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INTRODUCTION |
Endothelium-derived nitric oxide (NO) is a potent signaling
molecule in the cardiovascular system participating in many processes such as vascular relaxation, inhibition of platelet aggregation, regulation of endothelial cell adhesivity, and preservation of the
normal vessel wall structure (1). NO is generated from the conversion
of L-arginine to L-citrulline by the enzymatic action of an NADPH-dependent NO synthase
(NOS)1 that requires
Ca2+/calmodulin, FAD, FMN, and tetrahydrobiopterin as
cofactors (2). The endothelial NOS isoform (eNOS) is constitutively
expressed and activated upon an increase of intracellular
Ca2+ following cell stimulation with agonists such as
thrombin and bradykinin or through serine phosphorylation subsequent to
cell stimulation with shear stress or insulin (3, 4).
Evidence is accumulating that NO determines the antiatherosclerotic
properties of the endothelium (5). All major risk factors for
atherosclerosis including hypercholesterolemia, hypertension, and
smoking have been associated with impaired vascular NO synthesis (6).
The underlying mechanisms are thought to involve reduced formation of
NO due to a decrease in NOS expression or a limited availability of
L-arginine, as well as increased degradation of NO by
reaction with superoxide anions or oxidized low density lipoproteins
(5, 6). Recent studies indicate that under certain pathological
conditions, decreased availability of tetrahydrobiopterin may also be
responsible for dysfunction of endothelial nitric-oxide synthase. A
close link between cellular tetrahydrobiopterin levels and NO synthesis
was demonstrated for a number of different cell types including
endothelial cells (7-10), suggesting that an optimal concentration of
tetrahydrobiopterin is essential for agonist-induced production of NO.
Furthermore, tetrahydrobiopterin induced vasodilation in isolated
arteries (11-13) and inhibition of tetrahydrobiopterin biosynthesis
impaired endothelium-dependent relaxation in canine basilar
artery (14). Accordingly, tetrahydrobiopterin supplementation was
capable of restoring endothelium-dependent vasodilation in experimental diabetes and reperfusion injury as well as in patients with hypercholesterolemia, coronary artery disease and in cigarette smokers (15-20). Although the reason for a reduced availability of
tetrahydrobiopterin is not clear, it might be related to an impaired
synthesis, to a decreased affinity of the enzyme for its cofactor or to
prolonged oxidative stress. Since tetrahydrobiopterin has profound
effects on the structure of NOS including the ability to shift the heme
iron to its high spin state, the promotion of arginine binding and the
stabilization of the active dimeric form of the enzyme (21), a lack of
this cofactor may decrease NOS activity. There is also increasing
evidence that NOS-bound tetrahydrobiopterin acts as a redox-active
cofactor (22-24), but, unlike aromatic amino acid hydroxylases where
the fully reduced pterin serves as a reductant for oxygen, NOS is not
coupled to the dihydropteridine reductase as a
tetrahydrobiopterin-regenerating system (25). Interestingly, a
decreased availability of tetrahydrobiopterin may cause a shift in the
balance between the production of NO and oxygen free radicals by NOS.
Several biochemical studies indicated that activation of purified eNOS
in the presence of suboptimal levels of tetrahydrobiopterin results in
uncoupling of oxygen reduction and arginine oxidation, thereby
generating superoxide anions and subsequently hydrogen peroxide
(26-28). Thus, deficiency of tetrahydrobiopterin may cause both
impaired NO formation and increased oxygen radical formation with the
latter leading to increased NO inactivation.
Recently, we were able to demonstrate that preincubation of human
endothelial cells from umbilical veins and coronary arteries with
ascorbic acid led to an up to 3-fold increase of agonist-induced NO
synthesis (29). The ascorbate effect was specific, saturated at 100 µM, and was dependent on cellular uptake. Ascorbic acid induced neither eNOS expression nor L-arginine uptake.
Since the potentiating ascorbate effect was minimal, when NOS activity
was measured in lysates of ascorbate-pretreated cells in the presence of exogenous tetrahydrobiopterin, we suggested that ascorbic acid may
either enhance the availability of tetrahydrobiopterin or increase its
affinity for endothelial NOS. The present study was designed to
investigate the mechanisms underlying the effect of ascorbic acid on
endothelial NO synthesis. We demonstrate that ascorbate treatment of
endothelial cells causes an increase of intracellular
tetrahydrobiopterin levels and that this effect is based on a chemical
stabilization of the fully reduced form of the pterin.
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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 (FCS), collagenase, and human serum albumin (HSA) were from BioWhittaker Europe (Verviers, Belgium).
L-[2,3,4,5-3H]Arginine monohydrochloride (61 Ci/mmol), [3H]cGMP Biotrak radioimmunoassay systems, and
NAP-5 columns were purchased from Amersham Pharmacia Biotech (Freiburg,
Germany). Tumor necrosis factor-
(TNF-
) and interferon-
(IFN-
) were from Pharma Biotechnology (Hannover, Germany); NADPH,
tetrahydrobiopterin, sepiapterin and L-nitroarginine
methylester (L-NAME) were obtained from Alexis Corp.
