We investigated hydrogen peroxide production in mitochondria with low (liver, heart, brain) and high (brown adipose tissue, BAT) content of glycerophosphate dehydrogenase (mGPDH). ROS production at state 4 due to electron backflow from mGPDH was low, but after inhibition of electron transport with antimycin A high rates of mGPDH-dependent ROS production were observed in liver, heart and brain mitochondria. When this ROS production was related to activity of mGPDH, many-fold higher ROS production was found in contrast to succinate- (39-, 28-, 3-fold) or pyruvate plus malate-dependent ROS production (32-, 96-, 5-fold). This specific rate of mGPDH-dependent ROS production was also exceedingly higher (28-, 66-, 22-fold) compared to that in BAT. mGPDH-dependent ROS production was localized to the dehydrogenase+CoQ and complex III, the latter being the highest in all mitochondria but BAT. Our results demonstrate high efficiency of mGPDH-dependent ROS production in mammalian mitochondria with a low content of mGPDH and suggest its endogenous inhibition in BAT.
- MeSH
- Antimycin A pharmacology MeSH
- Financing, Organized MeSH
- Glycerolphosphate Dehydrogenase metabolism MeSH
- Adipose Tissue, Brown metabolism MeSH
- Mitochondria, Liver metabolism drug effects MeSH
- Cricetinae MeSH
- Rats MeSH
- Succinic Acid metabolism MeSH
- Pyruvic Acid metabolism MeSH
- Mitochondria metabolism drug effects MeSH
- Brain metabolism MeSH
- Hydrogen Peroxide metabolism MeSH
- Rats, Wistar MeSH
- Reactive Oxygen Species metabolism MeSH
- Electron Transport Complex III metabolism MeSH
- Mitochondria, Heart metabolism drug effects MeSH
- In Vitro Techniques MeSH
- Electron Transport MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Rats MeSH
- Male MeSH
- Animals MeSH
Whether active UCP1 can reduce ROS production in brown-fat mitochondria is presently not settled. The issue is of principal significance, as it can be seen as a proof- or disproof-of-principle concerning the ability of any protein to diminish ROS production through membrane depolarization. We therefore undertook a comprehensive investigation of the significance of UCP1 for ROS production, by comparing the ROS production in brown-fat mitochondria isolated from wildtype mice (that display membrane depolarization) or from UCP1(-/-) mice (with a high membrane potential). We tested the significance of UCP1 for glycerol-3-phosphate-supported ROS production by three methods (fluorescent dihydroethidium and the ESR probe PHH for superoxide, and fluorescent Amplex Red for hydrogen peroxide), and followed ROS production also with succinate, acyl-CoA or pyruvate as substrate. We studied the effects of the reverse electron flow inhibitor rotenone, the UCP1 activity inhibitor GDP, and the uncoupler FCCP. We also examined the effect of a physiologically induced increase in UCP1 amount. We noted GDP effects that were not UCP1-related. We conclude that only ROS production supported by exogenously added succinate was affected by the presence of active UCP1; ROS production supported by any other tested substrate (including endogenously generated succinate) was unaffected. This conclusion indicates that UCP1 is not involved in control of ROS production in brown-fat mitochondria. Extrapolation of these data to other tissues would imply that membrane depolarization may not necessarily decrease physiologically relevant ROS production. This article is a part of a Special Issue entitled: 18th European Bioenergetics Conference (Biochim. Biophys. Acta, Volume 1837, Issue 7, July 2014).
