A TWO-STEP PATHWAY THAT FORMS DIACYLGLYCEROL IN MAST CELLS

肥大对炎症介质的形成和/或胞吐 细胞在即刻超敏反应中发挥核心作用 并导致与这两种特应性疾病相关的慢性炎症 和非特应性炎症性疾病。我们的长期目标是 理解受体调节内源性蛋白的作用(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 研究试图探索磷脂酸的作用 磷酸水解酶(PA相)和磷脂酶D(PLD) 它们在信号转导和直接作用下产生的脂类 细胞融合过程中膜生化事件的调控 肾小管周围膜相互连接以及与质膜连接 在胞吐过程中。数据有力地表明，增加的质量 刺激Fc后的第二信使1，2-二酰甘油(DAG)。 肥大细胞中的Epsilon-RI主要来源于 卵磷脂(PC)。我们还展示了强大的 存在依赖Fc-epsilon-RI的PLD活性。这些观察结果 来自我们实验室的数据强烈支持这一观点 PLD激活可能最终导致肥大细胞的增加 作为受体依赖刺激的结果的相关DAG 一条同时涉及PLD和PH期的间接通路(PC--&GT；PA-- &gt；DAG)。拟议的研究试图描述以下方面的特征 这条路。具体地说，我们提出的研究试图：i)克隆 使用基于表达式的方法的PLD，该方法利用 我们实验室取得的新技术进展，使检测 PLD的含量极低，以及ii)在生化方面进行探索， 功能和药理学术语肥大细胞PA- 阶段及其在间接两步中作为第二步的作用 受体依赖性DAG的形成途径。关于PA- 阶段，各种秒的直接或间接影响 将根据信使对短期工作的贡献进行评估 调节，而不同的细胞因子环境在 调节PA-期的长期表达也将被探索。 这些研究将产生与以下关键要素有关的见解 DAG形成的间接途径及其亚细胞 位置、监管和可能在控制中的作用 肥大细胞激活和/或生理上的重要方面 胞吐作用胞吐作用的过程这一知识将有助于促进 治疗慢性阻塞性肺疾病的新药理学方法的发展 过敏性疾病。

The formation and/or exocytosis of inflammatory mediators by mast cells play central roles in immediate hypersensitivity reactions and contribute to 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 phosphatidic acid phosphohydrolase (PA-PHase) and phospholipase D (PLD) and the lipids they generate 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 during exocytosis. Data strongly suggest that the increased mass of the second messenger 1,2-diacylglycerol (DAG) after stimulating Fc- epsilon-RI in mast cells is largely derived from phosphatidylcholine (PC). We have also demonstrated the robust presence Fc-epsilon-RI dependent PLD activity. These observations are linked by data from our lab that strongly encourage the view that activation of PLD may culminate in an increase in mast cell associated DAG as the result of the receptor dependent stimulation of an Indirect Pathway involving both PLD and PH-PHase (PC-->PA-- >DAG). The proposed studies seek to characterize certain aspects of this pathway. Specifically, we propose studies seeking to: i) clone PLD using an expression based approach that takes advantage of a novel technical development made by our lab that permits detection of PLD in extremely low quantity and ii) to explore in biochemical, functional and pharmacologic terms the activity of mast cell PA- PHase and its role as the second step in the two step Indirect Pathway of receptor dependent DAG formation. With regard to PA- PHase, the direct or indirect effects of a variety of second messengers will be evaluated for their contributions to short term regulation, while the role of the varying cytokine milieu in regulating long term expression of PA-PHase will also be explored. These studies will generate insights pertaining to key elements of the Indirect Pathway of DAG formation and their subcellular location, regulation and likely roles in controlling physiologically important aspects of mast cell activation and/or the process of exocytosis. This knowledge will help to facilitate development of novel pharmacologic approaches to the therapy of allergic disorders.