Biomedical subjects

B Hagenbuch

Publications and source records attributed to B Hagenbuch.

At least 55 records · Page 3Linked to original sources

PubMed1997

Chlorambucil-taurocholate is transported by bile acid carriers expressed in human hepatocellular carcinomas.

BACKGROUND & AIMS: Chemotherapy of hepatocellular carcinomas is hampered by the insufficient accumulation of cytostatic drugs within the tumor cells. The aim of this study was to evaluate the feasibility of therapeutic strategies using antineoplastic agents coupled to bile acids. METHODS: Expression of the Na(+)-taurocholate-cotransporting polypeptide (NTCP) was analyzed in six hepatocellular carcinomas and in nonmalignant liver tissue. Uptake of the cytostatic drug [3H]-chlorambucil-taurocholate (S2676) was measured in Xenopus laevis oocytes injected with total messenger RNA (mRNA) from the carcinomas or peritumor tissue or with complementary RNA encoding the NTCP or the organic anion-transporting polypeptide (OATP) of human liver. RESULTS: Expression of hepatocellular carcinoma mRNA in oocytes resulted in mainly Na(+)-dependent uptake of chlorambucil-taurocholate. The level of NTCP mRNA in carcinomas amounted to 56% +/- 27% compared with peritumor tissue. Immunofluorescence studies confirmed the expression of NTCP on the surface of hepatocellular carcinoma cells. OATP expression, determined by immunoblotting, was similar in hepatocellular carcinomas and surrounding liver tissue (n = 3). NTCP mediated Na(+)-dependent uptake of chlorambucil-taurocholate (Michaelis constant, 11 mumol/L), whereas OATP mediated Na(+)-independent uptake. CONCLUSIONS: Hepatocellular carcinomas express the Na(+)-dependent bile acid transporter NTCP. Because NTCP mediates high-affinity uptake of chlorambucil-taurocholate, targeting of cytostatic bile acids to hepatocellular carcinomas could become a feasible therapeutic strategy.

Animals↗

PubMed1997

Regulation of hepatocyte bile salt transporters during hepatic regeneration.

Bile formation is an essential liver-specific function, and the hepatic regeneration that occurs in response to hepatocellular injury is often associated with cholestasis. We have employed a partial hepatectomy model to examine the effect of hepatic regeneration on tissue-specific bile salt transporters and on Na(+)-K(+)-adenosinetriphosphatase (ATPase). Liver-specific sodium-dependent taurocholate uptake by basolateral plasma membrane vesicles was undetectable 24 h after hepatectomy. Basolateral membrane protein expression of the sodium-taurocholate cotransporter and gene expression of Ntcp were decreased by > 90% 24 h after partial hepatectomy. In vitro transcription assays demonstrated that Ntcp gene transcription was also markedly reduced. In contrast, hepatic Na(+)-K(+)-ATPase activity, protein expression, and gene expression were unaffected by partial hepatectomy. Similarly, protein and gene expression of the ectoATPase, a putative canalicular bile salt transporter, and canalicular ATP-dependent taurocholate uptake remained unchanged. Partial hepatectomy results in a marked reduction in the gene transcription and expression of the liver-specific Ntcp, as well as a decrease in protein expression and loss of transport activity. These changes provide a potential mechanism for the decrease in hepatocellular bile salt transport that is associated with hepatic regeneration.

Adenosine Triphosphatases↗

PubMed1997

Uptake of 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate into isolated rat hepatocytes by three transport systems.

