Fluoxetine induced suicidal erythrocyte death

Abstract

The antidepressant fluoxetine inhibits ceramide producing acid sphingomyelinase. Ceramide is in turn known to trigger eryptosis the suicidal death of erythrocytes characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Ceramide is effective through sensitizing the erythrocytes to the pro-eryptotic effect of increased cytosolic Ca2+ activity ([Ca2+]i). In nucleated cells, fluoxetine could either inhibit or stimulate suicidal death or apoptosis. The present study tested whether fluoxetine influences eryptosis. To this end cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release and [Ca2+]i from Fluo-3 fluorescence intensity. As a result, a 48 h exposure of erythrocytes to fluoxetine (≥25 µM) significantly decreased forward scatter, increased annexin V binding and enhanced [Ca2+]i. The effect on annexin V binding was significantly blunted, but not abolished, in the absence of extracellular Ca2+. In conclusion, fluoxetine stimulates eryptosis, an effect at least in part due to increase of cytosolic Ca2+ activity.

Figure 1

Effect of fluoxetine on erythrocyte forward scatter. (A) Original histogram of forward scatter in erythrocytes following exposure for 48 h to Ringer solution without (grey shadow) and with (black line) presence of 50 µM fluoxetine; (B) Arithmetic means ± SEM (n = 12) of the normalized erythrocyte forward scatter (FSC) following incubation for 48 h to Ringer solution without (white bar) or with (black bars) fluoxetine (1–50 µM). * (p < 0.05) and *** (p < 0.001) indicate significant difference from the absence of fluoxetine (ANOVA).

Figure 2

Effect of fluoxetine on phosphatidylserine exposure and hemolysis. (A) Original histogram of annexin V binding of erythrocytes following exposure for 48 h to Ringer solution without (grey shadow) and with (black line) presence of 50 µM fluoxetine; (B) Arithmetic means ± SEM (n = 12) of erythrocyte annexin V binding following incubation for 48 h to Ringer solution without (white bar) or with (black bars) presence of fluoxetine (1–50 µM). For comparison, arithmetic means ± SEM (n = 4) of the percentage of hemolysis is shown as grey bars. ** (p < 0.01) and *** (p < 0.001) indicate significant differences from the absence of fluoxetine (ANOVA).

Figure 3

Effect of fluoxetine on erythrocyte cytosolic Ca²⁺ concentration. (A) Original histogram of Fluo-3 fluorescence intensity (arbitrary units) in erythrocytes following exposure for 48 h to Ringer solution without (grey shadow) and with (black line) presence of 50 µM fluoxetine; (B) Arithmetic means ± SEM (n = 12) of the Fluo-3 mean fluorescence intensity (MFI) (arbitrary units) in erythrocytes exposed for 48 h to Ringer solution without (white bar) or with (black bars) fluoxetine (1–50 µM). *** (P < 0.001) indicate significant difference from the absence of fluoxetine (ANOVA).

Figure 4

Effect of Ca²⁺ withdrawal on fluoxetine induced annexin V binding. Arithmetic means ± SEM (n = 4) of the percentage of annexin V binding erythrocytes after a 48 h treatment with Ringer solution without (white bar) or with (black bars) 50 µM fluoxetine in the presence (left bars, + Calcium) and absence (right bars, − Calcium) of calcium. *** (p < 0.001) indicate significant difference from the absence of fluoxetine (ANOVA), # (p < 0.05) indicates significant difference from the respective values in the presence of Ca²⁺.