ALBERT

Description: In the course of experiments designed to study the patterns and distribution of dehydration rates of RBCs permeabilized to K+, we made the surprising observation that isotonic dehydration of RBCs elicits a spontaneous rehydration process by activating a non-selective cation permeability pathway which is highly stimulated by elevated [Ca2+]i. RBCs suspended in plasma-like, high-Na+, low-K+ buffers at 37°C and uniformly permeabilized to K+ either with valinomycin or by maximal Gardos channel activation (with ionophore A23187 + Ca2+) sustained net loss of KCl and water. The dehydration was maximal in 4–6 min when 10 mM SCN− replaced 10 mM Cl− to avoid the anion permeability rate limitation. The extent and distribution of RBC hydration was followed in time by the changes in osmotic lysis curves or by flow cytometry (Advia 120 hematology system). In all experimental conditions tested (varying Hct (

Description: The plasma membrane calcium pump (PMCA) is the only active Ca2+ transporter in human red blood cells (RBCs). Previous measurements of maximal Ca2+ extrusion rates (Vmax) reported only mean values in the RBC population. Despite early evidence for differences in Ca2+ extrusion capacity among RBCs, the precise Vmax distribution remained unknown. It was important to characterize this distribution to assess the range and modality (uni- or multimodal) of PMCA Vmax variation and the likelihood of RBCs with elevated [Ca2+]i in the circulation participating in physiologic and pathologic processes. We report here the application of a new method to investigate the detailed distribution of PMCA Vmax activity in RBCs. The migrating profile of osmotic lysis curves was used to identify and quantify the fraction of cells that extrude a uniform Ca2+ load at different rates. The results revealed that RBCs from single donors have large variations in PMCA activity that follow a unimodal, broad distribution pattern consistently skewed toward higher Vmax values, suggesting an excess of cells with Vmax higher than the mean value. The method applied may provide a way of evaluating whether the observed variation in PMCA Vmax is related to cell age. (Blood. 2003;102:4206-4213)

Description: Phosphatidylserine (PS), exclusively present in the inner monolayer of the normal red blood cell (RBC) membrane, is exposed in subpopulations of sickle cells. PS-exposing RBCs were found predominantly among the densest and the very light sickle cells. Within the light RBC fraction, PS exposure was found on reticulocytes, transferrin receptor–expressing reticulocytes, and mature RBCs. The last subset contained low-density valinomycin-resistant RBCs, previously shown to have high Na+ and low K+content. This subpopulation contained the highest percentage of PS-exposing cells. The PS-exposing sickle cells did not show the sustained high cytosolic Ca++ levels that have been shown to activate scramblase activity. Data from this study indicate that PS exposure can occur at different stages in the life of the sickle RBC and that it correlates with the loss of aminophospholipid translocase activity, the only common denominator of the PS-exposing cells. The additional requirement of scramblase activation may occur during transient increases in cytosolic Ca++.

Description: Normal high-K+, low-Na+ RBCs, suspended in low-K+ media and permeabilized to K+ with valinomycin, become dehydrated from net loss of KCl and water. A very small fraction of light, normal RBC and larger fractions of light, sickle cell anemia (SCA) and beta-thalassemia RBC were found to be “valinomycin-resistant” (val-res) due to their Na+/K+ gradient dissipation (PNAS2000;97: 8050; BLOOD2000;96:24b). In thalassemia and SCA, although the primary lesions involve the globin genes, the major damage to the RBC membranes is mediated by oxidative stress. We previously showed (Cytometry2004;60:73) that thalassemic RBC have higher reactive oxygen species (ROS) and lower reduced glutathione (GSH) levels than normal RBC before or after in vitro oxidant stress (treatment with hydrogen peroxide). Here, we examined the oxidative status of val-res RBC from normal and beta-thalassemia major blood. RBC suspended in a plasma-like buffer containing 15 mM KCl and 10 mM valinomycin for 45 min were then layered on arabinogalactone (Larex) with density δ=1.091, and spun at 15,000 g for 30 min. Val-res cells were identified as the low density (δ

