Exercise training decreases rat heart mitochondria free radical generation but does not prevent Ca2+-induced dysfunction.

Exercise provides cardioprotection against ischemia-reperfusion injury, a process involving mitochondrial reactive oxygen species (ROS) generation and calcium overload. This study tested the hypotheses that isolated mitochondria from hearts of endurance-trained rats have decreased ROS production and improved tolerance against Ca(2+)-induced dysfunction. Male Fischer 344 rats were either sedentary (Sed, n = 8) or endurance exercise trained (ET, n = 11) by running on a treadmill for 16 wk (5 days/wk, 60 min/day, 25 m/min, 6 degrees grade). Mitochondrial oxidative phosphorylation measures were determined with glutamate-malate or succinate as substrates, and H(2)O(2) production and permeability transition pore (PTP) opening were determined with succinate. All assays were carried out in the absence and presence of calcium. In response to 25 and 50 microM CaCl(2), Sed and ET displayed similar decreases in state 3 respiration, respiratory control ratio, and ADP:O ratio. Ca(2+)-induced PTP opening was also similar. However, H(2)O(2) production by ET was lower than Sed (P < 0.05) in the absence of calcium (323 +/- 12 vs. 362 +/- 11 pmol.min(-1).mg protein(-1)) and the presence of 50 microM CaCl(2) (154 +/- 3 vs. 197 +/- 7 pmol.min(-1).mg protein(-1)). Rotenone, which blocks electron flow from succinate to complex 1, reduced H(2)O(2) production and eliminated differences between ET and Sed. Mitochondrial superoxide dismutase and glutathione peroxidase were not affected by exercise. Catalase activity was extremely low but increased 49% in ET (P < 0.05). In conclusion, exercise reduces ROS production in myocardial mitochondria through adaptations specific to complex 1 but does not improve mitochondrial tolerance to calcium overload.     

Resistance to Ca2+-induced opening of the permeability transition pore differs in mitochondria from glycolytic and oxidative muscles

This study determined whether susceptibility to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the slow oxidative soleus (Sol) and superficial white gastrocnemius (WG). Threshold for Ca2+-induced mitochondrial Ca2+ release following PTP opening was determined with a novel approach using permeabilized ghost myofibers. Threshold values for PTP opening were approximately threefold higher in fibers from WG compared with those from Sol (124+/-47 vs. 30.4+/-6.8 pmol Ca2+/mU citrate synthase). A similar phenomenon was also observed in isolated mitochondria (threshold: 121+/-60 vs. 40+/-10 nmol Ca2+/mg protein in WG and Sol), indicating that this was linked to differences in mitochondrial factors between the two muscles. The resistance of WG fibers to PTP opening was not related to the expression of putative protein modulators (cyclophilin D, adenylate nucleotide translocator-1, and voltage-dependent anion channels) or to difference in respiratory properties and occurred despite the fact that production of reactive oxygen species, which promote pore opening, was higher than in the Sol. However, endogenous matrix Ca2+ measured in mitochondria isolated under resting baseline conditions was approximately twofold lower in the WG than in the Sol (56+/-4 vs. 111+/-11 nmol/mg protein), which significantly accounted for the resistance of WG. Together, these results reveal fiber type differences in the sensitivity to Ca2+-induced PTP opening, which may constitute a physiological mechanism to adapt mitochondria to the differences in Ca2+ dynamics between fiber types.     

