title: eNOS-beta-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 β-actin increases eNOS activity and NO production in lung endothelial cells. To study whether eNOS-β-actin association contributes to increased peroxynitrite production, eNOS-β-actin interaction were inhibited by reducing β-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-β-actin interaction prevented hyperoxia-induced increases in eNOS-β-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-β-actin association, eNOS activity, and protein tyrosine nitration in the lungs. Our data indicate that increased association of eNOS with β-actin in PAEC contributes to hyperoxia-induced increase in the production of peroxynitrite which may cause nitrosative stress in pulmonary vasculature.
authors.affiliation: Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912, USA.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Kondrikov
authors.forename: Shawn
authors.initials: S
authors.lastname: Elms
authors.forename: David
authors.initials: D
authors.lastname: Fulton
authors.forename: Yunchao
authors.initials: Y
authors.lastname: Su
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018496:Hyperoxia / Q000503:physiopathology; D015151:Immunoblotting; D008168:Lung / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000096:biosynthesis*; D011485:Protein Binding / Q000187:drug effects; D011651:Pulmonary Artery / Q000166:cytology; D034622:RNA Interference; D013481:Superoxides / Q000378:metabolism; D013552:Swine; D014443:Tyrosine / Q000378:metabolism
chemical_list: D000199:Actins; D009842:Oligopeptides; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D014443:Tyrosine; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen
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