Regulating Cell Volume
Maintaining a consistent cellular volume may contribute to a cell's metabolism as well as to its shape
Biologists have often noted that, by its very nature, the cell is in constant danger of losing its life. The
cell is a semiporous sac of dissolved chemicals floating in a soup of dissolved chemicals-a situation that
makes it vulnerable to the effects of osmosis. Any imbalance in the number of dissolved particles
between a cell's interior and exterior can cause water either to rush in and burst the cell's membrane, or
to seep out, causing the cell to shrink.
Cell physiologists are coming to appreciate that changes in a cell's volume compromise more than just
the shapeor even the integrity-of a cell. In some instances, alterations in cell volume threaten the proper
functioning of an organ or tissue. The brain's function becomes impaired, for example, if its cells swell
beyond the capacity of the skull's hard shell. But in other cases, the consequences are somewhat more
subtle. Consider a flat cell, such as the fibroblast. Such a cell could take up many times its original
volume and become spherical without much danger of bursting. Yet even these cells respond quickly to
rectify even minor changes in volume. What could be the reason?
The answer, we are starting to discover, is that alterations in cell volume may have large-scale effects on
the cell's overall function. In order to avoid volume alterations, all cells have adapted mechanisms to
equalize the osmotic activity between their internal and external environments. For many years,
scientists believed these mechanisms relied mainly on the influx and outflow of small, charged inorganic
particles, primarily the ions of sodium, potassium, hydrogen and chloride. Organic molecules that
traverse membranes during the course of normal cellular function can also be osmotically active and so
affect cell volume. The reverse is true, too. Alterations in cell volume can affect the rate and even the
direction of metabolism. In recent studies in our laboratory and in others, biologists are coming to
recognize that cell-volume changes and metabolism are much more intertwined than previously thought.
In part, this new understanding comes from a re-examination of the osmotic pressures exerted by the
normal substrates and by-products of everyday metabolism. For example, amino acids, the components
of proteins, are transported across cell membranes and can therefore affect the dynamics of cell-volume
regulation. The incorporation of amino adds into proteins or the breakdown of proteins into their
constituent amino acids can have profound-and opposite effects on cell volume. In our own laboratory
and in collaboration with the laboratory of Dieter Haussinger at the University of Dusseldorf we are
finding that cells actually exploit cell-volumeregulatory ion transport to provoke cellvolume alterations
in order to affect other cellular functions, such as protein synthesis and protein breakdown.
One of the cellular functions affected by cell-volume changes has only recently been appreciated.
Studies have led investigators to find that alterations in cell volume have consequences that reach into
the very nucleus of the cell. Changes in cell volume can alter the activities of certain genes. In some
instances, the affected genes code for components of the cell-volume regulatory system, so the
connection is somewhat obvious. But in other instances, the genes affected participate in metabolic
activities that until now did not appear to have a relation to cell volume. Such findings hint at some very
subtle and previously underappredated interactions between a cell's physical and biochemical status.
Overcoming Osmosis
At its core, the problem of cell-volume regulation is the problem of overcoming osmosis. Osmosis is
the flow of water into or out of compartments separated by a semipermeable barrier in order to equalize
the concentration of osmotically active particles-dissolved particles that provoke the osmotic flux of
water-in the two compartments. This is the situation presented by a membrane-bound cell floating in a
watery medium.
Lang, F., and Waldegger, S.. (1997). Regulating cell volume American Scientist, 85(5) 456-463.
The relationship between osmosis and cell volume has been understood for over two centuries, thanks to
the experiments of an 18th-century English clergyman named Stephen Hales. In 1733, Hales injected
large volumes of water into an animal's veins and noted a marked swelling of the liver, kidneys and
other organs. This swelling, he saw, could be reversed by injecting salt water into the animal's veins.
