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Am J Physiol Heart Circ Physiol 288: H1893-H1899, 2005. First published December 2, 2004; doi:10.1152/ajpheart.01110.2003
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N-methyl-D-aspartate receptor activation in human cerebral endothelium promotes intracellular oxidant stress

Christopher D. Sharp,1 J. Houghton,1 J. W. Elrod,1 A. Warren,1 T. H. Jackson, IV,1 A. Jawahar,2 A. Nanda,2 A. Minagar,3 and J. S. Alexander1

Departments of 1Molecular and Cellular Physiology, 2Neurosurgery, and 3Neurology, Louisiana State University Health Sciences Center, Shreveport, Louisiana

Submitted 20 November 2003 ; accepted in final form 29 November 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 Ca2+ antagonist; 3) LaCl3, an extracellular Ca2+ 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 Ca2+ 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 glutamate accumulation and overstimulation of N-methyl-D-aspartate (NMDA) receptors, referred to as glutamate excitotoxicity (14, 32, 36).

In addition to glutamate excitotoxicity in neurons, extracellular glutamate may also cause an endothelial excitotoxicity associated with a loss of the cerebral endothelial barrier dysfunction (41). This endothelial excitotoxicity appears to be NMDA receptor (NMDAR) dependent and may help explain the increased microvascular solute permeability, which temporally parallels neuronal excitotoxicity (1, 20, 37).

Previously, we (41) demonstrated human cerebral endothelial expression of functional NMDARs and that activation of these receptors leads to a loss of cerebral endothelial barrier. We (41) found that this endothelial dysfunction was blocked by at least one intracellular antioxidant [N-acety-L-cysteine (NAC)], suggesting that glutamate-induced oxidant stress is from intracellular rather than extracellular sources. Whereas oxidants are recognized as central mediators of postischemic injury, sources of oxidants in the postischemic cerebral microcirculation have so far only implicated activated/adherent neutrophils (10), xanthine oxidase-derived oxidants (8), and the mitochondria (1) as mediators of increased edema but have not considered excitotoxic mechanisms.

We (41) previously showed that glutamate provokes a loss of brain cerebral endothelial barrier without cell death, lysis, or necrosis; however, it does appear to reflect some form of oxidant-mediated stress. Therefore, our present study examines the hypothesis that excitotoxic levels of glutamate activate NMDARs on brain endothelial cells, causing Ca2+ influx with the result that increased intracellular Ca2+ triggers the production of intracellular reactive oxygen species (ROS), possibly derived from mitochondrial respiration, reduced nicotinamide adenine dinucleotide [NADPH] oxidase, or arachidonic acid (AA) metabolism. Our current results extend on our previous findings that brain capillary endothelium exposed to glutamate form ROS from multiple sources. Therefore, this glutamate-induced oxidant stress in brain endothelium may lead to the activation of secondary messages resulting in disruption of endothelial tight junctions, barrier failure, and increased permeability and/or edema formation seen in stroke/ischemic brain injury.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. Medium 199 (M-199), insulin, transferrin, selenium, heparin, HEPES, MK-801, L-glutamic acid, 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8), NMDA, and NAC were purchased from Sigma (St. Louis, MO). Cyclosporine A (CSA) was purchased from Bedford Laboratories (Bedford, OH), diphenyleiodonium (DPI) was purchased from BioMol (Plymouth Meeting, PA), and itraconazole was purchased from Ortho Biotech (Bridgewater, NJ). All experiments were performed at 37°C, 7.5% CO2, and in HBSS (Sigma) plus glucose with 15 mM HEPES (pH 7.4).

Cell culture. Immortalized human brain endothelial cells (IHEC) (supplied by Dr. D. Stanimirovic, Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON, Canada) were maintained in M-199 (with 10% FCS, 1% antibiotic/antimycotic, 5 g/ml insulin, 5 g/ml transferrin, 5 ng/ml selenium, and 600 USP units/1 heparin) and grown to 100% confluency. IHEC were seeded onto Cytodex 3 microcarrier beads (382 cm2/ml; Sigma) for the fluorescent indicator studies. All endothelial cultures were used on reaching confluency 5 days after seeding.

