Absence of the intestinal microbiota exacerbates hepatobiliary disease in a murine model of primary sclerosing cholangitis.

Unlabelled: Primary sclerosing cholangitis (PSC) is a chronic, idiopathic, fibroinflammatory cholangiopathy. The role of the microbiota in PSC etiopathogenesis may be fundamentally important, yet remains obscure. We tested the hypothesis that germ-free (GF) mutltidrug resistance 2 knockout (mdr2(-/-) ) mice develop a distinct PSC phenotype, compared to conventionally housed (CV) mdr2(-/-) mice.

Micro-computed tomography and nuclear magnetic resonance imaging for noninvasive, live-mouse cholangiography.

The cholangiopathies are a diverse group of biliary tract disorders, many of which lack effective treatment. Murine models are an important tool for studying their pathogenesis, but existing noninvasive methods for assessing biliary disease in vivo are not optimal. Here we report our experience with using micro-computed tomography (microCT) and nuclear magnetic resonance (MR) imaging to develop a technique for live-mouse cholangiography.

Cholangiocyte cilia express TRPV4 and detect changes in luminal tonicity inducing bicarbonate secretion.

Cholangiocytes, epithelial cells lining the biliary tree, have primary cilia extending from their apical membrane into the ductal lumen. Although important in disease, cilia also play a vital role in normal cellular functions. We reported that cholangiocyte cilia are sensory organelles responding to mechanical stimuli (i.

Isolation and characterization of cholangiocyte primary cilia.

Primary cilia are distinct organelles expressed by many vertebrate cells, including cholangiocytes; however, their functions remain obscure. To begin to explore the physiological role of these organelles in the liver, we described the morphology and structure of cholangiocyte cilia and developed new approaches for their isolation. Primary cilia were present only in bile ducts and were not observed in hepatocytes or in hepatic arterial or portal venous endothelial cells.

Cryptosporidium parvum infects human cholangiocytes via sphingolipid-enriched membrane microdomains.

Cryptosporidium parvum attaches to intestinal and biliary epithelial cells via specific molecules on host-cell surface membranes including Gal/GalNAc-associated glycoproteins. Subsequent cellular entry of this parasite depends on host-cell membrane alterations to form a parasitophorous vacuole via activation of phosphatidylinositol 3-kinase (PI-3K)/Cdc42-associated actin remodelling. How C.

Cholangiocyte biology.

Purpose Of Review: Cholangiocytes are increasingly recognized as biologically important epithelia because of the diverse array of cellular processes in which they participate. Collectively, these processes define normal function and, when disturbed, account for abnormalities that cause disease. Advances in animal models and sophisticated technology in imaging and gene silencing have led to substantial progress in defining the roles that cholangiocytes play in signaling, transport of water, ions and solutes, and alterations that result in cholestasis.

Cytoskeletal and motor proteins facilitate trafficking of AQP1-containing vesicles in cholangiocytes.

Background Information: We have previously showed that: (i) cholangiocytes contain AQP1 (aquaporin 1) water channels sequestered in intracellular vesicles; and (ii) upon stimulation with choleretic agonists such as secretin or dibutyryl-cAMP (dbcAMP), the AQP1 vesicles move via microtubules to the apical cholangiocyte membrane to facilitate osmotically driven, passive water movement (i.e. ductal bile secretion).

Multiple TLRs are expressed in human cholangiocytes and mediate host epithelial defense responses to Cryptosporidium parvum via activation of NF-kappaB.

Infection of epithelial cells by Cryptosporidium parvum triggers a variety of host-cell innate and adaptive immune responses including release of cytokines/chemokines and up-regulation of antimicrobial peptides. The mechanisms that trigger these host-cell responses are unclear. Thus, we evaluated the role of TLRs in host-cell responses during C.

Expression and subcellular localization of aquaporin water channels in the polarized hepatocyte cell line, WIF-B.

Background: Recent data suggest that canalicular bile secretion involves selective expression and coordinated regulation of aquaporins (AQPs), a family of water channels proteins. In order to further characterize the role of AQPs in this process, an in vitro cell system with retained polarity and expression of AQPs and relevant solute transporters involved in bile formation is highly desirable. The WIF-B cell line is a highly differentiated and polarized rat hepatoma/human fibroblast hybrid, which forms abundant bile canalicular structures.

Cholangiocyte biology.

Purpose Of Review: Cholangiocytes are increasingly recognized as biologically important because of the diverse array of cellular processes in which they participate. Collectively, these processes define normal function and, when disturbed, account for abnormalities that cause disease. Advances in animal models and sophisticated technology in imaging and gene silencing have allowed progress in defining the roles that cholangiocytes play in signaling, transport of water, ions and solutes, and alterations that result in cholestasis.

Cholangiocyte biology.

Purpose Of Review: Cholangiocytes are increasingly recognized as biologically important because of the diversity of cellular processes in which they participate. Collectively, these processes define normal function and, when disturbed, account for abnormalities that cause disease. Advances in animal models of disease, sophistication of technology in imaging, and gene silencing have allowed progress in defining the roles that cholangiocytes play in signaling; transport of water, ions, and solutes; and alterations that result in cholestasis.

Phosphatidylinositol 3-kinase and frabin mediate Cryptosporidium parvum cellular invasion via activation of Cdc42.

