Transport of taurocholate by mutants of negatively charged amino acids, cysteines, and threonines of the rat liver sodium-dependent taurocholate cotransporting polypeptide Ntcp.

The relevance of functional amino acids for taurocholate transport by the sodium-dependent taurocholate cotransporting polypeptide Ntcp was determined by site-directed mutagenesis. cRNA from 28 single-points mutants of the rat liver Ntcp clone was expressed in Xenopus laevis oocytes. Mutations were generated in five conserved negatively charged amino acids (aspartates and glutamates) which were present in nine members of the SBAT-family, in two nonconserved negatively charged amino acids, in all eight Ntcp-cysteines, and in two threonines from a protein kinase C consensus region of the Ntcp C-terminus. Functional amino acids were Asp115, Glu257, and Cys266, which were found to be essential for the maintenance of taurocholic acid transport. Asp115 is located in the large intracellular loop III, whereas Glu257 and Cys266 are located in the large extracellular loop VI. Four mutations of threonines from the C-terminus of the Ntcp by alanines or tyrosines showed no effects on sodium-dependent taurocholate transport. Introduction of the FLAG(R) motif into several transport negative point mutations demonstrated that all mutated proteins besides one were present within the cell membrane of the oocytes and provided proof that an insertion defect has not caused transport deficiency by these Ntcp mutants. The latter was observed only with the transport negative mutant Asp24Asn. In conclusion, loop amino acids are required for sodium-dependent substrate translocation by the Ntcp.     

Identification of a sodium-dependent organic anion transporter from rat adrenal gland

In this study, a novel sodium-dependent organic anion transporter (Soat) was identified. Soat is expressed in rat brain, heart, kidney, lung, muscle, spleen, testis, adrenal gland, small intestine, and colon. The Soat protein consists of 370 amino acids and shows 42% and 31% overall amino acid sequence identity to the ileal sodium-dependent bile acid transporter (Isbt) and the Na(+)/taurocholate cotransporting polypeptide (Ntcp), respectively. Soat is predicted to have nine transmembrane domains, with an N-terminus outside the cell and an intracellular C-terminus. The Soat gene is localized on chromosome 14 and is coded by six exons mapped in region 14p22. When expressed in Xenopus laevis oocytes, Soat shows transport function for estrone-3-sulfate (Km = 31 microM, Vmax = 5557 fmol/oocyte/30 min) and dehydroepiandrosterone sulfate (Km = 30 microM, Vmax = 5682 fmol/oocyte/30 min). Soat does not transport taurocholate, estradiol-17beta-glucuronide, nor ouabain.     

Multihormonal regulation of hepatic sinusoidal Ntcp gene expression

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.     

The rat liver Na(+)/bile acid cotransporter. Importance of the cytoplasmic tail to function and plasma membrane targeting

To understand the potential functions of the cytoplasmic tail of Na(+)/taurocholate cotransporter (Ntcp) and to determine the basolateral sorting mechanisms for this transporter, green fluorescent protein-fused wild type and mutant rat Ntcps were constructed and the transport properties and cellular localization were assessed in transfected COS 7 and Madin-Darby canine kidney (MDCK) cells. Truncation of the 56-amino acid cytoplasmic tail demonstrates that the cytoplasmic tail of rat Ntcp is involved membrane delivery of this protein in nonpolarized and polarized cells and removal of the tail does not affect the bile acid transport function of Ntcp. Using site-directed mutagenesis, two tyrosine residues, Tyr-321 and Tyr-307, in the cytoplasmic tail of Ntcp have been identified as important for the basolateral sorting of rat Ntcp in transfected MDCK cells. Tyr-321 appears to be the major basolateral-sorting determinant, and Tyr-307 acts as a supporting determinant to ensure delivery of the transporter to the basolateral surface, especially at high levels of protein expression. When the two Tyr-based basolateral sorting motifs have been removed, the N-linked carbohydrate groups direct the tyrosine to alanine mutants to the apical surface of transfected MDCK cells. The major basolateral sorting determinant Tyr-321 is within a novel beta-turn unfavorable tetrapeptide Y(321)KAA, which has not been found in any naturally occurring basolateral sorting motifs. Two-dimensional NMR spectroscopy of a 24-mer peptide corresponding to the sequence from Tyr-307 to Thr-330 on the cytoplasmic tail of Ntcp confirms that both the Tyr-321 and Tyr-307 regions do not adopt any turn structure. Since the major motif YKAA contains a beta-turn unfavorable structure, the Ntcp basolateral sorting may not be related to the clathrin-adaptor complex pathway, as is the case for many basolateral proteins.     