Properties of Ca(2+) transport in mitochondria of Drosophila melanogaster

We have studied the pathways for Ca(2+) transport in mitochondria of the fruit fly Drosophila melanogaster. We demonstrate the presence of ruthenium red (RR)-sensitive Ca(2+) uptake, of RR-insensitive Ca(2+) release, and of Na(+)-stimulated Ca(2+) release in energized mitochondria, which match well characterized Ca(2+) transport pathways of mammalian mitochondria. Following larger matrix Ca(2+) loading Drosophila mitochondria underwent spontaneous RR-insensitive Ca(2+) release, an event that in mammals is due to opening of the permeability transition pore (PTP). Like the PTP of mammals, Drosophila Ca(2+)-induced Ca(2+) release could be triggered by uncoupler, diamide, and N-ethylmaleimide, indicating the existence of regulatory voltage- and redox-sensitive sites and was inhibited by tetracaine. Unlike PTP-mediated Ca(2+) release in mammals, however, it was (i) insensitive to cyclosporin A, ubiquinone 0, and ADP; (ii) inhibited by P(i), as is the PTP of yeast mitochondria; and (iii) not accompanied by matrix swelling and cytochrome c release even in KCl-based medium. We conclude that Drosophila mitochondria possess a selective Ca(2+) release channel with features intermediate between the PTP of yeast and mammals.     

Calcium-induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43

Carbenoxolone (Cbx), a substance from medicinal licorice, is used for antiinflammatory treatments. We investigated the mechanism of action of Cbx on Ca(2+)-induced permeability transition pore (PTP) opening in synaptic and nonsynaptic rat brain mitochondria (RBM), as well as in rat liver mitochondria (RLM), in an attempt to identify the molecular target of Cbx in mitochondria. Exposure to threshold Ca(2+) load induced PTP opening, as seen by sudden Ca(2+) efflux from the mitochondrial matrix and membrane potential collapse. In synaptic RBM, Cbx (1 μM) facilitated the Ca(2+)-induced, cyclosporine A-sensitive PTP opening, while in nonsynaptic mitochondria the Cbx threshold concentration was higher. A well-known molecular target of Cbx is the connexin (Cx) family, gap junction proteins. Moreover, Cx43 was previously found in heart mitochondria and attributed to the preconditioning mechanism of protection. Thus, we hypothesized that Cx43 might be a target for Cbx in brain mitochondria. For the first time, we detected Cx43 by Western blot in RBM, but Cx43 was absent in RLM. Interestingly, two anti-Cx43 antibodies, directed against amino acids 252 to 270 of rat Cx43, abolished the Cbx-induced enhancement of PTP opening in total RBM and in synaptic mitochondria, but not in RLM. In total RBM and in synaptic mitochondria, PTP caused dephosphorylation of Cx43 at serine 368. The phosphorylation level of serine 368 was decreased at threshold calcium concentration and additionally in the combined presence of Cbx in synaptic mitochondria. In conclusion, active mitochondrial Cx43 appears to counteract the Ca(2+)-induced PTP opening and thus might inhibit the PTP-ensuing mitochondrial demise and cell death. Consequently, we suggest that activity of Cx43 in brain mitochondria represents a novel molecular target for protection.     

Apoptosis driven by IP(3)-linked mitochondrial calcium signals

Increases of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) evoked by calcium mobilizing agonists play a fundamental role in the physiological control of cellular energy metabolism. Here, we report that apoptotic stimuli induce a switch in mitochondrial calcium signalling at the beginning of the apoptotic process by facilitating Ca(2+)-induced opening of the mitochondrial permeability transition pore (PTP). Thus [Ca(2+)](m) signals evoked by addition of large Ca(2+) pulses or, unexpectedly, by IP(3)-mediated cytosolic [Ca(2+)] spikes trigger mitochondrial permeability transition and, in turn, cytochrome c release. IP(3)-induced opening of PTP is dependent on a privileged Ca(2+) signal transmission from IP(3) receptors to mitochondria. After the decay of Ca(2+) spikes, resealing of PTP occurs allowing mitochondrial metabolism to recover, whereas activation of caspases is triggered by cytochrome c released to the cytosol. This organization provides an efficient mechanism to establish caspase activation while mitochondrial metabolism is maintained to meet ATP requirements of apoptotic cell death.     

