Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-α is mediated by ceramide
Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-α is mediated by ceramide
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DOI：
10.1165/ajrcmb.24.6.4228
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发表时间：
2001-06-01
影响因子：
6.4
通讯作者：
Duranteau, J
中科院分区：
文献类型：
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作者：
Corda, S;Laplace, C;Duranteau, J
肿瘤坏死因子(TNF)-α可增加肿瘤细胞和肝细胞线粒体活性氧簇(ROS)的产生。然而，肿瘤坏死因子-α是否刺激内皮细胞线粒体ROS的产生尚未见报道，我们研究了肿瘤坏死因子-α对内皮细胞线粒体ROS产生的影响及其信号转导途径。用荧光显微镜观察培养的人脐静脉内皮细胞(HUVEC)，以二氯二氢荧光素二乙酸酯(DCFH-DA)作为ROS产生的标志物和摄取碘化丙啶作为细胞存活率的指标。TNF-α可剂量依赖地增加HUVEC对DCFH的氧化作用。为了确定ROS的来源，使用线粒体呼吸链抑制剂鱼藤酮+噻吩甲酰三氟丙酮(TTFA)和乌司匹林的阻断剂抗霉素A(AA)，鱼藤酮和TTFA被抑制(n=7，P<；0.05)，而AA在3分钟内增加(118%，P<；n=4，P<；相反，依赖于磷酸的烟酰胺腺嘌呤二核苷酸氧化酶抑制剂二苯基碘、黄嘌呤氧化酶抑制剂别嘌醇、一氧化氮和环氧合酶途径抑制剂N-欧米伽-硝基-L-精氨酸和甲芬那酸都不能抑制HUVEC产生ROS。此外，神经酰胺激活的蛋白激酶(CAPK)抑制剂二甲氨基嘌呤(1 mM；n=6，P<；0.05)可完全阻断TNF-α诱导的ROS的产生。因此，肿瘤坏死因子-α诱导HUVEC线粒体ROS的产生，主要发生在乌比西喹酮位点，并通过涉及CAPK的神经酰胺依赖的信号通路来介导。
Tumor necrosis factor (TNF)-alpha increases mitochondrial reactive oxygen species (ROS) production in tumor cells and hepatocytes. However, whether TNF-alpha stimulates mitochondrial ROS production in endothelial cells (EC) has not yet been reported, We studied the effect of TNF-alpha on mitochondrial ROS generation in EC and the signaling pathways involved. Cultured human umbilical vein EC (HUVEC) were studied by fluorescence microscopy, using dichlorodihydrofluorescein diacetate (DCFH-DA) as a marker of ROS production and propidium iodide uptake for cell viability. TNF-alpha increased DCFH oxidation in HUVEC dose-dependently. To determine the source of ROS, the mitochondrial respiratory chain inhibitors rotenone + thenoyltrifluoroacetone (TTFA), which inhibit electron entry to ubiquinone, and antimycin A (AA), a blocker of ubisemiquinone, were used, Rotenone and TTFA inhibited (n = 7, P < 0.05), whereas AA increased (118% in 3 min; n = 4, P < 0.01) ROS generation in HUVEC, In contrast, ROS production was not abolished by the nicotinamide adenine dinucleotide phosphate-dependent oxidase inhibitor diphenylene iodonium, by the xanthine oxidase inhibitor allopurinol, nor by the nitric oxide and cyclooxygenase pathway inhibitors N-omega-nitro-L-arginine and mefenamic acid. In addition, TNF-alpha -induced ROS production was inhibited by the acidic sphingomyelinase inhibitor desipramine (5 muM; -80%, n = 4, P < 0.01) and totally blocked by the ceramide-activated protein kinase (CAPK) inhibitor dimethylaminopurine (1 mM; n = 6, P < 0.05). Thus, TNF-alpha induces mitochondrial ROS production in HUVEC that primarily occurs at the ubisemiquinone site and is mediated by ceramide-dependent signaling pathways involving CAPK.