| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
, ionic
strength, and volume changes
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
 |
ABSTRACT |
|---|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
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
K+-Cl
cotransporter. Each was activated by a specific signal: the channel by
a decrease in intracellular ionic strength and the
K+-Cl
cotransporter by cell enlargement. A decrease in ionic strength also
affected
K+-Cl
cotransport activity, but by acting as a negative modulator of the
cotransport. Thus cells swollen by salt accumulation respond by
activating exclusively the
K+-Cl
cotransport, leading to a
Cl-dependent
K+ 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.
volume regulation; taurine; volume-activated transports; anion channel; potassium-chloride cotransport
| |
INTRODUCTION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
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
K+ and
Cl) 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 K+ and
Cl transport mechanisms
have been described: a
K+-Cl
cotransporter, separate K+ and
Cl channels, and a
K+/H+
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
Na+,
K+,
Cl, and taurine account for
up to 90% of trout erythrocyte osmolality, with the remaining
osmolality due to the presence of greatly or totally impermeant solutes
such as ATP, ADP, PO34, 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 K+,
Cl, 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
K+ and
Cl. 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
Cl-dependent pathway that
displays the characteristics of a
K+-Cl
cotransport (4, 37). Such an "isosmotic" swelling can be induced
by hormonal stimulation of an
Na+/H+
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
"Cl-dependent"
component and a
"Cl-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
Na+, choline, or
tetramethylammonium (TMA) (20, 37). However, although activation of the
taurine, K+, and
Cl transport systems will
tend to correct volume changes, the simultaneous activations of other
pathways may partly counteract RVD [net entry of
Na+ 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
K+ (6, 9, 29, 32) and diverse
organic solutes (15, 17, 21, 23, 30, 31, 46).
|
|
Thus trout erythrocytes possess multiple swelling-sensitive 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).
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 |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Materials. Ouabain, DIDS, and N-ethylmaleimide (NEM) were obtained from Sigma Chemical (St. Louis, MO), [methyl-14C]choline, 86Rb+, [14C]sorbitol, and 22Na+ 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
Cl-dependent
K+ 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
K+ 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-N'-3-propanesulfonic acid (pH 7.95, 320 mosmol/kgH2O).
In some experiments, Na+ was
replaced by 145 mM choline. For
Cl-free solutions,
Cl
was replaced by nitrate (NO3) or
methylsulfate (MeSO4). All
solutions contained ouabain to give a final concentration of
104 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-N'-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
NH+4 salts (0-50 mM) substituted for
0-50 mM Na+ salts. Cell
swelling results from an uptake of
NH+4 salts due to the simultaneous diffusion of
NH3 into the cell and the
transmembrane exchanges of anion (intracellular
OH/extracellular
Cl or
NO3), mediated
by the band 3 protein, which practically abolishes pH changes (<0.1
pH unit with 50 mM NH+4 salts).
3) Cells were suspended in isosmotic
(320 mosmol/kgH2O) saline
solutions containing 180 mM urea or 50 mM
NH+4 salts or mixtures of
NH+4 salts and urea. Osmolality was
maintained constant by replacing 90 mM
Na+ salts of the basic solution
with 180 mM urea or 50 mM NH+4 salts plus 40 mM N-methyl-D-glucamine salts or appropriate
mixtures, e.g., (in mM) 100 urea, 20 NH+4
salts, and 20 N-methyl-D-glucamine salts. Cell
swelling results from the penetration of urea and/or
NH+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 22Na+ (45 s), and a correction of 3.5% was applied for this to all calculations.
Transport measurements. Unidirectional K+ transport was measured using 86Rb+ as convenient congener. [14C]choline, [14C]taurine, [14C]sorbitol, and 22Na+ 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 Na+, 145 choline, 5 taurine, 5 sorbitol, and 4 K+.
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,
Cl, 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 + 0.5% BSA followed by 2 additional rinses without albumin). Swelling was subsequently induced by suspending the cells in a DIDS-free hypotonic medium.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Swelling-activated transports of
K+.
