Interaction of genistein with the mitochondrial electron transport chain results in opening of the membrane transition pore

Genistein, a natural isoflavone present in soybeans, is a potent agent in the prophylaxis and treatment of cancer. Addition of genistein to isolated rat liver mitochondria (RLM) induces swelling, loss of membrane potential and release of accumulated Ca2+. These changes are Ca2+-dependent and are prevented by cyclosporin A (CsA) and bongkrekic acid (BKA), two classical inhibitors of the mitochondrial permeability transition (MPT). Induction of the MPT by genistein is accompanied by oxidation of thiol groups and pyridine nucleotides. The reducing agent dithioerythritol and the alkylating agent N-ethylmaleimide (NEM) completely prevent the opening of the transition pore, thereby emphasizing that the effect of the isoflavone correlates with the mitochondrial redox state. Further analyses showed that genistein induces the MPT by the generation of reactive oxygen species (ROS) due to its interaction with the respiratory chain at the level of mitochondrial complex III.     

The Coenzyme Q10 analog decylubiquinone inhibits the redox-activated mitochondrial permeability transition: role of mitcohondrial [correction mitochondrial] complex III

The mitochondrial permeability transition (MPT) is a key event in apoptotic and necrotic cell death and is controlled by the cellular redox state. To further investigate the mechanism(s) involved in regulation of the MPT, we used diethylmaleate to deplete GSH in HL60 cells and increase mitochondrial reactive oxygen species (ROS) production. The site of mitochondrial ROS production was determined to be mitochondrial respiratory complex III (cytochrome bc1), because 1). stigmatellin, a Qo site inhibitor, blocked ROS production and prevented the MPT and cell death and 2). cytochrome bc1 activity was abolished in cells protected from the redox-dependent MPT by stigmatellin. We next investigated the effect of pretreating cells with coenzyme Q10 analogs decylubiquinone (dUb) and ubiquinone 0 (Ub0) on the redox-dependent MPT. Pretreatment of HL60 cells with dUb blocked ROS production induced by GSH depletion and prevented activation of the MPT and cell death, whereas Ub0 did not. Since we also found that dUb did not inhibit cytochrome bc1 activity, the mechanism of protection against redox-dependent MPT by dUb may depend on its ability to scavenge ROS generated by cytochrome bc1. These results indicate that dUb, like the clinically used ubiquinone analog idebenone, may serve as a candidate antioxidant compound for the development of pharmacological agents to treat diseases where there is an oxidative stress component.     

Microcalorimetric study of the effect of Ce(III) on metabolic activity of mitochondria isolated from indice rice 9311

Rare earth elements (REEs) have beneficial influence on plant growth and are widely used in agriculture practice, but little is known about behavior of the REEs on mitochondria in plant cells. Thermogenic metabolic curves were determined by the ampoule method at 303 K using a TAM air isothermal microcalorimeter in mitochondria isolated from indice rice 9311 (Oryza sativa L.), and the effect of Ce(III) on mitochondrial metabolism was investigated. By analyzing the obtained heat flux curves, the crucial parameters such as activity recovery rate constant (k) and maximum heat power (P(m)) were investigated. Application of Ce3+ in concentrations ranging from 0 to 120 microg/ml significantly increased k and P(m) values, with the maximum reaching 261 and 180% of the control, respectively. Concentrations from 140 to 150 microg/ml had the opposite effect. These results were consistent with previous reports on the effects of REEs on plant growth. It was concluded that the Ce(III)-induced change of mitochondrial metabolic activity is a possible mechanism by which Ce(III) influenced indice rice 9311 growth.     

