代谢型谷氨酸受体在脑缺血耐受中的研究进展

代谢型谷氨酸受体（metabotropicglutamatereceptors。mGluRs）是脑内广泛存在的受体。通过与G蛋白偶联调节膜上离子通道、第二信使生成、参与诸多的生理及病理过程。目前为止已克隆出8种mGhRs，分别为mGluRl，mGluR2。mGluR3，mGluR4，mGluR5，mGluR6。mGluR7。mGluR8。其中．多种mGluRs又有剪接变异。根据氨基酸的序列相似性，信号转导机制及激动剂的选择性不同。这些mGluRs又分为G-Ⅰ，GⅡ。GⅢ三组：G-Ⅰ组（mGluRl，mGluR5）主要通过活化磷脂酶C（PLC），将膜内的磷酸肌醇（P1）水解；G-Ⅱ组（mGhR2。mGluR3）和GIⅡ组（mGluR4,mGluR6,mGluR7,mGluR8）受体激活时，抑制由毛候萜（forskolin）诱发的胞内cAMP累积。mGluRs可作为突触后成分或者突触前自身受体而调节中枢神经系统的突触传递。不同的mGluRs的转导机制不同。因此mGluRs对谷氨酸介导的突触传递可以是增强作用。可以表现为抑制效应，并参与调节膜上酶及离子受体的功能及参与海马突触可塑性，影响海马突触传递效能，影响神经系统的发育、突触形成。现将这些受体在脑缺血缺氧及脑耐受形成中的作用做一综述。     

缺血再灌注大鼠脑内代谢型谷氨酸受体1和代谢型谷氨酸受体5的mRNA水平变化

目的:利用缺血再灌注大鼠模型研究代谢型谷氨酸受体1(mGluRl)和代谢型谷氨酸受体5(mGluR5)的mRNA表达水平在缺血再灌注后的变化,探讨其在缺血后脑损伤中的作用。方法:采用线栓法制备缺血再灌注大鼠模型,运用RT-PCR技术半定量分析脑组织内mGluRl和mGluR5的mRNA表达水平。结果:以β-Actin为参照,实验组缺血2h再灌注24h后缺血侧mGluRl和mGluR5的mRNA相对值较假手术对照组均显著上升(P<0.05);非缺血侧两者的mRNA相对值与假手术对照组相比,无显著差异(P/0.05)。结论:代谢型谷氨酸受体l和代谢型谷氨酸受体都参与了缺血再灌注后的病理过程。     

代谢型谷氨酸受体1亚型选择性拮抗剂LY367385对抗大鼠缺血性脑水肿

目的研究代谢型谷氨酸受体1亚型（mGluR1）选择性拮抗剂LY367385对大鼠缺血性脑水肿的影响.方法Wistar雄性大鼠（280～320g）线栓法复制大脑中动脉闭塞（MCAO）脑缺血模型.动物随机分为生理盐水（NS）对照组、LY367385给药组及MK-801给药组,于MCAO后1min,侧脑室内注射NS或LY367385（500nmol）5μl,或腹腔注射MK-801（1mg/kg）.各组动物分别于MCAO 6、24h进行神经病学评分、脑含水量测定及脑梗死面积测定.结果LY367385明显改善大鼠脑缺血引起的神经症状,而MK-801在MCAO 6h增加神经病学评分.LY367385降低大鼠MCAO引起的脑含水量增加,MK-801无明显作用.LY367385及MK-801均降低脑梗死面积百分率,且LY367385作用优于MK-801.结论LY367385能对抗大鼠脑缺血性脑水肿,作用明显优于MK-801.     

代谢型谷氨酸受体5对创伤性神经元损伤保护作用研究

目的研究代谢型谷氨酸受体5（mGluR5）的特异性激动剂对创伤性脑损伤后神经元的保护作用,并初步探讨其作用机制。方法原代培养2W大鼠皮层神经元细胞,采用神经元机械性损伤模型,加入两种mGluR5特异性激动剂2-氯-5羟苯基甘氨酸（CHPG）和3-氰．氮苯甲酰胺（CDPPB）,通过乳酸脱氢酶（LDH）释放率、caspase-3活性检测,研究mGluR5激动剂对神经元损伤可能具有的保护作用;通过western—blot检测细胞外信号调节激酶（ERK）、c—Jun氨基末端激酶（JNK）、p38表达变化并研究这种保护作用的产生机制。结果损伤后LDH释放率和easpase-3活性显著增加,CHPG和CDPPB明显抑制了LDH的释放率和easpase-3的活性,并呈剂量依赖性;Westernblot结果提示,CHPG和CDPPB显著增加了ERK的磷酸化水平,而对JNK、p38无影响。结论mGluR5的激动剂CHPG和CDPPB对机械性神经元损伤具有保护作用,这种保护作用可能是通过激活ERK通路实现。     

腺苷2A受体、亲代谢谷氨酸受体对异动症可塑性改变的影响

腺苷2A受体(A2AR)大量分布于脑基底节区,和多巴胺D2受体(D2R)及亲代谢型谷氨酸受体(mGluR5)形成异聚体,共同调节纹状体突触前后功能。研究显示左旋多巴诱发异动症PD动物模型纹状体A2AR、mGluR5表达增加,而两受体拮抗剂应用可改善异动症PD动物模型异常行为,从而提出A2AR和mGlu5参与了异动症突触可塑性的改变。     

促代谢型谷氨酸受体激活的分子开关调节LTP的诱导

促代谢型谷氨酸受体激活的分子开关调节ＬＴＰ的诱导已知在海马ＣＡＩ区突触激活ＮＭＤＡ受体与促代谢型谷氨酸受体（ｍＧｌｕＲ）是诱导ＬＴＰ所必不可少的。英国的Ｌｏｌｌｉｎｇｒｉｄｅｙ实验室最近报道：在ＬＴＰ的形成过程中ＮＭＤＡ受体与ｍＧ１ｕＲ的基本作用是不...     

组代谢型谷氨酸受体通过抑制凋亡参与脑缺血耐受的诱导

目的观察组代谢型谷氨酸受体阻断剂α-methyl-(4-tetrazolyl-phenyl)glycine(MTPG)对脑缺血预处理(cerebralischemicpreconditioning,CIP)抗损伤性缺血所致海马神经元凋亡作用的影响。方法24只SD大鼠采用四血管闭塞法制造大鼠全脑缺血模型。采用Tunel法检测海马CA1区锥体细胞凋亡情况。结果假手术组几乎无阳性细胞表达;损伤性缺血组可见明显的阳性细胞表达和核固缩,阳性细胞数、总面积、平均光密度明显增加(P<0.05);CIP 损伤性缺血组阳性染色细胞数、总面积、平均光密度较损伤性缺血组明显降低(P<0.05);MTPG CIP 损伤性缺血组阳性染色细胞数、总面积、平均光密度与CIP 损伤性缺血组相比明显增加(P<0.05)。结论mGluR2/3通过抑制凋亡,参与BIT的诱导。     

腺苷A2A受体拮抗剂对左旋多巴诱发异动症大鼠行为、纹状体A2A受体和代谢型谷氨酸受体5亚型蛋白表达的影响

目的 评价腺苷A2A受体拮抗剂(CSC)对左旋多巴(L-DOPA)诱发异动症大鼠行为、纹状体A2A受体和代谢型谷氨酸受体5亚型(mGluR5)蛋白表达的影响.方法 6-羟多巴胺(6-OHDA)立体定向损毁大鼠右内侧前脑束,建立单侧损毁帕金森病(PD)大鼠模型.采用随机数字表法将40只成功PD大鼠随机分为4组(每组10只):生理盐水组;L-DOPA 25 mg/kg+苄丝肼6.25 mg/kg组;CSC 2.5 mg/kg组;L-DOPA 25 mg/kg+苄丝肼6.25 mg/kg联合CSC 2.5 mg/kg组.给予大鼠每日2次腹腔注射,持续21 d.在治疗第2、9、11、18、21天观察大鼠行为学变化,Western blot检测纹状体区腺苷A2A受体和mGluR5的蛋白表达水平.结果 L-DOPA联合CSC组PD大鼠损毁对侧前肢跨步数显著增加,与治疗前比较差异有统计学意义,与L-DOPA组相比,前肢功能改善程度不随时间延长而减弱.单独CSC组治疗后对侧前肢跨步数明显增加,与治疗前比较差异有统计学意义,有疗效逐渐增加至稳定趋势.L-DOPA联合CSC组[(11±5)分]部分口颌及肢体异常不自主运动评分较L-DOPA组[(17±4)分]显著减少,差异有统计学意义(t=2.44,P＜0.05).L-DOPA联合CSC治疗逆转了L-DOPA诱导的对侧旋转反应时间缩短和腺苷A2A受体、mGluR5蛋白表达的上调,差异均有统计学意义.结论 腺苷A2A受体与mGluR5均参与了L-DOPA诱发的异动症的发生发展,A2A受体拮抗剂能够改善PD运动症状,增强L-DOPA的抗PD效应且部分减轻异常不自主运动,对L-DOPA诱发的异动症的治疗有着较好的应用前景.

Abstract：

Objective To study the behavioural changes and biological effects of selective adenosine A2A receptor antagonist (CSC) in a rat model of levodopa(L-DOPA) -induced dyskinesia (LID).Methods The hemi-parkinsonian rat model was produced by stereotaxically injecting 6-OHDA to the right medial forebrain bundle. Rats were randomly divided into 4 treatment groups with a random number generating program to receive intraperitoneal injections twice daily for 21 days (n = 10): saline, L-DOPA at 25 mg/kg with benserazide at 6. 25 mg/kg, CSC at 2. 5 mg/kg alone and CSC at 2.5 mg/kg with L-DOPA at 25 mg/kg plus benserazide at 6. 25 mg/kg. Forepaw adjusting steps, abnormal involuntary movements (AIM) and rotational response duration were observed on 2, 9, 11,18 and 21 d. After sacrifice, the expression of adenosine A2A R and mGluR5 was observed by Western blot. Results Co-administration of LDOPA with CSC significantly increased the forelimb adjusting steps of parkinsonian rats during 21 days of treatment when compared to L-DOPA alone. CSC treatment alone increased the forelimb adjusting steps significantly. Co-administration of L-DOPA with CSC ( ( 11 ± 5 ) score) significantly decreased the AIM scores of limb and orolingual muscles when compared to L-DOPA alone (( 17 ± 4) score; t = 2. 44, P ＜0. 05). The subchronic L-DOPA treatment upregulated the striatal expression of adenosine A2A R and mGluR5. However, co-administration of L-DOPA with CSC reversed the shortening of the rotational motor response duration induced by L-DOPA administration during the period of the treatment and attenuated the LDOPA-induced upregulation of adenosine A2A R and mGluR5 expressions. Conclusions CSC improves motor function in a hemi-parkinson rat model, potentiates the antiparkinsonian effects with L-DOPA and partly attenuates LID. Co-administration of L-DOPA with CSC reverses the L-DOPA-induced upregulated expression of A2A R and mGluR5, indicating the involvement of both A2A R and mGluR5 in the onset and progression of LID. Adenosine A2AR antagonists may be promising drugs for treatment of LID.     

