Developmental Programming of eNOS Uncoupling and Enhanced Vascular Oxidative Stress in Adult Rats After Transient Neonatal Oxygen Exposure

Abstract

The authors have previously shown that neonatal hyperoxic stress leads to high blood pressure, impaired endothelium-mediated vasodilatation, and increased vascular production of superoxide anion by NAD(P)H oxidase in adulthood. However, it is unknown whether changes in nitric oxide (NO) production and/or bioinactivation prevail and whether NO synthase (NOS) is also a source of superoxide. The purpose of this study was to evaluate whether adult animals exposed to neonatal hyperoxic stress have impaired vascular NO production associated with NOS uncoupling participating to vascular superoxide production and vascular dysfunction. In adult male rats exposed to 80% oxygen from day 3 to 10 of life (H, n = 6) versus room air controls (CTRL, n = 6), vascular (aorta) NO production is decreased at baseline (CTRL: 21 ± 1 vs. H: 16 ± 2 4,5-diaminofluorescein diacetate fluorescence intensity arbitrary units; P < 0.05) and after carbachol stimulation (acetylcholine analog; CTRL: 26 ± 2 vs. H: 18±2; P < 0.05). Pretreatment with L-arginine (CTRL: 32 ± 4 vs. H: 31 ± 5) and L-sepiapterine [analog of key NOS cofactor tetrahydro-L-biopterin (BH4)] (CTRL: 30 ± 3 vs. H: 29 ± 3) normalizes NO production after carbachol. L-Sepiapterine also normalizes impaired vasodilatation to carbachol. Vascular endothelial NO synthase (eNOS) immunostaining is reduced, whereas total eNOS protein expression is increased in H (CTRL: 0.76 ± 0.08 vs. H: 1.76± 0.21; P < 0.01). The significantly higher superoxide generation (CTRL: 20 ± 2 vs. H: 28 ± 3 hydroethidine fluorescence intensity arbitrary units; P < 0.05) is prevented by pretreatment with the eNOS inhibitor N-nitro-L-arginine methyl ester (CTRL: 21 ± 4 vs. H: 22 ± 4). Taken together, the current data indicate a role for eNOS uncoupling in enhanced vascular superoxide, impaired endothelium-mediated vasodilatation, and decreased NO production in adult animals with programmed elevated blood pressure after a brief neonatal oxygen exposure.