Induced Permeability Transition Pore Opening Research Articles

Cysteine 202 of cyclophilin D is a site of multiple post-translational modifications and plays a role in cardioprotection.

Cyclophilin-D is a well-known regulator of the mitochondrial permeability transition pore (PTP), the main effector of cardiac ischaemia/reperfusion injury. However, the binding of CypD to the PTP is poorly understood. Cysteine 202 (C202) of CypD is highly conserved among species and can undergo redox-sensitive post-translational modifications. We investigated whether C202 regulates the opening of PTP. We developed a knock-in mouse model using CRISPR where CypD-C202 was mutated to a serine (C202S). Infarct size is reduced in CypD-C202S Langendorff perfused hearts compared to wild type (WT). Cardiac mitochondria from CypD-C202S mice also have higher calcium retention capacity compared to WT. Therefore, we hypothesized that oxidation of C202 might target CypD to the PTP. Indeed, isolated cardiac mitochondria subjected to oxidative stress exhibit less binding of CypD-C202S to the proposed PTP component F1F0-ATP-synthase. We previously found C202 to be S-nitrosylated in ischaemic preconditioning. Cysteine residues can also undergo S-acylation, and C202 matched an S-acylation motif. S-acylation of CypD-C202 was assessed using a resin-assisted capture (Acyl-RAC). WT hearts are abundantly S-acylated on CypD C202 under baseline conditions indicating that S-acylation on C202 per se does not lead to PTP opening. CypD C202S knock-in hearts are protected from ischaemia/reperfusion injury suggesting further that lack of CypD S-acylation at C202 is not detrimental (when C is mutated to S) and does not induce PTP opening. However, we find that ischaemia leads to de-acylation of C202 and that calcium overload in isolated mitochondria promotes de-acylation of CypD. Furthermore, a high bolus of calcium in WT cardiac mitochondria displaces CypD from its physiological binding partners and possibly renders it available for interaction with the PTP. Taken together the data suggest that with ischaemia CypD is de-acylated at C202 allowing the free cysteine residue to undergo oxidation during the first minutes of reperfusion which in turn targets it to the PTP.

The mitomiR/Bcl-2 axis affects mitochondrial function and autophagic vacuole formation in senescent endothelial cells

During senescence, cells undergo distinctive biochemical and morphological changes and become dysfunctional. MiRNAs are involved in the senescence process and specific miRNAs can localize to mitochondria (mitomiRs). We hypothesized that part of the typical alterations of senescence may depends on mitomiRs deregulation. Therefore, we thoroughly explored the phenotype of human endothelial cells undergoing replicative senescence (sHUVECs) and observed elongated/branched mitochondria, accumulation of autophagic vacuoles (AVs), increased ROS and IL-1β production and reduced expression of Bcl-2 compared to younger cells (yHUVECs). Despite these pro-apoptotic features, sHUVECs are more resistant to serum deprivation, conceivably due to development of pro-survival strategies such as upregulation of Bcl-xL and Survivin. We demonstrate that mitomiR-181a, -34a, and -146a, are overexpressed and localize to mitochondria in sHUVECs compared with yHUVECs and that they: i) down-regulate Bcl-2, ii) induce permeability transition pore opening and activation of caspase-1 and 3, iii) affect sensitivity to apoptosis and iv) promote the conversion of LC3-I to LC3-II. Overall, we document for the first time that some mitomiRs can act as mediators of the multiple but functionally linked biochemical and morphological changes that characterize aging cells and that they can promote different cellular outcomes according to the senescence status of the cell.

\u03b1-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson\u2019s disease

Protein aggregation causes α-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric α-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric α-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate α-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of α-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson’s disease.

Mersalyl prevents the Tl+-induced permeability transition pore opening in the inner membrane of Ca2+-loaded rat liver mitochondria

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 resulted in the Tl+-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca2+ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl+-induced MPTP opening in the inner membrane of Ca2+-loaded rat liver mitochondria.

