The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release

This study assesses the controversial role of the mitochondrial permeability transition (MPT) in apoptosis. In primary rat hepatocytes expressing an IkappaB superrepressor, tumor necrosis factor alpha (TNFalpha) induced apoptosis as shown by nuclear morphology, DNA ladder formation, and caspase 3 activation. Confocal microscopy showed that TNFalpha induced onset of the MPT and mitochondrial depolarization beginning 9 h after TNFalpha treatment. Initially, depolarization and the MPT occurred in only a subset of mitochondria; however, by 12 h after TNFalpha treatment, virtually all mitochondria were affected. Cyclosporin A (CsA), an inhibitor of the MPT, blocked TNFalpha-mediated apoptosis and cytochrome c release. Caspase 3 activation, cytochrome c release, and apoptotic nuclear morphological changes were induced after onset of the MPT and were prevented by CsA. Depolarization and onset of the MPT were blocked in hepatocytes expressing DeltaFADD, a dominant negative mutant of Fas-associated protein with death domain (FADD), or crmA, a natural serpin inhibitor of caspases. In contrast, Asp-Glu-Val-Asp-cho, an inhibitor of caspase 3, did not block depolarization or onset of the MPT induced by TNFalpha, although it inhibited cell death completely. In conclusion, the MPT is an essential component in the signaling pathway for TNFalpha-induced apoptosis in hepatocytes which is required for both cytochrome c release and cell death and functions downstream of FADD and crmA but upstream of caspase 3.     

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.     

Calcium, free radicals and excitotoxins in neuronal apoptosis

Apoptosis is an active process of cell destruction, characterized by cell shrinkage, chromatin aggregation with extensive genomic fragmentation, and nuclear pyknosis. In contrast, necrosis is characterized by passive cell swelling, intense mitochondrial damage with rapid energy loss, and generalized disruption of internal homeostasis. This swiftly leads to membrane lysis, release of intracellular constituents that evoke a local inflammatory reaction, edema, and injury to the surrounding tissue. In collaboration, our two research laboratories have been defining excitotoxic signals that lead to apoptosis versus necrosis via, among other pathways, Ca2+ signaling mechanisms; this is the subject of this brief review.     

The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins

Early in programmed cell death (apoptosis), mitochondrial membrane permeability increases. This is at least in part due to opening of the permeability transition (PT) pore, a multiprotein complex built up at the contact site between the inner and the outer mitochondrial membranes. The PT pore has been previously implicated in clinically relevant massive cell death induced by toxins, anoxia, reactive oxygen species, and calcium overload. Here we show that PT pore complexes reconstituted in liposomes exhibit a functional behavior comparable with that of the natural PT pore present in intact mitochondria. The PT pore complex is regulated by thiol-reactive agents, calcium, cyclophilin D ligands (cyclosporin A and a nonimmunosuppressive cyclosporin A derivative), ligands of the adenine nucleotide translocator, apoptosis-related endoproteases (caspases), and Bcl-2-like proteins. Although calcium, prooxidants, and several recombinant caspases (caspases 1, 2, 3, 4, and 6) enhance the permeability of PT pore-containing liposomes, recombinant Bcl-2 or Bcl-XL augment the resistance of the reconstituted PT pore complex to pore opening. Mutated Bcl-2 proteins that have lost their cytoprotective potential also lose their PT modulatory capacity. In conclusion, the PT pore complex may constitute a crossroad of apoptosis regulation by caspases and members of the Bcl-2 family.     

The thiol-specific antioxidant enzyme prevents mitochondrial permeability transition. Evidence for the participation of reactive oxygen species in this mechanism

Mitochondrial swelling and membrane protein thiol oxidation associated with mitochondrial permeability transition induced by Ca2+ and inorganic phosphate are inhibited in a dose-dependent manner either by catalase, the thiol-specific antioxidant enzyme (TSA), a protein recently demonstrated to present thiol peroxidase activity, or ebselen, a selenium-containing heterocycle which also possesses thiol peroxidase activity. This inhibition of mitochondrial permeability transition is due to the removal of mitochondrial-generated H2O2 which can easily diffuse to the extramitochondrial space. Whereas ebselen required the presence of reduced glutathione as a reductant to grant its protective effect, TSA was fully reduced by mitochondrial components. Decrease in the oxygen concentration of the reaction medium also inhibits mitochondrial permeabilization and membrane protein thiol oxidation, in a concentration-dependent manner. The results presented in this report confirm that mitochondrial permeability transition induced by Ca2+ and inorganic phosphate is reactive oxygen species-dependent. The possible importance of TSA as an intracellular antioxidant, avoiding the onset of mitochondrial permeability transition, is discussed in the text.     

