Figure 9.

Extracellular-regulated kinase 1/2 (ERK1/2) upstream inhibitor, PD98059, attenuates the mercury-induced phospholipase D (PLD) activation, ERK1/2 phosphorylation, and PLD₁ phosphorylation in mouse aortic endothelial cells (MAECs). The MAECs (5 × 10⁵ cells/ 35 mm dish) were labeled with [³²P]orthophosphate in phosphate-free Dulbecco-modified Eagle medium (DMEM) for 12 hours. Following [³²P]orthophosphate labeling, cells were pretreated with minimal essential medium (MEM) alone or MEM containing ERK1/2 upstream inhibitor, PD98059 (10 μmol/L), for 1 hour. Following pretreatment, cells were treated with MEM alone or MEM containing methylmercury (10 μmol/L) for 1 hour. At the end of the incubation period, [³²P]phosphatidylbutanol ([³²P]PBt) formed was determined (A). B, MAECs (5 × 10⁵ cells/35 mm dish) pretreated with MEM alone or MEM containing PD98059 (10 μmol/L) for 1 hour were treated with MEM alone or methylmercury (10 μmol/L) for 1 hour. Following treatment, proteins in cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with phospho-ERK1/2-specific, ERK1/2-specific, (C) phosphotheo-nine-PLD₁-specific, and PLD₁-specific monoclonal antibodies. The intensity of the protein bands were digitally determined as described in Materials and Methods. Data represent mean ± standard deviation (SD) calculated from 3 independent experiments. *Significantly different at P < .05 as compared to cells treated with MEM alone. **Significantly different at P < .05 as compared to cells treated with MEM containing mercury alone.