l-Glutamate is a major excitatory neurotransmitter that binds ionotropic and metabotropic glutamate receptors. Cerebral endothelial cells from many species have been shown to express several forms of glutamate receptors; however, human cerebral endothelial cells have not been shown to express either the N-methyl-D-aspartate (NMDA) receptor message or protein. This study provides evidence that human cerebral endothelial cells express the message and protein for NMDA receptors. Human cerebral endothelial cell monolayer electrical resistance changes in response to glutamate receptor agonists, antagonists, and second message blockers were tested. RT-PCR and Western blot analysis were used to demonstrate the presence of the NMDA receptor. Glutamate and NMDA (1 mM) caused a significant decrease in electrical resistance compared with sham control at 2 h postexposure; this response could be blocked significantly by MK-801 (an NMDA antagonist), 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethyoxybenzoate (an intracellular Ca2+ antagonist), and N-acetyl-L-cystein (an antioxidant). Trans(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid, a metabotropic receptor agonist (1 mM), did not significantly decrease electrical resistance. Our results are consistent with a model where glutamate, at excitotoxic levels, may lead to a breakdown in the blood brain barrier via activation of NMDA receptors.
. 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...
The dextran sulfate (DSS) model of colitis causes intestinal injury sharing many characteristics with inflammatory bowel disease, e.g., leukocyte infiltration, loss of gut epithelial barrier, and cachexia. These symptoms are partly mediated by entrapped leukocytes binding to multiple endothelial adhesion molecules (MAdCAM-1, VCAM-1, ICAM-1, and E-selectin). Pravastatin, an 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor, has anti-inflammatory potency in certain inflammation models; therefore, in this study, we measured the effects of pravastatin in DSS-induced colitis. The administration of pravastatin (1 mg/kg) relieved DSS-induced cachexia, hematochezia, and intestinal epithelial permeability, with no effect on serum cholesterol. Histopathologically, pravastatin prevented leukocyte infiltration and gut injury. Pravastatin also blocked the mucosal expression of MAdCAM-1. DSS treatment promoted mucosal endothelial nitric-oxide synthase (eNOS) mRNA degradation, an effect that was blocked by pravastatin. Importantly, the protective effects of pravastatin in DSS-induced colitis were not found in eNOS-deficient mice. Our results demonstrate that HMG-CoA reductase inhibitors preserve intestinal integrity in colitis, most likely via increased eNOS expression and activity, independent of cholesterol metabolism.Inflammatory bowel disease (IBD) (Crohn's colitis and ulcerative colitis) is characterized by tissue edema, increased gut epithelial permeability, and extensive infiltration of the gut by leukocytes. The general morbidity and weight loss in individuals with IBD can be attributed to leukocyte sequestration in the gut in this condition (Perkal and Seashore, 1989;Shanahan, 2002). The current literature suggests that multiple immune, genetic, and environmental factors influence both the initiation and progression of colitis (Farrell and Peppercorn, 2002). Despite the fact that the normal intestinal mucosa maintains a high density of leukocytes compared with most tissues, it is not typically inflamed or edematous. However, during active periods of colitis, the colon is even more extensively colonized by lymphocytes and neutrophils that promote extensive oxidant and protease-dependent injury to the gut. Therefore, it is assumed that the intestine has several specialized mechanisms that normally contain these immune responses and that the impairment of these immune-limiting processes causes the entrapment and activation of leukocytes seen in IBD injury. Among the several endogenous agents that control inflammation, nitric oxide has received a great deal of interest as a factor that can limit forms of inflammation. Endothelial cells release nitric oxide (NO) through both by the "constitutive" (eNOS and NOS3) and inducible nitric oxide synthases (iNOS and NOS1). NO released by microvascular endothelial cells reduces several indices of inflammation in vivo and in vitro. NO is a potent reactive oxygen species scavenger and can block many oxidant-mediated inflammatory responses including leukocyte and platel...
Recent clinical trials indicate the efficacy of interferon (IFN)-beta 1b in reducing relapse rate in relapsing-remitting multiple sclerosis (MS), whereas a surge of IFN-gamma precedes and provokes acute relapses. Disruption of the cerebral endothelial barrier and transendothelial migration of inflammatory cell migration into the brain play a significant role in pathogenesis of MS and may be driven by this surge in IFN-gamma. However, the molecular mechanisms underlying the beneficial effects of IFN-beta 1b against the deleterious effects of IFN-gamma on the barrier formed by the junctional proteins remain to be characterized. The authors investigated the effects of IFN-beta 1b, IFN-beta 1a, and IFN-gamma on the integrity of two endothelial junctional proteins, occludin and vascular endothelial-cadherin (VE-cadherin). Human umbilical vein endothelial cell (HUVEC) layers were treated with IFN-beta 1b, IFN-beta 1a, IFN-gamma, IFN-beta 1b plus IFN-gamma, or IFN-beta 1a plus IFN-gamma. IFN-beta 1b, IFN-beta 1a, and IFN-gamma effects on occludin and VE-cadherin integrity and electrical resistance were assessed by Western blotting and immunofluorescence. IFN-gamma significantly reduced occludin expression and produced gaps in endothelial monolayers. VE-cadherin expression was decreased to a lesser extent in endothelial cells exposed to IFN-gamma. IFN-beta 1b significantly attenuated the IFN-gamma-induced decrease in occludin and VE-cadherin expression. The protective effects of IFN-beta 1a on IFN-gamma-treated endothelial cells were similar to those of IFN-beta 1b. IFN-gamma also significantly reduced endothelial monolayer electrical resistance; this effect was blocked by either IFN-beta 1a or IFN-beta 1b. IFN-beta 1a and IFN-beta 1b effectively prevent the IFN-gamma-induced disintegration of the endothelial tight junctions and sustain barrier against the effects of IFN-gamma. The protective effects of IFN-beta on occludin and VE-cadherin stability appear to represent molecular mechanisms for the therapeutic effects of the IFN-beta on blood brain barrier in MS.
L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1) cerebral endothelial barrier and 2) cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells) have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR) expression via immunohistochemistry and murine neuroepithelial cell line (V1) were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.
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