Amiloride derivatives modulate PS externalization in neutrophil-like PLB-985 cells.
During brain or cardiac ischemia/reperfusion neutrophils are recruited and activated contributing to inflammation and tissue damage. Neutrophils are removed from inflamed tissues by phosphatidylserine-dependent phagocytosis. Production of reactive oxygen species by the neutrophil NADPH-oxidase is known to affect phosphatidylserine externalization. Amiloride derivatives are inhibitors of the sodium-proton exchanger providing substantial protection in animal models of brain and cardiac ischemia/reperfusion injury; however their effects on neutrophils remain incompletely known. We investigated the effect of 5-(N,N-hexomethylene)amiloride (HMA) on phosphatidylserine externalization in wild type and NADPH-oxidase deficient PLB-985 cells differentiated into neutrophils. We show that HMA had a dual effect: (1) 60 microM HMA induced phosphatidylserine externalization in at least 40% of the cells; (2) 20 microM HMA had no direct effect but enhanced phosphatidylserine externalization induced by cell activation with PMA or calcium ionophore A23187. Both effects were independent of the NADPH-oxidase and were not due to changes in intracellular pH. 60 microM HMA induced a capacitative calcium entry which was necessary for phosphatidylserine externalization. The HMA-induced PS externalization was inhibited by salubrinal, an inhibitor of ER-stress-linked apoptosis. Lower HMA concentration enhanced PMA or A23187 effects through PKC and calcium dependent pathways. The caspase inhibitor Z-VAD-FMK weakly diminished phosphatidylserine externalization, suggesting that activation of caspases 7, 8, 9 and 3 was not involved. Increasing phosphatidylserine externalization by low concentrations of HMA improved the engulfment of PMA-activated PLB-985 cells by macrophages, providing a novel therapeutic strategy to limit the accumulation of neutrophils in injured tissues.
Bourge
Mickael
M
Univ Paris-Sud, Orsay F-91405, France.
Tlili
Asma
A
Dupré-Crochet
Sophie
S
Nüsse
Oliver
O
Sulpice
Jean-Claude
JC
D000584:Amiloride / Q000378:metabolism / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D053179:Caspase 7 / Q000378:metabolism; D002477:Cells / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000378:metabolism / Q000502:physiology; D019255:NADPH Oxidases / Q000378:metabolism / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology*; D010587:Phagocytosis; D010718:Phosphatidylserines / Q000378:metabolism / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D000584:Amiloride; D019255:NADPH Oxidases; D053179:Caspase 7; D013755:Tetradecanoylphorbol Acetate