Journal of Hepatology
Volume 31, Issue 1 , Pages 179-191, July 1999

Regulation of cholangiocyte bile secretion

  • Leonardo Baiocchi

      Affiliations

    • Department of Medical Physiology, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, USA
    • ,
  • Gene LeSage

      Affiliations

    • Department of Internal Medicine, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, USA
    • ,
  • Shannon Glaser

      Affiliations

    • Division of Research and Education, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, USA
    • ,
  • Gianfranco Alpini

      Affiliations

    • Corresponding Author InformationGianfranco Alpini, Assistant Professor, Internal Medicine and Medical Physiology, The Texas A & M University System, Health Science Center, College of Medicine and Central Texas Veterans Health Care System, 1901 South 1st Street, Bldg. 147, Temple, TX, 76504, USA. Tel: 254 774 8742. Fax: 254 771 5725 and 254 724 7113.
    • Department of Internal Medicine, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, USA
    • Department of Medical Physiology, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, USA
    • Central Texas Veterans Health Care System, Temple, TX, USA

Article Outline

 

The intrahepatic biliary system is comprised of a set of anastomosing and progressively larger tubes resembling a tree lined by cholangiocytes 1., 2.. The major function of the biliary system is modification of canalicular bile by secretory and reabsorptive processes in cholangiocytes as bile passes through bile ducts before reaching the duodenum 1., 3., 4., 5., 6., 7., 8.. In contrast to hepatocytes, where secretion is primarily unregulated and constant (9), bile duct secretion is modified by hormones, peptides, nerves and biliary constituents 1., 3., 4., 5., 6., 7., 8., 10., 11., 12., 13., 14., 15., 16., 17., 18., 19., 20., 21., 22., 23., 24.. The bile duct system attracts attention from clinicians, pathologists, physiologists and cell biologists because it is the target for a variety of liver diseases including primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), graft vs. host disease, cystic fibrosis and idiopathic ductopenic disorders 1., 2..

This review focuses on the mechanisms regulating ductal bile secretion. The structural and morphologic features of the biliary system will first be reviewed along with a discussion of newly developed experimental models of isolated cells 1., 5., 6., 7., 8., 10., 11., 12., 25., 26., isolated intrahepatic bile ducts (IBDUs) 4., 24., 27., 28., 29. and cultured cell systems 17., 23., 30., 31.. The mechanisms of fluid and solute transport in cholangiocytes will also be discussed. Gastrointestinal hormones, peptides and neuropeptides work together to finely tune ductal bile secretion by balancing control through mutual stimulation or inhibition.

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Morphologic Features of the Intrahepatic Biliary System 

Classically, the intrahepatic biliary system has been divided into three segments, based upon duct size 1., 2., 32., 33., 34.. These include the extrahepatic bile duct, large bile ducts (visible to the naked eye) and intrahepatic small bile ducts that are visualized by microscopy 1., 32., 33., 34.. Further segmentation of bile ducts may be defined upon regionalization of secretory functions 4., 10., 11., 27. (see below). From a functional point of view, bile is transferred from the canalicular space to the smallest bile ducts (approximately 5 μm in diameter) via the duct of Hering 32., 33., 34.. Small bile ducts, lined with 4–5 cholangiocytes, are characterized by the presence of a basement membrane, tight junctions between cells and microvilli projecting into the bile duct lumen 32., 33.. As small branches of a tree, small bile ducts join into intralobular ducts, which range from 20–100 μm in cross-sectional diameter 32., 33., 34.. As bile ducts become larger, the lining cholangiocytes are progressively larger and more columnar in shape 32., 33.. In situ, one bile duct is usually observed in each portal tract; however, in disease states bile ducts may become more numerous (ductal proliferation) or more rare (ductopenia) 1., 2., 32., 33..

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Experimental Models for Studying the Biliary System 

Although much can be understood from the in situ morphologic evaluation of intrahepatic bile ducts, comprehension of their function has required the development of in vivo and in vitro experimental models. In vivo models induce an increase in the numbers of cholangiocytes by proliferation 1., 3., 5., 6., 8., 11., 12., 35., 36.. In these ductal hyperplastic models cholangiocyte secretion markedly increases 1., 3., 5., 6., 8., 11., 27., 37.. The in vitro models are based on techniques for separating IBDUs 4., 24., 27., 28., 29. or isolated cholangiocytes 1., 5., 6., 7., 8., 10., 11., 16., 25., 26., 37., 38. from the remainder of the liver cell populations (Fig. 1).

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  • Fig. 1. 

    Pure subpopulations of small (a, ∼8 μm in diameter) and large (b, ∼15 μm in diameter) cholangiocytes and small (c, <15 μm in diameter) and large (d, >15 μm in diameter) IBDUs obtained from different portions of the intrahepatic biliary tree. Small and large cholangiocytes were obtained by counterflow elutriation (Reproduced with permission from ref. 10). Small ducts were pruned off from large ducts by a pulsed nitrogen laser beam (Reproduced with permission from ref. 4). Note that small and large cholangiocytes (orig. magn., ×625) and small and large IBDUs (orig. magn., ×2100) differ in size and morphological appearance.

