Robert M. Bookchin

Evidence for a direct reticulocyte origin of dense red cells in sickle cell anemia.

To explore our hypothesis of a direct reticulocyte origin of irreversibly sickled cells (ISCs), we fractionated light, reticulocyte-rich, and discocyte-rich sickle anemia red cells on Stractan gradients, and examined the effects of deoxygenation-induced sickling, external Ca2+, acidification, and replacing external Na+ by impermeant N-methyl-D-glucamine (NMG+). Sickling permeabilized light reticulocyte-rich cells to cations (Na+, K+, and Ca2+) more than discocytes; without external Ca2+, Na+ influx matched K+ efflux, with stable cell volume; with Ca2+, many light, low hemoglobin (Hb) F reticulocytes dehydrated rapidly (preventable by quinine, a Ca2(+)-dependent K+ channel inhibitor). Acidification of oxygenated discocytes (high mean Hb F) and reticulocyte-rich fractions yielded denser, reticulocyte-enriched cells with lower Hb F (as in light reticulocyte or dense ISC-rich fractions). Light cells shrank when NMG+ replaced Na+, supporting predictions of a Na(+)-dependent volume control system. Demonstration of sickling-induced, Ca2(+)-dependent dehydration of Hb F-free reticulocytes, and conservation of acid-stimulated K:Cl cotransport among low Hb F, reticulocyte-enriched cells in discocyte fractions support the hypothesis. Ancillary new findings included heparin stimulation of sickling-induced Na+ and K+ permeabilizations, and Ca2+ inhibition of the Na+ leak.

Ligand-induced conformational dependence of hemoglobin in sickling interactions☆

Abstract It is generally accepted that the deoxy-conformation is required for the polymerization of hemoglobin S which results in gelation and sickling, and that Hb F does not interact with Hb S. Gelation experiments performed on mixtures of deoxy-Hb S or deoxy-Hb C Harlem ( α 2 β 6val,73Asn with various cyanmethemoglobins (which have a fixed oxy-confornation regardless of the presence of oxygen) show that (1), although cyanmet-Hb S does not gel on deoxygenation, cyanmet-Hb A participates equally as well as deoxy-Hb A does in gelation with deoxy-Hb S; this indicates that the deoxy-conformation is necessary for the Hb contributing the β 6Val -determined binding site, but is not required for the participatory role of non-S hemoglobins in gelation; (2) unlike deoxy-Hb F, cyanmet-Hb F does participate in gelation with deoxy-Hb S, as well as do deoxy- or cyanmet-Hb A; this constitutes a surprising reversal of the conformational dependence seen with Hb S in gelation, and suggests that the non-S hemoglobins provide complementary and/or secondary interaction sites whose conformational requirements are different from that of the β 6Val -determined site; (3) deoxy-Hb C Harlem (which alone interacts much less than pure deoxy-Hb S, but gels in mixtures with deoxy-Hb A as readily as does deoxy-Hb S) does not participate in gelation with cyanmet-hemoglobins A, F or S; this (together with earlier findings that Hb Korle-Bu ( α 2 β 2 73Asn ) participates very little in gelation with Hb S or Hb C Harlem ) suggests that β 73Asn may interfere with gelation in more than one manner, depending on the conformational state of the non-S hemoglobin and on whether the substitution is present on the same chain as the primary ( β 6Val ) site or on a separate β chain. In view of recent ultrastructural data, theoretical considerations of Pauling concerning liquid crystals, and model building, the present data are viewed in terms of a suggested polymeric structure: a packed helix with a low pitch, having about six molecules per turn, which permits the existence of primary and secondary interacting sites.

The binding of hemoglobin to membranes of normal and sickle erythrocytes.

The binding of hemoglobins A, S, and A2 to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with 14C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A2, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4-22 degrees C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied. An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites. These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.

Effects of deoxygenation on active and passive Ca2+ transport and on the cytoplasmic Ca2+ levels of sickle cell anemia red cells.

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.

A mathematical model of the volume, pH, and ion content regulation in reticulocytes. Application to the pathophysiology of sickle cell dehydration.

We developed a mathematical model of the reticulocyte, seeking to explain how a cell with similar volume but much higher ionic traffic than the mature red cell (RBC) regulates its volume, pH, and ion content in physiological and abnormal conditions. Analysis of the fluxbalance required by reticulocytes to conserve volume and composition predicted the existence of previously unsuspected Na(+)-dependent Cl- entry mechanisms. Unlike mature RBCs, reticulocytes did not tend to return to their original state after brief perturbations. The model predicted hysteresis and drift in cell pH, volume, and ion contents after transient alterations in membrane permeability or medium composition; irreversible cell dehydration could thus occur by brief K+ permeabilization, transient medium acidification, or the replacement of external Na+ with an impermeant cation. Both the hysteresis and drift after perturbations were shown to depend on the pHi dependence of the K:Cl cotransport, a major reticulocyte transporter. This behavior suggested a novel mechanism for the generation of irreversibly sickled cells directly from reticulocytes, rather than in a stepwise, progressive manner from discocytes. Experimental tests of the models predictions and the hypothesis are described in the following paper.

Deoxygenation permeabilizes sickle cell anaemia red cells to magnesium and reverses its gradient in the dense cells.

