(Received for publication, October 27, 1995; and in revised form, December 27, 1995)
From the
Receptor-stimulated arachidonic acid (AA) mobilization in
P388D macrophages consists of a transient phase in which AA
accumulates in the cell and a sustained phase in which AA accumulates
in the incubation medium. We have shown previously that a secretory
group II phospholipase A
(sPLA
) is the enzyme
responsible for most of the AA released to the incubation medium. By
using selective inhibitors for each of the PLA
s present in
P388D
macrophages, we demonstrate herein that the cytosolic
group IV PLA
(cPLA
) mediates accumulation of
cell-associated AA during the early steps of P388D
cell
activation. The contribution of both cPLA
and sPLA
to AA release can be distinguished on the basis of the different
spatial and temporal characteristics of activation and substrate
preferences of the two phospholipase A
s (PLA
s).
Furthermore, the results suggest the possibility that a functionally
active cPLA
may be necessary for sPLA
to act.
cPLA
action precedes that of sPLA
, and
overcoming cPLA
inhibition by artificially increasing
intracellular free AA levels restores extracellular AA release.
Although this suggests cross-talk between cPLA
and
sPLA
, selective inhibition of one other PLA
present in these cells, namely the
Ca
-independent PLA
, does not block, but
instead enhances receptor-coupled AA release. These data indicate that
Ca
-independent PLA
does not mediate AA
mobilization in P388D
macrophages. Collectively, the
results of this work suggest that each of the PLA
s present
in P388D
macrophages serves a distinct role in cell
activation and signal transduction.
Phospholipase A (PLA
) (
)enzymes play a fundamental role in numerous cellular
processes by generating an array of metabolites with various biological
functions. PLA
-mediated hydrolysis of glycerophospholipids
results in the release of arachidonic acid (AA) and lysophospholipids,
which may either exert direct effects or serve as substrates for the
generation of other lipid messengers such as the eicosanoids or
platelet-activating factor (PAF)(1) .
Mammalian cells
contain multiple PLA forms(1) , and there is
considerable interest in determining the role that each PLA
plays in mediating cellular functions. At least three different
cellular PLA
s have been proposed to play a role in the
mobilization of AA from phospholipids. These are the cytosolic group IV
PLA
(cPLA
)(2, 3, 4) ,
the secretory group II PLA
(sPLA
)(5, 6, 7) , and a
cytosolic Ca
-independent PLA
(iPLA
)(8, 9) . Involvement of one or
another PLA
form appears to depend on the cell type and
agonist involved.
Our laboratory has been examining the molecular
mechanisms involved in AA mobilization in murine P388D macrophage-like cells (6, 10, 11, 12) . Stimulation of
these cells with nanomolar quantities of the receptor agonist PAF
results in a very modest mobilization of free AA. However,
preincubation of the cells with bacterial lipopolysaccharide (LPS)
prior to stimulation with PAF increases the release of AA by these
cells by about 2-3-fold(10) . Recently, we have
demonstrated that AA mobilization in response to LPS/PAF involves
participation of a sPLA
localized at the outer surface of
the cell and that this enzyme accounts for the majority of the AA
released to the extracellular medium(6, 12) . In the
current study, we have obtained further evidence using chemical
inhibitors for the role of sPLA
and have aimed at defining
the roles played by the other two PLA
s present in
P388D
macrophages, namely cPLA
and
iPLA
.
Figure 1:
Different AA pools in
P388D cells. LPS-treated cells labeled with both
[
H]AA and [
C]AA were
stimulated with 100 nM PAF for 10 min to measure extracellular
AA or for 1.5 min to measure cell-associated AA. The ratio
C/
H of extracellular AA (E) or
cell-associated AA (C) was quantitated and is shown. The
C/
H ratio for the major AA-containing
phospholipid classes in these cells is also shown for comparison. PS, phosphatidylserine.
