Blood samples (≈30-40 μL) were collected at 2, 5, 11, 21, 31, and 41 minutes after BA administration into heparinized tubes. Total tissue RNA was extracted using RNA-Bee reagent (Tel-Test, Inc., Friendswood, TX) according to the manufacturer’s protocol. Each RNA pellet was redissolved in 0.2 mL of diethyl pyrocarbonate-treated water. RNA concentrations were quantified by way of ultraviolet absorbance Autophagy activator at 260 nm. RNA integrity was confirmed by way of agarose gel electrophoresis

of 5 μg of total RNA and visualization of the intact 18S and 28S bands by way of ethidium bromide staining. The messenger RNA (mRNA) expression of genes in liver and ileum samples was determined using Quantigene Plex 2.0 (Panomics/Affymetix, check details Inc., Fremont, CA). Individual bead-based oligonucleotide probe sets, specific for each gene examined, were developed by Panomics/Affymetix, Inc. Genes and accession numbers are freely available at http://www.panomics. com (sets #21021 and #21151). Samples were analyzed using a Bio-Plex 200 System Array reader with Luminex 100 xMAP, and data were acquired using Bio-Plex Data Manager version 5.0 (Bio-Rad, Hercules, CA). Assays were performed according to each manufacturers’ protocol. All data were standardized

to the internal control glyceraldehyde 3-phosphate dehydrogenase. The mRNA of farnesoid X receptor (FXR) and small heterodimer partner (SHP) was quantified with MCE公司 QuantiGene 1.0 (Panomics) as described.8 The probe set for SHP has also been described.9 Probe sets for FXR (Supporting Information Table 1) were designed using ProbeDesigner 1.0 (Bayer Corp., Emeryville,

CA) and synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Internal standards, as well as bile, plasma, and liver samples, were prepared for bile-acid speciation as described by Alnouti et al.10 with modifications.11 Briefly, plasma samples were deproteinized with ice-cold acetonitrile containing internal standards (d4-G-CDCA, d4-CDCA). The supernatants were removed, dried under vacuum, and reconstituted in 50% methanol. For extraction of bile acids from liver, 100-110 mg of livers were homogenized in 500 μL water, and an additional 1 volume of 50% methanol. The liver homogenates (600 μL) were transferred to a new tube and 10 μL of internal standard, and 3 mL of ice-cold acetonitrile was added. The mixtures were shaken vigorously for 1 hour and centrifuged at 11,000g for 10 minutes. The supernatants were transferred to a glass tube. The pellets were re-extracted with another 1 mL of methanol. Resultant supernatants from two extractions were combined, evaporated under vacuum for 3 hours at 50°C, and reconstituted in 100 μL of 50% methanol.

” It would be nice to have something to tell them. “
“Objective.— To demonstrate that occipital nerve injury is associated with

chronic postoperative headache in patients who have undergone acoustic neuroma excision and to determine whether occipital nerve excision is an effective treatment for these headaches. Background.— Few previous reports have discussed the role of occipital nerve injury in the pathogenesis of the postoperative headache noted to commonly occur following the retrosigmoid approach to acoustic neuroma resection. No studies have supported a direct etiologic link between the two. The authors report on a series of acoustic neuroma patients with postoperative headache presenting as occipital neuralgia who were found to have occipital nerve injuries and were treated see more for chronic headache by excision of the injured nerves. Methods.— Records were reviewed to identify patients who had undergone surgical excision of the greater and lesser occipital nerves for refractory chronic postoperative headache APO866 research buy following acoustic neuroma resection. Primary outcomes examined were change in migraine headache index, change in number of pain medications used, continued use of narcotics, patient satisfaction,

and change in quality of life. Follow-up was in clinic and via telephone interview. Results.— Seven patients underwent excision of the greater and lesser occipital nerves. All met diagnostic criteria for occipital neuralgia and failed conservative management. Six of 7 patients experienced pain reduction of greater than 80% on the migraine index. Average pain medication use decreased from 6 to 2 上海皓元 per patient; 3 of 5 patients achieved independence

