Bile acid transport across the hepatocyte canalicular membrane (original) (raw)
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Bile acid transport across the hepatocyte canalicular meni.brane
2000
Bile acids, which are present at physiological pH val- ues as bile salts, are synthesized in the hepatocyte from cholesterol and secreted across the canalicular (apical) membrane into bile. Bile salts are taken up from blood across the sinusoidal (basal) membrane, in part metabolized within the hepatocyte, and se- creted into bile. The uphill bile salt transport across the canalicular
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.
Journal of Biological Chemistry, 2001
ABC transporter trafficking in rat liver induced by cAMP or taurocholate and [ 35 S]methionine metabolic labeling followed by subcellular fractionation were used to identify and characterize intrahepatic pools of ABC transporters. ABC transporter trafficking induced by cAMP or taurocholate is a physiologic response to a temporal demand for increased bile secretion. Administration of cAMP or taurocholate to rats increased amounts of SPGP, MDR1, and MDR2 in the bile canalicular membrane by 3-fold; these effects abated after 6 h and were insensitive to prior treatment of rats with cycloheximide. Half-lives of ABC transporters were 5 days, which suggests cycling of ABC transporters between canalicular membrane and intrahepatic sites before degradation. In vivo [ 35 S]methionine labeling of rats followed by immunoprecipitation of (sister of Pglycoprotein) (SPGP) from subcellular liver fractions revealed a steady state distribution after 20 h of SPGP between canalicular membrane and a combined endosomal fraction. After mobilization of transporters from intrahepatic sites with cAMP or taurocholate, a significant increase in the amount of ABC transporters in canalicular membrane vesicles was observed, whereas the decrease in the combined endosomal fraction remained below detection limits in Western blots. This observation is in accordance with relatively large intracellular ABC transporter pools compared with the amount present in the bile canalicular membrane. Furthermore, trafficking of newly synthesized SPGP through intrahepatic sites was accelerated by additional administration of cAMP but not by taurocholate, indicating two distinct intrahepatic pools. Our data indicate that ABC transporters cycle between the bile canaliculus and at least two large intrahepatic ABC transporter pools, one of which is mobilized to the canalicular membrane by cAMP and the other, by taurocholate. In parallel to regulation of other membrane transporters, we propose that the "cAMP-pool" in hepatocytes corresponds to a recycling endosome, whereas recruitment from the "taurocholate-pool" involves a hepatocyte-specific mechanism.
Hepatology, 1994
To study the effect of bile acids on P-glycoproteinmediated drug transport, we performed experiments using multidrug resistant cells and rat canalicular membrane vesicles. Cellular accumulation and efflux of rhodamine 123 were measured in drug-resistant cells by means of computerized quantitative image analysis and fluorescence microscopy. ATP-dependent [3H]daunomycin transport was studied by means of rapid filtration in canalicular membrane vesicles prepared from normal rats. Doxorubicin-sensitive (PSI-2) and-resistant (PNlA) 3T3 cells and human-derived hepatocellular carcinoma doxorubicin-sensitive and-resistant cells were used. Taurochenodeoxycholate and glycochenodeoxycholate, taurolithocholate and ursodeoxycholate (50 to 200 Fmol/L) inhibited rhodamine 123 and [SH]daunomycin transport in multidrug-resistant cells and canalicular membrane vesicles, respectively, whereas taurocholate, taurodeoxycholate and tauroursodeoxycholate did not. Primary and secondary unconjugated bile acids had no effect. These results reveal that taurolithocholate, taurochenodeoxycholate and glycochenodeoxycholate and ursodeoxycholate inhibit P-glycoprotein-mediated drug transport function in multidrug resistant cell lines and in canalicular membrane vesicles. These results suggest possible interaction between P-glycoprotein function and bile acids in cholestasis and after treatment of patients with ursodeoxycholic or chenodeoxycholic acid. (HEPATOLOGY 1994;20:170-176.) Multidrug resistance (MDR) frequently occurs during cancer chemotherapy and can be produced in cell lines in
Second International Ringberg Conference: "Cell Biology and Molecular Basis of Liver Transport
Hepatology, 1996
