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Protein tyrosine kinases (PTK) of the Src family are thought to suppress K-Cl cotransport (KCC) activity via negative regulation of protein phosphatases. However, some PTK inhibitors reduce KCC activity, suggesting opposite regulation by different PTK families. We have reported previously that deoxygenation of sickle cells stimulates KCC and activates Syk (a Syk family PTK), but not Lyn (an Src family PTK). In this study the same results were obtained when PTK activities were measured under the conditions used to measure KCC activity and which prevent any change in intracellular [Mg(2+)]. Methyl-2,5-dihydroxycinnamate (DHC), a PTK inhibitor, was more selective for Syk than Lyn, while staurosporine (ST), a broad-specificity protein kinase inhibitor, inhibited Lyn more than Syk. Deoxygenation or 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d] pyrimidine (pp2, a specific Src inhibitor) stimulated KCC independently. These effects were not additive and were inhibited by DHC. In contrast, ST-induced KCC activation was resistant to DHC, suggesting a different pathway of activation. Overall, these data indicate that Syk activity is required for KCC activation, either induced by deoxygenation of sickle cells, or mediated by Src inhibition in oxygenated cells, and that Syk and Src PTKs exert opposing and interconnected regulatory effects on the activity of the transporter.

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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)

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Sickle red blood cells (RBCs) become depleted of potassium, leading to dehydration and abnormally elevated cellular density. The increased sickling that results is important for both hemolysis and vasocclusion. In this study, sickle cells were subjected to high-speed centrifugation, and the bottom 15% were isolated. This procedure removed light cells and to a variable degree enriched cells that were denser than normal to produce a high-density-enriched (HDE) population of sickle cells. Autologous HDE cells from 3 subjects were labeled with biotin and re-infused. The following determinations were performed: (1) the survival and density changes of HDE cells; (2) the amount of fetal hemoglobin (HbF) in labeled cells after magnetic isolation; (3) the percentage of labeled F cells; (4) the percentage of labeled cells displaying external phosphatidylserine (PS). For patients with 3.5%, 4.5%, and 24% HbF in the HDE RBCs, the circulation half-time was 40, 80, and 180 hours, respectively. The percentage of HbF (measured in all 3 subjects) and of F cells (measured in 2 subjects) in labeled RBCs increased with time after re-infusion, indicating that HDE F cells have longer in vivo survival than HDE non-F cells. The percentage of PS(+), biotin-labeled HDE cells showed no consistent increase or decrease with time after re-infusion. These data provide evidence that HDE sickle cells, especially those that do not contain HbF, have a very short in vivo survival, and that the percentage of PS(+) cells in a re-infused HDE population does not change in a consistent manner as these cells age in the circulation.

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Sickle red blood cells (RBC) become dehydrated as a consequence of potassium loss. This process depends at least partly on deoxygenation and may be influenced by the presence of oxygenation/deoxygenation cycles and the frequency of cycling. In this study, sickle RBC were subjected to approximately 180 oxygenation/deoxygenation cycles during 4 hours to evaluate RBC dehydration with cycle periods more similar to in vivo cycles than those in previous studies. A continuous-flow, steady-state apparatus circulated a dilute RBC suspension through gas-permeable silicone tubing with segments that were exposed to either nitrogen or ambient oxygen. The percentage of sickling and partial pressure of oxygen were measured by means of sampling ports in the deoxygenation and oxygenation regions. The density increase (dehydration) of young (transferrin receptor-positive) and mature (transferrin receptor-negative) RBC and the requirements for calcium and chloride were evaluated. Density increase correlated with the percentage of sickled cells at the deoxygenation sampling port and was observed only in the presence of calcium, thereby implicating the calcium-dependent potassium channel (Gardos pathway). Density increase was not dependent on the presence of chloride, making it unlikely that KCl cotransport was an important pathway under these conditions. (Blood. 2000;95:2164-2168)

