Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin
Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin
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DOI：
10.1074/jbc.m002248200
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发表时间：
2000-06-09
影响因子：
4.8
通讯作者：
Keaney, JF
中科院分区：
文献类型：
--
作者：
Huang, A;Vita, JA;Keaney, JF
抗坏血酸增强血管疾病患者NO生物活性的机制尚不清楚。我们研究了细胞内抗坏血酸在内皮源性NO生物活性中的作用。抗坏血酸与猪主动脉内皮细胞（PAECs）孵育产生时间和剂量依赖性的细胞内抗坏血酸积累，通过a23187诱导的cGMP积累，使NO的生物活性提高了70%。这种效应是由于抗坏血酸促进了NO的产生，因为抗坏血酸刺激了PAEC氮氧化物（NO2- + NO3-）的产生和l -精氨酸到l -瓜氨酸的转化，分别增加了59%和72%，而没有改变cGMP对真实NO的反应。抗坏血酸还能刺激PAEC膜组分或杆状病毒感染的Sf9细胞的eNOS的催化活性。抗坏血酸在不改变l -精氨酸K-m的情况下，使牛eNOS V-max提高了50%。抗坏血酸的作用依赖于四氢生物蝶呤（BH4），因为抗坏血酸在BH4浓度为10 μ M时无效，或者在paec中用sepapterin处理以增加细胞内BH4。抗坏血酸的作用也具有特异性，因为a23187刺激的paec中cGMP积累对细胞内谷胱甘肽操作不敏感，并且只有抗坏血酸而不是谷胱甘肽增加了细胞内BH4的浓度。这些数据表明抗坏血酸通过增加细胞内BH4含量以BH依赖的方式增强NO的生物活性。
Ascorbic acid enhances NO bioactivity in patients with vascular disease through unclear mechanism(s). We investigated the role of intracellular ascorbic acid in endothelium-derived NO bioactivity. Incubation of porcine aortic endothelial cells (PAECs) with ascorbic acid produced time- and dose-dependent intracellular ascorbic acid accumulation that enhanced NO bioactivity by 70% measured as A23187-induced cGMP accumulation. This effect was due to enhanced NO production because ascorbate stimulated both PAEC nitrogen oxide (NO2- + NO3-) production and L-arginine to L-citrulline conversion by 59 and 72%, respectively, without altering the cGMP response to authentic NO. Ascorbic acid also stimulated the catalytic activity of eNOS derived from either PAEC membrane fractions or baculovirus-infected Sf9 cells. Ascorbic acid enhanced bovine eNOS V-max by similar to 50% without altering the K-m for L-arginine. The effect of ascorbate was tetrahydrobiopterin (BH4)-dependent, because ascorbate was ineffective with BH4 concentrations >10 mu M or in PAECs treated with sepiapterin to increase intracellular BH4. The effect of ascorbic acid was also specific because A23187-stimulated cGMP accumulation in PAECs was insensitive to intracellular glutathione manipulation and only ascorbic acid, not glutathione, increased the intracellular concentration of BH4. These data suggest that ascorbic acid enhances NO bioactivity in a BH,-dependent manner by increasing intracellular BH4 content.