From the
Phosphatidylcholine synthesis and degradation are tightly
regulated to assure a constant amount of the phospholipid in cellular
membranes. The chemotactic peptide fMLP and the phorbol ester, phorbol
12-myristate 13-acetate, are known to stimulate phosphatidylcholine
degradation by phospholipase D in human neutrophils. fMLP alone
triggered phosphatidylcholine breakdown into phosphatidic acid, but did
not stimulate phosphatidylcholine synthesis or activation of the
rate-limiting enzyme CTP:phosphocholine cytidylyltransferase. Adding
cytochalasin B to fMLP led to some conversion of phosphatidic acid into
diglyceride, and fMLP was then able to trigger choline incorporation
into phosphatidylcholine, and cytidylyltransferase translocation from
cytosol to membranes. Inhibition of phosphatidylcholine-phospholipase D
activation with tyrphostin led to inhibition of choline incorporation.
Therefore, phosphatidic acid-derived diglyceride but not phosphatidic
acid alone was effective to promote cytidylyltransferase translocation.
With phorbol 12-myristate 13-acetate as agonist, and by selective
labeling of phosphatidylinositol and phosphatidylcholine, we
demonstrated that only phosphatidylcholine-derived diglyceride
participated in cytidylyltransferase translocation. Oleic acid
stimulated phosphatidylcholine synthesis, but induced a weak increase
in diglyceride and a slight cytidylyltransferase translocation, and did
not stimulate phospholipase D activity. Our data established that only
diglyceride derived from phosphatidylcholine degradation by the
phospholipase D/phosphatidate phosphatase pathway are required for
agonist-induced cytidylyltransferase translocation and subsequent
choline incorporation into phosphatidylcholine.
The presence of a phosphatidylcholine cycle related to the
sn-2 position of the glycerol backbone through the
deacylation/reacylation pathway has been characterized in the human
neutrophil
(1) . Evidence for cycling degradation/resynthesis
reactions concerning the sn-3 position have also been reported
in the work of Daniel et al.(2) and proposed by Pelech
and Vance (3) as a basal turnover pathway, but were rarely investigated
upon cell activation. The CDP-choline pathway accounts for the main
production of phosphatidylcholine in mammalian cells:
phosphatidylcholine is synthesized from external choline in three
enzymatic steps involving, respectively, choline kinase (EC 2.7.1.32),
CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15), and choline
phosphotransferase (EC 2.7.8.2). Cytidylyltransferase is the key
regulatory enzyme of the pathway and its activity can be regulated by
different factors
(4) . Two forms of the enzyme are present in
cells: a non-active cytosolic form and a highly active membrane-bound
form
(5) . Diglyceride, formed in cells challenged with
PMA,
The present investigation has been
undertaken on human neutrophils whose phosphatidylcholine degradation
at the sn-3 position by phospholipase D has been
characterized
(8, 9, 10, 11) . Earlier
studies on phosphatidylcholine synthesis in this cell model were mainly
related to methylation of
phosphatidylethanolamine
(12, 13) , to the effect of
anti-tubulin or anti-microfilament agents
(14, 15) , or
to phosphatidylcholine turnover during phagocytosis
(16) . In the
latter study, activation of choline phosphotransferase was reported.
However, cytidylyltransferase has not been studied yet in a
hematopoietic cell line. This study has been conducted using three
different compounds: oleic acid, the phorbol ester PMA, and the
receptor-dependent chemotactic peptide, fMLP. Oleic acid has been shown
to be a potent activator of cytidylyltransferase translocation and has
been studied as such in various models
(17, 18) . PMA
stimulates phosphatidylcholine turnover in several cell
types
(2, 19, 20) . Stimulation of
phosphatidylcholine synthesis appears to involve protein kinase
C
(21) , without implicating a direct regulation of
cytidylyltransferase by this kinase
(6, 22) . Moreover,
PMA stimulates phosphatidylcholine degradation via phospholipase D,
whose activation partly involves protein kinase C
(23) . fMLP
binds to a specific seven-pass transmembrane receptor on human
neutrophils and has been shown to induce phosphatidylcholine
degradation through phospholipase D activation
(11) , since
neutrophils are lacking phosphatidylcholine-specific phospholipase
C
(24) . Although the fMLP receptor is coupled to G-proteins,
phospholipase D activation is dependent upon the activity of tyrosine
kinases
(25) .
We now report that stimulation of
[
When
neutrophils were labeled either with
[
Phospholipase D activity was also monitored
with the choline-labeled lyso-PAF, which was reacylated in cells into
alkylacyl-glycerophospho[
Effect of Oleic Acid and Phorbol Ester on
[
In the presence of PMA,
a lower amount of cytidylyltransferase was recovered in the cytosolic
fraction of permeabilized cells, as compared to controls.
(Fig. 4A). By contrast the activity in the particulate
was increased by 16-fold (Fig. 4B). However, the sum of
total activity in particulate plus cytosol was higher than the value in
non-treated cells (Fig. 4C), indicating that binding of
cytidylyltransferase on membranes leads to an overactivation of the
enzyme, about 50% above the basal value. Nevertheless, PMA induced the
translocation to membranes of a high amount of cytidylyltransferase.
