Taurocholate-cotransporting Polypeptide Research Articles

The hepatocellular bile acid transporter Ntcp facilitates uptake of the lethal mushroom toxin ?-amanitin

Hepatotoxicity caused by the mushroom poison alpha-amanitin is an unusual but serious cause of death and liver transplantation. Understanding the mechanisms of alpha-amanitin uptake may lead to rational therapeutic approaches. Because older data suggested that a sodium-dependent bile acid transporter is responsible for alpha-amanitin uptake, we tested the hypothesis that Na(+)-taurocholate cotransporter polypeptide (Ntcp) facilitates hepatocellular alpha-amanitin uptake. Human hepatoblastoma cells (HepG2), cells that have lost native Ntcp expression, were stably transfected with the rat Ntcp gene. Taurocholate uptake by the transfected cells exhibited a physiologically normal K(m) and V(max). A toxicologically relevant functional assay for alpha-amanitin uptake was developed by measuring its ability to block cytokine-induced synthesis of interleukin-1 receptor antagonist (IL-1Ra) mRNA. Treatment with interleukin-1beta (10 ng/ml) and interleukin-6 (100 ng/ml) increased IL-1Ra mRNA abundance 8.6-fold and 15.6-fold in HepG2 cells and Ntcp-transfected cells, respectively. Pretreatment of transfected cells with 1 micro M alpha-amanitin for 6-10 h almost completely blocked induction of IL-1Ra mRNA (1.9-fold induction) whereas pretreatment of non-transfected cells did not block induction of IL-1Ra mRNA (21.6-fold induction, P<0.02 compared with stimulated transfected cells without alpha-amanitin). These findings demonstrate that Ntcp may be an important mediator of alpha-amanitin uptake by the liver.

The Presence of Xenobiotic Transporters in Rat Placenta

Understanding the role of transporters in placental handling of xenobiotics across the maternal-fetal interface is essential to evaluate the pharmacological and toxicological potential of therapeutic agents, drugs of abuse, and other xenobiotics to which the mother is exposed during pregnancy. Therefore, the purpose of this study was to assess mRNA levels of various transporters in placenta and to compare these to levels in maternal liver and kidney, predominant organs of excretion, to determine which transporters are likely to have a role in xenobiotic transfer within the placenta. During late stage pregnancy, relative amounts of mRNA levels of 40 genes representing 11 families/group of transporters were assessed in placenta with respect to relative maternal liver and kidney mRNA levels. Members of the following transporter families were assessed: three multidrug resistance (Mdr), six multidrug resistance-associated protein (Mrp), eight organic anion-transporting polypeptide (Oatp), three organic anion transporters (Oat), five organic cation transporters (Oct), two bile acid transporters (Na(+)/taurocholate-cotransporting polypeptide and bile salt export protein), four metal (ZnT1, divalent metal transporter 1, Menkes and Wilsons), a prostaglandin, two peptide, two sterolin, and four nucleoside transporters. Of the 40 genes evaluated, 16 [Mdr1a and 1b, Mrp1 and 5, Oct3 and Octn1, Oatp3 and 12, four metal, a prostaglandin, AbcG8, equilibrative nucleoside transporter 1 (ENT1), and ENT2] were expressed in placenta at concentrations similar to or higher than in maternal liver and kidney. The abundance of these mRNA transcripts in placenta suggests a role for these transporters in placental transport of xenobiotics and supports their role in the transport of endogenous substances.

Effects of thyroid state on the expression of hepatic thyroid hormone transporters in rats

