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Membrane depolarization and depletion of intracellular calcium stores are associated with delay of apoptosis in human neutrophils

Apoptosis occurs rapidly in human polymorphonuclear leukocytes (PMN) after exposure to 1 mM cycloheximide (CHX). We examined whether this form of stimulated apoptosis altered either resting cytosolic free Ca2+ concentrations ([free Ca]) or membrane potential (Ψ) in PMN and found no significant effects. However, manipulation of either PMN intracellular Ca2+ stores or Ψ was found to delay CHX‐induced apoptosis. Depletion of PMN intracellular Ca2+ stores with thapsigargin caused membrane depolarization and significantly delayed CHX‐induced apoptosis based on both morphological and annexin‐V‐fluorescein isothiocyanate binding criteria. Short‐term suspension (4 h) of PMN in Ca2+‐free buffer depleted internal Ca2+ stores, induced membrane depolarization at 2.5 h, and delayed spontaneous (24 h) apoptosis but had no effect on CHX‐induced apoptosis. Rapid membrane depolarization with 150 mM KCl buffer significantly delayed CHX‐induced apoptosis, suggesting that depolarization rather than Ca2+ stores depletion was the crucial event. Timing experiments revealed that depolarization within 12 min of CHX exposure significantly delayed apoptosis. Collectively, these observations suggest an early Ψ‐sensitive step in the apoptosis pathway initiated by CHX. CHX exposure alone does not alter either resting PMN [free Ca] or Ψ; accompanying depolarization of plasma membrane (either electrochemically or via depletion of internal Ca2+ stores) delays CHX‐induced apoptosis in a time‐dependent manner. J. Leukoc. Biol. 64: 759–766; 1998.