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 •B (NF-KB) 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 jaM) and chrysin (50 jaM) had no effect, while NAS (1 mM) abolished the increase in nitrine production and iNOS protein abundance in cultured VSMC exposed to interleukin-li~ (IL-I~, 60 U/ml) or the adenylyl cyclase activator forskolin (10 jaM). DCI also revealed a marked inhibitory effect in IL-1/$-stimulated VSMC, but was less effective in cells treated with forskolin. DCI, but not NAS, also suppressed the activation of NF-IcB in VSMC exposed to IL-I~, while no significant NF-KB 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-KB 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-KB by IL-I~ in VSMC, and that transeripton factors other than NF-KB mediate the induction of iNOS expression by elevating the intracellular concentration of cyclic AMP.

Expression of Inducible Nitric-oxide Synthase and Intracellular Protein Tyrosine Nitration in Vascular Smooth Muscle Cells

“…The proinflammatory cytokines (TNF-␣, IL-1␤, and IFN-␥) and LPS utilized in this study have been previously shown to induce expression of iNOS in vascular smooth muscle cells (1)(2)(3)(4)(5), and the increased production of nitric oxide that accompanies the induction of iNOS is thought to contribute in the pathophysiology of sepsis and other inflammatory disorders (1)(2)(3)(4)(5). The expression of iNOS was significantly increased after inhibition of nitric oxide production (Fig.…”

“…3). NF-B is one of the transcriptional factors that mediates transcription of iNOS in rat smooth muscle cells (2)(3)(4), and numerous studies have implicated reactive oxygen species in the regulation of NF-B activity under inflammatory conditions (11)(12)(13)(14)(15). The data in this cell model system indicate that upon stimulation with cytokines and LPS, endogenous H 2 O 2 prevents the activation of this transcription factor, consistent with observations in alve- Cells stably transfected with catalase demonstrated cytokine-mediated intracellular protein nitration comparable to that of the vector-transfected control cells (Fig.…”

“…1 has been shown to induce the expression of inducible nitric-oxide synthase (iNOS), which may account for the vasorelaxation and hypotension associated with inflammatory and septic conditions (1)(2)(3)(4)(5). A number of transcriptional factors including nuclear transcription factor B (NF-B) have been shown to mediate the expression of iNOS in vascular smooth muscle cells (2)(3)(4).…”

“…A number of transcriptional factors including nuclear transcription factor B (NF-B) have been shown to mediate the expression of iNOS in vascular smooth muscle cells (2)(3)(4). In addition to nitric oxide, exposure of vascular smooth muscle cells to cytokines such as TNF-␣ or IL-1␤ also results in the generation of reduced oxygen species such as superoxide and hydrogen peroxide (5)(6)(7)(8)(9).…”

Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases

“…In rat thoracic VSM cells, pharmacological antioxidants have been shown to modulate NF-κB activation [64,70,88]. We as well as others have shown that NF-κB activity is potentiated by increased protein levels of catalase, although in this case the mechanisms of action have not been delineated [42,60].…”