Dephosphorylation of the Rieske iron-sulfur protein after induction of the mitochondrial permeability transition

In the mitochondrial permeability transition (MPT), MPT pores open to cause the mitochondrial inner membrane to become non-selectively permeable to molecules of mass up to 1500 Da. In this study, we used proteomics to investigate protein changes after MPT induction. Isolated rat liver mitochondria were incubated with various MPT inducers, including CaCl2, tert-butylhydroperoxide, and phenylarsine oxide, in the presence and absence of the MPT inhibitor, cyclosporin A. MPT induction was confirmed by an absorbance swelling assay. Mitochondrial membrane proteins prepared from control and treated mitochondria were separated by two-dimensional (2D) gel electrophoresis and stained with SyproRuby or Coomassie blue. Proteins of interest were further identified by mass spectrometry. 2D gel electrophoresis by isoelectric focusing and SDS-PAGE consistently showed a protein spot that shifted to a more basic isoelectric point after the MPT. This shift was prevented by CsA but did not occur after protonophoric uncoupling. Mass spectrometry identified this protein as the Rieske iron-sulfur protein (RISP) of ubiquinol-cytochrome c reductase (Complex III). Phosphatase treatment of sonicated mitochondria caused the same shift in RISP as occurred in MPT inducer-treated mitochondria. 2D gel electrophoresis by blue-native-PAGE and SDS-PAGE showed that RISP existed as an apparent monomer in mitochondrial membranes in addition to forming a complex with ubiquinol-cytochrome c reductase. These findings suggest that RISP may be part of MPT pores and that dephosphorylation of RISP may play a role in regulation of the MPT.     

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.     

Cobalt induces oxidative stress in isolated liver mitochondria responsible for permeability transition and intrinsic apoptosis in hepatocyte primary cultures

It is well established that cobalt mediates the occurrence of oxidative stress which contributes to cell toxicity and death. However, the mechanisms of these effects are not fully understood. This investigation aimed at establishing if cobalt acts as an inducer of mitochondrial-mediated apoptosis and at clarifying the mechanism of this process. Cobalt, in the ionized species Co(2+), is able to induce the phenomenon of mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) with the opening of the transition pore. In fact, Co(2+) induces mitochondrial swelling, which is prevented by cyclosporin A and other typical MPT inhibitors such as Ca(2+) transport inhibitors and bongkrekic acid, as well as anti-oxidant agents. In parallel with mitochondrial swelling, Co(2+) also induces the collapse of electrical membrane potential. However in this case, cyclosporine A and the other MPT inhibitors (except ruthenium red and EGTA) only partially prevent DeltaPsi drop, suggesting that Co(2+) also has a proton leakage effect on the inner mitochondrial membrane. MPT induction is due to oxidative stress, as a result of generation by Co(2+) of the highly damaging hydroxyl radical, with the oxidation of sulfhydryl groups, glutathione and pyridine nucleotides. Co(2+) also induces the release of the pro-apoptotic factors, cytochrome c and AIF. Incubation of rat hepatocyte primary cultures with Co(2+) results in apoptosis induction with caspase activation and increased level of expression of HIF-1alpha. All these observations allow us to state that, in the presence of calcium, Co(2+) is an inducer of apoptosis triggered by mitochondrial oxidative stress.     

Inhibition of mitochondrial respiration mediates apoptosis induced by the anti-tumoral alkaloid lamellarin D

