calcimycin and Anemia--Sickle-Cell

Increased endothelin-1 (ET-1) levels, disordered thiol protein status, and erythrocyte hydration status play important roles in sickle cell disease (SCD) through unresolved mechanisms. Protein disulfide isomerase (PDI) is an oxidoreductase that mediates thiol/disulfide interchange reactions. We provide evidence that PDI is present in human and mouse erythrocyte membranes and that selective blockade with monoclonal antibodies against PDI leads to reduced Gardos channel activity (1.6±0.03 to 0.56±0.02 mmol·10(13) cell(-1)·min(-1), P<0.001) and density of sickle erythrocytes (D50: 1.115±0.001 to 1.104±0.001 g/ml, P=0.012) with an IC50 of 4 ng/ml. We observed that erythrocyte associated-PDI activity was increased in the presence of ET-1 (3.1±0.2 to 5.6±0.4%, P<0.0001) through a mechanism that includes casein kinase II. Consistent with these results, in vivo treatment of BERK sickle transgenic mice with ET-1 receptor antagonists lowered circulating and erythrocyte associated-PDI activity (7.1±0.3 to 5.2±0.2%, P<0.0001) while improving hematological parameters and Gardos channel activity. Thus, our results suggest that PDI is a novel target in SCD that regulates erythrocyte volume and oxidative stress and may contribute to cellular adhesion and endothelial activation leading to vasoocclusion as observed in SCD.

Topics: Anemia, Sickle Cell; Animals; Antibodies, Monoclonal; Calcimycin; Calcium Ionophores; Casein Kinase II; Cell Membrane; Endothelin A Receptor Antagonists; Enzyme Inhibitors; Erythrocyte Volume; Erythrocytes; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Mice; Oxidative Stress; Protein Disulfide-Isomerases; Sulfhydryl Compounds

No specific inhibitors of the plasma membrane Ca(2+) pump have been found to date, limiting research on the particular contribution of this pump to the Ca(2+) homeostasis of animal cells. The search for Ca(2+) pump inhibitors may have been hampered by the lack of an efficient screening method to measure pump activity that would provide an alternative to the lengthy and costly adenosine triphosphatase or Ca(2+)-flux measurements. We propose here a novel screening method in which Ca(2+) pump inhibition is translated into easily measurable cell dehydration. Intact human red cells, suspended in Ca(2+)-containing, low-K(+) buffers were exposed to sequential additions of (1) ionophore A23187 (t = 0) to load the cells with Ca(2+); (2) CoCl(2) (t = 1 minute) to block ionophore-mediated Ca(2+) transport and to allow complete extrusion of the Ca(2+) load by the pump in less than 5 minutes; and (3) NaSCN (t = 6 minutes) to accelerate cell dehydration via Ca(2+)-sensitive K(+) channels when the Ca(2+) load is retained as a result of Ca(2+) pump inhibition. Samples were taken at 10 to 25 minutes after ionophore addition and delivered into hypotonic media containing about 45 mmol/L NaCl. Non-dehydrated cells-with normal, uninhibited pumps-instantly underwent lysis, whereas dehydrated cells-with inhibited pumps-resisted lysis, resulting in translucent or opaque samples, respectively, which were quantifiable by light-absorption measurements. Vanadate was used as a test substance to assess the effect of putative pump inhibitors. This method offers a cost-efficient and easily automated alternative for testing large numbers of natural or synthetic agents.

