Taurolithocholic Acid Exerts Cholestatic Effects via Phosphatidylinositol 3-Kinase-dependent Mechanisms in Perfused Rat Livers and Rat Hepatocyte Couplets

Abstract

Taurolithocholic acid (TLCA) is a potent cholestatic agent. Our recent work suggested that TLCA impairs hepatobiliary exocytosis, insertion of transport proteins into apical hepatocyte membranes, and bile flow by protein kinase C⑀ (PKC⑀)-dependent mechanisms. Products of phosphatidylinositol 3-kinases (PI3K) stimulate PKC⑀. We studied the role of PI3K for TLCA-induced cholestasis in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets (IRHC). In IPRL, TLCA (10 mol/liter) impaired bile flow by 51%, biliary secretion of horseradish peroxidase, a marker of vesicular exocytosis, by 46%, and the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione, by 95% and stimulated PI3K-dependent protein kinase B, a marker of PI3K activity, by 154% and PKC⑀ membrane binding by 23%. In IRHC, TLCA (2.5 mol/liter) impaired canalicular secretion of the fluorescent bile acid, cholylglycylamido fluorescein, by 50%. The selective PI3K inhibitor, wortmannin (100 nmol/liter), and the anticholestatic bile acid tauroursodeoxycholic acid (TUDCA, 25 mol/liter) independently and additively reversed the effects of TLCA on bile flow, exocytosis, organic anion secretion, PI3K-dependent protein kinase B activity, and PKC⑀ membrane binding in IPRL. Wortmannin also reversed impaired bile acid secretion in IRHC. These data strongly suggest that TLCA exerts cholestatic effects by PI3K-and PKC⑀-dependent mechanisms that are reversed by tauroursodeoxycholic acid in a PI3K-independent way.

Tauroursodeoxycholic acid inserts the bile salt export pump into canalicular membranes of cholestatic rat liver

“…The anticholestatic bile acid, TUDCA, reversed the effect of TLCA on Bsep density in canalicular membranes and mediated insertion of Bsep into canalicular membranes of cholestatic rat liver along with stimulation of bile flow, vesicular exocytosis, and organic anion secretion in the same experimental model as reported recently. 7,8 These data complement and corroborate our previous immunoelectronmicroscopic findings which showed that TUDCA stimulates insertion of another key apical carrier, the conjugate export pump, Mrp2, into its target membrane in bile acid-induced cholestasis. 7 Bile acids and Mrp2 substrates such as glutathione conjugates represent the major driving forces of bile formation.…”

“…The exact molecular mechanism by which TUD-CA stimulates exocytotic fusion of vesicles at the apical membrane 7,25 and apical carrier insertion 7,12,13 remains elusive. Various signaling molecules including isoforms of PKC, 7,9,13 cytosolic-free calcium (Ca 2 þ ) i , 9,25 mitogen-activated protein (MAP) kinases p38 MAPK6,12,13 and p44/42 MAPK , 11,14 src kinase 6 as well as PI3K 8,[26][27][28] have been shown to be involved in bile acid-dependent hepatobiliary exocytosis and carrier insertion/retrieval in apical membranes under various experimental conditions. 29 In the normal rat liver, TUDCA may trigger insertion of Bsep into canalicular membranes by a dual integrin-dependent signaling pathway which includes activation of p44/42 MAPK on one hand and activation of p38 MAPK on the other hand 6,[11][12][13] Preliminary evidence suggests that this pathway may be less relevant for the anticholestatic effect of TUDCA in bile acid-induced cholestasis 14,15 In TLCA-induced cholestasis, the anticholestatic effect of TUDCA appears to be mediated in part by PKC-dependent mechanisms although the exact isoform involved has not yet been determined.…”

“…[1][2][3][4][5][6] We have recently reported that the cholestatic bile acid taurolithocholic acid (TLCA) reduces the density of the apical conjugate export pump, Mrp2 (ABCC2), in canalicular membranes of rat livers and markedly impairs organic anion secretion into bile, 7 in part by phosphatidylinositol 3-kinase (PI3K)-dependent mechanisms. 8 The taurine conjugate of UDCA (TUDCA) reversed the cholestatic effect of TLCA on bile formation, normalized Mrp2 density in canalicular membranes of cholestatic hepatocytes, and stimulated organic anion secretion into bile in part by protein kinase C (PKC)-dependent mechanisms. 7 In line with these observations, TUDCA also protected against TLCA-induced impairment of bile acid secretion in hepatocyte couplets by a PKC-dependent mechanism.…”

cAMP-GEF cytoprotection by Src tyrosine kinase activation of phosphoinositide-3-kinase p110 β/α in rat hepatocytes

“…In nonhepatic cells, cAMP can activate Src-TYK (30), and Src-TYK are known to upregulate PI3K/Akt signaling (2,9,38,39,48). In addition, Src-TYK are well-known prosurvival signaling molecules in many cell types including hepatocytes (5,14,44,55). Thus Src-TYK may also be involved in cAMP-GEF-mediated cytoprotection and PI3K activation in hepatocytes.…”

Cyclic AMP stimulates Mrp2 translocation by activating p38α MAPK in hepatic cells

“…For example, transcription of Mrp2 is downregulated in cholestatic liver diseases (41), and Mrp2 is retrieved from the canalicular membrane during cholestasis induced by taurolithocholate (TLC) (2) or estradiol-17␤-glucuronide (E 2 17G) (25). The TLC-induced retrieval of Mrp2 may be mediated via a phosphoinositide-3-kinase (PI3K)/PKC⑀-dependent mechanism (3). Taurocholate (TC), tauroursodeoxycholate (TUDC), and cAMP, on the other hand, increase biliary excretion of Mrp2 substrates and induce Mrp2 translocation to the canalicular membrane (2,9,33), and cAMP can reverse E 2 17G-induced retrieval of Mrp2 (25).…”