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INTRODUCTION |
During the past 10 years, a great amount of evidence has
demonstrated that ceramide
(Cer),1 a key molecule in
sphingolipid metabolism, represents a crucial modulator of cell life,
being involved in signal transduction mechanisms that control cell
growth arrest, differentiation, and apoptosis (reviewed in Refs. 1-3).
Changes in Cer levels can be induced by a large number of stimuli
(including growth factors, hormones, and cytokines), cytotoxic and
infection agents, as well as different environmental conditions (1, 3,
4). The mechanisms through which stimuli control Cer intracellular
levels involve the regulation of multiple enzymes with different
subcellular localization: sphingomyelinase, sphingomyelin-synthase,
glucosylceramide synthase, Cer-synthase, ceramidase, and serine
palmitoyltransferase (3, 5-7). In addition, in the same cell type,
different isoforms of some of these enzymes may be present, and a
single stimulus may have more than one target enzyme. Moreover, it has
been demonstrated that Cer interaction with multiple targets can result
in different biological responses (1). A key element in defining the
role of Cer in cell signaling is its hydrophobic nature, and its
consequent inability to spontaneously move among different subcellular
sites where the enzymes of its metabolism and its molecular targets are
located. As a consequence, the biological effects of Cer may also
depend on the regulation of its intracellular traffic and the presence
of specific signaling pools of this bioactive sphingoid in the cell
(7-9).
The role of Cer as a mediator in signal transduction pathways governing
life has also been demonstrated in cells of the nervous system
(reviewed in Refs. 10-12). In particular, recent studies indicate
that, through the regulation of sphingomyelinase, sphingomyelin synthase, or serine palmitoyl transferase activities, the modulation of
Cer levels in glial cells plays a fundamental role in the control of
cell proliferation, differentiation, and apoptosis (13-16). This
crucial role of Cer is strongly supported by recent evidence about Cer
mediation in the apoptotic and antitumoral activity of cannabinoids in
gliomas (14) and Cer tumor levels inversely correlated with human
astrocytoma malignancy and poor prognosis (17).
Among the molecules that can regulate growth, differentiation, and
apoptosis in different cell types, nitric oxide (NO) has emerged as a
key element in its role as an inter- and intra-cellular mediator of
physiological and pathophysiological events in the nervous system
(18-21). The major enzymes responsible for NO production are the
constitutive neuronal NO synthase in neurons and the inducible one in
glial cells (20, 21). Various stimuli are able to regulate the activity
and/or expression of these enzymes. Noteworthy, in both astrocytes and
glioma cells, Cer is involved in cytokine-mediated NO synthase
induction (22, 23).
The role of NO in the control of glial cell growth is documented by
studies demonstrating the antiproliferative action of NO in both
astrocytes and glioma cell lines (24, 25). Notwithstanding this
evidence, the molecular mechanisms underlying these effects on glial
cell growth are still largely unknown. Furthermore, in some extraneural
cells, recent evidence shows that NO may exert an apoptotic effect by
increasing Cer level through the regulation of the metabolic pathways
involved in its generation or removal (26, 27).
In this study we investigate the possible involvement of Cer in the
inhibitory role exerted by NO on C6 glioma cell proliferation. In
particular, we focus on the effects of NO on the molecular mechanisms
involved in the control of cellular Cer levels. Here, we present
evidence that in C6 glioma cells NO promotes a Cer increase by
inhibiting its transport, possibly vesicle-mediated, from ER to Golgi
apparatus. As a consequence, newly synthesized Cer accumulates and
appears to act as a mediator to the antiproliferative effect of NO.
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EXPERIMENTAL PROCEDURES |
Materials--
Dulbecco's modified Eagle's medium (DMEM),
fetal calf serum (FCS), brefeldin A (BFA), bovine serum albumin
fraction V (BSA), fatty acid free-BSA, sphingomyelinase (SMase) from
Staphylococcus aureus, UDP-glucose (UDP-Glc), fumonisin
B1 (FB1),
N-acetyl-D-erythro-sphingosine (C2-Cer),
N-hexanoyl-D-erythro-sphingosine
(C6-Cer), and N-acetyl-DL-dihydrosphingosine (C2-DHCer) were from Sigma Chemical Co. (St. Louis, MO).
[methyl-3H]Thymidine (20 Ci/mmol),
[3H]choline (19 Ci/mmol), [3H]serine (23 Ci/mmol),
D-erythro-[3-3H]sphingosine (Sph)
(19.7 Ci/mmol), [Sph-3H]C6-Cer, labeled at C-3 of the
long chain base (19 Ci/mmol) and [
-32P]ATP (3000 Ci/mmol) were from PerkinElmer Life Sciences (Boston, MA).
[Sph-3H]sphingomyelin (SM) (0.4 Ci/mmol) was obtained as
previously described (28). PAPANONOate was from Alexis-Italia (Firenze, Italy).
6-((N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl) sphingosine (NBD-C6Cer) and
N-(4,4-difluoro-5,7-dimethyl-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingosine (BODIPY-C5Cer) were from Molecular Probes
Europe (Leiden, The Netherlands). High performance thin layer
chromatography (HPTLC) silica gel plates were from Merck (Darmstadt, Germany).
Cell Cultures--
The C6 glioma cell line was obtained from
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia
(Brescia, Italy). Cells were routinely maintained in DMEM supplemented
with 10% FCS (DMEM plus 10% FCS), 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in an atmosphere of 5%
CO2 and 95% humidified air. Cell viability was assessed
with the trypan blue exclusion test.
Proliferation Assays--
For proliferation assays, cells were
plated in 35-mm dishes (104 cells/cm2) and
maintained in DMEM plus 10% FCS for 24 h. Then, the plates were
washed twice with DMEM containing 0.25% FCS and incubated for 48 h in the same medium before use as quiescent cells. Cells were then
incubated in DMEM plus 10% FCS for 24 h with or without the
following molecules: 100-400 µM PAPANONOate, 10 µM-1 mM cGMP, 10 µM C2-Cer or
C6-Cer or C2-DHCer, and 0.25 unit/ml SMase. 1 µCi/ml
[3H]thymidine was added to each plate 4 h before
cell harvesting, and the thymidine incorporation in
trichloroacetic-insoluble material was determined.
