谷氨酸致人神经母细胞瘤细胞兴奋性毒损伤的机制(英文)

目的探讨谷氨酸导致人神经母细胞瘤细胞(SH-SY5Y cells)兴奋性毒损伤的机制。方法MTT法检测SH-SY5Y细胞存活率;测定乳酸脱氢酶释放量观察细胞损伤程度;DAPI染色法观察细胞凋亡形态学特点;钙流法检测胞浆钙离子浓度变化 ;以胞内谷胱甘肽、超氧化物歧化酶活性和胞外丙二醛含量检测谷氨酸引发SH-SY5Y细胞的氧化应激状态。结果谷氨酸导致SH-SY5Y细胞受损,包括存活率下降、乳酸脱氢酶释放量增多及形态结构发生改变;谷氨酸处理 20 min 后,胞浆钙离子浓度无显著改变,而处理 24 h 后,胞浆钙离子大量增加,且 MK801 (NMDA受体拮抗剂)及LY341495 (代谢型谷氨酸受体拮抗剂)均不能抑制钙离子内流的增多;谷氨酸可导致SH-SY5Y氧化损伤,包括胞内谷胱甘肽含量减少、超氧化物歧化酶活性降低、胞外脂质过氧化产物丙二醛水平升高等,而丹参酮IIA (一种抗氧化剂)可减轻这些氧化损伤。结论谷氨酸导致SH-SY5Y细胞兴奋性毒损伤可能是通过氧化损伤产生的,而不依赖于 NMDA 受体介导的钙稳态的破坏。     

LTP的研究进展(Ⅰ)

LTP（长时程动作电位增强，或称长时程增强）是目前神经科学热点课题。各方面研究支持LTP与学习及记忆过程相关。基于其发生机制，LTP可分为NMDA受体及受体依赖性和Mossy fiber LTP两类。前一类由突触后NMDA受体激活，导致钙离子内流，钙浓度升高而引发，后一类则是蛋白激酶A活动引起突触前膜内钙离子浓度升高。结果神经递质释放增强，最终引起LTP。本概述了脑片技术对LTP研究的贡献，LTP发生与维持的相关因素，以及最新LTP研究的有趣发现。下期继续LTP话题，我们将介绍最近与LTP相关的BDNF（脑组织神经生长因子）及基因遗传学研究。     

凝血酶对原代培养海马神经元游离钙浓度的影响

目的研究凝血酶对原代培养海马神经元内游离Ca^2 水平的影响。方法采用原代培养大鼠海马神经元方法，用钙离子指示剂Fura-2双波长荧光检测凝血酶对海马神经元内游离钙浓度的影响。结果(1～40)U／ml凝血酶可使海马神经元内游离Ca^2 水平显著升高，且呈剂量依赖性。凝血酶受体激活肽可明显升高细胞内游离[Ca^2 ]i。当胞外Ca^2 为1．3mmol／L时，40U／ml凝血酶可使海马神经元游离[Ca^2 ]i明显增加；而当胞外Ca^2 为0．0mmol／L时，40U／ml凝血酶不影响海马神经元游离[Ca^2 ]i。预先加入MK-801可显著降低凝血酶的升钙作用。结论凝血酶使神经细胞内游离Ca^2 浓度异常升高的作用机制可能是通过激活凝血酶受体，继而激活NMDA受体门控的Ca^2 通道介导胞外Ca^2 大量内流。     

缺血性脑卒中发病机制研究新进展

短暂或持久的局灶性脑缺血可引起一系列病理生理变化导致脑损害 ,且随时间和缺血程度增加而加重。本文就中枢神经系统中兴奋性氨基酸 (谷氨酸 )三种受体 :NMDA、AMPA、metabotropic (促代谢 )受体 ,神经元及神经胶质细胞的去极复极化 ,缺血后的炎症反应及细胞凋亡几方面在缺血性脑卒中中的作用机制作一综述。     

少突胶质细胞的钙信号

钙是细胞内重要的第二信使,参与机体内多种生理过程,其胞内的钙信号传递依赖于胞内外的钙离子(Ca2+)浓度差,表现为胞质内钙的变化.Ca2+进入细胞内与钙的受体如钙调素(calmodulin,CaM)结合后发挥系列效应.以往认为Ca2+仅在兴奋性细胞中起传递信号作用,但近年来随着研究的深入,发现无论是兴奋性还是非兴奋性细胞,钙都作为第二信使参与信号转导及基因的表达调控.少突胶质细胞是中枢神经系统髓鞘形成细胞,被认为是中枢神经系统中一种非兴奋性细胞,其对于细胞外的刺激可表现为胞内钙浓度的变化从而调控细胞活动.     

