C and phospholipase. D are activated independently of each other in chemotactic peptide-stimulated human neutrophils. Theodore. J. Mullmann,. Boonlert.
Phospholipase
C and phospholipase
independently human
of each
in chemotactic
peptide-stimulated
neutrophils Theodore J. Mullmann, Robert W. Egan, and Schering-Plough
Abstract:
other
D are activated
cytochalasin
Institute,
Cheewatrakoolpong, BiIlah
Kenilworth,
B-treated
New
C. Anthes,
Marvin
I. Siegel,
Jersey
late
most PLC,
[2].
In
containing phosphoglycerides to form choline and diradyl-sn-glycero-3-phosphate (phosphatidic acid), suggesting a possible link between PLC and PLD. However, in the absence of cytochalasin B or extracellular Ca2, PLC was fully activated by fMLP with minimal activa-
potent
tion of PLD, sufficient for
down might be necessary for PLD activation. Because PKC inhibitors such as K252a and staurosporine do not inhibit PLD in fMLP-stimulated neutrophils [9-il], it is likely that diacylglycerol and Ca2 mobilized during fMLP stimulation act directly at the level of PLD and not through PKC. Subse-
indicating that PLD activation.
required
the
PLC Full
simultaneous
neutrophils
John
stimulated with fMet-Leu-Phe (fMLP) in the presence of Ca24, phospholipase C (PLC) activity, as measured by inositol-1,4,5-trisphosphate (1P3) formation, preceded phospholipase D (PLD)-catalyzed breakdown of choline-
fMLP
When
Research
Boonlert M. Motasim
activation activation
were
alone is not of PLD by
presence
of both
Ca24
and cytochalasin B, a condition that caused no further enhancement of PLC. This result suggests that PLD products are not involved in the regulation of PLC activation. tion there
activate PLD in the absence of PLC. Treatment of intact neutrophils with pertussis toxin inhibited both PLC and PLD, with PLC inhibition occurring at lower concentrations than PLD inhibition. These differential effects of pertussis toxin and the observed lack of inhibition of
distinct
G-proteins.
Taken
together,
suggests that, in fMLP-stimulated PLD are activated through Leukoc. Biol. 53: 630-635; Key Words: G-proieins calcium diglycerides
pert
is believed activation, receptors
these
to inimply through
observations
neutrophils, PLC independent mechanisms. 1993. ussis
toxin
signal
are
phorbol
Ca24
ionophore
diacylglycerol
ester
activators
phorbol
A23187,
the
are
with the phospholipase
in many
cells
accumulations
of PLD
(PMA)
including
neutrophils
products,
PA and
rapidly substrates.
Biology
pepand
activated to hydrolyze PLC hydrolyzes (PIP2) to produce 1,2-
Volume
53, June
perand
13-acetate
[2, 6, 9]. More important, 1-oleolyl-2-acetylglycerol PMA act synergistically with A23187 to activate granulocytes [9], raising the possibility that initial in diacylglycerol and Ca2 due to phosphoinositide
are
and PLD in increases break-
DG,
in large
quantities, may, in turn, exert modulatory effects on PLC. This possibility is indicated by the fact that the addition of PA to fibroblasts activates PLC [12, 13] and that PMA, a diacylglycerol mimic, inhibits PLC in many cells including neutrophils [14, 151. The objective of the present study was to examine the relationship between PLC and PLD in fMLP-stimulated neutrophils. By using both kinetic and pharmacologic proaches, we have obtained data suggesting that activated in the absence of PLC, that PLD products involved in PLC activation, and that PLC and PLD pled to fMLP receptors through distinct G-proteins.