(Läufelfingen, Switzerland); LiChrosphere RP-18 columns were from
Merck (Darmstadt, Germany), Nucleosil® 10 SA columns from
Macherey-Nagel (Düren, Germany) and Duralon-UV membranes from
Stratagene (La Jolla, CA). Endothelial cell growth supplement, FAD,
FMN, calmodulin, ionomycin, EDTA, trypsin/EDTA solution (0.05/0.02%,
v/v), dimethyl sulfoxide (Me2SO), GTP,
2,4-diamino-6-hydroxypyrimidine (DAHP), L-ascorbic acid,
lipopolysaccharide (LPS) from Escherichia coli,
E-TOXATE® kit, and other reagents were purchased from
Sigma (Deisenhofen, Germany).
The GTP-cyclohydrolase I cDNA probe (550 base pairs) was obtained
by polymerase chain reaction using consensus primers to GTP
cyclohydrolase I from E. coli, mouse, and man. The identity with GTP cyclohydrolase I was confirmed by sequencing. Recombinant murine sepiapterin reductase was expressed in E. coli using
a maltose-binding protein fusion expression system kindly provided by
Irmgard Ziegler (Munich, 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.
Cell Cultures--
Human umbilical cord vein endothelial cells
(HUVEC) were prepared with 0.05% collagenase and cultured in
75-cm2 plastic flasks in M199 containing 15% FCS, 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, on
60-mm-diameter dishes for the measurement of citrulline formation, and
on 90-mm-diameter dishes for the investigation of the other parameters.
Experiments were carried out with monolayers of the first to second passage.
Experimental Incubations--
Preincubations of HUVEC with
L-ascorbic acid, sepiapterin, DAHP, and the combination of
TNF-
, IFN-
, and LPS were performed in culture medium for 24 h. To improve the detection of biopterin derivatives in cell
supernatants, in some of the experiments culture medium containing
serum dialyzed against Hanks' balanced salt solution was used. 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. Endotoxin
contamination of ascorbic acid solutions was measured with the
coagulation Limulus amebocyte lysate assay and was proven to
be below the detection limit of the kit (0.05 unit/ml). DAHP was added
to the culture medium to give the indicated final concentrations.
Sepiapterin was dissolved in Me2SO, and stock solutions of
the other compounds were prepared in M199 containing 10% FCS.
Stimulation of cells with the Ca2+ ionophore, ionomycin,
was performed in the absence of ascorbic acid, sepiapterin, or DAHP.
Ionomycin was dissolved at 1 mM in Me2SO and
stored at
20 °C until use. The final concentration of
Me2SO during experimental incubations and cell stimulation 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.
Measurement of Citrulline Synthesis--
Citrulline synthesis
was measured by a modification of a previously described technique
(30). Cell monolayers were incubated at 37 °C for 30 min in 1.5 ml
of Hepes buffer (pH 7.4) containing 0.25% HSA. Subsequently, cells
were stimulated with 2 µM ionomycin in the presence of 10 µM L-arginine and 3.3 µCi/ml
L-[3H]arginine. 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 AG50WX-8
(Na+ form). The radioactivity corresponding to the
[3H]citrulline content of the eluate was quantified by
liquid scintillation counting. Agonist-induced
[3H]citrulline production was calculated from the
difference in radioactivity from ionomycin-stimulated cells and the
corresponding unstimulated cells, and was expressed in femtomoles/mg of
cell protein. Basal [3H]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 and stimulated with 2 µM ionomycin
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) were 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 picomoles/mg of cell protein. The agonist-induced cGMP
production was determined from the difference of cGMP content in
ionomycin-stimulated cells and the corresponding unstimulated cells.
Determination of eNOS Activity--
Experiments were performed
with tetrahydrobiopterin-free eNOS that was expressed in yeast
Pichia pastoris and purified as described (28). The assay
solution (100 µl) contained 50 mM Tris-HCl buffer (pH
7.4), 0.3 µg of eNOS, 100 µM
L-[3H]arginine (100,000 cpm), 0.5 mM CaCl2, 0.2 mM NADPH, 5 µM FAD, 5 µM FMN, 10 µg/ml calmodulin, 10 nM to 100 µM tetrahydrobiopterin, and 0.2 mM CHAPS. Incubations without Ca2+ served as a
blank. The enzyme assay was performed in the presence or absence of 100 µM ascorbic acid. After 10 min 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 AG50WX-8 (Na+ form). The enzyme activity
was calculated from the [3H]citrulline content of the
eluate and expressed in nanomoles/mg/min.
Measurement of Intracellular Biopterin Derivatives--
HUVEC
monolayers were detached with trypsin/EDTA and resuspended in Hepes
buffer (pH 7.4). Aliquots of 2-5 × 106 cells were
centrifuged (500 × g, 6 min), and 100 µl of oxidant solution (0.02 M KI/I2 in 0.1 M HCl
or 0.02 M KI/I2 in 0.1 M NaOH) were
added to the cell pellets. After sonication on ice, aliquots for the
determination of proteins were taken and the homogenates were incubated
for 1 h in the dark at room temperature. Subsequently, 10 µl of
HCl (1 M) were added to samples oxidized in base, the precipitates were removed by centrifugation and excess iodine was
destroyed by the addition of 10 µl of ascorbic acid (0.2 M). Quantification of biopterin in supernatants was
performed as described (31). Briefly, 10 µl of the cell extracts were
injected onto a 250-mm-long, 4-mm inner diameter column filled with
5-µm particles of LiChrosphere RP-18 and protected with a 4-mm-long
precolumn. Biopterin was eluted with 15 mM potassium
phosphate buffer (pH 6.4) at a flow rate of 0.8 ml/min and detected by
fluorescence at excitation of 350 nm and emission of 440 nm using a FP
920 fluorescence detector (Jasco, Tokyo, Japan). Tetrahydrobiopterin degradation products generated by the loss of the side chain at C6
(pterin and isoxanthopterin) were separated on Nucleosil®
10 SA columns, eluted with 50 mM potassium phosphate buffer
(adjusted to pH 2.8 with H3PO4) at a flow rate
of 1.5 ml/min, and detected by fluorescence as described above. The
amount of 5,6,7,8-tetrahydrobiopterin was calculated from the
difference in biopterin concentrations measured after oxidation in acid
(total biopterins) and base (7,8-dihydrobiopterin + biopterin).
Intracellular levels of 7,8-dihydrobiopterin + biopterin were generally
low and not always detectable in non-cytokine-treated cells. Pteridine
levels were expressed in picomoles/mg of cell protein or in
picomoles/90-mm-diameter dish.
Measurement of Biopterin Derivatives in Cell
Supernatants--
Following experimental incubations, cell
supernatants were collected and oxidized with 0.02 M
KI/I2 in 0.1 M HCl or 0.02 M KI/I2 in 0.1 M NaOH to detect total biopterins
or 7,8-dihydrobiopterin + biopterin, respectively. The processing of
oxidized supernatants; the measurement of biopterin, pterin, and
isoxanthopterin; and the calculation of the biopterin derivatives were
performed as described for cell extracts. Additionally, non-oxidized
supernatants were used to determine biopterin. Samples of the
respective culture medium that contained small amounts of serum-derived
biopterin served as blanks. Pteridines released by the cells into the
medium of a 90-mm-diameter dish were expressed in picomoles/dish.
Determination of GTP Cyclohydrolase I mRNA Levels by Northern
Blot Analysis--
Total RNA from HUVEC was extracted according to
Chirgwin et al. (32). RNA was electrophoretically resolved
on 1% agarose, 6% formaldehyde gels and blotted to nylon membranes
(Duralon-UV). After binding of RNA to the membranes by UV irradiation
(Stratalinker; Stratagene, La Jolla, CA), blots were hybridized
overnight with 106 cpm/ml [32P]dCTP-labeled
probe for human GTP cyclohydrolase I at 65 °C according to standard
protocols. Radioactivity was visualized by a PhosphorImager. As a
control, membranes were probed for human glyceraldehyde-3-phosphate dehydrogenase.
Measurement of GTP Cyclohydrolase I Activity--
The GTP
cyclohydrolase I assay was performed as described recently (31). Cell
extracts depleted of membranes were freed from low molecular weight
compounds by NAP-5 columns. The reaction mixture consisted of 50 mM Tris-HCl (pH 7.8) containing 0.3 M KCl, 2.5 mM EDTA, and 10% (v/v) glycerol, ~1-4 mg/ml cytosolic protein, and 2 mM GTP in a total volume of 300 µl. In
some experiments 100 µM ascorbic acid was added to the
test. After incubation for 1 h at 37 °C in the dark, the
reaction was terminated by adding 10 µl 1 M HCl.
Subsequently, the 7,8-dihydroneopterin triphosphate formed was oxidized
to neopterin triphosphate by the addition of 10 µl of 0.1 M I2 solubilized in 0.25 M KI.
After 1 h in the dark, the precipitate was removed by
centrifugation and 10 µl of ascorbic acid (0.1 M) were
added to destroy excess iodine in the supernatant. The mixture was
neutralized with NaOH, and the phosphates were cleaved by alkaline
phosphatase (10 units/assay). The resulting neopterin was then
quantified by reversed phase HPLC with fluorescence detection as
described above for biopterin. GTP cyclohydrolase I activities were
expressed in picomoles of neopterin/mg of cytosolic protein/min.
