Inhibition of K+ efflux prevents mitochondrial dysfunction, and suppresses caspase-3-, apoptosis-inducing factor-, and endonuclease G-mediated constitutive apoptosis in human neutrophils.
Neutrophils die rapidly via apoptosis and their survival is contingent upon rescue from constitutive programmed cell death by signals from the microenvironment. In these experiments, we investigated whether prevention of K(+) efflux could affect the apoptotic machinery in human neutrophils. Disruption of the natural K(+) electrochemical gradient suppressed neutrophil apoptosis (assessed by annexin V binding, nuclear DNA content and nucleosomal DNA fragmentation) and prolonged cell survival within 24-48 h of culture. High extracellular K(+) (10-100 mM) did not activate extracellular signal-regulated kinase (ERK) and Akt, nor affected phosphorylation of p38 MAPK associated with constitutive apoptosis. Consistently, pharmacological blockade of ERK kinase or phosphatidylinositol 3-kinase (PI 3-kinase) did not affect the anti-apoptotic action of KCl. Inhibition of K(+) efflux effectively reduced, though never completely inhibited, decreases in mitochondrial transmembrane potential (DeltaPsi(m)) that preceded development of apoptotic morphology. Changes in DeltaPsi(m) resulted in attenuation of cytochrome c release from mitochondria into the cytosol and decreases in caspase-3 activity. Culture of neutrophils in medium containing 80 mM KCl with the pan-caspase inhibitor Z-VAD-FMK resulted in slightly greater suppression of apoptosis than KCl alone. High extracellular KCl also attenuated translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) from mitochondria to nuclei. The DNase inhibitor, aurintricarboxylic acid (ATA) partially inhibited nucleosomal DNA fragmentation, and the effects of ATA and 80 mM KCl were not additive. These results show that prevention of K(+) efflux promotes neutrophil survival by suppressing apoptosis through preventing mitochondrial dysfunction and release of the pro-apoptotic proteins cytochrome c, AIF and EndoG independent of ERK, PI 3-kinase and p38 MAPK. Thus, K(+) released locally from damaged cells may function as a survival signal for neutrophils.
El Kebir
Driss
D
Research Center, Maisonneuve-Rosemont Hospital, University of Montréal, 5415 boulevard de l'Assomption, Montréal, QC, Canada H1T 2M4.
József
Levente
L
Khreiss
Tarek
T
Filep
János G
JG
D000328:Adult; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051033:Apoptosis Inducing Factor / Q000037:antagonists & inhibitors / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D061945:Caspase Inhibitors; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008875:Middle Aged; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011188:Potassium / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D051033:Apoptosis Inducing Factor; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D061945:Caspase Inhibitors; D004706:Endodeoxyribonucleases; D004791:Enzyme Inhibitors; D048049:Extracellular Signal-Regulated MAP Kinases; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011188:Potassium; D011189:Potassium Chloride; D051057:Proto-Oncogene Proteins c-akt; D048051:p38 Mitogen-Activated Protein Kinases; C052391:endonuclease G