Asya Makhro

Calcium in red blood cells-a perilous balance.

Ca2+ is a universal signalling molecule involved in regulating cell cycle and fate, metabolism and structural integrity, motility and volume. Like other cells, red blood cells (RBCs) rely on Ca2+ dependent signalling during differentiation from precursor cells. Intracellular Ca2+ levels in the circulating human RBCs take part not only in controlling biophysical properties such as membrane composition, volume and rheological properties, but also physiological parameters such as metabolic activity, redox state and cell clearance. Extremely low basal permeability of the human RBC membrane to Ca2+ and a powerful Ca2+ pump maintains intracellular free Ca2+ levels between 30 and 60 nM, whereas blood plasma Ca2+ is approximately 1.8 mM. Thus, activation of Ca2+ uptake has an impressive impact on multiple processes in the cells rendering Ca2+ a master regulator in RBCs. Malfunction of Ca2+ transporters in human RBCs leads to excessive accumulation of Ca2+ within the cells. This is associated with a number of pathological states including sickle cell disease, thalassemia, phosphofructokinase deficiency and other forms of hereditary anaemia. Continuous progress in unravelling the molecular nature of Ca2+ transport pathways allows harnessing Ca2+ uptake, avoiding premature RBC clearance and thrombotic complications. This review summarizes our current knowledge of Ca2+ signalling in RBCs emphasizing the importance of this inorganic cation in RBC function and survival.

S-glutathionylation of the Na,K-ATPase catalytic α subunit is a determinant of the enzyme redox-sensitivity

Background: Na,K-ATPase activity is extremely sensitive to changes in the redox state. Results: Binding of glutathione to the regulatory cysteine residues of the catalytic subunit completely inhibits the Na,K-ATPase by blocking the ATP-binding site. Conclusion: S-Glutathionylation of the catalytic subunit is revealed as a mechanism controlling the Na,K-ATPase function. Significance: Regulatory S-glutathionylation adjusts Na,K-ATPase activity to the changes in intracellular redox state and ATP levels. Na,K-ATPase is highly sensitive to changes in the redox state, and yet the mechanisms of its redox sensitivity remain unclear. We have explored the possible involvement of S-glutathionylation of the catalytic α subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the α subunit in duck salt glands, rabbit kidneys, and rat myocardium. Exposure of the Na,K-ATPase to oxidized glutathione (GSSG) resulted in an increase in the number of S-glutathionylated cysteine residues. Increase in S-glutathionylation was associated with dose- and time-dependent suppression of the enzyme function up to its complete inhibition. The enzyme inhibition concurred with S-glutathionylation of the Cys-454, -458, -459, and -244. Upon binding of glutathione to these cysteines, the enzyme was unable to interact with adenine nucleotides. Inhibition of the Na,K-ATPase by GSSG did not occur in the presence of ATP at concentrations above 0.5 mm. Deglutathionylation of the α subunit catalyzed by glutaredoxin or dithiothreitol resulted in restoration of the Na,K-ATPase activity. Oxidation of regulatory cysteines made them inaccessible for glutathionylation but had no profound effect on the enzyme activity. Regulatory S-glutathionylation of the α subunit was induced in rat myocardium in response to hypoxia and was associated with oxidative stress and ATP depletion. S-Glutathionylation was followed by suppression of the Na,K-ATPase activity. The rat α2 isoform was more sensitive to GSSG than the α1 isoform. Our findings imply that regulatory S-glutathionylation of the catalytic subunit plays a key role in the redox-induced regulation of Na,K-ATPase activity.

Red cell investigations: Art and artefacts

Red blood cell research is important for both, the clinical haematology, such as transfusion medicine or anaemia investigations, and the basic research fields like exploring general membrane physiology or rheology. Investigations of red blood cells include a wide spectrum of methodologies ranging from population measurements with a billion cells evaluated simultaneously to single-cell approaches. All methods have a potential for pitfalls, and the comparison of data achieved by different technical approaches requires a consistent set of standards. Here, we give an overview of common mistakes using the most popular methodologies in red blood cell research and how to avoid them. Additionally, we propose a number of standards that we believe will allow for data comparison between the different techniques and different labs. We consider biochemical analysis, flux measurements, flow cytometry, patch-clamp measurements and dynamic fluorescence imaging as well as emerging single-cell techniques, such as the use of optical tweezers and atomic force microscopy.

