- Email: [email protected]
Membrane Transport in Sickle Cell Disease
Gibson and Ellory
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Membrane Transport in Sickle Cell Disease Submitted 03/18/02 (Communicated by J. Hoffman, M.D., 03/21/02)
J. S. Gibson1 and J. C. Ellory2 ABSTRACT: We have reviewed here a number of membrane transport events in red cells from normal individuals and sickle cell patients which respond to changes in O2 tension. Some deoxygenation-induced changes in membrane permeability are unique to HbS cells and contribute to their dehydration and subsequent sickling. Polymerization of HbS, or specific oxidant damage (or altered redox potential), is a likely factor underlying the abnormal behavior. The key regulatory sites within the membrane or associated proteins remain uncertain and their identity will form the focus of future research. A model for sickle cell dehydration is presented. Inhibition of these permeability changes represents possible avenues for future chemotherapy to ameliorate the condition. © 2002 Elsevier Science (USA)
OVERVIEW OF SICKLE CELL DISEASE
acid substitution and gene mutation responsible for most cases of SCD were described (46, 65). Sickle Hb (HbS) usually has a single amino acid substitution at position 6 on the  chain, with glutamic acid replaced by valine. The loss of a negative charge at this site on the outside of the HbS molecule (cf. normal HbA), enables neighboring molecules to aggregate upon deoxygenation, eventually forming long polymers (12). (Here we refer to HbS-containing red cells as HbS cells; HbA-containing ones as HbA cells.) It is these polymers which distort the red cell shape into sickles, and other bizarre shapes. The polymerization and shape change is, at first, reversible upon oxygenation but eventually, and in some cells quite quickly, irreversible shape change occurs. A number of factors induce sickling. These include hypoxia, acid pH, urea, high temperature and low intracellular [Mg2⫹] (21). Cell volume is particularly critical, however, polymerization of HbS on deoxygenation occurs with a lag time which is inversely proportional to a very high power (30th is sometimes quoted) of [HbS] (19). Thus, if the red cell shrinks, so that [HbS] rises a little, then polymerization and shape change are
Almost 100 years ago, a laboratory report on a blood sample taken from the Grenadan Walter Noel, then a dental student studying in Chicago, described the presence of abnormally shaped red cells (83). It contained a sketch depicting elongated, sickled cells, a characteristic feature of sickle cell disease (SCD). This case heralded the impact of SCD in Western medicine. Later, it became apparent that the disease was genetic: only homozygous (HbSS; cf. normal HbAA) individuals developed the disease, while heterozygotes (HbAS, or so-called sickle trait individuals), usually lacked symptoms, although their cells could be induced to sickle ex vivo. About 50 years ago, Linus Pauling uncovered the molecular defect in SCD (79). He used the then novel technique of gel electrophoresis to reveal a difference in mobility of hemoglobin (Hb) from normal individuals, and those with SCD or heterozygotes with sickle trait. His paper, entitled “Sickle cell anemia, a molecular disease,” concludes that the protein structure of Hb must differ between normal individuals and sickle cell patients. Over the ensuing two decades, the amino
Correspondence and reprint requests to: J. C. Ellory or J. S. Gibson. Fax: ⫹44 (0) 1865 272488. E-mail: [email protected] (J. C. Ellory) or [email protected] (J. S. Gibson). 1 Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, United Kingdom. 2 University Laboratory of Physiology, University of Oxford, Oxford OX1 3PT, United Kingdom. 1079-9796/02 $35.00 © 2002 Elsevier Science (USA)
All rights reserved.
303
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Gibson and Ellory
late or inhibit transport, and, conversely, some transporters, such as the Na⫹/K⫹ pump and the anion exchanger (AE1) appear to be unaffected by O2 tension. In fact, O2 exerts an exquisite regulatory influence on red cell membrane transport, acting to co-ordinate the regulation of different transporters for a number of different physiological roles. We have reviewed O2-dependent transport in red cells elsewhere (30), and shall deal only briefly with the subject here, pointing out its relevance to the behavior of sickle cells. The K⫹-Cl⫺ cotransporter (KCC) is well represented as an example of an O2-sensitive membrane transport system in the red cells of vertebrates (Figs. 1a and 2a). Motais and colleagues (7) showed that high PO2 stimulated KCC in trout red cells. Subsequently, their work has been extended to mammals, particularly horse (31, 86), sheep (14) and human (35). The transporter is only fully active at high PO2 levels; as PO2 is reduced transporter activity declines; and, at low PO2 levels, the transporter is inactivated. Half maximal activity of KCC occurs at approximately the PO2 levels found in mixed venous blood. As for many other influences acting on KCC, that of O2 is mediated by regulatory protein kinase and phosphatase enzymes (17, 18, 48). In addition, for many stimuli usually considered to regulate KCC activity (notably the more physiological ones of cell volume, H⫹ ions and urea), at low PO2 levels, the transporter is refractory. When inactivated by low PO2, only high concentrations of urea (⬎500 mM) (85) and unphysiologically low pH (⬍6.7) (13) affect its activity. Interestingly, transporters with reciprocal actions—like the Na⫹-K⫹-Cl⫺ cotransporter (NKCC), which in many cells mediates the opposite volume regulatory response to KCC, i.e., volume regulatory increase as opposed to volume regulatory decrease—are inhibited by high PO2 levels and stimulated by low PO2 levels (30, 70). When the O2 dependence of these two electroneutral cation-coupled Cl⫺ cotransporters is compared, they form a striking mirror image of each other, with similar P50 levels, but one (KCC) stimulated at high PO2, the other (NKCC) at low PO2 (Fig. 1a). How O2 controls transporter activity is not known. A number of different lines of evidence,
much more likely to occur as the cell traverses hypoxic regions of the microvasculature (87), and, as explained later, unfortunately HbS cells are liable to rapid, irreversible shrinkage. Hypoxic capillaries in active muscle beds, at low pH, and in the renal medulla with high levels of urea, are obvious vulnerable regions for sickling. Notwithstanding its simple etiology, SCD is a markedly heterogeneous condition (e.g., 12, 84). The basis for this is not clear, but it probably involves epistatic or modifying genes (for example, variants or mutations in the transporters outlined below, or in their regulatory proteins). Red cell heterogeneity is also manifest within a single SCD patient. A spectrum of cell densities is seen and it is possible to fractionate the total red cell population by density gradient separation [for example, using arabinogalactan or Percoll (16, 26, 77)]. The transport phenotype, and other cell parameters (for example concentration of organic phosphates), differ between different cell densities (e.g., 5, 25, 78). As red cells dehydrate, their density becomes greater—and this is also a normal maturation event in HbA cells (44). Most dense cells are therefore among the oldest of the cell population, but, in addition, there is a fraction of HbS cells which dehydrate very rapidly, the so-called “fast-track” dehydration (6, 27). Thus, within the densest cells, a small population are very young (28), these may be most significant in the pathogenesis of SCD. The importance of cell density and different cell fractions has been well reviewed (e.g., 6, 50), although we shall only touch on it in the following account. O2-SENSITIVE MEMBRANE TRANSPORT IN RED CELLS Before examining the abnormal transport phenotype of HbS cells in detail, it is useful to summarize first the ways in which O2 tension controls the activity of membrane transporters in normal red cells. This property is very widespread amongst vertebrate red cells, affecting a range of transporters involving both ions and nonelectrolytes [see Table 1 in (30)]. The effect of O2 is specific. Thus, high O2 tension can either stimu304
Gibson and Ellory
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
FIG. 1. Evidence that regulation of red cell membrane permeability by O2 involves a subfraction of total haemoglobin. (a) Comparison of the activities of KCC in equine red cells and NKCC in turkey red cells showing a sigmoidal, but reciprocal, response of both to changes in PO2. (b) Treating cells with carbon monoxide prevents deoxygenation-induced inhibition of volume-sensitive KCC. (c) Pink ghosts prepared from human red cells retain an O2-dependent KCC activity, white ones do not. (d) The substituted benzaldehyde, 12C79, prevents inhibition of KCC at low PO2 levels, but not desaturation of Hb. Data taken from Speake et al. (86), Muzyamba et al. (70, 73), Gibson et al. (36), and Khan et al. (55).
however, indicate a role for membrane-bound Hb (see 30). Thus, for several transporters, carbon monoxide and nitrite treatment mimic the action of O2 (49, 68, 73) (Fig. 1b). These reagents result in formation of either carboxyHb or metHb, both of which have an oxy conformation of Hb irrespective of PO2, but they can also interact with other hemoproteins beside Hb. That Hb is the likely target is suggested by the sigmoidal nature of the response of transporters, such as KCC and NKCC, to changes in PO2 (70, 86). If Hb is involved, it is unlikely to be the total, or bulk, red
cell Hb. Thus, pink ghosts (45, 82) with only about 5% total Hb remaining have an O2-dependent KCC, while white ghosts, prepared by the gel filtration method (54, 94) and stripped of ⬎99% Hb, have lost the ability to respond (55) (Fig. 1c). In addition, KCC in cells treated with the substituted benzaldehyde, 12C79, a reagent which stabilizes Hb in the oxy form and thereby increases O2 affinity (3), becomes largely O2 insensitive (36) (Fig. 1d). Nevertheless, in these cells, O2 saturation at low PO2 levels is very minimal, so most Hb is in the deoxy conformation, again 305
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Gibson and Ellory
FIG. 2. Evidence that some of the abnormal cation permeability of red cells from sickle cell patients involves Hb polymerization. (a) Normal response of KCC in HbA cells to changes in O2 tension showing inhibition at low PO2 levels. (b) Activity of Psickle, determined as a Cl⫺-independent K⫹ flux, is induced by deoxygenation. (c) Unlike KCC in HbA cells, by contrast, KCC in HbS cells remains active at low PO2 levels. (d) Sickling is also induced by deoxygenation. (b), (c), and (d) also show the effect of 12C79 (5 mM): the abnormal changes in permeability are shifted toward lower PO2 levels—activity of Psickle and deoxygenation-induced KCC correlate with the sickling shape change. Data are taken from Gibson et al. (32).
arguing against the involvement of bulk Hb. These findings would also exclude changes secondary to deoxygenation of bulk Hb, i.e., in free [organic phosphates], free intracellular [Mg2⫹] or pH, in the signaling pathway. They imply that a subfraction of Hb is likely to participate, and, in ghosts, much of this is membrane-bound. We have reviewed elsewhere (30) the evidence that the target for this fraction of Hb may be the cytoplasmic N-terminus [the so-called cdb3 (64)] of the anion exchanger, AE1.
