Cation transport and cell volume changes in maturing rat
reticulocytes
Heimo
Mairbäurl1,
Susanne
Schulz1, and
Joseph
F.
Hoffman2
1 Department of Sports Medicine, University of Heidelberg,
69115 Heidelberg, Germany; and 2 Department of
Cellular and Molecular Physiology, Yale University School of
Medicine, New Haven, Connecticut 06520
 |
ABSTRACT |
During maturation, reticulocytes lose membrane material,
including transporters, and this is accompanied by a loss of cell water
and volume. Here we determined a possible role of ion transport in
adjusting cell volume during maturation. Reticulocytes and red blood
cells of different ages were prepared from erythropoietin-treated rats
by density gradient fractionation. Cell volume and ion transport were
measured in freshly prepared cells and in reticulocytes during in vitro
maturation. Reticulocytes had an increased K content and cell volume,
whereas intracellular Na was decreased. All parameters approached whole
blood values after 2 days in culture. Na-K pump was elevated in
reticulocytes and decreased during maturation. Na-K-2Cl cotransport
(NKCC) activity was lower in reticulocytes and was activated 8- and
20-fold by shrinkage and okadaic acid, respectively, whereas
stimulation was barely detectable in high-buoyant density red blood
cells. The ouabain- and bumetanide-insensitive Na flux in reticulocytes
decreased on maturation. Most of it was inhibited by amiloride,
indicating the presence of Na/proton exchange. Our results show that,
although the Na-K-pump activity in reticulocytes is very much
increased, the enhanced capacity of NKCC is essentially cryptic until
stimulated. Both types of capacities (activities) decrease during
maturation, indicating a possible loss of transport protein. The
decrease was constrained to the period of reticulocyte maturation. Loss
of transport capacity appears to exceed the loss of membrane surface
area. Reticulocyte age-related changes in the net electrochemical
driving force indicate that the increasing NKCC activity might
contribute to the reduction in cell water.
erythropoietin; red blood cells; cell aging; Na-K pump; Na-K-2Cl
cotransport
 |
INTRODUCTION |
DURING DIFFERENTIATION OF
RED BLOOD CELLS significant functional and structural changes
occur (for review see Ref. 38). Structural changes include
enucleation, loss of organelles, and changes in composition and
membrane surface area with subsequent changes in cellular metabolism,
e.g., loss of the capability of protein synthesis and oxidative
metabolism. The decrease in the membrane surface area (38,
42) has been explained by a "shedding" of membrane material
as exosomes, which are vesicles that can be collected by
high-speed centrifugation from the supernatant of in vitro
maturing reticulocytes (22) and from plasma after phlebotomy (23). Exosome membranes show the typical
composition of the red blood cell membrane but exhibit high
concentrations of selected membrane proteins characteristic of immature
red blood cells such as the transferrin receptor (24),
which is barely detectable in plasma membranes of mature red blood
cells. Therefore, a selective extrusion mechanism for the removal of
certain membrane proteins has been proposed (9, 22, 23).
Interestingly, exosomes do not seem to contain cytosolic proteins
(22).
The decrease in cell volume during maturation of reticulocytes can be
attributed in part to the loss of membrane and intracellular constituents. However, red blood cell volume is also determined by the
cellular cation content that, in turn, is controlled by specific
transport systems (for review see Ref. 39). In red blood
cell subpopulations of different ages, separated according to their
buoyant density (4, 18), an inverse relationship was found
between cell density and cell volume as well as cation content
(1, 7, 10, 18, 27). Under steady-state
conditions, cell volume and cation content are set by a balance between
the activity of the Na-K pump, cation leaks, and other transporters such as Na-K-2Cl cotransport (NKCC) (12, 35, 41). A
decrease in the activity of the Na-K pump during reticulocyte
maturation has been described in a variety of species. In sheep red
blood cells, which show a dimorphism with regard to their cation
content, Na-K pumps are inactivated to a greater extent from
reticulocytes during maturation from low-K sheep than during maturation
from their high-K counterparts. A significant amount of those lost Na-K
pumps was found to be shed into exosomes or was degraded by
energy-dependent mechanisms (3). Mature red blood cells from carnivores lack the Na-K pump (13, 19, 40), although it still can be found in their reticulocytes (40), again
indicating specific inactivation or extrusion mechanisms. Changes in
the Na-K-pump activity during reticulocyte maturation also have been described by others (e.g., Refs. 5 and 15). The activity of other transporters possibly involved in the control of red blood
cell volume, such as NKCC, Na/Li exchange, Na/H exchange, and K-Cl
cotransport, was also found to be increased in fractions of young red
blood cells (5, 6, 8, 10, 16, 37).
In the present study changes in cell volume, cation content, and cation
transport were evaluated during maturation of rat reticulocytes to
evaluate a possible role of changes in transport activity in
determining the cell volume during reticulocyte maturation and the role
of matured red blood cells. A method suitable to enhance reticulocyte
production in rats by injection of erythropoietin (EPO) and their
isolation by density gradient centrifugation is described for
confirmation of previous studies. It was found that the Na-K-pump
capacity decreased with maturation. NKCC activity was low in normal
reticulocytes and increased slightly with maturation and cell aging. In
contrast, reticulocyte NKCC capacity evidenced by maximal activation
with okadaic acid was lost during in vivo and in vitro maturation.
Preliminary results have been presented in abstract form (30, 31,
33).
 |
MATERIALS AND METHODS |
Treatment of animals and preparation of red blood cells with
different buoyant density.
Male Sprague-Dawley rats with an average weight of 450 g were
injected intraperitoneally with a single dose of 200 U/kg recombinant human EPO (Recormon; Boehringer Mannheim, Mannheim, Germany). Control
rats were injected with an equivalent volume of 150 mM NaCl solution.
