Heller R, Münscher-Paulig F, Gräbner R, Till U
Center of Vascular Biology and Medicine, Friedrich-Schiller-University of Jena, 99089 Erfurt, Germany.
J Biol Chem. 1999 Mar 19;274(12):8254-60. doi: 10.1074/jbc.274.12.8254.
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.
已证明，抗坏血酸可通过一种被认为涉及保护一氧化氮（NO）免受自由基失活影响的机制，增强动脉粥样硬化患者受损的内皮依赖性血管舒张功能。本研究以人脐静脉和冠状动脉内皮细胞为对象，探究L-抗坏血酸是否还会影响细胞内NO的合成。将内皮细胞与0.1 - 100微摩尔抗坏血酸孵育24小时，随后在无细胞外抗坏血酸的情况下，用离子霉素（2微摩尔）或凝血酶（1单位/毫升）刺激15分钟。抗坏血酸预处理使细胞产生的NO量增加了3倍，这一增加量通过其副产物瓜氨酸的形成以及其效应分子cGMP的积累来衡量。该效应在100微摩尔时达到饱和，且其动力学与抗坏血酸进入细胞的摄取情况相似。对前体分子L-古洛糖酸内酯和不同抗坏血酸衍生物的研究表明，抗坏血酸的烯二醇结构对其影响NO合成的作用至关重要。抗坏血酸既不诱导一氧化氮合酶（NOS）蛋白的表达，也不增强NOS底物L-精氨酸进入内皮细胞的摄取。当在存在已知NOS活性辅助因子的情况下，从抗坏血酸预处理细胞的细胞裂解物中测量瓜氨酸形成时，抗坏血酸的作用极小。然而，当测定中省略辅助因子四氢生物蝶呤时，抗坏血酸预处理显示出与在完整细胞中所见相似的增强作用，这表明抗坏血酸可能会提高四氢生物蝶呤的可用性或增加其对内皮NOS的亲和力。我们的数据表明，细胞内抗坏血酸可增强内皮细胞中NO的合成，这可能部分解释了抗坏血酸对血管的有益作用。
