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-γ (IFN-γ)-activated macrophage J774.1 cells through the inducible nitric oxide synthase (iNOS) pathway. The iNOS gene was synergistically induced by LPS and IFN-γ. The inductive mechanism of ascorbate on the iNOS gene was studied by examining the degradation of IκBα by Western blotting, activation of the nuclear factor kappa B (NF-κB) by gel shift assays, and protein levels of interferon regulatory factor 1 (IRF-1) in LPS- and IFN-γ-activated cells. Ascorbate had no effect on the onset of either IκBα degradation or the nuclear translocation of NF-κB, but it delayed the recovery of IκBα. The prolonged degradation of IκBα caused by ascorbate in LPS- and IFN-γ-activated cells paralleled elevated NF-κB binding to DNA, which led to an increase in the iNOS protein level. Ascorbate alone did not induce IκBα degradation or NF-κB activation. Furthermore, ascorbate exerted no effect on the expression of IκBα and ubiquitin genes in the activated cells. Ascorbate could modulate NF-κB DNA binding activity in response to combined LPS and IFN-γ activation, which increases NO production in activated macrophages.