I. Martin,* John. Wentland,*. Luke Akard,* Jan Jansen,*. James Thompson,* and Joe G.N. Garcia. *Bo. Marrow. Transplantation. Laboratoiy,. Methodist. Hospital.
Phorbol ester-induced priming of superoxide generation by phosphatidic acid-stimulated neutrophils and granule-free neutrophil cytoplasts Rafat A. Siddiqui,* Denis Wentland,* Luke Akard,*
English,*t Kevin Harvey,* Yi Cui,* Margaret I. Martin,* Jan Jansen,* James Thompson,* and Joe G.N. Garcia
*Bo
Laboratoiy,
Marrow
Sciences,
Transplantation
Indiana
Medicine/Critical
University
School
Methodist
ofMedicine,
Hospital
Indianapolis,
This study was undertaken to examine the mechanisms involved in polymorphonuclear leukocyte superoxide release stimulated by exogenous phosphatidic acid (PA). Unlike the immediate burst of superoxide release affected by membrane-permeable dioctanoylglycerol (DiC8-DAG), dioctanoyl phosphatidic acid (DiC8-PA) induced superoxide release after a lag period of 5-20 mm. This period was considerably reduced or eliminated when cells were primed by substimulatory levels of phorbol myristate acetate (PMA). Granule-depleted neutrophil cytoplasts also responded to DiC8-PA with a burst of superoxide generation. Activation of the cytoplast superoxide generating system in response to DiC8-PA was also significantly faster after cells had been preexposed to substimulatory levels of PMA, indicating that at least a portion of the priming mechanism was independent of PMA-induced degranulation. To further examine the potential mechanism of PMA priming of responses to PA, we evaluated the activity of neutrophil ecto-phosphatidic acid phosphohydrolase (edo-PA phosphohydrolase), which generates diacylglycerol from exogenous PA. PMA priming had no discernable effect on the activity of this enzyme. In addition, propranolol, an inhibitor of PA phosphohydrolase, did not selectively inhibit PMA priming ofneutrophil responses to DiC8-PA, indicating that priming did not result from acceleration of DiC8-PA hydrolysis. We therefore investigated the possibility that activation of protein kinase C was the basis of the primed response. Several semiselective protein kinase C inhibitors (caiphostin C, H-7, and acylmethylglycerol) inhibited DiC8-DAGand DiC8-PAinduced superoxide release as well as PMA-primed responses to approximately the same extent. These results are consistent with the hypothesis that neutrophil responses to phosphatidate are mediated by diglyceride generated by the action of ecto-PA phosphohydrolase. PMA priming does not result from increased catalytic activity of ecto-PA phosphohydrolase but rather seems to result from potentiation of an intermediate involved in the cells’ response to multiple stimuli. J. Leukoc. Bwl.
Key
Words:
nose
C
PMA
#{149}
1995. second
priming
Indiana,
Indianapolis, Department
Indiana;
School
ofMedicine,
ofAllted
Division
Health
of Pulmonaiy
Care
Abstract:
58: 189-195;
oflndiana,
John
INTRODUCTION Phosphatidic acid potentially exerts important effects on cellular activation. On receptor-mediated activation of phospholipase D this phospholipid is generated within plasma
Abbreviations: phosphohydrolase, phosphohydrolase; phosphatidic
.
phospholipid
.
protein
ki-
where
it acts
as a second
messenger
in
Received 1995.
Journal
cal C, calphostin C; ecto-PA neutrophil plasma membrane ecto-phosphatidic acid DiC8-DAG, dioctanoyl glycerol; DiC8-PA, dioctanoyl FMLP, formyl-methionyl-leucyl-phenylalanine; PKC,
acyl MG, acylmethylglycerol;
acid;
protein kinase ered saline. Reprint Laboratory,
messenger
membranes
key signal transduction pathways [1, 2]. Encountered on the surface of activated targets, such as endothelial cells, phosphatidic acid may directly stimulate several effector cell functions [3]. Indeed, exogenous phosphatidic acid has profound mitogenic and biological effects, including growth stimulation of fibroblasts [4, 5] and T cell clones [6], induction of mRNA for proto-oncogenes and growth factors [4, 5, 7], activation of the neutrophil NADPH oxidase [8], promotion of Ca2 entry into cells and mobilization of intracellular Ca2 [9-1 1], inhibition of CAMP formation [12], delaying G2 to mitosis transition in A431 cells [13], and phosphorylation of cellular proteins [14, 15]. In addition to these direct effects, cellular processes induced by exogenous phosphatidic acid may be regulated by phosphatidic acid phosphohydrolase, which converts phosphatidic acid into diacylglycerol. Thus, phosphatidic acid potentially exerts a wide range of effects on cellular processes by activating protein kinase C (PKC) [l6J. A neutrophil plasma membrance ecto-phosphatidic acid phosphohydrolase (ecto-PA phosphohydrolase), which regulates responses of human neutrophilic leukocytes to exogenous phosphatidic acid, has been recently identified [8]. This enzyme allows delayed but sustained availability of diglycerides to the cells’ interior where the lipid initiates several responses, including activation of the superoxide
C; PMA,
requests:
phorbol
Rafat
Methodist December
of Leukocyte
A.
Hospital 3, 1994;
myristate Siddiqui,
acetate; Bone
PBS, phosphate-buff-
Marrow
Transplantation
of Indiana, Indianapolis, IN 46202. revised March 9, 1995; accepted April
Biology
Volume
58,
August
1995
13,
189
MATERIALS Dioctanoyl
DAG)
AND METHODS
phosphatidic
were
obtained
acylmethylglycerol chem Nudear other
I
(actl
Avanti
MG),
and
and
Polar
H-7
dioctanoyl
Lipids.
were
Calphostin
purchased
glycerol
(DiC8-
C (cal
from
isolation
Neutrophils were isolated from healthy donors by using Ficoll-Hypaque density gradient centrifugation, as described previously [22]. After mentation of erythrocytes the buffy layer was layered over a cushion
10
Fig. 1. Induction phils (1 x 106)
ofneutrophil were incubated
containing
30
20
11n
(mm)
superoxide in a final
release volume
sediof
Ficoll-Hypaque The neutrophils
(3 ml) in 16 x 25-mm disposable were pelleted by centrifugation
Contaminating ride. Neutrophils of 1 x 107/ml mM KC1, 1.5
erythrocytes were lysed with isotonic ammonium chin. were washed and suspended at a final concentration in phosphate-buffered saline (PBS)/glucose buffer (2.6 mM KH2PO4, 0.5 mM MgCl2, 136 mM NaCl, 8 mM
NaHPO4,
5.5 mM glucose,
1 mM EGTA,
borosilicate glass tubes. at 800 g for 20 mm.
pH 7.4).
by DiC8-DAG. Neutroof I ml of PBS/glucose
final concentrations
indicated
C),
Calbio.
(LaJolla, CA). [ p1 ATP (3000 Ci/mmol) was from New England (Boston, MA) and diacylglycerol kinase was from Lipidex. All reagents were from Sigma Chemical Co. (St. Louis, MO).
Neutrophil
0
buffer
acid (DiC8-PA) from
of an aqueous
suspen-
of DiC8-DAG. with I mg/mI cytochrome c, as described in Materials Methods. The change in the absorbance due to reduction of cytochrome C by the released superoxide was monitored continuously at 550 sion and
nm by using
a temperature
(37C)-controlled
spectrophotometer.
