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 farmakologie   MeSH
• financování organizované MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• hnědá tuková tkáň metabolismus   MeSH
• jaterní mitochondrie metabolismus   účinky léků   MeSH
• křečci praví MeSH
• krysa rodu rattus MeSH
• kyselina jantarová metabolismus   MeSH
• kyselina pyrohroznová metabolismus   MeSH
• mitochondrie metabolismus   účinky léků   MeSH
• mozek metabolismus   MeSH
• peroxid vodíku metabolismus   MeSH
• potkani Wistar MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• respirační komplex III metabolismus   MeSH
• srdeční mitochondrie metabolismus   účinky léků   MeSH
• techniky in vitro MeSH
• transport elektronů MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata 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
• elektronová paramagnetická rezonance MeSH
• glycerolfosfáty farmakologie   MeSH
• guanosindifosfát farmakologie   MeSH
• hnědá tuková tkáň metabolismus   MeSH
• imunoblotting MeSH
• iontové kanály genetika   metabolismus   MeSH
• karbonylkyanid-p-trifluormethoxyfenylhydrazon farmakologie   MeSH
• kyselina jantarová farmakologie   MeSH
• kyselina pyrohroznová farmakologie   MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie účinky léků   metabolismus   fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• nízká teplota MeSH
• peroxid vodíku metabolismus   MeSH
• protonové ionofory farmakologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• spotřeba kyslíku účinky léků   MeSH
• superoxidy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• adenosintrifosfát biosyntéza   MeSH
• adenosintrifosfatasy * nedostatek   MeSH
• energetický metabolismus * MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové potenciály MeSH
• mitochondrie * metabolismus   MeSH
• reaktivní formy kyslíku * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem 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 analogy a deriváty   farmakologie   MeSH
• buněčné dýchání MeSH
• elektronová paramagnetická rezonance MeSH
• ethidium analogy a deriváty   chemie   MeSH
• ferrikyanidy farmakologie   MeSH
• financování organizované MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• glycerolfosfáty metabolismus   MeSH
• hnědá tuková tkáň enzymologie   účinky léků   ultrastruktura   MeSH
• křečci praví MeSH
• mitochondrie enzymologie   metabolismus   účinky léků   MeSH
• reaktivní formy kyslíku analýza   metabolismus   MeSH
• respirační komplex III metabolismus   MeSH
• spotřeba kyslíku MeSH
• transport elektronů MeSH
• ubichinon metabolismus   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• mužské pohlaví MeSH
• zvířata 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
• analýza rozptylu MeSH
• benzimidazoly MeSH
• fluorescenční barviva MeSH
• inhibiční koncentrace 50 MeSH
• karbocyaniny MeSH
• kardiomyocyty metabolismus   MeSH
• krysa rodu rattus MeSH
• mitochondrie metabolismus   MeSH
• molekulární struktura MeSH
• potkani Wistar MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• rotenon toxicita   MeSH
• silymarin chemie   farmakologie   MeSH
• spotřeba kyslíku účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• buňky NK MeSH
• interleukin-2 MeSH
• lidé MeSH
• lymfocyty MeSH
• lymfokiny krev   MeSH
• nádory děložního čípku imunologie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví 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
• hypertenze chemicky indukované   metabolismus   MeSH
• krevní tlak účinky léků   MeSH
• krysa rodu rattus MeSH
• kuchyňská sůl * MeSH
• ledviny účinky léků   metabolismus   MeSH
• mozek metabolismus   MeSH
• orgánová specificita účinky léků   MeSH
• oxidační stres účinky léků   MeSH
• potkani inbrední Dahl MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• tkáňová distribuce MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• acetofenony farmakologie   MeSH
• antioxidancia farmakologie   MeSH
• aorta metabolismus   účinky léků   MeSH
• fagocyty enzymologie   MeSH
• fibroblasty účinky léků   MeSH
• NADPH-oxidasy antagonisté a inhibitory   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

The innate immune response represents the first-line of defense against invading pathogens. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in various aspects of innate immune function, which involves respiratory bursts and inflammasome activation. These reactive species widely distributed within the cellular environment are short-lived intermediates that play a vital role in cellular signaling and proliferation and are likely to depend on their subcellular site of formation. NADPH oxidase complex of phagocytes is known to generate superoxide anion radical (O2•-) that functions as a precursor for antimicrobial hydrogen peroxide (H2O2) production, and H2O2 is utilized by myeloperoxidase (MPO) to generate hypochlorous acid (HOCl) that mediates pathogen killing. H2O2 modulates the expression of redox-responsive transcriptional factors, namely NF-kB, NRF2, and HIF-1, thereby mediating redox-based epigenetic modification. Survival and function of immune cells are under redox control and depend on intracellular and extracellular levels of ROS/RNS. The current review focuses on redox factors involved in the activation of immune response and the role of ROS in oxidative modification of proteins in macrophage polarization and neutrophil function.

• MeSH
• kyselina chlorná MeSH
• oxidace-redukce MeSH
• oxidační stres MeSH
• peroxid vodíku * MeSH
• přirozená imunita MeSH
• superoxidy * MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.

• MeSH
• ferrikyanidy metabolismus   MeSH
• glycerolfosfátdehydrogenasa chemie   metabolismus   MeSH
• glycerolfosfáty metabolismus   MeSH
• krysa rodu rattus MeSH
• mitochondrie enzymologie   metabolismus   MeSH
• peroxid vodíku metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• savci MeSH
• sukcinátdehydrogenasa chemie   metabolismus   MeSH
• transport elektronů * MeSH
• ubichinon metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH