Induction of cAMP synthesis by iloprost or forskolin and the effect of the pretreatments on the synthesis in HEL or NS-Meg cells. Each value (mean ± SE, n = 3) shows the amount of cAMP synthesized by 1 × 105 cells. Cells were first preincubated with PMA or respective cytokines for 24 hours, then incubated in a 24-well microplate (1 × 105 cells/well) in 500 μL of RPMI 1640 containing 0.5 mmol/L 3-isobutyl-1-methylxanthine and 0.1% BSA at 37°C for 10 minutes and then iloprost or forskolin was added to each well. Incubation was continued for another 30 minutes. (A) and (B) Addition of iloprost increased significantly (**, P < .001) the synthesis of cAMP in each cell line. Addition of iloprost to the cells that had been preincubated for 24 hours with PMA or cytokines further increased (**, P < .001) cAMP synthesis as compared with the untreated cells of each cell line. The pretreatments with the cytokines (without iloprost) yielded no significant increase of cAMP in the cells from each cell line compared with nontreated cells (data not shown). (C) Forskolin increased cAMP synthesis without significant difference between PMA- or cytokine-pretreated cells and nontreated ones (control).