generating enzyme. Consistent with this hypothesis, superoxide release initiated by exogenous phosphatidate starts after a lag phase or activation period of 5-20 mm at 37CC, whereas responses to diacylglycerol commence much earher [8]. However, it remains unknown whether phosphatidic acid exerts effects independent of diacylglycerol that participate in the activation of the superoxide generating system or induction ofother cellular functions. Important information regarding the events involved in neutrophil activation have been obtained from studies of neutrophil priming where cells are exposed to suboptimal levels of one agonist before addition of a second stimulating ligand. For example, priming of neutrophils with relalively
low
concentrations
of
phorbol
myristate
I
acetate
(PMA) [17-19] or diacylglycerol [20] enhances superoxide release subsequently stimulated by formyl-methionylleucyl-phenylalanine (FMLP) and leukotriene Ri. By altering incubation conditions during priming or assessing the influence of priming on individual cellular components, it is possible to derive information pertinent to both the mechanism of priming and the mechanism by which the secondary has been
stimulus
exerts
its direct
effects.
For
demonstrated that cytokine priming phil responses to FMLP correlate well with tion of FMLP receptor levels [21]. PMA responses in other systems has been found to with activation of neutrophil protein kinase enzyme involved in the stimulus-response ated by a number ofagents [20]. In this study oxidative
responses
of
neutrophils
and
example,
it
of neutrothe up-regulapriming of be associated C (PKC), an pathway initiwe found that
neutrophil
cyto-
plasts induced by phosphatidic acid were markedly amplifled when the cells were preexposed to substimulatory levels of PMA. We therefore investigated the potential role of ecto-PA phosphohydrolase and PKC activation in PMAinduced priming ofthese responses to phosphatidic acid.
190
Journal
of Leukocyte
Biology
Volume
58,
August
1995
0
10
20
30
40
Time (mm) Fig.
2.
Priming of DiC8-PA-induced superoxide release. Superoxide release was assayed continuously at 37’C. In the top panel cells were stimulated with the indicated concentrations of D1C8-PA, dispersed into aqueous suspension. In the bottom panel cells were primed with I nM PMA before stimulation with indicated concentrations ofdispersed DiC8PA. Superoxide release induced by priminglevels ofPMA alone are shown for comparison (#{149}-S).
1.
TABLE
Rate
and
Activation
Time
of Superoxide
Release
by Neutrophils
and
Neutrophil
Cytoplasts
Superoxide Activation
Neutrophils
25 sM
-
Cytoplasts
21.5
DiC8-PA
25 tM
+
100
sM
DiC8-PA
+
100
jiM
DiC8-PA
(nmol/inin)
± 2.00
2.05
(2) 4.5 ±0.71c (2) 7.9 ± 1.80 (10) 2.0 ± 0.7i (4)
DiC8-PA
-
100 jsM D1C8-PA
-
Rate
(mm)
Stimulus
Priming”
Cell preparation
releaseb
time
347
±
100 sM
1.80 ± 0.48
1.30 ± 0.25
#{176}Where indicated (+), cells or cytoplasts ,v alues are mean ± SD of the number induced alterations in rates and activation P < . 001. dp
were preincubated of determinations times of superoxide
DiC8-PA
with I nM PMA shown in parentheses. release.
0.73
before stimulation The Student’s
(5)
± 0.16 (8)
with t-test
014d
(2) 5.43 ± 1.30 (10) 11.9 ± 2.85c (4)
(5) +
± 0.20 (2)
2.47 ± 0.97e (8)
either 25 or 100 sM was used to determine
DiC8-PA. the significance
of
.oi.
Isolation
of neutrophil
Enudeated neutrophil [23]. Briefly, neutrophils
cytoplasts
Phosphatidic later use.
cytoplasts were prepared as described previously were suspended in a prewarmed (37’C) 12.5%
Ficoll solution containing 20 lsM cytochalasin B and preincubated for 30 mm at 37’C. This neutrophil suspension (4 ml) was then layered over a prewarmed (37C) discontinuous Ficoll density gradient consisting of 4 ml of 25% and 4 ml of 16% Ficoll and containing 20 jsM cytochalasin B throughout the gradient. The gradient was centrifuged at 37’C for 30
mm
in an ultracentrifuge
at the plaits
interface
were washed remove cytochalasin 1 x 107/ml
(Beckman)
between
12.5%
at 81,000
and
16%
extensively (five times) B and finally suspended
in PBS
glucose
g (r”)
Ficoll
was
and
the fraction
collected.
The
cyto-
with PBS/glucose buffer at a final concentration
to of
buffer.
Assays of superoxide release were conducted by continuously monitoring the change in absorbance at 550 nm due to reduction of cytochrome C by released superoxide, as described previously [22]. Neutrophils or cytoplasts (1 x 106) were incubated in a final volume of 1 ml of prewarmed PBS/glucose buffer containing 1 mg/mI cytochrome c. The entire reaction was conducted in a temperature-controlled (37’C) spectro-
photometer.
After
was
by adding
initiated
a short
were also dissolved of DiC8-DAG and tions
were
prepared
prepared
exceed
0.2%,
release.
In these
were
by
sonication
that
had
experiments in
the
the time
stimulus peroxide response reduced ments;
preincubation
superoxide
DiC8-PA,
in
sulfoxide,
a level
included
indicates
(2 mm)
DiC8-DAG,
or PMA.
production
Inhibitors
of PKC
in dimethyl sulfoxide. In these experiments DiC8-PA were dried under nitrogen and
in dimethyl
control
required
PBS.
PMA
stock
solutions
the final concentration no
influence
equal
on
assays.
The
to produce
of which
priming
concentrations
aliquots stock solu-
or
superoxide
of dimethyl
activation
absorbance
time
were
did not sulfoxide
(lag
the
of [P]
nents
in
[24]. the
Phosphatidic
organic
chloroform/methanol/20% acid
band
was
eluted
with
Radioactive
DiC8-PA,
methanol
and
stored
in aliquots
for
assay
generated
as described
above,
was sonicated
into
phosphatidic acid phosphohydrolase assay buffer (150 mM NaCI, I mM EGTA, 10 mM HEPES, pH 7.2) along with nonradioactive DiC8-PA, which was added to the radioactive material at a final concentration of 10 mM. Phosphatidic acid phosphohydrolase assays were conducted by incubating 100 jsM of this substrate with 1 x 106 neutrophils in PBS/glucose buffer at 37C for 15 mm. The reaction was stopped by adding a mixture of chloroforn/methanol/HCl (1:2:0.03) and then separated
organic
and
aqueous
phases
by adding
equal
amounts
of chloro-
form and water. The aqueous phase was washed twice with chloro. form/methanol (2:1), and the release of [52p] inorganic phosphate resulting from the action of ecto-PA phosphohydrolase [8] was determined by Cherenkov counting of aqueous phase in a scintillation counter. Preliminary experiments* verified that under the conditions used 32Pi release effected by intact cells was linear over a range of 2.5 x iO-i.5 x 106 cells/assay. With 1 x 106 cells/assay phosphatidic acid hydrolysis increased linearly with incubation periods up to 60 mm at 37’C. The rate of phosphatidic acid hydrolysis followed classic Michaelis Menton kinetics as substrate concentration was varied over a range of 0-2 mM, with half-maximal hydrolysis obtained with a concentration of
approximately rates
1 mM
decreased
DiCS-PA.
linearly
with
no
At lower
PA concentrations
apparent
cooperativity
reaction
observed.
Cell viability of cells
determined an equal (Sigma) pressed
in the presence
of various
agonists
and
inhibitors
was
by trypan blue exclusion. Cell suspensions were mixed with volume of 0.4% trypan blue in Hanks’ balanced salt solution and examined microscopically for dye uptake. Results are cxas the percentage of cells (±sD) that excluded dye.
