Different Concentrations Of Glutamate Research Articles

Effects of Oxytocin on Glutamate Mediated Neurotoxicity in Neuroblastoma Cell Culture.

We aimed to investigate the effects of oxytocin on neurite growth, cell viability, cell proliferation and apoptosis to demonstrate its neuroprotective effect on glutamate induced neurotoxicity in human neuroblastoma SH-SY5Y cell culture. The effect of oxytocin on the toxic effects of glutamate in human neuroblastoma SH-SY5Y cell line with the Neurotoxicity Screening Test (NTT), apoptotic effects by Terminal Transferase dUTP Nick End Labeling (TUNEL) method and cell viability test by 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) method. In the NTT test; Neurotoxicity was induced by adding glutamate at a concentration of 32 μM to the cell culture. Oxytocin was added at 1, 3, 10, 30 and 100 μM concentrations and its effect on neurite elongation was investigated. It was demonstrated by TUNEL method that application of glutamate caused apoptosis. Afterwards, when glutamate and different doses of oxytocin were given, antiapoptotic effect was evaluated with the apoptotic index. Glutamate was found to have a dose-dependent neurotoxic effect and reduced neurite elongation by 50% at a concentration of 32 μM. It was shown that the inhibition of neurite elongation caused by glutamate decreased in a dose-dependent manner by applying oxytocin. Especially oxytocin was found to significantly reduce neurite inhibition and show a neuroprotective effect starting from 10 μM concentrations. The concentration at which glutamate reduces cell proliferation by 50% was determined as 54 μM in MTT. Subsequently, it was observed that the adverse effect of glutamate on cell proliferation significantly decreased with oxytocin administration, depending on the dose. It was found that different concentrations of glutamate have a significant toxic effect on cell proliferation and viability, glutamate inhibits neurite elongation in a dose-dependent manner; oxytocin reduces neurite inhibition caused by glutamate, has a neuroprotective effect, increases cell viability and has antiapoptotic effects.

Glutamate as a neural factor for the ex vivo release of catecholamines from the rabbit hippocampus

The aim of the research was to confirm the hypothesis about the direct influence of different concentrations of glutamate (Glu; 5, 50 and 200 μM) on the release of catecholamines (CA’s) from hippocampal slices incubated for 90 min. The hippocampus is the central structure of the motivational system and, along with the hypothalamus, amygdala, and medial prefrontal cortex (mPFC), is responsible for memory, learning, and the initiation and course of the reaction to stressoric factors. In our research, we focused on the direct effect of different Glu concentrations on CA release without the involvement of other neural cerebral structures and humoral responses. The effect of Glu added to the incubation fluid was found to be inconsistent. When added at the lowest concentration, Glu inhibited the release of CA from the incubated slices, and at higher concentrations tended to increase the release of CA. Notably, Glu significantly increased the release of epinephrine (E) from incubated slices of the hippocampus. These effects may suggest that physiological concentrations of Glu inhibit CA release from hippocampal neurons in vitro and therefore enhance its excitatory effects in vivo.

SLC38A10 Transporter Plays a Role in Cell Survival Under Oxidative Stress and Glutamate Toxicity.

Solute carrier (SLC) transporters regulate amino acids, glucose, ions, and metabolites that flow across cell membranes. In the brain, SLCs are the key regulators of neurotransmission, in particular, the glutamate/GABA-glutamine (GGG) cycle. Genetic mutations in SLCs are associated with various neurodevelopmental and neurodegenerative diseases. In this study, we have investigated the role of SLC38A10 under acute oxidative and glutamate stress in mouse primary cortical cells from SLC38A10 knockout (KO) mice. The ER/golgi localized transporter, SLC38A10, transports glutamate, glutamine, and alanine in brain cells, and the aim of this study was to determine the possible effects of removal of SLC38A10 in primary cortical cells under glutamate and oxidative challenges. Primary cortical neuronal cultures of wild-type (WT) cell and SLC38A10 KO mice were subjected to different concentrations of glutamate and hydrogen peroxide. There was no morphological change observed between KO and WT cortical neurons in culture. Interestingly, KO cells showed significantly lower cell viability and higher cell death compared to WT cells under both glutamate and hydrogen peroxide exposure. Further, we evaluated the possible role of p53 in neuronal cell apoptosis in KO cells. We found decreased intracellular p53 protein levels under glutamate and hydrogen peroxide treatment in KO cortical cells. In contrast, caspase 3/7 activity remains unaltered under all conditions. These results demonstrate an indirect relationship between the expression of SLC38A10 and p53 and a role in the cell defense mechanism against neurotoxicity.

