Zuo Li, Clanton Thomas L
Division of Pulmonary, Critical Care & Sleep Medicine, Department of Internal Medicine, College of Medicine and Public Health, 201 Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, 473 W. 12th Ave., Columbus, Ohio 43210, USA.
Am J Physiol Cell Physiol. 2005 Jul;289(1):C207-16. doi: 10.1152/ajpcell.00449.2004. Epub 2005 Mar 23.
Many tissues produce reactive oxygen species (ROS) during reoxygenation after hypoxia or ischemia; however, whether ROS are formed during hypoxia is controversial. We tested the hypothesis that ROS are generated in skeletal muscle during exposure to acute hypoxia before reoxygenation. Isolated rat diaphragm strips were loaded with dihydrofluorescein-DA (Hfluor-DA), a probe that is oxidized to fluorescein (Fluor) by intracellular ROS. Changes in fluorescence due to Fluor, NADH, and FAD were measured using a tissue fluorometer. The system had a detection limit of 1 microM H2O2 applied to the muscle superfusate. When the superfusion buffer was changed rapidly from 95% O2 to 0%, 5%, 21%, or 40% O2, transient elevations in Fluor were observed that were proportional to the rise in NADH fluorescence and inversely proportional to the level of O2 exposure. This signal could be inhibited completely with 40 microM ebselen, a glutathione peroxidase mimic. After brief hypoxia exposure (10 min) or exposure to brief periods of H2O2, the fluorescence signal returned to baseline. Furthermore, tissues loaded with the oxidized form of the probe (Fluor-DA) showed a similar pattern of response that could be inhibited with ebselen. These results suggest that Fluor exists in a partially reversible redox state within the tissue. When Hfluor-loaded tissues were contracted with low-frequency twitches, Fluor emission and NADH emission were significantly elevated in a way that resembled the hypoxia-induced signal. We conclude that in the transition to low intracellular P(O2), a burst of intracellular ROS is formed that may have functional implications regarding skeletal muscle O2-sensing systems and responses to acute metabolic stress.
许多组织在缺氧或缺血后的复氧过程中会产生活性氧（ROS）；然而，在缺氧期间是否会形成ROS仍存在争议。我们测试了这样一个假设，即在复氧前急性缺氧暴露期间，骨骼肌中会产生活性氧。将分离的大鼠膈肌条加载二氢荧光素 - DA（Hfluor - DA），这是一种被细胞内ROS氧化为荧光素（Fluor）的探针。使用组织荧光计测量由于Fluor、NADH和FAD引起的荧光变化。该系统对施加于肌肉灌流液中的H2O2的检测限为1 microM。当灌流缓冲液从95% O2迅速变为0%、5%、21%或40% O2时，观察到Fluor的短暂升高，其与NADH荧光的升高成正比，与O2暴露水平成反比。该信号可被40 microM依布硒仑（一种谷胱甘肽过氧化物酶模拟物）完全抑制。在短暂缺氧暴露（10分钟）或暴露于短暂的H2O2后，荧光信号恢复到基线。此外，加载探针氧化形式（Fluor - DA） 的组织表现出类似的反应模式，也可被依布硒仑抑制。这些结果表明Fluor在组织内以部分可逆的氧化还原状态存在。当加载Hfluor的组织进行低频抽搐收缩时，Fluor发射和NADH发射以类似于缺氧诱导信号的方式显著升高。我们得出结论，在向低细胞内P（O2）转变时，会形成一阵细胞内ROS，这可能对骨骼肌氧传感系统和对急性代谢应激的反应具有功能意义。
