Departments of Medicine, 1 University of Colorado Health Sciences Center, Denver, Colorado 80262; 2 Duke University Medical Center, Durham, North Carolina 27710; and 3 Mayo Clinic, Rochester, Minnesota 55905
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ABSTRACT |
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To evaluate whether ATP in bile serves as a
signaling factor regulating ductular secretion, voltage-clamp studies
were performed using a novel normal rat cholangiocyte (NRC) model. In
the presence of amiloride (100 µM) to block
Na+ channels, exposure of the
apical membrane to ATP significantly increased the short-circuit
current (Isc)
from 18.2 ± 5.9 to 52.8 ± 12.7 µA
(n = 18). The response to
ATP is mediated by basolateral-to-apical Cl transport because it is
inhibited by 1) the
Cl
channel blockers
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (1 mM),
diphenylanthranilic acid (1.5 mM), or
5-nitro-2-(3-phenylpropylamino)benzoic acid (50 or 100 µM) in the
apical chamber, 2) the
K+ channel blocker
Ba2+ (5 mM), or
3) the
Na+-K+-2Cl
cotransport inhibitor bumetanide (200 µM) in the basolateral chamber.
Other nucleotides stimulated an increase in
Isc with a rank
order potency of UTP = ATP = adenosine 5'-O-(3)-thiotriphosphate, consistent with P2u purinergic
receptors. ADP, AMP, 2-methylthioadenosine 5'-triphosphate, and
adenosine had no effect. A cDNA encoding a rat
P2u receptor
(rP2uR) was isolated from a liver
cDNA library, and functional expression of the corresponding mRNA in
Xenopus laevis oocytes resulted in the
appearance of ATP-stimulated currents with a similar pharmacological
profile. Northern analysis identified hybridizing mRNA transcripts in
NRC as well as other cell types in rat liver. These findings indicate
that exposure of polarized cholangiocytes to ATP results in luminal
Cl
secretion through
activation of P2u receptors in the
apical membrane. Release of ATP into bile may serve as an autocrine or
paracrine signal regulating cholangiocyte secretory function.
liver; chloride channel; ion transport; bile
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INTRODUCTION |
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THE FORMATION OF BILE by the liver depends on
complementary interactions between two distinct cell types, including
hepatic parenchymal cells, which account for ~80% of liver mass, and
bile duct epithelial cells (cholangiocytes), which account for
~2-5% of liver mass (45). Secretion is initiated by hepatocytes
that actively transport bile acids and other organic solutes into the canalicular space between cells (32). Subsequently, canalicular bile
enters the lumen of the extensive network of intrahepatic ducts where
it undergoes dilution and alkalization as a result of cholangiocyte
Cl and
secretion (10, 31). Despite the comparatively small number of duct cells, cholangiocyte secretion is
thought to account for up to 40% of human bile flow (32). Moreover,
intrahepatic ducts represent an important target of injury in several
disease states, including sclerosing cholangitis, primary biliary
cirrhosis, and liver transplant rejection. However, the small size and
intrahepatic location of cholangiocytes have limited investigation of
the cellular mechanisms involved in ductular secretion.
Recent studies of isolated cholangiocytes and intrahepatic duct units
from rat liver have focused on the role of secretin-stimulated bile
flow (19, 31, 38). Secretin binds to receptors in the basolateral
membrane (13) that are positively coupled to adenylyl cyclase and
stimulates exocytosis (21),
Cl/
exchange (31), and opening of
Cl
channels (29). These
Cl
channels are localized
to the apical membrane and are associated with expression of cystic
fibrosis transmembrane conductance regulator (CFTR) (2, 10, 48), the
protein product of the cystic fibrosis (CF) gene (2). Defective
regulation of Cl
channels
in the genetic disease CF is likely to contribute to the cholestasis
and biliary obstruction observed in 5-30% of these patients (15).
These and other findings provide indirect support for the assumption
that transepithelial transport of
Cl
represents an important
driving force for fluid and electrolyte secretion across intrahepatic
bile ducts (10, 15).
