title: EPR quantification of vascular nitric oxide production in genetically modified mouse models.
abstract: 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.
authors.affiliation: Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Khoo
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Alp
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Bendall
authors.forename: Seinosuke
authors.initials: S
authors.lastname: Kawashima
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Yokoyama
authors.forename: Yin-Hua
authors.initials: YH
authors.lastname: Zhang
authors.forename: Barbara
authors.initials: B
authors.lastname: Casadei
authors.forename: Keith M
authors.initials: KM
authors.lastname: Channon
mesh_terms: D000818:Animals; D001011:Aorta / Q000737:chemistry / Q000378:metabolism*; D001013:Aorta, Thoracic / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D023421:Models, Animal; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D019001:Nitric Oxide Synthase