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Anisosmotic

cell volume regulation:

a comparative

view

M. E. CHAMBERLIN
AND K. STRANGE
Department
of Zoological and Biomedical Sciences and College of Osteopathic Medicine,
Ohio University,
Athens 45701; and Department
of Physiology and Biophysics,
Wright State University
School of Medicine, Dayton, Ohio 45435

CHAMBERLIN, M. E., AND K. STRANGE. Anisosmotic ceZZuohne regulation: a comparative view. Am. J. Physiol. 257 (Cell Physiol. 26): Cl59-Cl73,
1989.-A
variety of organisms and cell types spanning the five taxonomic
kingdoms are exposed, either naturally or through experimental
means, to
osmotic stresses. A common physiological
response to these challenges is
maintenance
of cell volume through changes in the concentration
of intracellular inorganic
and organic solutes, collectively termed osmolytes. Research on the mechanisms by which the concentration
of these solutes is
regulated has proceeded along several experimental
lines. Extensive studies
on osmotically activated ion transport pathways have been carried out in
vertebrate cells and tissues. Much of our knowledge on organic osmolytes
has come from investigations
on invertebrates,
bacteria, and protists. The
relative simplicity of bacterial genetics has provided a powerful and elegant
tool to explore the modifications
of gene expression during volume regulation. An implication
of this diverse experimental
approach is that phylogenetically divergent organisms employ uniquely adapted mechanisms
of
cell volume regulation.
Given the probability
that changes in extracellular
osmolality were physiological
stresses faced by the earliest organisms, it is
more likely that cell volume regulation
proceeds by highly conserved
physiological
processes. We review volume regulation
from a comparative
perspective, drawing examples from all five taxonomic kingdoms. Specifically, we discuss the role of inorganic
and organic solutes in volume
maintenance
and the mechanisms
by which the concentrations
of these
osmolytes are regulated. In addition,
the processes that may transduce
volume perturbations
into regulatory responses, such as stretch activation
of ion channels, intracellular
signaling, and genomic regulation,
are discussed. Throughout
this review we emphasize areas we feel are important
for future research.
osmotic stress; osmolytes;

membrane

OF cell volume is a fundamental physiological process. Under isosmotic conditions cell swelling
tends to occur because of the presence of negatively
charged macromolecules in the cytoplasm. This swelling,
however, is counteracted by active extrusion of solutes,
such as Na+ via the Na+-K+-ATPase. In contrast, exposure to anisosmotic media drives water into or out of the
cell due to alterations in extracellular osmolality. Many
cells studied to date respond to these anisosmotically
induced volume changes by modulating membrane transport and/or metabolic pathways that alter the concentration of intracellular solutes. The processesresponsible
for returning cell volume toward its original value after
osmotic swelling or shrinkage are termed regulatory volMAINTENANCE

0363-6143/89

$1.50

Copyright

transport;

cellular

metabolism

ume decrease (RVD) and regulatory volume increase


(RVI), respectively.
Anisosmotic cell volume regulation is mediated by
changes in the concentration of intracellular osmotically
active solutes. These solutes consist of inorganic ions
and small organic molecules and are collectively referred
to as osmolytes. The mechanisms of volume regulatory
inorgan ic ion tra nsport have been studi .ed extensively in
cells of animals, bacteria, and protists (for reviews see
Refs. 47, 48, 50, 56, 65, 69, 72, 79, 80, 99, 128, 140, 143).
In addition, numerous investigations have examined the
role played by organic molecules in volume maintenance
of plant, animal, protist, fungal, and bacterial cells (for
reviews see Refs. 22, 26, 27, 35, 36, 58, 78,128,156).
One

0 1989 the American

Physiological

Society

Cl59

Cl60

INVITED

of our main intentions in the writing of this review was


to determine if there are general patterns of volume
regulatory responses for all cells and whether these responses are controlled by common mech.anisms. The
conclusion th .at we reached, however, is not so much that
there are many obvious similarities between diverse cell
types but instead that various groups of organisms have
been used as model systems to answer specific questions.
For example, genetic mechanisms of volume regulation
have been studied extensively in bacteria, whereas much
of what we know about organi .c osmolytes has come from
investigations on particular species of invertebrates, protists, and bacteria. This had made it difficult to easily
compare and contrast volume regulatory processes in
diverse organisms. Nevertheless, it is clear that much of
what is learned from studies on one group of organisms
can and, in some cases, is being applied to investigations
of other cell types. Throughout this review we speculate
on what we feel are important directions for a.dditional
studies of v.olume regulatory mechanisms. It is hoped
that such an approach will stimulate further thinking
and experi .mentation on this important topic.

REVIEW

TABLE 1. Examples of volume regulatory ion transport


pathways identified in various organisms
Organism

Mechanism

Regulatory
K+-H+ exchangers
Parallel Cl--HCO?
exchangers
K+ channels

and K+-H+

K+ or KC1 loss

Unidentified
Ca2+-dependent
pathways

NaCl loss pathways


Na+ loss
Regulatory

Parallel
Na-H
exchangers

and Cl--HCOc

NaCl cotransporters
Na+-K+-2Clcotransporters

K+ pumps
Cl- pumps

red blood

cells

Bacteria
Amphibian
urinary
bladder
Amphibian
gallbladder
Lymphocytes
Ehrlich
ascites cells
Mammalian
renal tubules
Lymphocytes
Ehrlich
ascites cells
Teleost red blood cells
Avian red blood cells
Mammalian
red blood cells
Annelid
coelomocytes
Molluscan
red blood cells
Crustacean
nerves and
muscle fibers
Horseshoe
crab myocardium
Canine red blood cells

KC1 cotransporters

Unidentified
pathways

Type

decrease
Bacteria
Amphibian

Cl- channels

OSMOLYTES

Inorganic ions. Inorganic ion transport has been implicated in cell volume regulati .on in a wide of variety
organisms (Table 1). The extent of information available
on volume regulatory ion transport pathways in animals,
plants, moneran .S fungi, and protists is, however, extremely variable Ion transport pathways activated bY
changes in environmental osmolality have been examined in some detail in protists (algae) and monerans
(bacteria and cyanobacteria). Species in these two kingdoms that are surrounded by rigid cell walls maintain a
internal
hydrostatic
pressure
significant
positive
gradient. This so-called turgor pressure is regulated
within narrow physiological limits and is generated by
the accumulation of solutes and water in the cytoplasm.
In organisms with rigid cell walls, external osmolality
has variable effects on cell volume. Exposure to hypertonic stress causes cell shrinkage and loss of turgor
pressure, whereas hypotonicity results in increased turgor pressure but may have little or no effect on cell
volume. The presence of a cell wall will obviously limit
or prevent cell swelling. This dual nature of cell volume
change in walled organ isms makes it difficult to separate
the process of turgor regulation from that of volume
regulation per se. What is clear, however, is that variations in transmembrane hydrostatic and osmotic pressure gradients result in the activation of specific ion
transport pathways that return these parameters to their
original values.
Detailed studies on volume regulatory ion transport
have been conducted in giant algal cells, such as Valonia,
Nitella, and Halicystis. When exposed to hypertonic
stress, these organisms activate K+ or Cl- pumps that
drive net salt and water uptake and that are responsible
for restoration of cell volume and turgor (reviewed in
Ref. 69). The mechanisms responsible for reducing cell
volume and turgor in hypotonically stressed algal cells
have not been studied in any detail.

volume

or Cell

volume

increase
Amphibian
red blood cells
Amphibian
gallbladder
Lymphocytes
Mammalian
renal tubules
Mammalian
red blood cells
Ehrlich
ascites cells
Amphibian
skin
Avian red blood cells
Ehrlich
ascites cells
Cultured
canine kidney
cells
(MDCK)
Bacteria
Algae
Algae

See Refs. 47, 48, 50, 56, 65, 69, 72, 79, 80, 99, 128, 140,
detailed discussions
of these pathways.

