A reactive oxygen-generating system activates nuclear Factor-kappa B and releases tumor necrosis factor-alpha in coronary smooth muscle cells

Background. Recently we reported that bacterial lipopolysaccharide (LPS) st imulates release of tumor necrosis factor alpha (TNF-alpha) from porcine co ronary arteries and smooth muscle cells cultured from those vessels. It has also been reported that plasma levels of TNF-a are elevated after myocardi al infarction. Since it is known that the production of reactive oxygen int ermediates (ROI) occurs during ischemia and ROI are suggested activators of the nuclear regulatory factor kappa B (NF-kappa B), we tested the hypothes is that release of TNF-alpha from smooth muscle cells could also be stimula ted with a ROI-generating system, Materials and Methods. Smooth muscle cells were isolated from porcine coron ary arteries. Confluent cells in 48-well culture dishes were treated for 30 min with 0.003 units/ml xanthine oxidase (XO) and 2 mM hypoxanthine (IM) a dded to the culture medium. The medium was then removed and the cells were washed three times and fresh medium without HX-XO was added. Then, at 1, 3, and 6 h the medium was removed and analyzed for biologically active TNF-al pha In other experiments, smooth muscle cells were treated with 20 mu g/ml LPS for 6 h and aliquots of medium analyzed for TNF-alpha. Untreated cells served as controls. Data were analyzed by two-way ANOVA with repeated measu res. Extracts of total cell protein were prepared and activation of NF-kapp a B was determined by electrophoretic mobility shift assay. Results. Treatment of cells with HX-XO stimulated release of TNF-alpha whic h rose to a maximum of 17.5 +/- 1.7 units/mg cell protein at 6 h, This was significantly higher (P < 0.05) than release stimulated by LPS (10.2 +/- 1. 0 units/mg at 6 h) or TNF-alpha detected in the culture medium from untreat ed control cells (4.2 +/- 0.9 units/mg protein at 6 h). Both HX/XO and LPS activated NF-kappa B. Conclusions, These results support the conclusion that coronary smooth musc le cells are a potential source of TNF-alpha during events that are associa ted with formation of ROI such as myocardial ischemia. (C) 1999 Academic Pr ess.