Vepa S, Scribner W M, Natarajan V
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-2879, USA.
Free Radic Biol Med. 1997;22(1-2):25-35. doi: 10.1016/s0891-5849(96)00241-9.
Oxidants play a significant role in endothelial cell dysfunction through modulation of diverse biochemical reactions and signal transduction pathways. Towards understanding the role of oxidants in vascular injury, we studied the effect of hydrogen peroxide (H2O2), vanadate, and pervanadate (V(4+)-OOH) on [32Pi] uptake and protein phosphorylation in bovine pulmonary artery endothelial cells (BPAEC). The incorporation of labelled [32Pi] into BPAEC was dependent on the concentration of the oxidant employed and time of incubation. Of the oxidants tested, pervanadate (10 microM) induced maximum incorporation of [32Pi] into cells (two- to threefold over control) followed by H2O2 (1 mM) and vanadate (100 microM) and clear differences in labeled protein profiles were noticed between control and oxidant treated cells. The proteins, analyzed by SDS-PAGE, showed distinct increases in labeling patterns ranging from 21-205 kDa, as evidenced by autoradiography. While the majority of the incorporated [32Pi] was in serine/threonine residues, immunoprecipitation and immunoblotting of cell lysates, using an antiphosphotyrosine antibody, revealed that oxidant treatment resulted in significant increases in total protein tyrosine phosphorylation. Most significantly, immunoprecipitation of cell lysates, from pervanadate treatment showed distinct tyrosine phosphorylation of 22 kDa protein, which was identified as caveolin, a marker of caveolae. Pervanadate-mediated phosphorylation was effectively inhibited by staurosporine (5 microM), while genistein showed only partial attenuation. Furthermore, H2O2 treatment resulted in enhanced phosphorylation of 24 kDa protein, which was attenuated by genistein. In addition, oxidant-treated cells exhibited increased tyrosine kinase activity and decreased phosphatase activity. These data show differences in labeling profiles of proteins in response to different oxidants, suggesting differential modulation of distinct protein kinases/phosphatases.
氧化剂通过调节多种生化反应和信号转导途径，在内皮细胞功能障碍中发挥重要作用。为了了解氧化剂在血管损伤中的作用，我们研究了过氧化氢（H2O2）、钒酸盐和过氧钒酸盐（V(4+)-OOH）对牛肺动脉内皮细胞（BPAEC）中[32Pi]摄取和蛋白质磷酸化的影响。标记的[32Pi]掺入BPAEC取决于所用氧化剂的浓度和孵育时间。在所测试的氧化剂中，过氧钒酸盐（10 microM）诱导[32Pi]最大程度地掺入细胞（比对照高两到三倍），其次是H2O2（1 mM）和钒酸盐（100 microM），并且在对照细胞和经氧化剂处理的细胞之间观察到标记蛋白质谱的明显差异。通过SDS-PAGE分析的蛋白质显示，自显影证明标记模式在21-205 kDa范围内有明显增加。虽然大部分掺入的[32Pi]存在于丝氨酸/苏氨酸残基中，但使用抗磷酸酪氨酸抗体对细胞裂解物进行免疫沉淀和免疫印迹显示氧化剂处理导致总蛋白质酪氨酸磷酸化显著增加。最显著的是，来自过氧钒酸盐处理的细胞裂解物的免疫沉淀显示22 kDa蛋白质有明显的酪氨酸磷酸化，该蛋白质被鉴定为小窝蛋白，是小窝的标志物。星形孢菌素（5 microM）有效抑制了过氧钒酸盐介导的磷酸化，而染料木黄酮仅显示部分减弱作用。此外，H2O2处理导致24 kDa蛋白质的磷酸化增强，该磷酸化被染料木黄酮减弱。此外，经氧化剂处理的细胞表现出酪氨酸激酶活性增加和磷酸酶活性降低。这些数据显示了不同蛋白质对不同氧化剂反应的标记谱差异，表明不同的蛋白激酶/磷酸酶受到不同的调节。
