Although the functional response of neutrophils, the attack on invading entitities by products of the oxidative burst and by the contents of the granules, has been studied in great detail, the mechanism by which this response follows upon exposure of the cells to their specific stimuli remains incompletely understood. It has been shown that, upon contact with soluble stimuli such as chemoattractants or phorbol esters, or with particulate agonists such as opsonized particles or immune complexes, granulocytes undergo rapid changes in membrane cation permeability, in cytoplasmic concentrations of Ca++, H+, Na+ and K+ ions, activation of an oxidative burst, release of granule contents and, for certain stimuli, enhanced phosphoinositide turnover and/or chemotaxis. The temporal sequence of these events, and their inter-dependence, as well as the nature of the transduction signals, remain unelucidated. While undifferentiated precursor cells appear unable to mount any of these responses except chemotaxis, their capability to do so rises as the cells mature into granulocytes (from human bone marrow myeloid precursor cell) or granulocytoids (from the human leukemic HL-60 cell line). The mechanism of the acquisition of this response capability is unknown. We plan to address these questions by examining neutrophils, as well as the precursor cells as a function of their stage of differentiation. We shall be concerned solely with the initial stages of the response (within the first 90 seconds) and will hence be investigating the kinetics of changes in the various cation gradients, in transmembrane potential, in intracellular Ca++ and H+, as a function of the stimulus and dose used, of the presence of specific cation channel blocking agents (such as dimethyl amiloride), of certain ionophores (such as valinomycin, ionomycin, nigericin), of functional blocking agents such as B. pertussis toxin, of the external Na+ and K+ concentrations (altered by altering the buffers), and of internal Ca++ (adjusted by using a cytoplasmic chelator such as BAPTA) and H+ (adjusted with NH4Cl or CH3COOH) concentrations. Because the reactions are rapid we shall used fluorescent indicators for most of the studies. Since there are advantages and disadvantages to each, we shall investigate not only the fluorescence changes in suspensions, which are averaged over the cell population, but also in single cells, as observed in a fluorescence activated cell sorter, which allows us to explore the possibility that our measurements reflect properties of only a portion of the cells.
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