l-Ascorbic Acid Potentiates Nitric Oxide Synthesis in Endothelial Cells

Abstract

Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether l-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1–100 μm ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 μm) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 μm and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor moleculel-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate l-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid. Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether l-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1–100 μm ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 μm) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 μm and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor moleculel-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate l-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid. nitric oxide NO synthase endothelial cell NOS medium 199 human serum albumin inducible NOS l-nitroarginine methyl ester phenylmethylsulfonyl fluoride dithiothreitol human umbilical vein endothelial cells coronary artery endothelial cells phosphate-buffered saline polyacrylamide gel electrophoresis Endothelium-derived nitric oxide (NO)1 exerts several vasoprotective activities including smooth muscle relaxation, inhibition of platelet activation, and regulation of endothelial cell permeability and adhesivity (1Furchgott R.F. Zawadski J.V. Nature. 1980; 288: 373-376Crossref PubMed Scopus (9908) Google Scholar, 2Alheid U. Frölich J.C. Förstermann U. Thromb. Res. 1987; 47: 561-571Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 3Westendorp R.G.J. Draijer R. Meinders A.E. van Hinsberg V.W.M. J. Vasc. Res. 1994; 31: 42-51Crossref PubMed Scopus (91) Google Scholar, 4Heller R. Bussolino F. Ghigo D. Pescarmona G.P. Calvino R. Gasco A. Till U. Bosia A. Agents Actions. 1994; 45: 177-181Google Scholar). NO is generated from the conversion of l-arginine to l-citrulline by the enzymatic action of an NADPH-dependent NO synthase (NOS) that requires tetrahydrobiopterin, FAD, and FMN as cofactors (5Palmer R.M.J. Ashton D.S. Moncada S. Nature. 1988; 133: 664-666Crossref Scopus (4095) Google Scholar). The endothelial NOS isoform (ecNOS) is constitutively expressed and is activated upon an increase of intracellular calcium following cell stimulation with receptor-dependent stimuli such as thrombin and bradykinin or with receptor-independent stimuli like calcium ionophore (6Busse R. Mülsch A. FEBS Lett. 1990; 265: 133-136Crossref PubMed Scopus (431) Google Scholar).Since NO interferes with key processes in atherogenesis (7Bult H. Mol. Med. Today. 1996; 2: 510-518Abstract Full Text PDF PubMed Scopus (43) Google Scholar), a lack of NO might promote the development of atherosclerosis. Indeed, clinical studies have confirmed an impairment of vascular NO synthesis in patients with atherosclerosis or with increased atherogenic risk factors (8Cooke J.P. Dzau V.J. Annu. Rev. Med. 1997; 48: 489-509Crossref PubMed Scopus (644) Google Scholar). The dysfunction of the endothelial NO pathway may involve impaired signal transduction mechanisms, decreased ecNOS activity, reduced intracellular availability of l-arginine, or increased inactivation of NO by superoxide anions or oxidized low density lipoproteins (7Bult H. Mol. Med. Today. 1996; 2: 510-518Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 8Cooke J.P. Dzau V.J. Annu. Rev. Med. 1997; 48: 489-509Crossref PubMed Scopus (644) Google Scholar). Accordingly, initial therapeutic strategies for an improvement of endothelial vasodilator function include the application of angiotensin-converting enzyme inhibitors that stimulate NO synthesis through the local accumulation of bradykinin as well as the supplementation of l-arginine and the use of antioxidants (7Bult H. Mol. Med. Today. 1996; 2: 510-518Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 9Moncada S. Higgs E.A. FASEB J. 1995; 9: 1319-1330Crossref PubMed Scopus (720) Google Scholar).Ascorbic acid is the most important water-soluble antioxidant in human plasma (10Frei B. England L. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6377-6381Crossref PubMed Scopus (1696) Google Scholar). It effectively scavenges superoxide and other reactive oxygen species and protects lipids against peroxidation (11Som S. Raha C. Chatterjee I.B. Acta Vitaminol. Enzymol. Google Scholar, B. J. Full Text PDF PubMed Google Scholar). Ascorbic acid is thought to a in atherogenesis studies have that plasma ascorbic acid to the from coronary A. J. Res. 1996; Google Scholar). a of known to with an increased risk for atherosclerosis have been with plasma of ascorbic acid J.C. J. 1989; PubMed Scopus Google Scholar, S. J. 1996; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. Full Text PDF PubMed Scopus Google Scholar). several studies have shown that an application of ascorbic acid endothelium-dependent vasodilation in patients with coronary artery or and in J. 1996; PubMed Scopus Google Scholar, B. 1996; PubMed Scopus Google Scholar, U. B. H. 1997; PubMed Scopus Google Scholar, E.A. 1997; PubMed Scopus Google Scholar, B. C. H. PubMed Scopus Google Scholar, H. 1996; PubMed Scopus Google Scholar). were to the of ascorbic acid may NO from effects of ascorbic acid on endothelial NO synthesis have not been present study was to whether ascorbic acid affects NO production in human endothelial cells. The synthesis of NO was measured as the formation of its co-product citrulline and as the accumulation of its effector molecule ascorbate-pretreated cells with the ionomycin and the of ascorbic acid on citrulline formation in cell lysates and on the of ecNOS protein was that ascorbic acid NO synthesis in endothelial and suggest that this action may an important of this to its beneficial effects in the vascular present study that l-ascorbic acid in J. PubMed Scopus Google Scholar, C. A. J. L. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google endothelial NO synthesis in a and was shown by of the formation of the NO co-product citrulline and the accumulation of the NO effector molecule the might additionally an increase in of ascorbate effect on NO synthesis was most to an increase of the intracellular ascorbic acid cell stimulation was in the absence of extracellular ascorbate. cells to saturated with ascorbic acid its in is Accordingly, an of the cells with ascorbate may to an intracellular accumulation of the Indeed, 100 ascorbate an uptake of the into endothelial cells was and saturated and 24 The of this uptake is as the of an mechanism has been in endothelial cells A. 1995; PubMed Scopus Google suggest that the of ascorbic acid into endothelial cells in the present study may at for the of the ascorbate effect on endothelial NO synthesis. The followed a kinetics similar to the ascorbate uptake a of cells with 100 μm ascorbic acid and and 24 a of the ascorbate uptake into endothelial cells might 100 and μm A. 1995; PubMed Scopus Google the lack of NO synthesis with ascorbic acid 100 100 μm is in the of plasma of J. PubMed Scopus Google Scholar), and similar have been in a study that the ascorbic acid as a function of in C. A. J. L. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google structure of l-ascorbic acid of an with an enediol with a Our data suggest that this structure may essential for the potentiating effect of ascorbate on NO an ascorbic acid precursor is the enediol and into ascorbic acid in human cells to the absence of the enzyme Accordingly, did not citrulline or synthesis. the other acid is to ascorbate by Med. 1994; PubMed Scopus Google a and ascorbate known to by to the enediol structure F. J. PubMed Scopus Google Scholar), was as as ascorbate in potentiating NO synthesis. ascorbate a action of the ascorbate might to a lack of the enzyme H. PubMed Scopus Google and to studies that have shown that the ascorbic acid of ascorbate was low in J. Sci. Vitaminol. PubMed Scopus Google ascorbic acid NO production in endothelial did not induce the expression of ecNOS The of ecNOS with in cell lysates or in cells was not by ascorbate ascorbic acid did not induce synthesis of NOS the protein as ecNOS by was not different in and cells. The potentiating effect of ascorbic acid on NO synthesis was not to a supplementation of the NOS substrate l-arginine ascorbic acid did not increase the cellular uptake of this acid. the endothelial NO formation is by the intracellular of cofactors such as FAD, tetrahydrobiopterin, and Annu. Rev. 1997; PubMed Scopus Google Scholar), that ascorbate might through effects on the availability of Indeed, when the NO formation was in cell lysates from cells and ascorbate-pretreated cells saturated and in the presence of NOS the potentiating effect of ascorbic acid was this was most to the of tetrahydrobiopterin a potentiating