2. FUNCTIONAL HETEROGENEITY OF INTRAHEPATIC CHOLANGIOCYTES

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FUNCTIONAL HETEROGENEITY OF INTRAHEPATIC CHOLANGIOCYTES

Gene D. LeSage, Shannon S. Glaser, Heather Francis, Jo Lynne Phinizy and Gianfranco Alpini INTRODUCTION The aim of this chapter is to summarize recent findings that support the concept that the biliary epithelium is morphologically and functionally heterogeneous. The knowledge of cholangiocyte functions is rapidly accumulating largely because of technical advances and more investigative work that has led to the recognition that cholangiocytes are almost always either primarily or secondarily involved in human liver diseases (Alpini et al., 2002; Roberts et al., 1997). The development and introduction of experimental models, the identification and characterization of transport systems, and their second messenger systems has enhanced our understanding of cholangiocyte pathobiology. In this overview we will describe the morphology of intrahepatic bile ducts and their blood supply in the liver. Next, we will summarize overall bile duct function, and then link the structural differences between large and small ducts with their functional differences. We also will review the physiological advantages of having regional distribution in secretion and absorption in the biliary tree. We also will describe the models for bile duct injury, and the mechanisms underlying restricted size-dependent bile duct injury. Correlations to human liver diseases in which

The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 21–48 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15002-2

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injury is restricted to limited ranges of bile duct sizes will be examined. This will be followed by a review of the repair of bile ducts by cholangiocyte proliferation, and mechanisms by which bile duct structure is restored. And finally, we will review the capacity of cholangiocytes lining small bile ducts to differentiate, and consider the role pluripotent liver cells play within small bile ducts or closely adjacent to them in response to liver injury.

MORPHOLOGY Only a resum´e needs to be given here since this subject is dealt with fully in Chapter 1. Biliary epithelial cells (cholangiocytes) line the intra- and extrahepatic bile ducts of the liver (Alpini et al., 2002; Kanno et al., 2000; Ludwig, 1987; Marzioni et al., 2002; Sasaki et al., 1967; Schaffner & Popper, 1961). Bile duct structure comprises an anastomosing network of small bile ducts, which coalesce into progressively larger ducts (Alpini et al., 2002; Kanno et al., 2000; Ludwig, 1987; Marzioni et al., 2002). As these ducts become larger, the lining cholangiocytes change from cuboidal to columnar cell morphology (Sasaki et al., 1967; Schaffner & Popper, 1961). Progressively, larger ducts merge to form the hepatic duct, and then the common bile duct, which drains into the intestinal tract (Kanno et al., 2000; Ludwig, 1987; Marzioni et al., 2002). When the three-dimensional formation of the intrahepatic bile duct is complete (Masyuk et al., 2001), it closely resembles a tree, with the common and hepatic ducts corresponding to the trunk, the intrahepatic bile ducts corresponding to the large branches and the small ducts corresponding to the smallest tree limbs (Masyuk et al., 2001). The structure of the bile duct system is well suited for its primary function, which is to produce and transfer bile from the liver to the intestinal tract (Alpini et al., 1988, 2002; Cho et al., 1995; Cho & Boyer, 1999).

BILE FORMATION AND COMPOSITION Bile is composed of bile acids, cholesterol, phospholipids, bile pigments and inorganic electrolytes (Alpini et al., 2002; Tietz et al., 1995). Bile secretion is initiated by the movement of these substances from hepatocytes into the bile canaliculi (Nathanson & Boyer, 1991) and during the transfer from hepatocytes to the intestine. Bile is subsequently modified in the bile ducts by cholangiocytes (Alpini et al., 1988, 2002; Cho et al., 1995; Cho & Boyer, 1999; Kanno et al., 2000; Marzioni et al., 2002; Tietz et al., 1995). Three primary steps lead to bile generation. The first is active transport of bile acids from blood into bile canaliculi

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(Nathanson & Boyer, 1991). And the second is a canalicular, bile acid-independent secretion representing 30–60% of basal bile flow (Nathanson & Boyer, 1991). The third step in bile formation is absorption and secretion of fluid and inorganic electrolytes by bile ducts (Alpini et al., 1988, 2002; Tietz et al., 1995). Ductal bile flow is primarily regulated by the hormone secretin functioning to produce a bicarbonate-rich bile secretion (Alpini et al., 1989, 2002; Alvaro et al., 1993, 1997) and represents 30–40% of basal bile flow in humans, and 10% in rats (Alpini et al., 1989). Secretin stimulates ductal bile flow by increasing intracellular cyclic adenosine monophosphate (cAMP) levels (Kato et al., 1992; LeSage et al., 1996, 1999), which promotes biliary HCO− 3 secretion (Alpini et al., 1988, 1989, 1996; Alvaro et al., 1993, 1997; LeSage et al., 1996; Tietz et al., 1995), by stimulating apical cystic fibrosis transmembrane regulator (CFTR) Cl− channels (Alpini, Glaser et al., 1997; Fitz et al., 1993), in addition to the Cl− /HCO− 3 exchanger (Alpini et al., 1996; Alvaro et al., 1993, 1997; LeSage et al., 1996; Strazzabosco et al., 1991). For a comprehensive treatment of this subject, see Chapter 4 by Anwer.

ABNORMALITIES OF BILE DUCT FUNCTION Abnormalities of bile duct structure-function, or gene expression are the primary cause of liver diseases that target biliary epithelium such as in cystic fibrosis (CF), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and the idiopathic ductopenia syndrome (Alpini et al., 2002). In the other forms of liver disease (e.g. cirrhosis, and chronic hepatitis C), bile ducts are secondarily involved as the result of cell proliferation caused by chronic liver inflammation (Alpini et al., 2002). Present understanding of the pathophysiology of cholangiocytes in these disorders is rather limited or not infrequently non-existent.

