Vitamin A and retinoic acid have previously been shown to confer some protection against a severe course of malaria by fostering the phagocytosis of parasitized erythrocytes. Phagocytosis of erythrocytes is stimulated by phosphatidylserine exposure at the cell surface. The present study has thus been performed to explore the effect of retinoic acid and the specific retinoic acid receptor (RAR) agonist 4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB) on erythrocyte annexin V binding, which reflects phosphatidylserine exposure at the cell surface. A 24 hours exposure to either, retinoic acid (3 μM) or TTNPB (3 μM), indeed significantly increased annexin binding, an effect paralleled by decrease of forward scatter reflecting cell shrinkage. According to Fluo3 fluorescence, exposure to either, retinoic acid (10 μM, 24 hours) or TTNPB (10 μM, 6 hours), significantly increased cytosolic Ca2+-activity, a known trigger of phosphatidylserine exposure. Infection of erythrocytes with Plasmodium falciparum increased phosphatidylserine exposure, an effect increased in the presence of TTNPB. In conclusion, retinoid acid and TTNPB trigger phosphatididylserine exposure and cell shrinkage of erythrocytes, typical features of suicidal erythrocyte death or eryptosis. The eryptosis could participate in the accelerated clearance of parasitized erythrocytes from circulating blood following treatment with retinoids.
Background: Novobiocin, an aminocoumarin antibiotic, interferes with heat shock protein 90 and hypoxia inducible factor dependent gene expression and thus compromises cell survival. Similar to survival of nucleated cells, erythrocyte survival could be disrupted by eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phospholipd scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i). The Ca2+ sensitivity of phospholipid scrambling is enhanced by ceramide. The present study explored, whether novobiocin elicits eryptosis. Methods: [Ca2+]i was estimated from Fluo3-fluorescence, ceramide abundance utilizing fluorescent antibodies, cell volume from forward scatter, phosphatidylserine-exposure from annexin V binding. Results: A 48 hours exposure to novobiocin (500 µM) was followed by a significant increase of [Ca2+]i, decrease of forward scatter, increase of annexin-V-binding and enhanced ceramide formation. Removal of extracellular Ca2+ virtually abrogated the increase of annexin-V-binding following novobiocin exposure. Conclusions: Novobiocin stimulates eryptosis, an effect at least in part due to entry of extracellular Ca2+ and formation of ceramide.
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