Ⅱ组代谢型谷氨酸受体通过抑制凋亡参与脑缺血耐受的诱导

目的观察Ⅱ组代谢型谷氨酸受体阻断剂α-methyl-(4-tetrazolyl-phenyl) glycine(MTPG)对脑缺血预处理(cerebral ischemic preconditioning,CIP)抗损伤性缺血所致海马神经元凋亡作用的影响.方法 24只SD大鼠采用四血管闭塞法制造大鼠全脑缺血模型.采用Tunel法检测海马CA1区锥体细胞凋亡情况.结果假手术组几乎无阳性细胞表达;损伤性缺血组可见明显的阳性细胞表达和核固缩,阳性细胞数、总面积、平均光密度明显增加(P<0.05);CIP+损伤性缺血组阳性染色细胞数、总面积、平均光密度较损伤性缺血组明显降低(P<0.05);MTPG+CIP+损伤性缺血组阳性染色细胞数、总面积、平均光密度与CIP+损伤性缺血组相比明显增加(P<0.05).结论 mGluR2/3通过抑制凋亡,参与BIT的诱导.     

Divalent cations modulate the activity of metabotropic glutamate receptors

Metabotropic glutamate receptors (mGluRs) and calcium receptors (CaR) are closely related G protein-coupled receptors (GPCRs). The similar structural and functional properties of mGluRs and CaRs include conserved amino acid residues involved in glutamate binding in mGluRs and Ca2+ binding in the CaR. Furthermore, recent findings have demonstrated that mGluRs can respond to high extracellular Ca2+ (Ca2+(o)) whereas CaR activity is potentiated by L-amino acids. We show that both mGluR1 and mGluR2 are activated by Ca2+(o) in the absence of glutamate in the extracellular media. This activation by Ca2+(o) is antagonized by Mg2+(o). Unlike the CaR, in which the intracellular carboxyl tail has been reported to be involved in Ca2+(o)-dependent activity, the carboxyl tail of mGluRs does not seem to play a role in mediating Ca2+(o) actions. On the other hand, we find that preservation of disulfide bonds in the N-terminal extracellular domain of mGluRs is essential for stimulation by Ca2+(o) as well as glutamate. Because the mGluR1 EC50 for Ca2+(o) is within the physiologic range of Ca2+ in the synaptic cleft, mGluR function is likely regulated by changes in divalent cations caused by synaptic activity under normal or pathologic conditions.     

Immunochemical localization of the metabotropic glutamate receptors in the rat heart

The localization of the glutamate receptor outside of the central nervous system is becoming more evident. These receptors have been implicated in brain function and pathology. It can also be envisioned that they play a vital role in the physiology of other organs and systems. We recently reported the presence of ionotropic glutamate receptors in the rat heart. These were distributed differentially in specific cardiac structures, including nerve terminals, ganglion cells, and the conducting system. In this study, we investigated the presence and localization of the metabotropic glutamate receptors (mGluRs) in the rat heart by immunohistochemistry. The experimental data show that the mGluR 1alpha, mGLuR 2/3, and mGluR 5 are present in the rat heart. Their preferential localization includes nerve terminals, ganglion cells, and elements of the conducting system. The mGluR 5 was the only receptor located in the intercalated disks of the cardiac muscle and in the endothelial lining of the blood vessels. This preferential localization to the different components of the conducting system and cardiac neural structures suggest that they play a role in the physiology of the heart.     

Reversal of glutamate excitotoxicity by activation of PKC-associated metabotropic glutamate receptors in cerebellar granule cells relies on NR2C subunit expression.

