A fluorescence-based method for measuring nitric oxide in extracts of skeletal muscle

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 (NO S) 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 displaye d 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 acti vity by 47% to an intermediate value consistent with the associated convers ion of the muscle phenotype from fast to slow. (C) 2001 Academic Press.