Mechanisms of Endocytosis in Adrenal Chromaffin Cells.

肾上腺嗜铬细胞内吞作用的机制Corey B.SmithCase西部储备大学应激是对恶劣环境的一种生理反应。压力会导致肾上腺嗜铬细胞向血液中释放更多的化学递质，包括肾上腺素。这种释放是通过含有信息包的递质(称为分泌颗粒)与细胞内部表面融合实现的，使它们的内容物从细胞中排出。在正常情况下，嗜铬细胞释放低水平的肾上腺素，通过引导血液流向内脏，将眼睛聚焦在附近的物体上，并增加胰腺释放的胰岛素，使机体处于能量储存的“繁殖和喂养”状态。在应激状态下，嗜铬细胞释放高水平的肾上腺素，导致“要么战斗要么逃跑”的反应。在这种状态下，升高的肾上腺素将血流转移到骨骼肌，使他们专注于远处的物体，并提高血糖水平。此外，应激激活导致嗜铬细胞释放被称为脑啡肽的止痛化合物。这些信号分子共同将生物体置于逃避或防御的状态。因此，嗜铬细胞的调节递质释放在决定生物体的生理和代谢状态中起着至关重要的作用。本实验室的总体目标是提供对嗜铬细胞的基本了解，嗜铬细胞是“繁殖和喂养”以及“战斗或逃跑”状态的主要贡献者。这项建议着重于嗜铬细胞在基础激活与应激激活下释放递质的机制。持续的递质释放需要含有递质的分泌颗粒的循环利用。如果不回收，电池将很快耗尽其供应。专门的颗粒成分从细胞表面回收，并用递质分子重新填充以供重复使用。假设在所有条件下，这种回收都是通过相同的机制进行的。然而，最近的研究表明，在基础激活和应激激活下，再循环过程存在差异。这项提案概述了一系列旨在测试签名事件的实验，这些事件描述了小鼠肾上腺嗜铬细胞在基础激活和应激激活条件下的循环机制。它提供的实验将测试在递质释放过程中颗粒的融合和崩溃程度，以及颗粒和细胞表面成分的混合程度，从而确定在重新生成活性分泌颗粒之前所需的回收过程的复杂性。它还将确定参与循环机制的关键分子的作用，从而为分泌颗粒的重新生成确定潜在的调节点。这些实验中采用的技术包括电生理测量单个细胞的表面积，电化学测定释放的递质数量，细胞表面成分的定量荧光成像和分子破坏循环机制。因此，将提供肾上腺嗜铬细胞在正常“繁殖和进食”以及“战斗或逃跑”应激激活状态下持续释放肾上腺素和脑啡肽的基本机制的描述。

Mechanisms of Endocytosis in Adrenal Chromaffin CellsCorey B. SmithCase Western Reserve UniversityStress is a physiological response to a hostile environment. Stress causes chromaffin cells of the adrenal gland to increase their release of chemical transmitters, including adrenaline, into the blood stream. This release is achieved by fusion of transmitter containing packets, called secretory granules, with the inside cell surface, causing their contents to be expelled from the cell. Under normal conditions, chromaffin cells release low levels of adrenaline and set the organism in a 'breed and feed' state of energy storage by directing blood flow to the internal organs, focusing the eye on nearby objects and increasing insulin release from the pancreas. With stress, chromaffin cells release elevated levels of adrenaline, contributing to the 'fight or flight' response. In this state, the elevated adrenaline diverts blood flow to skeletal muscle, focuses they eye on distant objects and increases blood sugar levels. In addition, stress-activation causes chromaffin cells to release analgesic compounds, termed enkephalins. Together these signaling molecules place the organism into a state for escape or defense. Thus, regulated transmitter release from chromaffin cells plays a critical role in determining the physiological and metabolic status of an organism. It is the overall goal of this laboratory to provide a fundamental understanding of the chromaffin cell as a primary contributor to the 'breed and feed' as well as the 'fight or flight' state. This proposal focuses on the mechanisms utilized by chromaffin cells for transmitter release under basal versus stress activation.Sustained transmitter release requires the recycling of the transmitter-containing secretory granules. Without recycling the cell would soon exhaust its supply. Specialized granule components are retrieved from the cell surface and refilled with transmitter molecules for re-use. It was assumed that this recycling occurred by the same mechanism under all conditions. However, it was recently demonstrated that differences in recycling processes exist under basal versus stress-activation. This proposal outlines a series of experiments designed to test signature events that delineate recycling mechanisms under conditions of basal versus stress activation in mouse adrenal chromaffin cells. It provides experiments that will test the degree of granule fusion and collapse during transmitter release and the degree to which the granule and cell surface components mix, thus defining the complexity of the recycling process required prior to re-generation of viable secretory granules. It will also determine the role of key molecules involved in the recycling mechanism, thus defining points of potential regulation for the re-generation of secretory granules. Techniques employed in these experiments include electrophysiological measures of the surface area of single cells, electrochemical determination of the amount of transmitter released, quantitative fluorescence imaging of cell surface components and molecular disruption of the recycling mechanism. Thus a fundamental description of the mechanisms utilized by adrenal chromaffin cells for the sustained release of adrenaline and enkephalin during the normal 'breed and feed' as well as the 'fight or flight' stress-activated state will be provided.