Induction of permeability transition in pancreatic mitochondria by cerulein in rats

Hyperstimulation with cholecystokinin analogue cerulein induces a mild edematous pancreatitis in rats. There is evidence for a diminished energy metabolism of acinar cells in this experimental model. The aim of this study was to demonstrate permeability transition of the mitochondrial inner membrane as an early change in mitochondrial function and morphology. As functional parameters, the respiration and membrane potential of mitochondria isolated from control and cerulein-treated animals were measured, and changes in volume and morphology were investigated by swelling experiments and electron microscopy. Five hours after the first injection of cerulein, the leak respiration was nearly doubled and the resting membrane potential was decreased by about 17 mV. These alterations were reversed by extramitochondrial ADP or did not occur when cyclosporin A was added to the mitochondrial incubation. A considerable portion of the mitochondria isolated from cerulein-treated animals was swollen and showed dramatic changes in morphology such as a wrinkled outer membrane and the loss of a distinct cristae structure. These data provide evidence for the opening of the mitochondrial permeability transition pore at an early stage of cerulein induced pancreatitis. This suggests that the permeability transition is an initiating event for lysis of individual mitochondria and the initiation of apoptosis and/or necrosis, as had been shown to occur in this experimental model.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.     

Role of mitochondrial permeability transition pores in mitochondrial autophagy

During autophagy, cells rid themselves of damaged and superfluous mitochondria, as well as other organelles. This activation of mitochondrial turnover could be the result of changes in the physiological state of mitochondria. Confocal microscopy and fluorescence techniques indicate that onset of mitochondrial permeability transition is one such change. The mitochondrial permeability transition is a reversible phenomenon whereby the mitochondrial inner membrane becomes freely permeable to solutes of less than 1500 Da. At onset of the mitochondrial permeability transition, mitochondria depolarize, uncouple, and undergo large amplitude swelling due to opening of permeability transition pores, which may form by aggregation of damaged, misfolded membrane proteins. When injurious cellular stresses occur, cells may protect themselves using autophagy to remove damaged mitochondria and mutated mitochondrial DNA. Ca²⁺ overloading, reactive oxygen and nitrogen species, decreased mitochondrial membrane potential, and oxidation of pyridine nucleotides and glutathione all promote mitochondrial damage and onset of the mitochondrial permeability transition. The mitochondrial permeability transition is also associated with necrosis and apoptosis after a variety of stimuli. This review emphasizes the role of the mitochondrial permeability transition as a key event in mitochondrial autophagy.     

Release of mitochondrial DNA fragments from brain mitochondria of irradiated mice

Recently, we demonstrated that the release of mtDNA fragments from mitochondria occurs as a result of the opening of a non-specific pore in the inner mitochondrial membrane. Here, we show that irradiation of mice stimulates the appearance of mtDNA fragments in the cytosolic fractions of the brain. The fragments of mtDNA were found as early as 1h after irradiation, when no observable alteration of mitochondrial functioning occurred. The involvement of mitochondrial permeability transition in this process is discussed.     

Adenine nucleotide translocator 1 deficiency increases resistance of mouse brain and neurons to excitotoxic insults

The mitochondrial adenine nucleotide translocators (Ant) are bi-functional proteins that transport ADP and ATP across the mitochondrial inner membrane, and regulate the mitochondrial permeability transition pore (mtPTP) which initiates apoptosis. The mouse has three Ant isoforms: Ant1 expressed in heart, muscle, and brain; Ant2 expressed in all tissues but muscle; and Ant4 expressed primarily in testis. Ant1-deficient mice manifest muscle and heart but not brain pathology. Brain Ant1 is induced by stress, while Ant2 is not. Ant1-deficient mice are resistant to death induced by systemic exposure to the brain excitotoxin, kainic acid (KA), and their hippocampal and cortical neurons are significantly more resistant to neuronal death induced by glutamate, KA, and etoposide. The mitochondrial membrane potential of Ant1-deficient brain mitochondria is increased and the mtPTP is more resistance to Ca⁺⁺ induced permeability transition. Hence, Ant1-deficiency may protect the brain from excitotoxicity by desensitizing the mtPTP and by blocking the pro-apoptotic induction of Ant1 by stress.     

