首页> 外文期刊>Endothelium: Journal of endothelial cell research >Identification of a functional leukocyte-type NADPH oxidase in human endothelial cells :a potential atherogenic source of reactive oxygen species.
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Identification of a functional leukocyte-type NADPH oxidase in human endothelial cells :a potential atherogenic source of reactive oxygen species.

机译:人内皮细胞中功能性白细胞型NADPH氧化酶的鉴定:活性氧的潜在动脉粥样硬化源。

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Cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein levels have increased reactive oxygen species (ROS) generation. The enzyme responsible for this ROS production elevation is unknown. We have examined for the presence of a functional leukocyte-type NADPH oxidase in EC to elucidate whether this enzyme could be the ROS source. The plasma membrane fraction of disrupted EC showed a reduced-minus-oxidized difference spectra with absorption peaks identical to those observed in the spectra of the leukocyte NADPH oxidase component, cytochrome b558. Western-blot analysis, using anti-gp91 -phox. anti -p22-phox. anti -p47-phox. and anti -p67-phox antibodies, demonstrated the protein expression of NADPH oxidase subunits in EC. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed the mRNA expression of gp91-phox, p22-phox, p47-phox, and p67-phox in EC. Sonicates from unstimulated EC produced no measurable superoxide; whereas, exogenously applied arachidonic acid activated superoxide generation in a manner that was dependent upon the presence of NADPH and both membrane and cytosolic fractions combined. Apocynin, a specific leukocyte NADPH oxidase inhibitor, was shown by Western-blot analysis of membrane and cytoplasmic fractions to inhibit the translocation of p47-phox to the membrane of stimulated EC. These findings support the presence of a functionally active leukocyte-type NADPH oxidase in EC. NADPH oxidase could be the major cellular ROS source in EC perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.
机译:暴露于致动脉粥样硬化的低密度脂蛋白水平的培养的人内皮细胞(EC)具有增加的活性氧(ROS)生成。造成这种ROS产生增加的酶未知。我们检查了EC中是否存在功能性白细胞型NADPH氧化酶,以阐明该酶是否可能是ROS来源。被破坏的EC的质膜部分显示出减少的负氧化差异光谱,其吸收峰与白细胞NADPH氧化酶组分细胞色素b558的光谱中观察到的吸收峰相同。使用抗gp91 -phox的蛋白质印迹分析。抗-p22-phox。抗-p47-phox。和抗-p67-phox抗体,证明了EC中NADPH氧化酶亚基的蛋白表达。逆转录聚合酶链反应(RT-PCR)显示EC中gp91-phox,p22-phox,p47-phox和p67-phox的mRNA表达。来自未刺激的EC的声波产物未产生可测量的超氧化物。然而,外源施加的花生四烯酸以取决于NADPH的存在以及膜和胞质组分结合的方式激活了超氧化物的产生。 Apocynin是一种特定的白细胞NADPH氧化酶抑制剂,通过膜和细胞质组分的Western印迹分析显示,抑制了p47-phox向刺激的EC膜的转运。这些发现支持在EC中存在功能活性白细胞型NADPH氧化酶。 NADPH氧化酶可能是EC摄动中主要的细胞ROS来源,据推测这是导致动脉粥样硬化发病的主要因素。

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