Effect of radical scavengers on TNF alpha-mediated activation of the uPA in cultured cells.

Active oxygen, produced by cultured cells following stimulation with various growth factors, seems to be involved in signal transduction leading to cellular responses such as gene expression and growth modulation. In the present study, the intracellular oxidation state was measured in immortalized human endothelial cells (ECV304) after treatment with tumor necrosis factor (TNF) alpha, using a fluorescent dye and a laser-scanning confocal microscope. The intracellular oxidation state was increased 60 min after the addition of TNF alpha, and this increase was abolished by a radical scavenger, N-acetylcysteine (NAC), which is also a precursor of glutathione, and by pyrrolidine dithiocarbamate (PDTC). TNF alpha increased the steady state level of urokinase-type plasminogen activator (uPA), and NAC inhibited this increase at a dose that also inhibited the increase in the intracellular oxidation state. PDTC, on the other hand, did not affect the induction of the uPA gene by TNF alpha. These results suggest that intracellular glutathione level rather than the oxidation state is necessary for the induction of the uPA gene by TNF alpha.