Reactive oxygen species, but not Ca2+ overloading, trigger pH- and mitochondrial permeability transition-dependent death of adult rat myocytes after ischemia-reperfusion

We investigated the role of pH, reactive oxygen species (ROS), Ca2+, and the mitochondrial permeability transition (MPT) in pH-dependent ischemia-reperfusion injury to adult rat myocytes. Myocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 3 h to simulate ischemia. To simulate reperfusion, myocytes were reoxygenated at pH 6.2 or 7.4 for 2 h. Some myocytes were treated with MPT blockers (cyclosporin A and N-methyl-4-isoleucine cyclosporin) and antioxidants (desferal, diphenylphenylene diamine, and 2-mercaptopropionyl glycine). Mitochondrial membrane potential, inner membrane permeabilization, and ROS formation were imaged with tetramethylrhodamine methyl ester, calcein, and chloromethyldichlorofluorescein diacetate, respectively. For Ca2+ imaging, myocytes were coloaded with rhod-2 and fluo-4 to evaluate mitochondrial and cytosolic Ca2+, respectively. After 10 min of reperfusion at pH 7.4, calcein redistributed across the mitochondrial inner membrane, an event preceded by mitochondrial ROS formation and accompanied by hypercontracture, mitochondrial depolarization, and then cell death. Acidotic reperfusion, antioxidants, and MPT blockers each prevented the MPT, depolarization, hypercontraction, and cell killing. Antioxidants, but neither MPT blockers nor acidotic reperfusion, inhibited ROS formation after reperfusion. Furthermore, anoxic reperfusion at pH 7.4 prevented cell death. Both mitochondrial and cytosolic Ca2+ increased during ischemia but recovered in the first minutes of reperfusion. Mitochondrial and cytosolic Ca2+ overloading again occurred late after reperfusion. This late Ca2+ overloading was blocked by MPT inhibition. Intramitochondrial Ca2+ chelation by cold loading/warm incubation of BAPTA did not prevent cell death after reperfusion. In conclusion, mitochondrial ROS, together with normalization of pH, promote MPT onset and subsequent myocyte death after reperfusion. In contrast, Ca2+ overloading appears to be the consequence of bioenergetic failure after the MPT and is not a factor promoting MPT onset.     

Partial contribution of mitochondrial permeability transition to t-butyl hydroperoxide-induced cell death

Mitochondrial permeability transition (MPT) is thought to determine cell death under oxidative stress. However, MPT inhibitors only partially suppress oxidative stress-induced cell death. Here, we demonstrate that cells in which MPT is inhibited undergo cell death under oxidative stress. When C6 cells were exposed to 250 μM t-butyl hydroperoxide (t-BuOOH), the loss of a membrane potential-sensitive dye (tetramethylrhodamine ethyl ester, TMRE) from mitochondria was observed, indicating mitochondrial depolarization leading to cell death. The fluorescence of calcein entrapped in mitochondria prior to addition of t-BuOOH was significantly decreased to 70% after mitochondrial depolarization. Cyclosporin A suppressed the decrease in mitochondrial calcein fluorescence, but not mitochondrial depolarization. These results show that t-BuOOH induced cell death even when it did not induce MPT. Prior to MPT, lactate production and respiration were hampered. Taken together, these data indicate that the decreased turnover rate of glycolysis and mitochondrial respiration may be as vital as MPT for cell death induced under moderate oxidative stress.     

Mitochondrial permeability transition in rat hepatocytes after anoxia/reoxygenation: role of Ca2+-dependent mitochondrial formation of reactive oxygen species

