MECHANISMS OF AIRWAY HYPERRESPONSIVENESS

建议继续进行研究，以确定分泌型磷脂酶（g）IIa和gVPLA 2在调节人嗜酸性粒细胞中支气管活性白三烯合成和分泌中的分子和结构。第一个目的是检查这两种重组人（h）亚型的功能作用，以确定其在质膜上的活性位点。 将使用定点诱变来确定活性位点，并且将测试该假设以确定活性位点，并且将测试该假设，即hVPL 2与其紧密同源物gIIaPLA 2不同，引起质膜外层的直接水解和通过不涉及胞质（c）PLA 2或MAPK活化的途径活化LTC 4合成。这些研究假设，AA通量诱导的优先亲和力的gVPLA 2的质膜的外信封占其实质性的水解活性;这种水解活性被假定为导致激活的5-LO细胞内以及合成cysLT。在第二个目的中，将通过免疫组织化学评估气道中产生和分泌这两种14 kDa同种型的位点，并通过Western印迹分析确认。通过新开发的夹心ELISA评估各组织的含量，并使用计算机视频显微测量法评估气道上皮、内皮和巨噬细胞分泌物在引起人气道外植体收缩中的生理意义。本提案的最终（第三）目的将研究sPLA 2与质膜结合的分子机制。据推测，硫酸乙酰肝素蛋白聚糖（HSPG）迅速内化的gIIaPLA 2，它具有优先的嗜酸性粒细胞膜的内表面的结合亲和力，因此具有较低的水解活性。提出研究如何在活性位点和hVPLA 2的界面结合位点的定点后生改变其结合到质膜的外被膜的能力，减少由HSPG内化。据推测，这些研究将确定两种机制，通过这两种机制，具有高度同源性的分泌型磷脂酶以不同的方式起作用，以启动或增加AA水解和cysLT合成：1）通过直接诱导A合成和cysLT从外膜产生，2）通过增加由外源刺激激活的嗜酸性粒细胞的可用底物。从这些研究中获得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花生酸合成的机制。

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.