Lamellarin D (Lam D), a marine alkaloid, exhibits a potent cytotoxicity against many different tumors. The pro-apoptotic function of Lam D has been attributed to its direct induction of mitochondrial permeability transition (MPT). This study was undertaken to explore the mechanisms through which Lam D promotes changes in mitochondrial function and as a result apoptosis. The use of eight Lam derivatives provides useful structure-apoptosis relationships. We demonstrate that Lam D and structural analogues induce apoptosis of cancer cells by acting directly on mitochondria inducing reduction of mitochondrial membrane potential, swelling and cytochrome c release. Cyclosporin A, a well-known inhibitor of MPT, completely prevents mitochondrial signs of apoptosis. The drug decreases calcium uptake by mitochondria but not by microsomes indicating that Lam D-dependent permeability is specific to mitochondrial membranes. In addition, upon Lam D exposure, a rapid decline of mitochondrial respiration and ATP synthesis occurs in isolated mitochondria as well as in intact cells. Evaluation of the site of action of Lam D on the electron-transport chain revealed that the activity of respiratory chain complex III is reduced by a half. To determine whether Lam D could induce MPT-dependent apoptosis by inhibiting mitochondrial respiration, we generated respiration-deficient cells (ρ0) derived from human melanoma cells. In comparison to parental cells, ρ0 cells are totally resistant to the induction of MPT-dependent apoptosis by Lam D. Our results indicate that functional mitochondria are required for Lam D-induced apoptosis. Inhibition of mitochondrial respiration is responsible for MPT-dependent apoptosis of cancer cells induced by Lam-D.     

Effects of isocoumarins isolated from Paepalanthus bromelioides on mitochondria: uncoupling, and induction/inhibition of mitochondrial permeability transition

Isolated mitochondria may undergo uncoupling, and in presence of Ca(2+) at different conditions, a mitochondrial permeability transition (MPT) linked to protein thiol oxidation, and demonstrated by CsA-sensitive mitochondrial swelling; these processes may cause cell death either by necrosis or by apoptosis. Isocoumarins isolated from the Brazilian plant Paepalanthus bromelioides (Eriocaulaceae) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin were assayed at 1-50 microM on isolated rat liver mitochondria, for respiration, MPT, protein thiol oxidation, and interaction with the mitochondrial membrane using 1,6-diphenyl-1,3,5-hexatriene (DPH). The isocoumarins did not significantly affect state 3 respiration of succinate-energized mitochondria; they did however, stimulate 4 respiration, indicating mitochondrial uncoupling. Induction of MPT and protein thiol oxidation were assessed in succinate-energized mitochondria exposed to 10 microM Ca(2+); inhibition of these processes was assessed in non-energized organelles in the presence of 300 microM t-butyl hydroperoxide plus 500 microM Ca(2+). Only paepalantine was an effective MPT/protein thiol oxidation inducer, also releasing cytochrome c from mitochondria; the protein thiol oxidation, unlike mitochondrial swelling, was neither inhibited by CsA nor dependent on the presence of Ca(2+). Vioxanthin was an effective inhibitor of MPT/protein thiol oxidation. All isocoumarins inserted deeply into the mitochondrial membrane, but only paepalantine dimer and vioxantin decreased the membrane's fluidity. A direct reaction with mitochondrial membrane protein thiols, involving an oxidation of these groups, is proposed to account for MPT induction by paepalantine, while a restriction of oxidation of these same thiol groups imposed by the decrease of membrane fluidity, is proposed to account for MPT inhibition by vioxanthin.     

Cryopreservation induces mitochondrial permeability transition in a bovine sperm model

When the mitochondria of somatic cells are exposed to pathological calcium overload, these trigger mitochondrial permeability transition (MPT) leading to mitochondrial dysfunction and cell death. Cryopreservation procedures expose mammalian spermatozoa to physical and chemical stressors, which affect plasma membrane integrity and induce a pathological calcium overload that gradually promotes loss of sperm quality and ultimately function. Although several studies highlight the role of calcium in many physiological and pathological processes, the MPT induced by an intracellular calcium increase and its effect on the cell quality of mammalian spermatozoa are unknown. The aim of this study was to evaluate the effects of cryopreservation on MPT and its relationship with the deterioration of sperm quality in a bovine model. To do this, frozen bovine spermatozoa were thawed and adjusted to 2 × 10⁶ mL⁻¹ and incubated for 4 h at 38 °C. Using flow cytometry, we evaluated MPT by the calcein-AM and cobalt chloride method, intracellular Ca²⁺ level using FLUO3-AM, plasma membrane integrity by exclusion of propidium iodide, mitochondrial membrane potential (ΔΨm) with tetramethylrhodamine methyl ester perchlorate and intracellular ROS production with dihydroethidium. ATP levels were assessed by a chemiluminiscent method. The results showed that thawed spermatozoa trigger MPT associated with an intracellular calcium increase and that this was accompanied by ΔΨm dissipation, decrease of ATP levels and ROS production, and deterioration of plasma membrane integrity. In conclusion, cryopreservation induces MPT and this is associated with a loss of sperm quality.     

