(Received for publication, March 13, 1995; and in revised form, August 24, 1995)
From the
Addition of submicromolar concentrations of arachidonic acid
(AA) to human neutrophils induced a 2-fold increase in the activity of
a cytosolic phospholipase A (PLA
) when measured
using sonicated vesicles of
1-stearoyl-2-[
C]arachidonoylphosphatidylcholine
as substrate. A similar increase in cytosolic PLA
activity
was induced by stimulation of neutrophils with leukotriene
B
(LTB
), 5-oxoeicosatetraenoic acid, or
5-hydroxyeicosatetraenoic acid (5-HETE). LTB
was the most
potent of the agonists, showing maximal effect at 1 nM.
Inhibition of 5-lipoxygenase with either eicosatetraynoic acid or
zileuton prevented the AA-induced increase in PLA
activity
but had no effect on the response induced by LTB
.
Furthermore, pretreatment of neutrophils with a
LTB
-receptor antagonist, LY 255283, blocked the AA- and
LTB
-induced activation of PLA
but did not
influence the action of 5-HETE. Treatment of neutrophils with
pancreatic PLA
also induced an increase in the activity of
the cytosolic PLA
; this response was inhibited by both
eicosatetraynoic acid or LY 255283.
The increases in PLA activity in response to stimulation correlated with a shift in
electrophoretic mobility of the 85-kDa PLA
, as determined
by Western blot analysis, suggesting that phosphorylation of the 85-kDa
PLA
likely underlies its increase in catalytic activity.
Although stimulation of neutrophils with individual lipoxygenase
metabolites did not induce significant mobilization of endogenous AA,
they greatly enhanced the N-formylmethionyl-leucyl-phenylalanine-induced mobilization of
AA as determined by mass spectrometry analysis. Our findings support a
positive-feedback model in which stimulus-induced release of AA or
exocytosis of secretory PLA
modulate the activity of the
cytosolic 85-kDa PLA
by initiating the formation of
LTB
. The nascent LTB
is then released to act on
the LTB
receptor and thereby promote further activation of
the 85-kDa PLA
. Since 5-HETE and LTB
are known
to prime the synthesis of platelet-activating factor, the findings
suggest that 85-kDa PLA
plays a role in platelet-activating
factor synthesis.
Arachidonic acid (AA) ()is the precursor for
prostaglandins and leukotrienes (collectively named eicosanoids), which
together with platelet-activating factor (PAF) are important lipid
mediators involved in allergic and inflammatory reactions. The initial
step in the production of eicosanoids is hydrolysis of sn-2-arachidonate from membrane phospholipids generating free
AA; phospholipase A
s (PLA
) with different
characteristics have been implicated in this
hydrolysis(1, 2, 3) . The 14-kDa
PLA
s (type I and II) are secretory enzymes, and although
they show no apparent preference for hydrolysis of AA-containing
phospholipids(4, 5) , they have been suggested to
participate in the generation of eicosanoids after first being secreted
by mobilizing AA from phospholipids on the outer leaflet of the plasma
membrane(6, 7, 8, 9, 10, 11, 12) .
The 85-kDa PLA
(13, 14) is an
intracellular enzyme with clear preference for AA-containing
phospholipids(5, 15, 16) . Furthermore, the
85-kDa PLA
translocates to membranes in response to
submicromolar concentrations of
Ca
(13, 17, 18) and is also
regulated by phosphorylation, which results in an increase in its
catalytic activity(19, 20, 21) . These
characteristics make the 85-kDa PLA
a likely candidate
responsible for the stimuli-induced mobilization of AA and the
subsequent generation of eicosanoids.
While many cell types release
both cyclooxygenase and lipoxygenase metabolites, human neutrophils
(PMN) release predominantly the 5-lipoxygenase products, leukotriene
B (LTB
) and
5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), upon stimulation.
