Physiological significance of volume-regulatory
transporters
W. Charles O'Neill
Am J Physiol Cell Physiol 276:995-1011, 1999.
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invited review
Physiological significance of
volume-regulatory transporters
W. CHARLES O’NEILL
Renal Division, Department of Medicine, and Department of Physiology,
Emory University School of Medicine, Atlanta, Georgia 30322
cell volume; regulatory volume increase; regulatory volume decrease;
sodium-potassium-chloride cotransporter; sodium-proton antiporter
THE NECESSITY TO RAPIDLY REGULATE volume after shrinkage or swelling is obvious in cells from lower organisms,
which are continually exposed to osmotic stress. Yet, in
mammals, where osmolarity of the internal milieu is
carefully regulated, cells also exhibit rapid volumeregulatory responses. Are volume-regulatory pathways
in mammalian cells merely a vestige that have no
significant physiological role? The answer to this question lies in the realization that mammalian cells are
still subject to volume changes, despite a relatively
constant osmolarity. This is most apparent in transporting epithelia, where slight changes in the large apical
and basolateral fluxes could lead to rapid changes in
cell volume. Furthermore, even if cell volume were not
threatened, mechanisms are still needed to alter cell
volume, such as during hypertrophy and atrophy, apoptosis, and differentiation. Thus mammalian cells clearly
require volume-regulatory mechanisms, despite their
isosmotic existence. Although the principal volumeregulatory transporters have been identified and characterized, their activation by changes in cell volume is
poorly understood and their physiological role is largely
unexplored. This review focuses on the salient points of
volume-regulatory ion transporters that have direct
relevance to mammalian physiology, with particular
emphasis on the distinction between anisosmotic and
isosmotic volume regulation, the role of intracellular
Cl⫺, and the need for cell volume regulation in vivo.
Space does not permit a detailed discussion of the
intracellular sensing and signaling mechanisms, and
readers are referred to a recent, comprehensive review
on this subject (94).
DETERMINANTS OF CELL VOLUME
In discussing cell volume it is important to distinguish between factors that determine steady-state volume and mechanisms that correct acute perturbations
in steady-state volume. This has been a source of
confusion, and it is best to consider these as entirely
separate processes. Transporters responsible for correcting acute changes in cell volume are usually not active
at steady-state volume. Thus steady-state cell volume
is not a balance between their ongoing, opposing actions. Rather, cells appear to establish a steady-state
volume and then activate volume-regulatory transporters only when cell volume deviates from this ‘‘set point.’’
0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society
C995
Downloaded from ajpcell.physiology.org on October 17, 2007
O’Neill, W. Charles. Physiological significance of volume-regulatory
transporters. Am. J. Physiol. 276 (Cell Physiol. 45): C995–C1011, 1999.—
Research over the past 25 years has identified specific ion transporters
and channels that are activated by acute changes in cell volume and that
serve to restore steady-state volume. The mechanism by which cells sense
changes in cell volume and activate the appropriate transporters remains
a mystery, but recent studies are providing important clues. A curious
aspect of volume regulation in mammalian cells is that it is often absent
or incomplete in anisosmotic media, whereas complete volume regulation
is observed with isosmotic shrinkage and swelling. The basis for this may
lie in an important role of intracellular Cl⫺ in controlling volumeregulatory transporters. This is physiologically relevant, since the principal threat to cell volume in vivo is not changes in extracellular osmolarity
but rather changes in the cellular content of osmotically active molecules.
Volume-regulatory transporters are also closely linked to cell growth and
metabolism, producing requisite changes in cell volume that may also
signal subsequent growth and metabolic events. Thus, despite the
relatively constant osmolarity in mammals, volume-regulatory transporters have important roles in mammalian physiology.
C996
INVITED REVIEW
This is far more efficient than having opposing volumeregulatory transporters continuously active.
Steady-State Cell Volume
The steady-state disequilibrium that maintains cell
volume can be threatened by changes in extracellular
osmolarity, but a far more common threat is a change in
the cellular content of osmotically active molecules,
so-called isosmotic volume change. Cell volume can be
viewed as a balance between the osmotic effects of high
concentrations of impermeant anions and low concentrations of Cl⫺ within cells, and changes in either
without offsetting changes in the other can alter cell
volume. Changes in the former occur through synthesis, degradation, and fluxes, whereas changes in intracellular Cl⫺ concentration ([Cl⫺]i ) arise from altered
fluxes or membrane potential. Do the mechanisms
described above for maintenance of steady-state cell
volume protect cells from acute changes in cell volume
as well? Cell shrinkage can reduce K⫹ conductance (37,
166), which should increase cell volume. However, the
response would be slow and does not appear to contribute substantially to volume recovery (166). Although
cell swelling has the opposite effect of increasing K⫹
conductance, this occurs through channels distinct
from those responsible for basal K⫹ conductance.
Cells instead recruit additional mechanisms to correct acute deviations in their volume. These processes,
termed regulatory volume increase (RVI) and regulatory volume decrease (RVD), occur through activation
of specific transporters in the plasma membrane that
mediate net fluxes of osmotically active molecules (and
therefore water). Not surprisingly, both [Cl⫺]i and
macromolecule concentration play key roles in governing these volume-regulatory responses. A further adaptation to osmotic stress is the accumulation of organic
osmolytes such as sorbitol, sn-glycero-3-phosphorylcholine, betaine, and taurine, which occurs in response to
increased ionic strength rather than cell shrinkage (70,
207) and therefore is not truly a volume-regulatory
mechanism. Instead, these compounds replace high
intracellular salt concentrations that can perturb protein structure and impair enzyme function (18, 70),
enabling cells to function in high osmolarity, such as in
the renal medulla. Organic osmolytes also appear in
the brain during chronic hypernatremia and comprise
the so-called idiogenic osmoles (195). Organic osmolytes do have an impact on volume regulation, because
they must be rapidly removed when normal osmolarity
is restored in order to prevent cell swelling.
The mechanism by which cells sense changes in their
volume is unknown but clearly is not through changes
in osmolarity, ionic strength, or ion concentration, since
isosmotic volume changes also activate RVI and RVD.
Cell volume could be sensed chemically as changes in
the intracellular concentration of impermeant molecules or mechanically through some form of stretch
receptor. The former possibility, proposed by Minton et
al. (129) and based on the concept of macromolecular
crowding, is supported by direct experimental data.
According to this theory, it is not cell volume per se that
is regulated but rather the state of intracellular water.
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Mere existence in an isosmotic environment represents a continual threat to cell volume and integrity
due to the large concentration of impermeant, negatively charged molecules within cells, such as proteins,
nucleic acids, and other organophosphates. In addition
to the osmotic force generated by the molecules themselves, there is an additional osmotic force due to an
asymmetrical distribution of permeant ions created by
the impermeant anions. Known as the Gibbs-Donnan
equilibrium (65, 115), this is actually a steady-state
disequilibrium. Unless the osmotic gradient is counterbalanced, there would be continuous influx of water
and ions. In theory this counterbalancing could be
accomplished by hydrostatic pressure, but it is highly
unlikely that the plasma membrane can generate the
tension required to balance the Donnan effect (115). In
the absence of an opposing force, cells must either
restrict permeability or actively extrude fluid. Because
mammalian cells are generally quite permeable to
water and there is no evidence for active transport of
water, it must be solute that is actively transported and
has restricted diffusion. This is illustrated by ionophores and other compounds that increase membrane
permeability to Na⫹ and K⫹, which produce cell swelling without altering the Gibbs-Donnan ‘‘equilibrium.’’
