Role of Ca²⁺-independent phospholipase A₂γ in Ca²⁺-induced mitochondrial permeability transition

Our laboratory previously demonstrated Ca²⁺-independent phospholipase A₂γ (iPLA₂γ) is localized to mitochondria and that iPLA₂ inhibition blocks cisplatin-induced caspase-mediated apoptosis. Whereas the mitochondrial permeability transition (MPT) is a key control point for apoptosis, the role of mitochondrial iPLA ₂γ in MPT has not been established. In the present study, we addressed this issue. Ca²⁺-induced renal cortex mitochondrial (RCM) swelling was blocked by the MPT inhibitor cyclosporine A. The R-isomer of bromoenol lactone (R-BEL), which enantiospecifically inhibits iPLA ₂γ, inhibited Ca²⁺-induced RCM MPT, whereas S-BEL (negative control) had no effect. Ca²⁺ treatment resulted in a significant increase in free arachidonic acid (AA) (>50 μM) in the RCM suspension that was blocked by pretreatment with BEL. No increases in free myristic, palmitic, stearic, oleic, linoleic, or docosahexaenoic acid were detected after Ca²⁺ treatment. The addition of AA (18 μM) to Ca²⁺-treated RCM with inhibited iPLA₂γ activity restored MPT. We also determined that RCM iPLA₂γ displays higher activity against plasmenylcholine with AA in the sn-2 position than oleic acid. Ca²⁺ exposure significantly increased RCM iPLA ₂γ activity; however, the Ca²⁺-induced activation of iPLA₂γ was not the result of mitochondrial membrane potential dissipation, opening of the MPT pore, or mitochondrial swelling. Taken together these findings provide strong evidence that Ca²⁺-induced RCM MPT is mediated by iPLA₂γ-catalyzed AA liberation.