Glutamate-induced Increase Research Articles

Aminoglutethimide prevents excitotoxic and ischemic injuries in cortical neurons.

Aminoglutethimide is a clinically available drug that suppresses steroid biosynthesis by inhibiting enzymes such as cytochrome P450scc and aromatase. Because several members of neurosteroids regulate glutamate receptors, we investigated the effect of aminoglutethimide on cell death induced by overactivation of glutamate receptors in CNS neurons. Long-term pretreatment of organotypic cerebrocortical slice cultures with aminoglutethimide (100-1000 microM) for 6 days or over resulted in concentration-dependent suppression of neuronal cell death induced by NMDA. Aminoglutethimide (1000 microM) also inhibited neurotoxicity of AMPA and kainate, but not of ionomycin or staurosporine. The protective effect of aminoglutethimide against NMDA cytotoxicity was not mimicked by other steroid synthesis inhibitors including trilostane and exemestane, and was not reversed by concurrent application of steroids such as pregnenolone, estrone, 17beta-estradiol and estriol. In dissociated rat cerebrocortical cell cultures, long-term treatment with aminoglutethimide (10-1000 microM) attenuated NMDA receptor-mediated glutamate cytotoxicity but produced no significant effect on glutamate-induced increases in intracellular Ca2+. Brief as well as long-term pretreatment with aminoglutethimide (30-1000 microM) prevented NMDA receptor-dependent ischemic neuronal injury in organotypic cerebrocortical slice cultures, which was associated with suppression of glutamate release during the ischemic insult. These results indicate that aminoglutethimide, irrelevant to its actions on neurosteroid synthesis, protects CNS neurons from excitotoxic and ischemic injuries. Development of aminoglutethimide analogs possessing neuroprotective properties may be of therapeutic value.

15-Methoxypinusolidic acid from Biota orientalis attenuates glutamate-induced neurotoxicity in primary cultured rat cortical cells

15-Methoxypinusolidic acid (15-MPA), a pinusolide derivative isolated from Biota orientalis (Cupressaceae) leaves prevented glutamate-induced excitotoxicity in primary cultured rat cortical cells in vitro. 15-MPA had more selectivity in protecting neurons against N-methyl- d-aspartate (NMDA)-induced neurotoxicity than that induced by kainic acid (KA). The glutamate-induced increase of intracellular calcium ([Ca 2+] i) in cortical cells was effectively reduced by 15-MPA. Moreover, 15-MPA could successfully reduce the subsequent overproduction of nitric oxide (NO) and the level of cellular peroxide, and inhibit glutathione (GSH) depletion and lipid peroxidation induced by glutamate in our cultures. Collectively, these results suggested that 15-MPA attenuated glutamate-induced excitotoxicity via stabilization of [Ca 2+] i homeostasis and suppression of oxidative stress possibly through the actions on the NMDA receptors.

NMDA and group I metabotropic glutamate receptors activation modulates substance P release from the arcuate nucleus and median eminence

Glutamate participates in the regulation of secretion of several neuropeptides, including substance P (SP). Glutamate acts through ionotropic (iGluR) and metabotropic (mGluR) receptors. We have investigated whether glutamate receptor agonists and antagonists could affect SP release from the arcuate nucleus and the median eminence (ARC/ME). An increase in SP-like immunoreactivity (SP-LI) release from ARC/ME was induced by glutamate and N-methyl- d-aspartate (NMDA). This increase was prevented by d-(−)-2-amino-5-phosphono pentanoic acid (DAP5) (0.1 mM), a specific NMDA antagonist and by (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) (0.1 mM), a selective antagonist of group I mGluR. The selective non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3(1H-4H)-dione (DNQX) (0.1 mM) and (RS)-α-methyl-4-tetrazolylphenylglycine (MTPG) (0.1 mM), a group II and III mGluRs antagonist, did not affect the stimulatory effect of glutamate. A group I selective agonist, ( S)-3,5-dihydroxyphenylglycine (DHPG) induced a significant increase in SP-LI release. Supporting the participation of nitric oxide (NO) in the effect of glutamate on SP-LI release, NAME (0.5 mM), a NO synthase inhibitor, reduced the glutamate-induced increase in SP-LI release from ARC/ME. Similarly, glutamate did not induce an increase in SP-LI release in the presence of meloxicam (0.1 mM) (a cyclooxygenase-2 (COX-2) specific inhibitor) indicating that prostaglandins production may also be involved in the glutamate effect. These data indicate that glutamate increases SP-LI release from the ARC/ME by acting through NMDA and group I mGluRs in the male rat. This stimulatory effect could be mediated by nitric oxide and prostaglandin production.

