Arachidonic Acid Activates K-Cl-cotransport in HepG2 Human Hepatoblastoma Cells

Abstract

K(+)-Cl(-)-cotransport (KCC) has been reported to have various cellular functions, including proliferation and apoptosis of human cancer cells. However, the signal transduction pathways that control the activity of KCC are currently not well understood. In this study we investigated the possible role of phospholipase A(2) (PLA(2))-arachidonic acid (AA) signal in the regulatory mechanism of KCC activity. Exogenous application of AA significantly induced K(+) efflux in a dose-dependent manner, which was completely blocked by R-(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl]oxy]acetic acid (DIOA), a specific KCC inhibitor. N-Ethylmaleimide (NEM), a KCC activator-induced K(+) efflux was significantly suppressed by bromoenol lactone (BEL), an inhibitor of the calcium-independent PLA(2) (iPLA(2)), whereas it was not significantly altered by arachidonyl trifluoromethylketone (AACOCF(3)) and p-bromophenacyl bromide (BPB), inhibitors of the calcium-dependent cytosolic PLA(2) (cPLA(2)) and the secretory PLA(2) (sPLA(2)), respectively. NEM increased AA liberation in a dose- and time-dependent manner, which was markedly prevented only by BEL. In addition, the NEM-induced ROS generation was significantly reduced by DPI and BEL, whereas AACOCF(3) and BPB did not have an influence. The NEM-induced KCC activation and ROS production was not significantly affected by treatment with indomethacin (Indo) and nordihydroguaiaretic acid (NDGA), selective inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Treatment with 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, markedly produced ROS and activated the KCC. Collectively, these results suggest that iPLA(2)-AA signal may be essentially involved in the mechanism of ROS-mediated KCC activation in HepG2 cells.

Keywords: Arachidonic acid; HepG2 cells; K+-Cl--cotransport; N-Ethylmaleimide; Phospholipase A2; Reactive oxygen species.

Fig. 1

AA activates KCC in HepG2 human hepatoblastoma cells. The data (A) show changes in [K⁺]_i as a function of time, measured by using the K⁺-sensitive fluorescent dye PBFI/AM. In the data, PBFI fluorescence ratios are directly proportional to [K⁺]_i. The arrow shows the time point for addition of AA (10 µM). DIOA (100 µM), a KCC inhibitor, was added 10 min before AA treatment. Quantitative changes (B) were expressed as percent changes of the maximum decrease in PBFI fluorescence ratio induced by the drug compared to control condition in which the cells were treated with a drug-free vehicle. Each column represents the mean value of four replications with bars indicating SEM. ^*p<0.05 compared to control, ^#p<0.05 compared to AA alone.

Fig. 2

Effects of PLA₂ inhibitors on the KCC activation induced by NEM in HepG2 human hepatoblastoma cells. The data (A) show changes in [K⁺]_i as a function of time, measured by using the K⁺-sensitive fluorescent dye PBFI/AM. The arrows show the time points for addition of NEM (100 µM). AACOCF₃ (10 µM), BEL (10 µM) and BPB (10 µM) were added 10 min before NEM treatment. Quantitative changes (B) were expressed as percent changes of the maximum decrease in PBFI fluorescence induced by the drug compared to control condition in which the cells were treated with a drug-free vehicle. Each column represents the mean value of four replications with bars indicating SEM. ^*p<0.05 compared to control, ^#p<0.05 compared to NEM alone.

Fig. 3

Effects of inhibitors of COX and LOX on the KCC activation induced by NEM in HepG2 human hepatoblastoma cells. The data (A) show changes in [K⁺]_i as a function of time, measured by using the K⁺-sensitive fluorescent dye PBFI/AM. The arrows show the time points for addition of NEM (100 µM). Indo (30 µM) and NDGA (50 µM) were added 10 min before NEM treatment. Quantitative changes (B) were expressed as percent changes of the maximum decrease in PBFI fluorescence induced by the drug compared to control condition in which the cells were treated with a drug-free vehicle. Each column represents the mean value of four replications with bars indicating SEM. ^*p<0.05 compared to control.

Fig. 4

Time-course of [³H]AA release induced by NEM (A) and the effects of PLA₂ inhibitors on the NEM-induced [³H]AA release (B) in HepG2 human hepatoma cells. (A) HepG2 cells were labeled with medium containing [³H]AA and then treated with either vehicle or NEM (100 µM) for a designated time. Assay for [³H]AA release was done by scintillation counting method as described in Method section. (B) NEM was treated with or without various drugs for 60 min. In these experiments AACOCF₃ (10 µM), BEL (10 µM) and BPB (10 µM) were used as a specific inhibitor of the cPLA₂, iPLA₂ and sPLA₂, respectively. These inhibitors were added 10 min before NEM treatment. Results are expressed as percent change of control condition in which cells were treated with a drug-free vehicle. All the data points represent the mean values of four replications with bars indicating SEM.

Fig. 5

Effects of PLA₂ inhibitors and DPI on the ROS generation induced by NEM in HepG2 human hepatoblastoma cells. The data (A) show changes in ROS levels as a function of time, which was measured by DCF fluorescence method. The arrows show the time points for addition of NEM (100 µM). DPI (50 µM), an inhibitor of NADPH oxidase, and PLA₂ inhibitors, AACOCF₃ (10 µM), BEL (10 µM) and BPB (10 µM) were added 10 min before NEM treatment. In the data (B) results are expressed as fold increase compared to the initial DCF fluorescence intensity. Data points represent the mean values of four replications with bars indicating SEM. ^*p<0.05 compared to control condition in which the cells were incubated with NEM-free medium, ^#p<0.05 compared to NEM alone.

Fig. 6

Effects of inhibitors of COX and LOX on the ROS generation induced by NEM in HepG2 human hepatoblastoma cells. The data (A) show changes in ROS levels as a function of time, which was measured by DCF fluorescence method. The arrows show the time points for addition of NEM (100 µM). Indo (30 µM), a COX inhibitor and NDGA (50 µM), a LOX inhibitor were added 10 min before NEM treatment. In the data (B) results are expressed as fold increase compared to the initial DCF fluorescence intensity. Data points represent the mean values of four replications with bars indicating SEM.

Fig. 7

Effects of ETYA, a non-metabolizable analogue of AA, on the KCC activation (A, C) and ROS generation (B, D) in HepG2 human hepatoblastoma cells. The data (A, B) show changes in [K⁺]_i and ROS levels as a function of time, respectively. The arrows show the time points for addition of ETYA (10 µM). In these experiments DIOA (100 µM) and DPI (50 µM), were added 10 min before ETYA treatment. Quantitative changes were expressed as percent changes of the maximum decrease in PBFI fluorescence (C) and fold increase compared to the initial DCF fluorescence intensity (D) compared to control condition in which the cells were treated with a drug-free vehicle. Each column represents the mean value of four replications with bars indicating SEM. ^*p<0.05 compared to ETYA alone.