PHYSIOLOGICAL REVIEWS   Vol. 78 No. 1 January 1998, pp. 247-306
Copyright ©1998 by the American Physiological Society

Functional Significance of Cell Volume Regulatory Mechanisms

FLORIAN LANG, GILLIAN L. BUSCH, MARKUS RITTER, HARALD VÖLKL, SIEGFRIED WALDEGGER, ERICH GULBINS, AND DIETER HÄUSSINGER

Institute of Physiology, University of Tübingen, Tübingen; Department of Internal Medicine, University of Düsseldorf, Düsseldorf, Germany; and Department of Internal Medicine and Institute of Physiology, University of Innsbruck, Innsbruck, Austria

I. INTRODUCTION
II. CELL VOLUME REGULATORY MECHANISMS
    A. Ions in Steady-State Maintenance of Cell Volume
    B. Volume Regulatory Ion Transport
    C. Osmolytes
    D. Further Metabolic Pathways Contributing to Cell Volume Regulation
III. INTRACELLULAR SIGNALING OF CELL VOLUME REGULATION
    A. Macromolecular Crowding
    B. Cytoskeleton
    C. Cell Membrane Stretch
    D. Cell Membrane Potential
    E. Cytosolic pH
    F. Calcium
    G. G Proteins
    H. Protein Phosphorylation
    I. Chloride
    J. Magnesium
    K. Eicosanoids
    L. pH in Acidic Cellular Compartments
    M. Gene Expression
IV. CHALLENGES OF CELL VOLUME CONSTANCY
    A. Alterations of Extracellular Osmolarity
    B. Alterations of Extracellular Ion Composition
    C. Energy Depletion
    D. Ion Transport Altered by Hormones and Transmitters
    E. Substrate Transport
    F. Metabolism
    G. Others
V. ROLE OF CELL VOLUME REGULATORY MECHANISMS IN CELL FUNCTIONS
    A. Erythrocyte Function
    B. Epithelial Transport
    C. Regulation of Metabolism
    D. Receptor Recycling
    E. Hormone and Transmitter Release
    F. Excitability and Contraction
    G. Migration
    H. Pathogen Host Interactions
    I. Cell Proliferation
    J. Cell Death
    K. Others
REFERENCES

	ABSTRACT

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Lang, Florian, Gillian L. Busch, Markus Ritter, Harald Völkl, Siegfried Waldegger, Erich Gulbins, and Dieter Häussinger. Functional Significance of Cell Volume Regulatory Mechanisms. Physiol. Rev. 78: 247-306, 1998.  To survive, cells have to avoid excessive alterations of cell volume that jeopardize structural integrity and constancy of intracellular milieu. The function of cellular proteins seems specifically sensitive to dilution and concentration, determining the extent of macromolecular crowding. Even at constant extracellular osmolarity, volume constancy of any mammalian cell is permanently challenged by transport of osmotically active substances across the cell membrane and formation or disappearance of cellular osmolarity by metabolism. Thus cell volume constancy requires the continued operation of cell volume regulatory mechanisms, including ion transport across the cell membrane as well as accumulation or disposal of organic osmolytes and metabolites. The various cell volume regulatory mechanisms are triggered by a multitude of intracellular signaling events including alterations of cell membrane potential and of intracellular ion composition, various second messenger cascades, phosphorylation of diverse target proteins, and altered gene expression. Hormones and mediators have been shown to exploit the volume regulatory machinery to exert their effects. Thus cell volume may be considered a second message in the transmission of hormonal signals. Accordingly, alterations of cell volume and volume regulatory mechanisms participate in a wide variety of cellular functions including epithelial transport, metabolism, excitation, hormone release, migration, cell proliferation, and cell death.

	I. INTRODUCTION

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With only few exceptions (416), the membranes of animal cells are highly permeable to water (212, 776). Animal cell membranes cannot tolerate substantial hydrostatic pressure gradients, and water movement across those membranes is in large part dictated by osmotic pressure gradients (406, 445, 608, 985, 1043). Thus any imbalance of intracellular and extracellular osmolarity is paralleled by respective water movement across cell membranes and subsequent alterations of cell volume.

