. N-methyl-D-aspartate receptor activation in human cerebral endothelium promotes intracellular oxidant stress. Am J Physiol Heart Circ Physiol 288: H1893-H1899, 2005. First published December 2, 2004; doi:10.1152/ajpheart.01110.2003.-Cerebral endothelial cells in the rat, pig, and, most recently, human have been shown to express several types of receptors specific for glutamate. High levels of glutamate disrupt the cerebral endothelial barrier via activation of N-methyl-D-aspartate (NMDA) receptors. We have previously suggested that this glutamate-induced barrier dysfunction was oxidant dependent. Here, we provide evidence that human cerebral endothelial cells respond to glutamate by generating an intracellular oxidant stress via NMDA receptor activation. Cerebral endothelial cells loaded with the oxidant-sensitive probe dihydrorhodamine were used to measure intracellular reactive oxygen species (ROS) formation in response to glutamate receptor agonists, antagonists, and second message blockers. Glutamate (1 mM) significantly increased ROS formation compared with sham controls (30 min). This ROS response was significantly reduced by 1) MK-801, a noncompetitive NMDA receptor antagonist; 2) 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate, an intracellular Ca 2ϩ antagonist; 3) LaCl3, an extracellular Ca 2ϩ channel blocker; 4) diphenyleiodonium, a hemeferryl-containing protein inhibitor; 5) itraconazole, a cytochrome P-450 3A4 inhibitor; and 6) cyclosporine A, which prevents mitochondrial membrane pore transition required for mitochondrial-dependent ROS generation. Our results suggest that the cerebral endothelial barrier dysfunction seen in response to glutamate is Ca 2ϩ dependent and may require several intracellular signaling events mediated by oxidants derived from reduced nicotinamide adenine dinucleotide oxidase, cytochrome P-450, and the mitochondria. reactive oxygen species; mitochondria; reduced nicotinamide adenine dinucleotide oxidase; arachidonic acid; human; brain THE BLOOD-BRAIN BARRIER (BBB) selectively regulates the exchange of solutes between the vascular and cerebral interstitial space, effectively protecting the brain against blood-borne neurologically active and potentially damaging substances (2, 3). The BBB is created by adherens junctions and tight junctions that tightly seal cerebral endothelial cells together to create a greatly reduced paracellular rate of exchange compared with that seen in other vascular beds (2, 9, 37). During stroke and trauma, the cerebrum is injured by cerebral oxygen and glucose deprivation, and this hypoxic and glucose-free environment is associated with a massive release of glutamate into the synaptic space with a loss of this barrier. This excessive glutamate release reflects a loss in cellular ATP, dissipation of membrane ion gradients, cell potassium efflux, opening of voltage-dependent sodium channels, and membrane depolarization. This membrane depolarization leads to even more glutamate being released by exocytosis at synapses and causes a massive gluta...