HETEROGENEITY IN OTHER EPITHELIA Heterogeneity of epithelial cell function, reactions to injury or capacity to differentiate may depend upon the cells’ position within the structure of the organ (Cohn et al., 1992; Katz & Jungermann, 1993; Nielsen et al., 1993). Examples include: (i) differences in water permeability and expression of aquaporins in the epithelial cells lining the distal and proximal tubules in the kidney (Nielsen et al., 1993); (ii) differing absorptive and secretory capacity in cells along the villous crypt axis in intestinal epithelium (Cohn et al., 1992); and (iii) differing transport and metabolic capacities of hepatocytes in the periportal and perivenular zones

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in the liver (Katz & Jungermann, 1993). Different functions in various regions inside the organ are considered as physiologic advantages as well illustrated by the dependency of the urine concentrating ability of the kidney on the heterogeneity of kidney epithelial cells, and aquaporin expression in different tubular segments (Nielsen et al., 1993). Epithelial heterogeneity is also reflected in the response to disease as evidenced in liver injury. Damage of hepatocytes is limited to certain zones of the liver lobule (Katz & Jungermann, 1993).

Heterogeneity of Cholangiocytes During the last ten years, our interest has centered on the heterogeneity of cholangiocyte secretory functions, bile duct reactions to injury, and cholangiocyte proliferation and differentiation (Alpini et al., 1996, 1997, 1998, 2001, 2002; LeSage et al., 1999, 2001). We have been able to show that in large intrahepatic bile ducts (exceeding 15 ␮m in diameter) compared to small intrahepatic bile ducts (less than 15 ␮m) there are: (i) clearly distinguishable secretory functions (Alpini et al., 1996, 1997); (ii) different capacities for differentiation and degrees (Alpini et al., 2001); (iii) varying sensitivity to injury (LeSage et al., 1999, 2001); and (iv) different proliferative capacities (Alpini et al., 1998; LeSage et al., 1999, 2001). The overall view of the model for cholangiocyte heterogeneity that we have developed based on experimental findings is shown in Fig. 1. As it turns out, the fundamental difference between large and small intrahepatic bile duct function is attributable to differences in gene expression (Alpini et al., 1996, 1998; Alpini, Glaser, Robertson, Phinizy et al., 1997; Alpini, Glaser, Robertson, Rodgers et al., 1997), with large ducts expressing the secretin receptor (Alpini et al., 1996, 1998; Alpini, Elias et al., 1997; Alpini, Glaser, Robertson, Rodgers et al., 1997; LeSage, Glaser et al., 1999; LeSage, Alvaro et al., 1999; LeSage et al., 2001), apical CFTR Cl− channels (Alpini et al., 1997) and Cl− /HCO− 3 exchanger (Alpini et al., 1996). This difference results in only large intrahepatic bile ducts secreting fluid in response to secretin (Alpini, Glaser, Robertson, Phinizy et al., 1996; Alpini, Glaser, Robertson, Rodgers et al., 1997; Alpini et al., 1997). The function of small ducts, in terms of secretion, remains to be determined. Increased sensitivity of large bile ducts to injury is due to the differential expression of drug metabolizing enzymes, and pro-apoptosis proteins in large and small ducts (LeSage, Alvaro et al., 1999; LeSage, Benedetti et al., 1999; LeSage et al., 2001). This difference results in primarily large bile duct damage in response to toxic injury from the administration of carbon tetrachloride (CCl4 ) or ␣-naphthylisothiocyanate (ANIT) (LeSage, Benedetti et al., 1999; LeSage, Glaser et al., 1999; LeSage et al., 2001). Differences in cholangiocyte

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Fig. 1. Overall View of the Model for Cholangiocyte Heterogeneity. Note: Large intrahepatic bile ducts (>15 ␮m in diameter) secrete a bicarbonate rich fluid, are lined by cholangiocytes, which are the only cells in the liver that express secretin receptors and are sensitive to injury from either toxins or drugs. Small intrahepatic bile ducts (<15 ␮m in diameter) have an unknown secretory function, are resistant to drug or toxin induced liver injury and have the capacity to proliferate and differentiate with injury of large intrahepatic bile ducts.

proliferative capacity depend on the nature of bile duct injury. In the classic bile duct injury model, the bile duct ligated (BDL) rat, only large bile ducts proliferate (Alpini et al., 1998). In contrast, when large bile ducts are injured by CCl4 or ANIT administration, a loss of large bile duct secretory capacity is associated with small bile duct proliferation and de novo expression of the secretin receptor, apical CFTR Cl− channels and Cl− /HCO− 3 exchanger in small ducts, to compensate for the loss of large cholangiocyte functions (LeSage, Glaser, LeSage, Glaser, Alvaro et al., 1999; LeSage et al., 2001; Marucci et al., 1999). Proliferation of small cholangiocytes is also induced by the feeding of the bile acid, taurocholate, which also causes proliferation of large cholangiocytes (Alpini et al., 2001). We propose that cholangiocytes lining small ducts have the capacity to differentiate into secreting epithelium, primarily as a response to loss of large bile duct function.

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BILE DUCT STRUCTURE Intrahepatic bile ducts are lined by columnar cells, which posses a basement membrane (Benedetti et al., 1996; Schaffner & Popper, 1961). There is a conspicuous ER and Golgi as well as a vacuolar compartment, but they are less well developed compared to hepatocytes (Benedetti et al., 1996; Sasaki et al., 1967; Schaffner & Popper, 1961). The basolateral membrane has an underlying basement membrane (Benedetti et al., 1996; Schaffner & Popper, 1961). Functional tight junctions have been identified between cholangiocytes (LaRusso et al., 1991). The apical membrane has microvilli, thereby increasing the effective surface area (LaRusso et al., 1991). Intrahepatic bile ducts are classified by diameter: hepatic ducts (>800 ␮m), segmental ducts (400–800 ␮m), area ducts (300–400 ␮m), septal bile ducts (100–200 ␮m), interlobular ducts (15–100 ␮m), and bile ductules (<15 ␮m) (Kanno et al., 2000; Ludwig, 1987; Marzioni et al., 2002). Small bile ducts are lined by 4 to 5 cuboidal cholangiocytes and larger bile ducts consist of 10 to100 cholangiocytes (Kanno et al., 2000; Ludwig, 1987; Marzioni et al., 2002). In cholangiocytes lining large bile ducts, the Golgi apparatus is well developed and is located between the apical pole and the nucleus (Benedetti et al., 1996). In contrast, cholangiocytes lining small bile ducts have less cytoplasm and the nucleus/cytoplasm ratio is high (Benedetti et al., 1996). The different nucleus/cytoplasm ratio observed between small and large bile ducts (Benedetti et al., 1996) may represent cholangiocytes in large ducts being more differentiated cells, which express membrane receptor, transporters, and channels that are responsible for ductal secretion. In contrast, the smaller cytoplasm in small cholangiocytes suggests that small cholangiocytes may be undifferentiated primitive cells.