Measurement of 6-Pyruvoyl-tetrahydropterin Synthase
Activity--
The enzyme assay was performed as described (31). 100 µl of the following mixture were incubated for 1 h at 37 °C:
100 mM Tris-HCl (pH 7.4), 20 mM
MgCl2, 2 mM NADPH, 2 milliunits of sepiapterin reductase, 40 µM 7,8-dihydroneopterin triphosphate
(enzymatically prepared from GTP using recombinant GTP cyclohydrolase
I), ~1 mg of cytosolic protein freed from low molecular weight
compounds by NAP-5 columns. Subsequently, the tetrahydrobiopterin
formed was oxidized by the addition of 5 µl of 1 M HCl
and 5 µl of 0.1 mM I2 dissolved in 0.25 mM KI for 1 h in the dark. The precipitates were then
removed by centrifugation, and excessive iodine was destroyed by the
addition of 10 µl of 0.1 M ascorbic acid. Biopterin thus
formed was quantified by reversed phase HPLC as described above. The
activity of 6-pyruvoyl-tetrahydropterin synthase was expressed in
picomoles of biopterin/mg of cytosolic protein/min.
Measurement of Tetrahydrobiopterin in Aqueous Solution--
To
measure tetrahydrobiopterin stability in aqueous solution, the
pteridine was added to PBS and incubated at room temperature. After
various times aliquots were taken and oxidized with 0.01 KI/I2 in 0.1 M HCl or 0.1 M NaOH.
The measurement of biopterin levels and the calculation of
tetrahydrobiopterin concentration were performed as described above.
Protein Determination--
After lysing the HUVEC monolayers or
cell homogenates with solubilization buffer (100 mM NaOH,
2% Na2CO3, and 1% SDS), proteins were
measured according to Lowry using the Bio-Rad DC protein microassay and
bovine serum albumin as standard. Protein determination in cytosolic
fractions was performed by the Bradford method applying the same standard.
Statistical Analysis--
Each experimental point was performed
in duplicate (citrulline, cGMP) or triplicate (pteridines). All data
are given as means ± S.E. of three to five 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 paired data
were performed. A p value of < 0.05 was accepted as
statistically significant.
 |
RESULTS |
Effect of Sepiapterin on Ascorbic Acid-induced Potentiation of
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. Preincubation of the
cells with ascorbic acid (100 µM, 24 h) led to a
2.7-fold increase in ionomycin-triggered citrulline formation and a
2.8-fold potentiation of Ca2+-dependent cGMP
accumulation (Fig. 1) thus confirming the
results of a recent study reported by our group (29). The effects of ascorbate were mimicked by pretreatment of the cells with increasing concentrations of sepiapterin (0.001-10 µM, 24 h;
Fig. 1), which is intracellularly converted into tetrahydrobiopterin
via a salvage pathway (33). When HUVEC were coincubated with ascorbic
acid (100 µM, 24 h) and sepiapterin (0.001-10
µM, 24 h), a decrease of the ascorbic acid-mediated
potentiation of ionomycin-induced citrulline and cGMP formation
occurred that was dependent on the sepiapterin concentration. The
ascorbic acid effect was minimal in cells coincubated with 10 µM sepiapterin and 100 µM ascorbate (Fig.
1). Both ionomycin-stimulated citrulline and cGMP production in
untreated HUVEC and cells preincubated with ascorbic acid and/or sepiapterin were entirely blocked by a 30-min preincubation with 1 mM of the NOS inhibitor L-NAME (data not
shown).

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Fig. 1.
Influence of sepiapterin on ascorbic
acid-induced potentiation of citrulline and cGMP formation. HUVEC
were preincubated for 24 h with 0.001-10 µM
sepiapterin in culture medium in the absence or presence of 100 µM ascorbic acid. Subsequently, cells were stimulated in
Hepes buffer (pH 7.4) for 15 min with 2 µM ionomycin and
processed for either citrulline or cGMP measurement. Data are shown as
agonist-induced [3H]citrulline formation or cGMP
production calculated from the differences between stimulated and
unstimulated cells and expressed in femtomoles/mg or picomoles/mg of
cell protein, respectively (mean ± S.E., n = 4);
cells with and without ascorbate pretreatment were compared (*,
p < 0.05).
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Effect of Ascorbic Acid on the Activation of Purified eNOS by
Tetrahydrobiopterin--
Tetrahydrobiopterin-free eNOS was inactive in
the absence of exogenous tetrahydrobiopterin. The pteridine (1 nM to 100 µM) stimulated the formation of
citrulline in a concentration-dependent manner with an
EC50 of 0.31 ± 0.036 µM and a maximal
effect at about 100 µM (Fig.
2). The presence of 100 µM
ascorbic acid in the assay solution resulted in a slight decrease of
the EC50 to 0.16 ± 0.014 µM
(n = 3, p < 0.05) without significant
increase in maximal enzyme activity. Ascorbate had no effect on eNOS
activity in the absence of exogenous tetrahydrobiopterin.

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Fig. 2.
