The mitochondrial permeability transition

This review summarizes recent work on the regulation of the permeability transition pore, a cyclosporin A-sensitive mitochondrial channel that may play a role in intracellular calcium homeostasis and in a variety of forms of cell death. The basic bioenergetics aspects of pore modulation are discussed, with some emphasis on the links between oxidative stress and pore dysregulation as a potential cause of mitochondrial dysfunction that may be relevant to cell injury.     

Two modes of activation of the permeability transition pore: the role of mitochondrial cyclophilin

Mitochondria possess an inner membrane channel, the permeability transition pore, which is inhibited by cyclosporin A (CsA) and by matrix protons. As suggested recently by our laboratory, pore closure by these inhibitors may be due to dissociation of mitochondrial cyclophilin (CyP-M), a matrix peptidyl-prolyl-cis-trans isomerase, from its putative binding site on the pore. Unbinding of CyP-M would follow a CsA-dependent or proton-dependent change in conformation of the CyP-M molecule. It is interesting that upon binding of CsA the enzymatic activity of CyP-M is inhibited, but it is not clear whether this event plays a role in pore inhibition. Here we report experiments designed to further test the role of CyP-M in pore function. Our results indicate that CyP-M-dependent and independent mechanisms of pore activation may exist, and that the peptidylprolyl-cis-trans-isomerase activity of CyP-M is not necessarily involved in pore modulation by CyP-M.     

Imaging the mitochondrial permeability transition pore in intact cells

The involvement of mitochondrial permeability transition pore (MTP) in cellular processes is generally investigated by indirect means, such as changes in mitochondrial membrane potential or pharmacological inhibition. However, such effects could not be related univocally to MTP. In addition, source of errors could be represented by the increased retention of membrane potential probes induced by cyclosporin A (CsA) and the interactions between fluorescent probes. We developed a direct technique for monitoring MTP. Cells were co-loaded with calcein-AM and CoCl2, resulting in the quenching of the cytosolic signal without affecting the mitochondrial fluorescence. MTP inducers caused a rapid decrease in mitochondrial calcein fluorescence which, however, was not completely prevented by CsA. Besides the large and rapid efflux of calcein induced by MTP agonists, we also observed a constant and spontaneous decrease of mitochondrial calcein which was completely prevented by CsA. Thus, MTP likely fluctuates between open and closed states in intact cells.     

Modulation of the mitochondrial permeability transition by nitric oxide

The influence of nitric oxide on mitochondrial permeability transition (MPT) phenomenon was studied. NO was generated by photolysis of S-nitroso-N-acetylcysteine, AcCys(NO), with green light (lambda = 550 nm). Two distinct effects of nitric oxide on rat liver mitochondria were identified. First, NO accelerated an onset of swelling in Ca2(+)-loaded mitochondria in a cyclosporin-A-sensitive manner acting as an inducer of permeability transition. This was, apparently, a result of irreversible alteration of mitochondrial function accompanying the inhibition of respiratory chain in the presence of calcium. Formation of ESR-visible iron-sulfur dinitrosyl complexes (g = 2.041) could also contribute to the irreversible changes resulting in MPT induction. Second, NO changed significantly the response of mitochondria to Ca2+/phosphate-induced MPT, acting as a regulator of permeability transition. In this case the action of nitric oxide led to division of the mitochondria into two subpopulations: one which underwent the rapid permeability transition and another in which the MPT was inhibited. The effect of NO on Ca2+/Pi-induced MPT was transient and resulted from reversible inhibition of cytochrome oxidase followed by the changes in transmembrane potential and Ca2+ distribution. The characteristic time of duration of these NO modulated effects depended on nitric oxide as well as on oxygen concentrations. With increasing NO at fixed oxygen concentrations, this time levelled off to reach a maximum value which was inversely related to the oxygen concentration. It is concluded that under physiological condition the duration of reversible NO effects on mitochondrial function could be determined by oxygen concentration.     

