(Received for publication, March 25, 1996, and in revised form, November 26, 1996)
From the Cardiology Section, Department of Medicine, Veterans
Administration Medical Center, Baylor College of Medicine,
Houston, Texas 77030 and the Division of Cardiology,
Department of Medicine, University of Cincinnati,
Cincinnati, Ohio 45267
To determine whether activation of the neutral
sphingomyelinase pathway was responsible for the immediate (<30 min)
negative inotropic effects of tumor necrosis factor- (TNF-
), we
examined sphingosine levels in diluent and TNF-
-stimulated cardiac
myocytes. TNF-
stimulation of adult feline cardiac myocytes provoked
a rapid (<15 min) increase in the hydrolysis of
[14C]sphingomyelin in cell-free extracts, as well as an
increase in ceramide mass, consistent with cytokine-induced activation of the neutral sphingomyelinase pathway. High performance liquid chromatographic analysis of lipid extracts from TNF-
-stimulated cardiac myocytes showed that TNF-
stimulation produced a rapid (<30
min) increase in free sphingosine levels. Moreover, exogenous D-sphingosine mimicked the effects of TNF-
on
intracellular calcium homeostasis, as well as the negative inotropic
effects of TNF-
in isolated contracting myocytes; time course
studies showed that exogenous D-sphingosine produced
abnormalities in cell shortening that were maximal at 5 min. Finally,
blocking sphingosine production using an inhibitor of ceramidase,
n-oleoylethanolamine, completely abrogated the negative
inotropic effects of TNF-
in isolated contracting cardiac myocytes.
Additional studies employing biologically active ceramide analogs and
sphingosine 1-phosphate suggested that neither the immediate precursor
of sphingosine nor the immediate metabolite of sphingosine,
respectively, were likely to be responsible for the immediate negative
inotropic effects of TNF-
. Thus, these studies suggest that
sphingosine mediates the immediate negative inotropic effects of
TNF-
in isolated cardiac myocytes.
Tumor necrosis factor-alpha (TNF-)1
is a proinflammatory cytokine that has been implicated as a potential
pathogenetic mechanism for cardiac disease states wherein left
ventricular dysfunction supervenes, including systemic sepsis (1),
acute viral myocarditis (2), cardiac allograft rejection (3),
myocardial reperfusion injury (4), and congestive heart failure (5).
The long-standing interest in defining the mechanisms responsible for
the cardiodepressant effects of TNF-
has been intensified recently
by experimental studies that have shown that TNF-
produces negative
inotropic effects in the intact left ventricle (6, 7), in thin strips of myocardial tissue (8), and in isolated contracting cardiac myocytes
(6). Although the exact cellular signaling pathways that are
responsible for the negative inotropic effects of TNF-
are not
known, a careful inspection of the literature suggests that TNF-
modulates myocardial function through at least two different
pathways.
It is quite clear, for example, that TNF- can produce immediate
negative inotropic effects in myocardial tissue within 10-30 min (6,
8). Similarly, it is equally clear that TNF-
exerts delayed effects
on myocardial function that appear to be related to uncoupling of the
-adrenergic receptor from cyclic AMP, rather than from a direct
depression in basal myocardial contractility per se;
moreover, these effects occur only after prolonged TNF-
exposure
(24-72 h) (9, 10). Given the recognition that TNF-
increases nitric
oxide (NO) levels in myocardial tissue through increased transcription
of the inducible Ca2+-independent form of nitric oxide
synthase (NOS) (11, 12), given that NO directly mediates myocardial
depression (13, 14), and given that NO is likely responsible for the
uncoupling of the
-adrenergic receptor following TNF-
stimulation
(15), the logical assumption has been that NO mediates the full
spectrum of cytokine-induced cardiodepressant effects. However, no
previous report to date has provided direct evidence that shows that
TNF-
stimulates NO production in cardiac myocytes with a time course that is rapid enough to explain the immediate negative inotropic effects of TNF-
(6). As a case in point, a recent study in which
thin strips of myocardial tissue from Syrian hamsters were treated with
TNF-
provided indirect evidence that suggested that the immediate
(<5 min) negative inotropic effects "appear(ed) to result from
enhanced activity of a constitutive
(Ca2+-dependent) NO synthase enzyme in the
myocardium." (8) Nonetheless, although combinations of cytokines may
increase Ca2+-dependent NOS activity indirectly
over 24 h by increasing the synthesis of NOS cofactors (16), the
demonstration of a rapid increase in
Ca2+-dependent NOS activity by TNF-
, or by
any other cytokine, has not been observed thus far (17). Moreover, we
have found that the immediate negative inotropic effects of TNF-
were not abrogated by NOS inhibition (6), suggesting that TNF-
may
produce myocardial depression through a NOS-independent pathway.
