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

Previously suggested antioxidant mechanisms for mitochondria-targeted plastoquinone SkQ1 included: i) ion-pairing of cationic SkQ1+ with free fatty acid anions resulting in uncoupling; ii) SkQ1H2 ability to interact with lipoperoxyl radical; iii) interference with electron flow at the inner ubiquinone (Q) binding site of Complex III (Qi), involving the reduction of SkQ1 to SkQ1H2 by ubiquinol. We elucidated SkQ1 antioxidant properties by confocal fluorescence semi-quantification of mitochondrial superoxide (Jm) and cytosolic H2O2 (Jc) release rates in HepG2 cells. Only in glycolytic cells, SkQ1 prevented the rotenone-induced enhancement of Jm and Jc but not basal releases without rotenone. The effect ceased in glutaminolytic aglycemic cells, in which the redox parameter NAD(P)H/FAD increased after rotenone in contrast to its decrease in glycolytic cells. Autofluorescence decay indicated decreased NADPH/NADH ratios with rotenone in both metabolic modes. SkQ1 did not increase cell respiration and diminished Jm established high by antimycin or myxothiazol but not by stigmatellin. The revealed SkQ1 antioxidant modes reflect its reduction to SkQ1H2 at Complex I IQ or Complex III Qi site. Both reductions diminish electron diversions to oxygen thus attenuating superoxide formation. Resulting SkQ1H2 oxidizes back to SkQ1at the second (flavin) Complex I site, previously indicated for MitoQ10. Regeneration proceeds only at lower NAD(P)H/FAD in glycolytic cells. In contrast, cyclic SkQ1 reduction/SkQ1H2 oxidation does not substantiate antioxidant activity in intact cells in the absence of oxidative stress (neither pro-oxidant activity, representing a great advantage). A targeted delivery to oxidative-stressed tissues is suggested for the effective antioxidant therapy based on SkQ1.

• MeSH
• antimycin A analogy a deriváty   farmakologie   MeSH
• antioxidancia farmakologie   MeSH
• buňky Hep G2 MeSH
• flavinadenindinukleotid metabolismus   MeSH
• glykolýza MeSH
• lidé MeSH
• methakryláty farmakologie   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• NAD metabolismus   MeSH
• oxidace-redukce MeSH
• oxidační stres MeSH
• oxidativní fosforylace * MeSH
• plastochinon analogy a deriváty   farmakologie   MeSH
• polyeny farmakologie   MeSH
• respirační komplex I metabolismus   MeSH
• respirační komplex III metabolismus   MeSH
• rotenon farmakologie   MeSH
• superoxidy metabolismus   MeSH
• thiazoly farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH