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  • This study compared PMS ascorbate

    2021-09-08

    This study compared PMS+ascorbate and t-BHP in terms of their effects on the volume-dependent parameters of normal human RBCs. Incubation with both oxidative systems resulted in the RBC density and osmotic resistance distribution shifts. Clotrimazole or Са2+ added into the medium, as well as high extracellular K+ levels, modulated the redistribution effects, suggesting that the effects observed are mediated to some extent by Gardos channel activation.
    Materials and methods
    Results
    Discussion The mechanisms whereby PMS+ascorbate and t-BHP induce oxidative stress in RBCs are different. The primary response of RBCs to PMS+ascorbate is the generation of superoxide radicals [32], [33], whereas the first to form during incubation of RBCs with t-BHP are alkoxyl and peroxyl radicals of hydroperoxides [34]. Both oxidative systems were shown to produce similar effects on the cells: reduced glutathione is rapidly depleted, xpo 1 undergoes oxidation, and passive K+ permeability of cell membranes increases [22], [24], [34], [35], [36], [37]. This study examined the effects of two oxidants – phenazine methosulfate (50–1500 µM)+10 mM ascorbate and t-BHP (1–3 mM) – on the volume-related characteristics of normal human red blood cells: their density (Fig. 1) and osmotic resistance (Fig. 2, Fig. 3, Fig. 4) distributions. A 20–30-min incubation at 37 °C with the oxidants studied produced opposite effects on the cell volume. If the suspending medium contained Са2+, incubation of RBSs with PMS+ascorbate led to a considerable increase in their density (shrinking), whereas a reduction in their density (swelling) was observed after incubation with t-BHP. If the suspending medium contained no Са2+, PMS+ascorbate failed to induce RBC shrinking (Fig. 1, Fig. 2), whereas the t-BHP-induced swelling was much more pronounced (Fig. 1, Fig. 2). As shown in our earlier study, PMS+ascorbate causes RBCs to shrink in a Са2+-containing medium by activating the Gardos channel [22]. Suspecting that the reduction in the extent of t-BHP-induced swelling in a Са2+-containing medium is also due to the Gardos effect, we studied whether the PMS+ascorbate and t-BHP-induced osmotic resistance distribution shift is affected by (i) clotrimazole and (ii) high extracellular K+. The data shown in Fig. 3, Fig. 4 provide evidence suggesting the involvement of Gardos channel activation in the changes of the volume-related characteristics of RBCs induced by the oxidants used. In our experiments with oxidation treatment, no vesiculation was observed (data not shown), suggesting that the cell surface area remained constant. Therefore, from the shift in Mc determined experimentally, it was possible to quantitatively estimate the change in the cell volume (see Аppendix). The maximum shrinking (minimum Мс of about 80 mOsm/kg) was observed at the PMS concentration of 100 µM and amounted to more than 20% of the volume of an intact RBC. With a further increase in the PMS concentration to 1.5 mM, the extent of shrinking decreased (Fig. 3A, curve 1) and partial hemolysis was observed. At a concentration of 10 μM, the specific inhibitor of Gardos channels clotrimazole largely abolished the shrinking effect PMS+ascorbate in a Са2+-containing medium. A statistically significant difference of Мс from its control value was obtained only for 100 μM PMS (Fig. 3A, curve 2). In the concentration range from to 3 mM, incubation with t-BHP in a Са2+-containing medium for 30 min resulted in a dose-dependent RBC swelling. The greatest effect (volume increase of about 8%) was observed at a concentration of 3 mM. If the medium was either free of Са2+, contained clotrimazole, or contained K+ at a high concentration, the greatest effect was as large as 14–15% (Fig. 3, Fig. 4). In conclusion, in our experiments the two oxidants studied produced opposite effects on the RBC density and osmotic resistance distributions. Specifically, PMS+ascorbate caused RBC shrinking, whereas incubation with t-BHP resulted in RBC swelling. However, in both cases, there was activation of Са2+-dependent K+ channels (Gardos-effect). It is Gardos channel activation that mediates RBC dehydration in response to PMS+ascorbate treatment [22] and reduces the extent of t-BHP-induced swelling. The differences observed are obviously due to different mechanisms of action of PMS+ascorbate and t-BHP on xpo 1 ion channels of the RBC membrane [2], [34]. In Ca2+-containing media, PMS+ascorbate raises intracellular Са2+ concentration in RBCs (probably by inhibiting Ca2+-ATPase [29]), modifies and activates Gardos channels, causing the cells to shrink [21], [22], [33]. At the same time, there is considerable evidence that incubation of RBCs with t-BHP leads to activation of nonselective cation channels [38], [39], causing not only intracellular Ca2+, but also Na+ to rise. As a result, swelling is observed, which is partially offset by the Gardos effect. These findings are consistent with the suggestion that Gardos-channel activation is some general property of the cell response to oxidative treatment, which provides for RBC volume regulation in vivo [12], [22], [40].