title: Effect of chloride channel inhibitors on cytosolic Ca2+ levels and Ca2+-activated K+ (Gardos) channel activity in human red blood cells.
abstract: DIDS, NPPB, tannic acid (TA) and AO1 are widely used inhibitors of Cl(-) channels. Some Cl(-) channel inhibitors (NPPB, DIDS, niflumic acid) were shown to affect phosphatidylserine (PS) scrambling and, thus, the life span of human red blood cells (hRBCs). Since a number of publications suggest Ca(2+) dependence of PS scrambling, we explored whether inhibitors of Cl(-) channels (DIDS, NPPB) or of Ca(2+)-activated Cl(-) channels (DIDS, NPPB, TA, AO1) modified intracellular free Ca(2+) concentration ([Ca(2+)]i) and activity of Ca(2+)-activated K(+) (Gardos) channel in hRBCs. According to Fluo-3 fluorescence in flow cytometry, a short treatment (15 min, +37 °C) with Cl(-) channels inhibitors decreased [Ca(2+)]i in the following order: TA > AO1 > DIDS > NPPB. According to forward scatter, the decrease of [Ca(2+)]i was accompanied by a slight but significant increase in cell volume following DIDS, NPPB and AO1 treatments. TA treatment resulted in cell shrinkage. According to whole-cell patch-clamp experiments, TA activated and NPPB and AO1 inhibited Gardos channels. The Cl(-) channel blockers further modified the alterations of [Ca(2+)]i following ATP depletion (glucose deprivation, iodoacetic acid, 6-inosine), oxidative stress (1 mM t-BHP) and treatment with Ca(2+) ionophore ionomycin (1 μM). The ability of the Cl(-) channel inhibitors to modulate PS scrambling did not correlate with their influence on [Ca(2+)]i as TA and AO1 had a particularly strong decreasing effect on [Ca(2+)]i but at the same time enhanced PS exposure. In conclusion, Cl(-) channel inhibitors affect Gardos channels, influence Ca(2+) homeostasis and induce PS exposure of hRBCs by Ca(2+)-independent mechanisms.
authors: Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, Kharkov, Ukraine.
authors: Yuliya V
authors: YV
authors: Kucherenko
authors: Lisa
authors: L
authors: Wagner-Britz
authors: Ingolf
authors: I
authors: Bernhardt
authors: Florian
authors: F
authors: Lang
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects* / Q000378:metabolism*; D004594:Electrophysiology; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D009579:Nitrobenzoates / Q000494:pharmacology; D013634:Tannins / Q000494:pharmacology
chemical_list: D018118:Chloride Channels; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D009579:Nitrobenzoates; D013634:Tannins; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium