Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells.
ABSTRACT We have investigated the effects of inhibiting sphingomyelin (SM) biosynthesis on cellular diacylglycerol (DAG) content and protein kinase C (PKC) activation during growth initiation in Madin-Darby canine kidney cells. We utilized beta-chloroalanine (BCA) to inactivate serine C -palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway. This inactivation prevented growth, but did not affect viability. When the inhibitor was replaced with fresh culture medium, the cells continued their proliferation in a normal way. BCA (2 mM) inhibited [(32)P]P(i), [(3)H]palmitic acid and [ methyl -(3)H]choline incorporation into SM, but did not influence the synthesis of other major phospholipids. SM synthesis and DAG generation were decreased by 51% and 47.6% respectively. Particulate PKC activity was not observed in cells incubated with BCA, in contrast with a 5-fold increase in control cells. BCA inhibited 75% of the [(3)H]thymidine incorporation, and the cells were arrested before the S phase of the cell cycle. Moreover, exogenous D-erythrosphingosine restored SM synthesis, DAG generation and cell proliferation. These data indicate that the contribution of DAG generated during SM synthesis plays an important role in PKC activation and cell proliferation.
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ABSTRACT: Heart disease is widely believed to develop from two pathological processes. Circulating lipoproteins containing the nondegradable lipid, cholesterol, accumulate within the arterial wall and perhaps are oxidized to more toxic lipids. Both lipid accumulation and vascular reaction to the lipids lead to the gradual thickening of the vascular wall. A second major process that in some circumstances is a primary event is the development of a local inflammatory reaction. This might be a reaction to vessel wall injury that accompanies infections, immune disease, and perhaps diabetes and renal failure. In this chapter, we will focus on the relationship between de novo synthesis of sphingolipids and lipid metabolism, atherosclerosis, and cardiomyopathy.Advances in experimental medicine and biology 01/2011; 721:19-39. · 1.83 Impact Factor
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ABSTRACT: Expressions of cell-cycle regulating proteins are altered after stroke. Cell-cycle inhibition has shown dramatic reduction in infarction after stroke. Ceramide can induce cell-cycle arrest by up-regulation of cyclin-dependent kinase (Cdk) inhibitors p21 and p27 through activation of protein phosphatase 2A (PP2A). Tricyclodecan-9-yl-xanthogenate (D609)-increased ceramide levels after transient middle cerebral artery occlusion (tMCAO) in spontaneously hypertensive rat (SHR) probably by inhibiting sphingomyelin synthase (SMS). D609 significantly reduced cerebral infarction and up-regulated Cdk inhibitor p21 and down-regulated phospho-retinoblastoma (pRb) expression after tMCAO in rat. Others have suggested bFGF-induced astrocyte proliferation is attenuated by D609 due to an increase in ceramide by SMS inhibition. D609 also reduced the formation of oxidized phosphatidylcholine (OxPC) protein adducts. D609 may attenuate generation of reactive oxygen species and formation of OxPC by inhibiting microglia/macrophage proliferation after tMCAO (please also see note added in proof: D609 may prevent mature neurons from entering the cell cycle at the early reperfusion, however may not interfere with later proliferation of microglia/ macrophages that are the source of brain derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF-1) in offering protection). It has been proposed that D609 provides benefit after tMCAO by attenuating hypoxia-inducible factor-1alpha and Bcl2/adenovirus E1B 19 kDa interacting protein 3 expressions. Our data suggest that D609 provides benefit after stoke through inhibition of SMS, increased ceramide levels, and induction of cell-cycle arrest by up-regulating p21 and causing hypophosphorylation of Rb (through increased protein phosphatase activity and/or Cdk inhibition).Molecular Neurobiology 02/2010; 41(2-3):206-17. · 5.47 Impact Factor
