Apoptotic Cell Death and Caspase-3 Activation Induced by N-Methyl-d-Aspartate Receptor Antagonists and Their Prevention by Insulin-Like Growth Factor I

The effect of N-methyl-D-aspartate (NMDA) receptor antagonists on cell viability was studied in rat primary cortical cells. NMDA antagonists [MK-801 and 2-amino-5-phosphonovalerate (APV)] induced cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation. Treatment of cells with MK-801 (an NMDA antagonist) for 1-2 days induced apoptotic cell death in a dose-dependent manner (1 nM to 10 microM). NMDA (25 microM), however, inhibited the MK-801 (0.1 microM)-induced apoptotic cell death. MK-801 and APV decreased the concentration of intracellular calcium ion. Activation of caspase-3 was accompanied by MK-801-induced cell death in a dose-dependent manner, and an inhibitor of caspase-3 reduced the cell death. Further, cycloheximide (0.2 microg/ml) completely protected the cells from MK-801-induced apoptotic cell death and caspase-3 activation. Insulin-like growth factor I completely attenuated MK-801-induced apoptotic cell death and caspase-3 activation. These results demonstrated that the moderate NMDA receptor activation is probably involved in the survival signal of the neuron.     

Regulation of homocysteine-induced MMP-9 by ERK1/2 pathway

Homocysteine (Hcy) induces matrix metalloproteinase (MMP)-9 in microvascular endothelial cells (MVECs). We hypothesized that the ERK1/2 signaling pathway is involved in Hcy-mediated MMP-9 expression. In cultured MVECs, Hcy induced activation of ERK, which was blocked by PD-98059 and U0126 (MEK inhibitors). Pretreatment with BAPTA-AM, staurosporine (PKC inhibitor), or Gö6976 (specific inhibitor for Ca²⁺-dependent PKC) abrogated ERK phosphorylation, suggesting the role of Ca²⁺ and Ca²⁺-dependent PKC in Hcy-induced ERK activation. ERK phosphorylation was suppressed by pertussis toxin (PTX), suggesting the involvement of G protein-coupled receptors (GPCRs) in initiating signal transduction by Hcy and leading to ERK activation. Pretreatment of MVECs with genistein, BAPTA-AM, or thapsigargin abrogated Hcy-induced ERK activation, suggesting the involvement of the PTK pathway in Hcy-induced ERK activation, which was mediated by intracellular Ca²⁺ pool depletion. ERK activation was attenuated by preincubation with N-acetylcysteine (NAC) and SOD, suggesting the role of oxidation in Hcy-induced ERK activation. Pretreatment with an ERK1/2 blocker (PD-98059), staurosporine, folate, or NAC modulated Hcy-induced MMP-9 activation as measured using zymography. Our results provide evidence that Hcy triggers the PTX-sensitive ERK1/2 signaling pathway, which is involved in the regulation of MMP-9 in MVECs. calcium signaling; protein kinase C; Src; G protein-coupled receptor; nonreceptor tyrosine kinase; protein G_i; protein G_q; protein tyrosine kinase 2; microvascular endothelial cell; cardiovascular remodeling     

NMDA receptor activation induces mitochondrial dysfunction, oxidative stress and apoptosis in cultured neonatal rat cardiomyocytes

Glutamate is a well-characterized excitatory neurotransmitter in the central nervous system (CNS). Recently, glutamate receptors (GluRs) were also found in peripheral tissues, including the heart. However, the function of GluRs in peripheral organs remains poorly understood. In the present study, we found that N-methyl-D-aspartate (NMDA) could increase intracellular calcium ([Ca(2+)]i) level in a dose-dependent manner in cultured neonatal rat cardiomyocytes. NMDA at 10(-4) M increased the levels of reactive oxygen species (ROS), cytosolic cytochrome c (cyto c), and 17-kDa caspase-3, but depolarized mitochondrial membrane potential, leading to cardiomyocyte apoptosis. In addition, NMDA treatment induced an increase in bax mRNA but a decrease in bcl-2 mRNA expression in the cardiomyocytes. The above effects of NMDA were blocked by the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), and by ROS scavengers glutathione (GSH) and N-acetylcystein (NAC). These results suggest that stimulation of NMDA receptor in the cardiomyocyte may lead to apoptosis via a Ca(2+), ROS, and caspase-3 mediated pathway. These findings suggest that NMDA receptor may play an important role in myocardial pathogenesis.     