Three to five days later, the rats were anesthetized with ether and
exsanguinated by cardiac puncture. The blood of up to six animals was
pooled for one experiment. Aliquots (5 ml) of whole blood were layered
on 26-ml density gradient medium composed of 10% 10-fold concentrated
phosphate-buffered saline (GIBCO-BRL, Grand Island, NY), 68% Percoll
(Sigma), and 22% deionized water in polycarbonate centrifuge tubes.
This medium had a density of ~1.095 g/ml and an osmolality of ~285
mosmol/kg (34). The tubes were centrifuged for 30 min at
20,000 g at 12°C in a Sorvall RC-5B or R28S centrifuge
using the SS-34 Rotor (Sorvall, Du Pont). Several fractions of cells
were collected by aspiration, beginning at the top after the
supernatant plasma and buffy coat were removed. Contaminating
leukocytes were removed from each fraction of red blood cells by
filtration through a mixture of
-cellulose and microcrystalline
cellulose. The top fraction, formed of red blood cells of the lowest
buoyant density, usually contained >90% reticulocytes; a fraction
taken from the middle of the tube contained ~2.5% reticulocytes; and
the bottom fraction, formed of red blood cells with the highest buoyant
density, usually had <1% reticulocytes (see Table 1).
In vitro maturation of reticulocytes.
Red blood cells from the top fraction were suspended [hematocrit (Hct)
~0.3%] in culture medium (RPMI 1640; GIBCO-BRL) substituted with
7% fetal calf serum, 2 mM glutamine, 10 mM HEPES (pH 7.3 at room
temperature), and antibiotics and were incubated for several days under
tissue culture conditions in a humidified 5% CO2-balance air atmosphere.
Flux measurements.
For the measurement of the unidirectional efflux of 22Na,
the concentrations of intracellular Na (Nai) and K
(Ki) were optimized by adjustment to ~50 mmol per liter
of cells with the nystatin technique as described previously
(32), except that all steps were carried out at room
temperature. During this procedure the cells were also loaded with
trace amounts of 22Na. The loading medium was composed of
(in mM) 70 NaCl, 70 KCl, 1 NaH2PO4, 1 MgSO4, 23 sucrose, and 20 HEPES (pH 7.2 at room
temperature). The content of NaCl and KCl of the loading medium was
altered when different Nai and Ki
concentrations were required. After the cells were loaded, nystatin was
removed by seven washes with loading medium containing 0.3% bovine
serum albumin (Sigma).
The flux medium contained (in mM) 100 NaCl, 5 KCl, 10 glucose, 80 sucrose, and 20 HEPES (pH 7.4 at 37°C). The sucrose concentration in
the flux medium was modified to adjust the cell volume. Fluxes (Hct
~2%) were measured over a period of 90 s in the absence of ouabain but over a period of 5 min in the presence of ouabain. Fluxes
were started by adding cells to the flux medium and were terminated by
packing the cells in a microfuge for 10 s. Radioactivity was
determined in the original cell suspension and in the supernatant medium after centrifugation. Fluxes were calculated from the initial Nai concentration and the efflux rate constant
(okNa). The fluxes were
linear over the indicated time periods as determined in separate experiments.
The activity of the Na-K pump was taken as the portion of
22Na-efflux inhibitable with 5 mM ouabain
(oMNaouab), whereas NKCC activity was the
portion inhibited with 10 µM bumetanide in the presence of ouabain
(oMNabumet). Okadaic acid (Calbiochem, La
Jolla, CA) was used to inhibit protein-phosphatase activity to maximize
NKCC activity. The effectiveness of concentrations of inhibitors used
in flux experiments was tested with dose-response curves.
Hct was determined after microcentrifugation, and the hemoglobin
concentration was measured spectrophotometrically after conversion to
cyanmethemoglobin. Reticulocytes were counted on air-dried smears of
cells after they were stained (reticulocyte stain, Sigma).
Nai and Ki concentrations were determined by
flame photometry (Corning 410) in cell lysates after being packed to a
Hct >97% in narrow-bore 300-µl microcentrifuge tubes. The
distribution ratio of Cl (rCl) was determined
after red blood cells were incubated with tissue culture medium to
which trace amounts of 36Cl were added (20).
The activity of 36Cl was measured in a beta counter (model
TR2100, Packard) after the medium and cells were separate by
microcentrifugation. In some experiments cell water content was
determined from the wet weight-to-dry weight ratio, and the volume of
water in the cells was calculated according to Tosteson and Hoffman
(41). For presentation, cell water was calculated from
mean cellular hemoglobin concentration (MCHC) (36).
The activity of the Na-K-ATPase was measured according to Forbush
(14) on membranes prepared from red blood cells after lysis in and four washes with 5 mM Tris-phosphate buffer (pH 7.3 at
room temperature). Na-K-ATPase activity was taken from the time course
of phosphate release that was sensitive to inhibition by 5 mM ouabain.
Statistical evaluation.
Results are presented as means ± SD and are from a series of
experiments in which individual parameters were determined. Not all
measurements could be made on each set of samples because of the small
number of cells obtained in individual preparations. All measurements
were performed in duplicate or triplicate. Differences among parameters
determined in fractions of red blood cells of different buoyant density
and among reticulocytes kept in culture for various periods of time
were evaluated by one-way analysis of variance. The level of
statistical significance was P < 0.05.
 |
RESULTS |
Treatment of rats with EPO caused an increase in whole blood
reticulocyte counts from ~2.5% up to 10%. In the fraction of cells
with the lowest buoyant density, reticulocytes could be enriched to a
purity >90% by density gradient centrifugation compared with
preparations from the blood of untreated rats in which the yield was
only ~50%. Reticulocytes obtained after EPO treatment had a lower
buoyant density than those obtained from untreated rats, and the
density of new methylene blue-stained material was higher than, and
often showed patterns similar to, those seen in red blood cell
precursors just after enucleation. These observations probably indicate
that reticulocytes obtained after EPO treatment are on average younger
than normal and may represent reticulocytes that have undergone
accelerated release (38).
