Publications

• Membrane transporters for sulfated steroids in the human testis--cellular localization, expression pattern and functional analysis.

  Fietz D., Bakhaus K., Wapelhorst B., Grosser G., Guenther S., Alber J., Doering B., Kliesch S., Weidner W., Galuska C.E., Hartmann M.F., Wudy S.A., Bergmann M., Geyer J.

  Sulfated steroid hormones are commonly considered to be biologically inactive metabolites, but may be reactivated by the steroid sulfatase into biologically active free steroids, thereby having regulatory function via nuclear androgen and estrogen receptors which are widespread in the testis. Howe ... >> More

  Sulfated steroid hormones are commonly considered to be biologically inactive metabolites, but may be reactivated by the steroid sulfatase into biologically active free steroids, thereby having regulatory function via nuclear androgen and estrogen receptors which are widespread in the testis. However, a prerequisite for this mode of action would be a carrier-mediated import of the hydrophilic steroid sulfate molecules into specific target cells in reproductive tissues such as the testis. In the present study we detected predominant expression of the Sodium-dependent Organic Anion Transporter (SOAT), the Organic Anion Transporting Polypeptide 6A1, and the Organic Solute Carrier Partner 1 in human testis biopsies. All of these showed significantly lower or even absent mRNA expression in severe disorders of spermatogenesis (arrest at the level of spermatocytes or spermatogonia, Sertoli cell only syndrome). Only SOAT was significantly lower expressed in biopsies showing hypospermatogenesis. By use of immunohistochemistry SOAT was localized to germ cells at various stages in human testis biopsies showing normal spermatogenesis. SOAT immunoreactivity was detected in zygotene primary spermatocytes of stage V, pachytene spermatocytes of all stages (I-V), secondary spermatocytes of stage VI, and round spermatids (step 1 and step 2) in stages I and II. Furthermore, SOAT transport function for steroid sulfates was analyzed with a novel liquid chromatography tandem mass spectrometry procedure capable of profiling steroid sulfate molecules from cell lysates. With this technique, the cellular inward-directed SOAT transport was verified for the established substrates dehydroepiandrosterone sulfate and estrone-3-sulfate. Additionally, β-estradiol-3-sulfate and androstenediol-3-sulfate were identified as novel SOAT substrates. << Less

  PLoS ONE 8:e62638-e62638(2013) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Expression and transport properties of the human ileal and renal sodium-dependent bile acid transporter.

  Craddock A.L., Love M.W., Daniel R.W., Kirby L.C., Walters H.C., Wong M.H., Dawson P.A.

  The enterohepatic circulation of bile acids is maintained by Na(+)-dependent transport mechanisms. To better understand these processes, a full-length human ileal Na(+)-bile acid cotransporter cDNA was identified using rapid amplification of cDNA ends and genomic cloning techniques. Using Northern ... >> More

  The enterohepatic circulation of bile acids is maintained by Na(+)-dependent transport mechanisms. To better understand these processes, a full-length human ileal Na(+)-bile acid cotransporter cDNA was identified using rapid amplification of cDNA ends and genomic cloning techniques. Using Northern blot analysis to determine its tissue expression, we readily detected the ileal Na(+)-bile acid cotransporter mRNA in terminal ileum and kidney. Direct cloning and mapping of the transcriptional start sites confirmed that the kidney cDNA was identical to the ileal Na(+)-bile acid cotransporter. In transiently transfected COS cells, ileal Na(+)-bile acid cotransporter-mediated taurocholate uptake was strictly Na+ dependent and chloride independent. Analysis of the substrate specificity in transfected COS or CHO cells showed that both conjugated and unconjugated bile acids are efficiently transported. When the inhibition constants for other potential substrates such as estrone-3-sulfate were determined, the ileal Na(+)-bile acid cotransporter exhibited a narrower substrate specificity than the related liver Na(+)-bile acid cotransporter. Whereas the multispecific liver Na(+)-bile acid cotransporter may participate in hepatic clearance of organic anion metabolites and xenobiotics, the ileal and renal Na(+)-bile acid cotransporter retains a narrow specificity for reclamation of bile acids. << Less

  Am. J. Physiol. 274:G157-G169(1998) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Cloning and functional characterization of human sodium-dependent organic anion transporter (SLC10A6).

  Geyer J., Doering B., Meerkamp K., Ugele B., Bakhiya N., Fernandes C.F., Godoy J.R., Glatt H., Petzinger E.

