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INTRODUCTION |
ATP plays an important role as an extracellular agonist that
mediates its various effects by acting on specific membrane P2 receptor
subtypes (1, 2). P2 receptors comprise receptors of the ligand-gated
ion channel type, as well as of the G-protein-linked superfamily,
termed P2X and P2Y, respectively (3). At present, seven genes for P2X
receptors have been cloned (4-8), but their physiological significance
has not been fully established yet. One of the most recently cloned P2X
receptors (the P2X7) has a pharmacological profile typical
of the receptor previously termed P2Z (9) with the
photoactivable analog of ATP,
Bz-ATP,1 the most potent
agonist. The P2Z receptor induces the formation of pores
when exposed to concentrations of extracellular ATP in the 100 µM to 1 mM range (Refs. 10-12, but see also
Ref. 13). In contrast to other P2X receptors, the P2X7
receptor has a long COOH-terminal intracellular chain, which is by
itself not responsible for the lytic properties of this receptor (14)
but probably induces the formation of a second messenger involved in
the lysis (12). In summary, the
P2X7(P2Z)-receptors share with the other P2X
receptors the ability to open a non-selective channel and with
P2Z receptors the induction of cell lysis by repeated
applications of the agonist (8).
Since the pioneering work of Gallacher (15), ATP has been recognized as
a major non-adrenergic non-cholinergic stimulus of saliva secretion.
Salivation, like other exocrine secretions, occurs in two steps;
(a) acinar cells secrete an isotonic plasma-like fluid, and
(b) the electrolyte composition of this primary secretion is
modified during its transfer to the mouth by the ductal tree (16). The
ducts reabsorb Na+ and Cl
and secrete
K+ and HCO3 (17). The
study of these two phases of the secretory process has been facilitated
by the description of an improved technique to separate ducts and acini
(18). It could be observed that extracellular ATP increased the
intracellular concentration of calcium ([Ca2+]i)
both in rat submandibular gland (RSMG) acini and ducts. This result
suggested that ATP might regulate both phases of secretion. In acini,
ATP increased the intracellular concentration of Na+ (19),
activated the Na+/H+ exchanger, and opened a
chloride channel (20). It also inhibited the response to agonists
activating the L-
-phosphatidylinositol 4,5-bisphosphate-selective phospholipase C (21). Two purinergic responses were reported in pure RSMG ducts. At low concentrations, ATP
activated a P2Y1 receptor, while at high concentrations, it stimulated a P2X receptor (18, 22). The ionotropic receptor triggered
the secretion of kallikrein. The molecular characterization of the
ionotropic receptor present on RSMG ductal cells has not been achieved
yet. Both P2X4 and P2X7 transcripts have been
observed in rat submandibular glands (23), and, according to Buell
et al. (6), P2X4 are expressed in RSMG acini but
not in RSMG ducts. It can thus be speculated that the P2X receptors
expressed in ducts are of the P2X7 type.
The functional consequences of the activation of this receptor have not
been determined. The coupling of some P2Y receptors to phospholipases C
and phospholipases A2 has been clearly established (24-26). Reports on the regulation of the activity of phospholipases by P2X receptors are more scarce (27-29). The purpose of this work was
to study the contribution of ionotropic purinergic receptors to the
release of arachidonic acid by RSMG ductal cells. The phospholipases A2 involved in this response and their role in the
secretion in response to purines were also explored.
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EXPERIMENTAL PROCEDURES |
Materials--
Fura-2-acetoxymethyl ester (AM) was from
Molecular Probes (Eugene, OR). 2-Methylthioadenosine 5'-triphosphate
(2-MeSATP) was purchased from ICN Biochemicals (Costa Mesa, CA).
Collagenase P, bovine serum albumin (BSA, fraction V), Coomassie
Brilliant Blue, adenosine 5'-triphosphate (ATP, sodium salt), and
ATP
S were from Boehringer Mannheim (Mannheim, Germany).
2,3-Dialdehyde ATP (periodate-oxidized ATP), Bz-ATP, thapsigargin,
A23187, diethylenetriaminepentaacetic acid (DTPA),
N-benzoyl-DL-arginine
p-nitroanilide, HEPES, EGTA, DNase I (from bovine pancreas),
digitonin, carbamylcholine chloride, epinephrine, isoproterenol,
ionomycin, phospholipid, neutral lipid and fatty acid standards, and
the other purines and ATP analogues were obtained from Sigma.
Arachidonyl trifluoromethylketone (AACOCF3), 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082),
and suramin were supplied by Alexis Biochemicals (Woburn, MA) and (E)-6-(bromoethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (bromoenol lactone, BEL) by Cayman Chemical (Ann Arbor, MI).
[methyl-14C]Choline chloride (200 µCi/ml, 55 mCi/mmol) and myo-[2-3H]inositol (1 mCi/ml,
18.3 Ci/mmol) were purchased from Amersham International
(Buckinghamshire, UK) and
[5,6,8,9,11,12,14,15-3H]arachidonic acid (0.1 mCi/ml,
60-100 Ci/mmol) from NEN Life Science Products (Bruxelles, Belgium).
Avian myeloblastosis virus reverse transcriptase was from Amersham
Pharmacia Biotech (Barcelona, Spain), and GStractTM RNA
isolation kit II was from Maxim Biotech, Inc. Percoll was obtained from
Pharmacia (Uppsala, Sweden) and the amino acid mixture (without
glutamine) from Life Technologies, Inc. (Uxbridge, UK). Analytical
grade anion exchange resin AG® 1-X8 was obtained from
Bio-Rad. Silica Gel-60 F254 plates were from Merck
(Darmstadt, Germany). The other materials were purchased from
various sources and were reagent grade.
Isolation of RSMG Ducts--
Male Sprague-Dawley rats (200 g)
fed ad libitum and given free access to water were used. The
rats were killed by exposure to diethylether, and the glands were
immediately dissected and finely minced. The suspension of ductal cells
was prepared as described previously (18) with some modifications.
Briefly, the minced glands of one rat were digested in the presence of 2.6 mg of collagenase P (1.52 units/mg) for 20 min at 37 °C under constant shaking (90 cycles/min) in 10 ml of HEPES-buffered saline (HBS) containing (mM): 24.5 HEPES, 96 NaCl, 6 KCl, 2.5 NaH2PO4, 11.5 glucose, 5 pyruvate, 5 glutamate,
5 fumarate, 1% (v/v) glutamine-free amino acid mixture (Life
Technologies, Inc.), and 0.125% (w/v) bovine serum albumin (BSA). The
pH was adjusted to 7.4 with NaOH. Ten minutes after the beginning of
the digestion and at the end of the digestion, the cells were aspirated
several times with 10-, 5-, and 2-ml glass pipettes. The suspension was
then washed three times with an isotonic saline solution, resuspended
in 10 ml of collagenase-free HBS and incubated for 10 min in the
presence of 0.06 mg of DNase I (420 units/mg). The crude suspension was pipetted again, filtered through a nylon mesh and washed five times in
a saline solution. Cells were resuspended in 4 ml of HBS and
distributed in two tubes containing 6 ml of an isotonic Percoll
solution (40%). The tubes were centrifuged at 4,000 × g at 4 °C for 10 min. At the end of the centrifugation,
one population of cells had sedimented while the other had remained on
the top of the Percoll. The microscopic examination revealed that the upper band was a ductal suspension while acini had sedimented through
the Percoll. The protein content of the ductal suspension was 3.6 ± 0.6 mg/pair of glands (n = 10) measured with a
Bio-Rad protein assay kit using -globulin as standard. The assay of
kallikrein (a ductal marker) confirmed that the upper layer was
enriched 4.4-fold in kallikrein activity with respect to the crude
cellular suspension. Ducts were aspirated, washed, and resuspended in
HBS buffer for further experiments.