Onset of the mitochondrial permeability transition (MPT) is the penultimate event leading to lethal cellular ischemia-reperfusion injury, but the mechanisms precipitating the MPT after reperfusion remain unclear. Here, we investigated the role of mitochondrial free Ca(2+) and reactive oxygen species (ROS) in pH- and MPT-dependent reperfusion injury to hepatocytes. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 h and then reoxygenated at pH 7.4 to simulate ischemia-reperfusion. Some cells were loaded with the Ca(2+) chelators, BAPTA/AM and 2-[(2-bis-[carboxymethyl]aono-5-methoxyphenyl)-methyl-6-methoxy-8-bis[carboxymethyl]aminoquinoline, either by a cold loading protocol for intramitochondrial loading or by warm incubation for cytosolic loading. Cell death was assessed by propidium iodide fluorometry and immunoblotting. Mitochondrial Ca(2+), inner membrane permeability, membrane potential, and ROS formation were monitored with Rhod-2, calcein, tetramethylrhodamine methylester, and dihydrodichlorofluorescein, respectively. Necrotic cell death increased after reoxygenation. Necrosis was blocked by 1 μM cyclosporin A, an MPT inhibitor, and by reoxygenation at pH 6.2. Confocal imaging of Rhod-2, calcein, and dichlorofluorescein revealed that an increase of mitochondrial Ca(2+) and ROS preceded onset of the MPT after reoxygenation. Intramitochondrial Ca(2+) chelation, but not cytosolic Ca(2+) chelation, prevented ROS formation and subsequent necrotic and apoptotic cell death. Reoxygenation with the antioxidants, desferal or diphenylphenylenediamine, also suppressed MPT-mediated cell death. However, inhibition of cytosolic ROS by apocynin or diphenyleneiodonium chloride failed to prevent reoxygenation-induced cell death. In conclusion, Ca(2+)-dependent mitochondrial ROS formation is the molecular signal culminating in onset of the MPT after reoxygenation of anoxic hepatocytes, leading to cell death.     

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 4 h 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 1 h 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.     

Mitochondrial Dysfunction in the Pathogenesis of Necrotic and Apoptotic Cell Death

Mitochondria are frequently the target of injury after stresses leading to necrotic and apoptoticcell death. Inhibition of oxidative phosphorylation progresses to uncoupling when opening ofa high conductance permeability transition (PT) pore in the mitochondrial inner membraneabruptly increases the permeability of the mitochondrial inner membrane to solutes of molecularmass up to 1500 Da. Cyclosporin A (CsA) blocks this mitochondrial permeability transition(MPT) and prevents necrotic cell death from oxidative stress, Ca²⁺ ionophore toxicity,Reye-related drug toxicity, pH-dependent ischemia/reperfusion injury, and other models of cell injury.Confocal fluorescence microscopy directly visualizes onset of the MPT from the movementof green-fluorescing calcein into mitochondria and the simultaneous release from mitochondriaof red-fluorescing tetramethylrhodamine methylester, a membrane potential-indicatingfluorophore. In oxidative stress to hepatocytes induced by tert-butylhydroperoxide, NAD(P)Hoxidation, increased mitochondrial Ca²⁺, and mitochondrial generation of reactive oxygen speciesprecede and contribute to onset of the MPT. Confocal microscopy also shows directly thatthe MPT is a critical event in apoptosis of hepatocytes induced by tumor necrosis factor-.Progression to necrotic and apoptotic cell killing depends, at least in part, on the effect theMPT has on cellular ATP levels. If ATP levels fall profoundly, necrotic killing ensues. If ATPlevels are at least partially maintained, apoptosis follows the MPT. Cellular features of bothapoptosis and necrosis frequently occur together after death signals and toxic stresses. A newterm, necrapoptosis, describes such death processes that begin with a common stress or deathsignal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmedcellular resorption (apoptosis) depending on modifying factors such as ATP.     

Role of the mitochondrial permeability transition in apoptotic and necrotic death after ischemia/reperfusion injury to hepatocytes

Reperfusion of ATP-depleted tissues after warm or cold ischemia causes pH-dependent necrotic and apoptotic cell death. In hepatocytes and other cell types as well, the mechanism underlying this reperfusion-induced cell death involves onset of the mitochondrial permeability transition (MPT). Opening of permeability transition (PT) pores in the mitochondrial inner membrane initiates the MPT, an event blocked by cyclosporin A (CsA) and pH less than 7.4. Thus, both acidotic pH and CsA prevent MPT-dependent reperfusion injury. Glycine also blocks reperfusion-induced necrosis but acts downstream of PT pore opening by stabilizing the plasma membrane. After the MPT, ATP availability from glycolysis or other source determines whether cell injury after reperfusion progresses to ATP depletion-dependent necrosis or ATP-requiring apoptosis. Thus, apoptosis and necrosis after reperfusion share a common pathway, the MPT. Cell injury progressing to either necrosis or apoptosis by shared pathways can be more aptly termed necrapoptosis.     

