Roy Shukla, Parinandi Narasimham, Zeigelstein Roy, Hu Qinghua, Pei Yong, Travers Jeffrey B, Natarajan Viswanathan
Department of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, MD 21224, USA.
Antioxid Redox Signal. 2003 Apr;5(2):217-28. doi: 10.1089/152308603764816578.
We investigated the effect of hyperoxia on phospholipase D (PLD) activation in bovine lung microvascular endothelial cells (BLMVECs). Generation of intracellular reactive oxygen species in BLMVECs exposed to hyperoxia for 2 or 24 h was three-fold higher compared with normoxic cells as measured by dichlorodihydrofluorescein di(acetoxymethyl ester) fluorescence. Exposure of BLMVECs to hyperoxia for 2 or 24 h attenuated 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated PLD activation compared with normoxic cells, however, hyperoxia did not alter basal PLD activity. Antioxidants, such as propyl gallate and pyrrolidine dithiocarbamate, reversed the effect of hyperoxia on TPA-induced PLD activity. Furthermore, the TPA-induced PLD activation was inhibited not only by the protein kinase C inhibitor, Go6976, but also by the tyrosine kinase inhibitor, genistein, and by the Src kinase specific inhibitor, PP-2, suggesting the involvement of protein kinase C and also tyrosine kinases in TPA-induced PLD activation. Western blot analysis of cell lysates from the hyperoxic (2 or 24 h) BLMVECs stimulated with TPA with anti-phosphotyrosine antibody showed an attenuation in overall tyrosine phosphorylation of proteins. In conclusion, we have demonstrated that hyperoxia enhanced the generation of reactive oxygen species in lung microvascular endothelial cells and attenuated TPA-induced protein tyrosine phosphorylation and PLD activation. As protein tyrosine phosphorylation and PLD play important roles in inflammatory responses, this could provide a mechanism for the regulation of endothelial barrier function during hyperoxic lung injury.
我们研究了高氧对牛肺微血管内皮细胞（BLMVECs）中磷脂酶D（PLD）激活的影响。通过二氯二氢荧光素二（乙酰氧基甲基酯）荧光测量，暴露于高氧环境2小时或24小时的BLMVECs中细胞内活性氧的生成量比常氧细胞高三倍。与常氧细胞相比，将BLMVECs暴露于高氧环境2小时或24小时会减弱12 - O - 十四烷酰佛波醇13 - 乙酸酯（TPA）介导的PLD激活，然而，高氧并未改变基础PLD活性。抗氧化剂，如没食子酸丙酯和吡咯烷二硫代氨基甲酸盐，可逆转高氧对TPA诱导的PLD活性的影响。此外，TPA诱导的PLD激活不仅受到蛋白激酶C抑制剂Go6976的抑制，还受到酪氨酸激酶抑制剂染料木黄酮以及Src激酶特异性抑制剂PP - 2的抑制，这表明蛋白激酶C和酪氨酸激酶均参与了TPA诱导的PLD激活。用抗磷酸酪氨酸抗体对经TPA刺激的高氧（2小时或24小时）BLMVECs的细胞裂解物进行蛋白质印迹分析显示，蛋白质的总体酪氨酸磷酸化程度降低。总之，我们已经证明高氧增强了肺微血管内皮细胞中活性氧的生成，并减弱了TPA诱导的蛋白质酪氨酸磷酸化和PLD激活。由于蛋白质酪氨酸磷酸化和PLD在炎症反应中起重要作用，这可能为高氧性肺损伤期间内皮屏障功能的调节提供一种机制。