Changes in calcium-dependent membrane permeability properties in mitochondria of livers from arthritic rats

The involvement of the mitochondrial permeability transition pore (PTP) in the responses of mitochondria from adjuvant-induced arthritic rats to Ca(2+) addition was investigated. The respiratory activity, the Ca(2+)-induced osmotic swelling and the electrophoretic (45)Ca(2+) uptake were evaluated in the absence and in the presence of cyclosporin A (CsA), a well-known inhibitor of the mitochondrial PTP. The Ca(2+)-induced mitochondrial permeability transition (MPT) process occurred in mitochondria from arthritic rats even in the presence of a low Ca(2+) concentration. Whereas in the normal condition, the Ca(2+)-induced uncoupling of oxidative phosphorylation and osmotic swelling was observed in the presence of 10 or 20 microM Ca(2+) concentration, in the arthritic condition, these events occurred at 1.0 microM concentration. In addition, mitochondria from arthritic rats presented an impaired ability to accumulate (45)Ca(2+). All these effects were completely prevented by the administration of CsA. The results of the present study suggest that the higher sensitivity of mitochondria from arthritic rats to Ca(2+)-induced MPT may be an important factor in the pathogenesis of the arthritis disease.     

High-affinity peripheral benzodiazepine receptor ligand, PK11195, regulates protein phosphorylation in rat brain mitochondria under control of Ca(2+)

The effects of PK11195, a high-affinity peripheral benzodiazepine receptor (PBR) ligand, on protein phosphorylation in isolated purified rat brain mitochondria were investigated. The isoquinoline carboxamide ligand of PBR, PK11195, but not the benzodiazepine ligand Ro5-4864, in the nanomolar concentration range strongly increased the phosphorylation of 3.5 and 17 kDa polypeptides. The effect of PK11195 was seen in the presence of elevated Ca(2+) levels (3 x 10(-7) to 10(-6) m), but not at very low Ca(2+) levels (10(-8) to 3 x 10(-8) m). This indicates that PBR involves Ca(2+) as a second messenger in the regulation of protein phosphorylation. Staurosporine, an inhibitor of protein kinase activity was able to suppress the PK11195-promoted protein phosphorylation. When the permeability transition pore (PTP) was opened by threshold Ca(2+) load, phosphorylation of the 3.5-kDa polypeptide was diminished, but strong phosphorylation of the 43-kDa protein was revealed. The 43-kDa protein appears to be a PTP-specific phosphoprotein. If PTP was opened, PK11195 did not increase the phosphorylation of the 3.5 and 17-kDa proteins but suppressed the phosphorylation of the PTP-specific 43-kDa phosphoprotein. The ability of PK11195 to increase the protein phosphorylation, which was lost under Ca(2+)-induced PTP opening, was restored again in the presence of calmidazolium, an antagonist of calmodulin and inhibitor of protein phosphatase PP2B. These results show a tight interaction of PBR with the PTP complex in rat brain mitochondria. In conclusion, a novel function of PBR in brain mitochondria has been revealed, and the PBR-mediated protein phosphorylation has to be considered an important element of the PBR-associated signal transducing cascades in mitochondria and cells.     

Cyclosporin A-insensitive permeability transition in brain mitochondria: inhibition by 2-aminoethoxydiphenyl borate

The mitochondrial permeability transition pore (PTP) may operate as a physiological Ca2+ release mechanism and also contribute to mitochondrial deenergization and release of proapoptotic proteins after pathological stress, e.g. ischemia/reperfusion. Brain mitochondria exhibit unique PTP characteristics, including relative resistance to inhibition by cyclosporin A. In this study, we report that 2-aminoethoxydiphenyl borate blocks Ca2+-induced Ca2+ release in isolated, non-synaptosomal rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+. Ca2+ release was not mediated by the mitochondrial Na+/Ca2+ exchanger or by reversal of the uniporter responsible for energy-dependent Ca2+ uptake. Loss of mitochondrial Ca2+ was accompanied by release of cytochrome c and pyridine nucleotides, indicating an increase in permeability of both the inner and outer mitochondrial membranes. Under these conditions, Ca2+-induced opening of the PTP was not blocked by cyclosporin A, antioxidants, or inhibitors of phospholipase A2 or nitric-oxide synthase but was abolished by pretreatment with bongkrekic acid. These findings indicate that in the presence of adenine nucleotides and Mg2+,Ca2+-induced PTP in non-synaptosomal brain mitochondria exhibits a unique pattern of sensitivity to inhibitors and is particularly responsive to 2-aminoethoxydiphenyl borate.     