When cells from the top fraction (>95% reticulocytes) were kept in
culture for in vitro maturation, the number of reticulocytes decreased
by ~5-15% within the first 24-36 h (Fig.
1A). A rapid drop to <10%
was then seen over the following 2 days, whereas barely any
reticulocytes were detectable after 4 days in culture. Loss of stained
material was associated with an increase in MCHC by ~25% (Fig.
1B).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
Changes in reticulocyte counts and mean cellular
hemoglobin concentration (MCHC) during in vitro maturation of
reticulocytes. Reticulocytes were prepared from whole blood of
erythropoietin (EPO)-treated rats by density gradient centrifugation
and cultured in RPMI-1640 at 37°C at 5% CO2.
A: individual values from 3-5 days in culture.
B: means ± SD from different experiments;
n = 5 for each day in culture. The line in A
was drawn from a fit to a sigmoid-shaped curve. *P < 0.05 compared with MCHC on day 0.
|
|
Cell water and cation content.
As a measure of changes in the cell volume of reticulocytes, the change
in cell water content was estimated from the changes in MCHC (Table
1). This assumes that the amount
of hemoglobin per cell was the same in the different fractions of red
blood cells separated by density gradient centrifugation and does not change on subsequent incubation. MCHC was ~25% lower initially in
the reticulocyte-rich fractions than in the unfractionated cell
population (Table 2) but approached the
latter level during the 3 days of incubation.
The concentration of Nai per liter of cells was lower in
reticulocytes than in unfractionated cells, whereas the concentration of Ki was elevated (Table 1). These results are similar to
those observed in pig reticulocytes (26) but are in
contrast to the results of Furukawa et al. (15), who found
that Nai and Ki in rat reticulocytes obtained
after phenylhydrazine treatment were essentially the same as those in
mature erythrocytes of control rats. Our results show that
Nai increases and Ki decreases to values
similar to those found in unfractionated red blood cells as MCHC
increases in red blood cells of increasing buoyant density (Table 1).
The small difference in Ki between red blood cells from the
middle and bottom fractions was not statistically significant, although
Nai was significantly increased in cells from the bottom fraction. There was no consistent reticulocyte age-related change in
rCl, but in the most dense (bottom) red blood
cell population rCl was slightly higher
(P < 0.15) than in the reticulocyte-rich (top)
fraction (results not shown).
Reticulocyte maturation-related changes of Nai,
Ki, MCHC, and cell water are summarized in Table 2. In the
course of 3 days of in vitro maturation, the percentage of
reticulocytes decreased from initially >95% to ~2.2%. As the
percentage of reticulocytes decreased, both MCHC and Nai
increased, whereas Ki decreased. No significant change in
rCl was found. It is also evident that, as cells
matured in culture (i.e., day 3 compared with fresh whole blood), there was a lower MCHC and higher values of Nai and
Ki.
Na-K pump.
To assess whether the Na-K pumps of reticulocytes compared with those
of matured erythrocytes had the same or different sensitivities to
ouabain, dose-response curves were performed (Fig.
2). The results indicate that the
sensitivity of the Na-K pump to inhibition by ouabain was similar in
the reticulocyte-rich fraction (top) and in the fraction with mature
erythrocytes (middle and bottom fractions). The Na-K-pump activity was
measured at an extracellular K concentration of 2.5 mM. It should be
recognized that pump inhibition in all red blood cell fractions was
incomplete even at an ouabain concentration of 5 mM. Higher
concentrations of ouabain were avoided because of the possibility of
nonspecific effects. However, the main conclusion concerning changes in
pump capacity would not be statistically altered had full pump
inhibition been obtained.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2.
Ouabain sensitivity of 22Na efflux in rat red
blood cells of different buoyant density. Rats were treated with EPO,
and red blood cells of different buoyant density were prepared by
density gradient centrifugation (see MATERIALS AND
METHODS). Three fractions of cells were prepared: the top
fraction contained 98.2%, the middle fraction contained 6%, and the
bottom fraction contained 0.1% reticulocytes. Intracellular Na
(Nai) and K (Ki) were adjusted with the
nystatin technique. 22Na efflux was measured in a medium
containing (in mM) 140 choline-Cl, 2.5 KCl, 9 glucose, 20 HEPES (pH 7.4 at 37°C), and varying concentrations of ouabain. Each flux was
measured in duplicate. The ouabain-sensitive flux
(oMNaouab), taken as the
Na+-K+ pump flux, represents the difference in
Na+ efflux obtained in the absence and presence of ouabain
at each concentration. Data are from 1 of 2 experiments with similar
results.
|
|
In freshly isolated reticulocytes, when Nai had been
increased, the activity of the Na-K pump [maximum volume
(Vmax) for Nai] in the top fraction was
3-5 times higher than in the middle or bottom red blood cell
populations (Fig. 3A),
confirming the results indicated in the dose-response experiments shown
in Fig. 2. Similar observations were made previously on reticulocytes
from high- and low-K+ sheep (3), rats
(15, 31), pigs (26), and humans
(32). In addition, during in vitro maturation of
reticulocytes, the magnitude of the Na-K pump, studied at
Vmax, decreased with the disappearance of reticulocytes
(Fig. 3B) and reached values representative of
unfractionated cells (data not shown) on day 2 in culture. These results indicate the time course of pump capacity found in
maturing reticulocytes. Results shown in Fig. 3, A and
B, were obtained in different series of experiments, which
explains the difference in the magnitude of the pump fluxes between
cells in the top fraction of fractionated cells (Fig. 3A)
and day 0 in unfractionated cells in culture (Fig.
