A contribution of calmodulin to cellular deformability of calcium-loaded human erythrocytes

The effect of intracellular calcium on the deformability of human erythrocytes was studied with a rheoscope, especially in relation to the dynamic structure of membrane cytoskeleton. The appropriate calcium-loading and calcium-depletion were performed to intact erythrocytes with A23187 in potassium buffer. The total calcium content was varied in the range of 0.25 to 3 times as much as normal content, without complete ATP depletion and shape change (the reduction of mean cell volume and the condensation of hemoglobin due to dehydration were avoided). Increasing the intracellular calcium content by about 1.5 times of normal, the deformability was distinctly decreased, while calcium depletion did not affect the deformability. Reduced deformability of the calcium-loaded erythrocytes was restored by the treatment with calmodulin inhibitors, W-7 or trifluoperazine. However, such an effect by calmodulin inhibitors was not detected on normal or calcium-depleted erythrocytes. In conclusion, the interaction between calcium-calmodulin complex and cytoskeletal proteins may affect the membrane stiffness which is regulated through the change of the cytoskeletal structure, and contributes to the deformability of erythrocytes.     

Changes of erythrocyte deformability induced by calcium accumulation and calmodulin inhibitors

The deformability of human erythrocytes was investigated with a rheoscope to study the role of intracellular calcium in the dynamic cytoskeletal structure. Calcium was loaded to or depleted from erythrocytes with a calcium ionophore (A 23187) in a Na- or a K-HEPES buffer. (1) After calcium loading in the Na-HEPES buffer, the cell volume of erythrocytes was greatly reduced due to dehydration. On the contrary, upon calcium-loading or -depletion in the K-HEPES buffer, the intracellular calcium content could be varied in the range of 1/4 to 3 times as much as that of control cells without the reduction of mean cell volume. Further incubation without A 23187 and calcium in the K-HEPES buffer enabled the calcium-loaded erythrocytes to restore the cell shape and the ATP concentration. (2) When intracellular calcium content was increased to above 1.5 times of the normal value, the deformability was distinctly decreased. On the other hand, the deformability was unchanged when the intracellular calcium content was reduced below the normal level. (3) The deformability, once decreased due to the calcium accumulation, was recovered by the treatment with a calmodulin inhibitor, W-7 or trifluoperazine, while these drugs were not effective on the deformability of control or calcium-depleted erythrocytes. We conclude that the membrane stiffness which influence the deformability of erythrocytes, is modulated by the intracellular calcium content through the interaction between the calcium-calmodulin complex and the cytoskeletal proteins.     

Effect of intracellular organic phosphates on erythrocyte deformability

Organic phosphates in human erythrocytes were selectively varied by incubating fresh human erythrocytes in phosphate-buffered saline containing inosine, pyruvate, adenine, and/or adenosine in various concentrations. The deformability of erythrocytes was measured at 24 degrees C with a rheoscope under shear stress of 8-82 dyn/cm2. (1) With increasing 2, 3-DPG (5 approximately 15 mM/l cells), undeformable erythrocytes increased due to the increased mean corpuscular hemoglobin concentration (MCHC). However, these cells became deformable, when the MCHC was reduced by suspending in hypotonic medium. (2) At the same MCHC, the deformability of 2, 3-DPG-enriched erythrocytes was still reduced, compared with that of control erythrocytes, probably due to altered membrane viscoelastic properties. (3) 2, 3-DPG-reduced erythrocytes (2.2 mM/l cells) was not altered in their deformability. (4) Deformability of 2, 3-DPG-enriched erythrocytes was not changed by lowering oxygen tension. (5) Deformability of erythrocytes was not affected by varying intracellular ATP in the range of 0.5 approximately 2.2 mM/l cells (ATP in control cells was 1 mM/l cells). (6) Increment of IMP (approximately 0.9 mM/l cells) and ITP (approximately 0.5 mM/l cells) did not alter the deformability of erythrocytes. (7) Interaction of intracellular organic phosphates with membrane proteins was discussed.     

The decreased membrane fluidity of in vivo aged, human erythrocytes. A spin label study.

