The production of oxygen radicals by human blood neutrophils is critical for the success of host defense, but also may be detrimental to the host in certain inflammatory processes, i.e. in the adult respiratory distress syndrome, arthritis, etc. Understanding the regulation of oxygen radical production is the long-term goal of this project and is important for developing methods of controlling various disease states. The enzyme system in neutrophils responsible for the conversion of oxygen to toxic products appears to be an activatable membrane-bound NADPH oxidase, and its mechanism of activation is not yet known. A variety of stimulating agents (i.e. phagocytic particles, chemoattractants, calcium ionophores, and phorbol esters) activate NADPH oxidase. Previous data suggest that the pathways leading to NADPH oxidase activation are complex, with both stimulus-specific and shared components, and that the shared pathway consists of at least two separately regulated steps. Recent observations have implicated protein kinase C as having a major role in receptor-mediated cellular regulation. Several events are triggered by stimulation of neutrophils that could cause activation of protein kinase C, including an increase in calcium concentration, the release of diacylglycerol, and the release of arachidonate. In addition, protein kinase C appears to be the receptor for phorbol esters, which are potent stimulants for neutrophils.
Specific Aim 1 of this project will test the hypothesis that protein kinase C is the central, shared intermediate in the pathway of NADPH oxidase activation. Preliminary data indicate that stimulation of neutrophils increases protein kinase C activity in a particulate fraction of the cell and that the increase correlates with oxidase activation. Further experiments to test the correlation between the two events will be performed. The principal investigator has recently shown that arachidonate activates NADPH oxidase in subcellular fractions from unstimulated human neutrophils and that separate, inactive fractions can be reconstituted to form an active system. This cell-free activation system will be utilized in Specific Aim 2 to determine specific biochemical mechanisms (i.e. phosphorylation of proteins, membrane phospholipid changes, membrane biophysical changes) which correlate with NADPH oxidase activation and to identify required co-factors.
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