Biomedical subjects

W Honscha

Publications and source records attributed to W Honscha.

At least 19 recordsLinked to original sources

PubMed2000

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

We have cloned two complementary DNAs (cDNAs), RL-Mtx-1 and RL-Mtx-2, corresponding to the bile acid- sensitive methotrexate carrier from rat liver by direct full-length rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) using degenerated primers that were deduced from published sequences of tumor cell methotrexate transporters. When expressed in Xenopus laevis oocytes and cosM6 cells, both clones mediate methotrexate and bumetanide transport. RL-Mtx-1 consists of 2,445 bp with an open reading frame of 1,536 bp. The corresponding protein with 512 amino acids has a molecular weight of 58 kd. RL-Mtx-2 (2,654 bp) differs by an additional insert of 203 bp. This insert is located in frame at position 1,196 of the RL-Mtx-1 and contains the typical splice junction sites at the 5' and 3' end, indicating that the RL-Mtx-2 messenger RNA (mRNA) is generated by alternative splicing. The insert contains a stop codon that shortens the RL-Mtx-2 protein to 330 amino acids (38 kd). Both cDNAs contain the binding site sequence for the dioxin/nuclear translocator responsive element (Ah/Arnt-receptor) in conjunction with a barbiturate recognition sequence (Barbie box). Preliminary results show that the Barbie box acts as a negative regulatory element. The two liver cDNA clones show homologies to the published sequences of folate and the reduced folate carriers, but no homology is found to the transport systems for organic anions like the Ntcp1, oatp1, OAT-K1, and OAT1. Expression of the mRNA for the methotrexate carrier is found in liver, kidney, heart, brain, spleen, lung, and skeletal muscle, but not in the testis as revealed by Northern blot analysis. The highest abundance of the mRNA is found in the kidney.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed1999

Characterization of the bile acid sensitive methotrexate carrier of rat liver cells.

The chemotherapeutic agent methotrexate is widely used in tumor therapy for different forms of leukemia and for the therapy of arthritis. Methotrexate is eliminated from systemic blood circulation by the liver and its transport into hepatocytes is therefore described in detail in this paper. Methotrexate uptake is energy- and sodium-dependent. The Km and the Vmax are 23 microM and 36 pmol/mg protein min, respectively. The apparent activation energy (E(app)) of methotrexate uptake (5 microM [3H]methotrexate) is 53.73 kJ/mol, which indicates an energy-dependent carrier-mediated process. Although methotrexate is a folate derivative, folate itself does not inhibit methotrexate uptake, whereas the reduced folates, dihydrofolate and tetrahydrofolate are weak uncompetitive inhibitors. In contrast, the bile acids taurocholate and cholate are effective competitive inhibitors of methotrexate uptake into hepatocytes. Further strong inhibitors are the loop diuretic bumetanide, the mycotoxin ochratoxin A and bromosulfophthalein. Because tumor patients develop drug resistance during methotrexate therapy, the uptake of methotrexate was tested in different hepatoma cell lines. In HepG2-cells and Reuber hepatoma Fao-cells the transport was non-existent or very small. However, the hepatocytoma fusion cell line HPCT-1E3, a hybrid cell line between primary rat hepatocytes and rat Reuber Fao-cells, shows an intermediate transport activity with a threefold increase of the methotrexate uptake. These results indicate the presence of a bile acid sensitive methotrexate carrier in hepatocytes which is absent in dedifferentiated hepatoma cells. The carrier differs from previously described transporters for the uptake of organic anions.

Animals↗

PubMed1999

[Transgenic animals as bioreactors in the service of the pharmaceutical industry].

A modern pharmacotherapy without the use of proteins as drugs is not more practicable. In the clinic blood coagulation factors (factor VIII and IX), growth hormones (human growth hormone), enzymes (1-antitrysin, insulin), and cytokines are currently used. At the beginning, the proteins were isolated from biological sources or expressed in vitro by the use of E. coli or eukaryotic cell lines. At the moment efforts were undertaken to express these proteins in the milk of transgenic animals. This review article describes the methods for the generation of transgenic animals, the benefits and drawbacks of this new technique in comparison to established methods for the production of proteins as pharmaceuticals. At the end of the review possible improvements of the method are described.