Influence of ascorbic acid on
tetrahydrobiopterin dependence of eNOS activity. Enzyme assays
were performed in the absence or presence of 100 µM
ascorbate with tetrahydrobiopterin-free recombinant eNOS purified from
the yeast P. pastoris. The assay solution contained 50 mM Tris-HCl buffer (pH 7.4), 0.3 µg/100 µl of eNOS, 100 µM L-[3H]arginine (100,000 cpm), 0.5 mM CaCl2, 0.2 mM NADPH, 5 µM FAD, 5 µM FMN, 10 µg/ml calmodulin, 10 nM to 100 µM tetrahydrobiopterin, and 0.2 mM CHAPS. After 10 min at 37 °C, the mixture was
subjected to cation exchange chromatography and
[3H]citrulline was quantified by liquid scintillation
counting. The Ca2+-dependent citrulline
formation was expressed in nanomoles/mg/min (mean ± S.E.,
n = 3).
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Effect of Ascorbic Acid on Intracellular Tetrahydrobiopterin
Levels--
The preincubation of HUVEC with ascorbic acid (100 µM, 24 h) led to an increase of intracellular
tetrahydrobiopterin levels from 0.38 ± 0.04 pmol/mg of protein to
1.14 ± 0.09 pmol/mg (n = 20). The effect of
ascorbic acid was concentration-dependent between 1 and 100 µM and saturable since concentrations above 100 µM did not induce a further increase of
tetrahydrobiopterin levels (Fig. 3). In
contrast, the effect of 1 mM ascorbic acid was somewhat
lower (
9.3% ± 2.72% compared with the effect of 100 µM, n = 5, not significant), suggesting
that at unphysiologically high concentrations additional, possibly
pro-oxidant effects of ascorbate might interfere with the mechanisms
leading to increased levels of tetrahydrobiopterin.

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Fig. 3.
Effect of ascorbic acid on intracellular
tetrahydrobiopterin levels. HUVEC were preincubated for 24 h
with 1 µM to 1 mM ascorbic acid in culture
medium. Aliquots of 5 × 106 cells were oxidized with
0.02 M KI/I2 in 0.1 M HCl or 0.1 M NaOH, and the resulting biopterin was quantified by
reversed phase HPLC. Tetrahydrobiopterin was calculated from the
difference in biopterin concentration after oxidation in acid and base
and expressed in picomoles/mg of protein. Data are shown as mean ± S.E. from five 5 experiments. *, p < 0.05 versus untreated control cells.
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Effect of Ascorbic Acid on the Expression and Activity of GTP
Cyclohydrolase I--
To investigate whether the ascorbic
acid-mediated increase of intracellular tetrahydrobiopterin levels was
due to increased pterin biosynthesis, we performed studies on the
expression and activity of GTP cyclohydrolase I, the first and
rate-limiting enzyme of pteridine synthesis. Since the expression of
GTP cyclohydrolase I in HUVEC is generally low, experiments were
carried out with cells pretreated without and with cytokines to induce
enzyme expression. Fig. 4 shows the
effects of cytokines (250 units/ml TNF-
, 250 units/ml IFN-
, and 1 µg/ml LPS, 24 h) and ascorbate (100 µM, 24 h)
on GTP cyclohydrolase I mRNA expression. In accordance with previous reports (34, 35), cytokines had a profound effect on GTP
cyclohydrolase mRNA levels, but no differences were seen between
ascorbate-treated cells and their respective controls.

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Fig. 4.
Effect of ascorbic acid on GTP cyclohydrolase
mRNA expression. Total RNA was harvested from HUVEC
preincubated for 24 h with 100 µM ascorbic acid in
the absence or presence of a mixture of cytokines (250 units/ml
TNF- , 250 units/ml IFN- ) and LPS (1 µg/ml). After
electrophoresis on 1% agarose, 6% formaldehyde gels (20 µg/lane)
the RNA was blotted on nylon membranes and hybridized overnight with
[32P]dCTP-labeled probes for human GTP cyclohydrolase I
(GTP-CH I) and human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). One typical experiment out of three
is shown.
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The GTP cyclohydrolase I activity in cytosolic fractions from
cytokine-treated HUVEC was 0.52 ± 0.05 pmol/mg/min
(n = 3). The addition of 100 µM ascorbic
acid to the assay had no effect on enzyme activity (0.54 ± 0.04 pmol/mg/min), suggesting that the compound does not act as a direct
cofactor of the enzyme. The GTP cyclohydrolase activity of cytosolic
fractions of non-cytokine-treated HUVEC was below the detection limit
of the method (0.02 pmol/mg/min) and was not increased to detectable
levels by 100 µM ascorbate, added either to the cell
culture media 24 h prior to measurements or added directly to the
enzyme assays (data not shown).
Effect of Ascorbic Acid on the Activity of
6-Pyruvoyl-tetrahydropterin Synthase--
We also studied the effect
of ascorbic acid on the activity of 6-pyruvoyl-tetrahydropterin
synthase, the second enzyme in the de novo synthesis of
tetrahydrobiopterin. Enzyme activity was 1.53 ± 0.13 pmol/mg/min
in control cells and not changed when HUVEC were preincubated with 100 µM ascorbic acid for 24 h (1.67 ± 0.24 pmol/mg/min, n = 4). When ascorbic acid was added to
6-pyruvoyl-tetrahydropterin synthase incubations of cellular extracts
or recombinant enzymes, no increase of tetrahydrobiopterin levels was
observed (data not shown).