As outlined below, most mammalian cells are bathed in extracellular fluid with almost constant osmolarity. Nevertheless, considerable alterations of extracellular osmolarity are encountered in a variety of diseases. Excessive alterations of extracellular osmolarity occur in kidney medulla during transition between antidiuresis and diuresis (63).

Even at constant extracellular osmolarity, cell volume constancy is compromised by alterations of intracellular osmolarity. A wide variety of metabolic pathways leads to cellular formation or dissipation of osmotically active substances. Moreover, transport across the cell membrane modifies cellular osmolarity and thus cell volume.

To avoid excessive alterations of cell volume, cells have developed and utilize a multitude of volume regulatory mechanisms including transport across the cell membrane and metabolism. These mechanisms are triggered by minute alterations of cell volume. They not only serve to readjust cell volume but profoundly modify a wide variety of cellular functions. Thus cell volume is an integral element within the cellular machinery regulating cellular performance.

It is the aim of this review to illustrate the functional significance of cell volume. To this end, a description of the volume regulatory mechanisms and the cellular functions sensitive to cell volume is followed by a discussion of the role of cell volume in several integrated cell functions.

This review does not consider volume regulatory mechanisms in prokaryotic cells or comparative aspects of cell volume regulation, and the reader may refer to the respective pertinent literature (84, 96, 128, 175, 225, 232, 236, 309, 336, 387-391, 395, 400, 604, 698, 763, 792, 852, 970, 1121, 1138, 1168).

Instead, the discussion focuses on the significance of cell volume for the performance of mammalian cells. Moreover, the paper stresses recent developments. For a more complete coverage of earlier literature, the reader may refer to previous reviews on cell volume regulation (114, 425, 539, 545, 682, 776, 778, 815, 817, 843, 911a, 971, 1061, 1168), osmolytes (63, 142, 144, 370), and the role of cell volume in regulation of cell function (495, 693).

For various functions, experimental evidence pointing to the involvement of cell volume and cell volume regulatory mechanisms is intriguing but far from conclusive. It is hoped that this review stimulates further experimental effort in this exciting area of research to clarify the many remaining questions.

	II. CELL VOLUME REGULATORY MECHANISMS

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Rapid changes of cell volume are usually caused by movement of water across the cell membrane (J_v), which is driven by a hydrostatic (p) and osmotic () pressure gradient and depends on the hydraulic conductivity of the cell membrane (L_p) depends on the effective concentration difference across the cell membrane (c) and the reflection coefficient () for each solute i where R and T are the gas constant and the absolute temperature, respectively. Application of the equations would require that the cytosol behaves as a dilute solution. This is not entirely true, as discussed elsewhere in detail (194-196, 357, 722). The cell membrane does not withstand hydrostatic pressure gradients exceeding 2 kPa (445), which is equivalent to 1 mmol/l c. Even though interaction of the cell membrane with the cytoskeleton may allow the hydrostatic gradient to become larger (382, 577, 828), the movement of water is mainly dictated by osmotic gradients across the cell membrane.

The L_p depends on the presence of water channels (aquaporins) in the cell membrane, a family of molecules, which are inserted into the cell membrane and allow the passage of water (6, 214, 270, 300, 349, 356, 494, 536, 565, 773, 980). The aquaporins are especially important in water transporting epithelia but may be expressed in nonepithelial cells. Even though osmotically driven water transport is an obvious requirement for osmotic cell swelling and cell volume regulation, water movement across the cell membrane is rarely a limiting factor in cell volume changes. Thus alterations of intra- or extracellular osmolarity are in general followed by the respective movements of water and alterations of cell volume.