Liver uptake of thyroxine (T4) is mediated by transporters and is rate limiting for hepatic 3,3',5-triiodothyronine (T3) production. We investigated whether hepatic mRNA for T4 transporters is regulated by thyroid state using Xenopus laevis oocytes as an expression system. Because X. laevis oocytes show high endogenous uptake of T4, T4 sulfamate (T4NS) was used as an alternative ligand for the hepatic T4 transporters. Oocytes were injected with 23 ng liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats, and after 3-4 days uptake was determined by incubation of injected and uninjected oocytes for 1 h at 25 degrees C or for 4 h at 18 degrees C with 10 nM [125I]T4NS. Expression of type I deiodinase (D1), which is regulated by thyroid state, was studied in the oocytes as an internal control. Uptake of T4NS showed similar approximately fourfold increases after injection of liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats. A similar lack of effect of thyroid state was observed using reverse T3 as ligand. In contrast, D1 activity induced by liver mRNA from hyperthyroid and hypothyroid rats in the oocytes was 2.4-fold higher and 2.7-fold lower, respectively, compared with euthyroid rats. Studies have shown that uptake of iodothyronines in rat liver is mediated in part by several organic anion transporters, such as the Na+/taurocholate-cotransporting polypeptide (rNTCP) and the Na-independent organic anion-transporting polypeptide (rOATP1). Therefore, the effects of thyroid state on rNTCP, rOATP1, and D1 mRNA levels in rat liver were also determined. Northern analysis showed no differences in rNTCP or rOATP1 mRNA levels between hyperthyroid and hypothyroid rats, whereas D1 mRNA levels varied widely as expected. These results suggest little effect of thyroid state on the levels of mRNA coding for T4 transporters in rat liver, including rNTCP and rOATP1. However, they do not exclude regulation of hepatic T4 transporters by thyroid hormone at the translational and posttranslational level.

PRL, Placental Lactogen, and GH Induce Na+/Taurocholate-Cotransporting Polypeptide Gene Expression by Activating Signal Transducer and Activator of Transcription-5 in Liver Cells

We investigated the transcriptional regulation of the Na+/taurocholate cotransporting polypeptide gene by PRL, placental lactogen, and GH. In primary hepatocytes, ovine PRL induced a dose-dependent phosphorylation and nuclear translocation of signal transducers and activators of transcription-5a and -5b, but not -1 or -3, whereas mouse placental lactogen I and rat GH activated -5a, -5b, and -1. In EMSAs, ovine PRL, mouse placental lactogen I, and rat GH increased the specific DNA binding of nuclear signal transducer and activator of transcription-5 to its consensus element in both transfected HepG2 cells and primary hepatocytes. PRL, placental lactogen I, and GH also increased Na+/taurocholate cotransporting polypeptide mRNA expression in hepatocytes from control and pregnant (mouse placental lactogen I) rats. Genistein, a phosphotyrosine kinase inhibitor, inhibited PRL-induced signal transducer and activator of transcription-5 activation and Na+/taurocholate-cotransporting polypeptide mRNA. In HepG2 cells transiently cotransfected with either the long form of the rat PRL receptor or rat GH receptor, signal transducer and activator of transcription-5a and a -5-responsive luciferase expression vector containing the Na+/taurocholate-cotransporting polypeptide promoter, mouse placental lactogen I, like ovine PRL, activated -5a via the long form of the rat PRL receptor; whereas rat GH activated -5a via rat GH receptor, leading to transactivation of the Na+/taurocholate-cotransporting polypeptide promoter. These data establish that PRL and placental lactogen I induce Na+/taurocholate-cotransporting polypeptide gene expression via signal transducer and activator of transcription-5 proteins in liver, and indicate that these hormones play an important role in regulating liver metabolic function.

PRL, placental lactogen, and GH induce NA(+)/taurocholate-cotransporting polypeptide gene expression by activating signal transducer and activator of transcription-5 in liver cells.

We investigated the transcriptional regulation of the Na(+)/taurocholate cotransporting polypeptide gene by PRL, placental lactogen, and GH. In primary hepatocytes, ovine PRL induced a dose-dependent phosphorylation and nuclear translocation of signal transducers and activators of transcription-5a and -5b, but not -1 or -3, whereas mouse placental lactogen I and rat GH activated -5a, -5b, and -1. In EMSAs, ovine PRL, mouse placental lactogen I, and rat GH increased the specific DNA binding of nuclear signal transducer and activator of transcription-5 to its consensus element in both transfected HepG2 cells and primary hepatocytes. PRL, placental lactogen I, and GH also increased Na(+)/taurocholate cotransporting polypeptide mRNA expression in hepatocytes from control and pregnant (mouse placental lactogen I) rats. Genistein, a phosphotyrosine kinase inhibitor, inhibited PRL-induced signal transducer and activator of transcription-5 activation and Na(+)/taurocholate-cotransporting polypeptide mRNA. In HepG2 cells transiently cotransfected with either the long form of the rat PRL receptor or rat GH receptor, signal transducer and activator of transcription-5a and a -5-responsive luciferase expression vector containing the Na(+)/taurocholate-cotransporting polypeptide promoter, mouse placental lactogen I, like ovine PRL, activated -5a via the long form of the rat PRL receptor; whereas rat GH activated -5a via rat GH receptor, leading to transactivation of the Na(+)/taurocholate-cotransporting polypeptide promoter. These data establish that PRL and placental lactogen I induce Na(+)/taurocholate-cotransporting polypeptide gene expression via signal transducer and activator of transcription-5 proteins in liver, and indicate that these hormones play an important role in regulating liver metabolic function.

