(Received for publication, October 7, 1994; and in revised form, November 18, 1994)
From the
Ceramide, a product arising from sphingomyelinase activity, has
been shown to act as an intracellular second messenger in effecting
growth inhibition, cellular differentiation, and apoptosis. In the
present study, the relative effects of cell-permeable ceramides, N-acetylsphingosine (C-ceramide) and N-hexanoylsphingosine (C
-ceramide), on neutrophil
responses were measured. When cells were activated with fMet-Leu-Phe,
C
-ceramide both potentiated (<1 µM) and
inhibited (>1 µM) superoxide generation. C
-
and C
-ceramide inhibited phorbol ester-induced superoxide
release from neutrophils at IC
values of 5 and 120
µM, respectively. C
-ceramide had no effect on
semipurified protein kinase C activity. Neither ceramide affected
significantly the general level of phosphorylated proteins in phorbol
ester-treated cells. C
-ceramide (1-20
µM) alone did not change cytosolic free Ca
levels but inhibited Ca
and Mn
influx in fMet-Leu-Phe-activated neutrophils. In contrast,
sphingosine enhanced Ca
entry; thus, ceramide
conversion to sphingosine was not significant. Unlike
C
-ceramide, C
-dihydroceramide failed to block
superoxide generation or Ca
influx. Preincubation of
cells with 10 nM okadaic acid reversed slightly the effects of
C
-ceramide. Calyculin A, tautomycin, and much higher
concentrations of okadaic acid inhibited agonist-induced Ca
influx. We postulate that C
-ceramide may inhibit
neutrophil superoxide release by activation of type 2A protein
phosphatases. Results suggest that protein phosphatase type 1
up-regulates Ca
entry, whereas type 2A (or a
ceramide-activated subtype) forestalls Ca
entry by
inactivating a calcium influx factor.
Recent studies have shown that sphingoid lipids function as
second messengers in signal
transduction(1, 2, 3, 4, 5, 6) .
Among other effects, sphingosine inhibits protein kinase C
(PKC)()(1, 2) , and sphingosine 1-phosphate
mobilizes intracellular Ca
stores(6) . The
biological effects of 1
,25-dihydroxyvitamin D
, tumor
necrosis factor-
, interferon-
, and interleukin-1 correlate
with early activation of a neutral sphingomyelinase and elevations of
ceramide levels in target cells(3, 4, 5) .
Cell-permeable synthetic ceramides were shown to mimic hormonal
effects. For example, C
-ceramide, tumor necrosis
factor-
, or vitamin D
each induces differentiation of
HL-60 cells (a leukemic cell line) into monocyte-like
cells(3, 4) . A downstream effect in each case was the
down-regulation of c-myc mRNA. Other studies report that
ceramides induce growth arrest, differentiation, and apoptosis in
fibroblasts, myeloid, and lymphoid
cells(3, 4, 7) . On a biochemical level,
Kolesnick and co-workers showed that ceramides activate
proline-directed serine/threonine kinases in A431 and HL-60
cells(3, 8) . Hannun and co-workers in turn have
advocated a mediator role for protein phosphatases
(PPases)(5, 9) . Working with extracts from rat brain
and T9 glioma cells, they showed that ceramides activate both
heterotrimeric PPase-2A and a related subtype they called
ceramide-activated protein phosphatase (CAPP). In vitro, CAPP
activity is inhibited by okadaic acid (OA)(5) . The relative
primacy of ceramide-activated kinases and PPases remains to be defined
in different cell types.
We have reported that exogenous sphingosine
mobilizes Ca in human neutrophils
(PMNs)(10) . Our results suggest that sphingosine acts
indirectly through a metabolite, a likely candidate being sphingosine
1-phosphate. In this study, we address the question of whether the
cell-permeable ceramides N-acetylsphingosine
(C
-ceramide) and N-hexanoylsphingosine
(C
-ceramide) can modulate cellular activation and
Ca
homeostasis in PMNs. The results show that
C
-ceramide effectively dampened specific signaling
pathways. We hypothesize that PPases may mediate the observed ceramide
effects.
Stock solutions of ceramides were made up in dimethyl sulfoxide and stored at -80 °C; when added to aqueous reaction mixtures, the final concentration of the carrier solvent did not exceed 0.5%.
