Modulation of cell surface GABA B receptors by desensitization,trafficking and regulated degradation

Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system,and mediates its effects via two classes of receptors:the GABA A and GABA B receptors.GABA A receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission.GABA B receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission.The extent of inhibitory neurotransmission is determined by a variety of factors,such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g.,phosphorylation),as well as by the number of receptors present in the plasma membrane available for signal transduction.The level of GABA B receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation.In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA B receptors in the plasma membrane,and thereby signaling strength.

Electroacupuncture (EA) Speeds Up the Regulation of Hypothalamic Pituitary Adrenal Axis Dysfunction in Acute Surgical Trauma Rats: Mediated by Hypothalamic Gamma-Aminobutyric Acid (GABA)_AReceptors

Hypothalamic Corticotropin-releasing factor (CRF) directly activates the hypothalamic pituitary adrenal axis (HPA axis) during the surgical trauma induced stress response. Electroacupuncture (EA) has been demonstrated to have stress relieving effects in breast surgery, colorectal surgery, prostatectomy and craniotomy. This study was aimed to investigate the hypothesis that EA could regulate hypothalamic CRF in surgical trauma rats. In experiment one, Sprague-Dawley (SD) male rats were divided into intact, model (10% partial hepatectomy), sham EA and EA group. Rats from the Sham EA and EA group were stimulated at ST36-Zusanli and SP6-Sanyiniiao acupoints twice, 24 hours before the surgery and immediately after the surgery. Expressions of hypothalamic CRF and CRFR, GABA receptors, glutamate decarboxylase (GAD), serum adrenocorticotropic hormone (ACTH) and Corticosterone (CORT) were observed at 2, 4, 8 and 24 h after the surgery by radioimmunoassay (RIA), western blot, real-time PCR and immunohistochemistry. In the experiment two, SD male rats were divided into the intact, model, model + vehicle, model + L-838,417 EA and EA + L838,417 group. It was found that hypothalamus CRF, serum ACTH and CORT levels were increased in model group compared with the intact group, and those in the EA group decreased in comparison with the model group. Compared with the model group, hypothalamus-aminobutyric acid (GABA) receptor Aα3 mRNA and protein expressions of the EA group raised strikingly. In conclusion, EA alleviated surgical stress response by improving the GABA synthesis in hypothalamus, thus enhancing GABA receptors’ inhibitory regulation of the HPA axis dysfunction in rats with acute surgical trauma.

Modulation of firing activity by endogenous GABA_A receptors in the globus pallidus of MPTP-treated parkinsonian mice

The globus pallidus in rodents,equivalent to the external segment of the globus pallidus in primates,plays an important role in movement regulation.Previous studies have shown abundant γ-aminobutyric acid（GABA）ergic innervation and GABAA receptors in the globus pallidus.In this study,we investigated the effects of endogenous GABAA receptors on the spontaneous firing activity of pallidal neurons in both normal and MPTP-treated mice using multi-barrel electrodes extracellular recordings in vivo.We found that in normal mice,pressure ejection of 0.1 mmol/L gabazine,a specific GABA A receptor antagonist,increased the spontaneous firing rate of globus pallidus neurons by 27.6 ± 5.6%.Furthermore,in MPTP mice（14 days after MPTP treatment）,0.1 mmol/L gabazine increased the firing rates by 51.0 ± 7.9%,significantly greater than in normal mice.These results suggest that endogenous GABAA receptors modulate the activity of globus pallidus neurons.The present findings may provide a rationale for investigations into the potential role of GABAA receptors in Parkinson’s disease.

