Rat brain cortex mitochondria release group II secretory phospholipase A2 under reduced membrane potential

Activation of brain mitochondrial phospholipase(s) A2 (PLA2) might contribute to cell damage and be involved in neurodegeneration. Despite the potential importance of the phenomenon, the number, identities, and properties of these enzymes are still unknown. Here, we demonstrate that isolated mitochondria from rat brain cortex, incubated in the absence of respiratory substrates, release a Ca2-dependent PLA2 having biochemical properties characteristic to secreted PLA2 (sPLA2) and immunoreacting with the antibody raised against recombinant type IIA sPLA2 (sPLA2-IIA). Under identical conditions, no release of fumarase in the extramitochondrial medium was observed. The release of sPLA2 from mitochondria decreases when mitochondria are incubated in the presence of respiratory substrates such as ADP, malate, and pyruvate, which causes an increase of transmembrane potential determined by cytofluorimetric analysis using DiOC6(3) as a probe. The treatment of mitochondria with the uncoupler carbonyl cyanide 3-chlorophenylhydrazone slightly enhances sPLA2 release. The increase of sPLA2 specific activity after removal of mitochondrial outer membrane indicates that the enzyme is associated with mitoplasts. The mitochondrial localization of the enzyme has been confirmed by electron microscopy in U-251 astrocytoma cells and by confocal laser microscopy in the same cells and in PC-12 cells, where the structurally similar isoform type V-sPLA2 has mainly nuclear localization. In addition to sPLA2, mitochondria contain another phospholipase A2 that is Ca2-independent and sensitive to bromoenol lactone, associated with the outer mitochondrial membrane. We hypothesize that, under reduced respiratory rate, brain mitochondria release sPLA2-IIA that might contribute to cell damage.