MATERIALS
transduction
chemotactic C (PLC)
cell
1-oleolyl-2-acetylglycerol,
12-myristate
of PLD
PLD also stimuCa2 mobilization
apPLD is are not are cou-
AND METHODS
Materials 1-O-[ 3H]Alkyl-2-lyso-sn-glycero-3-phosphocholine mmol) and I-O-alkyl-2-lyso-sn-glycero-3-phospho[3H]choline (79 Ci/mmol) were purchased from Dupont-NEN.
stimulated (fMLP),
of Leukocyte
the
activate and
J.
diacylglycerol and inositol-1,4,5-trisphosphate (1P3) [1], and choline-containing phosphoglycerides (PCs) are hydrolyzed by PLD to produce diradyl-sn-glycero-3-phosphate (phosphatidic acid, PA) and choline [2, 3]. This PA is then metabolized by PA phosphohydrolase to l,2-diradyl-snglycerol (DG) [4-6]. IP3 mobilizes Ca2 from intracellular stores [7], and DG acts in concert with Ca2 to activate protein kinase C (PKC) [8].
Journal
cells, agents that to DG production
addition, synthetic
binding
phospholipase D (PLD) specific phospholipid phosphatidylinositol-4,5-bisphosphate
630
the
other leading
and
INTRODUCTION When neutrophils tide fMet-Leu-Phe
meant
quent
Furthermore, under conditions of complete inhibiof PLC by phorbol 12-myristate 13-acetate (PMA), was no inhibition of PLD, showing that fMLP can
fMLP-stimulated PLD by PMA, which activate G-proteins involved in PLC that PLC and PLD are linked to fMLP
In
1993
lipids
kits were
were purchased
obtained from
from Avanti
(100
1P3
Amersham.
Unlabeled
Polar
Precoated
Lipids.
Ci/
Abbreviations: fMLP, formyl-Met-Leu-Phe; CB, cytochalasin B; PLC, phospholipase C; 1P3, inositol-1,4,5-trisphosphate; PLD, phospholipase D; PC, choline-containing phosphoglycerides; PA, diradyl-sn-glycerol-3-3phosphate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PT, pertussis toxin; DG, diradyiglycerol; alkylacyl-GP, [1-O-alkyl]-2-acyl-snglycero-3-phosphate; alkylacyl-GPC, l-O-alkyl-2-acyl-sn-glycero-3-phosphocholine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EGTA, jethylene-bis(oxyethylenenitrilo)ltetraacetic acid. Reprint requests: TheodoreJ. Mullman, Schering-Plough tute, 2015 Galloping Hill Road, Kenilworth, NJ 07033-0539. Received January 7, 1993; accepted February 5, 1993.
Research
Insti-
silica
gel
Pertussis
G (0.25 toxin
purchased
mm thick) was purchased (PT) was from Calbiochem.
from
Pharmacia-LKB
cytochalasin B (CB), PMA, and all other Chemical Co.
Isolation
Biotechnology.
fatty acid-free reagents were
and labeling
from Whatman. Ficoll-Paque was
of human
bovine purchased
Assays
fMLP,
serum
albumin, from Sigma
the 1-O-alkyl chain or the choline moiety as described previously [10]. Briefly, cell mixed with either 1-O-alkyl-2-lyso-sn-glycero-3(1 tCi/107 cells) or 1-O-alkyl-2-lyso-sn-
glycero-3-phospho[ tion for 45 HEPES-saline
beled
cells
Incubation
of sus-
5H]choline (2 1tCi/107). Following incubaat 37#{176}C, cells were washed twice with buffer to remove unincorporated label. La-
then
resuspended
in the
done
same
buffer.
in
duplicate
was performed representative
or
triplicate.
at least experiments.
twice.
