Stimulation of Eryptosis by Narasin

Abstract

Background/Aims: Narasin, an ionophore used for the treatment of coccidiosis, has been shown to foster apoptosis of tumor cells. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by Ca²⁺ entry with subsequent increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), and by ceramide. The present study explored, whether and how narasin induces eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to narasin (10 and 25 ng/ml) significantly increased the percentage of annexin-V-binding cells. Forward scatter was decreased by 1 ng/ml narasin but not by higher narasin concentrations (10 and 25 ng/ml). Narasin significantly increased Fluo3-fluorescence (10 and 25 ng/ml) and slightly, but significantly increased ceramide abundance (25 ng/ml). The effect of narasin on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca²⁺. Conclusions: Narasin triggers phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled and partially dependent on Ca²⁺ entry. Narasin further leads to cell shrinkage and slight increase of ceramide abundance.