Department of Medicine, Division of Gastroenterology, University of Washington and Department of Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98108
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ABSTRACT |
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Pancreatic duct
epithelial cells (PDEC) mediate the exocrine secretion of fluid and
electrolytes. We previously reported that ATP and UTP interact with
P2Y2 receptors on nontransformed canine PDEC to increase
intracellular free Ca2+ concentration
([Ca2+]i) and stimulate
Ca2+-activated Cl and K+
channels. We now report that ATP interacts with additional
purinergic receptors to increase cAMP and activate Cl
channels. ATP, 2-methylthio-ATP, and ATP-
-S stimulated a 4- to
10-fold cAMP increase with EC50 of 10-100 µM.
Neither UTP nor adenosine stimulated a cAMP increase, excluding a role
for P2Y2 or P1 receptors. Although UTP stimulated an
125I
efflux that was fully inhibited by
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
tetra(acetoxymethyl) ester (BAPTA-AM), ATP stimulated a partially resistant efflux, suggesting activation of additional Cl
conductances through P2Y2-independent and
Ca2+-independent pathways. In Ussing chambers, increased
cAMP stimulated a much larger short-circuit current
(Isc) increase from basolaterally permeabilized
PDEC monolayers than increased [Ca2+]i.
Luminal ATP and UTP and serosal UTP stimulated a small
Ca2+-type Isc increase, whereas
serosal ATP stimulated a large cAMP-type Isc
response. Serosal ATP effect was inhibited by P2 receptor blockers and
unaffected by BAPTA-AM, supporting ATP activation of Cl
conductances through P2 receptors and a Ca2+-independent
pathway. RT-PCR confirmed the presence of P2Y11 receptor mRNA, the only P2Y receptor acting via cAMP.
iodide efflux; adenosine 5'-triphosphate; Ussing chamber
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INTRODUCTION |
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THE EXTRACELLULAR
FUNCTION of ATP and other nucleotides as messenger agents in
regulating cellular metabolism and ion transport is now well
established. The effects of these agents are mediated through specific
P2 receptors composed of two major subclasses, the P2X receptors
possessing two transmembrane domains and intrinsic ion channel activity
and the P2Y receptors possessing seven transmembrane domains (8,
23, 24). The P2Y family includes selective purinoceptors
(P2Y1 receptor activated preferentially by ADP and ATP),
nucleotide receptors responsive to adenine and uracil nucleotides (P2Y2 receptor activated equipotently by ATP and UTP and
P2Y8 receptor activated equally by all triphosphate
nucleotides), and pyrimidinoceptors (P2Y3 and
P2Y6 receptors activated preferentially by UDP and
P2Y4 receptor activated preferentially by UTP). Although these P2Y receptor subtypes couple to phospholipase C to stimulate an
increase in intracellular free Ca2+ concentration
[Ca2+]i (1, 9, 23), the recently
cloned P2Y11 receptor has the unique property of activating
adenylyl cyclase in addition to phospholipase C (4). This
receptor exhibits 33% amino acid identity with the P2Y1
receptor, its closest homolog. When this receptor was expressed in
1321N1 astrocytoma cells and in CHO-K1 cells, the rank order of agonist
potency was ATP--S = 2' and 3'-O-(4-benzoylbenzoyl)-ATP > ATP > 2-methylthio-ATP > ADP; UTP and UDP were inactive (4,
5). Northern blot probing showed the P2Y11
receptor to be present in HL-60 leukemia cells and, weakly, in the
small intestine. Madin-Darby canine kidney (MDCK) cells are the
only other known cell type that expresses these receptors
(22). Although P2Y11 receptors may mediate
cAMP-dependent differentiation in HL-60 cells, the expression and
function of these receptors in different epithelial cells remain to be
further defined (6, 11, 27). In this report, we define a
secretory role for P2Y11 receptors on pancreatic duct
epithelial cells (PDEC).
PDEC mediate secretion of fluid and electrolytes and, together with
acinar cells, which mediate secretion of digestive proenzymes, constitute pancreatic exocrine secretion. The secretory function of
PDEC results from many ion transport pathways, including, on the apical
surface of the cell, the cystic fibrosis transmembrane conductance
regulator (CFTR), a cAMP-activated Cl channel, a distinct
Ca2+-activated Cl
channel, and a
HCO
exchanger, and, on the
basolateral surface of the cell, K+ channels, a
Na+-(HCO3)n cotransporter, and a
Na+/H+ antiporter. We previously reported
(16) that ATP and UTP interact with specific
P2Y2 receptors expressed on both apical and basolateral surfaces of cultured dog PDEC to promote an increase in
[Ca2+]i, stimulate mucin secretion, and
activate both Cl
and K+ conductances. We now
report that ATP also interacts with a P2Y receptor (most likely the
P2Y11 receptor) on the basolateral surface of these cells
to activate cAMP-dependent Cl
channels. To our knowledge,
this is the first description of a secretory function for these
receptors in epithelial cells.
