Brueckl Corinna, Kaestle Stephanie, Kerem Alexander, Habazettl Helmut, Krombach Fritz, Kuppe Hermann, Kuebler Wolfgang M
Institute of Physiology Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, Arnimallee 22, 14195 Berlin, Germany.
Am J Respir Cell Mol Biol. 2006 Apr;34(4):453-63. doi: 10.1165/rcmb.2005-0223OC. Epub 2005 Dec 15.
Lung capillary endothelial cells (ECs) are a critical target of oxygen toxicity and play a central role in the pathogenesis of hyperoxic lung injury. To determine mechanisms and time course of EC activation in normobaric hyperoxia, we measured endothelial concentration of reactive oxygen species (ROS) and cytosolic calcium (Ca(2+)) by in situ imaging of 2',7'-dichlorofluorescein (DCF) and fura 2 fluorescence, respectively, and translocation of the small GTPase Rac1 by immunofluorescence in isolated perfused rat lungs. Endothelial DCF fluorescence and Ca(2+) increased continuously yet reversibly during a 90-min interval of hyperoxic ventilation with 70% O(2), demonstrating progressive ROS generation and second messenger signaling. ROS formation increased exponentially with higher O(2) concentrations. ROS and Ca(2+) responses were blocked by the mitochondrial complex I inhibitor rotenone, whereas inhibitors of NAD(P)H oxidase and the intracellular Ca(2+) chelator BAPTA predominantly attenuated the late phase of the hyperoxia-induced DCF fluorescence increase after > 30 min. Rac1 translocation in lung capillary ECs was barely detectable at normoxia but was prominent after 60 min of hyperoxia and could be blocked by rotenone and BAPTA. We conclude that hyperoxia induces ROS formation in lung capillary ECs, which initially originates from the mitochondrial electron transport chain but subsequently involves activation of NAD(P)H oxidase by endothelial Ca(2+) signaling and Rac1 activation. Our findings demonstrate rapid activation of ECs by hyperoxia in situ and identify mechanisms that may be relevant in the initiation of hyperoxic lung injury.
肺毛细血管内皮细胞（ECs）是氧中毒的关键靶点，在高氧性肺损伤的发病机制中起核心作用。为了确定常压高氧条件下EC激活的机制和时间进程，我们分别通过2',7'-二氯荧光素（DCF）和fura 2荧光的原位成像测量了活性氧（ROS）的内皮浓度和胞质钙（Ca(2+)），并通过免疫荧光法检测了分离的灌注大鼠肺中小GTP酶Rac1的转位。在70% O(2)的高氧通气90分钟期间，内皮DCF荧光和Ca(2+)持续但可逆地增加，表明ROS的逐渐生成和第二信使信号传导。ROS的形成随着O(2)浓度的升高呈指数增加。ROS和Ca(2+)反应被线粒体复合物I抑制剂鱼藤酮阻断，而NAD(P)H氧化酶抑制剂和细胞内Ca(2+)螯合剂BAPTA主要在30分钟后减弱高氧诱导的DCF荧光增加的后期阶段。在常氧条件下，肺毛细血管ECs中的Rac1转位几乎检测不到，但在高氧60分钟后很明显，并且可以被鱼藤酮和BAPTA阻断。我们得出结论，高氧诱导肺毛细血管ECs中ROS的形成，其最初起源于线粒体电子传递链，但随后涉及内皮Ca(2+)信号传导和Rac1激活对NAD(P)H氧化酶的激活。我们的研究结果表明高氧在原位快速激活ECs，并确定了可能与高氧性肺损伤起始相关的机制。
