Mechanism of endothelial cell NADPH oxidase activation by angiotensin II -: Role of the p47Phox subunit
Mechanism of endothelial cell NADPH oxidase activation by angiotensin II -: Role of the p47Phox subunit
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DOI：
10.1074/jbc.m209793200
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发表时间：
2003-04-04
影响因子：
4.8
通讯作者：
Shah, AM
中科院分区：
文献类型：
--
作者：
Li, JM;Shah, AM
内皮细胞表达一种组成型活性吞噬细胞型NADPH氧化酶，其活性通过血管紧张素II等激动剂增强。我们最近报道了李，j - m。（2002） [j]。化学，277,19952-19960)，与中性粒细胞相反，在未受刺激的内皮细胞中，NADPH氧化酶的很大一部分以预组装的细胞内复合物的形式存在。在这里，我们研究了血管紧张素ii诱导内皮细胞NADPH氧化酶激活的机制。在p47（phox）敲除小鼠分离的冠状微血管内皮细胞中，血管紧张素II （100 nmol/l）诱导的活性氧产生（通过二氯氢荧光素或荧光素化学发光测量）完全不存在。将p47(phox) cDNA转染到p47（phox-/-）细胞中可以恢复血管紧张素II的应答，而将反义p47(phox) cDNA转染到野生型细胞中则可以减少p47（phox）并抑制血管紧张素II的应答。在未受刺激的人微血管内皮细胞中，p47(phox)-p22（phox）复合物形成显著，但p47（phox）磷酸化极少。血管紧张素II诱导P47（phox）的丝氨酸快速磷酸化（在1分钟内，在15分钟内达到峰值），P47(phox) -p22（phox）复合物形成增加1.9 +/- 0.1倍，nadph依赖性O-2（自由基阴离子）产生增加1.6 +/- 0.2倍（p < 0.05）。P47 （phox）被重新分配到“核”和膜富集的细胞部分。这些数据表明，血管紧张素ii刺激的内皮细胞NADPH氧化酶活性是通过丝氨酸磷酸化p47（phox）及其增强与p22（phox）的结合来调节的。
Endothelial cells express a constitutively active phagocyte-type NADPH oxidase whose activity is augmented by agonists such as angiotensin II. We recently reported (Li, J.-M., and Shah, A. M. (2002) J. Biol. Chem. 277, 19952-19960) that in contrast to neutrophils a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as preassembled intracellular complexes. Here, we investigate the mechanism of angiotensin II-induced endothelial NADPH oxidase activation. Angiotensin II (100 nmol/liter)-induced reactive oxygen species production (as measured by dichloro-hydrofluorescein fluorescence or lucigenin chemiluminescence) was completely absent in coronary microvascular endothelial cells isolated from p47(phox) knockout mice. Transfection of p47(phox) cDNA into p47(phox-/-) cells restored the angiotensin II response, whereas transfection of antisense p47(phox) cDNA into wild-type cells depleted p47(phox) and inhibited the angiotensin II response. In unstimulated human microvascular endothelial cells, there was significant p47(phox)-p22(phox) complex formation but minimal detectable p47(phox) phosphorylation. Angiotensin II induced rapid serine phosphorylation of P47(phox) (within 1 min, peaking at similar to15 min), a 1.9 +/- 0.1-fold increase in p47(phox)-p22(phox) complex formation and a 1.6 +/- 0.2-fold increase in NADPH-dependent O-2(radical anion) production (p < 0.05). p47(phox) was redistributed to "nuclear" and membrane-enriched cell fractions. These data indicate that angiotensin II-stimulated endothelial NADPH oxidase activity is regulated through serine phosphorylation of p47(phox) and its enhanced binding to p22(phox).