MECHANISMS OF AIRWAY HYPERRESPONSIVENESS

在竞争继续的研究中，提出了确定分泌磷脂酶(s)的分子和结构，组(g) IIa和gVPLA2调节人嗜酸性粒细胞支气管活性白三烯的合成和分泌。第一个目的是研究这两种重组人(h)亚型的功能作用，以确定它们在质膜上的活性位点。位点定向诱变将用于确定活性位点，并将测试该假设以确定活性位点，并且将测试hVPL2与其相近的同源物gIIaPLA2不同，通过不涉及细胞质(c) PLA2或MAPK激活的途径引起质膜外层的直接水解和LTC4合成的激活的假设。这些研究假设，由gVPLA2对质膜外包膜的优先亲和力诱导的AA通量是其大量水解活性的原因；这种水解活性被认为会引起细胞内5-LO的活化以及cysLT的合成。在第二个目标中，将通过免疫组织化学评估气道中这两个14kDa亚型产生和分泌的位点，并通过Western blot分析确认。每个组织的含量将通过新开发的夹心ELISA进行评估，并使用计算机视频显微法评估气道上皮、内皮和巨噬细胞分泌在诱导人气道移植体收缩中的生理意义。本研究的最后（第三）目的是研究sPLA2s与质膜结合的分子机制。假设硫酸肝素蛋白多糖（HSPG）能快速内化gIIaPLA2，而gIIaPLA2对嗜酸性细胞膜的内表面具有优先的结合亲和力，因此具有较低的水解活性。研究人员提出要研究hVPLA2活性位点和界面结合位点的位点定向突变如何改变其与质膜外包膜结合的能力，从而减少HSPG的内化。据推测，这些研究将确定具有高同源性的分泌磷脂酶以不同方式启动或增强AA水解和cysLT合成的两种机制：1)通过直接诱导外膜A合成和cysLT生成；2)通过增加外源刺激激活的嗜酸性粒细胞的可用底物。来自这些研究的数据将阐明潜在的细胞-细胞相互作用以及新定义的调节支气管活性类二十烷醇合成的机制。

Studies are proposed in competing continuation to determine the molecular and structural of secretory(s) phospholipases, groups (g) IIa and gVPLA2 in regulating the synthesis and secretion of bronchoactive leukotrienes in human eosinophils. The first aim examines the functional roles of these two recombinant human (h) isoforms to determine the locus of their activity on the plasma membrane. Site directed mutagenesis will be used to determine the active site, and the hypothesis will be tested to determine the active site, and the hypothesis will be tested that hVPL2, unlike its close homologue gIIaPLA2, causes direct hydrolysis of the outer layer of the plasma membrane and activation of LTC4 synthesis through a pathway that does not involve either cytosolic (c) PLA2 or MAPK activation. These investigations hypothesize that AA flux induce by the preferential affinity of gVPLA2 for the outer envelope of the plasma membrane accounts for its substantial hydrolytic activity; this hydrolytic activity is postulated to cause activation of 5-LO intracellularly as well as synthesis of cysLT. In the second aim, the sites in the airway at which these two 14kDa isoforms are produced and secreted will be assessed by immunohistochemistry and confirmed by Western blot analysis. The content of each tissue will be assessed by a newly developed sandwich ELISA, and the physiological significance of secretion from airway epithelium, endothelium and macrophages in eliciting contraction of human airway explants will be assessed using computer videomicrometry. The final (third) aim of this proposal will examine the molecular mechanism of binding of sPLA2s to the plasma membrane. It is hypothesized that heparan sulfate proteoglycan (HSPG) rapidly internalizes gIIaPLA2, which has preferential binding affinity for the inner surface of the eosinophil membrane and hence has a low hydrolytic activity. Studies are proposed to examine how site-directed metagenesis at the active site and interfacial binding sites of hVPLA2 alters its ability to bind to the outer envelope of the plasma membrane, diminishing internalization by HSPG. It is postulated that these studies will define two mechanisms by which secretory phospholipases having high homologies act in different manners to either initiate or augment AA hydrolysis and cysLT synthesis: 1) by direct induction of A synthesis and cysLT production from the outer membrane and 2) by increasing available substrate for eosinophils activated by exogenous stimuli. Data derived from these studies will elucidate potential cell-cell interaction as well as newly defined mechanisms regulating synthesis of bronchoactive eicosanoids.