Vol. 281, Issue 3, G612-G625, September 2001
INVITED REVIEW
Regulation of cholangiocyte bicarbonate secretion
Noriatsu
Kanno1,2,
Gene
LeSage1,
Shannon
Glaser1, and
Gianfranco
Alpini1,2,3
Departments of 1 Internal Medicine and 2 Medical
Physiology, Scott & White Hospital and Texas A&M University System
Health Science Center, College of Medicine, and 3 Central
Texas Veterans Health Care System, Temple, TX 76504
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ABSTRACT |
The objective
of this review article is to discuss the role of secretin and its
receptor in the regulation of the secretory activity of intrahepatic
bile duct epithelial cells (i.e., cholangiocytes). After a brief
overview of cholangiocyte functions, we provide an historical
background for the role of secretin and its receptor in the regulation
of ductal secretion. We review the newly developed experimental in vivo
and in vitro tools, which lead to understanding of the mechanisms of
secretin regulation of cholangiocyte functions. After a description of
the intracellular mechanisms by which secretin stimulates ductal
secretion, we discuss the heterogeneous responses of different-sized
intrahepatic bile ducts to gastrointestinal hormones. Furthermore, we
outline the role of a number of cooperative factors (e.g., nerves,
alkaline phosphatase, gastrointestinal hormones, neuropeptides, and
bile acids) in the regulation of secretin-stimulated ductal secretion.
Finally, we discuss other factors that may also play an important role
in the regulation of secretin-stimulated ductal secretion.
bile flow; adenosine 3',5'-cyclic monophosphate; cystic fibrosis
transmembrane regulator; chloride/bicarbonate exchanger; gastrointestinal hormones; intrahepatic biliary epithelium; peptides; nerves
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INTRODUCTION |
IN THE LIVER TWO TYPES of epithelia
(i.e., hepatocytes and cholangiocytes) contribute to bile secretion
(12, 27, 34, 73, 105, 132, 135, 136, 140).
Separate hepatic (105) and ductular transport mechanisms
(6, 8, 10, 12, 13, 16, 18, 20, 27, 34, 43-46, 63, 75)
allow for regulation of bile volume and composition required for
changing physiological needs. The bile acid-dependent flow derived from
hepatocyte canalicular secretion accounts for 30-60% of
spontaneous basal bile flow (105). A canalicular bile
acid-independent secretion, probably caused by transport into bile of
organic solutes (glutathione) and inorganic electrolytes, accounts for
30-60% of basal bile flow (105). At the level of the
bile ducts, both secretion and reabsorption of fluid and inorganic
electrolytes modify canalicular bile (6, 10-13, 16, 22, 27,
34, 43-46, 63, 75, 93-95, 119, 120, 135, 143). Ductal
secretion chiefly occurs in response to secretin and represents 30% of
basal bile flow in humans and 10% in rats (11, 140).
Glutathione is present in bile but is almost quantitatively broken down
within the biliary tree by hepatic 
-glutamyltransferase (1,
90, 112). Cholangiocytes possess specific membrane transport systems for a large number of substrates. For example, cholangiocytes absorb glucose (81), bile acids (8, 51, 80),
and amino acids (50, 125) from bile. Human cholangiocytes
are also involved in the transport of secretory component and IgA into
bile (137).
The secretory/absorptive properties of the intrahepatic biliary
epithelium are supported by the presence of microvilli on their apical
pole (31, 71, 79). Ductal bile secretion is coordinately
regulated by a number of factors including nerves (16,
83), enzymes [e.g., alkaline phosphatase (AP)
(17)], gastrointestinal hormones [e.g., secretin
(10, 143), somatostatin (143), and gastrin
(63)], peptides [endothelin-1 (ET-1) (38), bombesin, and vasoactive intestinal polypeptide (VIP) (43,
46)], and bile salts (5, 7) (Table
1).
Pathologically, cholangiocytes are the target cells in a number of
chronic cholangiopathies including primary biliary cirrhosis (PBC) and
primary sclerosing cholangitis (PSC), diseases that are associated with
cholangiocyte proliferation and/or loss (12, 120). In
rodents, cholangiocyte proliferation/loss is achieved by a number of
pathological maneuvers including bile duct ligation (BDL) (10,
12, 62, 63, 83, 85, 143), experimental cirrhosis [induced by
chronic administration of CCl4 (4) or phenobarbital in conjunction with CCl4 (76)],
experimental fascioliasis [induced by oral administration of 20 metacercariae of Fascioliasis hepatica (88)],
partial hepatectomy (84), acute CCl4
administration (85, 86), vagotomy (83), and
chronic feeding of bile acids (7) or the toxin
-naphthylisothiocyanate (ANIT) (11, 32) (Table
2). These models of bile duct
hyperplasia/ductopenia are closely associated with increases (e.g.,
after BDL, ANIT feeding, oral administration of F. hepatica,
cirrhosis, or partial hepatectomy; Refs. 7,
9-11, 32, 62,
63, 83, 84, 88,
143) or decreases (e.g., after CCl4
administration or vagotomy; Refs. 83, 85,
86) in ductal secretion (Table 2).
