Antioxidants differentially affect nuclear factor κB-mediated nitric oxide synthase expression in vascular smooth muscle cells

Abstract

Increased ‘oxidative stress’ resulting in the activation of nuclear factor κB (NF-κB) is thought to play a crucial role in the cytokine-mediated expression of the inducible isoform of nitric oxide synthase (iNOS) in different cell types. Therefore, the effects of four different antioxidants, carbocromen, chrysin, 3,4-dichloroisocoumarin (DCI) and N-acetylserotonin (NAS), on iNOS expression were investigated in vascular smooth muscle cells (VSMC). All antioxidants strongly reduced the phorbol ester-stimulated superoxide anion formation in native VSMC. Carbocromen (200 μM) and chrysin (50 μM) had no effect, while NAS (1 mM) abolished the increase in nitrine production and iNOS protein abundance in cultured VSMC exposed to interleukin-1β (IL-1β, 60 U/ml) or the adenylyl cyclase activator forskolin (10 μM). DCI also revealed a marked inhibitory effect in IL-1β-stimulated VSMC, but was less effective in cells treated with forskolin. DCI, but not NAS, also suppressed the activation of NF-κB in VSMC exposed to IL-1β, while no significant NF-κB activation was detected in forskolin-treated cells. These findings demonstrate that antioxidants differentially affect iNOS expression in VSMC both at the transcriptional level by preventing the activation of NF-κB and at the post-transcriptional level, presumably by promoting iNOS mRNA or protein degradation. They also suggest that reactive oxygen intermediates do not play a role in the activation of NF-κB by IL-1β in VSMC, and that transcripton factors other than NF-κB mediate the induction of iNOS expression by elevating the intracellular concentration of cyclic AMP.