Heterogeneity of the intrahepatic biliary epithelium

Abstract

Heterogeneity of the intrahepatic biliary epithelium

Shannon Glaser1, Heather Francis2, Sharon DeMorrow3, Gene LeSage4, Giammarco Fava5, Marco Marzioni6, Julie Venter7, Gianfranco Alpini8 

1Department of Medicine, Division of Research and Education, Scott & White Memorial Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TX, United States
2Division of Research and Education, Scott & White Memorial Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TX, United States
3, 4University of Texas at Houston Medical School, Houston, TX. United States
5, 6Department of Gastroenterology, Università Politecnica delle Marche, Azienda Ospedaliera “Ospedali Riuniti di Ancona”, Ancona, Italy
7Department of Medicine, Scott & White Memorial Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TX, United States
8Central Texas Veterans Health Care System, Department of Medicine, Systems Biology and Translational Medicine, Scott & White Memorial Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TX, United States

Supported by a grant award from Scott & White Hospital and The Texas A&M University System Health Science Center, a VA Merit Award, a VA Research Scholar Award and the NIH grants DK58411 and DK062975 to Dr. Alpini, by grant awards to Shannon Glaser and Heather Francis from Scott & White Hospital.

Correspondence to: Shannon Glaser, MS, Department of Medicine, Division of R&E, Scott and White Memorial Hospital and The Texas A&M University System Health Science Center College of Medicine, MRB, 702 South West H.K. Dodgen Loop, Temple, Texas 76504, United States. sglaser@neo.tamu.edu


Abstract

The objectives of this review are to outline the recent findings related to the morphological heterogeneity of the biliary epithelium and the heterogeneous pathophysiological responses of different sized bile ducts to liver gastrointestinal hormones and peptides and liver injury/toxins with changes in apoptotic, proliferative and secretory activities. The knowledge of biliary function is rapidly increasing because of the recognition that biliary epithelial cells (cholangiocytes) are the targets of human cholangiopathies, which are characterized by proliferation/damage of bile ducts within a small range of sizes. The unique anatomy, morphology, innervation and vascularization of the biliary epithelium are consistent with function of cholangiocytes within different regions of the biliary tree. The in vivo models [e.g., bile duct ligation (BDL), partial hepatectomy, feeding of bile acids, carbon tetrachloride (CCl4) or a-naphthylisothiocyanate (ANIT)] and the in vivo experimental tools [e.g., freshly isolated small and large cholangiocytes or intrahepatic bile duct units (IBDU) and primary cultures of small and large murine cholangiocytes] have allowed us to demonstrate the morphological and functional heterogeneity of the intrahepatic biliary epithelium. These models demonstrated the differential secretory activities and the heterogeneous apoptotic and proliferative responses of different sized ducts. Similar to animal models of cholangiocyte proliferation/injury restricted to specific sized ducts, in human liver diseases bile duct damage predominates specific sized bile ducts. Future studies related to the functional heterogeneity of the intrahepatic biliary epithelium may disclose new pathophysiological treatments for patients with cholangiopathies.


Key words: cAMP; Gastrointestinal hormones; Growth factors; Mitosis; Nerves

 

Source: World J Gastroenterol 2006; 12(22): 3523-3536


Anatomical and morpholical characteristicsof the biliary epithelium

 
Two kinds of epithelial cells, hepatocytes and cho-langiocytes, are present in the liver[1-3]. While hepatocytes initially secrete bile into the bile canaliculus[4], cholangiocytes modify bile of canalicular origin by a series of coordinated spontaneous and hormone/peptide regulated secretion/reabsorption of water and electrolytes before it reaches the small intestine[3,5-7]. For more information on the mechanisms of bile formation we refer to recent reviews[4,5]. The human biliary system is divided into extrahepatic bile ducts and intrahepatic bile ducts, the latter further sub-divided into large and small bile ducts[2,3,8]. The intrahepatic bile ducts represent that part of the biliary tree proximal to the confluence of the hepatic ducts[9]extending from the canals of Hering to the large extrahepatic ducts[2,3,8]. In human liver, a study by Ludwig classified the intrahepatic bile duct system upon duct diameter[8], small bile ductules (< 15 mm), interlobular ducts (15-100 mm), septal ducts (100-300 mm), area ducts (300-400 mm), segmental ducts (400-800 mm) and hepatic ducts (> 800 mm)[8] (Table 1). Small ductules are lined by 4-5 cholangiocytes, have a basement membrane, tight junctions between cells and microvilli projecting into the bile duct lumen[10,11]. Cholangiocytes are progressively larger and more columnar in shape in larger bile ducts (lined by 10-12 cholangiocytes)[10,11].
 
In rats, morphological studies in liver sections and small and large intrahepatic bile duct units (IBDU) have shown[2,12-14] that the intrahepatic biliary tree is divided into: (1) small ducts (< 15 mm in external diameter) lined by small cholangiocytes (approximately 8 mm in diameter)[12,13]; and (2) and large ducts (> 15 mm in diameter) lined by large cholangiocytes (approximately 15 mm in diameter)[12,13] (Figure 1, Table 1). Specifically, we have shown[12] that the rat intrahepatic biliary epithelium is formed by ducts of different sizes (5 to 200 mm in external diameter) and cholangiocytes of different cell areas (3 to 80 mm2).  Furthermore, a direct relationship exists between cholangiocyte area and external duct diameter, a finding that demonstrates that small ducts are lined by small cholangiocytes, whereas larger ducts are lined by larger cholangiocytes[12-14]. The fact that small and large ducts are lined by small and large cholangiocytes, respectively, is important since it allows for the assignment of the secretory, apoptotic and proliferative functions (achieved in isolated small and large cholangiocytes) within the different portions of the intrahepatic biliary epithelium. Recently, Masyuk et al[15] have reconstructed the intrahepatic biliary epithelium that 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 of a tree.
 
       Studies by Phillips et al[16] have shown that no major ultrastructural differences exist among cholangiocytes lining small and large bile ducts. However, in support of the concept that the intrahepatic biliary epithelium is morphologically heterogeneous, electron microscopic studies by Benedetti et al[14] in rat liver sections and IBDU have demonstrated that large bile ducts are lined by 8-15 cholangiocytes and small ducts by 4-5 cholangiocytes. The studies also showed that small and large cholangiocytes have a multilobulated nucleus, numerous vesicles at the subapical region, tight junctions, high density of microvilli and lysosomes and a few mitochondria[14]. Other studies have shown the presence of microvilli and cilia in the apical plasma membrane of cholangiocytes[17,18], cilia that play an important role in the regulation of cholangiocyte functions[19,20]. While large cholangiocytes are columnar in shape, small cholangiocytes have a cuboidal shape[14].  Abundant Golgi apparatus was observed between the apical pole and the nucleus[14]. Rough endoplasmic reticulum was inconspicuous in the smallest ducts and increased only slightly in the largest[14]. While large cholangiocytes display a small nucleus and conspicuous cytoplasm, small cholangiocytes possess a high nucleus/cytoplasm ratio[14]. Cholangiocytes have distinct apical and basolateral membranes[14,17,18].Coated pits have also been observed on the apical and basolateral membranes of cholangiocytes, a finding suggesting receptor-mediated endocytosis at both domains of cholangiocytes[21]. Functional tight junctions are located between adjacent cholangiocytes in proximity to the apical domain[17].
 

Innervation

 
There is growing information regarding the role of the nervous system in the regulation of the pathophysiology of the biliary epithelium[3,22-27]. In the liver, adrenergic and cholinergic nerves are located around the hepatic artery, portal vein, and the biliary epithelium[28,29]. The intrahepatic arteries, veins, bile ducts and hepatocytes are also innervated[28,29]. In the autonomic nervous system, there are a number of regulatory peptides including neuropeptide tyrosine (NPY)[30,31], calcitonin gene related peptide (CGRP), somatostatin, vasoactive intestinal polypeptide (VIP) (mostly associated with parasympathetic fibers), enkephalin and bombesin[31-35]. NPY-positive nerves are present in extrahepatic bile ducts[36] and have been suggested to regulate bile flow by autocrine/paracrine mechanisms[37]. We have shown that NPY inhibits cholangiocarcinoma growth by interaction with a G-protein coupled receptor by Ca2+-dependent modulation of Src/ERK1/2 phosphorylation[38]. Nerve fibers containing CGRP and substance P are present around blood vessels and bile duct radicles within portal tracts[39,40]. VIP-positive nerve fibers are located in the walls of hepatic arteries, portal veins and bile ducts[41].

Vascularization

 
The intrahepatic and extrahepatic bile ducts are nourished by a complex network of minute vessels [i.e., peribiliary vascular plexus (PBP)], which originate from branches of the hepatic artery and flow principally into the hepatic sinusoids, either directly (lobular branch) or by portal vein branches (prelobular branches)[42,43]. Since the blood flows in the opposite direction (from the large towards the small ducts) to bile flow, the PBP presents a counter-current stream of biliary reabsorbed substances to hepatocytes[44,45]. We have previously shown that the function of the intrahepatic biliary tree is linked to its vascular supply sustained by the PBP[44]. Changes in intrahepatic bile duct mass are associated with changes of the PBP architecture[44]. Following BDL, the PBP undergoes hyperplasia, thus supporting the increased nutritional and functional demands from the proliferating bile ducts[44]. In support of this concept, studies[46] have shown that following chronic feeding of ANIT (which induces increases in both cholangiocyte proliferation/apoptosis)[47], the hepatic artery and portal vein undergo marked proliferation, presumably to support the increased nutritional and functional demands of the proliferated bile ducts[44,46]. However, the proliferation of the PBP occurs only after the hyperplasia of bile ducts[44]. Recent studies have shown that small and large rat bile ducts have a different vascular supply[44]. The PBP is primarily present around large bile ducts and less visible around small bile ducts[44], a finding that may partly explain why large but not small cholangiocytes proliferate following BDL in rats[48] and why small and large ducts differentially proliferate or are damaged in other experimental models of cholangiocyte proliferation/loss including chronic feeding of certain bile acids (e.g., taurocholate and taurolithocholate)[49], ANIT[47] or acute gavage administration of CCl4[50,51] (Figure 2) or partial hepatectomy[52].

