Correlation between chemotactic peptide-induced changes in chlorotetracycline fluorescence and F-actin content in human neutrophils: A role for membrane-associated calcium in the regulation of actin polymerization?

Abstract

Several observations indicate that the triggering event for receptor-mediated actin polymerization takes place in or close to the plasma membrane. Stimulation of human neutrophils with the chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMetLeuPhe) causes rapid and transient changes in both chlorotetracycline (CTC) fluorescence and the cellular content of filamentous actin (F-actin), thus suggesting a regulatory role for membrane-bound calcium in actin polymerization. In the present study, tetracaine, a proposed antagonist to membrane-bound calcium, totally inhibited the rebinding of the membrane calcium released by fMetLeuPhe. This was accompanied by a magnified and sustained increase in the cellular content of F-actin. In agreement, N-ethylmaleimide, an inhibitor of motile functions, completely abolished the fMetLeuPhe-triggered changes in both CTC fluorescence and F-actin content and rapidly reversed the responses when added after the peptide. The tumor promoter phorbol-12-myristate-13-acetate, caused only small changes in CTC fluorescence and F-actin content, and reduced a subsequent fMetLeuPhe-induced CTC response and actin polymerization. Inhibition of the breakdown of phosphatidylinositol 4,5-bisphosphate, by calcium depletion, had no significant effects on the fMetLeuPhe-induced CTC response and alterations in F-actin content, whereas pretreatment with pertussis toxin totally inhibited both these responses. Consequently, the strong correlation between changes in CTC fluorescence and F-actin content, found in this study, suggests a triggering or modulating role of membrane-associated calcium on actin polymerization in human neutrophils.