br Conflict of interest br

Conflict of interest
Introduction Since its discovery in 1958 [1]the Ca2+-dependent K+ efflux in human red blood YAP-TEAD Inhibitor 1 (RBC) (further referred to as the Gárdos effect) has been extensively studied (see 2, 3for review) and has been helpful in understanding the role of the Ca2+-activated K+ channels (K(Ca)), especially in excitable cells. The role of K(Ca) in non-excitable cells is much less understood. A feature of the Gárdos effect is its inducibility by various agents and/or treatments. In addition to NaF and ATP depletion 1, 4, propranolol 5, 6, 7, NaVO3[8], lead [10]or redox modification 9, 11and, probably, also PGE2 [12]have been used as inducers. Despite the different molecular character of the individual inducers, their action involves the translocation of extracellular Ca2+ across the RBC membrane which, finally, leads to the activation of K(Ca) and massive K+ loss. Therefore, it is feasible that the common denominator of all inducers (with lead as a possible exception) is their ability to perturb the RBC Ca2+ homeostasis which leads to (transient or permanent) accumulation of Ca2+ in the RBC cytoplasm. As the RBC Ca2+ homeostasis presumably comprises only two components, the low-capacity Ca2+ influx pathway and the high-capacity Ca2+ efflux pathway represented by the Ca2+ pump, the inducers of the Gárdos effect could serve as tools to characterize the Ca2+ influx pathway(s) and, thereby, to unravel the nature of the Gárdos effect. Extensive characteristics of the Ca2+ influx induced by ATP depletion [4]and by NaVO38, 13, 14, 15, 16are available and suggest that the Ca2+ influx is mediated by a carrier. The question of whether NaF and NaVO3 activate the same or different Ca2+ influx pathway(s) was addressed in this work.
Materials and methods Blood from healthy volunteers of both sexes was withdrawn by venipuncture into EDTA-containing anticoagulant (5 mmol/l), and was used within 3 days, stored at 0–4°C. RBC were isolated after centrifugation of the blood (10 min at 600×g) and aspiration of the supernatant with the buffy coat, and three-fold washing with and, finally, suspending into a medium containing (in mmol/l): 20 Tris-HCl (pH 7.3), 130 NaCl, 5 KCl, 10 glucose (further referred to as the suspension medium), to a hematocrit of 30%, and immediately used for experiments. Pig blood was obtained from the local slaughterhouse and was used for experiments on the same day. The influx of Ca2+ was measured with the radionuclide 45Ca, after repetitive washing for removal of extracellular radioactivity, as described previously [8]. Aliquots of 30% suspension were preincubated with 1 mmol/l NaVO3 for 15 min or for 3 min with 10 mmol/l NaF at 25°C, and 45CaCl2 (2.5 mmol/l final) was added and incubated for 60 min at the same temperature unless indicated otherwise. The incubation was stopped by addition of the same volume of stopping medium containing (in mmol/l): 20 Tris-HCl (pH 7.3), 75 KCl, 60 NaCl, 10 glucose, and 1 EDTA, and by rapid centrifugation of the sample in a microcentrifuge. The supernatant was sucked off and the pellet was washed with the stopping medium three more times. Finally, the pellet was precipitated with 10% perchloric acid containing 20 mmol/l LaCl3, the precipitate was centrifuged and the pellet was used for liquid scintillation counting. Control cells without NaVO3 were treated in parallel. When inhibitors were tested, the same volume of solvent (DMSO, methanol, max. 0.5% v/v) was added to the control samples. All samples were done in duplicate and the average value of parallel samples (±standard error) is given in the figures. The standard error is indicated by bars when it exceeded the dimension of the symbol. The results presented in the figures represent typical experiments obtained independently from each other. The number of experiments performed varied from three to nine. The values were corrected for the radioactivity obtained in control cells without inducer.