Figure 1:
Effect of
C-ceramide (C-2) on fMLP-induced
O
generation by human neutrophils. Panel A, effect of incubation time on
O
release. Superoxide release was
measured by ferricytochrome c reduction as described under
``Experimental Procedures.'' Neutrophils (1
10
cells/ml, at 37 °C) were preincubated with 10 µM C
-ceramide for the times indicated in the figure
before fMLP (1 µM) was added. Successive traces have been
superimposed for better comparison. Panel B, the dose effect
of C
-ceramide on the kinetics of O
generation. Cells were incubated for 1 min with various
concentrations of C
-ceramide before the addition of fMLP;
other conditions were similar to panel A. Panel C,
average dose-response effect of a 1-min preincubation with
C
-ceramide on neutrophil O
release. Initial linear rates of O
generation were calculated as percentage of control
(vehicle-treated cells). Results are means ± S.E. of nine
experiments. * indicates significantly difference from control values
with p < 0.05 by the Bonferroni t test method
after analysis of variance.
Similar results were obtained
when neutrophils were stimulated with PMA, a PKC activator. The
respiratory burst triggered by PMA is characterized by a lag of about 1
min and prolonged kinetics of O release(13) . When neutrophils were pretreated with
C
-ceramide (1-10 µM) for 1 min, lag
times were increased slightly, and initial linear rates of
O
release were depressed dose dependently (Fig. 2A). Although not shown, the total amount of
O
generated after 30 min was also
decreased proportionately. In contrast to C
-ceramide,
C
-dihydroceramide, a negative
control(4, 9) , was without effect at 50
µM. The duration of incubation was not a factor since
cells treated for 30 min with C
-dihydroceramide before PMA
was added continued to express O
rates
similar to those of controls. Negative results were also obtained using
80 µM C
-dihydroceramide (not shown).
Figure 2:
Effect of ceramide on PMA-induced
O generation. Panel A, the dose
effect of C
-ceramide on the kinetics of
O
release. Neutrophils were preincubated
with various doses of C
-ceramide for 1 min before PMA (1
µM) was added to cell suspensions. The time course of
O
release was recorded continuously as in Fig. 1. Similar studies were performed using
C
-ceramide. Panel B, the initial linear rates of
O
generation were calculated as a
percentage of control rates and the results plotted against ceramide
concentration (C-6 = C
-ceramide). Results
are means ± S.E. of four
experiments.
Under
similar conditions (1-min incubation before the addition of PMA),
C-ceramide was less potent than C
-ceramide in
blocking free radical formation. The IC
at a cell density
of 1
10
/ml was estimated to be 5 and 120
µM, respectively for C
- and
C
-ceramide (Fig. 2B). Incubation time
influenced C
-ceramide effects moderately. Cells treated for
1 min with 30 µM C
-ceramide released
O
at the same rate as controls; if they
were treated for 30 min with 30 µM C
-ceramide
before PMA was added, the expressed rates were 75 ± 3.8% (n = 3) of controls.
Figure 3:
Effect of C- and
C
-ceramide on Ca
/phospholipid-dependent
PKC activity. PKC semipurified from rat brain was assayed according to
methods described under ``Experimental Procedures.''
P
incorporation into a peptide substrate was
expressed as a percentage of control radioactivity in which dimethyl
sulfoxide vehicle was added instead of ceramide. Staurosporine (10
nM) was used as a positive control. Results shown are means
± S.E. of four experiments.
Figure 4:
Effect of ceramides on protein
phosphorylation. Neutrophils prelabeled with P
according to the methods described under ``Experimental
Procedures'' were incubated for 6 min at 37 °C with vehicle
(control, lane a), 50 nM calyculin A (lane
c), 10 µM C
-ceramide (lane e),
or 150 µM C
-ceramide (lane g) before
reactions were stopped by 15% trichloroacetic acid. About 50 µg of
protein/sample was loaded. In lanes b, d, f,
and h, the same reagents were added to cells except that 100
nM PMA was added to cells 1 min into the incubation. Total
protein was separated by sodium dodecyl sulfate-gel electrophoresis and
autoradiograms made from dried gels.
We
found that 1-20 µM C-ceramide alone had
little or no effect on neutrophil
[Ca
]
under short term
(5-10-min) incubations. This agent did modulate changes in
[Ca
]
induced by fMLP (Fig. 5). In panels A-C, control traces show the
time course of elevation of [Ca
]
triggered by fMLP. As reported by this group and
others(11, 12) , the rise in
[Ca
]
was biphasic. Agonist
activation of phospholipase C led to the hydrolysis of
phosphatidylinositol 4,5-bisphosphate and formation of inositol
1,4,5-trisphosphate (Ins(1,4,5)P
); the latter mobilized
internally stored Ca
and produced a transient
elevation of [Ca
]
to micromolar
levels. The primary Ca
spike was succeeded by a
secondary influx of extracellular Ca
which maintained
[Ca
]
between 0.2 and 0.5
µM for 6-10 min. The addition of 3 mM EGTA
to cells reduced extracellular Ca
to submicromolar
levels, and, by eliminating Ca
influx, isolated the
internal Ca
release component (Fig. 5B). A similar trace was obtained by treating
neutrophils with PMA before the addition of fMLP, a result that is in
keeping with a negative feedback role for PKC on Ca
influx (Fig. 5A)(11, 12) .
Figure 5:
Ceramide inhibition of store-regulated
Ca entry. Neutrophils were loaded with fura-2 as
described under ``Experimental Procedures,'' and the
fluorescence signal was recorded continuously. PMNs from three
different donors were used in panels A, B, and C (the control traces illustrate the variability in experiments). In
each, traces were recorded consecutively and overlaid afterward for
better comparison. In panels A and B, vehicle
(control), PMA (100 nM), EGTA (3 mM),
C
-dihydroceramide (50 µM), or
C
-ceramide (20 µM or at indicated
concentrations) were added to neutrophil suspensions 1 min before fMLP
(1 µM). On the right in panel A, the dotted line retraces the control trace shown on the left. In panel C, 10 nM OA was added 30 min
and/or 20 µM C
-ceramide 2 min before fMLP.
Results are representative of at least three
experiments.
When PMNs were pretreated with C-ceramide for 1 min
before fMLP was added, the ensuing fura-2 traces were similar to those
obtained in the presence of EGTA or PMA; that is,
C
-ceramide dose dependently inhibited the secondary phase
of Ca
influx (Fig. 5A). By
comparison, C
-ceramide (20 µM) depressed the
peak height of the Ca
spike to a lesser extent. As a
control, C
-dihydroceramide (50 µM) failed to
alter fMLP-induced rises in [Ca
]
(Fig. 5B). C
-ceramide applied at
concentrations similar to those for C
-ceramide was also
inactive.
C-ceramide inhibition of Ca
influx was corroborated by Mn
influx studies (Fig. 6). Mn
entering fura-2-loaded
neutrophils is detected by its quenching of the dye fluorescence and is
used as a measure of the permeability of cation
channels(10, 11) . Results show that
C
-ceramide alone failed to change background rates of
Mn
entry (left panel, Fig. 6). In
agreement with previous reports, fMLP increased the rate of
Mn
influx after a short delay of about 30-60 s (right panel). In contrast, in cell suspensions treated
consecutively with C
-ceramide and fMLP, Mn
entry was blocked completely for several min before resuming at a
rate similar to background controls.
Figure 6:
Effects of C-ceramide on the
kinetics of Mn
entry in fura-2-loaded neutrophils.
Cell suspensions were excited at 360 nm, the isosbestic wavelength for
Ca
-fura-2 interactions. Mn
influx
was indicated by quenching of fura-2 fluorescence. Neutrophils were
incubated with C
-ceramide for 1 min before MnCl
(left panel) or MnCl
together with fMLP (right panel) were added at points marked by arrows.
Mn
interaction with a small amount of extracellular
fura-2 caused the abrupt but limited quenching following the addition
of MnCl
. Results are representative of two
experiments.
A working model considered in
this study is the activation of PPase by ceramides. Since in vitro experiments show that CAPP activation is reversed by
OA(5) , we investigated the effect of OA on
C-ceramide inhibition of Ca
entry (Fig. 5C). We found over three experiments that a
30-min preincubation of cells with 10 nM OA had little or no
effect on fMLP-induced secondary Ca
influx (the small
decrease in the extent of the Ca
spike was not
reproducible). In cells treated with OA, C
-ceramide, and
fMLP in the order shown, OA alleviated C
-ceramide
inhibition of Ca
entry by a modest extent (Fig. 3C).