Increased mitochondrial calcium coexists with decreased reperfusion injury in postconditioned (but not preconditioned) hearts

Ca(2+) is the main trigger for mitochondrial permeability transition pore opening, which plays a key role in cardiomyocyte death after ischemia-reperfusion. We investigated whether a reduced accumulation of mitochondrial Ca(2+) might explain the attenuation of lethal reperfusion injury by postconditioning. Anesthetized New Zealand White rabbits underwent 30 min of ischemia, followed by either 240 (infarct size protocol) or 60 (mitochondria protocol) min of reperfusion. They received either no intervention (control), preconditioning by 5-min ischemia and 5-min reperfusion, postconditioning by four cycles of 1-min reperfusion and 1-min ischemia at the onset of reflow, or pharmacological inhibition of the transition pore opening by N-methyl-4-isoleucine-cyclosporin (NIM811; 5 mg/kg iv) given at reperfusion. Area at risk and infarct size were assessed by blue dye injection and triphenyltetrazolium chloride staining. Mitochondria were isolated from the risk region for measurement of 1) Ca(2+) retention capacity (CRC), and 2) mitochondrial content of total (atomic absorption spectrometry) and ionized (potentiometric technique) calcium concentration. CRC averaged 0.73 +/- 0.16 in control vs. 4.23 +/- 0.17 mug Ca(2+)/mg proteins in shams (P < 0.05). Postconditioning, preconditioning, or NIM811 significantly increased CRC (P < 0.05 vs. control). In the control group, total and free mitochondrial calcium significantly increased to 2.39 +/- 0.43 and 0.61 +/- 0.10, respectively, vs. 1.42 +/- 0.09 and 0.16 +/- 0.01 mug Ca(2+)/mg in sham (P < 0.05). Surprisingly, whereas total and ionized mitochondrial Ca(2+) decreased in preconditioning, it significantly increased in postconditioning and NIM811 groups. These data suggest that retention of calcium within mitochondria may explain the decreased reperfusion injury in postconditioned (but not preconditioned) hearts.     

Role of calcium and superoxide dismutase in sensitizing mitochondria to peroxynitrite-induced permeability transition

The mitochondrial permeability transition pore (PTP) is a membrane protein complex assembled and opened in response to Ca(2+) and oxidants such as peroxynitrite (ONOO(-)). Opening the PTP is mechanistically linked to the release of cytochrome c, which participates in downstream apoptotic signaling. However, the molecular basis of the synergistic interactions between oxidants and Ca(2+) in promoting the PTP are poorly understood and are addressed in the present study. In isolated rat liver mitochondria, it was found that the timing of the exposure of the isolated rat liver mitochondria to Ca(2+) was a critical factor in determining the impact of ONOO(-) on PTP. Specifically, addition of Ca(2+) alone, or ONOO(-) and then Ca(2+), elicited similar low levels of PTP opening, whereas ONOO(-) alone was ineffective. In contrast, addition of Ca(2+) and then ONOO(-) induced extensive PTP opening and cytochrome c release. Interestingly, Cu/Zn-superoxide dismutase enhanced pore opening through a mechanism independent of its catalytic activity. These data are consistent with a model in which Ca(2+) reveals a molecular target that is now reactive with ONOO(-). As a test of this hypothesis, tyrosine nitration was determined in mitochondria exposed to ONOO(-) alone or to Ca(2+) and then ONOO(-), and mitochondrial membrane proteins were analyzed using proteomics. These studies suggest protein targets revealed by Ca(2+) include dehydrogenases and CoA - containing enzymes. These data are discussed in the context of the role of mitochondria, Ca(2+), and ONOO(-) in apoptotic signaling.     

Complex contribution of cyclophilin D to Ca2+-induced permeability transition in brain mitochondria, with relation to the bioenergetic state

Cyclophilin D (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca(2+), and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca(2+) caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca(2+) in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca(2+) deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli.     

Rotenone inhibits the mitochondrial permeability transition-induced cell death in U937 and KB cells

The permeability transition pore (PTP) is a mitochondrial inner membrane Ca(2+)-sensitive channel that plays a key role in different models of cell death. Because functional links between the PTP and the respiratory chain complex I have been reported, we have investigated the effects of rotenone on PTP regulation in U937 and KB cells. We show that rotenone was more potent than cyclosporin A at inhibiting Ca(2+)-induced PTP opening in digitonin-permeabilized cells energized with succinate. Consistent with PTP regulation by electron flux through complex I, the effect of rotenone persisted after oxidation of pyridine nucleotides by duroquinone. tert-butyl hydroperoxide induced PTP opening in intact cells (as shown by mitochondrial permeabilization to calcein and cobalt), as well as cytochrome c release and cell death. All these events were prevented by rotenone or cyclosporin A. These data demonstrate that respiratory chain complex I plays a key role in PTP regulation in vivo and confirm the importance of PTP opening in the commitment to cell death.     

The participation of pyridine nucleotides redox state and reactive oxygen in the fatty acid-induced permeability transition in rat liver mitochondria

The ability of low concentrations (5-15 microM) of long-chain fatty acids to open the permeability transition pore (PTP) in Ca(2+)-loaded mitochondria has been ascribed to their protonophoric effect mediated by mitochondrial anion carriers, as well as to a direct interaction with the pore assembly [M.R. Wieckowski and L. Wojtczak, FEBS Lett. 423 (1998) 339-342]. Here, we have compared the PTP opening ability of arachidonic acid (AA) with that of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) at concentrations that cause similar quantitative dissipation of the membrane potential (DeltaPsi) in Ca(2+)-loaded rat liver mitochondria respiring on succinate. The initial protonophoric effects of AA and FCCP were only slightly modified by carboxyatractyloside and were followed by PTP opening, as indicated by a second phase of DeltaPsi disruption sensitive to EGTA, ADP, dithiothreitol and cyclosporin A. This second phase of DeltaPsi dissipation could also be prevented by rotenone or NAD(P)H-linked substrates which decrease the pyridine nucleotide (PN) oxidation that follows the stimulation of oxygen consumption induced by AA or FCCP. These results suggest that, under the experimental conditions used here, the PTP opening induced by AA or FCCP was a consequence of PN oxidation. Exogenous catalase also inhibited both AA- and FCCP-induced PTP opening. These results indicate that a condition of oxidative stress associated with the oxidized state of PN underlies membrane protein thiol oxidation and PTP opening.     

The peripheral-type benzodiazepine receptor is involved in control of Ca2+-induced permeability transition pore opening in rat brain mitochondria

