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Irwin M Arias - One of the best experts on this subject based on the ideXlab platform.
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lkb1 ampk and pka control abcb11 trafficking and polarization in hepatocytes
PLOS ONE, 2014Co-Authors: Jennifer Lippincottschwartz, Laszlo Homolya, Prabuddha Sengupta, Michal Jarnik, Jeanpierre Gillet, Lynn Vitalecross, Silvio J Gutkind, Irwin M AriasAbstract:Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, Taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by Taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not Taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.
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bile acid stimulates hepatocyte polarization through a camp epac mek lkb1 ampk pathway
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Dong Fu, Yoshiyuki Wakabayashi, Jennifer Lippincottschwartz, Irwin M AriasAbstract:This study describes a unique function of Taurocholate in bile canalicular formation involving signaling through a cAMP-Epac-MEK-Rap1-LKB1-AMPK pathway. In rat hepatocyte sandwich cultures, polarization was manifested by sequential progression of bile canaliculi from small structures to a fully branched network. Taurocholate accelerated canalicular network formation and concomitantly increased cAMP, which were prevented by adenyl cyclase inhibitor. The cAMP-dependent PKA inhibitor did not prevent the Taurocholate effect. In contrast, activation of Epac, another cAMP downstream kinase, accelerated canalicular network formation similar to the effect of Taurocholate. Inhibition of Epac downstream targets, Rap1 and MEK, blocked the Taurocholate effect. Taurocholate rapidly activated MEK, LKB1, and AMPK, which were prevented by inhibition of adenyl cyclase or MEK. Our previous study showed that activated-LKB1 and AMPK participate in canalicular network formation. Linkage between bile acid synthesis, hepatocyte polarization, and regulation of energy metabolism is likely important in normal hepatocyte development and disease.
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Taurocholate transport by rat liver sinusoidal membrane vesicles evidence of sodium cotransport
Hepatology, 2007Co-Authors: Masayasu Inoue, Rolf K H Kinne, Thao Tran, Irwin M AriasAbstract:To elucidate the first step in the vectorial transport of bile acids by the liver, plasma membrane vesicles were isolated from rat liver by differential and sucrose-Ficoll density gradient centrifugation. The membranes were selectively enriched 20-fold in Na+,K+−ATPase activity, a marker of sinusoidal plasma membranes. Electron microscopy of pellets from sinusoidal membrane fraction did not reveal other organelles. The initial rate of Taurocholate uptake by the membrane vesicles was stimulated by a sodium gradient but not by a potassium gradient (Naoutside> Nainside). Sodium-dependent uptake of Taurocholate was inhibited at low temperature (0oC), reduced by decreasing intravesicular space, and showed transstimulation in tracer exchange experiments indicating that uptake into vesicles, rather than binding to membranes, was occurring. Sodium-dependent Taurocholate transport by the vesicles exhibited saturation kinetics with respect to Taurocholate (apparent Km = 56 μM, Vmax = 0.65 nmoles per mg × 15 sec at 100 mM NaNO3 and 25oC) and sodium (apparent Km = 48 mM at 50 μM Taurocholate). Other cations, such as lithium and choline, did not replace sodium in its stimulatory action. Sodium-dependent Taurocholate uptake was selectively inhibited by cholic acid and probenecid. When the electrical potential difference across the vesicle membrane was altered by anion replacement, a more negative intravesicular potential inside the vesicles stimulated and a more positive potential inhibited sodium-dependent Taurocholate transport. These data indicate the presence of a Taurocholate-sodium cotransport system in the sinusoidal membranes of rat hepatocytes. This sodium-cotransport system probably participates in sodium-dependent uptake of bile acids into the hepatocyte. Bile acid secretion in the liver can be classified as secondary active transport.