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ABSTRACT: Lon is an ATP-dependent serine protease that plays a significant role in the quality control of proteins in cells, degrading misfolded proteins and certain short-lived regulatory proteins under stresses as such heat-shock and UV irradiation. It is known that some polymers containing phosphate groups regulate enzymatic activity by binding with Lon. We focused on the phospholipids of biological membrane components such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and cardiolipin (CL), and examined whether or not liposomes containing these phospholipids regulate the enzymatic activity of Lon. CL-containing liposomes specifically inhibited both the proteolytic and ATPase activities of Lon in a dose-dependent manner. In addition, on pull-down assay, we found that CL-containing liposomes selectively bound to Lon. The interaction between CL-containing liposomes and Lon changed with the order of addition of Mg(2+)/ATP. When CL-containing liposomes were added after the addition of Mg(2+)/ATP to Lon, the binding of CL-containing liposomes to Lon was significantly decreased as compared with the reversed order. In fact, we found that CL-containing liposomes bound to Lon, resulting in inhibition of the enzymatic activity of Lon. These results suggest that Lon interacts with CL in biological membranes, which may regulate the functions of Lon as a protein-degrading centre in accordance with environmental changes inside cells.Journal of Biochemistry 03/2011; 149(5):519-27. · 3.07 Impact Factor
Biochem. J. (2003) 373, 917–924 (Printed in Great Britain)
Diacylglycerol generated during sphingomyelin synthesis is involved
in protein kinase C activation and cell proliferation in Madin–Darby
canine kidney cells
Jorge CERB´ON1and Rosa del Carmen L´OPEZ-S´ANCHEZ
Department of Biochemistry, Centro de Investigaci´ on y de Estudios Avanzados del Instituo Polit´ enico Nacional, P.O. Box 14-740, M´ exico City, M´ exico
kinase C (PKC) activation during growth initiation in Madin–
Darby canine kidney cells. We utilized β-chloroalanine (BCA) to
inactivate serine C-palmitoyltransferase, the first enzyme in the
sphingolipid biosynthesis pathway. This inactivation prevented
growth, but did not affect viability. When the inhibitor was re-
placed with fresh culture medium, the cells continued their
proliferation in a normal way. BCA (2 mM) inhibited [32P]Pi,
[3H]palmitic acid and [methyl-3H]choline incorporation into
SM, but did not influence the synthesis of other major
by 51% and 47.6% respectively. Particulate PKC activity was
not observed in cells incubated with BCA, in contrast with
a 5-fold increase in control cells. BCA inhibited 75% of
the [3H]thymidine incorporation, and the cells were arrested
before the S phase of the cell cycle. Moreover, exogenous
generated during SM synthesis plays an important role in PKC
activation and cell proliferation.
Key words: diacylglycerol (DAG) generation, protein kinase C
(PKC) regulation, sphingomyelin synthesis.
In the majority of cells, agonist-stimulated diacylglycerol (DAG)
production is generated by the activation of several signalling
pathways. Changes in DAG mass and phospholipid turnover sug-
majority of DAG in cells . Three pathways of PC metabolism
are potentially linked to DAG generation: one catalysed by PC-
specific phospholipase C (PC-PLC); a second pathway catalysed
by PC-specific phospholipase D (PC-PLD), followed by phos-
phatidic acid phosphatase (PAP) activation, and the third the
ceramide choline phosphotransferase (SM synthase; EC 2.7.8.-).
Numerous studies have investigated the generation of DAG by
the PC-PLC- and PC-PLD–PAP-mediated signalling pathways
, and the signalling role of ceramides, sphingosine and
their derivatives produced through the degradation of membrane
sphingolipids are currently under intense investigation [3–5]. In
contrast, signalling mediated by the generation of DAG during
the synthesis of SM has not been reported. We have studied the
and subsequent protein kinase C (PKC) activation during growth
in Madin–Darby canine kidney (MDCK) cells. MDCK cells were
chosen because they possess DAG-sensitive conventional PKCα
and β isoenzymes, which translocate from cytosol to plasma
membrane upon activation with DAG [6,7].
We took advantage of the availability of three metabolic
inhibitors: β-chloroalanine (BCA), D609 and fumonisin B1.