The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability

Mitochondria are well known as sites of electron transport and generators of cellular ATP. Mitochondria also appear to be sites of cell survival regulation. In the process of programmed cell death, mediators of apoptosis can be released from mitochondria through disruptions in the outer mitochondrial membrane; these mediators then participate in the activation of caspases and of DNA degradation. Thus the regulation of outer mitochondrial membrane integrity is an important control point for apoptosis. The Bcl-2 family is made up of outer mitochondrial membrane proteins that can regulate cell survival, but the mechanisms by which Bcl-2 family proteins act remain controversial. Most metabolites are permeant to the outer membrane through the voltage dependent anion channel (VDAC), and Bcl-2 family proteins appear to be able to regulate VDAC function. In addition, many Bcl-2 family proteins can form channels in vitro, and some pro-apoptotic members may form multimeric channels large enough to release apoptosis promoting proteins from the intermembrane space. Alternatively, Bcl-2 family proteins have been hypothesized to coordinate the permeability of both the outer and inner mitochondrial membranes through the permeability transition (PT) pore. Increasing evidence suggests that alterations in cellular metabolism can lead to pro-apoptotic changes, including changes in intracellular pH, redox potential and ion transport. By regulating mitochondrial membrane physiology, Bcl-2 proteins also affect mitochondrial energy generation, and thus influence cellular bioenergetics. Cell Death and Differentiation (2000) 7, 1182 - 1191     

BH3 death domain peptide induces cell type-selective mitochondrial outer membrane permeability

The BH3 domain is essential for the release of cytochrome c from mitochondria by pro-apoptotic Bcl-2 family proteins during apoptosis. This study tested the hypothesis that a Bax peptide that includes the BH3 domain can permeabilize the mitochondrial outer membrane and release cytochrome c in the absence of a permeability transition at the mitochondrial inner membrane. BH3 peptide (0.1-60 microm) released cytochrome c from mitochondria in the presence of physiological concentrations of ions in a cell type-selective manner, whereas a BH3 peptide with a single amino acid substitution was ineffective. The release of cytochrome c by BH3 peptide correlated with the presence of endogenous Bax at the mitochondria and its integral membrane insertion. Cytochrome c release was accompanied by adenylate kinase release, was not associated with mitochondrial swelling or substantial loss of electrical potential across the inner membrane, and was unaffected by inhibitors of the permeability transition pore. Cytochrome c release was, however, inhibited by Bcl-2. Although energy-coupled respiration was inhibited after the release of cytochrome c, mitochondria maintained membrane potential in the presence of ATP due to the reversal of the ATP synthase. Overall, results support the hypothesis that BH3 peptide releases cytochrome c by a Bax-dependent process that is independent of the mitochondrial permeability transition pore but regulated by Bcl-2.     

Peroxynitrite and Brain Mitochondria: Evidence for Increased Proton Leak

Abstract: Peroxynitrite has been reported to inhibit irreversibly mitochondrial respiration. Here we show that three sequential additions of 200 µ M peroxynitrite (initial concentration) to rat brain mitochondria (0.2 mg of protein/ml) significantly stimulated state 4 respiration and that further additions progressively inhibited it. No stimulation of state 3 respiration or of the maximal enzymatic activities of the respiratory chain complexes was observed on identical peroxynitrite exposure. State 4 respiration is a consequence of the proton permeability of the mitochondrial inner membrane, and we demonstrate that the peroxynitrite-induced stimulation of state 4 respiration is accompanied by a decreased mitochondrial membrane potential, suggesting an increase in this proton leak. Cyclosporin A did not affect the stimulation, suggesting no involvement of the mitochondrial permeability transition pore. The stimulation was prevented by the lipid-soluble vitamin E analogue Trolox, suggesting the involvement of lipid peroxidation, a proposed mechanism of peroxynitrite cytotoxicity. Lipid peroxidation has previously been reported to increase membrane bilayer proton permeability. The high polyunsaturate content of brain mitochondrial phospholipids may predispose them to peroxidation, and thus a peroxynitrite-induced, lipid peroxidation-mediated increase in proton leak may apply particularly to brain mitochondria and to certain neurodegenerative disorders thought to proceed via mechanisms of mitochondrial oxidative damage.     

Molecular basis of morphological changes in mitochondrial membrane accompanying induction of permeability transition, as revealed by immuno-electron microscopy

The mitochondrial inner membrane typically shows a condensed structure when examined by electron microscopy. However, this typical structure is known to disappear upon induction of the mitochondrial permeability transition (PT). This change in the appearance of the mitochondrial membrane structure that accompanies the induction of PT is thought to reflect changes in the permeability of inner mitochondrial membrane; however, its molecular basis has remained uncertain. In the present study, changes in membrane status were examined by immuno-electron microscopy using antibodies against the voltage-dependent anion channel (VDAC), beta-subunit of F1-ATPase (F1beta), and cytochrome c (cyt. c). In control mitochondria, antibody against VDAC was observed at the rim of the mitochondria, whereas antibodies against F1beta and cytochrome c bound these molecules inside of the mitochondria. However, in PT-induced mitochondria, all three antibodies were observed at the mitochondrial rim. These results strongly suggest that the inner mitochondrial membrane is shoved to the rim region of mitochondria upon induction of mitochondrial PT.     

