Am J Physiol Gastrointest Liver Physiol
281: G1059–G1067, 2001.
Polarized expression and function of P2Y
ATP receptors in rat bile duct epithelia
JONATHAN A. DRANOFF,1 ANATOLY I. MASYUK,2 EMMA A. KRUGLOV,1
NICHOLAS F. LARUSSO,2 AND MICHAEL H. NATHANSON1
1
Yale University School of Medicine, New Haven, Connecticut 06520;
and 2Mayo Clinic and Foundation, Rochester, Minnesota 55905
Received 3 February 2001; accepted in final form 26 April 2001
Dranoff, Jonathan A., Anatoly I. Masyuk, Emma A.
Kruglov, Nicholas F. LaRusso, and Michael H.
Nathanson. Polarized expression and function of P2Y ATP
receptors in rat bile duct epithelia. Am J Physiol Gastrointest
Liver Physiol 281: G1059–G1067, 2001.— Extracellular nucleotides may be important regulators of bile ductular secretion, because cholangiocytes express P2Y ATP receptors and
nucleotides are found in bile. However, the expression, distribution, and function of specific P2Y receptor subtypes in
cholangiocytes are unknown. Thus our aim was to determine
the subtypes, distribution, and role in secretion of P2Y receptors expressed by cholangiocytes. The molecular subtypes
of P2Y receptors were determined by RT-PCR. Functional
studies measuring cytosolic Ca2⫹ (Ca2⫹
i ) signals and bile
ductular pH were performed in isolated, microperfused intrahepatic bile duct units (IBDUs). PCR products corresponding to P2Y1, P2Y2, P2Y4, P2Y6, and P2X4 receptor
subtypes were identified. Luminal perfusion of ATP into
IBDUs induced increases in Ca2⫹
that were inhibited by
i
apyrase and suramin. Luminal ATP, ADP, 2-methylthioadenosine 5⬘-triphosphate, UTP, and UDP each increased
Ca2⫹
i . Basolateral addition of adenosine 5⬘-O-(3-thiotriphosphate) (ATP-␥-S), but not ATP, to the perifusing bath increased Ca2⫹
i . IBDU perfusion with ATP-␥-S induced net bile
ductular alkalization. Cholangiocytes express multiple P2Y
receptor subtypes that are expressed at the apical plasma
membrane domain. P2Y receptors are also expressed on the
basolateral domain, but their activation is attenuated by
nucleotide hydrolysis. Activation of ductular P2Y receptors
induces net ductular alkalization, suggesting that nucleotide
signaling may be an important regulator of bile secretion by
the liver.
purinoceptor; nucleotide; nucleoside triphosphate diphosphohydrolase; calcium; secretion; chloride
such as ATP mediate a
number of cell processes through interaction with specific P2Y receptors (33, 45). P2Y receptors are expressed by a number of tissues and regulate diverse
processes including vasomotor responses (37), neurotransmission (3), platelet aggregation (16, 23), and
secretion (13, 38, 42). In epithelial cells, P2Y receptors
are linked specifically to fluid and electrolyte secretion
EXTRACELLULAR NUCLEOTIDES
Address for reprint requests and other correspondence: J. A.
Dranoff, Section of Digestive Diseases, Yale Univ. School of Medicine, 333 Cedar St., PO Box 208019, New Haven, CT 06520-8019
(E-mail: [email protected]).
http://www.ajpgi.org
independent of the cystic fibrosis (CF) transmembrane
conductance regulator (CFTR)(12, 49). For this reason,
activation of P2Y receptors has been proposed as a
therapy for CF (19, 20).
Multiple subtypes of P2Y receptors are found in
mammals, and they are distinguished both by molecular identity and by pharmacological activation (33). In
the rat, four P2Y receptors have been identified, designated P2Y1 (43), P2Y2 (10), P2Y4 (4), and P2Y6 (8).
Although P2Y2 and P2Y4 are activated by both ATP
and UTP, P2Y1 is activated by ADP (and to a lesser
extent ATP) and P2Y6 is activated only by UDP. Distinct combinations of P2Y subtypes are found in different epithelial tissues and may allow tissue-specific
regulation of nucleotide responses. Moreover, some tissues differentially express specific P2Y receptor subtypes at apical and/or basolateral plasma membranes
(22, 25, 47).
Several lines of evidence indirectly suggest that
stimulation of nucleotide receptors may mediate secretion in the liver. Nucleotide release mediates communication from hepatocytes to cholangiocytes in cocultures of isolated cells (39). Furthermore, ATP and
other nucleotides are found in bile at concentrations
that can activate P2Y receptors (9). Finally, P2Y receptors are found on apical membranes in biliary epithelial cell lines (36, 38). Together, these data suggest that
hepatocytes may signal to cholangiocytes through release of nucleotides into bile followed by activation of
P2Y receptors at the luminal (apical) membrane. In
addition, cholangiocytes also express basolateral P2Y
receptors (29), which may mediate signaling from neural or vascular tissue. Thus the goal of this study was
to define the expression and subcellular distribution of
P2Y subtypes in bile duct epithelia and to determine
the relative roles of apical and basolateral receptors in
mediating functional activation of cholangiocytes.