Topics: Anemia, Sickle Cell; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Membrane; Dehydration; Enzyme Inhibitors; Erythrocyte Membrane; Homeostasis; Humans; In Vitro Techniques; Ionophores; Mass Screening; Potassium Channels; Potassium Chloride; Sodium Cyanide; Vanadates

We describe a population of sickle cell anemia red cells (SS RBCs) ( approximately 4%) and a smaller fraction of normal RBCs (<0.03%) that fail to dehydrate when permeabilized to K(+) with either valinomycin or elevated internal Ca(2+). The nonshrinking, valinomycin-resistant (val-res) fractions, first detected by flow cytometry of density-fractionated SS RBCs, constituted up to 60% of the lightest, reticulocyte-rich (R1) cell fraction, and progressively smaller portions of the slightly denser R2 cells and discocytes. R1 val-res RBCs had a mean cell hemoglobin concentration of approximately 21 g of Hb per dl, and many had an elongated shape like "irreversibly sickled cells," suggesting a dense SS cell origin. Of three possible explanations for val-res cells, failure of valinomycin to K(+)-permeabilize the cells, low co-ion permeability, or reduced driving K(+) gradient, the latter proved responsible: Both SS and normal val-res RBCs were consistently high-Na(+) and low-K(+), even when processed entirely in Na-free media. Ca(2+) + A23187-induced K(+)-permeabilization of SS R1 fractions revealed a similar fraction of cal-res cells, whose (86)Rb uptake showed both high Na/K pump and leak fluxes. val-res/cal-res RBCs might represent either a distinct erythroid genealogy, or an "end-stage" of normal and SS RBCs. This paper focuses on the discovery, basic characterization, and exclusion of artifactual origin of this RBC fraction. Many future studies will be needed to clarify their mechanism of generation and full pathophysiological significance.

Topics: Anemia, Sickle Cell; Bumetanide; Calcimycin; Calcium; Cell Membrane Permeability; Drug Resistance; Electron Probe Microanalysis; Erythrocytes; Ionophores; Ouabain; Potassium; Potassium Channels; Rubidium; Sodium; Spectrophotometry, Atomic; Valinomycin

We investigated a transgenic mouse model of sickle cell disease, homozygous for deletion of mouse beta-globin and containing transgenes for human beta(S) and beta(S-antilles) globins linked to the transgene for human alpha-globin. In these mice, basal cGMP production in aortic rings is increased, whereas relaxation to an endothelium-dependent vasodilator, A-23187, is impaired. In contrast, aortic expression of endothelial nitric oxide synthase (NOS) is unaltered in sickle mice, whereas expression of inducible NOS is not detected in either group; plasma nitrate/nitrite concentrations and NOS activity are similar in both groups. Increased cGMP may reflect the stimulatory effect of peroxides (an activator of guanylate cyclase), because lipid peroxidation is increased in aortae and in plasma in sickle mice. Despite increased vascular cGMP levels in sickle mice, conscious systolic blood pressure is comparable to that of aged-matched controls; sickle mice, however, evince a greater rise in systolic blood pressure in response to nitro-L-arginine methyl ester, an inhibitor of NOS. Systemic concentrations of the vasoconstrictive oxidative product 8-isoprostane are increased in sickle mice. We conclude that vascular responses are altered in this transgenic sickle mouse and are accompanied by increased lipid peroxidation and production of cGMP; we suggest that oxidant-inducible vasoconstrictor systems such as isoprostanes may oppose nitric oxide-dependent and nitric oxide-independent mechanisms of vasodilatation in this transgenic sickle mouse. Destabilization of the vasoactive balance in the sickle vasculature by clinically relevant states may predispose to vasoocclusive disease.

Topics: Anemia, Sickle Cell; Animals; Aorta; Autoantibodies; Blood Pressure; Calcimycin; Cyclic GMP; Disease Models, Animal; Endothelium, Vascular; Globins; Hemoglobin, Sickle; Humans; In Vitro Techniques; Lipid Peroxidation; Mice; Mice, Transgenic; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Papaverine; Vasoconstriction; Vasodilation