Glutamate up-regulates P-glycoprotein expression in rat brain microvessel endothelial cells by an NMDA receptor-mediated mechanism

The accumulation of glutamate in the extracellular space in the central nervous system (CNS) plays a major part in ischemic and anoxic damage. In this study, we examined the effect of glutamate on the expression and activity of P-glycoprotein (P-gp) in rat brain microvessel endothelial cells (RBMECs) making up the blood-brain barrier (BBB). The level of P-gp expression significantly increased in RBMECs after the treatment of 100 microM glutamate. At this concentration, glutamate also enhanced rat mdr1a and mdr1b mRNA levels determined by RT-PCR analysis. Flow cytometry was used to study P-gp activity by analysis of intracellular rhodamine123 (Rh123) accumulation. Overexpression of P-gp resulted in a decreased intracellular accumulation of Rh123 in RBMECs. Glutamate-induced increase of intracellular reactive oxygen species (ROS) was observed by using the 2',7'-dichlorofluorescein (2',7'-DCF) assay. MK-801, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, and ROS scavenger N-acetylcysteine obviously blocked ROS generation and attenuated the changes of both expression and activity of P-gp induced by glutamate in RBMECs. These data suggested that glutamate up-regulated P-gp expression in RBMECs by an NMDA receptor-mediated mechanism and that glutamate-induced generation of ROS was linked to the regulation of P-gp expression. Therefore, transport of P-gp substrates in BBB appears to be affected during ischemic and anoxic injury.     

MK-801抑制福尔马林实验引起的大鼠脊髓背角环氧合酶-2的表达

应用免疫组织化学方法,观察鞘内注射N-methyl—D—aspartate(NMDA)受体拮抗剂MK-801对福尔马林实验引起的大鼠脊髓背角环氧合酶-2(cyclooxygenase-2,COX-2)表达的影响。结果表明：MK-801对福尔马林实验引起的第1相缩足反射仅有一定抑制作用,但对第2相缩足反射有显著的抑制作用,且呈剂量依赖性。与这种行为学的变化相对应,MK-801可显著抑制福尔马林实验引起的脊髓背角COX-2表达的增加,并且这种抑制作用与MK-801的剂量呈正相关。这些结果表明,在福尔马林实验中,NMDA受体的活动是引起脊髓背角COX-2表达增加的原因之一。     

Ouabain-sensitive and ouabain-resistant isoforms of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase in cerebellar granule cells control MAP kinase activity

Three-hour incubation of rat cerebellar granule cells with 0.1 μM ouabain increases intracellular levels of Ca²⁺ ions and reactive oxygen species (ROS) resulting in pronounced activation of Mitogen-Activated Protein Kinase (MAPK). Higher concentrations of ouabain induce further increases in MAPK activity. The activating effect of ouabain is attenuated by the NMDA-receptor antagonists MK-801 and D-AP5. The data obtained suggest that similar to NMDA receptors ouabain-sensitive and ouabain-resistant isoforms of Na⁺,K⁺-ATPase are actively involved in intracellular signaling cascades controlling proliferative activity of neuronal cells.     

Rodent lymphocytes express functionally active glutamate receptors

RT-PCR demonstrated that ionotropic (iGluR NR1) and metabotropic (mGluR Group III) glutamate receptors are expressed in rodent lymphocytes. Flow cytometry showed that activation of iGluR NR1 by N-methyl-D-aspartate (NMDA) increased intracellular free calcium and reactive oxygen species (ROS) levels and activated caspase-3. The latter effect was attenuated by the NMDA antagonist, 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), by the antioxidant N-acetylcysteine and by cyclosporin A. Treatment with L-2-amino-4-phosphonobutyric acid (L-AP4), an mGluR Group III agonist, increased lymphocyte ROS levels but to a lower extent than did NMDA. Activation of lymphocytes with both NMDA and L-AP4 caused a synergistic increase in ROS levels and induced necrotic cellular death without elevating the caspase-3 activation observed in the presence of NMDA alone. These results show that lymphocyte iGluR NR1 and mGluR Group III receptors may be involved in controlling rodent lymphocyte functions and longevity as they regulate events in cell proliferation, maturation, and death.     

NMDA receptor blockade attenuates CCK-induced reduction of real feeding but not sham feeding

Systemic injection of MK-801, a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor ion channels, increases meal size and delays satiation. We examined whether MK-801 increases food intake by directly interfering with actions of cholecystokinin (CCK). Prior administration of MK-801 (100 microg/kg ip) reversed the inhibitory effects of CCK-8 (2 and 4 microg/kg ip) on real feeding of both liquid and solid foods. MK-801 alone did not alter 30-min sham intake of 15% sucrose compared with intake after saline. Furthermore, while CCK-8 (2 or 4 microg/kg ip) reduced sham intake, this reduction was not attenuated by MK-801 pretreatment. To ascertain whether MK-801 attenuation of CCK-induced reduction of real feeding was associated with attenuated inhibition of gastric emptying, we tested the effect of MK-801 pretreatment on CCK-induced inhibition of gastric emptying of 5-ml saline loads. Ten-minute gastric emptying was accelerated after MK-801 (3.9 +/- 0.2 ml) compared with saline vehicle (2.72 +/- 0.2 ml). CCK-8 (0.5 microg/kg ip) reduced 10-min emptying to 1.36 +/- 0.3 ml. Pretreatment with MK-801 did not significantly attenuate CCK-8-induced reduction of gastric emptying (0.9 +/- 0.4 ml). This series of experiments demonstrates that blockade of NMDA ion channels reverses inhibition of real feeding by CCK. However, neither inhibition of sham feeding nor inhibition of gastric emptying by CCK is attenuated by MK-801. Therefore, increased food intake after NMDA receptor blockade is not caused by a direct interference with CCK-induced satiation. Rather, increased real feeding, either in the presence or absence of CCK, depends on blockade of NMDA receptor participation in other post-oral feedback signals such as gastric sensation or gastric tone.     

Homocysteine-Induced Apoptosis in Endothelial Cells Coincides With Nuclear NOX2 and Peri-nuclear NOX4 Activity

Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01–2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47^phox expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ _m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ _m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47^phox, and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47^phox in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47^phox was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.     

Stimulation of Dopamine Release from Cultured Rat Mesencephalic Cells by Naturally Occurring Excitatory Amino Acids: Involvement of Both N-Methyl-D-Aspartate (NMDA) and Non-NMDA Receptor Subtypes

In rat mesencephalic cell cultures, L-glutamate at concentrations ranging from 100 microM to 1 mM stimulated release of [3H]dopamine that was attenuated by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6,7-dinitroquinoxalinedione, but not by the selective NMDA receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801; 10 microM) and 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (300 microM). Even at 1 mM glutamate, this release was Ca2+ dependent. These observations suggest that the release was mediated by a non-NMDA receptor. Only release stimulated by a lower concentration (10 microM) of glutamate was inhibited by MK-801 (10 microM), indicating that glutamate at this concentration activates the NMDA receptor. By contrast, L-aspartate at concentrations of 10 microM to 1 mM evoked [3H]dopamine release that was completely inhibited by MK-801 (10 microM) and was also Ca2+ dependent (tested at 1 and 10 mM aspartate). Thus, effects of aspartate involved activation of the NMDA receptor. Sulfur-containing amino acids (L-homocysteate, L-homocysteine sulfinate, L-cysteate, L-cysteine sulfinate) also evoked [3H]dopamine release. Release evoked by submillimolar concentrations of these amino acids was attenuated by MK-801 (10 microM), indicating involvement of the NMDA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMDA glutamate receptor antagonist MK-801 induces proteome changes in adult human brain slices which are partially counteracted by haloperidol and clozapine

