title: Stimulation of Suicidal Erythrocyte Death by Rottlerin.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Cardiology, Vascular Medicine and Physiology, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany.
authors.forename: Morena
authors.initials: M
authors.lastname: Mischitelli
authors.forename: Mohamed
authors.initials: M
authors.lastname: Jemaà
authors.forename: Mustafa
authors.initials: M
authors.lastname: Almasry
authors.forename: Caterina
authors.initials: C
authors.lastname: Faggio
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000098:Acetophenones / Q000494:pharmacology*; D001578:Benzopyrans / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D000072817:Eryptosis / Q000187:drug effects*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism; D012542:Scattering, Radiation
chemical_list: D000098:Acetophenones; D001578:Benzopyrans; D002518:Ceramides; D010718:Phosphatidylserines; C085746:rottlerin; D002118:Calcium