title: Hyperoxia-induced reactive oxygen species formation in pulmonary capillary endothelial cells in situ.
abstract: 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+)](i)) 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+)](i) 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+)](i) 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+)](i) 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.
authors.affiliation: Institute of Physiology Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, Arnimallee 22, 14195 Berlin, Germany.
authors.forename: Corinna
authors.initials: C
authors.lastname: Brueckl
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kaestle
authors.forename: Alexander
authors.initials: A
authors.lastname: Kerem
authors.forename: Helmut
authors.initials: H
authors.lastname: Habazettl
authors.forename: Fritz
authors.initials: F
authors.lastname: Krombach
authors.forename: Hermann
authors.initials: H
authors.lastname: Kuppe
authors.forename: Wolfgang M
authors.initials: WM
authors.lastname: Kuebler
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology; D002196:Capillaries / Q000378:metabolism; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016257:Fura-2; D018496:Hyperoxia / Q000378:metabolism* / Q000473:pathology; D066298:In Vitro Techniques; D008168:Lung / Q000098:blood supply*; D008297:Male; D008833:Microcirculation / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000502:physiology; D010100:Oxygen / Q000502:physiology*; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; C037631:2',7'-dichlorofluorescein; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein; D042967:Electron Transport Complex I; D010100:Oxygen; D002118:Calcium; D016257:Fura-2