143 for

Bacterial osmoregulatory ion fluxes have been studied


extensively in Escherichia coli. Exposure of E. coli to
hypertonic environments results in activation of two K+
uptake pathways responsible for increasing turgor pressure and cell volume (47). The Trk pathway has a low
K+ affinity and high maximum transport rate (Lax),
appears to be driven by the transmembrane proton electrochemical gradient, and may be regulated by phosphorylation (47, 48). The Kdp system consists of a K+stimulated ATPase with an extremely high affinity for
K+ (47,48). The anions accompanying K+ transport have
not been completely identified, but uptake of negatively
charged amino acids is at least partially responsible for
charge balance (47,98). Hypotonic stress is accompanied
by loss of intracellular K+. The nature of the K+ efflux
pathways involved in cell volume and turgor reduction is
uncertain at present but may involve either K+ channels
(113) or K+-H+ exchangers (25, 47). Presumably in the
case of K+-H+ exchange, protons taken up by the cell are
buffered, and therefore this process results in a net loss
of osmotically active particles. Interestingly, these K+efflux pathways are activated by thiol reagents (13, 112),
as is the volume regulatory N-ethylmaleimide (NEM)-

INVITED

sensitive KC1 pathway in sheep red blood cells (99). This


suggests that the control of volume regulation in these
two diverse organisms may be similar.
Few studies have been conducted on the cell volume
regulatory role played by inorganic ions in the invertebrates. Studies on crustacean
muscle fibers (115) and
nerves (88) and on annelid coelomocytes
(38) have suggested that RVD is partially mediated by net K+ loss.
RVD in molluscan red blood cells is at least partially
mediated by loss of KC1 (141). Warren and Pierce (155)
have shown that the myocardium
of the horseshoe crab
loses significant
quantities of NaCl on exposure to hyposmotic media. The transport
mechanisms
mediating
volume regulatory
ion losses in these invertebrate
cells
have not been elucidated.
In sharp contrast to the invertebrates,
extensive studies of volume regulatory
ion transport
have been conducted in vertebrate
cells and tissues. RVD in most
vertebrate cells is mediated to a large extent by passive
KC1 loss through parallel K+ and Cl- channels, parallel
K+-H+ and Cl--HCO;
exchangers
or coupled KC1 cotransporters
(reviewed in Refs. 50, 80, 140). A noteworthy exception to this generalization
is the dog red blood
cell, which lacks a functional Na+-K+-ATPase
and, as a
consequence, has a high Na+ and a low K+ content (122).
RVD occurs in these cells by active Na+ extrusion (121),
possibly mediated by a Na+-Ca2 exchanger (123). A role
for Na+ extrusion
in RVD has also been suggested by
studies on collapsed renal tubules and renal cortical slices
(55, 62, 138). Recent studies by Proverbio
et al. (133)
have shown that cell membranes from rat kidney cortex
contain a ouabain- and K+-insensitive
Na+ pump that is
activated by cell swelling. Because all renal tubule cells
studied to date have typically low intracellular
Na+ concentrations
(lo-20 mM; see Ref. 153), the extent to which
Na+ loss can mediate anisosmotic
RVD is limited.
RVI in most vertebrate cells studied to date is mediated
primarily by one of two mechanisms.
Volume regulatory
Na+-K+-2Clcotransport
pathways have been described
in avian red blood cells, frog skin, Ehrlich ascites tumor
cells, and Madin-Darby
canine kidney (MDCK)
cells
(reviewed in Refs. 50,80, 140). Parallel Na+-H+ and Cl-HCO; exchangers mediate RVI in human lymphocytes,
Necturus gallbladder, and mouse medullary thick ascending limb (reviewed in Refs. 50, 80, 140). Hoffmann
et al.
(83) have also described a NaCl cotransporter
activated
in Ehrlich cells after cell shrinkage. Interestingly,
Hoffmann (79) has suggested that this transport
pathway
and the Na+-K+-2Clcotransporter
described by Geck
and Pfeiffer (56) are actually the same system functioning in different modes. Eveloff and Calamia (49) have
suggested a similar interchange
between the NaCl and
the Na+-K+-2Clcotransporters
found in rabbit renal
medullary thick ascending limb cells. In these cells the
mode of the transporter
is dependent on extracellular
osmolality.
Organic osmolytes. Organic osmolytes have been implicated in cell volume regulation of animal, plant, bacterial, fungal, and protist cells exposed to anisosmotic
environments.
Although the accumulation
or loss of any
organic solute would serve to bring the cell to osmotic

Cl61

REVIEW

equilibrium with its environment,


only a restricted number of organic osmolytes are used in biological systems.
The general classes of organic osmolytes
are sugars,
polyols, amino acids, methylamines,
and urea (Table 2).
The monosaccharides,
glucose, mannose, and fructose,
are used in freshwater, cyanobacteria,
diatoms, and fungi
(22). Disaccharides,
such as sucrose and trehalose, are
found in algae, plants, animals, nonmarine cyanobacteria
(22)) and eubacteria (98). Linear and cyclic polyols, such
as sorbitol, arabitol, mannitol, and inositol, are used by
marine cyan .obacteria, algae, fungi, and plants (22). Polyols are also accumulated in renal medullary cells during
hypertonic
stress (118). Complex polyhydric
compounds
(heterosides)
are accumulated
by marine cyanobacteria
and algae (22). Glutamate and proline (98, 102) as well
as the cyclic amino acid ectoine (137, 151) are used in
bacterial cell volume regulation. Proline is also concentrated by plant cells during hypertonic
stress (22). Marine invertebrates
use several amino acids, including
glycine, serine, proline, alanine, taurine, and ,&alanine
(58, 160). Taurine is involved in volume regulation of
fish red blood cells (51, 53, 101) and Ehrlich ascites

Sugars
Monosaccharides
(e.g., glucose,
mannose,
fructose)
Disaccharides
trehalose)

(e.g., sucrose,

Polyols (e.g., sorbitol,


arabitol,
mannitol,
heterosides)

inositol,

no acids (e, ,g., proline,


glutamate,
taurine,
ectoine)

Methylamines
(e.g., betaine,
sarcosine,
glycerophosphorylcholine)

Urea

Cyanobacteria
Diatoms
Fungi
Cyanobacteria
Eubacteria
Algae
Plants
Insects
Crustaceans
Cyanobacteria
Algae
Fungi
Plants
Insects
Crustaceans
Mammalian
renal cells
Mammalian
brain cells
Eubacteria
Plants
Annelids
Mollusks
Crustaceans
Elasmobranch
red blood
Teleost red blood cells
Ehrlich
ascites cells
Cyanobacteria
Eubacteria
Plants
Mollusks
Echinoderms
Holocephalans
Agnathans
Elasmobranchs
Teleosts
Mammalian
renal cells
Elasmobranchs
Amphibians
Mammalian
renal cells

cells

See Refs. 10, 18, 22, 26, 27, 35, 36, 46, 51, 53, 58, 78,81, 98, 101, 102,
106, 118, 128, 129, 137, 151, 156, 159, 160 for detailed discussions
of
intracellular

organic

osmolytes.