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Sickle red blood cells (RBC) are subject to a number of important cellular changes and selection pressures. In this study, we validated a biotin RBC label by comparison to the standard 51Cr label, and used it to study changes that occur in sickle cells as they age. Sickle RBC had a much shorter lifespan than normal RBC, but the two labels gave equivalent results for each cell type. A variable number of sickle, but not normal, RBC disappeared from the circulation during the first few hours after reinfusion. The number of biotinylated sickle reticulocytes was decreased by 50% after 24 h and 75% after 48 h, with a gradual decrease in the amount of reticulum per cell. The labeled sickle cells exhibited major density increases during the first 4-6 d after reinfusion, with smaller changes thereafter. A small population of very light, labeled sickle RBC was essentially constant in number after the first few days. Fetal hemoglobin (HbF) content was determined in isolated biotinylated sickle RBC after reinfusion, allowing an estimate of lifespan for RBC containing HbF (F cells) and non-F cells. The lifespan of sickle biotinylated RBC lacking HbF was estimated to be approximately 2 wk, whereas F cells survived 6-8 wk.

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KCl cotransport activated by swelling of sickle red blood cells (SS RBC)is inhibited by deoxygenation. Yet recent studies found a Cl--dependent increase in sickle reticulocyte density with cyclic deoxygenation. This study sought to demonstrate cotransporter stimulation by deoxygenation of SS RBC in isotonic media with normal pH. Low-density SS RBC exhibited a Cl--dependent component of the deoxygenation-induced net K+ efflux, which was blocked by two inhibitors of KCl cotransport, [(dihydroindenyl)oxy]alkanoic acid and okadaic acid. Cl--dependent K+ efflux stimulated by deoxygenation was enhanced 2.5-fold by clamping of cellular Mg2+ at the level in oxygenated cells using ionophore A-23187. Incubating cells in high external K+ or Rb+ minimized inhibition of KCl cotransport by internal Mg2+, and under these conditions deoxygenation markedly stimulated KCl cotransport in the absence of ionophore. Activation of KCl cotransport by deoxygenation of SS RBC in isotonic media at normal pH is consistent with the generalized dephosphorylation of membrane proteins induced by deoxygenation and activation of the cotransporter by a dephosphorylation mechanism. Na+/H+ exchange activity, known to be modulated by cytosolic Ca2+ elevation and cell shrinkage, remained silent under deoxygenation conditions.

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Erythrocyte dehydration is an important feature of sickle cell disease, leading to increased sickle hemoglobin polymerization and decreased red blood cell survival. Substantial in vivo dehydration appears to occur in reticulocytes or in an even younger subset of reticulocytes that are positive for transferrin receptor. Previous studies have suggested both sickling-dependent and sickling-independent components of dehydration for these cells. Two types of investigations are reported here. The first series of experiments explored the possibility that fetal hemoglobin (HbF) content influences the in vivo dehydration of very young, transferrin receptor-positive (T+) cells. These studies confirmed that in most patients the T+ cells in the densest fraction lacked HbF (T+ F-). However, T+ F- and T+ F+ cells appeared to have the same tendency to become moderately dense. The second type of investigation examined moderately dense T+ cells with normalized K+ content and determined the effect of HbF content on KCl cotransport-mediated dehydration in oxygenated incubations. Under these conditions, both T+ F- and T+ F+ cells had an equal tendency to become more dense by this pathway. Taken together, these studies indicate that at least some young sickle cells become moderately dense due to higher KCl cotransport activity independent of HbF content (and by inference, independent of sickling). However, to become very dense, it appears that further dehydration through a sickling-mediated pathway is required. We suggest that the dehydration of young sickle cells occurs in two steps, with the first dominated by KCl cotransport and the second having an important sickling-dependent component.

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The K+ efflux that mediates sickle-cell dehydration may occur through several pathways, including two with a high capacity for mediating rapid K+ loss, KCl cotransport and the Ca(2+)-dependent K+ channel [K(Ca2+)]. The rate and pathway of red blood cell (RBC) dehydration most likely depends on cell age and hemoglobin (Hb) composition, with the presence of HbF playing an important role. Oxygenated sickle RBCs have relatively stable cell volume during incubation in vitro, whereas deoxygenated cells become dehydrated, and therefore more dense, due to activation of one or more K+ efflux pathways. In this investigation, sickle RBCs were deoxygenated either continuously or in 15-minute cycles for 4 hours, and the density increases of very young, transferrin receptor-positive (TfR+) cells and the remaining TfR- cells were determined. The contribution of KCl cotransport was estimated by replacing Cl- with NO3-. K(Ca2+) was inhibited by removal of Ca2+ or addition of charybdotoxin (ChTX). For both continuous and cyclic deoxygenation, TfR+ cells had a greater density increase when compared with TfR- cells. The lower percentage of HbF found in the TfR+ population may contribute to this difference. With continuous deoxygenation, the density shift was decreased by inhibition of K(Ca2+), but not by inhibition of KCl cotransport. With cyclic deoxygenation, the density shift was decreased in an independent, additive manner by inhibition of both pathways. Thus, cyclic deoxygenation of sickle cells under these conditions appears to activate both K(Ca2+) and the KCl cotransporter.