Cell volume regulatory mechanisms are thus most conveniently disclosed by exposing cells to abrupt changes of extracellular osmolarity. If cells are exposed to hypotonic extracellular fluid, they initially swell as more or less perfect osmometers but then approach the original cell volume by so-called regulatory cell volume decrease (RVD). If cells are exposed to hypertonic extracellular fluid, they initially shrink like almost perfect osmometers but then may approach the original cell volume by so-called regulatory cell volume increase (RVI). It should be kept in mind, however, that exposure of cells to anisotonic extracellular fluid does not only modify cell volume but also the volume of intracellular organelles such as mitochondria (969). Furthermore, in parallel to cellular osmolarity, cellular ionic strength is altered even if extracellular ionic strength is kept constant. Thus the sequelae of osmotic alterations of cell volume are not necessarily identical to the consequences of isotonic alterations of cell volume.

Alterations of cell volume may be limited by constraints from extracellular tissue, as shown for the brain (1220), the heart (972), and renal proximal tubules (753, 755). More important, however, is the ability of cells to adjust intracellular osmolarity by ion movement across the cell membrane and by generation, breakdown, uptake, or release of organic substances.

Ions contribute the bulk of intracellular (mainly K⁺) and extracellular (mainly NaCl) osmolarity. Furthermore, ions contribute some two-thirds to cell volume regulation after rapid alterations of extracellular osmolarity (331, 1266). Thus ion transport across the cell membrane is of paramount importance for the regulation of cell volume. Largely because of volume regulatory ion transport, RVD and RVI are accomplished within minutes after exposure to anisotonic media.

To maintain their metabolic functions, cells have to accumulate a number of substances, such as proteins, amino acids, or carbohydrate metabolites. The concentration of these substances is higher within the cells than in extracellular fluid. The excess cellular concentrations of these organic substances are counterbalanced by lower intracellular ion concentration. Most cells extrude Na⁺ in exchange for K⁺ by the Na⁺-K⁺-ATPase. The cell membrane is only poorly permeable to Na⁺, and the exclusion of impermeable Na⁺ outweighs the cellular osmolarity created by impermeant organic solutes (double Donnan hypothesis; Refs. 721, 776). On the other hand, the cell membrane is highly permeable to K⁺. The exit of K⁺ creates an outside-positive cell membrane potential, which drives Cl out of the cell. The low intracellular Cl concentration compensates for the excess intracellular concentration of organic substances.

Inhibition of the Na⁺-K⁺-ATPase with ouabain eventually leads to cell swelling (see Table 2) because of dissipation of the Na⁺ and K⁺ gradients, depolarization of the cell membrane, and subsequent entry of Cl into the cells (688, 779). However, inhibition of the Na⁺-K⁺-ATPase does not always lead to a rapid increase of cell volume, which may remain constant (407, 779, 924, 1039) or even transiently decrease (16, 631, 919, 1131). A sequence of events leading to cell shrinkage after inhibition of the Na⁺-K⁺-ATPase includes increase of intracellular Na⁺ activity, reversal of Na⁺/Ca²⁺ exchange, increase of intracellular Ca²⁺ activity, subsequent activation of Ca²⁺-sensitive K⁺ channels, hyperpolarization (despite decrease of intracellular K⁺ activity), and cellular KCl loss. Obviously, the time required by ouabain to eventually cause cell swelling depends on Na⁺ entry. In thick ascending limb (519, 520, 1178) or diluting segment (444) of the nephron, for instance, swelling can be delayed by inhibition of Na⁺-K⁺-2Cl cotransport.

Under the influence of ouabain, hepatocytes and renal cortical cells are apparently able to maintain their cell volume by electrolyte accumulation in intracellular vesicles, which are subsequently expelled by exocytosis (1038, 1039, 1257-1260). The electrolyte accumulation is accomplished by a H⁺-ATPase in parallel to Cl channels (1039). At least theoretically, a H⁺-ATPase in the plasma membrane could similarly maintain cell volume by creating a cell negative potential difference across the cell membrane, thus driving Cl extrusion.

