Formyl-methionylleucylphenylalanine (fMLP) activation of neutrophils causes an increase in intracellular Ca2+, activation of protein kinase C and an increase in F-actin content. To examine the role of Ca2+ and protein kinase C activation as determinants of change in F-actin content of neutrophils, we used the NBD-phallacidin extraction assay to compare the kinetics and extent of change in F-actin content of cells activated with fMLP, the calcium ionophore A23187 or phorbol myristate acetate (PMA). All stimuli increase the F-actin content in a dose-dependent manner; however, the rate of increase is slower and the maximum F-actin content is less for calcium ionophore and PMA than for fMLP-activated cells. The A23187-induced increase in F-actin content, but not that of fMLP, depends upon external free [Ca2+]. In A23187-activated cells, F-actin content increases at [Ca2+]free greater than or equal to 5 microM, is maximal at [Ca2+]free greater than or equal to 10 microM and is negligible at physiologic free [Ca2+] (10(-7)-10(-6) M). Combinations of PMA with A23187 or fMLP inhibit the A23187, but not the fMLP, activated actin polymerization. Comparison and combination of these activators shows that neither Ca2+-dependent activation with A23187 nor activation with PMA alone or in combination mimic the fMLP-induced changes in cytoskeleton organization of neutrophils.
T H Howard, D Wang