Swelling activation of transport pathways in erythrocytes:
effects of Cl2, ionic strength, and volume changes
HÉLÈNE GUIZOUARN AND RENÉ MOTAIS
Laboratoire J. Maetz, Département de Biologie Cellulaire et Moléculaire, Commissariat à l’Energie
Atomique, and Unité de Recherche Associée 1855, Centre National de la Recherche Scientifique,
06238 Villefranche-sur-Mer Cedex, France
volume regulation; taurine; volume-activated transports; anion channel; potassium-chloride cotransport
MOST CELLS RESPOND to swelling by activating membrane transport systems that mediate the net loss of
osmotically active, cytoplasmic solutes, thereby allowing the cell to undergo a regulatory volume decrease
(RVD). Inorganic ions (mainly K1 and Cl2 ) and small
organic solutes (amino acids, polyols, and methylamines) are used by cells for such volume control. There
is now substantial evidence from a number of mammalian cell types that the volume-regulated efflux of
organic osmolytes such as taurine, sorbitol, and myoinositol occurs via a swelling-activated anion channel (2,
18, 44, 45), the volume-sensitive organic osmolyte
anion channel, which has not been characterized at the
molecular level. For inorganic ions, a number of different swelling-activated K1 and Cl2 transport mechanisms have been described: a K1-Cl2 cotransporter,
separate K1 and Cl2 channels, and a K1/H1 antiporter
(for review see Refs. 24, 34, and 42).
Fish red blood cells have proved to be a useful model
system for the study of aspects of cell volume regulation. Table 1 shows that Na1, K1, Cl2, and taurine
account for up to 90% of trout erythrocyte osmolality,
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C210
with the remaining osmolality due to the presence of
greatly or totally impermeant solutes such as ATP,
ADP, PO32
4 , and proteins (mainly Hb). Because cell
volume is regulated by the net loss of osmotically active
solutes, RVD of trout erythrocytes can only be achieved
by the loss of K1, Cl2, and taurine down their electrochemical gradients. However, we previously showed
(37) that, depending on how its volume has been
altered, the erythrocyte will or will not involve taurine
in its volume correction and will choose between different transport systems to jettison K1 and Cl2. As
illustrated in Fig. 1, when the volume increase is due to
a net uptake of salts and obliged water, i.e., resulting in
an increase in cell electrolyte concentration, the regulatory response involves only a KCl loss mediated by a
strictly Cl2-dependent pathway that displays the characteristics of a K1-Cl2 cotransport (4, 37). Such an
‘‘isosmotic’’ swelling can be induced by hormonal stimulation of an Na1/H1 exchange or by exposure of the
erythrocytes to a solution containing NH4Cl. Conversely, when a volume increase of the same magnitude
occurs as a result of water entering the cell along its
chemical gradient (‘‘hyposmotic’’ swelling) or dragged
by an uncharged solute (e.g., urea), leading to a dilution
of cell electrolytes, RVD occurs by a loss of taurine,
which accounts for as much as 50% of the total RVD,
and by a loss of KCl. The loss of KCl is then mediated by
two different systems: a ‘‘Cl2-dependent’’ component
and a ‘‘Cl2-independent’’ component, the relative magnitude of the two components varying widely between
experiments (20). Furthermore, hyposmotic swelling
also activates pathways permeable to diverse cations
such as Na1, choline, or tetramethylammonium (TMA)
(20, 37). However, although activation of the taurine,
K1, and Cl2 transport systems will tend to correct
volume changes, the simultaneous activations of other
pathways may partly counteract RVD [net entry of Na1
down its electrochemical gradient (20)] or appear physiologically irrelevant (transport of choline or TMA). In
several other fish species, a similar hyposmotic regulatory pattern has been described involving K1 (6, 9, 29,
32) and diverse organic solutes (15, 17, 21, 23, 30, 31,
46).
Thus trout erythrocytes possess multiple swellingsensitive transport pathways. The fact that they adopt
different regulatory patterns after isosmotic and hyposmotic swellings of similar magnitude shows that the
cells are responding to more than just simple swelling
and suggests that cell electrolyte concentration could
be involved in regulation of the different pathways. For
taurine the pathway activity is related to intracellular
electrolyte concentration (37).
0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society
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Guizouarn, Hélène, and René Motais. Swelling activation of transport pathways in erythrocytes: effects of Cl2,
ionic strength, and volume changes. Am. J. Physiol. 276 (Cell
Physiol. 45): C210–C220, 1999.—If swelling of a cell is
induced by a decrease in external medium tonicity, the
regulatory response is more complex than if swelling of
similar magnitude is due to salt uptake. The present results
provide an explanation. In fish erythrocytes, two distinct
transport pathways were swelling activated: a channel of
broad specificity and a K1-Cl2 cotransporter. Each was
activated by a specific signal: the channel by a decrease in
intracellular ionic strength and the K1-Cl2 cotransporter by
cell enlargement. A decrease in ionic strength also affected
K1-Cl2 cotransport activity, but by acting as a negative
modulator of the cotransport. Thus cells swollen by salt
accumulation respond by activating exclusively the K1-Cl2
cotransport, leading to a Cl2-dependent K1 loss. By contrast,
cells swollen by electrolyte dilution respond by activating
both pathways, leading to a reduced loss of electrolytes and a
large loss of taurine. Thus two swelling-sensitive pathways,
differently regulated, would allow control of the ionic composition of a cell exposed to different volume perturbations.
SWELLING ACTIVATION OF TRANSPORT PATHWAYS
Table 1. Na1, K1, Cl2, and taurine contents
in trout erythrocytes
Content,
µmol/g dcs
Na1
K1
Cl2
Taurine
Water content, gH2O/g dcs
12.3 6 1.5
270.9 6 2.9
114.9 6 3.9
91.5 6 3.4
Osmolality,
mosmol/kgH2O
7.1 6 0.9
156.6 6 1.7
66.4 6 2.3
52.9 6 2.0
1.73 6 0.02
The purpose of the present study was to gain a better
understanding of this complex situation by analyzing
how all the different pathways are stimulated and the
nature of their relationship. The study involved modifying trout erythrocyte volume by degrees in various
ways and monitoring the solute movements that were
induced.
