Molecular control of differential peptide transmitter exocytosis.

描述(申请人提供)：环境威胁、体力消耗或损伤以及心理紧张都会导致交感-肾上腺“不战即逃”应激反应的启动。神经内分泌肾上腺髓质嗜铬细胞从交感内脏神经接受兴奋性突触输入。内脏激活导致肾上腺嗜铬细胞释放儿茶酚胺以及一系列不同的神经和血管活性多肽递质进入血液循环。不同水平的压力导致不同的释放 儿茶酚胺与多肽递质形成适当的生理反应。所有分泌的激素、儿茶酚胺以及多种多肽递质都被共同包装在相同的分泌颗粒中。因此，差异激素的释放是在颗粒融合后的一个步骤中调节的。在交感神经刺激的基础嗜铬细胞刺激下，自由溶儿茶酚胺的选择性释放通过短暂的融合事件发生，其特征是颗粒腔和细胞外间隙之间有一个狭窄的、有结构的胞外融合孔。在交感神经张力下，选择性和适度的儿茶酚胺释放在能量储存的“休息和消化”代谢状态中发挥重要作用，调节包括胰腺胰岛素分泌、增加内脏血流量和维持基础心脏活动在内的动态平衡生理功能。作为对应激的反应，嗜铬细胞刺激的增加调节了分泌颗粒融合的方式，导致胞外融合孔的扩张，以最大限度地释放儿茶酚胺，促进共包装的肾上腺肽递质的胞吐作用。血浆儿茶酚胺水平升高，再加上肾上腺衍生的多肽递质，是交感神经性“非战即逃”应激反应的核心效应因子。它们共同调节为防御或逃跑做准备的多个过程，包括全身性镇痛(脑啡肽)、心输出量增加(儿茶酚胺增加)、流向骨骼肌的血液(心房利钠因子、神经肽Y)和血糖(胰岛抑素)。因此，分泌融合孔的调节扩张是急性应激反应的关键因素。了解导致气孔膨胀的分子机制是我们的总体目标。我们总结了前人的工作，并提供了初步的数据来制定3个特定的目标，以测试活性依赖的Dynamin I去磷酸化事件，随后多聚体扩孔复合体的招募，以及在融合孔扩张调节中对肌球蛋白马达活性的要求。为了实现这些目标，我们使用了最先进的电生理、电化学和定量荧光显微镜以及最新开发的硅纳米线基场效应晶体管(SiNW-FET)生物传感器来测量特定的多肽释放。所获得的数据将提供对急性交感-肾上腺应激反应的关键调节因子的分子理解，以及由于其调节不当而导致的病理改变。

DESCRIPTION (provided by applicant): Environmental threat, physical exertion or injury and psychological strain all lead to the initiation of the sympatho-adrenal "fight or flight" stress response. Neuroendocrine adrenal medullary chromaffin cells receive excitatory synaptic input from the sympathetic splanchnic nerve. Splanchnic activation causes adrenal chromaffin cells to release catecholamines as well as a diverse array of neuro- and vaso-active peptide transmitters into the circulation. Different levels of stress result in the differential release of catecholamine versus peptide transmitters to formulate the appropriate physiological response. All secreted hormones, catecholamines as well as multiple species of peptide transmitters, are co-packaged in the same secretory granules. Thus, differential hormone release is regulated at a step after granule fusion. Under basal chromaffin cell excitation, set by the sympathetic tone, selective release of freely-soluble catecholamine occurs through a transient fusion event, characterized by a narrow, structured exocytic fusion pore between the granule lumen and the extracellular space. Under sympathetic tone, selective and modest catecholamine release plays an important role in the "rest and digest" metabolic status of energy storage, regulating homeostatic physiological functions including pancreatic insulin secretion, increased blood flow to the viscera and maintenance of basal cardiac activity. In response to stress, increased chromaffin cell stimulation modulates the mode of secretory granule fusion, leading to the expansion of the exocytic fusion pore to maximize catecholamine release and facilitate exocytosis of the co-packaged adrenal peptide transmitters. Elevated serum catecholamine levels, in combination with adrenal-derived peptide transmitters, are core effectors of the sympathetic "fight or flight" stress response. Together they regulate multiple processes that prepare for defense or escape, including generalized analgesia (enkephalin), increased cardiac output (elevated catecholamines), blood flow to skeletal muscle (atrial natriuretic factor, Neuropeptide Y) and blood glucose (pancreastatin). Thus, regulated expansion of the secretory fusion pore represents a key element of the acute stress response. It is our overall goal to understand the molecular mechanism responsible for pore expansion. We summarize previous work and provide preliminary data to formulate 3 specific aims to test an activity-dependent dynamin I de- phosphorylation event, subsequent recruitment of a multimeric pore-expansion complex, and requirement for myosin motor activity in the regulation of fusion pore expansion. In the execution of these aims, we employ state of the art electrophysiological, electrochemical and quantitative fluorescence microscopy as well as a newly-developed silicon nanowire-based field effect transistor (SiNW-FET) biosensor to measure specific peptide release. The data obtained will provide a molecular understanding of the key regulators of the acute sympatho-adrenal stress response as well as pathologies resulting from its improper regulation.