Uptake of norcholansulfonate (3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate), an isogeometric analogue of cholate into isolated rat liver hepatocytes occurs only by saturable transport. In order to identify the transport systems involved, uptake of norcholansulfonate was studied using 7 beta-NBD-NCT ({N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl})-2'-aminoethanesulfonate) as a competing substrate. For transport of both bile salt derivatives, which mutually inhibit their mediated transport competitively, the existence of at least three transport systems must be assumed. Uptake studies using the cloned hepatic Na+/cholyltaurine cotransporting polypeptide stably expressed in CHO cells (Chinese hamster ovary cells) showed that both bile salt derivatives were transported and furnished the definite KT values of this single transport system and the ratio of the maximal uptake velocities. On the basis of these data, uptake of both bile salt derivatives into rat hepatocytes and their mutual competitive inhibition could be analyzed for three transport systems. The maximal flux rates J2 and the half-saturation constants KT2 in the presence of Na+ (143 mM) are for norcholansulfonate: J1(Na+ 143) = 1.0 +/- 0.2 nmol/(min . mg protein), KT1(Na+ 143) = 15 +/- 4 microM, J2(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT2(Na+ 143) = 15 +/- 2 microM, J3(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT3(Na+ 143) = 60 +/- 15 microM, and for 7 beta-NBD-NCT J1(Na+ 143) = 0.14 +/- 0.04 nmol/(min.mg protein), KT1(Na+ 143) = 3.1 +/- 0.5 microM, J2(Na+ 143) = 0.014 +/- 0.005 nmol/(min.mg protein), KT2(Na+ 143) = 21 +/- 2 microM, J3(Na+ 143) = 1.0 +/- 0.1 nmol/(min.mg protein), KT3(Na+ 143) = 190 +/- 25 microM. The kinetic parameters are in accordance with the assumptions that the cloned Na+/cholyltaurine cotransporting polypeptide represents transport system 2 and that the kinetically identified additional transport system 1 is either strictly or partially Na(+)-dependent.

Animals↗

PubMed1996

Multiple factors regulate the rat liver basolateral sodium-dependent bile acid cotransporter gene promoter.

The hepatic uptake of bile acids from the portal circulation is primarily dependent upon a sodium-dependent basolateral membrane transporter. In order to begin to investigate the factors controlling rat liver sodium-dependent bile acid cotransporter (ntcp) gene expression, we isolated approximately 30 kilobase pairs of rat genomic DNA in three overlapping lambdaphage clones. The rat ntcp gene is distributed over 16.5 kilobase pairs as five exons. Primer extension analysis revealed two closely spaced transcription initiation sites, 27 and 41 nucleotides downstream of a TATA sequence. Regulation of transcription was investigated first by transfection of primary rat hepatocytes by a series of 5'-deleted rat ntcp promoter-driven luciferase constructs (from approximately -6 kilobase pairs to -59 base pairs of upstream sequences, terminating at nucleotide +47), identifying a minimal promoter element: nucleotide -158 to +47. This minimal promoter was active in transfected HepG2, but inactive in NIH3T3, Caco-2, and Madin-Darby canine kidney cells, indicating that the determinants of hepatocyte-specific expression reside within this region. The individual elements within the minimal promoter were investigated via transfection of HepG2 cells by a series of 20 mutant plasmids, each containing a 10-base pair sequential block mutation. Eight mutant constructs profoundly suppressed promoter activity; encompassing sequences from -66 to +4 nt, and +15 to +24 nucleotides, while no other 10-base pair mutation significantly interfered with minimal promoter activity. Deoxyribonuclease I footprint analysis of the minimal promoter revealed three bound regions; -92 to -74 (footprint C), -50 to -37 (footprint B), and -17 to +12 (footprint A). Gel mobility shift assays provided evidence for hepatocyte nuclear factor 1 binding within footprint A and a liver-enriched factor(s) that binds within a novel palindrome in footprint B. These studies indicate that three elements direct the basal and tissue-restricted expression of the rat ntcp promoter; a TATA element, the liver-enriched transcription factor hepatocyte nuclear factor 1, and an unknown liver-enriched factor that binds within a novel palindrome in footprint B.

Alternative Splicing↗

PubMed1996

Effect of antisense oligonucleotides on the expression of hepatocellular bile acid and organic anion uptake systems in Xenopus laevis oocytes.

