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INTRODUCTION |
Arachidonic acid (AA)1
and its eicosanoid metabolites (e.g. prostaglandins and
leukotrienes) play critical roles in the initiation or modulation of a
broad spectrum of biological responses, including many inflammatory
processes. In mammalian cells AA is normally stored in membrane
phospholipids and released primarily by phospholipase A2
(PLA2). Among several types of mammalian PLA2s,
the 85-kDa cytosolic form (cPLA2) appears to specifically
catalyze receptor-promoted AA release. Considerable efforts have been
made in recent years to study the mechanism for the activation of
cPLA2 and the subsequent release of AA. In a variety of
cell types, cPLA2 activation occurs as a result of
phosphorylation by mitogen-activated protein (MAP) kinase (1).
We have recently demonstrated that in MDCK-D1 cells
P2Y2 (previously termed P2U) purinergic
receptor-promoted AA release is mediated through cPLA2
activation by MAP kinase (2). Other data have shown that stimulation of
P2Y2 purinergic receptors in these cells also stimulates
adenylyl cyclase (AC) activity, increasing cellular cAMP levels through
an autocrine/paracrine mechanism involving prostaglandin E2
(PGE2) production subsequent to cPLA2-mediated
AA release (3, 4). In the present study we sought to assess the
relationship of these two signaling pathways activated by
P2Y2 purinergic receptors. We found that activation of the
AC system can inhibit cPLA2-mediated AA release by
P2Y2 purinergic receptors through the inhibition of MAP
kinase in MDCK-D1 cells. Therefore, we define a negative
feedback mechanism via an autocrine/paracrine cycle in which
P2Y2 receptors can attenuate the activation of
PLA2 and AA release initiated by receptor agonists.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
MDCK-D1 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% serum and
passaged every 3-4 days by trypsinization using trypsin/EDTA. Cells
were used for experiments when grown to approximately 70% confluence.
Assay of cAMP--
Growth medium was removed from cells, and
cells were equilibrated for 30 min at 37 °C in serum-free
Dulbecco's modified Eagle's medium containing 20 mM Hepes
buffer (DMEH, pH 7.4). Subsequently cells were incubated in fresh DMEH
with PGE2 for 5 min at 37 °C in the presence of 200 µM isobutylmethylxanthine or 100 µM
Ro20-1724, two different phosphodiesterase inhibitors. Reactions were
terminated by aspiration of medium and addition of 7.5%
trichloroacetic acid. Intracellular cAMP levels were determined by
radioimmunoassay (Calbiochem, CA) of trichloroacetic acid extracts
following acetylation, as described previously (3).
[3H]AA Release in Intact Cells--
Cells were
labeled with [3H]AA by incubation with 0.5 µCi of
[3H]AA (specific activity, 100 Ci/mmol; NEN Life Science
Products) per ml for approximately 20 h in 24-well plates. Cells
were washed four times with DMEH supplemented with 5 mg/ml bovine serum
albumin and then incubated in the same medium at 37 °C for 15-20
min to equilibrate the temperature. Agents of interest were then added in 1 ml of 37 °C medium after removing equilibration medium. Release of [3H]AA was assayed and normalized to the percentage of
incorporated radioactivity, as described previously (2).
cPLA2 Activity Assay of Cell
Lysates--
cPLA2 activity was assayed in lysates
prepared from cells incubated with various agents, as described
previously (2). Briefly, cells were incubated with indicated agonists
in DMEH for 10 min at 37 °C, washed with ice-cold buffer containing
250 mM sucrose, 50 mM Hepes, pH 7.4, 1 mM EGTA, 1 mM EDTA, phosphatase inhibitors (200 µM Na3VO4, 1 mM
levamisole) and protease inhibitors (500 µM
phenylmethylsulfonyl fluoride, 8 µM pepstatin, 16 µM leupeptin, and 1 mM diisopropyl
fluorophosphate), and then scraped into ice-cold buffer identical to
the washing buffer except that sucrose was omitted but 100 nM okadaic acid and 1 mM dithiothrietol were
added. Scraped cells were sonicated, and cell lysates (supernatants
after centrifugation at 4 °C for 10 min at 500 × g)
were assayed for cPLA2 using
1-stearoyl-2[14C]arachidonyl-L-3-phosphatidyl
choline as substrate in the assay buffer described above containing 10 mg/ml bovine serum albumin and 10 mM CaCl2.
Cell lysates (100 µl) were added to an equal volume of substrate in a
shaking 37 °C water bath so that final concentrations were, in
addition to phosphatase and protease inhibitors, 20 µM
1-stearoyl-2[14C]arachidonyl-L-3-phosphatidyl
choline, 5 mM CaCl2, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothrietol, 50 mM
Hepes, pH 7.4, and 10-30 µg of protein (determined by a Bradford
protein assay kit (Bio-Rad) with a bovine serum albumin standard).
Reactions were stopped by adding 0.75 ml of 1:2 (v/v)
chloroform/methanol. Samples were processed and assayed for
[14C]AA by thin layer chromatography, as described
previously (2).
Assay of Phospholipase D Activation--
MDCK-D1
cells were labeled by an overnight incubation with [3H]
palmitate. Washed cells were then incubated for 1 h with 20 µM indomethacin (to block PGE2 formation),
with 0.5 µM isobutylmethylxanthine for 20 min, with
20 µM PGE2 or 50 µM
isoproterenol for 20 min, and then with either 300 µM ATP
or 300 µM UTP for 10 min. Cells were lysed and
phosphatidylethanol was resolved by thin layer chromatography, as
described previously (5). Phosphatidylethanol was expressed as the
percentage of total cellular radioactivity.
Phosphorylation-induced Mobility Shift, SDS-Polyacrylamide Gel
Electrophoresis, and Immunoblotting of MAP Kinase--
Cells were
washed five times with DMEH supplemented with 2 mg/ml bovine serum
albumin, incubated in this medium at 37 °C for 1 h, and then
with specified agonists for 3 min. Reactions were stopped by aspiration
of medium and washing of cells four times with ice-cold buffer (62.5 mM Tris HCI, pH 6.8, plus 10% glycerol), and protease and
phosphatase inhibitors were used for PLA2 activity assays.
Scraped cells were lysed in SDS-polyacrylamide gel electrophoresis loading buffer and boiled for five min, and samples were
electrophoresed on SDS-polyacrylamide gel electrophoresis using 7.5%
acrylamide. Following transfer to an Immobilon-P polyvinylidene
fluoride membrane (Millipore) and blocking for 1 h with 5% nonfat
dry milk dissolved in phosphate-buffered saline, membranes were
incubated with 1:2000-3000 diluted anti-p42 MAP kinase rabbit serum
for 90 min and then with 1:2000 diluted horseradish-peroxidase-linked
donkey anti-rabbit immunoglobulin for 1 h (both in 5% nonfat dry
milk dissolved in phosphate-buffered saline) and then washed four times
with phosphate-buffered saline (5 min each wash). MAP kinase bands were
visualized using ECL immunoblotting detection reagents (Amersham
Pharmacia Biotech).
Data Presentation--
Unless otherwise specified, the data
shown in figures are the means ± S.D. of triplicate measurements
and are representative of results obtained in two to four experiments.
Results were analyzed for statistical significance by one-way analysis
of variance (with Bonferroni's correction, where appropriate).
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RESULTS |
Increase in Cellular cAMP Inhibits P2Y2
Receptor-promoted AA Release and cPLA2 Activation in
MDCK-D1 Cells--
We have previously demonstrated in
MDCK-D1 cells that activation of P2Y2
purinergic receptors results in AA release via activation of
cPLA2 (2) and that stimulation of P2Y2
purinergic receptors in these cells can increase cellular cAMP levels
via the action of the PGE2 generated from AA (3, 4). To
further investigate the relationship of these two signaling pathways
activated by P2Y2 receptors, we examined the effects of
cAMP-increasing agents on P2Y2 receptor-promoted AA
release. As shown in Fig. 1, agents that
increase cAMP in MDCK- D1 cells (PGE2,
forskolin, and Ro20-1724) inhibited AA release stimulated by the
P2 receptor agonists ATP and UTP. Isoproterenol also
inhibited the AA release stimulated by these purinergic agonists (data
not shown).

