Mitochondrial uncoupling protein-2 (UCP2) has been suggested to participate in the attenuation of the reactive oxygen species production, but the mechanism of action and the physiological significance of UCP2 activity remain controversial. Here we tested the hypothesis that UCP2 provides feedback downregulation of oxidative stress in vivo via synergy with an H2O2-activated mitochondrial calcium-independent phospholipase A2 (mt-iPLA2). Tert-butylhydroperoxide or H2O2 induced free fatty acid release from mitochondrial membranes as detected by gas chromatography/mass spectrometry, which was inhibited by r-bromoenol lactone (r-BEL) but not by its stereoisomer s-BEL, suggesting participation of mt-iPLA2γ isoform. Tert-butylhydroperoxide or H2O2 also induced increase in respiration and decrease in mitochondrial membrane potential in lung and spleen mitochondria from control but not UCP2-knockout mice. These data suggest that mt-iPLA2γ-dependent release of free fatty acids promotes UCP2-dependent uncoupling. Upon such uncoupling, mitochondrial superoxide formation decreased instantly also in the s-BEL presence, but not when mt-iPLA2 was blocked by R-BEL and not in mitochondria from UCP2-knockout mice. Mt-iPLA2γ was alternatively activated by H2O2 produced probably in conjunction with the electron-transferring flavoprotein:ubiquinone oxidoreductase (ETFQOR), acting in fatty acid β-oxidation. Palmitoyl-d,l-carnitine addition to mouse lung mitochondria, respiring with succinate plus rotenone, caused a respiration increase that was sensitive to r-BEL and insensitive to s-BEL. We thus demonstrate for the first time that UCP2, functional due to fatty acids released by redox-activated mt-iPLA2γ, suppresses mitochondrial superoxide production by its uncoupling action. In conclusion, H2O2-activated mt-iPLA2γ and UCP2 act in concert to protect against oxidative stress.

Department of Membrane Transport Biophysics No 75 Institute of Physiology Academy of Sciences Vídeňská 1083 Prague 14220 Czech Republic

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