During the course of previous studies we observed that TNF--induced
activation of the type 1 TNF receptor (TNFR1) resulted in reversible
negative inotropic effects in isolated cardiac myocytes as a direct
result of alterations in intracellular calcium homeostasis (6, 18).
Insofar as concentrations of TNF-
that produced negative inotropic
effects did not produce discernible changes in the voltage-sensitive
inward calcium current, we suggested that the TNF-
-induced
alterations in intracellular calcium homeostasis were secondary to
alterations in sarcoplasmic reticular handling of calcium (6).
Encouraged by the observation that TNF-
-induced oligomerization of
TNFR1 leads to the rapid degradation of sphingomyelin (19) with the
resultant generation of a sphingoid base termed sphingosine (20), as
well as by the observation that sphingosine was not only present in
cardiac and skeletal muscle (21), but was also capable of blocking
calcium release from the ryanodine receptor (22, 23), we investigated
whether the immediate negative inotropic effects of TNF-
were
mediated by sphingosine. In the present brief report we demonstrate
that sphingosine is both necessary and sufficient to produce the
negative inotropic effects of TNF-
in isolated cardiac myocytes,
thus suggesting that TNF-
-induced activation of the neutral
sphingomyelinase pathway is responsible for the immediate negative
inotropic effects of this proinflammatory cytokine.
Acidic (pH 5.0) and
neutral (pH 7.5) sphingomyelinase activity were measured in adult
feline cardiac myocytes according to the method described by Machleidt
et al. (24). Briefly, feline cardiac myocytes were isolated,
and a 2-ml suspension of cells was plated at a final concentration of
5 × 104 cells·ml1 onto laminin-coated
(20 µg·ml
1) polystyrene Petri dishes as described
previously (25, 26). On the 1st day in culture the M199 medium was
changed, and the cells were treated for 0, 5, 15, 30, and 60 min with
diluent (endotoxin-free 0.1% human serum albumin) or with recombinant
human TNF-
(200 units·ml
1). The cells were then
lysed, and the resultant cell supernatants were incubated at 37 °C
with 1 µl of [methyl-14C]sphingomyelin (25 µCi·ml
1). The reaction was stopped after 2 h by
the addition of chloroform:methanol (2:1). After thorough mixing by
inversion and vortexing, ddH2O was added, and the two
phases were separated by centrifugation. The upper aqueous phase
containing [14C]phosphorylcholine was removed and counted
in a liquid scintillation counter. Final results were expressed as fold
increase in [14C]phosphorylcholine levels relative to
base-line levels.
Ceramide mass
was measured according to the method of Preiss et al. (27)
Briefly, isolated cardiac myocytes were prepared as described above and
stimulated for 30 min with diluent, TNF- (200 units·ml
1), either in the presence or absence of a
specific inhibitor of ceramidase: n-oleoylethanolamine (NOE)
(28). The lipid fractions from cell extracts were extracted with
chloroform/methanol (1:1 (v/v)), the samples vortexed, and then
separated by centrifugation. The upper aqueous phase was aspirated and
used for determination of the protein concentration, whereas the
chloroform phase was dried in vacuo. The extracts were
resuspended and incubated with Escherichia coli
diacylglycerol and [
-32P]ATP using a commercial
diacylglycerol kinase assay system (Amersham Corp.). Ceramide
1-phosphate was then isolated by thin layer chromatography (TLC) using
chloroform/methanol/glacial acetic acid (65:15:5 (v/v/v)) in the
presence of a ceramide standard, which was run simultaneously along
with the unknown samples. Authentic ceramide 1-phosphate was identified
by autoradiography, and the spots corresponding to ceramide 1-phosphate
were scraped from the plates and then counted in a scintillation
counter. Final results were expressed as cpm/mg protein.