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INVITED

tumor cells (81). The methylamine,


betaine, is found in
saline (nonmarine)
cyanobacteria
(22)) eubacteria (98,
102), plants (160), mammalian renal cells (10, 118, X9),
and marine invertebrates
(see Ref. 58). Proline betaine
is found in the opisthobranch
mollusk Elysia chLorotica
(129) and in bacteria (98). High concentrations
of trimethylamine
oxide (TMAO)
are found in several vertebrates, such as hagfish, skates, rays, chimaeras,
and
coelocanths
(see Refs. 18, 46, 160). Relatively
lower
concentrations
are found in teleost fishes and some invertebrates
(46). Another
methylamine,
glycerophosphorylcholine,
is found in mammalian renal medulla (10,
68, 158, 159). Urea is an important
intracellular
solute
in some vertebrates,
most notably elasmobranchs,
coelocanths, amphibia, and mammals (renal medulla) (10,
159, 160). A role for this compound in RVD has been
postulated (18).
Properties
of organic osmolytes. Intracellular
solutes
can be grouped into two broad categories: destabilizing
and stabilizing
solutes (35, 142, 160). The stabilizing
solutes are generally those that do not perturb protein
structure and function even at high concentrations.
Destabilizing solutes, such as inorganic ions, can, however,
alter protein structure
and function. Most organic osmolytes, with the exception of urea, are considered to be
stabilizing solutes. Stabilizing organic solutes are also
referred to as compatible
solutes, since they are compatible with enzyme function (142, 160).
What properties
differentiate
compatible from destabilizing solutes ? As discussed by Yancey et al. (160),
certain solutes may perturb enzyme activity by binding
to ligands (substrates,
cofactors, modulators,
etc.) or to
active sites on the protein surface. Most compatible
solutes are neutral at physiological
pH and therefore
would not bind to ligands and active sites. Destabilizing
solutes may impair enzyme function by disrupting
the
hydration layer surrounding
the protein. This would lead
to an expansion of the enzymes compact globular structure and a possible loss of activity. In contrast, it appears
that compatible solutes are excluded from the protein
surface so that it remains hydrated by bulk water (107).
The mechanisms of solute exclusion are not fully understood, but three hypotheses
have been proposed
(reviewed in Ref. 107). First, protein surface groups may
repel particular
solutes because of charge incompatibility. Second, some solutes, such as sucrose, may be steritally restricted from interacting with the protein surface.
Third, Arakawa and Timasheff (5,6) and Low (107) have
postulated that many of these compounds increase waterwater interactions
in the vicinity of the solute. Because
it is energetically
unfavorable
to disrupt water-water
interactions,
bulk unstructured
water is available for
protein hydration. Structured
water and its accompanying solute are thus excluded from the protein surface.
Destabilizing
solutes can have minimal effects on intracellular proteins if stabilizing solutes are present. This
balance between the effects of destabilizing
and stabilizing solutes is the basis of the counteracting
solute strategy (160). Counteracting
solutes are most effective when
they appear in a particular
ratio, a ratio that promotes
the optimal conformational
state of the enzyme. For

REVIEW

example, several enzymes display normal activity and


structure when the destabilizing effects of urea are offset
by methylamines.
The optimal ratio of urea to methylamines is 2:1, which is the ratio found in elasmobranch
tissues (162,163). A variation on the counteracting
solute
strategy is that of compensating
solutes (36). In this
scheme, stabilizing solutes appear to offset the detrimental effects of inorganic ions (35, 142). This has been
suggested as a role for intracellular
glycerol in unicellular
algae (1) and betaine in E. cob (148), organisms
that
accumulate K+ during RVI.
The grouping of organic solutes into the categories of
compatible or counteracting
solutes may not be entirely
valid. There are several situations
where compatible
solutes inhibit enzyme activity (108, llO), urea activates
enzymes (161), or solutes that should counteract
urea
fail to do so (71, 139, 161). In addition, the ability of a
solute to promote a compact protein conformation
may
not predict its effect on enzyme activity. For example,
Mishino and Fridovich
(110) have shown that TMAO
stabilizes catalase yet inhibits
its enzymatic
activity.
These exceptions should not be viewed as a repudiation
of the compatible/counteracting
solute hypothesis but a
testimony to the complex nature of solute-protein
interactions. Obtaining a coherent picture of these interactions is difficult, in part, because experimental
conditions
may alter the stabilizing
and destabilizing
effects of
solutes. Solutes that interact with hydrophobic
regions
of proteins, and thus disrupt them, can stabilize these
same proteins
at low temperatures
when hydrophobic
interactions
are minimized (4). Furthermore,
the destabilizing or stabilizing effects of solutes may be influenced
by the solute concentration
(32).
As can be seen from the above discussion,
organic
osmolytes
can have variable effects on proteins.
The
effects of these solutes on protein structure and function
have been examined in a relatively few enzymes, and it
is unlikely that all enzymes respond to all organic osmolytes in the same way. A more thorough understanding
of the interactions
between organic osmolytes and protein function requires further experimentation.
Regulation of intracellular organic solute concentration.
The accumulation or loss of organic osmolytes during
cell volume regulation is mediated by changes in cellular
transport and/or metabolism. As discussedabove, polyols
are involved in cell volume regulation in a variety of
organisms. The regulation of polyol concentrations has
been most thoroughly studied in algal and renal medullary cells. In the single cell alga, DunalieZla teriolecta,
glycerol is synthesized during RVI by the breakdown of
large, osmotically inactive starch molecules (61). This
process is rapid, with glycerol concentrations reaching
maximal levels after a 30-min exposure to hypertonic
saline (20). De novo protein synthesis is not required for
this response (23). Instead, it appears that glycerol-3phosphate dehydrogenase is stimulated by low ATP levels that are observed in the first 5 min of hypertonic
shock (20). During RVD the cells lose glycerol to the
external medium as well as metabolize this osmolyte
(164).
In another alga, Poterioochromonas malhamensis, iso-