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EXPERIMENTAL PROCEDURES |
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Chemicals and reagents.
ATP, UTP, ATP--S, 2-methylthio-ATP,
,
-methylene ATP,
adenosine, suramin, reactive blue, and tissue culture medium and
supplements were from Sigma (St. Louis, MO), and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
tetra(acetoxymethyl) ester (BAPTA-AM) was from Calbiochem (La Jolla,
CA). Na125I (16 mCi/mg iodide) was purchased from Amersham
(Arlington Heights, IL).
Cell culture.
Canine PDEC isolated from the accessory pancreatic duct were cultured
on Transwell inserts (Costar, Cambridge, MA) coated with Vitrogen
(Collagen Corporation, Palo Alto, CA) and suspended over a feeder layer
of normal human gallbladder myofibroblasts. This system exposes the
PDEC to growth factors from myofibroblasts that are necessary for their
propagation and differentiation (20). We previously
demonstrated (15, 17) that these PDEC have many of the
characteristics expected of pancreatic duct cells, such as mucin
secretion and expression of Ca2+-activated K+
channels, Ca2+-activated Cl channels, and
cAMP-activated CFTR Cl
channels. These cells were also
used successfully to examine the secretory effects of agents acting via
cAMP, such as vasoactive intestinal polypeptide (15, 20),
and increased [Ca2+]i, such as histamine and
trypsin (18, 19). The cells used in this report were
between passages 9 and 30. Unless specified otherwise, each experiment and corresponding comparisons used monolayers seeded, cultured, and studied at the same time.
Determination of intracellular cAMP. Confluent monolayers of PDEC grown on Transwell inserts were washed with (in mM) 140 NaCl, 4.7 KCl, 1.2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4, and treated with ATP or analogs for 20 min. The cells were then scraped and transferred (using 1 ml of water) to a test tube containing 1 ml of 5% perchloric acid. After 15 min at 4°C, the samples were centrifuged at 1,500 g for 10 min and 0.4 ml of 30% potassium bicarbonate was added to the supernatant to precipitate potassium perchlorate. After 45 min at 4°C the samples were again centrifuged at 1,500 g for 10 min, and the supernatant was lyophilized overnight. The dried sample was resuspended in 1.2 ml of buffer, and the cAMP concentration was determined by radioimmunoassay using a kit from Diagnostic Products (Los Angeles, CA).
Efflux studies.
The use of cellular 125I effluxes to study
the activation of Cl
conductances has been validated
(28) and was effective for characterizing Cl
conductances on canine PDEC (15, 16, 18, 19).
Ussing chamber studies. Confluent monolayers of PDEC and their supporting membrane were excised from the Transwell system and mounted in modified Ussing chambers with an aperture area of 0.95 cm2. In this system, the luminal compartment is in contact with the apical surface of the PDEC and the serosal compartment is in contact with the basolateral surface of the cell.
To study apical ClIdentification of P2Y11 mRNA by RT-PCR. Confluent PDEC were harvested from Transwell inserts by trypsinization for 30 min at 37°C followed by centrifugation at 500 g for 10 min at 4°C. The cells were washed once with PBS, and total cellular RNA was extracted and purified according to the guanidinium-isothiocyanate method (3) with reagents supplied by Qiagen (Valencia, CA). Typically, 3 mg of total RNA was isolated from 12 Transwell inserts. RNA was further purified by DNaseI treatment for 30 min at 37°C with an absorbance ratio at 260 nm/280 nm of 2.0. A sample of 0.15 mg of RNA was reverse-transcribed in (mM) 10 Tris · HCl, 50 KCl, and 1.5 MgCl2, pH 8.3, supplemented with 0.0125 U/ml of placental RNase inhibitor, 1.25 mM random decamer, 50 mM dNTP, and 100 U of Moloney murine leukemia virus reverse transcriptase (Ambion, TX). Reverse transcription was allowed to occur for 1 h at 42°C, followed by inactivation for 10 min at 92°C. The cDNA was next PCR-amplified using 1 U of SuperTaq DNA polymerase (Ambion) in 50 µl of reaction mixture containing 125 µM dNTP and 0.5 µM reverse/forward primer mix, using the following thermocycling profile: one 10-min cycle at 95°C, followed by 40 cycles of 30 s at 95°C, 90 s at 60°C, and 90 s at 72°C, and a final holding at 72°C for 10 min. The primers, derived from the human DNA sequence of the P2Y11 receptor and synthesized by IDT (Coralville, IO), were forward 5'-CTGGTGGTTGAGTTCCTGGT-3' and reverse 5'-GTTGCAGGTGAAGAGGAAGC-3' (expected human product size: 234 bp) (22). The PCR product was then visualized by ultraviolet lighting after 8% polyacrylamide gel electrophoresis at 100 V for 30 min. The DNA in the dominant band at ~220 bp was eluted from the gel and sequenced using an ABI 373 sequencer (Applied Biosystems, Foster City, CA) and an ABI prism dye terminator sequencing protocol. Sequence homology was determined using the advanced BLAST program from the National Center for Biotechnology Information.
