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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 2164-2168
RED CELLS
Dehydration of mature and immature sickle red blood cells during
fast oxygenation/deoxygenation cycles: role of KCl cotransport and
extracellular calcium
Anthony J. McGoron,
Clinton H. Joiner,
Mary B. Palascak,
William J. Claussen, and
Robert
S. Franco
From the Department of Radiology and the Division of
Hematology/Oncology, Department of Internal Medicine, University of
Cincinnati College of Medicine; Children's Hospital Research
Foundation; and the Comprehensive Sickle Cell Center, Cincinnati, OH.
 |
Abstract |
Sickle red blood cells (RBC) become dehydrated as a consequence of
potassium loss. This process depends at least partly on deoxygenation
and may be influenced by the presence of oxygenation/deoxygenation cycles and the frequency of cycling. In this study, sickle RBC were
subjected to approximately 180 oxygenation/deoxygenation cycles during
4 hours to evaluate RBC dehydration with cycle periods more similar to
in vivo cycles than those in previous studies. A continuous-flow,
steady-state apparatus circulated a dilute RBC suspension through
gas-permeable silicone tubing with segments that were exposed to either
nitrogen or ambient oxygen. The percentage of sickling and partial
pressure of oxygen were measured by means of sampling ports in the
deoxygenation and oxygenation regions. The density increase
(dehydration) of young (transferrin receptor-positive) and mature
(transferrin receptor-negative) RBC and the requirements for calcium
and chloride were evaluated. Density increase correlated with the
percentage of sickled cells at the deoxygenation sampling port and was
observed only in the presence of calcium, thereby implicating the
calcium-dependent potassium channel (Gardos pathway). Density increase
was not dependent on the presence of chloride, making it unlikely that
KCl cotransport was an important pathway under these conditions.
(Blood. 2000;95:2164-2168)
© 2000 by The American Society of Hematology.
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Introduction |
Dehydration of sickle red blood cells (RBC), which is
caused by intracellular depletion of potassium (K+),
appears to be an important pathophysiologic factor in sickle cell
disease.1,2 Both mature and immature (transferrin
receptor-positive [TfR+]) sickle RBC are found in the
abnormally dense fractions, indicating that loss of K+ is a
rapid process for at least some cells.3,4 The
K+ efflux that mediates sickle RBC dehydration and shifts
in cell density may occur through 2 high-capacity pathways, KCl
cotransport and the calcium (C++)-dependent K+
channel.1 A relatively nonselective increase in
cation permeability also occurs as a direct result of sickling.
Although cation fluxes due to this deoxygenation-induced mechanism are
most likely too small to cause measurable density changes directly
during short in vitro experiments, this pathway is thought to be
responsible for the influx of Ca++ that activates the
C++-dependent K+ channel if intracellular
Ca++ exceeds a threshold value of approximately 40 nmol/L.5,6
Previous studies showed that KCl cotransport was active in sickle RBC
at low pH (maximal at pH 6.8) and under hypotonic
conditions.7 There is also evidence that sickle RBC KCl
cotransport activity is dependent on oxygen in a complex way, with
higher activity at a low and high partial pressure of oxygen
(pO2) and lower activity at an intermediate
pO2.8 This activity may contribute to the dehydration of sickle RBC, especially younger cells, under oxygenated conditions. Apovo et al9 and Franco et al10
found that during relatively slow oxygenation/deoxygenation cycles,
both KCl cotransport and the Ca++-dependent K+
channel were important in the dehydration of sickle RBC
but that hydration changes during continuous deoxygenation depended
only on the presence of Ca++. Horiuchi and
Asakura11 studied a reticulocyte-enriched light fraction
subjected to deoxygenation cycles and found that extracellular Ca++ was required for net cation depletion and
density increase. However, the role of KCl cotransport was not investigated.
The goal of the research presented here was to determine the pathway or
pathways responsible for dehydration of mature (transferrin receptor-negative [TfR ]) sickle RBC and immature
(TfR+) sickle RBC during a large number of relatively fast
oxygenation/deoxygenation cycles (180 cycles at 80 seconds per cycle).
