ION CHANNELS AND MONONUCLEAR PHAGOCYTE ACTIVATION

描述：这是一个持续努力的更新应用程序， 确定离子通道在巨噬细胞功能中的作用， 特别是颗粒释放和随后炎性释放 细胞因子 通过电生理学，包括单通道， 全细胞和电容测量，显微荧光和分子 在生物学方面，研究人员提出了以下具体目标。 一、离子 通道激活的结果，自由基释放以下 将鉴定单核吞噬细胞中的颗粒摄取。 出版并 这个实验室的初步工作已经确定了一种超氧化物产生的 非选择性去极化膜电流 电生理研究 选择性、门控和药理学将用于识别通道或 产生自由基诱导电流的通道。 二是 膜电容，离子通道激活，变化 在颗粒摄取后的细胞内Ca和细胞内pH中， 测定 吞噬作用和胞吐作用将直接测定为变化 在膜电容中。 内吞事件将与 钙的变化，电导的变化，以及细胞内pH的变化， 原代人源性巨噬细胞系和巨噬细胞样细胞系 转化细胞系 目标是确定干预点， 其吞噬作用可以与分泌分离。 第三和最后 具体的目标是针对确定的作用， 内向整流钾通道IRK 1在细胞功能中起重要作用。 的 研究者假设呼吸爆发反应以及 促炎细胞因子分泌将在细胞中下调， 由于IRK 1表达缺失而经历慢性去极化 或功能。 对这一假设的调查将通过以下方式完成： 几种方法。 Ba2+（50 - 250 1细胞系中呼吸爆发活性的影响。 测定 显性阴性，无功能孔突变体，如果IRK 1将 注射到巨噬细胞（培养的原代和转化细胞 线）以检查对单个细胞中的功能的影响。 稳定型J774.1 其中IRK 1被敲除或外向整流Kv1.5被敲除的细胞系 通过四环素调节的逆转录病毒载体系统过表达， 将用于评估IRK 1表达对晚期肿瘤的影响。 巨噬细胞功能，包括颗粒释放和基因激活。 最后，含有基因设计的显性基因的转基因 将构建IRK 1阴性突变体。 巨噬细胞特异性启动子 将用于指导表达到粒细胞群体中， 转基因小鼠，其中IRK 1的作用将被评估。

DESCRIPTION: This is a renewal application of an ongoing effort to determine the role that ion channels play in macrophage function, specifically particle release and subsequent release of inflammatory cytokines. By means of electrophysiology, including single-channel, whole-cell, and capacitance measurements, microfluorimetry and molecular biology, the investigator proposes the following specific aims. First, ion channels activated as a consequence of free radical release following particle uptake in mononuclear phagocytes will be identified. Published and preliminary work from this laboratory have identified a superoxide-generated nonselective, depolarizing membrane current. Electrophysiologic studies of selectivity, gating and pharmacology will be used to identify the channel or channels that produce the free radical-induced current. Second, the relationship between membrane capacitance, ion channel activation, changes in intracellular Ca and intracellular pH following particle uptake will be determined. Phagocytosis and exocytosis will be assayed directly as changes in membrane capacitance. The endocytic event will be correlated with changes in Ca, changes in conductance, and changes in intracellular pH in both primary human-derived macrophage cell lines and a macrophage-like transformed cell line. The goal will be to determine intervention points at which phagocytosis could be uncoupled from secretion. The third and final specific aim is directed toward the determination of the role that the inwardly rectifying K+ channel IRK1 plays in cellular function. The investigator hypothesizes that respiratory burst response as well as pro-inflammatory cytokine secretion will be down-regulated in cells that undergo chronic depolarization as a consequence of a loss of IRK1 expression or function. Investigation of this hypothesis will be accomplished through several approaches. The effect of IRK1 inactivation by Ba2+ (50 - 250 micromolar) on respiratory burst activity in the J774.1 cell line will be determined. Dominant negative, nonfunctional pore mutants if IRK1 will be injected into macrophage cells (both cultured primary and transformed cell lines) to examine the effect on function in single cells. Stable J774.1 cell lines in which IRK1 is knocked out or the outwardly rectifying Kv1.5 is overexpressed by means of a tetracyline-regulated retroviral vector system, will be used to assess the effects of IRK1 expression on later stages of macrophage function, including granule release and gene activation. Finally, transgenes individually containing a genetically designed dominant negative IRK1 mutant will be constructed. A macrophage specific promoter will be used to direct expression to the granulocyte cell population in transgenic mice, in which the role of IRK1 will be assessed.