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: 5215320
• 负责人: JARDA T WROBLEWSKI
• 金额: --
• 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/5215320
• 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（mGluRs）通过G蛋白偶联至 细胞内第二信使的形成。 这些的耦合 磷脂酶C（PLC）受体导致膜的水解 磷脂酰肌醇（PI）和两个第二信使分子的产生 肌醇-1，4，5-三磷酸（IP 3）和甘油二酯（DG）。 证据 这表明存在多种亚型的 PI偶联mGluR。 这种异质性是基于 不同脑区中激动剂和拮抗剂作用的药理学 和选定的神经元群体，以及在发展过程中， 分子克隆的基础。 许多研究表明，mGluRs 偶联PI水解发挥至关重要的作用，在脑功能有关 突触传递、发育和神经元损伤。 为此 原因，并鉴于可能的治疗影响，它是 对于理解这些受体的功能异质性至关重要， 它们识别位点的药理学作用以及第二个 它们激活后产生的信使在细胞内神经元中发挥作用， 信号装置. 本研究项目的长期目标是在体外探索， 使用原代培养的神经元，PI偶联的mGluRs的异质性， 在与受体相关的分子机制方面- 介导的PI水解激活，mGluR- 刺激PI水解神经元功能，药理学和 mGluR诱导的PI反应的调节特性。 因此，委员会认为， 小脑颗粒细胞的原代培养物将用于：1。评价 PI偶联mGluRs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， 通过建立这些药物的激动剂和拮抗剂药理学， 受体。 2.通过以下方法确定mGluRs在PI代谢中的作用： 鉴定与之偶联的PLC的特定底物和产物， mGluRs和建立mGluRs的相对贡献 两个第二信使IP 3和DG的形成。3.确定的作用 Fura-2介导的PI偶联mGluRs在细胞内Ca ~（2+）稳态中的作用 成像以评估IP 3敏感性Ca 2+商店的大小， mGluR激动剂和拮抗剂对调节 细胞内Ca 2+浓度。 这种方法，结合mRNA 测量，将被扩展到识别的功能表达 从其他脑组织制备的神经元培养物中存在的PI偶联mGluRs 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水解的刺激和抑制以及 mGluRs和AMPA受体之间的相互作用。 这些研究将 提供基本信息，以了解 PI偶联mGluRs的药理学和神经化学性质， 在神经元功能中的作用，以及未来治疗药物的开发 战略布局