Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury. (1/1245)

We investigated mechanisms of cell death during hypoxia/reoxygenation of cultured kidney cells. During glucose-free hypoxia, cell ATP levels declined steeply resulting in the translocation of Bax from cytosol to mitochondria. Concurrently, there was cytochrome c release and caspase activation. Cells that leaked cytochrome c underwent apoptosis after reoxygenation. ATP depletion induced by a mitochondrial uncoupler resulted in similar alterations even in the presence of oxygen. Moreover, inclusion of glucose during hypoxia prevented protein translocations and reoxygenation injury by maintaining intracellular ATP. Thus, ATP depletion, rather than hypoxia per se, was the cause of protein translocations. Overexpression of Bcl-2 prevented cytochrome c release and reoxygenation injury without ameliorating ATP depletion or Bax translocation. On the other hand, caspase inhibitors did not prevent protein translocations, but inhibited apoptosis during reoxygenation. Nevertheless, they could not confer long-term viability, since mitochondria had been damaged. Omission of glucose during reoxygenation resulted in continued failure of ATP production, and cell death with necrotic morphology. In contrast, cells expressing Bcl-2 had functional mitochondria and remained viable during reoxygenation even without glucose. Therefore, Bax translocation during hypoxia is a molecular trigger for cell death during reoxygenation. If ATP is available during reoxygenation, apoptosis develops; otherwise, death occurs by necrosis. By preserving mitochondrial integrity, BCL-2 prevents both forms of cell death and ensures cell viability.  (+info)

Beneficial effects of raxofelast (IRFI 016), a new hydrophilic vitamin E-like antioxidant, in carrageenan-induced pleurisy. (2/1245)

1. Peroxynitrite is a strong oxidant that results from reaction between NO and superoxide. It has been recently proposed that peroxynitrite plays a pathogenetic role in inflammatory processes. Here we have investigated the therapeutic efficacy of raxofelast, a new hydrophilic vitamin E-like antioxidant agent, in rats subjected to carrageenan-induced pleurisy. 2. In vivo treatment with raxofelast (5, 10, 20 mg kg(-1) intraperitoneally 5 min before carrageenan) prevented in a dose dependent manner carrageenan-induced pleural exudation and polymorphonuclear migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels, as well as histological organ injury were significantly reduced by raxofelast. 3. Immunohistochemical analysis for nitrotyrosine, a footprint of peroxynitrite, revealed a positive staining in lungs from carrageenan-treated rats. No positive nitrotyrosine staining was found in the lungs of the carrageenan-treated rats, which received raxofelast (20 mg kg 1) treatment. 4. Furthermore, in vivo raxofelast (5, 10, 20 mg kg(-1)) treatment significantly reduced peroxynitrite formation as measured by the oxidation of the fluorescent dihydrorhodamine 123, prevented the appearance of DNA damage, the decrease in mitochondrial respiration and partially restored the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy. 5. In conclusion, our study demonstrates that raxofelast, a new hydrophilic vitamin E-like antioxidant agent, exerts multiple protective effects in carrageenan-induced acute inflammation.  (+info)

Cytosolic Ca2+ movements of endothelial cells exposed to reactive oxygen intermediates: role of hydroxyl radical-mediated redox alteration of cell-membrane Ca2+ channels. (3/1245)

1. The mode of action of reactive oxygen intermediates in cysosolic Ca2+ movements of cultured porcine aortic endothelial cells exposed to xanthine/xanthine oxidase (X/XO) was investigated. 2. Cytosolic Ca2+ movements provoked by X/XO consisted of an initial Ca2+ release from thapsigargin-sensitive intracellular Ca2+ stores and a sustained Ca2+ influx through cell-membrane Ca2+ channels. The Ca2+ movements from both sources were inhibited by catalase, cell-membrane permeable iron chelators (o-phenanthroline and deferoxamine), a *OH scavenger (5,5-dimethyl-1-pyrroline-N-oxide), or an anion channel blocker (disodium 4, 4'-diisothiocyano-2, 2'-stilbenedisulphonic acid), suggesting that *O2- influx through anion channels was responsible for the Ca2+ movements, in which *OH generation catalyzed by intracellular transition metals (i.e., Haber-Weiss cycle) was involved. 3. After an initial Ca2+ elevation provoked by X/XO, cytosolic Ca2+ concentration decreased to a level higher than basal levels. Removal of X/XO slightly enhanced the Ca2+ decrease. Extracellular addition of sulphydryl (SH)-reducing agents, dithiothreitol or glutathione, after the removal of X/XO accelerated the decrement. A Ca2+ channel blocker, Ni2+, abolished the sustained increase in Ca2+, suggesting that Ca2+ influx through cell-membrane Ca2+ channels was extracellularly regulated by the redox state of SH-groups. 4. The X/XO-provoked change in cellular respiration was inhibited by Ni2+ or dithiothreitol as well as inhibitors of Haber-Weiss cycle, suggesting that Ca2+ influx was responsible for *OH-mediated cytotoxicity. We concluded that intracellular *OH generation was involved in the Ca2+ movements in endothelial cells exposed to X/XO. Cytosolic Ca2+ elevation was partly responsible for the oxidants-mediated cytotoxicity.  (+info)

Myogenin induces a shift of enzyme activity from glycolytic to oxidative metabolism in muscles of transgenic mice. (4/1245)

