NMDA受体阻滞剂治疗癫痫的研究进展

脑癫痫放电也与Ｎ－甲基－Ｄ－天冬氨酸（ＮＭＤＡ）受体异常兴奋相关，其受体兴奋能被ＮＭＤＡ受体阻滞剂竞争性与非竞争性地阻断。动物实验和临床研究均显示，ＮＭＤＡ受体阻滞剂呈延缓或防止痫性惊厥的发展，药物作用机制有别于抗癫痫药。     

Antagonistic effect of nerve growth factor on neuronal injury induced by hypoxia in cultured cerebral cortical neurons of rats

目的：以连二亚硫酸钠造成原代培养的大鼠胎鼠脑皮质神经元缺氧损伤,观察神经生长因子（ＮＧＦ）对缺氧损伤神经元的影响．方法：测定神经元生存力及细胞外液乳酸脱氢酶（ＬＤＨ）的活性来分析ＮＧＦ的作用,脑皮质细胞匀浆用于测定丙二醛（ＭＤＡ）含量及超氧化物歧化酶（ＳＯＤ）和谷胱甘肽过氧化物酶（ＧＳＨＰｘ）活性．结果：缺氧后,ＬＤＨ释放及细胞生存力降低．ＮＧＦ（１－１００μｇ·Ｌ－１）浓度依赖地减少ＬＤＨ的释放及ＭＤＡ的生成,ＮＧＦ１００μｇ·Ｌ－１显著提高细胞生存力及提高ＳＯＤ和ＧＳＨＰｘ活性２７倍．结论：ＮＧＦ通过减少脂质过氧化物生成及提高ＳＯＤ和ＧＳＨＰｘ活性来保护大脑皮质细胞抗缺氧损伤     

NMDA和非NMDA受体介导猫脊髓伤害性和非伤害性信息传递

用多管微电极离子电泳技术研究猫脊髓背角神经元活动。８０％的神经元被兴奋性氨基酸ＮＭＤＡ、使君子氨酸、卡因酸或ＤＬ－高磺丙氨酸（ＤＬＨ）激活。ＮＭＤＡ受体拮抗剂ＡＰＶ和氯受酮，非ＮＭＤＡ受体拮抗剂ＤＮＱＸ以及广谱拮抗剂４－羟基喹啉酸（Ｋｙｎ）均抑制神经元的伤害性反应。Ｋｙｎ抑制非伤害性反应大大强于氯胺酮。ＮＭＤＡ和非ＮＭＤＡ受体均参与介导脊髓伤害性反应，非伤害性信息主要由非ＮＭＤＡ受体介导。     

用放射自显影术研究人参总皂甙对大鼠海马NMDA受体的影响

采用〔３Ｈ〕ＴＣＰ放射配基作海马ＮＭＤＡ受体的自显影，研究人参总皂甙在海马齿状回颗粒细胞层诱发ＬＴＰ效应和促进大鼠记忆保持能力时，海马内此受体的变化。给人参总皂甙的大鼠海马ＣＡ１、ＣＡ３和ＤＧ（ｄｅｎｔａｔｅｇｙｒｕｓ齿状回）的ＮＭＤＡ受体银粒数较给生理盐水大鼠的平均分别增多４２．８４％±２．４％，３８．５７％±３．１３％，３５．８１％±１．４６％（Ｐ＜０．０５、Ｐ＜０．０１），表明人参总皂甙在诱发海马ＬＴＰ和促进大鼠记忆行为时，可增加海马的ＮＭＤＡ受体数目和活性。     

Protective Effect of Tetrandrine against Excitatory Amino Acids induced Neuronal Injury in Cortical Culture

