Ascorbate stimulates endothelial nitric oxide synthase enzyme activity by rapid modulation of its phosphorylation status.
Ascorbate stimulates endothelial nitric oxide synthase enzyme activity by rapid modulation of its phosphorylation status.
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DOI：
10.1016/j.freeradbiomed.2012.03.022
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发表时间：
2012-05-15
影响因子：
7.4
通讯作者：
Heiss, Elke H.
中科院分区：
文献类型：
--
作者：
Ladurner, Angela;Schmitt, Christoph A.;Schachner, Daniel;Atanasov, Atanas G.;Werner, Ernst R.;Dirsch, Verena M.;Heiss, Elke H.
关键词：
已知长期暴露于抗坏血酸盐通过稳定eNOS辅因子四氢生物蝶呤（BH 4）来增强内皮型一氧化氮合酶（eNOS）活性。我们研究了抗坏血酸对原代（HUVEC）和永生化人内皮细胞（EA.hy926）中eNOS功能的急性影响，旨在为抗坏血酸给药后在体内观察到的快速血管舒张提供分子解释。eNOS的酶活性和细胞内BH 4水平分别通过精氨酸-瓜氨酸转化试验和HPLC分析进行评估。在一段时间的4小时，抗坏血酸稳定地增加eNOS活性，虽然内皮BH 4水平保持不变相比，未经处理的对照细胞。免疫印迹分析显示，早在5分钟后，抗坏血酸治疗剂量依赖性增加磷酸化eNOS-丝氨酸1177和伴随降低磷酸化eNOS-苏氨酸495，磷酸化模式的eNOS活性增加的指示。通过采用药理学抑制剂，siRNA介导的敲低方法，和蛋白磷酸酶2A（PP 2A）的催化亚基的过表达，我们表明，这种效果至少部分是由于减少PP 2A活性和随后的AMP激活激酶的激活。在这份报告中，我们揭示了一种新的机制，如何抗坏血酸快速激活eNOS独立的BH 4稳定的影响。抗坏血酸可在数分钟内增强内皮型一氧化氮合酶（eNOS）的活性。这种作用的发生与四氢生物蝶呤的稳定无关。抗坏血酸以PP 2A和AMPK依赖的方式调节eNOS磷酸化。
Long-term exposure to ascorbate is known to enhance endothelial nitric oxide synthase (eNOS) activity by stabilizing the eNOS cofactor tetrahydrobiopterin (BH4). We investigated acute effects of ascorbate on eNOS function in primary (HUVEC) and immortalized human endothelial cells (EA.hy926), aiming to provide a molecular explanation for the rapid vasodilatation seen in vivo upon administration of ascorbate. Enzymatic activity of eNOS and intracellular BH4 levels were assessed by means of an arginine–citrulline conversion assay and HPLC analysis, respectively. Over a period of 4 h, ascorbate steadily increased eNOS activity, although endothelial BH4 levels remained unchanged compared to untreated control cells. Immunoblot analyses revealed that as early as 5 min after treatment ascorbate dose-dependently increased phosphorylation at eNOS-Ser1177 and concomitantly decreased phosphorylation at eNOS-Thr495, a phosphorylation pattern indicative of increased eNOS activity. By employing pharmacological inhibitors, siRNA-mediated knockdown approaches, and overexpression of the catalytic subunit of protein phosphatase 2A (PP2A), we show that this effect was at least partly owing to reduction of PP2A activity and subsequent activation of AMP-activated kinase. In this report, we unravel a novel mechanism for how ascorbate rapidly activates eNOS independent of its effects on BH4 stabilization. ► Ascorbate can enhance the activity of endothelial NO synthase (eNOS) within minutes. ► This effect occurs independent of stabilization of tetrahydrobiopterin. ► Ascorbate modulates eNOS phosphorylation in a PP2A- and AMPK-dependent manner.
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影响因子：
5.1
作者：
FAVIER, A;SAPPEY, C;MICOUD, M
通讯作者：
MICOUD, M
DOI：
10.1016/j.mrfmmm.2004.03.006
发表时间：
2004-07-13
影响因子：
2.3
作者：
Choi, SW;Benzie, IFF;Strain, JJ
通讯作者：
Strain, JJ
DOI：
10.1152/ajpheart.2000.279.3.h1172
发表时间：
2000-09-01
影响因子：
4.8
作者：
Hirai, N;Kawano, H;Yasue, H
通讯作者：
Yasue, H
影响因子：
10.8
作者：
Garry A;Edwards DH;Fallis IF;Jenkins RL;Griffith TM
通讯作者：
Griffith TM
影响因子：
4.8
作者：
Huang, A;Vita, JA;Keaney, JF
通讯作者：
Keaney, JF