Oxidative stress increases synthesis of big endothelin-1 by activation of the endothelin-1 promoter

Modulation of the biosynthesis of the vasoconstrictor peptide endothelin-1 by oxygen-derived free radicals generated by xanthine oxidase or hydrogen p eroxide was studied in cultured endothelial cells. Endothelin-1 metabolism was investigated at the level of endothelin-1 promoter, preproendothelin-1 mRNA and intracellular big endothelin-1. Endothelin-1 mRNA, as characterize d by Northern blotting, was increased both time-and dose-dependently by xan thine oxidase to up to 500% above baseline. Analysis of endothelin-1 promot er activity using a construct containing 1329 bp of the endothelin-1 promot er revealed that promoter activity was increased up to eight-fold by incuba tion with xanthine oxidase. Specificity was ascertained by co-incubation wi th superoxide dismutase and catalase leading to inhibition of the effect of xanthine oxidase. A significant contribution of nitric oxide was ruled out , since NOS III-mRNA transcription remained unchanged and L-NAME did not si gnificantly alter endothelin-l promoter activity. Synthesis of intracellula r big endothelin-1 protein was increased dose-dependently by xanthine oxida se. Our results indicate that oxidative stress leads to increased endotheli al synthesis of big endothelin-1, which is a previously unknown mechanism a nd may help to understand the detrimental association of increased oxidativ e stress and elevated endothelin-1 levels in pathophysiological conditions promoting atherosclerosis. (C) 2000 Academic Press.