D—Ser—NMDA受体的新调控因子

最近研究证实哺乳动物神经系统中存在内源性的D－Ser。这种内源性D－Ser在神经系统中的分布与NMDA受体的分布相平行。进一步的研究表明，D－Ser由突触旁星形胶质细胞产生，而作用于突触后NMDA受体上的Gly结合位点，对NMDA受体的功能进行调控。本文将综述D－Ser在神经系统中的分布，合成及其生理机能。     

Kainate受体与癫痫的研究进展

Kainate(KA)受体属于离子型谷氨酸受体(iGluRs)的一类亚型受体,与其它两种iGluRs,NMDA受体(N-甲基-D-门冬氨酸)和AMPA受体(氨基-3-羟基-5甲基-4-异恶丙酸)相比,具有其自身的生物学特性.近几年随着对KA受体研究的深入,发现KA受体与癫痫病的发病机制有一定的内在联系,因此对KA受体与癫痫关系的研究,可能为癫痫的临床治疗及新药研发开辟一条新的途径.本文介绍KA受体近年来的药理学特性、生理学功能及其在癫痫发病过程中的作用机制的研究进展.     

兴奋性氨基酸毒性与肌萎缩侧索硬化症的研究进展

兴奋性氨基酸毒性是肌萎缩侧索硬化症的主要发病机制之一,主要涉及以下几个环节:突触间隙兴奋性氨基酸水平的升高,谷氨酸转运蛋白异常导致的谷氨酸摄取减少,谷氨酸受体缺陷及钙离子结合蛋白的异常导致的运动神经元细胞内钙离子稳态失衡。对于各环节在发病机制中的具体作用仍存在争论。     

氯胺酮对NMDA诱导大鼠脊髓背角星形胶质细胞损伤的保护作用

目的观察氯胺酮对N-甲基-D-天冬氨酸(NMDA)受体过度激活诱导大鼠脊髓背角星形胶质细胞凋亡影响,并探讨其可能的作用机制.方法取新生2～3 d wistar大鼠T11-L6脊髓背角星形胶质细胞原代纯化培养,GFAP鉴定星形胶质细胞纯度达98%后用于实验.将细胞随机分6组:对照组(C组),NMDA组(N组),氯胺酮组(K组)和三种不同浓度氯胺酮加NMDA组(0.1,0.5,1 mmol/L,标记为NK1～NK3组),再培养24 h后检测SOD活性和MDA含量,免疫组化HE复染观察Bcl-2蛋白和形态学变化,流式细胞仪检测星形胶质细胞凋亡率.结果N组细胞发生了大量凋亡,SOD活性显著降低,MDA含量明显增加,Bcl-2蛋白表达不明显;NK3组细胞凋亡被显著抑制,Bcl-2蛋白强阳性表达,SOD活性明显增加和MDA含量低.结论NMDA受体过度激活可诱导大鼠脊髓背角星形胶质细胞大量凋亡,适量氯胺酮显著抑制了细胞凋亡,其机制可能是增强了星形胶质细胞Bcl-2蛋白表达,同时抑制了自由基的产生和增强了SOD活性.     

LRIG1相关功能片段对EGFR活性和胶质瘤细胞增殖的影响

目的 探讨人多亮氨酸重复区免疫球蛋白样蛋白1（LRIG1）相关功能片段对表皮生长因子受体（EGFR）下游信号通路的影响及对人脑胶质瘤细胞增殖活性的影响。方法 构建缺失LRIG1胞内段（LRIG1-ET）的和缺失LRIG1胞外段（LRIG1-TC）的真核表达质粒,将这两个质粒及LRIG1全长（LRIG1-FL）质粒分别转染脑胶质瘤U251细胞系和原代星形胶质细胞瘤细胞,用蛋白质印迹技术检测EGFR下游信号蛋白有丝分裂原活化蛋白激酶激酶（MAPKK/MEK）的磷酸化水平,用MTT法检测转染细胞的增殖活性。结果 全长LRIG1、LRIG1胞外段、LRIG1胞内段对人脑U251细胞和原代胶质瘤细胞的增殖活性均有抑制作用;全长LRIG1蛋白的抑制作用最强,其次为胞内段,再次为胞外段。全长LRIG1、LRIG1胞外段、LRIG1胞内段均使原代星形胶质细胞瘤细胞的MEK蛋白磷酸化水平下降。结论 LRIG1全长、胞外段、胞内段均能通过抑制EGFR下游信号抑制U251细胞和原代星形胶质瘤细胞的细胞增殖;LRIG1胞外段和胞内段具有独立的功能,均有可能对人脑胶质瘤的治疗发挥重要作用。     