Ceramide Quantification--
Total sphingolipids of C6 glioma
cells (6 × 105 cells) were labeled at equilibrium by
incubating cells with 1 µCi of [3H]Sph (20 Ci/mmol) in
DMEM plus 10% FCS for 24 h. At the end of incubation, the plates
were washed twice with fresh medium and chased for 3 h in DMEM
plus 10% FCS. [3H]Sph-labeled cells were then stimulated
with 400 µM PAPANONOate in DMEM plus 10% FCS or
incubated in DMEM plus 10% FCS. At different intervals, the cells were
washed twice with PBS at 4 °C and harvested, and total lipids were
extracted as recently described (28). The total lipid extract was
partitioned, and the organic phase was subjected to mild alkaline
methanolysis, a 1-h treatment at 37 °C with 0.1 M KOH in
methanol. The methanolyzed organic phase and the aqueous phase were
analyzed by HPTLC using as solvent systems chloroform/methanol/water
(55:20:3, by volume) and butanol/acetic acid/water (3:1:1, by
volume), respectively.
[3H]Choline Metabolism in Intact Cells--
C6
glioma cells plated at 1.5 × 104 cell/cm2
in 35-mm dishes were maintained 48 h in DMEM plus 10%FCS and then
incubated with fresh medium containing [3H]choline (2 µCi/ml) with or without 400 µM PAPANONOate. At
different pulse times, the cells were washed twice with PBS at 4 °C
and harvested, and total lipids were extracted and partitioned (28). The organic phase was analyzed by HPTLC using
chloroform/methanol/acetic acid/water (25:15:4:2, by volume) as the
solvent system.
[3H]Serine Metabolism in Intact Cells--
C6
glioma cells plated at 1.5 × 104 cell/cm2
in 35-mm dishes were maintained 48 h in DMEM plus 10%FCS and then
incubated with fresh medium containing [3H]serine (2 µCi/ml). In pulse experiments cells were fed with [3H]serine in the presence or absence of 400 µM PAPANONOate. In chase experiments, after a 1-h pulse
with the radiolabeled compound (2 µCi/ml), the cells were submitted
to a period of chase in DMEM plus 10%FCS with or without 400 µM PAPANONOate. At appropriate pulse or chase times, the
cells were washed twice with PBS at 4 °C and harvested, and total
lipids were extracted and processed as previously described (28).
[Sph-3H]Sphingomyelin,
[3H]Sphingosine, and
N-Hexanoyl-[3H]sphingosine Metabolism in Intact
Cells--
C6 glioma cells plated at 1.5 × 104
cell/cm2 in 35-mm dishes were maintained 48 h in DMEM
plus 10%FCS. Stock solutions of [Sph-3H]SM,
[3H]Sph, and [Sph-3H]C6-Cer in absolute
ethanol were prepared and added to fresh medium. In all cases the final
concentration of ethanol never exceeded 0.1% (v/v). The cells were
pulsed for different times with [Sph-3H]SM (1 µCi/ml),
[3H]Sph (0.3 µCi/ml), or [Sph-3H]C6-Cer
(0.6 µCi/ml) with or without 400 µM PAPANONOate. To
evaluate the effect of BFA and ATP depletion on [3H]Sph
metabolism, the cells were preincubated for 30 min at 37 °C with 1 µg/ml BFA or 20 mM 2-deoxy-D-glucose, 2 mM NaN3 (28); the same conditions were
maintained during the [3H]Sph pulse. At different
pulse times the cells were washed twice with PBS at 4 °C and
harvested, and total lipids were extracted and processed as previously
described (28).
In Vitro Enzyme Assays--
SMases, SM synthase, and GlcCer
synthase activities were assayed using as enzyme source cell homogenate
(obtained by sonication in H2O three times, 10 s at
4 °C) of control and PAPANONOate-treated cells.
Mg2+-dependent neutral sphingomyelinase
(N-SMase) and acidic sphingomyelinase (A-SMase) were assayed using
[Sph-3H]SM as substrate as previously described (29). The
N-SMase incubation mixture contained 20 mM Tris-Cl (pH
7.4), 10 mM MgCl2, 0.1% Triton X-100, 250 µM [Sph-3H]SM (0.1 µCi), and 5-20 µg
of cell protein in a final volume of 25 µl. The A-SMase incubation
mixture contained 200 mM acetate buffer (pH 5.0), 10 mM EDTA, 0.1% Triton X-100, 250 µM
[Sph-3H]SM (0.1 µCi), and 2-10 µg of cell protein in
a final volume of 25 µl. After 30-min incubation at 37 °C, the
reactions were stopped by adding 100 µl of chloroform/methanol (2:1,
by volume) at 4 °C.
SM-synthase activity was assayed as previously described (16), with
minor modifications. The incubation mixture contained 50 mM
Tris-Cl (pH 7.4), 25 mM KCl, 0.5 mM EDTA, 2 nmol (0.05 µCi) of [Sph-3H]C6-Cer (as 1:1 complex with
fatty acid-free BSA), and 20 µg of cell protein in a final volume of
50 µl. After 15-min incubation at 37 °C, the reaction was stopped
by adding 150 µl of chloroform/methanol (1:2, by volume) at
4 °C.
GlcCer synthase activity was assayed as previously described (30) with
minor modifications. The incubation mixture contained 50 mM
Tris-Cl (pH 7.4), 25 mM KCl, 10 mM
MnCl2, 5 mM UDP-Glc, 2 nmol (0.05 µCi) of
[Sph-3H]C6-Cer (as 1:1 complex with fatty acid-free BSA),
and 15 µg of cell protein in a final volume of 50 µl. After 15-min
incubation at 37 °C, the reaction was stopped by adding 150 µl of
chloroform/methanol (1:2, by volume) at 4 °C. In all cases, after
lipid extraction and phase separation, the [3H]lipids
were resolved by HPTLC as previously described (16).
Sphingosine kinase activity was assayed as described (31). Briefly,
control and PAPANONOate-treated cells were washed with PBS and scraped
in Sph kinase buffer (20 mM Tris-Cl (pH 7.4) containing 20% glycerol, 1 mM mercaptoethanol, 1 mM
orthovanadate, 1 mM EDTA, 15 mM NaF, 0.5 mM 4-deoxypyridoxine, 0.4 mM
phenylmethylsulfonyl fluoride, 40 mM
-glycerophosphate,
and 10 µg/ml each of leupeptin, aprotinin, and trypsin inhibitor).