The
Each
set
data
presented
of ex-
AND DISCUSSION
PLC and PLD are activated independently in fMLP-stimulated neutrophils
of each other
To examine the temporal relationship between the activation of PLC and PLD, human neutrophils were labeled in alkylacyl-GPC with 3H in the 1-O-alkyl chain. To measure simultaneously the two products of PLC and PLD, [3H]alkylacyl-GPC-labeled cells were stimulated, cell reac-
tions
mm were
were
periments are from
RESULTS
neutrophils
Neutrophils were obtained from human blood that was collected in the anticoagulant acid-citrate-dextrose as described [16]. Isolated neutrophils were suspended in HEPES-saline buffer containing 125 mM NaC1, 0.7 mM MgC12, 10 mM glucose, 0.5 mM EGTA, 1.0 mg/mI fatty acid-free bovine serum albumin, and 25 mM HEPES, pH 7.4, and labeled with 3H in either alkylacyl-GPC pensions were phosphocholine
Data presentation
were
stopped
with
perchloric
acid,
and
the
resulting
precipitate was separated from the supernatant. The lipidcontaining pellet was analyzed for [3H]alkylacyl-GP (PLD activity) and the supernatant was analyzed for IP3 mass (PIP2-specific PLC activity). Activation of PC-specific PLC has been shown in other systems [2]. However, by labeling
conditions
In some experiments, cells were incubated with PT for 2 h at the indicated concentrations. Assay conditions were essentially as described [4-6]. Briefly, assay mixtures in a final volume of 250 1d contained 1.5 mM CaCl2 and 5 tM CB (unless indicated otherwise). In some experiments, PMA was also added to the assay mixture. To these mixtures were then added prelabeled cells (5 x 106) and the suspensions were incubated for 5 mm (37#{176}C) before initiating the reactions by adding either 100 nM fMLP or buffer. At the appropriate times, the reactions were stopped by either perchloric acid for IP3 measurements or chloroform-methanol (1:2 by volume) for lipid analyses. The final concentration of dimethyl sulfoxide in the assays did not exceed 0.1%, a concentration that had no discernible effects.
Simultaneous
measurement
G) 0 F-
a 1
0
E 1) 0
of PLC and PLD
To measure IP3 mass and 1-O-[3H]alkyl-2-acyl-sn-glycero-3phosphate ([3H]alkylacyl-GP) simultaneously, reaction mixtures containing [3H]alkylacyl-GPC-labeled cells and other appropriate additions were stopped with 50 d of ice-cold 20% perchloric acid. After 20 mm on ice, these centrifuged at 2000g for 30 mm. The resulting were then analyzed for IP3 mass as described lipid-containing extracted with
Cl)
pellet was suspended chloroform-methanol
in 250 (1:2 by
samples
were
E
a-
supernatants
below
and
jd of water volume).
the and
1P3 mass assay
10000
>. 0 15
=
The supernatants of perchloric acid-treated cells were neutralized by the addition of 16 il of 10 N KOH followed by 25 d of 1 M Tris, pH 9.0. The pH of the samples was measured and samples that remained acidic were adjusted to pH 9.0. The precipitates of these neutralized samples were removed by centrifugation for 30 mm at 2000g. Aliquots (100 d) of these supernatants were then analyzed for 1P3 quantification, using 1P3 binding kits.
Assay of PLD products
C,,
TIME, Fig.
1.
Time
stimulated described labeled
by the and
Mullmann
the
cells,
IP,
CaCl2, of buffer (0)
1.5 mM
were
reactions
were quantified raphy, respectively
ci aL
of
neutrophils. in Materials
fore the addition Phases from the lipid-containing pellet were separated procedure of Bligh and Dyer [17]. [3H]Alkylacyl-GP [3H]choline were measured as described [10].
and Neutrophils and Methods.
course
stopped
and or
with
were 5 iM 100
CB nM
perchloric
Phospholipase
described
in
Materials
D/phospholipase
formation
labeled
Samples
by an IP, receptor-binding as
sec
(3H]alkylacyl-GP
in
IMLP-
in 5H]alkylacyl-GPC (250
were
pi)
containing
preincubated
as 5
for
5 mm
x
106 be-
(#{149}). At the indicated times, acid. IP, and (‘Hlalkylacyl-GP kit and by thin-layer chromatog-
IMLP
and
Methods.