from narcotics. Six patients experienced 80% or greater improvement in quality of life at an average follow-up of 32 months. There was one treatment failure. Occipital nerve neuroma or nerve entrapment was identified during surgery in all cases where treatment was successful but not in the treatment failure. Conclusion.— In contradistinction to previous reports, we have identified a subset of patients in whom the syndrome of postoperative headache appears directly related to the presence of occipital nerve injuries. In patients with postoperative headache meeting diagnostic criteria for occipital neuralgia, occipital nerve excision appears to provide relief of the headache syndrome and meaningful improvement in quality of life. Further studies are needed to confirm these results and to determine whether occipital nerve injury may present as headache types other than occipital neuralgia. These findings suggest that patients presenting with chronic postoperative headache should be screened for the presence of surgically treatable occipital nerve injuries. “
“Objective.

Furthermore,

platelet inhibition may block the release of important growth factors, such as FGF, HGF, ILF, VEGF, PDGF, and serotonin, that play a role in www.selleckchem.com/products/abc294640.html HCC development and growth. Unfortunately, Sahasrabuddhe et al.’s epidemiological study did not provide data on the stratification of the protective effect according to the causes of CLD and HCC, hence lacking confirmation that the aspirin effect is selective for HBV-related liver disease. However, an antiviral activity of aspirin against HCV or other flaviruses has already been suggested, by way of COX-2 inhibition,[3] and by way of the induction of Cu/Zn-SOD expression as well as direct antioxidant properties.[4] COX-independent, platelet independent and antioxidant-independent protective effects of aspirin against liver injury have also been reported. Imaeda et al.,[5] using an acetaminophen-induced acute liver injury model, showed that low-dose aspirin inhibits inflammasome-mediated pathways, thereby reducing the transcription of inflammatory cytokines. Chemoprevention of cancer with aspirin is not a novel concept. It has been investigated in the setting of colorectal cancer through multiple cohort and case control studies, demonstrating benefit; however

two large randomized TAM Receptor inhibitor controlled trials (RCTs)[6, 7] that included more than 22,000 and 39,000 patients, respectively, did not show significant benefit in reducing colorectal cancer incidence. Thus, a final judgment on the effect of aspirin on colorectal cancer prevention is still pending.[8] Similarly, the evidence presented in Sahasrabuddhe et al.’s[2] study is not robust enough to recommend the use of aspirin in the prevention of HCC. The strengths of the study included the statistical power from the large cohort with many events, the ability to separate

aspirin from nonaspirin NSAIDs, and the robustness of results to sensitivity analyses addressing protopathic bias and confounding by indication. Despite the striking results from this study as well as other studies providing support for biological plausibility, caution must be taken in their interpretation. There are several well-documented examples of the inability to reproduce associations from observational studies into clinical trial settings, including 上海皓元医药股份有限公司 the Women’s Health Study[7] which was unable to detect a benefit of low-dose aspirin on cancer. In other words, observing that aspirin users are less likely to develop HCC than nonusers does not necessarily mean that giving aspirin to patients will reduce their likelihood of HCC. Several factors may underlie this discrepancy, including variability in study populations, dose and duration of intervention, and differential measurement. However, a fundamental challenge for observational studies is the opportunity for selection bias.

This suggests that transport of D4TCA is a limiting factor at both the peroxisomal membrane and the plasma membrane.

Only small amounts of D4TCA were detected in peroxisomes. This may be expected, as D4TCA only transiently resides in peroxisomes. Moreover, D4TCA may disappear from the peroxisomal fraction during their isolation PF-01367338 price due to (1) mechanical rupture of the peroxisomal membrane, and (2) maintained export of D4TCA without new production in peroxisomes. However, at present we are not able to discriminate between tauro/glyco-CA formed in the peroxisomes followed by transport to the cytosol and tauro/glycol-CA that is formed in the cytosol directly. This requires manipulation of the peroxisomal bile salt shuttle, either by inhibiting the to-be-identified-peroxisomal bile salt transporters or manipulating peroxisome biogenesis. It is relevant to note

