The role of the mitochondrial permeability transition in cell death

The mitochondrial permeability transition (MPT) is a non-selective inner membrane permeabilization that occurs in response to increased calcium load and redox stress. Currently, two models of the MPT exist including the, largely hypothetical, native proteinaceous pore model and the oxidized inner membrane protein model which may reflect the extremes in a continuum of changes that occur to the inner membrane prior to its permeabilization. Here I discuss evidence that the MPT per se leads to necrosis, but not cytochrome c release and apoptosis. However, data also suggest that signaling crosstalk between the MPT and Bcl-2 family proteins occurs indicating an important role for the MPT in apoptosis.     

The still uncertain identity of the channel-forming unit(s) of the mitochondrial permeability transition pore

Mitochondria from different organisms can undergo a sudden process of inner membrane unselective leakiness to molecules known as the mitochondrial permeability transition (MPT). This process has been studied for nearly four decades and several proteins have been claimed to constitute, or at least regulate the usually inactive pore responsible for this transition. However, no protein candidate proposed as the actual pore-forming unit has passed rigorous gain- or loss-of-function genetic tests. Here we review evidence for -and against- putative channel-forming components of the MPT pore. We conclude that the structure of the MPT pore still remains largely undefined and suggest that future studies should follow established technical considerations to unambiguously consolidate the channel forming constituent(s) of the MPT pore.     

Limited mitochondrial permeabilization is an early manifestation of palmitate-induced lipotoxicity in pancreatic beta-cells

Involvement of the mitochondrial permeability transition (MPT) pore in early stages of lipotoxic stress in the pancreatic beta-cell lines MIN6 and INS-1 was the focus of this study. Both long term (indirect) and acute (direct) effects of fatty acid (FA) application on beta-cell susceptibility to Ca(2+)-induced MPT induction were examined using both permeabilized and intact beta-cells. Long term exposure to moderate (i.e. below cytotoxic) levels of the saturated FA palmitate sensitized beta-cell mitochondria to MPT induced by Ca(2+). Long term exposure to palmitate was significantly a more efficient inducer of MPT than the unsaturated FA oleate, although upon acute application both caused similar MPT activation. Application of antioxidants, inhibitors of the ceramide pathway, or modifiers of membrane fluidity did not protect beta-cell mitochondria from FA exposure. However, significant protection was provided by co-application of the unsaturated FA oleate in a phosphatidylinositol 3-kinase-dependent manner. Characterization of MPT pore opening in response to moderate palmitate treatment revealed the opening of a unique form of MPT in beta-cells as it encompassed features of both low and high conductance MPT states. Specifically, this MPT showed solute selectivity, characteristic of a low conductance MPT; however, it affected mitochondrial respiration and membrane potential in a way typical of a high conductance MPT. Activation of the full-size/high conductance form of MPT required application of high levels of FA that reduced growth and initiated apoptosis. These findings suggest that in the beta-cell, MPTs can act as both initiators of cell death and as versatile modulators of cell metabolism, depending on the mode of the MPT pore induced.     

Beta-phenylethyl isothiocyanate mediated apoptosis; contribution of Bax and the mitochondrial death pathway

The initiating events that lead to the induction of apoptosis mediated by the chemopreventative agent beta-phenyethyl isothiocyanate (PEITC) have yet to be elucidated. In the present investigation, we examined the effects of PEITC on mitochondrial function and apoptotic signaling in hepatoma HepG2 cells and isolated rat hepatocyte mitochondria. PEITC induced a conformational change in Bax leading to its translocation to mitochondria in HepG2 cells. Bax accumulation was associated with a rapid loss of mitochondrial membrane potential (Deltapsim), impaired respiratory chain enzymatic activity, release of mitochondrial cytochrome c and the activation of caspase-dependent cell death. Caspase inhibition did not prevent Bax translocation, the release of cytochrome c or the loss of Deltapsim, but blocked caspase-mediated DNA fragmentation and cell death. To determine whether PEITC dependent Bax translocation caused loss of Deltapsim by the activation of the mitochondrial permeability transition (MPT), we examined the effects of PEITC in isolated rat hepatocyte mitochondria. Interestingly, PEITC did not induce MPT in isolated rat mitochondria. Accordingly, using pharmacological inhibitors of MPT namely cyclosporine A, trifluoperazine and Bongkrekic acid we were unable to block PEITC mediated apoptosis in HepG2 cells, this suggesting that mitochondrial permeablisation is a likely consequence of Bax dependent pore formation. Taken together, our data suggest that mitochondria are a key target in PEITC induced apoptosis in HepG2 cells via the pore forming ability of pro-apoptotic Bax.     

