Activation of Mast Cells and Eosinophils: Studies at the Single-Cell Level

不可兴奋细胞的激活导致细胞内钙的增加和其他细胞内信号的产生，这些信号共同启动生理反应。以肥大细胞和嗜酸性粒细胞两种免疫效应细胞为模型系统，提出了非兴奋性细胞中刺激-反应耦合的单细胞研究。肥大细胞多年来一直是一个有价值的模型。相比之下，尽管嗜酸性粒细胞在各种免疫反应中很重要，但对它们的信号转导知之甚少。肥大细胞的研究将继续利用已建立的大鼠嗜碱性白血病细胞系。豚鼠嗜酸性粒细胞将用于更多的嗜酸性粒细胞信号转导的初步研究。定量图像增强显微镜将用于测量Ca2+，膜电位，胞外分泌和趋化性在单个细胞。在许多情况下，在许多单个细胞中可以同时监测这些参数中的一个以上。电生理测量，利用全细胞记录技术的穿孔贴片配置，将用于识别钙电流或任何其他电导，这些电导在响应各种刺激时被激活。同时测量细胞内Ca2+，离子电流和胞吐也计划。长期目标是确定细胞内Ca2+增加的机制，并确定Ca2+信号的哪些方面在细胞的胞外和/或趋化反应中是重要的。当肥大细胞或嗜酸性粒细胞与某些蛋白质或肽效应器（如免疫球蛋白）相互作用时，效应器与这些细胞表面的受体结合并导致特定的生理反应（如分泌特定储存颗粒的内容物）。这些研究解决的问题是细胞外部受体的结合是如何导致生理反应的，这种现象被称为刺激-反应耦合。我们已经确定，细胞内钙离子的增加是刺激的结果，而钙离子水平的增加会导致其他细胞内信号的产生，最终导致反应。本项目将探索钙在刺激-反应耦合中的作用，使用新颖和成熟的技术（有时同时）相结合，以扩展我们对这种形式的跨膜信号转导机制的理解。

Activation of non-excitable cells leads to an increase in intracellular calcium and the generation of other intracellular signals which together initiate the physiological response. Single-cell studies of stimulus-response coupling in non-excitable cells are proposed, using two immune effector cells, the mast cell and the eosinophil, as model systems. The mast cell has been a valuable model for many years. In contrast, remarkably little is known about signal transduction in eosinophils, in spite of their importance in a variety of immne responses. Studies on mast cells will continue to utilize the well-established rat basophilic leukemia cell line. Guinea pig eosinophils will be used for more preliminary studies of signal transduction in eosinophils. Quantitative image enhanced microscopy will be used to measure Ca2+, membrane potential, exocytosis and chemotaxis in single cells. In many cases more than one of these parameters can be monitored simultaneously in many individual cells. Electrophysiologcal measurements, utilizing the perforated patch configuration of the whole cell recording technique, will be used to identify calcium currents or any other conductances that are activated in response to various stimuli. Simultaneous measurements of intracellular Ca2+, ionic current, and exocytosis are also planned. The long term goal is to identify the mechanisms by which intracellular Ca2+ is increased and to determine what aspects of the Ca2+ signal are important in the exocytotic and/or chemotactic response of a cell. When mast cells or eosinophils interact with certain protein or peptide effectors (such as immunoglobulins), the effectors bind to receptors on the surfaces of these cells and result in a specific physiological response (such as secretion of the contents of specific storage granules). The question addressed in these studies is how the binding of a receptor at the outside of the cell results in the physiological response, a phenomenon termed stimulus-response coupling. It has already been established that intracellular calcium is increased as a result of stimulation, and that the increased calcium levels leads to the generation of other intracellular signals culminating in the response. This project will probe the role of calcium in stimulus-response coupling, using a combination of novel and established techniques (sometimes simultaneously) in order to extend our understanding of the mechanism of this form of transmembrane signal transduction.