MATERIALS AND METHODS
Materials. Ouabain, DIDS, and N-ethylmaleimide (NEM)
were obtained from Sigma Chemical (St. Louis, MO), [methyl14C]choline, 86Rb1, [14C]sorbitol, and 22Na1 from Amersham
(Little Chalfont, UK), and [14C]taurine from NEN Life Science (Boston, MA). All other chemicals were reagent grade.
Cell preparation. Rainbow trout (Onchorhynchus mychiss,
200–250 g) were obtained from a commercial hatchery and
kept for at least 1 wk in the laboratory. They were stunned by
a sharp blow on the head, and blood was obtained by caudal
venipuncture by using heparinized syringes. The red blood
cells were washed four times in standard saline solution, and
the buffy coat was removed by suction. They were then
suspended at 20% hematocrit, oxygenated, and incubated
overnight at 4°C with 5 mM glucose to ensure that they
reached a steady state with respect to ion and water contents
before experimentation. We showed previously (5), and it has
been confirmed by others (28, 39), that oxy-deoxyhemoglobin
transition in fish erythrocytes regulates a Cl2-dependent K1
transport under isosmotic conditions, with deactivation of the
pathway occurring in anoxia or at a very low PO2. Therefore,
experiments at high or variable PO2 do not allow a clear
characterization of the volume-sensitive K1 fluxes in these
cells. Thus, before experiments the cells were again washed
four times in an N2 atmosphere in the appropriate saline
solution, and experiments were performed under an N2
atmosphere.
Solutions. All experiments were performed in solutions
flushed with N2 and maintained under an O2- and CO2-free N2
atmosphere at 15°C. The basic solution used throughout the
experiments contained (in mM) 145 NaCl, 4 KCl, 5 CaCl2, 1
MgSO4, and 15 N-2-hydroxyethylpiperazine-N8-3-propanesulfonic acid (pH 7.95, 320 mosmol/kgH2O). In some experiments, Na1 was replaced by 145 mM choline. For Cl2-free
solutions, Cl2 was replaced by nitrate (NO2
3 ) or methylsulfate
(MeSO4 ). All solutions contained ouabain to give a final
concentration of 1024 M. Swelling was induced by different
procedures (37). 1) Cells were exposed to media of various
tonicities; the basic solution (320 mosmol/kgH2O) was diluted
by addition of different volumes of buffered water with 15 mM
15 N-2-hydroxyethylpiperazine-N8-3-propanesulfonic acid, pH
7.95, depending on the desired final osmolality. The osmolality was measured by a Wescor vapor pressure osmometer. 2)
Cells were exposed to isotonic media containing various
concentrations of NH1
4 salts (0–50 mM) substituted for 0–50
mM Na1 salts. Cell swelling results from an uptake of NH1
4
salts due to the simultaneous diffusion of NH3 into the cell
and the transmembrane exchanges of anion (intracellular
2
OH2/extracellular Cl2 or NO3 ), mediated by the band 3
protein, which practically abolishes pH changes (,0.1 pH
unit with 50 mM NH1
4 salts). 3) Cells were suspended in
Fig. 1. Isosmotic swelling is induced by
a net uptake of electrolytes and osmotically obliged water. Hormonal stimulation of Na1/H1 exchange or suspension
of erythrocytes in an isotonic solution
containing NH4Cl promotes an isosmotic swelling, which is characterized
by a slight increase of intracellular
electrolyte concentration, i.e., ionic
strength (µ; see Table 2). Cells respond
by releasing K1 via a Cl2-dependent
pathway (K1-Cl2 cotransport). Hyposmotic swelling is induced by a decrease
in extracellular osmolality (or by diffusion of an uncharged solute such as
urea), generating an osmotic inflow of
water, which decreases intracellular
ionic strength (see Table 2). Volumeregulatory response is then much more
complex, involving taurine loss and K1
loss via 2 distinct (Cl2-dependent and
Cl2-independent) pathways. Other
pathways are activated, mediating
downhill uptake of Na1 (which counteracts regulatory volume decrease) and
movements of diverse structurally unrelated compounds [e.g., choline, tetramethylammonium (TMA), sorbitol].
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 18, 2017
Values are means 6 SE; n 5 17. Water content was measured as
difference between wet and dry cell weight. Amounts of Na1, K1, Cl2,
and taurine were measured on extracts from dry cells. Osmolality of
Na1, K1, Cl2, and taurine was calculated from their concentration in
cell water. Sum of osmolalities calculated in this way (283 mosmol/
kgH2O) represents ,90% of total cell osmolality (320 mosmol/
kgH2O), if it is assumed that cell and external saline osmolalities are
identical. dcs, Dry cell solids.
C211
C212
SWELLING ACTIVATION OF TRANSPORT PATHWAYS
RESULTS
Swelling-activated transports of K1. First, the effect
of various degrees of swelling on K1 fluxes was investigated. In Fig. 2A, trout erythrocytes were swollen to
various degrees by exposure to isotonic saline (320
mosmol/kgH2O) containing various amounts of NH1
4
salts in replacement of Na1 salts. Such a swelling,
termed isosmotic, results from a net uptake of NH41
salts and osmotically obliged water (37). The media
contained Cl2 or NO2
3 as anion. Isosmotic swelling of
cells in Cl2-containing media was followed by a quite
linear increase in the ouabain-insensitive K1 influx
as a function of cell volume. By contrast, in NO2
3containing media there is no discernible influence of
cell volume on the K1 flux. Thus trout erythrocytes
responded to isosmotic swelling by activating exclusively a Cl2-dependent K1 flux, regardless of the magnitude of the volume increase. In Fig. 2B, trout erythrocytes were swollen to various degrees by exposure to
hypotonic media of various osmolalities. In media with
and without Cl2 (with NO32), hyposmotic swelling was
followed by an increase in the ouabain-insensitive K1
flux. The fluxes measured in Cl2-containing media
Fig. 2. K1 flux as a function of cell volume (expressed as cell water
content). A: erythrocytes isosmotically swollen by exposure to isotonic media containing various concentrations of NH1
4 salts (0–50
1
mM), with Cl2 (k) or NO2
3 (j) as permeant anion. NH4 salts were
added in replacement of same amount of Na1 salts in saline. Thus
osmolality of medium was constant (320 mosmol/kgH2O). Cell volume is expressed as grams of cell water per gram of dry cell solids
(dcs) and was determined by weighing cells before and after drying at
80°C. Values are averages of 3 similar experiments (error bars are
within symbols) and show that K1 flux (measured as Rb1 influx)
