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 (NO·)-mediated BBB disruption was evaluated. Endothelial cell cultures were exposed to NO· gas, in the presence or absence of the OONO− blocker FeTPPS. Separate exposure to NO· 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 μM and above. At 250 μM (the highest concentration usable), FeTPPS displayed a trend toward prevention of NO· elicited perturbation of barrier integrity. Dichlorofluorescein diacetate is oxidized to fluorescent dichlorofluorescein by OONO− but only marginally by NO· or O2·−. We observed large and rapid increases in fluorescence in ECV304 preloaded cells following NO· exposure, which were blocked by FeTPPS. Furthermore, using an antinitrotyrosine antibody we detected the nitration of endothelial cell proteins following NO· exposure and conclude that NO·-mediated BBB dysfunction is predominantly elicited by OONO− and not NO·. Proposed mechanisms of NO·-mediated OONO− elicited barrier dysfunction and damage are discussed.