DEVELOPMENT OF A FLUORESCENT INDICATOR FOR NITRIC-OXIDE BASED ON THE FLUORESCEIN CHROMOPHORE

Endogenous nitric oxide (NO) appears to modulate many physiological an d pathophysiological processes. In order to obtain direct evidence for NO functions in vivo, we have developed 4,5-diaminofluorescein (DAF-2 ) as a novel fluorescent indicator for NO. Green-fluorescent triazolof luorescein formed by the reaction of NO and DAF-2 affords high sensiti vity for NO (detection limit: 5 nM). Membrane-permeable DAF-2 diacetat e (DAF-2 DA) was loaded into activated rat aortic smooth muscle cells, where the eater bonds are hydrolyzed by intracellular esterase, gener ating DAF-2. The fluorescence in the cells increased in a NO concentra tion-dependent manner. This imaging method should be useful for studie s of the dynamic biological actions of NO at the molecular level with fine temporal and spatial resolution.