Cholinergic systems in the posterior hypothalamic nucleus are involved in blood pressure decrease-induced excitation of anterior hypothalamic area neurons in rats

Previously, we have demonstrated that decreases in blood pressure induced by intravenous nitroprusside increase the firing rate of angiotensin II-sensitive neurons in the anterior hypothalamic area (AHA) of rats and that this increase of neural firing rate is blocked by the pressure application of losartan onto the same neurons. It has been suggested that acetylcholine in the posterior hypothalamic nucleus (PHN) serves as a neurotransmitter in a pathway which can modulate baroreceptor reflexes. In the present study, we examined whether acetylcholine in the PHN is involved in the nitroprusside-induced increase of the firing of angiotensin II-sensitive neurons in the AHA of rats. Male Wistar rats were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Decreases in blood pressure induced by intravenous nitroprusside (100 ug/kg) increased the firing rate of AHA angiotensin II-sensitive neurons. The nitroprusside-induced increase of firing rate of AHA neurons was inhibited by PHN microinjection of the cholinoceptor antagonist scopolamine and potentiated by PHN microinjection of the cholinesterase inhibitor physostigmine. Microinjections of carbachol and glutamate into the PHN caused increases of firing rate of AHA neurons. The carbachol-induced but not glutamate-induced increase of unit firing was abolished by the pre-microinjection of scopolamine into the same sites of the PHN. These findings suggest that the nitroprusside-induced increase of firing of AHA neurons is mediated via acetylcholine at the level of the PHN.

Cholinergic stimulation in the posterior hypothalamic nucleus activates angiotensin II-sensitive neurons in the anterior hypothalamic area of rats

We have previously reported that some neurons in the anterior hypothalamic area (AHA) are tonically activated by endogenous angiotensins in rats and that activities of these AHA angiotensin II-sensitive neurons are enhanced in spontaneously hypertensive rats (SHR). Acetylcholine in the posterior hypothalamic nucleus (PHN) has been implicated in hypertension in SHR. It is suggested that there exist neuronal projections from the PHN to the AHA in rats. In this study, we examined whether cholinergic stimulation in the PHN activates AHA angiotensin II-sensitive neurons. Male Wistar rats were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Microinjection of carbachol, physostigmine and glutamate into the PHN caused an increase in firing rate of AHA angiotensin II-sensitive neurons in anesthetized rats. The carbachol-induced increase of firing rate was inhibited by pressure application of the AT1 receptor antagonist losartan onto AHA angiotensin II-sensitive neurons. The glutamate-induced increase of firing rate was also inhibited by the pressure application of losartan. PHN microinjections of carbachol and glutamate did not affect blood pressure in these anesthetized rats. In conscious rats, PHN microinjection of carbachol produced an increase of blood pressure and the carbachol-induced pressor response was inhibited by bilateral microinjections of losartan into the AHA. These findings indicate that cholinergic stimulation in the PHN activates AHA angiotensin II-sensitive neurons. It seems likely that the activation of AHA angiotensin II-sensitive neurons induced by PHN cholinergic stimulation is partly mediated via release of angiotensins at AHA angiotensin II-sensitive neuron levels.