Cell Swelling-induced Translocation of Rat Liver Na+/Taurocholate Cotransport Polypeptide Is Mediated via the Phosphoinositide 3-Kinase Signaling Pathway

Cell swelling stimulates phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) in hepatocytes, and the PI3K signaling pathway is involved in cAMP-mediated translocation of sinusoidal Na(+)/taurocholate (TC) cotransporter (Ntcp) to the plasma membrane. We determined whether cell swelling also stimulates TC uptake and Ntcp translocation via the PI3K and/or MAPK signaling pathway. All studies were conducted in isolated rat hepatocytes. Hepatocyte swelling induced by hypotonic media resulted in: 1) time- and medium osmolarity-dependent increases in TC uptake, 2) an increase in the V(max) of Na(+)/TC cotransport, and 3) wortmannin-sensitive increases in TC uptake and plasma membrane Ntcp mass. Hepatocyte swelling also induced wortmannin-sensitive activation of PI3K, protein kinase B, and p70(S6K). Rapamycin, an inhibitor of p70(S6K), inhibited cell swelling-induced activation of p70(S6K) but failed to inhibit cell swelling-induced stimulation of TC uptake. Because PD98095, an inhibitor of MAPK, did not inhibit cell swelling-induced increases in TC uptake, it is unlikely that the effect of cell swelling on TC uptake is mediated via the MAPK signaling pathway. Taken together, these results indicate that 1) cell swelling stimulates TC uptake by translocating Ntcp to the plasma membrane, 2) this effect is mediated via the PI3K, but not MAPK, signaling pathway, and 3) protein kinase B, but not p70(S6K), is a likely downstream effector of PI3K.

Short-term regulation of bile acid uptake by microfilament-dependent translocation of rat ntcp to the plasma membrane.

The Na+-taurocholate cotransport polypeptide (ntcp) is the primary transporter for the uptake of bile acids in the liver. The second messenger adenosine 3':5'-cyclic monophosphate (cAMP) rapidly increases ntcp protein concentration in the plasma membrane, yet the mechanism is unknown. To investigate this, HepG2 cells were transiently transfected with a carboxy-terminal-tagged green fluorescence protein (GFP) conjugate of ntcp, and then examined by confocal video microscopy. Transporter activity was directly assayed with 3H-taurocholic acid (TC) scintigraphy. ntcp-GFP targeted to the plasma membrane in transfected cells, and the conjugate protein transported 3H-TC as effectively as unmodified rat ntcp. Stimulation of ntcp-GFP cells with cAMP increased GFP fluorescence in the plasma membrane by 40% (P <.0001) within 2.5 minutes and by 55% within 10 minutes. Similarly, cAMP increased transport of bile acids by 30%. Cytochalasin D, an inhibitor of microfilaments, did not prevent ntcp-GFP from targeting to the plasma membrane, but completely abolished the increase in GFP fluorescence seen in response to cAMP. In contrast, the microtubule inhibitor, nocodazole, prevented development of membrane fluorescence in 48 (96%) of 50 cells. Cells regained plasma membrane fluorescence within 2 hours after nocodazole removal. These findings suggest that targeting of ntcp to the plasma membrane consists of 2 steps: 1) delivery of ntcp to the region of the plasma membrane via microtubules; and 2) insertion of ntcp into the plasma membrane, in a microfilament- and cAMP-sensitive fashion.