Deciphering the molecular pathways associated with N-methyl-D-aspartate receptor (NMDAr) hypofunction and its interaction with antipsychotics is necessary to advance our understanding of the basis of schizophrenia, as well as our capacity to treat this disease. In this regard, the development of human brain-derived models that are amenable to studying the neurobiology of schizophrenia may contribute to filling the gaps left by the widely employed animal models. Here, we assessed the proteomic changes induced by the NMDA glutamate receptor antagonist MK-801 on human brain slice cultures obtained from adult donors submitted to respective neurosurgery. Initially, we demonstrated that MK-801 diminishes NMDA glutamate receptor signaling in human brain slices in culture. Next, using mass-spectrometry-based proteomics and systems biology in silico analyses, we found that MK-801 led to alterations in proteins related to several pathways previously associated with schizophrenia pathophysiology, including ephrin, opioid, melatonin, sirtuin signaling, interleukin 8, endocannabinoid, and synaptic vesicle cycle. We also evaluated the impact of both typical and atypical antipsychotics on MK-801-induced proteome changes. Interestingly, the atypical antipsychotic clozapine showed a more significant capacity to counteract the protein alterations induced by NMDAr hypofunction than haloperidol. Finally, using our dataset, we identified potential modulators of the MK-801-induced proteome changes, which may be considered promising targets to treat NMDAr hypofunction in schizophrenia. This dataset is publicly available and may be helpful in further studies aimed at evaluating the effects of MK-801 and antipsychotics in the human brain.     

Chronic Dizocilpine (MK-801) Reversibly Delays GABAA Receptor Maturation in Cerebellar Granule Neurons In Vitro

Abstract: We investigated the effect of chronically blocking NMDA receptor stimulation to examine changes in GABA_A receptor expression and pharmacology in cerebellar granule cells at different stages of maturation. We have previously shown that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunits. When NMDA receptor stimulation is blocked with MK-801 during the first week in vitro, a decrease in the α1, γ2S, and γ2L receptor subunit mRNAs occurred. When present only during the second week, changes were limited to the α1 and γ2L mRNAs. Finally, if MK-801 was present during the first week and removed during the second week, these changes reversed. Whole-cell voltage-clamp recordings showed that treatment with MK-801 during either the first or second week increased the EC₅₀ of the receptors for GABA and attenuated the potentiation mediated by flunitrazepam. Last, these properties were reversed if MK-801 was removed after the first week in vitro. Our results suggest that MK-801 reversibly inhibits GABA_A receptor maturation by modulating receptor subunit expression and that the altered pharmacological responses appear to be dominated by changes in the levels of allosteric modulation mediated by the γ2 receptor subunit.     

Reactive Transformation and Increased BDNF Signaling by Hippocampal Astrocytes in Response to MK-801

MK-801, also known as dizocilpine, is a noncompetitive N-methyl-D-aspartic acid (NMDA) receptor antagonist that induces schizophrenia-like symptoms. While astrocytes have been implicated in the pathophysiology of psychiatric disorders, including schizophrenia, astrocytic responses to MK-801 and their significance to schizotypic symptoms are unclear. Changes in the expression levels of glial fibrillary acid protein (GFAP), a marker of astrocyte activation in response to a variety of pathogenic stimuli, were examined in the hippocampus of rats treated with the repeated MK-801 injection (0.5 mg/10ml/kg body weight for 6 days) and in primary cultured hippocampal astrocytes incubated with MK-801 (5 or 20 μM for 24 h). Moreover, the expression levels of BDNF and its receptors TrkB and p75 were examined in MK-801-treated astrocyte cultures. MK-801 treatment enhanced GFAP expression in the rat hippocampus and also increased the levels of GFAP protein and mRNA in hippocampal astrocytes in vitro. Treatment of cultured hippocampal astrocytes with MK-801 enhanced protein and mRNA levels of BDNF, TrkB, and p75. Collectively, our results suggest that hippocampal astrocytes may contribute to the pathophysiology of schizophrenia symptoms associated with NMDA receptor hypofunction by reactive transformation and altered BDNF signaling.     

