ION CHANNELS AND MONONUCLEAR PHAGOCYTE ACTIVATION

描述：这是正在进行的努力的续订申请 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的炎性释放 细胞因子。通过电生理手段，包括单通道， 全电池、电容测量、显微荧光法和分子 生物学方面，研究人员提出了以下具体目标。第一，离子 因自由基释放而激活的通道 单核吞噬细胞中的颗粒摄取将被确定。出版并出版 该实验室的初步工作已经确定了一种由超氧化物产生的 非选择性、去极化的膜电流。脑电生理学研究 将使用选择性、门控和药理学来识别通道或 产生自由基诱导电流的通道。第二， 膜电容与离子通道激活、变化的关系 摄取颗粒后的细胞内钙和细胞内pH将 下定决心。吞噬作用和胞吐作用将作为变化直接检测 以膜电容表示。内吞事件将与 细胞内钙离子、电导和细胞内pH的变化 原代人源性巨噬细胞系和巨噬细胞样细胞 转化的细胞系。目标将是确定干预点在 这种吞噬作用可以从分泌物中解脱出来。第三次也是最后一次 具体目标是确定 内向整流K+通道IRK1在细胞功能中发挥作用。这个 研究人员假设呼吸爆发反应以及 促炎细胞因子的分泌将在以下细胞中下调 由于IRK1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 有几种方法。Ba2+(50-250)对IRK1失活的影响 微摩尔)对J774.1细胞呼吸爆发活性的影响 下定决心。如果IRK1将是显性负的、无功能的孔突变体 注射到巨噬细胞(培养的原代细胞和转化的细胞 行)，以检查对单个细胞功能的影响。稳定的J774.1 IRK1被敲除或Kv1.5向外整流的细胞系 通过四环素调控的逆转录病毒载体系统过度表达， 将被用来评估IRK1表达对卵巢癌后期阶段的影响 巨噬细胞功能，包括颗粒释放和基因激活。 最后，单独含有基因设计的显性基因的转基因 将构建IRK1阴性突变体。巨噬细胞特异性启动子 将被用来将其直接表达到 转基因小鼠，其中IRK1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.