increased as a function of cell volume in Cl2-containing, but not
24 M ouabain.
NO2
3 -containing, media. All solutions contained 10
B: erythrocytes hyposmotically swollen by exposure to media of
various tonicities (from 320 to 220 mosmol/kgH2O) and containing
Cl2 (k) or NO2
3 (j) as permeant anion. Appropriate 320-mosmol
saline solutions were diluted by addition of different volumes of
buffered water. Values are averages of 4 similar experiments and
show that K1 flux increased as a function of cell volume in Cl2- and
24 M ouabain.
NO2
3 -containing media. All solutions contained 10
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isosmotic (320 mosmol/kgH2O) saline solutions containing
1
180 mM urea or 50 mM NH1
4 salts or mixtures of NH4 salts
and urea. Osmolality was maintained constant by replacing
90 mM Na1 salts of the basic solution with 180 mM urea or 50
mM NH1
4 salts plus 40 mM N-methyl-D-glucamine salts or
appropriate mixtures, e.g., (in mM) 100 urea, 20 NH1
4 salts,
and 20 N-methyl-D-glucamine salts. Cell swelling results
from the penetration of urea and/or NH1
4 salts and osmotically obliged water.
Determination of water content. Just before swelling and
then at various time intervals, samples of cell suspension
were poured into three nylon tubes and centrifuged for 10 min
at 30,000 g in a refrigerated centrifuge. The red cell pellet was
separated from the supernatant by slicing the tube with a
razor blade. It was extracted with a close-fitting plastic rod
onto a piece of weighed aluminum foil; after the pellet was
weighed wet, it was dried to a constant weight for 10 h at 80°C
and reweighed. Cell water content is expressed as grams of
water per gram of dry cell solids. The extracellular space was
measured after a very short exposure of cells to 22Na1 (45 s),
and a correction of 3.5% was applied for this to all calculations.
Transport measurements. Unidirectional K1 transport was
measured using 86Rb1 as convenient congener. [14C]choline,
[14C]taurine, [14C]sorbitol, and 22Na1 were used to measure
other unidirectional transports. Uptake measurements refer
to values of solutes determined in cells as a function of time.
Influx rates were estimated from the amount of radioactivity
accumulated within a fixed incubation period that fell within
the initial linear phase of the uptake time courses. Radioisotopes were added to the cell suspension just after swelling;
thus the same batch of swelled cells could be used to
determine different solute uptakes. Samples were poured into
three nylon tubes and centrifuged (30,000 g, 10 min, 4°C); the
supernatant was kept to measure external radioactivity, and
the pellet was weighed wet and dry for cell water determination. For beta-radioactivity counting, the dry pellet was
suspended in 1 ml of distilled water overnight, and radioactivity in the extract was measured by liquid scintillation in a
beta counter (Packard). For determination of gamma radioactivity, the pellet was counted dry (Kontron gamma counter).
Uptakes are expressed in micromoles per gram of dry cell
solids and fluxes in micromoles per gram of dry cell solids per
hour. In these experiments the external concentrations of the
solutes were generally (in mM) 145 Na1, 145 choline, 5
taurine, 5 sorbitol, and 4 K1.
Ion content and concentration. The dry cells were suspended in 5 ml of distilled water overnight; then 100 µl of 70%
(vol/vol) perchloric acid were added to the suspension. After
centrifugation at 30,000 g for 10 min the clear supernatant
was saved for analysis of cations, Cl2, and amino acids. Ions
were measured as previously described (20). A trapping
correction of 3.5% was routinely applied to the final calculation. Ion contents were expressed in micromoles per gram of
dry cell solids. Ion concentrations in cell water were calculated from ion contents and cell water contents and expressed
as millimoles per liter of cell water.
Covalent fixation of DIDS. DIDS was covalently bound to
the red cell membrane by incubation in the basic isotonic
saline (10% hematocrit with 100 µM DIDS at 15°C for 90
min). Then the unreacted DIDS was washed away (1 rinse
with 25 vol of saline 1 0.5% BSA followed by 2 additional
rinses without albumin). Swelling was subsequently induced
by suspending the cells in a DIDS-free hypotonic medium.
C213
SWELLING ACTIVATION OF TRANSPORT PATHWAYS
were substantially greater than those in
NO2
3 -containing media, the difference between the two
curves corresponding to the Cl2-dependent component
of the K1 flux (Fig. 3).
Two main observations emerged from these data.
The Cl2-dependent K1 component, considered a K1Cl2 cotransport, was always activated when cell volume increased, but the pattern of activation as a
function of cell volume was different depending on the
way cells were swollen (Fig. 3). When cell swelling was
isosmotically induced, the flux increased considerably
Table 2. Examples of changes in intracellular electrolyte concentration and cell volume induced
by different swelling protocols
Time, min
V, gH2O/g dcs
[Na1 ], mM
[K1 ], mM
[Cl2 ], mM
[NH1
4 ], mM
SE, mmol/l
Isosmotic swelling induced by activation of Na1/H1 exchange
0
26
1.87 6 0.01
2.54 6 0.02
0
3
1.84 6 0.01
2.53 6 0.01
0
3
1.73 6 0.02
2.36 6 0.02
0
3
1.66 6 0.01
2.37 6 0.00
6.97 6 0.22
69.08 6 0.43
146.82 6 1.42
92.66 6 1.16
78.67 6 0.36
96.14 6 2.42
232.46
257.88
Isosmotic swelling induced by exposure to medium containing 50 mM NH4Cl
3.81 6 0.50
2.62 6 0.12
148.50 6 1.67
104.48 6 0.41
79.25 6 0.62
101.08 6 0.82
0.00
43.36
231.56
251.54
Hyposmotic swelling induced by exposure to hypotonic medium
7.10 6 0.83
4.83 6 0.59
157.28 6 1.48
113.64 6 1.04
66.33 6 1.94
46.89 6 1.42
230.71
165.32
Hyposmotic swelling induced by exposure to isosmotic urea-containing medium
4.93 6 0.18
2.41 6 0.33
163.32 6 1.63
115.31 6 1.37
69.59 6 0.45
46.38 6 1.24
237.84
164.10
Values are means 6 SE of 3 paired determinations (17 different experiments for hypotonic medium). Measurements of ion (µmol/g dcs) and
water (gH2O/g dcs) contents (see MATERIAL AND METHODS) allow calculation of ion concentrations ([Na1 ], [K1 ], [Cl2 ], and [ NH1
4 ]) in cell water.
