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

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 o...