title: Novel Insights in the Regulation of Phosphatidylserine Exposure in Human Red Blood Cells.
abstract: OBJECTIVE
  In previous publications we were able to demonstrate the exposure of phosphatidylserine (PS) in the outer membrane leaflet after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA), phorbol-12 myristate-13acetate (PMA), or 4-bromo-A23187 (A23187). It has been concluded that three different mechanisms are responsible for the PS exposure in human RBCs: (i) Ca2+-stimulated scramblase activation (and flippase inhibition) by A23187, LPA, and PMA; (ii) PKCα activation by LPA and PMA; and (iii) enhanced lipid flip flop caused by LPA. Further studies aimed to elucidate interconnections between the increased Ca2+ content, scramblase- and PKCα-activation. In addition, the role of the Ca2+-activated K+ channel (Gardos channel) activity in the process of PS exposure needs to be investigated.
  
  
  METHODS
  The intracellular Ca2+ content and the PS exposure of RBCs have been investigated after treatment with LPA (2.5 µM), PMA (6 µM), or A23187 (2 µM). Fluo-4 and annexin V-FITC has been used to detect intracellular Ca2+ content and PS exposure, respectively. Both parameters (Ca2+ content, PS exposure) were studied using flow cytometry. Inhibitors of the scramblase, the PKCα, and the Gardos channel have been applied.
  
  
  RESULTS
  The percentage of RBCs showing PS exposure after activation with LPA, PMA, or A23187 is significantly reduced after inhibition of the scramblase using the specific inhibitor R5421 as well as after the inhibition of the PKCα using chelerythrine chloride or calphostin C. The inhibitory effect is more pronounced when the scramblase and the PKCα are inhibited simultaneously. Additionally, the inhibition of the Gardos channel using charybdotoxin resulted in a significant reduction of the percentage of RBCs showing PS exposure under all conditions measured. Similar results were obtained when the Gardos channel activity was suppressed by increased extracellular K+ content.
  
  
  CONCLUSIONS
  PS exposure is mediated by the Ca2+-dependent scramblase but also by PKCα activated by LPA and PMA in a Ca2+-dependent and a Ca2+-independent manner. Furthermore, we hypothesize that a hyperpolarisation of RBCs caused by the opening of the Gardos channel is essential for the scramblase activity as well as for a fraction of the LPA-induced Ca2+ entry.
authors: Laboratory of Biophysics, Faculty of Natural and Technical Sciences III, Saarland University, Saarbrücken, Germany.
authors: Mauro C
authors: MC
authors: Wesseling
authors: Lisa
authors: L
authors: Wagner-Britz
authors: Duc Bach
authors: DB
authors: Nguyen
authors: Salome
authors: S
authors: Asanidze
authors: Judy
authors: J
authors: Mutua
authors: Nagla
authors: N
authors: Mohamed
authors: Benjamin
authors: B
authors: Hanf
authors: Mehrdad
authors: M
authors: Ghashghaeinia
authors: Lars
authors: L
authors: Kaestner
authors: Ingolf
authors: I
authors: Bernhardt
mesh_terms: D017304:Annexin A5 / Q000235:genetics / Q000378:metabolism; D053119:Benzophenanthridines / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D018999:Charybdotoxin / Q000494:pharmacology; D004906:Erythrocyte Count; D004912:Erythrocytes; D005786:Gene Expression Regulation; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008246:Lysophospholipids / Q000494:pharmacology*; D008724:Methomyl / Q000031:analogs & derivatives / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D017304:Annexin A5; D053119:Benzophenanthridines; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D008246:Lysophospholipids; D009281:Naphthalenes; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; C111182:ethanimidothioic acid N-((N-butylthio-N-methylamino)-carbonyloxy)-methylester; D018999:Charybdotoxin; D008724:Methomyl; D000001:Calcimycin; C016299:chelerythrine; D051571:Protein Kinase C-alpha; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; C032881:lysophosphatidic acid; D002118:Calcium