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Human BSEP (ABCB11) mutations are the molecular basis for at least three clinical forms of liver disease, progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), and intrahepatic cholestasis of pregnancy (ICP). To better understand the pathobiology of these disease phenotypes, we hypothesized that different mutations may cause significant differences in protein defects. Therefore we compared the effect of two PFIC2 mutations (D482G, E297G) with two BRIC2 mutations (A570T and R1050C) and one ICP mutation (N591S) with regard to the subcellular localization, maturation, and function of the rat Bsep protein. Bile salt transport was retained in all but the E297G mutant. Mutant proteins were expressed at reduced levels on the plasma membrane of transfected HEK293 cells compared with wild-type (WT) Bsep in the following order: WT > N591S > R1050C ~ A570T ~ E297G >> D482G. Total cell protein and surface protein expression were reduced to the same extent, suggesting that trafficking of these mutants to the plasma membrane is not impaired. All Bsep mutants accumulate in perinuclear aggresome-like structures in the presence of the proteasome inhibitor MG-132, suggesting that mutations are associated with protein instability and ubiquitin-dependent degradation. Reduced temperature, sodium butyrate, and sodium 4-phenylbutyrate enhanced the expression of the mature and cell surface D482G protein in HEK293 cells. These results suggest that the clinical phenotypes of PFIC2, BRIC2, and ICP may directly correlate with the amount of mature protein that is expressed at the cell surface and that strategies to stabilize cell surface mutant protein may be therapeutic. ATP-binding cassette; progressive familial intrahepatic cholestasis type 2; benign recurrent intrahepatic cholestasis type 2; intrahepatic cholestasis of pregnancy; taurocholate transport

Phloracetophenone (2,4,6-trihydroxyacetophenone, THA) is a potent choleretic in the bile fistula rat, although the mechanism is unknown. In the present study, we examined how THA enhances bile secretion. Stepwise infusions of THA (1-4 [micro]mol/min) in the isolated perfused rat liver resulted in an immediate and dose-dependent increase in bile flow (BF), which reached saturation. The increase in BF was not associated with a change in the excretion of bile acids, suggesting that THA stimulated bile acid-independent bile flow. To further define the mechanism, the effect of THA on the excretion of sulfohromophthalein (BSP) and disulfobromophthalein (DBSP), typical multidrug resistance protein-2 (Mrp2) substrates was examined. THA inhibited the biliary excretion of both substrates. Because DBSP is excreted without conjugation to glutathione, in contrast to BSP, the findings suggest that THA might compete with DBSP and BSP metabolites at a common canalicular transport site, presumably Mrp2. THA infusions had no effect on the subcellular localization and distribution of either Mrp2 or the bile salt export pump (Bsep), nor the integrity of the tight junction. In contrast, the choleretic activity of THA was completely absent in the TR--rat, an animal model that lacks Mrp2, directly implicating this canalicular export pump as the mechanisms by which THA is excreted in bile. THA also partially reversed the cholestatic effects of estradiol-17[beta]-D-glucuronide, a process also dependent on Mrp2. In conclusion, the choleretic activity of THA and its possible metabolites is dependent on Mrp2. THA appears to stimulate BF by its osmotic effects and may attenuate the cholestatic effects of hepatotoxins undergoing biotransformation and excretion via similar pathways. hepatobiliary excretion; multidrug-resistance-associated protein 2; bile secretion; ABC transporters doi:10.1152/ajpgi.00578.2006