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Fig. 1.
Differential effects of PGE2 and
forskolin on AA release promoted by ATP, UTP, bradykinin, or
epinephrine. MDCK-D1 cells labeled with
[3H]AA were incubated with 10 µM
PGE2 (A) or 30 µM forskolin and/or
100 µM Ro20-1724 (B) for 20 min and then with
either medium (basal), 300 µM ATP (A only),
300 µM UTP, 50 µM epinephrine, or 1 µM bradykinin for 10 min to measure [3H]AA
release as described under "Experimental Procedures." *,
p < 0.05.
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PGE2-mediated stimulation of cAMP and inhibition of
P2Y2 receptor-promoted AA release displayed a similar
concentration-response relationship, with a nearly maximal effect of
PGE2 achieved at 1-10 µM both for production
of cAMP and inhibition of AA release (Fig.
2). Because P2Y2
receptor-promoted AA release in MDCK-D1 cells is mediated
by activation of cPLA2 (2), we tested whether an increase
in cAMP would blunt activation of this lipase. Indeed, as shown in Fig.
3, activation of PLA2
activity in cell lysates by the specific P2Y2 agonist UTP
was substantially inhibited by treatment of cells with
PGE2. Therefore, increases in cellular cAMP decrease AA
release and cPLA2 activation by P2Y2 receptors in MDCK-D1 cells.

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Fig. 2.
Concentration response of effects of
PGE2 on cAMP levels and 300 µM UTP-promoted AA release of
MDCK-D1 cells. Cells were assayed for
[3H]AA or in parallel for cAMP content as described under
"Experimental Procedures" and in the legend to Fig. 1. , AA;
, cAMP.
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Fig. 3.
Effect of PGE2 on UTP-promoted
activation of PLA2 in MDCK-D1
cells. MDCK-D1 cells were incubated with or
without 10 µM PGE2 for 20 min and then with
300 µM UTP for 10 min. Cell lysates were prepared, and
PLA2 activity was assayed as described under
"Experimental Procedures." The effect of PGE2 on
UTP-promoted PLA2 activity was statistically significant.
*, p < 0.05.
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MDCK-D1 cells possess
1-adrenergic receptors
and B2-bradykinin receptors that promote AA release in
response to stimulation by epinephrine and bradykinin, respectively
(8-10). As do P2Y2 receptors,
1-adrenergic
receptors and bradykinin receptors promote AA release through
activation of cPLA2 in MDCK-D1 cells (2, 3, 7).
Therefore, we tested whether increases in cAMP would regulate AA
release by these different receptors. Unlike the results obtained for
P2Y2 receptors, AA release elicited by
1-adrenergic receptors and bradykinin receptors was not
inhibited by forskolin, Ro20-1724 (an inhibitor of phosphodiesterase),
or both (Fig. 1B). Treatment with PGE2 alone or
with PGE2 and Ro20-1724 yielded similar results (data not
shown). Neither forskolin nor PGE2 stimulation of cAMP
accumulation was diminished by the presence of various concentrations
of epinephrine or bradykinin, as compared with the presence of ATP or
UTP (data not shown).
Activation of PKA and Inhibition of MAP Kinase Are Responsible for
the cAMP-mediated Inhibition of P2Y2 Receptor-promoted AA
Release--
To test whether the inhibitory effect of the increase in
cellular cAMP on P2Y2 receptor-promoted AA release and
cPLA2 activation in MDCK-D1 cells is mediated
by the activation of PKA, we examined the effect of the PKA inhibitor
H89 on PGE2-mediated inhibition of AA release. As shown in
Fig. 4, PGE2-mediated
inhibition of ATP- or UTP-stimulated AA release was completely
prevented by pretreatment of cells with H89, whereas H89 had no
statistically significant effect on basal or P2Y2
receptor-promoted AA release. These data suggest that activation of PKA
by cAMP is responsible for the inhibitory effects of increased cellular
cAMP levels on P2Y2 receptor-promoted AA release.

