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Radiation-inactivation analysis of the Na+/bile acid co-transport system from rabbit ileum.

ABSTRACT: The functional-unit molecular size of the Na+/bile acid cotransport system and the apparent target size of the bile-acid-binding proteins in brush-border membrane vesicles from rabbit ileum were determined by radiation inactivation with high-energy electrons. The size of the functional transporting unit for Na(+)-dependent taurocholate uptake was determined to 451 +/- 35 kDa, whereas an apparent molecular mass of 434 +/- 39 kDa was measured for the Na(+)-dependent D-glucose transport system. Proteins of 93 kDa and 14 kDa were identified as putative protein components of the ileal Na+/bile acid cotransporter in the rabbit ileum, whereas a protein of 87 kDa may be involved in passive intestinal bile acid uptake. Photoaffinity labelling with 3- and 7-azi-derivatives of taurocholate revealed a target size of 229 +/- 10 kDa for the 93 kDa protein, and 132 +/- 23 kDa for the 14 kDa protein. These findings indicate that the ileal Na+/bile acid co-transport system is in its functional state a protein complex composed of several subunits. The functional molecular sizes for Na(+)-dependent transport activity and the bile-acid-binding proteins suggest that the Na+/bile acid co-transporter from rabbit ileum is a homotetramer (AB)4 composed of four AB subunits, where A represents the integral 93 kDa and B the peripheral 14 kDa brush-border membrane protein.

SUBMITTER: Kramer W

PROVIDER: S-EPMC1136507 | biostudies-other | 1995 Feb

REPOSITORIES: biostudies-other

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Inhibition of Na+-taurocholate Co-transporting polypeptide-mediated bile acid transport by cholestatic sulfated progesterone metabolites.

Project description:Sulfated progesterone metabolite (P4-S) levels are raised in normal pregnancy and elevated further in intrahepatic cholestasis of pregnancy (ICP), a bile acid-liver disorder of pregnancy. ICP can be complicated by preterm labor and intrauterine death. The impact of P4-S on bile acid uptake was studied using two experimental models of hepatic uptake of bile acids, namely cultured primary human hepatocytes (PHH) and Na(+)-taurocholate co-transporting polypeptide (NTCP)-expressing Xenopus laevis oocytes. Two P4-S compounds, allopregnanolone-sulfate (PM4-S) and epiallopregnanolone-sulfate (PM5-S), reduced [(3)H]taurocholate (TC) uptake in a dose-dependent manner in PHH, with both Na(+)-dependent and -independent bile acid uptake systems significantly inhibited. PM5-S-mediated inhibition of TC uptake could be reversed by increasing the TC concentration against a fixed PM5-S dose indicating competitive inhibition. Experiments using NTCP-expressing Xenopus oocytes confirmed that PM4-S/PM5-S are capable of competitively inhibiting NTCP-mediated uptake of [(3)H]TC. Total serum PM4-S + PM5-S levels were measured in non-pregnant and third trimester pregnant women using liquid chromatography-electrospray tandem mass spectrometry and were increased in pregnant women, at levels capable of inhibiting TC uptake. In conclusion, pregnancy levels of P4-S can inhibit Na(+)-dependent and -independent influx of taurocholate in PHH and cause competitive inhibition of NTCP-mediated uptake of taurocholate in Xenopus oocytes.

| S-EPMC2878045 | biostudies-literature

Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the ileal Na+-dependent bile-acid transporter.

Project description:Many of the transporters involved in the transport of bile acids in the enterohepatic circulation have been characterized. The basolateral bile-acid transporter of ileocytes and cholangiocytes remains an exception. It has been suggested that rat multidrug resistance protein 3 (Mrp3) fulfills this function. Here we analyse bile-salt transport by human MRP3. Membrane vesicles from insect ( Spodoptera frugiperda ) cells expressing MRP3 show time-dependent uptake of glycocholate and taurocholate. Furthermore, sulphated bile salts were high-affinity competitive inhibitors of etoposide glucuronide transport by MRP3 (IC50 approximately 10 microM). Taurochenodeoxycholate, taurocholate and glycocholate inhibited transport at higher concentrations (IC50 approximately 100, 250 and 500 microM respectively). We used mouse fibroblast-like cell lines derived from mice with disrupted Mdr1a, Mdr1b and Mrp1 genes to generate transfectants that express the murine apical Na+-dependent bile-salt transporter (Asbt) and MRP3. Uptake of glycocholate by these cells is Na+-dependent, with a K(m) and V(max) of 29+/-7 microM and 660 +/- 63 pmol/min per mg of protein respectively and is inhibited by several organic-aniontransport inhibitors. Expression of MRP3 in these cells limits the accumulation of glycocholate and increases the efflux from cells preloaded with taurocholate or glycocholate. In conclusion, we find that MRP3 transports both taurocholate and glycocholate, albeit with low affinity, in contrast with the high-affinity transport by rat Mrp3. Our results suggest that MRP3 is unlikely to be the principal basolateral bile-acid transporter of ileocytes and cholangiocytes, but that it may have a role in the removal of bile acids from the liver in cholestasis.