A Na(+)-dependent bile acid (Na+/taurocholate co-transporting polypeptide; Ntcp) and a Na(+)-independent bromosulphophthalein (BSP)/bile acid uptake system (organic-anion-transporting polypeptide; oatp) have been cloned from rat liver by using functional expression cloning in Xenopus laevis oocytes. To evaluate the extent to which these cloned transporters could account for overall hepatic bile acid and BSP uptake, we used antisense oligonucleotides to inhibit the expression of Ntcp and oatp in Xenopus laevis oocytes injected with total rat liver mRNA. An Ntcp-specific antisense oligonucleotide co-injected with total rat liver mRNA blocked the expression of Na(+)-dependent taurocholate uptake by approx. 95%. In contrast, an oatp-specific antisense oligonucleotide when co-injected with total rat liver mRNA had no effect on the expression of Na(+)-dependent taurocholate uptake, but it blocked Na(+)-independent uptake of taurocholate by approx. 80% and of BSP by 50%. Assuming similar expression of hepatocellular bile acid and organic anion transporters in Xenopus laevis oocytes, these results indicate that Ntcp and oatp respectively represent the major, if not the only, Na(+)-dependent and Na(+)-independent taurocholate uptake systems in rat liver. By contrast, the cloned oatp accounts for only half of BSP transport, suggesting that there must be additional, non-bile acid transporting organic anion uptake systems in rat liver.

Analysis of Variance↗

PubMed1996

Expression of a rat liver phosphatidylcholine translocator in Xenopus laevis oocytes.

A phospholipid translocating protein from rat liver has been expressed in Xenopus laevis oocytes. Injection of oocytes with total rat liver messenger RNA (mRNA) resulted in the function expression of saturable uptake of the water soluble phophatidylcholine derivative L-alpha-dibutyroylglycero-3-phophatidylcholine (diC4PC), Kinetic studies revealed an apparent Km value of approximately 10 mmol/L, which is similar to the value previously obtained in isolated rat liver canalicular plasma membrane vesicles for an adenosine triphosphate (ATP)-independent phosphatidylcholine translocator. Size fractionation of total rat liver mRNA yielded an active mRNA species between 1.8 and 2.6 kb, that stimulated the expressed phophatidylcholine uptake activity approximately fivefold as compared with differently sized mRNA subfractions. This active mRNA size class is too small to code for the mdr2 P-glycoprotein, which has been suggested to function as an ATP-dependent canalicular phosphatidylcholine translocator. Hence, the data indicate that there are at least two separate polypeptides involved in phospholipid translocation from hepatocytes into bile.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1996

The peptide-based thrombin inhibitor CRC 220 is a new substrate of the basolateral rat liver organic anion-transporting polypeptide.

The peptidomimetic thrombin inhibitor CRC 220, 4-methoxy-2,3,6-trimethylphenylsulfonyl-L-aspartyl-D-4-amidinop henylalanyl- piperidide, is taken up into isolated rat hepatocytes through active, carrier-mediated transport. This uptake is inhibited by bile acids. Functional expression in Xenopus laevis oocytes was performed to identify the transport system responsible for the hepatocellular CRC 220 uptake. Injection of poly(A)+RNA in X. laevis oocytes resulted in a two- to three-times higher uptake of CRC 220, compared with uninjected or water-injected control oocytes. Taurocholate (200 mumol/L) inhibited this uptake completely. No uptake of the peptidomimetic thrombin inhibitor was observed, when X. laevis oocytes were injected with complementary RNA (cRNA) encoding either the cloned rat liver Na(+)-dependent taurocholate transporter Ntcp, the renal oligopeptide carrier rhaPT or the intestinal oligopeptide transporter PepT1. However, after injection of cRNA of the cloned rat liver Na(+)-independent organic anion transporting polypeptide oatp, a specific and saturable CRC 220 uptake was observed (Michaelis-Menten constant 29.5 mumol/L). Cis-inhibition with known oatp-substrates, e.g., 20 mumol/L Bromsulphalein (BSP), 2007 mumol/L taurocholate and 2007 mumol/L cholate, occurred in oatp-expressing X. laevis oocytes, whereas substrates of the two peptide carriers as well as dipeptide- and single-amino acid constituents of the thrombin inhibitor itself lacked any significant inhibitory effects. These data show that the modified dipeptide CRC 220 is a highly selective substrate of the organic anion transporting polypeptide oatp in the basolateral plasma membrane of rat hepatocytes.