Statistics. Unless specified otherwise, results are expressed as means ± SE. When appropriate, statistical significance was determined with Student's t-test using the Statview 512+ software program (Brainpower, Calabasas, CA).
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RESULTS |
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Determination of intracellular cAMP.
As shown in Fig. 1A, compared
with a control incubation, a 5.5-fold increase in intracellular cAMP,
from 0.178 ± 0.039 to 1.002 ± 0.246 (relative units;
n = 6 for each), was observed in PDEC treated with 100 µM of ATP for 20 min (P < 0.01, unpaired 2-tailed
t-test with 10 df). When 14 experiments (each with
n 3) were combined, the increase in cAMP produced by 100 µM ATP was 5.0 ±1.5-fold greater than that in the control
incubation. A similar increase was also observed with the ATP analog
2-methylthio-ATP (1.083 ± 0.181, increased over control with
P < 0.001). In contrast, treatment with UTP showed no
significant increase in cAMP (0.213 ± 0.020, P = 0.441 compared with control).
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Iodide efflux studies.
To correlate the ATP-stimulated increase in cAMP with a biological
event, activation of Cl conductances was assessed through
studies of 125I
efflux. We previously
established (15) that activation of either the
cAMP-activated CFTR or the Ca2+-activated Cl
channel is reflected by an increase in the
125I
efflux rate coefficient. As shown in
Fig. 3, 100 µM of either UTP (Fig.
3A) or ATP (Fig. 3B) stimulated an increase in
125I
efflux. In Fig. 3A, the
125I
efflux increase stimulated by UTP was
totally abolished by preincubation with the cell-permeant
Ca2+ chelator BAPTA-AM (50 µM), suggesting that this
efflux was totally mediated through an increase in
[Ca2+]i. Indeed, in the control experiments
shown in Fig. 3, C and D, the
125I
effluxes resulting from either
Ca2+ influx (treatment with 1 µM of the calcium ionophore
A-23187) or mobilization of intracellular calcium (treatment with 2 µM thapsigargin) were also completely inhibited by BAPTA-AM. In
contrast, the efflux rate increase stimulated by ATP was only partially inhibited by the same calcium chelator (manifested by a delayed and
smaller peak response; Ref. 15), suggesting that this
efflux was only partially mediated by increased
[Ca2+]i (Fig. 3B). As previously
reported (16), 100 µM adenosine did not stimulate an
increase in 125I
efflux (Fig. 3E);
this finding is consistent with the earlier absence of adenosine effect
on cAMP production.
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Ussing chamber studies.
To examine further the ATP activation of Cl conductances
on PDEC, confluent monolayers of PDEC were mounted in Ussing chambers and their basolateral membranes were permeabilized to small monovalent ions with 0.36 mg/ml nystatin. This procedure allowed examination of
ion transport through the apical membranes of PDEC. A serosal
luminal Cl
concentration gradient of 135 mM was also
maintained using appropriate buffers to drive Cl
through
activated Cl
conductances, an event reflected by an
increase in Isc. Under these conditions, both
forskolin (100 µM), acting through cAMP, and the calcium ionophore
A-23187 (1 µM), acting through increased [Ca2+]i, stimulated an increase in
Isc. However, the Isc
increase stimulated through the cAMP pathway (Fig.
4B) was much larger than the
Isc stimulated by increased
[Ca2+]i (Fig. 4A). Indeed, the
average maximal Isc increase after forskolin was
67.5 ± 3.3 µA/cm2 (n = 6) vs.