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Cellular cation homeostasis in mouse erythrocytes with defective membrane skeletons was examined in three mouse mutants, hemolytic anemia (sphha/sphha), spherocytosis (sph/sph), and normoblastosis (nb/nb), and compared with reticulocytes produced by repetitive bleeding of congenic normal mice. To assess reticulocyte maturity, nucleic acid and transferrin receptor contents were measured by fluorescence flow cytometry; mutant cells were somewhat more mature than normal reticulocytes by these criteria. Red blood cell (RBC) sodium contents (Nac+) in homozygous sphha/sphha, sph/sph, and nb/nb animals were 30.1 +/- 0.9, 28.9 +/- 0.3, and 26.9 +/- 1.5 mmol/L cell, respectively, whereas cellular potassium (Kc+) was 102 +/- 2.6, 101 +/- 7.8, and 97.4 +/- 3.0. Nac+ and Kc+ in normal reticulocyte preparations were 11.3 +/- 0.7 and 123 +/- 10, respectively. Net Na+ and K+ fluxes in the presence of ouabain were markedly increased in mutant RBCs. Sodium uptake was 14.8 +/- 1.6, 15.4 +/- 3.3, and 14.7 +/- 3.1 mmol/L cell/h in sphha/sphha, sph/sph, and nb/nb mutants, respectively, whereas K+ loss was 17.0 +/- 4.0, 15.0 +/- 3.8, and 14.1 +/- 2.6. Normal mouse reticulocytes gained Na+ at a rate of 3.9 +/- 1.0 mmol/L cell/h and lost K+ at 6.0 +/- 2.1, rates indistinguishable from those in mature mouse RBCs. Potassium loss from sphha/sphha and nb/nb cells was not dependent on the presence of a Na+ gradient, and net cation movements were insensitive to bumetanide (sphha/sphha and nb/nb RBCs) and to chloride replacement with sulfamate (nb/nb cells). We conclude that mutant mouse RBCs with dysfunctional membrane skeletons have increased passive permeability to monovalent cations. These findings support a role of the membrane skeleton in the maintenance of the membrane permeability barrier and suggest that the abnormal permeability associated with human hereditary spherocytosis and elliptocytosis may be a consequence of the membrane skeleton defects reported in these disorders.

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Changes in a membrane sterol exchange of sickle red blood cells (SS RBC) induced by deoxygenation were studied using the fluorescent cholesterol analogue dehydroergosterol (DHE). DHE uptake by SS RBC membrane was measured by the incubation of SS RBC with small unilamellar vesicles (SUV) containing DHE. Deoxygenation of SS RBC, but not normal RBC, increased the rate of DHE uptake. DHE membrane content after 5 h of incubation with SUV in the cell-to-SUV ratio of 1:1 (mol lipid) was 16.25 +/- 0.94 and 12.22 +/- 0.85% of total sterol for deoxygenated and oxygenated cells, respectively. Membrane spicules isolated from these deoxygenated SS RBC had three-fold higher DHE content, suggesting that the increased sterol exchange was localized to spicules. When isolated spicules were incubated with DHE-SUV directly, 91 +/- 3% of membrane sterol was rapidly exchanged, in contrast to intact RBC, in which a maximum of 33% of sterol could be exchanged. The results suggest that spicule formation in SS RBC alters membrane cholesterol structure, such that a domain of cholesterol that is normally nonexchangeable becomes readily exchangeable with exogenous sterol.