Fluorescent analysis of oxidants. When confluency was reached, the beads were washed with HBSS (15 mM HEPES, pH 7.36), allowed to equilibrate for 40 min without any fluorescent probe, and then washed an additional 20 min with 5 µM dyhidrorhodamine (DHR) (for a total of 1 h). Pretreatments, if needed, were also added during the 20-min DHR equilibration time. Cells were then gently washed three times in HBSS-HEPES, dispersed equally into a 96-well plate (150 µl of packed beads per well, equivalent to ~50-cm2 surface area). Monolayers were then exposed to 1 mM glutamate in HBSS-HEPES. A FLUOstar Optima fluorometer (BMG Lab Technologies) was used to read the emission of 529 nm resulting from 509-nm excitation of DHR every 5 min (for 30 min) following glutamate exposure. The data from the fluorometer converted into percent increase in rhodamine 123 signal over time. This conversion allowed collection of a large sample size (at least an n = 6) for statistically comparing data from different cell passages and treatments.

Statistical analysis. All values are expressed as means ± SD. Data were analyzed by using one-way ANOVA with Bonferroni's correction for multiple comparisons. Significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Glutamate-induced oxidant stress is NMDAR dependent. On the basis of our previous findings that the endothelial barrier dysfunction caused by 1 mM glutamate exposure was blocked by 4 mM NAC (41), and because oxidant stress can lead to endothelial barrier failure (5, 6, 27), we hypothesized that the loss in endothelial monolayer resistance was due to stimulation/activation of an NMDAR and subsequent intracellular ROS formation. To test this, we pretreated IHEC with DHR (an intracellular ROS indicator) (47). DHR is converted to the fluorescent indicator rhodamine 123 by only peroxynitrite or hydroxyl radical (34). After the IHEC were loaded with DHR, the cells were exposed to 1 mM glutamate [concentrations of 1–10 mM glutamate are commonly used to model stroke or excitotoxic events (11, 12, 17, 47)], and the change in fluorescent intensity was read with a fluorometer over time (for 30 min at 37°C).

Within 30 min of glutamate exposure, there was a significant increase in rhodamine 123 fluorescent intensity compared with sham controls (i.e., increase in intracellular ROS concentration) (Fig. 1). To verify that this oxidant stress was NMDAR dependent, the IHEC were pretreated with 10 µM MK-801, 100 µM TMB-8, and 0.5 mM LaCl3 (41). MK-801 completely blocked the increase in intracellular ROS seen in response to 1 mM glutamate exposure. Both the intracellular Ca2+ antagonist TMB-8 (Fig. 2) and the Ca2+ channel blocker LaCl3 significantly decreased the oxidant formation induced by 1 mM glutamate (Fig. 3), but TMB-8 still allowed a significant amount of ROS to be formed by glutamate compared with controls (Fig. 2). The finding that TMB-8 was unable to completely block the intracellular ROS formation and that LaCl3 could suggests that the influx of Ca2+ (through a ligand-gated calcium channel such as the NMDAR-1) is the main response of this model. Interestingly, the cell iron chelator desferrioxamine (desferal; 0.1 mM) also significantly attenuated the production of DHR in response to glutamate, measured as relative fluorescence units (control: 2,286 ± 1,891; glutamate: 41,376 ± 2,186; desferal + glutamate: 8,686 ± 280; P < 0.01). These findings and the fact that MK-801 blocked the glutamate response support a role of NMDARs in glutamate-induced endothelial oxidant stress (41). Parallel tests with hydroethidine, an O2–· selective fluorometric probe (39), failed to reveal evidence of significant superoxide production, which could reflect either production of superoxide below the limit of detection of hydroethidine or such a rapid conversion of superoxide to another oxidant that is not detected by hydroethidine (data not shown).



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  		Fig. 1. Increases in rhodamine 123 fluorescent intensity [increase in intracellular reactive oxygen species (ROS)] in response to 1 mM glutamate ± 10 µM MK-801 [a specific N-methyl-D-aspartate receptor (NMDAR)1 antagonist, 20-min pretreatment]. Immortalized human brain endothelial cells (IHEC) were grown to confluency on Cytodex 3 microcarrier beads, and exposure to 1 mM glutamate was observed to cause a significant (P < 0.001) increase in rhodamine 123 fluorescent intensity in vitro compared with sham control with in 30 min of glutamate exposure. The increase in intracellular ROS caused by 1 mM glutamate exposure was significantly blocked with MK-801 (P < 0.001).

 


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  		Fig. 2. Increases in rhodamine 123 fluorescent intensity (increase in intracellular ROS) in response to 1 mM glutamate ± 10–4 M 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular Ca2+ antagonist (20-min pretreatment). IHEC were grown to confluency on Cytodex 3 microcarrier beads, and exposure to 1 mM glutamate was observed to cause a significant (P < 0.001) increase in rhodamine 123 fluorescent intensity in vitro compared with sham control within 30 min of glutamate exposure. The increase in intracellular ROS caused by 1 mM glutamate exposure was significantly decreased with TMB-8 (P < 0.05).

 


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  		Fig. 3. Increases in rhodamine 123 fluorescent intensity (increase in intracellular ROS) in response to 1 mM glutamate ± 0.5 mM LaCl3, a calcium channel blocker (20-min pretreatment). IHEC were grown to confluency on Cytodex 3 microcarrier beads, and exposure to 1 mM glutamate was observed to cause a significant (P < 0.001) increase in rhodamine 123 fluorescent intensity in vitro compared with sham control with in 30 min of glutamate exposure. The increase in intracellular ROS caused by 1 mM glutamate exposure was significantly blocked with LaCl3 (P < 0.001).

 
Sources of NMDAR-induced oxidant stress. NMDARs are ligand-gated Ca2+ channels and may activate several Ca2+-dependent formation of intracellular ROS from multiple sources (mitochondrial respiration, NADPH oxidase, and AA metabolism). Therefore, we sought to identify the exact source(s) of the NMDAR-dependent intracellular oxidant stress. IHEC were pretreated with (in µM) 5 CSA, 10 DPI, 1 itraconazole, and 1 bromophenacylbromide (BPB). CSA (Fig. 4), DPI (Fig. 5), and itraconazole (Fig. 6) significantly blocked the glutamate oxidant stress. BPB did not block the NMDAR-dependent intracellular oxidant stress (Fig. 7). CSA, which prevents the release of ROS by the mitochondria, was the only pharmacological blocker that could completely block glutamate-induced ROS (no significant difference in rhodamine 123 intensity between CSA + 1 mM glutamate cells and sham control cells). Therefore, on the basis of these data, our conclusion is that, although multiple source(s) [mitochondrial respiration, NADPH oxidase, and cytochrome P-450 (CYP450)] contribute to the glutamate-induced oxidant stress, the main source appears to be the mitochondria.



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  		Fig. 4. Increases in rhodamine 123 fluorescent intensity (increase in intracellular ROS) in response to 1 mM glutamate ± 5 µM cyclosporine A (CSA), a mitochondrial ROS-releasing inhibitor (20-min pretreatment). IHEC were grown to confluency on Cytodex 3 microcarrier beads, and exposure to 1 mM glutamate was observed to cause a significant (P < 0.001) increase in rhodamine 123 fluorescent intensity in vitro compared with sham control within 30 min of glutamate exposure. The increase in intracellular ROS caused by 1 mM glutamate exposure was significantly blocked with CSA (P < 0.001).