In addition, this confirms that membrane tension is not
responsible for maintaining steady-state cell volume.
Another simple maneuver that causes cell swelling is
replacement of extracellular Na⫹ with K⫹. Because
neither the Gibbs-Donnan equilibrium nor other osmotic forces are altered (115), elimination of the outward K⫹ gradient is directly implicated in cell swelling.
By virtue of the selective permeability of the plasma
membrane to K⫹ over Na⫹, this gradient results in an
outward K⫹ current and a negative membrane potential that dictates a low intracellular concentration of
permeant anions, primarily Cl⫺. The source of energy is
ultimately the Na⫹-K⫹ pump, explaining why its inhibition often leads to cell swelling. This ability to extrude
Cl⫺ (coupled with extrusion of Na⫹ by the Na⫹-K⫹
pump) appears to be the principal mechanism that
maintains steady-state cell volume. In cells with high
anion permeability and low membrane potential, such
as erythrocytes, it is the net cation extrusion by the
Na⫹-K⫹ pump coupled with low membrane permeability to both Na⫹ and K⫹ that limits Cl⫺ entry. In
erythrocytes from carnivores, which lack Na⫹-K⫹
pumps, net cation extrusion occurs through combined
action of Ca2⫹ pumps (Ca2⫹-ATPases) and Na⫹/Ca2⫹
exchangers. The former generates a very large inward
Ca2⫹ gradient that is then used to pump out Na⫹ via the
latter (127). Cl⫺ accompanies Na⫹ efflux by virtue of the
electrogenicity of both transporters.
Acute Changes in Steady-State Volume
INVITED REVIEW
Consequences of Cell Shrinkage and Swelling
What exactly are the detrimental effects of changes
in cell volume that volume-regulatory mechanisms are
designed to prevent? Aside from extreme results such
as cell lysis, this is an area that has received little
attention. The most obvious effects of changing cell
volume are mechanical, since the function of many cells
depends on their architecture. Close examination reveals blebbing of the plasma membrane after severe
hypotonic swelling (217). In addition to direct distortion, shrinkage and swelling also induce rapid changes
in the actin cytoskeleton (Ref. 66 and P. B. Perry and
W. C. O’Neill, unpublished observations), presumably
as a protective measure to ease tension on the plasma
membrane. At the tissue level, swelling of parenchymal
cells can compromise regional blood flow, possibly aggravated by swelling of vascular endothelium and smooth
muscle (17). Due to its rigid confines, the brain is the
predominant site of morbidity from hyponatremia and
hypernatremia, and, accordingly, is the tissue that
exhibits the most complete volume regulation. At the
cellular level, swelling and shrinkage could alter metabolic reactions through changes in the concentrations
of enzymes and substrates. In this regard, cell swelling
is more of a problem than cell shrinkage, which is well
tolerated. For instance, in alveolar macrophages, hypotonic swelling decreases O2 consumption and increases
lactate production while there is little effect of hypertonic shrinkage (169). In mouse L cells, halving cell
volume with extracellular sorbitol does not alter growth
or the oxidation or metabolism of glucose (119). In both
these studies and in other studies, severe hypertonic
shrinkage (osmolarity ⬎700 mosM) does impair cell
growth and metabolism, but this appears to be an effect
of increased ionic strength rather than cell shrinkage
(18, 70).
REGULATORY VOLUME INCREASE
Volume-Regulatory Transporters
A seemingly simple way for cells to increase volume
after shrinkage is by the opening of Na⫹ channels, with
Cl⫺ following, but this appears to be a rare event (214).
Instead, volume regulation occurs primarily through
electroneutral transporters, principally the Na⫹-K⫹2Cl⫺ cotransporter (NKCC1) and the Na⫹/H⫹ antiporter (NHE1). NKCC1 mediates coupled influx of Na⫹,
K⫹, and Cl⫺ and is found in virtually all cells. It is
distinguished by its sensitivity to sulfamoylbenzoic
acid derivatives, the so-called ‘‘loop’’ diuretics such as
furosemide and bumetanide (56). NHE1, the ubiquitous NHE isoform, produces a net inward movement of
ions by coupling with Cl⫺/HCO3⫺ exchange (Fig. 1). The
combined effect is influx of Na⫹ and Cl⫺ with efflux of
H⫹ and HCO3⫺. The latter two combine to form CO2,
which diffuses back into the cell to regenerate H⫹ and
HCO3⫺. The net result is influx of NaCl. The Na⫹-K⫹
pump is a necessary participant in RVI, exchanging
Na⫹ for K⫹. The pump also provides the thermodynamic energy for RVI by maintaining a low intracellular Na⫹ concentration and, indirectly, a low [Cl⫺]i.
Amino acid uptake can also contribute to RVI (30), but
this is probably minor at normal extracellular amino
acid concentrations.
The mechanism by which cell shrinkage activates
NKCC1 or NHE1 is unknown. Because activation
occurs within minutes, it cannot be due to increased
synthesis of transporters. Current data suggest that
protein phosphorylation is involved. Activation of
NKCC1 by shrinkage is associated with phosphorylation of the transporter (on serine and threonine, but not
tyrosine) and can be blocked by kinase inhibitors and
mimicked by inhibitors of protein phosphatases (90,
163). Although these agents have a similar effect on
NHE1 activity (9), cell shrinkage does not increase
phosphorylation of the transporter (53). The data imply
the existence of a volume-sensitive protein kinase (or
more than one) that phosphorylates NKCC1 and a
Fig. 1. Transporters involved in regulatory volume increase. NKCC1,
Na⫹-K⫹-2Cl⫺ transporter; NHE1, Na⫹/H⫹ antiporter.
Downloaded from ajpcell.physiology.org on October 17, 2007
In erythrocytes, volume-regulatory transporters can be
activated in the absence of changes in cell volume by
altering intracellular protein concentration or by adding agents that perturb protein hydration (157). These
findings have been extended to nonerythroid cells,
specifically renal tubular cells (83) and barnacle muscle
(200). The question that remains is the nature of the
sensor for macromolecular crowding. Mechanical sensing of cell volume is an attractive theory, since cell
structure is directly threatened, but the evidence is
indirect. Agents that perturb the actin cytoskeleton can
inhibit RVD (24, 25, 42, 49, 184), and channels activated directly by membrane stretch can also be activated by cell swelling (176). Mechanical sensing of
changes in cell volume remains speculative since no
role for the cytoskeleton has been demonstrated in RVI,
and the role of stretch-activated channels in RVD
remains unproven. The issue of how cells sense their
volume is far from resolved, and it is quite possible that
both chemical and mechanical sensors are employed.
C997
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INVITED REVIEW
Table 1. Volume-regulatory response to hypertonic shrinkage in various mammalian tissues and cells
Cell or Tissue
Transporter
No
No
No
Yes
No
No
No
No
No
No
Yes
No
No
No
Yes
No
Yes
No
Yes
Osmotic Agent
Mannitol
NaCl
NaCl
NHE
NaCl
NaCl
NaCl, sucrose
NaCl
NaCl
NaCl
?
NKCC, NHE
NHE
?
