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

描述（由申请人提供）：本研究的长期目标是阐明乙醇对中枢神经系统中环磷酸腺苷（cAMP）信号通路影响的分子和细胞机制。cAMP信号转导被认为在动物对乙醇的生理和行为反应中以及在人类酒精中毒的发展和易感性中起关键作用。cAMP信号系统可以通过急性和慢性乙醇暴露来调节。以往的研究多采用免疫学和放射化学方法来测定乙醇对cAMP水平的影响，时间分辨率差，无空间分辨率。近年来，人们开发了单链cAMP传感器分子，它可以通过荧光共振能量转移（FRET）来监测cAMP水平。使用这些传感器分子，它是可能的，以高时空分辨率的亚细胞水平上的cAMP浓度的实时动态变化的研究。新出现的实验证据支持亚细胞区室化对cAMP信号传导至关重要的概念。我们将利用这项技术研究乙醇对cAMP信号系统的影响，通过测试的假设，乙醇影响cAMP代谢的亚细胞区室特异性的方式。在具体目标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Epac 1-camp，在不存在和存在乙醇的情况下，在单个细胞水平上实时监测HeLa细胞中的cAMP水平。过去通过放射化学方法研究的乙醇对cAMP代谢影响的关键观察结果将得到证实。epac 1-camps将被基因改造，使传感器可以针对细胞核，质膜和细胞质。将检查乙醇对这三个隔室中cAMP的影响。在具体目标2中，我们将确定磷酸二酯酶，蛋白激酶A和A-激酶锚定蛋白对乙醇所观察到的影响的贡献。在存在对这些蛋白质特异性的药理学试剂的情况下，将通过FRET传感器检查乙醇对三个隔室中的cAMP代谢的影响。在具体目标3中，我们将确定乙醇对原代培养的神经元细胞亚细胞区室中cAMP代谢的影响。将从大脑皮层和纹状体分离的原代大鼠神经元用靶向不同亚细胞区室（细胞核、质膜和细胞质）的Epac 1-camp转染。在不存在和存在乙醇的情况下，将以高时空分辨率监测cAMP水平。我们将在本研究过程中获得的知识和技术专长对于未来研究乙醇对大脑中cAMP代谢的影响是不可或缺的。项目叙述环磷酸腺苷（cAMP）信号传导被认为是人类酗酒发展和易感性的一个促成因素。我们将在本研究过程中获得的知识和技术专长对于理解酒精对大脑中cAMP代谢的影响是不可或缺的，并可能导致新一代组织和细胞特异性药物的开发。

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.