Oleic acid treatment had only a slight effect on cytidylyltransferase
translocation. When the cells were stimulated with fMLP plus
cytochalasin B, we observed the binding of cytidylyltransferase to
membranes (Fig. 4B), and no variation in the sum of
total activity between cytosol and particulate as compared to control
cells (Fig. 4C). Thus, our data show a modification of
cytidylyltransferase distribution between cytosol and membranes induced
with PMA and fMLP, in contrast to oleic acid treatment that induced a
weak redistribution of the enzyme. Relationship between Phosphatidylcholine Breakdown and Resynthesis
(Fig. 5-8)-To investigate the mechanisms involved in
the regulation of cytidylyltransferase translocation, we have analyzed
the amount of diglyceride produced in the different experimental
conditions (Fig. 5). Basal amounts of diglyceride varied from
about 100 to 300 pmol/10
To better assess whether
phosphatidylcholine-derived diglyceride alone controlled
cytidylyltransferase translocation, we differentially labeled
phosphatidylinositol and phosphatidylcholine, and followed their
respective conversion into diglyceride upon cell stimulation with PMA
(Fig. 7). Cell labeling with [
In the present work we have investigated activation of a
phosphatidylcholine cycle triggered with agonists and oleic acid in a
terminally differentiated cell. Hydrolysis of phosphatidylcholine,
induced with a variety of agonists, has been well characterized in this
cell type
(8, 9, 10, 11) .
Phosphatidylcholine has been identified as an alternative source for
phosphatidic acid and diglyceride as second
messengers
(5, 8, 9, 10, 11) ,
and an activated phosphatidylcholine cycle, analogous to that of
phosphoinositide, appears necessary to reconstitute the initial amount
of cell phosphatidylcholine
(3) . However, resynthesis of
phosphatidylcholine from diglyceride is regulated by the amount of
CDP-choline, which is produced by the enzyme cytidylyltransferase.
Therefore, the present study has been focused on cytidylyltransferase
activation and its possible relationship to phosphatidylcholine
degradation.
We show that oleic acid and PMA stimulate
[
Preincubation of the cells with the labeled choline prior to
addition of the agonist is required to observe a net effect of fMLP as
well as PMA. It suggests that a specific pool of choline might be
involved, or that some channeling would be required
(41) . The
increase of phosphatidylcholine radioactivity in fMLP-stimulated
neutrophils was strictly dependent on the presence of cytochalasin B
(Fig. 3A). Cytochalasin B has been widely used in
neutrophil studies, since it enhances cellular responses such as
degranulation or oxidative burst
(42) . In the presence of
cytochalasin B, the fMLP-induced phosphatidylcholine synthesis appears
to level off rapidly, whereas with PMA, enhanced
[
Activation of phosphatidylcholine synthesis by
receptor-mediated agonists has been rarely considered. We show that
fMLP triggers a typical cytidylyltransferase translocation
(Fig. 4). In the case of PMA an additional activation of the
membrane form of cytidylyltransferase occurs, which accounts for an
increase of about 50% of total activity (Fig. 4C). The
mechanisms leading to such activation remain to be established.
Surprisingly, only a weak difference in membrane-bound
cytidylyltransferase is observed after oleic acid treatment as compared
to control, whereas oleic acid has been reported so far as the most
potent inducer of cytidylyltransferase translocation (4). Since we
could observe a net oleic acid-induced cytidylyltransferase
translocation in undifferentiated HL-60 cells (37), but not in
neutrophils, we can suggest that the fatty acid efficiency depends upon
the stage of cell differentiation.
Among the regulators of
cytidylyltransferase activity, diglyceride has been reported to control
cytidylyltransferase translocation
(5) . At variance with all the
reports to date
(4, 6) , the fatty acid has a weak effect
on mass diglyceride formation (Fig. 5A). This is
consistent with the slight cytidylyltransferase translocation to
membranes. In contrast with growing cells
(6) , oleic acid
appears poorly metabolized into diglyceride in the human neutrophil. It
should be noticed that the effect of oleic acid and PMA on the
diglyceride generation in human neutrophil, i.e. in a
differentiated cell, is exactly the opposite of what we observed
previously in a tumor cell
(43) .
