Structure of the Human Cholesterol Transporter ABCG1.

ABCG1 is an ATP binding cassette (ABC) transporter that removes excess cholesterol from peripheral tissues. Despite its role in preventing lipid accumulation and the development of cardiovascular and metabolic disease, the mechanism underpinning ABCG1-mediated cholesterol transport is unknown. Here we report a cryo-EM structure of human ABCG1 at 4 Å resolution in an inward-open state, featuring sterol-like density in the binding cavity. Structural comparison with the multidrug transporter ABCG2 and the sterol transporter ABCG5/G8 reveals the basis of mechanistic differences and distinct substrate specificity. Benzamil and taurocholate inhibited the ATPase activity of liposome-reconstituted ABCG1, whereas the ABCG2 inhibitor Ko143 did not. Based on the structural insights into ABCG1, we propose a mechanism for ABCG1-mediated cholesterol transport.

Computationally guided high-throughput design of self-assembling drug nanoparticles.

Nanoformulations of therapeutic drugs are transforming our ability to effectively deliver and treat a myriad of conditions. Often, however, they are complex to produce and exhibit low drug loading, except for nanoparticles formed via co-assembly of drugs and small molecular dyes, which display drug-loading capacities of up to 95%. There is currently no understanding of which of the millions of small-molecule combinations can result in the formation of these nanoparticles. Here we report the integration of machine learning with high-throughput experimentation to enable the rapid and large-scale identification of such nanoformulations. We identified 100 self-assembling drug nanoparticles from 2.1 million pairings, each including one of 788 candidate drugs and one of 2,686 approved excipients. We further characterized two nanoparticles, sorafenib-glycyrrhizin and terbinafine-taurocholic acid both ex vivo and in vivo. We anticipate that our platform can accelerate the development of safer and more efficacious nanoformulations with high drug-loading capacities for a wide range of therapeutics.

Aß aggregation behavior at interfaces with switchable wettability: a bioinspired perspective to understand amyloid formation.

An amphiphilic taurocholic acid (TCA) doped polypyrrole (PPy) film (PPy/TCA) was used as a dynamic mimic membrane model to explore how switchable surface wettability influences amyloid aggregation. Our results indicate that the hydrophobic surface, not the hydrophilic surface, plays important roles in Aß40 adsorption and aggregation.

Role of Sodium Taurocholate Cotransporting Polypeptide as a New Reporter and Drug-Screening Platform: Implications for Preventing Hepatitis B Virus Infections.

PURPOSE: Sodium taurocholate cotransporting polypeptide (NTCP) is a transmembrane protein responsible for delivering indocyanine green (ICG), an ideal infrared fluorescent dye, from extracellular space into the cytoplasm. Additionally, NTCP located in the hepatocyte membrane is the portal for hepatitis B and D virus (HBV/HDV) infections. This study verified the feasibility of NTCP as a reporter and further established a drug-screening platform for HBV/HDV infections. PROCEDURES: NTCP was transduced into HT-29, a colorectal cancer cell line. To examine the use of NTCP as a reporter, NTCP-expressing cells were treated with ICG and examined through flow cytometry, an in vivo imaging system (IVIS), and confocal microscopy. Furthermore, ICG was administrated to NTCP-expressing tumor-bearing nude mice and examined using the IVIS. To study the drug-screening platform, NTCP-expressing cells were treated with cyclosporin A, an NTCP inhibitor, and ICG, and examined using a multimode detection platform. Moreover, nude mice were injected with NTCP inhibitors and ICG, and subsequently, their ICG signal was examined in vivo and in the blood. RESULTS: In the reporter study, the ICG signal was higher in NTCP-expressing cells/tumors than in control cells/tumors after ICG treatment. In the drug-screening platform study, NTCP-expressing cells had decreased ICG intensity after treatment with NTCP inhibitors and ICG. Nude mice that were administered cyclosporin A had lower ICG intensity in the liver and higher intensity in the peripheral tissue and blood. CONCLUSIONS: NTCP and ICG form an ideal reporter system with extensive applications in cancer biology, robust drug-drug interactions, and drug screening in HBV/HDV infections.