Description: Nitric oxide (NO) is produced from arginine by nitric oxide synthase (NOS) and is essential to the maintenance of vascular tone. Plasma arginine levels are reduced in sickle cell anemia patients, and we have previously reported that S+S− Antilles mice have lower plasma arginine than control mice (C57BL). Long term arginine supplementation (5% arginine in chow) restored plasma arginine levels in S+S− Antilles mice to that found in C57BL mice, and, in S+S− Antilles mice, MCHC and the percent high density red cells were reduced. Our observation that Ca++-activated K+ channel [K(Ca) channel or Gardos channel] activity is reduced in supplemented vs non-supplemented S+S− Antilles mice can account for reduced red cell density and dense cell formation (Blood99: 1103, 2002). The time course of arginine induced changes in red cell indices in sickle transgenic mice has not yet been studied. Using the Advia 120 Hematology System, we found that, within 7 days after the onset of arginine supplementation, MCHC* (CHCM in the Advia system) fell from 33.4±0.5 g/dL to 31.0±0.2 (mean ±SD, P37 g/dL) decreased from 10.5±4.3 % to 4.5±1.6 (P

Description: We described earlier a small fraction of sickle (SS) and normal RBC that resisted dehydration when K+-permeabilized in low-K+ media with valinomycin (val) or Ca2+ + A23187 (“valres” and “calres”, together “CVres”) (Bookchin et al, PNAS, 97:8045, 2000). Their resistance to dehydration was shown to result from their high-Na+ and low-K+ contents, but the mechanism of generation of these CVres RBCs and their (patho)- physiological relevance were unknown. We noted then that SS-valres RBCs included a large number of ISC shapes, suggesting that their dehydration and/or membrane damage might play a role. We report now that these RBCs also show a relatively high %Hb-F, as measured by HPLC, comparable to Hb F levels in the corresponding denser SS discocyte fractions; this suggested that the oldest SS RBCs, the SS F-cells, were also more likely to become CVres. Franco et al (Blood, 96:3610, 2000) reinfused biotin-labeled high-density-enriched SS RBCs and detected an older population of light, labeled RBCs, supporting our hypothesis that these cells are derived from the dense cell population and represent a terminal state. To test the possible age-related origin of normal calres and valres RBCs, we isolated these cells, now defined as those retaining a density lower than 1.117 after K+-permeabilization, by spinning the val- or Ca2+ + A23187-treated RBCs through diethylphthalate oil (D=1.117 g/ml) to separate the dehydrated cells (pellets) from the CVres cells (on top of the oil). The fraction of RBCs in pellets and on top of the oil was estimated in each sample from Hb measurements, and comprised ~0.2–0.4% (valres) or 1.0–5.0% (calres) of normal RBCs. The higher yield of calres RBCs was attributed to additional net Na+ gain by RBCs during the isolation procedures by activation of the non-selective cation permeability pathway Pcat induced by elevated [Ca2+]i (Bookchin et al, Blood, 104:439a, 2004). Their Hb A1c content was measured by HPLC to serve as a reliable age-marker (in non-diabetic normal RBCs). The fraction of Hb A1c was 7.08 ± 0.25%[SD] for valres and 8.19 ± 0.35% for calres RBCs (total RBC = 5.77 ± 0.25%), consistent with an advanced age of these CVres RBCs. We recently reported that normal RBCs show an age-dependent decline in Ca2+-pump Vmax (Lew et al, Blood, 102:4206, 2003). Together, these finding are consistent with the following mechanisms involved in CVres RBC generation: The age-decline in PMCA activity results in an increasing density of aging RBCs, by allowing intermittent episodes of [Ca2+]i elevations in the circulation, with transient Gardos channel activation and gradual dehydration by net KCl and water loss. The variable increase in [Ca2+]i together with the resulting RBC dehydration then activates Pcat, resulting in Na+ gain and further K+ loss, with a net gain in NaCl and water and cell swelling overtaking the previous dehydrated state. The resulting state of the cells mirrors the features of CVres RBCs. Our recent findings of generation of valres RBCs from normal RBCs following exposure to various oxidants in vitro (Amer et al, BBA, 1760:793, 2006) raises the possibility that oxidant damage to the RBC membranes may play a contributory role to the above age-related functional alterations, and we are currently testing for such effects.