We then questioned the
origin of the diglyceride involved in cytidylyltransferase
translocation. First, with fMLP as agonist, the absence of cytochalasin
B blocks phosphatidylcholine breakdown at the phophatidic acid step
(Fig. 6A), with no subsequent effect on
phosphatidylcholine resynthesis and probably on cytidylyltransferase
translocation. This result indicates that in vivo generation
of an anionic phospholipid, such as phosphatidic acid, is not involved
in cytidylyltransferase translocation. When phosphatidic acid is
converted into diglyceride in the presence of cytochalasin B
(Fig. 6B), then cytidylyltransferase translocation
occurs. The alkaloid compound cytochalasin B, has been shown to release
calcium from an intracellular pool
(44) which might activate
phosphatidate phosphatase
(45) . The chemotactic peptide fMLP
induces the formation both of phosphatidylinositol-derived and
phosphatidylcholine-derived diglyceride, through
phosphoinositide-phospholipase C
(39) and
phosphatidylcholine-phospholipase D/phosphatidate phosphatase pathways,
respectively
(9) . Another report
(46) demonstrates that
in the absence of cytochalasin B, fMLP induces a weak and transient
formation of phosphoinositide-derived diglyceride, since no diglyceride
can be recovered after 1-min of cell activation. Because no stimulation
of [
Second, an additional argument arises from the
use of PMA, which has been shown to inhibit phosphoinositide
phospholipase C
(47) . Therefore diglyceride generated with PMA
stimulation can only originate from phosphatidylcholine. We have
confirmed that PMA inhibits phosphoinositide-derived diglyceride
formation in neutrophils (Fig. 7A), and we demonstrate
that the membrane-associated cytidylyltransferase parallels the
kinetics of phosphatidylcholine-derived diglyceride. It is noteworthy
that phosphatidylcholine-derived diglyceride is generated only after a
5-min lag time (Fig. 7B), the same lag time also being
noticed in the time course of cytidylyltransferase association with
membranes (Fig. 7C), in the mass increase of diglyceride
(Fig. 5B), in the [
Third, the results
obtained with the non-agonist compound oleic acid further strengthens a
relationship between phospholipase D and cytidylyltransferase when
cells are activated by agonists. The fatty acid could lead to
diglyceride synthesis either by incorporation of oleic acid into
monoglyceride
(45) , or by activation of the phospholipase
D/phosphatidate phosphatase pathway. Oleic acid has been shown to
stimulate invitro phospholipase D from
brain
(48) , but only one report demonstrates invivo activation of the enzyme in hepatocytes
(49) .
In the neutrophil we demonstrate that oleic acid is unable to stimulate
the phosphatidylcholine-phospholipase D, as assessed by the absence of
phosphatidate or phosphatidylethanol formation (Fig. 6). We
propose that only diglyceride formed through monoglyceride acylation
plays a role in the slight cytidylyltransferase translocation induced
by oleic acid.
To assess the point that phospholipase D activation
is absolutely required for further phosphatidylcholine resynthesis, we
have blocked fMLP-induced phospholipase D activation with the tyrosine
kinase inhibitor tyrphostin (Fig. 8). Tyrphostin has no effect on
the phosphoinositide-phospholipase C
The human neutrophil appears, therefore, to be a good model
for studying cytidylyltransferase activation in a differentiated cell,
and for investigating the signaling events leading to the enzyme
phosphorylation or dephosphorylation
(50) . Potential sites for
phosphorylation with mitogen-activated protein kinases have been
recently identified in the enzyme sequence
(51) . The human
neutrophil which contains agonist-activable p40
(
)
has been pointed out to control
cytidylyltransferase translocation
(6) . Whether
phosphatidylcholine degradation into diglyceride occurs prior or
subsequent to enhanced [
H]choline incorporation
via the CDP-choline pathway has been a matter of
debate
(6, 7) .
H]choline incorporation into
phosphatidylcholine, triggered by PMA and fMLP, depends only on the
presence of diglyceride formed from phosphatidylcholine-derived
phosphatidic acid. Our data provide evidence for an
``activated'' phosphatidylcholine cycle in human neutrophils
triggered by receptor and non-receptor agonists, and indicates a
coupling between phospholipase D and cytidylyltransferase activation.
Such a coupling is not required for oleic acid which triggers
[
H]choline incorporation without phospholipase D
activation.
Chemicals and
Products
[methyl-H]Choline
chloride (2.89 TBq (78Ci)/mmol),
phospho[methyl-
C]choline-ammonium salt
(2.22 GBq (60 mCi)/mmol), [
-
P]ATP (110 TBq
(3000 Ci)/mmol),
[5,6,8,9,11,12,14,15-
H]arachidonic acid (7.73 TBq
(209Ci)/mmol), and
1-O-[
H]alkyl-2-lyso-sn-glycero-3-phosphocholine
([
H]alkyl-labeled lyso-PAF) (80 Ci/mmol) were
purchased from the Radiochemical Center (Amersham, Bucks, United
Kingdom). The [
H]choline-labeled lyso-PAF,
i.e. 1-O-hexadecyl-2-lyso-sn-glycero-3-phospho[N-methyl-
H]choline
(3.07 TBq (83 Ci)/mmol) was specially prepared by Amersham (UK).
Percoll was obtained from Pharmacia (Uppsala, Sweden). PMA,
4-O-methyl-PMA, fMLP, cytochalasin B, tyrphostin 47, oleic
acid, digitonin, and all other chemicals were from Sigma. All stock
solutions were prepared in dimethyl sulfoxide whose final
concentrations in cell incubation and assays never exceeded 0.02%
(v/v). When oleic acid was dissolved in ethanol, final ethanol
concentration was 1% (v/v).