RESULTS
AND DISCUSSION
DiC8-PA
D1C8-DAG (10 nmol) was dried along with 100 jimol of phosphatidylethanolamine and 100 jsmol of cardiolipin under nitrogen gas. The dried material was suspended in 350 sl of reaction buffer (100 mM NaCl, 50 mM Tris base, 10 mM MgCl2, I mM -mercaptoethanol, 0.7% Triton X-100, pH 6.6) by brief sonication. Phosphorylation of DAG was initiated by adding 10 sg ofDAG kinase and 250 isCi ofcarner-free [32P1 ATP. After 30 mm at 37C the reaction was stopped with chloroform/methanol/HC1 (1:2:0.03) and the lipids extracted as described
previously
was
phosphohydrolase
Viability
period)
at 550 nm after
was added. After, the activation period rates of sustained surelease were calculated from the initial linear region of the curve by using an extinction coefficient of 21.1/mM/cm for cytochrome c. The illustrated results are representative experisimilar results were obtained in at least two other assays.
Synthesis
ecto-PA
into
release
Superoxide
acid
located
phase
acid by
was
using
methylamine by autoradiography
separated
from
a solvent
system
(60:35:10). and
scraped
The
other
Effects
release
by neutrophils
and DiC8-PA
on superoxide
and enucleated
neutrophil
cytoplasts The addition sulted in an
of exogenous DiC8-DAG to neutrophils immediate burst of superoxide release
rein a
compo-
consisting
of
*Man,
phosphatidic from
of DiC8-DAG
the
silica.
Siddiqui
MI.,
Siddiqui,
R. A., Garcia,J.G.N.,
English,
D. Manuscript
in
preparation.
et al.
Phosphatidic
acid
stimulation
of superoxide
release
191
[23]. Neutrophil cytoplasts responded to 100 with an activation period that was considerthan the activation time of unprimed neutro1). This decrease may have been the result of fusion of granule material with the plasma membrane during cytoplast preparation. Moreover, the activation penod of cytoplasts was further shortened by PMA priming. However, unlike results obtained with intact cells, PMA priming had no significant influence on the rate of superoxide generation by cytoplasts stimulated with DiC8-PA (Table 1). responses
U)
a)
.tM DiC8-PA ably shorter phils (Table
3.0
0
E
2.5
C 0 ti) N
2.0
>‘
0
1 .5
0 >‘
I
1 .0 0.D
0.5 0.0
0
5
10
15
Time
Fig. 3. Effects of PMA on ecto-PA phils. Enzyme assays were conducted with intact resting or PMA-stimulated
The
results
reflect
25
30
(mm)
phosphohydrolase by incubating neutrophils
stopped by addingchloroform/methanol/HC1(1:2:0.03)and of radioactivity in the aqueous phase was
spectrometry.
20
the mean
activity in neutro100 jsM [51P1 DiC8-PA at 37C. Reactions were
determined ± SEM of three
the amount by scintillation experiments.
0
It)
It)
4
dose-dependent manner up to 5 j.tM (Fig. 1). Concentrations of DiC8-DAG greater than 5 j.tM had inhibitory effects on superoxide release. In contrast, DiC8-PA-induced superoxide release began after an activation time of 5-20 mm at 37’C (Fig. 2). Higher concentrations of DiC8-PA resulted in a shorter activation time as well as a faster rate of superoxide release (Table 1). When neutrophils were first primed with substimulatory concentrations of PMA ( 1 nM), the burst of superoxide released on treatment with DiC8-PA began after a considerably shorter activation time. As shown in Figure 2, after priming the activation time required for superoxide release was almost eliminated when cells were stimulated with 50-200 p.tM DiC8-PA. In addition, the rate of superoxide release evoked by DiC8-PA in PMA-primed neutrophils was considerably faster than the rate evoked by DiC8-PA in neutrophils that were not preexposed to DiC8-PA (Fig. 2). Table 1 presents a quantitative comparison of superoxide generation by PMA-primed and otherwise untreated neutrophils in response to 25 and 100 tM phosphatidic acid. PMA priming significantly shortened the activation time and increased the rate of superoxide release by neutrophils stimulated with either concentration of agonist. In ancillary experiments we used neutrophil cytoplasts to assess the possible role of degranulation in the priming of responses to phosphatidic acid. Although these cellular organelles
possess
a limited
amount
of granular
material,
they neither up-regulate the membrane content ule markers nor do they expel granule contents bolic stimulation and have been successfully assess the role of degranulation, per se, in specific
192
Journal
of Leukocyte
Biology
Volume
58,
August
of granon metaused to cellular
1995
C) C (5 .
0 U)
4
Time Fig. 4. Inhibitory superoxide release
effects
of
propranolol
(mm) on
superoxide
release.
in
A
was stimulated by the addition of 100 tM DiC8-PA to PMA-primed neutrophils. In B superoxide generation in previously untreated neutrophils was stimulated with 100 sM D1C8-PA. C depicts the influence of propranolol on superoxide generation stimulated with 5 pM DiC8-DAG.
Stimulus
.
Di C8-PA’
Di C8-PA
Di C8-DAG
Inhibitor
*CaIC
0 It)
In
4 (5 C)
C (5 .
0
4
.
,
ff
0.8
iI
0.4.
A
.
Af
!
02 .
A
f
#{149},,
A
I
j
I
4aA
-
A
#{231}P5OgJM
!
A
-.*
Acyl MG
4
dia
,I_-
.1
#{149}‘#{231}A
f#{149} Control
cf#{243}OO
I
ro
06
.T
I
4 t
0
10
20
30
40
0
10
20
30
40
0
10
20
30
40
Time (mm) Fig. 5. Effects of PKC inhibitors release was assayed by incubating untreated cells.
Effects
of PMA on neutrophil
on superoxide PMA-primed
ecto-PA
release. neutrophils
Cells
were preincubated with 100 jsM DiC8-PA
phosphohydrolase
with cal C (top), H-7 (A), 100 sM DiC8-PA
ing of superoxide increased catalysis plasma membrane
activity The above results are consistent with the hypothesis that PMA priming is the result of activation of ecto-PA phosphohydrolase, resulting in accelerated conversion of DiC8-PA into DiC8-DAG. We tested this hypothesis by directly examining the influence of PMA priming on the activity of phosphatidic acid phosphohydrolase in intact neutrophils. PMA-stimulated or resting cells were incubated with 2P] DiC8-PA for various time periods and release of [S P] Pi was quantitated. As shown in Figure 3, hydrolysis of [32P] DiC8-PA effected by resting neutrophils proceded at a rate similar to those affected by neutrophils preexposed to 1 nM PMA. Preincubation of neutrophils with higher concentrations of PMA (10 nM) also had no effect on hydrolytic activity. Thus, PMA prim-
Siddiqui
(middle), (B), or
or acyl MG (bottom). 5 sM D1C8-DAG (C)
Superoxide in previously
release does not appear to result from of phosphatidic acid by neutrophil phosphatidic acid phosphohydrolase.
Effects of propranolol
on superoxide
release
We next examined the influence of propranolol on the priming of neutrophil responses to phosphatidic acid because this agent is an effective inhibitor of cellular phosphatidic acid phosphohydrolase. We reasoned that if PMA enhanced neutrophil responses to phosphatidic acid by accelerating its conversion to diglycerides, phosphatidic acid phosphohydrolase inhibitors, such as propranolol, should inhibit priming but not necessarily effect direct responses to other stimuli, such as DiC8-DAG. Indeed, as shown in Figure 4, propranolol effectively inhibited PMA priming of superoxide release. Propranolol also
et al.
Phosphatidic
acid
stimulation
of
superoxide
release
193
TABLE
2.