Organic solute transporter (OST[alpha]-OST[beta]) is a novel heteromeric bile acid and sterol transporter expressed at the basolateral membranes of epithelium in the ileum, kidney, and liver. To detennine whether OST[alpha]-OST[beta] undergoes farnesoid X receptor (FXR)-dependent adaptive regulation following cholestatic liver injury, mRNA and protein expression levels were analyzed in patients with primary biliary cirrhosis (PBC) and following common bile duct ligation (CBDL) in rats and Fxr null and wild-type mice. Hepatic OST[alpha] and OST[beta] mRNA increased 3- and 32-fold, respectively, in patients with PBC compared with controls, whereas expression of Ost[alpha] and Ost[beta] also increased in the liver of vats and mice following CBDL. In contrast, expression of Ost[alpha] and Ost[beta] mRNA was generally lower in Fxr null mice, and CBDL failed to enhance expression of Ost[alpha] and Ost[beta] compared with wild-type mice. HepG2 cells treated for 24 h with chenodeoxycholic acid, a selective FXR ligand, had higher levels of OST[alpha] and OST[beta] mRNA and protein. Increases in OST protein were visualized by confocal microscopy at the plasma membrane. These results indicate that expression of Ost[alpha] and Ost[beta] are highly regulated in response to cholestasis and that this response is dependent on the FXR bile acid receptor. bile acid and steroid transporter; primary biliary cirrhosis; cholangiocytes; kidney; bile acid reabsorption

The mechanisms by which fluid moves across the luminal membrane of cholangiocyte epithelia are uncertain. Previous studies suggested that aquaporin-1 (AQP1) is an important determinant of water movement in rat cholangiocytes and that cyclic AMP mediates the movement of these water channels from cytoplasm to apical membrane, thereby increasing the osmotic water permeability. To test this possibility we measured agonist-stimulated fluid secretion and osmotically driven water transport in isolated bile duct units (IBDUs) from AQP1 wild-type (+/+) and null (-/-) mice. AQP1 expression was confirmed in a mouse cholangiocyte cell line and +/+ liver. Forskolin-induced fluid secretion, measured from the kinetics of IBDU luminal expansion, was 0.05 fl/min and was not impaired in -/- mice. Osmotic water permeability (Pf), measured from the initial rate of IBDU swelling in response to a 70-mosM osmotic gradient, was 11.1 x [10.sup.-4] cm/s in +/+ mice and 11.5 x [10.sup.-4] cm/s in -/- mice. [P.sub.f] values increased by ~50% in both +/+ and -/- mice following preincubation with forskolin. These findings provide direct evidence that AQP1 is not rate limiting for water movement in mouse cholangiocytes and does not appear to be regulated by cyclic AMP in this species. aquaporins; cholangiocytes; bile formation; adenosine 3',5'-cyclic monophosphate

The influences of bombesin on ion transport processes implicated in the control of intracellular pH (pHi) and HCO3- secretion in polarized cholangiocytes were studied. Results indicated that bombesin induces biliary secretion from cholangiocytes by triggering luminal Cl-/HCO3- exchange, which may be related to basolateral electrogenic Na+-HCO3- symport.

Experiment was conducted to study the role of protein kinase A (PKA), protein kinase C (PKC) and protein phosphatases in the process of secretin stimulation of fluid and bicarbonate secretion from biliary epithelium using a novel isolated bile duct unit (BDU) model from rat liver. Results suggest that PKA but not PKC is involved in the signal transduction of secretin-stimulated fluid secretion and Cl-/HCO-3 exchange activity in rat bile duct epithelium, a process inactivated by dephosphorylation by protein phosphatases 1 and/or 2A.

The role of hormone-induced intracellular calcium (Ca2+) signals in the secretion of bile by the bile duct cells was investigated in a rat model. Acetylcholine (ACh) and ATP increased Ca2+ signaling in a dose dependent manner. Hepatocyte Ca2+ agonists vasopressin, angiotensin and phenylephrine, or by exocrine pancreas agonists cholecystokinin andd bombesin did not increase Ca2+ in isolated bile duct units, whereas ACh did not increase Ca2+ in isolated hepatocytes. These results suggest that Ca2+ signaling depend on the activation of muscarinic and purinergic receptors found in bile duct cells.

The cytosolic calcium organizes as calcium waves among hepatocytes in the intact tissues in response to the hormonal stimuli. The initiation of vasopressin is investigated for the calcium waves in hepatocytes within the rat liver. The confocal studies show that increase in calcium began as waves in hepatocytes near central venules and then spread opposite to the direction of blood flow to hepatocytes near portal venules. The pericentral-to-periportal calcium waves helps in the peristaltic flow of bile.