Role of Protein Phosphatases in Cyclic AMP-mediated Stimulation of Hepatic Na+/Taurocholate Cotransport

Cyclic AMP has been proposed to stimulate Na+/taurocholate (TC) cotransport in hepatocytes by translocating Na+/TC cotransport polypeptide (Ntcp) to the plasma membrane and to induce Ntcp dephosphorylation. Whether protein phosphatases 1 and 2A (PP1/2A) are involved in the regulation of Na+/TC cotransport by cAMP was investigated in the present study. Okadaic acid and tautomycin, inhibitors of PP1/2A, inhibited cAMP-mediated increases in TC uptake and cytosolic [Ca2+], and only tautomycin inhibited basal TC uptake. Removal of cAMP reversed cAMP-mediated increases in TC uptake and plasma membrane Ntcp mass. Okadaic acid alone increased Ntcp phosphorylation without affecting Ntcp mass in plasma membranes and homogenates. In the presence of okadaic acid, cAMP failed to increase plasma membrane Ntcp mass, induce Ntcp dephosphorylation, and decrease endosomal Ntcp mass. Phosphorylated Ntcp was detectable in endosomes isolated from okadaic acid-treated hepatocytes but not in endosomes from control and cAMP-treated hepatocytes. PP1 was found to be enriched in plasma membranes, whereas PP2A was mostly in the cytosol. Cyclic AMP did not activate either PP1 or PP2A, whereas okadaic acid inhibited primarily PP2A. These results suggest that 1) the effect of cAMP on Na+/TC cotransport is not mediated via either PP1 or PP2A; rather, cAMP-mediated signaling pathway is maintained by PP2A and inhibition of PP2A overrides cAMP-mediated effects, and 2) okadaic acid, by inhibiting PP2A, inhibits cAMP-mediated increases in Na+/TC cotransport by decreasing the ability of cAMP to increase cytosolic [Ca2+]. It is proposed that cAMP-mediated dephosphorylation of Ntcp leads to an increased retention of Ntcp in the plasma membrane, and okadaic acid, by inhibiting PP2A, inhibits cAMP-mediated stimulation of Na+/TC cotransport by reversing the ability of cAMP to increase cytosolic [Ca2+] and to induce Ntcp dephosphorylation.

Partial maintenance of taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich configuration.

This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions. Hepatocytes isolated from male Wistar rats (230-280 g) were cultured on a simple collagen film, on a substratum of gelled collagen or between two layers of gelled collagen (sandwich configuration). Hepatocyte morphology, taurocholate uptake properties, and expression of the sinusoidal transport protein. Na+/taurocholate-cotransporting polypeptide (Ntcp) were examined in these cultures at day 0 and day 5. By day 5, monolayer integrity had deteriorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At day 5, taurocholate accumulation at 5 min in hepatocytes cultured on a simple collagen film, on a substratum of gelled collagen, and in a sandwich configuration was approximately 13%, 20% and 35% of day-0 levels, respectively, and occurred predominately by a Na+-dependent mechanism. The initial taurocholate uptake rate vs. concentration (1-200 microM) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent Km values were comparable for day-0 and day-5 hepatocytes (3241 microM). In contrast, the Vmax values of hepatocytes cultured on a simple collagen film, on gelled collagen and in a sandwich configuration were approximately 5, 6 and 14% of the values at day 0, respectively; values for the first-order rate constant were 5-, 3- and 2-fold lower, respectively. Immunoblot analysis indicated that at day 5 Ntcp expression in hepatocytes cultured in a sandwich configuration was greater than in hepatocytes cultured on a simple collagen film. A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid uptake properties in primary cultures of rat hepatocytes.

Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter.

