Bradykinin inhibits ceramide production and activates
phospholipase D in rabbit cortical collecting duct cells
Gele
Liu1,
Leonard
Kleine2,
Rania
Nasrallah1, and
Richard L.
Hébert1,3
1 Departments of Cellular and
Molecular Medicine,
2 Biochemistry, Microbiology
and Immunology, and 3 Medicine,
University of Ottawa, Ottawa, Ontario, Canada K1H 8M5
 |
ABSTRACT |
Recent reports suggest that inflammatory cytokines, growth
factors, and vasoconstrictor peptides induce sphingomyelinase (SMase) activity. This results in the hydrolysis of sphingomyelin (SM) into
ceramide, which is implicated in various cellular functions. Although
ceramide regulates phospholipase D (PLD) activity, there is controversy
about this relationship. Thus we investigated whether the effect of
bradykinin (BK), a proinflammatory factor and vasodilator, was mediated
by ceramide signal transduction and by PLD. In rabbit cortical
collecting duct (RCCD) cells, BK increased SM levels and decreased
ceramide levels in a time-dependent manner. Thus SMase activity was
inhibited by BK. Also, the production of ceramide was regulated in a
concentration-dependent manner. The
BK-B1 antagonist [Lys-des-Arg9,Leu8]BK
did not affect ceramide signal transduction but the
BK-B2 antagonist (Hoe-140) blocked
the effect of BK on SMase, suggesting that the
BK-B2 receptor mediates BK-induced
inhibition of ceramide generation. Our results show that exogenous
SMase significantly hydrolyzed endogenous SM to form ceramide and
weakly activated PLD. In contrast, BK induced a significant activation
of PLD. However, additive effects of BK and ceramide on PLD activity
were not observed. We concluded that in RCCD cells, the BK-induced second messengers ceramide and phosphatidic acid were generated by
distinct signal transduction mechanisms, namely the SMase and PLD pathways.
sphingomyelin; sphingomyelinase
 |
INTRODUCTION |
CERAMIDE, A KEY PRODUCT of sphingolipids, is rapidly
emerging as a component of a wide variety of signal transduction
pathways, which produce specific biological effects. Sphingomyelin (SM) is composed of a very long chain, saturated or mono-unsaturated fatty
acid, a sphingosin base backbone (predominantly sphingosine), and a
phosphocholine headgroup (1, 3, 30). The headgroup appears
to come from phosphatidylcholine (PC), but other sources have not been
ruled out. SM turnover involves removal of the headgroup and
amide-linked fatty acid by sphingomyelinases (SMase) and ceramidases, respectively. SMase action is the only known mechanism for SM hydrolysis in mammalian cells. However, most, if not all, mammalian cells appear capable of signaling through the SM pathway (36). The
action of certain extracellular agents results in SM hydrolysis by
SMase and the concomitant generation of ceramide (23, 31, 36). Indeed,
ceramide is emerging as a putative second messenger, mediating the
effects of extracellular agents on cell growth, differentiation, and
apoptosis (38, 50).
Endogenous SMases can be placed into three classes: acidic SMase
(aSMase), neutral SMase (nSMase), and alkaline SMase (23). Apparently,
nSMase and aSMase play important roles in ceramide signal transduction.
Exogenous SMase, mainly from bacteria, is an important tool used in
investigating ceramide signal transduction (6, 51). Addition of
exogenous SMase decreases endogenous SM (12). The ceramide that is
generated by exogenous SMase induces apoptosis in human myeloid
leukemia cells (18). The synthetic, cell-permeable, short-chain
ceramides such as
C2-(N-acetylsphingosine), C6-(N-hexanoylsphingosine),
and
C8-(N-octanoylsphingosine)
also have been used as tools in investigating and understanding the pathway of ceramide signal transduction (20, 44).
Interestingly, phospholipase D (PLD) promotes the apoptotic DNA
fragmentation induced by tumor necrosis factor-
(TNF-
) (19). Ceramides can modify cell signaling via PLD (14). Meacci et al. (29)
suggests that ceramide can activate PLD in human fibroblasts, an effect
that was mimicked by treatment with Staphylococcus
aureus SMase. In contrast, ceramide inhibited
IgE-mediated activation of PLD in rat basophilic leukemia cells (34).
Adding C6-ceramide to
undifferentiated cells resulted in the inhibition of PLD activity and
reductions in diacylglycerol (DAG) and phosphatidate (PA) levels in the
rat neuroblastoma N1E-115 (8). In general, ceramide is
considered to be a negative modulator of PLD (3, 53); however, ceramide
is also able to activate PLD in specific cell types (29). Therefore,
the relationship between ceramide and PLD activity requires further investigation.
Recent results suggest that inflammatory cytokines such as TNF-
(9,
26, 27), growth factors such as nerve growth factor (NGF) (11), and
vasoconstrictor peptides such as endothelin-1 (ET-1) (5, 46, 48) induce
SMase activity, resulting in SM hydrolysis to produce ceramide. Kidney,
in particular the collecting duct segment of the nephron, represents a
major site of renal sodium and water reabsorption. In the present
study, we have used a recently characterized rabbit cortical collecting
duct (RCCD) cell line (4) to study the regulation of SMase and PLD by
BK in this rabbit nephron segment. These RCCD cells represent a
heterogenous population of principal,
-intercalated, and
-intercalated epithelial cell types as assessed by specific
monoclonal antibodies (Mab 703 and Mab 503) and
Arachis hypogea lectin
staining. They have retained both their morphological and
hormonal response properties and possess the signaling machinery
present downstream of angiotensin signaling (4). However, it is not
known whether the effects of BK involve ceramide signal transduction or
whether there is cross talk between BK- and ceramide-induced PLD
activity. Therefore, we studied the cellular signaling of SM-ceramide
and PLD activity induced by BK in RCCD cells.
 |
MATERIALS AND METHODS |
BK, S. aureus SMase,
N-acetyl-D-sphingosine
(C2-ceramide), adenosine
5'-trisphosphate, phosphatidylethanol (PEt), PC,
1,2-dioleoyl-rac-glycerol (C18:1[cis]-9),
L-
-phosphatidic acid,
cardiolipin, and all other drugs, standards, and salts were purchased
from Sigma. DMEM-F12 media and newborn calf serum were obtained from
GIBCO-BRL. Escherichia coli DAG kinase
(specific activity: 13 U/mg protein) and D609 (tricyclodecan-9-yl-xanthogenate, potassium salt) were purchased from
Calbiochem.
[Methyl-14C]choline
chloride (specific activity: 55.0 mCi/mmol) and
[
-32P]ATP (specific
activity: >3,000 Ci/mmol) were supplied by Amersham. [14C]myristic acid
(specific activity: 40-60 mCi/mmol) was obtained from DuPont-New
England Nuclear. Thin-layer chromatography (TLC) plates (0.25 mm thick)
were purchased from Whatman. HPLC grade solvents were supplied by
British Drug House.
