1 Cincinnati Comprehensive Sickle Cell Center and Divisions of 2 Pediatric and 4 Medical Hematology/Oncology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039; and 3 Laboratoire de Biomembranes et Messagers Cellulaires, Centre National de la Recherche Scientifique ERS 571, Université Paris XI, Orsay, France
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ABSTRACT |
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KCl cotransport activated by swelling of sickle red blood cells
(SS RBC) is inhibited by deoxygenation. Yet recent studies found a
Cl-dependent increase in
sickle reticulocyte density with cyclic deoxygenation. This study
sought to demonstrate cotransporter stimulation by deoxygenation of SS
RBC in isotonic media with normal pH. Low-density SS RBC exhibited a
Cl
-dependent component of
the deoxygenation-induced net K+
efflux, which was blocked by two inhibitors of KCl cotransport, [(dihydroindenyl)oxy]alkanoic acid and okadaic acid.
Cl
-dependent
K+ efflux stimulated by
deoxygenation was enhanced 2.5-fold by clamping of cellular
Mg2+ at the level in oxygenated
cells using ionophore A-23187. Incubating cells in high external
K+ or
Rb+ minimized inhibition of KCl
cotransport by internal Mg2+, and
under these conditions deoxygenation markedly stimulated KCl
cotransport in the absence of ionophore. Activation of KCl cotransport
by deoxygenation of SS RBC in isotonic media at normal pH is consistent
with the generalized dephosphorylation of membrane proteins induced by
deoxygenation and activation of the cotransporter by a
dephosphorylation mechanism.
Na+/H+
exchange activity, known to be modulated by cytosolic
Ca2+ elevation and cell shrinkage,
remained silent under deoxygenation conditions.
potassium; sodium; erythrocyte; reticulocyte; volume regulation
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INTRODUCTION |
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DEHYDRATION OF RED BLOOD CELLS containing sickle hemoglobin (Hb S) (SS RBC) contributes to the pathophysiology of sickling because of the high dependence of polymerization on the concentration of Hb S (19). Although the mechanism of dehydration in vivo is not entirely clear, cation loss from sickle cells in vitro appears to involve both sickling-dependent and sickling-independent mechanisms (35).
Sickling-dependent cation loss results from changes in membrane permeability to Na+, K+, and Ca2+ associated with formation of membrane spicules induced by ordered Hb S polymer. In the presence of physiological external Ca2+ concentrations, sickling-induced K+ loss exceeds Na+ gain, as a result of three different mechanisms: 1) imbalance between Na+ influx and K+ efflux via the sickling-induced pathway per se (4, 37), 2) unbalanced compensation of the Na+/K+ pump (40), and 3) activation of Ca2+-dependent K+ channels by Ca2+ influx via the sickling-induced pathway (4, 6). The relative contribution of these pathways to SS RBC dehydration in vivo is not known and may, in fact, vary among different patients and among SS RBC subpopulations in the same patient.
Sickling-independent cation loss from SS RBC may be mediated by the KCl cotransporter, a volume regulatory pathway exhibiting high activity in reticulocytes and capable of rapid K+ efflux (5, 10). The cotransporter probably contributes to the volume reduction that accompanies normal reticulocyte maturation. KCl cotransport activity is markedly increased in SS RBC (5), perhaps as a consequence of the young age of these cells (10) or possibly secondary to specific interactions of Hb S with the transporter or its regulators (47). The importance of KCl cotransport in dehydration of SS reticulocytes was suggested by our recent finding that reticulocytes that had been dehydrated in vivo had higher apparent KCl cotransport activity than reticulocytes that had remained normally hydrated in vivo (28).
Under isotonic conditions at pH 7.3-7.4, no
Cl-dependent component of
K+ loss from oxygenated SS RBC can
be demonstrated (7, 10, 36, 39). KCl cotransport is known to be
activated in swollen or acidified cells by mechanisms involving
dephosphorylation of the transporter or its regulator(s) (3, 25, 32,
33). KCl cotransport activation in SS RBC has generally been considered independent of sickling because deoxygenation inhibits
swelling-activated KCl cotransport (11). Binding to deoxygenated Hb of
the major cellular Mg2+ buffers,
2,3-diphospho-D-glycerate and
ATP, results in increased intracellular
Mg2+ (24, 48), which inhibits the
activated cotransporter (8, 18, 43, 49). In this context, it was not
surprising that previous studies found no
Cl
-dependent
K+ efflux in SS RBC subjected to
prolonged, continuous deoxygenation (2, 39). These results were
difficult to reconcile, however, with later studies showing that the
K+ efflux and/or density
changes of SS RBC subjected to cyclic deoxygenation under isotonic
conditions at normal pH were inhibited by the KCl cotransport inhibitor
[(dihydroindenyl)oxy]alkanoic acid (DIOA) (1, 46,
52). Recently we also reported that SS RBC reticulocytes exhibited a significant
Cl
-dependent change in
density when subjected to cyclic deoxygenation in vitro, but not under
continuous deoxygenation or oxygenation (29).
