Opposing effects of reactive oxygen species and cholesterol on endothelial nitric oxide synthase and endothelial cell caveolae.
Opposing effects of reactive oxygen species and cholesterol on endothelial nitric oxide synthase and endothelial cell caveolae.
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DOI：
10.1161/01.res.85.1.29
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发表时间：
1999-07
影响因子：
20.1
通讯作者：
Timothy E. Peterson;Veronica Poppa;H. Ueba;A. Y. Wu;C. Yan;B. Berk
中科院分区：
文献类型：
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作者：
Timothy E. Peterson;Veronica Poppa;H. Ueba;A. Y. Wu;C. Yan;B. Berk
内皮型一氧化氮合酶（eNOS）合成一氧化氮（NO）对正常血管稳态至关重要。eNOS功能受到激动剂和血流的快速调节，并长期受到调节mRNA稳定性和基因转录的因子的调节。最近，eNOS定位到被称为小泡的特殊质膜内陷，被认为是eNOS活性最大化的必要条件。由于小窝富含胆固醇，高胆固醇血症与一氧化氮生成增加有关，我们首先研究了胆固醇负荷对培养牛主动脉内皮细胞（BAECs） eNOS定位和一氧化氮生成的影响。采用OptiPrep梯度密度离心法制备微囊泡富集组分。用30微克/毫升胆固醇处理BAECs 24小时，eNOS蛋白总表达量显著增加（1.50倍），eNOS与小泡富集膜相关（2.23倍），钙离子载体刺激NO生成（1.56倍）。由于活性氧（ROS）有助于高胆固醇血症的内皮功能障碍，我们接下来研究了ROS对eNOS定位和小泡数量的影响。1微mol/L LY83583（一种产生超氧化物的萘喹喹啉二酮）处理BAECs 24小时，可减少电镜观察到的小泡数量，并阻止胆固醇介导的eNOS表达增加。在体外，将富含小窝蛋白的膜暴露于ROS（黄嘌呤加黄嘌呤氧化酶）中，比eNOS更容易将小窝蛋白从膜上解离。这些结果表明，胆固醇处理增加了eNOS表达，而ROS处理降低了eNOS表达以及eNOS与小窝蛋白在小窝富集膜中的关联。我们的数据表明，氧化应激通过调节小泡形成、eNOS表达和eNOS-小泡蛋白相互作用来调节内皮功能。
Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular homeostasis. eNOS function is rapidly regulated by agonists and blood flow and chronically by factors that regulate mRNA stability and gene transcription. Recently, localization of eNOS to specialized plasma membrane invaginations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO production, we first studied the effects of cholesterol loading on eNOS localization and NO production in cultured bovine aortic endothelial cells (BAECs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifugation. Treatment of BAECs with 30 microgram/mL cholesterol for 24 hours stimulated significant increases in total eNOS protein expression (1.50-fold), eNOS associated with caveolae-enriched membranes (2.23-fold), and calcium ionophore-stimulated NO production (1.56-fold). Because reactive oxygen species (ROS) contribute to endothelial dysfunction in hypercholesterolemia, we next studied the effects of ROS on eNOS localization and caveolae number. Treatment of BAECs for 24 hours with 1 micromol/L LY83583, a superoxide-generating napthoquinolinedione, decreased caveolae number measured by electron microscopy and prevented the cholesterol-mediated increases in eNOS expression. In vitro exposure of caveolae-enriched membranes to ROS (xanthine plus xanthine oxidase) dissociated caveolin more readily than eNOS from the membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveolae formation, eNOS expression, and eNOS-caveolin interactions.