NaCl
NaCl
NaCl
NaCl, mannitol
Mannitol
Mannitol
Mannitol
NaCl
Raffinose
Raffinose
OMCD (rabbit)
IMCD (rat)
Yes
Yes
NHE1
NKCC, NHE
Sucrose
NaCl
IMCD (rat)
Yes
NHE
Sucrose
MDCK cells
MDCK
MDCK
MDCK
OK cells
IMCD-3 cells
Other epithelia
Eccrine (monkey)
No
Yes
No
No
No
Yes
No
NKCC
Sucrose
Lacrimal acinar (rat)
Jejunal villus cells (guinea
pig)
Jejunal crypts (guinea pig)
Salivary acinar cells (rat)
Nervous system
Whole brain (rabbit)
Whole brain (rabbit)
Whole brain (rat)
Neurons, ganglia (mouse)
Nerve terminals, brain (rat)
Astrocytes (rat)
Astrocytes (rat)
Astrocytes (rat)
Neuroblastoma
C6 glioma cells
C6 glioma cells
C6 glioma cells
C6 glioma cells
Mesenchymal
Osteosarcoma
Chondrocytes
Gingival fibroblasts
Fibroblasts, human skin
Myogenic L6 cells
Skeletal muscle (dog)
Skeletal muscle (rabbit)
Yes
Yes
NKCC, NHE, amino acid
NKCC
Mannitol
NaCl
Yes
No
NKCC
NaCl
NaCl
Skeletal muscle (rabbit)
CHO cells
HeLa cells
Cardiovascular
Heart (cat)
Lung (rabbit)
No
No
No
Glucose
NaCl
Mannitol
No
No
Sucrose
NaCl
Yes
Yes
Yes
No
Yes
No
No
Yes
No
No
Yes
Yes
No
No
No
No
Yes
No
No
Yes
NaCl ⫹ osmolyte
?
?
NKCC
?
NKCC, NHE
? Amino acid
Mannitol
NaCl
Balanced salt
Mannitol
NaCl, NMDG, sucrose
NaCl
NaCl, sucrose
Glucose
NaCl, sucrose, mannitol
Choline chloride
Sucrose
Mannitol
NaCl
Sucrose
NaCl
NaCl, mannitol
NaCl
NaCl
Mannitol
Sodium gluconate
NaCl, sucrose
D-glucose
Sucrose
Sucrose
Sucrose, NaCl
NaCl
Notes
Some RVI over many hours
Some RVI over many hours
Neonatal
Some RVI over many hours
Only with gradual shrinkage
Requires ADH, cAMP
With or without ADH
Requires butyrate
Principal cells
Intercalated cells (subpopulation)
Faster with ADH
Requires stimulation with
cAMP
Requires stimulation with
cAMP
Requires butyrate
Mannitol in isosmotic medium
Reference
216
158
165
186
*
50
73
102
67
104
104
88
44
118
202
202
135
197
197
43
54
201
219
170
174
189
130
220
Requires stimulation with
cAMP
NHE in presence of CO2 /HCO3
206
But not with mannitol
138
168
Choline in isosmotic medium
Slow, incomplete
Declining baseline
Requires additives
Additives present
Occasional RVI in situ
Slow, amino acids required
Minimal RVI; no RVI with
sucrose
Mannitol in isosmotic medium
Sucrose in isosmotic medium
32
116
192
3
29
75
4
87
141a
36
114
85
133
124
21
86
64
10
30
185
68
192
3
179
205
151
213
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Hematopoietic
Erythrocytes (dog)
Erythrocytes (dog)
Erythrocytes (human
neonatal)
Erythrocytes (human)
Erythrocytes (guinea pig)
Lymphocytes (human)
Lymphocytes (human)
Platelets (human)
HL-60 cells
Renal epithelia
Proximal tubule (rabbit)
Proximal tubule (rabbit)
Proximal tubule (rabbit)
Proximal tubule (rabbit)
Proximal tubule (rabbit)
mTAL (mouse)
cTAL (mouse)
CCD (rabbit)
CCD (rabbit)
CCD (rabbit)
RVI
C999
INVITED REVIEW
Table 1.—Continued
Cell or Tissue
RVI
Smooth muscle cells (rat
aorta)
Endothelial cells (BAEC)
Endothelial cells (BPAEC)
Endothelial cells (HUVEC)
Endothelial cells (BAEC)
Lung endothelium (rabbit)
Cardiac myocytes (rabbit)
Gastrointestinal
Perfused liver (rat)
Isolated hepatocytes (rat)
Isolated hepatocytes (rat)
Cultured hepatocytes (rat)
Pancreatic B cells (rat)
Pancreatic B cells (mouse)
Ehrlich ascites cells
No
Sucrose
150
No
No
No
Yes
No
No
Sucrose, NaCl
Mannitol
Mannitol
Sucrose
NaCl
Mannitol
143
187
120
141
213
33
Yes
No
No
Yes
Yes
No
No
Transporter
NKCC
? NHE
Na⫹ channels
NKCC, NHE
Osmotic Agent
Raffinose
Sucrose
NaCl
Sucrose
Mannitol?
NaCl
NaCl, sucrose
Notes
Mannitol in isosmotic medium
Reference
96
23
5
214
128
35
101
regulatory protein that activates NHE1. Hopefully, the
recent demonstration of a volume-sensitive kinase that
phosphorylates NKCC1 in vitro will lead to identification of this kinase (145). Although the identity of this
kinase is unknown, other volume-sensitive kinases
have been tentatively identified. One of these is myosin
light chain kinase (MLCK), which is activated by
shrinkage in vascular endothelial and smooth muscle
cells (89), mesangial cells (203), and glial cells (188).
Inhibition of MLCK blocks activation of NKCC1 in
shrunken endothelial cells (89) and activation of NHE1
in shrunken astrocytes (188). However, MLCK is not
the volume-sensitive kinase that phosphorylates
NKCC1, and the mechanism by which myosin light
chain phosphorylation influences NKCC1 and NHE1 is
unknown. Hypertonicity increases phosphorylation of
stress-activated protein kinases and mitogen-activated
protein (MAP) kinases (45, 69). It is not known whether
this is an effect of hypertonicity or cell shrinkage, and
phosphorylation of MAP kinase also occurs after hypotonicity, suggesting a nonspecific stress response rather
than a specific response to cell volume. However, phosphorylation and activation of the tyrosine kinases
p59fgr and p56/59hck occurred in neutrophils shrunken
both hypertonically and isosmotically (91), directly
implicating cell shrinkage.
Hypertonic vs. Isosmotic Shrinkage
It is important to distinguish between these two
fundamentally different types of cell shrinkage because
the response frequently differs, a fact that is often not
appreciated. Hypertonic shrinkage removes only water
from cells, whereas isosmotic shrinkage results from
loss of osmotically active molecules, usually KCl. Examples of isosmotic shrinkage include returning cells to
isotonic medium after volume regulation in hypotonic
medium, substituting external Na⫹ or Cl⫺ with impermeant molecules, and increasing intracellular Ca2⫹
concentration ([Ca2⫹]i ) to open K⫹ and Cl⫺ channels.
RVI almost always occurs after isosmotic shrinkage but
is infrequent after hypertonic shrinkage. Although
occasionally noted, absence of hypertonic RVI has
largely been ignored and never systematically examined. Table 1 is an attempt at a complete listing of
mammalian cells and tissues in which hypertonic RVI
has been examined. For this purpose, RVI was defined
as a significant volume increase occurring within 1 h.
