Objective: Ursodeoxycholic acid (UDCA) exerts anticholestatic effects in part by PKC-dependent mechanisms. Its taurine conjugate, TUDCA, is a cPKCα agonist. We tested whether PKA might contribute to the anticholestatic action of TUDCA via cooperative cPKCα-/PKA-dependent mechanisms in taurolithocholic acid (TLCA)-induced cholestasis.
Methods: In perfused rat liver, bile flow was determined gravimetrically, organic anion secretion spectrophotometrically, LDH release enzymatically, CREB phosphorylation by immunoblotting, cAMP by immunoassay. PKC/PKA inhibitors were tested radiochemically. In vitro phosphorylation of the conjugate export pump, Mrp2/Abcc2, was studied in rat hepatocytes and human Hep-G2 hepatoma cells.
Results: In livers treated with TLCA(10μmol/l)+TUDCA(25μmol/l), combined inhibition of cPKC by the cPKC-selective inhibitor Gö6976 (100nmol/l) or the nonselective PKC inhibitor staurosporine (10nmol/l) and of PKA by H89 (100nmol/l) reduced bile flow by 36% (p<0.05) and 48% (p<0.01), and secretion of the Mrp2/Abcc2 substrate, 2,4-dinitrophenyl-S-glutathione, by 31% (p<0.05) and 41% (p<0.01), respectively; bile flow was unaffected in control livers or livers treated with TUDCA only or TLCA+taurocholic acid. Inhibition of cPKC or PKA alone did not affect the anticholestatic action of TUDCA. Hepatic cAMP levels and CREB phosphorylation as readout of PKA activity were unaffected by bile acids tested suggesting a permissive effect of PKA for the anticholestatic action of TUDCA. Rat and human hepatocellular Mrp2 were phosphorylated by phorbolester pretreatment and recombinant cPKCα, nPKCδ, and PKA, respectively, in a staurosporine-sensitive way.
Conclusion: UDCA conjugates exert their anticholestatic action in bile acid-induced cholestasis in part via cooperative posttranslational cPKCα-/PKA-dependent mechanisms. Hepatocellular Mrp2 may be one target of bile acid-induced kinase activation.