Upregulation of aldose reductase by homocysteine in type II alveolar epithelial cells

Homocysteine (Hcy) has recently been recognized as an integral component of several disorders. However, the association between hyperhomocysteinemia (HHcy) and pulmonary disease is not well understood. The combination of two-dimensional electrophoresis and tandem mass spectrometry detected and identified proteins that are differentially expressed in human type II alveolar epithelial cells (A549 cells) treated by Hcy. We found that aldose reductase (AR) showed more abundant expression in the cells. Further, Hcy (100-500microM) could induce a time- and dose-dependent upregulation of AR protein levels. Immunohistochemical staining of cross-sections from HHcy mice lungs also revealed increased expression of AR protein. Intracellular levels of reactive oxygen species (ROS) were remarkably elevated in A549 cells treated with Hcy. Pretreatment of A549 cells with catalase and SOD significantly suppressed the Hcy-induced AR expression, which suggests the involvement of ROS in this process. The major signaling pathway mediating the upregulation of AR was demonstrated to be the Ras/Raf/ERK1/2 pathway. In addition, Hcy might reduce surfactant protein B (SP-B) expression in the cells, which could be significantly attenuated by Alrestatin, an AR inhibitor, indicating a damaging role of Hcy-induced AR elevation in the lung. These results show a novel and unanticipated link between HHcy and AR upregulation that may be a risk factor in pulmonary disease of patients with HHcy.     

Activity-dependent NMDA receptor-mediated activation of protein kinase B/Akt in cortical neuronal cultures

The serine/threonine protein kinase B (PKB)/Akt is a phosphoinositide 3-kinase (PI3K) effector that is thought to play an important roll in a wide variety of cellular events. The present study examined whether PKB activation in cortical neuronal cultures is coupled with synaptic activity. A 1-h incubation of neuronal cultures with tetrodotoxin (TTX), the PI3K inhibitor wortmannin, the NMDA receptor antagonist MK-801 or removal of extracellular calcium significantly reduced basal levels of phospho(Ser473)-PKB, indicating that activity-dependent glutamate release maintains PKB activation through an NMDA receptor-PI3K pathway. A 5-min exposure to NMDA (50 micro m) in the presence of TTX increased phospho-PKB back to levels observed in the absence of TTX. NMDA stimulation of phospho-PKB was blocked by wortmannin, the CaMKII inhibitor KN-93, MK-801, and removal of extracellular calcium. We have previously shown that NMDA receptors can bi-directionally regulate activation of extracellular-signal regulated kinase (ERK), and NMDA receptor stimulation of PKB in the present study appeared to mirror activation of ERK. These results suggest that in cultured cortical neurons, PKB activity is dynamically regulated by synaptic activity and is coupled to NMDA receptor activation. In addition, NMDA receptor activation of ERK and PKB may occur through overlapping signaling pathways that bifurcate at the level of Ras.     

