Wakabayashi Y, Fujita H, Morita I, Kawaguchi H, Murota S
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan.
Biochim Biophys Acta. 1995 Mar 16;1265(2-3):103-9. doi: 10.1016/0167-4889(94)00202-p.
We have demonstrated that the endothelial cell-derived superoxide anion is deeply involved in the endothelial cell injury induced by activated neutrophils (Fujita, H., Morita, I. and Murota, S. (1994) Arch. Biochem. Biophys. 309, 62-69). To clarify the mechanism underlying the increase in the endothelial cell-derived superoxide anion induced by activated neutrophils, the conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO) in cultured endothelial cells isolated from bovine carotid arteries was investigated. Although the endothelial cells expressed both XD and XO activity, the XO activity of unstimulated cells comprised about 12% of the total (XD + XO) activity. When endothelial cells were exposed to neutrophils activated with phorbol 12-myristate 13-acetate (PMA), XO activity rapidly increased about 3-fold over the control. Whereas treatment of endothelial cells with PMA alone or unstimulated neutrophils alone did not increase the XO activity at all. The increase in XO activity in endothelial cells was also observed on the treatment of the cells with neutrophils activated with leukotriene B4 or thrombin. To determine whether or not proteases released from activated neutrophils are involved in the increased conversion of XD to XO in endothelial cells, the effects of the elastase specific inhibitor, ONO-5046, and protease inhibitors, such as aprotinin, gabexate mesylate and urinastatin, were examined. However, these protease inhibitors did not suppress the conversion of XD to XO induced by PMA-activated neutrophils. Moreover, the treatment of endothelial cells with purified human neutrophil elastase and H2O2 also did not affect the conversion at all. In contrast, monoclonal antibodies against CD11a and CD18 significantly inhibited the increased conversion of XD to XO induced by PMA-activated neutrophils. Moreover, tyrosine kinase inhibitors such as staurosporin and herbimysine also inhibited the increased conversion of XD to XO induced by PMA-activated neutrophils. These results indicate that the adhesion of activated neutrophils to endothelial cells via CD11a/CD18-ICAM-1 is involved in the conversion of XD to XO in endothelial cells induced by activated neutrophils.
我们已经证明，内皮细胞衍生的超氧阴离子与活化的中性粒细胞诱导的内皮细胞损伤密切相关（藤田浩、森田一和室田秀，（1994年）《生物化学与生物物理学文献》309卷，62 - 69页）。为了阐明活化的中性粒细胞诱导内皮细胞衍生的超氧阴离子增加的机制，我们研究了从牛颈动脉分离的培养内皮细胞中黄嘌呤脱氢酶（XD）向黄嘌呤氧化酶（XO）的转化。尽管内皮细胞同时表达XD和XO活性，但未受刺激细胞的XO活性约占总（XD + XO）活性的12%。当内皮细胞暴露于用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）活化的中性粒细胞时，XO活性迅速增加，比对照增加约3倍。而单独用PMA处理内皮细胞或单独用未受刺激的中性粒细胞处理内皮细胞根本不会增加XO活性。在用白三烯B4或凝血酶活化的中性粒细胞处理细胞时，也观察到内皮细胞中XO活性增加。为了确定活化的中性粒细胞释放的蛋白酶是否参与内皮细胞中XD向XO转化的增加，我们检测了弹性蛋白酶特异性抑制剂ONO - 5046以及蛋白酶抑制剂如抑肽酶、甲磺酸加贝酯和尿抑胃素的作用。然而，这些蛋白酶抑制剂并未抑制PMA活化的中性粒细胞诱导的XD向XO的转化。此外，用纯化的人中性粒细胞弹性蛋白酶和H2O2处理内皮细胞也根本不影响这种转化。相反，抗CD11a和CD18的单克隆抗体显著抑制了PMA活化的中性粒细胞诱导的XD向XO转化的增加。此外，诸如星形孢菌素和赫比霉素等酪氨酸激酶抑制剂也抑制了PMA活化的中性粒细胞诱导的XD向XO转化的增加。这些结果表明，活化的中性粒细胞通过CD11a/CD18 - ICAM - 1与内皮细胞的黏附参与了活化的中性粒细胞诱导的内皮细胞中XD向XO的转化。