METHODS
Animals and materials. Male Sprague-Dawley rats (180–
250 g; Harlan Sprague-Dawley, Indianapolis, IN) were used
for cholangiocyte isolations. Male Fisher 344 rats (225–250 g;
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0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society
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P2Y RECEPTORS IN RAT CHOLANGIOCYTES
Harlan Sprague-Dawley) were used for intrahepatic bile duct
isolation. ATP, ADP, 2-methylthioadenosine 5⬘-triphosphate
(2-MeS-ATP), adenosine 5⬘-O-(3-thiotriphosphate) (ATP-␥S), ␣,␤-methyleneadenosine 5⬘-triphosphate (␣,␤-MeATP),
␤,␥-methyleneadenosine 5⬘-triphosphate (␤,␥-MeATP), 2⬘and 3⬘-O-(4-benzoylbenzoyl)adenosine 5⬘-triphosphate (BzATP),
UTP, UDP, ACh, suramin, apyrase, and 1,2-bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid (BAPTA)-AM were obtained from Sigma Chemical (St. Louis, MO). Fluo 4-AM and
2⬘,7⬘-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) dextran were obtained from Molecular Probes (Eugene, OR). All
other chemicals were of the highest quality commercially available.
Buffer composition. The composition of isotonic (290
mosmol/kgH2O) Krebs-Ringer-bicarbonate buffer (KRB) was
(in mM) 120.0 NaCl, 5.9 KCl, 1.2 Na2HPO4, 1.0 MgSO4, 25.0
NaHCO3, 1.25 CaCl2, and 5.0 glucose. For HCO3⫺ transport
experiments, the perfusion buffer contained 140 mM NaCl, 5
mM Na2HPO4, and 25–100 ␮M BCECF dextran, pH 7.2.
Preparation of single rat bile duct cells. Single cholangiocytes were prepared and characterized in the Cell Isolation
and Morphology Core Facilities of the Yale Liver Center as
described previously (40). This preparation results in a bile
duct epithelial preparation that is ⬃98% pure as assessed by
positive staining for the biliary epithelial markers ␥-glutamyl transpeptidase, cytokeratin-19, and cytokeratin-7 (1,
34). Experiments using single bile duct cells were performed
12 h after plating.
RT-PCR. P2Y receptor subtypes in cholangiocytes were
detected using two-step RT-PCR performed on RNA from
freshly isolated bile duct cells. Bile duct cell total RNA was
extracted using TRIzol reagent (Life Technologies, Rockville,
MD). RNA was analyzed by electrophoresis and spectrophotometry and treated with RNase-free DNase (RQ1, Promega,
Madison, WI) for 15 min at 37° before RT-PCR. First-strand
cDNA was prepared according to manufacturer’s instructions
using Moloney murine leukemia virus reverse transcriptase
and a 1:1 mixture of random hexamer and oligo(dT)18 primers (Advantage RT-for-PCR; Clontech, Palo Alto, CA). Firststrand cDNA was used as a template for specific amplification of primers for P2Y subtypes P2Y1, P2Y2, P2Y4, and P2Y6
using the following primer pairs: P2Y1, 5⬘-TGG CGT GGT
GCT GCA CCC TCT CAA GCT-3⬘ and 5⬘-CGG GAC AGT
CTC CTT CTG AAT GTA-3⬘; P2Y2, 5⬘-CTG CCA GGC ACC
CGT GCT CTA CTT-3⬘ and 5⬘-CTG AGG TCA AGT GAT
CGG AAG GAG-3⬘; P2Y4, 5⬘-CAC CGA TAC CTG GGT ATC
TGC CAC-3⬘ and 5⬘-CAG ACA GCA AAG ACA GTC AGC
ACC-3⬘; P2Y6, 5⬘-GGA GAC CTT GCC TGC CGC TGC CGC
CTG GTA-3⬘ and 5⬘-TAC CAC GAC AGC CAT ACG GGC
CGC-3⬘(44); P2X1, 5⬘-AGG CCG TGT GGG GTG TTC ATC
TC-3⬘ and 5⬘-ACC TTG GGC TTT CCT TTC TGC TTT TC-3⬘;
P2X2, 5⬘-GAG GCG GGT CAA GGG CGG TCT G-3⬘ and
5⬘-GGG GTC TTG GGA TCC TGC ATT ACT TG-3⬘; P2X3,
5⬘-ACC GGC CGC TGC GTG AAC TAC A-3⬘ and 5⬘-AAG
GCC GCC ACC GAG CTG ATA TG-3⬘; P2X4, 5⬘-GAT CTG
GGA CGT GGC GGA CTA TGT GA-3⬘ and 5⬘-TAT GGG GCA
GAA GGG ATC CGT TTG AG-3⬘; P2X5, ATG GCG AGT GTT
CTG AGG ACC ATC AC-3⬘ and 5⬘-TGC CCC TGC CCA GCG
GAC AAT AGA C-3⬘; P2X6, 5⬘-CGC ATC GGG GAC CTT GTG
G-3⬘ and 5⬘-ATC CCG GCA TCA GCA GAC G; P2X7, CTT
CGG CGT GCG TTT TGA CAT CC-3⬘ and 5⬘-AGG GCC CTG
CGG TTC TCT GGT AGT T-3⬘. PCR reactions were amplified
with Advantage cDNA polymerase mix (Clontech) using the
following cycling parameters: 94° ⫻ 1 min; 30 cycles [94° ⫻
30 s, 60° (63° for P2Y4) ⫻ 30 s, 72° ⫻ 1 min]; 72° ⫻ 5 min.