Sphingosine levels in adult cardiac myocytes were determined according to the method of Merrill et al. (29). Briefly, lipid extracts were obtained from freshly isolated cardiac myocytes by thoroughly mixing the cells with chloroform/methanol (1:2) for 5 min. In order to account for variability in the lipid extraction process, 0.6 nmol of tetradecylamine was added to the unknown samples as an internal standard (30). Equal volumes of chloroform and 1 M NaCl were then added, and the two phases were separated by centrifugation. The upper aqueous phase was discarded, and the chloroform phase was washed twice with 1 M NaCl and vacuum-dried for 30 min. The dried lipid extracts were then subjected to a mild alkaline hydrolysis by resuspending them in 0.1 M KOH in methanol for 1 h (37 °C), in order to remove ester-containing glycerolipids. After cooling to room temperature the samples were re-extracted as described above, and the free long chain sphingoid bases recovered in the chloroform phase, washed 2 × with 1 M NaCl, and then vacuum-dried. Thereafter, samples were derivatized with o-phthaldialdehyde, exactly as described by Merrill and colleagues (29).
Sphingosine Analysis by Liquid ChromatographyO-Phthaldialdehyde derivatives of the
unknown samples were separated by reverse phase HPLC using an isocratic
elution with methanol, 5 mM potassium phosphate (pH 7.0)
(90:10), employing a Beckman 112 solvent delivery module, a 250 × 4.6 mm C18 (5 µm) Brownlee column with a Dynamax C18 guard column;
all samples were run at 1 ml/min. Fluorescent derivatives were detected
using Waters 420-AC fluorescent detector with excitation and emission
wavelengths of 340 and 455 nm, respectively. Under the above
conditions, incorporated fluorescence is linearly related to sphingoid
base levels between 2 to 400 pmol. To determine the absolute level of
sphingosine in unknown samples, the amount of fluorescence was compared
with a standard curve generated from known quantities of sphingoid base. To compare the level of sphingosine in diluent and
TNF--stimulated samples, we expressed the amount of free sphingosine
present in the unknown samples as the ratio of the area under the
sphingosine peak to the area under the tetradecylamine peak.
To determine
whether stimulation with TNF- would increase the level of free
sphingosine in isolated adult cardiac myocytes, freshly isolated cells
were treated for 30 min with TNF-
(20 and 200 units·ml
1), as well as TNF-
mutants that bind
selectively either to the type 1 (TNFR1) TNF receptor (corresponding
mutant = TNFM1) or the type 2 (TNFR2) TNF receptor (corresponding
mutant = TNFM2). Both the TNFM1 and TNFM2 mutants were the
generous gifts of W. Lesslauer (F. Hoffman-La Roche, Basel,
Switzerland) (31). The specificity of the mutated TNF ligands for
binding to feline TNFR1 and TNFR2 has been validated previously (18).
For these studies we used a single concentration of TNFM1(2
ng·ml
1) and TNFM2 (2 ng·ml
1), since
this concentration is equivalent to the concentration of wild-type
TNF-
(200 units·ml
1) that produces well-defined
negative inotropic effects in adult feline cardiac myocytes (6)
Cell motion was characterized by
video-edge detection at a stimulation frequency of 0.25 Hz, using
experimental conditions identical to those we have described elsewhere
(6, 32). Four separate series of experiments were performed. First, to
determine whether blocking the conversion of ceramide to sphingosine
would abrogate the negative inotropic effects of TNF-, freshly
isolated feline cardiac myocytes were allowed to stabilize for 1 h
and were then treated for 30 min at 37 °C with 200 units·ml
1 of TNF-
, either in the presence or absence
of a specific inhibitor of ceramidase, n-oleoylethanolamine
(NOE) (28). In preliminary control experiments we determined that
concentrations of NOE >10.0 µM completely inhibited cell
motion; therefore, 1.0 µM NOE was chosen as the maximal
concentration of NOE to inhibit ceramidase. As an additional control
for the above experiments NOE-pretreated cells (1.0 µM)
were stimulated with 200 units·ml
1 of TNF-
and 1 µM D-sphingosine for 30 min. Second, to
determine whether exogenous D-sphingosine would mimic the
negative inotropic effects of TNF-
, cardiac myocytes were incubated
for 30 min with a broad range of concentrations (0.1 nM to
10 µM) of D-sphingosine or a closely related
sphingoid base, 10 µM dihydrosphingosine. To determine
the time course for the effects of D-sphingosine (1 µM), cell shortening was examined at 5, 15, and 30 min.