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The ontogeny of Na(+)-K(+)-adenosinetriphosphatase (ATPase) mRNA in the mouse lung was examined, using alpha- and beta-isoform-specific probes in Northern blot assays and for in situ hybridization analysis. Northern blot assays demonstrated an increase in Na(+)-K(+)-ATPase expression in the perinatal period, peaking at birth (D1), with alpha 1- and beta 1-isoform levels reaching six to eight times adult levels. In situ alpha 1-isoform hybridization signals were localized primarily to developing airway epithelium and were most intense on D1. Postnatally, alpha 1-isoform hybridization signals persisted in airway epithelium, although progressively diminishing in intensity relative to perinatal levels. In developing alveolar regions, alpha 1-isoform hybridization signals remained slightly above background during this period. beta 1-Isoform hybridization signals increased dramatically during the perinatal period in both developing airway and alveolar epithelia. Postnatally, beta 1-isoform hybridization signals declined slightly in airway epithelium and developed a punctate pattern in alveolar epithelium. These data indicate that the perinatal increase in Na(+)-K(+)-ATPase expression observed in the developing mouse lung is localized primarily to epithelial structures and is therefore likely to be related to the changes in transepithelial ion and fluid transport known to occur in the perinatal period.

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Net cation movements were measured in low-density sickle red blood cells (SS RBC) in the presence and absence of oxygen. External Ca2+ (Ca2+o) partially inhibited deoxygenation-induced fluxes of both Na+ and K+. Deoxygenation-induced Na+ influx was reduced by 2 mM Ca2+o to 0.71 +/- 0.04 (SE) of its value in Ca(2+)-free solutions, whereas this ratio was 0.90 +/- 0.05 for K+ efflux (P < 0.01 by paired t-test). Because Ca2+o inhibited Na+ influx more than K+ efflux, net cation loss in deoxygenated SS RBC was higher in the presence of Ca2+o. In separate experiments, Ca2+o reduced deoxygenation-induced Na+ influx to 0.66 +/- 0.03 of its Ca(2+)-free value compared with 0.77 +/- 0.03 for Rb+ influx (P < 0.001), indicating relative selectivity of this effect for Na+ over Rb+. However, this effect is not specific for Ca2+ because other divalent cations also inhibited deoxygenation-induced Na+ and K+ fluxes. Under the conditions of these experiments, no evidence for K+ channel activation was found, indicating that K+ loss measured in deoxygenated SS RBC was mediated by the deoxygenation-induced pathway. These studies show that in the presence of Ca2+o deoxygenation-induced Na+ influx and K+ efflux are unbalanced. This pathway can, therefore, mediate cation loss and contribute directly to cellular dehydration in SS RBC.

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A subset of sickle cells becomes K(+)-depleted and dehydrated before or soon after leaving the bone marrow. These young cells may be identified in blood as transferrin receptor-positive (TfR+) dense reticulocytes. KCl cotransport, which is normally active in young erythroid cells with a maximum at pH 6.8, is a candidate pathway for K+ depletion of sickle reticulocytes. In this investigation, KCl cotransport activity was evaluated in young, TfR+ cells which had become dense in vivo and in age-matched cells which had retained normal hydration. Sickle erythrocytes were first separated into three primary density fractions, with care taken to preserve the in vivo hydration state. After normalization of intracellular hemoglobin concentration with nystatin, the cells were incubated at 37 degrees C for 20 min at pH 6.8 and 7.4. Before and after incubation, each primary fraction was separated into four secondary density fractions. The percentage of TfR+ cells in each secondary fraction was measured and a density distribution for TfR+ cells was determined for each primary fraction before and after incubation. The density shift during incubation was a measure of KCl cotransport. TfR+ cells from the denser primary fractions II and III had significantly more density shift than TfR+ cells from the light fraction I. Although the shifts were larger at low pH, differences between primary fractions were also observed at pH 7.4. These data indicate that the cells which become dense quickly in vivo have more KCl cotransport activity than those which remain light in vivo, and support this pathway as a primary mechanism for dehydration of young sickle cells.