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Animal Models of Duct Hyperplasia and Ductopenia 

Cholangiocyte hyperplasia is induced in rats by a number of pathological maneuvers including bile duct ligation (BDL) 1., 3., 5., 8., 36., 39., 70% liver hepatectomy (6) and α-naphthylisothiocyanate (ANIT) feeding (39). In these models, cholangiocytes proliferate increasing their number markedly in the liver 1., 3., 25., 39.. In animals with ductal hyperplasia, a marked increase in ductal secretion is observed after the administration of secretin, whereas in normal rats secretin induces no choleresis 3., 5., 6., 8., 16., 35., 38.. The increase in ductal bile secretion in BDL and ANIT-fed rats is primarily due to both more secreting ducts 3., 5., 6., 8., 16., 35., 38. and an increased responsiveness of cholangiocytes to secretin due to upregulation of secretin receptor (SR) 1., 5., 6., 8., 11., 37., 40.. These models are also important since larger numbers of cholangiocytes can be isolated from hyperplastic as compared to normal liver 3., 7., 11., 25., 26..

In vivo models of bile duct loss would be extremely useful in the study of the cholangiopathies associated with ductopenia (e.g., PBC). We have demonstrated that acute carbon tetrachloride (CCl4) gavage administration induces transient bile duct loss in both normal and BDL rats 41., 42.. Furthermore, CCl4-induced duct loss is primarily due to cholangiocyte apoptosis and the duct damage is associated with transient loss of secretory function 41., 42.. Animal models of persistent ductopenia have not been developed as of yet.

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In Vitro Cholangiocyte Experimental Models 

The understanding of bile duct function leapt forward when a number of groups developed techniques to isolate pure populations of cholangiocytes 1., 5., 6., 7., 8., 10., 11., 12., 15., 16., 25., 26., 37., 43. or IBDUs 4., 13., 14., 15., 24., 27., 28., 29. or to grow cholangiocytes in culture 17., 23., 31., 44., 45., 46.. The techniques for isolating cholangiocytes and IBDUs are reviewed in detail elsewhere 4., 24., 26., 29.. Biliary physiologists have studied cyclic adenosine 3′, 5′-monophosphate (cAMP) synthesis, Cl− channel activity, Cl−/HCO3− exchanger activity, H+/Na− exchanger, water channel activity in isolated cholangiocytes and polarized IBDUs 1., 6., 7., 8., 10., 11., 12., 13., 14., 15., 17., 22., 23., 24., 29., 30., 37., 45., 47., 48., 49., 50., 51.. Polarized IBDUs have the additional advantage of direct assessment of ductal secretion by observation of expansion of the lumen 4., 24., 29.. Direct microinjection of molecules into the duct lumen is also feasible in this model 4., 24., 29.. Pure populations of cholangiocytes and IBDUs have also allowed investigators to demonstrate the presence of receptors (e.g. SR, somatostatin (SSTR2), acetylcholine, estrogen, and gastrin receptors) and transporters or channels (e.g., cystic fibrosis transmembrane regulator (CFTR), Cl−/HCO3−, apical bile acid transporter and water channels) in cholangiocytes. These transporters and receptors are important in ductal bile secretion 1., 4., 5., 6., 7., 8., 10., 11., 12., 13., 17., 22., 24., 29., 30., 37., 38., 43., 45., 47., 48., 50., 51., 52., 53..

Normal rat cholangiocytes have been maintained in long-term cultures 31., 46.. The cell lines originate from enzymatic digests of portal tract and are cultured on specialized media to maintain their growth. These cells have been particularly useful for transport studies since they maintain polarity on a semi-permeable membrane, which allows studies of vectorial transport across both the cholangiocyte basolateral and apical membranes (46). Cholangiocarcinoma cell lines or immortalized human cholangiocytes are also widely employed by investigators in those cases where the function of interest is maintained in transformed cells 17., 23., 44., 45..

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Intracellular Mechanisms of Cholangiocyte Secretion 

Although cholangiocytes comprise only 3–5% of the total liver cell population 1., 3., 25., they secrete up to 10% of total bile flow in rats and 40% in humans (39). The contribution of bile ducts to overall bile flow is usually estimated from choleresis following administration of secretin 3., 5., 6., 8., 39.. The increment in bile flow is associated with an increase in bicarbonate ions in bile and a reciprocal decline in Cl− ions 3., 8., suggesting the Cl−/HCO3− exchanger on the biliary epithelium plays an important role in secretion 6., 10., 13., 15., 24., 29., 30.. Cl− channels are also present on cholangiocytes 11., 17., 23., 45.. These channels appear critical for ductal secretion and may be a rate-limiting step in cholangiocyte secretion 11., 17., 23., 45.. Finally, water channels on the biliary epithelium may also regulate bile secretion through changes in ductal water permeability 22., 49., 50..

