O-HEXADECYL-2-O-METHYL-SN-GLYCERO-3-PHOSPHOCHOLINE INHIBITS DIACYLGLYCEROL KINASE IN WEHI-3B CELLS

The effects of O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET- 16-OCH3-GPC) and its metabolite 1-O-hexadecyl-2-O-methyl-sn-glycerol ( AMG) on the activity of diacylglycerol kinase (DGK) in WEHI-3B cells w ere investigated. Treatment of WEHI-3B cells with 200 nM 12-O-tetradec anoylphorbol-13-acetate (TPA) for 5 min leads to the activation of cyt osolic DGK without significant effect on microsomal DGK. When these ce lls were first exposed to 50 muM ET-16-OCH3-GPC for 30 min prior to ac tivation with TPA, the activity of DGK was inhibited by about 70%, as measured by the ability of enzyme to form P-32!phosphatidic acid (P- 32!PA). Addition of either ET-16-OCH3-GPC or AMG to the preparation of enzyme in vitro also inhibited 1,2-dioleoyl-sn-glycerol (DG) phosphor ylation in the presence of gamma-P-32!ATP. The IC50 value for inhibit ion of cytosolic DGK by ET-16-OCH3-GPC and AMG were about 8.5 and 15 m uM, respectively. ET-16-OCH3-GPC also inhibited the ability of guanosi ne 5'-O-(3-thiophosphate) (GTP-gammaS) to activate DGK in vitro. The p otency of ET-16-OCH3-GPC at 10 muM in inhibiting DGK was greater than that of sphingosine at 50 muM, but less than that of R59022 (a specifi c DGK inhibitor) at 10 muM. The abilities of ET-16-OCH3-GPC and AMG to inhibit cytosolic DGK in intact WEHI-3B cells and enzyme preparations in vitro suggest that the cytotoxic activity of ether lipids may in p art result from interference with this vital enzyme involved in the sy nthesis of phospholipids from DG and in cell-signaling systems.