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The gallbladder stores and concentrates bile between meals. Gallbladder motor function is regulated by bile acids via the membrane bile acid receptor, TGR5, and by neurohormonal signals linked to digestion, for example, cholecystokinin and FGF15/19 intestinal hormones, which trigger gallbladder emptying and refilling, respectively. The cycle of gallbladder filling and emptying controls the flow of bile into the intestine and thereby the enterohepatic circulation of bile acids. The gallbladder also largely contributes to the regulation of bile composition by unique absorptive and secretory capacities. The gallbladder epithelium secretes bicarbonate and mucins, which both provide cytoprotection against bile acids. The reversal of fluid transport from absorption to secretion occurs together with bicarbonate secretion after feeding, predominantly in response to an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent pathway triggered by neurohormonal factors, such as vasoactive intestinal peptide. Mucin secretion in the gallbladder is stimulated predominantly by calcium-dependent pathways that are activated by ATP present in bile, and bile acids. The gallbladder epithelium has the capacity to absorb cholesterol and provides a cholecystohepatic shunt pathway for bile acids. Changes in gallbladder motor function not only can contribute to gallstone disease, but also subserve protective functions in multiple pathological settings through the sequestration of bile acids and changes in the bile acid composition. Cholecystectomy increases the enterohepatic recirculation rates of bile acids leading to metabolic effects and an increased risk of nonalcoholic fatty liver disease, cirrhosis, and small-intestine carcinoid, independently of cholelithiasis. Among subjects with gallstones, cholecystectomy remains a priority in those at risk of gallbladder cancer, while others could benefit from gallbladder-preserving strategies. © 2016 American Physiological Society. Compr Physiol 6:1549-1577, 2016., (Copyright © 2016 John Wiley & Sons, Inc.)

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) anchors and regulates apical membrane proteins in epithelia. EBP50 is inducible by estrogen and may affect cell proliferation, although this latter function remains unclear. The goal of this study was to determine whether EBP50 was implicated in the ductular reaction that occurs in liver disease. EBP50 expression was examined in normal human liver, in human cholangiopathies (ie, cystic fibrosis, primary biliary cirrhosis, and primary sclerosing cholangitis), and in rats subjected to bile-duct ligation. The regulation of EBP50 by estrogens and its impact on proliferation were assessed in both bile duct-ligated rats and Mz-Cha-1 human biliary epithelial cells. Analyses of cell isolates and immunohistochemical studies showed that in normal human liver, EBP50 is expressed in the canalicular membranes of hepatocytes and, together with ezrin and cystic fibrosis transmembrane conductance regulator, in the apical domains of cholangiocytes. In both human cholangiopathies and bile duct-ligated rats, EBP50 was redistributed to the cytoplasmic and nuclear compartments. EBP50 underwent a transient increase in rat cholangiocytes after bile-duct ligation, whereas such expression was down-regulated in ovariectomized rats. In addition, in Mz-Cha-1 cells, EBP50 underwent up-regulation and intracellular redistribution in response to 17beta-estradiol, whereas its proliferation was inhibited by siRNA-mediated EBP50 knockdown. These results indicate that both the expression and distribution of EBP50 are regulated by estrogens and contribute to the proliferative response in biliary epithelial cells.

Despite evidence that mucin overproduction is critical in the pathogenesis of gallstones, the mechanisms triggering mucin production in gallstone disease are unknown. Here, we tested the potential implication of an inflammation-dependent epidermal growth factor receptor (EGF-R) pathway in the regulation of gallbladder mucin synthesis. In gallbladder tissue sections from subjects with cholesterol gallstones, mucus accumulation was associated with neutrophil infiltration and with increased expressions of EGF-R and of tumor necrosis factor-alpha (TNF-alpha). In primary cultures of human gallbladder epithelial cells, TNF-alpha induced EGF-R overexpression. In the presence of TNF-alpha, EGF-R ligands (either EGF or transforming growth factor-alpha) caused significant increases in MUC5AC mRNA and protein production, whereas expression of the other gallbladder mucins MUC1, MUC3, and MUC5B was unchanged. In addition, on gallbladder tissue sections from subjects with gallstones, increased MUC5AC immunoreactivity was detected in the epithelium and within mucus gel in the lumen. Studies in primary cultures demonstrated that MUC5AC up-regulation induced by the combination of TNF-alpha with EGF-R ligands was completely blunted by inhibitors of EGF-R tyrosine kinase and mitogen-activated protein/extracellular signal-related kinase kinase. In conclusion, an inflammation-dependent EGF-R cascade causes overproduction of the gel-forming mucin MUC5AC, which accumulates in cholesterol gallstone disease. The ability to interrupt this cascade is of potential interest in the prevention of cholesterol gallstones.