ABNORMAL TRANSPORT IN SICKLE CELLS Our understanding of the altered membrane transport of HbS cells dates from the seminal work of Tosteson and co-workers in the early 1950s (90 –92). They showed that change in O2 tension had little affect on cation balance in normal HbA cells. Oxygenated HbS cells also maintained the usual cation distribution, with high intracellular levels of K⫹ and low intracellular 306
Gibson and Ellory
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Na⫹. By contrast, deoxygenated HbS cells gained Na⫹ and lost K⫹. This exchange was abrogated by first treating cells with carbon monoxide, so it was not due to PO2 per se, but more likely to the conformation of oxyHb/carboxyHb (or possibly interaction with some other heme). Tosteson also demonstrated that, under certain conditions, HbScontaining red cells shrink, thereby eliciting the deleterious sequelae outlined above. These observations underlie much of our appreciation of altered membrane transport in sickle cell disease, and considerable work over the past 50 years has been concerned with explaining the underlying physiological and molecular mechanisms. Normal HbA cells slowly lose intracellular solutes and water as they mature over their life span of 120 days (becoming more dense as they do so, see above) (44). HbS cells, however, are liable to rapid irreversible loss of K⫹ and Cl⫺, with water following osmotically. These shrunken cells are more likely to be removed from the circulation by the reticulo-endothelial system, or other processes. They are also much more likely to sickle and become trapped in the microvasculature. Their life span is very much reduced to about 1/10th that of HbA cells, some 10 –20 days. Three membrane transport systems, abnormally active in HbS cells, participate in this shrinkage: the deoxygenation-induced cation channel (termed Psickle); the Gardos channel or Ca2⫹-activated K⫹; and the K⫹-Cl⫺ cotransporter (KCC). The behavior of these pathways is discussed below.
the Na⫹/K⫹ pump, there will be a net loss of cations as this active transporter uses energy from ATP hydrolysis in an attempt restore normal Na⫹ and K⫹ gradients, exchanging intracellular Na⫹ for extracellular K⫹ (53). The main importance of Psickle, however, is that it allows entry of Ca2⫹ (81). The latter contributes to activation of the second transport pathway, the Gardos channel. THE GARDOS CHANNEL The Gardos channel is a Ca2⫹-activated K⫹ channel of intermediate conductance, recently cloned and ascribed IK1 (or its synonym, SK4) (6, 9, 29, 47, 93). There are about 150 channels per cell and, once activated, they mediate K⫹ efflux at high rates, with Cl⫺ following, coupled indirectly via the membrane potential. Deoxygenation-induced Ca2⫹ entry via Psickle is the main precipitating factor for Gardos channel activation (81), although there is also modest inhibition of the plasma membrane Ca2⫹ pump (PMCA) (24), a high capacity ATPase which normally keeps intracellular [Ca2⫹] below the threshold for Gardos channel activity. Note that it is free intracellular [Ca2⫹] which regulates channel activity, and this should not be confused with total intracellular [Ca2⫹] (77). Thus, sickle cells contain inside-out vesicles of plasma membrane which sequester Ca2⫹, which is unavailable for Gardos channel activation but which results in a high total intracellular [Ca2⫹] (61). THE K⫹-Cl⫺ COTRANSPORTER (KCC)
THE DEOXYGENATION-INDUCED CATION PATHWAY (Psickle)
Finally, directly coupled K⫹ and Cl⫺ fluxes across the membrane of HbS cells are also high. These are mediated by KCC, probably the KCC1 isoform (8, 10, 15, 80). This transporter is a member of the cation-coupled Cl⫺ cotransporter superfamily, which includes NKCC and the thiazide-sensitive Na⫹-Cl⫺ cotransporter (NCC) (69). The cotransporter is regulated by phosphorylation via a cascade of protein kinases and phosphatases, with a final serine–threonine dephosphorylation associated with activity (48). An upstream tyrosine residue phosphorylation may also be involved with transporter stimulation (18). It is reg-
Psickle is a fairly nonspecific cation channel or channels of unknown identity which opens stochastically on deoxygenation (4, 51, 62, 78, 90) (see Fig. 2b). Much of the early work on this entity was carried out by Joiner and colleagues (50, 51) who used passive Na⫹ and K⫹ fluxes to show activation of Psickle as PO2 falls below about 40 mmHg. Psickle can mediate both entry of Na⫹ and loss of K⫹, and in the presence of extracellular divalent cations, K⫹ loss is greater than Na⫹ entry, hence it can mediate dehydration directly (52). In addition, due to the 3:2 stoichiometry of 307
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Gibson and Ellory
FIG. 3. Model for dehydration of sickle cells and stabilization of cell volume using transport inhibitors. (a) Under oxygenated conditions, cells can lose K⫹ and Cl⫺ through KCC should cells experience low pH or urea; Psickle and the Gardos channel are inactive. (b) When deoxygenated, changes in membrane permeability are exacerbated and can interact to produce rapid dehydration and sickling. Thus, HbS polymerizes, Psickle is activated, intracellular Ca2⫹ levels become sufficient for activation of the Gardos channel; KCC remains active despite deoxygenation and can be stimulated by low pH and urea. Isosmotic loss of K⫹ and Cl⫺ will elevate [HbS] and promote sickling, and also acidify the cell and further stimulate KCC. (c) Eventually, but much more rapidly under hypoxic conditions, solute loss and dehydration results in irreversible sickling. (d) Finally, partial inhibition of these events, perhaps by combinations of inhibitors, allow stabilization of cell volume and prevention of sickling: clotrimazole (CLT) and new agents like ICA17403 inhibit the Gardos channel; intracellular Mg2⫹ and Cl⫺ channel blockers like dihydroindenyloxyalkanoic acid (DIOA) inhibit KCC; 12C79 and DMA inhibit HbS polymerization; and activation of Psickle; NS3623 reduces Cl⫺ permeability and thereby also inhibits conductive loss of cations via the Gardos channel.
renal medulla), thereby increasing the contribution of KCC to cell dehydration.
ulated by swelling, reduction in pH (from about 7.4 to 7.0), urea, and high temperature, with H⫹ ions likely to be the most important stimulus in vivo (21, 22). As noted in the previous section, it is also O2-dependent, so that in normal red cells, KCC1 is quiescent in the absence of sufficiently high levels of O2 tension (35) (Fig. 2a). The O2 response in HbS cells, however, is different. In these cells, the transporter is highly active at high, arterial PO2 levels, starts to deactivate as PO2 levels are lowered, but then at tensions below about 40 mmHg activity increases again (Fig. 2c). In fully deoxygenated cells, activity is not dissimilar to that in fully oxygenated ones, and, in fact, on average, is about 10% higher (35). This abnormal O2 response is critical. It means that HbS cells, but not HbA ones, have a KCC1 that can respond to H⫹ ions (and urea) in hypoxic regions of the circulation, such as active muscle beds (or
INTERACTION OF THE VARIOUS PERMEABILITY PATHWAYS Together, these three pathways mediate solute loss, water will follow osmotically, with subsequent cell shrinkage. Their relative contribution, however, remains controversial (e.g., 2, 28, 74), and probably varies with PO2 and cell fraction, as well as between individuals (50). In addition, this array of transporters provides considerable scope for volume instability, notably positive feedback with a potential for rapid dehydration (6) (see Fig. 3). Thus, solute loss via any of the pathways will elevate [HbS], promote HbS polymerization and further activation of Psickle (and probably KCC— see later); isosmotic efflux of KCl will reduce 308
Gibson and Ellory
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
intracellular [Cl⫺], via activation of Cl⫺ influx and HCO⫺ 3 efflux via AE1, thereby acidify the cell, providing another stimulus for KCC activity; and, even simple oxy-deoxy transitions of Hb as the cells circulate between the arterial and venous vessels will also produce pH transients (60), which again can potentially stimulate KCC (see Fig. 3). Finally, since there is no obvious mechanism by which shrunken cells can regain lost solutes, solute efflux need not be a large all-ornone event, rather it may be intermittent and of relatively modest magnitude during each episode, before cumulative loss places cell volume at a critical level.
transporter in the membrane, the transduction pathway—the regulatory protein kinase/phosphatase enzymes—maintains the transporter in its inactive state, for example through steric hindrance, or insufficient or abnormal levels of the participating enzymes. TRANSPORT OF AMINO ACIDS Transport of organic solutes is also different in HbS cells compared to HbAs. Thus, we have examined several amino acid transporters including system gly and ASC (57). Both transport small uncharged amino acids: system ASC is a Na⫹-dependent transporter, typically transporting alanine, serine, and cysteine (hence the acronym), but whose main function is probably transport of cysteine; system gly is both Na⫹- and Cl⫺-dependent transporter, whose main substrate is glycine. In the red cell, these systems are important as they provide two of the three precursors of reduced glutathione (GSH) (23), a tripeptide of glycine, cysteine and glutamic acid, which forms a major part of the anti-oxidant defense of the red cell. In HbS cells, levels of GSH are lower than normal (58), although it is not clear whether this follows reduced synthesis or increased consumption (see later). In HbS cells, we have shown that systems ASC and gly are upregulated compared to HbA cells (57). Furthermore, both systems are stimulated by oxygenation. The kinetics are complicated and differ between the two: for example, oxygenation increases Vmax of system gly with no change in Km; for system ASC, Vmax is unaffected but Km halves on oxygenation. The significance of these effects has not been fully investigated.
COMPARISON WITH NORMAL RED CELLS The three pathways responsible for HbS cell dehydration remain cryptic in normal HbA cells. Psickle is not seen, although notwithstanding its apparent absence from HbA cells, the gene or genes encoding for it must be present. The Gardos channel is observed (29), but normally remains inactive because the high capacity PCMA, coupled with relatively low passive Ca2⫹ permeability, maintain intracellular Ca2⫹ below the threshold (63, 90). Ca2⫹ levels will rise and stimulate the Gardos channel should the pump be inhibited, for example by metabolic depletion, as in the original experiments of Gardos (29). Finally, while KCC is also present in the membrane, it has a much lower capacity than in HbS cells (20). The reason for the smaller maximal activity is not entirely clear. KCC activity is higher in younger HbA cells, and certainly the average cell population is younger in SCD (15, 40). That does not appear to be the entire explanation, however. Recently, it has been shown that expression of KCC is higher in HbS cells than HbA ones (88), although again it is not known why. Finally, as well as having a reduced maximal activity, KCC in HbA cells is largely quiescent and unresponsive to physiological stimuli (such as H⫹ ions and swelling), although it is responsive to pharmacological manipulations such as treatment with Nethylmaleimide, ionophore-induced Mg2⫹ depletion or high hydrostatic pressure (11, 20, 39). Presumably, although there is expression of the
MECHANISMS RESPONSIBLE FOR THE ALTERED TRANSPORT PHENOTYPE OF SICKLE CELLS There are many reasons why the membrane of HbS cells may differ from those of normal HbA cells, we discuss some below. First, the cell population is much younger in sickle cell disease, as evident from the reduced life span of affected cells. Young HbA cells per se, however, isolated as the least dense fraction on 309
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Gibson and Ellory
density gradients, do not share the same properties as HbS cells. There is no evidence of Psickle on deoxygenation, nor of an active KCC at low PO2 levels (32, 56). Second, red cells from patients with SCD contain HbS, rather than the normal HbA. This has a lower O2 affinity (12), and may also have different affinity for key regulatory sites controlling membrane permeability. It certainly has altered charge. Loss of negative charges at key residues on the  chain (mainly around positions 6 and 7) is associated with increased activity of KCC (76). The O2 response of these hemoglobinopathies, aside from sickle cells, remains poorly investigated. We have shown recently, however, that red cells containing HbSC, although they sickle at low PO2 levels and develop a pathway like that of Psickle, have KCC which is still inactivated by deoxygenation (34). This uncoupling of Psickle and sickling from low PO2-induced KCC is intriguing. Deoxy HbS also polymerizes. These polymers of HbS distort the membrane, and also sequester cell water and alter cell metabolism (12, 19, 59). Treatment of cells with the substituted benzaldehyde, 12C79, or the crosslinking agent dimethyl adipimidate (DMA), increases the O2 affinity of HbS (3, 36, 37). Both reagents also shift the deoxy-induced changes in shape and membrane permeability—i.e., sickling, and activation of Psickle and the deoxy-component of KCC—toward lower PO2 levels (32, 37) (Figs. 2b and 2c). Correlation of these transport events with the sickling shape change (Fig. 2d) implies that both result from polymerization of HbS, or a secondary effect of polymerization. The different response of HbSC-containing red cells can be rationalized if deoxygenated HbC, unincorporated into polymer, remains free and is thus able to act, like deoxyHbA, as an inhibitor of KCC activity (34). A third difference is the presence of higher protein tyrosine kinase (PTK) activity in HbS cells (66, 67). On deoxygenation, PTK activity is elevated further. Although inhibition of KCC is associated with a serine-threonine phosphorylation event, phosphorylation of tyrosine residues appears to operate in the opposite way, i.e., to stimulate transport (see above). One target for red cell PTK is cdb3 of AE1, and its phosphorylation
is indeed increased in HbS cells (67). It may be that this is sufficient to displace deoxy Hb, or other proteins which bind to this site (64), and thereby moderate the inhibitory effect of deoxygenation on KCC activity. Finally, oxidant damage or changes in redox potential may be important. Sickle cells contain lower levels of reduced glutathione (GSH) than normal red cells (58). They are also subject to the oxidative effects of hemichromes, free heme and iron, and possibly other species because of the relative instability of HbS (41, 42). We, and other groups, have investigated the action of a number of oxidants including chloro-2,4-dinitrobenzene, phenazine methosulfate (phenazine), menadione sodium bisulfite, and nitrite on HbA and HbS cells (33, 38, 71, 72, 75). Results show that all of these oxidants stimulate KCC activity, with stimulation correlating with metHb formation rather than GSH depletion (cf. 75). They also reduce inhibition of KCC activity in deoxygenated cells. Furthermore, phenazine in particular also activates the Gardos channel (71). For phenazine, stimulation of both KCC and the Gardos is more marked in deoxygenated cells compared with oxygenated ones, a transport phenotype which begins to resemble that of deoxygenated HbS cells. The effects of phenazine on deoxygenated red cells depends on the presence of extracellular Ca2⫹. It appears to inhibit PMCA, and to a greater extent on dexoygenation (33). This effect, coupled with a possible increased sensitivity of the Gardos channel for intracellular Ca2⫹ (1), may account for its action on the channel. In the case of KCC, calyculin A, a serine-threonine phosphatase inhibitor, abolishes the action of phenazine when added prior to the oxidant, but has only modest inhibitory effects when added afterwards, implying decreased activity of an inhibitory protein kinase (33). A fall in [ATP] could explain both inhibition of PMCA and the kinase, but bulk ATP levels remain high. It may be that a privileged pool of ATP is involved, responsible for regulation of membrane pumps and other transporters (43), and that this is affected by oxidants. A further possibility is specific effects of changes in redox potential. We are investigating some of these possibilities. 310
Gibson and Ellory
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
ACKNOWLEDGMENTS
11.
