The long term objectives of this proposal are to elucidate the exact sequence of biochemical events that are involved in the priming and stimulation of superoxide (02-) production by neutrophils. Superoxide is a key component of the oxygen-dependent antimicrobial mechanisms of these cells which provide a major defense against infectious organisms. Certain cytokines prime neutrophils to release increased amounts of 02-. Therapeutic value of these factors is now being tested clinically in a variety of cancer and AIDS patients with promising results. Thus, experiments outlined herein are relevant to an understanding of host- defense mechanisms and immune deficiency diseases. Specifically, this project addresses two largely unknown areas. These are: (1) the mechanism(s) of action of the hydroxylated eicosatetraenoic acids (HETE) in modulating 02- production, and (2) the link between protein phosphorylation and 02- generation. Neutrophils produce 5- and 15-HETE under various circumstances. 5-HETE dramatically potentiates 02- release, whereas 15-HETE inhibits it. We propose that 5- HETE primes neutrophils by increasing the flux of Ca2+ across the plasmalemma, perhaps by opening a channel for this cation. This will be measuring the rates of 45Ca2+ uptake by these cells in the presence of different concentrations of 5-HETE. The effects of a variety of calcium channel antagonists on this process will be evaluated. The relationships between the rates of 45Ca2+ uptake and 02- generation will be defined, and possible antagonistic effects of 15-HETE will be sought. With regard to protein phosphorylation, we have observed that stimulation of 02- release by neutrophils is always accompanied by an intense phosphorylation of two proteins with molecular weights of ca. 47 and 49 kDa. Quantitatively, these are the two major proteins which are phosphorylated upon stimulation of these cells. While the 47 kDa protein has been extensively characterized, virtually nothing is known of the 49 kDa species. Experiments are detailed herein to identify, characterize and purify this protein. Techniques of biochemistry and cell biology will be employed. We will determine whether the 49 kDa protein is a component of the 02- generating system, or is involved in some other fashion in the mechanism of stimulation of these cells (e.g., phospholipose D). Changes in the activity of the 49 kDa protein after phosphorylation will be sought to forge a link between this modification reaction and cell stimulation.
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