Materials and Methods

Materials. Medium 199 (M199), human serum (HS), fetal calf serum (FCS), and collagenase were from BioWhittaker Europe (Verviers, Belgium). 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), and [3H]cGMP Biotrak radioimmunoassay systems were purchased from Amersham Pharmacia Biotech (Freiburg, Germany). Tumor necrosis factor-a (TNF-a) and interferon-y (IFN-y) were from Pharma Biotechnology (Hannover, Germany). NADPH, tetrahydrobiopterin, sepiapterin, and L-nitroarginine methylester (l-NAME) were obtained from Alexis Corporation (Läufelfingen, Switzerland). All other biochemical reagents were purchased from Sigma Chemical (Deisenhofen, Germany). Endotoxin contamination of ascorbic acid solutions was measured with the coagulation Limulus amebocyte lysate assay and was proved to be below the detection limit of the kit (0.05 u/mL).

Cell cultures. Human umbilical cord vein endothelial cells (HUVEC) were prepared with 0.05% collagenase and cultured in M199 containing 15% FCS, 5% HS, and 7.5 ^g/mL endothelial cell growth supplement. Experiments were carried out with monolayers of the first to second passage. Preincubation of cells with L-ascorbic acid, L-gulonolactone, dehydroascorbic acid, sepiapterin, 2,4-diamino-6-hydroxypyrimidine (DAHP), or the combination of TNF-a, IFN-y, and lipopolysaccharide (LPS) was performed in culture medium. Cell stimulation with ionomycin (2 ^mol/L, 15 min) or thrombin (1 U/mL, 15 min) was carried out in Hepes buffer (10 mmol/L Hepes, 145 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgSO4, 10 mmol/L glucose, 1.5 mmol/L CaCl2, pH 7.4) in the absence of ascorbic acid, sepiapterin, DAHP, or cytokines.

Measurement of citrulline and cGMP formation. Endothelial cells were stimulated with ionomycin or thrombin in Hepes buffer (pH 7.4) containing 10 ^mol/L

L-[3H]arginine (0.33 Ci/mmol) for the measurement of citrulline formation or 0.5 mmol/L isobutylmethylxanthine for cGMP determinations. The [3H]citrulline generated was separated from [3H]arginine by cation exchange chromatography [Dowex AG50WX-8 (Na+ form)] and quantified by liquid scintillation counting (84,85). The cGMP accumulated was measured in cellular extracts by radioim-munoassay following the instructions of the manufacturer.

[14C]Ascorbic acid uptake in endothelial cells. Endothelial cells were incubated in culture medium containing 100 ^mol/L [14C]ascorbic acid (16 mCi/mmol). After various times, cells were washed with cold Hepes buffer (pH 7.4) containing 100 ^mol/L phloretin and lysed with 100 mmol/L NaOH, 2% Na2CO3, and 1% sodium dodecylsulfate. The radioactivity of cell lysates was measured by liquid scintillation counting.

Determination of eNOS activity. Experiments were performed with tetrahydro-biopterin-free eNOS that was expressed in yeast Pichia pastoris and purified as described (86). The assay solution (100 ^L) contained 50 mmol/L Tris-HCl buffer (pH 7.4), 0.3 ^g eNOS, 100 ^mol/L L-[3H]arginine (100,000 cpm), 0.5 mmol/L CaCl2, 0.2 mmol/L NADPH, 5 ^mol/L FAD, 5 ^mol/L FMN, 10 ^g/mL calmodulin, 10 nmol/L-100 ^mol/L tetrahydrobiopterin, and 0.2 mmol/L CHAPS. The [^citrulline generated was separated from [3H]arginine by ion exchange columns and quantified as described above.

Measurement of biopterin derivatives. Culture medium was collected and endothelial monolayers were detached with trypsin/EDTA (0.05%/0.02%, vol/vol). Aliquots of 5 x 106 cells and 1-mL aliquots of medium were oxidized with 0.02 mol/L KI/I2 in 0.1 mol/L HCl or in 0.1 mol/L NaOH for 1 h in the dark. The precipitates were removed by centrifugation and excess iodine was destroyed by the addition of 0.02 mol/L ascorbic acid. Quantification of biopterin in supernatants was performed by high-performance liquid chromatography (HPLC) as described (87). 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). Additionally, nonoxidized supernatants were used to determine biopterin.

Determination of GTP cyclohydrolase I (GTP-CH I) expression and activity. Extraction of total RNA from endothelial cells, electrophoresis on 1% agarose/6% formaldehyde gels, Northern blotting, and hybridization of the blots with 106 cpm/mL [32P]dCTP-labeled probe for human GTP cyclohydrolase I, obtained by polymerase chain reaction using consensus primers to GTP cyclohydrolase I from Escherichia coli, mouse and human, were performed according to standard protocols. GTP-CH I activity in cytosolic fractions from endothelial cells was measured as described (87).

Statistical analysis. All data are given as means ± SEM, n = 3-5 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 was performed. A P value of < 0.05 was accepted as significant.

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