Animals↗

PubMed1996

Bumetanide is not transported by the Ntcp or by the oatp: evidence for a third organic anion transporter in rat liver cells.

The loop diuretic bumetanide which inhibits hepatic bile acid uptake competitively according to its transport kinetics has been proposed to serve as a substrate of a multispecific bile acid transport system in liver parenchymal cells. However, when the in vitro transcripts of two cloned hepatic bile acid uptake carriers, the Ntcp (Na+/taurocholate cotransporting polypeptide) and the oatp (organic anion transporting polypeptide), was expressed for three days in Xenopus laevis oocytes [3H]bumetanide uptake was not increased although bile acid uptake was stimulated. The data presented show that bumetanide is taken up by a third organic anion transport system which is different from the cloned bile acid transporters.

ATP-Binding Cassette Transporters↗

PubMed1996

Functional characterization of the hepatic sodium-dependent taurocholate transporter stably transfected into an immortalized liver-derived cell line and V79 fibroblasts.

Bile acids are taken up into liver parenchymal cells by active, carrier-mediated transport. This transport is lost during cell transformation in permanent growing liver tumor cell lines. In order to establish bile acid uptake in a permanent mammalian cell culture system, we transfected the cDNA from the cloned rat liver Na(+)-taurocholate cotransporting polypeptide (Ntcp) in Chinese hamster lung fibroblasts (V79 cells) and in a "hepatocyte-like" cell line HPCT-1F3 with three different gene transfer methods (calcium phosphate precipitation, lipofection, electroporation). A stable integration of the cDNA in both cell genomes was observed. However, in V79 fibroblasts, a permanent functional expression of taurocholate transport was not achieved. The sodium-dependent uptake of taurocholate was expressed permanently only in HPCT-1E3 cells, if the Ntcp was transfected by electroporation. In this cell line (HPCT-1E3-TC-6/2), substrate specificity, sodium- and energy dependence, as well as the kinetic parameters of the transfected single transporter were measured. The sodium-dependent taurocholate uptake was inhibited by addition of non-labeled bile acids, bumetanide, sulfobromophthalein and oligomycin. Pretreatment with 10 mM Na(+)-butyrate of this cell culture for 22 h stimulated taurocholate uptake twofold. Neither butyrate-stimulated cells nor unstimulated cells transport glycocholate or cholate. Besides taurocholate a fluorescence-labeled taurocholate derivative, NBD-taurocholate, was taken up by the HPCT-1E3-TC cells. In conclusion, the specific gene transfer with the electroporation technique in combination with the "right" cell line, HPCT-1E3, has been successful for the permanent and functional expression of the Ntcp. This allowed direct monitoring of the solitary sodium-dependent taurocholate transport system in a "liver cell-like" environment.

Animals↗

PubMed1996

What we have learned about bumetanide and the concept of multispecific bile acid/drug transporters from the liver.

Bumetanide is a weak organic acid which is transported into hepatocytes by a transport system that is related neither to the cloned sodium-dependent taurocholate cotransporting polypeptide Ntcp nor to the cloned organic anion transporting polypeptide oatp. Bumetanide is known to be transported in the kidney by a multispecific organic anion transporter which is the pAH-transporter from the proximal tubule cell. In the liver, bumetanide uptake competes with bile acid uptake, indicating a functionally related multispecific transporter for bile acids and drugs in hepatocytes. This multispecific bile acid transporter MBAT has not been cloned yet. When basolateral membranes were photoaffinity labeled with [3H]bumetanide, several bumetanide binding proteins were separated and identified after protein sequencing from two-dimensional electrophoresis gels.

Adenosine Triphosphate↗

PubMed1995

Relationship between the microsomal epoxide hydrolase and the hepatocellular transport of bile acids and xenobiotics.