兴奋性氨基酸类神经毒剂与粉防己碱（ｔｅｔｒａｎｄｒｉｎｅ，Ｔｅｔ）共同作用于原代培养胎鼠大脑皮层神经元２４小时，发现１０７，１０６ｍｏｌ·Ｌ１Ｔｅｔ明显降低５０μｍｏｌ·Ｌ１谷氨酸（ｇｌｕｔａｍａｔｅ，Ｇｌｕ），３００μｍｏｌ·Ｌ１βＮｍｅｔｈｙｌａｍｉｎｏＬａｌａｎｉｎｅ（ＢＭＡＡ，ＮＭＤＡ受体激动剂）和２０μｍｏｌ·Ｌ１βＮｏｘａｌｙｌａｍｉｎｏＬａｌａｎｉｎｅ（ＢＯＡＡ，ｎｏｎＮＭＤＡ受体激动剂）导致的培养液乳酸脱氢酶（ｌａｃｔａｔｅｄｅｈｙｄｒｏｇｅｎａｓｅ，ＬＤＨ）活性的增高；细胞形态损害减轻，细胞数量增加。对２０μｍｏｌ·Ｌ１ＮＭＤＡ介导的神经元损伤改变无影响。提示Ｔｅｔ对某些Ｇｌｕ类神经毒剂引起的胎鼠大脑皮层神经元损伤有一定保护作用，其机制可能是抑制细胞膜上的Ｎａ＋通道开放，阻止膜去极化而影响电压依赖性Ｃａ２＋通道启动。对ＮＭＤＡ受体可能亦有一定作用。     

Modulation of cyclothiazide on the glutamate receptor/NO/cyclic GMP pathway in the hippocampus of freely-moving rats

通过活体微透析的方法研究了环噻嗪对大鼠海马谷氨酸受体／ＮＯ／ｃＧＭＰ通路的影响．局部灌流α－氨基羟甲基异唑丙酸（ＡＭＰＡ）受体脱敏阻断剂环噻嗪能引起细胞外ｃＧＭＰ水平的提高．环噻嗪的这种作用能够被ＮＯ合酶抑制剂Ｎ－硝基－Ｌ－精氨酸（Ｌ－ＮＮＡ）或选择性的可溶性鸟苷酸环化酶抑制剂１Ｈ－［１，２，４］二唑［４，３－ａ］喹喔啉－１－酮（ＯＤＱ）所阻断．在环噻嗪灌流过程中，大鼠呈现明显的痉挛前的行为变化湿狗样反应（ＷＤＳ）．由环噻嗪引起的ｃＧＭＰ增加和ＷＤＳ反应能够被Ｎ－甲基－Ｄ－天冬氨酸（ＮＭＤＡ）受体通道阻断剂甲基二苯并环庚烯亚胺（ＭＫ－８０１）或镁离子所阻断．ＡＭＰＡ受体拮抗剂６，７－二硝基喹喔啉－２，３－二酮（ＤＮＱＸ）和２，３－二羟基－６－硝基－７－氨磺酰基－苯并（ｆ）－喹喔啉（ＮＢＱＸ）可拮抗ＷＤＳ反应，但不能阻断环噻嗪引起的ｃＧＭＰ反应．这种结果表明：（１）在海马内与ＮＯ－ｃＧＭＰ通路有关的ＡＭＰＡ受体由于内源性谷氨酸的存在保持部分脱敏状态．（２）环噻嗪对ＡＭＰＡ受体脱敏的阻断作用可导致内源性ＮＭＤＡ受体的激活．     

(-),( )黄皮酰胺对鼠脑内 NMDA- 受体的影响

用［３Ｈ］ＭＫ８０１放射配体竟争结合法测定了（－），（＋）黄皮酰胺对大鼠前脑，海马，皮层等部位突触膜的ＮＭＤＡＲ的作用，以探讨其促智机制。同时用饱和实验分析ｐｏ给药１０ｄ后，小鼠脑内该受体密度的变化。结果表明：（－），（＋）黄皮酰胺对脑内各部位的ＮＭＤＡ受体均无特异亲和力。但（－）黄皮酰胺在体给药１０ｄ后能使小鼠脑内ＮＭＤＡ受体密度显著增高，并呈一定的量效关系。提示黄皮酰胺的药理作用有光学选择性；（－）黄皮酰胺增加脑内ＮＭＤＡ受体密度为其促智作用提供了重要理论依据。     

Modulation of intrathecal morphine-induced immunosuppression by microinjection of naloxone into periaqueductal gray

目的：观察中脑导水管周围灰质（ＰＡＧ）的阿片受体和促皮质素（ＡＣＴＨ）在鞘内吗啡影响免疫功能时的作用和变化．方法：用铕标的靶细胞检测大鼠脾脏自然杀伤（ＮＫ）细胞活性，ＭＴＴ和结晶紫蓝法分析ＣｏｎＡ诱生的脾细胞白细胞介素２（ＩＬ２），肿瘤坏死因子ＴＮＦβ活性和血清ＴＮＦα水平，放免法测定血清促皮质素（ＡＣＴＨ）水平．结果：鞘内注射吗啡抑制脾ＮＫ细胞活性，ＣｏｎＡ诱生的ＩＬ２和ＴＮＦβ活性，并伴有血清ＡＣＴＨ水平的上升．ＰＡＧ微量注射纳洛酮部分拮抗吗啡引起的ＮＫ细胞活性的抑制，伴ＡＣＴＨ水平恢复．结论：ＰＡＧ内的阿片受体参与鞘内注射吗啡引起的ＮＫ细胞活性的抑制，同时伴有ＨＰＡ轴的激活     

粉防己碱对体外培养大鼠脑皮层神经元损伤的保护作用

兴奋性氨基酸类神经毒剂与粉防己碱（Ｔｅｔ）共同作用于原代培养胎鼠大脑皮层神经元２４ｈ，发现１０－７，１０－６ｍｏｌ·Ｌ－１Ｔｅｔ明显降低谷氨酸（Ｇｌｕ），β－Ｎ－ｏｘａｌｙｌａｍｉｎｏ－Ｌ－ａｌａｎｉｎｅ（ＢＯＡＡ）和β－Ｎ－ｍｅｔｈｙｌａｍｉｎｏ－Ｌ－ａｌａｎｉｎｅ（ＢＭＡＡ）导致的培养液乳酸脱氢酶（ＬＤＨ）活性增高，细胞形态损害减轻，细胞数量增加。对ＮＭＤＡＡ介导的神经元损伤改变无影响。提示Ｔｅｔ对某些Ｇｌｕ类神经毒剂引起的胎鼠大脑皮层神经元损伤有一定保护作用，其机制可能是抑制细胞膜Ｎａ＋通道开放，阻止膜去极化而影响电压依赖性Ｃａ２＋通道启动。对ＮＭＤＡ受体可能亦有一定作用。     

三种阿片受体激动剂对突触传递的抑制作用

目的：比较不同浓度三种阿片受体激动剂对突触传递的抑制作用．方法：用细胞内记录和细胞外微电泳技术，于大鼠伏核脑片制备上记录神经元的兴奋性突触后电位和谷氨酸钠所致细胞膜去极化．结果：（ＤＡｌａ２，ＮＭｅＰｈｅ４，Ｇｌｙｏｌ）ｅｎｋｅｐｈａｌｉｎ（ＤＡＧＯ），（ＤＰｅｎ２，５）ｅｎｋｅｐｈａｌｉｎ（ＤＰＥＮ），ａｎｄｔｒａｎｓ（±）３，４ｄｉｃｈｌｏｒｏＮｍｅｔｈｙｌＮ［２（１ｐｙｒｏｌｉｄｉｎｙｌ）ｃｙｃｌｏｈｅｘｙｌ］ｂｅｎｚｅｎｅａｃｅｔａｍｉｄｅｍｅｔｈａｎｅｓｕｌｆｏｎａｔｅ（Ｕ５０４８８Ｈ）（１μｍｏｌ·Ｌ－１）均降低突触传递，尤以ＤＡＧＯ抑制作用最著．结论：三种阿片受体激动剂对突触传递的抑制作用均具有浓度依赖性，其作用机制与谷氨酸介导的突触传递受抑制有关．     

Differences in efficacy and Na(+) sensitivity between alpha(2B) and alpha(2D) adrenergic receptors: implications for R and R* states

