Cytosolic phospholipase A₂α inhibition prevents neuronal NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not subsequent cell death

Phospholipase A₂ (PLA₂) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [³H]-AA released into the extracellular medium following NMDA receptor stimulation (100 μM) increased with time and was completely prevented by the addition of the NMDA receptor antagonist MK-801 (10 μM) or by removal of extracellular Ca²⁺. Neither diacylglycerol lipase inhibition (RHC-80267; 10 μM) nor selective inhibition of Ca²⁺-independent PLA₂ [bromoenol lactone (BEL); 10 μM] alone had an effect on NMDA receptor-stimulated release of [ ³H]-AA. Release was prevented by methyl arachidonyl fluorophosphonate (MAFP) (5 μM) and AACOCF₃ (1 μM), inhibitors of both cytosolic PLA₂ (cPLA₂) and Ca²⁺-independent PLA ₂ isozymes. This inhibition effectively translated to block of NMDA-induced prostaglandin (PG) production. An inhibitor of p38MAPK, SB 203580 (7.5 μM), also significantly reduced NMDA-induced PG production providing suggestive evidence for the role of cPLA₂α. Its involvement in release was confirmed using cultures derived from mice deficient in cPLA ₂α, which failed to produce PGs in response to NMDA receptor stimulation. Interestingly, neither MAFP, AACOCF₃ nor cultures derived from cPLA₂α null mutant animals showed any protection against NMDA-mediated neurotoxicity, indicating that inhibition of this enzyme may not be a viable protective strategy in disorders of the cortex involving over-activation of the NMDA receptor.