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

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 K(m) value of 13.9 +/- 4.7 microM for cells expressing Ntcp alone, which is comparable with 22.2 +/- 4.5 microM for cells expressing both Ntcp and Bsep and V(max) 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.     

Degradation of the sodium taurocholate cotransporting polypeptide (NTCP) by the ubiquitin-proteasome system

The sodium taurocholate cotransporting polypeptide (Ntcp, Slc10a1) is the major uptake system for bile acids into liver cells. This study investigated the degradation of rat Ntcp and human NTCP by the ubiquitin-proteasome system (UPS). In stably transfected HepG2 cells, rat Ntcp was complex-glycosylated and localized at the plasma membrane. Inhibition of proteasomes by MG-132 or lactacystin led to the accumulation of intracellular Ntcp, a process dependent on de novo protein synthesis. Intracellular Ntcp was core-glycosylated, indicating an endoplasmic reticulum (ER) origin. Core-glycosylated Ntcp was found in cytosolic, detergent-insoluble deposits with characteristics of aggresomes: they co-localized with ubiquitin at the microtubule organization center and Ntcp from these deposits was polyubiquitinated. Transient transfections of Ntcp/NTCP induced intracellular deposits that co-localized with ubiquitin, even in the absence of proteasome inhibitors. Similarly, in livers of patients with progressive familial intrahepatic cholestasis, NTCP could be detected co-localized with ubiquitin in hepatocytes. We conclude that maturing Ntcp/NTCP is degraded by the ubiquitin-proteasome system at the level of ER-associated degradation (ERAD). An imbalance in the synthesis and degradation of NTCP at the level of the ER or alterations in the ERAD machinery might be the cause of intracellular NTCP deposits in transient transfections and in cholestatic livers.     

Molecular cloning and functional characterization of two alternatively spliced Ntcp isoforms from mouse liver1

To isolate the murine Na+/taurocholate cotransporting polypeptide (Ntcp), we screened a mouse liver cDNA library and identified Ntcp1, encoding a 362 amino acid protein and Ntcp2, encoding a 317 amino acid protein which had a shorter C-terminal end. Both isoforms mediated saturable Na+-dependent transport of taurocholate when expressed in Xenopus laevis oocytes. Analysis of the gene revealed that Ntcp2 is produced by alternative splicing where the last intron is retained.     

Localization of the Sodium-Taurocholate cotransporting polypeptide in membrane rafts and modulation of its activity by cholesterol in vitro

BACKGROUND: The relevance of discrete localization of hepatobiliary transporters in specific membrane microdomains is not well known. AIM: To determine whether the Na+/taurocholate cotransporting polypeptide (Ntcp), the main hepatic sinusoidal bile salt transporter, is localized in specific membrane microdomains. METHODS: Presence of Ntcp in membrane rafts obtained from mouse liver was studied by immunoblotting and immunofluorescence. HEK-293 cells stably transfected with rat Ntcp were used for in vitro studies. Expression, localization and function of Ntcp in these cells were assessed by immunoblotting, immunofluorescence and biotinylation studies and Na+ -dependent taurocholate uptake assays, respectively. The effect of cholesterol depletion/repletion assays on Ntcp function was also investigated. RESULTS: Ntcp localized primarily to membrane rafts in in vivo studies and localized partially in membrane rafts in transfected HEK-293 cells. In these cells, membrane cholesterol depletion resulted in a shift of Ntcp localization into non-membrane rafts, which correlated with a 2.5-fold increase in taurocholate transport. Cholesterol repletion shifted back part of Ntcp into membrane rafts, and normalized taurocholate transport to values similar to control cells. CONCLUSION: Ntcp localizes in membrane rafts and its localization and function are regulated by membrane cholesterol content. This may serve as a novel regulatory mechanism of bile salt transport in liver.     

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 β₁-integrin-dependent TUDC action, which is frequently used in the treatment of cholestatic liver disease.     

