© 1999 American Heart Association, Inc.
Molecular Medicine |
Sphingosylphosphorylcholine Induces a Hypertrophic Growth Response Through the Mitogen-Activated Protein Kinase Signaling Cascade in Rat Neonatal Cardiac Myocytes
From the Second Department of Internal Medicine (K.S., T.Y., M.K., R.N.), Gunma University School of Medicine, and Laboratory of Signal Transduction (F.O.), Institute for Molecular and Cellular Regulation, Gunma University School of Medicine, Maebashi, Japan.
Correspondence to Tomoyuki Yokoyama, MD, Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-22, Showa-machi, Maebashi, 371, Japan. E-mail yokoyamt{at}news.sb.gunma-u.ac.jp
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Abstract |
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Abstract—The sphingolipid metabolites, sphingosine (SPH), SPH 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC), can act as intracellular as well as extracellular signaling molecules. These compounds have been implicated in the regulation of cell growth, differentiation, and programmed cell death in nonmyocytes, but the effects of sphingolipid metabolites in cardiac myocytes are not known. Cultured neonatal rat cardiac myocytes were stimulated with SPH (1 to 10 µmol/L), S1P (1 to 10 µmol/L), or SPC (0.1 to 10 µmol/L) for 24 hours to determine the effects of sphingolipid metabolites on the rates of protein synthesis and degradation. Stimulation with SPC led to an increase in the total amount of protein, an accelerated rate of total protein synthesis, and a decrease in protein degradation in a dose-dependent manner. However, S1P had little effect and SPH had no effect on total protein synthesis. In addition, stimulation with SPC led to a 1.4-fold increase in myocardial cell size and enhanced atrial natriuretic factor gene expression. Pretreatment of the cardiac myocytes with pertussis toxin or PD98059 attenuated the SPC-induced hypertrophic growth response. Further, stimulation with SPC increased phosphorylation of mitogen-activated protein kinase (MAPK) and stimulated MAPK enzyme activity. Finally, endothelin-1 stimulated the generation of SPC in cardiac myocytes. The observation that SPC induces a hypertrophic growth response in cardiac myocytes suggests that SPC may play a critical role in the development of cardiac hypertrophy. The effects of SPC could be mediated, in part, by activation of a G protein–coupled receptor and a MAPK signaling cascade.
Key Words: sphingolipid • cardiac myocyte • hypertrophy • receptor • mitogen-activated protein kinase
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Introduction |
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Sphingolipids were once thought to be the inert structural components of cell membranes. However, recent studies have shown that sphingolipids are important participants in the regulation of a variety of cellular processes.1 Sphingosine (SPH), a metabolite of ceramide, has been shown to stimulate cell proliferation2 and regulate intracellular Ca2+ mobilization.3 4 Exogenous SPH 1-phosphate (S1P) has also been reported to regulate Ca2+ mobilization,5 6 microfilament reorganization,7 cell migration,8 and fibroblast proliferation.9 10 Further, sphingosylphosphorylcholine (SPC), another metabolite of sphingomyelin, has been shown to be a potent mitogen in a variety of cell types.11 12 More recently, plasma membrane receptors coupled to G proteins with a high affinity for S1P and/or SPC have been described in various cell types.13 These sphingolipid metabolites can therefore act as intracellular and extracellular signaling molecules in the regulation of cell growth and differentiation.
In rat ventricular myocytes, SPH has been shown to modulate
myocyte contractile behavior by inhibiting intracellular
Ca2+ transients and L-type
Ca2+ channel conductance.14 15 In
contrast, SPC causes an increase in the diastolic
Ca2+ concentration in rat cardiac myocytes by
activating the ryanodine receptor and possibly another intracellular
Ca2+-release channel.16 Further,
both S1P and SPC are capable of activating muscarinic
K+ current channels
(Ik(Ach)) in guinea pig atrial
myocytes.17 Activation of
Ik(Ach) by SPC suggests that there is
signaling via the pertussis toxin (PTX)–sensitive G protein–coupled
pathway. Recently, Oral et al18 reported that the SPH
content of adult feline cardiac myocytes increases with stimulation of
tumor necrosis factor-
and that both SPH and S1P have negative
inotropic effects on myocytes. However, the role of sphingolipid
metabolites in the development of cardiac hypertrophy is
not known.
