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 NMDA-R 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. RT-PCR 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 fluorescence 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.
Betzen
Christian
C
Institute of Physiology and Pathophysiology, Universitätsmedizin, Johannes Gutenberg-University of Mainz, 55128 Mainz, Germany.
White
Robin
R
Zehendner
Christoph M
CM
Pietrowski
Eweline
E
Bender
Bianca
B
Luhmann
Heiko J
HJ
Kuhlmann
Christoph R W
CR
D000818:Animals; D017209:Apoptosis; D001812:Blood-Brain Barrier / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D051379:Mice; D016202:N-Methylaspartate / Q000494:pharmacology; D018384:Oxidative Stress; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D016202:N-Methylaspartate