谷氨酶传感器及在流动注射分析中的应用

利用谷氨酸氧化酶共价偶联于硅烷化铂化铂丝表面。构建一种简单的微酶电极,该电极具有良好的操作性能,应用于流动注射分析系统,可用来测量谷氨酸含量,测量范围０－２．０ｍｍｏｌ／Ｌ,精度、响应时间小于６０秒,使用寿命大于２０天,实际测量发酵液中谷氨酸含量,回收率为９８．７％－１０７．５％。     

以二茂铁为介质的谷氨酸电流型传感器的研究

文研究了一种用于测定发酵液谷氨酸含量的介质电流型谷氨酸电极。这种电极是由谷氨酸氧化酶同以二茂铁为介质的化学修饰电极复合构成。研究了各种因素对此电极响应的影响和电极本身的性能与实测验证,结果表明本电极在搅拌反应室(CSR)系统中响应的最适pH为6．8,最适温度为30"C,使用寿命为6天,电极专一性较好。在流通注射分析(FIA)系统中响应的最适pH为7．0,最适温度为27℃,测量范围为0．0125－O．125mol／L(射(20μ1),精度(CV)为1．3％,响应时间为Imin,校正曲线相关系数r=0．998,使用寿命为8天,经实际测量发酵液中谷氨酸含量,与瓦氏呼吸仪对照,相对误差为±6%。     

谷氨酸氧化酶电极的研究

用戊二醛作交联剂将谷氨酸氧化酶和牛血清白蛋白共交联,置于内层醋酸纤维素抗干扰膜和外层聚碳酸酯扩散控制膜之间,制成酶膜,将其与过氧化氢探头复合制成了扩散控制型谷氨酸氧化酶电极,并构建了采用该电极的流动注射分析芽纺。酶电极红性范围0—1000mg／L。50s响应电流达稳态值的95％。流动注射分析系统响应时间20s,检测速度60次／h,线性范围5—8 000mg,L,酶膜使用寿命两周以上。系统选择性好,仅对干扰物L－谷氨酰胺和L－天冬氨酸有微弱响应。对同一样品连续测定41次的变异系数2．8％。测量味精发酵液和瓦勃呼吸计的结果相吻合。可望应用于味精发酵及食品工业中。     

测定谷氨酸的微生物传感器的研究

本文研究了测定谷氨酸的微生物传感器。用琼脂固定化的大肠杆菌(Escherichia coli As 1．505)和二氧化碳电极制成膜状传感器,用聚丙烯酰胺固定化的大肠杆菌和二氧化碳电极制成柱状传感器。结果表明,膜状传感器在25℃测定谷氨酸的范围为60－1200mg／I,响应时间为1020min,柱状传感器在30℃测定谷氨酸的范围为60－800mg／1,响应时间为5—7 min。两种传感器测定误差为2—3％以下,20天内使用60多次,不见其活力下降。     

急性低血压对前庭神经内侧核区谷氨酸和牛磺酸含量的影响

本实验用脑部微量透析法和高效液相色谱法观察急性失血诱发大鼠低血压时前庭神经内侧核(medial vestibular nucleus,MVN)内氨基酸类神经递质谷氨酸(glutamate,Glu)和牛磺酸(tautine,Tau)含量的变化,以了解MVN内这些氨基酸类递质是否参与血压的调节。实验观察到,正常血压大鼠MVN内Glu和Tau浓度在透析探针植入90 min后趋于稳定,此时 Glu的平均含量为(18．96±0．27)pmol／8 μl透析液,Tau的平均含量为(7．73±0．05)pmol/8μl透析液。动脉抽血1．5 ml诱发急性低血压时,同侧MVN区Glu含量增加(P<0．05),Tau含量减少(P<0．05)。用利多卡因(lidocaine)短时阻断前庭传入及用化学方法永久破坏单侧前庭器官后,动脉抽血1.5 ml使血压降低30％,同侧MVN内氨基酸类神经递质无明显改变。结果提示,急性失血引起的低血压可通过前庭器官影响MVN的活动,该区内氨基酸类递质可能参与这一活动的调节。     

固定化谷氨酸氧化酶管流动注射测定谷氨酸的研究

多孔玻璃珠固定谷氪酸氧化酶与过氧化氢酶分别制成相应的固定化酶管,结合流动注射分析系统测定谷氨酸的含量。测定线性范围在0．1—2．O mmol／L．精度(c．V)O．7％．测定速率每小时80样以上．使用寿命至少4个月。在各氨酸浓度低于2．5mmol／L时．pH在6．5—8．0．温度20—35℃．磷酸盐浓度在0.05—0.25mol／L范围内对测定几乎无影响．对不同发酵时间的谷氨酸发酵液测定,测定的结果与酶试剂盒及瓦氏法比较,结果一致．说明该方法已具有实际应用价值。     