Mevalonolactone disrupts mitochondrial functions and induces permeability transition pore opening in rat brain mitochondria: Implications for the pathogenesis of mevalonic aciduria

Mevalonic aciduria (MVA) is caused by severe deficiency of mevalonic kinase activity leading to tissue accumulation and high urinary excretion of mevalonic acid (MA) and mevalonolactone (ML). Patients usually present severe neurologic symptoms whose pathophysiology is poorly known. Here, we tested the hypothesis that the major accumulating metabolites are toxic by investigating the in vitro effects of MA and ML on important mitochondrial functions in rat brain and liver mitochondria. ML, but not MA, markedly decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and the capacity to retain Ca2+ in the brain, besides inducing mitochondrial swelling. These biochemical alterations were totally prevented by the classical inhibitors of mitochondrial permeability transition (MPT) cyclosporine A and ADP, as well as by ruthenium red in Ca2+-loaded mitochondria, indicating the involvement of MPT and an important role for mitochondrial Ca2+ in these effects. ML also induced lipid peroxidation and markedly inhibited aconitase activity, an enzyme that is highly susceptible to free radical attack, in brain mitochondrial fractions, indicating that lipid and protein oxidative damage may underlie some of ML-induced deleterious effects including MTP induction. In contrast, ML and MA did not compromise oxidative phosphorylation in the brain and all mitochondrial functions evaluated in the liver, evidencing a selective toxicity of ML towards the central nervous system. Our present study provides for the first time evidence that ML impairs essential brain mitochondrial functions with the involvement of MPT pore opening. It is therefore presumed that disturbance of brain mitochondrial homeostasis possibly contributes to the neurologic symptoms in MVA.

FOF1-ATP Synthase Dimers and The Mitochondrial Permeability Transition Pore from Yeast to Mammals

The mitochondrial permeability transition pore (PTP) is a voltage-dependent channel that allows solutes of molecular mass ≤ 1.5 kDa to equilibrate across the inner membrane. Matrix Ca2+ accumulation, together with Pi and a set of compounds collectively called “inducers”, is necessary to induce PTP opening. In mammals cyclosporin (Cs) A desensitizes the PTP through its binding to cyclophilin D, a matrix protein that facilitates PTP opening. Yeast and Drosophila mitochondria also possess Ca2+-activated channels which, at variance from the mammalian PTP, are insensitive to CsA and inhibited rather than activated by Pi. We show (i) that the permeability properties of the Drosophila channel, which displays selectivity toward Ca2+ and H+, are not modified by expression of human cyclophilin D; and (ii) that, in keeping with our recent demonstration that the mammalian PTP forms from dimers of the FOF1-ATP synthase, Ca2+-dependent currents can be elicited in reconstitution experiments with purified dimers of the yeast enzyme. We are currently investigating the effect of genetic ablation of FOF1-ATP synthase subunits that mediate dimerization on PTP opening in yeast, Drosophila and mammalian mitochondria. Our findings suggest that the PTP-forming ability of FOF1-ATP synthase has been conserved in evolution, and that the channels display species-specific features.

Elevated skeletal muscle apoptotic signaling following glutathione depletion

Oxidative stress has a well-established role in numerous intracellular signaling pathways, including apoptosis. Glutathione is an important cellular antioxidant and is the most abundant low molecular weight thiol in the cell. Although previous work has shown a link between glutathione and apoptosis, this relationship has not been defined in skeletal muscle. The present investigation examined the effect of glutathione depletion on skeletal muscle apoptotic signaling, and mitochondrial apoptotic-susceptibility. Administration of L: -buthionine-[S,R]-sulfoximine (BSO; 30mM in drinking water for 10days) caused glutathione depletion in whole muscle and isolated mitochondria, as well as elevated muscle catalase protein content and reactive oxygen species (ROS) generation. Glutathione depletion was associated with elevated DNA fragmentation, mitochondrial Bax levels, Poly(ADP-ribose) polymerase (PARP) cleavage, and calpain activity; however, caspase-3, -8, and -9 activity were not altered. BSO administration was also associated with higher cytosolic and nuclear protein levels of apoptosis-inducing factor (AIF), but not cytochrome c, second mitochondria-derived activator of caspase (Smac), or endonuclease G (EndoG). In addition, isolated mitochondria from BSO animals demonstrated significantly lower membrane potential, increased Ca(2+)-induced permeability transition pore opening, and greater basal and ROS-induced AIF and cytochrome c release. These results demonstrate that glutathione depletion in skeletal muscle increases caspase-independent signaling, as well as augments mitochondrial-associated apoptotic events to subsequent cell death stimuli.