Cell culture.
The RCCD cells (4) from passages 3-25 were grown in DMEM-F12
medium containing 10% fetal calf serum, 0.4% penicillin/streptomycin solution, 15 mM HEPES, 50 nM hydrocortisone, 44 mM sodium bicarbonate, and ITS (insulin-transferrin-sodium selenite). Cells were
grown to confluence in an atmosphere of 5%
CO2 at 37°C and then routinely incubated in standard medium without serum for 24 h before treatment.
Treatment and solvent extraction of cell
lipids.
The cells were washed three times with DMEM-F12 medium and incubated
for 30 min before treatment. The experiments were performed on RCCD
cells cultured in 100 × 20-mm plates, and three dishes were
pooled per sample. After different treatments, the reaction was
terminated by three washings with ice-cold DMEM-F12 media. The lipid
extraction method was based on that of Bligh and Dyer (2). After
centrifugation at 500 g for 2 min at
4°C, the cells were transferred to an ice-cold ground glass
homogenizer containing 0.5 ml chloroform/methanol/HCl (20:40:1,
vol/vol/vol), and homogenized on ice. The homogenate was left on ice
for 30 min. The mixture was then transferred to a microcentrifuge tube,
and the homogenizer was rinsed with 1 ml chloroform. The washings and
0.3 ml 1 M NaCl were added to the microcentrifuge tube, vortexed, and
spun at 11,000 g for 5 min at 4°C.
The upper aqueous layer was discarded, and the lower lipid-containing
layer was transferred to a 1-ml glass Chrompack vial, dried under a
stream of O2-free
N2 gas, and redissolved in 200 µl chloroform. The samples were stored at
20°C until
analyzed. The particulate protein interface was air-dried, dissolved in
0.5 ml 2 M NaOH, and assayed for protein according to Lowry's method
(24).
Measurement of radioactivity.
[14C]SM,
[32P]ceramide,
[14C]ceramide, and
[14C]PEt were measured
using the Molecular Dynamics System with the ImageQuaNT computer software provided by Molecular Dynamics. The TLC plates were exposed to
phosphor screens for 48 h (32P) or
72 h (14C) in exposure cassettes
and were scanned with the phosphor imager. Results were obtained as
volume per spot and expressed as volumes per milligrams protein. Volume
calculations include the area and density of the
radioactivity-generated spots. Results are expressed as percentage of control.
Measurement of sphingomyelin levels.
Because choline is a component of sphingolipids (43), cells were
prelabeled with 1 µCi/ml
[methyl-14C]choline
chloride in DMEM-F12 media 24 h before the treatment. The cells were
then stimulated with or without 100 nM BK (for 1, 5, 10 and 15 min) or
10 µM (for 1, 5, 10, 15, 30, 60, 90, and 120 min). Lipids were then
extracted, and 20 µl (10 mg/ml in chloroform) of SM + PC were added
to each sample as internal standards. The TLC plates were heated to
80°C for 30 min. The samples (25 µl in chloroform) were spotted
onto the plates. SM + PC (20 µl of 10 mg/ml in chloroform) were added
as external standards. The plates were developed in
chloroform/methanol/acetic acid/water (50:30:8:5, vol/vol/vol/vol). TLC
plates were dried, and I2 staining was used to mark the areas corresponding to SM and PC.
Quantitation of ceramide by the
[
-32P]ATP
and DAG kinase method.
We quantified ceramide using the
[
-32P]ATP and DAG
kinase method, as described by Preiss et al. (37) and modified by
Wright et al. (49). Briefly, cells were stimulated with BK (100 nM) for
5, 10, 20, and 30 min or with S. aureus SMase (0.1 U/ml) for 5 min. The lipids were
extracted and stored in chloroform at
20°C until used. The
chloroform was then evaporated under
N2. A blank tube and a standard
ceramide tube were always included. For each 10 µl of DAG kinase (20 mU), 50 µl of reaction buffer, 10 µl of 20 mmol/l dithiothreitol,
and 10 µl of
[
-32P]ATP (2.5 × 105 dpm/nmol) were added,
and the mixture was incubated at 25°C for 30 min. Reaction buffer
contained 25 mmol/l MgCl2 and 2 mmol/l EGTA were dissolved in 50 ml of 100 mmol/l imidazole (pH 6.6), and the pH was readjusted to 6.6. The reaction was terminated by the
addition of 0.5 ml ice-cold chloroform/methanol (1:2 vol/vol). The
lipids were extracted by the addition of 0.5 ml chloroform and 0.5 ml 1 M NaCl. The mixture was spun at 12,000 g for 3 min, and the upper aqueous
phase was discarded. To improve the extraction of ceramide and to
remove the excess
[
-32P]ATP, we
modified the procedure as follows: the lower organic phase was
sequentially washed with 0.75 ml of 1% perchloric acid with 0.3 ml
chloroform/methanol (1:2 vol/vol), 0.2 ml chloroform, and 0.2 ml water.
The resultant organic phase containing the radioactive ceramide was
dried under a stream of N2 and
reconstituted in 25 µl chloroform/methanol (95:5, vol/vol). The
reconstituted sample was spotted onto a Silica Gel 60 TLC plate, which
had been previously heat activated, and developed in a solvent mixture
of chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1,
vol/vol/vol/vol/vol).
Direct measurement of ceramide production using
[14C]myristic
acid.
To directly measure ceramide, we have developed a method using
[14C]myristic
acid-labeled cells and a TLC solvent of ethyl acetate/acetic acid/trimethylpentane. RCCD cells were labeled with
[14C]myristic acid for
24 h in the cell culture environment. After the extraction of lipids,
the samples were spotted on an oxalate-coated Silica Gel 60 TLC plate.
The plate was developed with a solvent of ethyl acetate/acetic
acid/trimethylpentane (EAT solvent; 9:2:5, vol/vol/vol).
With this method, we were able to separate
[14C]ceramide from
other
[14C]myristate-containing
neutral lipids. The external standard ceramide and endogenous ceramide
produced by exogenous SMase served to confirm the position of
[14C]ceramide.
To investigate the ceramide concentration response to BK, we stimulated
[14C]myristic
acid-labeled RCCD cells with BK (from
10
9 M to
10
5 M) for 20 min. Lipids
were extracted from cells and the
14C-labeled ceramide was separated
from other 14C-containing lipids
by TLC with the EAT solvent (see Fig. 3). To determine which BK
receptor subtype mediates BK-induced ceramide signal transduction, we
stimulated
[14C]myristic
acid-labeled RCCD cells with BK (100 nM) for 20 min with or without the
BK-B1 receptor antagonist
[Lys-des-Arg9,Leu8]BK
(10 µM) or the BK-B2 receptor
antagonist Hoe-140 (10 µM). Then lipids were extracted from cells,
and the 14C-labeled ceramide was
separated from other
14C-containing lipids on TLC
plates (see Fig. 4). These experiments were repeated three times.