应用FIA型微生物传感器测定谷氨酸含量的研究

目前应用酶或微生物细胞作为分子识别元件构建生物传感器测定谷氨酸含量的研究引起了广泛的兴趣,并陆续有实例报道。在这些报道中人们依据不同的酶催反应选择相应的离子选择性电极,如CO₂电极、NH₄⁺电极等,而测量对象有直接的测定谷氨酸,也有测定谷氨酸单钠的间接测定法。本文选用了可固定大量细胞的固定化细胞柱与流动注入法相结合的测量方法,并根据细胞柱的动力学模型分析动态响应曲线,计算测量结果,提高了测量精度,扩大了测量范围。     

谷氨酸生产菌S9114中的谷氨酸脱氢酶的研究

谷氨酸脱氢酶 (GDH)是谷氨酸生物合成的关键酶 ,谷氨酸棒杆菌S91 1 4是目前我国味精工业应用最广泛的生产菌种 ,其谷氨酸脱氢酶的研究尚未见报道。分离纯化该菌中的谷氨酸脱氢酶 ,研究其辅酶组成 ,对揭示谷氨酸脱氢酶的分子结构和性质 ,提高谷氨酸产率很有必要。将培养至对数期中期的细胞离心收集并用含适量DTT、ED TA的Tris_HCl缓冲液 (pH 7 5 )洗涤 ,用Frenchpressurecellpress破碎 ,离心去除菌体碎片得无细胞抽提液。然后使用 KTA_10 0快速纯化系统经DEAE_纤维素柱、疏水柱 (HIC)、G_2 0 0凝胶过滤柱层析得到纯化大约 70倍的以NAD PH为辅酶的GDH和部分纯化的以NADH辅酶的GDH。这两个酶分别对NADPH、NADH高度专一 ,不能相互代替。经HPLC和SDS_PAGE测得前一种酶的分子量和亚基分子量分别为 188kD和 32kD ,表明该酶为具有相同亚基的六聚体。酶活性测定使用HITACHIU_30 0 0分光光度计利用NAD(P)H在 340nm氧化的初速度进行。蛋白质含量测定利用Bradford方法进行 ,并以牛血清白蛋白为标准蛋白。纯化结果表明S91 1 4中确实存在两种GDH ,其中以NADH为辅酶的GDH尚未见报道。和某些具有两种GDH的微生物一样 ,S91 1 4可能也是以NADPH为辅酶的GDH参与谷氨酸的合成代谢 ,以NADH为辅酶的GDH参与谷氨酸的分解代谢。     

天津短杆菌T6-13谷氨酸脱氢酶的研究

本文研究了天津短杆菌(Brevibacterium tianjinese)T6-13谷氨酸脱氢酶(GDH)[EC1.4．1．4]的纯化和性质。该酶以辅酶11(NADP)为其专一性辅酶,正、逆反应酶活力最适pH分别为7．5和8．9—9 9,对热较敏感。 该酶对还原型辅酶11(NADPH)、α-酮戊二酸(α-KG)、NH,、NADP和L-谷氨酸(GA)的Km值分别为0．076、3．23、4．0、0．02和120．48 mmol／L。该酶受反应产物的抑制,逆反应受NADPH、α-KG和NH+4的抑制,正反应受NADP和谷氨酸的抑制,但该酶所催化的逆反应既不受三羧酸循环代谢中间产物的抑制,也不受氨基酸的抑制和氨基酸的积累抑制。对发酵过程中谷氨酸脱氢酶活力变化的研究表明,前期酶活力逐渐上升,当发酵至16小时左右酶活力最高,其后酶活力逐渐下降;二级种子的酶活力与发酵过程中酶活力最高时相当。     

内循环气升式发酵罐的特性及其用于谷氨酸发酵的研究

对50L内循环气升式发酵罐的混合、传质、气含率、液体循环等性能进行了系统的研究,优选出设备的最佳结构及得出氧的体积传质系数(k_La)关联式。在此基础上,进行了谷氨酸的一次低糖发酵试验,得出适用于此设备的谷氨酸发酵最佳工艺。实验结果的最高产酸率为7．69％,转化率为58．8％,比使用同一菌种的用机械搅拌罐发酵的产酸率(6．O％)及转化率(44％)分别提高28．2％和33．6％。     