This work was supported by Action Research and The Wellcome Trust. The text is based on the Jacobs– Parpart–Ponder Lecture given by Professor Ellory to the American Red Cell Club, San Francisco, California, 24 February, 2002.
12.
13.
14.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Alvarez, J., Garcia-Sancho, J., and Herreros, B. (1984) Effects of electron donors on Ca2⫹-dependent K⫹ transport in one-step inside-out vesicles from the human erythrocyte membrane. Biochim. Biophys. Acta 771, 23–27. Apovo, M., Beuzard, Y., Galacteros, F., Bachir, D., and Giraud, F. (1994) The involvement of the Cadependent K channel and of the KCl cotransport in sickle cell dehydration during cyclic deoxygenation. Biochim. Biophys. Acta 1225, 255–258. Beddell, C. R., Goodford, P. J., Kneen, G., White, R. D., Wilkinson, S., and Wootton, R. (1984) Substituted benzaldehydes designed to increase the oxygen affinity of human haemoglobin and inhibit the sickling of sickle erythrocytes. Br. J. Pharmacol. 82, 397– 407. Berkowitz, L. R., and Orringer, E. P. (1985) Passive sodium and potassium movements in sickle erythrocytes. Am. J. Physiol. 249, C208 –C214. Bookchin, R. M., Etzion, Z., Sorette, M. P., Mohandas, N., and Lew, V. L. (1993) Heterogeneity of ion transport in sickle cell anemia reticulocytes revealed by patterns of acid-induced dehydration. Blood 82, 350a. Bookchin, R. M., Ortiz, O. E., and Lew, V. L. (1991) Evidence for a direct reticulocyte origin of dense red cells in sickle cell anemia. J. Clin. Invest. 87, 113– 124. Borgese, F., Motais, R., and Garcia-Romeu, F. (1991) Regulation of Cl-dependent K transport by oxy-deoxyhemoglobin transitions in trout red cells. Biochim. Biophys. Acta 1066, 252–256. Brugnara, C., Bunn, H. F., and Tosteson, D. C. (1986) Regulation of erythrocyte cation and water content in sickle cell anemia. Science 232, 388 –390. Brugnara, C., de Franceschi, L., and Alper, S. L. (1993) Inhibition of Ca⫹⫹-dependent K⫹ transport and cell dehydration in sickle erythrocytes by clotrimazole and other imidazole derivatives. J. Clin. Invest. 92, 520 –526. Brugnara, C., Kopin, A. S., Bunn, H. F., and Tosteson, D. C. (1985) Regulation of cation content and cell volume in hemoglobin erythrocytes from patients with homozygous hemoglobin C disease. J. Clin. Invest. 75, 1608 –1617.
15.
16.
17.
18.
19. 20.
21.
22.
23.
24.
25.
26. 311
Brugnara, C., and Tosteson, D. C. (1987) Inhibition of K transport by divalent cations in sickle erythrocytes. Blood 70, 1810 –1815. Bunn, H. F., and Forget, B. G. (1986) Hemoglobin: Molecular, Genetic and Clinical Aspects. Saunders, Philadelphia. Campbell, E. H., and Gibson, J. S. (1998) Magnesium and the O2 dependence of KCl cotransport in LK sheep red cells. J. Physiol. 506(3), 679 – 688. Campbell, E. H., and Gibson, J. S. (1997) Oxygenation and KCl cotransport in sheep red cells. J. Physiol. 501, P154 –P155. Canessa, M., Fabry, M. E., Blumenfeld, N., and Nagel, R. L. (1987) Volume-stimulated, Cl-dependent K efflux is highly expressed in young human red cells containing normal hemoglobin or HbS. J. Membr. Biol. 97, 97–105. Corash, L. M., Piomelli, S., Chen, H. C., Seaman, C., and Gross, E. (1984) Separation of erythrocytes according to age on a simplified density gradient. J. Lab. Clin. Med. 84, 147–151. Cossins, A. R., and Gibson, J. S. (1997) Volumesensitive transport systems and volume homeostasis in vertebrate red blood cells. J. Exp. Biol. 200, 343–352. Cossins, A. R., Weaver, Y. R., Lykkeboe, G., and Nielsen, O. B. (1994) Role of protein phosphorylation in control of K flux pathways of trout red blood cells. Am. J. Physiol. 267, C1641–C1650. Eaton, W. A., and Hofrichter, J. (1987) Hemoglobin S gelation and sickle cell disease. Blood 70, 1245–1266. Ellory, J. C., Dunham, P. B., Logue, P. J., and Stewart, G. W. (1982) Anion-dependent cation transport in erythrocytes. Phil. Trans. Roy. Soc. London B 299, 483– 495. Ellory, J. C., Gibson, J. S., and Stewart, G. W. (1998) Pathophysiology of abnormal cell volume in human red cells. Contrib. Nephrol. 123, 220 –239. Ellory, J. C., Hall, A. C., and Ody, S. A. (1989) Is acid a more potent activator of KCl co-transport than hypotonicity in human red cells? J. Physiol. 420, 149P. Ellory, J. C., Preston, R. L., Osotimehin, B., and Young, J. D. (1983) Transport of amino acids for glutathione synthesis in human and dog red cells. Biomed. Biophys. Acta 42, 48 –52. Etzion, Z., Tiffert, T., Bookchin, R. M., and Lew, V. L. (1993) Effects of deoxygenation on active and passive Ca2⫹ transport and on the cytoplasmic Ca2⫹ levels of sickle cell anemia red cells. J. Clin. Invest. 92, 2489 –2498. Fabry, M. E., Mears, J. G., Patel, P., Schaefer-Rego, K., Carmichael, L. D., Martinez, G., and Nagel, R. L. (1984) Dense cells in sickle cell anaemia: The effects of gene interaction. Blood 64, 1042–1048. Fabry, M. E., and Nagel, R. L. (1982) Heterogeneity of red cells in the sickler: A characteristic with prac-
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
Gibson and Ellory
tical clinical and pathophysiological implications. Blood Cells 8, 9 –15. Fabry, M. E., Romero, J. R., Buchanan, I. D., Suzuka, S. M., Stamatoyannopoulos, G., Nagel, R. L., and Canessa, M. (1991) Rapid increase in red blood cell density driven by K:Cl cotransport in a subset of sickle cell anemia reticulocytes and discocytes. Blood 78, 217–225. Franco, R. S., Palascak, M., Thompson, H., Rucknagel, D. L., and Joiner, C. H. (1996) Dehydration of transferrin receptor-positive sickle reticulocytes during continuous or cyclic deoxygenation: Role of KCl cotransport and extracellular calcium. Blood 88, 4359 – 4365. Gardos, G. (1958) The function of calcium and potassium permeability of human erythrocytes. Biochim. Biophys. Acta 30, 653– 654. Gibson, J. S., Cossins, A. R., and Ellory, J. C. (2000) Oxygen-sensitive membrane transporters in vertebrate red cells. J. Exp. Biol. 203, 1395–1407. Gibson, J. S., Honess, N. A., Cossins, A. R., Ellory, J. C., and Schwabe, A. E. (1994) Oxygen tension and KCl co-transport in equine red cells. J. Physiol. 476, 60P. Gibson, J. S., Khan, A., Speake, P. F., and Ellory, J. C. (2001) O2 dependence of K⫹ transport in sickle cells: The effects of different cell populations and the substituted benzaldehyde, 12C79. FASEB J. 15, 823– 832. Gibson, J. S., and Muzyamba, M. C. (2002) The effect of phenazine metosulphate on Ca2⫹ transport in human red blood cells. J. Physiol., in press. Gibson, J. S., Muzyamba, M. C., Ball, S. E., and Ellory, J. C. (2001) K⫹ transport in HbSC-containing human red blood cells. J. Physiol., in press. Gibson, J. S., Speake, P. F., and Ellory, J. C. (1998) Differential oxygen sensitivity of the K⫹-Cl⫺ cotransporter in normal and sickle human red blood cells. J. Physiol. 511, 225–234. Gibson, J. S., Speake, P. F., and Ellory, J. C. (1999) The effect of the substituted benzaldehyde, 12C79, on K⫹ transport in human red blood cells. Eur. J. Physiol. 437, 498 –500. Gibson, J. S., Stewart, G. W., and Ellory, J. C. (2000) Effect of dimethyl adipimidate on K⫹ transport and shape change in red blood cells from sickle cell patients. FEBS Lett. 480, 179 –183. Gusev, G. P., and Bogdanova, A. Y. (1992) Evidence for stimulation of the K-Cl cotransport system by phenazine methosulphate. Biochem. Pharmacol. 43, 2275–2279. Hall, A. C., and Ellory, J. C. (1986) Effect of high hydrostatic pressure on ‘passive’ monovalent cation transport in human red cells. J. Membr. Biol. 94, 1–17. Hall, A. C., and Ellory, J. C. (1986) Evidence for the presence of volume-sensitive KCl transport in ‘young’
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55. 312
