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 (cPLA2) 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-[C]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, p67 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 cPLA2 was induced by Ang II in human neutrophils. In addition, Ang II induced translocation of the cytosolic oxidase components, detected by translocation of p47, 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.
Hazan-Halevy
Inbal
I
Hypertension Unit, Soroka Medical Center and Ben-Gurion University, Beer Sheva, Israel. inbal@bgu.ac.il
Levy
Tal
T
Wolak
Talya
T
Lubarsky
Ira
I
Levy
Rachel
R
Paran
Esther
E
D000328:Adult; D000804:Angiotensin II / Q000494:pharmacology*; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010586:Phagocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D017382:Reactive Oxygen Species / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D017382:Reactive Oxygen Species; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D019255:NADPH Oxidases; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2