PHOSPHOLIPASE A(2) STIMULATION BY METHYL MERCURY IN NEURON CULTURE

In primary prelabeled cultures of cerebellar granule cells, methyl mer cury (MeHg) induced a concentration- and time-dependent release of [H- 3]arachidonic acid. MeHg-induced [H-3]arachidonate release was partial ly dependent on the extracellular Ca2+ concentration. MeHg at 10-20 mu M also stimulated basal Ca-45(2+) uptake after 20 min of incubation a t 37 degrees C, and at 10 mu M inhibited K+ depolarization-stimulated uptake. MeHg stimulated [H-3]arachidonate uptake, but had no effect on the rate of phospholipid reacylation. Phospholipase A(2) (PLA(2)) act ivation preceded cytotoxicity, but at higher concentrations of MeHg su ch dissociation was not evident. Inhibition of MeHg-induced PLA(2) act ivation by 100 mu M mepacrine failed to modify cytotoxicity. MeHg-indu ced lipoperoxidation, measured as the production of thiobarbituric aci d-reacting products, was inhibited by alpha-tocopherol without inhibit ion of [H-3]arachidonate release. The absence of alpha-tocopherol inhi bition of MeHg-induced arachidonate release precludes a causal role fo r lipoperoxide-induced PLA(2) activation in this system. Moreover, MeH g induced an increased susceptibility of unilamellar vesicles to exoge nous PLA(2) in the presence of low Ca2+ concentrations without evidenc e of lipid peroxidation. [H-3]Arachidonate incorporation into granule neuron phospholipids was analyzed by isocratic HPLC analysis. Relative ly high proportional incorporation was found in the combined phosphati dylcholine fractions and phosphatidylinositol. With MeHg, an increase in the relative specific activity of incorporation was found in the ph osphatidylinositol fraction, indicating a preferential turnover in thi s phospholipid species in the presence of MeHg.