Experimental models

 
A number of in vivo models (e.g., BDL, acute admini-stration of CCl4, partial hepatectomy, chronic feeding of ANIT or bile salts)[47-52] demonstrated that the intrahepatic biliary epithelium is functionally heterogeneous, with specific sized bile ducts (i.e., small and large) differentially responding to liver injury/toxins with changes in proliferative, apoptotic and secretory activities[2,3,12,47-52,54,62,71-73]. A number of in vitro experimental models (i.e., small and large cholangiocytes and IBDU and small and large immortalized normal murine cholangiocytes) (Figure 1)[12,13,47,48,50,51,75] have allowed us to suggest that the intrahepatic biliary epithelium is morphologically and functionally heterogeneous[2, 3,12,47-52,54,62,71-73]. The very first approach that was employed and that significantly contributed to lay down the basis of this field of research was the purification of small and large cholangiocytes from rat liver by counterflow elutriation[12,54,76]. Coupling such a technique to immunoaffinity separation[12,18,54], it was possible to isolate two distinct subpopulations of small (approximately 8 mm in diameter, obtained at the centripetal flow rate of 25 ml/min) and large (approximately 14 mm in diameter, collected at the flow rate of 55 mL/min) cholangiocytes (Figure 1)[12,54]. The two subpopulations of small and large cholangiocytes are further purified by immunoaffinity separation[18] using an antibody against an unidentified antigen (expressed by all intrahepatic cholangiocytes)[18] and characterized morphologically (by computerized image analysis) (Figure 1)[12,54], phenotypically (expression of g-glutamyltransferase and cytokeratin-19 genes)[12,54] and functionally (by measurement of gene expression of secretin receptor, CFTR and Cl-/HCO3- exchanger and basal and secretin-stimulated cAMP levels, Cl- efflux and Cl-/HCO3- exchanger activity)[12,54].
 
In addition, we have developed a technique for isolating small (diameter smaller than 15 mm) and large (diameter greater than 15 mm) IBDU from small and large bile ducts, respectively (Figure 1)[13]. This important tool allowed us to directly evaluate the differential secretory responses of different portions of the biliary epithelium to selected gastrointestinal hormones/peptides[13,25,65,77]. As shown in Figure 1, the small duct was pruned off from the large duct by a brief exposure of a laser focused on the junction between large and small ducts (arrow) leading to separation of small from large ducts[13]. Small and large IBDU were characterized by morphometric analysis, gene expression for secretin receptor, CFTR and Cl-/HCO3- exchanger, secretin-induced cAMP levels, and secretion by change in luminal size in response to agonists including secretin, insulin, the a1-adrenergic receptor agonist, the a2-adrenergic receptor agonist, UK14,304 and the D2 dopaminergic receptor agonist, quinelorane[13,25,65,66,77].
 
Most recently. we have immortalized, from normal mice (BALB/c), small and large cholangiocytes by the introduction of the SV40 large T antigen gene, that allowed, after cloning, to establish small and large cholangiocyte cell lines[75]. The characteristics of the two subpopulations were evaluated by electron microscopy (EM) and measurement of trans-epithelial electrical resistance (TER), and secretin-stimulated cAMP levels[75]. EM, TER and differential cAMP response to secretin are consistent with the concept that small and large immortalized cholangiocytes originate from small and large ducts, respectively[75]. Microarray successfully displayed characteristic differential cDNA expression between small and large cholangiocytes[75]. Using the above described methods individually or in tandem, has allowed us to clearly demonstrate heterogeneity of the intrahepatic biliary epithelium and to dissect the differential physiological responses of these distinct subpopulations of cholangiocytes to endogenous stimuli.
 

Heterogeneous expression of proteins

 
The heterogeneous expression of some enzymes/proteins and membrane transporters/receptors in small and large ducts from mice, rats and humans is summarized in Table 2. In human liver, large septal bile ducts mainly express the sialylated Lewisa blood group antigen[78]. In normal and diseased human livers, hepatic, segmental, area, and septal bile ducts, and peribiliary glands express pancreatic enzymes such as pancreatic lipase, pancreatic a-amylase, and trypsin[79,80]. By microarray of RNA from small and large immortalized murine cholangiocytes, we have demonstrated the heterogeneous expression of approximately 80 proteins between small and large cholangiocytes[75]. The pathophysiological relevance of the differential expression of these messages remains to be addressed.
 
Secretory activity
Recent studies have demonstrated that large bile ducts are the major anatomical sites of cAMP-dependent ductal secretion by activation of cAMP/PKA/CFTR/Cl-/HCO3- exchanger (Figure 3)[3,12,13,48,54]. Specifically, studies in isolated small and large cholangiocytes and IBDU from normal and BDL rats have shown that large (but not small) cholangiocytes express the messages for secretin receptor, CFTR and Cl-/HCO3- exchanger and respond to secretin with increases in cAMP levels, Cl- efflux and Cl-/HCO3- exchanger activity and IBDU lumen expansion (Figure 3)[12,13,48,54]. In rat liver, large ducts express alkaline phosphatase and g-glutamyltranspeptidase[81]. The expression of alkaline phosphatase in large ducts is consistent with our previous studies[81] showing that alkaline phosphatase inhibits secretin-stimulated choleresis by blockage of CFTR activity, which is expressed only in large ducts (Figure 3)[54]. Furthermore, large cholangiocytes (which is the only cholangiocyte subpopulation expressing the somatostatin receptor, SSTR2)[48] are the major anatomical sites of somatostatin inhibition of secretin-stimulated ductal secretion (Figure 3)[48,55]. The inhibitory effects of somatostatin on secretin-stimulated secretion in large cholangiocytes are associated with reduced cAMP levels, Cl- efflux and Cl-/HCO3- exchanger activity[48,55,82]. The counter-regulatory effect of somatostatin on the choleretic effect of secretin is important in modulating ductal secretion in pathological conditions associated with cholangiocyte proliferation/loss[3]. Parallel with the findings observed in rat bile ducts[3,12,13,48, 4], in human liver secretin-stimulated duct secretory activity is heterogeneous, since only large bile interlobular ducts express the Cl-/HCO3- exchanger[83].
  
We have demonstrated the presence of insulin and CCK-B/gastrin receptors in large cholangiocytes from normal and BDL rats and have shown that these two hormones inhibit secretin-stimulated ductal secretion of BDL rats by IP3/Ca2+/PKCa-dependent decrease of cAMP levels[7,72,77]. Similarly, we found that ETA and ETB receptors are expressed by large cholangiocytes and that ET-1 inhibits secretin-stimulated cAMP levels and ductal bile secretion of BDL rats by interaction with ETA but not ETB receptors[59]. Furthermore, recent data have shown that: (1) the D2 dopaminergic receptors are expressed by large BDL cholangiocytes; and (2) the D2 dopaminergic receptor agonist, quinelorane, inhibits secretin-stimulated ductal secretion by activation of the Ca2+-dependent PKCg[25]. The a2-adrenergic receptor agonist, UK14, 304, inhibits secretin-stimulated cAMP-dependent Cl- efflux and Cl-/HCO3- in large cholangiocytes and secretin-stimulated lumen expansion in large IBDU of BDL rats[66]. The a1-adrenergic receptor agonist, phenylephrine, stimulates cAMP levels and secretin-stimulated secretion of large BDL cholangiocytes by IP3/Ca2+-dependent activation of PKCa and PKCbⅡ[65]. We have recently demonstrated[26] that acetylcholine, by interacting with M3 receptor subtypes, potentiates secretin-stimulated cAMP levels and Cl-/HCO3- exchanger activity in IBDU and purified cholangiocytes by a Ca2+-calcineurin mediated but PKC independent modulation of adenylyl cyclase.
 
Following hepatocyte secretion[84], bile acids are reabsorbed by the biliary epithelium[85], then they return via the PBP to the hepatocytes for secretion into bile (cholehepatic shunting)[86]. As a mechanism for bile acids entry into cholangiocytes, the apical Na+-dependent bile transporter, ASBT (structurally identical to the ileal bile acid transporter) is expressed on the apical membranes of large cholangiocytes[87]. Consistent with functional activity for ASBT in cholangiocytes, studies have shown Na+-dependent and saturable uptake of taurocholate in normal cholangiocyte cultures[88] and large cholangiocytes[89]. These data suggests that after taurocholate and taurolithocholate enter into large cholangiocytes by ABAT, they stimulate secretin-stimulated ductal bile flow in these cholangiocyte subpopulations[89,90]. Other studies have shown that both taurocholate and taurolithocholate increase secretin-stimulated cAMP levels in large but not small cholangiocytes[90]. Chronic feeding of ursodeoxycholate and tauroursodeoxycholate to BDL rats inhibits secretin-stimulated ductal secretion in large cholangiocytes[62].
 
As evidence against the notion that small cholangio-cytes may be primitive, undifferentiated cells that do not display secretory activity, recent studies have shown that in pathological conditions associated with damage of large cAMP-responsive ducts (e.g., after acute CCl4 administration) (Figure 3)[50,51], small cholangiocytes transiently compensate for large cholangiocyte damage by de novo activation of secretory (including expression of secretin receptor and secretin-stimulated cAMP response)[50,51] and proliferative[50,51] (see below) activities.  Following ANIT feeding and partial hepatectomy, small cholangiocytesproliferate and secrete by the de novo expression of secretin receptor and activation of cAMP response[47,52]. Since preliminary data and unpublished observations (Alpini, 2005) show that small rat and mouse cholangiocytes express receptors (ETA, CCK-B/gastrin, a1-adrenergic, D2 dopaminergic, insulin, H1 histamine) signaling by activation of IP3/Ca2+/PKC[59,91], we propose that there is a secretory gradient in the intrahepatic biliary tree with small cholangiocytes secreting water and electrolytes by activation of the IP3/Ca2+/PKC pathway, whereas large cholangiocytes secrete bile by activation of the cAMP/PKA/CFTR/Cl-/HCO3- exchanger[2,5,2,13,48,54].