Measurement of PLD activity.
To measure PLD activity, we labeled RCCD cells with 1 µCi/ml
[14C]myristic acid
(47) in DMEM-F12 media 24 h before the treatment. In the presence of
ethanol, the formation of
[14C]PEt, a specific
product of PLD activity, was determined (3). The cells were treated
with 100 nM BK with or without S. aureus SMase (0.1 U/ml) or
C2-ceramide (50 µM), or with
S. aureus SMase (0.1 U/ml) with or
without D609 (10 µM) in the presence of 1% ethanol. D609, a PLC
inhibitor (33) specific for phosphatidylcholine-phospholipase C
(PC-PLC) (42), does not inhibit phosphatidylinositol-PLC, PLA2, or PLD in U937
cells. D609 has been used to establish the functional
coupling of PC-PLC with SMase (42). After extraction of lipids, the
samples were spotted on an oxalate-coated Silica Gel 60 TLC plate. The
plate was developed with a solvent of ethyl acetate/acetic
acid/trimethylpentane (9:2:5, by volume). With this method, we were
able to separate
[14C]PEt from other
[14C]myristate-containing
neutral lipids. These experiments were repeated four times.
Statistics.
To determine the statistical significance of differences between more
than two groups, we used ANOVA and the Bonferroni multiple comparison
tests (4, 21). Differences of P < 0.05 were considered statistically significant. Results were presented
as means ± SE of three or four independent experiments.
 |
RESULTS |
BK increases the SM level.
SM is hydrolyzed by SMase to produce ceramide. To determine the SM pool
response to BK, we stimulated cells with BK (100 nM or 10 µM) for the
times indicated in Fig. 1,
A-C. For both concentrations of
BK, [14C]SM levels
increased for 5 min, at which time they were significantly different
from the control level (39 ± 7%,
n = 3, P < 0.01 for 100 nM; 45 ± 5%,
n = 3, P < 0.01 for 10 µM). The levels of
SM then reached a plateau and remained at that level for up to 15 min
(100 nM) or 120 min (10 µM). The increased
[14C]SM levels in the
presence of 10 µM BK were always higher than those with 100 nM BK.
Thus the [14C]SM
levels increased in a time-dependent manner.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1.
Bradykinin (BK) increases sphingomyelin (SM) level.
[Methyl-14C]choline
chloride-labeled rabbit cortical collecting duct (RCCD) cells were
stimulated with 10 µM BK for 30-120 min
(A and
B) or with 100 nM or 10 µM BK for
1-15 min (C). Lipids were
extracted from cells, and labeled SM was separated from other
14C-containing lipids by
thin-layer chromatography (TLC). A
represents typical image, whereas B
and C represent results obtained after
scanning. The 2 bands represent 2 isoforms of SM. Results were obtained
as volume per spot (vol/mg protein). Volume calculations include area
and density of radioactivity-generated spots with phosphor imager. Data
are expressed as means ± SE from 3 independent experiments. , 10 µM BK; , 100 nM BK. * P < 0.01.
|
|
Time-dependent reduction in ceramide production by
BK.
To further determine whether BK inhibits SMase activity, ceramide
production was measured (Fig. 2,
A and
B) by an indirect method, whereby
ceramide generation was phosphorylated in the presence of
sn-1,2-diacylglycerol kinase (DGK) and
[
-32P]ATP to form
[32P]ceramide
phosphate. Low levels of ceramide were present in the unstimulated control cells. BK (100 nM) decreased the ceramide levels
by 25 ± 3%, 48 ± 5%, and 52 ± 4% at 10, 20, and 30 min, respectively (n = 3, P < 0.02) (Fig.
2B). This decrease in ceramide started after 5 min and lasted up to 30 min. These changes reflect the
time-dependent increase in
[14C]SM levels that
were obtained in Fig. 1. The exogenous standard ceramide and
[32P]ceramide
phosphate produced by exogenous SMase further confirmed that the
[32P]ceramide
phosphate induced by BK was coming from the SM pool (Fig.
2A).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Time-dependent reduction in ceramide production by BK. RCCD cells were
stimulated with 100 nM BK for 5, 10, 20, and 30 min, and lipids were
extracted. Lipid samples were treated with reaction solution containing
Escherichia coli diacylglycerol (DAG)
kinase and
[ -32P]ATP. Blank
and exogenous standard ceramide tubes were similarly treated with
reaction solution. As a separate control, cells were treated with
exogenous sphingomyelinase (SMase; 0.1 U/ml) for 15 min and extracted
lipids were treated with reaction solution.
[32P]ceramide-phosphate
was separated from other lipids by TLC.
A: representative image of
[32P]ceramide-phosphate
shows blank; standard ceramide; control; 5, 10, 20, and 30 min of BK
stimulation; and exogenous SMase stimulation.
B: results obtained after scanning.
The 2 bands represent 2 isoforms of ceramide. Results were obtained as
volume per spot (vol/mg protein). Volume calculations include area and
density of radioactivity-generated spots with phosphor imager. Data are
expressed as means ± SE from 3 independent experiments.
* P < 0.05;
** P < 0.02.
|
|
Concentration-dependent reduction in ceramide
production by BK.
To confirm whether BK inhibits SMase activity and to investigate the
concentration response to BK, we directly measured ceramide in
[14C]myristic
acid-labeled RCCD cells after stimulation with different concentrations
of BK. The data (Fig. 3,
A and
B) suggest that within 20 min, BK
causes a concentration-dependent decrease in ceramide levels (39 ± 3%, 47 ± 2%, 55 ± 6%, 60 ± 4%, and 65 ± 5% for the
concentrations of BK from
10
9 M to
10
5 M, respectively).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 3.
Concentration-response curve for BK-induced reduction of ceramide
levels. [14C]myristic
acid-labeled RCCD cells were stimulated with BK (between
10 9 and
10 5 M) for 20 min. Lipids
were extracted from cells and
14C-labeled ceramide was separated
from other 14C-containing lipids
by TLC. A: representative image of
[14C]ceramide shows
control and BK stimulation from
10 9 to
10 5 M. B: results obtained after scanning.
The 2 bands represent 2 isoforms of ceramide. Results were obtained as
volume per spot (vol/mg protein). Volume calculations include area and
density of radioactivity-generated spots with phosphor imager. Data are
expressed as means ± SE from 3 independent experiments.
* P < 0.01.
|
|
BK-B2
receptor mediates effect of BK on SMase.