Cells were disrupted by freeze and thawing and centrifuged at
105,000 × g for 90 min. An aliquot of the supernatant
(20-50 µg of protein) was diluted in Sph kinase buffer, then Sph (50 µM final concentration as a 1:1 complex with fatty
acid-free BSA) was added. The reaction was started by the addition of
10 µCi of [
-32P]ATP (1 mM final
concentration) containing MgCl2 (10 mM final concentration) in a final volume of 200 µl. After incubation at 37 °C for 30 min, the reaction was stopped by the addition of 1 M HCl, and radiolabeled sphingosine 1-phosphate was
resolved by HPTLC (31). After visualization by autoradiography, the
radioactive spots were scraped off from the plate and counted for radioactivity.
Analysis of the Intracellular Distribution of Fluorescent
Ceramides--
C6 glioma cells plated at 1.5 × 104 cell/cm2 were grown on a glass coverslip
and maintained 48 h in DMEM plus 10%FCS. Then cells were
incubated with 5 µM BODIPY-C5-Cer or
NBD-C6-Cer (as 1:1 complex with fatty acid free BSA) in
DMEM at 4 °C for 30 min (32). After washing (three times with DMEM
plus 10%FCS and 0.34 mg/ml fatty acid-free BSA), cells were incubated
1 h at 37 °C in DMEM plus 10%FCS with or without 400 µM PAPANONOate. Cells were then washed (three times with
PBS) and fixed with 0.5% glutaraldehyde solution in PBS for 10 min at
4 °C. The specimens were immediately observed and analyzed with a
fluorescence microscope (Olympus BX-50) equipped with a fast high
resolution charge-coupled device camera (Colorview 12) and a image
analytical software (Analysis from Soft Imaging System GmbH).
Preparation of Cytosolic Fractions--
C6 glioma cells plated
at 6 × 104 cell/cm2 in 100-mm dishes were
maintained 48 h in DMEM plus 10%FCS and then incubated for 1 h in the same medium with or without 400 µM PAPANONOate.
At the end of incubation, the cells were washed twice with PBS at 4 °C and gently harvested in 0.75 ml of PBS. Cytosolic fractions were prepared as previously described (33) with some modifications. All
manipulations were carried out at 4 °C or on ice. The cells were
precipitated by centrifugation at 250 × g for 10 min,
suspended in 4 volumes of 10 mM Tris-Cl, pH 7.4, 0.25 M sucrose, and homogenized by 3 cycles of freezing in
liquid nitrogen and thawing at 37 °C. The homogenate was centrifuged
at 900 × g for 10 min to precipitate nuclei. The
postnuclear supernatant was centrifuged twice at 100,000 × g for 1 h to remove the particulate fraction
completely. The supernatant obtained from the second centrifugation was
rapidly frozen in liquid nitrogen and stored at
80 °C until use as
a cytosolic fraction. The protein concentration of the cytosolic fraction obtained was about 1.8 mg/ml.
Preparation of [3H]Cer-labeled Semi-intact
Cells--
Cells plated at 6 × 104
cell/cm2 in 100-mm dishes were maintained 48 h in DMEM
plus 10% FCS and then incubated for 1 h with or without 400 µM PAPANONOate. To prepare semi-intact cells loaded with
[3H]Cer, cells were then washed twice with DMEM plus 10%
FCS and pulsed in the same medium with 1 µM
[3H]Sph (1.43 µCi/dish) for 30 min. To minimize Cer
utilization for complex sphingolipids, incubation was performed at
10 °C. At the end of pulse, [3H]Sph-labeled cells were
incubated in the same medium containing 5 µM
FB1 (an inhibitor of Cer synthase) for 15 min. Hereafter, all manipulations were carried out at 4 °C. Semi-intact C6 glioma cells were prepared as previously described (33) with several modifications. In particular, cells were washed twice in a
hypotonic buffer (10 mM Hepes-KOH, 40 mM KCl,
0.1 mM MgCl2, pH 7.3) and incubated in the same
buffer for 10 min. Then, the hypotonic buffer was replaced by an
isotonic one (10 mM Hepes-KOH, 115 mM KCl, pH
7.3), and the cells were rapidly, gently scraped and collected by low
speed centrifugation at 250 × g for 5 min with the
brake off. After washing with the isotonic buffer, the pellets were resuspended in the isotonic buffer (about 120 µl/dish) and
immediately used as [3H]Cer-labeled semi-intact cell
preparations. With this procedure, more than 80% C6 glioma cells were
trypan blue-positive. The enzymatic analysis of both precipitate and
supernatant fractions showed that more than 85% lactate dehydrogenase
activity was recovered in the supernatant. The protein concentration of
the semi-intact cell preparations from both control and NO-treated
cells was in the 1.2-1.4 mg of protein/ml range.
In Vitro Assay of Cer Transport from ER to the Site of GlcCer and
SM Synthesis--
The Cer transport assay was performed (33) by
incubating [3H]Cer-labeled semi-intact cells (13 µg of
protein) and the cytosol (32.5 µg of protein) in 100 µl (final
volume) of a transport assay mixture containing, as final
concentrations: 20 mM Hepes-KOH, pH 7.0, 70 mM
KCl, 2.5 mM magnesium acetate, 250 µM GTP,
0.2 mM dithiothreitol, 0.5 mM UDP-Glc, 0.5 mM MnCl2, an ATP-regenerating system (50 µM ATP, 2 mM creatine phosphate, 8 units/ml
creatine phosphokinase), and 5 µM FB1. In
some experiments, the transport assay was performed in the absence of
the ATP-regenerating system or in 20 mM Hepes-KOH, pH 7.0, and 70 mM KCl, with or without 0.5 mM UDP-Glc.
In the cytosol exchange experiments, semi-intact cells from control or
NO-treated cells were incubated in the presence of the cytosol from
NO-treated or control cells, respectively. Mixtures were incubated 30 min at 37 °C and stopped by the addition of 780 µl of
chloroform/methanol (1:2, by volume) and partitioned as described (33).