Effect of phosphatidylserine on the binding properties of glutamate receptors in brain sections from adult and neonatal rats

The effects of phosphatidylserine (PS) on the binding properties of the AMPA (-amino-3-hydroxy-5-methylisoxazolepropionic acid) and NMDA ( N-methyl-d-aspartate) subtypes of glutamate receptors were analyzed by quantitative autoradiography of [³H]AMPA, [³H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [³H]glutamate binding on at brain tissue sections. Preincubation of brain sections with PS produced an increase in [³H]AMPA binding without modifying the binding properties of [3H]CNQX, an antagonist of AMPA receptors. This effect of PS appeared to be specific for the AMPA subtype of glutamate receptors as the same treatment did not modify [³H]glutamate binding to the NMDA receptors. Furthermore, the PS-induced increase in [3H]AMPA binding was different in various brain structures, being larger in the molecular layer of the cerebellum and almost absent in the striatum. Preincubation with calcium also augmented [³H]AMPA binding, and the lack of additivity of the effects of calcium and PS on [³H]AMPA binding strongly suggests that both treatments share a common mechanism(s) for producing increased agonist binding. Finally, the effect of PS on AMPA receptor properties was markedly reduced in rat brain sections prepared from neonatal rats at a developmental stage that is normally characterized by the absence of LTP expression in certain brain regions. The present data are consistent with the hypothesis that alteration in the lipid composition of synaptic membranes may be an important mechanism for regulating AMPA receptor properties. which could be involved in producing long-lasting changes in synaptic operation.     

Testing NMDA receptor block as a therapeutic strategy for reducing ischaemic damage to CNS white matter

Damage to oligodendrocytes caused by glutamate release contributes to mental or physical handicap in periventricular leukomalacia, spinal cord injury, multiple sclerosis, and stroke, and has been attributed to activation of AMPA/kainate receptors. However, glutamate also activates unusual NMDA receptors in oligodendrocytes, which can generate an ion influx even at the resting potential in a physiological [Mg2+]. Here, we show that the clinically licensed NMDA receptor antagonist memantine blocks oligodendrocyte NMDA receptors at concentrations achieved therapeutically. Simulated ischaemia released glutamate which activated NMDA receptors, as well as AMPA/kainate receptors, on mature and precursor oligodendrocytes. Although blocking AMPA/kainate receptors alone during ischaemia had no effect, combining memantine with an AMPA/kainate receptor blocker, or applying the NMDA blocker MK-801 alone, improved recovery of the action potential in myelinated axons after the ischaemia. These data suggest NMDA receptor blockers as a potentially useful treatment for some white matter diseases and define conditions under which these blockers may be useful therapeutically. Our results highlight the importance of developing new antagonists selective for oligodendrocyte NMDA receptors based on their difference in subunit structure from most neuronal NMDA receptors.     

Role of zinc influx via AMPA/kainate receptor activation in metabotropic glutamate receptor-mediated calcium release

The uptake of free zinc into CA3 pyramidal cells and its significance was examined in rat hippocampal slices with ZnAF-2DA, a membrane-permeable zinc indicator. Intracellular ZnAF-2 signal in the CA3 pyramidal cell layer was increased during delivery of tetanic stimuli to the dentate granule cell layer. This increase was completely blocked in the presence of CNQX, an AMPA/kainate receptor antagonist. These results suggest that free zinc is taken up into CA3 pyramidal cells via activation of AMPA/kainate receptors. The effect of free zinc levels in the CA3 pyramidal cells on the increase in intracellular calcium via Group I metabotropic glutamate receptors was examined by regional delivery of tADA, a Group I metabotropic glutamate receptor agonist, to the stratum lucidum after blockade of AMPA/kainate receptor-mediated calcium and zinc influx. Intracellular calcium orange signal in the CA3 pyramidal cell layer was increased by tADA, whereas intracellular ZnAF-2 signal was not increased even in the presence of 100 muM zinc, suggesting that tADA induces calcium release from internal stores in CA3 pyramidal cells and is not involved in zinc uptake. The increase in calcium orange signal by tADA was enhanced by perfusion with pyrithione, a zinc ionophore that decreased basal ZnAF-2 signal in the CA3 pyramidal cell layer. It was blocked by perfusion with pyrithione and zinc that increased basal ZnAF-2 signal. The present study indicates that the increase in free calcium levels via the metabotropic glutamate receptor pathway is inversely related to free zinc levels in CA3 pyramidal cells.     