Inhibition of mitochondrial respiratory chain complex I by TNF results in cytochrome c release, membrane permeability transition, and apoptosis

Mitochondria have been shown to play a key role in apoptosis induction. However, the sequence of changes that occur in the mitochondria in the initial step of apoptosis has not been clearly elucidated. Here, we showed that mitochondrial respiratory chain (MRC) complex I was inhibited during the early phase of TNF- or serum withdrawal apoptosis. The importance of complex I inhibition in apoptosis is also supported by the observation that rotenone, an inhibitor of complex I but not that of other complexes, could induce apoptosis in a manner comparable to TNF. We hypothesized that inhibition of complex I could affect electron flow through other complexes leading to cytochrome c release by an antioxidant-sensitive pathway and caspase 3 activation followed by the induction of membrane permeability transition (MPT). This hypothesis is supported by the following observations: (1) TNF and rotenone induced MPT and cytochrome c release; (2) TNF-induced complex I inhibition was observed prior to cytochrome c release and MPT induction; (3) MPT induction was inhibited by a caspase 3 inhibitor, z-DEVD-CH2F, and an antioxidant pyrrolidine dithiocarbamate (PDTC), whereas cytochrome c release was only inhibited by PDTC. Thus, these results suggest that MRC complex I plays a key role in apoptosis signalings.     

Inhibition of mitochondrial permeability transition by polyamines and magnesium: importance of the number and distribution of electric charges

The protective effects of Mg2+ and various natural and synthetic polyamines on the permeability transition of isolated rat liver mitochondria have been compared. The permeability transition was induced by incubating the mitochondria in a sucrose medium at pH 7.4 in the presence of 100 microM Ca2+ and 1 mM phosphate and was monitored via the release of endogenous Mg2+, sucrose permeation, mitochondria swelling and the fall of transmembrane potential. By all of these parameters (only the traces of delta psi have been reported) spermine fully inhibited the transition at 25 microM concentration, spermidine and caldine at 250 microM and Mg2+ at 500 microM concentration. Both putrescine and dien exhibited only a partial protection even at 2.5 mM concentration. The protective action resulted strictly dependent on the number of the positive charges of each cation. In the case of polyamines this number is also determined by the nature of the methylene carbon chains of each compound.     

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.     

From calcium signaling to cell death: two conformations for the mitochondrial permeability transition pore. Switching from low- to high-conductance state

The permeability transition pore (PTP) is a channel of the inner mitochondrial membrane that appears to operate at the crossroads of two distinct physiological pathways, i.e., the Ca2+ signaling network during the life of the cell, and the effector phase of the apoptotic cascade during Ca2+-dependent cell death. Correspondingly, two open conformations of the PTP can also be observed in isolated organelles. A low-conductance state, that allows the diffusion of small ions like Ca2+, is pH-operated, promoting spontaneous closure of the channel. A high-conductance state, that allows the unselective diffusion of big molecules, stabilizes the channel in the open conformation, disrupting in turn the mitochondrial structure and causing the release of proapoptotic factors. Our current results indicate that switching from low- to high-conductance state is an irreversible process that is strictly dependent on the saturation of the internal Ca2+-binding sites of the PTP. Thus, the high-conductance state of the PTP, which was shown to play a pivotal role in the course of excitotoxic and thapsigargin-induced cell death, might result from a Ca2+-dependent conformational shift of the low-conductance state, normally participating in the regulation of cellular Ca2+ homeostasis as a pH-operated channel. These observations lead us to propose a simple biophysical model of the transition between Ca2+ signaling and Ca2+-dependent apoptosis.     

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.     

