PHOSPHOLIPASE D AND MAST CELL ACTIVATION

肥大细胞对炎症介质的形成和/或胞吐 在即刻超敏反应中发挥核心作用并做出贡献 与慢性炎症的发展和/或持久有关 既有特应性和非特应性炎症性疾病。我们的远距离飞行 目的是了解受体调节内源性蛋白的功能(S) 生物信息性脂质分子在启动、调节和/或 影响肥大细胞介体释放。为此，拟议的 研究试图探索磷脂酶D(PLD)和脂质的作用 它产生的产物，磷脂酸(PA)，在两个信号转导中 和直接调节参与的膜生化事件。 肾小管周围膜相互融合及与血浆融合 薄膜。初步数据显示，在纯化的大鼠中存在强健的 Fcepsilon-RI依赖的血清肥大细胞对PLD活性的评估 两种独立的分析方法(其中一种是新颖的)。尽管1，2- 二酰甘油(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的激活对 形成一大部分DAG，作为以下结果积累 一种间接通路的受体依赖性刺激 PLD(PC PLD&GT；PA PA相&GT；DAG)。拟议的研究旨在 肥大细胞PLD和TO受体依赖性激活的特征 探索这一途径在胞吐作用中的调节重要性。 具体地说，使用两种独立的方法来量化关联的细胞 对于PLD，我们建议：i)详细描述PLD的变化 在完整的肥大细胞中的活动是生理上的结果 Fcepsilon-RI交联剂和 非免疫激动剂；ii)确定PLD的亚细胞位置 激活；以及iii)评估PA在启动过程中所起的作用， 持续和/或终止胞吐作用。每个主题都有一个主要的共同主题 这些目标之一是确定信号传递元件/秒 参与PLD调控的信使/效应器系统 活动。具体地说，GTP结合蛋白，Ca+2，蛋白质的作用 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 各种激动剂和拮抗剂被选择来最大限度地利用 要采用的不同的桅杆单元模型系统(完整的桅杆 细胞、通透性细胞、细胞质和分离的分泌颗粒 和质膜)。 关于PLD的性质、调节和亚细胞定位的研究 PA的活化、生成及其衍生产物的生成 PA的其他生物信息重要代谢物(DAG和Lyso-PA) --与肥大细胞介质释放密切相关的过程-- 应该有助于更清楚地理解免疫学 肥大细胞的激活。这一知识将有助于促进 治疗变态反应的新药理方法的发展 精神错乱。

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.