  We have cloned human sodium-dependent organic anion transporter (SOAT) cDNA, which consists of 1502 bp and encodes a 377-amino acid protein. SOAT shows 42% sequence identity to the ileal apical sodium-dependent bile acid transporter ASBT and 33% sequence identity to the hepatic Na(+)/taurocholate- ... >> More

  We have cloned human sodium-dependent organic anion transporter (SOAT) cDNA, which consists of 1502 bp and encodes a 377-amino acid protein. SOAT shows 42% sequence identity to the ileal apical sodium-dependent bile acid transporter ASBT and 33% sequence identity to the hepatic Na(+)/taurocholate-cotransporting polypeptide NTCP. Immunoprecipitation of a SOAT-FLAG-tagged protein revealed a glycosylated form at 46 kDa that decreased to 42 kDa after PNGase F treatment. SOAT exhibits a seven-transmembrane domain topology with an outside-to-inside orientation of the N-terminal and C-terminal ends. SOAT mRNA is most highly expressed in testis. Relatively high SOAT expression was also detected in placenta and pancreas. We established a stable SOAT-HEK293 cell line that showed sodium-dependent transport of dehydroepiandrosterone sulfate, estrone-3-sulfate, and pregnenolone sulfate with apparent K(m) values of 28.7, 12.0, and 11.3 microm, respectively. Although bile acids, such as taurocholic acid, cholic acid, and chenodeoxycholic acid, were not substrates of SOAT, the sulfoconjugated bile acid taurolithocholic acid-3-sulfate was transported by SOAT-HEK293 cells in a sodium-dependent manner and showed competitive inhibition of SOAT transport with an apparent K(i) value of 0.24 mum. Several nonsteroidal organosulfates also strongly inhibited SOAT, including 1-(omega-sulfooxyethyl)pyrene, bromosulfophthalein, 2- and 4-sulfooxymethylpyrene, and alpha-naphthylsulfate. Among these inhibitors, 2- and 4-sulfooxymethylpyrene were competitive inhibitors of SOAT, with apparent K(i) values of 4.3 and 5.5 microm, respectively, and they were also transported by SOAT-HEK293 cells. << Less

  J. Biol. Chem. 282:19728-19741(2007) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Ethnicity-dependent polymorphism in Na+-taurocholate cotransporting polypeptide (SLC10A1) reveals a domain critical for bile acid substrate recognition.

  Ho R.H., Leake B.F., Roberts R.L., Lee W., Kim R.B.

  The key transporter responsible for hepatic uptake of bile acids from portal circulation is Na+-taurocholate cotransporting polypeptide (NTCP, SLC10A1). This transporter is thought to be critical for the maintenance of enterohepatic recirculation of bile acids and hepatocyte function. Therefore, f ... >> More

  The key transporter responsible for hepatic uptake of bile acids from portal circulation is Na+-taurocholate cotransporting polypeptide (NTCP, SLC10A1). This transporter is thought to be critical for the maintenance of enterohepatic recirculation of bile acids and hepatocyte function. Therefore, functionally relevant polymorphisms in this transporter would be predicted to have an important impact on bile acid homeostasis/liver function. However, little is known regarding genetic heterogeneity in NTCP. In this study, we demonstrate the presence of multiple single nucleotide polymorphisms in NTCP in populations of European, African, Chinese, and Hispanic Americans. Specifically four nonsynonymous single nucleotide polymorphisms associated with a significant loss of transport function were identified. Cell surface biotinylation experiments indicated that the altered transport activity of T668C (Ile223-->Thr), a variant seen only in African Americans, was due at least in part to decreased plasma membrane expression. Similar expression patterns were observed when the variant alleles were expressed in HepG2 cells, and plasma membrane expression was assessed using immunofluorescence confocal microscopy. Interestingly the C800T (Ser267-->Phe) variant, seen only in Chinese Americans, exhibited a near complete loss of function for bile acid uptake yet fully normal transport function for the non-bile acid substrate estrone sulfate, suggesting this position may be part of a region in the transporter critical and specific for bile acid substrate recognition. Accordingly, our study indicates functionally important polymorphisms in NTCP exist and that the likelihood of being carriers of such polymorphisms is dependent on ethnicity. << Less

  J. Biol. Chem. 279:7213-7222(2004) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Substrate specificity of the rat liver Na(+)-bile salt cotransporter in Xenopus laevis oocytes and in CHO cells.

