Cl- channel blockers NPPB and niflumic acid blunt Ca(2+)-induced erythrocyte 'apoptosis'.
Exposure to Ca2+ ionophore ionomycin, osmotic shock, oxidative stress and glucose depletion trigger cell shrinkage and scramblase-mediated phosphatidylserine exposure at the outer leaflet of the erythrocyte cell membrane. The effects are partially due to activation of GARDOS channels and subsequent cellular K+ loss leading not only to cell shrinkage but also participating in the triggering of erythrocyte scramblase. As conductive loss of K+ would depend on the parallel loss of anions we hypothesised that activation of scramblase is similarly dependent on the activity of Cl- channels. To test this hypothesis, we used Cl- channel blockers NPPB and niflumic acid. It is shown here that treatment of erythrocytes with 1 microM ionomycin leads to cellular K+ loss, decrease of hematocrit and decrease of forward scatter in FACS analysis reflecting cell shrinkage as well as increase of annexin positive cells reflecting phosphatidylserine exposure. Those events were significantly blunted in the presence of 100 microM NPPB by 34% (K+ loss), 45% (hematocrit), 32% (forward scatter) and 69% (annexin binding), or in the presence of 100 microM niflumic acid by 15% (forward scatter) and 45% (annexin binding), respectively. Moreover, oxidative stress triggered annexin binding which was again significantly inhibited (by 51%) in the presence of 100 microM NPPB. In conclusion, Cl- channels presumably participate in the regulation of erythrocyte 'apoptosis'.
Department of Physiology, University of Tübingen, Germany.
Svetlana
S
Myssina
Philipp A
PA
Lang
Daniela S
DS
Kempe
Stefanie
S
Kaiser
Stephan M
SM
Huber
Thomas
T
Wieder
Florian
F
Lang
D017302:Annexins / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000037:antagonists & inhibitors*; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000502:physiology*; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006400:Hematocrit; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008565:Membrane Proteins / Q000502:physiology; D009544:Niflumic Acid / Q000494:pharmacology*; D009579:Nitrobenzoates / Q000494:pharmacology*; D009997:Osmotic Pressure; D018384:Oxidative Stress; D049294:Phospholipid Transfer Proteins / Q000502:physiology; D011188:Potassium / Q000378:metabolism
D017302:Annexins; D018118:Chloride Channels; D007476:Ionophores; D008565:Membrane Proteins; D009579:Nitrobenzoates; D049294:Phospholipid Transfer Proteins; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D009544:Niflumic Acid; D015759:Ionomycin; D011188:Potassium; D002118:Calcium