All of these findings could be explained by a model in which
deoxygenation induces a change in the phosphorylation-dephosphorylation equilibrium of the cell, thereby activating KCl cotransport. Such a
deoxygenation-induced change in phosphorylation was recently reported
for several red cell membrane proteins and shown to result from
activation of okadaic acid-sensitive phosphatase(s) (22). The model
predicts that the "activated" cotransporter would, nevertheless, be inhibited by elevated internal
Mg2+ during deoxygenation. On
reoxygenation, with lowering of cellular Mg2+, the activated KCl
cotransporter would be released from inhibition and mediate
K+ and
Cl efflux until the slower
process of deactivation of the transporter occurred. The present study
was undertaken to test the hypothesis that the KCl cotransporter is
activated by deoxygenation, by measuring transport under conditions
that controlled cellular Mg2+. In
addition, we examined whether the
Na+/H+
exchanger could contribute to the deoxygenation-induced
Na+ influx, since deoxygenation
may produce an elevation of cytosolic Ca2+ (21) as well as cell
shrinkage, both of which could result in the activation of
Na+/H+
exchange (12, 20). A preliminary report of these studies has appeared
in abstract form (38).
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MATERIALS AND METHODS |
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Incubation media.
Unless otherwise noted, chemicals were obtained from Sigma (St. Louis,
MO) and were reagent grade or better. HEPES-buffered saline (HBS)
contained (in mM) 140 NaCl, 20 HEPES (pH 7.4 at 37°C with NaOH),
0.1 EDTA, and 10 glucose. In experiments in which cellular
Mg2+ was manipulated with A-23187,
EGTA replaced EDTA, and total external Mg2+ concentration was 0.15 mM,
unless indicated otherwise. This concentration of external
Mg2+ has been shown to maintain
constant internal Mg2+ and cell
volume in ionophore-treated RBC (8, 26). HEPES-buffered K+ medium and
Rb+ medium were identical to HBS,
except that K+ and
Rb+ salts, respectively, replaced
Na+ salts. In
NO3 media,
NO
3 salts replaced
Cl
salts. Ouabain was
present at 0.1 mM in all solutions. DIOA and okadaic acid were
purchased from Research Biochemicals International (Natick, MA).
Blood samples and density separations.
After informed consent, blood was obtained by venipuncture into
heparinized tubes from volunteers homozygous for Hb S. Unless otherwise
stated, all experiments were performed on the least dense 15-25%
of the SS RBC population, since these cells have the highest KCl
cotransport activity (5, 10). Fresh whole blood was washed in HBS at
4°C without removing the buffy coat and then applied to
discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradients. After
centrifugation at 3,000 g for 20 min, cells with density () < 1.085 were isolated as described
previously (28, 30), washed, and filtered through glass wool to remove white blood cells. When stored overnight, cells were suspended in
HEPES-buffered solution containing 15 mM NaCl and 125 mM KCl. Spun
hematocrit values were measured on oxygenated samples in microhematocrit tubes after centrifugation for 5 min at 13,000 g in a hematocrit centrifuge.
Cellular cation measurements.
Cellular cations were measured by methods described in detail
previously (37, 39). Briefly, samples containing 5-10 µl of
cells were taken into iced tubes containing 110 mM
MgCl2 layered over dibutyl
phthalate (Fisher, Pittsburgh, PA). Cells exposed to ionophore were not
washed before exposure to MgCl2,
but there was no indication of cell swelling or hemolysis before
centrifugation through phthalate oil. There was also no difference in
Na+ or
K+ fluxes in
NO3 media between cells incubated with
or without ionophore, indicating that the short exposure of cells to
the combination of isotonic MgCl2
and ionophore at 0°C did not affect the monovalent cation content
of the cells. After the tubes had been washed, the oil was removed and
the cells were lysed for analysis of cations by flame emission
spectroscopy (Perkin-Elmer model 370 atomic absorption
spectrophotometer, Norwalk, CT). Hemoglobin was measured at 540 nm
using a Beckman DU spectrophotometer (Beckman Instruments). Cation
content was calculated as millimoles per kilogram hemoglobin; all
cation measurements were made in triplicate, unless otherwise noted.
Net cation fluxes. As in previous studies (37-40), SS RBC were suspended at 2% hematocrit in appropriate media, and ionophore or drugs were added as needed in stock solutions in DMSO. DMSO was present in a final concentration of 0.4-1 vol% and was added to controls. Cells were warmed to 37°C for 10 min, and an initial cation sample was taken. Oxygenated samples were capped, whereas deoxygenated samples were flushed with humidified N2 for 1 h, after which the flasks remained sealed. This deoxygenation protocol did not change the osmolality of media, indicating that N2 humidification was adequate. A second cation sample was taken after 2 h of incubation, and the net flux was calculated from the change in cation content.
Na+/H+
exchange measurements.
Blood was centrifuged on Percoll gradients, and a density fraction
(1.076 < < 1.106) was isolated. Cells were washed in solution
A [in mM: 140 NaCl, 5 KCl, 1 MgCl2, 1 NaH2PO4-Na2HPO4 (20:80), 10 HEPES-Tris (pH 7.4 at 37°C), and 10 glucose] and
stored at 4°C for 12-36 h. Just before the experiments, cells
were washed once in solution
A and incubated at 15% hematocrit at
37°C for 45 min in the same solution containing 2 mM adenine and 10 mM inosine. Cells were then washed four times in
solution
B
(solution A without
PO3
4 and supplemented with 1 mM
CaCl2, 0.1 mM ouabain, and 0.01 mM
bumetanide) and resuspended in the same solution at 10% hematocrit.
Cells were incubated at 37°C for 5 min with 40 µM dimethyl
amiloride (DMA) and then for 10 min with 0.4 µM phorbol 12-myristate
13-acetate (PMA). DMSO from the stock solutions of DMA and PMA was
present at a final concentration of 0.4-0.8 vol% and was added to
control suspensions. Cells were then either kept oxygenated or flushed
with humidified N2, as described
previously (22, 23). After 90 min, cells were washed twice in a
solution containing 110 mM MgCl2
and 10 mM HEPES-Tris (pH 7.4 at 4°C). Pellets were lysed, and
lysate was analyzed for hemoglobin and
Na+.