Human, Bovine, and Rabbit Retinal Glutamate-Induced [3H]D-Aspartate Release: Role in Excitotoxicity

The pharmacological basis of glutamate-induced [³H]D-aspartate release was investigated in isolated human, bovine and rabbit retinas. Isolated mammalian retinas were preloaded with [³H]D-aspartate and then prepared for studies of neurotransmitter release using the superfusion method. Release of [³H]D-aspartate was elicited by K⁺ (50 mM) or by L-glutamate. In bovine retinas, L-glutamate, but not D-glutamate induced an overflow of [³H]D-aspartate that was partially inhibited by low external calcium, -conotoxin (10 nM) or nitrendipine (1 M). Metabotropic glutamate receptor (GLUR) agonists also evoked [³H]D-aspartate release in both bovine and human retinas whereas polyamines only enhanced the excitatory effects of L-glutamate on [³H]D-aspartate release. Antagonists of GLURs and the polyamine site inhibited L-glutamate evoked [³H]D-aspartate overflow with the following rank order of potency: MCPG >ifenprodil > AP-5 > arcaine> MK-801. In conclusion, L-glutamate-induces a stereoselective, calcium-dependent release of [³H]D-aspartate from isolated mammalian retinas that can be mimicked by GLUR agonists (and blocked by both receptor and polyamine site antagonists).     

Possible linkage between glutamate transporter and mitogen-activated protein kinase cascade in cultured rat cortical astrocytes

The mitogen-activated protein kinases (MAPKs) play a pivotal role in the mediation of cellular responses to a variety of signalling molecules. In the present study, we investigated possible linkage between glutamate signalling and the MAPK cascade in cultured rat cortical astrocytes. Exposure of the cells to L-glutamate (100-1000 microM) resulted in an increase in phosphorylated p44/42 MAPK (ERK1/2) in a concentration- and time-dependent manner. The glutamate-induced ERK1/2 phosphorylation was blocked by U0126 and PD98059, specific inhibitors of the MAPK-activating enzyme MEK. Furthermore, L-glutamate-induced ERK1/2 phosphorylation was not mimicked by glutamate receptor agonists and was not blocked by glutamate receptor antagonists. In contrast, the effect of L-glutamate was mimicked by D- and L-aspartate and transportable glutamate uptake inhibitors. These results suggest that the MEK/ERK cascade is activated by a mechanism related to glutamate transporters. We propose that the glutamate transporter functions as a receptor transmitting extracellular glutamate signal to intracellular messengers.     

Vesicular storage and secretion of L-glutamate from glucagon-like peptide 1-secreting clonal intestinal L cells

Vesicular glutamate transporter (VGLUT) is responsible for the vesicular storage of l-glutamate, and plays an essential role in glutamate-mediated intercellular signal transmission in the CNS and in some neuroendocrine cells. Intestinal L cells are the glucose-responsive neuroendocrine cells responsible for the secretion of glucagon-like peptide 1 (GLP-1). We have shown that intestinal L cells express VGLUT2, a VGLUT isoform, which suggests that L cells secrete L-glutamate. In the present study, we investigated this possibility using GLUTag mouse clonal L cells. RT-PCR and northern blot analyses revealed expression of the VGLUT1 and VGLUT2 genes, but not of the VGLUT3 gene. Western blot analysis revealed immunological counterparts for VGLUT2, whereas an immunological counterpart of VGLUT1 was not detected. Indirect immunofluorescence microscopy revealed a punctate distribution of VGLUT2 immunoreactivity throughout the cells, which co-localized with GLP-1. Double-labeling immunoelectronmicroscopy confirmed the association of VGLUT2 with GLP-1-containing secretory granules. The membrane fraction exhibited ATP-dependent L-glutamate uptake, which was sensitive to bafilomycin A1 (a vacuolar proton ATPase inhibitor) and Evans blue (a VGLUT inhibitor) but insensitive to D,L-aspartate. Upon depolarization with KCl, GLUTag cells secreted appreciable amounts of L-glutamate and GLP-1. D-Glucose and methyl-alpha-D-glucopyranoside, stimulators of exocytosis of GLP-1, also triggered the secretion of L-glutamate. The L-glutamate secretion was partially dependent on Ca2+ and sensitive to bafilomycin A1. These results demonstrated that GLUTag cells stored L-glutamate in secretory granules and secreted it with GLP-1 by exocytosis. As GLUTag cells and intestinal L cells express kainate receptors and plasma membrane glutamate transporters, these results support the concept of L-glutamate-mediated intercellular signaling in the vicinity of intestinal L cells.     

