(St. Louis, MO), unless otherwise indicated. BAPTA/AM (1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester; intracellular Ca2+ chelator)4 and N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7; a calmodulin antagonist that binds to calmodulin and inhibits Ca2+/calmodulin-regulated enzyme activities, such as CaMK protein kinase)4 were purchased from Calbiochem Biotechnology (San Diego, CA). Primers for real-time polymerase chain reaction (PCR) were purchased from SABiosciences (Valencia, CA). The RNeasy Mini Kit (to purify total RNA) was purchased from Qiagen Inc. (Valencia, CA). The radioimmunoassay (RIA) kits, for the measurement of cAMP (cAMP [125I]

Biotrak Assay System, RPA509) and IP3 (IP3 [3H] Biotrak Assay System, TRK1000) levels, were purchased from GE Healthcare (Piscataway, NJ). Antibodies (Abs) were purchased from Santa Cruz Biotechnology (Santa Cruz, PF-6463922 CA), unless otherwise indicated. The CFTR monoclonal Ab (immunoglobulin G1) was purchased from Thermo Fisher Scientific (Fremont, CA). The anti learn more Cl−/HCO3− AE2 Ab was obtained from Alpha Diagnostic International (San Antonio, TX). Male C57/BI6N mice (20-25 g) were purchased from Charles River Laboratories (Wilmington, MA), kept

in a temperature-controlled environment with 12-hour light-dark cycles and free access to water and standard chow. Studies were performed in normal mice, and mice that, immediately after BDL,3 were treated with daily intraperitoneal (IP) injections of (1) 0.9% saline (vehicle) or (2) GABA (50 mg/kg body weight; b.w.)15 in the absence or presence of BAPTA/AM (6 mg/kg b.w.)16 or W7 (50 μmol/kg b.w.)17 for 7 days. Animal surgeries and anesthesia (50 mg/kg b.w., IP) were performed in accord with protocols approved by the Scott & White and Texas A&M HSC Institutional Animal Care and Use

Committee (Temple, TX). In vitro studies were performed in immortalized small and large cholangiocyte lines, which display morphological and functional characteristic similar to that of find more freshly isolated small and large cholangiocytes.4, 18 GABA receptor expression (GABAA, GABAB, and GABAC) was evaluated by immunohistochemistry (IHC) in liver sections (4-5 μm thick). After IHC, sections were analyzed by two board-certified researchers in a blinded fashion using a BX-51 light microscope (Olympus, Tokyo, Japan) with a video camera (Spot Insight; Diagnostic Instrument, Inc., Sterling Heights, MI) and evaluated with an Image Analysis System (IAS 2000; Delta Sistemi, Rome, Italy). Expression of GABA receptors was evaluated in small and large cholangiocytes by real-time PCR and immunofluorescence (IF).19 The primers (from SABiosciences) used are described in the Supporting Materials. A delta delta threshold cycle analysis was obtained using small cholangiocytes as control samples.