Dietary Supplementation with Docosahexanoic Acid or Arachidonic Acid Delay Ca2+ ‐Induced Permeability Transition Pore Opening in Cardiac Mitochondria

Supplementation with docosahexanoic acid (DHA, commonly found in fish oil) in clinically relevant amounts causes a 2-fold increase in DHA in cardiac phospholipids, and delays Ca2+-induced mitochondrial permeability transition pore (MPTP) opening. The mechanism for this effect is not clear, however the increase in DHA is accompanied by a decrease in the ù-6 PUFA arachidonic acid (ARA), which could be responsible for the effects on the MPTP. We determined the role of ARA on MPTP opening by feeding rats DHA, ARA or DHA+ARA. Male Wistar rats were fed either a los DHA+AHA diet(CTRL), a diet containing DHA or ARA at 2.5% of energy intake, or both DHA and ARA at 2.5% each, for 10 wks. Cardiac mitochondria were isolated. All three diets profoundly altered the phospholipid composition of mitochondria compared to CTRL. Both the DHA and ARA groups delayed Ca2+-induced MPTP opening vs. CTRL (p<0.05) while DHA+ARA increased sensitivity to MPTP opening (p<0.05 vs CTRL, DHA or ARA). Furthermore, both diets containing ARA significantly increased the pro-inflammatory marker TBX B2 by 2- to 3-fold compared to CTRL and DHA diets (p<0.05). DHA+ARA also increased lipid peroxidation compared to ARA alone (p=0.008) and to CTRL (p=0.034). This suggests that the effect of DHA on MPTP is not due to depletion of ARA. DHA+ARA showed no added benefit, but rather sensitized MPTP to opening, suggesting that a very large dose of PUFA might be detrimental.

Protection of pancreatic INS-1 β-cells from glucose- and fructose-induced cell death by inhibiting mitochondrial permeability transition with cyclosporin A or metformin

Hyperglycemia is detrimental to β-cell viability, playing a major role in the progression of β-cell loss in diabetes mellitus. The permeability transition pore (PTP) is a mitochondrial channel involved in cell death. Recent evidence suggests that PTP inhibitors prevent hyperglycemia-induced cell death in human endothelial cells. In this work, we have examined the involvement of PTP opening in INS-1 cell death induced by high levels of glucose or fructose. PTP regulation was studied by measuring the calcium retention capacity in permeabilized INS-1 cells and by confocal microscopy in intact INS-1 cells. Cell death was analyzed by flow cytometry. We first reported that metformin and cyclosporin A (CsA) prevented Ca2+-induced PTP opening in permeabilized and intact INS-1 cells. We then showed that incubation of INS-1 cells in the presence of 30 mM glucose or 2.5 mM fructose induced PTP opening and led to cell death. As both metformin and CsA prevented glucose- and fructose- induced PTP opening, and hampered glucose- and fructose- induced cell death, we conclude that PTP opening is involved in high glucose- and high fructose- induced INS-1 cell death. We therefore suggest that preventing PTP opening might be a new approach to preserve β-cell viability.

Cyclophilin‐D is dispensable for atrophy and mitochondrial apoptotic signalling in denervated muscle

In the present study, we specifically determined whether the regulatory protein cyclophilin-D (CypD), and by extension opening of the permeability transition pore (PTP), is involved in the activation of mitochondria-derived apoptotic signalling previously described in skeletal muscle following loss of innervation. For this purpose, CypD-defficient (CypD-KO) mice and their littermate controls were submitted to unilateral sciatic nerve transection, and mitochondrial resistance to Ca2+-induced opening of the PTP, and muscle apoptotic signalling were investigated 14 days post-surgery. Denervation caused atrophy, facilitated Ca2+-induced opening of the PTP in vitro in permeabilized muscle fibres, and activation of the apoptotic proteolytic cascade in the whole muscle of both mouse strains. In CypD-KO mice, mitochondrial resistance to Ca2+-induced PTP opening was greater than in WT mice, in both the normal and the denervated state, indicating that lack of CypD desensitized to PTP opening. However, denervation in CypD-KO mice still resulted in a facilitation of PTP opening compared to normally innervated contralateral muscle, indicating that in vitro additional factors could poise mitochondria from denervated muscle toward PTP opening. At the whole muscle level, lack of CypD, despite conferring greater resistance to PTP opening, did not protect against atrophy, release of mitochondrial pro-apoptotic factors and activation of caspases following denervation. Altogether, these results provide direct evidence that CypD-dependent PTP opening is dispensable for atrophy and apoptotic signalling in skeletal muscle following denervation.