Deoxy-stimulated cation fluxes have been implicated in the generation of the dense and irreversibly sickled red blood cells (RBCs) in patients homozygous for hemoglobin S (SS). We now report on the effect of short term deoxygenation on K+ and Na+ transport in RBCs from control mice (C57Bl/6J) and a transgenic (alphaHbetaS[betaMDD]) mouse line that expresses high levels of human alphaH and betaS-chains and has a small percent dense cells but does not exhibit anemia. In transgenic mouse RBCs (n = 5) under oxygenated conditions, K+ efflux was 0.22 +/- 0.01 mmol/L cell x min and Na+ influx was 0.17 +/- 0.02 mmol/L cell x min. Both fluxes were stimulated by 10 min deoxygenation in transgenic but not in control mice. The deoxy-stimulated K+ efflux from transgenic mouse RBCs was about 55% inhibited by 5 nm charybdotoxin (CTX), a blocker of the calcium activated K+-channel. To compare the fluxes between human and mouse RBCs, we measured the area of mouse RBCs and normalized values to area per liter of cells. The deoxy-simulated CTX-sensitive K+ efflux was larger than the CTX-sensitive K+ efflux observed in RBCs from SS patients. These results suggest that in transgenic mice, deoxygenation increases cytosolic Ca2+ to levels which open Ca2+-activated K+ channels. The presence of these channels was confirmed in both control and transgenic mice by clamping intracellular Ca2+ at 10 microM with the ionophore A23187 and measuring Ca2+-activated K+ efflux. Both types of mouse had similar maximal rates of CTX-sensitive, Ca2+-activated K+ efflux that were similar to those in human SS cells. The capacity of the mouse red cell membrane to regulate cytosolic Ca2+ levels was examined by measurements of the maximal rate of calmodulin activated Ca2+-ATPase activity. This activity was 3-fold greater than that observed in human RBCs thus indicating that mouse RBC membranes have more capacity to regulate cytosolic Ca2+ levels. In summary, transgenic mouse RBCs exhibit larger values of deoxy-stimulated K+ efflux and Na+ influx when compared to human SS cells. They have a similar Ca2+-activated K+ channel activity to human SS cells while expressing a very high Ca2+ pump activity. These properties may contribute to the smaller percent of very dense cells and to the lack of adult anemia in this animal model.

Topics: Adult; Anemia, Sickle Cell; Animals; Calcimycin; Calcium-Transporting ATPases; Charybdotoxin; Erythrocytes; Hemoglobin, Sickle; Humans; Ionophores; Mice; Mice, Inbred C57BL; Mice, Transgenic; Potassium; Potassium Channel Blockers; Sodium

We have previously shown that a pretreatment with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), reduced deoxygenation-induced K+ loss and Ca2+ uptake and prevented cell dehydration in sickle anemia red blood cells (SS cells) (H. Fathallah, E. Coezy, R.-S. De Neef, M.-D. Hardy-Dessources, and F. Giraud. Blood 86: 1999-2007, 1995). The present study explores the detailed mechanism of this PMA-induced inhibition. The main findings are, first, the detection of PKC alpha and PKC zeta in normal red blood cells and the demonstration that both isoforms are expressed at higher levels in SS cells. The alpha-isoform only is translocated to the membrane and activated by PMA and by elevation of cytosolic Ca2+. Second, PMA is demonstrated to activate Ca2+ efflux in deoxygenated SS cells by a direct stimulation of the Ca2+ pump. PMA, moreover, inhibits deoxygenation-induced, charybdotoxin-sensitive K+ efflux in SS cells. This inhibition is partly indirect and explained by the reduced deoxygenation-induced rise in cytosolic Ca2+ resulting from Ca2+ pump stimulation. However, a significant inhibition of the Ca2+-activated K+ channels (K(Ca) channels) by PMA can also be demonstrated when the channels are activated by Ca2+ plus ionophore, under conditions in which the Ca2+ pump is operating near its maximal extrusion rate, but swamped by Ca2+ plus ionophore. The data thus suggest a PKC alpha-mediated phosphorylation both of the Ca2+ pump and of the K(Ca) channel or an auxiliary protein.