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Fig. 4.
Effect of H89 on PGE2-mediated
inhibition of AA release promoted by ATP and UTP.
[3H]AA-labeled MDCK-D1 cells were incubated
with either 10 µM PGE2 (20 min), 1.33 µM H89 (1 h prior to PGE2 incubation),
PGE2 plus H89, or with neither agent, and then with 300 µM ATP or UTP for 10 min to measure [3H]AA
release, as described under "Experimental Procedures." *,
p < 0.05 relative to control; #, p < 0.05 relative to PGE2 alone.
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Because MAP kinase plays a critical role in the activation of
cPLA2 and AA release in MDCK-D1 cells (2), we
next assessed whether activation of MAP kinase by P2Y2
receptors was inhibited by pretreatment of cells with PGE2.
Consistent with this idea were results with epinephrine, which also
activates MAP kinase activity in MDCK-D1 cells (7).
PGE2 blocked the MAP kinase activation by UTP but not that
by epinephrine (Fig. 5). As shown in Fig.
6, the UTP-induced gel shift of MAP
kinase was inhibited by incubation of cells with PGE2. This
PGE2-mediated inhibition of MAP kinase was reversed by
treatment of cells with H89, suggesting that activation of PKA is
responsible for both the cAMP-mediated inhibition of MAP kinase
activation and inhibition of AA release and cPLA2
activation. Based on these and previous data related to the role of MAP
kinase on P2Y receptor-promoted activation of
cPLA2 in MDCK-D1 cells, we conclude that the
cAMP/PKA system negatively regulates cPLA2 activated by
P2Y2 receptors through the inhibition of MAP kinase
activation.

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Fig. 5.
Effect of PGE2 on activation of
MAP kinase by UTP and epinephrine. MDCK-D1 cells were
incubated in the absence or presence of 10 µM
PGE2 for 20 min and then with 50 µM
epinephrine (Epi) or 300 µM UTP for 3 min. MAP
kinase activation was assessed as described under "Experimental
Procedures."
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Fig. 6.
Effects of PGE2 and H89 on
UTP-promoted activation of MAP kinase. MDCK-D1 cells
were incubated with 10 µM PGE2, 1.33 µM H89, or both as stated in the legend to Fig. 4 and
then with or without 300 µM UTP for 3 min. MAP kinase
activation was assessed as described under "Experimental
Procedures."
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P2Y Receptor Activation of Phospholipase D Activity Is
Not Inhibited by Elevation of cAMP--
In addition to activation
cPLA2, P2Y receptors can also increase
phospholipase D activity in MDCK-D1 cells (5). To determine whether the inhibitory effect of cAMP on cPLA2 activation
occurs at more upstream levels of the signaling cascade, such as at the level of receptor or G protein, we measured the effect of increasing cAMP on phospholipase D activity. The inability of increases in cAMP to
blunt ATP- and UTP-mediated phospholipase D activity (Fig. 7) argues that a more distal, nonshared
component, such as MAP kinase (Figs. 5 and 6), is the site of negative
regulation of cPLA2/AA release by increases in cAMP.

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Fig. 7.
Lack of effect of PGE2 and
isoproterenol on ATP- and UTP-promoted PLD activation.
MDCK-D1 cells were labeled with [3H]palmitate
and then incubated with 20 µM PGE2 and 50 µM isoproterenol prior to incubation with 300 µM ATP or 300 µM UTP and assayed for PLD
activity, as described under "Experimental Procedures."
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DISCUSSION |
Inhibitory effects of cAMP-increasing agents on AA release have
been observed in several previous studies (e.g. Refs.
