Fujii Y, Goldberg P, Hussain S N
Department of Anesthesiology and Critical Care Medicine, Tokyo Medical and Dental University, School of Medicine, Japan.
Chest. 1998 Aug;114(2):569-76. doi: 10.1378/chest.114.2.569.
Nitric oxide (NO), a highly reactive species produced by the activity of NO synthases (NOS), is normally present in the exhaled air of humans and animals. Exhaled NO concentration increases significantly in humans with sepsis and animals, but neither the source nor NOS isoforms responsible for this rise in pulmonary NO production are known. The main objective of this study is to determine the sites and the mechanisms of enhanced NO production in the exhaled air of endotoxemic pigs.
Randomized, controlled, animal study.
University-based animal research facility.
Thirteen pathogen-free adult female pigs (22 to 27 kg).
Anesthetized pigs were divided into two groups: control and lipopolysaccharides (LPS) (septic) groups. In both groups, extrathoracic (upper airways, nasal, and paranasal) and intrathoracic (bronchi, bronchioles, and alveoli) compartments were ventilated equally with two separate ventilators connected to two tracheal tubes. The LPS group received slow infusion (over 2 h) of Escherichia coli endotoxin (10 microg/kg/h), whereas saline solution was infused into the control group. Expired air of the two compartments was collected throughout the 2-h observation period. The animals were then killed and the lungs were quickly excised and frozen.
Hemodynamic variables were measured in both groups. NO concentration in the exhaled air of both compartments was measured with a chemiluminescence analyser. Pulmonary NOS activity was evaluated by measuring the conversion of L-[2,3H]-arginine to L-[2,3H]-citrulline, and pulmonary expression of NOS was evaluated by immunoblotting.
Baseline NO concentration in both groups was significantly higher in the extrathoracic vs intrathoracic compartment (average of 5.2 vs 3.4 parts per billion). Endotoxin infusion elicited a significant and early (after 45 min) rise in exhaled NO concentration in the extrathoracic compartment. Exhaled NO in the intrathoracic compartment also rose significantly but after 90 min of endotoxin infusion. Measurement of lung NOS activity showed a substantial rise in Ca++/calmodulin-dependent activity in the LPS group with no rise in Ca++/calmodulin-independent activity. Immunoblotting of lung tissue samples indicated the absence of the inducible isoform in both groups of animals. Moreover, LPS injection elicited no significant alterations in the pulmonary expression of the endothelial and the neuronal isoforms.
Both extrathoracic and intrathoracic compartments contribute to the rise in exhaled NO production in experimental septic shock. The rise in exhaled NO production is due to increased activity of constitutive NOS isoforms as a result of increased cofactor availability and/or downregulation of the endogenous inhibitors of NOS.
一氧化氮（NO）是由一氧化氮合酶（NOS）的活性产生的一种高反应性物质，通常存在于人和动物的呼出气体中。脓毒症患者和动物呼出的NO浓度显著升高，但肺部NO产生增加的来源和负责的NOS亚型均未知。本研究的主要目的是确定内毒素血症猪呼出气体中NO产生增加的部位和机制。
随机、对照动物研究。
大学动物研究设施。
13只无特定病原体的成年雌性猪（22至27千克）。
将麻醉的猪分为两组：对照组和脂多糖（LPS）（脓毒症）组。两组均通过连接到两根气管导管的两台独立呼吸机对胸外（上呼吸道、鼻腔和鼻旁窦）和胸内（支气管、细支气管和肺泡）腔室进行同等通气。LPS组接受大肠杆菌内毒素缓慢输注（2小时以上）（10微克/千克/小时），而对照组输注生理盐水。在整个2小时观察期内收集两个腔室的呼出气体。然后处死动物，迅速切除肺并冷冻。
两组均测量血流动力学变量。用化学发光分析仪测量两个腔室呼出气体中的NO浓度。通过测量L-[2,3H]-精氨酸向L-[2,3H]-瓜氨酸的转化来评估肺NOS活性，通过免疫印迹法评估肺中NOS的表达。
两组胸外腔室的基线NO浓度显著高于胸内腔室（平均分别为5.2和3.4十亿分之一）。内毒素输注导致胸外腔室呼出的NO浓度显著且早期（45分钟后）升高。胸内腔室呼出的NO也显著升高，但在内毒素输注90分钟后。肺NOS活性测量显示LPS组中Ca++/钙调蛋白依赖性活性大幅升高，而Ca++/钙调蛋白非依赖性活性未升高。肺组织样本的免疫印迹表明两组动物均不存在诱导型亚型。此外，LPS注射未引起内皮型和神经元型亚型在肺中的表达发生显著改变。
胸外和胸内腔室均导致实验性脓毒症休克中呼出NO产生增加。呼出NO产生增加是由于组成型NOS亚型活性增加，这是由于辅因子可用性增加和/或NOS内源性抑制剂下调所致。
