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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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Bile Acid Interactions with Cholangiocytes

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Bile acids interact with cholangiocytes numerous ways. A specific bile acid transporter (ASBT) is localized on the apical membrane posed to absorb biliary bile acids. On the basolateral membrane three transport systems have been identified (t-ASBT, MDR3 and an anion exchanger system). Studies in cultured cholangiocytes show one-way transport bile acids from the apical to the basolateral membrane. Indirect evidence for a cholehepatic shunt pathway initiated by bile acid absorption by ASBT from bile that obligates bile acids to return via the peribiliary plexus to hepatocytes for resecretion into bile. The contribution of the cholehepatic shunt pathway in overall hepatobiliary transport of bile acids and the role of the cholehepatic shunt pathway in the adaptation to chronic cholestasis due to extrahepatic obstruction remain to be determined. ASBT is both acutely regulated by a cAMP-dependent translocation to the apical membrane and chronically regulated by changes in gene expression in response to biliary bile acid concentration. Biliary bile acid concentration and composition may regulate cholangiocyte functions. After uptake by ASBT, bile acids signal intracellular calcium protein kinase C (PKC), phosphoinositide 3-kinase (PI3K), mitogen-activated protein (MAP) kinase and extracellular signal-regulated protein kinase (ERK) intracellular signals in cholangiocytes with resultant changes in cholangiocyte secretion proliferation and survival. Different bile acids have differential effects on cholangiocyte intracellular signals resulting in opposite effects on cholangiocyte secretion, proliferation and survival.

Overview

In earlier studies of hepatobiliary physiology it was generally concluded that bile salts do not appreciably interact with bile ducts.1 Twenty years ago the cholehepatic shunt pathway was proposed to explain the hypercholeretic nature of certain bile acids.2,3 This hypothesis suggested that bile acids in a protonated, uncharged form undergo passive biliary absorption followed by transfer of bile acids back to hepatocytes for resecretion into bile (see fig. 1).2 Later other studies suggested that in the presence of bile duct obstruction bile acids may enter cholangiocytes.4 Following the discovery of the expression of a bile acid transporter on the apical membrane of cholangiocytes (apical sodium-dependent bile acid transporter or ASBT)5-8 there has been renewed interest in the potential of cholehepatic shunting of bile acids. More recent studies have shown that adaptation of cholangiocyte apical bile acid uptake mechanisms occurs in both physiologic and pathophysiologic conditions.7,8 For instance cholangiocyte ASBT adapts to chronic cholestasis induced by bile duct obstruction by upregulation of cholangiocyte transport capacity potentially leading to augmentation cholehepatic shunting of bile acids.9 Other studies have shown that bile acid efflux mechanisms (truncated ASBT and MDR3) are present on the basolateral membrane of cholangiocytes providing a route for bile acid entry into the circulation.10,11

Figure 1. Classic cholehepatic shunt pathway (shown in black) compared to direct biliary bile acid excretion (shown in white).

Figure 1

Classic cholehepatic shunt pathway (shown in black) compared to direct biliary bile acid excretion (shown in white). Bile acids that are poor substrates for coenzyme A synthetase and inefficiently conjugated by hepatocytes are passively absorbed by biliary (more...)

Bile acids also interact with cholangiocytes leading to alteration of cholangiocyte secretion, proliferation, apoptosis and differentiation.7,8,12-14 Bile acids following entry into cholangiocytes by ASBT, may alter Ca+2, cAMP, PKC, and PI3K intracellular signaling systems.8,12,15-18 Recent data suggest that bile acids may stimulate cholangiocyte proliferation and secretion.13,14 and thus accumulating bile acids due to chronic cholestasis may promote ductal hyperplasia in chronic cholestatic liver disease. Alternatively, changes in bile acid composition (enrichment with ursodeoxycholate) leads to diminished cholangiocyte proliferation and reduction of bile mass in animal models of bile duct hyperplasia.8 Changes in biliary bile acid composition or concentration may also modulate cholangiocyte survival.19,20 Increasing biliary bile acid concentration by bile acid feeding reduces cholangiocyte apoptosis induced by CCl4 or vagotomy in rats.19,20 Finally bile acids may alter cholangiocyte differentiation since small bile ducts following chronic exposure to bile acids begin expressing proteins and functions normally only present in large intrahepatic bile ducts.7

The purpose of this review is to provide a comprehensive summary of the current understanding on how bile acids interact with cholangiocytes. First, the mechanisms for bile acid uptake at the cholangiocyte apical membrane will be reviewed. Second, basolateral bile acid efflux mechanisms are outlined. Third, the current evidence for cholehepatic shunting of bile acids is summarized. Next, the mechanisms responsible for acute and chronic regulation of ASBT activity in cholangiocytes are reviewed. Finally, the concept of intracellular bile acids acting as signaling molecules in cholangiocytes will be developed and the evidence for bile acids regulation of cholangiocyte secretion proliferation and survival will be summarized.