The decreased membrane fluidity of the in vivo aged, human erythrocytes is found, by monitoring the electron paramagnetic resonance (EPR) spectra of fatty acid spin labels incorporated into the membrane. In addition, the decreased cell sizes and the decreased cholesterol and phospholipids contents, without significant changes of the quantity of the membrane proteins, also the decrease of ATP and 2,3-diphosphoglycerate and the increase of ADP and AMP, in the aged cells, were observed. Further the functional impairments of the aged cells, i.e. the increased oxygen affinity and the decreased deformability, were shown. On the basis of these quantitative data, the alteration of the protein-lipid organization, due to decreased lipid/protein ratio, the modified protein-lipid interaction and/or the influences of the diminished ATP content, is suggested to contribute towards the decreased membrane fluidity of the in vivo aged erythrocytes.     

The decreased membrane fluidity of in vivo aged,human erythrocytes. A spin label study

The decreased membrane fluidity of the in vivo aged, human erythrocytes is found, by monitoring the electron paramagnetic resonance (EPR) spectra of fatty acid spin labels incorporated into the membrane.In addition, the decreased cell sizes and the decreased cholesterol and phospholipids contents, without significant changes of the quantity of the membrane proteins, also the decrease of ATP and 2,3-diphosphoglycerate and the increase of ADP and AMP, in the aged cells, were observed. Further the functional impairments of the aged cells, i.e. the increased oxygen affinity and the decreased deformability, were shown.On the basis of these quantitative data, the alteration of the protein-lipid organization, due to decreased lipid/protein ratio, the modified protein-lipid interaction and/or the influences of the diminished ATP content, is suggested to contribute towards the decreased membrane fluidity of the in vivo aged erythrocytes.     

Measurement of the distribution of red blood cell deformability using an automated rheoscope

BACKGROUND: Red blood cells (RBCs) have to deform markedly to pass through the smallest capillaries of the microcirculation. Techniques for measuring RBC deformability often result in an indication of the mean value. A deformability distribution would be more useful for studying diseases that are marked by subpopulations of less deformable cells because even small fractions of rigid cells can cause circulatory problems. METHODS: We present an automated rheoscope that uses advanced image analysis techniques to determine a RBC deformability distribution (RBC-DD) by analyzing a large number of individual cells in shear flow. The sensitivity was measured from density-separated fractions of one blood sample and from cells rendered less deformable by heat treatment. A preliminary experiment included the RBC-DDs of a patient with sickle cell anemia, one on dialysis and being treated with erythropoietin, and one with elliptocytosis. RESULTS: Measurement of the RBC-DD was highly reproducible. The sensitivity test showed markedly different deformability distributions of density-separated cells and yielded distinct RBC-DDs after each additional minute of heat treatment. CONCLUSION: The automated rheoscope enabled the determination of RBC-DDs from which less deformable subpopulations can be established. The shape of an RBC-DD may be valuable in assessing cell fractions with normal and anomalous deformability within pathologic blood samples.     

Reduced erythrocyte deformability associated with calcium accumulation

Erythrocytes exposed to subhemolytic shear stress in vitro exhibit decreased deformability as determined by a filtration method. Intracellular calcium content of these cells has been measured by atomic absorption spectroscopy and found to be 35 and 55% higher than controls (0.0157 μmol/ml packed red blood cells) after shear stress levels of 100 and 130 N/cm², respectively. These alterations occur without significant changes in ATP level, intracellular magnesium content, cell volume, or morphology, and without large associated sodium and potassium fluxes. Results indicate that calcium may be responsible for or associated with changes in the viscoelastic properties of the red cell membrane caused by sublytic mechanical trauma.     

The Rho kinase inhibitor Y-27632 increases erythrocyte deformability and low oxygen tension-induced ATP release

Low oxygen (O(2)) tension and mechanical deformation are stimuli for ATP release from erythrocytes. It has been shown previously that rabbit erythrocytes made less deformable with diamide, a thiol cross-linking agent, release less ATP in response to low O(2) tension, suggesting a link between these two stimuli. In nonerythroid cells, activation of the Rho/Rho kinase signaling pathway has been reported to decrease cell deformability by altering Rho kinase-dependent cytoskeleton-protein interactions. We investigated the hypothesis that the Rho kinase inhibitor Y-27632 would increase erythrocyte deformability and thereby increase low O(2) tension-induced ATP release from erythrocytes. Here we show that Y-27632 (1 μM) increases erythrocyte deformability (5%) and increases low O(2) tension-induced ATP release (203%) from healthy human erythrocytes. In addition, we found that, when erythrocytes were made less deformable by incubation with diamide (100 μM), Y-27632 restored both deformability and low O(2) tension-induced ATP release to levels similar to those measured in the absence of diamide. These findings suggest that the Rho kinase inhibitor Y-27632 is able to reverse the diamide-induced decrease in erythrocyte deformability and rescue low O(2) tension-induced ATP release. These results further support a link between erythrocyte deformability and ATP release in response to low O(2) tension.     