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mechanisms by which camp increases bile acid secretion in rat liver and canalicular membrane vesicles
American Journal of Physiology-gastrointestinal and Liver Physiology, 2003Co-Authors: Suniti Misra, Lyuba Varticovski, Irwin M AriasAbstract:Bile acid secretion induced by cAMP and Taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of Taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent Taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canalicular membrane vesicles increased ATP-dependent transport above baseline activity. In contrast to effects induced by Taurocholate, cAMP-stimulated intracellular trafficking of the canalicular ABC transporters was unaffected by wortmannin, and recruitment of multidrug resistance protein 2, but not bile salt excretory protein (bsep), was partially decreased by colchicine. These studies indicate that trafficking of bsep and other canalicular ABC transporters to the canalicular membrane in response to cAMP is independent of PI3K activity. In addition, PI3K lipid products are required for activation of bsep in the canalicular membrane. These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep, and bile acid secretion.
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phosphoinositide 3 kinase lipid products regulate atp dependent transport by sister of p glycoprotein and multidrug resistance associated protein 2 in bile canalicular membrane vesicles
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Suniti Misra, Peter Ujhazy, Lyuba Varticovski, Irwin M AriasAbstract:Bile acid transport and secretion in hepatocytes require phosphatidylinositol (PI) 3-kinase-dependent recruitment of ATP-dependent transporters to the bile canalicular membrane and are accompanied by increased canalicular PI 3-kinase activity. We report here that the lipid products of PI 3-kinase also regulate ATP-dependent transport of Taurocholate and dinitrophenyl-glutathione directly in canalicular membranes. ATP-dependent transport of Taurocholate and dinitrophenyl-glutathione in isolated canalicular vesicles from rat liver was reduced 50–70% by PI 3-kinase inhibitors, wortmannin, and LY294002, at concentrations that are specific for Type I PI 3-kinase. Inhibition was reversed by addition of lipid products of PI 3-kinase (PI 3,4-bisphosphate and, to a lesser extent, PI 3-phosphate and PI 3,4,5-trisphosphate) but not by PI 4,5-bisphosphate. A membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and leads to activation of PI 3-kinase in macrophages doubled PI 3-kinase activity in canalicular membrane vesicles and enhanced Taurocholate and dinitrophenyl-glutathione transport in canalicular membrane vesicles above maximal ATP-dependent transport. The effect of the peptide was blocked by wortmannin and LY294002. PI 3-kinase activity was also necessary for function of the transporters in vivo. ATP-dependent transport of Taurocholate and PI 3-kinase activity were reduced in canalicular membrane vesicles isolated from rat liver that had been perfused with Taurocholate and wortmannin. PI 3,4-bisphosphate enhanced ATP-dependent transport of Taurocholate in these vesicles above control levels. Our results indicate that PI 3-kinase lipid products are necessary in vivo and in vitro for maximal ATP-dependent transport of bile acid and nonbile acid organic anions across the canalicular membrane. Our results demonstrate regulation of membrane ATP binding cassette transporters by PI 3-kinase lipid products.
Peter J. Meier - One of the best experts on this subject based on the ideXlab platform.
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Stable expression and functional characterization of a Na+-Taurocholate cotransporting green fluorescent protein in human hepatoblastoma HepG2 cells
Cytotechnology, 2000Co-Authors: Gerd A. Kullak-ublick, Bruno Stieger, Manfred G. Ismair, Ralf Kubitz, Marcus Schmitt, Dieter Häussinger, Bruno Hagenbuch, Peter J. Meier, Ulrich Beuers, Gustav PaumgartnerAbstract:Sodium-dependent uptake of bile acids from blood is aliver-specific function which is mediated by theNa^+-Taurocholate cotransporting polypeptide(Ntcp). We report the stable expression of aNa^+-Taurocholate cotransporting green fluorescentfusion protein in the human hepatoblastoma cell lineHepG2, normally lacking Ntcp expression. Ntcp-EGFPassociated green fluorescence colocalized with Ntcpimmunofluorescence in the plasma membrane. Intransfected HepG2 cells, the fusion protein mediatedthe sodium-dependent uptake of the bile acidTaurocholate (K_m: 24.6 μmol/l) and of the anionicsteroids estrone-3-sulfate and dehydroepiandrosteronesulfate. We conclude that the Ntcp-EGFP fusion proteinfollows the sorting route of Ntcp, is functionallyidentical to Ntcp and could be used to monitor proteintrafficking in living HepG2 cells.
