Sequential formation of reactive oxygen species by mitochondria and NAD(P)H oxidase in lung endothelial cells during hyperoxia

Formation of reactive oxygen species (ROS) in lung capillary endothelial cells contributes crucially to the pathogenesis of hyperoxic lung injury. To determine mechanisms of endothelial ROS formation in normobaric hyperoxia, we measured endothelial concentration of reactive oxygen species (ROS) and cytosolic calcium ([Ca 2+ ] i ) by imaging of 2′,7′dichlorofluorescein (DCF), ethidium, and fura 2 fluorescence, respectively, and translocation of the small GTPase Rac1 in isolated perfused rat lungs. During 90 min of hyperoxic ventilation with 70% O 2 , endothelial DCF and ethidium fluorescence and [Ca 2+ ] i increased continuously, demonstrating progressive superoxide formation and second messenger responses. ROS and [Ca 2+ ] i responses were completely blocked by the mitochondrial complex I inhibitor rotenone, whereas the NAD(P)H oxidase inhibitors diphenyleneiodonium and AEBSF and the intracellular Ca 2+ chelator BAPTA predominantly attenuated ROS production 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 Ca 2+ dependent translocation of Rac1. Our findings demonstrate sequential activation of ROS forming pathways by hyperoxia in situ which may be relevant in the initiation of hyperoxic lung injury. Supported by DFG GRK 865