Vasoactive intestinal peptide receptor-1 (VPAC1) is the high-affinity receptor of vasoactive intestinal peptide (VIP), a major regulator of bile secretion. To better define the level at which VPAC1 stimulates bile secretion, we examined its expression in the different cell types participating in bile formation (i.e., hepatocytes, bile duct, and gallbladder epithelial cells). Because VPAC1 expression was previously shown to be regulated by nuclear receptors, we tested the hypothesis that it may be regulated by the farnesoid X receptor (FXR). Quantitative RT-PCR and immunoblot analyses of cell isolates indicated that VPAC1 is expressed in all three cell types lining the human biliary tree, with predominant expression in the gallbladder. In primary cultures of human gallbladder epithelial cells, VIP induced cAMP production and chloride secretion. Analysis of the VPAC1 gene revealed the presence of potential FXR response element sequences, and both FXR and RXRalpha expressions were detected in gallbladder epithelial cells. In these cells, the FXR pharmacological agonist GW4064 upregulated VPAC1 expression in a dose-dependent manner, and this effect was antagonized by the RXRalpha ligand, 9-cis retinoic acid. Chenodeoxycholate activated endogenous FXR in gallbladder epithelial cells, as ascertained by electromobility shift assay and upregulation of the FXR target gene, small heterodimer partner. Chenodeoxycholate also provoked an increase in VPAC1 mRNA and protein content in these cells. In conclusion, FXR agonists may increase gallbladder fluid secretion through transcriptional activation of VPAC1, which may contribute to the regulation of bile secretion by bile salts and to a protective effect of FXR pharmacological agonists in gallstone disease.

Fluid and ion secretion in the gallbladder is mainly triggered by the intracellular second messenger cAMP. We examined the action of bile salts on the cAMP-dependent pathway in the gallbladder epithelium. Primary cultures of human gallbladder epithelial cells were exposed to agonists of the cAMP pathway and/or to bile salts. Taurochenodeoxycholate and tauroursodeoxycholate increased forskolin-induced cAMP accumulation to a similar extent, without affecting cAMP basal levels. This potentiating effect was abrogated after PKC inhibition, whereas both taurochenodeoxycholate and tauroursodeoxycholate induced PKC-alpha and -delta translocation to cell membranes. Consistent with a PKC-mediated stimulation of cAMP production, the expression of six adenylyl cyclase isoforms, including PKC-regulated isoforms 5 and 7, was identified in human gallbladder epithelial cells. cAMP-dependent chloride secretion induced by isoproterenol, a beta-adrenergic agonist, was significantly increased by taurochenodeoxycholate and by tauroursodeoxycholate. In conclusion, endogenous and therapeutic bile salts via PKC regulation of adenylyl cyclase activity potentiate cAMP production in the human gallbladder epithelium. Through this action, bile salts may increase fluid secretion in the gallbladder after feeding.

Tissue expression of drug-metabolizing enzymes influences susceptibility to drugs and carcinogens. Because the biliary epithelium, exposed to bile-borne chemicals, may give rise to drug-induced cholangiopathies and to cholangiocarcinomas, we determined the pattern of expression of drug-metabolizing enzymes in this epithelium. We first demonstrated by blot analyses that biliary epithelial cells (BEC) isolated from human gallbladders display cytochrome P450 (CYP) 1A, 2E1, and 3A, microsomal epoxide hydrolase (mEH), alpha, mu, and pi glutathione S-transferase (GST), transcripts and proteins. We also identified CYP-associated steroid 6beta-hydroxylase activity in BEC. CYP and mEH expression was 5- to 20-fold lower in BEC than in autologous hepatocytes, and further differed by a higher ratio of CYP3A5/CYP3A4, and by CYP1A1 predominance over CYP1A2. alphaGST was highly expressed in both hepatocytes and BEC, while piGST was restricted to BEC. In approximately 50% of individuals, muGST was expressed in hepatocytes and at lower levels in BEC. By using the same antibodies as those used in immunoblots, we could show by immunohistochemistry that CYP2E1, CYP3A, mEH, alpha, mu, and piGST immunoreactivities are expressed and display a heterogeneous distribution in the epithelium lining the entire biliary tract except for small intrahepatic bile ducts that were devoid of CYP3A and alphaGST immunoreactivities. In conclusion, BEC contribute to phase II, and although to a lesser extent than hepatocytes, to phase I biotransformation. The distribution of drug-metabolizing enzymes in BEC suggest that they are heterogeneous in their ability to generate and detoxicate reactive metabolites, which may contribute to specific distributions of cholangiopathies.

Cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, functions as an adenosine 3', 5'-cyclic monophosphate (cAMP)-regulated chloride channel in the apical membrane of biliary epithelial cells, including gallbladder epithelial cells. It has been shown that triangle upF508, the most common CF mutation, impedes CFTR trafficking to the apical surface of epithelial cells. To elucidate the mechanisms of CF biliary disease, we examined structural features, CFTR expression, and chloride transport properties in gallbladder epithelial cells from nine triangle upF508 homozygous liver transplant recipients. Three CF patients had microgallbladders, characterized by severe histological abnormalities. Microgallbladder epithelial cells displayed aberrant immunolocalization of CFTR and of other normally apical proteins in the lateral domain of their plasma membrane and in their cytoplasm. This pattern was mimicked by chronic cholecystitis in non-CF patients. In the 6 remaining CF patients, CFTR was predominantly apical in the gallbladder epithelium, consistent with the detection of a fully glycosylated form by Western blot. In CF as compared with non-CF gallbladder epithelial cells in primary culture, chloride efflux was lower in response to cAMP and tended to be higher in response to exogenous adenosine 5'-triphosphate (ATP). The CF cells exhibited a residual cAMP-dependent chloride secretion that was inversely correlated with ATP-induced chloride secretion, and almost completely blunted in the cells derived from microgallbladders. Our results suggest that epithelial structural alterations aggravate triangle upF508 CFTR mislocalization in the gallbladder epithelium. The associated decrease in residual cAMP-dependent chloride secretion may contribute to biliary damage despite the up-regulation of alternative chloride transport pathways.

Fluid and ion transport across biliary epithelium contributes to bile flow. Alterations of this function may explain hepatobiliary complications in cystic fibrosis (CF). We investigated electrogenic anion transport across intact non-CF and CF human gallbladder mucosa in Ussing-type chambers. In non-CF tissues, baseline transmural potential difference (PD), short-circuit current (Isc), and resistance (R) were -2.2 +/- 0.3 mV (lumen negative), 40.7 +/- 7.8 microA/cm2, and 66.5 +/- 9.6 Omega. cm2, respectively (n = 14). The addition of forskolin (10(-5) mol/L) to the apical and basolateral baths and that of adenosine 5'-triphosphate (ATP) (10(-4) mol/L) to the apical bath induced significant increases in Isc by 8.0 +/- 1.4 and 10.3 +/- 1.8 microA/cm2, respectively. Depletion of bathing solutions in Cl- and HCO3- significantly reduced baseline Isc and the forskolin- and ATP-induced increases in Isc. Anion secretion was stimulated by extracellular ATP via P2Y2 purinoceptors, as indicated by the effects of different nucleotides on Isc and on 36Cl efflux in cultured gallbladder epithelial cells. This effect was mediated by cytosolic calcium increase and Ca2+/calmodulin-dependent protein kinase II, as ascertained by using inhibitors. In CF preparations, basal PD and Isc were lower than in non-CF, and the response to forskolin was abolished, whereas the response to ATP was enhanced (P <.05 for all). We conclude that electrogenic anion secretion occurs in human gallbladder mucosa under basal state and is stimulated by an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent pathway mediated by cystic fibrosis transmembrane conductance regulator (CFTR), and by exogenous ATP via a CFTR-independent pathway that is up-regulated in CF and involves P2Y2 purinoceptors and a calcium-dependent pathway.

Ion and fluid transport across the biliary epithelium contributes to bile secretion. Since endothelin (ET)-1 affects ion transport activities and is released by human gallbladder- derived biliary epithelial cells in primary culture, we examined the expression of ET peptides and ET receptors and the influence of ET-1 on ion transport in this epithelium ex vivo. In freshly isolated gallbladder epithelial cells, preproET-1, -2, and -3 mRNAs were detected by reverse transcription PCR and ET-1 isopeptide was identified by chromatography. The cells also displayed ET receptor mRNAs and high-affinity binding sites for ET-1, mostly of the ETB type. Electrogenic anion secretion across intact gallbladder mucosa was stimulated by forskolin, secretin, and exogenous ATP, as assessed by short-circuit current (Isc) increases in Ussing-type chambers. ET-1 inhibited forskolin- and secretin-induced changes in Isc, without affecting baseline Isc or ATP-induced changes. Accordingly, ET-1 significantly reduced the accumulation of intracellular cAMP elicited by forskolin and secretin in the epithelial cells, and this effect was abolished by pertussis toxin. This is the first evidence that ET-1 is synthesized and inhibits, via a Gi protein-coupled receptor, cAMP-dependent anion secretion in human gallbladder epithelium, indicating a role in the control of bile secretion by an autocrine/paracrine mechanism.