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 membrane permeability transition and cell death

Mitochondria are important organelles for energy production, Ca2+ homeostasis, and cell death. In recent years, the role of the mitochondria in both apoptotic and necrotic cell death has received much attention. In apoptotic and necrotic death, an increase of mitochondrial membrane permeability is considered to be one of the key events, although the detailed mechanism remains to be elucidated. The mitochondrial membrane permeability transition (MPT) is a Ca2+-dependent increase in the permeability of the mitochondrial membrane that leads to loss of Deltapsi, mitochondrial swelling, and rupture of the outer mitochondrial membrane. The MPT is thought to occur after the opening of a channel, which is termed the permeability transition pore (PTP) and putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), cyclophilin D (Cyp D: a mitochondrial peptidyl prolyl-cis, trans-isomerase), and other molecule(s). Our studies of mice lacking Cyp D have revealed that it is essential for occurrence of the MPT and that the Cyp D-dependent MPT regulates some forms of necrotic cell death, but not apoptotic death. We have also shown that two anti-apoptotic proteins, Bcl-2 and Bcl-x(L), block the MPT by directly inhibition of VDAC activity. Here we summarize a role of the MPT in cell death.     

Tetrandrine concentrations not affecting oxidative phosphorylation protect rat liver mitochondria from oxidative stress

The effects of tetrandrine (6,6', 7,12-tetramethoxy-2, 2'-dimethyl-berbaman) on the mitochondrial function were assessed on oxidative stress, mitochondrial permeability transition (MPT), and bioenergetics of rat liver mitochondria. At concentrations lower than 100nmol/mg protein, tetrandrine decreased the hydrogen peroxide formation, the extent of lipid peroxidation, the susceptibility to Ca(2+)-induced opening of MPT pore, and inhibited the inner membrane anion channel activity, not significantly affecting the mitochondrial bioenergetics. High tetrandrine concentrations (100-300nmol/mg protein) stimulated succinate-dependent state 4 respiration, while some inhibition was observed for state 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations. The respiratory control ratio and the transmembrane potential were depressed but the adenosine diphosphate to oxygen (ADP/O) ratio was less affected. A slight increase of the inner mitochondrial membrane permeability to H(+) and K(+) by tetrandrine was also observed. It was concluded that low concentrations of tetrandrine afford protection against liver mitochondria injury promoted by oxidative-stress events, such as hydrogen peroxide production, lipid peroxidation, and induction of MPT. Conversely, high tetrandrine concentrations revealed toxicological effects expressed by interference with mitochondrial bioenergetics, as a consequence of some inner membrane permeability to H(+) and K(+) and inhibition of the electron flux in the respiratory chain. The direct immediate protective role of tetrandrine against mitochondrial oxidative stress may be relevant to clarify the mechanisms responsible for its multiple pharmacological actions.     

Electrophysiology clarifies the megariddles of the mitochondrial permeability transition pore

After a brief review of the early history of mitochondrial electrophysiology, the contribution of this approach to the study of the mitochondrial permeability transition (MPT) is recapitulated. It has for example provided evidence for a dimeric nature of the MPT pore, allowed the distinction between two levels of control of its activity, and underscored the relevance of redox events for the phenomenon. Single-channel recording provides a means to finally solve the riddle of the biochemical entity underlying it by comparing the characteristics of the pore with those of channels formed by candidate molecules or complexes. The possibility that this entity may be the protein import machinery of the inner mitochondrial membrane is emphasized.     