Straightforward interpretations of
experiments using higher concentrations of OA were thwarted by the
finding that 50-200 nM OA inhibited Ca entry. This echoes previous reports showing that 1 µM OA dampened store-operated Ca
entry in
neutrophils and HeLa cells (17, 18) . Calyculin A and
tautomycin were more potent than OA (Fig. 7). Maximal inhibition
of fMLP-triggered Ca
influx occurred after a
10-20-min incubation with 2 nM calyculin A or 1 min with
30 nM tautomycin. OA applied under similar conditions had no
effect (not shown in Fig. 7).
Figure 7:
Effect of PPase-1/PPase-2A inhibitors on
fMLP-induced Ca influx. Fura-2-loaded neutrophils
were pretreated for 10 min (trace a) or 20 min (trace
b) with 2 nM calyculin A before the addition of 1
µM fMLP. In trace c, 30 nM tautomycin
was added to cells 1 min before fMLP.
Although ceramides do not
inhibit PKC directly, the possibility exists that ceramide were
converted to sphingosine, a PKC inhibitor. For comparison, Fig. 8shows the effect of sphingosine pretreatment on
fMLP-induced elevations of
[Ca]
. As shown, 0.5 and 1
µM sphingosine alone mediated minor but significant
elevation of [Ca
]
. This was
attributed to metabolism of some of the sphingosine to sphingosine
1-phosphate and the mobilization of Ca
stores by the
latter(6, 10) . When fMLP was added after 2 min, the
secondary influx of Ca
was enhanced. In this respect
sphingosine mediated the same effect as other PKC inhibitors such as
staurosporine and auranofin; that is, alleviation of the feedback
effect of PKC(11, 12) . In short, sphingosine exerted
an effect opposite to that shown for C
-ceramide. These
results imply that significant conversion of C
-ceramide to
sphingosine did not occur over the duration of the experiments.
Figure 8:
Effect of sphingosine on fMLP-induced
Ca influx. Neutrophil
[Ca
]
was monitored as
outlined in Fig. 5. Sphingosine (0.5 or 1 µM) and
fMLP (0.2 µM) were added to fura-2-loaded neutrophils at
points indicated by arrows. The dotted line on the rightmost two traces outlines the superimposition of the fMLP
control trace on the left. These results were collected in a
Perkin Elmer (MKF-4) fluorescence
spectrophotometer.
Accelerated Ca efflux may contribute to apparent
declines in [Ca
]
. To assess
this possibility,
Ca
-loaded neutrophils
were treated with fMLP ± ceramide and
Ca
release monitored (Fig. 9). As
reported previously, the initial rapid phase of release of internal
Ca
was matched by an equally rapid efflux of
Ca
in the first 30 s(10, 11) . The
results show that the rates of
Ca
efflux
were similar in PMNs stimulated by fMLP with or without
C
-ceramide preincubation. In all cases, accelerated
Ca
transport occurred in the first 30 s after fMLP
addition. After 30 s, Ca
efflux was similar to
background. C
-ceramide alone had the same effect as
dimethyl sulfoxide vehicle controls.
Figure 9:
Efflux of Ca
from neutrophils treated with sphingosine. Neutrophils were
loaded with
Ca
, incubated with reagents
for times shown in the figure at 37 °C, and the supernatant
recovered as outlined under ``Experimental Procedures.'' The
radioactivity in the supernatant was expressed as a percentage of the
total activity in supernatant and pellet fractions.
, fMLP;
, 10 µM C
-ceramide added 1 min before
fMLP;
, 20 µM C
-ceramide added 1 min
before fMLP;
, 20 µM C
-ceramide;
, dimethyl sulfoxide vehicle control. Where fMLP was applied,
timing started with the addition of this reagent. Results are
representative of two experiments.
Present results show that C-ceramide at
concentrations >1 µM inhibited
O
production by human neutrophils in a
time- and dose-dependent manner. Below 1 µM,
C
-ceramide potentiated or primed the
O
response induced by fMLP (Fig. 1C). This finding raises the possibility that
agonists such as tumor necrosis factor-
, platelet-activating
factor, leukotriene B
etc. (20) may prime
neutrophils by activation of sphingomyelinase. The linkage of tumor
necrosis factor-
to the sphingomyelinase pathway has been
demonstrated in leukemic cell lines (4) . Whether this occurs
in neutrophils remains to be established.
The preceding results
differ from those reported by Yanaga and Watson (21) , who
found C-ceramide had not affected the formation of reactive
oxygen species by neutrophils. In their experiments, PMNs were
incubated for 10 min with 30 µM C
-ceramide,
followed by 10 min with fMLP; cells were then scored for reduction of
nitroblue tetrazolium. This is an either-or test and is less sensitive
than the continuous cytochrome c reduction assay. In our hands
30 µM C
-ceramide completely inhibited
O
production whether PMNs were
preincubated for 2 or 10 min with this agent.