Besides release of these eicosanoids, exogenously added 5-HETE has been
shown to ``prime'' PMN for release of AA and 5-lipoxygenase
metabolites (22) as well as PAF (22, 23, 24) when challenged with another
stimuli. Furthermore, ionophore-induced mobilization of cellular AA (25) and production of LTB
(26) have been
shown to be enhanced by exogenously added AA and LTB
,
respectively. Although an increase in activity of one or more PLA
types was assumed in the above studies, no measurements of the
activity of PLA
were performed. In the present study, we
have examined the effect of exogenously added free fatty acids and
5-lipoxygenase metabolites on the activity and phosphorylation, as
determined by mobility shifts on Western blots, of the intracellular
85-kDa PLA
in PMN.
PMN (0.75-1.1
10
cells/ml; 4 ml total volume) were incubated at 37 °C
in Dulbecco's phosphate-buffered saline (PBS) containing 1
mM CaCl
. Free fatty acids were dissolved in
ethanol and diluted in PBS prior to the addition to cell suspensions,
resulting in a final concentration of ethanol of <0.1%.
LTB
, 5-oxoETE, 15-oxoETE, 5-HETE, and compound I, dissolved
in methanol, were dried under nitrogen and resuspended in 25 µl of
BSA (2.5 mg/ml) prior to addition to the cells. Zileuton and ETYA were
dissolved in Me
SO and added to cells resulting in a final
Me
SO concentration of 0.1%. LY 255283 was dissolved in
water. Pancreatic PLA
was diluted in PBS prior to the
addition to cell suspensions. Control cells received appropriate
vehicle (Me
SO and/or BSA), which had no effect on the
PLA
activity compared with no addition. Ten volumes of
ice-cold PBS without CaCl
were added to terminate
incubations before suspensions were centrifuged at 300
g for 10 min. When cells were treated with exogenous
PLA
, an additional wash of the cells with 40 ml of PBS was
included. All subsequent procedures were performed at 4 °C. Cells
were resuspended in 1 ml of 80 mM KCl, 10 mM Hepes
(pH 7.4), 1 mM EDTA, 1 mM EGTA, 40 µg/ml
leupeptin, 25 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl
fluoride, 10 mM NaF, 0.2 mM
NH
VO
, and 4 mM dithiothreitol (Buffer
A) and broken by sonication for 2
5 s at a power setting of 2
and 10% output with a probe sonicator (Heat System Inc.). Broken cells
were centrifuged at 10
g for 10 min, and
the residual supernatant was further centrifuged at 10
g for 60 min to obtain cytosol and membrane
fractions. Glycerol was added to the cytosol fraction to a final
concentration of 10% (v/v). Membrane fractions were resuspended by
sonication for 5 s in 0.35 ml of Buffer A containing 10% glycerol. The
cytosol fraction could be stored at either 4 °C or -20 °C
for 3 weeks without any major loss of PLA
activity.
Protein content of the subcellular fractions from PMN was determined according to the method of Bradford (30) using BSA as standard.
The cytosol fraction from PMN, disrupted in the presence of
Ca-chelators, contains the predominant part of the
PLA
activity when assayed with sonicated vesicles of
arachidonoylphosphatidylcholine as substrate. Under these assay
conditions (see ``Experimental Procedures''), the cytosol
fraction from control cells showed a specific PLA
activity
of 0.26 ± 0.04 pmol/µg/10 min (mean ± S.D. from 20
different PMN preparations); however, some variations between different
preparations were seen (discussed below). While the 10
g pellet contained some activity, it amounted to
only 2-4% of the activity found in the cytosol fraction. We were
unable to detect any PLA
activity in the 10
g pellet. The activity of PLA
, both
in the cytosol and the 10
g pellet, was
totally Ca
-dependent and resistant to dithiothreitol.
These characteristics indicated that the PLA
we assay is
similar or identical to the 85-kDa PLA
, which has
previously been identified in several different cell types including
PMN(34) .
Addition of submicromolar concentrations of free
AA to PMN resulted in a 1.8-2-fold increase in PLA activity in the cytosol fraction compared with that from control
cells (Fig. 1). The AA response was maximal at 0.5 µM with no further increase seen using 10 µM AA. In
contrast to the response seen with AA, no increase in PLA
activity was observed with linolenic acid (Fig. 1),
linoleic acid, or oleic acid (not shown) in the range from 0.5 to 50
µM. While Fig. 1data were obtained with fatty
acids added to the cells in ethanol (<0.1%), almost identical data
were obtained when fatty acids were added to cells dissolved in
Me
SO or complexed to BSA (not shown).
