Induction Of Mitochondrial Permeability Transition Research Articles

Induction of Mitochondrial Permeability Transition by the DNA-intercalating Cationic Dye Ethidium Bromide

This work shows that the DNA cationic probe, ethidium bromide (EtBr), induces the transition from selective to non-selective mitochondrial permeability. This statement is based on the findings, indicating: (i) EtBr induced the release of accumulated Ca(2+) through a mechanism sensitive to cyclosporin A and octylguanidine; (ii) EtBr induced the release of cytochrome c and (iii) EtBr induced mitochondrial swelling. Interestingly, mersalyl inhibited, in a non-competitive fashion, EtBr uptake, which would indicate that the uptake may be carried out through a protein membrane system. This work also shows that the effect of the dye on permeability transition was stimulated by carboxyatractyloside. Taking into account the facts that EtBr inhibited the ADP exchange reaction and increased the binding of the fluorescent probe eosin-5-maleimide to adenine nucleotide translocase, it is tempting to assume a possible interaction between EtBr and the ADP/ATP carrier.

Superoxide anion mediated mitochondrial dysfunction leads to hepatocyte apoptosis preferentially in the periportal region during copper toxicity in rats

Chronic exposure to copper induces hepatocellular apoptosis with greater injury in the periportal region compared to the perivenous region. Here we have identified the factors responsible for the development of regional damage in the liver under in vivo conditions. Enhanced production of reactive oxygen species (ROS) with predominance of superoxide radical (O 2 −) indicates the contribution of redox imbalance in the process. This may be linked with copper catalyzed oxidation of GSH to GSSG resulting in the generation of O 2 −. Downregulation of Cu-Zn SOD in consequence of the degradation of this enzyme, causes decreased dismutation of O 2 −, that further contributes to the enhanced level of O 2 − in the periportal region. Decreased functioning of Mn SOD activity, reduction in mitochondrial thiol/disulphide ratio and generation of O 2 − were much higher in the mitochondria from periportal region, which point to the involvement of this organelle in the regional hepatotoxicity observed during copper exposure. This was supported by copper-mediated enhanced mitochondrial dysfunction as evident from ATP depletion, collapse of mitochondrial membrane potential (MMP) and induction of mitochondrial permeability transition (MPT). Results suggest the active participation of O 2 − in inducing mitochondrial dysfunction preferentially in the periportal region that eventually leads to the development of hepatotoxicity due to copper exposure under in vivo condition.

Prevention of I/R injury in fatty livers by ischemic preconditioning is associated with increased mitochondrial tolerance: the key role of ATPsynthase and mitochondrial permeability transition

Ischemia/reperfusion (I/R) injury is a commonly encountered clinical problem and occurs probably as a consequence of irreversible mitochondrial injury. The increased susceptibility of fatty livers to ischemic injury is associated with depletion of adenosine triphosphate (ATP) content, which is preserved by preconditioning. Mitochondria being the main ATP production source for the cell, we aimed to evaluate whether ischemic preconditioning (IPC) of fatty livers prevents the impairment in mitochondrial function induced by I/R. Lean and steatotic animals were subjected to 90 min of hepatic warm ischemia and 12 h of reperfusion. IPC effect was tested in fatty livers. After reperfusion, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured. Mitochondrial membrane potential, mitochondrial respiration and susceptibility to mitochondrial permeability transition (MPT) were evaluated, as well as ATPase activity and adenine nucleotides. IPC of fatty livers decreased serum AST and ALT levels. Fatty animals subjected to I/R exhibited decreased mitochondrial membrane potential and a delay in the repolarization after a phosphorylation cycle, associated with increased state 4 respiration. Increased tolerance to MPT induction, preservation of F(1)F(o)-ATPsynthase activity and mitochondrial bioenergetics were observed in ischemic preconditioned fatty livers. Thus, IPC is an endogenous protecting mechanism that preserves mitochondrial function and bioenergetics in fatty livers.

