Regulation of hepatic EAAT-2 glutamate transporter expression in human liver cholestasis

Figure 4 Effect of long-term treatment with phorbol 12-myristate 13-acetate on EAAT2 expression in HepG2 cells. A: Effect of phorbol 12-myristate 13-acetate (PMA) on multidrug resistance protein 2 (MRP2) cell surface distribution. Cells were treated with 500 nmol/L PMA (lower panels) or vehicle (upper panels) for 4 h, fixed and incubated with anti-EAAT2 and anti-MRP2 primary antibody. Immunoreactivity was visualized using FITC-conjugated rabbit anti-goat and cyanine-3-conjugated goat anti-mouse antibodies respectively. Cell nuclei appear in blue (DNA staining with DAPI). In control HepG2 cells, the MRP2 transporter is mainly localized at the canalicular side. Such organization is absent in PMA-treated cells. Images were representative of several fields examined from 3 independent experiments (original magnification × 200); B: Effect of pre-treating HepG2 cells with PMA for 1, 2 and 4 h on D-[³H]-aspartate uptake. Data are expressed as percent of control and correspond to mean ± SE of three independent experiments performed in sextuplicate. Inset shows the data obtained after the analysis of extracellular LDH levels in HepG2 cell cultures treated with 500 nmol/L PMA or vehicle for 4 h. Data shown correspond to mean values ± SE of typical experiments performed four times; C: Effect of PMA treatment on EAAT2 immunostaining distribution in HepG2 cells was analyzed by confocal microscopy. Cells were treated with 500 nmol/L PMA or vehicle for 4 h, fixed and incubated with anti-EAAT2 primary antibody. Immunoreactivity was visualized using a fluorescein isothiocyanate-conjugated goat anti-rabbit antibody. Images (single optical slides through the cells) were representative of several fields examined from 3 independent experiments. Original magnification is × 200 for controls and × 400 for PMA treated groups. Scale bar, 20 μm. DAPI: 4’,6-diamidino-2-phenylindole; LDH: Lactate dehydrogenase.