Although hypertonic RVI does correlate somewhat with
tissue type or origin, being more common in glial,
neural, and epithelial cells, there are clearly discrepancies between studies on the same tissue or cell type.
Absence of hypertonic RVI cannot be explained by lack
of volume-regulatory transporters, because most of the
cells listed undergo RVI after isosmotic shrinkage. One
exception is mature human erythrocytes, which do
have a low complement of transporters.
Can thermodynamics explain the lack of hypertonic
RVI? When hypertonicity is produced with an impermeant molecule such as sucrose or mannitol, the concentration of extracellular ions is unchanged while intracellular ions increase in concentration, thereby reducing
the driving force for influx. This can actually lead to
further cell shrinkage (101) and is accentuated by the
occasional use of impermeant molecules in place of
NaCl in the reference isotonic solution. However, hypertonic RVI is still infrequent when NaCl is the osmotic
agent. Given reasonable estimates of intracellular ion
activities, activation of NKCC1 should result in net
influx of ions, primarily because of the inwardly directed Cl⫺ gradient. In the absence of accurate determinations of intracellular ion activities, it can always be
argued that NKCC1 is at equilibrium. However, substitution of external Na⫹ with K⫹ to equalize their concentrations still does not result in net influx and RVI,
despite the fact that the driving force for influx is
increased sevenfold (143, 168). Furthermore, thermodynamics cannot explain a lack of hypertonic RVI by
NHE1 and anion exchange, provided some CO2 and
Downloaded from ajpcell.physiology.org on October 17, 2007
mTAL, medullary thick ascending limb of Henle; cTAL, cortical thick ascending limb of Henle; CCD, cortical collecting duct; ADH,
antidiuretic hormone; OMCD, outer medullary collecting duct; IMCD, inner medullary collecting duct; BAEC, bovine aortic endothelial cells;
BPAEC, bovine pulmonary artery endothelial cells; HUVEC, human umbilical vein endothelial cells; NKCC, Na⫹-K⫹-2Cl⫺ cotransport; NHE,
Na⫹/H⫹ exchange; NMDG, N-methyl-D-glucamine; RVI, regulatory volume increase; MDCK, Madin-Darby canine kidney; OK, opossum
kidney; CHO, Chinese hamster ovary. * J. Willis, personal communication.
C1000
INVITED REVIEW
Fig. 2. Theoretical relationship between intracellular Cl⫺ concentration ([Cl⫺]i ) and cell water during isosmotic shrinkage, where steadystate [Cl⫺]i comprises one third of intracellular anions and volume
changes are mediated by isosmotic fluxes of KCl.
rect effect on NKCC1. The inhibition of NKCC1 by Cl⫺
in internally perfused squid axons (16) is consistent
with this hypothesis. In a sense this represents classic
feedback inhibition of NKCC1 and provides an ideal yet
simple mechanism for regulating cell volume. The
nature of this Cl⫺ effect is unclear. In endothelial cells,
hypertonic and isosmotic shrinkage activate unidirectional influx through NKCC1 to about the same degree,
yet net influx only occurs after the latter (143). This
suggests that intracellular Cl⫺ blocks net influx while
allowing the partial reaction [K⫹(Rb⫹ )/K⫹ exchange] to
continue. Evidence for inhibition of NKCC1 by internal
Cl⫺ has also been obtained in Ehrlich ascites cells (101),
salivary acinar cells (168), and tracheal epithelial cells
(58). In many cells NKCC1 apparently functions in a
similar mode at steady-state volume as well (21, 46,
101, 116, 143, 150), essentially ‘‘running in neutral’’ due
to inhibition by intracellular Cl⫺, yet poised for rapid
influx when [Cl⫺]i decreases. Because cells already
expend energy to counteract swelling due to the GibbsDonnan equlibrium, it makes little sense to allow net
influx via NKCC1 or NHE1 at steady-state volume.
The mechanism by which hypertonic shrinkage prevents RVI by NHE1 has not been examined. In the
absence of CO2/HCO3⫺, RVI could be limited by the
alkalinization that accompanies Na⫹ influx and rapidly
shuts off NHE1. For instance, if shrinkage produces a
0.2 unit alkaline shift in the pH set point for NHE1 and
the internal buffering capacity is 30 mM/pH unit,
NHE1 shuts off after an influx of only 6 mM Na⫹. The
fact that more substantial RVI via NHE1 can occur in
the nominal absence of CO2/HCO3⫺ (after isosmotic
shrinkage) indicates that there is still enough CO2/
HCO3⫺ to permit some Cl⫺/HCO3⫺ exchange. Because
hypertonic RVI requires more Na⫹ influx, it may be
substantially slower under these conditions. However,
there appears to be an additional mechanism since
hypertonic shrinkage can actually suppress activation
of NHE1, presumably due to an effect of internal Cl⫺
(168).
Inhibition of RVI by intracellular Cl⫺ may explain
the variable responses to hypertonic shrinkage noted in
Table 1. Epithelial cells, which frequently exhibit hypertonic RVI, may have a significantly lower [Cl⫺]i that is
below the threshold for inhibition of RVI even after
hypertonic shrinkage. Some epithelia that do not exhibit RVI under standard hypertonic conditions do so in
the presence of agents that raise cAMP levels (43, 54,
202), most likely via activation of Cl⫺ channels and
reduction of [Cl⫺]i. Hypertonic RVI by C6 glioma cells
appears to require additives as well, including insulin
and PGE1 (133). This could also be due to a decrease in
[Cl⫺]i, but the possibility that these agents might
modulate the inhibitory effect of Cl⫺ cannot be ruled
out. An additional additive that confers hypertonic RVI
on renal epithelial cells is butyrate (135, 170). The
mechanism is not known, but the RVI is only partly
accounted for by monovalent ions (170), suggesting
that butyrate stimulates the synthesis of organic osmolytes. The occasional observation of hypertonic RVI in
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HCO3⫺ are present. These results indicate that the lack
of RVI in hypertonic medium is a regulatory event
rather than a thermodynamic effect. This could be
because the responses to the two types of shrinkage are
inherently different (40) or could be explained by
superimposed regulatory effects of ionic strength or
intracellular ion concentrations. In either event it is
clear that cells can sense how they are being shrunk.
Although the difference between isosmotic and hypertonic shrinkage could be sensed as a change in ionic
strength or an agonist-induced signal, the most logical
candidate is [Cl⫺]i. Cl⫺ is a minority anion in most cells,
yet it is the major permeant anion, with the majority of
anions being impermeant proteins and organophosphates. While hypertonic shrinkage increases [Cl⫺]i in
parallel with all other cytoplasmic constituents, [Cl⫺]i
decreases with isosmotic shrinkage, since the fluid
moving out of cells has a higher Cl⫺ concentration than
cytoplasm. As with hypertonic shrinkage, the concentration of all other cytoplasmic constituents increases
(except for K⫹, which should change little since it
comprises the vast majority of intracellular cations).
The decrease in [Cl⫺]i is actually greater than the
decrease in cell volume, making Cl⫺ concentration a
more sensitive indicator of changes in cell volume than
cell volume itself under isosmotic conditions. As shown
in Fig. 2, if Cl⫺ accounts for one third of cellular anions
and a cell loses one sixth of its volume via loss of KCl,
half the cell Cl⫺ is lost and [Cl⫺]i theoretically decreases
by 40% (1/2 the original Cl⫺ content in 5/6 the original
volume). It is not surprising then that [Cl⫺]i has
important effects on volume-regulatory transporters.
