Peroxynitrite mediates nitric oxide-induced blood-brain barrier damage

Using the in vitro blood-brain barrier (BBB) model ECV304/C6, which consists of cocultures of human umbilical vein endothelial-like cells (ECV304) and rat glioma cells (C6), the role of peroxynitrite (OONO-) in nitric oxide (NOcircle)-mediated BBB disruption was evaluated. Endothelial cell cultures were exposed to NOcircle gas, in the presence or absence of the OONO- blocker FeTPPS. Separate exposure to NOcircle and OONO- resulted in endothelial cell cytotoxicity and a decline in barrier integrity. Unfortunately, FeTPPS induced significant detrimental effects on model BBB integrity at a concentration of 300 muM and above. At 250 muM (the highest concentration usable), FeTPPS displayed a trend toward prevention of NOcircle elicited perturbation of barrier integrity. Dichlorofluorescein diacetate is oxidized to fluorescent dichlorofluorescein by OONO- but only marginally by NOcircle or O-2(circle-). We observed large and rapid increases in fluorescence in ECV304 preloaded cells following NOcircle exposure, which were blocked by FeTPPS. Furthermore, using an antinitrotyrosine antibody we detected the nitration of endothelial cell proteins following NOcircle exposure and conclude that NOcircle-mediated BBB dysfunction is predominantly elicited by OONO- and not NOcircle. Proposed mechanisms of NOcircle-mediated OONO- elicited barrier dysfunction and damage are discussed.