Melatonin inhibits cardiolipin peroxidation in mitochondria and prevents the mitochondrial permeability transition and cytochrome c release

Cardiolipin oxidation is emerging as an important factor in mitochondrial dysfunction as well as in the initial phase of the apoptotic process. We have previously shown that exogenously added peroxidized cardiolipin sensitizes mitochondria to Ca 2+-induced mitochondrial permeability transition (MPT) pore opening and promotes the release of cytochrome c. In this work, the effects of intramitochondrial cardiolipin peroxidation on Ca 2+-induced MPT and on the cytochrome c release from mitochondria were studied. The effects of melatonin, a compound known to protect the mitochondria from oxidative damage, on both of these processes were also tested. tert-Butylhydroperoxide (t-BuOOH), a lipid-soluble peroxide that promotes lipid peroxidation, was used to induce intramitochondrial cardiolipin peroxidation. Exposure of heart mitochondria to t-BuOOH resulted in the oxidation of cardiolipin, associated with an increased sensitivity of mitochondria to Ca 2+-induced MPT and with the release of cytochrome c from the mitochondria. All these processes were inhibited by micromolar concentrations of melatonin. It is proposed that melatonin inhibits cardiolipin peroxidation in mitochondria, and this effect seems to be responsible for the protection afforded by this agent against the MPT induction and cytochrome c release. Thus, manipulating the oxidation sensitivity of cardiolipin with melatonin may help to control MPT and cytochrome c release, events associated with cell death, and thus, be used for treatment of those disorders characterized by mitochondrial cardiolipin oxidation and Ca 2+ overload.

Drugs targeting the mitochondrial pore act as citotoxic and cytostatic agents in temozolomide-resistant glioma cells

BackgroundHigh grade gliomas are one of the most difficult cancers to treat and despite surgery, radiotherapy and temozolomide-based chemotherapy, the prognosis of glioma patients is poor. Resistance to temozolomide is the major barrier to effective therapy. Alternative therapeutic approaches have been shown to be ineffective for the treatment of genetically unselected glioma patients. Thus, novel therapies are needed. Mitochondria-directed chemotherapy is an emerging tool to combat cancer, and inner mitochondrial permeability transition (MPT) represents a target for the development of cytotoxic drugs. A number of agents are able to induce MPT and some of them target MPT-pore (MPTP) components that are selectively up-regulated in cancer, making these agents putative cancer cell-specific drugs.ObjectiveThe aim of this paper is to report a comprehensive analysis of the effects produced by selected MPT-inducing drugs (Betulinic Acid, Lonidamine, CD437) in a temozolomide-resistant glioblastoma cell line (ADF cells).MethodsEGFRvIII expression has been assayed by RT-PCR. EGFR amplification and PTEN deletion have been assayed by differential-PCR. Drugs effect on cell viability has been tested by crystal violet assay. MPT has been tested by JC1 staining. Drug cytostatic effect has been tested by mitotic index analysis. Drug cytotoxic effect has been tested by calcein AM staining. Apoptosis has been assayed by Hoechst incorporation and Annexine V binding assay. Authophagy has been tested by acridine orange staining.ResultsWe performed a molecular and genetic characterization of ADF cells and demonstrated that this line does not express the EGFRvIII and does not show EGFR amplification. ADF cells do not show PTEN mutation but differential PCR data indicate a hemizygous deletion of PTEN gene. We analyzed the response of ADF cells to Betulinic Acid, Lonidamine, and CD437. Our data demonstrate that MPT-inducing agents produce concentration-dependent cytostatic and cytotoxic effects in parallel with MPT induction triggered through MPTP. CD437, Lonidamine and Betulinic acid trigger apoptosis as principal death modality.ConclusionThe obtained data suggest that these pharmacological agents could be selected as adjuvant drugs for the treatment of high grade astrocytomas that resist conventional therapies or that do not show any peculiar genetic alteration that can be targeted by specific drugs.

Timosaponin A-III Induces Autophagy Preceding Mitochondria-Mediated Apoptosis in HeLa Cancer Cells

Timosaponin A-III (TAIII), a saponin isolated from the rhizome of Anemarrhena asphodeloides, exhibits potent cytotoxicity and has the potential to be developed as an anticancer agent. Here, we provide evidence that TAIII induces autophagy in HeLa cells followed by apoptotic cell death. TAIII-induced autophagy was morphologically characterized by the formation of membrane-bound autophagic vacuoles recognizable at the ultrastructural level. TAIII-treated cells expressing green fluorescent protein (GFP)-labeled microtubule-associated protein 1 light chain 3 (LC3) displayed punctate fluorescence indicative of LC3 recruitment to the autophagosome. This was associated with the conversion of LC3-I (the cytosolic form) into LC3-II (the lipidated form located on the autophagosome membrane). TAIII treatment also induced mitochondrial dysfunction involving overproduction of reactive oxygen species and reduction of mitochondrial membrane potential accompanied by induction of mitochondrial permeability transition. Prolonged exposure to TAIII resulted in cytochrome c release and caspase-3 activation, events that signified the onset of apoptotic cell death. TAIII-induced autophagy preceded apoptosis, as evidenced by early autophagic vacuole formation, GFP-LC3 translocation, and LC3-II increase in the absence of caspase-3 cleavage. Notably, TAIII-mediated apoptotic cell death was potentiated by treatment with autophagy inhibitor 3-methyladenine or small interfering RNA against the autophagic gene beclin 1. These findings suggest that TAIII-elicited autophagic response plays a protective role that impedes the eventual cell death. In terms of structure-activity relationship, the sugar chain in TAIII is indispensable to the drug action, as the sugar-lacking aglycone sarsasapogenin did not induce autophagy and exhibited weaker cytotoxicity.