Cholangiocyte Bile Acid Uptake

Although earlier studies have suggested bile acid uptake mechanisms were present in cholangiocytes, the identification of ASBT by Lazaridis et al21 and Alpini et al5 brought to the forefront the interest in bile acid transport in the biliary system. ASBT had been previously been identified in ileum and kidney tubules22 and ASBT has been proposed as the major transporter involved in the reclamation of bile acids in the intestine and in the nephron respectively.22 Studies by Alpini et al5 showed the presence of gene expression for both the ASBT and ileal bile acid binding protein (IBABP) in cholangiocytes. Immunofluorescence studies showed that ABAT protein is expressed on the apical membrane of isolated cholangiocytes and isolated bile duct units (IBDU).5 Uptake studies using [3H]-taurocholate showed the majority of taurocholate uptake is Na+-dependent with a Km of 43 μM and a Vmax of 190 pmol/min.5 These values were lower compared to measurements by Lazaridis et al21 however studies by Alpini et al5 were performed in freshly isolated cholangiocytes (which may be a more physiological model) whereas those by Lazaridis were done in a rat cholangiocyte cell line. In addition the kinetics for taurocholate uptake in freshly isolated cholangiocytes has a similar Km and Vmax as reported by other investigators for ASBT-mediated uptake in the ileum.23,24 The Km for a transporter is generally similar to the physiologic concentration of the transported substrate. The markedly lower Km for ASBT in cholangiocytes compared to biliary bile acid concentration may be due to the effect of unstirred layer adjacent bile duct lumen membrane which would reduce the effective bile acid concentration immediately adjacent to the cholangiocyte apical membrane.25 Lazaridis et al21 demonstrated vectorial transport of bile acids from apical to basolateral direction and an absence of transport in the basolateral to apical direction in a normal rat cholangiocyte cell line in a polarized culture system. No additional bile acid uptake proteins have as of yet been identified in the cholangiocyte apical membrane.

Intracellular Bile Acid Movement in Cholangiocytes

In hepatocytes cytosolic binding proteins have been shown to sequester bile acids in a bound state which may prevent the cytotoxicity of free intracellular bile acids.26,27 The presence of a high affinity binding sites would significantly reduce the rate of transcellular transport of bile acids. Indeed previous studies have shown that the transcellular transport comprises the greatest proportion of time in the overall transcellular transport of conjugated bile acids in hepatocytes.28

IBABP is expressed in cholangiocytes.5 Very little is know whether it functions to prevent intracellular toxicity, modulates transcellular transport or it changes in expression in response to increased bile acid flux in cholangiocytes or the presence of cholestasis. Recent studies show expression of IBABP in the ileum is regulated by bile acid concentrations through the effects of farnesoid X receptor (FXR).29

Bile Acid Efflux in Cholangiocytes

Since Lazaridis et al studies21 have shown that bile acid transport in cholangiocytes is vectorial (e.g., apical-to-basolateral) mechanisms are likely present in the basolateral membrane that facilitates the efflux of bile acids into peribiliary plexus circulation. Three mechanisms have been identified in previous studies that together likely account for bile acid efflux in cholangiocytes. The first mechanism was identified employing bile duct fragments. The studies showed that fluorescent bile acid analogs can be taken up across the cholangiocyte basolateral membrane.30 The uptake process involved an anion exchanger mechanism that was identified by inhibition of bile acid uptake in the absence of Cl- or HCO3- or the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.30 Although the authors studied uptake across the basolateral membrane, in their model, they proposed that their studies reflected physiologically an anion exchanger that effluxes bile acids out of cholangiocytes subsequent to apical uptake. The protein responsible for this anion exchanger was not identified.

The second mechanism for bile acid efflux was identified by Lazaridis et al as an exon-2 skipped alternatively spliced form of ASBT designated t-ASBT.6 Alternative splicing causes a frame shift that produces a 154-aa protein. t-ASBT is expressed in rat cholangiocytes ileum and kidney and is localized to the basolateral domain of cholangiocytes.6 Transport studies in Xenopus oocytes revealed that t-ASBT functions as a bile acid efflux protein. Compared to ASBT alternative splicing changes the cellular targeting of ASBT and provides a mechanism for rat cholangiocytes to efflux bile acids at the basolateral membrane.6 The regulation of t-ASBT expression in response to biliary or circulatory bile acid concentrations or in the presence of cholestasis has not been determined. The regional distribution of t-ASBT in large and small ducts is not known.

The third mechanism for bile acid efflux in cholangiocytes is MDR3. Previous studies have shown that MDR3 is expressed on the basolateral membrane of cholangiocytes and MDR3 is upregulated in chronic cholestasis associated with type 3 progressive familial intrahepatic cholestasis.31 It has been proposed that up-regulation of MDR3 may promote cholehepatic shunting in chronic cholestasis thus preventing the toxic effects of accumulating bile acids in cholangiocytes.31 Similar upregulation of MDR3 in hepatocyte basolateral membrane has been proposed to pump bile acids out of hepatocytes with canalicular cholestasis32 and MDR3 is upregulated in patients with Dubin-Johnson syndrome.33 There is no direct evidence that shows MDR3 effluxes bile acids in cholangiocytes or provides a mechanism for prevention of accumulation of bile acids in cholangiocytes during cholestasis.

Cholehepatic Shunting of Bile Acids

Hoffman proposed bile acids may cycle between cholangiocytes and hepatocytes through a cholehepatic shunt pathway.3 Unconjugated bile acids34 or noncharged bile acids (norursodeoxycholate)34 were observed to induce a greater degree of bile flow per bile acid molecule excreted in bile. To account for this hypercholeretic effect it was proposed that unconjugated bile acids may be passively absorbed by bile ducts, enter the peribiliary plexus adjacent to intrahepatic bile ducts then forwarded to the hepatic sinusoids to be returned to cholangiocytes by hepatocyte secretion.3 Typically, cholehepatic shunting initiated by passive absorption of nonionized bile salt results in the generation of bicarbonate molecule in bile which then increases biliary bicarbonate excretion. Other criteria for cholehepatic shunting besides hypercholeresis and alkalinization of bile have been identified. With the hypercholeresis the biliary transit time for the unconjugated or non charged bile acids was greater than expected which would be predicted by longer retention time in the liver due to more than one passage through the hepatobiliary axis.34 Back perfusion of the isolated perfused liver (infusion into the hepatic vein) a route where the blood from the peribiliary plexus does not appreciably enter the hepatic sinusoids reduced the hypercholeretic effect of ursodeoxycholic acid35 Increased number of bile ducts in animal models of cirrhosis was associated with increased hypercholeresis due to ursodeoxycholic acid infusion an observation that was attributed to increased cholehepatic shunting due to increase bile duct mass.36 The magnitude of absorption of bile acids under physiologic or pathophysiologic conditions in man is not known.

With the identification of apical and basolateral bile acid transport proteins in cholangiocytes there is renewed interest in the cholehepatic shunt pathway functioning in bile secretion and how it may adapt to cholestasis. From a functional point of view, the pathway provides a mechanism to enhance bile acid-dependent bile flow and biliary lipid excretion. With multiple passages of bile acids through the canalicular membrane (as a result of recycling through cholangiocytes), the cholehepatic shunt pathway has the potential to increase the efficiency of bile acid-induced biliary lipid excretion and bile acid-dependent bile flow.3 From the pathophysiologic point of view, the pathway provides an alternative route for continuation of hepato-cholangiocyte flux of bile acids despite the presence of complete bile duct obstruction. 37 The latter may well be an important pathophysiologic response of the liver to bile duct obstruction.

In support of ASBT initiating cholehepatic shunting, our studies38 have shown that following the administration of secretin to bile duct ligated rats there is acute upregulation of ASBT in cholangiocytes (as described in the next section). With secretin stimulation of ASBT activity in cholangiocytes there is in a marked increase in taurocholate-induced choleresis (12+2 μl per μmol bile acid excreted with basal cholangiocyte ASBT activity compared to 38+6 μl per μmol bile acid excreted during high cholangiocyte ASBT activity). Similarly with experimental augmentation of cholangiocyte ASBT activity taurocholate induces a much greater increase in biliary phospholipid (1.5-fold increase in μmol phospholipid per μmol bile acid excreted compared to basal) and cholesterol secretion (2-fold increase in μmol cholesterol per μmol bile acid excreted compared to basal). Finally, following the administration of secretin, the taurocholate transit time is increased by 7 minutes. The taurocholate-induced hypercholeresis, increased biliary lipid excretion and increased taurocholate transit time are consistent with enhanced taurocholate cholehepatic shunting due to up regulation of ASBT by secretin (fig. 2). Additional studies will be needed to establish the degree of cholehepatic shunting of conjugated bile acids in normal rats.

Figure 2. Cholehepatic shunt pathway initiated by ductal absorption by cholangiocyte ASBT.

Figure 2

Cholehepatic shunt pathway initiated by ductal absorption by cholangiocyte ASBT. In contrast to the classic cholehepatic shunt pathway (shown in fig. 1) conjugated (rather than unconjugated) bile acids are absorbed by cholangiocytes to be returned to (more...)

Overview of the Regulation of ASBT Expression in Cholangiocytes

Alteration of bile acid transporter activity may occur physiologically in response to local bile acid concentrations to fine tune bile acid transport capacity. For instance ASBT has been shown by some studies to be upregulated in the ileum with increased intestinal bile concentration39 so that to provide increased intestinal bile acid transport activity in response to increased intestinal bile acid load. Alternatively bile acid transporter expression may be chronically modified in pathologic conditions to prevent intracellular accumulation of toxic bile acids due to altered bile acid metabolism or retention.40 For instance in chronic cholestasis due to bile duct obstruction there is downregulation of hepatocyte sinusoid transporters and upregulation of hepatocyte canalicular transporters which provides the combined effect to reduce hepatocyte intracellular retention of bile acids that occurs with cholestasis.41 Regulation of bile acid transporters may also occur regionally within anatomical confines of an organ. For instance ASBT expression is present exclusively in the distal ileum (and not proximal small bowel)42 and sinusoidal transporters are present to a greater degree in the periportal region compared to the pericentral region of the hepatic lobule.41 Temporally regulation of bile acid transporters has been shown to occur gradually through changes in gene expression39,40,43,44 or acutely by changes in cell membrane transporter content by transporter translocation.45,46

Acute Regulation of ASBT in Cholangiocytes

Previous studies have shown that ASBT transport activity acutely increases in cholangiocytes and ileal epithelial cells by a cAMP-dependent mechanism.38,47,48 In cholangiocytes increased cAMP induced by secretin doubles Na+-dependent bile acid uptake in isolated cholangiocytes48 and in perfused bile duct fragments.38 This effect is likely due to protein translocation since pretreatment of cholangiocytes with the microtubule inhibitor colchicine prevents the cAMP-induced increase of Na+-dependent bile acid transport.38,48 The effects of secretin on protein translocation of ASBT to the apical membrane of cholangiocytes were studied employing isolated apical membranes from cholangiocytes.48 These studies showed that cAMP increases apical membrane ASBT only in the absence of colchicine. A model for cAMP-dependent recycling of ASBT in the cholangiocyte apical membrane is shown in Figure 3. The model shows that cAMP increases apical ASBT membrane content but also suggests that ASBT recycles back to latent intracellular stores once the secretin/cAMP stimulus has abated. This mechanism for acute induction of ASBT activity in cholangiocytes has been proposed to provide an accentuation of cholehepatic bile acid shunting in the postprandial period thus accentuating bile flow and biliary lipid secretion (see cholehepatic shunting section above).38

Figure 3. Membrane recycling of ASBT.

Figure 3

Membrane recycling of ASBT. Increased cAMP enhances bile acid uptake in cholangiocytes. In cholangiocyte label number 1 latent ASBT resides in an inactive position within the cytoplasm of cholangiocytes. In cholangiocyte labeled 2 activation of the secretin (more...)

Chronic Regulation of ASBT Expression in Cholangiocytes

ASBT expression in cholangiocytes changes chronically in response to biliary bile acid concentrations and the presence of cholestasis.7-9,19 With increase in biliary bile acid concentration due to feeding taurocholate to rats there is an increase in total liver ASBT.7 In this model the increased ASBT is due to both increased number of cholangiocytes in the liver and the maintenance of ASBT expression per cell. In bile duct ligated rats depleted of biliary bile acids by 12 hours external biliary drainage there is a marked decrease in cholangiocyte ASBT gene and protein expression and transport activity.49 ASBT gene and protein expression and transport activity can be restored in bile depleted rats by infusion of taurocholate to maintain biliary bile acid concentration.49 These studies employing bile acid feeding and bile acid depletion show that ASBT expression in cholangiocytes is chronically regulated in a direct proportion to biliary bile acid concentration.