human red cells. Biochim. Biophys. Acta 858, 317– 320. Hebbel, R. P., Eaton, J. W., Balasingam, M., and Steinberg, M. H. (1982) Spontaneous oxygen radical generation by sickle erythrocytes. J. Clin. Invest. 70, 1253–1259. Hebbel, R. P., Morgan, W. T., Eaton, J. W., and Hedlund, B. E. (1988) Accelerated autoxidation and heme loss due to instability of sickle hemoglobin. Proc. Natl. Acad. Sci. USA 85, 237–241. Hoffman, J. F. (1997) ATP compartmentalization in human erythrocytes. Curr. Opin. Hematol. 4, 112– 115. Hoffman, J. F. (1958) On the relationship of certain erythrocyte characteristics of their physiological age. J. Cell. Comp. Physiol. 51, 415– 423. Hoffman, J. F. (1958) Physiological characteristics of human red blood cell ghosts. J. Gen. Physiol. 42, 9 –28. Ingram, V. M. (1957) Gene mutations in human haemoglobin, the chemical difference between normal and sickle cell haemoglobin. Nature 180, 326 –328. Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) A human intermediate conductance calcium-activated potassium channel. Proc. Natl. Acad. Sci. USA 94, 11651–11656. Jennings, M. L., and Al-Rohil, N. (1990) Kinetics of activation and inactivation of swelling-stimulated K/Cl transport: The volume-sensitive parameter is the rate constant for inactivation. J. Gen. Physiol. 95, 1021–1040. Jensen, F. B. (1990) Nitrite and red cell function in carp: Control factors for nitrite entry, membrane potassium ion permeation, oxygen affinity and methaemoglobin formation. J. Exp. Biol. 152, 149 –166. Joiner, C. H. (1993) Cation transport and volume regulation in sickle red blood cells. Am. J. Physiol. 264, C251–C270. Joiner, C. H., Dew, A., and Ge, D. L. (1988) Deoxygenation-induced fluxes in sickle cells. I. Relationship between net potassium efflux and net sodium influx. Blood Cells 13, 339 –348. Joiner, C. H., Jiang, M., and Franco, R. S. (1995) Deoxygenation-induced cation fluxes in sickle cells. IV. Modulation by external calcium. Am. J. Physiol. 269, C403–C409. Joiner, C. H., Platt, O. S., and Lux, S. E. (1986) Cation depletion by the sodium pump in red cells with pathological cation leaks: Sickle cells and xerocytes. J. Clin. Invest. 78, 1487–1496. Kaplan, J. H. (1982) Sodium pump-mediated ATP: ADP exchange. The sided effects of sodium and potassium ions. J. Gen. Physiol. 80, 915–937. Khan, A., Gibson, J. S., and Ellory, J. C. (2000)
Gibson and Ellory
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Oxygen-dependent KCl cotransport in ghosts from normal human red blood cells. J. Physiol. 527P, 38P. Khan, A., Speake, P. F., Gibson, J. S., and Ellory, J. C. (2000) Density gradient separation of sickle cells. J. Physiol. 525, 25P. Kiessling, K., Roberts, N., Gibson, J. S., and Ellory, J. C. (2000) A comparison in normal individuals and sickle cell patients of reduced glutathione precursors and their transport between plasma and red cells. Hematol. J. 1, 243–249. Lachant, N. A., Davidson, W. D., and Tanaka, K. R. (1983) Impaired pentose phosphate shunt function in sickle cell disease: A potential mechanism for increased Heinz body formation and membrane lipid peroxidation. Am. J. Hematol. 15, 1–13. Lew, V. L., Balazs, T., and Bookchin, R. M. (1995) Osmotic effects of water compartments in protein polymers. Cell Biochem. Funct. 13, 173–176. Lew, V. L., Freeman, C. J., Ortiz, O. E., and Bookchin, R. M. (1991) A mathematical model of the volume, pH and ion content regulation in reticulocytes. J. Clin. Invest. 87, 100 –112. Lew, V. L., Hockaday, A., Sepulveda, M. I., Somlyo, A. P., Somlyo, A. V., Ortiz, O. E., and Bookchin, R. M. (1985) Compartmentalization of sickle cell calcium in endocytic inside-out vesicles. Nature 315, 586 –589. Lew, V. L., Ortiz, O. E., and Bookchin, R. M. (1997) Stochastic nature and red cell population distribution of the sickling-induced Ca2⫹ permeability. J. Clin. Invest. 99, 2727–2735. Lew, V. L., Tsien, R. Y., Miner, C., and Boockchin, R. M. (1982) Physiological [Ca2⫹]i level and pumpleak turnover in intact red cells measured using an incorporated Ca chelator. Nature 298, 478 – 481. Low, P. S. (1986) Structure and function of the cytoplasmic domain of band 3: Center of erythrocyte membrane–peripheral protein interactions. Biochim. Biophys. Acta 864, 145–167. Marotta, C. A., Wilson, J. T., Forget, B. J., and Weissman, S. M. (1977) Human beta-globin messenger RNA. III. Nucleotide sequences derived from complementary DNA. J. Biol. Chem. 252, 5040 –5051. Merciris, P., Hardy-Dessources, M. D., and Giraud, F. (2001) Deoxygenation of sickle cells stimulates Syk tyrosine kinase and inhibits a membrane tyrosine phosphatase. Blood 98, 3121–3127. Merciris, P., Hardy-Dessources, M. D., Sauvage, M., and Giraud, F. (1998) Involvement of deoxygenationinduced increase in tyrosine kinase activity in sickle cell dehydration. Pfluger’s Arch. Eur. J. Physiol. 436, 315–322. Motais, R., Garcia-Romeu, F., and Borgese, F. (1987) The control of Na/H exchange by molecular oxygen in trout erythrocytes. J. Gen. Physiol. 90, 197–207.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
313
Mount, D. B., Delpire, E., Gamba, G., Hall, A. E., Poch, E., Hoover, R. S. J., and Hebert, S. C. (1998) The electroneutral cation-chloride cotransporters. J. Exp. Biol. 201, 2091–2102. Muzyamba, M. C., Cossins, A. R., and Gibson, J. S. (1999) Regulation of Na⫹-K⫹-2Cl⫺ cotransport in turkey red cells: The role of oxygen tension and protein phosphorylation. J. Physiol. 517, 421– 429. Muzyamba, M. C., and Gibson, J. S. (2001) The effect of phenazine methosulphate on K⫹ transport in human red blood cells. J. Physiol., in press. Muzyamba, M. C., Speake, P. F., and Gibson, J. S. (2001) The effect of oxidants on O2-dependent K⫹ transport in normal human red blood cells. J. Physiol. 531, 124P. Muzyamba, M. C., Speake, P. F., and Gibson, J. S. (2000) Oxidants and regulation of KCl cotransport in equine red blood cells. Am. J. Physiol. 279, C981– C989. Ohnishi, S. T., Horiuchi, K. Y., and Horiuchi, K. (1986) The mechanism of in vitro formation of irreversibly sickled cells and modes of action of its inhibitors. Biochim. Biophys. Acta 886, 119 –129. Olivieri, O., Bonollo, M., Friso, S., Girelli, D., Corrocher, R., and Vettore, L. (1993) Activation of K⫹/ Cl⫺ cotransport in human erythrocytes exposed to oxidative agents. Biochim. Biophys. Acta 1176, 37– 42. Olivieri, O., Vitoux, D., Galacteros, F., Bachir, D., Blouquit, Y., Beuzard, Y., and Brugnara, C. (1992) Hemoglobin variants and activity of (K⫹Cl⫺) cotransport system in human erythrocytes. Blood 79, 793– 797. Ortiz, O. E., Lew, V. L., and Bookchin, R. M. (1986) Calcium accumulated by sickle cell anaemia red cells does not affect their potassium (86Rb) flux components. Blood 67, 710 –715. Ortiz, O. E., Lew, V. L., and Bookchin, R. M. (1990) Deoxygenation permeabilizes sickle cell anaemia red cells to magnesium and reverses its gradient in the dense cells. J. Physiol. 427, 211–226. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C. (1949) Sickle cell anemia, a molecular disease. Science 110, 1141–1152. Pellegrino, C. M., Rybicki, A. C., Musto, S., Nagel, R. L., and Schwartz, R. S. (1998) Molecular identification of erythroid K:Cl cotransporter in human and mouse erythroleukemic cells. Blood Cells Mol. Dis. 24, 31– 40. Rhoda, M. D., Apovo, M., Beuzard, Y., and Giraud, F. (1990) Ca2⫹ permeability in deoxygenated sickle cells. Blood 75, 2453–2458. Schwoch, G., and Passow, H. (1973) Preparation and properties of human erythrocyte ghosts. Mol. Cell. Biochem. 2, 197–218.
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
83. 84. 85.
86.
87.
88.
89.
Gibson and Ellory
Serjeant, G. R. (2001) The emerging understanding of sickle cell disease. Br. J. Haematol. 112, 3–18. Serjeant, G. R. (1997) Sickle-cell disease. Lancet 350, 725–730. Speake, P. F., and Gibson, J. S. (1997) Urea-stimulated K-Cl cotransport in equine red blood cells. Pfluger’s Arch. Eur. J. Physiol. 434, 104 –112. Speake, P. F., Roberts, C. A., and Gibson, J. S. (1997) Effect of changes in respiratory blood parameters on equine red blood cell K-Cl cotransporter. Am. J. Physiol. 273, C1811–C1818. Stuart, J., and Ellory, J. C. (1988) Rheological consequences of erythrocyte dehydration. Br. J. Haematol. 69, 1– 4. Su, W., Shmukler, B. E., Chernova, M. A., StuartTilley, A. K., De Franceschi, L., Brugnara, C., and Alper, S. L. (1999) Mouse K-Cl cotransporter KCC1: Cloning, mapping, pathological expression, and functional regulation. Am. J. Physiol. 277, C899 –C912. Tiffert, T., Etzion, Z., Bookchin, R. M., and Lew, V. L. (1993) Effects of deoxygenation on active and passive
90.
91.
92.
93.
94.
314
Ca2⫹ transport and cytoplasmic Ca2⫹ buffering in normal human red cells. J. Physiol. 464, 529 –544. Tosteson, D. C. (1955) The effects of sickling on ion transport. II. The effect of sickling on sodium and cesium transport. J. Gen. Physiol. 39, 55– 67. Tosteson, D. C., Carlsen, E., and Dunham, E. T. (1955) The effects of sickling on ion transport. I. Effect of sickling on potassium transport. J. Gen. Physiol. 39, 31–53. Tosteson, D. C., Shea, E., and Darling, R. C. (1952) Potassium and sodium of red blood cells in sickle cell anaemia. J. Clin. Invest. 48, 406 – 411. Wolff, D., Cecchi, X., Spalvins, A., and Canessa, M. (1988) Charybdotoxin blocks with high affinity the Ca-activated K⫹ channel of HbA and HbS red cells: Individual differences in the number of channels. J. Membr. Biol. 106, 243–252. Wood, P. G. (1989) Preparation of resealable membranes maximally depleted of cytoplasmic components by gel filtration and tryptic digestion: Resealable white ghosts. Methods Enzymol. 173, 346 –355.
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Membrane Transport in Sickle Cell Disease Submitted 03/18/02 (Communicated by J. Hoffman, M.D., 03/21/02)
J. S. Gibson1 and J. C. Ellory2 ABSTRACT: We have reviewed here a number of membrane transport events in red cells from normal individuals and sickle cell patients which respond to changes in O2 tension. Some deoxygenation-induced changes in membrane permeability are unique to HbS cells and contribute to their dehydration and subsequent sickling. Polymerization of HbS, or specific oxidant damage (or altered redox potential), is a likely factor underlying the abnormal behavior. The key regulatory sites within the membrane or associated proteins remain uncertain and their identity will form the focus of future research. A model for sickle cell dehydration is presented. Inhibition of these permeability changes represents possible avenues for future chemotherapy to ameliorate the condition. © 2002 Elsevier Science (USA)
OVERVIEW OF SICKLE CELL DISEASE
acid substitution and gene mutation responsible for most cases of SCD were described (46, 65). Sickle Hb (HbS) usually has a single amino acid substitution at position 6 on the  chain, with glutamic acid replaced by valine. The loss of a negative charge at this site on the outside of the HbS molecule (cf. normal HbA), enables neighboring molecules to aggregate upon deoxygenation, eventually forming long polymers (12). (Here we refer to HbS-containing red cells as HbS cells; HbA-containing ones as HbA cells.) It is these polymers which distort the red cell shape into sickles, and other bizarre shapes. The polymerization and shape change is, at first, reversible upon oxygenation but eventually, and in some cells quite quickly, irreversible shape change occurs. A number of factors induce sickling. These include hypoxia, acid pH, urea, high temperature and low intracellular [Mg2⫹] (21). Cell volume is particularly critical, however, polymerization of HbS on deoxygenation occurs with a lag time which is inversely proportional to a very high power (30th is sometimes quoted) of [HbS] (19). Thus, if the red cell shrinks, so that [HbS] rises a little, then polymerization and shape change are
Almost 100 years ago, a laboratory report on a blood sample taken from the Grenadan Walter Noel, then a dental student studying in Chicago, described the presence of abnormally shaped red cells (83). It contained a sketch depicting elongated, sickled cells, a characteristic feature of sickle cell disease (SCD). This case heralded the impact of SCD in Western medicine. Later, it became apparent that the disease was genetic: only homozygous (HbSS; cf. normal HbAA) individuals developed the disease, while heterozygotes (HbAS, or so-called sickle trait individuals), usually lacked symptoms, although their cells could be induced to sickle ex vivo. About 50 years ago, Linus Pauling uncovered the molecular defect in SCD (79). He used the then novel technique of gel electrophoresis to reveal a difference in mobility of hemoglobin (Hb) from normal individuals, and those with SCD or heterozygotes with sickle trait. His paper, entitled “Sickle cell anemia, a molecular disease,” concludes that the protein structure of Hb must differ between normal individuals and sickle cell patients. Over the ensuing two decades, the amino
Correspondence and reprint requests to: J. C. Ellory or J. S. Gibson. Fax: ⫹44 (0) 1865 272488. E-mail: [email protected] (J. C. Ellory) or [email protected] (J. S. Gibson). 1 Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, United Kingdom. 2 University Laboratory of Physiology, University of Oxford, Oxford OX1 3PT, United Kingdom. 1079-9796/02 $35.00 © 2002 Elsevier Science (USA)
All rights reserved.
303
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Gibson and Ellory
late or inhibit transport, and, conversely, some transporters, such as the Na⫹/K⫹ pump and the anion exchanger (AE1) appear to be unaffected by O2 tension. In fact, O2 exerts an exquisite regulatory influence on red cell membrane transport, acting to co-ordinate the regulation of different transporters for a number of different physiological roles. We have reviewed O2-dependent transport in red cells elsewhere (30), and shall deal only briefly with the subject here, pointing out its relevance to the behavior of sickle cells. The K⫹-Cl⫺ cotransporter (KCC) is well represented as an example of an O2-sensitive membrane transport system in the red cells of vertebrates (Figs. 1a and 2a). Motais and colleagues (7) showed that high PO2 stimulated KCC in trout red cells. Subsequently, their work has been extended to mammals, particularly horse (31, 86), sheep (14) and human (35). The transporter is only fully active at high PO2 levels; as PO2 is reduced transporter activity declines; and, at low PO2 levels, the transporter is inactivated. Half maximal activity of KCC occurs at approximately the PO2 levels found in mixed venous blood. As for many other influences acting on KCC, that of O2 is mediated by regulatory protein kinase and phosphatase enzymes (17, 18, 48). In addition, for many stimuli usually considered to regulate KCC activity (notably the more physiological ones of cell volume, H⫹ ions and urea), at low PO2 levels, the transporter is refractory. When inactivated by low PO2, only high concentrations of urea (⬎500 mM) (85) and unphysiologically low pH (⬍6.7) (13) affect its activity. Interestingly, transporters with reciprocal actions—like the Na⫹-K⫹-Cl⫺ cotransporter (NKCC), which in many cells mediates the opposite volume regulatory response to KCC, i.e., volume regulatory increase as opposed to volume regulatory decrease—are inhibited by high PO2 levels and stimulated by low PO2 levels (30, 70). When the O2 dependence of these two electroneutral cation-coupled Cl⫺ cotransporters is compared, they form a striking mirror image of each other, with similar P50 levels, but one (KCC) stimulated at high PO2, the other (NKCC) at low PO2 (Fig. 1a). How O2 controls transporter activity is not known. A number of different lines of evidence,
much more likely to occur as the cell traverses hypoxic regions of the microvasculature (87), and, as explained later, unfortunately HbS cells are liable to rapid, irreversible shrinkage. Hypoxic capillaries in active muscle beds, at low pH, and in the renal medulla with high levels of urea, are obvious vulnerable regions for sickling. Notwithstanding its simple etiology, SCD is a markedly heterogeneous condition (e.g., 12, 84). The basis for this is not clear, but it probably involves epistatic or modifying genes (for example, variants or mutations in the transporters outlined below, or in their regulatory proteins). Red cell heterogeneity is also manifest within a single SCD patient. A spectrum of cell densities is seen and it is possible to fractionate the total red cell population by density gradient separation [for example, using arabinogalactan or Percoll (16, 26, 77)]. The transport phenotype, and other cell parameters (for example concentration of organic phosphates), differ between different cell densities (e.g., 5, 25, 78). As red cells dehydrate, their density becomes greater—and this is also a normal maturation event in HbA cells (44). Most dense cells are therefore among the oldest of the cell population, but, in addition, there is a fraction of HbS cells which dehydrate very rapidly, the so-called “fast-track” dehydration (6, 27). Thus, within the densest cells, a small population are very young (28), these may be most significant in the pathogenesis of SCD. The importance of cell density and different cell fractions has been well reviewed (e.g., 6, 50), although we shall only touch on it in the following account. O2-SENSITIVE MEMBRANE TRANSPORT IN RED CELLS Before examining the abnormal transport phenotype of HbS cells in detail, it is useful to summarize first the ways in which O2 tension controls the activity of membrane transporters in normal red cells. This property is very widespread amongst vertebrate red cells, affecting a range of transporters involving both ions and nonelectrolytes [see Table 1 in (30)]. The effect of O2 is specific. Thus, high O2 tension can either stimu304
Gibson and Ellory
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
FIG. 1. Evidence that regulation of red cell membrane permeability by O2 involves a subfraction of total haemoglobin. (a) Comparison of the activities of KCC in equine red cells and NKCC in turkey red cells showing a sigmoidal, but reciprocal, response of both to changes in PO2. (b) Treating cells with carbon monoxide prevents deoxygenation-induced inhibition of volume-sensitive KCC. (c) Pink ghosts prepared from human red cells retain an O2-dependent KCC activity, white ones do not. (d) The substituted benzaldehyde, 12C79, prevents inhibition of KCC at low PO2 levels, but not desaturation of Hb. Data taken from Speake et al. (86), Muzyamba et al. (70, 73), Gibson et al. (36), and Khan et al. (55).
however, indicate a role for membrane-bound Hb (see 30). Thus, for several transporters, carbon monoxide and nitrite treatment mimic the action of O2 (49, 68, 73) (Fig. 1b). These reagents result in formation of either carboxyHb or metHb, both of which have an oxy conformation of Hb irrespective of PO2, but they can also interact with other hemoproteins beside Hb. That Hb is the likely target is suggested by the sigmoidal nature of the response of transporters, such as KCC and NKCC, to changes in PO2 (70, 86). If Hb is involved, it is unlikely to be the total, or bulk, red
cell Hb. Thus, pink ghosts (45, 82) with only about 5% total Hb remaining have an O2-dependent KCC, while white ghosts, prepared by the gel filtration method (54, 94) and stripped of ⬎99% Hb, have lost the ability to respond (55) (Fig. 1c). In addition, KCC in cells treated with the substituted benzaldehyde, 12C79, a reagent which stabilizes Hb in the oxy form and thereby increases O2 affinity (3), becomes largely O2 insensitive (36) (Fig. 1d). Nevertheless, in these cells, O2 saturation at low PO2 levels is very minimal, so most Hb is in the deoxy conformation, again 305
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Gibson and Ellory
FIG. 2. Evidence that some of the abnormal cation permeability of red cells from sickle cell patients involves Hb polymerization. (a) Normal response of KCC in HbA cells to changes in O2 tension showing inhibition at low PO2 levels. (b) Activity of Psickle, determined as a Cl⫺-independent K⫹ flux, is induced by deoxygenation. (c) Unlike KCC in HbA cells, by contrast, KCC in HbS cells remains active at low PO2 levels. (d) Sickling is also induced by deoxygenation. (b), (c), and (d) also show the effect of 12C79 (5 mM): the abnormal changes in permeability are shifted toward lower PO2 levels—activity of Psickle and deoxygenation-induced KCC correlate with the sickling shape change. Data are taken from Gibson et al. (32).
arguing against the involvement of bulk Hb. These findings would also exclude changes secondary to deoxygenation of bulk Hb, i.e., in free [organic phosphates], free intracellular [Mg2⫹] or pH, in the signaling pathway. They imply that a subfraction of Hb is likely to participate, and, in ghosts, much of this is membrane-bound. We have reviewed elsewhere (30) the evidence that the target for this fraction of Hb may be the cytoplasmic N-terminus [the so-called cdb3 (64)] of the anion exchanger, AE1.
