Effects of resveratrol on the rat brain respiratory chain

The aim of this work was to investigate the possible effects of resveratrol on the mitochondrial respiratory chain in rat brains. Isolation of mitocho ndria was performed at 4 degrees C using differential centrifugation. Mitoc hondrial respiration rate (0.4 mg of protein/ml) was determined by measurin g mitochondrial oxygen consumption with a Clark electrode at 37 degrees C. Respiratory control ratio (RCR) was evaluated as the state 3/state 4 ratio of oxidative phosphorylation with substrates adenosine 5'-diphosphate (ADP) and malate plus glutamate, respectively in the presence and in the absence of resveratrol. The rate of oxygen consumption by the different complexes was checked using rotenone (2 mu M), malonate (10 mM), antimycin A (1 mu M) , potassium cyanide (KCN) (0.3 mM) and oligomycin (10 mu M) to inhibit comp lexes II, III, IV, V and I, respectively. Moreover enzyme activity determin ations were checked as follows: the activities of complexes ii-iii were mea sured as the rate of cytochrome c reduction at 550 nm (37 degrees C) succes sively triggered either by succinate (complexes II and III) or by decylubiq uinol (DUQH(2)) (complex III), in the presence and in the absence of resver atrol. Adenosine 5'-triphosphate (ATP) synthase activity was checked as ATP hydrolysis (ATPase) at 37 degrees C for 10 min from purified mitochondria on Percoll gradient. The inorganic phosphate (PI) concentration was measure d by the Fiske and Subbarow method. When complexes I to V were activated by glutamate plus malate, resveratrol (10(-11) - 10(-4) M) significantly decr eased RC (p < 0.001) following a biphasic curve with two EC50 values, 0.162 +/- 0.072 mu M and 24.5 +/- 4.0 mu M, representing about 56% of total oxyg en consumption inhibition. We also observed a concentration-dependent effec t on state 3 with two EC50 values, 2.28 +/- 0.87 nM and 27 +/- 5 mu M respe ctively On the other hand, resveratrol inhibited state 4 following a concen tration-dependent curve with an EC50 of 37 +/- 11 mu M. When complex IV ope rated alone resveratrol (100 mu M) did not modify oxygen consumption compar ed with control, indicating that this molecule did not inhibit complex IV. Thus resveratrol inhibits the mitochondrial respiratory chain through compl exes I to III. in order to confirm these data, we measured the enzymatic ac tivity of ubiquinol cytochrome c reductase alone and in the presence of res veratrol. in the presence of disrupted mitochondria, after freeze thawing c ycles (three times), resveratrol inhibited about 20% of complex ill activit y These results suggest that resveratrol and DUQH(2) could be competitive o n complex III. Resveratrol significantly inhibited ATPase activity (p < 0.0 01) following a biphasic curve with two EC50 values, 0.39 +/- 0.15 nM and 2 3.1 +/- 6.4 mu M, both representing about 80% of oligomycin-dependent ATPas e total activity. Resveratrol was effective as a protecting agent on the th ree models of oxidation. On lipid peroxidation of brain synaptosomes induce d by the Fenton reaction, it was three times more potent than DUQH(2). Its effectiveness in reducing 1,1-diphenyl-2-picryl hydrazyl radical (DPPH degr ees) showed a stoichiometry of two, indicating that two hydrogen atoms of r esveratrol were abstracted by the process. Resveratrol was also able to sca venge the superoxide anion (O(2)(-)degrees) generated from rat forebrain mi tochondria in a concentration dependent manner In conclusion, resveratrol c an decrease complex III activity by competition with coenzyme Q. This prope rty is especially interesting as this complex is the site where reactive ox ygen substances (ROS) are generated. By decreasing the activity of complex III, resveratrol cannot only oppose t he production of ROS but can also scavenge them.