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

描述(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磷酸腺苷(CAMP)信号通路影响的分子和细胞机制。CAMP信号转导通路在动物对乙醇的生理和行为反应以及人类酒精中毒的发生和易感性中起关键作用。急性和慢性乙醇暴露均可调节cAMP信号系统。以往的研究多采用免疫和放射化学方法测定乙醇对脑组织cAMP水平的影响，但时间分辨率和空间分辨率较低。最近发展起来的单链多肽链cAMP传感器分子，可以通过荧光共振能量转移(FRET)来监测cAMP水平。利用这些传感器分子，可以在高时空分辨率的亚细胞水平上研究cAMP浓度的实时动态变化。新出现的实验证据支持亚细胞区隔对cAMP信号转导至关重要的观点。我们将利用这项技术来研究乙醇对cAMP信号系统的影响，方法是检验乙醇以亚细胞室特异性方式影响cAMP代谢的假设。在特定的目标1中，我们将确定乙醇对HeLa细胞cAMP代谢的影响。使用基于FRET的cAMP传感器Epac1-cAMPS，在没有乙醇和存在乙醇的情况下，将在单细胞水平上实时监测HeLa细胞中的cAMP水平。过去用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。Epac1-cAMPS将经过基因改造，这样传感器就可以定位到细胞核、质膜和细胞质。乙醇对这三个隔室内cAMP的影响将被检测。在特定目标2中，我们将确定磷酸二酯酶、蛋白激酶A和A-激酶锚定蛋白在乙醇所观察到的效应中的作用。在存在针对这些蛋白质的特定药理试剂的情况下，乙醇对三个隔室内cAMP代谢的影响将通过FRET传感器进行检测。在具体目标3中，我们将确定乙醇对原代培养的神经细胞亚细胞室内cAMP代谢的影响。从大脑皮层和纹状体分离的原代大鼠神经元将被靶向不同亚细胞室(核、质膜和细胞质)的Epac1-cAMP基因导入。在没有乙醇和存在乙醇的情况下，将以高时空分辨率监测cAMP水平。我们在这项研究过程中将获得的知识和技术专长，对于未来研究乙醇对大脑cAMP代谢的影响是不可或缺的。项目叙述环磷酸腺苷(CAMP)信号转导被认为是人类酒精中毒发生和易感性的一个促成因素。我们在这项研究过程中将获得的知识和技术专长，对于了解酒精对大脑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.