11-13). However, the molecular mechanism for this phenomenon was not
defined in these earlier studies. We recently showed that
P2Y2 receptors utilize MAP kinase and protein kinase C
as parallel pathways for the activation of cPLA2 in
MDCK-D1 cells; blockade of either of these two pathways
impairs the activation of cPLA2 by P2Y2 receptor agonists (2). In the present study, we found that activation
of the AC/PKA system can inhibit P2Y2 receptor-promoted cPLA2 activation by inhibiting the MAP kinase signaling
pathway. This finding is consistent with the reports that cAMP and
cAMP-mediated activation of PKA can inhibit MAP kinase activation in
other types of cells (14-16).
AA release activated by P2Y2 receptors, but not activated
that by
1-adrenergic or B2-bradykinin
receptors, was inhibited by the AC/PKA system (Fig. 1). Because we have
found that MAP kinase appears not to be involved in the regulation of
AA release/cPLA2 activation by bradykinin receptors in
MDCK-D1 cells (6), we were not surprised by the absence of
inhibition of bradykinin receptor-promoted AA release by the AC/PKA
system. Another group has used MDCK-D1 cells and observed
slight inhibition of bradykinin receptor-promoted AA release by cAMP
(17). However, a similar inhibition also was noted of "basal" AA
release in that study. The absence of inhibition by the AC/PKA system
on
1-adrenergic receptor-promoted AA release was
unexpected, because MAP kinase activation is responsible for AA
release/cPLA2 activation by
1-adrenergic receptors in MDCK-D1 cells (7). The ability of the AC/PKA
system to inhibit P2Y2 receptor-mediated, but not
1-adrenergic receptor-mediated activation of MAP kinase
(Fig. 4), leads us to conclude that the differential inhibitory effects
of the AC/PKA system on AA release lies in differences in MAP kinase
activation by the two receptors. This conclusion is also supported by
studies showing a lack of inhibition by cAMP of P2
receptor-mediated activation of PLD activity (Fig. 7). Other data
indicate that MAP kinase activation is not required for PLD activation
by P2 receptors or
1-adrenergic receptors in
MDCK cells (18).2 We
speculate that P2Y2 purinergic and
1b-adrenergic receptors are coupled to cPLA2
and MAP kinase through different signaling pathways (perhaps via
different G proteins) that are differentially sensitive to increases in
cAMP. Alternatively, signaling components utilized by the two types of
receptors are compartmentalized such that increases in cAMP
selectively regulate components unique to the P2Y2 receptor pathway.
In conclusion, the present study demonstrates that the AC/PKA
system plays a negative role in the regulation of AA
release/cPLA2 activation by P2Y2 receptors
through inhibition of MAP kinase activation. This negative regulation
occurs for P2Y2 receptors but not for two other classes of
receptors coupled to cPLA2/AA release and is apparently
secondary to effects of cAMP/PKA to inhibit MAP kinase activation.
Because the P2Y2 receptor can activate the AC/PKA system by
promoting cPLA2-mediated release of AA and its subsequent
conversion to PGE2 (3, 4), our results define a feedback
cycle whereby P2Y2 receptors in MDCK-D1 cells
activate AA release and production of PGE2.
PGE2, in turn, activates the AC/PKA system and then
inhibits MAP kinase to decrease the AA signaling cascade. Such a cycle
could serve to produce homologous desensitization of the purinergic
receptor pathway in response to nucleotides and thus would blunt
ongoing production of AA and AA metabolites. Moreover, the cross-talk
that occurs between AC-stimulating pathways and the purinergic pathway
also represents a mechanism for the heterologous desensitization of the
P2Y purinergic receptor pathway. The feedback cycle
described herein may contribute to both physiologic and pharmacologic
regulation of the P2Y purinergic receptor signaling.
Overall, these results, together with evidence that P2Y2
receptors in MDCK-D1 cells are coupled to cAMP production via release of AA and its conversion to PGE2, define a
potentially important feedback loop for regulation of AA formation.