Two different mRNAs from rat liver code for the transport of bumetanide and taurocholate in Xenopus laevis oocytes

Honscha, Walther; Schulz, Kirsten; Müller, Doris; Petzinger, Ernst

doi:10.1016/0922-4106(93)90035-8

Cited by 17 publications

(12 citation statements)

References 13 publications

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“…Studies utilizing X. laevis oocytes have also suggested the existence of a mRNA fraction that upon oocyte injection results in the expression of a protein that mediates the sodium-dependent uptake of cholate and bumetanide as well as taurocholate. Oocytes expressing Ntcp, in contrast, preferentially transport taurocholate, have a reduced transport capacity for cholate and a negligible uptake of bumetanide, clearly establishing the existence of two distinct transporters (4). Further evidence that the observed sodium-dependent uptake of bile acids is a result of the contribution from more than one transport system is derived from the antibody protection experiments described in Table I.…”

Section: Discussionmentioning

confidence: 72%

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The Functional Expression of Sodium-dependent Bile Acid Transport in Madin-Darby Canine Kidney Cells Transfected with the cDNA for Microsomal Epoxide Hydrolase

Dippe

Amoui

Stellwagen

et al. 1996

Journal of Biological Chemistry

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Previous studies have suggested that the enzyme microsomal epoxide hydrolase (mEH) is able to mediate sodium-dependent transport of bile acids such as taurocholate into hepatocytes (von Dippe, P., Amoui, M., Alves, C., and Levy, D. (1993) Am. J. Physiol. 264, G528 -G534). In order to characterize directly the putative transport properties of the enzyme, a pCB6 vector containing the cDNA for this protein (pCB6-mEH) was transfected into Madin-Darby canine kidney (MDCK) cells, and stable transformants were isolated that could express mEH at levels comparable with the levels expressed in hepatocytes. Sodium-dependent transport of taurocholate was shown to be dependent on the expression of mEH and to be inhibited by the bile acid transport inhibitor 4,4-diisothiocyanostilbene-2,2disulfonic acid (DIDS), as well as by other bile acids. Kinetic analysis of this system indicated a K m of 26.3 M and a V max of 117 pmol/mg protein/min. The K m value is essentially the same as that observed in intact hepatocytes. The transfected MDCK cells also exhibited sodium-dependent transport of cholate at levels 150% of taurocholate in contrast to hepatocytes where cholate transport is only 30% of taurocholate levels, suggesting that total hepatocyte bile acid transport is a function of multiple transport systems with different substrate specificities, where mEH preferentially transports cholate. This hypothesis is further supported by the observation that a monoclonal antibody that partially protects (26%) taurocholate transport from inhibition by DIDS in hepatocytes provides almost complete protection (88%) from DIDS inhibition of hepatocyte cholate transport, suggesting that taurocholate is also taken up by an alternative system not recognized by this antibody. Additional support for this concept is provided by the observation that the taurocholate transport system is almost completely protected (92%) from DIDS inhibition by this antibody in MDCK cells that express mEH as the only bile acid transporter. These results demonstrate that mEH is expressed on the surface of hepatocytes as well as on transfected MDCK cells and is able to mediate sodiumdependent transport of taurocholate and cholate.

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Section: Discussionmentioning

confidence: 72%

“…These systems have been studied in perfused liver, intact hepatocytes, and sinusoidal plasma membrane vesicles. Kinetic studies have suggested the parallel functioning of several distinct sodium-dependent organic anion transporters with different substrate specificity profiles (3)(4)(5).…”

mentioning

confidence: 99%

The Functional Expression of Sodium-dependent Bile Acid Transport in Madin-Darby Canine Kidney Cells Transfected with the cDNA for Microsomal Epoxide Hydrolase

Dippe

Amoui

Stellwagen

et al. 1996

Journal of Biological Chemistry

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“…Although cis-inhibition studies in hepatocytes revealed an interaction between the transport of TC and bumetanide (Petzinger et al, 1989;Blitzer et al, 1982), the transport of bumetanide was found to be predominantly mediated by other transporter(s); using oocytes, it was found that Na + -dependent transport of TC and bumetanide is coded by different mRNA fractions in rat liver (Honscha et al, 1993). In Ntcptransfected CHO cells, the uptake of bumetanide was not significant, although the Ntcp-mediated transport of TC was inhibited by this ligand (Schroeder et al, 1998).…”

