EXOCYTOSIS FROM ADRENAL CHROMAFFIN CELLS

肾上腺儿茶酚胺的钙依赖性分泌物 髓质嗜铬细胞不仅是 肾上腺髓质的功能，但也是一个模型 儿茶酚胆胺和其他物质的胞吐释放 神经末梢和细胞的神经递质和激素。 研究的持续重点是确定和调查 培养细胞单层胞吐作用的生化基础 肾上腺髓质嗜铬细胞。短暂的孵化 含有低浓度洋地黄素的嗜铬细胞使 质膜对Ca~(2+)、ATP和蛋白质的通透性 允许微摩尔的钙离子直接刺激胞吐。我们 最近证明，不仅是低分子量 物种，但也包括蛋白质，如胰酶(27 KDa)和肌球蛋白 洋地黄处理后可引入130 kDa的链激酶 细胞及其对细胞内生化过程的影响 分泌物调查。在一些拟议的研究中 经过数字处理的细胞将被用于研究直接 不同手法对大鼠钙离子依赖性分泌的影响 蛋白质等细胞内生化反应的研究 磷酸化(以伽马-32P)ATP为底物)。可溶的 洋地黄治疗后产生的包括蛋白质在内的胞浆因子 伴随钙依赖性丧失的嗜铬细胞 分泌物。拟议研究的一个重要目标是 确定钙离子依赖的分泌是否可以 通过添加回可溶的、胞浆成分而重构的 包括蛋白质。初步数据表明，这种方法是 可行。在该提案中，既有完整的，也有洋地黄素渗透的 嗜铬细胞将被用来研究：1)蛋白质的作用 分泌物中的激酶C；2)ATP非依赖性分泌物及其 与ATP依赖性分泌的关系；3)机制 嗜铬颗粒的原位渗透稳定性， 分泌小泡；4)胞浆蛋白在细胞中的作用 胞吐作用；5)嗜铬颗粒膜蛋白的作用 是否使用针对蛋白质的抗体进行胞吐作用；以及6) 溶酶体酶解酶的释放和构成的调控 通过不涉及的途径进行的蛋白质分泌 嗜铬颗粒。

The Ca2+-dependent secretion of catecholamines from adrenal medullary chromaffin cells is not only fundamental to the function of the adrenal medulla, but also serves as a model for exocytotic release of catechocholamines and other neurotransmitters and hormones from nerve terminals and cells. The continuing focus of the reserch is to identify and investigate the biochemical basis for exocytosis using monolayers of cultured adrenal medullary chromaffin cells. Brief incubation of chromaffin cells with low concentrations of digitonin renders the plasma membrane permeable to Ca2+, ATP and proteins and allows micromolar Ca2+ to stimulate directly exocytosis. We have recently demonstrated that not only low molecular weight species but also proteins such as trypsin (27 kDa) and myosin light chain kinase (130 kDa) can be introduced into digitonintreated cells and their effects on intracellular biochemical processes and secretion investigated. In some of the proposed studies digitomintreated cells will be used to investigate the direct effects of various manipulations on Ca2+-dependent secretion and on intracellular biochemical reactions such as protein phosphorylation (with gamma-32P)ATP as a substrate). Soluble cytosolic factors including proteins exit from digitonintreated chromaffin cells with a concomittant loss of Ca2+-dependent secretion. One important goal of the proposed studies is to determine whether Ca2+-dependent secretion can be reconstituted by adding back soluble, cytosolic constituents including proteins. Preliminary data suggest that this approach is feasible. In the proposal both intact and digitonin-permeabilized chromaffin cells will be used to investigate: 1) the role of protein kinase C in secretion; 2) ATPindependent secretion and its relationship to ATPdependent secretion; 3) mechanisms underlying the in situ osmotic stability of chromaffin granules, the secretory vesicles; 4) the role of cytosolic protein in exocytosis; 5) the role of a chromaffin granule membrane protein is exocytosis using antibodies directed against the protein; and 6) the regulation of lysosomal enyzme release and constitutive protein secretion which occur via pathways not involving chromaffin granules.