Nox2-dependent glutathionylation of endothelial NOS leads to uncoupled superoxide production and endothelial barrier dysfunction in acute lung injury

Abstract

Microvascular barrier integrity is dependent on bioavailable nitric oxide (NO) produced locally by endothelial NO synthase (eNOS). Under conditions of limited substrate or cofactor availability or by enzymatic modification, eNOS may become uncoupled, producing superoxide in lieu of NO. This study was designed to investigate how eNOS-dependent superoxide production contributes to endothelial barrier dysfunction in inflammatory lung injury and its regulation. C57BL/6J mice were challenged with intratracheal LPS. Bronchoalveolar lavage fluid was analyzed for protein accumulation, and lung tissue homogenate was assayed for endothelial NOS content and function. Human lung microvascular endothelial cell (HLMVEC) monolayers were exposed to LPS in vitro, and barrier integrity and superoxide production were measured. Biopterin species were quantified, and coimmunoprecipitation (Co-IP) assays were performed to identify protein interactions with eNOS that putatively drive uncoupling. Mice exposed to LPS demonstrated eNOS-dependent increased alveolar permeability without evidence for altered canonical NO signaling. LPS-induced superoxide production and permeability in HLMVEC were inhibited by the NOS inhibitor nitro-l-arginine methyl ester, eNOS-targeted siRNA, the eNOS cofactor tetrahydrobiopterin, and superoxide dismutase. Co-IP indicated that LPS stimulated the association of eNOS with NADPH oxidase 2 (Nox2), which correlated with augmented eNOS S-glutathionylation both in vitro and in vivo. In vitro, Nox2-specific inhibition prevented LPS-induced eNOS modification and increases in both superoxide production and permeability. These data indicate that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to LPS. Furthermore, the results implicate Nox2-mediated eNOS-S-glutathionylation as a mechanism underlying LPS-induced eNOS uncoupling in the lung microvasculature.

The eNOS signalosome and its link to endothelial dysfunction

“…A role for NOX2 in this particular posttranslational modification has recently been reported [98]. NOX2-specific inhibition prevents lipopolysaccharideinduced eNOS S-glutathionylation and reduces O 2 − production and permeability in lung microvascular endothelial cells.…”

Hydrogen peroxide-induced calcium influx in lung microvascular endothelial cells involves TRPV4

“…Experimentally, increasing ROS by treatment with oxidants induces edema formation in isolated perfused lungs (6,52). Similarly, application of external oxidants or induction of intracellular ROS production worsens barrier function in vitro (5,40,67). Conversely, protective effects were reported with augmentation of endothelial antioxidant mechanisms (8,49,50).…”

Association between functional polymorphisms in the nitric oxide synthase 3 gene and pediatric acute respiratory distress syndrome

“…The role of eNOS in lung development, which may influence the clinical syndromes of neonatal respiratory distress, remains to be elucidated (Han et al, 2004). Wu et al (2014) suggested that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to lipopolysaccharides. The results of this study also implicated the Nox2-mediated eNOS-S-glutathionylation in LPS-induced eNOS uncoupling in the lung microvasculature (Wu et al, 2014).…”

“…Wu et al (2014) suggested that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to lipopolysaccharides. The results of this study also implicated the Nox2-mediated eNOS-S-glutathionylation in LPS-induced eNOS uncoupling in the lung microvasculature (Wu et al, 2014). A study of the SNP rs1799983 in NOS3 showed a significant increase in the GG genotype and G allele frequencies in groups with respiratory distress syndrome (Shen et al, 2014).…”