During the previous granting period, a unique biphasic organotypic culture system of guinea pig tracheal epithelial cells was developed. Exogenously- generated )2 metabolites were shown to stimulate mucin secretion by such airway epithelial cells in vitro. The mechanism of stimulation involved intracellular generation of additional O2 radicals, increased cyclooxygenase metabolism of arachidonic acid to PGF2a, and PGF2a - stimulated mucin secretion. Studies in this proposal will investigate further intracellular mechanisms involved in the response of respiratory epithelium to O2 metabolites. The hypothesis to be addressed is that the following sequence occurs within airway epithelial cells in response to oxidant stress. Exposure of airway epithelium to extracellular O2 metabolites results in peroxidation of membrane lipids. Intracellular lipid peroxides stimulate release of arachidonic acid from cellular stores by activation of phospholipase A2 (PLA2) and also """"""""initiate"""""""" activity of Prostaglandin H synthase (cyclooxygenase and peroxidase) resulting in increased production of PGF2a by epithelial cells. PGF2a thus synthesized then stimulates mucin secretion through an autocrine or paracrine mechanism involving activation of a guanine nucleotide regulatory (G) protein associated with a phosphatidylinositol (PI) - specific phospholipase C (PLC). Activated PLC catalyzes PI hydrolysis and intracellular production of inositol phosphates (IP's) and/or activation of protein kinase C (PKC), resulting in enhanced mucin secretion. There will be two phases to the proposed studies. The first will focus on defining mechanisms within epithelial cells leading from exposure to external oxidant stress to enhanced production of PGF2a. The second phase will examine intracellular mechanisms of PGF2a 0 stimulated mucin secretion, specifically the role of a putative G protein linked to a PI- specific PLC, intercellular production of IP s (utilizing HPLC with fluorometric and/or amperometric detection) and activation of PKC.
Showing the most recent 10 out of 40 publications
