title: Nitric oxide detection and visualization in biological systems. Applications of the FNOCT method.
abstract: Fluorescent Nitric Oxide Cheletropic Traps (FNOCTs) were applied to specifically trap nitric oxide (NO) with high sensitivity. The fluorescent o-quinoid pi-electron system of the FNOCTs (lambda(exc) = 460 nm, lambda(em) = 600 nm) reacts rapidly with NO to a fluorescent phenanthrene system (lambda(exc) = 380 nm, lambda(em) = 460 nm). The cyclic nitroxides thus formed react further to non-radical products which exhibit identical fluorescence properties. Using the acid form of the trap (FNOCT-4), NO release by spermine NONOate and by lipopolysaccharide (LPS)-activated alveolar macrophages were studied. A maximum extracellular release of NO of 37.5 nmol h(-1) (10(6) cells)(-1) from the macrophages was determined at 11 h after activation. Furthermore, intracellular NO release by LPS-activated macrophages and by microvascular omentum endothelial cells stimulated by the Ca2+ ionophore A-23187, respectively, was monitored on the single cell level by means of fluorescence microscopy. After loading the cells with the membrane-permeating acetoxymethylester derivative FNOCT-5, which is hydrolyzed to a non-permeating dicarboxylate by intracellular hydrolases, NO formation by the endothelial cells started immediately upon stimulation, whereas start of NO production by the macrophages was delayed with a variation between 4 and 8 h for individual cells. These results demonstrate that the FNOCTs can be used to monitor NO release from single cells, as well as from NO-donating compounds, with high sensitivity and with temporal and spatial resolution.
authors.affiliation: Institut für Organische Chemie, Universität Essen, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Meineke
authors.forename: U
authors.initials: U
authors.lastname: Rauen
authors.forename: H
authors.initials: H
authors.lastname: de Groot
authors.forename: H G
authors.initials: HG
authors.lastname: Korth
authors.forename: R
authors.initials: R
authors.lastname: Sustman
mesh_terms: D000818:Animals; D003470:Culture Media; D008262:Macrophage Activation / Q000187:drug effects; D016676:Macrophages, Alveolar / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods*; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity
chemical_list: D003470:Culture Media; D009569:Nitric Oxide