Abstract
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 microm 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 microm. Accordingly, the effect of ascorbic acid on Ca(2+)-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 microm, 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.
Highlights
Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentrationdependent fashion without affecting NO synthase (NOS) expression or L-arginine uptake
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 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
Summary
Materials—Plasticware for cell culture was from Greiner Labortechnik (Frickenhausen, Germany). 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). 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 ϩ biopterin, respectively. 100 l 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 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. A p value of Ͻ 0.05 was accepted as statistically significant
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