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.

Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite

“…Sheep pulmonary microvascular EC exposure to hyperoxia (100% O 2 , 30 min) increased the cellular O 2 ⅐ Ϫ production, as detected by EPR spectroscopy, and blocking the ETC with rotenone decreased the O 2 ⅐ Ϫ signal (67). Detection of ROS in capillary ECs by in situ imaging of 2Ј,7Ј-dichlorofluorescein fluorescence in perfused rat lungs showed that ROS formation increases with PO 2 (21-100% O 2 , 90 min) and originates from mitochondria (9).…”

“…Since it is known that 1-4% of O 2 reacting with the ETC is incompletely reduced to O 2 ⅐ Ϫ , and the O 2 ⅐ Ϫ formation rate by the ETC complexes III and I increases linearly with O 2 concentration (42,74), we hypothesized that, under flow, the relative "hyperoxic state" of 21% O 2 raises the O 2 concentration in EC mitochondria and enhances the formation of O 2 ⅐ Ϫ , which, via the reaction with shear-induced NO, increases the intramitochondrial ONOO Ϫ levels and potentiates the inhibition of respiration. Although it is known that 1) either hyperoxia (21-100% O 2 ) or H (1-3% O 2 )/RO (21% O 2 ) increases the mitochondrial O 2 ⅐ Ϫ generation and inhibits respiration (9,21,33,67,71) and 2) shear at 21% O 2 increases the EC ONOO Ϫ formation intramitochondrially (30), the respiratory function of sheared ECs at 21% O 2 and at lower, but physiological, O 2 levels has not been examined.…”

Nitric oxide-dependent inhibition of alveolar fluid clearance in hydrostatic lung edema

“…Experimental procedures have been described previously (7,48). For isolated perfusion, lungs from anesthetized Sprague-Dawley rats were continuously pump-perfused at 14 ml/min and 37°C.…”

Acid aspiration-induced lung inflammation and injury are exacerbated in NADPH oxidase-deficient mice

“…We considered the hypothesis that by increasing the level of ambient ROIs, hyperoxia may attenuate the excessive inflammation and injury observed in CGD mice following acid injury. Hyperoxia may also increase the level of NADPH oxidase-derived ROIs from lung endothelial cells (2,30). The effect of hyperoxia is to increase ambient levels of ROIs, whereas activation of the NADPH oxidase in phagocytes results in a rapid burst of superoxide anion generation and downstream ROI metabolites.…”