Published online before print April 13, 2001, doi: 10.1161/hh0801.089604
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© 2001 American Heart Association, Inc.
Article |
Ceramide Reduces Endothelium-Dependent Vasodilation by Increasing Superoxide Production in Small Bovine Coronary Arteries
From the Department of Pharmacology and Toxicology (D.X.Z., P.-L.L.) and the Department of Physiology (A.-P.Z.), Medical College of Wisconsin, Milwaukee.
Correspondence to Pin-Lan Li, MD, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail pli{at}mcw.edu
Abstract
Abstract—Ceramide serves as a second messenger in a variety of mammalian cells. Little is known regarding the role of ceramide in the regulation of vascular endothelial function. The present study was designed to determine whether ceramide affects endothelium-dependent vasodilation in coronary arteries and to explore the mechanism of action of ceramide. In isolated and pressurized small bovine coronary arteries, cell-permeable C2-ceramide (10-5 mol/L) markedly attenuated vasodilator responses to bradykinin and A23187 (by 40% and 60%, respectively). In the presence of NG-nitro-L-arginine methyl ester, ceramide produced no further inhibition on the vasodilation induced by these vasodilators. Ceramide had no effect on DETA NONOate–induced vasodilation. By use of a fluorescence NO indicator (4,5-diaminofluorescein diacetate), intracellular NO was measured in the endothelium of freshly isolated small coronary arteries. It was found that ceramide significantly inhibited bradykinin-induced NO increase within endothelial cells. However, it had no effect on the activity of arterial or endothelial NO synthase. Pretreatment of the arteries with sodium dihydroxybenzene disulfonate (Tiron, 10-3 mol/L), a cell-permeable superoxide scavenger, or polyethylene glycol superoxide dismutase (100 U/mL) largely restored the inhibitory effects of ceramide on the vasodilation and NO increase induced by bradykinin or A23187. Moreover, ceramide time-dependently increased intracellular superoxide (O2- · ) in the endothelium, as measured by a fluorescent O2- · indicator, dihydroethidium. These results demonstrate that ceramide inhibits endothelium-dependent vasodilation in small coronary arteries by decreasing NO in vascular endothelial cells and that this decrease in NO is associated with increased O2- · but not with the inhibition of NO synthase activity within these cells.
Key Words: ceramide • vasodilation • coronary vessels • nitric oxide • superoxide
Ceramide, a
sphingolipid, has emerged as an intracellular second messenger for
various cytokines and hormones, such as tumor necrosis factor
(TNF)-
, interleukin-1ß, 1,25-dihydroxy-vitamin
D3, and nerve growth
factor.1 2 3 4
The ceramide-mediated signaling exists in a variety of mammalian
tissues and cells and plays an important role in a number of cellular
processes, including cell growth and differentiation,
apoptosis, and inflammatory
responses.5 6
Recent studies have indicated that ceramide may also be involved in the
regulation of ion channel activity, intracellular
Ca2+ concentrations, and contractile
responses in vascular smooth
muscles.7 8 9 10 11 12
However, the role of ceramide in the regulation of vascular
endothelial function has yet to be
clarified.
It is well known that the
endothelium-dependent vasodilation (EDVD) in
coronary circulation is impaired during myocardial
ischemia and
reperfusion.13 14
The mechanism by which ischemia/reperfusion attenuates EDVD is
not completely understood but may involve the actions of increased
cytokines, such as TNF- and interleukin-1ß, in the
myocardial tissue during ischemia and
reperfusion.15 TNF- and
other cytokines have been reported to inhibit the release of
endothelial NO and EDVD in a variety of vascular
beds.16 17 18 19
However, the mechanism mediating these actions remains unclear. Recent
studies have indicated that these cytokines can increase
intracellular ceramide in vascular endothelial cells,
which then mediates a number of cellular responses to these
cytokines, such as inflammatory responses and
apoptosis.20 21
Given the importance of ceramide in the actions of these
cytokines, it is possible that increased ceramide in the
vascular endothelium also contributes to the impairment
of EDVD induced by these cytokines.
