From the Division of Basic Science, Department of Pediatrics,
National Jewish Medical and Research Center, Denver, Colorado 80206 and
the Department of Pathology, University of Colorado School of Medicine,
Denver, Colorado 80262
Arachidonic acid release is induced
in macrophages with diverse agonists including calcium ionophores,
phorbol myristate acetate (PMA), okadaic acid, and the phagocytic
particle, zymosan, and correlates with activation of cytosolic
phospholipase A2 (cPLA2). The role of
calcium and phosphorylation of cPLA2 in regulating arachidonic acid release was investigated. Zymosan induced a rapid and
transient increase in [Ca2+]i. This in itself is
not sufficient to induce arachidonic acid release since ATP and
platelet activating factor (PAF), agonists that induce transient
calcium mobilization in macrophages, induced little arachidonic acid
release. Unlike zymosan, which is a strong activator of
mitogen-activated protein kinase (MAPK), ATP and PAF were weak MAPK
activators and induced only a partial and transient increase in
cPLA2 phosphorylation (gel shift). However, ATP or PAF
together with colony stimulating factor-1 (CSF-1) synergistically stimulated arachidonic acid release. CSF-1 is a strong MAPK activator that induces a rapid and complete cPLA2 gel shift but not
calcium mobilization or arachidonic acid release. Arachidonic acid
release was more rapid in response to CSF-1 plus ATP or PAF than
zymosan and correlated with the time course of the cPLA2
gel shift. Although low concentrations of ionomycin induced a lower
magnitude of calcium mobilization than ATP, the response was more
sustained resulting in arachidonic acid release. A23187 and ionomycin
induced weak MAPK activation, and a partial and transient
cPLA2 gel shift. The MAPK kinase inhibitor, PD 98059 suppressed A23187-induced MAPK activation and cPLA2 gel
shift but had little effect on arachidonic acid release. These results
indicate that in macrophages a transient increase in
[Ca2+]i and sustained phosphorylation of
cPLA2 can act together to promote arachidonic acid release
but neither alone is sufficient. A sustained increase in calcium is
sufficient for inducing arachidonic acid release. However, PMA and
okadaic acid induce arachidonic acid release without increasing
[Ca2+]i, although resting levels of calcium are
required, suggesting alternative mechanisms of regulation.
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INTRODUCTION |
The production of the proinflammatory lipid mediators, the
eicosanoids (i.e. prostaglandins and leukotrienes), is
dependent on the availability of the precursor, free arachidonic acid.
The release of arachidonic acid from the sn-2 position of
membrane phospholipid is a highly regulated process that occurs in
response to cell activation. Phospholipase A2
(PLA2)1 enzymes,
which cleave fatty acid from the sn-2 position of
phospholipid, play a central role in controlling the release of
arachidonic acid. The importance of the arachidonic acid-selective,
85-kDa cytosolic PLA2 (cPLA2) in mediating
agonist-induced release of arachidonic acid is now well recognized (1,
2). cPLA2 is regulated post-translationally by both
phosphorylation and calcium. Calcium plays a role by promoting binding
of cPLA2 to membrane, which is mediated by a
calcium-phospholipid-binding domain at the amino terminus of the enzyme
(3-5). Treatment of cells with calcium-mobilizing agonists has been
shown to induce binding of cPLA2 to nuclear membrane and
endoplasmic reticulum (6-8). Stimulation of a variety of cell types
with diverse agonists that induce arachidonic acid release also has
been shown to promote serine phosphorylation of cPLA2 that
is accompanied by an increase in cPLA2 activity and a
decrease in electrophoretic mobility (gel shift) (1, 9, 10).
cPLA2 can be phosphorylated by protein kinase C, p42/p44
mitogen-activated protein kinases (MAPK), or protein kinase A in
vitro but only phosphorylation by MAPK results in a significant increase in cPLA2 activity and induces a cPLA2
gel shift (10, 11). MAPK phosphorylates cPLA2 at Ser-505
and phosphorylation of this site has been shown to be required for
cPLA2-mediated arachidonic acid release in Chinese hamster
ovary cells treated with a variety of agonists (12).
Current evidence indicates that cPLA2 phosphorylation at
Ser-505 by MAPK in itself is not sufficient for arachidonic acid release (1). It has been shown in Chinese hamster ovary cells that
phosphorylation of cPLA2 induced by PMA is not sufficient for inducing arachidonic acid release, but PMA acts synergistically with calcium ionophore (10). In macrophages, CSF-1 induces
cPLA2 phosphorylation but not arachidonic acid release
although it can act synergistically with calcium mobilizing agonists
(13). It has been suggested that an increase in intracellular calcium
concentration [Ca2+]i, but not phosphorylation of
cPLA2, is essential for arachidonic acid release in rat
liver macrophages (14). In platelets, thrombin-stimulated arachidonic
acid release does not require phosphorylation of cPLA2 on
Ser-505 (15). Although these studies indicate an important role for an
increase in [Ca2+]i in regulating arachidonic
acid release, alternative mechanisms are indicated by results showing
that cPLA2-mediated arachidonic acid release induced by
okadaic acid occurs without an increase in
[Ca2+]i (16). In light of these observations,
mouse peritoneal macrophages were used as a model to investigate the
role of calcium and phosphorylation of cPLA2 in regulating
arachidonic acid release.