Heterogeneity of the calcium-induced permeability transition in isolated non-synaptic brain mitochondria

Calcium overload of neural cell mitochondria plays a key role in excitotoxic and ischemic brain injury. This study tested the hypothesis that brain mitochondria consist of subpopulations with differential sensitivity to calcium-induced inner membrane permeability transition, and that this sensitivity is greatly reduced by physiological levels of adenine nucleotides. Isolated non-synaptosomal rat brain mitochondria were incubated in a potassium-based medium in the absence or presence of ATP or ADP. Measurements were made of medium and intramitochondrial free calcium, light scattering, mitochondrial ultrastructure, and the elemental composition of electron-opaque deposits within mitochondria treated with calcium. In the absence of adenine nucleotides, calcium induced a partial decrease in light scattering, accompanied by three distinct ultrastructural morphologies, including large-amplitude swelling, matrix vacuolization and a normal appearance. In the presence of ATP or ADP the mitochondrial calcium uptake capacity was greatly enhanced and calcium induced an increase rather than a decrease in mitochondrial light scattering. Approximately 10% of the mitochondria appeared damaged and the rest contained electron-dense precipitates that contained calcium, as determined by electron-energy loss spectroscopy. These results indicate that brain mitochondria are heterogeneous in their response to calcium. In the absence of adenine nucleotides, approximately 20% of the mitochondrial population exhibit morphological alterations consistent with activation of the permeability transition, but less than 10% exhibit evidence of osmotic swelling and membrane disruption in the presence of ATP or ADP.     

A role for mitochondrial aquaporins in cellular life-and-death decisions?

Mitochondria dominate the process of life-and-death decisions of the cell. Continuous generation of ATP is essential for cell sustenance, but, on the other hand, mitochondria play a central role in the orchestra of events that lead to apoptotic cell death. Changes of mitochondrial volume contribute to the modulation of physiological mitochondrial function, and several ion permeability pathways located in the inner mitochondrial membrane have been implicated in the mediation of physiological swelling-contraction reactions, such as the K+ cycle. However, the channels and transporters involved in these processes have not yet been identified. Osmotic swelling is also one of the fundamental characteristics exhibited by mitochondria in pathological situations, which activates downstream cascades, culminating in apoptosis. The permeability transition pore has long been postulated to be the primary mediator for water movement in mitochondrial swelling during cell death, but its molecular identity remains obscure. Inevitably, accumulating evidence shows that mitochondrial swelling induced by apoptotic stimuli can also occur independently of permeability transition pore activation. Recently, a novel mechanism for osmotic swelling of mitochondria has been described. Aquaporin-8 and -9 channels have been identified in the inner mitochondrial membrane of various tissues, including the kidney, liver, and brain, where they may mediate water transport associated with physiological volume changes, contribute to the transport of metabolic substrates, and/or participate in osmotic swelling induced by apoptotic stimuli. Hence, the recent discovery that aquaporins are expressed in mitochondria opens up new areas of investigation in health and disease.     

Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria

The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.     

Metaxin deficiency alters mitochondrial membrane permeability and leads to resistance to TNF-induced cell killing

Metaxin, a mitochondrial outer membrane protein, is critical for TNF-induced cell death in L929 cells. Its deficiency, caused by retroviral insertion-mediated mutagenesis, renders L929 cells resistance to TNF killing. In this study, we further characterized metaxin deficiency-caused TNF resistance in parallel with Bcl-X_L overexpression-mediated death resistance. We did not find obvious change in mitochondria membrane potential in metaxin-deficient (Met^mut) and Bcl-X_L-overexpressing cells, but we did find an increase in the release rate of the mitochondrial membrane potential probe rhodamine 123 (Rh123) that was preloaded into mitochondria. In addition, overexpression of a function-interfering mutant of metaxin (MetaDTM/C) or Bcl-X_L in MCF-7.3.28 cells also resulted in an acquired resistance to TNF killing and a faster rate of Rh123 release, indicating a close correlation between TNF resistance and higher rates of the dye release from the mitochondria. The release of Rh123 can be controlled by the mitochondrial membrane permeability transition (PT) pore, as targeting an inner membrane component of the PT pore by cyclosporin A (CsA) inhibited Rh123 release. However, metaxin deficiency and Bcl-X_L overexpression apparently affect Rh123 release from a site(s) different from that of CsA, as CsA can overcome their effect. Though both metaxin and Bcl-X_L appear to function on the outer mitochondrial membrane, they do not interact with each other. They may use different mechanisms to increase the permeability of Rh123, since previous studies have suggested that metaxin may influence certain outer membrane porins while Bcl-X_L may form pores on the outer membrane. The alteration of the mitochondrial outer membrane properties by metaxin deficiency and Bcl-X_L overespression, as indicated by a quicker Rh123 release, may be helpful in maintaining mitochondrial integrity.     