The reversibility of the effects of D-sphingosine (1 µM) was determined by treating the cells for 30 min and
then washing the cells free from exogenous D-sphingosine;
cell motion was examined 30 min after the D-sphingosine was
removed from the cells. Given that sphingosine 1-phosphate, the
phosphorylated metabolite of sphingosine, is thought to mediate many of
the biological effects of D-sphingosine (33), we performed
parallel experiments to those described above for sphingosine, by
incubating the cells for 30 min with 0.1-10 µM
sphingosine 1-phosphate. To determine the time course for the effects
of sphingosine 1-phosphate (1 µM), cell shortening was examined at 5, 15, 30, and 60 min after stimulation. Third, to determine whether exogenous ceramide would mimic the effects of TNF-
, the cells were treated for 30 min with two different ceramide analogs: C2-ceramide
(N-acetyl-D-sphingosine) and
C6-ceramide (N-hexanoyl-D-sphingosine). In preliminary
control experiments we established that concentrations
100
µM C2-ceramide and
10 µM
C6-ceramide were overtly toxic to the cells; therefore, we employed 10 µM C2-ceramide and 1 µM C6-ceramide for these studies. Fourth, to
determine whether TNF-
-induced stimulation of the phosphatidylcholine-dependent phospholipase C pathway
(PC-PLC), with resultant activation of protein kinase C (PKC), was
responsible for the immediate negative inotropic effects of TNF-
, we
pretreated the cells with D609 (60 min), an inhibitor of the PC-PLC
pathway (34). In preliminary control experiments we determined that concentrations of
1 µM D609 did not depress cell motion
significantly; therefore, we used 0.4 µM D609 to inhibit
PC-PLC activity. Since the maximal concentration of D609 used may not
have inhibited PC-PLC activity completely, we also pretreated the cells
with 20 µM sangivamycin, which has been shown to
significantly inhibit diacylglycerol-dependent protein
kinase C (PKC) activity (35); 20 µM sangivamycin did not
significantly inhibit cell motion in preliminary control experiments.
For the studies with D609 and sangivamycin, the cells were pretreated
with inhibitor for 60 min and were then stimulated with TNF-
or
diluent for an additional 30 min prior to assessing cell motion. In
addition to these indirect studies, we performed direct measurements of
PKC activity in TNF-
and sphingosine-stimulated cardiac myocytes.
Evidence for cytokine-induced activation of PKC (translocation) was
determined in freshly isolated myocytes stimulated for 1, 7.5, or 30 min either with 200 units·ml
1 TNF-
using
differential membrane centrifugation and immunoblot analysis, as
described previously (36); cells stimulated with 100 nM
phorbol 12-myristate 13-acetate (PMA) served as the appropriate positive controls. Two separate primary antibodies were employed for
immunoblotting; the first antibody was specific for PKC
, the major
PKC isoform in myocytes, whereas the second antibody recognized PKC
,
-
, -
, and -
. The relative amounts of protein kinase C in the
membrane and cytosolic fractions were assessed by laser densitometry
and expressed as a ratio of membrane-to-cytosolic PKC. Evidence for
inhibition of PKC activity was sought by determining the incorporation
of [32P]ATP into a PKC-specific pseudosubstrate peptide,
as described previously (37). Briefly, cardiac myocytes were
homogenized and then centrifuged for 1 h at 1,000 × g; the resultant crude homogenate was then separated into
cytosolic and membrane fractions by centrifugation at 70,000 × g. Insofar as preliminary studies showed that
75% of the
PKC activity resided in the cytosolic fraction, this fraction was
employed for all further studies. The PKC inhibitory activity of
TNF-
(200 units·ml
1) and sphingosine (0.1-10
µM) was compared with that of l00 nM staurosporine in assays wherein myocyte cytosolic extracts were stimulated with mixed micelles containing 0.3 mg/ml
phosphatidyl-L-serine and 24 µg/ml PMA, in order to
simulate PKC activity.