As indicated above, ion transport across the cell membrane is the most efficient and rapid means of altering cellular osmolarity. During cell swelling, cells extrude ions, thus accomplishing RVD, whereas during cell shrinkage, cells accumulate ions to achieve RVI. The activation of ion release during RVD is paralleled by inhibition of ion uptake mechanisms, and the ion uptake during RVI is paralleled by inhibition of ion release mechanisms. Thus the simultaneous stimulation of ionic mechanisms for RVD and RVI is largely avoided (927, 938). A tremendous amount of work has been dedicated to the elucidation of the ion transport systems in different tissues. A synopsis of tissue-specific transport systems is beyond the scope of this review and has been reviewed in detail elsewhere (682).

1. Regulatory cell volume decrease

The transport systems most often activated by cell swelling are separate K⁺ and anion channels. In several studies, the anion channels activated by cell swelling have been found to be nonselective, allowing the passage not only of Cl but also of HCO₃ (

2. Regulatory cell volume increase

The major ion transport systems accomplishing electrolyte accumulation in shrunken cells are the Na⁺-K⁺-2Cl cotransporter (

The cellular accumulation of electrolytes after cell shrinkage is limited because high ion concentrations interfere with structure and function of macromolecules, including proteins (25, 139, 192, 193, 413, 491, 564, 573, 1376, 1381). Furthermore, alterations of ion gradients across the cell membrane would affect the respective transporters. An increase of intracellular Na⁺ activity, for instance, would reverse Na⁺/Ca²⁺ exchange and thus increase intracellular Ca²⁺ activity, which would in turn affect a multitude of cellular functions (1226).

To circumvent the untoward effects of disturbed ion composition, cells produce so-called osmolytes, molecules specifically designed to create osmolarity without compromising other cell functions (37, 44, 141, 371, 384, 484, 629, 630, 714, 875, 1138, 1377, 1378). Unlike ions, organic osmolytes even at high concentrations are compatible with normal macromolecular function. Thus the term compatible osmolytes has been coined (129).

Three groups of osmolytes are used in mammalian cells: polyalcohols, such as sorbitol and inositol; methylamines, such as glycerophosphorylcholine and betaine; and amino acids and amino acid derivatives, such as glycine, glutamine, glutamate, aspartate, and taurine (141, 368, 370, 371, 629, 630, 714, 719, 724, 1077, 1381). Tissue-specific utilization of the various osmolytes has been reviewed elsewhere in detail (682).

Osmolytes are specifically important for cell volume regulation in renal medulla, where extracellular osmolarity may become more than fourfold that of isotonicity (62, 64-66, 99, 112, 141, 369, 399, 442, 452, 525, 666, 718, 724, 855, 1075, 1076, 1078, 1352, 1353, 1356, 1377, 1378), and in the brain, where cell volume alterations cannot be tolerated due to the rigid skull and where alterations of ion composition would affect excitability (453, 524, 715-717, 745, 746, 915, 1155, 1166, 1167, 1170, 1220, 1221, 1266, 1270).

Osmolytes not only replace ions as osmotically active species but also stabilize macromolecules, thus counteracting the adverse effects of inorganic (such as K⁺, Na⁺, and Cl) and organic (such as spermine) ions (26, 138, 139, 213, 334, 662, 1271, 1351). Furthermore, betaine and glycerophosphorylcholine, and to a lesser extent inositol, counteract the destabilizing effect of urea on proteins (145, 203, 384, 483, 750, 879, 966, 1119, 1380, 1382). For normal cell function, an appropriate balance must be maintained between destabilizing (i.e., ions, urea) and stabilizing (i.e., counteracting osmolytes) forces (19, 267, 807, 863, 1272, 1376, 1383). Accordingly, an increase of urea concentration specifically favors the parallel increase of glycerophosphorylcholine (723, 855, 966).

Beyond their function in cell volume regulation, osmolytes are protective against the destructive effects of excessive temperatures (34, 292, 352, 383, 534, 579, 759, 926, 989, 1058, 1160, 1193, 1200) and dessication (169, 232). Furthermore, they have been found to ease cell membrane assembly (642).

