PHOSPHOLIPASE D AND MAST CELL ACTIVATION

肥大细胞对炎症介质的形成和/或胞吐作用 在速发型超敏反应中发挥核心作用， 慢性炎症的发展和/或持续 过敏性和非过敏性炎症性疾病。 我们的远程 目的是了解受体调节的内源性 生物信息脂质分子在启动、调节和/或 影响肥大细胞介质释放。 为此，建议 研究试图探索磷脂酶D（PLD）和脂质 它产生的产物磷脂酸（PA），在两个信号转导 和直接调节膜生化事件参与的 颗粒周膜相互融合以及与血浆融合 膜的 初步数据表明，在纯化的大鼠中， 通过以下方法评估的Fc ε-RI依赖性PLD活性的血清肥大细胞 两个独立的试验（其中一个是新的）。 虽然1.2- 甘油二酯（DAG）不是当前提案的重点，数据来自 我们的实验室强烈建议PLD激活对于 形成大部分的DAG积累的结果， 间接途径的受体依赖性刺激， PLD（PC PLD> PA PA-相> DAG）。 拟议的研究旨在 表征肥大细胞PLD的受体依赖性活化， 探索这一途径在胞吐作用中的调节重要性。 具体地，使用两种独立的方法来定量细胞相关的 PLD，我们建议：i）详细描述PLD中的变化 在完整的肥大细胞中的活性是由于生理上的 通过Fcepsilon-RI交联和 非免疫激动剂; ii）确定PLD的亚细胞位点 激活;和iii）评估PA在引发， 持续和/或终止胞吐作用。 每个主题都有一个共同的主题， 这些目标之一是确定信号转换元件/秒 参与PLD调节的信使/效应子系统 活动 具体而言，GTP结合蛋白，Ca+2，蛋白 激酶C、cAMP/蛋白激酶A、酪氨酸蛋白磷酸化和 磷脂酶A2的脂肪酸产物将使用 选择多种激动剂和拮抗剂以最大限度地利用 不同的肥大细胞模型系统（完整的肥大细胞 细胞、透化细胞、细胞质和分离的分泌颗粒 和质膜）。 对PLD的性质、调控及亚细胞定位的研究 活化、PA的生成和衍生物的生成 PA的其他生物信息学重要代谢产物（DAG和lyso-PA） --与肥大细胞介质释放紧密相关的过程-- 应该有助于更清楚地了解免疫学 肥大细胞的活化。 这些知识将有助于促进 发展新的药理学方法来治疗过敏性 紊乱

The formation and/or exocytosis of inflammatory mediators by mast cell play central roles in immediate hypersensitivity reactions and contribute to the development and/or perpetuation of chronic inflammation associated with both atopic and nonatopic inflammatory diseases. Our long range goal is to understand the function(s) of receptor regulated endogenous bioinformational lipid molecules in initiating, regulating and/or effecting mast cell mediator release. Toward this end, the proposed studies seek to explore the roles of phospholipase D (PLD) and the lipid product it generates, phosphatidic acid (PA), in both signal transduction and the direct regulation of membrane biochemical events involved in the fusion of perigranular membranes with each other and with the plasma membrane. Preliminary data indicate the robust presence in purified rat serosal mast cells of Fcepsilon-RI dependent PLD activity as assessed by two independent assays (one of which is novel). Although 1,2- diacylglycerol (DAG) is not the focus of the current proposal, data from our laboratory strongly suggest that PLD activation is important to the formation of a large fraction of the DAG accumulating as the result of the receptor dependent stimulation of an Indirect Pathway initiated by PLD (PC PLD> PA PA-PHase> DAG). The proposed studies seek to characterize receptor-dependent activation of mast cell PLD and to explore the regulatory importance of this pathway in exocytosis. Specifically, using two independent methods to quantitate cell associated PLD, we propose: i) to characterize in detail the changes in PLD activity in intact mast cells occurring as the result of physiologically relevant stimulation both by Fcepsilon-RI crosslinking and by nonimmunologic agonists; ii) to determine the subcellular site of PLD activation; and iii) to assess the role that PA plays in initiating, perpetuating and/or terminating exocytosis. A major common theme in each of these aims is determining the signal transducing element/second messenger/effector systems that participate in the regulation of PLD activity. Specifically, the role of GTP binding proteins, Ca+2, protein kinase C, cAMP/protein kinase A, tyrosine protein phosphorylation and the fatty acid products of phospholipase A2 will be carefully evaluated using a variety of agonists and antagonists chosen to maximally take advantage of the different mast cell model systems to be employed (intact mast cells, permeabilized cells, cytoplasts and isolated secretory granules and plasma membranes). These studies of the nature, regulation and subcellular location of PLD activation, the resultant formation of PA and the derivative generation of other bioinformationally important metabolites of PA (DAG and lyso-PA) -- processes tightly associated with the mast cell mediator release -- should contribute to a much clearer understanding of immunologic activation of mast cells. This knowledge will help to facilitate the development of novel pharmacologic approaches to the therapy of allergic disorders.