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    • In order to test whether

    2022-02-09

    In order to test whether the enhanced hyperpolarization in the presence of increasing amounts of extracellular Ca2+ could be due to an ionophore effect of NS309, red cell uptake of 45Ca and partition of 45Ca into a water/n-octanol phase +/−100 μM NS309 were determined. However, no indication of ionophore properties was observed. When hRBCs are suspended in a sucrosed substituted medium, nominally Ca2+ free (about 4 μM as contamination), the initial depolarization activates the non-selective voltage dependent cation channel (the NSVDC channel) resulting in a repolarization. When stationarity is reached, after about 7 min, addition of Ca2+ (1 mM) gives rise to a hyperpolarization, due to Ca2+ entry through the NSVDC channel causing activation of the Gardos channel. This hyperpolarization is followed by a redepolarization, caused by the voltage dependent closing of the NSVDC channel mediated Ca2+ influx and the pump extrusion of the Ca2+ (see Fig. 3). If instead of Ca2+, 50 μM NS309 is added as hyperpolarization is again observed, but in this case not transient, which is the case too, if Ca2+ and NS 309 is combined, but the hyperpolarization reaches more negative values. In the presence of 100 μM NS309, the original monophasic osmotic fragility curve, which can be fitted to a complementary error function (Eq. 2), reflecting the underlying normal distribution, changes into a biphasic curve, which can be described by a sum of two weighted complementary error functions (Eq. 3), see Fig. 4, left panel. The fraction of the cells, which changes into the high resistance fraction (hrf) vs. time, can be fitted to a mono exponential function, see Fig. 4 right panel and Table 1. As illustrated in Fig. 4, left panel and Fig. 5, the Prednisolone sale either transit to a high resistance state, or retains relatively unchanged the osmotic resistance, corresponding to the initial (control) state, as illustrated by Fig. 5, right panel, which shows the % NaCl solution at which half of the remaining cells have hemolyzed and the interval in % NaCl in which 68% hemolysis occurs. An alternative approach to the determination of the fraction of osmotic high resistance cells in the presence of NS309, is an isolation of the dense cells by centrifugation of the cell suspension through a heavy phathalate oil, where the fraction of cells, which has experienced a massive loss of KCl and water forms a pellet below the oil. At the normal chloride conductance (20 μS/cm2) the first arrival of heavy cells is observed after a lag time of about 4 min, see Fig. 6. However, if the chloride conductance is lowered to about 10% of the normal value, the lag time before the first arrival is increased to about 30 min. In both cases, the increase of the heavy fraction with time can be fitted to mono exponential functions having nearly identical rate constants.
    Discussion Following the addition of NS309 to a suspension of red cells, a hyperpolarization, estimated by the CCCP method, is observed. This hyperpolarization, which is dose dependent, can be reversed by the action of the Gardos channel blockers nitrendipine [13] and clotrimazole [14], see Fig. 1. Since the hyperpolarization is dependent on the concentration of NS309, as well as the concentration of extracellular calcium, a possible explanation for at least part of this observation could be that NS309 had calcium ionophore properties. However, since NS309 neither caused an increase of Ca2+ influx into red cells nor gave rise to a 45Ca transport from a Ringer solution into an n-octanol phase, this possibility seems to be excluded. This demonstrates that NS309 acts as a Gardos channel activator in intact human red cells, as has previously been shown to be the case for hIK and SK channels in expression systems, where NS309 has been shown to increase the channel sensitivity towards Ca2+ [10]. In the human red cell two major transport systems are activated by Ca2+, the Gardos channel and the PMCA, both of which have Ca2+ sensitivities in the same range. It has previously been shown, that a non-selective cation pathway, the NSVDC channel, which is permeable to Ca2+ too, is activated by depolarization caused by suspension of the cells in a sucrose substituted Ringer. Subsequent addition of Ca2+ activates the Gardos channel, followed by deactivation, due to calcium extrusion by the PMCA [15]. Addition of NS309 to a suspension of cells in a sucrose substituted Ringer containing contaminating Ca2+ only, (about 4 μM), causes likewise a hyperpolarization, but contrary to the calcium induced hyperpolarization in the absence of NS309, this is not transient, see Fig. 3, indicating that the Ca2+ sensitivity of the PMCA is unaffected by NS309. This is further supported by the finding that NS309 did not enhance liberation of inorganic phosphate from inside-out vesicles in the presence of ATP and calmodulin.