The overall goal of this proposal is to characterize specific signal transduction mechanisms that are activated after neutrophil stimulation and are involved in heart injury. Neutrophils release lethal oxygen radicals (such as superoxide anions) that are capable of exacerbating tissue damage, as encountered after the infiltration of these cells during post-ischemic myocardial necrosis and reperfusion injury. The enzyme phospholipase D (PLD) is central to the production of PA, a putative second messenger involved in the release of superoxide anions by neutrophils. The authors found that a PLD activity can be specifically immunoprecipitated in its active form with anti-phospho-tyrosine antibodies from cell lysates. The molecular weight of neutrophil PLD as well as the intracellular localization are different from those reported for other mammalian cells. Additionally, the PLD activity is found increased in adherent neutrophils in conditions in which superoxide release is activated. As a result, it is now hypothesized that a novel isoform of PLD in human neutrophils is regulated through tyrosine phosphorylation, which allows the enzyme to become active and synthesize PA, that in turn triggers the release of oxygen radicals.
The Specific Aims of this proposal are: (1) Purify and characterize a novel granulocyte PLD isoform by column chromatography and by molecular biology techniques, using the sequenced purified protein (PY-PLD) as well as the existing mammalian PLD cDNA sequence (PLD1). (2) Determine the mechanism of granulocyte PLD regulation, particularly the phosphorylating kinase, the site(s) of phosphorylation and the role of small GTP-binding proteins, using immunoprecipitation with antibodies against epitope-tagged proteins and in vitro enzymatic assays. (3) Elucidate the role of PY-PLD in the NADPH oxidase system, by blocking superoxide release with PLD antibodies in vitro and in vivo. These studies will result in a clearer understanding of the oxidative processes involved in heart injury and suggest novel therapeutic interventions.
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