Physical training regulates muscle metabolic and contractile properties by altering gene expression. Electrical activity evoked in muscle fiber membrane during physical activity is crucial for such regulation, but the subsequent intracellular pathway is virtually unmapped. Here we investigate the ability of myogenin, a muscle-specific transcription factor strongly regulated by electrical activity, to alter muscle phenotype. Myogenin was overexpressed in transgenic mice using regulatory elements that confer strong expression confined to differentiated post-mitotic fast muscle fibers. In fast muscles from such mice, the activity levels of oxidative mitochondrial enzymes were elevated two- to threefold, whereas levels of glycolytic enzymes were reduced to levels 0.3-0.6 times those found in wild-type mice. Histochemical analysis shows widespread increases in mitochondrial components and glycogen accumulation. The changes in enzyme content were accompanied by a reduction in fiber size, such that many fibers acquired a size typical of oxidative fibers. No change in fiber type-specific myosin heavy chain isoform expression was observed. Changes in metabolic properties without changes in myosins are observed after moderate endurance training in mammals, including humans. Our data suggest that myogenin regulated by electrical activity may mediate effects of physical training on metabolic capacity in muscle.  (+info)

Mitochondrial group II introns, cytochrome c oxidase, and senescence in Podospora anserina. (5/1245)

Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (alpha) has been thought to play a prominent role in this syndrome. Intron alpha is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of the COX1 gene. We describe here the first mutant of P. anserina that has the alpha sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron alpha. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron alpha is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron alpha plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, "immortality" can be acquired not by the absence of intron alpha but rather by the lack of active cytochrome c oxidase.  (+info)

Oxygen-dependent inhibition of respiration in isolated renal tubules by nitric oxide. (6/1245)

BACKGROUND: The partial pressure (tension) of oxygen (PO2) in the kidney medulla has been established to be lower than that of the cortex. The kidney medulla has been shown to be particularly sensitive to hypoxia. However, the measured PO2 in the kidney medulla is sufficient to support maximal respiration. It has been recently shown that endogenously produced nitric oxide (NO) may inhibit oxygen consumption in the kidney. We studied whether NO plays a role in hypersensitivity of the kidney medulla to hypoxia. METHODS: We studied the effect of added NO on isolated cortical and outer medullary renal tubules in simultaneous oxygen consumption and NO measurements at different oxygen concentrations. RESULTS: We found that NO could potently and reversibly inhibit respiration at nanomolar concentrations. The inhibitory effect of NO was markedly increased at low physiological oxygen concentrations. The effect of NO was cGMP independent because the selective guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) at a 10 microM concentration had no effect on basal or NO-inhibited respiration. The value for half-maximal NO-mediated inhibition of respiration was virtually identical to that found in isolated mitochondria, and therefore, NO was most likely directly acting on mitochondria. Interestingly, we found no differences in sensitivity to NO-mediated inhibition between outer medullary and cortical tubules. CONCLUSIONS: We suggest that because of its low PO2, the renal outer medulla is more sensitive to hypoxia, not because of the low PO2 as such, but probably because of the competition between NO and oxygen to control respiration.  (+info)

Declines in mitochondrial respiration during cardiac reperfusion: age-dependent inactivation of alpha-ketoglutarate dehydrogenase. (7/1245)

We previously reported that cardiac reperfusion results in declines in mitochondrial NADH-linked respiration. The degree of inactivation increased with age and was paralleled by modification of protein by the lipid peroxidation product 4-hydroxy-2-nonenal. To gain insight into potential sites of oxidative damage, the present study was undertaken to identify specific mitochondrial protein(s) inactivated during ischemia and reperfusion and to determine which of these losses in activity are responsible for observed declines in mitochondrial respiration. Using a Langendorff rat heart perfusion protocol, we observed age-dependent inactivation of complex I during ischemia and complex IV and alpha-ketoglutarate dehydrogenase during reperfusion. Although losses in complex I and IV activities were found not to be of sufficient magnitude to cause declines in mitochondrial respiration, an age-related decrease in complex I activity during ischemia may predispose old animals to more severe oxidative damage during reperfusion. It was determined that inactivation of alpha-ketoglutarate dehydrogenase is responsible, in large part, for observed reperfusion-induced declines in NADH-linked respiration. alpha-Ketoglutarate dehydrogenase is highly susceptible to 4-hydroxy-2-nonenal inactivation in vitro. Thus, our results suggest a plausible mechanism for age-dependent, reperfusion-induced declines in mitochondrial function and identify alpha-ketoglutarate dehydrogenase as a likely site of free radical-mediated damage.  (+info)

Respiratory uncoupling induces delta-aminolevulinate synthase expression through a nuclear respiratory factor-1-dependent mechanism in HeLa cells. (8/1245)

Nuclear respiratory factor (NRF)-1 appears to be important for the expression of several respiratory genes, but there is no direct evidence that NRF-1 transduces a physiological signal into the production of an enzyme critical for mitochondrial biogenesis. We generated HeLa cells containing plasmids allowing doxycycline-inducible expression of uncoupling protein (UCP)-1. In the absence of doxycycline, UCP-1 mRNA and protein were undetectable. In the presence of doxycycline, UCP-1 was expressed and oxygen consumption doubled. This rise in oxygen consumption was associated with an increase in NRF-1 mRNA. It was also associated with an increase in NRF-1 protein binding activity as determined by electrophoretic mobility shift assay using a functional NRF-1 binding site from the delta-aminolevulinate (ALA) synthase promoter. Respiratory uncoupling also caused a time-dependent increase in protein levels of ALA synthase, an early marker for mitochondrial biogenesis. ALA synthase induction by respiratory uncoupling was prevented by transfecting cells with an oligonucleotide antisense to the region of the NRF-1 initiation codon; a scrambled oligonucleotide with the same base composition had no effect. Respiratory uncoupling increases oxygen consumption and lowers energy reserves. In HeLa cells, uncoupling also increases ALA synthase, an enzyme critical for mitochondrial respiration, but only if translatable mRNA for NRF-1 is available. These data suggest that the transcription factor NRF-1 plays a key role in cellular adaptation to energy demands by translating physiological signals into an increased capacity for generating energy.  (+info)

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