Although the hepatic manifestations of CF may occasionally be severe,
it is notable that the prevalence of biliary disease is much lower than
pancreatic or pulmonary injury in CF (15). The reasons for these
differences are not known. In the CF mouse model, the expression of
alternative Cl channels
unrelated to CFTR is an important determinant of organ-level disease
(9). Thus one potential explanation for these clinical differences is
that biliary secretion might also be regulated by secretin- and
CFTR-independent pathways. Indeed,
Cl
channels that are
distinct from CFTR have been identified in biliary cells and cell lines
(14, 28, 39, 40). Definition of their physiological role(s) has been
limited by the lack of information regarding their cellular
localization to the apical (luminal) or basolateral membranes, by the
inability to measure transepithelial potentials across polarized cell
monolayers, and by the limited information regarding the other
receptors and physiological factors involved in regulation of secretory
function.
Extracellular ATP is a potent signaling molecule that regulates many transport and metabolic pathways in the liver (17). These effects are mediated by activation of one or more purinergic (P2) receptors, which are expressed by hepatocytes (22), nonparenchymal liver cells (16), and intra- and extrahepatic biliary cells (11, 27, 33, 47). Based on signaling properties and agonist specificity, at least five subclasses of P2 receptors have been identified in different cell types (18), including P2T, P2U, and P2Y receptors, which signal through G proteins, P2X receptors, which are ligand-gated cation channels, and P2Z receptors expressed by mast cells.
In nonpolarized biliary cells, ATP and UTP are equally potent in their
ability to increase intracellular
Ca2+ concentration, consistent
with expression of receptors of the P2u subclass (27, 47). Studies in
isolated duct units indicate that
P2u receptors are localized in
part to the basolateral domain (33). However, there is no direct
information regarding the presence or function of apical receptors,
which would be of potential interest to biliary function for several
reasons. First, ATP is released by hepatocytes and is present in bile
in concentrations sufficient to activate
P2 receptors if they are present
in the apical region (7). Second, receptor binding increases the
Cl permeability of isolated
cholangiocytes (27), and Cl
efflux may represent one of the cellular mechanisms involved in
secretion. Third, stimulation of apical
P2u receptors in airway cells
elicits potent secretory responses (26). Consequently, in these studies
we have utilized a recently developed model of cultured normal rat
cholangiocytes (NRC) (46) to evaluate the hypothesis that apical
exposure to ATP represents an autocrine and/or paracrine signal
that modulates biliary secretion through receptor-mediated increases in
transepithelial Cl
transport.
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METHODS |
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Materials.
Purinergic receptor agonists included adenosine, adenosine
5'-monophosphate sodium salt (AMP), adenosine
5'-diphosphate potassium salt (ADP), adenosine
5'-triphosphate disodium salt (ATP), 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP), adenosine
5'-O-(3-thiotriphosphate) tetralithium salt (ATPS), and
uridine 5'-triphosphate sodium salt (UTP). These were made fresh
as stock solutions and added to the perfusion chamber to achieve the
final concentrations indicated. Transport inhibitors included amiloride
hydrochloride, 3-(aminosulfonyl)-5-(butylamino)4-phenoxybenzoic acid (bumetanide), niflumic
acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid
(DIDS), diphenylanthranilic acid (DPC; Fluka, Switzerland), and
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, Calbiochem, La
Jolla, CA). The last three were dissolved in dimethyl sulfoxide (DMSO)
and used in the concentrations indicated with a final DMSO concentration <0.01%. Other reagents were of high grade and were obtained from Sigma Chemical (St. Louis, MO) unless otherwise indicated.
Cell culture. NRC were cultured as recently described (46). These cells possess phenotypic features of biliary origin, including expression of the differentiated markers GT, CK-7, and CK-19, and form polarized monolayers with tight junctions between cells and microvilli in the apical membrane (46).
Voltage clamp measurements of short-circuit current.