Glutamate Chemical Exchange Saturation Transfer (GluCEST) Magnetic Resonance Imaging in Pre-clinical and Clinical Applications for Encephalitis.

BackgroundEncephalitis is a common central nervous system inflammatory disease that seriously endangers human health owing to the lack of effective diagnostic methods, which leads to a high rate of misdiagnosis and mortality. Glutamate is implicated closely in microglial activation, and activated microglia are key players in encephalitis. Hence, using glutamate chemical exchange saturation transfer (GluCEST) imaging for the early diagnosis of encephalitis holds promise.MethodsThe sensitivity of GluCEST imaging with different concentrations of glutamate and other major metabolites in the brain was validated in phantoms. Twenty-seven Sprague–Dawley (SD) rats with encephalitis induced by Staphylococcus aureus infection were used for preclinical research of GluCEST imaging in a 7.0-Tesla scanner. For the clinical study, six patients with encephalitis, six patients with lacunar infarction, and six healthy volunteers underwent GluCEST imaging in a 3.0-Tesla scanner.ResultsThe number of amine protons on glutamate that had a chemical shift of 3.0 ppm away from bulk water and the signal intensity of GluCEST were concentration-dependent. Under physiological conditions, glutamate is the main contributor to the GluCEST signal. Compared with normal tissue, in both rats and patients with encephalitis, the encephalitis areas demonstrated a hyper-intense GluCEST signal, while the lacunar infarction had a decreased GluCEST signal intensity. After intravenous immunoglobulin therapy, patients with encephalitis lesions showed a decrease in GluCEST signal, and the results were significantly different from the pre-treatment signal (1.34 ± 0.31 vs 5.0 ± 0.27%, respectively; p = 0.000).ConclusionGlutamate plays a role in encephalitis, and the GluCEST imaging signal has potential as an in vivo imaging biomarker for the early diagnosis of encephalitis. GluCEST will provide new insight into encephalitis and help improve the differential diagnosis of brain disorders.

Astrocytes enhance the tolerance of rat cortical neurons to glutamate excitotoxicity.

Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders, including stroke, trauma and neurodegenerative diseases. Astrocytes are the main cells for the removal of glutamate in the synaptic cleft and may affect the tolerance of neurons to the glutamate excitotoxicity. Therefore, the present study aimed to investigate the tolerance of rat cortical neurons to glutamate excitotoxicity in the presence and absence of astrocytes. Rat cortical neurons in the presence or absence of astrocytes were exposed to different concentrations of glutamate (10–2,000 µM) and 10 µM glycine for different incubation periods. After 24 h, the Cell Counting kit-8 (CCK-8) assay was used to measure the cytotoxicity to neurons in the presence or absence of astrocytes. According to the results, in the absence of astrocytes, glutamate induced a concentration-dependent decrease of neuronal survival rate compared with the control rat cortical neurons, and the neurotoxic half-maximal inhibitory concentration (IC50) at 15, 30 and 60 min was 364.5, 258.5 and 138.3 µM, respectively. Furthermore, in the presence of astrocytes, glutamate induced a concentration-dependent decrease of neuronal survival rate compared with the control rat cortical neurons, and the neurotoxic IC50 at 15, 30 and 60 min was 1,935, 932.8 and 789.3 µM, respectively. However, astrocytic toxicity was not observed when the rat cortical astrocytes alone were exposed to different concentrations of glutamate (500, 1,000 and 2,000 µM) for 6, 12 and 24 h. In conclusion, the glutamate-induced neurotoxic IC50 values at 15, 30 and 60 min were respectively higher in the presence of astrocytes as compared with those in the absence of astrocytes, suggesting that astrocytes can protect against rat cortical neuronal acute damage induced by glutamate.

