GUDC inhibits cytochrome c release from human cholangiocyte mitochondria

Although ursodeoxycholic acid (UDC) is considered effective treatment for p rimary biliary cirrhosis (PBC), its mechanism of action is unclear. We test ed the hypothesis that UDC is taken up by cholangiocytes and inhibits caspa se 3-dependent apoptosis. We used the human cholangiocyte cell line (H69) a nd assessed it for expression and function of an apical sodium-dependent bi le acid transporter (ASBT) by RT-PCR and uptake of tritiated taurocholic ac id. We experimentally induced apoptosis in H69 cells using beauvericin (BV) and determined caspase 3 activation using a fluorogenic substrate and mito chondrial 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 induce d apoptosis in 39.4 +/- 1.3% of cells at 2 h (0.23 +/- 0.2% in controls). I n 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 cholangi ocytes pretreated with GUDC for 24 h and those treated with BV for 2 h, cas pase 3-like activity was reduced by 79% and mitochondrial CC release was in hibited. In summary, the human cholangiocyte cell line H69 possesses a func tional bile acid transporter, and GUDC decreases BV-induced apoptosis and i nhibits activity of caspase 3 protease by blocking CC release from mitochon dria, 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. (C) 1999 Academic Press.