Incorporation of [3H]Arachidonic Acid,
[methyl-14C]Choline and myo-[2-3H]Inositol
to Ductal Cells--
The ducts from two glands of one rat were
resuspended in 1 ml of HBS in the presence of 0.5 mM
CaCl2. Three µCi/ml [3H]arachidonic acid
([3H]AA) were added, and the suspension was incubated at
25 °C with gentle shaking. At various times, 50-µl aliquots were
transferred to Eppendorf tubes and centrifuged at 16,000 × g for 1 min. The pellet was washed with 0.9% NaCl, the
lipids were extracted, and the incorporation of [3H]AA
was estimated. Labeling was very rapid, and steady state was reached
after approximately 60 min. At that time, 80 ± 3% of the total
cellular precursor was incorporated into the lipidic fraction. Most of
this radioactivity was distributed among various phospholipids;
phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol + phosphatidyl serine incorporated, respectively, 46 ± 1%,
18.8 ± 0.3%, and 5 ± 1% of the total lipidic fraction.
To study the metabolism of phosphatidylcholine and phosphatidyl
inositol, ductal cells from 1 or 3 rats, respectively, were incubated
for 90 min as described above in 1 ml of HBS containing 0.5 mM CaCl2 and 4 µCi/ml
[methyl-14C]choline or 25 µCi/ml
myo-[2-3H]inositol.
Assay for [3H]Arachidonic Acid Release--
At the
end of the isotopic labeling, ductal cells were washed and incubated
for 1 h in 6 ml of calcium-free HBS in the absence of the tracer.
After this incubation, the ducts were washed again and resuspended in 6 ml of calcium-free HBS. Assays were performed at 37 °C under
constant shaking and started by adding 100 µl of cell suspension to
400 µl of HBS containing CaCl2 (final concentration 0.5 mM) and the agonists. The reaction was stopped by
centrifugation for 30 s at 10,000 × g. Four
hundred µl of the supernatants were transferred to vials and mixed
with 4 ml of scintillation mixture. Radioactivity of the samples was
measured in a scintillation spectrometer (model 2000 CA, Tri-Carb,
Packard). In order to estimate the radioactivity already present in the
medium at the start of each assay, several samples of the cellular
suspension were taken during the experiments, mixed with 400 µl of
HBS, and directly centrifuged, and the radioactivity present in the
supernatant was counted. These results were used as blank values and
used to estimate the release of [3H]AA during the
incubation at 37 °C. Under these assay conditions, 1 mM
ATP or 100 µM Bz-ATP promoted the release up to 8 ± 1% (n = 10) and 14 ± 3% (n = 10), respectively, of the incorporated isotope after 20 min of
stimulation. In order to characterize the nature of the radioactive
compound released by the ductal cells, the supernatant was extracted
with chloroform:methanol:HCl (200:100:1). This extract was concentrated
under vacuum, and the lipids were analyzed on TLC plates using two
different elution systems: chloroform:methanol:acetic acid:water
(90:8:1:0.8) for the separation of AA and its oxidation metabolites and
petroleum ether:diethylether:acetic acid (60:45:1) for the separation
of AA and diacylglycerol. Unlabeled AA was used as standard
(RF = 0.72 and 0.43 for the above described elution
systems, respectively) and was visualized using iodine vapor. The bands
were scraped from the plates, and the radioactivity was measured by
liquid scintillation. The [3H]AA released represented
about 80% of the total counts found in the supernatant.
Extraction of Cellular Lipids and Their Separation by
TLC--
Total cellular lipids were extracted as described previously
(30) with minor modifications. Briefly, pellets from ductal cells were
resuspended in 0.9% NaCl and transferred to ice-cold glass tubes
containing 500 µl of chloroform:methanol:HCl (200:100:1). After 30 min in an ice bath, a final concentration of 0.1 M HCl was
added and the tubes were vortexed and let at room temperature. The
tubes were then centrifuged at 200 × g for 15 min; 200 µl of the organic phase were vacuum-dried in an automatic SpeedVac concentrator (Savant AS290), and resuspended in 40 µl chloroform. Aliquots (30 µl) were spotted on 20 × 20-cm Silica Gel 60 F254 plates. An ascending chromatography was performed
using chloroform:methanol:acetic acid:water (75:45:12:3) as the eluant
for the analysis of phospholipids. Spots were identified by comigration
with authentic standards, which were visualized by exposure to iodine.
The silica gel containing radioactivity was scraped into scintillation
vials and quantified by liquid scintillation.
RNA Extraction and Reverse Transcription-Polymerase Chain
Reaction--
Total RNA was extracted from gland tissue and purified
cell fractions using a genomic-free RNA extraction kit
(GStractTM RNA isolation kit II, Maxim Biotech, Inc.)
based on the Chomczynski and Sacchi method (31). First strand cDNA
was synthesized from 1.5 µg of RNA using random primers and avian
myeloblastosis virus reverse transcriptase in accordance with the
supplier's instructions. Specific primers for the detection of
P2X7 transcripts were designed using the Genetics Computer
Group program GCG Primer. Upstream and downstream primers were based on
unique sequences within exon 8 and exon 12, respectively, stretching
bases 958-980 (P2X7.up) and 1323-1341
(P2X7.down) of the P2X7 sequence (GenBank
accession no. X95882). The amplified product was thus predicted to be 384 base pairs in length. These regions correspond to the intracellular and carboxyl-terminal extracellular loops, respectively, of the corresponding amino acid sequence. PCR was performed as described previously (32) with some modifications. A hot-start touchdown PCR
protocol with thin-walled tubes was employed involving denaturation at
95 °C and extension at 72 °C. The annealing stages began at 65 °C, dropped by 1 °C every two cycles until 56 °C, at which all subsequent annealings were performed. Each step lasted for 50 s, and a total of 45 cycles were performed. The reaction mixture contained 1.5 mM magnesium. Amplified products were
analyzed by electrophoresis in 1.8% agarose gels and viewed with
ethidium bromide staining. A 
X174 HaeIII digest was used
as a size standard. In order to confirm that the amplicons observed
corresponded to RNA and not contaminating genomic DNA, we performed PCR
under identical conditions with rat genomic DNA. The amplicon in this case was 
1400 base pairs, indicating the presence of the four predicted introns in the gDNA between exons 8 and 12 (data not shown).
Thus, the PCR products obtained from the submandibular gland and from
the acinar and ductal cells were derived from expressed mRNA and
not genomic DNA.
