Protein Kinase Cα and P-Type Ca2+ Channel CaV2.1 in Red Blood Cell Calcium Signalling

Background/Aims: Protein kinase Cα (PKCα) is activated by an increase in cytosolic Ca2+ in red blood cells (RBCs). Previous work has suggested that PKCα directly stimulates the CaV2.1 channel, whereas other studies revealed that CaV2.1 is insensitive to activation by PKC. The aim of this study was to resolve this discrepancy. Methods: We performed experiments based on a single cell read-out of the intracellular Ca2+ concentration in terms of Fluo-4 fluorescence intensity and phosphatidylserine exposure to the external membrane leaflet. Measurement modalities included flow cytometry and live cell imaging. Results: Treatment of RBCs with phorbol 12-myristate 13-acetate (PMA) led to two distinct populations of cells with an increase in intracellular Ca2+: a weak-responding and a strong-responding population. The EC50 of PMA for the number of cells with Ca2+ elevation was 2.7±1.2 µM; for phosphatidylserine exposure to the external membrane surface, it was 2.8±0.5 µM; and for RBC haemolysis, it was 2.9±0.5 µM. Using pharmacological manipulation with the CaV2.1 inhibitor ω-agatoxin TK and the broad protein kinase C inhibitor Gö6983, we are able to show that there are two independent PMA-activated Ca2+ entry processes: the first is independent of CaV2.1 and directly PKCα-activated, while the second is associated with a likely indirect activation of CaV2.1. Further studies using lysophosphatidic acid (LPA) as a stimulation agent have provided additional evidence that PKCα and CaV2.1 are not directly interconnected in a signalling chain. Conclusion: Although we provide evidence for a lack of interaction between PKCα and CaV2.1 in RBCs, further studies are required to decipher the signalling relationship between LPA, PKCα and CaV2.1

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

Handling and protocols of LPA-induced Ca²⁺ influx.

<p>Fluorescence signals of single RBCs treated with 2.5 µM LPA (A) and 10 µM LPA (B). (C) Comparison of average Ca²⁺ signals induced by different concentrations of LPA [same data as in (A) and (B)]. (D) Average of the Ca²⁺ signals after their synchronization to the onset of the response.</p

Different parameters of single-cell response.

<p>(A) Definition of the different parameters related to the cellular response. (B)–(F) Statistical analysis of the parameters defined in (A); the colour-code for all diagrams is given in the right part of the panel. To avoid interindividual differences (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067697#pone.0067697.s003" target="_blank">Figure S3</a>), all measurements were performed with freshly prepared samples from a single healthy donor. (B) Maximal intensity of the cellular response within the period of measurement (max response). (C) Time interval between LPA application and the onset of the reaction (reaction time). (D) Value of the main plateau or the major peak of the Ca²⁺ response (amplitude). (E) Hill slope (steepness) of the Ca²⁺ increase (S_H). (F) The time point when the ratio (F/F_o) reached the value of half the amplitude (X_half). The values in (D)–(F) are extracted from a Hill-equation fitting. Parameters in (B) and (D) are derived from the total number of cells, while (C), (E) and (F) refer exclusively to responding cells and therefore do not give a number for the control condition. The numbers below the boxes give the cell numbers taken from three blood samples.</p

Ca²⁺ response of old RBCs to LPA stimulation.

<p>(A) Analysis of PKH26 fluorescence of 20,000 RBCs by flow cytometry before (upper left) and 1 hour after staining with PKH26 (upper right). After the staining procedure, the cells were reinjected into the same mice and analysed 7 days (lower left) and 43 days (lower right) later. The percentage of PKH26 labelled cells (PKH(+), region R2) is indicated. (B) Control and LPA stimulation experiments were performed on PKH26-positive (+) and PKH26-negative (–) RBCs. The maximal response under the different conditions is given. We discriminated between PKH(+) cells identified directly under the microscope (VIS, low in number) and RBCs sorted by FACS. The numbers below the boxes give the cell numbers taken from three mice. (C) Amplitude histogram of the RBC treated under the conditions mentioned in (B). (D) Representative intensity traces of PKH(+) cells stimulated with 5 µM LPA revealing a high heterogeneity also in old RBCs. (E) AChE activity in control and old RBCs with and without stimulation with 5 µM LPA for 15 min. The measurements comprise of a colorimetric assay based on 2×10⁶ cells per measurement and the data is the average of three mice.</p