Composition of Buffers
Buffer A contained 20
mM Hepes, pH 7.4, 137 mM NaCl, 2.6 mM KCl,
5.5 mM glucose, 10 µM choline; and buffer B
contained buffer A without choline. The digitonin buffer consisted of
0.2 mg/ml digitonin, 10 mM Tris-HCl, pH 7.4, 0.25 M
sucrose, 0.5 mM phenylmethylsulfonyl fluoride.
Neutrophil Isolation
Buffy coats were obtained
from healthy donors of the local blood bank (Centre Régional de
Transfusion Sanguine, Toulouse, France). Human neutrophils were
separated by using a slight modification of the method previously
described
(26) . Briefly, a leukocyte-rich supernatant was
obtained following addition of a 2% (w/v) dextran solution in isotonic
NaCl to a buffy coat. After centrifugation, remaining erythrocyte
contamination of the leukocyte pellet was eliminated with suspension
for 10 min in cold isotonic NHCl. Separation of leukocytes
across a Percoll gradient gave a final cell population of more than 98%
neutrophils, as assessed with May-Grünswald-Giemsa staining.
Cell Stimulation
In experiments dealing with
choline labeling, 10 cells were incubated in buffer A with
1 µCi of [
H]choline (final specific
radioactivity of 0.4 µCi/nmol) for 20 min prior addition of stimuli
(oleic acid, PMA, or 4-O-methyl-PMA, fMLP). At each incubation
time, 0.5 ml of cell suspension (2
10
cells) was
harvested and pelleted (2,800
g for 1 min) using an
MSE microcentrifuge (Kontron Instruments). The cell pellet was
extracted according to Bligh and Dyer (27) and radioactivity of the
organic phase was determined. This radioactivity corresponded to
phosphatidylcholine labeling only, as checked by analysis on thin layer
chromatography (TLC). In some experiments, lactate dehydrogenase
activity was measured in the supernatant of incubation.
H]arachidonic acid or
[
H]lyso-PAF, 10
cells were incubated
for 30 min with 1 µCi of the tritiated compound, washed with buffer
A containing 2.5% (w/v) bovine serum albumin, and finally resuspended
in buffer A.
Pulse-Chase Experiments
Neutrophils were
prelabeled in buffer A containing [H]choline (1
µCi/1
10
cells) for 30 min. After removal of
the labeling medium, cells were rinsed twice and incubated in medium
without labeled precursor and in the presence of the agonists. At each
incubation time, 0.5 ml of cell suspension was harvested, extracted
according to Bligh and Dyer
(27) , and radioactivity from the
organic phase was determined. The labeled aqueous choline metabolites
were separated by TLC in methanol, 0.6% NaCl, NH
OH
(50/50/5, v/v)
(28) . The spots were identified, scraped off, and
counted for radioactivity.
Digitonin Permeabilization of Cells
Digitonin
permeabilization was performed as described by Pelech et al. (29). Briefly, neutrophils were rinsed twice with cold incubation
buffer B after treatment with the agonists and permeabilized with
digitonin buffer at 4 °C for the indicated times. After
centrifugation at 13,000 g for 1 min, the supernatant
(released cytosolic content) was kept at 4 °C before
cytidylyltransferase assay. Meanwhile the pellet (containing cell
ghosts) was resuspended in 10 mM Tris-HCl, pH 7.4, 0.25
M sucrose, 0.5 mM phenylmethylsulfonyl fluoride, and
sonicated with three bursts of 1 s at 30% power output with a microtip
probe equipped-sonicator (Heat Systems, Ultrasonics Inc., model W-225
R).
Cytidylyltransferase Assay
CTP:phosphocholine
cytidylyltransferase activity was assayed as described
previously
(30) . The incubation mixture contained 20 mM
Tris succinate, pH 7.8, 6 mM MgCl, 8 mM
CTP, 4 mM
phospho-[methyl-
C]choline (0.5
mCi/mmol), and up to 300 µg of protein from cytosol or membrane
fractions. A sonicated suspension of total lipid extract (1 mM
lipid phosphorus) from Krebs-II cells was added to assay the cytosolic
enzyme. The incubation was carried out for 30 min at 37 °C and
stopped by boiling in the presence of unlabeled phosphocholine (200
mM final concentration). [
C]CDP-choline
was separated and measured as described (28).
Mass Measurement of Diglyceride
Neutrophils were
incubated in buffer B for the indicated times in the presence of
agonists and then extracted according to the method of Bligh and
Dyer
(27) . An aliquot of the lipid fraction was dried under
nitrogen and resolubilized with 20 µl of 7.5% (w/v)
octyl--D-glucoside, 5 mM cardiolipin, 1
mM diethylenetriaminepentaacetic acid. Diglyceride amounts
were determined as described by Wright et al.(31) , a
modification of the method of Preiss et al.(32) , using
Escherichia coli diglyceride kinase (Lipidex, Inc.) and
[
-
P]ATP.