Calcium Measurements--
[Ca2+]i was
determined as described previously (18). Briefly, ducts from one rat
were resuspended in 3 ml of HBS. Aliquots (1 ml) were incubated at
25 °C with 1 ml of HBS buffer in the presence of 0.5% (w/v) BSA,
0.25 mM CaCl2 and 2 µM Fura-2/AM. After 45 min, 1 ml of the suspension was removed, washed with isotonic
NaCl, and resuspended in 2 ml of magnesium-free HBS without BSA or
amino acids. The cells were constantly stirred in the cuvette, and the
excitation wavelength was switched every second from 340 nm to 380 nm
(slit width 4 nm). The light emitted at 505 nm was recorded (slit width
8 nm). The voltage of the photomultiplier was 700-750 V. At the start
of each assay, the signal observed after excitation at 345 nm was
arbitrarily set at 50% maximal scale by adjusting this voltage. At the
end of the assay, the traces were calibrated with the successive
addition of 0.5 mM digitonin and 40 mM EGTA (pH
8.5 with Tris). Autofluorescence was measured at both wavelengths in
cells that were not loaded with the Ca2+ indicator and was
subtracted from all the data before calculation of the ratios. The
calcium concentration was estimated by the ratio method (33), using a
Kd value of 263 nM.
Activity of the Polyphosphoinositide-specific Phospholipase
C--
The ductal cells obtained from 3 rats were labeled with
myo-[2-3H]inositol free of magnesium as
described previously (21). At the end of the incubation, the cells were
washed twice with 10 ml of HBS in the absence of labeled inositol and
finally resuspended in 4.5 ml of HBS in the presence of 10 mM LiCl. They were preincubated in this medium for 10 min
at 37 °C. Aliquots (100 µl) were incubated in a final 500 µl of
HBS medium in the presence of the tested agents for 5 min at 37 °C.
At the end of the incubation, the samples were centrifuged at
10,000 × g for 30 s. The supernatant was
discarded, and 500 µl of 10% (w/v) ice-cold trichloroacetic acid
were added to the pellet. The samples were centrifuged for 2 min at
10,000 × g, and the supernatant was transferred to
glass tubes. The pellets were washed again with 500 µl of 10%
trichloroacetic acid, centrifuged and the supernatants were pooled. The
trichloroacetic acid was extracted with diethylether saturated with
water. The extract was neutralized with 1 M KOH and diluted
with 8 ml of 10 mM HEPES, 2 mM EDTA (pH 7.4).
The inositol phosphates were isolated using a Dowex AG1-X8 column (34).
The glycerophosphoinositol was eluted with 12 ml of 30 mM
ammonium formate. The inositol derivatives (inositol mono-, bis-, and
trisphosphates) were eluted simultaneously from the Dowex column using
12 ml of 0.7 M ammonium formate and 0.1 M
formic acid. The radioactivity eluted off the column was quantified by
liquid scintillation counting. Each determination was performed in triplicate.
Kallikrein Secretion--
The ductal cells isolated from 1 rat
were resuspended in 4 ml of magnesium-free HBS. The stimulation of the
cells with the agonist was performed for 10 min as described for the
[3H]AA release assay. At the end of the incubation, the
cells were centrifuged for 30 s at 10,000 × g.
Four hundred µl of the supernatant were transferred to Eppendorf
tubes and kept on ice. The kallikrein assay was performed as described
previously (18). The assay was run in a 1-ml plastic cuvette using 300 µl of Tris buffer (0.5 M, pH 8.2), 300 µl of substrate
solution (2 mg/ml
N-benzoyl-DL-arginine p-nitroanilide), and 300 µl of the sample. The absorbance
was measured at 405 nm during 3 min in a Uvikon 943 spectrophotometer. The results were plotted as absorbance versus time, and the
activity of the enzyme was calculated using the slope of the linear
part of the curve. An aliquot of the ductal suspension was homogenized by sonication (2 pulses of 5 s at 8 µm with a Soniprep 150) and assayed for kallikrein content. This result gave an estimate of the
total content of kallikrein in the cell suspension. Results were
expressed as a percentage of the total kallikrein content released
during the incubation at 37 °C. These results were corrected with
regard to the kallikrein present in the medium prior to the incubation
of the cells with the agonists.
Statistical Analysis--
Unless otherwise indicated, values in
figures are given as means ± standard error (S.E.) of n
experiments performed in triplicate with n different cell
preparations. Statistical significance of the results was determined
using a Student's test and represented by *** (p 
0.001), ** (0.001 p 0.01), * (0.01 p 0.05), and NS (not significant) (0.05 p).
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RESULTS |
Release of [3H]AA from RSMG Ductal Cells in Response
to Purinergic Agonists--
RSMG ductal cells were prelabeled with
[3H]AA and incubated for various times in the presence of
either 1 mM ATP or 100 µM Bz-ATP. As shown in
Fig. 1, the two purinergic agonists
increased the release of [3H]AA from prelabeled cells.
The maximum stimulation was observed after 15 min. After 20 min of
exposure to ATP or Bz-ATP, the release of [3H]AA was
increased by 216 ± 13% and 310 ± 12%, respectively. The other purinergic agonists tested (2-MeSATP, AMP-PNP, ,
-methylene ATP, ,-methylene ATP, 5'-adenosine tetraphosphate, ATPS, ADP, and UTP) were unable to elicit a significant response in terms of
[3H]AA release (Table I).
Among the nonpurinergic secretagogues tested, only 100 µM
carbachol was able to induce a small effect (118 ± 8%). The
stimulation of the cells for 20 min with calcium ionophores (100 nM ionomycin or 1 µM A23187) or the depletion of the intracellular calcium stores in cells incubated for 20 min with
1 µM thapsigargin had no significant effect on the
release of [3H]AA from the cells (Table I). In separate
experiments, it was shown that all these agents (epinephrine,
carbachol, ionomycin, and thapsigargin) increased the
[Ca2+]i in the ductal suspension (data not
shown).
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Fig. 1.
Time course of the release of
[3H]AA by RSMG ductal cells in response to ATP and
Bz-ATP. [3H]AA-labeled ductal cells were washed and
resuspended in a magnesium-free HBS medium containing 0.125% BSA.
Cells were stimulated with 1 mM ATP ( ) or 100 µM Bz-ATP ( ) in the presence of 0.5 mM
CaCl2. The reaction was carried out at 37 °C and stopped
by centrifugation at indicated times. The radioactivity of the
supernatants was measured, and the results are expressed as percentage
of radioactivity with respect to unstimulated controls. Data represent
mean ± S.E. of three independent experiments performed in
triplicate.
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Table I
Effect of purinergic agonists and other Ca2+-mobilizing agents
on the release of [3H]arachidonic acid
[3H]AA-labeled ductal cells were washed and resuspended in a
magnesium-free HBS medium containing 0.125% BSA. Cells were stimulated
with different drugs in the presence of 0.5 mM
CaCl2. The reaction was carried out at 37 °C and stopped by
centrifugation 20 min after stimulation. The radioactivity of the
supernatants was measured and the results are expressed as percentage
of radioactivity with respect to unstimulated controls. Data represent
mean ± S.E. of at least three independent experiments performed
in triplicate.
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The release of [3H]AA in response to ATP and Bz-ATP was
dose dependent (Fig. 2). ATP
significantly increased the release of [3H]AA at 100 µM, its half-maximal and maximal effects being observed at 220 µM and 1 mM. The RSMG ductal cells
were more sensitive to Bz-ATP, which significantly increased the
release of [3H]AA at concentrations as low as 10 µM. The EC50 and the maximal concentration
for Bz-ATP were 15 and 100 µM.
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Fig. 2.
Dose-response curve for ATP and Bz-ATP on the
release of [3H]AA by RSMG ductal cells.