The peripheral-type benzodiazepine receptor (PBR) is an 18 kDa mitochondrial membrane protein with still elusive function in cell death. Here, we studied whether PBR is involved in Ca2+-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria (RBM). PTP opening is important in mitochondrial events leading to programmed cell death. Immunoblots revealed a single 18 kDa anti-PBR antibody-immunoreactive band in purified RBM. Adenine nucleotide transporter, a key PTP component, was found in the PBR-immunoprecipitate. In isolated intact RBM, addition of a specific anti-PBR antibody [H. Li, Z. Yao, B. Degenhardt, G. Teper, V. Papadopoulos, Cholesterol binding at the cholesterol recognition/interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 1267-1272] delayed Ca2+-induced dissipation of membrane potential (psi(m)) and diminished cyclosporine A-sensitive Ca2+ efflux, which are both indicative for the suppression of PTP opening. Moreover, anti-PBR antibody caused partial retention of Ca2+ in the mitochondrial matrix in spite of psi(m) dissipation, and reduced activation of respiratory rate at Ca2+-induced PTP opening. A release of pro-apoptotic factors, AIF and cytochrome c, from RBM was shown at threshold Ca2+ load. Anti-PBR antibody blocked the release of AIF but did not affect the cytochrome c release. Addition of ATP was able to initiate PTP closing, associated with psi(m) restoration and Ca2+ re-accumulation. At the same time mitochondrial protein phosphorylation (incorporation of 32P from [gamma-32P]ATP) occurred and anti-PBR antibody was able to inhibit phosphorylation of these proteins. The endogenous PBR ligand, protoporphyrin IX, facilitated PTP opening and phosphorylation of the mitochondrial proteins, thus, inducing effects opposite to anti-PBR antibody. This study provides evidence for PBR involvement in PTP opening, controlling the Ca2+-induced Ca2+ efflux, and AIF release from mitochondria, important stages of initiation of programmed cell death.     

Calcium release from the rat liver mitochondria during collapse of the membrane potential

Ca(2+)-release from rat liver mitochondria after protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP)-induced membrane depolarisation is studied. It is shown that the release of calcium is accompanied by an increase of the inner mitochondrial membrane permeability as the result of the opening of permeability transition pore (PTP). Calcium is released from mitochondria through the uniporter working in reverse mode and also by PTP mechanism which accounts for ruthenium red (RR)-insensitive component of total. Ca(2+)-release. Unlike Ca2+, the strontium release from the mitochondria is completely sensitive to RR, specific uniporter blocker, which shows the absence of rapid Sr(2+)-efflux mechanisms other than uniporter of bivalent cations. The data obtained also give an evidence that the lifetime of the open state of the pore is limited, and barrier properties of the mitochondrial membrane are restored after the closure of the pore.     

H2O2 generation is decreased by calcium in isolated brain mitochondria

Release of H(2)O(2) in response to Ca(2+) loads (1-100 microM) was investigated using Amplex red fluorescent assay in isolated guinea-pig brain mitochondria respiring on glutamate plus malate or succinate. In mitochondria challenged with Ca(2+) (10 microM), in the absence of adenine nucleotides and inhibitors of the respiratory chain, the rate of H(2)O(2) release, taken as an indication of H(2)O(2) production, was decreased by 21.8+/-1.6% in the presence of NADH-linked substrates and by 86.5+/-1.8% with succinate. Parallel with this, a Ca(2+)-induced loss in NAD(P)H fluorescence, sustained depolarization, decrease in fluorescent light scattering signal and in calcein fluorescence were detected indicating an increased permeability and swelling of mitochondria, which were prevented by ADP (2 mM). In the presence of ADP H(2)O(2) release from mitochondria was decreased, but Ca(2+) no longer influenced the generation of H(2)O(2). We suggest that the decreased H(2)O(2) generation induced by Ca(2+) is related to depolarization and NAD(P)H loss resulting from a non-specific permeability increase of the mitochondrial inner membrane.     