Chronic Treatment with Mood Stabilizers Lithium and Valproate Prevents Excitotoxicity by Inhibiting Oxidative Stress in Rat Cerebral Cortical Cells

Recent studies indicate that chronic treatment with the mood-stabilizing drugs lithium and valproate produces a neuroprotective effect against excitotoxicity. In this study, we aimed to determine whether inhibiting oxidative damage plays a role in a neuroprotective effect of lithium and valproate against excitotoxicity. Intracellular free calcium concentration was measured with the fluorescent calcium ion indicator fluo-3. Malondialdehyde, an end product derived from peroxidation of polyunsaturated fatty acid, and protein carbonyls were used to assess oxidative damage to lipid and protein. Excitotoxicity was assayed by measuring cell viability with the MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] method and by measuring deoxyribonucleic acid (DNA) fragmentation with TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling) staining. We found that chronic treatment with lithium and valproate at their therapeutically relevant concentrations significantly inhibited the glutamate-induced increase of intracellular free calcium concentration, lipid peroxidation, protein oxidation, DNA fragmentation, and cell death in primary cultured rat cerebral cortical cells. This treatment had no effect on basal intracellular free calcium concentration, lipid peroxidation, protein oxidation, DNA fragmentation, and cell death. Our results suggest that chronic treatment with lithium and valproate inhibits oxidative damage to lipid and protein and in turn produces a neuroprotective effect against excitotoxicity.

Calcium-mediated spine stem restructuring

A spine is a protrusion from the dendritic (or somatic) surface of a neuron. In recent experiments, caffeine-induced calcium released from internal stores was shown to cause elongation of dendritic spine stems in slice cultures. Still another experiment indicates that glutamate-induced increases in calcium may cause spine stem shortening. Harris draws a schematic model to explain these seemingly conflicting results, indicating that a small amount of activity may increase free calcium within the spines and cause spine stem elongation, but an excessive amount of activity may increase intraspine calcium beyond a critical level and cause spine stem shortening (see [1, Figure 2]). This paper develops a mathematical model for a fixed population of spines along the dendrite, each with a dynamic structure and calcium level. The system is integrated over time and space to observe an interdependent relationship between calcium, morphology and chemical/electrical activity. Results of simulation qualitatively capture phenomena observed in recent experiments and exhibit periodic oscillations in potential when the spines have excitable membrane properties by allowing spine structure to transition through threshold geometries for generation of action potentials in a bidirectional manner. As in recent experiments, a variety of chemical and structural profiles emerge, depending on membrane properties, patterns of synaptic input, and initial conditions considered.

Neuroprotective effects of novel small peptides in vitro and after brain injury

Thyrotropin-releasing hormone (TRH) and TRH analogues have been reported to be neuroprotective in experimental models of spinal cord injury and head injury. We have previously shown that a diketopiperazine structurally related to the TRH metabolite cyclo-his-pro reduces neuronal cell death in vitro and in vivo. Here we report the neuroprotective activity of other cyclic dipeptides in multiple in vitro models of neuronal injury and after controlled cortical impact (CCI) in mice. Using primary neuronal cultures, three novel dipeptides were compared to the previously reported diketopiperazine as well as to vehicle controls; each of the compounds reduced cell death after direct physical trauma or trophic withdrawal. Two of these peptides also protected against glutamate toxicity and β-amyloid-induced injury; the latter also strongly inhibited glutamate-induced increases in intracellular calcium. Treatment with each of the test compounds resulted in highly significant improvement of motor and cognitive recovery after CCI, as well as markedly reducing lesion volumes as shown by high field magnetic resonance imaging. DNA microarray studies following fluid percussion induced traumatic brain injury (TBI) in rats showed that treatment with one of these dipeptides after injury significantly down-regulated expression of mRNAs for cell cycle proteins, aquaporins, cathepsins and calpain in ipsilateral cortex and/or hippocampus, while up-regulating expression of brain-derived neurotrophic factor, hypoxia-inducible factor and several heat-shock proteins. Many of these mRNA expression changes were paralleled at the protein level. The fact that these small peptides modulate multiple mechanisms favoring neuronal cell survival, as well as their ability to improve functional outcome and reduce posttraumatic lesion size, suggests that they may have potential utility in clinical head injury.

