l-Ascorbic Acid Potentiates Endothelial Nitric Oxide Synthesis via a Chemical Stabilization of Tetrahydrobiopterin*

Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentration-dependent fashion without affecting NO synthase (NOS) expression or l-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 μm to 1 mm, 24 h) led to an up to 3-fold increase of intracellular tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 μm. Accordingly, the effect of ascorbic acid on Ca2+-dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001–100 μm, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of tetrahydrobiopterin. This was evident as an increase in the half-life of tetrahydrobiopterin in aqueous solution. Furthermore, the increase of tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.

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 Ca 2ϩ /calmodulin, FAD, FMN, and tetrahydrobiopterin as cofactors (2). The endothelial NOS isoform (eNOS) is constitutively expressed and activated upon an increase of intracellular Ca 2ϩ 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)(8)(9)(10), suggesting that an optimal concentration of tetrahydrobiopterin is essential for agonistinduced production of NO. Furthermore, tetrahydrobiopterin induced vasodilation in isolated arteries (11)(12)(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)(16)(17)(18)(19)(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 NOSbound tetrahydrobiopterin acts as a redox-active cofactor (22)(23)(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 M, 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.
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 MgSO 4 , 10 glucose, 1.5 CaCl 2 .
Cell Cultures-Human umbilical cord vein endothelial cells (HU-VEC) were prepared with 0.05% collagenase and cultured in 75-cm 2 plastic flasks in M199 containing 15% FCS, 5% human serum, and 7.5 g/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 M) 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 Me 2 SO, and stock solutions of the other compounds were prepared in M199 containing 10% FCS. Stimulation of cells with the Ca 2ϩ ionophore, ionomycin, was performed in the absence of ascorbic acid, sepiapterin, or DAHP. Ionomycin was dissolved at 1 mM in Me 2 SO and stored at Ϫ20°C until use. The final concentration of Me 2 SO 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 M ionomycin in the presence of 10 M L-arginine and 3.3 Ci/ml L-[ 3 H]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 ϩ form). The radioactivity corresponding to the [ 3 H]citrulline content of the eluate was quantified by liquid scintillation counting. Agonist-induced [ 3 H]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 [ 3 H]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 M 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 l 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 l) contained 50 mM Tris-HCl buffer (pH 7.4), 0.3 g of eNOS, 100 M L-[ 3 H]arginine (100,000 cpm), 0.5 mM CaCl 2 , 0.2 mM NADPH, 5 M FAD, 5 M FMN, 10 g/ml calmodulin, 10 nM to 100 M tetrahydrobiopterin, and 0.2 mM CHAPS. Incubations without Ca 2ϩ served as a blank. The enzyme assay was performed in the presence or absence of 100 M 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 ϩ form). The enzyme activity was calculated from the [ 3 H]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 ϫ 10 6 cells were centrifuged (500 ϫ g, 6 min), and 100 l of oxidant solution (0.02 M KI/I 2 in 0.1 M HCl or 0.02 M KI/I 2 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 l 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 l of ascorbic acid (0.2 M). Quanti-fication of biopterin in supernatants was performed as described (31). Briefly, 10 l of the cell extracts were injected onto a 250-mm-long, 4-mm inner diameter column filled with 5-m 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 H 3 PO 4 ) at a flow rate of 1.5 ml/min, and detected by fluorescence as described above. The amount of 5,6,7,8tetrahydrobiopterin was calculated from the difference in biopterin concentrations measured after oxidation in acid (total biopterins) and base (7,8-dihydrobiopterin ϩ biopterin). Intracellular levels of 7,8-dihydrobiopterin ϩ 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/I 2 in 0.1 M HCl or 0.02 M KI/I 2 in 0.1 M NaOH to detect total biopterins or 7,8-dihydrobiopterin ϩ 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 10 6 cpm/ml [ 32 P]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, ϳ1-4 mg/ml cytosolic protein, and 2 mM GTP in a total volume of 300 l. In some experiments 100 M 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 l 1 M HCl. Subsequently, the 7,8-dihydroneopterin triphosphate formed was oxidized to neopterin triphosphate by the addition of 10 l of 0.1 M I 2 solubilized in 0.25 M KI. After 1 h in the dark, the precipitate was removed by centrifugation and 10 l 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 l of the following mixture were incubated for 1 h at 37°C: 100 mM Tris-HCl (pH 7.4), 20 mM MgCl 2 , 2 mM NADPH, 2 milliunits of sepiapterin reductase, 40 M 7,8-dihydroneopterin triphosphate (enzymatically prepared from GTP using recombinant GTP cyclohydrolase I), ϳ1 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 l of 1 M HCl and 5 l of 0.1 mM I 2 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 l 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/I 2 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% Na 2 CO 3 , 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 Ϯ 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 Ͻ 0.05 was accepted as statistically significant.