Dephosphorylation of Ser-226 facilitates plasma membrane retention of Ntcp

Ntcp is a phosphoprotein, and its translocation by cAMP to the plasma membrane is associated with dephosphorylation. However, the phosphorylation site(s) of Ntcp is not known. Thus, the aim of the present study was to determine the potential Ntcp phosphorylation sites and whether any of these phosphorylation sites is involved in Ntcp translocation. To determine the potential phosphorylation sites, metabolically labeled [32P]Ntcp isolated from hepatocytes was digested with clostripain and then subjected to SDS-PAGE followed by autoradiography. Clostripain digestion resulted in two phosphorylated peptides, and cAMP decreased phosphorylation of one of the peptides (7.8 K(d)), which contains the putative third cytoplasmic loop with three serine (Ser-213, Ser-226, and Ser-227) and two threonine (Thr-219 and Thr-225) residues. To determine whether any one of these serine/threonine residues is phosphorylated and/or is involved in Ntcp translocation, each of these serine/threonine residues were mutated to alanine. HuH-7 cells were transiently transfected with the wild-type and the mutated Ntcps followed by determination of taurocholate uptake and Ntcp expression, translocation and phosphorylation. Mutation of only Ser-226 resulted in 30% decrease in Ntcp phosphorylation and in 2.5 and 3.2-fold increases in taurocholate uptake and Ntcp retention in the plasma membrane, respectively. Cyclic AMP failed to further decrease phosphorylation and increase translocation of S226A-Ntcp. Taken together, these results suggest that the Ser-226 in the third cytoplasmic loop of Ntcp is phosphorylated and cAMP may increase Ntcp translocation to the plasma membrane by dephosphorylating Ntcp at this site.     

Localization of the Sodium-Taurocholate cotransporting polypeptide in membrane rafts and modulation of its activity by cholesterol in vitro

Background

The relevance of discrete localization of hepatobiliary transporters in specific membrane microdomains is not well known.

Aim

To determine whether the Na⁺/taurocholate cotransporting polypeptide (Ntcp), the main hepatic sinusoidal bile salt transporter, is localized in specific membrane microdomains.

Methods

Presence of Ntcp in membrane rafts obtained from mouse liver was studied by immunoblotting and immunofluorescence. HEK-293 cells stably transfected with rat Ntcp were used for in vitro studies. Expression, localization and function of Ntcp in these cells were assessed by immunoblotting, immunofluorescence and biotinylation studies and Na⁺-dependent taurocholate uptake assays, respectively. The effect of cholesterol depletion/repletion assays on Ntcp function was also investigated.

Results

Ntcp localized primarily to membrane rafts in in vivo studies and localized partially in membrane rafts in transfected HEK-293 cells. In these cells, membrane cholesterol depletion resulted in a shift of Ntcp localization into non-membrane rafts, which correlated with a 2.5-fold increase in taurocholate transport. Cholesterol repletion shifted back part of Ntcp into membrane rafts, and normalized taurocholate transport to values similar to control cells.

Conclusion

Ntcp localizes in membrane rafts and its localization and function are regulated by membrane cholesterol content. This may serve as a novel regulatory mechanism of bile salt transport in liver.     

Physiological characteristics of allo-cholic acid

The physiological characterstics of allo-cholic acid (ACA), a typically fetal bile acid that reappears during liver regeneration and carcinogenesis were investigated. [(14)C] Tauro-ACA (TACA) uptake by Chinese hamster ovary cells expressing rat organic anion transporter polypeptide (Oatp)1 or sodium-taurocholate cotransporter polypeptide (Ntcp) was lower than that of [(14)C]taurocholic acid (TCA). Although TACA inhibited ATP-dependent TCA transport across plasma membrane vesicles from Sf9 cells expressing rat or mouse bile salt export pump (Bsep), no ATP-dependent TACA transport was found. In rats, TACA was secreted into bile with no major biotransformation and it had lower clearance and longer half-life than TCA. In mice, TACA bile output was lower (-50%) than that of TCA, whereas TACA induced 9-fold higher bile flow than TCA. Even though the intracellular levels were lower for TACA, translocation into the hepatocyte nucleus was higher for TACA than for TCA; however, rate of DNA synthesis, expression levels of alpha-fetoprotein, albumin, Ntcp, and Bsep, cell viability, and apoptosis in rat hepatocytes were similarly affected by both isomers. In conclusion, TACA partly shares hepatocellular uptake system(s) for TCA. Furthermore, in contrast to other "flat" bile acids, TACA is efficiently secreted into bile via transport system(s) other than Bsep and is highly choleretic, hence its appearance during certain situations may prevent accumulation of cholestatic precursors.     