A Novel Two-Component System, GluR-GluK, Involved in Glutamate Sensing and Uptake in Streptomyces coelicolor.

Two-component systems (TCSs), the predominant signal transduction pathways employed by bacteria, play important roles in physiological metabolism in Streptomyces Here, a novel TCS, GluR-GluK (encoded by SCO5778-SCO5779), which is located divergently from the gluABCD operon encoding a glutamate uptake system, was identified as being involved in glutamate sensing and uptake as well as antibiotic biosynthesis in Streptomyces coelicolor Under the condition of minimal medium (MM) supplemented with different concentrations of glutamate, deletion of the gluR-gluK operon (gluR-K) resulted in enhanced actinorhodin (ACT) but reduced undecylprodigiosin (RED) and yellow type I polyketide (yCPK) production, suggesting that GluR-GluK plays a differential role in antibiotic biosynthesis. Furthermore, we found that the response regulator GluR directly promotes the expression of gluABCD under the culture condition of MM with a high concentration of glutamate (75 mM). Using the biolayer interferometry assay, we demonstrated that glutamate acts as the direct signal of the histidine kinase GluK. It was therefore suggested that upon sensing high concentrations of glutamate, GluR-GluK would be activated and thereby facilitate glutamate uptake by increasing gluABCD expression. Finally, we demonstrated that the role of GluR-GluK in antibiotic biosynthesis is independent of its function in glutamate uptake. Considering the wide distribution of the glutamate-sensing (GluR-GluK) and uptake (GluABCD) module in actinobacteria, it could be concluded that the GluR-GluK signal transduction pathway involved in secondary metabolism and glutamate uptake should be highly conserved in this bacterial phylum.IMPORTANCE In this study, a novel two-component system (TCS), GluR-GluK, was identified to be involved in glutamate sensing and uptake as well as antibiotic biosynthesis in Streptomyces coelicolor A possible GluR-GluK working model was proposed. Upon sensing high glutamate concentrations (such as 75 mM), activated GluR-GluK could regulate both glutamate uptake and antibiotic biosynthesis. However, under a culture condition of MM supplemented with low concentrations of glutamate (such as 10 mM), although GluR-GluK is activated, its activity is sufficient only for the regulation of antibiotic biosynthesis. To the best of our knowledge, this is the first report describing a TCS signal transduction pathway for glutamate sensing and uptake in actinobacteria.

Glutamate Impairs Mitochondria Aerobic Respiration Capacity and Enhances Glycolysis in Cultured Rat Astrocytes

ObjectiveTo study the effect of glutamate on metabolism, shifts in glycolysis and lactate release in rat astrocytes. MethodsAfter 10 days, secondary cultured astrocytes were treated with 1 mmol/L glutamate for 1 h, and the oxygen consumption rates (OCR) and extra cellular acidification rate (ECAR) was analyzed using a Seahorse XF 24 Extracellular Flux Analyzer. Cell viability was then evaluated by MTT assay. Moreover, changes in extracellular lactate concentration induced by glutamate were tested with a lactate detection kit. ResultsCompared with the control group, treatment with 1 mmol/L glutamate decreased the astrocytes' maximal respiration and spare respiratory capacity but increased their glycolytic capacity and glycolytic reserve. Further analysis found that 1-h treatment with different concentrations of glutamate (0.1-1 mmol/L) increased lactate release from astrocytes, however the cell viability was not affected by the glutamate treatment. ConclusionThe current study provided direct evidence that exogenous glutamate treatment impaired the mitochondrial respiration capacity of astrocytes and enhanced aerobic glycolysis, which could be involved in glutamate injury or protection mechanisms in response to neurological disorders.

Chlorpyrifos Toxicity in Mouse Cultured Cerebellar Granule Neurons at Different Stages of Development: Additive Effect on Glutamate-Induced Excitotoxicity.

