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 microM and above. At 250 microM (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.
Tan
Kian H
KH
Centre for Research in Biomedicine, Faculty of Applied Sciences, University of the West of England, Bristol, United Kingdom BS16 1QY.
Harrington
Sian
S
Purcell
Wendy M
WM
Hurst
Roger D
RD
D001812:Blood-Brain Barrier / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008665:Metalloporphyrins / Q000494:pharmacology; D008954:Models, Biological; D009569:Nitric Oxide / Q000494:pharmacology*; D030421:Peroxynitrous Acid / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014471:Umbilical Veins
C466420:5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride; D008665:Metalloporphyrins; D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; D009569:Nitric Oxide