BDL, bile duct ligation; BSA, bovine serum albumin; cAMP, cyclic adenosine monophosphate; CCl4, carbon tetrachloride; ERK1/2, extracellular signal-regulated Cilomilast mw kinase; FACS, fluorescence-activated cell sorting; IBDM, intrahepatic bile duct mass; MEK, mitogen-activated protein kinase kinase;

PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reation; PKA, protein kinase A; SEM, standard error of the mean; SR, secretin receptor; WT, wild-type. Reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. The nuclear dye 4′,6-diamidino-2-phenylindole was obtained from Molecular Probes, Inc. (Eugene, OR). Porcine secretin was purchased from Peninsula Laboratories (Belmont, CA). The polyclonal SR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was raised against a peptide mapping at the C terminus of SR of human origin and cross-reacts with mouse.20 The antibody

against proliferating cell nuclear antigen (PCNA) was purchased from Santa BMS-907351 supplier Cruz Biotechnology. The mouse anti–cytokeratin-19 antibody was purchased from Caltag Laboratories Inc. (Burlingame, CA). Goat phosphorylated ERK1/2 and total ERK1/2 (44-42 kDa) polyclonal affinity purified antibodies were purchased from Santa Cruz Biotechnology. The RIA kits for the determination of intracellular cAMP levels in cholangiocytes were purchased from Perkin Elmer (Shelton, CT). All animal experiments (Table 1) were performed in accordance with a protocol approved by the Scott & White and Texas A&M Health Science Center Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals published

by the National Institutes of Health (Publication No. 85-23, revised 1996). Our SR+/+ (wild-type [WT]) or SR knockout (SR−/−)21 mice were maintained in a temperature-controlled environment (20-22°C) with a 12:12-hour light/dark cycle. We used adult male WT and SR−/− mice (approximately 25-30 g) of the N5 generation: (1) as normal treated with saline (0.9% NaCl) or secretin 上海皓元医药股份有限公司 (2.5 nmol/kg/day, a dose similar to that used by us for another gastrointestinal hormone, gastrin, in rodents)18 by way of intraperitoneally implanted Alzet osmotic minipumps (Alzet, CA) for 7 days; or (2) for sham operation or BDL (for 3 and 7 days).5, 20, 22 Because our previous studies21 showed that SR−/− mice have a renal defect in water reabsorption and associated polyuria and polydipsia, experiments were performed to determine whether the response of SR−/− mice to BDL was due to the lack of SR rather than severe dehydration. Thus, we evaluated changes in body weight and mortality rate in the experimental groups of Table 1. In addition, both WT and SR−/− mice (after BDL or administration of secretin) received oral hydration therapy, consisting of up to 1 ml of normal saline subcutaneously up to twice daily along with water in gel form on the ground and food supplements.