After counting the radioactivity, the organic phase was analyzed by
HPTLC using as solvent system chloroform/methanol/water (55:20:3, by
volume). To obtain quantitative data on the transport activities of
semi-intact cells, [3H]Cer-labeled semi-intact cells were
also extracted prior to the Cer transport assay, and the radioactivity
associated to Cer, SM, and GlcCer was used as the background.
Other Methods--
Total protein was assayed with the Coomassie
Blue-based Pierce Reagent, using BSA as standard. SM and GM3 contents
were determined as previously reported (34, 35). Lactate dehydrogenase
activity was measured according to Strorrie and Maddie (36).
Radioactivity was measured by liquid scintillation counting or
radiochromatoscanning. Digital autoradiography of HPTLC plates was
performed with a Beta-Imager 2000 (Biospace, France), and the
radioactivity associated with individual lipids was determined with
software provided with the instrument. The 3H-labeled
sphingolipids were recognized and identified as previously described
(28). Statistical significance of differences was determined by
Student's t test.
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RESULTS |
Effect of NO on C6 Glioma Cell Proliferation--
To investigate
the effect of exogenously delivered NO on C6 glioma cell proliferation,
cells were treated with different concentrations of the NO-releasing
molecule PAPANONOate. In the 100-400 µM range, PAPANONOate induced a dose-dependent inhibition of cell
proliferation (Fig. 1, left
panel). The maximal inhibitory effect on
[3H]thymidine incorporation of about 80% was reached at
400 µM PAPANONOate. In these experimental conditions, at
both 24 and 48 h after treatment with the NO donor, cell
viability, assessed by trypan blue exclusion, was not affected. This
indicates that the effect of the NO-releasing compound on
[3H]thymidine incorporation results from the inhibition
of cell growth and not from toxicity. Moreover, when PAPANONOate was
used after being decayed for 24 h, no effect on
[3H]thymidine incorporation was observed, indicating that
the effect of PAPANONOate on C6 glioma cell proliferation was due to NO
generation. As shown in Fig. 1 (right panel),
dibutyryl-cGMP (the membrane-permeant analog of cGMP)
administered in the 0.01-1 mM range, did not affect [3H]thymidine incorporation into proliferating glioma
cells.

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Fig. 1.
Effect of NO and cGMP on C6 glioma cell
proliferation. Quiescent C6 glioma cells were incubated 24 h
in DMEM plus 10% FCS with different concentrations of PAPANONOate
(left panel) or dibutyryl-cGMP (right panel). In
the last 4 h of incubation, cells were pulsed with 1 µCi of
[3H]thymidine, and the radioactivity associated with
trichloroacetic-insoluble material was determined. Results are shown as
a percentage of control, untreated cells. All values are the mean ± S.D. of at least three individual experiments. *, p < 0.001 versus control.
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Involvement of Cer in the NO-induced Cell Growth Inhibition of C6
Glioma Cells--
We next evaluated the possible involvement of Cer as
a mediator for the antiproliferative activity of NO in C6 glioma cells. To this purpose, we first measured intracellular Cer levels at various
intervals after the administration of PAPANONOate to cells labeled to
the equilibrium with [3H]Sph. In preliminary experiments,
we set up the labeling conditions so as to obtain a
[3H]GM3/[3H]SM ratio corresponding to that
of the endogenous compounds as an index for a steady-state labeling of
cell sphingolipids. As shown in Fig. 2,
in these conditions, exogenously delivered NO caused a rapid and
significant increase of the Cer cellular level. The maximum increase,
corresponding to 167% of the control levels, was reached 4 h
after NO treatment. Cer levels in NO-treated cells remained
significantly higher than in controls, even after 24 h.

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Fig. 2.
Effect of NO on Cer levels in C6 glioma
cells. 6 × 105 cells were labeled with 1 µCi
of [3H]Sph, in DMEM plus 10% FCS, for 24 h. After a
3-h chase in DMEM plus 10% FCS, cells were incubated in the same
medium with (<) or without ( ) 400 µM PAPANONOate. At
different intervals, cells were harvested and submitted to lipid
quantification as described under "Experimental Procedures."
Results are shown as percentage of control, untreated cells. All values
are the mean ± S.D. of at least three individual experiments. *,
p < 0.001 versus control.
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In addition, we obtained evidence that those treatments able to
increase Cer cellular levels mimicked the NO-induced cell growth
inhibition (Fig. 3). In particular, the
incubation of C6 cells with cell-permeable analogs C2- or C6-Cer as
well as the treatment with bacterial SMase resulted in a significant
reduction of [3H]thymidine incorporation into DNA. Under
the same conditions, the C2-Cer dihydro- derivative had no
effect.

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Fig. 3.
Effect of Cer on C6 glioma cells
proliferation. Quiescent C6 glioma cells were incubated 24 h
in DMEM plus 10% FCS with or without 10 µM C2-Cer,
C6-Cer, C2-DHCer, or 0.25 unit/ml bacterial SMase. During the last
4 h, cells were pulsed with 1 µCi of
[3H]thymidine, and the radioactivity associated with
trichloroacetic-insoluble material was determined. Results are shown as
percentage of control, untreated cells. All values are the mean ± S.D. of at least three individual experiments. *, p < 0.001 versus control.
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Effect of NO on [3H]SM and [3H]Choline
Metabolism and on SMase and SM Synthase Activities--
To investigate
the metabolic pathways involved in NO-induced modification of Cer
levels, we first examined the effect of NO on SM degradation and
biosynthesis. The possible contribution of SM degradation to the
increase of Cer levels observed in NO-treated cells was evaluated in
pulse experiments with [Sph-3H]SM, performed with or
without PAPANONOate. After a 1-h pulse, similar values of
[3H]SM and [3H]Cer were found in control
and treated cells (Fig. 4, upper
panel). In these conditions, [3H]Sph formed from
[3H]Cer hydrolysis was 3.3% total incorporated
radioactivity in control and 3.1% in treated cells, indicating that NO
did not modify Cer cleavage by ceramidases. We also evaluated the
effect of NO on in vitro activity of N-SMase and A-SMase.
Using homogenates obtained from control or NO-treated cells as the
enzyme source, and adding PAPANONOate to the homogenate of control
cells, no difference was observed in either N-SMase or A-SMase
activities (Fig. 4, lower panel).