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Cl−/HCO3− Exchanger 

The Cl−/HCO3− exchanger on the cholangiocyte apical membrane is thought to be primarily responsible for bicarbonate secretion in bile 6., 10., 14., 15., 24., 29., 30.. Cl−/HCO3− exchanger activity may be assessed in isolated cholangiocytes by a microfluorometric technique from the change in intracellular pH, measured following removal or addition of chloride into the media 6., 10., 14., 15., 24., 29., 30.. Employing this technique, we and others have shown that the Cl−/HCO3− exchanger is stimulated by the hormone secretin 6., 10., 13.. Stimulation of the Cl−/HCO3− exchanger by secretin requires activation of Cl− channels since the effects of secretin are blocked by 5-nitro-2-(3-phenylpro-pylamino) benzoic acid (NPPB), a specific inhibitor of the cAMP-dependent Cl− channels (23). The dependence of the Cl−/HCO3− exchanger activity on Cl− channels is likely due to the absolute requirement for lumenal directed Cl− secretion to replenish biliary Cl− internalized into cholangiocytes by the Cl−/HCO3− exchanger 13., 29., 30.. Alternatively, the depolarization induced by opening of Cl− channels would increase HCO3− internalization by the Na+-HCO3− symporter (51). The increase in intracellular HCO3− and pH directly activates the Cl−/HCO3− exchanger 10., 13., 19., 24., 29., 51.. As the third mechanism, insertion of CFTR or Cl−/HCO3− exchanger to the apical cholangiocyte membrane from intracellular cytoplasmic latent stores could activate their activity (30). Consistent with this latter hypothesis, secretin dependent increase in ductal bicarbonate excretion is inhibited by the microtubule inhibitor, colchicine (7). Finally, a fourth potential explanation for secretin-induced choleresis is the activation of a Na+/H+ exchanger by secretin (30). This exchanger would favor intracellular alkalinization in cholangiocytes (30). The Cl−/HCO3− exchanger is subsequently stimulated by high intracellular pH due to the presence of a pH-dependent domain on the exchanger 13., 30.. The finding that amiloride, an inhibitor of Na+/H+ exchanger, inhibits secretin-stimulated ductal secretion supports this hypothesis 13., 54., 55..

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Cl− Channels 

CFTR channels open in response to secretin 11., 23.. Cl− channel activation by secretin occurs by secretin stimulated cAMP synthesis, which activates protein kinase A (PKA), which subsequently activates, by phosphorylation, the Cl− channel CFTR (14). Two additional cAMP-independent Cl− channels have been identified in cholangiocytes 56., 57.. One is intracellular Ca2+-dependent, characterized electrophysiologically by outward rectification and K+ conductance and is inhibited by the Cl− channel blocker, 4,4′-diisothiocy-anatostibene-2,2′-disulfonic acid (DIDS) 56., 57.. The other chloride channel is Ca2+-insensitive and inhibited by pertussis toxin (58). The supplemental role of non-CFTR Cl− channels in ductal secretion is supported by findings that the presence of Ca2+-activated Cl− channels in CFTR (−/−) knockout mice prevents cystic fibrosis bile duct lesions (58). Although these channels have the potential to enhance ductal secretion as CFTR, the role of these two additional chloride channels in ductal secretion is unknown.

In addition to the cAMP activation of Cl− channels, other investigators have demonstrated that the stimulation of purinergic P2u receptors on cholangiocyte apical membranes activates Cl− channels (54). Physiologic concentrations of ATP in bile activate the P2u receptor, which mobilizes [Ca2+]i stores and activates Ca2+-dependent apical membrane Cl− channels 54., 56.. These findings raise the possibility that ATP secreted by hepatocytes into bile may control Ca2+-dependent Cl− channel and ductal bile secretion through activation of P2u receptors. This signaling through ATP may represent paracrine regulation of ductal secretion and crosstalk between hepatocytes and cholangiocytes.

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Water Channels 

As a complement to ion transporters/exchangers/channels in the biliary epithelium, the movement of fluid into ductal bile might involve water channels (aquaporins) 22., 49., 50.. Dr. LaRusso's laboratory has demonstrated the presence of aquaphorin-1 in biliary epithelium 22., 49., 50.. In the kidney-collecting tubule and bladder epithelium, water channel activity is regulated by vasopressin (59). In the biliary epithelium, secretin increases aquaphorin activity in the cholangiocyte apical membrane by stimulating the movement of latent inactive aquaphorin (associated with internal cholangiocyte cytoplasmic vesicles) to the cholangiocyte apical membrane where they become active water channels 22., 49.. It has been proposed that a portion of the secretin-induced choleresis is dependent upon activation of water channels in cholangiocytes 22., 49..

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Hormone/Peptide Regulation of Cholangiocyte Secretory Activity 

The role of various gastrointestinal hormones, peptides, cholinergic and purinergic pathways is summarized in Table 1.