Expected product size ranged from 300 to 600 bp. Control
reactions were performed with DNase-treated RNA that was
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treated either with RNA but not RT as a template (RNA
controls), to eliminate the possibility of genomic DNA contamination, or water rather than RNA as a template (water
controls), to eliminate the possibility of reagent contamination. Products were analyzed by gel electrophoresis and sequenced to verify their identity.
Microperfusion of microdissected rat intrahepatic bile duct
units. Individual microdissected rat intrahepatic bile duct
units (IBDUs) were prepared, perfused, and observed as
described previously (27). Briefly, concentric glass pipettes
were used to cannulate both lumens of the IBDU, and the
apparatus was transferred to a special stage for use with a
fluorescence microscope (see Measurement of nucleotide-induced changes in cytosolic Ca2⫹ in IBDUs). Solutions were
delivered near the tip of the perfusion pipette through a fluid
exchange pipette filled by a variable-speed syringe pump
(Harvard Apparatus, Holliston, MA) with a 1-ml gas-tight
syringe (Hamilton, Oceanside, CA). When perfusing solutions were switched for apical perfusion studies, the initial
perfusion apparatus was stopped, emptied, and then refilled
with the subsequent perfusion solution. The IBDU perfusion
rate was then recalibrated. IBDUs were perfused at rates of
from 10 to 120 nl/min.
The external surfaces of IBDUs were bathed simultaneously in a buffer whose composition was continuously
controlled. The bath solution was stirred and oxygenated
with a 5% O2-95% CO2 gas mixture. Fluid temperature was
maintained at 37° with a temperature controller (Digi-Sense;
Cole Parmer, Vernon Hills, IL). IBDUs were equilibrated for
up to 30 min before the start of each experimental protocol.
Measurement of nucleotide-induced changes in cytosolic
Ca2⫹ in IBDUs. Changes in cytosolic Ca2⫹ (Ca2⫹
i ) in cholangiocytes were detected using the cell-permeant Ca2⫹-sensitive fluorescent indicator fluo 4-AM. After dissection and
perfusion as described in Microperfusion of microdissected
rat intrahepatic bile duct units, IBDUs were luminally perfused with fluo 4-AM (24 ␮M) for 30 min at 37°C. A fluorescence illuminator (90720 TE epifluorescence attachment for
Nikon TE 300 microscope) was specially modified by the
Mayo Division of Engineering to detect fluorescence changes
from a fixed-size aperture. Fluorescence was detected from a
100-␮m-diameter circular spot within a region of cytosolic
fluorescence in an IBDU by a photosensor module (H5784;
Hamamatsu Photonics, Bridgewater, NJ). Background fluorescence and autofluorescence were negligible with this system. The signal was digitized by a data-acquisition board
(PCI-6023E; National Instruments, Austin, TX) using a PCtype computer. A custom-modified software application (LabView 5.1; National Instruments) was used to control data
acquisition and display data during each experiment and to
output the data to a spreadsheet for analysis. Data samples
were collected 10 times/s. Apical P2Y and P2X receptors were
stimulated by perfusion of nucleotides into the duct lumen,
whereas basolateral P2Y and P2X receptors were stimulated
by addition of nucleotides to the perifusing bath fluid. Fluorescence values were expressed as percentage of baseline.
Data analysis of Cai2⫹ changes. Each experiment was
graphed separately with a spreadsheet program, with time
as the x-axis and raw fluorescence as the y-axis. Baseline and
peak values were identified manually. Baseline fluorescence
was defined as a plateau of fluorescence lasting ⱖ5 s immediately before addition of agonist. Experiments were not
performed unless a constant baseline of 10–20 s was identified before experimental runs. Isolated fluorescence peaks
lasting ⱕ1 s were disregarded to eliminate the possibility of
artifactual fluorescence increases. Fluorescence increases for
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P2Y RECEPTORS IN RAT CHOLANGIOCYTES
each individual experiment were expressed as a peak-tobaseline fluorescence ratio.