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EXPERIMENTAL PROCEDURES |
Materials--
[5,6,8,9,11,12,14,15-3H]Arachidonic
acid (100 Ci/mmol) and [32P]orthophosphoric acid were
from NEN Life Science Products Inc. Pathogen-free female ICR mice (8 weeks old) were from Harlan Sprague-Dawley. Fura-2-AM, Fluo-3-AM,
Quin-2-AM, and pluronic F-127 were from Molecular Probes. Anti-rabbit
IgG horseradish peroxidase-linked F(ab')2 fragment,
[32P]ATP (3000 Ci/mmol), and the ECL detection kit for
immunoblotting were from Amersham. Zymosan (yeast cell walls), A23187,
phenylmethylsulfonyl fluoride, probenecid, leupeptin, aprotinin, and
fetal bovine serum were from Sigma. Zymosan was prepared as described
previously (9). Okadaic acid and PMA were from LC Services Co.
Ionomycin was from Calbiochem-Novabiochem Co. CSF-1 was a gift from The Genetics Institute (Cambridge, MA). The MAPK kinase (MAPKK) inhibitor, PD 98059, was kindly provided by Dr. Alan Saltiel (Parke Davis Research
Division, Warner Lambert Co.). PAF was from Biomol Research Labs, Inc.
Sequencing grade, modified trypsin was obtained from Promega.
Dulbecco's modified Eagle's medium (DMEM) and Hank's balanced salts
solution were from Whittaker Bioproducts. Glass coverslips (13 mm
diameter) were from Fisher. Protein concentrations were determined
using the BCA reagent from Pierce. Polyclonal antibody (11683) to
recombinant human cPLA2 was produced as described previously (17). MAPK antibodies to the p44 (C-16) and p42 (C-14) isoforms that were used for immunoprecipitation were obtained from
Santa Cruz Biochemicals. Antibody that recognizes
tyrosine-phosphorylated p42 and p44 MAPK was obtained from New England
Biolabs.
Arachidonic Acid Release--
Resident mouse peritoneal
macrophages were isolated and labeled with
[3H]arachidonic acid as described previously (9). After
labeling, the cells were washed 3 times with DMEM and stimulated in 1 ml of DMEM with various agonists as indicated for the specific
experiments. For some experiments the [3H]arachidonic
acid-labeled macrophages were depleted of calcium by incubation in DMEM
containing 7.5 mM EGTA and 40 µM Quin-2-AM for 60 min (18, 19). The cells were washed and then treated with
agonists in fresh DMEM containing 7.5 mM EGTA. The medium was removed and centrifuged at 1400 × g in a Sorvall
RT 6000 refrigerated centrifuge for 10 min, and the cells were scraped
into 1 ml of 0.1% Triton X-100. The amount of radioactivity in the
cells and medium was measured by liquid scintillation spectrometry.
Immunoblotting--
After stimulation, the macrophages (6 × 106/35-mm dish) were lysed on ice with 100 µl of lysis
buffer A (50 mM Hepes, pH 7.4, 150 mM NaCl, 1.0 mM EGTA, 1.0 mM EDTA, 10% glycerol, 1% Triton X-100, 100 µM sodium orthovanadate, 10 mM
tetrasodium pyrophosphate, 100 mM sodium fluoride, 300 nM p-nitrophenyl phosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1.0 mM
phenylmethylsulfonyl fluoride). After incubation on ice for 30 min the
lysates were centrifuged for 15 min. The supernatant was boiled for 5 min in Laemmli buffer (20). For analysis of MAPK using phosphospecific antibodies, samples (20-40 µg of protein) were run on 10%
polyacrylamide gels and immunoblotted according to the manufacturers
instructions. For analyzing cPLA2 gel shift, samples (20 µg of protein) were resolved on 20-cm 10% SDS-polyacrylamide gels
(1% bisacrylamide, pH 8.3) and then transferred to a nitrocellulose
membrane. After blocking with 5% milk for 1-2 h, the membrane was
incubated overnight at 4 °C with anti-cPLA2 polyclonal
antibody at 1:2000 dilution in 20 mM Tris, pH 7.6, 137 mM NaCl, and 0.05% Tween (TTBS buffer) containing 5%
milk, followed by incubation with anti-rabbit IgG horseradish
peroxidase antibody (1:5000 dilution in TTBS) for 30 min at 25 °C.
The immunoreactive protein was detected using the Amersham ECL
system.