Novel Dimeric Acetylcholinesterase Inhibitor Bis(7)-tacrine, but Not Donepezil, Prevents Glutamate-induced Neuronal Apoptosis by Blocking N-Methyl-d-aspartate Receptors

The neuroprotective properties of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons (CGNs). Exposure of CGNs to 75 mum glutamate resulted in neuronal apoptosis as demonstrated by Hoechst staining, TUNEL, and DNA fragmentation assays. The bis(7)-tacrine treatment (0.01-1 mum) on CGNs markedly reduced glutamate-induced apoptosis in dose- and time-dependent manners. However, donepezil and other AChE inhibitors, even at concentrations of inhibiting AChE to the similar extents as 1 mum bis(7)-tacrine, failed to prevent glutamate-induced excitotoxicity in CGNs; moreover, both atropine and dihydro-beta-erythroidine, the cholinoreceptor antagonists, did not affect the anti-apoptotic properties of bis(7)-tacrine, suggesting that the neuroprotection of bis(7)-tacrine appears to be independent of inhibiting AChE and cholinergic transmission. In addition, ERK1/2 and p38 pathways, downstream signals of N-methyl-d-aspartate (NMDA) receptors, were rapidly activated after the exposure of glutamate to CGNs. Bis(7)-tacrine inhibited the apoptosis and the activation of these two signals with the same efficacy as the coapplication of PD98059 and SB203580. Furthermore, using fluorescence Ca(2+) imaging, patch clamp, and receptor-ligand binding techniques, bis(7)-tacrine was found effectively to buffer the intracellular Ca(2+) increase triggered by glutamate, to reduce NMDA-activated currents and to compete with [(3)H]MK-801 with an IC(50) value of 0.763 mum in rat cerebellar cortex membranes. These findings strongly suggest that bis(7)-tacrine prevents glutamate-induced neuronal apoptosis through directly blocking NMDA receptors at the MK-801-binding site, which offers a new and clinically significant modality as to how the agent exerts neuroprotective effects.

Mode of action of taurine as a neuroprotector

Previously, it has been shown that taurine exerts its protective function against glutamate-induced neuronal excitotoxicity through its action in reducing glutamate-induced elevation of intracellular free calcium, [Ca 2+] i. Here, we report the mechanism underlying the effect of taurine in reducing [Ca 2+] i. We found that taurine inhibited glutamate-induced calcium influx through L-, P/Q-, N-type voltage-gated calcium channels (VGCCs) and NMDA receptor calcium channel. Surprisingly, taurine had no effect on calcium influx through NMDA receptor calcium channel when cultured neurons were treated with NMDA in Mg 2+-free medium. Since taurine was found to prevent glutamate-induced membrane depolarization, we propose that taurine protects neurons against glutamate excitotoxicity by preventing glutamate-induced membrane depolarization, probably through its effect in opening of chloride channels and, therefore, preventing the glutamate-induced increase in calcium influx and other downstream events.

Neuroprotective effects of atorvastatin against glutamate‐induced excitotoxicity in primary cortical neurones

Statins [3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors] exert cholesterol-independent pleiotropic effects that include anti-thrombotic, anti-inflammatory, and anti-oxidative properties. Here, we examined direct protective effects of atorvastatin on neurones in different cell damage models in vitro. Primary cortical neurones were pre-treated with atorvastatin and then exposed to (i) glutamate, (ii) oxygen-glucose deprivation or (iii) several apoptosis-inducing compounds. Atorvastatin significantly protected from glutamate-induced excitotoxicity as evidenced by propidium iodide staining, nuclear morphology, release of lactate dehydrogenase, and mitochondrial tetrazolium metabolism, but not from oxygen-glucose deprivation or apoptotic cell death. This anti-excitototoxic effect was evident with 2-4 days pre-treatment but not with daily administration or shorter-term pre-treatment. The protective properties occurred independently of 3-hydroxy-3-methylglutaryl-CoA reductase inhibition because co-treatment with mevalonate or other isoprenoids did not reverse or attenuate neuroprotection. Atorvastatin attenuated the glutamate-induced increase of intracellular calcium, which was associated with a modulation of NMDA receptor function. Taken together, atorvastatin exerts specific anti-excitotoxic effects independent of 3-hydroxy-3-methylglutaryl-CoA reductase inhibition, which has potential therapeutic implications.

Presynaptic and Postsynaptic Roles of NO, cGK, and RhoA in Long-Lasting Potentiation and Aggregation of Synaptic Proteins

Recent results suggest that long-lasting potentiation at hippocampal synapses involves the rapid formation of clusters or puncta of presynaptic as well as postsynaptic proteins, both of which are blocked by antagonists of NMDA receptors and an inhibitor of actin polymerization. We have investigated whether the increase in puncta involves retrograde signaling through the NO-cGMP-cGK pathway and also examined the possible roles of two classes of molecules that regulate the actin cytoskeleton: Ena/VASP proteins and Rho GTPases. Our results suggest that NO, cGMP, cGK, actin, and Rho GTPases including RhoA play important roles in the potentiation and act directly in both the presynaptic and postsynaptic neurons, where they contribute to the increase in puncta of synaptic proteins. cGK phosphorylates synaptic VASP during the potentiation, whereas Rho GTPases act both in parallel and upstream of cGMP, in part by maintaining the synaptic localization of soluble guanylyl cyclase.

The medial amygdaloid area is involved in activation of angiotensin II-sensitive neurons in the anterior hypothalamic area

We have previously reported that some neurons in the anterior hypothalamic area (AHA) are tonically activated by endogenous angiotensins in rats and that the activities of these AHA angiotensin II-sensitive neurons are enhanced in spontaneously hypertensive rats. It is suggested that there exist neural projections from the medial amygdala to the AHA in rats. In this study, we examined whether neurons in the medial amygdaloid area (MeA) are involved in the activation of AHA angiotensin II-sensitive neurons. Male Wistar rats were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Microinjection of glutamate into the MeA caused an increase in the firing rate of AHA angiotensin II-sensitive neurons. The glutamate-induced increase of firing rate was inhibited by pressure application of the AT1 receptor antagonist losartan onto AHA angiotensin II-sensitive neurons. The microinjection of glutamate into the central amygdaloid area also increased the firing rate of AHA angiotensin II-sensitive neurons, but the glutamate-induced increase of firing rate was not affected by pressure application of losartan onto AHA angiotensin II-sensitive neurons. The microinjection of corticotropin-releasing factor (CRF) into the MeA also increased the firing rate of AHA angiotensin II-sensitive neurons, but the CRF-induced increase of firing rate was not inhibited by pressure application of losartan onto AHA angiotensin II-sensitive neurons. Repeated microinjection of glutamate into the MeA caused an increase in the release of angiotensins in the AHA. These findings indicate that neurons in the MeA are involved in the activation of AHA angiotensin II-sensitive neurons. It seems likely that the activation of AHA angiotensin II-sensitive neurons induced by glutamate but not CRF is partly mediated via the release of angiotensins at AHA angiotensin II-sensitive neuron levels.

α-Synuclein produces a long-lasting increase in neurotransmitter release

Wild-type alpha-synuclein, a protein of unknown function, has received much attention because of its involvement in a series of diseases that are known as synucleinopathies. We find that long-lasting potentiation of synaptic transmission between cultured hippocampal neurons is accompanied by an increase in the number of alpha-synuclein clusters. Conversely, suppression of alpha-synuclein expression through antisense nucleotide and knockout techniques blocks the potentiation, as well as the glutamate-induced increase in presynaptic functional bouton number. Consistent with these findings, alpha-synuclein introduction into the presynaptic neuron of a pair of monosynaptically connected cells causes a rapid and long-lasting enhancement of synaptic transmission, and rescues the block of potentiation in alpha-synuclein null mouse cultures. Also, we report that the application of nitric oxide (NO) increases the number of alpha-synuclein clusters, and inhibitors of NO-synthase block this increase, supporting the hypothesis that NO is involved in the enhancement of the number of alpha-synuclein clusters. Thus, alpha-synuclein is involved in synaptic plasticity by augmenting transmitter release from the presynaptic terminal.

Rapid inhibition of the glutamate-induced increase of intracellular free calcium by magnesium in rat hippocampal neurons.