Intracellular calcium transients were determined in isolated contracting cardiac myocytes using the fluorescent indicator fluo-3 AM (20 µM), exactly as we have described previously (6). A time-intensity curve for fluorescence brightness was determined for a single cardiac myocyte contraction by measuring the total fluorescence brightness over the surface area of individual cell; the final time-intensity curves were determined as the average values for 10 consecutive contraction sequences after cell shortening had stabilized. For the purpose of comparison between D-sphingosine (1 µM) and diluent-treated cells, peak levels of intracellular fluorescence brightness were compared.
Statistical AnalysisData are expressed as mean ± S.E. Data were analyzed by one-way analysis of variance, with post hoc testing where appropriate (Dunnett's), or by non-paired t tests.
TNF--induced oligomerization of TNFR1 has been shown to
activate membrane-bound neutral sphingomyelinase, with the resultant generation of ceramide, which can then be deacylated to sphingosine by
the enzyme ceramidase (20). Three series of experiments were performed
to determine whether TNF-
activated this pathway in the adult
mammalian cardiac myocyte. Table I shows that TNF-
stimulation resulted in time-dependent increase in neutral
sphingomyelinase activation in adult cardiac myocytes. As shown,
[14C]phosphorylcholine levels were significantly
different from control values by 15 min (p < 0.05) and
were
2-fold greater than control values 60 min following
stimulation. TNF-
stimulation also activated the acidic
sphingomyelinase pathway, consistent with reports from other
laboratories (34). Next we measured ceramide mass in diluent and
TNF-
-stimulated cardiac myocytes (n = 4 dishes per
group). When compared with diluent-treated controls, TNF-
stimulation provoked a significant (p < 0.05) 1.8-fold
increase in ceramide mass (10,458 ± 571 versus
18,955 ± 831 cpm/µg protein). In contrast, stimulating the
cells with diluent in the presence of NOE resulted in
8-fold
increase in ceramide mass (82,998 ± 5,140 cpm/µg protein), whereas stimulating the cells with TNF-
in the presence of NOE resulted in
12-fold increase in ceramide mass (121,155 ± 24,086 cpm/µg protein) Thus, these studies are consistent with the
notion that NOE acts, at least in part, by inhibiting ceramidase (28). Finally, to determine whether TNF-
activation would stimulate increased levels of free sphingosine in cardiac myocytes, freshly isolated cells were stimulated with TNF-
for 30 min before lipid extraction. Fig. 1 shows the elution profiles for the
o-phthaldialdehyde derivatives of the lipid extracts from
cardiac myocytes stimulated with diluent (Fig. 1A) and 200 units·ml
1 of TNF-
(Fig. 1B). As shown, a
major sphingosine peak was resolved at 14 min in the diluent and
TNF-
-treated myocytes, consistent with previous studies that have
demonstrated constitutive levels of sphingosine in cardiac and skeletal
muscle (21). The identity of the sphingosine peak was confirmed by its
comigration on HPLC with authentic sphingosine standards, as well as by
the observation that closely related exogenous lipids
(dihydrosphingosine and psychosine) had different elution times when
added to the unknown samples. In preliminary control experiments we
established that the amount of free sphingosine present in isolated
cardiac myocytes was
23 pmol/106 cells, consistent with
reports in cardiac and skeletal muscle, as well as other cell types
(29, 38, 39). As shown in Fig. 1 the elution profile for
tetradecylamine, which was added as an internal standard, had a
retention time of 30 min. In the representative example depicted in
Fig. 1B, TNF-
stimulation led to a 1.4-fold increase in
the sphingosine/TDA ratio when compared with values obtained in
diluent-treated cardiac myocytes.
|
Fig. 2 shows two important findings with respect to
group data for sphingosine levels in isolated cardiac myocytes. First, stimulating the cells with a concentration of TNF- (200 units·ml
1) that consistently depresses cell shortening
(6, 32) resulted in a significant (p < 0.05) increase
in free sphingosine levels within 30 min, whereas stimulating the cells
with a concentration of TNF-
(20 units·ml
1) that
does not depress cell shortening (6) did not stimulate increased
(p > 0.05) levels of free sphingosine levels relative to control values. Moreover, the time course for the TNF-
-stimulated increase in sphingosine levels was sufficiently rapid to explain the
temporal development (i.e. < 30 min) of TNF-
-induced
contractile dysfunction in isolated cardiac myocytes (6), thin strips
of isolated muscle (8), as well as in the intact left ventricle (6).
