Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology · 2012
Inhibitory effect of furosemide on non-selective voltage-independent cation channels in human erythrocytes.
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Kucherenko Yuliya V, Lang Florian
Аннотация
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.
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.