Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling.
Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling.
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DOI：
10.1016/j.freeradbiomed.2013.04.035
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发表时间：
2013-10
影响因子：
7.4
通讯作者：
Vasquez-Vivar, Jeannette
中科院分区：
文献类型：
--
作者：
Whitsett, Jennifer;Range Filho, Artur;Sethumadhavan, Savitha;Celinska, Joanna;Widlansky, Michael;Vasquez-Vivar, Jeannette
关键词：
四氢生物蝶呤（BH 4）是NO合成和抑制eNOS释放超氧化物所必需的。使用BH 4治疗内皮功能障碍的临床试验产生了混合结果。不良结果可能是由于BH 4的快速全身和细胞氧化。BH 4的氧化产物之一，7，8-二氢生物蝶呤（7，8-BH 2）通过二氢叶酸还原酶（DHFR）再循环回BH 4。这种酶是无处不在的分布，并显示出广泛的活动，这取决于物种特异性因素和细胞类型。缺乏关于接受BH 4处理的人内皮细胞中BH 4再循环的动力学和效率的信息。为了表征该反应，我们应用了一种新的多电极库仑HPLC方法，该方法能够直接定量7，8-BH 2和BH 4，这是基于荧光的方法所不可能的。我们发现，基础未处理的BH 4和7，8-BH 2浓度在人EC低于牛和鼠内皮瘤细胞。用BH 4处理人EC瞬时增加细胞内BH 4，同时积累更稳定的7，8-BH 2。这与导致优先BH 4增加的牛或鼠EC不同。使用BH 4非对映体6S-BH 4和6 R-BH 4，证明了酶促DHFR再循环对总细胞内BH 4的狭窄贡献。7，8-BH 2还原为BH 4在细胞中以非常慢的速率发生，并且需要超生理水平的7，8-BH 2，表明该反应是动力学限制的。活性测定证实hDHFR对7，8-BH 2（DHF <KM>7，8-BH 2）具有非常低的亲和力，并且叶酸抑制7，8-BH 2再循环。我们的结论是，低活性的内皮DHFR是一个重要的因素，限制了BH 4疗法的好处，这可能会进一步加剧叶酸补充剂。
Tetrahydrobiopterin (BH4) is required for NO synthesis and inhibition of superoxide release from eNOS. Clinical trials using BH4 to treat endothelial dysfunction have produced mixed results. Poor outcomes may be explained by the rapid systemic and cellular oxidation of BH4. One of the oxidation products of BH4, 7,8-dihydrobiopterin (7,8-BH2), is recycled back to BH4 by dihydrofolate reductase (DHFR). This enzyme is ubiquitously distributed and shows a wide range of activity depending on species-specific factors and cell type. Information about the kinetics and efficiency of BH4 recycling in human endothelial cells receiving BH4 treatment is lacking. To characterize this reaction, we applied a novel multi-electrode coulometric HPLC method that enabled the direct quantification of 7,8-BH2 and BH4 which is not possible with fluorescent-based methodologies. We found that basal untreated BH4 and 7,8-BH2 concentrations in human ECs is lower than bovine and murine endothelioma cells. Treatment of human ECs with BH4 transiently increased intracellular BH4 while accumulating the more stable 7,8-BH2. This was different from bovine or murine ECs that resulted in preferential BH4 increase. Using BH4 diastereomers, 6S-BH4 and 6R-BH4, the narrow contribution of enzymatic DHFR recycling to total intracellular BH4 was demonstrated. Reduction of 7,8-BH2 to BH4 occurs at very slow rates in cells and needs supra-physiological levels of 7,8-BH2, indicating this reaction is kinetically limited. Activity assays verified that hDHFR has very low affinity for 7,8-BH2 (DHF<KM>7,8-BH2) and folic acid inhibits 7,8-BH2 recycling. We conclude that low activity of endothelial DHFR is an important factor limiting the benefits of BH4 therapies which may be further aggravated by folate supplements.
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DOI：
10.1161/atvbaha.107.142257
发表时间：
2007-06-01
影响因子：
8.7
作者：
Mayahi, Lila;Heales, Simon;Hingorani, Aroon D.
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DOI：
10.1152/ajpheart.00823.2007
发表时间：
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影响因子：
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影响因子：
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