Subsequent to
the labeling, the cells were activated with PAF, and the
[C]/[
H] ratios were
determined in the phospholipid classes as well as in the AA liberated
at the two different locations. Cell-associated free AA and
extracellular free AA had very different
[
C]/[
H] ratios, indicating
that the AA released at these two locations was derived from different
pools (Fig. 1). The
[
C]/[
H] ratio for
extracellular free AA had a ratio well below those of PC and
PI/phosphatidylserine, but close to that of PE (Fig. 1). This
suggested that PE may be a major source for the AA released to the
extracellular medium. Consistent with this view, the
C/
H ratio for extracellular free AA in
unstimulated cells was 0.7 ± 0.1, that is, slightly higher than
that observed in PAF-activated cells (0.4 ± 0.1). In contrast,
the [
C]/[
H] ratio for
cell-associated free AA was intermediate between that of PE and those
of PC and PI/phosphatidylserine (Fig. 1), suggesting that
cell-associated free AA has been derived from all of these phospholipid
classes. Within error, there was no difference between the
C/
H ratio for cell-associated AA in
unstimulated cells (1.0 ± 0.2) versus PAF-stimulated
cells (0.8 ± 0.1), suggesting that intracellular resting levels
of AA may also derive from all major phospholipid classes.
Using antisense RNA technology, we have previously demonstrated that
group II sPLA is responsible for at least 60-70% of
the AA released to the incubation medium but is not involved in raising
cellular AA levels shortly after cell activation with PAF(12) .
In the current study, pharmacological inhibition of sPLA
was accomplished by incubating the cells either with the
water-soluble phospholipid analog, diC
SNPE(14) , or
the indole derivative LY311727, which is an indomethacin
analogue(18) . diC
SNPE inhibits human synovial
group II PLA
with an IC
of 27 µM when assayed in a spectrophotometric assay with 2 mM substrate. (
)At concentrations up to 100
µM, diC
SNPE has no effect on pure human group
IV cPLA
, nor does it affect PLA
activity from
P388D
cell homogenates as measured toward
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine
vesicles in the presence of Ca
and
-mercaptoethanol. (
)In addition, diC
SNPE
does not inhibit pure Ca
-independent PLA
from P388D
cells(13) . The properties of the
indole derivative LY311727 as a potent and selective inhibitor of
sPLA
have recently been reported(18) .
sPLA inhibition by either diC
SNPE or
LY311727 markedly decreased the extracellular release of
[
H]AA from prelabeled P388D
cells (Fig. 2A and Fig. 3A). No effect of
these inhibitors was detected on the accumulation of cell-associated
free [
H]AA (Fig. 2B and
3B). These data are fully consistent with our previous data
using antisense RNA technology to block sPLA
activity (12) .
Figure 2:
Effect of diCSNPE on
PAF-stimulated [
H]AA mobilization in
P388D
cells. [
H]AA-labeled
LPS-treated cells were incubated with the indicated concentrations of
diC
SNPE for 15 min. Subsequently, the cells were incubated
with (
) or without (
) 100 nM PAF for either 10 (A) or 1.5 (B) min. Extracellular
[
H]AA release (A) and cell-associated
[
H]AA (B) were quantitated as described
under ``Experimental
Procedures.''
Figure 3:
Effect of LY311727 on PAF-stimulated
[H]AA mobilization in P388D
cells.
[
H]AA-labeled LPS-treated cells were incubated
with the indicated concentrations of LY311727 for 15 min. Subsequently,
the cells were incubated with (
) or without (
) 100 nM PAF for either 10 (A) or 1.5 (B) min.
Extracellular [
H]AA release (A) and
cell-associated [
H]AA (B) were
quantitated as described under ``Experimental
Procedures.''
Involvement of group IV cPLA was
initially investigated by using MAFP(19) . This compound is an
irreversible inhibitor of the cPLA
and has no effect on the
sPLA
(19) . We have confirmed in our laboratory
these findings and in addition have found that MAFP does not
appreciably affect arachidonoyl-CoA synthetase,
lysophosphatidylcholine:arachidonoyl-CoA acyltransferase, or
CoA-independent transacylase activities in homogenates from
MAFP-treated cells. Fig. 4shows that MAFP strongly inhibited AA
mobilization in PAF-activated cells. Whereas MAFP inhibited the
extracellular release of [
H]AA from prelabeled
cells by about 75% (Fig. 4A), the PAF-induced
accumulation of cellular [
H]AA was almost
completely blocked by the inhibitor (Fig. 4B).
Figure 4:
Effect of MAFP on PAF-stimulated
[H]AA mobilization in P388D
cells.
[
H]AA-labeled LPS-treated cells were incubated
with the indicated concentrations of MAFP for 15 min. Subsequently, the
cells were incubated with (
) or without (
) 100 nM PAF for either 10 (A) or 1.5 (B) min.
Extracellular [
H]AA release (A) and
cell-associated [
H]AA (B) were
quantitated as described under ``Experimental
Procedures.''