The involvement of sphingolipid metabolites in cell growth led us to hypothesize that sphingolipid metabolites trigger hypertrophic growth responses in cardiac myocytes. We therefore examined the effects of exogenous sphingolipid metabolites, SPH, S1P, and SPC, on the rate of protein synthesis and degradation in isolated rat neonatal cardiac myocytes. Additional studies were performed to evaluate whether the hypertrophic growth response induced by exogenous sphingolipid metabolites is mediated by activating a G protein–coupled receptor.
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Materials and Methods |
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Materials
D-SPH, S1P, SPC, PTX, and calphostin C were obtained from Sigma Chemical Co. PD98059 (2'-amino-3'-methoxyflavone) was obtained from BIOMOL Research Laboratories, Inc. [3H]Phenylalanine, [N-methyl-14C]sphingomyelin, and [9,10(n)-3H]palmitic acid were obtained from Amersham Co.
Protein Synthesis and Degradation
Primary neonatal rat cardiac ventricular myocyte
cultures were prepared as previously described.19 The
amount of newly synthesized total protein and protein degradation were
determined by the previously described methods.20
Measurements of Cell Surface Area
Cardiac myocyte cultures (2x105 cells per
35-mm culture dish) were fixed with 3%
paraformaldehyde and incubated with 3% BSA to block
nonspecific binding and then with a monoclonal antibody against
sarcomeric -actinin (Sigma). Antibody binding proteins were
visualized colorimetrically using a VECTATIN Elite ABC
kit (Vector Laboratories, Inc). Planimetry was performed using NIH
Image software, which automatically calculates cell area from a manual
tracing of the cell outline.
Northern Blot Analysis of Atrial Natriuretic
Factor (ANF) mRNA
Total RNA isolations and Northern hybridization with a
restriction fragment probe for mouse ANF DNA21 were
performed essentially as previously described.19
Identification of
Threonine/Tyrosine– Phosphorylated Extracellular
Signal–Regulated Kinases (ERKs) by Western Blot Analysis
To identify threonine/tyrosine–phosphorylated
forms (which are active forms) of p44 ERK and p42 ERK in cardiac
myocytes, Western blot analysis was performed using a
polyclonal rabbit anti-human phosphospecific mitogen-activated
protein kinase (MAPK) (Thr202/Try204) antibody, which detects
phosphorylated threonine 202 and tyrosine 204 of p44
and p42 ERKs (New England Biolabs, Inc).22
MAPK Activity Assay
MAPK activity was assayed by using a commercially available kit
(Amersham).
Assay of SPC Production
SPC generation was assessed by the method of Bowser and
Gray,23 with some modifications. Cardiac myocyte cultures
were labeled for 24 hours with 50 nmol of
[N-methyl-14C]sphingomyelin (55
mCi/mmol) or 20 pmol of [9,10(n)-3H]palmitic
acid (50 Ci/mmol). On day 2, the myocyte cultures were washed 3 times
with cold PBS and then exposed to DMEM supplemented with 0.1% BSA. The
cultures were then stimulated with diluent, endothelin-1 (ET-1;
0.1 µmol/L) or isoproterenol (1 µmol/L), for 24
hours. Culture medium and cells were collected separately. Samples were
dried with evaporator and dissolved in 20 µL of chloroform:methanol
(1:1, vol/vol). The sample solution was subjected to thin-layer
chromatography (TLC, HPTLC plate silica gel 50, Merck)
and developed twice with methanol:0.5% NaCl:13N ammonia solution
(50:50:1, vol/vol). The autoradiography of TLC was
quantified using a phosphor imager (Fujix BAS 2000).
Statistical Analysis
Values are mean±SE. One-way ANOVA was used to evaluate
differences between groups. Where appropriate, post hoc multiple
comparison tests were performed to evaluate differences between the
control and experimental groups. A P value <0.05 was
considered statistically significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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Effects of Sphingolipid Metabolites on Total Protein Synthesis in Cardiac Myocytes
We first examined the effects of sphingolipid metabolites, SPC, S1P, and SPH on the total protein contents of neonatal rat cardiac myocytes. As shown in Figure 1A
, stimulation with SPC for 24 hours increased the total cellular protein
content in a concentration-dependent manner. The increase in the total
protein content caused by incubation with SPC at a concentration of
5 µmol/L was greater than in control cells. In contrast,
stimulation for 24 hours with neither S1P nor SPH had effects on the
total cellular protein content (data not shown). Second, the effects of
sphingolipid metabolites on [3H]phenylalanine
incorporation in cardiac myocytes was examined. Figure 1B
demonstrates that stimulation with SPC for 24 hours increased the rate
of [3H]phenylalanine incorporation in myocytes
in a concentration-dependent manner. Treatment with 
0.5 µmol/L
SPC had no significant effect on
[3H]phenylalanine incorporation, whereas
stimulation with 
1 µmol/L SPC resulted in a significant
increase in the rate of [3H]phenylalanine
incorporation. Stimulation with S1P for 24 hours also increased
[3H]phenylalanine incorporation in myocytes in
a concentration-dependent manner. However, the effect of S1P on
[3H]phenylalanine incorporation was less than
that seen with SPC (Figure 1C). Only a high concentration
(10 µmol/L) of S1P resulted in a significant increase in the
rate of [3H]phenylalanine incorporation. In
contrast, incubation with SPH had no effect on either
[3H]phenylalanine incorporation or total
protein content (Figure 1D). These data indicate that SPC
increases total protein synthesis as well as total protein content.
