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Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 367, Issue 4, pp 391–396 | Cite as

Inhibition of erythrocyte cation channels and apoptosis by ethylisopropylamiloride

  • K. S. Lang
  • S. Myssina
  • V. Tanneur
  • T. Wieder
  • S. M. Huber
  • F. Lang
  • C. Duranton
Original Article

Abstract

Even though lacking mitochondria and nuclei erythrocytes do undergo apoptotic cell death which is characterized by breakdown of phosphatidylserine asymmetry (leading to annexin binding), membrane blebbing and cell shrinkage. Previously, we have shown that erythrocyte apoptosis is triggered by osmotic shrinkage at least in part through activation of cell volume-sensitive cation channels and subsequent Ca2+ entry. The channels could not only be activated by cell shrinkage but as well by replacement of Cl with gluconate. Both, channel activity and annexin binding were sensitive to high concentrations of amiloride (1 mM). The present study has been performed to search for more effective blockers. To this end channel activity has been evaluated utilizing whole-cell patch-clamp and annexin binding determined by FACS analysis as an indicator of erythrocyte apoptosis. It is shown that either, increase of osmolarity or replacement of Cl by gluconate triggers the activation of the cation channel which is inhibited by amiloride at 1 mM but not at 100 µM. Surprisingly, the cation channel was significantly more sensitive to the amiloride analogue ethylisopropylamiloride (EIPA, IC50=0.6±0.1 µM, n=5). Exposure of the cells to osmotic shock by addition of sucrose (850 mOsm) led to stimulation of annexin binding which was inhibited similarly by EIPA (IC50=0.2±0.2 µM, n=4). Moreover, annexin binding was inhibited by higher concentrations of HOE 642 (IC50=10±5 µM, n=5) and HOE 694 (IC50=12±6 µM, n=4). It is concluded that osmotic shock stimulates a cation channel which participates in the triggering of erythrocyte apoptosis. EIPA is an effective inhibitor of this cation channel and of channel mediated triggering of erythrocyte apoptosis.

Keywords

Cell volume Annexin binding Osmotic cell shrinkage Cation conductance Patch-clamp 

Notes

Acknowledgements

The authors acknowledge the technical assistance and the meticulous preparation of the manuscript by Tanja Loch and Marion Rabe. Christophe Duranton has been supported by a grant of the Alexander von Humboldt Foundation (Germany, IV-FRA1071599). This study was supported by the Deutsche Forschungsgemeinschaft, Nr. La 315/4-3 and La 315/6-1, the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Center for Interdisciplinary Clinical Research) 01 KS 9602 and the Biomed program of the EU (BMH4-CT96-0602).

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Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • K. S. Lang
    • 1
  • S. Myssina
    • 1
  • V. Tanneur
    • 1
  • T. Wieder
    • 1
  • S. M. Huber
    • 1
  • F. Lang
    • 1
  • C. Duranton
    • 1
  1. 1.Department of PhysiologyUniversity of TübingenTübingenGermany

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