Physiol Rev 85: 179 –200, 2005;
doi:10.1152/physrev.00052.2003.
Ion Transport Pathology in the Mechanism
of Sickle Cell Dehydration
VIRGILIO L. LEW AND ROBERT M. BOOKCHIN
Physiological Laboratory, University of Cambridge, Cambridge, United Kingdom;
and Department of Medicine, Albert Einstein College of Medicine, Bronx, New York
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I. Introduction
A. Sickle cell anemia: first “molecular disease”
B. The long path from molecular origin to pathophysiology
II. Basics of Red Blood Cell and Reticulocyte Homeostasis
A. Methodological points
B. Na⫹-K⫹ fluxes in RBCs and reticulocytes
C. Pathways of RBC dehydration
III. How Do Sickle Cells Dehydrate?
A. Early findings and puzzles
B. Discovery of the high calcium content of SS cells
C. Discovery of endocytic inside-out vesicles
D. Failings of “gradualism”
E. Multitrack dehydration as an alternative to gradualism
F. Original working hypothesis on the mechanism of fast-track dehydration
G. Experimental tests of the fast-track dehydration hypothesis
IV. Dehydration-Resistant Cells
V. Properties of Psickle
A. Ion selectivity of Psickle
B. The stochastic nature of the Psickle distribution
VI. Therapeutic Strategies Aimed at Ion Transport Targets to Prevent Sickle Cell Dehydration
A. Antisickling strategies
B. Psickle inhibition
C. Gardos channel inhibition
D. Anion conductance inhibition
E. Control of KCC
VII. Open Questions and the Direction of Future Research
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Lew, Virgilio L., and Robert M. Bookchin. Ion Transport Pathology in the Mechanism of Sickle Cell Dehydration.
Physiol Rev 85: 179 –200, 2005; doi:10.1152/physrev.00052.2003.—Polymers of deoxyhemoglobin S deform sickle cell
anemia red blood cells into sickle shapes, leading to the formation of dense, dehydrated red blood cells with a
markedly shortened life-span. Nearly four decades of intense research in many laboratories has led to a mechanistic
understanding of the complex events leading from sickling-induced permeabilization of the red cell membrane to
small cations, to the generation of the heterogeneity of age and hydration condition of circulating sickle cells. This
review follows chronologically the major experimental findings and the evolution of guiding ideas for research in this
field. Predictions derived from mathematical models of red cell and reticulocyte homeostasis led to the formulation
of an alternative to prevailing gradualist views: a multitrack dehydration model based on interactive influences
between the red cell anion exchanger and two K⫹ transporters, the Gardos channel (hSK4, hIK1) and the K-Cl
cotransporter (KCC), with differential effects dependent on red cell age and variability of KCC expression among
reticulocytes. The experimental tests of the model predictions and the amply supportive results are discussed. The
review concludes with a brief survey of the therapeutic strategies aimed at preventing sickle cell dehydration and
with an analysis of the main open questions in the field.
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0031-9333/05 $18.00 Copyright © 2005 the American Physiological Society
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VIRGILIO L. LEW AND ROBERT M. BOOKCHIN
I. INTRODUCTION
B. The Long Path From Molecular Origin
to Pathophysiology
A. Sickle Cell Anemia: First “Molecular Disease”
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The wealth of information on the molecular, genetic,
and epidemiological aspects of SS gathered in the 1950s
and 1960s did little to help explain its pathophysiology.
We briefly review here some relevant facts which illustrate the difficulties in connecting the basic molecular
defect with clinical events. SS patients suffer chronic and
acute organ failure and episodic pain attributable to vasoocclusive events in the microcirculation (18, 20, 172). SS
RBCs exhibit marked heterogeneity of age, morphology,
and hydration state (17, 98). A characteristic of the SS
hematological profile is the presence of a highly dehydrated, dense, and rigid subpopulation of cells, many of
which have a typical “elongated hard potato” appearance,
which changes little on deoxygenation. These cells were
termed “irreversibly sickled cells” (ISCs), since their abnormal shape persisted when the cells were fully reoxygenated in vitro, reversing all intracellular Hb polymerization; this indicated that their shape depended on membrane alterations, shown later to involve their
cytoskeletons (209, 210). The “hyperdense” SS cells (e.g.,
␦ ⬎ 1.118 or MCHC ⬎ 45 g/dl), comprised mostly of ISCs,
were shown to play a complex role in microvascular
occlusion (172). Also, their proportion among circulating
RBCs appeared reduced during painful vaso-occlusive crisis, consistent with their selective retention within
blocked vascular domains (18, 20). However, although the
proportion of circulating ISCs was found to correlate with
the severity of the anemia (241), no significant correlation
was found between the magnitude of the dense cell fraction and the frequency or severity of painful crises (19).
The propensity of deoxy-Hb S to polymerize is much
reduced in the presence of substantial fractions of Hb A
or Hb F within the RBCs (255). RBCs from heterozygous
AS subjects, with only ⬃40% Hb S, do not sickle in vivo at
normal oxygen tensions, except in the hypertonic environment of the renal medulla, where their cell Hb concentrations are markedly increased by osmotic shrinkage.
Persons with sickle trait have normal hematological profiles and are asymptomatic, except under the high stress
of maximum exercise or in low PO2 conditions (high
altitude or sudden decompression in aircraft) (54). Persons doubly heterozygous for Hb S and for hereditary
persistence of fetal Hb (SF), have normal hematological
profiles and no clinical evidence of sickling (54). The
increased number of Hb F-containing RBCs (40, 84) in SS
patients is most enriched among the normal-density SS
cells and are selectively excluded from the densest cell
fractions (17) (see Fig. 1). Thus Hb combinations that
prevent sickling in vivo also prevent the development of
the hematological and clinical manifestations of SS, including the formation of very dense, dehydrated RBCs,
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Sickle cell anemia (SS) is an inherited disease whose
origin and demographic roots may be traced to malaria
endemic areas. The name derives from the peculiar sicklelike shapes that SS red blood cells (RBCs) acquire on
deoxygenation. The first indication that this abnormal
response resulted from a primary hemoglobin (Hb) abnormality was the observation that formation of sicklelike
spicules on deoxygenation of RBCs from SS infants was
absent or minimal until fetal hemoglobin (Hb F, ␣2␥2) was
replaced by adult Hb (␣2␤2), at about 3– 6 mo of age (272).
In 1949, applying moving boundary electrophoresis to
hemolysates from normal, sickle cell trait (AS) and SS
subjects, Pauling et al. (221) showed that nearly all the Hb
in the RBCs of (homozygous) SS patients had a mobility
different from that of normal Hb (Hb A), and that heterozygous AS carriers had both types of Hb in their RBCs,
⬃40% of abnormal Hb S and 60% of normal Hb A. This
crucial experiment identified a molecular disease and also
explained its pattern of inheritance.
In one of the first applications of two-dimensional
peptide mapping, Ingram (145, 146) identified the precise
molecular difference between Hbs A and S as a single
amino acid substitution on the ␤-chain of Hb, valine for
glutamic acid in position 6. Epidemiological studies suggested that the persistence of the AS trait in malariainfested areas resulted from the protection it afforded to
AS carriers against severe malaria caused by Plasmodium falciparum (2, 3). Two general protective mechanisms had been considered in the literature: restricted
parasite growth and enhanced immune retrieval of the
parasitized RBCs. Much of the early work is conflictive
and confusing, mainly because of the limitations of in
vitro studies to reproduce the conditions in vivo (1).
Recent work (9, 59, 135) strongly suggests that resistance
in AS RBCs is caused by enhanced phagocytosis of RBCs
infected with ring stage parasites in response to alterations of the membrane surface associated with increased
hemichrome formation, aggregated band 3, elevated autologous IgG, and complement C3c fragments. Studies by
a number of investigators (133, 224, 246), also in the early
1950s, characterized the functional abnormality of Hb S
responsible for the sickle morphology: deoxygenated Hb
S (deoxy-Hb S) tetramers spontaneously aggregate to
form birefringent “tactoid bodies” composed of bundles
of polymer fibers, which in turn formed a gel network. In
the RBCs, these fiber bundles protrude into the plasma
membrane generating elongated spiculed projections, a
process described as “sickling.” For more detailed and
complete historical backgrounds, with insightful anecdotic accounts, see chapters 11 and 12 in Bunn and Forget
(54) and Serjeant’s historical review (240).
MECHANISMS OF SICKLE CELL DEHYDRATION
II. BASICS OF RED BLOOD CELL AND
RETICULOCYTE HOMEOSTASIS
Human RBCs are water-permeable sacs with the
highest soluble protein concentration of any cell, ⬃7.2
mmol Hb/l cell water. A two-dimensional, spectrin-actin
based, meshlike cytoskeleton, with highly structured
links to integral membrane proteins embedded in the lipid
bilayer, endows the RBCs their characteristic biconcave
shape and flexibility (41, 66, 67, 209, 219, 220, 250). The
high water permeability of the RBCs ensures their continued osmotic equilibrium in plasma so that they can shrink
or swell only by the loss or gain of a fluid isosmotic with
surrounding plasma. Since the plasma protein concentration is ⬍1 mM, there is a powerful oncotic pressure
driving water into the cells. To remain osmotically stable
over their ⬃120-day circulatory life-span, mature RBCs
evolved a strategy to maintain a nearly constant volume
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with minimal energy expenditure: their Na⫹ and K⫹ permeabilities are extremely low (10, 186) so that relatively
few metabolically fuelled Na⫹ pumps per cell suffice to
balance their cation gradient-driven “leaks.” As a result,
the anion permeability was freed from limiting constraints, allowing the RBCs to evolve an extremely efficient CO2 ferry between tissues and lungs, based on high
Cl⫺ and HCO⫺
3 exchange (the Jacobs-Stewart cycle; Refs.
147, 148, 188) and electrodiffusional fluxes (130, 143, 144,
174). This high electrodiffusional anion permeability set
the membrane potential very near the equilibrium potentials of Cl⫺ and HCO⫺
3 , about ⫺10 mV (110, 114, 138, 139,
176, 177, 267).
The fine homeostatic balance described above for
normal RBCs, which is remarkably constant for most of
their 120-day life-span, is markedly altered in SS cells,
whose ion fluxes, ion content regulation, and hydration
state become highly disrupted in the circulation.
A. Methodological Points
1. Density fractionation of SS RBCs
Density fractionation of SS cells, which allows the
separation and subsequent study of RBCs in different
hydration states, has become an essential tool for research on the mechanism of dehydration. The density
distribution of SS cells is much more heterogeneous than
that of normal RBCs (17), as illustrated in Figure 1. The
usual method for simple analysis of RBC density distributions is centrifugation through Percoll or Percoll/arabinogalactan gradients (17, 69, 76, 98, 202), whereas for ion
transport studies, isolation of SS RBCs by density fractionation on discontinuous arabinogalactan gradients is
preferred (37, 76, 96, 216). Figure 1 provides a general
guide of the distribution of each cell type as a function of
density, and of their contributions to the heterogeneity
expected within each density band. The cells that predominate in the lightest density ranges are reticulocytes
and dehydration-resistant cells (DRCs) which represent a
recently discovered (29), high-Na⫹, low-K⫹ group of cells
that resist dehydration when K⫹-permeabilized in plasmalike media, as detailed below. In SS blood, F cells are
distributed broadly among the light density fractions rich
in reticulocytes and among the light discocytes, but are
most enriched among denser and older discocytes. Hyperdense ISCs occur almost exclusively among RBCs exceeding a density of 1.118 g/ml.
2. Ion fluxes
Ion fluxes in RBCs are usually expressed in units (or
subunits) of moles per liter original cells per hour (mol ·
l original cells⫺1 · h⫺1). “Original” means that measured
fluxes are referred to as 1 liter of normal, packed RBCs at
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despite the presence of substantial proportions of Hb S
within the cells.
This brief overview emphasizes that in vivo sickling
and generation of hyperdense SS RBCs are necessary
conditions for the clinical manifestations of the disease
and that the mechanisms by which sickling leads to the
marked age and hydration heterogeneity of SS cells are
among the most important pathophysiological issues in
SS disease, and the specific subject of this review. Over
five decades of intense research in many laboratories
elucidated the complex links between the single molecular defect and the mechanisms of SS cell dehydration. As
described below, important breakthroughs were provided
by integrated approaches to cell physiology. In an era so
dominated by “omics” and reductionist approaches, the
development of this important connection between basic
science and clinical application exemplifies the need for
ongoing physiological expertise to bridge the gap between genes and molecules at one end and function or
dysfunction at the other.
In this review we focus on the ideas which guided
research at each stage, and on the puzzling and conflicting
observations which led to the formulation of new questions and hypotheses; we point out how these formulations eventually led to the answers that represent the
current consensus on the mechanisms of SS cell dehydration. We briefly survey the therapeutic strategies aimed at
preventing SS cell dehydration and conclude with an
overview of the open questions and future developments
in the field.
For the benefit of readers not familiar with RBC
physiology, the next section introduces some basic concepts and methodological considerations specific to RBC
and reticulocyte homeostasis, which should help understand how SS RBCs become dehydrated in vivo.
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VIRGILIO L. LEW AND ROBERT M. BOOKCHIN
the beginning of an experiment. Because one liter normal
RBCs contains a mean of 340 g Hb or ⬃1013 RBCs, fluxes
are sometimes expressed in equivalent units of moles per
340 g Hb per hour (mol · 340 g Hb⫺1 · h⫺1) or moles per
1013 cells per hour (mol · 1013 cells⫺1 · h⫺1). To compare
RBC fluxes with those expressed per unit area in other
cell types, the following conversion applies assuming a
mean RBC area of 155 ␮m2 for human RBCs (273): 1 mol ·
l original cells⫺1 · h⫺1 is equivalent to ⬃2 ⫻10⫺19 mol ·
␮m⫺2 · s⫺1. With unidirectional cation fluxes in the order
of 2–3 mmol · l original cells⫺1 · h⫺1 in normal RBCs, this
is equivalent to a few hundred ions per ␮m2 per second,
an extremely low flux value among eukaryotes.
3. Use of ionophores
Much of the information about RBC Ca2⫹ and Mg2⫹
homeostasis was obtained using the divalent cation ionophore A23187 (77, 100, 102, 115, 194, 217, 229, 233, 237,
258, 262, 264). This important tool provided the state-ofthe-art method to measure the activity of the plasma
membrane Ca2⫹ pump (PMCA Vmax) in intact RBCs and
helped characterize the cytoplasmic Ca2⫹ and Mg2⫹ buffering properties of normal and SS RBCs (77, 96, 103, 193,
262). All these methods have been described in detail
before (198) and detailed in a recent review (258); we
note here only some of the unique ionophore properties
used in studies of SS RBC Ca2⫹ homeostasis. Addition of
the ionophore A23187 to RBCs generates an instant and
remarkably uniform increase in divalent cation permeability (203, 204, 243–245). This uniformity results from a
high partition coefficient of the ionophore in the RBC
membrane, with high on-off rate constants, as shown by
the instant redistribution of ionophore when ionophorefree RBCs are rapidly mixed into the suspension. Careful
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adjustments of three experimental parameters, hematocrit (Hct), extracellular Ca2⫹ concentration, and final concentration of ionophore in the cell suspension allow precise control of Ca2⫹ or Mg2⫹ fluxes, over a wide range of
values. Addition of Co2⫹ in excess of Ca2⫹ in the media
instantly arrests the ionophore-mediated Ca2⫹ fluxes, allowing exposure of uphill Ca2⫹ extrusion by the PMCA
after a Ca2⫹ load and thus providing the basis for measurements of RBC PMCA Vmax (44, 77, 230, 261). Although
Co2⫹ is itself transported by the ionophore, its intracellular effects on the PMCA are those of a weak Mg2⫹ replacement (230). Finally, it is important to be aware that
PMCA-saturating Ca2⫹ loads generate progressive and
eventually irreversible ATP depletion because the rate at
which a Ca2⫹-saturated PMCA hydrolyzes ATP exceeds
the glycolytic capacity of a large fraction of RBCs (192).
Irreversibility results from the conversion of ATP to IMP,
a three-enzyme process triggered by PMCA ATPase activity and can thus be prevented by inhibition of the PMCA
(77, 263).
B. Naⴙ-Kⴙ Fluxes in RBCs and Reticulocytes
The unidirectional fluxes of Na⫹ or K⫹ in mature
normal RBCs are ⬃2–3 mmol · l original cells⫺1 · h⫺1,
whereas in reticulocytes they may be 10- to 30-fold higher
(10, 156 –159, 232, 274). This traffic represents the instant
pump-leak balance of these cells: the Na⫹ pump, with its
3:2 Na⫹-K⫹ stoichiometry, compensates for the combined
net passive Na⫹ and K⫹ fluxes through all the individual
passive transport pathways (197). Reticulocytes are the
enucleated erythroid cells released from the bone marrow
into the circulation where they differentiate into mature
RBCs within 2–3 days. The maturation process involves
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FIG. 1. Approximate distribution profile of each
sickle cell (SS) cell type as a function of cell density and
hemoglobin concentration; comparison with the density
distribution of normal red blood cells (RBCs). The relation between hemoglobin concentration ([Hb]; in g/dl)
and density (in g/ml) (standard units in current use) was
computed from [Hb] ⫽ 378(D ⫺ 1), derived by Lew et al.