The ability of Na(+) ions to modulate coupling of alpha(2B)- and alpha(2D)-adrenergic receptors to G proteins was investigated in isolated membranes from transfected PC12 and NIH 3T3 fibroblast cells. The initial rate of epinephrine-stimulated [(35)S]GTPgammaS binding was higher for alpha(2D)-receptors (the rat homolog of the alpha(2A)-receptor) in both cell types, whereas both alpha(2B)- and alpha(2D)-receptor responses were higher in PC12 cell membranes. Pertussis toxin completely blocked agonist-stimulated binding. Graded increases in Na(+) caused a progressive loss of basal GTP binding, indicative of its ability to reduce the level of the active R* state of the receptor. This inhibitory effect of Na(+) was more pronounced in PC12/alpha(2B) than PC12/alpha(2D) membranes. Epinephrine-stimulated GTP binding in PC12/alpha(2B) membranes was also more sensitive to Na(+) inhibition than in PC12/alpha(2D) membranes. In saturation [(35)S]GTPgammaS binding studies, the presence of Na(+) reduced apparent GTP affinity, and its effect was greater in PC12/ alpha(2B) membranes, consistent with a greater reduction in the active R* conformation of the receptor. The higher efficacy of epinephrine at alpha(2D) receptors and their lesser sensitivity to Na(+) are both indicative of a more stable R* state. Together these results suggest that differences in the modulatory influence of Na(+) within a family of G(i)-coupled receptors may reflect differences in the stability of the active R* state.     

槲皮素保护少突胶质前体细胞缺氧低糖损伤的体外研究

目的观察槲皮素(quercetin,QUE)对Na2S2O4联合低糖培养基诱导少突胶质前体细胞(oligodendrocyte precursor cells,OPCs)缺氧低糖损伤的保护作用。方法建立OPCs缺氧低糖损伤模型(OGD);在所建模型基础上,通过检测OPCs相对存活率、LDH漏出率及观察细胞形态学变化,研究槲皮素对OPCs缺氧低糖损伤的保护作用,并比较QUE共孵育和预孵育对OPCs的保护效果。结果随OPCs损伤时间延长细胞凋亡数量增多,而QUE能提高OPCs相对存活率、降低LDH漏出率并改善其形态学变化,其神经保护作用有剂量效应,且共孵育比预孵育的保护效果更明显。结论槲皮素可通过提高OPCs相对存活率及减少LDH漏出率对缺氧低糖损伤的OPCs发挥保护作用。     

沙棘总黄酮对改善神经细胞损伤的作用

目的探讨沙棘总黄酮(TFH)对神经细胞的保护作用及其机制。方法采用培养PC12细胞NaCN及缺糖引起的细胞损伤模型,以及Na2S2O4、KCl分别诱导PC12细胞缺氧损伤和Ca2+超载损伤模型,研究TFH对神经细胞损伤的保护作用。结果TFH对PC12细胞损伤有明显的保护作用。结论TFH具有保护神经细胞的作用。     

Pharmacological differences between muscarinic receptors coupled to phosphoinositide turnover and those coupled to adenylate cyclase inhibition

Pharmacological differences between muscarinic cholinergic receptors coupled to phosphoinositide turnover and those coupled to adenylate cyclase were studied. Stimulation of muscarinic receptors from SK-N-SH human neuroblastoma cells resulted in phosphoinositide hydrolysis, but not in inhibition of cAMP formation. As has been shown previously, stimulation of muscarinic receptors from NG108-15 neuroblastoma x glioma cells, on the other hand, resulted in inhibition of cAMP formation without any observable phosphoinositide hydrolysis. These two cell lines provide a useful model system in which to study differential coupling of muscarinic cholinergic receptors. Inhibition of [3H]N-methyl scopolamine [( 3H]NMS) binding and inhibition of carbachol-stimulated function by the antagonists pirenzepine, AF-DX 116, and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were studied in this system. Pirenzepine inhibited [3H]NMS binding in both cell lines with low affinity (Ki of 130 and 160 nM in NG108-15 and SK-N-SH cells respectively), indicating that both cell lines express M2 receptors. None of the three antagonists studied exhibited any clear selectivity for the receptors in one cell line over those of the other. In contrast, several agonists including acetylcholine, bethanechol and carbachol exhibited pronounced selectivity. These agonists inhibited [3H]NMS binding to membranes from SK-N-SH cells with IC50 values that were 17-, 3- and 38-fold higher, respectively, than those of NG108-15 cells. This selectivity was still observed when whole cells rather than membranes were studied. These findings indicate that pharmacological differences between receptors coupled to phosphoinositide turnover and those coupled to cAMP inhibition can be detected with certain agonists, but not with the antagonists pirenzepine, AF-DX 116 or 4-DAMP.     