NMR studies of intracellular free calcium, free magnesium and sodium in the guinea pig reticulocyte and mature red cell

During the maturation process reticulocytes lose their intracellular organelles and undergo changes in membrane lipid composition and ion transport properties. While several reports indicate differences in the levels of magnesium, sodium and calcium in reticulocytes and erythrocytes, controversy remains concerning the actual magnitude and direction of ionic alterations during reticulocyte maturation. One problem with all of these studies is that the techniques used are invasive and are limited to measuring only the total cell ion content. We have used 31P, 23Na and 19F nuclear magnetic resonance (NMR) spectroscopy to compare the intracellular free ion and phosphometabolite levels in guinea pig reticulocytes and mature red blood cells. In contrast to a sharply decreased concentration of ATP in erythrocytes in comparison to reticulocytes, the intracellular free magnesium, measured using 31P-NMR, was increased by about 65% upon maturation (150 mumol/l cell water in reticulocytes in comparison to 250 mumol/l cell water in erythrocytes). Sizeable but opposite changes in intracellular sodium (5.5 mumol/ml cells in reticulocytes vs. 8.5 mumol/ml cells in erythrocytes) and intracellular free calcium (99 nM vs. 31 nM in reticulocytes and mature red cells, respectively) were also observed, suggesting that alterations in the kinetics of membrane ion transport systems, accompanying changes in phospholipid and cholesterol content, occur during the process of red cell maturation. However, in contrast to dog red blood cells, there was no evidence for the presence of a Na+/Ca2+ exchanger in guinea pig reticulocytes or erythrocytes.     

Effect of cell shape, membrane deformability and phospholipid organization on phosphate-calcium-induced fusion of erythrocytes

Fusion of bovine and goat erythrocytes was studied using the phosphate-calcium protocol. Both bovine and goat red cells are resistant to fusion with phosphate and calcium, under conditions that promote fusion of normal human erythrocytes. Fusion resistance is not related to decreased (5%) membrane deformability of erythrocytes of these species, since chicken erythrocytes which are 40% less deformable than human erythrocytes undergo fusion with efficiency similar to human red blood cells. Incorporation of either phosphatidylcholine or phosphatidylserine into bovine erythrocytes mediated by lipid exchange/transfer protein, caused fusion of these erythrocytes. Fluorescence analysis of merocyanine 540 dye labeled erythrocytes, by flow cytometry, showed that the frequency of cells which exhibit dye binding was much less (35%) in dimyristoylphosphatidylcholine (DMPC) incorporated compared to untreated bovine erythrocytes (80%), indicating that incorporation of DMPC caused closed packing of lipids in the external leaflet of the bilayer. These studies show that fusion of bovine erythrocytes, mediated by phosphate and calcium, has a requirement for either specific phospholipids such as phosphatidylcholine, phosphatidylserine, or closed packing of lipids in the external leaflet of the bilayer.     

Effect of pH on the velocity of erythrocyte aggregation

The effect of pH on the velocity of aggregation of human erythrocytes was quantitatively examined with a rheoscope combined with a video-camera, an image analyzer and a computer, in relation to the morphological changes of erythrocytes and their aggregates. (i) With increasing pH of the medium, the velocity of erythrocyte aggregation increased. (ii) The rouleaux formed at high pH were longer in shape and more stable against the increase of shear rate than those formed at low pH. (iii) With increasing pH, the diameter of erythrocyte increased, the (maximum) thickness decreased, and the cell volume decreased. The pH dependency of erythrocyte aggregation may be mainly due to the morphological change of erythrocytes, and partly due to the changes of erythrocyte deformability and of interaction with macromolecules.     

