Regulation of trascription factor by GABA_B mechanisms in cultured neuronal cells

培养神经元细胞中 GABA_B 机制对转录因子的调节

基本信息

批准号：
08672537
负责人：
ISHIGE Kumiko
金额：
$ 0.9万
依托单位：
Nihon University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08672537/
关键词：
gamma-hydoxybutyric acid cyclic AMP responsive element activator protein 1 GABA_B receptors Ca^<2+>neuronal cells activator protein 1 DNA a ctivator proteinl 細胞内Ca^<2+>

项目摘要

It this study, the effect of gamma-hydroxybutyric acid (GHB) in nuclear cyclic AMP responsive element (CRE) -and activator protein 1 (AP-1) DNA-binding activities in primary neuronal cultured cellswas examined. Gel-shift assays showed that exposure of the cells to GHB (1mM) increased nuclear CRE-and AP-1 DNA-binding activities in cultured mouse cerebellar granule cells and cerebral cortical cells. These increases in nuclear DNA-binding activities in cultured mouse cerebellar granule cells were antagonized by specific GABA_B antagonists such as CGP 55845 (1muM) and CGP 35348 (1mM). In addition, treatment of the cells with 1,2-bis (2'-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), an intracellular Ca^<2+> chelating agent, or thapsigargin, an endoplasmic reticulum Ca^<2+>-ATPase inhibitor, suppressed the GHB-induced increases in nuclear DNA-binding activities. Furthermore, the GHB-induced increases in nuclear CRE-and AP-1 DNA-binding activities were suppressed by treatment with calmodulin kinase II inhibitors such as KN-62 (10muM) and KN-93 (10muM). In addition, the CRE-binding activity was completely supershifted by the CRE-binding protein (CREB) antibody, and was eliminated by the phospholyrated CREB antibody. The AP-1 DNA-binding activity was blocked by c-Jun and c-Fos antibodies. These results siggest that stimulation of GABA_B receptors by GHB activates intracellular Ca2+ storage sites and that increased intracellular Ca^<2+> plays an important role in elevation of nuclear CRE-and AP-1 DNA-binding activities and that calmodulin kinase II plays an important role in DHB-induced increases in both DNA-binding activities in cultured cerebellar granule cells. It is also suggested that GHB promotes the phosphorylation of CREB.

在本研究中，检测了 γ-羟基丁酸 (GHB) 对原代神经元培养细胞中核环 AMP 反应元件 (CRE) 和激活蛋白 1 (AP-1) DNA 结合活性的影响。凝胶迁移试验表明，在培养的小鼠小脑颗粒细胞和大脑皮层细胞中，将细胞暴露于 GHB (1mM) 会增加核 CRE 和 AP-1 DNA 结合活性。培养的小鼠小脑颗粒细胞中核 DNA 结合活性的增加被特定的 GABA_B 拮抗剂（例如 CGP 55845 (1μM) 和 CGP 35348 (1mM)）拮抗。此外，用细胞内Ca^2+螯合剂1,2-双(2'-氨基苯氧基)乙烷-N,N,N',N'-四乙酸四乙酰氧基甲酯(BAPTA-AM)处理细胞。药剂，或毒胡萝卜素，一种内质网Ca 2+ -ATP酶抑制剂，抑制GHB诱导的核DNA结合活性的增加。此外，GHB 诱导的细胞核 CRE 和 AP-1 DNA 结合活性的增加可通过钙调蛋白激酶 II 抑制剂（例如 KN-62 (10μM) 和 KN-93 (10μM)）治疗得到抑制。此外，CRE结合活性被CRE结合蛋白(CREB)抗体完全超移，并被磷酸化CREB抗体消除。 AP-1 DNA 结合活性被 c-Jun 和 c-Fos 抗体阻断。这些结果表明，GHB 对 GABA_B 受体的刺激激活了细胞内 Ca2+ 储存位点，并且细胞内 Ca^2+ 的增加在核 CRE-和 AP-1 DNA 结合活性的升高中发挥着重要作用，并且钙调蛋白激酶 II 发挥着重要作用。 DHB 诱导培养的小脑颗粒细胞 DNA 结合活性增加中发挥重要作用。还表明 GHB 促进 CREB 的磷酸化。