1 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235; and 2 Department of Pharmacology, Yonsei University College of Medicine, Seoul 120-752, Korea
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ABSTRACT |
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Purinergic receptors in the basolateral and luminal membranes of
the pancreatic duct can act by a feedback mechanism to coordinate transport activity in the two membranes during ductal secretion. The
goal of the present work was to identify and localize the functional P2
receptors (P2R) in the rat pancreatic duct. The lack of selective
agonists and/or antagonists for any of the cloned P2R dictated the use
of molecular and functional approaches to the characterization of
ductal P2R. For the molecular studies, RNA was prepared from
microdissected pancreatic intralobular ducts and was shown to be free
of mRNA for amylase and endothelial nitric oxide synthase (markers for
acinar and endothelial cells, respectively). A new procedure is
described to obtain an enriched preparation of single duct cells
suitable for electrophysiological studies. Localization of P2R was
achieved by testing the effect of various P2R agonists on intracellular
Ca2+ concentration
([Ca2+]i)
of microperfused intralobular ducts. RT-PCR analysis suggested the
expression of six subtypes of P2R in the pancreatic duct: three P2YR
and three P2XR. Activation of
Cl current by various
nucleotides and coupling of the receptors activated by these
nucleotides to G proteins confirmed the expression of multiple P2R in
duct cells. Measurement of
[Ca2+]i
in microperfused intralobular ducts suggested the expression of
P2X1R,
P2X4R, probably
P2X7R, and as yet unidentified
P2YR, possibly P2Y1R, in the
basolateral membrane. Expression of
P2Y2R, P2Y4R, and
P2X7R was found in the luminal
membrane. The unprecedented expression of such a variety of P2R in one
cell type, many capable of activating
Cl
channels, suggests that
these receptors may have an important role in pancreatic duct cell function.
rat pancreatic duct; purinergic receptors; intracellular calcium; chloride channels
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INTRODUCTION |
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THE PANCREATIC DUCT SECRETES most of the fluid and
determines the final electrolyte composition of the pancreatic juice
(3, 7). Acinar cells secrete digestive enzymes and a small volume of a
plasmalike isotonic fluid into the common acinar lumen, which then
flows into intercalated ducts. As the fluid passes through the ductal
system, duct cells absorb the
Cl and secrete
HCO
3 and fluid to the lumen.
Transcellular Cl
transport
by the duct is the major function controlling ductal secretion. This
activity is of particular interest because it is intimately regulated
by cystic fibrosis transmembrane conductance regulator (CFTR) (3). In
cystic fibrosis, Cl
absorption and HCO
3 secretion are
impaired, which results in hyperconcentration of digestive enzymes and
mucines and leads to obstruction of intralobular ducts (34).
Several hormones and neurotransmitters regulate pancreatic ductal secretion. The best studied so far are regulation by secretin and cholinergic stimulation. Secretin stimulates the generation of cAMP (12) to activate CFTR in the luminal membrane (LM) (13, 14). Cholinergic stimulation of the M3 muscarinic receptors (16) increases ductal intracellular Ca2+ concentration ([Ca2+]i) (4, 17, 31, 40), strongly depolarizes the basolateral membrane (BLM) potential (17), and stimulates fluid secretion (4).
Another agonist acting on pancreatic duct (8, 17) and other CFTR-expressing epithelial cells (20, 25, 30, 32, 33, 37) is ATP, which interacts with P2 receptors (P2R). P2R are unique in epithelial physiology because they are expressed in both the BLM and LM. This provides the cells with a receptor system to coordinate transepithelial function through central and paracrine/autocrine inputs. Thus the basolateral receptors are stimulated by ATP secreted by purinergic neurons. ATP secreted by acinar cells stored in secretory granules and by ductal ATP-binding cassette transporters such as CFTR to the duct lumen are likely the substrate for the luminal P2R. This form of regulation is attracting considerable attention, as it may be of clinical relevance (34, 35). Characterization of P2R expressed in epithelial cells is partial and not conclusive. Most published studies relied on a pharmacological approach of determining sequence of potency to several P2R agonists. On the basis of such studies, it has been suggested that airway epithelial cells express P2Y2R in the LM and P2Y3R in the BLM (20). Nasal epithelial cells were suggested to express P2Y2R in the BLM and P2Y6R in the LM (24). P2Y1R and/or P2Y2R were found in the BLM, and P2zR was found in the LM of submandibular duct cells (2, 25, 37). The response of isolated ducts to ATP (17) and other nucleotides led Christofferson et al. (8) to suggest the expression of P2uR and P2zR in pancreatic duct cells. Membrane localization of P2R was not determined. The only conclusive work was reported on submandibular acinar cells, in which molecular and functional studies reported the expression of P2X4R in these cells (5).
A potential problem with the pharmacological approach, which is
highlighted in several recent reviews and other articles (5, 6, 11, 19,
22, 23, 26, 29), is the uncertainty with which P2R can be identified
when more than one P2R is expressed in the same cell type. All
available agonists and antagonists show cross-reactivity and similar
affinity for at least two P2R types (1, 5, 11, 23). Furthermore, the
same P2R expressed in different cells can display a different profile
of responsiveness to P2 agonists (11, 23), and two P2R can assemble
into one functional unit (26). Molecular characterization together with functional studies can circumvent, in part, some of these difficulties (5, 23, 26). In the present work we prepared RNA from microdissected pancreatic intralobular ducts, which was free of mRNA for amylase and
endothelial nitric oxide synthase (eNOS; markers for acinar cells and
endothelial cells in blood capillaries, respectively) to analyze by
RT-PCR the P2R expressed in pancreatic duct cells. Surprisingly, the
pancreatic duct appears to express six subtypes of P2R: three P2YR and
three P2XR. Measurement of
Cl current activation by
various nucleotides and coupling of the receptors activated by these
nucleotides to G proteins confirmed the expression of multiple P2R in
duct cells. Finally, measurement of
[Ca2+]i
in microperfused intralobular ducts suggested the expression of several
P2XR and P2YR in the BLM and LM of the pancreatic duct. The expression
of such a variety of P2R in the duct suggests that these receptors have
an important role in pancreatic duct cell function.
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METHODS |
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Chemicals and solutions. All
nucleotides were purchased from Sigma, except 2-methylthioadenosine
5'-triphosphate (2-MeS-ATP) which was from RBI (Natick, MA). Fura
2-AM was from Molecular Probes (Eugene, OR). The standard perfusion
solution (bath and lumen) contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES
(pH 7.4 with NaOH). This solution was supplemented with 10 mM sodium
pyruvate, 0.02% soybean trypsin inhibitor, and 1 mg/ml BSA to form
pancreatic solution A (PSA). The
standard solution was the bath solution during current recording. The
pipette solution contained (in mM) 140 KCl, 10 HEPES (pH 7.3 with
NaOH), 1 MgCl2, 1 ATP, and 0.1 EGTA. These conditions were found as optimal to record the inward
current in duct cells, which was carried mostly by
Cl.
Microdissection of pancreatic duct and fluorescence recording. Intralobular ducts were microdissected from the rat pancreas and microperfused exactly as described before (40). Rats (male or female, Sprague-Dawley, 75-100 g) were anesthetized by intraperitoneal injection of 40 mg/kg pentobarbital sodium and killed by exposure to air saturated with methoxyflurane. The pancreas was removed and placed in PSA for microdissection. For measurement of [Ca2+]i, two segments of intralobular duct were microdissected. One was kept on ice until use and the other was transferred to a perfusion chamber. After insertion of the luminal perfusion micropipette, the duct was loaded with fura 2 by luminal perfusion for 10-15 min with a solution containing 2.5 µM fura 2-AM. Bath perfusion started before application of the dye to the lumen and, except during application of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) to the bath, the bath perfusions did not stop until the end of each experiment. In three experiments BzATP was applied to the bath, as were all other agonists, by continuous perfusion. Because of limited agonist availability and cost, in four experiments bath perfusion was stopped, and BzATP was applied to the bath by infusion of 1 ml solution (~13 chamber volumes) containing 1 mM BzATP. Bath stimulation was terminated by resumption of perfusion. In all experiments luminal BzATP was applied by perfusion. Fura 2 fluorescence was recorded at excitation wavelengths of 355 and 380 nm as detailed in Ref. 36. Results are expressed as the 355/380 fluorescence ratios because contamination with extracellular dye, which tended to remain in the connective tissue, prevented meaningful calibration (see also Ref. 40).
