GUDC inhibits cytochrome c release from human cholangiocyte mitochondria.
Although ursodeoxycholic acid (UDC) is considered effective treatment for primary biliary cirrhosis (PBC), its mechanism of action is unclear. We tested the hypothesis that UDC is taken up by cholangiocytes and inhibits caspase 3-dependent apoptosis. We used the human cholangiocyte cell line (H69) and assessed it for expression and function of an apical sodium-dependent bile acid transporter (ASBT) by RT-PCR and uptake of tritiated taurocholic acid. We experimentally induced apoptosis in H69 cells using beauvericin (BV) and determined caspase 3 activation using a fluorogenic substrate and mitochondrial cytochrome c release (CC) into the cytosol by immunoblot analysis. We found that a functional ASBT is expressed by H69 cells as demonstrated by RT-PCR and bile acid uptake studies. Exposure of H69 cells to BV induced apoptosis in 39.4 +/- 1.3% of cells at 2 h (0.23 +/- 0.2% in controls). In contrast, when H69 cells were preincubated with GUDC (50 mM) for 24 h and then exposed to BV, apoptosis was inhibited by 23% (P < 0.03). In cholangiocytes pretreated with GUDC for 24 h and those treated with BV for 2 h, caspase 3-like activity was reduced by 79% and mitochondrial CC release was inhibited. In summary, the human cholangiocyte cell line H69 possesses a functional bile acid transporter, and GUDC decreases BV-induced apoptosis and inhibits activity of caspase 3 protease by blocking CC release from mitochondria. These preliminary results are consistent with our hypothesis that the beneficial effect of UDC on PBC may involve decreased apoptosis after GUDC uptake by cholangiocytes.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis; D001652:Bile Ducts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003574:Cytochrome c Group / Q000378:metabolism*; D047630:Depsipeptides; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases; D008562:Membrane Glycoproteins; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010455:Peptides; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013656:Taurocholic Acid / Q000378:metabolism; D014580:Ursodeoxycholic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)