First, the effect of various degrees of swelling on
K+ 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 NH+4 salts in
replacement of Na+ salts. Such a
swelling, termed isosmotic, results from a net uptake of
NH+4 salts and osmotically obliged water (37). The media contained
Cl or
NO3 as anion. Isosmotic swelling
of cells in Cl-containing
media was followed by a quite linear increase in the ouabain-insensitive K+ influx
as a function of cell volume. By contrast, in
NO3containing media there is
no discernible influence of cell volume on the K+ flux. Thus trout erythrocytes
responded to isosmotic swelling by activating exclusively a
Cl-dependent
K+ 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
Cl (with
NO3), hyposmotic swelling was
followed by an increase in the ouabain-insensitive
K+ flux. The fluxes measured in
Cl-containing media were
substantially greater than those in
NO3-containing media, the
difference between the two curves corresponding to the
Cl-dependent component of
the K+ flux (Fig.
3).
|
|
-dependent
K+ component, considered a
K+-Cl
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 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
K+-Cl
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
Cl-independent
K+ 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
Cl-independent
K+ 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
Cl-independent
K+ flux. To investigate this
possibility further, two distinct experimental protocols were employed.
We compared the activation of the
Cl-independent
K+ 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).
|
|
-free,
NO3-containing media of
different tonicities, and the resulting
Cl-independent
K+ flux was measured. Cell volume
and Cl-independent
K+ flux are plotted as a function
of electrolyte dilution in Fig. 4A. An
electrolyte dilution of <10% had no discernible influence on
K+ 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
NO3-containing saline (320 mosmol/kgH2O) in which 90 mM
NaNO3 was replaced by urea,
NH4NO3,
or
urea-NH4NO3 mixtures (see MATERIALS AND
METHODS). 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
urea-NH4NO3 mixtures. Figure
4B shows that, at a constant cell
volume (40% increase in cell water relative to control erythrocytes), activation of the
Cl-independent
K+ 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 = 1)
or greater (dilution factor = 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 = 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, shows that the electrolyte set
point for activation of the
Cl-independent
K+ flux was very similar in the
two experiments. Because the concentration of cytoplasm-impermeant
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
Cl-dependent and
Cl-independent
K+ transport systems; the
transport of compounds as structurally diverse as amino acids (taurine)
and inorganic (Na+) 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 swollen in a
Cl-free,
NO3-containing saline,
K+ loss per hour was 33.81 ± 4.45 µmol/g dry cell solids and taurine loss was 37.97 ± 3.55 µmol/g dry cell solids, whereas
Na+ uptake was 29.10 ± 1.59 µmol/g dry cell solids (5 experiments). In other words, the net
Na+ uptake practically
counterbalanced the volume-regulatory
K+ loss mediated by the
Cl-independent component;
therefore, RVD in a Cl-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 Na+ (20).
-independent
K+ 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 (Na+) and organic
(choline) cations. These results, along with the data reported above
for the Cl-independent
K+ pathway, are shown in Fig.
5.
|
-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.
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 intracellular
Cl concentration (25). The
experiments described above were performed in
Cl-containing media, except
for those involved in the measurement of the
Cl-independent
K+ fluxes, which were performed in
Cl-free,
NO3-containing media. To test
whether a decrease in Cl
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
Na+ 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
Cl by
NO3 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 that taurine flux was similar in NO3 and
Cl media, i.e., when
intracellular Cl has been
totally replaced or only 40% diluted. Replacement of Cl 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 Cl. Because
Na+ and choline fluxes were
unaffected by the replacement of
Cl with
MeSO4 (not shown), activation of
these solute fluxes, like that of taurine, is dependent on a reduction
of cell electrolyte content.
|
-independent)
K+ 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
Cl-independent, but not the
Cl-dependent,
K+ 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 volume-dependent taurine transport
but also Na+, choline, sorbitol,
and Cl-independent
K+ transports in a similar manner.
Thus the pharmacological characteristics of all these pathways appear
strikingly similar.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
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, the specific intracellular signal(s) involved in its activation.
K+-Cl
cotransporter.
A Cl-dependent
K+ pathway with the
characteristics of a
K+-Cl
cotransporter (4, 6, 37) is activated in response to isosmotic and
hyposmotic swelling (Fig. 3). To define
Cl-dependent and
Cl-independent
K+ pathways,
NO3 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),
NO3 has been shown to be a less efficient substitute for Cl
than MeSO4 or sulfamate. However,
under the experimental conditions used in this study (maximum 40%
increase in cell water content), NO3 is a reasonably good
substitute, since the K+ fluxes
measured in NO3-containing media
were similar to or only very slightly higher than those in media
containing MeSO4 or sulfamate (not shown).
