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 peroxide 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 characterized by Northern blotting, was increased both time- and dose-dependently by xanthine oxidase to up to 500% above baseline. Analysis of endothelin-1 promoter activity using a construct containing 1329 bp of the endothelin-1 promoter revealed that promoter activity was increased up to eight-fold by incubation with xanthine oxidase. Specificity was ascertained by co-incubation with 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 significantly alter endothelin-1 promoter activity. Synthesis of intracellular big endothelin-1 protein was increased dose-dependently by xanthine oxidase. Our results indicate that oxidative stress leads to increased endothelial synthesis of big endothelin-1, which is a previously unknown mechanism and may help to understand the detrimental association of increased oxidative stress and elevated endothelin-1 levels in pathophysiological conditions promoting atherosclerosis.
Kähler
J
J
Department of Cardiology, University Hospital Hamburg, Germany.
Mendel
S
S
Weckmüller
J
J
Orzechowski
H D
HD
Mittmann
C
C
Köster
R
R
Paul
M
M
Meinertz
T
T
Münzel
T
T
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D015152:Blotting, Northern; D002374:Catalase / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000235:genetics* / Q000378:metabolism*; D016232:Endothelins / Q000096:biosynthesis*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000378:metabolism; D011863:Radioimmunoassay; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism; D014969:Xanthine Oxidase / Q000494:pharmacology
D019332:Endothelin-1; D016232:Endothelins; D004791:Enzyme Inhibitors; D005609:Free Radicals; D011498:Protein Precursors; D012333:RNA, Messenger; D006861:Hydrogen Peroxide; D002374:Catalase; D008156:Luciferases; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D019331:NG-Nitroarginine Methyl Ester