Probing the molecular interactions between pharmaceutical polymeric carriers and bile salts in simulated gastrointestinal fluids using NMR spectroscopy.

The number of poorly soluble new drugs is increasing and one of the effective ways to deliver such pharmaceutically active molecules is using hydrophilic polymers to form a solid dispersion. Bile salts play an important role in the solubilisation of poorly soluble compounds in the gastrointestinal tract (gut) prior to absorption. When a poorly water-soluble drug is delivered using a hydrophilic polymer based solid dispersion oral formulation, it is still unclear whether there are any polymer-bile salt interactions, which may influence the drug dissolution and solubilisation. This study, using two widely used hydrophilic model polymers, Hydroxypropyl methylcellulose (HPMC) and polyvynilpirrolidone (PVP), and sodium taurocholate (NaTC) as the model bile salt, aims to investigate the interactions between the polymers and bile salts in simulated fed state (FeSSIF) and fasted state (FaSSIF) gut fluids. The nature of the interactions was characterised using a range of NMR techniques. The results revealed that the aggregation behaviour of NaTC in FaSSIF and FeSSIF is much more complex than in water. The addition of hydrophilic polymers led to the occurrences of NaTC-HPMC and NaTC-PVP aggregation. For both systems, pH and ionic strength strongly influenced the aggregation behavior, while the ion type played a less significant role. The outcome of this study enriched the understanding of the aggregation behaviour of bile salts and typical hydrophilic pharmaceutical polymers in bio-relevant media. Due to the high surface-activity of the bile salts and their ability to interact with polymers, such aggregation behaviour is expected to play a role in drug solubilisation in the gut when the drug is delivered by hydrophilic polymer based dispersions.

Spectral-fluorescent study of the interaction of polymethine dye probes with biological surfactants - bile salts.

Spectral-fluorescent properties of polymethine dye probes anionic 3,3'-di(sulfopropyl)-4,5,4',5'-dibenzo-9-ethylthiacarbocyanine-betaine (DEC) and cationic 3,3',9-trimethylthiacarbocyanine iodide (Cyan 2) in the presence of biological surfactants, bile salts sodium cholate (NaC), sodium deoxycholate (NaDC) and sodium taurocholate (NaTC), as well as sodium dodecyl sulfate (SDS), have been studied in a wide range of surfactant concentrations. When a surfactant is introduced into a solution of DEC, changes of the spectral-fluorescent properties are observed due to decomposition of dye dimers into cis-monomers and cis-trans conversion of the resulting monomers. In the presence of SDS, both processes occur in parallel, caused by noncovalent interaction of dye monomers with micelles, and mainly occur near the critical micelle concentration (CMC). In contrast, upon the introduction of increasing concentrations of bile salts, decomposition of dye dimers into the monomers begins at lower concentrations than cis-trans conversion. The former process is almost completed at concentrations close to CMC of secondary micelles (CMC2), while the latter process occurs even at concentrations of bile salts much higher than CMC2. Hence, DEC can serve as a probe that permits estimating the value of CMC2 and is indicative of reorganization of secondary micelles upon an increase in bile salt concentration. Aggregation of DEC and Cyan 2 on bile salts is also observed. Since it is observed at relatively low concentrations of bile salts (

Oral siRNA delivery using dual transporting systems to efficiently treat colorectal liver metastasis.

