Submitted on April 6, 2006
Accepted on January 19, 2007
Enhanced suicidal death of erythrocytes from gene-targeted mice lacking the Cl-/HCO3- exchanger AE1
Ahmad Akel1, Carsten A Wagner2, Jana Kovacikova3, Ravi Sankar Kasinathan4, Valentin Kiedaisch1, Saisudha Koka1, Seth L. Alper5, Ingolf Bernhardt6, Thomas Wieder1, Stephan Huber1, and Florian Lang1*
1 Department of Physiology, University of Tuebingen, Tuebingen, Germany
2 Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland
3 Institute of Physiology and Center for Integrative Human Physiology, University of Zürich, Zurich, Switzerland
4 Physiology I, Institute of Physiology, Tuebingen, Germany
5 Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States
6 Zentrales Isotopenlabor/AG Biophysik, University of Saarbrücken, Saarbrücken, Germany
* To whom correspondence should be addressed. E-mail: florian.lang{at}uni-tuebingen.de.
Genetic defects of the anion exchanger AE1 may lead to spherocytic erythrocyte morphology, severe hemolytic anemia and/or cation leaks. In normal erythrocytes osmotic shock, Cl- removal and energy depletion activate Ca2+-permeable cation channels with Ca2+ induced eryptosis, i.e. surface exposure of phosphatidylserine, cell shrinkage and membrane blebbing, all features typical for apoptosis of nucleated cells. Eryptotic erythrocytes are engulfed by macrophages and cleared from circulating blood. The present experiments explored whether AE1 deficiency favors eryptosis. Peripheral blood erythrocyte number was significantly smaller in gene-targeted mice lacking AE1 (AE1-/- ) than in their wild type littermates ((AE1+/+), despite an increased percentage of reticulocytes ((AE1-/-: 49%, (AE1+/+: 2%), an indicator of enhanced erythropoiesis. Annexin binding reflecting phosphatidylserine exposure was significantly larger in (AE1-/-erythrocytes (~10 %) than in (AE1+/+erythrocytes (~1 %). Osmotic shock (addition of 400 mM sucrose), Cl- removal (replacement with gluconate) or energy depletion (removal of glucose) led to significantly stronger annexin binding in (AE1-/-erythrocytes than in (AE1+/+erythrocytes. The increase of annexin binding following exposure to the Ca2+ ionophore ionomycin (1 µM) was, however, similar in (AE1-/- and in (AE1+/+erythrocytes. Fluo3 fluorescence revealed markedly increased cytosolic Ca2+ activity in (AE1-/-erythrocytes. Clearance of CFSE-labelled erythrocytes from circulating blood was more rapid in (AE1-/- than in (AE1+/+mice and accelerated by ionomycin treatment in both genotypes. In conclusion, lack of AE1 is associated with enhanced Ca2+ entry and subsequent scrambling of cell membrane phospholipids.