Both the time course and absolute values for the TNF-
-induced increase in free sphingosine levels in intact myocytes are consistent with previously reported values for sphingosine levels obtained from
whole cell lipid extracts following TNF-
stimulation (40). A second
important point shown by Fig. 2 is that stimulating the myocytes with
the TNFM1 ligand produced a significant (p < 0.05)
1.4-fold increase in free sphingosine levels, similar to that which
was observed with the wild type TNF-
. As shown, the TNFM2 ligand,
which does not affect cell motion (18), did not increase levels of free
sphingosine in isolated contracting cardiac myocytes. The findings
obtained with the TNFM1 and TNFM2 ligands are consistent with previous
reports that have shown that TNFR1, as opposed to TNFR2, is coupled to
the neutral sphingomyelinase pathway (19) and that activation of TNFR1,
as opposed to TNFR2, produces negative inotropic effects in isolated
contracting cardiac myocytes (18). Next, to determine whether blocking
the conversion of ceramide to sphingosine would abrogate the negative
inotropic effects of TNF-
, the cells were pretreated (60 min) with a
specific inhibitor of ceramidase: n-oleoylethanolamine (NOE)
(28). The important finding shown by Fig. 3 is that the
negative inotropic effects of TNF-
were abrogated completely by
pretreatment with 1.0 µM NOE. To confirm that NOE blocked
the generation of free sphingosine, we measured free sphingosine levels
in TNF-
-stimulated cells in the presence and absence of NOE. HPLC
analysis showed that NOE pretreatment blunted the TNF-
-induced
increase in free sphingosine levels by
75%. Moreover, when the
NOE-pretreated cells were stimulated concurrently with sphingosine and
TNF-
, we observed a significant depression in cell shortening,
suggesting that NOE did not act by interfering with the negative
inotropic effects of sphingosine in isolating contracting cardiac
myocytes. Fig. 3 also shows that pretreating the cells with
C2 and C6-ceramide analogs had no significant effect on cell motion.
To determine whether sphingosine itself was sufficient to mimic the
effects of TNF- in isolated cardiac myocyte shortening, the cells
were treated with 0.1 nM to 10 µM
D-sphingosine. The salient finding shown by Fig.
4A is that treating the cells with exogenous
D-sphingosine for 30 min resulted in a
concentration-dependent decrease in myocyte shortening that
was significantly different from control for
0.001 µM
sphingosine. Importantly, the concentrations of exogenous
D-sphingosine that were necessary to depress cell shortening fell within the theoretically calculated range for sphingosine levels in TNF-
-stimulated cardiac myocytes (
0.1-1.0 µM, assuming a cell of density of 1.23 g·ml
1) (21, 38). To confirm the specificity of the
observed effects with D-sphingosine, the cells were treated
with dihydrosphingosine, which differs from sphingosine structurally by
the absence of a double bond in the carbon 4-5 position (41). Fig.
4A shows that dihydrosphingosine had no effect on isolated
cell shortening, thus arguing against a nonspecific lipid membrane
effect of sphingosine. The inset of Fig. 4A shows
that the sphingosine-induced (1 µM) depression in cell
shortening was maximal at
5 min, congruent with the overall rapid
time course for the development of the immediate negative inotropic
effects of TNF-
(6, 8). Finally, as shown in the inset,
the negative inotropic effects of D-sphingosine were shown
to be completely reversible within 30 min after
D-sphingosine was washed out of the cells (Fig. 4),
consistent with previous observations that the immediate negative
inotropic effects of TNF-
are completely reversible (6, 8). Fig.
