PLA₂ dependence of diaphragm mitochondrial formation of reactive oxygen species

Contraction-induced respiratory muscle fatigue and sepsis- related reductions in respiratory muscle force-generating capacity are mediated, at least in part, by reactive oxygen species (ROS). The subcellular sources and mechanisms of generation of ROS in these conditions are incompletely understood. We postulated that the physiological changes associated with muscle contraction (i.e., increases in calcium and ADP concentration) stimulate mitochondrial generation of ROS by a phospholipase A₂ (PLA₂)-modulated process and that sepsis enhances muscle generation of ROS by upregulating PLA₂ activity. To test these hypotheses, we examined H₂O₂ generation by diaphragm mitochondria isolated from saline-treated control and endotoxin-treated septic animals in the presence and absence of calcium and ADP; we also assessed the effect of PLA₂ inhibitors on H₂O₂ formation. We found that 1) calcium and ADP stimulated H₂O₂ formation by diaphragm mitochondria from both control and septic animals; 2) mitochondria from septic animals demonstrated substantially higher H₂O₂ formation than mitochondria from control animals under basal, calcium-stimulated, and ADP- stimulated conditions; and 3) inhibitors of 14-kDa PLA₂ blocked the enhanced H₂O₂ generation in all conditions. We also found that administration of arachidonic acid (the principal metabolic product of PLA₂ activation) increased mitochondrial H₂O₂ formation by interacting with complex I of the electron transport chain. These data suggest that diaphragm mitochondrial ROS formation during contraction and sepsis may be critically dependent on PLA₂ activation.