Proliferation and apoptosis

 
Cholangiocyte proliferation is coordinately regulated by a number of factors including gastrointestinal hormones/peptides, growth factors, cAMP and IP3/Ca2+/PKC pathways, nerves and bile acids[2,3,24,49,61,62,68,70-72,92-94]. Recent studies have shown that different sized cholangiocytes differentially proliferate or are damaged by apoptosis in response to injury, toxins, nerve resection and selected diets[2,26,48-52]. Following BDL, large but not small cholangiocytes proliferate with increases in basal and secretin-stimulated choleresis (Figure 3)[2,3,48]. We propose that large cholangiocytes selectively proliferate in response to BDL due to: (1) the predominant expression of VEGF in large compared to small cholangiocytes (Alpini et al, 2005, unpublished observation); and (2) the presence of the PBP mainly around large bile ducts, and less discernable around small bile ducts[44]. In support of this concept, in rats with BDL proliferation of the peribiliary plexus occurs only around large ducts[44]. Furthermore, we have recently demonstrated[94] that neutralization of VEGF levels of large cholangiocytes (by administration of a neutralizing anti-VEGF antibodies) reduces cholangiocyte growth typical of BDL rats[6]. In support of the concept that PBP and VEGF play a role in the regulation of large cholangiocyte function, hepatic artery ligation in BDL rats is associated with: (1) the disappearance of the PBP; (2) increased apoptosis and impaired proliferation of large cholangiocytes; and (3) decreased cholangiocyte VEGF secretion[93]. The effects of hepatic artery ligation on PBP and large cholangiocyte function were prevented by chronic administration of r-VEGF-A that, by maintaining the integrity of the PBP and large cholangiocyte proliferation, prevents bile duct damage following ischemic injury[93].
 
A number of gastrointestinal hormones/peptides have been shown to regulate the differential proliferative response of small and large cholangiocytes. We have shown that cholangiocytes express a1, a2, b1 and b2 thyroid hormone receptors and that the chronic administration of the thyroid hormone agonist, 3, 3¢, 5 L-tri-iodothyronine to BDL rats reduces in vivo the proliferation of large cholangiocytes[95], the only cholangiocyte subpopulation proliferating in this model[48]. In addition, in BDL rats we have shown that somatostatin inhibits the growth of large cholangiocytes by a decrease in cAMP levels[48]. Furthermore, gastrin inhibits large cholangiocyte proliferation in BDL rats by Ca2+/PKC-dependent inhibition of cAMP levels[72]
 
We have demonstrated that ovariectomy in BDL female rats reduces the proliferation of large cholangiocytes and induces a decrease in the expression of a and b estrogen receptors[69]. We propose that estrogens play a role in the management of chronic cholestatic liver diseases.
 
Recent studies have shown that nerves regulate the differential proliferative response of intrahepatic ducts.  We have shown that the activation of serotonin 1 A and 1 B receptors in cholangiocytes leads to the inhibition of large cholangiocyte proliferation in BDL rats[67]. Serotonin inhibition of large cholangiocyte proliferation was associated with activation of the IP3/Ca2+/PKC signaling pathway and the consequent inhibition of the cAMP/PKA/Src/ERK 1/2 pathway[67]. Since cholangiocytes secrete serotonin, we propose that serotonin limits the growth of intrahepatic bile ducts in the course of chronic cholestasis by an autocrine mechanism. Similarly, we have shown that cholangiocytes secrete NGF and that NGF secretion increases in proliferating BDL cholangiocytes compared to normal cholangiocytes[24]. In vivo, immunoneutralization of NGF (with an anti-NGF antibody) decreased large cholangiocyte proliferation[24]. The data suggest that NGF regulates cholangiocyte proliferation by an autocrine mechanism.
 
We have demonstrated that sensory innervation via α-calcitonin gene related peptide (a-CGRP) plays a role in adaptive proliferative responses of large cholangiocytes during cholestasis following BDL[96]. Specifically, we have shown that small and large murine cholangiocytes express the CGRP receptor components (calcitonin like receptor or CLR, receptor component protein or RCP and receptor activity modifying protein or RAMP1)[96]. Large, but not small, cholangiocytes proliferate in response to a-CGRP, proliferation that was blocked by CGRP[8-37], a-CGRP receptor antagonist[97]. a-CGRP stimulation of large cholangiocyte proliferation was associated with increased cAMP levels and phosphorylation of PKA and p38[97]. We observed a decrease in the number of proliferating large cholangiocytes in BDL knock-out mice (lacking a-CGRP) compared to BDL wild-type mice[96].
 
The role of the second messenger, cAMP, in the regulation of hepatic cell proliferation has been demonstrated in a number of animal models that stimulate hepatocyte and cholangiocyte proliferation via cAMP dependent mechanisms[26,50-52,54,98,99]. Following partial hepatectomy, there is an increase in intracellular cAMP levels in regenerating hepatocytes[100] and cholangiocytes[52].  Activation of Gas coupled receptors leads to activation of adenylyl cyclase and increased cAMP levels, whereas activation of Gai coupled receptors results in inhibition of AC activity and lowered intracellular cAMP levels[101]. cAMP response elements mediating transcriptional activation in response to increased intracellular cAMP levels have been identified[102]. In support of these findings, we have shown that chronic administration of forskolin to normal rats increased cAMP levels and the proliferation of large but not small cholangiocytes compared to rats receiving saline[70]. In purified cholangiocytes, forskolin increased large (but not small) cholangiocyte proliferation[70], which was blocked by Rp-cAMPs (a PKA inhibitor)[74], PP2 (a Src inhibitor)[103] and PD98059 (a MEK inhibitor)[104]. The effects of forskolin on large cholangiocyte proliferation were associated with increased phosphorylation of PKA, Src Tyr 139 and ERK1/2[70].  Maintenance of cAMP levels by forskolin administration prevents the effects of vagotomy on large cholangiocyte apoptosis (activation) and proliferation (inhibition)[26].
 
The acute administration of CCl4 to normal and BDL rats induces decreased cAMP levels and loss of function of large cholangiocytes at d 2 and transient elevation of cAMP levels in small cholangiocytes[50,51]. In these models, small cholangiocytes de novo express secretin receptors, a key component of the biliary proliferative and secretory mechanisms, suggesting that intracellular cAMP plays a key role in the: (1) de novo expression of large cholangiocyte phenotypes by small cholangiocytes (to compensate for loss of large cholangiocyte function); and (2) perhaps the differentiation of small cholangiocytes towards a cholangiocyte subpopulation that has the capacity to secrete and proliferate by cAMP-dependent pathway[50,51].
 
Following partial hepatectomy, both small and large cholangiocytes proliferate and participate in the regeneration of the intrahepatic biliary epithelium[52]. A single gavage dose of CCl4 to normal and BDL rats induces damage of large, cAMP-responsive cholangiocytes, whereas small cholangiocytes (resistant to CCl4) de novo proliferate and secrete (by the activation of the secretin receptor and secretin-stimulated cAMP levels) to compensate for the damage and loss of functional activity of large cholangiocytes[50,51]. The differential resistance of small and large cholangiocytes to CCl4 is presumably due to the presence of cytochrome P4502E1 (the enzyme that converts CCl4 to its radicals)[105]in large but not small cholangiocytes[50,51].  Chronic administration of the toxin, ANIT, induces proliferation of both small and large cholangiocytes, proliferation that (in contrast to other models including BDL)[26] was associated with enhanced apoptosis[47]. We propose that following ANIT or CCl4 feeding, the proliferation of small cholangiocytes may be due to the presence of cholangiocyte apoptosis in these models[47,50,51]. We also propose that the lack of small cholangiocyte proliferation in BDL rats may be due to the absence of cholangiocyte apoptosis in this model[26]. Similar to what is observed following acute CCl4 administration[50], the differential responses of small and large cholangiocytes to liver injury/toxins may be due to differential expression of other enzymes/proteins in small and large cholangiocytes. In support of this concept, phase I or mixed-function oxygenase 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 glutathione-S-transferase) drug-metabolizing enzymes are heterogeneously expressed by cholangiocytes[50,81,106]. Similarly, since small murine cholangiocytes express annexin-V[107] (that regulates cell apoptosis)[108], this finding may explain partly why small ducts are more resistant than large ducts to some hepatic injury/toxins[50, 51]. In support of this concept, recent studies have shown that bcl-2 (an anti-apoptotic protein)[109] is expressed by small bile ducts in normal human liver and human liver with cirrhosis and focal nodular hyperplasia[110], a finding that may also explain partly the greater resistance of small cholangiocytes to damage[3,50,51].
 
In vitro treatment of normal cholangiocytes with taurocholate and taurolithocholate increases the proliferation of large but not small cholangiocytes[90]. Chronic feeding of taurocholate and taurolithocholate to normal rats induces the de novo expression of ASBT and activation of proliferation of small cholangiocytes, which do not constitutively express ASBT and are mitotically quiescent, and increases the proliferation of large cholangiocytes[49]. Prolonged feeding of ursodeoxycholate and tauroursodeoxycholate to BDL rats reduces the growth of large cholangiocytes[62] that selectively proliferate in this hyperplastic model[48].  Furthermore, depletion of endogenous bile acids reduced large cholangiocyte proliferation compared with BDL rats[92]. Re-infusion of taurocholate to bile acid-depleted rats prevented the decrease in cholangiocyte proliferation that was maintained at levels similar to those of BDL rats[92].
 
