METABOTROPIC EXCITATORY AMINOACID RECEPTORS COUPLED W/PHOSPHOINOSITIDE HYDROLYSIS

与磷酸肌醇水解偶联的代谢兴奋性氨基酸受体

• 批准号: 3760940
• 负责人: JARDA T WROBLEWSKI
• 金额: --
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3760940
• 关键词: calcium flux; cellular pathology; developmental neurobiology; diacylglycerols; excitatory aminoacid; glutamate receptor; granule cell; hydrolysis; inositol phosphates; laboratory rat; neuropharmacology; phosphatidylinositols; phospholipase C; receptor coupling; receptor expression; tissue /cell culture

Excitatory amino acid (glutamate) receptors comprise a class of metabotropic glutamate receptors (mGluRs) coupled through G proteins to the formation of intracellular second messengers. The coupling of these receptors to phospholipase C (PLC) results in the hydrolysis of membrane phosphoinositides (PI) and generation of two second messenger molecules inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DG). Evidence is now accumulating which points to the existence of multiple subtypes of PI-coupled mGluRs. This heterogeneity has been postulated based on the pharmacology of agonist and antagonist action in different brain regions and selected neuronal populations, as well as during development, and on the basis of molecular cloning. Numerous studies suggest that mGluRs coupled to PI hydrolysis play a crucial role in brain function related to synaptic transmission, development and neuronal injury. For this reason, and in view of the possible therapeutic implications, it is essential to understand the functional heterogeneity of these receptors, the pharmacology of their recognition sites and the role that second messengers generated by their activation play in intracellular neuronal signalling. The long term objective of this research project is to explore in vitro, using primary cultures of neurons, the heterogeneity of PI-coupled mGluRs in terms of the molecular mechanisms associated with the receptor- mediated activation of PI hydrolysis, the significance of mGluR- stimulated PI hydrolysis in neuronal function, and the pharmacology and regulatory properties of mGluR-induced PI responses. Accordingly, primary cultures of cerebellar granule cells will be used to: 1. Evaluate the functional expression of PI-coupled mGluRs by determining the developmental pattern of expression of mRNA and the appearance of the PI response in neurons cultured in media enhancing receptor expression and by establishing the agonist and antagonist pharmacology of these receptors. 2. Determine the role of mGluRs in PI metabolism by identifying the specific substrates and products of PLCs coupled to mGluRs and establishing the relative contribution of mGluRs to the formation of two second messengers IP3 and DG. 3. Determine the role of PI-coupled mGluRs in intracellular Ca2+ homeostasis by using Fura-2 imaging to evaluate the magnitude of IP3-sensitive Ca2+ stores and the contribution of mGluR agonists and antagonists to the regulation of intracellular Ca2+ concentrations. This approach, combined with mRNA measurements, will be extended to identify the functional expression of PI-coupled mGluRs present in neuronal cultures prepared from other brain areas. 4. Determine the role of ionotropic glutamate receptors in the regulation of PI hydrolysis including the role of NMDA receptors in the stimulation and inhibition of PI hydrolysis and the molecular nature of interactions between mGluRs and AMPA receptors. These studies will provide information fundamental for the understanding of the pharmacological and neurochemical properties of PI-coupled mGluRs, their role in neuronal function, and for development of future therapeutic strategies.

兴奋性氨基酸（谷氨酸）受体包括一类 代谢型谷氨酸受体 (mGluR) 通过 G 蛋白偶联 细胞内第二信使的形成。 这些的耦合 磷脂酶 C (PLC) 受体导致膜水解 磷酸肌醇 (PI) 和两个第二信使分子的产生 肌醇-1,4,5-三磷酸 (IP3) 和二酰甘油 (DG)。 证据是 现在正在积累，这表明存在多种亚型 PI 耦合 mGluR。 这种异质性是基于以下假设的 不同脑区激动剂和拮抗剂作用的药理学 和选定的神经元群体，以及在发育过程中，以及 分子克隆的基础。 大量研究表明 mGluR 与 PI 水解耦合在脑功能相关中发挥至关重要的作用 突触传递、发育和神经元损伤。 为了这 原因，并考虑到可能的治疗意义，这是 了解这些受体的功能异质性至关重要， 它们的识别位点的药理学以及第二位点的作用 由其激活产生的信使在细胞内神经元中发挥作用 发出信号。 该研究项目的长期目标是探索体外、 使用神经元原代培养物，PI 偶联 mGluR 的异质性 就与受体相关的分子机制而言 介导的 PI 水解激活，mGluR- 的意义 刺激神经元功能中的 PI 水解，以及药理学和 mGluR 诱导的 PI 反应的调节特性。 因此， 小脑颗粒细胞的原代培养物将用于： 1. 评估 通过确定 PI 偶联 mGluR 的功能表达 mRNA 表达的发育模式和 PI 的出现 在培养基中培养的神经元的反应增强受体表达和 通过建立这些药物的激动剂和拮抗剂药理学 受体。 2. 确定 mGluRs 在 PI 代谢中的作用 识别与 PLC 耦合的特定基质和产品 mGluRs 并确定 mGluRs 对 形成两个第二信使IP3和DG。 3. 确定角色 使用 Fura-2 PI 偶联 mGluR 促进细胞内 Ca2+ 稳态 成像评估 IP3 敏感的 Ca2+ 储存量和 mGluR 激动剂和拮抗剂对调节的贡献 细胞内 Ca2+ 浓度。 这种方法与 mRNA 相结合 测量，将被扩展以识别函数表达 PI 偶联 mGluR 存在于从其他大脑制备的神经元培养物中 地区。 4. 确定离子型谷氨酸受体在 PI 水解的调节，包括 NMDA 受体在 PI水解的刺激和抑制及其分子性质 mGluR 和 AMPA 受体之间的相互作用。 这些研究将 提供理解的基本信息 PI 偶联 mGluR 的药理学和神经化学特性，它们的 在神经元功能中的作用，以及未来治疗方法的开发 策略。