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Fig. 4.
Effect of NO on [3H]SM
metabolism and SMase activity in C6 glioma cells. Upper
panel, cells grown in DMEM plus 10% FCS were incubated with
[Sph-3H]SM for 1 h with or without 400 µM PAPANONOate. Then, cell lipids were extracted and
analyzed as described under "Experimental Procedures." Open
bars, control cells; closed bars, NO-treated cells.
Lower panel, A-SMase and N-SMase activities were measured
using [Sph-3H]SM as substrate and cell homogenate
obtained from control (open bars) or NO-treated
(closed bars) cells, as enzyme source. The enzymatic
activity of control homogenates was also measured in the incubation
mixture containing 400 µM PAPANONOate (squared
bars). All values are the mean ± S.D. of at least three
individual experiments.
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The possible effect of NO on SM biosynthesis was then evaluated by
pulsing cells with [3H]choline with or without
PAPANONOate (Fig. 5, upper
panel). At 2-h pulse, the incorporation of
[3H]choline into SM in NO-treated cells was about 40% of
that measured in control cells, whereas the incorporation of the same
precursor into phosphatidylcholine, the phosphorylcholine donor for the conversion of Cer to SM, was not affected by NO. On the basis of this
evidence we assessed the effect of NO on in vitro activity of SM synthase. Here too, the homogenates obtained from control or
NO-treated cells were used as the enzyme source, and, in the case of
control cells, the enzymatic activity was also measured in the presence
of PAPANONOate. As shown in Fig. 5 (lower panel), neither NO
cell treatment nor NO addition in the enzymatic assay affected SM
synthase activity in C6 glioma cells.

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Fig. 5.
Effect of NO on [3H]choline
metabolism and SM synthase in C6 glioma cells. Upper
panel, cells grown in DMEM plus 10% FCS were pulsed with 2 µCi/ml [3H]choline for 2 h, with or without 400 µM PAPANONOate. At the end, cell lipids were extracted
and analyzed as described under "Experimental Procedures."
Open bars, control cells; closed bars, NO-treated
cells. PC, phosphatidylcholine. Lower panel, SM synthase
activity was measured using [Sph-3H]C6-Cer as substrate,
and cell homogenate from control (open bars) or NO-treated
cells (closed bars) was the enzyme source. The enzymatic
activity of control homogenates was also measured in the incubation
mixture containing 400 µM PAPANONOate (squared
bars). All values are the mean ± S.D. of at least three
individual experiments. *, p < 0.001 versus
control.
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Effect of NO on [3H]Sph Metabolism--
For further
information on the effect of NO on SM biosynthesis, taking into account
that in glial cells exogenous Sph is rapidly incorporated first in Cer
and then in SM and glycosphingolipids (37, 38), we performed an
additional metabolic study using [3H]Sph. When C6 glioma
cells were pulsed for 1 h with [3H]Sph with or
without PAPANONOate, the radioactive precursor was rapidly and
efficiently incorporated in both control and treated cells. In both
cases, [3H]Sph was mainly metabolized to
N-acylated compounds, most represented by Cer, SM, and, in
lower amounts, GlcCer and GM3 (Fig. 6,
upper panel). After 1-h pulse, the uptake of Sph and the
incorporation of radioactivity into N-acylated compounds (as
the sum of tritiated Cer, SM, GlcCer, and GM3) were very similar in
control and NO-treated cells. However, treatment with NO strongly
modified the distribution of radioactivity between the different Sph
metabolites; in fact, [3H]Cer was about 2-fold higher in
NO-treated than in control cells (Fig. 6, upper panel). At
the same time, in NO-treated cells, the radioactivity incorporated into
SM, GlcCer, and GM3 was 40, 35, and 34% less, respectively, than
in controls. In these conditions, treatment with NO donor did
not modify the amount of radioactivity incorporated into sphingosine
1-phosphate (Fig. 6, upper panel). As in the case of SM
synthase, the in vitro activity of GlcCer synthase was
similar in control (0.27 ± 0.04 nmol/mg of protein/min) and
NO-treated cells (0.26 ± 0.039 nmol/mg of protein/min), and this
was also found after addition of PAPANONOate to the incubation mixture
of control cell assay (0.28 ± 0.045 nmol/mg of protein/min). In
addition, the activity of Sph kinase assayed in vitro was
very similar in control (0.12 ± 0.013 nmol/min/mg of protein) and
NO-treated (0.13 ± 0.015 nmol/min/mg of protein) cells.

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Fig. 6.
Effect of NO on [3H]Sph and
[3H]serine metabolism in C6 glioma cells.
Upper panel, cells were pulsed with 0.3 µCi/ml
[3H]Sph for 1 h in DMEM plus 10% FCS with or
without 400 µM PAPANONOate SP-1P, sphingosine
1-phosphate. Lower panel, cells were pulsed with 2 µCi/ml
[3H]serine in DMEM plus 10% FCS for 1 h and chased
2 h in the same medium. 400 µM PAPANONOate was added
during pulse (left part) or chase (right part)
period. In all cases, at the end, cell lipids were extracted and
analyzed as described under "Experimental Procedures." Open
bars, control cells; closed bars, NO-treated cells. All
values are the mean ± S.D. of at least three individual
experiments. *, p < 0.001 versus
control.
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Effect of NO on Sphingolipid Metabolism from
[3H]Serine--
The possible effect of NO on the
de novo biosynthesis and metabolic processing of Cer was
investigated after administration of [3H]serine. After a
1-h pulse, the incorporation of radioactivity into total lipids and the
amount of radioactivity associated to sphingolipids were very similar
in control and NO-treated cells indicating that NO did not affect the
de novo biosynthesis of Cer. At this pulse time, NO
treatment promoted an increase of the radioactivity associated to Cer
with a concomitant decrease of that associated to SM (Fig. 6,
lower left panel). This effect was found to be more marked
when NO was administered during chase. In particular, as shown in Fig.
6 (lower right panel), NO induced a 40% increase in
[3H]Cer levels, paralleled by a significant decrease of
the radioactivity incorporated into SM.
Effect of NO on [Sph-3H]C6-Cer Metabolism--
On
the basis of these results we investigated the possibility that NO
impairs the availability of Cer for the biosynthesis of complex
sphingolipids. To this purpose we evaluated the effect of PAPANONOate
on [Sph-3H]C6-Cer metabolism. As shown in Fig.
