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

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.

However, the mechanisms of this cholestatic effect are not yet clear (3,4). TLCA induces cholestasis at low micromolar concentrations in vivo (1) as well as in the isolated perfused liver (5,6) and in isolated hepatocyte couplets (7) 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 (6). 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 Ca 2ϩ influx across hepatocellular membranes (8 -10), (ii) reduce hepatocellular membrane binding of the Ca 2ϩ -sensitive ␣-isoform of protein kinase C (PKC␣), a mediator of regulated exocytosis (6), and (iii) selectively translocate the Ca 2ϩ -independent ⑀-isoform of PKC to canalicular membranes and activate membrane-bound PKC (6,11). The role of PKC⑀ as a mediator of TLCA-induced cholestasis, however, remains elusive because specific PKC⑀ 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 (12,13). In hepatocytes, PI3K is involved in taurocholic acid (TCA)-induced biliary bile acid secretion (14,15). Interestingly, products of PI3K, phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate, are potent stimuli of the ⑀-isoform of PKC in transfected insect cells as well as in human hepatoma cells (16,17), possibly via binding and recruitment to membranes of phosphoinositide-dependent kinase-1 (PDK-1) (18) and subsequent PDK-1-dependent phosphorylation and autophosphorylation of PKC⑀ (19) in a way similar to activation of the best characterized PI3K effector, the proto-oncogene Akt/protein kinase B (PKB) (13). Therefore, we speculate that the TLCA cholestatic effects may be mediated by PI3K-and PKC⑀-dependent mechanisms. PI3K can be selectively blocked by specific PI3K inhibitors, among which wortmannin is the best characterized in vivo and in vitro (20).
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 (21,22).
In the present study we investigated the role of PI3K and PKC⑀ 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.
Animals-Male Sprague-Dawley rats (229 Ϯ 16 g) were obtained from Charles River (Sulzfeld, Germany). They were subjected to a 12-h day-night rhythm with free access to rodent food and water.
Isolated Rat Liver Perfusion-The technical procedure used has been described previously (6). Rats were anesthetized with sodium pentobarbital (50 mg/kg of body weight, intraperitoneal). After cannulation of the bile duct, the portal vein, and the inferior vena cava, the latter was ligated above the right renal vein. The liver was perfused with Krebs-Ringer bicarbonate solution (6) at 37°C at a constant flow rate of 4.0 -4.5 ml/min/g of liver. Temperature and perfusion pressure were continuously monitored and did not significantly change during any of the experimental conditions chosen in this study. Bile flow was measured gravimetrically in pretared tubes. Hepatovenous efflux of lactate dehydrogenase was measured as an indicator of liver cell damage by use of a standard enzymatic test (24). Two perfusion protocols were applied for determination of (i) hepatobiliary exocytosis and (ii) secretion of the model Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione (GS-DNP).
Protocol I; Hepatobiliary Exocytosis (6,25)-Livers were preloaded with 5 mg/dl HRP, 1 g/dl bovine serum albumin for 25 min in a recirculating Krebs-Ringer bicarbonate perfusion solution (40 ml/min). The perfusion was then switched to a non-recirculating HRP-and bovine serum albumin-free Krebs-Ringer bicarbonate perfusion, residual HRP in the vascular bed was washed out for 5 min, and the PI3K inhibitor, wortmannin (or the carrier Me 2 SO only, 0.001%, v/v), was continuously infused to reach a final concentration of 100 nmol/liter in the portal vein. After 5 min, bile acids (or the carrier Me 2 SO only; 0.1%, v/v) were infused for 50 min at a continuous rate into the perfusion medium to reach a final concentration of 10 or 25 mol/liter, respectively, in the portal vein. At the end of the experiments, the left anterior liver lobe was clamped and excised. A sample of about 200 mg wet weight was immediately transferred into ice-cold homogenization buffer and was homogenized for determination of PKC⑀ distribution (see below).
