Kondrikov, D; Elms, S; Fulton, D; Su, Y
Oxygen toxicity is the most severe side-effect of oxygen therapy in neonates and adults. Pulmonary damage of oxygen toxicity is related to over-production of reactive oxygen species (ROS). Reaction of ROS with nitric oxide (NO) results in the formation of reactive nitrogen species, such as peroxynitrite. It is not clear whether reactive nitrogen species are implicated in the pulmonary complications of oxygen toxicity. In the present study, we investigated the effect of hyperoxia on the production of peroxynitrite in pulmonary artery endothelial cells (PAEC). PAEC were exposed to normoxia (21% oxygen, 74% nitrogen, and 5% CO2) and hyperoxia (95 % oxygen and 5% CO2) at 37^C for 24 h, then peroxynitrite, NO release, endothelial NO synthase (eNOS) activity, superoxide production, and association of eNOS with beta-actin were measured. Incubation of PAEC with 95% oxygen for 24 h resulted in an increase in the intracellular level of peroxynitrite. The presence of uric acid, a peroxynitrite scavenger, prevented hyperoxia-induced increase in peroxynitrite. The increase in the production of peroxynitrite is accompanied by increases in eNOS activity, NO release, superoxide production, and association of eNOS with beta-actin in PAEC. Treatment of PAEC with a synthetic peptide with sequence corresponding to actin binding site on eNOS (aa 326-333 of human eNOS) prevented hyperoxia-induced increase in eNOS/beta-actin association, eNOS activity, NO release, and peroxynitrite production. Hyperoxia failed to induce the increases in eNOS activity, NO release, and peroxynitrite formation in COS-7 cells transfected with plasmids containing cDNA for eNOS mutant in which amino acids leucine and tryptophan were replaced with alanine in the actin binding site on eNOS. Taken together, our data indicate that increased association of eNOS with beta-actin in PAEC contributes to hyperoxia-induced increase in the production of peroxynitrite which may cause nitrosative stress in pulmonary vasculature.
Supported by NIH, AHA, and FAMRI.