7, after 2-h pulse, the biosynthesis of
[Sph-3H]C6-SM and [Sph-3H]C6-GlcCer (both
derived from the direct metabolic use of the short chain, free
diffusing Cer) was not affected by NO. On the contrary the utilization
of long-chain [3H]Cer, produced in the ER from
[3H]Sph derived from [Sph-3H]C6-Cer
catabolism (39), was strongly inhibited by NO. In fact, in NO-treated
cells [3H]Cer was about 2-fold higher, whereas
[3H]SM and [3H]GlcCer were 40 and 30%
less than those measured in control cells. Noteworthy, the amount of
[3H]Sph produced from the degradation of
[Sph-3H]C6-Cer by ceramidase and represented by the sum
of Sph and its sphingolipid metabolites was very similar in control and
NO-treated cells.

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Fig. 7.
Effect of NO on [Sph-3H]C6-Cer
metabolism in glioma cells. Cells grown in DMEM plus 10% FCS were
pulsed with 0.6 µCi/ml [Sph-3H]C6-Cer (as BSA complex,
1:1 mol/mol), for 2 h, with or without 400 µM
PAPANONOate. Lipids were then extracted and partitioned as described
under "Experimental Procedures." The organic phase was analyzed by
HPTLC using chloroform/methanol/water (55:20:3, by volume) and
chloroform/methanol/CaCl2 0.2% (50:42:11, by volume) as
solvent systems and the radioactivity associated to
N-hexanoyl sphingolipids, C6-Cer, C6-GlcCer, C6-SM, and long
chain sphingolipids, Cer, GlcCer, and SM measured. Open
bars, control cells; closed bars, NO-treated cells. All
values are the mean ± S.D. of at least three individual
experiments. *, p < 0.001 versus
control.
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Effect of NO on the Intracellular Distribution of
BODIPY-C5-Cer and NBD-C6-Cer--
The
transport of natural Cer from ER to the Golgi apparatus can be
qualitatively evaluated from the analysis of BODIPY-C5-Cer redistribution into cells (32, 40). We thus investigated the effect of
NO on the behavior of this fluorescent Cer into intact C6 glioma cells.
After labeling ER and other intracellular membranes, cells were
chased, in the presence or absence of 400 µM PAPANONOate to allow the redistribution of BODIPY-C5-Cer. In control
cells most of fluorescence accumulated in the perinuclear region (Fig. 8A), representative of the
Golgi apparatus (32), whereas in NO-treated cells the accumulation of
fluorescence in the Golgi region was strongly reduced (Fig.
8B). In contrast, when cells were labeled with
NBD-C6-Cer, which spontaneously moves between intracellular
membranes, NO did not appreciably modify the accumulation of NBD
fluorescence in the perinuclear Golgi region (Fig. 8, C and
D).

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Fig. 8.
Effect of NO on intracellular distribution of
BODIPY-C5Cer and NBD-C6Cer. C6 glioma
cells grown on a coverslip were incubated with 5 µM BODIPY-C5Cer (A and
B) or NBD-C6Cer (C and D)
for 30 min at 4 °C; then, prelabeled cells were incubated at
37 °C for 1 h with (B and D) or without
(A and C) 400 µM PAPANONOate. All
images were processed and printed identically. Scale bar, 20 µm.
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Effect of NO on SM and GlcCer Formation from [3H]Sph
in BFA-treated Cells--
To obtain further evidence for the effect of
NO on Cer traffic, we then investigated the effect of NO on
[3H]Sph metabolism in C6 glioma cells treated with BFA,
which leads to the fusion of the cis-Golgi membranes with
the ER (41). Our working hypothesis was that, if NO determines a defect
in ER to Golgi apparatus trafficking of Cer, treatment with BFA should make Cer metabolism insensitive to NO. After pretreatment with BFA for
30 min at 37 °C, cells were pulsed for 30 min with
[3H]Sph in the presence of BFA alone or with PAPANONOate.
Then, the conversion of [3H]Cer to [3H]SM
and [3H]GlcCer was compared in control and BFA-treated
cells (Fig. 9). In BFA-treated cells, the
amount of [3H]SM and [3H]GlcCer synthesized
from [3H]Cer was found to be about 2-fold higher than in
control ones. In the presence of BFA, NO was no more able to impair
[3H]Cer metabolic utilization, thus implying that NO
affects the translocation of Cer from the ER to the Golgi
apparatus.

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Fig. 9.
Effect of NO on [3H]Sph
metabolism in BFA-treated C6 glioma cells. Cells were preincubated
in DMEM plus 10% FCS containing BFA (1 µg/ml) for 30 min at
37 °C, and then labeled with 0.3 µCi/ml [3H]Sph for
30 min in BFA-containing medium with or without 400 µM
PAPANONOate. At the end, cell lipids were extracted and analyzed as
described under "Experimental Procedures." Closed bars,
control cells; open bars, BFA-treated cells; striped
bars, cells treated with BFA and NO. All values are the mean ± S.D. of at least three individual experiments.
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Effect of NO on SM Formation from [3H]Sph after ATP
Depletion--
To gain insight into the possible mechanisms of Cer
transport affected by NO, we next examined the effect of NO on
[3H]Cer metabolism, after promoting ATP depletion of
cells. After pulse with [3H]Sph in an ATP-depleting
medium, the incorporation of radioactivity into SM and GlcCer was found
to be about 70 and 30% less than controls (Fig.
10). This was paralleled by a 2-fold
increase of [3H]Cer levels. Moreover, ATP depletion did
not reduce either [3H]Sph uptake or its incorporation
into N-acylated compounds (as the sum of tritiated Cer SM
and GlcCer, Fig. 10). This ruled out the possibility that the decrease
in [3H]SM formation in ATP-depleted cells was due to a
decrease in the cellular uptake of [3H]Sph or its
utilization for [3H]Cer synthesis. In ATP-depleted cells,
NO treatment did not further modify the conversion of
[3H]Cer to either [3H]SM or
[3H]GlcCer. Thus, these results show that in C6 glioma
cells the trafficking of Cer to the site of SM and, in a lesser extent, of GlcCer biosynthesis is mainly ATP-dependent and that ATP
depletion mimicked the NO effect on Cer metabolism.