3B).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 3.
Activity of the Na+-K+ pump at
maximal cell volume (Vmax) for Nai of density
fractionated red blood cells (A) and reticulocytes in
culture (B). Density-fractionated red blood cells
(A) with increased Nai were prepared as
described in MATERIALS AND METHODS. The top, middle, and
bottom fractions had the same approximate reticulocyte content as that
indicated in Table 1. Reticulocyte measurements, as used in Fig.
1B, were similar to those referred to in Table 2, when the
fraction used contained >90% reticulocytes. 22Na efflux
was measured in the absence and presence of 5 mM ouabain, with the
difference representing the ouabain-sensitive or Na-K pump component of
the efflux. Date are means ± SD from triplicate flux measurements
from 1 of 4 experiments with similar results. *P < 0.05 compared with values from top fraction (A) and day
0, (B).
|
|
During the course of these experiments we realized that, when cells
were loaded with nystatin to achieve Vmax conditions with concentrations of Nai and Ki of ~50 mM, the
Nai of reticulocytes decreased during preparations for the
flux measurements, reaching ~35 mM. This was probably caused by the
high activity and capacity of the Na-K pump despite storage at 12°C.
Because we wanted to obtain an estimate of Na-K-pump capacity rather
than actual activity, it was important to know the dependency of the
pump-mediated Na efflux on Nai. Figure
4 shows that Vmax was
achieved at values of Nai of ~25 mmol per liter of packed
cells (31). Obviously the decrease in Nai did
not significantly affect the results on pump capacity measurements as
presented in Fig. 3B.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4.
Na dependency of the activity of the Na-K pump in
unfractionated red blood cells. Red blood cells were prepared as
described in MATERIALS AND METHODS. Nai was
altered with the nystatin technique by inversely altering the NaCl and
KCl concentrations of the nystatin loading medium to attain the
indicated concentrations of Nai. Na-K-pump activity
measured as ouabain (5 mM)-sensitive 22Na efflux is plotted
against the concentration of Nai. Data points are mean
values from triplicate flux measurements from 1 of 3 experiments with
similar results.
|
|
NKCC.
In freshly fractionated red blood cells, the ouabain-insensitive
22Na efflux was highest in reticulocytes and decreased with
increasing cell density. This kind of increased activity of ion
transport has also been found in human red blood cells from patients
with reticulocytosis (44). NKCC, measured in the presence
of ouabain as the bumetanide-sensitive efflux at optimized values of
Nai and Ki, was somewhat higher in
density-fractionated red blood cells taken from the middle and bottom
fractions compared with that in the reticulocyte-rich top fraction (the
context values in Fig. 5A and
Fig. 8). During the culture of reticulocytes for 4 days, the
bumetanide-sensitive efflux of 22Na appears to have
decreased slightly (Fig. 5B).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of okadaic acid on the activity of the Na-K-2Cl
cotransport (NKCC) at Vmax for Nai of
density-fractionated red blood cells (A) and reticulocytes
in culture (B). Reticulocytes and fractions of red blood
cells of different buoyant density were prepared from EPO-treated rats
as described in MATERIALS AND METHODS. After the cellular
content of Nai and Ki was modulated with the
nystatin technique, 22Na efflux was measured in a medium
containing (in mM) 140 choline-Cl, 2.5 KCl, 9 glucose, and 20 HEPES (pH
7.4 at 37°C) containing 5 mM ouabain in the absence and presence of
10 µM bumetanide. NKCC activity was taken as the bumetanide-sensitive
component of the efflux (oMNabumet). When
indicated, cells were pretreated with 1 µM okadaic acid before
measuring the flux. Data are means ± SD from 4 experiments.
*P < 0.05 compared with fluxes measured in freshly
prepared reticulocytes (Top) at equivalent experimental conditions;
#P < 0.05 between untreated (control) and okadaic
acid-treated cells.
|
|
To test whether the capacity of NKCC was different in different
portions of red blood cells, 22Na efflux was also measured
in these cells after treatment with okadaic acid to achieve maximal
NKCC stimulation by inhibition of the phosphatase activity
(29). Figure 5A indicates that NKCC activity of
reticulocytes (top portion) can be stimulated ~10-fold by okadaic
acid. In contrast, an approximately threefold activation of transport
was found in the middle and a twofold activation in the bottom
fraction. Measurements on cultured reticulocytes show that the
activation of NKCC by okadaic acid decreased significantly during
maturation of reticulocytes (Fig. 5B). Freshly isolated reticulocytes (Fig. 5A, top fraction) and reticulocytes
prepared for maturation studies were from different experimental
series, which explains the difference in the magnitude of NKCC fluxes between the types of experiments.
To test the responsiveness of NKCC to changes in cell volume, we
treated freshly fractionated red blood cells of different density (age)
with nystatin to increase Nai and then incubated them in
media with different sucrose concentrations to alter cell volume. The
results summarized in Fig. 6A
show that NKCC activity was very low in the reticulocyte-rich fraction
when the cells swelled in hypotonic medium (255 mosmol/kg). By
contrast, cell shrinkage in medium of 385 mmol/kg caused a 25-fold
activation relative to the that in the swollen state. In
swollen red blood cells of high buoyant density (bottom fraction), NKCC
transport activity was much higher than that seen in swollen
reticulocytes but was less respondent to cell shrinkage where the
transport rate was less than doubled.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of changes in cell volume on the activity of NKCC
(A) and on the activity of ouabain + bumetanide-insensitive (OBI) 22Na efflux (B) in
freshly prepared reticulocytes (top fraction) and dense (bottom
fraction) red blood cells. The reticulocyte-enriched fraction contained
~97% reticulocytes, whereas the most dense (bottom) fraction had
~0.2% reticulocytes. Cells were treated with nystatin to increase
Nai. Na effluxes (oMNa) were
measured in media with varying sucrose concentration to adjust the
osmolality to the indicated values and contained 5 mM ouabain ± 10 µM bumetanide. A: bumetanide-sensitive (NKCC) efflux of
Nai. B: efflux that remains after inhibition of
the Na-K pump and NKCC with ouabain + bumetanide
(oMNaOBI) as a function of flux medium
osmolality. Cells are at their normal cell volumes at medium osmolality
between 300 and 310 mosmol/kg. Data are mean values from 1 (triplicate
flux measurements, maximal experimental error ± 10%) of 3 experiments with similar results. OBI flux rates measured in
reticulocytes at the highest osmolalities were at best estimates
because the flux rate was outside the linear range.
|
|
During these experiments we observed that the efflux that remained
after exposure to both ouabain and bumetanide, that is, the
ouabain + bumetanide-insensitive (OBI) flux, or
oMNaOBI, was markedly stimulated by cell
shrinkage in the reticulocyte fraction (Fig. 6B). It is also
evident that, in shrunken reticulocytes, the OBI flux exceeded
considerably the NKCC activity shown in Fig. 6A. In the
older, more dense cells, the OBI efflux was lower and displayed only a
modest increase with decreasing cell volume. Preliminary results
indicate that activation of OBI by cell shrinkage was almost completely
inhibited by 100 µM amiloride and may therefore represent Na/proton
exchange (29). Figure 7
shows that okadaic acid, in addition to stimulating NKCC, also
dramatically stimulates the OBI Na efflux in the reticulocyte-rich
(top) fraction, while stimulation was only two- to fourfold in more
dense cells (bottom fraction). Thus cell shrinkage (Fig. 6B)
and okadaic acid treatment (Fig. 7) of cells at their normal volume
both resulted in stimulation of the OBI flux similar to their
stimulation of NKCC activity (Figs. 5A and 6A).
Whether the underlying mechanism(s) controlling these fluxes are the
same or related is not known.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 7.
Activation of OBI 22Na efflux by okadaic acid
of red blood cells of different buoyant density. The
reticulocyte-enriched top fraction contained 95%, the middle fraction
contained 6.5%, and the bottom fraction <0.1% reticulocytes. Fluxes
were measured after Nai and Ki were optimized
by adjustment with the nystatin technique as described in
MATERIALS AND METHODS. The concentration of okadaic acid
was 1 µM. Data are means + SD from 3 experiments.
*P < 0.05 compared with fluxes measured in freshly
prepared reticulocytes (Top) at equivalent experimental conditions;
#P < 0.05 between untreated (control) and okadaic
acid-treated cells.
|
|
Because NKCC can be stimulated by either okadaic acid (Fig.
5A) or cell shrinkage (Fig. 6A), the question can
be asked, what are the combined effects of okadaic acid and cell
shrinkage? Figure 8 shows that the
activation of NKCC by both shrinkage and okadaic acid was highest in
reticulocytes (top fraction) and decreased with increasing cell density
(middle and bottom fractions). However, in reticulocytes the
activation by okadaic acid exceeded considerably the activation by
shrinkage, whereas in the bottom cell fraction both okadaic acid and
shrinkage caused the same degree of stimulation. Shrinkage did not
affect NKCC activity significantly in okadaic acid-treated cells,
indicating that okadaic acid caused maximal transport activation in all
cell fractions. It should be added that the protein kinase inhibitor
staurosporine markedly reduced the NKCC activity of reticulocytes at
normal cell volume but had no effect on the middle and bottom fractions
of cells (results not shown).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 8.
Activation of NKCC by cell shrinkage and okadaic acid of
red blood cells of different buoyant density. The reticulocyte-enriched
top fraction contained 95%, the middle fraction 6.5%, and the bottom
fraction <0.1% reticulocytes. Fluxes were measured after
Nai and Ki were optimized by adjustment with
the nystatin technique under conditions described in MATERIALS
AND METHODS. The osmolality of the control and shrinkage media
was 300 and 380 mosmol/kg, respectively. The concentration of okadaic
acid was 1 µM. Data are means ± SD from 3 experiments.
*P < 0.05 compared with control reticulocytes (top
fraction) at equivalent experimental conditions.
#P < 0.05 between okadaic acid and
shrinkage activation.
|
|
 |
DISCUSSION |
The purpose of this study was to examine changes in the cation
transport activity, ion content, and volume during the maturation of
reticulocytes to determine at which stage of cell maturation these
changes occur and to test whether the measured changes in transport
might account for the reticulocyte maturation-related changes in cell
volume. The results show that the capacity of cation transport systems
such as the Na-K pump, NKCC, and, possibly, Na/H exchange is highest in
reticulocytes and decreases with increasing red blood cell buoyant
density. Concomitantly, Nai increases, whereas
Ki and cell volume decrease. Results from experiments on
cultured reticulocytes indicate that changes in transport and ionic
composition are constrained to the period of reticulocyte maturation
because no further changes were found in mature cells. These results
are in the line with other studies on maturation-dependent changes of
various ion transport systems of red blood cells (e.g., Refs.
2, 3, 10, 16, and
25) but add new insight into a possible contribution of NKCC in
maturation-dependent changes in cell volume.