Numerous studies have shown that a decrease in the bioavailability of NO plays a central role in the endothelial dysfunction or impairment of EDVD.22 Although reduced NO bioavailability may be associated with alterations of cellular NO signaling and with a lack of the substrate or cofactors for NO synthase (NOS), there is accumulating evidence suggesting that superoxide (O2- · ) and other reactive oxygen species can interact with NO and thereby regulate or modulate the biological actions of NO and endothelial function.23 24 It has been demonstrated that O2- · inactivates NO, which reduces arteriolar dilation to acetylcholine or other endothelium-dependent vasodilators. Superoxide dismutase significantly increases the half-life of NO and restores EDVD.25 26 27 28 Because ceramide has been found to increase the O2- · production in vascular tissues,29 we hypothesized that the ceramide-stimulated oxidant stress may impair EDVD through NO inactivation, thereby leading to endothelial dysfunction of coronary circulation induced by cytokines during myocardial ischemia/reperfusion. To test this hypothesis, the present study was designed to determine the effect of ceramide on EDVD and to explore the role of NO and O2- · interaction in mediating the action of ceramide by use of isolated and pressurized small bovine coronary arteries. Then, we directly measured NO and O2- · concentrations in the endothelium of these arteries and examined the effects of ceramide on NO and O2- · production with the use of fluorescence microscopy.
Materials and Methods
Isolated Small Coronary Artery
Preparation
Fresh bovine hearts were obtained from a local
abattoir. The left ventricular wall was rapidly dissected
and immersed in ice-cold physiological saline
solution (PSS) containing the following (in
10-3 mol/L):
NaCl 119, KCl 4.7, CaCl2 1.6,
MgSO4 1.17,
NaH2PO4 1.18,
NaHCO3 24, EDTA 0.026, and glucose 5.5, pH 7.4.
This myocardial section was transported immediately to the laboratory.
Small intramural coronary arteries from the left anterior
descending artery were carefully dissected and placed in cold PSS until
cannulation (up to 4 hours). Segments of small arteries (100- to
200-µm internal diameter) were transferred to a water-jacketed
perfusion chamber and cannulated with 2 glass micropipettes at their in
situ length, as we described
previously.10 30
The outflow cannula was clamped, and the arteries were pressurized to
60 mm Hg. The arteries were bathed in the PSS equilibrated with
95% O2/5% CO2 and
maintained at pH 7.4 and 37°C. The internal diameter of the arteries
was measured with a video system composed of a stereomicroscope (Leica
MZ8), a charge-coupled device camera (KP-MI AU, Hitachi), a video
monitor (VM-1220U, Hitachi), a video measuring apparatus
(VIA-170, Boeckeler Instrument), and a video printer (UP890 MD, Sony).
The arterial images were recorded continuously with a
videocassette recorder (M-674, Toshiba).
After a 1-hour equilibration period, the arteries were
precontracted by 
50% of their resting diameter with the
thromboxane A2 analogue U46619. Once
steady-state contraction was obtained, cumulative dose-response curves
to the endothelium-dependent vasodilators bradykinin
(BK, 10-10 to
10-6 mol/L) and
A23187 (10-9 to
10-5 mol/L) and
to the endothelium-independent vasodilator DETA NONOate
(10-7 to
10-4 mol/L)
were determined by measuring changes in the internal diameter. To study
the effect of ceramide on vasodilator response to BK, A23187, or DETA
NONOate, arteries were preincubated with cell-permeable
C2-ceramide
(10-5 mol/L)
for 30 minutes, and dose-response curves to the vasodilators were
further established. To examine the involvement of NO or
O2- · in
ceramide-induced endothelial dysfunction, the arteries
were preincubated with
NG-nitro-L-arginine
methyl ester (L-NAME,
10-4 mol/L; an
NO synthase inhibitor), sodium dihydroxybenzene disulfonate
(Tiron, 10-3
mol/L; a cell-permeable
O2- ·
scavenger), superoxide dismutase (SOD, 100 U/mL), or polyethylene
glycol SOD (PEG-SOD, 100 U/mL) in the presence or absence of
C2-ceramide
(10-5 mol/L)
for 30 minutes, and then dose responses to BK and A23187 were
determined. All drugs were added into the bath solution. Between
pharmacological interventions, the arteries were washed 3 times with
PSS and allowed to equilibrate in drug-free PSS for 20 to 30 minutes.
