Eryptosis, the suicidal death of erythrocytes, is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by several stress conditions including isotonic cell shrinkage (Cl- removal) and energy depletion (glucose removal). Both are effective through an increase in the cytosolic Ca2+ concentration. Phosphatidylserine-exposing erythrocytes are cleared from circulating blood. Enhanced eryptosis thus leads to anemia. Accordingly, drugs interfering with eryptosis may prove useful in the treatment of anemia. The present study explored, whether caffeine interferes with eryptosis. Erythrocyte phosphatidylserine exposure was estimated from annexin V-binding, cell volume from forward scatter and cytosolic Ca2+ activity from Fluo3 fluorescence. Under control conditions, eryptosis affected less than 5% of the erythrocytes and was not significantly modified by the presence of caffeine (50-500 µM).Glucose depletion (for 48 hours) significantly increased Fluo3 fluorescence and annexin V-binding and decreased forward scatter, effects partially reversed by caffeine (500 µM).Low Cl- solution (Cl- exchanged by gluconate for 48 hours) similarly increased annexin V-binding and decreased forward scatter, effects again reversed by caffeine (50-500 µM). In conclusion, caffeine inhibits Ca2+ entry following glucose depletion and thus counteracts eryptosis during isotonic cell shrinkage and energy depletion.

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