Influence of aging on membrane permeability transition in brain mitochondria

The mitochondrial inner membrane permeability transition (MPT) plays an important role in the pathophysiology of acute disorders of the central nervous systems, including ischemic and traumatic brain injury, and possibly in neurodegenerative diseases. Opening of the permeability transition pore (PTP) by a combination of abnormally elevated intramitochondrial Ca2+ and oxidative stress induces the collapse of transmembrane ion gradients, resulting in membrane depolarization and uncoupling of oxidative phosphorylation. This loss of ATP synthesis eventually results in cellular metabolic failure and necrotic cell death. Drugs, e.g., cyclosporin A, can inhibit the permeability transition through their interaction with the mitochondria-specific protein, cyclophilin D, and demonstrate neuroprotection in several animal models. These characteristics of the MPT were developed almost exclusively from experiments performed with young, mature rodents whereas the neuropathologies associated with the MPT are most prevalent in the elderly population. Some evidence indicates that the sensitivity of mitochondria to Ca2+-induced PTP opening is greater in the aged compared to the young mature brain; however, the basis for this difference is unknown. Based on knowledge of factors that regulate the MPT and on other comparisons between cells and mitochondria from young and old animals, several features may be important. These aging-related features include impaired neuronal Ca2+ homeostasis, increased oxidative stress, increased cyclophilin D protein levels, oxidative modification of the adenine nucleotide translocase and of cardiolipin, and changes in the levels of anti-death mitochondrial proteins, e.g., Bcl-2. The influence of aging on both the contribution of the MPT to neuropathology and the neuroprotective efficacy of MPT inhibitors is a substantial knowledge gap that requires extensive research at the subcellular, cellular, and animal model levels.

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.

Differential apoptosis-related protein expression, mitochondrial properties, proteolytic enzyme activity, and DNA fragmentation between skeletal muscles

Increased skeletal muscle apoptosis has been associated with a number of conditions including aging, disuse, and cardiovascular disease. Skeletal muscle is a complex tissue comprised of several fiber types with unique properties. To date, no report has specifically examined apoptotic differences across muscles or fiber types. Therefore, we measured several apoptotic indices in healthy rat red (RG) and white gastrocnemius (WG) muscle, as well as examined the expression of several key proteins across fiber types in a mixed muscle (mixed gastrocnemius). The protein content of apoptosis-inducing factor (AIF), apoptosis repressor with caspase recruitment domain (ARC), Bax, Bcl-2, cytochrome c, heat shock protein 70 (Hsp70), and second mitochondria-derived activator of caspases (Smac) were significantly (P < 0.05) higher in RG vs. WG muscle. Cytosolic AIF, cytochrome c, and Smac as well as nuclear AIF were also significantly (P < 0.05) higher in RG compared with WG muscle. In addition, ARC protein expression was related to muscle fiber type and found to be highest (P < 0.001) in type I fibers. Similarly, AIF protein expression was differentially expressed across fibers; however, AIF was correlated to oxidative potential (P < 0.001). Caspase-3, -8, and -9 activity, calpain activity, and DNA fragmentation (a hallmark of apoptosis) were also significantly higher (P < 0.05) in RG compared with WG muscle. Furthermore, total muscle reactive oxygen species generation, as well as Ca(2+)-induced permeability transition pore opening and loss of membrane potential in isolated mitochondria were greater in RG muscle. Collectively, these data suggest that a number of apoptosis-related indices differ between muscles and fiber types. Given these findings, muscle and fiber-type differences in apoptotic protein expression, signaling, and susceptibility should be considered when studying cell death processes in skeletal muscle.