Cryptosporidium parvum invades target epithelia via a mechanism that involves host cell actin reorganization. We previously demonstrated that C. parvum activates the Cdc42/neural Wiskott-Aldrich syndrome protein network in host cells resulting in actin remodeling at the host cell-parasite interface, thus facilitating C.

Water transporting properties of hepatocyte basolateral and canalicular plasma membrane domains.

Previous work from our laboratory supports an important role for aquaporins (AQPs), a family of water channel proteins, in bile secretion by hepatocytes. To further define the pathways and molecular mechanisms for water movement across hepatocytes, we directly assessed osmotic water permeability (Pf) and activation energy (Ea) in highly purified, rat hepatocytes basolateral membrane vesicles (BLMV) and canalicular membrane (CMV) vesicles by measuring scattered light intensity using stopped-flow spectrophotometry. The time course of scattered light for BLMV and CMV fit well to a single-exponential function.

Glucagon induces the plasma membrane insertion of functional aquaporin-8 water channels in isolated rat hepatocytes.

Although glucagon is known to stimulate the cyclic adenosine monophosphate (cAMP)-mediated hepatocyte bile secretion, the precise mechanisms accounting for this choleretic effect are unknown. We recently reported that hepatocytes express the water channel aquaporin-8 (AQP8), which is located primarily in intracellular vesicles, and its relocalization to plasma membranes can be induced with dibutyryl cAMP. In this study, we tested the hypothesis that glucagon induces the trafficking of AQP8 to the hepatocyte plasma membrane and thus increases membrane water permeability.

Rat hepatocyte aquaporin-8 water channels are down-regulated in extrahepatic cholestasis.

Hepatocytes express the water channel aquaporin-8 (AQP8), which is mainly localized in intracellular vesicles, and its adenosine 3',5'-cyclic monophosphate (cAMP)-induced translocation to the plasma membrane facilitates osmotic water movement during canalicular bile secretion. Thus, defective expression of AQP8 may be associated with secretory dysfunction of hepatocytes caused by extrahepatic cholestasis. We studied the effect of 1, 3, and 7 days of bile duct ligation (BDL) on protein expression, subcellular localization, and messenger RNA (mRNA) levels of AQP8; this was determined in rat livers by immunoblotting in subcellular membranes, light immunohistochemistry, immunogold electron microscopy, and Northern blotting.

Somatostatin stimulates ductal bile absorption and inhibits ductal bile secretion in mice via SSTR2 on cholangiocytes.

With an in vitro model using enclosed intrahepatic bile duct units (IBDUs) isolated from wild-type and somatostatin receptor (SSTR) subtype 2 knockout mice, we tested the effects of somatostatin, secretin, and a selective SSTR2 agonist (L-779976) on fluid movement across the bile duct epithelial cell layer. By RT-PCR, four of five known subtypes of SSTRs (SSTR1, SSTR2A/2B, SSTR3, and SSTR4, but not SSTR5) were detected in cholangiocytes in wild-type mice. In contrast, SSTR2A/2B were completely depleted in the SSTR2 knockout mice whereas SSTR1, SSTR3 and SSTR4 were expressed in these cholangiocytes.

Agonist-induced coordinated trafficking of functionally related transport proteins for water and ions in cholangiocytes.

We previously proposed that ductal bile formation is regulated by secretin-responsive relocation of aquaporin 1 (AQP1), a water-selective channel protein, from an intracellular vesicular compartment to the apical membrane of cholangiocytes. In this study, we immunoisolated AQP1-containing vesicles from cholangiocytes prepared from rat liver; quantitative immunoblotting revealed enrichment in these vesicles of not only AQP1 but also cystic fibrosis transmembrane regulator (CFTR) and AE2, a Cl- channel and a Cl-/HCO3- exchanger, respectively. Dual labeled immunogold electron microscopy of cultured polarized mouse cholangiocytes showed significant colocalization of AQP1, CFTR, and AE2 in an intracellular vesicular compartment; exposure of cholangiocytes to dibutyryl-cAMP (100 microm) resulted in co-redistribution of all three proteins to the apical cholangiocyte plasma membrane.

Intrahepatic bile ducts transport water in response to absorbed glucose.

The physiological relevance of the absorption of glucose from bile by cholangiocytes remains unclear. The aim of this study was to test the hypothesis that absorbed glucose drives aquaporin (AQP)-mediated water transport by biliary epithelia and is thus involved in ductal bile formation. Glucose absorption and water transport by biliary epithelia were studied in vitro by microperfusing intrahepatic bile duct units (IBDUs) isolated from rat liver.

Channel-mediated water movement across enclosed or perfused mouse intrahepatic bile duct units.

We previously reported the development of reproducible techniques for isolating and perfusing intact intrahepatic bile duct units (IBDUs) from rats. Given the advantages of transgenic and knockout mice for exploring ductal bile formation, we report here the adaptation of those techniques to mice and their initial application to the study of water transport across mouse intrahepatic biliary epithelia. IBDUs were isolated from livers of normal mice by microdissection combined with enzymatic digestion.

Experimental models to study cholangiocyte biology.

Cholangiocytes-the epithelial cells which line the bile ducts-are increasingly recognized as important transporting epithelia actively involved in the absorption and secretion of water, ions, and solutes. This recognition is due in part to the recent development of new experimental models. New biologic concepts have emerged including the identification and topography of receptors and flux proteins on the apical and/or basolateral membrane which are involved in the molecular mechanisms of ductal bile secretion.