Formation Of Mitochondrial Permeability Transition Research Articles

Translocation of iron from lysosomes to mitochondria during ischemia predisposes to injury after reperfusion in rat hepatocytes

The mitochondrial permeability transition (MPT) initiated by reactive oxygen species (ROS) plays an essential role in ischemia–reperfusion (IR) injury. Iron is a critical catalyst for ROS formation, and intracellular chelatable iron promotes oxidative injury-induced and MPT-dependent cell death in hepatocytes. Accordingly, our aim was to investigate the role of chelatable iron in IR-induced ROS generation, MPT formation, and cell death in primary rat hepatocytes. To simulate IR, overnight-cultured hepatocytes were incubated anoxically at pH 6.2 for 4h and reoxygenated at pH 7.4. Chelatable Fe2+, ROS, and mitochondrial membrane potential were monitored by confocal fluorescence microscopy of calcein, chloromethyldichlorofluorescein, and tetramethylrhodamine methyl ester, respectively. Cell killing was assessed by propidium iodide fluorimetry. Ischemia caused progressive quenching of cytosolic calcein by more than 90%, signifying increased chelatable Fe2+. Desferal and starch–desferal 1h before ischemia suppressed calcein quenching. Ischemia also induced quenching and dequenching of calcein loaded into mitochondria and lysosomes, respectively. Desferal, starch–desferal, and the inhibitor of the mitochondrial Ca2+ uniporter (MCU), Ru360, suppressed mitochondrial calcein quenching during ischemia. Desferal, starch–desferal, and Ru360 before ischemia also decreased mitochondrial ROS formation, MPT opening, and cell killing after reperfusion. These results indicate that lysosomes release chelatable Fe2+ during ischemia, which is taken up into mitochondria by MCU. Increased mitochondrial iron then predisposes to ROS-dependent MPT opening and cell killing after reperfusion.

Real-time concurrent monitoring of apoptosis, cytosolic calcium, and mitochondria permeability transition for hypermulticolor high-content screening of drug-induced mitochondrial dysfunction-mediated hepatotoxicity

A quantitative high-content screening (HCS) was suggested for the real-time monitoring of drug-induced mitochondrial dysfunction-mediated hepatotoxicity. This HCS is very advantageous in that it allows simultaneous observation of drug-induced activations of hepatotoxic pathways using hypermulticolor cellular imaging. The mitochondrial permeability transition (MPT), cytosolic calcium, and caspase-3 were selected as functional markers to verify drug-induced hepatotoxicity and were concurrently monitored in HepG2 cells in a real-time manner. Nefazodone, tolcapone, and troglitazone caused mitochondrial dysfunction and subsequent apoptotic HepG2 cell death in addition to marked cytosolic calcium increase. On the other hand, extrinsic pathway-mediated apoptotic cell death was monitored when HepG2 cells were treated with piroxicam. It was found that piroxicam-treated HepG2 cells showed apoptotic cell death without the MPT formation, while a cytosolic calcium increase was clearly observed. This finding was confirmed by the caspase-8 inhibition assay. These results demonstrated the unique potential of real-time hypermulticolor HCS to screen hepatotoxic drugs at the in vitro stage rather than the later in vivo stage based on an animal model and to ultimately reduce the probability of drug failure.

Ischemia‐induced mobilization of lysosomal iron predisposes rat hepatocytes to ischemia‐reperfusion (IR) injury