that this is the first report that demonstrates the presence of a specific product of peroxisomal metabolism in the peroxisome-enriched fractions after a full cell fractionation procedure. Mechanical breakage of peroxisomes was kept to a minimum by using optimized protocols that stabilize these organelles,13 which also further reconfirmed the predominant peroxisomal location of BAAT because it remained (almost) undetectable in the cytosol-enriched fractions after Nycodenz gradient centrifugation. Remarkably, significant amounts of BAAT, catalase, and PMP70 were also detected in low density gradient fractions cofractionating, in part, with mitochondria. In these fractions also D4TCA click here was detected. It remains to be determined whether these fractions contain a subpopulation of peroxisomes that may be involved in the bile salt conjugation as well. To obtain independent evidence for the peroxisomal shuttle we also analyzed the subcellular distribution of several variants of 4-nitrobenzo-2-oxa-1,3-diazole

(NBD)-labeled cholic acid (with the NBD group at the 3alpha, 3beta, 7alpha, and 7beta position, respectively)27 by fluorescence microscopy. Only 3alpha-NBD-cholate was taurine-conjugated and exported to the medium by cultured rat hepatocytes. However, the medchemexpress efficiency of conjugation is much lower (>90%) compared to D4CA. Interestingly, a clear accumulation of 3alpha-NBD-CA in subcellular structures was detected at early timepoints (see Supporting Fig. S2). Unfortunately, due to technical limitations we were unable so far to identify these subcellular structures (see Supporting data for details). Still, the detection of a clear punctuate staining pattern for 3alpha NBD-cholate in hepatocytes supports our data that bile salts (transiently) accumulate in membrane enclosed organelles. Remarkably, we did not detect D4GCA in the peroxisomal fractions. Still, BAAT is believed to be responsible for both taurine and glycine-conjugation of bile salts.3 This may indicate that the peroxisomal bile salt exporter in rat hepatocytes has a higher affinity for GCA compared to TCA.

This suggests that transport of D4TCA is a limiting factor at both the peroxisomal membrane and the plasma membrane.

Only small amounts of D4TCA were detected in peroxisomes. This may be expected, as D4TCA only transiently resides in peroxisomes. Moreover, D4TCA may disappear from the peroxisomal fraction during their isolation Y-27632 solubility dmso due to (1) mechanical rupture of the peroxisomal membrane, and (2) maintained export of D4TCA without new production in peroxisomes. However, at present we are not able to discriminate between tauro/glyco-CA formed in the peroxisomes followed by transport to the cytosol and tauro/glycol-CA that is formed in the cytosol directly. This requires manipulation of the peroxisomal bile salt shuttle, either by inhibiting the to-be-identified-peroxisomal bile salt transporters or manipulating peroxisome biogenesis. It is relevant to note

that this is the first report that demonstrates the presence of a specific product of peroxisomal metabolism in the peroxisome-enriched fractions after a full cell fractionation procedure. Mechanical breakage of peroxisomes was kept to a minimum by using optimized protocols that stabilize these organelles,13 which also further reconfirmed the predominant peroxisomal location of BAAT because it remained (almost) undetectable in the cytosol-enriched fractions after Nycodenz gradient centrifugation. Remarkably, significant amounts of BAAT, catalase, and PMP70 were also detected in low density gradient fractions cofractionating, in part, with mitochondria. In these fractions also D4TCA Sorafenib cost was detected. It remains to be determined whether these fractions contain a subpopulation of peroxisomes that may be involved in the bile salt conjugation as well. To obtain independent evidence for the peroxisomal shuttle we also analyzed the subcellular distribution of several variants of 4-nitrobenzo-2-oxa-1,3-diazole