[3H]AA-labeled ductal cells were washed and resuspended
in a magnesium-free HBS medium containing 0.125% BSA. Cells were
stimulated with different concentrations of ATP () or Bz-ATP ()
in the presence of 0.5 mM CaCl2. The reaction
was carried out at 37 °C and stopped by centrifugation 20 min after
stimulation. The radioactivity of the supernatants was measured, and
the results were corrected according to their basal values and are
expressed as percentage of radioactivity with respect to unstimulated
controls. The basal release of radioactivity averaged 2,000 ± 88 dpm (n = 10) at 20 min. Data represent mean ± S.E. of three independent experiments performed in triplicate.
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Effect of Purinergic Inhibitors and Antagonists on the Release of
[3H]AA--
To further characterize the purinergic
receptors coupled with the increased release of [3H]AA,
the effect of several antagonists was tested (Table
II). Among these antagonists, oxidized
ATP (oATP) is an inhibitor of ecto-ATPases and an irreversible
inhibitor of P2Z receptor in human lymphocytes (29) and in
mouse macrophages (35). The cells were preincubated with oATP for 60 min before exposure to the purinergic agonist. By itself, oATP did not
affect the basal release of [3H]AA. At a 100 µM concentration, oATP inhibited by 52 ± 6% and 46 ± 6% the [3H]AA release in response to 1 mM ATP and 100 µM Bz-ATP, respectively. At
500 µM oATP, the ATP and Bz-ATP purinergic responses were
inhibited by 77 ± 7% and 76 ± 9% (Table II). The second
antagonist tested was suramin. This inhibitor of ecto-ATPases is also
an inhibitor of purinergic receptors, which does not discriminate
between the P2X and the P2Y subtypes and an inhibitor of
P2Z receptors (29, 35). At a 500 µM
concentration, suramin inhibited by 50% the response to 100 µM Bz-ATP or 1 mM ATP. Coomassie Blue was the last inhibitor tested. It has been reported that Coomassie Blue blocks
the response of submandibular acini to Bz-ATP (21). At a 10 µM concentration, it inhibited the increase of the
[Ca2+]i in response to 100 µM
Bz-ATP and to 1 mM ATP in RSMG ductal cells (18). Ten
µM Coomassie Blue inhibited by 62 ± 8% and 40 ± 12% the release of [3H]AA induced by 1 mM
ATP and 100 µM Bz-ATP, respectively. The decreases
observed on cellular [3H]AA-labeled phospholipids in
Bz-ATP-stimulated cells at 20 min were also partially inhibited by 10 µM Coomassie Blue (data not shown). These observations
indicated that the [3H]AA release from the stimulated
cells could be at least in part secondarily coupled to the activation
of P2Z receptor.
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Table II
Effect of P2 receptor inhibitors and antagonists on the release of
[3H]arachidonic acid in rat submandibular ductal cells
stimulated with ATP or Bz-ATP
[3H]AA-labeled ductal cells were washed and resuspended in a
magnesium-free HBS medium containing 0.125% BSA. Cells were incubated
for indicated times with different inhibitors of the purinergic
response in the presence of 0.5 mM CaCl2. In the
experiments with oxidized ATP the cells were incubated for 60 min with
the inhibitor and then the cells were washed. The cells were further
stimulated with 1 mM ATP or 100 µM Bz-ATP.
The reaction was carried out at 37 °C and stopped by centrifugation
20 min after stimulation. The radioactivity of the supernatants was
measured and the results are expressed as percentage of inhibition.
Data represent mean ± S.E. of three independent experiments
performed in triplicate.
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Effect of Divalent Cations on the Release of [3H]AA
in Response to Purinergic Agonists--
It is generally recognized
that the active form of ATP on P2X7(P2Z)
receptors is the free tetraionic form (ATP4). The
complexation of ATP by divalent cations might thus decrease the
concentration of the active form. However, divalent cations might also
block the nonspecific cation channels coupled to the ionotropic
receptors. Magnesium (18) and nickel (36) were used in these studies.
The concentrations of the different forms of ATP in the presence of
either Ni2+ or 5 mM Mg2+ was
estimated using the Solgaswater program (37) and the equilibrium constants from different well documented sources (38, 39). According to
our calculations (Table III) in a
Ni2+ or Mg2+ free medium containing 1 mM ATP and 0.5 mM Ca2+, the
ATP4- concentration was 29.7 µM. The
ATP4- concentration was barely affected by the presence of
different concentrations of Ni2+. It was calculated that
the addition of 1 mM Ni2+ should decrease the
[ATP4-] to 25.8 µM, a concentration able to
activate the P2Z receptors of rat parotid gland (40). In
the presence of 5 mM Mg2+, the free
ATP4- drops dramatically to 7.4 µM. At this
concentration, ATP is not able to stimulate the low affinity receptor
described previously (18).
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Table III
Calculated concentrations of ATP4 and complexed ATP in the
presence and absence of Mg2+ and Ni2+ in the assay
media (in µM)
The concentration of different species of complexed ATP (in
µM) has been calculated with the computer program
Solgaswater, considering all the possible forms in a medium containing
1 mM ATP (total concentration) and 24.5 mM
HEPES, 0.5 mM CaCl2, 96 mM NaCl, 6 mM KCl, 2.5 mM NaH2PO4, 11.5 mM glucose, 5 mM pyruvate, 5 mM
glutamate, 5 mM fumarate, pH = 7.4, in the presence or
absence of 5 mM Mg2+ and different concentrations
of Ni2+. For clarity of the table, minor species (<1
µM) have been omitted.
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Five mM MgCl2 inhibited the release of
[3H]AA in response to 100 µM Bz-ATP and 1 mM ATP by 95 ± 4% and 91 ± 6%, respectively. It can be concluded that ATP4- is the form of ATP promoting
the release of [3H]AA from cellular phospholipids. The
release of [3H]AA in response to Bz-ATP and ATP was also
inhibited when various concentrations of Ni2+ were added to
the medium (Fig. 3). The half-maximal
inhibitory concentrations were 0.18 and 0.24 mM with
respect to 100 µM Bz-ATP and 1 mM ATP. The
response to the purinergic agonists was totally abolished in the
presence of concentrations of Ni2+ higher than 0.75 mM. The inhibition exerted by 0.75 mM
Ni2+ on the [3H]AA release in
Bz-ATP-stimulated cells was reversed by the addition of 1.5 mM DTPA (a chelator of metal ions) to the medium (Fig. 4, left panel). By
itself, DTPA slightly affected (0.05 < p) the maximal
release of [3H]AA in the absence of Ni2+.
This inhibition might be secondary to the chelation of external calcium
(see above). In order to maintain the availability of Ca2+
in the presence of 1.5 mM DTPA, these experiments were thus
performed in the presence of 1 mM calcium, instead of 0.5 mM. That is the reason why the maximal effect on the
[3H]AA release is higher in these experiments than the
results shown in Fig. 3. Considering that the addition of nickel in the
medium did not significantly decrease the [ATP4],
nickel could exert its inhibition on the nonspecific cation channels
coupled to the P2X7 (P2Z) receptors.
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Fig. 3.
Effect of various concentrations of
Ni2+ on the release of [3H]AA from RSMG
ductal cells stimulated with ATP or Bz-ATP.