Cellular osmolyte accumulation can be achieved by stimulated uptake, enhanced formation, or decreased degradation. Decrease of intracellular osmolyte concentration is accomplished by degradation or release. As compared with RVI accomplished by ions, accumulation of osmolytes is a slow process taking hours to days.

6. Amino acids

In addition to taurine, the cellular concentration of several other amino acids and amino acid metabolites is modified by cell volume, including glutamine, glutamate, glycine, proline, serine, threonine, -alanine, (N-acetyl)aspartate, and GABA (for review, see Refs.

In addition to amino acids, numerous organic metabolites contribute to cellular osmolarity. Several metabolic pathways known to be sensitive to cell volume may modify the concentrations of these metabolites and thus contribute to cell volume regulation. Cell swelling increases glycogen synthesis and inhibits glycolysis, thus decreasing the concentration of carbohydrate metabolites (12, 49, 50, 696, 819, 953). Furthermore, cell swelling has a relatively weak stimulatory effect on lipogenesis (51). As detailed in section VC, cell volume changes interfere with a great number of other metabolic functions that to some extent may modify cellular osmolarity. The overall impact of these effects on cellular osmolarity is probably modest, but the influence of cell volume on various metabolic pathways is of paramount importance for regulation of metabolic function (see sect. VC).

	III. INTRACELLULAR SIGNALING OF CELL VOLUME REGULATION

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Cell swelling and shrinkage exert profound effects on intracellular signaling mechanisms, which in turn modify a multitude of cellular functions including the volume regulatory mechanisms. A great deal of experimental effort has been spent in elucidating the intracellular machinery underlying cell volume regulation. Frequently, the result has been inconclusive for several reasons. 1) Not every effect of altered cell volume on intracellular signaling is related to regulation of cell volume. 2) Cells usually use several mechanisms in parallel, with different, partially overlapping cellular signaling mechanisms. 3) Different cells utilize distinct mechanisms, i.e., the information gained in any given cell cannot necessarily be generalized to other cells. 4) Volume regulation requires mechanisms that are themselves not modified by cell volume changes but rather permissive for activation of cell volume regulatory mechanisms.

In the simplest case, an intracellular mechanism serves cell volume regulation if it is modified by alterations of cell volume, and a qualitatively and quantitatively identical modification of this intracellular mechanism triggers the appropriate alterations of cell volume. This requirement frequently is not met. If the identical modification does not trigger respective alterations of cell volume, the participation of a mechanism in cell volume regulation still cannot be ruled out, since the mechanism may require the collaboration of other mechanisms to be effective. Similarly, the use of inhibitors, even if they are specific, does not lead to conclusive results. On the one hand, inhibition of cell volume regulation by elimination of a given element of intracellular signaling (e.g., inhibition of protein kinases or removal of Ca²⁺) does not discriminate between volume regulatory and permissive mechanisms. On the other hand, cell volume regulation may prove insensitive to elimination of a volume regulatory mechanism if other mechanisms operating in parallel are strong enough to replace the defect. Further examples could be given, each of which illustrates that the experimental elucidation of the complex machinery serving cell volume regulation is extremely difficult.

Even though our understanding of the intracellular machinery mediating cell volume regulation is still incomplete, knowledge of the interaction between cell volume, elements of cellular signaling, and cell volume regulatory mechanisms is mandatory for understanding the role of cell volume for cell function.

Cell swelling leads to dilution and cell shrinkage to concentration of cellular constituents including proteins. The concentration of intracellular proteins markedly influences their function (131, 353, 376, 834, 836, 937, 1011). In erythrocytes, the volume regulatory set point can indeed be varied by manipulation of intracellular protein concentration (835, 837, 1397). The set points of both the KCl symport (835, 838) and the Na⁺/H⁺ exchanger (209, 210, 940) appear to be determined by macromolecular crowding. It has been suggested that among the enzymes sensitive to ambient protein concentration is a kinase that is inactivated by protein dilution during cell swelling and activated by protein crowding during cell shrinkage (835, 838). This kinase may inhibit the volume regulatory KCl cotransport. Its inactivation during cell swelling would then disinhibit volume regulatory KCl efflux.