Preparation of RNA. To prepare RNA,
most of the pancreatic ductal tree was microdissected and carefully
cleaned from adherent cells and connective tissue as much as possible
(see Fig. 1 in Ref. 40). Then five to seven segments of the
intralobular duct were cut, inspected for absence of acinar cells,
blood capillaries, and cell debris (see Fig.
1, A and
B), and transferred to
a clean Eppendorf tube. The ducts were washed twice with PSA and packed and treated with Trizol for extraction of RNA. The ductal origin of the
RNA was verified by PCR analysis of marker mRNAs. RNA extracted from
submandibular gland tissue (all cells) was used as a source for
positive controls. As shown in Fig.
1C, pancreatic ductal RNA contained
mRNA for CFTR and was free from mRNA for amylase (marker for acinar
cells) and eNOS (marker for pancreatic blood capillaries; see Ref. 37).
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Preparation and enrichment of single pancreatic duct
cells. Our experience in isolation of single duct cells
from the submandibular gland (40) indicated that duct cells have lower
density than acinar cells. On the basis of this observation, we used
the following procedure to obtain a preparation enriched in single
pancreatic duct cells. Rats were killed as detailed above for
microdissection of intralobular ducts. The pancreas was minced and
digested with collagenase and trypsin by a standard published procedure
(39). Most of the acini were removed by a 10-s centrifugation at 500 rpm using a Sorvall centrifuge model RC-3B. The supernatant was removed
to a clean tube, and the cells were collected by a 3-min centrifugation
at 1,500 rpm. The pelleted cells were washed twice with PSA,
resuspended in 8 ml of ice-cold PSA containing 10 mg/ml BSA, and placed
in a standard 15-ml conical plastic tube. The low abundance of duct
cells in this preparation is shown in Fig. 1D. The tube was placed on ice, and
the cells were allowed to sediment at 1 g. Every 10 min the
pelleted cells were carefully collected from the bottom of the tube
with a Pasteur pipette for microscopic inspection to verify removal of
mostly acinar cells. After 7-10 collections, the remaining cells
were harvested by a 3-min centrifugation at 1,500 rpm, resuspended in
PSA, and kept on ice until use. Figure
1E shows the enrichment in duct cells relative to acinar cells in this preparation. Pancreatic duct cells
were distinguished from acinar cells by their size and appearance. The
secretory granules can be easily identified in the larger acinar cells,
whereas the smaller duct cells had a smooth appearance. In accordance
with their different sizes, during electrophysiological recordings duct
and acinar cells were distinguished by their different capacitance. In
a typical series of experiments, the capacitance of acinar cells
averaged 13.6 ± 2.6 pF (n = 11),
with a minimum and a maximum of 9.2 and 17.8 pF, respectively. In 14 duct cells from the same preparations, the average capacitance was 4.4 ± 0.8 pF, with a minimum and a maximum of 3.2 and 6.3 pF,
respectively. Finally, all 11 acinar cells strongly responded to 1 mM
carbachol stimulation, showed no response to 100 µM adenosine
5'-O-(3-thiotriphosphate) (ATPS), and subsequently strongly responded to 1 nM cholecystokinin octapeptide (CCK-8). All 14 duct cells strongly responded to
stimulation with carbachol and ATP
S (see
RESULTS for protocol) and showed very
small or no response to CCK-8. Based on these observations, we believe
that all responses (summarized in Table 2 below) are from
pancreatic duct cells and are not contaminated by recording from other
cell types.
RT-PCR analysis. Approximately 2 ng of
total RNA extracted with the RNAzol kit were used to perform the RT
reaction with the GeneAMP RNA-PCR kit (Perkin-Elmer) in a 20-µl
reaction volume as specified by the manufacturer. Random hexamers were
used as RT primers for cDNA synthesis by incubation for 15 min at
42°C, 5 min at 95°C, and 5 min at 4°C. PCR primers were
designed for mRNA of each gene product using Gene Runner software
version 3.00 as well as published sequences. All primers, reaction
conditions, and expected product sizes are listed in Table
1. PCRs were started by a 10-min hot start with a
Perkin-Elmer AmpliTaq Gold DNA polymerase. For all primers, 35 cycles
of cDNA amplifications were carried out in a PTC-100 thermal cycler (MJ
Research). The PCR products were separated on a 2% agarose gel
containing 0.1 µg/ml ethidium bromide. The identity of all amplified
products was verified by sequencing.
