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. 2007 Sep 12;407(Pt 1):129–140. doi: 10.1042/BJ20070606

Figure 7. Uncoupling effects of mitoBHT in rat liver mitochondria and rat thymocytes.

Figure 7

(A) Uptake and release of mitoBHT. The electrode was calibrated by five additions of 0.5 μM mitoBHT in the presence of mitochondria. Addition of 4 mM succinate induced membrane potential and caused a decrease in the external mitoBHT concentration. The external mitoBHT concentration was largely restored when the membrane potential was dissipated with 0.3 μM FCCP. (B) Representative traces of mitochondrial oxygen consumption in the presence of mitoBHT. DMSO, different concentrations of mitoBHT or 0.3 μM FCCP were present where indicated. (C) Double logarithmic plots of uncoupling in rat liver mitochondria. The titration of respiration rates was with mitoBHT (open circles), mitoDNP (open triangles) or TPMP (squares). Closed circles, respiration rate at each mitoBHT concentration after subsequent addition of 0.3 μM FCCP. Values are means±S.E.M. for four independent experiments. Lines were fitted by regression. (D) Effect of mitoBHT on the respiration of rat liver mitochondria in the presence of CAT. Double logarithmic plots of uncoupling in the presence of 2 nmol of CAT/mg of protein (added before succinate). Open circles, the titration of respiration rate with mitoBHT. Closed circles, respiration rate at each mitoBHT concentration after subsequent addition of 0.3 μM FCCP. Values are means±S.E.M. for four independent experiments. The line was fitted by regression. (E) CAT-sensitive stimulation of mitochondrial respiration rate by mitoBHT in linear co-ordinates. CAT-sensitive stimulation of respiration rate (open circles, shaded profile) was estimated as total uncoupling [filled circles; data from (C) where CAT was absent] minus CAT-insensitive respiration [open triangles; data from (D) where CAT was present]. Lines were fitted as in Figures 5(E)–5(H). (F) Kinetic response of proton leak to membrane potential of rat liver mitochondria incubated with 2 nmol of CAT/mg of protein, in the presence of DMSO and different concentrations of mitoBHT. Values are means±S.E.M. for four or five independent experiments. (G) Potential-independent uncoupling of rat liver mitochondria by mitoBHT. Data taken from (F) at the highest common membrane potential of 152 mV (solid line; slope=0.69±0.10; error bars for interpolated points are the weighted means of the error bars on flanking experimental points), or at the highest potential for each uncoupler concentration [broken line; slope=0.39±0.04; points and error bars as in (F) but omitted here for clarity]. The slopes are significantly different. (H) Effect of mitoBHT on the respiration of rat thymocytes, presented as a double logarithmic plot. Values are means±S.E.M. for three independent experiments. The slope of the regression line is 0.13±0.05.