The vasodilator response was expressed as percent relaxation of
U46619-induced precontraction on the basis of changes in the internal
diameter.
Measurement of Intracellular [NO] in the
Endothelium
A novel fluorescent NO indicator,
4,5-diaminofluorescein diacetate (DAF-2 DA), as recently
described by Kojima et al,31
was used to measure intracellular NO concentration ([NO]) within the
endothelial cells of freshly isolated small bovine
coronary arteries. DAF-2 DA can readily enter the cells and be
hydrolyzed by cytosolic esterases to the DAF-2 that is trapped inside
the cells. In the presence of NO and oxygen, a relatively
nonfluorescent DAF-2 is transformed into the highly green
fluorescent triazole form, DAF-2T. Thus, the increases in
DAF-2T fluorescence represent the elevation of [NO].
Small intramural arteries (300- to 500-µm internal diameter) were
carefully dissected as described above and transferred to a 35-mm
dissecting dish coated with Sylgard (World Precision
Instruments) and containing ice-cold HEPES-buffered PSS that consists
of the following (in
10-3 mol/L):
NaCl 140, KCl 4.7, CaCl2 1.6,
MgSO4 1.17,
NaH2PO4 1.18, glucose
5.5, and HEPES 10, pH 7.4. The arterial segment was cut
open along its longitudinal axis and pinned onto the dish with lumen
side upward. Care was taken not to disrupt the
endothelium. After 1 hour of equilibrium at 37°C, the
arterial segment was incubated with DAF-2 DA
(10-5 mol/L,
Calbiochem) in 1 mL PSS at room temperature for 30 minutes. The
segments were then rinsed 3 times with PSS, and the dish was mounted on
the stage of an epifluorescence microscope (Nicon E600)
equipped with a x20 objective and 490-nm excitation and a 510- to
560-nm emission filters. Digital images were captured and
analyzed by using a PC-controlled charge-coupled device camera
(Roper Scientific RTE/CCD-1300-Y/HS) and MetaMorph imaging and
analysis software (Universal Imaging Corp).
BK (10-6 mol/L) was added into the bath solution to stimulate NO production. To study the effect of ceramide on endothelial [NO], the arteries were incubated with C2-ceramide (10-5 mol/L) for 30 minutes before the response to BK was determined. To examine whether O2- · was involved in the effect of ceramide on endothelial [NO], the arteries were incubated with C2-ceramide and Tiron (10-3 mol/L) for 30 minutes, and then the response to BK was determined. NO fluorescence was measured every 5 minutes in a single area of endothelial layer. Results were expressed as the integrated fluorescence intensity within the area observed.
Activity of NOS
NOS activity was determined by measuring the
conversion of [3H]arginine to
[3H]citrulline32
and by using the isotopic NOS detection kit (Calbiochem) according to
manufacturer’s protocol. Briefly, the homogenates prepared
from cultured bovine coronary endothelial cells
(25 µg protein)33 or small
bovine coronary arteries (100 µg protein) were incubated in
50 µL reaction mixture containing the following (in
10-3 mol/L):
Tris-HCl 25 (pH 7.4), CaCl2 0.6, ß-NADPH 1,
tetrahydrobiopterin 0.003, flavin adenine dinucleotide
0.001, flavin mononucleotide 0.001, and cold
L-arginine 0.005, along
with 1.0 µCi [3H]arginine in the absence
or presence of C2-ceramide. After incubation for
15 or 60 minutes (for endothelial cell or
arterial homogenates, respectively) at 37°C,
the reaction was terminated by the addition of 400 µL of ice-cold
stop buffer containing the following (in
10-3 mol/L):
HEPES 50 (pH 5.5) and EDTA 5. Equilibrated cation exchange resin was
added to the samples, and they were then applied to spin columns. After
centrifugation, the eluate (containing
[3H]citrulline) was collected, and the
radioactivity was determined with a liquid scintillation counter. To
determine the effect of ceramide on NOS activity in intact
endothelial cells, the confluent
endothelial cell cultures in 150-mm dishes were treated
with C2-ceramide for 30 minutes, followed by
harvesting and homogenization. The formation of
citrulline was then assayed as described above. In these experiments,
the formation rate of citrulline represented NOS activity,
which was expressed as picomoles per milligram protein per
minute.
