title: Ascorbic acid synthesis due to L-gulono-1,4-lactone oxidase expression enhances NO production in endothelial cells.
abstract: 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.
authors.affiliation: Department of Molecular Medicine, Kyungpook National University School of Medicine, Junggu, Daegu, Republic of Korea.
authors.forename: Hyo Jung
authors.initials: HJ
authors.lastname: Kim
authors.forename: Sang Il
authors.initials: SI
authors.lastname: Lee
authors.forename: Duk-Hee
authors.initials: DH
authors.lastname: Lee
authors.forename: Debra
authors.initials: D
authors.lastname: Smith
authors.forename: Hanjoong
authors.initials: H
authors.lastname: Jo
authors.forename: Herb E
authors.initials: HE
authors.lastname: Schellhorn
authors.forename: Yong Chool
authors.initials: YC
authors.lastname: Boo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000096:biosynthesis* / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives / Q000096:biosynthesis; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015870:Gene Expression; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D050544:L-Gulonolactone Oxidase / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D013400:Sugar Acids / Q000378:metabolism; D014162:Transfection
chemical_list: D007476:Ionophores; D013400:Sugar Acids; D001708:Biopterins; D009569:Nitric Oxide; C007895:gulonolactone; D000001:Calcimycin; D050544:L-Gulonolactone Oxidase; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin; D001205:Ascorbic Acid