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INTRODUCTION |
Endothelium-derived nitric oxide
(NO)1 exerts several
vasoprotective activities including smooth muscle relaxation,
inhibition of platelet activation, and regulation of endothelial cell
permeability and adhesivity (1-4). NO is generated from the conversion
of L-arginine to L-citrulline by the enzymatic
action of an NADPH-dependent NO synthase (NOS) that
requires tetrahydrobiopterin, FAD, and FMN as cofactors (5). The
endothelial NOS isoform (ecNOS) is constitutively expressed and is
activated upon an increase of intracellular calcium following cell
stimulation with receptor-dependent stimuli such as
thrombin and bradykinin or with receptor-independent stimuli like
calcium ionophore (6).
Since NO interferes with key processes in atherogenesis (7), a lack of
NO might promote the development of atherosclerosis. Indeed, clinical
studies have confirmed an impairment of vascular NO synthesis in
patients with atherosclerosis or with increased atherogenic risk
factors (8). The dysfunction of the endothelial NO pathway may involve
impaired signal transduction mechanisms, decreased ecNOS activity,
reduced intracellular availability of L-arginine, or
increased inactivation of NO by superoxide anions or oxidized low
density lipoproteins (7, 8). Accordingly, initial therapeutic
strategies for an improvement of endothelial vasodilator function
include the application of angiotensin-converting enzyme inhibitors
that stimulate NO synthesis through the local accumulation of
bradykinin as well as the supplementation of L-arginine and
the use of antioxidants (7, 9).
Ascorbic acid is the most important water-soluble antioxidant in human
plasma (10). It effectively scavenges superoxide and other reactive
oxygen species and protects lipids against peroxidation (11, 12).
Ascorbic acid is thought to play a protective role in atherogenesis
since epidemiological studies have demonstrated that plasma ascorbic
acid levels are inversely related to the mortality from coronary heart
disease (13). Moreover, a number of conditions known to be associated
with an increased risk for atherosclerosis have been linked with lower
plasma levels of ascorbic acid (14-17). Recently, several studies have
shown that an acute application of ascorbic acid enhanced
endothelium-dependent vasodilation in patients with
diabetes, coronary artery disease, hypertension, hypercholesterolemia,
or chronic heart failure and in cigarette smokers (18-23). These
findings were attributed to the radical scavenging ability of ascorbic
acid which may protect NO from inactivation. So far, however, direct
effects of ascorbic acid on endothelial NO synthesis have not been examined.
The present study was designed to investigate whether ascorbic acid
affects NO production in human endothelial cells. The synthesis of NO
was measured as the formation of its co-product citrulline and as the
accumulation of its effector molecule cGMP after stimulating
ascorbate-pretreated cells with the calcium-increasing agonists
ionomycin and thrombin. Additionally, the influence of ascorbic acid on
calcium-dependent citrulline formation in cell lysates and
on the content of ecNOS protein was investigated. We report that
ascorbic acid stimulates NO synthesis in endothelial cells, and we
suggest that this action may be an important characteristic of this
agent to exert its beneficial effects in the vascular system.
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EXPERIMENTAL PROCEDURES |
Materials--
Plasticware for cell culture was from Greiner
Labortechnik (Frickenhausen, Germany). Medium 199 (M199), human serum,
fetal calf serum, collagenase, and human serum albumin (HSA) were from Boehringer Ingelheim Bioproducts (Heidelberg, Germany).
L-[2,3,4,5-3H]Arginine monohydrochloride (61 Ci/mmol), L-[U-14C]arginine monohydrochloride
(303 mCi/mmol),
L-[carboxyl-14C]ascorbic acid (16 mCi/mmol), [3H]cGMP Biotrak radioimmunoassay systems,
hyperfilm
max, ECL, and hyperfilm ECL were purchased
from Amersham Corp. (Bucks, UK). Tran35S-label and
methionine-free RPMI medium were from ICN Pharmaceuticals (Costa Mesa,
CA); nitrocellulose was from Millipore (Eschborn, Germany); specific
monoclonal antibodies against human ecNOS (clone 3) or against murine
macrophage-inducible NOS (iNOS, clone 6) were from Transduction
Laboratories (Lexington, KY); the fluorescein isothiocyanate-conjugated
secondary antibody (rabbit anti-mouse IgG (H + L)) with minimal
cross-reaction to human serum proteins was from Dianova (Hamburg,
Germany); NADPH, tetrahydrobiopterin, and L-nitroarginine
methyl ester (L-NAME) were from Alexis Corp. (Läufelfingen, Switzerland). Endothelial cell growth supplement, peroxidase-labeled anti-mouse IgG (Fab-specific), anti-mouse agarose, FAD, FMN, calmodulin, ionomycin, thrombin, EDTA, EGTA, trypsin/EDTA solution (0.05/0.02%, v/v), leupeptin, phenylmethylsulfonyl fluoride (PMSF), L-ascorbic acid, dehydro-L-ascorbic
acid, ascorbate 2-phosphate, ascorbate 2-sulfate,
L-gulonolactone, and other reagents were purchased from
Sigma (Deisenhofen, Germany).