Ca(2+)-induced high amplitude swelling and cytochrome c release from wheat (Triticum aestivum L.) mitochondria under anoxic stress

Under stress conditions, mitochondria sense metabolic changes, e.g. in pH, cytoplasmic Ca(2+), energy status, and reactive oxygen species (ROS), and respond by induction of the permeability transition pore (PTP) and by releasing cytochrome c, thus initiating the programmed cell death (PCD) cascade in animal cells. In plant cells, the presence of all the components of the cascade has not yet been shown. In wheat (Triticum aestivum L.) root mitochondria, the onset of anoxia caused rapid dissipation of the inner membrane potential, initial shrinkage of the mitochondrial matrix and the release of previously accumulated Ca(2+). Ca(2+) uptake by mitochondria was dependent on the presence of inorganic phosphate. Treatment of mitochondria with high micromolar and millimolar Ca(2+) (but not Mg(2+)) concentrations induced high amplitude swelling, indicative of PTP opening. Alterations in mitochondrial volume were confirmed by transmission electron microscopy. Mitochondrial swelling was not sensitive to cyclosporin A (CsA)-an inhibitor of mammalian PTP. The release of cytochrome c was monitored under lack of oxygen. Anoxia alone failed to induce cytochrome c release from mitochondria. Oxygen deprivation and Ca(2+) ions together caused cytochrome c release in a CsA-insensitive manner. This process correlated positively with Ca(2+) concentration and required Ca(2+) localization in the mitochondrial matrix. Functional characteristics of wheat root mitochondria, such as membrane potential, Ca(2+) transport, swelling, and cytochrome c release under lack of oxygen are discussed in relation to PCD.     

Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore by the proton electrochemical gradient. Evidence that the pore can be opened by membrane depolarization.

This paper reports an investigation on the relationship between the proton electrochemical gradient (delta mu H+) and the cyclosporin A-sensitive permeability transition pore (PTP) in rat liver mitochondria. Using the SH group cross-linker phenylarsine oxide as the inducer, we show that both matrix pH and the membrane potential can modulate the process of PTP induction independently of Ca2+. We find that membrane depolarization induces the PTP per se when pHi is above 7.0, while at acidic matrix pH values PTP induction is effectively prevented. Since Ca2+ uptake leads to major modifications of the delta mu H+ (i.e. matrix alkalinization and membrane depolarization), we have explored the possibility that the Ca(2+)-induced changes of the delta mu H+ may contribute to PTP induction by Ca2+. Our data in mitochondria treated with Ca2+ plus N-ethylmaleimide and Ca2+ plus phosphate show that membrane depolarization is a powerful inducer of the PTP. Taken together, our observations indicate that the PTP can be controlled directly by the delta mu H+ both in the absence and presence of Ca2+, and suggest that a collapse of the membrane potential may be the cause rather than the consequence of PTP induction under many experimental conditions. Thus, many inducers may converge on dissipation of the membrane potential component of the delta mu H+ by a variety of mechanisms.     

Opening of the mitochondrial permeability transition pore induces reactive oxygen species production at the level of the respiratory chain complex I

We have investigated the consequences of permeability transition pore (PTP) opening on the rate of production of reactive oxygen species in isolated rat liver mitochondria. We found that PTP opening fully inhibited H(2)O(2) production when mitochondria were energized both with complex I or II substrates. Because PTP opening led to mitochondrial pyridine nucleotide depletion, H(2)O(2) production was measured again in the presence of various amounts of NADH. PTP opening-induced H(2)O(2) production began when NADH concentration was higher than 50 microm and reached a maximum at over 300 microm. At such concentrations of NADH, the maximal H(2)O(2) production was 4-fold higher than that observed when mitochondria were permeabilized with the channel-forming antibiotic alamethicin, indicating that the PTP opening-induced H(2)O(2) production was not due to antioxidant depletion. Moreover, PTP opening decreased rotenone-sensitive NADH ubiquinone reductase activity, whereas it did not affect the NADH FeCN reductase activity. We conclude that PTP opening induces a specific conformational change of complex I that (i) dramatically increases H(2)O(2) production so long as electrons are provided to complex I, and (ii) inhibits the physiological pathway of electrons inside complex I. These data allowed the identification of a novel consequence of permeability transition that may partly account for the mechanism by which PTP opening induces cell death.