Florian Lang & Siegfried Waldegger. American Scientist. Volume 85, Issue 5. Sep/Oct 1997.
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.
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.
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. Following 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 particles-in 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.