Objective: Ursodeoxycholic acid (UDCA) exerts anticholestatic
effects in part by protein kinase C (PKC)-dependent mechanisms. Its
taurine conjugate, TUDCA, is a cPKCa agonist. We tested whether
protein kinase A (PKA) might contribute to the anticholestatic
action of TUDCA via cooperative cPKCa-/PKA-dependent mechanisms in
taurolithocholic acid (TLCA)-induced cholestasis. Methods: In
perfused rat liver, bile flow was determined gravimetrically,
organic anion secretion spectrophotometrically, lactate
dehydrogenase (LDH) release enzymatically, cAMP response-element
binding protein (CREB) phosphorylation by immunoblotting, and 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 (10 mmol/l)+TUDCA (25 mmol/l),
combined inhibition of cPKC by the cPKCselective inhibitor Go¨6976
(100 nmol/l) or the nonselective PKC inhibitor staurosporine (10
nmol/l) and of PKA by H89 (100 nmol/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 the bile acids tested,
suggesting a permissive effect of PKA for the anticholestatic
action of TUDCA. Rat and human hepatocellular Mrp2 were
phosphorylated by phorbol ester pretreatment and recombinant cPKCa,
nPKCe, and PKA, respectively, in a staurosporine-sensitive manner.
Conclusion: UDCA conjugates exert their anticholestatic action in
bile acid-induced cholestasis in part via cooperative
post-translational cPKCa-/PKA-dependent mechanisms. Hepatocellular
Mrp2 may be one target of bile acid-induced kinase activation.
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