 


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  		Fig. 5. Increases in rhodamine 123 fluorescent intensity (increase in intracellular ROS) in response to 1 mM glutamate ± 10 µM diphenyleiodonium (DPI), a reduced nicotinamide adenine dinucleotide [NADPH] oxidase and cytochrome P-450 (CYP450) inhibitor (20-min pretreatment). IHEC were grown to confluency on Cytodex 3 microcarrier beads, and exposure to 1 mM glutamate was observed to cause a significant (P < 0.001) increase in rhodamine 123 fluorescent intensity in vitro compared with sham control with in 30 min of glutamate exposure. The increase in intracellular ROS caused by 1 mM glutamate exposure was significantly decreased with DPI (P < 0.001).

 


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  		Fig. 6. Increases in rhodamine 123 fluorescent intensity (increase in intracellular ROS) in response to 1 mM glutamate ± 1 µM itraconazole (an NADPH oxidase and CYP450 inhibitor, 20-min pretreatment). IHEC were grown to confluency on Cytodex 3 microcarrier beads, and exposure to 1 mM glutamate was observed to cause a significant (P < 0.001) increase in rhodamine 123 fluorescent intensity in vitro compared with sham control with in 30 min of glutamate exposure. The increase in intracellular ROS caused by 1 mM glutamate exposure was significantly decreased with itraconazole (P < 0.01).

 


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  		Fig. 7. Increases in rhodamine 123 fluorescent intensity (increase in intracellular ROS) in response to 1 mM glutamate ± 1 µM bromophenacylbromide (BPB), a phospholipase A2 inhibitor (20-min pretreatment). IHEC were grown to confluency on Cytodex 3 microcarrier beads, and exposure to 1 mM glutamate was observed to cause a significant (P < 0.001) increase in rhodamine 123 fluorescent intensity in vitro compared with sham control with in 30 min of glutamate exposure. The increase in intracellular ROS caused by 1 mM glutamate exposure was not decreased with BPB.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Brain ischemia-reperfusion has been clearly demonstrated to injure cerebral neurons, the microvasculature, and parenchymal cells (21, 24, 25, 37, 48). At least two distinct phases of ROS-dependent injury are recognized in the ischemia-reperfusion exposed cerebrum: 1) a transcription-independent phase (30 min) in which injury is mediated by the production of extracellular ROS and signals through those pathways and 2) a transcription-dependent phase (>4 h), which can also involve the production of intracellular ROS but depends on activation of genes and transcription factors (i.e., NF-{kappa}B, AP-1) and protein translation (31).

Because the edema seen in stroke is reported to occur <3 h after the onset of cerebral ischemia, it may be less likely that transcription-dependent responses play dominant roles in the development of the initial (<3 h) cerebral edema (19, 22). The 3-h time frame for the development of microvascular edema matches our findings that cerebral endothelial cells exhibit stress mediated by activation of NMDARs, characterized by endothelial barrier dysfunction (within 2 h) (41). We also demonstrated in vitro that the glutamate-induced cerebral endothelial barrier breakdown was blocked by the antioxidant NAC (41).

The present study explored two questions: 1) is the barrier dysfunction seen in glutamate stress due to an NMDAR-dependent oxidant stress, and if so, 2) what is(are) the intracellular source(s) of these ROS? It is not surprising that glutamate stimulation of endothelial NMDARs causes an oxidant stress, because it is accepted that glutamate stimulates the production and release of ROS from the surrounding glial and neuronal cells (1–3, 37). However, the fact that the endothelial stress was blocked by pretreatment with the antioxidant NAC suggests that the oxidant stress originates intracellularly and is not derived from extraendothelial sources (i.e., glial or neuronal sources, xanthine oxidase, or active neutrophils).

To test whether cerebral endothelial cells exposed to glutamate exhibit an NMDAR-dependent intracellular oxidant stress, we loaded human brain capillary endothelial cells with the oxidant-sensitive fluorescent probe DHR and exposed them to 1 mM glutamate with/without a pre- and cotreatment with MK-801, TMB-8, and LaCl3. As seen in Fig. 1, 1 mM glutamate significantly increased rhodamine 123 fluorescent intensity, and this oxidant stress was blocked by MK-801 pretreatment, a specific noncompetitive inhibitor of the NMDAR (41). Similar tests on these cells with hydroethidine failed to reveal measurable production of superoxide in response to NMDA or glutamate (data not shown). Because DHR is a hydroxyl- and peroxynitrite-selective probe (34), our data suggest that either or both hydroxyl or peroxynitrite radicals, but probably not superoxide, could mediate these effects. Because desferal, an iron chelator, reduced oxidant stress (measured by DHR) in response to glutamate by ~84%, our findings are consistent with hydroxyl radical formation through classic Fenton chemistry.

