Todd A. Wyatt, Ph.D.

Dissertation research performed under the guidence of Dr. Katherine B. Pryswansky

ABSTRACT
    Neutrophil activation in response to chemotactic agents, phagocytic stimuli, and other endogenous regulators is a complex process involving migration, phagocytosis, degranulation, and generation of toxic oxygen metabolites. Modulation of the neutrophil response by intracellular second messengers, especially cyclic 3',5'-guanosine monophosphate (cGMP), has been proposed, but no mechanism of action of cGMP has been determined. This dissertation demonstrates that the major cellular receptor for cGMP, the cGMP-dependent protein kinase (G-kinase), is present in human neutrophils, and is compartmentalized to specific intracellular structures during cell activation. To further define the role of cGMP and G-Kinase in neutrophil functions, the following data are presented: (i) G-kinase is transiently localized to intracellular compartments containing intermediate filaments human neutrophils activated with the formyl-peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP), or the calcium ionophore, A23187. This response was transient in that the co-localization of G-kinase and the intermediate filaments is most significant at 3 min and was no longer detected by 5 min. This co-localization suggests that intermediate filaments are specific targeting proteins for G-kinase; (ii) An increase in the intracellular levels of cGMP, the specific activator of G-kinase, correlates with the time of co-localization of G-kinase and the intermediate filaments in neutrophils stimulated with fMLP or A23187. It is during this time of elevated cGMP levels and co-localized G-kinase and intermediate filaments that the neutrophil intermediate filament subunit protein, vimentin, is phosphorylated by G-kinase; and (iii) Degranulation of azurophil and specific granules in fMLP or A23187 stimulated neutrophils is shown to be controlled by increasing or decreasing cGMP-stimulated G-kinase activity. Treatment of cells with L-arginine, the precursor to nitric oxide formation which stimulates guanylate cyclase, increases degranulation of the neutrophil azurophil and specific granule marker proteins, myeloperoxidase and lactoferrin, respectively. Alternatively, decreasing cGMP-stimulated G-kinase activity with LY83583, an agent which lowers cGMP levels, or 8-Cl-Rp-cAMPS, a competitive antagonist to G-kinase, inhibits degranulation. This cellular and biochemical approach for understanding the role of G-kinase in neutrophil function defines an intracellular role of cGMP in neutrophils for the first time.