The elucidation of the mechanism by which the human neutrophil mounts the non-mitochondrial respiratory burst is crucial in understanding neutrophil-mediated lung damage and normal microbicidal function. This proposal focuses on defining an activation pathway of the NADPH-oxidase, the enzyme responsible for producing the toxic oxygen-derived species, superoxide (O2-) and ultimately other oxidative agents. By linking the specific phorbol ester (PMA) receptor to activation of the NADPH-oxidase, and identifying the receptor as protein kinase C (PK-C) several lines of inquiry to trace the PMA activation pathway were suggested. In recently published studies, a broken cell system employed a reconstituted endogenous PK-C system or purified PK-C to generate an active NADPH-oxidase. These studies were the first to demonstrate a direct functional role for PK-C in human neutrophil, and offers the opportunity to differentiate the relative importance of this system vis a vis other activation cascades. Specific goals include: 1) Differentiation of PMA stimulation from activation pathways independent of PK-C. The use of PK-C inhibition and the differentiation of activities selectively stimulated by various agonists, which are not activated by phorbol esters will define the assignment of the PK-C pathway in intact cells. 2) Characterization by immunoblotting techniques of human PK-C is important in comparing this ubiquitous enzyme to those found in other human tissues and to those previously isolated from sources. 3) Plasma membranes harvested from resting neutrophils, will be incubated with purified PK-C dependent products will direct investigation to isolate the crucial components of this activation pathway. These studies than represent a comprehensive approach to elucidating the role of PK-C in the activation of the human neutrophil respiratory burst, an issue of importance defining the role of phagocytic cells in a variety of inflammatory and toxic lung conditions. Further, this research serves as a model of ligand-receptor coupled reaction of secretory cells, and the definition of what must now be viewed as the """"""""distal"""""""" component of the activation cascade.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Hematology Subcommittee 2 (HEM)
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Boston University
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