Chlorpyrifos (CPF) is a neurotoxic organophosphorus (OP) insecticide. Its mechanism of action includes oxidative stress, excitotoxicity, and inhibition of the acetylcholinesterase enzyme (AChE). The aim of the present study is to investigate CPF toxicity in mature and immature cerebellar granule neurons (CGNs), as well as its effect on glutamate induced excitotoxicity. This study was an in vitro experimental study performed on mice cultured CGNs. Immature and mature neurons were exposed to different concentrations of CPF (1-1000 µM) and glutamate (10-600 µM) for 48 hours after which we used the MTT assay to measure cytotoxicity. Immature neurons had exposure to CPF for 5 days in order to evaluate the cytotoxic effect on developing neurons. Mature neurons received sub-lethal concentrations of CPF (10, 100 µM) combined with different concentrations of glutamate. AChE activity and reactive oxygen species (ROS) generation were assessed after treatments. Immature CGNs had increased sensitivity to CPF toxicity compared to mature neurons. We observed significantly greater ROS production in immature compared to mature neurons, however AChE activity was more inhibited in mature neurons. Although CPF toxicity was not well correlated with AChE inhibition, it correlated well with ROS production. Glutamate toxicity was potentiated by sub-lethal concentration of CPF, however glutamate induced ROS production was not affected. The results suggested that CPF potentiated glutamate toxicity by mechanisms other than oxidative stress. CPF toxicity differed in mature and immature neurons. Potentiated glutamate toxicity by CPF implied that CPF exposure might be a risk factor for neurodegenerative disease.

Paracrine Neuroprotective Effects of Neural Stem Cells on Glutamate-Induced Cortical Neuronal Cell Excitotoxicity

Glutamate is a major excitatory neurotransmitter in mammalian central nervous system. Excessive glutamate releasing overactivates its receptors and changes calcium homeostasis that in turn leads to a cascade of intracellular events causing neuronal degeneration. In current study, we used neural stem cells conditioned medium (NSCs-CM) to investigate its neuroprotective effects on glutamate-treated primary cortical neurons. Embryonic rat primary cortical cultures were exposed to different concentrations of glutamate for 1 hour and then they incubated with NSCs-CM. Subsequently, the amount of cell survival in different glutamate excitotoxic groups were measured after 24 h of incubation by trypan blue exclusion assay and MTT assay. Hoechst and propidium iodide were used for determining apoptotic and necrotic cell death pathways proportion and then the effect of NSCs-CM was investigated on this proportion. NSCs conditioned medium increased viability rate of the primary cortical neurons after glutamate-induced excitotoxicity. Also we found that NSCs-CM provides its neuroprotective effects mainly by decreasing apoptotic cell death rate rather than necrotic cell death rate. The current study shows that adult neural stem cells could exert paracrine neuroprotective effects on cortical neurons following a glutamate neurotoxic insult.

Glucose replaces glutamate as energy substrate to fuel glutamate uptake in glutamate dehydrogenase-deficient astrocytes.

Cultured astrocytes treated with siRNA to knock down glutamate dehydrogenase (GDH) were used to investigate whether this enzyme is important for the utilization of glutamate as an energy substrate. By incubation of these cells in media containing different concentrations of glutamate (range 100-500 µM) in the presence or in the absence of glucose, the metabolism of these substrates was studied by using tritiated glutamate or 2-deoxyglucose as tracers. In addition, the cellular contents of glutamate and ATP were determined. The astrocytes were able to maintain physiological levels of ATP regardless of the expression level of GDH and the incubation condition, indicating a high degree of flexibility with regard to regulatory mechanisms involved in maintaining an adequate energy level in the cells. Glutamate uptake was found to be increased in these cells when exposed to increasing levels of extracellular glutamate independently of the GDH expression level. Moreover, increased intracellular glutamate content was observed in the GDH-deficient cells after a 2-hr incubation in the presence of 100 µM glutamate. It is significant that GDH-deficient cells exhibited an increased utilization of glucose in the presence of 250 and 500 µM glutamate, monitored as an increase in the accumulation of tritiated 2-deoxyglucose-6-phosphate. These findings underscore the importance of the expression level of GDH for the ability to utilize glutamate as an energy source fueling its own energy-requiring uptake.

