Real-time measurement of ethanol's effect on cyclic AMP metabolism in live cells

实时测量乙醇对活细胞中环磷酸腺苷代谢的影响

• 批准号: 7660532
• 负责人: Masami Yoshimura
• 金额: $ 19.39万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-20 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7660532
• 关键词: A kinase anchoring protein; Acute; Adenylate Cyclase; Alcoholism; Animals; Behavioral; Brain; Camping; Cell Nucleus; Cell membrane; Cells; Cerebral cortex; Chronic; Corpus striatum structure; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; Development; Ethanol; Fluorescence Resonance Energy Transfer; Future; Generations; Goals; Hela Cells; Human; Knowledge; Lead; Life; Measurement; Measures; Metabolism; Methods; Molecular; Monitor; Neuraxis; Neurons; Pharmaceutical Preparations; Physiological; Play; Predisposition; Protein Isoforms; Proteins; Rattus; Reagent; Research; Resolution; Role; Signal Pathway; Signal Transduction; System; Technical Expertise; Technology; Testing; Time; Tissues; alcohol effect; alcohol exposure; alcohol response; base; metabolic abnormality assessment; phosphoric diester hydrolase; polypeptide; radiochemical; research study; sensor

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the molecular and cellular mechanisms underlying the effects of ethanol on the cyclic adenosine monophosphate (cAMP) signaling pathway in the central nervous system. cAMP signal transduction has been postulated to play a critical role in the physiological and behavioral responses to ethanol in animals and in the development of and predisposition to alcoholism in humans. The cAMP signaling system can be modulated by both acute and chronic ethanol exposure. The effect of ethanol on the levels of cAMP was measured by immunological and radiochemical methods in past studies, which had poor temporal resolution and no spatial resolution. Recently, single polypeptide chain cAMP sensor molecules have been developed, which can monitor cAMP levels by fluorescence resonance energy transfer (FRET). Using these sensor molecules it is possible to study real-time dynamic changes in the concentration of cAMP at a subcellular level with high spacio-temporal resolution. Emerging experimental evidences support the concept that subcellular compartmentalization is critical for cAMP signaling. We will utilize this technology for studying the effects of ethanol on the cAMP signaling system by testing the hypothesis that ethanol influences cAMP metabolism in a subcellular compartment specific manner. In Specific Aim 1, we will determine the effects of ethanol on cAMP metabolism in HeLa cells. Using a FRET-based cAMP sensor, Epac1-camps, the level of cAMP in HeLa cells will be monitored in real-time at the single cell level in the absence and presence of ethanol. Key observations of ethanol effects on cAMP metabolism studied by radiochemical methods in the past will be confirmed. Epac1-camps will be genetically modified so that the sensor can be targeted to the nucleus, plasma membrane, and cytoplasm. The effects of ethanol on cAMP in these three compartments will be examined. In Specific Aim 2, we will determine the contributions of phosphodiesterase, protein kinase A, and A-kinase anchoring proteins on the observed effects of ethanol. In the presence of pharmacological reagents specific to these proteins, ethanol's effect on cAMP metabolism in the three compartments will be examined by the FRET sensors. In Specific Aim 3, we will determine the effects of ethanol on cAMP metabolism in subcellular compartments of neuronal cells in primary culture. Primary rat neurons isolated from the cerebral cortex and striatum will be transfected with Epac1- camps targeted to different subcellular compartments (nucleus, plasma membrane, and cytoplasm). The levels of cAMP will be monitored in high spacio-temporal resolutions in the absence and presence of ethanol. The knowledge and technical expertise we will gain during the course of this study is indispensable for future studies of ethanol's effects on cAMP metabolism in the brain. Project Narrative Cyclic adenosine monophosphate (cAMP) signal transduction has been postulated to be a contributing factor to the development of and predisposition to alcoholism in humans. The knowledge and technical expertise we will gain during the course of this study is indispensable for understanding the effects of alcohol on cAMP metabolism in the brain, and may lead to the development of a new generation of tissue- and cell- specific drugs.

描述（由申请人提供）：这项研究的长期目标是阐明乙醇对中枢神经系统中环状腺苷单磷酸（CAMP）信号通路的影响的分子和细胞机制。据推测，CAMP信号转导在动物的生理和行为反应以及对人类酒精中毒的发展和倾向中起着至关重要的作用。营地信号系统可以通过急性和慢性乙醇暴露来调节。在过去的研究中，通过免疫和放射化学方法来衡量乙醇对cAMP水平的影响，这些方法的时间分辨率较差，没有空间分辨率。最近，已经开发了单多肽链cAMP传感器分子，该分子可以通过荧光共振能量转移（FRET）监测cAMP水平。使用这些传感器分子可以研究以高时空分辨率的亚细胞水​​平的cAMP浓度的实时动态变化。新兴的实验证据支持了亚细胞隔室化对于cAMP信号至关重要的概念。我们将利用这项技术来研究乙醇对cAMP信号系统的影响，通过测试乙醇以亚细胞室特定方式影响营地代谢的假设。在特定的目标1中，我们将确定乙醇对HeLa细胞中cAMP代谢的影响。使用基于FRET的CAMP传感器EPAC1训练营，在不存在乙醇的情况下，将在单细胞水平上实时监测HeLa细胞中的cAMP水平。过去将证实乙醇对乙醇对cAMP代谢的影响的主要观察结果将得到证实。 EPAC1训练营将进行遗传修饰，以便将传感器靶向核，质膜和细胞质。将检查乙醇对这三个隔室的cAMP的影响。在特定目标2中，我们将确定磷酸二酯酶，蛋白激酶A和A-激酶锚定蛋白对观察到的乙醇作用的贡献。在具有这些蛋白质特异性的药理试剂的存在下，乙醇对三个隔室中cAMP代谢的影响将由FRET传感器检查。在特定的目标3中，我们将确定乙醇对原发性培养的神经元细胞亚细胞隔室中cAMP代谢的影响。从脑皮质和纹状体中分离出的原发性大鼠神经元将用靶向不同的亚细胞室（核，质膜和细胞质）的EPAC1-营地转染。在不存在和存在乙醇的情况下，将在高时空的分辨率中监测cAMP的水平。在这项研究过程中，我们将获得的知识和技术专业知识对于将来对乙醇对大脑营地代谢的影响的影响来说是必不可少的。已经假定，项目叙事循环腺苷单磷酸（CAMP）信号转导是促成人类酒精中毒的发展和倾向的一个因素。在这项研究过程中，我们将获得的知识和技术专业知识对于理解酒精对大脑营地代谢的影响是必不可少的，并且可能导致新一代的组织和细胞特异性药物的发展。