Ⅱ、Ⅲ组亲代谢型谷氨酸受体激动剂逆转1-甲基-4-苯基吡啶离子抑制星形胶质细胞摄取谷氨酸

目的 :探讨Ⅱ、Ⅲ组亲代谢型谷氨酸受体(metabotropicglutamatereceptors ,mGluRs)激动剂对 1 甲基 4 苯基吡啶离子 (1 methyl 4 phenylpyridinium ,MPP )抑制星形胶质细胞摄取谷氨酸 (Glu)的影响。方法 :应用同位素标记法测定星形胶质细胞对培养液中 [3 H] D ,L 谷氨酸的摄取 ,应用四氮唑 (MTT)比色法检测星形胶质细胞的活性。结果 :MPP 15 0、2 0 0 μmol·L-1可以明显抑制星形胶质细胞摄取Glu ,抑制率达 5 8.3%和 70 .1% ,但并不影响细胞活性 ;Ⅱ组mGluRs激动剂 (2S ,2’R ,3’R) 2 (2’ ,3’ dicar boxycyclopropyl)glycine (DCG IV ) 0 .1、 1、 10 ,10 0 μmol·L-1和Ⅲ组mGluRs激动剂L( ) 2 amino 4 phosphonobutyricacid (L AP4 ) 1、10、10 0 μmol·L-1可以逆转MPP 对星形胶质细胞摄取Glu的抑制作用。结论 :MPP 抑制星形胶质细胞摄取Glu可能与直接影响谷氨酸转运体 (GluTs)的功能有关 ,激活星形胶质细胞上的Ⅱ、Ⅲ组mGluRs可以通过促进GluTs摄取Glu、进而降低细胞外液的Glu浓度而发挥神经保护作用。     

线粒体KATP开放剂二氮嗪促进星形胶质细胞摄取谷氨酸

目的研究线粒体ATP敏感性钾通道(mitochondrial ATP-sensitive potassium channel, mitoKATP)开放剂二氮嗪(diazoxide)对星形胶质细胞摄取谷氨酸(glutamate)的影响.方法取新生大鼠脑星形胶质细胞作原代培养,用液体闪烁计数仪测定[3H]-D,L-谷氨酸的摄入量判断细胞的谷氨酸摄取功能.结果二氮嗪呈浓度依赖性地促进星形胶质细胞摄取谷氨酸,且能抑制1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium,MPP+)对星形胶质细胞摄取谷氨酸的损伤作用;浓度在100μmol·L-1以上时,二氮嗪可完全逆转MPP+对星形胶质细胞摄取谷氨酸的抑制作用;二氮嗪的上述作用可被选择性mito KATP 阻断剂5-羟基癸酸 (5-hydroxydecanoate,5-HD)拮抗.结论二氮嗪通过开放mitoKATP增强星形胶质细胞谷氨酸转运体(glutamate transporters, GluTs)的功能.     

埃他卡林增强星形胶质细胞摄取谷氨酸的作用

目的研究新型ATP敏感性钾通道开放剂(KATPCO)埃他卡林(iptakalim,Ipt)对星形胶质细胞摄取谷氨酸的影响。方法取新生大鼠脑星形胶质细胞作原代培养，将Ipt直接作用于细胞，观察它对正常和6-羟基多巴胺(6-OHDA)损伤模型细胞摄取谷氨酸的影响；分别加入阳性对照药吡那地尔和非特异性钾通道阻断药格列本脲分析Ipt的作用机制。根据［³H］标记的D,L-谷氨酸摄入量判断细胞谷氨酸摄取作用强度。结果Ipt和吡那地尔都能增强星形胶质细胞的谷氨酸摄取作用、逆转6-OHDA引起的谷氨酸摄取抑制效应，预先加入格列本脲后，上述作用均被取消。结论埃他卡林可能通过促进钾通道开放增强星形胶质细胞摄取谷氨酸的作用。     

羟乙基葛根素对脑星形胶质细胞氧化性损伤的保护作用

目的研究羟乙基葛根素对大鼠脑星形胶质细胞氧化性损伤的保护作用。方法取第4代培养的星形胶质细胞，以比色法测定细胞培养液中乳酸脱氢酶(LDH)活性，流式细胞术测定细胞凋亡率，［³H］-谷氨酸摄取法测定细胞摄取功能，比色法测定细胞内超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量。结果羟乙基葛根素可明显降低过氧化氢(H₂O₂)损伤所致的星形胶质细胞LDH的释放、降低细胞凋亡率、增加谷氨酸摄取率、使细胞内MDA含量减少而SOD活性增加。结论羟乙基葛根素可改善星形胶质细胞的神经营养功能、抑制星形胶质细胞凋亡，其机制可能与其抗氧化作用有关。     

白藜芦醇对培养大鼠脑皮层星形胶质细胞增殖及谷氨酸代谢的影响

目的 探讨不同浓度向藜芦醇(resveratrol,Res)对培养大鼠脑皮层星形胶质细胞增殖及谷氨酸代谢功能的影响.方法 原代提取并纯化培养脑皮层星形胶质细胞.采用5-溴脱氧尿核苷(BrdU)掺入法检测细胞增殖:通过同位素掺人法和ELISA法检测谷氨酸代谢指标:谷氨酸摄取量、总谷胱苷肽(GSH)含量和谷氨酰胺合成酶(Glutamine Synthetase,GS)含量与活性.结果 Res抑制细胞增殖并随着浓度升高和培养时间延长而抑制增强.Res浓度为25μM和50μM时,谷氨酸摄取量和GSH含量显著增加(P<0.05).而大于250μM时,两者却降低,但在此高浓度下,GS的细胞含量与活性却显著升高(P<0.05),而在低浓度时无任何变化.结论 Res可抑制星形胶质细胞增殖;对其谷氨酸代谢具有二元效应,即低浓度可促进谷氨酸代谢,高浓度则降低谷氨酸代谢.     

高糖对高钾刺激引起新生大鼠星形胶质细胞谷氨酸释放和再摄取的影响

目的探讨高糖对高钾刺激引起星形胶质细胞谷氨酸释放和再摄取的影响。方法胶质原纤维酸性蛋白免疫染色法鉴定培养的星形胶质细胞纯度。星形胶质细胞分别用含葡萄糖5,10,20和40 mmol.L-1的细胞外液培养1 h,然后加入氯化钾75 mmol.L-1,于0,1,2,3,4和5 min后分别取细胞培养液。用柱前邻苯二甲醛和N-异丁酰基-D-半胱氨酸衍生高效液相色谱法测定细胞培养液中谷氨酸含量。结果培养的星形胶质细胞纯度在95%以上。不同浓度葡萄糖的细胞外液孵育星形胶质细胞0,1和3 h细胞培养液中谷氨酸含量无明显变化。氯化钾75 mmol.L-1孵育1,2,3,4和5 min后,含葡萄糖10,20和40 mmol.L-1组细胞培养液中谷氨酸含量均比对应时间点含葡萄糖5 mmol.L-1组明显升高(P<0.01),葡萄糖5和10 mmol.L-1组谷氨酸含量在3 min达到峰值,葡萄糖20 mmol.L-1组4 min达峰值;葡萄糖40 mmol.L-1组3 min谷氨酸含量升高最明显,4 min短暂下降后5 min又明显升高。结论高糖能够促进星形胶质细胞谷氨酸的释放和抑制再摄取。     

依达拉奉对H2O2致星形胶质细胞损伤的保护作用

目的:观察依达拉奉(EDA)对H2O2损伤后的星形胶质细胞活力的影响及其细胞内谷胱甘肽(GSH)含量、诱导型一氧化氮合酶(iNOS)表达的影响.方法:应用MTT法检测星形胶质细胞活力;Tietze法检测细胞内GSH含量;Western-blot法检测细胞内iNOS的表达.结果:H2O2作用24 h后可呈浓度依赖性地抑制星形胶质活力;EDA可逆转H2O2导致的星形胶质细胞活力的下降,胞内GSH含量的降低以及iNOS表达的增加.结论:EDA可改善H2O2所致的星形胶质细胞的氧化损伤.     

MPP+和6-羟基多巴胺对C6胶质瘤细胞摄取谷氨酸的影响

目的：研究1-甲基4-苯基-吡啶离子(MPP^ )和6-羟基多巴胺(6-OHDA)对C6胶质瘤细胞摄取谷氨酸(Glu)的影响。方法：应用放射免疫法测定C6胶质瘤细胞对培养液中[^3H]-D，L-谷氨酸的摄取；应用四氮唑(MTY)比色法检测胶质瘤细胞的活性。结果：6-OHDA和MPP^ 均不同程度地抑制C6胶质瘤细胞摄取Glu；6-OHDA显抑制C6胶质瘤细胞活性，而MPP^ 却不影响细胞活性；蛋白激酶C(PKC)激动剂TPA显增强C6细胞对Glu的摄取，并拮抗：MPP^ 对C6细胞摄取Glu的抑制。结论：6-OHDA抑制C6胶质瘤细胞对Glu的摄取，可能与其抑制细胞活性有关，而MPP^ 的抑制作用可能与直接影响转运体的摄取功能有关，并可能由PKC途径介导。     

小脑星形胶质细胞功能的最新进展

许多研究有助于我们了解小脑星形胶质细胞,特别是伯格曼细胞(BG)。但是,直到最近没人知道它们的功能。在完整小脑内,荧光钙指示器结合星形胶质的标志物SR101多细胞药物填充剂可使星形胶质细胞成像。另外,荧光钙蛋白标记星形胶质细胞的选择靶向作用能够研究它们的功能,不混淆其他神经纤维网的效应与多细胞药物填充和SR101的着色。小脑皮质的2种星形胶质细胞:BG细胞和有缘膜的原浆性星形细胞,在亚细胞水平上钙的浓度到钙的波形,它们显示不同的信号肽指令。许多星形胶质细胞是由嘌呤能物质释放和嘌呤能物质介导所触发的。通过神经元活动和全局血流量的改变证实大量星形胶质细胞的钙浓度是增加。在这篇综述中,概述BG细胞和原浆性胶质细胞的功能。希望一些工具研究它们的钙离子动力学及功能。     

Ⅱ、Ⅲ组亲代型谷氨酸受体激动剂对6-羟基多巴诱导的PC12细胞毒性无保护作用

阐明Ⅱ、Ⅲ组亲代谢型谷氨酸受体(metabotropic glutamate receptors,mGluRs)激动剂对6—羟基多巴(6-hydroxydopamine,6-OHDA)诱导的PC12细胞毒性是否具有保护作用。方法：应用高效液相色谱仪联用荧光检测技术测定谷氨酸浓度，应用四甲基偶氮唑盐(MTT)比色法测定PC12细胞活性。结果：6—OHDA剂量依赖性地诱导PC12细胞释放谷氨酸、减低细胞活性，Ⅱ组mGluRs激动剂DCG—IV和Ⅲ组mGluRs激动剂L-AP4对6—0HDA诱导的PC12细胞释放谷氨酸和细胞活性的降低均无显影响。结论：6—OHDA对多巴胺神经元的损伤作用与其诱导谷氨酸过度释放及其继发的兴奋性神经毒性有关，Ⅱ、Ⅲ组亲代谢型谷氨酸受体激动剂对6—OHDA诱导的PC12细胞毒性无保护作用。     

Inhibitory effects of ginseng total saponins on hypoxia-induced dysfunction and injuries of cultured astrocytes

The effects of ginseng total saponins (GTS) on hypoxic damage of primary cultures of astrocytes were studied. Hypoxia was created by placing cultures in an air tight chamber that was flushed with 95% N(2)/5% CO(2) for 15 min before being sealed. Cultures showed evidence of significant cell injury after 24 h of hypoxia (increased lactate dehydrogenase (LDH) content in the culture medium, cell swelling and decreased glutamate uptake and protein content). Addition of GTS (0.1, 0.3 mg/ml) to the cultures during the exposure to hypoxic conditions produced dose-dependent inhibition of the LDH efflux. GTS (0.1, 0.3 mg/ml) also produced significant inhibition of the increased cell volume of astrocytes measured by [(3)H]O-methyl-D-glucose uptake under the hypoxic conditions. Decreased glutamate uptake and protein content was inhibited by GTS. These data suggest that GTS prevents astrocytic cell injury induced by severe hypoxiain vitro.     

Inhibitory effects of 1-methyl-4-phenylpyridinium on glutamate uptake into cultured C6 glioma cells

INTRODUCTION Glutamate is a predominant excitatory neurotrans-mitter in the mammalian central neuron system[1]. En-hanced glutamatergic activity is implicated in the pa-thology of neurological diseases, such as Parkinson dis-ease (PD)[1]. PDis characterized by a progressive andselective loss of dopaminergic neurons in substantianigra pars compacta (SNpc) which is the site of a glialactivation in PD. Regulation of glutamate level in thesynaptic cleft by glutamate transporters loca…     

Influence of SIB 1893, a selective mGluR5 receptor antagonist, on the anticonvulsant activity of conventional antiepileptic drugs in two models of experimental epilepsy

SIB 1893, a non-competitive antagonist of group I metabotropic glutamate receptor subtype 5, administered at doses ranging from 0.25 to 10 mg/kg, failed to influence pentetrazole-induced convulsions in mice. Moreover, SIB 1893 (10 and 20 mg/kg) did not affect the protective action of valproate, ethosuximide, phenobarbital and clonazepam in this test. Similarly, the mGluR5 antagonist did not modulate the antiseizure activity of carbamazepine, diphenylhydantoin and phenobarbital against maximal electroshock in mice. The combined treatment of SIB 1893 with conventional antiepileptic drugs did not lead to motor impairment. Long-term memory disturbances were observed only in the case of the combination of SIB 1893 with phenobarbital.     

