Evidence that cytosolic calcium increases are not sufficient to stimulate phospholipid scrambling in human T-lymphocytes

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 [Ca2+]. 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 Ca2+-elevating agents ionomycin and thapsigargin, in imaging experiments to test the idea that increases in bulk cytosolic [Ca2+] stimulate scrambling. Intracellular Ca2+ increases of ≈ 2μM accompanied ionomycin-stimulated scrambling in ≈ 50% of cells, and scrambling occurred in > 99% of cells in which intracellular Ca2+ rose to 4μM. Chelating intracellular Ca2+ with bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid or EGTA suppressed both ionomycin-stimulated intra cellular Ca2+ increases and scrambling, demonstrating that intracellular Ca2+ increases are necessary for ionomycin-stimulated scrambling. However, elevating intracellular Ca2+ to 2–4μM with thapsigargin, a drug that depletes intracellular Ca2+ stores and triggers Ca2+ entry via Ca2+-release-activated Ca2+ channels, did not trigger scrambling, as assessed with either FM1-43 or FITC-labelled annexin V. These results suggest that increases in intracellular [Ca2+] are necessary but not sufficient to stimulate scrambling in lymphoyctes, and indicate that ionomycin has an additional effect that is required to stimulate scrambling.