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
K+-Cl
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
K+ 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
K+-Cl
cotransport, with ionic strength acting as a modulator of the activated
cotransporter;
K+-Cl
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
K+-Cl
cotransport (8), suggesting that in trout erythrocytes dilution of
cytosolic impermeant compounds could be the parameter controlling activation of the
K+-Cl
cotransport. Moreover, at any particular dog red cell volume, decreases
in ionic strength diminished
K+-Cl
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
K+-Cl
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
K+-Cl
cotransport and the transport of structurally unrelated compounds such
as K+ (as a
Cl-independent component),
taurine, Na+, 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 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
Cl or
K+ concentration, rather than the
electrolyte concentration as a whole, exerts such a control. However,
total replacement of Cl by
NO3 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
Na+/H+
exchange or exposure to NH+4 salts,
indicating that the nature of cations accumulated in the cell
(Na+ in the presence of ouabain,
K+ in its absence, or
NH+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."
-independent component),
Na+, 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 DIDS-sensitive 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, DIDS- and 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
Cl 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 support the view of a direct
implication of trout band 3 in the formation of an anion-selective
channel permeable to structurally unrelated compounds (19, 36). The
hypothesis of a single route for all solutes raises the question of how
cations might permeate such channels. Electrophysiological studies of
swelling-activated, taurine-permeable anion channels indicate a low
conductivity to monovalent cations in cultured mammalian cells (26) but
a relatively high cation permeability in skate hepatocytes (25). The
"background Cl
channel" of hippocampal neurons is also an anion-selective channel that has a significant permeability to monovalent cations, and Franciolini and Nonner (16) proposed a permeation model in which a
cation interacts with an anion to move across the membrane. A similar
proposal has been made by Bursell and Kirk (6) for the swelling-induced
transport of cations in fish erythrocytes.
The mechanism by which cell ionic strength can activate the putative
channel remains to be defined. When trout band 3 was expressed in
oocytes, the resulting anion current was a sigmoidal function of the
level of band 3 expression, consistent with the view that the
conductance pathway was formed by, or required, multimeric arrangements
of the protein (13). In skate erythrocytes, osmotic swelling resulted
in an increase in the relative proportion of band 3 dimers and
tetramers in the membrane, leading to the proposal that band 3 aggregation may be involved in osmolyte channel formation/activation
(38). Thus it can be envisioned that a change in intracellular ionic
strength, by altering cytoskeletal architecture and/or
interaction between band 3 and the cytoskeleton, allows rearrangement
of band 3 in the membrane. Alternatively, or perhaps additionally,
changes in ionic strength could modify the activity of a protein
involved in channel regulation, since, according to Parker (40),
decreasing cell electrolytes alters the thermodynamic activity of
cytoplasmic proteins via electrolyte-macromolecule interaction.
Why two RVD mechanisms in a single cell?
The question arises concerning the reasons why a single cell possesses
two different regulatory volume decrease transport pathways
specifically activated by distinct stimuli: one that mediates
selectively a loss of electrolytes (KCl) and is turned on by the volume
increase and another that is turned on by the decrease in cell
electrolyte concentration and mediates essentially a loss of taurine
(the loss of electrolytes occurring via this pathway as
Cl-independent
K+, being practically
counterbalanced by an entry of electrolytes such as NaCl). Clearly,
when swelling results from an uptake of electrolytes, the best way for
a cell to undergo volume regulation is to activate the pathway that
selectively mediates a loss of electrolytes. Conversely, when swelling
results from an entry of water, which dilutes cell electrolytes, the
best way is then to activate the taurine pathway by using organic
osmolytes to recover volume and by preventing an additional decrease of
electrolyte level.
cotransport and the
Na+-K+
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
K+-Cl
cotransporter (which is always activated by cell swelling). It also
makes sense that the signal that turns on the taurine channel and damps
the
K+-Cl
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.
| |
FOOTNOTES |
|---|
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. §1734 solely to indicate this fact.
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.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
1.
Armsby, C. C.,
C. Brugnara,
and
S. L. Alper.
Cation transport in mouse erythrocytes: role of K-Cl cotransport in regulatory volume decrease
Am. J. Physiol.
268 (Cell Physiol. 37):
C894-C902,
1995
2.
Banderali, U.,
and
G. Roy.
Anion channels for amino acids in MDCK cells.
Am. J. Physiol.
263 (Cell Physiol. 32):
C1200-C1207,
1992
3.