Histamine, an aminergic neurotransmitter, regulates many pathophysiological functions. Four G-protein coupled histamine receptors (H1, H2, H3 and H4) exist[111].  While H1 histamine receptors act via Gaq mobilizing [Ca2+]i[112], activation of H2 histamine receptors is modulated by Gas proteins, coupled to adenylyl cyclase[113]. H3 and H4 histamine receptors couple to Gai/o proteins that inhibit adenylyl cyclase[114]. Based upon our preliminary data, we propose a model in which the overall outcome of histamine on cholangiocyte growth is represented by a balance between its stimulatory (by activation of H1 and H2 histamine receptors)[91,115] and its inhibitory (by activation of H3 and H4 histamine receptors)[116, 117] actions on small and large cholangiocyte proliferation.  Specifically, we have shown that small but not large mouse cholangiocytes: (1) express the H1 histamine receptors and the calcium-dependent CaMK I (but not II or IV) protein kinase; and (2) proliferate in response to H1 histamine receptor agonists, proliferation that was blocked by BAPTA/AM, Gö6976 and W-7, a CAMK inhibitor[118]. IP3 (but not cAMP) levels were increased in small cholangiocytes treated with HTMT dimaleate. Chronic administration of the specific H3/H4R agonist (RAMH) to BDL rats decreased large cholangiocyte proliferation and cAMP levels compared to BDL rats treated with NaCl[116,117]. This inhibition is mediated through negative regulation of the cAMP-dependent PKA/ERK1/2 pathway[116,117].
 
The mechanisms by which different sized ducts proliferate or are damaged in response to various liver injury/toxins (e.g., BDL, partial hepatectomy, vagotomy, feeding of ANIT, bile acids or CCl4)[3,26,47-52] are unclear.  Furthermore, the pathophysiology of small cholangiocytes is undefined in these models. Based upon preliminary data and unpublished observations from our laboratory, we propose that neural/hormonal-dependent (cholinergic and adrenergic) activation of the Ca2+-dependent NFAT (Nuclear Factor of Activated T-lymphocytes) stimulates the proliferative response of small cholangiocytes, whereas neural/hormonal-dependent activation of the cAMP-dependent CREB stimulates the proliferation of large cholangiocytes. NFAT is a ubiquitous transcription factor that was initially described in T-lymphocytes.  Five isoforms of NFAT have been identified. Four of these isoforms (NFATc1 to c4) are regulated by Ca2+ signaling[119]. Preliminary data shows that Ca2+-dependent activation of NFATc1/c4 stimulates the proliferation of small cholangiocytes after CCl4-induced damage of cAMP-responsive large bile ducts[120]. Specifically, we have shown that small but not large normal rat cholangiocytes express the NFAT isoforms, NFAT c1 and c4[120]. CCl4 both in vivo and in vitro increased small cholangiocyte proliferation that was blocked by BAPTA/AM and 11R-VIVIT (NFAT inhibitor peptide)[121]. Furthermore, unpublished data from our laboratory show that the de novo growth of small cholangiocytes is regulated via adrenergic stimulation of Ca2+-dependent activation of NFATc1/c4 (Ca2+/calcineurin) and Sp1 (Ca2+/PKC). NFAT and Sp1 cooperatively interact to regulate proliferative phenotypes in other cell types[122].
 
Recent studies have shown that bile acids have cyto-protective effects against apoptosis in large cholangiocytes. Feeding of taurocholate to BDL rats (treated with a single dose of CCl4) prevents CCl4-induced damage of large cholangiocytes, whereas small cholangiocytes (which are de novo activated following CCl4-induced damage of large ducts)[50,51] remained mitotically dormant and unresponsive to secretin (Figure 3)[123]. In vitro, taurocholate prevented the inhibitory effects of CCl4 on apoptotic, proliferative and secretory capacity of large BDL cholangiocytes[123]. The protective effects of taurocholate against CCl4-induced damage of large BDL cholangiocytes are due to the activation of PI3-K and AKT expression[123]. Furthermore, feeding of taurocholate to BDL + vagotomy rats prevented vagotomy activation of large cholangiocyte apoptosis and inhibition of large cholangiocyte growth[124], effects that were abolished by wortmannin, a PI3-K inhibitor[125]. Functional ASBT expression as well as phosphorylation of Akt were reduced by vagotomy but restored by taurocholate feeding[124]. Chronic feeding of taurocholate prevented the increase in cholangiocyte apoptosis and the damage of large cholangiocyte proliferation induced by adrenergic denervation by 6-OHDA administration[126]. Taurocholate effects are mediated by the PI3K pathway, since the simultaneous administration of wortmannin reverses such effects[126]. In addition, the feeding of ursodeoxycholate and tauroursodeoxycholate to BDL + vagotomy rats prevented the activation of apoptosis and the loss of proliferation of large cholangiocytes observed in this model[127]. In this study[127], the protective effects of these two bile acids were neutralized by the simultaneous administration of BAPTA/AM (an intracellular Ca2+ chelator)[72] or Gö6976 (a PKC inhibitor)[65]. Both ursodeoxycholate and tauroursodeoxycholate increased IP3 and Ca2+ levels, together with enhanced phosphorylation of PKC-a[127]. The data suggests that bile acids are important in modulating large cholangiocyte proliferation in denervated livers. 

Heterogeneity in chlorangiopathies

 
Chronic cholestatic liver diseases (cholangiopathies), which target intrahepatic and extrahepatic bile ducts, are characterized by the coexistence of cholangiocyte growth/apoptosis, inflammation and fibrosis[3,128]. Cholangiopathies differentially target the biliary epithelium with heterogeneous proliferative and apoptotic responses of different sized ducts[3,47,50,129-131]. Primary biliary cirrhosis is characterized by the selective proliferation/loss of small interlobular bile ducts[3,132]. Some studies demonstrated that damage of interlobular bile ducts is immune mediated[3,133]. The origin of primary sclerosing cholangitis (PSC), which is associated with inflammation and fibrosis of bile ducts, originates from multiple factors including autoimmune, bacterial, congenital, drug, or viral agents[3,73]. PSC affects mainly extrahepatic and interlobular or septal bile ducts although smaller bile ducts can be affected[3,73]. Patients with small duct PSC seem to have a good prognosis in terms of survival and development of cholangiocarcinoma[134]. Cholangiocarcinoma occurs frequently in patients with PSC and targets mainly the major bile duct bifurcation[3,135]. Peripheral cholangiocarcinoma occur within the liver rather than within large bile ducts may arise from small bile ducts[3,135].  Mutations in the CFTR gene are responsible for causing the human biliary disease, cystic fibrosis, due to defective transport of water and chloride presumably by large cholangiocytes expressing CFTR[136]. Our previous studies in rodent liver has shown that CFTR is expressed principally in large cholangiocytes and in bile ducts greater than 15 mM diameter[12,13] but in studies of human liver of cystic fibrosis patients, CFTR was expressed in both large and small ducts[137].
 
Defective chloride transport and chloridemediated bile secretion by large cholangiocytes may be respon-sible for the reduced fluidity and alkalinity of bile, leading to bile duct damage. Ca2+-dependent Cl- cha-nnels[138,139] (presumably expressed by both small and large cholangiocytes) may be able to secrete bile, thus compensating for loss of CFTR functional activity of CFTR in large cholangiocytes[54]. In polycystic kidney liver disease (PKLD), the genetic defect results in the growth of multiple epithelial cysts within the renal, liver parenchyma and intrahepatic bile ducts[140]. The disease targets presumably large bile ducts since the cystic ductal cells also secrete Cl- and HCO3- (as normal large cholangiocytes)[2,3,54,71,73] but the secretion is diminished, likely due to reduced Cl-/HCO3- exchanger activity in cystic ductal cells as compared with normal cholangiocytes[140]. Biliary atresia, which is the most common reason of cholestasis in infants and children, is a destructive, inflammatory process of the extrahepatic bile ducts but as the disease progresses smaller intrahepatic bile ducts are also involved[141]. The pathogenesis of biliary atresia is unknown but infections or toxic agents combined with genetic/immunologic susceptibility have been proposed[3, 142, 143].

Summary

 
In this review, we have summarized the findings demon-strating that the intrahepatic biliary epithelium is heterogeneous regarding: (1) morphological characteristics, vascularization and innervation; (2) secretory activity in response to gastrointestinal hormones/peptides, nerve receptor agonists and bile salts; and (3) apoptotic and proliferative responses to liver injury/toxins and gastrointestinal hormones/peptides. Specifically, the intrahepatic biliary epithelium is formed by bile ducts of different sizes with small ducts lined by small cholangiocytes, whereas larger ducts are lined by larger cholangiocytes[12-14]. Following a general background on cholangiocyte functions, we discussed the in vivo and in vitro experimental models that allowed us to demonstrate that the biliary epithelium is morphologically and functionally heterogeneous. Following a brief review on the heterogeneous distribution of non-transport related proteins, we discussed the secretory functions of small and large cholangiocytes. While large cholangiocytes secrete water and electrolytes[12,13,48] by changes in cAMP/PKA/CFTR/Cl-/HCO3-, small cholangiocytes may secrete bile by a transduction pathway (different from that observed in large cholangiocytes)[12,13,48] involving activation of IP3/Ca2+/PKC. We have presented data demonstrating that small and large cholangiocytes differentially proliferate or are damaged in response to liver injury/toxins. Small and large ducts also differ regarding the proliferative and apoptotic responses to liver injury/toxins[2,71,73]. We propose that activation of the Ca2+-dependent NFAT stimulates the proliferation of small cholangiocytes, whereas neural/hormonal-dependent activation of the cAMP-dependent CREB stimulates the proliferation of large cholangiocytes.  In the last part of the review, we have briefly outlined the heterogeneity of the biliary epithelium in relationship to chronic cholestatic liver diseases targeting different sized ducts.
 
