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B Hagenbuch

Publications and source records attributed to B Hagenbuch.

69 records · Page 4Linked to original sources
PubMed1994

In situ localization of the hepatocytic Na+/Taurocholate cotransporting polypeptide in rat liver.

BACKGROUND/AIMS: An Na+/taurocholate cotransporting polypeptide (Ntcp) has recently been cloned from rat liver. The aim of this study was to directly characterize the native Ntcp on the protein level and study its in situ distribution in rat liver. METHODS: A rabbit antiserum was raised against a fusion protein containing the maltose-binding protein and the C terminus of Ntcp. Native Ntcp was localized in situ by immunofluorescent techniques. Expression of Ntcp was directly correlated with taurocholate uptake measurements in stably transfected Chinese hamster ovary cells. RESULTS: Native Ntcp showed an apparent molecular weight of 51,000. After deglycosylation of isolated basolateral rat liver plasma membranes, the apparent molecular weight of Ntcp decreased to 33,500. In intact rat liver, Ntcp was selectively localized at the basolateral surface domain of hepatocytes. In short-term cultured hepatocytes, a positive surface immunoreaction was only obtained in detergent-permeabilized cell cultures. In stably transfected Chinese hamster ovary cells, the surface expression of immunopositive Ntcp was associated with Na(+)-dependent taurocholate uptake activity. CONCLUSIONS: Native Ntcp represents a glycoprotein of the basolateral hepatocyte plasma membrane with its C-terminal end facing the intracellular compartment. Furthermore, surface expression of Ntcp is a prerequisite for Na(+)-dependent taurocholate uptake to occur, thus providing further proof for its bile acid transport function in rat liver.

Animals↗
PubMed1994

Expression and characterization of a functional rat liver Na+ bile acid cotransport system in COS-7 cells.

A cDNA for the rat liver sodium-dependent bile acid cotransporter was expressed in COS-7 cells to study the functional properties of the translated protein in a mammalian cell line. A 1.2-kb insert was ligated into a pMAMneo vector and transiently transfected using electroporation. After optimal conditions were established, the transiently transfected COS cells were screened with fluorescent-conjugated labeled bile acids for evidence of expression of the cotransporter after 48 h. The uptake of [3H]taurocholate ([3H]TC) was then determined in cells transfected with or without the bile acid insert. Progressive uptake of [3H]TC (0.45 microM) was observed for 30 min in the presence of sodium. In contrast, no uptake of [3H]TC was observed in the absence of sodium, in nontransfected COS cells, or in COS cells transfected with the empty plasmid. Kinetic studies revealed a Michaelis constant (Km) of 29 microM, essentially identical to the Km of this cotransporter described in intact rat hepatocytes and membrane vesicles. Uptake of [3H]TC (5.0 microM) at 5 min (n = 3-6) was inhibited by 100 microM taurochenodeoxycholic acid (81%), tauroursodeoxycholic acid (77%), cholic acid (55%), chenodeoxycholic acid (74%), and ursodeoxycholic acid (56%) but not by 100 microM taurodehydrocholate, 1 mM probenecid, or 100 microM bilirubin. In contrast, bumetanide (500 microM) inhibited [3H]TC uptake by 52%. These studies indicate that the isolated cDNA codes for a physiological bile acid transporter present in rat hepatocytes and that posttranslational factors present in mammalian cells may not be as important in defining properties of this cotransport system.

Animals↗
PubMed1994

Molecular cloning, chromosomal localization, and functional characterization of a human liver Na+/bile acid cotransporter.

We have used a cDNA probe from a cloned rat liver Na+/taurocholate cotransporting polypeptide (Ntcp) to screen a human liver cDNA library. A 1,599-bp cDNA clone that encodes a human Na+/taurocholate cotransporting polypeptide (NTCP) was isolated. The human NTCP consists of 349 amino acids (calculated molecular mass of 38 kD) and exhibits 77% amino acid homology with the rat Ntcp. In vitro translation experiments indicate that the protein is glycosylated and has a molecular weight similar to the rat Ntcp. Injection of in vitro transcribed cRNA into Xenopus laevis oocytes resulted in the expression of Na(+)-dependent taurocholate uptake. Saturation kinetics indicated that the human NTCP has a higher affinity for taurocholate (apparent Km = 6 microM) than the previously cloned rat protein (apparent Km = 25 microM). NTCP-mediated taurocholate uptake into oocytes was inhibited by all major bile acid derivatives (100 microM), bumetanide (500 microM), and bromosulphophthalein (100 microM). Southern blot analysis of genomic DNA from a panel of human/hamster somatic cell hybrids mapped the human NTCP gene to chromosome 14.