Ubiquinone Analogs: A Mitochondrial Permeability Transition Pore-Dependent Pathway to Selective Cell Death

BackgroundProlonged opening of the mitochondrial permeability transition pore (PTP) leads to cell death. Various ubiquinone analogs have been shown to regulate PTP opening but the outcome of PTP regulation by ubiquinone analogs on cell fate has not been studied yet.Methodology/Principal FindingsThe effects of ubiquinone 0 (Ub0), ubiquinone 5 (Ub5), ubiquinone 10 (Ub10) and decyl-ubiquinone (DUb) were studied in freshly isolated rat hepatocytes, cultured rat liver Clone-9 cells and cancerous rat liver MH1C1 cells. PTP regulation by ubiquinones differed significantly in permeabilized Clone-9 and MH1C1 cells from that previously reported in liver mitochondria. Ub0 inhibited PTP opening in isolated hepatocytes and Clone-9 cells, whereas it induced PTP opening in MH1C1 cells. Ub5 did not affect PTP opening in isolated hepatocytes and MH1C1 cells, but it induced PTP opening in Clone-9 cells. Ub10 regulated PTP in isolated hepatocytes, whereas it did not affect PTP opening in Clone-9 and MH1C1 cells. Only DUb displayed the same effect on PTP regulation in the three hepatocyte lines tested. Despite such modifications in PTP regulation, competition between ubiquinones still occurred in Clone-9 and MH1C1 cells. As expected, Ub5 induced a PTP-dependent cell death in Clone-9, while it did not affect MH1C1 cell viability. Ub0 induced a PTP-dependent cell death in MH1C1 cells, but was also slightly cytotoxic in Clone-9 by an oxidative stress-dependent mechanism.Conclusions/SignificanceWe found that various ubiquinone analogs regulate PTP in different ways depending on the cell studied. We took advantage of this unique property to develop a PTP opening-targeted strategy that leads to cell death specifically in cells where the ubiquinone analog used induces PTP opening, while sparing the cells in which it does not induce PTP opening.

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

Carbenoxolone (Cbx), a substance from the medicinal licorice is used for anti‐inflammatory treatments. Cbx was already shown to induce opening of the permeability transition pore (PTP) in rat liver mitochondria (RLM). We investigated the mechanism of action of Cbx on Ca2+‐induced PTP opening in rat brain mitochondria (RBM), and in rat heart mitochondria (RHM) and in RLM, in an attempt to identify the molecular target of Cbx in mitochondria. In RBM, RHM and RLM, threshold Ca2+ load induced PTP opening. This was manifested by sudden Ca2+efflux from the mitochondrial matrix, a drop in membrane potential, and swelling of mitochondria. At 1 μM, Cbx enhanced the Ca2+‐induced, Cyclosporine A‐sensitive PTP opening. A well‐known target of Cbx in cells is the connexin family of proteins, which form gap junctions. Moreover, Connexin43 (Cx43) was previously found in RHM and attributed to the protective mechanism induced by ischemic preconditioning. We hypothesized that Cx43 might be a target for Cbx in brain mitochondria. We clearly detected Cx43 by Western blot in RBM and in mitochondria isolated from astrocytes, less in RHM, but not in RLM. Interestingly, the anti‐Cx43 antibody abolished the Cbx‐enhanced PTP opening in RBM, but not in RLM. In RBM, PTP caused serine‐368 phosphorylation of Cx43. These results show that Cbx facilitates Ca2+‐induced PTP opening, addressing as possible molecular target Cx43 in brain mitochondria.

The brain‐specific protein, p42IP4(ADAP 1) is localized in mitochondria and involved in regulation of mitochondrial Ca2+