BCA inhibits serine C-palmitoyltransferase (SPT, E.C.22.214.171.124),
the first committed step of sphingolipid synthesis. SPT is a
Abbreviations used: BCA, β-chloroalanine, DAG, diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; MDCK, Madin–
Darby canine kidney; PAP, phosphatidic acid phosphatase; PC, phosphatidylcholine; PC-PLC, PC-specific phospholipase C; PC-PLD, PC-specific
phospholipase D; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PKC, protein kinase C; SM, sphingomyelin; SPT, serine
1To whom correspondence should be addressed (e-mail email@example.com).
that 5 mM BCA inhibits SPT completely in 15 min without
affecting cell viability for up to 6 h . β-F-Alanine inhibits
sphinganine synthesis and its incorporation into sphingomyelin
by at least 60%, and fumonisin B1 is an inhibitor of ceramide
synthase that decreased the SM mass by approx. 50% . In
the present study, when the synthesis of SM was inhibited with
2 mM BCA, the generation of DAG was decreased and there was
no PKC activation. DNA synthesis was also inhibited, suggesting
that cells were arrested before S phase, probably at the G1 to
S phase transition. Fumonisin B1 (0.14–1 µM) and D609 (20–
40 µg/ml) inhibited SM synthesis and decreased DAG levels to
a similar extent. Our data provide evidence that DAG generated
during SM synthesis participates in the activation of PKC and is
required for the normal transit through the cell cycle.
MATERIALS AND METHODS
[32P]Pi(carrier-free, 10 mCi/ml), [γ-32P]ATP (>5000 Ci/mmol;
3H]palmitic acid (54 Ci/mmol), [methyl-3H]choline chloride
(60–85 Ci/mmol) and the DAG assay system were purchased
from Amersham Biosciences. Fetal bovine serum (FBS), trypsin
and the PKC assay kit were obtained from Gibco; Dulbecco’s
modified Eagle’s medium (DMEM) was from Microlab; BCA,
fumonisin B1, D609, o-phthalaldehyde and other chemicals were
from Sigma; organic solvents were obtained from Merck, and
scintillation liquid was from New England Nuclear.
c ?2003 Biochemical Society
J. Cerb´ on and R. C. L´ opez-S´ anchez
MDCK cells between passages 65–75 were obtained from the
American Type Culture Collection and were a gift of Dr J. L.
Reyes (Department of Physiology, Centro Investigaciones y de
Estudios Avanzados, M´ exico). Cells were cultured in complete
medium [DMEM supplemented with 10% (v/v) FBS, 100 units/
ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine] in
a humidified air incubator containing 5% CO2 at 37◦C, and
cells were grown to confluence (5–6 days). The culture medium
was removed and cells were trypsinized for the minimum time
required for the cells to detach from the culture dish. Cells were
then washed and resuspended in PBS and viability was assessed
by the Trypan Blue exclusion method. All experiments were
initiated with cell suspensions obtained from confluent cultures.
After trypsinization, cells were washed once with PBS/DMEM
medium and allowed to adhere to 100 mm tissue-culture dishes
(Corning) at (5.5–6.0)×106cells/8 ml of complete medium and
incubated at 37◦C in 5% CO2.
MDCK cells (5×105cells) were seeded in complete medium on
35 mm plastic plates with or without BCA at various concen-
trations. At the indicated times, cells were trypsinized and the cell
of two independent experiments.
Labelling and analysis of phospholipids
Lipids were extracted using the methanol stop procedure .
The addition of methanol prior to scraping the cells from the
dish was shown to stop cellular metabolism and permit accurate
Directly after removal of the culture dishes from the incubator,
the complete medium was aspirated off as completely as possi-
ble, the cells were washed twice with PBS and 1 ml of methanol
was added. Cells were scraped off the dish and transferred to
a 13 mm×100 mm glass tube. Dishes were washed once with
1 ml of methanol, which was also transferred to the tubes. Phases
were split by the addition of chloroform (1 ml) and water to
obtain a chloroform/methanol/water ratio of 5:10:4 (by vol.). The
monolayer was vortex-mixed and left at room temperature. After
1 h, the cellular debris was pelleted by centrifugation and the su-
pernatant transferred to another tube (16 mm×125 mm). The
(5:10:4, by vol.) for 1 h. After centrifugation, the supernatants
were combined. Phospholipid phosphorus (nmol of Pi present
in the chloroform phase) was determined by the method of
Ames , and labelled lipids in the organic phase were
resolved by two-dimensional TLC on silica gel G plates and
developed in chloroform/methanol/acetic acid/water (85:20:8:4,
by vol.) in the first dimension and chloroform/methanol/28%
(v/v) ammonium hydroxide (81:31:5, by vol.) in the second
dimension. For the separation of DAG, petroleum ether/diethyl
ether/acetic acid (90:10:1, by vol.) was used. After separation,
were included. The individual areas were removed from the
plate and quantified by scintillation counting. Radioactivity was
corrected for the amount of phospholipids.
