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 (psi) in PMN and found no significant effects. However, manipulation of either PMN intracellular Ca2+ stores or psi 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 psi-sensitive step in the apoptosis pathway initiated by CHX. CHX exposure alone does not alter either resting PMN [free Ca] or psi; accompanying depolarization of plasma membrane (either electrochemically or via depletion of internal Ca2+ stores) delays CHX-induced apoptosis in a time-dependent manner.