In Situ Determination of Phospholipase D
Activity
To analyze the generation of labeled phosphatidic acid
and diglyceride, cells were labeled with
[H]alkyl-lyso-GPC (1 µCi/10
cells) for 30 min at 37 °C
(9, 11) , then
stimulated with the appropriate compound. Lipids were extracted using
Bligh and Dyer procedure
(27) , and phosphatidylcholine breakdown
products were separated on silica plates according to Olson et
al.(33) .
H]choline. Activation of
the phospholipase was represented by the release of free
[
H]choline in the upper phase of the Bligh and
Dyer extract. In that case, 10
cells were labeled with 1
µCi of the radioactive precursor for 30 min prior to stimulation.
Inhibition of phospholipase D activation by tyrphostin was obtained in
cells preincubated for 5 min with a final concentration of 100
µM of the compound before stimulation.
Miscellaneous Determinations
Protein was
determined according to the method of Lowry et al.(34) in the presence of sodium dodecyl sulfate (0.07%, w/v),
using bovine serum albumin as a standard. Lactate dehydrogenase
activity and N-acetyl--D-glucosaminidase
activity were measured as described previously
(35, 36) .
Data Presentation
Results are expressed as the
average ± S.E. of three separate experiments, or the average of
two experiments with less than 10% variance.
H]Choline Incorporation into
Phosphatidylcholine (Fig. 1)-Human neutrophils were
incubated with [
H]choline and various
concentrations of oleic acid or PMA. Results reported in
Fig. 1A indicate a sharp dose dependence of stimulated
[
H]choline incorporation when exposed to the
fatty acid. A 5-fold stimulation was obtained at concentrations of
oleic acid between 40 and 60 µM, within the range of
non-lytic concentrations. The dose dependence was very similar to our
previous results on HL-60 cells
(37) .
Figure 1:
Concentration-dependent incorporation
of [H]choline into phosphatidylcholine in the
presence of oleic acid (A) or PMA (B). Neutrophils
were incubated with [
H]choline for 30 min in the
presence of oleic acid (Panel A,
]), or for 20 min
in the presence of PMA (Panel B,
) or
4-O-methyl-PMA (Panel B,
). Radioactivity
incorporated into phosphatidylcholine was measured as described under
``Experimental Procedures.'' Cell integrity was monitored
with release of lactate dehydrogenase (
). Results are expressed
as average ±S.E. of three determinations (A), or
average of two determinations (B).
As shown in
Fig. 1B, PMA stimulated [H]choline
incorporation by 2.5-fold with an optimal effect between 100 and 500
nM, a concentration range used in many studies. On the other
hand, 4-O-methyl-PMA, a phorbol ester which does not activate
protein kinase C
(38) , did not produce any change in
phosphatidylcholine labeling.
Cytidylyltransferase Catalyzes the Rate-limiting Step of the
CDP-choline Pathway in Human Neutrophils (Fig. 2)
We next
investigated the labeling of the different choline derivatives in cells
challenged with oleic acid or PMA. As compared to untreated cells,
pulse-chase experiments performed with oleic acid revealed a strong
decrease in phosphocholine labeling, and an increase in CDP-choline
labeling (Fig. 2A). Free internalized
[H]choline also showed a time-dependent decrease
but no change between control and treated cells was observed. Labeling
of phosphatidylcholine was clearly increased in the presence of 40
µM oleic acid. The variations observed for phosphocholine
radioactivity were opposite to those for CDP-choline and
phosphatidylcholine. This is consistent with cytidylyltransferase being
the rate-limiting enzyme of the CDP-choline pathway in our cell
model
(30) .
Figure 2:
Pulse-chase experiments with
[H]choline in the presence of oleic acid
(A) or PMA (B). Neutrophils were prelabeled for 30
min with [
H]choline in Buffer A, separated in two
pools, rinsed twice, and incubated in the absence (
) or presence
of 40 µM oleate (Panel A,
) or 500
nM PMA (Panel B,
). At the indicated times,
cells were harvested and lipids extracted. Radioactivity of aqueous
choline metabolites and phosphatidylcholine was determined:
(a) free internalized choline; (b) phosphocholine;
(c) CDP-choline; and (d) phosphatidylcholine. Results
are expressed as average of two (A) or three determinations
(B) ±S.E.
A similar pattern was also observed using PMA as
an agonist (Fig. 2B). [H]Choline
levels remained constant for 30 min with no change between control and
treated cells. Phosphocholine labeling decreased after a lag period of
5 min. A similar lag time was noticed for the enhancement of
phosphatidylcholine radioactivity, whereas no clear-cut modification of
CDP-choline levels was evident. This might indicate a concomitant
stimulation of choline phosphotransferase, as we previously reported
for phospholipase C-treated Krebs-II cells
(30) . Similar results
to those reported with PMA were obtained with fMLP as an agonist (not
shown).
Comparative Effect of Oleic Acid and Agonists on
Phosphatidylcholine Synthesis and Cytidylyltransferase Translocation (
Fig. 3
and Fig. 4)
We have performed a time course
study of [H]choline incorporation in the presence
of the chemotactic peptide fMLP, PMA, or 4-O-methyl-PMA. The
stimulation of [
H]choline incorporation into
phosphatidylcholine was better observed when cells were preincubated
for 20 min with [
H]choline prior to the addition
of cytochalasin B and fMLP. Neither cytochalasin B nor fMLP alone had
any stimulating effect (Fig. 3A). The ratio of
[
H]choline incorporation in cells stimulated with
fMLP plus cytochalasin B versus control was maximal at 5 min.