CREB activation mediates plasticity in cultured hippocampal neurons

Activation of cyclic AMP dependent kinase is believed to mediate slow onset, long-term potentiation (LTP) in central neurons. Cyclic-AMP activates a cascade of molecular events leading to phosphorylation of the nuclear cAMP responsive element binding protein (pCREB). Whereas a variety of stimuli lead to activation of CREB, the molecular processes downstream of CREB, which may be relevant to neuronal plasticity, are yet largely unknown. We have recently found that following exposure to estradiol, pCREB mediates the large increase in dendritic spine density in cultured rat hippocampal neurons. We now extend these observations to include other stimuli, such as bicuculline, that cause the formation of new dendritic spines. Such stimuli share with estradiol the same mechanism of action in that both require activity-dependent CREB phosphorylation. Our observations suggest that CREB phosphorylation is a necessary, but perhaps not sufficient, step in the process leading to the generation of new dendritic spines and perhaps to functional plasticity as well.     

Dietary restriction augments protection against induction of the mitochondrial permeability transition

Exposure to oxidants or phosphate, especially in the presence of calcium, has been long known to lead to mitochondrial structural alteration and damage. In the past 15 years, it has become increasingly appreciated that this damage is often the result of a cyclosporin A-sensitive event, the "permeability transition" (PT). Using liver mitochondria isolated from male Fischer 344 rats of 6-24 months of age, we now present evidence that long-term, life-prolonging, dietary restriction regimens greatly delay induction of a PT following challenge. Dietary restriction slowed induction by 25 microM calcium, or by calcium in conjunction with the strong oxidant t-butyl hydroperoxide, by approximately 50%. The increased resistance to PT induction was maintained through 24 months of age. Dietary restriction also protected against t-butyl hydroperoxide in the presence of high calcium challenges (250 microM), although the extent of this protection was age-dependent. Induction by 2.5 mM phosphate alone was blocked in most 6-month-old dietary restricted animals and was slowed by 50-100% in animals 12-24 months of age. Susceptibility to 25 microM calcium in conjunction with phosphate varied in an age-dependent manner, ranging from 4-12 times slower in the dietary restricted animals than in their ad lib fed counterparts. Together, these data provide evidence that the factors regulating PT induction are affected by long-term physiological and environmental conditions such as age and diet. The observed effects represent one of the largest recognized dietary restriction-mediated increases in a parameter related to antioxidant defenses. These data also suggest that the endogenous defense systems that protect mitochondria from calcium in conjunction with inorganic phosphate differ from those that protect against calcium in conjunction with an oxidant.     

Src activation in the induction of long-term potentiation in CA1 hippocampal neurons

Long-term potentiation (LTP) is an activity-dependent strengthening of synaptic efficacy that is considered to be a model of learning and memory. Protein tyrosine phosphorylation is necessary to induce LTP. Here, induction of LTP in CA1 pyramidal cells of rats was prevented by blocking the tyrosine kinase Src, and Src activity was increased by stimulation producing LTP. Directly activating Src in the postsynaptic neuron enhanced excitatory synaptic responses, occluding LTP. Src-induced enhancement of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptor-mediated synaptic responses required raised intracellular Ca2+ and N-methyl-D-aspartate (NMDA) receptors. Thus, Src activation is necessary and sufficient for inducing LTP and may function by up-regulating NMDA receptors.     

Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes

The vesicle-associated membrane protein (VAMP) family is essential to vesicle-mediated protein transport. Three mammalian isoforms, VAMP-1, VAMP-2, and cellubrevin, play a role in protein transport to the plasma membrane. In this study, we describe a new rat VAMP-1 isoform produced by alternative pre-mRNA splicing. Only one VAMP-1 isoform dominates in each tissue. Analysis of the nucleotide sequence for the newly discovered isoform, VAMP-1b, reveals that its expression is determined by whether an intron is retained or removed. The predicted amino acid sequences for the VAMP-1 isoforms differ at the carboxy-terminal end of the protein. A similar process has been described for VAMPs in Drosophila melanogaster and suggests a conserved function for the carboxy-terminal domain that can be modulated.     