Effect of GTP Cyclohydrolase I Inhibition on Ascorbic Acid-mediated
Increase of Tetrahydrobiopterin Levels and eNOS Activity--
The GTP
cyclohydrolase I inhibitor DAHP inhibits de novo synthesis
of tetrahydrobiopterin by acting as an analogue of the first pyrimidine
intermediate formed in the GTP cyclohydrolase reaction (36).
Accordingly, preincubation of HUVEC with DAHP (1 mM,
24 h) caused a decrease of intracellular tetrahydrobiopterin levels from 0.61 ± 0.04 pmol/mg of protein to 0.13 ± 0.03 pmol/mg (n = 4) (Fig. 5).
In parallel, ionomycin-stimulated formation of citrulline and cGMP was
decreased upon pretreatment of the cells with DAHP (0.5-1
mM, 24 h) to 22% and 12% of controls, respectively (n = 4). DAHP also reduced tetrahydrobiopterin levels
as well as citrulline and cGMP formation in the presence of 100 µM ascorbate. However, the potentiating effect of
ascorbate was increased rather than decreased under these conditions,
i.e. at limited cellular availability of tetrahydrobiopterin
(Fig. 5).

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Fig. 5.
Influence of DAHP on ascorbic acid-mediated
increase of tetrahydrobiopterin levels and potentiation of citrulline
and cGMP formation. HUVEC were preincubated for 24 h with
0.5-1.0 mM DAHP in culture medium in the absence or
presence of 100 µM ascorbic acid. Subsequently, cells
were stimulated for 15 min in Hepes buffer (pH 7.4) with 2 µM ionomycin and processed for either citrulline or cGMP
measurement. Agonist-induced [3H]citrulline formation or
cGMP production were calculated from the differences between stimulated
and unstimulated cells and expressed in femtomoles/mg or picomoles/mg
of cell protein, respectively. Additionally, biopterin levels were
measured by reversed phase HPLC after oxidation of cells with 0.02 M KI/I2 in 0.1 M HCl or 0.1 M NaOH and tetrahydrobiopterin was calculated from the
difference. Data are shown as mean ± S.E. (n = 4). Controls and DAHP-treated cells (+) and cells with and without
ascorbate pretreatment (*) were compared (+ and *, p < 0.05).
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Effect of Ascorbic Acid on Tetrahydrobiopterin Stability in Aqueous
Solution--
To investigate whether ascorbic acid increased
tetrahydrobiopterin stability, 100 nM of the compound were
incubated at room temperature in PBS in the absence or presence of 100 µM ascorbic acid and pteridine levels were determined at
various times points. Fig. 6 shows that
incubation of tetrahydrobiopterin in aerobic buffer solution led to a
decay of the pterin below detectable concentrations within 20 min
suggesting that the half-life of tetrahydrobiopterin was
10 min. In
the presence of ascorbate the apparent half-life of tetrahydrobiopterin
was increased to approximately 70 min.

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Fig. 6.
Effect of ascorbic acid on
tetrahydrobiopterin stability in aqueous solution. 100 nM tetrahydrobiopterin were added to phosphate buffered
saline (pH 7.4) (PBS) and incubated at room temperature in the absence
or presence of 100 µM ascorbic acid. At the indicated
times, aliquots were oxidized with 0.01 M KI/I2
in 0.1 M HCl or 0.1 M NaOH and
tetrahydrobiopterin was calculated from the difference. Data are
expressed as percentage of values measured at zero time (mean ± S.E., n = 3).
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Effect of Ascorbic Acid on Tetrahydrobiopterin Stability in Intact
Cells--
5,6,7,8-Tetrahydrobiopterin is intracellularly oxidized to
the quinonoid 6,7-[8H]dihydrobiopterin, which spontaneously
rearranges to 7,8-dihydrobiopterin (37). The latter is further degraded to biopterin. Additionally, tetrahydrobiopterin can lose the side chain
at C6, thereby generating other pterins such as pterin and isoxanthopterin. To investigate a stabilizing effect of ascorbic acid
on the fully reduced biopterin in intact cells, the levels of
tetrahydrobiopterin and its derivatives in cells and cell supernatants were measured and balanced on the basis of picomoles of
pteridines/dish. The experiments were performed in
cytokine-preincubated cells (250 units/ml TNF-
, 250 units/ml
IFN-
, and 1 µg/ml LPS, 24 h) to increase the expression of
the GTP cyclohydrolase I and thus pteridine production.
