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We describe here a fluorescence assay for nitric oxide synthase activity in skeletal muscle based on a new indicator, 4,5-diaminofluorescein (DAF-2). The rapid and irreversible binding of DAF-2 to oxidized NO allows real-time measurement of NO production. The method is safer and more convenient than the usual citrulline radioassay and can be used with crude muscle extracts. Rabbit fast tibialis anterior (TA) muscle had a nitric oxide synthase (NOS) activity of 44.3 +/- 3.5 pmol/min/mg muscle. Addition of NOS blocker N(G)-allyl-L-arginine reduced this activity by 43%. Slow soleus muscle displayed NOS activity of 7.3 +/- 2.5 pmol/min/mg muscle, 16% that of the TA muscle. Continuous stimulation of TA muscle at 10 Hz for 3 weeks reduced NOS activity by 47% to an intermediate value consistent with the associated conversion of the muscle phenotype from fast to slow.

Original publication

DOI

10.1006/niox.2001.0374

Type

Journal article

Journal

Nitric oxide

Publication Date

2001

Volume

5

Pages

475 - 481

Keywords

Animals, Fluorescein, Indicators and Reagents, Mice, Mice, Inbred C57BL, Mice, Inbred mdx, Muscle, Skeletal, Nitric Oxide, Nitric Oxide Synthase, Rabbits, Sensitivity and Specificity, Spectrometry, Fluorescence