Distinct behaviors of adenylate kinase and cytochrome c observed following induction of mitochondrial permeability transition by Ca2+ in the absence of respiratory substrate

For induction of the mitochondrial permeability transition (PT) by Ca(2+), the addition of a respiratory substrate such as succinate is required. However, earlier studies indicated the possible induction of the mitochondrial PT by Ca(2+) in the absence of a respiratory substrate (Hunter, D.R., and Haworth, R.A. (1979) Arch. Biochem. Biophys. 195, 453-459). In the present study, we obtained clear evidence showing that the mitochondrial PT could be induced by Ca(2+) even in the absence of respiratory substrate. We next examined the protein release from mitochondria that accompanied the induction of PT in the absence of a respiratory substrate. Interestingly, distinct from the ordinary mitochondrial PT induced by Ca(2+) in the presence of a respiratory substrate, which is associated with the release of mitochondrial cytochome c and adenylate kinase, the mitochondrial PT occurring in the absence of a respiratory substrate was associated with release of mitochondrial adenylate kinase but not with that of mitochondrial cytochrome c. This experimental system should be quite useful for understanding the mechanisms of protein release from mitochondria.

High fluence low‐power laser irradiation induces mitochondrial permeability transition mediated by reactive oxygen species

High fluence low-power laser irradiation (HF-LPLI) can induce cell apoptosis via the mitochondria/caspase-3 pathway. Here, we further investigated the mechanism involved in the apoptotic process in human lung adenocarcinoma cells (ASTC-a-1) at a laser irradiation fluence of 120 J/cm(2) (633 nm). Cytochrome c release was ascribed to mitochondrial permeability transition (MPT) because the release was prevented by cyclosporine (CsA), a specific inhibitor of MPT. Furthermore, mitochondrial permeability for calcein (approximately 620 Da) was another evidence for the MPT induction under HF-LPLI treatment. A high-level intracellular reactive oxygen species (ROS) generation was observed after irradiation. The photodynamically produced ROS caused onset of MPT, as the ROS scavenger docosahexaenoic acid (DHA) prevented the MPT. However, CsA failed to prevented cell death induced by HF-LPLI, indicating the existence of other signaling pathways. Following laser irradiation, Bax activation occurred after mitochondrial depolarization and cytochrome c release, indicating Bax activation was a downstream event. In the presence of CsA, Bax was still activated at the end-stage of apoptotic process caused by HF-LPLI, suggesting that Bax was involved in an alternative-signaling pathway, which was independent of MPT. Under HF-LPLI treatment, cell viabilities due to pre-treatment with DHA, CsA, or Bax small interfering RNA (siRNA) demonstrated that the MPT signaling pathway was dominant, while Bax signaling pathway was secondary, and more importantly ROS mediated both pathways. Taken together, these results showed that HF-LPLI induced cell apoptosis via the CsA-sensitive MPT, which was ROS-dependent. Furthermore, there existed a secondary signaling pathway through Bax activation. The observed link between MPT and triggering ROS could be a fundamental phenomenon in HF-LPLI-induced cell apoptosis.

Resistance of Young Rat Hepatic Mitochondria to Bile Acid-Induced Permeability Transition: Potential Role of α-Tocopherol