However, S1P had little effect and SPH had no effect on total protein
synthesis.
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Effect of Sphingolipid Metabolites on Protein Degradation in
Cardiac Myocytes
Figure 1E
shows that after 24 and 48 hours of
stimulation with SPC, the release of incorporated
[3H]phenylalanine was lower for SPC-treated
cardiac myocytes than for control cells, suggesting that SPC
stimulation decreases protein turnover. Two-way ANOVA indicated that
there were both significant time-dependent (P<0.0001) and
group-dependent (P<0.013) differences in the release of
incorporated [3H]phenylalanine. In contrast,
stimulation with S1P for 24 and 48 hours had no significant effect on
the release of incorporated [3H]phenylalanine
(Figure 1F). In the present study, SPC increased the rate of
phenylalanine incorporation and reduced the rate of release of
incorporated phenylalanine. These facts show that SPC not only quickens
amino acid metabolism but also works to accumulate protein
in cardiac myocytes.
Effect of SPC on Cardiac Myocyte Size
After treatment with SPC (10 µmol/L) for 48 hours, the cell
area increased significantly compared with control cells (Figure 2). This increase in cell area after
stimulation with SPC was similar to the increase in area seen with ET-1
(0.1 µmol/L). ET-1 is known to induce hypertrophy in
cultured neonatal rat myocytes.24
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Effect of SPC on the Embryonic-Fetal Type Gene Expression in
Cardiac Myocytes
To determine whether sphingolipid metabolites induce expression of
embryonic-fetal type genes, the level of ANF mRNA expression was
determined by Northern blot analysis. As shown in Figure 3, stimulation with SPC (10
µmol/L) or ET-1 (0.1 µmol/L) for 24 hours increased ANF mRNA
expression in cardiac myocytes. However, ANF mRNA expression in
myocytes incubated with S1P (10 µmol/L) was not different from
ANF mRNA expression in control cells.
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Effect of PTX on Sphingolipid Metabolite-Induced Hypertrophic
Response in Cardiac Myocytes
To investigate the possibility that G proteins may be involved in
the hypertrophic growth response induced by SPC or S1P in cardiac
myocytes, myocyte cultures were treated with PTX (50 ng/mL) before the
addition of SPC (10 µmol/L) or S1P (10 µmol/L). PTX
pretreatment inhibited the SPC- or S1P-induced increase in total
protein synthesis by 60% to 78% (Figure 4). Furthermore, PTX partially, but
significantly, inhibited SPC-induced increase in cell size and ANF mRNA
expression (Figures 5 and 6). In the absence of SPC, PTX had no
effects on total protein synthesis (Figure 4) and cell size
(108±7% of control). These results suggest that the increase in
cardiac myocyte hypertrophy induced by SPC or S1P requires,
at least in part, PTX-sensitive G proteins.