Measurement of nucleotide-induced changes in pH in
IBDUs. The luminal pH of perfused IBDUs was measured by
using the cell-impermeant pH-sensitive dye BCECF dextran
and the quantitative epifluorescence approach described
above (27). The IBDUs were perfused with HCO3⫺-free buffer
containing BCECF dextran (pH 7.2) and simultaneously
bathed with KRB or HCO3⫺/CO2-free buffer (pH 7.4). Luminal
dye was excited alternately at 495 ⫾ 10 nm and 440 ⫾ 10 nm
wavelengths, and emission was monitored as described
above. In situ calibration curves were generated to convert
fluorescence excitation ratios (F495/F440) to pH values.
Statistics. Changes in fluo 4 fluorescence ratio and pH are
expressed as means ⫾ SE. Comparisons between groups
were made using Student’s t-test. A P value of ⬍0.05 was
taken as significant.
RESULTS
Rat cholangiocytes express multiple P2 receptor subtypes. Molecular expression of P2Y receptor subtypes
in freshly isolated rat cholangiocytes was determined
by RT-PCR. Oligonucleotide primers specific to the four
P2Y subtypes cloned in rat (33, 44) were used. Cholangiocytes expressed all four of these P2Y subtypes (Fig.
1), which were absent in RNA and water controls.
Similar results were found in freshly isolated rat hepatocytes (not shown). These results are in contrast to
findings in the normal rat cholangiocyte (NRC) cell
Fig. 1. A: rat cholangiocytes express P2Y receptor subtypes P2Y1,
P2Y2, P2Y4, and P2Y6. Molecular expression of P2Y subtypes by rat
cholangiocytes was evaluated by RT-PCR. Cholangiocytes were immunoisolated, and RNA was extracted. Cholangiocyte cDNA was
exposed to specific oligonucleotide primers for P2Y1 (lane 1), P2Y2
(lane 2), P2Y4 (lane 3), and P2Y6 (lane 4). Bands of predicted molecular weight are found for each subtype but not in RNA controls not
exposed to reverse transcriptase (⫺, lanes 5–8). Positive bands were
gel excised and sequenced and were found to have 100% identity to
published sequences. B: rat cholangiocytes express the P2X receptor
subtype P2X4. Molecular expression of subtypes P2X1–7 was examined using RT-PCR of immunoisolated rat cholangiocytes using specific oligonucleotide probes (P2Y2 oligonucleotides were used as a
positive control in lane 2). Cholangiocytes expressed subtype P2X4.
Positive bands were found in total liver (P2X1–7) and heart
(P2X1,2,4,5).
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line, in which the only P2Y receptor molecularly identified was the P2Y2 receptor (38). Because nucleotideinduced increases in Cai2⫹ could be induced by P2X
ligand-gated ion channels as well as P2Y G proteincoupled receptors, we tested for the presence of P2X
receptors in rat cholangiocytes. Molecular expression
of P2X receptors was examined using RT-PCR of immunoisolated rat cholangiocytes with oligonucleotide
probes specific for rat P2X subtypes P2X1–7 (see Fig.
6A). Cholangiocytes expressed only the P2X4 subtype.
Positive control PCR products were expressed using
either total liver (P2X1–7) or heart (P2X1,2,4,5) (not
shown). Together, these data show that rat cholangiocytes express P2Y1, P2Y2, P2Y4, and P2Y6 receptors
and P2X4 receptors.
Rat cholangiocytes express multiple P2Y subtypes at
apical plasma membrane. Experiments were performed in IBDUs to determine which of these P2Y
receptors were expressed at the apical membranes.
IBDUs were mounted on a specially designed stage for
visualization on a fluorescence microscope (Fig. 2A).
Because P2Y receptors couple to phospholipase C
(PLC)- and inositol trisphosphate (InsP3)-mediated increases in Cai2⫹, Cai2⫹ was monitored to indicate P2Y
receptor activation. IBDUs were loaded with the Ca2⫹sensitive dye fluo 4-AM by luminal perfusion with the
dye (Fig. 2B). Subsequent luminal perfusion with nucleotides induced serial increases in fluo 4 fluorescence,
reflecting increases in Cai2⫹ (Fig. 2C). These findings
suggest that P2Y receptors are expressed on the apical
membrane of bile duct cells.
