Dipyridamole inhibits sickling-induced cation fluxes in sickle red blood cells

Sickling-induced cation fluxes contribute to cellular dehydration of sickle red blood cells (SS RBCs), which in turn potentiates sickling. This study examined the inhibition by dipyridamole of the sickling-induced fluxes of Na(+), K(+), and Ca(++) in vitro. At 2% hematocrit, 10 microM dipyridamole inhibited 65% of the increase in net fluxes of Na(+) and K(+) produced by deoxygenation of SS RBCs. Sickle-induced Ca(++) influx, assayed as (45)Ca(++) uptake in quin-2-loaded SS RBCs, was also partially blocked by dipyridamole, with a dose response similar to that of Na(+) and K(+) fluxes. In addition, dipyridamole inhibited the Ca(++)-activated K(+) flux (via the Gardos pathway) in SS RBCs, measured as net K(+) efflux in oxygenated cells exposed to ionophore A23187 in the presence of external Ca(++), but this effect resulted from reduced anion conductance, rather than from a direct effect on the K(+) channel. The degree of inhibition of sickling-induced fluxes was dependent on hematocrit, and up to 30% of dipyridamole was bound to RBC membranes at 2% hematocrit. RBC membrane content of dipyridamole was measured fluorometrically and correlated with sickling-induced flux inhibition at various concentrations of drug. Membrane drug content in patients taking dipyridamole for other clinical indications was similar to that producing inhibition of sickling-induced fluxes in vitro. These data suggest that dipyridamole might inhibit sickling-induced fluxes of Na(+), K(+), and Ca(++) in vivo and therefore have potential as a pharmacological agent to reduce SS RBC dehydration. (Blood. 2001;97:3976-3983)     

Partially oxygenated sickled cells: sickle-shaped red cells found in circulating blood of patients with sickle cell disease.

A previously uncharacterized type of sickled cell was found in venous blood of patients with sickle cell disease when blood was collected without exposure to air and fixed immediately with 1% glutaraldehyde solution equilibrated with 5% oxygen. These cells were either elongated, resembling irreversibly sickled cells (ISCs), or nonelongated, with a raisin-like shape. Both types assumed a normal discoidal shape upon full oxygenation. Since these cells exist only under partially oxygenated conditions, they are described as partially oxygenated sickled cells (POSCs). POSCs are morphologically distinct from partially deoxygenated sickled cells formed during deoxygenation by having rounded edges, while the latter have sharp edges. Transmission electron microscopy of POSCs revealed various amounts of misaligned Hb S polymers. Investigations in vitro demonstrated the formation of POSC-like cells by partial oxygenation of deoxygenated cells. Since POSCs contain intracellular fibers and sickle readily upon deoxygenation, they may have clinical and pathological significance.     

Mechanical properties of oxygenated red blood cells in sickle cell (HbSS) disease

Little data exist for the mechanical properties of individual irreversible or reversible sickle cells (ISC and RSC, respectively), nor is the process of ISC formation well understood. For oxygenated ISC and density-fractionated RSC, we have used micropipette techniques to measure cell surface area (SA) and volume (V), membrane shear elastic modulus (mu), time constant for viscoelastic shape recovery (tc), and hence to calculate membrane surface viscosity (eta = mu X tc). Volume loss associated with increasing cell density was accompanied by a proportionately smaller surface area decrease; SA/V ratio thus increased for denser cells, with ISC having the highest values. Membrane area loss by fragmentation must thus be accompanied by an accelerated decrease in cell volume. ISC had relatively rigid membranes (mu 130% above normal controls) and tc close to normal values, so that their effective membrane viscosity was more than double control. RSC had viscoelastic properties close to control, but showed wider variation between sickle cell donors and within samples. Measurements on density-separated RSC showed that, on average, mu was nearly constant, but that tc was longer for the densest cells, with their eta approaching ISC levels. A small subpopulation of RSC were found that had mu close to ISC values. Hypotonically swollen ISC (with internal hemoglobin concentration decreased to normal levels) retained their increased membrane stiffness but had markedly decreased tc, so that their eta approached normal values. The results show that elevated hemoglobin concentration influences the viscoelastic behavior of ISC and RSC, but that an irreversible change in membrane elasticity also occurs for ISC. These data suggest that ISC formation occurs via a two- stage process: (1) accelerated volume loss leading to increased cytoplasmic and effective membrane viscosity; (2) a sharp rise in membrane rigidity, presumably linked to membrane structural alteration.     