(NBD)-labeled cholic acid (with the NBD group at the 3alpha, 3beta, 7alpha, and 7beta position, respectively)27 by fluorescence microscopy. Only 3alpha-NBD-cholate was taurine-conjugated and exported to the medium by cultured rat hepatocytes. However, the MCE公司 efficiency of conjugation is much lower (>90%) compared to D4CA. Interestingly, a clear accumulation of 3alpha-NBD-CA in subcellular structures was detected at early timepoints (see Supporting Fig. S2). Unfortunately, due to technical limitations we were unable so far to identify these subcellular structures (see Supporting data for details). Still, the detection of a clear punctuate staining pattern for 3alpha NBD-cholate in hepatocytes supports our data that bile salts (transiently) accumulate in membrane enclosed organelles. Remarkably, we did not detect D4GCA in the peroxisomal fractions. Still, BAAT is believed to be responsible for both taurine and glycine-conjugation of bile salts.3 This may indicate that the peroxisomal bile salt exporter in rat hepatocytes has a higher affinity for GCA compared to TCA.

Previously, it has been shown that toll-like receptor (TLR) signalling is impaired by HBsAg selleck compound leading to an attenuation of innate and adaptive immune responses which may possibly facilitate chronicity of infection. Here, we aimed to analyze immune activation in HBV transgenic mice lacking the HBs antigen (1.4 tgHBV-s-mut). Methods: Liver tissue of 1.4 tgHBV-s-mut mice, HBV negative littermates, TLR3-/- and TLR3-/-/1.4 tgHBV-s-mut animals was investigated. HBxAg-targeting siRNAs or ligand for TLR3 (poly I:C) were injected intravenously. Quantitative RT-PCR, Southern blot, ELISA and western blot were performed to detect levels of immune genes,

HBV DNA, HBeAg or HBcAg. Results: Hepadnaviral replication-dependent expression of interferon beta (IFN-p), interferon sensitive Selleck Napabucasin gene 15 (ISG15), interferon-induced protein with tetratricopeptide repeats 1 (IFI-T1) was observed in 1.4 tgHBV-s-mut mice. This immune response could be normalized by treatment with HBx-Ag-targeting siRNAs and was completely abrogated in TLR3-/-/1.4 tgHBV-s-mut animals. Suppression of these TLR3-mediated immune responses led to increased HBV replication. Non-pa-renchymal liver cells were identified as source of these antiviral

responses. Further application of TLR3 ligand poly I:C significantly induced the expression of ISG15, IFI-T1, IFN-p and suppressed HBV replication in vivo and in vitro. However, the fold induction of these immune genes was significantly lowered in 1.4 tgHBV-s-mut mice compared to control animals, reflecting a HBs-independent immune evasion. Conclusions: In contrast to data from HBV-infected patients, hepatic TLR signalling was not totally

abrogated MCE公司 in HBV transgenic mice that lack HBsAg. In 1.4 tgHBV-s-mut mice viral replication induced TLR3-mediated antiviral responses in non-parenchymal liver cells. We therefore hypothesize that HBsAg is a major component of HBV that attenuates TLR signaling thus leading to impairment of innate and adaptive immune responses in the liver. Disclosures: Hans-Peter Vornlocher – Management Position: Axolabs GmbH The following people have nothing to disclose: Catherine I. Real, Mengji Lu, Markus Hossbach, Kerstin Jahn-Hofmann, Ludger M. Ickenstein, Matthias J. John, Reinhold Schirmbeck, Melanie Lutterbeck, Kathrin Gibbert, Ulf Dittmer, Guido Gerken, Joerg F. Schlaak, Ruth Broering Background and Aim: Natural Killer (NK) cells play important roles in innate immune response in viral infection. The activation of NK cells are controlled by various activating and inhibitory NK cell receptors and many NK cell receptors have been identified. In this study, we focused on NKp46, an activating receptor, and NKG2A, an inhibitory receptor, and investigated the frequencies and function of NKp46 and NKG2A expressing NK cells in chronic hepatitis B (CHB) patients. Methods: Sixty six CHB patients and 32 healthy subjects (HS) were enrolled in this study.