INVITED
floridoside
(galactosylglycerol)
is accumulated
within
minutes of the onset of hyperosmotic
stress (reviewed in
Refs. 78, 156). This compound is synthesized
from a
storage compound,
chrysolaminarin.
The synthesis
of
this modified polyol depends on the action of isofloridoside phosphate synthetase,
an enzyme that is normally
stored in an inactive form. During hyperosmotic
stress,
cellular proteases activate the enzyme, and synthesis
of
isofloridoside
ensues. It is thought that calmodulin-dependent processes are involved in activating
the proteases. During hyposmotic
stress, the isofloridoside
is
reincorporated
into chrysolaminarin.
It has been suggested that sorbitol may play a role in
cell volume regulation in renal medullary cells. During
antidiuresis
the osmolality
in this region of the mammalian kidney can reach levels X,200 mosmol/kgH20
(90, 104) with a concomitant
increase in medullary sorbitol levels (68, 158, 159). Regulation of sorbitol levels
has been studied in cultured renal medullary cells. Unlike
algal cells, however, the accumulation
of polyols during
RVI in cultured medullary cells is quite slow, taking 1224 h before sorbitol levels rise (152). The carbon source
for sorbitol synthesis is thought to be exogenous glucose
(12, 118), although synthesis
from glycogen, which is
found in intact renal medulla (loo), has not been examined. Synthesis of sorbitol in cultured cells is dependent,
in part, on the induction
of aldose reductase,
which
converts glucose to sorbitol (l&12,19,152;
see SENSORS
AND TRANSDUCERS). In many cells, however, excess glucose is converted to glycogen. To assure that sorbitol is
synthesized from glucose, there must be low activities of
glucokinase and other enzymes involved in glycogen synthesis. This aspect of glucose metabolism
in cultured
renal cells has not been described. If sorbitol is synthesized from glycogen, glycolysis may be modified to increase the levels of glucose 6-phosphate,
which, in turn,
is converted to glucose and ultimately sorbitol. Funneling
carbons to sorbitol synthesis will be potentiated if there
is increased synthesis
and/or decreased degradation of
glucose 6-phosphate.
This aspect of sorbitol metabolism
has not been examined in renal cells but has been extensively studied in freeze-tolerant
insects that synthesize
sorbitol, a cryoprotectant,
from glycogen. During the
period of sorbitol accumulation in these organisms, there
is increased activity of glycogen phosphorylase
and hexokinase (see Ref. 144). At the same time, the catabolism
of glucose 6-phosphate
is restricted
because phosphofructokinase
is inhibited. This inhibition is due, in part,
to a fall in fructose 2,6-bisphosphate,
an activator
of
phosphofructokinase.
This modulator might be involved
in the regulation of renal glycolysis (85) and therefore
may ultimately
affect sorbitol synthesis
in renal cells.
Another point of control in the synthesis of renal sorbitol
may be the concentration
of urea in the medulla. Rabbit
muscle phosphofructokinase
is sensitive to urea and may
not be protected from ureas inhibitory
effects by trimethylamines (71). During antidiuresis,
medullary levels of
urea are very high. It is tempting to speculate that if
renal phosphofructokinase
is similar to muscle phosphofructokinase,
then the presence of high urea concentrations during antidiuresis
would inhibit phosphofructo-

REVIEW

Cl63

kinase and thus facilitate sorbitol synthesis.


In contrast to RVI, sorbitol levels are rapidly adjusted
during RVD. Bagnasco et al. (11) have shown that renal
medullary
cells transferred
from 600 to 300 mosmol/
kgH,O saline lose 50% of their sorbitol in 15 min. The
mechanisms
of this sorbitol efflux have not been elucidated.
In addition to sorbitol,
inositol levels in the renal
medulla fluctuate with the diuretic state of the animal
(68, 158, 159). Studies on cultured renal cells reveal that
hyperosmotic
media stimulate the Na+-dependent
uptake of inositol (119). Interestingly,
chronic hypernatremia is associated with elevated levels of renal inositol
(106) and may reflect the Na+ dependency of the uptake
mechanism.
Inositol appears to play an important role in the maintenance of brain cell volume. Lohr et al. (106) reported
a 60% increase in rat brain inositol levels during chronic
hypernatremia.
Cultured rat glial cells also show dramatic increases in inositol levels during exposure to a
hypertonic growth medium (K. Strange, R. Morrison,
C.
W. Heilig, and S. R. Gullans, unpublished
observations).
Amino acids are involved in volume regulation in a
wide variety of organisms, but most studies have focused
on animal cells. Euryhaline
marine invertebrates
regulate intracellular
levels of amino acids during osmotic
stress (reviewed in Ref. 58). Analogous to the process in
the alga, Dunaiella, some workers
have proposed that
amino acids may be derived from an osmotically inactive
macromolecule,
protein. Bishop et al. (21) suggest that
normal protein turnover
in conjunction
with decreased
amino acid efflux could account for amino acid accumulation in hyperosmotic
conditions.
Deaton (43) showed
that protease inhibitors
decreased the accumulation
of
free amino acids in isolated gills of the bivalve mollusk
Geukensia demissa (formally Modiolus demissus). In addition, Deaton et al. (44) demonstrated
a correlation
between aminopeptidase
activity
and accumulation
of
free amino acids in tissues of another bivalve, Mytilus
edulis, exposed to hyperosmotic
conditions.
In both of
these mollusks,
alanine is the predominant
amino acid
accumulated
during the first hours of hyperosmotic
stress. Most other amino acids do not change in concentration (43, 44). No specific alanine-rich
storage protein
has been described, and if general protein catabolism is
the source of amino acids, individual
free amino acid
levels should increase in proportion
to their representation in proteins. Bishop et al. (21) have suggested that
protein-derived
amino acids could be metabolized to alanine. The evidence for this process, however, is lacking.
Baginski and Pierce (9) have suggested that alanine is
synthesized
from glycogen during hyperosmotic
stress.
Therefore glycogen could be considered to be the osmotically inactive storage molecule for amino acid synthesis.
Nitrogen,
which is required for amino acid synthesis,
could be derived from transamination
reactions or uptake
of NH: from seawater. In support of this later mechanism, Armstrong
et al. (7) have shown that NH: uptake
was increased during hyperosmotic
stress in the prawn,
Machrobrachium
rosenbergii.
In addition to using intracellular
storage molecules as

Cl64

INVITED

a carbon source for amino acid synthesis, amino acids or


their precursors could be transported
into the cells during
hyperosmotic
stress. Relatively little is known about the
transport
of amino acids or their precursors
during RVI
in animal cells. The net uptake of alanine into isolated
crab axons during hyperosmotic
stress has been reported
(reviewed in Ref. 58). This increased uptake is due to
increased influx, with no change in efflux. It has been
suggested that uptake of amino acids from the environment or extracellular
fluids may be important
in shortterm acclimation
of euryhaline invertebrates
to hyperosmotic conditions (41, 145).
In contrast to RVI, the role of amino acids in RVD in
animal cells has been more thoroughly
explored. The
coelomocytes
of the bloodworm,
Glycera dibranchiata,
and the red blood cells of the blood clam, Noetia ponderosa, appear to be excellent systems to examine volume
regulatory processes in invertebrates.
Costa et al. (39)
demonstrated
a significant decline in free amino acids in
the coelomocytes of Glycera when the worms were acclimated to low salinities. Acute exposure of isolated cells
to low salinity resulted in the specific loss of proline and
taurine (39). Efflux of these amino acids is potentiated
by the removal of Ca2+ from the seawater and is unaffected by metabolic inhibitors
or ouabain (38). Taurine
efflux is dependent on Na+ and Cl-, and it appears that
more than one Na+ is transported
with each taurine
molecule (132). Pierce and co-workers
(2, 3, 141) have
studied volume regulatory processes in the red blood cells
of N. ponderosa. Although both Glycera and Noetia cells
lose amino acids during RVD, there appear to be several
differences between these two systems. Unlike Glycera
coelomocytes, no specific amino acid (e.g., taurine) is lost
from Noetia red blood cells. Instead individual
amino
acids are lost in proportion
to their original concentration in the cells (3). The presence of metabolic inhibitors
or deletion of extracellular
Ca2+ inhibits
amino acid
efflux from Noetia cells bathed in hyposmotic
seawater
(3
Taurine is involved in RVD in vertebrate cells. During
exposure to hypotonic solutions, there is a loss of taurine
from teleost (51) and elasmobranch
(101) red blood cells
as well as from Ehrlich ascites tumor cells (81). Similar
transport
mechanisms
have been described for taurine
efflux in teleost red blood cells and Ehrlich ascites tumor
cells, and an overview of only the fish red blood cell is
covered here. Fincham et al. (51) have examined volumesensitive taurine transport
in flounder and eel red blood
cells. During RVD, the NaCl-dependent
amino acid uptake pathway in these cells is inhibited while the NaClindependent amino acid permeability
is increased (51).
This results in a net loss of taurine from the cell. The
change in the NaCl-independent
pathway is selective for
small amino acids, including
glycine, y-aminobutyric
acid, alanine, and taurine and is thought to represent the
efflux pathway.
In addition to net efflux of amino acids during RVD,
amino acids are lost by oxidation.
There are several
reports of increased oxidation of 14C-labeled amino acids
and increased ammonium excretion during acclimation
of marine invertebrates
to dilute seawater (reviewed in

REVIEW

Ref. 58). Ballantyne


and co-workers
(15, 17, 117) have
demonstrated
that low osmolality stimulates
glutamate
oxidation in mitochondria
isolated from osmoconformers
and suggested that increased mitochondrial
oxidation
may be involved in acclimation to hyposmotic conditions.
An aspect of volume regulation that has received little
attention is the relative role of d- and L-amino acids in
RVI and RVD. Many studies employ chromatographic
techniques to determine the amino acids lost or accumulated during volume regulation.
These techniques,
however, do not separate isomers of amino acids. The disomers can be determined with d-amino oxidases, and
such analyses have revealed that some invertebrates
have
high levels of d-amino acids (131). Whether
these disomers are involved in cell volume regulation in invertebrates as well as other organisms remains to be determined.
Accumulation
of methylamines
has been most extensively studied in bacteria. During hyperosmotic
stress,
E. coZi take up betaine and proline betaine from the
medium (98). In addition, E. coli synthesize
betaine if
choline or glycine betaine aldehyde is present in the
medium (146). Control of these processes are discussed
below.
Metabolic constraints
on the use of organic osmolytes.
As can be seen from the previous section, a particular
organism uses only a few of the many suitable organic
osmolytes. In addition, cells of different tissues in multicellular organisms may use different organic osmolytes
for cell volume regulation. Because any organic osmolyte
could be involved in RVI or RVD, the use of a particular
osmolyte must occur for some reason. In this section we
speculate on the metabolic and environmental constraints that might determine the use of an individual or
specific class of organic osmolytes.
Several organic osmolytes, such as TMAO, urea, ,&
alanine, and taurine, are terminal products of metabolic
pathways. Other solutes, such as sorbitol, glycerol, and
proline, are produced by pathways that diverge from
mainstream metabolism, such as glycolysis or citric
acid cycle. In either case it is possible that these solutes
could be accumulated and lost without radically affecting
the overall flux through major metabolic pathways.
These processes do cost the cell energy, however, since
synthesis of organic osmolytes, which may be subsequently lost from the cells, diverts metabolites from
energy-producing pathways. There have been few studies
on the metabolic cost of using organic solutes in cell
volume regulation. Raven (134), however, has explored
this issue with regard to plant cell volume maintenance.
He concludes that the use of organic osmolytes costs the
cells (in units of photons) ten times more than the use
of inorganic ions. Presumably this high cost is offset by
the relative compatibility of many organic solutes with
protein structure and function. The alternative, volume
regulation with perturbing inorganic ions, requires the
costly synthesis of new proteins that would function in
an altered ionic environment (160).
The use of a particular osmolyte may be under some
environmental control. Raven (134) calculates that syn-

INVITED

thesis of proline or betaine by plants is more energy


efficient if the nitrogen source is NH: rather than NO;.
He also suggests that in low-nitrogen soils sorbitol may
be synthesized instead of nitrogenous solutes. In a similar
vein, animals that have a high-nitrogen
diet may be able
to afford the use of nitrogenous solutes in cell volume
regulation. For example, elasmobranchs are carnivorous
and utilize urea and methylamines
as osmolytes. In addition, urea is a waste product that is not oxidized and
therefore can be involved in two functions, osmoregulation and nitrogen balance. Another environmental
factor
that may affect solute accumulation
is oxygen tension.
For example, the solutes used by the amoeba, Acanthamoeba castellanii, for RVI are sensitive to environmental
oxygen levels. Under oxygenated conditions amino acids
are accumulated, whereas under anoxic conditions cell
volume appears to be regulated but amino acids are not
involved. It is suggested that synthesis of amino acids in
ATP-limited
anoxic conditions may be energetically unfeasible (57). Precursor availability
may be another factor that determines the specific organic osmolyte used
during cell volume regulation. Cultured renal (MDCK)
cells increase intracellular
inositol and glycerophosphorylcholine levels 1.5-fold when exposed to solutions made
hypertonic by the addition of 150 mM NaCl. Sorbitol
levels are unaffected by this treatment. If, however, the
solution is made hypertonic with 300 mM glucose, sorbitol and glycerophosphorylcholine
levels rise 1,200 and
685%, respectively. Inositol levels increase only 5% under
these conditions
(24). Freshly dissected rat papillary
tubules also increase sorbitol accumulation
in solutions
made hypertonic by high concentrations of glucose (158).
The specific organic osmolytes used by bacteria also
depend on precursors in the medium. In the absence of
exogenous choline or betaine, hypertonically
stressed E.
coli synthesize glutamate
and trehalose. If choline or
betaine is present in the medium, then hypertonic stress
stimulates the uptake and/or synthesis of betaine (146;
also

see

SENSORS

AND

REVIEW

Cl65

Discussion in the preceding sections indicates that an


important
area for further investigation
should involve
studies of the coordination
between inorganic and organic osmolyte regulatory mechanisms. Many organisms
and cell types, such as bacteria, invertebrates, algae, and
Ehrlich ascites cells, utilize both inorganic ions and
organic compounds for volume maintenance. For example, the unicellular
alga Platymonas subcordiformis appears to use mannitol, Na, K+, and Cl- for the regulation
of cell volume (89). Small hyposmotic shocks are compensated for in this organism by the loss of intracellular
Na, K+, and Cl-, whereas large hyposmotic stresses
result in the efflux of both inorganic ions and mannitol.
This observation suggests that there are different set
points for controlling organic and inorganic efflux pathways. Exposure of this organism to hy-perosmotic stress
results in a rapid, transitory increase in intracellular
NaCl and a slower permanent accumulation of mannitol.
These results suggest that readily available inorganic
ions mediate short-term volume regulation but that longterm volume maintenance depends on the accumulation
of more compatible organic solutes. Similar phenomena
have been observed in some euryhaline invertebrates (38,
128, 141, 155) and are likely to occur in many other
organisms and cell types, including cells of the mammalian renal medulla. Both intertidal organisms and renal
medullary cells can be exposed to relatively rapid and
large hyperosmotic
stresses, but the accumulation
of
organic solutes is in many cases rather slow (for examples
see Refs. 8,44,152). RVI could be similarly slow in these
cell types or it could initially be mediated by inorganic
ion transport pathways. Studies are needed in a variety
of cell types to determine the relative contribution
made
by organic and inorganic osmolytes to volume regulation,
the relative rates at which these two classes of osmolytes
are lost or accumulated during volume stress, and the
cellular signals that regulate the intracellular
concentration of these solutes.

TRANSDUCERS).

Another constraint on the particular osmolyte used is


the interaction of that solute with the metabolism of the
cell. As discussed earlier, osmolytes can alter enzyme
function, and there is evidence that mitochondria
are
sensitive to their solute environment
(reviewed in Ref.
14). Ballantyne and Moon (16) have shown that TMAO
inhibits mitochondrial
oxidation of the carnitine esters
of fatty acids, presumably because of the structural similarity between carnitine and TMAO. If this is a general
phenomenon, TMAO may not be an important osmolyte
in tissues that oxidize long-chain fatty acids. Interestingly,glycerophosphorylcholine,
which resembles TMAO
and carnitine, is found in renal medullary cells (10, 68,
159) that have a limited capacity for lipid oxidation (157).
Because organic osmolytes may affect some enzymes or
mitochondria,
their fluctuating
concentrations
during
cell volume regulation may modulate cellular metabolism. This aspect of metabolic regulation has not been
examined, and studies that correlate osmolyte effects on
isolated enzymes or on mitochondria
with their effects
on cellular metabolism need to be conducted.
Coordinated use of inorganic and organic osmolytes.