It is known that BK signals through two receptor subtypes,
BK-B1 and
BK-B2 (38, 49). To determine which
receptor subtype is responsible for ceramide signal transduction, we
stimulated [14C]myristic
acid-labeled RCCD cells with BK (100 nM) for 20 min with or without
BK-B1 receptor antagonist or
BK-B2 receptor antagonist; [14C]ceramide
production was directly measured by TLC using the EAT solvent. Figure
4 demonstrates that BK decreased ceramide
levels by 52% and that neither of the receptor antagonists have any
significant effect by themselves (
4 ± 0.2% and
8.8 ± 0.3%, respectively) on ceramide levels. The presence of the
BK-B1 receptor antagonist did not
affect the decrease in ceramide levels induced by BK (
56.7 ± 3%). In contrast, the BK-B2
receptor antagonist completely inhibited the effect of BK (
9.9 ± 0.2%), indicating that BK-B2
receptor mediated the effect of BK on ceramide production.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 4.
BK-B2 receptor mediates effect of
BK on SMase.
[14C]myristic
acid-labeled RCCD cells were stimulated with BK (100 nM) for 20 min
with or without the BK-B1 receptor
antagonist
[Lys-des-Arg9,Leu8]BK
(10 µM) or the BK-B2 receptor
antagonist Hoe-140 (10 µM). Lipids were extracted from cells and
14C-labeled ceramide was separated
from other 14C-containing lipids
by TLC. A: representative image of
[14C]ceramide shows
control, BK, BK-B1 receptor
antagonist, BK + BK-B1,
BK-B2 receptor antagonist, and BK + BK-B2.
B: results obtained after scanning.
The 2 bands represent 2 isoforms of ceramide. Results were obtained as
volume per spot (vol/mg protein). Volume calculations include area and
density of radioactivity-generated spots with phosphor imager. Data are
expressed as means ± SE from 3 independent experiments.
* P < 0.01.
|
|
Effect of BK and exogenous SMase on PLD
activity.
PLD activity was measured using a transphosphatidylation reaction
unique to the enzyme, in which the phospholipid headgroup is exchanged
for ethanol, producing PEt in preference to PA (47). We have determined
the effect of BK, with or without exogenous SMase, on PLD activity
(Fig. 5, A
and B). In the presence of 1% ethanol, BK (100 nM) stimulation of
[14C]myristate-labeled
cells resulted in a level of
[14C]PEt of 186 ± 57% (n = 4, P < 0.05) above the control levels at 10 min. Also, SMase (0.1 U/ml) increased
[14C]PEt production by
45 ± 8% above the control levels
(n = 4, P < 0.05). There was a slight
increase in [14C]PEt
formation (226 ± 32% above controls) when both BK and SMase were
present, but this difference was not statistically significant (NS;
n = 4), suggesting that exogenous
SMase did not enhance BK stimulation of
[14C]PEt formation.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of BK and exogenous SMase on phospholipase D (PLD) activity. In
presence of 1% ethanol,
[14C]myristic
acid-labeled RCCD cells were stimulated with BK (100 nM) for 10 min
with or without Staphylococcus aureus
SMase (0.1 U/ml) for 15 min. PLD activity was measured using a
transphosphatidylation reaction unique to the enzyme, in which
phospholipid headgroup is exchanged for ethanol, producing
phosphatidylethanol (PEt) in preference to phosphatidate (PA).
14C-labeled PEt was extracted from
cells and separated from other
14C-containing lipids on TLC
plates. A: representative image of
[14C]PEt shows
control, BK, exogenous SMase, and BK + exogenous SMase.
B: results obtained after scanning.
Results were obtained as volume per spot (vol/mg protein). Volume
calculations include area and density of radioactivity-generated spots
with phosphor imager. Data are expressed as means ± SE from 4 independent experiments. * P < 0.05.
|
|
Effect of BK on endogenous ceramide induced by
exogenous SMase.
The effect of BK on ceramide induced by exogenous SMase is shown in
Fig. 6, A
and B. Exogenous S. aureus SMase (0.1 U/ml) catalyses the hydrolysis of
endogenous SM to form ceramide. The [14C]ceramide, which
was produced in the presence of exogenous SMase, was significantly
higher than the levels present in untreated controls or in BK-treated
cells (668 ± 47% over the control level, n = 4, P < 0.01). However, BK (100 nM) did
not inhibit the
[14C]ceramide
formation (BK + SMase, 699 ± 65% over the control levels) that was
induced by exogenous SMase (n = 4, P = NS). Also, D609, a PLC
antagonist specific for PC-PLC (3, 21), did not block the increase in
[14C]ceramide (666 ± 72% over the control levels) induced by exogenous SMase
(n = 4, P = NS). These results suggested that
BK did not influence the formation of endogenous ceramide induced by
exogenous SMase, and that PC-PLC was not involved.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of BK on endogenous ceramide induced by exogenous SMase.
[14C]myristic
acid-labeled RCCD cells were stimulated with BK (100 nM) for 10 min
with or without S. aureus SMase (0.1 U/ml) for 15 min, or with SMase ± D609 (10 µM). Lipids were
extracted from cells and
14C-labeled ceramide was separated
from other 14C-containing lipids
on TLC plates. A: representative image
of [14C]ceramide shows
control, BK, exogenous SMase, exogenous SMase + D609, and BK + exogenous SMase. B: results obtained
after scanning. The 2 bands represent 2 isoforms of ceramide. Results
were obtained as volume per spot (vol/mg protein). Volume calculations
include area and density of radioactivity-generated spots with phosphor
imager. Data are expressed as means ± SE from 4 independent
experiments. * P < 0.05;
** P < 0.01.
|
|
BK-mediated interaction between endogenous or
exogenous ceramide and PLD.
To determine whether there is cross talk between the ceramide and PLD
pathways, we investigated whether there was an interaction between
endogenous or exogenous ceramide and BK-stimulated PLD. Cells were
preincubated with 50 µM
C2-ceramide for 1 h or with 0.1 U/ml exogenous SMase for 5 min. In the presence of 1% ethanol, cells
were either left untreated or treated with 100 nM BK for 10 min.
Exogenous C2-ceramide alone did
not affect PLD activity and did not influence BK-stimulated
[14C]PEt formation
(Fig. 7, A
and B). We were able to monitor the activity of PLD and SMase in the same sample. Prelabeling the cells
with [14C]myristic
acid labels both the SM pool and the PC pool. The
[14C]ceramide produced
by SMase, via hydrolysis of SM, and the
[14C]PEt produced from
PC, after stimulation of PLD, can be measured on the same TLC plate.
Figure 8,
A and
B, demonstrates that at the same time,
BK stimulates PLD activity to produce PEt and inhibits ceramide
formation. Similarly, exogenous SMase-stimulated ceramide formation was
accompanied by an increase in PLD activity. However, when both BK and
exogenous SMase were present, there was no apparent additive effect on
PLD activity or on ceramide production (Fig. 8,
A and
B). Figure
9 illustrates the distinct pathways by
which the effects of BK on SMase and PLD are mediated.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of BK and exogenous ceramide on PLD activity.