Determination of Intracellular Calcium by
Spectrofluorimetry--
Macrophages were plated on coverslips (13 mm
diameter) in 2 cm2 wells (24-well plate) at a density of
1 × 106 cells/well, and incubated for 3 h in a
humidified atmosphere of 10% CO2 in air at 37 °C. The
cells were washed twice with Ca2+- and
Mg2+-free Hank's balanced salts solution to remove
non-adherent cells and incubated overnight in DMEM containing 10%
fetal bovine serum. After rinsing twice with phosphate-buffered saline,
and once with phenol red-free DMEM containing 2.5 mM
probenecid (medium A), the cells were incubated for 1 h at
37 °C in medium A containing 5 µM Fura-2-AM and
0.025% pluronic F-127. The coverslips were rinsed twice with
phosphate-buffered saline and once with medium A, and kept in medium A
in the dark at room temperature until used for measuring
[Ca2+]i (within 1 h). Coverslips were placed
in a diagonal position in a standard 1-cm square quartz cuvette
containing 2 ml of Krebs-Ringer phosphate dextrose buffer (4.8 mM KCl, 0.93 mM CaCl2, 1.2 mM MgSO4, 3.1 mM
NaH2PO4, 12.5 mM
Na2HPO4, 120 mM NaCl, and 0.2%
dextrose) containing 2.5 mM probenecid. The cuvette was
fitted with a plastic O-ring to position the coverslip just above a
magnetic stirring bar. The cuvette was then placed in a SLM
8000TM C Photon Counting Spectrofluorimeter and maintained at 37 °C with continuous stirring. After equilibration for 5 min, the excitation ratio of 340/380 nm was recorded with an emission wavelength of 505 nm. [Ca2+]i was calculated
according to the equation, [Ca2+]i = Kd × b × (R-Rmin)/(Rmax-R),
where R is the measured fluorescence ratio (21).
Rmax, the maximum 340/380 nm ratio of Fura-2
fluorescence, was obtained by treating cells with 25 µM
digitonin. Rmin, the minimum 340/380 nm
fluorescence ratio was obtained by addition of 7.5 mM EGTA
to the medium. Kd is the dissociation constant of
the Fura-2·Ca2+ complex (225 nm), and b is the
ratio of fluorescence at 380 nm at 0 and saturating Ca2+
concentrations. The leakage of Fura-2 from the cells, which was determined by adding 4 mM MnCl2 to the assay
mixture, was found to be negligible.
Determination of Intracellular Calcium Changes by Confocal
Microscopy--
Macrophages were plated in a 24-well plate at a
density of 1 × 106/well, and loaded with 5 mM Fluo-3-AM under the same conditions as described above.
After loading, the cells were incubated in 200 µl of Krebs-Ringer
phosphate dextrose buffer containing 2.5 mM probenecid and
observed with a Bio-Rad Confocal microscope MIC 500 with Fitz filter.
After the basal calcium image was taken, 200 µl of Krebs-Ringer
phosphate dextrose containing agonists was added. Images were recorded
every 10 s.
MAPK Activity Assay--
Macrophages were plated at 10 × 106 cells/35-mm dish and stimulated as described above.
Cells were scraped on ice into 150 µl of lysis buffer: 20 mM Hepes, pH 7.4, 2 mM EGTA, 50 mM
-glycerophosphate, 1% Triton X-100, 10% glycerol, 200 µM sodium orthovanadate, 10 mM tetrasodium
pyrophosphate, 100 mM sodium fluoride, 3 µM
para-nitrophenyl phosphate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin. Lysates were precleared by incubation with 30 µl of
protein A-Sepharose beads (1:1 in lysis buffer) for 15 min at 4 °C.
Either p42 or p44 MAPKs were immunoprecipitated from the supernatants
by incubation with 0.5 µg of antibody and 12 µl of protein
A-Sepharose beads (1:1 in lysis buffer) for 2 h at 4 °C. The
beads were washed twice with lysis buffer and twice with 20 mM Hepes, pH 7.6, containing 200 µM sodium
vanadate, 20 mM magnesium chloride, and 2 mM
dithiothreitol (kinase buffer). Reactions were carried out in 50 µl
of kinase buffer containing 700 µM ATP, 200 µM epidermal growth factor receptor
peptide662-681 substrate (Macromolecular Resources, Fort
Collins, CO), 50 µg/ml cAMP-dependent protein kinase inhibitor IP-20,
and 20 µCi of [32P]ATP and incubated at 30 °C for 30 min. Reactions were stopped by brief centrifugation and addition of 13 µl of 50% trichloroacetic acid. A portion of the supernatant (30 µl) was spotted onto phosphocellulose P-81 filter discs, which were
then washed three times with 75 mM phosphoric acid (5 min)
and once with acetone, followed by Cerenkov counting.
Two-dimensional Phosphopeptide Mapping of 32P-Labeled
cPLA2 Tryptic Peptides--
32P-Labeled
cPLA2 was prepared in the Sf9 baculovirus expression
system as described previously (16). To prepare labeled
cPLA2 from macrophages, cells (40 × 106
cells/75 cm2 flask) were isolated and cultured overnight as
described previously (9). Two flasks were used for each treatment.
Cells were rinsed with phosphate-free minimal essential medium and then
incubated for 5 h in 10 ml of phosphate-free minimal essential
medium, containing 5% fetal bovine serum and
[32P]orthophosphoric acid (0.2 mCi/ml). The labeled cells
were treated with either vehicle (Me2SO) or okadaic acid
for 90 min. After stimulation, cells were rinsed with ice-cold
phosphate-buffered saline then lysed in 500 µl of ice-cold lysis
buffer A. Labeled cPLA2 was then immunoprecipitated from
cell lysates using a 1:50 dilution of antiserum for macrophage lysates
or a 1:7.5 dilution for Sf9 cell lysates as described previously
(16). Immunoprecipitated cPLA2 was separated on a 10%
SDS-polyacrylamide gel, detected by autoradiography, eluted from the
dried gel and precipitated from the gel elution buffer using
trichloroacetic acid as described previously (16). Recombinant human
cPLA2 (5 µg) was added as carrier protein to the
macrophage samples before trichloroacetic acid precipitation.
Trichloroacetic acid precipitates were resuspended in 200 µl of
freshly made 50 mM ammonium bicarbonate (pH 7.8-8.2), and
trypsin was added at a ratio of 1:10 (trypsin:cPLA2 by
weight). The samples were incubated for 3 h at 37 °C and then
washed by repeated addition of water and removal in a Speed Vac.
Samples were resuspended in acetic acid, and then thin layer
electrophoresis followed by ascending chromatography was carried out as
described previously (16).
 |
RESULTS |
Role of Calcium Mobilization in Regulating Arachidonic Acid
Release--
Arachidonic acid release can be induced in mouse
peritoneal macrophages by diverse agonists such as PMA, the calcium
ionophore, A23187, the phosphatase inhibitor, okadaic acid, and the
phagocytic stimulus, zymosan. This correlates with activation of
cPLA2 which becomes phosphorylated on serine residues
resulting in an increase in its activity. Experiments were carried out
to investigate the role of calcium in regulating arachidonic acid
release. Macrophages were loaded with Fluo-3-AM to investigate the
effect of agonists on changes in [Ca2+]i by
confocal microscopy. This technique allowed evaluation of individual
cells in the population. The effects of the agonists that induce
arachidonic acid release were compared with ATP, a known calcium
mobilizing agonist in macrophages (22, 23). Zymosan and ATP both
induced a rapid increase in [Ca2+]i as evidenced
by the increased fluorescence at 10 s (Fig.
1). The response of the population was
heterogeneous, with smaller changes evident by increases in blue
fluorescence to greater increases in [Ca2+]i in
the yellow to red range. By 50-90 s the [Ca2+]i
had diminished but still remained above control levels. As expected,
the response to A23187 was rapid, but in contrast to zymosan and ATP,
the increase in [Ca2+]i was more sustained and
had not diminished by 90 s. Neither okadaic acid nor PMA induced
an increase in [Ca2+]i (Fig. 1). The lack of
effect with PMA and okadaic acid was confirmed by spectrofluorimetric
measurements using Fura-2-AM loaded cells (data not shown).

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Fig. 1.
Increase of intracellular calcium in
macrophages determined by confocal microscopy. A basal level
calcium image of Fluo-3-labeled macrophages was taken and then the
cells were stimulated with zymosan (30 particles/cell), PMA (32 nM), okadaic acid (1 µM), A23187 (0.5 µg/ml), ATP (100 µM), PAF (100 nM), or
CSF-1 (1000 units/ml). An image of the same field of cells is shown at
various times after stimulation. As the intracellular calcium
concentration increases, the color changes to bright blue to
yellow-green to red. The results of a
representative experiment are shown and were verified in at least three
independent experiments.
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Although PMA and okadaic acid induced arachidonic acid release without
increasing intracellular calcium, it was possible that resting levels
of [Ca2+]i were required. Consequently, the
effect of depleting the cells of calcium by including EGTA in the
culture medium and loading the cells with Quin-2-AM on agonist-induced
arachidonic acid release was investigated (Fig.
2). Chelating just extracellular calcium
by incubating the macrophages in medium containing EGTA significantly
suppressed arachidonic acid release in response to all the agonists.
Loading the cells with Quin-2-AM in addition to chelating extracellular
calcium with EGTA further suppressed arachidonic acid release in
response to zymosan, A23187, and okadaic acid. The response to PMA was
not further affected in the Quin-2-AM loaded cells beyond the
suppression observed using EGTA alone. Since PMA and okadaic acid did
not induce an increase in [Ca2+]i, the inhibition
of arachidonic acid release by extracellular EGTA suggested that this
treatment was depleting the resting levels of Ca2+, and
that this was required for arachidonic acid release. Incubation of the
macrophages in medium containing EGTA was found to rapidly decrease
resting levels of [Ca2+]i when evaluated by
confocal microscopy or by fluorescence changes in Fura-2-AM loaded
cells (data not shown). These results suggest that although PMA and
okadaic acid do not induce an increase in
[Ca2+]i, maintaining the resting level of
[Ca2+]i is necessary for optimal arachidonic acid
release.

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Fig. 2.
Effect of chelating extracellular and
intracellular calcium on stimulated [3H]arachidonic acid
release. Macrophages labeled with [3H]arachidonic
acid were preincubated with 7.5 mM EGTA in the presence or
absence of 40 µM Quin-2-AM for 60 min followed by
stimulation with zymosan (30 particles/cell), PMA (32 nM),
or A23187 (0.5 µg/ml) for 60 min, or with okadaic acid (1 µM) for 90 min. The amount of
[3H]arachidonic acid released into the medium was
determined and expressed as a percentage of the total radioactivity
(cell-associated plus medium). Results are expressed as mean ± S.D. (n = 3) of a representative experiment and were
verified in three independent experiments.