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A subset of sickle cells have an increased density at the reticulocyte stage of development, indicating that they are either abnormally dense upon release from the bone marrow or become dense quickly in the circulation. These cells are of interest because they most likely have severely disrupted cation regulation and a short lifespan. Based on the distribution of fetal hemoglobin (HbF) in the density fractions of sickle red blood cells (RBCs) and in vitro studies of cellular K+ loss, it seems likely that HbF content is an important in vivo determinant of dense cell formation. In this study, we tested the hypothesis that young, dense cells have low HbF content. Sickle RBCs were first separated into light and dense fractions. Reticulocytes were isolated from unfractionated cells and from each density fraction with an immunomagnetic technique directed against transferrin receptors (TfR) and assayed for the percentage of HbF and K+/Hb ratio. TfR+ reticulocytes isolated from unfractionated cells had a much lower HbF content when compared with all the unfractionated RBCs. This is most likely caused by enrichment of F cells because of a longer circulation life span. Heavy TfR+ reticulocytes had a K+/Hb ratio similar to that measured in the entire dense population and contained very low levels of HbF, averaging 2.5% of the level in all RBCs, 11.7% of the level in all TfR+ reticulocytes, and 4.0% of the level in all dense RBCs. These findings suggest that TfR+ dense cells derive predominantly from non-F cells. Furthermore, the amount of HbF in the circulating dense cells suggests that many of these cells do not derive from the TfR+ dense cells.

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Sterols are not randomly distributed in membranes but appear to be localized in multiple kinetic domains. Factors that regulate these sterol domains are not well-understood. A recently developed fluorescence polarization assay that measures molecular sterol transfer [Butko, P., Hapala, I., Nemecz, G., of Schroeder, F. (1992) J. Biochem. Biophys. Methods 24, 15-37] was used to examine the mechanism whereby anionic phospholipids and liver sterol carrier protein-2 (SCP2) enhance sterol transfer. Two exchangeable and one very slowly or nonexchangeable sterol domain were resolved in phosphatidylcholine (POPC)/sterol small unilamellar vesicles (SUV). Inclusion of 10 mol % anionic phospholipids enhanced sterol exchange primarily by redistribution of sterol domain sizes rather than by alteration of half-times of exchange. This effect was dependent primarily on the percent content rather than the net charge per anionic phospholipid. In contrast, SCP2 simultaneously altered both the distribution of sterol molecules between kinetic domains and the exchange half-times of exchangeable sterol domains. The effects of SCP2 were much more pronounced when 10% acidic phospholipid was incorporated in the SUV. Compared to spontaneous sterol exchange, in the presence of 1.5 microM SCP2, the rapidly exchanging pool was increased by 36 to 330%, depending on the SUV phospholipid composition. Concomitantly, exchange half-times for rapidly and slowly exchangeable sterol were reduced by 60 to 98% for 1t1/2 and 14 to 85% for 2t1/2, respectively. The stimulatory effect of SCP2 was saturable and dependent both on protein concentration and on content of acidic phospholipids in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

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Structural domains of cholesterol and their regulation in the erythrocyte membrane are poorly understood. Dehydroergosterol fluorescence polarization change was used to continuously monitor the kinetics of sterol exchange and sterol domain size in erythrocyte ghost membranes. Direct correlation between molecular sterol exchange and steady-state dehydroergosterol fluorescence polarization measurements was obtained without separation of donor and acceptor membranes. Three important observations were made. First, sterol exchange between small unilamellar vesicles (SUV) with the same cholesterol/phospholipid ratio as the erythrocyte membrane (1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol = 1:1) was resolved into three kinetic cholesterol domains: 23 +/- 9% of total sterol was rapidly exchangeable, with t1/2 = 23 +/- 6 min; 59 +/- 9% of total sterol was slowly exchangeable, with t1/2 = 135 +/- 3 min; and 19 +/- 9% of total sterol was essentially nonexchangeable, with a t1/2 of days. Second, the substitution of erythrocyte ghosts for SUV as an acceptor significantly altered the kinetic parameters of sterol exchange from donor SUV, graphically showing that both the properties of the acceptor and spontaneous desorption of cholesterol from the donor SUV influenced spontaneous cholesterol transfer. Third, studies of exchange between erythrocyte ghosts revealed multiple kinetic pools of sterol differing from those in the SUV: 4 +/- 2% of total sterol was rapidly exchangeable, with t1/2 = 32 +/- 9 min; 29 +/- 3% of total sterol was very slowly exchangeable, with t1/2 = 23 +/- 7 h; and a surprisingly large 67 +/- 2% of total sterol was nonexchangeable, with a t1/2 of days.

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