Section: Molecular Cloning and Functional Analysis Of Ntcpmentioning

confidence: 94%

Transporters for Bile Acids and Organic Anions

Suzuki

Sugiyama

2002

Pharmaceutical Biotechnology

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“…The handling of the oocytes and the uptake experiments has been described. 53,54 Computer Programs. For the assembly of the sequencing files the program DNASTAR (Lasergene, Madison, WI) was used.…”

Section: Methodsmentioning

confidence: 99%

Cloning and functional characterization of the bile acid-sensitive methotrexate carrier from rat liver cells

Honscha¹,

Dötsch²,

Thomsen³

et al. 2000

Hepatology

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The antifolates methotrexate and trimetrexate are potent inhibitors of the dihydrofolate reductase, an enzyme that delivers by the metabolism of folate and its reduced forms the C1-groups for the synthesis of nucleotides and amino acids. Methotrexate, which has a comparatively moderate toxicity is one of the most important clinically used chemotherapeutics for the treatment of leukemias, such as acute lymphoblastic leukemia of children. It is also used in breast cancer, squamous tumors of the head and neck, lymphomas, and choriocarcinomas. 1,2 Uptake studies of methotrexate and the natural folic acid derivatives in different tumor and normal cells have shown that at least two different carrier systems mediate the uptake of these compounds. The 40 kd folate binding protein has a high affinity for folic acid but a low affinity for reduced folates. [3][4][5][6][7][8][9][10] The corresponding complementary DNAs (cDNAs) as well as genomic clones have been isolated from various sources, 11-19 but a detailed characterization of the transporter function is still missing.Different cDNAs and genomic clones for the reduced folate carrier, which has a high affinity for the reduced folates 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and methotrexate, but a low affinity for folate [20][21][22][23][24] have been also isolated from different immortal cell lines 25-31 but the clones were not characterized in detail.The expression and the abundance of the two carriers varied in different tumor and normal cells. From uptake studies it became evident that, first, in some cell systems both transport systems work in tandem 32 whereas two independently operating carrier systems were also described. 33,34 Second, tumor cells differ from normal cells in their transport properties. [35][36][37][38][39] Results from Chello et al. 40 showed, for example, a 30-fold higher affinity for methotrexate in the mouse leukemia cell line L1210 than in freshly isolated murine intestinal cells.Hepatocytes are exceptional with respect to folate transport. In contrast to most other cells, hepatocytes express a separate carrier system for 5-methyltetrahydrofolate, which is different from the reduced folate transporter. 41,42 We recently characterized the uptake of methotrexate into freshly isolated hepatocytes 43 and showed that this chemotherapeutic is taken up by a sodium-dependent active transport system, which is inhibited by the reduced folates, dihydrofolate, and tetrahydrofolate but not by folate itself. Moreover, substrates of two well-known organic anion transport systems in the liver, the Ntcp1 44,45 and the oatp1, 46 like the bile acids taurocholate and cholate as well as xenobiotics such as bromosulfophthalein, the loop diuretic bumetanide, and the mycotoxin ochratoxin A are potent inhibitors of the hepatocellular methotrexate carrier. Because some substrates were Abbreviations: cDNA, complementary DNA; RL-Mtx, rat liver methotrexate transporter; RACE-PCR, rapid amplification of cDNA ends polymerase chain reaction; DMEM, Dulbecco modi...

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Two different mRNAs from rat liver code for the transport of bumetanide and taurocholate in Xenopus laevis oocytes

Cited by 17 publications

References 13 publications

The Functional Expression of Sodium-dependent Bile Acid Transport in Madin-Darby Canine Kidney Cells Transfected with the cDNA for Microsomal Epoxide Hydrolase

The Functional Expression of Sodium-dependent Bile Acid Transport in Madin-Darby Canine Kidney Cells Transfected with the cDNA for Microsomal Epoxide Hydrolase

Transporters for Bile Acids and Organic Anions

Cloning and functional characterization of the bile acid-sensitive methotrexate carrier from rat liver cells

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