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.     

Cell age-dependent changes in deformability and calcium accumulation of human erythrocytes

The deformability of human erythrocytes was measured in a rheoscope, as a function of intracellular calcium content (varied with ionophore (A23187) and CaCl2) without complete ATP depletion and echinocytic transformation. Loading calcium into intact erythrocytes (calcium content: 16.8 mumol/1 packed cells = 1.48 amol per cell), the cell volume and energy charge gradually decreased. Further, the membrane fluidity of the lipid portion decreased without crosslinking of membrane proteins. A distinct transition from deformable to undeformable cells was observed by the rheoscope technique: i.e., 50% transition occurred at 40-50 mumol calcium/1 packed cells (= 3.5-4.0 amol per cell) and more than 90% above 100 mumol/1 packed cells (= 6.5 amol per cell) at a shear stress of 140 dyn/cm2. The deformable cells maintained their deformability to ellipsoidal disks independent of the average calcium content. The underformable cells, separated as high-density cells by density gradient centrifugation after calcium-loading, showed lower glucose-6-phosphate dehydrogenase activity than low-density-deformable cells; thus, the calcium-loaded, undeformable cells were presumably in vivo aged cells. The younger cells, fractionated as low-density cells from intact erythrocytes, were more deformable than aged cells. Upon calcium-loading, the younger cells restored their cell volume and deformability, while the aged cells, containing originally more calcium and less ATP, decreased their volume and became undeformable. Therefore, calcium accumulation by ionophore-CaCl2 takes place in preference to aged cells of lower energy metabolism, and leads to cellular dehydration and loss of deformability, due to condensed hemoglobin and altered membrane organization.     

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.     

Osmotic resistance of high-density erythrocytes in transglutaminase 2-deficient mice

Transglutaminase 2 (TGase 2) is a Ca(2+)-dependent enzyme responsible for the posttransttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Elevating the intracellular concentration of Ca(2+)-ions in human erythrocytes leads to the formation of cytoskeletal and cytoplasmic protein polymers. The Ca(2+)-dependent TGase 2-dependent cross-linking activity has been proposed for its involvement in erythrocyte aging, by inducing irreversible modification of their cell shape and deformability. Accordingly, we found that high-density ("old") TGase 2(minus sign/minus sign) red blood cells (RBCs) were more resistant to osmotic stress-induced hemolysis than those from wild type mice. In addition, elevating the intracellular concentration of Ca(2+) by treatment of total RBCs with ionophore A23187 resulted in enhanced resistance of TGase 2-deficient erythrocytes compared to their normal counterpart. These findings indicate that TGase 2 may have a role in regulating structural flexibility of RBCs, possibly affecting their life span in physiopathological conditions, such as erythrocyte senescence, which are accompanied by increases in intracellular Ca(2+) concentration.     

Biochemical,biophysical and haemorheological effects of dimethylsulphoxide on human erythrocyte calcium loading

The studies using dimethylsulphoxide (DMSO) and/or the 4-bromo-calcium ionophore A23187 (Br-A23187) often neglect the precise knowledge of some of their biochemical, biophysical and haemorheological effects. The aim of the present study was to evaluate these effects on erythrocytes after whole blood incubations with DMSO or Br-A23187 dissolved in DMSO. There were no significant differences between the different aliquots in the values of P(50), pH, erythrocyte deformability, erythrocyte membrane fluidity, haemoglobin and intracellular Ca(2+) concentrations ([Ca(2+)](i)). Aliquots with DMSO (independently of the presence of Br-A23187 or added Ca(2+)) had lower erythrocyte aggregation indexes and higher plasma concentrations of K(+)], Na(+)] and Ca(2+) than the aliquots without DMSO (independently of the presence of added Ca(2+)). Aliquots with added calcium (without the presence of Br-A23187 in DMSO) had a significantly higher erythrocyte acetylcholinesterase activity. Our data shows that calcium loading, the usual objective of Br-A23187 incubations, cannot be fulfilled with the studied experimental conditions. The coherence between our results and those obtained by other authors with different biological systems and different modulators of the rise on [Ca(2+)](i) suggests a non-specific effect of DMSO, disabling the action of the modulator. It can be reasoned that the decreased erythrocyte aggregation (without significant changes on the deformability or membrane fluidity) can result either from the decrease of the hydrogen bonding contribution to erythrocyte aggregation or the increased ionic strength influence on the erythrocyte membrane surface.     

