DETECTION AND IMAGING OF NITRIC-OXIDE WITH NOVEL FLUORESCENT INDICATORS - DIAMINOFLUORESCEINS

Nitric oxide is a gaseous, free radical which plays a role as an intra cellular second messenger and a diffusable intercellular messenger. To obtain direct evidence for NO functions in vivo, we have designed and synthesized diaminofluoresceins (DAFs) as novel fluorescent indicator s for NO. The fluorescent chemical transformation of DAFs is based on the reactivity of the aromatic vicinal diamines with NO in the presenc e of dioxygen, The N-nitrosation of DAFs, yielding the highly green-fl uorescent triazole form, offers the advantages of specificity, sensiti vity, and a simple protocol for the direct detection of NO (detection limit 5 nM). The fluorescence quantum efficiencies are increased more than 100 times after the transformation of DAFs by NO. Fluorescence de tection with visible light excitation and high sensitivity enabled the practical assay of NO production in living cells. Membrane-permeable DAF-2 diacetate (DAF-2 DA) can be used for real-time bioimaging of NO with fine temporal and spatial resolution. The dye was loaded into act ivated rat aortic smooth muscle cells, where the ester bonds are hydro lyzed by intracellular esterase, generating DAF-2. The fluorescence in the cells increased in a NO concentration-dependent manner.