2
2
2
In experiments in which NO2
3 replaced Cl , strictly parallel experiments were run in Cl media, and NO3 concentration was assumed to be
1
1
2
equal to Cl2 concentration. NH1
4 content was calculated as difference between changes in cation (Na 1 K ) and Cl contents with assumption
2
24 M) was present in all media. Osmotic pressure of isotonic media was 320 mosmol/kgH O. V,
that 1 NH1
2
4 is neutralized by 1 Cl . Ouabain (10
cell volume; SE, intracellular electrolyte concentration.
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Fig. 3. Cl2-dependent K1 fluxes as a function of cell volume (expressed as cell water content) in erythrocytes swollen isosmotically
(k) and hyposmotically (n). Data were obtained from Fig. 2, A and B,
respectively, by subtracting fluxes measured in NO2
3 -containing
media from those measured in Cl2-containing media.
and regularly at the first increase in volume. In other
words, any deviation from normal volume was immediately ‘‘sensed’’ by cells, which responded by activating
the K1-Cl2 cotransporter, and the greater the volume
increase the greater the activation. On the other hand,
when swelling was hyposmotically induced, the threshold for activation of the flux, called the set point, was
shifted to higher cell volume, and the maximal activity
was greatly reduced.
The Cl2-independent K1 component, which was activated as a function of cell volume under hyposmotic
swelling conditions (Fig. 2B), was not activated under
isosmotic cell-swelling conditions, irrespective of the
degree of swelling (Fig. 2A). In other words, activation
of the Cl2-independent K1 component is not simply
triggered by the cell volume increase but is dependent
on changes in the intracellular concentration of salts or
impermeant compounds. Because the cells were swollen to the same extent in isosmotic and hyposmotic
solutions, any changes in the concentration of cytoplasm-impermeant compound were the same with both
types of treatment; conversely, the concentration of
intracellular electrolytes decreased in the hyposmotically swollen cells and increased in the isosmotically
swollen cells (Table 2), indicating that intracellular
electrolyte concentration could play a role in the activation process of the Cl2-independent K1 flux. To investigate this possibility further, two distinct experimental
protocols were employed. We compared the activation
of the Cl2-independent K1 flux when dilution of electrolytes results from cell volume increase (Fig. 4A) and
when dilution of electrolytes occurs at a constant
swollen cell volume (Fig. 4B).
Erythrocytes exposed to hypotonic media of different
tonicities show a simultaneous increase in cell volume
and decrease in intracellular solute concentration. A
direct relationship exists between the two parameters:
C214
SWELLING ACTIVATION OF TRANSPORT PATHWAYS
Exposure of cells to urea was followed by diffusion of
urea and osmotically obliged water, inducing cell swelling and dilution of electrolytes (Table 2). By contrast, a
similar increase in volume induced by exposure to
NH4NO3 produced an increase in the cytoplasmic electrolyte concentration (Table 2). Exposure of cells to
urea-NH4NO3 mixtures induces intermediate changes
in intracellular electrolyte concentration. Thus, at a
constant cell volume, the cell electrolyte concentration
can be varied according to the composition of the
suspending media, with electrolytes becoming concentrated in NH4NO3 and diluted in urea and ureaNH4NO3 mixtures. Figure 4B shows that, at a constant
cell volume (40% increase in cell water relative to
control erythrocytes), activation of the Cl2-independent
K1 component is dependent on a reduction of the
intracellular electrolyte concentration; despite a large
cell volume increase, no discernible activation was
measured when electrolyte concentrations remained
equivalent to (dilution factor 5 1) or greater (dilution
factor 5 0.9) than that of control unswollen erythrocytes. Activation started when electrolytes were diluted
and increased progressively as a function of electrolyte
dilution. The set point for activation, visually estimated from four similar experiments, corresponded to
an ,10% dilution (dilution factor 5 1.1). Thus, when
the cell volume was increased to a constant value,
activation was only dependent on a reduction of electrolyte concentration. A comparison of Fig. 4, A and B,
Fig. 4. Swelling-activated Cl2-independent K1 flux (k) as a function
of intracellular electrolyte dilution. To measure Cl2-independent
2 substitute. A: hyposmotic swelling
fluxes, NO2
3 was used as a Cl
(electrolyte dilution varying as cell volume). Cells, previously incu2
bated in Cl2-free, NO2
3 -containing saline to replace internal Cl by
2-free, NO2-containing media of tonicities
NO2
,
were
suspended
in
Cl
3
3
varying from 320 (isotonic) to 220 mosmol/kgH2O. In these conditions, as long as external osmolality decreased, cell volume increased
and, simultaneously, cellular impermeant compounds and electrolytes were progressively diluted. Cell volume, measured as cell water
content per gram of dry cell solids, was expressed as percentage of
control, unswollen cells. Intracellular amounts of electrolytes were
measured; their concentrations in cell water (mM) were then calculated (see Table 2). Abscissa: changes in cell electrolyte concentration
expressed as dilution factor in comparison with electrolyte concentration before cells were swollen, i.e., 1. Cell volume (r; right ordinate)
and Cl2-independent K flux (k; left ordinate) are plotted as a function
of dilution factor of electrolytes. There is no discernible influence of
cell swelling on Cl2-independent K1 flux as long as water content of
swollen cells was ,110% of control cells, i.e., a 10% dilution of
electrolytes. Values are from 1 of 5 experiments giving similar
results. B: isosmotic swelling (electrolyte dilution obtained at a
constant cell volume). All cells were 40% swollen by suspension in
isosmotic Cl2-free, NO2
3 -containing media (320 mosmol/kgH2O) in
which 90 mM NaNO3 was replaced by urea, NH4NO3, or ureaNH4NO3 mixtures (see MATERIALS AND METHODS ). Intracellular
amounts of electrolytes (µmol/g dcs) were measured; their concentrations in cell water (mM) were then calculated (see Table 2). Abscissa:
changes in cell electrolyte concentration expressed as dilution factor
in comparison with electrolyte concentration before cells were swollen, i.e., 1 (vertical dashed line). In cells swollen in an isosmotic saline
containing 50 mM NH4NO3, electrolytes were not diluted but slightly
concentrated (dilution factor 0.93), as shown in Table 2. Cell volume
(r) and Cl2-independent K flux (k) are plotted as a function of
dilution factor of electrolytes. There was no discernible increase of
Cl2-independent K1 flux as long as dilution of electrolytes was 10%.