Recently two different bile-acid carriers for the hepatocellular sodium-dependent uptake of taurocholate have been described. The first transport system was isolated and characterized by functional expression cloning in Xenopus laevis oocytes. The corresponding cDNA clone, named Ntcp for Na+/taurocholate co-transporting polypeptide, codes for a protein of 362 amino acids and shows no similarity to previously known sequences. The transport function of this carrier system is well documented by expression in Xenopus laevis oocytes and by transient and stably transfected cell lines. In addition, several lines of evidence implied that the well-known xenobiotic-metabolizing enzyme microsomal epoxide hydrolase (mEH, EC 3.3.2.3) is also able to mediate sinusoidal uptake of taurocholate. Furthermore, it was claimed that the same enzyme also mediates the uptake of the conjugated bile acid into the smooth endoplasmic reticulum (ER). No direct proof of the transport function of mEH by its heterologous expression has yet been published. In the present work we used a stable transfected cell line that expressed high levels of heterologous mEH for uptake studies of various bile acids and the loop diuretic bumetanide. The uptake of the conjugated bile acid taurocholate, of the non-conjugated bile acid cholate and of the organic anion bumetanide was measured in the transfected as well as in the non-transfected parental cell line. These organic anions represent the main substrates of the known transport systems for organic anions in the rat liver. The results show that the microsomal epoxide hydrolase is unable to transport taurocholate, cholate or bumetanide. Furthermore, Western-blot analysis revealed the expression of mEH in hepatoma tumor cell lines, which show no transport activity for these organic anions. These results show that it is unlikely that mEH can mediate the transport of these substrates.

Animals↗

PubMed1993

A membrane-bound form of protein disulfide isomerase (PDI) and the hepatic uptake of organic anions.

Protein disulfide isomerase (PDI) was considered to be involved in the hepatic uptake of certain organic anions because the protein is photoaffinity labeled by photolabile derivatives of the bile acid taurocholate. Several lines of evidences including photoaffinity labeling experiments indicated a close relationship between the uptake of bile acids and the organic anion bumetanide. The possible involvement of PDI in hepatic transport processes of these organic anions was tested with polyclonal antibodies raised against a PDI-beta-galactosidase fusion protein. Western blot analysis and immunofluorescence of intact hepatocytes showed that protein disulfide isomerase is located in sinusoidal rat liver plasma membranes. This protein is immunologically identical with microsomal PDI prepared from bovine liver. The plasma membrane form of PDI is, however, not labeled by photoactivated bumetanide as revealed by two-dimensional gel electrophoresis. These results indicate that, although a membrane-bound form of the PDI is present in the sinusoidal plasma membrane of rat hepatocytes, this protein is not involved in the hepatocellular uptake of the organic anion bumetanide.

Animals↗

PubMed1993

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

The aim of this study was to elucidate whether bumetanide, which is a competitive inhibitor of carrier mediated bile acid uptake in liver cells, is transported by bile acid carriers. The expression of hepatocellular transport proteins for bile acid uptake and the uptake of the loop diuretic bumetanide was therefore studied in Xenopus laevis oocytes by injection of rat liver poly(A)(+)-RNA. Three hours after injection, a 70% increase in [3H]taurocholate uptake versus noninjected oocytes was accompanied by an increase in only 24% in the uptake of [3H]bumetanide. Size fractionation of the poly(A)(+)-RNA yielded 33 mRNA fractions of which fraction 21 accounted for an 800% increase of taurocholate transport with only a slight increase in bumetanide uptake. Bumetanide transport was coded by mRNA-fraction 18, which stimulated uptake by 160-200% with a concomitant small increase in taurocholate uptake. Uptake of cholate was induced by both mRNA fractions with almost 2.5 fold greater expression by the bumetanide fraction. Oocyte transport of taurocholate (expressed by fraction 21) and bumetanide transport (expressed by fraction 18) were characterized in terms of Na+ dependency, inhibition by 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (DIDS) and mutual competition. The results indicate that the bumetanide transporter mRNA is clearly different from the mRNA for the taurocholate transport protein. The mRNA fraction 18 was used for the construction of a cDNA library.

Animals↗

PubMed1992

Separation and purification by two-dimensional gel electrophoresis of a 52-54 kDa bumetanide binding protein from rat liver plasma membranes.