Aluminum as an inducer of the mitochondrial permeability transition

Treatment of rat liver mitochondria with aluminum in the presence of Ca2+ results in large amplitude swelling accompanied by loss of endogenous Mg2+ and K+ and oxidation of endogenous pyridine nucleotides. The presence of cyclosporin A, ADP, bongkrekic acid, N-ethylmaleimide and dithioerythritol prevent these effects, indicating that binding of aluminum to the inner mitochondrial membrane, most likely at the level of adenine nucleotide translocase, correlates with the induction of the membrane permeability transition (MPT). Indeed, aluminum binding promotes such a perturbation at the level of ubiquinol-cytochrome c reductase, which favors the production of reactive oxygen species. These metabolites generate an oxidative stress involving two previously defined sites in equilibrium with the glutathione and pyridine nucleotides pools, the levels of which correlate with the increase in MPT induction. Although the above-described phenomena are typical of MPT, they are not paralleled by other events normally observed in response to treatment with inducers of MPT (e.g., phosphate), such as the collapse of the electrochemical gradient and the release of accumulated Ca2+ and oxidized pyridine nucleotides. Biochemical and ultrastructural observations demonstrate that aluminum induces a pore opening having a conformation intermediate between fully open and closed in a subpopulation of mitochondria. While inorganic phosphate enhances the MPT induced by ruthenium red plus a deenergizing agent, aluminum instead inhibits this phenomenon. This finding suggests the presence of a distinct binding site for aluminum differing from that involved in MPT induction.     

Manganese induces the mitochondrial permeability transition in cultured astrocytes

Manganese is known to cause central nervous system injury leading to parkinsonism and to contribute to the pathogenesis of hepatic encephalopathy. Although mechanisms of manganese neurotoxicity are not completely understood, chronic exposure of various cell types to manganese has shown oxidative stress and mitochondrial energy failure, factors that are often implicated in the induction of the mitochondrial permeability transition (MPT). In this study, we examined whether exposure of cultured neurons and astrocytes to manganese induces the MPT. Cells were treated with manganese acetate (10-100 microM), and the MPT was assessed by changes in the mitochondrial membrane potential and in mitochondrial calcein fluorescence. In astrocytes, manganese caused a dissipation of the mitochondrial membrane potential and decreased the mitochondrial calcein fluorescence in a concentration- and time-dependent manner. These changes were completely blocked by pretreatment with cyclosporin A, consistent with induction of the MPT. On the other hand, similarly treated cultured cortical neurons had a delayed or reduced MPT as compared with astrocytes. The manganese-induced MPT in astrocytes was blocked by pretreatment with antioxidants, suggesting the potential involvement of oxidative stress in this process. Induction of the MPT by manganese and associated mitochondrial dysfunction in astrocytes may represent key mechanisms in manganese neurotoxicity.     

High Concentration of Gadolinium Ion Modifying Isolated Rice Mitochondrial Biogenesis

Mitochondria play an important role in plant growth and development, cooperating with the endoplasmic reticulum and nucleus. Gadolinium, one of the rare earth elements, is an inhibitor of stretch-activated calcium channels located on the endoplasmic reticulum and plasma membrane and has no effect on nuclear calcium variation in plant cells. We analyzed the effects of Gd³⁺ on mitochondria function by monitoring mitochondrial swelling, changes of membrane fluidity, and transmembrane potential collapse and by observing mitochondrial ultrastructure. We found that high concentration of Gd³⁺ induces rice mitochondrial dysfunction through mitochondrial permeability transition (MPT). The protection of DTT and EDTA demonstrate that Gd³⁺ blocks the inner membrane ion channel through thiol chelation.     