Topics: Anemia, Sickle Cell; Arachidonic Acid; Calcimycin; Calcium; Calcium-Transporting ATPases; Chromatography, DEAE-Cellulose; Cytosol; Diglycerides; Egtazic Acid; Enzyme Activation; Erythrocyte Membrane; Erythrocytes; Humans; In Vitro Techniques; Isoenzymes; Kinetics; Molecular Weight; Protein Kinase C; Protein Kinase C-alpha; Reference Values; Tetradecanoylphorbol Acetate

The phospholipids of the human red cell are distributed asymmetrically in the bilayer of the red cell membrane. In certain pathologic states, such as sickle cell anemia, phospholipid asymmetry is altered. Although several methods can be used to measure phospholipid organization, small organizational changes have been very difficult to assess. Moreover, these methods fail to identify subpopulations of cells that have lost their normal phospholipid asymmetry. Using fluorescently labeled annexin V in flow cytometry and fluorescent microscopy, we were able to identify and quantify red cells that had lost their phospholipid asymmetry in populations as small as 1 million cells. Moreover, loss of phospholipid organization in subpopulations as small as 0.1% of the total population could be identified, and individual cells could be studied by fluorescent microscopy. An excellent correlation was found between fluorescence-activated cell sorter (FACS) analysis results using annexin V to detect red cells with phosphatidylserine (PS) on their surface and a PS-requiring prothrombinase assay using similar red cells. Cells that bound fluorescein isothiocyanate (FITC)-labeled annexin V could be isolated from the population using magnetic beads covered with an anti-FITC antibody. Evaluation of blood samples from patients with sickle cell anemia under oxygenated conditions demonstrated the presence of subpopulations of cells that had lost phospholipid asymmetry. While only a few red cells were labeled in normal control samples (0.21% +/- 0.12%, n = 8), significantly increased (P < .001) annexin V labeling was observed in samples from patients with sickle cell anemia (2.18% +/- 1.21%, n = 13). We conclude that loss of phospholipid asymmetry may occur in small subpopulations of red cells and that fluorescently labeled annexin V can be used to quantify and isolate these cells.

Topics: Anemia, Sickle Cell; Annexin A5; Calcimycin; Calcium; Cell Separation; Erythrocyte Membrane; Erythrocytes; Erythrocytes, Abnormal; Ethylmaleimide; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Humans; Immunomagnetic Separation; Membrane Lipids; Microscopy, Fluorescence; Phosphatidylserines; Phospholipids

Elevated [Ca2+]i in deoxygenated sickle cell anemia (SS) red cells (RBCs) could trigger a major dehydration pathway via the Ca(2+)-sensitive K+ channel. But apart from an increase in calcium permeability, the effects of deoxygenation on the Ca2+ metabolism of sickle cells have not been previously documented. With the application of 45Ca(2+)-tracer flux methods and the combined use of the ionophore A23187, Co2+ ions, and intracellular incorporation of the Ca2+ chelator benz-2, in density-fractionated SS RBCs, we show here for the first time that upon deoxygenation, the mean [Ca2+]i level of SS discocytes was significantly increased, two- to threefold, from a normal range of 9.4 to 11.4 nM in the oxygenated cells, to a range of 21.8 to 31.7 nM in the deoxygenated cells, closer to K+ channel activatory levels. Unlike normal RBCs, deoxygenated SS RBCs showed a two- to fourfold increase in pump-leak Ca2+ turnover. Deoxygenation of the SS RBCs reduced their Ca2+ pump Vmax, more so in reticulocyte- and discocyte-rich than in dense cell fractions, and decreased their cytoplasmic Ca2+ buffering. Analysis of these results suggests that both increased Ca2+ influx and reduced Ca2+ pump extrusion contribute to the [Ca2+]i elevation.