Binding characteristics of the muscarinic receptor subtype of the NG108-15 cell line

Summary Kinetic, saturation and competition binding studies were conducted on the muscarinic receptor binding sites labeled by [³H]N-methylscopolamine ([³H]NMS) in membranes prepared from NG108-15 cells. The pharmacology of the NG108-15 cell muscarinic receptors was compared to that of the M₁ receptors of rat cortex labeled using [³H]pirenzepine, the M₂ and M₃ receptors of rat heart and submaxillary gland, respectively, labeled using [³H]NMS and the muscarinic receptors of the PC12 cell line also labeled using [³H]NMS.The rate of dissociation of [³H]NMS from the NG10815 cell muscarinic receptor was similar to that obtained at the M₃ receptor and at the muscarinic receptor of the P12 cells but was slower that the dissociation rate obtained at the M₂ cardiac muscarinic receptor. The Kd of [³H]NMS in the NG108-15 cells was significantly lower than that obtained at the M₂ and M₃ receptor but was similar to the Kd obtained in PC12 cells. In competition studies the affinity estimates for AF-DX 116, 4-DAMP, methoctramine and pirenzepine were not consistent with the presence of either an M₁, M₂ Or M₃ receptor but were identical to the affinity estimates obtained at the muscarinic receptor of the PC12 cell line.On the basis of these data we conclude that the muscarinic receptor present in the NG108-15 cells is different to the M₁, M₂ or M₃ subtypes already described but is similar to the muscarinic receptor present in the PC12 cell line. Since NG108-15 cells expresses mRNA for the m4 muscarinic receptor gene described by Bonner et al. (1987) we propose that the muscarinic receptors present in this cell line be denoted as M4 receptors.Send offprint requests to A. Michel at the above address     

黄芩素对过氧化氢致PC12细胞损伤的保护作用研究

目的:研究黄芩素(BAI)对过氧化氢(H2O2)损伤的神经细胞的保护作用。方法:用体外细胞培养法,预先孵育24h,以500μmol·L-1H2O2复制大鼠肾上腺髓质嗜铬瘤分化细胞株(PC12)不可逆氧化损伤模型,观察BAI对其影响,并在损伤时分为移除药物和不移除药物2种情况进行分析;通过MTT微量比色法测定细胞活度,并测定培养液中乳酸脱氢酶(LDH)的含量。结果:预先给予BAI作用24h后,在造模时不论是否移除药物,BAI对损伤细胞均有显著的保护作用,尤以不移除药物的效果更为显著。镜下观察,BAI组细胞形态出现一定程度上的边缘模糊,单个细胞体积变大,有少许细胞融合的状态发生;MTT法测定细胞活度升高,培养液中LDH含量降低,与模型组比较差异显著。结论:BAI能显著减轻PC12细胞损伤,其作用机制可能在氧化-抗氧化环节,与BAI能够减轻呼吸链阻断造成的自由基堆积、抗氧化和清除自由基的能力有关。     

