MK801 blocks hypoxic blood-brain-barrier disruption and leukocyte adhesion.
The aim of the present study was to examine the signaling pathways of hypoxia followed by reoxygenation (H/R)-induced disruption of the blood-brain-barrier (BBB) in a co-culture of astrocytes and brain endothelial cells (BEC) in vitro. We analyzed the possible stabilizing effect of MK801, a highly selective N-methyl-d-aspartate receptor (NMDAR) antagonist, on BBB integrity. Levels of reactive oxygen species (ROS), glutamate (Glut) release and monocyte adhesion were measured under normoxia and H/R. BBB integrity was monitored measuring the trans-endothelial electrical resistance (TEER). TEER values dropped under H/R conditions which was abolished by MK801. Glut release from astrocytes, but not from endothelial cells was significantly increased under H/R, as were ROS levels and monocyte adhesion. The oxidative stress was blocked by MK801 and the NAD(P)H-oxidase inhibitor apocynin. We observed that calcium (Ca(2+)) signaling plays a crucial role during ROS generation and monocyte adhesion under H/R. ROS levels were decreased by applying ryanodine, a blocker of Ca(2+) release from the endoplasmic reticulum (ER) and by lowering the extracellular Ca(2+) concentration. Xestospongin C, which blocks IP(3) mediated Ca(2+) release from the ER did not alter ROS production under H/R conditions. These findings indicate that both extracellular Ca(2+) influx and ryanodine-mediated intracellular Ca(2+) release from the ER during H/R contribute to ROS formation at the BBB. Blocking ROS or Ca(2+) signaling prevented H/R-induced monocyte adhesion to BEC. We conclude, that the activation of NMDAR under H/R by Glut increases intracellular Ca(2+) levels, contributes to BBB disruption, ROS generation and monocyte adhesion.
Kuhlmann
Christoph R W
CR
Institute of Physiology and Pathophysiology, Johannes Gutenberg University of Mainz, Duesbergweg 6, 55128 Mainz, Germany. SuC.Kuhlmann@t-online.de
Zehendner
Christoph M
CM
Gerigk
Marlis
M
Closhen
Dorothea
D
Bender
Bianca
B
Friedl
Peter
P
Luhmann
Heiko J
HJ
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes; D001812:Blood-Brain Barrier / Q000187:drug effects*; D001921:Brain / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects* / Q000502:physiology; D015687:Cell Hypoxia / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D016291:Dizocilpine Maleate / Q000494:pharmacology*; D017097:Electric Impedance; D042783:Endothelial Cells; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology*; D018698:Glutamic Acid / Q000378:metabolism; D007962:Leukocytes / Q000187:drug effects*; D047028:Macrocyclic Compounds / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D012433:Ryanodine / Q000494:pharmacology; D013552:Swine
D000098:Acetophenones; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D047028:Macrocyclic Compounds; D010080:Oxazoles; D017382:Reactive Oxygen Species; C511704:xestospongin C; D012433:Ryanodine; D018698:Glutamic Acid; D016291:Dizocilpine Maleate; C056165:acetovanillone; D010100:Oxygen; D002118:Calcium