An improved method for the detection of NO has been developed by Amano and Noda (30) based on the use of a nitric oxide radical scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (carboxy PTIO) and the Griess reagent. The addition of carboxy PTIO to stimulated cells increases the amount of NO2- because carboxy PTIO rapidly reacts with NO to form NO2- (and not NO3-), which is then assayed using the Griess reaction (31). Carboxy-PTIO has also been used as a scavenger of NO and, thereby, to test the roles of NO in various physiological functions. Nevertheless, the metabolic alterations induced by carboxy-PTIO have not been fully elucidated. It is known that carboxy-PTIO completely inhibits peroxynitrite-induced formation of 3-nitrotyrosine from free tyrosine as well as nitration of bovine serum albumin (32).
Figure 3 shows the effect of carboxy-PTIO (160 (M) on the secretion of NO2- (measured by the Griess assay) by unstimulated, LPS stimulated, IFN-y-stimulated, and lipopolysaccharide plus IFN-y-stimulated RAW264.7 murine macrophages as a function of time. Clearly, carboxy-PTIO almost doubles the sensitivity of the Griess assay. A 5 (L aliquot of stock carboxy-PTIO is added per 3 mL of the cell growth medium (final concentration of 53 (M) and, after incubation at 37°C aliquots of the medium are assayed as detailed in Subheading 3.1.
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