Hu Qinghua, Yu Zu-Xi, Ferrans Victor J, Takeda Kazuyo, Irani Kaikobad, Ziegelstein Roy C
Department of Medicine, Division of Cardiology, Johns Hopkins Bayview Medical Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
J Biol Chem. 2002 Sep 6;277(36):32546-51. doi: 10.1074/jbc.M201550200. Epub 2002 Jul 1.
There is increasing evidence that intracellular reactive oxygen species (ROS) play a role in cell signaling and that the NADPH oxidase is a major source of ROS in endothelial cells. At low concentrations, agonist stimulation of membrane receptors generates intracellular ROS and repetitive oscillations of intracellular Ca(2+) concentration (Ca(2+)) in human endothelial cells. The present study was performed to examine whether ROS are important in the generation or maintenance of Ca(2+) oscillations in human aortic endothelial cells (HAEC) stimulated by histamine. Histamine (1 microm) increased the fluorescence of 2',7'-dihydrodichlorofluorescin diacetate in HAEC, an indicator of ROS production. This was partially inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 microm), by the farnesyltransferase inhibitor H-Ampamb-Phe-Met-OH (2 microm), and in HAEC transiently expressing Rac1(N17), a dominant negative allele of the protein Rac1, which is essential for NADPH oxidase activity. In indo 1-loaded HAEC, 1 microm histamine triggered Ca(2+) oscillations that were blocked by DPI or H-Ampamb-Phe-Met-OH. Histamine-stimulated Ca(2+) oscillations were not observed in HAEC lacking functional Rac1 protein but were observed when transfected cells were simultaneously exposed to a low concentration of hydrogen peroxide (10 microm), which by itself did not alter either Ca(2+) or levels of inositol 1,4,5-trisphosphate (Ins-1,4,5-P(3)). Thus, histamine generates ROS in HAEC at least partially via NADPH oxidase activation. NADPH oxidase-derived ROS are critical to the generation of Ca(2+) oscillations in HAEC during histamine stimulation, perhaps by increasing the sensitivity of the endoplasmic reticulum to Ins-1,4,5-P(3).
越来越多的证据表明，细胞内活性氧（ROS）在细胞信号传导中发挥作用，并且NADPH氧化酶是内皮细胞中ROS的主要来源。在低浓度下，激动剂刺激膜受体可在人内皮细胞中产生细胞内ROS和细胞内Ca(2+)浓度（[Ca(2+)]i）的重复振荡。本研究旨在检查ROS在组胺刺激的人主动脉内皮细胞（HAEC）中[Ca(2+)]i振荡的产生或维持中是否重要。组胺（1微摩尔）增加了HAEC中2',7'-二氢二氯荧光素二乙酸酯的荧光，这是ROS产生的指标。这被NADPH氧化酶抑制剂二亚苯基碘鎓（DPI，10微摩尔）、法尼基转移酶抑制剂H-Ampamb-Phe-Met-OH（2微摩尔）以及在瞬时表达Rac1（N17）的HAEC中部分抑制，Rac1（N17）是蛋白质Rac1的显性负等位基因，对NADPH氧化酶活性至关重要。在负载indo 1的HAEC中，1微摩尔组胺引发的[Ca(2+)]i振荡被DPI或H-Ampamb-Phe-Met-OH阻断。在缺乏功能性Rac1蛋白的HAEC中未观察到组胺刺激的[Ca(2+)]i振荡，但当转染细胞同时暴露于低浓度过氧化氢（10微摩尔）时观察到，过氧化氢本身不会改变[Ca(2+)]i或肌醇1,4,5-三磷酸（Ins-1,4,5-P(3)）水平。因此，组胺至少部分通过NADPH氧化酶激活在HAEC中产生ROS。NADPH氧化酶衍生的ROS对于组胺刺激期间HAEC中[Ca(2+)]i振荡的产生至关重要，可能是通过增加内质网对Ins-1,4,5-P(3)的敏感性。
