Nitric oxide detection and visualization in biological systems. Applications of the FNOCT method

Fluorescent Nitric Oxide Cheletropic Traps (FNOCTs) were applied to specifi cally trap nitric oxide (NO) with high sensitivity. The fluorescent o-quino id pi-electron system of the FNOCTs(lambda(exc)= 460 nm, lambda(em) = 600 n m) reacts rapidly with NO to a fluorescent phenanthrene system (lambda(exc) = 380 nm, lambda(exc) = 460 nm). The cyclic nitroxides thus formed react f urther to non-radical products which exhibit identical fluorescence propert ies. Using the acid form of the trap (FNOCT-4), NO release by spermine NONO ate and by lipopolysaccharide (LPS)activated alveolar macrophages were stud ied. A maximum extracellular release of NO of 37.5 nmol h(-l) (10(6) cells) (-1) from the macrophages was determined at 11 h after activation. Furtherm ore, intracellular NO release by LPS-activated macrophages and by microvasc ular omentum endothelial cells stimulated by the Ca2+ ionophore A-23187, re spectively, was monitored on the single cell level by means of fluorescence microscopy. After loading the cells with the membrane-permeating acetoxyme thylester derivative FNOCT-5, which is hydrolyzed to a non-permeating dicar boxylate by intracellular hydrolases, NO formation by the endothelial cells started immediately upon stimulation, whereas start of NO production by th e 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 hig h sensitivity and with temporal and spatial resolution.