MONITORING INTRACELLULAR NITRIC-OXIDE FORMATION BY DICHLOROFLUORESCININ NEURONAL CELLS

A method for rapid fluorometric assay of intracellular nitric oxide (N O) formation was developed for use in cultured neuronal cells. In a ce ll-free system 2,7-dichlorofluorescin (DCF), a non-fluorescent species , is oxidized by NO to dichlorofluorescein, a fluorescent compound. Ad dition of NO to a solution containing DCF increased the fluorescent si gnal within 10 s and continued to increase slowly over a 10-min period . The intensity of the fluorescence was dependent upon the concentrati on of NO. In DCF-loaded PC12 cells, addition of NO markedly increased fluorescence (limit of detection = 16 mu M NO) and pretreatment with r educed hemoglobin (Hb) inhibited the NO-mediated increase of fluoresce nce in both the cell-free system and PC12 cells. In PC12 cells loaded with DCF, the NO generator sodium nitroprusside (SNP) produced a rapid increase of fluorescence. To rule out the possibility that reactive o xygen species (ROS) mediated the increased of fluorescence, superoxide dismutase (SOD) and catalase were added to the cuvette. The enzymes d id not alter the fluorescence generated after addition of NO to PC12 c ells. This assay was used to determine the ability of glutamate to sti mulate NO production in cerebellar granule cells. When 10 mu M glutama te was added to DCF-loaded cerebellar granule cells, a rapid increase in fluorescence was noted. The fluorescence was blacked approximately 50% after addition of either Hb or SOD, or by pretreatment with N-G-ni tro-L-arginine methyl ester (300 mu M), a nitric oxide synthase (NOS) inhibitor. It was concluded that glutamate stimulated intracellular ge neration of both NO and ROS, and at least 50% of the oxidation of DCF was attributed to intracellular generation of NO. These results demons trate that oxidation of DCF by NO can be used to measure intracellular generation of NO and by adding either Hb or SOD to the cell system, t he extent of oxidation of DCF attributed to NO and ROS can be determin ed.