Quercetin and dehydrosilybin are polyphenols which are known to behave like uncouplers of respiration in isolated mitochondria. Here we investigated whether the effect is conserved in whole cells. Following short term incubation, neither compound uncouples mitochondrial respiration in whole H9c2 cells below 50μM. However, following hypoxia, or long term incubation, leak (state IV with oligomycin) oxygen consumption is increased by quercetin. Both compounds partially protected complex I respiration, but not complex II in H9c2 cells following hypoxia. In a permeabilised H9c2 cell model, the increase in leak respiration caused by quercetin is lowered by increased [ADP] and is increased by adenine nucleotide transporter inhibitor, atractyloside, but not bongkrekic acid. Both quercetin and dehydrosilybin dissipate mitochondrial membrane potential in whole cells. In the case of quercetin, the effect is potentiated post hypoxia. Genetically encoded Ca++ sensors, targeted to the mitochondria, enabled the use of fluorescence microscopy to show that quercetin decreased mitochondrial [Ca++] while dehydrosilybin did not. Likewise, quercetin decreases accumulation of [Ca++] in mitochondria following hypoxia. Fluorescent probes were used to show that both compounds decrease plasma membrane potential and increase cytosolic [Ca++]. We conclude that the uncoupler-like effects of these polyphenols are attenuated in whole cells compared to isolated mitochondria, but downstream effects are nevertheless apparent. Results suggest that the effect of quercetin observed in whole and permeabilised cells may originate in the mitochondria, while the mechanism of action of cardioprotection by dehydrosilybin may be less dependent on mitochondrial uncoupling than originally thought. Rather, protective effects may originate due to interactions at the plasma membrane.

• MeSH
• buněčné linie MeSH
• digitonin farmakologie   MeSH
• fluorescenční mikroskopie MeSH
• konfokální mikroskopie MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• mitochondriální ADP/ATP-translokasy metabolismus   MeSH
• quercetin farmakologie   MeSH
• silymarin farmakologie   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dehydrosilybin MeSH Prohlížeč
• digitonin MeSH
• mitochondriální ADP/ATP-translokasy MeSH
• quercetin MeSH
• silymarin MeSH
• vápník MeSH

Digitonin solubilizes mitochondrial membrane, breaks the integrity of the respiratory chain and releases two mobile redox-active components: coenzyme Q (CoQ) and cytochrome c (cyt c). In the present study we report the inhibition of glycerol-3-phosphate- and succinate-dependent oxygen consumption rates by digitonin treatment. Our results show that the inhibition of oxygen consumption rates is recovered by the addition of exogenous synthetic analog of CoQ idebenone (hydroxydecyl-ubiquinone; IDB) and cyt c. Glycerol-3-phosphate oxidation rate is recovered to 148 % of control values, whereas succinate-dependent oxidation rate only to 68 %. We find a similar effect on the activities of glycerol-3-phosphate and succinate cytochrome c oxidoreductase. Our results also indicate that succinate-dependent oxidation is less sensitive to digitonin treatment and less activated by IDB in comparison with glycerol-3-phosphate-dependent oxidation. These findings might indicate the different mechanism of the electron transfer from two flavoprotein-dependent dehydrogenases (glycerol-3-phosphate dehydrogenase and succinate dehydrogenase) localized on the outer and inner face of the inner mitochondrial membrane, respectively.

• MeSH
• cytochromy c metabolismus   MeSH
• digitonin farmakologie   MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• glycerolfosfáty metabolismus   MeSH
• hypertyreóza metabolismus   MeSH
• jaterní mitochondrie účinky léků   metabolismus   MeSH
• kinetika MeSH
• krysa rodu Rattus MeSH
• kyselina jantarová metabolismus   MeSH
• mitochondriální membrány účinky léků   metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• obnova funkce MeSH
• oxidace-redukce MeSH
• potkani Wistar MeSH
• spotřeba kyslíku účinky léků   MeSH
• sukcinát: cytochrom c oxidoreduktasa metabolismus   MeSH
• ubichinon analogy a deriváty   farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytochromy c MeSH
• digitonin MeSH
• glycerolfosfátdehydrogenasa MeSH
• glycerolfosfáty MeSH
• idebenone MeSH Prohlížeč
• kyselina jantarová MeSH
• sukcinát: cytochrom c oxidoreduktasa MeSH
• ubichinon MeSH