What Hales's work hinted at, and what cell physiologists now take for granted, is that the cell's internal
disposition is determined in part by its relation to its external environment. When Hales injected water
into the animal's circulation, he was in actuality diluting the concentration of osmotically active particles
in the extracellular fluid surrounding the body's cells. Follow,ing the dilution, the cells contained more
osmotically active particles than the surrounding fluid, and osmotic pressure worked in the direction of
diluting the contents of the cells. To equalize the osmotic disparity, the cells had to admit water, which
caused the swelling. The effect was reversed when salt was put into the extracellular medium. Injecting
salt into the extracellular medium increased the number of osmotically active particles in the
environment of the cells. This restored the osmotic balance between the internal and external
environments of the cells. Under these circumstances, osmotic pressures dictated that the swollen cells
discharge water and shrink back to a normal size.
Hales's experiment served as a prototype for most experiments on cell volume to follow. Biologists still
test theories of cell volume by altering the osmolarity-the concentration of osmotically active particlesin the medium surrounding the cells. In the typical cell-volume experiment, a biologist will likely place
the cells in media containing various concentrations of osmotically active particles. When a cell is
placed in an isotonic solution-one whose concentration of osmotically active particles is equal to that of
the cell's interior-the cell's volume does not change. In a hypotonic solution, in which fewer osmotically
active particles are in the medium than in the cell's interior, the cell swells, as in Hales's initial
experiment. In contrast, in a hypertonic solution, the number of osmotically active particles in the
medium exceeds the number inside the cell, and water rushes out of the cell, thus concentrating the
osmotically active particles inside. In this instance, the cell shrinks.
Although it is experimentally expedient to move cells into solutions containing various concentrations of
osmotically active particles, it is not entirely realistic. Rarely are cells called on to respond to large
fluctuations of external osmolarity. The cells of certain fish that live at the boundaries between fresh
and salt water must respond to such fluctuations, as must some types of kidney cells. Other than these,
however, most cells face osmotic challenges not from changes in their external environments, but from
the normal cellular functions. For example, the synthesis of proteins from amino acids eliminates
osmotically active particles from the cell's interior by reducing the number of amino acids. This has
corresponding effects on cell volume. On the other hand, the breakdown of proteins increases the
number of amino acids inside the cell (and so the number of osmotically active particles), which affects
cell volume. Not only do internal changes result in changes in cell volume, but the traffic of particles
across the cell membrane also has effects on cell volume. The entrances and exits of osmotically active
particles required by the cell as it conducts its routine cellular functions also alter the relative number of
such particles inside and outside the cell, which in turn, affects cell volume.
Ions across the Membrane
During the course of normal cellular activity, cells take up and expel substances that can throw them into
osmotic imbalance. It appears that even the most routine interaction between a cell and its environment
can threaten this balance. An act as simple and necessary as taking in nutrients could imperil the cell,
were it not for the sophisticated mechanisms in place that restore ionic equilibrium.
For example, cells need to take up fuel in the form of carbohydrates and nutrients, such as amino acids.
But taking in these substances also increases the number of osmotically active particles inside the cell
and creates an osmotic imbalance. This influx of substances leads to cell swelling as water flows in,
owing to the osmotic imbalance. Since cell swelling compromises cellular function, excess cellular
osmolarity is corrected by a counterbalancing flux of inorganic ions. To compensate for the amino
acids, for example, the cell accumulates positively charged potassium ions in exchange for positively
charged sodium ions. At the same time, the cell membrane is made highly permeable to potassium ions,
which exit the cell, thus creating an excess in positive charge outside the cell. The surplus in external
positive charge drives negatively charged chloride ions out of the cell. At this point, the low
intracellular concentration of chloride outweighs the high concentration of organic substances.
Chloride-ion expulsion is followed by the outflow of water, which restores the cell to its proper volume.
The above illustrates a general rule of cell-volume regulation. The most powerful means available to the
cell for changing or correcting osmotic imbalances is an exchange of ions across the cell membrane. It
is no wonder, then, that so many different membrane proteins are devoted to shuttling inorganic ions
back and forth. Many different carrier or transporter proteins are embedded in the cell membrane in
order to bring about each type of exchange. For example, at least five different proteins exist just to
exchange sodium for hydrogen ions. Recently, four members of the sodium-hydrogen ion-exchanger
protein family were isolated and cloned, and their responsiveness to cell-volume changes was tested
directly.
Cell shrinkage activates three of these exchangers and inhibits one of them.