The composition of the Hepes buffer (pH 7.4) was as follows (in
mM): 10 Hepes, 145 NaCl, 5 KCl, 1 MgSO4, 10 glucose, 1.5 CaCl2. Hepes homogenization buffer (pH 7.2)
consisted of (in mM) 20 Hepes, 0.5 EDTA, 0.5 EGTA, 1 dithiothreitol (DTT) 1 PMSF, 0.001 pepstatin, and 0.002 leupeptin.
Hepes/sorbitol buffer (pH 7.4) contained (in mM) 10 Hepes,
340 sorbitol, 1 EDTA, 2 DTT, 1 PMSF, 0.6 leupeptin, and 0.025 pepstatin. The composition of the solubilization buffer was 100 mM NaOH, 2% Na2CO3, and 1% SDS.
Cell Cultures--
Human umbilical cord vein endothelial cells
(HUVEC) were prepared with 0.05% collagenase, and primary coronary
artery endothelial cells (CAEC) were obtained from Clonetics (San
Diego, CA). Cells were cultured in 75-cm2 plastic flasks in
M199 containing 15% fetal calf serum, 5% human serum, and 7.5 µg/ml
endothelial cell growth supplement. Confluent cultures were detached by
trypsin/EDTA and plated on 30-mm diameter wells for the purpose of cGMP
determination and on 60-mm diameter dishes for the investigation of the
other parameters. Experiments were carried out with monolayers of the
first to second passage (HUVEC) or third to fourth passage (CAEC).
Experimental Incubations--
Preincubations of HUVEC or CAEC
with L-ascorbic acid, L-gulonolactone,
dehydroascorbic acid, and different ascorbic acid derivatives were
performed in culture medium for 1-24 h. The low content of ascorbic
acid in M199 (0.3 µM) was neglected since the liquid media were stored between 1 and 3 weeks before use, and the compound is
extremely labile in solution. L-Ascorbic acid stock
solution was freshly prepared in M199 and neutralized with 200 mM NaOH before the addition to the cells. Stock solutions
of the other compounds were prepared in M199. Stimulation of cells with
ionomycin or thrombin was performed in the absence of ascorbic acid or
its derivatives. Ionomycin was dissolved at 1 mM in
Me2SO and stored at
20 °C until use. The final
concentration of Me2SO did not exceed 0.1%, and control
cells received the same volume addition of solvent. The viability of
cells was determined by trypan blue exclusion and ranged from 95 to
98% under the different conditions described. None of the tested
compounds altered the growth behavior of endothelial cells as evaluated
by [3H]thymidine incorporation and by cell counting.
Measurement of Citrulline Synthesis--
Citrulline synthesis
was measured by a modification of a previously described technique
(24). Cell monolayers were incubated at 37 °C for 30 min in 1.5 ml
of Hepes buffer (pH 7.4) containing 0.25% HSA. 10 µM
L-[3H]arginine (0.33 Ci/mmol) were added to
each dish, and 1 min later cells were stimulated with 2 µM ionomycin or 1 unit/ml thrombin. After 15 min the
reaction was stopped with cold phosphate-buffered saline (PBS)
containing 5 mM L-arginine and 4 mM
EDTA, and the cells were denaturated with 96% ethanol. After
evaporation, the soluble cellular components were dissolved in 20 mM Hepes-Na (pH 5.5) and applied to 2-ml columns of Dowex
AG-50W-X8 (Na+ form). The [3H]citrulline
content of the eluate was quantified by liquid scintillation counting.
Agonist-induced citrulline production was calculated from the
difference in radioactivity from ionomycin- or thrombin-stimulated cells and the corresponding unstimulated cells and was expressed in
pmol/mg cell protein. Basal citrulline synthesis was determined from
the L-NAME (1 mM, 30 min
preincubation)-inhibitable radioactivity in unstimulated cells and was
not always detectable.