Values are from 1 of 4 experiments giving similar results.
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an entry of water into the cell causes, e.g., a 10%
increase in cell volume (expressed as cell water content
per gram of dry cell solids) and a 10% dilution of
intracellular electrolytes. Figure 4A depicts a typical
experiment. Cell swelling was obtained by suspending
trout erythrocytes in hypotonic Cl2-free, NO2
3 -containing media of different tonicities, and the resulting
Cl2-independent K1 flux was measured. Cell volume
and Cl2-independent K1 flux are plotted as a function
of electrolyte dilution in Fig. 4A. An electrolyte dilution
of ,10% had no discernible influence on K1 flux. From
five similar experiments the set point for activation was
visually estimated and corresponded to an ,10% salt
dilution. As shown in Fig. 4A, above the set point the
flux increased rapidly and in a linear manner.
To induce electrolyte dilution at a constant cell
volume, the following protocol was adopted. Trout
erythrocytes were swollen to the same extent (40%
increase in cell water) by exposure to isosmotic
NO2
3 -containing saline (320 mosmol/kgH2O) in which
90 mM NaNO3 was replaced by urea, NH4NO3, or
urea-NH4NO3 mixtures (see MATERIALS AND METHODS ).
SWELLING ACTIVATION OF TRANSPORT PATHWAYS
Fig. 5. Comparison of taurine, Na1, choline, and Cl2-independent K1
fluxes as a function of electrolyte dilution. A: hyposmotic swelling.
Cells were suspended in media of different tonicities varying from
320 (isotonic) to 220 mosmol/kgH2O. In these conditions, dilution of
electrolytes varies as cell volume (indicated by dashed line for
clarity). Fluxes are expressed as percentage of flux measured when
cells are exposed to maximal hypotonicity (at 100% flux all signs are
superimposed). Abscissa: dilution of electrolytes as in Fig. 4A.
B: isosmotic swelling. All cells were 40% swollen by suspension in
isosmotic media (320 mosmol/kgH2O) containing mixtures of NH1
4
salts and urea (see Fig. 4B and MATERIALS AND METHODS ), leading to
dilution of electrolytes at a constant cell volume (cell volume indicated by a dashed line for clarity). Fluxes are expressed as percentage
of flux measured at maximal urea concentration, i.e., 180 mM (at
100% flux all signs are superimposed). Abscissa: dilution of electrolytes as in Fig. 4B.
3) The set points for activation of all solute fluxes
were similar (,10% dilution of electrolytes) when
electrolyte concentration varied with changes in cell
volume (Fig. 5A) or at a constant swollen cell volume
(Fig. 5B). In other words, the set points were insensitive to the concentration of impermeant compounds.
In skate hepatocytes it has been shown that activation of the taurine pathway is dependent on intracellu-
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shows that the electrolyte set point for activation of the
Cl2-independent K1 flux was very similar in the two
experiments. Because the concentration of cytoplasmimpermeant compounds, such as proteins, is strictly
related to volume change, i.e., progressively decreased
in Fig. 4A and maintained constant in Fig. 4B, the set
point appears insensitive to changes in the concentration of impermeant compounds.
Other swelling-dependent transport systems. In response to swelling, trout erythrocytes activate several
membrane pathways other than Cl2-dependent and
Cl2-independent K1 transport systems; the transport
of compounds as structurally diverse as amino acids
(taurine) and inorganic (Na1 ) and organic (choline)
cations is induced in a similar fashion (20). An organic
uncharged solute, sorbitol, can also be transported (see
Fig. 7A). It must be pointed out that activation of these
pathways allows a large amount of compounds to be
transported. For example, when hyposmotically swol1
len in a Cl2-free, NO2
3 -containing saline, K loss per
hour was 33.81 6 4.45 µmol/g dry cell solids and
taurine loss was 37.97 6 3.55 µmol/g dry cell solids,
whereas Na1 uptake was 29.10 6 1.59 µmol/g dry cell
solids (5 experiments). In other words, the net Na1
uptake practically counterbalanced the volume-regulatory K1 loss mediated by the Cl2-independent component; therefore, RVD in a Cl2-free medium results
exclusively from taurine loss. It is interesting to note
that permeability of choline is also very large, even
greater than that of Na1 (20).
However, as described above for the Cl2-independent
K1 transport, these pathways are activated when cells
are hypotonically swollen but remain inactivated when
cells are isosmotically swollen (37). We have shown
previously (37) with cells swollen at a constant volume
that activation of the taurine pathway is dependent on
a decrease in intracellular electrolyte concentration.
Experiments were then designed to compare activation
of the taurine pathway when dilution of electrolytes
occurs at a constant cell volume or results from cell
volume increase. Similarly, experiments were performed to analyze the putative role of electrolyte dilution on the volume-sensitive pathways mediating the
transport of inorganic (Na1 ) and organic (choline)
cations. These results, along with the data reported
above for the Cl2-independent K1 pathway, are shown
in Fig. 5.
From the analysis of Fig. 5, several observations can
be made.
1) For all compounds, i.e., Na1, choline, taurine, and
1
K (as the Cl2-independent component), activation of
fluxes and electrolyte concentration were inversely
related when cell volume was changing (Fig. 5A) or
kept constant (Fig. 5B). In other words, at a constant
cell volume, activation of all fluxes was strictly dependent on a reduction of cell electrolyte concentration.