Activation of muscarinic receptors inhibits glutamate-induced GSK-3β overactivation in PC12 cells

To investigate the actions of the muscarinic agonist carbachol on glutamate-induced neurotoxicity in PC12 cells, and the underlying mechanisms. PC12 cells were treated with different concentrations of glutamate for 24 or 48 h. The cell viability was measured using MTT assay, and the expression and activation of GSK-3β were detected with Western blot. β-Catenin translocation was detected using immunofluorescence. Luciferase reporter assay and real-time PCR were used to analyze the transcriptional activity of β-catenin. Glutamate (1, 3, and 10 mmol/L) induced PC12 cell death in a dose-dependent manner. Moreover, treatment of the cells with glutamate (1 mmol/L) caused significant overactivation of GSK-3β and prevented β-catenin translocation to the nucleus. Pretreatment with carbachol (0.01 μmol/L) blocked glutamate-induced cell death and GSK-3β overactivation, and markedly enhanced β-catenin transcriptional activity. Activation of muscarinic receptors exerts neuroprotection in PC12 cells by attenuating glutamate-induced GSK-3β overactivation, suggesting potential benefits of muscarinic agonists for Alzheimer's disease.

Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held

Establishing the spatiotemporal concentration profile of neurotransmitter following synaptic vesicular release is essential for our understanding of inter-neuronal communication. Such profile is a determinant of synaptic strength, short-term plasticity and inter-synaptic crosstalk. Synaptically released glutamate has been suggested to reach a few millimolar in concentration and last for <1 ms. The synaptic cleft is often conceived as a single concentration compartment, whereas a huge gradient likely exists. Modelling studies have attempted to describe this gradient, but two key parameters, the number of glutamate in a vesicle (N(Glu)) and its diffusion coefficient (D(Glu)) in the extracellular space, remained unresolved. To determine this profile, the rat calyx of Held synapse at postnatal day 12-16 was studied where diffusion of glutamate occurs two-dimensionally and where quantification of AMPA receptor distribution on individual postsynaptic specialization on medial nucleus of the trapezoid body principal cells is possible using SDS-digested freeze-fracture replica labelling. To assess the performance of these receptors as glutamate sensors, a kinetic model of the receptors was constructed from outside-out patch recordings. From here, we simulated synaptic responses and compared them with the EPSC recordings. Combinations of N(Glu) and D(Glu) with an optimum of 7000 and 0.3 μm(2) ms(-1) reproduced the data, suggesting slow diffusion. Further simulations showed that a single vesicle does not saturate the synaptic receptors, and that glutamate spillover does not affect the conductance amplitude at this synapse. Using the estimated profile, we also evaluated how the number of multiple vesicle releases at individual active zones affects the amplitude of postsynaptic signals.

Microaerobic dark fermentative hydrogen production by the photosynthetic bacterium,Rhodobacter capsulatusJP91

The photosynthetic bacterium Rhodobacter capsulatus produces hydrogen under nitrogen-limited, anaerobic, photosynthetic conditions. The present study examined whether R. capsulatus can produce hydrogen under microaerobic conditions in the dark with limiting amounts of O2 and fixed nitrogen. The relationship between hydrogen production, different O2 concentrations and carbon sources as well as two different N sources, glutamate and ammonium, were studied in batch culture using a Hup strain of R. capsulatus. The effect of different O2 concentrations, ranging from 0.5 to 20%, on hydrogen production was examined in dark batch cultures of R. capsulatus grown on RCV medium. Different carbon sources, e.g. glucose, succinate, lactate, acetate and malate, were used at various concentrations (20-40 mM). Similarly, different concentrations of glutamate and ammonium (2-9 mM) were examined for optimum microaerobic dark hydrogen production. Maximum hydrogen production was observed at an O2 concentration of 4-8%. There was a highly positive correlation between O2 and growth (r 2 ¼ 0.67), whereas O2 concentration and hydrogen productivity were negatively correlated (r 2 ¼ 20.3). Succinate (25 mM) together with glutamate (3.5 mM) gave the highest specific hydrogen productivity (5.61 mmol hydrogen/(mg cell dry weight/ml)). The maximum average hydrogen yield was 0.6 mol hydrogen/mol malate followed by 0.41 mol hydrogen/mol lactate, 0.36 mol hydrogen/mol succinate, whereas minimum amounts of hydrogen were produced from glucose and acetate (0.16 mol hydrogen/mol and 0.07 mol hydrogen/mol, respectively). The implications for developing a system capable of improved hydrogen production are discussed.

Measurement of Synchronous Activity by Microelectrode Arrays Uncovers Differential Effects of Sublethal and Lethal Glutamate Concentrations on Cortical Neurons

We grew cultures of rat cortical cells on microelectrode arrays to investigate the effects of glutamate-mediated neurotoxicity as a model of traumatic brain injury. Treatment with two different concentrations of glutamate, 175 and 250μM, led to different outcomes. Cultures treated with 250μM glutamate suffered a loss in overall activity that was not seen in cultures treated with 175μM glutamate. An analysis of the changes in the synchronization of action potential firing between electrodes, however, revealed a loss of synchronization in subsets of electrode pairs treated with both the higher and lower concentrations of glutamate. We found that this loss of action potential synchronization was dependent on the initial amount of synchronization prior to injury. Finally, our data suggest that the synchronization of electrical activity as well as the susceptibility to loss of firing synchrony is independent of the distance between neurons in a network.

Electrochemical sensor array for bioprocess monitoring

A silicon-based biosensor chip consisting of an array of amperometric enzyme sensors has been developed for monitoring the concentration of glutamate and glutamine in cell-culture fermentation processes. The glutamate sensor was constructed by immobilising glutamate oxidase, while the glutamine sensor by sequential coupling of glutaminase and glutamate oxidase. The enzymes were immobilised by means of cross-linking with glutaraldehyde on the surface of the patterned platinum thin-film electrodes. The developed biosensors have been electrochemically characterised in solutions with different concentrations of glutamate and glutamine in terms of sensitivity, response time, linear working range and lifetime. A high sensitivity of 96 nA/mM and 100 nA/mM was registered for the glutamate and glutamine sensors, respectively. The preliminary experiments in cell-culture medium have shown a good correlation between the glutamine and glutamate concentrations measured with the biosensor chip and the commercially available biochemistry analyser. The obtained results demonstrate the feasibility of the realised biosensor chip for monitoring the glutamine and glutamate concentrations in fermentation processes.

Sensory attributes of dishes containing shrimp paste with different concentrations of glutamate and 5′-nucleotides

The shrimp paste called belacan is a traditional umami taste condiment extensively used in Malaysia that is rich in glutamate and 5′-nucleotides. The aim of this study was to determine the concentration of glutamate and 5′-nucleotides of various types of foods prepared with belacan and to measure their sensory attributes. The concentration of free glutamic acid found in different brands of belacan was 180–530mg/100g and in local dishes 601–4207mg/100g. The total amount of 5′-nucleotides in belacan samples ranged from 0.85 to 42.25μg/g. A Quantitative Descriptive Analysis (QDA) using a list of 17 sensory attributes showed a good correlation between belacan concentration in the final food and a range of positive sensory attributes, except for bitter, sweet, sour taste and astringency. Belacan also contains bitter, sweet and sour compounds that change the positive attributes of belacan at higher concentrations. The highest aroma attributes were linked to nasi goreng belacan (belacan fried rice) while the highest flavour attributes were found in sambal belacan. There was a 32 folds significant increase of umami attributes with the addition of belacan to final foods. The optimum amount of belacan was 0.45% for asam pedas (tamarind flavoured dish with belacan), 18% for sambal belacan (chilli belacan), 1.5–2.5% for kangkong goreng belacan (stir fried water convolous with belacan), and 2% for nasi goreng belacan.