8.1 ± 1.9 (n = 7) µA/cm2 after
A-23187.
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RT-PCR.
Because the only P2 receptor to date known to be coupled to
adenylate cyclase is the P2Y11 receptor, expression of this
receptor in canine PDEC was studied through RT-PCR of the RNA from
these cells, using unique primers from the known sequence of the human P2Y11 receptor. As shown in Fig.
6A, a single band, with a size approximating the one expected for the fragment deduced from the human
P2Y11 receptor (234 bp), was observed (lane b).
Without reverse transcriptase, no signal was seen (Fig. 6A,
lane c), excluding possible genomic DNA contamination.
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DISCUSSION |
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The exocrine function of the pancreas is mediated by acinar cells,
which secrete digestive enzymes, and PDEC, which secrete electrolytes.
For PDEC, activation of Cl channels is a key event in
electrolyte secretion; in cystic fibrosis, for example, impaired
expression or function of CFTR, a cAMP-activated Cl
channel, results in decreased HCO
channels through
P2Y2 receptors. We now demonstrate that ATP, but not UTP,
can also activate the cAMP-stimulated CFTR Cl
channel via
increased cAMP and the P2Y11 receptor.
We first observed that cAMP in PDEC was increased after treatment with
ATP. In other epithelial cells, ATP can stimulate an increase in cAMP
indirectly. Indeed, in T84 cells, ATP partly stimulates secretion
through breakdown to AMP and adenosine and subsequent interaction with
the adenosine P1 receptor (7), whereas, in MDCK cells,
ATP, acting via P2Y2 receptors, stimulates the release of
arachidonic acid release, which in turn activates prostaglandin
receptors on conversion to prostaglandins (21). Mediation
of the ATP effect through either of these pathways was excluded for
PDEC. Response to ATP--S, a nonhydrolyzable analog of ATP, suggests
that ATP, and not a degradation product, is the bioactive agent.
Furthermore, adenosine itself did not stimulate an increase in cAMP,
excluding a role for the P1 receptor. With regard to the
P2Y2-arachidonic acid-prostaglandin pathway, the inability
of UTP to increase cAMP suggests that, unlike the case of MDCK cells,
P2Y2 receptors on PDEC do not indirectly mediate this
effect. In addition, blockade of arachidonic acid conversion to
prostaglandin by the cyclooxygenase inhibitor indomethacin did not
inhibit ATP-stimulated cAMP increase. By exclusion, this effect appears
to be mediated by a direct interaction between ATP and the PDEC, most
likely the P2Y11 receptor, the only P2Y receptor coupled to
adenylate cyclase.
Although the average fivefold increase in cAMP is modest (compared with
the 10-fold increase observed with forskolin; data not shown), it was
sufficient to produce a biological effect. Indeed, this increase in
cAMP caused an activation of the CFTR Cl
channel, a
crucial component for the secretory function of PDEC. This activation
was established using two different modalities, measurements of
125I
efflux and of Isc
in Ussing chambers. Monitoring 125I
efflux to
assess activation of Cl
conductances, first described by
Venglarik et al. (28), has been validated for the
Ca2+-activated Cl
channel and cAMP-activated
CFTR channel in these PDEC (15). As previously shown, UTP,
interacting with the P2Y2 receptor, stimulated
125I
efflux through the
Ca2+-activated Cl
channel (16);
as expected, this effect was completely inhibited by Ca2+
chelation with BAPTA-AM. ATP also stimulated an increase in
125I
efflux; however, only a portion of this
increase was inhibited by BAPTA-AM. These findings are consistent with
the hypothesis that although ATP, like UTP, may interact with the
P2Y2 receptor to increase [Ca2+]i
and stimulate 125I
efflux through the
Ca2+-activated Cl
channel, it also interacts
with the P2Y11 receptor to stimulate a BAPTA-resistant
125I
efflux through the cAMP-dependent CFTR
Cl
channel.
Activation of the CFTR Cl channel is further verified
with the use of confluent PDEC monolayers mounted in Ussing chambers. The protocol used in this report (basolateral permeabilization and
serosal-to-luminal Cl
gradient) allows Cl
flow through activated Cl
channels to be reflected as an
increased Isc. In addition, Cl
flow through the Ca2+-activated Cl
channel
can also be differentiated from flow through the cAMP-stimulated CFTR
Cl
channel on the basis of magnitude of the maximal
Isc increase elicited. Indeed, additional
experiments confirmed that the large Isc
increase stimulated by forskolin through increased cAMP is DIDS
insensitive, and thus mediated by the CFTR Cl
channel,
whereas the smaller Isc increase stimulated by
A-23187 through increased Ca2+ is DIDS sensitive, and thus
mediated through the Ca2+-activated Cl
channel (Nguyen et al., manuscript in preparation).