Although bile acid transport by bile duct epithelial cells, or cholangiocytes, has been postulated, the details of this process remain unclear. Thus, we performed transport studies with [3H]taurocholate in confluent polarized monolayers of normal rat cholangiocytes (NRC). We observed unidirectional (i.e., apical to basolateral) Na+-dependent transcellular transport of [3H]taurocholate. Kinetic studies in purified vesicles derived from the apical domain of NRC disclosed saturable Na+-dependent uptake of [3H]taurocholate, with apparent Km and Vmax values of 209+/-45 microM and 1.23+/-0.14 nmol/mg/10 s, respectively. Reverse transcriptase PCR (RT-PCR) using degenerate primers for both the rat liver Na+-dependent taurocholate-cotransporting polypeptide and rat ileal apical Na+-dependent bile acid transporter, designated Ntcp and ASBT, respectively, revealed a 206-bp product in NRC whose sequence was identical to the ASBT. Northern blot analysis demonstrated that the size of the ASBT transcript was identical in NRC, freshly isolated cholangiocytes, and terminal ileum. In situ RT-PCR on normal rat liver showed that the message for ASBT was present only in cholangiocytes. Immunoblots using a well-characterized antibody for the ASBT demonstrated a 48-kD protein present only in apical membranes. Indirect immunohistochemistry revealed apical localization of ASBT in cholangiocytes in normal rat liver. The data provide direct evidence that conjugated bile acids are taken up at the apical domain of cholangiocytes via the ASBT, and are consistent with the notion that cholangiocyte physiology may be directly influenced by bile acids.

CAMP increases liver Na+-taurocholate cotransport by translocating transporter to plasma membranes.

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.

Chlorambucil-taurocholate is transported by bile acid carriers expressed in human hepatocellular carcinomas

BACKGROUND & AIMS: Chemotherapy of hepatocellular carcinomas is hampered by the insufficient accumulation of cytostatic drugs within the tumor cells. The aim of this study was to evaluate the feasibility of therapeutic strategies using antineoplastic agents coupled to bile acids.METHODS: Expression of the Na(+)-taurocholate-cotransporting polypeptide (NTCP) was analyzed in six hepatocellular carcinomas and in nonmalignant liver tissue. Uptake of the cytostatic drug [3H]- chlorambucil-taurocholate (S2676) was measured in Xenopus laevis oocytes injected with total messenger RNA (mRNA) from the carcinomas or peritumor tissue or with complementary RNA encoding the NTCP or the organic anion-transporting polypeptide (OATP) of human liver.RESULTS: Expression of hepatocellular carcinoma mRNA in oocytes resulted in mainly Na(+)-dependent uptake of chlorambucil-taurocholate. The level of NTCP mRNA in carcinomas amounted to 56% +/- 27% compared with peritumor tissue. Immunofluorescence studies confirmed the expression of NTCP on the surface of hepatocellular carcinoma cells. OATP expression, determined by immunoblotting, was similar in hepatocellular carcinomas and surrounding liver tissue (n = 3). NTCP mediated Na(+)- dependent uptake of chlorambucil-taurocholate (Michaelis constant, 11 mumol/L), whereas OATP mediated Na(+)-independent uptake.CONCLUSIONS: Hepatocellular carcinomas express the Na(+)-dependent bile acid transporter NTCP. Because NTCP mediates high-affinity uptake of chlorambucil-taurocholate, targeting of cytostatic bile acids to hepatocellular carcinomas could become a feasible therapeutic strategy.(Gastroenterology 1997 Oct;113(4):1295-305)

Molecular and functional characterization of bile acid transport in human hepatoblastoma HepG2 cells

Bile acids are taken up into human liver by Na+-dependent and Na+-independent transport mechanisms. In hepatocarcinogenesis, numerous liver-specific functions are lost and the uptake of organic anions is markedly reduced. We have investigated the molecular and functional derangements of bile acid transport in the human hepatoblastoma cell line HepG2. Uptake of [3H]-taurocholic acid was saturable and entirely Na+ independent, with the kinetic characteristics of the human liver organic anion transporting polypeptide (OATP). OATP, but not the Na+-dependent bile acid transporter (Na+-taurocholate-cotransporting polypeptide [NTCP]), was detectable by reverse-transcription polymerase chain reaction (RT-PCR) analysis of HepG2 RNA. The level of OATP expression in HepG2 cells was determined by Northern blot analysis and was found to be 40% in comparison with normal liver. Transfection of an OATP-derived phosphorothioate (PTO)-antisense oligonucleotide into HepG2 cells resulted in 77% inhibition of temperature-dependent bile acid uptake. Injection of HepG2 messenger RNA (mRNA) into Xenopus laevis oocytes significantly stimulated Na+-independent taurocholate uptake, indicating the expression of a bile acid transport protein. We conclude that bile acid uptake into human hepatoblastoma HepG2 cells is mediated by the multi-specific organic anion transporting polypeptide OATP. Therapeutic strategies employing bile acid-derived cytostatic agents for the treatment of hepatocellular carcinomas may therefore depend upon the expression of the Na+-independent bile acid transporter OATP in hepatic malignancies.