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Effect of PD98059 on the SPC-Induced Hypertrophic Response in
Cardiac Myocytes
To investigate the possibility that the MAPK signaling cascade may
be involved in the hypertrophic growth response induced by SPC, myocyte
cultures were treated with PD98059 (50 µmol/L), an
inhibitor of p44 and p42 ERK, before the addition of SPC
(10 µmol/L). ERKs are protein serine/threonine kinases and are
members of the MAPK family. PD98059 pretreatment inhibited 74% of the
SPC-induced increase in total protein synthesis (Figure 4). As
shown in Figures 5 and 6, PD98059 pretreatment inhibited
64.5% of the SPC-induced increase in cell size and significantly
inhibited SPC-induced increase in ANF mRNA expression in cardiac
myocytes. In the absence of SPC, PD98059 had no effects on total
protein synthesis (Figure 4) and cell size (103±5% of
control). These results suggest that the increase in protein synthesis,
cell size, and ANF mRNA expression caused by SPC involves, at least in
part, the MAPK signaling cascade. Furthermore, both PTX and PD98059
pretreatment for 24 hours inhibited SPC-induced decrease in release of
incorporated phenylalanine by 58.5% and 53.8%, respectively (Figure 7). In the absence of SPC, neither
pretreatment with PTX nor PD98059 for 24 hours had an effect on the
release of incorporated phenylalanine (315.7±10.7 and 302.7±6.1
cpm/µg protein, respectively).
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Effect of SPC on the Threonine/Tyrosine
Phosphorylation of ERKs and MAPK Activity in
Cardiac Myocytes
To determine whether SPC activates p44 and/or p42 ERK in
cardiac myocytes, we examined the content of
threonine/tyrosine–phosphorylated p44 and p42 ERK with
Western blot analysis using a specific antibody recognizing
only threonine/tyrosine–phosphorylated p44 and p42
ERK. As shown in Figure 8, stimulation
with SPC (10 µmol/L) or ET-1 (0.1 µmol/L) for 5 or 15
minutes significantly increased the contents of
threonine/tyrosine–phosphorylated p44 and p42 ERK.
Thus, SPC stimulated threonine/tyrosine phosphorylation
of both ERK isoforms in cardiac myocytes. As shown in Figure 9A, stimulation with SPC (10
µmol/L) for 5 or 15 minutes significantly increased the MAPK
activity. These results were similar to the effect of SPC on
threonine/tyrosine phosphorylation of ERKs. Thus, SPC
stimulates a MAPK cascade in cardiac myocytes with increasing
phosphorylation of ERKs and enzyme activity of
MAPK.
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Effect of PD98059 on the SPC-Induced Increase in MAPK Activity in
Cardiac Myocytes
To confirm that 50 µmol/L of PD98059 inhibited MAPK
activity in cardiac myocytes, we examined the effect of PD98059 on
SPC-induced increase in MAPK activity. As shown in Figure 9B, 50 µmol/L of PD98059 inhibited 47.0% of the SPC-induced
increase in MAPK activity. Although 50 µmol/L of PD98059 should
be enough to inhibit MAPK activity, 50 µmol/L of PD98059 did not
completely inhibit the activation of MAPK by SPC. These results were
similar to a previous study in which PD98059 inhibited 
50% of the
nerve growth factor–induced increase in MAPK activity in PC-12
cells.25 Also, the degree of inhibition of MAPK activity
in Swiss 3T3 cells by PD98059 differed by the type of
agonists.26 Thus, we could not exclude the possibility
that the present results, in which PD98059 inhibited 64% to 74%
of the SPC-induced increase in protein synthesis and cell size, were
due to a partial inhibitory effect of PD98059 on MAPK
activity. This is a potential limitation of studies using PD98059. It
is also possible that MAPK-independent pathways are also involved in
SPC-induced increase in protein synthesis and cell size.
Effect of Calphostin C on the SPC-Induced Hypertrophic Response and
Increase in MAPK Activity in Cardiac Myocytes
To investigate the possibility that the protein kinase C
(PKC)–dependent signaling cascade may be involved in the hypertrophic
growth response induced by SPC, myocyte cultures were treated with
calphostin C (1 µmol/L), an inhibitor of PKC, before
the addition of SPC (10 µmol/L). Calphostin C pretreatment
inhibited 55.5% of SPC-induced increase in cell size (Figure 5). However, the effect of calphostin C on SPC-induced increase
in ANF mRNA expression was not significant (Figure 6). Further,
calphostin C and PTX inhibited the SPC-induced increase in MAPK
activity by 29.9% and 37.4%, respectively (Figure 9B). From
these results, SPC stimulates MAPK activity in part via a sphingolipid
receptor coupled to PTX-sensitive G proteins and PKC in cardiac
myocytes. However, SPC induces hypertrophic growth responses via both
PKC-dependent and PKC-independent signaling pathways.