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Electrophysiology. The whole cell
configuration of the patch-clamp technique (15) was used to record the
inward current at a holding potential of 40 mV. All current
recordings were performed at room temperature. Current was recorded
with a patch-clamp set up from Axon Instruments using pCLAMP 6 and a
DigiData 1,200 interface. The patch-clamp output was filtered at 10 Hz.
Leak current was subtracted from all records. The amplitude of the currents stimulated by carbachol and ATP
S was within 15% in any given cell preparation but varied considerably between cell
preparations (range 70-200 pA/pF); therefore, the results in Table
2 are given as number of responding cells.
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RESULTS AND DISCUSSION |
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Pancreatic duct cells express multiple
P2R. The first indication for the expression of
multiple P2R in pancreatic duct cells was obtained by measuring the
properties of the ATP-activated inward current. The inward current was
carried mostly by Cl. Hence
ATP and carbachol activated a nonselective cation current and a
Cl
current. Accordingly,
the reversal potential under the ionic composition of the solutions
used in the present work was close to 0 mV. Substitution of external
Na+ with
K+ and internal
K+ for
Na+ had minimal effect on the
current recorded at
40 mV. On the other hand, reducing external
Cl
concentration to 33 mM
(at internal Cl
of 150 mM)
resulted in a current with a reversal potential of
38 ± 3 mV, which is close to the Nernst reversal potential for Cl
. Stimulation of duct
cells by bath perfusion with a solution containing supramaximal
concentration of carbachol (1 mM) or application of ATP by puffing a
solution containing 5 mM ATP activated the Cl
current to a similar
extent (Fig.
2A). ATP
also activated the Cl
current when it was applied by bath perfusion (Fig.
2B). Removal and readdition of
Ca2+ to the perfusion medium
showed that ATP activated both
Ca2+-dependent and
Ca2+-independent
Cl
currents
(n = 9). These findings are similar to
those reported in several cell types (20, 24, 30, 32, 33, 37), in which
ATP binds to multiple P2R to activate at least two different Cl
channels.
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P2R are classified into two classes, metabotropic (P2Y) and ionotropic
(P2X) (1, 23, 29). Because P2YR, but not P2XR, are coupled to G
proteins (6, 22, 23, 29), a convenient test for the expression of P2XR
is to measure the effect of inhibition of G proteins with guanosine
5'-O-(2-thiodiphosphate)
(GDPS) on agonist-dependent activation of
Cl
current. Figure
2C shows that including 2 mM GDP
S
in the pipette solution abolished the response of the cells to
stimulation with carbachol, a
Gq-coupled receptor (39). The same
cells responded normally to stimulation with ATP. In fact, the
magnitude of the currents activated by ATP in control cells and cells
infused with GDP
S was similar in all experiments.
Activation of Ca2+-dependent and
Ca2+-independent
Cl currents by ATP shown in
Fig. 2 suggested expression of more than one type of P2R in these
cells. This excluded the ability to identify the type of P2R expressed
in pancreatic duct cells by binding or activity assays based on
determining the potency sequence of P2R agonists. Indeed, in
preliminary studies we measured the apparent affinity for the
nucleotides listed in Table 2 to activate
Cl
current in the presence
or absence of GDP
S and could not obtain definitive results.
Perfusion of several nucleotides such as 2-MeS-ATP and
-methylene-ATP (
-MeATP) resulted in variable stimulating signals and relatively rapid desensitization of the response to other
P2 agonists, including ATP. Therefore, in most experiments nucleotides
were applied by a puffer pipette. We noticed that in this setup ~5-
to 10-fold higher concentrations of ATP or ATP
S in the puffer
pipette were needed to obtain the same intensity of stimulation
observed when the agonists were applied by perfusion. In addition, the
need to change the puffer pipette for each change in agonist
concentration precluded obtaining accurate dose-response curves for the
agonists used. Nonetheless, half-maximal responses for the agonists
listed in Table 2 were obtained at concentrations between 25 µM
(
-MeATP) and 100 µM (BzATP). This profile did not conform to
any known P2R type. Thus we concluded that pharmacological characterization is unreliable in identifying the P2R expressed in
pancreatic duct cells.
