Flow cytometric kinetic measurements of neutrophil phospholipase A activation

• Published in: The Journal of biological chemistry 1992-10, Vol.267 (30), p.21465-21470
• Main Authors: Meshulam, T, Herscovitz, H, Casavant, D, Bernardo, J, Roman, R, Haugland, R.P., Strohmeier, G.S., Diamond, R.D., Simons, E.R.
• Format: Article
• Language: English
• Subjects: activation; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Boron Compounds; Calcium - metabolism; Cells, Cultured; Enzyme Activation; Enzymes and enzyme inhibitors; Flow Cytometry; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; Kinetics; leukocytes (neutrophilic); Liposomes; man; N-Formylmethionine Leucyl-Phenylalanine - pharmacology; Neutrophils - enzymology; Phosphatidylcholines - metabolism; phospholipase A; Phospholipases A - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)36632-3

The interrelationships between activation of phospholipases and neutrophil stimulus-induced Ca2+ responses remain unclear. We report here that immune complexes activate a phosphatidylcholine-specific phospholipase A in a neutrophil only after the cytoplasmic Ca2+ transient has been initiated in the same cell, while chemotactic peptide activation does not proceed via such a phospholipase A-mediated mechanism. Measurements of [Ca2+] changes and of phosphatidylcholine-specific phospholipase A activity were made by flow cytometry, using Indo-1 for Ca2+ indication, and a new fluorescent probe, bis-BODIPY-phosphatidylcholine, localized in the inner leaflet of the plasma membrane, to measure phospholipase A activation. Both 100 nM formyl-methionyl-leucyl-phenylalanine (with or without cytochalasin B) and 60 micrograms/ml insoluble immune complexes elicited cytoplasmic Ca2+ transients, but only insoluble immune complexes stimulated phospholipase A activation in a subpopulation of cells exhibiting an elevation of [Ca2+]in. Phospholipase A activation followed the Ca2+ transient, starting, in each cell, after [Ca2+]in had begun to decrease as Ca2+ redistributed in the activated cell. The products of this phospholipase activation were confirmed by thin layer chromatography. We conclude that neutrophils respond to immune complexes with an elevated cytoplasmic Ca(2+)-requiring phosphatidylcholine-specific phospholipase A activation and to chemotactic peptides by a different mechanism.