4B shows that when the cells were pretreated with
1
µM sphingosine 1-phosphate, the extent of cell shortening
was reduced significantly (p < 0.05) compared with
control values. The inset of Fig. 4B shows that the time course for the onset of negative inotropic effects with sphingosine 1-phosphate was delayed relative to that observed with
D-sphingosine and was significantly different
(p < 0.05) from control only after 30 min of
continuous stimulation. Thus, the time course for the onset of negative
inotropic effects in sphingosine 1-phosphate-treated myocytes is
inconsistent with the rapid onset of negative inotropic effects
observed with TNF-
(<10-15 min) (6). Moreover, two additional
lines of evidence suggest that the negative inotropic effects of
sphingosine are not mediated by sphingosine 1-phosphate. First, if the
conversion of sphingosine to sphingosine 1-phosphate is necessary for
negative inotropism in cardiac myocytes, then one would predict that
exogenous sphingosine 1-phosphate would depress cell shortening at an
earlier or at least equivalent time point to that observed with
exogenous D-sphingosine, particularly given that exogenous
sphingosine and sphingosine 1-phosphate are taken up rapidly by
mammalian cells (<1-5 min) (33, 42), and given that both amphipathic
molecules share a similar time to onset of action when applied
exogenously (42). However, as shown by Fig. 4, A and
B, the time course for the onset of action for sphingosine
1-phosphate was
30 min and was 6-fold slower than was observed when
the cells were stimulated with D-sphingosine (5 min).
Second, if sphingosine 1-phosphate mediates the negative inotropic
effects of sphingosine, then one would predict sphingosine 1-phosphate
would be more potent than sphingosine on a molar basis. However, as
shown by Fig. 4, A and B, sphingosine 1-phosphate
is 1000-fold less potent than sphingosine in terms of producing
negative inotropic effects in isolated cardiac myocytes. Thus, while we
cannot exclude a potential contributory role for sphingosine
1-phosphate in terms of mediating the negative inotropic effects of
TNF-
, the data do not support a primary role for this molecule.
We next examined the effects of exogenous D-sphingosine on
intracellular calcium transients in isolated contracting cardiac myocytes to determine whether sphingosine would mimic the effects of
TNF- on intracellular calcium homeostasis (6). Fig. 5
shows representative time-intensity curves for fluorescence brightness in cardiac myocytes treated either with 1 µM
D-sphingosine or with diluent. As shown, treatment with
D-sphingosine produced a striking decrease in the peak
levels of intracellular fluorescence brightness, consistent with
previous observations that 1 µM sphingosine is sufficient
to inhibit calcium release by the sarcoplasmic reticular ryanodine
receptor (22, 23). Similar findings with respect to the effects of
sphingosine on intracellular calcium homeostasis have also been
observed in neonatal cardiac myocytes (43). The inset of
Fig. 5 summarizes the results for the studies, wherein peak
fluorescence brightness was examined for groups of diluent and
D-sphingosine-treated cells. As shown, there was
50%
decrease (p < 0.05) in the peak intensity of
fluorescence brightness for the D-sphingosine (1 µM) -treated cells compared with diluent-treated controls, again consistent with the previous findings from this laboratory that have shown that TNF-
suppressed peak intracellular fluorescence brightness by
40% (6).