Calcium and the malaria parasite: parasite maturation and the loss of red cell deformability

In the studies reported here, we examined the role of calcium in the maturation of the human malaria parasite Plasmodium falciparum, and in the loss of red cell deformability associated with parasite maturation. P. falciparum alters the permeability of its host red cell, which normally maintains submicromolar cytoplasmic concentrations of calcium. Infection of the red cell and parasite maturation produce a 30-fold increase in calcium uptake. Both parasite maturation and the loss of red cell deformability are blocked by EGTA (by extracellular-free calcium concentrations less than or equal to 35 microM) and by other calcium antagonists. The loss of red cell deformability that occurs with parasite maturation is accompanied by alterations in the cytoskeletal proteins of parasitized red cells similar to those produced by the calcium ionophore A23187 (reductions in bands 2.1 [ankyrin], 4.1, and 5 [actin]). These results establish that parasite development and the loss of red cell deformability are calcium-dependent. They suggest that parasite-induced changes in the calcium permeability of the red cell activate endogenous transglutaminase activity by raising the free calcium concentration of the red cell cytoplasm.     

A calcium requirement for electric field-induced cell shape changes and preferential orientation

C3H/10T1/2 mouse embryo fibroblasts stimulated by a steady electric field (10 V/cm) for 30 min exhibited lamellar retraction on the sides facing the electrodes. Some cells elongated and preferentially oriented with their long axis perpendicular to the field direction. Depletion of external calcium or blockage of calcium influx with lanthanum or the calcium channel blocker D-600 resulted in a reduction of the field-induced response. When external calcium was elevated stepwise from 0 to 10 mM, the field-induced response increased correspondingly. Electric stimulation in the presence of the calcium ionophore A23187 resulted in an increase of spindle-shaped cells with no preferential orientation. This response was blocked by calcium depletion and lanthanum, but not by D-600. The anticalmodulin drug W-13 inhibited the field-induced responses observed in normal buffer as well as in the presence of A23187. Some cell death resulted from prolonged electric field exposure, and the mortality was reduced by calcium depletion, lanthanum or D-600, but was not affected by W-13. We postulate that local calcium influx through channels opened by the electric field produces areas of high intracellular calcium which stimulate the cytoskeletal network to induce lamellar retraction. Prolonged field-induced calcium influx may eventually overcome the cell's mitochondrial calcium-buffer system, leading to necrotic calcification.     

Effects of divalent cation ionophore A23187 on potassium permeability of rat erythrocytes.

A23187 transports calcium rapidly into rat erythrocytes, apparently by an electroneutral exchange for intracellular magnesium and protons. When red cells are incubated in the absence of any added divalent cations, A23187 transports internal magnesium out of the cells, in exchange for extracellular protons. Magnesium uptake into erythrocytes is produced by A23187, providing the extracellular concentration of this cation exceeds intracellular levels, and the ionophore also transports strontium, but not barium, into red cells. A23187 produces a rapid and extensive loss of intracellular potassium from erythrocytes during uptake of calcium or strontium, but not magnesium. When red cells are incubated in the absence of any exogenous divalent cations, A23187 still produces a potassium efflux and this is inhibited completely by small amounts of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and restored by the addition of calcium in excess of the chelator. Although EDTA enhances the extent of magnesium release from erythrocytes incubated with A23187, it prevents the potassium efflux. Dipyridamole and 4-acetamid-4'-isothiocyano-stilbene-2,5'-disulfonic acid, which decrease chloride premeability of erythrocytes, inhibit the A23187-induced potassium loss from red cells. Rutamycin, peliomycin, venturicidin, and A23668B also inhibit potassium efflux from intact cells incubated with A23187, but this effect is not correlated with their abilities to inhibit various ATPases in red cell membrane preparations. It is concluded that A23187 does not transport potassium directly across the erythrocyte plasma membrane, but permits small amounts of endogenous calcium to interact with some membrane component to enhance potassium permeability of the cell.     

Effect of ionophore A23187 upon membrane function and ion movement in human and toad erythrocytes