NRC were seeded at a density of 1 × 105
cells/cm2 on collagen-treated
polycarbonate filters with a pore size of 0.4 µm (Costar, Cambridge,
MA). Before study transmembrane resistance was estimated with an
epithelial tissue voltmeter [EVOHM; World Precision Instruments (WPI), Sarasota, FL], and only inserts with a resistance >1,000 · cm2 were
used for experiments. Transepithelial transport was
measured under voltage-clamp conditions using a DVC 1,000 voltage-
current clamp amplifier (WPI). Cells were mounted in a
Trans-24 mini-perfusion system for tissue culture cups (WPI). All
experiments were carried out at 37°C, and basolateral and apical
(luminal) sides were perfused continuously and independently in a
closed system with a standard NaCl-rich buffer (as will be described),
by bubbling O2 through air-lift
circulators. Test substrates were added to the apical or basolateral
buffer solution as indicated. Transepithelial voltage (Vt) was clamped to 0 mV, and
short-circuit current
(Isc) was recorded through agar bridges (3% agar in 3 M KCl) connected to Ag-AgCl electrodes (cartridge electrodes, WPI). Transepithelial resistance was calculated by measuring the current response to a 10 mV
change in Vt. Data were collected
in 0.5-min intervals or were digitized for storage on a computer. To
minimize potential effects of day-to-day variability in the
preparations, results are compared with same-day controls and presented
as means ± SD. Student's t-test
analysis for unpaired data was performed with SigmaPlot (Jandel
Scientific), and P < 0.05 was
considered significant.
Solutions.
The standard NaCl-rich extracellular solution (pH 7.35) contained (in
mM) 140 NaCl, 4 KCl, 1 KH2PO4,
2 MgCl2, 1 CaCl2, 5 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES)-NaOH. Solutions were preequilibrated with
O2 (100%) and maintained at 37°C. Electrogenic secretion
and
Cl
/
exchange would not be anticipated to contribute substantially to the
Isc response
because the solutions were nominally
free and because exchange
activity does not result in net transfer of charge.
cDNA cloning and expression. An oligo(dT) rat liver cDNA library in Zap II (Stratagene, La Jolla, CA) was screened by plaque hybridization according to the manufacturer's recommendation with a 700 bp Bsr DI restriction enzyme fragment encompassing the open reading frame from the cloned mouse P2u receptor cDNA (kindly provided by Dr. David Julius, Univ. of California, San Francisco). A single clone (rP2uR) was isolated, and both strands were sequenced by the dideoxy chain termination method using Sequenase 2.0 (Life Science, Arlington Heights, IL).
In vitro transcription. The rP2uR cDNA cloned into pBluescript SK (Stratagene) was linearized by restriction digest using Not I or Acc restriction enzymes and transcribed in vitro for expression in oocytes with T7 (sense cRNA) or with T3 (antisense cRNA) polymerase (Ambion, Austin, TX). The cRNA preparations were precipitated and resuspended in deionized water at a final concentration of 1 µg/µl. The integrity of the transcripts was verified by electrophoresis on agarose gel.
cDNA expression in oocytes. Oocytes were isolated from Xenopus laevis (Nasco, Racine, WI) using standard procedures (43), and the follicular cell layer was removed after treatment with collagenase (GIBCO-BRL, Gaithersburg, MD). Oocytes were incubated in ND-96 (in mM, 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH 7.5) (43) and maintained at 18°C. Twenty-four hours after isolation, healthy stage V and VI oocytes were microinjected with 50 nl of water or an equal volume containing rP2u receptor sense or antisense cRNA (1 µg/µl). Two electrode voltage clamp studies were performed after 48-72 h using an Axoclamp 2 amplifier (Axon Instruments, Foster City, CA) at room temperature under continuous superfusion. Data were digitized for storage on a personal computer. Digitized recordings were analyzed using Axotape software (Axon Instruments).