Reactive oxygen species (ROS) initiate onset of the mitochondrial permeability transition (MPT) and play a key role in IR injury. Iron is a critical catalyst for ROS generation. Accordingly, our aim was to investigate the role of chelatable iron in ROS‐dependent MPT formation and cell death during IR to rat hepatocytes. Cells were incubated anoxically at pH 6.2 for 4 h and re‐oxygenated at pH 7.4 to simulate IR. Chelatable iron, ROS, mitochondrial membrane potential and cell death were monitored by confocal imaging of calcein, chloromethyl dichlorofluorescein, tetramethylrhodamine methyl ester and propidium iodide, respectively. Ischemia caused progressive quenching of cytosolic calcein, indicating an increase of chelatable Fe2+, which was suppressed by desferal and starch‐desferal pretreatment. Quenching of calcein was also blocked by ebselen and ferristatin, inhibitors of lysosomal divalent metal transporter‐1. Ischemia induced quenching of calcein loaded into mitochondria, which was blocked by desferal, starch‐desferal and Ru360. These agents also decreased mitochondrial ROS formation, MPT onset and cell killing after reperfusion. In conclusion, lysosomal iron is mobilized into mitochondria during ischemia. Increased mitochondrial iron then predisposes cells for ROS‐mediated MPT opening and cell killing after reperfusion. (Supported by NIH grants DK073336 and DK37034)

Limited Mitochondrial Permeabilization Is an Early Manifestation of Palmitate-induced Lipotoxicity in Pancreatic β-Cells

Involvement of the mitochondrial permeability transition (MPT) pore in early stages of lipotoxic stress in the pancreatic beta-cell lines MIN6 and INS-1 was the focus of this study. Both long term (indirect) and acute (direct) effects of fatty acid (FA) application on beta-cell susceptibility to Ca(2+)-induced MPT induction were examined using both permeabilized and intact beta-cells. Long term exposure to moderate (i.e. below cytotoxic) levels of the saturated FA palmitate sensitized beta-cell mitochondria to MPT induced by Ca(2+). Long term exposure to palmitate was significantly a more efficient inducer of MPT than the unsaturated FA oleate, although upon acute application both caused similar MPT activation. Application of antioxidants, inhibitors of the ceramide pathway, or modifiers of membrane fluidity did not protect beta-cell mitochondria from FA exposure. However, significant protection was provided by co-application of the unsaturated FA oleate in a phosphatidylinositol 3-kinase-dependent manner. Characterization of MPT pore opening in response to moderate palmitate treatment revealed the opening of a unique form of MPT in beta-cells as it encompassed features of both low and high conductance MPT states. Specifically, this MPT showed solute selectivity, characteristic of a low conductance MPT; however, it affected mitochondrial respiration and membrane potential in a way typical of a high conductance MPT. Activation of the full-size/high conductance form of MPT required application of high levels of FA that reduced growth and initiated apoptosis. These findings suggest that in the beta-cell, MPTs can act as both initiators of cell death and as versatile modulators of cell metabolism, depending on the mode of the MPT pore induced.

7-Ketocholesterol enhances 1-methyl-4-phenylpyridinium-induced mitochondrial dysfunction and cell death in PC12 cells

The present study investigated the promoting effect of oxysterol 7-ketocholesterol against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP(+)) in differentiated PC12 cells. 7-Ketocholesterol significantly enhanced the MPP(+)-induced nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. N-Acetylcysteine, ascorbate, trolox, carboxy-PTIO and Mn-TBAP reduced the cytotoxic effect of MPP(+) in the presence of 7-ketocholesterol. The results indicate that 7-ketocholesterol shows a synergistic effect against the cytotoxic effect of MPP(+). 7-Ketocholesterol may enhance the MPP(+)-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of reactive oxygen species and depletion of GSH. The findings suggest that 7-ketocholesterol as a promoting agent for the formation of mitochondrial permeability transition may enhance the toxic neuronal cell injury.

Differential Involvement of Mitochondrial Permeability Transition in Cytotoxicity of 1-Methyl-4-Phenylpyridinium and 6-Hydroxydopamine

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the influence of the mitochondrial membrane permeability transition inhibition against the toxicity of 1-methyl-4-phenylpyridinium (MPP(+)) and 6-hydroxydopamine (6-OHDA) in relation to the mitochondria-mediated cell death process and role of oxidative stress. Both MPP(+) and 6-OHDA induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in differentiated PC12 cells. Cyclosporin A (CsA), trifluoperazine and aristolochic acid, inhibitors of mitochondrial permeability transition, significantly attenuated the MPP(+)-induced mitochondrial damage leading to caspase-3 activation, increased oxidative stress and cell death. In contrast to MPP(+), the cytotoxicity of 6-OHDA was not reduced by the addition of the mitochondrial permeability transition inhibitors. The results show that the cytotoxicity of MPP(+) may be mediated by the mitochondrial permeability transition formation, which is associated with formation of reactive oxygen species and the depletion of GSH. In contrast, the 6-OHDA-induced cell injury appears to be mediated by increased oxidative stress without intervention of the mitochondrial membrane permeability transition.

