title: Ascorbate enhances iNOS activity by increasing tetrahydrobiopterin in RAW 264.7 cells.
abstract: Studies on the effect of ascorbic acid on inducible nitric oxide synthase (iNOS) activity are few and diverse, likely to be dependent on the species of cells. We investigated a role of ascorbic acid in iNOS induction and nitric oxide (NO) generation in mouse macrophage cell line RAW 264.7. Although interferon- (IFN-) gamma alone produced NO end products, ascorbic acid enhanced NO production only when cells were synergistically stimulated with IFN-gamma plus Escherichia coli lipopolysaccharide (LPS). Ascorbate neither enhanced nor decreased the expression of iNOS protein in RAW 264.7 cells, in contrast to the reports that ascorbic acid augments iNOS induction in a mouse macrophage-like cell line J774.1 and that ascorbate suppresses iNOS induction in rat skeletal muscle endothelial cells. Intracellular levels of tetrahydrobiopterin (BH4), a cofactor for iNOS, were increased by ascorbate in RAW 264.7 cells. However, ascorbate did not increase GTP cyclohydrolase I mRNA, the main enzyme at the critical steps in the BH4 synthetic pathway, expression levels and activity. Sepiapterin, which supplies BH4 via salvage pathway, more efficiently enhanced NO production if ascorbate was added. These data suggest that enhanced activation of iNOS by ascorbic acid is mediated by increasing the stability of BH4 in RAW 264.7 cells.
authors.affiliation: Department of Dermatology, Kagawa Medical University, Kagawa, Japan.
authors.forename: Kozo
authors.initials: K
authors.lastname: Nakai
authors.forename: Masahide
authors.initials: M
authors.lastname: Urushihara
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Kubota
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kosaka
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology*; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism*; D002478:Cells, Cultured; D004357:Drug Synergism; D004926:Escherichia coli; D006136:GTP Cyclohydrolase / Q000235:genetics / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D011622:Pterins / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000970:Antineoplastic Agents; D000975:Antioxidants; D008070:Lipopolysaccharides; D011622:Pterins; D012333:RNA, Messenger; D001708:Biopterins; D009569:Nitric Oxide; D007371:Interferon-gamma; C016727:sepiapterin; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D006136:GTP Cyclohydrolase; C003402:sapropterin; D001205:Ascorbic Acid