Oral siRNA delivery is an ideal way to translate siRNA therapeutic effects in the clinic due to its ability to be administered in convenient and multiple dosages. However, an effective oral delivery system requires overcoming both a hostile gastrointestinal (GI) environment and non-specific targeting. Here, an HTsRP-NC system is a new oral siRNA delivery system consisting of a siRNA/protamine (sRP) nano-complex protected by a multi-functional hyaluronic acid-taurocholic acid (HA-TCA) conjugate. The HTsRP-NC promotes cell penetration and enhances endosomal escape in cancer cells. Moreover, protection of the sRP by HA-TCA from the hostile GI environment helps the AKT siRNA complex to reach the liver through the utilization of a TCA-mediated enterohepatic bile acid recycling system. AKT siRNA was released by 90% in presence of hyaluronidase in the tumor cells which indicate the potential use of HTsRP-NCs for siRNA delivery to treat tumor. After HA receptor (CD44)-mediated cellular uptake of the HTsRP-NC by the liver cancer cells, functional expression of AKT siRNA leads to the suppression of metastatic liver cancer growth in a colorectal liver metastasis (CLM) murine model. Tumor nodules were reduced by more than 1 mm size compared to control group and tumor cells were suppressed by 50% after HTsRP-NCs treatment with AKT siRNA. Overall, oral administration of the HTsRP-NC supports its potential in therapeutic applications for the effective treatment of CLM.

De Novo Macrocyclic Peptide Inhibitors of Hepatitis B Virus Cellular Entry.

Hepatitis B virus (HBV) constitutes a significant public health burden, and currently available treatment options are not generally curative, necessitating the development of new therapeutics. Here we have applied random non-standard peptide integrated discovery (RaPID) screening to identify small macrocyclic peptide inhibitors of HBV entry that target the cell-surface receptor for HBV, sodium taurocholate cotransporting polypeptide (NTCP). In addition to their anti-HBV activity, these molecules also inhibit cellular entry by the related hepatitis D virus (HDV), and are active against diverse strains of HBV (including clinically relevant nucleos(t)ide analog-resistant and vaccine escaping strains). Importantly, these macrocyclic peptides, in contrast to other NTCP-binding HBV entry inhibitors, exhibited no inhibition of NTCP-mediated bile acid uptake, making them appealing candidates for therapeutic development.

Preparation and in vitro and in vivo evaluation of quercetin-loaded mixed micelles for oral delivery.

Quercetin (QT) is a plant polyphenol with various pharmacological properties. However, the low water solubility limits its therapeutic efficacy. In the present study, QT-loaded sodium taurocholate-Pluronic P123 (QT-loaded ST/P123) mixed micelles were developed and characterized, and the effect of the formulation on improving the water solubility of QT was investigated. QT-loaded ST/P123 mixed micelles were prepared by thin film hydration-direct dissolution and optimized by uniform design. The optimal formulation possessed high drug loading (12.6%) and entrapment efficiency (95.9%) in small (16.20 nm) spherically-shaped micelles. A low critical micelle concentration indicated that the micelles were stable, and they showed a sustained release pattern, as determined in vitro in simulated gastric fluid and intestinal fluid. Pharmacokinetic evaluation showed the Cmax and AUC0-24 were 1.8-fold and 1.6-fold higher than the QT suspension. The present results indicate that QT-loaded ST/P123 micelles are potential candidates to improve the solubility and oral bioavailability of QT.

Oral siRNA Delivery to Treat Colorectal Liver Metastases.

Convenient multiple dosing makes oral administration an ideal route for delivery of therapeutic siRNA. However, hostile GI environments and nonspecific biological trafficking prevent achieving appropriate bioavailability of siRNA. Here, an orally administered AuNP-siRNA-glycol chitosan-taurocholic acid nanoparticle (AR-GT NPs) was developed to selectively deliver Akt2 siRNA and treat colorectal liver metastases (CLM). AR-GT NPs are dual padlocked nonviral vectors in which the initially formed AuNP-siRNA (AR) conjugates are further encompassed by bifunctional glycol chitosan-taurocholic acid (GT) conjugates. Covering the surface of AR with GT protected the Akt2 siRNA from GI degradation, facilitated active transport through enterocytes, and enhanced selective accumulation in CLM. Our studies in CLM animal models resulted in the reduction in Akt2 production, followed by initiation of apoptosis in cancer cells after oral administration of Akt2 siRNA-loaded AR-GT. This therapeutic siRNA delivery system may be a promising approach in treating liver-associated diseases.

Spectroscopic Investigation of the Interaction of the Anticancer Drug Mitoxantrone with Sodium Taurodeoxycholate (NaTDC) and Sodium Taurocholate (NaTC) Bile Salts.

The focus of the present work was to investigate the interaction of the anticancer drug mitoxantrone with two bile salts, sodium taurodeoxycholate (NaTDC) and sodium taurocholate (NaTC). Ultraviolet-visible (UV-Vis) absorption and electron paramagnetic resonance (EPR) spectroscopy were used to quantify the interaction and to obtain information on the location of mitoxantrone in bile salt micelles. The presence of submicellar concentrations of both bile salts induces mitoxantrone aggregation and the extent of drug aggregation in NaTDC is higher than in NaTC. For micellar bile salts concentrations, mitoxantrone monomers are entrapped in the micellar core. Binding constants, micelle/water partition coefficients and the corresponding thermodynamic parameters for binding and partitioning processes were estimated using the changes in monomer absorbance in the presence of bile salts. Binding interaction of mitoxantrone is stronger for NaTDC than NaTC micelles, whereas partitioning efficiency is higher for NaTC micelles for all investigated temperatures. Thermodynamic parameters indicate that both binding and partitioning processes are spontaneous and entropy controlled. The spectral behavior and thermodynamic parameters indicate distinct types of mitoxantrone interaction with NaTDC and NaTC micelles supported by the differences in nature and structure of bile salts micelles.

Differential interaction behaviors of an alkaloid drug berberine with various bile salts.

The present study reports a meticulous characterization of the interaction of a potent anti-cancer drug, berberine chloride (BR) with a series of bile salt aggregates having varying hydrophobicity (sodium deoxycholate (NaDC), sodium cholate (NaC), and sodium taurocholate (NaTC)). The absorption spectrum of BR reveals a complex profile comprised of four distinct peaks. Analysis of the modulations of the absorption spectral properties of BR following interaction with the bile salts raising deeper questions unveils a critical insight into the mode of interaction of the cationic drug (BR) with the bile salts; a greater degree of perturbation of the microenvironment of the isoquinolinic part of BR compared to the benzenoid part. The remarkable modulation of the fluorescence profile of BR with added bile salts provides a sensitive indicator for monitoring the interaction scenario. However, an intriguing observation in this context reveals differential fluorescence behavior of BR in various bile salt aggregates, that is, similar observations in NaDC and NaC (which are legitimately interpreted according to the 'two-step association model') in comparison to NaTC. Such contrasting behavior of BR in NaTC aggregates has been rationalized on the basis of the possibility of formation of dye aggregate facilitated because of the proximity of the cationic drug molecules to the anionic headgroup of NaTC bile salt. Surprisingly, our spectroscopic results evidence for binding location of the drug at the interfacial region in all the bile salt aggregates. To this end, the time-resolved fluorescence decay behavior of the drug within various bile salt aggregates has been meticulously studied. The fluorescence decay results are found to be highly sensitive to the structure and size of the bile salt aggregates eventually leading to characterization of the interaction of the drug with the bile salts in excellent corroboration with the steady-state data. Furthermore, the time-resolved fluorescence anisotropy decay measurements yielded insight into the modulation of rotational dynamical behavior of the drug within the bile salt aggregates.

N,N-Dimethyl Tertiary Amino Group Mediated Dual Pancreas- and Lung-Targeting Therapy against Acute Pancreatitis.

Acute pancreatitis (AP) is a sudden inflammation of the pancreas with high mortality rate worldwide. As a severe complication to AP, acute lung injury has been the major cause of death among patients with AP. Poor penetration across the blood pancreas barrier (BPB) and insufficient drug accumulation at the target site often result in poor therapeutic outcome. Our previous work successfully demonstrated a dual-specific targeting strategy to pancreas and lung using a phenolic propanediamine moiety. Inspired by this, a simplified ligand structure, N,N-dimethyl tertiary amino group, was covalently conjugated to celastrol (CLT) to afford tertiary amino conjugates via either an ester (CP) or an amide linkage (CTA). With sufficient plasma stability, CTA was subjected to the following studies. Compared to CLT, CTA exhibited excellent cellular uptake efficiency in both rat pancreatic acinar cell line (AR42J) and human pulmonary alveolar epithelial cell line (A549). Organic cation transporters were proven to be responsible for this active transport process. Given systemically, CTA specifically distributed to pancreases and lungs in rats thus resulting in a 2.59-fold and 3.31-fold increase in tissue-specific accumulation as compared to CLT. After CTA treatment, tissue lesions were greatly alleviated and the levels of proinflammatory cytokines were downregulated in rats with sodium taurocholate induced AP. Furthermore, CTA demonstrated marginal adverse effect against major organs with reduced cardiac toxicity compared to CLT. Together, tertiary amine mediated dual pancreas- and lung-targeting therapy represents an efficient and safe strategy for AP management.

Na+-taurocholate cotransporting polypeptide (NTCP/SLC10A1) ortholog in the marine skate Leucoraja erinacea is not a physiological bile salt transporter.

The Na+-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1) is a hepatocyte-specific solute carrier, which plays an important role in maintaining bile salt homeostasis in mammals. The absence of a hepatic Na+-dependent bile salt transport system in marine skate and rainbow trout raises a question regarding the function of the Slc10a1 gene in these species. Here, we have characterized the Slc10a1 gene in the marine skate, Leucoraja erinacea The transcript of skate Slc10a1 (skSlc10a1) encodes 319 amino acids and shares 46% identity to human NTCP (hNTCP) with similar topology to mammalian NTCP. SkSlc10a1 mRNA was mostly confined to the brain and testes with minimal expression in the liver. An FXR-bile salt reporter assay indicated that skSlc10a1 transported taurocholic acid (TCA) and scymnol sulfate, but not as effectively as hNTCP. An [3H]TCA uptake assay revealed that skSlc10a1 functioned as a Na+-dependent transporter, but with low affinity for TCA (Km = 92.4 µM) and scymnol sulfate (Ki = 31 µM), compared with hNTCP (TCA, Km = 5.4 µM; Scymnol sulfate, Ki = 3.5 µM). In contrast, the bile salt concentration in skate plasma was 2 µM, similar to levels seen in mammals. Interestingly, skSlc10a1 demonstrated transport activity for the neurosteroids dehydroepiandrosterone sulfate and estrone-3-sulfate at physiological concentration, similar to hNTCP. Together, our findings indicate that skSlc10a1 is not a physiological bile salt transporter, providing a molecular explanation for the absence of a hepatic Na+-dependent bile salt uptake system in skate. We speculate that Slc10a1 is a neurosteroid transporter in skate that gained its substrate specificity for bile salts later in vertebrate evolution.

In vitro inhibition of Clostridium difficile by commercial probiotics: A microcalorimetric study.

The aim of the study was to investigate the influence of some commercial probiotics on the growth of Clostridium difficile using isothermal microcalorimetry, a technique which can monitor the real time growth of bacteria. Commercial probiotic strains and products, Lactobacillus acidophilus LA-5®, Bifidobacterium lactis BB-12®, Probio 7® and Symprove™ were co-cultured with C. difficile in Brain Heart Infusion (BHI) broth supplemented with 0.1% (w/v) l-cysteine hydrochloride and 0.1% (w/v) sodium taurocholate and monitored in the microcalorimeter. Pseudomonas aeruginosa NCIMB 8628 was also co-cultured with C. difficile and studied. The results indicated inhibition of C. difficile by the probiotics. The inhibition of C. difficile was shown to be pH-dependent using neutralized and unmodified cell free supernatant (CFS) produced by the probiotic strains. However, concentrated CFS of the probiotics also inhibited the intestinal pathogen in a non pH-dependent manner, likely due to specific antimicrobial substances produced. The results also indicated that C. difficile growth was greatly influenced by the presence of sodium taurocholate and by the pH of the medium. A medium pH of between 6.45 and 6.9 demonstrated maximum growth of the organism in the microcalorimeter.

Analysis of amino acid residues in the predicted transmembrane pore influencing transport kinetics of the hepatic drug transporter organic anion transporting polypeptide 1B1 (OATP1B1).

The hepatic uptake transporters OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) mediate the uptake of endogenous metabolites and drugs from blood into hepatocytes. Alterations of transport function are accompanied with variations in drug plasma concentrations and the risk of adverse drug effects. Thus, knowledge on amino acids determining substrate recognition or transport kinetics are important to predict alterations in transport kinetics. Therefore, we analyzed the charged amino acids His54 and Tyr169, both located at the extracellular entry of the predicted transmembrane pore of OATP1B1. Based on a computational analysis we established HEK293 cell lines overexpressing the mutant OATP1B1 proteins HEK-OATP1B1p.H54Q, -p.H54A, -p.Y169H and -p.Y169A and analyzed protein expression, localization and transport kinetics of the four OATP1B1 substrates bromosulfophthalein, estradiaol-17ß-glucuronide, taurocholate and pravastatin. Consequences on transport were detected for all mutants and these were different for each amino acid exchange and for each substrate tested. For example, the exchange H54Q resulted in reduced transport for BSP (78% of wildtype OATP1B1 transport at 0.05µM, P<0.01) with reduced affinity to this substrate (Km value increases from 0.76µM to 8.04µM) but in stimulated E217ßG transport (138% compared to wildtype transport at 10µM, P<0.001). Investigating amino acid exchanges located at the extracellular entry of the transport pore of the OATP1B1 protein we demonstrated that these residues are involved in modulating transport kinetics and this participation strongly depends on the substrate and not on the physicochemical character of the investigated amino acid.

Anti-inflammatory effect of taurocholate on TNBS-induced ulcerative colitis in mice.

Taurocholate is a natural conjugated bile acid. The aim of this study was to evaluate the anti-inflammatory effect of taurocholate in TNBS-induced ulcerative colitis in mice. The colitis were induced by rectal administration of TNBS. After 24h, the experimental animals were treated with sulfasalazine (SASP, 500mg/kg/day) and taurocholate (20, 40 and 60mg/kg) for 7 consecutive days. The anti-inflammatory effects of taurocholate for colitis were assessed by body weight, colonic weight and length, macroscopic scores, and histopathological examinations. In addition, the colonic tissue levels of myeloperoxidase (MPO) activity, interleukin (IL)-1ß, interferon (IFN-γ) and tumour necrosis factor-α (TNF-α) were also determined to assess the effect of taurocholate. Compared with the model group, treatment with taurocholate (20, 40 and 60mg/kg) significantly inhibited the body weight loss, improved colonic weight and length, and decreased macroscopic and histopathological scores. Furthermore, the activity accumulation of MPO and the colonic tissue levels of IL-1ß, IFN-γ and TNF-α were also decreased by administration of taurocholate. All the findings of this study suggested that taurocholate has the anti-inflammatory effect in ulcerative colitis in mice and indicated it as a good candidate to treat inflammatory bowel disease.

Lipid nanoparticles enhance the absorption of cyclosporine A through the gastrointestinal barrier: In vitro and in vivo studies.

In the present work, the feasibility of cyclosporine A lipid nanoparticles (CsA LN) for oral administration was investigated. Three CsA LN formulations were developed using Precirol as lipid matrix, one stabilized with Tween(®) 80 (Tw) and the other two with mixtures of phosphatidylcholine or Pluronic(®) F127 with taurocholate (Lec:TC and PL:TC, respectively). The physical characteristics of the LN were studied under gastrointestinal pH and their integrity was found to be dependent on the stabilizers. The in vitro intestinal permeability was assessed with a human colon adenocarcinoma cell model and in vivo pharmacokinetic and biodistribution studies were performed in Balb/c mice using Sandimmune Neoral(®) as reference. In vitro results showed the highest CsA permeability with the LN containing Lec:TC. In contrast, the best in vivo performance was achieved from the LN containing Tw. The bioavailability of CsA was matched and even enhanced with Precirol nanoparticles. This study suggests the suitability of LN as promising vehicles for CsA oral delivery.

In vitro and in vivo study of Baicalin-loaded mixed micelles for oral delivery.

The aim of this work was to research the potential functions and the mechanism of absorption of the baicalin (BC)-loaded micelle system that contained Pluronic P123 copolymer (P123) and sodium taurocholate (ST) as carrier materials via oral delivery. Based on the numerous advantages of oral administration, such as cost-effectiveness, flexible and accommodated dosing regimen, and improved compliance for patients, the ST-P123-MMs system would be evaluated as oral delivery vehicle of BC. In this study, X-ray powder diffractometer analysis confirmed the phase change of BC after being incorporated in mixed micelles. The release study in simulated gastric fluid/simulated intestinal fluid exhibited that BC-loaded ST-P123-MMs presented a sustained drug release behavior. Compared with coumarin-6 solution, higher cellar uptake efficiency was achieved for coumarin-6 loaded ST-P123-MMs towards Caco-2 cell lines. The in situ perfusion test in rat indicated that the absorption of BC-loaded ST-P123-MMs in intestinal tract was stronger than BC solution. After oral administration, the Cmax and AUC of BC-loaded ST-P123-MMs were 1.77 times and 1.54 times as high as those of BC suspension in rat, respectively. Promisingly, the formulated BC exhibited a prolonged circulation time with the oral bioavailability increased to 1.54-fold compared with the control group. These results all suggested that P123 and ST mixed micelles could serve as a promising approach to oral administration of BC.

Regulation of plasma membrane localization of the Na+-taurocholate cotransporting polypeptide (Ntcp) by hyperosmolarity and tauroursodeoxycholate.

In perfused rat liver, hepatocyte shrinkage induces a Fyn-dependent retrieval of the bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane (Cantore, M., Reinehr, R., Sommerfeld, A., Becker, M., and Häussinger, D. (2011) J. Biol. Chem. 286, 45014-45029) leading to cholestasis. However little is known about the effects of hyperosmolarity on short term regulation of the Na(+)-taurocholate cotransporting polypeptide (Ntcp), the major bile salt uptake system at the sinusoidal membrane of hepatocytes. The aim of this study was to analyze hyperosmotic Ntcp regulation and the underlying signaling events. Hyperosmolarity induced a significant retrieval of Ntcp from the basolateral membrane, which was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes but not of c-Src. Hyperosmotic internalization of Ntcp was sensitive to SU6656 and PP-2, suggesting that Fyn mediates Ntcp retrieval from the basolateral membrane. Hyperosmotic internalization of Ntcp was also found in livers from wild-type mice but not in p47(phox) knock-out mice. Tauroursodeoxycholate (TUDC) and cAMP reversed hyperosmolarity-induced Fyn activation and triggered re-insertion of the hyperosmotically retrieved Ntcp into the membrane. This was associated with dephosphorylation of the Ntcp on serine residues. Insertion of Ntcp by TUDC was sensitive to the integrin inhibitory hexapeptide GRGDSP and inhibition of protein kinase A. TUDC also reversed the hyperosmolarity-induced retrieval of bile salt export pump from the canalicular membrane. These findings suggest a coordinated and oxidative stress- and Fyn-dependent retrieval of sinusoidal and canalicular bile salt transport systems from the corresponding membranes. Ntcp insertion was also identified as a novel target of ß1-integrin-dependent TUDC action, which is frequently used in the treatment of cholestatic liver disease.