[Cl⫺]i inhibits NKCC1, and this occurs through inhibition of phosphorylation and probably through a direct
effect on the transporter as well. Phosphorylation of
NKCC1 is stimulated by lowering [Cl⫺]i and inhibited
by raising [Cl⫺]i (57, 111), probably via effects on the
same kinase activated by cell shrinkage (110). However, this cannot be the principal mechanism that
blocks hypertonic RVI, since there is still a substantial
increase in NKCC1 phosphorylation with hypertonic
shrinkage, suggesting that Cl⫺ has an additional, di-
INVITED REVIEW
other cells and the discrepancies between studies employing similar cells could be due to subtle differences
in culture conditions or the inadvertent presence of
agonists or growth factors, perhaps autocrine in nature. Hypertonic RVI may also depend on the rate of
shrinkage. In renal proximal tubules, no shrinkage
occurred when tonicity was increased 1.5 mosM/min,
whereas the cells behaved as perfect osmometers at 3
mosM/min (104). A similar response has been described
in C6 glioma cells (134), but these cells do exhibit some
RVI after acute hypertonic shock (133). The basis for
this so-called isovolumetric regulation is unknown but
could relate to the fact that [Cl⫺]i is not markedly
elevated during shrinkage. This phenomenon is probably not widespread, since most tissues do not exhibit
RVI during hypertonicity in vivo, which also develops
gradually.
Hypertonic Shrinkage In Vivo
after 8 days in kangaroo rats (193), 4 days in dogs (224),
and 3 days in Moroccan goats (76). In humans, strenuous exercise in hot conditions without water can result
in water losses up to 5% of body weight (8% of total body
water, 7% increase in osmolarity) without functional
impairment (26, 100). Because periods of water deprivation are probably common in mammals, hypertonicity
appears to be a routine occurrence that is well tolerated.
The lack of hypertonic RVI may instead be due to
potential detrimental effects on the organism. Substantial water losses are often well tolerated, without
circulatory collapse, because the loss is distributed
across all compartments (intracellular, interstitial, and
intravascular). If RVI occurred, the water loss would be
concentrated in the one third of body water that is not
intracellular. A 10% loss of body water would instead
result in a 30% decrease in extracellular volume,
including intravascular volume. Thus the absence of
RVI helps maintain vascular volume during water
deprivation, essentially allowing tissues to serve as a
water reservoir (112). Another serious consequence of
RVI would be the profound transcellular shift of K⫹.
Correction of a mere 1% cell shrinkage via cellular
uptake of KCl could decrease plasma K⫹ concentration
as much as 3 mM.
Because of the potential detrimental effects of hypertonic RVI and the seemingly minimal detrimental
effects of mild hypertonic shrinkage, RVI may be sacrificed for the benefit of the whole organism. Exceptions
appear to be intestinal epithelium (138) and brain (3,
28, 29, 137, 192) and cells cultured from these sites
(116, 133). Because intestinal epithelium is one of the
few tissues routinely exposed to sudden changes in
osmolarity, hypertonic RVI would be appropriate, particularly since cell shrinkage could disrupt the mucosal
barrier. The brain is a critical organ with limited
capacity to shrink without structural damage because
of the inelasticity of the skull. Another exception may
be chondrocytes. The extracellular fluid in cartilage is
hypertonic compared with plasma, owing to the high
concentration of fixed, polyanionic macromolecules,
and this can change with applied load (64). Hypertonic
RVI is occasionally observed in chondrocytes in situ but
not in isolated chondrocytes (Ref. 63 and A. C. Hall,
personal communication). The specific mechanism that
allows hypertonic RVI to proceed in some tissues and
cells but not in others is unknown.
Isosmotic Shrinkage In Vivo
It appears that volume-regulatory pathways in mammalian cells are designed with the dual purpose of
avoiding RVI after hypertonic shrinkage yet mediating
rapid volume recovery after isosmotic shrinkage. So
when does the need for isosmotic RVI arise? Isosmotic
shrinkage results from cellular loss of osmotically
active molecules, usually K⫹ and Cl⫺, since they are the
principal permeant ions. This can occur rapidly during
agonist stimulation (58, 146, 168). In cultured vascular
endothelial cells, cell volume decreases 16% over 10
min in response to Ca2⫹-mobilizing agonists by virtue of
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The lack of RVI in most isolated or cultured cells is
supported by data obtained in whole tissue and in vivo.
During infusion of mannitol into dogs, the total intracellular water compartment behaves as a perfect osmometer (216), and in perfused skeletal muscle, which
accounts for 75% of total intracellular water, there is no
recovery of intracellular water after hypertonic shrinkage (68). A 6% increase in osmolarity in rats produced
by dehydration decreased total intracellular water by
11% (137), with a decrease noted in muscle and several
other tissues except brain. In dehydrated humans, loss
of cellular water from skeletal muscle was precisely the
inverse of the 7% increase in osmolarity (26), with no
increase in the content of K⫹ or Cl⫺. Hypertonic infusions of NaCl, sucrose, or mannitol in rabbits also
produced the expected decrease in total intracellular
water and muscle cell water with no increase in K⫹
content (3, 192), again indicating absence of RVI. In
hypertonically perfused lung, total alveolar cell volume
decreased in exact inverse proportion to osmolarity
(213), indicating that the lack of RVI extends to other
tissues as well. The absence of hypertonic RVI in vivo is
consistent with the clinical dictum that water deficits
are distributed equally across all compartments.
Based on both in vitro and in vivo data, it appears
that mammalian cells are poised to undergo RVI after
isosmotic shrinkage but not hypertonic shrinkage. This
might seem appropriate since these cells reside in an
environment of constant osmolarity. But is the osmolarity of mammalian plasma and interstitial fluid truly
that constant? Despite the best efforts of the hypothalamus, kidneys, colon, and skin, significant water losses
still occur during water deprivation. Thus osmolarity
fluctuates with the frequency of water intake. In various rodents and ungulates, particularly those that
reside in arid areas, water loss that exceeds 20% of
body weight is well tolerated (112, 181). Deprivation for
as short as 1 day can lead to a 6% or greater weight loss
(112, 181). Some of this fluid comes from the intestinal
lumen (113) and there is also excretion of electrolytes,
so the corresponding rise in osmolarity is not as great.
Deprivation of water increases plasma osmolarity 6–7%
C1001
C1002
INVITED REVIEW
REGULATORY VOLUME DECREASE
Volume-Regulatory Transporters
The predominant pathway for RVD is the opening of
K⫹ channels and anion channels. The K⫹ channels
involved are usually large-conductance, Ca2⫹-activated
channels (so-called BK channels). The molecular basis
of the anion conductance is unknown and may comprise
more than one channel (196). The conductance can be
quite nonselective and can include a variety of organic
anions in addition to Cl⫺ (196), hence the term volumesensitive organic anion channel (VSOAC). VSOACs are
quite ubiquitous, but differences exist between cell
types, particularly in the pharmacology of inhibition.