Molecular and functional characterization of bile acid transport in human hepatoblastoma HepG2 cells

Bile acids are taken up into human liver by Na+-dependent and Na+-independent transport mechanisms. In hepatocarcinogenesis, numerous liver-specific functions are lost and the uptake of organic anions is markedly reduced. We have investigated the molecular and functional derangements of bile acid transport in the human hepatoblastoma cell line HepG2. Uptake of [3H]-taurocholic acid was saturable and entirely Na+ independent, with the kinetic characteristics of the human liver organic anion transporting polypeptide (OATP). OATP, but not the Na+-dependent bile acid transporter (Na+-taurocholate-cotransporting polypeptide [NTCP]), was detectable by reverse-transcription polymerase chain reaction (RT-PCR) analysis of HepG2 RNA. The level of OATP expression in HepG2 cells was determined by Northern blot analysis and was found to be 40% in comparison with normal liver. Transfection of an OATP-derived phosphorothioate (PTO)-antisense oligonucleotide into HepG2 cells resulted in 77% inhibition of temperature-dependent bile acid uptake. Injection of HepG2 messenger RNA (mRNA) into Xenopus laevis oocytes significantly stimulated Na+-independent taurocholate uptake, indicating the expression of a bile acid transport protein. We conclude that bile acid uptake into human hepatoblastoma HepG2 cells is mediated by the multi-specific organic anion transporting polypeptide OATP. Therapeutic strategies employing bile acid-derived cytostatic agents for the treatment of hepatocellular carcinomas may therefore depend upon the expression of the Na+-independent bile acid transporter OATP in hepatic malignancies. (Hepatology 1996 May;23(5):1053-60)

Molecular mechanisms of hepatic bile salt transport from sinusoidal blood into bile.

An increasingly complex picture has emerged in recent years regarding the bile salt transport polarity of hepatocytes. At the sinusoidal (or basolateral) plasma membrane two bile salt-transporting polypeptides have been cloned. The Na(+)-taurocholate-cotransporting polypeptide (Ntcp) can account for most, if not all, physiological properties of the Na(+)-dependent bile salt uptake function in mammalian hepatocytes. The cloned organic anion-transporting protein (Oatp1) can mediate Na(+)-independent transport of bile salts, sulfobromophthalein, estrogen conjugates, and a variety of other amphipathic cholephilic compounds. Hence, Oatp1 appears to correspond to the previously suggested basolateral multispecific bile sale transporter. Intracellular bile salt transport can be mediated by different pathways. Under basal bile salt flux conditions, conjugated trihydroxy bile salts bind to cytoplasmic binding proteins and reach the canalicular plasma membrane predominantly via cytoplasmic diffusion. More hydrophobic mono- and dihydroxy and high concentrations of trihydroxy bile salts associate with intracellular membrane-bound compartments, including transcytotic vesicles, endoplasmic reticulum (ER), and Golgi complex. A facilitated bile salt diffusion pathway has been demonstrated in the ER. The exact role of these and other (e.g., lysosomes, "tubulovesicular structures") organelles in overall vectorial transport of bile salts across hepatocytes is not yet known. Canalicular bile salt secretion is mediated by two ATP-dependent transport systems, one for monovalent bile salts and the second for divalent sulfated or glucuronidated bile salt conjugates. The latter is identical with the canalicular multispecific organic anion transporter, which also transports other divalent organic anions, such as glutathione S-conjugates. Potential dependent canalicular bile salt secretion has also been suggested to occur, but its exact mechanism and physiological significance remain unclear, since a potential driven bile salt uptake system has also been identified in the ER. Hypothetically, and similar to changes in cell volume, the intracellular potential could also play a role in the regulation of the number of bile salt carriers at the canalicular membrane and thereby indirectly influence the maximal canalicular bile salt transport capacity of hepatocytes.

