Granulocytes produce substantial quantities of superoxide (02-) and hydrogen peroxide (H202) during phagocytosis, or when they are stimulated by a variety of agents (immune complexes, chemotactic peptides, phorbol esters). The overall objectives of this application are to determine whether stimulation of granulocytes, as described involves the transient production of diacylglycerol and inositol phosphates. If so, we aim to establish the source(s) of the diglyceride (e.g., phosphatidylinositol and triglyceride). Both diglyceride and inositol (1,4,5)-trisphophate (Ins(1,4,5)P3) may constitute important signals in the generation of the cellular response. Diacylglycerol is the physiological activator of the calcium-activated phospholipid-dependent protein kinase (protein kinase C). Inositol (1,4,5)-trisphosphate, which may be liberated with diglyceride, could be involved in the mobilization of calcium in the cells. Studies on the diacylglycerol and inositol phosphate phenomena in other systems have usually depended on radioactive measurements. We believe that mass measurements are essential for a real quantitative resolution of these questions, and anticipate devising additional methods to quantify diacylglycerol, in particular. The time-course and dose-response curves for the formation of diglyceride and each of the inositol phosphates will be determined for a wide variety of stimulating agents, to establish which phenomena are common to the overall mechanism of stimulation and which are specific to a particular stimulus. A special interest of this laboratory is the stimulation of 02- release from granulocytes by agents that induce phase transitions in the lipid matrix of the plasmalemma. The studies described here may enable us to determine if such physical alterations in the membrane ultimately induce 02- release by activating protein kinase C or phospholipase C. Such phase transitions could occur in vivo, and have not been given significant attention. The project is health-related because of its connection to the role of oxygen radicals in killing bacteria, and in tumoricidal action. Further, there are indications that a constant exposure to oxygen radicals may effect tissue damage during chronic inflammation. Finally, some agents that stimulate the cells above (e.g., phorbol esters, retinoids) have been implicated in phenomena that underlie tumorigenesis and its control.
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