In contrast to taurocholate feeding which increases cholangiocyte ASBT expression, feeding ursodeoxycholic acid to bile duct ligated rats markedly reduces cholangiocyte ASBT gene and protein expression and taurocholate transport activity.8 Although the mechanism for differential effects of different bile acids on ASBT expression in cholangiocytes has been not defined, they are consistent with our studies showing differential effects of different bile acids on cholangiocyte secretion and proliferation (see below).

With chronic cholestasis due to bile duct ligation, intrahepatic bile ducts markedly increase in number (approximately 10 fold increase after 1 week). In this model, ASBT expression per cholangiocyte is maintained9 so overall there is effective increase in biliary bile acid absorptive capacity in BDL rats. We propose that the increased ASBT in BDL rats provides an alternative excretory pathway in the presence of biliary obstruction so that to prevent the bile acid stasis in the liver and the subsequent accumulation of toxic bile acids in hepatocytes.9

Recently, it was demonstrated that bile acids modulate ASBT expression through activation of the peroxisome proliferator-activated receptor alpha (PPARalpha)22 and Activator protein 1 (AP-1) element regulates the transcription of the rat ASBT gene.44 It remains to be determined if regulation of ASBT expression in cholangiocytes occurs through PPARalpha or AP-1.

Regional ABAT Expression in the Biliary Tree

Regionalization of bile duct functions occur in rat liver.5 Large (greater than 20 μm diameter bile ducts) which are lined by large cholangiocytes contribute to hormone-induced ductal secretion whereas small (smaller than 20 μm diameter bile ducts) do not contribute to hormone-induced ductal secretion.50,51 Studies by Alpini et al5 showed that ASBT is expressed in large cholangiocytes but not small cholangiocytes. The absence of ABST expression in small intrahepatic bile ducts may lead to more efficient hepatobiliary excretion of bile acids since bile acid uptake in small ducts closely adjacent to the canalicular bile acid secretion process may hinder the post canalicular assembly of polymolecular bile acid-lipid micelles and vesicles. Recently experimental models have been developed where ASBT gene and protein expression and transport activity have been shown to extend into small bile ducts.7 In the taurocholate feed rat, a model where biliary bile acid concentration increases approximately two fold, Alpini et al7 found de novo ASBT expression in small ducts. The authors suggested that with expansion of bile acid pool and increased biliary bile acid concentration, the extension of ASBT expression into small ducts leads to enhanced cholehepatic shunting of bile acids. Whether ASBT expression in small ducts alters bile acid-lipid micelles or vesicle formation has not been determined.

Bile Acid Signaling in Cholangiocytes

Overview

In a variety of cells, bile acids have been shown to function as intracellular signals and to profoundly alter cellular functions such as proliferation, differentiation, secretion and apoptosis.52-57 These studies have shown that cellular uptake is required for bile acids to signal cellular processes. In cells not expressing a bile acid transporter (which normally do not respond to the presence of bile acids in the media) experimentally expression of a bile acid transporter activates de novo bile acid signaling.58 Once intracellular, bile acids at concentrations of less that 1 μM have been shown to alter intracellular Ca2+, PKC, MEK, ERK and PI3K pathways in hepatocytes or cholangiocytes.8,16,20,55,57,59-62 Through these downstream signals, bile acids have been shown to alter cell proliferation, secretion, apoptosis and gene expression.8,16,20,55,57,59-62 In addition, bile acids have been shown to induce activation (by phosphorylation) of the EGF receptor in hepatocytes60 and cholangiocytes.63 Finally, a specific bile acid nuclear ligand (FXR) has been shown to regulate bile acid and lipid synthesis in hepatocytes64,65 but the signaling properties of FXR in cholangiocytes has not been defined. Bile acid intracellular signaling is summarized in Figure 4. The signaling effects of bile acids should be distinguished from the toxic effects of bile acids where bile acid in high concentration, through changes in membrane lipids, induces cellular damage in a nonspecific manner.66

Figure 4. Paradigm for bile acid signaling in cholangiocytes.

Figure 4

Paradigm for bile acid signaling in cholangiocytes. After internalization by a bile acid transporter intracellular bile acids may signal cell transduction pathways shown in white resulting in changes in secretion proliferation and differentiation.

