Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry

A simple and sensitive assay and a cellular bioimaging method for nitric ox ide (NO) were developed using a novel diaminofluorescein DAF-FM and its dia cetate. DAF-FM is converted via an NO-specific mechanism to an intensely fl uorescent triazole derivative. For the measurement of NO, the triazole deri vative of OAF-FM was determined by reversed-phase high-performance liquid c hromatography with fluorescence detection. In the presence of 1 muM DAF-FM, the concentrations of NOR-1, an NO donor, in the range of 2-200 nM were li nearly related to the fluorescence intensity. This sensitive NO assay enabl ed us to detect the spontaneous and substance P-induced NO release from iso lated porcine coronary arteries, both of which were dependent entirely on t he NO synthase activity in vascular endothelial cells. We also obtained flu orescence images of cultured smooth muscle cells of the rat urinary bladder after loading with DAF-FM diacetate. In the cells pretreated with cytokine s, the fluorescence intensity increased with time after DAF-FM loading. Thi s increase in the fluorescence intensity was blocked by prior treatment of the muscle cells with an NO synthase inhibitor, N-G-nitro-L-arginine methyl ester. Therefore, the present novel diaminofluorescein fluorometry should be useful not only for sensitive NO assay, but also for NO imaging in a var iety of biological specimens. (C) 2000 Academic Press.