ASPECTS OF THE VASCULAR SUPPLY The intrahepatic bile ducts are nourished by the peribiliary plexus, which originates from the hepatic artery (Gaudio et al., 1996; Ohtani et al., 1983; Terada et al., 1989; Yamamoto & Phillips, 1984). The peribiliary plexus is most notable around large bile ducts, and less discernible around small bile ducts (Gaudio et al., 1996). Blood flowing through the peribiliary plexus enters the periportal sinusoids (Gaudio et al., 1996; Ohtani et al., 1983; Terada et al., 1989; Yamamoto & Phillips, 1984). These anatomic relationships have two potential physiologic implications. First, substances absorbed from intrahepatic bile ducts can be transferred back to the hepatic sinusoids, returned to hepatocytes, and then potentially secreted into bile (Gaudio et al., 1996; Yamamoto & Phillips, 1984). This exchange between cholangiocytes and hepatocytes has been termed “cholehepatic shunting” and

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has been suggested to occur for both bile acids and certain drugs (see below) (Hofmann, 1989). Second, blood flow is countercurrent to the direction of bile flow (Ohtani et al., 1983). Countercurrent bile and blood flow may be physiologically advantageous for bile formation (vid´e infra).

DUCTAL BILE SECRETION Studies using isolated cholangiocytes and isolated intrahepatic bile duct units (IBDU) have revealed the nature of the transport mechanisms underlying secretinstimulated ductal secretion (Alpini et al., 1996, 1998; Alpini, Glaser, Robertson et al., 1997; Alpini, Glaser, Rodgers et al., 1997; Cho & Boyer, 1999; Cho et al., 1995; Mennone et al., 1995; Roberts et al., 1993). Cholangiocytes modify canalicular bile by secretion of Cl− and HCO− 3 (Alpini et al., 1988, 1989, 1996, 1997; Alvaro et al., 1993, 1997; Fitz et al., 1993; LeSage et al., 1996; Mennone et al., 1995; Roberts et al., 1993). On the basolateral membrane, Na+ /H+ exchanger − and the Na+ : HCO− 3 symporter mediate HCO3 uptake (Alvaro et al., 1993), and on the apical membrane, cAMP-activated CFTR Cl− channel (Alpini et al., 1997; Fitz et al., 1993) and Cl− /HCO− 3 exchanger (Alpini et al., 1996, 1997; Alvaro et al., 1993, 1997; LeSage et al., 1996; Mennone et al., 1995; Roberts et al., 1993) secrete bicarbonate into the lumen. Both cAMP and Ca2+− activated Cl− channels are present on the apical membrane (Alpini et al., 1997; Fitz et al., 1993). Cholangiocyte secretory functions are increased by the hormones secretin (Alpini et al., 1988, 1989, 1996, 1997, 1998, 2002; Alvaro et al., 1993; Kato et al., 1992; LeSage et al., 1996), bombesin (Cho et al., 1995) and vasoactive intestinal peptide (VIP) (Cho & Boyer, 1999), whereas the gastrointestinal hormones somatostatin (Tietz et al., 1995), gastrin (Glaser et al., 1997) and insulin (LeSage et al., 2002) decrease secretion due to up and down regulation of cAMP, respectively (Alpini et al., 1996, 1997, 1998; Glaser et al., 1997; LeSage et al., 2002). ATP (Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998) and bile acids (Alpini et al., 1997, 1999, 2001) regulate ductal secretion by signaling events at the apical membrane of the cholangiocytes by the Na+ - dependent bile acid transporter, ASBT (Alpini et al., 1997; Lazaridis et al., 1997). It will be recalled that ATP signals through purinergic receptors located on the apical membrane of cholangiocytes (Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998). Regional Distribution of Bile Ductal Secretion Secretin-regulated secretion by cholangiocytes occurs exclusively in large bile ducts (>15 ␮m in diameter) and cholangiocytes (>13 ␮m in diameter) in rats

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(Alpini et al., 1996, 1997). Previous studies using rats have shown that an isolated cholangiocyte population rich in large cholangiocytes (>13 ␮m in diameter) derives principally from large intrahepatic bile ducts (Alpini et al., 1996 (see Fig. 2, upper panel). In sharp contrast, an isolated cholangiocyte population rich in small cholangiocytes (<8 ␮m in diameter) derives from small intrahepatic bile ducts (Fig. 2, lower panel). Large (but not small) cholangiocytes, and IBDU isolated from rats expressing sensitivity to secretin, somatostatin (SSTR2 ) receptors, and the Cl− /HCO− 3 exchanger respond to secretin and somatostatin by manifesting changes in cAMP levels (Alpini et al., 1996, 1997). As a direct demonstration of regionalization of secretory events in the biliary tree, large but not small isolated intrahepatic IBDU respond to secretin by showing an increase in duct lumen (Alpini et al., 1997). Similar to rats, human bile duct secretion is also regional in distribution since only large-size bile ducts express the Cl− /HCO− 3 exchanger (Martinez-Anso et al., 1994). Although one cannot draw a direct correlation between IBDU size reported in our previous study and the bile duct diameter in human liver sections, as defined by Ludwig’s classification (Ludwig, 1987), it would seem reasonable to suggest that the small IBDU would best be characterized as ductules in the Ludwig classification and the larger IBDU would represent the interlobular ducts (Kanno et al., 2000). The exclusivity of secretion to only large ducts is possibly due to the presence of the peribiliary plexus, the vascular element for cholangiocytes being limited to ducts greater than 15 ␮m in diameter (Gaudio et al., 1996).

