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

Abstract

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.

Does Erythropoietin Regulate TRPC Channels in Red Blood Cells?

“…The results are shown in Figure 2. The heterogeneity of the data is not caused by inter-individual differences but by a heterogeneous response of the cells similar as reported before for the stimulation with Lysophospatidic acid [21]. In murine RBCs, Epo as well as PGE 2 induced a clear increase in the intracellular Ca…”

“…Wild-type mice were C57Bl6/N, the same as the background of the dKO mice. For enriching reticulocytes (RNA) in the blood samples, mice were treated for an induction of a reticulocytosis as described before [21]. These experiments were conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.…”

“…Then RBCs were plated on coverslips and 20 min were allowed for sedimentation and deesterification of the Fluo-4. Data analysis was performed as previously described [21], data extraction (defining the regions of interest) was done in ImageJ (Wayne Rasband, National Institute of Health, USA), further data analysis and determination of the maximal cellular response was processed in Igor Pro 6.2 (WaveMetrics, Portland, Oregon, USA) with custom made macros and statistical analysis was performed in Prism 6 (GraphPad Software, La Jolla, California, USA). The physiological solution, in which RBCs were kept during the staining procedure and the experiments was Tyrode solution containing in mM: 135 NaCl, 5.4 KCl, 10 Glucose, 10 HEPES, 1.8 CaCl 2 , 1 MgCl 2 , pH 7.35 adjusted with NaOH, 300 mOsmol/ kg H 2 O.…”

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

“…Comparison of RBC activation using LPA from different companies In our experiments described in previous papers [3,15,16,21] we realised that RBC stimulation with LPA from different batches obtained from Sigma-Aldrich led to significant different results of the intracellular Ca 2+ content as well as PS exposure. To investigate this phenomenon in more detail we performed experiments with LPA from 4 different batches, 2 from Sigma-Aldrich, one from Cayman Chemical Company, and one from Santa Cruz Biotechnology Inc. All 4 LPA probes were freshly ordered, re-frozen only once and were applied to the same blood on the same day and using the same HPS.…”

Involvement of Calcium, Reactive Oxygen Species, and ATP in Hexavalent Chromium-Induced Damage in Red Blood Cells

“…Thus far, no direct evidence has been reported that increased intracellular Ca 2+ levels are vital to eryptosis. However, a study has reported the heterogeneity between the elevation of intracellular Ca 2+ concentrations and increased eryptosis [41] where RBCs eryptosis occurs without the involvement of Ca 2+ [20]. In this study, we observed that Ca 2+ influx occurred within 6 h after Cr 6+ exposure.…”