To verify that Cai2⫹ increases induced by luminal
nucleotides were specific to P2Y receptor activation,
experiments were performed in the presence of either
the ATP diphosphohydrolase apyrase or the P2Y inhibitor suramin (Fig. 3). ATP (100 ␮M) increased fluo 4
fluorescence by 78 ⫾ 10% (n ⫽ 5), whereas ATP (100
␮M) ⫹ apyrase (3 U/ml) increased fluorescence by only
5 ⫾ 0.8% (n ⫽ 5; P ⬍ 0.01 by 1-tailed t-test), and ATP
(100 ␮M) ⫹ suramin (50 ␮M) increased fluorescence by
only 9 ⫾ 2.7% (n ⫽ 5; P ⬍ 0.02 by 1-tailed t-test). These
findings demonstrate that increases in Cai2⫹ induced
by luminal perfusion of nucleotides are caused by activation of apical P2Y receptors.
To determine whether apical cholangiocyte P2Y receptors are activated in a concentration-sensitive fashion, we perfused IBDUs with ATP at a concentration
range of 10 nM–1 mM. Perfusion with 10 nM ATP did
not increase fluo 4 fluorescence; 100 nM ATP increased
fluo 4 fluorescence by ⬃22%; 1 ␮M and 10 ␮M ATP
increased fluo 4 fluorescence by 41% and 46%, respectively; and 100 ␮M ATP increased fluo 4 fluorescence
by ⬃88% and was the maximum concentration of ATP
found to increase fluo 4 fluorescence. Perfusion with 1
mM ATP decreased fluo 4 fluorescence by ⬃5%. This
may have been caused by hyperstimulation of G protein-coupled receptors leading to nonspecific G protein
cross talk (32) or, alternatively, by acidification of the
cytosol and subsequent pH-dependent decrease in fluo
4 fluorescence (Molecular Probes). Together, these
findings suggest that cholangiocytes are sensitive to
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P2Y RECEPTORS IN RAT CHOLANGIOCYTES
Fig. 2. P2Y receptors are expressed on
apical cholangiocyte membranes. A:
representative transmission image of a
microperfused intrahepatic bile duct
unit (IBDU). B: fluorescence image of
the same IBDU loaded with fluo 4-AM.
A segment of bile duct was manually
dissected and cannulated and then
loaded with the Ca2⫹-sensitive dye fluo
4. C: representative tracing of a cytosolic Ca2⫹ (Cai2⫹) increase in an IBDU. A
100-␮m pinhole was placed over an intracellular area of fluorescence, and
then serial changes in fluorescence
were monitored over time. Fluo 4 fluorescence remains stable during perfusion with buffer alone, but a rapid increase in fluo 4 fluorescence is seen
after addition of ATP (100 ␮M). Similar
findings were seen with 2-methylthioadenosine 5⬘-triphosphate (2-MeSATP), ADP, UDP, and UTP. f/f0, Peakto-baseline fluorescence ratio.
ATP over the range 100 nM–100 ␮M, which includes
the concentrations of nucleotides found in bile (9).
To investigate whether apical cholangiocyte membranes express multiple subtypes of P2Y receptors, we
perfused IBDUs with nucleotides that are specific to
each particular P2Y receptor subtype. ATP, ADP,
2-MeS-ATP, UTP, and UDP each increased fluo 4 fluorescence by 41 ⫾ 4 to 62 ⫾ 11% at concentrations of
1–10 ␮M and by 88 ⫾ 20 to 100 ⫾ 22% at a concentration of 100 ␮M (Fig. 4B). Pretreatment of ducts with
ATP, ADP, UTP, or UDP had no effect on subsequent
nucleotide responses (ATP, n ⫽ 6; ADP, n ⫽ 6; UTP,
n ⫽ 7; UDP, n ⫽ 7). Because ADP, ATP, and 2-MeSATP act at the P2Y1 receptor, UTP and ATP act at the
P2Y2 and P2Y4 receptors, and UDP acts at the P2Y6
receptor, these findings suggest that rat cholangiocytes
express all four of these P2Y subtypes at the apical
membrane. Because nucleotide pretreatment had no
effect on subsequent nucleotide responses, this sug-
gests that these responses to different nucleotides are
mediated by expression of multiple P2Y receptor subtypes rather than nucleotide interconversion.
Rat cholangiocytes express P2Y receptors at basolateral plasma membrane. Additional experiments were
performed in IBDUs to investigate whether P2Y receptors are expressed at the basolateral membranes of
cholangiocytes. The presence of basolateral P2Y receptors was investigated by addition of nucleotides to the
perifusing bath. ACh was used as a positive control
because cholangiocytes are known to express M3 muscarinic receptors at the basolateral membrane (2, 29).
ACh (100 ␮M) increased fluo 4 fluorescence by 24 ⫾ 1%
(n ⫽ 4; P ⬍ 0.05 vs. ATP), but only minimal increases
in fluorescence (2–10%) were evoked by ATP (100 ␮M;
n ⫽ 5), ADP (100 ␮M; n ⫽ 5), UTP (100 ␮M; n ⫽ 5), or
UDP (100 ␮M; n ⫽ 5). However, basolateral addition of
the nonhydrolyzable ATP analog ATP-␥-S (100 ␮M)
increased fluo 4 fluorescence by 30 ⫾ 2% (n ⫽ 3; P ⬍
Fig. 3. Cai2⫹ increases induced by apical nucleotides result from activation of P2Y receptors.