A reliable approach to identify P2R expression is PCR analysis of P2R
in a defined cell type. To achieve that, we prepared RNA from
microdissected intralobular ducts. Because smooth muscle and
endothelial cells in blood vessels express several P2R (6, 23), it was
important to verify the absence of contaminating blood capillaries in
our preparation. eNOS protein is abundantly and exclusively expressed
in pancreatic capillaries (37) and is therefore a good marker for
contaminating capillaries. As described in
METHODS and Fig. 1, the RNA prepared
from microdissected intralobular ducts contained mRNA for CFTR (ducts)
but not mRNA for eNOS (blood capillaries) or amylase (acinar cells).
Figure 3 shows the results of RT-PCR
analysis of P2R expressed in pancreatic duct using this mRNA
preparation. Pancreatic ducts were found to express at least three P2XR
(P2X1,
P2X4, and
P2X7) and four P2YR
(P2Y1, P2Y2, P2Y4, and
P2Y5). The ability of the primers that gave negative
results in pancreatic ducts to amplify the expected PCR product was
ascertained by using mRNA preparation from a submandibular gland tissue
(Fig. 3, bottom). Although no
mammalian homolog of P2Y3R has
been identified, Li et al. (27) provided evidence that
P2Y3R is the functional avian
homolog of mammalian P2Y6R.
Nevertheless, primers for P2Y3R and P2Y6R were used to probe the
expression of this gene in pancreatic duct. With both sets of primers
no product was amplified. It is also important to note at this stage
that, despite extensive efforts, no response could be found for the
cloned and expressed P2Y5R when
stimulated with various P2 agonists (21, 28). Thus the P2Y5R may be an orphan G
protein-coupled receptor for a nonnucleotide agonist. Consequently,
although P2Y5R is expressed in
pancreatic duct cells, the possible function of this receptor is not
considered further in the present work. The surprising number of P2R
expressed in one cell type prompted several verifications of these
findings (between 3 and 6 RNA preparations from different animals).
Positive signals like those shown in Fig. 3 were obtained in all
experiments. In addition, the identity of all RT-PCR products was
verified by sequencing. The multiple P2Rs expressed in pancreatic duct cells explain well the failure of the pharmacological approach to
identify the P2R expressed in these cells.
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G protein-coupled and G protein-independent
receptors. A critical question raised by the PCR
analysis is whether all P2R expressed in duct cells are functional.
Considering the number of P2R expressed in duct cells, the only
feasible approach was to test the effect of agonists reported to be at
least partially selective for defined P2R. These agonists could be
divided into two groups: those that activate the ionotropic, G
protein-independent P2R and those that activate the metabotropic, G
protein-coupled P2R. Figure 4 shows the
protocol used to identify the agonists activating the G
protein-independent P2R. Duct cells were dialyzed through the patch
pipette with solutions that were with or without 2 mM GDPS to
inhibit all G proteins. Inhibition of G protein-dependent signaling was
always verified by the complete inhibition of the response to
muscarinic stimulation with 1 mM carbachol. Stimulation by ATP
S,
BzATP, and
-MeATP was not inhibited by GDP
S. Cells were
stimulated with ATP
S by perfusion of a solution containing the
maximal effective concentration of 100 µM. In these and all other
current recording experiments, all other nucleotides were applied by
puffing. A concentration of 1 mM in the puffing pipette solution was
found to be maximal for all agonists. Because the response to ATP
S
and carbachol was used as a control, their effect was tested in all
cells. The response to these agonists was highly consistent where 141 out of 154 (92%) cells responded to carbachol, and 131 out of these 154 cells (85%) responded to ATP
S. Of the 141 cells responding to
carbachol, 131 (93%) responded to ATP
S. Finally, 133 out of 147 cells (90%) infused with GDP
S responded to ATP
S (Table
2).
