}], which was reversed by cyclosporin A. Rhod-2 staining showed decreased mitochondrial [Ca^{2 +}] after PK11195 treatment. PK11195 potentiated glucose-induced insulin secretion probably due to the increased cytoplasmic [Ca^{2 +}]. Calpain was activated following Ca^{2 +} release, and calpain inhibitors attenuated death of insulinoma cells by PK11195. These results suggest that PK11195 induces mitochondrial potential loss, cytochrome c translocation, increased insulin secretion in conjunction with an increase in cytoplasmic [Ca^{2 +}] and calpain activation, which collectively leads to apoptosis of insulinoma cells.     

Oxidative Stress Underlies the Mechanism for Ca2+-induced Permeability Transition of Mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca²⁺ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca²⁺ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca²⁺ increased the generation of H²O² by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca²⁺ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca²⁺-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca²⁺ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.     

Cyclosporin A binding in mitochondrial cyclophilin inhibits the permeability transition pore and protects hearts from ischaemia/reperfusion injury

When loaded with high (pathological) levels of Ca²⁺, mitochondria become swollen and uncoupled as the result of a large non-specific increase in membrane permeability. This process, known as the mitochondrial permeability transition (MPT), is exacerbated by oxidative stress and adenine nucleotide depletion. These conditions match those that a heart experiences during reperfusion following a period of ischaemia. The MPT is caused by the opening of a non-specific pore that can be prevented by sub-micromolar concentrations of cyclosporin A (CsA). A variety of conditions that increase the sensitivity of pore opening to [Ca²⁺], such as thiol modification, oxidative stress, increased matrix volume and chaotropic agents, all enhance the binding of matrix cyclophilin (CyP) to the inner mitochondrial membrane in a CsA-sensitive manner. In contrast, ADP, membrane potential and low pH decrease the sensitivity of pore opening to [Ca²⁺] without affecting CyP binding. We present a model of pore opening involving CyP binding to a membrane target protein followed by Ca²⁺-dependent triggering of a conformational change to induce channel opening. Using the ischaemic/reperfused rat heart we have shown that the mitochondrial pore does not open during ischaemia, but does do so during reperfusion. Recovery of heart during reperfusion is improved in the presence of 0.2 µM CsA, suggesting that the MPT may be critical in the transition from reversible to irreversible reperfusion injury. (Mol Cell Biochem 174: 167–172, 1997)     

Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ efflux rates through Na⁺-dependent and Na⁺-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

On the Opening of an Insensitive Cyclosporin A Non-specific Pore by Phenylarsine Plus Mersalyl

The purpose of this work was addressed to provide new information on the effect of thiol reagents on mitochondrial non-specific pore opening, and its response to cyclosporin A (CSA). To meet this proposal phenylarsine oxide (PHA) and mersalyl were employed as tools to induce permeability transition and CSA to inhibit it. PHA-induced mitochondrial dysfunction, characterized by Ca²⁺ efflux, swelling, and membrane de-energization, was inhibited by N-ethylmaleimide and CSA. Conversely, mersalyl failed to inhibit the inducing effect of phenylarsine oxide, it rather strengthened it. In addition, the effect of mersalyl was associated with cross-linking of membrane proteins. The content of membrane thiol groups accessible to react with PHA, mersalyl, and PHA plus mersalyl was determined. In all situations, permeability transition was accompanied by a significant decrease in the whole free membrane thiol content. Interestingly, it is also shown that mersalyl hinders the protective effect of cyclosporin A on PHA-induced matrix Ca²⁺ efflux.     

