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.

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.

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.

The 60-kDa bumetanide-binding protein from rat liver membranes is a catalase.

A hepatic bumetanide-binding protein of molecular mass 60 kDa was isolated from rat liver sinusoidal plasma membranes after photoaffinity labelling with [3H]bumetanide. The protein was purified by non-equilibrium pH gel electrophoresis/two-dimensional gel electrophoresis. The amino acid sequences of two internal fragments share 67% and 89% similarity with rat liver catalase, which has a molecular mass of 59.758 kDa. With H2O2 as a substrate, the catalytic activity was measured in rat liver plasma membrane preparations. This activity was blocked by bumetanide and aminobumetanide. Polyclonal antibodies were raised against the purified 60-kDa membrane bumetanide-binding protein. The antibody anti-Bum-Ab 60 immunoprecipitated a 60-kDa protein from rat hepatocytes. Immunoblot analysis of SDS/PAGE and two-dimensional PAGE gels confirmed that the antibody was specific for the 60-kDa bumetanide-binding protein and cross-reacted with commercially available purified bovine liver catalase. Immunofluorescence showed the presence of the 60-kDa antigen in the plasma membrane of intact hepatocytes. Western-blot analysis revealed that the protein was present in rat kidney cortex homogenate but was lacking in hepatoma cells AS-30 D, Reuber H35 FAO and HPCT cells (clone 1E3), in spleen, and in ileum. These results indicate that a plasma-membrane-derived catalase binds bumetanide in rat liver.