Regulatory Hormones and Neurotransmitters Secretin, bombesin and VIP increase ductal secretion (Alpini et al., 1988, 1989, 1996, 1997, 2002; Cho & Boyer, 1999; Cho et al., 1995; Kato et al., 1992). Insulin, gastrin and somatostatin decrease cholangiocyte secretion (Glaser, 1997; LeSage, 2002; Tiets, 1995). Acetylcholine (ACh) and ␣−1 adrenergic agonists potentiate (Alvaro et al, 1997; LeSage et al., 2001), whereas insulin, dopamine and −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Fig. 2. [Upper Panel] Small [A] and Large [B] IBDU were Isolated as Described in Materials and Methods and Cultured in MEM for 12–24 Hours at 37 ◦ C. Note: A lumen is clearly visible (arrow), with surrounding epithelial cells lining the duct lumen. The photomicrogram was obtained with DIC optics to enhance image contrast and, due to a narrow depth of focus, provides a clear outline of the duct lumen. Original magn., X2100. [lower panel] Frequency distribution of diameters of small and large cholangiocytes purified by counterflow elutriation and immunoaffinity separation from normal rat liver (A–D). Note that cells differ in size and morphological appearance (A–B). Orig. magn., X625.

Functional Heterogeneity of Intrahepatic Cholangiocytes

Fig. 2.

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endothelin agonists inhibit (Caligiuri et al., 1998; Glaser et al., 2003; LeSage et al., 2002) secretin-stimulated ductal secretion. Secretin, gastrin, and somatostatin receptors are present only in large cholangiocytes (Alpini et al., 1996, 1997, 1998), whereas endothelins ETA and ETB receptors are expressed by both small and large cholangiocytes (Caligiuri et al., 1998). Secretin binds to secretin receptor on the basolateral membrane of cholangiocytes (Alpini et al., 1994) resulting in increased cAMP levels (Alpini et al., 1996, 1997; Kano et al., 1992). Bombesin and VIP increase duct secretion independent of cAMP synthesis (Cho et al., 1995; Cho & Boyer, 1999) and the origin of ductal choleresis (small vs. large ducts) is unknown. In vivo, secretin induces a much larger bicarbonate-rich chloresis in rats with enhanced ductal hyperplasia induced by BDL (Alpini et al., 1988; Glaser et al., 1997; Tietz et al., 1995), cirrhosis (Alpini et al., 1997), or chronic ANIT feeding (LeSage et al., 2001) compared to normal rats where secretin-induced choleresis is minimal (Alpini et al., 1988, 1989; LeSage, 1996). Similarly, bombesin and VIP increase bile flow and bicarbonate to a much greater degree in BDL rats compared to controls (Cho et al., 1995; Cho & Boyer, 1999). Increased secretion in response to secretin, bombesin and VIP is consistent with an increased number of cholangiocytes in BDL rats producing more ductal bile flow (Alpini et al., 1988, 1989). Insulin, gastrin and somatostatin inhibit the expression of secretin receptor and secretin-stimulated cAMP synthesis, thus preventing secretin’s choleric effects (Glaser et al., 1997; LeSage et al., 2002; Tietz et al., 1995). Both gastrin and insulin increase intracellular Ca2+ and PKC-␣ activity in cholangiocytes (Glaser et al., 1997; LeSage et al., 2002), which are required for inhibition of secretin-stimulated cAMP synthesis and ductal secretion. ACh enhances secretin-stimulated ductal bile secretion through interaction with M3 ACh receptor subtypes on cholangiocytes (Alvaro et al., 1997). ACh increases secretin-stimulated (but not basal) activity of the Cl− /HCO− 3 exchanger in IBDU and secretin-stimulated cAMP synthesis in isolated cholangiocytes (Alvaro et al., 1997). The potentiation of secretinstimulated ductal bile secretion is dependent on Ca2+ but not PKC (Alvaro et al., 1997). FK-506 and cyclosporine inhibit ACh potentiation of secretin-stimulated Cl− /HCO− 3 exchanger, demonstrating that calcineurin most likely mediates the cross-talk between calcium and adenylyl cyclase pathways (Alvaro et al., 1997).

Paracrine and Autocrine Control of Ductal Secretion Extracellular ATP and adenosine have well defined roles as stimulants of electrolyte and fluid secretion in bile ducts (Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998). In cholangiocytes, ATP binds to apical P2Y2 (P2U)

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receptors (Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998). ATP, released from hepatocytes, is capable of stimulating receptors on both adjacent hepatocytes and after reaching the biliary tree, stimulating receptors on the apical domain of cholangiocytes (Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998). Thus, ATP may play a role in coordinating the hepatocyte and ductal components of bile formation, a process that has been termed “hepatobiliary coupling” (Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998). ATP in bile is a potent stimulus for cholangiocyte Cl− and fluid secretion and activates basolateral NHE (Elsing et al., 1996; Zsembery et al., 1998). ATP also increases Cl− /HCO− 3 exchanger activity in cholangiocytes pretreated with cAMP analogs (Melero et al., 2002; Strazzabosco et al., 1997). Consequently, the release of ATP from cholangiocytes may regulate secretion of cholangiocytes downstream (paracrine signaling). It remains to be determined if cholangiocytes lining small bile ducts can release ATP and signal downstream cholangiocytes in large ducts, thus providing a mechanism for the regulation of ductal secretion by cross-talk between small and large cholangiocytes. The presence of multiple stimulatory (i.e. secretin, bombesin, vasoactive, ACh, and ATP) (Elsing et al., 1996; Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998) and inhibitory (i.e. endothelin, insulin, gastrin and somatostatin) (Alpini et al., 1998; Caligiuri et al., 1998; Glaser et al., 1997; LeSage et al., 2002; Tietz et al., 1995) stimuli to cholangiocyte secretion likely reflects the need for fine regulation of cholangiocyte secretion from both circulatory and nerve inputs. The presence of these hormone receptors primarily in large bile ducts supports the concept that ductal secretion originates primarily from large ducts (Alpini et al., 1996, 1997).

BILE ACID TRANSPORT Until recently it was assumed that bile acids, after hepatocyte secretion, were simply conducted to the intestine by bile ducts (Hofmann, 1989). Studies with bile duct ligated rats suggest that bile acids may move across the biliary epithelium (Lamri et al., 1992). The absorbed bile acids return via the peribiliary plexus to the hepatocytes for secretion into bile (Gurantz & Hofmann, 1984; Palmer et al., 1987). This shunting of bile acids back and forth between hepatocytes and cholangiocytes has been termed “cholehepatic shunting” (Gurantz & Hofmann, 1984; Palmer et al., 1987). More recently, interest in bile acid transport in bile ducts has risen sharply due to the identification of bile acid transport in cholangiocytes (Alpini, Glaser, Rodgers et al., 1997; Lazaridis et al., 1997). Studies by us (Alpini et al., 1997)

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showed genetic and protein expression for the apical Na+ -dependent ASBT and the 14-KD ileal cytosolic binding protein (IBABP) in cholangiocytes. ASBT is structurally identical to the ileal bile acid transporter which is the sole transporter involved in the reclamation of bile acids from the ileum (Aldini et al., 1992). ASBT is expressed in large but not small (less than 15 ␮m) bile ducts (Alpini et al., 1997). We have suggested that the presence of ASBT in small ducts would counterpoise secretion of bile acids at the level of the canalicular membrane, thus reducing overall bile acid-induced bile flow (Alpini et al., 1997). Bile acids interact with cholangiocytes, both in vitro (Alpini, Glaser, Robertson, Phinizy, Rodgers et al., 1997) and in vivo in bile acid fed animals (Alpini, Glaser, Ueno et al., 1999; Alpini, Ueno et al., 2001) resulting in cholangiocyte proliferation and increases in ductal bile secretion of cholangiocytes (Alpini, Glaser, Robertson, Phinizy, Rodgers et al., 1997; Alpini, Glaser, Ueno et al., 1999; Alpini et al., 2001). Corresponding to the presence of ASBT only in large cholangiocytes (Alpini et al., 1997), bile acids stimulate proliferation and secretion only in large cholangiocytes in vitro (Alpini et al., 1997). This observation is probably ascribable to the requirement of bile acid uptake by ASBT, since only intracellular bile acids can signal cholangiocyte proliferation and secretion (Alpini et al., 1997). Chronic feeding of taurocholate and taurolithocholate induces de novo expression of ASBT, and activation of proliferative and secretin-stimulated secretory capacity of small cholangiocytes (Alpini et al., 2001), which normally do not express ASBT (Alpini et al., 1997) and are unresponsive to secretin (Alpini et al., 1996, 1997) and mitotically dormant (Alpini et al., 1998).

Physiologic Advantages of Cholangiocyte Heterogeneity in Ductal Secretion Limiting secretin-stimulated ductal secretion only to large ducts (Alpini et al., 1996, 1997) may provide the liver with a number of theoretical physiological advantages. First, secretion from large ducts might provide a washing effect to prevent retention of precipitable material or mucus proteins in larger intrahepatic bile ducts. Second, limited secretion in large ducts theoretically takes advantage of the counterflow effect of the opposite direction of blood and bile flow within the biliary system. With blood flow directed from large to small ducts, concentration gradients created between large ducts and the circulation due to secretion would not be dissipated in small ducts, which do not express secretory transporters (Alpini et al., 1996, 1997). Third, enhanced cholehepatic shunting of solutes (e.g. bile acids) would be more effective since bile acids absorbed by large bile ducts (Alpini et al., 1997) could enter the peribiliary plexus, where small ducts, with a minimal

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peribiliary plexus could not participate in cholehepatic shunting. These concepts concerning physiologic advantages of cholangiocyte heterogeneity are conjectural in nature and need to be experimentally tested.

Cholangiocyte Proliferative and Repair Responses Whereas cholangiocytes are mitotically dormant (Alpini et al., 1998; LeSage et al., 1996), they proliferate in most human liver diseases (Alpini et al., 2002). They proliferate following experimental bile duct injury caused by BDL (Alpini et al., 1988, 1989) or ANIT feeding (Alpini et al., 1988; Kossor et al., 1995; LeSage et al., 2001). Cholangiocytes also proliferate following partial hepatectomy, and restore bile duct mass to normal within one week (LeSage et al., 1996). Thus far, all models of cholangiocyte proliferation show an associated increase in ductal secretion (Alpini et al., 1988, 1989, 1999; LeSage et al., 1996, 2001).

Regional Distribution of Bile Duct Proliferation In the intrahepatic biliary epithelium, there are specific compartments from which cholangiocytes proliferate (i.e. small and large sized ducts) and that differentially respond to injury, hepatic toxins or diets (Alpini et al., 1998; LeSage, Benedetti et al., 1999; LeSage, Glaser et al., 1999; LeSage et al., 2001). Following BDL, large cholangiocytes existing in large bile ducts proliferate, thus resulting in a 10 to 20-fold increase in the number of intrahepatic bile ducts (Alpini et al., 1998). By contrast, following partial hepatectomy, the regrowth of bile ducts occurs as the result of proliferation of both small and large bile ducts (LeSage et al., 1996). Administration of a single dose of CCl4 to normal or BDL rats and chronic ANIT feeding to normal rats induces a transient reduction of bile ducts (ductopenia) due to cholangiocyte loss (by apoptosis) (LeSage, Glaser et al., 1999; LeSage, Penedetti et al., 1999; LeSage et al., 2001). In large bile ducts, there is a loss of secretory and proliferative activities, whereas small bile ducts are more resistant to CCl− 4 and ANIT-induced damage. They proliferate and secrete to compensate for loss of large cholangiocyte function. The differential response of small and large ducts to CCl4 is likely due to the presence of cytochrome P450 2E1, the enzyme that initiates CCl4 hepatoxicity (Clawson et al., 1989; Handler & Goldstein, 1996) in large but not small cholangiocytes (LeSage, Benedetti et al., 1999; LeSage, Glaser et al., 1999). Variable sensitivities of large and small ducts to other forms of injury or carcinogens may be due to differential expression of other enzymes or proteins in large and small cholangiocytes. Phase I or mixed-function oxygenase

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enzymes e.g. microsomal cytochrome P-450, aminopyrine-N-demethylases, G-6 PO4 , and NADPH cytochrome C reductase, and phase II or glutathione redox cycle enzymes (e.g. GSH-peroxidase, UDP-glucuronosyltransferase, and glutathioneS-transferase), and drug-metabolizing enzymes are heterogeneously expressed by cholangiocytes (Lakehal et al., 1999; LeSage et al., 1999; Mathis et al., 1989). Proliferative stimuli can trigger a focal proliferative response in other epithelia besides cholangiocytes. For example, with prolonged hormonal stimulation, replication of pancreatic cells is restricted to intralobular but not interlobular ducts (Elsasser et al., 1990). EGF-induced proliferation in the kidney is restricted to proximal tubular cells (Han et al., 2002). In the liver, periportal hepatocytes have a greater proliferative capacity than perivenous hepatocytes following partial hepatectomy (Lee et al., 1998). It could well be that the differential blood supply with possibly its circulating growth factors such as vascular endothelial growth factor (VEGF) may be considered as an additional explanation for the differential proliferation of small and large ducts. Keeping in mind that the peribiliary plexus surrounds solely large ducts, proliferation of the plexus occurs only around the large ducts following BDL (Gaudio et al., 1996). It therefore has been postulated that VEGF which is released by only large cholangiocytes supports proliferation of large bile ducts after BDL and also (by a paracrine mechanism) supports the growth of the peribiliary plexus lying adjacent to the large ducts. This implies that small cholangiocytes do not play a role in the proliferation of adjacent blood vessels, which is consistent with the expansion of small bile ducts in the BDL model. When hepatocyte regeneration in the experimental animal is impaired, small bile ducts proliferate and invade adjacent hepatocyte parenchyma. These ductal cells are referred to as oval cells; their association with defective regeneration has led to the view that they are the progeny of facultative stem cells (Alison et al., 1998).

Cholangiocyte Apoptosis Apoptosis or programmed cell death is activated in an organism as a defense mechanism against the accumulation of damaged cells (Guicciardi & Gores, 2002). Apoptosis is considered an important mechanism in cholangiocyte death, leading to ductopenia (Guicciardi & Gores, 2002). In ANIT or CCl4 treated rats, the triggering of small bile duct proliferation may depend on the presence of cholangiocyte apoptosis in these models (LeSage, Benedetti et al., 1999; LeSage, Glaser et al., 1999; LeSage, Glaser, Ueno et al., 2001), whilst the failure of proliferation of small bile ducts to occur in BDL rats may be due to the absence of cholangiocyte apoptosis in this model (LeSage, Glaser, Ueno et al., 1999).

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It is now known that bile acids are cytoprotective, and anti-apoptotic in relation to cholangiocytes. For example, after feeding taurocholate to BDL, CCl4 -treated rats (Marucci et al., 2003), small cholangiocytes [which are de novo activated in this model of CCl4 -induced damage of large ducts (LeSage, Benedetti et al., 1999; LeSage, Glaser et al., 1999)] are found to be mitotically quiescent and unresponsive to secretin. Studies done in vitro using large cholangiocytes obtained from BDL rats show that taurocholate stops the inhibitory effects of CCl4 on apoptotic, proliferative and secretory capacities of large cholangiocytes from occurring (Marucci et al., 2003). Taurocholate protects against the effects of apoptosis and loss of function in large cholangiocyte. These functions depend on the activation of PI3-K and AKT expression (Marucci et al., 2003). As will be recalled, Bcl-2 modulates apoptosis in the liver and is upregulated in BDL rats (Kurosawa et al., 1997), an effect that protects hepatocytes from bile salt induced apoptosis (Kurosawa et al., 1997). Liver Bcl-2 is particularly expressed in cholangiocytes. This observation has led Que et al. (1997) to postulate that Bcl protects cholangiocytes against exposure to bile salt in high concentrations. It is also noteworthy that Bcl-2 expression in small bile ducts exceeds that in larger ducts, suggesting the presence of greater resistance in smaller ducts to apoptosis (Charlotte et al., 1994). A parallel situation exists in respect of annexin-V in murine liver: it is expressed mainly by small intrahepatic bile ducts and plays a role in the regulation of apoptosis (Diakonova et al., 1997). Such findings could partly account for the higher resistance found in small ducts to agents/drugs that induce apoptosis. Cholangiocyte Heterogeneity: Non-transport Related Proteins In normal and cholestatic human liver bile ducts, whether hepatic, segmental, area, or septal, and peribiliary glands express pancreatic enzymes such as lipase, ␣-amylase, and trypsin (Terada et al., 1992, 1994). The human bile duct system has its own pattern of blood group antigen expression with sialylated Lewisa antigens present primarily in large septal bile ducts (Okada et al., 1988). Very recently, the microarray technique has been used to demonstrate the existence of over 80 other proteins that are differentially expressed by large and small cholangiocytes (Ueno et al., 2003). Both the physiological and pathological significance of differential expression of this wide variety of proteins has not yet been explored.

DISEASE STATES Damage to bile ducts causes several chronic cholestatic disorders (cholangiopathies) (Alpini et al., 2002; Roberts et al., 1997). In these cholangiopathies,

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there often coexists cholangiocyte death and proliferation, ductal remodeling, inflammation and fibrosis (Alpini et al., 2002; Roberts et al., 1997). Cholestasis is almost always present and may be the cause or promoter for the progression of the disease (Alpini et al., 2002; Roberts et al., 1997). CF is an example of biliary cirrhosis secondary to a dysfunction of cholangiocyte ion transport (Alpini et al., 2002; Roberts et al., 1997). Thus, dysfunctional biliary electrolyte transport may also promote the cholestasis in other cholangiopathies (Alpini et al., 2002; Roberts et al., 1997). Most primary cholangiopathies appear to be due to an autoimmune induced process (Alpini et al., 2002; Roberts et al., 1997). Cytokines and proinflammatory mediators likely induce apoptotic and proliferative responses in cholangiocytes, activate fibrogenesis, and alter the transport functions of cholangiocytes (Alpini et al., 2002; Roberts et al., 1997). Cholangiopathies differentially affect the biliary epithelium, leading to selective alteration and destruction of specific sized ducts (Alpini et al., 2002; Roberts et al., 1997).