 
Future Perspectives
 
The concept that the biliary epithelium is functionally heterogeneous is clinically relevant since in chronic cholestatic liver diseases cholangiocyte proliferation/damage is an event restricted to a specific duct size. Further studies are needed for understanding the pathophysiology of small cholangiocytes in the overall contribution of the functions of the biliary epithelium. However, some preliminary studies from our laboratory suggest that small cholangiocytes secrete bile (by a IP3/Ca2+/PKC-dependent mechanism) and proliferate by activation of the Ca2+-dependent transcription factor, NFAT. Further studies are necessary to evaluate the role of the nervous system in the regulation of the heterogeneous secretory, apoptotic and proliferative responses of different sized bile ducts to gastrointestinal hormones, injury/toxins and viruses. Since PBP proliferation is observed only in large proliferating cholangiocytes from BDL rats, we propose that blood supply and circulating factors (e.g., vascular endothelial growth factor and placental growth factor) may be important in the regulation of the heterogeneous response of cholangiocytes to liver injury/toxins.

References

 
1     Yokoyama HO, Wilson ME, Tsuboi KK, Stowell RE. Regeneration of mouse liver after partial hepatectomy. Cancer Res 1953; 13: 80-85  
2     Kanno N, LeSage G, Glaser S, Alvaro D, Alpini G. Functional heterogeneity of the intrahepatic biliary epithelium. Hepatology 2000; 31: 555-561  
3     Alpini G, Prall RT, LaRusso NF. The pathobiology of biliary epithelia. In: Arias IM, Boyer JL, Chisari FV, Fausto N, Jakoby W, Schachter D, and Shafritz DA, eds.The Liver; Biology & Pathobiology, 4th ed.Philadelphia, PA: Lippincott Williams & Wilkins 2001: 421-435  
4     Nathanson MH, Boyer JL. Mechanisms and regulation of bile secretion. Hepatology 1991; 14: 551-566  
5     Kanno N, LeSage G, Glaser S, Alpini G. Regulation of cholangiocyte bicarbonate secretion. Am J Physiol Gastrointest Liver Physiol 2001; 281: G612-625  
6     Alpini G, Lenzi R, Sarkozi L, Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules. J Clin Invest 1988; 81: 569-578  
7     Glaser SS, Rodgers RE, Phinizy JL, Robertson WE, Lasater J, Caligiuri A, Tretjak Z, LeSage GD, Alpini G. Gastrin inhibits secretin-induced ductal secretion by interaction with specific receptors on rat cholangiocytes. Am J Physiol 1997; 273: G1061-1070   
8     Ludwig J. New concepts in biliary cirrhosis. Semin Liver Dis 1987; 7: 293-301  
9     Ludwig J, Ritman EL, LaRusso NF, Sheedy PF, Zumpe G. Anatomy of the human biliary system studied by quantitative computer-aided three-dimensional imaging techniques. Hepatology 1998; 27: 893-899  
10   Schaffner F, Popper H. Electron microscopic studies of normal and proliferated bile ductules. Am J Pathol 1961; 38: 393-410  
11   Carruthers JS, Steiner JW. Studies on the fine structure of proliferated bile ductules. I. Changes of cytoarchitecture of biliary epithelial cells. Can Med Assoc J 1961; 85: 1223-1236  
12   Alpini G, Roberts S, Kuntz SM, Ueno Y, Gubba S, Podila PV, LeSage G, LaRusso NF. Morphological, molecular, and functional heterogeneity of cholangiocytes from normal rat liver. Gastroenterology 1996; 110: 1636-1643  
13   Alpini G, Glaser S, Robertson W, Rodgers RE, Phinizy JL, Lasater J, LeSage GD. Large but not small intrahepatic bile ducts are involved in secretin-regulated ductal bile secretion. Am J Physiol Gastrointest Liver Physiol 1997; 272: G1064-1074  
14   Benedetti A, Bassotti C, Rapino K, Marucci L, Jezequel AM. A morphometric study of the epithelium lining the rat intrahepatic biliary tree. J Hepatol 1996; 24: 335-342  
15   Masyuk TV, Ritman EL, LaRusso NF. Quantitative assessment of the rat intrahepatic biliary system by three-dimensional reconstruction. Am J Pathol 2001; 158: 2079-2088  
16   Phillips MJ, Poucell S, Patterson J, Valencia P. The normal liver. In: Phillips MJ, Powell S, Patterson S, Valencia P, eds. The liver: an atlas and text of ultrastructural pathology. New York, NY: Raven Press, 1987: 1-35  
17   LaRusso NF, Ishii M, Vroman BT. The ins and outs of membrane movement in biliary epithelia. Trans Am Clin Climatol Ass 1990; 102: 245-258; discussion 258-259  
18   Ishii M, Vroman B, LaRusso NF. Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver. Gastroenterology 1989; 97: 1236-1247  
19   Vroman B, LaRusso NF. Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells. Lab Invest 1996; 74: 303-313  
20   Masyuk TV, Huang BQ, Ward CJ, Masyuk AI, Yuan D, Splinter PL, Punyashthiti R, Ritman EL, Torres VE, Harris PC, LaRusso NF. Defects in cholangiocyte fibrocystin expression and ciliary structure in the PCK rat. Gastroenterology 2003; 125: 1303-1310  
21   Ishii M, Vroman B, LaRusso NF. Morphologic demonstration of receptor-mediated endocytosis of epidermal growth factor by isolated bile duct epithelial cells. Gastroenterology 1990; 98: 1284-1291  
22   Elsing C, Hubner C, Fitscher BA, Kassner A, Stremmel W. Muscarinic acetylcholine receptor stimulation of biliary epithelial cells and its effect on bile secretion in the isolated perfused liver [corrected]. Hepatology 1997; 25: 804-813  
23   Alvaro D, Alpini G, Jezequel AM, Bassotti C, Francia C, Fraioli F, Romeo R, Marucci L, Le Sage G, Glaser SS, Benedetti A. Role and mechanisms of action of acetylcholine in the regulation of rat cholangiocyte secretory functions. J Clin Invest 1997; 100: 1349-1362  
24   Gigliozzi A, Alpini G, Baroni GS, Marucci L, Metalli VD, Glaser SS, Francis H, Mancino MG, Ueno Y, Barbaro B, Benedetti A, Attili AF, Alvaro D. Nerve growth factor modulates the proliferative capacity of the intrahepatic biliary epithelium in experimental cholestasis. Gastroenterology 2004; 127: 1198-1209  
25   Glaser S, Alvaro D, Roskams T, Phinizy JL, Stoica G, Francis H, Ueno Y, Barbaro B, Marzioni M, Mauldin J, Rashid S, Mancino MG, LeSage G, Alpini G. Dopaminergic inhibition of secretin-stimulated choleresis by increased PKC-gamma expression and decrease of PKA activity. Am J Physiol Gastrointest Liver Physiol 2003; 284: G683-694  
26   LeSage G, Alvaro D, Benedetti A, Glaser S, Marucci L, Baiocchi L, Eisel W, Caligiuri A, Phinizy JL, Rodgers R, Francis H, Alpini G. Cholinergic system modulates growth, apoptosis, and secretion of cholangiocytes from bile duct-ligated rats. Gastroenterology 1999; 117: 191-199  
27   Barbaro B, Glaser S, Francis H, Taffetani S, Marzioni M, LeSage G, Alpini GG. Nerve regulation of cholangiocyte functions. In: Alpini G, Alvaro D, LeSage G, Marzioni M, and LaRusso NF eds. Pathophysiology of the Bile Duct System. Georgetown, Texas, USA: Landes Biosciences,2004: 199-209  
28   Reilly FD, McCuskey PA, McCuskey RS. Intrahepatic distribution of nerves in the rat. Anat Rec 1978; 191: 55-67  
29   Tsuneki K, Ichihara K. Electron microscope study of vertebrate liver innervation. Arch Histol Jpn 1981; 44: 1-13  
30   Gulbenkian S, Wharton J, Hacker GW, Varndell IM, Bloom SR, Polak JM. Co-localization of neuropeptide tyrosine (NPY) and its C-terminal flanking peptide (C-PON). Peptides 1985; 6: 1237-1243  
31   Lundberg JM, Terenius L, Hokfelt T, Martling CR, Tatemoto K, Mutt V, Polak J, Bloom S, Goldstein M. Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function. Acta Physiol Scand 1982; 116: 477-480  
32   Costa M, Furness JB. Somatostatin is present in a subpopulation of noradrenergic nerve fibres supplying the intestine. Neuroscience 1984; 13: 911-919  
33   Gibbins IL, Furness JB, Costa M, MacIntyre I, Hillyard CJ, Girgis S. Co-localization of calcitonin gene-related peptide-like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea pigs. Neurosci Lett 1985; 57: 125-130  
34   Jule Y, Clerc N, Niel JP, Condamin M. [Met]- and [Leu]enke-phalin-like immunoreactive cell bodies and nerve fibres in the coeliac ganglion of the cat. Neuroscience 1986; 18: 487-498  
35   Schultzberg M, Dalsgaard CJ. Enteric origin of bombesin immunoreactive fibres in the rat coeliac-superior mesenteric ganglion. Brain Res 1983; 269: 190-195  
36   Burt AD, Tiniakos D, MacSween RN, Griffiths MR, Wisse E, Polak JM. Localization of adrenergic and neuropeptide tyrosine-containing nerves in the mammalian liver. Hepatology 1989; 9: 839-845  
37   el-Salhy M, Stenling R, Grimelius L. Peptidergic innervation and endocrine cells in the human liver. Scand J Gastroenterol 1993; 28: 809-815  
38   Fava G, Glaser S, Francis H, Phinizy JL, Venter J, Reichenbach R, Taffetani S, Marzioni M, Marucci L, Benedetti A, Alpini G. Neuropeptide Y (NPY) inhibits cholangiocarcinoma growth by interaction with a G-protein coupled receptor by Ca2+-dependent modulation of Src/ERK1/2 phosphorylation. Gastroenterology 2004; 126: AT926  
39   Inoue N, Sakai H, Magari S, Sakanaka M. Distribution and possible origins of substance P-containing nerve fibers in the rat liver. Ann Anat 1992; 174: 557-560  
40   Goehler LE, Sternini C, Brecha NC. Calcitonin gene-related peptide immunoreactivity in the biliary pathway and liver of the guinea-pig: distribution and colocalization with substance P. Cell Tissue Res 1988; 253: 145-150  
41   Akiyoshi H, Gonda T, Terada T. A comparative histochemical and immunohistochemical study of aminergic, cholinergic and peptidergic innervation in rat, hamster, guinea pig, dog and human livers. Liver 1998; 18: 352-359  
42   Ohtani O, Kikuta A, Ohtsuka A, Taguchi T, Murakami T. Microvasculature as studied by the microvascular corrosion casting/scanning electron microscope method. I. Endocrine and digestive system. Arch Histol Jpn 1983; 46: 1-42  
43   Terada T, Ishida F, Nakanuma Y. Vascular plexus around intrahepatic bile ducts in normal livers and portal hypertension. J Hepatol 1989; 8: 139-149  
44   Gaudio E, Onori P, Pannarale L, Alvaro D. Hepatic microcirculation and peribiliary plexus in experimental biliary cirrhosis: a morphological study. Gastroenterology 1996; 111: 1118-1124  
45   Yamamoto K, Phillips MJ. A hitherto unrecognized bile ductular plexus in normal rat liver. Hepatology 1984; 4: 381-385  
46   Masyuk TV, Ritman EL, LaRusso NF. Hepatic artery and portal vein remodeling in rat liver: vascular response to selective cholangiocyte proliferation. Am J Pathol 2003; 162: 1175-1182  
47   LeSage G, Glaser S, Ueno Y, Alvaro D, Baiocchi L, Kanno N, Phinizy JL, Francis H, Alpini G. Regression of cholangiocyte proliferation after cessation of ANIT feeding is coupled with increased apoptosis. Am J Physiol Gastrointest Liver Physiol 2001; 281: G182-190  
