The long-term goal of this proposal is to understand the mechanism of canalicular bile formation and cholestasis. Our present understanding of the pathogenesis of cholestasis is based on studies to define the physiological regulation of transporters involved in bile formation and their deregulation in cholestasis. Cyclic AMP stimulates bile formation by translocating solute transporters to the plasma membrane and reverses acute cholestasis associated transporter dislocation. The focus of the present proposal will be to further characterize the signaling pathways involved in cAMP-mediated transporter translocation and reversal of transporter dislocation in acute cholestasis. The following hypotheses are proposed: A) cAMP stimulates Ntcp translocation by dephosphorylating Ntcp at S226 via activation of PP2B and/or inhibition of ERK1/2. B) PKC-zeta mediates cAMP-induced Ntcp translocation by facilitating PKB activation in hepatocytes. C) cAMP stimulates translocation of Ntcp, Bsep and Mrp2 by activating PKC-delta and/or p38 MAPK. D) cAMP reverses TLC-induced retrieval of Bsep and Mrp2 by reversing the effect of TLC on PKC-alpha and/or PKCE. E) cAMP stimulates net translocation of Ntcp by stimulating Rab4-mediated exocytotic insertion and/or by inhibiting Rab5-mediated endocytic internalization. Proposed studies will be conducted in perfused rat livers, rat hepatocytes and HUH-7 cell lines. Hepatocytes and HUH-7 cells transfected with wild-type and mutant Ntcp will be used to determine the role of phosphorylation in Ntcp translocation. Role of various kinases will be evaluated by manipulating their activity using chemical inhibitors, wild-type and dominant negative plasmids and siRNA. Immunofluorescence and co-immunoprecipiation studies will be used to determine colocalization of Rab proteins with Ntcp. Collectively, proposed studies should provide further insights into signaling pathways by which cAMP stimulates bile formation and reverses acute cholestasis. In addition, these studies should help define the role of Rab proteins in cAMP-induced vesicle trafficking. ? ? ?
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