Abstract 974: Endothelial NOS (eNOS) Regulates Mitochondrial Oxidative Stress in Fetal Pulmonary Artery Endothelial Cells (PAEC)

Abstract

Background : Endothelial NOS regulates pulmonary vasodilation at birth by synthesis and release of NO. Exposure of fetal lung to O 2 results in increased oxidative phoshporylation and ATP release at birth. ATP stimulates NO release from eNOS. Superoxide (O 2 − ) is generated by mitochondria as a byproduct of oxidative phosphorylation. The regulation of mitochondrial O 2 during this transition and its role in impaired vasodilation that occurs in persistent pulmonary hypertension of newborn (PPHN) remain unknown. Objective : We investigated the hypothesis that targeting of eNOS to mitochondrial outer membrane regulates O 2 − in fetal PAEC in response to ATP and O 2 . Methods : Studies were done in isolated PAEC and pulmonary arteries (PA) from fetal lambs. PPHN was induced by prenatal ductal constriction for 8 days. Association of eNOS with outer membrane protein, porin and chaperone, hsp90 was determined by immunoprecipitation and double label immuno-fluorescence. Mitochondrial NO and O 2 − levels were determined by DAF-2 and mito-HE reagents. Relaxation responses of PA rings to ATP and NO were determined in the presence/absence of decoy peptides that inhibit eNOS association with hsp90 and porin and mitochondrial targeted ubiquinones. Results : Mitochondria from PAEC showed presence of eNOS on outer membrane but no nNOS. Exposure of PAEC to ATP or O 2 increased the association of eNOS with porin and hsp90 in the mitochondria. ATP increased mitochondrial NO release. Inhibition of eNOS-hsp90 association or eNOS-porin association with decoy peptides that mimic specific eNOS domains required for these interactions resulted in increased mitochondrial O 2 − and attenuated the relaxation response of PA rings to ATP and NO. PAEC from PPHN lambs showed increased basal O 2 consumption, decreased NO-regulation of O 2 consumption and increased mitochondrial O 2 − . PA rings from PPHN lambs showed impaired relaxation responses to ATP and NO and improved relaxation when treated with mitochondrial targeted ubiquinone. Conclusion : Interaction of eNOS with mitochondria plays an important role in the regulation of O 2 − in PAEC during postnatal transition to the O 2 rich environment. Pulmonary hypertension is associated with impaired targeting of eNOS and increase in mitchondrial O 2 − .