Mass measurement of cellular phospholipids
To label phospholipids pools uniformly for steady-state labelling,
cells were seeded at 6×106cells (in 100 mm dishes) in complete
medium containing [32P]Pi (10 µCi/ml) or [3H]palmitic acid
(5 µCi/ml) and grown to confluence. The use of large dishes
ensured that the cells had adequate room to divide several times
before contact inhibition occurred. Knowing the amount (nmol)
composition in the steady-state labelled cells, the amount of each
phospholipid was determined and expressed as nmol/mg of
protein. The protein concentration was measured by the method
of Bradford  using BSA as the standard. To monitor if there
was any phospholipid accumulation during growth re-initiation
and DAG were determined.
DAG mass measurements
A radioenzymic assay employing Escherichia coli DAG kinase
(Amersham Biosciences) was used to quantify DAG production
under defined mixed micelle conditions [7.5% (w/v) n-octyl-
β-glucopyranoside and 5 mM cardiolipin in 1 mM diethylene-
thyamine pentacetic acid] to solubilize the DAG. In the presence
of[γ-32P]ATP (>5000 Ci/mmol), the enzymeconvertsDAG into
[32P]phosphatidic acid. The assay was performed according to the
manufacturer’s instructions. [32P]Phosphatidic acid was extracted
and subsequently separated from other lipids and residual [γ-
32P]ATP by TLC using phosphatidic acid (Sigma) as reference.
The radioactivity of [32P]phosphatidic acid was estimated by
liquid-scintillation counting. The DAG/phospholipid ratios were
calculated and expressed as pmol of DAG/nmol of phosphate.
using the DAG kinase method.
Measurement of phospholipid turnover
Confluent cultures of MDCK cells were subcultured in DMEM
alone containing 10 µCi/ml [32P]Piin the absence or presence
of 2 mM BCA. At t=0, the average cell number per 100 mm
dish was (5.8–6.0)×106cells. After 2 h, the cells were washed
with PBS, and fresh medium in the absence or presence of
2 mM BCA was added. At the indicated times up until 10 h,
duplicate dishes of cells were harvested by washing twice with
ice-cold PBS and the lipids were extracted, separated by TLC and
quantified as described above. At the 2 h pulse time, there were
226700 c.p.m./dish in the control and 200000 c.p.m./dish in the
cells treated with BCA.
MDCK cells were also labelled with [3H]choline (5 µCi/ml) in
the absence or presence of BCA. At the indicated times, duplicate
MDCK cells were grown to confluence and 6.0×106cells were
subcultured on 100 mm tissue-culture plates containing complete
medium in the absence (control) or presence of 2 mM BCA.
After incubation for the indicated times, cells were washed twice
with PBS, scraped from dishes in extraction buffer [20 mM
Tris/HCl (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA and 25 µg/ml
c ?2003 Biochemical Society
Sphingomyelin synthesis and cell proliferation
each of aprotinin and leupeptin], disrupted by freeze-thawing and
the cytosolic and membrane fractions were separated by centri-
fugation (100000 g, 1 h, 4◦C) in a Beckman L-37 ultracen-
and the pellet was resuspended in extraction buffer containing
0.5% Triton X-100. Cytosolic and membrane fractions were
assayed for PKC activity using a PKC assay kit (Gibco). The
PKC assay kit is based on measurement of the phosphorylation of
acetyl-myelin basic protein(4–14) as described by Yasuda et al.
, and results are expressed as pmol of phosphate/min per
6×106cells. Protein levels was corrected on the basis of protein
content of the dish analysed.