[3H]AA-labeled ductal cells were washed and resuspended
in a magnesium-free HBS medium containing 0.125% BSA. Cells were
stimulated with 1 mM ATP () or 100 µM
Bz-ATP () in the presence of 0.5 mM CaCl2
and increasing concentrations of Ni2+. The reaction was
carried out at 37 °C and stopped by centrifugation 20 min after
stimulation. The radioactivity of the supernatants was measured, and
the results are expressed as percentage of radioactivity with respect
to unstimulated controls as stated in Fig. 2. Data represent mean ± S.E. of three independent experiments performed in triplicate.
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Fig. 4.
Reversal by DTPA of the effect of
Ni2+ and DTPA on the release of [3H]AA and on
the [Ca2+]i in Bz-ATP-stimulated RSMG ductal
cells. Left panel,
[3H]AA-labeled ductal cells were washed and resuspended
in a magnesium-free HBS medium 0.125% BSA. Cells were stimulated with
100 µM Bz-ATP in the presence of 1 mM
CaCl2 and in the absence (circles) or presence
(triangles) of 0.75 mM Ni2+. At 10 min 1.5 mM DTPA (open symbols) or
vehicle (closed symbols) were added. The reaction
was carried out at 37 °C and stopped by centrifugation at indicated
times. The radioactivity of the supernatants was measured, and the
results are expressed as percentage of radioactivity with respect to
unstimulated controls as stated in Fig. 2. Data represent mean ± S.E. of three independent experiments performed in triplicate.
Right panel, Fura-2-loaded cells were resuspended in a HBS
medium in the absence of magnesium, BSA or amino acids and in the
absence or presence of 0.75 mM Ni2+. Two min
before stimulation, 1 mM CaCl2 was added. At
time 0, cells were stimulated with 100 µM Bz-ATP, and 10 min later 1.5 mM DTPA or vehicle was added (see legend).
The fluorescence was recorded as described under "Experimental
Procedures." The traces represent the ratio
F340/F380 and are the
mean of three experiments with different cell preparations. For clarity
of the figure, error bars have been omitted.
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This hypothesis was tested in parallel experiments, where the effect of
nickel on the [Ca2+]i in response to purines was
tested. RSMG ductal cells were loaded with Fura-2 and resuspended in
HBS medium in the presence of 1 mM CaCl2. When
the cells were incubated in control conditions, the addition of 100 µM Bz-ATP to the medium sharply increased the
[Ca2+]i from 202 ± 41 to 646 ± 94 nM (n = 3, p < 0.01) and the [Ca2+]i continued to rise slowly thereafter
(Fig. 4, right panel). The addition of 1.5 mM DTPA at 10 min caused a slight drop on the calcium level
(from 1264 ± 42 to 1067 ± 112 nM, 0.05 < p). This decrease might be due to the binding of some
calcium to DTPA and the subsequent decrease of the free calcium
concentration in the medium. When the RSMG ductal cells were
resuspended in the presence of 0.75 mM Ni2+,
the initial response to 100 µM Bz-ATP was completely
inhibited (Fig. 4, right panel). The
[Ca2+]i increased slowly reaching 367 ± 95 nM at 10 min. It can also be noted that the intracellular
Fura-2 was not quenched by nickel, confirming that at the opposite to
calcium and manganese (18), this divalent cation cannot enter the RSMG
ductal cells via the nonspecific cation channels coupled to the
P2X7 receptors. The chelator added 10 min after Bz-ATP
almost totally reversed, for the first 10 s, the inhibition
exerted by Ni2+ on the response to Bz-ATP. Five minutes
after the addition of DTPA, the [Ca2+]i increased
to 550 nM.
Expression of P2X7 Receptor mRNA in RSMG
Cells--
The P2Z receptor has been recently cloned in
rat brain by Surprenant et al. (8) and termed
P2X7 according to the current classification. The
expression of the P2X7-receptor mRNA in ducts and acini
was demonstrated by RT-PCR using rat whole brain as a positive control.
As shown in Fig. 5 P2X7
transcripts were detected in the whole RSMG as well as in both acinar
and ductal purified fractions.
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Fig. 5.
RT-PCR detection of P2X7 mRNA
in rat submandibular gland cells. The P2X7 transcript
is expressed in the rat submandibular gland (SMG), and in
both acinar (A) and ductal (D) cells purified
from the same. RT-PCR detection of mRNA transcripts encoding this
receptor is illustrated in an ethidium bromide-stained, 1.8% agarose
gel of the corresponding PCR products. Rat whole brain (WB)
RNA was used as a positive control. The ladder () is the X174
HaeIII digest, with corresponding sizes in base pairs
indicated to the left. The predicted size of the amplified
product is 384 base pairs.
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Origin of the [3H]AA Released in Response to ATP and
Bz-ATP--
As stated under "Experimental Procedures,"
[3H]AA was preferentially incorporated into
phosphatidylcholine (PC), which represented 46% of the total
lipid-labeled fraction, whereas the incorporation into
phosphatidylethanolamine (PE) and phosphatidylserine + phosphatidyl inositol (PS+PI) was 18.8% and 5%, respectively. When
[3H]AA-labeled cells were stimulated with 1 mM ATP or 100 µM Bz-ATP, we observed at 10 and 20 min a 22% and 32% decrease into the content of
[3H]AA-labeled PC pool, respectively (Table
IV). At 20 min a significant 23%
decrease of the [3H]AA-labeled PE pool was also observed
in the ATP-stimulated cells. The response to ATP was completely blocked
by the addition of 5 mM MgCl2 to the medium
(data not shown), confirming the contribution of P2X7
receptors in this response. The PS+PI [3H]AA-labeled pool
was not significantly affected by ATP or Bz-ATP (data not shown). No
significant variations of the [3H]AA-labeled phosphatidic
acid and diacylglycerol pools in response to ATP or Bz-ATP could be
observed and the amount of dpm lost by the phospholipid fraction
correlated well with the radioactivity released in the medium. Thus,
the radioactivity lost from the total phospholipid fraction in
Bz-ATP-stimulated ducts with respect to controls at 10 and 20 min was
2038 ± 165 and 4036 ± 456 dpm, respectively. By comparison,
the radioactivity found in the supernatants in the same conditions was
1865 ± 889 and 4883 ± 156 dpm. The effect of purinergic
agonists on the metabolism of PC was further characterized using
[14C]choline-labeled cells. One hundred µM
Bz-ATP time-dependently decreased the content of
[14C]choline-labeled PC with a maximal 33 ± 2%
decrease observed after 20 min (data not shown), a value similar to the
value observed with [3H]AA-labeled PC. PC provided most
of the [3H]AA released in response to purinergic
agonists, suggesting that a phospholipase A2 could be
activated by P2X7 agonists in RSMG ductal cells either
directly or after the activation of phospholipase D. The purpose of the
next experiments was to further characterize the mechanisms involved in
the release of [3H]AA from the ATP-stimulated RSMG ductal
cells.
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Table IV
Effect of Bz-ATP on the degradation of phospholipids in RSMG ductal
cells
[3H]AA-labeled ductal cells were washed and resuspended in
magnesium-free HBS medium containing 0.125% BSA. Cells were stimulated
with 100 µM Bz-ATP in the presence of 0.5 mM
CaCl2 in a final volume of 500 µl. The reaction was carried
out at 37 °C and stopped by centrifugation at indicated times. The
lipids extracted from the pellets were separated by TLC as described
under "Experimental Procedures" and the radioactivity present in
the bands of PC, PE, and PS+PI was determined. Data represent mean ± S.E. of three independent experiments performed in triplicate. The
percentage of 3H content with respect to unstimulated controls
and statistical significance are also shown.
