REGULATION OF NEURONAL RECEPTORS

神经元受体的调节

• 批准号: 2273425
• 负责人: EUGENE M BARNES
• 金额: $ 18.26万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2273425
• 关键词: GABA receptor; benzodiazepine receptor; benzodiazepines; chick embryo; drug tolerance; gene expression; inhibitor /antagonist; laboratory mouse; messenger RNA; molecular cloning; muscimol; neurons; neuropharmacology; northern blottings; nuclear runoff assay; nucleic acid hybridization; nucleic acid metabolism; nucleic acid sequence; protein metabolism; radiotracer; receptor binding; receptor expression; receptor sensitivity; restriction mapping; solution hybridization; stimulant /agonist; tissue /cell culture

The GABA-A/benzodiazepine receptor (GBZR) complex on vertebrate neurons plays a key role in inhibitory neurotransmission. The long-term goal of this project is to understand the neuronal regulation of GBZR receptor number and subcellular distribution. We have recently demonstrated that chronic exposure of chick cortical neurons to GABA produces a down-regulation of GBZR3. In order to study the underlying regulatory mechanisms, three specific objectives are developed in this proposal. 1. To test the hypothesis that exposure of cortical neurons to GABA and benzodiazepine agonists accelerates internalization of GBZRs. A novel membrane-impermeant benzodiazepine, SPTC-1012S, which is a potent displacer of [35S]flunitrazepam binding to intact neurons, will be used in an assay for receptor sequestration. This will allow us to study the effects of GABA and benzodiazepine agonists, both alone and in combination, on this process. Possible up-regulation by benzodiazepine antagonists and reverse agonists, as well as GABA antagonists, will also be evaluated. 2. To test the hypothesis that exposure of neurons to GABA and benzodiazepine agonists produces a reduction in ligand binding sites and GBZR peptides. Our approach involves metabolic labeling of GBZR peptides with [35S]methionine and determining their rates of degradation by pulse-chase techniques. In addition, the effects of down-regulation on ligand binding density, affinity, and-specificity will be determined. 3. To test the hypothesis that agonist exposure reduces the level of GBZR transcripts. The GBZR alphasubunit mRNAs which are subject to down-regulation will be identified by Northern hybridization and their rates of degradation quantified by DNA-excess solution hybridization. Furthermore, nuclear run-on transcription assays will be used to determine the rates of GBZR alpha-subunit mRNA synthesis and to examine the role of receptor agonists in repression. It is suggested that this combination of techniques from pharmacology, biochemistry, and molecular biology will provide new insights into pathways which modulate synaptic function. By means of these regulatory mechanisms, cell-cell communication and drug-cell interaction could produce persistent changes in neuronal excitability. Furthermore, these may represent molecular mechanisms which could establish tolerance and habituation to benzodiazepines.

脊椎动物上的GABA-A/苯二氮卓受体（GBZR）复合物 神经元在抑制性神经传递中起关键作用。 长期 该项目的目标是了解GBZR的神经元调节 受体数和亚细胞分布。 我们最近有 证明鸡皮神经元长期暴露于GABA 产生GBZR3的下调。 为了研究基础 监管机制，在此制定了三个特定目标 提议。 1。检验以下假设：皮质神经元暴露于 GABA和苯二氮卓激动剂加速GBZR的内在化。 一个 新颖的膜 - 覆盖苯二氮卓，SPTC-1012S，这是一个有效的 [35s]氟尼氏酶与完整神经元结合的位移器将被使用 在受体隔离的测定中。 这将使我们能够研究 GABA和苯二氮卓激动剂的影响，无论 在此过程中的结合。 苯二氮卓类药物可能上调 拮抗剂和反向激动剂以及GABA拮抗剂也将 进行评估。 2。检验神经元暴露于GABA的假设 和苯二氮卓激动剂产生的配体结合位点降低 和GBZR肽。 我们的方法涉及GBZR的代谢标签 具有[35s]甲氨酸的肽并确定其降解速率 通过脉冲练习技术。 另外，下调的影响 将确定配体结合密度，亲和力和特异性。 3。检验以下假设，即激动剂暴露会降低GBZR的水平 成绩单。 gbzr alphasubunit mRNA受 下调将通过北部杂交及其 通过DNA筛选溶液杂交量化的降解速率。 此外，核跑步转录分析将用于 确定GBZR Alpha-Subunit mRNA合成的速率并检查 受体激动剂在抑制中的作用。 建议从 药理学，生物化学和分子生物学将提供新的 深入了解调节突触功能的途径。 通过 这些调节机制，细胞 - 细胞通信和药物细胞 相互作用可能会导致神经元兴奋性的持续变化。 此外，这些可能代表可能的分子机制 建立对苯二氮卓类药物的耐受性和习惯。