Membranetransport proteins have to be highly responsive to changes in cell volume, and their activity is
regulated accordingly
In addition to transporter proteins, ions and water traverse the membrane through channels, protein pores
inserted into the membrane when a substance needs to pass through the membrane. The pores are closed
when the need for transport is gone.
As cell physiologists continue to identify transporters and channels and to describe their properties, they
may inch closer to a question that has remained unsolved for all of these years. It is obvious to anyone
looking at a random collection of cells that they come in a variety of shapes and sizes. Fibroblasts and
red blood cells are flat or disc-shaped, respectively, whereas the white blood cells called lymphocytes
are plump and round. Clearly, the normal volume and shape for any given cell is dependent on its type.
Scientists have wished to know for years how any particular cell determines what its normal volume
should be and how departures from that norm are sensed. The answer may lie in part with the
differential regulation of the various transporters and carriers in different cell types.
Cell Volume and Metabolism
Whereas ion transport offers a rapid and powerful means to adjust intracellular osmolarity, the use of
ions is limited by their destabilizing influence on intracellular proteins. Since excessive accumulation of
ions during cell shrinkage jeopardizes the function of proteins, cells adjust intracellular osmolarity with
so-called osmolytes, organic substances that participate in metabolism or are specifically designed to
alter intracellular osmolarity without compromising other cellular functions. Among the osmolytes are a
class of compounds called polyols, which include inositol and sorbitol, methylamines, such as betaine
and glycerophosphorylcholine, as well as amino acids and their derivatives, such as taurine.
Beyond that, cell volume governs a wide variety of metabolic pathways, and biologists have become
increasingly aware that cell volume has a profound influence on the biochemistry of the cell.
As we have previously noted, the disposition of amino acids and proteins affects cell volume. By
breaking down proteins into amino acids, cells increase the internal concentration of osmotically active
particles. The reverse is achieved when proteins are formed from amino acids-the number of
osmotically active particles is decreased. The same is true of the synthesis and breakdown of many
large biological molecules. For example, the synthesis of glycogen, a large carbohydrate, from its
constituent glucose molecules or the breakdown of glycogen into glucose molecules also has
corresponding effects on cell volume.
Work in our laboratory and in that of others suggests that cells actively stimulate protein and glycogen
synthesis during cell swelling. This has the effect of reducing the number of osmotically active particles
inside the cell. In contrast, the dynamics in a shrunken cell are just the opposite-the breakdown of large
macromolecules, such as glycogen and proteins, is actually promoted in an attempt to increase the
internal concentration of osmotically active particles.
We think that cells must have acquired the ability to adjust their metabolism to cell-volume changes
very early in evolution. Chemical-messenger molecules, such as hormones, which appear much later in
the history of cellular evolution, may exploit these archaic patterns of cellular behavior to regulate cell
function. Insulin, which is secreted into the blood stream in response to high blood levels of the sugar
glucose, for example, stimulates the cotransport of sodium, potassium and chloride ions into liver cells
as well as the exchange of sodium and hydrogen ion. These ionic fluxes swell the cells, which in turn,
inhibits the breakdown of proteins and of glycogen inside of the cell.
The hormone glucagon is the functional opposite of insulin, and it initiates the opposite sequence of
events inside the cell. Glucagon activates ion channels in liver cells, shrinks cells and stimulates the
breakdown of proteins and glycogen. This is but one example of how hormones induce cell-volume
changes, which become, in effect, another message involved in the regulation of cellular function. In a
similar manner, cell-volume alterations help to signal the need for either cell proliferation, which is
preceded by cell swelling, or programmed cell death, which is paralleled by cell shrinkage.
In addition to the synthesis and breakdown of macromolecules, cell biologists are starting to understand
that volume changes can exert an influence on metabolism through a phenomenon called
"macromolecular crowding.” The cell is not just a sac filled with a dilute solution, but the enzymes and
other macromolecules are densely packed, hence the notion of crowding. Since the function of cellular
macromolecules crucially depends on their concentration, alterations of cell volume perturb cellular
function by diluting or concentrating macromolecules.