Determination of cGMP--
HUVEC monolayers were incubated for
30 min in M199 containing 0.25% HSA and 0.5 mM
isobutylmethylxanthine. Subsequently, the cells were stimulated with 2 µM ionomycin or 1 unit/ml thrombin for 15 min. The
reaction was stopped with 96% ethanol. When the ethanol had
evaporated, 0.3 ml of buffer (50 mM Tris, 4 mM
EDTA (pH 7.5)) was applied. The cGMP content of 100 µl of cellular extract was measured by radioimmunoassay following the instructions of
the manufacturer. The intracellular cGMP concentration was expressed in
pmol/mg cell protein. The agonist-induced cGMP production was
determined from the difference of cGMP content in ionomycin- or
thrombin-stimulated cells and the corresponding unstimulated cells.
[14C]Ascorbic Acid Uptake in Endothelial
Cells--
HUVEC were incubated in culture medium containing 100 µM [14C]ascorbic acid (16 mCi/mmol). After
various times, incubations were stopped by washing the cells with cold
Hepes buffer (pH 7.4) containing 100 µM phloretin which
had been shown to prevent the efflux of
[14C]dehydroascorbic acid (25). HUVEC were lysed with
solubilization buffer; an aliquot of the lysate was taken to determine
the protein content (26), and the radioactivity of the remaining sample was measured by liquid scintillation counting.
[14C]Arginine Uptake into Endothelial
Cells--
HUVEC were incubated in culture medium containing 335 µM L-[14C]arginine (3 mCi/mmol). After various times, incubations were stopped by washing the
cells with cold Hepes buffer (pH 7.4) containing 5 mM
L-arginine. The solubilization of the cells and the
measurement of radioactivity were performed as described above.
SDS Electrophoresis and Immunoblotting--
HUVEC were detached
by trypsin/EDTA and suspended in a small volume of Hepes/sorbitol
buffer (pH 7.4). For preparation of subcellular fractions, cell
suspensions were sonicated on ice and centrifuged for 60 min at
100,000 × g and 4 °C. Proteins were solubilized by
boiling the cells as well as the soluble and particulate fractions in
Laemmli sample buffer. The samples were separated by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) on 7% gels (50 µg of protein per
lane), blotted onto nitrocellulose membranes, and subsequently
subjected to immunostaining with primary antibodies against human ecNOS
or against murine iNOS (1:250, 1.5 h). After an incubation with a
peroxidase-conjugated secondary antibody (1:1000, 1.5 h),
visualization of NOS was achieved using the ECL technique.
Flow Cytometric Measurement of ecNOS Protein--
HUVEC were
fixed with 0.5% formaldehyde in PBS for 2.5 min, dissociated by a
combination of trypsinization (2 min) and scraping, and finally
suspended in PBS containing 0.1% HSA. Samples of 5 × 105 cells/50 µl were labeled with a monoclonal antibody
to ecNOS (1:25, 30 min) in the presence of 0.06% saponin (w/v) and
subsequently incubated with fluorescein isothiocyanate-conjugated
anti-mouse IgG(H + L) (1:50, 30 min). The cell-associated fluorescence
of 5000 cells per sample was determined in a FACScan flow cytometer (Becton/Dickinson) using the CellQuest software and reported as mean
fluorescence intensity in the FL1 channel. The mean fluorescence intensity values were corrected for nonspecific staining. Calibration of the flow cytometer and calculation of the numbers of ecNOS molecules
per cell from the corrected mean fluorescence intensity values were
done using Dako FluoroSpheres.
Metabolic Labeling and Immunoprecipitation of ecNOS--
HUVEC
were incubated with 100 µM ascorbic acid for 6 h.
Subsequently, 50 µCi of Tran35S-label reagent in
methionine-free RPMI medium containing 100 µM ascorbate
was added for 3 h. Untreated control cells were processed in
parallel. The labeled cells were lysed by the addition of Hepes buffer
(pH 7.4) containing 0.5% Triton X-100, 0.05% SDS, and protease inhibitors (1 mM PMSF, 0.6 mM leupeptin, 0.025 mM pepstatin). Immunoprecipitation was performed with a
monoclonal anti-ecNOS antibody (8 µg per 500 µg of lysate protein,
4 °C, 2 h) which was followed by a 1-h incubation with
anti-mouse agarose. The precipitates were washed with PBS containing
0.1% Triton X-100, 0.1% SDS, and 0.2% HSA and eluted by boiling in
Laemmli sample buffer. The samples were separated, blotted, and
immunostained as described above. The autoradiography was performed
with hyperfilm
max at
20 °C for 2-10 days.