The Mitochondrial Permeability Transition Is Required for Tumor Necrosis Factor Alpha-Mediated Apoptosis and Cytochrome c Release

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

Mitochondrial permeability transition: a common pathway to necrosis and apoptosis

Opening of high conductance permeability transition pores in mitochondria initiates onset of the mitochondrial permeability transition (MPT). The MPT is a causative event, leading to necrosis and apoptosis in hepatocytes after oxidative stress, Ca(2+) toxicity, and ischemia/reperfusion. CsA blocks opening of permeability transition pores and protects cell death after these stresses. In contrast to necrotic cell death which is a consequence of ATP depletion, ATP is required for the development of apoptosis. Reperfusion and the return of normal pH after ischemia initiate the MPT, but the balance between ATP depletion after the MPT and ATP generation by glycolysis determines whether the fate of cells will be apoptotic or necrotic death. Thus, the MPT is a common pathway leading to both necrotic and apoptotic cell death after ischemia/reperfusion.     

Flurbiprofen,a Cyclooxygenase Inhibitor,Protects Mice from Hepatic Ischemia/Reperfusion Injury by Inhibiting GSK-3β Signaling and Mitochondrial Permeability Transition

Flurbiprofen acts as a nonselective inhibitor for cyclooxygenases (COX-1 and COX-2), but its impact on hepatic ischemia/reperfusion (I/R) injury remains unclear. Mice were randomized into sham, I/R and flurbiprofen (Flurb) groups. The hepatic artery and portal vein to the left and median liver lobes were occluded for 90 min and unclamped for reperfusion to establish a model of segmental (70%) warm hepatic ischemia. Pretreatment of animals with flurbiprofen prior to I/R insult significantly decreased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), and prevented hepatocytes from I/R-induced apoptosis/necrosis. Moreover, flurbiprofen dramatically inhibited mitochondrial permeability transition (MPT) pore opening, and thus prevented mitochondrial-related cell death and apoptosis. Mechanistic studies revealed that flurbiprofen markedly inhibited glycogen synthase kinase (GSK)-3β activity and increased phosphorylation of GSK-3β at Ser9, which, consequently, could modulate the adenine nucleotide translocase (ANT)–cyclophilin D (CyP-D) complex and the susceptibility to MPT induction. Therefore, administration of flurbiprofen prior to hepatic I/R ameliorates mitochondrial and hepatocellular damage through inhibition of MPT and inactivation of GSK-3β, and provides experimental evidence for clinical use of flurbiprofen to protect liver function in surgical settings in addition to its conventional use for pain relief.     

Necrosis and apoptosis: sequence of liver damage following reperfusion after 60 min ischemia in rats

This study evaluated the time-dependent modes of cell death that occur during the course of reperfusion after 60 min ischemia. The serum ALT level increased immediately after reperfusion, peaked at 6 h and then declined gradually thereafter. This was supported by the H&E staining of the liver tissues taken at 2 h reperfusion, which revealed massive peri-portal necrosis. The succinate driven mitochondrial-swelling rate, release of cytochrome c into the cytoplasm, increase in caspase-3 activity and TUNEL stained tissue were measured to determine the changes in the biochemical markers of apoptosis. The biochemical markers of apoptosis increased by 2 h of reperfusion, peaked at 6 h and remained elevated throughout the 24 h reperfusion period. Cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT), inhibited MPT opening, the release of cytochrome c and caspase-3 activation. This indicates that necrotic death occurs particularly in the peri-portal region in the initial period of reperfusion, and delayed apoptotic death occurs primarily in the peri-central region in the liver tissues undergoing I/R.     

Induction of the mitochondrial permeability transition in cultured astrocytes by glutamine

Ammonia is a toxin that has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE), and astrocytes appear to be the principal target of ammonia toxicity. Glutamine, a byproduct of ammonia metabolism, has been implicated in some of the deleterious effects of ammonia on the CNS. We have recently shown that ammonia induces the mitochondrial permeability transition (MPT) in cultured astrocytes, but not in neurons. We therefore determined whether glutamine is also capable of inducing the MPT in cultured astrocytes. Astrocytes were treated with glutamine (4.5 mM) for various time periods and the MPT was assessed by changes in 2-deoxyglucose (2-DG) mitochondrial permeability, calcein fluorescence assay, and by changes in cyclosporin A (CsA)-sensitive inner mitochondrial membrane potential (deltapsi(m)) using the potentiometric dye, JC-1. Astrocytes treated with glutamine significantly increased 2-DG permeability (120%, P<0.01), decreased mitochondrial calcein fluorescence, and concomitantly dissipated the deltapsi(m). All of these effects were blocked by CsA. These data indicate that glutamine induces the MPT in cultured astrocytes. The induction of the MPT by glutamine in astrocytes, and the subsequent development of mitochondrial dysfunction, may partially explain the deleterious affects of glutamine on the CNS in the setting of hyperammonemia.     

Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion

To clarify the relationship between reactive oxygen species (ROS) and cell death during ischemia-reperfusion (I/R), we studied cell death mechanisms in a cellular model of I/R. Oxidant stress during simulated ischemia was detected in the mitochondrial matrix using mito-roGFP, a ratiometric redox sensor, and by Mito-Sox Red oxidation. Reperfusion-induced death was attenuated by over-expression of Mn-superoxide dismutase (Mn-SOD) or mitochondrial phospholipid hydroperoxide glutathione peroxidase (mito-PHGPx), but not by catalase, mitochondria-targeted catalase, or Cu,Zn-SOD. Protection was also conferred by chemically distinct antioxidant compounds, and mito-roGFP oxidation was attenuated by NAC, or by scavenging of residual O₂ during the ischemia (anoxic ischemia). Mitochondrial permeability transition pore (mPTP) oscillation/opening was monitored by real-time imaging of mitochondrial calcein fluorescence. Oxidant stress caused release of calcein to the cytosol during ischemia, a response that was inhibited by chemically diverse antioxidants, anoxia, or over-expression of Mn-SOD or mito-PHGPx. These findings suggest that mitochondrial oxidant stress causes oscillation of the mPTP prior to reperfusion. Cytochrome c release from mitochondria to the cytosol was not detected until after reperfusion, and was inhibited by anoxic ischemia or antioxidant administration during ischemia. Although DNA fragmentation was detected after I/R, no evidence of Bax activation was detected. Over-expression of the anti-apoptotic protein Bcl-X_L in cardiomyocytes did not confer protection against I/R-induced cell death. Moreover, murine embryonic fibroblasts with genetic depletion of Bax and Bak, or over-expression of Bcl-X_L, failed to show protection against I/R. These findings indicate that mitochondrial ROS during ischemia triggers mPTP activation, mitochondrial depolarization, and cell death during reperfusion through a Bax/Bak-independent cell death pathway. Therefore, mitochondrial apoptosis appears to represent a redundant death pathway in this model of simulated I/R. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Cell death during ischemia: relationship to mitochondrial depolarization and ROS generation

Ischemia-reperfusion injury induces cell death, but the responsible mechanisms are not understood. This study examined mitochondrial depolarization and cell death during ischemia and reperfusion. Contracting cardiomyocytes were subjected to 60-min ischemia followed by 3-h reperfusion. Mitochondrial membrane potential (DeltaPsi(m)) was assessed with tetramethylrhodamine methyl ester. During ischemia, DeltaPsi(m) decreased to 24 +/- 5.5% of baseline, but no recovery was evident during reperfusion. Cell death assessed by Sytox Green was minimal during ischemia but averaged 66 +/- 7% after 3-h reperfusion. Cyclosporin A, an inhibitor of mitochondrial permeability transition, was not protective. However, pharmacological antioxidants attenuated the fall in DeltaPsi(m) during ischemia and cell death after reperfusion and decreased lipid peroxidation as assessed with C11-BODIPY. Cell death was also attenuated when residual O(2) was scavenged from the perfusate, creating anoxic ischemia. These results suggested that reactive oxygen species (ROS) were important for the decrease in DeltaPsi(m) during ischemia. Finally, 143B-rho(0) osteosarcoma cells lacking a mitochondrial electron transport chain failed to demonstrate a depletion of DeltaPsi(m) during ischemia and were significantly protected against cell death during reperfusion. Collectively, these studies identify a central role for mitochondrial ROS generation during ischemia in the mitochondrial depolarization and subsequent cell death induced by ischemia and reperfusion in this model.     