Figure 1:
Effect of AA and linolenic acid on the
activity of PLA in PMN. PMN (3
10
cells/4 ml) were treated for 5 or 10 min with the indicated
concentration of AA (open circles) or linolenic acid (closed circles). Cytosol fractions from control and
stimulated cells (30-40 µg of protein; 1
10
cell equivalents) were assayed for PLA
activity as
described under ``Experimental Procedures.'' Results are mean
values ± S.D. from three or more PMN preparations and expressed
as -fold increase above the activity in control cells, which averaged
0.26 ± 0.04 pmol/µg/10 min as described in the
text.
We next
investigated the actions of selected AA metabolites. Stimulation of PMN
with either LTB, 5-HETE, or 5-oxoETE induced an increase in
PLA
(Fig. 2). The potency among these agents varied
with LTB
, the most potent of the metabolites, showing
maximal effect at 1 nM. 5-oxoETE and 5-HETE were at least 50
times less active than LTB
, with 5-oxoETE being about
5-fold more potent than 5-HETE. In studies not shown, we found that 5
µM 15-oxoETE and 10 nM compound I (an isomer of
LTB
) did not stimulate PMN to alter cytosolic PLA
activity. Likewise, 0.5 µM prostaglandins
D
, E
, and I
were inactive.
15-oxoETE, compound I and the prostaglandins do not stimulate PMN
functional responses(27, 35) . Finally, pretreatment
of PMN with 0.5 µM prostaglandin E
for 2 min
did not affect the ability of LTB
to activate PLA
(data not shown).
Figure 2:
Effect
of 5-lipoxygenase metabolites on the activity of PLA in
PMN. Cells were stimulated with the indicated concentration of
LTB
(closed circles), 5-oxoETE (open
squares), or 5-HETE (closed squares) for 5 min, and
PLA
activity was determined in the cytosol fraction.
Results are mean values ± S.D. from three different PMN
preparations and expressed as -fold increase above the activity in
control cells.
The time course of AA and
LTB-induced activation of PLA
is shown in Fig. 3. While the LTB
response was almost fully
developed after 1 min of stimulation, the AA response was only marginal
at this time point but reached the maximal response by 2 min. Thus, the
pathway mediating PMN responses to AA is distinctly slower acting than
that for LTB
.
Figure 3:
Time course of AA- and
LTB-induced activation of PLA
. Cells were
treated with 0.5 µM AA (open circles) or 1 nM LTB
(closed circles) for the indicated time.
Cytosol fraction from control and treated cells were assayed for
PLA
activity. Results are mean values ± S.D. from
three different PMN preparations and expressed as -fold increase above
the activity in control cells.
Activation of protein kinase C by
stimulation with phorbol myristate acetate has been shown to lead to an
increase in the activity of the 85-kDa PLA in PMN (36) as well as several other cell
types(19, 37, 38, 39) . Since
unsaturated fatty acids can activate protein kinase
C(40, 41) , we considered the possibility that the
AA-induced increase in PLA
activity might reflect the
direct action of AA on protein kinase C. Alternatively, AA might act
indirectly by first being converted to a lipoxygenase product and then
triggering activation of PLA
via LTB
or
putative 5-HETE receptors. We therefore investigated the effect of two
competitive inhibitors of lipoxygenase on the AA-induced activation of
PLA
. Pretreatment of PMN with either ETYA, an inhibitor of
both cyclooxygenase and lipoxygenase, or zileuton, a selective
inhibitor of 5-lipoxygenase, abolished the AA-induced activation of
PLA
(Fig. 4). Neither of these inhibitors, at a
concentration of 5 µM, altered the basal activity of
PLA
or the LTB
-induced activation of
PLA
.