TABLE 1. Effect of gastrointestinal hormones, peptides, and innervation on ductal bile secretion
Hormones/peptidesReceptorSecond messengerEffect on ductal bile secretion
SecretinSecretin receptorcAMP ↑Induces a bicarbonate-rich choleresis in BDL rats
SomatostatinSSTR2cAMP ↓Inhibition of basal and secretin-induced choleresis in BDL rats
GastrinGastrin/CCK B* IP3, [Ca2+]i, PKC ↑* No effect on basal bile flow
* No effect on basal cAMP* Inhibits secretin-induced bicarbonate-rich choleresis in BDL rats
* Inhibits secretin-induced cAMP synthesis
Bombesin* Likely* No effect on cAMP, cGMP or [Ca2+]IIncreases bile flow through stimulation of Cl−/HCO3− exchanger
* Direct localization to be defined* To be defined
Substance P??* No effect on ductal fluid secretion
VIP* LikelycAMP?Increases bile flow through stimulation of Cl−/HCO3− exchanger
* Direct localization to be defined
InsulinInsulin receptor* No effect on basal cAMP* No effect on basal bile secretion
* Inhibits secretin-induced cAMP synthesisInhibits secretin-induced bicarbonate-rich choleresis
EndothelinET a, ET b* IP3, [Ca2+]I* No effect on basal bile flow
* No effect on basal cAMP* Inhibits secretin-induced bicarbonate-rich choleresis
* Inhibits secretin-induced cAMP synthesis
AcetylcholineM3 ACh* IP3, [Ca2+]IIncreases basal and secretin-stimulated bicarbonate secretion
* Cross-talking between [Ca2+]I and cAMP
* Increases secretin stimulated cAMP synthesis
Purinergic modulationPurinergic receptor* [Ca2+]ICa2+-dependent Cl− secretion in bile
* Independent on CFTR pathway

Secretin 

Secretin is a 27-aminoacid peptide produced from S cells located in the mucosa of the proximal part of the small intestine (60). Its physiological functions during digestion include stimulation of bicarbonate-enriched pancreatic and duodenal secretion and inhibition of gastric HCl release (61). Secretin plays a key role in the regulation of secretory processes of the intrahepatic biliary epithelium by interaction with specific receptors located solely on cholangiocytes in rat liver (37), which induces an increase in intracellular cAMP levels 5., 6., 7., 8., 12.. Increased cAMP levels lead to opening of low conductance Cl− channels (i.e., CFTR) 17., 23., and activation of the apically located Cl−/HCO3− exchanger 6., 13., 24., 30. which results in bicarbonate secretion in bile 3., 5., 8.. The effect of secretin on cholangiocyte secretion is depicted in Fig. 2.

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  • Fig. 2. 

    Schematic representation of the effect of secretin on the cholangiocyte secretion. Secretin interacts with basolateral receptors and increases intracellular cAMP synthesis, which acts via PKA to phosphorylate and activate CFTR. CFTR functions as a Cl− channel. CFTR, a member of the ATP-binding cassette proteins, has two ATP binding sites, and ATP-binding is required for Cl− channel opening. Maintenance of the Cl−/HCO3− exchanger is dependent upon the Cl− channel function of CFTR, presumably due to the requirement for CFTR to return Cl− to the duct lumen that is internalized into cholangiocytes by the Cl−/HCO3− exchanger.

The second messenger systems involved in secretin-stimulated cholangiocyte secretion have been partially elucidated (14). Studies by Alvaro et al. (14), have shown that the PKA signaling system plays an important role in the regulation of secretin-stimulated Cl−/HCO3− exchanger and ductal fluid secretion. In these studies (14), Sp-adenosine 3′,5′-cyclic monophosphothiolate (Sp-cAMPs), a PKA-specific agonist, markedly increased bicarbonate secretion in cholangiocytes whereas Rp-cAMPs, a specific PKA inhibitor (14), decreased secretin-induced stimulation of fluid secretion and Cl−/HCO3− exchanger. The protein kinase C (PKC) system did not alter basal or secretin-stimulated fluid secretion and Cl−/HCO3− exchanger activity (14). Once CFTR is activated by cAMP-PKA-dependent phosphorylation, the cascade of the secretory processes occurs by a dephosphorylation step driven by endogenous protein serine/threonine protein phosphatases 1 and 2A (14). These phosphatases, by dephosphorylating the regulatory domain of CFTR, promote conformational changes opposite to those induced by PKA, which lead to occlusion of the Cl− conductance pathway, thus restoring the basal quiescent state (14). This is a further example of how an important cellular function is regulated, at the level of a protein (i.e., CFTR), by a balance between the activities of kinases (activation) and phosphatases (inactivation). In this regulatory balance an important role may be played by alkaline phosphatase 62., 63., which is a protein phosphatase (non-specific) markedly expressed in the liver 3., 64.. Alkaline phosphatase inhibits CFTR in biliary cell lines (65); however, its role in the regulation of cholangiocyte functions is still unknown. Since cholangiocytes are continuously exposed in their apical membrane to high concentrations of alkaline phosphatase in bile (3) and, since this enzyme specifically increases in diseases which target the biliary tree, its impact in the development and progression of these diseases should merit extensive investigation. Recent preliminary data from our group seem to support this concept 62., 63.. These studies show that administration of alkaline phosphatase inhibits ductal bile secretion in BDL rats (20). Kato et al. (7) have also shown that secretin stimulates exocytosis in purified cholangiocytes via a cAMP-dependent, but cGMP-, IP3- and Ca2+-independent mechanism and suggest that exocytosis may play an important role in secretin-stimulated ductal bile secretion.

Heterogeneity of the expression of secretin receptor. Intrahepatic ducts greater than 15 μm in diameter are the major sites where secretin stimulates ductal bile secretion 4., 10., 11.. We isolated pure preparations of small (∼8 μm in diameter) and large (∼15 μm in diameter) cholangiocytes and small (<15 μm in diameter) and large (>15 μm in diameter) IBDUs obtained from different sized segments of the intrahepatic biliary tree 4., 10., 11., 12.. These studies have shown that small cholangiocytes purified from small bile ductules (<15 μm in diameter) do not express SR, CFTR, and Cl−/HCO3− exchanger, whereas medium- and large-sized cholangiocytes (purified from medium and large ducts) express the SR, CFTR, and Cl−/HCO3− exchanger 4., 10., 11., 12.. The studies also indicate that cholangiocytes from small ductules (<15 μm in diameter) do not respond to secretin with increases in intracellular cAMP levels and activation of the Cl−/HCO3− exchanger and chloride efflux which occurs in large cholangiocytes from large (>15 μm in diameter) interlobular bile ducts 4., 10., 11., 12.. In BDL rats SR, CFTR, and Cl−/HCO3− exchanger gene expression, cAMP synthesis, Cl− efflux and Cl−/HCO3− exchanger activity are also restricted to large IBDUs and large isolated cholangiocytes 11., 27.. The latter finding indicates functional heterogeneity is maintained in rats with ductal hyperplasia.

Role of secretin receptor in the regulation of cholangiocyte growth. Recent findings from our laboratory suggest that the expression of the SR in cholangiocytes may also have important pathophysiological implications in the progression of cholestatic liver diseases. For example, marked downregulation of SR gene expression is observed when cholangiocyte proliferation is reduced in BDL rats by vagotomy (18) or bile acid depletion (66). In contrast, SR is upregulated with all forms of cholangiocyte proliferation studied to date 1., 3., 5., 6., 8., 11., 12., 27., 37., 38., 43.; thus cholangiocyte stimulation by secretin could be a potential pathway for the modulation of cholangiocyte proliferation. We have also shown that cholangiocyte cAMP levels [a functional assay for the SR 4., 5., 6., 7., 8., 10., 11., 12., 38., 43.] are increased with cholangiocyte proliferation 5., 6., 8., 11., 12., 43. and are depressed with reduced cholangiocyte proliferation 18., 66.. These findings are consistent with studies showing that the cAMP system regulates cellular growth in other cell systems 67., 68..

Somatostatin 

Somatostatin is a cyclic tetradecapeptide secreted by pancreatic islets (D-cells), gastrointestinal tract, nervous system and thyroid gland (61). Somatostatin has antisecretory properties in a number of organs including stomach (69), intestine (70), and pancreas (71). In the liver, somatostatin decreases both basal and secretin-stimulated ductal bile secretion and bicarbonate secretion in BDL rats by interacting with a subtype of somatostatin (SSTR2) receptor expressed only by cholangiocytes in rat liver (8). The interaction of somatostatin with its receptor leads to a decrease in the second messenger system, cAMP 8., 12., and membrane Cl− efflux (72). In support of the concept of the inhibitory effects of somatostatin on cholangiocyte secretory processes, Tietz et al. have shown that somatostatin inhibits secretin-stimulated exocytic insertion of vesicles in purified cholangiocytes (8), which may play an important role in the secretion of ductal bile. This inhibitory effect of somatostatin on cholangiocyte bile secretion is more evident in animal models of ductal hyperplasia (e.g., BDL) 8., 12. where cholangiocyte secretory activity is increased 1., 3., 5., 8., 11., 37., 39..

The anatomical sites of somatostatin inhibition of cholangiocyte secretory processes are mainly large cholangiocytes in large ducts (12). To obtain these findings, we have isolated two distinct subpopulations of small (∼ mean diameter 8 μm) and large (∼ mean diameter 15 μm) cholangiocytes from different portions of the biliary tree of BDL rats and shown that: (i) SSTR2 receptor is only expressed by large cholangiocytes; and (ii) somatostatin decreases ductal secretory activity [evidenced by depressed SR gene expression, cAMP synthesis (12)] only in large cholangiocytes Fig. 3, Fig. 4 that express the SSTR2 receptor. These findings demonstrated that a cooperative regulation of ductal bile secretion exists between secretin and SSTR2 receptors 8., 12..

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  • Fig. 3. 

    Cartoon showing the heterogeneity of the secretin and somatostatin (SSTR2) receptor in different-sized (small and large) cholangiocytes along the length of the intrahepatic biliary tree. Note that small cholangiocytes in small ducts do not express the secretin or SSTR2 receptor and do not respond physiologically to either secretin or somatostatin. In contrast, large cholangiocytes contain the SR and respond to secretin with increases in cAMP levels, opening of Cl− channels opening and activation of Cl−/HCO3− exchanger activity. Stimulation of the SSTR2 receptor in large cholangiocytes leads to depressed cAMP synthesis and to a decrease in Cl− channels opening and Cl−/HCO3− exchanger activity.

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  • Fig. 4. 

    (Top) Genetic expression of SSTR2 mRNA in small and large cholangiocytes purified from BDL rats by counterflow elutriation. Note that SSTR2 gene expression is restricted to large cholangiocytes in large bile ducts. (Bottom) Effect of somatostatin on basal and secretin-induced cAMP levels in small and large cholangiocytes from BDL rats. Note that somatostatin induces a decrease in secretin-stimulated cAMP synthesis.* p<0.05 vs.secretin-stimulated cAMP levels. (Reproduced with permission from reference 12).

In addition to inhibition of cholangiocyte secretion, somatostatin also inhibits cholangiocyte proliferation by specifically interacting with SSTR2 receptor 8., 12. in BDL rats 12., 36. and prevents the growth of human cholangiocarcinoma cells in vitro or in vivo when implanted in athymic mice (73). Moreover, we have shown (12) that somatostatin inhibition of cholangiocyte growth is restricted to large cholangiocytes expressing the SSTR2 receptor), but not in small cholangiocytes where SSTR2 is absent.

Gastrin 

Gastrin is a gastrointestinal hormone secreted by antral G cells in the stomach that stimulates gastric acid secretion (74). Gastrin receptors belong to the superfamily of cholecystokinin (CCK-A and CCK-B) receptors (75). CCK modulates secretion and growth in a number of epithelia by interaction with CCK-A receptors through the cAMP PKA-dependent system (76), whereas gastrin exerts its physiological functions by interaction with gastrin/CCK-B receptors that activate IP3 and intracellular Ca2+ ([Ca2+]I) transduction pathways through PKC-, IP3-dependent mechanisms 77., 78.. In the liver, gastrin has no effect on basal bile flow or bicarbonate secretion of both normal and BDL rats (5). However, gastrin inhibits in vivo secretin-induced bicarbonate-rich choleresis (Fig. 5) and depresses SR gene expression and cAMP synthesis in cholangiocytes by selectively interacting with specific gastrin/CCK-B receptor (5). To extend these findings we have evaluated gastrin transduction pathway in the intrahepatic biliary epithelium and showed that the inhibitory effect of gastrin on secretin-stimulated ductal bile secretion is dependent on [Ca2+]i and PKC pathways (Glaser, LeSage and Alpini, unpublished observations, 1999). The inhibitory effects of gastrin on secretin-stimulated cholangiocyte secretion were blocked by L-362,260 [a specific inhibitor of CCK-B receptor (79)], H7 [a specific PKC inhibitor (80)] and BAPTA/AM [a chelator of intracellular Ca2+ (15)] but not L-364,718 [a specific inhibitor of CCK-A receptor (80)].

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  • Fig. 5. 

    Effect of secretin and gastrin on bile secretion (A) and biliary bicarbonate excretion (B) in normal and BDL rats. (A) Hormones were infused for 30 min via jugular vein cannula after a 60-min equilibration period with Krebs-Henseleit bicarbonate solution (KRH).* p< 0.05 vs. its basal value. ▴ p<0.05 vs.secretin-stimulated bile secretion. (B) Bicarbonate concentration was measured as total CO2.). * p< 0.05 vs. its corresponding value from normal rat liver ▴ p<0.05 vs. secretin-stimulated bicarbonate secretion of BDL rats. ** p< 0.05 vs. its basal value. (Reproduced with permission from reference 5).

Similar to the antiproliferative and antisecretory effects of somatostatin 12., 36., 73., gastrin administration ablates cholangiocyte growth in BDL rats 20., 21.. We have also shown (Glaser, LeSage and Alpini, unpublished observations, 1999) that gastrin inhibits the growth of cholangiocarcinoma cell lines, Mz-ChA-1 (a gift from Dr. G. J. Fitz, University of Colorado). The inhibitory effect of gastrin on cholangiocarcinoma growth occurs by specific interaction with the gastrin/CCK-B (but not CCK-A) receptor and is dependent on [Ca2+]i and PKC pathways (Glaser, LeSage and Alpini, unpublished observations, 1999) since the inhibitory effects of gastrin on cholangiocyte proliferation are blocked by L-362,260, H7, BAPTA/AM but not L-364,718. The data support the concept that gastrointestinal hormones may play an important role in the regulation of cholangiocyte growth in both ductal hyperplasia and cholangiocarcinoma. The data suggest that modulation of gastrin blood levels may be used as a therapeutic approach to reduce cholangiocyte proliferation present in cholestasis or in neoplastic diseases of bile ducts.

Bombesin 

Bombesin is a tetradecapeptide that induces bowel motility (81) and gallbladder contraction (82) and increases secretion in the stomach and pancreas (83). Bombesin receptors are present in the gastrointestinal tract (81). Some studies 84., 85. indicate that in vivo infusion of bombesin induces bicarbonate-enriched choleresis in dogs. Furthermore, Dr. Boyer's laboratory has demonstrated that cholangiocytes are the major anatomical site of bombesin action in the liver 47., 48.. This study shows that bombesin stimulates the activity of Cl−/HCO3− exchanger in cholangiocytes, which is simultaneously counterbalanced by an increase in Na+/HCO3− symporter activity to allow a constant pH in the cell. The stimulatory effect of bombesin on cholangiocyte secretory processes presumably occurs by interaction with specific receptors, since the dose-dependent increase in secretion in IBDU was inhibited almost completely by a specific bombesin receptor inhibitor, [Tyr4, D-Phe12]-bombesin at 1 μM (48). In these studies 47., 48., stimulation of the Cl−/HCO3− exchanger activity by bombesin was independent of the increase in the second messengers cAMP, cGMP or [Ca2+]i. The demonstration of bombesin secretory activity on cholangiocytes is important since it introduces the novel concept of neuromodulation of cholangiocyte functions by this neuropeptide. Also, the data support the concept that other neuropeptides may play an important regulatory role in biliary transport and secretion. Taking into account that bombesin alters growth and enzyme activities in the pancreas and induces hyperplasia of the fundic mucosa, and stimulates gastrin cell proliferation in the antral mucosa (86), we suggest a possible role of bombesin in the regulation of cholangiocyte growth.

Substance P 

Immunoreactivity for substance P, an 11-aminoacid peptide, is present in nervous fibers of the gastrointestinal tract (87). Previous studies (88) on isolated pancreatic ducts showed that substance P decreased basal and secretin-stimulated fluid secretion. Similarly, in vivo studies in dogs have shown that substance P inhibits the choleretic effect of secretin (89). In contrast, substance P does not seem to play a major role in the regulation of cholangiocyte secretory functions 90., 91.. For example, in purified IBDUs substance P does not increase fluid and electrolyte transport and does not modulate the effect of other gastrointestinal hormones such as secretin, VIP or bombesin.

Vasoactive Intestinal Peptide (VIP) 

VIP is a 28-aminoacid peptide present in central and peripheral nervous tissue 92., 93.. VIP is responsible for several physiologic effects including increased pancreatic and intestinal secretion, inhibition of gastric secretion, and vasodilatation (94). There is evidence that VIP increases bile secretion in humans by directly interacting with bile ducts (95). Preliminary data on isolated IBDUs indicate that VIP induces a bicarbonaterich fluid secretion by activating the Cl−/HCO3− exchanger changer 90., 91., a process that is partially inhibited by the Cl− channel inhibitor, NPPB (96). The choleretic effect induced by VIP is more potent than that induced by secretin and is not inhibited by somatostatin, indicating a signaling pathway different from that of secretin and somatostatin 1., 4., 5., 6., 8., 10., 11., 37.. Although VIP modulates secretory processes in other cell systems via the cAMP-PKA transduction pathway 92., 93., no information exists regarding the intracellular signaling and the signal transduction pathway of VIP in cholangiocytes.

Insulin 

A choleretic effect of insulin on bile secretion has been described in several mammals including dogs, rats, and guinea pigs 97., 98., 99.. Recently, in our laboratory we have begun to address the role of insulin in ductal bile secretion (19). Similar to the effects of gastrin (5) and endothelin (16) on ductal bile secretion, we have shown (19) that insulin has no effect on spontaneous secretory activities of cholangiocytes from normal or BDL rats, but markedly inhibits secretin-induced ductal bile secretion. Specifically, insulin decreased: (i) SR gene expression and both secretin-stimulated cAMP levels and Cl−/HCO3− exchanger activity in purified cholangiocytes; (ii) secretin-stimulated lumen expansion in IBDUs; and (iii) secretin-induced bicarbonate-rich choleresis. The effects of insulin on secretin-induced ductal secretion occur by interaction with specific receptors present on cholangiocytes that are overexpressed after BDL (19). It is possible that the upregulation of insulin receptors and decrease in ductal secretion counterpoises the other factors leading to increased ductal secretion following BDL. In a fashion similar to that observed in hepatocytes, where insulin stimulates cell growth (68), we suggest that the insulin receptor pathway may play a role in the regulation of cholangiocyte growth.

Endothelin 

Endothelins (ET-1, ET-2, and ET-3) are a superfamily of vasoactive peptides that interact with at least two subtypes of receptors, ETA and ETB, that are present in several organs (100). ET-1, a potent vasoconstrictor of 21 amino acids, exerts a number of functions in a variety of organs (101). Human gallbladder epithelial cells in culture (102) and human bile ducts express ET-1 (103). In the liver, ET-1 induces cholestasis, which is associated with an increase in portal pressure when administered to the isolated perfused rat liver 104., 105..