Northern blot analysis. Total RNA from freshly isolated rat hepatocytes, HTC rat hepatoma cells, and NRC cells was prepared using the guanidine thiocyanate and phenol method (Tel-Test, Friendswoods, TX). RNA preparations were separated on formaldehyde-containing agarose gels (1%) and then blotted on nylon membranes (Schleicher-Schuell, Keene, NH) and ultraviolet cross-linked for 2 min. A probe spanning nucleotides 933 to 1,690 from the rP2uR cDNA clone was obtained by polymerase chain reaction amplification and was radiolabeled with [32P]dCTP using Klenow (Life Science, Arlington Heights, IL). The blot was probed in QuickHyb (Stratagene) according to the manufacturer's instructions. The membrane was then prehybridized for 15 min in QuickHyb at 68°C. Two washes in 2× saline-sodium citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) for 15 min at room temperature each were followed by a wash in 0.1× SSC, 0.1% SDS for 30 min at 60°C.
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RESULTS |
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Basal properties of NRC monolayers.
NRC cells plated on collagen-coated inserts formed polarized monolayers
after 8 to 14 days (Fig. 1). As cells
became confluent there was an increase in transepithelial resistance
indicating formation of tight junctions. Inserts with values >1,000
· cm2 as
measured with an EVOHM voltmeter produced reliable and reproducible results in voltage-clamp studies in which transepithelial ion transport
could be evaluated more directly (20). When mounted in the recording
chamber, inserts rapidly (<7 min) equilibrated and developed a basal
Isc of 18.2 ± 5.9 µA (n = 18) with a
lumen-negative potential of
3.2 ± 1.9 mV. The average
transepithelial resistance measured in the Ussing chamber of 809 ± 436
· cm2
was consistently lower than values measured with the EVOHM voltmeter, as reported for other cell types (20).
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Nucleotides stimulate transepithelial transport through activation
of receptors in apical membrane.
To assess whether exposure of the apical membrane to nucleotides
regulates transepithelial transport, ATP (0.1-100 µM) was added
selectively to the solution bathing the luminal surface. Exposure to
ATP caused a rapid increase in
Isc in all
monolayers tested. The response occurred rapidly and peaked within ~5
min as shown in Fig. 2. The increase in
Isc
(Isc) of
34.6 ± 12.2 µA (n = 18) was
temporally associated with development of more negative luminal
potentials of
12.0 ± 7.3 mV and a decrease in transepithelial resistance by 11 ± 16%, consistent with opening of
conductance pathways. The
Isc response
tended to return toward basal values over 15-60 min despite the
continued presence of ATP and
V recovered to
5.1 ± 5.9 mV
over the same period. To minimize any effect of day-to-day variability
in the magnitude or time course of the response, subsequent studies
using test reagents were compared with control values measured on the
same study day and are presented as relative values.
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Pharmacological properties of apical receptor.
These findings are consistent with the presence of ATP receptors in the
apical membrane that stimulate transepithelial ion transport. A similar
response was observed with the nonhydrolyzable analog ATPS,
indicating that receptor activation does not depend on ATP hydrolysis
and that the breakdown products of ATP are not responsible for the
effect. To further evaluate the pharmacological properties of the
receptor, other test nucleotides were added to the apical bath at a
concentration of 50 µM. The rank order potency for stimulation of
Isc showed
approximately equal responses to ATP
S, UTP, and ATP, and the
concentration of UTP required to produce half-maximal increases in
Isc was ~2
µM (Fig. 3). ATP showed a similar
concentration dependence (data not presented). Other nucleotides,
including ADP, AMP, 2-MeS-ATP, and adenosine, failed to increase
Isc above basal
levels. These findings are consistent with the pharmacological
properties of receptors of the P2u
subclass (18).
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Identification of conductances contributing to
Isc.
The ATP-induced increase in
Isc was
associated with generation of a lumen-negative potential, consistent
with activation of apical-to-basolateral
Na+ or basolateral-to-apical
Cl transport, or both.
