Sympathetic Outflow to Catecholamine Storage Vesicles

描述(由申请人提供)： 高血压患者，节后交感神经和嗜铬细胞的传出交感神经活性增加，导致血管收缩、钠滞留和血压升高。嗜铬颗粒素/分泌颗粒素(Chromoranin A[CGA].嗜铬粒蛋白B[CGB]和分泌性颗粒蛋白II[SgII]，是一类酸性蛋白，存在于胺、肽类激素和神经递质分泌小泡的核心，如嗜铬颗粒和节后交感神经(去甲肾上腺素能)轴突的大而致密的核心小泡。CGA片段抑制儿茶酚胺的释放，CGA片段血管抑素扩张阻力血管，CGA片段胰岛抑素拮抗胰岛素分泌和血糖升高。这项建议开发了三个目标，利用新的靶向光蛋白结构(荧光素酶或绿色荧光蛋白[GFP])或新表达的cDNA，来阐明交感神经外流如何改变儿茶酚胺储存囊泡的组成。在目标1中，我们利用新型转基因小鼠品系，携带CgA或SgII启动子/荧光素酶报告基因，在体内探索交感肾上腺系统刺激/转录(刺激/分泌/合成)耦合的机制。在目标2中，我们使用一系列新的靶向CGA结构域/绿色荧光蛋白(CGA/EGFP)嵌合体来发现儿茶酚胺储存囊泡蛋白一级结构中的信息，这些信息解释了它们进入调节的分泌途径。在目标3中，我们使用一种新的cDNA表达克隆方法来鉴定分泌器官中反式蛋白，这些反式蛋白与CGA相互作用，从而为其运输到受调控的分泌途径奠定了基础。这些研究将加深我们对儿茶酚胺储存囊泡的生物合成以及在交感神经刺激时其分泌蛋白的补充的理解。最后，这些光蛋白和cDNA试剂可以被其他研究者广泛用于阐明交感神经嗜铬细胞系统中的生物合成、运输和离子通量事件。

DESCRIPTION (provided by applicant): In hypertension, efferent sympathetic activity to post-ganglionic sympathetic nerves and chromaffin cells is increased, contributing to vasoconstriction, sodium retention, and elevations in blood pressure. The chromogranins/secretogranins (chromogranin A [Cga]. Chromogranin B [Cgb]. And secretogranin II [SgII], are a family of acidic proteins found in cores of amine and peptide hormone and neurotransmitter secretory vesicles, such as chromaffin granules and large dense core vesicles of post-ganglionic sympathetic (noradrenergic) axons. Their biologically active fragments act on hormone or neurotransmitter release as well as on target cells at several sites: CgA fragment catestatin inhibits catecholamine release, CgA fragment vasostatin dilates resistance vessels, CgA fragment pancreastatin antagonizes insulin secretion and elevates blood glucose. This proposal develops 3 aims, employing novel targeted photoprotein constructs (luciferase or green fluorescent protein [GFP]) or novel expressed cDNAs, to elucidate how sympathetic outflow changes the composition of catecholamine storage vesicles. In Aim 1, we employ novel transgenic mouse strains, harboring CgA or SgII promoter/luciferase reporters, to probe mechanisms of stimulus/transcription (stimulus/secretion/synthesis) coupling in the sympathoadrenal system in vivo. In Aim 2, we use a series of novel, targeted CgA domain/green fluorescent protein (CgA/EGFP) chimeras to discover information within the primary structure of catecholamine storage vesicle proteins that accounts for their trafficking into the regulated secretory pathway. In Aim 3, we use a novel cDNA expression cloning approach to identify proteins in trans, within the secretory apparatus, which interact with CgA and thereby underlie its trafficking into the regulated secretory pathway. These studies will enhance our understanding of the biosynthesis of catecholamine storage vesicles and the replenishment of their secretory proteins during sympathetic stimulation. Finally, these photoprotein and cDNA reagents can be widely employed by other investigators in elucidating biosynthetic, trafficking, and ion flux events in the sympathochromaffin system.