| S-EPMC1223054 | biostudies-other

Increased Bile Acid Synthesis and Impaired Bile Acid Transport in Human Obesity.

Project description:ContextAlterations in bile acid (BA) synthesis and transport have the potential to affect multiple metabolic pathways in the pathophysiology of obesity.ObjectiveThe objective of the study was to investigate the effects of obesity on serum fluctuations of BAs and markers of BA synthesis.DesignWe measured BA fluctuations in 11 nonobese and 32 obese subjects and BA transporter expression in liver specimens from 42 individuals and specimens of duodenum, jejunum, ileum, colon, and pancreas from nine individuals.Main outcome measuresWe analyzed serum BAs and markers of BA synthesis after overnight fasting, during a hyperinsulinemic-euglycemic clamp, or a mixed-meal tolerance test and the association of BA transporter expression with body mass index.ResultsBA synthesis markers were 2-fold higher (P < .01) and preferentially 12α-hydroxylated (P < .05) in obese subjects, and both measures were correlated with clamp-derived insulin sensitivity (r = -0.62, P < .0001, and r = -0.39, P = .01, respectively). Insulin infusion acutely reduced serum BAs in nonobese subjects, but this effect was blunted in obese subjects (δBAs -44.2% vs -4.2%, P < .05). The rise in serum BAs postprandially was also relatively blunted in obese subjects (δBAs +402% vs +133%, P < .01). Liver expression of the Na+-taurocholate cotransporting polypeptide and the bile salt export pump were negatively correlated with body mass index (r = -0.37, P = .02, and r = -0.48, P = .001, respectively).ConclusionsObesity is associated with increased BA synthesis, preferential 12α-hydroxylation, and impaired serum BA fluctuations. The findings reveal new pathophysiological aspects of BA action in obesity that may lend themselves to therapeutic targeting in metabolic disease.

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Na+ - and Cl- -coupled active transport of nitric oxide synthase inhibitors via amino acid transport system B(0,+).

Project description:Nitric oxide synthase (NOS) inhibitors have therapeutic potential in the management of numerous conditions in which NO overproduction plays a critical role. Identification of transport systems in the intestine that can mediate the uptake of NOS inhibitors is important to assess the oral bioavailability and therapeutic efficacy of these potential drugs. Here, we have cloned the Na+ - and Cl- -coupled amino acid transport system B(0,+) (ATB(0,+)) from the mouse colon and investigated its ability to transport NOS inhibitors. When expressed in mammalian cells, ATB(0,+) can transport a variety of zwitterionic and cationic amino acids in a Na+ - and Cl- -coupled manner. Each of the NOS inhibitors tested compete with glycine for uptake through this transport system. Furthermore, using a tritiated analog of the NOS inhibitor N(G)-nitro-L-arginine, we showed that Na+ - and Cl- -coupled transport occurs via ATB(0,+). We then studied transport of a wide variety of NOS inhibitors in Xenopus laevis oocytes expressing the cloned ATB(0,+) and found that ATB(0,+) can transport a broad range of zwitterionic or cationic NOS inhibitors. These data represent the first identification of an ion gradient-driven transport system for NOS inhibitors in the intestinal tract.

| S-EPMC199559 | biostudies-literature

Functional expression cloning and characterization of the hepatocyte Na+/bile acid cotransport system.

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| S-EPMC52983 | biostudies-other

NPY receptor subtype in the rabbit isolated ileum.

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12α-Hydroxylated bile acid enhances accumulation of adiponectin and immunoglobulin A in the rat ileum.

