Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential - PubMed (original) (raw)

Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential

Nadeene Parker et al. Biosci Rep. 2008 Apr.

Abstract

Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of ROS (reactive oxygen species). This proton leak can be induced by the lipid peroxidation products of ROS, such as HNE (4-hydroxynonenal). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and ANT (adenine nucleotide translocase), thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We have found that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent on incubation with respiratory substrate. In the presence of HNE, mitochondria energized with succinate became progressively more leaky to protons over time compared with mitochondria in the absence of either HNE or succinate. Energized mitochondria must attain a high membrane potential to allow HNE to activate uncoupling: a drop of 10-20 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11%), ANT (64%) and other pathways (25%). Our findings have shown that exogenous HNE only activates uncoupling at high membrane potential. These results suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production.

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Figures

Figure 1. Energization-dependent stimulation of proton leak in rat skeletal muscle mitochondria by 50 μM HNE

Proton leak kinetics were measured in rat skeletal muscle mitochondria as described in the Experimental section. (A) Timings of additions to the incubation chamber. Mitochondria plus inhibitors (M) were added at _t_=−10 min, 50 μM HNE (where present) was added at _t_=−9 min, 4 mM succinate (S) was added at the times indicated, then leak kinetics (LK) were measured between _t_=0 and _t_=3 min by titration of membrane potential and oxygen consumption rate with malonate from 0.14 to 2.3 mM. The kinetic response of mitochondrial proton leak rate (measured as oxygen consumption rate) to membrane potential measured after energization with 4 mM succinate for 0.75 (circles), 2.5 (triangles) or 5.0 min (squares) was measured without (B) or with (C) 50 μM HNE. (D) Proton leak rate interpolated at 166 mV [continuous vertical lines in (B) and (C)]; the average highest protonmotive force common to all curves generated in the experiment. The results are the means±S.E.M. of duplicate experiments performed with five separate preparations. One-way ANOVA was performed for (D); _P_=0.0001, with Tukey's multiple comparison test used post-hoc; significant differences, *P<0.05, **P<0.001.

Figure 2. Sensitivity of HNE-stimulated proton leak in rat skeletal muscle mitochondria to membrane potential

(A–C) Effect of malonate, (D–F) effect of KCN and (A, B, D, E) proton leak kinetics. Succinate (4 mM) was added at _t_=− 1.5 min (see Figure 1A) in the absence (open symbols) or presence (closed symbols) of 50 μM HNE. Proton leak kinetics were measured between _t_=0 and _t_=3 min after no treatment (A, D) or the addition of 430 μM malonate (B) or 43 μM KCN (E) immediately prior to addition of succinate. (C, F) Inhibition of HNE-stimulated proton leak by malonate and KCN. The increase in proton conductance at _t_=0 caused by addition of 50 μM HNE was interpolated (continuous vertical lines in A, B, D, E) at the highest common membrane potential, 164 mV in (C) and 169 mV in (F). ‘X’ indicates the drop in membrane potential measured at _t_=0 caused by pre-addition of malonate or KCN. (G) Effect of this drop in membrane potential on HNE-stimulated proton conductance. The increases in proton leak rate (C and results not shown) using 140 μM and 290 μM malonate (circles), and using or 14 μM and 29 μM KCN (triangles) (F and results not shown) are plotted against the decrease (‘X’) in membrane potential at _t_=0 in the absence of HNE. The results are the means±S.E.M. for five (A–C) or six (D–F) experiments each performed in duplicate. Significantly different from zero addition by paired Student's t test: **P<0.01, ***P<0.001. Lines in (G) are linear regressions.

Figure 3. Contribution of UCP3 to HNE-stimulated proton leak after 2.5 min energization in mouse skeletal muscle mitochondria

Proton leak kinetics in mitochondria from wild-type (A) and _Ucp3_-KO (B) mice. Succinate (4 mM) was added at _t_=− 2.5 min, without (open symbols) or with (closed symbols) 50 μM HNE. (C) Proton leak due to UCP3 measured by interpolation at 160 mV (continuous vertical lines in A, B) as (A) minus (B). The results are the means±S.E.M. for 12 experiments, each performed in duplicate. *Significantly different from control (paired Student's t test; P<0.05).

Figure 4. Contribution of ANT to HNE-stimulated proton leak after 2.5 min energization in _Ucp3_-KO mouse skeletal muscle mitochondria

(A, B) Proton leak kinetics in mitochondria from _Ucp3_-KO mice. Succinate (4 mM) was added at _t_=− 5 min, without (open symbols) or with (closed symbols) 50 μM HNE in the absence (A) or presence (B) of 7 nmol of carboxyatractylate/mg of protein. (C) Proton leak due to ANT measured by interpolation at 157 mV (continuous vertical lines in A, B) as (A) minus (B). The results are the means±S.E.M. for 6 experiments, each performed in duplicate. *Significantly different from control (paired Student's t test; P<0.03). CAT, carboxyatractylate.

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Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential - PubMed (original) (raw)