Topics: Anemia, Sickle Cell; Biological Transport; Biological Transport, Active; Buffers; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Hypoxia; Cobalt; Cytoplasm; Erythrocytes; Humans; Hydrogen-Ion Concentration; Ions; Models, Chemical; Reticulocytes

A four-fold increase in the binding of 3H-PDBu by red cell membrane ghosts isolated from sickle red cells compared to that from normal controls is presented. Phosphorylation studies with gamma-32P-ATP indicate a similar (two to three-fold) increase in the radiolabelling of the acid-precipitable membrane proteins in sickle red cells. When red cells were loaded with Ca2+ using Ionophore A23187, both normal and sickle red cells enhanced their phosphorylation and sickle red cells to a greater extent than normal red cells. Polyacrylamide slab gel electrophoretic separation of the phosphoproteins and autoradiography also reveal phosphorylation, predominantly of protein bands 3, 4.1 and 4.9 which are known in the red cells as specific substrates for the PDBu receptor, protein kinase C. These results indicate that membrane association of protein kinase C in sickle red cells is increased, possibly as a consequence of the pathological change in their ability to accumulate intracellular calcium.

Topics: Anemia, Sickle Cell; Caenorhabditis elegans Proteins; Calcimycin; Calcium; Carrier Proteins; Erythrocyte Membrane; Humans; Membrane Proteins; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phosphoproteins; Phosphorylation; Protein Kinase C; Receptors, Drug; Receptors, Immunologic

Erythrocytes from 14 patients with homozygous sickle cell anaemia were treated with the calcium ionophore A23187 to induce loss of cellular potassium and water. The dehydrated cells showed a decrease in filterability (loss of deformability) through pores of 5 micron diameter. Oxpentifylline and cetiedil citrate, which preserve erythrocyte cation and water content, had a significant (p less than 0.01) protective effect against loss of deformability at a concentration of 1 mumol/l. Oxpentifylline showed no adverse effect on the rheology, morphology, or haemolysis of sickle cells at concentrations up to 500 mumol/l. Drugs that act on the erythrocyte membrane to maintain cell hydration are of potential rheological benefit in sickle cell anaemia.

Topics: Anemia, Sickle Cell; Antisickling Agents; Azepines; Calcimycin; Dose-Response Relationship, Drug; Erythrocyte Deformability; Erythrocytes, Abnormal; Humans; Pentoxifylline; Theobromine

In order to determine the capacity of sickle cells to undergo transglutaminase-catalyzed cross-linking of membrane proteins, human normal and sickle erythrocytes were incubated with [ring-2-14C]histamine in the presence of Ca2+ and ionophore A23187. The [14C]histamine incorporation into membrane components was observed in freshly prepared erythrocytes. Incorporation of radioactivity into spectrin and Band 3 membrane components was significantly (P less than 0.001) less in sickle erythrocytes than in normal cells. Transglutaminase deficiency was excluded by the finding of increased activity of this enzyme in sickle cells from patients with reticulocytosis. The incorporation of [3H]spermine into red cell membranes was also less in sickle erythrocytes than in normal cells under the same conditions of incubation used for [ring-2-14C]histamine. Sickle erythrocytes were more permeable to these amines than normal cells. It is proposed that the gamma-glutamyl sites of membrane proteins in sickle erythrocytes are less accessible for transglutaminase-catalyzed cross-linking to histamine and polyamines in vitro, perhaps due to prior in vivo activation of this enzyme by the increased calcium in sickle cells and/or shielding secondary to altered membrane organization.

Topics: Acyltransferases; Anemia, Sickle Cell; Calcimycin; Calcium; Cell Membrane Permeability; Erythrocyte Membrane; Histamine; Humans; In Vitro Techniques; Membrane Proteins; Polyamines; Spermine; Transglutaminases

Spicules from sickle red cells were examined for their effects on the clotting activity of blood. The spicules were obtained from the sickle red cells after deoxygenation and oxygenation and were tested for clotting activity with Russell's viper venom assay. A marked increase in clotting activity was observed when spicules were added to the system. The increase was distinctly greater than that observed after the addition of sickle red cells while normal red cells had little effect. Vesicles prepared from sickle or normal red cells by incubation with the ionophore A-23187+Ca2+ also markedly increased clotting activity. The effect of spicules or vesicles on the clotting system may be related to reorganization of phospholipid in the spectrin-poor membrane of the spicules or vesicles. Because of these effects, the spicules from the sickle red cells may contribute to the hypercoagulable state in these patients and possibly to their vaso-occlusive crises since free spicules are present in their plasma. Vesicles from red cells from other types of anaemia with hypercoagulability may have a similar effect on coagulation.