1. Our findings of a low total magnesium content in the dense fraction (over 1.118 g ml‐1) of sickle cell anaemia (SS) red cells seemed inconsistent with the low Mg2+ permeability and outward Mg2+ gradient seen in normal red cells, and prompted studies of the Mg2+ permeability and equilibria in the SS cells. 2. Deoxygenation and sickling induced Mg2+ permeabilization in SS cells, supporting non‐specificity of the sickling‐induced cation permeabilization, previously described for Na+, K+ and Ca2+. The extent of Mg2+ permeabilization was comparable in SS cells with normal or high density. 3. Compared with normal‐density SS cells and normal red cells, the dense SS cells showed a much larger increase in the fraction of ionized magnesium ([Mg2+]i) on deoxygenation, resulting in [Mg2+]i levels sufficient to reverse the normal inward direction of the transmembrane Mg2+ gradient. 4. The molar ratio of 2,3‐diphosphoglycerate (2,3‐DPG) to haemoglobin was markedly reduced in the dense SS cells. Since 2,3‐DPG and ATP are the main cytoplasmic Mg2+ buffers, their further reduction upon binding to deoxyhaemoglobin accounts for the high [Mg2+]i in the deoxygenated dense SS cells; the resulting outward electrochemical Mg2+ gradient, together with sickling‐induced Mg2+ permeabilization, could explain the decreased total magnesium content of these cells. 5. The above findings suggested that the documented low sodium pump fluxes in dense SS cells may result from an increased Mg2+:ATP ratio, which is known to inhibit Na(+)‐K+ exchange fluxes through the sodium pump. If so, deoxygenation, by increasing the Mg2+:ATP ratio, should inhibit the pump further, whereas increasing ATP should relieve the inhibition. Experiments designed to test this possibility showed that in these dense SS cells, the ouabain‐sensitive K(86Rb) influx was low in oxygenated cells, was reduced further by deoxygenation, but was substantially increased after treatment with inosine, pyruvate and phosphate to increase their organic phosphate pool. These results were thus consistent with such a mechanism for Na+ pump inhibition in the dense SS cells.

PATHOPHYSIOLOGY OF SICKLE CELL ANEMIA

The anemia results from the markedly shortened circulatory survival of SS cells, together with a limited erythropoietic response. Both independent properties of Hb S-polymerization of the deoxy-Hb and instability of the oxy-Hb-contribute to early red cell destruction by effects on the Hb and on the red cell membranes. The erythroid response is limited mainly by the low oxygen affinity of SS cells, caused by the polymer and the increased 2,3-DPG. But the worst culprits in these processes are the dense, dehydrated SS cells (including the ISCs), most of which are formed rapidly from non-Hb F-reticulocytes by cation transport mechanisms triggered by polymerization. Since the clinical consequences of microvascular occlusion far exceed those of anemia per se, measures to lessen the anemia must also inhibit polymerization and sickling.

Polymerisation of haemoglobin SA hybrid tetramers.

ERYTHROCYTE sickling and gelation of concentrated solutions of deoxyhaemoglobin (Hb) S (α2β26 Val) results from helical polymerisation of the tetramers, with a spatial orientation approximated by recent ultrastructural and optical studies. Earlier observations of the gelling behaviour of Hb S or Hb C Harlem (α2β26Va1, 73Asn) mixed with Hb A or other haemoglobins gave indirect evidence concerning intertetrameric contact sites, and we proposed that only one β6 valine-determined site was active per tetramer, the other β chain providing different polymer contacts1,2. Our arguments required that asymmetrical hybrids (for example α2βAβS) occurred in Hb mixtures; their long-suspected presence has recently been established3–5, but the dissociation equilibria by which these hybrids form (Fig. 1) hinders their isolation and direct testing of their ability to polymerise. To circumvent this difficulty we have prepared Hb SA hybrids cross-linked intratetramerically to prevent dissociation, and have found them capable of polymer and gel formation quite similar to that of Hb S.

Nitrogen mustard: An “in vitro” inhibitor of erythrocyte sickling

Abstract Sickling of erythrocytes from patients with sickle cell anemia can be completely inhibited by exposure to nitrogen mustard (HN2). Reaction of hemoglobin (Hb) S with increasing quantities of HN2 produces a progressive rise in the minimum concentration of hemoglobin required for gelation on deoxygenation. Quantities of HN2 sufficient to alter gelation cause no observable change in oxygen affinity or in heme:heme interaction. Preliminary studies of osmotic fragility and autohemolysis showed no abnormalities after erythrocytes were treated with HN2. Due to the known toxicity of HN2, no “in vivo” intravenous therapy with this agent can be considered.

Age Decline in the Activity of the Ca2+-sensitive K+ Channel of Human Red Blood Cells

The Ca2+-sensitive K+ channel of human red blood cells (RBCs) (Gardos channel, hIK1, hSK4) was implicated in the progressive densification of RBCs during normal senescence and in the mechanism of sickle cell dehydration. Saturating RBC Ca2+ loads were shown before to induce rapid and homogeneous dehydration, suggesting that Gardos channel capacity was uniform among the RBCs, regardless of age. Using glycated hemoglobin as a reliable RBC age marker, we investigated the age–activity relation of Gardos channels by measuring the mean age of RBC subpopulations exceeding a set high density boundary during dehydration. When K+ permeabilization was induced with valinomycin, the oldest and densest cells, which started nearest to the set density boundary, crossed it first, reflecting conservation of the normal age–density distribution pattern during dehydration. However, when Ca2+ loads were used to induce maximal K+ fluxes via Gardos channels in all RBCs (F max), the youngest RBCs passed the boundary first, ahead of the older RBCs, indicating that Gardos channel F max was highest in those young RBCs, and that the previously observed appearance of uniform dehydration concealed a substantial degree of age scrambling during the dehydration process. Further analysis of the Gardos channel age–activity relation revealed a monotonic decline in F max with cell age, with a broad quasi-Gaussian F max distribution among the RBCs.