Retention of bile acids within the liver is a primary factor in the pathogenesis of cholestatic liver disorders, which are more common in human infants. The objective of this study was to evaluate developmental changes in mitochondrial factors involved in bile acid-induced hepatocyte injury. Hepatic mitochondria from adult rats (aged 9 wk) underwent a mitochondrial permeability transition (MPT) and release of cytochrome c upon exposure to glycochenodeoxycholic acid. In contrast, mitochondria from young rats (age 6-36 d) were resistant to MPT induction and cytochrome c release. Neither mitochondrial levels of MPT-associated proteins (voltage-dependent anion channel, cyclophilin D, or adenine nucleotide translocase), Bcl-2 family proteins, nor antioxidant enzymes explained this resistance. Mitochondria from young rats contained 2- to 3-fold higher alpha-tocopherol (alpha-TH). In vivo alpha-TH enrichment of adult hepatic mitochondria increased their MPT resistance. Tetra-linoleoyl cardiolipin (TL-CL), the primary molecular species of CL, was reduced in mitochondria of the young rat; however, enrichment with CL and TL-CL only modestly increased their MPT susceptibility. In conclusion, we observed an unexpected resistance in young rats to bile acid induction of mitochondrial cell death pathways, which may be related to developmental differences in membrane composition.

Involvement of cyclophilin D in mitochondrial permeability transition induction in intact cells

The mitochondrial permeability transition (MPT) is involved in both Ca 2+ signaling and cell death. The present study aimed to clarify the involvement of cyclophilin D, a peptidyl prolyl cis– trans isomerase (PPIase), in MPT induction in intact cells. To achieve this, we used C6 cells overexpressing wild-type or PPIase-deficient cyclophilin D, and measured the inner mitochondrial membrane permeability to calcein, a 623-Da hydrophilic fluorescent molecule, to evaluate MPT induction. In vector control cells, the percentage of MPT induction by ionomycin increased as the Ca 2+ concentration in the extracellular medium increased. This result indicates that the present method is valid for numerical evaluation of MPT induction. In C6 cells expressing the PPIase-deficient mutant, the percentage of MPT induction was significantly decreased compared with wild-type CypD-overexpressing cells or vector control cells. These results suggest that cyclophilin D is involved in MPT induction by Ca 2+ in intact cells.

Cobalt induces oxidative stress in isolated liver mitochondria responsible for permeability transition and intrinsic apoptosis in hepatocyte primary cultures

It is well established that cobalt mediates the occurrence of oxidative stress which contributes to cell toxicity and death. However, the mechanisms of these effects are not fully understood. This investigation aimed at establishing if cobalt acts as an inducer of mitochondrial-mediated apoptosis and at clarifying the mechanism of this process.Cobalt, in the ionized species Co2+, is able to induce the phenomenon of mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) with the opening of the transition pore. In fact, Co2+ induces mitochondrial swelling, which is prevented by cyclosporin A and other typical MPT inhibitors such as Ca2+ transport inhibitors and bongkrekic acid, as well as anti-oxidant agents. In parallel with mitochondrial swelling, Co2+ also induces the collapse of electrical membrane potential. However in this case, cyclosporine A and the other MPT inhibitors (except ruthenium red and EGTA) only partially prevent ΔΨ drop, suggesting that Co2+ also has a proton leakage effect on the inner mitochondrial membrane. MPT induction is due to oxidative stress, as a result of generation by Co2+ of the highly damaging hydroxyl radical, with the oxidation of sulfhydryl groups, glutathione and pyridine nucleotides. Co2+ also induces the release of the pro-apoptotic factors, cytochrome c and AIF. Incubation of rat hepatocyte primary cultures with Co2+ results in apoptosis induction with caspase activation and increased level of expression of HIF-1α.All these observations allow us to state that, in the presence of calcium, Co2+ is an inducer of apoptosis triggered by mitochondrial oxidative stress.

Zfra is an inhibitor of Bcl-2 expression and cytochrome c release from the mitochondria

Zfra is a small size 31-amino-acid C2H2 zinc finger-like protein, which is known to interact with c-Jun N-terminal kinase 1 (JNK1), WW domain-containing oxidoreductase (WWOX, FOR or WOX1), TNF receptor-associated death domain protein (TRADD) and nuclear factor kappaB (NF-κB) during stress response. Here, we show that Zfra became phosphorylated at Ser8 (as determined by specific antibody) and translocated to the mitochondria in response to inducers of mitochondrial permeability transition (MPT) (e.g. staurosporine and betulinic acid). Overexpressed Zfra induced cell death. This event is associated, in part, with increased dissipation of mitochondrial membrane potential (MMP) and increased chromosomal DNA fragmentation. Intriguingly, Zfra significantly downregulated Bcl-2 and yet blocked cytochrome c release from the mitochondria. Overexpression of an S8G-Zfra mutant (Ser8 to Gly8 alteration) could not induce cell death, probably due to its failure of translocating to the mitochondria and causing MMP dissipation. Over-expressed proapoptotic WOX1 induced cytochrome c release from the mitochondria. Zfra bound and blocked the effect of WOX1. Taken together, Ser8 is essential for overexpressed Zfra to exert cell death via the mitochondrial pathway. Zfra downregulates Bcl-2 and induces MMP dissipation but causes no cytochrome c release, indicating a novel death pathway from the mitochondria.

