Role of primary bile salts in the regulation of sinusoidal substrate uptake in rat liver (original) (raw)
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Expression and regulation of hepatic drug and bile acid transporters
Toxicology, 2000
Transport across hepatocyte plasma membranes is a key parameter in hepatic clearance and usually occurs through different carrier-mediated systems. Sinusoidal uptake of compounds is thus mediated by distinct transporters, such as Na + -dependent or Na + -independent anionic transporters and by some cationic transporters. Similarly, several membrane proteins located at the apical pole of hepatocytes have been incriminated in the excretion of compounds into the bile. Indeed, biliary elimination of anionic compounds, including glutathione S-conjugates, is mediated by MRP2, whereas bile salts are excreted by a bile salt export pump (BSEP) and Class I-P-glycoprotein (P-gp) is involved in the secretion of amphiphilic cationic drugs, whereas class II-P-gp is a phospholipid transporter. The expression of hepatic transporters and their activity are regulated in various situations, such as ontogenesis, carcinogenesis, cholestasis, cellular stress and after treatment by hormones and xenobiotics. Moreover, a direct correlation between a defect and the absence of transporter with hepatic disease has been demonstrated for BSEP, MDR3-P-gp and MRP2.
Hepatology, 2005
The cellular and subcellular localization and mechanism of transport of the heteromeric organic solute transporter (OST) OST␣-OST was examined in human and rodent epithelia. The two subunits of the transporter were expressed together in human small intestine, kidney, and liver, tissues that also express the apical sodium-dependent bile acid uptake transporter ASBT (SLC10A2). Indirect immunofluorescence microscopy localized OST␣ and OST to the basolateral membrane of mouse, rat, and human ileal enterocytes, renal proximal tubular cells, and cholangiocytes. Transport in OST␣-OSTexpressing Xenopus laevis oocytes was unaffected by depletion of intracellular adenosine triphosphate, or by changes in transmembrane Na ؉ , K ؉ , H ؉ , or Cl ؊ concentration gradients. However, the oocytes demonstrated robust substrate efflux and trans-stimulation, indicating that transport occurs by facilitated diffusion. Madin Darby canine kidney cells coexpressing mouse Ost␣ and Ost exhibited enhanced apical to basolateral transport of the major glycine and taurine conjugated bile acid species. In conclusion, the selective localization of OST␣ and OST to the basolateral plasma membrane of epithelial cells responsible for bile acid and sterol reabsorption, the substrate selectivity of the transporter, and the facilitated diffusion transport mode collectively indicate that OST␣-OST is a key basolateral transporter for the reabsorption of these important steroid-derived molecules. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/ 0270-9139/suppmat/index.html). (HEPATOLOGY 2005;42:1270-1279.) B ile acid and sterol reabsorption by ileal enterocytes, renal proximal tubular cells, and biliary epithelial cells is essential for cholesterol homeostasis, the absorption of dietary fats and vitamins, and proper regulation of bile flow and biliary lipid secretion. 1-4 The initial step in bile acid reabsorption by these epithelial cells-namely, uptake across the apical membrane-is mediated in large part by the apical sodium bile acid transporter (ASBT; gene name SLC10A2). ASBT is most abundant on the apical (luminal) surface of ileal enterocytes, renal proximal tubular cells, and hepatic cholangiocytes. 4 Loss-of-function mutations in the human ASBT gene are associated with bile acid malabsorption, 5 and targeted deletion of the ASBT gene eliminates enterohepatic cycling of bile acids in mice. 2 In the bile duct-obstructed rat, downregulation of renal Asbt facilitates renal excretion of bile acids. 6 In contrast to the apical uptake step, the mechanism for sterol export across the basolateral membrane of these cells is not well defined. However, a recent study demonstrated that ileal basolateral bile acid export may be mediated by the Ost␣-Ost heteromeric transporter, 7 a transporter that was initially identified in the liver of the little skate (Leucoraja erinacea). 8,9 In contrast to all other organic anion transporters identified to date, transport activity requires the coexpression of two distinct gene products: a predicted 340-amino acid, 7-transmembrane
Applied In Vitro Toxicology
The hepatocyte adaptive response has been postulated to play a protective role in cholestatic disease states, including primary biliary cirrhosis where bile acid biliary efflux is reduced. Regulation of bile acid synthesis by farnesoid X receptor (FXR) has been well defined; however, bile acid transport has not. Utilizing sandwichcultured human hepatocytes (SCHH) and B-CLEAR Ò technology, we demonstrated that basolateral efflux and not canalicular efflux (bile salt export pump [BSEP]) was the primary driver of bile acid intracellular accumulation. Following 72 hours of exposure to CDCA, decreases of total endogenous bile acid mass and CYP7A1 mRNA content were observed in SCHH consistent with FXR activation. No marked changes were observed in mRNA content of bile acid uptake transporters, however, induction of bile acid efflux transporters OSTa (3.1-6.8 •), OSTb (21-187 •), and BSEP (2.2-7.5 •) mRNA content was observed. While decreases of d8-TCA biliary clearance were inconsistent with the increases in BSEP mRNA content, decreases in the intracellular concentrations of the model bile acid, d 8-TCA, were observed in SCHH following CDCA exposure. Overall, these data suggest that basolateral efflux of bile acids via OSTa/b is a potentially important compensatory mechanism to prevent cholestatic hepatotoxicity.