Measurement of Citrulline Formation in Cell Lysates--
HUVEC
were detached by trypsin/EDTA, resuspended in a small volume of
homogenization buffer, and sonicated on ice. The assay solution
contained Hepes homogenization buffer (pH 7.2), 1 mg/ml lysate
protein, 20 µM L-[14C]arginine
(303 mCi/mmol), 3 µM tetrahydrobiopterin, 1 µM FAD, 1 µM FMN, 100 nM
calmodulin, 2 mM NADPH, and 2.5 mM
CaCl2. Incubations without calcium served as a blank. In
some experiments tetrahydrobiopterin, FAD, FMN, or calmodulin were
omitted from the test. After a 15-min incubation at 37 °C, the
reaction was stopped by adding Hepes-Na (20 mM, pH 5.5)
containing 2 mM EDTA, and the reaction mixture was applied
to 2-ml columns of Dowex AG-50W-X8 (Na+ form). The
[3H]citrulline content of the eluate was quantified by
liquid scintillation counting and after correction for basal values
expressed in pmol/mg lysate protein. In previous experiments we have
demonstrated that citrulline synthesis is a function of lysate protein
up to 2.5 mg/ml and is linear for at least 20 min. Furthermore,
half-maximal citrulline formation was detectable at 3.4 ± 0.3 µM L-arginine (results not shown).
Protein Determination--
After lysing the cells with
solubilization buffer or with Laemmli sample buffer, proteins were
measured according to Lowry et al. (26). Protein
determination in cell lysates containing DTT was performed according to
Bradford using the Bio-Rad protein microassay.
Statistical Analysis--
Each experimental point was performed
in duplicate. All data are given as means ± S.E. of three to six
independent experiments. To determine the statistical significance of
the described results, analysis of variance with Bonferroni's
correction for multiple comparisons or Student's t test for
unpaired data were performed. A p value of <0.05 was
accepted as statistically significant.
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RESULTS |
Effect of Ascorbic Acid on Citrulline and cGMP Formation in
Endothelial Cells--
NO production upon endothelial cell stimulation
is accompanied by an increased synthesis of citrulline which is
produced stoichiometrically with NO, and by an accumulation of
intracellular cGMP which is generated when NO activates the soluble
guanylate cyclase of the cells. Accordingly, both parameters indicate
the formation of NO. Stimulation of HUVEC with ionomycin led to a
citrulline formation of 37 ± 4 pmol/mg cellular protein
(n = 6). 24 h pretreatment of HUVEC with 0.1-100
µM ascorbic acid potentiated ionomycin-stimulated citrulline production in a dose-dependent manner up to
73 ± 19 pmol/mg (Fig. 1). Similar
results were obtained when HUVEC were stimulated with thrombin although
the thrombin response was generally lower. Additionally, ascorbic acid
potentiated ionomycin-induced citrulline formation in CAEC up to
2.3-fold (Fig. 1). The agonist-induced citrulline formation in both
untreated and ascorbic acid-pretreated endothelial cells was completely
inhibited when cells were preincubated with 1 mM of the NOS
inhibitor L-NAME 30 min before stimulation. The basal
citrulline synthesis was generally lower than 5 pmol/mg protein and was
not enhanced by ascorbate incubation.

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Fig. 1.
Influence of ascorbic acid on agonist-induced
citrulline formation in endothelial cells. HUVEC or CAEC were
preincubated for 24 h with 0.1-100 µM ascorbic acid
in culture medium. Subsequently, cells were stimulated for 15 min with
ionomycin or thrombin in Hepes buffer (pH 7.4) containing 1.5 mM calcium and 10 µM
L-[3H]arginine (0.33 Ci/mmol). The generated
[3H]citrulline was separated from
[3H]arginine by cation exchange chromatography and
measured by liquid scintillation counting. Data are shown as
agonist-induced citrulline production calculated from the difference in
radioactivity from stimulated and unstimulated cells and expressed in
pmol/mg cell protein (mean ± S.E., n = 6);
untreated and ascorbate-treated cells were compared, *p < 0.05.
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The effect of ascorbic acid on ionomycin-stimulated citrulline
synthesis was saturable since concentrations above 100 µM
did not induce further amplification. 24 h of preincubation of
HUVEC with 100, 200, 500, and 1000 µM ascorbate followed
by a stimulation with ionomycin caused a citrulline production (in
pmol/mg cell protein) of 84 ± 6, 81 ± 8, 88 ± 10, and
86 ± 6, respectively (n = 3).
