MECHANISMS OF AIRWAY HYPERRESPONSIVENESS

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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.