Figure 4:
Effect of lipoxygenase inhibitors on the
AA- and LTB-induced activation of PLA
. PMN were
pretreated with 5 µM zileuton (Zil), 5 µM ETYA, or vehicle (Me
SO) for 10 min prior to
stimulation with either 0.5 µM AA or 1 nM
LTB
for an additional 5 min. Cytosol fraction from control
and treated cells were assayed for PLA
activity. Results
are mean values ± S.D. from different PMN preparations (n) and expressed as -fold increase above the activity in
control cells.
In another set of experiments, we examined the
effect of LY 255283, a competitive LTB receptor antagonist,
on the increase in PLA
activity induced by AA and
LTB
. Preincubation of PMN with 10 µM LY 255283
completely abolished both the AA- and LTB
-induced
activation of PLA
, yet it had no effect on the basal
activity of PLA
or the increase in PLA
activity
induced by 5-HETE (Fig. 5) or 5-oxoETE (not shown). As little as
0.5 µM LY 255283 was sufficient to block the AA-induced
activation of PLA
.
Figure 5:
Effect of the LTB receptor
antagonist LY 255283 on AA-, LTB
- and 5-HETE-induced
activation of PLA
. PMN were pretreated with 10 µM LY 255283 for 2 min or subjected to no pretreatment followed by
stimulation with either 0.5 µM AA, 1 nM
LTB
, or 0.5 µM 5-HETE for 5 min. Cytosol
fraction from control and treated cells were assayed for PLA
activity. Results are mean values ± S.D. from three
different PMN preparations and expressed as -fold increase above the
activity in control cells.
The increase in catalytic activity of
the 85-kDa PLA has been linked to phosphorylation of the
enzyme(19, 20, 21, 36) , which
results in reduced mobility upon electrophoresis(19) . When the
cytosol fraction from control PMN was subjected to electrophoresis and
immunoblotted with an antibody against the 85-kDa PLA
, two
bands were detected with the lower band (higher mobility) being the
more prominent (Fig. 6Fig. 7Fig. 8). It should be
pointed out that the distribution of immunodetected 85-kDa PLA
between the two bands in the cytosol fraction from different
control preparations varied. In one control PMN preparation, an equal
amount of protein was seen in the two bands, and this was accompanied
by a somewhat higher specific activity of PLA
(0.32
pmol/µg/10 min versus 0.23 pmol/µg/10 min in Fig. 6) as well as a lower -fold increase in response to
stimulation (1.55-fold versus 1.9-fold in Fig. 6).
These differences between PMN preparations could be due to partially
activated cells in some preparations, resulting in a partial shift in
mobility (phosphorylation) of the 85-kDa PLA
. Cytosol
fractions from PMN stimulated with optimally active amounts of either
LTB
, 5-HETE, or AA (as defined in PLA
assays)
resulted in all of the immunodetected 85-kDa PLA
migrating
with reduced mobility (Fig. 6). This suggests that the three
stimuli induce phosphorylation of the 85-kDa PLA
. Fig. 6also shows that the AA-induced shift in mobility
(phosphorylation) could be blocked by pretreatment of the cells with
the LTB
-receptor antagonist LY 255283; the blocking of the
shift in mobility correlates with the loss of stimulation of the
PLA
activity by AA (Fig. 5). The
LTB
-induced shift in mobility of the 85-kDa PLA
was also inhibited by LY 255283, while that induced by 5-HETE was
not affected (Fig. 7). Immunoblotting of the 10
g pellet from control and stimulated PMN
revealed the same pattern as seen in the cytosol, i.e. two
bands from control cells and one band with reduced mobility from
stimulated cells (Fig. 6). We emphasize that the amount of
10
g pellet subjected to immunoblotting
was from 3
10
cell equivalents, whereas the amount
from the cytosol fraction was from 6
10
cell
equivalents. This indicates that the 10
g pellet constitutes only a minor part of the immunodetectable
85-kDa PLA
when PMN are broken in the presence of
Ca
-chelators. This is in agreement with our PLA
activity data. No immunodetectable 85-kDa PLA
could
be found in the 10
g pellet (results not
shown).