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oatp2 mediates bidirectional organic solute transport a role for intracellular glutathione
Molecular Pharmacology, 2000Co-Authors: Peter J. Meier, Nazzareno BallatoriAbstract:One member of the OATP family of transporters, rat Oatp1, functions as an anion exchanger that is driven in part by the glutathione (GSH) electrochemical gradient, indicating that other OATP-related transporters may also be energized by this mechanism. The present study examined whether rat Oatp2 is also an anion exchanger, and, if so, whether it is energized by the GSH electrochemical gradient. As with Oatp1, uptake of 10 microM [(3)H]Taurocholate in Oatp2-expressing Xenopus laevis oocytes was trans-stimulated by intracellular 0.2 mM unlabeled Taurocholate, indicating bidirectional transport. Interestingly, [(3)H]Taurocholate uptake in Oatp2-expressing oocytes was also trans-stimulated when oocytes were preloaded with GSH, S-methylglutathione, S-sulfobromophthalein-glutathione, S-dinitrophenyl glutathione, or ophthalmic acid (a GSH analog) but not by glutarate or N-acetylcysteine, suggesting that GSH derivatives and conjugates may function as intracellular substrates for Oatp2. Support for this hypothesis was provided by the demonstration of enhanced [(3)H]GSH and [(3)H]S-(2,4-dinitrophenyl)-glutathione efflux in Oatp2-expressing oocytes. However, in contrast to Oatp1, extracellular GSH failed to cis-inhibit uptake of [(3)H]Taurocholate or [(3)H]digoxin in Oatp2-expressing oocytes, indicating that the stimulatory effect of high intracellular GSH concentrations is not due to a coupled exchange mechanism. Taken together, the results indicate that Oatp2 mediates bidirectional transport of organic anions by a GSH-sensitive facilitative diffusion mechanism and suggest that this transporter may play a role in cellular export of specific organic molecules.
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rifamycin sv and rifampicin exhibit differential inhibition of the hepatic rat organic anion transporting polypeptides oatp1 and oatp2
Hepatology, 2000Co-Authors: Karin Fattinger, Peter J. Meier, Bruno Hagenbuch, Valentino Cattori, Bruno StiegerAbstract:The antibiotics, rifamycin SV and rifampicin, are known to interfere with hepatic bile salt and organic anion uptake. The aim of this study was to explore which transport systems are affected. In short-term-cultured rat hepatocytes, low concentrations (10 micromol/L) of both compounds inhibited mainly sodium-independent Taurocholate uptake, whereas higher concentrations (100 micromol/L) also inhibited sodium-dependent Taurocholate uptake. In Xenopus laevis oocytes expressing the Na(+)/Taurocholate cotransporting polypeptide (Ntcp), high rifamycin SV and rifampicin concentrations were required for inhibition of Taurocholate uptake. In contrast, sodium-independent Taurocholate uptake mediated by the organic anion transporting polypeptides, Oatp1 and Oatp2, was already substantially inhibited by 10 micromol/L rifamycin SV. Rifampicin potently inhibited Oatp2-mediated Taurocholate uptake, but did not interfere with Oatp1-mediated Taurocholate uptake. Similar effects of rifamycin SV and rifampicin were found for Oatp1- and Oatp2-mediated estradiol-17beta-glucuronide transport. Dixon plot analysis yielded a pattern compatible with competitive inhibition of estradiol-17beta-glucuronide transport with K(i) estimates of 6.6 micromol/L and 7.3 micromol/L for rifamycin SV-induced inhibition of Oatp1 and Oatp2, respectively, and of 1.4 micromol/L for rifampicin-induced inhibition of Oatp2. These results demonstrate that rifamycin SV and rifampicin exhibit differential inhibition on Oatp1 and Oatp2, and identify rifampicin as a selective Oatp2 inhibitor. The data indicate that these inhibitors can be used to determine the in vivo relevance of Oatp1 and Oatp2 for the overall bioavailability and disposition of drugs and other Oatp1/2 substrates.