Summary Addition of 0.1–0.3 m A23187, a divalent cation ionophore, to human erythrocytes suspended in a 1.0mm ⁴⁵Ca²⁺-containing buffer results in a small ( two fold) increase in [Ca²⁺] _i , a significant decrease in osmotic fragility, and a decrease in intracellular K⁺ (100 mmoles/liter of cells to 70 mmoles/liter cells) without significant alteration of intracellular [Na⁺]. This decrease in [K⁺] _i is associated with a significant decrease in packed cell volume and correlates directly with the observed alteration is osmotic fragility. Increasing extracellular K⁺ to 125mm prevents the A23187-induced changes in osmotic fragility, K⁺ content and cell volume, but does not prevent the ionophore-induced uptake of⁴⁵Ca²⁺. Addition of 0.1–0.3 m A23187 to toad erythrocytes leads to an increase in⁴⁵Ca²⁺ uptake comparable to that observed in human erythrocytes, but does not alter osmotic fragility, cell volume or K⁺ content. Higher concentrations of ionophore (3.0–10.0 m) cause a 30- to 50-fold increase in⁴⁵Ca²⁺ uptake and concomitant change in K⁺ content, cell volume and osmotic fragility. These changes in cell properties can be prevented by increasing extracellular [K⁺] to 90mm. The difference in sensitivity of the two cell types to A23187 is attributed to the presence of additional intracellular calcium pools within toad erythrocytes that prevent an increase in cytoplasmic Ca²⁺ until Ca²⁺ uptake is increased substantially at the higher concentrations of A23187.     

Is a rise in intracellular concentration of free calcium necessary or sufficient for stimulated cytoskeletal-associated actin?

The addition of the calcium ionophore A23187 to rabbit neutrophils increases the amount of actin associated with the cytoskeleton regardless of the presence or absence of calcium in the incubation medium. In the presence of extracellular calcium, the effect of A23187 is biphasic with respect to concentration. The action of the ionophore is rapid, transient, and is inhibited by pertussis toxin, hyperosmolarity, and quinacrine. On the other hand, the addition of pertussis toxin or hyperosmolarity has small if any, effect on the rise in intracellular calcium produced by A23187. While quinacrine does not affect the fMet-Leu-Phe-induced increase in cytoskeletal actin and the polyphosphoinositide turnover, its addition inhibits completely the stimulated increase in Ca-influx produced by the same stimulus. The results presented here suggest that a rise in the intracellular concentration of free calcium is neither necessary nor sufficient for the stimulated increase in cytoskeletal-associated actin. A possible relationship between the lipid remodeling stimulated by chemoattractants and the increased cytoskeletal actin is discussed.     

Effect of L-carnitine and acetyl-L-carnitine on the human erythrocyte membrane stability and deformability

In this study we examined the effect of carnitine and acetylcarnitine on the human erythrocyte membrane stability and membrane deformability. Since erythrocyte membranes are impermeable to these compounds, we resealed erythrocyte ghosts in the presence of different concentrations of carnitine or acetylcarnitine. Resealed ghosts can be adequately studied in their cellular deformability and membrane stability properties by means of ektacytometry. Both carnitine and acetylcarnitine alter the membrane stability but not membrane deformability of the red cell membrane. Resealed ghosts containing 20, 50, 150, and 300 microM carnitine had 1.1, 1.6, 0.9, and 0.7 times the normal stability. While resealed ghosts containing 20, 50, 150, and 300 microM acetylcarnitine had 1.1, 1.5, 1.3, and 1.2 times the normal stability. Such changes were found to be reversible. We also conducted SDS PAGE of cytoskeletal membrane proteins from membrane fragments and residual membranes produced during membrane stability analysis, and unsheared resealed membranes in those samples where we observed an increase or a decrease of membrane stability. No changes in the cytoskeletal membrane proteins were noticed, even when the samples, prior SDS PAGE analysis, were treated with or without dithiothreitol. In addition, fluorescence steady state anisotropy of DPH in the erythrocyte membrane treated with carnitine or acetylcarnitine shows no modification of the lipid order parameter. Our results would suggest that both carnitine and its acetyl-ester, at physiological concentrations, may increase membrane stability in mature erythrocytes, most likely via a specific interaction with one or more cytoskeletal proteins, and that this effect would manifest when the erythrocytes are subjected to high shear stress.     

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.     

Differential Erythropoietin Action upon Cells Induced to Eryptosis by Different Agents

Eryptosis is a process by which mature erythrocytes can undergo self-destruction sharing several features with apoptosis. Premature programmed erythrocyte death may be induced by different agents. In this study, we compared mechanisms involved in two eryptotic models (oxidative stress and cell calcium overload) so as to distinguish whether they share signaling pathways and could be prevented by erythropoietin (Epo). Phosphatidylserine (PS) translocation and increased calcium content were common signs in erythrocytes exposed to sodium nitrite plus hydrogen peroxide or calcium ionophore A23187 (CaI), while increased ROS and decreased GSH levels were detected in the oxidative model. Protein kinase activation seemed to be an outstanding feature in eryptosis induced by oxidative stress, whereas phosphatase activation was favored in the CaI model. Cell morphology and membrane protein modifications were also differential signs between both models. Epo was able to prevent cell oxidative imbalance, thus blunting PS translocation. However, the hormone favored intracellular calcium influx which could be the reason why it could not completely counteract the induction of eryptosis. Instead, Epo was unable to inhibit PS externalization in the CaI model. The different mechanisms involved in the eryptotic models may explain the differential action of Epo upon erythrocytes induced to eryptosis by different agents.     

