eNOS function is developmentally regulated: uncoupling of eNOS occurs postnatally.
At birth, the transition to gas breathing requires the function of endothelial vasoactive agents. We investigated the function of endothelial nitric oxide synthase (eNOS) in pulmonary artery (PA) vessels and endothelial cells isolated from fetal and young (4-wk) sheep. We found greater relaxations to the NOS activator A-23187 in 4-wk-old compared with fetal vessels and that the NOS inhibitor nitro-L-arginine blocked relaxations in both groups. Relaxations in 4-wk vessels were not blocked by an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, but were partially blocked by catalase. We therefore hypothesized that activation of eNOS produced reactive oxygen species in 4-wk but not fetal PA. To address this question, we studied NO and superoxide production by endothelial cells at baseline and following NOS stimulation with A-23187, VEGF, and laminar shear stress. Stimulation of NOS induced phosphorylation at serine 1177, and this event correlated with an increase in NO production in both ages. Upon stimulation of eNOS, fetal PA endothelial cells (PAEC) produced only NO. In contrast 4-wk-old PAEC produced superoxide in addition to NO. Superoxide production was blocked by L-NAME but not by apocynin (an NADPH oxidase inhibitor). L-Arginine increased NO production in both cell types but did not block superoxide production. Heat shock protein 90/eNOS association increased upon stimulation and did not change with developmental age. Cellular levels of total and reduced biopterin were higher in fetal vs. 4-wk cells. Sepiapterin [a tetrahydrobiopterin (BH4) precursor] increased basal and stimulated NO levels and completely blocked superoxide production. We conclude that the normal function of eNOS becomes uncoupled after birth, leading to a developmental adaptation of the pulmonary vascular system to produce oxygen species other than NO. We speculate this may be related to cellular production and/or maintenance of BH4 levels.
Mata-Greenwood
Eugenia
E
Dept. of Pediatrics, Northwestern Univ., Ward 12-191, 303 E. Chicago Ave., Chicago, IL 60611-008, USA.
Jenkins
Chrystal
C
Farrow
Kathryn N
KN
Konduri
G Ganesh
GG
Russell
James A
JA
Lakshminrusimha
Satyan
S
Black
Stephen M
SM
Steinhorn
Robin H
RH
D000375:Aging; D000818:Animals; D001120:Arginine / Q000494:pharmacology; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation; D005333:Fetus; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D011651:Pulmonary Artery / Q000187:drug effects / Q000201:enzymology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D012756:Sheep / Q000254:growth & development; D013314:Stress, Mechanical; D013481:Superoxides / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D014664:Vasodilation / Q000187:drug effects
D018841:HSP90 Heat-Shock Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D013481:Superoxides; D001708:Biopterins; D000001:Calcimycin; D001120:Arginine; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin