Oxidative stress in glial cultures: detection by DAF-2 fluorescence used as a tool to measure peroxynitrite rather than nitric oxide.
4,5-diaminofluorescein diacetate (DAF-2DA) is widely used as a fluorescent probe to detect endogenously produced nitric oxide (NO). Recent reports that refer to the high sensitivity of DAF-2 toward NO prompted us to test its efficiency and specificity in a mixed murine primary glial culture model, in which the NO-synthesizing enzyme inducible nitric oxide synthase (iNOS) is expressed by stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Cultures were loaded with DAF-2DA and the fluorescence was measured using confocal microscopy. NO production in the cultures was determined using the ozone/chemiluminescence technique. Due to the extremely high photosensitivity of DAF-2, low laser intensities were used to avoid artifacts. No difference in DAF-2 fluorescence was observed in NO-producing cultures compared to control cultures, whereas the NO/peroxynitrite-sensitive dye 2,7-dihydrodichlorofluorescein (DCF) showed a significant fluorescence increase specifically in microglia cells. A detectable gain in fluorescence was seen when NO-containing buffer was added to the DAF-2DA-loaded cells with a minimum NO concentration at 7.7 microM. An additional gain of DAF-2 fluorescence was obtained when the cells were depleted of glutathione (GSH) with L-buthionine S,R-sulfoximine (BSO). Hence, we monitored the change in DAF-2 fluorescence intensity in the presence of NO and O(-*)(2) in a cell-free solution. The fluorescence due to NO was indeed larger when O(-*)(2) was added, implying a higher sensitivity of DAF-2 for peroxynitrite. Nevertheless, our results also indicate that measurement of DCF fluorescence is a better tool for monitoring intracellular changes in the levels of NO and/or peroxynitrite than DAF-2.
Roychowdhury
Sanjoy
S
Otto-von-Guericke University, Institute for Medical Neurobiology, Magdeburg, Germany.
Luthe
Andreas
A
Keilhoff
Gerburg
G
Wolf
Gerald
G
Horn
Thomas F W
TF
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002474:Cell-Free System; D002478:Cells, Cultured; D019793:Fluorescein; D005452:Fluoresceins; D005978:Glutathione / Q000378:metabolism; D007202:Indicators and Reagents; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D030421:Peroxynitrous Acid / Q000032:analysis / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013481:Superoxides / Q000378:metabolism
C414689:4,5-diaminofluorescein diacetate; D000970:Antineoplastic Agents; D005452:Fluoresceins; D007202:Indicators and Reagents; D008070:Lipopolysaccharides; D013481:Superoxides; D030421:Peroxynitrous Acid; C029569:diacetyldichlorofluorescein; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D005978:Glutathione; D019793:Fluorescein