The influence of ursodeoxycholic acid (UDCA) on H+ and HCO3- transporters in bile duct epithelial cells separated from the normal rat liver was studied to assess the possible functions for the activation of biliary HCO3- release by UDCA. A quick intracellular acidification in separated BDE cells is generated by UDCA. Enhancement in HCO3-secretion through the Cl--HCO3-exchange does not cause the acidification induced by UDCA.

The intercellular barrier between bile and blood is created by the hepatocyte tight junctions. Structural changes in the tight junction (TJ) and an easily observable paracellular leak is caused by common bile duct (CBDL) cholestasis. The influence of CBDL on ZO-1alpha+ immunofluorescence (IF) localization and a ZO-1alpha+ mRNA, and protein expression during 2 weeks of CBDL are studied by the use of the epithelial isoform of the TJ protein ZO-1alpha+ as a monomolecular changes masker in hepatocyte TJs. A considerable increase in the levels of hepatic ZO-1alpha+ mRNA levels is revealed by the quantitative immunoblotting and ribonuclease protection respectively.

Abstract Background The organic solute transporter (OSTα-OSTβ) is a heteromeric transporter that is expressed on the basolateral membrane of epithelium in intestine, kidney, liver, testis and adrenal gland and facilitates efflux of bile acids and other steroid solutes. Both subunits are required for plasma membrane localization of the functional transporter but it is unclear how and where the subunits interact and whether glycosylation is required for functional activity. We sought to examine these questions for the human OSTα-OSTβ transporter using the human hepatoma cell line, HepG2, and COS7 cells transfected with constructs of human OSTα-FLAG and OSTβ-Myc. Results Tunicamycin treatment demonstrated that human OSTα is glycosylated. In COS7 cells Western blotting identified the unglycosylated form (~31 kD), the core precursor form (~35 kD), and the mature, complex glycoprotein (~40 kD). Immunofluorescence of both cells indicated that, in the presence of OSTβ, the alpha subunit could still be expressed on the plasma membrane after tunicamycin treatment. Furthermore, the functional uptake of 3H-estrone sulfate was unchanged in the absence of N-glycosylation. Co-immunoprecipitation indicates that the immature form of OSTα interact with OSTβ. However, immunoprecipitation of OSTβ using an anti-Myc antibody did not co-precipitate the mature, complex glycosylated form of OSTα, suggesting that the primary interaction occurs early in the biosynthetic pathway and may be transient. Conclusion In conclusion, human OSTα is a glycoprotein that requires interaction with OSTβ to reach the plasma membrane. However, glycosylation of OSTα is not necessary for interaction with the beta subunit or for membrane localization or function of the heteromeric transporter.

Cholangiocytes absorb and secrete fluid, modifying primary canalicular bile. In several [Cl.sup.-]-secreting epithelia, [Na.sup.+][-K.sup.+]-2[Cl.sup.-] cotransport is a basolateral [Cl.sup.-] uptake pathway facilitating apical [Cl.sup.-] secretion. To determine if cholangiocytes possess similar mechanisms independent of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] we assessed [Cl.sup.-]-dependent secretion in rat liver isolated polarized bile duct units (IBDUs) by using videomicroscopy. Without [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], forskolin (FSK) stimulated secretion entirely dependent on [Na.sup.+] and [Cl.sup.-] and inhibited by [Na.sup.+][-K.sup.+]-2[Cl.sup.-] inhibitor bumetanide. Carbonic anhydrase inhibitor ethoxyzolamide had no effect on FSK-stimulated secretion, indicating negligible endogenous [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] transport. In contrast, FSK-stimulated secretion was inhibited ~85% by [K.sup.+] channel inhibitor [Ba.sup.2+] and blocked completely by bumetanide plus [Ba.sup.2+]. IBDU [Na.sup.+][-K.sup.+]-2[Cl.sup.-] cotransport activity was assessed by recording intracellular pH during [NH.sub.4]Cl exposure. Bumetanide inhibited initial acidification rates due to [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] entry in the presence and absence of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. In contrast, when stimulated by FSK, a 35% increase in [Na.sup.+][-K.sup.+]-2[Cl.sup.-] cotransport activity occurred without [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. These data suggest a cellular model of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]-independent secretion in which [Na.sup.+][-K.sup.+]-2[Cl.sup.-] cotransport maintains high intracellular [Cl.sup.-] concentration. Intracellular cAMP concentration increases activate basolateral [K.sup.+] conductance, raises apical [Cl.sup.-] permeability, and causes transcellular [Cl.sup.-] movement into the lumen. Polarized IBDU cholangiocytes are capable of vectorial [Cl.sup.-]-dependent fluid secretion independent of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. Bumetanide-sensitive [Na.sup.+][-K.sup.+]-2[Cl.sup.-] cotransport, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange, and [Ba.sup.2+] sensitive [K.sup.+] channels are important components of stimulated fluid secretion in intrahepatic bile duct epithelium. ammonia; cholangiocyte; bumetanide; barium; pH