Gender-divergent profile of bile acid homeostasis during aging of mice

Aging is a physiological process with a progressive decline of adaptation and functional capacity of the body. Bile acids (BAs) have been recognized as signaling molecules regulating the homeostasis of glucose, lipid, and energy. The current study characterizes the age-related changes of individual BA concentrations by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in serum and liver of male and female C57BL/6 mice from 3 to 27 months of age. Total BA concentrations in serum increased 340% from 3 to 27 months in female mice, whereas they remained relatively constant with age in male mice. During aging, male and female mice shared the following changes: (1) BA concentrations in liver remained relatively constant; (2) the proportions of beta-muricholic acid (βMCA) increased and deoxycholic acid (DCA) decreased between 3 and 27 months in serum and liver; and (3) total BAs in serum and liver became more hydrophilic between 3 and 27 months. In female mice, (1) the mRNAs of hepatic BA uptake transporters, the Na(+)/taurocholate cotransporting polypeptide (Ntcp) and the organic anion transporting polypeptide 1b2 (Oatp1b2), decreased after 12 months, and similar trends were observed for their proteins; (2) the mRNA of the rate-limiting enzyme for BA synthesis, cholesterol 7α-hydroxylase (Cyp7a1), increased from 3 to 9 months and remained high thereafter. However, in male mice, Ntcp, Oatp1b2, and Cyp7a1 mRNAs remained relatively constant with age. In summary, the current study shows gender-divergent profiles of BA concentrations and composition in serum and liver of mice during aging, which is likely due to the gender-divergent expression of BA transporters Ntcp and Oatp1b2 as well as the synthetic enzyme Cyp7a1.     

Stable expression and functional characterization of a Na+-taurocholate cotransporting green fluorescent protein in human hepatoblastoma HepG2 cells

Sodium-dependent uptake of bile acids from blood is aliver-specific function which is mediated by theNa⁺-taurocholate cotransporting polypeptide(Ntcp). We report the stable expression of aNa⁺-taurocholate cotransporting green fluorescentfusion protein in the human hepatoblastoma cell lineHepG2, normally lacking Ntcp expression. Ntcp-EGFPassociated green fluorescence colocalized with Ntcpimmunofluorescence in the plasma membrane. Intransfected HepG2 cells, the fusion protein mediatedthe sodium-dependent uptake of the bile acidtaurocholate (K_m: 24.6 mol/l) and of the anionicsteroids estrone-3-sulfate and dehydroepiandrosteronesulfate. We conclude that the Ntcp-EGFP fusion proteinfollows the sorting route of Ntcp, is functionallyidentical to Ntcp and could be used to monitor proteintrafficking in living HepG2 cells.     

Hepatocyte nuclear factor-4alpha is a central transactivator of the mouse Ntcp gene

Sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake system for conjugated bile acids. Deletions of hepatocyte nuclear factor (HNF)-1alpha and retinoid X receptor-alpha:retinoic acid receptor-alpha 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-4alpha, 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-1alpha, HNF-4alpha cotransfection led to a robust 20-fold activation. Deletion analysis of mouse and rat Ntcp promoters mapped a conserved HNF-4alpha consensus site at -345/-326 and -335/-316 bp, respectively. p-475bpmNtcpLUC is not transactivated by HNF-1alpha but shows a 50-fold enhanced activity upon cotransfection with HNF-4alpha. Gel mobility shift assays demonstrated a complex of the HNF-4alpha-element formed with liver nuclear extracts that was blocked by an HNF-4alpha specific antibody. HNF-4alpha binding was confirmed by chromatin immunoprecipitation. Using Hepa 1-6 cells, HNF-4alpha-knockdown resulted in a significant 95% reduction in NTCP mRNA. In conclusion, mouse Ntcp is regulated by HNF-4alpha via a conserved distal cis-element independently of HNF-1alpha.     