(202; Eq. 12), where D is density. The various types of SS
cells are described in the text. Reticulocytes, enucleated
RBC precursors recently released from the bone marrow
and retaining intracellular RNA; DRCs, dehydration-resistant cells; ISCs, irreversibly sickled cells; F cells, SS cells
with a high content of fetal Hb; discocytes, SS cells
retaining discoid shapes in the oxygenated state. The
diagram illustrates the different types of SS cells likely to
be encountered within each density band and also the
trend of variation of each cell type as a function of
density. These trends are largely conserved despite the
wide interpatient variations in the proportions of each
type of cell in whole blood and within each density band.
Line types and corresponding cell types: long dash,
DRCs; short dash, reticulocytes; dotted, F cells; dash-dot,
light discocytes; dash-double dot, dense discocytes; continuous, ISCs; light gray, normal RBCs.
MECHANISMS OF SICKLE CELL DEHYDRATION
C. Pathways of RBC Dehydration
Dehydration of human RBCs in vivo may result from
the activation of one or more of three transporters expressed in their plasma membranes: 1) the Ca2⫹-sensitive,
small-conductance, K⫹-selective channel (Gardos channel, IK1 or hSK4; Refs. 116, 137, 195, 269), also expressed
in many other cell types (225); 2) a K⫹-Cl⫺ cotransporter
(KCC), regulated by internal pH and cell volume (46, 87,
107, 121, 181, 184), functionally active in reticulocytes and
much less so in mature AA and SS RBCs; and 3) the Na⫹
pump (125, 227, 247). The three transporters differ considerably in their dehydrating modalities, potencies, and
distributions among RBCs.
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1. RBC calcium and the Gardos channels
At physiological intracellular Ca2⫹ concentration
([Ca ]i) levels of ⬃20 –50 nM, Gardos channels are inactive, but they can be activated when [Ca2⫹]i levels are
increased in pathological and experimental conditions.
The [Ca2⫹]i activation threshold of Gardos channels in
intact RBCs is probably ⬃150 nM (242, 264). The mean
number of Gardos channels per RBC is not yet established, but the most reliable estimates are ⬃100 –200 (4,
48, 129, 137, 200, 205, 276). When the Gardos channels are
maximally activated by a uniform, saturating Ca2⫹ load in
the RBCs, flow-cytometry measurements show that during dehydration, the original volume distribution of the
RBCs is conserved (205). This indicates that each donor’s
RBCs do not differ much in the number or functional
expression of their Gardos channels and that they all
dehydrate fast when their channels are maximally activated. Unlike erythroid cells from other mammalian species, whose Gardos channel activity is lost during maturation (43), mature human RBCs show persistent Gardos
channel activity. The single Gardos channel conductance
is ⬃10 pS in physiological conditions (128). The openstate probability of the channels at saturating Ca2⫹ levels
has a peculiar temperature dependence. At 35°C it is very
low (between 0.01 and 0.1); it increases sharply at 25°C
and then falls slowly with decreasing temperature (127,
137). Thus, for experimental purposes, Gardos-mediated
dehydration persists at low temperatures at which the
PMCA is markedly inhibited (115, 201). In low-K⫹ media,
activation of Gardos channels hyperpolarizes the cell
(177), generating a favorable gradient for co-ion (Cl⫺ or
HCO⫺
3 ) exit via parallel voltage-sensitive pathways. The
resulting net loss of KCl and KHCO3 is tightly coupled to
osmotic-driven water loss, leading to cell dehydration. At
saturating [Ca2⫹]i levels for the Gardos channels, above 2
␮M, the anion permeability becomes rate-limiting for net
KCl loss (144, 173); at intermediate activation levels, either cation or anion permeabilities may limit the rate of
dehydration. Dehydration via Gardos channels may be
reduced by direct inhibition of the channels with charybdotoxin (IC50 ⬃1.2 nM) or clotrimazole (IC50 ⬃51 nM) (5,
49), or indirectly, by inhibition of the anion permeability
(12, 13, 50). The capacity of Gardos channels to mediate
rapid RBC dehydration when fully activated far exceeds
that of the other transporters. Gardos channel-mediated
42
K or 86Rb fluxes may reach mean values of ⬃1 mol · l
original cells⫺1 · h⫺1 (194). Such fluxes reflect mean permeability values on the order of 2 ⫻ 10⫺7 cm/s, equivalent
to rate constants of ⬃10 h⫺1 (111). At physiological Cl⫺
and HCO⫺
3 concentrations, full dehydration by maximally
activated Gardos channels may take over 1 h, the ratelimiting anion permeability being on the order of 2 ⫻ 10⫺8
2⫹
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not only the loss of protein synthesis along with residual
RNA, but also reduction or loss of many metabolic functions including a gradual decline in monovalent cation
transport activity and the reduction or inactivation of
selected transporters such as Na⫹ pumps and K⫹-Cl⫺
cotransporters. Johnstone (155) showed that during maturation of reticulocytes, there was formation of exosomes
containing proteins and lipids characteristic of the plasma
membrane and that the transferrin receptor, known to be
lost during red cell maturation, was contained in these
exosomes. Intrinsic membrane proteins, such as the anion transporter, were retained, but there was no further
information about other ion transporters. More recent
studies on cation transport in rat reticulocytes (211)
showed that their increased Na⫹ pump activity was considerably reduced during maturation, to a greater extent
than their loss of membrane surface area, suggesting
more specific loss of transport protein. Because reticulocyte volume decreases only by ⬃13% during normal maturation (231, 232), there must be a coordinated and compensated reduction in pump and leak cation flux components (202). Circulating reticulocytes represent an
extremely heterogeneous population of young cells, in a
continuum of progressive maturation with decreasing Hb
synthesis and cation turnover rates. Because of their
larger volumes and incomplete Hb complement, normal
reticulocytes are lighter than mature RBCs and can be
enriched in the upper layers of density-fractionated RBC
samples. Clearly, however, cation flux measurements in
reticulocyte-rich RBC fractions represent mean values of
broad distributions, from the low values of the mature
RBCs to the much higher but less clearly determined
upper values of the youngest reticulocytes. It is important
to bear in mind the limitations this functional and cellular
heterogeneity imposes on the interpretation of experimental flux measurements in reticulocyte-rich cell fractions, when only mean values are obtained.
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VIRGILIO L. LEW AND ROBERT M. BOOKCHIN
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关Ca 2⫹ 兴 i ⬇ ␣ 关Ca T 兴 i
where [Ca2⫹]i is expressed in ␮mol/l cell water, ␣ ⬃0.2–
0.3 over a wide range of experimentally induced [CaT]i
values, and [CaT]i is expressed in units of ␮mol/l original
cells. Thus ⬃20 –30% of the total mobilizable RBC Ca2⫹
content is in ionized form in cell water. This is particularly
important because it indicates that only minute net total
Ca2⫹ gains are required for [Ca2⫹]i to reach threshold
activation levels of Gardos channels. Physiological
[Ca2⫹]i levels (⬃20 –50 nM) represent the balance between passive Ca2⫹ influx and active Ca2⫹ extrusion, with
a pump-leak turnover rate of ⬃50 ␮mol · l original cells⫺1 ·
h⫺1 (83, 206). As in many other cell types, active Ca2⫹
extrusion is mediated by a large-capacity (high Vmax),
ATP-fuelled, P-type pump, the PMCA (60, 257). In human
RBCs, the PMCA is expressed with genomic isoforms 1
and 4 (60, 61, 234, 238, 239, 271). The mean Vmax of the
PMCA varies from 5 to 25 mmol · l original cells⫺1 · h⫺1,
over two orders of magnitude higher than the mean pumpleak turnover rate of Ca2⫹ (77). Recent measurements
revealed a large variation (over an order of magnitude) in
PMCA Vmax among the RBCs within individual blood samples (192). The distribution followed a broad unimodal
pattern, with the lower Vmax cells perhaps reflecting a
declining Ca2⫹ extrusion capacity among aging RBCs. An
important implication of this finding is that individual
RBCs, comparably permeabilized to Ca2⫹, may reach balancing pump-leak steady states with [Ca2⫹]i levels below
or above the activation threshold of Gardos channels,
leading to markedly different dehydration responses. Deoxygenation of RBCs was also found to induce a ⬃20%
reduction in PMCA Vmax in both AA and SS RBCs, with
detectable effects on Ca2⫹-induced dehydration of SS
cells (96, 260).
2. The KCC; dehydration-induced acidification
KCCs are expressed in a large variety of cell types,
including erythroid precursors (121, 180, 181). In a pioneering study, Anderson et al. (6) detected transcripts for
three of the four known KCC isoforms (KCC1, KCC3, and
KCC4), and for two additional splicing isotypes, in RNA
obtained from human erythroid cells derived from AA and
SS blood. However, which of these KCC isoforms and
alternative spliced variants participate in the functional
expression of KCCs in human reticulocytes is still unknown. In normal and SS RBCs, KCC activity is substantially reduced during maturation but can be partially reactivated experimentally in mature RBCs by thiol-reactive
agents such as N-ethylmalemide (183) or by high hydrostatic pressure (131). In reticulocytes, most of the passive
K⫹ traffic is mediated by KCC (46, 57, 132). KCC is a
multiregulated transporter, influenced by the oxygenation
state of Hb (120, 166), and stimulated by intracellular
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cm/s, an order of magnitude lower than that through fully
activated Gardos channels. When Cl⫺ and HCO⫺
3 are partially replaced by the more permeable SCN⫺ or HNO⫺
3
anions in experimental conditions, full K⫹ equilibration
and dehydration may be attained within 3– 4 min (111,
115, 259). It is important to bear this in mind as it illustrates the powerful restrictive effects of the Cl⫺ and
HCO⫺
3 permeabilities on the speed and extent of RBC
dehydration attainable during the brief intermittent periods of Gardos channel activation induced by sickling in
the circulation, as discussed below. Under whole cell
patch-clamp conditions, the anion conductance of RBCs
was estimated to be below 100 pS/cell (82, 91), well within
the order of magnitude expected if the Cl⫺ permeability
were through a conductive pathway. However, inhibitors
of anion exchange also inhibit the Cl⫺ permeability,
prompting the prevailing view that the Cl⫺ permeability
represents a ⬃1/10,000 slippage through the anion exchange carrier rather than a separate conductive pathway
(112, 113, 118, 173). This view is indirectly supported by
the fact that Cl⫺ channels have thus far eluded detection
in excised RBC membrane patches. However, certain kinetic and experimental difficulties (beyond the scope of
the present review) led Bennekou and collaborators (12,
13) to support the view that the Cl⫺ permeability, though
still mediated by the same band 3 as the anion exchanger,
is through a diffusional configuration of the protein. This
group pioneered the use of anion permeability inhibitors
as a promising new therapeutic strategy in sickle cell
disease; their description of the anion permeability as a
conductance is therefore included in section VID. In the
present context, we would emphasize that it is only the
magnitude, not the precise mechanism, of the anion permeability path that is an important determinant of the rate
of dehydration induced by Gardos channel activation.
Because the activity of Gardos channels is tightly
controlled by [Ca2⫹]i, it is necessary to review briefly the
main processes involved in Ca2⫹ regulation in normal
RBCs. Mature RBCs lack specialized Ca2⫹ accumulating
organelles and Ca2⫹ signaling functions. Their total calcium content ([CaT]i) is ⬍5 ␮mol/l original cells (30, 93,
134), of which ⬍1 ␮mol/l original cells can be extracted
by treatment with Ca2⫹ ionophores in the presence of
high Ca2⫹ chelator concentrations in the medium (30).
This represents a maximal estimate, limited by measurement error, so that the true exchangeable Ca2⫹ is probably much lower. The constitutive Ca2⫹ permeability of
RBCs (PCa) is extremely low, and Ca2⫹ ionophores have
become essential tools in investigations requiring Ca2⫹
loading of the cells. Compared with other cell types, RBCs
have minimal cytoplasmic Ca2⫹ buffering capacity (100,
260, 262). Cytoplasmic Ca2⫹ buffering can be approximated by the equation
MECHANISMS OF SICKLE CELL DEHYDRATION
Physiol Rev • VOL
the cell, and this necessarily dilutes their intracellular
concentrations as dehydration proceeds. At approximately constant external concentrations of Cl⫺ ⫹ HCO⫺
3,
intracellular dilution causes the [Cl⫺]o/[Cl⫺]i and
⫺
[HCO⫺
3 ]o/[HCO3 ]i concentration ratios to increase. The
parallel joint operation of the anion exchanger (AE1;
Refs. 126, 175, 256) and CO2 shunt in the RBC membrane,
known as the Jacobs-Stewart mechanism (136, 147, 148),
ensures rapid equilibrium given by
关H ⫹ 兴 i 关Cl ⫺ 兴 o 关HCO 3⫺ 兴 o
⫽
⫽
关H ⫹ 兴 o 关Cl ⫺ 兴 i
关HCO 3⫺ 兴 i
with cell acidification resulting from the increased inward
anion concentration ratio. Cell acidification was also
found associated with regulatory volume decrease processes in other cell types and was assumed to operate by
the same mechanism as in RBCs (140).
3. The Na⫹ pump
The Na⫹-K⫹ flux ratio through the Na⫹ pump is 3:2
(117, 228). Because of the high anion permeability of
RBCs and reticulocytes, electroneutrality is maintained
mainly by anion efflux balancing the extra Na⫹ efflux. If
the Na⫹ pump is stimulated by elevated internal Na⫹/K⫹
concentration ratios, the resulting increased NaCl efflux,
if not compensated by changes in passive fluxes, would
induce cell dehydration (70, 123, 170). But with only ⬃400
copies of the Na⫹ pump protein per cell (85, 125, 168), the
effect is bound to be rather slow, as confirmed with model
simulations (36). Moreover, despite the elevated internal
Na⫹/K⫹ in dense SS cells, their Na⫹ pumps were found to
be substantially inhibited (73). The inhibition was attributed to the abnormally high Mg/ATP ratio in these cells
(217), as it could be partially relieved by increasing ATP
levels towards the optimal ratio for the Na⫹ pump (88,
103, 104). Na⫹ pump fluxes in dense SS cells were found
to be inhibited even more by deoxygenation because
Mg2⫹ released from 2,3-diphosphoglycerate (2,3-DPG) elevated the Mg/ATP ratio further away from optimal (217).
Thus Na⫹ pump-mediated fluxes may contribute only
marginally to SS cell dehydration.
III. HOW DO SICKLE CELLS DEHYDRATE?
A. Early Findings and Puzzles
The relation between sickling and SS cell transport
abnormalities was first investigated by Tosteson and coworkers (265, 266, 268). They showed (268) that upon
deoxygenation of fresh, heparinized whole blood, SS
RBCs had a much larger gain of Na⫹ and loss of K⫹ than
RBCs from normal controls. On reoxygenation, SS cells
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acidification, by cell swelling and to a lesser extent, by
low intracellular Mg2⫹ concentrations (53, 150, 152, 178,
182, 249). Its functional state appears to be regulated by
phosphorylation and dephosphorylation of serine/threonine and tyrosine residues; it has not yet been determined
whether these residues are located on the KCC transporter itself or on one or more regulatory proteins. Jennings and al-Rohil (150, 152) described indirect evidence
that in rabbit RBCs, swelling activation of KCC occurs via
inactivation of a serine/threonine kinase, and Bize and
co-workers (23, 26) found that hypotonic swelling of
RBCs was associated with increased activities of protein
phosphatases (PP1 and PP2A), that PP1 increased in
RBCs whose ionic strength was lowered at constant volume, and that PP1 appears to mediate part of urea-stimulated KCC activity. Activation of KCC follows dephosphorylation of a serine/threonine residue by PP1 (153,
249) and/or PP2A (23, 26). As pointed out by Joiner et al.
(171), additional evidence of activation by some tyrosine
kinase inhibitors ( 22, 80) and inhibition by others (101,
213) suggests multiple control points, including PP1 itself
(25, 154). Proposed models of the biochemical activation
pathways continue to be updated with the latest data (21),
but the complex interrelations between the different aspects of activation or inhibition (changes in cell volume,
pH, urea, oxygenation state, [Mg2⫹]i, and phosphorylation/dephosphorylation) have yet to be determined.
