Involvement of the mitogen-activated protein kinase cascade in peroxynitrite-mediated arachidonic acid release in vascular smooth muscle cells.
Eicosanoid production is reduced when the nitric oxide (NO.) pathway is inhibited or when the inducible NO synthase gene is deleted, indicating that the NO. and arachidonic acid pathways are linked. We hypothesized that peroxynitrite, formed by the reaction of NO. and superoxide anion, may cause signaling events leading to arachidonic acid release and subsequent eicosanoid generation. Western blot analysis of rat arterial smooth muscle cells demonstrated that peroxynitrite (100-500 microM) and 3-morpholinosydnonimine (SIN-1; 200 microM) stimulate phosphorylation of extracellular signal-regulated kinase (ERK), p38, and cytosolic phospholipase A(2) (cPLA(2)). We found that peroxynitrite-induced arachidonic acid release was completely abrogated by the mitogen-activated protein/ERK kinase (MEK) inhibitor U0126 and by calcium chelators. With the p38 inhibitor SB-20219, we demonstrated that peroxynitrite-induced p38 phosphorylation led to minor arachidonic acid release, whereas U0126 completely blocked p38 phosphorylation. Addition of arachidonic acid caused p38 phosphorylation, suggesting that arachidonic acid or its metabolites are responsible for p38 activation. KN-93, a specific inhibitor of Ca(2+)/calmodulin-dependent kinase II (CaMKII), revealed no role for this kinase in peroxynitrite-induced arachidonic acid release in our cell system. Together, these results show that in response to peroxynitrite the cell initiates the MEK/ERK cascade leading to cPLA(2) activation and arachidonic acid release. Thus studies investigating the role of the NO. pathway on eicosanoid production must consider the contribution of signaling pathways initiated by reactive nitrogen species. These findings may provide evidence for a new role of peroxynitrite as an important reactive nitrogen species in vascular disease.
Upmacis
Rita K
RK
Deptartment of Biochemistry, Center of Vascular Biology, Weill Medical College of Cornell University, New York, NY 10021, USA. rupmacis@med.cornell.edu
Deeb
Ruba S
RS
Resnick
Matthew J
MJ
Lindenbaum
Rochelle
R
Gamss
Caryn
C
Mittar
Dev
D
Hajjar
David P
DP
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism* / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D015232:Dinoprostone / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D054513:Group IV Phospholipases A2; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism* / Q000494:pharmacology; D010741:Phospholipases A / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D048051:p38 Mitogen-Activated Protein Kinases
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D030421:Peroxynitrous Acid; D016718:Arachidonic Acid; D009569:Nitric Oxide; C002385:linsidomine; D008981:Molsidomine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; D015232:Dinoprostone