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Fig. 10.
Effect of NO on [3H]Sph
metabolism in ATP-depleted C6 glioma cells. Cells were
preincubated in DMEM plus 10% FCS, containing 20 mM
2-deoxy-glucose and 2 mM NaN3 for 30 min at
37 °C, and then labeled with 0.3 µCi/ml [3H]Sph for
1 h in ATP-depleting medium with or without 400 µM
PAPANONOate. After 1 h, lipids were extracted and analyzed
as described under "Experimental Procedures." Closed
bars, control cells; open bars, ATP-depleted cells;
stripped bars, ATP-depleted and NO-treated cells. All values
are the mean ± S.D. of at least three individual
experiments.
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Cer Transport from the ER to the Site of SM and GlcCer Biosynthesis
in Control and NO-treated Semi-intact Cells--
[3H]Cer
enriched semi-intact cells obtained from control and NO-treated cells
were used to obtain further insights on the effect of NO on Cer
transport from ER to the site of SM and GlcCer synthesis. When control
semi-intact cells were incubated in the buffer, [3H]SM
represented about 4% and [3H]GlcCer 1% of
N-acylated lipids (Fig. 11,
upper left panel). In the transport mixture containing the
cytosol obtained from control cells, the formation of both
[3H]SM and [3H]GlcCer was increased by
about 4-fold; the same increase in [3H]GlcCer was
observed after adding UDP-Glc to the buffer (Fig. 11, upper left
panel). The removal of the ATP-regenerating system from the
transport mixture led to a strong reduction of SM but not GlcCer
formation (Fig. 11, upper left panel).

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Fig. 11.
Metabolism of [3H]Cer to
[3H]SM and [3H]GlcCer in control and
NO-treated semi-intact cells. Upper panels,
[3H]Cer-labeled semi-intact cells from control
(left panel) and NO-treated (right panel) cells
were incubated at 37 °C for 30 min in the buffer (open
bars), in the transport mixture containing the corresponding
cytosol (striped bars), in the absence of the
ATP-regenerating system (squared bars), or in the buffer
containing 0.5 mM UDP-Glc (closed bars). At the
end of incubation, lipids were extracted and analyzed as described
under "Experimental Procedures." Lower panel, effect of
the cytosol exchange between control and NO-treated semi-intact cells.
[3H]SM (open bars) and
[3H]GlcCer (closed bars) were evaluated in the
transport assay conditions. After 30-min incubation at 37 °C, lipids
were extracted and analyzed as described under "Experimental
Procedures." All values are the mean ± S.D. of at least three
individual experiments.
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As shown in Fig. 11 (upper right panel), incubation of
NO-treated semi-intact cells with buffer alone led to
[3H]SM and [3H]GlcCer biosynthesis similar
to that observed in control semi-intact cells. The addition
of NO-treated cytosol and the transport mixture promoted a nearly
3-fold increase in [3H]GlcCer formation without any
significant change in [3H]SM; in the absence of the
ATP-regenerating system, SM formation was not modified. Here too, the
presence of UDP-Glc in the buffer was sufficient to promote
[3H]GlcCer biosynthesis. Thus, these results confirm
that, in C6 glioma cells, NO impairs the ATP,
cytosol-dependent, inter-membrane Cer traffic needed mainly
for SM biosynthesis and, to a lesser extent, for GlcCer.
To see if any specific cellular component is involved in the NO-induced
defective Cer translocation, we carried out fraction exchange
experiments in the transport assay (Fig. 11, lower panel). The use of NO-treated cytosol with control membranes did not
significantly modify the synthesis of [3H]SM or
[3H]GlcCer. Moreover, the use of cytosol obtained from
control cells in combination with NO-treated cells did not restore the
capacity to synthesize [3H]SM and
[3H]GlcCer (Fig. 11, lower panel) thus
suggesting that NO exerts its effect on Cer transport at the level of
the membranous compartment involved in Cer traffic.
 |
DISCUSSION |
The first evidence provided by this study is that NO exerts a
dose-dependent antiproliferative effect on C6 glioma cells. As observed in other cell types (42, 43), this effect was not mimicked
by a membrane-permeant non-hydrolyzable analog of cGMP, supporting the
evidence that also in these cells the antimitogenic effect of NO is
independent from the activation of guanylate cyclase, and thus from
cGMP. Instead, we found that NO induces an early and
significant increase in Cer levels and that treatments resulting in the
increase of cell Cer are able to mimic the antiproliferative effect of
NO in C6 glioma cells. Altogether, these data strongly suggest that Cer
may be a mediator of the antiproliferative effect of NO. In this
context, it is noteworthy that Cer has been recently indicated as a
mediator of the apoptotic response to NO in glomeruloendothelial as
well as mesangial and HL60 cells (26, 27). Furthermore, these results
strengthen the general role of Cer in the control of cell proliferation
(5) and, in particular, as a negative regulator of glial growth (13,
29, 44).
Evidence obtained from non-neural cells indicates that the modulation
of Cer levels following NO treatment can depend on both the activation
of N- and A-SMase and the inhibition of ceramidases (26, 27). The
studies here performed to single out the metabolic pathway responsible
for NO-dependent Cer accumulation in C6 glioma cells
indicate that the degradation of both SM and Cer is not involved in the
NO-induced regulation of Cer levels. By using different experimental
approaches, the major pathway responsible for the
NO-dependent Cer increase was found to involve a reduced utilization of Cer for the biosynthesis of complex sphingolipids, mainly SM. Thus, unlike what was observed in other cell types (26, 27),
in C6 glioma cells SM or Cer cleavage does not appear to be involved in
the modulation of Cer levels by NO. Instead, metabolic experiments
performed with [3H]choline, [3H]serine, and
[3H]Sph, all indicate that NO strongly reduces the
conversion of Cer to SM without affecting the biosynthesis of
[3H]Cer and [3H]phosphatidylcholine, the
two immediate precursors for SM formation. These results strongly
support that Cer utilization for SM biosynthesis represents a key
element/module in the Cer signaling involved in the control of glial
cell growth (16, 44) and more in general in cell proliferation (for a
review see Ref. 5). Notwithstanding the evidence of a
NO-dependent inhibition of both SM and GlcCer biosynthesis,
no direct effect of NO on the enzymatic activity of either SM or GlcCer
synthase was detected. Thus, differently from what occurs in cerebellar
astrocytes and hippocampal neurons after stimulation with basic
fibroblast growth factor (16, 45), the control of Cer levels might be
achieved by a mechanism other than the direct regulation of SM- and/or
GlcCer-synthase. A reasonable possibility is that NO acts by reducing
the availability of Cer as substrate for both enzymes, possibly
inducing a defect in the translocation of Cer, synthesized in the ER,
to the sites where SM-synthase and GlcCer-synthase are localized. Very
convincing support to this hypothesis was obtained by treating C6
glioma cells with [Sph-3H]C6-Cer, with or without the
NO-releasing agent. In fact, in the presence of NO, the direct
utilization of radiolabeled C6-Cer for the biosynthesis of C6-SM and
C6-GlcCer, which does not require Cer exit from the ER, was unmodified.