Protein kinase B/Akt mediates cAMP- and cell swelling-stimulated Na+/taurocholate cotransport and Ntcp translocation

Cyclic AMP and cell swelling stimulate hepatic Na+/TC cotransport and Ntcp translocation via the phosphoinositide 3-kinase signaling pathway. To determine the downstream target of the phosphoinositide 3-kinase action, we examined the role of protein kinase B (PKB)/Akt using SB203580 in hepatocytes as well as by transfection with a dominant negative (DN-PKB) or a constitutively active (CA-PKB) form of PKB in HuH-Ntcp cells. Both cAMP and cell swelling stimulated p38 mitogen-activated protein (MAP) kinase as well as PKB activity. Although 100 microm SB203580 inhibited cell swelling- and 8-chlorophenylthio-cAMP-induced activation of both p38 MAP kinase and PKB, 1 microm SB203580 inhibited activation of p38 MAP kinase, but not of PKB, in hepatocytes. 100 microm, but not 1 microm SB203580, inhibited cell swelling- and cAMP-induced increases in taurocholate (TC) uptake and Ntcp translocation in hepatocytes. TC uptake in HuH-Ntcp cells was more than 90% dependent on extracellular Na+. Cyclic AMP and cell swelling increased TC uptake by 50-100% and PKB activity 2-4-fold in HuH-Ntcp cells transfected with the empty vector and failed to increase PKB activity, TC uptake, and Ntcp translocation in DN-PKB-transfected HuH-Ntcp cells. Transfection with CA-PKB increased PKB activity, TC uptake, and Ntcp translocation in HuH-Ntcp cells compared with cells transfected with the empty vector. In contrast, transfection with DN-PKB did not affect basal PKB activity, TC uptake, or Ntcp translocation. Taken together, these results strongly suggest that cell swelling and cAMP-mediated stimulation of hepatic Na+/TC cotransport and Ntcp translocation requires activation of PKB and is mediated at least in part via a phosphoinositide 3-kinase/PKB-signaling pathway.     

Cross-talk between protein kinases Czeta and B in cyclic AMP-mediated sodium taurocholate co-transporting polypeptide translocation in hepatocytes

Cyclic AMP stimulates taurocholate (TC) uptake and sodium taurocholate co-transporting polypeptide (Ntcp) translocation in hepatocytes via the phosphoinositide-3 kinase (PI3K) signaling pathway. The aim of the present study was to determine whether protein kinase (PK) Czeta, one of the downstream mediators of the PI3K signaling pathway, is involved in cAMP-mediated stimulation of TC uptake. Studies were conducted in isolated rat hepatocytes and in HuH-7 cells stably transfected with rat liver Ntcp (HuH-Ntcp cells). Studies in hepatocytes showed that cAMP activates PKCzeta in a PI3K-dependent manner without inducing translocation of PKCzeta to the plasma membrane. Inhibition of cAMP-induced PKCzeta activity by myristoylated PKC (zeta/lambda) pseudosubstrate, a specific inhibitor of PKCzeta, and G? 6850, a PKC inhibitor, resulted in inhibition of cAMP-induced increases in TC uptake and Ntcp translocation. Studies in HuH-Ntcp cells showed that inhibition of cAMP-induced PKCzeta activation by dominant-negative (DN) PKCzeta resulted in inhibition of cAMP-induced increases in TC uptake and Ntcp translocation. DN PKCzeta also inhibited wild-type PKCzeta-induced increases in PKCzeta activity, TC uptake, and Ntcp translocation. Myristoylated PKC (zeta/lambda) pseudosubstrate and DN PKCzeta also inhibited cAMP-induced activation of PKB in hepatocytes and HuH-Ntcp cells, respectively. Neither DN PKB nor constitutively active PKB affected cAMP-induced activation of PKCzeta, and wild-type PKCzeta did not activate PKB. Taken together, these results suggest that cAMP-induced activation of PKB is dependent on cAMP-induced stimulation of PKCzeta. It is proposed that cAMP-induced Ntcp translocation involves the activation of the PI3K/PKCzeta signaling pathway followed by the activation of the PI3K/PKB signaling pathway.