Intracellular tetrahydrobiopterin and 7,8-dihydrobiopterin + biopterin
levels in HUVEC preincubated with the cytokine mixture were 30 ± 3.5 pmol/dish and 12 ± 1.8 pmol/dish, respectively (Fig. 7). Ascorbic acid (1-100
µM, 24 h) added to HUVEC simultaneously with the
cytokines caused a concentration-dependent increase of intracellular tetrahydrobiopterin levels up to 135 ± 16 pmol/dish and a decrease of dihydrobiopterin + biopterin levels down to 7 ± 1.1 pmol/dish (Fig. 7). Control HUVEC did not release
tetrahydrobiopterin but a considerable amount of 7,8-dihydrobiopterin
(100 ± 23 pmol/dish) and some biopterin (22 ± 5.5 pmol/dish) to the culture medium. Ascorbate (1-100 µM,
24 h) decreased this release to 33 ± 8 pmol/dish 7,8-dihydrobiopterin and 3 ± 2.5 pmol/dish biopterin, whereas tetrahydrobiopterin remained undetectable in culture supernatants. Interestingly, the total amount of biopterin derivatives in cells and
cell supernatants under the different experimental incubations was
maintained (Fig. 7). Intracellular levels of pterin and isoxanthopterin were below 3 pmol/dish and not altered by ascorbate treatment of cells.
Both compounds were not detectable in cell supernatants under the
various conditions described (data not shown).

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Fig. 7.
Effect of ascorbic acid on
tetrahydrobiopterin stability in intact cells. HUVEC were
preincubated for 24 h with 250 units/ml TNF- , 250 units/ml
IFN- , and 1 µg/ml LPS in the absence or presence of 100 µM ascorbic acid. Aliquots of 2 × 106
cells and 1-ml aliquots of cell supernatants were oxidized with 0.02 M KI/I2 in 0.1 M HCl or 0.1 M NaOH, and the resulting biopterin was quantified by
reversed phase HPLC. Biopterin levels after oxidation in base indicate
the amount of dihydrobiopterin + biopterin, whereas tetrahydrobiopterin
was calculated from the difference in biopterin concentration after
oxidation in acid and base. In order to balance biopterin derivatives
in cells and medium, pteridine levels were calculated in
picomoles/dish. Data are shown as mean ± S.E. from three
experiments. *, p < 0.05 versus levels of
biopterin derivatives in untreated control cells.
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DISCUSSION |
The present study demonstrates that the ascorbic acid-induced
potentiation of endothelial NO synthesis that has been described in a
previous paper (29) is due to an increase of intracellular tetrahydrobiopterin levels. Since ascorbic acid had only a marginal effect on the tetrahydrobiopterin concentration required for
half-maximal stimulation of recombinant eNOS, our data also suggest
that ascorbate does not modify the pterin affinity of the enzyme. The
effect of ascorbic acid on endothelial tetrahydrobiopterin levels was concentration-dependent in the physiological range and
saturated at 100 µM, corresponding to saturation of both
ascorbate uptake (38) and potentiation of NO synthesis (29) and
suggesting that intracellular tetrahydrobiopterin concentration and
thus NO formation are critically dependent on the tissue levels of ascorbate. Tetrahydrobiopterin levels in cultured endothelial cells
have already been shown to be insufficient to allow saturation of eNOS
with its cofactor and optimal NO synthesis (8, 10). This was confirmed
in our study since sepiapterin, which is intracellularly converted to
tetrahydrobiopterin (33), led to an increase of agonist-induced
citrulline and cGMP formation. Additionally, we demonstrated that
sepiapterin abolished the potentiating effect of ascorbic acid on NO
production in a concentration-dependent manner, suggesting that
ascorbate exerts its effect on NO synthesis only under suboptimal
intracellular tetrahydrobiopterin concentrations. From the data
presented here, we can speculate that tetrahydrobiopterin levels
between 1 and 2 pmol/mg of protein provide optimal reaction conditions
for NO formation in HUVEC.
Tetrahydrobiopterin is synthesized de novo from GTP by the
sequential action of three enzymes, GTP cyclohydrolase I,
6-pyruvoyl-tetrahydropterin synthase, and sepiapterin reductase. GTP
cyclohydrolase I has been shown to be the key enzyme of the de
novo pathway and to be regulated by cytokines such as TNF-
,
IFN-
, and interleukin-1
in a number of cell types including
endothelial cells (8, 10, 34). The cytokine-induced elevation of GTP
cyclohydrolase I activity in HUVEC has been related to an increased
transcription rate and an enhanced expression of the mRNA (35).
Likewise, but to a lower extent, 6-pyruvoyl-tetrahydropterin synthase
activity and mRNA abundance have been shown to be regulated by
inflammatory cytokines in HUVEC (39), although other studies have
reported a constitutive expression of the enzyme and no significant
changes upon treatment with these stimuli (8, 40). Our data confirm the
low activity and mRNA expression of GTP cyclohydrolase I in control
HUVEC underlining its rate-limiting role, and the up-regulation of both
parameters by a mixture of cytokines (TNF-
, IFN-
) and LPS.
