Zhang Qunwei, Matsuzaki Ikuo, Chatterjee Shampa, Fisher Aron B
Inst. for Environmental Medicine, University of Pennsylvania School of Medicine, 1 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068, USA.
Am J Physiol Lung Cell Mol Physiol. 2005 Dec;289(6):L954-61. doi: 10.1152/ajplung.00210.2005.
Previous studies have shown endothelial cell membrane depolarization and generation of reactive oxygen species (ROS) in endothelial cells with abrupt reduction in shear stress (ischemia). This study evaluated the role of ATP-sensitive potassium (K(ATP)) channels and NADPH oxidase in the ischemic response by using Kir6.2-/- and gp91(phox)-/- mice. To evaluate ROS generation, we subjected isolated perfused mouse lungs labeled with 2',7'-dichlorodihydrofluorescein (DCF), hydroethidine (HE), or diphenyl-1-pyrenylphosphine (DPPP) to control perfusion followed by global ischemia. In wild-type C57BL/6J mice, imaging of subpleural endothelial cells showed a time-dependent increase in intensity for all three fluorescence probes with ischemia, which was blocked by preperfusion with cromakalim (a K(ATP) channel agonist) or diphenyleneiodonium (DPI, a flavoprotein inhibitor). Endothelial cell fluorescence with bis-oxonol, a membrane potential probe, increased during lung ischemia indicating cell membrane depolarization. The change in membrane potential with ischemia in lungs of gp91(phox)-/- mice was similar to wild type, but ROS generation did not occur. Lungs from Kir6.2-/- showed marked attenuation of the change in both membrane potential and ROS production. Thus membrane depolarization during lung ischemia requires the presence of a K(ATP) channel and is required for activation of NADPH oxidase and endothelial ROS generation.
先前的研究表明，在内皮细胞中，随着剪切应力突然降低（缺血），会发生内皮细胞膜去极化并产生活性氧（ROS）。本研究通过使用Kir6.2基因敲除小鼠和gp91（phox）基因敲除小鼠，评估了ATP敏感性钾（K（ATP））通道和NADPH氧化酶在缺血反应中的作用。为了评估ROS的产生，我们对用2'，7'-二氯二氢荧光素（DCF）、氢乙啶（HE）或二苯基-1-芘基膦（DPPP）标记的离体灌注小鼠肺进行对照灌注，随后进行整体缺血处理。在野生型C57BL/6J小鼠中，对胸膜下内皮细胞的成像显示，随着缺血的发生，所有三种荧光探针的强度均呈时间依赖性增加，这被用克罗卡林（一种K（ATP）通道激动剂）或二苯碘铵（DPI，一种黄素蛋白抑制剂）预灌注所阻断。使用膜电位探针双苯磺酰草酰胺检测发现，肺缺血期间内皮细胞荧光增加，表明细胞膜去极化。gp91（phox）基因敲除小鼠肺缺血时膜电位的变化与野生型相似，但未发生ROS生成。Kir6.2基因敲除小鼠的肺显示膜电位变化和ROS产生均明显减弱。因此，肺缺血期间的膜去极化需要K（ATP）通道的存在，并且是激活NADPH氧化酶和内皮细胞ROS生成所必需的。