Bile Acid Signaling of Cholangiocyte Secretion

It had previously been observed that ursodeoxycholic acid increases biliary bicarbonate excretion into bile. Shimokura et al62 found that ursodeoxycholic acid directly increases cholangiocyte secretion. They demonstrated ursodeoxycholic acid increases intracellular Ca2+ and increases chloride channel activity in a malignant cholangiocyte cell line.62 The ursodeoxycholate effect on chloride channel activity was dependent on the increase in intracellular Ca2+. Our studies have shown that in freshly isolated cholangiocytes, taurocholic acid or taurolithocholic acid (1-20 μM) increases secretin-stimulated cAMP levels and secretin-stimulated Cl-/HCO3- exchanger activity.14 These effects were dependent on taurocholate uptake by ASBT since the stimulatory effect of taurocholate was not present in the absence of Na+.14 Also consistent for dependence of bile acid effects on cholangiocyte secretion on ASBT transport activity, we found the Km for ABAT in cholangiocytes to be close to the concentration where bile acids exert their maximum response in cholangiocytes.14 Similar to their effect in vitro, taurocholate or taurolithocholate feeding to normal rats for 7 days increased secretin-stimulated cAMP and Cl-/HCO3- exchanger activity in cholangiocytes and resulted in an increase of secretin-stimulated ductal bile flow in vitro.7 The studies show that, both in vitro and in vivo, bile acids can augment secretin-stimulated ductal bile flow but the intracellular signals responsible for this effect have not been completely elucidated. In contrast, to the stimulatory effects of taurocholate and taurolithocholate, ursodeoxycholate inhibits secretin-stimulated cAMP synthesis and Cl-/HCO3- exchanger activity in isolated cholangiocytes and secretin-stimulated ductal bile flow in vivo.8 Inhibition of cholangiocyte secretion by ursodeoxycholic acid was found to be dependent on the ability of ursodeoxycholic acid to increase intracellular calcium and activate PKC alpha in cholangiocytes.8 We have proposed that the stimulatory and inhibitory effects of taurocholate and ursodeoxycholate respectively on cholangiocyte secretion is due to their differing ability to activate intracellular Ca2+ and to activate different PKC isoforms.8

Bile Acid Signaling of Cholangiocyte Proliferation

In vitro taurocholate and taurolithocholate in 1-20 μM concentrations increase cholangiocyte proliferation.14 Feeding taurocholate or taurolithocholate to rats increases cholangiocyte proliferation in vivo, increases bile duct mass 2- to 3-fold13 and consistent with a ductal hyperplasia there is accentuation of secretin-stimulated cAMP synthesis and ductal bile flow (see above). The effects of taurocholate and taurolithocholate on bile duct proliferation in the bile acid feeding models occur in the absence of hepatic inflammation.13 Recent studies63 show that taurocholate can induce phosphorylation of EGF similar that reported in hepatocytes60 and taurocholate can activate signals downstream to EGF receptor (MEK and ERK) in cholangiocytes.16 Thus, taurocholate and taurolithocholate can function like EGF as growth factors in cholangiocytes.

In contrast to taurocholate and taurolithocholate, ursodeoxycholate inhibits cholangiocyte proliferation both in vitro in isolated cholangiocytes and in vivo in bile duct ligated rats.8 The inhibition of cholangiocyte proliferation was found to be dependent on activation of PKC alpha and calcium-dependent pathways.8 The inhibitory effect of ursodeoxycholate on cholangiocyte proliferation may be one mechanism for the histological and biochemical improvement of diseases targeting the biliary tree (e.g., primary biliary cirrhosis) with ursodeoxycholate treatment. Inhibition of cholangiocyte proliferation may reduce the number of proliferating cholangiocytes that release proinflammatory cytokines67 or profibrotic signaling molecules such as platelet derived growth factor.68 The lack of therapeutic effect for ursodeoxycholic acid in the late stage primary biliary cirrhosis may be at least partially related to lack of proliferating ducts (e.g., ductopenia) as the disease progresses.69 In a cholangiocarcinoma cell line we demonstrated that ursodeoxycholic acid inhibits growth by inhibition of Raf through PKC-dependent mechanism.18 This study supports the need for clinical trials examining the effect of ursodeoxycholic acid in the promotion and progression of cholangiocarcinoma in patients with primary sclerosing cholangitis.

Bile Acid Signaling of Cholangiocyte Death and Survival Pathways

Previous studies in hepatocytes have shown bile acids may be either cytotoxic70-73 or cytoprotective.74 Bile acid cytotoxicity may be induced by abrupt increased permeability of the inner mitochondrial membrane to ions leading to mitochondrial membrane permeability transition (MMPT), depolarization of the mitochondrial membrane potential, and uncoupling of oxidative phosphorylation.75 The uncoupling of oxidative phosphorylation, if extensive, results in ATP depletion and cellular death by necrosis.75 Furthermore the associated mitochondrial swelling has also been linked to redistribution of cytochrome c from the intermembrane space to the cytosol. In the cytosol, cytochrome c interacts with APAF-1 (apoptotic protease-activating factor 1) to activate caspase 9 and subsequently to cause apoptosis.76 Bile salt-induced hepatocyte apoptosis entails activation of the Fas death-receptor and subsequent activation of caspase 8 followed by activation of Bid which leads to mitochondrial dysfunction.70

Alternatively, bile acids may provide cytoprotective effects. Heuman et al77 proposed that the protective effect of ursodeoxycholic acid in opposing the hepatotoxicity of bile acids was due to its direct interaction with plasma membranes of hepatocytes. Ursodeoxycholic acid may provide membrane stability via a physicochemical effect by reducing the toxic bile salt disruption of cholesterol-rich model membranes.77 More recent studies revealed that ursodeoxycholic acid does not directly stabilize membranes but rather prevents hydrophobic bile acid-induced membrane disruption by alteration of the structure and composition of mixed micelles.78

In cholangiocytes, Benedetti et al73 showed that in vitro unconjugated but not conjugated bile acids induce ultrastructural evidence of cytotoxicity. These findings were not observed in vivo. Even in taurine depleted livers did not show evidence of cytotoxicity despite having high concentration of unconjugated bile acids in bile.73 The authors concluded that bile acid toxicity, although potentially present, in biliary epithelium is prevented in the intact liver by the presence of active transport processes.

Our studies have shown that taurocholate feeding is protective against cholangiocyte apoptosis induced by either CCl420 or vagotomy.19 In CCl4 treated animals cholangiocyte apoptosis, demonstrated by the presence of nuclear fragmentation, positive annexin staining and loss of cholangiocyte function (secretin-stimulated cAMP synthesis), was not observed in CCl4-treated rats that were fed taurocholate.20 Similarly, CCl4-induced apoptosis in vitro was ablated by pretreating cholangiocytes with 20 μM taurocholate. The taurocholate inhibition of CCl4-induced apoptosis required activation of PKC alpha.20 Taurocholate feeding also prevents vagotomy induced cholangiocyte apoptosis.19 In the vagotomy-induced model, apoptosis is associated with loss of PI3K activity and activation of caspase activities.19 Taurocholate feeding prevented cholangiocyte apoptosis, loss of PI3K activity and activation of caspase activity. Thus taurocholate is protective against CCl4- and vagotomy-induced cholangiocyte apoptosis by activation of the PKC and PI3K-dependent pathways respectively.

Feeding ursodeoxycholate inhibits the ductal hyperplasia in BDL rats, however, these studies showed that the effect of ursodeoxycholate was on inhibition of proliferation without increasing cholangiocyte apoptosis.8 In contrast, Que et al79 showed that ursodeoxycholate inhibits beauvericin-induced apoptosis in a cholangiocarcinoma cell line and that the inhibition was dependent on preventing cytochrome c release from mitochondria and subsequent activation of caspases.

Bile Acid Synthesis and Conjugation in Cholangiocytes

Bile acids, phospholipids and cholesterol are synthesized in a cholangiocarcinoma cell line.80 Cholangiocytes have also been shown to conjugate bile acids.80 The contribution of cholangiocyte bile acid synthesis and conjugation to the over all bile acid pool seems minimal since less that 3 percent of the total liver mass is composed of cholangiocytes.81 The mechanisms for regulation of bile acid synthesis in cholangiocytes have not been determined.

Gallbladder Epithelial Cells

Gallbladder epithelial cells and cholangiocytes share a number of functions, however, their primary role in the liver and their reaction to disease are quite different. Similar to cholangiocytes, gallbladder epithelial cells express ASBT and taurocholate have been shown to increase intracellular calcium, activate chloride channels and stimulate mucin secretion.82 In contrast, ursodeoxycholate through a PKC alpha-dependent pathway inhibits gallbladder mucin production.82 The authors proposed that ursodeoxycholate inhibition of gallbladder mucin product might provide benefit to biliary disorders such as cystic fibrosis.