N-methyl-d-aspartate receptors in human erythroid precursor cells and in circulating red blood cells contribute to the intracellular calcium regulation

The presence of N-methyl-d-aspartate receptor (NMDAR) was previously shown in rat red blood cells (RBCs) and in a UT-7/Epo human myeloid cell line differentiating into erythroid lineage. Here we have characterized the subunit composition of the NMDAR and monitored its function during human erythropoiesis and in circulating RBCs. Expression of the NMDARs subunits was assessed in erythroid progenitors during ex vivo erythropoiesis and in circulating human RBCs using quantitative PCR and flow cytometry. Receptor activity was monitored using a radiolabeled antagonist binding assay, live imaging of Ca(2+) uptake, patch clamp, and monitoring of cell volume changes. The receptor tetramers in erythroid precursor cells are composed of the NR1, NR2A, 2C, 2D, NR3A, and 3B subunits of which the glycine-binding NR3A and 3B and glutamate-binding NR2C and 2D subunits prevailed. Functional receptor is required for survival of erythroid precursors. Circulating RBCs retain a low number of the receptor copies that is higher in young cells compared with mature and senescent RBC populations. In circulating RBCs the receptor activity is controlled by plasma glutamate and glycine. Modulation of the NMDAR activity in RBCs by agonists or antagonists is associated with the alterations in whole cell ion currents. Activation of the receptor results in the transient Ca(2+) accumulation, cell shrinkage, and alteration in the intracellular pH, which is associated with the change in hemoglobin oxygen affinity. Thus functional NMDARs are present in erythroid precursor cells and in circulating RBCs. These receptors contribute to intracellular Ca(2+) homeostasis and modulate oxygen delivery to peripheral tissues.

Washing stored red blood cells in an albumin solution improves their morphologic and hemorheologic properties

Prolonged storage of red blood cells (RBCs) leads to storage lesions, which may impair clinical outcomes after transfusion. A hallmark of storage lesions is progressive echinocytic shape transformation, which can be partially reversed by washing in albumin solutions. Here we have investigated the impact of this shape recovery on biorheologic variables.

‘Gardos Channelopathy’: a variant of hereditary Stomatocytosis with complex molecular regulation

The Gardos channel is a Ca2+ sensitive, K+ selective channel present in several tissues including RBCs, where it is involved in cell volume regulation. Recently, mutations at two different aminoacid residues in KCNN4 have been reported in patients with hereditary xerocytosis. We identified by whole exome sequencing a new family with two members affected by chronic hemolytic anemia carrying mutation R352H in the KCNN4 gene. No additional mutations in genes encoding for RBCs cytoskeletal, membrane or channel proteins were detected. We performed functional studies on patients’ RBCs to evaluate the effects of R352H mutation on the cellular properties and eventually on the clinical phenotype. Gardos channel hyperactivation was demonstrated in circulating erythrocytes and erythroblasts differentiated ex-vivo from peripheral CD34+ cells. Pathological alterations in the function of multiple ion transport systems were observed, suggesting the presence of compensatory effects ultimately preventing cellular dehydration in patient’s RBCs; moreover, flow cytometry and confocal fluorescence live-cell imaging showed Ca2+ overload in the RBCs of both patients and hypersensitivity of Ca2+ uptake by RBCs to swelling. Altogether these findings suggest that the ‘Gardos channelopathy’ is a complex pathology, to some extent different from the common hereditary xerocytosis.

Red Cell Properties after Different Modes of Blood Transportation

Transportation of blood samples is unavoidable for assessment of specific parameters in blood of patients with rare anemias, blood doping testing, or for research purposes. Despite the awareness that shipment may substantially alter multiple parameters, no study of that extent has been performed to assess these changes and optimize shipment conditions to reduce transportation-related artifacts. Here we investigate the changes in multiple parameters in blood of healthy donors over 72 h of simulated shipment conditions. Three different anticoagulants (K3EDTA, Sodium Heparin, and citrate-based CPDA) for two temperatures (4°C and room temperature) were tested to define the optimal transportation conditions. Parameters measured cover common cytology and biochemistry parameters (complete blood count, hematocrit, morphological examination), red blood cell (RBC) volume, ion content and density, membrane properties and stability (hemolysis, osmotic fragility, membrane heat stability, patch-clamp investigations, and formation of micro vesicles), Ca2+ handling, RBC metabolism, activity of numerous enzymes, and O2 transport capacity. Our findings indicate that individual sets of parameters may require different shipment settings (anticoagulants, temperature). Most of the parameters except for ion (Na+, K+, Ca2+) handling and, possibly, reticulocytes counts, tend to favor transportation at 4°C. Whereas plasma and intraerythrocytic Ca2+ cannot be accurately measured in the presence of chelators such as citrate and EDTA, the majority of Ca2+-dependent parameters are stabilized in CPDA samples. Even in blood samples from healthy donors transported using an optimized shipment protocol, the majority of parameters were stable within 24 h, a condition that may not hold for the samples of patients with rare anemias. This implies for as short as possible shipping using fast courier services to the closest expert laboratory at reach. Mobile laboratories or the travel of the patients to the specialized laboratories may be the only option for some groups of patients with highly unstable RBCs.

