Abstract
Extracellular superoxide production in the Chinese hamster CCL39 fibroblast cell line was detected by direct counting of luminol-enhanced chemiluminescence in the presence of horseradish peroxidase (HRP). This superoxide production, which is directly related to cell density, can be inhibited by diphenylene iodonium, a specific inhibitor of NADPH-oxidase. The O−2generation is transiently inhibited following exposure of the cells to elevated temperatures. To study the possible relationship between HSP27 and its modulation of NADPH-oxidase activity we measured the generation of O−2activity in CCL39 cells containing either the gene coding for human HSP27 or a mutant form of this gene leading to HSP27 unable to be phosphorylated. Cells with the enhanced expression of normal human HSP27 as well as the non-phosphorylatable protein exhibited a 20-fold higher superoxide production than control CCL39 cells. Furthermore, we demonstrate that accumulation of normal HSP27 appears to play a role in prevention of NADPH-oxidase inhibition by heat.
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