Published ahead of print on December 15, 2005, doi:10.1165/rcmb.2005-0223OC
Am. J. Respir. Cell Mol. Biol., Volume 34, Number 4, April 2006, 453-463
A more recent version of this article appeared on April 1, 2006
Submitted on June 22, 2005
Revised on December 15, 2005
Hyperoxia-induced Reactive Oxygen Species Formation in Pulmonary Capillary Endothelial Cells in situ
Corinna Brueckl1, Stephanie Kaestle2, Alexander Kerem2, Helmut Habazettl3, Fritz Krombach1, Hermann Kuppe4, and Wolfgang M Kuebler3*
1 Institute for Surgical Research, University of Munich, Munich, Germany,
2 Institute of Physiology, Charite - Universitatsmedizin Berlin, Berlin, Germany,
3 Institute of Physiology, Charite - Universitatsmedizin Berlin, Berlin, Germany; Institute of Anesthesiology, Deutsches Herzzentrum Berlin, Berlin, Germany,
4 Institute of Anesthesiology, Deutsches Herzzentrum Berlin, Berlin, Germany
* To whom correspondence should be addressed. E-mail: wolfgang.kuebler{at}charite.de.
Lung capillary endothelial cells 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 endothelial cell 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. Both, 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. Both, ROS and [Ca2+]i responses were completely 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 endothelial cells was barely detectable at normoxia, yet prominent after 60 min of hyperoxia and could be blocked by both rotenone and BAPTA. We conclude that hyperoxia induces ROS formation in lung capillary endothelial cells 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 endothelial cells by hyperoxia in situ and identify mechanisms that may be relevant in the initiation of hyperoxic lung injury.
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