1 Yale University School of Medicine, New Haven, Connecticut 06520; and 2 Mayo Clinic and Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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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+ (Ca-S), but not ATP, to the
perifusing bath increased Ca
-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
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INTRODUCTION |
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EXTRACELLULAR NUCLEOTIDES 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 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.
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METHODS |
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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; 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 HCO
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). First-strand 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 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+ (Ca
Data analysis of Ca5 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
1 s
were disregarded to eliminate the possibility of artifactual
fluorescence increases. Fluorescence increases for each individual
experiment were expressed as a peak-to-baseline 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 HCO
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.
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RESULTS |
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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 line, in which the only P2Y receptor molecularly identified was the P2Y2 receptor
(38). Because nucleotide-induced increases in
Ca
|
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
Ca
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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 µM) 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 µM; n = 5), ADP (100 µM;
n = 5), UTP (100 µM; n = 5), or UDP
(100 µM; n = 5). However, basolateral addition of the
nonhydrolyzable ATP analog ATP--S (100 µM) increased fluo 4 fluorescence by 30 ± 2% (n = 3;
P < 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 µM,
consistent with previous observations in cholangiocytes
(29), whereas the lowest concentration of ATP-
-S
that induced an increase in fluo 4 fluorescence was 1 µM. The effect
of ATP-
-S was inhibited by both the P2Y antagonist suramin (50 µM)
and the Ca
|
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 Ca
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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 µM) induced a net increase in biliary pH
of 0.32 ± 0.078 units (P = 0.039). Basolateral
perfusion of ATP-
-S (100 µM) 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
Ca
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DISCUSSION |
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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 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
importif receptors are expressed 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
HCO
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
/HCO
channels and subsequent Cl
/HCO
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ACKNOWLEDGEMENTS |
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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 (DK-34989).
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FOOTNOTES |
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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: jonathan.dranoff{at}yale.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 February 2001; accepted in final form 26 April 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alpini, G,
Philips JO,
and
LaRusso NF.
The biology of biliary epithelia.
In: The Liver: Biology and Pathobiology (3rd ed.), edited by Arias IM,
et al. New York: Raven, 1994, p. 623-653.
2.
Alvaro, D,
Alpini G,
Jezequel AM,
Bassotti C,
Francia C,
Fraioli F,
Romeo R,
Marucci L,
LaSage G,
Glasser SS,
and
Benadetti A.
Role and mechanisms of action of acetylcholine in the regulation of rat cholangiocyte secretory functions.
J Clin Invest
100:
1349-1362,
1997
3.
Barnard, EA,
Simon J,
and
Webb TE.
Nucleotide receptors in the nervous system. An abundant component using diverse transduction mechanisms.
Mol Neurobiol
15:
103-129,
1997[ISI][Medline].
4.
Bogdanov, YD,
Wildman SS,
Clements MP,
King BF,
and
Burnstock G.
Molecular cloning and characterization of rat P2Y4 nucleotide receptor.
Br J Pharmacol
124:
428-430,
1998[Abstract].
5.
Cantiello, HF,
Jackson GR, Jr,
Prat AG,
Gazley JL,
Forrest JN, Jr,
and
Ausiello DA.
cAMP activates an ATP-conductive pathway in cultured shark rectal gland cells.
Am J Physiol Cell Physiol
272:
C466-C475,
1997
6.
Cantiello, HF,
Prat AG,
Reisin IL,
Ercole LB,
Abraham EH,
Amara JF,
Gregory RJ,
and
Ausiello DA.
External ATP and its analogs activate the cystic fibrosis transmembrane conductance regulator by a cyclic AMP-independent mechanism.
J Biol Chem
269:
11224-11232,
1994
7.
Chan, HC,
Cheung WT,
Leung PY,
Wu LJ,
Cheng-Chew SB,
Ko WH,
and
Wong PYD
Purinergic regulation of anion secretion by cystic fibrosis pancreatic duct cells.
Am J Physiol Cell Physiol
271:
C469-C477,
1996
8.
Chang, K,
Hanaoka K,
Kumada M,
and
Takuwa T.
Molecular cloning and functional analysis of a novel P2 nucleotide receptor.
J Biol Chem
270:
26152-26158,
1995
9.
Chari, RS,
Schutz SM,
Haebig JE,
Shimokura GH,
Cotton PB,
Fitz JG,
and
Meyers WC.
Adenosine nucleotides in bile.
Am J Physiol Gastrointest Liver Physiol
270:
G246-G252,
1996
10.
Chen, Z-P,
Krull N,
Xu S,
Levy A,
and
Lightman SL.
Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor.
Endocrinology
137:
1833-1840,
1996[Abstract].
11.
