Figure 6.

FTY720 inhibits cPLA₂ activity in vitro. cPLA₂ activity was determined in lysates from RBL-2H3 cells (A) or in lysates from HEK 293 cells overexpressing cPLA₂ (C) using a liposome assay with 1-palmitoyl-2-[¹⁴C]-arachidonyl-sn-glycerol-phosphatidylcholine (400 pmol) as substrate. Increasing concentrations of FTY720 were added separately. Data are expressed as picomole AA released per minute per milligram and are means ± SD of duplicate determinations. (B) RBL-2H3 or HEK 293 cells were pretreated without (□) or with FTY720 (■) for 18 hours, washed extensively, and lysed. cPLA₂ activity was determined in lysates. *P < .05. (D) sPLA₂ activity was determined in lysates from HEK 293 cells overexpressing sPLA₂ as described.³⁵ Data are expressed as percent hydrolysis of [³H]-oleate–labeled lipids. (E) iPLA₂ activity was determined with L-α-dipalmitoyl[2-palmitoyl-1-¹⁴C]phosphatidylcholine as substrate in liposomes containing dipalmitoylphosphatidylcholine and Triton X-100 as described.³⁶ Similar results were obtained in 3 independent experiments. Data are expressed as the means ± SD of duplicate determinations (D,E).