Upmacis Rita K, Deeb Ruba S, Resnick Matthew J, Lindenbaum Rochelle, Gamss Caryn, Mittar Dev, Hajjar David P
Deptartment of Biochemistry, Center of Vascular Biology, Weill Medical College of Cornell University, New York, NY 10021, USA.
Am J Physiol Cell Physiol. 2004 Jun;286(6):C1271-80. doi: 10.1152/ajpcell.00143.2003. Epub 2004 Jan 28.
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.
当一氧化氮（NO.）途径被抑制或诱导型一氧化氮合酶基因被敲除时，类花生酸生成减少，这表明NO.和花生四烯酸途径是相互关联的。我们推测，由NO.和超氧阴离子反应形成的过氧亚硝酸盐可能引发信号事件，导致花生四烯酸释放及随后的类花生酸生成。对大鼠动脉平滑肌细胞的蛋白质免疫印迹分析表明，过氧亚硝酸盐（100 - 500微摩尔）和3 - 吗啉代辛二酮（SIN - 1；200微摩尔）可刺激细胞外信号调节激酶（ERK）、p38和胞质磷脂酶A2（cPLA2）的磷酸化。我们发现，丝裂原活化蛋白/ERK激酶（MEK）抑制剂U0126和钙螯合剂可完全消除过氧亚硝酸盐诱导的花生四烯酸释放。使用p38抑制剂SB - 20219，我们证明过氧亚硝酸盐诱导的p38磷酸化导致少量花生四烯酸释放，而U0126完全阻断p38磷酸化。添加花生四烯酸会导致p38磷酸化，这表明花生四烯酸或其代谢产物是p38激活的原因。Ca(2+)/钙调蛋白依赖性激酶II（CaMKII）的特异性抑制剂KN - 93显示，在我们的细胞系统中该激酶在过氧亚硝酸盐诱导的花生四烯酸释放中不起作用。总之，这些结果表明，细胞对过氧亚硝酸盐的反应会启动MEK/ERK级联反应，导致cPLA2激活和花生四烯酸释放。因此，研究NO.途径在类花生酸生成中的作用时，必须考虑活性氮物质引发的信号通路的贡献。这些发现可能为过氧亚硝酸盐作为血管疾病中一种重要的活性氮物质的新作用提供证据。
