Role of reactive oxygen species generated by NADPH oxidase in the mechanism of activation of K+-Cl--cotransport by N-ethylmaleimide in HepG2 human hepatoma cells

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 hu man hepatoblastoma cells. N-ethylmaleimide (NEM), a KCC activator, induced Cl--dependent K+ efflux, which was markedly prevented by KCC inhibitors (ca lyculin-A, genistein and BaCl2), indicating that KCC is activated by NEM in the HepG2 cells. Treatment with NEM also induced a sustained increase in t he level of intracellular ROS assessed by 2',7'-dichlorofluorescein floures cence. Antioxidants, N-acetyl cysteine or N,N'-diphenyl-p-phenylenediamine significantly inhibited both ROS generation and KCC activation induced by N EM. The NEM-induced ROS production was significantly suppressed by inhibito rs of NADPH oxidase (diphenylene iodonium, apocynin and neopterine). These inhibitors also significantly inhibited the NEM-induced KCC activation. Tak en together, these results suggest that ROS generated by NADPH oxidase may mediate the NEM-induced activation of KCC in human hepatoma cells.