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1
Bai, Han, Li Wang, Wenhui Li, Xuhong Liu, Yaoxiong Xia, and Li Chang. "Test the Effectiveness of Quantitative Linear-Quadratic-Based (qLQB) Model on Evaluating Irradiation-Induced Liver Injury (ILI) Against Normal Tissue Complication Probability (NTCP)." Dose-Response 18, no. 3 (July 1, 2020): 155932582096172. http://dx.doi.org/10.1177/1559325820961721.
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Objectives: To test the effectiveness of quantitative linear-quadratic-based (qLQB) model on evaluating irradiation-induced liver injury (ILI) and establish the relation between the damaged ratio/percent (DRP) in qLQB model and normal tissue complication probility (NTCP). Materials and Methods: We established the qLQB model to calculate the ratio/percent (RP) between damaged cell/functional subunit (FSU) and entire cell/FSU of liver for radiation dose response, tested the qLQB against the Lyman-Kutcher-Burman (LKB) model, and established relation between the RP and NTCP through analyzing the dose of 32 patients with cancer of abdominal cavity who were treated with radiation therapy at our department. Based on varied α/β and varied parameters for NTCP, we put the calculated results into varied arrays for the next analysis. We named the 2 groups of RPs: RP1 (α/β = 3.0, α = 0.03) and RP2 (α/β = 8.0, α = 0.26), and named the 2 groups of NTCPs: NTCP1 (n = 0.32, m = 0.15, TD50(1) = 4000 cGy) and NTCP2 (n = 1.10, m = 0.28, TD50(1) = 4050 cGy). Results: Spearman correlation analysis was used to analyze the correlations among the groups, the results were as follows: RP1 vs NTCP1, rs = 0.83827, p < 0.0001; RP1 vs NTCP2, rs = 0.83827, p < 0.0001; RP2 vs NTCP2, rs = 0.79289, p < 0.0001; and RP2 vs NTCP1, rs = 0.79289, p < 0.0001. Conclusions: There is a significant correlation between RP value and NTCP for evaluating ILI, and there is no difference between qLQB model and LKB model on evaluating ILI.
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Hanada, Takashi, Yutaka Shiraishi, Toshio Ohashi, Junichi Fukada, Tomoki Tanaka, Atsunori Yorozu, and Naoyuki Shigematsu. "Variations in Rectal Volumes and Dosimetry Values Including NTCP due to Interfractional Variability When Administering 2D-Based IG-IMRT for Prostate Cancer." Journal of Radiotherapy 2014 (July 15, 2014): 1–7. http://dx.doi.org/10.1155/2014/298904.
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We estimated variations in rectal volumes and dosimetry values including NTCP with interfractional motion during prostate IG-IMRT. Rectal volumes, DVH parameters, and NTCPs of 20 patients were analyzed. For this patient population, the median (range) volume on the initial plan for the rectum was 45.6 cc (31.3–82.0), showing on-treatment spread around the initial prediction based on the initial plan. DVH parameters of on-treatment CBCT analyses showed systematic regularity shift from the prediction based on the initial plan. Using the Lyman-Kutcher-Burman model, NTCPs of predicted late rectal bleeding toxicity of rectal grade ≥ 2 (RTOG) and the QUANTEC update rectal toxicity for the prediction based on the initial plan were 0.09% (0.02–0.24) and 0.02% (0.00–0.07), respectively, with NTCPs from on-treatment CBCT analyses being 0.35% (0.01–6.16) and 0.12% (0.00–4.11), respectively. Using the relative seriality model, for grade ≥ 2 bleeding rectal toxicity, NTCP of the prediction based on the initial plan was 0.64% (0.15–1.22) versus 1.48% (0.18–7.66) for on-treatment CBCT analysis. Interfraction variations in rectal volumes occur in all patients due to physiological changes. Thus, rectal assessment during 2D-based IG-IMRT using NTCP models has the potential to provide useful and practical dosimetric verification.
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Chamunyonga, Crispen. "The impact of inter-fraction set-up errors on the probability of pulmonary and cardiac complication in left-sided breast cancer patients." Journal of Radiotherapy in Practice 13, no. 4 (February 17, 2014): 393–402. http://dx.doi.org/10.1017/s1460396914000065.
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AbstractPurposeThis study evaluated the impact of patient set-up errors on the probability of pulmonary and cardiac complications in the irradiation of left-sided breast cancer.Methods and materialsUsing the CMS XiO Version 4·6 radiotherapy planning system's normal tissue complication probability (NTCP) algorithm and the Lyman–Kutcher–Burman model, we calculated the dose–volume histograms (DVH) indices for the ipsilateral lung and heart and the resultant NTCP for radiation-induced pneumonitis and excess cardiac mortality in 12 left-sided breast cancer patients.ResultsIsocentric shifts in the posterior direction had the greatest effect on the lung V20, heart V25, and mean and maximum doses to the lung and the heart. DVH results show that the ipsilateral lung V20 tolerance was exceeded in 58% of the patients after 1 cm posterior shifts. Similarly, the heart V25 tolerance was exceeded after 1 cm antero-posterior and left–right isocentric shifts in 70% of the patients. The baseline NTCPs for radiation-induced pneumonitis ranged from 0·73% to 3·4%, with a mean value of 1·7%. The maximum reported NTCP for radiation-induced pneumonitis was 5·8% (mean 2·6%) after 1 cm posterior isocentric shift. The NTCP for excess cardiac mortality were 0% in 100% of the patients (n = 12) before and after set-up error simulations.ConclusionsSet-up errors in left-sided breast cancer patients have a statistically significant impact on the Lung NTCPs and DVH indices. However, with a central lung distance of 3 cm or less (CLD < 3 cm), and a maximum heart distance of 1·5 cm or less (MHD < 1·5 cm), the treatment plans could tolerate set-up errors of up to 1 cm without any change in the NTCP to the heart.
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Hytönen, Roni, Reynald Vanderstraeten, Max Dahele, and Wilko F. A. R. Verbakel. "Influence of Beam Angle on Normal Tissue Complication Probability of Knowledge-Based Head and Neck Cancer Proton Planning." Cancers 14, no. 12 (June 9, 2022): 2849. http://dx.doi.org/10.3390/cancers14122849.
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Knowledge-based planning solutions have brought significant improvements in treatment planning. However, the performance of a proton-specific knowledge-based planning model in creating knowledge-based plans (KBPs) with beam angles differing from those used to train the model remains unexplored. We used a previously validated RapidPlanPT model and scripting to create nine KBPs, one with default and eight with altered beam angles, for 10 recent oropharynx cancer patients. The altered-angle plans were compared against the default-angle ones in terms of grade 2 dysphagia and xerostomia normal tissue complication probability (NTCP), mean doses of several organs at risk, and dose homogeneity index (HI). As KBP could be suboptimal, a proof of principle automatic iterative optimizer (AIO) was added with the aim of reducing the plan NTCP. There were no statistically significant differences in NTCP or HI between default- and altered-angle KBPs, and the altered-angle plans showed a <1% reduction in NTCP. AIO was able to reduce the sum of grade 2 NTCPs in 66/90 cases with mean a reduction of 3.5 ± 1.8%. While the altered-angle plans saw greater benefit from AIO, both default- and altered-angle plans could be improved, indicating that the KBP model alone was not completely optimal to achieve the lowest NTCP. Overall, the data showed that the model was robust to the various beam arrangements within the range described in this analysis.
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Jung, Diana, Bruno Hagenbuch, Michael Fried, Peter J. Meier, and Gerd A. Kullak-Ublick. "Role of liver-enriched transcription factors and nuclear receptors in regulating the human, mouse, and rat NTCP gene." American Journal of Physiology-Gastrointestinal and Liver Physiology 286, no. 5 (May 2004): G752—G761. http://dx.doi.org/10.1152/ajpgi.00456.2003.
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Hepatic uptake of bile acids is mediated by the Na+-taurocholate cotransporting polypeptide (NTCP; SLC10A1) of the basolateral hepatocyte membrane. Several cis-acting elements in the rat Ntcp gene promoter have been characterized. However, little is known about the mechanisms that control the expression of the human or mouse NTCP/Ntcp. We, therefore, compared the transcriptional regulation of the human and mouse NTCP/Ntcp gene with that of the rat. By computer alignment, a sequence in the 5′-regulatory region that is conserved between species was identified near the transcription start site. Huh7 cells were transfected with luciferase constructs containing the conserved region from each species. The hepatocyte nuclear factors (HNF)1α and -4α and the retinoid X receptor/retinoic acid receptor dimer (RXRα/RARα) bound and transactivated the rat but not the human or mouse NTCP/Ntcp promoters. In contrast, activation by the CCAAT/enhancer binding protein-β was specific for human and mouse NTCP/Ntcp. The only consensus motif present in all three species was HNF3β. HNF3β formed a specific DNA-protein complex in electrophoretic mobility shift assays and inhibited NTCP/Ntcp promoter activity in cotransfection assays. Finally, a minor repressive effect of bile acids was only found for rat Ntcp. The transcriptional repressor small heterodimer partner (SHP) did not affect NTCP/Ntcp promoter activity. We conclude that 1) the transcriptional regulation of the conserved NTCP/Ntcp 5′-regulatory region differs considerably among human, mouse, and rat; and 2) the conserved NTCP/Ntcp regulatory region is not directly regulated by SHP. Bile acids may regulate NTCP/Ntcp indirectly by modulating the capacity of nuclear factors to activate gene expression.
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Bijsmans, Ingrid T. G. W., Rianne A. M. Bouwmeester, Joachim Geyer, Klaas Nico Faber, and Stan F. J. van de Graaf. "Homo- and hetero-dimeric architecture of the human liver Na+-dependent taurocholate co-transporting protein." Biochemical Journal 441, no. 3 (January 16, 2012): 1007–16. http://dx.doi.org/10.1042/bj20111234.
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The NTCP (Na+–taurocholate co-transporting protein)/SLC10A [solute carrier family 10 (Na+/bile acid co-transporter family)] 1 is tightly controlled to ensure hepatic bile salt uptake while preventing toxic bile salt accumulation. Many transport proteins require oligomerization for their activity and regulation. This is not yet established for bile salt transporters. The present study was conducted to elucidate the oligomeric state of NTCP. Chemical cross-linking revealed the presence of NTCP dimers in rat liver membranes and U2OS cells stably expressing NTCP. Co-immunoprecipitation of tagged NTCP proteins revealed a physical interaction between subunits. The C-terminus of NTCP was not required for subunit interaction, but was essential for exit from the ER (endoplasmic reticulum). NTCP without its C-terminus (NTCP Y307X) retained full-length wtNTCP (wild-type NTCP) in the ER in a dominant fashion, suggesting that dimerization occurs early in the secretory pathway. FRET (fluorescence resonance energy transfer) using fluorescently labelled subunits further demonstrated that dimerization persists at the plasma membrane. NTCP belongs to the SLC10A protein family which consists of seven members. NTCP co-localized in U2OS cells with SLC10A4 and SLC10A6, but not with SLC10A3, SLC10A5 or SLC10A7. SLC10A4 and SLC10A6 co-immunoprecipitated with NTCP, demonstrating that heteromeric complexes can be formed between SLC10A family members in vitro. Expression of SLC10A4 and NTCP Y307X resulted in a reduction of NTCP abundance at the plasma membrane and NTCP-mediated taurocholate uptake, whereas expression of SLC10A6 or NTCP E257N, an inactive mutant, did not affect NTCP function. In conclusion, NTCP adopts a dimeric structure in which individual subunits are functional. Bile salt uptake is influenced by heterodimerization when this impairs NTCP plasma membrane trafficking.