Kappa opioid agonists inhibit transmitter release from guinea pig hippocampal mossy fiber synaptosomes

Opioid agonists specific for the , , and opioid receptor subtypes were tested for their ability to modulate potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity from guinea pig hippocampal mossy fiber synaptosomes. The opioid agonists U-62,066E and (–) ethylketocyclazocine, but not the agonist [D-Ala²,N-MePhe⁴,Gly⁵-ol]-enkephalin (DAGO) nor the agonist [D-Pen^2,5]enkephalin (DPDE), inhibited the potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity. U-62,066E, but not DAGO or DPDE, also inhibited the potassium-evoked rise in mossy fiber synaptosomal cytosolic Ca²⁺ levels, indicating a possible mechanism for agonist inhibition of transmitter release. DAGO and DPDE were found to be without any effect on cytosolic Ca²⁺ levels or transmitter release in this preparation. The U-62,066E inhibition of the potassium-evoked rise in synaptosomal cytosolic Ca²⁺ levels was partially attenuated by the opioid antagonist quadazocine and insensitive to the -opioid specific antagonist ICI 174,864 and the opioid-preferring antagonists naloxone and naltrexone. Quadazocine also reversed U-62,066E inhibition of the potassium-evoked release of L-glutamate, but not dynorphin B-like immunoreactivity. These results suggest that opioid agonists inhibit transmitter release from mossy fiber terminals through both opioid and non- opioid receptor mediated mechanisms.     

Effects of L-glutamate/D-aspartate and monensin on lactic acid production in retina and cultured retinal Müller cells

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.     

Activation of p42 Mitogen-Activated Protein Kinase by Glutamate in Cultured Radial Glia

The effect of L-glutamate (Glu) and its structural analogs N-methyl-D-aspartate (NMDA), kainate (KA) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), on the activation of p42 mitogen activated protein kinase (MAPK) was examined in cultured chick radial glia cells, namely retinal Müller cells and cerebellar Bergmann cells. Glu, NMDA, AMPA and KA evoked a dose and time dependent increase in MAPK activity. AMPA and KA responses were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) whereas NMDA responses were sensitive to 3-[(RS)-2-carboxypiperazin-4-yl)]-propyl-1-phosphonate (CPP) indicating that the increase in MAPK activity is mediated by AMPA/low affinity KA and NMDA subtypes of Glu receptors. The present findings open the possibility of a MAPK cascade involvement in the regulation of Glu-induced gene expression in radial glia.     

L-Glutamate Supplementation Improves Small Intestinal Architecture and Enhances the Expressions of Jejunal Mucosa Amino Acid Receptors and Transporters in Weaning Piglets

L-Glutamate is a major oxidative fuel for the small intestine. However, few studies have demonstrated the effect of L-glutamate on the intestinal architecture and signaling of amino acids in the small intestine. The aim of this study was to investigate the effects of dietary L-glutamate supplementation on the intestinal architecture and expressions of jejunal mucosa amino acid receptors and transporters in weaning piglets. A total of 120 weaning piglets aged 35±1 days with an average body weight at 8.91±0.45 kg were randomly allocated to two treatments with six replicates of ten piglets each, fed with diets containing 1.21% alanine, or 2% L-glutamate. L-Glutamate supplementation increased the activity of glutamate oxaloacetate transaminase (GOT) in the jejunal mucosa. Also, the mRNA expression level of jejunal mucosa glutamine synthetase (GS) was increased by L-glutamate supplementation. The height of villi in duodenal and jejunal segments, and the relative mRNA expression of occludin and zonula occludens protein-1 (ZO-1) in jejunal mucosa were increased by dietary L-glutamate supplementation. L-Glutamate supplementation increased plasma concentrations of glutamate, arginine, histidine, isoleucine, leucine, methionine, phenylalanine and threonine. L-Glutamate supplementation also increased the relative mRNA expression of the jejunal mucosa Ca²⁺-sensing receptor (CaR), metabotropic glutamate receptor 1 (mGluR1) and metabotropic glutamate receptor 4 (mGluR4), and neutral amino acid transporter B⁰-like (SLC1A5) in the jejunal mucosa. These findings suggest that dietary addition of 2% L-glutamate improves the intestinal integrity and influences the expression of amino acid receptors and transporters in the jejunum of weaning, which is beneficial for the improvement of jejunal nutrients for digestion and absorption.     

