Specificity of endogenous fatty acid release during tumor necrosis factor-induced apoptosis in WEHI 164 fibrosarcoma cells

Recombinant tumor necrosis factor alpha (rTNF-alpha)-induced release of end ogenous fatty acids was examined in WEHI 164 clone 13 fibrosarcoma cells us ing a highly sensitive HPLC method. The initial rTNF-alpha-induced extracel lular release of endogenous fatty acids was dominated by 20:4n-6, 22:4n-6, 24:4n-6, and 18:1n-9 showing relative rates of 2.9, 0.9, 1.1, and 1.0, resp ectively. Release of endogenous AA and DNA fragmentation occurred simultane ously and preceded cell death by approx. 2 h, Methyl arachidonoyl fluoropho sphonate and LY311727, specific inhibitors of Ca2+-dependent cytosolic PLA( 2) (cPLA(2)) and secretory PLA(2) (sPLA(2)), respectively neither blocked r TNF-alpha-induced cytotoxicity or endogenous AA release. However, both inhi bitors reduced rTNF-alpha-induced release of other endogenous fatty acids. In comparison, the antioxidant butylated hydroxyanisole (BHA) completely in hibited the rTNF-alpha-induced cytotoxicity as well as AA release mediated through the TNF receptor p55, while the very similar antioxidant butylated hydroxytoluene had no effect. BHA did not inhibit recombinant cPLA(2) or sP LA2 enzyme activity in vitro. Furthermore, stimulation of cells with rTNF-a lpha for 4 h did not increase cPLA(2) enzyme activity. The data indicate th at neither cPLA(2) or sPLA(2) mediate rTNF-alpha-induced apoptosis and extr acellular AA release in WEHI cells. The results suggest that a BHA-sensitiv e signaling pathway coupled to AA release is a key event in TNF-induced cyt otoxicity in these cells.