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

  • Ulrich Beuers
    Correspondence
    To whom correspondence should be addressed: Dept. of Medicine II, Grosshadern, Klinikum of the University of Munich, Marchioninistrasse 15, D-81377 Munich, Germany. Tel.: 49-89-7095-2272; Fax: 49-89-7095-5271
    Footnotes
    Affiliations
    Department of Medicine II-Grosshadern, Klinikum of the University of Munich, 81377 Munich, Germany
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  • Gerald U. Denk
    Footnotes
    Affiliations
    Department of Medicine II-Grosshadern, Klinikum of the University of Munich, 81377 Munich, Germany
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  • Carol J. Soroka
    Affiliations
    Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510
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  • Ralf Wimmer
    Affiliations
    Department of Medicine II-Grosshadern, Klinikum of the University of Munich, 81377 Munich, Germany
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  • Christian Rust
    Affiliations
    Department of Medicine II-Grosshadern, Klinikum of the University of Munich, 81377 Munich, Germany
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  • Gustav Paumgartner
    Affiliations
    Department of Medicine II-Grosshadern, Klinikum of the University of Munich, 81377 Munich, Germany
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  • James L. Boyer
    Affiliations
    Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510
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  • Author Footnotes
    * This work was supported by Deutsche Forschungsgemeinschaft Grants Be 1242/5-3 and Be 1242/5-4 (to U. B.) and in part by National Institutes of Health Grants DK 25636 (to J. L. B.) and DK P30-34989 (to the Yale Liver Center). Some of the data were presented at the 36th Annual Meeting of the European Association for the Study of the Liver, Prague, Czechia, April 19–22, 2001 and at the 51st Annual Meeting of the American Association for the Study of Liver Disease, Dallas, TX, November 9–13, 2001 and were published in part in abstract form (Denk, G. U., Wimmer, R., Rust, C., Paumgartner, G., and Beuers, U. (2001) J. Hepatol. 34, Suppl. 1, 187 (abstr.) and Beuers, U., Denk, G. U., Soroka, C. J., Wimmer, R., Rust, C., Paumgartner, G., and Boyer, J. L. (2001)Hepatology34, 471 (abstr.)).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Both authors contributed equally to this work.
Open AccessPublished:March 07, 2003DOI:https://doi.org/10.1074/jbc.M209898200
      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 Cε (PKCε)-dependent mechanisms. Products of phosphatidylinositol 3-kinases (PI3K) stimulate PKCε. 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 μmol/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 PKCε membrane binding by 23%. In IRHC, TLCA (2.5 μmol/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 μmol/liter) independently and additively reversed the effects of TLCA on bile flow, exocytosis, organic anion secretion, PI3K-dependent protein kinase B activity, and PKCε 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 PKCε-dependent mechanisms that are reversed by tauroursodeoxycholic acid in a PI3K-independent way.
      TLCA
      taurolithocholic acid
      UDCA
      ursodeoxycholic acid
      TUDCA
      tauroursodeoxycholic acid
      IRHC
      isolated rat hepatocyte couplets
      AU
      arbitrary units
      CDNB
      1-chloro-2,4-dinitrobenzene
      CGamF
      cholylglycylamido fluorescein
      Me2SO
      dimethyl sulfoxide
      GS-DNP
      2,4-dinitro-S-glutathione
      HRP
      horseradish peroxidase
      Mrp2
      rat conjugate export pump
      PDK-1
      phosphoinositide-dependent kinase-1
      PI3K
      phosphoinositide 3-kinase
      PKB
      protein kinase B
      PKC
      protein kinase C
      TCA
      taurocholic acid
      TLC
      thin layer chromatography
      IPRL
      isolated perfused rat liver
      PtdIns
      phosphatidylinositol
      The hydrophobic bile acid taurolithocholic acid (TLCA)1 was identified as a potent cholestatic agent 35 years ago (
      • Javitt J.
      ,
      • Javitt N.B.
      • Emerman S.
      ). However, the mechanisms of this cholestatic effect are not yet clear (
      • Trauner M.