SENSORS

AND

TRANSDUCERS

The mechanisms

by which cells sense osmotically

in-

duced changes in turgor pressure, volume, and/or intra-

cellular composition and transduce those signals into a


regulatory response is an important and exciting area of
research. A study by Lohr and Grantham (105) demonstrated the exquisite sensitivity of volume sensors and/
or osmosensors in rabbit proximal tubules. These investigators demonstrated that proximal tubule cells could

sense alterations in cell volume and/or intracellular


composition of ~3% and/or transmembrane
osmotic gradients on the order of a few milliosmoles.
In contrast to
proximal tubule cells, which normally experience only
minor fluctuations in extracellular osmolality, other cells
may experience large anisosmotic stresses. For example,
euryhaline
osmoconformers
can experience seasonal,
daily, or hourly fluctuations
in the osmolality
of their
extracellular
fluids. Do the volume sensors and/or osmosensors in their cells display the same sensitivity as
proximal tubule cells or are larger osmotic gradients
needed to elicit a volume regulatory response? Further
studies are needed in this area.

Cl66

INVITED

Another important area in need of further research


concerns the role of volume sensors and/or osmosensors
in cell growth. Parmelee and Beebe (126) have demonstrated that K+ efflux is reduced in differentiating lens
fiber cells. This reduced K+ permeability is accompanied
by intracellular K+ accumulation, which causes water
influx, increased cell volume, and cell elongation. Does
the sensitivity and set point of volume sensors and/or
osmosensors change during cell growth? In the following
sections we review several possible mechanisms by which
a cell senses its volume and subsequently activates
RVD or RVI.
Stretch-activated channels. Stretch-activated (SA) ion
channels are a likely mechanism by which cells sense
and respond to volume changes. Patch-clamp techniques
have demonstrated that SA channels are activated by
small changes in hydrostatic pressure applied to the
patch pipette. These channels have been identified in a
wide variety of cells and tissues and have been postulated
to play a role cell volume regulation. Cahalan and Lewis
(28) have described a Cl- channel in T lymphocytes that
is activated by exposure of cells to a hypotonic medium
and inactivated by cell shrinkage and application of
suction to the patch pipette. This channel had an anion
selectivity sequence similar to the Cl- conductance previously shown to mediate RVD in these cells (63). In
giant spheroplasts of E. coli, Martinac et al. (109) have
observed a large-conductance (970 pS), voltage-sensitive
anion channel that is activated by hydrostatic pressure
as low as -10 mmHg. Patch-clamp studies in Necturus
choroid plexus have demonstrated the presence of an SA
Ca2+channel that is sensitive to hydrostatic pressures of
-5 to -15 mmHg (34). A stretch-insensitive Ca+-activated K+ channel has also been shown to be present in
these cells (34). This K+ channel is activated by cell
swelling in the presence but not in the absence of extracellular Ca2+ (34). Christensen (34) has postulated that
cell volume increase activates the Ca2+channel, resulting
in Ca2+ influx that in turn activates the K+ channel.
Loss of cellular K+ through the channel would presumably mediate RVD. Patch-clamp studies by Sackin (136)
have shown that Necturus proximal tubule cells possess
a basolateral K+ channel reversibly activated by negative
hydrostatic pressures of -9 to -15 mmHg. This channel
may the same as the Ba2+-inhibitable K+ channel postulated to be responsible for RVD in these cells (149).
The sensitivity of SA channels to membrane stretch
is noteworthy. Neglecting the contribution of membrane
folding, Sackin (136) calculated that the hydrostatic
pressures required to activate the K+ channel in Necturus
proximal tubule could be generated by cell volume increases as small as 1%. Hudson and Schultz (84) have
observed increased Cl- channel activity in Ehrlich ascites
cells swollen by as little as 5% during stimulation of
Na-coupled glycine uptake. These studies, together with
the work of Lohr and Grantham (105), clearly point to a
role for SA channels in sensing and responding to cellular
volume challenges.
It is not known whether activation of specific volumeregulatory channels is accompanied by modification of
resting membrane transport processes. Recently Morris

REVIEW

and Sigurdson (116) have described a new class of channels that is inactivated by membrane stretch. These
stretch-inactivated channels coexist with SA channels in
neurons. It is tempting to speculate that these channels
may indirectly play a role in volume regulation. For
example, cell swelling could activate specific volume regulatory K+ and Cl- channels and concomitantly inactivate resting K+ and Cl- conductances. Such a process
might allow the cell to more effectively control membrane function during volume regulation.
Cytoskeleton. Several investigations have implicated a
role for the cytoskeleton in cellular volume maintenance
(see Refs. 37, 52, 59). The function of the cytoskeleton
is likely to be indirect. One possibility is that cytoskeletal
elements may mediate the movement of cytoplasmic
vesicles containing volume regulatory transport proteins
to and from the plasma membrane. Endo- or exocytosis
of these proteins would then control volume regulatory
solute movements. Studies by Lewis and de Moura (103)
provide indirect support for a role of cytoskeleton-mediated vesicle translocation in anisosmotic volume regulation. These investigators demonstrated that exposure
of the rabbit urinary bladder to dilute salines caused a
dramatic increase in apical membrane area as measured
by changes in membrane capacitance. Reexposure of
bladders to isosmotic saline resulted in a return of apical
membrane area to control values. These area changes
appeared to be dependent on an intact microfilament
network, since they were blocked by cytochalasin B.
Whether these cells actually regulate their volume and
whether the observed changes in apical membrane area
are associated with RVD has not been shown.
An interesting possibility is that the cytoskeleton could
act as a sensor of volume-induced changes in membrane
tension and/or stretch. Sachs and co-workers (67, 135)
have attempted to elucidate the factors that determine
the stretch sensitivity of SA channels. These investigators postulated that the sensitivity of a SA channel is
increased when the force from a large membrane area is
gathered to a channel via attachment to submembrane
cytoskeletal elements. Guharay and Sachs (67) provided
experimental evidence supporting this idea by demonstrating that cytochalasins dramatically alter the tension
sensitivity of chick skeletal muscle SA channels.
Volume-induced membrane structural changes. Changes
in the volume of a cell should directly alter the structure
of the plasma membrane by changing the distribution
and packing of membrane lipids and proteins. Such
structural changes could, in theory, directly alter membrane transport pathways and/or signaling mechanisms
involved in volume regulation. Parker and co-workers
(reviewed in Ref. 125) have shown that volume perturbations alter the accessibility of reactive groups on dog
red blood cell membranes to glutaraldehyde or sulfhydryl
reagents. For example, exposure of shrunken cells to Nphenylmaleimide locks the volume regulatory Na+-H+
exchanger in the on mode so that the activity of the
transporter becomes independent of cell volume (124).
In walled organisms such as algal cells, changes in membrane structure have been proposed to be responsible for
activating turgor-regulating processes. Zimmerman et al.