The phorbol ester PMA was also clearly able to stimulate
[
H]choline incorporation into
phosphatidylcholine, after a lag time of 5 min
(Fig. 3B). The analog 4-O-methyl-PMA showed no
effect. The ratio of choline incorporation in cells stimulated with PMA
versus control continuously increased during the 20-min time
course.
Figure 3:
Time course of
[H]choline incorporation into phosphatidylcholine
in neutrophils stimulated with fMLP and PMA. Neutrophils were
preincubated with [
H]choline for 20 min, then
incubated in the absence (
) or presence of: Panel A, 1
µM fMLP (
), 5 µM cytochalasin B
(
), or 1 µM fMLP + 5 µM
cytochalasin B (
); Panel B, 500 nM PMA (
)
or 500 nM 4-O-methyl-PMA (
). Radioactivity in
the organic phase was determined, and results were expressed as average
of two determinations.
Figure 4:
Cytidylyltransferase redistribution upon
cell activation. Cell activation was performed for 20 min with optimum
concentration of agonists, and in the presence of cytochalasin B for
fMLP. Cytidylyltransferase activity was measured in the
digitonin-release medium (cytosolic form of the enzyme) and in the
presence of exogenous lipids (Panel A). The activity remaining
in the cell ghosts was determined in the absence of added lipids, and
accounted for the membranous form of the enzyme (Panel B). The
sum of cytosolic plus particulate activities is represented in
Panel C. Results are expressed as
picomolemin
and are mean ± S.E. of
three different experiments. Cont: control (untreated)
cells.
The relative distribution of cytidylyltransferase between
cytosol and membranes was investigated after 20 min incubation and
using two experimental approaches giving similar results: cell
disruption with nitrogen cavitation followed with centrifugation to
separate particulate and cytosolic fractions (not shown), or cell
permeabilization with digitonin (Fig. 4). Optimal digitonin
concentration and permeabilization time were determined (data not
shown) by measuring release of lactate dehydrogenase activity from
cytosol and by measuring
N-acetyl--D-glucosaminidase activity as a marker
of granule integrity (see ``Experimental Procedures'').
Results reported in Fig. 4display the amount of
cytidylyltransferase retained in the cell (particulate form) or
released into supernatant (cytosolic form).
cells, depending upon the batch of
neutrophils; such variations being in the range of literature data.
Oleic acid induced only a weak formation of diglyceride
(Fig. 5A). In contrast, diglyceride increased
continuously after a 5-min lag time in PMA-treated neutrophils, whereas
4-O-methyl-PMA had no effect (Fig. 5B). A rapid
2-fold increase in diglyceride mass was only noticed with fMLP plus
cytochalasin B, reaching a maximum at 5 min and decreasing slowly
thereafter (Fig. 5C). In this case, the maximum of
diglyceride formation corresponded to the maximum enhancement of
[
H]choline incorporation, as determined by the
ratios between stimulated and resting cells for diglyceride and
phosphatidylcholine labeling (not shown). Also, the kinetics of
PMA-induced diglyceride formation (Fig. 5B) paralleled
that of [
H]choline incorporation in cells
stimulated with the phorbol ester (Fig. 3B).
Figure 5:
Time
course of diglyceride formation in activated neutrophils. Neutrophils
were incubated for the indicated times in the absence () or
presence of: Panel A, 40 µM oleic acid (
);
Panel B, 500 nM PMA (
) or 500 nM
4-O-methyl-PMA (
); Panel C, 1 µM
fMLP (
), 5 µM cytochalasin B (
), or 1
µM fMLP + 5 µM cytochalasin B (
).
Following lipid extraction, diglyceride amount was measured as
described under ``Experimental Procedures.'' Results are
expressed as average of two determinations.
Because
phosphatidylcholine degradation was well documented in human
neutrophils
(8, 9, 10, 11) , results from
Fig. 5
prompted us to investigate a possible relationship between
phosphatidylcholine degradation and synthesis through the generation of
diglyceride
(9, 10) . In Fig. 6A,
stimulation of neutrophils with fMLP led only to the formation of
phosphatidylcholine-derived phosphatidic acid. In contrast,
simultaneous addition of fMLP and cytochalasin B enhanced phosphatidic
acid generation, and triggered the formation of phosphatidic
acid-derived diglyceride (Fig. 6B). Cytochalasin B also
increased the release of water-soluble radioactivity from endogenous
alkylacyl-glycerophospho[H]choline
(Fig. 6C). When passed through an anion exchange column
(Dowex 50), the radioactivity was found to correspond to
[
H]choline only (not shown), which is in
agreement with the absence of a phospholipase C acting on
phosphatidylcholine in the human neutrophil
(24) . Therefore
addition of the priming compound cytochalasin B to the fMLP agonist
triggered both [
H]choline release and
phosphatidylcholine-derived diglyceride production, indicating an
activation of the phospholipase D/phosphatidic acid phosphatase
pathway.
Figure 6:
Kinetics of phospholipase D-mediated
phosphatidylcholine breakdown products from endogenously labeled
[H]alkylacyl-GPC or
alkylacylglycerophospho[
H]choline. Cells were
prelabeled with
[
H]alkyl-lyso-glycerophosphocholine (Panels
A, B, D, E, and F) or
alkyl-lyso-glycerophospho[
H]choline (Panel
C). Generation of [
H]alkyl-labeled
phosphatidic acid (
) and diglyceride (
) was monitored in
cells stimulated with: Panel A, 1 µM fMLP;
Panel B, 1 µM fMLP + 5 µM
cytochalasin B; Panel D, 100 nM PMA; Panel
E, 40 µM oleic acid. When oleic acid (40
µM) was added in the presence of ethanol (1% final v/v) no
alkyl-labeled phosphatidylethanol (Panel F) was recovered
(
), as compared to cells incubated with ethanol only (
).
Results are expressed as percent of total radioactivity from 10
cells (A, B, D-F) and are average of two determinations.
Panel C, [
H]choline release from
alkylacyl-glycerophospho[
H]choline was checked in
the presence of fMLP (
) or fMLP + cytochalasin B
(
), in the conditions of Panel B. Results are expressed
as disintegrations/min recovered in the water phase of lipid extract
from 10
labeled cells. Average of two
determinations.
When neutrophils were stimulated with the non-receptor
agonist PMA, both phosphatidic acid and diglyceride were produced
(Fig. 6D). Phosphatidic acid formation preceded that of
diglyceride whose onset time was between 2.5 and 5 min, a time
comparable to that required for PMA-induced
[H]choline incorporation
(Fig. 3B). The non-agonist compound, oleic acid, was not
an activator of phospholipase D, as monitored with the lack of
phosphatidic acid and phosphatidylethanol formation (Fig. 6,
E and F). As a consequence, no phosphatidic
acid-derived diglyceride was produced, which appeared consistent with
the weak changes in the mass amount of total diglyceride
(Fig. 5A) and the slight cytidylyltransferase
translocation (Fig. 4).
H]arachidonic
acid was distributed mainly into phosphatidylinositol as
reported
(16) . In contrast, 90% of
[
H]lyso-PAF was incorporated into
phosphatidylcholine as we previously observed
(9, 11) .
Addition of PMA induced no variation in the level of
arachidonyl-labeled diglyceride (Fig. 7A), at variance
with the results observed for [
H]alkyl-labeled
diglyceride, whose kinetics of labeling was similar to that of total
diglyceride (Fig. 5B). Comparison between Fig. 7,
B and C, showed that the association of
cytidylyltransferase with the membrane paralleled the
phosphatidylcholine-derived diglyceride formation.
Figure 7:
Comparison between time course generation
of [H]arachidonyl or
[
H]alkyl-labeled diglyceride and membrane
association of cytidylyltransferase. Cells were labeled with either
[
H]arachidonic acid or
[
H]alkyl-lyso-GPC, and stimulated with 100
nM PMA. Panel A,
[
H]arachidonyl-labeled diglyceride (
);
Panel B, [
H]alkyl-labeled diglyceride
(
) and control cells (
). Results are expressed as percent of
total radioactivity and are average of two determinations. Panel
C, specific activity of membrane-bound cytidylyltransferase in
PMA-treated cells (
) as compared to non-treated cells (
).
Average of two determinations.
With the receptor
agonist fMLP, phosphatidylinositol-derived diglyceride would also have
been produced since the agonist also stimulates the
phosphatidylinositol-phospholipase C
(39) . To monitor the
incidence of phospholipase D-mediated phosphatidylcholine hydrolysis on
the phospholipid resynthesis, we blocked phospholipase D activation
with a tyrosine kinase inhibitor, tyrphostin. This compound lead to a
strong inhibition of the [H]choline release
(Fig. 8A). The effect of fMLP plus cytochalasin B on
stimulation of [
H]choline incorporation was
monitored in a parallel series of samples. In control cells, time
course of [
H]choline incorporation was slower
than the phospholipase D-mediated release of the base, since maximum
incorporation occurred at 15 min (Fig. 8B), whereas
maximum release was noticed at 1 min (Fig. 8A). As a
consequence of phospholipase D inhibition,
[
H]choline incorporation in fMLP-stimulated cells
no longer occurred (Fig. 8B). Therefore, phospholipase
D-mediated choline release from phosphatidylcholine was required prior
to [
H]choline incorporation into this
phospholipid when neutrophils were activated with fMLP. The choline
released upon cell activation almost leveled off from 1 to 25 min
(Fig. 8A), suggesting that this pool of free choline was
not reutilized for the phosphatidylcholine resynthesis monitored in
Fig. 8B.
