Stimulation of erythrocyte phospholipid scrambling by enniatin A



Scope: Enniatin A, a peptide antibiotic and common food contaminant, triggers mitochondrial dysfunction and apoptosis. Even though lacking mitochondria, erythrocytes may similarly undergo suicidal cell death or eryptosis. Eryptosis is characterized by cell shrinkage and cell membrane phospholipid scrambling. Triggers of phospholipid scrambling include energy depletion and increase in cytosolic Ca2+ activity ([Ca2+]i). The present study explored whether enniatin A triggers phospholipid scrambling.

Methods and results: Phospholipid scrambling was estimated from annexin-V-binding, cell volume from forward scatter (FSC), [Ca2+]i from Fluo3-fluorescence, cytosolic ATP-concentration ([ATP]i) using a luciferase assay and hemolysis from hemoglobin release. Exposure of erythrocytes for 48 h to enniatin A (≥2.5 μM) significantly increased [Ca2+]i, decreased [ATP]i, decreased FSC, triggered annexin-V-binding and elicited hemolysis. Annexin-V-binding affected 25%, and hemolysis 2% of treated erythrocytes. Decreased [ATP]i by glucose depletion for 48 h was similarly followed by increased [Ca2+]i, decreased FSC and annexin-V-binding. Enniatin A augmented the effect on [Ca2+]i and annexin-V-binding, but not on FSC. Annexin-V-binding was blunted by Ca2+ removal, by the cation channel inhibitor amiloride (1 mM), by the protein kinase C inhibitor staurosporine (500 nM) but not by the pancaspase inhibitor zVAD (10 μM).

Conclusion: The food contaminant enniatin A triggers ATP depletion and increases cytosolic Ca2+ activity, effects resulting in suicidal erythrocyte death.