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RESULTS |
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Cl dependence of
K+ efflux in
deoxygenated SS RBC.
Figure 1 depicts a series of experiments in which ouabain-treated SS
RBC were incubated under oxygenated or deoxygenated conditions in
either Cl
or
NO
3 media and net fluxes of
Na+ and
K+ were measured. Figure
1A depicts cells under control
conditions (without ionophore). In oxygenated cells there was no
Cl
-dependent transport of
either Na+ or
K+; fluxes in
NO
3 exceed those in
Cl
, as reported by others
(7, 39). On deoxygenation, both
Na+ and
K+ fluxes increased, as expected
from previous studies (2, 4, 37, 39), and
Na+ influx was independent of
Cl
.
K+ efflux, however, was 12.5%
higher in Cl
compared with
NO
3 media
(P < 0.04), suggesting KCl
cotransport activity stimulated by deoxygenation. This result contrasts
with previous findings from our laboratory (36, 39) and by others (2)
that there was no
Cl
-dependent component to
the K+ efflux in deoxygenated SS
RBC. This difference may arise from the fact that the current studies
utilized a low-density fraction of SS RBC (least dense 25-50% of
cells) containing a higher number of young cells with high KCl
cotransport activity (10), whereas previous studies used unfractionated
cells. In addition, the deoxygenation-induced K+ efflux (difference between
fluxes in deoxygenated and oxygenated cells) in this fraction exceeded
deoxygenation-induced Na+ influx,
which is also in contrast to previous results obtained in
unfractionated SS RBC showing balanced sickling-induced
Na+ and
K+ fluxes (2, 36, 39, 40). The
activation of KCl cotransport in this low-density fraction appears to
account for this difference.
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Cl-dependent
K+ efflux
stimulated by deoxygenation is blocked by okadaic acid and DIOA.
In a separate series of experiments of similar design, we tested the
effects of okadaic acid, a serine/threonine protein phosphatase inhibitor that blocks the swelling-induced activation of KCl
cotransport (33), and DIOA, a compound that blocks the cotransport
pathway (46, 52). Figure 2 shows net
K+ efflux in deoxygenated SS RBC.
The small Cl
-dependent
component of K+ efflux in
deoxygenated SS RBC was apparent in the control (no drug) cells without
ionophore; this was not present in oxygenated cells (not shown) and was
therefore stimulated by deoxygenation. This
Cl
-dependent
K+ efflux was augmented by
clamping Mg2+, as before, and was
completely blocked by both okadaic acid and DIOA, supporting its
mediation by the KCl cotransporter.
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Stimulation of KCl cotransport by deoxygenation is not a consequence
of the Bohr effect.
The Bohr effect of hemoglobin (9) produces two changes in RBC on
deoxygenation that might have secondary effects on KCl cotransport.
Cellular pH is increased by 0.05-0.10 pH units (24), but this
should inhibit, rather than stimulate, the system (7, 43, 49). The
reduced negative charge on deoxygenated Hb leads to
Cl uptake and slight cell
swelling on deoxygenation. To assess the effect of deoxygenation on
cell volume, hematocrit was measured on paired suspensions of normal
(AA) RBC subjected to both oxygenated and deoxygenated conditions,
taking care to ensure that cells remained deoxygenated during
measurement. The cell volume of deoxygenated cells relative to
oxygenated cells, calculated as the ratio of hematocrit values, was
1.008 ± 0.0004 (mean ± SD; n = 3). Such a change in volume of <1% produces minimal activation of
KCl cotransport (41) and is insufficient to account for the magnitude
of the stimulatory effect of deoxygenation.
Cl-dependent
K+ efflux
stimulated by deoxygenation is a function of
Mg2+.
We predicted that, if the effect of A-23187 to augment deoxygenation
stimulation of KCl cotransport activity in SS RBC is due to clamping of
cellular Mg2+ at the normal
oxygenated level, raising external
Mg2+ in A-23187-treated cells
would blunt the deoxygenation stimulation. To test this prediction, SS
RBC were deoxygenated as before in the presence of A-23187 at total
external Mg2+ concentrations of
0.15 and 1.5 mM. Figure 4 illustrates that at 0.15 mM Mg2+ there was a marked
stimulation of Cl
-dependent
K+ efflux in deoxygenated cells,
as was apparent in previous experiments (Fig. 1). At high
Mg2+ (1.5 mM), however, the
stimulation of the
Cl
-dependent component of
the K+ efflux in deoxygenated SS
RBC was markedly reduced compared with stimulation at 0.15 mM
Mg2+. These data are consistent
with the notion that increased cellular Mg2+ in deoxygenated SS RBC (in
the absence of ionophore) counteracts the stimulation of KCl
cotransport activity by partially inhibiting the transporter under
continuous deoxygenation.
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KCl cotransport activity in isotonic
high-Rb+(K+)
buffers is stimulated by deoxygenation of SS RBC without manipulating
cellular Mg2+.
Figure 6 depicts an experiment in which
Rb+ influx was measured in SS RBC
incubated under oxygenated and deoxygenated conditions (without
addition of ionophore) in buffers containing high external Rb+. Cells were deoxygenated in
isotonic HBS or HEPES-buffered
NaNO3 medium containing 0.1 mM
ouabain and 0.1 mM EDTA, and at time 0 isotonic
Rb+ buffer (either
Cl or
NO
3) was added to give 70 mM
Rb+ (+ 70 mM
Na+). Cellular
Rb+ uptake was measured at various
time points, as depicted in Fig. 6; the slopes of the curves represent
flux rates. Under oxygenated conditions,
Rb+ influx was greater in
Cl
medium than in
NO
3 medium, indicating a
Cl
-dependent component in
this experiment; however, this was not a consistent finding, and in the
pooled data of Fig. 7 this is not apparent.
