Calcium imaging of individual erythrocytes: Problems and approaches

Kaestner, Lars; Tabellion, Wiebke; Weiss, Erwin; Bernhardt, Ingolf; Lipp, Peter

doi:10.1016/j.ceca.2005.09.004

Cited by 69 publications

(41 citation statements)

References 16 publications

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“…The first two methods have been well established for several decades [34]–[37], whereas live-cell imaging of RBCs for Ca 2+ was introduced just a couple of years ago [25], [26] and has been used scarcely ever since [38]. To compare the methods, human RBCs from a single donor were stimulated with 5 µM LPA for 15 min, and the signals obtained were normalized to controls treated with Tyrode (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

“…Intercellular RBC aggregation has been shown to be evoked by exposure to lysophosphatidic acid (LPA) [19]–[21], which is released from activated platelets [22], fibroblasts, adipocytes and cancer cells [23]. LPA stimulation of RBCs is linked to a substantial increase of cytosolic Ca 2+ [19], [24], which is readily detectable using fluorescent Ca 2+ indicator dyes [25], [26]. Initially, LPA was thought to directly activate a non-selective cation channel in the RBC membrane [19], [26], but recent findings suggest the involvement of G-protein-coupled receptor-mediated processes [27] that are believed to be involved in numerous pathologies, such as sickle cell disease [28], hemolytic uremic syndrome [29], iron deficiency [30] and β-thalassemia [31].…”

Section: Introductionmentioning

confidence: 99%

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Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation

et al. 2013

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Red blood cells (RBCs) are among the most intensively studied cells in natural history, elucidating numerous principles and ground-breaking knowledge in cell biology. Morphologically, RBCs are largely homogeneous, and most of the functional studies have been performed on large populations of cells, masking putative cellular variations. We studied human and mouse RBCs by live-cell video imaging, which allowed single cells to be followed over time. In particular we analysed functional responses to hormonal stimulation with lysophosphatidic acid (LPA), a signalling molecule occurring in blood plasma, with the Ca2+ sensor Fluo-4. Additionally, we developed an approach for analysing the Ca2+ responses of RBCs that allowed the quantitative characterization of single-cell signals. In RBCs, the LPA-induced Ca2+ influx showed substantial diversity in both kinetics and amplitude. Also the age-classification was determined for each particular RBC and consecutively analysed. While reticulocytes lack a Ca2+ response to LPA stimulation, old RBCs approaching clearance generated robust LPA-induced signals, which still displayed broad heterogeneity. Observing phospatidylserine exposure as an effector mechanism of intracellular Ca2+ revealed an even increased heterogeneity of RBC responses. The functional diversity of RBCs needs to be taken into account in future studies, which will increasingly require single-cell analysis approaches. The identified heterogeneity in RBC responses is important for the basic understanding of RBC signalling and their contribution to numerous diseases, especially with respect to Ca2+ influx and the associated pro-thrombotic activity.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation

et al. 2013

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“…While there are broad characterisations of A23187- [22,23,25,37] and LPA- [11,20] induced Ca 2+ influx into RBCs available in the literature, reports on the PMA stimulation of RBCs are rather sparse [22,27,38]. Therefore, a set of experiments was performed to investigate the effect of various doses of PMA on Ca 2+ content, PS exposure and RBC haemolysis, as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…RBC stimulation with oleoyl-L-α-lysophosphatidic acid (LPA) is an example of a physiological stimulation leading to an increase in intracellular Ca 2+ [19,20]. The characterisation of the Ca 2+ entry and the consequent cell behaviour have been described in numerous reports [21,22,23,24,25].…”

Section: Introductionmentioning

confidence: 99%

Protein Kinase Ca and P-Type Ca²⁺Channel Ca_V2.1 in Red Blood Cell Calcium Signalling

Wagner-Britz

Wang

Kaestner

et al. 2013

Cell Physiol Biochem

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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 Ca_V2.1 channel, whereas other studies revealed that Ca_V2.1 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 Ca_V2.1 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 Ca_V2.1 and directly PKCα-activated, while the second is associated with a likely indirect activation of Ca_V2.1. Further studies using lysophosphatidic acid (LPA) as a stimulation agent have provided additional evidence that PKCα and Ca_V2.1 are not directly interconnected in a signalling chain. Conclusion: Although we provide evidence for a lack of interaction between PKCα and Ca_V2.1 in RBCs, further studies are required to decipher the signalling relationship between LPA, PKCα and Ca_V2.1.

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“…As depicted in Figure 1A, at rest, RBCs from ESRD patients show higher Ca 2+ concentration compared with healthy donors, while EPO treatment let to a slightly decreased free internal Ca 2+ concentration, indicating an inhibition of constitutively active channels in resting RBCs. Although the histograms (Figure 1Ab) give an impression of the distribution, the method lacks quantitative information concerning the Ca 2+ concentration (Kaestner et al, 2006). However, when compared to control conditions, the width of the distribution of Ca 2+ content is wider in ESRD patients or EPO-treated ESRD patients, leading to the conclusion that the cellular heterogeneity is greater in patients than in healthy subjects.…”

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Calcium homeostasis in red blood cells of dialysis patients in dependence of erythropoietin treatment

Wang

Bentum²,

Sester

et al. 2014

Front. Physiol.

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Calcium imaging of individual erythrocytes: Problems and approaches

Cited by 69 publications

References 16 publications

Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation

Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation

Protein Kinase Ca and P-Type Ca²⁺Channel Ca_V2.1 in Red Blood Cell Calcium Signalling

Calcium homeostasis in red blood cells of dialysis patients in dependence of erythropoietin treatment

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Calcium imaging of individual erythrocytes: Problems and approaches

Cited by 69 publications

References 16 publications

Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation

Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation

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

Calcium homeostasis in red blood cells of dialysis patients in dependence of erythropoietin treatment

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Protein Kinase Ca and P-Type Ca²⁺Channel Ca_V2.1 in Red Blood Cell Calcium Signalling