KCC mediates a strictly coupled electroneutral transport of K⫹ and Cl⫺, independent of the membrane potential (151). Persistence of KCC activity in RBC samples
may be constitutive, as in certain hemoglobinopathies
(Hb C, Ref. 52), or associated with reticulocytosis, as with
SS blood (46). In RBCs with active KCC, acidification may
induce dehydration (37), whose extent may be limited by
the inhibitory effect of cell shrinkage. Joiner et al. (171)
have recently shown that acid activation of the KCC is
abnormally exaggerated in SS reticulocytes and may thus
easily overcome the intensity of the volume-regulatory
“brake.” Inhibitory agents may act directly on the KCC or
on its regulatory intermediates.
The driving force for RBC dehydration by K⫹ permeabilization, as when mediated by Gardos channels or
KCCs, is the outward electrochemical gradient of K⫹. An
important side effect of isotonic dehydration by KCl and
KHCO3 loss is cell acidification (188, 197). This effect was
predicted by the Lew-Bookchin red cell model (188; Fig.
2) and was fully confirmed experimentally (111). Its
mechanism is as follows. Within the RBCs, the positive
charges on Na⫹ ⫹ K⫹ ⫹ Mg2⫹, ⬃150 meq/l cell water, are
balanced by negative charges on Hb⫺, Cl⫺, and HCO⫺
3 . To
a gross approximation, Hb provides 50 meq/l cell water of
negative charges, and Cl⫺ ⫹ HCO⫺
3 100 meq/l cell water.
The effluent from isotonically dehydrating cells contains
⬃150 meq/l KCl ⫹ KHCO3 salts. Therefore, the concentrations of Cl⫺ ⫹ HCO⫺
3 in the effluent are higher than in
185
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VIRGILIO L. LEW AND ROBERT M. BOOKCHIN
gained K⫹ and lost Na⫹, indicating that the effects of
deoxygenation were reversible. Since CO treatment of SS
blood prevented both Hb S polymerization and Na⫹/K⫹
shifts on deoxygenation, the deoxygenation effect was
strictly the result of intracellular Hb S polymerization.
Further transport experiments with tracers of Na⫹, K⫹,
and Cs⫹ indicated that sickling induced a reversible and
poorly selective increase in the electrodiffusional cation
permeability of the RBC membrane (265, 266). In retrospect, this seminal work provided the first demonstration
of the sickling-induced permeability pathway that we currently designate Psickle (201). The nearly balanced Na⫹
gain and K⫹ loss during deoxygenation in vitro did not
account for SS cell dehydration, and the question of how
SS cells dehydrate was first formulated in the literature 12
years later, with the ground-breaking work of Bertles and
Milner (17).
Although the distinct morphological features of ISCs
were well known (the elongated potato-like shape mentioned above), Bertles and Milner (17) described some
distinctive properties of this SS cell subpopulation which
proved crucial and prescient of our current understanding
of their origin. They showed that ISCs accumulated at the
Physiol Rev • VOL
bottom of ultracentrifuged columns of RBCs, indicating
their high density. Because their Hb content was normal,
their high density reflected a dehydrated state. With various isotopic labeling techniques, they showed that ISCs
were a relatively young SS cell subpopulation which, after
release from the bone marrow as reticulocytes, was rapidly transformed into ISCs within ⬃4 –7 days, and then
disappeared from the circulation faster than other mature, non-ISC RBCs. ISCs were therefore a subpopulation
of SS RBCs in rapid circulatory turnover. The exclusion of
most high Hb F-containing SS cells (whose polymerization was reduced or inhibited) from the hyperdense, ISCrich cell fraction (Fig. 1) suggested that sickling was
necessary for ISC formation.
Bertles and Milner’s pioneering work focused much
of the subsequent research on the pathophysiology of
sickle cell anemia on the mechanism of SS cell dehydration. However, in the absence of a clear understanding of
the process, the relative young age and rapid turnover of
ISCs remained a largely ignored, isolated observation. For
the next two decades, research in the field was dominated
by the notion of gradual dehydration (“gradualism”), with
attention centered on the relative importance of the three
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FIG. 2. Main homeostatic components that participate in the regulation of RBC and reticulocyte volume, pH, ion content, membrane potential,
and intracellular Ca2⫹ concentration ([Ca2⫹]i). The diagram represents a normal human RBC or reticulocyte. Resistor arrows on the membrane
indicate electrodiffusional pathways. The thick double arrows for water and CO2 transport indicate rapid equilibration of these molecules across
the RBC membrane. Circles on the membrane represent carrier- or pump-mediated ion transport. The superscript “G” indicates permeabilities
G
described as “Goldmanian” in the RBC and reticulocyte models (188, 197). SITS/DIDS are inhibitors of the anion exchanger and of PCl
(55, 56).
Internal and external arrows denote inhibitory or stimulatory effects (⫺/⫹); double arrows represent chemical equilibria. HHb and Hb⫺ represent
c
protonated and unprotonated hemoglobin,respectively. Os lists the components required to compute internal osmolality: ¥Ci denotes the sum of
the intracellular concentrations of all transported solutes; Xi is the total intracellular concentration of impermeant solutes other than Hb (including
Mg2⫹), and fHb is the osmotic coefficient of Hb. E denotes membrane potential. CaB and MgB denote intracellular bound forms of Ca2⫹ and Mg2⫹,
respectively. The box on the top left summarizes Ca2⫹ homeostasis. The membrane circles represent the pump-leak circuit. The arrows indicate the
stimulatory effects of [Ca2⫹]i on the Gardos channels (K⫹), and on the PMCA, both in the plasma membrane and on endocytic inside-out vesicles
(IOV). The external box on the bottom right describes the equilibria prevailing during normal steady-states. The activities of the KCC (K-Cl
cotransporter, middle bottom) and Na⫹ pump decline sharply on maturation. Further details are described in the text.
MECHANISMS OF SICKLE CELL DEHYDRATION
transport systems potentially involved in SS cell dehydration: the Gardos channel, KCC, and the Na⫹ pump.
B. Discovery of the High Calcium Content
of SS Cells
C. Discovery of Endocytic Inside-Out Vesicles
In the early 1980s, available evidence suggested two
radically different alternatives to explain Ca2⫹ retention
without immediate dehydration in SS cells: either a profound failure of both Ca2⫹ pumps and Gardos channels
exclusive to intact SS RBCs (32, 33, 35, 187), or some sort
of Ca2⫹ compartmentalization that rendered Ca2⫹ inaccessible to these targets on the plasma membrane (35). So
strong was the general rejection of the idea of compartmentalization within RBCs at the time that not even its
mention as a remote alternative was acceptable in submitted manuscripts. It took a multidisciplinary approach
to resolve these options convincingly (34, 38, 199, 236,
275). Serial section analysis of SS RBCs clearly demonstrated the presence of intracellular compartments fully
enclosed within the cells. Surprisingly, a much smaller
number of fully enclosed vesicles was also found in a high
proportion of normal RBCs. With the application of procedures that destroyed the permeability barrier of the
RBC plasma membrane but retained intact vesicles inside,
it was possible to show that these vesicles had ATPdependent Ca2⫹-accumulating capacity, as expected from
Physiol Rev • VOL
endocytic (inside-out) vesicles (EIOVs) with the PMCA
pumping Ca2⫹ inwards (Fig. 2). X-ray microanalysis of the
elemental composition of intravesicular and cytoplasmic
compartments of cryosectioned SS cells showed clear
distinctions: calcium was located exclusively in the vesicles, which also contained high Na⫹ and low K⫹, suggesting an endocytic origin, and high phosphate, consistent
with intravesicular Ca2⫹ accumulation in the form of
amorphous hydroxyapatite crystals (34, 38). The intracellular vesicles could be coated with specific PMCA antibodies, and vesicular uptake could be inhibited by vanadate, but not by thapsigargin (A. Muoma and V. L. Lew,
unpublished observations). These results confirmed the
endocytic origin of the Ca2⫹-accumulating vesicles and
excluded the participation of residual, endoplasmic reticulum-derived organelles from earlier erythroid stages,
which express thapsigargin-sensitive SERCA-type Ca2⫹
pumps (277) instead of PMCA-type pumps.
There had been earlier reports showing vesicular
accumulation within RBCs of splenectomized persons
(141). This had been interpreted as reflecting a continuous process of endocytic vesiculation in circulating RBCs,
stabilized by extraction (“pitting”) during passage through
splenic sinusoids. Such an interpretation could also explain, at least in part, the unusually high accumulation of
EIOVs in RBCs of SS patients because of their known
functional asplenia (222, 223). In summary then, the high
(micromolar) Ca2⫹ content of SS RBCs is restricted to
EIOVs and results from a cumulative process initiated by
increased Ca2⫹ influx through Psickle. The resulting increase in [Ca2⫹]i stimulates the PMCA to accumulate
Ca2⫹ in EIOVs while also extruding it through the plasma
membrane. Ca2⫹ accumulation is thus the result of the
prevailing balance between EIOV Ca2⫹ uptake and Ca2⫹
extrusion through the plasma membrane (Fig. 2). With
hindsight, the long time it took to explain the significance
of the high [CaT]i of SS cells represents a cautionary tale
of “pride and prejudice” blocking the early consideration
of unconventional alternatives.
D. Failings of “Gradualism”
With the PMCA and Gardos channels of SS RBCs now
recognized as fully functional, the immediate question
was whether the transient [Ca2⫹]i elevations during deoxygenation were sufficient to trigger Gardos channel activation and explain SS cell dehydration. When Ca2⫹ influx
was uniformly elevated in AA cells to levels comparable
to those in Psickle-permeabilized SS cells (using Ca2⫹ ionophores), a small fraction of AA cells became rapidly dehydrated (264). This can now be explained by the wide
distribution of PMCA activity found in AA cells (192). At
low Ca2⫹ influx, only the cells with the weakest Ca2⫹
pump Vmax activities would allow [Ca2⫹]i to reach or
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In 1973, Eaton et al. (89) and Palek et al. (218)
independently reported that SS RBCs had a highly elevated total calcium content, which was highest in the
densest SS cell fraction, rich in ISCs. Ca2⫹ uptake was
found to be increased on deoxygenation, suggesting that
Ca2⫹ was also permeable through Psickle, a finding subsequently confirmed in a variety of experimental conditions
(31, 95). The Ca2⫹ link suggested that the Gardos channel
might be the main dehydration pathway, and much research during the 1970s and 1980s explored experimental
conditions that could generate ISCs in vitro by deoxygenation- and Ca2⫹-dependent processes (16, 33, 35, 46, 69 –
75, 97, 98, 122, 123, 160, 170, 214). However, rather than
providing clear answers, the high calcium content of SS
cells became a most confusing issue that took another
decade to resolve. Why was the Ca2⫹ taken up during
deoxygenation retained? Was the Ca2⫹ pump with its
normal high-Vmax somehow incapacitated? Did SS RBCs
have much higher cytoplasmic Ca2⫹ buffering capacity
than normal RBCs? Ca2⫹ was most elevated in the denser
cell fraction, but was also substantially increased in SS
cells with normal density (not dehydrated). What then
prevented dehydration of these high-Ca2⫹ cells? Were
their Gardos channels also inhibited?
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VIRGILIO L. LEW AND ROBERT M. BOOKCHIN
Physiol Rev • VOL
E. Multitrack Dehydration as an Alternative
to Gradualism
It became clear that although Psickle, [Ca2⫹]i, Gardos
channels, and KCC symporters were important participants in SS cell dehydration, neither gradualism nor the
idea of a dominant dehydration pathway would lead to an
explanation of the well-characterized heterogeneity of
age and hydration state of circulating SS cells. The specific issues that had to be addressed included the declining but still elevated proportion of reticulocytes observed
among progressively denser SS cell fractions, the relatively young age of ISCs, and the exclusion of F cells from
the hyperdense cell fraction. With large quantitative variations, these characteristics represent a consistent pattern among RBCs from all SS patients (Fig. 1).
A clear corollary of this formulation was that dehydration must not proceed uniformly and gradually from all
SS reticulocytes to ISCs but rather at different rates in
different SS RBCs, and fastest in the rapid-turnover ISCs.
The questions then were whether this multitrack conditioning started at the reticulocyte stage or later, and by
what mechanisms. At least for ISC formation, commitment to fast-track dehydration had to occur at a rather
early maturational stage. By the mid 1980s, little was
known about reticulocyte homeostasis, and it became
clear that ignorance in this area was a major obstacle to
progress. Our earlier experience with homeostatic modeling of Na⫹-transporting epithelia (196) and of mature
normal RBCs (188) generated predictions of such novel
and unexpected behavior, subsequently confirmed (111),
that it clearly exposed the limitations in our intuitive
understanding of complex systems. This experience encouraged a similar effort to develop a mathematical-computational model of reticulocyte homeostasis encoding all
the processes illustrated in Figure 2 (197).
The reticulocyte model (197) was developed with
two important assumptions based on good experimental
evidence: that the KCC, with intracellular pH and volume
regulatory properties as described by Brugnara et al. (46)
for the light reticulocyte-rich fraction of SS cells, constituted the main passive K⫹ transport pathway, and that the
pump-leak, steady-state Na⫹-K⫹ traffic across the reticulocyte membrane was at least one order of magnitude
higher than in mature RBCs (232, 274). This formulation
exposed a hitherto unsuspected need of high-capacity net
Na⫹ and Cl⫺ entry pathways in reticulocytes to balance
net Na⫹ efflux through the Na⫹ pump and net Cl⫺ efflux
through KCC. A simple numerical example may help to
illustrate this point. A reticulocyte with a KCC operating
at a rate of 10 mmol · l original cells⫺1 · h⫺1 in steady-state
will need a balancing K⫹ uptake of at least 10 mmol · l
original cells⫺1 · h⫺1 through the Na⫹ pump. Because of
its 3:2 Na⫹-K⫹ stoichiometry, net Na⫹ extrusion by the
pump will be at least 15 mmol · l original cells⫺1 · h⫺1. To
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exceed threshold activation levels of the Gardos channels. Results from different laboratories showed that applying single or cyclical deoxygenation to medium-density
SS RBCs (discocyte fractions) generated variable extents
of Ca2⫹-dependent SS cell dehydration. However, the observations were difficult to relate to physiological conditions, raising some doubt as to whether Gardos-mediated
dehydration played any role in vivo. This doubt was
reinforced in 1986 by the advent of a contender dehydrator, KCC.
Hall and Ellory (132) and Canessa et al. (57) each
reported a high level of KCC activity among light, reticulocyte-rich, and young SS RBC fractions. In detailed studies of the intracellular pH and volume regulatory properties of the KCC among different density fractions of SS
cells, Brugnara et al. (46, 47) showed that stimulation of
KCC-mediated 86Rb(K) fluxes by intracellular acidification was particularly steep in the lightest, reticulocyterich SS cell fraction, that inhibition by hypertonic-induced
shrinkage, though present, appeared relatively weak, and
that acidification of SS cells resulted in their dehydration.
Thus the KCC of SS RBCs shared many of the characteristics of the KCC characterized in other species (86, 150,
152, 180, 181).
“Gradualism,” by which we mean the notion of gradual, progressive dehydration of all SS cells, from reticulocytes to ISCs, was the guiding idea behind all experimental designs until the late 1980s. It also pervaded the
interpretation of all results, leaving an increasing body of
experimental data unexplained. Although it was clear that
both Gardos channels and KCC transporters had the potential to dehydrate SS cells, it was difficult to integrate
the numerous reported experiments within a pathophysiological scheme that could generate the observed heterogeneity of age and hydration of SS cells. One problem was
based on the uncertainty about whether [Ca2⫹]i levels in
deoxygenated SS cells ever reached Gardos channel activation thresholds. Another complexity was that Gardos
channel-mediated dehydration alone could not explain
the early observations (17) that the ISCs, the hyperdense,
most dehydrated SS RBCs, appeared to be selectively
generated from very young SS RBCs soon after their
release from the bone marrow, because, except for the
“dehydration-resistant cells” described below (DRCs; see
sect. IV), all reticulocytes and RBCs are similarly sensitive
to Ca2⫹-induced dehydration. KCC-mediated dehydration
alone also failed to explain ISC formation, particularly
why ISCs should have the highest Ca2⫹ content of all SS
cells and be relatively depleted of F cells. But the most
important objection to exclusive KCC-mediated dehydration was the need to postulate circulatory acidification as
the trigger, with no clear role for sickling-induced permeabilization.
MECHANISMS OF SICKLE CELL DEHYDRATION
cause further dehydration and further acidification (by
the mechanism described above as a side effect of dehydration by isotonic KCl loss of normal RBCs), thus perpetuating KCC stimulation, further KCl and water loss,
and further acidification until the driving K⫹ gradient was
dissipated. According to the model, immediately after the
perturbation, the dehydration effect would be hardly detectable, but its intensity would build up over a day or
two, depending on the KCC activity attributed to the cell.
The delayed dehydration response also indicated that in
SS reticulocytes with the KCC regulatory kinetics reported by Brugnara et al. (46), the inhibitory effects of
shrinkage on KCC activity would be insufficient to prevent progressive cell dehydration.
F. Original Working Hypothesis on the Mechanism
of Fast-Track Dehydration
The reticulocyte properties described above, if valid
for at least a subset of SS reticulocytes, were consistent
with a new hypothetic mechanism for fast-track dehydration and ISC formation in the circulation (190, 197). In
principle, the triggering perturbation in vivo could be
either sickling, followed by transient activation of Gardos
channels with associated cell acidification, or local RBC
acidification in the microcirculation. In addition, either
event could also stimulate further dehydration in propor-
FIG. 3. Working hypothesis on the mechanism
of generation of ISCs directly from SS reticulocytes, as formulated by Lew et al. (197). Arrows
indicate fluxes, stimulatory effects (⫹), or directions of change (up or down). Deoxygenation of SS
RBCs causes Hb S polymerization and membrane
permeabilization through Psickle, allowing passive
net Na⫹ and Ca2⫹ entry and K⫹ exit. Na⫹ entry is
balanced by K⫹ loss without immediate effect on
cell volume. Ca2⫹ entry elevates [Ca2⫹]i sufficiently
in some cells to activate their Gardos channels,
hyperpolarize the cell (E), and stimulate Cl⫺ efflux
through the available Cl⫺ permeability pathways
(band 3, Psickle; Ref. 164). Net loss of KCl causes
cell dehydration and acidification via the JacobsStewart mechanism (J-S), which operates like a
Cl⫺-H⫹ cotransport (188). Acidification, in turn,
stimulates KCC (K:Cl) causing further KCl loss,
dehydration, and acidification in a positive feedback loop which, unlike Psickle, can operate continuously in oxygenated and deoxygenated conditions, with a magnitude proportional to the KCC
expression within each reticulocyte. [From Lew et
al. (197).]
Physiol Rev • VOL
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sustain a steady-state ionic balance and constant volume,
such a reticulocyte will require net influxes of Na⫹ and
Cl⫺ of ⬃15 and 10 mmol · l original cells⫺1 · h⫺1, respectively. Thus, unlike with mature RBCs, a reticulocyte with
a high expression of KCC would be highly vulnerable to
rapid shrinkage in a low-Na⫹ or Na⫹-free medium, a
prediction confirmed by the experimental results (37).
However, the nature of the Na⫹ and Cl⫺ entry pathways
remains to be elucidated. Another important consideration to bear in mind is that because of their higher ionic
traffic and similar volume, comparable transport perturbations will generate faster volume shifts in reticulocytes
than in mature RBCs.
Model simulations of the dynamic behavior of reticulocytes in response to various stimuli revealed a surprising intrinsic instability: a brief and transient acidification
or K⫹ permeabilization episode, which would cause only
a minor, brief, and fully reversible change in the homeostatic variables of the mature RBC model, triggered a slow
but accelerating irreversible drift to a final hyperdense
steady-state in reticulocytes (197). This response resulted
from the intracellular pH sensitivity encoded for the KCC
in the model (Figs. 2 and 3), since it did not occur when
acid stimulation was removed from the kinetic definition
of the KCC. Analysis of this response revealed an unsuspected positive-feedback loop (Fig. 3, right): any transient perturbation that lowered intracellular pH would
stimulate the KCC; the resulting KCl and water loss would
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VIRGILIO L. LEW AND ROBERT M. BOOKCHIN
G. Experimental Tests of the Fast-Track
Dehydration Hypothesis
The experimental results (37) showed that deoxygenation of light, reticulocyte-rich SS cell fractions caused
morphological sickling as well as substantial shifts in cell
Na⫹, K⫹, and water content of the reticulocytes. Psickle in
reticulocytes exhibited a number of distinctive features,
absent in discocytes, the most surprising of which were
powerful stimulation by heparin and inhibition by divaPhysiol Rev • VOL
lent cations, effects which remain unexplained. Sicklinginduced features common to all samples tested were increased permeability to Na⫹, K⫹, and Ca2⫹; generation of
denser cell subpopulations due to net KCl loss in excess
of NaCl gain, only in the presence of Ca2⫹; and systematic
reticulocyte enrichment from 50 – 60% to over 90% in the
cell fractions that became denser on deoxygenation. As
we have learned more recently, most of the nonreticulocytes in the light SS cell fractions were DRCs (Fig. 1, Ref.
29), so the observed enrichment indicated that the sickling effects in these fractions were exclusive to reticulocytes. Thus, despite their relatively low cell [Hb], deoxygenation-induced sickling produced more dramatic homeostatic responses in SS reticulocytes than in the
discocytes, with measurable Ca2⫹-dependent dehydration
evident after a single deoxygenation episode. These results fulfilled a necessary condition for the hypothesis
outlined in Figure 3.
The hypothetical mechanism of fast-track dehydration (Fig. 3) generated several predictions amenable to
direct experimental tests. According to the hypothesis,
reticulocytes present among denser cell fractions should
represent rapidly dehydrating cells on the way to becoming ISCs. If the KCC-dependent positive-feedback loop
participates in fast-track dehydration, then SS reticulocytes of intermediate density should be susceptible to
further dehydration by acidification while oxygenated.
Acidification of oxygenated SS discocyte-rich cell fractions, containing 2–20% reticulocytes, generated fractions
of denser cells highly enriched (to over 80%) in reticulocytes (37). These results then confirmed that the reticulocytes present among SS discocytes represented cells
with the properties expected from fast-track dehydrating
cells.
Further insight into the participation of the KCCs and
Gardos channels in multitrack dehydration could be obtained by testing the responses of F reticulocytes among
the SS RBCs to acidification and deoxygenation. We
would expect the dehydration responses to acidification
of the high-F and low-F SS reticulocytes to be similar if
they exhibited similar KCC activities. The experimental
results fully confirmed that the KCC acid sensitivity was
retained in the F reticulocytes (37). On the other hand,
after deoxygenation, with sickling inhibited in the F reticulocytes, we would expect those reticulocytes that became denser to be largely depleted of Hb F. As illustrated
in Figure 1, Hb F cells tend to accumulate in the dense
discocyte fraction, whereas the hyperdense, ISC-rich fraction is virtually depleted of F cells. Partially protected
from the effects of sickling, the SS F cells have the longest
life-span (17, 106). Most of the F cells in the discocyte
fraction represent mature F cells whose KCC activity
(acid sensitivity) is lost; therefore, they should not be
included among the SS discocytes made denser by acidi-
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tion to its frequency. Based on these considerations, a
working hypothesis was proposed for a fast-track mechanism of generation of ISCs directly from SS reticulocytes, as illustrated in Figure 3: sickling of SS reticulocytes in the circulation activates Psickle. Psickle increases
the permeability of the membrane to Na⫹, K⫹, Ca2⫹, and
Mg2⫹ allowing increased net fluxes of these ions down
their electrochemical gradients. Net Na⫹ gain through
Psickle is largely balanced by net K⫹ loss with no immediate volume change (265, 266). Increased Ca2⫹ influx
would tend to elevate [Ca2⫹]i, exceeding the activation
threshold of Gardos channels in some cells within each
sickling episode, and thus triggering net KCl loss and
dehydration with associated cell acidification. Acidification, in turn, would stimulate KCC-mediated dehydration
with intensity proportional to the level of expression and
activity of KCC in each cell. Acid-stimulated KCC dehydration would operate continuously, both in oxy and deoxy conditions, thus speeding dehydration of high-KCC
reticulocytes to become dense reticulocytes and eventually young ISCs. KCC activity may be additionally stimulated by high urea in the renal medulla and by kinasemediated effects linked to the oxygenated state of the
RBCs (119, 163, 248).
The hypothesized preference for a dehydration trigger dependent on sickling rather than acidification (Fig. 3)
derived from the need to account for the high Ca2⫹ content of ISCs. However, at the time this hypothesis was
being developed, there had been no investigations of the
membrane-permeabilizing effects of sickling in light, reticulocyte-rich SS cell fractions. Guided by gradualist
ideas, the general experimental approach had been to
follow dehydration from the “normal” volume stage onwards, i.e., either with unfractionated SS cells in which
discocytes dominate, or with density-fractionated discocytes. It was unknown whether Psickle was activated in
reticulocytes, and there were reasons to doubt it. Because
of the high-power dependence of Hb S polymerization on
cell Hb concentration (90), reticulocytes, whose cell [Hb]
was relatively low, had not been considered important
participants in sickling or in sickling-induced effects.
Hence, the starting point of an investigation of this hypothesis focused on the effects of sickling in reticulocytes (37).
MECHANISMS OF SICKLE CELL DEHYDRATION
IV. DEHYDRATION-RESISTANT CELLS
Although the multitrack dehydration model offered a
satisfactory explanation of the main characteristics of SS
cell dehydration, new findings showed that among the
light, reticulocyte-rich SS cells, there were RBCs with
unusual characteristics not corresponding to any of the
known SS cell types or stages. In a study of the Ca2⫹independent K(86Rb) transport properties of the light,
reticulocyte-rich SS cell density fraction two distinct
K(86Rb) pools were identified which differed in their
K(86Rb) turnover rates (94): a rapid-turnover pool, inhibitable by high bumetanide concentrations, and a bumetanide-insensitive, slow-turnover pool, each corresponding
to a separate RBC subpopulation. The rapid-turnover
pool, which could be tracer-equilibrated within ⬃5 min,
suggested the presence among light SS cells of RBCs with
a high K⫹ permeability and low K⫹ content; the possible
nature and origin of these RBCs were puzzling.
The mystery of the cells with the rapid-turnover pool
of K(86Rb) was resolved with the discovery of DRCs (29).
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These cells were first recognized by their failure to dehydrate when exposed to the K⫹-selective ionophore valinomycin in low-K⫹, plasmalike media, a property enabling their detection by flow cytometry and their isolation by further density fractionation. They were found to
represent between 10 and 60% of the light, reticulocyterich fraction of SS cells (density ⬍1.091) in different
patients. Resistance to dehydration proved to result from
Na⫹-K⫹ gradient dissipation, with substantial reversal of
RBC Na⫹/K⫹ content ratios. (Ca2⫹ ⫹ A23187)-induced K⫹
permeabilization exposed similar fractions of DRCs as
with valinomycin. K(86Rb) flux studies identified DRCs as
the cells responsible for the bumetanide-sensitive, rapidturnover K(86Rb) pool. The morphology of DRCs showed
elongated shapes reminiscent of ISCs, suggesting a possible origin from dense ISCs. Such an origin would mean
that dense ISCs would become leaky to cations, dissipate
their residual K⫹ gradient while gaining more Na⫹ than
could be balanced by the Na⫹ pump, eventually swelling
and migrating to lower densities. Bottom-up migration
was supported by the work of Holtzclaw et al. (142), who
reinjected biotin-labeled dense SS discocytes and demonstrated their upward migration in vivo through the different SS cell density levels, with eventual accumulation in
the lightest density fraction. Together, these findings suggest that SS DRCs may represent the senescent condition
of ISCs and other dense SS RBCs before their removal
from the circulation.
Nothing is known about the putative mechanism of
bottom-up migration of ISCs, on their way to become
DRCs. The conversion of a K⫹-depleted, dehydrated RBC
to a high-Na⫹, swollen RBC must involve a sufficiently
large Na⫹ permeability increase for net NaCl gain to
overcome Na⫹ pump fluxes stimulated by the high
Na⫹/K⫹ ratio of the cells. What activates such a permeability pathway? The homeostatic environment within deoxygenated ISCs differs from those in normal RBCs or in
SS discocytes (189). In the shrunken ISCs, the volume of
cell water in the polymer water compartment (PWC) of
deoxy-Hb S polymers may exceed that remaining in the
cytosolic phase. Most small solutes partition within the
PWC, whereas all soluble deoxy-Hb S at equilibrium with
polymer, and all enzymes remain excluded from the PWC
(27, 191). Activation of the Na⫹ permeability pathway
responsible for the ISC-DRC transition may thus result in
some way from this special ISC environment, or simply
from the dehydration itself.
Investigation of the origin and mechanism of formation of DRCs must be considered work in progress, in its
early stages. DRCs were found in much smaller numbers
among normal RBCs (29), and in increased amounts
among RBCs from persons with ␤-thalassemia intermedia
and with Hb E/␤-thalassemia (28). Among SS cells they
constitute the vast majority of nonreticulocytes recovered
in the light, reticulocyte-rich fraction, accounting for most
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fication, an expectation fully confirmed by the experimental results (37).
From the theoretical analysis and the experimental
results, a multitrack-dehydration picture emerged as follows: SS reticulocytes released from the bone marrow are
variably endowed with KCC expression. Despite their low
cell [Hb], upon deoxygenation they are all exposed to
sickling-induced, Ca2⫹-dependent dehydration via Gardos
channels. Those with high KCC activity are preferentially
vulnerable to fast-track dehydration and ISC formation by
the combined effect of intermittent Gardos channel activation during deoxygenation and continuously stimulated
KCC activity; this explains the presence of acid-sensitive
reticulocytes among discocytes on their way to becoming
young ISCs. ISCs have the highest total Ca2⫹ levels of any
SS cells, and also a unique abundance of large EIOVs
(199), suggesting a disposition to stimulated endocytosis
associated with fast-track dehydration, not yet explained.
Those reticulocytes with lesser expression of KCCs dehydrate more slowly, almost exclusively via Gardos channels, and mature into KCC-silent, reticulum-free discocytes and dense discocytes. Sickling-resistant F reticulocytes are capable of dehydration by acidification, but this
capacity is largely lost by the time they reach the density
of discocytes and dense discocytes (Fig. 1). This multitrack-dehydration hypothesis provided a satisfactory explanation of the major heterogeneity features of SS cells
and supplied a mechanism for fast-track dehydration that
could finally explain the observations of Bertles and Milner (17). Multitrack dehydration now represents the general consensus on the origin of SS cell heterogeneity and
on the alternative rates of SS cell dehydration (99, 105–
109, 212).
191
192
VIRGILIO L. LEW AND ROBERT M. BOOKCHIN
of the RBC heterogeneity observed in this density fraction.
V. PROPERTIES OF PSICKLE
A. Ion Selectivity of Psickle
As noted above, Psickle increases the permeability to
Na⫹, K⫹, and Ca2⫹. Sickling was also shown to generate
a reversible increase in SS RBC Mg2⫹ permeability, allowing net down-gradient Mg2⫹ movements (217); this proved
to be an important basis for trials of Mg2⫹-based therapy
(39), as discussed below. The alkali metal cation selectivity of Psickle was studied by Joiner et al. (169), who found
no differences between Li⫹, Na⫹, Rb⫹, and Cs⫹; organic
cations such as tetramethylammonium, tetraethylammonium, and N-methylglucamine were excluded from the
RBCs. Psickle is thus best characterized as a poorly selective permeability pathway for small, inorganic monovalent and divalent cations. Investigations of the effects of
deoxygenation on electrodiffusional and self-exchange
anion fluxes by Joiner et al. (164) showed no effects of
deoxygenation on either flux component, whereas Clark
and Rossi (74) reported a two- to threefold increase in
tracer-Cl⫺ uptake by deoxygenated SS RBCs in the presence of DIDS. Thus anion movements through Psickle are
either absent or so small that they have no physiological
significance. As noted above, SS reticulocytes and discocytes differ in some Psickle properties. Particularly intriguing is the heparin sensitivity of Psickle exclusive to reticulocytes: a deoxygenation pulse with the RBCs suspended
in heparinized plasma dissipates their Na⫹-K⫹ gradients
much faster than in heparin-free serum (37). Also exclusive to reticulocytes is the inhibitory effect of divalent
cations on Psickle (not limited to Ca2⫹) (37, 167).
B. The Stochastic Nature of the Psickle Distribution
An important property of Psickle is its stochastic nature, which becomes manifest when following the sickling
responses of SS RBCs (193, 201). Psickle permeabilization
to Ca2⫹ was followed in SS discocytes using a method
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The most profound gap in our knowledge of the
mechanism of SS dehydration concerns the molecular
nature of Psickle. It seems ironic that over half a century
after the discovery of the “first molecular disease” we
should remain ignorant of the molecular nature of the
permeability pathway by which the basic Hb S abnormality leads to SS RBC dehydration and all its downstream
effects. Despite this important gap, a number of important
functional properties of Psickle have recently been characterized.
that allowed separation of RBCs whose [Ca2⫹]i levels
were below or above Gardos channel activation during
each sickling pulse (193). Removal of “activated” cells
from a previous pulse had little effect on the fraction of
activated cells in a subsequent pulse. The sickling-induced increase in Ca2⫹ permeability persisted for the
duration of each sickling episode and was fully reversed
on reoxygenation. These results, together with additional
experiments in which the fraction of activated cells were
studied as a function of the external Ca2⫹ concentration,
showed that sickling is a stochastic event of random
intensity among SS RBCs, capable of generating maximal
Gardos channel activation in a small fraction of cells
during each deoxygenation-sickling pulse. Consistent
with the stochastic nature of Psickle, repeated pulses led to
the progressive accumulation of dense cells, whereas single long pulses caused only an early production of a single
small fraction of dense RBCs. At physiological extracellular [Ca2⫹] levels, the mean Ca2⫹ influx increased from
its normal value of ⬃50 ␮mol · l original cells⫺1 · h⫺1 to as
much as ⬃300 ␮mol · l original cells⫺1 · h⫺1 (96). But it is
important to bear in mind that mean flux values in deoxygenated SS cells provide no information about the wide
range of Psickle values induced in individual cells. Because
Psickle and [Ca2⫹]i may transiently reach very high values
in some cells, Ca2⫹ effects previously considered of
doubtful relevance may in fact participate in the pathology of dense SS cells. Prominent among these are activation of Ca2⫹-sensitive proteases (62, 124, 149, 270) and
cross-linking of membrane proteins (207) with consequent increase in membrane rigidity (209, 210).
When electrophysiologists finally approach the study
of Psickle under patch clamp, they ought to bear in mind
the probabilistic nature of Psickle in each deoxygenation
pulse before consulting their psychiatrist for the lack of
reproducibility!
How could the stochastic behavior of Psickle be interpreted? Psickle must result from reversible interactions
between deoxy-Hb S polymers and membrane components that either generate a new transport pathway or
modify a preexisting one. If the polymers interact in some
way with the membrane cytoskeleton, they may induce
the formation of oligomeric aggregates of band 3 dimers,
which could function as a large ion channel (201), consistent with the observed inhibitory effect of stilbenes on
Psickle (161, 162). If the number of permeabilizing contacts
between deoxy-Hb S polymers and the membrane in each
deoxygenation pulse were small, random and variable
from pulse to pulse in each cell, and if the permeabilizing
structure remained stable for the duration of each deoxygenation episode, this could account for the observed
stochastic behavior of Psickle, for the wide cell to cell
variation in its magnitude, and for the constancy of Psickle
within each pulse (201). However, all such suggestions
remain highly speculative at present, and much more
MECHANISMS OF SICKLE CELL DEHYDRATION
work will be needed to advance our knowledge of the
molecular nature of Psickle.
VI. THERAPEUTIC STRATEGIES AIMED AT ION
TRANSPORT TARGETS TO PREVENT
SICKLE CELL DEHYDRATION
A. Antisickling Strategies
Deoxy-Hb S polymerization is directly responsible
for Psickle and is therefore a prime target. Of the many
antisickling strategies considered in the past, only the use
of the cytotoxic agent hydroxyurea proved both clinically
successful and extensively applied (63– 65, 251, 252). The
rationale for its use was based on the enhancement of Hb
F production it promoted in SS patients (226). However,
hydroxyurea proved to have many additional effects other
than enhanced Hb F production. Among many others, it
increased the mean cell volumes in reticulocytes and
mature SS cells (215), and this effect preceded the elevation in Hb F production (42), as did the clinical improvement in some patients. Thus, as originally suggested by
Orringer et al. (215), the beneficial effects of this agent
might be partly due to its direct effect on SS cell volume,
the origin and nature of which remain unexplained.
Several other pharmacological agents capable of inducing Hb F production are at various stages of development, including butyrate derivatives, decitabine, a derivative of 5-azacytidine, and various combinations of these
agents with hydroxyurea. Information about their mechanisms of action and current status is described in a
recent overview (8).
B. Psickle Inhibition
As discussed above, the molecular nature of Psickle
remains unknown. There is little doubt, however, that a
specific Psickle inhibitor would be well worth the investment of major effort. Promising future research lines in
this area would be to attempt patch-clamping deoxygenated SS cells in cell-attached, or ionophore-perforatedpatch configurations, to gain information about the
Physiol Rev • VOL
charge-transfer properties of Psickle, or the development
of a Psickle-screening assay, to allow the systematic search
for specific inhibitors. For such a search to be efficient,
we need at least some preliminary clues about the molecular nature of Psickle and its precise cation selectivity
properties. Though not particularly enlightening, the only
clues presently available are its differential properties in
SS reticulocytes and discocytes considered above, and
the inhibitory effect of stilbene disulfonates (161), suggesting band 3 protein participation in Psickle. Dipyridamole, a widely used antiplatelet agent, with limited side
effects, was also shown to inhibit Psickle (165), but no
trials have been reported to date.
C. Gardos Channel Inhibition
After the discovery of the inhibitory effect of clotrimazole on Gardos channels by Alvarez et al. (5), its therapeutic potential was immediately recognized (49, 92).
Brugnara and collaborators (81, 45, 235, 51) initiated a
series of clinical trials of oral administration of clotrimazole with pharmacokinetic studies and obtained promising results [for a recent review, see Steinberg and Brugnara (253)]. Clotrimazole has many effects unrelated to
Gardos channel inhibition (5, 15). A variety of adverse
effects associated with oral administration of clotrimazole prompted a search for more potent and specific
structural derivatives, leading to a novel Gardos channel
inhibitor, ICA-17043, 10 times more potent than clotrimazole and with no effect on cytochrome P-450 (254).
This agent is currently undergoing phase I/II clinical
trials (253).
D. Anion Conductance Inhibition
As discussed above, although the mean value of the
Psickle-mediated cation fluxes elicited in each sickling episode is not particularly high, the Ca2⫹ permeability
through Psickle reaches sufficiently high values in a small
fraction of RBCs for maximal activation of the Gardos
channels (201). Because of the stochastic nature of Psickle,
most (low-F) SS cells will eventually be exposed to transient periods of maximal Gardos channel stimulation in
the circulation. During such periods, the extent of dehydration could be effectively reduced by blocking the anion conductance, thus reducing net KCl or KHCO3 loss, a
therapeutic strategy developed by Bennekou and coworkers (11–14). A novel family of anion conductance
inhibitors, NS1652 (13) and NS3623 (12), appeared effective in reducing Gardos channel-mediated dehydration of
SS cells and offers a promising new approach in SS
therapy.
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Each of the components outlined in Figure 3 participates in SS RBC dehydration and is therefore a potential
therapeutic target. Following the sequence with which we
analyzed the original hypothesis, we list the following
specific targets: sickling (deoxy-Hb S polymerization),
Psickle (membrane permeabilization), Gardos channels,
the electrodiffusional anion conductance pathway, KCC
(K-Cl) and the Jacobs-Stewart mechanism (J-S). We will
consider each of these in turn.
193
194
VIRGILIO L. LEW AND ROBERT M. BOOKCHIN
E. Control of KCC
VII. OPEN QUESTIONS AND THE DIRECTION
OF FUTURE RESEARCH
Sickle cell anemia and related sickle cell disease
syndromes have long been models for the investigation of
RBC physiology and pathophysiology, and they remain an
open field for extensive further investigation. At this
stage, many of the fundamental issues underlying the
mechanisms of sickle cell dehydration have been addressed, as well as the explanation of the age and volume
heterogeneity of SS cells. Two major issues remain open
in this area of research. The first concerns the molecular
nature of Psickle, addressed above. The second concerns
Physiol Rev • VOL
We thank the Wellcome Trust (United Kingdom) and the
National Institutes of Health (United States) for funds.
Address for reprint requests and other correspondence: V.
L. Lew, Dept. of Physiology, Univ. of Cambridge, Downing
Street, Cambridge CB2 3EG, UK (E-mail: [email protected]).
REFERENCES
1. Akide-Ndunge OB, Ayi K, and Arese P. The Haldane malaria
hypothesis: facts, artifacts, and a prophecy. Redox Rep 8: 311–316,
2003.
2. Allison AC. Notes on sickle-cell polymorphism. Ann Hum Genet
19: 39 –51, 1954.
3. Allison AC. Protection afforded by sickle-cell trait against subtertian malareal infection. Br Med J 4857: 290 –294, 1954.
4. Alvarez J and Garcı́a-Sancho JJ. An estimate of the number of
Ca2⫹-dependent K⫹ channels in the human red cell. Biochim Biophys Acta 903: 543–546, 1987.
5. Alvarez J, Montero M, and Garcı́a-Sancho J. High affinity inhibition of Ca2⫹-dependent K⫹ channels by cytochrome P-450 inhibitors. J Biol Chem 267: 11789 –11793, 1992.
6. Anderson K, Crable SC, Gallagher PG, and Joiner CH. Expression of multiple KCl cotransporter isoforms in sickle and normal
erythroid cells. Blood 100: 666a, 2002.
7. Arese P, Turrini F, Bussolino F, Lutz HU, Chiu D, Zuo L,
Kuypers F, and Ginsburg H. Recognition signals for phagocytic
removal of favic, malaria-infected and sickled erythrocytes. Adv
Exp Med Biol 307: 317–327, 1991.
8. Atweh GF, DeSimone J, Saunthararajah Y, Fathallah H, Weinberg RS, Nagel RL, Fabry ME, and Adams RJ. Hemoglobinopathies. Hematology 2003: 14 –39, 2003.
9. Ayi K, Turrini F, Piga A, and Arese P. Enhanced phagocytosis of
ring-parasitized mutant erythrocytes. A common mechanism that
may explain protection against falciparum-malaria in sickle-trait
and beta-thalassemia-trait. Blood. In press.
10. Beaugé L and Lew VL. Passive fluxes of sodium and potassium
across red cell membranes. In: Membrane Transport in Red Cells,
edited by J. C. Ellory and V. L. Lew. London: Academic, 1977, p.
39 –51.
11. Bennekou P. The feasibility of pharmacological volume control of
sickle cells is dependent on the quantization of the transport pathways. A model study. J Theoret Biol 196: 129 –137, 1999.
12. Bennekou P, De Franceschi L, Pedersen O, Lian L, Asakura T,
Evans G, Brugnara C, and Christophersen P. Treatment with
NS3623, a novel Cl-conductance blocker, ameliorates erythrocyte
dehydration in transgenic SAD mice: a possible new therapeutic
approach for sickle cell disease. Blood 97: 1451–1457, 2001.
13. Bennekou P, Pedersen O, Moller A, and Christophersen P.
Volume control in sickle cells is facilitated by the novel anion
conductance inhibitor NS1652. Blood 95: 1842–1848, 2000.
14. Bennekou P and Stampe P. The effect of ATP, intracellular
calcium and the anion exchange inhibitor DIDS on conductive
anion fluxes across the human red cell membrane. Biochim Biophys Acta 942: 179 –185, 1988.
85 • JANUARY 2005 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.2 on July 4, 2017
No specific KCC inhibitors are currently available.
Increased intracellular [Mg2⫹] and cell alkalinization
partially inhibit KCC in SS cells (53, 58, 179). Based on
these effects and on the fact that Psickle permeabilizes
SS cells to Mg2⫹ (217), Bookchin et al. (39) proposed a
rationale for the possible benefits of controlled hypermagnesemia in the treatment of sickle cell anemia. By
setting the plasma extracellular [Mg2⫹] levels above the
electrochemical equilibrium of Mg2⫹ across SS cell
membranes, deoxygenated SS cells would gain MgCl2
and water, thus elevating [Mg2⫹]i and [Cl⫺]i, with secondary cell alkalinization due to reduction of the inward Cl⫺ concentration ratio. These effects should
tend to reduce KCC activity and KCC-mediated SS cell
dehydration. Additional beneficial effects, independent
of KCC mediation, were expected from reduced Ca2⫹
influx through Psickle by competition with plasma Mg2⫹,
from systemic vasodilatation, muscle relaxation, and
inhibition of platelet aggregation. Anecdotic reference
to beneficial Mg effects in preventing crisis in individual SS patients had been advanced many years before
by Lehman (185). De Franceschi and collaborators performed the first systematic studies of oral Mg2⫹ supplementation on transgenic sickle mice (79) and SS patients (78). In both studies, the Mg2⫹ and K⫹ content of
SS cells was substantially normalized, KCC activity was
reduced, and SS cell density was lowered. Prolonged
administration of oral Mg pidolate had the additional
effect of reducing the frequency or extent of painful
crises (78), thus confirming Lehman’s original observations. In experimental conditions it was possible to
delay or block dehydration-induced cell acidification
using anion exchange inhibitors (111). Such an effect
would prevent the operation of the KCC feedback loop
through the Jacobs-Stewart mechanism (Fig. 3). However, because of the multiple functions of the anion
exchanger, this may prove a dangerous strategy even if
suitable inhibitors became available.
the origin and mechanism of formation of DRCs. Do these
cells represent the homeostatic condition of senescent
ISCs, as the early observations suggested? By what mechanism do hyperdense ISCs become light (low density)
DRCs? Could this be a common final path for discocytes
and Hb F cells as well? Could DRCs from normal subjects
also suffer a similar fate? Is the DRC condition an epiphenomenon or an essential part of the mechanisms of programmed RBC senescence and circulatory removal (7, 68,
208)?
MECHANISMS OF SICKLE CELL DEHYDRATION
Physiol Rev • VOL
36. Bookchin RM, Ortiz OE, and Lew VL. Mechanisms of red cell
dehydration in sickle cell anemia. Application of an integrated red
cell model. In: The Red Cell Membrane: A Model for Solute Transport, edited by B. U. Raess and G. Tunnicliffe. Clifton, NJ: Humana,
1990, p. 443– 461.
37. Bookchin RM, Ortiz OE, and Lew VL. Evidence for a direct
reticulocyte origin of dense red cells in sickle cell anemia. J Clin
Invest 87: 113–124, 1991.
38. Bookchin RM, Ortiz OE, Somlyo AV, Somlyo AP, Sepulveda
MI, Hockaday A, and Lew VL. Calcium-accumulating inside-out
vesicles in sickle cell anemia red cells. Trans Assoc Am Phys 98:
10 –20, 1985.
39. Bookchin RM, Tiffert JT, Davies SC, Vichinsky E, and Lew VL.
Magnesium Therapy for Sickle Cell Anemia: A New Rationale,
edited by Y. Beuzard, B. Lubin, and J. Rosa. Paris: Libbey Eurotext,
1995, p. 545–546.
40. Boyer SH, Belding TK, Margolet L, and Noyes AN. Fetal hemoglobin restriction to a few erythrocytes (F cells) in normal
human adults. Science 188: 361–363, 1975.
41. Branton D, Cohen CM, and Tyler J. Interaction of cytoskeletal
proteins on the human erythrocyte membrane. Cell 24: 24 –32, 1981.
42. Bridges KR, Barabino GD, Brugnara C, Cho MR, Christoph
GW, Dover G, Ewenstein BM, Golan DE, Guttmann CR, Hofrichter J, Mulkern RV, Zhang B, and Eaton WA. A multiparameter analysis of sickle erythrocytes in patients undergoing hydroxyurea therapy. Blood 88: 4701– 4710, 1996.
43. Brown AM, Ellory JC, Young JD, and Lew VL. A calciumactivated potassium channel present in foetal red cells of the sheep
but absent from reticulocytes and mature red cells. Biochim Biophys Acta 511: 163–175, 1978.
44. Brown AM and Simonsen LO. Intracellular cobalt buffering by
intact human red cells: evidence for time-dependent and metabolism-dependent changes (Abstract). J Physiol 361: 26P, 1985.
45. Brugnara C, Armsby CC, Sakamoto M, Rifai N, Alper SL, and
Platt O. Oral administration of clotrimazole and blockade of human erythrocyte Ca2⫹-activated K⫹ channel: the imidazole ring is
not required for inhibitory activity. J Pharmacol Exp Ther 273:
266 –272, 1995.
46. Brugnara C, Bunn HF, and Tosteson DC. Regulation of erythrocyte cation and water content in sickle cell anemia. Science 232:
388 –390, 1986.
47. Brugnara C, Bunn HF, and Tosteson DC. Ion content and transport and the regulation of volume in sickle cells. Ann NY Acad Sci
565: 96 –103, 1989.
48. Brugnara C, De Franceschi L, and Alper SL. Ca2⫹-activated K⫹
transport in erythrocytes. Comparison of binding and transport
inhibition by scorpion toxins. J Biol Chem 268: 8760 – 8768, 1993.
49. Brugnara C, De Franceschi L, and Alper SL. Inhibition of
Ca2⫹-dependent K⫹ transport and cell dehydration in sickle erythrocytes by clotrimazole and other imidazole derivatives. J Clin
Invest 92: 520 –526, 1993.
50. Brugnara C, De Franceschi L, Bennekou P, Alper SL, and
Christophersen P. Novel therapies for prevention of erythrocyte
dehydration in sickle cell anemia. Drug News Perspect 14: 208 –220,
2001.
51. Brugnara C, Gee B, Armsby CC, Kurth S, Sakamoto M, Rifai
N, Alper SL, and Platt OS. Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease. J Clin Invest
97: 1227–1234, 1996.
52. Brugnara C, Kopin AS, Bunn HF, and Tosteson DC. Regulation
of cation content and cell volume in erythrocytes from patients
with homozygous hemoglobin C disease. J Clin Invest 75: 1608 –
1617, 1985.