Mitochondrial ROS-induced ROS release: an update and review

Unstable mitochondrial membrane potential and redox transitions can occur following insults including ischemia/reperfusion injury and toxin exposure, with negative consequences for mitochondrial integrity and cellular survival. These transitions can involve mechanisms such as the recently described process, "Reactive Oxygen Species (ROS)-induced ROS-release" (RIRR), and be generated by circuits where the mitochondrial permeability transition (MPT) pore and the inner membrane anion channel (IMAC) are involved. The exposure to excessive oxidative stress results in an increase in ROS reaching a threshold level that triggers the opening of one of the requisite mitochondrial channels. In turn, this leads to the simultaneous collapse of the mitochondrial membrane potential and a transient increased ROS generation by the electron transfer chain. Generated ROS can be released into cytosol and trigger RIRR in neighboring mitochondria. This mitochondrion-to-mitochondrion ROS-signaling constitutes a positive feedback mechanism for enhanced ROS production leading to potentially significant mitochondrial and cellular injury. This review and update considers a variety of RIRR mechanisms (involving MPT, IMAC and episodes of mitochondrial transient hyperpolarization). RIRR could be a general cell biology phenomenon relevant to the processes of programmed mitochondrial destruction and cell death, and may contribute to other mechanisms of post-ischemic pathologies, including arrhythmias.     

Role of calcium and cyclophilin D in the regulation of mitochondrial permeabilization induced by glutathione depletion

The mitochondrial permeability transition (MPT) is a calcium and oxidative stress sensitive transition in the permeability of the mitochondrial inner membrane that plays a crucial role in cell death. However, the mechanism regulating the MPT remains controversial. To study the role of oxidative stress in the regulation of the MPT, we used diethyl maleate (DEM) to deplete glutathione (GSH) in human leukemic CEM cells. GSH depletion increased mitochondrial calcium and reactive oxygen species (ROS) levels in a co-dependent manner causing loss of mitochondrial membrane potential (deltapsi(m)) and cell death. These events were inhibited by the calcium chelator BAPTA-AM and the antioxidants N-acetylcysteine (NAC) and the triphenyl phosphonium-linked ubiquinone derivative MitoQ. In contrast, the MPT inhibitor cyclosporine A (CsA) and small interference RNA (siRNA) knockdown of cyclophilin D (Cyp-D) were not protective. These results indicate that mitochondrial permeabilization induced by GSH depletion is not regulated by the classical MPT.     

Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells

Cholesterol enrichment of rat liver mitochondria (CHM) impairs atractyloside-induced mitochondrial permeability transition (MPT) due to decreased membrane fluidity. In this study we addressed the effect of cholesterol enrichment on MPT induced by reactive oxygen species (ROS). Superoxide anion generated by xanthine plus xanthine oxidase triggered mitochondrial swelling and cytochrome c release in CHM, which was prevented by butylated hydroxytoluene, an anti-voltage-dependent anion channel antibody, or cyclosporin A. Furthermore, hydrogen peroxide generated by the combination of ganglioside GD3 and mitochondrial GSH depletion elicited mitochondrial swelling and release of cytochrome c, Smac/Diablo and apoptosis-inducing factor in control mitochondria and CHM. Thus, ROS induce MPT and apoptosome activation regardless of decreased mitochondrial membrane dynamics due to cholesterol enrichment.     

Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader

Opening of permeability transition (PT) pores in the mitochondrial inner membrane causes the mitochondrial permeability transition (MPT) and leads to mitochondrial swelling, membrane depolarization, and release of intramitochondrial solutes. Here, our aim was to develop high-throughput assays using a fluorescence plate reader to screen potential inducers and blockers of the MPT. Isolated rat liver mitochondria (0.5 mg/ml) were incubated in multiwell plates with tetramethylrhodamine methyl ester (TMRM, 1 microM), a potential-indicating fluorophore, and Fluo-5N (1 microM), a low-affinity Ca(2+) indicator. Incubation led to mitochondrial polarization, as indicated by uncoupler-sensitive quenching of the red TMRM fluorescence. CaCl(2) (100 microM) addition led to ruthenium red-sensitive mitochondrial Ca(2+) uptake, as indicated by green Fluo-5N fluorescence. After Ca(2+) accumulation, mitochondria depolarized, released Ca(2+) into the medium, and began to swell. This swelling was monitored as a decrease in light absorbance at 620 nm. Swelling, depolarization, and Ca(2+) release were prevented by cyclosporin A (1 microM), confirming that these events represented the MPT. Measurements of Ca(2+), mitochondrial membrane potential, and swelling could be made independently from the same wells without cross interference, and all three signals could be read from every well of a 48-well plate in about 1 min. In other experiments, mitochondria were ester-loaded with carboxydichlorofluorescein (carboxy-DCF) during the isolation procedure. Release of carboxy-DCF after PT pore opening led to an unquenching of green carboxy-DCF fluorescence occurring simultaneously with swelling. By combining measurements of carboxy-DCF release, Ca(2+) uptake, membrane potential, and swelling, MPT inducers and blockers can be distinguished from uncouplers, respiratory inhibitors, and blockers of Ca(2+) uptake. This high-throughput multiwell assay is amenable for screening panels of compounds for their ability to promote or block the MPT.     

Vimang (Mangifera indica L. extract) induces permeability transition in isolated mitochondria, closely reproducing the effect of mangiferin, Vimang's main component

Mitochondrial permeability transition (MPT) is a Ca(2+)-dependent, cyclosporin A (CsA)-sensitive, non-selective inner membrane permeabilization process. It is often associated with apoptotic cell death, and is induced by a wide range of agents or conditions, usually involving reactive oxygen species (ROS). In this study, we demonstrated that Mangifera indica L. extract (Vimang), in the presence of 20 microM Ca(2+), induces MPT in isolated rat liver mitochondria, assessed as CsA-sensitive mitochondrial swelling, closely reproducing the same effect of mangiferin, the main component of the extract, as well as MPT-linked processes like oxidation of membrane protein thiols, mitochondrial membrane potential dissipation and Ca(2+) release from organelles. The flavonoid catechin, the second main component of Vimang, also induces MPT, although to a lesser extent; the minor, but still representative Vimang extract components, gallic and benzoic acids, show respectively, low and high MPT inducing abilities. Nevertheless, following exposure to H(2)O(2)/horseradish peroxidase, the visible spectra of these compounds does not present the same changes previously reported for mangiferin. It is concluded that Vimang-induced MPT closely reproduces mangiferin effects, and proposed that this xanthone is the main agent responsible for the extract's MPT inducing ability, by the action on mitochondrial membrane protein thiols of products arising as a consequence of the mangiferin's antioxidant activity. While this effect would oppose the beneficial effect of Vimang's antioxidant activity, it could nevertheless benefit cells exposed to over-production of ROS as occurring in cancer cells, in which triggering of MPT-mediated apoptosis may represent an important defense mechanism to their host.     

Exploiting the Role of Resveratrol in Rat Mitochondrial Permeability Transition

Resveratrol (RSV), a natural polyphenolic antioxidant, has been considered an anticarcinogenic agent as it triggers tumor cell apoptosis through activation of the mitochondrial pathway. In our study, the effects of RSV on mitochondria, especially on the mitochondrial permeability transition (MPT) process, were investigated by multiple methods. We found that RSV induced a collapse of membrane potential and matrix swelling related to MPT. We further demonstrated that Ca²⁺ was necessary for this RSV-induced MPT opening. In addition, RSV induced the inner membrane permeabilization to H⁺ and K⁺, the depression of respiration and changes in membrane fluidity. The results suggested that RSV-induced MPT was accompanied by mitochondrial dysfunction. But the prohibition on lipid peroxidation and different effects of low- and high-dose RSV on membrane fluidity and respiration showed that the interaction of RSV and the mitochondria could not be the result of a single simple mechanism.     

