Molecular control of differential peptide transmitter exocytosis.

描述（由申请人提供）：环境威胁、体力消耗或伤害以及心理紧张都会导致交感-肾上腺“战斗或逃跑”应激反应的启动。神经内分泌肾上腺髓质嗜铬细胞接受来自交感内脏神经的兴奋性突触输入。内脏激活导致肾上腺嗜铬细胞释放儿茶酚胺以及多种神经和血管活性肽递质进入循环。不同程度的压力导致不同的释放， 儿茶酚胺与肽类递质之间的相互作用，以形成适当的生理反应。所有分泌的激素、儿茶酚胺以及多种肽递质共同包装在相同的分泌颗粒中。因此，差异激素释放在颗粒融合后的步骤中被调节。在基础嗜铬细胞的兴奋，设置由交感神经紧张，选择性释放的自由可溶性的儿茶酚胺发生通过一个短暂的融合事件，其特征在于一个狭窄的，结构化的胞吐融合孔之间的颗粒腔和细胞外空间。在交感神经紧张下，选择性和适度的儿茶酚胺释放在能量储存的“休息和消化”代谢状态中起重要作用，调节稳态生理功能，包括胰腺胰岛素分泌、增加流向内脏的血流量和维持基础心脏活动。在应激反应中，增加的嗜铬细胞刺激调节分泌颗粒融合的模式，导致胞吐融合孔的扩张，以最大化儿茶酚胺释放并促进共包装的肾上腺肽递质的胞吐。升高的血清儿茶酚胺水平与肾上腺源性肽递质结合，是交感神经“战斗或逃跑”应激反应的核心效应物。它们共同调节多个过程，为防御或逃避做准备，包括全身镇痛（脑啡肽），增加心输出量（升高的儿茶酚胺），骨骼肌血流量（心房钠尿因子，神经肽Y）和血糖（胰蛋白酶抑制剂）。因此，分泌融合孔的调节扩张代表了急性应激反应的关键要素。我们的总体目标是了解负责孔扩张的分子机制。我们总结了以前的工作，并提供了初步的数据，制定3个具体的目标，以测试一个活动依赖的发动蛋白I去磷酸化事件，随后招聘的多聚体孔扩张复合物，并要求肌球蛋白运动活性的融合孔扩张的调节。在这些目标的执行中，我们采用最先进的电生理学，电化学和定量荧光显微镜，以及新开发的基于硅锗的场效应晶体管（SiNW-FET）生物传感器来测量特定的肽释放。所获得的数据将提供急性交感-肾上腺应激反应的关键调节因子的分子理解，以及由其不当调节引起的病理学。 公共卫生相关性：环境或身体威胁增加自主交感神经系统的放电，以激活急性“战斗或逃跑”应激反应。通过一个迄今为止知之甚少的过程，不同的交感神经活动水平导致独特的激素谱，以引起适当的生理反应。该建议将测试特定活性依赖性差异释放激素递质进入循环的分子信号级联，从而为交感-肾上腺急性应激反应提供机制基础。

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. PUBLIC HEALTH RELEVANCE: Environmental or physical threat increases firing of the autonomic sympathetic nervous system to activate the acute "fight or flight" stress response. Through an as-of-yet poorly understood process, different sympathetic activity levels result in unique hormonal profiles to elicit appropriate physiological responses. This proposal will test a molecular signaling cascade for the specific activity-dependent differential release of hormone transmitters into the circulation and thus provide the mechanistic basis for the sympatho-adrenal acute stress response.