Primary Biliary Cirrhosis PBC is the prototypic disease in humans of bile duct damage (Roberts et al., 1997). PBC is characterized by spotty rather than diffuse proliferation/loss of certain sized ducts (i.e. small interlobular bile ducts) (Alpini et al., 2002; Roberts et al., 1997). The etiology of PBC remains elusive. Current studies suggest that the interlobular bile duct destruction is immune based, and commonly associated with autoimmune diseases (Alpini et al., 2002; Roberts et al., 1997). Patients with PBC have autoantibodies that interreact with components of mitochondrial multi-enzyme complexes (Alpini et al., 2002; Roberts et al., 1997). In addition to binding to mitochondria, autoantibodies in the patients are thought to be against the autoantigen pyruvate dehydrogenase complex (PDC) dihydrolipoamide acetyltransferase (E2) which bind to the plasma membrane of cholangiocytes specifically in PBC (Alpini et al., 2002; Jones et al., 1995; Roberts et al., 1997).

Primary Sclerosing Cholangitis PSC is associated with inflammation and prominent fibrosis of intrahepatic and extrahepatic bile ducts (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). The sclerosis of the bile ducts may be the result of multiple factors, including autoimmune, bacterial, congenital, drug, or viral agents (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al.,

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1997). The etiology of PSC remains poorly understood, despite a large number of studies evaluating differing hypotheses. PSC has a propensity to primarily affect extrahepatic and large bile ducts in the liver, although a small duct variant of PSC has been described where the large ducts are spared (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997).

Cholangiocarcinoma Cholangiocarcinoma occurs somewhat frequently in patients with PSC (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). Cholangiocarcinoma has a strong predilection for involving the major bile duct bifurcation (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). Cholangiocarcinoma arising out of small bile ducts (peripheral cholangiocarcinoma) is rare (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). Cholangiocarcinoma is not seen in patients who have a small duct variant of PSC (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). Most of the other risk factors for cholangiocarcinoma have long-standing inflammation and injury of cholangiocytes (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). p53 overexpression and K-ras mutations occur commonly in patients with PSC and biliary tract cancer and are associated with a shortened life survival (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). The mechanisms responsible for the control of cholangiocarcinoma growth are poorly understood. However, there are studies showing that gastrin (by increasing PKC-␣) (Kanno et al., 2001) and ␣−2 adrenergic stimulation (through modulation of Raf-1 and B-Raf activities) (Kanno et al., 2002) are able to inhibit the growth of cholangiocarcinoma.

Cystic Fibrosis Although CF is primarily considered as a pulmonary disease, liver disease has been increasingly diagnosed during recent years, probably due to an increased suspicion that there is a connection between the two entities (Alpini et al., 2002; Roberts et al., 1997). Given that data assessing the effects of defective CFTR on cholangiocyte biology have not yet been obtained, it seems likely that impaired secretory function of cholangiocytes maybe responsible for reduced bile flow and alkalinity (Alpini et al., 2002; Roberts et al., 1997). Although a state of functional CFTR is absent or reduced in large bile ducts in the liver (Alpini et al., 2002;

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Roberts et al., 1997), there is also the distinct possibility that other Cl− channels (e.g. Ca2+ - dependent) (Roman et al., 1999; Schlenker et al., 1997) are present in both small and large cholangiocytes, and that these channels act to maintain bile duct function by substituting for CFTR activity. However, to date, no clear association between specific CFTR mutations and the presence of liver disease has been observed. Treatment with ursodeoxycholic acid, aimed at improving biliary secretion in terms of bile viscosity and bile acid composition, is currently the most effective therapeutic modality in CF-associated liver disease (Paumgartner & Beuers, 2002).

Polycystic Kidney Liver Disease (PKLD) In autosomal dominant PKLD the genetic defect results in the slow growth of multiple epithelial cysts within the renal and liver parenchyma (Perrone et al., 1997). Cysts appear in the intrahepatic biliary tree in PKLD (Perrone et al., 1997). The abnormality appears to develop out of large bile ducts since the cystic ductal cell also secretes Cl− and HCO− 3 like normal large cholangiocytes but secretion is diminished, probably as a result of reduced Cl− /HCO− 3 exchanger activity in the apical membrane of cystic ductal cells as compared with the normal cholangiocytes (Perrone et al., 1997).

Biliary Atresia Biliary atresia is a destructive, inflammatory process of the intrahepatic and extrahepatic bile ducts, which leads to obliteration of the biliary tract and to biliary cirrhosis. It is the most common cause of cholestasis in infants and children. The pathogenesis of biliary atresia is unknown. When larger ducts are involved in biliary atresia the prognosis is poor. Most recent studies have focused on dysregulation of ductal morphogenesis and environmental factors (viruses or metabolic insults) in combination with genetic or immunologic susceptibility. Reovirus type 3 and Group C rotavirus have been implicated in biliary atresia. A dysregulation of ductal morphogenesis is supported by the frequent coexistence with other developmental anomalies, particularly visceral organ symmetry. This association suggests abnormalities in developmental genes that cause the failure of the ductal remodeling process at the hilum. Recently, a mouse model with insertional mutation in the proximal region of mouse chromosome 4 has been described with features of biliary atresia and abdominal situs inversus.

