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 stim ulated apoptosis altered either resting cytosolic free Ca2+ concentrations ([free Ca]) or membrane potential (psi) in PMN and found no significant eff ects. However, manipulation of either PMN intracellular Ca2+ stores or psi was found to delay CHX-induced apoptosis, Depletion of PMN intracellular Ca 2+ stores with thapsigargin caused membrane depolarization and significantl y delayed CHX-induced apoptosis based on both morphological and annexin-v-f luorescein isothiocyanate binding criteria. Short-term suspension (4 h) of PMN in Ca2+-free buffer depleted internal Ca2+ stores, induced membrane dep olarization at 2.5 h, and delayed spontaneous (24 h) apoptosis but had no e ffect on CIM-induced apoptosis. Rapid membrane depolarization with 150 mM K CI buffer significantly delayed CIM-induced apoptosis, suggesting that depo larization rather than Ca2+ stores depletion was the crucial event. Timing experiments revealed that depolarization within 12 min of CHX exposure sign ificantly delayed apoptosis. Collectively, these observations suggest an ea rly psi-sensitive step in the apoptosis pathway initiated by CHX. CHX expos ure alone does not alter either resting PMN [free Ca] or psi; accompanying depolarization of plasma membrane (either electrochemically or via depletio n of internal Ca2+ stores) delays CHX-induced apoptosis in a time-dependent manner.