Kim J A, Lee Y S
College of Pharmacy, Yeungnam University, Kyongsan 712-749, Korea.
Free Radic Res. 2001 Jul;35(1):43-53. doi: 10.1080/10715760100300581.
K(+)-Cl(-)-cotransport (KCC) is ubiquitously present in all cells, and plays an essential role in ion and volume regulation. In this study we investigated the role of reactive oxygen species (ROS) in regulation of KCC in HepG2 human hepatoblastoma cells. N-ethylmaleimide (NEM), a KCC activator, induced Cl(-)-dependent K+ efflux, which was markedly prevented by KCC inhibitors (calyculin-A, genistein and BaCl2), indicating that KCC is activated by NEM in the HepG2 cells. Treatment with NEM also induced a sustained increase in the level of intracellular ROS assessed by 2',7'-dichlorofluorescein fluorescence. Antioxidants, N-acetyl cysteine or N,N'-diphenyl-p-phenylenediamine significantly inhibited both ROS generation and KCC activation induced by NEM. The NEM-induced ROS production was significantly suppressed by inhibitors of NADPH oxidase (diphenylene iodonium, apocynin and neopterine). These inhibitors also significantly inhibited the NEM-induced KCC activation. Taken together, these results suggest that ROS generated by NADPH oxidase may mediate the NEM-induced activation of KCC in human hepatoma cells.
钾氯共转运体（KCC）普遍存在于所有细胞中，在离子和容积调节中发挥着重要作用。在本研究中，我们调查了活性氧（ROS）在人肝癌细胞系HepG2中对KCC调节的作用。KCC激活剂N - 乙基马来酰亚胺（NEM）诱导了氯离子依赖性钾离子外流，而KCC抑制剂（花萼海绵诱癌素A、染料木黄酮和氯化钡）可显著抑制这种外流，这表明在HepG2细胞中NEM激活了KCC。用NEM处理还导致通过2',7'-二氯荧光素荧光评估的细胞内ROS水平持续升高。抗氧化剂N - 乙酰半胱氨酸或N,N'-二苯基对苯二胺显著抑制了NEM诱导的ROS生成和KCC激活。NADPH氧化酶抑制剂（二亚苯基碘鎓、鱼藤酮和新蝶呤）显著抑制了NEM诱导的ROS产生。这些抑制剂也显著抑制了NEM诱导的KCC激活。综上所述，这些结果表明NADPH氧化酶产生的ROS可能介导了NEM诱导的人肝癌细胞中KCC的激活。
