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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

The nuclear receptor FXR[alpha] (NR1H4) plays a pivotal role in maintaining bile salt and lipid homeostasis by functioning as a bile salt sensor in mammals. In contrast, FXR[beta] (NRIH5) from mouse is activated by lanosterol and does not share common ligands with FXR[alpha]. To further elucidate FXR ligand/receptor and structure/function relationships, we characterized a FXR gene from the marine skate, Leucoraja erinacea, representing a vertebrate lineage that diverged over 400 million years ago. Phylogenetic analysis of sequence data indicated that skate Fxr (sFxr) is a FXR[beta]. There is an extra sequence in the middle of the sFxr ligand binding domain (LBD) compared with the LBD of FXR[alpha]. Luciferase reporter assays demonstrated that sFxr responds weakly to scymnol sulfate, bile salts, and synthetic FXR[alpha] ligands, in striking difference from human FXR[alpha] (hFXR[alpha]). Interestingly, all-trans retinoic acid was capable of transactivating both hFXR[alpha] and sFxr. When the extra amino acids in the sFxr LBD were deleted and replaced with the corresponding sequence from hFXR[alpha], the mutant sFxr gained responsiveness to ursodeoxycholic acid, GW4064, and fexaramine. Surprisingly, chenodeoxycholic acid antagonized this activation. Together, these results indicate that FXR is an ancient nuclear receptor and suggest that FXR[alpha] may have acquired ligand specificity for bile acids later in evolution by deletion of a sequence from its LBD. Acquisition of this property may be an example of molecular exploitation, where an older molecule is recruited for a new functional role. nuclear receptor; structure/function relationship

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

Multidrug resistance-associated protein MRP3/Mrp3 (ABCC3) is upregulated in cholestasis, an adaptive response that may protect the liver from accumulation of toxic compounds, such as bile salts and bilirubin conjugates. However, the mechanism of this upregulation is poorly understood. We and others have previously reported that fetoprotein transcription factor/liver receptor homolog-1 is an activator of MRP3/Mrp3 expression. In searching for additional regulatory elements in the human MRP3 promoter, we have now identified nuclear receptor retinoic X receptor-ot:retinoic acid receptor-[alpha] (RXR[alpha]:RAR[alpha]) as a repressor of MRP3 activation by transcription factor Sp1. A luciferase reporter assay demonstrated that cotransfection of transcription factor Sp1 stimulates the MRP3 promoter activity and that additions of RXR[alpha]:RAR[alpha] abrogated this activation in a dose-dependent manner. Site mutations and gel shift assays have identified a Sp1 binding GC box motif at -113 to -108 nts upstream from the MRP3 translation start site, where RXR[alpha]:RAR[alpha] specifically reduced Sp1 binding to this site. Mutation of the GC box also reduced MRP3 promoter activity. The functional role of RXR[alpha]:RAR[alpha] as a repressor of MRP3 expression was further confirmed by RAR[alpha] small-interfering RNA knockdown in HepG2 cells, which upregulated endogenous MRP3 expression. In summary, our results indicate that activator Sp1 and repressor RXR[alpha]:RAR[alpha] act in concert to regulate MRP3 expression. Since RXR[alpha]:RAR[alpha] expression is diminished by cholestatic liver injury, loss of RXR[alpha]:RAR[alpha] may lead to upregulation of MRP3/Mrp3 expression in these disorders. gene expression; transporter; cholestasis; multidrug resistance-associated protein 3 doi:10.1152/ajpgi.00191.2006

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

Multidrug resistance protein Mrp2 (symbol Abcc2) in liver plays a significant role in the biliary excretion of organic anionic conjugates. Mutations in human MRP2 result in defects in excretion of conjugated bilirubin and other cholephiles known as the Dubin-Johnson syndrome. Previous studies indicate that transporters with Mrp2-like functions are present in ancient vertebrates. We have now characterized an Mrp2 ortholog at the molecular level from the liver of the small skate, Raja erinacea, a marine vertebrate that evolved ~200 million years ago. The full-length skate Mrp2 cDNA is 6 kb and encodes for a 1,564-amino acid peptide with 56% identity to human Mrp2. Northern blot analysis demonstrated that skate Mrp2 is abundantly expressed in skate liver, intestine, and kidney. Immunoblots reveal a 180-kDa protein in skate liver. Immunofluorescence studies locate skate Mrp2 to the apical membrane of hepatocytes, renal tubules, and intestine. A PDZ-interacting motif is also found at its COOH terminus. Further sequence analysis indicates that transmembrane domains 1, 9, 11, 16, and 17 are the most highly conserved transmembrane domains between skate Mrp2 and mammalian MRP2/Mrp2s. This analysis indicates that Mrp2 orthologs evolved early in vertebrate evolution and that conserved domains may be important determinants of Mrp2 substrate specificity. organic solute transporter; cloning; elasmobranch

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

Cellular uptake of organic solutes is mediated in large part by a gene family of membrane transporters called OATPs (SLC21A). To study the structural determinants and evolutionary development of the SLC21A family, we have cloned and functionally characterized a highly expressed evolutionarily primitive Oatp from the liver of the small skate, Raja erinacea. A full-length cDNA (2.3 kb) was obtained that encodes a protein of 689 amino acids. The characteristics of this novel skate Oatp, including tissue expression, subcellular localization, substrate selectivity, [Na.sup.+] dependence, and inhibitor selectivity were generally similar to liver-specific human OATP-C and rat Oatp4. However, sequence comparisons with other OATPs indicate that this skate Oatp shares only ~40-50% amino acid identity with the liver-specific OATPs/Oatps and with human OATP-F. Further computer analysis revealed that the highest amino acid identities reside in the first external (78%) and internal loops (75%) and transmembrane domains 2 (76%), 3 (62%), 4 (70%), and 11 (64%). We propose that the conserved regions of the SLC21A transporter family may be critical structural determinants of substrate specificity and function. skate Oatp; transporter; evolution; hepatic uptake

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.

Research in gene therapy could lead to a treatment for some familial forms of cholestasis. Cholestasis occurs when the body cannot produce bile. Bile is produced in the liver and bile ducts by cells called hepatocytes and cholangiocytes, respectively. The bile must then be transported out of the cells, where it collects in the gallbladder. A defect in the transport proteins can lead to cholestasis. Some cases of familial cholestasis have been linked to specific chromosome locations. Ursodiol may be effective in many patients and gene therapy is a promising treatment.

The mechanisms of action governing the hepatocellular transport of the sulfated bile alcohol scymnol sulfate and its role in bile formation in fish liver skate hepatocytes were investigated. Male skates were used in an experiment to examine uptake of bile salts into freshly isolated skate hepatocytes and bile production from isolated perfused skate livers. Results showed an enterohepatic circulation of the bile alcohol in the free-swimming skate. In addition, there was a significant absorption in the intestinal epithelium of the fish liver.

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 transcellular translocation of bile salt derivative (N-(7-(4-nitrobenzo-2-oxa-1,3-diazol-))-7beta-amino-3alpha,12aplha-dihydroxy -5beta-cholan-24-oyl-2-aminoethanesulfonate (NBD-TC)) in skate hepatocytes is microtubule dependent. This dependence is essential for canalicular secretion. The uptake of NBD-TC is active and carrier mediated, and independent of the sodium or the potential difference gradient.