      • Meier P.J.
      • Boyer J.L.
      ,
      • Kullak-Ublick G.A.
      • Beuers U.
      • Paumgartner G.
      ). TLCA induces cholestasis at low micromolar concentrations in vivo (
      • Javitt J.
      ) as well as in the isolated perfused liver (
      • Scholmerich J.
      • Baumgartner U.
      • Miyai K.
      • Gerok W.
      ,
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • Dombrowski F.
      ) and in isolated hepatocyte couplets (
      • Milkiewicz P.
      • Mills C.O.
      • Roma M.G.
      • Ahmed-Choudhury J.
      • Elias E.
      • Coleman R.
      ) in vitro. TLCA impairs hepatobiliary exocytosis, a key step for the insertion of apical carrier proteins into their target membrane, and lowers the density of the apical conjugate export pump, Mrp2, in canalicular membranes of liver cells in association with reduced canalicular excretion of organic anions (
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • Dombrowski F.
      ). In parallel, TLCA modulates a number of signaling events in liver cells that may contribute to membrane vesicle fusion and membrane protein insertion; TLCA may (i) impair Ca2+ influx across hepatocellular membranes (
      • Combettes L.
      • Berthon B.
      • Doucet E.
      • Erlinger S.
      • Claret M.
      ,
      • Beuers U.
      • Nathanson M.H.
      • Boyer J.L.
      ,
      • Beuers U.
      • Probst I.
      • Soroka C.
      • Boyer J.L.
      • Kullak-Ublick G.A.
      • Paumgartner G.
      ), (ii) reduce hepatocellular membrane binding of the Ca2+-sensitive α-isoform of protein kinase C (PKCα), a mediator of regulated exocytosis (
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • Dombrowski F.
      ), and (iii) selectively translocate the Ca2+-independent ε-isoform of PKC to canalicular membranes and activate membrane-bound PKC (
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • Dombrowski F.
      ,
      • Beuers U.
      • Probst I.
      • Soroka C.
      • Boyer J.L.
      • Kullak-Ublick G.A.
      • Paumgartner G.
      ). The role of PKCε as a mediator of TLCA-induced cholestasis, however, remains elusive because specific PKCε inhibitors for in vivo use are not available.
      Products of phosphatidylinositol-3 kinases (PI3Ks) are mediators of diverse cellular functions and may also modulate secretory activity of epithelial cells (
      • Rameh L.E.
      • Cantley L.C.
      ,
      • Toker A.
      ). In hepatocytes, PI3K is involved in taurocholic acid (TCA)-induced biliary bile acid secretion (
      • Misra S.
      • Ujhazy P.
      • Gatmaitan Z.
      • Varticovski L.
      • Arias I.M.
      ,
      • Misra S.
      • Ujhazy P.
      • Varticovski L.
      • Arias I.M.
      ). Interestingly, products of PI3K, phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate, are potent stimuli of the ε-isoform of PKC in transfected insect cells as well as in human hepatoma cells (
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ,
      • Moriya S.
      • Kazlauskas A.
      • Akimoto K.
      • Hirai S.
      • Mizuno K.
      • Takenawa T.
      • Fukui Y.
      • Watanabe Y.
      • Ozaki S.
      • Ohno S.
      ), possibly via binding and recruitment to membranes of phosphoinositide-dependent kinase-1 (PDK-1) (
      • Toker A.
      • Newton A.C.
      ) and subsequent PDK-1-dependent phosphorylation and autophosphorylation of PKCε (
      • Cenni V.
      • Doppler H.
      • Sonnenburg E.D.
      • Maraldi N.
      • Newton A.C.
      • Toker A.
      ) in a way similar to activation of the best characterized PI3K effector, the proto-oncogene Akt/protein kinase B (PKB) (
      • Toker A.
      ). Therefore, we speculate that the TLCA cholestatic effects may be mediated by PI3K- and PKCε-dependent mechanisms. PI3K can be selectively blocked by specific PI3K inhibitors, among which wortmannin is the best characterized in vivo and in vitro (
      • Powis G.