With this system, we previously demonstrated (16) the
presence of P2Y2 receptors on both apical and basolateral
membranes of PDEC. Indeed, either apical or serosal UTP stimulated a
small Isc increase compatible with activation of
the Ca2+-activated Cl channel. As shown in
Fig. 4C, apical P2Y2 receptors interacted with
luminal ATP to activate Ca2+-activated Cl
channels, resulting in a small Isc increase that
was inhibited by BAPTA (Fig. 5A, top). In
contrast, as shown in Fig. 4D, serosal ATP elicited a large
Isc increase, as seen with activation of the
cAMP-activated Cl
channel. Mediation of this large
Isc increase through the cAMP pathway is further
supported by its resistance to BAPTA (Fig. 5A), a
Ca2+ chelator that previously abolished the
Ca2+-type Isc increase elicited by
UTP acting via the P2Y2 receptor (16). The
basolateral receptor responsible for the large
Isc increase elicited by serosal ATP is a P2
receptor, because it was inhibited by both suramin and reactive blue.
Of the P2 receptors, we previously observed (16) that
stimulation of the basolateral P2Y2 receptor by serosal UTP
resulted in only a small Isc increase that was
inhibited by BAPTA, compatible with activation of the Ca2+-activated Cl
channel. Thus, although the
P2Y2 receptor may account for a small component of the
Isc response to serosal ATP, it cannot account for the large Isc increase resistant to BAPTA.
Serosal ATP-
-S and 2-methylthio-ATP both elicited the same type of
large Isc response, consistent with the agonist
profile for the stimulation of cAMP production. Therefore, the large
Isc response from serosal ATP is most likely
mediated by the P2Y11 receptor.
Many P2Y and P2X receptors have been demonstrated on PDEC (10,
14). P2Y receptors are classically coupled to phospholipase C,
the only exception being the recently cloned P2Y11
receptor, which is also coupled to adenylate cyclase (4).
Pharmacologically, the P2Y11 receptor is the likely
candidate to mediate the stimulation of cAMP increases in PDEC by ATP.
Indeed, ATP, ATP--S, 2-methylthio-ATP, and ADP, but not UTP,
stimulated an increase in cAMP and a large cAMP-type
Isc increase in Ussing chambers consistent with
the agonist profile of P2Y11 receptors expressed in
transfected CHO cells (5). For the putative
P2Y11 receptor on HL-60 leukemia cells and for the
P2Y11 receptor expressed on transfected CHO cells, the
relative potency of the different nucleotides are ATP-
-S > ATP > 2-methylthio-ATP > ADP (4-6). As
shown in Fig. 1, testing at 100 µM concurrently, we obtained an
efficiency profile of ATP-
-S > ATP = 2-methylthio-ATP > ADP, similar to that observed with the human
P2Y11 receptor except for the equivalence between ATP and
2-methylthio-ATP. As shown in Fig. 2, the EC50 for ATP,
ATP-
-S, and 2-methylthio-ATP were all between 10 and 100 µM,
values intermediate between those observed for the HL-60
P2Y11 receptor (EC50 of 30, 100, and 300 µM
for ATP-
-S, ATP, and 2-methylthio-ATP, respectively) and the
transfected human P2Y11 receptors (EC50 of
~3, 10, and 30 µM for ATP-
-S, ATP, and 2-methylthio-ATP,
respectively). It is possible that these differences reflect variance
between human and canine P2Y11 receptors, the cell model
used (HL-60 vs. PDEC vs. transfected cells), or the partial degradation
of ATP or analogs by nucleotidases present on the surfaces of these
cells. The latter possibility is further supported by the strongest
response to ATP-
-S. Through RT-PCR followed by sequencing, we
verified the presence of mRNA for P2Y11 receptors on these
PDEC. Of note, the canine cDNA fragment sequenced showed 84% homology
with its human counterpart.
P2X4, P2X7, and P2X1 are also expressed on the basolateral (P2X1 and P2X4) and/or apical (P2X7) membranes of rat PDEC (10, 14). Although characterization of the secretory effects of P2X receptors is not within the scope of this report, the possibility that these receptors may mediate some of the findings in this report should be addressed.
