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Published ahead of print on December 15, 2005, doi:10.1165/rcmb.2005-0223OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 453-463, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0223OC

Hyperoxia-Induced Reactive Oxygen Species Formation in Pulmonary Capillary Endothelial Cells In Situ

Corinna Brueckl, Stephanie Kaestle, Alexander Kerem, Helmut Habazettl, Fritz Krombach, Hermann Kuppe and Wolfgang M. Kuebler

Institute for Surgical Research, University of Munich, Munich; Institute of Physiology, Charité – Universitätsmedizin, and Institute of Anesthesiology, Deutsches Herzzentrum, Berlin, Germany

Correspondence and requests for reprints should be addressed to Prof. Dr. Wolfgang M. Kuebler, Institute of Physiology Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Arnimallee 22, 14195 Berlin, Germany. E-mail: wolfgang.kuebler{at}charite.de

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 ([Ca2+]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 [Ca2+]i increased continuously yet reversibly during a 90-min interval of hyperoxic ventilation with 70% O2, demonstrating progressive ROS generation and second messenger signaling. ROS formation increased exponentially with higher O2 concentrations. ROS and [Ca2+]i responses were blocked by the mitochondrial complex I inhibitor rotenone, whereas inhibitors of NAD(P)H oxidase and the intracellular Ca2+ 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 [Ca2+]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.

Key Words: endothelium • hyperoxia • mitochondria • NAD(P)H oxidase • reactive oxygen species




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