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 throug h the inducible nitric oxide synthase (iNOS) pathway. The iNOS gene was syn ergistically induced by LPS and IFN-gamma. The inductive mechanism of ascor bate 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-ka ppa B) by gel shift assays, and protein levels of interferon regulatory fac tor 1 (IRF-1) in LPS- and IFN-gamma-activated cells. Ascorbate had no effec t on the onset of either I kappa B alpha degradation or the nuclear translo cation of NF-kappa B, but it delayed the recovery of I kappa B alpha. The p rolonged 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 indu ce I kappa B alpha degradation or NF-kappa B activation. Furthermore, ascor bate exerted no effect on the expression of I kappa B alpha and ubiquitin g enes in the activated cells. Ascorbate could modulate NF-kappa B DNA bindin g activity in response to combined LPS and IFN-gamma activation, which incr eases NO production in activated macrophages.