Homocysteine activates T cells by enhancing endoplasmic reticulum-mitochondria coupling and increasing mitochondrial respiration

Hyperhomocysteinemia (HHcy) accelerates atherosclerosis by increasing proliferation and stimulating cytokine secretioninTcells.However,whetherhomocysteine (Hcy)-mediated T cell activation is associated with metabolic reprogramming is unclear. Here, our in vivoand in vitrostudies showed that Hcy-stimulated splenic T-cell activation in mice was accompanied by increased levels of mitochondrial reactive oxygen species (ROS) and calcium, mitochondrial mass and respiration. Inhibiting mitochondrial ROS production and calcium signals or blocking mitochondrial respiration largely blunted Hcy-induced T-cell interferon γ (IFN-γ) secretion and proliferation. Hcy also enhanced endoplasmic reticulum (ER) stress in T cells, and inhibition ofERstress with 4-phenylbutyric acid blocked Hcy-induced T-cell activation. Mechanistically, Hcy increased ER-mitochondria coupling, and uncoupling ER-mitochondria by the microtubule inhibitor nocodazole attenuated Hcy-stimulated mitochondrial reprogramming, IFN-γ secretion and proliferation in T cells, suggesting that juxtaposition of ER and mitochondria is required for Hcy-promoted mitochondrial function and T-cell activation. In conclusion, Hcy promotes T-cell activation by increasing ER-mitochondria coupling and regulating metabolic reprogramming.     

Intracranial pancreatic islet transplantation increases islet hormone expression in the rat brain and attenuates behavioral dysfunctions induced by MK-801 (dizocilpine)

The treatment of rodents with non-competitive antagonist of the N-Methyl-d-aspartate (NMDA) receptor, MK-801 (dizocilpine), induces symptoms of psychosis, deficits in spatial memory and impairment of synaptic plasticity. Recent studies have suggested that insulin administration might attenuate the cognitive dysfunctions through the modulatory effect on the expression of NMDA receptors and on the brain insulin signaling. Intrahepatic pancreatic islet transplantation is known as an efficient tool for correcting impaired insulin signaling. We examined the capacity of syngeneic islets grafted into the cranial subarachnoid cavity to attenuate behavioral dysfunctions in rats exposed to MK-801. Animals were examined in the open field (OF) and the Morris Water Maze (MWM) tests following acute or subchronic administration of MK-801. We found well-vascularized grafted islets expressing insulin, glucagon and somatostatin onto the olfactory bulb and prefrontal cortex. Significantly higher levels of insulin were detected in the hippocampus and prefrontal cortex of transplanted animals compared to the non-transplanted rats. All animals expressed normal peripheral glucose homeostasis for two months after transplantation. OF tests revealed that rats exposed to MK-801 treatment, showed hyper-responsiveness in motility parameters and augmented center field exploration compared to intact controls and these effects were attenuated by the grafted islets. Moreover, in the MWM, the rats treated with MK-801 showed impairment of spatial memory that were partially corrected by the grafted islets. In conclusion, intracranial islet transplantation leads to the expression of islet hormones in the brain and attenuates behavioral and cognitive dysfunctions in rats exposed to MK-801 administration without altering the peripheral glucose homeostasis.     

Homocysteine-evoked 45Ca release in the rabbit hippocampus is mediated by both NMDA and group I metabotropic glutamate receptors: in vivo microdialysis study

This in vivo microdialysis study compared the effects of NMDA and d,l-homocysteine (Hcy) administered via dialysis medium on ⁴⁵Ca efflux from prelabeled rabbit hippocampus. Application of these agonists evoked dose-dependent, and sensitive to MK-801, opposite effects: NMDA decreased the ⁴⁵Ca radioactivity in the dialysate, whereas Hcy induced the release of ⁴⁵Ca. The latter effect was potentiated by glycine, inhibited by the antagonist of group I metabotropic glutamate receptors (mGluR) LY367385, and mimicked by t-ADA, an agonist of these receptors. Electron microscopic examination of pyramidal neurones in the CA1 sector of the hippocampus in the vicinity of the microdialysis probe after NMDA application demonstrated swelling of mitochondria, which was prevented by cyclosporin A. This study shows, for the first time, Hcy-induced activation of both group I mGluR and NMDA receptors, which may play a role in acute Hcy neurotoxicity. We present new applications of brain microdialysis in studies on excitotoxicity and neuroprotection.     

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

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.