Measurement of Intracellular
[O2- · ]
in the Endothelium
The intracellular
O2- ·
concentration
([O2- · ])
was detected by measuring the fluorescence intensity resulting
from oxidation of dihydroethidium (DHE), as reported by Castilho
et al.34 DHE can enter the
cell and be oxidized by
O2- · to yield
ethidium. Ethidium binds to DNA, which produces strong red
fluorescence. Assays were performed on the
endothelium of small bovine coronary arteries
as prepared for NO measurement. The arterial segment was
incubated with 1 mL PSS containing
3x10-6 mol/L
DHE (Molecular Probes) purified with cationic resin (Dowex-50W-X8), and
the ethidium fluorescence was measured every 5 minutes for 30
minutes at 490-nm excitation and 610 nm-emission by using the same
imaging system as described above. To study the effect of ceramide on
[ O2-· ], the
arterial segment was incubated with
C2-ceramide
(5x10-6 mol/L)
in addition to DHE.
Statistical Analysis
Data are presented as mean±SEM. The
significance differences in mean values between and within multiple
groups were examined by using ANOVA for repeated measures, followed by
a Duncan multiple range test (Sigmastat). A value of
P<0.05 was considered
statistically significant.
Results
Effect of Ceramide on EDVD and
Endothelial [NO] in Small Coronary
Arteries
Endothelium-Dependent
Vasodilation
Concentration-response curves of the
endothelium-dependent vasodilators BK and A23187 were
determined before and after the treatment of arteries with
C2-ceramide
(10-5 mol/L for
30 minutes). As shown in
Figure 1
, BK and A23187 produced a concentration-dependent
vasodilation in small coronary arteries. Pretreatment of the
arteries with ceramide significantly attenuated the vasodilator
responses to BK
(Figure 1A) and A23187
(Figure 1B), with maximal inhibitions of 40% and 60%,
respectively. In contrast, ceramide had no effect on the vasodilation
induced by endothelium-independent vasodilator DETA
NONOate
(Figure 1C).
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To determine whether C2-ceramide acts
through an NO-dependent mechanism in the inhibition of EDVD, the
arteries were pretreated with the NOS inhibitor L-NAME
(10-4 mol/L) in
the absence or presence of C2-ceramide
(10-5 mol/L).
As shown in
Figure 2, L-NAME markedly attenuated the vasodilator
responses to BK and A23187. This confirmed that the response to these
vasodilators was NO dependent. In the presence of L-NAME, ceramide
produced no further inhibition of BK- or A23187-induced vasodilation.
The inhibition of vasodilation by ceramide and L-NAME was similar to
that by ceramide or L-NAME alone.
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Intracellular NO Concentration
The intracellular [NO] within the
endothelial cells was measured in the
endothelium of small coronary arteries.
Figure 3A presents typical fluorescence
microscopic images showing NO-induced DAF-2 green fluorescence
within endothelial cells. Incubation of the arteries
with BK (10-6
mol/L) produced a marked increase in NO fluorescence. In the
presence of ceramide
(10-5 mol/L),
this increase was significantly inhibited.
Figure 3B summarizes the results of these measurements. BK
induced a rapid and time-dependent increase in [NO] within
endothelial cells. No significant increase in [NO]
was observed in control arteries throughout 30 minutes. Addition of
C2-ceramide into bath solution caused a slight
decrease in basal [NO]. In the presence of
C2-ceramide, the BK-induced increase in [NO]
was significantly attenuated.
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To confirm that the NO fluorescence in these arteries is within endothelium, the denuded arteries were used. In these arteries, only weak and nonspecific fluorescence was observed, and there was no significant change in fluorescence after BK treatment. In addition, L-NAME was used to demonstrate the dependence of the NO increase on NOS activity. In the presence of L-NAME (10-4 mol/L), the BK-induced increase in fluorescence was markedly blocked, suggesting that the BK-induced increase in [NO] was through the activation of NOS.
