fm1-43 and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

Phospholipid scrambling, the disruption of normal plasma-membrane asymmetry, occurs during apoptotic and necrotic cell death and during the activation of platelets and neutrophils. It is currently believed that phospholipid scrambling is triggered simply by increases in bulk cytosolic [Ca(2+)]. We have presented evidence previously that the styryl dye FM1-43 is sensitive to phospholipid scrambling in Jurkat human leukaemic T-lymphocytes. Here we have used FM1-43, in combination with fura 2 and the Ca(2+)-elevating agents ionomycin and thapsigargin, in imaging experiments to test the idea that increases in bulk cytosolic [Ca(2+)] stimulate scrambling. Intracellular Ca(2+) increases of approximately 2 microM accompanied ionomycin-stimulated scrambling in approximately 50% of cells, and scrambling occurred in >99% of cells in which intracellular Ca(2+) rose to 4 microM. Chelating intracellular Ca(2+) with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid or EGTA suppressed both ionomycin-stimulated intra cellular Ca(2+) increases and scrambling, demonstrating that intracellular Ca(2+) increases are necessary for ionomycin-stimulated scrambling. However, elevating intracellular Ca(2+) to 2-4 microM with thapsigargin, a drug that depletes intracellular Ca(2+) stores and triggers Ca(2+) entry via Ca(2+)-release-activated Ca(2+) channels, did not trigger scrambling, as assessed with either FM1-43 or FITC-labelled annexin V. These results suggest that increases in intracellular [Ca(2+)] are necessary but not sufficient to stimulate scrambling in lymphoyctes, and indicate that ionomycin has an additional effect that is required to stimulate scrambling.

Topics: Annexin A5; Calcium; Calcium Chloride; Calcium-Transporting ATPases; Cytosol; Egtazic Acid; Fluoresceins; Fluorescent Dyes; Humans; Ionomycin; Jurkat Cells; Kinetics; Microscopy, Fluorescence; Phospholipids; Pyridinium Compounds; Quaternary Ammonium Compounds; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spectrometry, Fluorescence; T-Lymphocytes; Thapsigargin