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Neither a Transient Increase in Calcium nor cPLA2
Phosphorylation Are Sufficient for Arachidonic Acid
Release--
Experiments were carried out to determine the
contribution of calcium mobilization and cPLA2
phosphorylation in regulating arachidonic acid release. Agonists were
used that either induce strong MAPK activation leading to
cPLA2 phosphorylation, but with no calcium mobilization, or
conversely, that promote calcium mobilization but little MAPK
activation. A comparison of the effect of ATP and PAF on the magnitude
and time course of [Ca2+]i mobilization measured
spectofluorometrically in Fura-2-AM loaded macrophages is shown in Fig.
3. ATP induced an increase in
[Ca2+]i from 40 to 220 nM within
10 s but returned to baseline levels by 100 s. PAF also
induced a increase in [Ca2+]i to a slightly lower
magnitude than ATP but the response was consistently more sustained
(Fig. 3B). In contrast, CSF-1 did not promote an increase in
[Ca2+]i as measured both by confocal analysis
(Fig. 1) and spectrofluorometry (data not shown). The ability of these
agonists to activate p42 and p44 MAPKs and promote an increase in
cPLA2 phosphorylation was compared (Fig.
4). Activation of MAPKs was analyzed by
using antibody that recognizes the tyrosine-phosphorylated form of the
kinases. CSF-1 strongly activated p42 and p44 MAPKs by 5 min after
which there was a progressive decrease in activity although
considerable activation of p42 above control levels was still evident
by 60 min (Fig. 4A). In contrast, ATP and PAF weakly activated MAPKs by 5 min and activation was transient returning to near
control levels by 15 min. Consistent with the pattern of MAPK
activation, CSF-1 induced a rapid and complete gel shift of
cPLA2 that remained stable over time with only a small
portion of cPLA2 returning to the faster migrating form by
60 min (Fig. 4B). ATP and PAF did enhance cPLA2
phosphorylation by 5 min after stimulation but the gel shift was not
complete and was transient, with a significant portion of the
PLA2 returning to the faster migrating form by 30 min. In
comparison, zymosan induced a delayed increase in cPLA2
phosphorylation with a near complete gel shift evident by 30 min. This
is consistent with a delayed activation of MAPK by zymosan as
previously reported (13, 24). The effect of these agonists on the time
course of arachidonic acid release is shown in Fig.
5. CSF-1 alone did not stimulate
arachidonic acid release and the response to ATP was very weak. PAF
induced a small, consistent release of arachidonic acid. However, CSF-1 together with either ATP or PAF induced a rapid, synergistic release of
arachidonic acid that plateaued 5 min after agonist treatment. In
contrast, the response to zymosan was delayed by at least 10 min after
which arachidonic acid release continued to accumulate in the medium up
to 60 min. These results suggest that when there is a transient
increase in calcium the time course of arachidonic acid release
correlates with the time course of cPLA2 activation by
phosphorylation.

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Fig. 3.
Magnitude and time course of
[Ca2+]i mobilization by ATP and PAF.
Macrophages were loaded with Fura-2-AM and the change of
[Ca2+]i was measured spectrofluorometrically
after addition of: A, 100 µM ATP or
B, 100 nM PAF. A representative experiment is
shown. The results were verified in at least three independent
experiments.
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Fig. 4.
Time course of MAPK activation and
phosphorylation of cPLA2. Macrophages were stimulated
for the times indicated with ATP (100 µM), PAF (100 nM), CSF-1 (1000 units/ml), or zymosan (30 particles/cell).
Whole cell lysates were analyzed for p42 and p44 MAPK using
phosphospecific antibody (A) or cPLA2
(B) by Western blotting as described under "Experimental
Procedures."
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Fig. 5.
The time course of
[3H]arachidonic acid release.
[3H]Arachidonic acid-labeled macrophages were exposed to
zymosan (30 particles/cell), ATP (100 µM), PAF (100 nM), CSF-1 (1000 units/ml), ATP plus CSF-1, or PAF plus
CSF-1. The amount of label released into the medium at various times
after agonist treatment was determined and expressed as a percentage of
the total radioactivity (cells associated plus medium). The results are
expressed as mean ± S.D. (n = 3) of a
representative experiment and were verified in two independent
experiments.
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A Sustained Increase in Calcium Is Sufficient for Inducing
Arachidonic Acid Release--
In contrast to ATP and PAF, agonists
that induce a sustained increase in [Ca2+]i such
as the calcium ionophores A23187 and ionomycin promote relatively large
amounts of arachidonic acid release. Ionomycin (which does not
autofluoresce as is the case for A23187) was used to correlate the
concentration dependence of [Ca2+]i mobilization
versus arachidonic acid release in the macrophages (Fig.
6A). A concentration-dependent
accumulation of arachidonic acid in the medium occurred with ionomycin
from 5 to 500 nM that correlated with its ability to induce
an increase in [Ca2+]i (Fig. 6B).