In previous studies reticulocytes were prepared by long-term
centrifugation of whole blood of healthy donors and patients with
reticulocytosis (7). In animal experiments reticulocytosis was induced by repeated bleeding (3) or treatment with
phenylhydrazine (26). Both treatments put considerable
strain on the studied animals due to oxidative stress and hypoxia
caused by decreased oxygen transport capacity. In the present study
reticulocytes were separated from the whole blood of rats after
erythropoiesis was stimulated by the injection of human recombinant
EPO. This treatment, which was very well tolerated by the animals,
caused an increase in reticulocyte counts from ~2.5 to almost 10%
within the 4 days between EPO application and blood sampling but did not increase Hct. Density gradient fractionation resulted in
reticulocyte fractions with a purity of >90%, whereas the lowest
density fractions of red blood cells from untreated animals contained
only ~50% reticulocytes. Morphological studies of reticulocytes from
EPO-treated rats showed premature stages indicated by ~5% state 0 reticulocytes (17). These cells also had the highest
staining intensity as judged from gray-scale measurements of digitized
images (not shown). In the course of 3-4 days of in vitro
culturing, almost all material stained by new methylene blue had
disappeared, which is indicative of mature red blood cells.
An increased activity of transporters in reticulocyte-enriched
fractions of red blood cells has been observed repeatedly (for review
see Ref. 38). Yet, neither the mechanisms causing the transport activity to decrease nor their consequences on red blood cell
function are understood. In addition, it is important to study
reticulocytes that are formed under physiological conditions without
possible side effects of treatment. During reticulocyte maturation the
total cell surface area decreases by ~20-30% (38, 42), indicating a loss of membrane material. Some of this
material that was shed appears to be liberated in nanometer-sized
vesicles called "exosomes" (22), composed similarly to
the red blood cell membrane but distinctly differ in their content of
certain proteins (21, 22, 24). It was therefore speculated
that liberation of exosomes represents a mechanism for active extrusion of "unnecessary" membrane components (23).
Our results confirm earlier findings of a significant number of Na-K
pumps being lost during transition from the stage of reticulocyte to
mature red blood cell (3, 15). A portion of the lost pumps
was found in exosomes (22), and the remaining ones were
probably proteolyzed (43). Our results show further that
reticulocytes prepared after EPO treatment undergo changes similar to
those obtained with phenylhydrazine treatment or bleeding. This result
might indicate that treatment effects seem not to affect the equipping
of cells with certain transporters or their fate during reticulocyte
maturation. However, in addition to confirming previous findings on the
decrease in pump capacity, which indicates a decrease in the number of
pump copies during reticulocyte maturation (Figs. 2 and 3A),
our results provide no further insight into mechanisms of removal or inactivation.
NKCC activity at optimized Nai was lowest in reticulocytes
but increased as the buoyant density of cells increased. The highest cotransport activity was found in the most dense cells. Duhm
(10) also reported an increase in furosemide-sensitive Rb
uptake with increasing density of human red blood cells whose
Nai and Ki had not been modified but did not
specify the reticulocyte content of the samples studied. In this
(10) and other studies (11, 12) the
dependency of NKCC activity on the steady-state cell volume of red
blood cells of humans and rats was pointed out. A similar relation was
found in unfractionated cells of Nai and Ki
modified to optimize NKCC activity (32). Together, these results indicate a direct relation between the activity of NKCC and
cell volume that is valid not only when unfractionated red blood cells
from different donors are compared (12, 32) but also when
the red blood cell age-related variation in cell volume is considered
(this study and Refs. 10 and 32). In contrast, when NKCC
capacity was measured after full stimulation with okadaic acid, its
activity was highest in reticulocytes but decreased with maturation.
Also, the degree of NKCC stimulation by okadaic acid was much higher in
freshly prepared reticulocytes than in fresh red blood cells of high
buoyant density and in reticulocytes matured in culture. Shrinkage
activation of NKCC showed a similar pattern, although maximal shrinkage
stimulation was smaller than stimulation by okadaic acid. This result
indicates that the capacity of NKCC decreases as reticulocytes mature,
reflecting a decrease in the number of copies of NKCC protein. Another
possibility is, of course, that the protein kinase-protein phosphatase
system controlling NKCC activity undergoes a maturation-dependent
inactivation so that the NKCC phosphorylation potential decreases. NKCC
phosphorylation and activity also depend on cell volume and cytosolic
ion concentrations (28). Since cell volume decreases with
maturation of reticulocytes and red blood cell aging, cell
volume-related effects cannot account for the decreased phosphorylation
potential. The much smaller degree of stimulation by cell shrinkage
than by okadaic acid supports the idea of an age-related loss of cell
volume-sensitive NKCC phosphorylation systems. Another factor that
might account for NKCC changes during reticulocyte maturation might be
the cytosolic Mg concentration. However, although reticulocytes have a
higher total Mg content than mature red blood cells (38),
it is difficult to obtain a reasonable measure of intracellular Mg
relevant for NKCC activation (32) because of its binding
to organic phosphates and RNA, all of which change during reticulocyte maturation.
The maturation-dependent decrease in cell volume, together with the
lost membrane material, cannot explain the change in cellular ion
content (Tables 1 and 2). The ionic composition depends not only on the
content of nondiffusible ions but also on the activity of the Na-K pump
and of secondary active transporters such as NKCC and the leaks
(32, 35, 41). The decrease in pump capacity during
reticulocyte maturation might account for some of the increase in
Nai. The increased cell volume of reticulocytes might also
explain their low NKCC activity and the slight increase in NKCC
activity during reticulocyte maturation, when the cell volume
decreases. A high cotransport activity has been associated with a
decreased steady-state cell volume (12). This has been explained on the basis that an outwardly facing net electrochemical driving force, µnet, results in an NKCC-mediated net
solute extrusion that would tend to keep the cell volume small
(12, 32). Figure 9 shows
that µnet is directed inward in reticulocytes but outward in mature red cells. This could mean that, because of ion gradients, NKCC might also contribute to the decrease in cell water and volume that occurs during reticulocyte maturation.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 9.
Net electrochemical driving force for NKCC in
reticulocytes and red blood cells of different ages. The net
electrochemical driving force (µnet) for NKCC was
calculated from the equation shown in the inset
(11) from the values of red blood cell Na and K
concentrations shown in Tables 1 and 2 (after correction for cell water
content), extracellular concentrations of Na and K of 140 and 5 mM,
respectively, and a chloride ratio (rCl) of
0.67. In the formula, R and F refer to the gas
and Faraday constants, respectively; the subscripts o and i refer to
intracellular and extracellular concentrations, respectively, of
particular ions.
|
|
In conclusion, it is clear that maturing reticulocytes rapidly adjust
volume, ionic composition, and cation transport capacity to that
characteristic of mature red blood cells. Changes in transport activity
certainly contribute to the decrease in cell volume of reticulocytes.
The mechanisms causing the loss of transport capacity during
reticulocyte maturation are unclear. They appear to be independent of
the loss of membrane surface area, judging from the disproportionate
change of both parameters. Despite the maturation-dependent decrease in
the surface area, the amount of hemoglobin per cell remains unchanged.
This is of significance for maintaining a high oxygen transport
capacity without an increase in the number of red blood cells that
would be necessary if hemoglobin were also lost in this process.
Without the decrease in reticulocyte cell volume during maturation, the
Hct of whole blood would be significantly elevated, placing additional
strain on the cardiovascular system.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Heart, Lung, and Blood
Institute Grant HL-09906 and Deutsche Forschungsgemeinschaft Grant Ma
1503/6-1.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: H. Mairbäurl, Institut für Sportmedizin, Medizinische Klinik
und Poliklinik, Universität Heidelberg, Hospitalstrasse 3/4100,
69115 Heidelberg, Germany (E-mail:
heimo_mairbaeurl{at}med.uni-heidelberg.de).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 June 1999; accepted in final form 21 June 2000.
 |
REFERENCES |
1.
Astrup, J.
Sodium and potassium in human red cells. Variations among centrifuged cells.
Scand J Clin Lab Invest
33:
231-237,
1974[ISI][Medline].
2.
Benderoff, S,
Blostein R,
and
Johnstone RM.
Changes in amino acid transport during red cell maturation.
Membr Biochem
1:
89-106,
1978[ISI][Medline].
3.
Blostein, R,
Drapeau P,
Benderoff S,
and
Weigensberg AM.
Changes in Na+ ATPase and Na,K-pump during maturation of sheep reticulocytes.
Can J Biochem
61:
23-28,
1983[ISI].
4.
Borun, ER,
Figueroa WG,
and
Perry SM.
The distribution of Fe59 tagged human erythrocytes in centrifuged specimens as a function of cell age.
J Clin Invest
6:
676-679,
1957.
5.
Brugnara, C,
and
Defranceschi L.
Effect of cell age and phenylhydrazine on the cation transport properties of rabbit erythrocytes.
J Cell Physiol
154:
271-280,
1993[ISI][Medline].
6.
Canessa, M,
Fabry ME,
Suzuka SM,
Morgan K,
and
Nagel RL.
Na+/H+ exchange is increased in sickle cell anemia and young normal red cells.
J Membr Biol
116:
107-115,
1990[ISI][Medline].
7.
Chalfin, D.
Differences between young and mature rabbit erythrocytes.
J Cell Comp Physiol
47:
215-239,
1956[ISI].
8.
Chipperfield, AR,
and
Mangat DS.
(Na + K) co-transport in human red cells increases with cell age.
J Physiol (Lond)
380:
67P,
1986.
9.
Davis, JQ,
Dansereau D,
Johnstone RM,
and
Bennett V.
Selective externalization of an ATP-binding protein structurally related to the clathrin-uncoating ATPase/heat shock protein in vesicles containing terminal transferrin receptors during reticulocyte maturation.
J Biol Chem
261:
15368-15371,
1986[Abstract/Free Full Text].
10.
Duhm, J.
Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: modes of operation, dependence on extracellular and intracellular Na+, kinetics, pH dependency and the effect of cell volume and N-ethylmaleimide.
J Membr Biol
98:
15-32,
1987[ISI][Medline].
11.
Duhm, J,
and
Göbel BO.
Na+-K+ transport and volume of rat erythrocytes under dietary K+ deficiency.
Am J Physiol Cell Physiol
246:
C20-C29,
1984[Abstract/Free Full Text].
12.
Duhm, J,
and
Göbel BO.
Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytes in vitro and in vivo.
J Membr Biol
77:
243-254,
1984[ISI][Medline].
13.
Flatman, PW.
Sodium and potassium transport in ferret red blood cells.
J Physiol (Lond)
341:
545-557,
1983[Abstract].
14.
Forbush, B.
Assay of Na,K-ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecyl sulfate buffered with bovine serum albumin.
Anal Biochem
128:
159-163,
1983[ISI][Medline].
15.
Furukawa, H,
Bilezikian JP,
and
Loeb JN.
Potassium fluxes in the rat reticulocyte: ouabain sensitivity and changes during maturation.
Biochim Biophys Acta
649:
625-632,
1981[ISI][Medline].
16.
Hall, AC,
and
Ellory JC.