BMAP-28, an antibiotic peptide of innate immunity, induces cell death through opening of the mitochondrial permeability transition pore

BMAP-28, a bovine antimicrobial peptide of the cathelicidin family, induces membrane permeabilization and death in human tumor cell lines and in activated, but not resting, human lymphocytes. In addition, we found that BMAP-28 causes depolarization of the inner mitochondrial membrane in single cells and in isolated mitochondria. The effect of the peptide was synergistic with that of Ca(2+) and inhibited by cyclosporine, suggesting that depolarization depends on opening of the mitochondrial permeability transition pore. The occurrence of a permeability transition was investigated on the basis of mitochondrial permeabilization to calcein and cytochrome c release. We show that BMAP-28 permeabilizes mitochondria to entrapped calcein in a cyclosporine-sensitive manner and that it releases cytochrome c in situ. Our results demonstrate that BMAP-28 is an inducer of the mitochondrial permeability transition pore and that its cytotoxic potential depends on its effects on mitochondrial permeability.     

The mitochondrial permeability transition from in vitro artifact to disease target

The mitochondrial permeability transition pore is a high conductance channel whose opening leads to an increase of mitochondrial inner membrane permeability to solutes with molecular masses up to approximately 1500 Da. In this review we trace the rise of the permeability transition pore from the status of in vitro artifact to that of effector mechanism of cell death. We then cover recent results based on genetic inactivation of putative permeability transition pore components, and discuss their meaning for our understanding of pore structure. Finally, we discuss evidence indicating that the permeability transition pore plays a role in pathophysiology, with specific emphasis on in vivo models of disease.     

The voltage-dependent anion channel: an essential player in apoptosis

The increase of outer mitochondrial membrane permeability is a central event in apoptotic cell death, since it releases several apoptogenic factors such as cytochrome c into the cytoplasm that activate the downstream destructive processes. The voltage-dependent anion channel (VDAC or mitochondrial porin) plays an essential role in the increase of mitochondrial membrane permeability, and it is regulated by the Bcl-2 family of proteins via direct interaction. Anti-apoptotic Bcl-2 family members close the VDAC, whereas some (but not all) pro-apoptotic members interact with the VDAC to generate a protein-conducting channel through which cytochrome c can pass. Although the VDAC is directly involved in the apoptotic increase of mitochondrial membrane permeability and is known to be a component of the permeability transition pore complex, its role in the regulation of outer membrane permeability can be separated from the occurrence of permeability transition events, such as mitochondrial swelling followed by rupture of the outer mitochondrial membrane. The VDAC not only interacts with Bcl-2 family members, but also with other proteins, and probably acts as a convergence point for a variety of life-or-death signals.     

Role of the mitochondrial permeability transition and cytochrome C release in hydrogen peroxide-induced apoptosis

We investigated the role of the mitochondrial inner membrane permeability transition and subsequent release of cytochrome c into the cytosol during oxidative stress-evoked apoptosis. Sublethal oxidative stress was applied by treating L929 cells with 0.5 mM H2O2 for 90 min. Then the cellular localization of cytochrome c was examined by immunofluorescent staining and Western blotting. H2O2 treatment caused the permeability transition and pore formation, resulting in membrane depolarization and translocation of cytochrome c from the mitochondria into the cytosol. Pretreatment with cyclosporin A and aristolochic acid (to inhibit pore formation) significantly attenuated a reduction of the mitochondrial membrane potential, as well as signs of apoptosis such as DNA fragmentation, increased plasma membrane permeability, and chromatin condensation. Therefore, exposure to H2O2 caused the opening of permeability transition pores in the inner mitochondrial membrane. An essential role of cytosolic cytochrome c in the execution of apoptosis was demonstrated by its direct microinjection into the cytosol, thus bypassing the need for cytochrome c release from the mitochondrial intermembrane space. Microinjection of cytochrome c caused caspase-dependent apoptosis.     