ABNORMAL TRANSPORT IN SICKLE CELLS Our understanding of the altered membrane transport of HbS cells dates from the seminal work of Tosteson and co-workers in the early 1950s (90 –92). They showed that change in O2 tension had little affect on cation balance in normal HbA cells. Oxygenated HbS cells also maintained the usual cation distribution, with high intracellular levels of K⫹ and low intracellular 306
Gibson and Ellory
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Na⫹. By contrast, deoxygenated HbS cells gained Na⫹ and lost K⫹. This exchange was abrogated by first treating cells with carbon monoxide, so it was not due to PO2 per se, but more likely to the conformation of oxyHb/carboxyHb (or possibly interaction with some other heme). Tosteson also demonstrated that, under certain conditions, HbScontaining red cells shrink, thereby eliciting the deleterious sequelae outlined above. These observations underlie much of our appreciation of altered membrane transport in sickle cell disease, and considerable work over the past 50 years has been concerned with explaining the underlying physiological and molecular mechanisms. Normal HbA cells slowly lose intracellular solutes and water as they mature over their life span of 120 days (becoming more dense as they do so, see above) (44). HbS cells, however, are liable to rapid irreversible loss of K⫹ and Cl⫺, with water following osmotically. These shrunken cells are more likely to be removed from the circulation by the reticulo-endothelial system, or other processes. They are also much more likely to sickle and become trapped in the microvasculature. Their life span is very much reduced to about 1/10th that of HbA cells, some 10 –20 days. Three membrane transport systems, abnormally active in HbS cells, participate in this shrinkage: the deoxygenation-induced cation channel (termed Psickle); the Gardos channel or Ca2⫹-activated K⫹; and the K⫹-Cl⫺ cotransporter (KCC). The behavior of these pathways is discussed below.
the Na⫹/K⫹ pump, there will be a net loss of cations as this active transporter uses energy from ATP hydrolysis in an attempt restore normal Na⫹ and K⫹ gradients, exchanging intracellular Na⫹ for extracellular K⫹ (53). The main importance of Psickle, however, is that it allows entry of Ca2⫹ (81). The latter contributes to activation of the second transport pathway, the Gardos channel. THE GARDOS CHANNEL The Gardos channel is a Ca2⫹-activated K⫹ channel of intermediate conductance, recently cloned and ascribed IK1 (or its synonym, SK4) (6, 9, 29, 47, 93). There are about 150 channels per cell and, once activated, they mediate K⫹ efflux at high rates, with Cl⫺ following, coupled indirectly via the membrane potential. Deoxygenation-induced Ca2⫹ entry via Psickle is the main precipitating factor for Gardos channel activation (81), although there is also modest inhibition of the plasma membrane Ca2⫹ pump (PMCA) (24), a high capacity ATPase which normally keeps intracellular [Ca2⫹] below the threshold for Gardos channel activity. Note that it is free intracellular [Ca2⫹] which regulates channel activity, and this should not be confused with total intracellular [Ca2⫹] (77). Thus, sickle cells contain inside-out vesicles of plasma membrane which sequester Ca2⫹, which is unavailable for Gardos channel activation but which results in a high total intracellular [Ca2⫹] (61). THE K⫹-Cl⫺ COTRANSPORTER (KCC)
THE DEOXYGENATION-INDUCED CATION PATHWAY (Psickle)
Finally, directly coupled K⫹ and Cl⫺ fluxes across the membrane of HbS cells are also high. These are mediated by KCC, probably the KCC1 isoform (8, 10, 15, 80). This transporter is a member of the cation-coupled Cl⫺ cotransporter superfamily, which includes NKCC and the thiazide-sensitive Na⫹-Cl⫺ cotransporter (NCC) (69). The cotransporter is regulated by phosphorylation via a cascade of protein kinases and phosphatases, with a final serine–threonine dephosphorylation associated with activity (48). An upstream tyrosine residue phosphorylation may also be involved with transporter stimulation (18). It is reg-
Psickle is a fairly nonspecific cation channel or channels of unknown identity which opens stochastically on deoxygenation (4, 51, 62, 78, 90) (see Fig. 2b). Much of the early work on this entity was carried out by Joiner and colleagues (50, 51) who used passive Na⫹ and K⫹ fluxes to show activation of Psickle as PO2 falls below about 40 mmHg. Psickle can mediate both entry of Na⫹ and loss of K⫹, and in the presence of extracellular divalent cations, K⫹ loss is greater than Na⫹ entry, hence it can mediate dehydration directly (52). In addition, due to the 3:2 stoichiometry of 307
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Gibson and Ellory
FIG. 3. Model for dehydration of sickle cells and stabilization of cell volume using transport inhibitors. (a) Under oxygenated conditions, cells can lose K⫹ and Cl⫺ through KCC should cells experience low pH or urea; Psickle and the Gardos channel are inactive. (b) When deoxygenated, changes in membrane permeability are exacerbated and can interact to produce rapid dehydration and sickling. Thus, HbS polymerizes, Psickle is activated, intracellular Ca2⫹ levels become sufficient for activation of the Gardos channel; KCC remains active despite deoxygenation and can be stimulated by low pH and urea. Isosmotic loss of K⫹ and Cl⫺ will elevate [HbS] and promote sickling, and also acidify the cell and further stimulate KCC. (c) Eventually, but much more rapidly under hypoxic conditions, solute loss and dehydration results in irreversible sickling. (d) Finally, partial inhibition of these events, perhaps by combinations of inhibitors, allow stabilization of cell volume and prevention of sickling: clotrimazole (CLT) and new agents like ICA17403 inhibit the Gardos channel; intracellular Mg2⫹ and Cl⫺ channel blockers like dihydroindenyloxyalkanoic acid (DIOA) inhibit KCC; 12C79 and DMA inhibit HbS polymerization; and activation of Psickle; NS3623 reduces Cl⫺ permeability and thereby also inhibits conductive loss of cations via the Gardos channel.
renal medulla), thereby increasing the contribution of KCC to cell dehydration.
ulated by swelling, reduction in pH (from about 7.4 to 7.0), urea, and high temperature, with H⫹ ions likely to be the most important stimulus in vivo (21, 22). As noted in the previous section, it is also O2-dependent, so that in normal red cells, KCC1 is quiescent in the absence of sufficiently high levels of O2 tension (35) (Fig. 2a). The O2 response in HbS cells, however, is different. In these cells, the transporter is highly active at high, arterial PO2 levels, starts to deactivate as PO2 levels are lowered, but then at tensions below about 40 mmHg activity increases again (Fig. 2c). In fully deoxygenated cells, activity is not dissimilar to that in fully oxygenated ones, and, in fact, on average, is about 10% higher (35). This abnormal O2 response is critical. It means that HbS cells, but not HbA ones, have a KCC1 that can respond to H⫹ ions (and urea) in hypoxic regions of the circulation, such as active muscle beds (or
INTERACTION OF THE VARIOUS PERMEABILITY PATHWAYS Together, these three pathways mediate solute loss, water will follow osmotically, with subsequent cell shrinkage. Their relative contribution, however, remains controversial (e.g., 2, 28, 74), and probably varies with PO2 and cell fraction, as well as between individuals (50). In addition, this array of transporters provides considerable scope for volume instability, notably positive feedback with a potential for rapid dehydration (6) (see Fig. 3). Thus, solute loss via any of the pathways will elevate [HbS], promote HbS polymerization and further activation of Psickle (and probably KCC— see later); isosmotic efflux of KCl will reduce 308
Gibson and Ellory
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
intracellular [Cl⫺], via activation of Cl⫺ influx and HCO⫺ 3 efflux via AE1, thereby acidify the cell, providing another stimulus for KCC activity; and, even simple oxy-deoxy transitions of Hb as the cells circulate between the arterial and venous vessels will also produce pH transients (60), which again can potentially stimulate KCC (see Fig. 3). Finally, since there is no obvious mechanism by which shrunken cells can regain lost solutes, solute efflux need not be a large all-ornone event, rather it may be intermittent and of relatively modest magnitude during each episode, before cumulative loss places cell volume at a critical level.
transporter in the membrane, the transduction pathway—the regulatory protein kinase/phosphatase enzymes—maintains the transporter in its inactive state, for example through steric hindrance, or insufficient or abnormal levels of the participating enzymes. TRANSPORT OF AMINO ACIDS Transport of organic solutes is also different in HbS cells compared to HbAs. Thus, we have examined several amino acid transporters including system gly and ASC (57). Both transport small uncharged amino acids: system ASC is a Na⫹-dependent transporter, typically transporting alanine, serine, and cysteine (hence the acronym), but whose main function is probably transport of cysteine; system gly is both Na⫹- and Cl⫺-dependent transporter, whose main substrate is glycine. In the red cell, these systems are important as they provide two of the three precursors of reduced glutathione (GSH) (23), a tripeptide of glycine, cysteine and glutamic acid, which forms a major part of the anti-oxidant defense of the red cell. In HbS cells, levels of GSH are lower than normal (58), although it is not clear whether this follows reduced synthesis or increased consumption (see later). In HbS cells, we have shown that systems ASC and gly are upregulated compared to HbA cells (57). Furthermore, both systems are stimulated by oxygenation. The kinetics are complicated and differ between the two: for example, oxygenation increases Vmax of system gly with no change in Km; for system ASC, Vmax is unaffected but Km halves on oxygenation. The significance of these effects has not been fully investigated.
COMPARISON WITH NORMAL RED CELLS The three pathways responsible for HbS cell dehydration remain cryptic in normal HbA cells. Psickle is not seen, although notwithstanding its apparent absence from HbA cells, the gene or genes encoding for it must be present. The Gardos channel is observed (29), but normally remains inactive because the high capacity PCMA, coupled with relatively low passive Ca2⫹ permeability, maintain intracellular Ca2⫹ below the threshold (63, 90). Ca2⫹ levels will rise and stimulate the Gardos channel should the pump be inhibited, for example by metabolic depletion, as in the original experiments of Gardos (29). Finally, while KCC is also present in the membrane, it has a much lower capacity than in HbS cells (20). The reason for the smaller maximal activity is not entirely clear. KCC activity is higher in younger HbA cells, and certainly the average cell population is younger in SCD (15, 40). That does not appear to be the entire explanation, however. Recently, it has been shown that expression of KCC is higher in HbS cells than HbA ones (88), although again it is not known why. Finally, as well as having a reduced maximal activity, KCC in HbA cells is largely quiescent and unresponsive to physiological stimuli (such as H⫹ ions and swelling), although it is responsive to pharmacological manipulations such as treatment with Nethylmaleimide, ionophore-induced Mg2⫹ depletion or high hydrostatic pressure (11, 20, 39). Presumably, although there is expression of the
MECHANISMS RESPONSIBLE FOR THE ALTERED TRANSPORT PHENOTYPE OF SICKLE CELLS There are many reasons why the membrane of HbS cells may differ from those of normal HbA cells, we discuss some below. First, the cell population is much younger in sickle cell disease, as evident from the reduced life span of affected cells. Young HbA cells per se, however, isolated as the least dense fraction on 309
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Gibson and Ellory
density gradients, do not share the same properties as HbS cells. There is no evidence of Psickle on deoxygenation, nor of an active KCC at low PO2 levels (32, 56). Second, red cells from patients with SCD contain HbS, rather than the normal HbA. This has a lower O2 affinity (12), and may also have different affinity for key regulatory sites controlling membrane permeability. It certainly has altered charge. Loss of negative charges at key residues on the  chain (mainly around positions 6 and 7) is associated with increased activity of KCC (76). The O2 response of these hemoglobinopathies, aside from sickle cells, remains poorly investigated. We have shown recently, however, that red cells containing HbSC, although they sickle at low PO2 levels and develop a pathway like that of Psickle, have KCC which is still inactivated by deoxygenation (34). This uncoupling of Psickle and sickling from low PO2-induced KCC is intriguing. Deoxy HbS also polymerizes. These polymers of HbS distort the membrane, and also sequester cell water and alter cell metabolism (12, 19, 59). Treatment of cells with the substituted benzaldehyde, 12C79, or the crosslinking agent dimethyl adipimidate (DMA), increases the O2 affinity of HbS (3, 36, 37). Both reagents also shift the deoxy-induced changes in shape and membrane permeability—i.e., sickling, and activation of Psickle and the deoxy-component of KCC—toward lower PO2 levels (32, 37) (Figs. 2b and 2c). Correlation of these transport events with the sickling shape change (Fig. 2d) implies that both result from polymerization of HbS, or a secondary effect of polymerization. The different response of HbSC-containing red cells can be rationalized if deoxygenated HbC, unincorporated into polymer, remains free and is thus able to act, like deoxyHbA, as an inhibitor of KCC activity (34). A third difference is the presence of higher protein tyrosine kinase (PTK) activity in HbS cells (66, 67). On deoxygenation, PTK activity is elevated further. Although inhibition of KCC is associated with a serine-threonine phosphorylation event, phosphorylation of tyrosine residues appears to operate in the opposite way, i.e., to stimulate transport (see above). One target for red cell PTK is cdb3 of AE1, and its phosphorylation
is indeed increased in HbS cells (67). It may be that this is sufficient to displace deoxy Hb, or other proteins which bind to this site (64), and thereby moderate the inhibitory effect of deoxygenation on KCC activity. Finally, oxidant damage or changes in redox potential may be important. Sickle cells contain lower levels of reduced glutathione (GSH) than normal red cells (58). They are also subject to the oxidative effects of hemichromes, free heme and iron, and possibly other species because of the relative instability of HbS (41, 42). We, and other groups, have investigated the action of a number of oxidants including chloro-2,4-dinitrobenzene, phenazine methosulfate (phenazine), menadione sodium bisulfite, and nitrite on HbA and HbS cells (33, 38, 71, 72, 75). Results show that all of these oxidants stimulate KCC activity, with stimulation correlating with metHb formation rather than GSH depletion (cf. 75). They also reduce inhibition of KCC activity in deoxygenated cells. Furthermore, phenazine in particular also activates the Gardos channel (71). For phenazine, stimulation of both KCC and the Gardos is more marked in deoxygenated cells compared with oxygenated ones, a transport phenotype which begins to resemble that of deoxygenated HbS cells. The effects of phenazine on deoxygenated red cells depends on the presence of extracellular Ca2⫹. It appears to inhibit PMCA, and to a greater extent on dexoygenation (33). This effect, coupled with a possible increased sensitivity of the Gardos channel for intracellular Ca2⫹ (1), may account for its action on the channel. In the case of KCC, calyculin A, a serine-threonine phosphatase inhibitor, abolishes the action of phenazine when added prior to the oxidant, but has only modest inhibitory effects when added afterwards, implying decreased activity of an inhibitory protein kinase (33). A fall in [ATP] could explain both inhibition of PMCA and the kinase, but bulk ATP levels remain high. It may be that a privileged pool of ATP is involved, responsible for regulation of membrane pumps and other transporters (43), and that this is affected by oxidants. A further possibility is specific effects of changes in redox potential. We are investigating some of these possibilities. 310
Gibson and Ellory
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
ACKNOWLEDGMENTS
11.