      • Bonjouklian R.
      • Berggren M.M.
      • Gallegos A.
      • Abraham R.
      • Ashendel C.
      • Zalkow L.
      • Matter W.F.
      • Dodge J.
      • Grindey G.
      • Vlahos C.J.
      ).
      In contrast to TLCA, the hydrophilic bile acid ursodeoxycholic acid (UDCA) is a potent anticholestatic agent and is used for the treatment of a number of cholestatic disorders (
      • Lazaridis K.N.
      • Gores G.J.
      • Lindor K.D.
      ,
      • Beuers U.
      • Boyer J.L.
      • Paumgartner G.
      ). The taurine conjugate of UDCA (TUDCA) reverses TLCA-induced cholestasis by PKCα-dependent mechanisms (
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • Dombrowski F.
      ). Recently, PI3K was proposed to contribute to TUDCA-induced stimulation of bile acid secretion in normal rat liver (
      • Kurz A.K.
      • Block C.
      • Graf D.
      • Dahl S.V.
      • Schliess F.
      • Haussinger D.
      ).
      In the present study we investigated the role of PI3K and PKCε in TLCA-induced impairment of bile secretion in vivo in the model of the isolated perfused rat liver (IPRL) as well as in vitro in isolated rat hepatocyte couplets (IRHC) using the selective PI3K inhibitor wortmannin. We also investigated the role of PI3K in the ability of TUDCA to reverse TLCA-induced impairment of bile flow, organic anion secretion, and hepatobiliary exocytosis.

      DISCUSSION

      The present study indicates that the monohydroxy bile acid, TLCA, impairs bile flow, hepatobiliary exocytosis, and secretion of bile acids and other cholephiles by PI3K- and putatively PKCε-dependent mechanisms. The major finding of this study is that TLCA-induced cholestasis can be reversed by specific PI3K inhibitors. This is demonstrated by the reversal of TLCA-induced impairment of bile flow and HRP secretion in IPRL (Figs. 1 and 2) as well as the reversal of TLCA-induced impairment of CGamF secretion in IRHC (Fig. 6) after administration of wortmannin. Thus, this study confirms that an individual bile acid can modulate liver cell function including bile secretion by interacting with specific signal transduction pathways in hepatocytes.
      TLCA was the first human bile acid identified to cause cholestasis and jaundice (
      • Javitt J.
      ), yet the molecular mechanisms by which TLCA induces cholestasis have remained obscure. TLCA induces selective damage of canalicular membranes leading to an increase in membrane rigidity and loss of microvilli (
      • Kakis G.
      • Yousef I.M.
      ,
      • Kakis G.
      • Phillips M.J.
      • Yousef I.M.
      ). TLCA impairs transcellular movement of vesicles (
      • Marinelli R.A.
      • Roma M.G.
      • Pellegrino J.M.
      • Rodriguez Garay E.A.
      ) as well as vesicle fusion at the apical pole (
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • Dombrowski F.
      ) and inhibits secretion of organic anions and bile acids across the canalicular membrane (
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • Dombrowski F.
      ,
      • Milkiewicz P.
      • Mills C.O.
      • Roma M.G.
      • Ahmed-Choudhury J.
      • Elias E.
      • Coleman R.
      ,
      • Roma M.G.
      • Penalva G.L.
      • Aguero R.M.
      • Rodriguez Garay E.A.
      ). The recent finding that TLCA markedly reduces the density of the conjugate export pump, Mrp2, in the canalicular membrane (
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • Dombrowski F.
      ) strongly supports the concept that the mechanism of TLCA-induced cholestasis involves inhibition of vesicle-mediated carrier insertion in the apical liver cell membrane. This view is further supported by the present study demonstrating that the PI3K inhibitor, wortmannin, completely reverses TLCA-induced inhibition of hepatobiliary exocytosis in IPRL in vivo as well as canalicular bile acid secretion in IRHC in vitro.