As illustrated in Fig. 6, deoxygenation increased Rb+ influx approximately twofold
in NO
3 medium, reflecting the
sickling-induced increase in passive membrane permeability (39). In
Cl
media, however,
deoxygenation increased Rb+
influx even more strikingly.
Rb+ influx measured in saline
media with external Rb+ = 5.6 mM
showed no significant
Cl
dependent component
in either oxygenated or deoxygenated cells (not shown), indicating that
the deoxygenation stimulation of Cl
-dependent Rb+
influx was unique to the condition of high external
Rb+. These data demonstrate that
under conditions of high external Rb+, a substantial component of
the Rb+ influx stimulated by
deoxygenation of SS RBC was
Cl
dependent.
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Na+/H+
exchange is not activated by deoxygenation.
To investigate whether a component of the deoxygenation-induced
Na+ influx (Fig. 1) was mediated
by the
Na+/H+
exchanger, we examined the effect of DMA, an inhibitor of the antiporter (41). These experiments utilized a large density fraction of
SS RBC (1.076 < < 1.106);
Na+/H+
exchange has been found to be independent of density over this range
and decreased only in cells of higher density (12). Net Na+ influx was measured under
oxygenated and deoxygenated conditions in isotonic media at pH 7.4 as
described in MATERIALS AND METHODS, with and without DMA. DMA had no effect on
Na+ influx in oxygenated SS RBC or
in oxygenated or deoxygenated AA RBC (not shown), indicating that the
Na+/H+
exchanger was quiescent under these conditions. The
deoxygenation-induced Na+ flux was
calculated as the difference between the values in paired deoxygenated
and oxygenated samples, and was normalized to the value in control
cells (no added drugs). These normalized values are presented in Fig.
8 as averages of six experiments.
Deoxygenation-induced Na+ influx
was not inhibited by DMA, indicating that the
Na+/H+
exchanger was not activated by deoxygenation. In contrast,
deoxygenation-induced Na+ influx
was increased by 45% after a pretreatment of SS RBC with PMA, as
reported previously (22), and this PMA-stimulated component was
completely inhibited by DMA. These data demonstrate that the Na+/H+
exchanger is stimulated by PMA in deoxygenated SS red blood cells, as
previously observed in acid-loaded erythrocytes (14) and nucleated
cells (45). Interestingly, PMA had no effect on
Na+ influx under oxygenated
conditions in SS RBC or under oxygenated or deoxygenated conditions in
normal RBC (not shown). That activation of
Na+/H+
exchange in isotonic media at normal pH occurs only in SS RBC and
requires both deoxygenation and PMA implies a complex mechanism of
activation. In any case, the data indicate that the
Na+/H+
exchanger is present in SS RBC and can be stimulated in deoxygenated SS
RBC by PMA but that the exchanger is not activated by deoxygenation alone and thus is not likely to contribute to cation homeostasis under
physiological conditions.
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DISCUSSION |
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Early studies of cation fluxes in unfractionated SS RBC under
continuous deoxygenation in isotonic media at normal pH found no
Cl-dependent component to
the K+ efflux (2, 36, 39). This
was consistent with previous findings that the KCl cotransporter was
inactive under isotonic conditions at normal pH under oxygenated
conditions (7, 11), and, when activated by cell swelling or acid pH,
KCl cotransport was inhibited by deoxygenation (11). Canessa et al.
(11) showed that this inhibitory effect was a consequence of two
well-described phenomena: increased cellular
Mg2+ due to binding of organic
anions to deoxygenated Hb (24, 26, 48) and inhibition of the
cotransporter by internal Mg2+ (8,
11, 18). Deoxygenation also was shown to reduce KCl cotransport in
trout and horse RBC incubated in isotonic media at pH 7.0 (15, 31),
although the authors of these reports suggested that factors other than
cellular Mg2+ were involved.
Later studies, however, suggested that the
K+ loss from deoxygenated SS RBC
was mediated, at least in part, by the KCl cotransporter. Beuzard and
colleagues (46, 52) reported that a portion of the
K+ efflux from deoxygenated cells
was inhibited by DIOA. Apovo et al. (1) found that cyclic deoxygenation
of SS RBC resulted in dense cell formation that was independent of the
presence of Ca2+ and inhibited by
DIOA. Franco et al. (29) found that
Cl-dependent dense cell
formation in SS reticulocytes occurred with cyclic, but not continuous,
deoxygenation under isotonic conditions at normal pH. We proposed an
explanation for these findings that takes into account the increase in
Mg2+ associated with deoxygenation
(24, 48) and its effect on KCl cotransport (11, 18, 49) and the
alteration in protein phosphorylation equilibrium induced by
deoxygenation (22). Fathallah et al. (22) found that deoxygenation of
SS RBC, as well as AA RBC, resulted in nonspecific dephosphorylation of
RBC membrane proteins. We proposed (29) that the KCl cotransporter or
its regulators might be dephosphorylated on deoxygenation of SS RBC but
its activity attenuated by the accompanying increase in cellular Mg2+ (24, 26). On reoxygenation,
Mg2+ levels fall rapidly, whereas
dephosphorylation may persist for some period of time, during which KCl
cotransport could take place.
