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Stimulation of Suicidal Erythrocyte Death by Rottlerin
Morena Mischitelli1, Mohamed Jemaà, Mustafa Almasry
1Department of Cardiology, Vascular Medicine and Physiology, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany.
Background/Aims:
The phytochemical polyphenol rottlerin is a potent activator of diverse Ca2+ -sensitive K+ channels. Those channels play a decisive role in the execution of eryptosis, the suicidal death of erythrocytes, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i) and ceramide. The present study explored, whether rottlerin induces eryptosis and, if so, to test for the involvement of Ca2+ entry and ceramide.
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. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant.
Results:
A 48 hours exposure of human erythrocytes to rottlerin (1 - 5 µM) significantly increased the percentage of annexin-V-binding cells, an effect paralleled by significant decrease of forward scatter. Up to 5 µM rottlerin failed to significantly increase average Fluo3-fluorescence. Rottlerin (5 µM) did, however, significantly increase the ceramide abundance. Rottlerin (5 µM) further significantly increased hemolysis. The effect of rottlerin (5 µM) on annexin-V-binding was virtually abolished by removal of extracellular Ca2+.
Conclusions:
Rottlerin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry and ceramide.
Insights
Rottlerin induces suicidal erythrocyte death (eryptosis) by increasing cell shrinkage and membrane scrambling. This process involves calcium influx and ceramide production, highlighting rottlerin
Area of Science:
- Biochemistry
- Cell Biology
- Physiology
Background:
- Rottlerin, a phytochemical polyphenol, activates calcium-sensitive potassium channels.
- These channels are crucial for eryptosis, the programmed death of red blood cells.
- Eryptosis involves cell shrinkage and phosphatidylserine externalization, regulated by intracellular calcium and ceramide.
Purpose of the Study:
- To investigate if rottlerin induces eryptosis in human erythrocytes.
- To determine the role of calcium influx and ceramide in rottlerin-mediated eryptosis.
Main Methods:
- Flow cytometry was used to assess phosphatidylserine exposure (annexin-V binding) and cell volume (forward scatter).
- Intracellular calcium levels ([Ca2+]i) were measured using Fluo3-fluorescence.
- Ceramide abundance was quantified using specific antibodies, and hemolysis was measured spectrophotometrically.
Main Results:
- Rottlerin treatment (1-5 µM) significantly increased phosphatidylserine exposure and decreased cell volume.
- Rottlerin (5 µM) significantly elevated ceramide levels and induced hemolysis.
- The eryptosis-inducing effect of rottlerin was significantly reduced by removing extracellular calcium.
Conclusions:
- Rottlerin effectively stimulates eryptosis, characterized by erythrocyte shrinkage and membrane scrambling.
- Calcium influx and increased ceramide production are key mechanisms mediating rottlerin-induced eryptosis.