Project description:We previously reported that dietary supplementation with cholic acid (CA), the primary 12α-hydroxylated (12αOH) bile acid (BA), reduces plasma adiponectin concentration in rats. The aim of this study was to examine the distribution of adiponectin in the body of CA-fed rats and its influence on mucosal immunoglobulin A concentration in the intestine. Rats were fed a diet supplemented with or without CA (0.5 g CA/kg diet) for 13 weeks. A reduction in plasma adiponectin level was observed from week 3. At the end of the experiment, the CA diet reduced plasma adiponectin concentration both in the portal and aortic plasma. Accumulation of adiponectin was accompanied by an increase in cadherin-13 mRNA expression in the ileal mucosa of CA-fed rats. No increase was observed in adiponectin mRNA expression in the ileal and adipose tissues of the CA-fed rats. Immunoglobulin A concentration in the ileal mucosa was elevated in the CA-fed rats and was correlated with the ileal adiponectin concentration. 12αOH BAs may modulate mucosal immune response that are involved in the accumulation of adiponectin in the ileum.

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Structural determinants for transport across the intestinal bile acid transporter using C-24 bile acid conjugates.

Project description:The human apical sodium dependent bile acid transporter (hASBT) reabsorbs gram quantities of bile acid daily and is a potential prodrug target to increase oral drug absorption. In the absence of a high resolution hASBT crystal structure, 3D-QSAR modeling may prove beneficial in designing prodrug targets to hASBT. The objective was to derive a conformationally sampled pharmacophore 3D-QSAR (CSP-SAR) model for the uptake of bile acid conjugates by hASBT. A series of bile acid conjugates of glutamyl chenodeoxycholate were evaluated in terms of K(m) and normalized V(max) (normV(max)) using hASBT-MDCK cells. All monoanionic conjugates were potent substrates. Dianions, cations and zwitterions, which bound with a high affinity, were not substrates. CSP-SAR models were derived using structural and physicochemical descriptors, and evaluated via cross validation. The best CSP-SAR model for K(m) included two structural and two physiochemical descriptors, where substrate hydrophobicity enhanced affinity. A best CSP-SAR model for K(m)/normV(max) employed one structural and three physicochemical descriptors, also indicating hydrophobicity enhanced efficiency. Overall, the bile acid C-24 region accommodated a range of substituted anilines, provided a single negative charge was present near C-24. In comparing uptake findings to prior inhibition results, increased hydrophobicity enhanced activity, with dianions and zwitterions hindering activity.

| S-EPMC2997912 | biostudies-literature

Mutational Analysis of the GXXXG/A Motifs in the Human Na<sup>+</sup>/Taurocholate Co-Transporting Polypeptide NTCP on Its Bile Acid Transport Function and Hepatitis B/D Virus Receptor Function.

Project description:Homodimerization is essential for plasma membrane sorting of the liver bile acid transporter NTCP and its function as Hepatitis B/D Virus (HBV/HDV) receptor. However, the protein domains involved in NTCP dimerization are unknown. NTCP bears two potential GXXXG/A dimerization motifs in its transmembrane domains (TMDs) 2 and 7. The present study aimed to analyze the role of these GXXXG/A motifs for the sorting, function, and dimerization of NTCP. The NTCP mutants G60LXXXA64L (TMD2), G233LXXXG237L (TMD7) and a double mutant were generated and analyzed for their interaction with wild-type NTCP using a membrane-based yeast-two hybrid system (MYTH) and co-immunoprecipitation (co-IP). In the MYTH system, the TMD2 and TMD7 mutants showed significantly lower interaction with the wild-type NTCP. In transfected HEK293 cells, membrane expression and bile acid transport activity were slightly reduced for the TMD2 mutant but were completely abolished for the TMD7 and the TMD2/7 mutants, while co-IP experiments still showed intact protein-protein interactions. Susceptibility for in vitro HBV infection in transfected HepG2 cells was reduced to 50% for the TMD2 mutant, while the TMD7 mutant was not susceptible for HBV infection at all. We conclude that the GXXXG/A motifs in TMD2 and even more pronounced in TMD7 are important for proper folding and sorting of NTCP, and so indirectly affect glycosylation, homodimerization, and bile acid transport of NTCP, as well as its HBV/HDV receptor function.

| S-EPMC8257933 | biostudies-literature

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