2) The dependence of fluxes on electrolyte concentration was identical for all compounds, suggesting that
the different pathways are in some way regulated in a
coordinated fashion or that all solutes move via a
common pathway.
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SWELLING ACTIVATION OF TRANSPORT PATHWAYS
Fig. 6. Effect of Cl2 replacement on swelling-sensitive transport of
taurine. To obtain total Cl2 replacement, cells were first incubated in
a basic isotonic saline (320 mosmol/kgH2O) in which NO2
3 was used as
a substitute for Cl2. Intracellular Cl2 is thus replaced by NO2
3 via
anion exchanges. Then cells were suspended in appropriate Cl2-free,
NO2
3 -containing media. A: isosmotic swelling, i.e., swelling without
dilution of electrolytes. Cells preincubated in NO2
3 saline were
exposed to isotonic saline in which 50 mM NaNO3 was replaced by 50
mM NH4NO3, thus inducing 40% increase in cell volume in ,2 min.
Similarly, control cells (i.e., Cl2-containing cells) were exposed to
isotonic Cl2-containing saline in which 50 mM NaCl was replaced by
50 mM NH4Cl. Time course and magnitude of swelling were similar
for both batches of cells. Taurine flux measurements started at 5 min.
Total replacement of Cl2 did not induce activation of volumesensitive taurine transport. B: hyposmotic swelling, i.e., swelling
accompanied by a decrease in electrolyte concentration. Control cells
were suspended in a hyposmotic, Cl2-containing medium (220 mosmol/
kgH2O), whereas cells preincubated in NO2
3 saline were suspended in
2
a hyposmotic, NO2
3 -containing medium. Total replacement of Cl did
not affect volume-sensitive taurine transport.
DISCUSSION
Trout erythrocytes possess multiple swelling-sensitive transport pathways that, for swelling of similar
magnitudes, can be activated to different degrees according to the manner in which the volume change has been
brought about. Depending on whether cell electrolyte
concentration has been increased or decreased during
swelling, the red blood cell will activate only one
regulatory pathway (a KCl cotransport) or numerous
additional pathways, implicating cell electrolyte concentration in the control of volume-sensitive pathways.
To try to understand this complex regulatory mechanism, we have to identify, for each transport system,
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 18, 2017
lar Cl2 concentration (25). The experiments described
above were performed in Cl2-containing media, except
for those involved in the measurement of the Cl2independent K1 fluxes, which were performed in Cl2free, NO2
3 -containing media. To test whether a decrease
in Cl2 concentration, rather than dilution of electrolytes, is responsible, in trout erythrocytes, for activation of all these swelling-sensitive pathways, the following experiments were performed. First, isosmotic
swelling was induced by suspending red blood cells in
isosmotic media containing NH4Cl or NH4NO3 exchanged for the same amount of Na1 salt, a condition in
which swelling occurs without dilution of electrolytes
(Table 2). As indicated above, swelling in NH4Cl did not
induce activation of the taurine pathway. Figure 6A
shows that total replacement of Cl2 by NO2
3 also did not
induce any activation of the taurine flux. In a second
series of experiments, a similar swelling (40% increase
in cell water) was induced by suspending erythrocytes
in hypotonic, NaCl- or NaNO3-containing media, a
condition in which swelling is accompanied by a decrease in electrolyte concentration. Figure 6B shows
2
that taurine flux was similar in NO2
3 and Cl media,
i.e., when intracellular Cl2 has been totally replaced or
only 40% diluted. Replacement of Cl2 by MeSO4 gave
similar results (not shown). Thus activation of the
taurine pathway appears dependent on the concentration of intracellular electrolytes and not on the concentration of intracellular Cl2. Because Na1 and choline
fluxes were unaffected by the replacement of Cl2 with
MeSO4 (not shown), activation of these solute fluxes,
like that of taurine, is dependent on a reduction of cell
electrolyte content.
In conclusion, the volume-sensitive pathways for
taurine, Na1, choline, and (Cl2-independent) K1 were
activated by a reduction in electrolyte concentration,
and their patterns of activation, including the set
points, appeared identical.
All these pathways have been shown previously to be
inhibited to a similar extent by a series of anion
transport inhibitors [e.g., furosemide, niflumic acid,
DIDS, and 5-nitro-2-(3-phenylpropylamino)benzoic acid
(NPPB)], and in cases when the dose dependences were
measured, these were also found to be similar (6, 19,
20). Moreover, in trout erythrocytes (19), but not in eel
erythrocytes (35), DIDS, when covalently bound to the
membrane, inhibited all these pathways, including the
Cl2-independent, but not the Cl2-dependent, K1 pathway. Figure 7A shows that covalently bound DIDS
similarly inhibited the volume-induced transport of
sorbitol, one of the other organic solutes transported in
response to swelling. Recently, Bursell and Kirk (6)
found that NEM (2 mM) was effective as an inhibitor of
the swelling-activated taurine transport in eel erythrocytes. As illustrated in Fig. 7B, NEM (1 mM) applied to
trout erythrocytes inhibited not only the volumedependent taurine transport but also Na1, choline,
sorbitol, and Cl2-independent K1 transports in a similar manner. Thus the pharmacological characteristics
of all these pathways appear strikingly similar.
SWELLING ACTIVATION OF TRANSPORT PATHWAYS
the specific intracellular signal(s) involved in its activation.
K1-Cl2 cotransporter. A Cl2-dependent K1 pathway
with the characteristics of a K1-Cl2 cotransporter (4, 6,
37) is activated in response to isosmotic and hyposmotic
swelling (Fig. 3). To define Cl2-dependent and Cl22
independent K1 pathways, NO2
3 has been used as a Cl
substitute, similar to studies on sheep, human (10, 33,
34), and other fish (6, 32) red blood cells. However, in
mouse (1) and trout red blood cells exposed to extreme
prelytic cell volume (3), NO2
3 has been shown to be a
less efficient substitute for Cl2 than MeSO4 or sulfamate. However, under the experimental conditions
used in this study (maximum 40% increase in cell water
content), NO2
3 is a reasonably good substitute, since the
K1 fluxes measured in NO2
3 -containing media were
similar to or only very slightly higher than those in
media containing MeSO4 or sulfamate (not shown).