VDAC electronics: 5. Mechanism and computational model of hexokinase-dependent generation of the outer membrane potential in brain mitochondria

Glycolysis plays a key role in brain energy metabolism. The initial and rate-limiting step of brain glycolysis is catalyzed mainly by hexokinase I (HKI), the majority of which is bound to the mitochondrial outer membrane (MOM), mostly through the mitochondrial inter-membrane contact sites formed by the voltage-dependent anion channel (VDAC, outer membrane) and the adenine nucleotide translocator (ANT, inner membrane). Earlier, we proposed a mechanism for the generation of the mitochondrial outer membrane potential (OMP) as a result of partial application of the inner membrane potential (IMP) to MOM through the electrogenic ANT-VDAC-HK inter-membrane contact sites. According to this previous mechanism, the Gibbs free energy of the hexokinase reaction might modulate the generated OMP (Lemeshko, Biophys. J., 2002). In the present work, a new computational model was developed to perform thermodynamic estimations of the proposed mechanism of IMP-HKI-mediated generation of OMP. The calculated OMP was high enough to electrically regulate MOM permeability for negatively charged metabolites through free, unbound VDACs in MOM. On the other hand, the positive-inside polarity of OMP generated by the IMP-HKI-mediated mechanism is expected to protect mitochondria against elevated concentrations of cytosolic Ca²⁺. This computational analysis suggests that metabolically-dependent generation of OMP in the brain mitochondria, controlled by many factors that modulate VDAC1-HKI interaction, VDAC's voltage-gating properties and permeability, might represent one of the physiological mechanisms of regulation of the brain energy metabolism and of neuronal death resistance, and might also be involved in various neurodegenerative disorders, such as Alzheimer's disease.     

p-Bromophenacyl bromide prevents cumene hydroperoxide-induced mitochondrial permeability transition by inhibiting pyridine nucleotide oxidation

Mitochondrial permeability transition is commonly characterized as a Ca2+ -dependent non-specific increase in inner membrane permeability that results in swelling of mitochondria and their de-energization. In the present study, the effect of different inhibitors of phospholipase A2--p-bromophenacyl bromide, dibucaine, and aristolochic acid--on hydroperoxide-induced permeability transitions in rat liver mitochondria was tested. p-Bromophenacyl bromide completely prevented the hydroperoxide-induced mitochondrial permeability transition while the effects of dibucaine or aristolochic acid were negligible. Organic hydroperoxides added to mitochondria undergo reduction to corresponding alcohols by mitochondrial glutathione peroxidase. This reduction occurs at the expense of GSH which, in turn, can be reduced by glutathione reductase via oxidation of mitochondrial pyridine nucleotides. The latter is considered a prerequisite step for mitochondrial permeability transition. Among all the inhibitors tested, only p-bromophenacyl bromide completely prevented hydroperoxide-induced oxidation of mitochondrial pyridine nucleotides. Interestingly, p-bromophenacyl bromide had no affect on mitochondrial glutathione peroxidase, but reacted with mitochondrial glutathione that prevented pyridine nucleotides from being oxidized. Our data suggest that p-bromophenacyl bromide prevents hydroperoxide-induced deterioration of mitochondria via interaction with glutathione rather than through inhibition of phospholipase A2.     

Control of membrane permeability by external ATP in mammalian cells: isolation of an ATP-resistant variant from Chinese hamster ovary cells

External ATP causes a great increase in the passive permeability of the plasma membrane for phosphorylated metabolites and other small molecules in cultured mammalian cells. We previously demonstrated that in CHO-K1 cells an ATP-dependent permeability change was induced in the presence of a mitochondrial inhibitor (KCN or rotenone), a cytoskeleton-attacking agent (vinblastine) and a calmodulin antagonist (trifluoperazine). These permeability changes were reversible but long exposure, for 30-60 min, to ATP together with a mitochondrial inhibitor significantly reduced the cell viability of the treated cells. Since this cell lysis was shown to be due to the ATP-dependent permeability change, we could isolate several clones resistant to the action of the external ATP from CHO-K1 cells after repeated treatment with ATP and rotenone. In 9.1 cells, one of the isolated clones, little or no ATP-dependent permeability change was observed in the presence of either a mitochondrial inhibitor, vinblastine or trifluoperazine. This CHO variant could be specifically resistant as to the change in membrane permeability induced by external ATP, since the permeabilities for the 2-deoxyglucose and drugs used in the present studies were similar to those in the case of the parent cells. These results suggest that a specific defect or alteration in the plasma membrane is involved in the ATP-dependent permeability change. It is also reported that Mg2+-dependent ATPase activity was found on the cell surface of both CHO-K1 and 9.1 cells, and this activity was shown to be not involved in the permeability change controlled by external ATP.