Modification of the cadmium reduction assay for detection of nitrite production using fluorescence indicator 2,3-diaminonaphthalene.
The measurement of nitric oxide (NO) is important for characterizing the regulatory roles of NO in various biological systems. In this communication we report that cadmium (Cd) reduction of nitrate (NO(-)(3)) to nitrite (NO(-)(2)) can be quantitated by using the fluorescence indicator, 2,3-diaminonaphthalene (DAN) to detect the sum of NO(-)(3) and NO(-)(2) (NO(-)(x)) from endothelial cells. This assay is at least 10-fold more sensitive than when Cd reduction is coupled with the spectrophotometric Greiss reaction and can be used to quantitate the small amounts of NO(-)(x) generated from the constitutive form of endothelial nitric oxide synthase (eNOS). In addition various P(2) purinoceptor agonists and antagonists do not interfere the Cd reduction/DAN assay. Thus the Cd reduction/DAN assay can be used not only to characterize P(2) purinoceptor release of NO(-)(x) from cultured endothelial cells but also to quantitate NO(-)(x) levels in serum.
Casey
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TE
Department of Microbiology/Molecular Medicine, Clemson University, Clemson, South Carolina 29634-1903, USA.
Hilderman
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RH
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000818:Animals; D002104:Cadmium / Q000737:chemistry*; D002417:Cattle; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes; D009573:Nitrites / Q000378:metabolism*; D010084:Oxidation-Reduction; D015032:Zinc / Q000737:chemistry
D005456:Fluorescent Dyes; D009573:Nitrites; D002104:Cadmium; C026373:2,3-diaminonaphthalene; D015081:2-Naphthylamine; D015032:Zinc