GetArtemisininsByAnymeans (GABA)： artemisinins path to conquer diabetes go through GABA receptors

Diabetes is a chronic disease of elevated blood glucose levels that affects more than 400 million people worldwide.Complications of diabetes affect the neuronal and the vascular systems and causes diabetic kidney disease,cardiovascular disease,hypertension,retinopathy,peripheral neuropathy,and diabetic food diseases.Diabetes is caused by either the body’s inability to

Effects of dopamine D_(1) receptor blockade on the ERG b- and d-waves during blockade of ionotropic GABA receptors

Background:Some data indicate that the dopaminergic and GABAergic systems interact in the vertebrate retina,but the type of interactions is not well understood.Methods:In this study we investigated the effect of dopamine D_(1) receptor blockade by 75μM SCH 23390 on the electroretinographic ON(b-wave)and OFF(d-wave)responses in intact frog eyecup preparations and in eyecups where the ionotropic GABA receptors were blocked by 50μM picrotoxin.Student’s t-test,One-way repeated measures ANOVA with Bonferroni post-hoc test and Two-way ANOVA were used for statistical evaluation of the data.Results:We found that SCH 23390 alone significantly enhanced the amplitude of the b-and d-waves without altering their latency.The effect developed rapidly and was fully expressed within 8-11 min after the blocker application.Picrotoxin alone also markedly enhanced the amplitude of the ERG ON and OFF responses and increased their latency significantly.The effect was fully expressed within 25-27 min after picrotoxin application and remained very stable in the next 20 min.The effects of SCH 23390 and picrotoxin are similar to that reported in our previous studies.When SCH 23390 was applied on the background of the fully developed picrotoxin effect,it diminished the amplitude of the b-and d-waves in comparison to the corresponding values obtained during application of picrotoxin alone.Conclusion:Our results demonstrate that the enhancing effect of D_(1) receptor blockade on the amplitude of the ERG b-and d-waves is not evident during the ionotropic GABA receptor blockade,indicating an interaction between these neurotransmitter systems in the frog retina.We propose that the inhibitory effect of endogenous dopamine mediated by D_(1) receptors on the ERG ON and OFF responses in the frog retina may be due to the dopamine-evoked GABA release.

Suppression of Human Liver Cancer Cell Migration and Invasion via the GABA_A Receptor

Objective To investigate the roles of theγ-aminobutyric acid(GABA) in the metastasis of hepatocellular carcinoma(HCC) and to explore the potential of a novel therapeutic approach for the treatment of HCC. Methods The expression levels of GABA receptor subunit genes in various HCC cell lines and patients’ tissues were detected by quantitative real-time polymerase chain reaction and Western blot analysis.Transwell cell migration and invasion assays were carried out for functional analysis.The effects of GABA on liver cancer cell cytoskeletal were determined by immunofluorescence staining. And the effects of GABA on HCC metastasis in nude mice were evaluated using an in vivo orthotopic model of liver cancer. Results The mRNA level of GABA receptor subunits varied between the primary hepatocellular carcinoma tissue and the adjacent non-tumor liver tissue.GABA inhibited human liver cancer cell migration and invasion via the ionotropic GABAa receptor as a result of the induction of liver cancer cell cytoskeletal reorganization.Pretreatment with GABA also significantly reduced intrahepatic liver metastasis and primary tumor formation in vivo. Conclusions These findings introduce a potential and novel therapeutic approach for the treatment of cancer patients based on the modulation of the GABAergic system.

Preliminary Study on γ-Aminobutyric Acid (GABA_A) Like Receptor in Rat Testis

For making it clear whether GABAA-like receptor is in cells or rat testis and how itsgene expresses in tissue cells, the mRNA of rat testis was microinjected into Xenopusoocyte. The membrane electrobiological results showed that an inward 30 nA micro-currentwas elicited, under two-electrode voltageclamped configuration, by extracellular GABA(500 μmol/L), and both Bicuculline and Picrotoxin could inhibit this effect. It is an indication that the mRNA of rat testis can express GABA receptor in the membrane of Xenonpus oocyte. Using Muscimol as a excitant of GABA receptor type A, the micro--currentwas also elicited to generate; however, using Baclofen as a excitant of GABA receptortype B, the micro-current could not be induced. These results indicate that GABA receptorexpressed in Xenopus oocyte membrane is not type B but for type A. On the basis of the research result above, 24-mer oligonucleotides to be complementary to the high conservativeregion of the cDNAs of neur-GABA receptors were synthesized as probes to hybridize respectively with the RNAs Of brain and testis of adult rat, and of rat testes of the variousages. The dot hybridizations showed that the hybrid signal of rat testis was stronger thanthat of rat brain, meaning that, compared with rat brain, the RNA of rat testis has morehomologous regions with probes, and also implying that the GABA receptor from theRNA Of rat testis may be similar to neural-GABA receptor. This receptor therefore iscalled as GABA_A-like receptor. The dot blot analysis above also showed the testis GABA_Alike receptor. The dot blot analysis above also showed testis GABA_A like receptor mRNAcontent changed with the develOPmental age of rats. There is a very low level of gene expression in the rat testis on day 5 postnatal; there is the highest expression of GABA_A-likereceptor gene in rat testes on day 30to 100 after birth; the expression begins to drop on day200 after birth.

表皮穿透和GABA_A受体不敏感性在小菜蛾对阿维菌素抗性中的作用

用氚标记阿维菌素点滴处理阿维菌素敏感 (ABM S)和抗性 (ABM R)种群小菜蛾幼虫 ,结果显示 ,在 5～ 36 0min内的 7个不同处理时间 ,ABM R种群的平均表皮穿透量比ABM S种群少 1 5倍 ,处理 2 4h后 ,ABM R种群仍有 45 9%的3 H 阿维菌素滞留于体表 ,而ABM S种群却有 98 4%的药剂穿透表皮。放射配体结合分析表明 ,GABAA 受体结合性质的改变是小菜蛾对阿维菌素的另一抗性机制 ,ABM S种群 (Kd=10 936 8± 0 4374nmol L)和ABM R种群 (Kd=9 832 8± 0 3933nmol L)的受体亲和力无显著差异 ,但抗性种群的最大结合量 (Bmax =71 2 842± 4 9910fmol mg蛋白 )比敏感种群 (Bmax =112 0 2 5 5± 7 8418fmol mg蛋白 )降低 6 3 6 % ,即抗性是受体数目的减少而非结构上的改变。

羟丁酸钠对大鼠原代培养皮层神经元缺氧复氧损伤的保护作用与GABA_A受体的关系

目的 :探讨羟丁酸钠 (SO)对大鼠皮层神经元缺氧复氧损伤的保护作用与GABAA 受体的关系。方法 :采用原代培养大鼠皮层神经元建立缺氧复氧损伤模型 ,观察细胞的形态学 ,测定其LDH漏出率、MDA含量、SOD、GPx活力。结果 :缺氧复氧可引起神经元损伤 ,LDH漏出率增加 ,MDA含量升高 (P <0 .0 1) ,SOD、GPx活力降低 (P <0 .0 1)。SO能显著减少损伤神经元的LDH漏出率及MDA的生成 (P <0 .0 1) ,升高SOD、GPx(P <0 .0 1)的活力 ,这种作用可被GABAA 受体阻断剂seurinine减弱 (P <0 .0 1)。结论 :SO对缺氧复氧神经元损伤的保护作用可能与其激动GABAA 受体有关。

吸入麻醉药镇痛、催眠作用与GABA_A受体的关系

目的 分析吸入麻醉药安氟醚、异氟醚和七氟醚催眠、镇痛作用与GABAA 受体的关系。方法 建立小鼠催眠、镇痛模型后 ,在小鼠催醒、热板、扭体实验中 ,分别观察用药后小鼠翻正反射消失持续时间、痛阈或扭体次数的变化。结果 一叶秋碱和荷包牡丹碱对上述 3种吸入麻醉药的催眠、镇痛作用的影响均无显著性 (P >0 0 5 )。结论 GABAA 受体并非安氟醚、异氟醚和七氟醚催眠。

黄芩茎叶黄酮作为GABA_A受体BZD结合部位配体(英文)