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identification of glutathione as a driving force and leukotriene c4 as a substrate for oatp1 the hepatic sinusoidal organic solute transporter
Journal of Biological Chemistry, 1998Co-Authors: Thomas K Lee, Peter J. Meier, Nazzareno BallatoriAbstract:oatp1 is an hepatic sinusoidal organic anion transporter that mediates uptake of various structurally unrelated organic compounds from blood. The driving force for uptake on oatp1 has not been identified, although a role for bicarbonate has recently been proposed. The present study examined whether oatp1-mediated uptake is energized by efflux (countertransport) of intracellular reduced glutathione (GSH), and whether hydrophobic glutathione S-conjugates such as leukotriene C4 (LTC4) and S-dinitrophenyl glutathione (DNP-SG) form a novel class of substrates for oatp1. Xenopus laevis oocytes injected with the complementary RNA for oapt1 demonstrated higher uptake of 10 nM [3H]LTC4 and 50 microM [3H]DNP-SG, and higher efflux of [3H]GSH (2.5 mM endogenous intracellular GSH concentration). The oatp1-stimulated LTC4 and DNP-SG uptake was independent of the Na+ gradient, cis-inhibited by known substrates of this transport protein and by 1 mM GSH, and was saturable, with apparent Km values of 0.27 +/- 0.06 and 408 +/- 95 microM, respectively. Uptake of [3H]Taurocholate, an endogenous substrate of oatp1, was competitively inhibited by DNP-SG. Of significance, oatp1-mediated Taurocholate and LTC4 uptake was cis-inhibited and trans-stimulated by GSH, and [3H]GSH efflux was enhanced in the presence of extracellular Taurocholate or sulfobromophthalein, indicating that GSH efflux down its large electrochemical gradient provides the driving force for uptake via oatp1. The stoichiometry of GSH/Taurocholate exchange was 1:1. These findings identify a new class of substrates for oatp1 and provide evidence for GSH-dependent oatp1-mediated substrate transport.
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the sister of p glycoprotein represents the canalicular bile salt export pump of mammalian liver
Journal of Biological Chemistry, 1998Co-Authors: Thomas Gerloff, Bruno Stieger, Bruno Hagenbuch, Jerzy Madon, Lukas Landmann, Jurgen Roth, Alan F Hofmann, Peter J. MeierAbstract:Canalicular secretion of bile salts is a vital function of the vertebrate liver, yet the molecular identity of the involved ATP-dependent carrier protein has not been elucidated. We cloned the full-length cDNA of the sister of P-glycoprotein (spgp; Mr approximately 160,000) of rat liver and demonstrated that it functions as an ATP-dependent bile salt transporter in cRNA injected Xenopus laevis oocytes and in vesicles isolated from transfected Sf9 cells. The latter demonstrated a 5-fold stimulation of ATP-dependent Taurocholate transport as compared with controls. This spgp-mediated Taurocholate transport was stimulated solely by ATP, was inhibited by vanadate, and exhibited saturability with increasing concentrations of Taurocholate (Km approximately 5 microM). Furthermore, spgp-mediated transport rates of various bile salts followed the same order of magnitude as ATP-dependent transport in canalicular rat liver plasma membrane vesicles, i.e. taurochenodeoxycholate > tauroursodeoxycholate = Taurocholate > glycocholate = cholate. Tissue distribution assessed by Northern blotting revealed predominant, if not exclusive, expression of spgp in the liver, where it was further localized to the canalicular microvilli and to subcanalicular vesicles of the hepatocytes by in situ immunofluorescence and immunogold labeling studies. These results indicate that the sister of P-glycoprotein is the major canalicular bile salt export pump of mammalian liver.
Laura Dominguez - One of the best experts on this subject based on the ideXlab platform.
