National Heart and Lung Institute, Imperial College London, UK.
Am J Respir Crit Care Med. 2010 Jan 1;181(1):21-30. doi: 10.1164/rccm.200904-0493OC. Epub 2009 Sep 24.
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.
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.
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.
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.
一氧化氮（NO）在慢性阻塞性肺疾病（COPD）患者的肺外周增加。然而，在 COPD 患者的肺外周组织中尚未鉴定出导致 NO 升高的一氧化氮合酶（NOS）的表达。
通过 Western blot 和逆转录-聚合酶链反应分别定量测定手术切除的肺组织标本中一氧化氮合酶 I 型（神经元 NOS [nNOS]）、II 型（诱导型 NOS [iNOS]）和 III 型（内皮 NOS [eNOS]）的蛋白和 mRNA 表达，包括非吸烟者对照、COPD 严重程度不同的患者和无 COPD 的吸烟者，并在肺上皮细胞系（A549）中进行。还在体外评估了 A549 细胞中硝化/氧化应激对 NOS 表达和活性的影响。通过免疫沉淀定量测定 nNOS 的硝化，在体外和体内，通过低温 SDS-PAGE/Western blot 检测过氧亚硝酸盐生成剂 3-吗啉代-sydnonimine-N-乙基碳酰胺（SIN1）存在时 nNOS 的二聚化。
与非吸烟者和正常吸烟者相比，严重和非常严重的 COPD 患者的肺组织中 nNOS（mRNA 和蛋白）呈分级增加。与未处理的细胞相比，过氧化氢（H2O2）和 SIN1 以及细胞因子混合物（IFN-γ、IL-1β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 活性增加，但不影响其二聚化。
COPD 患者的 nNOS 表达和活性显著增加，反映了疾病的严重程度，可能是氧化应激的结果。