[14C]myristic
acid-labeled RCCD cells were preincubated with
C2-ceramide (50 µM) for 1 h, and
then were stimulated with BK (100 nM) for 10 min in the presence of 1%
ethanol. 14C-labeled PEt was
extracted from cells and separated from other
14C-containing lipids by TLC.
A: representative image of
[14C]PEt shows
control, BK, exogenous
C2-ceramide, and BK + exogenous
C2-ceramide.
B: results obtained after scanning.
Results were obtained as volume per spot (vol/mg protein). Volume
calculations include area and density of radioactivity-generated spots
with phosphor imager. Data are expressed as means ± SE from 4 independent experiments. * P < 0.05.
|
|

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of BK and SMase on ceramide production and PLD activity.
[14C]myristic
acid-labeled RCCD cells were stimulated with BK (100 nM) for 10 min ± S. aureus SMase 0.1 U/ml for 15 min in presence of 1% ethanol. Lipids were extracted and both
14C-labeled ceramide and
14C-labeled PEt were separated by
TLC and identified on same plate. A:
representative image of
[14C]ceramide and of
[14C]PEt shows
control, BK, exogenous SMase, exogenous SMase + D609, and BK + exogenous SMase. B: results obtained
after scanning
[14C]PEt and
[14C]ceramide bands.
The 2 bands for ceramide represent 2 isoforms of ceramide. Results were
obtained as volume per spot (vol/mg protein). Volume calculations
include area and density of radioactivity-generated spots with phosphor
imager. Data are expressed as means ± SE from 4 independent
experiments. Open bars, PEt; shaded bars, ceramide.
* P < 0.05;
** P < 0.01..
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 9.
Effects of BK on SMase and on PLD are mediated by distinct pathways. BK
inhibited SMase activity and induced PLD activity. Endogenous and
exogenous ceramide did not influence PLD activity induced by BK. Cross
talk between BK-inhibited SMase activity and BK-induced PLD activity
was not observed. In RCCD cells, BK does not activate PLD via
production of ceramide. Effect of BK on SMase-ceramide pathway is
distinct from its effect on PLD-PA pathway. PC, phosphatidylcholine;
solid line, positive response; dotted line, negative response.
|
|
 |
DISCUSSION |
The interest in ceramide stems from the fact that it is involved in the
control of cell differentiation, proliferation, and apoptosis (7, 26,
50). It also seems to be involved in many other cellular activities,
making ceramide an important component of signal transduction pathways
(3). Furthermore, ceramide is a precursor for ceramide-1-phosphate,
sphingosine, and sphingosine-1-phosphate, which are now also recognized
as important second messengers.
SMase action is the only known mechanism for SM hydrolysis in mammalian
cells (36). However, most, if not all, mammalian cells appear capable
of signaling through the SM pathway (36). Our results provide evidence
that there is SMase activity in RCCD cells and that this enzyme is
inhibited by BK. The possibility that BK exerts some of its effects
through modulation of the SM-ceramide signaling pathway was provided by
several reports that suggest that inflammatory cytokines such as
TNF-
(26, 27) and the vasoconstrictor peptide endothelin-1 (ET-1)
(5, 46, 48) act via the activation of SMase. Thus we considered the
possibility that the proinflammatory and vasodilatory effects of BK
could (10, 45) be mediated by the inhibition of SMase. The present study indicates that BK was able to decrease SM hydrolysis and ceramide
production by inhibition of SMase activity in RCCD cells. This provides
for the possibility that BK might operate, in part, by opposing the
SMase-activating effect of other agents. Because the SM-ceramide
pathway has been implicated in these events, it is possible that BK is
involved in counteracting the ceramide-generating signals initiated by
a variety of agents. This suggests that BK may play a role in
regulating the overall balance of events such as vasoconstriction and vasodilatation.
Very few studies have investigated the effect of BK on ceramide
signaling. Meacci et al. (28, 29) showed that BK is able to activate
sphingolipid metabolism in human fibroblasts. BK provoked a rapid and
significant increase in ceramide followed by a transient rise in
sphingosine. However, BK neither decreased the SM pool nor activated
neutral or acidic SMases, thus the possibility remains that
glycosphingolipids might be the source of ceramide in human fibroblasts. Actually, ceramide may come from a variety of metabolic pathways such as de novo synthesis (16), dihydroceramide (17), glycerophospholipids (3), and sulfatides (13). However, SM is the main
source of ceramide, and this is the pathway being most investigated at
the present time. The different effects of BK on RCCD cells and human
fibroblasts can probably be attributed to differences in cell or tissue
specificity. Our data clearly suggest that the increase in SM levels
occurred concomitantly with a decrease in ceramide after BK stimulation.
The measurement of ceramide is usually done using the
[
-32P]ATP and DAG
kinase method, as described by Preiss et al. (37) and modified by
Wright et al. (49). However, this is an indirect method for the
measurement of ceramide. It requires the phosphorylation of ceramide to
[32P]ceramide-phosphate,
which is separated and measured on TLC plates. This method requires a
long time to complete and is complex and expensive. During the
experiments, we have developed a new method for the direct measurement
of ceramide production using
[14C]myristic acid and
a solvent of ethyl acetate/acetic acid/trimethylpentane for TLC
development. With this method, we were able to separate [14C]ceramide from
other
[14C]myristate-containing
neutral lipids. The external standard ceramide and endogenous ceramide
produced by exogenous SMase were used to confirm the position of
[14C]ceramide on the
plates. Both methods gave the same results. BK induced time- and
concentration-dependent increases in SM levels and a decrease in
ceramide production. Although SMase activity was not measured directly,
the results point convincingly to the inhibition of SMase by BK.
BK receptors are generally divided into two major subtypes,
BK-B1 and
BK-B2 (40, 52).
BK-B1 mediates the rapid, acute response (smooth muscle contraction or relaxation) as well as some
effects occurring more slowly (e.g., collagen synthesis) (40). The
BK-B2 receptor is responsible for
most of the biological effects of the kinins, including arterial
vasodilatation, plasma extravasation, venoconstriction, activation of
sensory fibers (e.g., fibers for pain), and stimulation of the release
of prostaglandins, endothelium-dependent relaxing factor (from
endothelia), norepinephrine (from nerve terminals and adrenals), and
other endogenous agents (25, 40, 41). It was reported that in rat
medullary collecting duct, the
BK-B2 antagonist Hoe-140 increased
chloride and water absorption (32), suggesting that
BK-B2 is the constitutive subtype in the kidney collecting duct. To determine which BK-receptor subtype
affects ceramide signal transduction, we stimulated
[14C]myristic
acid-labeled RCCD cells with BK in the presence of BK receptor
B1 or
B2 antagonists. The
BK-B1 antagonist
[Lys-des-Arg9,Leu8]BK
had no direct effect on ceramide production and did not affect the
inhibition of ceramide production induced by BK. In contrast, although
the BK-B2 antagonist Hoe-140 did
not affect ceramide levels by itself, it completely blocked the effect
of BK. These results suggest that the BK receptor
B2, but not
B1, mediates BK-induced inhibition
of ceramide signal transduction.
At present there are no specific inhibitors for SMase activity, but
exogenous SMase (6, 51) and short-chain ceramides such as
C2-ceramide (22) are widely used
as tools to investigate ceramide signal transduction. Exogenous SMase
is extensively used in the literature to mimic the effect of ceramide
generating signals (6, 11, 51). It is not clear how exogenous SMase
functions to generate an intercellular signal. Presumably, at least
some of the ceramide that is generated on the cell surface enters into the cell. Thus we used both exogenous SMase and
C2-ceramide in our experiments.
Exogenous SMase induced the production of very high levels of
intracellular ceramide. That exogenously added SMase is very effective
in hydrolysing endogenous SM has been widely reported in the literature
(6, 51). BK did not decrease the amount of ceramide that was elicited
by exogenous SMase. This is not unexpected because the concentration of
exogenous SMase was very high. These studies using exogenous SMase
served mainly as control to generate high levels of ceramide and to
study the effect of ceramide on PLD (next series of experiments). We
have previously demonstrated that BK activates PC-PLC in Madin-Darby canine kidney cells (21). The DAG that is produced due to action of
PC-PLC has been reported to be an activator of SMase (42). Thus we took
advantage of the fact that D609 is a potent inhibitor of PC-PLC (42).
Our results showed that D609 did not have any effect on the levels of
ceramide elicited by exogenous SMase.
Our data shows that BK induced a dramatic activation of PLD. In the
same samples, a significant decrease in ceramide levels was observed.
In contrast, exogenous SMase, which causes the production of very high
levels of ceramide, elicits only a very slight, albeit significant,
activation of PLD. The presence of exogenous SMase did not have any
effect on the BK-induced PLD activity. It is possible that exogenous
SMase would have a direct effect on hydrolysis of PC to PEt. Because we
observed production of very high levels of ceramide and activation of
PLD simultaneously in the same samples, it is probable that the
activation of PLD by exogenous SMase was mediated by ceramide. Whether
ceramide activates PLD directly or indirectly is not clear, in light of
the fact that sphingosine, the hydrolysis product of ceramide, is a
potent activator of PLD (3, 29). The cell-permeable short chain
C2 ceramide does not, by itself,
activate PLD, and does not blunt the activation of PLD due to BK. These
results would tend to support the possibility that exogenous SMase
might be acting directly on PLD. However, much evidence indicates that
short chain ceramides do not mimic all of the effects of endogenous
ceramide (8, 35, 39). Thus it was not surprising that exogenous
C2-ceramide did not affect PLD.
Because exogenous C2-ceramide,
which would also be hydrolysed to sphingosine, did not affect PLD, we
suggest that it was ceramide and not sphingosine that directly
activates PLD. Our results indicating ceramide is an activator of PLD
are in agreement with those of Meacci (28, 29). Having obtained
evidence that ceramide activates PLD, we would have expected that the
decrease in ceramide levels induced by BK would lead to less activation of PLD. Because PLD is activated by ceramide we suggest that activation of PLD by BK is not mediated via the
BK-B2 receptor/SMase/ceramide pathway but involves the activation of PLD by a different signaling pathway, possibly mediated by a different receptor subtype. It is known
that, in a number of different cell types, BK activates PLD through
G-protein coupled receptors, an effect mediated in part by PKC (21, 28,
29). That BK can activate PLD in kidney cells has been previously shown
by us (21) and others (28, 29), however, the relationship between BK's
effect on ceramide signaling and PLD activity had not been studied
previously. The involvement of ceramide in the regulation of PLD
activity is controversial. Ceramide has been previously described as a
negative modulator of PLD activity (3). Gomez-Munoz et al. (15) showed
that C2- and
C6-ceramides blocked the
activation of PLD by sphingosine 1-phosphate in rat fibroblasts.
However, Meacci et al. (28, 29) suggested that ceramide is an activator
of BK-mediated PLD activity. When BK and exogenous SMase were present
at the same time, no synergistic or additive effects were observed. The
activation of PLD was generally higher than that obtained with BK
alone, but this increase was not statistically significant.
In the present study, we have demonstrated that exogenous SMase, whose
only known activity is to hydrolyse SM to ceramide and phosphocholine,
activated PLD. We have also shown that BK inhibits SMase and activates
PLD in RCCD cells. These two observation strongly suggest two separate
signaling pathways. The inhibition of SMase was mediated by the
BK-B2 receptor subtype. Although both BK and ceramide activate PLD, no synergism or additive effect was
noted between the two PLD activators. We speculate that BK function, in
part, by counteracting the effects of SMase-activating pathways.
 |
ACKNOWLEDGEMENTS |
This research was supported by the Kidney Foundation of Canada and
Medical Research Council of Canada (MT-14103). The BK-B1 and BK-2
receptor antagonists were a generous gift from Dr. Domenico Regoli
(Department of Pharmacology, University of Sherbrooke).
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. L. Hébert, Dept. of Cellular and Molecular Medicine, Univ. of
Ottawa, 451 Smyth Road, Ottawa, Ontario, K1H 8M5, Canada (E-mail:
rhebert{at}uottawa.ca).
Received 30 June 1998; accepted in final form 23 December 1998.
 |
REFERENCES |
1.
Ballou, L. R.,
S. J. Laulederkind,
E. F. Rosloniec,
and
R. Raghow.
Ceramide signalling and the immune response.
Biochim. Biophys. Acta
1301:
273-287,
1996[Medline].
2.
Bligh, E. G.,
and
W. J. Dyer.
A rapid method of total lipid extraction and purification.
Can. J. Biochem. Physiol.
37:
911-917,
1959.
3.
Brindley, D. N.,
A. Abousalham,
Y. Kikuchi,
C. N. Wang,
and
D. W. Waggoner.
"Cross talk" between the bioactive glycerolipids and sphingolipids in signal transduction.
Biochem. Cell Biol.
74:
469-476,
1996[Medline].
4.
Burns, K. D.,
L. Regnier,
A. Roczniak,
and
R. L. Hébert.
Immortalized rabbit cortical collecting duct cells express AT1 angiotensin II receptors.
Am. J. Physiol.
271 (Renal Fluid Electrolyte Physiol. 40):
F1147-F1157,
1996[Abstract/Free Full Text].
5.
Catalan, R. E.,
M. D. Aragones,
A. M. Martinez,
and
I. Fernandez.
Involvement of sphingolipids in the endothelin-1 signal transduction mechanism in rat brain.
Neurosci. Lett.
220:
121-124,
1996[Medline].
6.