Because of interaction of proteins with ambient electrolytes, macromolecular crowding is reduced by increasing ionic strength, which indeed shifts the volume regulatory set point to smaller volumes (942).

Similarly, urea decreases the thermodynamic activity of proteins and thus reduces macromolecular crowding (211, 574, 723, 837, 978, 984, 1376). Urea has been shown to activate erythrocyte KCl transport (293, 596, 936), erythrocyte Na⁺/Ca²⁺ exchange (936), and hepatocyte K⁺ channels (474) and to inhibit Na⁺/H⁺ exchanger in erythrocytes (936) and adenosine 3',5'-cyclic monophosphate (cAMP) production (58), Na⁺/H⁺ exchanger (732), and Na⁺-K⁺-2Cl cotransport (595) in thick ascending limb cells, thus leading to cell shrinkage. In erythrocytes, the effect of urea was reversed by okadaic acid, pointing to the involvement of phosphorylation (936).

A variety of ion channels are activated by cell membrane stretch, i.e., increased tension of the cell membrane (1040, 1043). Stretch increases the open probability of the channels without affecting single-channel conductance or selectivity of the channels (1043).

The stretch-activated channels may be selective for K⁺ or for anions, thus directly serving cell volume regulation (857, 1043). Most of these channels, however, are nonselective cation channels, allowing the passage of K⁺, Na⁺, and Ca²⁺ (for review, see Refs. 682, 1043). Because of the cell-negative cell membrane potential, the respective electrochemical gradients favor the cellular accumulation of Na⁺ and Ca²⁺ rather than cellular loss of K⁺. Thus these channels are not likely to directly serve cell volume regulation, and inhibition of these channels by gadolinium has been shown to decrease osmotic swelling and favor regulatory decrease of cell volume (1173). On the other hand, Ca²⁺ entering the cells through these channels is thought to activate Ca²⁺-sensitive K⁺ channels (187, 1194, 1234).

The mechanism linking membrane stretch to activation of the channels has not been clearly defined (1043). Under discussion are 1) release of fatty acids from the stretched membrane and subsequent activation of stretch-sensitive channels by these fatty acids (917) and 2) stretch-induced activation of some element of the cytoskeleton, such as spectrin (1133). Because stretch enhances channel open probability in the cell-free excised patch configuration (1043), cytosolic components are apparently not required for channel activation.

It is debatable whether stretch-activated channels participate in the fine-tuning of cell volume, since considerable stretch is required to activate these channels (1043). Possibly, these channels may represent a last line of defense against excessive cell swelling but are not involved in the response to moderate changes of cell volume.

The influence of cell swelling on cell membrane potential depends on the ion channels preferentially activated or inactivated and on the potential difference before cell swelling. Activation of K⁺ channels and a low initial cell membrane potential favor hyperpolarization, whereas activation of anion or nonselective cation channels and a high initial cell membrane potential would favor depolarization. After cell swelling, hyperpolarization of the cell membrane is seen in hepatocytes (406), depolarization of the cell membrane in Ehrlich ascites tumor cells (680, 691), Madin-Darby canine kidney (MDCK) cells (947), opossum kidney cells (1235), lymphocytes (418, 419, 1060), pancreatic -cells (124), astrocytes (627), neuroblastoma cells (313), and vascular smooth muscle cells (685). In some cells, a transient hyperpolarization due to activation of K⁺ channels is followed by a more sustained depolarization due to activation of anion channels (516-518, 1002).

The alteration of cell membrane potential may influence the activity of additional ion channels. A depolarization of the cell membrane may open voltage-sensitive ion channels. In lymphocytes, RVD involves n-type K⁺ channels (273, 429), which are activated by cell membrane depolarization. Depolarization of the cell membrane may further activate voltage-sensitive Ca²⁺ channels, as observed in pancreatic -cells (124) and vascular smooth muscle cells (685). The increase of intracellular Ca²⁺ could trigger a variety of further mechanisms, as illustrated in section V, E and F.