In addition to citrulline formation, ionomycin and thrombin induced an
accumulation of intracellular cGMP in HUVEC of 5.4 ± 1.3 pmol/mg
cell protein and 2.8 ± 1.3 pmol/mg, respectively (n = 6). 24 h preincubation of HUVEC with 0.1-100
µM ascorbic acid potentiated both ionomycin- and
thrombin-stimulated cGMP formation up to 2.9- and 2.7-fold,
respectively, thus confirming the effects on citrulline production
(Fig. 2). Again, the agonist-stimulated cGMP increase in both untreated and ascorbic acid-preincubated cells
was entirely blocked by a 30-min preincubation with 1 mM L-NAME. Ascorbic acid did not affect the basal cGMP
formation that was 1.9 ± 0.2 pmol/mg in control cells and
2.0 ± 0.4 pmol/mg in cells pretreated with 100 µM
ascorbic acid for 24 h (n = 20).

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Fig. 2.
Influence of ascorbic acid on agonist-induced
cGMP formation in endothelial cells. HUVEC were preincubated for
24 h with 0.1-100 µM ascorbic acid in culture
medium. Subsequently, cells were stimulated for 15 min with ionomycin
or thrombin in M199 containing 0.5 mM
isobutylmethylxanthine (30 min preincubation). The accumulated cGMP was
measured in cellular extracts by radioimmunoassay. Data are shown as
agonist-induced cGMP production calculated from the difference in cGMP
content from stimulated and unstimulated cells and expressed in pmol/mg
cell protein (mean ± S.E., n = 6); untreated and
ascorbate-treated cells were compared, *p < 0.05.
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Time Dependence of the Ascorbic Acid Effect--
Preincubation of
HUVEC with 100 µM L-ascorbic acid for
different periods followed by stimulation with ionomycin revealed that the potentiating effect on citrulline and cGMP formation was related to
the incubation time. A maximal amplification of ionomycin-induced citrulline and cGMP production was achieved after an 18-h pretreatment (Fig. 3). After a 6-h preincubation, 46 and 30% of the maximal effects on citrulline and cGMP synthesis,
respectively, were measured, whereas a 1-h treatment with ascorbic acid
did not cause any potentiation of calcium-dependent
citrulline and cGMP formation (Fig. 3).

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Fig. 3.
Time dependence of the ascorbic acid effect
on ionomycin-induced citrulline and cGMP formation. HUVEC were
preincubated with 100 µM ascorbic acid for the indicated
times, stimulated with 2 µM ionomycin for 15 min, washed,
and processed for either citrulline or cGMP measurement. Data are shown
as agonist-induced increases in citrulline or cGMP production
calculated from the differences between stimulated and unstimulated
cells (mean ± S.E., n = 4;, *p < 0.05 versus untreated control cells.
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Transport of Ascorbic Acid into Endothelial Cells--
To measure
the [14C]ascorbic acid uptake into HUVEC, cells were
incubated with 100 µM [14C]ascorbate (16 mCi/mmol) for 1-24 h. About 17, 36, 47, and 75% of the maximal
ascorbic acid uptake were achieved after 1, 2, 3, and 6 h,
respectively, while saturation occurred between 12 and 24 h (Fig.
4). At 24 h the cellular ascorbic
acid level calculated from the specific radioactivity of the added
compound was 21.5 ± 3.7 nmol/mg protein assuming that ascorbate
in non-supplemented cells was negligible (27).

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Fig. 4.
Uptake of [14C]ascorbic acid
into endothelial cells. HUVEC were preincubated with 100 µM [14C]ascorbic acid (16 mCi/mmol) for the
indicated times, washed, and subsequently solubilized. The
radioactivity of cell lysates was measured by liquid scintillation
counting. Data are shown as counts/min of incorporated
[14C]ascorbic acid per mg of cell protein (mean ± S.E., n = 3). *p < 0.05 versus control values at zero time.
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Effect of Ascorbic Acid Derivatives on Citrulline and cGMP
Formation in Endothelial Cells--
In addition to
L-ascorbic acid, we investigated how the ascorbate
precursor molecule L-gulonolactone, the oxidation product dehydro-L-ascorbic acid, as well as the derivatives
ascorbate 2-phosphate and ascorbate 2-sulfate (Fig.
5) influence ionomycin-stimulated NO
production. All compounds were applied to HUVEC cultures at a
concentration of 100 µM and for a preincubation period of
24 h. L-Gulonolactone did not affect either
calcium-dependent citrulline or cGMP synthesis (Fig.
6). Dehydro-L-ascorbic acid
increased ionomycin-stimulated citrulline and cGMP production by 62 and 73%, respectively, and was less effective than L-ascorbic
acid (Fig. 6). Ascorbate 2-phosphate potentiated ionomycin-induced citrulline and cGMP formation to a similar extent as
L-ascorbic acid, whereas ascorbate 2-sulfate, another
derivative with a substituted enediol lactone ring, showed 27 (citrulline) or 20% (cGMP) of the ascorbate effect (Fig. 6).