Berenbrink, M.,
Y. R. Weaver,
and
A. R. Cossins.
Defining the volume dependence of multiple K flux pathways of trout red blood cells.
Am. J. Physiol.
272 (Cell Physiol. 41):
C1099-C1111,
1997
4.
Borgese, F.,
F. Garcia-Romeu,
and
R. Motais.
Control of cell volume and ion transport by 
-adrenergic catecholamines in erythrocytes of rainbow trout Salmo gairdneri.
J. Physiol. (Lond.)
382:
123-144,
1987
5.
Borgese, F.,
R. Motais,
and
F. Garcia-Romeu.
Regulation of Cl-dependent K transport by oxy-deoxyhemoglobin transitions in trout red cells.
Biochim. Biophys. Acta
1066:
252-256,
1991[Medline].
6.
Bursell, J. D. H.,
and
K. Kirk.
Swelling-activated K+ transport via two functionally distinct pathways in eel erythrocytes.
Am. J. Physiol.
270 (Regulatory Integrative Comp. Physiol. 39):
R61-R70,
1996
7.
Claireaux, G.,
S. Thomas,
B. Fievet,
and
R. Motais.
Adaptative respiratory responses to acute hypoxia. II. Blood oxygen carrying properties during hypoxia.
Respir. Physiol.
74:
91-98,
1988[Medline].
8.
Colclausure, G. C.,
and
J. C. Parker.
Cytosolic protein concentration is the primary volume signal for swelling-induced KCl cotransport in dog red cells.
J. Gen. Physiol.
100:
1-10,
1992
9.
Cossins, A. R.,
Y. R. Weaver,
G. Lykkeboe,
and
O. B. Nielsen.
Role of protein phosphorylation in control of K flux pathways of trout red blood cells.
Am. J. Physiol.
267 (Cell Physiol. 36):
C1641-C1650,
1994
10.
Dunham, P. B.,
J. Klimczak,
and
P. J. Logue.
Swelling activation of K-Cl cotransport in LK sheep erythrocytes: a three state process.
J. Gen. Physiol.
101:
733-765,
1993
11.
Egée, S.,
B. J. Harvey,
and
S. Thomas.
Volume-activated DIDS-sensitive whole-cell chloride currents in trout red blood cells.
J. Physiol. (Lond.)
504:
57-63,
1997
12.
Emma, F.,
M. McManus,
and
K. Strange.
Intracellular electrolytes regulate the volume set point of the organic osmolyte/anion channel VSOAC.
Am. J. Physiol.
272 (Cell Physiol. 41):
C1766-C1775,
1997
13.
Fiévet, B.,
N. Gabillat,
F. Borgese,
and
R. Motais.
Expression of band 3 anion exchanger induces chloride current and taurine transport: structure-function analysis.
EMBO J.
14:
5158-5169,
1995[Medline].
14.
Fiévet, B.,
R. Motais,
and
S. Thomas.
Role of adrenergic-dependent H+ release from red cells in acidosis induced by hypoxia in trout.
Am. J. Physiol.
252 (Regulatory Integrative Comp. Physiol. 21):
R269-R275,
1987
14a.
Fiévet, B.,
F. Perset,
N. Gabillat,
H. Guizouarn,
F. Borgese,
P. Ripoche,
and
R. Motais.
Transport of uncharged solutes in Xenopus oocytes expressing red cell anion exchangers (AE1s).
Proc. Natl. Acad. Sci. USA
95:
10996-11001,
1998
15.
Fincham, D. A.,
M. W. Wolowyk,
and
J. D. Young.
Volume-sensitive taurine transport in fish erythrocytes.
J. Membr. Biol.
96:
45-56,
1987[Medline].
16.
Franciolini, F.,
and
W. Nonner.
Anion and cation permeability of a chloride channel in rat hippocampal neurons.
J. Gen. Physiol.
90:
453-478,
1987
17.
Fugelli, K.,
and
S. M. Thoroed.
Taurine transport associated with cell volume regulation in flounder erythrocytes under anisosmotic conditions.
J. Physiol. (Lond.)
37:
245-261,
1986.
18.
García, J. J.,
R. Sanchez-Olea,
and
H. Pasantes-Morales.
Taurine release associated to volume regulation in rabbit lymphocytes.
J. Cell. Biochem.
45:
207-212,
1991[Medline].
19.
