Role of Ion Channels in Mononuclear Phagocyte Activation

描述（由申请方提供）：巨噬细胞是单核吞噬细胞系统的初级分化细胞，在宿主防御微生物感染中起主要作用。吞噬细胞具有多种质膜受体，其识别并结合体液中发现的颗粒和可溶性刺激物。活化的巨噬细胞分泌大量的小分子和大分子产物，包括细胞因子、趋化因子、生长因子和氧化代谢物。虽然众所周知，受体-配体结合是细胞内信号转导级联的初始步骤，其最终导致分泌和/或吞噬作用，但将受体结合和交联连接到囊泡融合的途径的钙和G蛋白依赖性仍不清楚。本申请中的研究针对在用可溶性和颗粒刺激物两者刺激后吞噬细胞中分泌的模型的开发。分泌过程的调节，所涉及的蛋白质，以及囊泡本身的身份仍然知之甚少。我们将通过独立操纵内部钙浓度和GTP-γ-S来检查分泌过程的调节。初步研究强烈支持的模型中，钙和G蛋白的作用相互独立的调节粒细胞分泌。这是我们的目标，在这个应用程序中，以确定G蛋白和胞浆钙离子的调节分泌铁受体交联诱导的互补作用。我们将使用单细胞电容测量结合电流分析法和荧光分析法来确定两种途径的贡献，并确定可能的作用机制。这是我们的假设，胞质钙有助于动员囊泡从储备池，并促进对接在质膜，而G蛋白触发的实际融合步骤。在一个全面的模型的发展，我们将（1）检查细胞溶质的钙和G蛋白在分泌过程中的动员和对接步骤的互补作用，（2）调查的Ca受体在分泌反应的身份，和（3）确定G蛋白参与囊泡融合过程。

DESCRIPTION (provided by applicant): The macrophage is the primary differentiated cell of the mononuclear phagocyte system and plays a major role in host defense against microbial infections. Phagocytes possess a diversity of plasma membrane receptors which recognize and bind both particulate and soluble stimuli found in body fluids. The activated macrophage secretes a large array of small and macromolecular products including, cytokines, chemokines, growth factors, and oxidative metabolites. While it is well known that receptor-ligand binding is the initial step in the intracellular signal transduction cascades that eventuate secretion and/or phagocytosis, the calcium and G protein dependence of the pathway(s) which links receptor binding and cross-linking to vesicle fusion is still unclear. Studies in this application are directed at the development of a model for secretion in phagocytic cells following stimulation with both soluble and particulate stimuli. The regulation of the secretory process, the proteins involved, and the identity of the vesicles themselves remain poorly understood. We will examine the regulation of the secretory process by independent manipulation of internal calcium concentration and GTP-gamma-S. Preliminary studies strongly support a model in which Ca and G proteins act independently of one another in the regulation of granulocyte secretion. It is our goal in this application to determine the complementary roles of G proteins and cytosolic calcium in the modulation of secretion induced by Fe-receptor cross linking. We will use a combination of single cell capacitance measurements coupled with amperometry and fluorimetry to determine the contribution of the two pathways and determine a possible mechanism of action. It is our hypothesis that cytosolic Ca helps mobilize vesicles from a reserve pool and promotes docking at the plasma membrane while G proteins trigger the actual fusion step. In the development of a comprehensive model, we will (1) examine the complementary roles of cytosolic Ca and G proteins in the mobilization and docking steps of the secretory process, (2) investigate the identity of the Ca receptor in the secretory response, and (3) identify the G proteins involved in the vesicle fusion process.