48   Alpini G, Glaser SS, Ueno Y, Pham L, Podila PV, Caligiuri A, LeSage G, LaRusso NF. Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation. Am J Physiol Gastrointest Liver Physiol 1998; 274: G767-775  
49   Alpini G, Ueno Y, Glaser S, Marzioni M, Phinizy JL, Francis H, LeSage G. Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes. Hepatology 2001; 34: 868-876  
50   LeSage GD, Glaser S, Marucci L, Benedetti A, Phinizy JL, Rodgers R, Caligiuri A, Papa E, Tretjak Z, Jezequel AM, Holcomb LA, Alpini G. Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver. Am J Physiol 1999; 276: G1289-1301  
51   LeSage GD, Benedetti A, Glaser S, Marucci L, Tretjak Z, Caligiuri A, Rodgers R, Phinizy JL, Baiocchi L, Francis H, Lasater J, Ugili L, Alpini G. Acute carbon tetrachloride feeding selectively damages large, but not small, cholangiocytes from normal rat liver. Hepatology 1999; 29: 307-319  
52   LeSage G, Glaser S, Robertson W, Phinizy JL, Rodgers R, Alpini G. Partial hepatectomy induces proliferative and secretory events in small cholangiocytes. Gastroenterology 1996; 110: A1250  
53   Alpini G, Ulrich CD 2nd, Phillips JO, Pham LD, Miller LJ, LaRusso NF. Upregulation of secretin receptor gene expression in rat cholangiocytes after bile duct ligation. Am J Physiol 1994; 266: G922-928  
54   Alpini G, Ulrich C, Roberts S, Phillips JO, Ueno Y, Podila PV, Colegio O, LeSage GD, Miller LJ, LaRusso NF. Molecular and functional heterogeneity of cholangiocytes from rat liver after bile duct ligation. Am J Physiol 1997; 272: G289-297  
55   Tietz PS, Alpini G, Pham LD, Larusso NF. Somatostatin inhibits secretin-induced ductal hypercholeresis and exocytosis by cholangiocytes. Am J Physiol 1995; 269: G110-118  
56   Cho WK, Mennone A, Rydberg SA, Boyer JL. Bombesin stimulates bicarbonate secretion from rat cholangiocytes: implications for neural regulation of bile secretion. Gastroenterology 1997; 113: 311-321  
57   Cho WK. Role of the neuropeptide, bombesin, in bile secretion. Yale J Biol Med 1997; 70: 409-416  
58   Cho WK, Boyer JL. Vasoactive intestinal polypeptide is a potent regulator of bile secretion from rat cholangiocytes. Gastroenterology 1999; 117: 420-428  
59   Caligiuri A, Glaser S, Rodgers RE, Phinizy JL, Robertson W, Papa E, Pinzani M, Alpini G. Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes. Am J Physiol 1998; 275: G835-846  
60   Alvaro D, Benedetti A, Marucci L, Delle Monache M, Monterubbianesi R, Di Cosimo E, Perego L, Macarri G, Glaser S, Le Sage G, Alpini G. The function of alkaline phosphatase in the liver: regulation of intrahepatic biliary epithelium secretory activities in the rat. Hepatology 2000; 32: 174-184  
61   Alpini G, Glaser SS, Ueno Y, Rodgers R, Phinizy JL, Francis H, Baiocchi L, Holcomb LA, Caligiuri A, LeSage GD. Bile acid feeding induces cholangiocyte proliferation and secretion: evidence for bile acid-regulated ductal secretion. Gastroenterology 1999; 116: 179-186  
62   Alpini G, Baiocchi L, Glaser S, Ueno Y, Marzioni M, Francis H, Phinizy JL, Angelico M, LeSage G. Ursodeoxycholate and tauroursodeoxycholate inhibit cholangiocyte growth and secretion of BDL rats through activation of PKC alpha. Hepatology 2002; 35: 1041-1052  
63   Baiocchi L, Alpini G, Glaser S, Angelico M, Alvaro D, Francis H, Marzioni M, Phinizy JL, Barbaro B, LeSage G. Taurohyodeoxycholate- and tauroursodeoxycholate-induced hypercholeresis is augmented in bile duct ligated rats. J Hepatol 2003; 38: 136-147  
64   Baiocchi L, LeSage G, Glaser S, Alpini G. Regulation of cholangiocyte bile secretion. J Hepatol 1999; 31: 179-191  
65   LeSage GD, Alvaro D, Glaser S, Francis H, Marucci L, Roskams T, Phinizy JL, Marzioni M, Benedetti A, Taffetani S, Barbaro B, Fava G, Ueno Y, Alpini G. Alpha-1 adrenergic receptor agonists modulate ductal secretion of BDL rats via Ca(2+)- and PKC-dependent stimulation of cAMP. Hepatology 2004; 40: 1116-1127  
66   Francis H, Glaser S, Alvaro D, Taffetani S, Marucci L, Benedetti A, Ueno Y, Marzioni M, LeSage G, Venter J, Baumann B, Phinizy JL, Alpini G. The α-2 adrenergic receptor agonist, UK14,304, inhibits secretin-stimulated ductal secretion of bile duct ligated (BDL) rats by activation of the G-protein Gαi. Hepatology 2003; 38: A1088  
67   Marzioni M, Glaser S, Francis H, Marucci L, Benedetti A, Alvaro D, Taffetani S, Ueno Y, Roskams T, Phinizy JL, Venter J, Fava G, LeSage GD, Alpini G. Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin. Gastroenterology 2005; 128: 121-137  
68   LeSage G, Glaser S, Alpini G. Regulation of cholangiocyte proliferation. Liver 2001; 21: 73-80  
69   Alvaro D, Alpini G, Onori P, Franchitto A, Glaser S, Le Sage G, Gigliozzi A, Vetuschi A, Morini S, Attili AF, Gaudio E. Effect of ovariectomy on the proliferative capacity of intrahepatic rat cholangiocytes. Gastroenterology 2002; 123: 336-344  
70   Francis H, Glaser S, Ueno Y, LeSage G, Marucci L, Benedetti A, Taffetani S, Marzioni M, Alvaro D, Venter J, Reichenbach R, Fava G, Phinizy JL, Alpini G. cAMP stimulates the secretory and proliferative capacity of the rat intrahepatic biliary epithelium through changes in the PKA/Src/MEK/ERK1/2 pathway. J Hepatol 2004; 41: 528-537  
71   Glaser S, Francis H, Marzioni M, Taffetani S, Phinizy JL, LeSage G, Alpini G. Functional heterogeneity of the intrahepatic biliary epithelium. In: Alpini G, Alvaro D, LeSage G, Marzioni M, and LaRusso NF eds. Pathophysiology of the Bile Duct System. Georgetown, Texas, USA: Landes Biosciences 2004: 245-254  
72   Glaser S, Benedetti A, Marucci L, Alvaro D, Baiocchi L, Kanno N, Caligiuri A, Phinizy JL, Chowdury U, Papa E, LeSage G, Alpini G. Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-B/Gastrin receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C alpha-dependent mechanisms. Hepatology 2000; 32: 17-25  
73   Marzioni M, Glaser SS, Francis H, Phinizy JL, LeSage G, Alpini G. Functional heterogeneity of cholangiocytes. Semin Liver Dis 2002; 22: 227-240  
74   Alvaro D, Mennone A, Boyer JL. Role of kinases and phosphatases in the regulation of fluid secretion and Cl-/HCO3- exchange in cholangiocytes. Am J Physiol 1997; 273: G303-313  
75   Ueno Y, Alpini G, Yahagi K, Kanno N, Moritoki Y, Fukushima K, Glaser S, LeSage G, Shimosegawa T. Evaluation of differential gene expression by microarray analysis in small and large cholangiocytes isolated from normal mice. Liver Int 2003; 23: 449-459  
76   Alpini G, Phillips JO, Vroman B, LaRusso NF. Recent advances in the isolation of liver cells. Hepatology 1994; 20: 494-514  
77   LeSage GD, Marucci L, Alvaro D, Glaser SS, Benedetti A, Marzioni M, Patel T, Francis H, Phinizy JL, Alpini G. Insulin inhibits secretin-induced ductal secretion by activation of PKC alpha and inhibition of PKA activity. Hepatology 2002; 36: 641-651  
78   Okada Y, Jinno K, Moriwaki S, Shimoe T, Tsuji T, Murakami M, Thurin J, Koprowski H. Blood group antigens in the intrahepatic biliary tree. I. Distribution in the normal liver. J Hepatol 1988; 6: 63-70  
79   Terada T, Kono N, Nakanuma Y. Immunohistochemical and immunoelectron microscopic analyses of alpha-amylase isozymes in human intrahepatic biliary epithelium and hepatocytes. J Histochem Cytochem 1992; 40: 1627-1635  
80   Terada T, Morita T, Hoso M, Nakanuma Y. Pancreatic enzymes in the epithelium of intrahepatic large bile ducts and in hepatic bile in patients with extrahepatic bile duct obstruction. J Clin Pathol 1994; 47: 924-927  
81   Mathis GA, Walls SA, D’Amico P, Gengo TF, Sirica AE. Enzyme profile of rat bile ductular epithelial cells in reference to the resistance phenotype in hepatocarcinogenesis. Hepatology 1989; 9: 477-485  
82   Alpini G, Ulrich C, Roberts S, Phillips JO, Ueno Y, Podila PV, Colegio O, LeSage GD, Miller LJ, LaRusso NF. Molecular and functional heterogeneity of cholangiocytes from rat liver after bile duct ligation. Am J Physiol 1997; 272: G289-297  
83   Martinez-Anso E, Castillo JE, Diez J, Medina JF, Prieto J. Immunohistochemical detection of chloride/bicarbonate anion exchangers in human liver. Hepatology 1994; 19: 1400-1406  
84   Hofmann AF. Current concepts of biliary secretion. Dig Dis Sci 1989; 34: 16S-20S  
85   Lamri Y, Erlinger S, Dumont M, Roda A, Feldmann G. Immunoperoxidase localization of ursodeoxycholic acid in rat biliary epithelial cells. Evidence for a cholehepatic circulation. Liver 1992; 12: 351-354  
86   Gurantz D, Hofmann AF. Influence of bile acid structure on bile flow and biliary lipid secretion in the hamster. Am J Physiol 1984; 247: G736-748  
87   Aldini R, Roda A, Lenzi PL, Ussia G, Vaccari MC, Mazzella G, Festi D, Bazzoli F, Galletti G, Casanova S. Bile acid active and passive ileal transport in the rabbit: effect of luminal stirring. Eur J Clin Invest 1992; 22: 744-750  
88   Lazaridis KN, Pham L, Tietz P, Marinelli RA, deGroen PC, Levine S, Dawson PA, LaRusso NF. Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter. J Clin Invest 1997; 100: 2714-2721  
89   Alpini G, Glaser S, Rodgers R, Phinizy JL, Robertson WE, Lasater J, Caligiuri A, Tretjak Z, LeSage GD. Functional expression of the apical Na+-dependent bile acid transporter in large but not small rat cholangiocytes. Gastroenterology 1997; 113: 1734-1740  
90   Alpini G, Glaser S, Robertson W, Phinizy JL, Rodgers RE, Caligiuri A, LeSage G. Bile acids stimulate proliferative and secretory events in large but not small cholangiocytes. Am J Physiol 1997; 273: G518-529  
91   Francis H, Glaser S, Ueno Y, Venter J, Reichenbach R, Summers R, Alpini G. Novel evidence for the activation of the growth of small (but not large) murine cholangiocytes by Interaction with H1 histamine Receptors. Hepatology 2005; 42: A1145  
92   Alpini G, Glaser S, Alvaro D, Ueno Y, Marzioni M, Francis H, Baiocchi L, Stati T, Barbaro B, Phinizy JL, Mauldin J, LeSage G. Bile acid depletion and repletion regulate cholangiocyte growth and secretion by a phosphatidylinositol 3-kinase-dependent pathway in rats. Gastroenterology 2002; 123: 1226-1237  
93   Gaudio E, Barbaro B, Alvaro D, Glaser S, Francis H, Franchitto A, Onori P, Ueno Y, Marzioni M, Fava G, Venter J, Reichenbach R, Summers R, Alpini G. Administration of r-VEGFA prevents hepatic artery ligation induced bile duct damage in bile duct ligated rats. Am J Physiol 2006; [Epub ahead of print]  
