Weissmann Norbert, Kuzkaya Nermin, Fuchs Beate, Tiyerili Vedat, Schäfer Rolf U, Schütte Hartwig, Ghofrani Hossein A, Schermuly Ralph T, Schudt Christian, Sydykov Akylbek, Egemnazarow Bakytbek, Seeger Werner, Grimminger Friedrich
Department of Internal Medicine II, Klinikstrasse 36, Justus-Liebig University, 35392 Giessen, Germany.
Respir Res. 2005 Jul 31;6(1):86. doi: 10.1186/1465-9921-6-86.
The sources and measurement of reactive oxygen species (ROS) in intact organs are largely unresolved. This may be related to methodological problems associated with the techniques currently employed for ROS detection. Electron spin resonance (ESR) with spin trapping is a specific method for ROS detection, and may address some these technical problems.
We have established a protocol for the measurement of intravascular ROS release from isolated buffer-perfused and ventilated rabbit and mouse lungs, combining lung perfusion with the spin probe 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) and ESR spectroscopy. We then employed this technique to characterize hypoxia-dependent ROS release, with specific attention paid to NADPH oxidase-dependent superoxide formation as a possible vasoconstrictor pathway.
While perfusing lungs with CPH over a range of inspired oxygen concentrations (1-21 %), the rate of CP* formation exhibited an oxygen-dependence, with a minimum at 2.5 % O2. Addition of superoxide dismutase (SOD) to the buffer fluid illustrated that a minor proportion of this intravascular ROS leak was attributable to superoxide. Stimulation of the lungs by injection of phorbol-12-myristate-13-acetate (PMA) into the pulmonary artery caused a rapid increase in CP* formation, concomitant with pulmonary vasoconstriction. Both the PMA-induced CPH oxidation and the vasoconstrictor response were largely suppressed by SOD. When the PMA challenge was performed at different oxygen concentrations, maximum superoxide liberation and pulmonary vasoconstriction occurred at 5% O2. Using a NADPH oxidase inhibitor and NADPH-oxidase deficient mice, we illustrated that the PMA-induced superoxide release was attributable to the stimulation of NADPH oxidases.
The perfusion of isolated lungs with CPH is suitable for detection of intravascular ROS release by ESR spectroscopy. We employed this technique to demonstrate that 1) PMA-induced vasoconstriction is caused "directly" by superoxide generated from NADPH oxidases and 2) this pathway is pronounced in hypoxia. NADPH oxidases thus may contribute to the hypoxia-dependent regulation of pulmonary vascular tone.
完整器官中活性氧（ROS）的来源和测量方法在很大程度上尚未得到解决。这可能与当前用于ROS检测技术相关的方法学问题有关。自旋捕捉电子自旋共振（ESR）是一种用于ROS检测的特定方法，可能解决其中一些技术问题。
我们建立了一种测量从分离的经缓冲液灌注和通气的兔和小鼠肺中血管内ROS释放的方案，将肺灌注与自旋探针1-羟基-3-羧基-2,2,5,5-四甲基吡咯烷（CPH）和ESR光谱相结合。然后我们采用该技术来表征缺氧依赖性ROS释放，特别关注作为一种可能的血管收缩途径的NADPH氧化酶依赖性超氧化物形成。
在一系列吸入氧浓度（1-21%）下用CPH灌注肺时，CP形成速率表现出氧依赖性，在2.5% O₂时最低。向缓冲液中添加超氧化物歧化酶（SOD）表明，这种血管内ROS泄漏的一小部分归因于超氧化物。通过向肺动脉注射佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）刺激肺，导致CP形成迅速增加，同时伴有肺血管收缩。PMA诱导的CPH氧化和血管收缩反应在很大程度上被SOD抑制。当在不同氧浓度下进行PMA刺激时，最大超氧化物释放和肺血管收缩发生在5% O₂时。使用NADPH氧化酶抑制剂和NADPH氧化酶缺陷小鼠，我们证明PMA诱导的超氧化物释放归因于NADPH氧化酶的刺激。
用CPH灌注分离的肺适用于通过ESR光谱检测血管内ROS释放。我们采用该技术证明：1）PMA诱导的血管收缩是由NADPH氧化酶产生的超氧化物“直接”引起的；2）该途径在缺氧时明显。因此，NADPH氧化酶可能有助于缺氧依赖性肺血管张力调节。