This work was supported by Action Research and The Wellcome Trust. The text is based on the Jacobs– Parpart–Ponder Lecture given by Professor Ellory to the American Red Cell Club, San Francisco, California, 24 February, 2002.
12.
13.
14.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Alvarez, J., Garcia-Sancho, J., and Herreros, B. (1984) Effects of electron donors on Ca2⫹-dependent K⫹ transport in one-step inside-out vesicles from the human erythrocyte membrane. Biochim. Biophys. Acta 771, 23–27. Apovo, M., Beuzard, Y., Galacteros, F., Bachir, D., and Giraud, F. (1994) The involvement of the Cadependent K channel and of the KCl cotransport in sickle cell dehydration during cyclic deoxygenation. Biochim. Biophys. Acta 1225, 255–258. Beddell, C. R., Goodford, P. J., Kneen, G., White, R. D., Wilkinson, S., and Wootton, R. (1984) Substituted benzaldehydes designed to increase the oxygen affinity of human haemoglobin and inhibit the sickling of sickle erythrocytes. Br. J. Pharmacol. 82, 397– 407. Berkowitz, L. R., and Orringer, E. P. (1985) Passive sodium and potassium movements in sickle erythrocytes. Am. J. Physiol. 249, C208 –C214. Bookchin, R. M., Etzion, Z., Sorette, M. P., Mohandas, N., and Lew, V. L. (1993) Heterogeneity of ion transport in sickle cell anemia reticulocytes revealed by patterns of acid-induced dehydration. Blood 82, 350a. Bookchin, R. M., Ortiz, O. E., and Lew, V. L. (1991) Evidence for a direct reticulocyte origin of dense red cells in sickle cell anemia. J. Clin. Invest. 87, 113– 124. Borgese, F., Motais, R., and Garcia-Romeu, F. (1991) Regulation of Cl-dependent K transport by oxy-deoxyhemoglobin transitions in trout red cells. Biochim. Biophys. Acta 1066, 252–256. Brugnara, C., Bunn, H. F., and Tosteson, D. C. (1986) Regulation of erythrocyte cation and water content in sickle cell anemia. Science 232, 388 –390. Brugnara, C., de Franceschi, L., and Alper, S. L. (1993) Inhibition of Ca⫹⫹-dependent K⫹ transport and cell dehydration in sickle erythrocytes by clotrimazole and other imidazole derivatives. J. Clin. Invest. 92, 520 –526. Brugnara, C., Kopin, A. S., Bunn, H. F., and Tosteson, D. C. (1985) Regulation of cation content and cell volume in hemoglobin erythrocytes from patients with homozygous hemoglobin C disease. J. Clin. Invest. 75, 1608 –1617.
15.
16.
17.
18.
19. 20.
21.
22.
23.
24.
25.
26. 311
Brugnara, C., and Tosteson, D. C. (1987) Inhibition of K transport by divalent cations in sickle erythrocytes. Blood 70, 1810 –1815. Bunn, H. F., and Forget, B. G. (1986) Hemoglobin: Molecular, Genetic and Clinical Aspects. Saunders, Philadelphia. Campbell, E. H., and Gibson, J. S. (1998) Magnesium and the O2 dependence of KCl cotransport in LK sheep red cells. J. Physiol. 506(3), 679 – 688. Campbell, E. H., and Gibson, J. S. (1997) Oxygenation and KCl cotransport in sheep red cells. J. Physiol. 501, P154 –P155. Canessa, M., Fabry, M. E., Blumenfeld, N., and Nagel, R. L. (1987) Volume-stimulated, Cl-dependent K efflux is highly expressed in young human red cells containing normal hemoglobin or HbS. J. Membr. Biol. 97, 97–105. Corash, L. M., Piomelli, S., Chen, H. C., Seaman, C., and Gross, E. (1984) Separation of erythrocytes according to age on a simplified density gradient. J. Lab. Clin. Med. 84, 147–151. Cossins, A. R., and Gibson, J. S. (1997) Volumesensitive transport systems and volume homeostasis in vertebrate red blood cells. J. Exp. Biol. 200, 343–352. Cossins, A. R., Weaver, Y. R., Lykkeboe, G., and Nielsen, O. B. (1994) Role of protein phosphorylation in control of K flux pathways of trout red blood cells. Am. J. Physiol. 267, C1641–C1650. Eaton, W. A., and Hofrichter, J. (1987) Hemoglobin S gelation and sickle cell disease. Blood 70, 1245–1266. Ellory, J. C., Dunham, P. B., Logue, P. J., and Stewart, G. W. (1982) Anion-dependent cation transport in erythrocytes. Phil. Trans. Roy. Soc. London B 299, 483– 495. Ellory, J. C., Gibson, J. S., and Stewart, G. W. (1998) Pathophysiology of abnormal cell volume in human red cells. Contrib. Nephrol. 123, 220 –239. Ellory, J. C., Hall, A. C., and Ody, S. A. (1989) Is acid a more potent activator of KCl co-transport than hypotonicity in human red cells? J. Physiol. 420, 149P. Ellory, J. C., Preston, R. L., Osotimehin, B., and Young, J. D. (1983) Transport of amino acids for glutathione synthesis in human and dog red cells. Biomed. Biophys. Acta 42, 48 –52. Etzion, Z., Tiffert, T., Bookchin, R. M., and Lew, V. L. (1993) Effects of deoxygenation on active and passive Ca2⫹ transport and on the cytoplasmic Ca2⫹ levels of sickle cell anemia red cells. J. Clin. Invest. 92, 2489 –2498. Fabry, M. E., Mears, J. G., Patel, P., Schaefer-Rego, K., Carmichael, L. D., Martinez, G., and Nagel, R. L. (1984) Dense cells in sickle cell anaemia: The effects of gene interaction. Blood 64, 1042–1048. Fabry, M. E., and Nagel, R. L. (1982) Heterogeneity of red cells in the sickler: A characteristic with prac-
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
Gibson and Ellory
tical clinical and pathophysiological implications. Blood Cells 8, 9 –15. Fabry, M. E., Romero, J. R., Buchanan, I. D., Suzuka, S. M., Stamatoyannopoulos, G., Nagel, R. L., and Canessa, M. (1991) Rapid increase in red blood cell density driven by K:Cl cotransport in a subset of sickle cell anemia reticulocytes and discocytes. Blood 78, 217–225. Franco, R. S., Palascak, M., Thompson, H., Rucknagel, D. L., and Joiner, C. H. (1996) Dehydration of transferrin receptor-positive sickle reticulocytes during continuous or cyclic deoxygenation: Role of KCl cotransport and extracellular calcium. Blood 88, 4359 – 4365. Gardos, G. (1958) The function of calcium and potassium permeability of human erythrocytes. Biochim. Biophys. Acta 30, 653– 654. Gibson, J. S., Cossins, A. R., and Ellory, J. C. (2000) Oxygen-sensitive membrane transporters in vertebrate red cells. J. Exp. Biol. 203, 1395–1407. Gibson, J. S., Honess, N. A., Cossins, A. R., Ellory, J. C., and Schwabe, A. E. (1994) Oxygen tension and KCl co-transport in equine red cells. J. Physiol. 476, 60P. Gibson, J. S., Khan, A., Speake, P. F., and Ellory, J. C. (2001) O2 dependence of K⫹ transport in sickle cells: The effects of different cell populations and the substituted benzaldehyde, 12C79. FASEB J. 15, 823– 832. Gibson, J. S., and Muzyamba, M. C. (2002) The effect of phenazine metosulphate on Ca2⫹ transport in human red blood cells. J. Physiol., in press. Gibson, J. S., Muzyamba, M. C., Ball, S. E., and Ellory, J. C. (2001) K⫹ transport in HbSC-containing human red blood cells. J. Physiol., in press. Gibson, J. S., Speake, P. F., and Ellory, J. C. (1998) Differential oxygen sensitivity of the K⫹-Cl⫺ cotransporter in normal and sickle human red blood cells. J. Physiol. 511, 225–234. Gibson, J. S., Speake, P. F., and Ellory, J. C. (1999) The effect of the substituted benzaldehyde, 12C79, on K⫹ transport in human red blood cells. Eur. J. Physiol. 437, 498 –500. Gibson, J. S., Stewart, G. W., and Ellory, J. C. (2000) Effect of dimethyl adipimidate on K⫹ transport and shape change in red blood cells from sickle cell patients. FEBS Lett. 480, 179 –183. Gusev, G. P., and Bogdanova, A. Y. (1992) Evidence for stimulation of the K-Cl cotransport system by phenazine methosulphate. Biochem. Pharmacol. 43, 2275–2279. Hall, A. C., and Ellory, J. C. (1986) Effect of high hydrostatic pressure on ‘passive’ monovalent cation transport in human red cells. J. Membr. Biol. 94, 1–17. Hall, A. C., and Ellory, J. C. (1986) Evidence for the presence of volume-sensitive KCl transport in ‘young’
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55. 312