94   Gaudio E, Barbaro B, Alvaro D, Glaser S, Francis H, Ueno Y, Meininger CJ, Franchitto A, Onori P, Marzioni M, Taffetani S, Fava G, Stoica G, Venter J, Reichenbach R, De Morrow S, Summers R, and Alpini G. Vascular endothelial growth factor stimulates rat cholangiocyte proliferation via an autocrine mechanism. Gastroenterology 2006; 130:1270-1282  
95   Fava G, Glaser S, Phinizy JL, Francis H, Marucci L, Benedetti A, Taffetani S, Venter J, Baumann B, Reichenbach R, Alpini G. Thyroid hormone inhibits cAMP dependent proliferation of cholangiocytes from bile duct ligated rats by a IP3/Ca2+/PKC-dependent mechanism. Hepatology 2003; 38: A1097  
96   Glaser S, Katki K, Supowit S, Francis H, Ueno Y, Venter J, Reichenbach R, Dickerson I, Summers R, Chiasson V, DiPette DJ, Alpini G. Alpha-calcitonin gene-related peptide (Alpha-CGRP) stimulates the proliferation of large cholangiocytes during obstructive cholestasis induced by bile duct ligation (BDL) via cAMP-dependent activation of MAPK p38. Hepatology 2005; 42: A13  
97   Supowit SC, Zhao H, DiPette DJ. Nerve growth factor enhances calcitonin gene-related peptide expression in the spontaneously hypertensive rat. Hypertension 2001; 37: 728-732  
98   Servillo G, Della Fazia MA, Sassone-Corsi P. Coupling cAMP signaling to transcription in the liver: pivotal role of CREB and CREM. Exp Cell Res 2002; 275: 143-154  
99   Moriuchi A, Ido A, Nagata Y, Nagata K, Uto H, Hasuike S, Hori T, Hirono S, Hayashi K, Tsubouchi H. A CRE and the region occupied by a protein induced by growth factors contribute to up-regulation of cyclin D1 expression in hepatocytes. Biochem Biophys Res Commun 2003; 300: 415-421  
100Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997; 276: 60-66  
101Choi EJ, Xia Z, Villacres EC, Storm DR. The regulatory diversity of the mammalian adenylyl cyclases. Curr Opin Cell Biol 1993; 5: 269-273  
102Andrisani OM. CREB-mediated transcriptional control. Crit Rev Eukaryot Gene Expr 1999; 9: 19-32  
103Alvaro D, Onori P, Metalli VD, Svegliati-Baroni G, Folli F, Franchitto A, Alpini G, Mancino MG, Attili AF, Gaudio E. Intracellular pathways mediating estrogen-induced cholangiocyte proliferation in the rat. Hepatology 2002; 36: 297-304  
104Seto-Young D, Zajac J, Liu HC, Rosenwaks Z, Poretsky L. The role of mitogen-activated protein kinase in insulin and insulin-like growth factor I (IGF-I) signaling cascades for progesterone and IGF-binding protein-1 production in human granulosa cells. J Clin Endocrinol Metab 2003; 88: 3385-3391  
105Clawson GA. Mechanisms of carbon tetrachloride hepatotoxicity. Pathol Immunopathol Res 1989; 8: 104-112  
106Lakehal F, Wendum D, Barbu V, Becquemont L, Poupon R, Balladur P, Hannoun L, Ballet F, Beaune PH, Housset C. Phase I and phase II drug-metabolizing enzymes are expressed and heterogeneously distributed in the biliary epithelium. Hepatology 1999; 30: 1498-1506  
107Katayanagi K, Van de Water J, Kenny T, Nakanuma Y, Ansari AA, Coppel R, Gershwin ME. Generation of monoclonal antibodies to murine bile duct epithelial cells: identification of annexin V as a new marker of small intrahepatic bile ducts. Hepatology 1999; 29: 1019-1025  
108Diakonova M, Gerke V, Ernst J, Liautard JP, van der Vusse G, Griffiths G. Localization of five annexins in J774 macrophages and on isolated phagosomes. J Cell Sci 1997; 110 (Pt 10): 1199-1213  
109Kurosawa H, Que FG, Roberts LR, Fesmier PJ, Gores GJ. Hepatocytes in the bile duct-ligated rat express Bcl-2. Am J Physiol 1997; 272: G1587-1593  
110Charlotte F, L’Hermine A, Martin N, Geleyn Y, Nollet M, Gaulard P, Zafrani ES. Immunohistochemical detection of bcl-2 protein in normal and pathological human liver. Am J Pathol 1994; 144: 460-465  
111Nguyen T, Shapiro DA, George SR, Setola V, Lee DK, Cheng R, Rauser L, Lee SP, Lynch KR, Roth BL, O’Dowd BF. Discovery of a novel member of the histamine receptor family. Mol Pharmacol 2001; 59: 427-433  
112Dickenson JM. Stimulation of protein kinase B and p70 S6 kinase by the histamine H1 receptor in DDT1MF-2 smooth muscle cells. Br J Pharmacol 2002; 135: 1967-1976  
113Mitsuhashi M, Mitsuhashi T, Payan D. Multiple signaling pathways of histamine H2 receptors. Identification of an H2 receptor-dependent Ca2+ mobilization pathway in human HL-60 promyelocytic leukemia cells. J Biol Chem 1989; 264: 18356-18362  
114Garcia-Sainz JA, Macias-Silva M, Olivares-Reyes A, Romero-Avila MT. Histamine activates phosphorylase and inositol phosphate production in guinea pig hepatocytes. Eur J Pharmacol 1992; 227: 325-331  
115Francis H, Taffetani S, Glaser S, Venter J, Phinizy JL, Reichenbach R, Fava G, Alvaro D, Marucci L, Benedetti A, Marzioni M, Alpini G. Histamine stimulates cholangiocyte proliferation through transduction pathways involving the H1 and H2 histamine receptor subtypes. Gastroenterology 2004; 126: AT925  
116Francis H, Glaser S, Venter J, Reichenbach R, Fava G, Alpini G. H3/4 histamine receptor agonists inhibit cholangiocyte growth in bile duct ligated (BDL) rats by negative regulation of the cAMP-dependent PKA/ERK1/2 pathway. FASEB J 2005; 19: 480.417  
117Francis H, Glaser S, Venter J, Taffetani S, Reichenbach R, Fava G, Marucci L, Benedetti A, Alvaro D, Summers R, Wyndham M, Vaculin S, Alpini G. The specific H3/H4 histamine receptor (HR) agonist, RAMH, inhibits cholangiocyte proliferation in bile duct ligated (BDL) rats by negative regulation of the cAMP- dependent PKA/ERK1/2 pathway. Hepatology 2004; 40: 468  
118Hughes K, Antonsson A, Grundstrom T. Calmodulin dependence of NFkappaB activation. FEBS Lett 1998; 441: 132-136  
119Lipskaia L, Lompre AM. Alteration in temporal kinetics of Ca2+ signaling and control of growth and proliferation. Biol Cell 2004; 96: 55-68  
120Glaser S, Francis H, Venter J, Reichenbach R, Ueno Y, Summers R, Alpini G. De novo proliferation of small cholangiocytes requires the activation of the calcium-dependent transcription factor NFATc1/c4. Hepatology 2005; 42: A451  
121Cano E, Canellada A, Minami T, Iglesias T, Redondo JM. Depolarization of neural cells induces transcription of the Down syndrome critical region 1 isoform 4 via a calcineurin/nuclear factor of activated T cells-dependent pathway. J Biol Chem 2005; 280: 29435-29443  
122Santini MP, Talora C, Seki T, Bolgan L, Dotto GP. Cross talk among calcineurin, Sp1/Sp3, and NFAT in control of p21(WAF1/CIP1) expression in keratinocyte differentiation. Proc Natl Acad Sci U S A 2001; 98: 9575-9580  
123Marucci L, Alpini G, Glaser SS, Alvaro D, Benedetti A, Francis H, Phinizy JL, Marzioni M, Mauldin J, Venter J, Baumann B, Ugili L, LeSage G. Taurocholate feeding prevents CCl4-induced damage of large cholangiocytes through PI3-kinase-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 2003; 284: G290-301  
124Marzioni M, LeSage GD, Glaser S, Patel T, Marienfeld C, Ueno Y, Francis H, Alvaro D, Tadlock L, Benedetti A, Marucci L, Baiocchi L, Phinizy JL, Alpini G. Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct-ligated rats. Am J Physiol Gastrointest Liver Physiol 2003; 284: G837-852  
125Misra S, Ujhazy P, Gatmaitan Z, Varticovski L, Arias IM. The role of phosphoinositide 3-kinase in taurocholate-induced trafficking of ATP-dependent canalicular transporters in rat liver. J Biol Chem 1998; 273: 26638-26644  
126Marzioni M, Glaser S, Francis H, Taffetani S, Marucci L, Benedetti A, Alvaro D, Phinizy JL, Baumann B, Venter J, Ueno Y, Alpini G. Taurocholate feeding prevents the functional damage of intrahepatic bile ducts induced by adrenergic denervation in a PI3K dependent manner. Hepatology 2003; 38: A28  
127Marzioni M, Francis H, Benedetti A, Ueno Y, Fava G, Venter J, Reichenbach R, Mancino MG, Summers R, Alpini G, Glaser S. Ca2+-dependent cytoprotective effects of ursodeoxycholic and tauroursodeoxycholic acid on the biliary epithelium in a rat model of cholestasis and loss of bile ducts. Am J Pathol 2006; 168:398-409  
128Strazzabosco M, Fabris L, Spirli C. Pathophysiology of cholangiopathies. J Clin Gastroenterol 2005; 39: S90-102  
129Macdonald P, Palmer J, Kirby JA, Jones DE. Apoptosis as a mechanism for cell surface expression of the autoantigen pyruvate dehydrogenase complex. Clin Exp Immunol 2004; 136: 559-567  
130Adams DH, Afford SC. Effector mechanisms of nonsuppurative destructive cholangitis in graft-versus-host disease and allograft rejection. Semin Liver Dis 2005; 25: 281-297  
131Xu WH, Ye QF, Xia SS. Apoptosis and proliferation of intrahepatic bile duct after ischemia-reperfusion injury. Hepatobiliary Pancreat Dis Int 2004; 3: 428-432  
132Nakanuma Y. Necroinflammatory changes in hepatic lobules in primary biliary cirrhosis with less well-defined cholestatic changes. Hum Pathol 1993; 24: 378-383  
133Ishibashi H, Shimoda S, Gershwin ME. The immune response to mitochondrial autoantigens. Semin Liver Dis 2005; 25: 337-346  
134Bjornsson E, Boberg KM, Cullen S, Fleming K, Clausen OP, Fausa O, Schrumpf E, Chapman RW. Patients with small duct primary sclerosing cholangitis have a favourable long term prognosis. Gut 2002; 51: 731-735  
135Khan SA, Thomas HC, Davidson BR, Taylor-Robinson SD. Cholangiocarcinoma. Lancet 2005; 366: 1303-1314  
136Curry MP, Hegarty JE. The gallbladder and biliary tract in cystic fibrosis. Curr Gastroenterol Rep 2005; 7: 147-153  
137Kinnman N, Lindblad A, Housset C, Buentke E, Scheynius A, Strandvik B, Hultcrantz R. Expression of cystic fibrosis transmembrane conductance regulator in liver tissue from patients with cystic fibrosis. Hepatology 2000; 32: 334-340  
138Schlenker T, Romac JM, Sharara AI, Roman RM, Kim SJ, LaRusso N, Liddle RA, Fitz JG. Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes. Am J Physiol 1997; 273: G1108-1117  
139Roman RM, Feranchak AP, Salter KD, Wang Y, Fitz JG. Endogenous ATP release regulates Cl- secretion in cultured human and rat biliary epithelial cells. Am J Physiol 1999; 276: G1391-1400  
140Perrone RD, Grubman SA, Murray SL, Lee DW, Alper SL, Jefferson DM. Autosomal dominant polycystic kidney disease decreases anion exchanger activity. Am J Physiol 1997; 272: C1748-1756  
141Arima T, Suita S, Shono T, Shono K, Kinugasa Y. The progressive degeneration of interlobular bile ducts in biliary atresia: an ultrastructural study. Fukuoka Igaku Zasshi 1995; 86: 58-64   
142Desmet VJ. Vanishing bile duct disorders. Prog Liver Dis 1992; 10: 89-121  
143Poupon R, Chazouilleres O, Poupon RE. Chronic cholestatic diseases. J Hepatol 2000; 32: 129-140  
144Celli A, Que FG, Gores GJ, LaRusso NF. Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes. Am J Physiol 1998; 275: G749-757  