Figure 8:
Comparative effect of a tyrosine kinase
inhibitor on phosphatidylcholine breakdown and resynthesis upon cell
stimulation with fMLP. Panel A, release of
[H]choline from endogenous
alkylacyl-glycerophospho[
H]choline in cells
incubated with 1 µM fMLP + cytochalasin B (
) or
1 µM fMLP + cytochalasin B following a 5-min
treatment with 100 µM tyrphostin (
), as compared to
control cells (
). Data represent the radioactivity recovered in
the aqueous phase. Panel B, incorporation of free labeled
choline into phosphatidylcholine from another set of cells, in the
conditions of Panel A: fMLP-activated cells (
), or
fMLP-activated cells + tyrphostin (
), as compared to
control cells (
). Data represent the radioactivity recovered in
the organic phase. Results are average of two
determinations.
H]choline incorporation into phosphatidylcholine
(Fig. 1). Moreover, by performing pulse-chase experiments, we
demonstrate that cytidylyltransferase is the rate-limiting enzyme for
the CDP-choline pathway for phosphatidylcholine synthesis in human
neutrophils (Fig. 2). Two different patterns of CDP-choline
labeling are observed in Fig. 2and are consistent with previous
observations on growing cells
(30, 40) . So far, the
CDP-choline pathway has not been carefully studied in terminally
differentiated cells, and specifically in hematopoietic ones.
H]choline incorporation appears to be a long
lasting event (Fig. 3B). Such a difference between these
two agonists has often been observed in many responses of activated
neutrophils.
H]choline occurs in the absence of
cytochalasin B (Fig. 3A), phosphoinositide-derived
diglyceride could not be involved in cytidylyltransferase translocation
in our cell model.
H]choline
incorporation (Fig. 3B) and in the phosphocholine
decrease in the chase experiment (Fig. 2B, panel b). The
results we obtained using a differential labeling of phosphoinositide
and phosphatidylcholine strongly support the idea that PMA-induced
cytidylyltransferase translocation is related to the generation of
phosphatidylcholine-derived diglyceride only.
, which is activated with G
proteins from the 7-helix fMLP receptor
(8) , and therefore
cannot prevent phosphoinositide-derived diglyceride. Also, tyrphostin
does not affect cytidylyltransferase which is phosphorylated only on
serine residues (5). Since cell treatment with this inhibitor
effectively blocks both phosphatidylcholine degradation and
resynthesis, this demonstrates that only phospholipase D-mediated
phosphatidylcholine degradation is required for subsequent resynthesis
of the phospholipid. As summarized in Fig. 9, diglyceride coming
from phosphatidylcholine breakdown stimulates the reverse pathway by
activating the rate-limiting enzyme. Activation of phosphatidylcholine
turnover by the receptor-mediated agonist fMLP involves tyrosine
kinases, whereas activation appears mediated by protein kinase C with
the non-receptor agonist PMA, since the 4-O-methyl analog is
ineffective. The effect of PMA triggering the phospholipase
D/phosphatidate phosphatase pathway with subsequent enhancement on
choline incorporation could explain the results of several reports
showing a PMA-stimulated phosphatidylcholine synthesis, with a phorbol
ester target being necessarily distinct of
cytidylyltransferase
(5, 6, 20) .
Figure 9:
Relationship between cytidylyltransferase
and phospholipase D activation in agonist-stimulated human neutrophils.
Diglyceride coming from phosphatidylcholine breakdown activate the
reverse pathway by promoting cytidylyltransferase translocation. The
receptor-mediated agonist fMLP triggers a complete phosphatidylcholine
cycle only when phosphatidic acid is converted into diglyceride,
i.e. in the presence of cytochalasin B. In the absence of the
alkaloid compound, phosphatidylcholine breakdown stops at the
phosphatidic acid step, with no further effect on choline
incorporation. Receptor-mediated activation of phosphatidylcholine
cycle is mediated by tyrosine kinase-dependent phospholipase D
stimulation, whereas the non-receptor agonist PMA appears to act
through a protein kinase C-dependent phospholipase D activation. Oleic
acid does not trigger a phosphatidylcholine cycle, but stimulates only
the CDP-choline pathway.
Although the
maximum activation of fMLP-induced phospholipase D is reached within 1
min (Fig. 6A and 8A), choline incorporation is
maximum only after 15 min (Fig. 8B). This suggests many
steps between the two processes of phosphatidylcholine breakdown and
resynthesis. One of these could be that the choline pool used for
phosphatidylcholine synthesis requires some time for labeling, before
an effect of the agonist on choline incorporation could be noticed.
This pool appears distinct from the one corresponding to the
phospholipase D-mediated choline release. Effectively, about half of
the choline released in Fig. 8A is recovered outside the
cells (not shown). Such observations fit in with the scheme of George
and co-workers
(41) indicating that choline originating from
phospholipid degradation is released out of the cell, whereas choline
involved in synthesis is ``channeled'' up to the appropriate
enzymes.
and p42
isoforms
(52) , should be a
convenient model to investigate the relevance of cytidylyltransferase
regulation with mitogen-activated protein kinases invivo.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.