Peroxynitrite (PN), generated from the reaction of nitric oxide (NO) and superoxide, is implicated in the pathogenesis of ischemic and neurodegenerative brain injuries. Mitochondria produce NO from mitochondrial NO synthases and superoxide by the electron transport chain. Our objective was to detect the generation of PN of mitochondrial origin and characterize its effects on mitochondrial respiratory function. Freshly isolated brain nonsynaptosomal mitochondria from C57Bl/6 (wild type, WT) and endothelial NO synthase knockout (eNOS-KO) mice were treated with exogenous PN (0.1, 1, 5 µmol/L) or a PN donor (SIN-1; 50 µmol/L) or a PN scavenger (FeTMPyP; 2.5 µmol/L). Oxygen consumption rate (OCR) was measured using Agilent Seahorse XFe24 analyzer and mitochondrial respiratory parameters were calculated. Mitochondrial membrane potential, superoxide, and PN were determined from rhodamine 123, dihydroethidium, and DAX-J2 PON green fluorescence measurements, respectively. Mitochondrial protein nitrotyrosination was determined by Western blots. Both exogenous PN and SIN-1 decreased respiratory function in WT isolated brain mitochondria. FeTMPyP enhanced state III and state IVo mitochondrial respiration in both WT and eNOS-KO mitochondria. FeTMPyP also elevated state IIIu respiration in eNOS-KO mitochondria. Unlike PN, neither SIN-1 nor FeTMPyP depolarized the mitochondria. Although mitochondrial protein nitrotyrosination was unaffected by SIN-1 or FeTMPyP, FeTMPyP reduced mitochondrial PN levels. Mitochondrial superoxide levels were increased by FeTMPyP but were unaffected by PN or SIN-1. Thus, we present the evidence of functionally significant PN generation in isolated brain mitochondria. Mitochondrial PN activity was physiologically relevant in WT mice and pathologically significant under conditions with eNOS deficiency.NEW & NOTEWORTHY Mitochondria generate superoxide and nitric oxide that could potentially react with each other to produce PN. We observed eNOS and nNOS immunoreactivity in isolated brain and heart mitochondria with pharmacological inhibition of nNOS found to modulate the mitochondrial respiratory function. This study provides evidence of generation of functionally significant PN in isolated brain mitochondria that affects respiratory function under physiological conditions. Importantly, the mitochondrial PN levels and activity were exaggerated in the eNOS-deficient mice, suggesting its pathological significance.
Keywords: eNOS; mitochondrial nitric oxide synthase; nNOS; nitrotyrosination; oxygen consumption rate.