Measurement of levels of sphingoid bases
Sphingosine and sphinganine (natural inhibitors of PKC) were
quantified using HPLC as described by Merrill et al. .
Sphingosine and sphinganine standards were used for identifi-
of 5×106cells were required for sufficient cellular sphingosine
to be accurately determined by this method.
Measurement of [3H]thymidine incorporation
MDCK cells (1×105cells/well) were incubated in complete
medium. [3H]Thymidine (1 µCi/ml) was added to each well
2 h before the end of the incubation period. Cells were washed
twice with ice-cold PBS and fixed with 95% (v/v) methanol
(15 min). After being washed with PBS, the cells were lysed
with 0.2 M NaOH, neutralized with 0.2 M HCl and subjected to
Difference between means was compared using Student’s t test or
ANOVA using the SAS computer program.
Effect of BCA on MDCK cell viability and proliferation
It is known that there is a concentration- and time-dependence
of inhibition of long-chain base synthesis by BCA . In order
to explore the relevance of sphingolipid synthesis on cell prolifer-
ation, we examined the effect of BCA. Figure 1 shows that there
was a concentration-dependent inhibition of cell proliferation and
that 2 mM BCA resulted in a complete inhibition. After 10 h
of exposure to BCA, there was no loss of cell viability (>90%
moved and replaced with fresh medium, cell proliferation was
re-activated and the cultures reached confluence after 5–6 days
(results not shown).
Effect of BCA on phospholipid synthesis
Exogenous added [9,10(n)-3H]palmitic acid was used to estimate
the synthesis of phospholipids. After 2 h in complete medium
in the absence (control) or presence of 2 mM BCA, MDCK cells
were washed twice with DMEM to remove BCA and then were
labelled with [3H]palmitic acid (5 µCi/ml of culture medium)
for 3.5 or 5.5 h. Labelling was started after 2 h of incubation
because, at this time, the levels of endogenous sphingoid bases
were drastically decreased (, and see below). Table 1 shows
that BCA decreased the amount of [3H]palmitic acid in SM in
Figure 1Effect of BCA on MDCK cells proliferation and viability
MDCK cells were seeded at a density of 5×104cells/cm2(35 mm dish) in complete medium
in the absence (control, ?) or presence of 0.5 (?), 1.0 (?), 1.5 (?) and 2.0 (?) mM BCA.
Cell density was determined by trypsinizing duplicate dishes at the indicated times and viable
cells were counted. Results are means+
−S.D. of three independent experiments.
Table 1 Effects of BCA on de novo phospholipid synthesis
MDCK cells were incubated with 5 µCi/ml [3H]palmitic acid in culture medium for 3.5 and
5.5 h, after which cellular lipids were extracted and analysed as described in the Materials
and methods section. The cells were preincubated with 2 mM BCA for 2 h and washed out
before the pulse (−2 h), or BCA was present before and during the pulse (5.5 h). Values are
expressed as d.p.m./nmol of phospholipid and are means+
representative experiment. *P <0.05 and **P <0.01 compared with control
−S.D. of triplicate samples from a
5.5 h (3.5 h pulse)7.5 h (5.5 h pulse)
Lipid ControlBCA (−2 h) BCA (5.5 h)ControlBCA (−2 h)
the cells pulsed for 3.5 h and 5.5 h by 45 and 49% respect-
ively (Table 1, columns 2 and 5). In other experiments, cells were
exposed to BCA for 2 h before and for 3.5 h during the pulse.
The amount of [3H]palmitic acid incorporated into SM was de-
creased by 76% (Table 1, column 3). Under all conditions tested,
the incorporation of radiolabel into total lipid extract, PC, phos-
phatidylserine (PS)/phosphtidylinositol (PI) and phosphatidyl-
ethanolamine (PE) was not altered by exposure to BCA. These
data indicate that BCA does not interfere with the biosynthesis of
other phospholipids, at least during the first 7.5 h of incubation.
Similar results were obtained with 5 µCi/ml [methyl-3H]choline.