Choi, JY,
Muallem D,
Kiselyov K,
Lee MG,
Thomas PJ,
and
Muallem S.
Aberrant CFTR-dependent HCO
12.
Clarke, LL,
and
Boucher RC.
Chloride secretory response to extracellular ATP in human normal and cystic fibrosis nasal epithelia.
Am J Physiol Cell Physiol
263:
C348-C356,
1992
13.
Clarke, LL,
Chinet T,
and
Boucher RC.
Extracellular ATP stimulates K+ secretion across cultured human airway epithelium.
Am J Physiol Lung Cell Mol Physiol
272:
L1084-L1091,
1997
14.
Clarke, LL,
Harline MC,
Gawenis LR,
Walker NM,
Turner JT,
and
Weisman GA.
Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium.
Am J Physiol Gastrointest Liver Physiol
279:
G132-G138,
2000
15.
Cressman, VL,
Lazarowski E,
Homolya L,
Boucher RC,
Koller BH,
and
Grubb BR.
Effect of loss of P2Y(2) receptor gene expression on nucleotide regulation of murine epithelial Cl() transport.
J Biol Chem
274:
26461-26468,
1999
16.
Fabre, JE,
Nguyen M,
Latour A,
Keifer JA,
Audoly LP,
Coffman TM,
and
Koller BH.
Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.
Nat Med
5:
1199-1202,
1999[ISI][Medline].
17.
Hirata K and Nathanson MH. Bile duct epithelia regulate biliary
bicarbonate excretion in normal rat liver. Gastroenterology.
In press.
18.
Inoue, CN,
Woo JS,
Schwiebert EM,
Morita T,
Hanaoka K,
Guggino SE,
and
Guggino WB.
Role of purinergic receptors in chloride secretion in Caco-2 cells.
Am J Physiol Cell Physiol
272:
C1862-C1870,
1997
19.
Knowles, MR,
Clarke LL,
and
Boucher RC.
Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis.
N Engl J Med
325:
533-538,
1991[Abstract].
20.
Knowles, MR,
Clarke LL,
and
Boucher RC.
Extracellular ATP and UTP induce chloride secretion in nasal epithelia of cystic fibrosis patients and normal subjects in vivo.
Chest
101:
60S-63S,
1992[Medline].
21.
Lazarowski, ER,
Homolya L,
Boucher RC,
and
Harden TK.
Direct demonstration of mechanically induced release of cellular UTP and its implication for uridine nucleotide receptor activation.
J Biol Chem
272:
24348-24354,
1997
22.
Leipziger, J,
Kerstan D,
Nitschke R,
and
Greger R.
ATP increases Ca
23.
Leon, C,
Hechler B,
Freund M,
Eckly A,
Vial C,
Ohlmann P,
Dierich A,
LeMeur M,
Cazenave JP,
and
Gachet C.
Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice.
J Clin Invest
104:
1731-1737,
1999
24.
Leung, AY,
Wong PY,
Yankaskas JR,
and
Boucher RC.
cAMP- but not Ca2+-regulated Cl conductance is lacking in cystic fibrosis mice epididymides and seminal vesicles.
Am J Physiol Cell Physiol
271:
C188-C193,
1996
25.
Luo, X,
Zheng W,
Yan M,
Lee MG,
and
Muallem S.
Multiple functional P2X and P2Y receptors in the luminal and basolateral membranes of pancreatic duct cells.
Am J Physiol Cell Physiol
277:
C205-C215,
1999
26.
Mason, SJ,
Paradiso AM,
and
Boucher RC.
Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium.
Br J Pharmacol
103:
1649-1656,
1991[Abstract].
27.
Masyuk, AI,
Gong AY,
Kip S,
Burke MJ,
and
La RN.
Perfused rat intrahepatic bile ducts secrete and absorb water, solute, and ions.
Gastroenterology
119:
1672-1680,
2000[ISI][Medline].
28.
Middleton, JP,
Mangel AW,
Basavappa S,
and
Fitz JG.
Nucleotide receptors regulate membrane ion transport in renal epithelial cells.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F867-F873,
1993
29.
Nathanson, MH,
Burgstahler AD,
Mennone A,
and
Boyer JL.
Characterization of cytosolic Ca2+ signaling in rat bile duct epithelia.
Am J Physiol Gastrointest Liver Physiol
271:
G86-G96,
1996
30.
North, RA,
and
Surprenant A.
Pharmacology of cloned P2X receptors.
Annu Rev Pharmacol Toxicol
40:
563-580,
2000[ISI][Medline].
31.
Paradiso, AM,
Mason SJ,
Lazarowski ER,
and
Boucher RC.