Because P2X receptors function as ATP-gated channels and are not
coupled to G protein, they will not mediate the observed increases in
cAMP produced by ATP and analogs. Furthermore, P2X receptors are cation
conductances (Ca2+ >> Na+ > K+) and should not directly mediate
125I efflux. On the other hand, they may
mediate 125I
through increased
[Ca2+]i; this effect, however, should be
inhibited by BAPTA-AM. Indeed, the same BAPTA-AM pretreatment totally
inhibited the efflux stimulated by increased
[Ca2+]i obtained with either the
Ca2+ ionophore A-23187 (influx of extracellular
Ca2+) or thapsigargin (mobilization of Ca2+
from intracellular stores). Thus the BAPTA-resistant effect exhibited in Fig. 3D is unlikely to be mediated through P2X receptors.
With regard to the Ussing chamber experiments, the basolateral
membranes of the PDEC are already permeabilized to small monovalent
ions with nystatin. Therefore, the possible activation of the
Na+ and K+ conductances intrinsic to P2X
receptors on the basolateral surfaces of these cells by serosal ATP
should not affect Isc. Alternatively, activation
of P2X receptors may indirectly stimulate Ca2+-activated
Cl
channels through cellular influx of Ca2+.
Again, the corresponding Isc increase should be
inhibited by BAPTA-AM and exhibit the characteristics of a current
mediated by Ca2+-activated Cl
channels. In
the aggregate, although the role of functional P2X receptors in these
canine PDEC remains to be examined, it is unlikely that these receptors
account for all the findings attributed to P2Y11 receptors
in this report.
ATP mediates both autocrine and paracrine functions (8, 24-26). On PDEC, the differential polarity of ATP receptor subtypes coupled to distinct signaling pathways may allow ATP to produce different effects as it interacts with either the serosal or apical surface of these cells. Through P2Y2 receptors expressed on both surfaces of the PDEC, ATP present in either the pancreatic ductal lumen or serosal compartment will be able to stimulate phospholipase C to activate both the Ca2+ and protein kinase C signaling pathways. On the other hand, because functional P2Y11 receptors are only expressed on the basolateral surface of the PDEC, only serosal ATP will be able to stimulate adenylate cyclase and activate the cAMP signaling pathway. This distinction may be relevant because the sources of luminal and serosal ATP may differ. In the biliary system, ATP may be released by bile duct epithelial cells into the luminal compartment for a paracrine effect on biliary secretion or an autocrine effect on cell volume regulation (2, 24). The same mechanism may be operative for ATP release into the pancreatic juice. Of note, should ATP efflux from epithelial cells be mainly mediated through CFTR (26), the apical expression of this channel would favor efflux into the luminal compartment. In addition, because the common bile duct and main pancreatic duct share an outflow into the duodenal lumen, bile may be refluxed into the pancreatic duct lumen. The ATP contained in that bile may interact with the apical surface of PDEC.
Serosal ATP, on the other hand, may be derived from interstitial components such as nerve endings. Alternatively, during inflammation, ATP may be released from injured cells or from inflammatory cells. The restriction of P2Y11 receptors to the basolateral surface of PDEC will ensure that the serosal ATP from these sources effects a different response.
To our knowledge, demonstration of a secretory effect mediated by the P2Y11 receptor would be the first description of a function for this receptor in epithelial cells. Indeed, P2Y11 receptors have only been described in HL-60 leukemia (4) and MDCK (22) cells. In HL-60 cells P2Y11 receptors may mediate cAMP-stimulated differentiation (6, 11, 27), and their function in MDCK cells, beyond cAMP stimulation, is still undefined. The sparseness of information on P2Y11 receptors may relate to their recent identification; our report further supports their importance in epithelial cell biology.
In summary, we have ascribed a secretory function to the P2Y11 receptors in epithelial cells of pancreatic ductal origin. These findings support further studies to define a role for these receptors and for ATP in the (patho)physiology of epithelial cells.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Sum Lee and Chris Savard for advice in the culture of PDEC and the cAMP assays.
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FOOTNOTES |
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This research was partially funded by the Department of Veterans Affairs, Cystic Fibrosis Foundation (RDP R565), and the National Institute of Diabetes and Digestive and Kidney Diseases (RO1-DK-55885).
Address for reprint requests and other correspondence: T. D. Nguyen, GI Section (S-111-Gastro), Puget Sound VA Health Care System, 1660 S. Columbian Way, Seattle, WA 98108 (E-mail: t1nguyen{at}u.washington.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 May 2000; accepted in final form 8 December 2000.
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