Garcia-Romeu, F.,
F. Borgese,
H. Guizouarn,
B. Fievet,
and
R. Motais.
A role for the anion exchanger AE1 (band 3 protein) in cell volume regulation.
Cell. Mol. Biol.
42:
985-994,
1996[Medline].
20.
Garcia-Romeu, F.,
A. R. Cossins,
and
R. Motais.
Cell volume regulation by trout erythrocytes: characteristics of the transport systems activated by hypotonic swelling.
J. Physiol. (Lond.)
440:
547-567,
1991
21.
Goldstein, L.,
and
S. R. Brill.
Volume-activated taurine efflux from skate erythrocytes: possible band 3 involvement.
Am. J. Physiol.
260 (Regulatory Integrative Comp. Physiol. 29):
R1014-R1020,
1991
22.
Goldstein, L.,
and
E. M. Davis.
Taurine, betaine and inositol share a volume-sensitive transporter in skate erythrocyte cell membrane.
Am. J. Physiol.
267 (Regulatory Integrative Comp. Physiol. 36):
R426-R431,
1994
23.
Haynes, J. K.,
and
L. Goldstein.
Volume-sensitive amino acid transport in erythrocytes of the little skate, Raja erinacea.
Am. J. Physiol.
265 (Regulatory Integrative Comp. Physiol. 34):
R173-R179,
1993
24.
Hoffman, E.,
and
L. O. Simonsen.
Membrane mechanisms in volume and pH regulation in vertebrate cells.
Physiol. Rev.
69:
315-382,
1989
25.
Jackson, P. S.,
K. Churchwell,
N. Ballatori,
J. L. Boyer,
and
K. Strange.
Swelling-activated anion conductance in skate hepatocytes: regulation by cell Cl and ATP.
Am. J. Physiol.
270 (Cell Physiol. 39):
C57-C66,
1996
26.
Jackson, P. S.,
and
K. Strange.
Volume-sensitive anion channels mediate swelling-activated inositol and taurine efflux.
Am. J. Physiol.
265 (Cell Physiol. 34):
C1489-C1500,
1993
27.
Jennings, M. L.,
and
N. Al-Rohil.
Kinetics of activation and inactivation of swelling-stimulated KCl cotransport. Volume-sensitive parameter is the rate constant for inactivation.
J. Gen. Physiol.
95:
1021-1040,
1990
28.
Jensen, F. B.
Influence of haemoglobin conformation, nitrite and eicosanoids on the K transport across the carp red blood cell membrane.
J. Exp. Biol.
171:
349-371,
1992
29.
Jensen, F. B.
Regulatory volume decrease in carp red blood cells: mechanisms and oxygenation-dependency of volume-activated potassium and amino acid transport.
J. Exp. Biol.
198:
155-165,
1994[Abstract].
30.
Joyner, S. E.,
and
K. Kirk.
Two pathways for choline transport in eel erythrocytes: a saturable carrier and a volume-activated channel.
Am. J. Physiol.
267 (Regulatory Integrative Comp. Physiol. 36):
R773-R779,
1994
31.
Kirk, K.,
J. C. Ellory,
and
J. D. Young.
Transport of organic substrates via a volume-activated channel.
J. Biol. Chem.
267:
23475-23478,
1992
32.
Lauf, P. K.
Evidence for a chloride-dependent potassium and water transport induced by hyposmotic stress in erythrocytes of the marine teleost, Opsanus tao.
J. Comp. Physiol.
146:
9-16,
1983.
33.
Lauf, P. K.
Foreign anions modulate volume set point of sheep erythrocyte K-Cl cotransport.
Am. J. Physiol.
260 (Cell Physiol. 29):
C917-C932,
1991
34.
Lauf, P. K.,
J. Bauer,
N. C. Adragna,
H. Fujise,
A. M. M. Zade-Oppen,
K. H. Ryu,
and
E. Delpire.
Erythrocyte KCl cotransport: properties and regulation.
Am. J. Physiol.
263 (Cell Physiol. 32):
C917-C932,
1992
35.
Lewis, R. A.,
J. D. H. Bursell,
and
K. Kirk.
Anion-selectivity of the swelling-activated osmolyte channel in eel erythrocytes.
J. Membr. Biol.
149:
103-111,
1996[Medline].
36.
Motais, R.,
B. Fievet,
F. Borgese,
and
F. Garcia-Romeu.
Association of the band 3 protein with a volume-activated anion and amino acid channel: a molecular approach.
