The new, fluorescent Ca2+ indicator, fura-2, promises to expand our understanding of the role of subcellular changes in Ca2+ underlying cell function. During an investigation of the role of Ca2+ in the polarization response of human polymorphonuclear leukocytes to formyl-methionyl-leucyl-phenylalanine, we found that fura-2 trapped by cells incubated with the acetoxy-methyl ester of fura-2, F2-AM, yielded measurements of Ca2+ that were depressed at rest and during the response to formyl-methionyl-leucyl-phenylalanine. Fura-2, trapped by the cells, exhibited a spectrum in the presence of saturating Ca2+ that differed from that of fura-2 free acid. We have shown that the cellular fluorescence can be spectrally decomposed into two components: one with Ca2+ sensitivity identical to fully deesterified fura-2, and another which is Ca2+-insensitive. The Ca2+-insensitive component appears to be more fluorescent than F2-AM as well as spectrally different from F2-AM. The insensitive form probably results from incomplete deesterification of F2-AM by the cells. In order to accurately measure Ca2+ in polymorphonuclear leukocytes, it is imperative to check for the presence of Ca2+-insensitive fluorescence. The contribution of Ca2+-insensitive fura-2 fluorescence can be assessed routinely from spectral data obtained by calibration of intracellular fura-2 with known [Ca2+] using ionomycin. The end-of-experiment calibration step not only ensures accurate [Ca2+] measurements in polymorphonuclear leukocytes and in other cell types that display Ca2+-insensitive, contaminating fluorescence but also yields the spectral characteristics of the insensitive species.

The mechanism by which a cell translates a spatially oriented, extracellular signal into a change in morphology and behavior is the key to understanding many biological processes. In order to investigate this general phenomenon, I have studied the chemotactic response of human polymorphonuclear leukocytes (PMN's) to f-methionyl-leucyl-phenylalanine (fMLP). Stimulation of PMN's with fMLP produces a plethora of intracellular events, including increases in cytosolic Ca+2. PMN's are also morphologically and behaviorally polarized by stimulation with chemoattractant; the membrane components and cytosolic organelles of polarized PMN's become asymmetrically distributed. Polarization and subsequent orientation of PMN's in the direction of fMLP are steps which precede and are necessary for chemotaxis. I have chosen to examine the role of Ca+2, a ubiquitous second messenger, in the polarization of PMN's to fMLP. To accomplish this goal, Ca+2 has been measured in resting and polarized PMN's, utilizing the intracellular fluorescence of the Ca+2-sensitive dye, fura-2. Initial experiments have revealed a Ca+2-insensitive form of fura-2 associated with PMN's which, if uncorrected, would lead to erroneous measurements of [Ca+2]. I have suggested putative sources for the Ca+2-insensitive fluorescence in PMN's and have presented two methods for accurate calculation of [Ca+2] in spite of the additional component of fluorescence. As measured from the cell-associated fluorescence of fura-2, [Ca+2] increases without a detectable lag upon addition of fMLP to PMN's in suspension. The rise in [Ca+2] is associated with an increase in the percentage of cells which polarize to fMLP. The increases in [Ca+2] and in polarization are both directly related to increases in the concentration of chemoattractant. Inhibition of the rise in [Ca+2], by exposure of the human donor to aspirin or addition of EGTA to isolated cells, results in a concommitant reduction in the percentage of cells which polarize to fMLP. These findings are consistent with the hypothesis that Ca+2 acts as a second messenger in the pathway of transduction of the extracellular signal which results in polarization. However, addition of ionomycin, the Ca+2-selective ionophore, to PMN's did not induce polarization either in the presence or in the absence of fMLP. This result suggests that increases Ca+2, which appear to be necessary for polarization, are locally distributed within the fMLP-stimulated PMN. Examination of the subcellular distribution of Ca+2 using the digital imaging microscope reveals that Ca+2 is not uniformly distributed in the polarized PMN. Cells polarized by stimulation with fMLP often exhibit regional differences in [Ca+2] from front to tail. The magnitude and direction of the intracellular gradient varies among cells and suggests that within individual cells, the heterogeneity of [Ca+2] varies temporally and spatially as the cell chemotaxes. The results of the experiments conducted in this dissertation suggest that Ca+2 plays an important role as second messenger in fMLP-stimulated PMN's. I suggest that the morphological polarity of the chemotactic PMN is dependent upon the establishment and maintenance of an intracellular Ca+2 gradient.