By affinity labeling with photolabile [3H]bumetanide, a 52-54 kDa bumetanide binding protein was identified in the sinusoidal plasma membrane fraction from rat liver. The protein is assumed to represent the carrier for hepatic uptake of loop diuretics. By two-dimensional (2D) gel electrophoresis we have purified this protein from hepatocytes, sinusoidal plasma membranes and subfractions of associated and integral plasma membrane proteins. Amongst more than 20 protein spots, a single integral plasma membrane protein was detected. The apparent pI of this molecule is 6.7. Specific labeling of this protein was not found in the fraction of associated plasma membrane proteins. To detect possible binding of radioactive bumetanide to microsomal cytochrome P450s, photolabeling experiments with integral plasma membrane proteins were performed under nitrogen/carbon monoxide atmosphere and in the presence of piperonyl butoxide. Labeling of the 52-54 kDa protein was not affected by these inhibitors of P450 enzymes. Taken together, these results indicate that the bumetanide binding protein is very likely to be a non-microsomal integral plasma membrane protein.

Affinity Labels↗

PubMed1991

Photoaffinity labeling of plasma membrane proteins involved in the transport of loop diuretics into hepatocytes.

To identify proteins involved in the hepatocellular uptake of loop diuretics, [3H]bumetanide was photoactivated by light flash in the presence of either intact isolated rat hepatocytes, rat liver basolateral plasma membranes or integral membrane proteins extracted from the basolateral plasma membranes. Proteins of 52-54, 48, 33, 27, 25 and 23 kDa in sodium dodecyl sulfate (SDS) gel electrophoresis were radiolabeled on intact hepatocytes. On liver basolateral plasma membranes a 50-52 kDa protein was the most intensely labeled protein. After separation into integral and associated membrane proteins by extraction with Triton X-114, radioactive labeling was only found in integral membrane proteins with a molecular weight of 50-52 kDa. Photoactivated bumetanide irreversibly inhibited the hepatocellular uptake of cholate, taurocholate but not of serine. Binding proteins for photoactivated bumetanide were absent on AS 30-D ascites hepatoma cells. Labeling of all proteins was sodium dependent in intact hepatocytes but was sodium independent in plasma membranes. Labeling was prevented by non-labeled bumetanide and by the loop diuretics piretanide and furosemide. Labeling protection was further achieved with organic anions such as bromosulfophthalein, rifampicin, probenecid and by the bile acids taurocholate, deoxycholate and dehydrocholate. The radiolabeled proteins did not belong to the bumetanide-sensitive NaCl/KCl co-transport system which apparently does not occur in intact isolated rat hepatocytes.

Affinity Labels↗

PubMed1991

Tissue-specific expression and differential inducibility of several microsomal epoxide hydrolase mRNAs which are formed by alternative splicing.

mRNA was isolated from several rat tissues and subjected to either the nuclease S1 or the RNAseA protection assay with probes covering the 5' end, the middle part, and the 3' end of the microsomal epoxide hydrolase (mEHb) cDNA. Whereas probes directed against the latter two regions yielded a single protected fragment, a probe which covered base pairs -148 to +453 (+1 defines the start of protein biosynthesis) yielded two protected fragments. The degree of protection of the two fragments was strongly dependent on the tissue from which the mRNA had been isolated. Thus at least two mEHb mRNAs which differ at their 5' ends are differentially expressed in various tissues. In addition the mRNAs corresponding to the two protected fragments were clearly differentially inducible by Aroclor 1254 treatment of the animals. Primer extension analysis with hepatic RNA from untreated animals yielded three primer-extended products corresponding to three mRNAs which differ at their 5' ends. As already seen in the nuclease S1 protection assay, one of the mRNAs was induced by Aroclor 1254 treatment. The expression of the two other mRNAs was either repressed or stable. Thus besides the mRNA already characterized for mEHb, there are at least two other mEHb mRNAs. This result was confirmed by the isolation of a mEHb cDNA which is completely distinct in its sequence in a region just preceding the initiation codon for protein biosynthesis. From that point on, the sequence of our cDNA becomes identical to the published mEHb cDNA. This point corresponds exactly to the start of exon 2 as determined from the genomic sequence. Thus the region where both mEHb cDNAs differ is encoded by two different exons 1, which are joined to exon 2 by alternative splicing. The tissue-specific expression and the different inducibility of the various mEHb mRNAs might indicate that their expression is governed by different promoters.

Animals↗

PubMed1989

Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2.

Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.

Animals↗

PubMed1978