目的从黄芩地上部分中寻找活性成分。方法利用各种柱色谱 (硅胶、SephadexLH 2 0、D10 1大孔吸附树脂和聚酰胺 )分离黄芩地上部分的活性成分。结果从黄芩地上部分的水提取物中分离得到 7种黄酮类化合物。其中白杨素对GABAA 受体的BZD结合部位有较高的亲和力 ,其IC50 值为 0 6 7μmol/L。 7种黄酮类化合物亲和力的排列顺序为 :白杨素 >汉黄芩苷元 >红花素 >黄芩素 >5 ,6 ,7 三羟基 4′ 甲氧基黄酮 >异红花素 >白杨素 7 葡萄糖醛酸苷。

Mg^(2+)对神经元保护机制的研究-Mg^(2+)对培养的大鼠海马神经元GABA_A受体介导电流的影响

目的 研究镁离子(Mg2+)对神经元保护的机制。方法 采用全细胞膜片钳技术,研究Mg2+对培养的大鼠海马神经元GABA_A受体介导电流的作用。结果 (1)在钳制电压为-40mV时,GABA可通过GABA_A受体介导产生内向电流;(2)当GABA和Mg2+同时作用时,该电流峰值增高;(3)在Mg2+存在的情况下,GABA的浓度-效应曲线平行左移。结论 Mg2+通过正性变构调节机制影响GABA_A受体的功能,达到保护神经元的作用。

大鼠中枢运动核团内谷氨酸受体、GABA_A受体、甘氨酸受体和阿片μ受体的定位分布

目的观察大鼠中枢运动核团内谷氨酸受体、γ－氨基丁酸受体（ＧＡＢＡＡＲ）、甘氨酸受体（ＧｌｙＲ）和阿片μ受体（ＭＯＲ）的定位分布，搞清谷氨酸、ＧＡＢＡ、甘氨酸和脑啡肽在中枢运动核团内的作用部位。方法免疫组织化学染色技术。结果在动眼神经核、滑车神经核、三叉神经运动核、展神经核、面神经核、疑核、舌下神经核、Ｅ－Ｗ核、上涎核、迷走神经背核和脊髓前角内均可见ＮＭＤＡＲ１样和ＧｌｕＲ２／３样阳性胞体，但ＮＭＤＡＲ１样和ＧｌｕＲ２／３样阳性纤维和终末仅见于动眼神经核、Ｅ－Ｗ核、滑车神经核、三叉神经运动核和迷走神经背核，除三叉神经运动核、疑核和脊髓前角能见到少量ＧＡＢＡＡＲβ亚单位样阳性胞体外，上述各核团也可见ＧＡＢＡＡＲβ亚单位样阳性的纤维和终末，而ＧＡＢＡＡＲα亚单位样阳性胞体主要见于疑核、舌下神经核、迷走神经背核和脊髓前角；中枢运动核团内均可见ＧｌｙＲ和ＭＯＲ样阳性的纤维和终末。结论谷氨酸、ＧＡＢＡ、甘氨酸和脑啡肽在中枢运动核团可能通过突触前和突触后机制，对中枢运动神经元发挥调控作用。

大鼠三叉神经尾侧亚核和脊髓内离子型谷氨酸受体、GABA_A受体和甘氨酸受体的分布

为了搞清兴奋性和抑制性氨基酸受体在伤害性刺激信息传递和调制初级门户内的精确定位，本研究用免疫组织化学技术观察了大鼠三叉神经尾侧亚核（Ｖｃ）和脊髓内离子型谷氨酸受体（ＮＭ－ＤＡ受体、ＡＭＰＡ受体和Ｋａｉｎａｔｅ受体）、ＧＡＢＡＡ受体和甘氨酸（Ｇｌｙ）受体的分布。ＮＭＤＡ受体１亚型分布于Ｖｃ和脊髓的各层，ＮＭＤＡ受体２Ａ／Ｂ亚型、Ｋａｉｎａｔｅ受体和ＧＡＢＡＡ受体α亚单位散在性地分布于Ｖｃ和脊髓后角的Ⅰ层，ＧＡＢＡＡ受体α亚单位仅见于Ｖｃ和脊髓Ⅰ层内轴突样结构。ＡＭＰＡ受体、ＧＡＢＡＡ受体β亚单位和Ｇｌｙ受体较密集地分布于Ｖｃ和脊髓的各层，尤其是它们的浅层（Ⅰ和Ⅰ层）。结合机能学研究的结果，讨论了这些受体阳性神经元在伤害性刺激信息传递和调制方面的作用。