Therefore, in selected studies, the
Na+ channel blocker amiloride (100 µM) was added to the apical chamber. Preincubation in amiloride did
not block nucleotide-stimulated increases in
Isc
(n = 81). Similarly, exposure to
amiloride after stimulation of
Isc by ATP had
no significant inhibitory effect (n = 10). These findings indicate the ATP-stimulated increase in
Isc is not due to
opening of amiloride-sensitive Na+
channels. It is notable, however, that amiloride decreased basal Isc values in
~30% of monolayers. The magnitude of the decrease tended to be
larger in monolayers with higher initial
Isc values, suggesting that apical Na+ uptake
may contribute to
Isc under basal
conditions. Consequently, subsequent studies were performed in the
presence of amiloride (100 µM) to minimize any potential
contribution of apical Na+
conductances.
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Molecular cloning of rat liver P2u receptor. Complementary DNAs encoding P2u receptors have recently been identified in mouse brain (24), rat alveolar type II cells (37), rat pituitary gland (8), and human tissues (34). To further characterize the receptor involved in hepatobiliary signaling, a rat liver cDNA library was screened by plaque hybridization using a Bsr D1 restriction digest cDNA fragment of the P2u receptor from mouse brain as a probe (24). A single clone of 2,164 bp was plaque purified (rP2uR), and sequence analysis revealed a 1,125-bp open reading frame, 444-bp 5' untranslated region, and 592-bp 3' untranslated region. The largest open reading frame encoded a putative protein of 374 amino acids with a predicted molecular mass of 42 kDa. Seven putative transmembrane domains were identified from the deduced sequence, typical for the G protein-coupled class of receptors. The liver amino acid sequence was identical to other rat P2u proteins obtained from pituitary gland or alveolar type II cell cDNA. Sequence comparison with the cloned murine and human P2u receptors showed >95% homology at the amino acid level (Fig. 6). However, the 5' untranslated region of the rP2uR cDNA harbors a 200 bp fragment immediately upstream of the putative AUG start codon that is not present in the mouse or the human P2u receptor.
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Identification of P2u mRNA by Northern analysis. To assess whether mRNA corresponding to rP2uR is present in hepatobiliary cells, Northern analysis was performed under high stringency conditions using a probe encompassing nucleotides 933 to 1,690. P2u mRNA was abundant in NRC, and was also detected as a 33-kb band in RNA from hepatocytes and HTC hepatoma cells (Fig. 7). Analysis of RNA from other rat tissues revealed hybridizing transcripts in lung, skeletal muscle, and liver tissue but not in brain or kidney tissues (data not shown).
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Functional characterization of cloned
rP2uR.
The functional properties of the protein encoded by
rP2uR were assessed by
heterologous expression. cRNA was prepared by in vitro transcription,
and after injection of rP2uR cRNA
(50 ng) into Xenopus oocytes, the
response to extracellular nucleotides was assessed by two electrode
voltage clamp studies. Exposure to ATP or UTP (100 µM) elicited peak
inward currents of ~200 nA (holding potential 50 mV) in
oocytes injected with rP2uR cRNA but not in control oocytes or oocytes injected with water (Fig. 8A) or
antisense cRNA.
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DISCUSSION |
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These studies of NRC monolayers provide direct support for the concept
that transepithelial transport of Cl represents an
important determinant of fluid and electrolyte secretion across
intrahepatic biliary epithelium (10) and identify extracellular
nucleotides as potent regulatory factors modulating biliary secretory
function through activation of P2u
receptors in the apical (luminal) membrane. Thus ATP released into bile may represent an autocrine and/or paracrine factor coupling the separate hepatic and ductular components of bile formation through stimulatory effects on duct cells.