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In one series of experiments 10/10 cells responded to ATPS, and in
the same series 20/20 cells dialyzed with 2 mM GDP
S responded to
stimulation with ATP
S (Table 2). Although ATP
S was proposed as a
selective agonist for P2X2R and
P2X4R (1), it was shown to be a
partial agonist for P2X2R (11) and
P2X3R (26). Pancreatic duct cells
express P2X1R and
P2X4R. Thus the effect of ATP
S
indicates expression of functional
P2X4R and possibly
P2X1R in duct cells. BzATP is a potent agonist for
P2X7R (1, 6, 22, 29). It is also a
partial agonist for P2X1 and
P2X2R (11). Thus the response of
duct cells to BzATP suggests that
P2X7R and
P2X1R are functional in pancreatic
duct cells. These findings are in agreement with a recent study
reporting an effect of BzATP on [Ca2+]i
of duct cells (8). The response of duct cells to
-MeATP that was
not sensitive to GDP
S inhibition suggests that the response was due
to an active P2X1R in duct cells.
-MeATP is a potent agonist for
P2X1R and
P2X3R (5, 9). Of the
-MeATP-sensitive P2R, pancreatic duct cells express only
P2X1R. Furthermore, extensive studies showed that this nucleotide does not activate
P2X2R,
P2X4R, P2X5R, and
P2X6R when the clones are
expressed in HEK-293 cells (5, 9, 11, 26). However, in several tissues,
P2X1R could be activated by
2-MeS-ATP as well as by
-MeATP (29, 22). In 14 of 14 experiments,
duct cells did not respond to 2-MeS-ATP when GDP
S was included in
the pipette solution (Table 2). Activation of
P2X4R by
-MeATP cannot
explain this discrepancy, since
P2X4R is also more sensitive to
2-MeS-ATP stimulation than to
-MeATP (22, 29). Although likely,
the absence of a GDP
S-insensitive 2-MeS-ATP response does not allow
us to state with certainty whether P2X1R expressed in the pancreatic
duct cell (Fig. 3) is functional.
The response of a second group of P2R agonists was completely inhibited
by GDPS, indicating coupling of these receptors to heterotrimeric G
proteins. Figure 5 shows individual
examples, and Table 2 summarizes the results of multiple experiments.
These agonists consistently activated pancreatic duct cells. The
response to UTP, 2-MeS-ATP, and ADP was observed in 17 of 19, 13 of 15, and 6 of 7 cells, respectively. In all experiments (13 with UTP, 14 with 2-MeS-ATP, and 11 with ADP), infusion of 2 mM GDP
S into the
cells prevented the response to these agonists. In further controls,
the same cells did not respond to muscarinic stimulation but showed a
completely normal response to ATP
S (Table 2). In eight of eight
experiments, we could not observe an effect of UDP on
Cl
current whether UDP was
applied by perfusion or by puffing. These observations should be
contrasted with the findings below showing the effect of UDP on
[Ca2+]i
of microperfused pancreatic ducts. The reason for a lack of response of
single duct cells to UDP is not clear at present. One possibility is
that the
[Ca2+]i
increase evoked by activation of the UDP-sensitive P2R is spatially segregated from the Cl
channels.
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Cross-reactivity of most P2R agonists with the various P2YR makes it
difficult to determine with any certainty which P2YR are functional in
duct cells. The very high sensitivity of
P2Y1R to stimulation by 2-MeS-ATP
(22, 29), the inhibition of the response to this agonist by GDPS
(Table 2), and the high potency for this agonist to increase
[Ca2+]i in
microperfused ducts (see Fig. 8) can be considered good evidence for a functional P2Y1R in
pancreatic duct cells. Similar arguments can be made for the effect of
UTP on P2Y2R and
P2Y4R expressed in duct cells.
These are the only P2R to show high affinity for UTP. We can be more
certain of the expression of a functional P2Y4R in pancreatic duct cells,
since the cells responded to UDP (see Fig. 8), an agonist for this
receptor (22, 29).
Sidedness of agonist response.
Physiologically and possibly clinically relevant information is the
cellular and membrane localization of the P2R in pancreatic duct cells.
Because activation of all known P2R increases
[Ca2+]i
(6, 22, 23, 29), an initial and partial answer to this question can be
obtained by measuring the effect of P2R agonists on
[Ca2+]i
of the microperfused pancreatic duct. In the first set of experiments we tested the effect of luminal and basolateral ATP on
[Ca2+]i.
