The aim of this study was to determine whether taurocholate prevents vagotomy-induced cholangiocyte apoptosis. After bile duct ligation (BDL) ϩ vagotomy, rats were fed taurocholate for 1 wk in the absence or presence of wortmannin. Caspase involvement was evaluated by measurement of caspase 8, 9, and 3 activities. Proliferation was determined by morphometry and PCNA immunoblots. Changes in phosphatidylinositol 3-kinase (PI3-kinase) activity were estimated by the expression of the phosphorylated

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... Akt protein. Apically located Na ϩdependent bile acid transporter (ABAT) expression and activity were evaluated by immunoblots and [ 3 H]taurocholate uptake, respectively. Cholangiocyte apoptosis increased, whereas proliferation decreased in BDL ϩ vagotomy rats. Taurocholate feeding prevented vagotomy effects on cholangiocyte functions, which were abolished by wortmannin. ABAT expression and activity as well as phosphorylated Akt protein expression were reduced by vagotomy but restored by taurocholate. The activities of caspase 8, 9, and 3 increased in BDL ϩ vagotomy rats but were restored by taurocholate. The protective effect of taurocholate was associated with maintenance of ABAT activity, downregulation of caspase 8, 9, and 3, and activation of PI3-kinase. Bile acids are important in modulating cholangiocyte proliferation in denervated livers. apoptosis; bile flow; intrahepatic biliary epithelium; proliferation; secretin CHOLANGIOCYTES ARE THE TARGET cells in several human cholestatic liver diseases (i.e.G837 example, the hydrophobic bile acid taurochenodeoxycholate activates PI3-kinase and inhibits caspase 8 activity in hepatocytes (45, 49), which prevent its inherent toxicity (45). cAMP protects cultured rat hepatocytes from apoptosis in a PI3-kinase-dependent manner (52, 53). In previous studies (2-4, 7), we have shown that taurocholate increases both basal and secretin-stimulated intracellular cAMP levels in cholangiocytes, both in vitro and in vivo, when given to rats by a dietary regimen containing this bile acid. We posed the following questions: first, does taurocholate feeding prevent vagotomy-induced apoptosis of cholangiocytes, vagotomy inhibition of cholangiocyte proliferation, and secretin-stimulated ductal secretion? Second, are taurocholate effects on vagotomy-induced bile duct damage mediated by the PI3-kinase/Akt pathway? And third, are changes in cholangiocyte apoptosis, proliferation, and secretion (following vagotomy and taurocholate feeding) dependent on ABAT activity? MATERIALS AND METHODS Materials. Reagents were purchased from Sigma (St. Louis, MO) unless otherwise indicated. Rat chow, containing 1% taurocholate or control diet (AIN-76), was prepared from Dyets (Bethlehem, PA). Control chow (AIN-76) has the same composition of the chow containing 1% taurocholate, but it does not contain taurocholate. The substrate for ␥-glutamyltranspeptidase (␥-GT), N-(␥-L-glutamyl)-4-methoxy-2-naphthylamide, was purchased from Polysciences (Warrington, PA). The PKA inhibitor, , and the caspase 8 inhibitor, Z-IETD-fmk (44), were purchased from Calbiochem (San Diego, CA). RIA kits for the determination of intracellular cAMP levels were purchased from Amersham (Arlington Heights, IL). [ 3 H]taurocholate (3.47 Ci/mmol) was purchased from New England Nuclear (Boston, MA). The antibodies vs. total and phosphorylated Akt (Ser 473 ) were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). The antibody vs. ABAT (rabbit anti-rat ABAT) was a gift from Dr. P. Dawson (Bowman Gray School of Medicine, Winston-Salem, NC). Animal models. Male Fischer 344 rats (150-175 g) were purchased from Charles River (Wilmington, MA), kept in a temperature-controlled environment (20-22°C) with a 12: 12-h light-dark cycle, and fed ad libitum with the selected diet. The majority of the studies were performed in the following four groups of animals: 1) rats that, immediately after BDL (for cell isolation) (1, 3, 6-8, 10, 17, 24, 28, 29, 32) or bile duct incannulation (BDI; for bile collection) (8), were fed control diet or 1% taurocholate for 1 wk; and 2) rats that (immediately after BDL or BDI) underwent vagotomy and, subsequently, were fed control diet or 1% taurocholate diet for 1 wk. To demonstrate the link between cholangiocyte apoptosis and the PI3-kinase-signaling pathway, rats, immediately after BDL ϩ vagotomy, were fed taurocholate and subsequently treated with wortmannin, a specific PI3-kinase inhibitor (35) (1 daily intraperitoneal injection of 0.7 mg kg/ body wt) (38) in DMSO for 1 wk. To demonstrate the link between cholangiocyte apoptosis and the ERK-signaling pathway, rats, immediately after BDL ϩ vagotomy, were fed taurocholate and subsequently treated with 1,4-diamino-2,3dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U-0126; an MEK inhibitor, 1 daily intraperitoneal injection of 5 mg G838 BILE ACID AND NERVE INTERACTION