Effect of lysine succinylation on the regulation of 2-oxoglutarate dehydrogenase inhibitor,OdhI, involved in glutamate production in Corynebacterium glutamicum

In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase (ODH) complex is negatively regulated by the unphosphorylated form of OdhI protein, which is critical for L-glutamate overproduction. We examined the potential impact of protein acylation at lysine (K)-132 of OdhI in C. glutamicum ATCC13032. The K132E succinylation-mimic mutation reduced the ability of OdhI to bind OdhA, the catalytic subunit of the ODH complex, which reduced the inhibition of ODH activity. In vitro succinylation of OdhI protein also reduced the ability to inhibit ODH, and the K132R mutation blocked the effect. These results suggest that succinylation at K132 may attenuate the OdhI function. Consistent with these results, the C. glutamicum mutant strain with OdhI-K132E showed decreased L-glutamate production. Our results indicated that not only phosphorylation but also succinylation of OdhI protein may regulate L-glutamate production in C. glutamicum.     

Effects of Metabotropic Glutamate Receptor Agonists and Antagonists on D-Aspartate Release from Mouse Cerebral Cortical and Striatal Slices

The cytosolic release of L-glutamate has been held to be responsible for the increase in extracellular glutamate to toxic levels in the brain. The mechanism and regulation of this release was now studied in cerebral cortical and striatal slices with D-[³H]aspartate, a non-metabolized analogue of L-glutamate and a poor substrate for vesicular uptake. L-Glutamate and D-aspartate strongly stimulated the release in a concentration-dependent manner. Of the ionotropic glutamate receptor agonists, only kainate enhanced the basal release in the striatum. Of the metabotropic glutamate receptor ligands, the group I agonist (S)-3,5-dihydroxyphenylglycine (S-DHPG) failed to affect the basal release but inhibited the D-aspartate-evoked release in the striatum. The group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) had no effect on the basal release in either preparation but enhanced the L-glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum, not however in the cerebral cortex. The group II agonist (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG IV) and the group II antagonist (2S)-2-ethylglutamate (EGLU) were without effect on the basal, D-aspartate- and L-glutamate-evoked releases of D-[³H]aspartate in either preparation. The group III agonist L-serine-O-phosphate (L-SOP) failed to affect the basal release but reduced the D-aspartate-evoked release in the striatum. The group III antagonist (RS)-methylserine-O-phosphate (MSOP) failed to affect the basal release but increased the glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum. Both L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) and (2S, 1S, 2R)-2-carboxycyclopropyl)glycine (L-CCG-III), transportable inhibitors of the high-affinity glutamate uptake, enhanced the basal release, more strongly in the striatum than in the cerebral cortex. L-CCG-III also increased the L-glutamate-evoked release in the striatum. Nontransportable dihydrokainate enhanced the basal release much less and failed to affect the glutamate-evoked release. The results indicate that the release of glutamate from cytosolic pools is carrier-mediated via homoexchange. This process is regulated in the striatum by metabotropic group I and group III receptors in a manner different from the regulation of the vesicular release of glutamate from presynaptic terminals.     

Evaluation of Drugs Acting at Glutamate Transporters in Organotypic Hippocampal Cultures: New Evidence on Substrates and Blockers in Excitotoxicity

Removal of L-glutamate (Glu) from the synapse is critical to maintain normal transmission and to prevent excitotoxicity, and is performed exclusively by excitatory amino acid transporters (EAATs). We investigated the effects of substrates and blockers of EAATs on extracellular Glu and cellular viability in organotypic cultures of rat hippocampus. Seven-day treatment with a range of drugs (L-trans-pyrrolidine-2,4-dicarboxylate, (2S,4R)-4-methyl-glutamate, (±)-threo-3-methylglutamate and DL-threo--benzyloxyaspartate), in the presence of 300 M added Glu, resulted in increased extracellular Glu and a significant correlation between Glu concentration and cellular injury (as indicated by lactate dehydrogenase release). In contrast, (2S,3S,4R)-2-(carboxycyclopropyl)glycine (L-CCG-III) exerted a novel neuroprotection against this toxicity, and elevations in extracellular Glu were not toxic in the presence of this compound. Similar results were obtained following two-week treatment of cultures without added Glu. Whilst blockade of GLT-1 alone was relatively ineffective in producing excitotoxic injury, heteroexchange of Glu by EAAT substrates may exacerbate excitotoxicity.