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FORMATION OF ADRENAL MEDULLA CHROMAFFIN VESICLES

肾上腺髓质嗜铬囊泡的形成

基本信息

批准号：
3152691
负责人：
NORMAN KIRSHNER
金额：
$ 9.61万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-12-01 至 1986-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3152691
关键词：
adrenal medulla autoradiography catecholamines chromaffin cells chromatography density gradient ultracentrifugation dexamethasone dopamine beta monooxygenase gel electrophoresis immunochemistry insulin methionine neurotrophic factors radiotracer secretion synaptic vesicles tissue /cell culture

项目摘要

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.

这些研究的长期目标是通过以下方式了解这一过程：形成嗜铬小泡。建议的具体目标研究的目的是获得关于生物起源和周转的信息，嗜铬小泡和胰岛素，神经生长因子，地塞米松和细胞刺激对这些过程的影响。的原代培养物肾上腺髓质细胞将被用于研究特定的可溶性蛋白质（嗜铬粒蛋白和可溶性多巴胺-β-羟化酶 [S-DBH]，其与儿茶酚胺一起释放，分泌和合成特定的膜蛋白（膜结合的 DBH [M-DBH]，其由细胞保留，并且它们并入嗜铬小泡由于DBH是可溶性含量的一个组成部分，而囊泡的膜则是这两者的标志分数将细胞与[35 S]甲硫氨酸及其掺入嗜铬小泡的S-DBH、M-DBH和嗜铬粒蛋白和其他亚细胞组分，通过免疫沉淀法测定，特异性抗血清，然后进行凝胶电泳。新生嗜铬细胞通过离心将囊泡与成熟囊泡分离，连续蔗糖密度梯度。标记蛋白的周转在成熟的囊泡中，将作为培养中时间的函数进行研究。囊泡膜的再利用以形成新生囊泡将在双同位素标记实验中进行检查。细胞具有将[35 S]蛋氨酸掺入成熟的嗜铬小泡中，用分泌物处理，并在暴露于 [3 H]蛋氨酸。 M-DBH、S-DBH和嗜铬粒蛋白中的~（35）S/~ 3 H比值将确定新生囊泡中存在的蛋白质，并与未刺激的细胞胰岛素、地塞米松对神经生长的影响因子对特定蛋白质的合成，对它们的掺入进入初生囊泡和囊泡的成熟速度将是测定这些研究将提供关于生命周期的基本信息嗜铬囊泡的。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于儿茶酚胺，预计这些研究也将与交感神经的突触囊泡及其在调节心血管系统和其他方面的生理作用自主功能