The mitochondrial permeability transition from yeast to mammals

Regulated permeability changes have been detected in mitochondria across species. We review here their key features, with the goal of assessing whether a “permeability transition” similar to that observed in higher eukaryotes is present in other species. The recent discoveries (i) that treatment with cyclosporin A (CsA) unmasks an inhibitory site for inorganic phosphate (Pi) [Basso, E., Petronilli, V., Forte, M.A. and Bernardi, P. (2008) Phosphate is essential for inhibition of the mitochondrial permeability transition pore by cyclosporin A and by cyclophilin D ablation. J. Biol. Chem. 283, 26307-26311], the classical inhibitor of the permeability transition of yeast and (ii) that under proper experimental conditions a matrix Ca²⁺-dependence can be demonstrated in yeast as well [Yamada, A., Yamamoto, T., Yoshimura, Y., Gouda, S., Kawashima, S., Yamazaki, N., Yamashita, K., Kataoka, M., Nagata, T., Terada, H., Pfeiffer, D.R. and Shinohara Y. (2009) Ca²⁺-induced permeability transition can be observed even in yeast mitochondria under optimized experimental conditions. Biochim. Biophys. Acta 1787, 1486-1491] suggest that the mitochondrial permeability transition has been conserved during evolution.     

On the Protection by Inorganic Phosphate of Calcium-Induced Membrane Permeability Transition

The role of inorganic phosphate as inhibitor of mitochondrial membrane permeability transition was studied. It is shown that in mitochondria containing a high phosphate concentration, i.e., 68 nmol/mg, Ca²⁺ did not activate the pore opening. Conversely, at lower levels of matrix phosphate, i.e., 38 nmol/mg, Ca²⁺ was able to induce subsequent pore opening. The inhibitory effect of phosphate was apparent in sucrose-based media, but it was not achieved in KCl media. The matrix free Ca²⁺ concentration and matrix pH were lowered by phosphate, but they were always higher in K⁺-media. In the absence of ADP, phosphate strengthened the inhibitory effect of cyclosporin A on carboxyatractyloside-induced Ca²⁺ efflux. Acetate was unable to replace phosphate in the induction of the aforementioned effects. It is concluded that phosphate preserves selective membrane permeability by diminishing the matrix free Ca²⁺ concentration.     

Interactions of melatonin with mammalian mitochondria. Reducer of energy capacity and amplifier of permeability transition

Melatonin, a metabolic product of the amino acid tryptophan, induces a dose-dependent energy drop correlated with a decrease in the oxidative phosphorylation process in isolated rat liver mitochondria. This effect involves a gradual decrease in the respiratory control index and significant alterations in the state 4/state 3 transition of membrane potential (ΔΨ). Melatonin, alone, does not affect the insulating properties of the inner membrane but, in the presence of supraphysiological Ca²⁺, induces a ΔΨ drop and colloid-osmotic mitochondrial swelling. These events are sensitive to cyclosporin A and the inhibitors of Ca²⁺ transport, indicative of the induction or amplification of the mitochondrial permeability transition. This phenomenon is triggered by oxidative stress induced by melatonin and Ca²⁺, with the generation of hydrogen peroxide and the consequent oxidation of sulfydryl groups, glutathione and pyridine nucleotides. In addition, melatonin, again in the presence of Ca²⁺, can also induce substantial release of cytochrome C and AIF (apoptosis-inducing factor), thus revealing its potential as a pro-apoptotic agent.     

Bile Acid-Induced Ca<Superscript>2+</Superscript> Efflux from Liver Mitochondria as a Factor Preventing the Formation of Mitochondrial Pores

The effect of bile acids as inducers of Ca²⁺ efflux from the matrix was studied on isolated rat liver mitochondria. Mitochondria in the presence of cyclosporin A (CsA) were energized by succinate, then loaded with Ca²⁺ and after the addition of the calcium uniporter inhibitor ruthenium red were de-energized by malonate. It was shown that under these conditions hydrophobic bile acids lithocholic and chenodeoxycholic at concentrations of 10 and 30 μM respectively and hydrophilic bile acids ursodeoxycholic and cholic at a concentration of 400 μM induce Ca²⁺ efflux from the mitochondrial matrix. It is noted that the efflux of these ions is not associated with damage of the inner mitochondrial membrane by bile acids, since it is accompanied by the generation of Δψ, i.e., the formation of the diffusion potential. It is assumed that along with induction of calcium efflux from the matrix, bile acids are also capable of transporting hydrogen and potassium ions in the opposite direction, i.e., perform H⁺/Ca²⁺ and K⁺/Ca²⁺ exchange. It was found that ruthenium red added to Ca²⁺-loaded energized mitochondria prevents the return of these ions to the matrix and weakens the effect of chenodeoxycholic acid as an inducer of the CsA-sensitive mitochondrial pore and the effect of ursodeoxycholic acid as an inducer of CsA-insensitive permeability of the inner mitochondrial membrane. We conclude that in the conditions of the calcium uniporter activity decrease, Ca²⁺ efflux from the matrix induced by bile acids can be considered as one of the mechanisms reducing their effectiveness as inducers of the Ca²⁺-dependent CsA-sensitive pore and CsA-insensitive permeability transition in mitochondria.     