effect of an ascorbate pretreatment was seen in cell lysates when tetrahydrobiopterin was omitted from the the of tetrahydrobiopterin the ascorbate effect in the absence of the cofactors FAD, or and when tetrahydrobiopterin was the potentiating effect of an ascorbate pretreatment on citrulline synthesis seen whether FAD, and were to the cell lysates not that ascorbic acid may at through an effect on the availability of tetrahydrobiopterin or enhance the affinity of this cofactor increase of intracellular tetrahydrobiopterin has been shown to stimulate the NOS suggesting that the ecNOS is saturated with this cofactor B. H. J. PubMed Scopus Google Scholar, D. A. H. J. Full Text PDF PubMed Google Scholar). is thought to the NOS B. C. B. J. 1997; PubMed Scopus Google and as a cofactor of l-arginine and an effector of the enzyme protein B. 1995; Scopus Google Scholar). The that ascorbic acid may enhance the affinity of tetrahydrobiopterin for the NOS isoform has been H. 1995; PubMed Scopus Google vascular effects of ascorbic acid have been to its may to a protection of NO from inactivation by superoxide and to an increase of NO may and may the improvement of endothelium-dependent vasodilation in patients by an ascorbic acid application J. 1996; PubMed Scopus Google Scholar, B. 1996; PubMed Scopus Google Scholar, U. B. H. 1997; PubMed Scopus Google Scholar, E.A. 1997; PubMed Scopus Google Scholar, B. C. H. PubMed Scopus Google Scholar, H. 1996; PubMed Scopus Google Scholar). The in this study a mechanism for vascular protection by ascorbic acid may when plasma of ascorbic acid saturated intracellular ascorbate suggest that with ascorbic acid the for NO synthesis in endothelial cells and that a in the cellular ascorbic acid may the development of endothelial clinical studies ascorbate effects on endothelial include that NO formation in this study that l-ascorbic acid NO formation in endothelial cells in a The effect was to an increase of intracellular ascorbate and saturated Ascorbic acid did not induce the expression of ecNOS protein and may at through an effect on the availability or affinity of tetrahydrobiopterin for studies in to The in this study suggest that NO formation in endothelial cells on ascorbate and that with ascorbic acid may to the strategies for an improvement of endothelial vasodilator function in Endothelium-derived nitric oxide (NO)1 exerts several vasoprotective activities including smooth muscle relaxation, inhibition of platelet activation, and regulation of endothelial cell permeability and adhesivity (1Furchgott R.F. Zawadski J.V. Nature. 1980; 288: 373-376Crossref PubMed Scopus (9908) Google Scholar, 2Alheid U. Frölich J.C. Förstermann U. Thromb. Res. 1987; 47: 561-571Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 3Westendorp R.G.J. Draijer R. Meinders A.E. van Hinsberg V.W.M. J. Vasc. Res. 1994; 31: 42-51Crossref PubMed Scopus (91) Google Scholar, 4Heller R. Bussolino F. Ghigo D. Pescarmona G.P. Calvino R. Gasco A. Till U. Bosia A. Agents Actions. 1994; 45: 177-181Google Scholar). NO is generated from the conversion of l-arginine to l-citrulline by the enzymatic action of an NADPH-dependent NO synthase (NOS) that requires tetrahydrobiopterin, FAD, and FMN as cofactors (5Palmer R.M.J. Ashton D.S. Moncada S. Nature. 1988; 133: 664-666Crossref Scopus (4095) Google Scholar). The endothelial NOS isoform (ecNOS) is constitutively expressed and is activated upon an increase of intracellular calcium following cell stimulation with receptor-dependent stimuli such as thrombin and bradykinin or with receptor-independent stimuli like calcium ionophore (6Busse R. Mülsch A. FEBS Lett. 1990; 265: 133-136Crossref PubMed Scopus (431) Google Scholar). NO interferes with key processes in atherogenesis (7Bult H. Mol. Med. Today. 1996; 2: 510-518Abstract Full Text PDF PubMed Scopus (43) Google Scholar), a lack of NO might promote the development of atherosclerosis. Indeed, clinical studies have confirmed an impairment of vascular NO synthesis in patients with atherosclerosis or with increased atherogenic risk factors (8Cooke J.P. Dzau V.J. Annu. Rev. Med. 1997; 48: 489-509Crossref PubMed Scopus (644) Google Scholar). The dysfunction of the endothelial NO pathway may involve impaired signal transduction mechanisms, decreased ecNOS activity, reduced intracellular availability of l-arginine, or increased inactivation of NO by superoxide anions or oxidized low density lipoproteins (7Bult H. Mol. Med. Today. 