Confocal microscopy of the mitochondrial permeability transition in necrotic cell killing, apoptosis and autophagy

Onset of the cyclosporin-A-sensitive mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by laser scanning confocal microscopy. The MPT is a causative event in many types of necrotic and apoptotic cell death, including oxidative stress, ischemia/reperfusion injury, Ca2+ ionophore toxicity and tumor necrosis factor alpha (TNF alpha) induced apoptosis, and may contribute to Reye's-related drug toxicity. Pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and increased mitochondrial Ca2+ and pH can each promote onset of the MPT in situ. The MPT can also be directly visualized during TNF alpha-induced apoptosis to hepatocytes. Mitochondria spontaneously depolarize in situ after nutrient deprivation before entering an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. We propose a model in which onset of the MPT to increasing numbers of mitochondria leads progressively to autophagy, apoptosis and necrotic cell death.     

The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition

Stably transfected Jurkat T cells were produced in which Bax expression is inducible by muristerone A. The cell death resulting from induction of the overexpression of Bax was prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A2 inhibitor aristolochic acid (ArA). The caspase-3 inhibitor Z-Asp-Glu-Val aspartic acid fluoromethylketone (Z-DEVD-FMK) had no effect on the loss of viability. The MPT was measured as the CyA plus ArA-preventable loss of the mitochondrial membrane potential (DeltaPsim). The MPT was accompanied by the release of cytochrome c from the mitochondria, caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. Z-DEVD-FMK had no effect on the loss of DeltaPsim and the redistribution of cytochrome c but did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. It is concluded that Bax induces the MPT, a critical event in the loss of cell viability. In addition to the cell death, the MPT mediates other typical manifestations of apoptosis in this model, namely release of cytochrome c, caspase activation with PARP cleavage, and DNA fragmentation.     

Cyclosporin A, but not FK 506, protects mitochondria and neurons against hypoglycemic damage and implicates the mitochondrial permeability transition in cell death

Induction of the mitochondrial permeability transition (MPT) has been implicated in cellular apoptosis and in ischemia-reperfusion injury. During MPT, a channel in the inner mitochondrial membrane, the mitochondrial megachannel, opens and causes isolated mitochondria to swell. MPT and mitochondrial swelling is inhibited by cyclosporin A (CsA), which may also inhibit apoptosis in some cells. Treatment with CsA (50 mg/kg, i.v.) showed a robust reduction of brain damage when administered 30 min before insulin-induced hypoglycemic isoelectricity of 30 min duration. Ultrastructural examination of the dentate gyrus revealed a marked swelling of dendrites and mitochondria during the hypoglycemic insult. In CsA-treated animals, mitochondria resumed a normal and contracted appearance during and after the hypoglycemic insult. Treatment with FK 506 (2 mg/kg, i.v.), a compound with immunosuppressive action similar to that of CsA, was not protective. Studies on the swelling kinetics of isolated mitochondria from the hippocampus showed that CsA, but not FK 506, inhibits calcium ion-induced MPT. We conclude that CsA treatment during hypoglycemic coma inhibits the MPT and reduces damage and that mitochondria and the MPT are likely to be involved in the development of hypoglycemic brain damage in the rat.     

Relationship between calcium permeability and rectification properties of AMPA receptors in cultured rat hippocampal neurons

Cultured rat hippocampal neurons were classified into three groups on the basis of the functional properties of their AMPA-subtype glutamate receptors. The type I neuron had AMPA receptors with an outwardly rectifying I-V relation and little permeability to Ca2+ whereas the AMPA receptors in the type II neuron were characterized by marked inward rectification and high Ca2+ permeability. In the third type of neuron, the responses of AMPA receptors exhibited intermediate properties in both I-V relation and Ca2+ permeability. We suggest that these intermediate properties in the third type of neuron reflect the coexistence of Ca(2+)-permeant and Ca(2+)-impermeant AMPA receptors.     

Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence

The structures of glycans N-linked to Arabidopsis proteins have been fully identified. From immuno- and affinodetections on blots, chromatography, nuclear magnetic resonance, and glycosidase sequencing data, we show that Arabidopsis proteins are N-glycosylated by high-mannose-type N-glycans from Man5GlcNAc2 to Man9GlcNAc2, and by xylose- and fucose (Fuc)-containing oligosaccharides. However, complex biantenary structures containing the terminal Lewis a epitope recently reported in the literature (A. -C. Fitchette-Lainé, V. Gomord, M. Cabanes, J.-C. Michalski, M. Saint Macary, B. Foucher, B. Cavalier, C. Hawes, P. Lerouge, and L. Faye [1997] Plant J 12: 1411-1417) were not detected. A similar study was done on the Arabidopsis mur1 mutant, which is affected in the biosynthesis of L-Fuc. In this mutant, one-third of the Fuc residues of the xyloglucan has been reported to be replaced by L-galactose (Gal) (E. Zablackis, W.S. York, M. Pauly, S. Hantus, W.D. Reiter, C.C.S. Chapple, P. Albersheim, and A. Darvill [1996] Science 272: 1808-1810). N-linked glycans from the mutant were identified and their structures were compared with those isolated from the wild-type plants. In about 95% of all N-linked glycans from the mur1 plant, L-Fuc residues were absent and were not replaced by another monosaccharide. However, in the remaining 5%, L-Fuc was found to be replaced by a hexose residue. From nuclear magnetic resonance and mass spectrometry data of the mur1 N-glycans, and by analogy with data reported on mur1 xyloglucan, this subpopulation of N-linked glycans was proposed to be L-Gal-containing N-glycans resulting from the replacement of L-Fuc by L-Gal.     

Chemical modification of arginines by 2,3-butanedione and phenylglyoxal causes closure of the mitochondrial permeability transition pore

We have investigated the role of arginine residues in the regulation of the mitochondrial permeability transition pore, a cyclosporin A-sensitive inner membrane channel. Isolated rat liver mitochondria were treated with the arginine-specific chemical reagent 2, 3-butanedione or phenylglyoxal, followed by removal of excess free reagent. After this treatment, mitochondria accumulated Ca2+ normally, but did not undergo permeability transition following depolarization, a condition that normally triggers opening of the permeability transition pore. Inhibition by 2,3-butanedione and phenylglyoxal correlated with matrix pH, suggesting that the relevant arginine(s) are exposed to the matrix aqueous phase. Inhibition by 2,3-butanedione was potentiated by borate and was reversed upon its removal, whereas inhibition by phenylglyoxal was irreversible. Treatment with 2,3-butanedione or phenylglyoxal after induction of the permeability transition by Ca2+ overload resulted in pore closure despite the presence of 0.5 mM Ca2+. At concentrations that were fully effective at inhibiting the permeability transition, these arginine reagents (i) had no effect on the isomerase activity of cyclophilin D and (ii) did not affect the rate of ATP translocation and hydrolysis, as measured by the production of a membrane potential upon ATP addition in the presence of rotenone. We conclude that reaction with 2,3-butanedione and phenylglyoxal results in a stable chemical modification of critical arginine residue(s) located on the matrix side of the inner membrane, which, in turn, strongly favors a closed state of the pore.     

