QUANTITATION OF NITROTYROSINE LEVELS IN LUNG SECTIONS OF PATIENTS AND ANIMALS WITH ACUTE LUNG INJURY
QUANTITATION OF NITROTYROSINE LEVELS IN LUNG SECTIONS OF PATIENTS AND ANIMALS WITH ACUTE LUNG INJURY
复制标题
DOI：
10.1172/jci117607
复制
发表时间：
1994-12-01
影响因子：
15.9
通讯作者：
MATALON, S
中科院分区：
文献类型：
--
作者：
HADDAD, IY;PATAKI, G;MATALON, S
活化的肺泡巨噬细胞和肺泡II型上皮细胞释放一氧化氮和超氧化物，两者以接近扩散限制的速率(6.7x10(9)M(-1)S(-1))反应形成过氧亚硝酸盐，过氧亚硝酸盐是一种能够破坏肺泡上皮和肺表面活性物质的强氧化剂。过氧亚硝酸盐，而不是一氧化氮或超氧化物，很容易硝化包括酪氨酸在内的酚环。我们用定量免疫荧光法测定了成人呼吸窘迫综合征(ARDS)患者肺和高氧暴露(100%O-2 60h)大鼠肺组织中硝基酪氨酸的含量。新鲜冰冻或石蜡包埋的肺切片仅与硝基酪氨酸多克隆抗体孵育，然后山羊抗兔抗体与罗丹明偶联。来自ARDS患者(n=5)或暴露于高氧环境的大鼠(n=4)的切片显示，特异性结合比对照组增加了两倍。这种结合被过量的硝基酪氨酸阻断，当硝基酪氨酸抗体被非免疫的免疫球蛋白取代时，这种结合就消失了。在其他实验中，我们证明了在体外与过氧亚硝酸盐孵育的大鼠肺切片中形成了硝基酪氨酸，但没有一氧化氮或活性氧物种。这些数据表明，急性肺损伤患者的肺中可能形成了毒性水平的过氧亚硝酸盐。
Activated alveolar macrophages and epithelial type II cells release both nitric oxide and superoxide which react at near diffusion-limited rate (6.7 x 10(9) M(-1)s(-1)) to form peroxynitrite, a potent oxidant capable of damaging the alveolar epithelium and pulmonary surfactant. Peroxynitrite, but not nitric oxide or superoxide, readily nitrates phenolic rings including tyrosine. We quantified the presence of nitrotyrosine in the lungs of patients with the adult respiratory distress syndrome (ARDS) and in the lungs of rats exposed to hyperoxia (100% O-2 for 60 h) using quantitative immunofluorescence. Fresh frozen or paraffin-embedded lung sections mere incubated with a polyclonal antibody to nitrotyrosine, followed by goat anti-rabbit IgG coupled to rhodamine. Sections from patients with ARDS (n = 5), or from rats exposed to hyperoxia (n = 4), exhibited a twofold increase of specific binding over controls. This binding was blocked by the addition of an excess amount of nitrotyrosine and was absent when the nitrotyrosine antibody was replaced with nonimmune IgG. In additional experiments we demonstrated nitrotyrosine formation in rat lung sections incubated in vitro with peroxynitrite, but not nitric oxide or reactive oxygen species. These data suggest that toxic levels of peroxynitrite may be formed in the lungs of patients with acute lung injury.