Considerably more arachidonic acid was released with 20-50
nM ionomycin compared with ATP (or PAF) even though the
magnitude of the calcium change at these concentrations was less than
with ATP. However, the increase in [Ca2+]i with
ionomycin was considerably more sustained than with ATP, which is
consistent with the influx of extracellular Ca2+ triggered
by the ionophore. Ionomycin also induced a partial gel shift of
cPLA2 (Fig. 6C). This is consistent with our
previous observation that A23187 treatment induced an increase in
cPLA2 activity in macrophages which was reversed by
phosphatase treatment, although the magnitude of cPLA2
activation was less than with other arachidonic acid mobilizing
agonists (PMA, zymosan, and okadaic acid) (9). Consequently the ability
of A23187 to induce a cPLA2 gel shift at various times
after agonist treatment was determined and compared with the time
course of arachidonic acid release (Fig.
7). A23187 induced a partial
cPLA2 gel shift by 5 min that increased slightly by 10 min,
however, the gel shift was not complete and it was transient.
Arachidonic acid release induced by A23187 was evident by 5 min and
continued to accumulate in the medium up to 60 min.

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Fig. 6.
Concentration-dependent stimulation of
arachidonic acid release, calcium mobilization, and cPLA2
gel shift by ionomycin. A, [3H]arachidonic
acid-labeled macrophages were exposed to the indicated concentrations
of ionomycin for 60 min. The amount of label released into the medium
was determined and expressed as a percentage of the total radioactivity
(cells associated plus medium). Results are expressed as mean ± S.E. of three independent experiments assayed in triplicate.
B, adherent macrophages were loaded with Fura-2-AM and the
change of [Ca2+]i was measured
spectrofluorometrically after addition of the indicated concentrations
of ionomycin. A representative experiment is shown and the results were
verified in two independent experiments. C, macrophages were
stimulated with ionomycin for 30 min and lysates analyzed for
cPLA2 by Western blotting as described under
"Experimental Procedures." A representative experiment is shown and
the results were verified in two independent experiments.
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Fig. 7.
The time course of A23187-induced arachidonic
acid release and cPLA2 phosphorylation.
[3H]Arachidonic acid-labeled macrophages were exposed to
A23187 (0.5 µg/ml) for the indicated times and the amount of label
released into the medium was determined and expressed as a percentage
of the total radioactivity (cell associated plus medium). The
inset shows a Western blot of cPLA2 at various
times after treatment of the macrophages with and without A23187 (0.5 µg/ml). A representative experiment is shown and the results were
verified in two independent experiments.
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Although a cPLA2 gel shift is characteristic of
phosphorylation by MAPK, we had previously reported that A23187 induced
only a very low, inconsistent activation of MAPK (24). This was
investigated in greater detail using a more specific and sensitive
assay for measuring p42 and p44 MAPK activity which involved
immunoprecipitating the specific kinases and measuring kinase activity
in an in vitro assay using an epidermal growth factor
receptor peptide substrate (Fig.
8A). Zymosan, PMA, and okadaic
acid all induced a 25-fold or greater increase in p42 MAPK activity and
a 5-12-fold increase in p44 MAPK activation. A23187 induced a
relatively low increase in p42 MAPK activation but no detectable
increase in p44 MAPK above unstimulated controls. The time course of
p42/p44 activation was evaluated by Western blot analysis using an
antibody specific for tyrosine-phosphorylated MAPK (Fig.
8B). Consistent with the results above, A23187 primarily
activated p42 MAPK, and the response was weak compared with MAPK
activation by PMA. In addition, MAPK activation by A23187 was transient
and returned to near baseline by 30 min. The weak activation of p42
MAPK by A23187 could be quantitatively inhibited by the MAPKK
inhibitor, PD 98059 (90 and 100% inhibition in two experiments).
Consistent with this observation, PD 98059 inhibited the
cPLA2 gel shift induced by A23187 (Fig.
9). However, arachidonic acid release
induced by A23187 was only slightly inhibited by PD 98059 (15%
inhibition at 10 µM PD98059). At 50 µM
PD98059, arachidonic acid release was slightly enhanced. These results
suggest that phosphorylation at Ser-505 is not necessary for
arachidonic acid release in these cells when there is a sustained
increase in [Ca2+]i.

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Fig. 8.
Activation of p42 and p44 MAPKs in
macrophages. A, macrophages were treated with zymosan (30 particles/cell, 15 min), PMA (32 nM, 5 min), okadaic acid
(1 µM, 90 min), or A23187 (0.5 mg/ml, 5 min). The p42 and
p44 MAPK isoforms were immunoprecipitated from cell lysates and their
activity measured in an in vitro kinase assay with epidermal
growth factor receptor peptide as a substrate. Phosphorylated peptide
was quantitated on phosphocellulose filter discs as described under
"Experimental Procedures." B, macrophages were treated
with A23187 (0.5 µg/ml) or PMA (32 nM) for the indicated
times and lysates analyzed for p42 and p44 MAPK by Western blotting
using phosphospecific antibody as described under "Experimental
Procedures." Representative experiments are shown and the results
were verified in at least two independent experiments.
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Fig. 9.