Figure 6:
Immunoblotting of 85 kDa PLA from cytosol and membrane fractions of PMN treated with
LTB
, 5-HETE or AA: effect of LY 255283 on AA-induced
mobility shift. The six bands from left to right represent the cytosol (upper set) or 10
g pellet (lower set) of cells treated as follows: left lane,
control unstimulated cells; second lane, 1 nM LTB
for 5 min; third lane, 0.5
µM 5-HETE for 5 min; fourth lane, 0.5 µM AA for 5 min; fifth lane, cells were pretreated with 10
µM LY 255283 for 2 min followed by treatment with 0.5
µM AA for 5 min; right lane, cells were treated
with 10 µM LY 255283 alone for 7 min. Cytosol fraction (upper set) and 10
g pellet (lower set) were prepared as described under
``Experimental Procedures.'' Twenty µg of protein from
cytosol fractions (6
10
cell equivalents) and 6
µg of protein from the 10
g pellets (3
10
cell equivalents) were subjected to
SDS-polyacrylamide gel electrophoresis. After electrotransfer to
nitrocellulose membrane and immunoblotting with a polyclonal antibody
against the 85-kDa PLA
, protein was detected with
Amersham's enhanced chemiluminescence detection system. All 12
samples were run on the same gel and split into two parts after
detection. Cont., control; LY, LY 255283; PLA
and PLA
-P depict
the unphosphorylated and phosphorylated enzyme, respectively, as
suggested by the shift in mobility.
Figure 7:
Immunoblotting of 85-kDa PLA in cytosol from PMN: effect of LY 255283 on LTB
- and
5-HETE-induced mobility shifts. Cells (1.1
10
/ml; 4
ml) were treated with either 10 µM LY 255283 for 7 min (LY), 10 µM LY 255283 for 2 min prior to 1 nM LTB
for 5 min (LY + LTB
), 10 µM LY 255283 for 2
min prior to 0.5 µM 5-HETE for 5 min (LY + 5-HETE). Twenty µg of protein from cytosol fractions (0.6
10
cell equivalents) were subjected to
SDS-polyacrylamide gel electrophoresis. After electrotransfer to
nitrocellulose membrane and immunoblotting with a polyclonal antibody
against the 85-kDa PLA
, protein was detected with
Amersham's enhanced chemiluminescence detection system. Positive
controls for the LTB
- and 5-HETE-induced shifts in mobility
were shown in Fig. 6.
Figure 8:
Effect of pancreatic PLA on
PMN 85-kDa PLA
. Cells (1.1
10
/ml; 4 ml
total) were pretreated with 10 µM LY 255283, 5
µM ETYA, or no pretreatment for 3 min prior to addition of
pancreatic PLA
(2 µg/ml) and incubation for 12 min.
Cells were washed twice with 30 ml of PBS followed by resuspension in
buffer A, sonication, and preparation of cytosol fraction. A,
cytosol fraction was assayed for PLA
activity. Results are
mean values ± S.D. from four different PMN preparations and are
expressed as -fold increase above the activity in cytosol fraction from
control cells. B, 20 µg of protein (6
10
cell equivalents) from cytosol fractions were subjected to
SDS-polyacrylamide gel electrophoresis, electrotransferred to
nitrocellulose membrane, and immunoblotted with a polyclonal antibody
against the 85-kDa PLA
followed by detection of protein
with Amersham's enhanced chemiluminescence detection system.
Representative results of three independent experiments are shown. Cont., control; LY, LY 255283; pan
PLA
, pancreatic
PLA
.
Having established that exogenously added AA induced
activation of cytosolic PLA, we hypothesized that an
extracellular PLA
added to PMN could mimic the effect of
AA. As seen in Fig. 8A, addition of pancreatic
PLA
to PMN results in an increase in the activity of
cytosolic PLA
; this increase was to a large extent
inhibited by pretreatment of the cells with LY 255283 or ETYA. In
accordance with these activity results, pancreatic PLA
also
induced a shift in mobility of the 85-kDa PLA
upon
electrophoresis, and this shift was partially inhibited by pretreatment
with LY 255283 and completely inhibited by ETYA (Fig. 8B).