Previous studies of biliary cells and cell lines have provided
pharmacological support for expression of purinergic receptors. In
isolated cells, exposure to nucleotides increases intracellular Ca2+ concentrations and membrane
Cl permeability (27, 30,
47). In isolated bile duct units, P2u receptors are present in the
basolateral membrane (33). However, basolateral receptor stimulation
has no apparent effect on secretion or cell volume as assessed by
optical techniques. Further definition of the physiological role(s) of
purinergic signaling has been limited in part by the lack of access to
the apical domain of polarized cells and by the lack of molecular insights regarding the properties of biliary purinergic receptors. A
role in regulation of secretion has been postulated because 1) in gallbladder from the amphibian
Necturus, ATP added to the apical
(mucosal) surface of the epithelium stimulates ionic conductances (11),
2) ATP is present in mammalian bile
in concentrations sufficient to activate receptors (7), and
3) ATP and UTP act as potent
secretagogues in airway and certain other epithelial cell types that
express apical receptors (26, 34). The mechanisms involved are complex,
with evidence for expression of receptors in both the apical and
basolateral membranes, and for coupling of receptors to both
Ca2+-dependent and
Ca2+-independent signaling
pathways according to the model under investigation (6, 44). However,
in airway epithelium, stimulation of
Cl
secretion by
extracellular nucleotides involves channels other than CFTR and has
been proposed as an approach to treatment of the secretory defect
associated with CF (23, 34).
NRC monolayers spontaneously developed high transepithelial resistances
and, under voltage-clamp conditions, exhibited basal Isc values of
10-30 µA and lumen-negative transepithelial potentials. In the
nominal absence of , basal transport was attributable for the most part to parallel function in the apical
membrane of an amiloride-sensitive
Na+ conductance and NPPB-sensitive
Cl
conductance(s). The
relative contribution of the Na+
conductance varied among monolayers, with increases in the
amiloride-sensitive component at higher basal values of
Isc.
Exposure to nucleotides elicited a rapid increase in
Isc that was
mediated by an increase in apical
Cl permeability and
stimulation of transepithelial
Cl
transport. In selected
studies, the increase in
Isc was
associated with lumen-negative potentials of
10 mV or more and
was unaffected by amiloride, consistent with net movement of
Cl
into the lumen.
Moreover, inhibitors of Cl
transport, including putative
Cl
channel blockers in the
apical solution, and inhibitors of
K+ channels and
Na+-K+-2Cl
cotransport in the basolateral solution significantly inhibited the
response to ATP. These findings indicate that purinergic stimulation is
positively coupled to transport mechanisms in both the apical and
basolateral membranes of biliary cells. It is interesting that NPPB and
DPC returned Isc
to basal values when added after ATP stimulation, but the inhibitory
effects of Cl
channel
blockers were incomplete in preincubation studies. The reasons for the
partial inhibition are not known and may reflect impaired access of
blockers to channels caused by the dense microvilli in the apical
membrane (46) or intrinsic differences in the pharmacological
properties of the channels themselves. In other epithelia the response
to purinergic stimulation involves several channel types, including
both CFTR and outwardly rectifying
Cl
channels, which differ
in their sensitivity to blockers (6, 42). Alternatively, a contribution
of other electrogenic transport mechanisms cannot be excluded.
Both molecular and functional criteria indicate that purinergic
receptors of the P2u subclass are
involved in the secretory response. When applied to the apical
membrane, ATP, UTP, and ATPS were equally potent in their effects on
Isc. Moreover,
rP2uR mRNAs are abundant in NRC
cells, rat hepatocytes, and liver cell lines, and expression of
rP2uR in oocytes gave similar
results. The presence in liver differs from results obtained by Rice et al. (37) but confirms the Northern blot analysis performed by Lustig et
al. (24) on mouse tissue. In addition, there is abundant physiological
evidence for the presence of purinergic receptors in hepatobiliary
cells (5, 17, 22, 27, 47). The
rP2uR cDNA identified here shows
95% homology at the amino acid level with the mouse brain receptor
and exhibits similar functional properties when expressed in oocytes
(24). Moreover, current activation in oocytes is mediated through a
Ca2+-dependent pathway that
appears analogous to the
Ca2+-mobilizing effects of UTP in
biliary cells (47).