In brain, p42(IP4) (centaurin-alpha1; recently named ADAP 1, which signifies ADP ribosylation factor GTPase activating protein with dual PH domains 1, within the large family of Arf-GTPase activating proteins) is mainly expressed in neurons. p42(IP4) operates as a dual receptor recognising two second messengers, the soluble inositol(1,3,4,5)tetrakisphosphate and the lipid phosphatidylinositol(3,4,5)trisphosphate. We show here for the first time that p42(IP4) is localized in mitochondria, isolated from rat brain and from cells transfected with p42(IP4). In rat brain mitochondria we additionally found interaction of p42(IP4) with 2', 3'-cyclic nucleotide 3'-phosphodiesterase and alpha-tubulin by pull-down binding assay and by immunoprecipitation. In mitochondria from Chinese hamster ovary cells, p42(IP4) is predominantly associated with the intermembrane space and the inner membrane. This localization of p42(IP4) indicates that p42(IP4) might have a still unknown mitochondrial function. We studied whether p42(IP4) is involved in Ca(2+)-induced permeability transition pore opening, which is important in mitochondrial events leading to programmed cell death. We used mouse neuroblastoma cells as a model for the functional studies of p42(IP4) in mitochondria. In mitochondria isolated from p42(IP4)-transfected mouse neuroblastoma cells, over-expression of p42(IP4) significantly decreased Ca(2+) capacity and lag time for Ca(2+) retention. Thus, we suggest that p42(IP4) is involved in the regulation of Ca(2+) transport in mitochondria. We propose that p42(IP4) promotes Ca(2+)-induced permeability transition pore opening and thus destabilizes mitochondria.

Effect of Transient and Permanent Permeability Transition Pore Opening on NAD(P)H Localization in Intact Cells

To study the effect of mitochondrial permeability transition pore (PTP) opening on NAD(P)H localization, intact cells were exposed to the Ca(2+) ionophore A23187. PTP opening, mitochondrial membrane potential, mitochondrial volume, and NAD(P)H localization were assessed by time-lapse laser confocal microscopy using the calcein-cobalt technique, tetramethylrhodamine methyl ester, MitoTracker, and NAD(P)H autofluorescence, respectively. Concomitant with PTP opening, NAD(P)H fluorescence increased outside mitochondria. These events occurred in all cells and were prevented by cyclosporin A. Mitochondrial membrane potential was not systematically collapsed, whereas mitochondrial volume did not change, confirming that A23187 induced transient PTP opening in a subpopulation of cells and suggesting that mitochondrial swelling did not immediately occur after PTP opening in intact cells. NAD(P)H autofluorescence remained elevated after PTP opening, particularly after membrane potential had been collapsed by an uncoupler. Extraction of nucleotide for NAD(P)H quantification confirmed that PTP opening led to an increase in NAD(P)H content. Because the oxygen consumption rate decreased, whereas the lactate/pyruvate ratio increased after PTP opening in intact cells, we conclude that PTP opening inhibits respiration and dramatically affects the cytosolic redox potential in intact cells.

Potential role of connexin43 in carbenoxolone‐promoted opening of the permeability transition pore in isolated brain and heart mitochondria

Carbenoxolone (Cbx) was reported to induce permeability transition pore (PTP) opening in isolated rat liver mitochondria (RLM), although the mitochondrial molecular target of Cbx has yet to be identified. Here, we investigated the effects of Cbx on Ca2+‐induced PTP opening in RLM, rat heart (RHM), and rat brain (RBM) mitochondria. In RLM, RHM, and RBM, threshold Ca2+ load induced PTP opening, as seen by sudden Ca2+efflux from the mitochondrial matrix and a drop in membrane potential. At about 1 μM, Cbx enhanced Ca2+‐induced CyclosporinA‐sensitive PTP opening. Since in isolated heart mitochondria Connexin43 (Cx43) was found, we suggested that Cbx might be target for Cx43 in mitochondria. We detected Cx43 by Western blot in the outer RBM membrane and in mitoplasts, but not in RLM. Anti‐Cx43 antibody abolished Cbx‐enhanced PTP opening in RBM but not in RLM, supposing possible involvement of another connexin type in RLM. Besides being a gap junction inhibitor, Cbx bears structural similarity to corticosteroids, indicating possible translocator protein (TSPO)/ peripheral‐type benzodiazepine receptor (PBR) targeting. Cbx‐enhanced PTP opening, which correlated with increased protein phosphorylation, was suppressed by an anti‐TSPO antibody. All these results indicate that Cbx facilitates Ca2+‐induced PTP opening having as possible targets in RHM and RBM both TSPO and Cx43.