In order to verify that the observed
increase of the 85-kDa PLA activity was due to the
exogenously added PLA
rather than possible
lipopolysaccharide (LPS) contamination in the pancreatic
PLA
preparation, we used a number of approaches. Polymyxin
B is an antibiotic that inactivates endotoxin by binding the lipid A
portion of LPS. Incubation of pancreatic PLA
(2 µg/ml)
with 1 mg/ml polymyxin B for 1 h did not decrease its ability to
activate the 85-kDa PLA
compared with untreated pancreatic
PLA
(data not shown). A second approach was to treat the
pancreatic lipase with Pronase, which consists of a mixture of several
proteolytic enzymes including endopeptidases and exopeptidases.
Therefore, Pronase should hydrolyze the PLA
enzyme while
leaving the endotoxin intact. Treatment of pancreatic PLA
(2 µg/ml) with 100 units of Pronase for 1 h resulted in a
total loss of exogenous PLA
ability to activate cytosolic
PLA
, indicating that the added PLA
was
responsible for the increase of 85-kDa PLA
activity (data
not shown). Finally, we were able to directly measure the amount of
endotoxin contamination in the pancreatic PLA
preparation
using a chromogenic limulus amebocyte lysate assay (Bio-Whitaker)
sensitive to 10 pg/ml LPS. The standard addition of 2 µg/ml
pancreatic PLA
was shown to contain less than 50 pg/ml LPS.
This amount of LPS has been shown to be below the threshold needed for
the activation of cytosolic PLA
in PMN(36) ,
strengthening the argument that the observed activation of cytosolic
PLA
was due to exogenously added PLA
, not LPS.
In addition to these treatments, boiling the pancreatic lipase for 15
min did not destroy its activity against phospholipid vesicles or its
ability to activate the 85-kDa PLA
in PMN.
Phosphorylation of the 85-kDa PLA and the concomitant
increase in activity, determined in an in vitro assay, may not
necessarily reflect a mobilization of cellular AA. We therefore
examined the effect of lipoxygenase metabolites on the mobilization of
AA from PMN using gas chromatography/mass spectrometry. In order to
prevent reacylation of mobilized AA, as well as metabolism to
lipoxygenase products, BSA (at a final concentration of 2 mg/ml) was
added to cells 15 s after stimuli. Although the lipoxygenase products
were added in BSA-containing vehicle (2.5 mg/ml), the final
concentration of BSA with the cells was only 0.03 mg/ml, which had no
significant effect on AA metabolism. In a pilot study, we found that a
powerful stimulus, 1 µM fMLP, did not result in any
detectable increase in free AA above that of control if BSA (final
concentration 2 mg/ml) was added to the cells 3 min after stimulation
(data not shown). However, using the same protocol altered by the
addition of BSA (final concentration 2 mg/ml) 15 s after fMLP
stimulation resulted in accumulation of AA (Table 1). PMN
stimulated with either LTB
, 5-HETE, or 5-oxoETE did not
induce any major mobilization of AA when added alone. This was true
whether these stimuli were added 3.25 min (Table 1) or 15 s (not
shown) before addition of BSA (final concentration, 2 mg/ml). On the
other hand, all three lipoxygenase metabolites substantially enhanced
the fMLP-induced mobilization of AA. The priming effect of LTB
on the fMLP-induced mobilization of AA was completely blocked by
LY 255283 (results not shown). Although the amount of mobilized AA mass
varied, the same pattern was seen in three separate experiments,
showing that 5-oxoETE and 5-HETE enhanced the fMLP-induced mobilization
of AA to a somewhat greater extent than LTB
. We also
examined the amounts of free linoleic and oleic acid in PMN. In
contrast to AA, these fatty acids were found in higher amounts in
control cells (25 and 400 ng/10
cells for linoleic acid and
oleic acid, respectively) and did not change significantly after
stimulation (results not shown).
A wide variety of different stimuli such as growth factors,
hormones, and cytokines have been shown to induce phosphorylation of
the 85-kDa PLA, which is accompanied by about a 2-fold
increase in the activity of the enzyme when assayed in
vitro(42, 43, 44, 45, 46, 47) .
