Proceedings of The Physiological Society

University of York (2002) J Physiol 539P, S016


Peroxynitrite elicits the damaging effect of nitric oxide on the blood-brain barrier

R.D. Hurst, S. Harrington and K.H. Tan

Centre for Research in Biomedicine, Faculty of Applied Sciences, University of West of England, Bristol, UK

Most blood-borne substances and cells do not readily cross the highly specialised endothelial cells of the blood-brain barrier (BBB). This protection of the central nervous system microenvironment is disrupted in many inflammatory neurological disorders and a role for pro-inflammatory cytokines and/or reactive oxygen species has been suggested (Merrill & Murphy, 1997). The free radical nitric oxide (NO·) has also been implicated in inflammatory-mediated BBB damage. The full mechanisms of NO·-mediated barrier damage and disruption remains unresolved, although evidence suggests that a breakdown in barrier integrity could be mediated by a mechanism of altered cellular energy homeostasis (Hurst & Clark, 1998 ; Hurst et al. 2001). NO· is relatively non-toxic but rapidly reacts with superoxide ions (O2·-) to form the extremely reactive radical peroxynitrite (OONO-). OONO- is able to mediate lipid peroxidation and protein tyrosine nitrosylation and hence our aim was to evaluate whether OONO- contributes to the disruption of the BBB following NO· exposure.

Using the in vitro BBB model ECV304/C6 (Hurst & Fritz, 1996) which consists of co-cultures of human umbilical vein endothelial-like cells (ECV304) and rat glioma cells (C6, both cell lines purchased from the European Collection of Animal Cell Cultures), cultures were exposed to authentic NO· gas, in the presence or absence of the NO· and OONO- scavengers PTIO (2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide) and FeTPPS (5, 10, 15, 20-tetrakis[4-sulfonato-phenyl]prophyrinato iron chloride), respectively. Confocal imaging enabled OONO- generation within ECV304 cells to be evaluated using non-fluorescent dichlorofluorescein diacetate (DCFH-DA) which is oxidised to generate the highly fluorescent compound 2fi,7fi-dichlorofluorescein by OONO- but only marginally by NO· or O2·- (Radi et al. 2001). Furthermore barrier endothelial cell viability and integrity were assessed by vital stain exclusion (trypan blue), and changes in transendothelial electrical resistance (TEER), respectively. Exposure to NO· gas (20 µM) resulted in a rapid and significant increase in fluorescence indicating intracellular OONO- formation. NO· gas also resulted in significant endothelial cell death (P < 0.001, Student's unpaired t test, n = 3 experiments) and a progressive decline in TEER. Cell death was dose-dependently prevented by FeTPPS but not PTIO (at a concentration shown, using the NO· electrode, to completely scavenge 20 µM NO·). While FeTPPS at a range of concentrations had no effect alone on endothelial cell viability it unfortunately induced detrimental effects on BBB model TEER at concentrations of 300 µM and above. At 250 µM FeTPPS demonstrated a trend (insignificant, Student's t test) towards prevention of NO· elicited perturbation of barrier integrity (TEER).

In summary, these data suggest that ONOO- is rapidly formed within endothelial cells and indeed elicits endothelial cell death and barrier dysfunction following NO· exposure.

Where applicable, experiments conform with Society ethical requirements