Amino Acid Sequence↗
PubMed1994

Functional characterization of the basolateral rat liver organic anion transporting polypeptide.

To characterize the transport functions of a recently cloned basolateral organic anion transporting polypeptide of rat hepatocytes we performed further kinetic transport and substrate cis-inhibition studies in organic anion-transporting polypeptide-cRNA injected Xenopus laevis oocytes. The studies demonstrate saturable Na(+)-independent sulfobromophthalein (Michaelis-Menten constant, 1.5 mumol/L) and taurocholate (Michaelis-Menten constant, 50 mumol/L) uptake by organic anion-transporting polypeptide. Sulfobromophthalein uptake was inhibited by the following organic anions: 0.01 mmol/L bilirubin (43%), 0.1 mmol/L indocyanine green (81%), 0.1 mmol/L 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 52%) and 1 mmol/L probenecid (74%). Competitive inhibition was shown for indocyanine green (inhibition constant about 1.3 mumol/L). Sulfobromophthalein and taurocholate uptakes were also inhibited by cholate, chenodeoxycholate, deoxycholate and ursodeoxycholate, as well as their glycine and taurine conjugates. Organic anion-transporting polypeptide also mediated uptake of glycocholate, tauroursodeoxycholate and taurochenodeoxycholate. No cis-inhibition of sulfobromophthalein uptake was seen in the presence of ATP, para-aminohippuric acid, bumetanide, digitoxin, reduced glutathione, leukotriene C4, nicotinic acid, ouabain, oxalate, rifampicin, succinate or sulfate. Furthermore, radioactively labeled para-aminohippuric acid, alpha-ketoglutarate and reduced glutathione were not taken up by organic anion-transporting polypeptide in cRNA-injected frog oocytes. These data confirm that organic anion-transporting polypeptide represents a novel hepatocellular organic anion uptake system that can mediate Na(+)-independent transport of monovalent (e.g., bile acids) and divalent (e.g., sulfobromophthalein and indocyanine green) cholephilic organic anions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Direct energization of bile acid transport into plant vacuoles.

Bile acids were shown to be transported into barley mesophyll vacuoles. Uptake of the cholate conjugates taurocholate and glycocholate is strictly ATP-dependent. Uptake of taurocholate is a saturable process (Km = 40 microM) and is inhibited by vanadate but not by bafilomycin, a specific inhibitor of the vacuolar H(+)-ATPase. Together with the observation that the non-hydrolyzable ATP analog AMPPNP (5'-adenylyl beta,gamma-imidodiphosphate) does not stimulate, but rather inhibits, the ATP-dependent uptake of taurocholate, and that a 3-fold accumulation of the bile acid is observed in the presence of bafilomycin, these results suggest that taurocholate is transported into the vacuole by a primary active process as is the case for its canalicular secretion in rat liver (Nishida, T., Gatmaitan, Z., Che, M., and Arias, I. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6590-6594). Taurocholate uptake is inhibited by other bile acids and is slightly stimulated by glutathione S-conjugates. The different responses of the glutathione S-conjugate (Martinoia, E., Grill, E., Tommasini, R., Kreuz, K., and Amrhein, N. (1993) Nature 364, 247-249) and the taurocholate transporters, respectively, to substrates, oligomycin, GTP, and UTP suggest the presence of at least two ATPases specifically involved in the transport of conjugates across the tonoplast. As cholate and its conjugates have so far not been reported to occur in plants, the physiological function of the novel transport ATPase described here is presently unknown.

Adenosine Triphosphatases↗
PubMed1993

Phylogenic and ontogenic expression of hepatocellular bile acid transport.

The phylogenic and ontogenic expression of mRNA for the Na+/bile acid cotransporter was determined by Northern analysis utilizing a full-length cDNA probe recently cloned from rat liver. mRNA was detected in several mammalian species, including rat, mouse, and man, but could not be found in livers from nonmammalian species, including chicken, turtle, frog, and small skate. When expression of the bile acid transporter in developing rat liver was studied, mRNA was detected between 18 and 21 days of gestation, at the time when Na(+)-dependent bile acid transport is first detected. Two hepatoma cell lines (HTC and HepG2), the latter of which is known to have lost the Na+/bile acid cotransport system, also did not express mRNA for this transporter. Finally, when mRNA from the lower vertebrate (the small skate) was injected into Xenopus oocytes, only a sodium-independent, chloride-dependent transport system for bile acids was expressed, confirming the integrity of the mRNA and consistent with prior functional studies of bile acid transport in this species. These findings establish that the Na+/bile acid cotransport mRNA is first transcribed in mammalian species, a process that is recapitulated late during mammalian fetal development in rat liver, and that this mRNA is lost in dedifferentiated hepatocytes. In contrast, the mRNA for a multispecific Na+/independent organic anion transport system is transcribed earlier in vertebrate evolution.