Animals↗

PubMed1996

Sinusoidal (basolateral) bile salt uptake systems of hepatocytes.

Sinusoidal (basolateral) bile sale uptake is mediated by Na(+)-dependent and Na(+)-independent transport systems. Two hepatocellular bile salt uptake systems have been cloned from rat and human livers. The Na(+)-taurocholate cotransporting polypeptides Ntcp and NTCP mediate strictly Na(+)-dependent bile salt uptake into rat and human hepatocytes, respectively. Extensive characterization of Ntcp expression and function in a variety of eukaryotic cell lines, cultured hepatocytes, and intact rat liver indicates that Ntcp can account for most, if not all, Na(+)-dependent bile salt transport functions in rat liver. Whether the same is also true for the human NTCP is less well understood. The Na(+)-independent organic anion transporting polypeptides oatpl (rat) and OATP (human) exhibit a wide and charge-independent substrate specificity. Their transported substrates include sulfobromophthalein, bile salts, estrone-3-sulfates, ouabain, and other neutral steroids, as well as certain amphipathic organic cations. Its broad and charge-independent substrate specificity indicates that oatpl represents the previously suggested hepatocellular "multispecific bile salt transporter.". Oatpl and OATP are also expressed in the kidney and brain. Whether there are additional oatpl- and OATP-related proteins involved in overall hepatic drug and steroid clearance remains to be determined.

Animals↗

PubMed1996

Polyspecific drug and steroid clearance by an organic anion transporter of mammalian liver.

An organic anion-transporting polypeptide that mediates sodium-independent uptake of negatively charged sulfobromophthalein and bile salts has recently been cloned from rat liver (Jacquemin et al., 1994). In this study we have extended the substrate specificity studies to neutral and positively charged organic compounds with use of the Xenopus laevis oocyte expression systems. We found that the same transporting polypeptide can also transport the neutral cardiac glycoside ouabain (apparent Km approximately 1.7mM); the endogenous steroids aldosterone (Km approximately 15nM), cortisol (Km approximately 13 microM) and dexamethasone; the anionic steroid-conjugates estrone-3-sulfate (Km approximately 4.5 microM) and estradiol-17-glucuronide (Km approximately 3.0 microM) and the exogenous amphipathic organic cation N-(4,4-azo-n-pentyl)-21-deoxyajmalinium, a permanently charged photolabile derivative of the antiarrhythmic drug N-propylajmaline. These data demonstrate that the previously cloned hepatic organic anion-transporting polypeptide can in fact transport a wide range of differently charged lipophilic organic compounds including exogenous and endogenous organic anions, neutral steroids and organic cations. Hence, a single sinusoidal (or basolateral) transporting polypeptide can account, at least in part, for charge-independent steroid and drug clearance in mammalian liver.

Aldosterone↗

PubMed1996

Uptake of the mycotoxin ochratoxin A in liver cells occurs via the cloned organic anion transporting polypeptide.

Ochratoxin A (OTA) is a mycotoxin produced by mold. It mainly causes nephropathies in humans and domestic animals as a major pathogenic contaminant of cereals and animal feed. Upon p.o. uptake and intestinal absorption, a large part of OTA is taken up by hepatocytes and eliminated into bile. In the present study, hepatocellular uptake of radiolabeled [3H]OTA in isolated rat hepatocytes was characterized; a saturable (K(m) = 18.9 microM, Vmax = 473 pmol/mg/min), temperature (Aapp = 30.4 and 76.6 kilo Joule/mol) and energy-dependent mycotoxin transport was found. This OTA uptake was inhibited by various bile acids, sulfobromophthalein and the thrombin inhibitor CRC 220. Because all inhibitors are substrates of the organic anion-transporting polypeptide (oatp), a recently cloned hepatic carrier, uptake experiments were performed in oatp-cRNA-injected Xenopus laevis oocytes. These studies revealed an oatp-specific OTA uptake (K(m) = 16.6 microM). In contrast, OTA was not transported by the hepatic Na+/taurocholate-cotransporting polypeptide. Known oatp substrates cis-inhibited OTA uptake in oatp-cRNA-injected oocytes in close correlation with the results derived from isolated hepatocytes. These results identify OTA as a new substrate for oatp. They further support the multispecific nature of oatp-mediated transport and stress the importance of this carrier for hepatic clearance of xenobiotics.