Autoinactivation of Neuronal AMPA Receptors via Glutamate-Regulated TARP Interaction

Neuronal AMPA receptors autoinactivate at high concentrations of glutamate, i.e., the current declines at glutamate concentrations above 10-100 microM. The mechanisms underlying this phenomenon are unclear. Stargazin-like TARPs are AMPA receptor auxiliary subunits that modulate receptor trafficking and channel properties. Here, we found that neuronal AMPA receptors and recombinant AMPA receptors coexpressed with stargazin autoinactivate at high concentrations of glutamate, whereas recombinant AMPA receptors expressed alone do not. The reduction of currents at high glutamate concentrations is not associated with a reduction of AMPA receptor number, but rather with the loss of stargazin-associated allosteric modulation of channel gating. We show that receptor desensitization promotes the dissociation of TARP-AMPA receptor complexes in a few milliseconds. This dissociation mechanism contributes to synaptic short-term modulation. The results demonstrate a mechanism for dynamic regulation of AMPA receptor activity to tune synaptic strength.

Pinoline and N-acetylserotonin reduce glutamate-induced lipid peroxidation in retinal homogenates

Glutamate is a neurotransmitter associated with oxidative retinal disorders. Pinoline (PIN) and N-acetylserotonin (NAS) are newly identified neural protectors. We investigated the glutamate-induced lipid peroxidation (LPO) and the protective effects of PIN and NAS in the retina. Porcine retinal homogenates were treated with different concentrations of glutamate. The malondialdehyde (MDA) level per unit weight of protein was quantified spectro-photometrically as an index of LPO. The glutamate concentration that induced a significant increase in retinal MDA was determined. The glutamate-treated retinal homogenate was then co-incubated with 5 different concentrations (0, 35.7, 71.5, 143 and 286 μM) of PIN, NAS or their combinations (concentration corresponding to 25, 50 and 75% of protection). Glutamate induced a significant dose-dependent increase in retinal MDA ( p < 0.0001). Co-incubation with PIN or NAS significantly suppressed the glutamate-induced MDA ( p < 0.01) in a dose-dependent manner ( p < 0.0001). The concentrations to inhibit 50% of LPO were 132.8 and 98.6 μM for PIN and NAS, respectively. In summary, elevated glutamate induced retinal LPO. Both PIN and NAS suppressed the glutamate-induced LPO and a synergic protection was evident after incubation in PIN/NAS mixtures.

Modulation of store‐operated Ca 2+ entry in PC12 cells

Both endoplasmic reticulum (ER) Ca2+ release and Ca2+ influx are essential components of intracellular Ca2+ ([Ca2+]i) regulation in neuronal cells. Previous studies in non-neuronal cells have shown that an acidotic state inhibits Ca2+ influx via L-type Ca2+ channels, while alkalosis increases influx. Glutamate has also been shown to cause dysregulation of Ca2+ influx. Store operated calcium entry (SOCE) that occurs following depletion of ER Ca2+ stores is a newly defined mechanism in neurons. In this study, we examined the effects of altered pH and of glutamate on SOCE in differentiated rat pheochromocytoma (PC12) cells, a model for neuronal cells loaded with the Ca2+ indicator fura-2. Cells were exposed to zero Ca2+ Tyrode's solution (pH 7.4) with 5 μM cyclopiazonic acid (CPA) to deplete the ER. 20 μM KBR-7943 was used to inhibit Na+/Ca2+ exchange. SOCE was then triggered by reintroduction of 2.5 mM Ca2+ Tyrodes (pH 7.4). The cells were washed and the protocol repeated with altered pH (6.6–7.8; one pH change per protocol), altered temperature (35–39 °C) and with different concentrations of glutamate. SOCE was progressively inhibited with decreased pH or temperature, while alkalosis and increased temperature stimulated influx (p<0.05, ANOVA with repeated measures). Increasing glutamate concentrations progressively inhibited SOCE. These data show that SOCE in PC12 cells is susceptible to acidosis, temperature, and agonist stimulation: factors that can adversely affect neuronal function under pathological conditions. Accordingly, altered SOCE function may contribute to neuronal Ca2+ overload under such conditions. Supported by the Departments of Anesthesiology, and Physiology and Biomedical Engineering, Mayo Clinic.

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.