Natarajan V, Vepa S, Shamlal R, Al-Hassani M, Ramasarma T, Ravishankar H N, Scribner W M
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-2879, USA.
Mol Cell Biochem. 1998 Jun;183(1-2):113-24. doi: 10.1023/a:1006872230910.
Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [32P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [32P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.
活性氧（ROS）介导的信号转导通路调节是导致血管疾病发生的细胞损伤和屏障功能障碍的重要机制。为了了解ROS在血管功能障碍中的作用，我们研究了由过氧化氢和钒酸盐混合得到的过氧钒酸盐（DPV）对牛肺动脉内皮细胞（BPAECs）中磷脂酶D（PLD）激活的影响。在存在0.05%丁醇的情况下，向BPAECs中添加DPV会导致[32P]磷脂酰丁醇（PBt）以剂量和时间依赖性方式积累。通过用抗磷酸酪氨酸抗体进行蛋白质印迹分析确定，DPV还导致几条蛋白条带（分子量20 - 200 kD）的酪氨酸磷酸化增加。DPV诱导的[32P] PBt积累受到诸如染料木黄酮、除莠霉素、 tyrphostin等酪氨酸激酶抑制剂以及用EGTA或BAPTA螯合Ca2+的抑制，然而，用抑制剂PKC双吲哚马来酰胺预处理BPAECs显示出最小的抑制作用。同样，用佛波酯（100 nM，18小时）下调BPAECs中PKC的主要亚型PKCα和ε，并未减弱DPV诱导的PLD激活。通过比较DPV和佛波酯之间的[32P] PBt形成来确定，酪氨酸激酶和PKC抑制剂的作用是特异性的。除了酪氨酸激酶抑制剂外，抗氧化剂如N - 乙酰半胱氨酸和吡咯烷二硫代氨基甲酸盐也减弱了DPV诱导的蛋白质酪氨酸磷酸化和PLD刺激。这些结果表明，用硫醇化合物还原可防止的氧化参与了DPV依赖性蛋白质酪氨酸磷酸化和PLD激活。