Hypothermia impedes calcium induced mitochondrial permeability transition pore opening in mitochondria harvested after cold ischemia of isolated hearts

Hypothermia decreases ischemia‐reperfusion (IR) injury. IR injury has been associated with mitochondrial permeability transition pore (PTP) opening, and decreased activity of the electron transport chain. We studied mitochondria from isolated hearts subjected to normothermic and hypothermic ischemia. Guinea pig isolated hearts were perfused at constant pressure with Krebs‐Ringer's solution at 37 ºC and subjected to 30 min of ischemia at 37ºC or 17ºC. After 5 min of reperfusion mitochondria were isolated. Mitochondrial [Ca2+]m, pH, membrane potential (ΔΨm) and NADH were measured spectrophotometrically with indo 1, BCECF, rhodamine 123 and autofluorescence. After energizing with pyruvic acid, [CaCl2] (0‐100 μM) and ADP were added. Adding CaCl2 caused a dose dependent [Ca2+]m increase. Ca2+ induced PTP opening, assessed by collapse of ΔΨm, and prevented by cyclosporine A, occurred after adding 50 μM CaCl2 after 37ºC IR, and adding 100 μM CaCl2 after 17ºC IR. ADP decreased ΔΨm, pH and NADH and increased [Ca2+]m in all mitochondria. These bioenergetic markers recovered after 17ºC IR, but not after 37ºC IR. Our study shows that hypothermia protects mitochondria against IR injury as shown by resistance to Ca2+ induced PTP opening and appropriate responses to ADP. This study emphasizes the importance of mitochondrial function in protecting against IR injury, and gives insight into how hypothermia reduces IR injury.

In brain mitochondria, calcium and cell death-associated permeability transition are controlled by possibly associated proteins, 2',3'-CNPase, Centaurin-alpha1 and peripheral benzodiazepine receptor, and their substrates/ligands

Mitochondria play a central role in calcium homeostasis and cellular calcium signaling. During cellular calcium overload, mitochondria take up cytosolic calcium, which, in turn, induces opening of the permeability transition pore (PTP), disruption of mitochondrial membrane potential and cell death. PTP is a protein complex changing according to the needs of the cell and responding to different external and internal stimuli. The identity of the PTP is still unresolved. 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and p42(IP4) (centaurin-alpha1) have been shown to be associated with rat brain mitochondria (RBM), but the exact role of these proteins in mitochondria is still obscure. Localization of p42(IP4) and CNP within the inner membrane and contact sites indicates further functions for these proteins. We found interaction of p42(IP4) with CNP by pull-down binding assay and by immunoprecipitation. Since PTP opening is important in mitochondrial events leading to programmed cell death, we studied whether p42(IP4) and CNP are involved in calcium-induced calcium release and consequently PTP. Simultaneous measurements of the respiratory rate, trans-membrane potential and calcium transport in the mitochondrial suspension were performed. We also developed the method of isolation of functionally active mitochondria from several cell types. We determined the calciumcapacity and lag-phase for PTP opening in mitochondria isolated from p42(IP4)-transfected and from control neuroblastoma cells. Overexpression of p42(IP4) led to promotion of calcium-induced PTP opening. The enzymatic activity of CNP was reduced under PTP opening, whereas the level of CNP detected in RBM before and after PTP opening were unchanged. Involvement of CNP in PTP operation was confirmed in further experiments using mitochondria isolated from CNP-knock-down oligodendrocytes (OLN93 cells). In mitochondria isolated from OLN93 cells transfected with CNP-targeting siRNA, CNP reduction was correlated with facilitation of calcium-induced PTP opening. The CNP substrates, 2',3'-cAMP and 2',3'-cNADP, induced PTP opening in RBM. The peripheral-type benzodiazepine receptor (PBR) is an 18 kDa mitochondrial membrane protein with still elusive functions. A release of pro-apoptotic factors, AIF and cytochrome c, from RBM was shown at threshold calcium load. Anti-PBR antibody blocked the release of AIF but did not affect the cytochrome c release. 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 calcium-induced calcium efflux, and AIF release from mitochondria. In summary, our results provide evidence that PBR, CNP and p42(IP4) are involved in regulation of calcium-induced PTP opening, important stage of initiation of programmed cell death.