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

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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Vectorial transport of bile salts across MDCK cells expressing both rat Na+-taurocholate cotransporting polypeptide and rat bile salt export pump

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00360.2003 ◽

2005 ◽

Vol 288 (1) ◽

pp. G159-G167 ◽

Cited By ~ 45

Author(s):

Sachiko Mita ◽

Hiroshi Suzuki ◽

Hidetaka Akita ◽

Bruno Stieger ◽

Peter J. Meier ◽

...

Keyword(s):

Bile Salt ◽

Bile Salts ◽

Mdck Cells ◽

Bile Salt Export Pump ◽

Madin Darby Canine Kidney ◽

Transcellular Transport ◽

Physiological Conditions ◽

Vectorial Transport ◽

Rat Bile ◽

Darby Canine Kidney

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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Partial replacement of bile salts causes marked changes of cholesterol crystallization in supersaturated model bile systems

Biochemical Journal ◽

10.1042/bj3400445 ◽

1999 ◽

Vol 340 (2) ◽

pp. 445-451 ◽

Cited By ~ 6

Author(s):

Tomoji NISHIOKA ◽

Susumu TAZUMA ◽

Gunji YAMASHITA ◽

Goro KAJIYAMA

Keyword(s):

Bile Salt ◽

Bile Salts ◽

Rank Order ◽

Gallstone Formation ◽

Partial Replacement ◽

Salt Composition ◽

Human Bile ◽

Identical Composition ◽

Model Bile ◽

Conjugated Bile Salts

Cholesterol crystallization is a key step in gallstone formation and is influenced by numerous factors. Human bile contains various bile salts having different hydrophobicity and micelle-forming capacities, but the importance of lipid composition to bile metastability remains unclear. This study investigated the effect of bile salts on cholesterol crystallization in model bile (MB) systems. Supersaturated MB systems were prepared with an identical composition on a molar basis (taurocholate/phosphatidylcholine/cholesterol, 152 mM:38 mM: 24 mM), except for partial replacement of taurocholate (10, 20, and 30%) with various taurine-conjugated bile salts. Cholesterol crystallization was quantitatively estimated by spectrophotometrically measuring crystal-related turbidity and morphologically scanned by video-enhanced microscopy. After partial replacement of taurocholate with hydrophobic bile salts, cholesterol crystallization increased dose-dependently without changing the size of vesicles or crystal morphology and the rank order of crystallization was deoxycholate > chenodeoxycholate > cholate (control MB). All of the hydrophilic bile salts (ursodeoxycholate, ursocholate and β-muricholate) inhibited cholesterol precipitation by forming a stable liquid-crystal phase, and there were no significant differences among the hydrophilic bile-salt species. Cholesterol crystallization was markedly altered by partial replacement of bile salts with a different hydrophobicity. Thus minimal changes in bile-salt composition may dramatically alter bile lipid metastability.

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Molecular Dynamics Simulation of the Aggregation Patterns in Aqueous Solutions of Bile Salts at Physiological Conditions

The Journal of Physical Chemistry B ◽

10.1021/acs.jpcb.5b07063 ◽

2015 ◽

Vol 119 (51) ◽

pp. 15631-15643 ◽

Cited By ~ 23

Author(s):

Fatmegyul Mustan ◽

Anela Ivanova ◽

Galia Madjarova ◽

Slavka Tcholakova ◽

Nikolai Denkov

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulation ◽

Aqueous Solutions ◽

Bile Salts ◽

Dynamics Simulation ◽

Physiological Conditions

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Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii

Canadian Journal of Microbiology ◽

10.1139/m68-005 ◽

1968 ◽

Vol 14 (1) ◽

pp. 25-31 ◽

Cited By ~ 169

Author(s):

G. W. Strandberg ◽

P. W. Wilson

Keyword(s):

Color Change ◽

Azotobacter Vinelandii ◽

Nitrogenase Activity ◽

Color Difference ◽

Potassium Nitrate ◽

Whole Cells ◽

System A ◽

Growth System ◽

A Cell ◽

Enzyme Formation

The formation and activity of nitrogenase2 in Azotobacter vinelandii OP was examined using a cell-free assay system. A lag period of about 30 min occurred between the exhaustion of the combined nitrogen source and growth on N2. Cells grown on ammonium acetate or potassium nitrate had no detectable nitrogenase activity. Nitrogenase activity appeared in cells, grown under a flowing gas phase of 20% O2 – 60% He, about 45 min after the exhaustion of ammonia. Nitrogenase formation was inhibited in a closed system with an atmosphere containing 40% O2 but not by one containing 20% O2. Hydrogen did not inhibit enzyme formation. The question of whether N2 is required for the formation of the enzyme could not be answered as this gas could not be completely eliminated from the growth system. Chloramphenicol prevented the formation of the enzyme and inhibited nitrogen fixation in whole cells, but had no effect on cell-free enzyme activity. A brief rise in turbidity which occurred during nitrogenase formation appeared to be due to a color change in the cells from reddish brown to dark brown. Spectrophotometric examination of extracts from ammonia- and N2-grown cells did not reveal any components responsible for this color difference, but this result may reflect only the presence of interfering substances in the crude extract.