Conductance of organic anions in addition to Cl⫺ is
appropriate for a volume-regulatory channel because
[Cl⫺]i is often low and could reach equilibrium across
the cell membrane before RVD is complete. Furthermore, there is a much greater outward electrochemical
gradient for organic anions than for Cl⫺. However,
organic osmolytes are very expensive metabolically,
since they must be replaced by synthesis or by uptake
from extracellular fluid against a steep concentration
gradient. It would be best to use organic anions for RVD
only when [Cl⫺]i is low and this may explain the
inhibition of VSOACs by Cl⫺. When [Cl⫺]i is above a
certain level, VSOACs are inhibited and RVD occurs by
Cl⫺ loss through a separate pathway (34, 196). Only
when [Cl⫺]i is low do cells open VSOACs and resort to
organic anions for RVD. The ‘‘willingness’’ of cells to
sacrifice these compounds attests to the importance of
preventing cell swelling. Inhibition of VSOACs by Cl⫺
not only conserves Cl⫺ but also ensures that cells
respond appropriately to the cause of swelling. When
swelling is due to Cl⫺ uptake, RVD occurs via Cl⫺ loss.
In contrast, when swelling is due to accumulation of
organic compounds (such as amino acids), Cl⫺ is lost to
a certain level (to accommodate the organic compounds) below which the organic osmolytes are lost.
An additional transporter that mediates RVD is the
K⫹-Cl⫺ cotransporter (KCC), which transports K⫹ and
Cl⫺ stoichiometrically in either direction across the
plasma membrane (98). Although structurally related
to NKCC1 and inhibited by furosemide, this transporter is distinguished by its Na⫹ independence and
insensitivity to bumetanide. Among physiological anions, it has a strict requirement for Cl⫺ and, not
surprisingly, is the principal mechanism for RVD in
cells with a high [Cl⫺]i such as erythrocytes. Studies of
KCC have been hampered by the lack of a specific
inhibitor, but, based on mRNA abundance, the transporter is probably more prevalent than previously
thought (47, 48, 132). For instance, it appears to be
responsible for about one-half the RVD in aortic endothelial cells (160).
The mechanisms by which cell swelling activates
volume-sensitive channels and KCC are poorly understood. The nature of the K⫹ channels involved suggests
Ca2⫹ as a signal, and evidence in support of this has
been obtained primarily in epithelial cells (22, 31, 72,
121, 122, 172, 173, 189, 221). The hypothesis based on
these data is that cell swelling induces Ca2⫹ influx,
possibly via stretch-activated channels, leading to a
rise in [Ca2⫹]i and subsequent activation of BK channels. However, in many other cells only a small rise in
[Ca2⫹]i occurs and is not required for activation of K⫹
efflux (52, 74, 125, 161, 222). One possible explanation
for this discrepancy is the distinction between dependence on Ca2⫹ and activation by Ca2⫹. The K⫹ channels
may require Ca2⫹ for activity, but swelling could open
them through a separate mechanism. Maneuvers that
block increases in [Ca2⫹]i (such as removing extracellular Ca2⫹ ) often lower basal [Ca2⫹]i, thereby preventing
channel activation by any mechanism. Further evidence against the Ca2⫹ theory is that Ca2⫹ is not
involved in the activation of anion channels or K⫹-Cl⫺
cotransport in swollen cells. Because the evidence for
Ca2⫹ signaling of RVD is quite compelling in epithelial
cells, it is reasonable to hypothesize two pathways for
activation of K⫹ channels by cell swelling: a mechanosensitive Ca2⫹ influx that is the primary pathway in
epithelia and a Ca2⫹-independent pathway operational
in nonepithelial cells. The nature of the Ca2⫹-independent signal is unknown, but phospholipase A2 has been
implicated in volume-sensitive K⫹ and anion channels
(136, 147), whereas protein kinase inhibition has been
implicated in the activation of KCC in swollen erythrocytes (11, 82, 84). Unfortunately, it has not yet been
possible to determine whether the cotransporter itself
is phosphorylated.
Hypotonic vs. Isosmotic Swelling
Essentially all cells (with the exception of some
erythrocytes) respond to hypotonic swelling with an
RVD, but the response is usually incomplete and cells
remain swollen. The failure to complete RVD has re-
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Ca2⫹-dependent K⫹ channels (and probably Cl⫺ channels as well). Inhibition of NKCC1 doubles the shrinkage, indicating that RVI is required to stabilize cell
volume during agonist stimulation. In addition to maintaining cell volume, this process also prevents large
decreases in [Cl⫺]i that could impair agonist-induced
Cl⫺ currents. RVI also occurs during stimulation of
secretory epithelia (41, 58, 211) where large solute
losses across the apical membrane would lead to rapid
shrinkage if not compensated by influx across the
basolateral membrane. Not surprisingly, NKCC1 and
NHE1 are components of the basolateral membrane
and become quite active when apical Cl⫺ channels are
open (57, 58, 168). This coupling of apical efflux and
basolateral influx via cell volume provides a simple
mechanism for maintaining cell volume during large
transepithelial fluxes, a process that has been termed
homocellular volume regulation (183). Another example of isosmotic shrinkage may be the decrease in
mucosal volume of the intestine during fasting (61). In
rats, a 15% volume decrease occurs during a 48-h fast
and is rapidly corrected within minutes after exposure
to an electrolyte solution. The restoration of cell volume
after refeeding is associated with stimulation of
NHE1 (61).
INVITED REVIEW
Hypotonic Swelling In Vivo
Despite the fact that mammalian cells regulate their
volume in hypotonic medium, the need rarely arises in
vivo. At the level of the whole organism, hypotonicity
can only occur from excess water intake (in the absence
of pathological states), which is possible since thirst is
not driven exclusively by osmolarity. For instance, mild
hypotonicity will develop when water is provided to
Fig. 3. Regulatory volume decrease (RVD) in aortic endothelial cells,
measured as changes in cell content of Na⫹ and K⫹. Top: cells placed
in medium made hypotonic by removal of NaCl. Middle: RVD after
isosmotic swelling. Cells were preincubated in isosmotic KCl medium
and then placed back in standard isosmotic medium. Bottom: Cells
were placed in hypotonic medium with 50 µM bumetanide. Dashed
line in each panel represents expected values for complete RVD.
[Adapted from O’Neill and Klein (143).]
rats deprived of water for 10 h (182). As a localized
phenomenon, hypotonicity may be more frequent. Ingestion of water can render the intestinal lumen hypotonic, and this can even extend to the liver since portal
blood can become hypotonic after water ingestion (71).
The RVD exhibited by isolated cells appears to accurately reflect the occurrence of RVD in vivo. In rats
made hyponatremic through administration of water
and vasopressin, total intracellular water increased far
less than predicted and total body K⫹ decreased (208,
209), consistent with RVD. This is indicative of RVD in
skeletal muscle, since it contains most of the cellular
water, but hypotonic RVD has also been demonstrated
in specific organs including liver (59, 96), heart (151),
and brain (195).
Isosmotic Swelling In Vivo
Isosmotic swelling is a much more common occurrence and results from an increase in the cellular
content of osmotically active molecules. Examples include uptake of nutrients, acidosis, or depolarization of
the cell membrane potential. Uptake of nutrients,
particularly amino acids and sugars, can and must
occur rapidly in tissues responsible for their assimilation after meals such as intestinal epithelium, liver,
and skeletal muscle. In Ehrlich ascites cells, for instance, 10 mM glycine, a physiological postprandial
concentration, produces a 17% increase in cell volume
through Na⫹-coupled uptake (77), and a similar response has been noted in intact, perfused liver (71).
