Mechanism of inhibition of taurolithocholate-induced retrieval of plasma membrane MRP2 by cyclic AMP and tauroursodeoxycholate.

Taurolithocholate (TLC) produces cholestasis by inhibiting biliary solute secretion in part by retrieving MRP2 from the plasma membrane (PM). Tauroursodeoxycholate (TUDC) and cAMP reverse TLC-induced cholestasis by inhibiting TLC-induced retrieval of MRP2. However, cellular mechanisms for this reversal are incompletely understood. Recently, we reported that TLC decreases PM-MRP2 by activating PKCε followed by phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS). Thus, cAMP and TUDC may reverse TLC-induced cholestasis by inhibiting the TLC/PKCε/MARCKS phosphorylation pathway. We tested this hypothesis by determining whether TUDC and/or cAMP inhibit TLC-induced activation of PKCε and phosphorylation of MARCKS Studies were conducted in HuH-NTCP cell line and rat hepatocytes. Activation of PKCε was determined from the translocation of PKCε to PM using a biotinylation method. Phosphorylation of MARCKS was determined by immunoblotting with a phospho-MARCKS antibody. TLC, but not cAMP and TUDC, activated PKCε and increased MARCKS phosphorylation in HuH-NTCP as well in rat hepatocytes. Treatment with TUDC or cAMP inhibited TLC-induced activation of PKCε and increases in MARCKS phosphorylation in both cell types. Based on these results, we conclude that the reversal of TLC-induced cholestasis by cAMP and TUDC involves, at least in part, inhibition of TLC-mediated activation of the PKCε/MARCKS phosphorylation pathway.