Use of chlortetracycline fluorescence for the detection of Ca storing intracellular vesicles in normal human erythrocytes

The uptake of chlortetracycline (CTC) and the nature of the fluorescence of CTC was studied in intact human erythrocytes from apparently healthy donors. The uptake of CTC at 22 degrees C proceeded with a t1/2 of about 3 min, and after 15 min a stable equilibrium was achieved with an intracellular accumulation by a factor of 5-6 relative to the medium concentration. The accumulation did not change in the range of CTC concentrations tested (20-500 microM). The Ca specificity of the CTC fluorescence spectrum was confirmed by Ca depletion of red cells using A23187 in the presence of EGTA and 0.2 mM Mg. This procedure decreased the total intracellular calcium content by about 70% and reduced the fluorescence intensity to one-fourth. Fluorescence microscopy of red cells incubated with 100 microM CTC at 22 degrees C showed that the fluorescence originated mainly from the red cell membrane. In addition, in about 15% of erythrocytes one or more fluorescent dots (diameter greater than 0.2 less than 1 microns) were detected. The fluorescence of the dots and membranes was related to calcium, as evidenced by the reduction of their intensity in Ca depleted cells. The number of erythrocytes with fluorescent dots and the frequency of the dots per cell was largely unaffected by lowering the incubation temperature to 0 degrees C, indicating that the dots most probably do not represent endocytotic artifacts induced by CTC. The number of dots was increased in erythrocytes preincubated with primaquine, demonstrating that CTC fluorescence can be applied to monitor the appearance of intracellular Ca storing vesicles. It is concluded that in (at least) 15% of erythrocytes obtained from apparently healthy donors intracellular vesicles containing Ca can be detected by CTC fluorescence microscopy.     

Localization of calcium and microfilament changes in mechanically stressed cells.

We combined fluorescence labeling, digital image processing, and micromanipulation to investigate the intracellular events induced by inflicting a mechanical stress on rat basophilic leukemia cells. Our findings were as follows: 1. Most cells displayed a localized calcium rise in response to micropipet aspiration. This represented an average threefold increase as compared to resting level, and it was observed during the first 10 s following aspiration. A slow return to initial level occurred within about 3 min. Further, this calcium rise involved a mobilization of intracellular stores, since it was not prevented by adding a calcium chelator into the extracellular medium. 2. All micropipet-aspirated cells displayed a local accumulation of microfilaments, with a preferential localization in the cell protrusions or near the pipet tips. 3. No absolute correlation was found between the localization of calcium rise and cytoskeletal accumulation. 4. Cell deformability was decreased when intracellular calcium was maintained at a constant (high or low) level with ionomycin and/or EGTA. It is concluded that cells have a general ability to respond to mechanical stimulation by a coordinated set of events. More parameters must be studied before the mechanisms of cell shape regulation are fully understood.     

In vitro effects of thyroxine on the mechanical properties of erythrocytes

In vitro effects of thyroxine on erythrocyte deformability and mechanical fragility were observed. Deformability of erythrocytes was improved in a dose dependent manner by thyroxine. Mechanical hemolysis was found to be lower if thyroxine was included in erythrocyte suspensions at concentrations close to the physiological levels (10(-9)M). These changes might be related to the alterations of intracellular calcium concentration, as in the erythrocyte suspensions containing 10(-9)M thyroxine, intracellular calcium concentration was found to be 30 times lower than the control suspensions which did not contain thyroxine. Thyroxine also reduced the mechanical hemolysis ratio in calcium loaded cells. These observations suggest that thyroxine might play some role in the regulation of the mechanical properties of erythrocytes which might be mediated via the effects on calcium metabolism.     