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Simon, Francis R., John Fortune, Mieko Iwahashi, Ishtiaq Qadri, and Eileen Sutherland. "Multihormonal regulation of hepatic sinusoidalNtcpgene expression." American Journal of Physiology-Gastrointestinal and Liver Physiology 287, no. 4 (October 2004): G782—G794. http://dx.doi.org/10.1152/ajpgi.00379.2003.
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Bile acids are efficiently removed from sinusoidal blood by a number of transporters including the Na+-taurocholate-cotransporting polypeptide (Ntcp). Na+-dependent bile salt uptake, as well as Ntcp, are expressed twofold higher in male compared with female rat livers. Also, estrogen administration to male rats decreases Ntcp expression. The aims of this study were to determine the hormonal mechanism(s) responsible for this sexually dimorphic expression of Ntcp. We examined castrated and hypophysectomized rats of both sexes. Sex steroid hormones, growth hormone, thyroid, and glucocorticoids were administered, and livers were examined for changes in Ntcp messenger RNA (mRNA). Ntcp mRNA and protein content were selectively increased in males. Estradiol selectively decreased Ntcp expression in males, whereas ovariectomy increased Ntcp in females, confirming the importance of estrogens in regulating Ntcp. Hypophysectomy decreased Ntcp mRNA levels in males and prevented estrogen administration from decreasing Ntcp, indicating the importance of pituitary hormones. Although constant infusion of growth hormone to intact males reduced Ntcp, its replacement alone after hypophysectomy did not restore the sex differences. In contrast, thyroid hormone and corticosterone increased Ntcp mRNA in hypophysectomized rats. Sex differences in Ntcp mRNA levels were produced only when the female pattern of growth hormone was administered to animals also receiving thyroid and corticosterone. Thyroid and dexamethasone also increased Ntcp mRNA in isolated rat hepatocytes, whereas growth hormone decreased Ntcp. These findings demonstrate the essential role that pituitary hormones play in the sexually dimorphic control of Ntcp expression in adult rat liver and in the mediation of estrogen effects.
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Bhardwaj, Ashok Kumar. "Tuberculosis control programme from NTCP to RNTCP to NTEP." Indian Journal of Community Health 32, no. 3 (September 30, 2020): 469–70. http://dx.doi.org/10.47203/ijch.2020.v32i03.001.
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India, the highest TB burden country in the world, is having an estimated incidence of 26.9 lakh cases in 2019 (WHO). The year 2019 marks another milestone year for TB surveillance effort in India, with a record high notification of 24 lakhs cases; an increase of over 12% as compared to 2018. Of the 24 lakhs TB cases,(1) 21.6 lakhs were incident TB cases (New and Relapse/ Recurrent). This amount to an incident notification rate of approximately 159 cases/lakh against the estimated incidence rate of 199 cases/ lakh population; thus, closing the gap between the estimated and notified incident cases to just 40 Cases per lakh population, or an approximate of 5.4 lakh missing cases across India.
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Ramasamy, Umadevi, M. Sawkat Anwer, and Christopher M. Schonhoff. "Cysteine 96 of Ntcp is responsible for NO-mediated inhibition of taurocholate uptake." American Journal of Physiology-Gastrointestinal and Liver Physiology 305, no. 7 (October 1, 2013): G513—G519. http://dx.doi.org/10.1152/ajpgi.00089.2013.
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The Na+taurocholate (TC) cotransporting polypeptide Ntcp/NTCP mediates TC uptake across the sinusoidal membrane of hepatocytes. Previously, we demonstrated that nitric oxide (NO) inhibits TC uptake through S-nitrosylation of a cysteine residue. Our current aim was to determine which of the eight cysteine residues of Ntcp is responsible for NO-mediated S-nitrosylation and inhibition of TC uptake. Thus, we tested the effect of NO on TC uptake in HuH-7 cells transiently transfected with cysteine-to-alanine mutant Ntcp constructs. Of the eight mutants tested, only C44A Ntcp displayed decreased total and plasma membrane (PM) levels that were also reflected in decreased TC uptake. C266A Ntcp showed a decrease in TC uptake that was not explained by a decrease in total expression or PM localization, indicating that C266 is required for optimal uptake. We speculated that NO would target C266 since a previous report had shown the thiol reactive compound [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET) inhibits TC uptake by wild-type NTCP but not by C266A NTCP. We confirmed that MTSET targets C266 of Ntcp, but, surprisingly, we found that C266 was not responsible for NO-mediated inhibition of TC uptake. Instead, we found that C96 was targeted by NO since C96A Ntcp was insensitive to NO-mediated inhibition of TC uptake. We also found that wild-type but not C96A Ntcp is S-nitrosylated by NO, suggesting that C96 is important in regulating Ntcp function in response to elevated levels of NO.
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Zakrzewicz, Dariusz, Regina Leidolf, Sebastian Kunz, Simon Franz Müller, Anita Neubauer, Silke Leiting, Nora Goldmann, Felix Lehmann, Dieter Glebe, and Joachim Geyer. "Tyrosine 146 of the Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) Is Essential for Its Hepatitis B Virus (HBV) Receptor Function and HBV Entry into Hepatocytes." Viruses 14, no. 6 (June 9, 2022): 1259. http://dx.doi.org/10.3390/v14061259.
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Na+/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1) is a hepatic bile acid uptake carrier participating in the enterohepatic circulation of bile acids. Apart from its transporter function, NTCP acts as the high-affinity liver-specific receptor for the hepatitis B virus (HBV), which attaches via its preS1-peptide domain of the large surface protein to NTCP, subsequently leading to endocytosis of the virus/NTCP-receptor complex. Although the process of NTCP-dependent HBV infection of hepatocytes has received much attention over the last decade, the precise molecular sites of the virus/NTCP interaction have not been fully identified. Inspection of the primary protein sequence of human NTCP revealed 139YIYSRGIY146 as a highly conserved tyrosine-rich motif. To study the role of Y139, Y141 and Y146 amino acids in NTCP biology, the aforementioned residues were substituted with alanine, phenylalanine or glutamate (mimicking phosphorylation) using site-directed mutagenesis. Similar to wt NTCP, the Y139A, Y141A, Y146A, Y141F, Y146F, and Y146E mutants were expressed at the plasma membrane of HEK293 cells and exhibited intact bile acid transport function. Y146A, Y146E, and Y146F demonstrated transport kinetics comparable to wild-type NTCP with Km values of 57.3–112.4 µM and Vmax values of 6683–7579 pmol/mg protein/min. Only Y141E was transport deficient, most likely due to an intracellular accumulation of the mutant protein. Most importantly, Y146A and Y146E mutation completely abrogated binding of the viral preS1-peptide to NTCP, while the Y146F mutant of NTCP showed some residual binding competence for preS1. Consequently, the NTCP mutants Y146A and Y146E, when expressed in HepG2 hepatoma cells, showed complete loss of susceptibility for in vitro HBV infection. In conclusion, tyrosine 146, and to some extent tyrosine 141, both belonging to the tyrosine-rich motif 139YIYSRGIY146 of human NTCP, are newly identified amino acid residues that play an essential role in the interaction of HBV with its receptor NTCP and, thus, in the process of virus entry into hepatocytes.
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Stross, Claudia, Stefanie Kluge, Katrin Weissenberger, Elisabeth Winands, Dieter Häussinger, and Ralf Kubitz. "A dileucine motif is involved in plasma membrane expression and endocytosis of rat sodium taurocholate cotransporting polypeptide (Ntcp)." American Journal of Physiology-Gastrointestinal and Liver Physiology 305, no. 10 (November 15, 2013): G722—G730. http://dx.doi.org/10.1152/ajpgi.00056.2013.
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The sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake transporter for bile salts into liver parenchymal cells, and PKC-mediated endocytosis was shown to regulate the number of Ntcp molecules at the plasma membrane. In this study, mechanisms of Ntcp internalization were analyzed by flow cytometry, immunofluorescence, and Western blot analyses in HepG2 cells. PKC activation induced endocytosis of Ntcp from the plasma membrane by ∼30%. Endocytosis of Ntcp was clathrin dependent and was followed by lysosomal degradation. A dileucine motif located in the third intracellular loop of Ntcp was essential for endocytosis but also for processing and plasma membrane targeting, suggesting a dual function of this motif for intracellular trafficking of Ntcp. Mutation of two of five potential phosphorylation sites surrounding the dileucine motif (Thr225 and Ser226) inhibited PKC-mediated endocytosis. In conclusion, we could identify a motif, which is critical for Ntcp plasma membrane localization. Endocytic retrieval protects hepatocytes from elevated bile salt concentrations and is of special interest, because NTCP has been identified as a receptor for the hepatitis B and D virus.
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Park, Se Won, Christopher M. Schonhoff, Cynthia R. L. Webster, and M. Sawkat Anwer. "Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane." American Journal of Physiology-Gastrointestinal and Liver Physiology 303, no. 5 (September 1, 2012): G657—G665. http://dx.doi.org/10.1152/ajpgi.00529.2011.
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Cyclic AMP stimulates translocation of Na+/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.
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Mita, Sachiko, Hiroshi Suzuki, Hidetaka Akita, Bruno Stieger, Peter J. Meier, Alan F. Hofmann, and Yuichi Sugiyama. "Vectorial transport of bile salts across MDCK cells expressing both rat Na+-taurocholate cotransporting polypeptide and rat bile salt export pump." American Journal of Physiology-Gastrointestinal and Liver Physiology 288, no. 1 (January 2005): G159—G167. http://dx.doi.org/10.1152/ajpgi.00360.2003.
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Bile salts are predominantly taken up by hepatocytes via the basolateral Na+-taurocholate cotransporting polypeptide (NTCP/SLC10A1) and secreted into the bile by the bile salt export pump (BSEP/ABCB11). In the present study, we transfected rat Ntcp and rat Bsep into polarized Madin-Darby canine kidney cells and characterized the transport properties of these cells for eight bile salts. Immunohistochemical staining demonstrated that Ntcp was expressed at the basolateral domains, whereas Bsep was expressed at the apical domains. Basal-to-apical transport of taurocholate across the monolayer expressing only Ntcp and that coexpressing Ntcp/Bsep was observed, whereas the flux across the monolayer of control and Bsep-expressing cells was symmetrical. Basal-to-apical transport of taurocholate across Ntcp/Bsep-coexpressing monolayers was significantly higher than that across monolayers expressing only Ntcp. Kinetic analysis of this vectorial transport of taurocholate gave an apparent Km value of 13.9 ± 4.7 μM for cells expressing Ntcp alone, which is comparable with 22.2 ± 4.5 μM for cells expressing both Ntcp and Bsep and Vmax values of 15.8 ± 4.2 and 60.8 ± 9.0 pmol·min−1·mg protein−1 for Ntcp alone and Ntcp and Bsep-coexpressing cells, respectively. Transcellular transport of cholate, glycocholate, taurochenodeoxycholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, ursodeoxycholate, and glycoursodeoxycholate, but not that of lithocholate was also observed across the double transfectant. This double-expressing system can be used as a model to clarify vectorial transport of bile salts across hepatocytes under physiological conditions.
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Schonhoff, Christopher M., Umadevi Ramasamy, and M. Sawkat Anwer. "Nitric oxide-mediated inhibition of taurocholate uptake involves S-nitrosylation of NTCP." American Journal of Physiology-Gastrointestinal and Liver Physiology 300, no. 2 (February 2011): G364—G370. http://dx.doi.org/10.1152/ajpgi.00170.2010.
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The sodium-taurocholate (TC) cotransporting polypeptide (NTCP) facilitates bile formation by mediating sinusoidal Na+-TC cotransport. During sepsis-induced cholestasis, there is a decrease in NTCP-dependent uptake of bile acids and an increase in nitric oxide (NO) levels in hepatocytes. In rat hepatocytes NO inhibits Na+-dependent uptake of taurocholate. The aim of this study was to extend these findings to human NTCP and to further investigate the mechanism by which NO inhibits TC uptake. Using a human hepatoma cell line stably expressing NTCP (HuH-NTCP), we performed experiments with the NO donors sodium nitroprusside and S-nitrosocysteine and demonstrated that NO inhibits TC uptake in these cells. Kinetic analyses revealed that NO significantly decreased the Vmax but not the Km of TC uptake by NTCP, indicating noncompetitive inhibition. NO decreased the amount of NTCP in the plasma membrane, providing a molecular mechanism for the noncompetitive inhibition of TC uptake. One way that NO can modify protein function is through a posttranslational modification known as S-nitrosylation: the binding of NO to cysteine thiols. Using a biotin switch technique we observed that NTCP is S-nitrosylated under conditions in which NO inhibits TC uptake. Moreover, dithiothreitol reversed NO-mediated inhibition of TC uptake and S-nitrosylation of NTCP, indicating that NO inhibits TC uptake via modification of cysteine thiols. In addition, NO treatment led to a decrease in Ntcp phosphorylation. Taken together these results indicate that the inhibition of TC uptake by NO involves S-nitrosylation of NTCP.
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Appelman, Monique D., Marion J. D. Robin, Esther W. M. Vogels, Christie Wolzak, Winnie G. Vos, Harmjan R. Vos, Robert M. Van Es, Boudewijn M. T. Burgering, and Stan F. J. Van de Graaf. "The Lipid Raft Component Stomatin Interacts with the Na+ Taurocholate Cotransporting Polypeptide (NTCP) and Modulates Bile Salt Uptake." Cells 9, no. 4 (April 16, 2020): 986. http://dx.doi.org/10.3390/cells9040986.
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The sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the basolateral membrane of hepatocytes, where it mediates the uptake of conjugated bile acids and forms the hepatocyte entry receptor for the hepatitis B and D virus. Here, we aimed to identify novel protein–protein interactions that could play a role in the regulation of NTCP. To this end, NTCP was precipitated from HA-tagged hNTCP-expressing HepG2 cells, and chloride channel CLIC-like 1 (CLCC1) and stomatin were identified as interacting proteins by mass spectrometry. Interaction was confirmed by co-immunoprecipitation. NTCP, CLCC1 and stomatin were found at the plasma membrane in lipid rafts, as demonstrated by a combination of immunofluorescence, cell surface biotinylation and isolation of detergent-resistant membranes. Neither CLCC1 overexpression nor its knockdown had an effect on NTCP function. However, both stomatin overexpression and knockdown increased NTCP-mediated taurocholate uptake while NTCP abundance at the plasma membrane was only increased in stomatin depleted cells. These findings identify stomatin as an interactor of NTCP and show that the interaction modulates bile salt transport.
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Nuttens, V. E., A. E. Nahum, and S. Lucas. "An NTCP Analysis of Urethral Complications from Low Doserate Mono- and Bi-Radionuclide Brachytherapy." Prostate Cancer 2011 (2011): 1–9. http://dx.doi.org/10.1155/2011/128360.
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Urethral NTCP has been determined for three prostates implanted with seeds based on125I (145 Gy),103Pd (125 Gy),131Cs (115 Gy),103Pd-125I (145 Gy), or103Pd-131Cs (115 Gy or 130 Gy). First,DU20, meaning that 20% of the urhral volume receive a dose of at leastDU20, is converted into an I-125 LDR equivalentDU20in order to use the urethral NTCP model. Second, the propagation of uncertainties through the steps in the NTCP calculation was assessed in order to identify the parameters responsible for large data uncertainties. Two sets of radiobiological parameters were studied. The NTCP results all fall in the 19%–23% range and are associated with large uncertainties, making the comparison difficult. Depending on the dataset chosen, the ranking of NTCP values among the six seed implants studied changes. Moreover, the large uncertainties on the fitting parameters of the urethral NTCP model result in large uncertainty on the NTCP value. In conclusion, the use of NTCP model for permanent brachytherapy is feasible but it is essential that the uncertainties on the parameters in the model be reduced.
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Sun, An-Qiang, I'Kyori Swaby, Shuhua Xu, and Frederick J. Suchy. "Cell-specific basolateral membrane sorting of the human liver Na+-dependent bile acid cotransporter." American Journal of Physiology-Gastrointestinal and Liver Physiology 280, no. 6 (June 1, 2001): G1305—G1313. http://dx.doi.org/10.1152/ajpgi.2001.280.6.g1305.
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The human Na+-taurocholate cotransporting polypeptide (Ntcp) is located exclusively on the basolateral membrane of hepatocyte, but the mechanisms underlying its membrane sorting domain have not been fully elucidated. In the present study, a green fluorescent protein-fused human NTCP (NTCP-GFP) was constructed using the polymerase chain reaction and was stably transfected into Madin-Darby canine kidney (MDCK) and Caco-2 cells. Taurocholate uptake studies and confocal microscopy demonstrated that the polarity of basolateral surface expression of NTCP-GFP was maintained in MDCK cells but was lost in Caco-2 cells. Nocodazole (33 μM), an agent that causes microtubular depolymerization, partially disrupted the basolateral localization of NTCP-GFP by increasing apical surface expression to 33.5% compared with untreated cells ( P < 0.05). Brefeldin A (BFA; 1–2 μM) disrupted the polarized basolateral localization of NTCP, but monensin (1.4 μM) had no affect on NTCP-GFP localization. In addition, low-temperature shift (20°C) did not affect the polarized basolateral surface sorting of NTCP-GFP and repolarization of this protein after BFA interruption. In summary, these data suggest that the polarized basolateral localization of human NTCP is cell specific and is mediated by a novel sorting pathway that is BFA sensitive and monensin and low-temperature shift insensitive. The process may also involve microtubule motors.
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Li, Jisu, Li Zong, Camille Sureau, Luke Barker, Jack R. Wands, and Shuping Tong. "Unusual Features of Sodium Taurocholate Cotransporting Polypeptide as a Hepatitis B Virus Receptor." Journal of Virology 90, no. 18 (July 6, 2016): 8302–13. http://dx.doi.org/10.1128/jvi.01153-16.
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ABSTRACTCell culture (cc)-derived hepatitis B virus (HBV) can infect differentiated HepaRG cells, but efficient infection requires addition of polyethylene glycol (PEG) during inoculation. Identification of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor enabled ccHBV infection of NTCP reconstituted HepG2 cells, although very little hepatitis B surface antigen (HBsAg) is produced. We found infection by patient serum-derived HBV (sHBV), which required purification of viral particles through ultracentrifugation or PEG precipitation, was PEG independent and much more efficient in HepaRG cells than in HepG2/NTCP cells. In contrast to hepatitis B e antigen (HBeAg), HBsAg was not a reliable marker of productive sHBV infection at early time points. A low HBsAg/HBeAg ratio by ccHBV-infected HepG2/NTCP cells was attributable to dimethyl sulfoxide (DMSO) in culture medium, NTCP overexpression, and HBV genotype D. HepG2/NTCP cells released more viral antigens than HepG2 cells after HBV genome delivery by adeno-associated virus, and stable expression of NTCP in a ccHBV producing cell line increased viral mRNAs, proteins, replicative DNA, and covalently closed circular DNA. NTCP protein expression in HepG2/NTCP cells, despite being driven by the cytomegalovirus promoter, was markedly increased by DMSO treatment. This at least partly explains ability of DMSO to promote ccHBV infection in such cell lines. In conclusion, NTCP appeared inefficient to mediate infection by serum-derived HBV. It could promote HBV RNA transcription while inhibiting HBsAg secretion. Efficient PEG-independent sHBV infection of HepaRG cells permits comparative studies of diverse clinical HBV isolates and will help identify additional factors on virion surface promoting attachment to hepatocytes.IMPORTANCECurrentlyin vitroinfection with hepatitis B virus (HBV) depends on cell culture-derived HBV inoculated in the presence of polyethylene glycol. We found patient serum-derived HBV could efficiently infect differentiated HepaRG cells independent of polyethylene glycol, which represents a more physiological infection system. Serum-derived HBV has poor infectivity in HepG2 cells reconstituted with sodium taurocholate cotransporting polypeptide (NTCP), the currently accepted HBV receptor. Moreover, HepG2/NTCP cells secreted very little hepatitis B surface antigen after infection with cell culture-derived HBV, which was attributed to NTCP overexpression, genotype D virus, and dimethyl sulfoxide added to culture medium. NTCP could promote HBV RNA transcription, protein expression, and DNA replication in HepG2 cells stably transfected with HBV DNA, while dimethyl sulfoxide could increase NTCP protein level despite transcriptional control by a cytomegalovirus promoter. Therefore, this study revealed several unusual features of NTCP as an HBV receptor and established conditions for efficient serum virus infectionin vitro.
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Palatini, Massimo, Simon Franz Müller, Michael Kirstgen, Silke Leiting, Felix Lehmann, Lena Soppa, Nora Goldmann, et al. "IFITM3 Interacts with the HBV/HDV Receptor NTCP and Modulates Virus Entry and Infection." Viruses 14, no. 4 (March 30, 2022): 727. http://dx.doi.org/10.3390/v14040727.
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The Na+/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of the virus/NTCP complex involves co-factors, but this process is not fully understood. As part of the innate immunity, interferon-induced transmembrane proteins (IFITM) 1–3 have been characterized as virus entry-restricting factors for many viruses. The present study identified IFITM3 as a novel protein–protein interaction (PPI) partner of NTCP based on membrane yeast-two hybrid and co-immunoprecipitation experiments. Surprisingly, IFITM3 knockdown significantly reduced in vitro HBV infection rates of NTCP-expressing HuH7 cells and primary human hepatocytes (PHHs). In addition, HuH7-NTCP cells showed significantly lower HDV infection rates, whereas infection with influenza A virus was increased. HBV-derived myr-preS1 peptide binding to HuH7-NTCP cells was intact even under IFITM3 knockdown, suggesting that IFITM3-mediated HBV/HDV infection enhancement occurs in a step subsequent to the viral attachment to NTCP. In conclusion, IFITM3 was identified as a novel NTCP co-factor that significantly affects in vitro infection with HBV and HDV in NTCP-expressing hepatoma cells and PHHs. While there is clear evidence for a direct PPI between IFITM3 and NTCP, the specific mechanism by which this PPI facilitates the infection process remains to be identified in future studies.
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Wang, Ying, Matthew Wilkerson, Jibin Li, Wei Zhang, Albert Owens, Stephen Wright, and Ismael Hidalgo. "Assessment of Statin Interactions With the Human NTCP Transporter Using a Novel Fluorescence Assay." International Journal of Toxicology 39, no. 6 (October 20, 2020): 518–29. http://dx.doi.org/10.1177/1091581820953066.
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Sodium taurocholate cotransporting polypeptide (NTCP), which is highly expressed in the sinusoidal membrane of hepatocytes, maintains bile acid homeostasis and participates in the hepatic disposition of a variety of endogenous substances as well as xenobiotics. Manifested by the involvement of organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) in the hepatic uptake of statin drugs, sinusoidal membrane transporters play an important role in the pharmacokinetics and pharmacodynamics of these agents. It has been speculated that NTCP may function as an alternative pathway for statin hepatic uptake, complementary to OATP1B1 and OATP1B3. In the current study, we produced stable NTCP-expressing human embryonic kidney 293 (HEK293) cells and developed a fluorescence-based assay using flow cytometry for measuring NTCP transport with chenodeoxycholyl-(Nε-7-nitrobenz-2-oxa-1,3-diazole)-lysine (CDCA-NBD) as the substrate. NTCP-mediated CDCA-NBD transport was time-dependent and exhibited typical Michaelis–Menten kinetics, with a K m of 6.12 µM. Compounds known to interact with NTCP, including chenodeoxycholic acid and taurocholic acid, displayed concentration-dependent inhibition of NTCP-mediated CDCA-NBD transport. We report here a systematic evaluation of the interaction between statins and the NTCP transporter. Utilizing this system, several statins were either found to inhibit NTCP-dependent transport or act as substrates. We find a good correlation between the reported lipophilicity of statins and their ability to inhibit NTCP. The objective was to develop a higher-throughput system to evaluate potential inhibitors such as the statins. The in vitro assays using CDCA-NBD as fluorescent substrate are convenient, rapid, and have utility in screening drug candidates for potential drug–NTCP interactions.
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Murray, John W., Amar J. Thosani, Pijun Wang, and Allan W. Wolkoff. "Heterogeneous accumulation of fluorescent bile acids in primary rat hepatocytes does not correlate with their homogenous expression of ntcp." American Journal of Physiology-Gastrointestinal and Liver Physiology 301, no. 1 (July 2011): G60—G68. http://dx.doi.org/10.1152/ajpgi.00035.2011.
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Sodium taurocholate-cotransporting polypeptide (ntcp) is considered to be a major determinant of bile acid uptake into hepatocytes. However, the regulation of ntcp and the degree that it participates in the accumulation of specific substrates are not well understood. We utilized fluorescent bile acid derivatives and direct quantitation of fluorescent microscopy images to examine the regulation of ntcp and its role in the cell-to-cell variability of fluorescent bile acid accumulation. Primary-cultured rat hepatocytes rapidly accumulated the fluorescent bile acids, chenodeoxycholylglycylamidofluorescein (CDCGamF), 7-β- nitrobenzoxadiazole 3-α hydroxy 5-β cholan-24-oic acid (NBD-CA), and cholyl-glycylamido-fluorescein (CGamF). However, in stably transfected HeLa cells, ntcp preferred CDCGamF, whereas the organic anion transporter, organic anion transporting polypeptide 1 (oatp1a1), preferred NBD-CA, and neither ntcp nor oatp1a1 showed strong accumulation of CGamF by these methods. Ntcp-mediated transport of CDCGamF was inhibited by taurocholate, cyclosporin, actin depolymerization, and an inhibitor of atypical PKC-ζ. The latter two agents altered the cellular distribution of ntcp as visualized in ntcp-green fluorescent protein-transfected cells. Although fluorescent bile acid accumulation was reproducible by the imaging assays, individual cells showed variable accumulation that was not attributable to changes in membrane permeability or cell viability. In HeLa cells, this was accounted for by variable levels of ntcp, whereas, in hepatocytes, ntcp expression was uniform, and low accumulation was seen in a large portion of cells despite the presence of ntcp. These studies indicate that single-cell imaging can provide insight into previously unrecognized details of anion transport in the complex environment of polarized hepatocytes.
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Stross, Claudia, Angelika Helmer, Katrin Weissenberger, Boris Görg, Verena Keitel, Dieter Häussinger, and Ralf Kubitz. "Protein kinase C induces endocytosis of the sodium taurocholate cotransporting polypeptide." American Journal of Physiology-Gastrointestinal and Liver Physiology 299, no. 2 (August 2010): G320—G328. http://dx.doi.org/10.1152/ajpgi.00180.2010.
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Bile salts influence signaling and metabolic pathways. In hepatocytes, the sodium taurocholate cotransporting polypeptide (Ntcp) is a major determinant of intracellular bile salt levels. Short-term downregulation of Ntcp is not well characterized to date. FLAG and enhanced green fluorescent protein (EGFP) tags were cloned to the extra- and intracellular termini of Ntcp. Endocytosis of Ntcp in transfected HepG2 cells was visualized by fluorescence of EGFP, and membrane surface expression of Ntcp was quantified by flow cytometry with fluorochrome-labeled FLAG antibodies. Activation of protein kinase C (PKC) by phorbolester or thymeleatoxin an activator of Ca2+-dependent conventional PKCs (cPKCs), induced endocytosis of Ntcp, whereas the Na+-K+-ATPase remained in the plasma membrane. The PKC inhibitor BIM I and the cPKC-selective inhibitor Gö6976 abolished PMA-induced endocytosis. Because of this internalization, cell surface expression of Ntcp was reduced by 36 ± 7%, bile salt uptake was decreased by 25%, and taurolithocholate sulfate-induced cell toxicity was prevented. In conclusion, Ca2+-dependent PKCs induce vesicular retrieval of Ntcp, thereby reducing bile salt uptake. This mechanism may protect hepatocytes from toxic intracellular bile salt concentrations.
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Iwamoto, Masashi, Wakana Saso, Ryuichi Sugiyama, Koji Ishii, Mio Ohki, Shushi Nagamori, Ryosuke Suzuki, et al. "Epidermal growth factor receptor is a host-entry cofactor triggering hepatitis B virus internalization." Proceedings of the National Academy of Sciences 116, no. 17 (April 5, 2019): 8487–92. http://dx.doi.org/10.1073/pnas.1811064116.
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Sodium taurocholate cotransporting polypeptide (NTCP) is a host cell receptor required for hepatitis B virus (HBV) entry. However, the susceptibility of NTCP-expressing cells to HBV is diverse depending on the culture condition. Stimulation with epidermal growth factor (EGF) was found to potentiate cell susceptibility to HBV infection. Here, we show that EGF receptor (EGFR) plays a critical role in HBV virion internalization. In EGFR-knockdown cells, HBV or its preS1-specific fluorescence peptide attached to the cell surface, but its internalization was attenuated. PreS1 internalization and HBV infection could be rescued by complementation with functional EGFR. Interestingly, the HBV/preS1–NTCP complex at the cell surface was internalized concomitant with the endocytotic relocalization of EGFR. Molecular interaction between NTCP and EGFR was documented by immunoprecipitation assay. Upon dissociation from functional EGFR, NTCP no longer functioned to support viral infection, as demonstrated by either (i) the introduction of NTCP point mutation that disrupted its interaction with EGFR, (ii) the detrimental effect of decoy peptide interrupting the NTCP–EGFR interaction, or (iii) the pharmacological inactivation of EGFR. Together, these data support the crucial role of EGFR in mediating HBV–NTCP internalization into susceptible cells. EGFR thus provides a yet unidentified missing link from the cell-surface HBV–NTCP attachment to the viral invasion beyond the host cell membrane.
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Noppes, Saskia, Simon Franz Müller, Josefine Bennien, Matthias Holtemeyer, Massimo Palatini, Regina Leidolf, Jörg Alber, and Joachim Geyer. "Homo- and heterodimerization is a common feature of the solute carrier family SLC10 members." Biological Chemistry 400, no. 10 (October 25, 2019): 1371–84. http://dx.doi.org/10.1515/hsz-2019-0148.
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AbstractThe solute carrier family SLC10 consists of seven members, including the bile acid transporters Na+/taurocholate co-transporting polypeptide (NTCP) and apical sodium-dependent bile acid transporter (ASBT), the steroid sulfate transporter SOAT as well as four orphan carriers (SLC10A3, SLC10A4, SLC10A5 and SLC10A7). Previously, homodimerization of NTCP, ASBT and SOAT was described and there is increasing evidence that carrier oligomerization is an important regulatory factor for protein sorting and transport function. In the present study, homo- and heterodimerization were systematically analyzed among all SLC10 carriers (except for SLC10A3) using the yeast-two-hybrid membrane protein system. Strong homodimerization occurred for NTCP/NTCP, ASBT/ASBT and SLC10A7/SLC10A7. Heterodimerization was observed for most of the SLC10 carrier combinations. Heterodimerization of NTCP was additionally investigated by co-localization of NTCP-GFP and NTCP-mScarlet with respective SLC10 carrier constructs. NTCP co-localized with SLC10A4, SLC10A5, SOAT and SLC10A7. This co-localization was most pronounced for SLC10A4 and was additionally confirmed by co-immunoprecipitation. Interestingly, SLC10 carrier co-expression decreased the taurocholate transport function of NTCP for most of the analyzed constructs, indicating that SLC10 carrier heterodimerization is of functional relevance. In conclusion, homo- and heterodimerization is a common feature of the SLC10 carriers. The relevance of this finding for regulation and transport function of the SLC10 carriersin vivoneeds further investigation.
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He, Wenhui, Zhiliang Cao, Fengfeng Mao, Bijie Ren, Yunfei Li, Dan Li, Huiyu Li, et al. "Modification of Three Amino Acids in Sodium Taurocholate Cotransporting Polypeptide Renders Mice Susceptible to Infection with Hepatitis D VirusIn Vivo." Journal of Virology 90, no. 19 (July 27, 2016): 8866–74. http://dx.doi.org/10.1128/jvi.00901-16.
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ABSTRACTSodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for hepatitis D virus (HDV) and its helper hepatitis B virus (HBV). In cultured cell lines, HDV infection through mouse NTCP is restricted by residues 84 to 87 of the receptor. This study shows that mice with these three amino acids altered their corresponding human residues (H84R, T86K, and S87N) in endogenous mouse NTCP supportde novoHDV infectionin vivo. HDV infection was documented by the presence of replicative forms of HDV RNA and HDV proteins in liver cells at day 6 after viral inoculation. Monoclonal antibody specifically binding to the motif centered on K86 in NTCP partially inhibited HDV infection. These studies demonstrated specific interaction between the receptor and the viral envelopesin vivoand established a novel mouse model with minimal genetic manipulation for studying HDV infection. The model will also be useful for evaluating entry inhibitors against HDV and its helper HBV.IMPORTANCENTCP was identified as a functional receptor for both HDV and HBV in cell cultures. We recently showed that neonatal C57BL/6 transgenic (Tg) mice exogenously expressing human NTCP (hNTCP-Tg) in liver support transient HDV infection. In this study, we introduced alterations of three amino acids in the endogenous NTCP of FVB mice through genome editing. The mice with the humanized NTCP residues (H84R, T86K, and S87N) are susceptible to HDV infection, and the infection can be established in both neonatal and adult mice with this editing. We also developed a monoclonal antibody specifically targeting the region of NTCP centered on lysine residue 86, and it can differentiate the modified mouse NTCP from that of the wild type and partially inhibited HDV infection. These studies shed new light on NTCP-mediated HDV infectionin vivo, and the NTCP-modified mice provide a useful animal model for studying HDV infection and evaluating antivirals against the infection.
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Zollner, Gernot, Peter Fickert, Dagmar Silbert, Andrea Fuchsbichler, Conny Stumptner, Kurt Zatloukal, Helmut Denk, and Michael Trauner. "Induction of short heterodimer partner 1 precedes downregulation of Ntcp in bile duct-ligated mice." American Journal of Physiology-Gastrointestinal and Liver Physiology 282, no. 1 (January 1, 2002): G184—G191. http://dx.doi.org/10.1152/ajpgi.00215.2001.
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Cholestasis is associated with retention of bile acids and reduced expression of the Na+/taurocholate cotransporter (Ntcp), the major hepatocellular bile acid uptake system. This study aimed to determine whether downregulation of Ntcp in obstructive cholestasis 1) is a consequence of bile acid retention and 2) is mediated by induction of the transcriptional repressor short heterodimer partner 1 (SHP-1). To study the time course for the changes in serum bile acid levels as well as SHP-1 and Ntcp steady-state mRNA levels, mice were subjected to common bile duct ligation (CBDL) for 3, 6, 12, 24, 72, and 168 h and compared with sham-operated controls. Serum bile acid levels were determined by radioimmunoassay. SHP-1 and Ntcp steady-state mRNA expression were assessed by Northern blotting. In addition, Ntcp protein expression was studied by Western blotting and immunofluorescence microscopy. Increased SHP-1 mRNA expression paralleled elevations of serum bile acid levels and was followed by downregulation of Ntcp mRNA and protein expression in CBDL mice. Maximal SHP-1 mRNA expression reached a plateau phase after 6-h CBDL (12-fold; P < 0.001) and preceded the nadir of Ntcp mRNA levels (12%, P < 0.001) by 6 h. In conclusion, bile acid-induced expression of SHP-1 may, at least in part, mediate downregulation of Ntcp in CBDL mice. These findings support the concept that downregulation of Ntcp in cholestasis limits intracytoplasmatic accumulation of potentially toxic bile acids.
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Ruggiero, Melissa J., Haley Miller, Jessica Y. Idowu, Jeremiah D. Zitzow, Shu-Ching Chang, and Bruno Hagenbuch. "Perfluoroalkyl Carboxylic Acids Interact with the Human Bile Acid Transporter NTCP." Livers 1, no. 4 (October 18, 2021): 221–29. http://dx.doi.org/10.3390/livers1040017.
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Na+/taurocholate cotransporting polypeptide (NTCP) is important for the enterohepatic circulation of bile acids, which has been suggested to contribute to the long serum elimination half-lives of perfluoroalkyl substances in humans. We demonstrated that some perfluoroalkyl sulfonates are transported by NTCP; however, little was known about carboxylates. The purpose of this study was to determine if perfluoroalkyl carboxylates would interact with NTCP and potentially act as substrates. Sodium-dependent transport of [3H]-taurocholate was measured in human embryonic kidney cells (HEK293) stably expressing NTCP in the absence or presence of perfluoroalkyl carboxylates with varying chain lengths. PFCAs with 8 (PFOA), 9 (PFNA), and 10 (PFDA) carbons were the strongest inhibitors. Inhibition kinetics demonstrated competitive inhibition and indicated that PFNA was the strongest inhibitor followed by PFDA and PFOA. All three compounds are transported by NTCP, and kinetics experiments revealed that PFOA had the highest affinity for NTCP with a Km value of 1.8 ± 0.4 mM. The Km value PFNA was estimated to be 5.3 ± 3.5 mM and the value for PFDA could not be determined due to limited solubility. In conclusion, our results suggest that, in addition to sulfonates, perfluorinated carboxylates are substrates of NTCP and have the potential to interact with NTCP-mediated transport.
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Wu, Menq-Rong, Yi-You Huang, and Jong-Kai Hsiao. "Use of Indocyanine Green (ICG), a Medical Near Infrared Dye, for Enhanced Fluorescent Imaging—Comparison of Organic Anion Transporting Polypeptide 1B3 (OATP1B3) and Sodium-Taurocholate Cotransporting Polypeptide (NTCP) Reporter Genes." Molecules 24, no. 12 (June 21, 2019): 2295. http://dx.doi.org/10.3390/molecules24122295.
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Molecular and cellular imaging in living organisms have ushered in an era of comprehensive understanding of intracellular and intercellular events. Currently, more efforts have been focused on the infrared fluorescent dyes that facilitate deeper tissue visualization. Both sodium taurocholate cotransporting polypeptide (NTCP) and organic-anion-transporting polypeptide 1B3 (OATP1B3) are capable of carrying indocyanine green (ICG) into the cytoplasm. We compared the feasibility of NTCP and OATP1B3 as reporter genes in combination with ICG. NTCP and OATP1B3 were transduced into HT-29 cells. Genetically modified HT-29 cells were inoculated into nude mice. ICG was administered in vitro and in vivo and the signals were observed under confocal microscopy, flow cytometry, multimode microplate reader, and an in vivo imaging system. Both NTCP- and OATP1B3-expressing cells and xenografts had higher ICG intensities. The OATP1B3-expressing xenograft has a higher ICG uptake than the NTCP-expressing xenograft. NTCP or OATP1B3 combined with ICG could serve as a noninvasive imaging modality for molecular and cellular imaging. OATP1B3 outperforms NTCP in terms of in vivo imaging.
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Zollner, Gernot, Martin Wagner, Peter Fickert, Andreas Geier, Andrea Fuchsbichler, Dagmar Silbert, Judith Gumhold, et al. "Role of nuclear receptors and hepatocyte-enriched transcription factors for Ntcp repression in biliary obstruction in mouse liver." American Journal of Physiology-Gastrointestinal and Liver Physiology 289, no. 5 (November 2005): G798—G805. http://dx.doi.org/10.1152/ajpgi.00319.2004.
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Expression of the main hepatic bile acid uptake system, the Na+-taurocholate cotransporter (Ntcp), is downregulated during cholestasis. Bile acid-induced, farnesoid X receptor (FXR)-mediated induction of the nuclear repressor short heterodimer partner (SHP) has been proposed as a key mechanism reducing Ntcp expression. However, the role of FXR and SHP or other nuclear receptors and hepatocyte-enriched transcription factors in mediating Ntcp repression in obstructive cholestasis is unclear. FXR knockout (FXR−/−) and wild-type (FXR+/+) mice were subjected to common bile duct ligation (CBDL). Cholic acid (CA)-fed and LPS-treated FXR−/− and FXR+/+ mice were studied for comparison. mRNA levels of Ntcp and SHP and nuclear protein levels of hepatocyte nuclear factor (HNF)-1α, HNF-3β, HNF-4α, retinoid X receptor (RXR)-α, and retinoic acid receptor (RAR)-α and their DNA binding were assessed. Hepatic cytokine mRNA levels were also measured. CBDL and CA led to Ntcp repression in FXR+/+, but not FXR−/−, mice, whereas LPS reduced Ntcp expression in both genotypes. CBDL and LPS but not CA induced cytokine expression and reduced levels of HNF-1α, HNF-3β, HNF-4α, RXRα, and RARα to similar extents in FXR+/+ and FXR−/−. DNA binding of these transactivators was unaffected by CA in FXR+/+ mice but was markedly reduced in FXR−/− mice. In conclusion, Ntcp repression by CBDL and CA is mediated by accumulating bile acids via FXR and does not depend on cytokines, whereas Ntcp repression by LPS is independent of FXR. Reduced levels of HNF-1α, RXRα, and RARα in CBDL FXR−/− mice and reduced DNA binding in CA-fed FXR−/− mice, despite unchanged Ntcp levels, indicate that these factors may have a minor role in regulation of mouse Ntcp during cholestasis.
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Kirstgen, Michael, Kira Alessandra Alicia Theresa Lowjaga, Simon Franz Müller, Nora Goldmann, Felix Lehmann, Dieter Glebe, Karl-Heinz Baringhaus, and Joachim Geyer. "Hepatitis D Virus Entry Inhibitors Based on Repurposing Intestinal Bile Acid Reabsorption Inhibitors." Viruses 13, no. 4 (April 12, 2021): 666. http://dx.doi.org/10.3390/v13040666.
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Identification of Na+/taurocholate co-transporting polypeptide (NTCP) as high-affinity hepatic entry receptor for the Hepatitis B and D viruses (HBV/HDV) opened the field for target-based development of cell-entry inhibitors. However, most of the HBV/HDV entry inhibitors identified so far also interfere with the physiological bile acid transporter function of NTCP. The present study aimed to identify more virus-selective inhibitors of NTCP by screening of 87 propanolamine derivatives from the former development of intestinal bile acid reabsorption inhibitors (BARIs), which interact with the NTCP-homologous intestinal apical sodium-dependent bile acid transporter (ASBT). In NTCP-HEK293 cells, the ability of these compounds to block the HBV/HDV-derived preS1-peptide binding to NTCP (virus receptor function) as well as the taurocholic acid transport via NTCP (bile acid transporter function) were analyzed in parallel. Hits were subsequently validated by performing in vitro HDV infection experiments in NTCP-HepG2 cells. The most potent compounds S985852, A000295231, and S973509 showed in vitro anti-HDV activities with IC50 values of 15, 40, and 70 µM, respectively, while the taurocholic acid uptake inhibition occurred at much higher IC50 values of 24, 780, and 490 µM, respectively. In conclusion, repurposing of compounds from the BARI class as novel HBV/HDV entry inhibitors seems possible and even enables certain virus selectivity based on structure-activity relationships.
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Tremmel, Roman, Anne T. Nies, Barbara A. C. van Eijck, Niklas Handin, Mathias Haag, Stefan Winter, Florian A. Büttner, et al. "Hepatic Expression of the Na+-Taurocholate Cotransporting Polypeptide Is Independent from Genetic Variation." International Journal of Molecular Sciences 23, no. 13 (July 5, 2022): 7468. http://dx.doi.org/10.3390/ijms23137468.
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The hepatic Na+-taurocholate cotransporting polypeptide NTCP/SLC10A1 is important for the uptake of bile salts and selected drugs. Its inhibition results in increased systemic bile salt concentrations. NTCP is also the entry receptor for the hepatitis B/D virus. We investigated interindividual hepatic SLC10A1/NTCP expression using various omics technologies. SLC10A1/NTCP mRNA expression/protein abundance was quantified in well-characterized 143 human livers by real-time PCR and LC-MS/MS-based targeted proteomics. Genome-wide SNP arrays and SLC10A1 next-generation sequencing were used for genomic analyses. SLC10A1 DNA methylation was assessed through MALDI-TOF MS. Transcriptomics and untargeted metabolomics (UHPLC-Q-TOF-MS) were correlated to identify NTCP-related metabolic pathways. SLC10A1 mRNA and NTCP protein levels varied 44-fold and 10.4-fold, respectively. Non-genetic factors (e.g., smoking, alcohol consumption) influenced significantly NTCP expression. Genetic variants in SLC10A1 or other genes do not explain expression variability which was validated in livers (n = 50) from The Cancer Genome Atlas. The identified two missense SLC10A1 variants did not impair transport function in transfectants. Specific CpG sites in SLC10A1 as well as single metabolic alterations and pathways (e.g., peroxisomal and bile acid synthesis) were significantly associated with expression. Inter-individual variability of NTCP expression is multifactorial with the contribution of clinical factors, DNA methylation, transcriptional regulation as well as hepatic metabolism, but not genetic variation.
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Hinuma, Shuji, Kazuyo Fujita, and Shun’ichi Kuroda. "Specific Binding and Endocytosis of Liposomes to HEK293T Cells via Myrisoylated Pre-S1 Peptide Bound to Sodium Taurocholate Cotransporting Polypeptide." Vaccines 10, no. 12 (November 30, 2022): 2050. http://dx.doi.org/10.3390/vaccines10122050.
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(1) Background: Sodium taurocholate cotransporting polypeptide (NTCP) functions as a key receptor for the hepatitis B virus (HBV) infection. Analyzing HBV and NTCP interaction is an important issue not only for basic research but also for the development of anti-HBV therapeutics. We developed here a novel model system to analyze the interaction of NTCP with liposomes instead of HBV. (2) Methods: Liposomal binding and endocytosis through NTCP in HEK293T cells were achieved by serial treatments of HEL293T cells transiently expressing NTCP-green fluorescence protein (GFP) fusion protein with a synthetic biotinylated pre-S1 peptide (Myr47-Bio) and streptavidin (SA) complex (i.e., Myr47-Bio+SA) followed by biotinylated liposomes. By this procedure, binding of [biotinylated liposomes]-[Myr47-Bio+SA]-[NTCP-GFP] was formed. (3) Results: Using this model system, we found that liposomal binding to NTCP on the cell surface via Myr47-Bio+SA was far more efficient than that to scavenger receptor class B type 1 (SR-B1). Furthermore, liposomes bound to cell surface NTCP via Myr47-Bio+SA were endocytosed into cells after cells were cultured at 37 °C. However, this endocytosis was suppressed by 4 °C or cytochalasin B treatment. (4) Conclusions: This model system will be useful for not only analyzing HBV entry mechanisms but also screening substances to prevent HBV infection.
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Zhao, Xiaoyu, Waqas Iqbal, Pingnan Sun, and Xiaoling Zhou. "Na+-Taurocholate Co-Transporting Polypeptide (NTCP) in Livers, Function, Expression Regulation, and Potential in Hepatitis B Treatment." Livers 1, no. 4 (November 4, 2021): 236–49. http://dx.doi.org/10.3390/livers1040019.
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Chronic hepatitis B virus (HBV) infection has become one of the leading causes of liver cirrhosis and hepatocellular carcinoma globally. The discovery of sodium taurocholate co-transporting polypeptide (NTCP), a solute carrier, as a key receptor for HBV and hepatitis D virus (HDV) has opened new avenues for HBV treatment. Additionally, it has led researchers to generate hepatoma cell lines (including HepG2-NTCP and Huh-7-NTCP) susceptible to HBV infection in vitro, hence, paving the way to develop and efficiently screen new and novel anti-HBV drugs. This review summarizes the history, function and critical findings regarding NTCP as a viral receptor for HBV/HDV, and it also discusses recently developed drugs targeting NTCP.
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Prayongrat, Anussara, Natchalee Srimaneekarn, Sira Sriswasdi, Yoichi M. Ito, Norio Katoh, Masaya Tamura, Yasuhiro Dekura, et al. "Assessment of the confidence interval in the multivariable normal tissue complication probability model for predicting radiation-induced liver disease in primary liver cancer." Journal of Radiation Research 62, no. 3 (April 24, 2021): 483–93. http://dx.doi.org/10.1093/jrr/rrab011.
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Abstract We developed a confidence interval-(CI) assessing model in multivariable normal tissue complication probability (NTCP) modeling for predicting radiation-induced liver disease (RILD) in primary liver cancer patients using clinical and dosimetric data. Both the mean NTCP and difference in the mean NTCP (ΔNTCP) between two treatment plans of different radiotherapy modalities were further evaluated and their CIs were assessed. Clinical data were retrospectively reviewed in 322 patients with hepatocellular carcinoma (n = 215) and intrahepatic cholangiocarcinoma (n = 107) treated with photon therapy. Dose–volume histograms of normal liver were reduced to mean liver dose (MLD) based on the fraction size-adjusted equivalent uniform dose. The most predictive variables were used to build the model based on multivariable logistic regression analysis with bootstrapping. Internal validation was performed using the cross-validation leave-one-out method. Both the mean NTCP and the mean ΔNTCP with 95% CIs were calculated from computationally generated multivariate random sets of NTCP model parameters using variance–covariance matrix information. RILD occurred in 108/322 patients (33.5%). The NTCP model with three clinical and one dosimetric parameter (tumor type, Child–Pugh class, hepatitis infection status and MLD) was most predictive, with an area under the receiver operative characteristics curve (AUC) of 0.79 (95% CI 0.74–0.84). In eight clinical subgroups based on the three clinical parameters, both the mean NTCP and the mean ΔNTCP with 95% CIs were able to be estimated computationally. The multivariable NTCP model with the assessment of 95% CIs has potential to improve the reliability of the NTCP model-based approach to select the appropriate radiotherapy modality for each patient.
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Mita, Sachiko, Hiroshi Suzuki, Hidetaka Akita, Hisamitsu Hayashi, Reiko Onuki, Alan F. Hofmann, and Yuichi Sugiyama. "Vectorial transport of unconjugated and conjugated bile salts by monolayers of LLC-PK1 cells doubly transfected with human NTCP and BSEP or with rat Ntcp and Bsep." American Journal of Physiology-Gastrointestinal and Liver Physiology 290, no. 3 (March 2006): G550—G556. http://dx.doi.org/10.1152/ajpgi.00364.2005.
Full textAbstract:
Na+-taurocholate-cotransporting peptide (NTCP)/SLC10A1 and bile salt export pump (BSEP)/ABCB11 synergistically play an important role in the transport of bile salts by the hepatocyte. In this study, we transfected human NTCP and BSEP or rat Ntcp and Bsep into LLC-PK1 cells, a cell line devoid of bile salts transporters. Transport by these cells was characterized with a focus on substrate specificity between rats and humans. The basal to apical flux of taurocholate across NTCP- and BSEP-expressing LLC-PK1 monolayers was 10 times higher than that in the opposite direction, whereas the flux across the monolayer of control and NTCP or BSEP single-expressing cells did not show any vectorial transport. The basal to apical flux of taurocholate was saturated with a Km value of 20 μM. Vectorial transcellular transport was also observed for cholate, chenodeoxycholate, ursodeoxycholate, their taurine and glycine conjugates, and taurodeoxycholate and glycodeoxycholate, whereas no transport of lithocholate was detected. To evaluate the respective functions of NTCP and BSEP and to compare them with those of rat Ntcp and Bsep, we calculated the clearance by each transporter in this system. A good correlation in the clearance of the examined bile salts (cholate, chenodeoxycholate, ursodeoxycholate, and their taurine or glycine conjugates) was observed between transport by human and that of rat transporters in terms of their rank order: for NTCP, taurine conjugates > glycine conjugates > unconjugated bile salts, and for BSEP, unconjugated bile salts and glycine conjugates > taurine conjugates. In conclusion, the substrate specificity of human and rat NTCP and BSEP appear to be very similar at least for monovalent bile salts under physiological conditions.
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Zahoor, Muhammad Atif, Adrian Kuipery, Alexander I. Mosa, Adam J. Gehring, and Jordan J. Feld. "HepG2-NTCP Subclones Exhibiting High Susceptibility to Hepatitis B Virus Infection." Viruses 14, no. 8 (August 17, 2022): 1800. http://dx.doi.org/10.3390/v14081800.
Full textAbstract:
HepG2 cells reconstituted with Hepatitis B virus (HBV) entry receptor sodium taurocholate co-transporting polypeptide (NTCP) are widely used as a convenient in vitro cell culture infection model for HBV replication studies. As such, it is pertinent that HBV infectivity is maintained at steady-state levels for an accurate interpretation of in vitro data. However, variations in the HBV infection efficiency due to imbalanced NTCP expression levels in the HepG2 cell line may affect experimental results. In this study, we performed single cell-cloning of HepG2-NTCP-A3 parental cells via limiting dilution and obtained multiple subclones with increased permissiveness to HBV. Specifically, one subclone (HepG2-NTCP-A3/C2) yielded more than four-fold higher HBV infection compared to the HepG2-NTCP-A3 parental clone. In addition, though HBV infectivity was universally reduced in the absence of polyethylene glycol (PEG), subclone C2 maintained relatively greater permissiveness under PEG-free conditions, suggesting the functional heterogeneity within parental HepG2-NTCP-A3 may be exploitable in developing a PEG-free HBV infection model. The increased viral production correlated with increased intracellular viral antigen expression as evidenced through HBcAg immunofluorescence staining. Further, these subclones were found to express different levels of NTCP, albeit with no remarkable morphology or cell growth differences. In conclusion, we isolated the subclones of HepG2-NTCP-A3 which support efficient HBV production and thus provide an improved in vitro HBV infection model.
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Webster, Cynthia R. L., Christopher Blanch, and M. Sawkat Anwer. "Role of PP2B in cAMP-induced dephosphorylation and translocation of NTCP." American Journal of Physiology-Gastrointestinal and Liver Physiology 283, no. 1 (July 1, 2002): G44—G50. http://dx.doi.org/10.1152/ajpgi.00530.2001.
Full textAbstract:
cAMP-mediated stimulation of hepatic bile acid uptake is associated with dephosphorylation and translocation of Na+-taurocholate (TC) cotransporting peptide (NTCP) to the plasma membrane. Although translocation of NTCP may be facilitated by dephosphorylation, the mechanism of dephosphorylation is unknown. The ability of cAMP to translocate and dephosphorylate NTCP is, in part, dependent on cAMP-mediated increases in cytosolic Ca2+concentration ([Ca2+]), indicating that a Ca2+/calmodulin-dependent protein phosphatase (PP2B) may be involved. Thus we studied the role of PP2B using the inhibitor cypermethrin (CM). Freshly isolated hepatocytes were pretreated with 1–5 nM CM for 30 min followed by 15 min incubation with 10 μM 8-(4-chlorophenylthio)cAMP. CM (5 nM) and FK-506 (5 μM) inhibited cAMP-stimulated TC uptake by 80 and 75%, respectively, without affecting basal TC uptake. CM also reversed cAMP-mediated NTCP dephosphorylation and translocation to 80 and 15% of the basal level, respectively. cAMP stimulated PP2B activity by 60%, and this effect was completely inhibited by 5 nM CM. PP2B dephosphorylated NTCP immunoprecipitated from control but not from cAMP-treated hepatocytes. The effect of CM was not due to any changes in cAMP-mediated increases in cytosolic [Ca2+] or decreases in mitogen-activated protein kinase (extracellular regulated kinases 1 and 2) activity. Taken together, these results suggest that cAMP dephosphorylates NTCP by activating PP2B in hepatocytes, and PP2B-mediated dephosphorylation of NTCP may be involved in cAMP-mediated NTCP translocation to the plasma membrane.
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Hata, Soichiro, Pijun Wang, Nicole Eftychiou, Meenakshisundaram Ananthanarayanan, Ashok Batta, Gerald Salen, K. Sandy Pang, and Allan W. Wolkoff. "Substrate specificities of rat oatp1 and ntcp: implications for hepatic organic anion uptake." American Journal of Physiology-Gastrointestinal and Liver Physiology 285, no. 5 (November 2003): G829—G839. http://dx.doi.org/10.1152/ajpgi.00352.2002.
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Transport of a series of3H-radiolabeled C23, C24, and C27bile acid derivatives was compared and contrasted in HeLa cell lines stably transfected with rat Na+/taurocholate cotransporting polypeptide (ntcp) or organic anion transporting polypeptide 1 (oatp1) in which expression was under regulation of a zinc-inducible promoter. Similar uptake patterns were observed for both ntcp and oatp1, except that unconjugated hyodeoxycholate was a substrate of oatp1 but not ntcp. Conjugated bile acids were transported better than nonconjugated bile acids, and the configuration of the hydroxyl groups (α or β) had little influence on uptake. Although cholic and 23 norcholic acids were transported by ntcp and oatp1, other unconjugated bile acids (chenodeoxycholic, ursodeoxycholic) were not. In contrast to ntcp, oatp1-mediated uptake of the trihydroxy bile acids taurocholate and glycocholate was four- to eightfold below that of the corresponding dihydroxy conjugates. Ntcp mediated high affinity, sodium-dependent transport of [35S]sulfobromophthalein with a Kmsimilar to that of oatp1-mediated transport of [35S]sulfobromophthalein ( Km= 3.7 vs. 3.3 μM, respectively). In addition, for both transporters, uptake of sulfobromophthalein and taurocholic acid showed mutual competitive inhibition. These results indicate that the substrate specificity of ntcp is considerably broader than previously suspected and caution the extrapolation of transport data obtained in vitro to physiological function in vivo.
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Le, Connie, Reshma Sirajee, Rineke Steenbergen, Michael A. Joyce, William R. Addison, and D. Lorne Tyrrell. "In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide." Viruses 13, no. 1 (January 12, 2021): 97. http://dx.doi.org/10.3390/v13010097.
Full textAbstract:
An estimated two billion people worldwide have been infected with hepatitis B virus (HBV). Despite the high infectivity of HBV in vivo, a lack of easily infectable in vitro culture systems hinders studies of HBV. Overexpression of the sodium taurocholate co-transporting polypeptide (NTCP) bile acid transporter in hepatoma cells improved infection efficiency. We report here a hepatoma cell culture system that does not require dimethyl sulfoxide (DMSO) for HBV infection. We overexpressed NTCP in Huh7.5 cells and allowed these cells to differentiate in a medium supplemented with human serum (HS) instead of fetal bovine serum (FBS). We show that human serum culture enhanced HBV infection in Huh7.5-NTCP cells, e.g., in HS cultures, HBV pgRNA levels were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. Human serum culture increased levels of hepatocyte differentiation markers, such as albumin secretion, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by culture in human serum may contribute to viral entry. Our study demonstrates an in vitro HBV infection of Huh7.5-NTCP cells without the use of potentially toxic DMSO.
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Evripioti, Antonia Alexandra, Ana Maria Ortega-Prieto, Jessica Katy Skelton, Quentin Bazot, and Marcus Dorner. "Phosphodiesterase-induced cAMP degradation restricts hepatitis B virus infection." Philosophical Transactions of the Royal Society B: Biological Sciences 374, no. 1773 (April 8, 2019): 20180292. http://dx.doi.org/10.1098/rstb.2018.0292.
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Hepatitis B virus (HBV) entry into hepatocytes is mediated via a high-affinity interaction between the preS1 glycoprotein and sodium/bile acid cotransporting polypeptide (NTCP). To date, in vitro model systems rely on high multiplicities of infection to achieve infection of cell lines overexpressing human NTCP. This study investigates a novel regulatory pathway for NTCP trafficking to the cell surface, induced by DMSO-mediated cellular differentiation. DMSO rapidly induces high cell surface expression of NTCP and results in increased susceptibility of cells to HBV infection. Additionally, DMSO treatment induces actin, as well as Tubulin reshaping within the cells. We show that direct disruption of the actin and Tubulin network directly enhances NTCP expression and the subsequent susceptibility of cells to HBV infection. DMSO induces these changes via alterations in the levels of cyclic (c)AMP, which participates in the observed actin rearrangements. Blocking of phosphodiesterases (PDEs), which degrade accumulated cAMP, had the same effect as DMSO differentiation and demonstrates that DMSO prevents phosphodiesterase-mediated cAMP degradation. This identifies adenylate cyclase as a novel target for blocking the entry of HBV via targeting the cell surface accumulation of NTCP. This article is part of the theme issue ‘Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses’.
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Mukhopadhayay, Sunil, M. Ananthanarayanan, Bruno Stieger, Peter J. Meier, Frederick J. Suchy, and M. Sawkat Anwer. "cAMP increases liver Na+-taurocholate cotransport by translocating transporter to plasma membranes." American Journal of Physiology-Gastrointestinal and Liver Physiology 273, no. 4 (October 1, 1997): G842—G848. http://dx.doi.org/10.1152/ajpgi.1997.273.4.g842.
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Adenosine 3′,5′-cyclic monophosphate (cAMP), acting via protein kinase A, increases transport maximum of Na+-taurocholate cotransport within 15 min in hepatocytes (S. Grüne, L. R. Engelking, and M. S. Anwer. J. Biol. Chem. 268: 17734–17741, 1993); the mechanism of this short-term stimulation was investigated. Cycloheximide inhibited neither basal nor cAMP-induced increases in taurocholate uptake in rat hepatocytes, indicating that cAMP does not stimulate transporter synthesis. Studies in plasma membrane vesicles showed that taurocholate uptake was not stimulated by the catalytic subunit of protein kinase A but was higher when hepatocytes were pretreated with cAMP. Immunoblot studies with anti-fusion protein antibodies to the cloned Na+-taurocholate cotransport polypeptide (Ntcp) showed that pretreatment of hepatocytes with cAMP increased Ntcp content in plasma membranes but not in homogenates. Ntcp was detected in microsomes, endosomes, and Golgi fractions, and cAMP pretreatment resulted in a decrease only in endosomal Ntcp content. It is proposed that cAMP increases transport maximum of Na+-taurocholate cotransport, at least in part, by translocating Ntcp from endosomes to plasma membranes.
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Geier, Andreas, Ina V. Martin, Christoph G. Dietrich, Natarajan Balasubramaniyan, Sonja Strauch, Frederick J. Suchy, Carsten Gartung, Christian Trautwein, and Meenakshisundaram Ananthanarayanan. "Hepatocyte nuclear factor-4α is a central transactivator of the mouse Ntcp gene." American Journal of Physiology-Gastrointestinal and Liver Physiology 295, no. 2 (August 2008): G226—G233. http://dx.doi.org/10.1152/ajpgi.00012.2008.
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Sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake system for conjugated bile acids. Deletions of hepatocyte nuclear factor (HNF)-1α and retinoid X receptor-α:retinoic acid receptor-α binding sites in the mouse 5′-flanking region corresponding to putatively central regulatory elements of rat Ntcp do not significantly reduce promoter activity. We hypothesized that HNF-4α, which is increasingly recognized as a central regulator of hepatocyte function, may directly transactivate mouse ( mNtcp). A 1.1-kb 5′-upstream region including the mouse Ntcp promoter was cloned and compared with the rat promoter. In contrast to a moderate 3.5-fold activation of mNtcp by HNF-1α, HNF-4α cotransfection led to a robust 20-fold activation. Deletion analysis of mouse and rat Ntcp promoters mapped a conserved HNF-4α consensus site at −345/−326 and −335/−316 bp, respectively. p-475bp mNtcpLUC is not transactivated by HNF-1α but shows a 50-fold enhanced activity upon cotransfection with HNF-4α. Gel mobility shift assays demonstrated a complex of the HNF-4α-element formed with liver nuclear extracts that was blocked by an HNF-4α specific antibody. HNF-4α binding was confirmed by chromatin immunoprecipitation. Using Hepa 1–6 cells, HNF-4α-knockdown resulted in a significant 95% reduction in NTCP mRNA. In conclusion, mouse Ntcp is regulated by HNF-4α via a conserved distal cis-element independently of HNF-1α.
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Kirstgen, Michael, Simon Franz Müller, Kira Alessandra Alicia Theresa Lowjaga, Nora Goldmann, Felix Lehmann, Sami Alakurtti, Jari Yli-Kauhaluoma, et al. "Identification of Novel HBV/HDV Entry Inhibitors by Pharmacophore- and QSAR-Guided Virtual Screening." Viruses 13, no. 8 (July 29, 2021): 1489. http://dx.doi.org/10.3390/v13081489.
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The hepatic bile acid transporter Na+/taurocholate co-transporting polypeptide (NTCP) was identified in 2012 as the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Since then, this carrier has emerged as promising drug target for HBV/HDV virus entry inhibitors, but the synthetic peptide Hepcludex® of high molecular weight is the only approved HDV entry inhibitor so far. The present study aimed to identify small molecules as novel NTCP inhibitors with anti-viral activity. A ligand-based bioinformatic approach was used to generate and validate appropriate pharmacophore and QSAR (quantitative structure–activity relationship) models. Half-maximal inhibitory concentrations (IC50) for binding inhibition of the HBV/HDV-derived preS1 peptide (as surrogate parameter for virus binding to NTCP) were determined in NTCP-expressing HEK293 cells for 150 compounds of different chemical classes. IC50 values ranged from 2 µM up to >1000 µM. The generated pharmacophore and QSAR models were used for virtual screening of drug-like chemicals from the ZINC15 database (~11 million compounds). The 20 best-performing compounds were then experimentally tested for preS1-peptide binding inhibition in NTCP-HEK293 cells. Among them, four compounds were active and revealed experimental IC50 values for preS1-peptide binding inhibition of 9, 19, 20, and 35 µM, which were comparable to the QSAR-based predictions. All these compounds also significantly inhibited in vitro HDV infection of NTCP-HepG2 cells, without showing any cytotoxicity. The best-performing compound in all assays was ZINC000253533654. In conclusion, the present study demonstrates that virtual compound screening based on NTCP-specific pharmacophore and QSAR models can predict novel active hit compounds for the development of HBV/HDV entry inhibitors.
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Fukano, Kento, Senko Tsukuda, Koichi Watashi, and Takaji Wakita. "Concept of Viral Inhibitors via NTCP." Seminars in Liver Disease 39, no. 01 (January 17, 2019): 078–85. http://dx.doi.org/10.1055/s-0038-1676804.
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AbstractIdentification of sodium taurocholate cotransporting polypeptide (NTCP) as an entry receptor for hepatitis B and D viruses (HBV and HDV) has not only promoted our understanding of the mechanism underlying the viral entry process, but also provided cell culture models supporting viral infection. These models have greatly facilitated cell-based chemical screening for the discovery of entry inhibitors, and mode of action studies using such inhibitors have shown the advantages of NTCP as a drug target. Furthermore, in vitro chemical screening by application of high-throughput affinity-based technologies that target NTCP has identified a variety of unique small molecules that interfere with viral entry. This review summarizes this hot topic in the development of HBV/HDV entry inhibitors, with special focus on the use of NTCP as a drug target.
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Lee, Jong-Won, Young-Il Jang, Wan-Shin Park, Sun-Woo Kim, and Byung-Jae Lee. "Photocatalytic and Pozzolanic Properties of Nano-SiO2/Al2O3-TiO2 Powder for Functional Mortar." Materials 12, no. 7 (March 28, 2019): 1037. http://dx.doi.org/10.3390/ma12071037.
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The present study intended to find a way to use TiO2, one of the most widely used photocatalysts, as a construction material. To that end, nano-SiO2/Al2O3-TiO2 powder (NTCP) was synthesized by coating SiO2 and Al2O3 support materials with TiO2. The NTCP was anatase phase spherical particles, specific surface areas were 319 m2/g and 267 m2/g for the SiO2-TiO2 powder and Al2O3-TiO2 powder. UV absorption test results showed the developed NTCP had a light absorption peak at wavelengths of 380 nm or below, and its absorbance was much larger than that of commercial TiO2. The NTCP formed smaller pores on the surface than commercial TiO2. As a result, the flow of the mortar decreased as the adsorption strength increased and combined a large number of water molecules. In addition, the Pozzolanic reaction by SiO2 and Al2O3 used as support materials produced many calcium silicate hydrate (C-S-H) and calcium aluminate hydrate (C-A-H). This has shown an increased strength of mortar mixed with the NTCP by promoting a nucleation effect and reducing the filling effect and the number of harmful holes in the mortar.
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Yan, Huan, and Wenhui Li. "Sodium Taurocholate Cotransporting Polypeptide Acts as a Receptor for Hepatitis B and D Virus." Digestive Diseases 33, no. 3 (2015): 388–96. http://dx.doi.org/10.1159/000371692.
Full textAbstract:
Infection of hepatitis B virus (HBV) remains a major public health problem worldwide. Understanding the viral infection and developing antivirals against HBV have been hampered by the lack of convenient culture systems and animal models for the infection. Sodium taurocholate cotransporting polypeptide (NTCP), a key bile acid transporter expressed in liver, was recently identified as a critical receptor for viral entry of HBV and its satellite virus hepatitis D virus (HDV). This finding enabled a reliable cell culture system for the viruses. Detailed studies have shown that NTCP is the major determinant for the species specificity of HBV and HDV at entry level. NTCP is responsible for most sodium-dependent bile salt uptake in liver. The molecular determinant critical for HBV/HDV infection overlaps with that for bile acids transporting on NTCP. We evaluated bile acids as potential antivirals for HBV and HDV infection, and developed bile acid derivatives that effectively block taurocholate transporting as well as viral infections. The discovery that NTCP acts as a receptor for HBV has opens a new door for future studies towards the ultimate goal of curative treatment of HBV infection.
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Webster, Cynthia R. L., and M. Sawkat Anwer. "Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp." American Journal of Physiology-Gastrointestinal and Liver Physiology 277, no. 6 (December 1, 1999): G1165—G1172. http://dx.doi.org/10.1152/ajpgi.1999.277.6.g1165.
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cAMP stimulates Na+-taurocholate (TC) cotransport by translocating the Na+-TC-cotransporting peptide (Ntcp) to the plasma membrane. The present study was undertaken to determine whether the phosphatidylinositol-3-kinase (PI3K)-signaling pathway is involved in cAMP-mediated translocation of Ntcp. The ability of cAMP to stimulate TC uptake declined significantly when hepatocytes were pretreated with PI3K inhibitors wortmannin or LY-294002. Wortmannin inhibited cAMP-mediated translocation of Ntcp to the plasma membrane. cAMP stimulated protein kinase B (PKB) activity by twofold within 5 min, an effect inhibited by wortmannin. Neither basal mitogen-activated protein kinase (MAPK) activity nor cAMP-mediated inhibition of MAPK activity was affected by wortmannin. cAMP also stimulated p70S6K activity. However, rapamycin, an inhibitor of p70S6K, failed to inhibit cAMP-mediated stimulation of TC uptake, indicating that the effect of cAMP is not mediated via p70S6K. Cytochalasin D, an inhibitor of actin filament formation, inhibited the ability of cAMP to stimulate TC uptake and Ntcp translocation. Together, these results suggest that the stimulation of TC uptake and Ntcp translocation by cAMP may be mediated via the PI3K/PKB signaling pathway and requires intact actin filaments.
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Elinger, Serge. "HBV: Stowaway of NTCP." Clinics and Research in Hepatology and Gastroenterology 38, no. 6 (December 2014): 661–63. http://dx.doi.org/10.1016/j.clinre.2014.07.009.
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Mirandola, Alfredo, Stefania Russo, Maria Bonora, Barbara Vischioni, Anna Maria Camarda, Rossana Ingargiola, Silvia Molinelli, et al. "A Patient Selection Approach Based on NTCP Models and DVH Parameters for Definitive Proton Therapy in Locally Advanced Sinonasal Cancer Patients." Cancers 14, no. 11 (May 28, 2022): 2678. http://dx.doi.org/10.3390/cancers14112678.
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(1) Background: In this work, we aim to provide selection criteria based on normal tissue complication probability (NTCP) models and additional explanatory dose-volume histogram parameters suitable for identifying locally advanced sinonasal cancer patients with orbital invasion benefitting from proton therapy. (2) Methods: Twenty-two patients were enrolled, and two advanced radiation techniques were compared: intensity modulated proton therapy (IMPT) and photon volumetric modulated arc therapy (VMAT). Plans were optimized with a simultaneous integrated boost modality: 70 and 56 Gy(RBE) in 35 fractions were prescribed to the high risk/low risk CTV. Several endpoints were investigated, classified for their severity and used as discriminating paradigms. In particular, when NTCP models were already available, a first selection criterion based on the delta-NTCP was adopted. Additionally, an overall analysis in terms of DVH parameters was performed. Furthermore, a second selection criterion based on a weighted sum of the ΔNTCP and ΔDVH was adopted. (3) Results: Four patients out of 22 (18.2%) were suitable for IMPT due to ΔNTCP > 3% for at least one severe toxicity, 4 (18.2%) due to ΔNTCP > 20% for at least three concurrent intermediate toxicities and 16 (72.7%) due to the mixed sum of ΔNTCP and ΔDVH criterion. Since, for some cases, both criteria were contemporary fulfilled, globally 17/22 patients (77.3%) would benefit from IMPT. (4) Conclusions: For this rare clinical scenario, the use of a strategy including DVH parameters and NTCPs when comparing VMAT and IMPT is feasible. We showed that patients affected by sinonasal cancer could profit from IMPT compared to VMAT in terms of optical and central nervous system organs at risk sparing.
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Dong, Zhongqi, Sean Ekins, and James E. Polli. "Quantitative NTCP pharmacophore and lack of association between DILI and NTCP Inhibition." European Journal of Pharmaceutical Sciences 66 (January 2015): 1–9. http://dx.doi.org/10.1016/j.ejps.2014.09.005.
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