异亚丙基莽草酸对H2O2损伤血管内皮细胞的保护作用

目的研究异亚丙基莽草酸(ISA)对血管内皮细胞损伤的保护作用。方法倒置显微镜下观察细胞形态学改变,MTT比色法检测细胞活性,用硝酸还原酶法测定细胞培养液中NO的含量。放射免疫法测定细胞培养液中前列环素代谢物6-酮-前列腺素F_1α(6-keto-PGF_1α)含量,比色法测定培养液及细胞裂解液的乳酸脱氢酶(LDH)活性并计算LDH的释放率。结果ISA可明显改善H₂O₂所致的内皮细胞变形、皱缩等损伤表现。1～100 μmol·L^-1 ISA可浓度依赖性的减轻H₂O₂引起的细胞活性降低和LDH释放,并促进H₂O₂诱导的血管内皮细胞释放NO和前列环素。结论异亚丙基莽草酸对H₂O₂损伤血管内皮细胞具有保护作用。     

Impaired glutamate clearance as a consequence of energy failure caused by MPP(+) in astrocytic cultures.

Astrocytes are the site of bioactivation of the parkinsonism-inducing agent 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) into its toxic 1-methyl-4-phenylpyridinium (MPP(+)) metabolite. The mechanism by which MPP(+) is capable of decreasing astrocytic glutamate uptake was evaluated in this study using primary cultures of astrocytes. Addition of glutamate to these cultures was followed by its efficient clearance from the extracellular space. However, when astrocytes were preincubated with MPP(+), glutamate clearance was significantly impaired. This effect was concentration-dependent, became more pronounced by prolonging the incubation in the presence of MPP(+) and occurred at a time when cell membrane integrity was still preserved. No evidence was found that reactive oxygen species contributed to MPP(+)-induced decrease in glutamate clearance. Indeed, neither the spin trapping agent alpha-phenyl-tert-butyl nitrone, the lazaroid antioxidant U-74389G, nor the disulfide-reducing agent dithiothreitol was capable of restoring glutamate net uptake. The effect of MPP(+) on glutamate clearance: (i) was accompanied by a decrease in cellular ATP; (ii) could be enhanced by withdrawing glucose from the incubation medium or by inhibiting glycolysis with 2-deoxyglucose, and (iii) could be reproduced using the mitochondrial complex I inhibitor rotenone. Taken together, these results indicate that, by acting as a mitochondrial poison, MPP(+) impairs energy metabolism of astrocytes and significantly reduces their ability to maintain low levels of extracellular glutamate.     

Human Herpesvirus 6 (HHV-6) Induces Dysregulation of Glutamate Uptake and Transporter Expression in Astrocytes

Human herpesvirus 6 (HHV-6) infects and establishes latency in the central nervous system (CNS). Reactivation of latent HHV-6 has been associated with neurologic diseases including epilepsy and multiple sclerosis (MS). In vivo, HHV-6 has been localized to astrocytes and can infect human astrocytes in vitro, suggesting that this virus may have a tropism for glial cells and may affect glial cell function. An essential role of astrocytes in the CNS is active maintenance of the excitatory neurotransmitter glutamate. Dysregulation of glutamate has been implicated as a potential mechanism of disease in both epilepsy and MS. Both disorders have demonstrated elevated glutamate in CSF and may be associated with dysregulation of glutamate signaling, uptake, and metabolism. This study demonstrates dysregulation of glutamate uptake in human astrocytes infected with both variants of HHV-6, A and B, with differential effects of HHV-6 in acute and persistently infected cells. Whereas astrocytes acutely infected with HHV-6 demonstrated increased glutamate uptake, cells persistently infected with HHV-6A and HHV-6B demonstrated impaired glutamate uptake. Functional dysregulation of glutamate uptake was associated with early increases in mRNA and protein expression of the glial glutamate transporter EAAT-2 followed by a sustained decrease in mRNA expression in astrocytes infected with both HHV-6A and HHV-6B. Dysregulated glutamate uptake and transporter expression suggests a mechanism for dysregulation of glutamate levels in vivo and a potential mechanism for virus-associated neurologic disease. This work was supported by the National Institutes of Health/National Institute of Neurological Disorders and Stroke. JF was supported by the MS Society of Canada.