Animals↗

PubMed1995

Stable inducible expression of a functional rat liver organic anion transport protein in HeLa cells.

Recently we expression cloned a rat liver organic anion transport protein in Xenopus laevis oocytes (Jacquemin, E. Hagenbuch, B, Stieger, B., Wolkoff, A.W., and Meier, P.J.,(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 133-137). In the present study, we have stably transfected the cDNA encoding this protein into HeLa cells by using a vector containing a zinc-inducible promoter. The parent cells have virtually no baseline transport of [35S]sulfobromophthalein, whereas the induced transfected cells express a novel 74-kDa protein and avidly transport this ligand. Transport by these cells is saturable (Km = 3.3 microM, Vmax = 257 pmol/min/mg protein), bidirectional, and highly temperature-dependent. In the presence of albumin, uptake of [35S]sulfobromophthalein requires the presence of extracellular Cl, whereas in the absence of albumin, this C1- dependence is not seen. These studies indicate that cellular uptake of sulfobromophthalein does not result from direct interaction with the plasma membrane lipid bilayer but rather requires the presence of a specific plasma membrane transporter.

Albumins↗

PubMed1995

Characterization of the endogenous carnitine transport and expression of a rat renal Na(+)-dependent carnitine transport system in Xenopus laevis oocytes.

L-Carnitine transport was characterized in Xenopus laevis oocytes before and after injection of mRNA isolated from rat renal cortex. Non-injected oocytes revealed endogenous Na(+)-dependent transport of L-carnitine. After injection of 15 ng of rat kidney mRNA, the Na(+)-dependent L-carnitine transport increased 2-3-fold, reaching maximal activity after 5-6 days. The expressed carnitine transport was maximal at pH 7.5, whereas the endogenous transport showed no clear maximum between pH 6.0 and 8.5. Kinetic analysis revealed apparent Km values for L-carnitine of 66 microM for the endogenous and 149 microM for the expressed transport. Trimethyl-lysine and D-carnitine inhibited both the endogenous and the expressed transport. In contrast, L-acetylcarnitine, L-isovalerylcarnitine, L-palmitoylcarnitine and butyrobetaine inhibited predominantly the expressed transport, whereas glycinebetaine had no inhibitory effect on either transport system. Size-fractionated rat renal-cortex mRNA (median size 2 kb) induced a 3-fold higher L-carnitine transport than did unfractionated mRNA. These studies demonstrate that Xenopus laevis oocytes exhibit Na(+)-dependent L-carnitine transport and provide the basis for expression-cloning of a rat renal Na(+)-dependent L-carnitine transport system.

Animals↗

PubMed1995

Molecular and functional characterization of an organic anion transporting polypeptide cloned from human liver.

BACKGROUND & AIMS: Based on a recently cloned rat liver organic anion transporter, we attempted to clone the corresponding human liver organic anion transporting polypeptide. METHODS: A human liver complementary DNA library was screened with a specific rat liver complementary DNA probe. The human liver transporter was cloned by homology with the rat protein and functionally characterized in Xenopus laevis oocytes. RESULTS: The cloned human liver organic anion transporting polypeptide consists of 670 amino acids and shows a 67% amino acid identity with the corresponding rat liver protein. Injection of in vitro transcribed complementary RNA into frog oocytes resulted in the expression of sodium-independent uptake of [35S]bromosulfophthalein (Michaelis constant [Km], approximately 20 mumol/L), [3H]cholate (Km, approximately 93 mumol/L), [3H]taurocholate (Km, approximately 60 mumol/L), [14C]glycocholate, [3H]taurochenodeoxycholate, and [3H]tauroursodeoxycholate (Km, approximately 19 mumol/L). Northern blot analysis showed cross-reactivity with messenger RNA species from human liver, brain, lung, kidney, and testes. Polymerase chain reaction analysis of genomic DNA from a panel of human-rodent somatic cell hybrids mapped the cloned human organic anion transporter to chromosome 12. CONCLUSIONS: These studies show that the cloned human liver organic anion transporter is closely related to, but probably not identical to, the previously cloned rat liver transporter. Furthermore, its additional localization in a variety of extrahepatic tissues suggests that it plays a fundamental role in overall transepithelial organic anion transport of the human body.