Regulation of the NA +/Taurocholate cotransport polypeptide (NTCP) by cyclic amp: Role for translocation and phosphorylation : [formula omitted], M. Ananthanarayanan, B. Stieger, P.J. Meier, F.J. Suchy, M.S. Anwer. Dept. of Veterinary Medicine, Tufts University, N. Grafton, MA, Dept. of Pediatrics, Yale University School of Medicine, New Haven, CT, Divn. of Clinical Pharmacology and Toxicology, University Hospital, Zürich, Switzerland

Regulation of the NA +/Taurocholate cotransport polypeptide (NTCP) by cyclic amp: Role for translocation and phosphorylation : [formula omitted], M. Ananthanarayanan, B. Stieger, P.J. Meier, F.J. Suchy, M.S. Anwer. Dept. of Veterinary Medicine, Tufts University, N. Grafton, MA, Dept. of Pediatrics, Yale University School of Medicine, New Haven, CT, Divn. of Clinical Pharmacology and Toxicology, University Hospital, Zürich, Switzerland

Prolactin increases mRNA encoding Na(+)-TC cotransport polypeptide and hepatic Na(+)-TC cotransport.

We have shown that prolactin (Prl) increases the transhepatic transport of taurocholate (TC) in postpartum rats and following treatment of ovariectomized (Ovx) rats with ovine Prl (oPrl). The present studies were designed to determine if treatment of Ovx rats with oPrl (100, 300, or 600 micrograms/day, 7 days iv) 1) increases Na(+)-TC cotransport in basolateral plasma membrane vesicles (bLPM), 2) induces a corresponding increase in the steady-state levels of Na(+)-TC cotransport polypeptide (Ntcp mRNA), and 3) if the oPrl-mediated increase in Na(+)-TC cotransport activity is blocked by cycloheximide, an inhibitor of protein synthesis. oPrl (300 micrograms/day) induced a twofold increase in the maximal velocity for Na(+)-TC cotransport in both hepatocytes and bLPM vesicles with little change in the Michaelis constant. Infusion of oPrl at a dose of 100, 300, or 600 micrograms/day increased steady-state Ntcp mRNA four-, ten-, and twofold, respectively. Finally, cycloheximide blocked the oPrl-mediated increase in Na(+)-TC cotransport but did not affect basal activity. These data support the hypothesis that an increase in Ntcp mRNA followed by increased synthesis and incorporation of Ntcp in the plasma membrane is responsible for the oPrl-mediated increase in Na(+)-TC cotransport in the basolateral plasma membrane domain of the hepatocyte.

Parallel decrease of Na+ -taurocholate cotransport and its encoding mRNA in primary cultures of rat hepatocytes

We investigated the molecular mechanism underlying the progressive loss of Na(+)-dependent bile salt uptake in primary cultured rat hepatocytes. A specific cDNA probe was used to quantitate the levels of mRNA encoding the Na(+)-taurocholate-cotransporting polypeptide at various culture times. Hepatocytes were cultured on collagen in the presence of insulin (10(-7) mol/L), dexamethasone (10(-7) mol/L) and 10% fetal calf serum for up to 72 hr. During this time period the dissociation constant of Na(+)-dependent taurocholate uptake remained stable (19 to 39 mumol/L), whereas the maximum velocity values decreased from 100% at 3 hr to 55%, 22% and 4% at 24, 48 and 72 hr, respectively. Concomitantly the levels of the Na(+)-taurocholate-cotransporting polypeptide mRNA also decreased from 100% at 3 hr to 41%, 24% and 4% at the later time points. In contrast, Northern hybridization with complementary DNA probes for three common housekeeping gene products revealed a 1.8- to 3.4-fold increase in the levels of mRNA encoding the alpha-subunit of the Na+K(+)-ATPase, beta-actin and glycerol-3-phosphate dehydrogenase. These data indicate that the loss of Na(+)-dependent bile salt uptake in primary cultures of rat hepatocytes is caused by decreased levels of its specific mRNA. Hence the studies further confirm that without specific measures (primary) cultured rat hepatocytes reverse their liver-specific phenotype to a more fetal pattern of gene expression.

Canalicular bile production in dogs.

Canalicular bile production in dogs.