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.