From these examples, it becomes increasingly clear that the physical state of the cell exerts some control
over biochemical processes. The exact way in which cell-volume changes exert their influence is as yet
poorly understood. In other instances, this connection is starting to come to light.
Mechanical Stress as Regulator
A cell's geometry is determined by much more than how much water it retains. Since the membranes of
animal cells offer very little in the way of support, cellular structure comes from an internal network of
protein fibers and tubules that forms a sort of cellular skeleton, the so-called cytoskeleton. The
cytoskeleton is likely hooked into the membrane and may even undergird at least part of its structure.
As such, it is conceivable that alterations in cell volume alter the stresses on the cytoskeleton.
In turn, the cytoskeleton may tug on or push into the membrane in such a way as to alter the ability of
embedded protein transporters and channels to admit and extrude osmotically active substances. This
picture has been brought into sharper focus recently by the discovery of mechanosensitive ion channels,
protein receptors embedded in the membrane whose activity can be altered by physical rather than
chemical pressures. Much of the evidence, however, suggests that many mechanosensitive channels are
fairly nonspecific and handle equally the flows of many positively charged ions, such as those of
potassium, sodium and calcium. This leads cell physiologists to conclude that these channels do not by
themselves contribute to cell-volume regulation. Other types of protein transporters, such as the
sodium-hydrogen exchanger, seem to have sites to which cytoskeletal components can bind.
Perturbations in the cytoskeleton may therefore physically alter the activity of such transporter
molecules.
Alterations in the cytoskeleton may themselves bring about changes in cell volume. Many studies on
the relation between the cytoskeleton and cell-volume regulation focus on fibers made of the protein
actin. Large actin filaments are composed of smaller actin molecules. Through the course of daily
cellular activity, actin filaments assemble and disassemble routinely. The disassembly of actin filaments
has been shown to coincide with a deactivation of potassium channels and an increase in the activity of a
protein that simultaneously transports sodium, potassium and chloride ions. Furthermore, the
cytoskeleton may play a role in inserting channels into the cell membrane. Finally, researchers have
found that the cytoskeleton provides tracks on which vesicles carrying membrane components travel to
and from the cell membrane. It is possible that mechanical forces that alter the cytoskeleton also alter
the intracellular transport of these membrane components.
Cell Volume and Genes
Many lines of evidence lately have indicated that alterations in cell volume result in alterations in the
activities of certain genes. Many of these genes code for proteins that are transporters of ions or
osmolytes. Others code for components of the cytoskeleton. But in several other cases, the genes
activated have no obvious or direct connection to cell-volume control. These include genes encoding
the so-called heat-shock proteins, which chaperone proteins around the cell or facilitate the proper
folding of others. It may be that the heat-shock proteins also serve to stabilize proteins in the face of
alterations in cell volume and volumedependent entry of salt. Several of the genes activated when the
volume is altered are of a general regulatory type and influence the activity of a wide variety of other
genes.
What is intriguing about these findings, in addition to the identification of specific genes sensitive to
volume changes, is the mechanism by which genes can be so affected. The problem of genetic
responses to volume change is especially perplexing, since the genes of animal cells are kept far away
from the general hubbub of cellular metabolism. Genes, in the form of DNA, are housed in a special
membrane-bound compartment called the nucleus.
Contact between a cell and its nucleus is generally carried out by chemical and ionic messengers, just as
the cell communicates with its exterior.
Many laboratories are now engaged in learning whether cell-volume changes result in molecular
dispatches to the nucleus, which in turn alter gene expression. It would certainly seem likely, and some
potential candidates have been identified, but as yet no definitive link has been made. It would also
seem likely, although it is not proved, that particular genes possess regulatory regions, tentatively called
cell-volume response elements, to which the volume-activated chemical messengers bind. As with other
compounds that alter gene activity, binding of the messenger to the response element alters gene
activity, either positively or negatively
In all, it must also be noted that issues surrounding cell-volume regulation, raised centuries ago by
Stephen Hales, seem to become increasingly complex with modernity. Alterations in cell volume have
cellular consequences more far-reaching than Stephen Hales could have ever imagined.
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