Measurements of Intracellular Ca2+ Content and Phosphatidylserine Exposure in Human Red Blood Cells: Methodological Issues.

BACKGROUND/AIMS: The increase of the intracellular Ca2+ content as well as the exposure of phosphatidylserine (PS) on the outer cell membrane surface after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA) has been investigated by a variety of research groups. Carrying out experiments, which we described in several previous publications, we observed some discrepancies when comparing data obtained by different investigators within our research group and also between batches of LPA. In addition, we found differences comparing the results of double and single labelling experiments (for Ca2+ and PS). Furthermore, the results of PS exposure depended on the fluorescent dye used (annexin V-FITC versus annexin V alexa fluor® 647). Therefore, it seems necessary to investigate these methodological approaches in more detail to be able to quantify results and to compare data obtained by different research groups. METHODS: The intracellular Ca2+ content and the PS exposure of RBCs separated from whole blood have been investigated after treatment with LPA (2.5 µM) obtained from three different companies (Sigma-Aldrich, Cayman Chemical Company, and Santa Cruz Biotechnology Inc.). Fluo-4 and x-rhod-1 have been used to detect intracellular Ca2+ content, annexin V alexa fluor® 647 and annexin V-FITC have been used for PS exposure measurements. Both parameters (Ca2+ content, PS exposure) were studied using flow cytometry and fluorescence microscopy. RESULTS: The percentage of RBCs showing increased intracellular Ca2+ content as well as PS exposure changes significantly between different LPA manufacturers as well as on the condition of mixing of LPA with the RBC suspension. Furthermore, the percentage of RBCs showing PS exposure is reduced in double labelling compared to single labelling experiments and depends also on the fluorescent dye used. Finally, data on Ca2+ content are slightly affected whereas PS exposure data are not affected significantly by the measuring method (flow cytometry, fluorescence microscopy). CONCLUSION: The LPA batch used and the mixing procedure of LPA and the RBC suspension has to be taken into consideration when comparing results of intracellular Ca2+ content and PS exposure of RBCs after LPA activation. In addition, one should consider that the results of single and double labelling experiments might be different depending on the fluorescent dyes used.

Phosphatidylserine Exposure in Human Red Blood Cells Depending on Cell Age.

BACKGROUND/AIMS: The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for suicidal erythrocyte death or eryptosis, which may be of importance for cell clearance from blood circulation. PS externalisation is realised by the scramblase activated by an increase of intracellular Ca2+ content. It has been described in literature that RBCs show an increased intracellular Ca2+ content as well as PS exposure when becoming aged up to 120 days (which is their life span). However, these investigations were carried out after incubation of the RBCs for 48 h. The aim of this study was to investigate this effect after short-time incubation using a variety of stimulating substances for Ca2+ uptake and PS exposure. METHODS: We separated RBCs by age in five different fractions by centrifugation using Percoll density gradient. The intracellular Ca2+ content and the PS exposure of RBCs with different age has been investigated after treatment with lysophosphatidic acid (LPA) as well as after activation of protein kinase C (PKC) using phorbol-12 myristate-13 acetate (PMA). For positive control RBCs were treated with 4-bromo-A23187. Measurement techniques included flow cytometry and live cell imaging (fluorescence microscopy). RESULTS: The percentage of RBCs showing increased Ca2+ content as well as the PS exposure did not change significantly in dependence on cell age after short-time incubation in control experiments (without stimulating substances) or using LPA or PMA. However, we confirm findings reported that Ca2+ content and the PS exposure of RBCs increased after 48 h incubation. CONCLUSION: No significant differences of intracellular Ca2+ content and PS exposure can be seen for RBCs of different age in resting state or after stimulation of Ca2+ uptake at short-time incubation.

Novel Insights in the Regulation of Phosphatidylserine Exposure in Human Red Blood Cells.

BACKGROUND/AIMS: In previous publications we were able to demonstrate the exposure of phosphatidylserine (PS) in the outer membrane leaflet after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA), phorbol-12 myristate-13acetate (PMA), or 4-bromo-A23187 (A23187). It has been concluded that three different mechanisms are responsible for the PS exposure in human RBCs: (i) Ca2+-stimulated scramblase activation (and flippase inhibition) by A23187, LPA, and PMA; (ii) PKCα activation by LPA and PMA; and (iii) enhanced lipid flip flop caused by LPA. Further studies aimed to elucidate interconnections between the increased Ca2+ content, scramblase- and PKCα-activation. In addition, the role of the Ca2+-activated K+ channel (Gardos channel) activity in the process of PS exposure needs to be investigated. METHODS: The intracellular Ca2+ content and the PS exposure of RBCs have been investigated after treatment with LPA (2.5 µM), PMA (6 µM), or A23187 (2 µM). Fluo-4 and annexin V-FITC has been used to detect intracellular Ca2+ content and PS exposure, respectively. Both parameters (Ca2+ content, PS exposure) were studied using flow cytometry. Inhibitors of the scramblase, the PKCα, and the Gardos channel have been applied. RESULTS: The percentage of RBCs showing PS exposure after activation with LPA, PMA, or A23187 is significantly reduced after inhibition of the scramblase using the specific inhibitor R5421 as well as after the inhibition of the PKCα using chelerythrine chloride or calphostin C. The inhibitory effect is more pronounced when the scramblase and the PKCα are inhibited simultaneously. Additionally, the inhibition of the Gardos channel using charybdotoxin resulted in a significant reduction of the percentage of RBCs showing PS exposure under all conditions measured. Similar results were obtained when the Gardos channel activity was suppressed by increased extracellular K+ content. CONCLUSION: PS exposure is mediated by the Ca2+-dependent scramblase but also by PKCα activated by LPA and PMA in a Ca2+-dependent and a Ca2+-independent manner. Furthermore, we hypothesize that a hyperpolarisation of RBCs caused by the opening of the Gardos channel is essential for the scramblase activity as well as for a fraction of the LPA-induced Ca2+ entry.

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

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.