Detection of endothelial nitric oxide release with the 2,3-diaminonapthalene assay

The reliable measurement of nitric oxide (NO) production by endothelial cells in vitro has become an important tool for investigating mechanisms of endothelial dysfunction. This study evaluates measuring NO production by cultured porcine pulmonary artery endothelial cells (PAEC) using the assay based on the fluorometric detection of 1-(H)-naphthotriazole, the fluorescent product of the reaction between nitrite (NO2-) and 2,3-diaminonapthalene (DAN). To stimulate NO production, PAEC were treated for 60 min with agonists known to stimulate endothelial NO production. The DAN assay was unable to detect NO production from agonist-stimulated PAEC. In contrast, chemiluminescence analysis, which detects NO, NO2-, and nitrate (NO3-) (collectively referred to as NO(x)), detected significant increases in NO(x) from stimulated PAEC. Nitrate reductase-mediated reduction of NO3-to NO2- in media from stimulated PAEC enhanced the ability of the DAN assay to detect NO release from PAEC. These results provide the first direct comparison of the sensitivity of these two commonly employed assays. Our findings emphasize that NO3-reduction may be required to enable the DAN assay to detect small amounts of NO produced by cultured endothelial cells.