eNOS-β-Actin Interaction Contributes to Increased Peroxynitrite Formation during Hyperoxia in Pulmonary Artery Endothelial Cells and Mouse Lungs

Abstract

Oxygen toxicity is the most severe side effect of oxygen therapy in neonates and adults. Pulmonary damage of oxygen toxicity is related to the overproduction of reactive oxygen species (ROS). In the present study, we investigated the effect of hyperoxia on the production of peroxynitrite in pulmonary artery endothelial cells (PAEC) and mouse lungs. Incubation of PAEC under hyperoxia (95% O 2 ) for 24 h resulted in an increase in peroxynitrite formation. Uric acid, a peroxynitrite scavenger, prevented hyperoxia-induced increase in peroxynitrite. The increase in peroxynitrite formation is accompanied by increases in nitric oxide (NO) release and endothelial NO synthase (eNOS) activity. We have previously reported that association of eNOS with ␤-actin increases eNOS activity and NO production in lung endothelial cells. To study whether eNOS-␤-actin association contributes to increased peroxynitrite production, eNOS-␤-actin interaction were inhibited by reducing ␤-actin availability or by using a synthetic peptide (P326TAT) containing a sequence corresponding to the actin binding site on eNOS. We found that disruption of eNOS-␤-actin interaction prevented hyperoxia-induced increases in eNOS-␤-actin association, eNOS activity, NO and peroxynitrite production, and protein tyrosine nitration. Hyperoxia failed to induce the increases in eNOS activity, NO and peroxynitrite formation in COS-7 cells transfected with plasmids containing eNOS mutant cDNA in which amino acids leucine and tryptophan were replaced with alanine in the actin binding site on eNOS. Exposure of mice to hyperoxia resulted in significant increases in eNOS-␤-actin association, eNOS activity, and protein tyrosine nitration in the lungs. Our data indicate that increased association of eNOS with ␤-actin in PAEC contributes to hyperoxia-induced increase in the production of peroxynitrite which may cause nitrosative stress in pulmonary vasculature.Oxygen therapy is an important element of the management of various conditions such as adult or neonatal respiratory distress syndrome, circulatory shock, infection, multiple-organ failure syndrome, and pulmonary hypertension (1-6). However, prolonged exposure to increased concentrations of oxygen induces diffuse pulmonary injuries, excessive inflammation, and lung fibrosis. The hyperoxia-induced damages to lung cells have been attributed to the generation of reactive oxygen species (ROS) 2 and subsequent formation of more potent oxidants such as peroxynitrite (ONOO Ϫ ) (7,8). Several studies have suggested that endothelial nitric-oxide synthase (eNOS) plays an important role in the pathogenesis of oxygen toxicity (8, 9). It has been reported that hyperoxia increases eNOS activity and nitric oxide (NO) release from endothelial cells (9, 10). Inhibition of eNOS using L-NAME or knock-out of eNOS reduces peroxynitrite-mediated cytotoxicity in hyperoxic cellular damage in retina (9, 11). However, the mechanism for hyperoxia-induced increases in eNOS activity and NO release has not been clarified. In the present study,...

Redox Proteomics in Selected Neurodegenerative Disorders: From Its Infancy to Future Applications

“…This specific isoform has been found to interact with b-actin, vascular endothelial growth factor, and caveolin 1 (231,323), which bolsters the role of eNOS in muscle contraction, cell proliferation, and apoptosis. Most recently, eNOS has been associated with heat shock proteins (HSPs) 70 and 90, which act as molecular chaperones by guiding damaged proteins to the proteasome for protein degradation.…”

Novel Peptide for Attenuation of Hyperoxia-induced Disruption of Lung Endothelial Barrier and Pulmonary Edema via Modulating Peroxynitrite Formation

“…Apoptosis-Exposure of lung endothelial cells to high concentration of oxygen leads to peroxynitrite formation (12). To investigate whether hyperoxia-induced disruption of lung endothelial barrier is caused by peroxynitrite, PAECs were exposed to hyperoxia or normoxia for 48 h in the presence and absence of peroxynitrite scavenger uric acid (3 mM).…”

“…Co-immunoprecipitation of eNOS and ␤-Actin-lung homogenates were used for immunoprecipitation as described previously (12,16). Lysates were incubated with anti-eNOS antibody or nonimmune IgG at 4°C overnight.…”

“…We have previously reported that hyperoxia increases the formation of NO and peroxynitrite in lung endothelial cells via increased interaction of eNOS 2 with ␤-actin in oxygen toxicity (12). The actin binding site on eNOS protein has been identified at amino acid residues 326 -333.…”

“…Mutation of hydrophobic residues leucine 326, leucine 328, tryptophan 330, and leucine 333 resulted in dissociation of eNOS from ␤-actin, indicating that these residues are critical for actin binding (13). A peptide with amino acid sequence corresponding to the actin binding region of eNOS residues 326 -333 has been shown to reduce the hyperoxia-induced formation of NO and peroxynitrite in lung endothelial cells (12). In the present study, a peptide (P326TAT) with amino acid sequence corresponding to the actin binding region of eNOS residues 326 -333 linked to a 10-amino acid transduction domain of HIV-tat (RKKRRQRRRA) was synthesized, and its effects on hyperoxiainduced disruption of lung endothelial barrier were studied using cultured endothelial cells and animal model.…”