Inhibitory Effects of Catecholamines and Anti-Oxidants on the Fluorescence Reaction of 4,5-Diaminofluorescein, DAF-2, a Novel Indicator of Nitric Oxide

4,5-Diaminofluorescein (DAF-2) is a newly developed indicator of nitric oxide (NO). Two amino groups of DAF-2 are oxidized by NO. We investigated the effects of reducers on the NO-induced oxidation of DAF-2. NOC-5 (0.1-10 μM), a NO-donor, concentration-depen-dently elicited fluorescence with 10 μM DAF-2. The rate of the fluorescence reaction was dependent on the width of the excitation band path. The presence of catecholamines (1 μM), but not tyrosine or phenylephrine, attenuated the fluorescence induced by NOC-5. Ascor-bate and other reducers like dithiothreitol, 2-mercaptoethanol, or glutathione (all 1 mM) abolished the fluorescence. These results suggest that reducers attenuate the NO-induced fluorescence of DAF-2 mainly through an anti-oxidative action.