Vaso-occlusion by sickle cells: evidence for selective trapping of dense red cells

We have characterized the type of red cells from sickle cell patients that were trapped in the course of sickle-cell vaso-occlusion. In addition, the perfusion conditions (arterial perfusion pressure [Pa] and oxygen tension [PO2]) leading to experimentally induced vaso-occlusion in the artificially perfused, innervated mesocecum microvascular preparation were determined. Microvascular obstruction was induced by decrease in Pa; the lower the Pa, the greater the peripheral resistance as well as the extent of obstruction. The cells involved in the obstruction were recovered by vasodilation (secondary to denervation) and increase in Pa. Densitometric analysis of density gradient-separated infused and trapped cells was supplemented with morphological analysis to ascertain the involvement of density classes as well as morphological types seen in oxy and deoxy sickle blood. The trapping phenomenon was sensitive to PO2. Percentage of densest gradient classes, ie, fraction 3 (F3; mainly dense unsicklable SS discocytes [USDs]) and fraction 4 (F4; irreversibly sickled cells [ISCs] and the densest discocytes), showed a significant increase in trapping when perfusion was switched from oxy to deoxy perfusate. Morphological analysis revealed that unsicklable SS discocytes are more effectively trapped when deoxygenated. The deoxygenation of infused cells did not further change the percentage of ISCs trapped, suggesting that ISCs are equally capable of sequestration in the oxy and the deoxy states. The venous effluent showed a selective and significant depletion of dense cells (F4) and ISC counts at all Pa. We conclude that the progressive obstruction of the microcirculation by sickle cells involves selective sequestration of the densest classes of cells and that this mechanism might explain their partial disappearance during painful sickle cell crisis.     

The monovalent cation leak in overhydrated stomatocytic red blood cells results from amino acid substitutions in the Rh-associated glycoprotein

Overhydrated hereditary stomatocytosis (OHSt) is a rare dominantly inherited hemolytic anemia characterized by a profuse membrane leak to monovalent cations. Here, we show that OHSt red cell membranes contain slightly reduced amounts of Rh-associated glycoprotein (RhAG), a putative gas channel protein. DNA analysis revealed that the OHSt patients have 1 of 2 heterozygous mutations (t182g, t194c) in RHAG that lead to substitutions of 2 highly conserved amino acids (Ile61Arg, Phe65Ser). Unexpectedly, expression of wild-type RhAG in Xenopus laevis oocytes induced a monovalent cation leak; expression of the mutant RhAG proteins induced a leak about 6 times greater than that in wild type. RhAG belongs to the ammonium transporter family of proteins that form pore-like structures. We have modeled RhAG on the homologous Nitrosomonas europaea Rh50 protein and shown that these mutations are likely to lead to an opening of the pore. Although the function of RhAG remains controversial, this first report of functional RhAG mutations supports a role for RhAG as a cation pore.     

Membrane protein interactions in sickle red blood cells: evidence of abnormal protein 3 function

The pattern of membrane abnormalities in sickle red blood cells suggests that sickle hemoglobin damages membrane proteins. We have previously shown a functional defect in sickle ankyrin, poor spectrin- binding ability. Here we examine the other major binding interactions of sickle membrane proteins including spectrin self-association, binding of ankyrin and protein 4.1 to protein 3, and the formation of the spectrin-actin-protein 4.1 complex. We found that sickle spectrin was normal in self-association and ability to participate in the spectrin-actin-protein 4.1 complex. Sickle protein 4.1 bound normally to protein 3 and formed normal complexes with actin and spectrin, even when sickle spectrin was used. The only major abnormality we found was a reduced ability of sickle protein 3 to bind ankyrin. This functional defect could not be explained experimentally on the basis of cysteine modification or enhanced tyrosine phosphorylation. We conclude that damage of sickle membrane proteins is not a diffuse scattershot process, but is largely confined to regions near membrane-associated hemoglobin, the spectrin-binding domain of ankyrin and the ankyrin- binding domain of protein 3. The mechanism and consequences of this damage continues to be investigated.     

Morphological features of red blood cells in subjects with sickle cell trait: changes during exercise.

Occasional complications and even death in subjects with sickle cell trait have been attributed to severe physical exertion. However, the extent to which sickling actually occurs during exercise has not been reported. This study examined the red blood cell morphological features immediately following near maximal upright graded bicycle exercise in five asymptomatic black subjects with hemoglobin AS. Exercise produced minimal sickling in vivo, which was not proportional to the intensity of exercise. The amount of sickling in vivo was small in comparison to that observed in the presence of severe hypoxia in vitro, never exceeding 0.75%. in seven normal subjects with hemoglobin AA, exercise did not cause changes in red blood cell morphological features. We conclude that exercise may initiate sickling in subjects with sickle cell trait.     

Elastic properties of stored red blood cells from sickle trait donor units

BACKGROUND AND OBJECTIVES: Red blood cells (RBCs) from patients with sickle cell disease present reduced deformability. The aim of this study was to analyse the elasticity of stored RBCs from patients with the sickle cell trait (AS). MATERIALS AND METHODS: The cell elasticity was studied, using laser optical tweezers, on storage days 1, 14, 21, 28 and 35. RESULTS: The elasticity of RBC from AS units stored for 1, 14 and 21 days was significantly greater compared with that of control RBC cells stored for the same time-period. More than 30% of the cells from AS units stored for 28 or 35 days were very rigid and escaped from the optical trap. CONCLUSIONS: RBCs became rigid during storage, suggesting that haemoglobin S might compromise the cell elasticity.     

Hypoxic vasodilation by red blood cells: evidence for an s-nitrosothiol-based signal

Red blood cells (RBCs) have been ascribed an essential role in matching blood flow to local metabolic demand during hypoxic vasodilation. The vasodilatory function of RBCs evidently relies on the allosteric properties of hemoglobin (Hb), which couple the conformation of Hb to tissue oxygen tension (Po(2)) and thereby provide a basis for the graded vasodilatory activity that is inversely proportional to Hb oxygen saturation. Although a large body of evidence indicates that the Po(2)-coupled allosteric transition from R (oxy)-state to T (deoxy)-state subserves the release from Hb of vasodilatory nitric oxide (NO) bioactivity, it has not yet been determined whether the NO-based signal is a necessary and sufficient source of RBC-mediated vasoactivity and it has been suggested that ATP or nitrite may also contribute. We demonstrate here by bioassay that untreated human RBCs rapidly and substantially relax thoracic aorta from both rabbit and mouse at low Po(2) (approximately 1% O(2)) but not at high Po(2) (approximately 21% O(2)). RBC-mediated vasorelaxation is inhibited by prior depletion of S-nitroso-Hb, potentiated by low-molecular-weight thiols, and dependent on cGMP. Furthermore, these relaxations are largely endothelium-independent and unaffected by NO synthase inhibition or nitrite. Robust relaxations by RBCs are also elicited in the absence of endothelial, neuronal or inducible NO synthase. Finally, contractions that appear following resolution of RBC-mediated relaxations are dependent on NO derived from RBCs as well as the endothelium. Our results suggest that an S-nitrosothiol-based signal originating from RBCs mediates hypoxic vasodilation by RBCs, and that vasorelaxation by RBCs dominates NO-based vasoconstriction under hypoxic conditions.     

Temperature dependence and bidirectional cation fluxes in red blood cells from spontaneously hypertensive rats

The net passive influx of Na+ and efflux of K+ (orthodirection) through the red blood cell membranes from spontaneously hypertensive rats (SHR) were observed to be significantly higher (p less than 0.05) than those of three strains of normotensive rats when the measurements were made at 4 degrees C. Similar comparative studies, carried out at 37 degrees C, in the absence or presence of ouabain, showed no difference in these fluxes through this membrane from SHR compared to those from Wistar-Kyoto (WKY) rats, one of the normotensive strains. A study was undertaken to determine the temperature at which the greater cation fluxes in SHR red blood cells occurred. The net fluxes of Na+ and K+ decreased as the temperature was reduced from 37 degrees to 15 degrees C, but a paradoxical increase in the fluxes was observed as the temperature was decreased from 15 degrees to 4 degrees C. Only with this temperature shift (15 degrees to 4 degrees C) was the increase in flux significantly greater in SHR than in WKY cells. Subsequent studies were designed to determine whether the difference in the transport systems of red blood cells of SHR and WKY could be observed in fluxes of these cations in either direction across the membrane. For "reverse direction" flux studies, red blood cells were loaded with Na+ (to 130 mEq/liter cell water) and depleted of K+ (to 30 mEq/liter cell water) by incubation with the ionophore monensin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deoxygenation-induced cation fluxes in sickle cells: II. Inhibition by stilbene disulfonates

