Modulation of nitric oxide production in human EA.Hy926 cells as measured by 3-amino, 4-aminomethyl-2',7'-difluorofluorescein dyes
Human EA.hy926 cells, a hybridoma of human umbilical vein endothelial and A549 cells, were chosen as a model to study modulation of endothelial nitric oxide synthase (eNOS) activity because of their retention of endothelial morphology and function as well as superior growth characteristics. the fluorescence of nitric oxide (NO)-specific difluorofluorescein dyes, DAF-FM and DAF-FM-DA (RTCalbiobiochem) was used to monitor extracellular and intracellular NO production, respectively, as an index of eNOS activity. Specificity and sensitivity of the assay were validated using a spontaneous NO donor, detaNONOate after labeling of cells with 5 μM of the intracellular form or coincubation with 1 μM of the extracellular form of DAF-FM. DAF-FM fluorescence was linear for both (r2 of 0.98 and 0.89, respectively) with increasing amounts of NO. the limit of detection for the intracellular form was < 34 nM NO and 68 nM NO for the extracellular form. Basal cell-mediated NO production was negligible; however, cellular oxidative stress, induced by BSO pretreatment and measured by dichlorofluorescin fluorescence, increased intracellular NO to about 50 nM; this was further enhanced by co-incubation with 100 uM H2O2. in contrast, pretreatment with vitamin C or co-incubation with 5-30 μM menadione increased cellular oxidative stress but reduced intracellular NO production. in the presence of tiron, a cell permeable SOD mimic, both basal and menadione-reduced intracellular NO production was enhanced. Cell pretreatment with tiron had no effect. Neither cell pretreatment with sepiapterin, a substrate in tetrahydrobiopterin synthesis, nor co-incubation with arginine, the substrate for eNOS, had any effect on NO production. a competitive inhibitor of eNOS, L-NAME, at 3-100 μM reduced basal NO production by ~35 %. in summary, EA.hy926 cells exhibit little to no basal NO production as measured by DAF FM dyes. However, this apparent NO production can be enhanced by certain forms of oxidative stress.
