The objective of this project is to determine the external ionic factors and granule and organelle related factors which modulate the transduction of the activation signal subsequent to membrane stimulation in human polymorphonuclear leukocytes. Through the use of curve fitting techniques, we will develop methods to characterize and statistically test changes in the shape of response curves obtained using fluorescent probes of transmembrane potential, cytosolic free calcium, and membrane bound calcium, as well as the optical parameters of forward and 90 degree light scatter. These analyses will be used to investigate the effects of ion transport, mitochondrial function, and degranulation upon various phases of activation as defined by the fluorescent and optical probes. A neutrophil devoid of nucleus, mitochondria and granules (neutrophil cytoplast) will be used as a model in order to explore the role of granules and organelles in the activation process. Internal pools of calcium will be defined in terms of their ability to be released by the mitochondrial coupler carbonyl cyanide P-trifluoromethoxyphenylhydrozone or the calcium ionophore A23187. The fluorescent probes will be localized to regions within the neutrophil by fluorescence microscopy and the source of changes in fluorescence signal will be quantitated using low light level video techniques and video image analysis. These studies will not only provide new information as to the sequence of the activation responses monitored by these fluorescent probes but will also enhance our understanding of the source of the fluorescent signals from the neutrophil.
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