In the present study, we have demonstrated a small
Cl-dependent
K+ efflux in continuously
deoxygenated, low-density SS RBC (Fig. 1A). The
Cl
-dependent component of
the deoxygenation-induced K+
efflux was augmented more than twofold in cells treated with ionophore
A-23187 to minimize the increase in cellular
Mg2+ associated with deoxygenation
(Fig. 1B). This supports the
proposed mechanism whereby the activated KCl cotransporter
is partially inhibited by elevated cellular
Mg2+ in continuously deoxygenated
SS RBC. Inhibition of the
Cl
-dependent component of
the deoxygenation-induced flux by DIOA (Fig. 2) identifies it as a
manifestation of KCl cotransport activity. Blockade by the phosphatase
inhibitor okadaic acid further supports the idea that the stimulation
is due to dephosphorylation of the cotransporter (or its regulators) by
deoxygenation.
Thus stimulation of KCl cotransport does indeed occur on deoxygenation
of low-density SS RBC. The activation of KCl cotransport in a
population of cells may be influenced by a variety of factors, including the age and density distribution of the cell population studied, cellular Mg2+ content and
buffering capacity, pH, conditions of deoxygenation, and flux
measurement techniques. Differences in these variables may explain why
a Cl-dependent component of
the deoxygenation-induced flux was not apparent in some previous
studies (2, 11, 39).
Stimulation of KCl cotransport by deoxygenation could also be
demonstrated by flux measurements in
high-Rb+(K+)
medium. Under these conditions of
trans-stimulation, it was shown that
the transporter was more resistant to inhibition by cellular
Mg2+, presumably due to the
kinetic alterations induced by the
trans-stimulation of the transporter
in high-K+ medium (41). In terms
of a kinetic model for KCl cotransport activity, this phenomenon could
be explained if the return of the empty carrier (outside to inside)
were rate limiting to KCl cotransport activity at low external
K+ and were also inhibited by
internal Mg2+. In high external
K+(Rb+),
if return of the filled carrier were no longer rate limiting (hence,
the trans-stimulation), transport
inhibition by Mg2+ might be
reduced. The data in Fig. 5 are consistent with this hypothesis, but
further experiments will be required to provide rigorous kinetic
support. Nevertheless, the stimulation of
Cl-dependent
K+ and
Rb+ fluxes by deoxygenation of SS
RBC is clear in
high-Rb+(K+)
medium (Figs. 6 and 7). As with the
K+ flux in
Mg2+-clamped cells in
low-K+ medium, these fluxes were
inhibited by okadaic acid and DIOA, indicating respectively their
activation by a dephosphorylation mechanism and mediation by KCl
cotransport.
Stimulation of KCl cotransport in these experiments occurred in the absence of Ca2+, indicating that sickling-induced Ca2+ influx, with K+ channel activation, cellular dehydration, and acidification as proposed by Bookchin et al. (4), was not the trigger for cotransport activation by deoxygenation under these conditions. The simplest explanation of our data is the direct activation of KCl cotransport by deoxygenation of SS RBC, presumably by a dephosphorylation event. Blockade of this activation by okadaic acid implicates a serine/threonine phosphatase in the process, probably protein phosphatase 1 (PP1), at least in the case of swelling-activated KCl cotransport (33). Involvement of tyrosine kinases and/or phosphatases in the regulation of KCl cotransport has recently been suggested. However, depending on species, tyrosine kinase inhibitors were reported either to inhibit (25, 50, 53) or to stimulate (3, 17, 25) cotransport. In fact, Flatman et al. (25) reported that the stimulation of KCl cotransport by one kinase inhibitor, staurosporine, was blocked by another inhibitor, genistein, suggesting two separate tyrosine phosphorylation sites. Inhibition of PP1 activity and direct activation of the cotransporter have been proposed to account for these effects, although the precise targets of tyrosine phosphorylation are not known and may vary among species. The involvement of both tyrosine kinase and PP1 in the activation of KCl cotransport implies the existence of two additional regulatory proteins: a tyrosine phosphatase and a serine/threonine kinase. Each of these enzymes is a potential target for modulators of KCl cotransport activation such as cell swelling, pH, Mg2+, oxidation, and phosphorylation-dephosphorylation. Furthermore, interactions among these modulators are likely to be quite complex. It is known that cell swelling, pH, and Mg2+ levels alter the effect of each other on KCl cotransport activity (8, 18, 43, 49). In addition, there is evidence for dual effects of Mg2+ on the cotransporter, both altering phosphorylation (18) and inhibiting the transporter directly (49). The interactions of multiple phosphorylation-dephosphorylation events (25, 50) in the activation of KCl cotransport and how they are affected by deoxygenation remain to be determined.
Our data suggest a new stimulus for activation of the KCl cotransporter, deoxygenation, which does not require the abnormal conditions of cell swelling or acidification and may be important both physiologically in normal RBC and pathologically in SS RBC. The magnitude of the K+ efflux via the KCl cotransporter stimulated by cyclic deoxygenation of RBC in vivo would be a complex function of circulatory transit times, dynamics of dephosphorylation-rephosphorylation of target proteins in relation to changing cellular Mg2+ levels, and the number of transporter molecules in individual cells. In vitro studies of the activation and inactivation of KCl cotransport in SS RBC by swelling and shrinking revealed delay times for activation of 1.7 ± 0.3 min and for inactivation of 3.6 ± 0.4 min (13). These delay times are somewhat longer than overall circulatory transit time (total blood volume divided by cardiac output), estimated at ~1 min in the healthy adult (44). However, transit times vary considerably among different vascular beds, bone and bone marrow having transit times 10-fold longer than kidney, brain, and lung (44). In addition, the adherence of SS reticulocytes to the endothelium of postcapillary venules (42) may further prolong circulatory times. Thus it is quite possible that individual cells might experience, at least intermittently, oxygenation-deoxygenation cycles in vivo that could activate the KCl cotransporter, with important effects in these cells on volume regulation.