Effect of the MAPKK inhibitor, PD98059, on
inhibition of A23187-induced cPLA2 gel shift and
arachidonic acid release. [3H]Arachidonic
acid-labeled macrophages were preincubated with various concentrations
of PD98059 for 30 min and then treated with A23187 (0.5 µg/ml) for 60 min. The amount of label released into the medium was determined and
expressed as a percentage of the total radioactivity (cell associated
plus medium). The results are expressed as mean ± S.E. of three
independent experiments. Inset, macrophages were
preincubated with 50 µM PD 98059 for 1 h and then
treated with A23187 (0.5 µg/ml) for 15 min. Cell lysates were
prepared and analyzed by Western blotting as described under
"Experimental Procedures." PD 98059 at 10 µM
similarly inhibited the cPLA2 gel shift. A representative
experiment is shown and the results were verified in three independent
experiments.
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Okadaic Acid Induces Phosphorylation of Unique Sites on
cPLA2 in Macrophages--
The results with PMA and okadaic
acid suggest that alternative mechanisms can regulate arachidonic acid
release in macrophages since these agonists act without increasing
[Ca2+]i. We have previously reported that when
cPLA2 is expressed in insect cells using baculovirus,
cPLA2-mediated arachidonic acid release can be induced by
okadaic acid and this occurs without an increase in
[Ca2+]i (16). In the Sf9 model, okadaic
acid induces a predominant increase in phosphorylation of
cPLA2 on Ser-727. In the macrophage model, okadaic acid
induced a greater decrease in electrophoretic mobility of
cPLA2 than the characteristic shift due to phosphorylation of Ser-505 seen with other agonists such as PMA (Fig.
10). This suggests that okadaic acid
induces phosphorylation of additional sites on cPLA2 in
macrophages. Experiments were carried out to investigate whether
okadaic acid induced phosphorylation of Ser-727 in macrophages. Cells
were labeled with [32P]orthophosphate, stimulated with
okadaic acid, and tryptic peptides of the immunoprecipitated
32P-labeled cPLA2 analyzed by two-dimensional
phosphopeptide mapping. Three predominant cPLA2
phosphopeptides were evident from unstimulated macrophages (Fig.
11A). Analysis of
32P-labeled cPLA2 from okadaic acid-stimulated
macrophages showed the appearance of new phosphopeptides, particularly
peptide 1, and an increase in labeling of other peptides. The
Ser-727-containing phosphopeptide from okadaic acid-treated Sf9
cells (peptide 1, Fig. 11C) was found to comigrate with
peptide 1 from okadaic acid-treated macrophages (Fig. 11D).
These results suggest that Ser-727 is phosphorylated on
cPLA2 in response to okadaic acid in the macrophages. The
sequence of the tryptic peptide containing Ser-727 is identical in
cPLA2 from mouse and human allowing this comparison to be
made.

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Fig. 10.
Agonist induced gel shift of
cPLA2. Macrophages were stimulated with okadaic acid
(1 µM) for 90 min or PMA (32 nM) for 5 min
and lysates analyzed for cPLA2 by Western blotting as
described under "Experimental Procedures." A representative
experiment is shown and the results were verified in at least three
independent experiments.
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Fig. 11.
Comparison of two-dimensional tryptic
phosphopeptide maps of 32P-labeled cPLA2 from
Sf9 cells and macrophages. Tryptic digests of
immunoprecipitated, gel-purified cPLA2 from unstimulated
macrophages (MØ/US), okadaic acid-stimulated macrophages
(MØ/OA), okadaic-stimulated Sf9 cells
(Sf9/OA), or from both okadaic-stimulated Sf9
cells and okadaic acid-stimulated macrophages co-spotted on the same
plate (Sf9/MØ/OA) were separated by two-dimensional
phosphopeptide mapping as described under "Experimental
Procedures." Electrophoresis was run in the horizontal
dimension with the anode on the left and chromatography was
run in the vertical dimension. The sample origin was below
the lower right-hand corner of the chromatographs (not
shown).