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Fig. 5.
Molecular structures of
L-gulonolactone, L-ascorbic acid, ascorbate
2-phosphate, ascorbate 2-sulfate, and dehydro-L-ascorbic
acid.
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Fig. 6.
Comparison of the effects of ascorbic acid,
dehydroascorbic acid, L-gulonolactone, ascorbate
2-phosphate, and ascorbate 2-sulfate on ionomycin-induced citrulline
and cGMP formation. HUVEC were preincubated for 24 h with 100 µM of the respective compounds, stimulated for 15 min
with 2 µM ionomycin, and processed for either citrulline
or cGMP measurement. Data are shown as agonist-induced increases in
citrulline or cGMP production calculated from the differences between
stimulated and unstimulated cells (mean ± S.E., n = 4); *p < 0.05 versus untreated control
cells.
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Effect of Ascorbic Acid on Arginine Uptake into Endothelial
Cells--
To determine if ascorbic acid affects the transport of the
NOS substrate arginine into the cells, HUVEC were incubated in culture
medium containing 335 µM
L-[14C]arginine (3 mCi/mmol) in the presence
or absence of 100 µM ascorbate for 1-24 h.
Table I shows that the
[14C]arginine uptake was time-dependent but
was not modified by ascorbic acid.
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Table I
Effect of ascorbic acid on arginine uptake into endothelial cells
HUVEC were incubated in culture medium containing 335 µM
L-[14C]arginine (3 mCi/mmol) in the presence or absence
of 100 µM ascorbic acid for the indicated times. The
radioactivity of cell lysates was measured by liquid scintillation
counting. Control values were obtained by adding and immediately
removing the [14C]arginine medium. Data are shown as cpm of
incorporated [14C]arginine per mg of cell protein (mean ± S.E., n = 3).
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Effect of Ascorbic Acid on ecNOS Protein Content in Endothelial
Cells--
To determine ecNOS expression, cell lysates and subcellular
fractions from untreated and ascorbate (100 µM, 24 h)-treated HUVEC were separated by SDS-PAGE and subjected to Western
blot analysis using an anti-ecNOS antibody. Fig.
7 shows that the ecNOS was mainly located
in the particulate fraction. Differences in ecNOS expression were not
seen in whole lysates or in membrane or cytosolic fractions between
untreated and ascorbic acid-incubated cells. Western blots from control
cells and ascorbate-treated cells were also labeled with a monoclonal
antibody against iNOS, but no staining could be detected (not
shown).

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Fig. 7.
Western blot analysis of NO synthase
protein. HUVEC were preincubated for 24 h in the absence or
presence of 100 µM ascorbic acid and detached with
trypsin/EDTA. Intact cells as well as subcellular fractions were
solubilized with Laemmli buffer. SDS-PAGE was performed on 7% gels (50 µg of protein/lane). Western blotting analysis was accomplished using
a monoclonal antibody against human ecNOS. One typical experiment out
of four is shown. C, control; AA, ascorbic
acid.
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The expression of ecNOS protein was also measured in permeabilized
HUVEC using a flow cytometric technique. The comparison of cellular
fluorescence signals with signals from calibration beads revealed about
105 molecules ecNOS per cell. The amount of ecNOS was not
changed when HUVEC were incubated with 100 µM ascorbic
acid for 24 h as the corrected mean fluorescence intensity was
14.5 ± 2.2 in control cells and 14.6 ± 2.3 in
ascorbate-treated cells (n = 10).
Effect of Ascorbic Acid on de Novo Synthesis of ecNOS
Protein--
To investigate if ascorbic acid leads to an enhanced
de novo synthesis of ecNOS protein in HUVEC, lysates from
biosynthetically labeled control cells and cells treated with 100 µM ascorbate (6 h preincubation and 3 h coincubation
with [35S]methionine medium) were immunoprecipitated with
an anti-human ecNOS antibody, resolved by SDS-PAGE, and subjected to
autoradiography and subsequent immunostaining. Fig.
8 shows that the 35S-labeled
band running at 135 kDa was specific for ecNOS and was not different
between control and ascorbate-pretreated cells suggesting that ecNOS
synthesis was not altered by ascorbic acid.

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Fig. 8.
Effect of ascorbic acid on de novo
synthesis of NO synthase protein. HUVEC were incubated with
100 µM ascorbic acid for 6 h and subsequently
incubated for 3 h with 50 µCi of Tran35S-label
reagent in methionine-free RPMI medium containing 100 µM
ascorbate. Labeled cells were immunoprecipitated with a monoclonal
antibody against human endothelial NO synthase (ecNOS). The
immunoprecipitates were separated by SDS-PAGE on 7% gels, blotted onto
nitrocellulose membranes, and subjected for 2-10 days to
autoradiography and subsequent immunostaining. One typical experiment
out of three is shown. C, control; AA, ascorbic
acid.