Figures

 

Figure 1 [Top] Isolation of small (A), approximately 8 mm diameter] and large (B), approximately 14 mm diameter] cholangiocytes from small and large ducts, respectively, from normal rats. Small and large cholangiocytes were purified by counterflow elutriation followed by immunoaffinity purification. Original magn., × 625. Reproduced with permission from Ref[12]. [Bottom] Isolation of small (C) and large (D) IBDU from normal rat liver. Small (< 15 mm in diameter) and large (> 15 mm in diameter) IBDU were pruned off from large ducts by a nitrogen pulsed dye laser and subsequently separated (D) by picking up IBDU with a micromanipulator micropipet. Original magnification × 2000. Reproduced with permission from Ref 13.

Figure 1

 

 


 

Figure 2 Measurement of H3 histone gene expression in small and large cholangiocytes from 1-wk BDL rats and 1-wk BDL rats treated with CCl4 or mineral oil. H3 histone gene expression in large cholangiocytes decreased on d 2 before returning to control values on d 7 after CCl4 treatment. H3 histone gene expression (which was absent in small cholangiocytes from BDL rats) was expressed by small cholangiocytes on d 1 and 2 before returning to control undetectable values on d 7 after CCl4 treatment. Administration of mineral oil to 1-wk BDL rats did not alter H3 histone gene expression in large cholangiocytes. The message for H3 histone gene was absent in small cholangiocytes from oil-treated rats. Comparability of RNA used was assessed by hybridization for GAPDH (housekeeping gene). Autoradiograms were quantified by densitometry. Densitometric values are means of 2 experiments. Reproduced with permission from Ref 50.

Figure 2

 

 

 


Figure 3 Working model for the heterogeneity of the intrahepatic biliary epithelium. The model proposes that: (1) bile ducts are morphologically heterogeneous with small ducts lined by small cholangiocytes and large ducts lined by large cholangiocytes; (2) small and large ducts similarly express both g-GT and cytokeratin-19; and (3) large (but not small) ducts express the secretin and somatostatin receptor, CFTR and Cl-/HCO3- and respond physiologically to these two hormones. The model also shows that following BDL, only large cholangiocytes proliferate and that a single dose of CCl4 induces damage and loss of large duct function, whereas small cholangiocytes (resistant to CCl4) de novo proliferate and secrete to compensate for the loss of large duct function. Reproduced with permission from Ref. 73.

AN-heterogeneityF03.jpg 



Tables

Table 1 Terminology and relationship between human and rat intrahepatic bile ducts

Table 1

 

 

 


 

Table 2 Expression and function of proteins and surface transporters in small and large ducts from rats and human

Table 2


http://www.biology-online.org/articles/heterogeneity_intrahepatic_biliary_epithelium/abstract.html