Role of the mitochondrial membrane permeability transition in cell death

In recent years, the role of the mitochondria in both apoptotic and necrotic cell death has received considerable attention. An increase of mitochondrial membrane permeability is one of the key events in apoptotic or necrotic death, although the details of the mechanism involved remain to be elucidated. The mitochondrial membrane permeability transition (MPT) is a Ca²⁺-dependent increase of mitochondrial membrane permeability that leads to loss of Δψ, mitochondrial swelling, and rupture of the outer mitochondrial membrane. The MPT is thought to occur after the opening of a channel that is known as the permeability transition pore (PTP), which putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), cyclophilin D (Cyp D: a mitochondrial peptidyl prolyl-cis, trans-isomerase), and other molecule(s). Recently, significant progress has been made by studies performed with mice lacking Cyp D at several laboratories, which have convincingly demonstrated that Cyp D is essential for the MPT to occur and that the Cyp D-dependent MPT regulates some forms of necrotic, but not apoptotic, cell death. Cyp D-deficient mice have also been used to show that the Cyp D-dependent MPT plays a crucial role in ischemia/reperfusion injury. The anti-apoptotic proteins Bcl-2 and Bcl-x_L have the ability to block the MPT, and can therefore block MPT-dependent necrosis in addition to their well-established ability to inhibit apoptosis.     

Oxidative stress and adenine nucleotide control of mitochondrial permeability transition

Mitochondria can initiate apoptosis by releasing cytochrome c after undergoing a calcium-dependent permeability transition (MPT). Although the MPT is enhanced by oxidative stress and prevented by adenine nucleotides such as adenosine 5'-diphosphate (ADP), the hypothesis has not been tested that oxidants regulate the effects of exogenous adenine nucleotides on the MPT and cytochrome c release. We found that cytochrome c release from intact rat liver mitochondria depended strictly on pore opening and not on membrane potential, and that MPT-enhancing oxidative stress also augmented cytochrome c release. At low oxidative stress, micromolar (ADP) and low adenosine 5'-triphosphate (ATP)/ADP ratio inhibited the MPT and cytochrome c release, whereas ATP or high ATP/ADP had only a slight effect. In freshly isolated mitochondria, the time to half-maximal MPT was related to the log of the ATP/ADP ratio. This function was shifted to shorter times by oxidative stress which decreased ADP protection and caused ATP to accelerate the calcium-dependent MPT. By comparison, mitochondria treated with reducing agents and those isolated from septic rats were protected from the MPT by both nucleotides. These results indicate that oxidation-sensitive site(s) in the membrane regulate the effects of adenine nucleotides on the MPT. The oxidant-based differences in the effects of ADP and ATP on the pore support the novel hypothesis that failure of the cell to consume ATP and provide adequate ADP at the adenine nucleotide transporter during oxidative stress predisposes to cytochrome c release and initiation of apoptosis.     

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.     

Ce(III)-Induced Rice Mitochondrial Permeability Transition Investigated by Spectroscopic and Microscopic Studies

Cerium has been widely used as fertilizer and feed additives in agriculture, but it might finally impair human health by food chain accumulation with its dosage increased in environmental and crops samples. To resolve the conflict, we investigated the effects of Ce(III) on isolated rice mitochondrial permeability transition (MPT) by examining mitochondrial swelling, transmembrane potential, membrane fluidity with spectroscopy, and observing the mitochondrial ultrastructure, meanwhile, the interaction site(s) and mechanism between Ce(III) and mitochondria were also studied. The results showed that the low level of Ce(III) had little effect on rice MPT, however, the higher level of Ce(III) could induce rice MPT, and the thiol (?SH) groups of membrane proteins (defined as “S” site) matched by Ce(III)-triggered rice MPT pore opening.     

Cytochrome bc(1) regulates the mitochondrial permeability transition by two distinct pathways

The mitochondrial permeability transition (MPT) pore is a calcium-sensitive channel in the mitochondrial inner membrane that plays a crucial role in cell death. Here we show that cytochrome bc(1) regulates the MPT in isolated rat liver mitochondria and in CEM and HL60 cells by two independent pathways. Glutathione depletion activated the MPT via increased production of reactive oxygen species (ROS) generated by cytochrome bc(1). The ROS producing mechanism in cytochrome bc(1) involves movement of the "Rieske" iron-sulfur protein subunit of the enzyme complex, because inhibition of cytochrome bc(1) by pharmacologically blocking iron-sulfur protein movement completely abolished ROS production, MPT activation, and cell death. The classical inhibitor of the MPT, cyclosporine A, had no protective effect against MPT activation. In contrast, the calcium-activated, cyclosporine A-regulated MPT in rat liver mitochondria was also blocked with inhibitors of cytochrome bc(1). These results indicate that electron flux through cytochrome bc(1) regulates two distinct pathways to the MPT, one unregulated and involving mitochondrial ROS and the other regulated and activated by calcium.     