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Contribution of Various Phospholipases A2 on the
Release of [3H]AA--
Various inhibitors were used to
study the implication of a phospholipase A2 in the release
of [3H]AA from RSMG ductal cells. Cells were preincubated
for 5 min with ONO-RS-082 (a nonspecific inhibitor of PLA2
since it is able to inhibit both the cytosolic and the secreted forms
of the enzyme; Ref. 41) before exposure to either 1 mM ATP
or 100 µM Bz-ATP. Five hundred µM
ONO-RS-082 fully inhibited the response to the two agonists
(IC50 67 and 85 µM for the response to ATP
and Bz-ATP, respectively). In the absence of agonists, ONO-RS-082 had
no significant effect on the release of [3H]AA. Two other
more specific inhibitors were tested next. AACOCF3 is a
potent, slow, tight-binding, reversible inhibitor of cPLA2 and it proved to be highly specific for the 85-kDa cytosolic
calcium-dependent PLA2 (cPLA2)
(42). In our conditions, incubation of the cells with
AACOCF3 for 2 min prior to the addition of the purinergic agonists produced a concentration-dependent decrease in the
stimulated [3H]AA release (Fig.
6, left panel),
whereas it had no effect on the basal [3H]AA release. The
inhibition averaged 15 ± 3% at a 300 nM
AACOCF3 concentrations. Half-maximal inhibition could be
observed at 2 µM and at a maximal 100 µM
concentration, AACOCF3 inhibited by 84 ± 4% the
response to 1 mM ATP (data not shown) and by 73 ± 6%
the response to 100 µM Bz-ATP (Fig. 6, left
panel). A full inhibition could never be observed. These
results suggested that a cPLA2 was at least partly
responsible for the release of [3H]AA in response to
purinergic agonists but that some other PLA2 might also be
involved. We examined the possibility that a calcium-independent PLA2 was present in RSMG ducts. The release of
[3H]AA was measured in the absence of calcium but in the
presence of 0.5 mM EGTA. In these conditions, Bz-ATP could
not increase the [Ca2+]i (18, data not shown).
The release of [3H]AA in response to Bz-ATP was inhibited
by 66 ± 3% but the stimulatory effect of Bz-ATP on the release
of [3H]AA remained highly significant when compared with
control (p < 0.001). The addition of various
concentrations of AACOCF3 to the medium did not affect this
calcium-insensitive release of [3H]AA in response to
Bz-ATP (Fig. 6, left panel). This result
confirmed that AACOCF3 inhibited the cPLA2,
which is a calcium-dependent PLA2. The presence
of a calcium-insensitive (iPLA2) in RSMG ducts was
confirmed with BEL, an inhibitor of the iPLA2 (43). In the presence of extracellular calcium, BEL slightly inhibited the basal
release of [3H]AA by 18% and 11% at 3 and 10 µM, respectively, whereas it had no significant effect at
30 and 100 µM. BEL dose-dependently inhibited the release of [3H]AA in response to either ATP
(IC50 1 µM; data not shown) or Bz-ATP
(IC50 2 µM; Fig. 6, right
panel). At a maximal 100 µM concentration of
BEL, the response to the two purinergic agonists was inhibited by
71 ± 3%. In the absence of extracellular calcium, 30 µM BEL fully inhibited the residual response to Bz-ATP
(Fig. 6, right panel).
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Fig. 6.
Effect of AACOCF3 and BEL on the
release of [3H]arachidonic acid in rat submandibular
ductal cells stimulated with Bz-ATP. [3H]AA-labeled
ductal cells were washed and resuspended in a magnesium-free HBS medium
containing 0.125% BSA in the presence of 0.5 mM
CaCl2 () or in the presence of 0.5 mM EGTA
(). The cells were preincubated for 2 min with various
concentrations of AACOCF3 (left panel) or for 5 min with various concentrations of BEL (right
panel). They were then stimulated with 100 µM
Bz-ATP in the presence of each inhibitor. The reaction was carried out
at 37 °C and stopped by centrifugation 20 min after stimulation. The
radioactivity of the supernatants was measured, and the results are
expressed as percentage of radioactivity with respect to unstimulated
controls. Data represent mean ± S.E. of three to six independent
experiments performed in triplicate.
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BEL and AACOCF3 were also used to test the role of the
iPLA2 and the cPLA2 in the remodeling of
cellular phospholipids. RSMG ductal cells were preincubated for 5 min
either in basal conditions or in the presence of 100 µM
BEL or AACOCF3 and then incubated in the same conditions
but in the presence of [3H]AA. After 60 min, the
radioactivity present in the phospholipid fraction was estimated. The
presence of BEL and AACOCF3 decreased by, respectively,
90 ± 1% and 51 ± 3% the incorporation of
[3H]AA in the phospholipids (data not shown). These data
suggest that iPLA2 (which is not specific for
arachidonyl-containing phospholipids) has a constitutive activity which
participates in the selective incorporation of arachidonic acid in phospholipids.
Lack of Correlation between the Activation of a Phospholipase C and
the Release of [3H]AA--
As reported previously, ATP-
and Bz-ATP-stimulated [3H]AA release could reflect
phospholipase C activation by the purinergic receptors and subsequent
stimulation of a calcium- or protein kinase C- dependent phospholipase
A2 (26, 30). In order to determine the relationship between
the [3H]AA release and the phospholipase C activation,
the cells were labeled with
myo-[2-3H]inositol. The stimulatory effect of
Bz-ATP on [3H]AA release from ducts was not secondary to
the activation of phospholipase C, since 100 µM Bz-ATP,
which was the best agonist on [3H]AA release, did not
significantly increase the liberation of inositol phosphates; the
radioactivity present in the fraction containing
IP1+IP2+IP3 increased from 200 ± 47 dpm in the absence of Bz-ATP to 263 ± 73 dpm in its
presence (NS, 0.05 < p). At the opposite, 100 µM carbachol, which did not significantly increase the
release of [3H]AA (Table I), increased the radioactivity
present in the inositol phosphate fraction to 1,366 ± 152 dpm
(p < 0.001 when compared with control). These results
confirmed that the increase in the [Ca2+]i
through the phospholipase C activation was not responsible for the
Bz-ATP-stimulated [3H]AA release.
Role of the Two Phospholipases A2 in the Physiology of
the Ductal Cells--
It has been reported previously that the
activation of ionotropic receptors increases the release of kallikrein
by RSMG ductal cells (18). The role of the cPLA2 and
iPLA2 in this response was tested next (Fig.
7). The ducts were preincubated with 100 µM AACOCF3 or BEL in a medium containing 0.5 mM Ca2+, and the release of kallikrein in the
medium was measured after incubation either in basal conditions or in
the presence of 100 µM Bz-ATP. The two inhibitors had
divergent effects on the secretion of kallikrein both in basal
conditions and in the presence of the purinergic agonist.
AACOCF3 increased the basal release of kallikrein from
18 ± 2% to 50 ± 7% (n = 3). It also
enhanced the secretory response to Bz-ATP. Bz-ATP increased the
secretion of kallikrein to 42 ± 1% in the absence of
AACOCF3 and to 75 ± 4% in its presence. Taking into
account that AACOCF3 is a structural analogue of AA, we
tested whether exogenous AA could also increase kallikrein secretion.