Effect of Ceramide on the Activity of
Arterial and Endothelial NOS
By determining the formation rate of
[3H]citrulline, the NOS activity in small
coronary arterial homogenates was found
to be 0.67±0.09 pmol/mg protein per minute. The addition of EDTA
(10-3 mol/L)
largely inhibited the formation of
[3H]citrulline (by 80%). However,
C2-ceramide at concentrations up to
4x10-5 mol/L
had no effect on the activity of NOS in these arteries. To further
examine the effect of ceramide on NOS activity in
endothelial cells, C2-ceramide
was incubated with endothelial cell
homogenates
(Figure 4A) or intact cells
(Figure 4B). Under both conditions, the NOS activity was not
altered. However, L-NAME
(10-4 mol/L) or
EDTA (10-3
mol/L) significantly inhibited the NOS activity of these
endothelial cell preparations.
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Contribution of
O2- · to
Ceramide-Induced Endothelial Dysfunction
Endothelium-Dependent
Vasodilation
To explore whether ceramide-induced NO decrease
and endothelial dysfunction are associated with
O2- · , the
arteries were pretreated with Tiron
(10-3 mol/L).
Tiron had no effect on either basal tone or vasodilator responses to BK
and A23187. However, it largely reversed the inhibitory
effect of ceramide on the vasodilation to BK or A23187
(Figure 5). The inhibitory effect of ceramide was
also restored by PEG-SOD (100 U/mL) but not by SOD (100 U/mL)
(Figure 6).
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Intracellular
O2- ·
Concentration
To provide direct evidence for the role of
O2- · in the
action of ceramide, the intracellular
[O2- · ] was
measured in the endothelium of small coronary
arteries.
Figure 7A presents the typical fluorescence
microscopic images showing
O2- · -induced
red fluorescence within endothelial cells. The
nuclei were the primary fluorescent structures labeled.
Incubation of the arteries with C2-ceramide
(5x10-6 mol/L)
produced a significant increase in
O2- ·
fluorescence compared with control conditions. Under resting
conditions, Tiron
(10-3 mol/L)
was without effect on
O2- ·
fluorescence, and removal of the endothelium
resulted in the loss of cell-specific fluorescence, confirming
that the O2- ·
detected was within the endothelium.
Figure 7B summarizes the results of these measurements.
C2-ceramide induced a time-dependent increase in
endothelial
[O2- · ]
compared with control conditions. Tiron was without effect on
endothelial
[O2- · ].
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Effect of Endogenous and Exogenous
O2- · on
Endothelial [NO]
To determine whether
O2- ·
interacts with NO and thereby reduces DAF-2 fluorescence and
whether ceramide reduces [NO] through
O2- · , the
endothelial [NO] was measured in the presence of
Tiron or an
O2- · -producing
system. Consistent with the results in arterial
preparations, the inhibitory effect of ceramide on the
BK-induced increase in [NO] was largely restored by pretreatment with
10-3 mol/L
Tiron
(Figure 8A), suggesting that
O2- · can
decrease [NO] within endothelial cells.
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To further confirm this interaction of
O2- · and NO,
the arteries were treated with xanthine
(2.5x10-5
mol/L)/purified xanthine oxidase (0.1 U/mL), a well-known
O2- · -producing
system, 15 minutes before BK
(10-6 mol/L).
It was found that xanthine/xanthine oxidase significantly attenuates
the BK-induced increase in endothelial [NO]
(Figure 8B
).
Discussion
The present study demonstrated that ceramide inhibited BK- and A23187-induced vasodilation in small coronary arteries. In the presence of L-NAME, ceramide had no further inhibition on the responses to these vasodilators. However, ceramide was without effect on the vasodilation induced by the endothelium-independent vasodilator DETA NONOate. These results suggest that ceramide has a detrimental effect on NO-mediated EDVD in coronary microcirculation and thereby may lead to endothelial dysfunction.
