Kim Hyo Jung, Lee Sang Il, Lee Duk-Hee, Smith Debra, Jo Hanjoong, Schellhorn Herb E, Boo Yong Chool
Department of Molecular Medicine, Kyungpook National University School of Medicine, Junggu, Daegu, Republic of Korea.
Biochem Biophys Res Commun. 2006 Jul 14;345(4):1657-62. doi: 10.1016/j.bbrc.2006.05.090. Epub 2006 May 24.
As a primary antioxidant, ascorbic acid (AA) provides beneficial effects for vascular health mitigating oxidative stress and endothelial dysfunction. However, the association of intracellular AA with NO production occurring inside the endothelial cells remains unclear. In the present study, we addressed this issue by increasing intracellular AA directly through de novo synthesis. To restore AA synthesis pathway, bovine aortic endothelial cells were transfected with the plasmid vector encoding L-gulono-1,4-lactone oxidase (GULO, EC 1.1.3.8), the missing enzyme converting L-gulono-1,4-lactone (GUL) to AA. Functional expression of GULO was verified by Western blotting and in vitro enzyme activity assay. GULO expression alone did not lead to AA synthesis but the supply of GUL resulted in a marked increase of intracellular AA. When the cells were stimulated with calcium ionophore, A23187, NO production was more active in the GULO-expressing cells supplied with GUL, in comparison with the cells without GULO expression or without GUL supply, indicating that intracellular AA regulated NO production. Enhancement of NO production by intracellular AA was further verified in aortic endothelial cells obtained from eNOS knockout mice that were cotransfected with eNOS and GULO constructs. GULO-dependent AA synthesis also elevated intracellular tetrahydrobiopterin content, implicating that this essential cofactor of endothelial nitric oxide synthase (eNOS) might mediate the AA effect. The present study strongly suggests that intracellular AA plays critical roles in vascular physiology through enhancing endothelial NO production.
作为一种主要的抗氧化剂，抗坏血酸（AA）通过减轻氧化应激和内皮功能障碍，为血管健康带来有益影响。然而，内皮细胞内细胞内抗坏血酸与一氧化氮（NO）生成之间的关联仍不清楚。在本研究中，我们通过从头合成直接增加细胞内抗坏血酸来解决这个问题。为了恢复抗坏血酸合成途径，用编码L-古洛糖酸-1,4-内酯氧化酶（GULO，EC 1.1.3.8）的质粒载体转染牛主动脉内皮细胞，该酶是将L-古洛糖酸-1,4-内酯（GUL）转化为抗坏血酸所缺失的酶。通过蛋白质免疫印迹法和体外酶活性测定验证了GULO的功能表达。单独的GULO表达不会导致抗坏血酸合成，但GUL的供应导致细胞内抗坏血酸显著增加。当用钙离子载体A23187刺激细胞时，与未表达GULO或未供应GUL的细胞相比，在供应GUL的表达GULO的细胞中，NO生成更活跃，表明细胞内抗坏血酸调节NO生成。在从eNOS基因敲除小鼠获得的主动脉内皮细胞中，共转染eNOS和GULO构建体，进一步验证了细胞内抗坏血酸对NO生成的增强作用。GULO依赖性抗坏血酸合成还提高了细胞内四氢生物蝶呤含量，这意味着内皮型一氧化氮合酶（eNOS）的这种必需辅因子可能介导了抗坏血酸的作用。本研究强烈表明，细胞内抗坏血酸通过增强内皮细胞NO生成在血管生理学中发挥关键作用。