The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death

Mitochondrial alterations including permeability transition (PT) constitute critical events of the apoptotic cascade and are under the control of Bcl-2 related gene products. Here we show that induction of PT is sufficient to activate CPP32-like proteases with DEVDase activity and the associated cleavage of the nuclear DEVDase substrate poly(ADP-ribose) polymerase (PARP). Thus, direct intervention on mitochondria using a ligand of the mitochondrial benzodiazepin receptor or a protonophore causes DEVDase activation. In addition, the DEVDase activation triggered by conventional apoptosis inducers (glucocorticoids or topoisomerase inhibitors) is prevented by inhibitors of PT. The protease inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) completely prevents the activation of DEVDase and PARP cleavage, as well as the manifestation of nuclear apoptosis (chromatin condensation, DNA fragmentation, hypoploidy). In addition, Z-VAD.fmk delays the manifestation of apoptosis-associated changes in cellular redox potentials (hypergeneration of superoxide anion, oxidation of compounds of the inner mitochondrial membrane, depletion of non-oxidized glutathione), as well as the exposure of phosphatidylserine residues in the outer plasma membrane leaflet. Although Z-VAD.fmk retards cytolysis, it is incapable of preventing disruption of the plasma membrane during protracted cell culture (12-24 h), even in conditions in which it completely blocks nuclear apoptosis (chromatin condensation and DNA fragmentation). Electron microscopic analysis confirms that cells treated with PT inducers alone undergo apoptosis, whereas cells kept in identical conditions in the presence of Z-VAD.fmk die from necrosis. These observations are compatible with the hypothesis that PT would be a rate limiting step in both the apoptotic and the necrotic modes of cell death. In contrast, it would be the availability of apoptogenic proteases that would determine the choice between the two death modalities.     

The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition

In several different cell lines, Bcl-2 prevents the induction of apoptosis (DNA fragmentation, PARP cleavage, phosphatidylserine exposure) by the pro-oxidant ter-butylhydroperoxide (t-BHP) but has no cytoprotective effect when apoptosis is induced by the thiol crosslinking agent diazenedicarboxylic acid his 5N,N-dimethylamide (diamide). Both t-BHP and diamide cause a disruption of the mitochondrial transmembrane potential delta psi(m) that is not inhibited by the broad spectrum caspase inhibitor z-VAD.fmk, although z-VAD.fmk does prevent nuclear DNA fragmentation and poly(ADP-ribose) polymerase cleavage in these models. Bcl-2 stabilizes the delta psi(m) of t-BHP-treated cells but has no inhibitory effect on the delta psi(m) collapse induced by diamide. As compared to normal controls, isolated mitochondria from Bcl-2 overexpressing cells are relatively resistant to the induction of delta psi(m) disruption by t-BHP in vitro. Such Bcl-2 overexpressing mitochondria also fail to release apoptosis-inducing factor (AIF) and cytochrome c from the intermembrane space, whereas control mitochondria not overexpressing Bcl-2 do liberate AIF and cytochrome c in response to t-BHP. In contrast, Bcl-2 does not confer protection against diamide-triggered delta psi(m) collapse and the release of AIF and cytochrome c. This indicates that Bcl-2 suppresses the permeability transition (PT) and the associated release of intermembrane proteins induced by t-BHP but not by diamide. To further investigate the mode of action of Bcl-2, semi-purified PT pore complexes were reconstituted in liposomes in a cell-free, organelle-free system. Recombinant Bcl-2 or Bcl-X(L) proteins augment the resistance of reconstituted PT pore complexes to pore opening induced by t-BHP. In contrast, mutated Bcl-2 proteins which have lost their cytoprotective potential also lose their PT-modulatory capacity. Again, Bcl-2 fails to confer protection against diamide in this experimental system. The reconstituted PT pore complex itself cannot release cytochrome c encapsulated into liposomes. Altogether these data suggest that pro-oxidants, thiol-reactive agents, and Bcl-2 can regulate the PT pore complex in a direct fashion, independently from their effects on cytochrome c. Furthermore, our results suggest a strategy for inducing apoptosis in cells overexpressing apoptosis-inhibitory Bcl-2 analogs.     