Gastroenterology, 1996
The molecular regulation of headenosine triphosphate-dependent and potential-driven patic bile acid transporters during cholestasis is largely bile acid transporters, 4-9 although a recent study suggests unknown. Cloning of complementary DNAs for the sinuthat the latter system may be confined to the endoplasmic soidal sodium-dependent taurocholate cotransporting reticulum. 10 polypeptide (ntcp), the cytosolic bile acid-binding pro-Recently, complementary DNAs (cDNAs) encoding tein 3a-hydroxysteroid dehydrogenase (3a-HSD), and a for a sodium-dependent taurocholate cotransporting putative canalicular bile acid transporter Ca 2/ , Mg 2/polypeptide have been cloned from rat (ntcp) and human ecto-adenosine triphosphatase, now facilitates such liver. 11,12 Expression of the rat liver ntcp in oocytes, transtudies. Methods: Protein mass, steady-state messensiently transfected COS cells, or stably transfected CHO ger RNA (mRNA) levels, and gene transcription were cells resulted in a strictly sodium-dependent saturable assessed in rat livers after common bile duct ligation uptake of taurocholate with a similar Michaelis constant (CBDL) from 1-7 days, and taurocholate uptake was (K m ) of 30-40 mmol/L as previously determined in isodetermined in isolated hepatocytes. Results: After CBDL, Na / -dependent taurocholate uptake (V max ) delated hepatocytes and basolateral plasma membrane prepclined by 70%. The levels of ntcp protein were reduced Abbreviations used in this paper: CBDL, common bile duct ligation;
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2008
Background: The relevance of discrete localization of hepatobiliary transporters in specific membrane microdomains is not well known. Aim: To determine whether the Na + /taurocholate cotransporting polypeptide (Ntcp), the main hepatic sinusoidal bile salt transporter, is localized in specific membrane microdomains. Methods: Presence of Ntcp in membrane rafts obtained from mouse liver was studied by immunoblotting and immunofluorescence. HEK-293 cells stably transfected with rat Ntcp were used for in vitro studies. Expression, localization and function of Ntcp in these cells were assessed by immunoblotting, immunofluorescence and biotinylation studies and Na +-dependent taurocholate uptake assays, respectively. The effect of cholesterol depletion/repletion assays on Ntcp function was also investigated. Results: Ntcp localized primarily to membrane rafts in in vivo studies and localized partially in membrane rafts in transfected HEK-293 cells. In these cells, membrane cholesterol depletion resulted in a shift of Ntcp localization into non-membrane rafts, which correlated with a 2.5-fold increase in taurocholate transport. Cholesterol repletion shifted back part of Ntcp into membrane rafts, and normalized taurocholate transport to values similar to control cells. Conclusion: Ntcp localizes in membrane rafts and its localization and function are regulated by membrane cholesterol content. This may serve as a novel regulatory mechanism of bile salt transport in liver.
Pharmaceutical Research, 2006
Purpose The human apical sodium-dependent bile acid transporter (hASBT) is a potential target for drug delivery, but an understanding of hASBT substrate requirements is lacking. The objective of this study was to characterize hASBT interaction with its native substrates, bile acids, and to evaluate C-24 conjugation and steroidal hydroxylation on transport affinity and inhibition potency. Methods Transport and inhibition kinetics of 15 bile acids were evaluated (cholate, chenodeoxycholate, deoxycholate, ursodeoxycholate, and lithocholate, including their glycine and taurine conjugates) with an hASBT–Madin-Darby canine kidney (MDCK) monolayer assay. Samples were analyzed via liquid chromatography–mass spectrometry (LC-MS) or chromatography–mass spectrometry–mass spectrometry (LC-MS-MS). Results C-24 conjugation improved the inhibitory potency of all native bile acids. There was an inverse association between number of steroidal hydroxyl groups and inhibitory potency. Glycolithocholate and taurolithocholate were the most potent inhibitors. Results from transport studies followed trends from inhibition studies. Conjugated dihydroxy and monohydroxy bile acids exhibited the highest hASBT-mediated transport (i.e., lower K t and higher J max). Across the 15 bile acids, K t generally followed K i. Additionally, J max correlated with K i, where greater inhibition potency was associated with higher transport capacity. Conclusion C-24 conjugation and steroidal hydroxylation pattern modulated native bile acid interaction with hASBT, with C-24 effect dominating steroidal hydroxylation effect. Results indicate that bile acid binding to hASBT may be the rate-limiting step in the apical transport of bile acids.