Oxidative stress upregulates the NMDA receptor on cerebrovascular endothelium

N-methyl-D-aspartate receptor (NMDA-R)-mediated oxidative stress has been implicated in blood-brain barrier (BBB) disruption in a variety of neuropathological diseases. Although some interactions between both phenomena have been elucidated, possible influences of reactive oxygen species (ROS) on the NMDAR itself have so far been neglected. The objective of this study was to examine how the cerebroendothelial NMDA-R is affected by exposure to oxidative stress and to assess possible influences on BBB integrity. RTPCR confirmed several NMDA-R subunits (NR1, NR2B-D) expressed in the bEnd3 cell line ( murine cerebrovascular endothelial cells). NR1 protein expression after exposure to ROS was observed via in-cell Western. The functionality of the expressed NMDA-R was determined by measuring DiBAC influorescence in ROS-preexposed cells upon stimulation with the specific agonist NMDA. Finally, the effects on barrier integrity were evaluated using the ECIS system to detect changes in monolayer impedance upon NMDA-R stimulation after exposure to ROS. The expression of NR1 significantly (p<0.001) increased 72 h after 30 min exposure to superoxide (+ 33.8 +/- 7.5%), peroxynitrite (+ 84.9 +/- 10.7%), or hydrogen peroxide (+ 92.8 +/- 7.6%), resulting in increased cellular response to NMDA-R stimulation and diminished monolayer impedance. We conclude that oxidative stress upregulates NMDA-R on cerebrovascular endothelium and thus heightens susceptibility to glutamate-induced BBB disruption. (C) 2009 Elsevier Inc. All rights reserved.