Fig. 8.

Pertussis toxin (Ptx) and MAP kinase inhibitors decrease S1P-induced AA release or calcium mobilization in A549 cells. The time course of S1P-induced cPLA₂α, ERK, and p38 phosphorylation was shown in A. Following ³H-AA labeling, cells were pretreated with Ptx (100 ng/ml) for 18 h, or U0126 (10 μM), PD-98059 (10 μM), SB-203580/SB35 (10 μM), or SB-202190/SB21 (3 μM) for 30 min followed by stimulation with 500 nM S1P for 10 min. The AA release assay is represented as percentage of control cells AA release (B). The calcium flux assay was performed in 96-well plates, and cells were stimulated with 500 nM S1P (C, ○: S1P; □: vehicle; ▵: Ptx-treated cells with S1P). The combined effect of Ptx and U0126 on S1P-induced AA release is represented in D. The AA release data represent the means ± SD from 3 independent experiments, *P < 0.001 by Student's t-test compared with control cells stimulated with S1P. **P < 0.001 by Student's t-test in comparison of U0126/Ptx-treated cells with U0126 or Ptx-treated cells. The calcium flux analysis gave similar results across 3 independent experiments.