Identification of calcium-independent phospholipase A₂γ in mitochondria and its role in mitochondrial oxidative stress

Oxidant-induced lipid peroxidation and cell death mediate pathologies associated with ischemia-reperfusion and inflammation. Our previous work in rabbit renal proximal tubular cells (RPTC) demonstrated that inhibition of Ca²⁺-independent phospholipase A2 (iPLA₂) potentiates oxidant-induced lipid peroxidation and necrosis, implicating iPLA₂ in phospholipid repair. This study was conducted to identify a RPTC mitochondrial PLA₂ and determine the role of PLA₂ in oxidant-induced mitochondrial dysfunction. iPLA₂ activity was detected in Percoll-purified rabbit renal cortex mitochondria (RCM) and in isolated mitochondrial inner membrane fractions from rabbit and human RCM. Immunoblot analysis and inhibitor sensitivity profiles revealed that iPLA ₂γ is the RCM iPLA₂ activity. RCM iPLA₂ activity was enhanced in the presence of ATP and was blocked by the PKCε V1-2 inhibitor. Oxidant-induced mitochondrial lipid peroxidation and swelling were accelerated by pretreatment with R-BEL, but not S-BEL. Furthermore, oxidant treatment of isolated RCM resulted in decreased iPLA₂γ activity. These results reveal that RCM iPLA₂ is iPLA ₂γ, RCM iPLA₂γ is regulated by phosphorylation by PKCε, iPLA₂γ protects RCM from oxidant-induced lipid peroxidation and dysfunction, and that a strategy to preserve or enhance iPLA₂γ activity may be of therapeutic benefit.