Aging↗
PubMed1993

Parallel decrease of Na(+)-taurocholate cotransport and its encoding mRNA in primary cultures of rat hepatocytes.

We investigated the molecular mechanism underlying the progressive loss of Na(+)-dependent bile salt uptake in primary cultured rat hepatocytes. A specific cDNA probe was used to quantitate the levels of mRNA encoding the Na(+)-taurocholate-cotransporting polypeptide at various culture times. Hepatocytes were cultured on collagen in the presence of insulin (10(-7) mol/L), dexamethasone (10(-7) mol/L) and 10% fetal calf serum for up to 72 hr. During this time period the dissociation constant of Na(+)-dependent taurocholate uptake remained stable (19 to 39 mumol/L), whereas the maximum velocity values decreased from 100% at 3 hr to 55%, 22% and 4% at 24, 48 and 72 hr, respectively. Concomitantly the levels of the Na(+)-taurocholate-cotransporting polypeptide mRNA also decreased from 100% at 3 hr to 41%, 24% and 4% at the later time points. In contrast, Northern hybridization with complementary DNA probes for three common housekeeping gene products revealed a 1.8- to 3.4-fold increase in the levels of mRNA encoding the alpha-subunit of the Na+K(+)-ATPase, beta-actin and glycerol-3-phosphate dehydrogenase. These data indicate that the loss of Na(+)-dependent bile salt uptake in primary cultures of rat hepatocytes is caused by decreased levels of its specific mRNA. Hence the studies further confirm that without specific measures (primary) cultured rat hepatocytes reverse their liver-specific phenotype to a more fetal pattern of gene expression.

Animals↗
PubMed1991

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

Liver parenchymal cells continuously extract high amounts of bile acids from portal blood plasma. This uptake process is mediated by a Na+/bile acid cotransport system. A cDNA encoding the rat liver bile acid uptake system has been isolated by expression cloning in Xenopus laevis oocytes. The cloned transporter is strictly sodium-dependent and can be inhibited by various non-bile-acid organic compounds. Sequence analysis of the cDNA revealed an open reading frame of 1086 nucleotides coding for a protein of 362 amino acids (calculated molecular mass 39 kDa) with five possible N-linked glycosylation sites and seven putative transmembrane domains. Translation experiments in vitro and in oocytes indicate that the transporter is indeed glycosylated and that its polypeptide backbone has an apparent molecular mass of 33-35 kDa. Northern blot analysis with the cloned probe revealed crossreactivity with mRNA species from rat kidney and intestine as well as from liver tissues of mouse, guinea pig, rabbit, and man.

Amino Acid Sequence↗
PubMed1991

Expression of the hepatocellular chloride-dependent sulfobromophthalein uptake system in Xenopus laevis oocytes.

The expression of the basolateral chloride-activated organic anion uptake system of rat hepatocytes has been studied in Xenopus laevis oocytes. Injection of oocytes with rat liver poly(A)+RNA resulted in the functional expression of chloride-dependent sulfobromophthalein (BSP) uptake within 3-5 d. This expressed chloride-dependent BSP uptake system exhibited saturation kinetics (apparent Km approximately 6.2 microM) and efficiently extracted BSP from its binding sites on BSA. Furthermore, the chloride-activated portion of BSP uptake was inhibited by bilirubin (10 microM; inhibition 53%), 4,4'-diisothiocyano-2,2-disulfonic acid stilbene (DIDS, 100 microM; 80%), taurocholate (100 microM; 80%), and cholate (200 microM; 95%). In contrast to results with total rat liver mRNA, injection of mRNA derived from the Na+/bile acid cotransporter cDNA (Hagenbuch, B., B. Stieger, M. Foguet, H. Lübbert, and P. J. Meier. 1991. Proc. Natl. Acad. Sci. USA. In press.) had no effect on BSP uptake into oocytes. Size fractionation of total rat liver mRNA revealed that a 2.0- to 3.5-kb size-class mRNA was sufficient to express the hepatic chloride-dependent BSP uptake system. These data indicate that "expression cloning" in oocytes represents a promising approach to ultimately clone the cDNA coding for the hepatocyte high affinity, chloride-dependent organic anion uptake system. Furthermore, the results confirm that the Na+/bile acid cotransport system does not mediate BSP uptake.

Animals↗
PubMed1990

Expression of the hepatocyte Na+/bile acid cotransporter in Xenopus laevis oocytes.