Evidence that mitochondrial respiration is a source of potentially toxic oxygen free radicals in intact rabbit hearts subjected to ischemia and reflow

Previous in vitro studies have shown that isolated mitochondria can generate oxygen radicals. However, whether a similar phenomenon can also occur in intact organs is unknown. In the present study, we tested the hypothesis that resumption of mitochondrial respiration upon reperfusion might be a mechanism of oxygen radical formation in postischemic hearts, and that treatment with inhibitors of mitochondrial respiration might prevent this phenomenon. Three groups of Langendorff-perfused rabbit hearts were subjected to 30 min of global ischemia at 37 degrees C, followed by reflow. Throughout ischemia and early reperfusion the hearts received, respectively: (a) 5 mM KCl (controls), (b) 5 mM sodium amobarbital (Amytal, which blocks mitochondrial respiration at Site I, at the level of NADH dehydrogenase), and (c) 5 mM potassium cyanide (to block mitochondrial respiration distally, at the level of cytochrome c oxidase). The hearts were then processed to directly evaluate oxygen radical generation by electron paramagnetic resonance spectroscopy, or to measure oxygen radical-induced membrane lipid peroxidation by malonyl dialdehyde (MDA) content of subcellular fractions. Severity of ischemia, as assessed by 31P-nuclear magnetic resonance measurements of cardiac ATP, phosphocreatine, and pH, was similar in all groups. Oxygen-centered free radical concentration averaged 3.84 +/- 0.54 microM in reperfused control hearts, and it was significantly reduced by Amytal treatment (1.98 +/- 0.26; p < 0.05), but not by KCN (2.58 +/- 0.96 microM; p = not significant (NS)), consistent with oxygen radicals being formed in the mitochondrial respiratory chain at Site I. Membrane lipid peroxidation of reperfused hearts was also reduced by treatment with Amytal, but not with KCN. MDA content of the mitochondrial fraction averaged 0.75 +/- 0.06 nM/mg protein in controls, 0.72 +/- 0.06 in KCN-treated hearts, and 0.54 +/- 0.05 in Amytal-treated hearts (p < 0.05 versus both groups). Similarly, MDA content of lysosomal membrane fraction was 0.64 +/- 0.09 nM/mg protein in controls, 0.79 +/- 0.15 in KCN-treated hearts, and 0.43 +/- 0.06 in Amytal-treated hearts (p < 0.05 versus both groups). Since the effects of Amytal are known to be reversible, in a second series of experiments we investigated whether transient mitochondrial inhibition during the initial 10 min of reperfusion was also associated with beneficial effects on subsequent recovery of cardiac function after wash-out of the drug. At the end of the experiment, recovery of left ventricular end-diastolic and of developed pressure was significantly greater in those hearts that had been treated with Amytal during ischemia and early reflow, as compared to untreated hearts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for a role of oxygen-derived free radicals and protein kinase C in nitrate tolerance

The anti-ischemic effects of organic nitrates are rapidly attenuated due to the development of nitrate tolerance. The mechanisms underlying this phenomenon likely involve several independent factors. As a vasodilator, nitroglycerin activates compensatory neurohumoral mechanisms such as the renin-angiotensin system and increases catecholamine and vasopressin levels, all of which may attenuate its vasodilator potency. Tolerance may be also due to the inability of the vessel to dilate after prolonged treatment with the nitrate. More recent experimental studies have challenged traditional tolerance concepts by demonstrating that tolerance is not associated with sulfhydryl group depletion, reduced nitroglycerin biotransformation, or desensitization of the target enzyme guanylyl-cyclase. Experimental and clinical observations suggest that tolerance may be the consequence of intrinsic abnormalities of the vasculature, including enhanced endothelial production of oxygen-derived free radicals secondary to an activation of NAD(P)H-dependent oxidases and an activation of PKC. Superoxide degrades nitric oxide derived from nitroglycerin (NTG) while C activation causes enhanced sensitivity of the vasculature to circulating neurohormones such as catecholamines, angiotensin II, and serotonin, all of which may compromise the vasodilator potency of NTG. Interestingly, these vascular consequences of in vivo NTG treatment such as superoxide production and PKC activation can be mimicked in vitro by incubating cultured endothelial and smooth muscle cells with angiotensin II. Furthermore, nitrate tolerance and rebound following sudden cessation of prolonged NTG therapy can be prevented by concomitant treatment with high-dose angiotensin-converting enzyme inhibition, angiotensin type 1 receptor blockade, or antioxidants such as hydralazine. Thus one can conclude that neurohumoral counterregulatory mechanisms such as increased circulating levels of angiotensin II may be at least in part responsible for tolerance mechanisms at the cellular level.     