Recently we reported that KCl cotransport activity was higher in SS reticulocytes that had become moderately dense in vivo relative to cells that retained normal density as reticulocytes (28), suggesting that KCl cotransport plays a role in reticulocyte dehydration in vivo. The fact that moderately dehydrated reticulocytes have the same levels of fetal hemoglobin (Hb F) as normally hydrated reticulocytes (27) implies that KCl cotransport, which is independent of Hb F (30), may be more important in the process of moderate reticulocyte dehydration than sickling-dependent mechanisms, which are inhibited by Hb F (19). Sickling-dependent processes, facilitated by moderate dehydration of reticulocytes, may contribute to more severe dehydration of certain reticulocytes (particularly those without Hb F), and to dehydration of more mature cells.
The abnormalities of volume regulation in SS RBC could theoretically
arise from perturbations of Na+
transport pathways. The
Na+/H+
exchanger is an important Na+
uptake pathway in human red blood cells (20), and pharmacological and
kinetic data suggest that red blood cells express at least the NHE1
isoform (14, 20). SS RBC exhibit elevated
Na+/H+
exchange activity (12), and we hypothesized that deoxygenation might
stimulate this pathway. The
Na+/H+
exchanger in RBC is activated by cell shrinkage (12) or, in the
presence of a pH gradient, by incubation with PMA or with Ca2+ and ionophore (20), both
treatments resulting in protein kinase C (PKC
)
translocation to the membrane (23). Deoxygenation of SS RBC in the
presence of external Ca2+ leads to
transient elevation of cytosolic
Ca2+ and subsequent cell volume
reduction (via activation of
Ca2+-dependent
K+ channels) (4, 6). The present
data, however, show that Na+/H+
exchange activity was not stimulated by deoxygenation alone. The
transient rise in Ca2+ associated
with deoxygenation is not sufficient to induce PKC
translocation (23), which may explain the failure of
deoxygenation to activate the transporter. Likewise, the addition of
PMA to oxygenated SS RBC was insufficient to activate the transporter, although under these conditions PMA induces translocation of
PKC
to the membrane (23). The combined requirement
for deoxygenation of SS RBC and the presence of PMA implies that both
elevated Ca2+ and
PKC
activation are required for stimulation of
Na+/H+
exchange, in agreement with the proposed mechanism of NHE1 activation in other cells (45). Nevertheless, it does not appear that the Na+/H+
exchanger mediates a component of deoxygenation-induced
Na+ influx under physiological
conditions.
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ACKNOWLEDGEMENTS |
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The technical assistance of Mary Palascak and graphic skills of William Claussen are gratefully acknowledged. We thank Dr. Donald Rucknagel for many useful discussions during the course of this work and for obtaining blood samples from the University of Cincinnati sickle cell clinic. We are also grateful to Drs. D. Bachir and F. Galacteros for providing blood samples from the Centre de la Drépanocytose et des Thalassémies (Hopital Henri Mondor, Créteil, France).
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-37515 and HL-57614 (to C. H. Joiner) and HL-51174 (to R. S. Franco) and by Fondation pour la Recherche Médicale Grant 40000 187S.05 to F. Giraud.
Address for reprint requests: C. H. Joiner, Comprehensive Sickle Cell Center, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039.
Received 3 November 1997; accepted in final form 12 February 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Apovo, M.,
Y. Beuzard,
F. Galacteros,
D. Bachir,
and
F. Giraud.
The involvement of the Ca-dependent K channel and of KCl cotransport in sickle cell dehydration during cyclic deoxygenation.
Biochim. Biophys. Acta
1225:
255-258,
1994[Medline].
2.
Berkowitz, L. R.,
and
E. P. Orringer.
Passive sodium and potassium movements in sickle erythrocytes.
Am. J. Physiol.
249 (Cell Physiol. 18):
C208-C214,
1985[Abstract].
3.
Bize, I.,
and
P. B. Dunham.
Staurosporine, a protein kinase inhibitor, activates K-Cl cotransport in LK sheep erythrocytes.
Am. J. Physiol.
266 (Cell Physiol. 35):
C759-C770,
1994
4.
Bookchin, R. M.,
O. E. Ortiz,
and
V. L. Lew.
Evidence for a direct reticulocyte origin of dense red cells in sickle cell anemia.
J. Clin. Invest.
87:
113-124,
1991[Medline].
5.
Brugnara, C.,
H. F. Bunn,
and
D. C. Tosteson.
Regulation of erythrocyte cation and water content in sickle cell anemia.
Science
232:
388-390,
1986[Medline].
6.
Brugnara, C.,
L. De Franceschi,
and
S. L. Alper.
Inhibition of Ca++-dependent K+ transport and cell dehydration in sickle erythrocytes by clotrimazole and other imidazole derivatives.
J. Clin. Invest.
92:
520-526,
1993[Medline].
7.
Brugnara, C.,
T. V. Ha,
and
D. C. Tosteson.
Acid pH induces formation of dense cells in sickle erythrocytes.
Blood
74:
487-495,
1989[Abstract].
8.
Brugnara, C.,
and
D. C. Tosteson.
Inhibition of K transport by divalent cation in sickle erythrocytes.
Blood
70:
1810-1815,
1987[Abstract].
9.
Bunn, H. F.,
and
B. G. Forget.
Hemoglobin: Molecular, Genetic, and Clinical Aspects. Philadelphia: Saunders, 1986.