J. Exp. Biol.
200:
361-367,
1996[Abstract].
37.
Motais, R.,
H. Guizouarn,
and
F. Garcia-Romeu.
Red cell volume regulation: the pivotal role of ionic strength in controlling swelling-dependent transport systems.
Biochim. Biophys. Acta
1075:
169-180,
1991[Medline].
38.
Musch, M. W.,
E. M. Davis,
and
L. Goldstein.
Oligomeric forms of skate erythrocyte band 3. Effect of volume expansion.
J. Biol. Chem.
269:
19683-19686,
1994
39.
Nielsen, O. B.,
G. Lykkeboe,
and
A. R. Cossins.
Oxygenation-activated K fluxes in trout red blood cells.
Am. J. Physiol.
263 (Cell Physiol. 32):
C1057-C1064,
1992
40.
Parker, J. C.
In defense of cell volume?
Am. J. Physiol.
265 (Cell Physiol. 34):
C1191-C1200,
1993
41.
Parker, J. C.,
P. B. Dunham,
and
A. P. Minton.
Effect of ionic strength on the regulation of Na/H exchange and K-Cl cotransport in dog red blood cells.
J. Gen. Physiol.
105:
677-699,
1995
42.
Sarkadi, B.,
and
J. C. Parker.
Activation of ion transport pathways by changes in cell volume.
Biochim. Biophys. Acta
1071:
407-427,
1991[Medline].
43.
Strange, K.
Are all cell volume changes the same?
News Physiol. Sci.
9:
223-228,
1994.
44.
Strange, K.,
F. Emma,
and
P. S. Jackson.
Cellular and molecular physiology of volume-sensitive anion channels.
Am. J. Physiol.
270 (Cell Physiol. 39):
C711-C730,
1996
45.
Strange, K.,
and
P. S. Jackson.
Swelling-activated organic osmolyte efflux: a new role for anion channels.
Kidney Int.
48:
994-1003,
1995[Medline].
46.
Thoroed, S. M.,
and
K. Fugelli.
The Na-independent taurine influx in flounder erythrocytes and its association with the volume regulatory taurine efflux.
J. Exp. Biol.
186:
245-268,
1994[Abstract].
This article has been cited by other articles:
|
|
 |
|
  S. K. Parks, M. Tresguerres, and G. G. Goss Cellular mechanisms of Cl- transport in trout gill mitochondrion-rich cells Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2009; 296(4): R1161 - R1169. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. K. Hoffmann, I. H. Lambert, and S. F. Pedersen Physiology of Cell Volume Regulation in Vertebrates Physiol Rev, January 1, 2009; 89(1): 193 - 277. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Lytle and T. McManus Coordinate modulation of Na-K-2Cl cotransport and K-Cl cotransport by cell volume and chloride Am J Physiol Cell Physiol, November 1, 2002; 283(5): C1422 - C1431. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. Goss, L. Jiang, D. H. Vandorpe, D. Kieller, M. N. Chernova, M. Robertson, and S. L. Alper Role of JNK in hypertonic activation of Cl--dependent Na+/H+ exchange in Xenopus oocytes Am J Physiol Cell Physiol, December 1, 2001; 281(6): C1978 - C1990. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.-L. T. Koomoa, M. W. Musch, A. Vaz MacLean, and L. Goldstein Volume-activated trimethylamine oxide efflux in red blood cells of spiny dogfish (Squalus acanthias) Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2001; 281(3): R803 - R810. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Wittels, E. M. Hubert, M. W. Musch, and L. Goldstein Osmolyte channel regulation by ionic strength in skate RBC Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2000; 279(1): R69 - R76. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Bize, B. Guvenc, A. Robb, G. Buchbinder, and C. Brugnara Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes Am J Physiol Cell Physiol, November 1, 1999; 277(5): C926 - C936. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Brochiero, B. Wallendorf, D. Gagnon, R. Laprade, and J.-Y. Lapointe Cloning of rabbit Kir6.1, SUR2A, and SUR2B: possible candidates for a renal KATP channel Am J Physiol Renal Physiol, February 1, 2002; 282(2): F289 - F300. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
) or
NO
) as permeant anion.
NH+4 salts were added in replacement of same
amount of Na+ 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 K+ flux
(measured as Rb+ influx) increased
as a function of cell volume in
Cl
). Data were
obtained from Fig. 
; right
ordinate) and Cl