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Effect of Ascorbic Acid on Citrulline Formation in Endothelial Cell
Lysates--
When the calcium-dependent citrulline
formation was analyzed in cell lysates under optimal conditions and
with the addition of all known NOS cofactors, the potentiating effect
of ascorbate pretreatment (100 µM, 24 h) as seen in
intact cells was largely not observed (Fig.
9). This was most probably due to the
addition of tetrahydrobiopterin since the measurement in the presence
of all cofactors except tetrahydrobiopterin revealed a 2.5-fold higher citrulline formation in lysates from ascorbate-pretreated cells (Fig.
9). Furthermore, the potentiating effect of ascorbic acid was not
observed in reactions where tetrahydrobiopterin was present, but other
cofactors (FAD, FMN, and calmodulin) were omitted (results not shown).
The citrulline formation in lysates from both untreated and ascorbic
acid-preincubated cells was completely blocked by the addition of 1 mM L-NAME.

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Fig. 9.
Effect of ascorbic acid on citrulline
formation in cell lysates. HUVEC were preincubated for 24 h
with 100 µM ascorbic acid and subsequently trypsinized
and sonicated. Citrulline formation was measured in Hepes buffer (pH
7.2) with 1 mg/ml lysate protein, 20 µM
L-[14C]arginine (303 mCi/mmol), 1 µM FAD, 1 µM FMN, 100 nM
calmodulin, 2 mM NADPH, and 2.5 mM
CaCl2 in the presence (left panel) or absence
(right panel, note the different scale units of the
x axis) of 3 µM tetrahydrobiopterin
(BH4). After 15 min at 37 °C, the mixture was subjected
to cation exchange chromatography, and [3H]citrulline was
quantified by liquid scintillation counting. The
calcium-dependent citrulline formation was expressed in
pmol/mg lysate protein (mean ± S.E., n = 6).
C, control; AA, ascorbic acid. *p < 0.05 versus untreated control cells.
|
|
 |
DISCUSSION |
The present study demonstrates that L-ascorbic acid in
physiologically relevant concentrations (28, 29) potentiates
agonist-induced endothelial NO synthesis in a dose- and
time-dependent fashion. This was shown by concomitant
changes of both the formation of the NO co-product citrulline and the
accumulation of the NO effector molecule cGMP, although the latter
might additionally indicate an increase in biological activity of NO.
The ascorbate effect on NO synthesis was most likely due to an increase
of the intracellular ascorbic acid concentration since cell stimulation
was performed in the absence of extracellular ascorbate. Under normal
culture conditions cells are unlikely to be saturated with ascorbic
acid because its concentration in culture media is generally low.
Accordingly, an incubation of the cells with ascorbate may lead to an
intracellular accumulation of the compound. Indeed, using 100 µM 14C-labeled ascorbate we found an uptake
of the compound into endothelial cells which was
time-dependent and saturated between 12 and 24 h. The
nature of this uptake is as yet unknown, although the involvement of an
active transport mechanism has been demonstrated in endothelial cells
(27).
We suggest that the observed rate of ascorbic acid transport into
endothelial cells demonstrated in the present study may at least
partially account for the time dependence of the ascorbate effect on
endothelial NO synthesis. The latter followed a kinetics similar to the
ascorbate uptake being about half-maximal after a 6-h incubation of
cells with 100 µM ascorbic acid and maximal between 18 and 24 h. Moreover, a saturation of the ascorbate uptake into
endothelial cells which might occur between 100 and 200 µM (27) could explain the lack of further NO synthesis
potentiation with ascorbic acid concentrations above 100 µM. Interestingly, 100 µM is in the range
of plasma levels of healthy individuals (28), and similar
concentrations have been found in a recent pharmacokinetic study that
described the ascorbic acid concentration as a function of dose in
healthy volunteers (29).