Theoretical model and experimental study of red blood cell (RBC) deformation in microchannels

The motion and deformation of red blood cells (RBCs) flowing in a microchannel were studied using a theoretical model and a novel automated rheoscope. The theoretical model was developed to predict the cells deformation under shear as a function of the cells geometry and mechanical properties. Fluid dynamics and membrane mechanics are incorporated, calculating the traction and deformation in an iterative manner. The model was utilized to evaluate the effect of different biophysical parameters, such as: inner cell viscosity, membrane shear modulus and surface to volume ratio on deformation measurements. The experimental system enables the measurement of individual RBCs velocity and their deformation at defined planes within the microchannel. Good agreement was observed between the simulation results, the rheoscope measurements and published ektacytometry results. The theoretical model results imply that such deformability measuring techniques are weakly influenced by changes in the inner viscosity of the cell or the ambient fluid viscosity. However, these measurements are highly sensitive to RBC shear modulus. The shear modulus, estimated by the model and the rheoscope measurements, falls between the values obtained by micropipette aspiration and laser trapping. The study demonstrates the integration of a theoretical model with a microfabricated device in order to achieve a better understanding of RBC mechanics and their measurement using microfluidic shear assays. The system and the model have the potential of serving as quantitative clinical tools for diagnosing deformability disorders in RBCs.     

Alteration of rheological properties of human erythrocytes by crosslinking of membrane proteins

The crosslinking of membrane proteins of human erythrocytes by diamide (diazene dicarboxylic acid bis(N,N-dimethylamide) ) was quantified by 4% polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. The relation between the crosslinking of membrane proteins and erythrocyte functions (rheological and oxygen transporting) was quantitatively examined. (i) The crosslinking of membrane protein was induced by diamide, without changing the shape and the contents of intracellular organic phosphates (adenylates and 2,3-diphosphoglycerate). The intensity of spectrin 2 in SDS-polyacrylamide gel electrophoresis decreased proportionally to diamide concentration. The percentage decrease in spectrin 2 (using band 3 as an internal standard) was the most appropriate indicator for crosslinking ("% crosslinking'). (ii) The suspension viscosity of erythrocytes increased in proportion to the percentage of crosslinking, in the range of applied shear rates of 3.76-752 s-1. (iii) Erythrocyte deformability (measured by a high-shear rheoscope) was reduced by the crosslinking. The change was detectable even at 5% crosslinking. (iv) Rouleaux formation (measured by a television image analyzer combined with a low-shear rheoscope) was inhibited by the crosslinking. The inhibition was also sensitively detected at more than 5% crosslinking. (v) Hemoglobin in erythrocytes was chemically modified by higher dose of diamide (probably by the binding of diamide with sulfhydryl groups). Also the oxygen affinity of hemoglobin increased and the heme-heme interaction decreased. (vi) The reduction of the crosslinking of membrane proteins by dithiothreitol apparently reversed the intensity of spectrin bands in SDS-polyacrylamide gel electrophoresis and the erythrocyte functions (the suspension viscosity and the deformability), though not completely.     

Effects of calcium and A23187 on deformability and volume of human red blood cells

Human red blood cells treated in vitro with Ca2+ plus A23187 in low K+ medium exhibited significantly decreased cell volume and deformability, the latter determined by ektacytometry. These effects of Ca2+ plus A23187 were prevented in the presence of high K+ medium. Increased K+ permeability mediated by increased intracellular Ca2+ (Gardos effect) was apparently responsible for decreased cell volume and deformability in low K+ medium. Although it is commonly accepted that Ca2+ accumulation and/or ATP depletion per se cause decreased red blood cell deformability, the present results demonstrate that acutely induced changes in red blood cell volume as promoted by Ca2+ are a more important determinant of red blood cell deformability.     

Erythrocyte adenosine triphosphate depletion during voluntary hyperventilation

Chronic hypophosphatemia in humans is associated with a slow depletion of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) in erythrocytes, combined with shape alteration, impaired deformability, and viability of the cells. Likewise, incubation of erythrocytes in alkaline solution is associated with ATP depletion. Since in hyperventilation both hypophosphatemia and alkalosis are present, we have investigated red cell organic phosphates, shape, deformability, and osmotic fragility before, during, and after 20 min of voluntary hyperventilation. On the average, red cell ATP decreased by 42%, the blood pH increased by 0.2 units, and plasma inorganic phosphorus decreased by 46% compared with the initial values. Red cell 2,3-DPG, shape, deformability, and osmotic fragility remained unchanged. After the end of hyperventilation ATP increased rapidly to control values in parallel with the normalization of the blood pH, whereas inorganic plasma phosphorus remained at the low level observed during hyperventilation. It is concluded that the combined effects of hypophosphatemia and alkalosis in acute hyperventilation lead to an isolated fall of red cell ATP, which occurs as rapid as after total inhibition of red cell glycolysis in vitro.     