The mitochondrial permeability transition regulates cytochrome c release for apoptosis during endoplasmic reticulum stress by remodeling the cristae junction

The role of the mitochondrial permeability transition (MPT) in apoptosis and necrosis is controversial. Here we show that the MPT regulates the release of cytochrome c for apoptosis during endoplasmic reticulum (ER) stress by remodeling the cristae junction (CJ). CEM cells, HCT116 colon cancer cells, and murine embryo fibroblast cells were treated with the ER stressor thapsigargin (THG), which led to cyclophilin D-dependent mitochondrial release of the profusion GTPase optic atrophy 1 (OPA1), which controls CJ integrity, and cytochrome c, leading to apoptosis. Interference RNA knockdown of Bax blocked OPA1 and cytochrome c release after THG treatment but did not prevent the MPT, showing that Bax was essential for the release of cytochrome c by MPT. In isolated mitochondria, MPT led to OPA1 and cytochrome c release independently of voltage-dependent anion channel and the outer membrane, indicating that the MPT is an inner membrane phenomenon. Last, the MPT was regulated by the electron transport chain but not mitochondrial reactive oxygen species, since THG-induced cell death was not blocked by antioxidants and did not occur in cells lacking mitochondrial DNA. Our results show that the MPT regulates CJ remodeling for cytochrome c-dependent apoptosis induced by ER stress and that mitochondrial electron transport is indispensable for this process.     

The oat mitochondrial permeability transition and its implication in victorin binding and induced cell death

The mitochondrion has emerged as a key regulator of apoptosis, a form of animal programmed cell death (PCD). The mitochondrial permeability transition (MPT), facilitated by a pore-mediated, rapid permeability increase in the inner membrane, has been implicated as an early and critical step of apoptosis. Victorin, the host-selective toxin produced by Cochliobolus victoriae, the causal agent of victoria blight of oats, has been demonstrated to bind to the mitochondrial P-protein and also induces a form of PCD. Previous results suggest that a MPT may facilitate victorin's access to the mitochondrial matrix and binding to the P-protein: (i) victorin-induced cell death displays features similar to apoptosis; (ii) in vivo, victorin binds to the mitochondrial P-protein only in toxin-sensitive genotypes whereas victorin binds equally well to P-protein isolated from toxin-sensitive and insensitive oats; (iii) isolated, untreated mitochondria are impermeable to victorin. The data implicate an in vivo change in mitochondrial permeability in response to victorin. This study focused on whether oat mitochondria can undergo a MPT. Isolated oat mitochondria demonstrated high-amplitude swelling when treated with spermine or Ca2+ in the presence of the Ca2+-ionophore A23187, and when treated with mastoparan, an inducer of the MPT in rat liver mitochondria. In all cases, swelling demonstrated size exclusion in the range 0.9-1.7 kDa, similar to that found in animal mitochondria. Further, MPT-inducing conditions permitted victorin access to the mitochondrial matrix and binding to the P-protein. In vivo, victorin treatment induced the collapse of mitochondrial transmembrane potential within 2 h, indicating a MPT. Also, the victorin-induced collapse of membrane potential was clearly distinct from that induced by uncoupling respiration, as the latter event prevented the victorin-induced PCD response and binding to P-protein. These results demonstrate that a MPT can occur in oat mitochondria in vitro, and are consistent with the hypothesis that an MPT, which allows victorin access to the mitochondrial matrix and binding to the P-protein, occurs in vivo during victorin-induced PCD.