Membrane-restricted regulation of Ca2+ release and influx in polarized epithelia.
Nature
377:
643-646,
1995[ISI][Medline].
32.
Quitterer, U,
and
Lohse MJ.
Crosstalk between G(i)- and G
(q)-coupled receptors is mediated by G
exchange.
Proc Natl Acad Sci USA
96:
10626-10631,
1999
33.
Ralevic, V,
and
Burnstock G.
Receptors for purines and pyrimidines.
Pharmacol Rev
50:
413-492,
1998
34.
Roberts, SK,
Ludwig J,
and
LaRusso NF.
The pathobiology of biliary epithelia.
Gastroenterology
112:
269-279,
1997[ISI][Medline].
35.
Roman, RM,
Feranchak AP,
Salter KD,
Wang Y,
and
Fitz JG.
Endogenous ATP release regulates Cl secretion in cultured human and rat biliary epithelial cells.
Am J Physiol Gastrointest Liver Physiol
276:
G1391-G1400,
1999
36.
Salter, KD,
Fitz JG,
and
Roman RM.
Domain-specific purinergic signaling in polarized rat cholangiocytes.
Am J Physiol Gastrointest Liver Physiol
278:
G492-G500,
2000
37.
Sauzeau, V,
Le Jeune H,
Cario-Toumaniantz C,
Vaillant N,
Gadeau AP,
Desgranges C,
Scalbert E,
Chardin P,
Pacaud P,
and
Loirand G.
P2Y1, P2Y2, P2Y4, and P2Y6 receptors are coupled to Rho and Rho kinase activation in vascular myocytes.
Am J Physiol Heart Circ Physiol
278:
H1751-H1761,
2000
38.
Schlenker, T,
Joelle M,
Romac J,
Sharara AI,
Roman RM,
Kim SJ,
LaRusso N,
Liddle RA,
and
Fitz JG.
Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes.
Am J Physiol Gastrointest Liver Physiol
273:
G1108-G1117,
1997
39.
Schlosser, SF,
Burgstahler AD,
and
Nathanson MH.
Isolated rat hepatocytes can signal to other hepatocytes and bile duct cells by release of nucleotides.
Proc Natl Acad Sci USA
93:
9948-9953,
1996
40.
Strazzabosco, M,
Mennone A,
and
Boyer JL.
Intracellular pH regulation in isolated rat bile duct epithelial cells.
J Clin Invest
87:
1503-1512,
1991[ISI][Medline].
41.
Stutts, MJ,
Chinet TC,
Mason SJ,
Fullton JM,
Clarke LL,
and
Boucher RC.
Regulation of Cl channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP.
Proc Natl Acad Sci USA
89:
1621-1625,
1992[Abstract].
42.
Stutts, MJ,
Fitz JG,
Paradiso AM,
and
Boucher RC.
Multiple modes of regulation of airway epithelial chloride secretion by extracellular ATP.
Am J Physiol Cell Physiol
267:
C1442-C1451,
1994
43.
Tokuyama, Y,
Hara M,
Jones EMC,
Fan Z,
and
Bell GI.
Cloning of rat and mouse P2Y purinoceptors.
Biochem Biophys Res Commun
211:
211-218,
1995[ISI][Medline].
44.
Webb, TE,
Boluyt MO,
and
Barnard EA.
Molecular biology of P2Y purinoceptors: expression in rat heart.
J Auton Pharmacol
16:
303-307,
1996[ISI][Medline].
45.
Williams, M.
Purines: from premise to promise.
J Auton Nerv Syst
81:
285-288,
2000[ISI][Medline].
46.
Wilson, PD,
Hovater JS,
Casey CC,
Fortenberry JA,
and
Schwiebert EM.
ATP release mechanisms in primary cultures of epithelia derived from the cysts of polycystic kidneys.
J Am Soc Nephrol
10:
218-229,
1999
47.
Yen, PT,
Herman P,
Van den Abbeele T,
Tan CT,
Bordure P,
Marianowski R,
Friedlander G,
and
Tran Ba Huy P.
Extracellular ATP modulates ion transport via P2Y purinoceptors in a middle-ear epithelial cell line.
ORL J Otorhinolaryngol Relat Spec
59:
170-175,
1997[ISI][Medline].
48.
Zeng, W,
Lee MG,
and
Muallem S.
Membrane-specific regulation of Cl channels by purinergic receptors in rat submandibular gland acinar and duct cells.
J Biol Chem
272:
32956-65,
1997
49.
Zsembery, A,
Strazzabosco M,
and
Graf J.
Ca2+-activated Cl channels can substitute for CFTR in stimulation of pancreatic duct bicarbonate secretion.
FASEB J
14:
2345-2356,
2000