Glutamate-stimulated peroxynitrite production in a brain-derived endothelial cell line is dependent on N-methyl-D-aspartate (NMDA) receptor activation

There is accumulating and convincing evidence indicating a role for glutamate in the pathogenesis of the human demyelinating disease multiple sclerosis (MS). Studies in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, demonstrate that pharmacological inhibition of specific glutamate receptors suppresses neurological symptoms and prevents blood-brain barrier (BBB) breakdown. The mechanisms through which glutamate influences BBB function during EAE remain unclear. Glutamate triggers the production of nitric oxide and superoxide, which can lead to the formation of peroxynitrite (ONOO-). Recent studies have implicated ONOO- in the loss of neurovascular integrity during EAE. We propose that glutamate contributes to BBB breakdown via the actions of ONOO-. The present investigation examined glutamate-induced ONOO- formation in the b.End3 brainderived endothelial cell line. b.End3 cells were incubated with a concentration range of glutamate and ONOO- production was assessed over time. Results showed a concentration and time-dependent increase in ONOO- levels in glutamate-treated cells that were suppressed by selective and non-selective inhibitors of ONOO--mediated reactions. Specific activation of b.End3-associated NMDA receptors also resulted in a concentration-dependent increase in ONOO- production. The ability of b.End3 cells to respond to the presence of glutamate was confirmed through the detection of NMDA receptor immnuoreactivity in cell extracts. In addition, the use of the NMDA receptor antagonists MK-801 and memantine reduced glutamate-mediated ONOO- generation from b.End3 cells. The data reinforce the important relationship between glutamate and the NMDA receptor, positioned at neurovascular sites, which maybe of particular relevance to the pathogenesis of demyelinating disease. (c) 2006 Elsevier Inc. All rights reserved.