Tauroursodeoxycholic acid mobilizes alpha-PKC after uptake in human HepG2 hepatoma cells.
BACKGROUND
Tauroursodeoxycholic acid (TUDCA) may exert anticholestatic effects via Ca(++)- and alpha-protein kinase C (alpha-PKC)-dependent apical vesicular insertion of canalicular transporters in cholestatic hepatocytes (Hepatology 2001; 33: 1206-16). Tauroursodeoxycholic acid is mainly taken up into liver cells by Na(+)-taurocholate cotransporting polypeptide (Ntcp). Tauroursodeoxycholic acid selectively translocates alpha-PKC, a key mediator of regulated exocytosis, to hepatocellular membranes. It is unclear whether TUDCA exerts its effects on alpha-PKC after carrier-mediated uptake into liver cells or by interaction with extracellular/membraneous structures.


METHODS
Human hepatoblastoma HepG2 cells lacking Ntcp were stably transfected with pcDNA3.1/Ntcp or sham-transfected with pcDNA3.1 [+]. Distribution of alpha-PKC was studied using a Western blotting technique. Uptake of [(3)H]taurocholic acid (TCA) was determined radiochemically.


RESULTS
[(3)H]taurocholic acid uptake was approximately 180-fold higher in Ntcp-transfected than in sham-transfected cells. Phorbol 12-myristate 13-acetate (1 micromol L(-1); positive control) increased membrane binding of alpha-PKC by 34% in Ntcp-transfected and by 37% in sham-transfected cells. Tauroursodeoxycholic acid (10 micromol L(-1)) increased membrane-associated alpha-PKC by 19% in Ntcp-transfected, but not in sham-transfected cells (-13%). Taurocholic acid (10 micromol L(-1)) did not affect the distribution of alpha-PKC.


CONCLUSIONS
Carrier-mediated uptake is a prerequisite for TUDCA-induced translocation of alpha-PKC to hepatocellular membranes.
Glasova
Helena
H
Department of Medicine II, Klinikum Grosshadern, University of Munich, Germany.
Berghaus
Thomas M
TM
Kullak-Ublick
Gerd A
GA
Paumgartner
Gustav
G
Beuers
Ulrich
U
D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002458:Cell Fractionation; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D026901:Membrane Transport Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D027981:Symporters; D013655:Taurochenodeoxycholic Acid / Q000378:metabolism* / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D014162:Transfection; D014316:Tritium / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D007527:Isoenzymes; D026901:Membrane Transport Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; D014316:Tritium; C071908:sodium-bile acid cotransporter; D013655:Taurochenodeoxycholic Acid; C031655:ursodoxicoltaurine; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate