PKCε-dependent and -independent effects of taurolithocholate on PI3K/PKB pathway and taurocholate uptake in HuH-NTCP cell line

Schonhoff, Christopher M.; Yamazaki, Ai; Hohenester, Simon; Webster, Cynthia R. L.; Bouscarel, Bernard; Anwer, M. Sawkat

doi:10.1152/ajpgi.00177.2009

Cited by 22 publications

(43 citation statements)

References 42 publications

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“…Based on perfusion experiments in dog liver it was suggested that TCDC inhibited TC-uptake by substrate competition. 39 In Huh7 cells expressing human NTCP, TLC competitively inhibits TC uptake, 40 whereas in rat hepatocytes a noncompetitive inhibition of bile salt uptake by TLC has been described. 41 In our setup, simple substrate competition was ruled out, because net bile salt uptake was also reduced by TCDC-stimulation when TC and 3 [H]-TC were replaced by TCDC and 3 [H]-TCDC, respectively (Supporting Fig.…”

Section: Discussionmentioning

confidence: 99%

Short-term feedback regulation of bile salt uptake by bile salts in rodent liver

et al. 2012

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The sodium taurocholate cotransporting polypeptide (Ntcp) is the major bile salt uptake transporter at the sinusoidal membrane of hepatocytes. Short-term feedback regulation of Ntcp by primary bile salts has not yet been investigated in vivo. Subcellular localization of Ntcp was analyzed in Ntcp-transfected HepG2-cells by flow cytometry and in immunofluorescence images from tissue sections by a new automated image analysis method. Net bile salt uptake was investigated in perfused rat liver by a pulse chase technique. In Flag-Ntcp-EGFP (enhanced green fluorescent protein) expressing HepG2-cells, taurochenodeoxycholate (TCDC), but not taurocholate (TC), induced endocytosis of Ntcp. TCDC, but not TC, caused significant internalization of Ntcp in perfused rat livers, as shown by an increase in intracellular Ntcp immunoreactivity, whereas Bsep distribution remained unchanged. These results correlate with functional studies. Rat livers were continuously perfused with 100 mu mol/L of TC. 25 mu mol/L of TCDC, taurodeoxycholate (TDC), tauroursodeoxycholate (TUDC), or TC were added for 30 minutes, washed out, followed by a pulse of (3)[H]-TC. TCDC, but not TDC, TUDC, or TC significantly increased the amount of (3)[H]-TC in the effluent, indicating a reduced sinusoidal net TC uptake. This effect was sensitive to chelerythrine (protein kinase C inhibitor) and cypermethrin (protein phosphatase 2B inhibitor). Phosphoinositide 3-kinase (PI3K) inhibitors had an additive effect, whereas Erk1/2 (extracellular signal activated kinase 1/2), p38MAPK, protein phosphatase 1/2A (PP1/2A), and reactive oxygen species (ROS) were not involved. Conclusion: TCDC regulates bile salt transport at the sinusoidal membrane by protein kinase C-and protein phosphatase 2B-mediated retrieval of Ntcp from the plasma membrane. During increased portal bile salt load this mechanism may adjust bile salt uptake along the acinus and protect periportal hepatocytes from harmful bile salt concentrations

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Section: Discussionmentioning

confidence: 99%

Short-term feedback regulation of bile salt uptake by bile salts in rodent liver

et al. 2012

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“…Previous studies showed that NO inhibits TC uptake in rat hepatocytes (37). However, it is not known whether TC uptake by human NTCP is also affected similarly by NO, and recent studies suggest differences in the type of inhibition between human NTCP and rat Ntcp (21,35). Moreover, we needed to establish inhibition characteristics of TC uptake by NO in HuH-NTCP cells to relate the effect on NTCP Snitrosylation.…”

Section: S-nitrosylation Of Ntcp In Snoc-treated Huh-ntcp Lysatesmentioning

confidence: 99%

Nitric oxide-mediated inhibition of taurocholate uptake involvesS-nitrosylation of NTCP