Figure 3 shows that activation of the K1-Cl2 cotransport as a function of cell volume was different in
isosmotically and hyposmotically swollen cells. Isosmotic swelling induced a strong KCl activation as soon
as a slight increase in volume occurred, and the KCl
activity continued to increase linearly as a function of
cell volume. Hyposmotic swelling also induced KCl
activation, but under these conditions the efficiency of
the K1-Cl2 cotransport was clearly reduced: 1) the
sensitivity to small changes in cell volume was much
lower, suggesting that the set point is shifted to higher
cell volumes; and 2) the volume dependence of the K1
flux was damped, displaying a rather sigmoidal relationship. Hyposmotic swelling promotes an increase in cell
volume and a dilution of impermeant compounds, as
does isosmotic swelling, but hyposmotic swelling also
promotes a dilution of intracellular electrolytes (i.e., a
decrease in ionic strength). Thus the volume increase
and/or the decrease in impermeant compound concentration appears to be the primary activator of the
K1-Cl2 cotransport, with ionic strength acting as a
modulator of the activated cotransporter; K1-Cl2 cotransport is more efficient at higher than at lower ionic
strengths, this parameter acting partly by altering the
set point.
Studies on dog red cell ghosts have shown that
dilution of cytoplasmic proteins, regardless of cell volume, activated K1-Cl2 cotransport (8), suggesting that
in trout erythrocytes dilution of cytosolic impermeant
compounds could be the parameter controlling activation of the K1-Cl2 cotransport. Moreover, at any particular dog red cell volume, decreases in ionic strength
diminished K1-Cl2 cotransport activity and shifted the
set point of the cotransporter to higher cell volume (41).
The authors interpreted these results as implying that
interaction of cell electrolytes with some intracellular
macromolecules induced changes in the activity of a
putative regulatory protein (41), such as perhaps the
inhibitory, volume-sensitive kinase (27). Our data in
Fig. 3 show that the set point for activation of the
K1-Cl2 cotransport was shifted to a higher cell volume
when swelling was hypotonically induced (i.e., when
ionic strength was decreased); this is in agreement with
the results obtained in dog red blood cells. However,
ionic strength not only altered the set point for activation but also decreased the maximal activity, indicating
a more complex effect of ionic strength than that
suggested for dog red blood cells.
Pathways induced by hyposmotic swelling. Swelling
of trout erythrocytes, when accompanied by a dilution
of intracellular electrolytes (hyposmotic swelling), simultaneously activates a K1-Cl2 cotransport and the
transport of structurally unrelated compounds such as
K1 (as a Cl2-independent component), taurine, Na1,
choline, and sorbitol. The data in Fig. 5B clearly
demonstrate that the dilution of intracellular electrolytes is the factor that tightly controls activation of all
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Fig. 7. A: effect of covalently bound DIDS on volume-activated influx
of sorbitol. Hyposmotically induced swelling resulted in a large
increase in sorbitol permeability, which was inhibited by covalently
bound DIDS. Values are averages of 3 experiments. DIDS was
covalently bound to membrane of red blood cells suspended in a basic
isotonic saline. Then unreacted DIDS was washed away. Swelling
was subsequently induced by suspending cells in a DIDS-free hypotonic medium (220 mosmol/kgH2O). B: effect of 1 mM N-ethylmaleimide (NEM) on volume-activated fluxes of Na1, choline, sorbitol,
taurine, and (Cl2-independent) K1 (Cl2-ind K1 ). Cells were not
preincubated with sulfhydryl reagent; NEM, added with radioisotopes, was present throughout flux period in hypotonic media (220
mosmol/kgH2O). For (Cl2-independent) K1 flux, cells were hyposmotically swollen in a Cl2-free, NO2
3 -containing medium. Hatched bars,
with NEM; open bars, without NEM.
C217
C218
SWELLING ACTIVATION OF TRANSPORT PATHWAYS
reach the critical ionic strength threshold. Thus only a
direct measurement of intracellular electrolyte concentration at each level of cell swelling would confirm that
the taurine channel is differently activated in C6 glioma
cells and in trout red blood cells. Nevertheless, it
remains possible that the nature of the taurine pathway may be different in the two cell types.
The present results showing that several pathways
induced by hyposmotic swelling are similarly activated
by ionic strength raise two main questions: 1) What is
the relationship between these pathways? 2) How does
ionic strength affect them?
The fact that fluxes of solutes so structurally diverse
as K1 (as Cl2-independent component), Na1, choline,
taurine, and sorbitol are similarly controlled by ionic
strength suggests that their movements are in some
way regulated in a coordinated fashion. This linkage
was first proposed (20) when, having shown that hyposmotic swelling activated several transports of structurally unrelated compounds, we noted 1) that all the
fluxes were inhibited by DIDS and other inhibitors of
the band 3 anion exchanger (e.g., niflumate and furosemide) and 2) that the IC50 for inhibition of cations and
taurine was the same. We suggested that the DIDSsensitive band 3 anion exchanger would control the
activity of multiple transport systems. Later, a series of
studies performed by various investigators on erythrocytes from other fish species (flounder, skate, and eel)
confirmed that hyposmotic swelling led to an increase
in the membrane transport of a wide range of solutes
(with the addition of new compounds such as betaine,
polyols, and nucleosides) and that these fluxes were
similarly inhibited by several blockers of the anion
exchanger (including NPPB). None of the inhibitors are
highly specific, and it is possible that their identical
effects on the different volume-activated transport pathways are coincidental. However, certain kinetic properties of these transports were studied and were found to
be similar. It was then suggested (22, 23, 31, 46) that all
these solutes share a common pathway with the characteristics of an anion channel, displaying considerable
similarity to channels mediating the volume-regulatory efflux of organic osmolytes from mammalian cells
(44). The recent results of Kirk and collaborators (6, 30,
35), obtained with eel erythrocytes, further support
this hypothesis of a single swelling-activated, DIDSand NPPB-sensitive channel of broad specificity. Likewise, it has been observed, using the patch-clamp
technique, that in isotonic conditions trout red blood
cells possess a significant DIDS-sensitive Cl2 conductance that is reversibly stimulated by hyposmotic cell
swelling (11). An additional argument in favor of the
single-pathway hypothesis is provided by the results
obtained on Xenopus oocytes expressing red cell band 3
anion exchangers (AE1). When expressed in oocytes,
the trout AE1 can function as an anion channel mediating the movements of taurine (13) and uncharged
solutes such as sorbitol (14a), whereas the highly
homologous isoform from a mature mammalian erythrocyte, a cell that has lost the capacity to regulate its
volume, fails to function as an anion channel and to
transport taurine and sorbitol. Thus these data strongly
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these pathways, as we have previously shown for
taurine (37); when erythrocytes were kept swollen at a
constant volume (40%), no activation occurred as long
as the concentration of intracellular electrolytes remained higher than or equivalent to that of control,
unswollen cells. Some activation was discernible when
the intracellular electrolytes were diluted by ,10%. On
greater dilution, the flux increased rapidly and linearly.
