Cholangiocyte bile acid uptake
- Bile acid interactions with cholangiocytes

Although earlier studies have suggested bile acid uptake mechanisms were present in cholangiocytes, the identification of ASBT by Lazaridis et al[17] and Alpini et al[5] brought to the forefront the interest in bile acid transport in the biliary system. ASBT had previously been identified in ileum and kidney tubules[18] and ASBT has been proposed as the major transporter involved in the reclamation of bile acids in the intestine and in the nephron, respectively[18]. Studies by Alpini et al[5] 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]. Our studies showed the majority of [3H]-taurocholate uptake is Na+-dependent with a Km of 43 mmol/L and a Vmax of 190 pmol/min[5]. These values were lower compared to measurements by Lazaridis et al[17], however studies by Alpini et al[5] 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[19,20]. 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 that would reduce the effective bile acid concentration immediately adjacent to the cholangiocyte apical membrane[21]. Lazaridis et al[17] 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. 
The natural substrate specificity of ASBT is narrow and restricted to unconjugated bile acids as well as their glycine- and taurine-conjugates[22]. ASBT appears to play a major role in the enterohepatic circulation of bile acids since dysfunctional mutations in the mouse or human ASBT genes cause profound bile acid malabsorption in humans[23]. In the renal tubule ASBT acts as a salvage mechanism to prevent urinary excretion of bile acids that undergo glomerular filtration. The role of ASBT in bile ducts is not as obvious as in the intestine and kidney. We have proposed that ASBT functions in a cholehepatic shunt where bile acids are absorbed in the biliary tract, secreted into the periductular capillary plexus, and carried directly back to the hepatocyte for secretion, thereby promoting bile flow. Alternatively, ASBT may function to sense bile acid concentration in bile, a mechanism that is dependent on bile acid uptake by ASBT and bile acid-dependent activation of intracellular signaling systems in cholangiocytes. The principles of cholehepatic shunting and bile acid signaling are discussed in a later section.
Intracellular bile acid movement in cholangiocytes
In hepatocytes, cytosolic binding proteins have been shown to sequester bile acids in a bound state that may prevent the cytotoxicity of free intracellular bile acids[24,25]. The presence of 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[26].
The ileal bile acid binding protein (IBABP) is expressed in cholangiocytes[5]. Very little is known as to 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 in 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[27].
Bile acid efflux in cholangiocytes
Since Lazaridis et al[17] studies 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. Four mechanisms have been identified in previous studies that together 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[28]. 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[28]. 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[6]as an exon-2 skipped, alternatively spliced form of ASBT, designated t-ASBT. Alternative splicing causes a frameshift 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 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[29]. 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[29]. Similar upregulation of MDR3 in hepatocyte basolateral membranes has been proposed to pump bile acids out of hepatocytes with canalicular cholestasis[30] and MDR3 is upregulated in patients with Dubin-Johnson syndrome[31]. 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.
Recently the Osta-Ostb heteromeric transporter was initially identified in the liver, ileum, and kidney[32]. In contrast to all other organic anion transporters identified to date, transport activity requires the coexpression of both Osta and Ostb proteins. Substrates for this transporter include the bile acid taurocholate, other steroids (estrone 3-sulfate and digoxin), and prostaglandin E2[33]. Osta and Ostb mRNA expression along the mouse gastrointestinal tract mirrors that of ASBT, and both Osta and Ostb proteins are localized to the basolateral surface of ileal, renal and bile duct cells[32]. Studies of bile acid transport in Osta and Ostb expressing Xenopus laevis oocytes showed bile acid efflux and trans-stimulation, indicating that transport occurs by facilitated diffusion. The selective localization of Osta and Ostb to the basolateral plasma membrane of epithelial cells responsible for bile acid and sterol reabsorption, the substrate selectivity of the transporter, suggest that heteromeric Osta and Ostb is an important basolateral bile acid transporter in biliary, ileal and renal epithelial cells.
Cholehepatic shunting of bile acids
Hoffman proposed bile acids may cycle between cho-langiocytes and hepatocytes through a cholehepatic shunt pathway[3]. Unconjugated bile acids[34] or non-charged 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 non-ionized bile salt results in the generation of bicarbonate molecules 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 acid[35]. 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]. Similarly, bile duct proliferation in Mdr2(-/-) mice is associated with a disproportionably high bile flow in response to tauroursocholate acid infusion, a finding that was interpreted as due to enhanced cholehepatic shunting of bile salts due to increased number of bile ducts. The magnitude of absorption of bile acids under physiologic or pathophysiologic conditions in man is not known.
Identification of apical and basolateral bile acid transport proteins in cholangiocytes, points to the possibility that the cholehepatic shunt pathway functions in bile secretion and may adapt in response 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 studies[38] 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 a marked increase in taurocholate-induced choleresis (12 ± 2 mL per mmol bile acid excreted with basal cholangiocyte ASBT activity compared to 38 ± 6 mL per mmol 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 mmol phospholipid per mmol bile acid excreted compared to basal) and cholesterol secretion (2-fold increase in mmol cholesterol per mmol bile acid excreted compared to basal). Finally, following the administration of secretion, the taurocholate transit time is increased by 7 min. 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. Additional studies will be needed to establish the degree of cholehepatic shunting of conjugated bile acids in normal rats.

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