Nutrient-induced swelling followed by RVD is also
observed in mammalian intestine (117). RVD after
nutrient uptake is incomplete, probably due to dilution
of intracellular Cl⫺.
Acidosis can also increase cell volume, a response
that is distinct and opposite from the effect of pH on the
Gibbs-Donnan equilibrium. By titrating negative
charges on impermeant molecules, a reduction in pH
decreases the tendency of cells to swell. However, this is
more than offset by accumulation of the anions that
accompany the H⫹ and replace impermeant anions
titrated by the H⫹ (79). If intracellular buffering capacity is 30 mM/pH unit, lowering cell pH by 0.5 with a
monovalent acid would lead to accumulation of 15 mM
anion. Cell swelling occurs during all three types of
acidosis: organic, inorganic, and respiratory. With organic acids such as lactic acid, the anion accumulates
not only within cells in which they are synthesized but
also in other cells, because the acids are weak and
permeant and because the anions can be transported
(105). The resulting swelling has been demonstrated in
astrocytes (80, 81, 105) and erythrocytes (191). Hydrochloric acidosis, the principal inorganic acidosis in vivo,
can be created in vitro by replacing HCO3⫺ with Cl⫺. The
assumption is that HCO3⫺ leaves cells in exchange for
Cl⫺, transmitting the acidosis to the cytoplasm. Because of intracellular buffering, the amount of HCO3⫺
leaving the cell and replacement anions entering will
be greater than the drop in HCO3⫺ concentration,
leading to an increase in osmotically active molecules.
In renal proximal tubular cells, a decrease in HCO3⫺
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mained a mystery, but recent studies in endothelial
cells provide an explanation (Fig. 3). When these cells
are swollen isosmotically by loading them with KCl,
which raises [Cl⫺]i as opposed to the decrease in [Cl⫺]i
after hypotonic swelling, there was a complete and far
more rapid RVD. In fact there was an overshoot that
was gradually corrected. Complete RVD could also be
accomplished in hypotonic medium by adding bumetanide to inhibit NKCC1. These data indicate that
incomplete RVD in hypotonic medium was due to a
decrease in [Cl⫺]i that resulted in net influx via NKCC1.
Hypotonic RVD in the presence of bumetanide is still
slower than isosmotic RVD, which may be a manifestation of the smaller outward Cl⫺ gradient or stimulation
of RVD pathways by Cl⫺. A similar phenomenon has
been described in Ehrlich ascites cells, in which swelling activates Na⫹/H⫹ exchange and amiloride enhances
RVD (103). Thus incomplete RVD in hypotonic medium
does not represent inadequacy of RVD pathways or
their sensitivity to cell volume but rather represents a
new steady-state volume at which efflux through RVD
pathways equals influx through RVI pathways that are
activated by a decrease in [Cl⫺]i. Thus, as with RVI,
RVD also depends on how volume is altered and centers
on [Cl⫺]i and its conservation.
C1003
C1004
INVITED REVIEW
reperfusion and the role of volume-regulatory transporters are unknown.
CHANGES IN THE SET POINT
FOR STEADY-STATE CELL VOLUME
Steady-state volume varies over the life span of a cell,
particularly during growth and differentiation. This
obviously requires suppression of normal volumeregulatory mechanisms, but volume-regulatory transporters may also play a more direct role. Activation of
NHE1 or NKCC1 in the absence of cell shrinkage could
lead to cell enlargement, whereas volume-independent
activation of RVD pathways could lead to cell shrinkage. Although the role of volume-regulatory transporters in adjusting steady-state cell volume has not been
established, the rapid activation of NKCC1 (152, 153,
155, 156, and W. C. O’Neill, J. D. Klein, and S. T.
Lamitina, unpublished observations) and NHE1 (60,
175) by growth factors lends credence to this concept.
Addition of serum produces a rapid increase in cell
volume (15% over 10 min) in endothelial cells, which is
mediated by NKCC1 (W. C. O’Neill, J. D. Klein, and
S. T. Lamitina, unpublished observations), and a similar response occurs in growth-stimulated fibroblasts
(126, 152, 153). A more chronic stimulation of NKCC1 is
also seen with growth factors (Ref. 19 and W. C. O’Neill,
J. D. Klein, and S. T. Lamitina, unpublished observations) and contributes to the increase in cell volume
during the growth cycle (19). This switch in NKCC1
from its normal mode of K⫹/K⫹ exchange at steadystate volume to a net influx mode implies that growth
stimuli can modulate or overcome the putative inhibitory effect of intracellular Cl⫺. Activation of NHE1 by
growth factors also increases cell volume (51), which,
like NKCC1, requires disinhibition of the transporter
at steady-state volume, in this case by shifting the pH
dependence to the right (51). The mechanism by which
growth factors activate NHE1 and NKCC1 is not
understood. Phosphorylation of NHE1 is increased
(178), but a mutated antiporter lacking phosphorylation sites is still activated by mitogens, although to a
lesser degree (210). Regulation of NKCC1 by growth
factors has not been examined at the molecular level
and, whatever the mechanism, it must overcome the
normal inhibition of net influx (presumably due to
intracellular Cl⫺ ) to increase cell volume. Volumeregulatory transporters may also be responsible for cell
enlargement during transformation. Fibroblasts expressing the ras oncogene were found to be 32% larger
than control cells, with upregulation of both NKCC1
and NHE1 (95, 126), whereas cell enlargement accompanying cytomegalovirus infection is associated with
enhanced activity of NHE1 (27).
Cell shrinkage is an early and dramatic event during
apoptosis (6, 7, 149). The mechanism is unknown but
appears to be biphasic. In dexamethasone-treated lymphoid cells, shrinkage begins in 12 h and progresses
until chromatin condensation (36 h). Although this is
associated with a net loss of K⫹, other cytoplasmic
components are lost as well, since buoyant density does
not change (7). The second phase of shrinkage after
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concentration from 25 to 5 mM increased cell volume by
6% (198), but there was no accumulation of Cl⫺ (199),
suggesting a different counteranion. In respiratory
acidosis CO2 diffuses into cells and dissociates into H⫹,
which are buffered, and HCO3⫺, which accumulates and
is partly exchanged for extracellular Cl⫺. Accordingly,
an increase in CO2 from 5 to 15% produced swelling of
renal proximal tubules accompanied by an increase in
the cellular content of both HCO3⫺ and Cl⫺ (199). An
additional mechanism of swelling in acidosis is activation of counterregulatory pathways. In astrocytes exposed to organic acids, activation of NHE1 by intracellular acidification leads to a second phase of swelling
(80, 81). It is not known whether RVD occurs during
acidification in vitro, and limited data are available in
vivo. Respiratory acidosis (10% CO2 for 3 h) did not
increase cell water in rat heart, brain, spleen, liver, or
skeletal muscle (171), implying that RVD had occurred.
In contrast, exposure of rat skeletal muscle to 30% CO2
for 95 min increased cell water by 11% (177), but such
severe acidosis may have overwhelmed or inhibited
RVD. In muscle biopsies from humans during vigorous
exercise, there was a small, insignificant increase in
intracellular water despite substantial cellular lactate
accumulation (190), again implying some volume correction.
Because the negative membrane potential is largely
responsible for maintaining cell volume by limiting Cl⫺
accumulation, depolarization can lead to cell swelling.
