L-Arg-NO通路对大鼠延髓腹面加压区心血管活动的影响及与L-Glu通路的关系

目的：研究一氧化氮（ＮＯ）前体Ｌ－精氨酸（Ｌ－Ａｒｇ）单侧微量注入大鼠延髓腹面加压区（ＶＳＭｐ）对动脉血压（ＡＰ）、心率（ＨＲ）、肾灌流压（ＰＰｋ）的影响及与Ｌ－谷氨酸（Ｌ－Ｇｌｕ）升压作用的关系。方法 采用延髓腹外侧部微量注射法,以整体灌流肾为模型观察与ＮＯ有关药物对心血管活动的影响。结果 （１）ＶＳＭｐ内微量注入Ｌ－Ａｒｇ（４０～１００ｎｍｏｌ）,ＡＰ和ＨＲ呈剂量依赖性下降,与生理盐水注入后的变化相比较,差异均有显著性。如预先注入ＮＯ合酶抑制剂Ｌ－硝基－精氨酸甲酯（Ｌ－ＮＡＭＥ）或鸟苷酸环化酶抑制剂甲基蓝,Ｌ－Ａｒｇ的降压效应被衰减。（２）ＶＳＭｐ内微量注入Ｌ－Ａｒｇ（１００ｎｍｏｌ）,ＰＰｋ与ＡＰ同步下降,与基础值比较,差异有显著性。（３）ＶＳＭｐ内微量注入Ｌ－Ｇｌｕ（３５０ｎｍｏｌ）,ＡＰ上升。如预先注     

清开灵对脑组织谷氨酸含量及NMDA受体的影响

观察清开灵对脑组织中谷氨酸 (Glu)和γ 氨基丁酸 (GABA)含量及NMDA受体的数目、亲和力的影响。结果发现 ,清开灵对脑组织中Glu ,GABA含量及Glu GABA值无明显影响 (P >0 .0 5 ) ;能明显下调神经细胞膜上NMDA受体的数目 (P <0 .0 5 )。提示清开灵的脑保护机制与下调NMDA受体的数目有关     

Pharmacological characterization of type 1α metabotropic glutamate receptor-stimulated [35S]-GTPγS binding

1. The activation of G proteins by type 1α metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1α receptor has been studied by use of a [³⁵S]-guanosine 5′-[γ-thio]triphosphate ([³⁵S]-GTPγS) binding assay.
2. L-Glutamate increased the rate of [³⁵S]-GTPγS binding in a concentration-dependent manner (−logEC₅₀ (M) 5.25±0.07), with an optimal (62.4±1.6%) increase over basal binding being observed following 60 min incubation at 30°C with 70 pM [³⁵S]-GTPγS, 1 μM GDP, 10 mM MgCl₂, 100 mM NaCl and 100 μg membrane protein ml⁻¹. The L-glutamate (100 μM)-stimulated increase in [³⁵S]-GTPγS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 μM).
3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K_D) for [³⁵S]-GTPγS binding of 0.76±0.20 nM and a maximal number of GTPγS-liganded G proteins (B_max) of 361±30 fmol mg⁻¹ protein.
4. Metabotropic glutamate receptor agonists, quisqualate (−logEC₅₀ (M) 6.74±0.06), 1S,3R-ACPD (4.64±0.08) and (S)-3,5-dihydroxyphenylglycine (5.16±0.23) also increased [³⁵S]-GTPγS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists.
5. (+)-α-Methylcarboxyphenylglycine (300 μM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [³⁵S]-GTPγS binding, allowing an antagonist equilibrium dissociation constant (K_D) of 34.0±7.8 μM to be calculated for this mGluR antagonist.
6. Pretreatment of BHK-mGluR1α cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml⁻¹, 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [³⁵S]-GTPγS binding (by 66.0±0.9%), and an altered concentration-effect relationship for agonist-stimulated [³⁵S]-GTPγS binding by the residual PTX-insensitive G-protein population.
7. The modulation of [³⁵S]-GTPγS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.

