Secretion From Individual Vesicles

描述（由申请人提供）：化学通讯包括从生物细胞释放化学信使，扩散到靶细胞，与特定受体结合，以及从细胞外空间清除。将碳纤维微电极（外加电位足以氧化儿茶酚胺）放置在孤立的分泌细胞旁，通过电氧化检测到的由离散的儿茶酚胺包组成的一系列电流峰值，可以跟踪胞吐作用。为了评估胞吐后的事件，碳纤维微电极可以放置在保持在生理缓冲液中的组织切片中。单细胞测量允许评估单个胞外事件，而组织切片测量提供释放物质扩散和清除的关键信息。在这个建议中，我们提出了一个计划，利用我们已经开发的电化学方法来研究一些生化和物理化学方面，这些方面是化学通信的核心。在这些研究中，我们将在单细胞和完整组织切片中探索释放事件的动力学。因此，这一建议扩展了我们之前的工作，提供了一个更完整的观点，从胞外作用到它们从细胞外液中去除的化学信使浓度的调节。三个特定的蛋白质，每个位于中心调控位置，将成为靶标；对于每一种蛋白质，我们都可以使用转基因小鼠来删除或添加这些蛋白质。第一种是解偶联蛋白2 (UCP2)，这是一种在神经元中发现的线粒体蛋白，是三磷酸腺苷（ATP）产生的关键调节因子。第二种是突触蛋白，一种对神经末梢的水疱定位至关重要的蛋白质。第三种是多巴胺转运蛋白（DAT），这种蛋白质负责将多巴胺重新整合回神经元中。因此，本建议的具体目的是：检查UCP2的作用，UCP2是一种线粒体蛋白，可以减少ATP的产生。我们将研究它对神经递质储存和释放的影响。2. 检查突触素在决定囊泡释放的可用性中的作用。突触蛋白是一种丰富的囊泡膜蛋白，已被提出调节囊泡释放的可用性。3. 检查过表达DAT的小鼠大脑中多巴胺的细胞外寿命。我们将研究这种蛋白过表达对多巴胺能神经传递的影响。4. 检测在含有酪氨酸羟化酶（TH）的神经元中表达绿色荧光蛋白（GFP）的转基因小鼠大脑中多巴胺的释放。5. 评估肾上腺释放儿茶酚胺的控制机制。通过在肾上腺片内精确放置碳纤维微电极，我们将监测儿茶酚胺从胞外释放到运输到血管的全过程。这项研究将为中枢神经和神经内分泌系统的化学通讯提供一个前所未有的视角。公共卫生相关性：化学通讯包括从生物细胞释放化学信使、分泌物质向靶细胞扩散、与特定受体结合以及从细胞外空间清除。在拟议的研究中，这些过程将通过放置在分离细胞附近或植入脑组织切片的化学传感微电极进行研究。

DESCRIPTION (provided by applicant): Chemical communication involves release of chemical messengers from biological cells, their diffusion to target cells, binding to specific receptors, and clearance from the extracellular space. Amperometry at a carbon-fiber microelectrode (with an applied potential sufficient to oxidize catecholamines) placed next to an isolated secretory cell can follow exocytosis as a series of current spikes composed of discrete packets of catecholamines detected by their electrooxidation. To evaluate events after exocytosis, carbon-fiber microelectrodes can be placed in tissue slices maintained in physiological buffer. The single cell measurements allow evaluation of individual exocytotic events while the tissue slice measurements provide key information on the diffusion and clearance of the released substances. In this proposal we set forth a plan to investigate some biochemical and physiochemical aspects that are central to chemical communication using the electrochemical approaches that we have developed. In these investigations we will probe the dynamics of release events both at single cells and in intact tissue slices. Thus, this proposal expands upon our prior work by providing a more complete view of the regulation of concentrations of chemical messengers from exocytosis to their removal from the extracellular fluid. Three specific proteins, each positioned at a central regulatory location, will be targets; for each protein, transgenically altered mice are available to us with deletions or additions of these proteins. The first is uncoupling protein 2 (UCP2), a mitochondrial protein found within neurons that is a key regulator of adenosine triphospate (ATP) production. The second is synapsin, a protein that is central to vesicular localization within nerve terminals. The third is the dopamine transporter (DAT), the protein responsible for reincorporation of dopamine back into its neurons. Thus, the specific aims of this proposal are: 1. Examine the role of UCP2, a mitochondrial protein that can diminish ATP production. We will examine its effects on neurotransmitter storage and release. 2. Examine the role of synapsin in determining the availability of vesicles for release. Synapsin, an abundant vesicular membrane protein, has been proposed to regulate the availability of vesicles for release. 3. Examine the extracellular lifetime of dopamine in the brains of mice that overexpress the DAT. We will examine the consequences of overexpression of this protein on dopaminergic neurotransmission. 4. Examine dopamine release in the brains of transgenic mice that express green-fluorescent protein (GFP) in neurons containing tyrosine hydroxylase (TH). 5. Evaluate control mechanisms of released catecholamines within the adrenal gland. Through precise placement of carbon-fiber microelectrodes within adrenal slices, we will monitor catecholamines from their exocytotic release to their transport to the blood vessels. The proposed research will provide an unprecedented view of chemical communication in the central nervous and neuroendocrine systems. PUBLIC HEALTH RELEVANCE: Chemical communication involves release of chemical messengers from biological cells, diffusion of secreted substances to target cells, binding to specific receptors, and clearance from the extracellular space. In the proposed research, these processes will be investigated with chemical sensing microelectrodes placed adjacent to isolated cells or implanted in slices of brain tissue.