Detection of nitric oxide production in cell cultures by luciferin-luciferase chemiluminescence

作者：Woldman Yakov Y^*; Eubank Tim D; Mock Andrew J; Stevens Natalia C; Varadharaj Saradhadevi; Turco Jenifer; Gavrilin Mikhail A; Branchini Bruce R; Khramtsov Valery V

来源：Biochemical and Biophysical Research Communications, 2015, 465(2): 232-238.

DOI：10.1016/j.bbrc.2015.08.001

• Nitric oxide
• Guanylyl cyclase
• Pyrophosphate
• Macrophages
• Endothelial cells
• Thermostable luciferase

A chemiluminescent method is proposed for quantitation of NO generation in cell cultures. The method is based on activation of soluble guanylyl cyclase by NO. The product of the guanylyl cyclase reaction, pyrophosphate, is converted to ATP by ATP sulfurylase and ATP is detected in a luciferin luciferase system. The method has been applied to the measurement of NO generated by activated murine macrophages (RAW 264.7) and bovine aortic endothelial cells. For macrophages activated by lipopolysaccharide and gamma-interferon, the rate of NO production is about 100 amol/(cell - min). The rate was confirmed by the measurements of nitrite, the product of NO oxidation. For endothelial cells, the basal rate of NO generation is 5 amol/(cell min); the rate approximately doubles upon activation by bradykinin, Ca2+ ionophore A23187 or mechanical stress. For both types of cells the measured rate of NO generation is strongly affected by inhibitors of NO synthase. The sensitivity of the method is about 50 pM/min, allowing the registration of NO generated by 10(20)-10(4) cells. The enzyme-linked chemiluminescent method is two orders of magnitude more sensitive than fluorescent detection using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM).

• 出版日期2015-9-18