Busu C, Atanasiu V, Caldito G, Aw T Y
"Carol Davila" University of Medicine and Pharmacy, Medical School, Biochemistry Department, Bucharest, Romania ; Department of Molecular & Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA.
"Carol Davila" University of Medicine and Pharmacy, Medical School, Biochemistry Department, Bucharest, Romania.
J Med Life. 2014 Oct-Dec;7(4):611-8.
Pathological conditions states such as stroke, diabetes mellitus, hypertension, dyslipidemia are associated with increased levels of free radicals that alter normal function of the vascular endothelium and perturb vascular homeostasis. The redox couples reduced glutathione (GSH)/oxidized glutathione (GSSG), NADH/NAD+, and NADPH/NADP+ play major functions in the intracellular redox balance. Any decrease in tissue or systemic GSH levels under the aforementioned pathologies would enhance oxidative damage to the vascular endothelium. Beside their role as coenzyme that participate in cellular metabolism, pyridine nucleotides serve also as substrate for enzymes involved in DNA repair and longevity. There is scant data on NAD+/NADH kinetics and distribution during human cells proliferation. Here, we determined the influence of cellular GSH status on the early dynamics of nuclear-to-cytosol (N-to-C) NAD+ and nuclear NADH kinetics (6 h interval) over 72 h of endothelial cell proliferation. The IHEC cell line was used as a surrogate for human brain micro vascular endothelial cells. Inhibition of GSH synthesis by buthionine sulfoximine (BSO) and sustained low cellular GSH significantly increased nuclear NADH levels (p<0.01), which correlated with lower nuclear GSH and prolonged cell cycle S-phase. When BSO was removed the pattern of nuclear NAD+ resembled that of control group, but nuclear NADH concentrations remained elevated, as in GSH deficient cells (p<0.01). The coincidence of high nuclear NADH and lower nuclear NAD+ with S-phase prolongation are suggestive of CtBP and NAD+-dependent DNA repair enzyme activation under conditions of decreased cellular GSH. These results provide important insights into GSH control of vascular endothelial growth and restitution, key processes in the restoration of the endothelium adjacent to the post-injury lesion site.
诸如中风、糖尿病、高血压、血脂异常等病理状态与自由基水平升高有关，自由基会改变血管内皮的正常功能并扰乱血管稳态。氧化还原对还原型谷胱甘肽（GSH）/氧化型谷胱甘肽（GSSG）、NADH/NAD⁺和NADPH/NADP⁺在细胞内氧化还原平衡中起主要作用。在上述病理状态下，组织或全身GSH水平的任何降低都会增强对血管内皮的氧化损伤。除了作为参与细胞代谢的辅酶的作用外，吡啶核苷酸还作为参与DNA修复和寿命的酶的底物。关于人类细胞增殖过程中NAD⁺/NADH动力学和分布的数据很少。在这里，我们确定了细胞GSH状态对内皮细胞增殖72小时内核-胞质（N-to-C）NAD⁺早期动力学和核NADH动力学（6小时间隔）的影响。IHEC细胞系用作人脑微血管内皮细胞的替代物。丁硫氨酸亚砜胺（BSO）抑制GSH合成并持续低细胞GSH显著增加核NADH水平（p<0.01），这与较低的核GSH和延长的细胞周期S期相关。当去除BSO时，核NAD⁺的模式类似于对照组，但核NADH浓度仍然升高，如同在GSH缺乏的细胞中一样（p<0.01）。高核NADH和低核NAD⁺与S期延长的同时出现提示在细胞GSH降低的情况下CtBP和NAD⁺依赖性DNA修复酶被激活。这些结果为GSH对血管内皮生长和修复的控制提供了重要见解，血管内皮生长和修复是损伤后病变部位附近内皮恢复的关键过程。