human red cells. Biochim. Biophys. Acta 858, 317– 320. Hebbel, R. P., Eaton, J. W., Balasingam, M., and Steinberg, M. H. (1982) Spontaneous oxygen radical generation by sickle erythrocytes. J. Clin. Invest. 70, 1253–1259. Hebbel, R. P., Morgan, W. T., Eaton, J. W., and Hedlund, B. E. (1988) Accelerated autoxidation and heme loss due to instability of sickle hemoglobin. Proc. Natl. Acad. Sci. USA 85, 237–241. Hoffman, J. F. (1997) ATP compartmentalization in human erythrocytes. Curr. Opin. Hematol. 4, 112– 115. Hoffman, J. F. (1958) On the relationship of certain erythrocyte characteristics of their physiological age. J. Cell. Comp. Physiol. 51, 415– 423. Hoffman, J. F. (1958) Physiological characteristics of human red blood cell ghosts. J. Gen. Physiol. 42, 9 –28. Ingram, V. M. (1957) Gene mutations in human haemoglobin, the chemical difference between normal and sickle cell haemoglobin. Nature 180, 326 –328. Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) A human intermediate conductance calcium-activated potassium channel. Proc. Natl. Acad. Sci. USA 94, 11651–11656. Jennings, M. L., and Al-Rohil, N. (1990) Kinetics of activation and inactivation of swelling-stimulated K/Cl transport: The volume-sensitive parameter is the rate constant for inactivation. J. Gen. Physiol. 95, 1021–1040. Jensen, F. B. (1990) Nitrite and red cell function in carp: Control factors for nitrite entry, membrane potassium ion permeation, oxygen affinity and methaemoglobin formation. J. Exp. Biol. 152, 149 –166. Joiner, C. H. (1993) Cation transport and volume regulation in sickle red blood cells. Am. J. Physiol. 264, C251–C270. Joiner, C. H., Dew, A., and Ge, D. L. (1988) Deoxygenation-induced fluxes in sickle cells. I. Relationship between net potassium efflux and net sodium influx. Blood Cells 13, 339 –348. Joiner, C. H., Jiang, M., and Franco, R. S. (1995) Deoxygenation-induced cation fluxes in sickle cells. IV. Modulation by external calcium. Am. J. Physiol. 269, C403–C409. Joiner, C. H., Platt, O. S., and Lux, S. E. (1986) Cation depletion by the sodium pump in red cells with pathological cation leaks: Sickle cells and xerocytes. J. Clin. Invest. 78, 1487–1496. Kaplan, J. H. (1982) Sodium pump-mediated ATP: ADP exchange. The sided effects of sodium and potassium ions. J. Gen. Physiol. 80, 915–937. Khan, A., Gibson, J. S., and Ellory, J. C. (2000)
Gibson and Ellory
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
Oxygen-dependent KCl cotransport in ghosts from normal human red blood cells. J. Physiol. 527P, 38P. Khan, A., Speake, P. F., Gibson, J. S., and Ellory, J. C. (2000) Density gradient separation of sickle cells. J. Physiol. 525, 25P. Kiessling, K., Roberts, N., Gibson, J. S., and Ellory, J. C. (2000) A comparison in normal individuals and sickle cell patients of reduced glutathione precursors and their transport between plasma and red cells. Hematol. J. 1, 243–249. Lachant, N. A., Davidson, W. D., and Tanaka, K. R. (1983) Impaired pentose phosphate shunt function in sickle cell disease: A potential mechanism for increased Heinz body formation and membrane lipid peroxidation. Am. J. Hematol. 15, 1–13. Lew, V. L., Balazs, T., and Bookchin, R. M. (1995) Osmotic effects of water compartments in protein polymers. Cell Biochem. Funct. 13, 173–176. Lew, V. L., Freeman, C. J., Ortiz, O. E., and Bookchin, R. M. (1991) A mathematical model of the volume, pH and ion content regulation in reticulocytes. J. Clin. Invest. 87, 100 –112. Lew, V. L., Hockaday, A., Sepulveda, M. I., Somlyo, A. P., Somlyo, A. V., Ortiz, O. E., and Bookchin, R. M. (1985) Compartmentalization of sickle cell calcium in endocytic inside-out vesicles. Nature 315, 586 –589. Lew, V. L., Ortiz, O. E., and Bookchin, R. M. (1997) Stochastic nature and red cell population distribution of the sickling-induced Ca2⫹ permeability. J. Clin. Invest. 99, 2727–2735. Lew, V. L., Tsien, R. Y., Miner, C., and Boockchin, R. M. (1982) Physiological [Ca2⫹]i level and pumpleak turnover in intact red cells measured using an incorporated Ca chelator. Nature 298, 478 – 481. Low, P. S. (1986) Structure and function of the cytoplasmic domain of band 3: Center of erythrocyte membrane–peripheral protein interactions. Biochim. Biophys. Acta 864, 145–167. Marotta, C. A., Wilson, J. T., Forget, B. J., and Weissman, S. M. (1977) Human beta-globin messenger RNA. III. Nucleotide sequences derived from complementary DNA. J. Biol. Chem. 252, 5040 –5051. Merciris, P., Hardy-Dessources, M. D., and Giraud, F. (2001) Deoxygenation of sickle cells stimulates Syk tyrosine kinase and inhibits a membrane tyrosine phosphatase. Blood 98, 3121–3127. Merciris, P., Hardy-Dessources, M. D., Sauvage, M., and Giraud, F. (1998) Involvement of deoxygenationinduced increase in tyrosine kinase activity in sickle cell dehydration. Pfluger’s Arch. Eur. J. Physiol. 436, 315–322. Motais, R., Garcia-Romeu, F., and Borgese, F. (1987) The control of Na/H exchange by molecular oxygen in trout erythrocytes. J. Gen. Physiol. 90, 197–207.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
313
Mount, D. B., Delpire, E., Gamba, G., Hall, A. E., Poch, E., Hoover, R. S. J., and Hebert, S. C. (1998) The electroneutral cation-chloride cotransporters. J. Exp. Biol. 201, 2091–2102. Muzyamba, M. C., Cossins, A. R., and Gibson, J. S. (1999) Regulation of Na⫹-K⫹-2Cl⫺ cotransport in turkey red cells: The role of oxygen tension and protein phosphorylation. J. Physiol. 517, 421– 429. Muzyamba, M. C., and Gibson, J. S. (2001) The effect of phenazine methosulphate on K⫹ transport in human red blood cells. J. Physiol., in press. Muzyamba, M. C., Speake, P. F., and Gibson, J. S. (2001) The effect of oxidants on O2-dependent K⫹ transport in normal human red blood cells. J. Physiol. 531, 124P. Muzyamba, M. C., Speake, P. F., and Gibson, J. S. (2000) Oxidants and regulation of KCl cotransport in equine red blood cells. Am. J. Physiol. 279, C981– C989. Ohnishi, S. T., Horiuchi, K. Y., and Horiuchi, K. (1986) The mechanism of in vitro formation of irreversibly sickled cells and modes of action of its inhibitors. Biochim. Biophys. Acta 886, 119 –129. Olivieri, O., Bonollo, M., Friso, S., Girelli, D., Corrocher, R., and Vettore, L. (1993) Activation of K⫹/ Cl⫺ cotransport in human erythrocytes exposed to oxidative agents. Biochim. Biophys. Acta 1176, 37– 42. Olivieri, O., Vitoux, D., Galacteros, F., Bachir, D., Blouquit, Y., Beuzard, Y., and Brugnara, C. (1992) Hemoglobin variants and activity of (K⫹Cl⫺) cotransport system in human erythrocytes. Blood 79, 793– 797. Ortiz, O. E., Lew, V. L., and Bookchin, R. M. (1986) Calcium accumulated by sickle cell anaemia red cells does not affect their potassium (86Rb) flux components. Blood 67, 710 –715. Ortiz, O. E., Lew, V. L., and Bookchin, R. M. (1990) Deoxygenation permeabilizes sickle cell anaemia red cells to magnesium and reverses its gradient in the dense cells. J. Physiol. 427, 211–226. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C. (1949) Sickle cell anemia, a molecular disease. Science 110, 1141–1152. Pellegrino, C. M., Rybicki, A. C., Musto, S., Nagel, R. L., and Schwartz, R. S. (1998) Molecular identification of erythroid K:Cl cotransporter in human and mouse erythroleukemic cells. Blood Cells Mol. Dis. 24, 31– 40. Rhoda, M. D., Apovo, M., Beuzard, Y., and Giraud, F. (1990) Ca2⫹ permeability in deoxygenated sickle cells. Blood 75, 2453–2458. Schwoch, G., and Passow, H. (1973) Preparation and properties of human erythrocyte ghosts. Mol. Cell. Biochem. 2, 197–218.
Blood Cells, Molecules, and Diseases (2002) 28(3) May/June: 303–314 doi:10.1006/bcmd.2002.0515
83. 84. 85.
86.
87.
88.
89.
Gibson and Ellory
Serjeant, G. R. (2001) The emerging understanding of sickle cell disease. Br. J. Haematol. 112, 3–18. Serjeant, G. R. (1997) Sickle-cell disease. Lancet 350, 725–730. Speake, P. F., and Gibson, J. S. (1997) Urea-stimulated K-Cl cotransport in equine red blood cells. Pfluger’s Arch. Eur. J. Physiol. 434, 104 –112. Speake, P. F., Roberts, C. A., and Gibson, J. S. (1997) Effect of changes in respiratory blood parameters on equine red blood cell K-Cl cotransporter. Am. J. Physiol. 273, C1811–C1818. Stuart, J., and Ellory, J. C. (1988) Rheological consequences of erythrocyte dehydration. Br. J. Haematol. 69, 1– 4. Su, W., Shmukler, B. E., Chernova, M. A., StuartTilley, A. K., De Franceschi, L., Brugnara, C., and Alper, S. L. (1999) Mouse K-Cl cotransporter KCC1: Cloning, mapping, pathological expression, and functional regulation. Am. J. Physiol. 277, C899 –C912. Tiffert, T., Etzion, Z., Bookchin, R. M., and Lew, V. L. (1993) Effects of deoxygenation on active and passive
90.
91.
92.
93.
94.
314
Ca2⫹ transport and cytoplasmic Ca2⫹ buffering in normal human red cells. J. Physiol. 464, 529 –544. Tosteson, D. C. (1955) The effects of sickling on ion transport. II. The effect of sickling on sodium and cesium transport. J. Gen. Physiol. 39, 55– 67. Tosteson, D. C., Carlsen, E., and Dunham, E. T. (1955) The effects of sickling on ion transport. I. Effect of sickling on potassium transport. J. Gen. Physiol. 39, 31–53. Tosteson, D. C., Shea, E., and Darling, R. C. (1952) Potassium and sodium of red blood cells in sickle cell anaemia. J. Clin. Invest. 48, 406 – 411. Wolff, D., Cecchi, X., Spalvins, A., and Canessa, M. (1988) Charybdotoxin blocks with high affinity the Ca-activated K⫹ channel of HbA and HbS red cells: Individual differences in the number of channels. J. Membr. Biol. 106, 243–252. Wood, P. G. (1989) Preparation of resealable membranes maximally depleted of cytoplasmic components by gel filtration and tryptic digestion: Resealable white ghosts. Methods Enzymol. 173, 346 –355.