Stimulation of NADPH oxidase by angiotensin II in human neutrophils is mediated by ERK, p38 MAP-kinase and cytosolic phospholipase A₂

Objective: The present research was designed to study the involvement of ERK and p38 MAP-kinase in cytosolic phospholipase A₂ (cPLA ₂) and NADPH-oxidase activation by angiotensin II (Ang II) in human neutrophils. Methods: NADPH-oxidase activity was measured by reduction of cytochrome C. cPLA₂ activity was measured in cell lysate using sonicated dispersions of 1-stearoyl-2-[¹⁴C]arachidonyl phosphatidylcholine. Cells were incubated with MEK inhibitor UO126 or with p38 MAP-kinase inhibitor SB202190 prior to stimulation with Ang II. Translocation of p47^phox, p67^phox and CPLA₂ and phosphorylation of ERK and p38 MAP-kinase were measured by immunoblot analysis. Results: Ang II induced a dose-dependent activation of NADPH oxidase in neutrophils and monocytes as well as in differentiated PLB-985 cells towards neutrophil or monocyte lineages, but not in cPLA₂-deficient differentiated PLB-985 cells. An immediate activation of both ERK and p38 MAP-kinase and of cPLA ₂ was induced by Ang II in human neutrophils. In addition, Ang II induced translocation of the cytosolic oxidase components, detected by translocation of p47^phox, which preceded the translocation of cPLA₂ induced by this agonist. The p38 MAP-kinase inhibitor SB202190 or the MEK-ERK pathway inhibitor UO126 totally inhibited the activation of both NADPH oxidase and CPLA₂ as well as the translocation of cytosolic oxidase components and of CPLA₂ to the membrane fractions. Conclusions: These results suggest that either ERK or p38 MAP-kinase are involved in the activation of both cPLA₂ and NADPH oxidase, and that cPLA₂ is required for activation of the NADPH oxidase by Ang II in human neutrophils.