oligomycins and 6-hydroxy-2-5-7-8-tetramethylchroman-2-carboxylic-acid

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of cellular responses to reduced oxygen availability. The contribution of mitochondria in regulation of HIF-1alpha in hypoxic cells has received recent attention. We demonstrate that inhibition of electron transport complexes I, III, and IV diminished hypoxic HIF-1alpha accumulation in different tumor cell lines. Hypoxia-induced HIF-1alpha accumulation was not prevented by the antioxidants Trolox and N-acetyl-cysteine. Oligomycin, inhibitor of F(0)F(1)-ATPase, prevented hypoxia-induced HIF-1alpha protein accumulation and had no effect on HIF-1alpha induction by hypoxia-mimicking agents desferrioxamine or dimethyloxalylglycine. The inhibitory effect of mitochondrial respiratory chain inhibitors and oligomycin on hypoxic HIF-1alpha content was pronounced in cells exposed to hypoxia (1.5% O(2)) but decreased markedly when cells were exposed to severe oxygen deprivation (anoxia). Taken together, these results do not support the role for mitochondrial reactive oxygen species in HIF-1alpha regulation, but rather suggest that inhibition of electron transport chain and impaired oxygen consumption affect HIF-1alpha accumulation in hypoxic cells indirectly via effects on prolyl hydroxylase function.

Topics: Acetylcysteine; Amino Acids, Dicarboxylic; Cell Hypoxia; Chromans; Deferoxamine; DNA-Binding Proteins; Electron Transport; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Mitochondria; Nuclear Proteins; Oligomycins; Proton-Translocating ATPases; Reactive Oxygen Species; Transcription Factors; Tumor Cells, Cultured

Preadipocytes are present and can proliferate to increase fat mass throughout adult life. The importance of mitochondria in these cells has never been investigated, although we recently reported that mitochondrial oxidative metabolism is non-negligible in white preadipocytes. Mitochondrial reactive oxygen species generation is intimately associated with respiratory chain function. An increasing number of reports support their role as signalling molecules. The aim of this work was to study the effects of mitochondrial reactive oxygen species on proliferation of white preadipocytes. Rotenone and oligomycin, inhibitors of complex I and of ATP synthase respectively, increased H(2)O(2) and inhibited cell growth of preadipocytes (without inducing necrosis or apoptosis). These effects were partly prevented by addition of radical scavengers. A chemical uncoupler had opposite effects on reactive oxygen species generation and cell growth. Propofol, which inhibits complex I but also scavenges free radicals, had effects similar to those of the uncoupler on both parameters. Thus, mitochondrial reactive oxygen species can influence development of adipose tissue by affecting the size of the white preadipocyte pool.

Topics: 3T3 Cells; Adipocytes; Animals; Butylated Hydroxyanisole; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Division; Cells, Cultured; Chromans; Enzyme Inhibitors; Free Radical Scavengers; Humans; Hydrogen Peroxide; Mice; Mitochondria; Oligomycins; Propofol; Reactive Oxygen Species; Rhodamines; Rotenone; Stromal Cells; Uncoupling Agents

In this study, we investigated the involvement of reactive oxygen species (ROS) and calcium in staurosporine (STS)-induced apoptosis in cultured retinal neurons, under conditions of maintained membrane integrity. The antioxidants idebenone (IDB), glutathione-ethylester (GSH/EE), trolox, and Mn(III)tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) significantly reduced STS-induced caspase-3-like activity and intracellular ROS generation. Endogenous sources of ROS production were investigated by testing the effect of the following inhibitors: 7-nitroindazole (7-NI), a specific inhibitor of the neuronal isoform of nitric oxide synthase (nNOS); arachidonyl trifluoromethyl ketone (AACOCF(3)), a phospholipase A(2) (PLA(2)) inhibitor; allopurinol, a xanthine oxidase inhibitor; and the mitochondrial inhibitors rotenone and oligomycin. All these compounds decreased caspase-3-like activity and ROS generation, showing that both mitochondrial and cytosolic sources of ROS are implicated in this mechanism. STS induced a significant increase in intracellular calcium concentration ([Ca(2+)](i)), which was partially prevented in the presence of IDB and GSH/EE, indicating its dependence on ROS generation. These two antioxidants and the inhibitors allopurinol and 7-NI also reduced the number of TdT-mediated dUTP nick-end labeling-positive cells. Thus, endogenous ROS generation and the rise in intracellular calcium are important inter-players in STS-triggered apoptosis. Furthermore, the antioxidants may help to prolong retinal cell survival upon apoptotic cell death.

Topics: Adenine; Allopurinol; Animals; Antioxidants; Apoptosis; Arachidonic Acids; Benzoquinones; Blotting, Western; Calcium; Carbon; Caspase 3; Caspases; Cell Death; Cell Survival; Chick Embryo; Chromans; Coloring Agents; Cytosol; DNA Fragmentation; Enzyme Inhibitors; Glutathione; In Situ Nick-End Labeling; Indazoles; Metalloporphyrins; Mitochondria; Neurons; Nitric Oxide Synthase; Oligomycins; Protein Isoforms; Reactive Oxygen Species; Retina; Rotenone; Staurosporine; Tetrazolium Salts; Thiazoles; Time Factors; Ubiquinone; Uncoupling Agents; Xanthine Oxidase

2-Oxoglutarate-supported rat liver mitochondria were loaded with moderate amounts of calcium and submitted to O2 deprivation and reoxygenation. In the presence of acetoacetate, anaerobic energy production maintained Ca2+ retention by mitochondria during the anoxia period unless the Pi concentration of the incubation solution was raised to 4-6 mM. Acetoacetate prompted Ca2+ release from O2-deprived mitochondria at elevated Pi levels, presumably due to occurrence of a permeability transition of the inner membrane. Providing 3-hydroxybutyrate and malate, together with acetoacetate, was found to delay the permeability transition until O2 was reintroduced, i.e., O2 triggered a paradoxical release of Ca2+ from mitochondria under these conditions. Whether initiated by O2 in the presence of Pi or by Pi under aerobic conditions, Ca2+ release was initially activated and subsequently inhibited or reversed in the presence of alpha-tocopherol (10-90 mumol.g protein-1). Similar effects were exerted by alpha-tocopherol during Pi-induced Ca2+ release from oligomycin-treated mitochondria supported by succinate (+ rotenone). In addition, the permeability transition was delayed by retinol (3-30 mumol.g protein-1) while beta-carotene, ubiquinone, and water-soluble antioxidants, including Trolox C, were ineffective. Other observations suggest that the Ca(2+)-releasing and/or -retaining effects of alpha-tocopherol and retinol may be independent from pro- and/or antioxidant activities. Effects resembling those of alpha-tocopherol were exerted by alpha-tocopherol succinate, which is devoid of antioxidant activity. Our data indicate that the permeability transition of Ca(2+)-loaded liver mitochondria may be triggered by O2, in the presence of ketone bodies, and affected by lipid-soluble antioxidants through mechanisms seemingly unrelated to free-radical generation or scavenging.

Topics: Acetoacetates; Anaerobiosis; Animals; Antioxidants; beta Carotene; Calcium; Carotenoids; Chromans; Hypoxia; Kinetics; Mitochondria, Liver; Oligomycins; Oxygen; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Ubiquinone; Vitamin A; Vitamin E

Addition of iron(III)-gluconate complex to isolated rat liver mitochondria resulted in an increased iron content of mitochondria. Iron was accumulated through a relatively fast process (maximal uptake in less than 2 min incubation) by an energy-independent mechanism. The in vitro iron overload of mitochondria was associated with enhancement in the oxygen consumption, which was due to the induction of lipoperoxidative processes catalyzed by iron. It was found that a concentration of iron as low as 0.1 mM elicits a consistent production of malondialdehyde in mitochondria. Concomitant with the induction of lipoperoxidation a progressive fall in the mitochondrial membrane potential was observed. The occurrence of energy-consuming processes as a consequence of iron addition, and particularly the enhancement of endogenous Ca2+ cycling across the membrane, was suggested as the cause of the membrane potential drop.

Topics: Animals; Butylated Hydroxytoluene; Chromans; Deferoxamine; Dose-Response Relationship, Drug; Edetic Acid; Electrochemistry; Energy Metabolism; Ferric Compounds; Iron; Malondialdehyde; Membrane Potentials; Mitochondria, Liver; Oligomycins; Oxygen Consumption; Rats

It has been found that addition of iron(III)-gluconate complex to rat liver mitochondria disturbed the mitochondrial Ca2+ transport. Indirect evidence when the changes in the membrane potential during the transport of Ca2+ were followed, as well as direct evidence, when the fluxes of Ca2+ were monitored by a Ca2+-selective electrode, indicated that this iron complex induced an efflux of Ca2+ from liver mitochondria. The mechanisms by which iron induced Ca2+ release appeared to be linked to the induction of lipoperoxidation of mitochondrial membrane. The mitochondrial membrane, however, did not become irreversibly damaged under these conditions, as indicated by its complete repolarization. It was also shown that the induction by iron of lipoperoxidation brought about an efflux of K+ from mitochondria.

Topics: Animals; Calcium; Chromans; Deferoxamine; Edetic Acid; Ferric Compounds; Iron; Magnesium; Membrane Potentials; Mitochondria, Liver; Oligomycins; Potassium; Rats; Succinates; Succinic Acid