Recovery of donor hearts after circulatory death with normothermic extracorporeal machine perfusion.

OBJECTIVES A severe donor organ shortage leads to the death of a substantial number of patients who are listed for transplantation. The use of hearts from donors after circulatory death could significantly expand the donor organ pool, but due to concerns about their viability, these are currently not used for transplantation. We propose short-term ex vivo normothermic machine perfusion (MP) to improve the viability of these ischaemic donor hearts. METHODS Hearts from male Lewis rats were subjected to 25 min of global in situ warm ischaemia (WI) (37°C), explanted, reconditioned for 60 min with normothermic (37°C) MP with diluted autologous blood and then stored for 4 h at 0-4°C in Custodiol cold preservation solution. Fresh and ischaemic hearts stored for 4 h in Custodiol were used as controls. Graft function was assessed in a blood-perfused Langendorff circuit. RESULTS During reconditioning, both the electrical activity and contractility of the ischaemic hearts recovered rapidly. Throughout the Langendorff reperfusion, the reconditioned ischaemic hearts had a higher average heart rate and better contractility compared with untreated ischaemic controls. Moreover, the reconditioned ischaemic hearts had higher tissue adenosine triphosphate levels and a trend towards improved tissue redox state. Perfusate levels of troponin T, creatine kinase and lactate dehydrogenase were not significantly lower than those of untreated ischaemic controls. The micro- and macroscopic appearance of the reconditioned ischaemic hearts were improved compared with ischaemic controls, but in both groups myocardial damage and oedema were evident. CONCLUSIONS Our results indicate that functional recovery from global WI is possible during short-term ex vivo reperfusion, allowing subsequent cold storage without compromising organ viability. We expect that once refined and validated, this approach may enable safe transplantation of hearts obtained from donation after circulatory death.

Cardiac N-methyl D-aspartate receptors as a pharmacological target

Abstract: This study focuses on characterization of the cardiac N-methyl D-aspartate receptors (NMDARs) as a target for endogenous and synthetic agonists and antagonists. Using isolated perfused rat hearts, we have shown that intracoronary administration of the NMDAR agonists and antagonists has a pronounced effect on autonomous heart function. Perfusion of rat hearts with autologous blood supplemented with NMDAR agonists was associated with induction of tachycardia, sinus arrhythmia, and ischemia occurring within physiological plasma concentration range for glutamate and glycine. Intracoronary administration of the NMDAR antagonists exerted an antiarrhythmic effect and resulted in bradycardia and improvement of capillary perfusion. Action of antagonists eliprodil, Ro25-6981, memantine, ketamine, and MK-801 on autonomous heart function diverged strikingly from that of L-type Ca2+ channel blockers. Cardiac NMDAR subunit composition differed from that of neuronal receptors and was age specific and chamber specific. Transcripts of the GluN3A and GluN2D were found in all heart chambers, whereas expression of GluN1 and GluN2A and 2C was restricted to the atria. Expression of the GluN2B protein in ventricles increased markedly with age of the animals. The obtained data reveal that NMDARs are expressed in rat heart contributing to the autonomic heart rate regulation and the function of the cardiac conduction system.

Comparing the impact of an acute exercise bout on plasma amino acid composition, intraerythrocytic Ca2+ handling, and red cell function in athletes and untrained subjects

The N-methyl d-aspartate receptors (NMDARs) mediating Ca(2+) uptake upon stimulation with glutamate and glycine were recently discovered in red blood cells (RBC) of healthy humans. Activation of these receptors with agonists triggered transient Ca(2+)-dependent decrease in hemoglobin oxygen affinity in RBC suspension. The aim of this study was to assess the potential physiological relevance of this phenomenon. Two groups formed by either healthy untrained volunteers or endurance athletes were subjected to a stepwise incremental cycling test to exhaustion. Plasma glutamate levels, activity of the NMDARs, and hemoglobin O2 affinity were measured in blood samples obtained before and after the exercise in both groups. Increase in plasma glutamate levels following exercise was observed in both groups. Transient Ca(2+) accumulation in response to the NMDAR stimulation with NMDA and glycine was followed by facilitated Ca(2+) extrusion from the RBC and compensatory decrease in cytosolic Ca(2+) levels. Short-term activation of the receptors triggered a transient decrease in O2 affinity of hemoglobin in both groups. These exercise-induced responses were more pronounced in athletes compared to the untrained subjects. Athletes were initially presented with lower basal intracellular Ca(2+) levels and hemoglobin oxygen affinity compared to non-trained controls. High basal plasma glutamate levels were associated with induction of hemolysis and formation of echinocytes upon stimulation with the receptor agonists. These findings suggest that glutamate release occurring during exhaustive exercise bouts may acutely facilitate O2 liberation from hemoglobin and improve oxygen delivery to the exercising muscle.