By using Fura-2/AM, the effects of magnesium (Mg2+) on the glutamate-induced increase of intracellular free calcium ([Ca2+]i) in the cultured hippocampal neurons and the features were investigated by integrated photoelectric detecting system. The experiments were designed to three groups (The drug was spit to the cells for 20 s): Group A receiving 1 x 10(-5) mol/L glutamate; Group B receiving 1 x 10(-5) mol/L glutamate and 1 x 10(-5) mol/L Mg2+ simultaneously; Group C receiving 1 x 10(-5) mol/L glutamate again after [Ca2+]i in group B back to the baseline. The results showed that in group A, [Ca2+]i was obviously increased. In group B, the changes in [Ca+] i and the peak value were significantly decreased. Moreover, the elevation of Phase 1 was slowed down and Phase 2 was shortened to some extent, and the plateau phase between them was relatively prolonged. In group C, calcium oscillation similar to that in group A occurred, but both the Phase 1 and Phase 2 were shortened and the delta[Ca2+]i was slightly decreased. It was suggested that Mg2+ could quickly inhibit the rise of [Ca2+]i induced by glutamate in the cultured hippocampal neurons in rats.

Adenosine A1-receptor modulation of glutamate-induced calcium influx in rat retinal ganglion cells.

Although adenosine receptors (A(1)-Rs and A(2)-Rs) have been identified in the mammalian retina, the role of adenosine in this tissue is not fully understood. The purpose of this work was to investigate the action of adenosine on glutamate-induced calcium influx in rat retinal ganglion cells (RGCs) and to determine whether adenosine modulates RGC voltage-gated calcium channels. Purified RGC cultures were generated from neonatal rats with a two-step panning procedure. Isolated RGCs were loaded with the ratiometric calcium-indicator dye fura-2, and the effect of adenosine (and related agonists and antagonists) on intracellular calcium levels ([Ca(2+)](i)) during exposure to glutamate (10 microM with 10 microM glycine) was assessed. The effect of adenosine on calcium channel currents was also studied in isolated RGCs with whole-cell patch-clamp techniques. In addition, the effect of adenosine on [Ca(2+)](i) was investigated in fura dextran-loaded RGCs in an intact adult rat retina preparation. In isolated RGCs, adenosine (10 and 100 microM) significantly reduced the glutamate-induced increase in [Ca(2+)](i) ( approximately 30%). The effect of adenosine was blocked by the A(1)-R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), but not by the A(2)-R antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). Adenosine (10 microM) inhibited calcium channel currents by 43%, and again this effect was blocked by DPCPX, but not DMPX. Adenosine (100 microM) also significantly reduced the elevation of [Ca(2+)](i) in RGCs in the intact retina during exposure to N-methyl-d-aspartate (NMDA; 100 microM). Adenosine can inhibit glutamate-induced calcium influx and voltage-gated calcium currents in rat RGCs through A(1)-R activation. This work supports a role for adenosine as a neuromodulator of mammalian RGCs.

Glutamate-induced glutamine synthetase expression in retinal Muller cells after short-term ocular hypertension in the rat.

To determine the effect of intraocular pressure (IOP) elevation on glutamate-induced expression of glutamine synthetase (GS) in retinal Müller cells of rat eyes. Six groups of three rats each were studied. Group I was a normal control group. In Group II, one eye received an intravitreal glutamate injection (75 nmoles) while the contralateral eye served as control. In Groups III and IV, IOP was raised in one eye by episcleral vein cauterization. Moderately elevated IOP was maintained for 1 day in Group III or 1 week in Group IV (35 +/- 1.9-45 +/- 5.2 mm Hg). An additional two groups of rats received bilateral intravitreal glutamate injections (75 nmoles) immediately (Group V), or 6 days (Group VI), after induction of IOP elevation in one eye. One day after glutamate injection the rats in all groups were killed, and the eyes enucleated and fixed. Retinas from left and right eyes of each animal were embedded together in LR White resin (Ted Pella, Redding, CA). Sections were processed for GS immunolabeling with antibodies to GS by two-stage immunogold labeling with silver enhancement. Images of labeled retinas from the two eyes were captured under identical light microscopic conditions and the GS immunoreactivity in Müller cells was compared between the left and right retinas in the same section by image analysis. An additional five rats were included in Group II and the retinas were analyzed by Western blot analysis to confirm immunohistochemical findings. Similar to the finding in the control group (Group I), the GS immunoreactivity of the left and right eyes of Group III and IV remained unchanged even though the right eyes in the two groups had elevation of IOP lasting for 24 hours and 1 week, respectively. However, GS levels were significantly increased by 40 +/- 5.7% in normotensive eyes 24 hours after intravitreal injection of glutamate (Group II). The rise in GS immunoreactivity was abolished in eyes with acute IOP elevation (Group V). In contrast, when the eyes were exposed to high IOP for 1 week (Group VI), the glutamate-induced increase in GS immunoreactivity was restored. Elevated levels of vitreal glutamate can increase the expression of GS in retinal Müller cells. This increase was blocked if IOP was acutely elevated for 24 hours but was restored if IOP remained elevated for 1 week. This finding suggests that moderate elevation of IOP causes only short-term functional changes of glutamate metabolism (amidation) by retinal Müller cells. However, it is not known to what extent endogenous extracellular glutamate can regulate GS expression in normal eyes or in eyes with glaucoma.