Figure 6A
shows that application of 1 mM ATP to the BLM or LM side of the duct
was equally effective in increasing
[Ca2+]i.
In all experiments tested (n = 16),
cells responding to bath ATP also responded to bath carbachol, which
usually increased [Ca2+]i
to a level somewhat higher than that induced by ATP. Activation of P2XR
increases Ca2+ influx, whereas
activation of P2YR increases
[Ca2+]i
by activation of Ca2+ release from
internal stores and Ca2+ influx
(6). This distinction can be used to determine the type of P2R
expressed on each side of the pancreatic duct. Figure 6,
B and
C, shows that removal of extracellular
Ca2+ from the luminal and bath
solutions reduced, but did not prevent, the effect of either
basolateral or luminal ATP on
[Ca2+]i.
Readdition of Ca2+ to the bath
following basolateral stimulation and to the lumen following luminal
stimulation caused a marked increase in
[Ca2+]i,
indicative of maintained activation of
Ca2+ influx during P2R
stimulation. Because ATP can activate P2YR and P2XR present in both
membranes (see Figs. 7 and 8), it is not clear which
pathway mediates the influx. However,
Ca2+ mobilization from internal
stores by basolateral or luminal ATP clearly indicates that P2YR are
expressed in both membranes.
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The agonists characterized in Table 2 in terms of their sensitivity to
GDPS inhibition were used to determine the side from which they
stimulate the pancreatic duct. Figure 7
shows that ATP
S and
-MeATP were effective only from the
basolateral side. ATP
S was the most effective and potent agonist
acting from the basolateral side of pancreatic duct
(n = 13/13 experiments alone and
n = 6/6 experiments when compared with
ATP in the same duct). The response of the duct to
-MeATP was
less consistent. The effect of 1 mM basolateral
-MeATP was
observed in three of five ducts that responded to basolateral ATP. The
same five ducts did not respond to luminal
-MeATP whether the
lumen was exposed to
-MeATP before (not shown) or after (Fig.
7B) stimulation of the ducts with
basolateral nucleotides. This analysis suggests that
P2X4R and P2R activated by
-MeATP (probably P2X1) are
expressed only in the BLM of pancreatic duct cells.
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The response of the pancreatic duct to agonists acting only from the
luminal side is illustrated in Fig. 8. In
all experiments (n = 7/7 in the
presence of external Ca2+ and 3/3
in the absence of external
Ca2+), the perfused duct
responded to luminal but not basolateral UTP. Figure
8A shows the effect of 100 µM UTP.
However, luminal UTP at a concentration as low as 1 µM increased
[Ca2+]i,
whereas up to 1 mM basolateral UTP had no effect. Thus UTP acts
exclusively from the luminal side of the duct.
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The effect of UDP was tested only at a single concentration of 1 mM.
Basolateral UDP had no effect on
[Ca2+]i
in four of four ducts that responded to bath ATP. In three of four of
these ducts luminal UDP increased
[Ca2+]i,
but the effect was variable (0, 0.3, 0.6, and 0.75 ratio units). Another agonist acting exclusively from the luminal side is 2-MeS-ATP (Fig. 8C). The response of the duct
to this agonist was consistent (5/6 of the ducts that responded to
basolateral and/or luminal ATP), although, unlike UTP (Fig.
8A), it tended to increase
[Ca2+]i
less than ATP. The maximal effective concentration of 2-MeS-ATP was
between 10 and 100 µM. Because of the uncertainty as to the exact P2R
subtype activated by each of the luminal agonists, it is not clear
which P2YR are expressed in the luminal membrane of the pancreatic
duct. However, this membrane expresses most of the P2YR types since
1) the effect of all luminal
agonists was inhibited by GDPS (Table 2) and
2) they had no effect on [Ca2+]i
when applied to the basolateral side at concentrations 10- to 100-fold
higher than those effective from the luminal side.
The last group of P2R agonists are those effective from both sides.