Previously, it has been shown that toll-like receptor (TLR) signalling is impaired by HBsAg Belnacasan cost leading to an attenuation of innate and adaptive immune responses which may possibly facilitate chronicity of infection. Here, we aimed to analyze immune activation in HBV transgenic mice lacking the HBs antigen (1.4 tgHBV-s-mut). Methods: Liver tissue of 1.4 tgHBV-s-mut mice, HBV negative littermates, TLR3-/- and TLR3-/-/1.4 tgHBV-s-mut animals was investigated. HBxAg-targeting siRNAs or ligand for TLR3 (poly I:C) were injected intravenously. Quantitative RT-PCR, Southern blot, ELISA and western blot were performed to detect levels of immune genes,

HBV DNA, HBeAg or HBcAg. Results: Hepadnaviral replication-dependent expression of interferon beta (IFN-p), interferon sensitive selleck chemical gene 15 (ISG15), interferon-induced protein with tetratricopeptide repeats 1 (IFI-T1) was observed in 1.4 tgHBV-s-mut mice. This immune response could be normalized by treatment with HBx-Ag-targeting siRNAs and was completely abrogated in TLR3-/-/1.4 tgHBV-s-mut animals. Suppression of these TLR3-mediated immune responses led to increased HBV replication. Non-pa-renchymal liver cells were identified as source of these antiviral

responses. Further application of TLR3 ligand poly I:C significantly induced the expression of ISG15, IFI-T1, IFN-p and suppressed HBV replication in vivo and in vitro. However, the fold induction of these immune genes was significantly lowered in 1.4 tgHBV-s-mut mice compared to control animals, reflecting a HBs-independent immune evasion. Conclusions: In contrast to data from HBV-infected patients, hepatic TLR signalling was not totally

abrogated 上海皓元 in HBV transgenic mice that lack HBsAg. In 1.4 tgHBV-s-mut mice viral replication induced TLR3-mediated antiviral responses in non-parenchymal liver cells. We therefore hypothesize that HBsAg is a major component of HBV that attenuates TLR signaling thus leading to impairment of innate and adaptive immune responses in the liver. Disclosures: Hans-Peter Vornlocher – Management Position: Axolabs GmbH The following people have nothing to disclose: Catherine I. Real, Mengji Lu, Markus Hossbach, Kerstin Jahn-Hofmann, Ludger M. Ickenstein, Matthias J. John, Reinhold Schirmbeck, Melanie Lutterbeck, Kathrin Gibbert, Ulf Dittmer, Guido Gerken, Joerg F. Schlaak, Ruth Broering Background and Aim: Natural Killer (NK) cells play important roles in innate immune response in viral infection. The activation of NK cells are controlled by various activating and inhibitory NK cell receptors and many NK cell receptors have been identified. In this study, we focused on NKp46, an activating receptor, and NKG2A, an inhibitory receptor, and investigated the frequencies and function of NKp46 and NKG2A expressing NK cells in chronic hepatitis B (CHB) patients. Methods: Sixty six CHB patients and 32 healthy subjects (HS) were enrolled in this study.

To elucidate the mechanisms leading from obesity and steatosis to HCC, Park et al. in their recent article, applied the well-established DEN model for selleck inhibitor tumor induction in wild-type mice.9 They first demonstrated that mice kept on a HFD exhibited greatly enhanced HCC development compared to nonobese mice when treated with DEN. In line with these findings, Park et al. described that also leptin-deficient obese mice display greatly enhanced HCC development relative to wild-type mice after administration

of DEN. DEN-related tumor induction was previously linked to enhanced hepatocyte death and thereby compensatory hepatocyte proliferation.10 Conversely, Park et al. describe reduced apoptosis and enhanced cell proliferation in HCC of obese mice as compared to HCC of mice placed on a low-fat diet. In line with these findings, transplantation of hepatoma

cells into lean mice that were placed on low-fat diet/HFD after inoculation of the cells revealed that the degree of host obesity determined tumor growth. This suggests that alterations in signal transduction pathways that modulate tumor cell proliferation independently of liver damage and compensatory proliferation may underlie the tumor-promoting effect of obesity. Indeed, Park et al. describe elevated c-Jun N-terminal kinase (JNK) activity and increased phosphorylation of the mammalian target of rapamycin (mTOR) target S6 kinase and its substrate ribosomal protein S6 in obese mice. Furthermore, HCCs in obese mice exhibited greatly elevated activity of both pro-oncogenic and inflammatory pathways such as extracellular

