EXOCYTOSIS FROM ADRENAL CHROMAFFIN CELLS

肾上腺分泌儿茶酚胺的Ca ~（2+）依赖性 髓质嗜铬细胞不仅是 肾上腺髓质的功能，但也作为一个模型， 儿茶酚胆碱和其他的胞吐释放 神经末梢和细胞的神经递质和激素。 研究的持续重点是识别和调查 利用培养的单层细胞进行胞吐作用的生化基础 肾上腺髓质嗜铬细胞 短暂孵育 低浓度毛地黄皂苷的嗜铬细胞使 质膜可渗透Ca 2+、ATP和蛋白质， 允许微摩尔Ca 2+直接刺激胞吐作用。 我们 最近证明，不仅低分子量 此外，蛋白质如胰蛋白酶（27 kDa）和肌球蛋白轻 链激酶（130 kDa）可被引入毛地黄皂苷处理的 细胞及其对细胞内生化过程的影响， 研究分泌物。 在一些拟议的研究中， 洋地黄素处理的细胞将用于研究直接 各种操作对Ca 2+依赖性分泌的影响， 细胞内的生化反应，如蛋白质 磷酸化（用γ-32 P）ATP作为底物）。 可溶性 细胞溶质因子包括蛋白质从洋地黄皂苷处理的 嗜铬细胞伴钙依赖性 分泌物 拟议研究的一个重要目标是 确定Ca 2+依赖性分泌是否可以 通过添加可溶性细胞溶质成分 包括蛋白质。 初步数据表明，这种方法是 可行 在提案中，完整和毛地黄皂苷透化 嗜铬细胞将用于研究：1）蛋白质的作用 2）ATP非依赖性分泌及其与细胞内分泌的关系 与ATP依赖性分泌的关系; 3）机制 作为嗜铬颗粒原位渗透稳定性的基础， 分泌囊泡; 4）胞浆蛋白在 胞吐作用; 5）嗜铬颗粒膜蛋白的作用 是使用针对蛋白质的抗体的胞吐作用;以及6） 溶酶体酶释放调节与组成型 蛋白质分泌通过不涉及 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.