Angiotensin II stimulates p38MAPK and ERK1/2 phosphorylation and p38 kinase activity in human neutrophils. Neutrophils (9 × 106 cells/mL) were preincubated at 37°C for 7 hours in KR-HEPES without additions. Next, they were treated with Ang II (100 nM) alone for the indicated times (A, B), or they were incubated with PD098059 (50 μM) for 30 minutes (C) or with DPI (10 μM) (D) for 30 minutes and then with Ang II for another 30 minutes. Cells were lysed, and protein extracts (50 μg/lane) were subjected to SDS-PAGE, transferred to PVDF, and probed with antiphospho-p38MAPK (A) or antiphospho-ERK1/2 (B-D), and the corresponding phosphorylated bands were detected by means of luminol-enhanced chemiluminescence. To verify even protein loading, the blots were subsequently stripped and reprobed with antibodies against unphosphorylated p38MAPK (A) or ERK1/2 (B-D). (E) Neutrophils were previously incubated with 5 μM SB203580 for 30 minutes and then with 100 nM Ang II. After cell lysis, p38MAPK activity was assessed by immune complex kinase assay, and intensities of the 28-kDa MBP bands in the autoradiogram were measured by densitometry scanning. Numbers represent the average activity increase from 3 independent experiments. Each blot is representative of a set of 3 experiments that yielded similar results.
