Abstract 974: Endothelial NOS (eNOS) Regulates Mitochondrial Oxidative Stress in Fetal Pulmonary Artery Endothelial Cells (PAEC)
Background: Endothelial NOS regulates pulmonary vasodilation at birth by synthesis and release of NO. Exposure of fetal lung to O2 results in increased oxidative phoshporylation and ATP release at birth. ATP stimulates NO release from eNOS. Superoxide (O2−) is generated by mitochondria as a byproduct of oxidative phosphorylation. The regulation of mitochondrial O2 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 O2− in fetal PAEC in response to ATP and O2.
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 O2− 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 O2 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 O2− and attenuated the relaxation response of PA rings to ATP and NO. PAEC from PPHN lambs showed increased basal O2 consumption, decreased NO-regulation of O2 consumption and increased mitochondrial O2−. 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 O2− in PAEC during postnatal transition to the O2 rich environment. Pulmonary hypertension is associated with impaired targeting of eNOS and increase in mitchondrial O2−.