In vivo neutrophil sequestration within lungs of humans is determined by in vitro "filterability".

Neutrophils are normally delayed in transit through the lung microcirculation, relative to the passage of erythrocytes. This sequestration contributes to a pulmonary pool of neutrophils that may relate to the relative inability of neutrophils to deform compared with erythrocytes when in transit in the pulmonary capillaries. A micropore membrane was used to model the human pulmonary microcirculation, in which cell deformability was measured as the pressure developed during filtration of the cells through the membrane at a constant flow. We demonstrated a significant correlation between in vitro deformability and in vivo lung sequestration of indium-111-labeled neutrophils in 10 normal subjects (r = 0.69, P less than 0.02). In eight patients with stable chronic obstructive pulmonary disease, this relationship was not significant (r = -0.2, P greater than 0.05). Furthermore, in a subject with microscopic pulmonary telangiectasia known to allow significant passage of 30-microns microspheres, neutrophils passed through the lungs without delay. Moreover, neutrophils from patients studied acutely with an exacerbation of chronic obstructive pulmonary disease were temporarily less deformable (P less than 0.01). These studies confirm that cell deformability is an important determinant of the normal neutrophil sequestration within the lungs. Changes in cell deformability may alter the extent of this sequestration.     

Intracellular calcium distribution in pigeon erythrocytes

Subcellular compartmentation of calcium has been studied in digitonin-treated pigeon erythrocytes. The following calcium pools could be detected: A non-vesicular and tightly bound pool of calcium able to reach values equivalent to 5 mumol calcium/ml cells that required millimolar calcium concentrations. A vesicular calcium pool with high calcium affinity that had properties similar to mitochondrial calcium transport. Extracellularly added ATP was strongly hydrolyzed by intact pigeon erythrocytes in the presence of either magnesium or calcium. The hydrolysis of ATP was not coupled to fluxes of either 45Ca2+ or 86Rb+ and most probably took place inside the cells.     

The binding of calcium ions by erythrocytes and `ghost''-cell membranes

1. Washed human erythrocytes, suspended in iso-osmotic sucrose containing 2.5mm-calcium chloride, bind about 400mug-atoms of calcium/litre of packed cells. Sucrose may be replaced by other sugars. 2. Partial replacement of sucrose by iso-osmotic potassium chloride diminishes the uptake of calcium, 50% inhibition occurring at about 50mm-potassium chloride. 3. Other univalent cations behave like potassium, whereas bivalent cations are much more inhibitory. The tervalent cations, yttrium and lanthanum, however, are the most effective inhibitors of calcium uptake. 4. An approximate correlation exists between the calcium uptake and the sialic acid content of erythrocytes of various species and of human erythrocytes that have been partially depleted of sialic acid by treatment with neuraminidase. However, even after complete removal of sialic acid, human erythrocytes still bind about 140mug-atoms of calcium/litre of packed cells. 5. A Scatchard (1949) plot of calcium uptake at various Ca(2+) concentrations in the suspending media shows the presence of three different binding sites on the external surface of the human erythrocyte membrane. 6. Erythrocyte ;ghost' cells, the membranes of which appear to be permeable to Ca(2+) ions, can bind about 1000mug-atoms of calcium per ;ghost'-cell equivalent of 1 litre of packed erythrocytes. This indicates that there are also binding sites for calcium on the internal surface of the erythrocyte membrane.     

Effects of the calcium-mediated enzymatic cross-linking of membrane proteins on cellular deformability

Summary Excess calcium binding affects the shape and dynamics of cellular deformation of human erythrocytes. It may be hypothesized that incorporation of calcium may modify cellular deformability by processes which include specific cross-linking of membrane proteins with resultant changes in cell shape and deformability. Since previous studies indicate that accumulation of calcium ions causes development of -glutamyl--lysine bridges in membrane proteins, under control of a membrane transamidating enzyme which specifically requires calcium ions for activation, experiments were devised to examine the relationship between cross-linking and deformability and to determine the effects of specific inhibitor of membrane protein cross-linking on the calcium-dependent modification of erythrocyte to the echinocytic shape. The elastic shear modulus of the membrane was not significantly affected by calcium-induced cross-linking, indicating that induced shape change, not altered elasticity, causes the observed reduction in cellular deformability. These findings support the interpretation that Ca⁺⁺-induced and transamidase-catalyzed cross-linking of membrane proteins contributes to fixation of altered cellular shape and decreased cellular deformability.