     

Glutamatergic regulation of striatal peptide gene expression in rats

Summary The mRNA levels encoding enkephalin and substance P were measured in the rat striatum following cortical ablation, blockade of N-methyl-D-aspartate (NMDA) receptors or inhibition of glutamate release by lamotrigine. Unilateral ablation of the cerebral cortex resulted in a decrease of substance P mRNA levels particularly in the rostral dorsolateral and dorsomedial striatum ipsilateral to the lesion. There was a similar trend for a reduction in levels of enkephalin mRNA. Continuous, intrastriatal infusion of the competitive NMDA receptor antagonist, 3-((±)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, (CPP, 0.12 and 1.2g/day) decreased both enkephalin mRNA and substance P mRNA in dose-dependent manner evenly throughout the striatum adjacent to the infusion site. Following subchronic administration of the presumed glutamate release inhibitor, lamotrigine (5 and 20 mg/kg IP) there was no significant alterations in either enkephalin mRNA or substance P mRNA levels in the striatum. Both enkephalin mRNA and substance P mRNA expression in the rat striatum appear tonically stimulated through postsynaptic NMDA receptor mediated mechanisms. This contrasts with differential dopaminergic modulation of peptides in striatal output neurons.     

Excitatory amino acid release from astrocytes during energy failure by reversal of sodium-dependent uptake

Non-synaptic release may be the major route of excitatory amino acid (EAA) efflux during cerebral ischemia. Possible routes of non-synaptic release include non-specific anion channels, reversal of Na⁺-, CI^?-, or Ca²⁺-dependent uptake, and cell lysis. In the present study we employ a novel approach to show reversal of Na⁺-dependent uptake as a major route of EAA efflux from astrocyte cultures under conditions of energy failure. Primary rat astrocyte cultures were subjected to combined blockade of glycolytic and oxidative metabolism after incubation with [³H]-D-aspartate (D-ASP). Energy failure produced an efflux of D-ASP that was maximal by 90 minutes. The efflux over this period was reduced by more than 50% in cells that had been pre-loaded with PDC (L-transpyrrolidine-2,4-dicarboxylic acid) or TBHA (threo-β-hydroxyaspartic acid), compounds that are competitive inhibitors of Na⁺-dependent glutamate uptake. The effect of pre-loading with the inhibitors was concentration dependent. No effect was seen if the inhibitors were added after induction of energy failure, suggesting that the attenuation of D-ASP efflux resulted from binding of the inhibitors to an intracellular site. These results provide strong evidence that EAA efflux from astrocytes under conditions of energy failure occurs largely through reversal of Na⁺-dependent uptake. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

AMPA receptors shape Ca2+ responses in cortical oligodendrocyte progenitors and CG-4 cells

Intracellular calcium signals triggered by glutamate receptor activation were studied in primary cortical oligodendrocyte lineage cells and in the oligodendrocyte cell line CG-4. Glutamate, kainate, and AMPA (30-300 μM) increased [Ca ²⁺]_i in both types of cells at the stage of oligodendrocyte progenitors (O-2A; GD3⁺) or pro-oligodendroblasts (04⁺). The peak amplitude of Ca²⁺ responses to glutamate receptor agonists was significantly larger in cortical cells. In CG-4 and in cortical cells, the majority (more than 90%) of bipolar GD3⁺ or multipolar 04⁺ cells responded to kamate. In all the cells analyzed, kainate was more efficacious than AMPA and glutamate. The percentage of bipolar or multipolar cells responding to glutamate was significantly lower in the CG-4 cell line than in primary cultures. Cellular responses typical of metabotropic glutamate receptor activation were observed in 20% of the cortical O-2A progenitors, but in none of the CG-4 cells. The AMPA-selective antagonist GYKI 52466 blocked kainate-induced Ca²⁺ responses in cortical O-2A cells. The selective AMPA receptor modulator cyclothiazide (30 μM) greatly potentiated the effects of AMPA (30-100 μM) on [Ca ²⁺]_i in cortical and CG-4 cells. Our findings indicate that Ca²⁺ responses in cells of the oligodendrocyte lineage are primarily shaped by functional AMPA receptors. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

镁对脑缺血早期脑组织中谷氨酸及一氧化氮含量的影响

为研究镁在脑缺血早期对神经元保护作用的机制,用电凝法建立大鼠大脑中动脉缺血模型,在造模前给大鼠注射２０ｇ／Ｌ硫酸镁,观察镁对脑组织中谷氨酸（Ｇｌｕ）及一氧化氮（ＮＯ）含量的影响。结果显示,高浓度镁可明显降低脑缺血皮质中Ｇｌｕ、ＮＯ含量,在缺血组Ｇｌｕ、ＮＯ的峰值浓度分别为９．８７、４４．９７ｎｍｏｌ／ｇ,而在加镁组两者浓度分别为７．６８、３３．９２ｎｍｏｌ／ｇ。提示：镁对脑缺血早期神经元的保护作用与其降低脑组织中Ｇｌｕ及ＮＯ含量有关。     