C relationship
631
TABLE
1.
Effect
of
Extracellular
Ca2
[5Hlalkylacyl-GP
on
fMLP-Stimulated
IP,
120
and
C
Accumulation.
0
Additions
IP, (pmol/IO’
cells)
‘H]Alkylacyl-GP
(dpm
x
100
10’)
E EGTA EGTA EGTA EGTA
0.8
±
0.2
2.7
±
0.2
+
IMLP
5.0
±
0.4
4.3
±
0.4
+
Ca2
1.0
±
0.3
3.3
±
0
+
Ca2
6.1
±
0.2
12.0
±
0.2
IMLP
+
0 80 C’)
a60
Using
the
samples
and
containing 5 sM no added Ca’
either
fMLP.
GP.
methodology
Reactions
These
were
products
described in the were preincubated
CB or
1.5
stopped
were
mM
Ca2,
after
then
legend
for
10 s for
IP,
and
30 s for
as described
0
1, duplicate 0.5 mM EGTA
5 mm with adding buffer
before
quantified
of Figure
or
100
C
nM
[‘H]alkylacyl-
for
Figure
.2
4#{176}
1. 20
studies lation PLC PLC In
[4] [18],
lack of phosphocholine concluded that PC
in fMLP-stimulated neutrophils. activity was measured. the
presence
produced
of
neither
of fMLP, mum
and by the it has been
1 mM
1P3
and
as
early
rapidly
mass accumudegraded by
only
0
PIP2-specifIc
PMA,
CB, neutrophils Upon addition
5 M
[3H]alkylacyl-GP.
1P3 accumulated 10 s and declining
at
Thus
Ca24
nor
is not
as 5 s, reaching
a maxi-
thereafter
1).
(Fig.
Fig. 3. PMA
inhibition
the methodology described in taining 1.5 mM CaCI,, 5 sM were preincubated for 5 mm After 10 s, the reactions were Figure 1.
The
IP3,
0
reaching
followed
a maximum
PLC-derived Ca2 in PLD On the stimulated Ca2, small PLC
within
diacylglycerol activation.
30-60
Using
the
of
accumulation
s. These
data
confirm
intracellular
IP3
was
produced in
to a near-maximal (Table is not sufficient to
alone
In
experiment,
addition
neutrophils and no
EGTA
level with 1), suggesting
[3H]alkylacyl-GP
activation this
mobilization
fully
of Ca2
activate
in excess
of were added only a that PLD
of EGTA
enhanced [3H]alkylacyl-GP formation sixfold with only minor increases in 1P3 accumulation, further indicating that PLDderived products are inconsequential to PLC modulation. Additional support for lack of PLC modulation by PLD products comes from the observations that wortmannin, protein tyrosine kinase inhibitors, and agents that elevate cellular cyclic AMP levels inhibit PLD [11, 20, 21] while having little or no
E
a. 10000
effect
on
more,
PLC
(ref.
-I
C’) -S
but also by fMLP 0
2
4
6
CYTOCHALASIN Fig. 2. Effect of CB
on
neutrophils.
IP,
and
mass
Using
the
Figure 1, duplicate samples containing concentrations of CB were preincubated buffer (0) or 100 nM fMLP (#{149}). After
I‘Hlalkylacyl-GP,
the
as described
Journal
reactions
in Materials
of
Leukocyte
were
1 2
B, tM
[‘Hlalkylacyl-GP methodology
1 0
8
in IMLP-
formation described
in
the
legend
of
1.5 mM CaCl, and the indicated for 5 mm before the addition of 10 s for IP, mass and 30 s for stopped
and
Biology
and
these
products
were
Volume
53, June
1993
and
of
of PLC. observation of PLC,
our
unpublished
neutrophils
with
These that needed
CB (Fig. 2).Thus, in Ca2-containing
when neutrophils buffer in the
like
PMA
fMLP,
was
added
as
protein
activation Ca2
kinase
are
IMLP-induced the presence
maximal with a formation. CB augmented a dose-dependent manner accumulation (see also ref. that the effects of CB and are not attributable to potent activator of PLD in
ogenously
data). PA
conclusions
production [3H]alkylacyl-GP generation in effects on IP3 ther indicate PLD activation PMA is a
such
Methods.
