Phospholipase A2 activation in cultured mouse hepatocytes exposed to tumor necrosis factor-alpha.
High concentrations of tumor necrosis factor alpha (TNF alpha) 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 A2 activation in TNF alpha-induced toxicity in mouse hepatocytes, since it has been reported to play a key role in TNF alpha cytolytic activity in other cell types. Recombinant murine TNF alpha (0.1 microgram/mL) caused a dose-dependent increase in PLA2 activity in cultured mouse hepatocytes. The increase in PLA2 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, TNF alpha-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 PLA2 inhibitors, chlorpromazine (CPZ) and 4-bromophenacyl bromide (BPB), both prevented the TNF alpha-induced increase in PLA2 activity. They also reduced ATP depletion, GSSG efflux, and cytotoxicity. The PLA2 inhibitor, manoalide (a natural marine product), completely prevented PLA2 activation and cytotoxicity induced by TNF alpha. Pretreatment of hepatocytes with cycloheximide, to inhibit protein synthesis, increased TNF alpha-induced cytotoxicity. Cycloheximide pretreatment also potentiated PLA2 activation, ATP depletion, and GSSG efflux. CPZ and BPB both reduced the extent of PLA2 activation, ATP depletion, GSSG formation, and cytotoxicity in the cycloheximide pretreated cells exposed to TNF alpha. Taken together, these results demonstrate that TNF alpha activates PLA2, which occurs prior to other deleterious events in hepatocytes, and that inhibition of PLA2 activity reduces cell injury by TNF alpha. This suggests that PLA2 activation may lead to impairment of energy metabolism, an oxidant stress, and cytotoxicity in cells exposed to TNF alpha. Additionally, protein synthesis inhibition potentiates TNF alpha induction of PLA2 and toxicity, suggesting that there is a protein-synthesis-dependent protective mechanism in hepatocytes which ameliorates the effects induced by PLA2. These findings provide strong evidence that PLA2 activation plays an important role in the pathogenesis of toxicity induced by TNF alpha in cultured mouse hepatocytes.
Department of Environmental Health, College of Veterinary Medicine & Biomedical Sciences, Colorado State University, Fort Collins 80523.
G M
GM
Adamson
T J
TJ
Carlson
R E
RE
Billings
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D015777:Eicosanoids / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D051379:Mice; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D015777:Eicosanoids; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D013729:Terpenes; D014409:Tumor Necrosis Factor-alpha; D016718:Arachidonic Acid; D003513:Cycloheximide; C045673:manoalide; D010741:Phospholipases A; D054467:Phospholipases A2