Topics: Anemia, Sickle Cell; Blood Coagulation; Blood Coagulation Tests; Calcimycin; Erythrocyte Membrane; Erythrocytes, Abnormal; Humans; Oxygen; Vacuoles

The formation of irreversibly sickled red cells has been studied by inducing cell shrinkage, ion loss, Ca2+ accumulation and membrane loss either singly or in combination. Valinomycin, A23187+Ca2+ or hypertonic saline caused shrinkage of the cells with retention of the sickled form after reoxygenation. The cells which had retained the sickle shape after treatment with the ionophores and reoxygenation remained sickled after exposure to hypotonic media. These cells were also osmotically insensitive. Retention of the sickled form was not dependent upon membrane loss as induced by repeated sickle-unsickle cycles or by A23187+Ca2+ treatment although repetitive sickling did give rise to shorter, stubbier spicules. Sickled red cells, either the endogenous irreversibly sickled cells or the sickled cells induced by deoxygenation, did not lose membrane by vesicle or spicule loss as normal cells or oxygenated sickle red cells do. Cell water loss without cell membrane loss appears to be an important factor in the irreversible sickling of red cells.

Topics: Anemia, Sickle Cell; Anti-Bacterial Agents; Calcimycin; Calcium; Erythrocyte Membrane; Erythrocytes, Abnormal; Humans; Saline Solution, Hypertonic; Valinomycin

Pre-treatment of normal erythrocytes with micromolar Ca2+ and ionophore A23187 induces abnormal phosphorylation of membrane polypeptides, as determined by labeling with exogenous 32Pi. The Ca2+ -induced effects, which include increased incorporation of 32P into acid-stable linkages and increased labeling in the Band 3 and 4.5-4.9 regions of SDS gels, are similar to those seen in untreated sickle erythrocytes. Part of the abnormal phosphorylation of sickle cells may be caused by their elevated intracellular Ca2+ levels.

Topics: Adenosine Triphosphate; Anemia, Sickle Cell; Anti-Bacterial Agents; Calcimycin; Calcium; Erythrocyte Membrane; Erythrocytes; Humans; Membrane Proteins; Phosphorylation

Topics: Anemia, Sickle Cell; Calcimycin; Calcium; Carbachol; Cell Membrane Permeability; Erythrocyte Membrane; Erythrocytes; Erythrocytes, Abnormal; Humans; Magnesium; Membrane Lipids; Phosphatidic Acids; Phosphatidylinositol Phosphates; Phosphatidylinositols; Polymers

Simultaneous measurements of Ca content and 42K+ influx in sickle cell anaemia red cells confirm predictions from earlier data in the literature that the increased Ca content of sickle cell anaemia cells which are not metabolically depleted does not cause a quinine-sensitive increase in K+ permeability. It is shown that the ionophore, A23187, can cause the Ca contained inside sickle cell anaemia cells to activate the quinine-sensitive K+-permeability mechanism. This demonstrates the existence of a Ca2+-refractory state of the K+ channel in sickle cell anaemia cells and a direct stimulatory effect of the ionophore A23187 on its Ca sensitivity.