Chen, J.,
M. Nikolova-Karakashian,
A. H. Merrill, Jr.,
and
E. T. Morgan.
Regulation of cytochrome P450 2C11 (CYP2C11) gene expression by interleukin-1, sphingomyelin hydrolysis, and ceramides in rat hepatocytes.
J. Biol. Chem.
270:
25233-25238,
1995[Abstract/Free Full Text].
7.
Chmura, S. J.,
E. Nodzenski,
M. A. Beckett,
D. W. Kufe,
J. Quintans,
and
R. R. Weichselbaum.
Loss of ceramide production confers resistance to radiation-induced apoptosis.
Cancer Res.
57:
1270-1275,
1997[Abstract].
8.
Clejan, S.,
R. S. Dotson,
E. W. Wolf,
M. P. Corb,
and
C. F. Ide.
Morphological differentiation of N1E-115 neuroblastoma cells by dimethyl sulfoxide activation of lipid second messengers.
Exp. Cell Res.
224:
16-27,
1996[Medline].
9.
Coroneos, E.,
Y. Wang,
J. R. Panuska,
D. J. Templeton,
and
M. Kester.
Sphingolipid metabolites differentially regulate extracellular signal-regulated kinase and stress-activated protein kinase cascades.
Biochem. J.
316:
13-17,
1996[Medline].
10.
Deng, X.,
X. Wang,
and
R. Anderson.
Influence of anti-inflammatory and antioxidant agents on endothelial permeability alterations induced by bradykinin.
J. Invest. Surg.
9:
337-349,
1996[Medline].
11.
Dobrowsky, R. T.,
M. H. Werner,
A. M. Castellino,
M. V. Chao,
and
Y. A. Hannun.
Activation of the sphingomyelin cycle through the low-affinity neurotrophin receptor.
Science
265:
1596-1599,
1994[Medline].
12.
Field, F. J.,
H. Chen,
E. Born,
B. Dixon,
and
S. Mathur.
Release of ceramide after membrane sphingomyelin hydrolysis decreases the basolateral secretion of tria-cylglycerol and apolipoprotein B in cultured human intestinal cells.
J. Clin. Invest.
92:
2609-2619,
1993[Medline].
13.
Ghosh, S.,
J. C. Strum,
and
R. M. Bell.
Lipid biochemistry: function of glycerolipids and sphingolipids in cellular signaling.
FASEB J.
11:
45-50,
1997[Abstract/Free Full Text].
14.
Gomez-Munoz, A.,
A. Martin,
L. O'Brien,
and
D. N. Brindley.
Cell-permeable ceramides inhibit the stimulation of DNA synthesis and phospholipase D activity by phosphatidate and lysophosphatidate in rat fibroblasts.
J. Biol. Chem.
269:
8937-8943,
1994[Abstract/Free Full Text].
15.
Gomez-Munoz, A.,
D. W. Waggoner,
L. O'Brien,
and
D. N. Brindley.
Interaction of ceramides, sphingosine, and sphingosine 1-phosphate in regulating DNA synthesis and phospholipase D activity.
J. Biol. Chem.
270:
26318-26325,
1995[Abstract/Free Full Text].
16.
Heape, A. M.,
F. Boiron,
J. J. Bessoule,
and
C. Cassagne.
Peripheral nerve sphingomyelin and cerebroside are both formed via two metabolically and kinetically distinct pathways in vivo.
Eur. J. Biochem.
226:
491-504,
1994[Abstract].
17.
Hirschberg, K.,
J. Rodger,
and
A. H. Futerman.
The long-chain sphingoid base of sphingolipids is acylated at the cytosolic surface of the endoplasmic reticulum in rat liver.
Biochem. J.
290:
751-757,
1993[Medline].
18.
Jarvis, W. D.,
F. A. Fornari,
R. S. Traylor,
H. A. Martin,
L. B. Kramer,
R. K. Erukulla,
R. Bittman,
and
S. Grant.
Induction of apoptosis and potentiation of ceramide-mediated cytotoxicity by sphingoid bases in human myeloid leukemia cells.
J. Biol. Chem.
271:
8275-8284,
1996[Abstract/Free Full Text].
19.
Jarvis, W. D.,
R. N. Kolesnick,
F. A. Fornari,
R. S. Traylor,
D. A. Gewirtz,
and
S. Grant.
Induction of apoptotic DNA damage and cell death by activation of the sphingomyelin pathway.
Proc. Natl. Acad. Sci. USA
91:
73-77,
1994[Abstract].
20.
Kanety, H.,
R. Hemi,
M. Z. Papa,
and
A. Karasik.
Sphingomyelinase and ceramide suppress insulin-induced tyrosine phosphorylation of the insulin receptor substrate-1.
J. Biol. Chem.
271:
9895-9897,
1996[Abstract/Free Full Text].
21.
Kennedy, C. R.,
P. R. Proulx,
and
R. L. Hébert.
Role of PLA2, PLC, and PLD in bradykinin-induced release of arachidonic acid in MDCK cells.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1064-C1072,
1996[Abstract/Free Full Text].
22.
Komori, H.,
and
M. Ito.
Conversion of short-chain ceramides to short-chain ceramide GM3 in B16 melanoma cells.
FEBS Lett.
374:
299-302,
1995[Medline].
23.
Liu, B., L. M. Obeid, and Y. A. Hannun.
Sphingomyelinases in cell regulation. Sem. Cell & Dev. Biol. 311-322, 1997.
24.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951[Free Full Text].
25.
Mantelli, L.,
S. Amerini,
and
F. Ledda.
Bradykinin-induced vasodilation is changed to a vasoconstrictor response in vessels of aged normotensive and hypertensive rats.
Inflamm. Res.
44:
70-73,
1995[Medline].
26.
Mathias, S.,
and
R. Kolesnick.
Ceramide: a novel second messenger.
Adv. Lipid Res.
25:
65-90,
1993[Medline].
27.
Mathias, S.,
A. Younes,
C. C. Kan,
I. Orlow,
C. Joseph,
and
R. N. Kolesnick.
Activation of the sphingomyelin signaling pathway in intact EL4 cells and in a cell-free system by IL-1
.
Science
259:
519-522,
1993[Medline].
28.
Meacci, E.,
V. Vasta,
M. Farnararo,
and
P. Bruni.
Bradykinin increases ceramide and sphingosine content in human fibroblasts: possible involvement of glycosphingolipids.
Biochem. Biophys. Res. Commun.
221:
1-7,
1996[Medline].
29.
Meacci, E.,
V. Vasta,
S. Neri,
M. Farnararo,
and
P. Bruni.
Activation of phospholipase D in human fibroblasts by ceramide and sphingosine: evaluation of their modulatory role in bradykinin stimulation of phospholipase D.
Biochem. Biophys. Res. Commun.
225:
392-399,
1996[Medline].
30.
Merrill, A. H., Jr.,
S. Lingrell,
E. Wang,
M. Nikolova-Karakashian,
T. R. Vales,
and
D. E. Vance.
Sphingolipid biosynthesis de novo by rat hepatocytes in culture. Ceramide and sphingomyelin are associated with, but not required for, very low density lipoprotein secretion.
J. Biol. Chem.
270:
13834-13841,
1995[Abstract/Free Full Text].
31.
Merrill, A. H., Jr.,
E. M. Schmelz,
D. L. Dillehay,
S. Spiegel,
J. A. Shayman,
J. J. Schroeder,
R. T. Riley,
K. A. Voss,
and
E. Wang.
Sphingolipids-the enigmatic lipid class: biochemistry, physiology, and pathophysiology.
Toxicol. Appl. Pharmacol.
142:
208-225,
1997[Medline].
32.
Mukai, H.,
W. R. Fitzgibbon,
G. Bozeman,
H. S. Margolius,
and
D. W. Ploth.
Bradykinin B2 receptor antagonist increases chloride and water absorption in rat medullary collecting duct.
Am. J. Physiol.
271 (Regulatory Integrative Comp. Physiol. 40):
R352-R360,
1996[Abstract/Free Full Text].
33.
Muller-Decker, K.
Interruption of TPA-induced signals by an antiviral and antitumoral xanthate compound: inhibition of a phospholipase C-type reaction.
Biochem. Biophys. Res. Commun.
162:
198-205,
1989[Medline].
34.
Nakamura, Y.,
S. Nakashima,
K. Ojio,
Y. Banno,
H. Miyata,
and
Y. Nozawa.
Ceramide inhibits IgE-mediated activation of phospholipase D, but not of phospholipase C, in rat basophilic leukemia (RBL-2H3) cells.
J. Immunol.
156:
256-262,
1996[Abstract].
35.
Olivera, A.,
A. Romanowski,
C. S. Rani,
and
S. Spiegel.
Differential effects of sphingomyelinase and cell-permeable ceramide analogs on proliferation of Swiss 3T3 fibroblasts.
Biochim. Biophys. Acta
1348:
311-323,
1997[Medline].
36.
Pena, L. A.,
Z. Fuks,
and
R. Kolesnick.
Stress-induced apoptosis and the sphingomyelin pathway.
Biochem. Pharmacol.
53:
615-621,
1997[Medline].
37.
Preiss, J.,
C. R. Loomis,
W. R. Bishop,
R. Stein,
J. E. Niedel,
and
R. M. Bell.
Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells.
J. Biol. Chem.
261:
8597-8600,
1986[Abstract/Free Full Text].
38.
Pushkareva, M.,
L. M. Obeid,
and
Y. A. Hannun.
Ceramide: an endogenous regulator of apoptosis and growth suppression.
Immunol. Today
16:
294-297,
1995[Medline].
39.
Pyne, S.,
J. Chapman,
L. Steele,
and
N. J. Pyne.
Sphingomyelin-derived lipids differentially regulate the extracellular signal-regulated kinase 2 (ERK-2) and c-Jun N-terminal kinase (JNK) signal cascades in airway smooth muscle.
Eur. J. Biochem.
237:
819-826,
1996[Abstract].
40.
Regoli, D.,
D. Jukic,
F. Gobeil,
and
N. E. Rhaleb.
Receptors for bradykinin and related kinins: a critical analysis.
Can. J. Physiol. Pharmacol.
71:
556-567,
1993[Medline].
41.
Schindelholz, B.,
and
B. F. X. Reber.
Bradykinin-induced collapse of rat pheochromocytoma (PC12) cell growth cones: a role for tyrosine kinase activity.
J. Neurosci.
17:
8391-8401,
1997[Abstract/Free Full Text].
42.
Schütze, S.,
K. Potthoff,
T. Machleidt,
D. Berkovic,
K. Wiegmann,
and
M. Kronke.
TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown.
Cell
71:
765-776,
1992[Medline].
43.
Sheard, N. F.,
and
S. H. Zeisel.
Choline: an essential dietary nutrient?
Nutrition
5:
1-5,
1989[Medline].
44.
Sjoholm, A.
Ceramide inhibits pancreatic
-cell insulin production and mitogenesis and mimics the actions of interleukin-1
.
FEBS Lett.
367:
283-286,
1995[Medline].
45.
van der Zee, D. C.,
E. de Heer,
J. Piersma,
and
C. Vermeij-Keers.
Ultrastructural alterations caused by immunological reactions after intracardiac injection of allogeneic antibodies against blood group antigens: an experimental study using the in vitro whole-rat embryo culture.
Teratology
52:
57-70,
1995[Medline].
46.
Viani, P.,
I. Zini,
G. Cervato,
G. Biagini,
L. F. Agnati,
and
B. Cestaro.
Effect of endothelin-1 induced ischemia on peroxidative damage and membrane properties in rat striatum synaptosomes.
Neurochem. Res.
20:
689-695,
1995[Medline].
47.
Ward, D. T.,
J. Ohanian,
A. M. Heagerty,
and
V. Ohanian.
Phospholipase D-induced phosphatidate production in intact small arteries during noradrenaline stimulation: involvement of both G-protein and tyrosine-phosphorylation-linked pathways.
Biochem. J.
307:
451-456,
1995[Medline].
48.
Wright, H. M.,
and
K. U. Malik.
Prostacyclin formation elicited by endothelin-1 in rat aorta is mediated via phospholipase D activation and not phospholipase C or A2.
Circ. Res.
79:
271-276,
1996[Abstract/Free Full Text].
49.
Wright, T. M.,
L. A. Rangan,
H. S. Shin,
and
D. M. Raben.
Kinetic analysis of 1,2-diacylglycerol mass levels in cultured fibroblasts. Comparison of stimulation by
-thrombin and epidermal growth factor.
J. Biol. Chem.
263:
9374-9380,
1988[Abstract/Free Full Text].
50.
Yamamoto, H.
Interrelation of differentiation, proliferation and apoptosis in cancer cells.
Journal of Osaka Dental University
29:
51-60,
1995[Medline].
51.
Yanaga, F.,
and
S. P. Watson.
Ceramide does not mediate the effect of tumour necrosis factor alpha on superoxide generation in human neutrophils.
Biochem. J.
298:
733-738,
1994[Medline].
52.
Yoshida, H.,
N. Ura,
and
K. Shimamoto.
Prostaglandin and kallikrein-kinin systems.
Nippon Rinsho
55:
1909-1914,
1997[Medline].
53.
Yoshimura, S.,
H. Sakai,
K. Ohguchi,
S. Nakashima,
Y. Banno,
Y. Nishimura,
N. Sakai,
and
Y. Nozawa.
Changes in the activity and mRNA levels of phospholipase D during ceramide-induced apoptosis in rat C6 glial cells.
J. Neurochem.
69:
713-720,
1997[Medline].
Am J Physiol Renal Physiol 276(4):F589-F598
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society