10.
Canessa, M.,
M. E. Fabry,
N. Blumenfeld,
and
R. L. Nagel.
Volume-stimulated, Cl-dependent K+ efflux is highly expressed in young human red cells containing normal hemoglobin or HbS.
J. Membr. Biol.
97:
97-105,
1987[Medline].
11.
Canessa, M.,
M. E. Fabry,
and
R. L. Nagel.
Deoxygenation inhibits the volume-stimulated, Cl-dependent K+ efflux in SS and young AA cells: a cytosolic Mg2+ modulation.
Blood
70:
1861-1866,
1987[Abstract].
12.
Canessa, M.,
M. E. Fabry,
S. M. Suzuka,
K. Morgan,
and
R. L. Nagel.
Na+/H+ exchange is increased in sickle cell anemia and young normal red cells.
J. Membr. Biol.
116:
107-115,
1990[Medline].
13.
Canessa, M.,
J. R. Romero,
C. Lawrence,
R. L. Nagel,
and
M. E. Fabry.
Rate of activation and deactivation of K:Cl cotransport by changes in cell volume in hemoglobin SS, CC, and AA red cells.
J. Membr. Biol.
142:
349-362,
1994[Medline].
14.
Ceolotto, G.,
P. Conlin,
G. Clari,
A. Semplicini,
and
M. Canessa.
Protein kinase C and insulin regulation of red blood cell Na+/H+ exchange.
Am. J. Physiol.
272 (Cell Physiol. 41):
C818-C826,
1997
15.
Cossins, A. R.,
Y. R. Weaver,
G. Lykkeboe,
and
O. B. Nielsen.
Role of protein phosphorylation in control of K flux pathways in trout red blood cells.
Am. J. Physiol.
267 (Cell Physiol. 36):
C1641-C1650,
1994
17.
De Francheschi, L.,
L. Fumagalli,
O. Olivieri,
R. Corrocher,
C. A. Lowell,
and
G. Berton.
Deficiency of Src family kinases Fgr and Hck results in activation of erythrocyte K/Cl cotransport.
J. Clin. Invest.
99:
220-227,
1997
18.
Delpire, E.,
and
P. K. Lauf.
Magnesium and ATP dependence of K-Cl cotransport in low K sheep red blood cells.
J. Physiol. (Lond.)
441:
219-231,
1991[Abstract].
19.
Eaton, W. A.,
and
J. Hofrichter.
Hemoglobin S gelation and sickle cell disease.
Blood
70:
1245-1266,
1987[Medline].
20.
Escobales, N.,
and
M. Canessa.
Ca++-activated Na+ fluxes in human red cells: amiloride sensitivity.
J. Biol. Chem.
260:
11914-11923,
1985
21.
Etzion, Z.,
T. Tiffert,
R. M. Bookchin,
and
V. L. Lew.
Effects of deoxygenation on active and passive Ca++ transport and on the cytoplasmic Ca++ levels of sickle cell anemia red cells.
J. Clin. Invest.
92:
2489-2498,
1993[Medline].
22.
Fathallah, H.,
E. Coezy,
R.-S. De Neef,
M.-D. Hardy-Dessources,
and
F. Giraud.
Inhibition of deoxygenation-induced membrane protein dephosphorylation and cell dehydration by phorbol esters and okadaic acid in sickle cells.
Blood
86:
1999-2007,
1995
23.
Fathallah, H.,
M. Sauvage,
J. R. Romero,
M. Canessa,
and
F. Giraud.
Effects of protein kinase C activation on Ca pump and KCa channel in deoxygenated sickle cells.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1206-C1214,
1997
24.
Flatman, P. W.
The effect of buffer composition and deoxygenation on the concentration of ionized magnesium inside human red blood cells.
J. Physiol. (Lond.)
300:
19-30,
1980[Abstract].
25.
Flatman, P. W.,
N. C. Adragna,
and
P. K. Lauf.
Role of protein kinases in regulating sheep erythrocyte K-Cl cotransport.
Am. J. Physiol.
271 (Cell Physiol. 40):
C255-C261,
1996
26.
Flatman, P. W.,
and
V. L. Lew.
Magnesium buffering in intact human red blood cells measured using the ionophore A23187.
J. Physiol. (Lond.)
305:
13-30,
1980[Abstract].
27.
Franco, R. S.,
R. Barker-Gear,
M. A. Miller,
S. M. Williams,
C. H. Joiner,
and
D. L. Rucknagel.
Fetal hemoglobin and potassium in isolated transferrin receptor-positive dense sickle reticulocytes.
Blood
84:
2013-2020,
1994
28.
Franco, R. S.,
M. Palascak,
H. Thompson,
and
C. H. Joiner.
KCl cotransport activity in light and dense transferrin receptor-positive sickle reticulocytes.
J. Clin. Invest.
95:
2573-2580,
1995[Medline].
29.
Franco, R. S.,
M. Palascak,
H. Thompson,
D. L. Rucknagel,
and
C. H. Joiner.
Dehydration of TfR+ sickle reticulocytes during continuous or cyclic deoxygenation: role of KCl cotransport and extracellular calcium.
Blood
88:
4359-4365,
1996
30.
Franco, R. S.,
H. Thompson,
M. Palascak,
and
C. H. Joiner.
The formation of transferrin receptor-positive sickle reticulocytes with intermediate density is not determined by HbF content.
Blood
90:
3195-3203,
1997
31.
Gibson, J. S.,
H. Godart,
J. C. Ellory,
H. Staines,
N. A. Honess,
and
A. R. Cossins.
Modulation of K+-Cl cotransport in equine red blood cells.
Exp. Physiol.
79:
997-1009,
1995.
32.
Jennings, M. L.,
and
N. Al-Rohil.
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[Abstract].
33.
Jennings, M. L.,
and
R. K. Schulz.
Okadaic acid inhibition of KCl cotransport: evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethyl-maleimide.
J. Gen. Physiol.
97:
799-818,
1991[Abstract].
34.
Joiner, C. H.
Deoxygenation-induced fluxes in sickle cells: II. Inhibition by stilbene disulfonates.
Blood
76:
212-220,
1990[Abstract].
35.
Joiner, C. H.
Cation transport and volume regulation in sickle red blood cells.
Am. J. Physiol.
264 (Cell Physiol. 33):
C251-C270,
1993
36.
Joiner, C. H.,
A. Dew,
and
D. L. Ge.
Deoxygenation-induced fluxes in sickle cells: I. Relationship between net potassium efflux and net sodium influx.
Blood Cells
13:
339-348,
1988[Medline].
37.
Joiner, C. H.,
M. Jiang,
and
R. S. Franco.
Deoxygenation-induced cation fluxes in sickle cells. IV. Modulation by external calcium.
Am. J. Physiol.
269 (Cell Physiol. 38):
C403-C409,
1995
38.
Joiner, C. H.,
M. Jiang,
and
R. S. Franco.
KCl cotransport is activated by deoxygenation of sickle red blood cells (Abstract).
Blood
88:
649a,
1996.
39.
Joiner, C. H.,
C. L. Morris,
and
E. S. Cooper.
Deoxygenation-induced cation fluxes in sickle cells: III. Cation selectivity and response to pH and membrane potential.
Am. J. Physiol.
264 (Cell Physiol. 33):
C734-C744,
1993
40.
Joiner, C. H.,
O. S. Platt,
and
S. E. Lux.
Cation depletion by the sodium pump in red cells with pathological cation leaks: sickle cells and xerocytes.
J. Clin. Invest.
78:
1487-1496,
1986[Medline].
41.
Kaji, D. M.
Kinetics of volume-sensitive K transport in human erythrocytes: evidence for asymmetry.
Am. J. Physiol.
256 (Cell Physiol. 25):
C1214-C1223,
1989
42.
Kaul, D. K.,
M. E. Fabry,
and
R. L. Nagel.
Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: pathophysiological implications.
Proc. Natl. Acad. Sci. USA
86:
3356-3362,
1989[Abstract].
43.
Lauf, P. K.,
A. Erdmann,
and
N. C. Adragna.
K-Cl cotransport, pH, and role of Mg in volume-clamped low-K sheep erythrocytes: three equilibrium states.
Am. J. Physiol.
266 (Cell Physiol. 35):
C95-C103,
1994
44.
Leggett, R. W.,
and
L. R. Williams.
A proposed blood circulation model for Reference Man.
Health Phys.
69:
187-201,
1995[Medline].
45.
Noël, J.,
and
J. Pouysségur.
Hormonal regulation, pharmacology, and membrane sorting of the vertebrate Na/H exchanger isoforms.
Am. J. Physiol.
268 (Cell Physiol. 37):
C283-C296,
1995
46.
Olivieri, O.,
D. Vitoux,
D. Bachir,
and
Y. Beuzard.
K+ efflux in deoxygenated sickle cells in the presence or absence of DIOA, a specific inhibitor of the [K-Cl] cotransport system.
Br. J. Haematol.
77:
117-120,
1991[Medline].
47.
Olivieri, O.,
D. Vitoux,
F. Galacteros,
D. Bachir,
Y. Blouquit,
Y. Beuzard,
and
C. Brugnara.
Hemoglobin variants and activity of the (K+Cl) cotransport system in human erythrocytes.
Blood
79:
793-797,
1992[Abstract].
48.
Ortiz, O. E.,
V. L. Lew,
and
R. M. Bookchin.
Deoxygenation permeabilizes sickle cell anaemia red cells to magnesium and reverses its gradient in the dense cells.
J. Physiol. (Lond.)
427:
211-226,
1990[Abstract].
49.
Ortiz-Carranza, O.,
N. C. Adragna,
and
P. K. Lauf.
Modulation of K-Cl cotransport in volume-clamped low-K sheep erythrocytes by pH, magnesium, and ATP.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1049-C1058,
1996
50.
Sachs, J. R.,
and
D. W. Martin.
The role of ATP in swelling-stimulated K-Cl cotransport in human red cell ghosts. Phosphorylation-dephosphorylation events are not in the signal transduction pathway.
J. Gen. Physiol.
102:
551-560,
1993[Abstract].
51.
Sardet, C.,
I. Counillon,
A. Franchi,
and
J. Pouysségur.
Growth factors induce phosphorylation of the Na/H antiporter, a glycoprotein of 110 kD.
Science
247:
123-126,
1990.
52.
Vitoux, D.,
O. Olivieri,
R. P. Garay,
E. J. Cragoe, Jr.,
F. Galacteros,
and
Y. Beuzard.
Inhibition of K+ efflux and dehydration of sickle cells by [(dihydroindenyl)oxy]alkanoic acid: an inhibitor of the K+Cl cotransport system.
Proc. Natl. Acad. Sci. USA
86:
4273-4276,
1989[Abstract].
53.
Weaver, Y. R.,
and
A. R. Cossins.
Protein tyrosine phosphorylation and the regulation of KCl cotransport in trout erythrocytes.
Pflügers Arch.
432:
727-734,
1996[Medline].