Figure 9 shows that these are ADP and
BzATP. The effect of ADP was observed in two of three ducts responding
to ATP, and in the two responding ducts ADP had a similar effect on
[Ca2+]i
when applied to the bath or the luminal sides. Note that the effect of
ADP was completely sensitive to GDPS (Table 2), indicating that ADP
activated only P2YR. In fact, ADP is the only agonist activating P2YR
found to be effective from the basolateral side. Whether ADP activates
the same P2YR on both sides is not known at present, since at least two
of the P2YR expressed in pancreatic duct cells (Fig. 3) can be
activated by ADP (22, 29).
|
The effect of basolateral and luminal BzATP was observed in all five of the seven ducts that responded to bath and/or luminal ATP. However, as illustrated in Fig. 9A, luminal BzATP was more effective than bath BzATP in increasing ductal [Ca2+]i. Although we were unsuccessful in demonstrating a response to bath BzATP in ducts continuously stimulated with 1 mM luminal BzATP, we do not believe that the effect of bath BzATP on [Ca2+]i is due to leakage into and activation of luminal receptors, since the same was not noted with any of the nucleotides acting only from the luminal side (Fig. 8). Hence it seems likely that the P2X7R identified in the present work is expressed in both the LM and BLM of the pancreatic duct. Expression of P2X7R was also found in the LM of submandibular duct cells (37, 38).
The myriad P2R expressed in each side of the pancreatic duct raises two
questions. 1) Does each pancreatic
duct cell express all P2R? 2) Why
does the pancreatic duct express so many P2R? At present, we can only
partially address the first question and only speculate with regard to
the second question. The only approach currently available to address
the number of P2R types expressed in each cell is the functional
approach. This approach is limited to measurements from single
dissociated cells, like those summarized in Table 2. Although the
pancreatic duct contains at least two major cell types and probably
several subtypes (10), we are quite confident that all duct cells
express the P2R activated by ATPS. Of the 141 single duct cells that
responded to carbachol, 131 responded to ATP
S, and all cells
responding to any of the agonists listed in Table 2 also responded to
ATP
S. Because the sample is not as large with the other agonists, it
is not clear how many P2R types each cell expresses. However, we note
that with all agonists the percentage of responding cells was very high, approaching 100%. This was also the case for single cells stimulated with UTP (17/19 responding). This finding is somewhat different from a recent study, which reported that approximately one-third of the ducts that showed a normal response to ATP did not
respond to UTP (8). Considering the different experimental systems and
cell preparation used in the two studies, the different observations
can be explained in many ways.
The high percentage of cells responding to all P2R agonists tested
suggests that each duct cell expresses most, if not all, the P2R
identified and the expression of multiple P2R in each membrane. This is
not without precedent. Molecular analysis showed expression of multiple
R2R in all regions of the brain (5). Measurement of short-circuit
current in primary cultures of nasal and airway epithelial cells in
monolayers suggested expression of at least three P2R in these cells
(20, 24, 30, 32, 33). Several studies showed that submandibular duct
cells express at least three P2R (2, 37, 38). A combined molecular and functional analysis, similar to that reported here for the pancreatic duct, is likely to show that the cells mentioned above and other epithelial cells express more than the three suggested P2R. Preliminary studies with isolated submandibular gland ducts suggest that this is
indeed the case (not shown). Expression of such a high number and
diverse P2R types in secretory epithelia suggests that P2R plays an
important role in controlling epithelial cell function. This is further
supported by the finding that P2R is expressed in both membranes. The
P2R may specialize in activating particular ion channels to control
cell activity. So far there are two examples of such a specialization.
In airway epithelia luminal ATP activates the outward rectifier
Cl channel (20, 30), and in
submandibular gland cells P2X7R activate a CFTR-like Cl
channel (37). Additional specialization may be achieved by expression
of selective P2R in microdomains of the LM and BLM to allow a localized
secondary regulation of transepithelial transport. Further refinement
of cell preparation and recording techniques is needed to probe the
physiological significance of the multitude of P2R in pancreatic duct
cells and other epithelia.
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ACKNOWLEDGEMENTS |
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We thank Roshi Mehdibeigi for technical support and Karen Miler for administrative assistance.
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FOOTNOTES |
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This work was funded by National Institutes of Health Grants DE-12309 and DK-38938.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Muallem, The Univ. of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9040 (E-mail: smuall{at}mednet.swmed.edu).
Received 4 January 1999; accepted in final form 28 April 1999.
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