The 85-kDa PLA
has been shown to act as a substrate for
protein kinase C in vitro; however, such phosphorylation does
not result in any increase in catalytic
activity(45, 48) . Instead, mitogen-activated protein
kinase has been proposed to be the kinase responsible for the
phosphorylation resulting in an increase in activity of the 85-kDa
PLA
(45, 49) . Activation of the 85-kDa
PLA
has been linked to release of eicosanoids in several
different cell
types(19, 21, 36, 42, 43) ,
which emphasizes the importance of this PLA
. The 85-kDa
PLA
of PMN has been examined in only a few studies, even
though the cells are prominent sources of eicosanoids in host-defense
and inflammatory reactions.
In the present study, we have
demonstrated that stimulation of PMN with 5-lipoxygenase metabolites
results in a 2-fold increase in the activity of a cytosolic PLA when assayed using sonicated vesicles of
1-stearoyl-2-[
C]arachidonoylphosphatidylcholine
as substrate. Immunoblot analysis of cytosol fractions from
LTB
- and 5-HETE-stimulated PMN using a polyclonal antibody
against the 85 kDa PLA
revealed a shift in electrophoretic
mobility of the 85-kDa PLA
. This shift has been shown to be
due to phosphorylation of the enzyme(19) . The decrease in
electrophoretic mobility of the 85-kDa PLA
in response to
stimulation of PMN correlated in all cases with an increase in the
enzymatic activity of PLA
in the cytosol fraction. This
correlation, together with the characteristics of the PLA
, i.e. its total Ca
-dependence and resistance
to dithiothreitol, indicate that the activity measured in the cytosol
fraction is due solely to the 85-kDa PLA
.
LTB(50) and apparently 5-HETE (51) bind to G-protein
coupled plasma membrane receptors. Several lines of evidence suggest
that these two receptors are distinctly
different(50, 51) . Indeed, we find that the
LTB
-induced activation of PLA
was inhibited by
pretreatment of PMN with a LTB
-receptor antagonist LY
255283, whereas the 5-HETE-induced activation of PLA
was
unaffected by this pretreatment. Our results thus support the previous
conclusion that the PMN recognition systems for 5-HETE and LTB
are distinctive. We presume, as indicated in earlier work (28, 52) that 5-HETE and 5-oxoETE operate through an
identical cell-activating mechanism. Maximal increase in PLA
activity was seen with 1 nM LTB
, while 500
nM 5-HETE and between 50 and 500 nM 5-oxoETE were
required to achieve the same level of response. These differences in
concentration required to activate the 85-kDa PLA
correlate
well with their potencies in stimulating PMN
function(28, 51, 52) .
AA as well as other
unsaturated fatty acids such as linolenic and linoleic acid have been
shown to activate protein kinase C(40, 41) . The
increase in PLA activity seen in the cytosol fraction of
PMN stimulated with AA could therefore be due to a direct activation of
protein kinase C. However, the fact that linolenic acid, even at
concentrations 100 times that of AA, did not result in any increase in
PLA
activity argues that AA does not act here through
protein kinase C. Instead, our results strongly suggest that the
increase in PLA
activity induced by exogenous AA is
mediated via metabolism of AA to a 5-lipoxygenase product prior to
activation of PLA
. This conclusion is supported by results
showing that either ETYA or zileuton, both competitive inhibitors of
5-lipoxygenase, completely suppress the AA response. Furthermore, the
AA response was also inhibited by the LTB
receptor
antagonist LY 255283. The time-course of PLA
activation in
response to LTB
and AA, in which maximal responses were
observed at 1 and 2 min, respectively, is also consistent with a model
in which conversion of AA to a 5-lipoxygenase metabolite is required
for the activation of the PLA
. AA can be rapidly
incorporated into neutral lipids and phospholipids and could become
unavailable for metabolism by lipoxygenase. However, since 1 nM LTB
was sufficient to induce a maximal response, only
a small fraction (<0.1%) of the added AA needs to be converted to
LTB
in order to account for the AA-induced activation of
the 85-kDa PLA
.
