Heller R, Unbehaun A, Schellenberg B, Mayer B, Werner-Felmayer G, Werner E R
Center of Vascular Biology and Medicine, Friedrich-Schiller-University of Jena, D-99089 Erfurt, Germany.
J Biol Chem. 2001 Jan 5;276(1):40-7. doi: 10.1074/jbc.M004392200.
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.
已表明抗坏血酸能以时间和浓度依赖性方式刺激内皮细胞一氧化氮（NO）的合成，而不影响一氧化氮合酶（NOS）的表达或L-精氨酸的摄取。本研究调查其潜在机制是否与NOS辅因子四氢生物蝶呤有关。用抗坏血酸盐（1微摩尔至1毫摩尔，24小时）预处理人脐静脉内皮细胞导致细胞内四氢生物蝶呤水平最多增加3倍，这是浓度依赖性的，在100微摩尔时达到饱和。因此，当通过内皮细胞与蝶酰三谷氨酸（0.001 - 100微摩尔，24小时）共同孵育使细胞内四氢生物蝶呤水平升高时，抗坏血酸对Ca(2+)依赖性瓜氨酸（NO的副产物）和cGMP（NO激活的可溶性鸟苷酸环化酶的产物）形成的影响被消除。相反，抗坏血酸并未改变内皮NOS对蝶呤的亲和力，这是在使用纯化的无四氢生物蝶呤酶的测定中测得的。抗坏血酸盐诱导的内皮细胞四氢生物蝶呤增加并非由于该化合物合成增强。抗坏血酸盐既未改变四氢生物蝶呤生物合成限速酶GTP环化水解酶I的mRNA表达，也未改变GTP环化水解酶I或6 - 丙酮酰 - 四氢蝶呤合酶（从头合成途径中的第二种酶）的活性。我们的数据表明抗坏血酸导致四氢生物蝶呤的化学稳定。这表现为四氢生物蝶呤在水溶液中的半衰期增加。此外，与细胞因子和抗坏血酸共同孵育的完整内皮细胞中四氢生物蝶呤水平的增加与细胞及细胞上清液中氧化程度更高的蝶呤衍生物（7,8 - 二氢生物蝶呤和生物蝶呤）的减少相关。本研究表明内皮细胞中饱和的抗坏血酸水平对于保护四氢生物蝶呤免受氧化以及为细胞NO合成提供最佳条件是必要的。