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the toxicity of praziquantel against mesocestoides vogae syn corti tetrathyridia can be assessed using a novel in vitro system
Parasitology Research, 2003Co-Authors: Jenny Saldana, Monica Marin, Cecilia Fernandez, Cecilia Casaravilla, Laura DominguezAbstract:We recently standardised Mesocestoides vogae (syn. corti) tetrathyridia cultures in the presence of sodium Taurocholate. Parasite clustering and segmentation were observed as Taurocholate-dependent effects in biphasic and monophasic media, respectively, and both were inhibited by a specific minimum inhibitory concentration (m.i.c.) of the cestocidal drugs albendazol and praziquantel. In the present study, we analysed the relationship between clustering inhibition and drug toxicity using praziquantel and a mouse experimental infection. In an "in vitro–in vivo" trial, a significant (ANOVA, P<0.05) reduction was observed in the infectivity of tetrathyridia previously cultured with praziquantel m.i.c. (0.06 µg/ml) for 10 days. In an "in vivo–in vitro" trial, the clustering of tetrathyridia recovered from mice treated with praziquantel was found to be markedly reduced: 22%, compared with 83% cluster-containing wells of parasites from control mice. These results show that the outcome of infection and the suppression of Taurocholate-induced clustering provide consistent indications of praziquantel toxicity against M. vogae, an observation confirmed by histological studies. The easily recorded clustering inhibition of M. vogae tetrathyridia in biphasic medium is a potentially useful system for the assessment of drug toxicity against cestode larvae..
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in vitro Taurocholate induced segmentation and clustering of mesocestoides vogae syn corti tetrathyridia cestoda inhibition by cestocidal drugs
Parasitology Research, 2001Co-Authors: Jenny Saldana, Monica Marin, Cecilia Fernandez, Laura DominguezAbstract:Mesocestoides vogae (syn. M. corti) tetrathyridia were cultured in the presence of sodium Taurocholate, for the purpose of exploring the suitability of this organism for the in vitro assay of cestocidal drugs. Parasite clustering and segmentation were observed as Taurocholate-dependent effects in biphasic and monophasic media, respectively. Interestingly, representative members of two major classes of known cestocidal agents (namely, albendazole and praziquantel) blocked these effects. Furthermore, it was possible to determine a specific concentration of the drugs that inhibited clustering and segmentation (minimum inhibitory concentration). In contrast, no inhibition was obtained in the presence of anthelmintics without cestocidal activity. These observations open the way for further studies focused at understanding how the activity of the drugs is involved in the suppression of the Taurocholate-induced effects.
Bruno Stieger - One of the best experts on this subject based on the ideXlab platform.
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transport of estradiol 17β glucuronide estrone 3 sulfate and Taurocholate across the endoplasmic reticulum membrane evidence for different transport systems
Biochemical Pharmacology, 2014Co-Authors: Katrin Wlcek, Lia Hofstetter, Bruno StiegerAbstract:Important reactions of drug metabolism, including UGT mediated glucuronidation and steroidsulfatase mediated hydrolysis of sulfates, take place in the endoplasmic reticulum (ER) of hepatocytes. Consequently, UGT generated glucuronides, like estradiol-17β-glucuronide, have to be translocated back into the cytoplasm to reach their site of excretion. Also steroidsulfatase substrates, including estrone-3-sulfate, have to cross the ER membrane to reach their site of hydrolysis. Based on their physicochemical properties such compounds are not favored for passive diffusion and therefore likely necessitate transport system(s) to cross the ER membrane in either direction. The current study aims to investigate the transport of Taurocholate, estradiol-17β-glucuronide, and estrone-3-sulfate in smooth (SER) and rough (RER) endoplasmic reticulum membrane vesicles isolated from Wistar and TR− rat liver. Time-dependent and bidirectional transport was demonstrated for Taurocholate, showing higher uptake rates in SER than RER vesicles. For estradiol-17β-glucuronide a fast time-dependent efflux with similar efficiencies from SER and RER but no clear protein-mediated uptake was shown, indicating an asymmetric transport system for this substrate. Estrone-3-sulfate uptake was time-dependent and higher in SER than in RER vesicles. Inhibition of steroidsulfatase mediated estrone-3-sulfate hydrolysis decreased estrone-3-sulfate uptake but had no effect on Taurocholate or estradiol-17β-glucuronide transport. Based on inhibition studies and transport characteristics, three different transport mechanisms are suggested to be involved in the transport of Taurocholate, estrone-3-sulfate and estradiol-17β-glucuronide across the ER membrane.