Effect
of Protein
Kinase
C Inhibitors
on
Neutrophil
Viability Inhibitor
No inhibitor Neutrophil
pM
(%
DiC8-PA
95.0
± 1.4
84.5
± 2.4
5 pM
DiC8-DAG
92.5
± 1.5
I
PMA
92.5
± 2.1
nM
aResults treated
with
are
mean
10 sM
and
SD of 4 determinations
calphostin
C (cal-C),
250
90.4 91.1 90.0 87.8 made
pM
H-7,
with or
either
100
pM
inhibited direct stimulation of superoxide release phosphatidic acid, consistent with the hypothesis phosphohydrolase-mediated conversion of phosphatidic acid to diacylglycerol is required for this response as However, the drug also strongly inhibited superoxide lease stimulated by DiC8-DAG. Thus, the influence propranolol on superoxide release is not specific and sumably does not result from selective inhibition of PA phosphohydrolase.
Effects of PKC inhibitors phosphatidic acid
on priming
of responses
Journal
of
Leukocyte
Biology
Volume
58,
August
viable
80.4
± 0.84
82.8
± 2.0 ± 0.96
88.5
acylmethyiglycerol
by that well. reof preecto-
to
1995
± 1.3
86.4
± 2.7
± 0.96
80.3
± 2.4
± 1.8
85.0
± 2.5
± 0.58
80.0
± 2.0
91.2
neutrophils
with the
stimulated (acyl
Acyl MG
cells)
± 3.2
untreated
Phosphatidic acid is known to directly activate PKC [25] and its hydrolysis product, diacylglycerol, is a second messenger that potently activates this enzyme [16]. Inhibition of superoxide release with propranolol may have resulted from the drug’s effect on PKC, because propranolol inhibits PMA-mediated release of superoxide in human neutrophils by inhibiting PKC [26]. To further explore the role of PKC in the response of neutrophils to phosphatidic acid, we used the PKC inhibitors cal C, H-7, and acyl MG. Each of these agents inhibited superoxide release elicited by DiC8-PA in cells previously exposed to low levels of PMA (Fig. 5). Their effects in this regard were similar to their effects on superoxide release stimulated by either phosphatidic acid or diacylglycerol mdividually. Each of the three PKC inhibitors used had discernable effects on neutrophil viability assessed by exclusion of trypan blue dye (Table 2). However, even at the highest concentration used, these effects were relatively low and not markedly enhanced by concurrent cellular stimulation. PKC may therefore be involved as a common intermediate in the signal transduction pathway activated by each of the stimuli. In conclusion, this investigation demonstrates that suboptimal concentrations of PMA potentiate neutrophil oxidative responses to phosphatidic acid, a response that is due in large part to diacylglycerides generated when phosphatidic acid is hydrolyzed by ecto-PA phosphohydrolase. PMA priming, however, does not result from increased activity of this enzyme but rather seems to result from potentiation of an intermediate involved in the cells’ response to multiple stimuli. The effects of PKC inhibitors on superoxide release stimulated by both phosphatidic acid and diacylglycerol are consistent with the view that a common signal transduction pathway involving PKC is activated by each stimulus. Thus, phosphatidic acid may exert its effects after hydrolysis to DAG by plasma membrane phosphatidic acid phosphohydrolase, as proposed by Perry et al. [8]. It is also possible that phosphatidic acid has some direct effects on neutrophils
194
H-7
stimulus
None 100
cal-C
MG)
and
that involve PKC further investigation.
then
indicated
with
incubated
activation
or with
agonists
and
the
neutrophils
indicated
this
agonists.
possibility
merits
ACKNOWLEDGMENTS This
research was supported by a Grant-in Aid to R.A.S. American Heart Association, Indiana Affiliate, by a grant to D.E. from the Phi Beta Psi Sorority, by grants from the Veterans Administration and National Institutes of Health to J.G.N.G., and by National Institutes of Health grant Al 25656 awarded to D.E. The authors thank Stephanie McGillem for secretarial support.
by the
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