In the 7 years since the 1st Ringberg conference on ''He-observed system is a Cif-transporter, which means that it is patic Transport of Organic Substances'' was held at Ringbergspecific for purines and uridine. The transporter was cloned Castle near Tegernsee in Bavaria, Germany, the molecular very recently by expression cloning in Xenopus laevis oobasis of transport in the liver has been investigated with cytes. 2 The 2.9-kb complementary DNA of the Na /-dependent great success. Several carrier proteins could be cloned and purine nucleoside transporter SPNT encodes a protein of 659 the molecular properties of hepatic transport systems are amino acid residues and an estimated molecular mass of 72 now better understood, e.g., their function, architecture, kd. Hydropathy analysis reveals 14 putative membrane spanmembrane insertion, expression, and subcellular localization. ning segments. In addition, new insights into the cell biology of hepatocellu-Mdr transporters, synonym P-glycoproteins, were discovlar membrane transport were obtained, including transport ered as plasma membrane transport ATPases that are overregulation by signal-transduction cascades and the involveexpressed in multidrug-resistant tumor cells. Mdr2 is a phosment of transport systems in pH homeostasis and cell volume pholipid translocator, whereas mdr1 and mdr3 confer the regulation. These items were the topics of the 2nd Internatransport of drugs into bile. S. Ruetz (Montréal, Quebec, Cantional Ringberg Conference on ''Cell Biology and Molecular ada) presented results regarding the detailed characteriza-Basis of Liver Transport.'' tion of mdr2-mediated phosphatidylcholine (PC) translocation in the canalicular membrane. Yeast mutants of the sec BIOCHEMISTRY AND MOLECULAR BIOLOGY 6-4 strain, which expressed the mdr2 protein in the mem-OF TRANSPORT brane of yeast secretory vesicles, were generated. By use of the fluorescent PC label 7-nitrobenzoxa-1,3-diazol phosphati-The recent discovery of canalicular primary active transdylcholine, he finds that the mdr2 protein translocates specifport systems that are involved in the vectorial transport of ically PC but not phosphatidylethanolamine or phosphatidylcholephilic compounds into bile has markedly stimulated the serine from the outer leaflet to the inner leaflet of the vesicles. understanding of bile formation. Previous to this era, the This translocation is affected by vanadate and verapamil, but adenosine triphosphatase (ATPase) activity of canalicular it is insensitive to vesicle acidification or membrane depolarmembranes was mainly attributed to a Ca 2/ Mg 2/-ATPase, ization. 3 When the vesicles accumulate taurocholate, PC an ectoenzyme cloned in 1989 by Lin and Guidotti. 1 As retranslocation is stimulated. Fifty micromoles of the bile acid viewed by I. M. Arias (Boston, MA), these enzymes degrade can act as a potential lipid solubilizer, extracting 7-nitroextracellular (intraluminal) adenosine triphosphate (ATP) to benzoxa-1,3-diazol phosphatidylcholine molecules from the adenosine diphosphate and adenosine monophosphate exposed secretory vesicle membrane bilayers, resulting in the (AMP). AMP might be further processed by an ecto-adenoformation of taurocholate/7-nitrobenzoxa-1,3-diazol phosphasine monophosphatase to yield adenosine, which is then tidylcholine aggregates. taken up back into the cell. This chain of substrate degrada-R. P. J. Oude Elferink (Amsterdam, The Netherlands) retion has also been observed in the liver, but, until recently, ported on recent investigations on the function of mouse mdr2 it remained unclear how ATP reaches the canalicular lumen. and mdr1a P-glycoproteins in the canalicular membrane by ATP appears to be transported into bile by the mdr1 transthe use of knockout mouse strains. 4,5 Mdr1a knockout mice port system (see below) and by vesicular exocytosis. Adenoshow normal secretion of bile acids, cholesterol, and phosphosine, the final degradation product of ATP, is then absorbed lipids into bile. The animals suffer from an increased sensitivby a Na /-dependent purine transporter; in the liver, this ity to amphipathic drugs, like ivermectine and vinblastine, transporter is only found in the canalicular membrane. It is which accumulate in the brain. In the mdr2 knockout mice, not inhibited by dipyridamole, but by nucleoside analogues bile contains almost no cholesterol, phospholipids, and glutaincluding arabinoside A, formycin B, zidovudine, and Ara B. thione; however, bile salt secretion is normal. The author According to the classification of nucleoside transporters, the hypothesizes that the mdr2 P-glycoprotein acts as a flippase that translocates PC from the inner (cytosolic) leaflet of the canalicular membrane to specific microdomains in the outer Abbreviations: ATPase, adenosine triphosphatase; ATP, adenosine triphosphate; AMP, leaflet. 6 From there, PC is released into bile by luminal bile adenosine monophosphate; PC, phosphatidylcholine; cMOAT, canalicular multispecific orsalt micelles. This would explain why bile contains PC almost ganic anion transporter; Ntcp, Na /-taurocholate cotransporting polypeptide; mEH, microexclusively and no other membrane phospholipids. Secretion somal epoxide hydrolase; BSP, bromosulfophthalein ; GLUT, glucose transporter; MAP, mitogen-activated protein; RVI, regulatory volume increase; cAMP, cyclic adenosine mono-of both PC and cholesterol are impaired in homozygous mdr2 phosphate; TNF-a, tumor necrosis factor a; BEC, biliary epithelial cells.