Pilot studies with brain endothelial cells exposed to nitric oxide (NO) synthase (NOS) inhibition suggests that these cells were actually stressed by exposure to NOS inhibitors (e.g., N-iminoethyl-L-ornithine or N-nitro-L-arginine methyl ester). Therefore, it is possible tonic NO production by these brain endothelium is protective and its reduction is stressful. Because this glutamate stress appears to be largely hydroxyl radical dependent, peroxynitrite could play a relatively minor role in the oxidant/barrier dysfunction described in this response to glutamate (41).

NMDARs are ligand-gated Ca2+ channels; therefore, if this glutamate-induced increase in intracellular ROS concentration is NMDAR dependent, then the oxidant stress should be Ca2+ dependent. Blocking intracellular Ca2+ with TMB-8 (an intracellular Ca2+ antagonist) significantly decreased the amount of intracellular ROS produced in response to glutamate (Fig. 2), but inhibition of Ca2+ influx with LaCl3 (a Ca2+ channel inhibitor) completely blocked the glutamate-induced oxidant stress (Fig. 3). These findings support our previous observations (41) that human cerebral endothelial cells can respond to glutamate via NMDARs in an oxidant-dependent manner; however, that report did not identify the sources of these ROS.

Because mitochondria are important sources of oxidants in neuronal excitotoxicity, we speculated that they might also be a source of ROS in endothelial cells. However, mitochondria are not the only sources of ROS that can be activated by Ca2+ elevation in cells. Other sources that have been suggested to play a role in the development of cerebral microvascular edema associated with excitotoxicity are NADPH oxidase, arachidonic acid metabolism, including cyclooxygenases (COX), lipoxygenases, and CYP450 monooxygenases.

When cells are made ischemic and reoxygenated, they often activate protective mechanisms that preserve the integrity of the mitochondria to maintain cell viability; however, this is accomplished at the expense of normal mitochondrial functions (7). During reoxygenation, mitochondrial membrane permeability increases and is characterized by a Ca2+-dependent collapse of the ion gradient mitochondrial depolarization, uncoupling of oxidative phosphorylation, and results in cellular ATP depletion (18). Therefore, ischemia compromises the normal mitochondrial function, altering the cellular redox status and leading to a cellular metabolic imbalance (8, 13, 16).

Studies on stroke suggest that mitochondrial generation of ROS contributes to endothelial following ischemia reperfusion. The role of the mitochondria in producing ROS is a difficult subject to study, because several inhibitors of mitochondrial respiration are prooxidant and/or proapoptotic (39). It was recently reported that CSA prevents the opening of mitochondrial membrane pores, a step required for ROS generation by mitochondria. Therefore, to test whether mitochondria were a source of oxidants in this response, we pretreated human cerebral endothelial cells with CSA before glutamate exposure. CSA completely inhibited glutamate-induced endothelial intracellular ROS development (Fig. 4). However, CSA is also capable of blocking heme proteins (4) e.g., CYP450; therefore, the complete inhibition of glutamate-induced ROS development by CSA only allows us to conclude that the mitochondria might be only one source of oxidant stress.