1996; 2: 510-518Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 8Cooke J.P. Dzau V.J. Annu. Rev. Med. 1997; 48: 489-509Crossref PubMed Scopus (644) Google Scholar). Accordingly, initial therapeutic strategies for an improvement of endothelial vasodilator function include the application of angiotensin-converting enzyme inhibitors that stimulate NO synthesis through the local accumulation of bradykinin as well as the supplementation of l-arginine and the use of antioxidants (7Bult H. Mol. Med. Today. 1996; 2: 510-518Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 9Moncada S. Higgs E.A. FASEB J. 1995; 9: 1319-1330Crossref PubMed Scopus (720) Google Scholar). Ascorbic acid is the most important water-soluble antioxidant in human plasma (10Frei B. England L. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6377-6381Crossref PubMed Scopus (1696) Google Scholar). It effectively scavenges superoxide and other reactive oxygen species and protects lipids against peroxidation (11Som S. Raha C. Chatterjee I.B. Acta Vitaminol. Enzymol. Google Scholar, B. J. Full Text PDF PubMed Google Scholar). Ascorbic acid is thought to a in atherogenesis studies have that plasma ascorbic acid to the from coronary A. J. Res. 1996; Google Scholar). a of known to with an increased risk for atherosclerosis have been with plasma of ascorbic acid J.C. J. 1989; PubMed Scopus Google Scholar, S. J. 1996; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. Full Text PDF PubMed Scopus Google Scholar). several studies have shown that an application of ascorbic acid endothelium-dependent vasodilation in patients with coronary artery or and in J. 1996; PubMed Scopus Google Scholar, B. 1996; PubMed Scopus Google Scholar, U. B. H. 1997; PubMed Scopus Google Scholar, E.A. 1997; PubMed Scopus Google Scholar, B. C. H. PubMed Scopus Google Scholar, H. 1996; PubMed Scopus Google Scholar). were to the of ascorbic acid may NO from effects of ascorbic acid on endothelial NO synthesis have not been The present study was to whether ascorbic acid affects NO production in human endothelial cells. The synthesis of NO was measured as the formation of its co-product citrulline and as the accumulation of its effector molecule ascorbate-pretreated cells with the ionomycin and the of ascorbic acid on citrulline formation in cell lysates and on the of ecNOS protein was that ascorbic acid NO synthesis in endothelial and suggest that this action may an important of this to its beneficial effects in the vascular present study that l-ascorbic acid in J. PubMed Scopus Google Scholar, C. A. J. L. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google endothelial NO synthesis in a and was shown by of the formation of the NO co-product citrulline and the accumulation of the NO effector molecule the might additionally an increase in of ascorbate effect on NO synthesis was most to an increase of the intracellular ascorbic acid cell stimulation was in the absence of extracellular ascorbate. cells to saturated with ascorbic acid its in is Accordingly, an of the cells with ascorbate may to an intracellular accumulation of the Indeed, 100 ascorbate an uptake of the into endothelial cells was and saturated and 24 The of this uptake is as the of an mechanism has been in endothelial cells A. 1995; PubMed Scopus Google suggest that the of ascorbic acid into endothelial cells in the present study may at for the of the ascorbate effect on endothelial NO synthesis. The followed a kinetics similar to the ascorbate uptake a of cells with 100 μm ascorbic acid and and 24 a of the ascorbate uptake into endothelial cells might 100 and μm A. 1995; PubMed Scopus Google the lack of NO synthesis with ascorbic acid 100 100 μm is in the of plasma of J. PubMed Scopus Google Scholar), and similar have been in a study that the ascorbic acid as a function of in C. A. J. L. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google structure of l-ascorbic acid of an with an enediol with a Our data suggest that this structure may essential for the potentiating effect of ascorbate on NO an ascorbic acid precursor is the enediol and into ascorbic acid in human cells to the absence of the enzyme Accordingly, did not citrulline or synthesis. the other acid is to ascorbate by Med. 1994; PubMed Scopus Google a and ascorbate known to by to the enediol structure F. J. PubMed Scopus Google Scholar), was as as ascorbate in potentiating NO synthesis. ascorbate a action of the ascorbate might to a lack of the enzyme H. PubMed Scopus Google and to studies that have shown that the ascorbic acid of ascorbate was low in J. Sci. Vitaminol. PubMed Scopus Google ascorbic acid NO production in endothelial did not induce the expression of ecNOS The of ecNOS with in cell lysates or in cells was not by ascorbate ascorbic acid did not induce synthesis of NOS the protein as ecNOS by was not different in and cells. The potentiating effect of ascorbic acid on NO synthesis was not to a supplementation of the NOS substrate l-arginine ascorbic acid did not increase the cellular uptake of this acid. the endothelial NO formation is by the intracellular of cofactors such as FAD, tetrahydrobiopterin, and Annu. Rev. 1997; PubMed Scopus Google Scholar), that ascorbate might through effects on the availability of Indeed, when the NO formation was in cell lysates from cells and ascorbate-pretreated cells saturated and in the presence of NOS the potentiating effect of ascorbic acid was this was most to the of tetrahydrobiopterin a potentiating effect of an ascorbate pretreatment was seen in cell lysates when tetrahydrobiopterin was omitted from the the of tetrahydrobiopterin the ascorbate effect in the absence of the cofactors FAD, or and when tetrahydrobiopterin was the potentiating effect of an ascorbate pretreatment on citrulline synthesis seen whether FAD, and were to the cell lysates not that ascorbic acid may at through an effect on the availability of tetrahydrobiopterin or enhance the affinity of this cofactor increase of intracellular tetrahydrobiopterin has been shown to stimulate the NOS suggesting that the ecNOS is saturated with this cofactor B. H. J. PubMed Scopus Google Scholar, D. A. H. J. Full Text PDF PubMed Google Scholar). is thought to the NOS B. C. B. J. 1997; PubMed Scopus Google and as a cofactor of l-arginine and an effector of the enzyme protein B. 1995; Scopus Google Scholar). The that ascorbic acid may enhance the affinity of tetrahydrobiopterin for the NOS isoform has been H. 1995; PubMed Scopus Google vascular effects of ascorbic acid have been to its may to a protection of NO from inactivation by superoxide and to an increase of NO may and may the improvement of endothelium-dependent vasodilation in patients by an ascorbic acid application J. 1996; PubMed Scopus Google Scholar, B. 1996; PubMed Scopus Google Scholar, U. B. H. 1997; PubMed Scopus Google Scholar, E.A. 1997; PubMed Scopus Google Scholar, B. C. H. PubMed Scopus Google Scholar, H. 1996; PubMed Scopus Google Scholar). The in this study a mechanism for vascular protection by ascorbic acid may when plasma of ascorbic acid saturated intracellular ascorbate suggest that with ascorbic acid the for NO synthesis in endothelial cells and that a in the cellular ascorbic acid may the development of endothelial clinical studies ascorbate effects on endothelial include that NO formation in this study that l-ascorbic acid NO formation in endothelial cells in a The effect was to an increase of intracellular ascorbate and saturated Ascorbic acid did not induce the expression of ecNOS protein and may at through an effect on the availability or affinity of tetrahydrobiopterin for studies in to The in this study suggest that NO formation in endothelial cells on ascorbate and that with ascorbic acid may to the strategies for an improvement of endothelial vasodilator function in The present study that l-ascorbic acid in J. PubMed Scopus Google Scholar, C. A. J. L. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google endothelial NO synthesis in a and was shown by of the formation of the NO co-product citrulline and the accumulation of the NO effector molecule the might additionally an increase in of The ascorbate effect on NO synthesis was most to an increase of the intracellular ascorbic acid cell stimulation was in the absence of extracellular ascorbate. cells to saturated with ascorbic acid its in is Accordingly, an of the cells with ascorbate may to an intracellular accumulation of the Indeed, 100 ascorbate an uptake of the into endothelial cells was and saturated and 24 The of this uptake is as the of an mechanism has been in endothelial cells A. 1995; PubMed Scopus Google Scholar). suggest that the of ascorbic acid into endothelial cells in the present study may at for the of the ascorbate effect on endothelial NO synthesis. The followed a kinetics similar to the ascorbate uptake a of cells with 100 μm ascorbic acid and and 24 a of the ascorbate uptake into endothelial cells might 100 and μm A. 1995; PubMed Scopus Google the lack of NO synthesis with ascorbic acid 100 100 μm is in the of plasma of J. PubMed Scopus Google Scholar), and similar have been in a study that the ascorbic acid as a function of in C. A. J. L. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). The structure of l-ascorbic acid of an with an enediol with a Our data suggest that this structure may essential for the potentiating effect of ascorbate on NO an ascorbic acid precursor is the enediol and into ascorbic acid in human cells to the absence of the enzyme Accordingly, did not citrulline or synthesis. the other acid is to ascorbate by Med. 1994; PubMed Scopus Google a and ascorbate known to by to the enediol structure F. J. PubMed Scopus Google Scholar), was as as ascorbate in potentiating NO synthesis. ascorbate a action of the ascorbate might to a lack of the enzyme H. PubMed Scopus Google and to studies that have shown that the ascorbic acid of ascorbate was low in J. Sci. Vitaminol. PubMed Scopus Google Scholar). ascorbic acid NO production in endothelial did not induce the expression of ecNOS The of ecNOS with in cell lysates or in cells was not by ascorbate ascorbic acid did not induce synthesis of NOS the protein as ecNOS by was not different in and cells. The potentiating effect of ascorbic acid on NO synthesis was not to a supplementation of the NOS substrate l-arginine ascorbic acid did not increase the cellular uptake of this acid. the endothelial NO formation is by the intracellular of cofactors such as FAD, tetrahydrobiopterin, and Annu. Rev. 1997; PubMed Scopus Google Scholar), that ascorbate might through effects on the availability of Indeed, when the NO formation was in cell lysates from cells and ascorbate-pretreated cells saturated and in the presence of NOS the potentiating effect of ascorbic acid was this was most to the of tetrahydrobiopterin a potentiating effect of an ascorbate pretreatment was seen in cell lysates when tetrahydrobiopterin was omitted from the the of tetrahydrobiopterin the ascorbate effect in the absence of the cofactors FAD, or and when tetrahydrobiopterin was the potentiating effect of an ascorbate pretreatment on citrulline synthesis seen whether FAD, and were to the cell lysates not that ascorbic acid may at through an effect on the availability of tetrahydrobiopterin or enhance the affinity of this cofactor increase of intracellular tetrahydrobiopterin has been shown to stimulate the NOS suggesting that the ecNOS is saturated with this cofactor B. H. J. PubMed Scopus Google Scholar, D. A. H. J. Full Text PDF PubMed Google Scholar). is thought to the NOS B. C. B. J. 1997; PubMed Scopus Google and as a cofactor of l-arginine and an effector of the enzyme protein B. 1995; Scopus Google Scholar). The that ascorbic acid may enhance the affinity of tetrahydrobiopterin for the NOS isoform has been H. 1995; PubMed Scopus Google Scholar). vascular effects of ascorbic acid have been to its may to a protection of NO from inactivation by superoxide and to an increase of NO may and may the improvement of endothelium-dependent vasodilation in patients by an ascorbic acid application J. 1996; PubMed Scopus Google Scholar, B. 1996; PubMed Scopus Google Scholar, U. B. H. 1997; PubMed Scopus Google Scholar, E.A. 1997; PubMed Scopus Google Scholar, B. C. H. PubMed Scopus Google Scholar, H. 1996; PubMed Scopus Google Scholar). The in this study a mechanism for vascular protection by ascorbic acid may when plasma of ascorbic acid saturated intracellular ascorbate suggest that with ascorbic acid the for NO synthesis in endothelial cells and that a in the cellular ascorbic acid may the development of endothelial clinical studies ascorbate effects on endothelial include that NO formation in this study that l-ascorbic acid NO formation in endothelial cells in a The effect was to an increase of intracellular ascorbate and saturated Ascorbic acid did not induce the expression of ecNOS protein and may at through an effect on the availability or affinity of tetrahydrobiopterin for studies in to The in this study suggest that NO formation in endothelial cells on ascorbate and that with ascorbic acid may to the strategies for an improvement of endothelial vasodilator function in and for