Intracellular Ca2+ Concentration and Phosphatidylserine Exposure in Healthy Human Erythrocytes in Dependence on in vivo Cell Age

Abstract

After about 120 days of circulation in the blood stream, erythrocytes are cleared by macrophages in the spleen and the liver. The "eat me" signal of this event is thought to be the translocation of phosphatidylserine from the inner to the outer membrane leaflet due to activation of the scramblase, while the flippase is inactivated. Both processes are triggered by an increased intracellular Ca²⁺ concentration. Although this is not the only mechanism involved in erythrocyte clearance, in this minireview, we focus on the following questions: Is the intracellular-free Ca²⁺ concentration and hence phosphatidylserine exposure dependent on the erythrocyte age, i.e. is the Ca²⁺ concentration, progressively raising during the erythrocyte aging in vivo? Can putative differences in intracellular Ca²⁺ and exposure of phosphatidylserine to the outer membrane leaflet be measured in age separated cell populations? Literature research revealed less than dozen of such publications with vastly contradicting results for the Ca²⁺ concentrations but consistency for a lack of change for the phosphatidylserine exposure. Additionally, we performed reanalysis of published data resulting in an ostensive illustration of the situation described above. Relating these results to erythrocyte physiology and biochemistry, we can conclude that the variation of the intracellular free Ca²⁺ concentration is limited with 10 μM as the upper level of the concentration. Furthermore, we propose the hypothesis that variations in measured Ca²⁺ concentrations may to a large extent depend on the experimental conditions applied but reflect a putatively changed Ca²⁺ susceptibility of erythrocytes in dependence of in vivo cell age.

Keywords: Ca2+ content; aging; flow cytometry; lysophosphatidic acid; phosphatidylserine exposure; red blood cells.

Figure 1

Reanalysis of data initially presented in Wesseling et al. (2016b). In the original publication, all fractions were only compared in pairs, while here we followed the approach to plot (and analyze) the measured effect in dependence of the cell age. Both panels present the situation under control conditions (without pharmalogical stimulation). (A) The percentage of erythrocytes showing increased Ca²⁺ content as well as phosphatidylserine exposure depicts a linear behavior in dependence of cell age with a very good regression, R² given in the figure. However, the slope of these linear regressions failed to be significantly different from zero, i.e., failed to show a significant change. (B) The percentage of erythrocytes showing decreased Ca²⁺ content, while phosphatidylserine exposure depicts a linear increase in dependence of cell age, again, with a very good regression, R² given in the figure. The slope of both linear regressions also failed to be significantly different from zero, i.e., failed to show a significant change. Furthermore, comparing the particular fractions between the two measurements (A,B) applying an unpaired t-test with Welch’s correction (unequal SD values), none of the fractions showed a significant difference (p > 0.05). Both measurements (A,B) were performed in the same laboratory. Blood samples were given from healthy sportsmen (Department of Sports Medicine) with an age between 18 and 36 years. Each data point consists of three donors (not identical between A and B) and for each donor and condition, 90,000 cells were analyzed by flow cytometry. The only obvious difference is the composition of the solutions. [(A) in mM]: 145 NaCl, 7.5 KCl, 2 CaCl₂, 10 glucose, 10 HEPES, pH 7.4 (Tyrode solution); [(B) in mM]: 125 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 5 glucose, 32 HEPES, pH 7.4 (Ringer solution). (A) A reprint from Bernhardt et al. (2019).

Figure 2

Reanalysis of data initially presented in Wesseling et al. (2016b). In the original publication, all fractions were only compared in pairs, while here we followed the approach to plot (and analyze) the measured effect in dependence of the cell age. (A) Presents the situation after 15 min of stimulation with lysophosphatidic acid (LPA). While the Ca²⁺ concentration seems to relate inversely proportional to erythrocyte age (slope is significant different from zero, p < 0.05 is marked with *), phosphatidylserine positive cells show a rather quadratic dependence on cell age. (B) Presents exclusively the phosphatidylserine exposure under more direct stimulations (15 min), namely the direct increase in intracellular Ca²⁺ in all cells by application of the Ca²⁺ ionophore A23187 in 2 mM Ca²⁺ containing solutions (dark red circles) and by a direct Ca²⁺-independent activation of protein kinase Cα (PKCα, orange triangles) by phorbol-12 myristate-13 acetate (PMA, an unspecific activator of conventional and novel PKCs but PKCα is the sole PKC of these 2 groups found in erythrocytes). Although different in amplitude, both stimulations result in a quadratic dependence on cell age. Blood samples were given from healthy sportsmen (Department of Sports Medicine) with an age between 18 and 36 years. Each data point consists of three donors and for each donor and condition 90,000 cells were analyzed by flow cytometry. For a complete dataset on the different stimulation modes, see Wesseling et al. (2016b). (A) A reprint from Bernhardt et al. (2019).