It is conceivable, as in skate hepatocytes (25), that
intracellular Cl2 or K1 concentration, rather than the
electrolyte concentration as a whole, exerts such a
control. However, total replacement of Cl2 by NO2
3 or
MeSO4 did not alter the patterns of activation. Moreover, we previously showed (37) that the taurine pathway remained inactive when swelling was induced by
catecholamine stimulation of Na1/H1 exchange or exposure to NH1
4 salts, indicating that the nature of cations
accumulated in the cell (Na1 in the presence of ouabain, K1 in its absence, or NH1
4 ) also did not control
pathway activation. Thus it is the concentration of
intracellular electrolytes (i.e., ionic strength), and not
their nature, that is the factor controlling activation.
Moreover, a comparison of Fig. 5, A and B, supplies
additional and important information: fluxes of all
solutes are controlled by the changes in cell electrolyte
concentration (i.e., ionic strength) but are independent
of the degree of cell volume increase. For example, at
1.3 electrolyte dilution, fluxes represent 30–35% of
maximal value when cell enlargement is 12% (Fig. 5A)
or 40% (Fig. 5B). This is a similar feature for each point
on the curves. In particular, activation occurs at the
same degree of electrolyte dilution when the cell volume is only slightly enlarged (2%, Fig. 5A) or greatly
enlarged (40%, Fig. 5B). Thus activation of all these
transport pathways is triggered by the decrease in ionic
strength and is independent of the degree of cell volume
increase. It means that activation is characterized by
an ‘‘ionic strength set point’’ and not by a ‘‘volume set
point.’’
Studying the regulation of the volume-sensitive taurine channel in C6 glioma cells, Emma et al. (12)
reached a different conclusion: activation of the channel would be controlled by a volume set point that is
modulated by changes in intracellular electrolyte concentration. In these experiments, acclimatization of C6
cells to hypertonic media allowed an increase in intracellular electrolyte level. Cell swelling was then induced by reducing bath osmolality (i.e., hypotonic
swelling). It was found that ‘‘at high intracellular
concentration a larger degree of cell swelling is needed
to activate a given amount of organic osmolyte efflux
compared with cells that have normal or below normal
inorganic ion levels,’’ leading to the conclusion that
intracellular electrolyte level modifies the volume set
point. This interpretation is satisfying, if it is assumed
that activation is triggered first by a volume set point.
However, if we assume that activation is primarily
triggered by an ionic strength threshold, as shown in
trout red blood cells, the results will be identical: at
high intracellular electrolyte concentration a larger
degree of hypotonically induced swelling (i.e., a larger
amount of water diluting electrolytes) is needed to
SWELLING ACTIVATION OF TRANSPORT PATHWAYS
exposed to deep hypoxia, catecholamines are released
that stimulate erythrocyte Na1/H1 exchangers, leading
to an accumulation of electrolytes (NaCl) in erythrocytes and cell swelling (4, 14). This sequence of events,
by increasing O2 content of erythrocytes at low PO2 (7),
allows the fish to survive in deep hypoxia. A return to
normoxic conditions, which is accompanied by deactivation of Na1/H1 exchanges, is followed by a recovery of
electrolyte content and cell volume (14). The most
efficient way to recover volume and normal electrolyte
content is then to specifically extrude salts in excess by
the simultaneous functioning of the K1-Cl2 cotransport
and the Na1-K1 pump and to repress the taurine efflux
pathway. However, trout, a quite euryhaline fish, can
also be exposed to various salinities, leading to hyposmotic swelling of red blood cells and a decrease in the
intracellular electrolyte concentration. Then it makes
sense that the cell uses organic osmolytes such as
taurine to undergo volume regulation and simultaneously reduce the loss of electrolytes occurring via the
K1-Cl2 cotransporter (which is always activated by cell
swelling). It also makes sense that the signal that turns
on the taurine channel and damps the K1-Cl2 cotransport is the dilution of cell electrolytes.
Thus when cells are physiologically subjected to
isotonic and hypotonic swelling conditions, the presence of two pathways appears beneficial, since they
play a complementary role in the simultaneous regulation of cell volume and cell electrolyte content. Although most studies of RVD are performed on cells
hypotonically swollen, swelling of many epithelial cell
types is physiologically achieved by a net salt uptake
(isotonic swelling). Then it is likely that cell types other
than trout erythrocytes possess two RVD mechanisms.
This possibility is supported by the recent findings
obtained in a mammalian cell line (12). In these cells
the swelling-induced channel mediating taurine efflux
was inhibited when the intracellular electrolyte concentration was initially elevated. Despite this inhibition,
however, cells were still able to undergo an RVD,
suggesting involvement of another regulatory pathway.
Moreover, as proposed by Strange and collaborators
(12, 25, 43), this inhibition of the taurine channel by an
elevated electrolyte content would have a physiological
significance, i.e., to avoid a loss of organic solutes that
could cause electrolytes to rise to toxic levels.
Address for reprint requests: R. Motais, CEA (DBCM) and CNRS
(URA 1855), BP 68, 06238 Villefranche-sur-Mer Cedex, France.
Received 12 March 1998; accepted in final form 29 September 1998.
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