Amino Acid Sequence↗

PubMed1995

Expression of sodium-independent organic anion uptake systems of skate liver in Xenopus laevis oocytes.

The expression of the basolateral sodium-independent organic anion uptake system of the little skate (Raja erinacea) has been studied in Xenopus laevis oocytes. Injection of oocytes with skate liver poly(A)+ RNA resulted in the functional expression of chloride-dependent sulfobromophthalein (BSP) uptake and sodium-independent taurocholate uptake within 3-5 days. The expressed chloride-dependent BSP uptake activity exhibited saturation kinetics [apparent Michaelis constant (Km) 1.8 microM] and efficiently extracted BSP from its binding sites on bovine serum albumin. The chloride-sensitive portion of BSP uptake was inhibited by bilirubin (10 microM; 27%), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (100 microM; 57%), bumetanide (100 microM; 48%), taurocholate (200 microM; 51%), and cholate (200 microM; 45%). Size fractionation of total skate liver mRNA revealed that a 1.8- to 2.9-kb size class mRNA was sufficient to express chloride-dependent BSP uptake and sodium-independent taurocholate uptake. In addition, a 1- to 1.7-kb size class mRNA expressed sodium-independent taurocholate uptake but had no effects on BSP uptake. This study confirms that an organic anion transport system for chloride-dependent BSP uptake, with characteristics similar to rat liver, is already expressed in the liver of lower vertebrates and thus represents a phylogenetically old system. Sodium-independent taurocholate uptake in skate liver may be mediated by two different transport systems.

Animals↗

PubMed1994

Prolactin increases hepatic Na+/taurocholate co-transport activity and messenger RNA post partum.

We have shown that Na+/taurocholate co-transport activity is decreased in pregnancy, but rebounds post partum relative to non-pregnant controls, and that activity can be increased by treatment with ovine prolactin [Ganguly, Hyde and Vore (1993) J. Pharmacol. Exp. Ther. 267, 82-87]. To determine the basis for these effects, Na+/taurocholate co-transport was determined in purified basolateral liver plasma-membrane (bLPM) vesicles and compared with steady-state mRNA levels encoding the Na+/taurocholate-co-transporting polypeptide (Ntcp) in non-pregnant controls, pregnant rats (19-20 days pregnant), rats post partum (48 h post partum) and rats post partum treated with bromocriptine to inhibit prolactin secretion. Na+/taurocholate co-transport activity (nmol/5 s per mg of protein) in bLPM was decreased from 10.4 +/- 1.8 in non-pregnant controls to 7.9 +/- 0.6 in bLPM in pregnant rats, but rebounded to 17.5 +/- 1.3 post partum; treatment of rats post partum with bromocriptine to inhibit prolactin secretion decreased activity to 14.1 +/- 0.9. Northern and slot-blot analyses revealed similar changes in mRNA for Ntcp, so that a positive correlation was observed between Na+/taurocholate co-transport activity and Ntcp mRNA. Furthermore, treatment of ovariectomized rats with ovine prolactin increased Ntcp mRNA 10-fold compared with solvent-treated controls, consistent with the 2-fold increase in Vmax, for Na+/taurocholate co-transport in isolated hepatocytes. These data are the first to demonstrate endogenous physiological regulation by prolactin of Ntcp mRNA in parallel with Na+/taurocholate co-transport activity.

Animals↗

PubMed1994

Functional expression cloning of the canalicular sulfate transport system of rat hepatocytes.