53. Brugnara C and Tosteson DC. Inhibition of K transport by divalent cations in sickle erythrocytes. Blood 70: 1810 –1815, 1987.
54. Bunn HF and Forget BG. Hemoglobin: Molecular, Genetic and
Clinical Aspects. Philadelphia, PA: Saunders, 1986.
55. Cabantchik ZI, Knauf PA, and Rothstein A. The anion transport
system of the red blood cell. The role of membrane protein evaluated by the use of “probes.” Biochim Biophys Acta 515: 239 –302,
1978.
85 • JANUARY 2005 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.2 on July 4, 2017
15. Benzaquen LR, Brugnara C, Byers HR, Gatton-Celli S, and
Halperin JA. Clotrimazole inhibits cell proliferation in vitro and in
vivo. Nat Med 1: 534 –540, 1995.
16. Berkowitz LR and Orringer EP. Effect of cetiedil, an in vitro
antisickling agent, on erythrocyte membrane cation permeability.
J Clin Invest 68: 1215–1220, 1981.
17. Bertles JF and Milner PFA. Irreversibly sickled erythrocytes: a
consequence of the heterogeneous distribution of hemoglobin
types in sickle cell anemia. J Clin Invest 47: 1731–1741, 1968.
18. Billett HH, Fabry ME, and Nagel RL. Hemoglobin distribution
width: a rapid assessment of dense red cells in the steady state and
during painful crisis in sickle cell anemia. J Lab Clin Med 112:
339 –344, 1988.
19. Billett HH, Kim K, Fabry ME, and Nagel RL. The percentage of
dense red cells does not predict incidence of sickle cell painful
crisis. Blood 68: 301–303, 1986.
20. Billett HH, Nagel RL, and Fabry ME. Evolution of laboratory
parameters during sickle cell painful crisis: evidence compatible
with dense red cell sequestration without thrombosis. Am J Med
Sci 296: 293–298, 1988.
21. Bize I. Theoretical validation for a model of KCC regulation in
human erythrocytes. Blood Cells Mol Dis 27: 121–126, 2001.
22. Bize I and Dunham PB. Staurosporine, a protein kinase inhibitor,
activates K-Cl cotransport in LK sheep erythrocytes. Am J Physiol
Cell Physiol 266: C759 –C770, 1994.
23. Bize I, Guvenc B, Robb A, Buchbinder G, and Brugnara C.
Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes. Am J Physiol Cell Physiol 277:
C926 –C936, 1999.
24. Bize I, Guvenc B, Robb A, Buchbinder G, and Brugnara C.
Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes. Am J Physiol Cell Physiol 277:
C926 –C936, 1999.
25. Bize I, Munoz P, Canessa M, and Dunham PB. Stimulation of
membrane serine-threonine phosphatase in erythrocytes by hydrogen peroxide and staurosporine. Am J Physiol Cell Physiol 274:
C440 –C446, 1998.
26. Bize I, Taher S, and Brugnara C. Regulation of K-Cl cotransport
during reticulocyte maturation and erythrocyte aging in normal and
sickle erythrocytes. Am J Physiol Cell Physiol 285: C31–C38, 2003.
27. Bookchin RM, Balazs T, and Lew VL. Measurement of the hemoglobin concentration in deoxyhemoglobin S polymers and characterization of the polymer water compartment. J Mol Biol 244:
100 –109, 1994.
28. Bookchin RM and Etzion Z. Increase in high-Na⫹, low-K⫹, “valinomycin-resistant” red cells in Hb E/beta-thalassemia, beta-thalassemia intermedia, and sickle cell anemia: a possible result of
oxidative membrane damage? Proc Symp Oxidative Stress in
Thalassemia: Etiology and Prevention Petchburi Thailand 2002.
29. Bookchin RM, Etzion Z, Sorette M, Mohandas N, Skepper JN,
and Lew VL. Identification and characterization of a newly recognized population of high-Na⫹, low-K⫹, low-density sickle and normal red cells. Proc Natl Acad Sci USA 97: 8045– 8050, 2000.
30. Bookchin RM and Lew VL. Progressive inhibition of the Ca pump
and Ca:Ca exchange in sickle red cells. Nature 284: 561–563, 1980.
31. Bookchin RM and Lew VL. Effect of a “sickling pulse” on calcium
and potassium transport in sickle cell trait red cells. J Physiol 312:
265–280, 1981.
32. Bookchin RM and Lew VL. Calcium transport abnormalities in
sickle-cell anemia red cells: Ca-pump failure or concealed IOVS?
Acta Physiol Latinoam 32: 224 –226, 1982.
33. Bookchin RM and Lew VL. Red cell membrane abnormalities in
sickle cell anemia. Prog Hematol 13: 1–23, 1983.
34. Bookchin RM, Ortiz OE, Hockaday A, Sepulveda MI, and Lew
VL. The state of calcium in sickle cell anemia red cells. In: Intracellular Calcium Regulation, edited by H. Bader, K. Gietzen, J.
Rosenthal, R. Rudel, and H. U. Wolf. Wolfeboro, NH: Manchester
Univ. Press, 1986, p. 327–334.
35. Bookchin RM, Ortiz OE, and Lew VL. Silent intracellular calcium
in sickle cell anemia red cells. In: The Red Cell. Proceedings of the
Sixth International Conference on Red Cell Metabolism and Function, edited by G. J. Brewer. New York: Liss, 1984, p. 17–28.
195
196
VIRGILIO L. LEW AND ROBERT M. BOOKCHIN
Physiol Rev • VOL
78. De Franceschi L, Bachir D, Galacteros F, Tchernia G, Cynober T, Neuberg D, Beuzard Y, and Brugnara C. Oral magnesium
pidolate: effects of long-term administration in patients with sickle
cell disease. Br J Haematol 108: 284 –289, 2000.
79. De Franceschi L, Beuzard Y, Jouault H, and Brugnara C.
Modulation of erythrocyte potassium chloride cotransport, potassium content, and density by dietary magnesium intake in transgenic SAD mouse. Blood 88: 2738 –2744, 1996.
80. De Franceschi L, Fumagalli L, Olivieri O, Corrocher R, Lowell
CA, and Berton G. Deficiency of Src family kinases Fgr and Hck
results in activation of erythrocyte K/Cl cotransport. J Clin Invest
99: 220 –227, 1997.
81. De Franceschi L, Saadane N, Trudel M, Alper SL, Brugnara C,
and Beuzard Y. Treatment with oral clotrimazole blocks Ca2⫹activated K⫹ transport and reverses erythrocyte dehydration in
transgenic SAD mice. A model for therapy of sickle cell disease.
J Clin Invest 93: 1670 –1676, 1994.
82. Desai SA, Bezrukov SM, and Zimmerberg J. A voltage-dependent channel involved in nutrient uptake by red blood cells infected
with the malaria parasite. Nature 406: 1001–1005, 2000.
83. Desai SA, Schlesinger PH, and Krogstad DJ. Physiologic rate of
carrier-mediated Ca2⫹ entry matches active extrusion in human
erythrocytes. J Gen Physiol 98: 349 –364, 1991.
84. Dover GJ, Boyer SH, Charache S, and Heintzelman K. Individual variation in the production and survival of F cells in sickle-cell
disease. N Engl J Med 299: 1428 –1435, 1978.
85. Dunham PB and Hoffman JF. Partial purification of the ouabainbinding component and of Na,K-ATPase from human red cell membranes. Proc Natl Acad Sci USA 66: 936 –943, 1970.
86. Dunham PB, Klimczak J, and Logue PJ. Swelling activation of
K-Cl cotransport in LK sheep erythrocytes: a three-state process.
J Gen Physiol 101: 733–765, 1993.
87. Dunham PB, Stewart GW, and Ellory JC. Chloride-activated
passive potassium transport in human erythrocytes. Proc Natl Acad
Sci USA 77: 1711–1715, 1980.
88. Dunn MJ. Red blood cell calcium and magnesium: effects upon
sodium and potassium transport and cellular morphology. Biochim
Biophys Acta 352: 97–116, 1974.
89. Eaton JW, Skelton TD, Swofford HS, Koplin CE, and Jacob
HS. Elevated erythrocyte calcium in sickle cell disease. Nature
246: 105–106, 1973.
90. Eaton WA and Hofrichter J. Sickle cell hemoglobin polymerization. Ad Protein Chem 40: 63–279, 1990.
91. Egee S, Lapaix F, Decherf G, Staines HM, Ellory JC, Doerig C,
and Thomas SL. A stretch-activated anion channel is up-regulated
by the malaria parasite Plasmodium falciparum. J Physiol 542:
795– 801, 2002.
92. Ellory JC, Culliford SJ, Stone PCW, and Stuart J. Clotrimazole, a potent Gardos channel inhibitor, protects sickle cells from
K⫹ loss and dehydration (Abstract). J Physiol 467: 384P, 1993.
93. Engelmann B and Duhm J. Intracellular calcium content of human erythrocytes: relation to sodium transport systems. J Membr
Biol 98: 79 – 87, 1987.
94. Etzion Z, Lew VL, and Bookchin RM. K(86Rb) transport heterogeneity in the low-density fraction of sickle cell anemia red blood
cells. Am J Physiol Cell Physiol 271: C1111–C1121, 1996.
95. Etzion Z, Tiffert JT, Lew VL, and Bookchin RM. Deoxygenation
increases [Ca2⫹]i in sickle cell anemia (SS) discocytes by Ca2⫹
pump inhibition as well as increased Ca2⫹ permeability (PCa) (Abstract). Blood 80: 77a, 1992.
96. Etzion Z, Tiffert T, Bookchin RM, and Lew VL. 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, 1993.
97. Fabry ME and Nagel RL. Heterogeneity of red cells in the sickler:
a characteristic with practical clinical and pathophysiological implications. Blood Cells 8: 9 –15, 1982.
98. Fabry ME and Nagel RL. The effect of deoxygenation on red cell
density: significance for the pathophysiology of sickle cell anemia.
Blood 60: 1370 –1377, 1982.
99. Fabry ME, Romero JR, Buchanan ID, Suzuka SM, Stamatoyannopoulos G, Nagel RL, and Canessa M. Rapid increase in red
85 • JANUARY 2005 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.2 on July 4, 2017
56. Cabantchik ZI and Rothstein A. The nature of the membrane
sites controlling anion permeability of human red blood cells as
determined by studies with disulfonic stilbene derivatives. J
Membr Biol 10: 311–330, 1972.
57. Canessa M, Fabry ME, Blumenfeld N, and Nagel RL. Volumestimulated, Cl⫺-dependent K⫹ efflux is highly expressed in young
human red cells containing normal hemoglobin or HbS. J Membr
Biol 97: 97–105, 1987.
58. Canessa M, Fabry ME, and Nagel RL. Deoxygenation inhibits
the volume-stimulated, Cl⫺-dependent K⫹ efflux in SS and young
AA cells: a cytosolic Mg2⫹ modulation. Blood 70: 1861–1866, 1987.
59. Cappadoro M, Giribaldi G, O’Brien E, Turrini F, Mannu F,
Ulliers D, Simula G, Luzzatto L, and Arese P. Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient
erythrocytes parasitized by Plasmodium falciparum may explain
malaria protection in G6PD deficiency. Blood 92: 2527–2534, 1998.
60. Carafoli E. Calcium pump of the plasma membrane. Physiol Rev
71: 129 –153, 1991.
61. Carafoli E. Plasma membrane calcium pump: structure, function
and relationships. Basic Res Cardiol 92 Suppl 1: 59 – 61, 1997.
62. Carafoli E and Molinari M. Calpain: a protease in search of a
function? Biochem Biophys Res Commun 247: 193–203, 1998.
63. Charache S, Barton FB, Moore RD, Terrin ML, Steinberg MH,
Dover GJ, Ballas SK, McMahon RP, Castro O, and Orringer
EP. Hydroxyurea and sickle cell anemia. Clinical utility of a myelosuppressive “switching” agent. The Multicenter Study of Hydroxyurea in Sickle Cell Anemia. Medicine 75: 300 –326, 1996.
64. Charache S, Terrin ML, Moore RD, Dover GJ, Barton FB,
Eckert SV, McMahon RP, and Bonds DR. Effect of hydroxyurea
on the frequency of painful crises in sickle cell anemia. Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia.
N Engl J Med 332: 1317–1322, 1995.
65. Charache S, Terrin ML, Moore RD, Dover GJ, McMahon RP,
Barton FB, Waclawiw M, and Eckert SV. Design of the multicenter study of hydroxyurea in sickle cell anemia. Investigators of
the Multicenter Study of Hydroxyurea. Control Clin Trials 16:
432– 446, 1995.
66. Chasis JA and Mohandas N. Erythrocyte membrane deformability and stability: two distinct membrane properties that are independently regulated by skeletal protein associations. J Cell Biol
103: 343–350, 1986.
67. Chasis JA, Prenant M, Leung A, and Mohandas N. Membrane
assembly and remodeling during reticulocyte maturation. Blood 74:
1112–1120, 1989.
68. Clark MR. Senescence of red blood cells: progress and problems.
Physiol Rev 68: 503–554, 1988.
69. Clark MR, Guatelli JC, Mohandas N, and Shohet SB. Influence
of red cell water content on the morphology of sickling. Blood 55:
823– 830, 1980.
70. Clark MR, Guatelli JC, White AT, and Shohet SB. Study on
dehydrating effect of the red cell Na⫹/K⫹-pump in nystatin-treated
cells with varying Na⫹ and water contents. Biochim Biophys Acta
646: 422– 432, 1981.
71. Clark MR, Mohandas N, Feo C, and Jacobs MS. Separate mechanisms of deformability loss in ATP-depleted and Ca-loaded erythrocytes. J Clin Invest 67: 531–539, 1981.
72. Clark MR, Mohandas N, and Shohet SB. Deformability of oxygenated irreversibly sickled cells. J Clin Invest 65: 189 –196, 1980.
73. Clark MR, Morrison CE, and Shohet SB. Monovalent cation
transport in irreversibly sickled cells. J Clin Invest 62: 329 –337,
1978.
74. Clark MR and Rossi ME. Permeability characteristics of deoxygenated sickle cells. Blood 76: 2139 –2145, 1990.
75. Clark MR, Ungar RC, and Shohet SB. Monovalent cation composition and ATP and lipid content of irreversibly sickled cells.
Blood 51: 1169 –1178, 1978.
76. Corash L. Density dependent red cell separation. In: Red Cell
Metabolism, edited by E. Beutler. New York: Churchill Livingstone,
1986, p. 90.
77. Dagher G and Lew VL. Maximal calcium extrusion capacity and
stoichiometry of the human red cell calcium pump. J Physiol 407:
569 –586, 1988.
MECHANISMS OF SICKLE CELL DEHYDRATION
100.
101.
102.
103.
104.
105.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
Physiol Rev • VOL
122.
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
rabbit, rat, and human. A new member of the cation-chloride
cotransporter family. J Biol Chem 271: 16237–16244, 1996.
Glader BE, Lux SE, Muller-Soyano A, Platt OS, Propper RD,
and Nathan DG. Energy reserve and cation composition of irreversibly sickled cells in vivo. Br J Haematol 40: 527–532, 1978.
Glader BE and Nathan DG. Cation permeability alterations during sickling: relation to cation composition and cellular hydration
of irreversibly sickled cells. Blood 51: 983–989, 1978.
Glaser T, Schwarz-Benmeir N, Barnoy S, Barak S, Eshhar Z,
and Kosower NS. Calpain (Ca2⫹-dependent thiol protease) in
erythrocytes of young and old individuals. Proc Natl Acad Sci USA
91: 7879 –7883, 1994.
Glynn IM and Karlish SJ. The sodium pump. Annu Rev Physiol
37: 13–55, 1975.
Groves JD and Tanner MJ. Glycophorin A facilitates the expression of human band 3-mediated anion transport in Xenopus oocytes. J Biol Chem 267: 22163–22170, 1992.
Grygorczyk R. Temperature dependence of Ca2⫹-activated K⫹
currents in the membrane of human erythrocytes. Biochim Biophys Acta 902: 159 –168, 1987.
Grygorczyk R and Schwarz W. Properties of the Ca2⫹-activated
K⫹ conductance of human red cells as revealed by the patch-clamp
technique. Cell Calcium 4: 499 –510, 1983.
Grygorczyk R, Schwarz W, and Passow H. Ca2⫹-activated K⫹
channels in human red cells. Comparison of single-channel currents with ion fluxes. Biophys J 45: 693– 698, 1984.
Gunn RB, Dalmark M, Tosteson DC, and Wieth JO. Characteristics of chloride transport in human red blood cells. J Gen Physiol
61: 185–206, 1973.
Hall AC and Ellory JC. Effect of high hydrostatic pressure on
“passive” monovalent cation transport in human red cells. J Membr
Biol 94: 1–17, 1986.
Hall AC and Ellory JC. Evidence for the presence of volumesensitive KCl transport in “young” human red cells. Biochim Biophys Acta 858: 317–320, 1986.
Harris JW. Studies on the destruction of red blood cells. VII.
Molecular orientation in sickle cell hemoglobin solutions. Proc Soc
Exp Biol Med 75: 197–201, 1950.
Harrison DG and Long C. The calcium content of human erythrocytes. J Physiol 199: 367–381, 1968.
Hebbel RP. Sickle hemoglobin instability: a mechanism for malarial protection. Redox Rep 8: 238 –240, 2003.
Hladky SB and Rink TJ. pH equilibrium across the red cell
membrane. In: Membrane Transport in Red Cells, edited by J. C.
Ellory and V. L. Lew. London: Academic, 1977, p. 115–135.
Hoffman JF, Joiner W, Nehrke K, Potapova O, Foye K, and
Wickrema A. The hSK4 (KCNN4) isoform is the Ca2⫹-activated K⫹
channel (Gardos channel) in human red blood cells. Proc Natl Acad
Sci USA 100: 7366 –7371, 2003.
Hoffman JF and Laris PC. Determination of membrane potentials in human and Amphiuma red blood cells by means of fluorescent probe. J Physiol 239: 519 –552, 1974.
Hoffman JF and Laris PC. Membrane electrical parameters of
normal human red blood cells. Soc Gen Physiol Ser 38: 287–293,
1984.
Hoffmann EK and Simonsen LO. Membrane mechanisms in
volume and pH regulation in vertebrate cells. Physiol Rev 69:
315–382, 1989.
Holroyde CP and Gardner FH. Acquisition of autophagic vacuoles by human erythrocytes. Physiological role of the spleen. Blood
36: 566 –575, 1970.
Holtzclaw JD, Jiang M, Yasin Z, Joiner CH, and Franco RS.
Rehydration of high-density sickle erythrocytes in vitro. Blood 100:
3017–3025, 2002.
Hunter MJ. A quantitative estimate of the non-exchange-restricted
chloride permeability of the human red cell (Abstract). J Physiol
218: 49P–50P, 1971.
Hunter MJ. Human erythrocyte anion permeabilities measured
under conditions of net charge transfer. J Physiol 268: 35– 49, 1977.
Ingram VM. A specific chemical difference between the globins of
normal human and sickle-cell anemia hemoglobin. Nature 178:
792–794, 1956.
85 • JANUARY 2005 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.2 on July 4, 2017
106.
blood cell density driven by K:Cl cotransport in a subset of sickle
cell anemia reticulocytes and discocytes. Blood 78: 217–225, 1991.
Ferreira HG and Lew VL. Use of ionophore A23187 to measure
cytoplasmic Ca buffering and activation of the Ca pump by internal
Ca. Nature 259: 47– 49, 1976.
Flatman PW, Adragna NC, and Lauf PK. Role of protein kinases
in regulating sheep erythrocyte K-Cl cotransport. Am J Physiol Cell
Physiol 271: C255–C263, 1996.
Flatman PW and Lew VL. Use of ionophore A23187 to measure
and to control free and bound cytoplasmic Mg in intact red cells.
Nature 267: 360 –362, 1977.
Flatman PW and Lew VL. Magnesium buffering in intact human
red blood cells measured using the ionophore A23187. J Physiol
305: 13–30, 1980.
Flatman PW and Lew VL. The magnesium-dependence of sodiumpump-mediated sodium-potassium and sodium-sodium exchange
in intact human red cells. J Physiol 315: 421– 446, 1981.
Franco RS, Barker-Gear R, Miller MA, Williams SM, Joiner
CH, and Rucknagel DL. Fetal hemoglobin and potassium in isolated transferrin receptor-positive dense sickle reticulocytes. Blood
84: 2013–2020, 1994.
Franco RS, Lohmann J, Silberstein EB, Mayfield-Pratt G,
Palascak M, Nemeth TA, Joiner CH, Weiner M, and Rucknagel
DL. Time-dependent changes in the density and hemoglobin F
content of biotin-labeled sickle cells. J Clin Invest 101: 2730 –2740,
1998.
Franco RS, Palascak M, Thompson H, and Joiner CH. KCl
cotransport activity in light versus dense transferrin receptor-positive sickle reticulocytes. J Clin Invest 95: 2573–2580, 1995.
Franco RS, Palascak M, Thompson H, Rucknagel DL, and
Joiner CH. Dehydration of transferrin receptor-positive sickle
reticulocytes during continuous or cyclic deoxygenation: role of
KCl cotransport and extracellular calcium. Blood 88: 4359 – 4365,
1996.
Franco RS, Thompson H, Palascak M, and Joiner CH. The
formation of transferrin receptor-positive sickle reticulocytes with
intermediate density is not determined by fetal hemoglobin content. Blood 90: 3195–3203, 1997.
Freedman JC and Hoffman JF. Ionic and osmotic equilibria of
human red blood cells treated with nystatin. J Gen Physiol 74:
157–185, 1979.
Freeman CJ, Bookchin RM, Ortiz OE, and Lew VL. K-permeabilized human red cells lose an alkaline, hypertonic fluid containing excess K over diffusible anions. J Membr Biol 96: 235–241, 1987.
Frohlich O. Relative contributions of the slippage and tunneling
mechanisms to anion net efflux from human erythrocytes. J Gen
Physiol 84: 877– 893, 1984.
Frohlich O, Leibson C, and Gunn RB. Chloride net efflux from
intact erythrocytes under slippage conditions. J Gen Physiol 81:
127–152, 1983.
Funder J and Wieth JO. Chloride and hydrogen ion distribution
between human red cells and plasma. Acta Physiol Scand 68:
234 –235, 1966.
Garcı́a-Sancho J and Lew VL. Detection and separation of human
red cells with different calcium contents following uniform calcium
permeabilization. J Physiol 407: 505–522, 1988.
Gardos G. The function of calcium in the potassium permeability
of human erythrocytes. Biochim Biophys Acta 30: 653– 654, 1958.
Garrahan PJ and Glynn IM. The stoichiometry of the sodium
pump. J Physiol 192: 217–235, 1967.
Gasbjerg PK, Funder J, and Brahm J. Kinetics of residual chloride transport in human red blood cells after maximum covalent
4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid binding. J Gen
Physiol 101: 715–732, 1993.
Gibson JS, Khan A, Speake PF, and Ellory JC. O2 dependence
of K⫹ transport in sickle cells: the effect of different cell populations and the substituted benzaldehyde 12C79. FASEB J 15: 823–
832, 2001.
Gibson JS, Speake PF, and Ellory JC. Differential oxygen sensitivity of the K⫹-Cl⫺ cotransporter in normal and sickle human red
blood cells. J Physiol 511: 225–234, 1998.
Gillen CM, Brill S, Payne JA, and Forbush B III. Molecular
cloning and functional expression of the K-Cl cotransporter from
197
198
VIRGILIO L. LEW AND ROBERT M. BOOKCHIN
Physiol Rev • VOL
168. Joiner CH and Lauf PK. The correlation between ouabain binding and potassium pump inhibition in human and sheep erythrocytes. J Physiol 283: 155–175, 1978.
169. Joiner CH, Morris CL, and Cooper ES. Deoxygenation-induced
cation fluxes in sickle cells. III. Cation selectivity and response to
pH and membrane potential. Am J Physiol Cell Physiol 264: C734 –
C744, 1993.
170. Joiner CH, Platt OS, and Lux SE. Cation depletion by the
sodium pump in red cells with pathologic cation leaks. Sickle cells
and xerocytes. J Clin Invest 78: 1487–1496, 1986.
171. Joiner CH, Rettig RK, Jiang M, and Franco RS. KCl cotransport
mediates abnormal, sulfhydryl-dependent volume regulation in
sickle reticulocytes. Blood. In press.
172. Kaul DK and Fabry ME. In vivo studies of sickle red blood cells.
Microcirculation 11: 1–13, 2004.
173. Knauf PA, Fuhrmann GF, Rothstein S, and Rothstein A. The
relationship between anion exchange and net anion flow across the
human red blood cell membrane. J Gen Physiol 69: 363–386, 1977.
174. Knauf PA, Law FY, and Marchant PJ. Relationship of net chloride flow across the human erythrocyte membrane to the anion
exchange mechanism. J Gen Physiol 81: 95–126, 1983.
175. Kopito RR. Molecular biology of the anion exchanger gene family.
Int Rev Cytol 123: 177–199, 1990.
176. Laris PC and Hoffman JF. Optical determination of electrical
properties of red blood cell and Ehrlich ascites tumor cell membranes with fluorescent dyes. Soc Gen Physiol Ser 40: 199 –210,
1986.
177. Lassen UV. Electrical potential and conductance of the red cell
membrane. In: Membrane Transport in Red Cells, edited by J. C.
Ellory and V. L. Lew. London: Academic, 1977, p. 137–172
178. Lauf PK. K⫹:Cl⫺ cotransport: sulfhydryls, divalent cations, and the
mechanism of volume activation in a red cell. J Membr Biol 88:
1–13, 1985.
179. Lauf PK. Passive K⫹-Cl⫺ fluxes in low-K⫹ sheep erythrocytes:
modulation by A23187 and bivalent cations. Am J Physiol Cell
Physiol 249: C271–C278, 1985.
180. Lauf PK and Adragna PK NC. K-Cl cotransport: properties and
molecular mechanism. Cell Physiol Biochem 10: 341–354, 2000.
181. Lauf PK, Bauer J, Adragna NC, Fujise H, Zade-Oppen AMM,
Ryu KH, and Delpire E. Erythrocyte K-Cl cotransport: properties
and regulation. Am J Physiol Cell Physiol 263: C917–C932, 1992.
182. Lauf PK, McManus TJ, Haas M, Forbush B, Duhm J III, Flatman PW, Saier MH Jr, and Russell JM. Physiology and biophysics of chloride and cation cotransport across cell membranes.
Federation Proc 46: 2377–2394, 1987.
183. Lauf PK, Perkins CM, and Adragna NC. Cell volume and metabolic dependence of NEM-activated K⫹-Cl⫺ flux in human red
blood cells. Am J Physiol Cell Physiol 249: C124 –C128, 1985.
184. Lauf PK and Theg BE. A chloride dependent K⫹ flux induced by
N-ethylmaleimide in genetically low K⫹ sheep and goat erythrocytes. Biochem Biophys Res Commun 92: 1422–1428, 1980.
185. Lehmann H. Treatment of sickle-cell anemia. Br Med J 5338:
1158 –1159, 1963.
186. Lew VL and Beaugé LA. Passive cation fluxes in red cell membranes. In: Transport Across Biological Membranes, edited by G.
Giebisch, D. C. Tosteson, and H. H. Ussing. Berlin: Springer-Verlag,
1979, vol. II, p. 85–115.
187. Lew VL and Bookchin RM. A Ca2⫹-refractory state of the Casensitive K permeability mechanism in sickle cell anemia red cells.
Biochim Biophys Acta 602: 196 –200, 1980.
188. Lew VL and Bookchin RM. Volume, pH and-ion content regulation in human red cells: analysis of transient behavior with an
integrated model. J Membr Biol 92: 57–74, 1986.
189. Lew VL and Bookchin RM. The osmotic effects of protein polymerization. Analysis of volume changes in sickle cell anemia red
cells following deoxy-hemoglobin S polymerization. J Membr Biol
122: 55– 67, 1991.
190. Lew VL and Bookchin RM. Role of reticulocyte transport heterogeneity in the generation of mature sickle cells with different
volumes. Biochem Soc Trans 20: 797– 800, 1992.
191. Lew VL and Bookchin RM. Osmotic effects of water compartments in protein polymers. Cell Biochem Funct 13: 173–176, 1995.
85 • JANUARY 2005 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.2 on July 4, 2017
146. Ingram VM. Gene mutations in human hemoglobin: the chemical
difference between normal and sickle cell hemoglobin. Nature 180:
326 –328, 1957.
147. Jacobs MH and Stewart DR. The role of carbonic anhydrase in
certain ionic exchanges involving the erythrocyte. J Gen Physiol
25: 539 –552, 1942.
148. Jacobs MH and Stewart DR. Osmotic properties of the erythrocyte. XII. Ionic and osmotic equilibria with a complex external
solution. J Cell Comp Physiol 30: 79 –103, 1947.
149. James P, Vorherr T, Krebs J, Morelli A, Castello G, McCormick DJ, Penniston JT, De Flora A, and Carafoli E. Modulation
of erythrocyte Ca2⫹-ATPase by selective calpain cleavage of the
calmodulin-binding domain. J Biol Chem 264: 8289 – 8296, 1989.
150. Jennings ML. Volume-sensitive K(⫹)/Cl(⫺) cotransport in rabbit
erythrocytes. Analysis of the rate-limiting activation and inactivation events. J Gen Physiol 114: 743–758, 1999.
151. Jennings ML and Adame MF. Direct estimate of 1:1 stoichiometry of K(⫹)-Cl(⫺) cotransport in rabbit erythrocytes. Am J Physiol
Cell Physiol 281: C825–C832, 2001.
152. Jennings ML and al Rohil N. 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, 1990.
153. Jennings ML and Schulz RK. Okadaic acid inhibition of KCl
cotransport. Evidence that protein dephosphorylation is necessary
for activation of transport by either cell swelling or N-ethylmaleimide. J Gen Physiol 97: 799 – 817, 1991.
154. Johansen JW and Ingebritsen TS. Phosphorylation and inactivation of protein phosphatase 1 by pp60v-src. Proc Natl Acad Sci
USA 83: 207–211, 1986.
155. Johnstone RM. The Jeanne Manery-Fisher Memorial Lecture
1991. Maturation of reticulocytes: formation of exosomes as a
mechanism for shedding membrane proteins. Biochem Cell Biol 70:
179 –190, 1992.
156. Johnstone RM, Adam M, Hammond JR, Orr L, and Turbide C.
Vesicle formation during reticulocyte maturation. Association of
plasma membrane activities with released vesicles (exosomes).
J Biol Chem 262: 9412–9420, 1987.
157. Johnstone RM and Ahn J. A common mechanism may be involved in the selective loss of plasma membrane functions during
reticulocyte maturation. Biomed Biochim Acta 49: S70 –S75, 1990.
158. Johnstone RM, Bianchini A, and Teng K. Reticulocyte maturation and exosome release: transferrin receptor containing exosomes shows multiple plasma membrane functions. Blood 74:
1844 –1851, 1989.
159. Johnstone RM, Mathew A, Mason AB, and Teng K. Exosome
formation during maturation of mammalian and avian reticulocytes: evidence that exosome release is a major route for externalization of obsolete membrane proteins. J Cell Physiol 147: 27–36,
1991.
160. Joiner CH. Studies on the mechanism of passive cation fluxes
activated by deoxygenation of sickle cells. Prog Clin Biol Res 240:
229 –235, 1987.
161. Joiner CH. Deoxygenation-induced cation fluxes in sickle cells. II.
Inhibition by stilbene disulfonates. Blood 76: 212–220, 1990.
162. Joiner CH. Cation transport and volume regulation in sickle red
blood cells. Am J Physiol Cell Physiol 264: C251–C270, 1993.
163. Joiner CH and Franco RS. The activation of KCl cotransport by
deoxygenation and its role in sickle cell dehydration. Blood Cells
Mol Dis 27: 158 –164, 2001.
164. Joiner CH, Gunn RB, and Frohlich O. Anion transport in sickle
red blood cells. Pediatr Res 28: 587–590, 1990.
165. Joiner CH, Jiang M, Claussen WJ, Roszell NJ, Yasin Z, and
Franco RS. Dipyridamole inhibits sickling-induced cation fluxes in
sickle red blood cells. Blood 97: 3976 –3983, 2001.
166. Joiner CH, Jiang M, Fathallah H, Giraud F, and Franco RS.
Deoxygenation of sickle red blood cells stimulates KCl cotransport
without affecting Na⫹/H⫹ exchange. Am J Physiol Cell Physiol 274:
C1466 –C1475, 1998.
167. Joiner CH, Jiang M, and Franco RS. Deoxygenation-induced
cation fluxes in sickle cells. IV. Modulation by external calcium.
Am J Physiol Cell Physiol 269: C403–C409, 1995.
MECHANISMS OF SICKLE CELL DEHYDRATION
Physiol Rev • VOL
213. Merciris P, Claussen WJ, Joiner CH, and Giraud F. Regulation
of K-Cl cotransport by Syk and Src protein tyrosine kinases in
deoxygenated sickle cells. Pflügers Arch 446: 232–238, 2003.
214. Ohnishi ST. Inhibition of the in vitro formation of irreversibly
sickled cells by cepharanthine. Br J Haematol 55: 665– 671, 1983.
215. Orringer EP, Blythe DS, Johnson AE, Phillips G Jr, Dover GJ,
and Parker JC. Effects of hydroxyurea on hemoglobin F and
water content in the red blood cells of dogs and of patients with
sickle cell anemia. Blood 78: 212–216, 1991.
216. Ortiz OE, Lew VL, and Bookchin RM. Calcium accumulated by
sickle cell anemia red cells does not affect their potassium (86Rb)
flux components. Blood 67: 710 –715, 1986.
217. Ortiz OE, Lew VL, and Bookchin RM. Deoxygenation permeabilizes sickle cell anemia red cells to magnesium and reverses its
gradient in the dense cells. J Physiol 427: 211–226, 1990.
218. Palek J. Calcium accumulation during sickling of hemoglobin S
red cells. Blood 42: 988 –1000, 1973.
219. Palek J and Lambert S. Genetics of the red cell membrane
skeleton. Semin Hematol 27: 290 –332, 1990.
220. Palek J and Liu SC. Membrane protein organization in ATPdepleted and irreversibly sickled cells. J Supramol Struct 10: 79 –
96, 1979.
221. Pauling L, Itano H, Singer SJ, and Wells IC. Sickle cell anemia:
a molecular disease. Science 110: 543–548, 1949.
222. Pearson HA, Cornelius EA, Schwartz AD, Zelson JH, Wolfson
SL, and Spencer RP. Transfusion-reversible functional asplenia in
young children with sickle-cell anemia. N Engl J Med 283: 334 –337,
1970.
223. Pearson HA, Spencer RP, and Cornelius EA. Functional asplenia in sickle-cell anemia. N Engl J Med 281: 923–926, 1969.
224. Perutz MF and Mitchison JM. State of hemoglobin in sickle-cell
anemia. Nature 166: 677– 679, 1950.
225. Petersen OH and Maruyama Y. Calcium-activated potassium
channels and their role in secretion. Nature 307: 693– 696, 1984.
226. Platt OS, Orkin SH, Dover G, Beardsley GP, Miller B, and
Nathan DG. Hydroxyurea enhances fetal hemoglobin production
in sickle cell anemia. J Clin Invest 74: 652– 656, 1984.
227. Post RL. A reminiscence about sodium, potassium-ATPase. Ann
NY Acad Sci 242: 6 –11, 1974.
228. Post RL, Albright CD, and Dayani K. Resolution of pump and
leak components of sodium and potassium ion transport in human
erythrocytes. J Gen Physiol 50: 1201–1220, 1967.
229. Pressman BC. Biological applications of ionophores. Annu Rev
Biochem 45: 501–530, 1976.
230. Raftos JE and Lew VL. Effect of intracellular magnesium on
calcium extrusion by the plasma membrane calcium pump of intact
human red cells. J Physiol 489: 63–72, 1995.
231. Rapoport S, Guest GM, and Wing M. Size, hemoglobin content,
and acid-soluble phosphorus of erythrocytes of rabbits with phenylhydrazine-induced reticulocytosis. Proc Soc Exp Biol Med 57:
334, 1944.
232. Rapoport SM. The Reticulocyte. Boca Raton, FL: CRC, 1986.
233. Reed PW and Lardy HA. A23187: a divalent cation ionophore.
J Biol Chem 247: 6970 – 6977, 1972.
234. Rega AF and Garrahan PJ. The Ca2⫹ Pump of Plasma Membranes. Boca Raton, FL: CRC, 1986.
235. Rifai N, Sakamoto M, Law T, Platt O, Mikati M, Armsby CC,
and Brugnara C. HPLC measurement, blood distribution, and
pharmacokinetics of oral clotrimazole, potentially useful antisickling agent. Clin Chem 41: 387–391, 1995.
236. Rubin E, Schlegel RA, and Williamson P. Endocytosis in sickle
erythrocytes: a mechanism for elevated intracellular Ca2⫹ levels.
J Cell Physiol 126: 53–59, 1986.
237. Sarkadi B, Szasz I, and Gardos G. The use of ionophores for
rapid loading of human red cells with radioactive cations for cation
pump studies. J Membr Biol 26: 357–370, 1976.
238. Schatzmann HJ. ATP-dependent Ca2⫹-extrusion from human red
cells. Experientia 22: 364 –365, 1966.
239. Schatzmann HJ and Vincenzi FF. Calcium movements across
the membrane of human red cells. J Physiol 201: 369 –395, 1969.
240. Serjeant GR. The emerging understanding of sickle cell disease.
Br J Haematol 112: 3–18, 2001.
85 • JANUARY 2005 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.2 on July 4, 2017
192. Lew VL, Daw N, Perdomo D, Etzion Z, Bookchin RM, and
Tiffert T. Distribution of plasma membrane Ca2⫹ pump activity in
normal human red blood cells. Blood 102: 4206 – 4213, 2003.
193. Lew VL, Etzion Z, and Bookchin RM. Dehydration response of
sickle cells to sickling-induced Ca(⫹⫹) permeabilization. Blood 99:
2578 –2585, 2002.
194. Lew VL and Ferreira HG. Variable Ca sensitivity of a K-selective
channel in intact red-cell membranes. Nature 263: 336 –338, 1976.
195. Lew VL and Ferreira HG. Calcium transport and the properties of
a calcium-activated potassium channel in red cell membranes. In:
Current Topics in Membranes and Transport, edited by A. Kleinzeller and F. Bronner. New York: Academic, 1978, vol. 10, p. 217–
277.
196. Lew VL, Ferreira HG, and Moura T. The behaviour of transporting epithelial cells. I. Computer analysis of a basic model. Proc R
Soc Lond B Biol Sci 206: 53– 83, 1979.
197. Lew VL, Freeman CJ, Ortiz OE, and Bookchin RM. A mathematical model on the volume, pH and ion content regulation of
reticulocytes. Application to the pathophysiology of sickle cell
dehydration. J Clin Invest 87: 100 –112, 1991.
198. Lew VL and Garcı́a-Sancho J. Measurement and control of intracellular calcium in intact red cells. In: Methods in Enzymology.
Biomembranes. Cellular and Subcellular Transport: Eukaryotic
(Nonepithelial) Cells, edited by S. Fleischer and B. Fleischer. San
Diego, CA: Academic, 1989, vol. 173, pt. T, p. 100 –112.
199. Lew VL, Hockaday A, Sepulveda MI, Somlyo AP, Somlyo AV,
Ortiz OE, and Bookchin RM. Compartmentalization of sickle cell
calcium in endocytic inside-out vesicles. Nature 315: 586 –589,
1985.
200. Lew VL, Muallem S, and Seymour CA. Properties of the Ca2⫹activated K⫹ channel in one-step inside-out vesicles from human
red cell membranes. Nature 296: 742–744, 1982.
201. Lew VL, Ortiz-Carranza OE, and Bookchin RM. Stochastic
nature and red cell population distribution of the sickling-induced
Ca2⫹ permeability. J Clin Invest 99: 2727–2735, 1997.
202. Lew VL, Raftos JE, Sorette MP, Bookchin RM, and Mohandas
N. Generation of normal human red cell volume, hemoglobin content and membrane area distributions, by “birth” or regulation?
Blood 86: 334 –341, 1995.
203. Lew VL and Simonsen LO. Ionophore A23187-induced calcium
permeability of intact human red blood cells (Abstract). J Physiol
308: 60P, 1980.
204. Lew VL and Simonsen LO. A23187-induced 45Ca-flux kinetics
reveals uniform ionophore distribution and cytoplasmic calcium
buffering in ATP-depleted human red cells. J Physiol 316: 6P–7P,
1981.
205. Lew VL, Tiffert T, Etzion Z, Perdomo D, Daw N, MacDonald L,
and Bookchin RM. Distribution of dehydration rates generated by
maximal Gardos channel activation in normal and sickle red blood
cells. Blood. In press.
206. Lew VL, Tsien RY, Miner C, and Bookchin RM. Physiological
[Ca2⫹]i level and pump-leak turnover in intact red cells measured
using an incorporated Ca chelator. Nature 298: 478 – 481, 1982.
207. Lorand L, Weissmann LB, Epel DL, and Bruner-Lorand J. Role
of intrinsic transglutaminase in the Ca2⫹-mediated crosslinking of
erythrocyte proteins. Biochemistry 75: 4479 – 4481, 1976.
208. Lutz HU. Erythrocyte clearance. In: Blood Cell Chemistry. Erythroid Cells, edited by J. R. Harris. New York: Plenum, 1990, vol. 1,
p. 81–120.
209. Lux SE. Spectrin-actin membrane skeleton of normal and abnormal red blood cells. Semin Hematol 16: 21–51, 1979.
210. Lux SE, John KM, and Karnovsky MJ. Irreversible deformation
of the spectrin-actin lattice in irreversibly sickled cells. J Clin
Invest 58: 955–963, 1976.
211. Mairbaurl H, Schulz S, and Hoffman JF. Cation transport and
cell volume changes in maturing rat reticulocytes. Am J Physiol
Cell Physiol 279: C1621–C1630, 2000.
212. McGoron AJ, Joiner CH, Palascak MB, Claussen WJ, and
Franco RS. Dehydration of mature and immature sickle red blood
cells during fast oxygenation/deoxygenation cycles: role of KCl
cotransport and extracellular calcium. Blood 95: 2164 –2168, 2000.
199
200
VIRGILIO L. LEW AND ROBERT M. BOOKCHIN
Physiol Rev • VOL
260. Tiffert T, Etzion Z, Bookchin RM, and Lew VL. Effects of
deoxygenation on active and passive Ca2⫹ transport and cytoplasmic Ca2⫹ buffering in normal human red cells. J Physiol 464:
529 –544, 1993.
261. Tiffert T, Garcı́a-Sancho J, and Lew VL. Irreversible ATP depletion caused by low concentrations of formaldehyde and of
calcium-chelator esters in intact human red cells. Biochim Biophys
Acta 773: 143–156, 1984.
262. Tiffert T and Lew VL. Cytoplasmic Ca2⫹ buffers in intact human
red cells. J Physiol 500: 139 –154, 1997.
263. Tiffert T and Lew VL. Kinetics of inhibition of the plasma membrane calcium pump by vanadate in intact human red cells. Cell
Calcium 30: 337–342, 2001.
264. Tiffert T, Spivak JL, and Lew VL. Magnitude of calcium influx
required to induce dehydration of normal human red cells. Biochim Biophys Acta 943: 157–165, 1988.
265. Tosteson DC. The effects of sickling on ion transport. II. The
effect of sickling on sodium and cesium transport. J Gen Physiol
39: 55– 67, 1955.
266. Tosteson DC, Carlsen E, and Dunham ET. The effects of sickling on ion transport. I. Effect of sickling on potassium transport.
J Gen Physiol 39: 31–53, 1955.
267. Tosteson DC and Hoffman JF. Regulation of cell volume by
active cation transport in high and low potassium sheep red cells.
J Gen Physiol 44: 169 –194, 1960.
268. Tosteson DC, Shea E, and Darling RC. Potassium and sodium of
red blood cells in sickle cell anemia. J Clin Invest 31: 406 – 411,
1952.
269. Vandorpe DH, Shmukler BE, Jiang L, Lim B, Maylie J, Adelman JP, De Franceschi L, Cappellini MD, Brugnara C, and
Alper SL. cDNA cloning and functional characterization of the
mouse Ca2⫹-gated K⫹ channel, mIK1. Roles in regulatory volume
decrease and erythroid differentiation. J Biol Chem 273: 21542–
21553, 1998.
270. Wang KKW, Roufogalis BD, and Villalobo A. Calpain I activates
Ca2⫹ transport by the reconstituted erythrocyte Ca2⫹ pump. J
Membr Biol 112: 233–245, 1989.
271. Wang KKW, Villalobo A, and Roufogalis BD. The plasma membrane calcium pump: a multiregulated transporter. Trends Cell Biol
2: 46 –52, 1992.
272. Watson J, Stahman AW, and Bilello FP. The significance of the
paucity of sickle cells in newborn Negro infants. Am J Med Sci 215:
419 – 423, 1948.
273. Whittam R. Transport and Diffusion in Red Blood Cells. London:
Arnold, 1964.
274. Wiley JS. Increased erythrocyte cation permeability in thalassemia and conditions of marrow stress. J Clin Invest 67: 917–922,
1981.
275. Williamson P, Puchulu E, Penniston JT, Westerman MP, and
Schlegel RA. Ca2⫹ accumulation and loss by aberrant endocytic
vesicles in sickle erythrocytes. J Cell Physiol 152: 1–9, 1992.
276. Wolff D, Spalvins A, and Canessa M. Charybdotoxin blocks with
high affinity the Ca-activated K⫹ channel of Hb A and Hb S red
cells: individual differences in the number of channels. J Membr
Biol 106: 243–252, 1988.
277. Xu C, Ma H, Inesi G, Al Shawi MK, and Toyoshima C. Specific
structural requirements for the inhibitory effect of thapsigargin on
the Ca2⫹ ATPase (SERCA). J Biol Chem 279: 17973–17979, 2004.
85 • JANUARY 2005 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.2 on July 4, 2017
241. Serjeant GR, Serjeant BE, and Milner PF. The irreversibly
sickled cell: a determinant of haemolysis in sickle cell anemia. Br J
Haematol 17: 527–533, 1969.
242. Simons TJB. Calcium-dependent potassium exchange in human
red cell ghosts. J Physiol 256: 227–244, 1976.
243. Simonsen LO. Binding of ionophore A23187 by bovine plasma
albumin. J Physiol 313: 34, 1981.
244. Simonsen LO, Gomme J, and Lew VL. Uniform ionophore
A23187 distribution and cytoplasmic calcium buffering in intact
human red cells. Biochim Biophys Acta 692: 431– 440, 1982.
245. Simonsen LO and Lew VL. The correlation between ionophore
A23187 content and calcium permeability of ATP-depleted human
red blood cells. In: Membrane Transport in Erythrocytes, edited
by U. V. Lassen, H. H. Ussing, and J. O. Wieth. Copenhagen:
Munksgaard, 1980, p. 208 –212.
246. Singer K and Singer L. Studies on abnormal hemoglobins. VIII.
The gelling phenomenon of sickle cell hemoglobin: its biologic and
diagnostic significance. Blood 8: 1008 –1023, 1953.
247. Skou JC and Esmann M. The Na,K-ATPase. J Bioenerg Biomembr 24: 249 –261, 1992.
248. Speake PF and Gibson JS. Urea-stimulated K-Cl cotransport in
equine red blood cells. Pflügers Arch 434: 104 –112, 1997.
249. Starke LC and Jennings ML. K-Cl cotransport in rabbit red cells:
further evidence for regulation by protein phosphatase type 1.
Am J Physiol Cell Physiol 264: C118 –C124, 1993.
250. Steck TL. The organization of proteins in the human red blood cell
membrane. J Cell Biol 62: 1–19, 1974.
251. Steinberg MH. Therapies to increase fetal hemoglobin in sickle
cell disease. Curr Hematol Rep 2: 95–101, 2003.
252. Steinberg MH, Barton F, Castro O, Pegelow CH, Ballas SK,
Kutlar A, Orringer E, Bellevue R, Olivieri N, Eckman J, Varma
M, Ramirez G, Adler B, Smith W, Carlos T, Ataga K, DeCastro
L, Bigelow C, Saunthararajah Y, Telfer M, Vichinsky E,
Claster S, Shurin S, Bridges K, Waclawiw M, Bonds D, and
Terrin M. Effect of hydroxyurea on mortality and morbidity in
adult sickle cell anemia: risks and benefits up to 9 years of treatment. JAMA 289: 1645–1651, 2003.
253. Steinberg MH and Brugnara C. Pathophysiological-based approaches to treatment of sickle cell disease. Annu Rev Med 54:
89 –112, 2003.
254. Stocker JW, De Franceschi L, McNaughton-Smith GA, Corrocher R, Beuzard Y, and Brugnara C. ICA-17043, a novel Gardos
channel blocker, prevents sickled red blood cell dehydration in
vitro and in vivo in SAD mice. Blood 101: 2412–2418, 2003.
255. Sunshine HR, Hofrichter J, and Eaton WA. Gelation of sickle
cell hemoglobin in mixtures with normal adult and fetal hemoglobins. J Mol Biol 133: 435– 467, 1979.
256. Tanner MJ. The major integral proteins of the human red cell.
Baillieres Clin Haematol 6: 333–356, 1993.
257. Tepikin AV, Voronina SG, Gallacher DV, and Petersen OH.
Pulsatile Ca2⫹ extrusion from single pancreatic acinar cells during
receptor-activated cytosolic Ca2⫹ spiking. J Biol Chem 267: 14073–
14076, 1992.
258. Tiffert T, Bookchin RM, and Lew VL. Calcium homeostasis in
normal and abnormal human red cells. In: Red Cell Membrane
Transport in Health and Disease, edited by I. Bernhardt and J. C.
Ellory. Berlin: Springer-Verlag, 2003, p. 373– 405.
259. Tiffert T, Daw N, Perdomo D, and Lew VL. A fast and simple
screening test to search for specific inhibitors of the plasma membrane calcium pump. J Lab Clin Med 137: 199 –207, 2001.