Alcohol and free radicals: from basic research to clinical prospects

An oxidative stress occurs in the liver of rats following various conditions of ethanol administration. The ethanol-inducible cytochrome P450 2E1 plays a key role in its generation, favoured itself by an increase in the "redox-active" fraction of intracellular non-heme iron. Administration of ethanol elicits the generation of the 1-hydroxyethyl radical, which has been identified in vivo. Its reactivity contributes to alcohol-induced immunological disturbances. Liver inflammatory and fibrotic disorders can be reproduced in rats by long-term ethanol administration associated with a high fat diet. The severity of these disorders is correlated to the intensity of the oxidative stress. Some conditions of ethanol administration to rats also elicit an oxidative stress in the myocardium and central nervous system. Through its inhibitory effect on glutamine synthetase activity and resulting excitotoxicity it may contribute to neuronal death and possibly to dependence on alcohol. Disorders related to an oxidative stress were also reported in the serum and erythrocytes as well as in liver biopsies from alcoholic individuals. Their detection may be useful to follow the evolution of alcoholic liver diseases. Supplementation with antioxidants such as vitamin E may be considered in the prevention of severe cellular disorders in individuals consuming large amounts of alcoholic beverages. An increase in free radical production is likely playing a role in the induction of severe cellular damage linked to repeated withdrawals occurring as a result of heavy and sporadic ethanol intake.     

The molecular structure of mitochondrial contact sites. Their role in regulation of energy metabolism and permeability transition

Contact sites between the outer and peripheral inner membrane of mitochondria are involved in protein precursor uptake and energy transfer. Hexokinase and mitochondrial creatine kinase could be attributed by different techniques to the energy transfer contacts. Kinetic analyses suggested a functional interaction between the kinases, outer membrane pore protein, and inner membrane adenylate translocator (ANT). This suggestion was strongly supported by isolation of hexokinase and creatine kinase complexes that were constituted of kinase oligomers, porin and ANT. Phospholipid vesicles carrying reconstituted kinase-porin-ANT complexes enclosed internal ATP in contrast to vesicles containing free porin only. This indicated that unspecific transport through porin was regulated by its interaction with a specific antiporter, ANT. A direct interaction between porin and ANT in the hexokinase complex conferred the reconstituted system with permeability properties reminiscent of the mitochondrial permeability transition (PT) pore. In the creatine kinase complex this interaction between porin and ANT was replaced by contact of both with the kinase octamer. Thus PT-pore-like functions were not observed unless the creatine kinase octamer was dissociated, suggesting that the ANT was locked in the antiporter state by interaction with the octamer. Indeed, reconstituted pure ANT showed PT-pore-like properties concerning Ca2+ sensitivity. However, as cyclophilin was missing, sensitivity against cyclosporin was not observed.     

Alveolar permeability enhancement by oleic acid and related fatty acids: evidence for a calcium-dependent mechanism

Pulmonary exposure to oleic acid (OA) is associated with permeability alterations and cellular damage; however, the exact relationship between these two effects has not been clearly established. Using cultured alveolar epithelial monolayers, we demonstrated that OA and some other fatty acids (< or = 50 microM) can induce permeability changes without detectable cellular damage. At higher concentrations, however, OA caused severe membrane damage and leakage to solute flux. The permeability enhancing effect of OA was observed with both the paracellular marker 3H-mannitol and the lipophilic transcellular indicator 14C-progesterone. While the effect of OA on transcellular permeability may be attributed to its known effect on membrane fluidity, the paracellular promoting effect of OA and its mechanism are not well established. We postulated that OA may increase paracellular permeability through a Ca(2+)-dependent tight junction mechanism. Using dual-excitation fluorescence microscopy, we demonstrated that OA can increase intracellular calcium, [Ca2+]i, in a dose-dependent manner. This effect was transient at low OA concentrations (< or = 50 microM) but became more pronounced and sustained at higher concentrations. Free hydroxyl and unsaturated groups were required for this activation since esterified OA (oleic methyl ester) and stearic acid (a saturated fatty acid with equal chain length) had much reduced effects on both the [Ca2+]i and the permeability alterations. Degree of unsaturation was unimportant since linolenic acid (18:3), linoleic acid (18:2), and OA (18:1) had similar and comparable effects on the two parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton selective substate of the mitochondrial permeability transition pore: regulation by the redox state of the electron transport chain

