Glasova Helena, Berghaus Thomas M, Kullak-Ublick Gerd A, Paumgartner Gustav, Beuers Ulrich
Department of Medicine II, Klinikum Grosshadern, University of Munich, Germany.
Eur J Clin Invest. 2002 Jun;32(6):437-42. doi: 10.1046/j.1365-2362.2002.01002.x.
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.
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.
[(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.
Carrier-mediated uptake is a prerequisite for TUDCA-induced translocation of alpha-PKC to hepatocellular membranes.
牛磺熊去氧胆酸（TUDCA）可能通过依赖钙离子（Ca(++)）和α-蛋白激酶C（α-PKC）的胆小管转运体顶端囊泡插入，对胆汁淤积性肝细胞发挥抗胆汁淤积作用（《肝脏病学》2001年；33卷：1206 - 16页）。牛磺熊去氧胆酸主要通过钠-牛磺胆酸盐共转运多肽（Ntcp）被摄取进入肝细胞。牛磺熊去氧胆酸选择性地将α-PKC（一种调节性胞吐作用的关键介质）转运至肝细胞膜。目前尚不清楚TUDCA是在通过载体介导摄取进入肝细胞后对α-PKC发挥作用，还是通过与细胞外/膜结构相互作用发挥作用。
将缺乏Ntcp的人肝癌HepG2细胞用pcDNA3.1/Ntcp进行稳定转染，或用pcDNA3.1[+]进行假转染。采用蛋白质印迹技术研究α-PKC的分布。通过放射化学方法测定[³H]牛磺胆酸（TCA）的摄取。
在Ntcp转染细胞中，[³H]牛磺胆酸的摄取比假转染细胞高约180倍。佛波醇12-肉豆蔻酸酯13-乙酸酯（1 μmol/L；阳性对照）使Ntcp转染细胞中α-PKC的膜结合增加34%，使假转染细胞中增加37%。牛磺熊去氧胆酸（10 μmol/L）使Ntcp转染细胞中膜相关α-PKC增加19%，但在假转染细胞中未增加（反而减少13%）。牛磺胆酸（10 μmol/L）不影响α-PKC的分布。
载体介导的摄取是TUDCA诱导α-PKC转位至肝细胞膜的前提条件。