神经慢性挤压伤疼痛模型大鼠脊髓GABA_A受体mRNA表达的变化

目的 :了解大鼠坐骨神经慢性挤压伤 (chronicconstrictioninjury ,CCI)模型脊髓GABAA受体α2 、γ2 亚型mRNA表达的变化。方法 :SD大鼠 2 0只 ,5只为正常对照 ,15只做成CCI模型。用VonFrey细丝和CO2 激光测定大鼠足爪的疼痛阈值变化 ,并于术后 3d、7d和 14d处死 (n =5每组 ) ,取L4～ 5脊髓用RT PCR方法测定不同时点大鼠脊髓GABAA受体α2 、γ2 亚型mRNA的表达量。结果 :CCI大鼠结扎侧在术后 3d即开始出现机械感觉异常和痛觉过敏 ,7d、14d组逐渐加重 (P <0 0 5 )。与正常大鼠比较 ,CCI大鼠GABAA受体α2 、γ2 亚单位mRNA表达量于术后 7d开始出现下降 (P <0 0 5 ) ,且术后 14d组进一步降低 (P <0 0 1)。结论 :CCI大鼠疼痛模型中脊髓GABAA受体α2 、γ2 亚单位的表达量明显下降 ,其在慢性神经性疼痛中发病机制中的确切作用尚待进一步研究。

海藻酸诱导复杂部分发作癫痫大鼠海马GABA_A受体α_1亚单位的表达

目的:研究海藻酸(KA)致痫大鼠海马GABAA受体α1亚单位的动态表达水平以及加巴喷丁(GBP)干预对它们的影响,探讨复杂部分发作癫痫(CPS)的发生机制,为开发针对GABA受体亚单位水平的新型抗癫痫药物(AEDs)提供理论依据。方法:成年雄性Wistar大鼠海马CA3区注射2μgKA建立复杂部分发作模型,采用免疫组化SABC法检测癫痫大鼠海马各区GABAA受体α1亚单位(GABAARα1)的动态表达水平,并用GBP干预癫痫的发作观察GBP对GABAARα1表达的影响。结果:KA致痫后海马CA1区、CA3区GABAARα1表达呈先升后降趋势,齿状回(DG)表达水平则逐渐增加。GBP对癫痫早期GABAARα1表达无直接影响。结论:在KA诱导的CPS模型中,GABAARα1表达减少和由此所引起的抑制功能减弱,可能是癫痫发生的一种分子机制。

大鼠三叉神经中脑核内GABA_A 受体和GABA_B受体的定位分布

目的 观察大鼠三叉神经中脑核 (Vme)内GABAA 受体 (GABAAR)和GABAB 受体 (GABABR)的定位分布 ,为研究GABA受体在面口部本体感觉信息传递和调控中的作用提供形态学依据。 方法 用免疫细胞化学技术 ,染色结果在光学显微镜下进行观察。 结果 在Vme内可观察到大量GABAAR和GABABR样免疫反应阳性的胞体、纤维和终末 ,其不同亚单位的分布具有一定差别 :1 GABAB R1亚单位主要分布于Vme神经元的胞体 ,呈强阳性反应 ,而GABABR2亚单位只见于Vme神经元胞体周围的纤维和终末内 ,呈弱阳性反应 ,Vme神经元为阴性 ;2 在Vme内亦可观察到大量GABAAR免疫反应阳性的胞体、纤维和终末 ,其主要亚单位有α1、α2、α3和 β。其中GABAARα1和GABAARα3亚单位只分布于神经元胞体 ,呈强阳性反应 ,GABAARα2和GABAARβ亚单位仅见于神经纤维和终末内 ,Vme神经元胞体为阴性 ;3 密集分布的GABABR2、GABAARα2和GABAARβ亚单位样免疫阳性纤维和终末似包绕在Vme神经元胞体的周围 ,其中以GABAARα2的免疫染色最强 ,分布也较为集中。 结论 GABAA 受体和GABAB 受体的不同亚单位在Vme内的定位分布有一定差异 ,提示GABA能神经终末对面口部本体感觉信息的传递发挥抑制性调控作用 ,可能主要是由位于Vme神经元胞体内的GABAARα1和GABAAR?