NADPH oxidase is a superoxide generating complex enzyme composed of at least five members: flavocytochrome b558 (the p21phox and gp91phox subunits), p47phox, p67phox, and Rac-2 (a GTP-binding protein). Ca2+ will activate Rac-2 and promote assembly of this NADPH oxidase complex. The activated NADPH oxidase complex has been shown to produce superoxide in endothelial cells (46), and, in fact, brain injury is reduced in mice lacking functional NADPH oxidase (45), documenting its role in injury. It is therefore possible that NADPH oxidase might be an additional source of endothelial ROS in glutamate stress. Pretreatment of cerebral endothelial cells with DPI (a flavin-containing protein inhibitor) will inhibit both NADPH oxidase and CYP450 (15). DPI significantly decreased the intracellular ROS development induced by glutamate (Fig. 5). Even so, inhibition of flavin-containing proteins with DPI did not completely block the increase in ROS. The observed decrease in oxidants caused by DPI pretreatment indicates that flavoproteins (i.e., NADPH oxidase and CYP450) may produce oxidants in this model.

Furthermore, because DPI block both NADPH oxidase and CYP450, we decided to treat the cerebral endothelial cells with itraconazole, a CYP450 3A4 inhibitor (38). Comparing the effects of pre- and cotreatment of brain endothelium with itraconazole and the DPI might discriminate between NADPH oxidase and CYP450 contributions in glutamate-induced stress. Itraconazole significantly blocked glutamate-induced ROS (Fig. 6). Because blockade of CYP450 does not completely block the formation of ROS suggests that both NADPH oxidase and CYP450 are equivalent sources of oxidants in the cerebral endothelial cells during glutamate stress.

Metabolism of arachidonate by three enzyme families including COX, lipoxygenases, and CYP450 can produce superoxide as a by-product and may augment cerebral ischemic injury (6, 12, 18, 26, 30, 30). Ca2+ will activate the oxidant-producing pathways of COX, lipoxygenase, and CYP450 via the activation of phospholipase A2 (PLA2); therefore, we pretreated the human cerebral endothelial cells with BPB, a PLA2 inhibitor to examine the role of arachidonate metabolism in ROS formation in response to glutamate. BPB did not decrease ROS production in response to glutamate exposure (Fig. 7). Therefore, because PLA2 inhibition did not decrease glutamate-induced ROS formation, our data suggest that AA metabolism is not related to oxidant stress in this response. However, as stated, itraconazole (a CYP450 inhibitor) did significantly decrease glutamate-induced oxidant stress. Although CYP450 is commonly associated with AA metabolism, there are other forms and locations of this enzyme [including the cytosol and the mitochondria (38)]. The results of the BPB and itraconazole experiments suggesting that CYP450 is involved in the glutamate response is unrelated to arachidonate metabolism.

Brain edema remains a life-threatening complication of stroke infarction with an 80% mortality rate (40). We previously reported (41) that human cerebral endothelial cells express NMDARs and that activation of these receptors results in glutamate stress and subsequent edema. Persistent stimulation of NMDARs during neuronal excitotoxicity is associated with a rapid and sustained elevation in cytoplasmic Ca2+ and production of ROS via several pathways (i.e., mitochondrial respiration, NADPH oxidase, and AA metabolism) (33). The results of these experiments indicate that glutamate can contribute to forms of brain injury via an NMDAR-mediated loss of endothelial barrier involving formation of ROS involving activation of the mitochondria, NADPH oxidase, and CYP450. The extent that each of these pathways contribute has not been determined exactly; but our data suggest that mitochondrial respiration is most likely a source (as is reported in neuronal excitotoxicity). We and others (6, 23, 27–30, 42–44) have shown that increased oxidants are linked to barrier dysfunction via the activation of PKC; we can only speculate currently as to whether this form of oxidant-mediated barrier dysfunction is also PKC dependent.

Future studies will follow the sources involved in the glutamate-induced cerebral endothelial oxidant stress by using brain endothelium isolated from gene knockout mice and via transfection. These methods will allow us to determine exactly the role of the mitochondria, NADPH oxidase, and CYP450. Although the data presented in this manuscript are pharmacological, they do suggest that both blockade of endothelial NMDARs and the use of antioxidants may be important prophylactic and therapeutic tools in the treatment of stroke and other associated cerebral traumas.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Danica Stanimirovic for supplying us with the IHEC used in this study.


   FOOTNOTES
 

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)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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