Deoxygenation-induced cation movements in sickle cells were inhibited 80% to 85% by the anion transport inhibitor, 4,4'-diisothiocyano-2,2'disulfostilbene (DIDS). Morphologic sickling was not altered by DIDS treatment, demonstrating that morphologic sickling was not sufficient to produce cation leaks in sickle cells. DIDS inhibition of deoxygenation-induced cation flux was not affected when l- replaced Cl-, indicating that conductive anion movements did not limit cation flux in deoxygenated cells treated with DIDS. Inhibition was irreversible after preincubation with DIDS at 37 degrees C for 20 minutes, and was not affected by the oxygenation state of cells at the time of drug exposure. Sulfate self-exchange was inhibited at lower DIDS concentrations than was deoxygenation-induced flux. Incubation of cells with DIDS at 4 degrees C produced progressive blockade of sulfate exchange, but did not alter deoxygenation-induced cation fluxes. Other stilbene disulfonates, including compounds incapable of covalent reactions, also inhibited deoxygenation-induced cation movements, although several other inhibitors of anion exchange did not. Dissociation of the inhibition of anion exchange and deoxygenation-induced cation flux indicates that the DIDS effect on deoxygenation-induced cation movements does not involve the well-characterized stilbene binding site of the anion exchanger. These data provide evidence for a membrane constituent on the external surface of oxygenated sickle cells capable of interacting with DIDS to prevent the increase in cation permeability associated with sickling.     

Integrin-associated protein is an adhesion receptor on sickle red blood cells for immobilized thrombospondin

The adhesive protein thrombospondin (TSP) potentially mediates sickle (SS) red blood cell (RBC) adhesion to the blood vessel wall, thereby contributing to vaso-occlusive crises in sickle cell disease. We previously reported that SS RBCs bind to immobilized TSP under flow conditions, whereas normal (AA) red cells do not. However, the SS RBC receptors that mediate this interaction are largely unknown. Here it is reported that integrin-associated protein (IAP), or CD47, mediates the adhesion of these cells to immobilized TSP under both flow and static conditions. A peptide derived from the C-terminal IAP binding site of TSP also supports sickle cell adhesion; adhesion to this peptide or to TSP is inhibited specifically by the anti-IAP monoclonal antibody, 1F7. Furthermore, these data suggest that IAP on SS RBCs is structurally different from that expressed on AA RBCs but that IAP expression levels do not vary between AA and SS RBCs. This structural difference may contribute to the enhanced adhesion of SS RBCs to immobilized TSP. These results identify IAP as a TSP receptor on SS RBCs and suggest that this receptor and its binding site within TSP represent potential therapeutic targets to decrease vaso-occlusion. (Blood. 2001;97:2159-2164)     

Normalizing effects of plasma on altered cation transport of red blood cells from essential hypertensive patients

Net Na+ and K+ fluxes were measured in Na+-loaded red cells from 19 normotensive control subjects, 22 essential hypertensive patients, and 8 secondary hypertensive patients. The ratio of Na+/K+ net fluxes was significantly lower in essential hypertensive patients than in normotensive control subjects. However, by the addition of the patients' own plasma, the net Na+ efflux rate was significantly increased in essential hypertensive patients, which caused the increment in the ratio of Na+/K+ net fluxes. This resulted in disappearance of the difference between normotensive and hypertensive subjects in the ratio of cation fluxes. It was possible that the abnormalities of cation transport in red cells from essential hypertensive patients might be compensated for by humoral factors in the plasma.     

Endothelial Lu/BCAM glycoproteins are novel ligands for red blood cell alpha4beta1 integrin: role in adhesion of sickle red blood cells to endothelial cells

The Lutheran (Lu) blood group and basal cell adhesion molecule (BCAM) antigens are both carried by 2 glycoprotein isoforms of the immunoglobulin superfamily representing receptors for the laminin alpha(5) chain. In addition to red blood cells, Lu/BCAM proteins are highly expressed in endothelial cells. Abnormal adhesion of red blood cells to the endothelium could potentially contribute to the vaso-occlusive episodes in sickle cell disease. Considering the presence of integrin consensus-binding sites in Lu/BCAM proteins, we investigated their potential interaction with integrin alpha(4)beta(1), the unique integrin expressed on immature circulating sickle red cells. Using cell adhesion assays under static and flow conditions, we demonstrated that integrin alpha(4)beta(1) expressed on transfected cells bound to chimeric Lu-Fc protein. We showed that epinephrine-stimulated sickle cells, but not control red cells, adhered to Lu-Fc via integrin alpha(4)beta(1) under flow conditions. Antibody-mediated activation of integrin alpha(4)beta(1) induced adhesion of sickle red cells to primary human umbilical vein endothelial cells; this adhesion was inhibited by soluble Lu-Fc and vascular cell adhesion molecule-1 (VCAM-1)-Fc proteins. This novel interaction between integrin alpha(4)beta(1) in sickle red cells and endothelial Lu/BCAM proteins could participate in sickle cell adhesion to endothelium and potentially play a role in vaso-occlusive episodes.     

The intriguing contribution of white blood cells to sickle cell disease - a red cell disorder

Sickle cell disease (SCD) is characterized by a point mutation that replaces adenine with thymidine in the sixth codon of the beta-globin gene, a unique morphological abnormality of red blood cells, vaso-occlusion with ischaemic tissue injury, and susceptibility to infections. Vascular lumen obstruction in SCD results from interaction of erythrocytes, leukocytes, platelets, plasma proteins, and the vessel wall. The disease phenotype is a product of various genes and environmental factors acting in concert with the protein lesion underlying the red cell anomaly. The severity of SCD increases with leukocyte count. The biological basis and therapeutic implications of this relationship are discussed. Leukocytes contribute to SCD by adhering to blood vessel walls and obstructing the lumen, aggregating with other blood cells with more effective blockage of the lumen, stimulating the vascular endothelium to increase its expression of ligands for adhesion molecules on blood cells, and causing tissue damage and inflammatory reaction which predispose to vaso-occlusion. Patients with impaired ability of leukocytes to kill microbes are more prone to infections; which precipitate sickle cell crisis. Reduction of leukocyte count ameliorates SCD. Similarly, targeted blockade or reduced synthesis of specific leukocyte adhesion molecules and their ligands might confer clinical benefit in SCD.     

Strong evidence for a weakly oxygenated ocean–atmosphere system during the Proterozoic

Earth’s surface has undergone a protracted oxygenation, which is commonly assumed to have profoundly affected the biosphere. However, basic aspects of this history are still debated—foremost oxygen (O₂) levels in the oceans and atmosphere during the billion years leading up to the rise of algae and animals. Here we use isotope ratios of iron (Fe) in ironstones—Fe-rich sedimentary rocks deposited in nearshore marine settings—as a proxy for O₂ levels in shallow seawater. We show that partial oxidation of dissolved Fe(II) was characteristic of Proterozoic shallow marine environments, whereas younger ironstones formed via complete oxidation of Fe(II). Regardless of the Fe(II) source, partial Fe(II) oxidation requires low O₂ in the shallow oceans, settings crucial to eukaryotic evolution. Low O₂ in surface waters can be linked to markedly low atmospheric O₂—likely requiring less than 1% of modern levels. Based on our records, these conditions persisted (at least periodically) until a shift toward higher surface O₂ levels between ca. 900 and 750 Ma, coincident with an apparent rise in eukaryotic ecosystem complexity. This supports the case that a first-order shift in surface O₂ levels during this interval may have selected for life modes adapted to more oxygenated habitats.

It is widely accepted that atmospheric oxygen (O₂) first accumulated to appreciable levels early in the Proterozoic Eon (2500 to 541 Ma), over a time interval known as the Great Oxidation Event (1, 2). A subsequent stepwise increase in atmospheric O₂ has been hypothesized to have paved the way for the emergence of animal life at some point in the late Proterozoic (3). However, the atmospheric O₂ levels (pO₂) of the ocean–atmosphere system during Earth’s intervening history (i.e., the middle Proterozoic) are highly debated: Despite suggestions that pO₂ remained relatively low during this time (4–6), recent work has argued that the O₂ demands of complex eukaryotes may have been exceeded well before their evolution (e.g., refs. 7–10). Therefore, the nature of the relationships between surface oxygenation and the evolutionary ecology of early complex life remain contentious (8, 11). Because surface redox conditions can affect the geochemistry of contemporaneous sediments, several geochemical indices from ancient sedimentary rocks have been employed as proxies for mid-Proterozoic atmospheric pO₂, predominantly the abundance or isotopic composition of redox-sensitive trace metals in marine carbonate rocks or shales (e.g., refs. 2, 6, 9, 10, 12). Atmospheric pO₂ estimates from these proxies are often characterized by large uncertainties, suffer from apparently conflicting inferred pO₂ histories, or both. A robust, mechanistically understood and sensitive proxy for mid-Proterozoic O₂ levels is therefore required.Iron (Fe) in seawater is soluble under anoxic conditions [as Fe(II)] and poorly soluble under oxic conditions [precipitating as Fe(III) oxyhydroxides]. Although oxidation of Fe(II) can also occur under anoxic conditions (via photo-oxidation or anoxygenic photosynthesis; ref. 13), the rate and extent of Fe(II) oxidation in aqueous environments—either abiotic or biologically mediated—will depend greatly on the abundance of O₂. Therefore, secular records of marine Fe(II) oxidation can potentially be used to track O₂ levels in seawater. At the low surface O₂ levels characteristic of early Earth, marine Fe(II) availability would have been comparatively high (sourced from hydrothermal inputs and the redox cycling of continental Fe), and partial oxidation of this seawater Fe(II) reservoir would have been common (14, 15). At higher O₂ levels characteristic of the modern oceans and atmosphere, any Fe(II) inputs to surface waters are quantitatively oxidized (16).Based upon the kinetics of Fe(II) oxidation and the associated Fe isotope systematics (16–18), it is possible to estimate the abundance of O₂ in seawater based upon the Fe isotope composition of chemical sediments (14). The partial oxidation of dissolved Fe(II) can lead to equilibrium fractionation whereby the produced Fe(III) (oxyhydr)oxides are enriched in ⁵⁶Fe by up to 1.0 to 3.2‰ (reported as the ratio of ⁵⁶Fe/⁵⁴Fe relative to the IRMM-014 standard reference material in parts per thousand; δ⁵⁶Fe) (17, 18). More rapid oxidation effectively lowers this overall fractionation (14, 18), and quantitative oxidation produces Fe(III) oxyhydroxides with an Fe isotope composition that mirrors that of the Fe(II) source. Biologically mediated Fe(II) oxidation (facilitated by anoxygenic phototrophs under anoxic conditions or microaerophilic chemotrophs under suboxic conditions) results in similar Fe isotope fractionations to that of abiotic Fe(II) oxidation (18) (SI Appendix). Compared to paleoredox proxies based on trace element geochemistry, the Fe isotopic composition of Fe-rich chemical sedimentary rocks is expected to be strongly rock-buffered, less likely to be affected by detrital contamination, and resistant to postdepositional alteration. Therefore, Fe-rich rocks can serve as robust Fe isotope archives in deep time (14, 18, 19) and can be used to constrain the redox state of ancient seawater.     

Evaluation of extracorporeal alkylation of red cells as a potential treatment for sickle cell anemia.

Nitrogen mustard and nor-nitrogen mustard inhibit sickling, but the concentrations required would be associated with unacceptable toxicity if these agents were administered to patients. Red cells could be treated extracorporeally and infused back into donors, if the alkylating agent could be removed or inactivated, if the treatment per se did not significantly shorten red cell survival, and if viable alkylated lymphocytes could be eliminated from the treated blood. To estimate whether these conditions could be met in a clinical trial, red cells from four dogs were alkylated at 6-wk intervals. No toxic reactions were observed, although not all nor-nitrogen mustard was removed by the washing procedure. Red cell survival was shortened to about half that of control cells, using concentrations of alkylating agent which reduce sickling by 50%. Lymphocytes from treated blood could still exclude trypan blue, but could not be shown to circulate after reinfusion into donor dogs. If alkylating agents are used to treat patients' cells, inhibition of sickling may outweigh the shortening of red cell life span induced by the treatment; blood should probably be irradiated before infusion to avoid administration of alkylated and potentially mutated, but viable, lymphocytes.     

Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation

Background

Cryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky.

Design and Methods

Cold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient’s erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient’s erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed.

Results

In the cold, the cryohydrocytosis patient’s erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO₃-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na⁺/K⁺ exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K⁺ uptake. Unidirectional K⁺ influx measurements showed that the patient’s cells have abnormally high activities of the cation-proton exchanger and the K⁺,Cl⁻ co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient’s erythrocytes differed from that of healthy donors.

Conclusions

These results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis.