cytochrome-c-t and myxothiazol

Visible spectroscopy was used to measure real-time changes in the oxidation state of cytochrome c (cyt c) and the a-cytochromes (cyt aa(3)) of cytochrome oxidase during mitochondrial outer membrane permeabilization (MOMP) initiated by anisomycin in HL-60 cells. The oxidation state of mitochondrial cyt c was found to be approximately 62% oxidized before MOMP and became approximately 70% oxidized after MOMP. In contrast, the cytosolic pool of cyt c was found to be almost fully reduced. This oxidation change allows cyt c release to be continuously and quantitatively monitored in real time. Anoxia and antimycin were used to fully reduce and fully oxidize, respectively, the mitochondrial pool of cyt c and it was found that the release of cyt c was independent of it oxidation state consistent with a simple model of cyt c passively diffusing down a concentration gradient through a pore or tear in the outer membrane. After MOMP was complete, the flux of cyt c diffusing back into the mitochondria was measured from the residual mitochondrial oxygen consumption after complete inhibition of the bc(1) with antimycin and myxothiazol. The outer membrane was found to be highly permeable after MOMP implying that the reduction of cyt c in the cytosol must be very rapid. The permeability of the outer membrane measured in this study would result in the release of cyt c with a time constant of less than 1 s.

Topics: Anisomycin; Antifungal Agents; Antimycin A; Caspase 3; Caspase 9; Cytochromes c; Cytosol; Electron Transport; Enzyme Activation; HL-60 Cells; Humans; Methacrylates; Mitochondrial Membranes; Oxidation-Reduction; Oxygen; Oxygen Consumption; Permeability; Protein Synthesis Inhibitors; Rotenone; Spectrum Analysis; Thiazoles; Uncoupling Agents

The effect of hypericin photoactivation on mitochondria of human prostate carcinoma cells was studied using a range of mitochondrial inhibitors. Oligomycin significantly enhanced hypericin phototoxicity while atractyloside and antymicin A conferred a significant protection. Use of myxothiazol did not affect cell survival following hypericin photoactivation. These results signify a protective role for F(1)F(0)-ATP synthase running in reverse mode, and a significant photodamage at the quinone-reducing site of mitochondrial complex III. In light of these results, we performed molecular modeling of hypericin binding to complex III. This revealed three binding sites, two of which coincided with the quinol-oxidizing and quinone-reducing centers. Using submitochondrial particles we examined hypericin as a possible substrate of complex III and compared this to its natural substrate, ubiquinone-10. Our results demonstrate uniquely that hypericin is an efficient substrate for complex III, and this activity is inhibited by myxothiazol and antimycin A. We further demonstrated that hypericin photosensitization completely inactivated complex III with ubiquinone as substrate. The ability to enhance HYP potency by inhibition of F(1)F(0)-ATP synthase or depress HYP efficacy by inhibition at the Qi site of complex III provides a potential to increase the therapeutic index of HYP and amplify its PDT action in tumor cells.

Topics: Anthracenes; Antimycin A; Binding Sites; Cell Line, Tumor; Cytochromes c; Electron Transport Complex III; Humans; Light; Methacrylates; Mitochondria; Mitochondrial Proton-Translocating ATPases; Models, Molecular; Perylene; Spectrophotometry, Ultraviolet; Submitochondrial Particles; Thiazoles; Ubiquinone

Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC-EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis.

Topics: Animals; Cytochromes c; Electron Transport; Electron Transport Complex III; Electron Transport Complex IV; Male; Methacrylates; Mitochondria, Liver; Mitochondria, Muscle; Mitochondrial Diseases; Oxygen Consumption; Potassium Cyanide; Rats; Rats, Wistar; Thiazoles; Ubiquinone

Although programmed cell death (PCD) is extensively studied in multicellular organisms, in recent years it has been shown that a unicellular organism, yeast Saccharomyces cerevisiae, also possesses death program(s). In particular, we have found that a high doses of yeast pheromone is a natural stimulus inducing PCD. Here, we show that the death cascades triggered by pheromone and by a drug amiodarone are very similar. We focused on the role of mitochondria during the pheromone/amiodarone-induced PCD. For the first time, a functional chain of the mitochondria-related events required for a particular case of yeast PCD has been revealed: an enhancement of mitochondrial respiration and of its energy coupling, a strong increase of mitochondrial membrane potential, both events triggered by the rise of cytoplasmic [Ca2+], a burst in generation of reactive oxygen species in center o of the respiratory chain complex III, mitochondrial thread-grain transition, and cytochrome c release from mitochondria. A novel mitochondrial protein required for thread-grain transition is identified.

Topics: Amiodarone; Antifungal Agents; Antimycin A; Apoptosis; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cytochromes c; DNA Fragmentation; Kinetics; Mating Factor; Membrane Potentials; Methacrylates; Mitochondria; Mitochondrial Proteins; Models, Biological; Oxygen; Peptides; Pheromones; Reactive Oxygen Species; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Thiazoles

The ubiquitously expressed mammalian thioredoxin reductases are selenoproteins that together with NADPH regenerate active reduced thioredoxins and are involved in diverse actions mediated by redox control. Two main forms of mammalian thioredoxin reductases have been isolated, one cytosolic (TrxR1) and one present in mitochondria (TrxR2). Although the principal target for TrxRs is thioredoxin, the cytosolic form can regenerate several important antioxidants such as ascorbic acid, lipoic acid, and ubiquinone. In this study we demonstrate that cytochrome c is a substrate for both TrxR1 and TrxR2. In addition, cells overexpressing TrxR2 are more resistant to impairment of complex III in the mitochondrial respiratory chain upon both antimycin A and myxothiazol treatments, suggesting a complex III bypassing function of TrxR2. Furthermore, we show that cytochrome c is reduced by TrxR2 in vitro, not only by using NADPH as an electron donor but also by using NADH, pointing at TrxR2 as an important redox protein on complex III impairment. These findings may be valuable in understanding respiratory disorders in mitochondrial diseases.

Topics: Anti-Bacterial Agents; Antifungal Agents; Antimycin A; Cell Division; Cell Survival; Cells, Cultured; Cytochromes c; Electron Transport; Electron Transport Complex III; Humans; Kidney; Methacrylates; Mitochondria; NAD; NADP; Selenocysteine; Thiazoles; Thioredoxin Reductase 1; Thioredoxin Reductase 2; Thioredoxin-Disulfide Reductase; Thioredoxins

In HeLa cells, complete inhibition of oxidative phosphorylation by oligomycin, myxothiazol or FCCP combined with partial inhibition of glycolysis by DOG resulted in a steady threefold decrease in the intracellular ATP level. The ATP level recovers when the DOG-containing medium was replaced by that with high glucose. In 48 h after a transient (3 h) [ATP] lowering followed by recovery of the ATP level, the majority of the cells commits suicide by means of apoptosis. The cell death does not occur if DOG or an oxidative phosphorylation inhibitor was added separately, treatments resulting in 10-35% lowering of [ATP]. Apoptosis is accompanied by Bax translocation to mitochondria, cytochrome c release into cytosol, caspase activation, reactive oxygen species (ROS) generation, and reorganization and decomposition of chromatin. Apoptosis appears to be sensitive to oncoprotein Bcl-2 and a pancaspase inhibitor zVADfmk. In the latter case, necrosis is shown to develop instead of apoptosis. The cell suicide is resistant to cyclosporine A, a phospholipase inhibitor trifluoroperazine, the JNK and p38 kinase inhibitors, oligomycin, N-acetyl cysteine and mitoQ, differing in these respects from the tumor necrosis factor (TNF)- and H(2)O(2)-induced apoptoses. It is suggested that the ATP concentration in the cell is monitored by intracellular "ATP-meter(s)" generating a cell suicide signal when ATP decreases, even temporarily, below some critical level (around 1 mM).

Topics: Adenosine Triphosphate; Apoptosis; bcl-2-Associated X Protein; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Caspases; Cytochromes c; Cytosol; Deoxyglucose; Enzyme Inhibitors; HeLa Cells; Humans; Intracellular Space; Methacrylates; Mitochondria; Oligomycins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Thiazoles