Protocol II; Organic Anion Secretion (6)-Livers were perfused in a non-recirculating fashion with HRP-and bovine serum albumin-free Krebs-Ringer bicarbonate for 95 min. After 15 min, the PI3K inhibitor, wortmannin, (or the carrier Me 2 SO only, 0.001%, v/v) was continuously infused for 80 min to reach a final concentration of 100 nmol/liter in the portal vein. After 20 min, the bile acids TLCA and TUDCA (or the carrier Me 2 SO only; 0.1%, v/v) were infused for 75 min at a continuous rate into the perfusion medium to reach a final concentration of 10 or 25 mol/liter, respectively, in the portal vein. After 45 min, CDNB, the precursor of GS-DNP, was infused for 10 min to reach a final concentration of 30 mol/liter in the portal vein, at which saturation of biliary GS-DNP secretion was observed in the perfused rat liver (26). At the end of the experiments, the left anterior liver lobe was clamped and excised. A sample of this lobe was immediately shock-frozen in liquid nitrogen and stored at Ϫ80°C for determination of PI3K activity and PKB (Ser-473) phosphorylation (see below).
Biliary HRP activity was determined spectrophotometrically using 4-aminoantipyrine as substrate and recording the linear change in absorption at 510 nm for 3 min at a constant temperature of 25°C (6,25). HRP (ng of protein/min/g of liver) was quantitated after establishing HRP standard curves. Biliary HRP secretion was expressed as % of secretion at min 45 (after HRP loading and washout period) to correct for differences in base-line total HRP activity.
Biliary secretion of GS-DNP was determined spectrophotometrically (6,26). 5 l of bile were added to 1000 l of H 2 O in a cuvette. Absorption was measured at 335 nm, and biliary GS-DNP levels (nmol/liter) were calculated using the formula c ϭ E 335 V total /⑀dV bile (E 335 , absorption at 335 nm; V total , 1005 l; ⑀, molar absorption coefficient 9.6 liters nmol Ϫ1 cm Ϫ1 ; d, 1 cm; V bile , 5 l). The low background absorption at 335 nm of the bile sample collected just before infusion of CDNB was set as 0.
PKB/Akt activity in hepatic tissue was determined by an immunoblotting technique (27,28). In brief, shock-frozen tissue (Ϫ80°C) was homogenized in ice-cold lysis buffer (27) (1 ml/100 mg of tissue) and processed as described (27,28). Aliquots were electrophoresed using sodium dodecyl sulfate, 10% polyacrylamide gel electrophoresis. Separated proteins were transferred to Immobilon-P membranes and probed with phospho-PKB (Akt Ser-473 ) antibodies at a dilution of 1:1000 overnight to detect the activated form of PKB. Then membranes were stripped and reprobed with a PKB antibody (1:1000) to detect total PKB in an identical procedure. After the use of a goat anti-rabbit IgG antibody (1:5000), a chemiluminescence reagent, and Hyperfilm ECL, the phospho-PKB and PKB bands were quantified by densitometry (NIH Image Densitometric Analysis 1.54; Bethesda, MD, 1994).
Distribution of the protein kinase C isoform ⑀ in hepatic tissue was determined by an immunoblotting technique exactly as described previously (6,11,29) using affinity-purified isoenzyme-specific antibodies for PKC⑀. The PKC bands at 90 kDa (⑀) were quantified by densitometry (NIH Image Densitometric Analysis 1.54; Bethesda, MD, 1994). Results were expressed as [(optical density of the particular fraction)/(total optical density of cytosol and membrane fraction)] ϫ 100 (%).
Isolation and culture of rat hepatocyte couplets was performed as previously described (30). Cells were plated in 100-mm Petri dishes (ϳ10 ϫ 10 4 cells/cm 2 ) containing glass coverslips and incubated at 37°C in L-15 medium for 4 h in an air atmosphere.