Synergistic effects of hydrogen peroxide and ethanol on cell viability loss in PC12 cells by increase in mitochondrial permeability transition

The promoting effect of ethanol against the cytotoxicity of hydrogen peroxide (H 2O 2) in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with H 2O 2 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. In PC12 cells and dopaminergic neuroblastoma SH-SY5Y cells, the promoting effect of ethanol on the H 2O 2-induced cell death was increased with exposure time. Ethanol promoted the nuclear damage, change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to H 2O 2 in PC12 cells. Catalase, carboxy-PTIO, Mn-TBAP, N-acetylcysteine, cyclosporin A and trifluoperazine inhibited the H 2O 2 and ethanol-induced mitochondrial dysfunction and cell injury. The results show that the ethanol treatment promotes the cytotoxicity of H 2O 2 against PC12 cells. Ethanol may enhance the H 2O 2-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of ROS and depletion of GSH. The findings suggest that ethanol as a promoting agent for the formation of mitochondrial permeability transition may enhance the neuronal cell injury caused by oxidants.

Modulation of tamoxifen-induced apoptosis by peripheral benzodiazepine receptor ligands in breast cancer cells

The peripheral benzodiazepine receptor (PBR), an integral protein of the mitochondrial membrane, is involved in the formation of mitochondrial permeability transition (MPT) pores. The opening of the MPT—leading to the dissipation of the inner-mitochondrial transmembrane potential (Δ Ψ m)—is considered to be an early apoptotic event. Therefore, we investigated the effect of the high-affinity PBR ligands Ro5-4684 and PK 11195 on tamoxifen (TAM)-induced apoptosis in MCF-7 and BT-20 breast cancer cell lines. Application of 100 nM TAM led to induction of apoptosis in both cell lines. Estrogene receptor (ER)-positive MCF-7 cells arrested in G 2/M by TAM treatment showed no general dissipation of Δ Ψ m, but reduction of Δ Ψ m was observed in a population of cells with high Δ Ψ m. In ER-negative BT-20 cells TAM treatment induced no arrest of the cell cycle but dissipation of Δ Ψ m. In both cell lines, nanomolar concentrations of the PBR ligands, which showed minor pro-apoptotic action themselves, reduced TAM-induced decrease of Δ Ψ m and apoptosis. In MCF-7 cells, a reduction of bcl-2 protein expression by TAM treatment was abolished by a combination of TAM with PBR ligands. Bax protein expression in BT-20 cells showed a significant increase in TAM-treated cells after 24 hr but was not increased when treated with TAM and PBR ligands. From these findings, we concluded that binding of PBR ligands in nanomolar concentrations protects cells against apoptosis.

Transient protection by peripheral benzodiazepine receptors during the early events of ultraviolet light-induced apoptosis.

The peripheral benzodiazepine receptor (PBR) is a mitochondrial protein involved in the formation of mitochondrial permeability transition (PT) pores which play a critical role during the early events of apoptosis. PBRs are located in many tissues and are strongly expressed in the superficial layers of human epidermis. PBRs play a protective role against free radical damage and PBR ligands modulate apoptosis. To investigate the role of PBR during the early events of ultraviolet (UV)-mediated apoptosis we compared the effects of UVB on PBR-transfected Jurkat cells and their wild type counterparts devoid of any PBR expression. Results indicate that early after UVB exposure (up to 4 h), PBR-transfected cells were more resistant to apoptosis and exhibited a delayed mitochondrial transmembrane potential drop, a diminished superoxide anions production, and a reduced caspase-3 activation. Taken together these findings suggest that PBR may regulate early death signals leading to UV induced apoptosis.