After 7.5 h of incubation in the presence of 2 mM BCA there was
a 42% decrease in labelled sphingomyelin (380+−5 compared
with 220+−17 d.p.m./nmol of lipid phosphorus) without a
significant difference in labelled PC (1291+−10 compared with
1179+−130 d.p.m./nmol of lipid phosphorus). [3H]Palmitic acid
incorporation into DAG was decreased by 60%.
c ?2003 Biochemical Society
J. Cerb´ on and R. C. L´ opez-S´ anchez
Figure 2 Effect of 2 mM BCA on [32P]-labelled phospholipid turnover
MDCKcells(5.6×106)wereseededincompletemedium(0 htimepoint)andpulse-labelledfor2 hwith10 µCi/ml[32P]Piintheabsence(control,?)orpresence(?)of2 mMBCA,washedtwice
with PBS, followed by culture medium (without isotope) with or without 2 mM BCA. At the indicated times, cells were harvested, lipids were extracted, separated by TLC and [32P] incorporated into
phospholipids was quantified. Results are expressed as c.p.m.×10−3/sample and are means+
−S.D. of duplicate dishes from five separate experiments. *P <0.05 and **P <0.01 compared with
Effect of 2 mM BCA on phospholipid turnover
We examined the effect of 2 mM BCA on phospholipidformation
(2 h pulse) and lipid turnover (2–10 h chase). The incorporation
of [32P]Piinto phospholipids was similar in both the control and
the BCA-treated cells (720+−77 and 615+−54 c.p.m.×10−3
respectively, after 5.5 h). The only significant difference observed
was a 35% inhibition in the labelling of SM after 5.5 h of culture
(Figure 2). The turnover of PC and PI occurred, since labelled PC
decreased by 50% (from 5–10 h) and labelled PI decreased by
31% (from 8–10 h). Removal of the choline or phosphocholine
moiety from PC, with a calculated half-life of 2–4 h when choline
was used as the radiolabel, has been found to be specific for
the G1phase of both the first and the second cell cycles .
Interestingly, labelled SM decreased by 75% in 2.5 h (between
5.5 and 7.5 h of culture). To our knowledge, this early and fast
SM turnover has not been observed before.
SM synthesis and DAG generation
To establish possible changes in the formation of DAG during
the synthesis of SM and to confirm their turnover, MDCK cells
were prelabelled with [3H]palmitic acid to steady state, washed
twice with DMEM and (5.5–6.0)×106cells were seeded in com-
plete medium without label. At the indicated times, lipids were
mitic acid into SM and DAG was determined. As shown in
Figure 3, there was a temporal correlation between SM synthesis
and the generation of labelled DAG. During the first 5.5 h of
incubation, SM and DAG increased approx. 2- and 2.5-fold
respectively. Between 5.5 h and 10 h of incubation both SM and
DAG decreased towards the levels found in confluent cells (0 h
time point). Such a decrease in labelled SM could result from the
activation of a reverse in SM synthesis, which has been found
Figure 3Time course of SM synthesis and DAG generation
Cells were prelabelled with [3H]palmitic acid for 5 days, washed and (5.5–6)×106cells
were seeded in complete medium without label. At the indicated times, lipids were extracted
and separated by TLC and label incorporated into SM and DAG was determined. The results
are expressed as d.p.m.×10−3per mg of protein. Results are the means+
determinations from a representative experiment of three separate experiments.
−S.D. of triplicate
in MDCK cells , or SM degradation. This reverse in SM
synthesis could explain the decrease in DAG and/or the ceramide
ceramide produced could be utilized for the synthesis of glyco-
sphingolipids or SM, or participate in the activation of protein
phosphatases (for reviews see [5,17]).
c ?2003 Biochemical Society
Sphingomyelin synthesis and cell proliferation
analysed by mass measurements
Influence of time of incubation on DAG levels in MDCK cells
MDCK cells derived from confluent cultures were seeded in complete medium in plastic dishes
at a cell density of (5.8–6)×106cells in the absence or presence of 2 mM BCA and directly
analysed for DAG content (DAG kinase) and total phospholipid phosphorus at the indicated
times of incubation. Values are means+
DAG at start was measured in confluent cells before trypsinization. −, not determined.
−S.D. from measurements on six separate dishes. The
DAG/phospholipid (pmol of DAG/nmol
of total phospholipid)
Time (h) Control BCA
Start (0 h)
Effect of time of incubation and BCA on cellular DAG mass
MDCK cells [(5.8–6.0)×106cells] were seeded in plastic dishes
containing 8 ml of complete medium and incubated at 37◦C in
the absence (control) or presence of 2 mM BCA. At the indicated
times, DAG (DAG kinase) and total phospholipid phosphorus
content were determined and DAG/phospholipid ratios (pmol of
DAG/nmol of total phospholipid) were calculated. Table 2 shows
that the amount of DAG increased 3.2 to 3.9 times after 2.0
and 5.5 h of incubation and then decreased (between 7.5 and
10 h) towards the level found in confluent cells (0 h time point).
This behaviour was similar to that found in cells prelabelled with
the amount of DAG measured after 2.0 and 5.5 h of incubation
was decreased 2.3 times when compared with the control values.
Effect of BCA on MDCK PKC
DAG-dependent activation of PKC is associated with a change
in its subcellular distribution from the cytosol to the membrane
fraction. Therefore in order to know if the DAG generated during
SM synthesis was involved in signalling, the activation of PKC
(particulate fraction) was determined at 2 and 5 h of incubation.
These time points were selected on the basis of the observed
increase in DAG and SM synthesis. The PKC activity in the
particulate fraction increased 5-fold in the control, whereas in
the BCA-treated cells no increase was detected (Table 3). This
supports further our proposal that DAG generated during SM
synthesis participates in signalling (PKC activation). Addition of
100 nMPMA30 minbeforeharvestingincreasedparticulatePKC
activity in the BCA-treated cells.
Content of sphingoid bases in MDCK cells
Sphingoid bases are reported to be inhibitors of PKC  and,
therefore, we determined the levels of sphingosine and sphin-
ganine. After 2 h of incubation in DMEM, the levels of sphingoid
goid bases determined at 0 h remained (Table 4). In cells exposed
bition of the synthesis of sphingoid bases, the decrease was even
greater. These results suggest a rapid metabolic transformation
following cell-cycle activation and minimize the possibility that
BCA-treated cells. In fact, it has been reported that sphingosine
Table 3 Inhibition of PKC activation by 2 mM BCA
Results are means+
(100 nM) was added 30 min before harvest.
−S.D. of three independent experiments performed in duplicate. PMA
Particulate PKC (pmol of phosphate/min per mg of protein)
Time (h)ControlBCA Control+PMABCA+PMA
Effect of time of incubation and BCA on cellular concentrations of
were extracted and their o-phthalaldehyde derivates were prepared and analysed by HPLC, as
described in the text. Values are means+
−S.D. of five separate experiments. ND, not detected.
Control Time (h)BCA
1-phosphate is synthesized rapidly in response to mitogenic
that this process was also taking place in MDCK cells.
Effect of D-erythrosphingosine on BCA-treated cells
, appears to inhibit various pyridoxal phosphate-dependent
eration of DAG. Table 5 shows that 1 and 2 µM sphingosine
restored the generation of DAG in the BCA-treated cells deter-
mined at 5.5 h of incubation, and 5 µM sphingosine did not
increase further the amount of DAG. When 2 mM BCA was pre-
sent throughout the treatment period, the addition of 1 and 5 µM
D-erythrosphingosine to the culture medium resulted in a 56 and
50% restoration of growth respectively, after 48 h of incubation.
Higher concentrations of sphingosine were less efficient (34.5%
growth recoveries were observed with 10 µM, and 20 µM was
cytotoxic). When added exogenously, sphingosine is a potent
inhibitor of PKC, and sphingosine was also found to inhibit the
activity of other enzymes, including Na+/K+ATPase, calcium/
CTP: phosphocholine cytidylyltransferase .
Effect of fumonisin B1 and D609 on the synthesis of SM, the
generation of DAG, ceramide levels and PKC activation
The effects of fumonisin B1 (inhibitor of ceramide synthesis) 
c ?2003 Biochemical Society