      Effects of the PI3K inhibitor, wortmannin, and of bile acids on total activity of class IA PI3K were determined in IPRL in the present study. Class IA PI3K are assumed to represent a predominant form of PI3K in secretory cells. Wortmannin inhibited PI3K activity in IPRL (Fig. 3), confirming that the effects of wortmannin on TLCA-induced cholestasis were mediated by PI3K in the present study. Bile acids at low micromolar concentrations did not induce significant changes of total class IA PI3K activity as determined by a PI3K assay in IPRL (Fig. 3), although the PI3K inhibitor, wortmannin, markedly affected TLCA-induced changes of secretion (Figs. 1 and 2). Thus, we speculate that low micromolar concentrations of bile acids may modulate PI3K activity in hepatocytes in vivo at a subcellular level that is not technically detectable when using a PI3K assay in liver homogenates.
      The serine/threonine protein kinase Akt/PKB is a well characterized target and effector of PI3K (
      • Toker A.
      ) and is used as a sensitive read-out of PI3K activity (
      • Webster C.R.
      • Anwer M.S.
      ,
      • Foukas L.C.
      • Daniele N.
      • Ktori C.
      • Anderson K.E.
      • Jensen J.
      • Shepherd P.R.
      ). Binding of lipid products of PI3K to the PKB pleckstrin homology domain is critical for PKB activation via phosphoinositide-dependent kinase-1 (PDK-1)-mediated phosphorylation (
      • Toker A.
      ). Accordingly, the specific PI3K inhibitor, wortmannin, reduced basal PKB activity in liver tissue in the present study (Fig. 4). Our new finding that the cholestatic bile acid TLCA and the anticholestatic bile acid TUDCA inversely regulate PKB activity in IPRL at low micromolar concentrations is of interest. TLCA-induced activation of PKB was completely reversed by wortmannin, further supporting activation of a PI3K-dependent signaling pathway by TLCA (Fig. 4). In contrast, TUDCA impaired PKB activity both under basal conditions and in livers treated with TLCA. The finding that wortmannin did not affect TUDCA-induced impairment of PKB activity under basal conditions and reversed TLCA-induced activation of PKB by effects that were additive to TUDCA supports the concept that TUDCA exerted its anticholestatic effects in the present model in a PI3K-independent manner, whereas cholestatic effects of TLCA were mediated by PI3K-dependent mechanisms. The exact molecular mechanisms by which bile acids inversely regulate PI3K and PKB activity remain to be elucidated.
      TLCA has already previously been shown to affect hepatocellular signaling cascades, which control vesicular exocytosis and membrane protein targeting in secretory cells. (i) TLCA specifically induces translocation of the ε-isoform of PKC to the canalicular membrane, increases intracellular levels of the physiological PKC activator,sn-1,2-diacylglycerol, and activates membrane-bound PKC (
      • Beuers U.
      • Probst I.
      • Soroka C.
      • Boyer J.L.
      • Kullak-Ublick G.A.
      • Paumgartner G.
      ,
      • Beuers U.
      • Throckmorton D.C.
      • Anderson M.S.
      • Isales C.M.
      • Thasler W.
      • Kullak-Ublick G.A.
      • Sauter G.
      • Koebe H.G.
      • Paumgartner G.
      • Boyer J.L.
      ). (ii) TLCA modulates [Ca2+]i (cytosolic free calcium) in isolated hepatocytes (
      • Combettes L.
      • Berthon B.
      • Doucet E.
      • Erlinger S.
      • Claret M.
      ,
      • Beuers U.
      • Nathanson M.H.
      • Boyer J.L.
      ,
      • Beuers U.
      • Probst I.
      • Soroka C.
      • Boyer J.L.
      • Kullak-Ublick G.A.
      • Paumgartner G.
      ,
      • Anwer M.S.
      • Engelking L.R.
      • Nolan K.
      • Sullivan D.
      • Zimniak P.
      • Lester R.
      ,
      • Combettes L.
      • Dumont M.
      • Berthon B.
      • Erlinger S.
      • Claret M.
      ) and may inhibit Ca2+ influx in vitro at concentrations ≥10 μmol/liter (
      • Combettes L.
      • Berthon B.
      • Doucet E.
      • Erlinger S.
      • Claret M.
      ,
      • Beuers U.
      • Nathanson M.H.
      • Boyer J.L.
      ,
      • Beuers U.
      • Probst I.
      • Soroka C.
      • Boyer J.L.
      • Kullak-Ublick G.A.
      • Paumgartner G.
      ). Both, activation of PKCε and impairment of Ca2+ influx have been related to impairment of exocytosis and membrane targeting of proteins (
      • Burgoyne R.D.
      • Morgan A.
      ,
      • Sapin C.
      • Baricault L.
      • Trugnan G.
      ).
      The ε-isoform of PKC is specifically activated in vitro by products of PI3K, PtdIns-3,4-bisphosphate and PtdIns-3,4,5-trisphosphate (
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ,
      • Moriya S.
      • Kazlauskas A.
      • Akimoto K.
      • Hirai S.
      • Mizuno K.
      • Takenawa T.
      • Fukui Y.
      • Watanabe Y.
      • Ozaki S.
      • Ohno S.
      ), possibly via phosphoinositide-dependent kinase I (PDK-1)-induced phosphorylation of Thr-566 in the activation loop and subsequent autophosphorylation of Ser-729 in the C-terminal hydrophobic motif (
      • Cenni V.
      • Doppler H.
      • Sonnenburg E.D.
      • Maraldi N.
      • Newton A.C.
      • Toker A.
      ). PDK-1 activity was not affected by wortmannin and bile acids in the present study (see “Results”). In human HepG2 hepatoma cells, activation of PI3K via stimulation of a mutant platelet-derived growth factor receptor led to specific translocation of PKCε from cytosol to membranes, a key step for activation of PKCε. This process was reversed by the addition of the PI3K inhibitor, wortmannin (
      • Moriya S.
      • Kazlauskas A.
      • Akimoto K.
      • Hirai S.
      • Mizuno K.
      • Takenawa T.
      • Fukui Y.
      • Watanabe Y.
      • Ozaki S.
      • Ohno S.
      ). Thein vivo findings in the present study are consistent with these observations. TLCA-induced translocation of PKCε to membranes was reversed by wortmannin and, as recently shown, by the anticholestatic bile acid TUDCA (
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • Dombrowski F.
      ). PKCε membrane binding was even more strongly inhibited when wortmannin was co-administered with TUDCA (Fig. 5). Thus, TLCA-induced membrane translocation of PKCε seems to be mediated by PI3K-dependent mechanisms.
      Interestingly, wortmannin and TUDCA exerted additive and independent anticholestatic effects on bile flow and organic anion secretion as well as hepatobiliary exocytosis in TLCA-treated IPRL in the present study. Submaximal dosing of wortmannin was virtually excluded as a potential explanation for these additive effects of wortmannin and TUDCA because administration of the PI3K inhibitor at doses of 100 and 500 nmol/liter resulted in comparable effects on TLCA-induced impairment of bile flow in IPRL (Fig. 1a). As shown previously, the anticholestatic effect of TUDCA on TLCA-induced impairment of organic anion secretion (and bile flow)
      U. Beuers, G. U. Denk, and R. Wimmer, unpublished observation.
      was mediated by PKCα- and putatively Ca2+-dependent mechanisms (
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • Dombrowski F.
      ) as documented by reversal of the anticholestatic effect of TUDCA by use of the PKC inhibitor, bisindolylmaleimide I. Bisindolylmaleimide I predominantly blocks the Ca2+-sensitive α-isoform of PKC. PKCα is selectively translocated by TUDCA to hepatocellular membranes (
      • Beuers U.
      • Throckmorton D.C.
      • Anderson M.S.
      • Isales C.M.
      • Thasler W.
      • Kullak-Ublick G.A.
      • Sauter G.
      • Koebe H.G.
      • Paumgartner G.
      • Boyer J.L.
      ,
      • Stravitz R.T.