Background & Aims: The cellular mechanisms that regulate biliary mucin secretion in humans are unknown. To address this question, human gallbladder epithelial cells were used in primary culture., Methods: [1-(14)C]-glucosamine-labeled glycoproteins secreted in vitro were analyzed and quantified after exposing cells to activators and inhibitors of the main transduction pathways and to potential biologically active secretagogues., Results: Secreted glycoproteins showed characteristics of biliary mucins. Activators of adenosine 3',5'-cyclic monophosphate-dependent pathway as well as secretin and vasoactive intestinal polypeptide did not significantly modify mucin secretion. By contrast, ionomycin and phorbol-12-myristate 13-acetate increased mucin secretion by 292% +/- 48% and 134% +/- 19% over basal level, respectively. The effects of these two agents were additive and were mediated by a calcium-dependent pathway implicating Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-kinase II) and by the activation of protein kinase C (PKC), respectively, as ascertained by using inhibitors. Mucin secretion was stimulated by extracellular adenosine 5'-triphosphate via P2U receptors, cytosolic calcium increase, and PKC and by taurochenodeoxycholate via cytosolic calcium increase and Ca2+/CaM-kinase II., Conclusions: Mucin secretion in human gallbladder is regulated predominantly by calcium-dependent pathways implicating Ca2+/CaM-kinase II and PKC. Extracellular adenosine 5'-triphosphate and taurochenodeoxycholate may play a role in the regulation of biliary mucin secretion by activating these different signaling pathways.

Background: Hepatobiliary complications in cystic fibrosis result predominantly from lesions of the biliary epithelium. These abnormalities affect the intrahepatic as well as extrahepatic bile ducts and the gallbladder. The protein cystic fibrosis transmembrane conductance regulator (CFTR), the gene product defective in cystic fibrosis, functions as a cAMP-activated chloride channel in the plasma membrane. As such, it may represent an important driving force for fluid transport across the epithelium., Experimental Design: The purpose of this study was to investigate the expression of CFTR in human gallbladder epithelial cells and to examine the chloride ion transport properties of these cells. Immunolocalization was performed on tissue sections. The reverse transcription-PCR was used to analyze the expression of CFTR mRNA in freshly isolated and cultured gallbladder epithelial cells. The CFTR protein was detected by Western blotting and immunoprecipitation. The chloride ion transport properties of the cells were determined by 36Cl efflux studies., Results: The CFTR protein was immunodetected in human gallbladder in situ and localized predominantly to the apical membrane of epithelial cells. High levels of CFTR mRNA and protein were maintained in gallbladder epithelial cells in primary cultured. Glycosylated forms of CFTR were present as confirmed by treatment with N-glycanase. Chloride efflux was stimulated by Ca(++)-dependent pathways but more intensely by cAMP-dependent pathways. Stimulation of chloride efflux by agonist of the cAMP-pathway was inhibited by diphenylamine carboxylic acid, a chloride channel blocker. Two physiologically active peptides--acting via cAMP, vasoactive intestinal peptide, and secretin--also stimulated chloride efflux in vitro., Conclusions: Our results are consistent with a high expression of endogenous functional CFTR protein in human gallbladder epithelial cells. Physiologically active peptides, vasoactive intestinal peptide and secretin, stimulate chloride conductance in these cells. These findings indicate that CFTR play an important role in the pathophysiology of the biliary epithelium, including the gallbladder epithelium.

Background: The three isoforms of endothelin (ET), ET-1, ET-2, and ET-3 are potent contractile agonists for smooth muscle in a wide variety of tissues including the gallbladder. There is increasing evidence that endothelin acts in a paracrine fashion, however, its cell source in the gallbladder is unclear., Experimental Design: To examine the production of ET by gallbladder and bile duct epithelium., Results: We show that human gallbladder epithelial cells in primary culture secrete endothelin. ET release was time-dependent, and intracellular ET was negligible, indicating de novo synthesis. Secretion was increased by physiologic concentrations of cholecystokinin. Epithelial cells lining hepatic cysts in primary culture also released ET, suggesting that the intrahepatic, as well as the extrahepatic biliary epithelium is a source of this cytokine. High performance liquid chromatography separation of the conditioned medium from both cell types showed a single peak corresponding to that of ET-1. In vivo, ET-1 was present in hepatic cyst fluid, but was not detectable in gallbladder or choledochal bile. On tissue sections, both intrahepatic and extrahepatic bile duct epithelial cells were labeled with an anti-"big" ET-1 polyclonal antibody., Conclusions: These results suggest that ET-1 is locally produced in the biliary tract and by a paracrine route, could play a role in choledochal motility and gallbladder contraction.