Using high-resolution oxygraphy, we tested the changes of various parameters characterizing the mitochondrial energy provision system that were induced by peroxidative damage. In the presence of succinate as respiratory substrate, 3 mM t-butyl hydroperoxide increased respiration in the absence of ADP, which indicated partial uncoupling of oxidative phosphorylation. Low activity of coupled respiration was still maintained as indicated by the ADP-activated and oligomycin-inhibited respiration. However, during the incubation the phosphorylative capacity decreased as indicated by the continuous decrease of the mitochondrial membrane potential. Under these experimental conditions the maximum capacity of the succinate oxidase system was inhibited by 50% in comparison with values obtained in the absence of t-butyl hydroperoxide. Our data thus indicate that the oxygraphic evaluation of mitochondrial function represents a useful tool for evaluation of changes participating in peroxidative damage of cell energy metabolism.

• MeSH
• chlorid draselný farmakologie   MeSH
• digitonin farmakologie   MeSH
• energetický metabolismus účinky léků   MeSH
• hepatocyty metabolismus   MeSH
• jaterní mitochondrie účinky léků   metabolismus   MeSH
• karbonylkyanid-p-trifluormethoxyfenylhydrazon farmakologie   MeSH
• krysa rodu Rattus MeSH
• membránové potenciály účinky léků   MeSH
• mitochondriální membrány účinky léků   MeSH
• oxidativní fosforylace účinky léků   MeSH
• potkani Wistar MeSH
• rozpřahující látky farmakologie   MeSH
• separace buněk MeSH
• spotřeba kyslíku účinky léků   MeSH
• techniky in vitro MeSH
• terc-butylhydroperoxid farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorid draselný MeSH
• digitonin MeSH
• karbonylkyanid-p-trifluormethoxyfenylhydrazon MeSH
• rozpřahující látky MeSH
• terc-butylhydroperoxid MeSH

Sensitivity of various mitochondrial enzymes to oxidative damage was tested on isolated rat liver hepatocytes permeabilized by digitonin. In permeabilized hepatocytes normal respiratory control values were obtained and mitochondrial membranes remained intact. Respiratory rates of NADH-dependent (glutamate + malate, palmitylcarnitine + malate) and flavoprotein-dependent (succinate) substrates were determined in hepatocytes exposed for 5 min to 0.5-3 mM tert-butyl hydroperoxide before addition of digitonin. Our data showed that oxidation of NADH-dependent substrates is much more sensitive to oxidative stress than oxidation of flavoprotein-dependent ones, evidently due to the modification of iron-sulfur clusters or SH groups in the NADH dehydrogenase enzyme complex (Complex I).

• MeSH
• aktivace enzymů účinky léků   MeSH
• digitonin MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• indikátory a reagencie MeSH
• jaterní mitochondrie účinky léků   enzymologie   MeSH
• krysa rodu Rattus MeSH
• NAD metabolismus   MeSH
• potkani Wistar MeSH
• spotřeba kyslíku účinky léků   MeSH
• substrátová specifita MeSH
• techniky in vitro MeSH
• terc-butylhydroperoxid farmakologie   MeSH
• transport elektronů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• digitonin MeSH
• indikátory a reagencie MeSH
• NAD MeSH
• terc-butylhydroperoxid MeSH

In contrast to the well-established anti-apoptotic effect of Bcl-2 protein, we have recently demonstrated that Bcl-2 overexpression by vaccinia virus causes apoptosis in BSC-40 cells, while it prevents apoptosis in HeLa G cells. Given the key role of mitochondria in the process of apoptosis, we focused on effects of Bcl-2 expression on mitochondrial energetics of these two cell lines. In this study we present data indicating that BSC-40 cells derive their ATP mainly from oxidative phosphorylation whereas HeLa G cells from glycolysis. More importantly, we show that in both cell lines, Bcl-2 inhibits mitochondrial respiration and causes a decrease of the ATP/ADP ratio. However, it appears that BSC-40 cells cannot sustain this decrease and die, while HeLa G cells survive, being adapted to the low ratio of ATP/ADP maintained by glycolysis. Based on this observation, we propose that the outcome of Bcl-2 expression is determined by the type of cellular ATP synthesis, namely that Bcl-2 causes apoptosis in cells relying on oxidative phosphorylation.