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MORPHOLOGY OF INTRAHEPATIC BILIARY EPITHELIUM |
Reviews on cholangiocyte secretory functions are available
(12, 19, 27, 34, 73, 120, 132, 135, 136, 140); however, none has specifically focused on the role and mechanisms of action of
secretin in the regulation of cholangiocyte secretion. The biliary duct
system has been classified into three segments based on duct size
(12, 89, 124). This classification includes extrahepatic
bile ducts, large bile ducts, and intrahepatic small bile ducts or
ductules (12, 89, 124). At the functional level, hepatocyte bile is transferred from the bile canaliculus to the smallest bile ducts (~5 µm in external diameter) through the duct of Hering (12, 89, 124). Small intrahepatic bile ducts
(lined by 4-5 cholangiocytes) are characterized by the presence of
a basement membrane, tight junctions between cells, and microvilli projecting into the duct lumen (12, 89, 124). Like small branches of a tree, small bile ducts join into intralobular ducts ranging from 20 to 100 µm in cross-sectional diameter (12, 89, 124). In larger bile ducts the lining cholangiocytes are
progressively larger and more columnar in shape (12, 89,
124).
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SECRETIN FUNCTIONS IN THE BODY |
Secretin [a 27-amino acid neuroendocrine peptide synthesized by
specific endocrine cells, S cells, localized mainly in the mucosa of
the duodenum and proximal jejunum (42, 152, 153)] regulates the physiological functions of many organs including brain
[e.g., activation of tyrosine hydroxylase activity
(69)], pancreas, intestine, and liver (9-11,
39, 85, 86, 153). Secretin stimulates the gastric secretion of
pepsin and inhibits the secretion of gastric acid and food-stimulated
gastrin from G cells in the gastric antrum (152, 153).
Furthermore, secretin affects the motility of the small intestine,
decreases lower esophageal sphincter pressure, relaxes the sphincter of
Oddi, and inhibits postprandial emptying (152, 153).
Secretin increases heart rate and causes dilatation of peripheral blood
vessels (29, 104).
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HISTORICAL BACKGROUND |
Secretin was originally discovered by Bayliss and Starling, who
demonstrated that this hormone stimulates pancreatic secretion and bile
flow in dogs (28). In vivo studies in dogs with BDL by
Rous and McMaster (122) showed that cholangiocytes secrete water and electrolytes in ductal bile. Andrews (23)
proposed a secretory model in which hepatocytes were responsible for
the hepatic metabolic activity, whereas the intrahepatic biliary tree represented the main secretory unit of the liver. On the basis of
measurement of electrolyte excretion in dog bile, Wheeler et al.
(156) identified two distinct anatomic secretory sites,
one responsible for bile acid-dependent bile flow and one (more distal) important for the choleretic effect of secretin. In support of the
secretory capacity of the ductal epithelium, studies in dogs and
rabbits have shown secretion of water and electrolytes by an isolated
segment of the extrahepatic bile duct in situ (103, 131)
and in vitro (40). Studies in dogs with bile fistula
(155) and isolated, perfused pig liver (66)
have shown that secretin increases bicarbonate-rich bile secretion and
that the increase in bile flow is proportional to the logarithm of the
dose of secretin. Furthermore, studies in isolated, perfused pig liver
demonstrated that the electrolyte composition of the fraction of bile
stimulated by secretin is independent of the bile flow before secretin
administration in the perfused preparation (67). Other
studies in dogs have shown that 1) infusion of secretin
via the hepatic artery [the major blood supply of the
intrahepatic biliary epithelium (37, 115)] induced
greater choleresis compared with secretin infusion through the splenic
vein and 2) the biliary tree volume was smaller during
secretin choleresis compared with taurocholate-induced choleresis
(154). These studies suggest that secretin-stimulated bicarbonate-rich bile flow derives from the interaction of this hormone
with intrahepatic bile ducts rather than hepatocytes. However, other
studies have shown that interruption of blood flow to the intrahepatic
biliary epithelium (by short-term ligation of the hepatic artery of
guinea pigs) does not alter cholangiocyte secretory activity
(142). Radiolabeled mannitol and erythritol (used with the
assumption that these carbohydrates are transported across the bile
canaliculus but not the biliary epithelium) have been used to define
the anatomic site of secretin-induced choleresis in guinea pigs
(55). However, recent studies in rats (128) and guinea pigs (141) have questioned the use of these two
molecules (to distinguish between hepatocyte and cholangiocyte bile
secretion), because they can cross the intrahepatic biliary epithelium.
For example, erythritol is not an ideal marker of canalicular bile flow
because it has been shown to cross the rat intrahepatic ductal epithelium, possibly via intercellular junctions (128). In
vivo studies in humans, baboons, and dogs have also shown that
intravenous infusion of secretin increases bile flow (87).
These studies also showed that secretin-induced choleresis is
associated with an increase in cAMP levels in extrahepatic bile duct
tissue in humans and baboons (but not dogs), suggesting that cAMP may
be a second messenger system for secretin (87). Conclusive
evidence for the role of secretin in directly stimulating ductal
secretion came from recent studies (2, 3, 10, 11, 76)
showing that in vivo secretin induces a massive bicarbonate-rich
choleresis [usually absent under normal conditions (10, 11, 63,
84, 143)] in rats with enhanced intrahepatic ductal mass
induced by BDL (2, 3, 10, 11), cirrhosis
(76), and chronic -naphthylisothiocyanate (ANIT)
feeding (11, 32). Secretin-stimulated choleresis results
from the direct interaction of secretin with secretin receptors [SR;
exclusively expressed by cholangiocytes in rat liver (15,
53)]. The interaction of secretin with its receptors induces an
increase in intracellular cAMP levels (6, 9, 13, 14, 62, 63, 75,
83-86, 143) and activation of the Cl
channel
cystic fibrosis transmembrane regulator (CFTR), with subsequent
activation of the Cl/HCO
exchanger
leading to bicarbonate secretion in ductal bile (Fig.
1).
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Fig. 1.
Schematic representation of the intracellular mechanisms by which
secretin, somatostatin, and gastrin coordinately regulate bicarbonate
secretion in ductal bile. Secretin interaction with its own receptor
induces an increase in cAMP-dependent protein kinase (PKA) activity.
Increased PKA activity activates (by phosphorylation) cystic fibrosis
transmembrane regulator (CFTR), leading to activation of the
Cl/HCO exchanger with subsequent
secretion of bicarbonate in water. Somatostatin (through interaction
with SSTR2 receptors) inhibits secretin-stimulated cAMP
levels, Cl channel activation, and
Cl/HCO exchanger activity, leading to
decreased ductal bicarbonate secretion. Gastrin inhibitory effect on
secretin-stimulated bicarbonate secretion is mediated by activation and
membrane translocation of the Ca2+-dependent protein kinase
C (PKC) pathway, leading (by cross talk with adenylate cyclase) to
decreased secretin-induced cAMP synthesis and CFTR and
Cl/HCO3 exchanger
activity.
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EXPERIMENTAL TOOLS FOR EVALUATING SECRETIN-STIMULATED DUCTAL
SECRETION |
In Vivo Models of Cholangiocyte Hyperplasia/Ductopenia
Recently, a number of animal models of ductal hyperplasia
[inducing an increase in the number of intrahepatic bile ducts
(7, 10, 11, 62, 83, 84) or loss of cholangiocytes
(85, 86)] have been developed and have allowed us to
increase our knowledge of the role and mechanisms of action of secretin
in the regulation of ductal secretion (Refs. 7,
10-13, 15, 62, 63, 76, 83-86,
88, 143; see Table 2). Rodent models of cholangiocyte hyperplasia include partial hepatectomy
(84), chronic feeding of bile acids [i.e., taurocholate
(TC) and taurolithocholate (TLC); Ref. 7] or ANIT
(11, 32), cirrhosis [induced by chronic administration of
phenobarbital in conjunction with CCl4 to rats
(76) or chronic CCl4 treatment in mice
(4)], experimental fascioliasis (88), and
BDL (9, 10) (Table 2). These models of duct hyperplasia
are closely associated with increased secretin-stimulated ductal
secretion evidenced by 1) increased SR gene expression (7, 9, 14, 15, 63, 84), secretin-stimulated cAMP levels
(7, 9, 14, 62, 63, 84, 143), and
Cl/HCO3 exchanger activity
(16, 84) in purified cholangiocytes and 2)
enhanced secretin-stimulated bicarbonate-rich choleresis in vivo
(10, 11, 63, 76, 83, 84, 88, 143).
Animal models of ductopenia [i.e., total vagotomy (83)]
or liver toxins [i.e., CCl4 (85, 86)] result
in a decrease or loss of secretin-stimulated ductal secretion.
Interruption of the cholinergic innervation (by total vagotomy of BDL
rats) inhibits SR gene expression, secretin-stimulated cAMP levels, and
secretin-induced bile flow and bicarbonate secretion (83).
Maintenance of cAMP levels by chronic forskolin administration to BDL
rats prevents the inhibitory effects of vagotomy on secretin-stimulated
cholangiocyte secretion (83). In rats, the toxin
CCl4 has been shown to selectively damage large
cholangiocytes (with loss of secretin-stimulated secretory responses),
whereas small cholangiocytes are resistant to
CCl4-induced duct damage and, de novo, express SR and
respond physiologically to secretin to compensate for the loss of
secretin-induced secretion of large ducts (85, 86).
In Vitro Tools
A major advancement in the understanding of the role and
mechanisms of action of secretin in the regulation of ductal secretion came from the development of sophisticated techniques [e.g.,
immunoaffinity separation (71, 75) and micropipetting
(6, 100, 119)] for the isolation and phenotypic
characterization of pure preparations of pooled small and large
cholangiocytes and intrahepatic bile duct units (IBDU) from normal and
cholestatic rat liver (6, 10, 11, 13, 45, 62, 63, 71, 75, 83, 84,
143). Isolated cholangiocytes and IBDU have allowed us to
evaluate the effect of secretin on cAMP levels (6, 9, 13, 14, 16, 62, 63, 75, 83-86, 98, 143), protein kinase A (PKA)
activity (16, 98), Cl channel conductance
(14, 54, 98),
Cl/HCO3 exchanger activity
(13, 16, 18, 84, 134), and water channel activity
(93, 95). IBDU have the advantage (over isolated cholangiocytes) of maintaining cell polarity, permitting direct assessment of secretin-stimulated ductal secretory activity by changes
in ductal lumen and the maintenance of polarized epithelial structure
(6, 38, 100, 119). Other advantages of the IBDU model
include the ability to microinject molecules directly into the duct
lumen or perfusion of the duct lumen (6, 97, 100, 119).
Primary cultures of rat cholangiocytes (151, 157) (which retain phenotypic and functional characteristics of biliary origin) are
pathophysiologically important tools and have allowed us to define the
role of cAMP after stimulation with forskolin (151), a
cAMP activator (77, 83). Studies by McGill et al.
(98) used a sophisticated approach (i.e., patch-clamp
recording technique in whole cells) to demonstrate cAMP-dependent
secretin stimulation of membrane Cl channel (i.e., CFTR)
activity in isolated rat cholangiocytes. Another important tool for
evaluating the mechanisms of secretin-stimulated ductal secretion is
represented by the isolation (by isopycnic centrifugation on sucrose
gradients) and characterization of apical and basolateral plasma
membrane vesicles from polarized normal rat cholangiocyte cultures
(144) and freshly isolated cholangiocytes isolated from
BDL rats (61, 145). The development of this tool has
allowed us to obtain important mechanistic information on the role of
secretin on ductal water channel activity (93-95).
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SECRETIN RECEPTOR |
Secretin receptors belong to a unique family of G protein-coupled
receptors including receptors for VIP, pituitary adenylate cyclase-activating polypeptide, gastric inhibitory peptide, glucagon, glucagon-like peptide 1, calcitonin, calcitonin gene-related peptide, parathyroid hormone, growth hormone-releasing factor, and
corticotropin-releasing factor (148). All the members of
this peptide family possess a remarkable amino acid sequence homology
and bind to G protein-coupled receptors, whose signaling mechanism
primarily involves adenylyl cyclase and/or PKA (148).
These receptors share homology with each other, indicating that
all originate from a common ancestral sequence through gene duplication
(148). SR has seven transmembrane-spanning helical domains
separating loop and tail domains (25). The SR extracellular domains contain sites for asparagine-linked glycosylation and a pair of cysteine residues linking the first and second
extracellular loops through a disulfide bond (25). The
confluence of the transmembrane helices and the extracellular loops
contribute to secretin binding to SR (25). The SR
intracellular domains include domains for G protein binding and sites
for phosphorylation (126). The SR (prototypic of the class
II family of G protein-coupled receptors) contains a long extracellular
amino-terminal domain containing six highly conserved Cys residues and
one Cys residue [Cys(11)] (25). Recent
studies have identified the specific structural and functional domain
of the secretin and its receptor (113). These studies have
shown that the amino-terminal 15 residues of secretin are critical for
the stimulation of SR (113). The studies have also shown
that the amino terminus of the SR is necessary, but not sufficient,
requiring the complementation of an extracellular loop domain for the
physiological response to secretin (113). Secretin binding
to the extracellular domain of SR results in coupling with
heterotrimeric G proteins at a cytosolic domain of SR
(58).
The G proteins consist of -, 
-, and 
-subunits
(114). G proteins are members of a superfamily of GTPases
that are fundamentally conserved from bacteria to mammals and play a
role in many aspects of cell regulation (114).
Ligand-bound SR activates G proteins, which in turn activate adenylyl
cyclase, leading to increased intracellular cAMP accumulation
(114). Characterization of hormone-binding domains of SR
and SR cloning and expression is beyond the scope of this review and
has been recently discussed in detail elsewhere (148).
Recently, the cDNA for the human (47), rat
(70), and rabbit (138) SR gene has been
cloned and functionally characterized. SR is widely distributed in the
body including brain, heart, stomach, intestine, and pancreas
(33, 57, 60, 70). In the liver, in situ autoradiographic
studies of liver sections (53) and in vitro studies in
purified cholangiocytes (15) and cholangiocyte membranes
(52) have shown that SR are exclusively expressed on the
basolateral membrane of rat cholangiocytes.
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MECHANISMS OF SECRETIN-REGULATED DUCTAL SECRETION |
Secretin stimulates ductal secretion by a series of sequential and
coordinated events (6, 10-16, 18, 22, 54, 63, 75, 84, 134,
143). First, secretin interacts with basolateral SR
(95), expressed only by cholangiocytes in rat liver
(15, 53). This interaction induces elevation of cytosolic
cAMP levels, activation of intracellular PKA (22, 41), and
opening of the cAMP-dependent Cl channels by
phosphorylation (41), leading to extrusion of
Cl ions with subsequent depolarization of the cell
membrane (54, 98) (Fig. 1). Opening of Cl
channels (14, 54, 98) induces a Cl gradient
favoring the activation of the apically located
Cl/HCO exchanger (13, 18, 84, 129, 134), which results in secretin-stimulated bicarbonate-rich choleresis (10, 11, 63, 84, 143) (Fig. 1). The
electrogenic Na+-HCO symport is
activated at the basolateral domain of a rat cholangiocyte cell line
(129), as a consequence of depolarization induced by
Cl efflux, and this causes the entry of
HCO into cells. Studies in purified rat
cholangiocytes have shown (18) that the
Na+-HCO symport counteracts bicarbonate excretion by working as an acid extruder, thus contributing to maintenance of intracellular pH. Secretin directly stimulates the
opening of the CFTR channel but does not directly regulate either the
Na+-HCO or the
Na+/H+ exchanger (NHE) of rat cholangiocytes
(18). The NHE3 isoform (which has been recently localized
to the apical membrane of mouse and rat cholangiocytes) plays an
important role in fluid secretion and absorption from bile duct lumen
(101). In pigs, cholangiocyte bicarbonate secretion is
also dependent on carbonic anhydrase II, an enzyme that catalyzes
hydration of carbon dioxide to bicarbonate and hydrogen ions
(36). Insertion of CFTR or
Cl/HCO exchanger into the apical
membrane domain of cholangiocytes from intracellular cytoplasmic stores enhances their activity (20). Other studies in guinea pigs
have shown that secretin-induced choleresis is caused by the activation of NHE by secretin, whose activation causes intracellular
alkalinization in cholangiocytes (68). Recent studies in
cultured human intrahepatic cholangiocytes have shown that human
cholangiocytes express two acid extruders (i.e.,
Na+/H+ exchanger and Na+-dependent
Cl/HCO exchanger) and an acid loader (i.e., Cl/HCO exchanger) but not the
cAMP-dependent H+-ATPase (133). These studies
show that HCO influx is regulated by
Cl/HCO exchange, whereas
Na+-HCO cotransport is inactive at
physiological pH (133). The studies have also shown that
stimulation of Na+-independent
Cl/HCO exchanger by cAMP does not
require activation of Cl conductance. Other studies in
pigs (64, 150) have proposed the role of
H+-ATPase in the regulation of ductal secretion, because
H+-ATPase is inserted into the basolateral cholangiocyte
membrane in response to secretin, thus working as an acid extruder.
However, recent studies in rats have shown that intrahepatic IBDU do
not express H+-ATPase (44).
Recent studies have shown that the PKA system regulates
secretin-stimulated bicarbonate-enriched ductal secretion
(22). These studies have shown that Sp-adenosine
3',5'-cyclic monophosphothiolate (Sp-cAMPS), a PKA-specific agonist,
stimulates ductal bicarbonate secretion, whereas Rp-cAMPS, a specific
PKA inhibitor, decreases secretin-stimulated ductal bicarbonate
secretion (22). The data suggest that secretin-induced
choleresis is regulated, at the level of CFTR, by a balance between the
activities of kinases [inducing activation (22)] and
phosphatases [causing inactivation (17)].
Other studies in rat cholangiocytes have shown that secretin stimulates
insertion of transporters into the apical membrane of cholangiocytes
via a cAMP-dependent but cGMP-, D-myo-inositol 1,4,5-trisphosphate (IP3)-, and
Ca2+-independent mechanism (75, 143).
The transport of water to ductal bile is regulated by membrane
aquaporin water channels (AQP) present in both the basolateral and
apical domains of rat (93, 95, 108, 121) and human
(107) intrahepatic cholangiocytes. In a fashion similar to
the urinary system, in which aquaporin activity in the collecting
tubule and bladder epithelium is modulated by vasopressin
(149), membrane water channels are also regulated in the
rat intrahepatic biliary epithelium (93, 95). In rats,
secretin increases water channel activity in the cholangiocyte apical
membrane by stimulating the movement of latent inactive AQP1
(associated with internal cholangiocyte cytoplasmic vesicles) to the
cholangiocyte apical membrane, where they become active water channels
(93, 95). Secretin-stimulated AQP1 insertion into rat
cholangiocyte apical membrane is microtubule dependent because it is
inhibited by pretreatment of cholangiocytes with colchicine (but not
with its inactive analog -lumicolchicine). These studies
demonstrated that secretin-induced secretion of water and electrolytes
is dependent on activation of latent AQP1 in rat cholangiocytes
(93, 95). Recent studies have also localized AQP4 in the
basolateral membrane of rat cholangiocytes (94). In
contrast to AQP1, which is targeted to the apical cholangiocyte membrane by secretin (93, 95), AQP4 is not regulated by
secretin (94). AQP4 may be important in facilitating
basolateral transport of water in cholangiocytes, an important step in
the regulation of bile secretion. Recent studies in Xenopus
oocytes injected with CFTR demonstrated the presence of CFTR in
vesicles and cAMP-dependent (by forskolin treatment) membrane insertion
of these CFTR-containing vesicles (139). The apical
membrane insertion of CFTR from these vesicles may provide a link
between activation of CFTR and cAMP-dependent (by secretin activation)
regulation of ductal secretion of water and electrolytes.
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FACTORS REGULATING SECRETIN-STIMULATED DUCTAL SECRETION |
Parasympathetic Innervation
In the liver, sympathetic and parasympathetic innervation
originate from the celiac ganglion (sympathetic) and from the vagus nerve (parasympathetic) (116, 147) and innervate the
hepatic artery, the portal vein, the intrahepatic and extrahepatic
biliary epithelium, and parenchymal cells (116, 147).
Recent studies in rats have shown that the parasympathetic system
regulates secretin-stimulated ductal secretion (16, 106).
Nathanson et al. (106) demonstrated that ACh elicits both
Ca2+ increase and oscillation in rat IBDU and isolated
cholangiocytes because of both influx of extracellular Ca2+
and mobilization of thapsigargin-sensitive Ca2+ stores.
Other studies have shown that intrahepatic parasympathetic terminations
release ACh, which interacts selectively with M3 ACh
receptors on cholangiocytes, inducing an increase in
secretin-stimulated cholangiocyte cAMP synthesis and
Cl/HCO3 exchanger activity by
Ca2+-calcineurin-mediated, PKC-independent modulation of
adenyl cyclase (16). In support of the concept that
cholinergic nerves regulate secretin-stimulated ductal secretion,
interruption of the parasympathetic innervation (by total vagotomy) in
BDL rats decreases SR gene expression and secretin-stimulated bile flow
and bicarbonate secretion through a decrease in M3 ACh
receptor expression (83).
Alkaline Phosphatase
AP is a nonspecific protein phosphatase whose precise function is
unknown. Elevated serum AP levels are observed in cholestatic liver
diseases (10, 123). Cholangiocytes are continuously
exposed at their apical membrane to high concentrations of AP in bile (74, 78). Recently, the effects of acute and chronic
administration of AP on secretin-stimulated ductal secretion were
evaluated in vivo in rats with bile fistula and in vitro in purified
rat IBDU (17). In vivo, acute and chronic administration
of AP decreased both basal and secretin-stimulated bile flow and
biliary bicarbonate secretion in BDL rats (17). In vitro,
basal and secretin-stimulated Cl/HCO
exchanger activity of rat IBDU was immediately inhibited by AP
intraluminal microinjection (apical exposure) but only after a
prolonged exposure to the basolateral domain of cholangiocytes
(17). The inhibitory effect of AP (which is transcytosed
from serum to cholangiocyte apical membrane) on secretin-stimulated
ductal secretion may be due to its capacity to block CFTR activity or
to hydrolyze ATP bonds (17). The findings suggest that
elevated serum and biliary AP levels may be not only the result of
cholestasis but also an adaptive reaction for decreasing secretory
activity during bile duct obstruction (17).
Gastrointestinal Hormones
Gastrin.
Gastrin modulates the functions of several epithelia by interaction
with CCK-B/gastrin receptors through Ca2+-,
IP3-, and protein kinase C (PKC)-dependent mechanisms (see Fig. 2) (159, 160). In the
liver, gastrin inhibits secretin-stimulated ductal secretion of BDL
rats at the physiological doses of
109-107 M (63). The
presence of an inhibitory effect of gastrin on secretin-induced ductal
secretion at a physiological dose [blood gastrin concentration of
109-1010 M in rats (82)]
supports the presence of specific, physiologically relevant receptors
for gastrin on cholangiocytes. We suggest that gastrin [similar to
somatostatin (9, 143)] may be physiologically important
in the regulation of enhanced secretin-stimulated ductal secretion in
cholestatic liver diseases by counterpoising the stimulatory effects of
secretin. The inhibitory effect of gastrin on secretin-stimulated
ductal secretion occurs through activation and membrane translocation
of the Ca2+-dependent PKC- (62). These data
suggest that cross-talk between the cAMP/PKA system [by which secretin
stimulates ductal secretion (9, 16, 62, 63, 84-86)]
and the IP3/PKC system [by which gastrin inhibits
secretion and proliferation (62, 159, 160)] may play an
important role in the regulation of overall cholangiocyte secretory
activity.
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Fig. 2.
Intracellular cAMP levels in cholangiocytes from 1-week
bile duct-ligated (BDL) rats. Cholangiocytes were incubated for 1 h at 37°C and subsequently-stimulated for 5 min at 22°C with
1) 0.2% BSA (basal), 2) 100 nM secretin,
3) 1 nM gastrin, 4) 1 nM gastrin + 100 nM
secretin in the presence or absence of L-365,260 (a
specific CCK-B/gastrin receptor antagonist; Ref. 30),
L-364,718 (a specific CCK-A receptor antagonist) (109),
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA)-AM (an intracellular Ca2+ chelator; Ref.
16), staurosporin, or H7 (PKC inhibitors; Ref.
16). Intracellular cholangiocyte cAMP levels were
determined by RIA. *P < 0.05 vs. basal
values; **P < 0.05 vs. secretin-stimulated
cAMP levels. Data are means ± SE of 3-6 experiments.
Reproduced with permission from Ref. 62.
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Somatostatin.