Bile acid secretion by IRHC was assessed by measuring the hepatocellular uptake and secretion of 1 mol/liter cholylglycylamido fluorescein (CGamF) into the canalicular space as previously described (30). CGamF was synthesized according to Schteingart et al. (31) and was kindly provided by Dr. Alan Hofmann. Four hours after isolation, hepatocytes (on coverslips) were briefly transferred to HEPES buffer (30). Then, cells were pretreated for 15 min at 37°C with (i) Me 2 SO (0.1%, v/v), (ii) 100 nmol/liter wortmannin and Me 2 SO, (iii) Me 2 SO for 5 min, and 2.5 M TLCA (in Me 2 SO, 0.1%, v/v) for 10 min, (iv) 100 nmol/liter wortmannin and Me 2 SO for 5 min, and 100 nmol/liter wortmannin and 2.5 M TLCA (in Me 2 SO, 0.1%, v/v) for 10 min, (v) Me 2 SO for 5 min and 5 mol/liter TLCA (in Me 2 SO, 0.1%, v/v) for 10 min, and (vi) 100 nmol/liter wortmannin and Me 2 SO for 5 min, and 100 nmol/liter wortmannin and 5 mol/liter TLCA for 10 min. Cells were then transferred for 5 min to HEPES buffer containing 1 mol/liter fluorescent CGamF at 37°C to allow adequate loading of the fluorescent bile acid and transferred back for 10 min to their previous dishes (i-vi). Hepatocyte secretion was stopped by placing coverslips in ice-cold HEPES buffer on ice, and cells were viewed immediately on a Zeiss LSM 510 microscope (Thornwood, NY). Laser settings were optimized for a dynamic range to avoid saturation of the fluorescence. The same settings were used for all conditions. Cells were analyzed on the confocal laser scanning microscope by one investigator (C. J. Soroka) who was blinded to the experimental conditions. Couplets were selected based upon the presence of a well defined canalicular space as determined under bright field optics. Images were then acquired with rapid scanning to avoid quenching of the fluorescence. Quantitation of uptake (uptake ϭ (F°cell ϩ F°can)/ m 2 ) and secretion (% secretion ϭ [F°can/(F°cell ϩ F°can)] ϫ 100) of CGamF was performed as previously published (30), except that NIH Image software was used.
Statistics-Data are expressed as the mean Ϯ S.D. Results were compared between groups using an unpaired two-tailed Student's t test or ANOVA when indicated. p Ͻ 0.05 was considered statistically significant.
Thus, as in the first perfusion protocol, wortmannin did not affect basal and TUDCA-induced bile flow but antagonized TLCA-induced impairment of bile flow. The anticholestatic effects of wortmannin and TUDCA on TLCA-induced impairment of bile flow were additive and independent.
Thus, wortmannin did not affect basal or TUDCA-induced secretion of the model Mrp2 substrate, GS-DNP, but partly antagonized TLCA-induced impairment of GS-DNP secretion. The anticholestatic effects of wortmannin and TUDCA were independent.
Hepatovenous lactate dehydrogenase release after 85 min was not affected by administration of wortmannin (100 nmol/ liter) or TUDCA (25 mol/liter). TLCA (10 mol/liter) alone or in combination with TUDCA (25 mol/liter) markedly increased lactate dehydrogenase release (Table I). These effects were reversed by wortmannin (100 nmol/liter) (Table I). Thus, wortmannin did not induce liver cell damage under the experimental conditions chosen but reversed liver cell damage induced by TLCA alone or by TLCA and TUDCA.
Kinase Activities in Tissue of Perfused Rat Livers-PI3K class IA activity, as determined by a PI3K assay after immunoprecipitation using an anti-PI3K p85 antibody, was reduced by wortmannin (100 nmol/liter) to 62% of controls (p Ͻ 0.01; Fig. 3). TLCA (10 mol/liter) tended to increase and TUDCA (25 mol/liter) tended to decrease total PI3K activity as determined by this methodological approach. Immunoprecipitation of PI3K class 1A isoforms using PI3K p110␣ or p110␤ antibodies revealed similar results (data not shown).
Thus, wortmannin inhibited PI3K activity in liver tissue. The limited sensitivity of the methodological approach may have prevented unequivocal disclosure of the effects of bile acids at low micromolar concentrations on PI3K activity.
Thus, TLCA markedly enhanced PKB activity, a sensitive marker of PI3K activity, in liver tissue, whereas both wortmannin and TUDCA impaired basal and TLCA-induced PKB activity in IPRL. The effects of wortmannin and TUDCA on TLCAinduced PKB activity were additive and independent.  Table I. PI3K activity was determined in shock-frozen liver tissue after immunoprecipitation using an anti-PI3K p85 antibody as described under "Experimental Procedures." The product of PI3K, phosphatidylinositol 3-phosphate (PtdIns(3)P), was identified by TLC and autoradiography. The bar graph represents the amount of phosphatidylinositol 3-phosphate formed by immunoprecipitates of liver tissue from experiments shown in Table I Table I. PKB activity was determined in shock-frozen liver tissue as the amount of pPKB(Ser-473) using a specific pPKB(Ser-473) antibody and a Western blotting technique as described under "Experimental Procedures." In parallel, total PKB mass was determined on each blot using a nonselective PKB antibody to prove that the total amount of PKB was identical on each lane. Panel A shows representative immunoblots of which the upper bands represent pPKB(Ser-473), and the lower bands represent total PKB under different experimental conditions. The bar graph in B represents activated PKB as determined by the amount of pPKB(Ser-473) in liver tissue from experiments shown in Table I The ⑀-isoenzyme of PKC was about equally distributed between cytosol (57.4 Ϯ 5.8%, n ϭ 5) and membranes (42.6 Ϯ 5.8%) in control livers treated with Me 2 SO. Neither wortmannin (100 nmol/liter) nor TUDCA (25 mol/liter) affected distribution of PKC⑀ (Fig. 5). In contrast, TLCA (10 mol/liter) significantly increased membrane binding of PKC⑀ by 23.0% (p Ͻ 0.05) as observed previously in isolated hepatocytes (11). Wortmannin (100 nmol/liter) as well as TUDCA (25 mol/liter) reversed the effect of TLCA on PKC⑀ membrane binding (Fig.  5). Interestingly, wortmannin significantly reduced PKC⑀ membrane binding by 28% also in livers treated with TLCA and TUDCA (Fig. 5).

FIG. 3. PI3K activity in liver tissue in the absence (white bars) or presence (black bars) of the PI3K inhibitor, wortmannin (Wo, 100 nmol/liter), under the experimental conditions described in
Thus, wortmannin did not affect membrane binding of PKC⑀ in liver tissue under basal conditions, but like TUDCA, reversed TLCA-induced membrane binding of PKC⑀. The effects of wortmannin and TUDCA on TLCA-induced membrane binding of PKC⑀ were independent and additive.

PI3K-dependent PKB (PKB/Akt) Activity in Isolated Rat
Hepatocytes-The amount of phospho-PKB(Ser-473), a sensitive read-out of the activation of the PI3K pathway (27,32), was markedly enhanced by TLCA (5 mol/liter) in hepatocytes in short term culture (Fig. 7) and reached levels up to 194 Ϯ 46% of controls after 60 min (p Ͻ 0.005 versus control; p Ͻ 0.05 versus TUDCA; p Ͻ 0.01 versus TCA). In contrast, TUDCA (10 mol/liter) only transiently increased PKB activity, whereas TCA (10 mol/liter) had no effect under the experimental conditions chosen (Fig. 7). Thus, TLCA markedly affected PI3K activity in isolated hepatocytes in vitro, whereas TUDCA exerted only minor transient effects on the PI3K pathway when administered at low micromolar concentrations. 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 PKC⑀-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 TLCAinduced 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 (1), 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 (33,34). TLCA impairs transcellular movement of vesicles (35) as well as vesicle fusion at the apical pole (6) and inhibits secretion of organic anions and bile acids across the canalicular membrane (6, 7, 36). The recent finding that TLCA markedly reduces the density of the conjugate export pump, Mrp2, in the canalicular membrane (6) strongly supports the concept that the mecha- nism 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 I A PI3K were determined in IPRL in the present study. Class I A 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 I A 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 (13) and is used as a sensitive read-out of PI3K activity (27,32). 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 (13). 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 TUDCAinduced 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 reg-ulate 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 ⑀-isoform of PKC to the canalicular membrane, increases intracellular levels of the physiological PKC activator, sn-1,2-diacylglycerol, and activates membrane-bound PKC (11,29). (ii) TLCA modulates [Ca 2ϩ ] i (cytosolic free calcium) in isolated hepatocytes (8 -10, 37, 38) and may inhibit Ca 2ϩ influx in vitro at concentrations Ն10 mol/liter (8 -10). Both, activation of PKC⑀ and impairment of Ca 2ϩ influx have been related to impairment of exocytosis and membrane targeting of proteins (39,40).