Effects of ciliary neurotrophic factor on excitotoxicity and calcium-ionophore A23187-induced cell death in cultured embryonic striatal neurons

Ciliary neurotrophic factor (CNTF) has a protective effect on the striatum in animal models of Huntington's disease. However, the mechanism through which it exerts its effect is not clear. In this study, we show that there is a concentration-dependent direct protective effect of CNTF against N-methyl-D-aspartate-mediated excitotoxicity on striatal neurons in vitro. The CNTF has to be added more than half an hour before the insult for the effect to occur and its effect is eliminated by the presence of the protein synthesis inhibitor cycloheximide. This suggests that the protective mechanism of CNTF does not involve acute interference with the glutamate receptors, but probably requires gene/protein expression. We have also shown that the effect of CNTF against glutamate-induced excitotoxicity is dependent on the concentration of glutamate with a protective effect more evident at a low grade excitotoxic insult. Finally, we saw no effect of CNTF on calcium ionophore A23187-induced toxicity in striatal cultures, indicating that the growth factor does not promote survival by enhancing general defenses against raised intracellular levels of calcium.     

Reduced platelet glutamate uptake in Parkinson's disease

Summary. Defects in mitochondrial enzymes have been found not only in substantia nigra, but also in platelets from Parkinson's Disease (PD) patients, suggesting a systemic impairment of energy metabolism. Since platelets present an energy-dependent glutamate uptake similar to that described in central nervous system, glutamate uptake was determined in platelets from 34 PD patients and 21 age-related normal controls, as Na⁺-dependent [³H]glutamate influx; glutamate level was also analyzed by reverse-phase HPLC. A 50% reduction of glutamate uptake (p < 0.001) was observed in idiopathic PD patients, respect to controls and secondary parkinsonian syndromes. The decrease correlated with the severity of PD, measured by the UPDRS (r = −0.54; P < 0.05). Glutamate level was increased in platelets of PD patients, but was not correlated to the uptake decrease. Both phoenomena may be explained by the modifications of mitochondrial enzymes described in platelets, which could be used as a peripheral model of glutamatergic function in PD. Received October 7, 1998; accepted January 7, 1999     

1-Aminoindan-1,5-dicarboxylic acid and (S)-(+)-2-(3'-carboxybicyclo[1.1.1] pentyl)-glycine, two mGlu1 receptor-preferring antagonists, reduce neuronal death in in vitro and in vivo models of cerebral ischaemia

Metabotropic glutamate (mGlu) receptors have been implicated in a number of physiological and pathological responses to glutamate, but the exact role of group I mGlu receptors in causing postischaemic injury is not yet clear. In this study, we examined whether the recently-characterized and relatively selective mGlu1 receptor antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) could reduce neuronal death in vitro, following oxygen-glucose deprivation (OGD) in murine cortical cell and rat organotypic hippocampal cultures, and in vivo, after global ischaemia in gerbils. When present in the incubation medium during the OGD insult and the subsequent 24 h recovery period, AIDA and CBPG significantly reduced neuronal death in vitro. The extent of protection was similar to that observed with the nonselective mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine [(+)MCPG] and with typical ionotropic glutamate (iGlu) receptor antagonists. Neuroprotection was also observed when AIDA or CBPG were added only after the OGD insult was terminated. Neuronal injury was not attenuated by the inactive isomer (-)MCPG, but was significantly enhanced by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid [(1S,3R)-ACPD] and the group I mGlu receptor agonist 3,5-dihydroxyphenylglycine (3,5-DHPG). The antagonists (+)MCPG, AIDA and CBPG were also neuroprotective in vivo, because i. c.v. administration reduced CA1 pyramidal cell degeneration examined 7 days following transient carotid occlusion in gerbils. Our results point to a role of mGlu1 receptors in the pathological mechanisms responsible for postischaemic neuronal death and propose a new target for neuroprotection.