Apical P2Y receptors were activated as described
in Fig. 2 with ATP (100 ␮M), ATP (100 ␮M) ⫹
apyrase (3 U/ml), or ATP (100 ␮M) ⫹ suramin
(50 ␮M). ATP alone nearly doubled fluo 4 fluorescence, whereas fluorescence was increased
only minimally by ATP in the presence of the
ATP diphosphohydrolase apyrase or the P2Y receptor antagonist suramin. *P ⬍ 0.01, **P ⬍ 0.02
vs. ATP by 1-tailed t-test. Similar findings were
seen on stimulation with 2-MeS-ATP, ADP,
UDP, and UTP (not shown).
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P2Y RECEPTORS IN RAT CHOLANGIOCYTES
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antagonist suramin (50 ␮M) and the Cai2⫹ chelator
BAPTA-AM (50 ␮M).
Together, these data suggest that cholangiocytes express P2Y receptors at the basolateral membrane, but
activation of these receptors by ATP and other nucleotides is attenuated in part by hydrolysis of nucleotides. Moreover, the decreased effect of basolateral
ATP-␥-S (relative to apical nucleotides) suggests that
the potency of nucleotides at the basolateral membrane
is weaker than at the apical membrane. Therefore,
expression of P2Y receptors at the basolateral plasma
membrane may be lower than that at the apical membrane. Alternatively, apical P2Y receptors may couple
more strongly to G proteins that link to PLC and InsP3
formation (31). A final possibility is that the unique
ability of basolateral ATP-␥-S to induce a relatively
weak Cai2⫹ transient is caused by phosphatase-resistant thiophosphorylation reactions catalyzed by ectokinases rather than activation of basolateral nucleotide
receptors. Direct measurement of basolateral ectonucleotidase activity would be needed to distinguish
among these possible explanations.
Functional expression of P2X receptors in rat cholangiocytes. Functional expression of P2X receptors was
examined using P2X-specific synthetic nucleotides ␣,␤MeATP (P2X agonist most potent at P2X1 and P2X2),
␤,␥-MeATP (P2X agonist most potent at P2X1), and
BzATP (P2X agonist most potent at P2X1 and P2X7)
(Fig. 6) (30). Stimulation of IBDUs with these agonists
at either the apical or basolateral membrane induced
minimal changes (⫺6% to ⫹8%) in Cai2⫹, yet apical
perfusion with ATP as a positive control yielded Cai2⫹
increase of 46 ⫾ 6.8%, similar to what was seen in prior
experiments. These data suggest that, although rat
cholangiocytes express the P2X receptor subtype P2X4,
they do not express P2X subtypes sensitive to P2XFig. 4. Multiple P2Y receptor subtypes are expressed on apical
cholangiocyte membranes. A: concentration-response curve for apical stimulation with ATP. Apical P2Y receptors were activated as
described in Fig. 2 for ATP at concentrations of 10 nM–1 mM. The
lowest concentration to activate apical ATP receptors was 100 nM,
and the greatest concentration to activate apical P2Y receptors was
100 ␮M. The apparent lack of response at 1 mM may be caused by
activation of other G protein-coupled receptor systems or by loss of
fluo 4 fluorescence caused by changes in cell pH. B: comparison of
nucleotide responses for apical nucleotides. Concentration responses
were determined for the nucleotides 2-MeS-ATP, ADP, ATP, UDP,
and UTP at concentrations of 1, 10, and 100 ␮M. At 1–10 ␮M, all
nucleotides induced a similar increase in fluo 4 fluorescence. At 100
␮M, all nucleotides induced a much greater increase in fluo 4 fluorescence. Nucleotide-induced increases in fluo 4 fluorescence were
roughly equal for each nucleotide at each concentration. Together,
these data suggest that apical cholangiocyte membranes express the
P2Y receptor subtypes P2Y1, P2Y2 or P2Y4, and P2Y6.
0.05 vs. ATP), similar to the increase induced by ACh
(Fig. 5). The lowest concentration of ACh that induced
an increase in fluo 4 fluorescence was 10 ␮M, consistent with previous observations in cholangiocytes
(29), whereas the lowest concentration of ATP-␥-S that
induced an increase in fluo 4 fluorescence was 1 ␮M.
The effect of ATP-␥-S was inhibited by both the P2Y
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Fig. 5. P2Y receptors are expressed on basolateral cholangiocyte
membranes. IBDU cells were loaded with fluo 4-AM and analyzed as
described in Fig. 2. Basolateral P2Y receptors were activated by
perifusing IBDUs with buffer containing ACh (100 ␮M), ATP (100
␮M), or adenosine 5⬘-O-(3-thiotriphosphate) (ATP-␥-S; 100 ␮M).