Mitochondrial impairment induced by poly(ADP-ribose) polymerase-1 activation in cortical neurons after oxygen and glucose deprivation

Neuronal cells injured by ischemia and reperfusion to a certain extent are committed to death in necrotic or apoptotic form. Necrosis is induced by gross ATP depletion or 'energy crisis' of the cell, whereas apoptosis is induced by a mechanism still to be defined in detail. Here, we investigated this mechanism by focusing on a DNA damage-sensor, poly(ADP-ribose) polymerase-1 (PARP-1). A 2-h oxygen and glucose deprivation (OGD) followed by reoxygenation (Reox) induced apoptosis, rather than necrosis, in rat cortical neurons. During the Reox, PARP-1 was much activated and autopoly(ADP-ribosyl)ated, consuming the substrate, NAD+. Induction of apoptosis by OGD/Reox was suppressed by overexpression of Bcl-2, indicating mitochondrial impairment in this induction process. Mitochondrial permeability transition (MPT), or membrane depolarization, and a release of proapoptotic proteins, i.e. cytochrome c, apoptosis-inducing factor and endonuclease G, from mitochondria were observed during the Reox. These apoptotic changes of mitochondria and the nucleus were attenuated by PARP-1 inhibitors, 1,5-dihydroxyisoquinoline and benzamide, and also by small interfering RNA specific for PARP-1. These results indicated that PARP-1 plays a principal role in inducing mitochondrial impairment that ultimately leads to apoptosis of neurons after cerebral ischemia.     

Detecting mitochondrial permeability transition by confocal imaging of intact cells pinocytically loaded with calcein.

When studied in vitro, mitochondrial permeability transition (MPT) is associated with an increase in mitochondrial permeability to solutes up to 1500 Da in mass and a loss of electrical potential difference across the inner mitochondrial membrane (Deltapsimit). The MPT has been implicated as being important in cellular calcium homeostasis, autophagy and cell death via necrosis and apoptosis. Thus, it is important to develop a valid technique for accurate measurement of this phenomenon in intact cells. We developed a procedure for the detection of MPT in intact cells that avoids the disadvantages associated with earlier approaches. In this new technique, unmodified (green-fluorescent) calcein is simultaneously introduced into the cytosol of millions of cells by the process of pinocytic loading and, to identify the position of individual mitochondria and to measure Deltapsimit, the cells are counter-stained with a red-fluorescing potentiometric dye. Using this approach with a variety of cell types, we demonstrate that cytosolic calcein is excluded from normal polarized mitochondria but enters them during MPT. This technique may be valuable in studies investigating the cellular functions of MPT.     

Cyclophilin D is required for mitochondrial removal by autophagy in cardiac cells

Autophagy is a highly regulated intracellular degradation process by which cells remove cytosolic long-lived proteins and damaged organelles. The mitochondrial permeability transition (MPT) results in mitochondrial depolarization and increased reactive oxygen species production, which can trigger autophagy. Therefore, we hypothesized that the MPT may have a role in signaling autophagy in cardiac cells. Mitochondrial membrane potential was lower in HL-1 cells subjected to starvation compared to cells maintained in full medium. Mitochondrial membrane potential was preserved in starved cells treated with cyclosporin A (CsA), suggesting the MPT pore is associated with starvation-induced depolarization. Starvation-induced autophagy in HL-1 cells, neonatal rat cardiomyocytes and adult mouse cardiomyocytes was inhibited by CsA. Starvation failed to induce autophagy in CypD-deficient murine cardiomyocytes, whereas in myocytes from mice overexpressing CypD the levels of autophagy were enhanced even under fed conditions. Collectively, these results demonstrate a role for CypD and the MPT in the initiation of autophagy. We also analyzed the role of the MPT in the degradation of mitochondria by biochemical analysis and electron microscopy. HL-1 cells subjected to starvation in the presence of CsA had higher levels of mitochondrial proteins (by Western blot), more mitochondria and less autophagosomes (by electron microscopy) than cells starved in the absence of CsA. Our results suggest a physiologic function for CypD and the MPT in the regulation of starvation-induced autophagy. Starvation-induced autophagy regulated by CypD and the MPT may represent a homeostatic mechanism for cellular and mitochondrial quality control.     

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/cm² (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 (～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. J. Cell. Physiol. 218: 603–611, 2009. © 2008 Wiley‐Liss, Inc.     

