Reactive oxygen species production by the mitochondrial respiratory chain in isolated rat hepatocytes and liver mitochondria: studies using myxothiazol
Section snippets
Materials
Collagenase type I was obtained from Worthington Biochemical Corp. (Lakewood, NJ). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) and MitoTracker Red CM-H2XRos (MTR) were obtained from Molecular Probes, (Eugene, OR) and dichlorofluorescein (DCF) was obtained from PolySciences, (Warrington, PA). Antimycin, Hepes, 1-malic acid, 1-glutamic acid, EGTA, rotenone, succinate, and ADP were obtained from Sigma Chemical (St. Louis, MO) and diphenyliodonium chloride was from Aldrich Chemical (Milwaukee,
Inhibition of hepatocyte respiration by myxothiazol is concentration and time dependent
The efficacy of myxothiazol to inhibit hepatocyte respiration was examined over a 1-h period in cells incubated with different concentrations of myxothiazol (Fig. 1). In this set of experiments, hepatocyte respiration was well maintained in untreated hepatocytes (i.e., control) with only a 10% decrease over the course of the hour. A 5-min exposure to all concentrations of myxothiazol was sufficient to significantly inhibit hepatocyte respiration to about 20% of the level measured in control
Discussion
The effects of myxothiazol on mitochondrial respiration are well documented. It effectively inhibits mitochondrial respiration by preventing an electron being transferred from the ubiquinol form of ubiquinone to the Rieske iron–sulfur protein [28]. This property has been previously demonstrated with liver, skeletal muscle, [21] and heart mitochondria [29], [30] and was verified in the present study for liver mitochondria. Myxothiazol is also an effective inhibitor of cellular respiration, as
Acknowledgements
The authors thank Mr. Ken Grant for his technical assistance with the confocal microscopy studies. These studies were supported by USPHS Grants AA02887 and AA00279 (to C.C.C.) and AA12058 and AA13682 (to S.M.B.) from the National Institute on Alcohol Abuse and Alcoholism.
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