Role of sodium/hydrogen exchanger isoform NHE3 in fluid secretion and absorption in mouse and rat cholangiocytes. Am J Physiol Gastrointest Liver Physiol 280: G247-G254, 2001.--[Na.sup.+]/[H.sup.+] exchanger (NHE) isoforms play important roles in intracellular pH regulation and in fluid absorption. The isoform NHE3 has been localized to apical surfaces of epithelia and in some tissues may facilitate the absorption of NaCl. To determine whether the apical isoform NHE3 is present in cholangiocytes and to examine whether it has a functional role in cholangiocyte fluid secretion and absorption, immunocytochemical studies were performed in rat liver with NHE3 antibodies and functional studies were obtained in isolated bile duct units from wild-type and NHE3 (-/-) mice after stimulation with forskolin, using video-microscopic techniques. Our results indicate that NHE3 protein is present on the apical membranes of rat cholangiocytes and on the canalicular membrane of hepatocytes. Western blots also detect NHE3 protein in rat cholangiocytes and isolated canalicular membranes. After stimulation with forskolin, duct units from NHE3 (-/-) mice fail to absorb the secreted fluid from the cholangiocyte lumen compared with control animals. Similar findings were observed in isolated bile duct units from wild-type mice and rats in the presence of the [Na.sup.+]/[H.sup.+] exchanger inhibitor 5-(N-ethyl-N-isopropyl)-amiloride. In contrast, we could not demonstrate absorption of fluid from the canalicular lumen of mouse or rat hepatocyte couplets after stimulation of secretion with forskolin. These findings indicate that NHE3 is located on the apical membrane of rat cholangiocytes and that this NHE isoform can function to absorb fluid from the lumens of isolated rat and mouse cholangiocyte preparations. bile secretion; bile duct epithelium; hepatocyte

Isolation of functional polarized bile duct units from mouse liver. Am J Physiol Gastrointest Liver Physiol 280: G241-G246, 2001.--The development of genetically altered murine animals has generated a need for in vitro systems in the mouse. We have now characterized a novel isolated bile duct unit (IBDU) preparation from the mouse to facilitate such studies. The mouse IBDU is isolated by portal perfusion of collagenase, blunt dissection, further enzymatic digestions, filtering through sized mesh, and culturing on Matrigel for 16-72 h. This mouse IBDU forms a central, enclosed lumen lined by polarized cytokeratin-19-positive cholangiocytes with numerous microvilli on the apical membrane. The IBDU responds to secretory stimuli, including secretin, vasoactive intestinal peptide, IBMX, and forskolin, resulting in expansion of the central lumen from secretion as quantified by videomicroscopy. The secretory response to secretin is dependent on [Cl.sup.-] and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in the perfusate. These findings indicate that mouse IBDUs are intact, polarized, functional bile duct secretory units that permit quantitative measurements of fluid secretion from mouse bile duct epithelium for the first time. This method should facilitate studies of cholangiocyte secretion in genetically altered murine animal models. isolated bile duct unit; biliary secretion; quantitative videomicroscopy; enzymatic isolation