Schonhoff

Ramasamy

Anwer

2011

American Journal of Physiology-Gastrointestinal and Liver Physi

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The sodium-taurocholate (TC) cotransporting polypeptide (NTCP) facilitates bile formation by mediating sinusoidal Na ϩ -TC cotransport. During sepsis-induced cholestasis, there is a decrease in NTCPdependent uptake of bile acids and an increase in nitric oxide (NO) levels in hepatocytes. In rat hepatocytes NO inhibits Na ϩ -dependent uptake of taurocholate. The aim of this study was to extend these findings to human NTCP and to further investigate the mechanism by which NO inhibits TC uptake. Using a human hepatoma cell line stably expressing NTCP (HuH-NTCP), we performed experiments with the NO donors sodium nitroprusside and S-nitrosocysteine and demonstrated that NO inhibits TC uptake in these cells. Kinetic analyses revealed that NO significantly decreased the V max but not the K m of TC uptake by NTCP, indicating noncompetitive inhibition. NO decreased the amount of NTCP in the plasma membrane, providing a molecular mechanism for the noncompetitive inhibition of TC uptake. One way that NO can modify protein function is through a posttranslational modification known as S-nitrosylation: the binding of NO to cysteine thiols. Using a biotin switch technique we observed that NTCP is S-nitrosylated under conditions in which NO inhibits TC uptake. Moreover, dithiothreitol reversed NO-mediated inhibition of TC uptake and S-nitrosylation of NTCP, indicating that NO inhibits TC uptake via modification of cysteine thiols. In addition, NO treatment led to a decrease in Ntcp phosphorylation. Taken together these results indicate that the inhibition of TC uptake by NO involves S-nitrosylation of NTCP. sodium-taurocholate cotransporting polypeptide TRANSPORT OF SOLUTES FROM the sinusoidal space to the canaliculus provides the driving force for bile formation (1). Bile formation, in turn, depends on the proper functioning of a number of hepatobiliary transporters present on the hepatic sinusoidal (basolateral) and canalicular (apical) membranes. NTCP, present at the basolateral membrane of hepatocytes, mediates the transport of conjugated bile salts such as taurocholate (TC) and taurochenodeoxycholate in a Na ϩ -dependent fashion using the sodium gradient produced by the Na , 13, 19, 39). Lipopolysaccharide (LPS)-induced cholestasis is associated with a decrease in TC uptake and an increase in proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-1␤ (IL-1␤) (6,11,19). At the same time, LPS also initiates a burst of NO production by inducible nitric oxide synthase in all major cell types of the liver (11).The roles for NO in the liver are diverse and varied. NO plays important roles in regulating hepatic blood flow including portal hypertension (11). NO has both proapoptotic and antiapoptotic actions in the liver (31). Additionally, NO plays important roles in alcoholic liver disease and ischemia/reperfusion injury (31). NO has also been implicated in regulating bile formation. Whereas lower levels of NO have been linked to increased bile flow, higher levels of NO are associate...

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“…Functional studies show that the underlying mutation results in a markedly reduced uptake activity of taurocholate (TC), which is due to an absence of the mutant protein in the plasma membrane of the hepatocyte (31). Recent studies suggest that the cholestatic bile salt taurolithocholate inhibits hepatic TC uptake and decreases plasma membrane levels of Ntcp (32). Also, taurochenodeoxycholate (TCDC) inhibits TC uptake at the sinusoidal membrane in a chelerythrine-and cypermethrin-dependent way (33).…”

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confidence: 99%

Regulation of Plasma Membrane Localization of the Na+-taurocholate cotransporting polypeptide (Ntcp) by Hyperosmolarity and Tauroursodeoxycholate

et al. 2015

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Background:Little is known about the effect of hyperosmotic hepatocyte shrinkage on Na ϩ -taurocholate cotransporting polypeptide (Ntcp) regulation. Results: Hyperosmolarity induces a Fyn-dependent retrieval of Ntcp, which is reversed by tauroursodeoxycholate via a ␤ 1 -integrin-mediated formation of cAMP. Conclusion: Fyn and ␤ 1 -integrins are novel regulators of Ntcp localization. Significance: The study provides new insights into the regulation of bile salt transport.

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PKCε-dependent and -independent effects of taurolithocholate on PI3K/PKB pathway and taurocholate uptake in HuH-NTCP cell line

Cited by 22 publications

References 42 publications

Short-term feedback regulation of bile salt uptake by bile salts in rodent liver

Short-term feedback regulation of bile salt uptake by bile salts in rodent liver

Nitric oxide-mediated inhibition of taurocholate uptake involvesS-nitrosylation of NTCP

Regulation of Plasma Membrane Localization of the Na+-taurocholate cotransporting polypeptide (Ntcp) by Hyperosmolarity and Tauroursodeoxycholate

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