19
premncubation
the activation rated by the PLD, but not
4
stimulated
and
other hand, when CB-treated in the presence of 0.5 mM
increase
activity.
632
neutrophils.
the legend of Figure 1, duplicate samples conCB, and the indicated concentrations of PMA before the addition of buffer or 100 nM fMLP. stopped and analyzed for IP, as described for
the previously reported conclusion that PLC precedes PLD in fMLP-stimulated cells [19]. This temporal relationship between PLC and PLD and the fact that the combined addition of the membrane-permeable diacylglycerol 1-oleoyl-2-acetylglycerol and the Ca2 ionophore A23187 to neutrophils activates PLD [9] are consistent with a role for
0
quantified
nM
fMLP-stimulated
of [3H]alkylacyl-GP
formation U)
>. 0 15
of IP, mass in
CB inhibitors
nor
of [9].
Further-
effect on further corroboactivation of of not only Ca24 had
were absence
no
stimulated of CB, 1P3
small
increase in [3H]alkylacyl-GP while having minimal 22). These data furextracellular Ca24 on PLC. neutrophils [9]. Un-
PLD requires neither exMore important, agents and PT exert differential
30000
pretreatment (Fig.
did
4),
enhance
fMLP’s
that PMA of its action of PLD
alkylacyl-GP)
ability
to activate
and products are incapable
PLD
(DG and of replacing
CB. These data support the view that PMA and fMLP utilize two distinct PLD activation mechanisms which can operate in parallel and that there is little crosstalk betwen these mechanisms. Thus, the observation that the fMLPstimulated mechanism of PLD activation remains fully func-
20000
E
not
indicating
tional mediated
10000
in
the absence mobilization
of PLC would of DG and Ca24
activation. suggested
Similar in other
-J
epidermal detectable
growth PLC
0
bryonic
C., I
neomycin, an aminoglycoside antibiotic that inhibits PLC by complexing with phosphoinositides, inhibited PLC completely while having only a minor effect on PLD [24]. However, incubation of neutrophils with neomycin (10 mM) for 2-4 h had no effect on fMLP-induced 1P3 accumulation or choline formation (our unpublished observation), presumably because neomycin did not permeate through the neutrophil plasma membrane. Although IP3-induced mobilization of Ca24 from intracellular pools appears to be unrelated to fMLP-induced PLD activation (Fig. 4), this PLD activation mechanism does require the simultaneous presence of both extracellular Ca2 and CB (Table 1 and Fig. 2). PLD activation by Ca24 and nonhydrolyzable GTP analogues in cell-free systems [25] is not affected by CB (our unpublished observation), suggesting that CB does not modify PLD’s interaction with Ca24 or G-proteins. It may, therefore, be hypothesized that, in intact cells, CB acts by facilitating the availability of extracellular
‘U
z = 15000
C,)
10000
5000
0
50
sec
TIME, Fig.
4.
Effect
of PMA
preincubation
on
the
I ‘H]cholmne
in neutrophils. In order to measure product that is metabolically much more
PLD the
cells
instead
were of
1.5 mM
CaCI2, for
dicated
times. (0),
5 tM
5 mm Buffer
()
addition;
with
preincubation
Duplicate
Aliquots
the
control;
PMA
samples
either
addition
from
preincubation with
and
CB,
before
IMLP-induced (‘H]choline, stable than
formation
of
the water-soluble l’Hlalkylacyl-GP,
1-O-a1kyl-2-lyso-glycero-3-phospho’HJcholine
[‘Ha1kylacyl-GPC.
cubated line.