The permeability transition pore of rat liver mitochondria can be closed by chelating free Ca2+, with respect to the passage of large molecules such as mannitol and sucrose. However, an apparent H+-conducting substate remains open under these conditions, as indicated by the persistence of maximal O2 consumption rates and by the failure to recover a membrane potential. Agents which favor a closed pore, such as cyclosporin A, ADP, Mg2+, or bovine serum albumin, do not close the H+-conducting substate, but it closes spontaneously when respiration becomes limited by the availability of O2. Closure provoked by an O2 limitation requires free Mg2+ in the sub-micromolar concentration range and becomes less efficient with increasing time spent in the presence of free Ca2+. The H+-conducting substate is apparently regulated by the redox status of the electron transport chain, with a reduced form favoring closure. A physical association (or equivalence) between the pore and one of the respiratory chain complexes is supported. These characteristics suggest that the transition is irreversible in vivo, if it involves a small fraction of total mitochondria, and would lead to their elimination and/or replacement by the cell. The implications of this proposal are considered, as they relate to a possible role for the transition in cellular apoptosis and the elimination of mitochondria containing mutated DNA.     

Three-dimensional spectral-spatial EPR imaging of free radicals in the heart: a technique for imaging tissue metabolism and oxygenation

It has been hypothesized that free radical metabolism and oxygenation in living organs and tissues such as the heart may vary over the spatially defined tissue structure. In an effort to study these spatially defined differences, we have developed electron paramagnetic resonance imaging instrumentation enabling the performance of three-dimensional spectral-spatial images of free radicals infused into the heart and large vessels. Using this instrumentation, high-quality three-dimensional spectral-spatial images of isolated perfused rat hearts and rabbit aortas are obtained. In the isolated aorta, it is shown that spatially and spectrally accurate images of the vessel lumen and wall could be obtained in this living vascular tissue. In the isolated rat heart, imaging experiments were performed to determine the kinetics of radical clearance at different spatial locations within the heart during myocardial ischemia. The kinetic data show the existence of regional and transmural differences in myocardial free radical clearance. It is further demonstrated that EPR imaging can be used to noninvasively measure spatially localized oxygen concentrations in the heart. Thus, the technique of spectral-spatial EPR imaging is shown to be a powerful tool in providing spatial information regarding the free radical distribution, metabolism, and tissue oxygenation in living biological organs and tissues.     

Norepinephrine inhibits a toxin resistant Ca2+ current in carotid body glomus cells: evidence for a direct G protein mechanism

The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible hinge: the NH2 half (site II part) containing antigenic site II up to the linear region (amino acids [aa] 253 to 275 encompassing epitope VI [aa 264]) and the COOH half (site III part) containing antigenic site III and the transmembrane and cytoplasmic domains. The structural and immunological roles of each part were investigated by cell transfection and mouse DNA-based immunization with homogeneous and chimeric G genes formed by fusion of the site II part of one genotype (GT) with the site III part of the same or another GT. Various site II-site III combinations between G genes of PV (Pasteur virus strain) rabies (GT1), Mokola (GT3), and EBL1 (European bat lyssavirus 1 [GT5]) viruses were tested. Plasmids pGPV-PV, pGMok-Mok, pGMok-PV, and pGEBL1-PV induced transient expression of correctly transported and folded antigens in neuroblastoma cells and virus-neutralizing antibodies against parental viruses in mice, whereas, pG-PVIII (site III part only) and pGPV-Mok did not. The site III part of PV (GT1) was a strong inducer of T helper cells and was very effective at presenting the site II part of various GTs. Both parts are required for correct folding and transport of chimeric G proteins which have a strong potential value for immunological studies and development of multivalent vaccines. Chimeric plasmid pGEBL1-PV broadens the spectrum of protection against European lyssavirus genotypes (GT1, GT5, and GT6).