Nitric Oxide Synthase Isoenzyme Expression and Activity in Peripheral Lung Tissue of Patients with Chronic Obstructive Pulmonary Disease
Nitric Oxide Synthase Isoenzyme Expression and Activity in Peripheral Lung Tissue of Patients with Chronic Obstructive Pulmonary Disease
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DOI：
10.1164/rccm.200904-0493oc
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发表时间：
2010-01-01
影响因子：
24.7
通讯作者：
Ito, Kazuhiro
中科院分区：
文献类型：
--
作者：
Brindicci, Caterina;Kharitonov, Sergei A.;Ito, Kazuhiro
原理：慢性阻塞性肺疾病（COPD）患者肺外周一氧化氮（NO）增加。然而，在不同严重程度的COPD患者的外周肺组织中尚未鉴定出导致NO升高的NO合酶的表达。I型一氧化氮合酶的蛋白质和mRNA表达（神经元NOS [nNOS]），II型（诱导型NOS [iNOS]）和III型分别通过Western印迹和逆转录聚合酶链反应定量测定非吸烟者对照组手术切除肺组织标本中内皮型一氧化氮合酶[eNOS]的含量，不同严重程度的COPD患者和无COPD的吸烟者，以及肺上皮细胞系（A549）。在体外A549细胞中还评价了硝化/氧化应激对NOS表达和活性的影响。nNOS硝化定量免疫沉淀和nNOS的二聚化检测低温SDS-PAGE/Western印迹中存在的过氧亚硝酸根发生器，3-morpholinosydnonimine-N-ethylcarbamide（SIN 1），在体外和在vivo. Measures和主要结果：从严重和非常严重的COPD患者的肺组织中nNOS（mRNA和蛋白质）的分级增加与非吸烟者和正常吸烟者相比。过氧化氢（H2 O2）和SIN 1以及细胞因子混合物（IFN-γ、IL-1 β和肿瘤坏死因子-α）以浓度依赖性方式增加A549细胞中nNOS的mRNA表达和活性。酪氨酸硝化导致nNOS活性在体外增加，但不影响其二聚化。结论：COPD患者有一个显着增加nNOS的表达和活性，反映了疾病的严重程度，可能是继发于氧化应激。
Rationale: 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.Objectives: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.Measurements and Main 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 (H2O2) and SIN1 as well as the cytokine mixture (IFN-gamma, IL-1 beta, 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.