The contribution of the Ca++-dependent K channel to sickle
RBC dehydration was determined by performing experiments with and
without Ca++. KCl cotransport was evaluated in experiments
with and without chloride (Cl).
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Materials and methods |
Informed consent was obtained under a protocol approved by the
University of Cincinnati Institutional Review Board. Subjects (genotype,  SS) had not undergone
transfusion for at least 3 months and were in a clinically stable
condition. Nine experiments were conducted. Blood was drawn from the
subjects into heparin-coated vacuum containers. After overnight storage
in plasma at 4°C, RBC were washed 3 times with the appropriate
buffer and suspended to a concentration of 50%. The buffers used were
HBIS (135 mmol/L of sodium chloride [NaCl], 20 mmol/L of HEPES, 5 mmol/L of KCl, 1 mmol/L of dibasic sodium phosphate
[Na2HPO4], 1.5 mmol/L of calcium chloride, 1 mmol/L of magnesium chloride, and 10 mmol/L of glucose; 290-300 mOsm/kg, pH 7.4, at 37°C); HBIN, which was the same as HBIS except that Cl was replaced with nitrate
(NO3); ethylene glycol tetraacetic acid
(EGTA), which was Ca++-free HBIS with 0.1 mmol/L of EGTA;
and phosphate-buffered saline (145 mmol/L of NaCl, 1.4 mmol/L of sodium
biphosphate, and 8.3 mmol/L of Na2HPO4;
pH 7.4).
Oxygenation/deoxygenation continuous-circulation system
The in vitro continuous-circulation system for RBC
oxygenation/deoxygenation (Figure 1) used
gas-permeable silicone tubing to transfer oxygen. For deoxygenation,
the tubing passed though a cylindrical gas-filled chamber that was
flushed with nitrogen. This cylinder was submerged in a
temperature-regulated (37°C) water bath that was continuously
stirred. For oxygenation, the gas-permeable tubing passed through
water-bath fluid that was exposed to ambient air. A peristaltic pump
circulated the RBC suspension through the oxygenation and deoxygenation
segments, which were connected by gas-impermeable tubing containing
oxygenation and deoxygenation sampling ports (Figure 1). The total
volume of the RBC suspension was 7.2 mL and the flow rate was 5.5 mL/min, yielding an average cycle time of approximately 80 seconds,
with 40 seconds in the deoxygenator and 22 seconds in the oxygenator. The suspension spent an additional 11 seconds in the oxygenation state
in the pump and associated tubing and 7 seconds in the deoxygenation state in the connecting tubing. The sampling ports, which consisted of
3-way valves, allowed samples (200 µL) to be withdrawn as fresh buffer was drawn into the system, thereby preserving the system volume.
The presence of parallel circuits permitted 2 RBC suspensions to be
cycled simultaneously. The calculated maximum shear stress on the cells
was < 1 dyne/cm2, which is much less than the threshold
value ( 100 dynes/cm2) for shear-dependent
K+-transport activation in normal RBC.12
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| Fig 1.
Two-channel fast oxygenation/deoxygenation circulation
system.
Oxygenated blood is pumped through the nitrogen chamber, outside the
water bath to access ports, to the oxygenator, and back to the pump.
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In most experiments, the gas-permeable silicone tubing had an inner
diameter (ID) of 2 mm and an outer diameter (OD) of 3 mm
(Specialty Manufacturing, Saginaw, MI). In some experiments, however,
thinner-walled silicone tubing (1.5-mm ID × 2-mm OD) was used to
provide a greater difference in pO2 values between the
oxygenation and deoxygenation states. The length of the tubing with the
smaller ID was increased to provide the same volume and circulation
time as the larger-ID tubing. The interaction between the oxygenation
and deoxygenation segments is complex, and the pO2 values
depend not only on tubing-wall thickness but also on hematocrit, flow
rate, and the relative lengths of tubing in the oxygenator and
deoxygenator. In preliminary experiments, values for these variables
that provided an adequate pO2 range while minimizing the
oxygenation/deoxygenation cycle time were determined.
The fast-cycle apparatus used in these experiments has several
advantages in addition to approximating in vivo kinetics more closely
than methods using slower cycles. There is no air-liquid interface,
which may cause lysis and lead to concentration of salts during long
incubation periods. Also, because the cells are in a flow system and
subjected to shear forces that tend to align them with the flow axis,
waves of polymer formation tend to be aligned in the same direction,
resulting in uniform, severely elongated sickle forms.
Experimental protocol
Buffer alone was circulated for approximately 20 minutes before 0.14 mL of the 50% RBC suspension was added during a 1.5-minute period to
distribute the cells evenly. The RBC suspensions (1%) were cycled for
4 hours. Oxygenation and deoxygenation samples were taken for
pO2 measurements (Blood Gas Analyzer, model 1304; Instrumentation Laboratories, Lexington, MA) at 30 minutes, 2 hours,
and 4 hours to verify pO2 stability. After 4 hours of
cycling, the 1% cell suspensions were centrifuged and the optical
density (415 nm) of the supernatants was determined. This value was
always < 5% of the corresponding optical density for a lysed 1%
cell suspension. A simultaneous oxygenated control was obtained by adding RBC (1% final concentration) to the same buffers used for oxygenation/deoxygenation cycling and incubating the mixture for 4 hours in a temperature-controlled (37°C) water bath. In 2 control experiments, cells were circulated without exposure to nitrogen, so
that no oxygenation/deoxygenation cycling occurred.
Approximately 20 minutes before the end of the cycle period, 200 µL
of cell suspension was removed and transferred, without exposure to the
atmosphere, to deoxygenated PBS containing 10% formalin. The
percentage of sickle RBC was determined by using Nomarsky optics to
assess duplicate, coded samples. A RBC was considered sickled if it had
at least 1 pointed projection. In these preparations, TfR+
and TfR cells were not distinguished, and all data on
cell sickling included both cell types.
After removal from the apparatus, the RBC suspensions were centrifuged
and the osmolality of the supernatant was determined. For each
experiment, the osmolality was found to be unchanged, thus eliminating
the possibility of a leak in the apparatus during cycling. All further
processing was done under ambient, oxygenated conditions. The cells
were washed once with HBIS and resuspended to a final RBC concentration
of 40%. An aliquot was placed in 10% buffered formalin to measure the
percentage of sickle RBC after reoxygenation.
Discontinuous arabinogalactan density gradients were prepared as
previously described4 by using densities of 1.086, 1.094, and 1.103 g/mL and a cushion > 1.15 g/mL. Ten microliters of each 40% cell suspension, including 2 cycled test samples and 2 noncycled controls, were separated at 22°C into 4 density fractions in a microultracentrifuge (Airfuge; Beckman, Palo Alto, CA). After the RBC
in each fraction were counted (CBC-5 counter; Coulter, Hialeah, FL),
the cells were allowed to react with mouse monoclonal fluorescein
isothiocyanate-conjugated anti-TfR (Dako, Carpenteria, CA) and an
appropriate idiotypic control antibody. The percentage of
TfR+ cells in each fraction was determined by flow
cytometry (XL-MCL flow cytometer, Coulter) as previously
described.4
TfR+ cells are a well-defined population that normally (ie,
in the absence of nucleated erythroid cells) contains the youngest erythroid cells in the circulation. TfR cells
represent a wide range of cell age, from reticulocytes that no longer
have detectable TfR to the oldest cells in the circulation. Usually
about one half of sickled reticulocytes are TfR+.
Therefore, in a typical patient with 10% reticulocytes, about 5% of
the TfR population are reticulocytes and 95% are
mature RBC.
After the percentage of total TfR+ cells in each fraction
was determined, a density score (range, 100-400) was calculated to characterize the density distribution of the RBC suspension
numerically.4 Density shift was defined as the difference
between the density score of the cycled RBC suspension and the density
score of the paired noncycled control RBC suspension. The role of
Ca++ in mediating the density shift of the cells was
evaluated by comparing the density shifts in parallel flow channels in
buffers containing either 1.5 mmol/L Ca++ or no
Ca++ (0.1 mmol/L EGTA). Activation of KCl cotransport was
determined by comparing buffers that contained either
Cl or NO3
in parallel flow chambers in the presence of Ca++.
The KCl cotransport pathway is dependent on the presence of Cl, whereas the Ca++-dependent
K+ channel is inactive in the absence of Ca++.
| |
Results |
A dilute suspension of sickle RBC (Figure
2) was repetitively cycled every 80 seconds
between oxygenation and deoxygenation states in a continuous-flow,
steady-state apparatus. The oxygenation and deoxygenation
pO2 values stabilized within 30 minutes after the RBC were
added and remained constant thereafter. The pO2 ranges were
25 to 37 mmHg in the deoxygenation port and 55 to 80 mmHg in the
oxygenation port. Because of the variability in pO2 values and differences in RBC properties among patients, there was a wide
range in the percentage of sickle RBC: 12% to 92% at the deoxygenation port and 4% to 59% at the oxygenation port. Morphologic sickling was uniform, with essentially all the sickle RBC extending in
2 directions along the same axis (Figure 2C). These cells appeared to
be morphologically similar to those in venous blood samples and were
distinctly different from the forms with more random polymer
orientation observed with deoxygenation in nonflow
systems.13 The cycling-induced sickling was
reversible, as indicated by an almost complete lack of sickle forms
after the cycled cells were washed in oxygenated buffer (Figure 2D).
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| Fig 2.
Photomicrographs of fixed sickle red blood cells from a
typical experiment.
(A) Results after 4 hours of incubation at 37°C under oxygenation
conditions. (B) Photomicrograph obtained from the oxygenation port
after 3.67 hours of cycling. (C) Photomicrograph obtained from the
deoxygenation port after 3.67 hours of cycling. (D) Results after 4 hours of cycling followed by oxygenation.
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After 4 hours of cycling, the cells were separated by density under
oxygenated conditions. An example of the distribution histograms for
cell density of control (incubated 4 hours under oxygenated conditions)
and cycled cells, with and without Ca++, is shown in Figure
3. Density distributions and scores are
given for the total RBC population and for the TfR+ and
TfR subpopulations. In the presence of
Ca++, the average density of all RBC increased during
cycling compared with the density in the paired noncycled controls. In
the absence of Ca++, there was no change in the density
score. As shown in Figure 3, there was a greater shift among
TfR+ cells than among
TfR cells in the presence of
Ca++. Cells that were circulated without
oxygenation/deoxygenation cycling, either with or without
Ca++, had no change in density score for either
TfR or TfR+ cells compared with
incubated, noncirculated controls.
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| Fig 3.
Typical cell-density distributions for control cells
(incubated 4 hours under oxygenation conditions) and for cells that
were cycled for 4 hours.
The cycled samples with and without calcium were run concurrently in
the 2 channels of the oxygenation/deoxygenation apparatus.
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It was expected that the amount of cycle-dependent dehydration would be
a function of the degree of sickling, which varied widely among
experiments. Figure 4 shows the change in
density score as a function of sickling (on the deoxygenation side)
after 4 hours of oxygenation/deoxygenation cycling. The trend lines were drawn by using only these experiments, with a direct comparison of
the effect of Ca++. For both TfR+ and
TfR cells, there was a linear relation between the
percentage of the sickle cells and the change in density in the
presence of Ca++. As was found in previous studies with
slower oxygenation/deoxygenation cycles,10 young
(TfR+) cells had a greater density shift than mature
(TfR) cells. In the absence of Ca++, no
consistent density shifts were observed, even with a high percentage
of sickle RBC. This lack of shift in the absence of Ca++ indicates that under these conditions there is
little or no dehydration that can be attributed to activation of KCl
cotransport.
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| Fig 4.
Change in density score (DS) compared with the percentage
of deoxygenation sickling for transferrin receptor-positive and
transferrin receptor-negative cells in the presence and absence of
extracellular calcium.