We have recently evaluated the effect of both ET-1 and ET-3 on ductal bile secretion in normal and BDL rat liver (16). In these studies, we found that ETA and ETB receptors are expressed by both small and large cholangiocytes 16., 106.. ET-1 did not alter spontaneous bile flow or bicarbonate secretion in normal or BDL rats but markedly decreased secretin-induced ductal bile secretion in BDL rats by specifically interacting with ETA (but not ETB) receptors (16), since the inhibitory effects of ET-1 on cholangiocyte secretory processes were completely blocked by BQ 610, a specific inhibitor of ETA receptor (107). We have also shown that ET-1 inhibition of ductal bile secretion is restricted to large cholangiocytes. ET-1 inhibited SR gene expression and secretin-stimulated cAMP synthesis in large cholangiocytes and secretin-induced lumenal expansion in large IBDUs from normal or BDL rats (16). In contrast, ET-3 did not alter cholangiocyte secretory processes (16). The inhibitory effects of ET-1 on secretin-induced ductal secretion is likely mediated by the Ca2+- and IP3-dependent transduction pathways (16). Finally, since we found both ETA and ETB receptors in small cholangiocytes as well 16., 106., we suggest the possible role of small cholangiocytes in the regulation of ductal bile secretion via Ca2+- and IP3-dependent mechanisms different from that shown for secretin 1., 3., 5., 7., 8., 10., 11., 23., 37., 39..

Acetylcholine 

The cholinergic pathway plays an important role in modulating gastrointestinal physiology through the regulation of vascular, metabolic and secretory processes as well as motility 108., 109.. In vivo studies in dogs have shown that distal stimulation of the vagus nerve increases bile secretion and biliary bicarbonate secretion, whereas interruption of the cholinergic innervation by vagotomy decreases basal bile secretion and biliary bicarbonate secretion (110). Recent studies from us 15., 18. and others (111) have shown that the cholinergic system is involved in the modulation of functions of the intrahepatic biliary epithelium. For example, acetylcholine (ACh) induces both [Ca2+]i increase and oscillation in isolated IBDUs and purified rat cholangiocytes due to both influx of extracellular Ca2+ and mobilization of thapsigargin-sensitive [Ca2+]i stores (111). Furthermore, we have demonstrated (15) that ACh, by specifically interacting with M3 ACh receptor subtype, increases secretin-stimulated cAMP synthesis and Cl−/HCO3− exchanger activity in both IBDUs and purified cholangiocytes by Ca2+-calcineurin-mediated, PKC-independent modulation of adenyl cyclase.

In support of the concept that the cholinergic system plays a key role in the regulation of cholangiocyte functions, preliminary data from our laboratory (18) show that interruption of the cholinergic innervation by vagotomy in BDL rats: (i) decreases intrahepatic duct mass due to both impaired cholangiocyte proliferation and enhanced cell death by apoptosis; and (ii) inhibits secretin-stimulated choleresis due to the loss of bile ducts and depression of SR gene expression and intracellular cAMP levels in cholangiocytes. Moreover, we have found (112) that maintenance of cholangiocyte cAMP levels, by in vivo forskolin administration to BDL rats, counterpoises the effects of vagotomy on proliferative, apoptotic and secretory cholangiocyte functions. Our data suggest that the cholinergic system, probably via regulation of adenyl cyclase, may be involved in the regulation of proliferative, apoptotic and secretory processes of the intrahepatic biliary epithelium.

Estrogens 

We have recently shown that rat cholangiocytes express estrogen receptors that are overexpressed during cholangiocyte growth following BDL (113). We have also shown that estrogen receptors play an important role in the regulation of cholangiocyte secretion in BDL but not normal rats (113). Specifically, we administered tamoxifen (an estrogen antagonist (114)) for 3 weeks to BDL rats and showed that tamoxifen treatment inhibited ductal bile secretion evidenced by: (i) depressed SR gene expression and both basal and secretin-stimulated intracellular cAMP levels in purified cholangiocytes; and (ii) spontaneous and secretin-stimulated increases in bile flow and biliary bicarbonate secretion (113).

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Conclusions and Future Perspectives 

Ductal bile secretion is the result of cooperative interactions between gastrointestinal hormones (some with stimulating action such as secretin, VIP, and bombesin and some with inhibitory effects such as somatostatin, and gastrin), peptides and nerves. The data related to the neuroregulation of cholangiocyte functions by acetylcholine and bombesin are perhaps the most interesting findings on the regulation of the functions of the intrahepatic biliary epithelium. In this context, preliminary data from our laboratory demonstrated the presence of adrenergic receptor Bl on cholangiocytes (Lesage, Glaser and Alpini, unpublished observations, 1999), which suggests a cooperative contribution of the cholinergic and adrenergic nerves in regulating cholangiocyte functions. Further studies are needed to elucidate the role of the adrenergic pathway in the regulation of ductal secretion. The role of gastrointestinal hormones in the regulation of cholangiocyte growth in cholestatic conditions should also merit attention. Further investigation is necessary to better understand the intracellular transduction pathways of the hormones and neuropeptides affecting biliary functions and the role of small cholangiocytes in the pathophysiology of the biliary epithelium.

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Acknowledgements 

We would like to thank Dr. Domenico Alvaro (University La Sapienza, Rome, Italy) for his constructive criticisms/suggestions during the preparation of this review article. We would also like to acknowledge Bryan Moss for his outstanding artwork and Jo Lynne Phinizy for her technical assistance.

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PII: S0168-8278(99)80180-9

Journal of Hepatology
Volume 31, Issue 1 , Pages 179-191, July 1999

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