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POTENTIAL FUNCTIONS OF SMALL CHOLANGIOCYTES We propose that small ducts act as a reserve cell population in the liver, which is ready to respond to injury. Following bile duct injury, small ducts proliferate to provide compensatory secretory function, whereas large ducts undergo repair (LeSage et al., 1999, 2001). Other investigators have proposed that small ducts function as progenitor cells for biliary cells and hepatocytes (Theise et al., 1999). Upon injury, this cell may be activated to proliferate and produce progeny, which can differentiate into either bile duct cells or hepatocytes (Theise et al., 1999). Although our studies with CCl4 , ANIT and partial hepatectomy failed to show that activation of small cholangiocytes produced hepatocyte precursor cells (e.g. expression of ␣-fetoprotein or albumin), other studies using the allyl alcohol model, Solt-Farber model and furan induced liver injury, show that cells within small ducts or closely adjacent to small ducts have the potential to differentiate into either bile duct cells and hepatocytes or develop into neuroendocrine cells, intestinal-type adenocarcinomas, hepatocellular carcinomas and cholangiocarcinomas (Edakuni et al., 2001; Factor et al., 1994; Novikoff & Yam, 1998; Pack et al., 1993; Petropoulos et al., 1985; Sell, 1990). These studies show that small cholangiocytes (or cells closely adjacent to small ducts) have great plasticity with capacities to differentiate into a variety of cell types (LeSage et al., 1999). Most of the secretory functions of cholangiocytes appear to be limited to cholangiocytes lining large bile ducts (Alpini et al., 1996, 1997, 1998), yet small ducts may also secrete based on mechanisms unrelated to cAMP/CFTR. Small cholangiocytes are rich in cytoplasmic vesicles (Benedetti et al., 1996) and vesicledependent fluid secretion is present in cholangiocytes (Marinelli et al., 1997, 1999). Annexin-V is also present in small cholangiocytes (Katayanagi et al., 1999); it has been shown to play a role in cytoskeletal-dependent vesicular transport. Small cholangiocytes have been shown to express ETA and ETB (Caligiuri et al., 1998). Since endothelin exerts its functions through activation of Ca2+ (Pinzani et al., 1996), we propose that small cholangiocytes participate in ductal bile secretory activity through a Ca2+ -regulated pathway that is cAMP-independent (Alpini et al., 1996; Alpini, Glaser, Robertson, Phinizy et al., 1997; Alpini, Glaser, Robertson, Rodgers et al., 1997; Glaser et al., 1997, 2003; LeSage, Alvaro, Benedetti et al., 1999; LeSage, Benedetti et al., 1999; LeSage et al., 1996, 2001) (e.g. calciumdependent Cl− channel). If this is the case, then cholangiocytes sequentially modify bile in spatially separate processes as it passes through the axis of the biliary tree.

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SUMMARY We have summarized the recent findings that demonstrate that cholangiocytes are heterogeneous with regard to morphology, secretory activity to gastrointestinal hormones/peptides and bile salts and proliferative/apoptotic responses to liver injury/toxins. On the basis of these findings, we propose a scheme for mapping the morphological, secretory, proliferative and apoptotic events in the intrahepatic biliary tree (Fig. 3). This model proposes that bile ducts are morphologically heterogeneous with small ducts lined by small cholangiocytes, and large ducts

Fig. 3. Mapping of the Morphological, Secretory, Proliferative, and Apoptotic Events in the Intrahepatic Biliary Tree. Note: This model proposes that bile ducts are morphologically heterogeneous with small ducts (on the left) lined by small cholangiocytes and large ducts (on the right) lined by large cholangiocytes. Large but not small bile ducts express secretin and somatostatin receptors, CFTR and Cl− /HCO− 3 , and respond to these two hormones with changes in ductal secretion in both normal and BDL rats. Small and large ducts differentially proliferate in response to BDL, partial hepatectomy, and CCl4 administration. In BDL rats, only the large ducts proliferate; in partial hepatectomy, both the small and large ducts proliferate, and in CCl4 -treated rats only the small bile ducts proliferate promptly. Cholangiocyte apoptosis is not observed in BDL rats, while in CCl4 -treated rats, cholangiocyte apoptosis is confined to the large bile ducts.

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lined by large cholangiocytes. Large but not small bile ducts, express secretin and somatostatin receptors, CFTR, and Cl− /HCO− 3 , and respond to these two hormones with changes in ductal secretion. Pathologically, small and large ducts respond differentially to specific injury/toxins. Following BDL, only large cholangiocytes proliferate, whereas following CCl4 administration, damage and loss of large duct function leads to de novo proliferation and secretion of small cholangiocytes (resistant to CCl4 ) in order to compensate for the loss of large duct function. In partial hepatectomy, both small and large cholangiocytes respond with increases in proliferation and secretion. The presence of cholangiocyte heterogeneity may well endow the biliary system with physiological advantages involving bile secretion, cholehepatic shunting, and countercurrent bile and blood flow. Finally, human cholangiopathies differentially target the small and large ducts leading to cholangiocyte proliferation/loss.

FUTURE DIRECTIONS Further studies are needed to determine why cholangiopathies are restricted to specific sized ducts. The observations of cholangiocyte heterogeneity in rat models need to be extended to include animal models that more closely resemble normal human biliary function under normal conditions and in cholangiopathies in disease. Further studies are also necessary for evaluating the role of small ducts in the overall contribution of ductal secretion during normal bile formation and as a compensatory role in diseases in which ductal bile secretion is reduced (e.g. CF). In ductopenia, the repair of bile ducts becomes critical, yet the mechanisms for duct morphogenesis are unknown. The roles of cholinergic, adrenergic, dopaminergic and serotoninergic innervation regulating the heterogeneous responses of bile ducts to agonists, injury/toxins and viruses may also be critical to the maintenance of bile duct structure after injury. Since microvascular proliferation may be important in repair of bile ducts after injury, studies are needed to evaluate the role of the blood supply and circulating factors (e.g. VEGF) in the regulation of cholangiocyte function. Since some cholangiopathies (e.g. PBC) likely develop by an immunological mechanism, it may be relevant to study the heterogeneous expression of specific antigens, which may regulate the pathogenesis of these immune mediated cholangiopathies.

ACKNOWLEDGMENTS Portions of the findings presented here were supported by a grant award to Dr. Alpini from Scott & White Hospital and Texas A&M University, by an NIH

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grant DK58411 and by VA Merit Award to Dr. Alpini, and by an NIH grant DK 54208 to Dr. LeSage.

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APPENDIX ANIT = ␣-naphthylisothiocyanate BDL = bile duct ligation CCl4 = carbon tetrachloride cAMP = adenosine 3 , 5 -monophosphate CF = cystic fibrosis CFTR = cystic fibrosis transmembrane regulator IBDU = intrahepatic bile duct units PBC = primary biliary cirrhosis PSC = primary sclerosing cholangitis