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.
Chacon-Cruz
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Center for Pediatric Research, Children's Hospital of The King's Daughters, Eastern Virginia Medical School, Norfolk 23510-1001, USA. ecruz@chkd.com
Oelberg
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DG
Davis
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P
Buescher
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ES
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D003513:Cycloheximide / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism / Q000502:physiology; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism* / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors
D017304:Annexin A5; D019284:Thapsigargin; D003513:Cycloheximide; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium