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

Figure 3 Mechanism pathways modulated by protein kinase C to regulate aspartate uptake capacity in HepG2 cells. A: Involvement of protein kinase C (PKC) pathway in the modulation of EAAT2 activity in HepG2 cells; Cells were pretreated with 1 μmol/L Ro-31-8220 or vehicle for 15 min. Thereafter, phorbol 12-myristate 13-acetate (PMA) (500 nmol/L) was added and the uptake assay was performed after 15 min. Data shown are means ± SE of three independent experiments performed in triplicate. PMA significantly inhibited D-[³H]-aspartate uptake, Ro-31-8220 had a non-intrinsic effect on aspartate uptake, and completely blocked the effect of PMA on aspartate uptake (Ro-31-8220 vs PMA plus Ro-31-8220 not significantly different). PD09859, a selective inhibitor of the MAPK pathway, failed to prevent the PMA-decrease in aspartate uptake; B: Effect of L-aspartate and phenylarsine oxide (PAO) on basal and PMA-decreased aspartate uptake in HepG2 cells. Cells were pretreated with PAO (10 μmol/L) or vehicle for 15 min. Thereafter, PMA (500 nmol/L) was added and the uptake assay was performed after 15 min. Results are expressed as percent of untreated cells and correspond to means ± SE of at least three independent experiments performed in triplicate. Statistical analysis was performed by One-way ANOVA followed by the Tukey’s test for multiple comparisons. ^aP < 0.05; ^bP < 0.001 vs the corresponding untreated control cells; and ^dP < 0.001 denotes a difference between PMA-treated cells with or without the pharmacological agent.