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.

Zfra is an inhibitor of Bcl‐2 expression and cytochrome c release from the mitochondria

Zfra is a small size 31‐amino‐acid C2H2 zinc finger‐like protein, which is known to interact with c‐Jun N‐terminal kinase 1 (JNK1), WW domain‐containing oxidoreductase (WWOX, FOR or WOX1), TNF receptor‐associated death domain protein (TRADD) and nuclear factor kappaB (NF‐kappaB) during stress response. Here, we show that Zfra became phosphorylated at Ser8 (as determined by specific antibody) and translocated to the mitochondria in response to inducers of mitochondrial permeability transition (MPT) (e.g. staurosporine and betulinic acid). Overexpressed Zfra induced cell death. This event is associated, in part, with increased dissipation of mitochondrial membrane potential (MMP) and increased chromosomal DNA fragmentation. Intriguingly, Zfra significantly downregulated Bcl‐2 and yet blocked cytochrome c release from the mitochondria. Overexpression of an S8G‐Zfra mutant (Ser8 to Gly8 alteration) could not result in cell death, due to its failure of translocating to the mitochondria and causing MMP dissipation. Proapoptotic WOX1 physically interacted with Zfra and induced cytochrome c release from the mitochondria. Zfra blocked the effect of WOX1. Taken together, Ser8 is essential for overexpressed Zfra to exert cell death via the mitochondrial pathway. Zfra downregulates Bcl‐2 and induces MMP dissipation but causes no cytochrome c release, indicating a novel death pathway from the mitochondria.

The Identity and Regulation of the Mitochondrial Permeability Transition Pore

The mitochondrial permeability transition (MPT) pore complex is a key participant in the machinery that controls mitochondrial fate and, consequently, cell fate. The quest for the pore identity has been ongoing for several decades and yet the main structure remains unknown. Established "dogma" proposes that the core of the MPT pore is composed of an association of voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT). Recent genetic knockout experiments contradict this commonly accepted interpretation and provide a basis for substantial revision of the MPT pore identity. There is now sufficient evidence to exclude VDAC and ANT as the main pore structural components. Regarding MPT pore regulation, the role of cyclophilin D is confirmed and ANT may still serve some regulatory function, although the involvement of hexokinase II and creatine kinase remains unresolved. When cell protection signaling pathways are activated, we have found that the Bcl-2 family members relay the signal from glycogen synthase kinase-3 beta onto a target at or in close proximity to the pore. Our experimental findings in intact cardiac myocytes and neurons indicate that the current "dogma" related to the role of Ca2+ in MPT induction requires reevaluation. Emerging evidence suggests that after injury-producing stresses, reactive oxygen species (but not Ca2+) are largely responsible for the pore induction. In this article we discuss the current state of knowledge and provide new data related to the MPT pore structure and regulation.

Mechanism of A23187-Induced Apoptosis in HL-60 Cells: Dependency on Mitochondrial Permeability Transition but Not on NADPH Oxidase

Calcium ions (Ca(2+)) are involved in a number of physiological cellular functions including apoptosis. An elevation in intracellular levels of Ca(2+) in A23187-treated HL-60 cells was associated with the generation of both intracellular and extracellular reactive oxygen species (ROS) and induction of apoptotic cell death. A23187-induced apoptosis was prevented by cyclosporin A, a potent inhibitor of mitochondrial permeability transition (MPT). The generation of extracellular ROS was suppressed by the NADPH oxidase inhibitor diphenylene iodonium, and by superoxide dismutase, but these agents had no effect on A23187-induced apoptosis. In contrast, the blocking of intracellular ROS by a cell-permeant antioxidant diminished completely the induction of MPT and apoptosis. In isolated mitochondria, the addition of Ca(2+) induced a typical MPT concomitant with the generation of ROS, which leads to augmentation of intracellular ROS levels. These results indicate that intracellular not extracellular ROS generated by A23187 is associated with the opening of MPT pores that leads to apoptotic cell death.

