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 cGMP has been proposed, but no mechanism of action of cGMP has emerged over the past two decades despite intensive investigation. Dr. Pryzwansky in collaboration with Dr. Thomas Lincoln has recently obtained evidence that the major cellular receptor for cGMP, the cGMP- dependent protein kinase (G-kinase) is present in neutrophils at low concentrations, and may be compartmentalized to specific intracellular structures during cell activation. To further define the role of cGMP and G-kinase in neutrophil functions, the goals of this research are: 1) to identify the intracellular compartments or structure(s) associated with G-kinase by immunocytochemistry, and to correlate its localization with cell function (motility and degranulation), 2) to investigate the physiologic role of cGMP and G-kinase by monitoring cGMP levels in response to chemoattractants and other modulators, by identifying specific cellular protein substrates for the kinase, and by studying the effects on neutrophil function of antibodies to G-kinase introduced into neutrophils by electroporation. Neutrophils play a vital role in host defense against microorganisms. These specialized cells move in response to a chemotactic gradient and phagocytose and kill foreign microorganisms. Although the regulatory mechanisms which control neutrophil activities have been the topic of investigation for a century, important gaps still remain in our understanding of how these physiologic processes are regulated. One area of potential importance, but uncertainty is the physiologic role of the small molecule, cyclic-GMP (cGMP), as a second messenger in controlling neutrophil functions. The combined cellular and biochemical approach proposed here should, for the first time, define the role of cGMP in neutrophils. An important key to understanding this pathway of cellular signal transduction may be to understand the subcellular distribution of the molecules involved. A unique feature of this project will be the use of state of the art techniques of light and electron microscopy to study the subcellular localization of key signaling molecules within neutrophils in various functional states.