Ascorbic acid treatment of cells, however, did not alter GTP
cyclohydrolase I mRNA levels nor enzyme activity, regardless whether it was added to the culture medium alone or together with cytokines. 6-Pyruvoyl-tetrahydropterin synthase activities in control
HUVEC were considerably higher than GTP cyclohydrolase I activities,
but were also not changed by preincubation of HUVEC with ascorbic acid.
Furthermore, ascorbate did not act as a direct cofactor of GTP
cyclohydrolase I since no increase in enzyme activity was measured when
the compound was added to the enzyme assay. Taken together, these
results suggest that ascorbic acid effects on intracellular
tetrahydrobiopterin levels are not due to an increased synthesis of the
compound. Accordingly, inhibition of tetrahydrobiopterin formation by
DAHP, an inhibitor of GTP cyclohydrolase I (36), did not prevent the
ascorbate-mediated increase of the pteridine although it substantially
decreased tetrahydrobiopterin levels in both control and ascorbic
acid-treated endothelial cells. Likewise, an inhibition of
Ca2+-dependent NO synthesis by DAHP was seen,
but the potentiating effect of ascorbate on ionomycin-stimulated
citrulline and cGMP formation was maintained.
Since ascorbic acid did not affect tetrahydrobiopterin synthesis, we
speculated that it might act by preventing the degradation of the
compound. Ascorbic acid has already been added to biological fluids to
increase tetrahydrobiopterin stability and to allow storage of the
samples before pteridine measurements (41). Accordingly, we found an
increase in tetrahydrobiopterin half-life when the compound was
dissolved in an aqueous solution in the presence of ascorbate. Our data
additionally demonstrate that ascorbic acid stabilizes
tetrahydrobiopterin in intact endothelial cells. The increase of
tetrahydrobiopterin in cytokine-stimulated cells treated with ascorbate
was paralleled by a decrease of 7,8-dihydrobiopterin and biopterin in
cells and cell supernatants, suggesting that a chemical stabilization
of the fully reduced pterin is the underlying mechanism for its
increased intracellular concentration. Since the total amount of
tetrahydrobiopterin, dihydrobiopterin, and biopterin remained constant
under the different experimental incubations, these data further
underline that ascorbate does not influence pterin formation. The
stabilizing function of ascorbate is most probably due to a chemical
reduction of the quinonoid 6,7-[8H]dihydrobiopterin to
tetrahydrobiopterin, which had already been shown for other reducing
compounds such as dithioerythritol and NADPH. We suggest that the
presence of the latter in the assay solution might also be responsible
for the minimal effect of ascorbic acid on the activation of purified
eNOS by tetrahydrobiopterin that was seen in our study.
The results presented here show that the oxidation of
tetrahydrobiopterin to the quinonoid 6,7-[8H]dihydrobiopterin
with a rearrangement to 7,8-dihydrobiopterin and further oxidation to biopterin is most likely the main pathway of tetrahydrobiopterin degradation in HUVEC. Other pterins generated by the loss of the side
chain at C6 could hardly be detected. Interestingly,
tetrahydrobiopterin remained intracellular under the different
experimental conditions investigated which is in contrast to previous
data obtained in human and murine endothelial cell lines (9, 42). Our
data show, however, that up to 91% of the dihydrobiopterin + biopterin formed in HUVEC was released into the medium, thereby preventing an
intracellular accumulation of dihydrobiopterin.
So far, beneficial vascular effects of ascorbic acid have been
attributed to its radical scavenging properties, which may lead to a
protection of NO from inactivation and may explain the improvement of
endothelium-dependent vasodilation in cardiovascular patients by an acute ascorbate application (43-48). Long term ascorbic acid administration also reversed endothelial vasomotor dysfunction in
patients, although the plasma levels reached might not have been high
enough to interfere with the reaction between superoxide anion and NO
(49). We suggest that the stabilization of the NOS cofactor
tetrahydrobiopterin resulting in an increased NO formation may
represent an additional mechanism of vascular protection by ascorbate,
which may be effective in vivo when plasma levels of
ascorbic acid supply saturated intracellular ascorbate concentrations. Interestingly, conditions that are thought to be associated with tetrahydrobiopterin deficiency (i.e. coronary artery disease
or smoking) have been characterized by low ascorbic acid levels in plasma or leukocytes (50-52), suggesting that cellular deficiency of
ascorbate may promote tetrahydrobiopterin oxidation and lead to
endothelial dysfunction.
In summary, this study shows that L-ascorbic acid in
physiologically relevant concentrations increases intracellular
tetrahydrobiopterin levels in endothelial cells in a
concentration-dependent and saturable fashion. Ascorbic
acid did not affect the synthesis of tetrahydrobiopterin but led to a
chemical stabilization of the compound. The results presented in this
study suggest that tissue saturation with ascorbic acid may maintain
tetrahydrobiopterin levels in endothelial cells in vivo,
thus providing optimal reaction conditions for NO synthesis and
preventing endothelial dysfunction.