Investigation of properties of the Ca2+ influx and of the Ca2+-activated K+ efflux (Gardos effect) in vanadate-treated and ATP-Depleted human red blood cells

In this study the properties of the Ca-45(2+) influx in human red blood cells (RBC) induced by NaVO3 or ATP-depletion were compared. Both NaNVO3-induced and ATP-depletion-induced Ca-45(2+) influxes were in the range 10(-6)-10(-5) mol Ca2+. l(cells)(-1) . h(-1). The saturatability of ATP-depletion-induced Ca-45(2+) with Ca2+ was much less pronounced than that of NaVO3-induced Ca-45(2+) influx. The NaVO3-induced Ca2+ influx was sensitive to nifedipine (IC50 = 50 mumol/l) and Cu2+ (IC50 = 9 mumol/l) but these inhibitors had only a marginal effect when ATP-depletion was used as the Ca2+ influx inducer. On the other hand, polymyxin B (PXB) (1-5 mg/ml) strongly stimulated the ATP-depletion-induced Ca-45(2+) influx whereas its effect on the NaVO3-induced Ca2+ influx was biphasic, with about 10% stimulation at lower PXB concentrations and an inhibition of 40% at higher concentrations. SDS-PAGE revealed that both NaVO3 and PXB induced changes in the protein phosphorylation pattern in the presence of Ca2+. NaVO3 stimulated the phosphorylation of several proteins and this effect was counteracted by PXB. The comparison of the kinetics and temperature dependencies of the Gardos effect induced by NaVO3 and the ATP-depletion showed marked differences. The ability of NaVO3 to induce the Gardos effect dramatically increased in ATP-depleted cells. These findings indicate that the Ca-45(2+) influxes preceding the activation of the Ca2+-activated K+ efflux (Gardos effect) stimulated by NaVO3 and by ATP-depletion, are mediated by different transport pathways. In addition, obtained re-sults demonstrate that ATP-depletion and NaVO3-treatment exert additive action in triggering the Gardos effect.