EXOCYTOSIS FROM ADRENAL CHROMAFFIN CELLS

激素和神经递质的胞吐释放是 细胞间通讯和突触传递。该过程 对内分泌、心血管和 神经系统在上一个资助期， 在定义参与Ca2+激活胞吐途径的步骤时， 使用完整的和毛地黄皂苷透化的牛嗜铬细胞。一个 竞争性更新的重要部分是延长 通过实施我们开发的两个新系统进行调查 来研究分泌。它们让我们可以更直接地操纵 迄今为止分泌途径的可能成分。我们已经开发 牛嗜铬细胞中的新型瞬时转染系统（原代， 非分裂细胞），以研究特定蛋白质在 分泌途径该方法的一个关键部分是与 目标蛋白，人生长激素（GH），其充当报告基因 在一小部分细胞中， 都被感染了我们还发现， 进入蛙卵母细胞进行胞吐作用。这种重建技术允许 我们第一次操纵分泌囊泡除了 细胞探索必要的分泌囊泡的组成部分， 胞吐作用这些新系统将使我们能够提出更精确的问题 关于该途径的生化机制。的 这两种系统的发展使我们能够使用功能测定， 研究嗜铬颗粒以及其他细胞蛋白成分， 胞吐作用将研究以下主题：1） GTP结合蛋白rab3a调节肾上腺嗜铬细胞胞吐作用 细胞2)突触结合蛋白（p65）在胞吐中的作用， 分泌囊泡的膜蛋白。3)的作用 多磷酸肌醇（PPI）在胞吐中的作用。4)的识别 嗜铬颗粒膜上的蛋白质成分在 青蛙卵母细胞的胞吐反应。

Exocytotic release of hormones and neurotransmitters is central to intercellular communication and synaptic transmission. The process is fundamental to the normal function of the endocrine, cardiovascular and nervous systems. In the previous grant period we made fundamental advances in defining the steps involved in the pathway of Ca2+-activated exocytosis using intact and digitonin permeabilized bovine chromaffin cells. An important part of the competing renewal is the extension of the investigations by implementation of two new systems that we have developed for studying secretion. They allow us to manipulate more directly than heretofore possible components of the secretory pathway. We have developed a novel transient transfection system in bovine chromaffin cells (primary, non-dividing cells) to study the role of specific proteins in the secretory pathway. A key part of the approach is to coexpress with a protein of interest, human growth hormone (GH), which serves as a reporter for the regulated secretory pathway in the small fraction of cells that are transfected. We also found that chromaffin granule membranes injected into frog oocytes undergo exocytosis. This reconstitution technique allows us for the first time to manipulate the secretory vesicle apart from the cell to explore components of the secretory vesicle necessary for exocytosis. These new systems will allows us to ask more precise questions concerning the biochemical mechanisms underlying the pathway. The development of both these systems allows us to use functional assays to study chromaffin granule as well as other cellular protein components in exocytosis. The following topics will be investigated: 1) The role of the GTP-binding protein rab3a in regulated exocytosis in adrenal chromaffin cells. 2) The role in exocytosis of synaptotagmin (p65), an integral membrane protein of secretory vesicles. 3) The role of polyphosphoinositides (PPIs) in exocytosis. 4) The identification of protein components on the chromaffin granule membrane important in the exocytotic response in frog oocytes.