|Budget Amount *help
¥9,400,000 (Direct Cost : ¥9,400,000)
Fiscal Year 1999 : ¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1998 : ¥6,200,000 (Direct Cost : ¥6,200,000)
We have developed diaminofluoresceins (DAFs), such as DAF-2, as fluorescent indicators for nitric oxide (NO). Under aerobic conditions, DAFs can trap NO to yield highly fluorescent triazolofluoresceins (DAF-Ts) by nitrosation and dehydaration. However, during the application of DAF-2 for NO imaging in vascular endothelial cells, we encountered the problem of pH dependency of the fluorescence intensity of DAF-Ts, which makes it difficult to monitor small changes in intracellular NO levels after a stimulus that shifts the intracellular pH level. Since we had anticipated this problem, we synthesized dichloro derivatives of DAF. The chlorine atoms lower the pKa value of the adjacent phenolic OH group due to their electronegativity. However, they were unexpectedly less useful than DAF-2 owing to their unstable fluorescence intensity around neutral pH. The instability may arise from a triazole proton, so we introduced a methyl group. As the resiults of various examinations, DAF-4 M2 was the
most sensitive to NO. The fluorescence intensity of the triazole form of DAF-4 M2 was stable above pH 5.8.
It was reported that fluorinated fluorescein derivatives are more resistant to photobleaching than fluorescein and can be efficiently excited with 488 nm spectral line of the argon ion laser used in confocal laser scanning microscopes. Therefore, we prepared DAF-FM, in which the chlorine atoms of DAF-4 M2 are replaced by fluorine atoms. Also, the effect of pH on the fluorescence intensity of DAF-FM T was examined. Judging from NO standard curves calibrated with 7 micromol DAF-FM, the detection limit of NO was 3 nM. The sensitivity of DAF-FM was 1.4 times higher than that of DAF-2. It is considered that this increase in sensitivity results from the higher rate of the reaction with NO+ equivalents due to the electron-donating effect of the methyl group.
For the bioimaging of NO, we synthesized DAF-FM DA, which permeates well into cells and is quickly transformed into water-soluble DAF-FM by esterases in the cytosol, where the dye can remain for a long time. The indicator DAF-FM DA was applied to the imagine of NO in cultured bovine aortic endothelial cells by using a fluorescence microscope. In conclusion, we suggest that DAF-FM is a useful tool for visualizing the temporal and spatial distribution of intracellular NO. Less