labeled
150
100
buffer
the aqueous (#{149}) preincubation with
followed
or
of buffer
100 or
100
nM nM
5 x PMA
106
were
fMLP
for
factor activity
kidney
Ca24
containing
dissociation systems.
indicate that PLCis irrelevant to PLD
cells
to cellular
of PLD instance,
For
from PLC in Swiss
has 3T3
been cells,
activated PLD in the absence [23]. Furthermore, in human
transfected
with
muscarinic
of any em-
receptors,
PLD.
cells, preinthe
in-
phase were counted for [‘H]chowith buffer followed by fMLP
PMA followed by buffer by IMLP addition.
addition;
(U)
80 effects on PLD activation by PMA or fMLP instance, PT and protein tyrosine kinase PLD activation by fMLP but not PMA, kinase C inhibitors act in a reverse manner. tions demonstrate that PMA activates PLD
[2, 9-11, 20]. For inhibitors inhibit whereas protein These observaby a mechanism
that is distinct from that utilized by fMLP. This knowledge and the fact that PMA is a potent inhibitor of fMLP-induced PLC activation [14, 15] provide the basis for the use of PMA as a tool to examine further the involvement of PLC in PLD activation. Under our assay conditions, preincubation with PMA for 5 mm inhibited fMLP-stimulated PLC activation (as measured by 1P3 accumulation) in a dose-dependent manner with complete inhibition occurring at 100 nM PMA (Fig. 3). Alkylacyl-sn-glycero-3-phospho[3H]choline-labeled cells incubated with PMA and CB for 5 mm produced [3H]choline (Fig. 4), a result consistent with our pervious findings [10]. When neutrophils were incubated with 100 nM PMA for 5 mm and subsequently stimulated with 100 nM fMLP for various greater due
times, the levels than those observed to
of [3H]choline with PMA
produced alone. This
were increase
fMLP
was approximately equal to that produced by fMLP in the absence of PMA exposure. Of note, the rate of fMLP-stimulated choline formation remained unaffected by PMA treatment. Furthermore, in the absence of CB, PMA
Mullmann
z 0 I-
60
I
z 40
20 0.13
0.50
0.25
TOXIN,
PERTUSSIS Fig.
5.
Effect
stimulated of Figure 2 h with buffer
of
on
IP, Using
and
13H]alkylacyl-GP
the
methodology
1, [3H]alkyl-GPC-labeled the and
indicated quadruplicate
and 5 M CB 100 nM IMLP. reactions
were
Materials
and
ci al.
PT
neutrophils.
amounts
human of PT.
samples
were preincubated After 10 s for 1P3 stopped and these
1.00
Cells
pg/mI formation
neutrophils were
containing
washed 5 x
106
twice
were
D/phospholipase
legend
cells,
1.5
quantified
C relationship
as
for
PTfree
with
for 5 mm before the addition (#{149}) and 30 s for [‘Hlalkylacyl-GP products
IMLP-
in the
were incubated
Methods.
Phospholipase
in
as described
mM
Ca2,
of buffer
or
(U),
the
described
in
TABLE
2.
Effect
of PT
on
the
fMLP-lnduced
Formation
of I’HlCholine
in Neutrophils
[‘HiCholine,
dpm
x
Preincubated
with
10’
Total Additions
-
None
PMA IMLP PMA
+
IMLP
“Using the methodology described or absence (-) of PT (1 ag/ml) for cubated for 5 mm before the addition counted for [‘H]choline.
PLC and PLD might G-proteins PT the
inhibits the receptor/G-protein
for 2 h.
PT
PLC and PT treatment
0.3
3.9
±
0.2
-
0.2
12.1
±
0.1
-
-
9.6
±
6.1
±
0.1
5.6
2.2
17.7
±
0.5 0.1
14.4
±
0.1
5.3
2.0
Figure
3, cells
labeled
of buffer
in l-O-alkyl-2-acyl-sn-glycero-3-phospho[’H]choline 1.5 mM CaCI,, 5.0 jaM CB, and fMLP. At the end of 30 s, the reaction was stopped
samples
or 100
nM
through
distinct
not
inhibit
caused activities
dependently enzymes proteins.