Description: We recently demonstrated that the activity of the PMCA varies greatly among the RBCs in a normal blood sample (Lew et al., Blood102:4206,2003). To test the possibility that these variations might be related to cell age, we designed a new experimental protocol to separate RBCs with different PMCA Vmax, which used glycosylated hemoglobin (Hb) A1c as an age marker for the normal RBCs, and avoided Co2+ (used in our usual PMCA activity assay) which we found to interfere with Hb A1c measurements: The ionophore A21837 was used to generate a high, rapid and uniform [CaT]i in RBCs, which were suspended in a 90mM-K+ buffer to prevent RBC dehydration by Gardos channel activation. These RBCs were then washed in high-K+ buffer (ice-cold to inhibit the Ca2+ pump) with 1% albumin to remove the ionophore. At t=0, the washed and packed ionophore-free cells were delivered into 20 volumes of high-K+ buffer at 37oC to initiate Ca2+ extrusion by the PMCA, and then sampled at 15 sec intervals into 25 vol of an ice-cold, K+-free, isotonic buffer containing 10 mM SCN−; these conditions were designed to trap un-extruded Ca2+ and to elicit rapid dehydration of those cells which had not yet pumped out all their Ca2+ load. After ~ 40 min at 0oC each sample was spun; the packed RBCs were resuspended in 10 vol of the same buffer and spun again through diethylphthalate oil (D=1.117 g/ml) to separate the dehydrated RBCs (pellets) from the non-dehydrated cells. The fraction of cells in pellets and on top of the oil was estimated in each sample from Hb measurements, and their Hb A1c content was measured by HPLC. Initially, almost all RBCs, except those with the most vigorous pumps, were recovered in the pellets, but with time, the fraction of cells which had fully extruded their Ca2+ load and were recovered on top of the oil approached 100%. The progressively smaller pellets contained RBCs with progressively weaker pumps; their Hb A1c fraction increased with decrease in the yield of RBCs in the pellet. This inverse correlation between yield and Hb A1c fraction in the pellets was observed in all six experiments performed with RBCs from four donors. Since the decline in PMCA activity correlated directly with an increase in Hb A1c, the observed population variation in PMCA activity reflects an age-related decline in PMCA activity. Whether this decline is due to progressive glycosylation of of a lysine residue near the catalytic ATP domain on the pump ATPase (Gonzalez Flecha et al., J Membr Biol171:25,1999) or to other mechanisms, remains to be determined. The age-decline in PMCA activity may be responsible for the increasing density of aging RBCs, by allowing intermittent episodes of [Ca2+]i elevations in the circulation, with transient Gardos channel activation and gradual dehydration by net KCl and water loss.

Description: The Ca2+-activated K+ channels of human red blood cells (RBCs) (Gardos channels, hIK1, hSK4) can mediate rapid cell dehydration, of particular relevance to the pathophysiology of sickle cell disease. Previous investigations gave widely discrepant estimates of the number of Gardos channels per RBC, from as few as 1 to 3 to as many as 300, with large cell-to-cell differences, suggesting that RBCs could differ extensively in their susceptibility to dehydration by elevated Ca2+. Here we investigated the distribution of dehydration rates induced by maximal and uniform Ca2+ loads in normal (AA) and sickle (SS) RBCs by measuring the time-dependent changes in osmotic fragility and RBC volume distributions. We found a remarkable conservation of osmotic lysis and volume distribution profiles during Ca2+-induced dehydration, indicating overall uniformity of dehydration rates among AA and SS RBCs. In light of these results, alternative interpretations were suggested for the previously proposed low estimates and heterogeneity of channel numbers per cell. The results support the view that stochastic Ca2+ permeabilization rather than Gardos-channel variation is the main determinant selecting which SS cells dehydrate through Gardos channels in each sickling episode. (Blood. 2005;105:361-367)

Description: Interaction of hemoglobin S polymers with the red blood cell (RBC) membrane induces a reversible increase in permeability (“Psickle”) to (at least) Na+, K+, Ca2+, and Mg2+. Resulting changes in [Ca2+] and [H+] in susceptible cells activate 2 transporters involved in sickle cell dehydration, the Ca2+-sensitive K+ (“Gardos”) channel (KCa) and the acid- and volume-sensitive K:Cl cotransport. We investigated the distribution of Psickle expression among deoxygenated sickle cell anemia (SS) RBCs using new experimental designs in which the RBC Ca2+ pumps were partially inhibited by vanadate, and the cells' dehydration rates were detected as progressive changes in the profiles of osmotic fragility curves and correlated with flow cytometric measurements. The results exposed marked variations in (sickling plus Ca2+)–induced dehydration rates within populations of deoxygenated SS cells, with complex distributions, reflecting a broad heterogeneity of their Psickle values. Psickle-mediated dehydration was inhibited by clotrimazole, verifying the role of KCa, and also by elevated [Ca2+]o, above 2 mM. Very high Psickle values occurred with some SS discocytes, which had a wide initial density (osmotic resistance) distribution. Together with its previously shown stochastic nature, the irregular distribution of Psickle documented here in discocytes is consistent with a mechanism involving low-probability, reversible interactions between sickle polymers and membrane or cytoskeletal components, affecting only a fraction of the RBCs during each deoxygenation event and a small number of activated pathways per RBC. A higher participation of SS reticulocytes in Psickle-triggered dehydration suggests that they form these pathways more efficiently than discocytes despite their lower cell hemoglobin concentrations.