P388D macrophages possess a third PLA
enzyme, namely a cytosolic iPLA
that shows no
preference for AA-containing phospholipids; in fact, it prefers
palmitoyl over arachidonoyl residues(20) . Recent evidence from
our laboratory indicates that MAFP also inhibits pure iPLA
from P388D
cells. (
)Therefore, at least
part of the MAFP-sensitive AA mobilization could be mediated by the
iPLA
in addition to the cPLA
. The iPLA
from P388D
macrophages is potently and irreversibly
inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one
(bromoenol lactone, BEL)(13) . This compound manifests over a
1000-fold selectivity for inhibition of the iPLA
s versus the Ca
-dependent sPLA
s (21) and has previously been used to investigate the role of
iPLA
in AA release in certain cell
types(8, 9) . In our laboratory, we have found that
BEL is a poor inhibitor of pure cPLA
. (
)This
inhibitor does not affect any of the following activities, measured in
homogenates from BEL-treated cells: cPLA
, sPLA
,
arachidonoyl-CoA synthetase, lysophosphatidylcholine:arachidonoyl-CoA
acyltransferase, and CoA-independent transacylase(22) .
The
effect of BEL on PAF-induced AA mobilization from LPS-primed
P388D cells is shown in Fig. 5. At concentrations up
to 50 µM, which totally block cellular
iPLA
(22) , BEL was ineffective in inhibiting either
the extracellular release of [
H]AA (Fig. 5A) or the accumulation of cellular free fatty
acid (Fig. 5B). Instead, BEL enhanced both basal and
PAF-stimulated [
H]AA mobilization, although the
ratio of stimulated versus unstimulated release remained the
same at all BEL concentrations. The enhancing effect of BEL on
P388D
macrophage AA release is probably related to its
inhibitory action on cellular fatty acid incorporation into
phospholipid(22) . The lack of any inhibitory effect of BEL on
[
H]AA release demonstrates that the iPLA
does not significantly contribute to this release. Therefore, the
MAFP-sensitive [
H]AA release should be ascribed
to the cPLA
.
Figure 5:
Effect of BEL on PAF-stimulated
[H]AA mobilization in P388D
cells.
[
H]AA-labeled LPS-treated cells were incubated
with the indicated concentrations of BEL for 30 min. Subsequently, the
cells were incubated with (
) or without (
) 100 nM PAF for either 10 (A) or 1.5 (B) min.
Extracellular [
H]AA release (A) and
cell-associated [
H]AA (B) were
quantitated as described under ``Experimental
Procedures.''
Figure 6:
Effect of okadaic acid (OkA) on
[H]AA mobilization in P388D
cells. A, [
H]AA-labeled cells were exposed to
the indicated amounts of okadaic acid for 30 min, washed, and incubated
for an additional 10-min period with (
) or without (
) 100
nM PAF. B and C,
[
H]AA-labeled cells were incubated with either
200 ng/ml LPS for 1 h, 1 µM okadaic acid for 30 min, or
both. In the LPS plus okadaic acid incubations, okadaic acid was
present only during the last 30 min of incubation. Subsequently, the
cells were washed and stimulated with 100 nM PAF. D,
cells labeled with [
H]choline were preincubated
with LPS, okadaic acid, or both as described above and then stimulated
with PAF for 1.5 min. Lyso-PC accumulation was determined as described
under ``Experimental
Procedures.''
Figure 7:
Effect of inhibiting either
cPLA or sPLA
on the time course of total
[
H]AA release from P388D
cells.
[
H]AA-labeled LPS-treated cells were preincubated
with MAFP (25 µM) (
), diC
SNPE (50
µM) (
), or neither (
) for 15 min.
Subsequently, the cells were incubated with 100 nM PAF for the
times indicated, except for the control (
), which also lacked
inhibitor. Afterwards, the supernatants were mixed with the cellular
homogenates obtained from scraping the cell monolayers with 0.5% Triton
X-100, and the resulting mix was subjected to lipid extraction. Free
[
H]AA was separated by thin-layer chromatography,
and radioactivity was determined by scintillation
counting.
We next explored whether the addition of
metabolites resulting from cPLA activity, i.e. free AA and lysophospholipids, could overcome the effect of MAFP
on extracellular [
H]AA release. As indicated
earlier, when [
H]AA-prelabeled LPS-treated cells
were stimulated for 15 min with PAF in the presence of MAFP (25
µM), [
H]AA release to the incubation
medium was strongly decreased (Fig. 8). If, however, P388D
cells were exposed to exogenous AA (1 µM) for 1 min
before PAF addition, the inhibitory effect of MAFP on extracellular
[
H]AA was greatly diminished (Fig. 8).
Preincubating the cells with lysophospholipids (i.e. lyso-PC,
lyso-PI, or lyso-PE) or other free fatty acids, whether saturated (i.e. palmitic, stearic, or arachidic acids) or unsaturated
(oleic or linoleic acids) did not overcome the inhibitory effect of
MAFP. Control experiments had shown that at the doses employed, none of
the above mentioned fatty acids or lysophospholipids exerted cytotoxic
effects or affected basal [
H]AA release. On the
other hand, preincubating the cells with exogenous AA prior to PAF
addition did not overcome the inhibitory effect of diC
SNPE
on extracellular [
H]AA release (Fig. 8).
Figure 8:
Exogenous AA overcomes the effect of MAFP
on extracellular [H]AA release.
[
H]AA-labeled LPS-treated cells were preincubated
with MAFP (25 µM), diC
SNPE (50
µM), or neither for 15 min, as indicated. Subsequently, 1
µM exogenous unlabeled AA was added 1 min before treatment
with PAF (100 nM), as indicated. After 15 min, extracellular
[
H]AA release was quantitated as described under
``Experimental Procedures.'' These data are the means
± S.E. of three experiments with duplicate incubations and are
expressed as a percentage of the response observed in the absence of
both inhibitor and exogenous AA. The 100% value corresponds to 2170
± 520 cpm.
The notion that both cPLA and sPLA
s mediate receptor activation of AA release
may represent a signaling mechanism common to agonists that elicit
short-term (i.e. PAF) or long-term responses (i.e. cytokines and growth factors). Work by Schalkwijk et al.(23, 24) in cytokine-stimulated rat mesangial
cells and by Murakami et al.(5) in
cytokine-stimulated human endothelial cells has also suggested that
both PLA
s may participate in regulating AA mobilization in
these cells, their relative contribution being dependent on the agonist
used. However, there are other cell systems such as platelets, in which
a role for sPLA
in AA release could not be demonstrated (25) . Moreover, AA release in thrombin-stimulated platelets
could be completely blocked by inhibiting the cPLA
,
suggesting that in this system, cPLA
is perhaps the only
effector involved in AA release(25) .
Domin and Rozengurt (26) have recently demonstrated that AA mobilization in Swiss
3T3 cells treated with platelet-derived growth factor follows a bimodal
kinetics. In this system, cPLA activation appears to be
responsible for the small burst of AA mobilization that occurs during
the first 20 min following agonist stimulation. However, the major
component of agonist-induced AA release was found to be due to another
unidentified effector. Because these data are very reminiscent of the
situation in PAF-stimulated LPS-primed P388D
cells, it is
tempting to speculate that the second effector involved in the system
studied by Domin and Rozengurt (26) is the sPLA
.
Although it is certain that the time course of the AA release responses
in platelet-derived growth factor-stimulated Swiss 3T3 cells and in
PAF-stimulated P388D
cells are clearly distinct, this is
most likely due to cell type differences and especially to the very
distinct nature of the agonists employed. As a matter of fact, when 3T3
cells are challenged with a short burst agonist such as bombesin, a
rapid and transient increase in cell-associated free AA is observed
shortly after receptor occupancy (27) .
By comparing the
[C]/[
H] ratios in free AA
with those in the phospholipids, we could delineate the origin of
cell-associated AA and extracellular AA. Although the interpretation of
these data may be complicated by the phenomenon of mixing AA pools as
well as the molecular heterogeneity of each phospholipid class, some
definite conclusions can be reached. The fact that the
[
C]/[
H] ratio for
extracellular AA is considerably lower than that of cell-associated AA
suggests that most of the extracellular free AA arises from PE, but
this is not the case for cell-associated AA. As a matter of fact, PE is
the only phospholipid whose
[
C]/[
H] ratio is
comparable with that of extracellular AA. Following a similar
rationale, it can be concluded that all major phospholipids contribute
to the early burst in cell-associated AA, although their relative
contribution cannot be estimated from our data. Because cPLA
is responsible for raising the levels of cell-associated free AA,
involvement of all major phospholipid classes in this process is
consistent with the notion that this enzyme does not distinguish among
phospholipid head groups(28, 29) .
