INTRACELLULAR HYDROGEN-PEROXIDE AND SUPEROXIDE ANION DETECTION IN ENDOTHELIAL-CELLS

One of the objectives of studying endothelial cells in vitro is to eva luate neutrophil-endothelial cell interactions including potential con sequences of oxidant-mediated damage to the endothelial cell. Current understanding of endothelial cell oxidative function is derived primar ily from the measurement of extracellular products. We utilized 2 dyes , 2',7'-dichlorofluorescin diacetate (DCFH-DA) and hydroethidine (HE), which measure hydrogen peroxide (H2O2) and superoxide anion (O-2(-)) respectively, for their suitability to monitor oxidative mechanisms in endothelial cells and to provide a reliable measure of intracellular oxidants. Endothelial cells stained with DCFH-DA and stimulated with H 2O2 exhibited an increase in the fluorescent product 2',7'-dichloroflu orescein (DCF) (measure of intracellular H2O2) which peaked at 10 min. Endothelial cells stained with HE and stimulated with H2O2 exhibited an increase in the fluorescent product ethidium bromide (EB) (measure of intracellular O-2(-)) which lasted for approximately 60 min. Supero xide dismutase increased DCF fluorescence in endothelial cells stimula ted with H2O2 by 158%. Allopurinol (xanthine oxidase inhibitor) reduce d DCF and EB fluorescence by 48% and 37% respectively in endothelial c ells stimulated with H2O2. Catalase completely inhibited an increase i n DCF or EB fluorescence in endothelial cells stimulated with H2O2 The re was a direct correlation between mean DCF and EB fluorescence inten sity and the concentration of H2O2 Or the number of phorbol 12-myrista te 13-acetate-activated neutrophils added to endothelial cells. We con clude from these studies that DCFH-DA and HE can be used to measure in tracellular H2O2 and O-2(-) in endothelial cells and that the xanthine oxidase pathway for intracellular O-2(-) production accounts for appr oximately 40% of the total intracellular O-2(-) generated in endotheli al cells after stimulation with H2O2. The combination of image cytomet ry and flow cytometry will be important for future evaluations of endo thelial cell function.