Stimulation of N-methyl-D-aspartate receptors, AMPA receptors or metabotropic glutamate receptors leads to rapid internalization of AMPA receptors in cultured nucleus accumbens neurons

In hippocampus and other regions, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors are inserted into synapses during long-term potentiation and removed during long-term depression. However, little is known about regulation of AMPA receptor trafficking in the nucleus accumbens (NAc), despite growing evidence that glutamate-dependent forms of plasticity in the NAc contribute to drug addiction. Using postnatal rat NAc cultures and an immunocytochemical method that selectively detects newly internalized GluR1, we studied the regulation of AMPA receptor internalization in NAc neurons by glutamate agonists. Newly internalized GluR1 was detected during 15 or 30 min of incubation at room temperature, indicating a basal rate of GluR1 turnover. The rate of GluR1 internalization was increased by glutamate (50 microM) within 5 min of its addition. Glutamate-induced GluR1 internalization was partially blocked by either an AMPA receptor antagonist (CNQX; 20 microM) or an N-methyl-D-aspartate (NMDA) receptor antagonist (APV; 50 microM). Both NMDA (50 microM) and AMPA (50 microM) increased GluR1 internalization in a Ca(2+)-dependent manner. The NMDA effect was blocked by APV while the AMPA effect was blocked by APV or CNQX. We interpret these findings to suggest that NMDA and AMPA ultimately trigger GluR1 internalization through the same NMDA receptor-dependent pathway. The effect of glutamate was also partially blocked by the group 1 metabotropic glutamate receptor antagonist N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC; 50 microM), while the group 1 agonist 3,5-dihydroxyphenylglycine (DHPG; 50 microM) stimulated GluR1 internalization. These data suggest that AMPA receptors on NAc neurons may be subject to rapid regulation of their surface expression in response to changes in the activity of glutamate inputs from cortical and limbic regions.     

Subunit selective decrease of AMPA and metabotropic glutamate receptor mRNA expression in rat brain by systemic administration of the NMDA receptor blocker MK-801

Glutamate mediates its effects in mammals through both ionotropic and metabotropic receptors. Antagonists of ionotropic N-methyl-d-aspartate (NMDA) glutamate receptors elicit neuroprotective and neurotropic effects that have been attributed to Ca²⁺ block through the membrane ion channel. Nonetheless, molecular and biochemical effects of NMDA receptor antagonism on other glutamate receptor subunits remain poorly understood. We investigated the effects of acute administration of the noncompetitive NMDA receptor antagonist MK-801 on the mRNA expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and metabotropic glutamate receptor (mGluR) subunits to determine the contribution of different glutamate receptors in response to blockade of NMDA receptor channels. In situ hybridization to rat brain sections revealed that AMPA receptor subunits GluR3 and GluR4, and mGluR3 were modestly but significantly decreased ∼10–20%, 8 h following 5 mg/kg MK-801 administration. A time course and dose response study revealed that the effect on mGluR3 was reversed by 24 h and occurred significantly at a dose range from 1 to 5 mg/kg. These results indicate that selected AMPA and mGluR subunit mRNAs respond at the RNA level to the blockade of NMDA receptors.     

Metabotropic glutamate receptors are involved in calcium-induced LTP of AMPA and NMDA receptor-mediated responses in the rat hippocampus

Effects of metabotropic glutamate (mGlu) receptors on calcium-induced long-term potentiation (LTP) of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) and N-methyl-d-aspartate (NMDA) receptor-mediated components were investigated in rat hippocampal slices using whole-cell patch-clamp recordings of excitatory postsynaptic currents (EPSCs). Calcium-induced LTP comprises a parallel, long-lasting increase of AMPA and NMDA receptor-mediated components. The calcium-induced LTP of the AMPA receptor-mediated component can be significantly attenuated by the use of a selective NMDA antagonist. (R.S)-α-methyl-4-carboxyphenylglycine (MCPG), a selective antagonist of mGlu receptors, abolished the long-lasting increase of both AMPA and NMDA receptor-mediated components observed in calcium-induced LTP. In current clamp mode, the application of a high calcium alone or Schaffer fiber stimulation alone (20 Hz) only generated a short-term increase in the firing rate of evoked action potentials. Conversely, a long-term increase in the firing rate was observed if Schaffer fiber stimulation (20 Hz) accompanied the perfusion of high calcium. These results suggest that calcium-induced LTP involves a parallel, long-lasting enhancement in ionotropic AMPA and NMDA receptor-mediated components. More importantly, the mGlu receptor plays a critical role in the establishment of both AMPA and NMDA receptor-mediated components underlying calcium-induced LTP. In addition, the present study also described an experimental condition in which the coapplication of the high calcium pulse and Schaffer fiber stimulation (20 Hz) can synergistically elicit a long-term increase of neuronal excitability.     

Mechanism of NMDA receptor contribution to axon-to-glia signaling in the crayfish medial giant nerve fiber

Electrical stimulation of crayfish giant axons at high frequency activates group II metabotropic and NMDA glutamate receptors on adjacent glial cells via release of N-acetylaspartylglutamate and glutamate formed upon its hydrolysis. This produces a transient depolarization followed by a prolonged hyperpolarization of glial cells that involves nicotinic acetylcholine receptor activation. The hyperpolarization is nearly completely blocked by antagonists of metabotropic glutamate receptors but only slightly reduced by inhibition of NMDA receptors. We report that the NMDA-induced hyperpolarization of glial cells is reduced by decreased calcium in the solution bathing the giant nerve fiber, while removal of sodium ions or block of voltage-dependent calcium channels completely prevents the glial response to NMDA. Inhibition of nicotinic acetylcholine receptors or removal of extracellular Cl(-) converts the glial response from a hyperpolarization to a depolarization that is sensitive to NMDA receptor antagonist. We propose that NMDA receptor activation by glutamate, formed from extracellular N-acetylaspartylglutamate during nerve stimulation, contributes to glial hyperpolarization by increasing intracellular Ca(2+) via opening of voltage-sensitive Ca(2+) channels. Based on our previous work, we propose further that the added Ca(2+) supplements that produced by N-acetylaspartylglutamate and glutamate acting on group II metabotropic glutamate receptors to cause an increased release of acetylcholine and a larger hyperpolarization.     

AMPA receptor modulation in previously frozen mouse brain sections: opposite effects of calcium in the cortex and hippocampus

Various forms of synaptic plasticity in the brain have been proposed to result from modifications in the properties of glutamate receptors by calcium-dependent mechanisms. In the present study, changes in glutamate receptors elicited by calcium treatment of previously frozen mouse brain sections were evaluated by qualitative as well as quantitative analysis of tritiated ligand binding to both alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and N-methyl-D-aspartate (NMDA) glutamate receptor subtypes. Quantitative analysis revealed that 3H-AMPA binding was reduced in a dose-dependent manner by calcium in the cerebral cortex and striatum formations. However, an opposite change in AMPA receptor properties was observed in the hippocampus, as calcium generated an increase of AMPA binding in all hippocampal fields. Analysis of the saturation kinetics of 3H-AMPA binding showed that the calcium-induced augmentation of AMPA binding in the stratum radiatum of the CA1 region was due to an alteration in the maximal number of sites, while the reduction of binding elicited by calcium in the cortex appeared to be due to modified AMPA receptor affinity. Calcium-induced downregulation of AMPA receptor affinity in the cortex and striatum was affected by baicalein, a selective inhibitor of the lipoxygenase pathways of arachidonic acid metabolism, whereas the same inhibitor did not modify calcium-mediated upregulation of receptor number in the CA1 region of the hippocampus. On the other hand, the effect of calcium appeared to be specific for the AMPA receptor, as the same treatment did not affect glutamate binding to the NMDA glutamate receptor subtype. Our results suggest the possibility that, depending on the brain regions, calcium ions may generate opposite modulation of AMPA receptor properties. Because the regulation of AMPA receptors by calcium-dependent enzymes has been implicated in synaptic plasticity, our results suggest that regional variations in the effect of calcium on AMPA binding account for differential plasticity at glutamatergic synapses.     

Acetylcholine enhances NMDA-evoked calcium rise in hippocampal neurons

The effects of acetylcholine (ACh) on changes in [Ca]i produced by the glutamate agonist N-methyl-D-aspartate (NMDA) were measured in cultured rat hippocampal neurons loaded with the fluorescent calcium indicator Fluo-3 in a confocal laser scanning microscope. NMDA produced a dose-dependent reversible rise in [Ca]i. ACh had a smaller and less consistent effect on [Ca]i but could cause a marked enhancement of the reactivity of neurons to NMDA. This effect was reversed by the presence of the muscarinic antagonist atropine. AMPA, another glutamate agonist which causes a rise in [Ca]i by activating voltage gated calcium influx was less affected by ACh. Caffeine, which releases calcium from intracellular stores also enhanced reactivity of these neurons to NMDA. It is suggested that ACh can enhance reactivity to NMDA by releasing calcium from internal stores.