Inhibitory effect of furosemide on non-selective voltage-independent cation channels in human erythrocytes.
BACKGROUND
Furosemide, a loop diuretic inhibiting the renal tubular Na(+),K(+),2Cl(-) cotransporter, has been shown to decrease cytosolic Ca(2+) concentration ([Ca(2+)](i)) in platelets and erythrocytes. [Ca(2+)](i) in erythrocytes is a function of Ca(2+) permeable cation channels. Activation of those channels e.g. by energy depletion or oxidative stress leads to increase of [Ca(2+)](i), which in turn triggers eryptosis, a suicidal erythrocyte death characterized by cell membrane scrambling. The present study was performed to explore whether furosemide influences the cation channels and thus influences eryptosis.


METHODS
Cation channel activity was determined by whole-cell patch clamp, [Ca(2+)](i) utilizing Fluo3 fluorescence and annexin V binding to estimate cell membrane scrambling with phosphatidylserine exposure.


RESULTS
A 45 min exposure to furosemide (10 and 100 µM) slightly, but significantly decreased cation channel activity and [Ca(2+)](i) in human erythrocytes drawn from healthy individuals. ATP-depletion (> 3 hours, +37°C, 6 mM ionosine and 6 mM iodoacetic acid) enhanced the non-selective cation channel activity, increased [Ca(2+)](i) and triggered cell membrane scrambling, effects significantly blunted by furosemide (10 - 100 µM). Oxidative stress by exposure to tert-butylhydroperoxide (0.1 -1 mM) similarly enhanced the non-selective cation channels activity, increased [Ca(2+)](i) and triggered cell membrane scrambling, effects again significantly blunted by furosemide (10 - 100 µM).


CONCLUSIONS
The present study shows for the first time that the loop diuretic furosemide applied at micromolar concentrations (10 - 100 µM) inhibits non-selective cation channel activity in and eryptosis of human erythrocytes.
Department of Physiology Institute I, Eberhard-Karls Universität Tübingen, Tübingen, Germany.
Yuliya V
YV
Kucherenko
Florian
F
Lang
D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000378:metabolism*; D016923:Cell Death; D004232:Diuretics / Q000494:pharmacology*; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D005665:Furosemide / Q000494:pharmacology*; D006801:Humans; D018384:Oxidative Stress / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010718:Phosphatidylserines / Q000378:metabolism
D027682:Cation Transport Proteins; D004232:Diuretics; D010718:Phosphatidylserines; D005665:Furosemide; D000255:Adenosine Triphosphate; D002118:Calcium