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

Abstract

. 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...

Cerebromicrovascular endothelial cells are resistant to l-glutamate

“…Furthermore, Andras et al reported a 20% drop of TEER even in control cultures after 24 h, in sharp contrast to our study, where TEER was maintained or even increased after 24-h incubation with L-glut or NMDA. The involvement of NMDA receptor activation in the observed L-glut effects reported in previous papers (1,36) is not supported by our negative findings showing that L-glut, but not calcymicin, fails to increase [Ca 2ϩ ] i in rat CMVECs, although L-glut results in a prompt increase in [Ca 2ϩ ] i in cultured rat neurons with the same technique (13).…”

“…In piglet CMVECs, the presence of NMDA receptors was suggested by [ 3 H]L-glut binding assays (34); however, these binding sites may not be ionotropic receptors but rather the well-known L-glut transporters of CMVECs. We restricted our efforts to seek L-glut receptor expression in CMVECs to the study of NMDA receptor subunit NR1 expression, since most of the positive studies demonstrating L-glut excitotoxicity emphasized the role of NMDA receptors in the mechanism of CMVEC injury (1,(33)(34)(35)(36)45). NR1 subunits are ubiquitous components of any functional NMDA receptors (6); thus the lack of NR1 immunopositivity in our CMVEC and CMVPC cultures is consistent with the insensitivity and resistance of these cells to the high doses of L-glut/ NMDA shown in the present study.…”

“…CMVECs have been reported to possess various L-glut receptor subunits or L-glut binding sites in rat, piglet, or human CMVECs (1,5,27,34,36,45). High concentrations of L-glut (1-3 mM), at least in vitro, are claimed to result in N-methyl-D-aspartate (NMDA) receptor activation with subsequent elevations in intracellular Ca 2ϩ levels ([Ca 2ϩ ] i ) and subsequent production of reactive oxygen species leading to cell dysfunction/death of CMVECs (33,35,36,45).However, the attractive CMVEC excitotoxicity hypothesis is challenged by several lines of evidence. First, many studies have failed to find any functional glutamate receptors in ovine, bovine, rat, and human cerebral microvessels or cerebral arteries (2, 31, 44).…”

Cerebroprotective Functions of HO-2

“…Neurons and glia are commonly recognized as the major targets for glutamate in the brain, but the existence of functional GluRs in cerebral vasculature has been long debated. However, the vast body of evidence accumulated over the recent years clearly demonstrates that GluRs are expressed in cerebral vascular endothelium and contribute to the endothelial functions in rats, humans, and newborn pigs [61,[84][85][86][87][88].…”

“…Ca 2+ overload of mitochondria leads to ROS generation, cytochrome c release and activation of Ca 2+ -dependent proteases that promote apoptosis [113,121]. Cerebrovascular endothelial cells are also major targets for glutamate toxicity [40,88]. In cerebral vascular endothelial cells from newborn pigs, glutamate (10 -4 -10 -3 M) increases superoxide production and causes oxidative stress-related cell death by apoptosis [40].…”