INVITED

(166) have suggested that changes in the compression of


the plasma membrane against the cell wall may block or
expose and/or activate volume regulatory solute transporters. Changes in the interaction of charged groups on
the cell wall and plasma membrane induced by osmotic
stress may also modulate volume regulatory mechanisms
(72). An electromechanical
model of turgor sensing
and signal transduction that could apply equally well to
both walled and nonwalled organisms has been proposed
by Zimmerman
and co-workers (reviewed in Ref. 165).
These investigators have shown that thinning of the cell
membrane in response to turgor and/or volume changes
results in a change in the internal electric field within
the membrane. Changes in electric field strength may
directly alter membrane functional properties (77).
Intracellular
signaling.
Several intracellular
signals
have been implicated in the control of cell volume regulation (Table 3). Intracellular
Ca2+ is thought to be
important
for activating volume regulatory K+-H+ exchange in Amphiuma red blood cells (31); taurine efflux
in molluscan (141) and elasmobranch
red blood cells
(101); and K+ conductances in lymphocytes
(65), Necturus gallbladder
(52, 54), and the urinary bladders of
frog (42) and toad (33). Both K+ and Cl- conductances
are activated by intracellular
Ca2+ in Ehrlich ascites cells
(82). Some studies have indicated that calmodulin
may
be involved in calcium-mediated
responses, since anticalmodulin drugs inhibit cell volume regulation (31, 52,
82). Studies on lymphocytes show that cell shrinkage or
increases in intracellular
Ca2+ stimulate
Na+-H+ exchange (64). Activation of this antiporter is not mediated
by inositol phosphates or protein kinase C. Nevertheless,
activation of another kinase related to protein kinase C
does occur during cell shrinkage (66). In elasmobranch
red blood cells, diacyl glycerol, but not inositol trisphosphate, is elevated during cell swelling, indicating that
activation of protein kinase C may control volume regulatory responses (111). Leukotrienes, which are metabolites of arachadonic acid, may also be involved in controlling volume regulation. Studies by Lambert et al. (96)
have demonstrated that cell swelling in Ehrlich ascites
cells stimulates leukotriene synthesis, which in turn activates volume regulatory K+ and Cl- efflux pathways.
In mouse kidney medullary thick ascending limbs, cell
shrinkage alone does not activate RVI processes (73). If,
TABLE

Cl67

REVIEW

however, the tubules are treated with vasopressin or an


analogue of cyclic AMP before cell shrinkage, parallel
volume regulatory Na+-H+ and Cl--HCOg
exchange
pathways are stimulated
(73, 74). These results imply
that vasopressin, acting via intracellular
cyclic AMP,
stimulates RVI mechanisms. Stimulation
of volume regulation under these conditions may be physiologically
important
in situ, since vasopressin titers are elevated
and medullary interstitial
osmolality is increased during
antidiuresis.
From the above discussion it is apparent that the
mechanisms that transduce cell volume changes to regulatory responses have been studied in relatively few cell
types. Janssens (86) postulated that intracellular
signals,
such as phosphoinositides,
diacylglycerol,
Ca2+, and
cyclic AMP arose early in eucaryotic evolution. Given
the probability
that osmotic stress was a physiological
challenge faced by the ancestral eucaryotes, it seems
likely that activation of volume regulatory mechanisms
may involve common signaling pathways in extant eucaryotes. Clearly, more work in this area is required.
Osmoregulatory
genes. Changes in gene transcription
and translation
are likely to play an important
role in
controlling
cell volume regulatory mechanisms.
Polymorphism of genes encoding for proteins involved in cell
volume regulation may also be crucial in determining the
adaptation and distribution
of a species throughout an
environmental
range with variable salinity (e.g., intertidal and estuarine organisms). Evidence for such genetic
control of cell volume maintenance has come from studies on mollusks, cultured renal papillary cells, and bacteria.
Studies on the intertidal
bivalve M. edulis have suggested an interesting means by which the genome may
play a role in cell volume regulation. Like many marine
invertebrates, M. edulis shows increased levels of intracellular amino acids in response to increases in external
salinity (44). As discussed earlier, one way in which
amino acid accumulation
may occur is by protein catabolism. In support of this hypothesis a number of investigators have shown that high salinity increases the
activity of the proteolytic enzyme aminopeptidase
I in
this organism (91, 114). Aminopeptidase
I is coded for
by the lap locus of the M. edulis genome. Natural populations of this bivalve show significant polymorphism
in

3. Intracellular signaling pathways implicated in control of cell volume


Signal

Ca2+

Pathway

Cell

K+-H+ exchange
Taurine
efflux
K+ conductance

Protein

kinases

Leukotrienes
Cyclic AMP

Cl- conductance
Na-H
exchange
Na+-H+
exchange
Taurine
efflux
K+ and Cl- conductances
Na+-H+
and Cl--HCO,

exchangers

Types

Amphibian
red blood cells
Molluscan
red blood cells
Elasmobranch
red blood cells
Amphibian
gallbladder
Amphibian
urinary
bladder
Ehrlich
ascites cells
Lymphocytes
Ehrlich
ascites cells
Lymphocytes
Lymphocytes
Elasmobranch
red blood cells
Ehrlich
ascites cells
Mammalian
renal cells

Refs.

31
141
101
52,54
33,42
82
65
82
64
66
111

96
73, 74

Cl68

INVITED

the lap gene (92). Genotypes that are either heterozygous


or homozygous for the lapg4 allele accumulate amino acids
faster when osmotically stressed than genotypes without
the allele (44, 76). The difference appears to be due to
the fact that the lapg4 allozyme has a 20% greater activity
per unit protein than other forms of the enzyme (94).
Populations
of 2M. edulis inhabiting
high-salinity
environments have been shown to have a higher frequency
of the lapg4 allele (91,93). The presence of this allele may
allow individuals
in these populations
to regulate cell
volume more effectively. Other possible adaptive roles of
this gene, such as nutritional
balance in different environmental settings, have, however, not been examined.
As described earlier, cultured renal papillary cells accumulate large amounts of sorbitol when exposed to
hypertonic media (26, 27). Sorbitol is synthesized from
glucose by the enzyme aldose reductase. Bedford et al.
(19) have shown that hypertonic stress causes a relatively
slow increase in the total amount of aldose reductase in
this cell line. After 3-4 days of exposure to a 600-mosM
medium, this enzyme accounted for ~10% of the total
soluble cellular protein. Induction
of aldose reductase
synthesis only occurred when extracellular
osmolality
was increased with impermeant
solutes that caused cell
shrinkage (152). Aldose reductase activity was strongly
correlated with intracellular
K+ concentration
as well as
with the sum of intracellular
K+ and Naf concentrations
(152). These results suggest that the trigger for induction
of aldose reductase synthesis is an increase in intracellular ionic strength.
Other than the studies on aminopeptidase
in mollusks
and on aldose reductase in renal cells, the role of protein
induction in cell volume regulation has not been extensively studied in many eucaryotes. There are, however,
several reports that implicate de novo protein synthesis
in volume regulatory responses. For example, Cronkite
et al. (40) found that the protist, Paramecium tetraurelia,
could not acclimate to hyperosmotic conditions if protein
synthesis was inhibited.
The accumulation
of proline
during salt stress in halophytes is thought to be due to
induction of A-pyrroline-5carboxylate
reductase (150).
Petronini
et al. (127) suggest that gene expression is
modulated when chick fibroblasts are acclimated to hyperosmotic conditions. During acclimation
to hyperosmotic stress, E. chlorotica slowly accumulates proline
betaine, and Pierce and Rowland (130) attribute this
slow time course to involvement
of the genome. Regulation of amino acid levels in the cells of marine invertebrates experiencing osmotic stress may take several days
or weeks (for examples see Refs. 3, 8, 44, 154). This slow
time course may indicate modulation of gene expression
during acclimation to new salinities. Studies of the role
of de novo protein synthesis during cell volume regulation seem warranted in these and other organisms.
In contrast to eucaryotic systems, extensive studies on
the genetic control of cell volume regulation have been
conducted on two closely related bacteria, E. coli and
Salmonella typhimurium.
Expression of a number of
genes in these organisms is regulated by extracellular
osmolality. Both ompF and ompC are genes that encode
for two outer membrane porins. Expression of ompF and