Moreover, treatment of C6 glioma cells with C6-Cer also resulted in the
generation of endogenous long-chain Cer, as in A549 cells (39). This
process most reasonably involves the recycling of Sph for the
biosynthesis of long-chain Cer at the ER level. Opposite to what was
observed for short-chain Cer, the utilization of long-chain Cer for the
biosynthesis of complex sphingolipids was strongly inhibited by NO.
This finding further confirms that newly synthesized Cer in the ER is
no longer available for complex sphingolipid biosynthesis in the
presence of NO.
The analysis of intracellular distribution of
BODIPY-C5-Cer, which mimics the intracellular movements of
natural Cer, provides additional evidence that NO can influence the
intracellular traffic of Cer from ER to the Golgi apparatus. Moreover,
results obtained with BFA demonstrate that, in C6 glioma cells, the
cis-Golgi represents the major subcellular site for both
GlcCer and SM biosynthesis and supports that NO induces a defect in Cer
translocation from ER to the Golgi apparatus. In fact, when cells were
treated with BFA, which causes Golgi disassembly and redistribution to
the ER (41), the conversion of Cer to both GlcCer and SM was strongly increased. The increase in GlcCer is in agreement with the generally observed cis-medial Golgi location of the
glucosyltransferase involved in its biosynthesis (46, 47). A different
consideration must be made for the BFA-dependent SM
increase in C6 glioma cells. As occurs in many cell types (48-50), the
results here obtained indicate that even in C6 glioma cells SM
biosynthesis occurs mainly in the cis-medial Golgi.
Nevertheless, in different cell types, SM synthesis can also occur at
different subcellular sites (51-53), and, in rat cerebellar
astrocytes, the activation of an SM synthase located in a compartment
other than cis-medial Golgi is involved in the basic
fibroblast growth factor signaling pathway involved in cell
proliferation (16). The direct availability of Cer for SM and GlcCer
synthases determined by BFA makes C6 glioma cells no more sensitive to
NO-dependent inhibition of Cer utilization. This evidence
further confirms that SM- and GlcCer-synthases are not the NO target
and strengthens the notion that the NO-dependent Cer
increase is mainly due to a defect in its translocation from ER to
cis-Golgi.
The way Cer moves from the ER to Golgi is still unclear but, on the
basis of many experimental results, two main mechanisms appear to be
involved and include an ATP-dependent vesicle-mediated Cer
transport, and a non-vesicular Cer translocation that can involve the
participation of transfer proteins as well as ER-Golgi membrane
contacts (54-56). Using a cell line with a specific defect in SM
biosynthesis, evidence was recently presented that Cer may preferentially use an ATP-independent non-vesicular pathway for glycosphingolipid production and an ATP-dependent
vesicle-mediated mechanism for the biosynthesis of SM (40). These same
researchers also demonstrated that the transport of Cer for SM
biosynthesis strongly depends on some unidentified cytosolic factors,
whereas the biosynthesis of GlcCer is mainly cytosol-independent (33). The results obtained in our present study on ATP-depleted cells and on
in vitro Cer transport assays are in agreement with this hypothesis. In fact, in C6 glioma cells, ATP depletion strongly affects
SM biosynthesis and, to a lesser extent, that of GlcCer. On the other
hand, the conversion of Cer into SM in semi-intact cells was strongly
dependent on the presence of ATP and cytosol, whereas that of GlcCer
appears to be mainly dependent on the availability of UDP-Glc. We also
obtained evidence that ATP depletion mimics the inhibitory effect of NO
on the biosynthesis of complex sphingolipids, in particular SM, and
that NO is no longer able to exert any effect in ATP-depleted cells.
Thus the ATP-dependent vesicle-mediated transport of Cer,
primarily for SM biosynthesis, is involved in the
NO-dependent Cer increase. Moreover, results obtained by
using semi-intact cells and cytosol derived from NO-treated cells, led us to exclude the involvement of a cytosolic factor as a NO target and
strongly suggest that the effect of NO on Cer traffic resides in the
membrane component involved in this process. To explain Cer
accumulation consequent to a NO-mediated impairment in its traffic, two
major mechanisms can be hypothesized. First, in the presence of NO,
newly synthesized Cer cannot move from the ER, thus accumulating in
this compartment where it may exert its biological effects (57).
Second, Cer-transferring structures, possibly vesicles, actually leave
the ER but, in the presence of NO, their Golgi-specific targeting is
impaired, thus determining an accumulation of Cer-containing structures
available for the interaction with Cer targets. Studies are in progress
in our laboratory to investigate these possibilities.
In conclusion, to the best of our knowledge this represents the first
evidence for an active role for Cer traffic in Cer signaling. The
results provided here demonstrate that NO is able to modulate the
intracellular traffic of Cer between the ER and the Golgi apparatus. As
a consequence, newly synthesized Cer accumulates and appears to act as
a mediator of the antiproliferative effect of NO in these cells. These
results point out the relevance of Cer intracellular movements and of
the role of specific signaling pools, in defining the biological
effects of Cer (7, 8). Altogether, this strengthens the paradigm that
the understanding of the complex signaling role of Cer cannot leave out
of consideration the metabolic origin, the topology of production, and
the intracellular traffic of this bioactive lipid and its interplay
with other signaling pathways.