ACh, which activates M3 receptors expressed on basolateral cholangiocyte membranes, induced an increase of ⬃25% in fluo 4 fluorescence. ATP, ADP, UDP, and UTP increased fluo 4 fluorescence
⬍10%; however, the nonhydrolyzable ATP analog ATP-␥-S induced
an increase in fluo 4 fluorescence roughly equal to that seen with
ACh.
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P2Y RECEPTORS IN RAT CHOLANGIOCYTES
Fig. 6. Functional analysis of bile ductular P2X
receptor expression. The functional expression of
P2X receptors in rat bile duct epithelia was accomplished by fluo 4 Cai2⫹ imaging of IBDUs
stimulated with P2X-specific agonists ␣,␤-methyleneadenosine 5⬘-triphosphate (MeATP) (␣,␤),
␤,␥-MeATP (␤,␥), and 2⬘- and 3⬘-O-(4-benzoylbenzoyl) ATP (BzATP) (Bz). Neither apical nor
basolateral stimulation with any of these agonists (100 ␮M each) induced an appreciable increase in Cai2⫹, whereas ATP (100 ␮M, apically
applied) induced an increase comparable to that
seen in Fig. 4.
specific nucleotides. Because there are no specific agonists available for the P2X4 receptor, the polarity of its
expression cannot be established.
Activation of bile duct epithelial P2Y receptors induces bile duct alkalization. To determine the functional role of cholangiocyte P2Y receptors in fluid and
electrolyte secretion, changes in biliary pH were monitored after addition of ATP-␥-S to either the apical or
the basolateral cell surface (Fig. 7). ATP-␥-S was used
to eliminate the role of nucleotide hydrolysis seen in
Fig. 5. Apical perfusion of ATP-␥-S (100 ␮M) induced a
net increase in biliary pH of 0.32 ⫾ 0.078 units (P ⫽
0.039). Basolateral perfusion of ATP-␥-S (100 ␮M) induced a trend toward increase in biliary pH of 0.10 ⫾
0.019 units (P ⫽ 0.058). The finding that apical ATP␥-S induced a larger increase in pH than basolateral
ATP-␥-S is consistent with the difference in Cai2⫹ increases seen with these agonists. For comparison,
stimulation with a maximal concentration of forskolin
increases luminal pH by ⬃0.6 units in this experimental system (27). These experiments demonstrate that
Fig. 7. Activation of P2Y receptors induces cholangiocyte HCO3⫺
secretion. Changes in ductular pH (⌬pH) induced by extracellular
nucleotides were examined using ductular perfusion with 2⬘,7⬘-bis(2carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-dextran. Apical stimulation with ATP-␥-S (100 ␮M) induced an approximate increase in duct
pH of 0.3 units (*P ⬍ 0.05), whereas basolateral stimulation with
ATP-␥-S (100 mM) induced an increase of 0.1 unit (**P ⫽ 0.058) relative
to nonstimulated controls perfused with identical buffer.
AJP-Gastrointest Liver Physiol • VOL
nucleotide signaling may contribute to regulation of
HCO3⫺ secretion in bile duct epithelia.
DISCUSSION
The role of extracellular nucleotides as signaling
molecules has now been shown in many tissue types
(15, 28, 42, 46). A specific role for nucleotides and their
Fig. 8. Proposed mechanism for activation of cholangiocyte P2Y
receptors. Bile duct epithelia (BDE) can be activated by either
luminal (apical) or basolateral nucleotides in the form of nucleotide
triphosphates (NTPs) or diphosphates (NDPs). At the apical membrane, P2Y receptor subtypes P2Y1, P2Y2, P2Y4, and P2Y6 are
expressed. P2Y receptors are also expressed at the basolateral membrane but are attenuated by endogenous nucleoside triphosphate
diphosphohydrolases or other mechanisms. Activation of either apical or basolateral P2Y receptors induces increases in Cai2⫹ and may
induce apical HCO3⫺ export, possibly by activating Ca2⫹-sensitive
Cl⫺ transport and subsequent Cl⫺/HCO3⫺ exchange.
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P2Y RECEPTORS IN RAT CHOLANGIOCYTES
receptors in liver, and in cholangiocytes in particular,
has been demonstrated in several ways. First, hepatocytes can signal to cholangiocytes over distances of
several hundred micrometers through nucleotide release, suggesting that P2Y receptors allow paracrine
signaling between these two liver cell types (39). Second, nucleotides are found in nanomolar to micromolar
concentrations in bile, suggesting that purinergic signaling from hepatocytes to cholangiocytes may occur
through release of nucleotides into bile (9). Third, expression of nucleotide receptors by bile duct-derived
cell lines is critical for autoregulation of cell volume,
demonstrating that P2Y receptors are important in
autocrine signaling in these cells (35). Finally, bile
duct-derived cell culture models express apical P2Y
receptors, which would be necessary for activation by
nucleotides released into bile (36, 38). Together, these
observations show an important potential role for purinergic signaling through P2Y receptors in bile ductular signaling.