REVIEW

ompC in E. coli is increased and decreased, respectively,

by external hypertonicity
(87). Cairney et al. (30) and
Dunlap and Csonka (45 ) have demonstrated that expression of the proU gene that encodes for a high-affinity
betaine transport system is dramatically
increased in S.
typhimurium
within minutes after exposure to hyperosmotic media. A proline and betai ne transport system
encoded for by the prop ge ne is induced in both S.
typhimurium
(29) and E. coli (60,87). In E. coli a choline
uptake system is induced by hypertonicity
as well (147).
Choline is converted to betaine in these organisms bY
the action of two dehydrogenases : ch .oline dehydrogen .ase
and glycine betaine aldehyde dehydrogenase. Both enzymes are induced in osmotically stressed cells (97) .
As discussed earlier, the Kdp system is partially responsible for osmoregulatory
K+ uptake in E. coli. -This
transport system is dependent on the expression of five
closely linked genes (reviewed in Ref. 47). Three inner
membrane proteins are encoded by the kdpA, kdpB, and
kdpC genes, which form an operon. Two positive regulators of these genes are encoded for by the adjacent
kdpDE operon. Laimins
et al. (95) have conducted a
series of elegant studies on the induction of the Kdp
system. The results of these investigations demonstrated
that neither alterations in intracellular
or extracellular
K+ affected gene expression unless external K+ dropped
to a point that limited cell growth. When external osmolality was increased at constant K .+ using impermeant
solutes, however, kdpABC expression was increased. Elevation of extracellular osmolality with the highly membrane permeable solute, glycerol, had no effect on expression. These results demonstrate that hypertonici .ty with
associated decreases in turgor pressure increases the
transcription
of the kdpA&operon.
The mechanisms
by which these diverse bacterial
genes are controlled by external osmolality are not completely understood. Several intriguing models have, however, been put forth. Epstein and co-workers (47, 95)
have postulated a mechanogenetic
control mechanism.
Studies by these investigators on the Kdp system have
indicated that the kdpD gene encodes -for a 70-kDa
protein that spans the periplasmic space, whereas kdpE
encodes for a 26-kDa soluble protein. The kdpD protein
1Medium

Osmolality
\1
\1 Turgor
I

d
?i&@

expression

? Intracellular

K+
? Betaine

& uptake

fTuY!gor
FIG. 1. Proposed
control of gene expression
during turgor regulation
in eubacteria.
Decreased
turgor
stimulates
ka! expression
and K+
accumulation.
Increased
intracellular
K+ stimulates
proU expression
and inhibits
kdp expression.
See text and Refs. 47, 75, 148 for detailed
descriptions
of these events.

INVITED

is thought to be a dimer associated with two kdpE protein


molecules. Changes in turgor pressure have been postulated to alter the conformation
of the kdpD-kdpE
protein
complex, allowing it to interact with the kdpABC promoter sequence, which in turn increases transcription
of
this operon and synthesis
of the high-affinity
Kdp K+
uptake system.
Hall and Silhavy (70) have shown that the expression
of ompC and ompF in E. coli is regulated by a single
operon composed of the ompR and erzuZ genes. The ompR
gene product is a soluble bifunctional
protein that can
exist as either a monomer or multimer. The multimeric
form is required for ompC expression, whereas the monomeric form is required to express ompF. Changes in
external osmolality
alter the equilibrium
between the
two forms. The function of the enuZ gene product is less
clear (see Ref. X20), although Hall and Silhavy (70) have
postulated that it is located in the outer membrane and
functions as an osmosensor.
An interesting
relationship
between K+ and betaine
transport
has been described in E. coli and S. typhimurium by Sutherland
et al. (148). As discussed above,
hypertonicity
induces the Kdp system for K+ uptake in
these organisms.
Elevation of intracellular
K+ in turn
stimulates pro U expression
and represses transcription
of the Kdp system. Presumably
the stimulation
of betaine transport
occurs at external osmolalities
where
continued K+ accumulation
would be detrimental
to enzyme function. Sutherland et al. (148) postulated that a
regulatory protein that undergoes conformational
change
in response to intracellular
K+ levels regulates proU
expression. More recently, elegant studies by Higgins et
al. (75) have shown that increases in the degree of DNA
supercoiling
dramatically
increases pro U expression.
These investigators
also demonstrated
that hypertonicity increases DNA supercoiling
and suggested that accumulation of intracellular
K+ may directly alter DNA
topology and gene expression (see Fig. 1 for a summary
of these interactions).
SUMMARY

Maintenance
of cell volume in the face of a changing
osmotic environment
was a physiological
problem faced
by the earliest life forms. It seems reasonable, therefore,
to hypothesize
that there are common mechanisms
underlying volume regulatory
processes
in extant organisms. It has been difficult,
however, to confirm this
hypothesis
in this review since specific aspects of cell
volume regulation have been studied in only a few species. Throughout
this article we tried to indicate areas
that may be particularly
important in understanding
the
mechanisms and control of cell volume regulation in all
organisms.
All cells experiencing
osmotic stress must
have some means of sensing their volume so that they
can initiate mechanisms
to adjust their intracellular
osmolality. The nature of these sensors and the level of
their sensitivity
are poorly understood, and further work
in this area is clearly needed. The intracellular
signals
that activate volume regulatory responses are just beginning to be explored, and this promises to be an exciting
area of research. Genetic aspects of cell volume regula-

REVIEW

Cl69

tion have been examined in bacteria, but an understanding of the role of the genome in eucaryotic cell volume
regulation is in its infancy. The coordinated use of inorganic and organic osmolytes during cell volume regulation deserves more attention, and the effect of fluctuating
osmolyte concentrations
on cellular metabolism needs to
be explored. Solute effects on protein structure and function have been investigated,
but these studies should be
expanded and the biological consequences
of proteinsolute interactions
need to be elucidated.
A coherent picture of the evolutionary,
physiological,
metabolic, and environmental
constraints
on the mechanism of cell volume regulation employed by a particular
species is dependent on a better understanding
of this
process in a wide variety of organisms.
It is hoped that
this review will stimulate
more studies on cell volume
regulation and has served to emphasize the value of a
comparative
approach to cellular physiology.
We wish to thank
Drs. J.S. Ballantyne,
M.A. Bisson,
J.H. Crowe,
S.R. Gullans,
S.H. Hebert,
and P.K. Lauf for their helpful comments
during the preparation
of this review.
This work was supported
in part by National
Institute
of Diabetes
and Digestive
and Kidney
Diseases
Grant DK-37317
and funds from
the Ohio University
College of Osteopathic
Medicine.
Address for reprint
requests: M. E. Chamberlin,
Dept. of Zoological
and Biomedical
Sciences,
Irvine
Hall, Ohio University,
Athens,
OH
45701.
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