In vivo, this would be most apparent in excitable
tissues such as nerve and muscle, particularly where
depolarization is due to opening of Na⫹ channels. The
expected swelling has in fact been demonstrated in
nerve fibers (78) and in cultured myogenic cells (185).
Although the Na⫹-K⫹ pump can rapidly export the
excess Na⫹, much of this is exchanged for K⫹, with the
resulting net efflux of only one osmotically active
molecule per cycle. Thus the Na⫹-K⫹ pump is an
inefficient volume-regulatory transporter that cannot
rapidly relieve cell swelling. Swelling of skeletal muscle
during repetitive contraction would be particularly
problematic since it could compromise regional blood
flow. Such swelling occurs during vigorous exercise,
accompanied by an increase in Cl⫺ content (123, 190),
but the swelling cannot be ascribed solely to repetitive
depolarization since acidosis could also contribute to
the swelling. The increase in intracellular water is
small, implying that volume regulation has occurred.
Volume regulation via Ca2⫹-activated K⫹ channels could
serve the dual purpose of repolarizing the membrane
potential (15). A dramatic example of isosmotic swelling
in vivo, possibly related to depolarization and acidosis,
occurs after ischemia (39, 215). Although ischemia
directly swells cells in vitro (92), in vivo this may
require reperfusion (215). The marked swelling of
endothelium probably contributes to reduced blood flow
and further ischemia, since perfusion with hypertonic
solutions maintains blood vessel patency and organ
perfusion (39), and prevents ischemic damage (2). The
cellular events responsible for cell swelling during
INVITED REVIEW
Fig. 4. Correlation between cell volume and proteolysis in perfused
rat liver. Perturbations included anisosmotic media, hormones, channel and transporter blockers, and nutrients. [Adapted from Haussinger (71).]
regulatory mechanism, since osmotically active molecules are consumed during synthesis of protein and
glycogen and released during proteolysis and gycogenolysis. In the liver, changes in cell volume serve to
couple metabolism to nutrient uptake and hormone
action (71). Nutrient uptake and insulin both cause cell
swelling (the latter via stimulation of NKCC1 and
NHE1), leading to an increase in the content of protein
and glycogen. Prevention of cell swelling by inhibiting
NKCC1 and NHE1 blocks this anabolic response. On
the other hand, glucagon causes cell shrinkage (probably via opening of K⫹ channels), thereby inducing a
catabolic response (proteolysis and glycogenolysis).
There is in fact a linear relationship between hepatocyte volume and protein synthesis and proteolysis
along which many anabolic and catabolic stimuli fall
(Fig. 4). The anabolic and catabolic effects of cell
volume may exist in skeletal muscle as well (106–108),
but the role of cell volume in the action of insulin in
target tissues other than liver is unclear (223). The
signaling mechanisms by which cell volume controls
cell metabolism and growth are unknown but could be
related to known growth signaling cascades. For instance, cell swelling has been shown to activate tyrosine kinases, phosphorylate (and activate) MAP kinases (180, 204), and to increase c-jun phosphorylation
(180), c-jun mRNA abundance (38), and phosphorylation of ribosomal protein S6 (109). To what extent any of
these is responsible for the metabolic effects of cell
volume is unknown.
SUMMARY
The purpose of this review is to dispel the notion that
cell volume regulation in higher organisms is a curious
but physiologically irrelevant phenomenon. Osmolarity
of the milieu interieure is not as constant as is often
assumed, and significant threats to cell volume still
exist in an isosmotic environment under both physiological and pathological conditions. In addition to maintaining cell volume, volume-regulatory mechanisms are
also employed to initiate changes in cell volume, as
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chromatin condensation is due to cellular fragmentation. Apoptotic shrinkage is inhibited by blocking K⫹
channels (6), but it is not known whether these are the
normal volume-regulatory channels. Cell shrinkage
also occurs during the latter stages of erythrocyte
maturation and may be mediated by the K⫹-Cl⫺ cotransporter (97, 99), which is expressed predominantly in
young erythrocytes (20, 62, 98, 142). Altered regulation
of this transporter may induce additional shrinkage
that contributes to sickling in hemoglobin SS disease
(13).
The mechanism that couples cell volume to cell
growth and differentiation is unknown. The macromolecular crowding theory of volume regulation could
account for the regulation of volume-regulatory transporters by cell growth. As cell content of protein and
other macromolecules changes, cell volume would be
perceived as too small or too large, and RVI or RVD
would be activated. This is consistent with the constant
relative content of cell water during the cell cycle in
HeLa cells (131) and the constant buoyant density
during early apoptosis in lymphoid cells (7), suggesting
a constant ratio of macromolecules to cell water. However, this scenario cannot account for the very early
activation of NKCC1 and/or NHE1 and the resulting
increase in cell volume produced by growth factors well
before any increase in protein content. These data are
more consistent with coupling in the other direction,
with the increase in cell volume stimulating growth.
Such coupling of cell growth to cell volume would
ensure, for instance, that cells reach a certain size
before mitosis. Consistent with this, inhibition of
NKCC1 reduces growth responses in endothelial cells
(155) and fibroblasts (19, 126, 139, 140, 154, 156) but
not in all studies (1). Inhibition of NHE1 also blunts
growth responses in a variety of cells (12, 60, 93, 164,
212), but the effect is variable and it is not clear
whether it is mediated through changes in cell volume
or intracellular pH (8). Because growth factors often
activate both NHE1 and NKCC1, dual inhibition may
be required to prevent cell swelling, possibly explaining
the failure of amiloride or loop diuretics alone to
completely inhibit growth. In fact, studies in fibroblasts
showed that both were required to completely block
DNA synthesis (154). These results suggest that activation of NHE1 and/or NKCC1 leads to an early increase
in cell volume that may signal other aspects of cell
growth. The role of cell shrinkage in apoptosis is even
less clear. Hypertonic shrinkage is reported to both
induce (14, 149) and inhibit (55) apoptosis, and prevention of cell shrinkage with K⫹ channel blockers does not
prevent apoptosis (6).
The concept that cell volume controls cell growth is
considerably strengthened by evidence that cell volume
also regulates cell metabolism. Swelling (either hypotonic or isosmotic) of hepatocytes (109, 167, 194) increases protein synthesis and decreases protein degradation, whereas hypertonic shrinkage has the opposite
effect (71) in liver and in other cells (162). Parallel
effects are seen on glycogen synthesis and breakdown
(71, 159). This process also represents a volume-
C1005
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INVITED REVIEW
required for cell growth and differentiation. The close
link between volume-regulatory transporters, cell volume, and cell growth and metabolism indicates that
regulation of cell volume is a fundamental and important property of mammalian cells. The sensing and
signaling mechanisms for cell volume regulation remain a mystery, and elucidation of these pathways will
provide important new information on other aspects of
cell physiology as well.
I thank Drs. Robert Gunn and Michael Jennings for their helpful
comments.
Studies from the author’s laboratory have been supported by
National Institutes of Health Grants DK-01643, HL-47449, and
DK-50268, the National Kidney Foundation of Georgia, and the
American Heart Association, Georgia Affiliate.
Address for reprint requests and other correspondence: W. C.
O’Neill, Renal Division, Emory Univ. School of Medicine, WMB 338,
1639 Pierce Dr., Atlanta, GA 30322 (E-mail: [email protected]).
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