Fig 1.
A scheme of the synthesis of mitoCCCP, a derivative of the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone) targeted to mitochondria by a decyl(triphenyl)phosphonium cation.
Fig 2.
Effect of mitoCCCP (2.5 μM) on the uncoupling activity of CCCP (A), SF6847 (B) or DNP (C) in rat liver mitochondria. Substrate: succinate. The membrane potential of mitochondria was estimated from changes in the absorbance of the potential sensitive dye safranine O (15 μM) at 555 nm and 523 nm. For other conditions, see Materials and methods.
Fig 3.
Effect of mitoCCCP (2.5 μM) on the stimulation of succinate-driven respiration of rat liver mitochondria by CCCP (100 nM, blue curve) or by DNP (10 μM, green curve).
The three top traces show the stimulation of respiration by 100 μM ADP due to ATP synthesis. Insert: Recoupling effect of mitoCCCP on the ADP/O ratio (amount of ADP phosphorylated/O atom consumed). Shown are Mean±S.D. (n = 4). For experimental conditions, see Materials and Methods.
Fig 4.
A. Effect of mitoCCCP (2.5 μM) on the CCCP (3 μM)—mediated electrical current through planar bilayer lipid membrane (BLM) made from DPhPC. The solution was 50 mM Tris, 50 mM MES, 10 mM KCl, 10 μg/ml EggPC liposomes, pH 7.4. The voltage applied to BLM was 50 mV. B. Effect of mitoCCCP (2 μM) on the CCCP (60 nM)–mediated proton fluxes through liposomes loaded with the pH-probe pyranine. Inner liposomal pH was estimated from the pyranine fluorescence intensities measured at 505 nm upon excitation at 455 nm. 1 μM lasalocid A was added at 600 s to equilibrate the pH. Other conditions: see Materials and Methods. Lipid concentration was 20 μg/ml.
Fig 5.
A. Dose dependence of respiratory stimulation by mitoCCCP with succinate as a substrate of mitochondria. Shown are Mean±S.D. (n = 4). For experimental conditions, see Materials and methods. B. Effect of mitoCCCP on the mitochondrial membrane potential in rat liver mitochondria (RLM) estimated by measuring absorbance changes of the potential-sensitive dye safranine O (15 μM). For experimental conditions, see Materials and methods. Insert: dose dependence of the effect of mitoCCCP on the membrane potential of RLM (Mean±S.D., n = 3).
Fig 6.
A. Concentration dependence of the effect of mitoCCCP (open circles) and CCCP (closed circles) on the NADH oxidation rate in submitochondrial particles (SMP). All the data are presented as mean ± SEM of at least 3 independent experiments. For experimental conditions, see Materials and methods. B. Traces of NADH oxidation by SMP in the presence of mitoCCCP and CCCP. Concentrations, left: mitoCCCP 0.45 μM, CCCP, 2.5 μM; right: CCCP, 1 μM, mitoCCCP, 0.45 μM. SMP concentration, 10 μg/ml. C. Effect of mitoCCCP and CCCP on the pH gradient across the membranes of submitochondrial particles (SMP), as measured by ACMA (0.5 μg/ml) fluorescence. Shown are traces of fluorescence at 480 nm (excited at 410 nm) in the medium containing 100 mM KCl, 10 mM HEPES, 5 mM MgCl2, 0.5 mM EGTA, pH 7.4. In the traces, 200 μM NADH was supplemented at t = 150 s. 1 μg/ml of gramicidin A was added at the end of each trace. Protein concentration was 25 μg/ml.