In addition to engaging the neutral sphingomyelinase pathway,
TNF--induced oligomerization of TNFR1 activates
phosphatidylcholine-specific phospholipase C (PC-PLC), with increased
activity of diacylglycerol-dependent PKC, as well as the
phospholipase A2 pathway, with increased formation of arachidonic acid
(44). Previously, we have shown that inhibiting arachidonic acid
cyclooxygenase did not abrogate the TNF-
-induced negative inotropic
effects (6), suggesting that prostaglandins were not responsible for
producing the negative inotropic effects of TNF-
. To determine
whether the PC-PLC pathway was important in terms of mediating the
negative inotropic effects of TNF-
, we stimulated isolated
contracting cardiac myocytes with TNF-
in the presence of specific
inhibitors of the PC-PLC and the diacylglycerol-dependent PKC pathways. These studies showed that the negative inotropic effects
of TNF-
were not abrogated by PC-PLC inhibition with D609 nor by PKC
inhibition with sangivamycin: that is TNF-
-induced a 21.2 ± 2% and 18 ± 6% decrease in myocyte shortening, respectively, in
cells pretreated with 0.4 µM D609 (p < 0.002 compared with control; n = 10 cells) and 20 µM sangivamycin (p < 0.009 compared with
control; n = 9 cells). We also directly measured PKC
activity in TNF-
and sphingosine-stimulated cells. In unstimulated
myocytes the PKC
membrane-to-cytosol ratio was
0.4. Stimulating
the cells with 100 nM PMA resulted in a rapid (<1 min)
increase in the PKC
membrane-to-cytosol ratio to 0.8, which was
maintained at 7.5 min. In contrast, there was no change in the
membrane-associated PKC
at any time point up to 30 min for the cells
stimulated with 200 units·ml
1 TNF-
. Identical
results were obtained when an antibody that recognized PKC
, -
,
-
, and -
was used. Insofar as sphingosine has been shown to
decrease PKC activity in certain cell types (41), we also measured
incorporation of 32P into a specific PKC pseudosubstrate
(n = 6 experiments/group) in PMA-stimulated cytosolic
extracts from cells that had been pretreated with TNF-
(200 units·ml
1) or sphingosine (0.1-10 µM).
This study showed that there was no significant difference in
radiolabeling of the pseudosubstrate in cytosolic extracts pretreated
with TNF-
(64 ± 4 pmol/min/mg) or with 0.1-10
µM sphingosine (76.0 ± 5.6, 72 ± 8.0, 76 ± 6.0 pmol/min/mg, respectively), when compared with the values
obtained in control cytosolic extracts (68 ± 3.2 pmol/min/mg). In
contrast, there was a significant decrease in PKC activity
(p < 0.05) in the cytosolic extracts that had been
pretreated with 100 nM staurosporine (32.0 ± 2 pmol/min/mg). Taken together, these latter studies suggest that neither
activation of PKC through the PC-PLC pathway nor inhibition of PKC
activity by sphingosine play a major role in mediating the negative
inotropic effects of TNF-
.
In summary, we have provided evidence that shows that TNF- and
sphingosine both control the same events in isolated contracting cardiac myocytes: that is alterations in intracellular calcium homeostasis and negative inotropism. We have also provided data that
show that the time course for and degree of free sphingosine production
following cytokine stimulation is sufficient to completely mimic the
negative inotropic effects of TNF-
in isolated cardiac myocytes.
Finally, we have shown that TNF-
-induced sphingosine production is
necessary for the negative inotropic effects of this cytokine, insofar
as blocking sphingosine production through inhibition of ceramidase
with NOE abrogates the negative inotropic effects of TNF-
. In
contrast to the findings for sphingosine, we have shown that ceramide,
the immediate precursor for sphingosine, is neither necessary nor
sufficient to mimic the negative inotropic effects of TNF-
and that
sphingosine 1-phosphate, the phosphorylated metabolite of sphingosine,
is 1000-fold less potent than sphingosine in terms of producing
negative inotropic effects; moreover, the delayed onset for the
biological effects of sphingosine 1-phosphate in cardiac myocytes is
inconsistent with the time course for the negative inotropic effects in
TNF-
. Taken together, the above results provide a rational basis for
concluding that sphingosine mediates the immediate negative inotropic
effects of TNF-
. Although we cannot exclude a potential contributory
role for NO in terms of modulating the immediate negative inotropic
effects of TNF-
, it bears reemphasis that TNF-
has not yet been
shown to produce NO with a sufficiently rapid time course to explain
the immediate negative inotropic effects of this cytokine (6). The
importance of the above issues notwithstanding, perhaps the more
intriguing biological issue that arises from these studies,
particularly in view of the pleiotropic nature of sphingosine signaling
in mammalian cells (20), is that of understanding what other role(s) sphingosine might play in cytokine-stimulated adult cardiac
myocytes.
We gratefully acknowledge the secretarial assistance of Jana Grana, as well as the technical assistance of Dorellyn Lee-Jackson. We also thank Dr. Emmanuel Coroneous and W. Robb MacLellan for their thoughtful suggestions and critical reviews of this manuscript as well as Dr. Andrew I. Schafer for his past and present guidance and support.