STIMULUS-EVENT COUPLING IN CHROMAFFIN SECRETORY GRANULES

说明(改编自应用程序) 在交感肾上腺嗜铬细胞分泌颗粒中，共存储 儿茶酚胺和多肽通过胞吐作用共同分泌 乙酰胆碱或新型非胆碱能神经递质垂体 腺苷环化酶激活肽(PACAP)。嗜铬颗粒/ 分泌颗粒蛋白(Cg/Sg)，一种在岩心中发现的酸性、可溶性蛋白质家族 胺和多肽荷尔蒙和神经递质分泌囊泡，具有 在神经内分泌中，这种小泡几乎无处不在。 系统。它们的蛋白分解片段对激素或 神经递质的释放以及不同部位的靶细胞。为 例如，嗜铬粒素A(CGA)片段血管抑素和儿茶素对照 血管反应性和儿茶酚胺释放与CGA片段胰腺抑素 会升高血糖。这项建议制定了3个具体目标，旨在寻求 了解因分泌而发生的分泌颗粒事件 刺激神经内分泌细胞，如嗜铬细胞。对以下问题的回应 被研究的是分泌物和基因表达(转录)。在目标1中，我们 将研究儿茶酚胺分泌和CGA基因反式激活 神经节前刺激嗜铬细胞后的反应减敏 以及这些反应的脱敏是否涉及G 蛋白偶联受体激酶(GRK)、β-阻滞素和/或第二 信使激活的激酶。在目标2中，我们将使用一部小说的荧光 CGA-EGFP嵌合体(增强型绿色荧光蛋白)原位检测 离散的CGA一级结构域是否对 CGA进入调节分泌途径的运输以及CGA是如何 与分泌颗粒的其他分泌或膜蛋白相互作用。 最后，我们将使用这个嵌合的CGA-EGFP蛋白作为校准的H+(PH) 传感器(目标3)，以原位调查，囊泡内pH和 它在生理促分泌器触发的分泌过程中的作用， 如PACAP或乙酰胆碱，或通过拟交感神经的胺。

DESCRIPTION (adapted from the application) In secretory granules of sympathoadrenal chromaffin cells, co-stored catecholamines and peptides are cosecreted by exocytosis in response to acetylcholine or to the novel non-cholinergic neurotransmitter pituitary adenylyl cyclase-activating polypeptide (PACAP). The chromogranins/ secretogranins (Cg/Sg), a family of acidic, soluble proteins found in the cores of amine and peptide hormone and neurotransmitter secretory vesicles, have a virtually ubiquitous occurrence in such vesicles throughout the neuroendocrine system. Their proteolytic fragments exert effects on hormone or neurotransmitter release as well as target cells at a variety of sites. For example, chromogranin A (CgA) fragments vasostatin and catestatin control vasoreactivity and catecholamine release, and CgA fragment pancreastatin elevates blood glucose. This proposal develops 3 specific Aims in seeking to understand secretory granule events which occur in response to secretory stimuli in neuroendocrine cells such as the chromaffin cell. The responses to be investigated are secretion and gene expression (transcription). In Aim 1, we will investigate how catecholamine secretion and CgA gene transactivation responses desensitize following pre-ganglionic stimulation of chromaffin cells by PACAP and whether desensitization of these responses involves G protein-coupled receptor kinases (GRK), beta-arrestins and/or second messenger-activated kinases. In Aim 2, we will use the fluorescence of a novel CgA-EGFP chimera (Enhanced Green Fluorescent Protein) to determine in situ whether discrete CgA primary structural domains are responsible for the trafficking of CgA into the regulated pathway of secretion, and how CgA interacts with other secreted or membrane proteins of the secretory granule. Finally, we will use this chimeric CgA-EGFP protein as a calibrated H+ (pH) sensor (Aim 3) to investigate in situ, the dynamics of intravesicular pH and its role in the secretory process triggered by the physiologic secretagogues, such as PACAP or acetylcholine, or by sympathomimetic amines.