Evidence for the presence of volume-sensitive KCl transport in "young" human red cells.
Biochim Biophys Acta
858:
317-320,
1986[ISI][Medline].
17.
Heilmeyer, L.
Blutfarbstoffwechselstudien I.
Dtsch Arch Klin Med
171:
123-153,
1931.
18.
Hoffman, JF.
On the relationship of certain erythrocyte characteristics to their physiological age.
J Cell Comp Physiol
51:
415-424,
1958[ISI].
19.
Hoffman, JF.
The red cell membrane and the transport of sodium and potassium.
Am J Med
41:
666-680,
1966[ISI][Medline].
20.
Hoffman, JF,
and
Laris PC.
Determination of membrane potentials in human and Amphiuma red blood cells by means of a fluorescent probe.
J Physiol (Lond)
239:
519-552,
1974[ISI][Medline].
21.
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[ISI][Medline].
22.
Johnstone, RM,
Adam M,
Hammond JR,
Orr L,
and
Turbide C.
Vesicle formation during reticulocyte maturation.
J Biol Chem
262:
9412-9420,
1987[Abstract/Free Full Text].
23.
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[ISI][Medline].
24.
Johnstone, RM,
Bianchini A,
and
Teng K.
Reticulocyte maturation and exosome release: transferrin receptors containing exosomes show multiple plasma membrane functions.
Blood
74:
1844-1851,
1989[Abstract].
25.
Kim, HD,
and
Luthra MG.
Pig reticulocytes. I. Transitory glucose permeability and metabolism.
Am J Physiol
230:
1668-1675,
1976[ISI][Medline].
26.
Kim, HD,
Luthra MG,
Hildenbrandt GR,
and
Zeidler RB.
Pig reticulocytes. II. Characterization of density-fractionated maturing reticulocytes.
Am J Physiol
230:
1676-1682,
1976[ISI][Medline].
27.
Lee, P,
Kirk RG,
and
Hoffman JF.
Interrelations among Na and K content, cell volume, and buoyant density in human red blood cell populations.
J Membr Biol
79:
119-126,
1984[ISI][Medline].
28.
Lytle, C.
A volume-sensitive protein kinase regulates the Na-K-2Cl cotransporter in duck red blood cells.
Am J Physiol Cell Physiol
274:
C1002-C1010,
1998[Abstract/Free Full Text].
29.
Mairbäurl, H,
and
Herth C.
Na+-K+-2Cl
cotransport, Na+/H+ exchange, and cell volume in ferret red cells.
Am J Physiol Cell Physiol
271:
C1603-C1611,
1996[Abstract/Free Full Text].
30.
Mairbäurl, H,
and
Hoffman JF.
Na-pump fluxes and cell composition of rat reticulocytes.
J Gen Physiol
96:
72a-162a,
1990.
31.
Mairbäurl, H,
and
Hoffman JF.
Na/K-pump activity and cell volume changes during maturation of rat reticulocytes.
In: The Sodium Pump: Recent Developments, edited by Kaplan J,
and DeWeer P.. New York: Rockefeller Univ. Press, 1991, p. 453-456.
32.
Mairbäurl, H,
and
Hoffman JF.
Internal magnesium, 2,3-diphosphoglycerate, and the regulation of the steady-state volume of human red blood cells by the Na/K/2Cl cotransport system.
J Gen Physiol
99:
721-746,
1992[Abstract].
33.
Mairbäurl, H,
and
Hoffman JF.
Na/K/Cl cotransport in rat red blood cells of different cell age.
J Gen Physiol
100:
57a-115a,
1992.
34.
Mairbäurl, H,
Humpeler E,
Schwaberger G,
and
Pessenhofer H.
Training dependent changes of red cell density and erythrocytic oxygen transport.
J Appl Physiol
55:
1403-1407,
1983[Abstract/Free Full Text].
35.
Milanick, M,
and
Hoffman JF.
Ion transport and volume regulation in red blood cells.
Ann NY Acad Sci
488:
174-186,
1986[ISI][Medline].
36.
Netter, H.
Theoretische Biochemie. Physikalisch-chemische Grundlagen der Lebensvorgänge. Heidelberg, Germany: Springer Verlag, 1959.
37.
O'Neill, WC.
Cl-dependent K transport in a pure population of volume-regulating human erythrocytes.
Am J Physiol Cell Physiol
256:
C858-C864,
1989[Abstract/Free Full Text].
38.
Rapoport, SM.
The Reticulocyte. Boca Raton, FL: CRC, 1986.
39.
Sarkadi, B,
and
Parker JC.
Activation of ion transport pathways by changes in cell volume.
Biochim Biophys Acta
1071:
407-427,
1991[ISI][Medline].
40.
Sha'afi, RI,
and
Pascoe E.
Further studies of sodium transport in feline red cells.
J Gen Physiol
61:
709-726,
1973[Abstract/Free Full Text].
41.
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[Abstract/Free Full Text].
42.
Waugh, RE,
McKenney JB,
Bauserman RG,
Brooks DM,
Valeri CR,
and
Snyder LM.
Surface area and volume changes during maturation of reticulocytes in the circulation of the baboon.
J Lab Clin Med
129:
527-535,
1997[ISI][Medline].
43.
Weigensberg, AM,
and
Blostein R.
Energy depletion retards the loss of membrane transport during reticulocyte maturation.
Proc Natl Acad Sci USA
80:
4978-4982,
1983[Abstract].
44.
Wiley, JS.
Increased erythrocyte cation permeability in thalassemia and conditions of marrow stress.
J Clin Invest
67:
917-922,
1981[ISI][Medline].
Am J Physiol Cell Physiol 279(5):C1621-C1630
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society