Bile Acid Effects on Cholangiocyte Function or Dysfunction in Humans

Compared to the understanding of the effects of bile acids on rodent cholangiocytes, very little is known regarding bile acid signaling of cholangiocyte function in health and disease in humans. Previous studies of biliary bicarbonate secretion in humans by employing PET scanning show biliary bicarbonate secretion is reduced in primary biliary cirrhosis, the prototypic disease of bile duct damage in humans.83 After treatment with ursodeoxycholate, biliary bicarbonate secretion in primary biliary cirrhosis patients is increased compared to the pretreatment values.83 It is not clear why ursodeoxycholic acid inhibits cholangiocyte secretion in bile duct ligated rats, but increases cholangiocyte secretion in primary biliary cirrhosis in humans. It is likely that pathophysiology of these two forms of biliary injury are different.

Cystic fibrosis also targets biliary epithelium in the liver.84 Clinical studies have shown that ursodeoxycholate may improve liver tests in cystic fibrosis patients.85 It has been proposed that the mechanism for action of ursodeoxycholate in cystic fibrosis is increased ductal bile flow [as demonstrated by the opening of chloride channels by Shimokura et al62] that reduces the bile plugs and obstruction due to thick biliary secretions.86 Ursodeoxycholate has been found to have measurable clinical effects in other diseases that target biliary epithelium (graft versus host disease involving the liver liver allograft rejection and bile-duct paucity syndromes).87 Finally ursodeoxycholate has been used in other chronic cholestatic liver disorders where biliary epithelium is not the primarily targeted (intrahepatic cholestasis of pregnancy, progressive familial intrahepatic cholestasis, nonalcoholic steatohepatitis, alcoholic liver disease and autoimmune hepatitis).87 The potential therapeutic effect of ursodeoxycholate in human liver diseases is reviewed elsewhere.87

Summary and Future Directions

Bile acids interact with cholangiocytes numerous ways. A specific bile acid transporter (ASBT) is localized on the apical membrane posed to absorb biliary bile acids.5,21 On the basolateral membrane three transport systems have been identified (t-ASBT, MDR3 and an anion exchanger system).6,30,31 Studies in cultured cholangiocytes show that cholangiocytes transport bile acids from apical to the basolateral membrane.21 There is indirect evidence for a cholehepatic shunt pathway initiated by bile acid absorption from bile by ASBT that leads to bile acids return via the peribiliary plexus to hepatocytes for secretion into bile.7,34,36-38,41 The contribution of the cholehepatic shunt pathway in overall hepatobiliary transport of bile acids and the role of the cholehepatic shunt pathway in the adaptation to chronic cholestasis due to extrahepatic obstruction remain to be determined. ASBT is both acutely regulated by a cAMP-dependent translocation to the apical membrane38 and chronically regulated by changes in gene expression in response to biliary bile acid concentration.9 Biliary bile acid concentration and composition may regulate cholangiocyte functions. After uptake by ASBT, bile acids signal calcium, PKC, PI3K, MEK and ERK intracellular signals in cholangiocytes with resultant changes in cholangiocyte secretion proliferation and survival.8,16,20,55,57,59-62 Different bile acids have differential effects on cholangiocyte intracellular signals resulting in opposite effects on cholangiocyte secretion proliferation and survival.14,49,88

In future studies the mechanisms explaining how different bile acids can differentially regulate different intracellular signals will be determined. To address the question of how chronic cholestatic liver disease may adapt by changes in cholehepatic shunting, new experimental paradigms to directly quantify bile acid absorption in bile ducts in experimental animals will be developed. Since multiple transporters with varying substrate specificity are present in the sinusoidal and canalicular membrane of hepatocytes, additional bile acid transporters may be found in cholangiocytes. When the mechanisms for bile acid cytoprotective effects in cholangiocytes are defined, a new therapeutic window in human biliary disorders may open that operates through modulation of biliary bile acid concentration and composition. Finally the role of cholangiocyte bile acid transport in the promotion of or adaptation to human liver disease needs to be determined.

Acknowledgments

Portions of the studies outlined in this chapter were supported by a grant award from Scott & White Hospital and Texas A&M University, by an NIH grant DK58411 and by VA Merit Award to Dr. Alpini.

References

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