Stimulation of metabotropic glutamate receptors (mGluRs) belonging to group I has been found to reduce N-methyl-D-aspartate (NMDA) receptor function in terms of both intracellular calcium concentration ([Ca2+]i) rise and neurotoxicity in cultured cerebellar granule cells. In the present study, we investigated whether the mGluR-elicited modulation of glutamate responses might rely on the heteromeric composition of NMDA receptor channel. NMDA receptors consist of two distinct groups of subunits: NR1, that is ubiquitously in the receptor complexes; and NR2A-D, that differentiate and potentiate NMDA receptor responses by assembling with NR1. Among NR2 subunits, only NR2A and NR2C mRNAs and relative proteins are detected in cerebellar granule cells at 10 days in vitro. To dissect the involvement of the two different subunits in making the NMDA receptor channel sensitive to modulation by group I mGluR agonists, expression of the NR2C subunit was prevented by treating the cells with specific antisense oligodeoxynucleotide (ODN). The capability of the mGluR agonists, trans-1-amino-cyclopentane-1,3-dicarboxylic acid (tACPD, 100 microM) or 3 hydroxyphenylglycine (3HPG, 100 microM), and the protein kinase C (PKC) activator, 4beta-phorbol-12,13-dibutyrate (PDBu, 1 microM), to inhibit the function of resultant NMDA receptors was then evaluated. We found that depletion of the NR2C subunit abolished the inhibitory effect of group I mGluR stimulation on glutamate-induced [Ca2+]i rise and neurotoxicity. The antisense ODN treatment also prevented the inhibitory effect of PDBu on glutamate responses. Conversely, in NR2C-lacking neurons, both group I mGluRs and PKC stimulation enhanced NMDA receptor-mediated effects. The present findings indicate that the capability of PKC-associated mGluRs to modulate native NMDA receptor function relies on the heteromeric configuration of the receptor-channel complex. Particularly, expression of the NR2C subunit is required to make the NMDA receptor sensitive to inhibitory modulation by mGluRs or PKC activation.     

Colocalization of metabotropic glutamate receptors in rat dorsal root ganglion cells

Glutamate is the main excitatory transmitter in both central and peripheral nervous systems. Discovery of metabotropic glutamate receptors (mGluRs) made it clear that glutamate can have excitatory or inhibitory effects on neuronal function, with group I mGluRs enhancing cell excitability but group II and III mGluRs decreasing excitability. The present study investigated the colocalization of mGluR subtypes representing groups I, II, or III in rat L5 dorsal root ganglion (DRG) cells. The analyses show that group III has the highest expression, with 75.0% of DRG cells expressing mGluR8, followed by group II, with 51.6% expressing mGluR2/3, followed by group I, with only 6.8% expressing mGluR1alpha. mGluR8 is expressed by small, medium, and large diameter cells. In contrast, mGluR1alpha and mGluR2/3 are expressed by mainly small and medium cells. Approximately half of cells expressing group I mGluR1alpha also express either group II mGluR2/3 or group III mGluR8. These mGluR1alpha double-labeled populations are not likely to overlap since >1.0% of mGluR1alpha are triple-labeled. As expected from the high percentage of single-labeled mGluR2/3 and mGluR8 cells, there is a considerable population of double-labeled cells with approximately 30% of each population expressing both receptors. Due to the fact that the number of mGluR1alpha-expressing cells in the DRG is low, the percentage of triple-labeled cells is also low ( approximately 1-2%). The prevalence of groups II and III indicate that glutamate could have a substantial inhibitory effect of primary afferent function, reducing and/or fine-tuning sensory input before transmission to the spinal cord. These anatomical data highlight the potential inhibitory role glutamate may play in peripheral sensory transmission.     

Activation by N-acetyl-L-aspartate of acutely dissociated hippocampal neurons in rats via metabotropic glutamate receptors

PURPOSE: We previously reported that an increase in the N-acetyl-L-aspartate (NAA) level due to the lack of aspartoacylase gene was found in the brain of the tremor rat (tm/tm), which is a mutant with a causative gene named tm that shows epileptic seizures. Therefore, NAA is suggested to be one of the factors involved in the induction of epileptic seizures. Patch-clamp studies were performed to determine whether NAA produces an excitatory effect on acutely dissociated rat hippocampal neurons. METHODS: Acutely dissociated hippocampal neurons were prepared from normal Wistar rats aged 3-4 weeks. NAA-induced currents were investigated by using the whole-cell voltage-clamp recording technique. RESULTS: Application of NAA at concentrations of 100 nM to 1 mM through a U-tube for 2 s produced an inward current in a concentration-dependent manner at a holding potential of -60 mV. When the current-voltage relation was examined, the reversal potential of the NAA-induced current was found to be approximately 0 mV. The NAA-induced current was inhibited by bath application of the metabotropic glutamate receptor (mGluR) antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG) and by intracellular application of guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS), a nonhydrolyzable GDP analogue. However, the NAA-induced current remained unaffected by glutamic acid diethyl ester, a non-N-methyl-D-aspartate (NMDA)-subtype ionotropic glutamate receptor antagonist, or the voltage-dependent ion channel blockers tetrodotoxin, CdCl2, and tetraethylammonium-chloride. Conversely, the mGluR agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) also induced an inward current, with a reversal potential of 0 mV. The ACPD-induced current also was inhibited by MCPG. CONCLUSIONS: These results suggest that NAA acts on the G protein-coupled mGluRs to induce an inward current that results in excitation of the neurons, thereby contributing to the occurrence of epileptic seizures.     

Activation of metabotropic glutamate receptors by repetitive stimulation in auditory cortex

To determine whether metabotropic glutamate receptors (mGluRs) contribute to the responses of neurons to repetitive stimulation in the rat auditory cortex in vitro, five stimulus pulses were delivered at 2-100 Hz which elicited five depolarizing synaptic responses, f-EPSPs: f-EPSPs(1-5). Stimulus pulses 2-5 delivered at low frequencies (2-10 Hz) elicited f-EPSPs(2-5) that were about 15% smaller than the response elicited by the first pulse (f-EPSP(1)). In the presence of the nonspecific mGluR agonist, ACPD, the amplitude of all f-EPSPs was 40% smaller than predrug responses. APV, CNQX, or bicuculline (antagonists of NMDA-, AMPA/kainate-, and GABA(A)-receptors, respectively) did not change this effect of ACPD. The mGluR antagonist, MCPG, had no effect on f-EPSPs but did reduce the effect of ACPD. High-frequency stimulation (50-100 Hz) elicited f-EPSPs that were smaller with each successive stimulus. In ACPD, f-EPSP(1) was 40% smaller than predrug, but f-EPSPs(3-5) were not changed compared to pre-ACPD f-EPSPs(3-5), indicating that ACPD occludes the effect of repetitive stimulation. MCPG increased f-EPSP(5) by 15%, indicating that a portion of the reduction of f-EPSPs during high-frequency stimulation is mediated by mGluRs. MCPG also partially blocked the effect of ACPD. In CNQX, ACPD only decreased EPSPs, but APV or bicuculline did not change the effect of ACPD. These results suggest that the successive reduction of f-EPSPs during a high-frequency train is partially a result of mGluR activation.     

Physiological role of group III metabotropic glutamate receptors in visually responsive neurons of the rat superficial superior colliculus

There is evidence from immunohistochemical and in situ hybridization studies for the presence of Group I, II and III metabotropic glutamate receptors (mGluRs) in the rat superficial superior colliculus (SSC). The purpose of this study was to investigate if manipulation of Group III mGluRs affects visual responses in the SSC. Drugs were applied by iontophoresis and single neuron activity was recorded extracellularly. L-AP4 (Group III agonist) resulted in a reduction of visual responses in most neurons, but also a potentiation in others. The effect of L-AP4 is drug- and stereospecific in that application of D-AP4 did not significantly affect visual responses. L-AP4 application also resulted in a potentiation of the response to iontophoretically applied NMDA. The effects of MPPG and CPPG (Group III antagonists) were compared with the effect of L-AP4 in the same neuron and were found to produce the opposite effect to L-AP4. Furthermore, the effect of L-AP4 could be blocked by coapplication of MPPG or CPPG. Presynaptic depression of glutamate release is a possible mechanism by which L-AP4 could reduce visual responses in the SSC whereas the potentiation of visual responses by L-AP4 could be due to a reduction of GABAergic inhibition. The finding that MPPG and CPPG, as well as antagonizing the L-AP4 effect, have a direct effect on visual responses suggests that Group III mGluRs are activated by endogenous transmitter released during visual stimulation.