The ⑀-isoform of PKC is specifically activated in vitro by products of PI3K, PtdIns-3,4-bisphosphate and PtdIns-3,4,5trisphosphate (16,17), 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 (19). 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 PKC⑀ from cytosol to membranes, a key step for activation of PKC⑀. This process was reversed by the addition of the PI3K inhibitor, wortmannin (17). The in vivo findings in the present study are consistent with these observations. TLCA-induced translocation of PKC⑀ to membranes was reversed by wortmannin and, as recently shown, by the anticholestatic bile acid TUDCA (6). PKC⑀ membrane binding was even more strongly inhibited when wortmannin was co-administered with TUDCA (Fig. 5). Thus, TLCA-induced membrane translocation of PKC⑀ 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 TLCAtreated 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) 2 was mediated by PKC␣-and putatively Ca 2ϩ -dependent mechanisms (6) as documented by reversal of the anticholestatic effect of TUDCA by use of the PKC inhibitor, bisindolylmaleimide I. Bisindolylmaleimide I predominantly blocks the Ca 2ϩ -sensitive ␣-isoform of PKC. PKC␣ is selectively translocated by TUDCA to hepatocellular membranes (29,41). TLCA impaired membrane binding of the Ca 2ϩ -sensitive PKC␣ (6), whereas TUDCA reversed TLCA-induced impairment of PKC␣ membrane binding (6). 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, PKC⑀ at the apical pole of the hepatocyte on one hand and impairment of Ca 2ϩ influx and PKC␣ 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. the two studies differed. Folli et al. (42) investigated the effect of wortmannin on hepatic uptake (endocytosis), transcellular trafficking, and biliary excretion of HRP in IPRL (42), whereas the present study mainly focused on the role of PI3K in exocytosis. In the previous study, inhibition of PI3K impaired endoand 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 (43). 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. (14,15) 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 (44). Class I PI3K are heterodimers made up of a 110-kDa catalytic subunit (p110) and an adaptor/regulatory subunit. Three p110 isoforms (␣, ␤, ␦) and at least seven adaptor proteins (p85, p55) may form class I A PI3K family members. In contrast, class I B PI3K (p110␥/ p101) are only abundant in mammalian white blood cells. Pt-dIns 4,5-bisphosphate appears to be the preferred substrate of class I PI3K in vivo, although these PI3K can also utilize PtdIns and PtdIns 4-phosphate as substrates in vitro (44). Three class II isoforms (PI3K-C2␣, -␤, -␥) have been detected in mammalian tissue. Their molecular mass is above 170 kDa, and their preferred substrate is PtdIns 4-phosphate. The ␥-isoform is mainly detected in liver (44). Class III PI3K are homologues of yeast vesicular-sorting protein Vsp34p and use only PtdIns as substrate (44). 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 mol/liter (23). The present study confirmed transient stimulation of PI3K by TUDCA at 10 mol/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 (45) as well as in vivo (Rust C, unpublished observation). Interestingly, the taurochenodeoxycholic acid-induced survival pathway did not involve PKB activation in vitro (45). 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 TLCAinduced 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 PKC⑀-dependent mechanisms. TUDCA reversed the inhibitory effects of TLCA on bile secretion by a PI3K-independent mechanism.