Topics: Anemia, Sickle Cell; Calcimycin; Calcium; Cell Membrane Permeability; Erythrocytes, Abnormal; Humans; Ion Channels; Potassium

Sickle cell anaemia red cells (SS) were reported to have a high Ca content and an increased Ca uptake on deoxygenation, but their Ca-pump activity was described as normal. This seemed puzzling because the saturated Ca-extrusion rate of the normal, high Ca-affinity Ca pump is about 10 mmol per 1 cells per h (refs 3, 4) and the highest sickling-induced Ca influx reported in SS cells and observed in ATP-depleted sickle-trait (SA) red cells never exceeded 0.2 mmol per 1 cells per h. Normal pump performance is, therefore, incompatible with Ca accumulation unless SS cells have abnormally high Ca-binding capacity. We provide here evidence which suggests that SS cells have normal Ca-buffering capacity and probably genetically normal Ca pumps, but that the sickling process causes progressive Ca-pump failure and a marked reduction in Ca:Ca exchange.

Topics: Anemia, Sickle Cell; Biological Transport; Calcimycin; Calcium; Calcium-Transporting ATPases; Erythrocytes, Abnormal; Humans

The response of sickle cells with varying water content to alterations in oxygen tension has been studied. Cells that were severely dehydrated while sickled retained the characteristic sickled morphology even after prolonged reoxygenation. When the cell water content was increased by reduction of the suspending medium osmolality, the cells unsickled. Cells that were dehydrated before deoxygenation were unable to assume the spiculated morphology typical of sicked cells. This was true both for high mean cell hemoglobin concentration (MCHC) discoid sickle cells and for irreversibly sickled cells. When such cells were resuspended in hypotonic medium before deoxygenation, they sickled with the characteristic morphology of sickle cells with normal MCHC. The morphological behavior of Ca-loaded sickled cells as well as irreversibly sickled cells showed a major influence of increased hemoglobin concentration and extremely high internal viscosity. Constraint on cell morphology by putative membrane rigidity was not observed.

Topics: Anemia, Sickle Cell; Calcimycin; Calcium; Erythrocytes, Abnormal; Hemoglobin, Sickle; Humans; Nystatin; Osmosis; Oxygen; Potassium; Sodium; Water

An ionophore specific for divalent cations has been used to load normal erythrocytes and erythrocytes from patients with sickle cell anaemia (Hb SS disease), with small amounts of calcium. Such calcium accumulation leads to decreased cellular water, potassium, adenosine triphosphate (ATP), and osmotic fragility, all characteristics of irreversibly sickled cells (ISCs). In addition, calcium loading of Hb SS, but not normal, erythrocytes causes a marked decrease of haemoglobin oxygen affinity; another, and specific, hallmark of ISCs. Ionophore-induced accumulation of calcium by deoxygenated Hb SS erythrocytes also leads to temporary retention of sickled shape following reoxygenation, despite the absence of detectable intracellular haemoglobin S fibres. All these effects require calcium in the incubation medium and support the idea that increased intracellular calcium is important in the formation of ISCs in patients with Hb SS disease.

Topics: Anemia, Sickle Cell; Calcimycin; Calcium; Erythrocytes; Erythrocytes, Abnormal; Hemoglobin, Sickle; Humans; In Vitro Techniques; Microscopy, Electron; Microscopy, Electron, Scanning; Oxygen

Evaluation of the role of calcium in irreversible sickling has been approached by treating sickled cells with calcium and the ionophore A23187. A calcium-dependent stabilization of the sickled cell shape was observed after reoxygenation of cells in the presence of ionophore. At low calcium concentrations, this retention of sickled shape was maintained for periods up to 1 hr. However, the morphology of the oxygen-stable sickled cells was like that of deoxygenated sickle cells and significantly different from the characteristic morphology of native irreversibly sickled cells (ISCs). Because the stabilized cells did not fulfill the morphological criterion for ISCs, the shape-stabilizing effect of calcium in this system did not provide additional support for the hypothesis that calcium accumulation was the determining factor in ISC generation.

Topics: Anemia, Sickle Cell; Anti-Bacterial Agents; Calcimycin; Calcium; Egtazic Acid; Erythrocytes, Abnormal; Humans; Magnesium; Oxygen Consumption; Time Factors