Recently, there has been accumulating evidence suggesting that ceramide and other sphingolipids play an important role in regulating vascular function. These sphingolipids may affect cell proliferation and differentiation, cell apoptosis, and vasomotion of different arteries.7 8 With respect to vasomotor regulation, cell-permeable ceramide and/or sphingomyelinase treatment has been shown to induce the relaxation of phenylephrine-contracted rat thoracic aorta9 11 but to produce contraction in canine cerebral arteries, rat mesenteric resistance and capacitance vessels, and bovine coronary resistance arteries.10 12 It seems that the effects of ceramide on vasomotor response are dependent on the species, vascular beds, or arterial size used in different studies. The present studies have demonstrated that ceramide attenuates L-NAME–sensitive EDVD, providing the first direct evidence that ceramide impairs NO-mediated EDVD in coronary resistant arteries. This effect of ceramide on EDVD is similar to that of another sphingolipid, sphingosine. It has been reported that sphingosine attenuates thrombin- and A23187-induced relaxation without affecting sodium nitroprusside–induced relaxation in pig coronary rings. The impairment of EDVD induced by sphingosine was shown to contribute to vascular dysfunction in atherosclerotic arteries.35 In those studies, sphingosine was proposed to inhibit endothelial NOS activity by a calmodulin-dependent mechanism, inasmuch as it may directly bind to calmodulin and thereby inhibit the activity of calmodulin-dependent enzymes. In addition, sphingosine may modify receptor–G-protein coupling by altering membrane fluidity, thus leading to impaired agonist-induced release of NO.35 However, there is no direct evidence supporting those mechanisms for the action of sphingosine.
In the present study, it was also found that ceramide
exhibits a greater inhibitory effect on A23187-induced EDVD
than on BK-induced EDVD. Treatment of the arteries with ceramide almost
completely abolished the vasodilator response to A23187, whereas it
inhibited BK-induced vasodilation by 40%. This difference in the
inhibitory effects of ceramide on vasodilator responses to
A23187 and BK was comparable to that obtained in L-NAME–pretreated
arteries. Previous studies have shown that BK stimulates
endothelial cells to release
endothelium-derived hyperpolarizing factor (EDHF) in
addition to NO,36 and it is
likely that A23187-induced EDVD is primarily mediated by NO, whereas
BK-induced EDVD is associated with both NO and EDHF in the present
study. These results provide further evidence that a deficiency of NO
is mainly responsible for ceramide-induced impairment of EDVD. This
view is also supported by the findings that ceramide did not
significantly affect the vasodilator response of coronary
arteries to arachidonic acid (authors’ unpublished
data, 2000), which primarily stimulates the release of EDHFs,
such as epoxyeicosatrienoic acids from endothelial
cells of the coronary
arteries.37 Although these
studies are preliminary, they suggest selective impairment of
NO-mediated vasodilation by ceramide in coronary
arteries.
To explore the mechanisms mediating the action of ceramide, we examined the effect of ceramide on intracellular [NO] and NOS activity in the endothelium. As expected, BK was found to induce a marked and time-dependent increase in [NO] in the endothelium of these freshly dissected arteries. In the presence of ceramide, the BK-induced [NO] increase was significantly attenuated. These results provide a direct evidence that the inhibitory effect of ceramide on EDVD is mediated by decreasing [NO] in the endothelium. However, a previous study has found that ceramide increases [NO] in cultured bovine aortic endothelial cells.38 The reason for this discrepancy is unclear. It is possible that compared with cultured aortic endothelial cells, the endothelium of freshly isolated small coronary arteries may exhibit different behavior in NO regulation. Moreover, the ceramide-induced activation of endothelial NOS in cultured cells38 did not occur in our freshly isolated coronary arteries. By measuring the conversion rate of L-arginine to citrulline, we found that ceramide had no effect on NOS activity. Our findings indicate that the inhibitory effect of ceramide on endothelial [NO] is not associated with the changes in NOS activity.
Numerous studies have been performed to examine the regulation of NO production in the arterial endothelium and to explore the mechanism of NO-mediated endothelial dysfunction in various pathological states, such as myocardial ischemia and reperfusion, atherosclerosis, and hypertension.23 24 However, most of those studies used pharmacological interventions to block or enhance NOS activity and then examined the alteration of endothelial function, such as EDVD. There were few studies to address these issues by directly measuring endothelial NO. By using a novel fluorescent NO indicator and freshly isolated coronary small arteries, we were able to detect intracellular [NO], whereby changes in [NO] within endothelial cells in response to endothelium-dependent vasodilators, such as BK and A23187, could be dynamically examined. This preparation provides a valuable model for studying NO regulation and NO-mediated endothelial dysfunction in the vasculature.