The permeability transition pore. Control points of a cyclosporin A-sensitive mitochondrial channel involved in cell death

The permeability transition pore (MTP) is a high conductance channel of the mitochondrial inner membrane inhibited by cyclosporin A. While the physiological role of the MTP has not been clarified yet, it is becoming clear that this channel plays an important role in the pathways leading to cell death. The recent demonstrations that the MTP is controlled by the membrane potential, that a variety of physiological and pathological effectors can modulate the threshold voltage at which pore opening occurs, and that surface potential may contribute to pore modulation provide a useful framework to describe the mechanistic aspects of pore function in isolated mitochondria. Here we (i) briefly review the key features of pore regulation, and report our recent progress on the role of oxidants and mitochondrial cyclophilin; and (ii) elaborate on how MTP regulation by cellular pathophysiological effectors (such as cytosolic [Ca2+] transients, oxidative stress, and changes in the concentration of polyamines, nitric oxide, and metabolites of both the sphingomyelin and phospholipase A2 pathways) might take place in vivo. Further definition of the MTP checkpoints should help in the design of specific modulators, and offers great promise for the development of new conceptual and pharmacological tools aimed at therapeutic intervention in pathological conditions where pore opening is a critical event.     

Effects of oxidants and glutamate receptor activation on mitochondrial membrane potential in rat forebrain neurons

Both glutamate and reactive oxygen species have been implicated in excitotoxic neuronal injury, and mitochondria may play a key role in the mediation of this process. In this study, we examined whether glutamate-receptor stimulation and oxidative stress interact to affect the mitochondrial membrane potential (delta psi). We measured delta psi in rat forebrain neurons with the ratiometric fluorescent dye JC-1 by using laser scanning confocal imaging. Intracellular oxidant levels were measured by using the oxidation-sensitive dyes 2',7'-dichlorodihydrofluorescein (DCFH2) and dihydroethidium (DHE). Application of hydrogen peroxide (0.3-3 mM) or 1 mM xanthine/0.06 U/ml xanthine oxidase decreased delta psi in a way that was independent of the presence of extracellular Ca2+ and was not affected by the addition of cyclosporin A, suggesting the presence of either a cyclosporin A-insensitive form of permeability transition, or a separate mechanism. tert-Butylhydroperoxide (730 microM) had less of an effect on delta psi than hydrogen peroxide despite similar effects on intracellular DCFH2 or DHE oxidation. Hydrogen peroxide-, tert-butylhydroperoxide-, and superoxide-enhanced glutamate, but not kainate, induced decreases in delta psi. The combined effect of peroxide or superoxide plus glutamate was Ca2+ dependent and was partially inhibited by cyclosporin A. These results suggest that oxidants and glutamate depolarize mitochondria by different mechanisms, and that oxidative stress may enhance glutamate-mediated mitochondrial depolarization.     

Imaging the permeability pore transition in single mitochondria

In mitochondria the opening of a large proteinaceous pore, the "mitochondrial permeability transition pore" (MTP), is known to occur under conditions of oxidative stress and matrix calcium overload. MTP opening and the resulting cellular energy deprivation have been implicated in processes such as hypoxic cell damage, apoptosis, and neuronal excitotoxicity. Membrane potential (delta psi(m)) in single isolated heart mitochondria was measured by confocal microscopy with a voltage-sensitive fluorescent dye. Measurements in mitochondrial populations revealed a gradual loss of delta psi(m) due to the light-induced generation of free radicals. In contrast, the depolarization in individual mitochondria was fast, sometimes causing marked oscillations of delta psi(m). Rapid depolarizations were accompanied by an increased permeability of the inner mitochondrial membrane to matrix-entrapped calcein (approximately 620 Da), indicating the opening of a large membrane pore. The MTP inhibitor cyclosporin A significantly stabilized delta psi(m) in single mitochondria, thereby slowing the voltage decay in averaged recordings. We conclude that the spontaneous depolarizations were caused by repeated stochastic openings and closings of the transition pore. The data demonstrate a much more dynamic regulation of membrane permeability at the level of a single organelle than predicted from ensemble behavior of mitochondrial populations.