(-)-Epigallocatechin gallate attenuates glutamate-induced cytotoxicity via intracellular Ca modulation in PC12 cells.

1. The effects of (-)-epigallocatechin gallate (EGCG), a green tea polyphenol, on glutamate-induced increases in intracellular Ca2+ concentrations ([Ca2+]i) and cytotoxicity in PC12 cells were investigated. 2. Changes in [Ca2+]i were measured using Fura-2/AM calcium indicator dye and cellular viabilities were determined by a viable cell count and a 3-(4,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. 3. Glutamate increased [Ca2+]i in PC12 cells in a dose-dependent manner. (-)-Epigallocatechin gallate attenuated this glutamate (30 mmol/L)-induced [Ca2+]i increase and EGCG (50 micromol/L) increased the viability of PC12 cells against glutamate-induced cytotoxicity. The EGCG effect was also found to be independent of its general anti-oxidant mechanism. In contrast, EGCG directly suppressed both N-methyl-D-aspartate (50 mmol/L)- and kainate (20 mmol/L)-mediated Ca2+ influx, but not metabotropic receptor-mediated Ca2+ release. 4. These results suggest that EGCG reduces the glutamate-induced [Ca2+]i increase by attenuating ionotropic Ca2+ influx and that this promotes the viability of PC12 cells.

Nutrition during brain activation: does cell-to-cell lactate shuttling contribute significantly to sweet and sour food for thought?

Functional activation of astrocytic metabolism is believed, according to one hypothesis, to be closely linked to excitatory neurotransmission and to provide lactate as fuel for oxidative metabolism in neighboring neurons. However, review of emerging evidence suggests that the energetic demands of activated astrocytes are higher and more complex than recognized and much of the lactate presumably produced by astrocytes is not locally oxidized during activation. In vivo activation studies in normal subjects reveal that the rise in consumption of blood-borne glucose usually exceeds that of oxygen, especially in retina compared to brain. When the contribution of glycogen, the brain’s major energy reserve located in astrocytes, is taken into account the magnitude of the carbohydrate-oxygen utilization mismatch increases further because the magnitude of glycogenolysis greatly exceeds the incremental increase in utilization of blood-borne glucose. Failure of local oxygen consumption to equal that of glucose plus glycogen in vivo is strong evidence against stoichiometric transfer of lactate from astrocytes to neighboring neurons for oxidation. Thus, astrocytes, not nearby neurons, use the glycogen for energy during physiological activation in normal brain. These findings plus apparent compartmentation of metabolism of glycogen and blood-borne glucose during activation lead to our working hypothesis that activated astrocytes have high energy demands in their fine perisynaptic processes (filopodia) that might be met by glycogenolysis and glycolysis coupled to rapid lactate clearance. Tissue culture studies do not consistently support the lactate shuttle hypothesis because key elements of the model, glutamate-induced increases in glucose utilization and lactate release, are not observed in many astrocyte preparations, suggesting differences in their oxidative capacities that have not been included in the model. In vivo nutritional interactions between working neurons and astrocytes are not as simple as implied by “sweet (glucose–glycogen) and sour (lactate) food for thought.”

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 μM 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.