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Effects of bile salt flux variations on the expression of hepatic bile salt transporters in vivo in mice
Journal of hepatology, 2002Co-Authors: Henk Wolters, Bruno Stieger, Baukje M. Elzinga, Julius F. W. Baller, Renze Boverhof, Margrit Schwarz, Henkjan J. VerkadeAbstract:Abstract Background/Aims : Expression of hepatic bile salt transporters is partly regulated by bile salts via activation of nuclear farnesoid X-activated receptor (Fxr). We investigated the physiological relevance of this regulation by evaluating transporter expression in mice experiencing different transhepatic bile salt fluxes. Methods : Bile salt flux was manipulated by dietary supplementation with Taurocholate (0.5% w/w) or cholestyramine (2% w/w) or by disruption of the cholesterol 7α-hydroxylase-gene ( Cyp7A −/− mice) leading to reduced bile salt pool size. Expression of hepatic transporters was assessed (polymerase chain reaction (PCR), immunoblotting, and immunohistochemistry). Results : Biliary bile salt secretion was increased (+350%) or decreased (−50%) after Taurocholate or cholestyramine feeding, respectively, but plasma bile salt concentrations and hepatic Fxr expression were not affected. The bile salt uptake system Na + -Taurocholate co-transporting polypeptide (Ntcp) and organic anion transporting polypeptide-1 (Oatp1) were down-regulated by Taurocholate and not affected by cholestyramine feeding. Cyp7A −/− mice did not show altered Ntcp or Oatp1 expression. Canalicular bile salt export pump (Bsep) was up-regulated by 65% in Taurocholate-fed mice, and slightly down-regulated in Cyp7A −/− mice. Conclusions : Large variations in hepatic bile salt flux have minor effects on expression of murine Ntcp and Bsep in vivo, suggesting that these transporters are abundantly expressed and able to accommodate a wide range of ‘physiological' bile salt fluxes.
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Stable expression and functional characterization of a Na+-Taurocholate cotransporting green fluorescent protein in human hepatoblastoma HepG2 cells
Cytotechnology, 2000Co-Authors: Gerd A. Kullak-ublick, Bruno Stieger, Manfred G. Ismair, Ralf Kubitz, Marcus Schmitt, Dieter Häussinger, Bruno Hagenbuch, Peter J. Meier, Ulrich Beuers, Gustav PaumgartnerAbstract:Sodium-dependent uptake of bile acids from blood is aliver-specific function which is mediated by theNa^+-Taurocholate cotransporting polypeptide(Ntcp). We report the stable expression of aNa^+-Taurocholate cotransporting green fluorescentfusion protein in the human hepatoblastoma cell lineHepG2, normally lacking Ntcp expression. Ntcp-EGFPassociated green fluorescence colocalized with Ntcpimmunofluorescence in the plasma membrane. Intransfected HepG2 cells, the fusion protein mediatedthe sodium-dependent uptake of the bile acidTaurocholate (K_m: 24.6 μmol/l) and of the anionicsteroids estrone-3-sulfate and dehydroepiandrosteronesulfate. We conclude that the Ntcp-EGFP fusion proteinfollows the sorting route of Ntcp, is functionallyidentical to Ntcp and could be used to monitor proteintrafficking in living HepG2 cells.
