title: Stimulation of Phospholipid Scrambling of the Erythrocyte Membrane by 9-Cis-Retinoic Acid.
abstract: OBJECTIVE
  The endogenous retinoid 9-cis-retinoic acid has previously been shown to trigger apoptosis in a wide variety of cells including several tumor cells and has thus been suggested for the treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms participating in the accomplishment of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) and formation of ceramide. The present study explored, whether 9-cis-retinoic acid induces eryptosis and whether the effect involves Ca2+ and/or ceramide.
  
  
  METHODS
  Flow cytometry was employed to estimate erythrocyte volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from hemoglobin concentration in the supernatant.
  
  
  RESULTS
  A 48 hours exposure of human erythrocytes to 9-cis-retinoic acid (≥ 0.5 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Exposure to 9-cis-retinoic acid (≥ 0.5 µg/ml) significantly increased Fluo3-fluorescence, and the effect of 9-cis-retinoic acid on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Exposure to 9-cis-retinoic acid (1 µg/ml) further significantly increased the ceramide abundance at the erythrocyte surface and significantly increased hemolysis.
  
  
  CONCLUSIONS
  9-cis-retinoic acid triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca2+ and ceramide.
authors.forename: Majed
authors.initials: M
authors.lastname: Abed
authors.forename: Kousi
authors.initials: K
authors.lastname: Alzoubi
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
authors.forename: Abdulla
authors.initials: A
authors.lastname: Al Mamun Bhuayn
mesh_terms: D000077556:Alitretinoin; D002118:Calcium / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D000072817:Eryptosis / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D006454:Hemoglobins / Q000032:analysis; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D002518:Ceramides; D006454:Hemoglobins; D010718:Phosphatidylserines; D000077556:Alitretinoin; D014212:Tretinoin; D002118:Calcium
keywords: 9-cis-retinoic acid; Calcium; Cell volume; Eryptosis; Phosphatidylserine