Elevated endothelin-1 levels impair nitric oxide homeostasis through a PKC-dependent pathway
Elevated endothelin-1 levels impair nitric oxide homeostasis through a PKC-dependent pathway
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DOI：
10.1161/circulationaha.105.001503
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发表时间：
2006-07-04
期刊：
影响因子：
37.8
通讯作者：
Ross, Heather J.
中科院分区：
文献类型：
--
作者：
Ramzy, Danny;Rao, Vivek;Ross, Heather J.
背景内皮素-1（ET-1）在维持血管张力和病理状态如缺血/再灌注（I/R）损伤、冠状动脉血管痉挛和心脏移植物血管病变中起重要作用。我们评估了I/R后ET-1水平升高的影响，以确定ET-1是否通过特异性蛋白激酶C（PKC）亚型的转位调节一氧化氮（NO）的产生。方法和结果-人大隐静脉内皮细胞（HSVECs）（n = 8）与ET-1或磷酸盐缓冲盐水（PBS）孵育24小时。通过测量硝酸盐/亚硝酸盐水平来确定上清液中的NO产生。检测内皮型一氧化氮合酶（eNOS）、诱导型一氧化氮合酶（iNOS）、小窝蛋白-1（caveolin-1）和蛋白激酶C（PKC）的蛋白表达。最后，在暴露于感兴趣的药物后评估PKC易位和活性。暴露于ET-1的HSVECs显示NO产生减少。PKC抑制减少NO的产生，而PKC激活增加生产。当暴露于ET-1的HSVECs用PKC激动剂PMA处理时，NO的产生得以维持。ET-1处理后eNOS蛋白表达减少。PKC抑制也下调eNOS蛋白表达，而PMA上调表达。与对照组相比，ET-1暴露导致PKC δ和PKC β易位显著增加，而PKC λ易位受到抑制。ET-1暴露显着降低整体PKC活性与control. Conclusions，我们的研究表明，高水平的ET-1损害内皮NO的生产通过异构体特异性PKC介导的抑制eNOS的表达。波生坦对ET-1的拮抗作用刺激PKC λ的易位，并导致PKC活性和NO产生增加。ET-1拮抗剂可能为改善血管稳态提供一种新的治疗策略。
Background-Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone and pathological states such as ischemia/reperfusion (I/R) injury, coronary vasospasm, and cardiac allograft vasculopathy. We assessed the effects of elevated ET-1 levels as seen after I/R to determine if ET-1 modulates nitric oxide ( NO) production via the translocation of specific protein kinase C (PKC) isoforms.Methods and Results-Human saphenous vein endothelial cells (HSVECs) (n = 8) were incubated with ET-1 or phosphate-buffered saline (PBS) for 24 hours. NO production was determined in the supernatant by measuring nitrate/nitrite levels. Protein expression of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), caveolin-1 and PKC were determined. Lastly, PKC translocation and activity were assessed after exposure to the drug of interest. HSVECs exposed to ET-1 displayed decreased NO production. PKC inhibition reduced NO production, whereas PKC activation increased production. NO production was maintained when HSVECs exposed to ET-1 were treated with the PKC agonist, PMA. eNOS protein expression was reduced after ET-1 treatment. PKC inhibition also downregulated eNOS protein expression, whereas PMA upregulated expression. ET-1 exposure led to a significant increase in PKC delta and PKC epsilon translocation compared with control, whereas translocation of PKC lambda was inhibited. ET-1 exposure significantly reduced overall PKC activity compared with control.Conclusions-Our study demonstrates that high levels of ET-1 impair endothelial NO production via an isoform-specific PKC-mediated inhibition of eNOS expression. ET-1 antagonism with bosentan stimulates translocation of PKC lambda and leads to increased PKC activity and NO production. ET-1 antagonism may provide a novel therapeutic strategy to improve vascular homeostasis.