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rifamycin sv and rifampicin exhibit differential inhibition of the hepatic rat organic anion transporting polypeptides oatp1 and oatp2
Hepatology, 2000Co-Authors: Karin Fattinger, Peter J. Meier, Bruno Hagenbuch, Valentino Cattori, Bruno StiegerAbstract:The antibiotics, rifamycin SV and rifampicin, are known to interfere with hepatic bile salt and organic anion uptake. The aim of this study was to explore which transport systems are affected. In short-term-cultured rat hepatocytes, low concentrations (10 micromol/L) of both compounds inhibited mainly sodium-independent Taurocholate uptake, whereas higher concentrations (100 micromol/L) also inhibited sodium-dependent Taurocholate uptake. In Xenopus laevis oocytes expressing the Na(+)/Taurocholate cotransporting polypeptide (Ntcp), high rifamycin SV and rifampicin concentrations were required for inhibition of Taurocholate uptake. In contrast, sodium-independent Taurocholate uptake mediated by the organic anion transporting polypeptides, Oatp1 and Oatp2, was already substantially inhibited by 10 micromol/L rifamycin SV. Rifampicin potently inhibited Oatp2-mediated Taurocholate uptake, but did not interfere with Oatp1-mediated Taurocholate uptake. Similar effects of rifamycin SV and rifampicin were found for Oatp1- and Oatp2-mediated estradiol-17beta-glucuronide transport. Dixon plot analysis yielded a pattern compatible with competitive inhibition of estradiol-17beta-glucuronide transport with K(i) estimates of 6.6 micromol/L and 7.3 micromol/L for rifamycin SV-induced inhibition of Oatp1 and Oatp2, respectively, and of 1.4 micromol/L for rifampicin-induced inhibition of Oatp2. These results demonstrate that rifamycin SV and rifampicin exhibit differential inhibition on Oatp1 and Oatp2, and identify rifampicin as a selective Oatp2 inhibitor. The data indicate that these inhibitors can be used to determine the in vivo relevance of Oatp1 and Oatp2 for the overall bioavailability and disposition of drugs and other Oatp1/2 substrates.
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the sister of p glycoprotein represents the canalicular bile salt export pump of mammalian liver
Journal of Biological Chemistry, 1998Co-Authors: Thomas Gerloff, Bruno Stieger, Bruno Hagenbuch, Jerzy Madon, Lukas Landmann, Jurgen Roth, Alan F Hofmann, Peter J. MeierAbstract:Canalicular secretion of bile salts is a vital function of the vertebrate liver, yet the molecular identity of the involved ATP-dependent carrier protein has not been elucidated. We cloned the full-length cDNA of the sister of P-glycoprotein (spgp; Mr approximately 160,000) of rat liver and demonstrated that it functions as an ATP-dependent bile salt transporter in cRNA injected Xenopus laevis oocytes and in vesicles isolated from transfected Sf9 cells. The latter demonstrated a 5-fold stimulation of ATP-dependent Taurocholate transport as compared with controls. This spgp-mediated Taurocholate transport was stimulated solely by ATP, was inhibited by vanadate, and exhibited saturability with increasing concentrations of Taurocholate (Km approximately 5 microM). Furthermore, spgp-mediated transport rates of various bile salts followed the same order of magnitude as ATP-dependent transport in canalicular rat liver plasma membrane vesicles, i.e. taurochenodeoxycholate > tauroursodeoxycholate = Taurocholate > glycocholate = cholate. Tissue distribution assessed by Northern blotting revealed predominant, if not exclusive, expression of spgp in the liver, where it was further localized to the canalicular microvilli and to subcanalicular vesicles of the hepatocytes by in situ immunofluorescence and immunogold labeling studies. These results indicate that the sister of P-glycoprotein is the major canalicular bile salt export pump of mammalian liver.
Richard B Kim - One of the best experts on this subject based on the ideXlab platform.