Effect of ET-1 and Isoproterenol on SPC Generation in Cardiac
Myocytes
To confirm whether SPC generation and release is increased in
cardiac myocytes in response to agonists, we assayed SPC generation in
cardiac myocytes and culture medium using
14C-labeled sphingomyelin. SPC generation was
assayed using radio-TLC. As shown in Figure 10, the cellular lysates labeled with
[14C]sphingomyelin were spotted on silica gel
plates, and autoradiograph showed specific bands corresponding to SPC.
Radioactivity quantified by BAS 2000 phosphor imager system
revealed that the samples treated with ET-1 had increased 1.8-fold
in radioactive SPC in comparison with that in control cells.
Isoproterenol was not increased in radioactive SPC. Further, there were
no bands corresponding to SPC in cellular lysates labeled with
[14C]palmitic acid (data not shown). These
results demonstrated that cardiac myocytes were capable of producing
SPC, and ET-1 stimulated the generation of SPC in myocytes. The
observation that cellular lysates labeled with
[14C]palmitic acid did not contain any SPC band
suggests that SPC is generated by the degradation of sphingomyelin but
is not produced by the pathway mediating SPH. We also attempt to
determine the release of SPC in culture medium from cardiac myocytes.
However, there was no band corresponding to SPC in medium labeled with
[14C]sphingomyelin or
[14C]palmitic acid (data not shown). This may
be a technical limitation of studies using radio-TLC assay.
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This simple experimental study shows for the first time that SPC induces a hypertrophic growth response in cardiac myocytes. The following 4 lines of evidence support this statement. First, when cardiac myocytes were stimulated with SPC, there were increases both in the rate of total protein synthesis and in the total cellular content of protein. Second, the stimulation of myocyte cultures with SPC resulted in a significant decrease in protein degradation to diluent-treated control cultures. Third, SPC stimulation produced a significant increase in cardiac myocyte cell size. Fourth, SPC induced the expression of ANF, which is one of the embryonic-fetal type genes in cardiac myocytes. Furthermore, the SPC-induced hypertrophic growth response was inhibited by a p44 and p42 ERK inhibitor, PD98059. SPC also stimulated threonine/tyrosine phosphorylation of ERKs and MAPK enzyme activity.
In previous studies, SPC has been shown to induce DNA synthesis and cellular proliferation in various cell lines.11 12 SPC-induced cellular proliferation is similar to the proliferation caused by serum and is much greater than that induced by insulin, fibroblast growth factor, epidermal growth factor, and 12-O-tetradecanoylphorbol-13-acetate (TPA) in fibroblasts.11 Recently, it has been shown in Swiss 3T3 fibroblast that SPC transiently activates p42 ERK and 90-kDa ribosomal S6 kinase (p90rsk) through a pathway dependent on PKC activity.27 Also, SPC stimulates PKC-mediated activation of p44 and p42 ERK in freshly isolated smooth muscle cells.28 Furthermore, SPC, as well as S1P, stimulates the DNA binding activity of the transcription activator protein AP-1.29
AP-1, which is one of the transcription factors activated by the Ras pathway and the MAPK cascade, consists of homo- and/or heterodimers of the fos and jun gene products and controls genes that are required for cell growth. In cardiac myocytes, the activation of the MAPK cascade is believed to be involved in the initiation of the hypertrophic response induced by ET-1, fibroblast growth factor, or mechanical stress.30 31 Furthermore, expression of the proto-oncogenes c-fos and c-jun is increased in cardiac myocytes stimulated with isoproterenol or stretch.32 33 The enhanced DNA binding activity of the AP-1 transcriptional factor complex is believed to regulate the hypertrophic response of myocytes. These previous studies support our present observations that SPC is a potent inducer of hypertrophic growth in ventricular myocytes.
The existence of sphingolipid receptors has been hypothesized on the basis of observations that the actions of SPH, S1P, or SPC can be attenuated by treatment with PTX. PTX prevents receptor coupling to Gi or G0 proteins, thereby blocking cellular signaling by these receptors.13 In Swiss 3T3 fibroblasts, SPC-induced activation of p42 ERK requires a PTX-sensitive G protein.27 On the basis of these previous studies, we examined the effect of PTX on S1P- or SPC-induced increase in protein synthesis, cell size, and ANF mRNA expression in cardiac myocytes. PTX attenuated 60% to 80% of the sphingolipid metabolite-induced effects. Therefore, exogenous administration of S1P or SPC may stimulate cardiac hypertrophy, at least in part, by activating sphingolipid receptors that are coupled to PTX-sensitive G proteins.