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 M, 24 h) led to a 2.7-fold increase in ionomycintriggered citrulline formation and a 2.8-fold potentiation of Ca 2ϩ -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 M, 24 h; Fig. 1), which is intracellularly converted into tetrahydrobiopterin via a salvage pathway (33). When HUVEC were coincubated with ascorbic acid (100 M, 24 h) and sepiapterin (0.001-10 M, 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 M sepiapterin and 100 M 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).
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 M) stimulated the formation of citrulline in a concentration-dependent manner with an EC 50 of 0.31 Ϯ 0.036 M and a maximal effect at about 100 M (Fig. 2). The presence of 100 M ascorbic acid in the assay solution resulted in a slight decrease of the EC 50 to 0.16 Ϯ 0.014 M (n ϭ 3, p Ͻ 0.05) without significant increase in maximal enzyme activity. Ascorbate had no effect on eNOS activity in the absence of exogenous tetrahydrobiopterin.
Effect of Ascorbic Acid on Intracellular Tetrahydrobiopterin Levels-The preincubation of HUVEC with ascorbic acid (100 M, 24 h) led to an increase of intracellular tetrahydrobiopterin levels from 0.38 Ϯ 0.04 pmol/mg of protein to 1.14 Ϯ 0.09 pmol/mg (n ϭ 20). The effect of ascorbic acid was concentrationdependent between 1 and 100 M and saturable since concentrations above 100 M did not induce a further increase of tetrahydrobiopterin levels (Fig. 3). In contrast, the effect of 1 mM ascorbic acid was somewhat lower (Ϫ9.3% Ϯ 2.72% compared with the effect of 100 M, n ϭ 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.
Effect of Ascorbic Acid on the Expression and Activity of GTP Cyclohydrolase I-To investigate whether the ascorbic acidmediated 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 g/ml LPS, 24 h) and ascorbate (100 M, 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. The GTP cyclohydrolase I activity in cytosolic fractions from cytokine-treated HUVEC was 0.52 Ϯ 0.05 pmol/mg/min (n ϭ 3). The addition of 100 M ascorbic acid to the assay had no effect on enzyme activity (0.54 Ϯ 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 noncytokine-treated HUVEC was below the detection limit of the method (0.02 pmol/mg/min) and was not increased to detectable levels by 100 M 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 Ϯ 0.13 pmol/mg/min in control cells and not changed when HUVEC were preincubated with 100 M ascorbic acid for 24 h (1.67 Ϯ 0.24 pmol/mg/min, n ϭ 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 Acidmediated 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 Ϯ 0.04 pmol/mg of protein to 0.13 Ϯ 0.03 pmol/mg (n ϭ 4) (Fig. 5). In parallel, ionomycinstimulated 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 ϭ 4). DAHP also reduced tetrahydrobiopterin levels as well as citrulline and cGMP formation in the presence of 100 M 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).
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 M 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 Յ10 min. In the presence of ascorbate the apparent half-life of tetrahydrobiopterin was increased to approximately 70 min.
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 g/ml LPS, 24 h) to increase the expression of the GTP cyclohydrolase I and thus pteridine production. Intracellular tetrahydrobiopterin and 7,8-dihydrobiopterin ϩ biopterin levels in HUVEC preincubated with the cytokine mixture were 30 Ϯ 3.5 pmol/dish and 12 Ϯ 1.8 pmol/dish, respectively (Fig. 7). Ascorbic acid (1-100 M, 24 h) added to HUVEC simultaneously with the cytokines caused a concentration-dependent increase of intracellular tetrahydrobiopterin levels up to 135 Ϯ 16 pmol/dish and a decrease of dihydrobiopterin ϩ biopterin levels down to 7 Ϯ 1.1 pmol/dish (Fig. 7). Control HUVEC did not release tetrahydrobiopterin but a considerable amount of 7,8-dihydrobiopterin (100 Ϯ 23 pmol/dish) and some biopterin (22 Ϯ 5.5 pmol/dish) to the culture medium. Ascorbate (1-100 M, 24 h) decreased this release to 33 Ϯ 8 pmol/dish 7,8-dihydrobiopterin and 3 Ϯ 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).

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 M, 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 FIG. 4. Effect of ascorbic acid on GTP cyclohydrolase mRNA expression. Total RNA was harvested from HUVEC preincubated for 24 h with 100 M ascorbic acid in the absence or presence of a mixture of cytokines (250 units/ml TNF-␣, 250 units/ml IFN-␥) and LPS (1 g/ml). After electrophoresis on 1% agarose, 6% formaldehyde gels (20 g/lane) the RNA was blotted on nylon membranes and hybridized overnight with [ 32 P]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.
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 Ca 2ϩ -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 ϩ 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)(44)(45)(46)(47)(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 concen-trations. 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. 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 g/ml LPS in the absence or presence of 100 M ascorbic acid. Aliquots of 2 ϫ 10 6 cells and 1-ml aliquots of cell supernatants were oxidized with 0.02 M KI/I 2 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 ϩ 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 Ϯ S.E. from three experiments. *, p Ͻ 0.05 versus levels of biopterin derivatives in untreated control cells.