Tjalkens Ronald B, Carbone David L, Wu Guoyao
Department of Environmental and Radiological Health Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.
Methods Mol Biol. 2011;758:267-77. doi: 10.1007/978-1-61779-170-3_18.
Nitric oxide (NO) is a free radical molecule with a short half-life (<5 s). Because its synthesis from L-arginine by constitutive NO synthase (NOS) is low in many cell types, including neurons and endothelial cells, direct detection of NO in biological systems is a difficult task. During pathological conditions in the CNS, the inducible form of NOS (iNOS or NOS2) is expressed in activated astrocytes and microglial cells and can result in higher levels of NO. However, it may still be difficult to detect NO in these cell types using typical spectrophotometric methods. Of particular note, NO is readily oxidized to nitrite and nitrate (relatively stable products) in cells and medium, which can be measured as a valid indicator of NO synthesis. The conversion of NO to peroxynitrite leads to the formation of stable protein adducts that can be detected by immunohistochemical or immunofluorescence methods. Additionally, intracellular levels of NO can be detected in real time using fluorescence imaging and NO-specific, cell permeable indicator dyes.
一氧化氮（NO）是一种半衰期较短（<5秒）的自由基分子。由于在包括神经元和内皮细胞在内的许多细胞类型中，由组成型一氧化氮合酶（NOS）从L-精氨酸合成NO的量较低，因此在生物系统中直接检测NO是一项艰巨的任务。在中枢神经系统的病理状态下，诱导型NOS（iNOS或NOS2）在活化的星形胶质细胞和小胶质细胞中表达，并可导致更高水平的NO。然而，使用典型的分光光度法在这些细胞类型中检测NO可能仍然很困难。特别值得注意的是，NO在细胞和培养基中很容易被氧化为亚硝酸盐和硝酸盐（相对稳定的产物），这可以作为NO合成的有效指标进行测量。NO转化为过氧亚硝酸盐会导致形成稳定的蛋白质加合物，可通过免疫组织化学或免疫荧光方法检测。此外，可使用荧光成像和NO特异性、细胞可渗透的指示染料实时检测细胞内的NO水平。