To further ascertain how ceramide reduces endothelial [NO] and consequently inhibits EDVD in coronary arteries, the role of O2- · was examined. Recent studies have indicated that ceramide and/or other sphingolipids may stimulate the production of O2- · in vascular cells.29 39 40 Because O2- · can interact with NO and thereby decrease [NO] within endothelial cells,25 26 27 28 we hypothesized that ceramide may stimulate the O2- · production in the vascular endothelium, which then interacts with NO and results in the impairment of EDVD in coronary circulation. The present study provided several lines of evidence to support this hypothesis. First, pretreatment of the arteries with Tiron, an antioxidant that is capable of scavenging O2- · from both the intracellular and extracellular environment,41 prevented the ceramide-induced inhibition of NO production and endothelial dysfunction. Second, ceramide significantly increased [O2- · ] in the endothelium, as measured by DHE fluorescence microscopy. Finally, exogenous O2- · produced by xanthine/xanthine oxidase inhibited the NO increase in the endothelium.
The cellular site at which O2- · is produced by ceramide and O2- · interacts with NO in the coronary arteries is still undetermined. Although pretreatment of the arteries with SOD, a cell-impermeable O2- · scavenger, did not prevent the inhibitory effect of ceramide on EDVD, PEG-SOD significantly blocked the action of ceramide. It seems that O2- · from either the intracellular or extracellular space contributes to the ceramide-induced impairment of EDVD. With respect to extracellular O2- · , it is possible that O2- · stimulated by ceramide is released or transferred into the vascular interstitium,42 43 44 where it interacts with NO and consequently blocks the action of NO. This interstitial O2- · can be scavenged by PEG-SOD, a chemically modified SOD that may exhibit enhanced adherence to the cell surface compared with native SOD adherence. It is also possible that the coupling of PEG may increase the cell permeability of SOD into endothelial cells, thereby increasing its efficiency in scavenging the O2-· induced by ceramide. Indeed, a previous study has shown that catalase and SOD can be effectively delivered into endothelial cells from the liver by chemical modification (ie, succinylation).45
In the cardiovascular system, there are several potential enzymatic sources of O2- · or other reactive oxygen species, including NAD(P)H oxidase, xanthine oxidase, NO synthase, and the mitochondrial respiratory chain.23 24 Previous studies have shown that ceramide activates NAD(P)H oxidase and increases the O2- · production in human aortic smooth muscle cells.29 Ceramide has also been shown to interact with the mitochondrial electron transport chain, leading to the generation of reactive oxygen species.46 47 Further studies are required to determine whether ceramide increases the production of O2- · in the coronary arterial endothelium through these or other mechanisms.
The present study did not attempt to determine the
pathogenic significance of ceramide-induced endothelial
dysfunction in the coronary circulation. Previous studies have
shown that ceramide or other sphingolipids are increased in cardiac
tissue during myocardial ischemia and reperfusion, which may
contribute to the detrimental effects of TNF- or other
cytokines under these
conditions.15 48
Given the important role of ceramide in mediating the effects of
cytokines, it is plausible that increased cytokines
during myocardial ischemia/reperfusion increase ceramide levels
in the coronary vascular endothelium or other
tissues and that ceramide, in turn, causes the impairment of EDVD. In
this respect, ceramide may represent an important signaling
molecule mediating endothelial dysfunction in the
coronary circulation during myocardial
ischemia/reperfusion.
In summary, the present study has demonstrated that
ceramide inhibits NO-mediated EDVD in small coronary arteries.
It has been suggested that the inhibitory effect of
ceramide is associated with increased
[O2- · ] and
the subsequent decrease in [NO] in endothelial cells
of the coronary arteries. These results led to a hypothesis
that ceramide-induced inhibition of EDVD may contribute to vascular
endothelial dysfunction of the coronary
circulation associated with cytokines, such as TNF-, during
myocardial ischemia and
reperfusion.
Acknowledgments
This study was supported by National Institutes of Health grants (HL-57244 to P.-L.L. and DK-54927 to A.-P.Z.) and the American Heart Association, an Established Investigator Grant (No. 9940167N to P.-L.L.) and a Predoctoral Fellowship (No. 0010185Z to D.X.Z.). The authors thank Gretchen Barg for secretarial assistance.
Footnotes
Original received December 4, 2000; revision received February 22, 2001; accepted March 2, 2001.
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