二苯乙烯苷对脑缺血啮齿动物脑NMDA受体及细胞内钙离子的影响

目的　研究二苯乙烯苷对脑缺血再灌注啮齿动物脑组织N 甲基 D 天 (门 )冬氨酸 (NMDA)受体结合力的影响 ,同时观察二苯乙烯苷对神经细胞内钙离子浓度的影响 ,初步探讨二苯乙烯苷对缺血再灌注动物的脑保护作用的机制。方法　麻醉后夹闭沙土鼠和小鼠双侧颈总动脉 10min后 ,小心撤去动脉夹制作缺血再灌注动物模型 :(1)沙土鼠再灌注 7d后处死 ,取前脑进行NMDA受体结合力实验 ;(2 )小鼠再灌注 15min后迅速断头取脑 ,切成脑片 ,加入Fluo 3/AM负载后 ,在激光扫描共聚焦显微镜上进行光切 ,观察不同层面皮层和海马区细胞内游离钙的荧光强度。结果　模型组的NMDA受体结合力比假手术组明显升高 ,而二苯乙烯苷治疗组可以降低模型动物的NMDA受体结合力 ;脑片的光切结果显示 ,脑缺血再灌注模型组神经细胞内游离钙离子浓度比假手术组明显升高 ;与模型组相比 ,二苯乙烯苷治疗组的细胞内游离钙离子浓度降低。结论　二苯乙烯苷能够抑制啮齿动物脑缺血再灌注所导致的脑组织NMDA受体结合力及神经细胞内钙离子浓度的升高 ,减轻钙超载导致的脑组织损伤 ,可能具有脑保护作用     

Genetical responses of neuronal cells to synaptic transmission]

To understand the cellular mechanisms that lead to the generation of synaptic plasticity of neuronal cells, it is important to understand the intracellular responses of neuronal cells stimulated via synaptic transmission. The stimulation of mouse cerebellar granule cells via NMDA (N-methyl-D-aspartate) receptors caused an increase in deoxyribonucleic acid (DNA)-binding activities to TRE (12-O-tetradecanoylphorbol-13-acetate-responsive element) and CRE (adenosine 3',5'-cyclic monophosphate-responsive element) motifs, depending upon the presence of extracellular Ca2+. The increases in TRE- and CRE-binding activities were also detected with the stimulation of non-NMDA receptors by kainate. The increases in TRE- and CRE-binding activities were both mediated by the same DNA-binding complexes whose binding affinity to CRE was about three-fold higher than that to TRE. On the other hand, the stimulation of neuroblastoma x glioma hybrid NG108-15 via muscarinic acetylcholine receptors, alpha 2-adrenergic receptors and bradykinin receptors caused a rapid induction of zif/268. An additive effect on the induction of zif/268 was observed when the different stimuli were simultaneously added. Thus, it is extremely likely that signals transduced via synaptic transmission are transferred to the level of gene expression and evoke some events which might contribute to the generation of synaptic plasticity in neuronal cells. In addition, we have found that the direct injection of plasmid DNAs into mouse skeletal muscle with fructose, glucose or NaCl solution led to a long-term expression of the introduced gene in muscle cells.     

梓醇对缺糖缺氧诱导PC12细胞损伤的保护作用研究

目的探讨梓醇对抗缺糖缺氧诱导的PCI2细胞损伤作用。方法梓醇预处理PCI2细胞,加入含连二亚硫酸钠的无糖Earle’s液制备损伤模型,检测细胞存活率,乳酸脱氢酶、超氧化物歧化酶、谷胱甘肽过氧化物酶、丙二醛水平,检测细胞内游离Ca2＋浓度。结果与模型组比较,梓醇可剂量依赖性的提高细胞存活率（P〈0．01）,减少乳酸脱氢酶漏出率（P〈0．05或〈0．01）,提高超氧化物歧化酶、谷胱甘肽过氧化物酶活性,拮抗丙二醛水平升高,降低细胞内游离Ca2＋浓度（P〈0．05或〈0．01）。结论梓醇对缺糖缺氧诱导的PCI2细胞损伤具有保护作用,作用机制可能与其清除自由基和抑制钙超载有关。     

益母草碱对缺糖/缺氧损伤PC12细胞的保护作用

目的研究盐酸益母草碱对缺糖/缺氧损伤大鼠肾上腺嗜铬瘤细胞株(PC12)的保护作用。方法采用连二亚硫酸钠(Na2S2O4)+缺糖建立PC12细胞缺糖/缺氧损伤模型(oxygen and glucose deprivation,OGD),MTT法检测各组细胞存活率,流式细胞仪检测各组细胞内活性氧含量。结果与模型组相比,25μmol·L-1盐酸益母草碱显著提高细胞存活率,降低细胞内的活性氧含量。结论盐酸益母草碱对PC12细胞缺糖/缺氧损伤模型有明显的保护作用。