Studies in dogs (117) and in rats (118, 143)
showed that somatostatin inhibits both basal and secretin-stimulated
bicarbonate-rich choleresis by inhibition of exocytic vesicle insertion
into cholangiocyte apical membranes (Refs. 75 and 143;
Fig. 2) through interactions with a subtype (i.e., SSTR2)
of somatostatin receptors (143). These studies also showed
that secretin-stimulated insertion of transporters into the apical
membrane of rat cholangiocytes is dependent on the microtubule system
because it is inhibited by pretreatment of cholangiocytes with
colchicine (75, 143). In rats, somatostatin inhibition of
secretin-stimulated ductal secretion is also associated with decreased
SR gene expression (9) and decreased secretin-stimulated
cAMP levels (9, 143). The inhibitory effect of
somatostatin on secretin-stimulated ductal secretion is more evident in
animal models of ductal hyperplasia (e.g., BDL), in which there is
upregulation of SR and enhanced secretin-stimulated cAMP levels. On the
basis of these findings (75, 143), the "membrane
microdomain recycling model" (Fig. 3)
has been proposed in rat liver to explain the cooperative interactions
between secretin and somatostatin in the regulation of ductal secretory
activity. In contrast to these findings, other studies in rats showed
that colchicine does not inhibit secretin-induced ductal secretion, thus providing evidence against a pivotal role of exocytic vesicle insertion into cholangiocyte apical membrane to explain the choleretic effect of secretin (49).
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Fig. 3.
The membrane microdomain recycling model for hormone-induced
alterations in ductular bile secretion. Circles with dots represent
hypothetical transport proteins or ion channels on vesicles, which can
be either inserted into or removed from the plasma membrane of
cholangiocytes by hormone-regulated exocytic and endocytic processes,
respectively. Reproduced with permission from Ref. 75.
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Peptides
Endothelin.
ET-1, a polypeptide containing 21 amino acids, has multifunctional
properties in several organs (127). Recent studies in rats
showed that ET-1 receptors ETA and ETB are
expressed by cholangiocytes and that ET-1 inhibits SR gene expression,
secretin-stimulated ductal lumen expansion, and secretin-induced cAMP
levels by selective interaction with ETA but not
ETB receptors (38). Furthermore, studies in
primary cultures of human gallbladder epithelial cells showed that
ET-1, via a Gi protein-coupled receptor, inhibits secretin-stimulated cAMP-dependent electrolyte secretion
(56).
Vasoactive intestinal peptide.
In vivo studies in humans showed that VIP potentiates the choleretic
effect of secretin on bile flow and bicarbonate secretion (110,
111). Variation among species may explain the different cooperative interactions between VIP and secretin in the regulation of
ductal bile secretion (43, 65, 92). In dogs, for example, VIP stimulated basal biliary secretion but did not alter the maximal effect of secretin on ductal secretion (92). In sheep
liver, whereas secretin stimulated bile flow, VIP had no effect on
ductal secretion (65). Although VIP regulates secretory
activity of other epithelia through the cAMP/PKA (35, 102)
or IP3/PKC pathway (130), recent studies in
rats showed that VIP stimulates basal (but not secretin-stimulated)
fluid and bicarbonate secretion via cAMP-independent pathways in IBDU
(43). Together, the data suggest that VIP regulates basal
and [possibly secretin stimulated (110, 111)] ductal
secretion through a signaling pathway different from that of secretin.
Bombesin.
In support of a possible interaction between bombesin and secretin,
Kaminski and Deshpande (72) showed in dogs that bombesin markedly increases the bicarbonate-rich choleresis produced by intraduodenal acid infusion through increased secretin release. Recent
studies in rats showed that bombesin increases ductal secretion and
that bombesin stimulation of Cl/HCO
exchanger activity was independent of the increase in the second
messengers cAMP, cGMP, and cytosolic Ca2+ (44,
46). Bombesin-stimulated biliary secretion is dependent on anion exchangers, Cl and K+ channels, and
carbonic anhydrase but not microtubules (46), through
mechanisms different from those established for secretin (6,
9-18, 22, 38, 62, 63, 83-87, 98, 143).
Substance P.
Studies in rats showed that substance P decreases basal and
secretin-stimulated secretion of pancreatic ducts (24).
Similarly, in vivo studies in dogs showed that substance P inhibits
secretin-stimulated choleresis (91). In contrast to these
observations, preliminary studies in rat IBDU showed that substance P
does not alter the effect of secretin on water and electrolyte
secretion (21).
Bile Acids
A number of studies in rats showed that certain bile acids enter
cholangiocytes through the Na+-dependent apical bile acid
transporter (ABAT) (8, 80), thus modifying
secretin-stimulated ductal bile secretion (5, 7, 8). For
example, recent studies showed that TC and TLC increased in vitro
(5) and in vivo (after chronic feeding) (7)
secretin SR gene expression, secretin-stimulated cAMP levels, and
secretin-stimulated bicarbonate-rich choleresis of normal rats.
Nitric Oxide
Recent studies by Trauner et al. (146) demonstrated
that nitric oxide and cGMP, which stimulate secretion of rat hepatocyte couplets, do not alter basal or secretin-stimulated ductal lumen volume
and Cl/HCO3 exchanger activity
of IBDU.