The molecular structure of L-ascorbic acid consists of an
unsaturated
-lactone ring with an enediol configuration (-COH=COH) conjugated with a carbonyl group (Fig. 5). Our data suggest that this
structure may be essential for the potentiating effect of ascorbate on
NO production. L-Gulonolactone, an ascorbic acid precursor
molecule, is lacking the enediol configuration and cannot be
transformed into ascorbic acid in human cells due to the absence of the
enzyme L-gulonolactone oxidase. Accordingly, it did not affect ionomycin-induced citrulline or cGMP synthesis. On the other
hand, dehydroascorbic acid which is partially converted back to
ascorbate by glutathione-dependent reactions (30) exerted a
partial stimulatory effect, and ascorbate 2-phosphate, known to be
hydrolyzed by phosphatases to restore the unsubstituted enediol ring
structure (31), was as active as ascorbate itself in potentiating NO
synthesis. Similarly, ascorbate 2-sulfate exerted a stimulatory action
which was, however, less than 30% of the ascorbate effect. This might
be due to a lack of the hydrolyzing enzyme arylsulfatase A (32) and
corresponds to studies that have shown that the ascorbic acid activity
of ascorbate 2-sulfate was low in humans (33).
Although ascorbic acid clearly enhanced agonist-induced NO
production in endothelial cells, it did not induce the expression of
ecNOS protein. The amount of ecNOS recognized with specific monoclonal
antibodies in cell lysates or in permeabilized cells was not altered by
ascorbate pretreatment. Likewise, ascorbic acid did not induce de
novo synthesis of NOS since the 35S-labeled protein
band which could be clearly identified as ecNOS by immunostaining was
not different in untreated and ascorbate-treated cells. The
potentiating effect of ascorbic acid on NO synthesis was also not due
to a supplementation of the NOS substrate L-arginine because ascorbic acid did not increase the cellular uptake of this
amino acid. Since the endothelial NO formation is influenced by the
intracellular amount of cofactors such as NADPH, FAD, FMN, tetrahydrobiopterin, and calcium/calmodulin (34), we suggested that
ascorbate might act through effects on the availability of these
compounds. Indeed, when the NO formation was determined in cell lysates
from control cells and ascorbate-pretreated cells under saturated
conditions and in the presence of all NOS cofactors, the potentiating
effect of ascorbic acid was largely lost. Interestingly, this was most
probably due to the addition of tetrahydrobiopterin since a
potentiating effect of an ascorbate pretreatment was seen in cell
lysates when only tetrahydrobiopterin was omitted from the assay.
Moreover, the addition of tetrahydrobiopterin reversed the ascorbate
effect in the absence of the cofactors FAD, FMN, or calmodulin, and
when tetrahydrobiopterin was omitted, the potentiating effect of an
ascorbate pretreatment on citrulline synthesis could be seen regardless
whether FAD, FMN, and calmodulin were added to the cell lysates (data
not shown). From these results we can speculate that ascorbic acid may
at least partially act through an effect on the availability of
tetrahydrobiopterin or enhance the affinity of this cofactor toward
ecNOS. An increase of intracellular tetrahydrobiopterin levels has
already been shown to stimulate the constitutive NOS activity
suggesting that the ecNOS is incompletely saturated with this cofactor
(35, 36). Tetrahydrobiopterin is thought to stabilize the active
homodimeric NOS (37) and probably acts as both a redox-active cofactor
of L-arginine oxidation and an allosteric effector of the
enzyme protein (38). The possibility that ascorbic acid may enhance the
apparent affinity of tetrahydrobiopterin for the neuronal NOS
isoform has also been suggested (39).
So far, protective vascular effects of ascorbic acid have been
attributed to its radical scavenging properties which may lead to a
protection of NO from inactivation by superoxide anion and to an
increase of NO bioavailability. These mechanisms may be important
in vivo and may explain the improvement of
endothelium-dependent vasodilation in cardiovascular patients
by an acute ascorbic acid application (18-23). The results presented
in this study describe a new mechanism for vascular protection by
ascorbic acid which may be effective when plasma levels of ascorbic
acid supply saturated intracellular ascorbate concentrations. We
suggest that tissue saturation with ascorbic acid provides the optimal
reaction conditions for adequate NO synthesis in endothelial cells and
that a decrease in the cellular ascorbic acid content may support the
development of endothelial dysfunction. Future clinical studies
investigating ascorbate effects on endothelial functions should thus
include parameters that evaluate NO formation in vivo.
In summary, this study demonstrates that L-ascorbic acid
potentiates agonist-induced NO formation in cultured endothelial cells
in a dose-dependent fashion. The effect was
time-dependent, related to an increase of intracellular
ascorbate levels, and saturated within physiologically relevant
concentrations. Ascorbic acid did not induce the expression of ecNOS
protein and may at least partially act through an effect on the
availability or affinity of tetrahydrobiopterin for ecNOS. Further
studies are in progress to clarify these suggested mechanisms. The
findings presented in this study suggest that NO formation in
endothelial cells depends on tissue ascorbate levels and that tissue
saturation with ascorbic acid may add to the strategies for an
improvement of endothelial vasodilator function in humans.