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differential inhibition of rat and human na dependent Taurocholate cotransporting polypeptide ntcp slc10a1 by bosentan a mechanism for species differences in hepatotoxicity
Journal of Pharmacology and Experimental Therapeutics, 2007Co-Authors: Elaine M Leslie, Paul B. Watkins, Richard B KimAbstract:Bile acid accumulation in hepatocytes due to inhibition of the canalicular bile salt export pump (BSEP/ABCB11) has been proposed as a mechanism for bosentan-induced hepatotoxicity. The observation that bosentan does not induce hepatotoxicity in rats, although bosentan has been reported to inhibit rat Bsep and cause elevated serum bile acids, challenges this mechanism. The lack of hepatotoxicity could be explained if bosentan inhibited hepatocyte uptake as well as canalicular efflux of bile acids. In the current study, bosentan was found to be a more potent inhibitor of Na(+)-dependent Taurocholate uptake in rat (IC(50) 5.4 microM) than human (IC(50) 30 microM) suspended hepatocytes. In addition, bosentan was a more potent inhibitor of Taurocholate uptake by rat Na(+)-dependent Taurocholate co-transporting polypeptide (Ntcp/Slc10a1) (IC(50) 0.71 microM) than human NTCP (SLC10A1) (IC(50) 24 microM) expressed in HEK293 cells. Thus, bosentan is a more potent inhibitor of Ntcp than NTCP, and this should result in less intrahepatocyte accumulation of bile acids in rats during bosentan treatment. To begin characterization of this species difference, two chimeric molecules were generated and expressed in HEK293 cells; NTCP(1-140)/Ntcp(141-362) and Ntcp(1-140)/NTCP(141-349). The mode of bosentan inhibition was noncompetitive for Ntcp, and competitive for NTCP (K(i) 18 microM) and NTCP(1-140)/Ntcp(141-362) (K(i) 1.7 microM); bosentan affected both the K(m) and V(max) of Ntcp(1-140)/NTCP(141-349) (K(i) 7.0 microM). The carboxyl portions of NTCP and Ntcp were found to confer species differences in basal Taurocholate transport V(max). In conclusion, differential inhibition of Ntcp and NTCP may represent a novel mechanism for species differences in bosentan-induced hepatotoxicity.
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ritonavir saquinavir and efavirenz but not nevirapine inhibit bile acid transport in human and rat hepatocytes
Journal of Pharmacology and Experimental Therapeutics, 2006Co-Authors: Marypeace Mcrae, Richard B Kim, Carolina M Lowe, Xianbin Tian, David L Bourdet, Brenda F Leake, Kim L R Brouwer, Angela D. M. KashubaAbstract:Human immunodeficiency virus-infected patients on antiretroviral drug therapy frequently experience hepatotoxicity, the underlying mechanism of which is poorly understood. Hepatotoxicity from other compounds such as bosentan and troglitazone has been attributed, in part, to inhibition of hepatocyte bile acid excretion. This work tested the hypothesis that antiretroviral drugs modulate hepatic bile acid transport. Ritonavir (28 μM), saquinavir (15 μM), and efavirenz (32 μM) inhibited [ 3 H]Taurocholate transport in bile salt export pump expressing Sf9-derived membrane vesicles by 90, 71, and 33%, respectively. In sandwich-cultured human hepatocytes, the biliary excretion index (BEI) of [ 3 H]Taurocholate was maximally decreased 59% by ritonavir, 39% by saquinavir, and 20% by efavirenz. Likewise, in sandwich-cultured rat hepatocytes, the BEI of [ 3 H]Taurocholate was decreased 100% by ritonavir and 94% by saquinavir. Sodium-dependent and -independent initial uptake rates of [ 3 H]Taurocholate in suspended rat hepatocytes were significantly decreased by ritonavir, saquinavir, and efavirenz. [ 3 H]Taurocholate transport by recombinant NTCP and Ntcp was inhibited by ritonavir (IC 50 = 2.1 and 6.4 μM in human and rat, respectively), saquinavir (IC 50 = 6.7 and 20 μM, respectively), and efavirenz (IC 50 = 43 and 97 μM, respectively). Nevirapine (75 μM) had no effect on bile acid transport in any model system. In conclusion, ritonavir, saquinavir, and efavirenz, but not nevirapine, inhibited both the hepatic uptake and biliary excretion of Taurocholate.