Non-conventional mitochondrial permeability transition: Its regulation by mitochondrial dynamics

Mitochondrial permeability transition (MPT) is a phenomenon that the inner mitochondrial membrane (IMM) loses its selective permeability, leading to mitochondrial dysfunction and cell injury. Electrophysiological evidence indicates the presence of a mega-channel commonly called permeability transition pore (PTP) whose opening is responsible for MPT. However, the molecular identity of the PTP is still under intensive investigations and debates, although cyclophilin D that is inhibited by cyclosporine A (CsA) is the established regulatory component of the PTP. PTP can also open transiently and functions as a rapid mitochondrial Ca²⁺ releasing mechanism. Mitochondrial fission and fusion, the main components of mitochondrial dynamics, control the number and size of mitochondria, and have been shown to play a role in regulating MPT directly or indirectly. Studies by us and others have indicated the potential existence of a form of transient MPT that is insensitive to CsA. This “non-conventional” MPT is regulated by mitochondrial dynamics and may serve a protective role possibly by decreasing the susceptibility for a frequent or sustained PTP opening; hence, it may have a therapeutic value in many disease conditions involving MPT.     

Role of the mitochondrial permeability transition in apoptotic and necrotic death after ischemia/reperfusion injury to hepatocytes

Reperfusion of ATP-depleted tissues after warm or cold ischemia causes pH-dependent necrotic and apoptotic cell death. In hepatocytes and other cell types as well, the mechanism underlying this reperfusion-induced cell death involves onset of the mitochondrial permeability transition (MPT). Opening of permeability transition (PT) pores in the mitochondrial inner membrane initiates the MPT, an event blocked by cyclosporin A (CsA) and pH less than 7.4. Thus, both acidotic pH and CsA prevent MPT-dependent reperfusion injury. Glycine also blocks reperfusion-induced necrosis but acts downstream of PT pore opening by stabilizing the plasma membrane. After the MPT, ATP availability from glycolysis or other source determines whether cell injury after reperfusion progresses to ATP depletion-dependent necrosis or ATP-requiring apoptosis. Thus, apoptosis and necrosis after reperfusion share a common pathway, the MPT. Cell injury progressing to either necrosis or apoptosis by shared pathways can be more aptly termed necrapoptosis.     

Cyclophilin D is required for mitochondrial removal by autophagy in cardiac cells

Autophagy is a highly regulated intracellular degradation process by which cells remove cytosolic long-lived proteins and damaged organelles. The mitochondrial permeability transition (MPT) results in mitochondrial depolarization and increased reactive oxygen species production, which can trigger autophagy. Therefore, we hypothesized that the MPT may have a role in signaling autophagy in cardiac cells. Mitochondrial membrane potential was lower in HL-1 cells subjected to starvation compared to cells maintained in full medium. Mitochondrial membrane potential was preserved in starved cells treated with cyclosporin A (CsA), suggesting the MPT pore is associated with starvation-induced depolarization. Starvation-induced autophagy in HL-1 cells, neonatal rat cardiomyocytes and adult mouse cardiomyocytes was inhibited by CsA. Starvation failed to induce autophagy in CypD-deficient murine cardiomyocytes, whereas in myocytes from mice overexpressing CypD the levels of autophagy were enhanced even under fed conditions. Collectively, these results demonstrate a role for CypD and the MPT in the initiation of autophagy. We also analyzed the role of the MPT in the degradation of mitochondria by biochemical analysis and electron microscopy. HL-1 cells subjected to starvation in the presence of CsA had higher levels of mitochondrial proteins (by Western blot), more mitochondria and less autophagosomes (by electron microscopy) than cells starved in the absence of CsA. Our results suggest a physiologic function for CypD and the MPT in the regulation of starvation-induced autophagy. Starvation-induced autophagy regulated by CypD and the MPT may represent a homeostatic mechanism for cellular and mitochondrial quality control.     