• MeSH
• adenosindifosfát metabolismus   MeSH
• adenosintrifosfát metabolismus   MeSH
• buněčné dýchání účinky léků   fyziologie   MeSH
• digitonin farmakologie   MeSH
• exprese genu MeSH
• HeLa buňky MeSH
• karbonylkyanid-p-trifluormethoxyfenylhydrazon farmakologie   MeSH
• kyselina jantarová farmakologie   MeSH
• lidé MeSH
• mitochondrie účinky léků   fyziologie   MeSH
• oligomyciny farmakologie   MeSH
• protoonkogenní proteiny c-bcl-2 genetika   fyziologie   MeSH
• rotenon farmakologie   MeSH
• spotřeba kyslíku účinky léků   MeSH
• transfekce MeSH
• transformované buněčné linie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• adenosindifosfát MeSH
• adenosintrifosfát MeSH
• digitonin MeSH
• karbonylkyanid-p-trifluormethoxyfenylhydrazon MeSH
• kyselina jantarová MeSH
• oligomyciny MeSH
• protoonkogenní proteiny c-bcl-2 MeSH
• rotenon MeSH

The paper presents a protocol for the evaluation of energy metabolism in hepatocytes with the aid of high-resolution oxygraphy in permeabilized cells. Optimal conditions are suggested for the isolation of hepatocytes from the hepatic tissue of the rat and the proper technique for control of their structural integrity. Our work describes in detail the experimental schemes for oxygraphic evaluation of the individual enzyme complexes of the respiratory chain (Complex I, II, IV) and methods for the assessment of the mitochondrial function of phosphorylation using the respiratory control index and oligomycin inhibitory effect on ADP-activated respiration. On the example of oxidative damage of hepatocytes by tert-butylhydroperoxide, we demonstrate the utility of oxygraphy in the diagnostics of pathologic conditions caused by disturbances in energy metabolism of the cell.

• MeSH
• buněčné dýchání MeSH
• digitonin farmakologie   MeSH
• energetický metabolismus * účinky léků   MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• jaterní mitochondrie metabolismus   MeSH
• krysa rodu Rattus MeSH
• oxidativní fosforylace MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• potkani Wistar MeSH
• spotřeba kyslíku účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• Názvy látek
• digitonin MeSH

A new method for cytofluorometric analysis of mitochondrial membrane potential deltapsi has been developed by using TMRM as a cationic, mitochondrial selective probe. The method is based on limited treatment of cultured cells with digitonin which permeabilises the plasma membrane and leaves mitochondria intact. The resulting signal of TMRM-stained cells thus represents only the probe accumulated in mitochondria. Fibroblasts and cybrids were used as a model cell systems and optimal conditions for digitonin treatment and staining by TMRM were described. The TMRM signal collapsed by valinomycin, KCN and antimycin A and FCCP titration was used to gradually lower deltapsi and characterise the stability of deltapsi. The method is suitable for sensitive measurement of deltapsi in different types of cultured cells.

• MeSH
• digitonin * MeSH
• fluorescenční barviva * MeSH
• fluorometrie metody   MeSH
• indikátory a reagencie MeSH
• intracelulární membrány fyziologie   MeSH
• lidé MeSH
• membránové potenciály MeSH
• mitochondrie fyziologie   MeSH
• rhodaminy * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• digitonin * MeSH
• fluorescenční barviva * MeSH
• indikátory a reagencie MeSH
• rhodaminy * MeSH