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Characteristics of bile salt uptake into skate hepatocytes

Biochemical Journal ◽

10.1042/bj2990665 ◽

1994 ◽

Vol 299 (3) ◽

pp. 665-670 ◽

Cited By ~ 14

Author(s):

G Fricker ◽

V Dubost ◽

K Finsterwald ◽

J L Boyer

Keyword(s):

Substrate Specificity ◽

Bile Salt ◽

Transport System ◽

Bile Salts ◽

Organic Anion ◽

Anion Transport ◽

Organic Anion Transport ◽

Salt Uptake ◽

Anion Transport System ◽

Alpha 7

The substrate specificity for the transporter that mediates the hepatic uptake of organic anions in freshly isolated hepatocytes of the elasmobranch little skate (Raja erinacea) was determined for bile salts and bile alcohols. The Na(+)-independent transport system exhibits a substrate specificity, which is different from the specificity of Na(+)-dependent bile salt transport in mammals. Unconjugated and conjugated di- and tri-hydroxylated bile salts inhibit uptake of cholyltaurine and cholate competitively. Inhibition is significantly greater with unconjugated as opposed to glycine- or taurine-conjugated bile salts. However, the number of hydroxyl groups in the steroid moiety of the bile salts has only minor influences on the inhibition by the unconjugated bile salts. Since the transport system seems to represent an archaic organic-anion transport system, other anions, such as dicarboxylates, amino acids and sulphate, were also tested, but had no inhibitory effect on bile salt uptake. To clarify whether bile alcohols, the physiological solutes in skate bile, share this transport system, cholyltaurine transport was studied after addition of 5 beta-cholestane-3 beta,5 alpha,6 beta-triol, 5 alpha-cholestan-3 beta-ol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. These bile alcohols inhibit cholyltaurine uptake non-competitively. In contrast, uptake of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, which is Na(+)-independent, is not inhibited by cholyltaurine. The findings further characterize a Na(+)-independent organic-anion transport system in skate liver cells, which is not shared by bile alcohols and has preference for unconjugated lipophilic bile salts.

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Selective recruitment of arrestin-3 to clathrin coated pits upon stimulation of G protein-coupled receptors

Journal of Cell Science ◽

10.1242/jcs.113.13.2463 ◽

2000 ◽

Vol 113 (13) ◽

pp. 2463-2470 ◽

Cited By ~ 1

Author(s):

F. Santini ◽

R.B. Penn ◽

A.W. Gagnon ◽

J.L. Benovic ◽

J.H. Keen

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Coated Pits ◽

Over Expression ◽

Physiological Conditions ◽

A Cell ◽

G Protein Coupled ◽

Comparable Magnitude

Non-visual arrestins (arrestin-2 and arrestin-3) play critical roles in the desensitization and internalization of many G protein-coupled receptors. In vitro experiments have shown that both non-visual arrestins bind with high and approximately comparable affinities to activated, phosphorylated forms of receptors. They also exhibit high affinity binding, again of comparable magnitude, to clathrin. Further, agonist-promoted internalization of many receptors has been found to be stimulated by exogenous over-expression of either arrestin2 or arrestin3. The existence of multiple arrestins raises the question whether stimulated receptors are selective for a specific endogenous arrestin under more physiological conditions. Here we address this question in RBL-2H3 cells, a cell line that expresses comparable levels of endogenous arrestin-2 and arrestin-3. When (beta)(2)-adrenergic receptors are stably expressed in these cells the receptors internalize efficiently following agonist stimulation. However, by immunofluorescence microscopy we determine that only arrestin-3, but not arrestin-2, is rapidly recruited to clathrin coated pits upon receptor stimulation. Similarly, in RBL-2H3 cells that stably express physiological levels of m1AChR, the addition of carbachol selectively induces the localization of arrestin-3, but not arrestin-2, to coated pits. Thus, this work demonstrates coupling of G protein-coupled receptors to a specific non-visual arrestin in an in vivo setting.

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