Lithocholic acid induces two different calcium-dependent inner membrane permeability systems in liver mitochondria

The effect of the most hydrophobic bile acid–lithocholic–as an inducer of two different Ca²⁺-dependent inner membrane permeability systems was studied on isolated rat liver mitochondria. It is shown that the addition of lithocholic acid at a concentration of 20 μM to the Ca²⁺-loaded mitochondria leads to swelling of the organelles, rapid release of Ca²⁺ from the matrix and almost complete collapse of Δψ. Mitochondrial pore blocker cyclosporin A (CsA) eliminates mitochondrial swelling but has no effect on the process of Ca²⁺ release and Δψ collapse. In the absence of Ca²⁺ lithocholic acid causes only a transient decrease of Δψ with subsequent complete recovery. Ruthenium red, inhibitor of mitochondrial Ca²⁺ uniporter, which blocks Ca²⁺ influx into the matrix, prevents mitochondrial swelling induced by lithocholic acid. At the same time, ruthenium red, which is added before lithocholic acid to the Ca²⁺-preloaded mitochondria, does not affect the swelling of the organelles but reduces the CsA-insensitive drop in Δψ. It is concluded that lithocholic acid is able to induce two Ca²⁺-dependent energy dissipation systems in the inner membrane of liver mitochondria: CsA-sensitive mitochondrial pore and CsA-insensitive permeability, which exhibits sensitivity to ruthenium red. It is found that the effect of this bile acid as an inductor of CsA-sensitive mitochondrial pore is not associated with the modulation of Pi effects. It is assumed that CsA-insensitive action of lithocholic acid is associated with the induction of Ca²⁺ efflux from the matrix in exchange for protons. In this case, the energy-dependent Ca²⁺ transport in the opposite direction with the participation of mitochondrial calcium uniporter sensitive to ruthenium red leads to the formation of calcium cycle and thereby to energy dissipation.     

Ascorbate and low concentrations of FeSO4 induce Ca2+-dependent pore in rat liver mitochondria

Oxidative stress is one of the most frequent causes of tissue and cell injury in various pathologies. The molecular mechanism of mitochondrial damage under conditions of oxidative stress induced in vitro with low concentrations of FeSO₄ and ascorbate (vitamin C) was studied. FeSO₄ (1-4 M) added to rat liver mitochondria that were incubated in the presence of 2.3 mM ascorbate induced (with a certain delay) a decrease in membrane potential and high-amplitude swelling. It also significantly decreased the ability of mitochondria to accumulate exogenous Ca²⁺. All the effects of FeSO₄ + ascorbate were essentially prevented by cyclosporin A, a specific inhibitor of the mitochondrial Ca²⁺-dependent pore (also known as the mitochondrial permeability transition). EGTA restored the membrane potential of mitochondria de-energized with FeSO₄ + ascorbate. We hypothesize that oxidative stress induced in vitro with FeSO₄ and millimolar concentrations of ascorbate damages mitochondria by inducing the cyclosporin A-sensitive Ca²⁺-dependent pore in the inner mitochondrial membrane.     

Triphenyltin as inductor of mitochondrial membrane permeability transition

The effect of triphenyltin on mitochondrial Ca²⁺ content was studied. It was found that this trialkyltin compound induces an increase in membrane permeability that leads to Ca²⁺ release, drop of the transmembrane potential, and efflux of matrix proteins. Interestingly, cyclosporin A was unable to inhibit triphenyltin-induced Ca²⁺ release. Based on these results it is proposed that the hyperpermeable state is produced by modification of 2.25 nmol of membrane thiol groups.