The electrophoretic method of Schägger and von Jagow (Anal. Biochem. 199, 233-231 (1991) was adapted to allow analysis of enzymes of the respiratory chain and the ATP-synthase in cultured human skin fibroblasts and amniocytes. The cells were fractionated with digitonin and mitoplasts were isolated and used for electrophoresis. The purification of mitoplasts and the resolution by electrophoresis of the oxidative phosphorylation complexes were optimal when 0.8-1.6 mg of digitonin/mg protein was used. Intact complexes I, III, IV, and V were clearly separated by blue native-polyacrylamide gel electrophoresis (PAGE) in the first dimension and their individual subunits by tricine-sodium dodecyl sulfate-PAGE in the second dimension. Approximately 10(6) fibroblasts or amniocytes (0.4-0.6 mg protein) were sufficient for complete analysis of the oxidative phosphorylation complexes using detection by staining and by Western blotting. Comparable resolution was obtained with other cell types. Studies of fibroblasts from patients with cytochrome c oxidase deficiency demonstrated the usefulness of the method for diagnosis of mitochondrial disorders.

• MeSH
• digitonin MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• enzymy analýza   MeSH
• fibroblasty metabolismus   MeSH
• kultivované buňky MeSH
• kůže metabolismus   MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• oxidativní fosforylace * MeSH
• plodová voda metabolismus   MeSH
• těhotenství MeSH
• transport elektronů MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• digitonin MeSH
• enzymy MeSH

Isolated rat heart cells permeabilised by digitonin were examined as an experimental model to study heart bioenergetics. The cells showed good indices of oxidative phosphorylation (acceptor control ratio about 8 with pyruvate plus malate). The adenosine triphosphatase activity detected in the cells was high and was calcium dependent (optimum [free calcium] about 400 nmol.litre-1); magnesium was necessary for its full activity. Double reciprocal plot l/v vs 1/[free calcium] at physiological free calcium concentrations was linear, thus showing free calcium to be a substrate for the adenosine triphosphatase (Km for calcium about 149 nmol.litre-1). Double reciprocal plot 1/v vs 1/[ATP] was also linear, thus showing that the adenosine triphosphatase activity could be ascribed to a single enzyme. Oxidative phosphorylation and the ATPase activity of the cells appeared to be functionally coupled. This was manifested by apparent preference by oxidative phosphorylation for adenosine diphosphate supplied by the adenosine triphosphatase activity (Km 45 mumol.litre-1) to external adenosine diphosphate (Km 152 mumol.litre-1; p less than 0.02). Apparent preference by the adenosine triphosphatase activity for adenosine triphosphate supplied by mitochondria (Km 74 mumol.litre-1) to external adenosine triphosphate (Km 169 mumol.litre-1) was also manifested by a significant difference in Km values (p less than 0.05).

• MeSH
• adenosindifosfát farmakologie   MeSH
• adenosintrifosfát farmakologie   MeSH
• Ca(2+)-Mg(2+)-ATPasa metabolismus   MeSH
• Ca2+-ATPasy metabolismus   MeSH
• digitonin farmakokinetika   MeSH
• hořčík farmakologie   MeSH
• kinetika MeSH
• krysa rodu Rattus MeSH
• myokard cytologie   enzymologie   MeSH
• oxidativní fosforylace * MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• spotřeba kyslíku účinky léků   MeSH
• srdce účinky léků   MeSH
• techniky in vitro MeSH
• vápník farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosindifosfát MeSH
• adenosintrifosfát MeSH
• Ca(2+)-Mg(2+)-ATPasa MeSH
• Ca2+-ATPasy MeSH
• digitonin MeSH
• hořčík MeSH
• vápník MeSH

The outer membrane resistance of brain and liver mitochondria to the action of digitonin was studied in 5- and 60-day-old rats. Disintegration of the outer membrane was evaluated by means of cytochrome oxidase activity. The mitochondria were isolated by the Ficoll gradient method. Brain mitochondria were found to be significantly more resistant to digitonin than liver mitochondria. It was also demonstrated that the resistance of the outer membrane of both brain and liver mitochondria rose approximately 1.3- to 1.4-fold during ontogenesis.

• MeSH
• digitonin farmakologie   MeSH
• intracelulární membrány účinky léků   MeSH
• jaterní mitochondrie účinky léků   MeSH
• krysa rodu Rattus MeSH
• léková rezistence MeSH
• mitochondrie účinky léků   MeSH
• mozek účinky léků   MeSH
• orgánová specificita MeSH
• respirační komplex IV metabolismus   MeSH
• věkové faktory MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• digitonin MeSH
• respirační komplex IV MeSH