However, previous studies failed to address several
key questions. First, the distribution and relative roles
of the different subtypes of P2Y receptors found in
cholangiocytes were unknown. The various subtypes of
P2Y receptors expressed in rat and other mammals
differ not only in molecular structure but also in nucleotide pharmacological preference. Here we found
that rat cholangiocytes express all four of the P2Y
subtypes previously identified in this species. Thus
these cells can be activated not only by ATP but also by
a variety of nucleotides. This finding is important for
several reasons. First, multiple P2Y subtypes have
been cloned only in the past few years. Although these
receptors share common tertiary structure and function, their primary structures vary greatly. A precise
understanding of P2Y receptor tissue distribution
would facilitate design of pharmacological agonists or
antagonists for these receptors. Moreover, endogenously occurring nucleotides found in body fluids include not only adenosine nucleotides but uridine nucleotides as well (21). Because all nucleotides are
equipotent in cholangiocyte activation, it is possible
that those nucleotides are also involved in signaling
responses for these cells.
A second question that has been addressed in the
present study is the distribution of P2Y receptors in
native cholangiocytes in particular. Although the demonstration of P2Y receptors has been examined in
cholangiocyte cell lines (36, 38), cell lines in general
have been inaccurate as models of P2Y receptor expression. Specifically, it has been found that expression and
polarized distribution of P2Y receptor subtypes depends on both number of days in culture and cell
support mechanisms (18). In fact, the NRC bile duct
cell line appears to express only P2Y2 receptors at the
apical membrane (38), whereas we report here that
native cholangiocytes also express P2Y1, P2Y4, and
P2Y6 apically. Thus cell culture models may provide an
insufficient means to determine the polarized distribution of P2Y receptor subtypes in native tissues. In the
liver, this is of critical import—if receptors are exAJP-Gastrointest Liver Physiol • VOL
G1065
pressed apically, they are activated by nucleotides released directly into bile, whereas if they are expressed
basolaterally, they are activated by nucleotides released into blood or by nerves. Our finding that P2Y
receptors are expressed both apically and basolaterally
shows that each mechanism may be operative in bile
duct epithelia. Our finding that basolateral nucleotide
signaling is attenuated at least in part by nucleotide
hydrolysis is consistent with the observation that ATP
degradation is biphasic during basolateral exposure to
NRC cells (36). This also suggests that nucleoside
triphosphate diphosphohydrolases may be localized to
the basolateral membranes of cholangiocytes rather
than to nearby cells or interstitium. Because nucleotides appear to be degraded more quickly basolaterally,
P2Y receptor activation at this plasma membrane domain may be more important for autocrine signaling,
whereas activation of apical P2Y receptors may be
more important for paracrine signaling (Fig. 8).
Because P2Y receptor activation can induce Cl⫺ and
HCO3⫺ secretion independent of CFTR, these receptors
are an attractive therapeutic target in secretory disorders such as CF. In fact, nucleotides activate Cl⫺ currents in multiple tissues affected by CF, including
respiratory, reproductive, and digestive epithelia (7,
12, 24, 26, 41, 48). In cholangiocytes, both CFTR and
P2Y-activated non-CFTR Cl⫺ channels are thought to
induce bulk fluid and electrolyte secretion through
apical Cl⫺/HCO3⫺ exchange, duct alkalization, and
paracellular water movement. Furthermore, second
messengers such as cAMP and Cai2⫹ may induce ATP
release (5, 6, 9) and thereby activate P2Y-mediated
secretion. Activation of Ca2⫹-sensitive Cl⫺ channels
and subsequent Cl⫺/HCO3⫺ exchange is a likely mechanism for nucleotide-induced bile duct alkalization.
However, duct alkalization may also work through
direct HCO3⫺ export (11, 14). Furthermore, studies in
the intact, bivascularly perfused liver suggest that
regulation of biliary HCO3⫺ excretion may be the principal function of cholangiocytes in vivo (17). The current work provides evidence that apical stimulation
with nucleotides may thus have the same physiological
effect as basolateral stimulation with secretin or ACh
(17). The mechanism by which extracellular nucleotides regulate bile duct alkalization should provide a
robust area for further research that may provide insights into the pathogenesis of CF and other cholestatic
conditions in which cholangiocytes are the target of
disease.
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-02739 (to J. A. Dranoff),
DK-24031 (to N. F. LaRusso), and DK-45710 (to M. H. Nathanson),
a Glaxo Wellcome Institute for Digestive Health Basic Research
Award (to J. A. Dranoff), and awards from the Cystic Fibrosis
Foundation (to M. H. Nathanson) and the Yale Liver Center (DK34989).
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