      • Rao Y.P.
      • Vlahcevic Z.R.
      • Gurley E.C.
      • Jarvis W.D.
      • Hylemon P.B.
      ). TLCA impaired membrane binding of the Ca2+-sensitive PKCα (
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • Dombrowski F.
      ), whereas TUDCA reversed TLCA-induced impairment of PKCα membrane binding (
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • Dombrowski F.
      ). Thus, we speculate that TLCA may impair targeting of apical carrier proteins and, thereby, hepatobiliary secretion by a dual mechanism that includes activation of PI3K and, subsequently, PKCε at the apical pole of the hepatocyte on one hand and impairment of Ca2+ influx and PKCα membrane binding on the other hand. Further work is needed to corroborate this assumption.
      In the present study wortmannin did not affect basal bile flow but stimulated biliary exocytosis in IPRL preloaded with HRP. These findings are in contrast to a study by Folli et al. (
      • Folli F.
      • Alvaro D.
      • Gigliozzi A.
      • Bassotti C.
      • Kahn C.R.
      • Pontiroli A.E.
      • Capocaccia L.
      • Jezequel A.M.
      • Benedetti A.
      ) who show that wortmannin reduces bile flow and biliary HRP secretion in IPRL. However, the experimental protocols of the two studies differed. Folli et al. (
      • Folli F.
      • Alvaro D.
      • Gigliozzi A.
      • Bassotti C.
      • Kahn C.R.
      • Pontiroli A.E.
      • Capocaccia L.
      • Jezequel A.M.
      • Benedetti A.
      ) investigated the effect of wortmannin on hepatic uptake (endocytosis), transcellular trafficking, and biliary excretion of HRP in IPRL (
      • Folli F.
      • Alvaro D.
      • Gigliozzi A.
      • Bassotti C.
      • Kahn C.R.
      • Pontiroli A.E.
      • Capocaccia L.
      • Jezequel A.M.
      • Benedetti A.
      ), whereas the present study mainly focused on the role of PI3K in exocytosis. In the previous study, inhibition of PI3K impaired endo- and transcytosis of fluid phase markers in IRHC and may, therefore, have impaired HRP uptake and transport across the hepatocyte in IPRL. In the present study, HRP was endocytosed before administration of wortmannin. Thus, in livers preloaded with HRP, stimulation of exocytosis by wortmannin may have antagonized a weak inhibiting effect of wortmannin on total bile flow, although the vesicular pathway may contribute less than 10% to total bile flow in IPRL (
      • Crawford J.M.
      ). Altogether, the findings of these two studies suggest that basolateral endocytosis is stimulated, and apical exocytosis is suppressed by intrinsic PI3K activity in IPRL.
      PI3K has also recently been demonstrated to be involved in regulation of canalicular bile acid secretion. Misra et al. (
      • Misra S.
      • Ujhazy P.
      • Gatmaitan Z.
      • Varticovski L.
      • Arias I.M.
      ,
      • Misra S.
      • Ujhazy P.
      • Varticovski L.
      • Arias I.M.
      ) observed that secretion of TCA by IPRL is mediated in part via PI3K-dependent mechanisms. Transport of TCA across the canalicular membrane was markedly reduced by wortmannin in IPRL and canalicular membrane vesicles. In contrast, the present study indicates that TLCA-induced impairment of bile acid secretion (Fig. 6) as well as bile flow, exocytosis, and organic anion secretion (Fig. 1, Table I) is reversed by wortmannin. Can these differences be explained? Different classes and subclasses of PI3K have been described that are all inhibited by the PI3K inhibitor, wortmannin (
      • Vanhaesebroeck B.
      • Waterfield M.D.
      ). Class I PI3K are heterodimers made up of a 110-kDa catalytic subunit (p110) and an adaptor/regulatory subunit. Three p110 isoforms (α, β, δ) and at least seven adaptor proteins (p85, p55) may form class IAPI3K family members. In contrast, class IB PI3K (p110γ/p101) are only abundant in mammalian white blood cells. PtdIns 4,5-bisphosphate appears to be the preferred substrate of class I PI3Kin vivo, although these PI3K can also utilize PtdIns and PtdIns 4-phosphate as substrates in vitro (
      • Vanhaesebroeck B.
