EPR quantification of vascular nitric oxide production in genetically modified mouse models.
Khoo JP., Alp NJ., Bendall JK., Kawashima S., Yokoyama M., Zhang YH., Casadei B., Channon KM.
With increasing use of genetically modified mice to study endothelial nitric oxide (NO) biology, methods for reliable quantification of vascular NO production by mouse tissues are crucial. We describe a technique based on electron paramagnetic resonance (EPR) spectroscopy, using colloid iron (II) diethyldithiocarbamate [Fe(DETC)2], to trap NO. A signal was seen from C57BL/6 mice aortas incubated with Fe(DETC)2, that increased 4.7-fold on stimulation with calcium ionophore A23187 [3.45+/-0.13 vs 0.73+/-0.13au (arbitrary units)]. The signal increased linearly with incubation time (r(2) = 0.93), but was abolished by addition of N(G)-nitro-l-arginine methyl ester (L-NAME) or endothelial removal. Stimulated aortas from eNOS knockout mice had virtually undetectable signals (0.14+/-0.06 vs 3.17+/-0.21 au in littermate controls). However, the signal was doubled from mice with transgenic eNOS overexpression (7.17+/-0.76 vs 3.37+/-0.43 au in littermate controls). We conclude that EPR is a useful tool for direct NO quantification in mouse vessels.