At 1, 10, and 100 µM, AA had no effect on the secretion
of kallikrein. BEL was a strong inhibitor of kallikrein secretion,
which decreased to 3 ± 1% in basal conditions and to 6 ± 2% in the presence of Bz-ATP (Fig. 7). The inhibitory effect of BEL
was dose-dependent (Fig. 8).
Half-maximal inhibitory concentrations were 10 µM both on
basal and on Bz-ATP-stimulated kallikrein release. Considering that BEL
was originally designed to inhibit the serine protease -chymotrypsin
(44), BEL could inhibit the activity of kallikrein rather that inhibit
its secretion. To test this hypothesis, RSMG ductal cells were
homogenized and the esterase activity of the homogenate was measured in
the absence or in the presence of increasing concentrations of BEL. At
a 100 µM concentration, the inhibition exerted by BEL on
the activity of kallikrein never exceeded 15% (data not shown). These
results confirmed that BEL inhibited the secretion of kallikrein rather
than the activity of the enzyme. The involvement of an
iPLA2 in the secretory response to Bz-ATP was further
confirmed by measuring the release of kallikrein in the absence of
extracellular calcium. In this condition, the basal release of
kallikrein was not significantly affected and the secretory response to
Bz-ATP was decreased from 46 ± 2% in the presence of 0.5 mM calcium to 36 ± 4% in the presence of 0.5 mM EGTA. The response was still highly significant when
compared with basal release (p < 0.01, n = 4).
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Fig. 7.
Effect of PLA2 inhibitors on the
secretion of kallikrein by RSMG ductal cells in response to
Bz-ATP. RSMG ductal cells were washed and resuspended in a
magnesium-free HBS medium containing 0.125% BSA and 0.5 mM
CaCl2. They were preincubated for 2 min with 100 µM AACOCF3 or for 5 min with 100 µM BEL. The cells were incubated with 100 µM Bz-ATP (closed bars) or vehicle
(open bars) in the presence of each inhibitor.
The incubation was carried out at 37 °C for 10 min and stopped by
centrifugation. The kallikrein secreted in the supernatant was measured
as described under "Experimental Procedures." Data are expressed as
percentage of kallikrein secreted when compared with the total cellular
content and are the mean ± S.E. of three independent experiments
performed in triplicate.
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Fig. 8.
Effect of BEL on the secretion of kallikrein
by RSMG ductal cells in response to Bz-ATP. RSMG ductal cells were
washed and resuspended in a magnesium-free HBS medium containing
0.125% BSA and 0.5 mM CaCl2. They were
preincubated for 5 min with increasing concentrations of BEL. The cells
were incubated with 100 µM Bz-ATP () or vehicle ()
in the presence of BEL. The incubation was carried out at 37 °C for
10 min and stopped by centrifugation. The kallikrein secreted in the
supernatant was measured as described under "Experimental
Procedures." Data are expressed as percentage of kallikrein secreted
when compared with the total cellular content and are the mean ± S.E. of three independent experiments performed in triplicate.
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DISCUSSION |
The present results constitute the first demonstration by RT-PCR
of the expression of P2X7 in ductal cells of RSMG. This
receptor is coupled with the activation of two distinct phospholipases A2. Indeed, ATP and Bz-ATP increased the release of
[3H]AA from prelabeled RSMG ductal cells. Based on the
response to various agonists, on the sensitivity to antagonists like
Coomassie Blue, or on the effect of divalent cations on the response to ATP, two types of purinergic receptors on RSMG ductal cells have been
described (18). At low concentrations, ATP stimulated a G-protein-coupled P2Y1 receptor that was very sensitive to
2-MeSATP. The occupancy of this receptor increased the intracellular
concentration of IP3, and an intracellular pool of calcium
was mobilized. The removal of calcium from the extracellular medium or
the addition of magnesium did not suppress this response to ATP. This
receptor is present in freshly isolated RSMG ductal cells, but its
coupling with an effector disappears during cell cultures (22). At high concentrations, ATP and its analog Bz-ATP both induced a massive increase of the [Ca2+]i, which was fully
dependent on the presence of calcium in the extracellular medium (18).
This response was blocked by Coomassie Blue or by the decrease of the
concentration of ATP4 secondary to the addition of
magnesium to the medium. The secretion of kallikrein by ductal cells
was increased in response to P2Z agonists (18). As shown in
this work, ATP and Bz-ATP, two agonists of the P2Z
receptors, increased the release of [3H]AA from RSMG
ductal cells. This response was not reproduced by agonists of the P2Y
receptors. The release of [3H]AA was not secondary to the
activation of a polyPI-specific phospholipase C since an agonist like
carbachol, which activated this enzyme, had no significant effect on
the release of [3H]AA. It was inhibited by oATP, suramin,
or Coomassie Blue, antagonists of the P2Z receptors. The
presence of magnesium in the medium also suppressed the release of
[3H]AA in response to ATP and Bz-ATP, further confirming
that P2Z receptors were involved in this response. Nickel
also inhibited the response to the purines. We could calculate that at
concentrations of nickel as high as 1 mM, the concentration
of ATP4 in the medium should be sufficient to fully
stimulate the P2Z receptors. RSMG ductal cells were
impermeant to nickel since the ion did not quench the fluorescence of
intracellular Fura-2 and since the inhibition by nickel was
instantaneously reversed by the addition of DTPA, a non-permeant
chelator of the ion. The inhibition by nickel could thus only be
explained by a direct blockade by this cation of the nonspecific cation
channel coupled to P2Z receptors. From these results, it
could be concluded that the activation of the nonspecific cation
channels coupled to P2Z receptors increased the release of
[3H]AA from RSMG ductal cells.
The [3H]AA released in the medium in response to
purinergic agonists originated from the cellular phospholipids, mainly
PC. Indeed, the decrement in the content of [3H]AA of
this class of phospholipids was in agreement with the increment of the
[3H]AA released into the extracellular medium. Several
lipases could be involved in the release of [3H]AA from
prelabeled phospholipids. It has been reported previously that
P2Z agonists can activate a phospholipase D in BAC1.2F5
macrophages (27) or in lymphocytes (29). The phosphatidic acid
generated by this enzyme could be hydrolyzed by a phospholipase
A2 or could generate diglycerides after hydrolysis by a
phosphatidate-phosphohydrolase. A diglyceride lipase would then
liberate [3H]AA from these diglycerides. Such a pathway
has been described in peritoneal mast cells (45) and accounts for the
release of [3H]AA in response to muscarinic agonists in
RSMG acinar cells (46). Preincubation of [3H]AA-labeled
RSMG ductal cells with propranolol (an inhibitor of the phosphatidate
phosphohydrolase, 47) did not affect the subsequent ATP- or
Bz-ATP-release of [3H]AA (data not shown). The release of
[3H]AA was inhibited by inhibitors of PLA2.
These results suggest that a PLA2 and not a diglyceride
lipase is responsible for the release of [3H]AA. At the
present time, we are unable to exclude the activation of a
phospholipase D previous to the activation of PLA2. It
should, however, be mentioned that: 1) the purines did not increase the release of free choline from phospholipids labeled with
[3H]choline; and 2) the activation of PLD by
P2Z agonists is dependent on bivalent cation influx (29),
but the release of [3H]AA in response to ATP could be
observed in the absence of divalent cation in the medium and in the
presence of EGTA, a chelator of these cations.