It is generally
assumed that the phospholipids are asymmetrically distributed in
cellular membranes, PC being localized primarily at the outer leaflet
and PE at the inner leaflet of the plasma membrane(30) . Thus
the notion that the extracellular AA release arises primarily from PE
would seem, at a first glance, unexpected. However, sPLA,
the enzyme primarily responsible for mobilizing AA to the extracellular
medium, has been reported to prefer PE over any other phospholipid when
these are presented in a natural membrane
system(29, 31) . It is possible that the sPLA
preference for PE in these studies could be caused by a higher
proportion of PE relative to other phospholipids in these membranes,
because studies with vesicles containing various kinds of phospholipids
have failed to detect any head group specificity(32) . However,
studies in platelets have demonstrated that during activation, a rapid
translocation of AA-containing PE from the inner to the outer leaflet
of the plasma membrane takes place(33) . Such a translocation
would permit the AA-containing PE to be readily accessible to the
extracellular sPLA
. Interestingly, recent work by Fourcade et al.(34) has suggested that loss of membrane
asymmetry resulting from movement of phospholipids from the inner to
the outer leaflet of the membrane may play a key role in regulating the
activity of extracellular sPLA
.
A perturbation of the lipid bilayer or
``membrane rearrangement,'' initiated by an agonist/receptor
interaction, appears to be required to activate sPLA at the
outer surface of the cell (35) The data reported herein suggest
that, in addition to phospholipid translocation(34) , such a
membrane rearrangement may involve a transient elevation of free AA
mediated by receptor-activated cPLA
. Thus, our results
appear to suggest a new role for free AA in cellular signaling, i.e. to help regulate the accessibility of sPLA
to
its substrate in the membrane.
However, such an intracellular
elevation of free AA is not itself sufficient to elicit the cellular
response. Therefore, other additional signals that occur at the
earliest stages of PAF activation, such as inositol phospholipid
turnover, Ca mobilization, or protein phosphorylation (11) are also required for the AA release process to fully take
place. When all of these signals are induced sufficiently, sPLA
begins to hydrolyze phospholipids at the outer surface of the
cell, and this results in full AA mobilization. According to this
model, augmentation of any of these early signals could result in an
increased liberation of AA to the extracellular medium. This is what
occurs in the experiments using okadaic acid, wherein augmentation of
cPLA
activity and hence cell-associated free AA levels
dramatically enhance the extracellular AA release, provided sPLA
is functional.
We should emphasize that the above model of
cross-talk between cPLA and sPLA
has to be
regarded as a working model and not as an established one. Much of our
evidence in favor of a causal relationship between cPLA
and
sPLA
rests on the use of the phosphonylfluoride MAFP, a
highly reactive compound. It cannot be ruled out at this time that MAFP
is exerting some other undesired effects or that the
sPLA
-activating effect of exogenous AA is unrelated to
cPLA
.
The fact that the
potentiating effect of BEL on extracellular AA release is still
observed in activated cells further indicates that the
receptor-regulated PLAs releasing AA during PAF stimulation
are distinct from the BEL-sensitive iPLA
. This view lends
support to a model whereby each of the distinct PLA
s
present in P388D
cells may play different roles in
regulating free AA availability during PAF-induced activation (Fig. 9). By interacting with its specific receptor at the
plasma membrane, PAF initiates the stimulation process by increasing
the intracellular Ca
concentration (step 1). PAF also
triggers a second as yet unknown signal (step 2)(11) . These
signals act in concert to initiate translational/post-translational
events that result in the activation of cPLA
and
sPLA
. The two enzymes, acting either intracellularly
(cPLA
) or extracellularly (sPLA
), are
responsible for mobilizing AA upon PAF receptor stimulation. On the
other hand, the iPLA
allows reincorporation of part of the
fatty acid previously liberated by its Ca
-dependent
counterparts and in this manner helps replenish cellular AA pools. If
the iPLA
were responsible for generating a significant
portion of lyso acceptors under activation conditions, this enzyme
might also play a role in eicosanoid metabolism by limiting the amount
of AA available for eicosanoid biosynthesis.
Figure 9:
Signal transduction model for
PAF-stimulated AA release in LPS-primed P388D macrophages.
The different roles of the iPLA
, cPLA
, and
sPLA
in AA incorporation and mobilization are indicated.
Inhibition (
) by pertussis toxin (PTX), BAPTA
(bis-(O-aminophenoxy)ethane-NNN`N`-tetracetic
acid), actinomycin D (ACTD), cyclohexamide (CHX), and
indomethacin (INDO) are also indicated. See text for further
details.