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

Abstract

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 γ-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 102–104 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).

N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress

“…It has been recently determined that activated mouse macrophages produced significant amount of NO (96 amol/(min. cell))17. When we compared the cytotoxic activity of activated mouse macrophages on control trophozoites and NAT, we found that NAT are more resistant to activated macrophages than the control trophozoites (Fig.…”

Hydrodynamic shear stress promotes epithelial-mesenchymal transition by downregulating ERK and GSK3β activities

“…ROS functions in redox signaling and oxidative stress, which regulate diverse physiological parameters such as the growth factor stimulation and inflammatory response generation [44]. NO triggers a signaling pathway regulated by SS [45, 46] and hematopoiesis [47]. These results demonstrate that +SS can trigger generation of free radical species, such as ROS and reactive nitrogen species.…”

Nitric Oxide Sensors for Biological Applications

“…Chemiluminescent probes can be used to detect NO in both liquid and gaseous samples [74][75][76][77]. The general scheme for the detection of NO in fluid samples via chemiluminescence is through the activation of guanylyl cyclase by NO exposure, causing guanosine triphosphate (GTP) to be converted to guanosine 3 ,5 -cyclic monophosphate (cGMP) which results in the release of pyrophosphate, which can then interact with adenosine triphosphate sulfurylase (ATP-sulfurylase) to form ATP [76,77].…”

“…The general scheme for the detection of NO in fluid samples via chemiluminescence is through the activation of guanylyl cyclase by NO exposure, causing guanosine triphosphate (GTP) to be converted to guanosine 3 ,5 -cyclic monophosphate (cGMP) which results in the release of pyrophosphate, which can then interact with adenosine triphosphate sulfurylase (ATP-sulfurylase) to form ATP [76,77]. The ATP that is formed through this series of reactions is then able to interact with a luciferin-luciferase system to emit light at 560 nm [76,77]. An advantage of the chemiluminescent system is that it is able to detect NO at nM concentrations, since the initial guanylyl cyclase interaction with NO leads to a 200-fold linear increase [76,77].…”

“…The ATP that is formed through this series of reactions is then able to interact with a luciferin-luciferase system to emit light at 560 nm [76,77]. An advantage of the chemiluminescent system is that it is able to detect NO at nM concentrations, since the initial guanylyl cyclase interaction with NO leads to a 200-fold linear increase [76,77]. Chemiluminescent probes have some disadvantages, including the inability to detect NO in vivo.…”