Usatyuk Peter V, Vepa Suryanarayana, Watkins Tonya, He Donghong, Parinandi Narasimham L, Natarajan Viswanathan
Department of Medicine, Division of Pulmonary and Critical Care, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA.
Antioxid Redox Signal. 2003 Dec;5(6):723-30. doi: 10.1089/152308603770380025.
Reactive oxygen species (ROS)-mediated compromise of endothelial barrier integrity has been implicated in a number of pulmonary disorders, including adult respiratory distress syndrome, pulmonary edema, and vasculitis. The mechanisms by which ROS increase endothelial permeability are unclear. We hypothesized that ROS-induced changes in cellular redox status (thiols) may contribute to endothelial barrier dysfunction. To test this hypothesis, we used N-acetylcysteine (NAC) and diamide to modulate intracellular levels of cellular glutathione (GSH) and investigated hydrogen peroxide (H(2)O(2))-mediated mitogen-activated protein kinase (MAPK) activation and transendothelial electrical resistance (TER). Exposure of bovine lung microvascular endothelial cells (BLMVECs) to H(2)O(2), in a dose- and time-dependent fashion, increased endothelial permeability. Pretreatment of BLMVECs with NAC (5 mM) for 1 h resulted in partial attenuation of H(2)O(2)-induced TER (a measure of increase in permeability) and GSH. Furthermore, treatment of BLMVECs with diamide, which is known to reduce the intracellular GSH, resulted in significant reduction in TER, which was prevented by NAC. To understand further the role of MAPKs in ROS-induced barrier dysfunction, we examined the role of extracellular signal-regulated kinase (ERK) and p38 MAPK on H(2)O(2)- and diamide-mediated permeability changes. Both H(2)O(2) and diamide, in a dose-dependent manner, activated ERK and p38 MAPK in BLMVECs. However, SB203580, an inhibitor of p38 MAPK, but not PD98059, blocked H(2)O(2)- and diamide-induced TER. Also, NAC prevented H(2)O(2)- and diamide-induced p38 MAPK, but not ERK activation. These results suggest a role for redox regulation of p38 MAPK in ROS-dependent endothelial barrier dysfunction.
活性氧（ROS）介导的内皮屏障完整性受损与多种肺部疾病有关，包括成人呼吸窘迫综合征、肺水肿和血管炎。ROS增加内皮通透性的机制尚不清楚。我们推测ROS诱导的细胞氧化还原状态（硫醇）变化可能导致内皮屏障功能障碍。为了验证这一假设，我们使用N-乙酰半胱氨酸（NAC）和二酰胺来调节细胞内谷胱甘肽（GSH）水平，并研究过氧化氢（H₂O₂）介导的丝裂原活化蛋白激酶（MAPK）激活和跨内皮电阻（TER）。将牛肺微血管内皮细胞（BLMVECs）暴露于H₂O₂，以剂量和时间依赖性方式增加内皮通透性。用NAC（5 mM）预处理BLMVECs 1小时导致H₂O₂诱导的TER（通透性增加指标）和GSH部分减弱。此外，用已知可降低细胞内GSH的二酰胺处理BLMVECs导致TER显著降低，而NAC可预防这种情况。为了进一步了解MAPKs在ROS诱导的屏障功能障碍中的作用，我们研究了细胞外信号调节激酶（ERK）和p38 MAPK在H₂O₂和二酰胺介导的通透性变化中的作用。H₂O₂和二酰胺均以剂量依赖性方式激活BLMVECs中的ERK和p38 MAPK。然而，p38 MAPK抑制剂SB203580可阻断H₂O₂和二酰胺诱导的TER，而PD9805（一种ERK抑制剂）则不能。此外，NAC可预防H₂O₂和二酰胺诱导的p38 MAPK激活，但不能预防ERK激活。这些结果表明p38 MAPK的氧化还原调节在ROS依赖性内皮屏障功能障碍中起作用。
