Nitric oxide synthase isoenzyme expression and activity in peripheral lung tissue of patients with chronic obstructive pulmonary disease.
BACKGROUND
Nitric oxide (NO) is increased in the lung periphery of patients with chronic obstructive pulmonary disease (COPD). However, expression of the NO synthase(s) responsible for elevated NO has not been identified in the peripheral lung tissue of patients with COPD of varying severity.


OBJECTIVE



METHODS
Protein and mRNA expression of nitric oxide synthase type I (neuronal NOS [nNOS]), type II (inducible NOS [iNOS]), and type III (endothelial NOS [eNOS]) were quantified by Western blotting and reverse transcription-polymerase chain reaction, respectively, in specimens of surgically resected lung tissue from nonsmoker control subjects, patients with COPD of varying severity, and smokers without COPD, and in a lung epithelial cell line (A549). The effects of nitrative/oxidative stress on NOS expression and activity were also evaluated in vitro in A549 cells. nNOS nitration was quantified by immunoprecipitation and dimerization of nNOS was detected by low-temperature SDS-PAGE/Western blot in the presence of the peroxynitrite generator, 3-morpholinosydnonimine-N-ethylcarbamide (SIN1), in vitro and in vivo.


RESULTS
Lung tissue from patients with severe and very severe COPD had graded increases in nNOS (mRNA and protein) compared with nonsmokers and normal smokers. Hydrogen peroxide (H(2)O(2)) and SIN1 as well as the cytokine mixture (IFN-gamma, IL-1beta, and tumor necrosis factor-alpha) increased mRNA expression and activity of nNOS in A549 cells in a concentration-dependent manner compared with nontreated cells. Tyrosine nitration resulted in an increase in nNOS activity in vitro, but did not affect its dimerization.


CONCLUSIONS
Patients with COPD have a significant increase in nNOS expression and activity that reflects the severity of the disease and may be secondary to oxidative stress.
National Heart and Lung Institute, Imperial College London, UK.
Caterina
C
Brindicci
Sergei A
SA
Kharitonov
Misako
M
Ito
Mark W
MW
Elliott
James C
JC
Hogg
Peter J
PJ
Barnes
Kazuhiro
K
Ito
D000328:Adult; D000368:Aged; D001944:Breath Tests; D016022:Case-Control Studies; D002460:Cell Line; D018450:Disease Progression; D004847:Epithelial Cells; D005260:Female; D005541:Forced Expiratory Volume; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D018384:Oxidative Stress; D029424:Pulmonary Disease, Chronic Obstructive / Q000201:enzymology*; D012042:Registries; D012907:Smoking; D015854:Up-Regulation
D007527:Isoenzymes; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II