- MeSH
- Electron Spin Resonance Spectroscopy MeSH
- Glycerophosphates pharmacology MeSH
- Guanosine Diphosphate pharmacology MeSH
- Adipose Tissue, Brown metabolism MeSH
- Immunoblotting MeSH
- Ion Channels genetics metabolism MeSH
- Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone pharmacology MeSH
- Succinic Acid pharmacology MeSH
- Pyruvic Acid pharmacology MeSH
- Membrane Potential, Mitochondrial drug effects MeSH
- Mitochondrial Proteins genetics metabolism MeSH
- Mitochondria drug effects metabolism physiology MeSH
- Mice, Inbred C57BL MeSH
- Mice, Knockout MeSH
- Cold Temperature MeSH
- Hydrogen Peroxide metabolism MeSH
- Proton Ionophores pharmacology MeSH
- Reactive Oxygen Species metabolism MeSH
- Oxygen Consumption drug effects MeSH
- Superoxides metabolism MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Isolated defects of mitochondrial ATPase due to diminished biosynthesis of the enzyme represent new class of severe mitochondrial diseases of nuclear origin. The primary cause of decreased cellular content of ATPase appears to be a problem in assembly of the F1 catalytic part of the enzyme. With the aim to elucidate how the low ATPase content affects mitochondrial energy provision and ROS production, we have investigated fibroblasts from patients with ATPase decrease to 10-30%. Measurements of cellular respiration showed pronounced decrease in ATPase capacity for basal respiration, mitochondrial ATP synthesis was decreased to 26-33%. Cytofluorometric analysis using TMRM revealed altered discharge of mitochondrial membrane potential (DeltaPsim) in patient cells, which was 20 mV increased at state 3-ADP. Analysis of ROS production by CM-H2DCFDA demonstrated 2-fold increase in ROS production in patient cells compared to controls. ROS production rate was sensitive to uncoupler (FCCP) and thus apparently related to increased DeltaPsim. Our studies clearly demonstrate that low ATPase content and decreased mitochondrial ATP production lead to high values of DeltaPsim and are associated with activation of ROS generation by the mitochondrial respiratory chain. In conclusion, both the energetic deprivation and increased oxidative stress are important components of the pathogenic mechanism of ATPase disorders.
- MeSH
- Adenosine Triphosphate biosynthesis MeSH
- Adenosine Triphosphatases * deficiency MeSH
- Energy Metabolism * MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Membrane Potentials MeSH
- Mitochondria * metabolism MeSH
- Reactive Oxygen Species * metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.
- MeSH
- Antimycin A analogs & derivatives pharmacology MeSH
- Cell Respiration MeSH
- Electron Spin Resonance Spectroscopy MeSH
- Ethidium analogs & derivatives chemistry MeSH
- Ferricyanides pharmacology MeSH
- Financing, Organized MeSH
- Glycerolphosphate Dehydrogenase metabolism MeSH
- Glycerophosphates metabolism MeSH
- Adipose Tissue, Brown enzymology drug effects ultrastructure MeSH
- Cricetinae MeSH
- Mitochondria enzymology metabolism drug effects MeSH
- Reactive Oxygen Species analysis metabolism MeSH
- Electron Transport Complex III metabolism MeSH
- Oxygen Consumption MeSH
- Electron Transport MeSH
- Ubiquinone metabolism MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Male MeSH
- Animals MeSH
Reactive oxygen species (ROS) originating from mitochondria are perceived as a factor contributing to cell aging and means have been sought to attenuate ROS formation with the aim of extending the cell lifespan. Silybin and dehydrosilybin, two polyphenolic compounds, display a plethora of biological effects generally ascribed to their known antioxidant capacity. When investigating the cytoprotective effects of these two compounds in the primary cell cultures of neonatal rat cardiomyocytes, we noted the ability of dehydrosilybin to de-energize the cells by monitoring JC-1 fluorescence. Experiments evaluating oxygen consumption and membrane potential revealed that dehydrosilybin uncouples the respiration of isolated rat heart mitochondria albeit with a much lower potency than synthetic uncouplers. Furthermore, dehydrosilybin revealed a very high potency in suppressing ROS formation in isolated rat heart mitochondria with IC(50) = 0.15 μM. It is far more effective than its effect in a purely chemical system generating superoxide or in cells capable of oxidative burst, where the IC(50) for dehydrosilybin exceeds 50 μM. Dehydrosilybin also attenuated ROS formation caused by rotenone in the primary cultures of neonatal rat cardiomyocytes. We infer that the apparent uncoupler-like activity of dehydrosilybin is the basis of its ROS modulation effect in neonatal rat cardiomyocytes and leads us to propose a hypothesis on natural ischemia preconditioning by dietary polyphenols.
- MeSH
- Analysis of Variance MeSH
- Benzimidazoles MeSH
- Fluorescent Dyes MeSH
- Inhibitory Concentration 50 MeSH
- Carbocyanines MeSH
- Myocytes, Cardiac metabolism MeSH
- Rats MeSH
- Mitochondria metabolism MeSH
- Molecular Structure MeSH
- Rats, Wistar MeSH
- Reactive Oxygen Species metabolism MeSH
- Rotenone toxicity MeSH
- Silymarin chemistry pharmacology MeSH
- Oxygen Consumption drug effects MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Enhanced production of superoxide radicals by nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase in the brain and/or kidney of salt hypertensive Dahl rats has been proposed to participate in the pathogenesis of this form of experimental hypertension. Most information was obtained in young Dahl salt-sensitive (DS) rats subjected to high salt intake prior to sexual maturation. Therefore, the aim of our study was to investigate whether salt hypertension induced in adult DS rats is also accompanied with a more pronounced oxidative stress in the brain or kidney as compared to Dahl salt-resistant (DR) controls. NADPH oxidase activity as well as the content of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes (oxidative index), which indicate a degree of lipid peroxidation, were evaluated in two brain regions (containing either hypothalamic paraventricular nucleus or rostral ventrolateral medulla) as well as in renal medulla and cortex. High salt intake induced hypertension in DS rats but did not modify blood pressure in DR rats. DS and DR rats did not differ in NADPH oxidase-dependent production of ROS, TBARS content or oxidative index in either part of the brain. In addition, high-salt diet did not change significantly any of these brain parameters. In contrast, the enhanced NADPH oxidase-mediated ROS production (without significant signs of increased lipid peroxidation) was detected in the renal medulla of salt hypertensive DS rats. Our findings suggest that there are no signs of enhanced oxidative stress in the brain of adult Dahl rats with salt hypertension induced in adulthood.
- MeSH
- Hypertension chemically induced metabolism MeSH
- Blood Pressure drug effects MeSH
- Rats MeSH
- Sodium Chloride, Dietary * MeSH
- Kidney drug effects metabolism MeSH
- Brain metabolism MeSH
- Organ Specificity drug effects MeSH
- Oxidative Stress drug effects MeSH
- Rats, Inbred Dahl MeSH
- Reactive Oxygen Species metabolism MeSH
- Tissue Distribution MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The aim of our study was to reveal the in vitro effects of Salvia officinalis L. (37.5, 75, 150, 200, 250, 300 and 600 µg/ml) extract on the TM3 Leydig cell viability, membrane integrity, steroidogenesis and reactive oxygen species production after 24 h and 48 h cultivation. For the present study, the extract prepared from Salvia officinalis L. leaves was analysed by high performance liquid chromatography (HPLC) for selected flavonoids and phenolic acids followed by a determination of its free radicals scavenging activity (DPPH). Furthermore, Leydig cell viability was assessed by the mitochondrial toxicity assay (MTT), while the membrane integrity was evaluated by 5- carboxyfluorescein diacetate-acetoxymethyl ester (5-CFDA-AM). The level of steroid hormones was performed by enzyme-linked immunosorbent assay (ELISA) from the culture media, while the superoxide radical generation was measured by the nitroblue tetrazolium chloride (NBT) assay. The results show that experimental concentrations did not damage the cell membrane integrity and viability when present at below 300 µg/ml, it was only at 600 µg/ml that a significant (P<0.05) cell viability decline was observed after a 48 h cultivation. A significant (P<0.05) stimulation of testosterone secretion was recorded at 250 µg/ml for 24 h, while the prolonged cultivation time significantly (P<0.05) increased the testosterone and progesterone production at 150, 200, 250 and 300 µg/ml. Furthermore, none of the selected doses exhibited significant ROS-promoting effects however, the highest dose of Salvia initiated the free radical scavenging activity in cultured mice Leydig cells.
- MeSH
- Cell Line MeSH
- Leydig Cells drug effects metabolism MeSH
- Plant Leaves chemistry drug effects MeSH
- Mice, Inbred BALB C MeSH
- Mice, Nude MeSH
- Mice MeSH
- Progesterone biosynthesis MeSH
- Reactive Oxygen Species metabolism MeSH
- Plant Extracts pharmacology MeSH
- Salvia officinalis chemistry MeSH
- In Vitro Techniques MeSH
- Testosterone biosynthesis MeSH
- Cell Survival drug effects MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
