© 1999 American Heart Association, Inc.
Original Contribution |
Chemiluminescent Detection of Oxidants in Vascular Tissue
Lucigenin But Not Coelenterazine Enhances Superoxide Formation
From the Departments of Anesthesiology (M.M.T., G.R., B.A.F.), Medicine (C.R.W.), and Biochemistry and Medical Genetics (B.A.F.), and the Center for Free Radical Biology (M.M.T., C.R.W., B.A.F.), University of Alabama at Birmingham, Birmingham, Ala, and Department of Biochemistry (E.S., R.R.), Universidad de la Republica, Montevideo, Uruguay.
Correspondence to Margaret M. Tarpey, Department of Anesthesiology, University of Alabama at Birmingham, 619 19th St S, Birmingham, AL 35233. E-mail margaret.tarpey{at}ccc.uab.edu
Abstract—Lucigenin-amplified chemiluminescence has frequently been used to assess the formation of superoxide in vascular tissues. However, the ability of lucigenin to undergo redox cycling in purified enzyme-substrate mixtures has raised questions concerning the use of lucigenin as an appropriate probe for the measurement of superoxide production. Addition of lucigenin to reaction mixtures of xanthine oxidase plus NADH resulted in increased oxygen consumption, as well as superoxide dismutase–inhibitable reduction of cytochrome c, indicative of enhanced rates of superoxide formation. Additionally, it was revealed that lucigenin stimulated oxidant formation by both cultured bovine aortic endothelial cells and isolated rings from rat aorta. Lucigenin treatment resulted in enhanced hydrogen peroxide release from endothelial cells, whereas exposure to lucigenin resulted in inhibition of endothelium-dependent relaxation in isolated aortic rings that was superoxide dismutase inhibitable. In contrast, the chemiluminescent probe coelenterazine had no significant effect on xanthine oxidase–dependent oxygen consumption, endothelial cell hydrogen peroxide release, or endothelium-dependent relaxation. Study of enzyme and vascular systems indicated that coelenterazine chemiluminescence is a sensitive marker for detecting both superoxide and peroxynitrite.
Key Words: superoxide • free radical • lucigenin • coelenterazine • peroxynitrite
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