Abstract
Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether L-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1-100 microM ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 microM) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 microM and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor molecule L-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate L-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid.
Highlights
Bition of platelet activation, and regulation of endothelial cell permeability and adhesivity [1,2,3,4]
Effect of Ascorbic Acid on Citrulline and cGMP Formation in Endothelial Cells—nitric oxide (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
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 NO synthase (NOS) inhibitor L-NAME 30 min before stimulation
Summary
Materials—Plasticware for cell culture was from Greiner Labortechnik (Frickenhausen, Germany). Cell monolayers were incubated at 37 °C for 30 min in 1.5 ml of Hepes buffer (pH 7.4) containing 0.25% HSA. Incubations were stopped by washing the cells with cold Hepes buffer (pH 7.4) containing 100 M 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. Incubations were stopped by washing the cells with cold Hepes buffer (pH 7.4) containing 5 mM L-arginine. 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). Measurement of Citrulline Formation in Cell Lysates—HUVEC were detached by trypsin/EDTA, resuspended in a small volume of homoge-
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