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Supplementary material for this article can be found on the H EPATOLOGY website ( http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html ).

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jws-hep.20942.fig1AB.tif65K Conjugated and unconjugated bile acids promote activation of intracellular signaling pathways in an ROS-dependent fashion. (A) Twenty-four hours after plating, primary rat hepatocytes were pretreated with vehicle (VEH) (DMSO), Trolox (TX) (10 μmol/L) or bongkrekic acid (BA) (50 μmol/L) for 30 minutes prior to addition of DCA (100 μmol/L). Cells were isolated 20 minutes after bile acid addition, lysed, and subjected to SDS-PAGE. Immunoblotting was performed to determine the tyrosine/threonine phosphorylation of ERK1/2. (B) Twenty-four hours after plating primary rat hepatocytes were pretreated with vehicle (VEH) (DMSO), cyclosporin A (CsA) (1 μmol/L), Trolox (TX) (10 μmol/L),N -acetyl cysteine (NAC) (20 mmol/L) or bongkrekic acid (BA) (50 μmol/L) for 30 minutes prior to addition of TDCA or TCA (100 μmol/L, each, as indicated). Cells were isolated 20 minutes after bile acid addition, lysed, and subjected to SDS-PAGE. Immunoblotting was performed to determine the tyrosine/threonine phosphorylation of ERK1/2.
jws-hep.20942.fig1C.tif60K Conjugated and unconjugated bile acids promote activation of intracellular signaling pathways in an ROS-dependent fashion. (C) Twenty-four hours after plating primary rat hepatocytes were pretreated with vehicle (VEH) (DMSO), Trolox (TX) (10 μmol/L) or bongkrekic acid (BA) (50 μmol/L) for 30 minutes prior to addition of DCA or TDCA (100 μmol/L each). Cells were isolated at the indicated times after bile acid addition, lysed, ERBB1 immunoprecipitated, and subjected to SDS-PAGE. Immunoblotting was performed to determine the tyrosine phosphorylation of ERBB1.
jws-hep.20942.fig2.TIF79KRegulation of cell signaling by bile acids in primary hepatocytes and established hepatocyte cell lines. Unconjugated bile acids ( e.g. , DCA) can enter hepatocytes either through membrane transporters ( e.g. , Ntcp) or diffuse through the lipid bilayer directly into the cell. In contrast, conjugated bile acids ( e.g. , TDCA) require expression of Ntcp or other transporters to rapidly enter hepatocytes, and thus in cells lacking transporter expression ( e.g. , McARH cells), conjugated bile acids do not significantly activate intracellular pathways. In the presence of a transporter, McARH cells become permissive for conjugated bile acids to enter these cells and to activate intracellular pathways in an ROS-dependent and PTX-independent manner. In primary hepatocytes that express endogenous bile acid transporters, conjugated bile acids activate intracellular pathways via two concerted mechanisms: by binding to a G i- coupled GPCR and by generating ROS.

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