MONITORING INTRACELLULAR NITRIC-OXIDE FORMATION BY DICHLOROFLUORESCIN IN NEURONAL CELLS
MONITORING INTRACELLULAR NITRIC-OXIDE FORMATION BY DICHLOROFLUORESCIN IN NEURONAL CELLS
复制标题
DOI：
10.1016/0165-0270(95)00018-p
复制
发表时间：
1995-09-01
影响因子：
3
通讯作者：
ISOM, GE
中科院分区：
文献类型：
--
作者：
GUNASEKAR, PG;KANTHASAMY, AG;ISOM, GE
开发了一种快速荧光测定细胞内一氧化氮 (NO) 形成的方法，用于培养的神经元细胞。在无细胞系统中，2,7-二氯荧光素 (DCF)（一种非荧光物质）被 NO 氧化为二氯荧光素（一种荧光化合物）。向含有 DCF 的溶液中添加 NO 会在 10 秒内增加荧光信号，并在 10 分钟内继续缓慢增加。荧光强度取决于 NO 的浓度。在负载 DCF 的 PC12 细胞中，添加 NO 显着增加荧光（检测限 = 16 μM NO），并且用还原血红蛋白 (Hb) 预处理可抑制无细胞系统和 PC12 细胞中 NO 介导的荧光增加。在负载 DCF 的 PC12 细胞中，NO 发生器硝普钠 (SNP) 产生的荧光快速增加。为了排除活性氧（ROS）介导荧光增加的可能性，向比色皿中添加超氧化物歧化酶（SOD）和过氧化氢酶。这些酶不会改变向 PC12 细胞添加 NO 后产生的荧光。该测定用于确定谷氨酸刺激小脑颗粒细胞中 NO 产生的能力。当将 10 μM 谷氨酸添加到负载 DCF 的小脑颗粒细胞中时，注意到荧光迅速增加。添加 Hb 或 SOD 后，或用一氧化氮合酶 (NOS) 抑制剂 N-G-硝基-L-精氨酸甲酯 (300 μM) 预处理后，荧光变黑约 50%。结论是谷氨酸刺激细胞内NO和ROS的产生，并且至少50%的DCF氧化归因于细胞内NO的产生。这些结果表明，NO 对 DCF 的氧化可用于测量细胞内 NO 的生成，并且通过向细胞系统中添加 Hb 或 SOD，可确定归因于 NO 和 ROS 的 DCF 氧化程度。
A method for rapid fluorometric assay of intracellular nitric oxide (NO) formation was developed for use in cultured neuronal cells. In a cell-free system 2,7-dichlorofluorescin (DCF), a non-fluorescent species, is oxidized by NO to dichlorofluorescein, a fluorescent compound. Addition of NO to a solution containing DCF increased the fluorescent signal within 10 s and continued to increase slowly over a 10-min period. The intensity of the fluorescence was dependent upon the concentration of NO. In DCF-loaded PC12 cells, addition of NO markedly increased fluorescence (limit of detection = 16 mu M NO) and pretreatment with reduced hemoglobin (Hb) inhibited the NO-mediated increase of fluorescence 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 oxygen species (ROS) mediated the increased of fluorescence, superoxide dismutase (SOD) and catalase were added to the cuvette. The enzymes did not alter the fluorescence generated after addition of NO to PC12 cells. This assay was used to determine the ability of glutamate to stimulate NO production in cerebellar granule cells. When 10 mu M glutamate 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-nitro-L-arginine methyl ester (300 mu M), a nitric oxide synthase (NOS) inhibitor. It was concluded that glutamate stimulated intracellular generation of both NO and ROS, and at least 50% of the oxidation of DCF was attributed to intracellular generation of NO. These results demonstrate 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, the extent of oxidation of DCF attributed to NO and ROS can be determined.