FORMATION OF ADRENAL MEDULLA CHROMAFFIN VESICLES

这些研究的长期目标是通过以下方式了解这一过程 这些嗜铬小泡是形成的。建议的具体目标是 研究是为了获得关于生物发生和更替的信息 嗜铬小泡及胰岛素、神经生长因子、 地塞米松和细胞刺激对这些过程的影响。的原生文化 肾上腺髓质细胞将被用于研究特定的 可溶性蛋白质(嗜铬颗粒蛋白和可溶性多巴胺-β-羟基糖酶 [S-DBH]与儿茶酚胺一起在 特定膜蛋白(膜结合蛋白)的分泌和合成 由细胞保留的DBH[M-DBH]，并将它们结合到 嗜铬小泡。由于胸径是这两种可溶物的组成部分 而囊泡的膜可以作为这两种情况的标志 分数。细胞将与[35S]蛋氨酸和它的 嗜铬小泡对S胸径、M胸径和嗜铬颗粒的掺入 和其他通过免疫沉淀测定的亚细胞组分 用凝胶电泳法检测特异性抗血清。初生嗜铬 通过离心法将成熟囊泡从成熟囊泡中分离出来。 连续的蔗糖密度梯度。标记蛋白质的周转 成熟的囊泡将作为培养时间的函数进行研究。 再利用囊泡膜形成新生囊泡 将在双同位素标记实验中进行检测。具有以下特性的细胞 将[35S]蛋氨酸掺入成熟的嗜铬小泡中将是 使用分泌物治疗，并在暴露于不同的时间后 [~H]蛋氨酸。M-胸径、S胸径和嗜铬粒体素的~(35)S/~3H比值 在新生的囊泡中的存在将被确定并与 未受刺激的细胞。胰岛素、地塞米松与神经生长的关系 影响特定蛋白质合成的因素，以及它们的掺入 新生的囊泡和成熟的囊泡的速度 下定决心。这些研究将提供有关生命周期的基本信息 嗜铬细胞囊泡的。因为肾上腺髓质和交感神经 神经元具有与合成、储存和分泌有关的共同特征 对儿茶酚胺，预计这些研究也将 与交感神经的突触小泡有关 在调节心血管系统和其他方面的生理作用 自主神经功能。

The long term objectives of these studies is to understand the process by which chromaffin vesicles are formed. The specific aim of the proposed studies are to obtain information on the biogenesis and turnover of chromaffin vesicle and the effects of insulin, nerve growth factor, dexamethasone and cell stimulation on these processes. Primary cultures of adrenal medulla cells will be used to study the biosynthesis of specific soluble proteins (chromogranins and soluble dopamine-Beta-hydroxglase [S-DBH] which are released together with the catecholamines during secretion and the synthesis of a specific membrane protein (membrane-bound DBH [M-DBH] which is retained by the cell, and their incorporation into chromaffin vesicles. Since DBH is a component of both the soluble content and the membrane of the vesicle it serves as a marker for both of these fractions. Cells will be incubated with [35S] methionine and its incorporation into S-DBH, M-DBH and chromogranins of chromaffin vesicles and other subcellular fractions determined by immunoprecipitation with specific anti-sera followed by gel electrophoresis. Nascent chromaffin vesicles will be separated from mature vesicles by centrifugation through continuous sucrose density gradients. The turnover of the labeled proteins in mature vesicles will be studied as a function of time in culture. Reutilization of vesicle membranes for the formation of nascent vesicles will be examined in double isotope labeling experiments. Cells which have incorporated [35S]methionine into mature chromaffin vesicles will be treated with secretogues and after various periods of time exposed to [3H]methionine. The 35S/3H ratios in M-DBH, S-DBH and chromogranins present in nascent vesicles will be determined and compared to those of unstimulated cells. The effects of insulin, dexamethasone and nerve growth factor on the synthesis of the specific proteins, on their incorporation into nascent vesicles and on the rate of maturation of vesicles will be determined. These studies will provide basic information on the life cycle of the chromaffin vesicle. Since the adrenal medulla and sympathetic neuron have common features related to the synthesis, storage and secretion of catecholamines, it is anticipated that these studies will also be relavant to the synaptic vesicle of the sympathetic nerve and its physiological role in the regulation of the cardiovascular system and other autonomic functions.