C-ceramide
was more potent than C
-ceramide in inhibiting
O
generation. Although C
- and
C
-ceramide were shown to be cell-permeable when applied to
other cell types(4, 5) , we have not directly measured
the relative rate of entry of these ceramides into human neutrophils. A
slower rate of uptake of C
-ceramide may partly account for
its lower potency. However, it is equally possible that differences
between C
- and C
-ceramide may reside in their
dissimilar effects on, or affinities for a putative cellular target.
The mechanism by which C-ceramide inhibited fMLP- and
PMA-induced O
in neutrophils remains to
be established. fMLP signals PMNs by activation of phospholipase C and
the generation of Ins(1,4,5)P
and
diacylglycerol(22) . The finding that C
-ceramide
inhibited weakly the initial Ca
transient induced by
fMLP (Fig. 5) suggests that Ins(1,4,5)P
formation or
Ins(1,4,5)P
release of intracellular Ca
was not the primary site of action.
Ceramide appears to
inhibit processes mediated by PKC, the cellular target of
diacylglycerol and PMA. Direct inhibition of PKC is unlikely as
C-ceramide failed to inhibit
Ca
-phospholipid-dependent PKC in vitro (Fig. 2) in agreement with other studies(5) .
C
-ceramide weakly inhibited PKC at concentrations >50
µM (Fig. 2B), but it is questionable
whether such high levels could be attained in intact cells under
physiological conditions. As we have argued under
``Results,'' the metabolism of ceramide to sphingosine, a PKC
inhibitor, was limited. Substantial sphingosine formation would have
enhanced fMLP-induced Ca
influx, not inhibited it ( Fig. 5and Fig. 8).
PKC-dependent reactions might be
countered by PPase-dependent dephosphorylation of substrate proteins. A
possible candidate is CAPP, characterized by Hannun's
group(5, 9) . In this study, the effective dose range
of C-ceramide and the inactivity of
C
-dihydroceramide are consistent with a role for CAPP. In vitro studies show that CAPP is a cation-independent,
serine/threonine PPase activated by 1-10 µM ceramide. Ceramide activation of CAPP is specific in that
C
-dihydroceramide is inactive and frequently applied in
experiments as a negative control for biological activity. CAPP is
related to and regarded as a subtype of the PPase-2A
family(5, 9) . More recently, Hannun's group
showed that C
-ceramide stimulates heterotrimeric forms of
PPase-2A up to 3.5-fold. By comparison C
-ceramide increases
5.5-fold the activity of CAPP isolated from T9 glioma
cells(9) .
In light of analyses of phosphorylated proteins
in PMNs, the PPase activation hypothesis must be qualified (Fig. 4). The autoradiograms of total proteins failed to show
significant dephosphorylation in cells treated with
C-ceramide alone or with concomitant PMA activation (Fig. 4). From this we conclude that C
-ceramide did
not activate PPases globally. However, this result does not necessarily
preclude the involvement of a CAPP that acts on selected substrates.
The gel system and labeling approach used in the present study are
insufficiently sensitive to discern subtle changes in phosphorylation
patterns. Before definitive conclusions can be made, more sensitive
methods must be used. Alternative approaches include two-dimensional
gel electrophoresis and autoradiography or pulse-chase experiments
after labeling electroporated cells with
[
-
P]ATP in the manner described by Lu et al.(15) .
A specific target of putative CAPP may
be p47-phox, a component of the
O-generating system, NADPH
oxidase(23) . Previous studies indicate that the assembly of
NADPH oxidase is associated with multiple phosphorylation of
p47-phox(24) . In experiments in which neutrophils
were stimulated with PMA for 5 min, eight distinct p47-phox phosphoproteins were detected in cytosolic and membrane fractions.
The possibility exists that CAPP may inhibit NADPH oxidase assembly and
activation by dephosphorylating a limited number of sites on
p47-phox. If true, the one-dimensional gel method may not
detect such changes as suggested earlier.