Novel substituted 1,4-anthracenediones with antitumor activity directly induce permeability transition in isolated mitochondria

Synthetic analogs of 1,4-anthraquinone (AQ code number), which block nucleoside transport, decrease DNA, RNA and protein syntheses, trigger cytochrome c release without caspase activation, induce apoptotic DNA fragmentation and inhibit the proliferation of wild-type and multidrug resistant tumor cells in the nM range in vitro, rapidly cause the collapse of mitochondrial transmembrane potential in cell and cell-free systems. Because mitochondrial permeability transition (MPT) requires more than depolarization to occur, antitumor AQs were tested for their ability to directly trigger specific markers of MPT in isolated mitochondria. In contrast to a spectrum of conventional anticancer drugs that are inactive, various AQs interact with isolated mitochondria in a concentration- and time-dependent manner to rapidly cause large amplitude swelling and Ca2+ release in relation with their effectiveness against L1210, HL-60 and LL/2 tumor cells in vitro. Indeed, the lead antitumor AQ8, AQ9 and AQ17 are also the most effective inducers of MPT in isolated mitochondria, whereas all AQ derivatives devoid of anti-proliferative activity also fail to trigger mitochondrial swelling and Ca2+ release. Moreover, the ability of 4 microM AQ17 to maximally induce mitochondrial swelling and Ca2+ release within 15 min is similar to that of classic MPT-inducing agents, such as 5 microg/ml alamethicin, 200 microM atractyloside, 5 microM phenylarsine oxide, 100 microM arsenic trioxide and a 100 microM Ca2+ overload. Interestingly, AQ17 requires a priming concentration of 20 microM Ca2+ to trigger mitochondrial swelling and Ca2+ release and these 0.1 microM ruthenium red-sensitive MPT events are abolished by 1 microM cyclosporin A, 2 mM ADP and 20 microM bongkrekic acid, which block components of the permeability transition pore (PTP), and also inhibited by 50-100 microM of various ubiquinones, which interact with the quinone binding site of the PTP and raise the Ca2+ load required for PTP opening. Hence, antitumor AQs that target isolated mitochondria and trigger MPT might directly interact with components of the PTP to induce conformational changes that increase its Ca2+ sensitivity and transition from the closed to the open state.

Catalase Takes Part in Rat Liver Mitochondria Oxidative Stress Defense

Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism with a rate constant of 0.0346 s(-1). The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 +/- 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue.

Peripheral benzodiazepine receptor ligand, PK11195 induces mitochondria cytochrome c release and dissipation of mitochondria potential via induction of mitochondria permeability transition

Mitochondrial permeability transition pore plays an important role in the processes of cell apoptosis and necrosis. The peripheral benzodiazepine receptor, a mitochondria outer-membrane protein, is involved in the regulation of mitochondrial permeability transition. In the present study, we test if PK11195, a peripheral benzodiazepine receptor ligand, can lead to the opening of mitochondrial permeability transition pores, and subsequently causes mitochondria cytochrome c loss and mitochondria uncoupling. In isolated cardiac mitochondria, PK11195 (50, 100, 200 μM) caused a dose-dependent mitochondrial swelling, cytochrome c loss, and the dissipation of mitochondrial potential. Cyclosporin A (0.2 μM), a specific inhibitor of mitochondrial permeability transition, completely prevented the mitochondrial swelling induced by PK11195, and maintained the cytochrome c content and membrane potential. These data suggest that peripheral benzodiazepine receptor ligand, PK11195 caused mitochondrial uncoupling and cytochrome c loss via induction of mitochondrial permeability transition.

Zfra invokes a novel mitochondrial pathway of cell death bypassing cytochrome c release

Zfra is a small size 31-amino-acid C2H2 zinc finger-like protein, and is known to regulate TNF-mediated cell death. Exposure of cells with inducers of mitochondrial permeability transition (MPT), such as staurosporine, atractyloside and betulinic acid, stimulated phosphorylation of Zfra at Ser8 and its translocation to the mitochondria. Overexpressed Zfra induced loss of mitochondrial membrane potential (LMMP), significantly downregulated MPT inhibitors Bcl-2 and Bcl-xL (without causing cytochrome c release), and eventually mediated cell death. In contrast, at low levels Zfra enhanced cell growth and blocked LMMP in dying cells treated with staurosporine. An S8G-Zfra mutant did not to translocate to the mitochondria during MPT and failed to induce cell death (when overexpressed). In comparison, T20G mutant translocated to the mitochondria during MPT but could not inhibit Bcl-2 expression. Together, phosphorylation at Ser8 is essential for overexpressed Zfra to exert its apoptotic function emanating from the mitochondria, where Zfra blocks cytochrome c release, downregulates the expression of Bcl-2 and Bcl-xL and induces LMMP.