Two major groups of intracellular PLA2 have been described.
The calcium-dependent cytosolic PLA2
(cPLA2) has a molecular mass of 85 kDa. This enzyme is
rather specific for 2-arachidonyl phospholipids. The NH2
terminus of this enzyme is a calcium-binding domain analogous to the C2
domain of protein kinase C. This enzyme cannot be activated in the
absence of an increase of [Ca2+]i. After
interacting with the C2 domain of the enzyme, calcium promotes its
translocation from the cytosol to intracellular membranes, bringing the
calcium-independent catalytic site to the membrane substrate (48).
Various calcium-independent phospholipases A2 have been
described (49). Ankyrin domains have been described in these
iPLA2, which could explain their self-association to form
catalytically competent species and their interaction with proteins
that may regulate their catalytic properties (50). Considering that no
inhibitor is fully specific of the iPLA2, the release of
[3H]AA in the absence of extracellular calcium is the
best evidence for the involvement of this enzyme in the hydrolysis of
cellular phospholipids (49). In our hands, the removal of extracellular calcium inhibited but did not suppress the release of
[3H]AA in response to Bz-ATP. This result suggested that
P2X7-receptors could activate both
calcium-dependent and calcium-independent phospholipases
A2. This was further confirmed by the use of
AACOCF3 (an inhibitor of cPLA2) and BEL (an
inhibitor of iPLA2). Neither of these inhibitors is fully
specific; it has been claimed that AACOCF3 could inhibit
iPLA2 (49, 51) and that BEL could inhibit the phosphatidate
phosphohydrolase (52). In the presence of extracellular calcium, none
of these two inhibitors could completely block the release of
[3H]AA. The inhibition exerted by AACOCF3 was
not observed in the absence of extracellular calcium, confirming that
this inhibitor blocked a calcium-sensitive PLA2. The
presence of a cPLA2 in RSMG ductal cells was confirmed by
Western blots using commercially available rabbit polyclonal antibodies
(data not shown). The poor quality of the gels (due to the high
background) precluded any study of the phosphorylated state of the
enzyme by the electrophoretic mobility shift method. We were also
unable to demonstrate the translocation of the enzyme from the cytosol
to a membrane compartment in the presence of Bz-ATP (data not shown).
The inhibition exerted by BEL on the release of [3H]AA
was still observed in the absence of extracellular calcium; the
stimulation of [3H]AA release by Bz-ATP was completely
blocked by a combination of calcium removal and BEL. This confirmed
that P2X7 agonists activated an iPLA2. The
mechanisms involved in the activation of iPLA2 remain
poorly understood. The enzyme has several ankyrin domains, which are
involved in the interaction with other proteins like
phosphofructokinase or in the interaction between several monomers of
iPLA2 (50). It has indeed been reported that the active
form of the enzyme has an oligomeric structure. More recently, it was
shown that the splicing of the introns can generate isoforms that have
no catalytic activity but can interact with the iPLA2 and
inhibit its activity (53). It has been recently reported that in
macrophages, P2Z agonists activate a transcription factor, NF-
B (54). Like iPLA2, this protein has several ankyrin
domains (55), and splice variants of a precursor behave as antagonists (54). The activation of iPLA2 and NF-B by
P2X7 agonists might involve similar mechanisms like
activation of the ubiquitin-proteasome pathway (56).
Considering that P2X7 receptors activate both a
cPLA2 and an iPLA2 and promote the secretion of
kallikrein, the correlation between these responses remained to be
established. BEL was a strong inhibitor of both basal and
Bz-ATP-stimulated release of kallikrein. Half-maximal concentrations of
BEL on [3H]AA release and on kallikrein secretion were
similar, suggesting that the two responses were coupled. Furthermore,
Ni2+ was able to block in a dose-dependent
manner the Bz-ATP-stimulated kallikrein secretion (data not shown).
These results suggest that iPLA2 was responsible for the
release of kallikrein in response to purinergic agonists. The
contribution of this phospholipase A2 to amylase secretion
from rat parotid acini (57) and to insulin secretion by pancreatic cells (58) has also been reported. This is consistent with the fact
that calcium is not required for the fusion of secretory granules with
the plasma membrane (59). Additionally, the presence of an
iPLA2 on the membrane of rat parotid secretory granules has
been reported (60). The stimulation by AACOCF3 of
kallikrein secretion by RSMG ductal cells (this report) or of amylase
by rat parotid acini (57) is more difficult to interpret. This response
was observed at concentrations higher than 10 µM. These
concentrations have no lytic effect (measured by the release of lactate
dehydrogenase or the release of Fura-2, data not shown). Considering
that 1) the activation of the two phospholipases A2 and the
hydrolysis of PC did not lead to an accumulation of intracellular
lysophospholipids, 2) lysophospholipids are known fusogens, 3)
cPLA2 is a strong lysophospholipase (it was recently shown
that cPLA2 could hydrolyze both sn1 and
sn2 isomers of palmitoylglycero-3-phosphocholine; Ref. 61),
4) AACOCF3 is an inhibitor of the lysophospholipase activity of cPLA2 (61), 5) the major role of
iPLA2 would be the remodeling of phospholipids in membranes
(49), and 6) macrophages expressing P2Z receptors
spontaneously fuse (62), we would like to propose the following model.
The iPLA2 would be activated by P2X7 agonists.
This enzyme, which has a weak specificity for arachidonyl-containing phospholipids, would release free fatty acids and sn1-acyl
lysophospholipids (probably sn1-acyl lysophosphorylcholine).
The lysophospholipids would favor the fusion between the membrane of
the secretory granule and the apical plasma membrane. These
lysophospholipids could be metabolized in two ways. 1) They might be
reacylated to phospholipids (the Lands cycle) by an acylase specific
for arachidonic acid. This would explain why a preincubation of the
RSMG ductal cells with BEL abolished the incorporation of
[3H]AA in the cellular phospholipids. 2) The
lysophospholipids might be hydrolyzed by the lysophospholipase activity
of cPLA2. According to this second hypothesis, the
secretory response to AACOCF3 could be secondary to the
accumulation of lysophospholipids generated by the constitutive
activity of iPLA2 and the inhibition of cPLA2 by AACOCF3. However, other hypotheses cannot be excluded.
It has been shown that AACOCF3 can be reduced to
AACOCH3, which, by itself, increased the stimulated level
of AA in cell-based assay and the production of
12-hydroxyeicosatetraenoic acid by ionophore-stimulated platelets (63).
The increased level of AA in response to these metabolites could
trigger exocytosis. Another hypothesis would be that
AACOCF3 behaves as an analog of AA and stimulates the secretory process, although in our hands the presence of AA did not
affect the basal secretion of kallikrein.
In conclusion, P2X7 agonists increase the release of
[3H]AA from RSMG ductal cells by activating two
phospholipases A2, a cPLA2 and an
iPLA2. The activation of the iPLA2 is
responsible for the secretion of kallikrein by ductal cells in response
to P2X7 activation.
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Abstract
Introduction
To whom all correspondence and reprint requests should be
addressed: Dept. de Bioquímica y Biología Molecular,
Facultad de Ciencias, Universidad del País Vasco/Euskal Herriko
Unibertsitatea, Apdo. 644, 48080 Bilbao, Spain. E-mail:
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