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DISCUSSION |
Arachidonic acid plays an important role as a second messenger and
as a precursor of inflammatory lipid mediators, consequently its levels
in cells are tightly regulated. The results of this study confirm that
arachidonic acid release can be regulated in an agonist-dependent
manner by diverse mechanisms in mouse peritoneal macrophages. Previous
work has shown that agonist-induced arachidonic acid release is largely
a PLA2-mediated process in these cells (25, 26). We have
previously reported that a variety of agonists that induce arachidonic
acid release in peritoneal macrophages activate cPLA2 by
serine phosphorylation through a MAPK-dependent mechanism
(9, 24). In this study, agonists that act by diverse mechanisms were
used to investigate the contribution of calcium and cPLA2
phosphorylation on Ser-505 in regulating arachidonic acid release in
this cell model. Phosphorylation of cPLA2 on Ser-505 was
evaluated by determining the ability of agonists to induce a
cPLA2 gel shift. Current evidence indicates that the gel
shift is due to phosphorylation on Ser-505 and that the extent of the gel shift can be used to evaluate the stoichiometry of phosphorylation at this MAPK site (10). We previously suggested that the okadaic acid-induced gel shift of cPLA2 in insect cells may be due
to phosphorylation of Ser-727. However, recent data shows that
expression in Sf9 cells of cPLA2 containing the
S727A mutation, but not cPLA2 containing the S505A
mutation, exhibits a gel shift in response to okadaic
acid.2
Using physiological agonists for macrophages, the results demonstrate
that neither a transient increase in [Ca2+]i, as
induced by ATP, nor phosphorylation of cPLA2 on Ser-505
alone, as induced by CSF-1, are sufficient for inducing arachidonic
acid release. We have previously shown that CSF-1 increases
phosphorylation and activity of cPLA2 in macrophages but is
not able on its own to induce arachidonic acid release (13). In the
present study, CSF-1 was confirmed not to induce an increase in
[Ca2+]i but did promote a sustained,
stoichiometric phosphorylation of cPLA2 on Ser-505. The
calcium-mobilizing agonists ATP and PAF were found to be weak MAPK
activators and induced only a partial and transient phosphorylation of
cPLA2 on Ser-505. ATP induces a transient increase in
[Ca2+]i and this together with its weak ability
to promote phosphorylation of cPLA2 were insufficient to
induce arachidonic acid release. However, the transient increase in
[Ca2+]i together with sustained, stoichiometric
phosphorylation of cPLA2 on Ser-505 as induced by CSF-1 act
together to synergistically promote arachidonic acid release. Zymosan
induces both a transient increase in calcium and stoichiometric
phosphorylation of cPLA2 on Ser-505 and is a potent inducer
of arachidonic acid release. In addition, the time course of
arachidonic acid release correlated with the time course of
cPLA2 phosphorylation on Ser-505 which was very rapid for
CSF-1 and delayed for zymosan.
Compared with ATP, PAF consistently induced a low level of arachidonic
acid release. PAF led to a similar degree of MAPK activation and
cPLA2 phosphorylation as ATP but it induced a more
sustained increase in [Ca2+]i. In macrophages,
PAF has been reported to induce a biphasic increase in
[Ca2+]i, an initial phase due to release from
intracellular stores and a second phase, due to influx of extracellular
calcium (27). The results suggest that the more sustained increase in calcium is contributing to the ability of PAF to induce a low level of
arachidonic acid release. In several models, arachidonic acid release
has been linked to the influx of extracellular calcium (28-31). From
results using rat liver macrophages, it has been suggested that an
increase in [Ca2+]i, but not cPLA2
phosphorylation, is necessary for arachidonic acid release (14).
However, our results show that cPLA2 phosphorylation on
Ser-505 is required in macrophages when there is a transient increase
in [Ca2+]i but that it is not essential when
there is a sustained increase in [Ca2+]i from
extracellular sources as induced by the calcium ionophores. It has been
shown in mast cells that a larger fraction of cPLA2
translocates to nuclear membrane in response to calcium ionophore, and
it remains on the membrane longer than when there is only a transient
increase in calcium as occurs with IgE/antigen (8). Although the
calcium ionophores induce a partial, transient cPLA2 gel
shift, the results suggest that this phosphorylation contributes little
to arachidonic acid release since inhibition of ionophore-induced MAPK
activation and phosphorylation of cPLA2 on Ser-505 by the
MAPKK inhibitor has no effect on arachidonic acid release. However, our
previous results have shown that CSF-1 can augment A23187-induced
arachidonic acid release in the macrophages (13). This suggests that
the phosphorylation of Ser-505 must be stoichiometric and more
sustained as occurs with CSF-1 to augment A23187-induced arachidonic
acid release. These results suggest that a sustained increase in
intracellular calcium induced by A23187 may be sufficient for
arachidonic acid release. Although we cannot rule out the possibility
that A23187 induces phosphorylation of a site that does not lead to a
gel shift but may contribute to activation, this is unlikely since we
have previously shown that A23187 induces only a small increase
(10-15%) in 32P labeling of cPLA2 in the
macrophages (9).
The results using PMA and okadaic acid demonstrate that arachidonic
acid release can be induced in macrophages without an increase in
[Ca2+]i. However, chelating extracellular and
intracellular calcium did suppress arachidonic acid release by these
agonists, suggesting that resting levels of calcium may be important.
Both of these agonists induce stoichiometric phosphorylation of
cPLA2 on Ser-505 but since this is not sufficient for
inducing arachidonic acid release without an increase in
[Ca2+]i, other regulatory events are implicated.
The ability of PMA on its own to induce arachidonic acid release is
unique to certain cell types such as macrophages and neutrophils (9, 32). In many cell types it is only effective when combined with a
calcium mobilizing agonist (10). The mechanisms involved in regulating
PMA-induced arachidonic acid release in macrophages are currently
unknown. In contrast, we have previously shown that okadaic acid
predominantly induces phosphorylation of cPLA2 on a novel
site (Ser-727) when it is expressed in insect cells. Okadaic acid
induces arachidonic acid release in the Sf9 model without an
increase in [Ca2+]i (16). In the macrophage
model, the gel shift pattern of cPLA2 from okadaic
acid-stimulated cells suggests that it is also phosphorylated on unique
sites in addition to Ser-505. In addition, analysis of
cPLA2 from okadaic acid-stimulated macrophages by
two-dimensional phosphopeptide mapping suggests that it is phosphorylated on Ser-727. Studies are underway to determine the functional relevance of phosphorylation on Ser-727.