The change in DS is defined as the DS after 4 hours of
oxygenation/deoxygenation cycling minus the DS of the appropriate
control incubated for 4 hours under oxygenation conditions. Open
symbols indicate experiments with ( ) or without ( ) calcium.
Shaded symbols indicate experiments with ( ) or without ( )
chloride in the presence of calcium. Symbols connected by a dotted or
broken line indicate paired experiments.
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Additional experiments (shaded symbols in Figure 4) compared
NO3 and
Cl buffers in parallel flow channels in the presence
of Ca++. For TfR+ cells, which have a high
potential KCl cotransport activity and therefore represent a sensitive
test for activation of this pathway, there was clearly no
Cl dependency for the observed density
shift. These results confirm that the KCl cotransport pathway was not
active under these conditions.
| |
Discussion |
Two major pathways have been proposed to mediate sickle RBC
dehydration: the Ca++-dependent K channel, which appears to
be activated by a sickling-induced increase in Ca++
permeability, and KCl cotransport, which is thought to
participate in the volume regulation of young erythroid cells but
may be overactive in sickle cells.
Our study showed that the Ca++-dependent K channel, but not
KCl cotransport, is activated in sickle RBC by fast
oxygenation/deoxygenation cycling at physiologic pH and osmolality and
that the resulting density shift is proportional to the percentage of
sickle cells. This is the first demonstration of sickle cell
dehydration mediated by oxygenation/deoxygenation cycles with periods
approaching those in vivo. As in previous studies with slower cycles,
the young TfR+ cells had a greater density shift than the
more mature RBC in the same cycled sample. Therefore, it appears that
the Ca++-dependent K channel is activated by both
continuous10 and cyclic deoxygenation conditions and that
either slow10 or fast cycles are effective in both mature
and immature sickle RBC. The greater density change of TfR+
cells could be due to either a quantitative or qualitative difference in sickling or to a greater sensitivity to sickling-induced dehydration in these cells. In the presence of Ca++, dehydration was
independent of Cl. Therefore, under fast-cycle
conditions, there was no evidence of KCl cotransport activation.
In young erythroid cells, KCl cotransport may be activated in vitro by
hypotonic swelling or low pH. However, the mechanism responsible for
activation of this pathway in vivo has not been determined. There is
evidence that cyclic deoxygenation can serve as an activating stimulus,
but this now appears to depend on the rate of cycling employed. Data
from Apovo et al9 and our laboratory10 showed
that under relatively slow cyclic, but not continuous, deoxygenation
conditions, KCl cotransport was activated. To explain the difference
between continuous and slow cyclic deoxygenation, we developed a model
in which activation depends on both the phosphorylation state of the
cell and the concentration of free magnesium
([Mg++]).10,14 Jennings and
Schulz15 showed that KCl cotransport activation is
associated with protein dephosphorylation, and deoxygenation results in
a general RBC protein dephosphorylation.16 However, there
is also an increase in [Mg++] on deoxygenation, since
2,3-diphosphoglycerate is bound to deoxyhemoglobin rather than
Mg++. Because [Mg++] inhibits KCl
cotransport,17-19 for the cotransporter to be active there
must be both dephosphorylation and low [Mg++], two
conditions not present during either continuous oxygenation or
continuous deoxygenation. When cells are cycled between oxygenation and
deoxygenation, however, it is possible that these two conditions exist
during part of the cycle.
It is reasonable to assume that changes in [Mg++] are
fast and follow the oxygenation/deoxygenation state of the hemoglobin. However, enzyme-mediated changes in phosphorylation state may be
slower. In fact, a delay time of several minutes for volume-stimulated KCl cotransport has been observed in sickle RBC.20 On the
basis of the slow-cycle data, we hypothesized that immediately after reoxygenation there is a period during which [Mg++] is
low but rephosphorylation lags behind, allowing both conditions to be
present simultaneously. It is during this phase of the cycle that KCl
cotransport may be active. This hypothesis is supported by recent
studies14 in which the ionophore A23187 and 0.15 mmol/L of
extracellular [Mg++] was used to clamp the intracellular
[Mg++] at oxygenated levels during continuous
deoxygenation of a light fraction of sickle RBC. This prevented the
usual increase in [Mg++] and resulted in a significant
increase in deoxygenation-induced, chloride-dependent potassium flux
(KCl cotransport). Therefore, these experiments showed that
deoxygenation, presumably through dephosphorylation, could activate the
cotransporter if [Mg++] is maintained at a low level.
The fast-cycle experiments described here examined this question from
another viewpoint. If KCl cotransport is active during slow cycling
only during the first part of the oxygenation phase, then it might be
expected that increasing the number of cycles in a given period would
result in greater activation, since the transition would occupy a
greater fraction of the cycle time. However, this assumes that there is
sufficient time during the deoxygenation phase for dephosphorylation to
occur. As noted above, evidence from studies in cells in which KCl
cotransport was activated by swelling indicated that dephosphorylation
takes minutes.20 If this were also
true of activation by deoxygenation, cells would not remain in the
deoxygenation state for a sufficient time during fast cycles for
subsequent activation upon oxygenation.
Although KCl cotransport was not active in our fast-cycle experiments,
there is good evidence that this pathway contributes to sickle RBC
dehydration in vivo. Oral administration of magnesium, an inhibitor of
this pathway, increases intracellular magnesium levels in sickle RBC
and has a beneficial effect on sickle cell hydration.21
Furthermore, rehydrated dense TfR+ sickle cells have been
found to have higher KCl cotransport activity than TfR+
sickle cells that remained light in vivo,4
suggesting that this pathway contributes to the fast dehydration that
occurs in some young sickle cells. KCl cotransport may be activated if
sickle cells are subjected to slower-than-normal deoxygenation cycles in vivo. For example, sickle reticulocytes adhere to postcapillary venules,22 presumably as a result of their high number of
adhesion molecules.23,24 The retention of these cells in
the circulation may be an important factor in activation of
sickle RBC KCl cotransport in vivo.
| |
Footnotes |
Submitted February 22, 1999; accepted November 12, 1999.
Supported by National Institutes of Health grant RO1 HL51174 and
Cincinnati Comprehensive Sickle Cell Center grant HL57614.
Reprints: Anthony J. McGoron, Florida International University
Biomedical Engineering Institute, 10555 W Flagler St, Miami, FL
33174; e-mail: mcgoron{at}eng.fiu.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Joiner CH.
Cation transport and volume regulation in sickle red blood cells.
Am J Physiol.
1993;264:C251[Abstract/Free Full Text].
2.
Bunn HF.
Pathogenesis and treatment of sickle cell disease.
N Engl J Med.
1997;337:762[Free Full Text].
3.
Bookchin RM, Ortiz OE, Lew VL.
Evidence for a direct reticulocyte origin of dense red cells in sickle cell anemia.
J Clin Invest.
1991;87:113.
4.
Franco RS, Palascak M, Thompson H, Joiner CH.
KCl cotransport activity in light versus dense transferrin receptor-positive sickle reticulocytes.
J Clin Invest.
1995;95:2573.
5.
Tiffert T, Spivak JL, Lew VL.
Magnitude of calcium influx required to induce dehydration of normal human red cells.
Biochim Biophys Acta.
1988;943:157[Medline]
[Order article via Infotrieve].
6.
Etzion Z, Tiffert T, Bookchin RM, Lew VL.
Effects of deoxygenation on active and passive Ca++ transport and on the cytoplasmic Ca++ levels of sickle cell anemia red cells.
J Clin Invest.
1993;92:2489.
7.
Brugnara C, Bunn HF, Tosteson DC.
Regulation of erythrocyte cation and water content in sickle cell anemia.
Science.
1986;232:388[Abstract/Free Full Text].
8.
Gibson JS, Speake PF, Ellory JC.
Differential oxygen sensitivity of the K+-Cl cotransporter in normal and sickle human red blood cells.
J Physiol (Lond).
1998;511:225[Abstract/Free Full Text].
9.
Apovo M, Beuzard Y, Galacteros F, Bachir D, Giraud F.
The involvement of the Ca-dependent K channel and of the KCl co-transport in sickle cell dehydration during cyclic deoxygenation.
Biochim Biophys Acta.
1994;1225:255[Medline]
[Order article via Infotrieve].
10.
Franco RS, Palascak M, Thompson H, Rucknagel DL, Joiner CH.
Dehydration of transferrin receptor-positive sickle reticulocytes during continuous or cyclic deoxygenation: role of KCl cotransport and extracellular calcium.
Blood.
1996;88:4359[Abstract/Free Full Text].
11.
Horiuchi K, Asakura T.
Formation of light irreversibly sickled cells during deoxygenation-oxygenation cycles.
J Lab Clin Med.
1987;110:653[Medline]
[Order article via Infotrieve].
12.
Ney PA, Christopher MM, Hebbel RP.
Synergistic effects of oxidation and deformation on erythrocyte monovalent cation leak.
Blood.
1990;75:1192[Abstract/Free Full Text].
13.
Joiner CH, Morris CL, Cooper ES.
Deoxygenation-induced cation fluxes in sickle cells III. Cation selectivity and response to pH and membrane potential.
Am J Physiol.
1993;264:C734[Abstract/Free Full Text].
14.
Joiner CH, Jiang M, Fathallah H, Giraud F, Franco RS.
Deoxygenation of sickle red blood cells stimulates KCl cotransport without affecting Na+/H+ exchange.
Am J Physiol.
1998;274:C1466[Abstract/Free Full Text].
15.
Jennings ML, 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.
1991;97:799[Abstract/Free Full Text].
16.
Fathallah H, Coezy E, De Neef RS, Hardy-Dessources MD, Giraud F.
Inhibition of deoxygenation-induced membrane protein dephosphorylation and cell dehydration by phorbol esters and okadaic acid in sickle cells.
Blood.
1995;86:1999[Abstract/Free Full Text].
17.
Brugnara C, Tosteson DC.
Inhibition of K transport by divalent cations in sickle erythrocytes.
Blood.
1987;70:1810[Abstract/Free Full Text].
18.
Canessa M, Fabry ME, Nagel RL.
Deoxygenation inhibits the volume-stimulated, Cl-dependent K+ efflux in SS and young AA cells: a cytosolic Mg2+ modulation.
Blood.
1987;70:1861[Abstract/Free Full Text].
19.
Delpire E, Lauf PK.
Magnesium and ATP dependence of K-Cl co-transport in low K+ sheep red blood cells.
J Physiol
1991;441:219[Abstract/Free Full Text].
20.
Canessa M, Romero JR, Lawrence C, Nagel RL, Fabry ME.
Rate of activation and deactivation of K:Cl cotransport by changes in cell volume in hemoglobin SS, CC and AA cells.
J Membr Biol.
1994;142:349[Medline]
[Order article via Infotrieve].
21.
De Franceschi L, Bachir D, Galacteros F, et al.
Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease.
J Clin Invest.
1887;100:1847[Medline]
[Order article via Infotrieve].
22.
Kaul DK, Fabry ME, Nagel RL.
Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: pathophysiological implications.
Proc Natl Acad Sci U S A.
1989;86:3356[Abstract/Free Full Text].
23.
Kumar A, Eckmam JR, Swerlick RA, Wick TM.
Phorbol ester stimulation increases sickle erythrocyte adherence to endothelium: a novel pathway involving  41 integrin receptors on sickle reticulocytes and fibronectin.
Blood.
1996;88:4348[Abstract/Free Full Text].
24.
Swerlick RA, Eckman JR, Kumar A, Jeitler M, Wick TM.
41-integrin expression on sickle reticulocytes: vascular cell adhesion molecule-1-dependent binding to endothelium.
Blood.
1993;82:1891[Abstract/Free Full Text].
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[Abstract]
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P. Bennekou, L. de Franceschi, O. Pedersen, L. Lian, T. Asakura, G. Evans, C. Brugnara, and P. Christophersen
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,
March 1, 2001;
97(5):
1451 - 1457.
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Abstract
Introduction