Studies on GABA_A receptor complexes in cultured cerebellar neuronal cells and synaptoneurosomes

培养小脑神经元细胞和突触神经体中 GABA_A 受体复合物的研究

• 批准号: 02671016
• 负责人: FUKUDA Hideomi
• 金额: $ 1.22万
• 依托单位: Nihon University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02671016/
• 关键词: GABA_Areceptor; GABA antagonist; SR 95531; ^<36>Cl^- uptake; ^<35>S-TBPS binding; Phospholipase A_2; Cultured cerebellar neuronal cells; synaptoneurosome; ピクロトキシン結合部位; ビククリンメチオダイド; IC_<50>値; アゴニスト作用; 受容体結合実験; ^<36>Clイオンの取込み; 培養神経細胞; アゴニスト /

In the present study, experiments were performed to characterize antagonistic activity and binding properties of SR 95531 [2-(3'carbethoxy-2'-propyl)-3-amino-6-Paramethoxy-phenyl-pyidaziniun bromide]. SR 95531 and bicuculline methiodide (BMI) inhibited muscimol-stimulated ^<36>Cl^- uptake into cortical synaptoneurosomes in a concentration-dependent manner. Inhibitory potency of SR 95531 for the muscimlstimlated ^<36>Cl^- uptake was 15 times higher than that of BMI. The IC_<50> value of SR 95531 for muscimol-stimulated ^<36>Cl^- uptake into cortical synaptoneurosomes was in close agreement with the K_D value of low-affinity binding sites of [ ^3H]SR 95531 in the FC. SR 95531 and BMI antagwonized GABA-induced inhibition of [ ^<35>S]TBPS binding in cortical membranes. SR 95531 was more potent than BMI. Pretreatment of the membranes with phospholipase A_2 (PLase A_2) invariably decreased [ ^3H]SR 95531 binding in the cerebral cortex and the cerebellum. On the other hand, preincubation with … More PLase A_2 significantly, increased the binding of [ ^3H]GABA in a concentration-dependent manner in cortical membranes. Although lower concentrations of PLase A_2 did not affect the binding of [ ^3H]GABA in the cerebellum, treatment with higher concentrations of PLase A_2 increased the binding in this region. In addition, PLase A_2-induced increase in the binding of [ ^3H]GABA in the cerebral cortex was greater than that in the cerebellum. Specific binding of [ ^3H]SR 95531 was also detected in cultured cerebellar neuronal cells. Pretreatment with PLase A_2 affected [ ^3H]GABA and [ ^3H]SR 95531 binding in the cultured cerebellar neuronal cells as in the case of the cerebellum. These results suggest that SR 95531 exerts GABA antagonistic action through the low-affinity binding site of SR 95531 and that exogenously added PLase A_2 induces the conversion of GABA_A receptors into the agonist preferential conformation by the products of catalytic activity of PLase A_2. In addition, cultured cerebellar neuronal cells may be a useful tool to investigate the long term effects of various drugs on GABA_A receptor complexes in vitro. Less

在本研究中，对SR 95531[2-(3‘carbethoxy-2’-propyl)-3-amino-6-Paramethoxy-phenyl-pyidaziniun溴化物的拮抗活性和结合特性进行了实验表征。SR 95531和甲基荷包牡丹碱以浓度依赖的方式抑制蝇毒酚刺激的皮质突触神经节摄取。SR 95531对肌间充质细胞摄取氯离子的抑制作用是BMI的15倍。SR 95531刺激皮层突触神经体摄取Cl~-的IC_(50)值与[~3H]SR_(95531)低亲和力结合部位的K_D值非常一致。SR 95531和BMI拮抗γ-氨基丁酸引起的皮质膜结合[^&t；35&gt；S]Tbps的抑制作用。SR 95531比BMI更有效。用磷脂酶A_2(PLASE A_2)处理膜后，大脑皮层和小脑的[~(3 H)]SR_(95531)结合量均减少。另一方面，与…预孵化更多的A_2以浓度依赖的方式增加皮质膜[~(3 H)]GABA的结合量。虽然低浓度的PLASE A_2不影响小脑[~(3 H)]GABA的结合，但高浓度的PLASE A_2处理增加了该区域的结合。此外，PLASE A_2引起大脑皮层[~(3 H)]GABA结合量的增加大于小脑。在培养的小脑神经细胞中也检测到[~(3 H)]SR-95531的特异性结合。与小脑一样，PLASE A_2对培养的小脑神经元细胞的[~(3 H)]-γ-氨基丁酸和[~(3 H)]SR-95531结合也有影响。这些结果表明，SR 95531通过SR 95531的低亲和力结合部位发挥GABA拮抗作用，外源加入PLASE A_2可通过PLASE A2催化活性的产物将GABA_A受体转化为激动剂优先构象。此外，体外培养的小脑神经元细胞可能是研究各种药物对GABA_A受体复合体长期作用的有用工具。较少

项目成果

伊藤 芳久: "Characterization and Binding properties of SR 95531,a pyridazinylーGABA derivative,in rat brain and cultured cerebllar neuronal cells." SYNAPSE. (1992)

Yoshihisa Ito：“SR 95531（一种哒嗪基-GABA 衍生物）在大鼠大脑和培养的小脑神经元细胞中的表征和结合特性”（1992 年）。

伊藤 芳久: "Characterization of antagonistic activity and binding property of SR 95531,a pyridazinylーGABA derivative,in rat brain and cultured cerebellar cells." Synapse.

Yoshihisa Ito：“SR 95531（一种哒嗪-GABA 衍生物）在大鼠大脑和培养的小脑细胞中的拮抗活性和结合特性的表征。”