Proteins are targets of reactive species and detection of oxidatively modified proteins is often used as an index of oxidative stress. Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide. Using fatty acid-free bovine serum albumin as a model we examined peroxynitrite-mediated protein modifications. The reaction of protein with peroxynitrite resulted in the oxidation of tryptophan and cysteine, in the nitration of tyrosine, in the formation of dityrosine, in the production of 2,4 dinitrophenylhydrazine-reactive carbonyls and in protein fragmentation. The formation of 3-nitrotyrosine represents a specific peroxynitrite-mediated protein modification that is different from modifications mediated by reactive oxygen species.

We have previously demonstrated the generation of reactive oxygen species (ROS) in cultured bovine pulmonary artery endothelial cells (BPAECs) and in isolated perfused rat lungs exposed to high K+ and during global lung ischemia. The present study evaluates the NADPH oxidase pathway as a source of ROS in these models. ROS production, detected by oxidation of the fluorophore, dichlorodihydrofluorescein, increased 2.5-fold in BPAECs and 6-fold in rat or mouse lungs exposed to high (24 mmol/L) K+. ROS generation was markedly inhibited by diphenyliodonium, a flavoprotein inhibitor, and by the synthetic peptide PR-39, an inhibitor of NADPH oxidase assembly, whereas allopurinol had no effect. With ischemia (1 hour), ROS generation by rat and mouse lungs increased 7-fold; PR-39 showed concentration-dependent inhibition of ROS production, with 50% inhibition at 3 micromol/L PR-39. ROS production in lungs exposed to high K+ or ischemia was essentially abolished in mice with a "knockout" of gp91(phox), a membrane-localized cytochrome component of NADPH oxidase; increased ROS production by these lungs after anoxia/reoxygenation was similar to control. PR-39 also inhibited ischemia and the high K+-mediated increase in lung thiobarbituric acid reactive substance. Western blotting of BPAECs and immunocytochemistry of BPAECs and rat and mouse lungs showed the presence of p47phox, a cytoplasmic component of NADPH oxidase and the putative target for PR-39 inhibition. In situ fluorescence imaging in the intact lung demonstrated that the increased dichlorofluorescein fluorescence in these models of ROS generation was localized primarily to the pulmonary endothelium. These studies demonstrate that ROS production in lungs exposed to ischemia or high K+ results from assembly and activation of a membrane-associated NAPDH oxidase of the pulmonary endothelium.

. TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs. Am J Physiol Lung Cell Mol Physiol 293: L923-L932, 2007. First published July 27, 2007; doi:10.1152/ajplung.00221.2007.-We have previously implicated calcium entry through stretch-activated cation channels in initiating the acute pulmonary vascular permeability increase in response to high peak inflation pressure (PIP) ventilation. However, the molecular identity of the channel is not known. We hypothesized that the transient receptor potential vanilloid-4 (TRPV4) channel may initiate this acute permeability increase because endothelial calcium entry through TRPV4 channels occurs in response to hypotonic mechanical stress, heat, and P-450 epoxygenase metabolites of arachidonic acid. Therefore, permeability was assessed by measuring the filtration coefficient (K f) in isolated perfused lungs of C57BL/6 mice after 30-min ventilation periods of 9, 25, and 35 cmH2O PIP at both 35°C and 40°C. Ventilation with 35 cmH2O PIP increased Kf by 2.2-fold at 35°C and 3.3-fold at 40°C compared with baseline, but Kf increased significantly with time at 40°C with 9 cmH2O PIP. Pretreatment with inhibitors of TRPV4 (ruthenium red), arachidonic acid production (methanandamide), or P-450 epoxygenases (miconazole) prevented the increases in Kf. In TRPV4 Ϫ/Ϫ knockout mice, the high PIP ventilation protocol did not increase Kf at either temperature. We have also found that lung distention caused Ca 2ϩ entry in isolated mouse lungs, as measured by ratiometric fluorescence microscopy, which was absent in TRPV4 Ϫ/Ϫ and ruthenium red-treated lungs. Alveolar and perivascular edema was significantly reduced in TRPV4 Ϫ/Ϫ lungs. We conclude that rapid calcium entry through TRPV4 channels is a major determinant of the acute vascular permeability increase in lungs following high PIP ventilation. pulmonary edema; P-450 epoxygenases; stretch-activated cation channel; vascular permeability; Ca 2ϩ channels; epoxyeicosatrienoic acids; temperature ACUTE LUNG INJURY (ALI) and the acute respiratory distress syndrome (ARDS) are life-threatening conditions caused by a variety of pathologic processes and affect over 200,000 patients in the United States each year (41). Although positive pressure mechanical ventilation is a life-saving intervention in the setting of ARDS and ALI, clinical trials have demonstrated that mechanical ventilation with excessive tidal volumes actually contributes to lung injury and increases mortality (6). Although clinicians and researchers have been interested in ventilator-induced lung injury (VILI) for decades, the molecular mechanisms driving this process remain incompletely understood (7,8,27,48).Many previous investigators have reported that high airway pressures and lung volumes can increase pulmonary endothelial and epithelial permeability (7,8,27). An altered ion channel activity occurs within seconds in response to mechanical stress, and an increase in intracellular Ca 2ϩ concentration ([C...