The expression of the basolateral Na+/bile acid (taurocholate) cotransport system of rat hepatocytes has been studied in Xenopus laevis oocytes. Injection of rat liver poly(A)+ RNA into the oocytes resulted in the functional expression of Na+ gradient stimulated taurocholate uptake within 3-5 days. This Na(+)-dependent portion of taurocholate uptake exhibited saturation kinetics (apparent Km approximately 91 microM) and could be inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene. Furthermore, the expressed taurocholate transport activity demonstrated similar substrate inhibition and stimulation by low concentrations of bovine serum albumin as the basolateral Na+/bile acid cotransport system previously characterized in intact liver, isolated hepatocytes, and isolated plasma membrane vesicles. Finally, a 1.5- to 3.0-kilobase size-class of mRNA could be identified that was sufficient to express the basolateral Na+/taurocholate uptake system in oocytes. These results demonstrate that "expression cloning" represents a promising approach to ultimately clone the gene and to further characterize the molecular properties of this important hepatocellular membrane transport system.

Animals↗
PubMed1987

Sodium-bicarbonate cotransport occurs in rat kidney cortical membranes but not in rat small intestinal basolateral membranes.

Basolateral membrane vesicles were isolated from rat kidney cortex and small intestinal enterocytes. Both membrane preparations show ATP-dependent calcium uptake and cytochalasin B-sensitive D-glucose transport. In renal membranes, sodium influx is stimulated by bicarbonate; bicarbonate-dependent sodium flux is membrane-potential-dependent and inhibited by 4,4'-di-isothiocyanato-2, 2'-stilbenedisulphanic acid ('DIDS'). Small intestinal basolateral membranes do not show bicarbonate-dependent sodium fluxes.

Adenosine Triphosphate↗
PubMed1986

Phosphate transport across the basolateral membrane from rat kidney cortex: sodium-dependence?

Basolateral membrane vesicles were isolated from rat renal proximal tubules by a Percoll-centrifugation method. Transport of phosphate could be stimulated by a sodium gradient but transport of D-glucose was not or only slightly affected by sodium. The Percoll-basolateral membrane fraction showed sodium-independent trans-stimulation of phosphate by itself. To test whether sodium stimulation of phosphate transport is influenced by the crosscontamination of basolateral with brush border membranes, the basolateral membrane fraction obtained by the Percoll-method was applied to free-flow electrophoresis for further purification. Thereby a separation of basolateral from brush border marker enzymes was obtained. It was possible to correlate net sodium-dependent phosphate transport with the brush border marker enzyme activity. ATP-dependent calcium uptake and cytochalasin B sensitive, sodium-independent D-glucose uptake followed basolateral, whereas phlorizin sensitive, sodium-dependent D-glucose followed brush border marker enzymes. Sodium dependent phosphate uptake was inhibited by D-glucose, this inhibition was released by phlorizin. It is concluded that the sodium-dependent phosphate uptake present in the Percoll-basolateral membrane fraction is due to crosscontamination with brush border membranes and that phosphate translocation across the basolateral membrane is carrier-mediated and sodium-independent.

Aminopeptidases↗
PubMed1985

Transport of sulphate in rat jejunal and rat proximal tubular basolateral membrane vesicles.

Basolateral membrane vesicles were isolated by a Percoll density gradient centrifugation method from small intestinal and renal proximal tubular epithelial cells. Transport of sulphate across the basolateral membrane was analyzed by measuring the uptake of tracer sulphate. In both membrane preparations, preloading the vesicles with sulphate- or hydroxyl-anions stimulated tracer sulphate uptake (trans-stimulation); an inwardly directed sodium gradient did not stimulate sulphate influx whether in the absence or in the presence of sulphate- or hydroxyl-ion-trans-stimulation. Under sulphate trans-stimulation conditions, DIDS (10(-4) mol/l) inhibited sulphate influx. In intestinal membranes, trans-stimulation of sulphate influx was obtained by preloading the vesicles with chloride, in renal membranes by preloading with bicarbonate. Under sulphate trans-stimulation conditions, in intestinal membranes, sulphate influx was strongly inhibited by chloride, in renal membranes, chloride inhibition was absent. Under bicarbonate trans-stimulation conditions, in renal membranes, sulphate transport was inhibited by lactate. It is concluded that small intestinal and renal proximal tubular basolateral membrane vesicles contain a transport mechanism for sulphate that cannot be energized by a sodium gradient. The transport system in small intestinal basolateral membranes seems to be different from that in renal membranes. It is suggested that the observed interaction between inorganic and organic anion transport in renal basolateral membranes is indirect.

Animals↗