C-ceramide
inhibition of fMLP-dependent Ca
influx resembled that
mediated by PMA. The latter finding confirms previous results
supporting a negative feedback role for PKC in pathways involving
phospholipase C activation and consequent diacylglycerol
formation(11, 12) . In neutrophils, the PKC-sensitive
components regulating Ca
permeability remain to be
identified. Diacylglycerol-activated PKC has been shown to enhance
plasma membrane Ca
pump activity and inhibit
phospholipase C(25, 26, 27) . The latter
effect is modest, and both effects do not account for the total
blockade of unidirectional Mn
influx(11, 12, 17) . It is unlikely
that C
-ceramide follows the same mechanism of action as
PKC. C
-ceramide neither stimulated significant kinase
activity in intact neutrophils (Fig. 4) nor increased
Ca
efflux (Fig. 9). Paradoxically, ceramide
blockade of PKC-mediated events should have enhanced, not depressed
Ca
entry. We interpreted the C
-ceramide
effect in the following manner.
Table 1summarizes PKC/PPase
regulation of Ca influx in fMLP-activated
neutrophils. Present and past results show that PKC inhibitors such as
staurosporine, auranofin, and sphingosine potentiate Ca
entry by suppressing the negative feedback effect of PKC (Fig. 7)(11, 12) . PPase-1/PPase-2A inhibitors
such as calyculin A, tautomycin, and OA cooperate with PKC to tilt the
balance in favor of protein phosphorylation thus decreasing
Ca
influx (Fig. 7). Calyculin A and tautomycin
are equally effective inhibitors of PPase-1 and PPase-2A, whereas OA is
50-100 times more effective against PPase-2A (19) . (
)The greater potency of calyculin A and tautomycin compared
with OA suggests that PPase-1 was involved in up-regulating
Ca
influx. Using these agents, Murata et al.(28) similarly concluded that PPase-1 regulates
thrombin-induced Ca
influx in human platelets.
Initially, we anticipate that ceramides, by stimulating PPase, would
follow the nascent pattern and enhance Ca
entry. The
contrary evidence points to targeting of specific PPases. We propose
that PPase-2A or a subtype stimulated by ceramide regulates signaling
component(s) different from that targeted by PPase-1.
We speculate
that the site affected by C-ceramide may be the putative
calcium influx factor recently implicated as a second messenger in
Ca
homeostasis(29, 30) . According
to the capacitative calcium entry model of Putney(31) , the
fill state of intracellular Ca
stores in many
nonexcitable tissues and cells regulates Ca
influx.
Emptying of such stores generates a physical change or a chemical
signal that opens Ca
channels on the plasma membrane.
Recently several groups have obtained results suggesting that a soluble
factor may be responsible for channel
opening(29, 30, 32, 33) .
Randriamampita and Tsien (29) isolated such a factor from
Jurkat cells and characterized it as a small (M
< 500) compound requiring a phosphate group for activity. We
hypothesize that CAPP may dephosphorylate calcium influx factor and
inactivate it. Calcium influx factor with M
<
500 would not be detected in the gel system used in this study (Fig. 4). At present we cannot rule out other mechanisms of
action for C
-ceramide. For example, C
-ceramide
may interfere with the generation or release of a soluble
Ca
influx factor. Alternatively,
C
-ceramide may inhibit the opening or operation of
Ca
influx channels on the plasma membrane.
Whatever its mechanisms of action, ceramide inhibition of
Ca influx may cause or contribute to the onset of
cell differentiation, inhibition of cell growth, and repression of
inflammatory responses. Ca
influx plays a role in
sustained directed motility of PMNs, is not needed to trigger the
respiratory burst by chemoattractants but does increase the maximum
response(34, 35) . In many secretory cells, including
PMNs, exocytosis induced by receptor activation is dependent on
Ca
influx and is eliminated when influx is blocked,
in spite of mobilization of intracellularly stored
Ca
(36, 37) . In fMLP-treated PMNs,
the bulk of diacylglycerol generated arises from phospholipase D
hydrolysis of phosphatidylcholine, a reaction that is dependent on
exogenous Ca
(34, 38) . Therefore,
reduced Ca
influx may be an indirect mechanism by
which ceramides inhibit PKC in receptor-activated neutrophils.
To
summarize, we found that 1-20 µM C-ceramide inhibited O
generation in PMA- and fMLP-activated neutrophils. The same doses
inhibited Ca
and Mn
influx in
fMLP-stimulated cells but exerted little effect on Ca
efflux. The mechanism of action of C
-ceramide remains
to be clarified but may involve the activation of more than one type of
protein phosphatase.