N-methyl-D-aspartate receptor activation in human cerebral endothelium promotes intracellular oxidant stress

• Published in: American journal of physiology. Heart and circulatory physiology Vol. 288; no. 4; pp. H1893 - H1899
• Main Authors: Sharp, Christopher D, Houghton, J, Elrod, J. W, Warren, A, Jackson, T. H., IV, Jawahar, A, Nanda, A, Minagar, A, Alexander, J. S
• Format: Journal Article
• Language: English
• Published: United States 01.04.2005
• Subjects: Brain - cytology; Calcium Channels - metabolism; Cell Line, Transformed; Endothelial Cells - cytology; Endothelial Cells - metabolism; Glutamic Acid - pharmacology; Humans; Ligands; Oxidative Stress - drug effects; Oxidative Stress - physiology; Reactive Oxygen Species - metabolism; Receptors, N-Methyl-D-Aspartate - metabolism; Second Messenger Systems - drug effects; Second Messenger Systems - physiology; Tight Junctions - metabolism
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.01110.2003

Departments of 1 Molecular and Cellular Physiology, 2 Neurosurgery, and 3 Neurology, Louisiana State University Health Sciences Center, Shreveport, Louisiana Submitted 20 November 2003 ; accepted in final form 29 November 2004 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 2+ antagonist; 3 ) LaCl 3 , an extracellular Ca 2+ channel blocker; 4 ) diphenyleiodonium, a heme-ferryl-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 2+ 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 Address for reprint requests and other correspondence: J. S. Alexander, Dept. of Molecular and Cellular Physiology, Louisiana State Univ. Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932 (E-mail: jalexa{at}lsuhsc.edu )