We have cloned a single cDNA encoding the canalicular sulfate transporter of rat liver using Xenopus laevis oocytes as a functional expression system. The cloned cDNA sulfate anion transporter-1 (sat-1) expresses saturable Na(+)-independent sulfate uptake (Km approximately 0.14 mM) that can be inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS, IC50 = 28 microM) and oxalate, but not by succinate or cholate. These properties are very similar to sulfate uptake expressed in oocytes injected with total rat liver mRNA and to the bicarbonate/sulfate exchange system previously characterized in canalicular rat liver plasma membrane vesicles. The cloned sat-1 cDNA has a total length of 3726 base pairs (bp) with an open reading frame encompassing 2109 bp, a 5'-untranslated region of 367 bp, and a 3'-untranslated region of 1250 bp. The coding region predicts a protein of 703 amino acids with a calculated molecular mass of 75.4 kDa. Computer-based hydrophobicity analysis suggests the presence of 12 putative transmembrane spanning domains. Furthermore, three potential glycosylation sites are detected (Asn-158, Asn-163, Asn-587). Northern blot analysis indicates that similar sulfate anion transporters are also present in the kidney, muscle, and brain of rat and in the liver of the mouse. Using antisense oligonucleotides the mRNA-species of the sat-1 analogue in rat kidney has been characterized by hybrid depletion experiments (Markovich, D., Bissig, M., Sorribas, V., Hagenbuch, B., Meier, P. J., and Murer, H. (1994) J. Biol. Chem. 269, 3022-3026).

Amino Acid Sequence↗

PubMed1994

Expression of rat renal sulfate transport systems in Xenopus laevis oocytes. Functional characterization and molecular identification.

Renal proximal tubular sulfate reabsorption is mediated by brush border membrane Na+/sulfate-cotransport and basolateral Na(+)-independent sulfate transport. Injection of rat kidney cortex mRNA into Xenopus laevis oocytes induced Na(+)-dependent as well as Na(+)-independent sulfate transport. The inhibition pattern of Na(+)-dependent uptake coincided with that known for the brush border membrane; the inhibition pattern of Na(+)-independent uptake suggested that this activity could be related to the basolateral cell surface. By Northern blot hybridization of size-fractionated mRNA, we provide evidence that the Na(+)-dependent uptake is induced by an mRNA species related to a recently cloned cDNA encoding rat renal cortex Na+/SO4 cotransport (NaSi-1; Markovich, D., Forgo, J., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8073-8077); the Na(+)-independent sulfate transport activity seems to be related to an mRNA species encoding a rat liver Na(+)-independent sulfate transporter (Bissig, M., Hagenbuch, B., Stieger, B., Koller, T., and Meier, P. J. (1994) J. Biol. Chem. 269, 3017-3021). Hybrid depletion experiments using antisense oligonucleotides provided further evidence for the association of the expressed transport activities to NaSi-1 and sat-1, respectively.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1994

Expression cloning of a rat liver Na(+)-independent organic anion transporter.

Using expression cloning in Xenopus laevis oocytes, we have isolated a cDNA encoding a rat liver organic anion-transporting polypeptide (oatp). The cloned oatp mediated Na(+)-independent uptake of sulfobromophthalein (BSP) which was Cl(-)-dependent in the presence of bovine serum albumin (BSA) at low BSP concentrations (e.g., 2 microM). Addition of increasing amounts of BSA had no effects on the maximal velocity of initial BSP uptake, but it increased the Km value from 1.5 microM (no BSA) to 24 microM (BSA/BSP molar ratio, 3.7) and 35 microM (BSA/BSP ratio, 18.4). In addition to BSP, the cloned oatp also mediated Na(+)-independent uptake of conjugated (taurocholate) and unconjugated (cholate) bile acids. Sequence analysis of the cDNA revealed an open reading frame of 2010 nucleotides coding for a protein of 670 amino acids (calculated molecular mass, 74 kDa) with four possible N-linked glycosylation sites and 10 putative transmembrane domains. Translation experiments in vitro indicated that the transporter was indeed glycosylated and that its polypeptide backbone had an apparent molecular mass of 59 kDa. Northern blot analysis with the cloned probe revealed crossreactivity with several mRNA species from rat liver, kidney, brain, lung, skeletal muscle, and proximal colon as well as from liver tissues of mouse and rabbit, but not of skate (Raja erinacea) and human.

Amino Acid Sequence↗