      • Waterfield M.D.
      ). Three class II isoforms (PI3K-C2α, -β, -γ) have been detected in mammalian tissue. Their molecular mass is above 170 kDa, and their preferred substrate is PtdIns 4-phosphate. The γ-isoform is mainly detected in liver (
      • Vanhaesebroeck B.
      • Waterfield M.D.
      ). Class III PI3K are homologues of yeast vesicular-sorting protein Vsp34p and use only PtdIns as substrate (
      • Vanhaesebroeck B.
      • Waterfield M.D.
      ). As cellular levels of PtdIns 3-phosphate are quite constant under physiological conditions, their role in short-term regulation of cellular metabolism is regarded as limited. Thus, it appears possible that different bile acids such as TCA or TLCA affect different subclasses of PI3K that are involved in regulation of biliary secretion. Future development of specific inhibitors may permit differentiation of the actions of these different PI3K isoforms.
      TUDCA has been shown to stimulate TCA secretion in normal IPRL in part by a PI3K-dependent mechanism and to stimulate PI3K activity at least transiently in isolated hepatocytes when administered at 500 μmol/liter (
      • Kurz A.K.
      • Block C.
      • Graf D.
      • Dahl S.V.
      • Schliess F.
      • Haussinger D.
      ). The present study confirmed transient stimulation of PI3K by TUDCA at 10 μmol/liter in isolated hepatocytes as determined by phosphorylation of PI3K-dependent PKB (Fig. 7). However, the present study did not reveal a role of PI3K in mediating choleretic and anticholestatic effects of TUDCA in vivo; bile flow, exocytosis, and organic anion secretion in IPRLs treated with TUDCA were not affected by wortmannin (Fig. 2, Table I). In addition, the anticholestatic effects of TUDCA in TLCA-treated livers were even enhanced when wortmannin was co-administered (Fig. 2). Thus, a mediator function of PI3K in TUDCA-induced bile secretion may be restricted to secretion of bile acids in normal liver.
      In the present study, co-administration of a PI3K inhibitor not only reversed TLCA-induced impairment of bile secretion but also cellular damage as determined by lactate dehydrogenase release (Table I). The improvement in bile flow alone could not account for this effect since TUDCA also improved secretion in TLCA-treated livers but failed to abolish the cell damage induced by TLCA in IPRL. Future studies will be necessary to elucidate the role of PI3K in TLCA-induced acute liver cell damage.
      The present data suggest that PI3K represents a potential target of future anticholestatic treatment strategies. It should be mentioned, however, that PI3K may activate a survival pathway in rat hepatocytes treated with the hydrophobic bile acid, taurochenodeoxycholic acid (TCDCA) which protects liver cells from TCDCA-induced damage in vitro (
      • Rust C.
      • Karnitz L.M.
      • Paya C.V.
      • Moscat J.
      • Simari R.D.
      • Gores G.J.
      ) as well as in vivo (Rust C, unpublished observation). Interestingly, the taurochenodeoxycholic acid-induced survival pathway did not involve PKB activation in vitro(
      • Rust C.
      • Karnitz L.M.
      • Paya C.V.
      • Moscat J.
      • Simari R.D.
      • Gores G.J.
      ). Thus, different bile acids may exert differential effects on PI3K- and PKB-mediated processes in liver cells. It remains to be clarified whether involvement of different PI3K isoforms or action in different subcellular compartments may contribute to these diverse effects of bile acids on PI3K and PKB.
      In summary, the present study demonstrates that TLCA-induced impairment of bile flow, hepatobiliary exocytosis, secretion of bile acids, and other organic anions as well as liver cell damage is mediated by PI3K- and putatively PKCε-dependent mechanisms. TUDCA reversed the inhibitory effects of TLCA on bile secretion by a PI3K-independent mechanism.

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