Cytokines
A number of studies indicate that the intrahepatic cholangiocytes
and peribiliary gland in normal human livers and in hepatolithiasis are
involved in local immunological responses through the transport of
secretory component and IgA into bile (137). Human
cholangiocytes also express ICAM-1 (26, 48), lymphocyte
function-associated antigen (LFA)-3 (48), CD40
(48), and human lymphocyte antigen (HLA) class 1 (26, 48). Human cholangiocytes secrete interleukin (IL)-6
and tumor necrosis factor- in bile (158). Furthermore, we showed previously (4) that interferon- inhibits SR
gene expression and secretin-stimulated bicarbonate-rich choleresis in
a murine model of cirrhosis induced by chronic CCl4
treatment. This observation suggests that cytokines produced by
cholangiocytes in an autocrine/paracrine manner may regulate ductal
secretion. Moreover, we recently showed (99) that human
biliary cell lines and murine and rat cholangiocytes express IL-5
receptors and that IL-5 inhibits Cl channel currents.
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HETEROGENEITY OF SECRETIN-INDUCED CHOLANGIOCYTE RESPONSES |
Recently, the isolation and phenotypic characterization of
distinct subpopulations of small (~8 µm in size) and large (~13 µm in size) cholangiocytes (13, 14) and small (<15 µm
in external diameter) and large (>15 µm in external diameter) IBDU
(6) allowed us to demonstrate that the intrahepatic
biliary epithelium is morphologically and functionally heterogeneous
(5, 6, 8, 13, 14, 85, 86). These studies showed that the
SR is solely expressed by large cholangiocytes in large ducts (6, 13), which respond physiologically to secretin with increases in
cAMP levels, Cl efflux, and
Cl/HCO exchanger activity (see Fig. 4). Other studies showed that
secretin-stimulated ductal secretion of large cholangiocytes is
inhibited by somatostatin, whose receptors (i.e., SSTR2)
are expressed by large but not small cholangiocytes (9).
Recent studies demonstrated that ET-1 decreases the secretin-induced secretion of large cholangiocytes by selectively interacting with ETA receptors (38). Consistent with this
concept, other studies showed that the
Cl/HCO exchanger [an important
component of secretin-stimulated bicarbonate- rich choleresis
(12, 13, 16, 18, 84, 119, 134)] is only expressed by
large bile ducts in humans (96).
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Fig. 4.
Schematic representation of the secretory heterogeneity
of the intrahepatic biliary tree. The cartoon shows that the major
sites of secretin- and somatostatin-regulated transport of water and
electrolytes involve cholangiocytes in large bile ducts (which contain
secretin and somatostatin receptors), whereas small cholangiocytes in
small ducts do not express secretin and somatostatin receptors and do
not participate in hormone-regulated ductal secretion.
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SUMMARY AND FUTURE PERSPECTIVES |
The findings discussed in this review emphasize that
secretin-stimulated ductal bile secretion is cooperatively regulated by
a number of factors, some with stimulatory effects [e.g., VIP, ACh,
the bile acids TC and TLC (5, 7, 16)] and some with inhibitory action [e.g., somatostatin, gastrin, ET-1, AP (9, 17,
38, 63, 143)]. The recent data related to the role and
mechanisms of action of ACh (16) and bile acids (5,
7) are the most interesting findings related to the modulation
of secretin-stimulated ductal secretion. The findings that rat
cholangiocytes of different sizes differentially respond to liver
injury and/or toxins has clinical relevance because a number of chronic
cholestatic liver diseases (e.g., PBC and PSC) are characterized by a
spotty rather than a diffuse proliferative response (12,
120). Studies are needed to evaluate the role and mechanisms of
action of the adrenergic and dopaminergic nerves in the regulation of
secretin-stimulated bicarbonate-rich choleresis. Taking into account
that after BDL microvascular proliferation occurs only adjacent to
large proliferating ducts (59) and that
secretin-stimulated secretion is only present in large cholangiocytes
(9, 13, 14, 85, 86), further studies are necessary to
understand the role of blood supply and vascularly derived factors in
the regulation of secretin-stimulated ductal bile secretion. On the
basis of these findings, it seems likely that our understanding of
secretin stimulation of ductal secretory functions will continue to
grow as a focus of increasing attention and importance.
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ACKNOWLEDGEMENTS |
We thank Dr. Domenico Alvaro (University of the Studies of Rome, La
Sapienza, Rome, Italy) for his suggestions during the preparation of
this review.
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FOOTNOTES |
Some of the work presented here was supported by a grant award to G. LeSage and G. Alpini from Scott & White Hospital and Texas A&M
University, by an American Association for the Studies of Liver
Diseases/Schering Advanced Hepatology Fellowship Program grant to N. Kanno, by National Institute of Diabetes and Digestive and Kidney
Diseases (NIDDK) Grant DK-54208 to G. LeSage, by a Department of
Veterans Affairs Merit Award and NIDDK Grant DK-58411 to G. Alpini, and
by a grant award from Scott & White Hospital to S. Glaser.
Address for reprint requests and other correspondence: G. Alpini, Depts. of Internal Medicine and Medical Physiology, Texas A&M
Univ. System Health Science Center, College of Medicine, Central Texas
Veterans HSC MRB, 702 South West H.K. Dodgen Loop, Temple, TX, 76504 (E-mail: galpini{at}tamu.edu).
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TOP
ABSTRACT
INTRODUCTION
