Pulmonary nitric oxide synthases and nitrotyrosine: findings during lung development and in chronic lung disease of prematurity.
BACKGROUND
Nitric oxide mediates and modulates pulmonary transition from fetal to postnatal life. NO is synthesized by 3 nitric oxide synthase isoforms. One key pathway of nitric oxide metabolism results in nitrotyrosine, a stable, measurable marker of nitric oxide production.


OBJECTIVE
The purpose of this study was to assess, by semiquantitative immunohistochemistry, nitric oxide synthase isoforms and nitrotyrosine at different airway and vascular tree levels in the lungs of neonates at different gestational ages and to compare results in control groups to those in infants with chronic lung disease.


METHODS
Formalin-fixed, paraffin-embedded, postmortem lung blocks were prepared for immunohistochemistry using antibodies to each nitric oxide synthase isoform and to nitrotyrosine. Blinded observers evaluated the airway and vascular trees for staining intensity (0-3 scale) at 5 levels and 3 levels, respectively. The control population consisted of infants from 22 to 42 weeks' gestation who died in < 48 hours. Results were compared with gestation-matched infants with varying severity of chronic lung disease.


RESULTS
In control and chronic lung disease groups, 22 to 42 weeks' gestation, staining for all 3 of the nitric oxide synthase isoforms was found in the airway epithelium from the bronchus to the alveolus or distal-most airspace. The abundance or distribution of nitric oxide synthase-3 staining in the airways did not show significant correlation with gestational age or severity of chronic lung disease. In the vascular tree, intense nitric oxide synthase-3 and moderate nitric oxide synthase-2 staining was found; nitric oxide synthase-1 was not consistently stained. Nitrotyrosine did stain in the pulmonary tree. Compared with controls where nitrotyrosine staining was minimal, regardless of gestation, in infants with chronic lung disease there was more than fourfold increase between severe chronic lung disease (n = 12) and either mild chronic lung disease or control infants (n = 16).


CONCLUSIONS
All 3 of the nitric oxide synthase isoforms and nitrotyrosine are detectable by immunohistochemistry early in lung development. Nitric oxide synthase ontogeny shows no significant changes in abundance or distribution with advancing gestational age nor with chronic lung disease. Nitrotyrosine is significantly increased in severe chronic lung disease.
Sheffield
Mark
M
Children's Mercy Hospitals and Clinics, Section of Neonatology, Department of Pediatrics, University of Missouri-Kansas City School of Medicine, Kansas City, Missouri 64108, USA.
Mabry
Sherry
S
Thibeault
Donald W
DW
Truog
William E
WE
D000367:Age Factors; D001344:Autopsy; D002908:Chronic Disease; D006801:Humans; D007150:Immunohistochemistry; D007231:Infant, Newborn; D007234:Infant, Premature; D008168:Lung / Q000254:growth & development / Q000378:metabolism*; D008171:Lung Diseases / Q000201:enzymology / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I / Q000032:analysis / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000032:analysis / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000032:analysis / Q000378:metabolism*; D012720:Severity of Illness Index; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
C002744:3-nitrotyrosine; D014443:Tyrosine; C496320:NOS1 protein, human; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III