CHARACTERIZATION OF EXOCYTOSIS IN ELECTROPERMEABILIZED NEUTROPHILS BYFLOW CYTOMETRIC ANALYSIS - DIFFERENCE IN SENSITIVITY TO CALCIUM AND GUANOSINE-5'-[GAMMA-THIO]TRIPHOSPHATE

When rabbit neutrophils were subjected to two electrical discharges of 4.75 kV/cm, the cells became permeable to propidium iodide. Measureme nt of propidium iodide fluorescence using flow cytometry showed that a ll cells in the suspension were permeabilized. The cells remained perm eable for > 20 min when the cells were stored at 0 degrees C. When exo cytosis was induced by Ca2+ alone, the orthogonal light scatter (a sen sitive parameter for cell granularity) of the complete population chan ged depending on the concentration. All the cells were equally sensiti ve to Ca2+ and showed a similar degree of exocytosis at the same time. In the presence of a fixed concentration of Ca2+ and a variable conce ntration of guanosine-5'-[nu-thio]triphosphate (GTP gamma S), a divisi on of the cell population was observed in the orthogonal light scatter histogram. At low GTP gamma S concentrations, a part of the populatio n showed complete exocytosis and a part of the population showed almos t no exocytosis. With increasing GTP gamma S concentrations, the light scatter pattern of the population changed indicating that the cells w ere gradually sensitive to GTP theta S. Electropermeabilized neutrophi ls showed an equal sensitivity to Ca2+ and a graded sensitivity to GTP gamma S. Flow cytometry is considered as an ideal tool to study such an effect on a cell-to-cell basis. (C) 1994 Wiley-Liss, Inc.