634
of each other might be linked The data further
Journal
of Leukocyte
data and
PLC is further subpreparations. For inGTP analogues to and not PLD [28],
indicate PLD
that, are
in fMLPactivated in-
and raise the possibility that to receptors through separate suggest that the availability
Biology
1. Rhee,
Volume
53, June
these 0of ex-
1993
S.G.,
inositol
Suh
P.-G.,
Ryu,
S-H.,
phospholipid-specific
that
this
Lee,
S.Y. (1989)
phospholipase
C.
Studies
of 244,
Science
546-550. 2. Billah,
MM.,
functions
Anthes,
of
J.C.
(1990)
The
phosphatidylcholine
regulation
hydrolysis.
and
cellular
Biochein.
269,
j
281-291.
J.
3. Exton, down.
j
4. Billah, MI. the
(1990) Signaling Biol. Che-m. 265,
through 1-4.
phosphatidycholine
MM., Eckel, S., Mullmann, T.J., (1989) Phosphatidylcholine hydrolysis
phosphatidate
and
stimulated
TJ.,
C5a
activation
major
route
to the
Immunol. 144, 6. Mullmann, T.J., PMA activation
levels
Biol.
production
R.W.,
by PLD
Siegel,
determines
in chemotactic
peptide
264, 17069-17077. Egan, R.W., Billah, M.M. (1990) D in human neutrophils: a Chem.
Egan,
Inositol
and
diglycerides.
R.W., Billah, MM. D in human neutrophils
of phosphatides 170, 1197-1202.
(1987) second
break-
Egan,
of phosphatides
1901-1908. Siegel, MI., of phospholipase
production Res. Commun.
7. Berridge, M.J. two interacting
j
Siegel, MI., of phospholipase
J.
to the ophys.
diglygeride
neutrophils.
5. Mullmann,
PMA-induced
suggesting that PLC and relevant 0-proteins reside in a membrane compartment. By contrast, PLD activation by nonhydrolyzable GTP analogues may require the presence of a factor [29, 30] that is likely to be a 0-protein. Additional reconstitution studies with purified 0-proteins and PLD are needed to demonstrate that the 0-proteins for PLC and present PLC
and
REFERENCES a
fMLP was reduced by 60%, a value identical to that observed for control cells stimulated with IMLP alone (Table 2). These results demonstrate that PMA does not modify PT sensitivity of 0-proteins involved in PLD activation. The notion of unique 0-proteins for PLD and stantiated by experiments with cell-free stance, addition of nonhydrolyzable neutrophil membranes activates PLC
incubated in the presence (+) or PMA (100 nM) were preinfrom the aqueous phase were
tracellular Ca2 to PLD is critical for activation Ca24 availability may be facilitated by CB.
by uncoupling uncoupling occurs [26]. PT and PLD
-
were buffer aliquots
either and
containing
[3H]choline formation (Table 2). However, PMA is believed to inhibit PLC by inactivating PLC-associated G-proteins. Therefore, lack of inhibition of fMLP-induced PLD by PMA (Fig. 4) would indicate that PLC-linked G-proteins might not be involved in PLD activation. Other possible interpretations exist for these results, including the one that PMA may regulate signaling events distal to G-proteins. In order to determine whether PMA modifies the PT effect on PLD, neutrophils were pretreated with PT (1 jzg/ml for 2 h) and then stimulated with PMA (100 nM for 5 mm), followed by stimulation with fMLP for an additional 30 s. Under this treatment protocol, the increase in PLD activity due to
PLD are distinct. In conclusion, our stimulated neutrophils,
PT
+
±
Quadruplicate
did
PT
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±
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PLD. of neutrophils
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4.0
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