Abstract
High concentrations of tumor necrosis factor α (TNFα) are cytotoxic to cultured hepatocytes. Impairment of energy metabolism and generation of an intracellular oxidant stress are important events in the pathogenesis of this toxicity (6). In the present study, we have examined the role of phospholipase A~2~ activation in TNFα‐in‐duced toxicity in mouse hepatocytes, since it has been reported to play a key role in TNFα cytolytic activity in other cell types. Recombinant murine TNFα (0.1 μg/mL) caused a dose‐dependent increase in PLA~2~ activity in cultured mouse hepatocytes. The increase in PLA~2~ activity was observed after only 0.5 hour of exposure (152 ± 10% of control), and continued to increased over the first 4 hours of exposure (292 ± 32%). However, TNFα‐induced GSSG efflux and ATP depletion did not occur until after 2 hours of exposure. Furthermore, a small level of cytotoxicity was observed after a 24 hour incubation period. Putative PLA~2~ inhibitors, chlorpromazine (CPZ) and 4‐bromophenacyl bromide (BPB), both prevented the TNFα‐induced increase in PLA~2~ activity. They also reduced ATP depletion, GSSG efflux, and cytotoxicity. The PLA~2~ inhibitor, manoalide (a natural marine product), completely prevented PLA~2~ activation and cytotoxicity induced by TNFα. Pretreatment of hepatocytes with cycloheximide, to inhibit protein synthesis, increased TNFα‐induced cytotoxicity. Cycloheximide pretreatment also potentiated PLA~2~ activation, ATP depletion, and GSSG efflux. CPZ and BPB both reduced the extent of PLA~2~ activation, ATP depletion, GSSG formation, and cytotoxicity in the cycloheximide pretreated cells exposed to TNFα. Taken together, these results demonstrate that TNFα activates PLA~2~ which occurs prior to other deleterious events in hepatocytes, and that inhibition of PLA~2~ activity reduces cell injury by TNFα. This suggests that PLA~2~ activation may lead to impairment of energy metabolism, an oxidant stress, and cytotoxicity in cells exposed to TNFα. Additionally, protein synthesis inhibition potentiates TNFα induction of PLA~2~ and toxicity, suggesting that there is a protein‐synthesis‐dependent protective mechanism in hepatocytes which ameliorates the effects induced by PLA~2~. These findings provide strong evidence that PLA~2~ activation plays an important role in the pathogenesis of toxicity induced by TNFα in cultured mouse hepatocytes.