Manganese induces the mitochondrial permeability transition in cultured astrocytes

Manganese is known to cause central nervous system injury leading to parkinsonism and to contribute to the pathogenesis of hepatic encephalopathy. Although mechanisms of manganese neurotoxicity are not completely understood, chronic exposure of various cell types to manganese has shown oxidative stress and mitochondrial energy failure, factors that are often implicated in the induction of the mitochondrial permeability transition (MPT). In this study, we examined whether exposure of cultured neurons and astrocytes to manganese induces the MPT. Cells were treated with manganese acetate (10-100 microM), and the MPT was assessed by changes in the mitochondrial membrane potential and in mitochondrial calcein fluorescence. In astrocytes, manganese caused a dissipation of the mitochondrial membrane potential and decreased the mitochondrial calcein fluorescence in a concentration- and time-dependent manner. These changes were completely blocked by pretreatment with cyclosporin A, consistent with induction of the MPT. On the other hand, similarly treated cultured cortical neurons had a delayed or reduced MPT as compared with astrocytes. The manganese-induced MPT in astrocytes was blocked by pretreatment with antioxidants, suggesting the potential involvement of oxidative stress in this process. Induction of the MPT by manganese and associated mitochondrial dysfunction in astrocytes may represent key mechanisms in manganese neurotoxicity.     

Silibinin treatment results in reducing OPA1&MFN1 genes expression in a rat model hepatic ischemia–reperfusion

The mitochondrial damage has a pivotal role in triggering apoptosis and cell death. This study assessed the effect of silibinin on optical atrophy-1 (OPA1) and mitofusin-1 (MFN1) gene expression in liver tissue during hepatic warm ischemia–reperfusion (IR). Four groups of rats, eight rats each were designed: Vehicle: the rats received normal saline and encountered to laparotomy, Sili: silibinin (60 mg/kg) was administered to animals, IR: the rats received the normal saline and insulted by liver IR procedure, and IR?+?Sili: silibinin was injected to rats. All groups were subjected to the same process of injection of the solvent or silibinin (30 min before laparotomy or ischemia and immediately after the reperfusion), intraperitoneally (IP). After 3 h of reperfusion, blood and liver tissue samples were collected for future examinations. Our results showed no significant differences between the Vehicle and Sili groups in all assessed parameters. In IR?+?Sili, the increased serum levels of AST and ALT in comparison with the control group were markedly reduced by silibinin treatment. Silibinin lowered the elevated expression of OPA1 and MFN1 mRNAs in the IR group. Histology revealed silibinin could decline tissue degeneration compared to the IR group. Electron microscopy of control and silibinin groups showed no fusion of mitochondria and tissue degradation both of which were observed in the IR group. The extent of tissue destruction and mitochondrial fusion decreased significantly with silibinin treatment. Silibinin has a protective effect on liver cells against IR induced injuries by preserving mitochondrial membrane.

     

A prolyl-hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate, induces haem oxygenase-1 expression in human cells through a mechanism independent of hypoxia-inducible factor-1alpha

Hypoxia-inducible factor (HIF)-1 is important for cellular homeostasis under hypoxia. Expression of haem oxygenase-1 (HO-1), an essential enzyme in haem catabolism, varies under hypoxia, depending on cell types. Here, we studied the role of HIF-1alpha, a component of HIF-1, in the regulation of HO-1 expression using three human cell lines: HeLa cervical cancer, and ARPE-19 and D407 retinal pigment epithelial cells. Under hypoxia (1% O(2)), the expression of HO-1 mRNA was decreased in HeLa cells, increased in D407 cells, and unchanged in ARPE-19 cells, while HIF-1alpha protein was accumulated in these cell lines. Thus, HIF-1alpha is unlikely to function as a key regulator for HO-1 expression under hypoxia. We then used ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor of prolyl hydroxylases, to accumulate HIF-1alpha protein under normoxia. Treatment with EDHB (250-500 microM) increased HIF-1alpha protein levels in HeLa and D407 cells, but not in ARPE-19 cells, whereas EDHB at lower concentrations (50-100 microM) consistently induced HO-1 mRNA expression (about 20-fold) in these three cell lines. Moreover, EDHB increased the HO-1 gene promoter activity via the enhancer that lacks a HIF-1-binding site. In conclusion, the signals evoked by hypoxia and after EDHB treatment differentially regulate HO-1 mRNA expression through HIF-1alpha-independent mechanisms.