Previous work has shown that 5-HETE
potentiates ionophore-induced formation of PAF, 5-HETE, and LTB in PMN(22) . Furthermore, synthesis of PAF, which in many
cases occurs in parallel with eicosanoids, can be induced by a
nonmetabolized bioactive analog of PAF and to a lesser extent by
LTB
(24) . In this context, it is noteworthy that
stimulation of PMN with PAF (10 nM, 5 min) induces a 2-fold
increase in the activity of PLA
in the cytosol fraction as
well as reduced mobility of 85 kDa PLA
upon
electrophoresis. (
)Using mass spectrometry, we have found
that LTB
, 5-oxoETE, and 5-HETE substantially enhance the
fMLP-induced mobilization of AA in intact PMN, while these
5-lipoxygenase products alone resulted in little or no mobilization of
AA. Since all three lipoxygenase products induced an increase in the
activity of PLA
when assayed in vitro, our data
suggest that phosphorylation alone is insufficient to mobilize any
major amount of AA in PMN. It appears that in addition to
phosphorylation, an increase in intracellular Ca
may
explain the striking increase in AA release observed when fMLP is added
as a second stimulus. This is in agreement with previous work on PMN
showing that tumor necrosis factor (53) and
granulocyte-macrophage colony-stimulating factor (54, 55) enhanced the fMLP-induced mobilization of AA
and formation of lipoxygenase products but had little effect alone.
Both of these cytokines have been shown, although not in PMN, to
phosphorylate the 85-kDa PLA
(43, 56) .
The involvement of secretory low molecular weight PLA in
the mobilization of AA and the subsequent generation of eicosanoids has
been suggested in several different cell types such as mast
cells(6, 9) , endothelial
cells(8, 10) , HL-60 cells (7) , mesangial
cells(10) , human neutrophils(12) , and in the
macrophage cell line P388D
(11) . Some of the
studies revealed formation of cyclooxygenase products in response to
treatment of intact cells with low molecular weight PLA
alone(8, 9, 10) , while others required
addition of a second stimulus such as antigen or
Ca
-ionophore to yield measurable
products(6, 7) . Our results demonstrate that addition
of low molecular weight PLA
can activate the intracellular
85-kDa PLA
in PMN. This activation could be inhibited to a
large extent by either LY 255283 or ETYA, suggesting that one or more
lipoxygenase metabolites is responsible for the communication between
the two types of PLA
. In PMN, Shimizu et al. (12) found no formation of lipoxygenase products in response to
addition of either pancreatic or venom PLA
(0.14 µg/ml;
1.5
10
cells), although each enhanced the
zymosan-induced formation of LTB
. Since we have shown (Fig. 2) that even small amounts of LTB
(4 pmol/4
10
cells) will activate the 85-kDa PLA
,
LTB
may have been formed also in the study by Shimizu et al. (12) but below the detection limit. In mast
cells, Fonteh et al. (9) has shown that addition of Naja naja PLA
resulted in low but significant
formation of LTB
(5-10 pmol/5
10
cells). Recently, Reddy et al.(57) reported that
neutrophils contain an LTB
-dependent PLA
that
requires extracellular Ca
for activation. These
findings, which were based on studies of intact cells, add new support
for our conclusions.
In summary, our results support a positive
feedback model for regulating the 85-kDa PLA. We propose
that 5-lipoxygenase products derived from initially released AA
activate the enzyme and prime for increased AA mass release. Secretory
PLA
, following its release by exocytosis, can liberate AA
from the plasma membrane and through its conversion to lipoxygenase
products can provide cross-talk between the secreted and cytosolic
PLA
s. In our experimental system, AA released by
extracellular PLA
appears to act mainly through formation
of LTB
and its receptor to activate the 85-kDa
PLA
, but 5-oxoETE and 5-HETE also activated the enzyme.
Further work will be necessary to determine the relative roles of
secretory and intracellular PLA
s in the mobilization of AA
and the subsequent formation of eicosanoids.