signal-regulated kinase (ERK) and signal transducer PR-171 supplier and activator of transcription 3 (STAT3). Obesity also enhanced interleukin-6 (IL-6) messenger RNA and tumor necrosis factor (TNF) 上海皓元 and IL-1b expression in both nontumor liver and HCC. As a corroboration of these data, growth of transplanted hepatoma cells can be slowed by administration of a JAK (Janus kinase) inhibitor that prevents STAT3 activation. Enhanced activity of STAT3, a major transcriptional target for IL-6, was previously linked to development of HCC in humans. Furthermore, He et al. demonstrated that IL-6 is required for HCC development and that circulating IL-6 is elevated in cirrhosis and HCC.11 In their study,9 Park et al. used IL-6−/− mice to elucidate whether IL-6 is an important component of tumor promotion in the context of obesity. As previously described, deletion of IL-6 protected mice from DEN-induced HCC development when the mice were kept on a low-fat diet. Furthermore, no increase in tumor formation and growth was observed when these mice were in kept on a HFD. Interestingly tumor load in male IL-6-/- mice was similar to that of wild-type female mice, but unlike wild-type females, which develop more HCC when rendered obese, no significant increase in tumor load was described in obese IL-6-/- males.

13, 37 In HCV-infected patients, increased hepatic SHP, MTP, NTCP, and CYP7A1 mRNA was observed, and FXR, G6P, and PEPCK mRNA levels did not change. This finding suggests that the FXR-SHP-CYP7A1 regulatory loop is totally compromised in HCV-infected Selleckchem R428 liver. The observed changes could be due to HCV infection. Alternatively, such changes could be adaptive host responses

in order to minimize liver injury. MTP is essential for hepatic lipoprotein assembly and secretion, and VLDL is important for HCV secretion from the infected cells.23 In addition, bile acid via FXR promotes genotype 1 HCV replication.38, 39 Thus, all these alterations are related to HCV life cycle. Activation of CAR ameliorates hyperglycemia by suppressing glucose production and stimulating glucose uptake and usage in the liver and improves steatosis by inhibiting hepatic lipogenesis and inducing β-oxidation.40 In our hepatitis C patients, CAR was significantly up-regulated and this was accompanied by decreased SREBP-1c and increased GLUT2 expression. This finding suggests that CAR may play a significant role in lipid and glucose metabolism in HCV-infected livers. In ethanol-fed mice, hepatic PPARα-mediated signaling Y-27632 molecular weight is decreased.41–43 In addition, AMPK activity and fatty acid synthesis-related genes are down-regulated.44 In the HCV-infected patients who had a history of drinking, our results showed that PPARα and RXRα expression levels were increased, with concomitant up-regulation

of their target genes involved in fatty acid oxidation and hepatic uptake and intracellular trafficking. Species difference may account for the differential findings. There were both current and noncurrent drinkers in group B, but no significant difference could be found in gene expression between the two groups (Supporting Table 2). This suggests the possibility of active drinking in “noncurrent drinkers”. In addition, the gene expression alteration does not seem to be caused 上海皓元医药股份有限公司 by differences in disease severity because there was no difference in liver panel, severity of fibrosis, or inflammation in these two cohorts (Supporting Table 3). Although PPARα and RXRα and their target genes were up-regulated in patients

with a history of alcohol drinking, the genes involved in antioxidant and inflammatory pathways did not change their expression level significantly (Supporting Fig. 2B). This result does not support the hypothesis that alcohol and HCV synergize through increasing PPARα activity, lipid peroxidation, oxidative stress, and thus liver injury. Other mechanisms have been proposed to explain the synergism of HCV infection and alcohol intake. For example, alcohol impairs the intracellular innate immune response in human hepatocytes and promotes HCV infection and replication.45 Multivariate analysis showed an independent association between the hepatic mRNA levels of FAS, FGF21, and IL-10 with HCV RNA. All these genes are regulated by nuclear receptors or coregulators.

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 Abiraterone purchase (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 Selleckchem STI571 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 MCE 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.