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2007
Organic anion transporting polypeptides (OATP/SLCO) are generally believed to function as electroneutral anion exchangers, but direct evidence for this contention has only been provided for one member of this large family of genes, rat Oatp1a1/Oatp1 (Slco1a1). In contrast, a recent study has indicated that human OATP1B3/OATP-8 (SLCO1B3) functions as a GSH-bile acid cotransporter. The present study examined the transport mechanism and possible GSH requirement of the two members of this protein family that are expressed in relatively high levels in the human liver, OATP1B3/OATP-8 and OATP1B1/OATP-C (SLCO1B1). Uptake of taurocholate in Xenopus laevis oocytes expressing either OATP1B1/OATP-C, OATP1B3/OATP-8, or polymorphic forms of OATP1B3/OATP-8 (namely, S112A and/or M233I) was cis-inhibited by taurocholate and estrone sulfate but was unaffected by GSH. Likewise, taurocholate and estrone sulfate transport were trans-stimulated by estrone sulfate and taurocholate but were unaffected by GSH. OATP1B3/OATP-8 also did not mediate GSH efflux or GSH-taurocholate cotransport out of cells, indicating that GSH is not required for transport activity. In addition, estrone sulfate uptake in oocytes microinjected with OATP1B3/OATP-8 or OATP1B1/OATP-C cRNA was unaffected by depolarization of the membrane potential or by changes in pH, suggesting an electroneutral transport mechanism. Overall, these results indicate that OATP1B3/ OATP-8 and OATP1B1/OATP-C most likely function as bidirectional facilitated diffusion transporters and that GSH is not a substrate or activator of their transport activity. membrane transport; anion exchange; driving force; glutathione; organic solute transporters THE CLEARANCE of endogenous substances and xenobiotics from blood is one of the vital functions of the liver. This elaborate system requires specific uptake, biotransformation, and excretion mechanisms. Hepatic uptake is mediated in large part by three types of transporters: the sodium-taurocholate cotransporter (SLC10) (9), organic anion or cation transporters (SLC22A) (6, 15), and organic anion transporting polypeptides (OATP/SLCO) (12, 20). Members of the OATP/SLCO family of solute carriers function as transporters for a large variety of amphipathic organic compounds. These proteins mediate Na ϩ-and ATP-independent transport of bile salts, steroids, thyroid hormones, anionic peptides, drugs, and xenobiotics (7, 8
Transport of bile acids in a human intestinal epithelial cell line, Caco-2
Biochimica et Biophysica Acta (BBA) - General Subjects, 1990
The transport of tanrocholic acid (TA) across Caco-2 cell monolayers was dependent on time in culture and reached a plateau after 28 days, at which time the apical (AP)-to-basolateral (131,) transport was 10-times greater than BL-to-AP transport. The amounts of TA inside the cells following application of 10 nM [I4C]TA to tile AP or BL side of the monolayers (30 min) were approximately equal (54.4 + 2.7 and 64.6 ± 2.8 fmol/mg protein, respectively). AP-to-BL transport of TA was saturable and temperature-dependent. Vm~ , and K m for transport were 13.7 pmol / mg protein per min and 49.7 pM, respectively. The transport of TA had an activation energy of 13.2 kcal-mo1-1, required Na + and glucose. AP-to-BL transport of |14C]TA was inhibited by the co-administration (on the AP side) of either unlabeled TA or deoxycholate, but it was not reduced by the presence of unlabeled TA on the BL side.