GABA_A五种亚型受体与BZ配基的3D-QSAR研究

GABAA受体是中枢神经系统内重要的抑制性受体,有广泛的神经生理活性.由于镇静/抗惊厥药物在临床上的广泛应用,使得其中苯并二氮杂作用位点尤为重要.我们用比较分子场法(CoMFA)对一系列咪唑苯并二氮杂类化合物(BZ)与五种重组受体亚型的亲和力进行了结构活性关系研究,得到的一组模型都有较高的交叉验证系数.并在此基础上,建立了非交叉验证的一组PLS模型.用该组模型对随机选择的6个化合物组成的测试集进行了预测,都得到了相当满意的结果,表明所建立的一组模型具有良好的预测能力.本研究对于设计高亲和力的BZ受体的配基和研究GABAA受体的模型有指导意义.

大鼠耳蜗螺旋神经节细胞GABA_A受体α_2 mRNA的表达及意义

目的 研究大鼠耳蜗螺旋神经节细胞中γ-氨基丁酸 (γ- aminobutyric acid,GABA) A受体α2 m RNA的表达并探讨其意义。方法 采用大鼠耳蜗石蜡切片 ,地高辛标记的 GABAAα2 c DNA探针 (5 4 9个碱基 ) ,抗地高辛的碱性磷酸酶检测系统 ,碱性磷酸酶显色剂 ,原位杂交检测大鼠耳蜗螺旋神经节细胞中 GABAAα2 m RNA受体的表达。结果 大鼠耳蜗中螺旋神经节细胞及其纤维均显示阳性杂交信号 ,表达 GABAAα2 受体 m RNA。骨螺旋板等其他结构不表达。作为阳性组织对照的小脑蒲肯野氏细胞和颗粒细胞的胞体及轴突表达 GABAAα2 受体。嗅上皮雌激素受体 c DNA探针阴性对照、无探针空白对照及无地高辛抗体对照的耳蜗螺旋神经节细胞及小脑组织均不表达GABAAα2 受体。结论 GABAAα2 受体位于听觉传导通路的第一级神经元——所有的 型螺旋神经节细胞及其纤维 ,强烈支持 GABA作为其递质 。

GABA_A和GABA_B受体介导的蟾蜍背根神经节神经元胞体膜反应

实验在蟾蜍离体背根神经节(DRG)标本进行细胞内记录。浴槽滴加10^(-4)-10^(-3)mol/LGABA引起膜电位改变如下:(1)去极化(79/100);(2)双相反应;先为去极化,继后为超极化(10/100);(3)无反应(11/100)。以上去极化反应均可为荷包牡丹碱所阻断。GABA-去极化时膜电导增加,逆转电位值为-15——25mV。低Cl^-和高Cl^-任氏液分别使GABA-去极化反应增大和减小。10^(-4)mol/Lbaclofen不引起膜电位改变。在GABA-去极化期间,观察到大部分细胞的动作电位时程(ApD)缩短。ApD的此种变化可为baclofen所模拟,但不为荷包牡丹碱所阻断。结果提示:蟾蜍DRG神经元胞体膜有GABA_A和GABA_B受体共存,前者介导膜电位的改变,后者介导ApD的缩短。本文并联系到初级传入终末的突触前抑制的产生机制进行了讨论。