4 We evaluated, by IF, whether small control vector- or CaMK I short hairpin RNA (shRNA)-transfected cholangiocytes express GABA receptors. Then, we performed studies to demonstrate that find protocol (1) GABA increases IP3 levels, mRNA, and/or protein expression for CaMK I and AC8 in small cholangiocytes4 and (2) silencing of CaMK I in small cholangiocytes prevents GABA-induced differentiation of small into large cholangiocytes and AC8 activation. The primers (from SABiosciences) used are described in the Supporting Materials. Knockdown

(∼70%)4 of the CaMK I gene in small cholangiocytes was established by a SureSilencing shRNA (SABiosciences) plasmid for mouse CaMK I, containing the gene for neomycin (geneticin) resistance for selection of transfected cells.4 Control or

CaMK I shRNA-transfected small cholangiocytes were incubated at 37°C with GABA (1 μM) for 3 days before evaluating (1) expression of GABA receptors by IF, (2) PCNA protein expression by immunoblottings, (3) expression of SR, CFTR, and Cl−/HCO3− AE2 by IF in a coded fashion, and (4) basal and secretin-stimulated cAMP levels by RIA.3, 22 Because AC8 regulates the function of large cholangiocytes,9 we proposed to demonstrated that IP3/Ca2+/CaMK buy Ku-0059436 I-dependent, GABA-induced differentiation of small into large cholangiocytes are dependent on the presence or activation of AC8. Thus, we studied: (1) biliary expression of AC8 (by IHC) in liver sections and small cholangiocytes from BDL mice treated with saline

or GABA for 1 week and (2) message expression of AC8 by real-time PCR4 in control vector- or CaMK I shRNA-transfected small cholangiocytes treated with 0.2% BSA GBA3 or GABA (1 μM) for 3 days. We studied the effect of in vitro GABA treatment (1 μM, 3 days) in the absence or presence of preincubation (2 hours) with the AC8 inhibitor, 2′-deoxyadenosine 3′-monophosphate (10 mM),23 on the differentiation of small into large cholangiocytes by measuring the semiquantitative expression of SR, CFTR, and Cl−/HCO3− AE2 by IF. The primers used are shown in the Supporting Materials. Data are expressed as mean ± SEM. Differences between groups were analyzed by the Student unpaired t test when two groups were analyzed and by analysis of variance when more than two groups were analyzed, followed by an appropriate post-hoc test. Mann-Whitney’s U test was used to determine ultrastructural differences between cells treated with BSA or GABA. For SEM, statistical analyses were performed using SPSS statistical software (SPSS, Inc., Chicago, IL). Both small (yellow arrows) and large (red arrows) bile ducts from normal (not shown) and BDL (treated with vehicle or GABA) mice express GABAA, GABAB, and GABAC receptors (Fig. 1A). By real-time PCR and IF (Fig.