Methylmercury enhances arachidonic acid release and cytosolic phospholipase A₂ expression in primary cultures of neonatal astrocytes

Cytosolic phospholipase A₂ (cPLA₂) stimulates the hydrolysis of sn-2 ester bond in membrane phospholipids releasing arachidonic acid (AA) and lysophospholipids. The present study examined the effect of methylmercury (MeHg) on cPLA₂ activation and AA release in primary cultures of neonatal rat cerebral astrocytes. Astrocytes were preloaded overnight at 37°C with ³H-AA to metabolically label phospholipids. The effect of MeHg on the activation of cPLA₂ was measured by the release of ³H-AA from astrocytes over 120 min. MeHg (5 μM) caused a significant increase in AA release at 10, 30, 60, and 120 min, whereas 2.5 μM MeHg significantly increased AA release only at 120 min. MeHg-induced increase in ³H-AA release was due to cPLA₂ activation, since arachidonyl trifluoromethyl ketone (AACOCF₃), a selective inhibitor of cPLA₂, completely abolished MeHg's effect. Consistent with these observations, MeHg (5.0 and 10.0 μM) increased cPLA₂ mRNA (6 h) and cPLA₂ protein expression (5.0 and 10.0 μM; 24 h). The time-course of these effects suggests an immediate direct or indirect effect of MeHg on cPLA₂ activation and ³H-AA release as well as a long-term effect involving the induction of cPLA₂. Thin layer chromatographic analysis of ³H-AA-labeled phospholipids showed that MeHg-stimulated astrocyte ³H-AA release was not due to increased incorporation of ³H-AA into the putative substrates of cPLA₂. These results invoke cPLA₂ as a putative target for MeHg toxicity, and support the notion that cPLA₂-stimulated hydrolysis and release of AA play a critical role in MeHg-induced neurotoxicity.