Molecular role of ascorbate in enhancement of NO production in activated macrophage-like cell line, J774.1.
Ascorbate-enhanced nitric oxide (NO) production in lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-activated macrophage J774.1 cells through the inducible nitric oxide synthase (iNOS) pathway. The iNOS gene was synergistically induced by LPS and IFN-gamma. The inductive mechanism of ascorbate on the iNOS gene was studied by examining the degradation of I kappa B alpha by Western blotting, activation of the nuclear factor kappa B (NF-kappa B) by gel shift assays, and protein levels of interferon regulatory factor 1 (IRF-1) in LPS- and IFN-gamma-activated cells. Ascorbate had no effect on the onset of either I kappa B alpha degradation or the nuclear translocation of NF-kappa B, but it delayed the recovery of I kappa B alpha. The prolonged degradation of I kappa B alpha caused by ascorbate in LPS- and IFN-gamma-activated cells paralleled elevated NF-kappa B binding to DNA, which led to an increase in the iNOS protein level. Ascorbate alone did not induce I kappa B alpha degradation or NF-kappa B activation. Furthermore, ascorbate exerted no effect on the expression of I kappa B alpha and ubiquitin genes in the activated cells. Ascorbate could modulate NF-kappa B DNA binding activity in response to combined LPS and IFN-gamma activation, which increases NO production in activated macrophages.
Mizutani
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Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Japan.
Tsukagoshi
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N
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D020672:I-kappa B Proteins; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010750:Phosphoproteins / Q000378:metabolism; D014452:Ubiquitins / Q000235:genetics
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D010750:Phosphoproteins; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D001205:Ascorbic Acid