Although sphingolipid receptors have not yet been characterized at the molecular level, 2 different G protein–coupled receptors for sphingolipid metabolites have been cloned. S1P activates the G protein–coupled receptor EDG-1, which was originally cloned as an immediate-early gene induced during differentiation of human endothelial cells.34 Other studies have shown that overexpression of the G protein–coupled receptors H218 and EDG-3, but not EDG-1, in Jurkat cells results in responsiveness to both S1P and SPC.35 There are also functional data supporting a pharmacological classification of the sphingolipid receptor family.13 The first class of receptors is characterized by their comparable sensitivities to nanomolar concentrations of both S1P and SPC. The second class includes receptors that are only activated by nanomolar concentrations of S1P but not by micromolar concentrations of SPC. The third class includes receptors that are only activated by nanomolar to low micromolar concentrations of SPC and not activated by micromolar concentrations of S1P. In our study, SPC induced cardiac hypertrophy at a concentration of 1 µmol/L. However, only high concentrations of S1P stimulated cardiac hypertrophy. Therefore, the sphingolipid receptors on cardiac myocytes that induce cardiac hypertrophy may belong to the third class of sphingolipid receptors.
Despite their structural relationship, the increase in total protein synthesis induced by S1P was less than that induced by SPC, and SPH had no effect on protein synthesis. Similar discrepant cellular responses have been reported. Specifically, SPC stimulates cellular proliferation of quiescent Swiss 3T3 fibroblasts to a greater extent than S1P or SPH.11 Furthermore, SPC elicits a rapid Ca2+ release from rat brain microsomes, whereas SPH and S1P do not cause Ca2+ release.36 Of the various sphingolipid metabolites, SPC is the only agent that can accelerate cutaneous wound healing in vivo in a diabetic mouse model.37 The effects of SPC that are distinct from those of other sphingolipid metabolites may be the result of the following 2 factors. First, our observations suggest that sphingolipid receptors of the third class are present on cardiac myocytes. This class of sphingolipid receptor has different sensitivities to SPC and S1P. Second, SPC may act via both PKC-dependent and PKC-independent signaling pathways, whereas the mitogenic activities of SPH and S1P are clearly independent of PKC activation.2 9 Furthermore, in contrast to the effects of SPH, SPC causes activation of one protein kinase and inhibition of another.38 Therefore, SPC may act through activation of signaling pathway(s) distinct from those utilized by SPH and S1P. In this study, SPC induces hypertrophic growth responses via both PKC-dependent and PKC-independent signaling pathways.
It is well known that SPC is present in patients with Niemann-Pick disease, a lipid storage disorder.39 40 Recent evidence based on nuclear magnetic resonance spectroscopy confirmed the existence of SPC in normal mouse tissue and human cancer cells.41 However, it is still unclear whether SPC concentrations are regulated by signaling molecules and whether SPC can be secreted and can act in an autocrine and/or paracrine manner. Recent studies have shown that a specific Pseudomonas strain secretes sphingolipid ceramide N-deacylase, which is an enzyme capable of cleaving sphingomyelin to produce SPC.42 Because sphingomyelin is a cellular membrane lipid, it is conceivable that SPC could be produced from sphingomyelin via activation of sphingolipid ceramide N-deacylase under pathophysiological conditions. Recently, Murata et al43 suggested that the activity of sphingomyelin acylase, which releases free fatty acid and SPC from sphingomyelin, is highly expressed in the stratum corneum from patients with atopic dermatitis.
In conclusion, the addition of exogenous SPC, a sphingolipid metabolite, induces a hypertrophic growth response in cardiac myocytes similar to that seen with ET-1. Furthermore, the effects of SPC may be mediated by a sphingolipid receptor coupled to PTX-sensitive G proteins and the MAPK pathway. Although we could not detect the release of SPC from cardiac myocytes, cardiac myocytes were capable of producing SPC by the degradation of sphingomyelin, and ET-1 stimulated the generation of SPC. Thus, our findings are the first to suggest a role for sphingolipid metabolites in the development of cardiac hypertrophy. Because SPC may potentially be used for clinical applications, such as a wound-healing agent,37 we believe that sphingolipid metabolites may play a critical role in the progression of cardiac hypertrophy as extracellular and intracellular signaling molecules. Further studies demonstrating quantitative changes in the concentration of SPC in cardiac myocytes or heart tissue under pathophysiological conditions, and identifying the source of extracellular SPC, are now in progress to confirm our hypothesis.
Received August 12, 1999; accepted September 10, 1999.
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