Calcium dynamically alters erythrocyte mechanical response to shear

Red blood cells (RBC) are constantly exposed to varying mechanical forces while traversing the cardiovascular system. Upon exposure to mechanical stimuli (e.g., shear stress), calcium enters the cell and prompts potassium-efflux. Efflux of potassium is accompanied by a loss of intracellular fluid; thus, the volume of RBC decreases proportionately (i.e., ‘Gárdos effect’). The mechanical properties of the cell are subsequently impacted due to complex interactions between cytosolic viscosity (dependent on cell hydration), the surface-area-to-volume ratio, and other molecular processes. The dynamic effects of calcium on RBC mechanics are yet to be elucidated, although accumulating evidence suggests a vital role. The present study thus examined the effects of calcium on contemporary biomechanical properties of RBC in conjunction with high-precision geometrical analyses with exposure to shear. Mechanical stimulation of RBC was performed using a co-axial Couette shearing system to deform the cell membrane; intracellular signaling events were observed via fluorescent imaging. Calcium was introduced into RBC using ionophore A23187. Increased intracellular calcium significantly impaired RBC deformability; these impairments were mediated by a calcium-induced reduction of cell volume through the Gárdos channel. Extracellular calcium in the absence of the ionophore only had an effect under shear, not at stasis. Under low shear, the presence of extracellular calcium induced progressive lysis of a sub-population of RBC; all remaining RBC exhibited exceptional capacity to deform, implying preferential removal of potentially aged cells. Collectively, we provide evidence of the mechanism by which calcium acutely regulates RBC mechanical properties.     

Production of 1,2-diacylglycerol and phosphatidate in human erythrocytes treated with calcium ions and ionophore A23187.

1. When the ionophore A23187 and Ca2+ were added to normal human erythrocytes, the incorporation of 32P into phosphatidate was enhanced within 1 min, but there was only slight labelling of other phospholipids. 2. Labelling of phosphatidate in these cells did not continue to increase after about 20min at 37 degrees C; by this time, radioactivity in phosphatidate was about ten times higher inionophore A23187-treated cells than in controls. A net synthesis of phosphatidate was measured in response to the increase in intracellular Ca2+ concentration; the content of this phospholipid in the cell was increased by approximately 50%. 3. In the presence of 2.5 mM-Ca2+ a maximum effect was seen with about 0.5 mug of ionophore/ml. 4. The concentration of Ca2+ giving half-maximal labelling of phosphatidate in the presence of 10 mug of ionophore A23187/ml was about 10 muM. 5. A rapid decrease of ATP content in the cell occurred in ionophore-treated cells. 6. Labelling of phosphatidate appeared to be secondary to the production of 1,2-diacylglycerol in the cells; accumulation of 1,2-diacylglycerol was only seen after about 15 min. After 60 min, the 1,2-diacylglycerol content of the cells was five to seven times that of untreated control cells. 7. The change in the shape of erythrocytes treated with Ca2+ and ionophore appeared to be related to accumulation of 1,2-diacylglycerol. 8. The source of 1,2-diacylglycerol has not been definitely identified, but its fatty acid compositon was similar to that of phosphatidylcholine. However, it has an unusually high content of hexadecenoic acid, a fatty acid not common in the major erythrocyte phospholipids. 9. Accumulation of 1,2-diacyglycerol also occurred in energy-starved cells, even in the absence of calcium; in this case it appeared to be produced by phosphatidate breakdown.     

Effects of chemotactic factors and other agents on the amounts of actin and a 65,000-mol-wt protein associated with the cytoskeleton of rabbit and human neutrophils

Stimulation of rabbit neutrophils by the chemotactic factors fMet-Leu-Phe and leukotriene B4, by platelet activating factor, or by arachidonic acid produces a rapid and dose-dependent increase in the amounts of actin and of a 65,000-mol-wt protein associated with the cytoskeleton. Phorbol 12-myristate, 13-acetate, the calcium ionophore A23187 in the presence or absence of EGTA, and the fluorescent calcium chelator quin-2 also cause an increase in cytoskeletal actin. The stimulated increases in the cytoskeletal actin are not dependent on a rise in the intracellular concentration of free calcium and are not mediated by an increase in the intracellular pH or activation of protein kinase C. The increases in the cytoskeletal actin produced by fMet-Leu-Phe and leukotriene B4, but not by phorbol 12-myristate, 13-acetate, are inhibited by high osmolarity. The effect of hyperosmolarity requires a decrease in cell volume, is not mediated by an increase in basal intracellular concentration of free calcium, and is not prevented by pretreating the cells with amiloride. Preincubation of the cells with hyperosmotic solution also inhibits degranulation produced by all the stimuli tested. The inhibitory action of high osmolarity on the fMet-Leu-Phe and leukotriene B4 induced stimulation of cytoskeletal actin is discussed in terms of the possibility that the addition of high osmolarity, either directly or through activation of protein kinase C, causes receptor uncoupling.