Secretion Coupled Endocytosis in Neuroendocrine Cells

神经内分泌细胞的分泌耦合内吞作用

• 批准号: 6579951
• 负责人: CRISTINA R ARTALEJO
• 金额: $ 6.93万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6579951
• 关键词: PC12 cells; calcium channel; dynamin; endocytosis; neuroendocrine system; protein isoforms; receptor mediated endocytosis; secretion

DESCRIPTION (Applicant's abstract): Regional deficits of monoamine transmitters are thought to play a critical role in the pathogenesis of various mental diseases, hence it is necessary to have detailed knowledge of the mechanisms underlying the secretion of these agents. The secretory cycle in neurons and neuroendocrine cells involves the release of neurotransmitters and hormones by exocytosis followed by recovery of vesicular membrane by endocytosis. We recently analyzed a novel form of endocytosis, called rapid endocytosis (RE), which occurs in a variety of secretory cells including neurons and is tightly coupled to secretion. RE is distinct from receptor-mediated endocytosis (RME) as it is a much faster process and does not involve clathrin. The protein dynamin is thought to participate in the vesicle fission reaction that is critical to various forms of endocytosis. Multiple forms of dynamin exist, and in this proposal we seek to establish if different isoforms are involved in distinct types of endocytosis as well as determine which domains of dynamin are essential to the function of the protein. For these studies we propose to use the PC12 cell line, a pheochromocytoma that exhibits properties of both adrenal chromaffin (AC) cells and sympathetic neurons. First, we will demonstrate that PC12 cells provide a useful model system for the molecular analysis of the secretory cycle in neuroendocrine cells. We will characterize the kinetics of exocytosis and endocytosis in these cells and assess whether the properties of these processes resemble those previously seen in AC cells. Second we will determine the dynamin isoforms expressed in these cells and their role in RE and RME. For this purpose we will employ a battery of molecular and immunological procedures, including isoform-specific antibodies. Thirdly, we will dissect which dynamin domains are essential for RE and RME, focussing on the pleckstrin-homology (PH) domain and the proline-rich C-terminal. We will introduce a variety of dynamin constructs into PC12 cells followed by measurement of RE and RME. In these studies we will use state-of-the-art electrophysiological recordings including measurements of membrane capacitance to directly monitor exocytosis and RE and amperometric detection of catecholamine release from single cells. These studies will have significance for secretory biology in general and should further our understanding of the cellular mechanisms of monoaminergic transmission so necessary for the development of novel therapeutic strategies.

描述（申请人摘要）：单胺递质的局部缺乏 被认为在各种精神疾病的发病机制中起着关键作用 疾病，因此有必要详细了解其机制 这些物质的分泌。神经元的分泌周期和 神经内分泌细胞涉及神经递质和激素的释放， 胞吐作用，随后通过胞吞作用恢复囊泡膜。我们 最近分析了一种新型的内吞作用，称为快速内吞作用（RE）， 它发生在包括神经元在内的各种分泌细胞中， 再加上分泌物RE不同于受体介导的内吞作用（RME） 因为这是一个快得多的过程并且不涉及网格蛋白。蛋白质 动力蛋白被认为参与囊泡分裂反应， 对各种形式的内吞作用至关重要。发动蛋白有多种形式， 在这个建议中，我们试图确定是否涉及不同的同种型， 不同类型的内吞作用，以及确定哪些领域的发动蛋白是 对蛋白质的功能至关重要。对于这些研究，我们建议使用 PC 12细胞系是一种嗜铬细胞瘤， 嗜铬（AC）细胞和交感神经元。首先，我们将证明， PC 12细胞提供了一个有用的模型系统，用于分子分析， 神经内分泌细胞的分泌周期。我们将描述 这些细胞中的胞吐和胞吞作用，并评估 这些过程类似于先前在AC细胞中看到的过程。第二，我们将 确定这些细胞中表达的动力蛋白亚型及其在RE中的作用 和RME。为此，我们将采用一系列分子和 免疫程序，包括同种型特异性抗体。三是 将剖析哪些发动蛋白域是RE和RME所必需的，重点是 pleckstrin-homology（PH）结构域和富含脯氨酸的C-末端。我们将 将多种发动蛋白构建体引入PC 12细胞， 测量RE和RME。在这些研究中，我们将使用最先进的 包括膜电容测量的电生理记录 直接监测胞吐作用和RE， 单细胞释放的儿茶酚胺。这些研究将具有重要意义 为分泌生物学的一般，并应进一步我们的理解， 单胺能传递的细胞机制， 开发新的治疗策略。