Mitochondrial oxidative stress induced by Ca2+ and monoamines: different behaviour of liver and brain mitochondria in undergoing permeability transition

Mitochondrial permeability transition (MPT) is correlated with the opening of a nonspecific pore, the so-called transition pore, that triggers bidirectional traffic of inorganic solutes and metabolites across the mitochondrial membrane. This phenomenon is caused by supraphysiological Ca(2+) concentrations and by other compounds leading to oxidative stress, while cyclosporin A, ADP, bongkrekic acid, antioxidant agents and naturally occurring polyamines strongly inhibit it. The effects of polyamines, including the diamine agmatine, have been widely studied in several types of mitochondria. The effects of monoamines on MPT have to date, been less well-studied, even if they are involved in a variety of neurological and neuroendocrine processes. This study shows that in rat liver mitochondria (RLM), monoamines such as tyramine, serotonin and dopamine amplify the swelling induced by calcium, and increase the oxidation of thiol groups and the production of hydrogen peroxide, effects that are counteracted by the above-mentioned inhibitors. In rat brain mitochondria (RBM), the monoamines do not amplify calcium-induced swelling, even if they demonstrate increases in the extent of oxidation of thiol groups and hydrogen peroxide production. In these mitochondria, the antioxidants are not at all or scarcely effective in suppressing mitochondrial swelling. In conclusion, we hypothesize that different mechanisms induce the MPT in the two different types of mitochondria evaluated. Calcium and monoamines induce oxidative stress in RLM, which in turn appears to induce and amplify MPT. This process is not apparent in RBM, where MPT seems resistant to oxidative stress.     

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.     

Gliotoxin induces Mg2+ efflux from intact brain mitochondria

Gliotoxin (GT) is a hydrophobic fungal metabolite of the epipolythiodioxopiperazine group which reacts with membrane thiols. When added to a suspension of energized brain mitochondria, it induces matrix swelling of low amplitude, collapse of membrane potential (DeltaPsi), and efflux of endogenous cations such as Ca2+ and Mg2+, typical events of mitochondrial permeability transition (MPT) induction. These effects are due to opening of the membrane transition pore. The addition of cyclosporin A (CsA) or ADP slightly reduces membrane potential collapse, matrix swelling and Ca2+ efflux; Mg2+ efflux is not affected at all. The presence of exogenous Mg2+ or spermine completely preserve mitochondria against DeltaPsi collapse, matrix swelling and Ca2+ release. Instead, Mg2+ efflux is only slightly affected by spermine. Our results demonstrate that, besides inducing MPT, gliotoxin activates a specific Mg2+ efflux system from brain mitochondria.     

Effect of Ca2+ on induction of the mitochondrial pore opening in the rat myocardium

The mitochondrial role opening (MPT) induced by Ca2+ has been studied in isolated rat heart mitochondria. MPT was characterized as cyclosporine A-inhibited swelling accompanied by the loss of membrane potential (deltapsim) and Ca2+ efflux after the Ca2+ -loading which was followed spectrophotometrically after the Ca2+ -arsenaso-III complex formation. It has been shown that in suspension of isolated mitochondria MPT was activated by low (with maximum at about 20 microM Ca2+) and high concentrations of Ca2+ (the concentration curve shows a saturation at about 1.0-1.5 mM). In all the cases an access of Ca2+ ions to the matrix space of the mitochondria was necessary for MPT induction. MPT activated by low concentrations of Ca2+ was accompanied by slow decrease of deltapsim and slow release of Ca2+, enhanced by ruthenium red (RR), and was independent of the substrate used (glutamate or succinate). It had not been observed if the respiratory chain was inhibited, even if the Ca2+ access to the inner mitochondrial membrane was provided by Ca2+ -ionophore A23187. At high Ca2+ concentrations rapid Ca2+ -uptake and release via Ca2+ -uniporter (inhibited by ruthenium red) followed by extensive swelling (pore formation) have been observed. It had been supposed that rapid MPT at high concentrations of Ca2+ was the result of Ca2+ entrance to the mitochondrial matrix and depolarisation of the mitochondrial membrane. The data obtained show two different mechanisms of Ca2+ -induced MPT. The one is sensitive to the redox-state of the electron transport chain and is abolished if the respiration is inhibited. The other is independent of mitochondrial respiration and needs only Ca2+ access to the inner mitochondrial membrane and Ca2+ binding to some specific sites leading to MPT opening.