The concentration of ATP required to stimulate Isc is similar to the average concentration of ATP in human bile of ~1.5 µM (7). Consequently, it is attractive to speculate that purinergic signaling mechanisms might contribute to regulation of secretion across intrahepatic ducts in vivo. If so, there are several important issues that remain to be addressed. First, the actual amount of ATP (or UTP) present within the lumen of intrahepatic ducts is not known, and the cellular origin and molecular mechanisms involved in ATP release into bile have not been established. Both hepatic and biliary cell lines in culture release nucleotides (7, 41), and express ATP-binding cassette (ABC) proteins, which have been implicated in transport of ATP in other cell types (1, 4, 36, 42). These ABC proteins include P-glycoproteins (mdr) in the apical (canalicular) membrane of hepatocytes (3) and CFTR in the apical membrane of intrahepatic duct cells (10). However, the role of ABC proteins in ATP transport remains controversial (35). In addition, ectonucleotidase activity and Na+-adenosine cotransport across the canalicular membrane could also modulate local nucleotide concentrations (3). Thus there are multiple potential sites for regulation of luminal nucleotide concentrations through variations in the rate of ATP release, ATP degradation, or bile flow. Any ATP escaping degradation would have direct access to the apical surface of duct cells as bile flows out of the canaliculus and into the lumen of intrahepatic ducts.
Second, recent studies indicate that
P2 receptors are also present in
the basolateral membrane of cholangiocytes (33). In isolated ducts,
basolateral receptor stimulation increases cytosolic Ca2+ concentration, but there is
no apparent effect on secretion or cell volume (33). Consistent with
these observations, we observed in preliminary studies that ATP added
to the basolateral solution induced relatively small
Isc responses
(4.5 ± 1.4 µA, n = 10), averaging only 10-15% of those induced by apical exposure.
ATPS (n = 3) and UTP
(n = 4) were without effect. Thus
apical receptors appear to contribute more importantly to local
regulation of secretion, and the additional type(s) and physiological
roles of basolateral purinergic receptors remain to be determined.
Finally, the intracellular signals that couple receptor binding to
channel opening have not been defined. In airway cells ATP stimulates
Cl secretion through
activation of multiple signal transduction pathways, including both
adenosine 3',5'-cyclic monophosphate (cAMP)-dependent and
independent mechanisms (44). In preliminary studies of NRC monolayers,
the cAMP inhibitor Rp-cAMPs failed to inhibit the response to ATP (data
not presented), and in isolated biliary cells, both cytosolic
Ca2+ concentration and
Ca2+-calmodulin-dependent kinases
increase membrane Cl
permeability (30, 40). If these findings are relevant to cholangiocytes
in vivo, it seems likely that apical ATP modulates Cl
secretion through one or
more cAMP-independent pathways not directly related to CFTR.
In summary, these studies of NRC cells indicate that extracellular ATP
may serve as an autocrine and/or paracrine factor that contributes to regulation of ductular secretion through activation of
P2u receptors in the apical
membrane. Local control of biliary function by factors present in bile
represents an important complement to the effects of secretin and other
basolateral signals. Moreover, apical
P2u receptors represent an
attractive therapeutic target for pharmacological approaches aiming to
modulate the volume and composition of bile and to bypass the
Cl secretory defect
associated with CF.
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ACKNOWLEDGEMENTS |
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This work was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46082 and DK-43278, and by the Deutsche Forschungsgemeinschaft Grant Schl 380/1-1.
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FOOTNOTES |
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Parts of this study have been published in abstract form (Hepatology 24: 514, 1996).
T. Schlenker, J. M.-J. Romac, and A. L. Sharara contributed equally to different aspects of this work.
Address for reprint requests: G. Fitz, 4200 East 9th Ave., Campus Box B-158, Univ. of Colorado Health Sciences Center, Denver, CO 80262.
Received 21 January 1997; accepted in final form 5 August 1997.
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