  Schroeder A., Eckhardt U., Stieger B., Tynes R., Schteingart C.D., Hofmann A.F., Meier P.J., Hagenbuch B.

  It has been proposed that the hepatocellular Na(+)-dependent bile salt uptake system exhibits a broad substrate specificity in intact hepatocytes. In contrast, recent expression studies in mammalian cell lines have suggested that the cloned rat liver Na(+)-taurocholate cotransporting polypeptide ( ... >> More

  It has been proposed that the hepatocellular Na(+)-dependent bile salt uptake system exhibits a broad substrate specificity in intact hepatocytes. In contrast, recent expression studies in mammalian cell lines have suggested that the cloned rat liver Na(+)-taurocholate cotransporting polypeptide (Ntcp) may transport only taurocholate. To characterize its substrate specificity Ntcp was stably transfected into Chinese hamster ovary (CHO) cells. These cells exhibited saturable Na(+)-dependent uptake of [3H]taurocholate [Michaelis constant (K(m)) of approximately 34 microM] that was strongly inhibited by all major bile salts, estrone 3-sulfate, bumetanide, and cyclosporin A. Ntcp cRNA-injected Xenopus laevis oocytes and the transfected CHO cells exhibited saturable Na(+)-dependent uptake of [3H]taurochenodeoxycholate (Km of approximately 5 microM), [3H]tauroursodeoxycholate (Km of approximately 14 microM), and [14C]glycocholate (Km of approximately 27 microM). After induction of gene expression by sodium butyrate, Na(+)-dependent transport of [3H]estrone 3-sulfate (Km of approximately 27 microM) could also be detected in the transfected CHO cells. However, there was no detectable Na(+)-dependent uptake of [3H]bumetanide or [3H]cyclosporin A. These results show that the cloned Ntcp can mediate Na(+)-dependent uptake of all physiological bile salts as well as of the steroid conjugate estrone 3-sulfate. Hence, Ntcp is a multispecific transporter with preference for bile salts and other anionic steroidal compounds. << Less

  Am. J. Physiol. 274:G370-G375(1998) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Functional and Pharmacological Comparison of Human and Mouse Na+/Taurocholate Cotransporting Polypeptide (NTCP).

  Floerl S., Kuehne A., Geyer J., Brockmoeller J., Tzvetkov M.V., Hagos Y.

  The Na⁺/taurocholate cotransporting polypeptide (NTCP) is located in the basolateral membrane of hepatocytes, where it transports bile acids from the portal blood back into hepatocytes. Furthermore, NTCP has a role for the hepatic transport of some drugs. Extrapolation of drug transport ... >> More

  The Na⁺/taurocholate cotransporting polypeptide (NTCP) is located in the basolateral membrane of hepatocytes, where it transports bile acids from the portal blood back into hepatocytes. Furthermore, NTCP has a role for the hepatic transport of some drugs. Extrapolation of drug transport data from rodents to humans is not always possible, because species differences in the expression level, localization, affinity, and substrate selectivity of relevant transport proteins must be considered. In the present study, a functional comparison of human NTCP (hNTCP) and mouse Ntcp (mNtcp) showed similar <i>K</i>_m values of 67 ± 10 µM and 104 ± 9 µM for the probe substrate estrone-3-sulfate as well as of 258 ± 42 µM and 199 ± 13 µM for the drug rosuvastatin, respectively. IC₅₀ values for the probe inhibitor cyclosporine A were 3.1 ± 0.3 µM for hNTCP and 1.6 ± 0.4 µM for mNtcp. In a drug and pesticide inhibitory screening on both transporters, 4 of the 15 tested drugs (cyclosporine A, benzbromarone, MK571, and fluvastatin) showed high inhibitory potency, but only slight inhibition was observed for the 13 tested pesticides. Among these compounds, only four drugs and three pesticides showed significant differences in their inhibition pattern on hNTCP and mNtcp. Most pronounced was the difference for benzbromarone with a fivefold higher IC₅₀ for mNtcp (27 ± 10 µM) than for hNTCP (5.5 ± 0.6 µM).In conclusion, we found a strong correlation between the transport kinetics and inhibition pattern among hNTCP and mNtcp. However, specific compounds, such as benzbromarone, showed clear species differences. Such species differences have to be considered when pharmacokinetic data are transferred from rodent to humans. << Less

  SLAS Discovery 26:1055-1064(2021) [PubMed] [EuropePMC]