As discussed in section IIB, increase of extracellular osmolarity may either depolarize or hyperpolarize cells due to activation of unspecific cation channels or inhibition of K⁺ and/or Cl channels.

Cell swelling leads to cytosolic acidification (216, 397, 479, 610, 616, 685, 757, 864, 1002, 1089, 1143, 1279), which has been explained by the exit of HCO₃ through anion channels (1334), by release of H⁺ from acidic intracellular compartments (see sect. IIIL), and by enhancement of Cl/HCO₃ exchange due to decreasing cellular Cl activity (757). This latter mechanism, however, should be impeded by inhibition of the exchanger at acidic cytosolic pH (645), and cellular acidosis is not inhibited by removal of extracellular Cl, at least in osteosarcoma cells (864).

Cell shrinkage is frequently observed to alkalinize cells, at least partially due to activation of volume regulatory Na⁺/H⁺ exchange (see sect. IIB).

The impact of intracellular pH on volume regulatory mechanisms has not been explored. After cell swelling, the cytosolic acidification may impede activation of volume regulatory K⁺ channels (783) and may contribute to inhibition of glycolysis and thus to the decreased release of lactic acid (696). The alkalinization after cell shrinkage should stimulate glycolysis (696).

After cell swelling, intracellular Ca²⁺ concentration ([Ca²⁺]_i) increases in a variety of cells, whereas it remains apparently constant in others (for review, see Refs. 682, 815). Swelling may increase [Ca²⁺]_i by both activation of Ca²⁺-permeable channels in the cell membrane and Ca²⁺ release from intracellular stores. Calcium-permeable channels may be activated by cell membrane stretch (see sect. IIIC ), cell membrane depolarization (see sect. IIID), and/or protein kinase C (1066). Calcium release from intracellular stores is presumably triggered by inositol phosphates (52, 72, 1180, 1288, 1289) or Ca²⁺-induced Ca²⁺ release (516, 518). The regulation of [Ca²⁺]_i by cell volume may interfere with signaling of Ca²⁺-recruiting hormones, as shown for gastric parietal cells (885) and HT-29 cells (330). In those cells, agonist-induced entry of Ca²⁺ was further stimulated by cell swelling (330, 885) and inhibited by cell shrinkage (885).

The increase of [Ca²⁺]_i accounts for the activation of Ca²⁺-sensitive K⁺ channels, as shown in Ehrlich ascites tumor cells (188), MDCK cells (1334), proximal tubule cells (290, 605), thick ascending limb cells (1194), choroid plexus epithelial cells (187), and neuroblastoma cells (313). In MDCK cells, [Ca²⁺]_i did not appreciably increase upon moderate osmotic cell swelling, even though the Ca²⁺-sensitive K⁺ channels were already activated by this treatment (1002). Possibly small, localized increases of [Ca²⁺]_i are sufficient to activate the K⁺ channels but are not detected by fluorescence measurements (1357).

Despite the activation of Ca²⁺-sensitive K⁺ channels, RVD is apparently not mediated by a rise of [Ca²⁺]_i in Ehrlich ascites tumor cells (1204), and swelling-induced K⁺ efflux was virtually unaffected by the inhibitors of the Ca²⁺-sensitive K⁺ channels, clotrimazole and charybdotoxin (489). Because these K⁺ channels are inwardly rectifying (1334), K⁺ exit through these channels during cell swelling may be limited by the depolarization of the cell membrane. Other less inwardly rectifying K⁺ channels may thus be more important for cell volume regulation in those cells (539). In a variety of tissues, increase of [Ca²⁺]_i was not required for RVD (for review, see Ref. 682). Clearly, activation of K⁺ channels by Ca²⁺ may contribute to, but is frequently not crucial for, RVD (539).

In contrast to volume regulatory K⁺ channels in many tissues, volume regulatory Cl channels have been found to be insensitive to Ca²⁺ in intestinal cells (517, 657), ciliary epithelial cells (1385), cardiac myocytes (1227), T84 colon carcinoma cells (1134, 1364), airway epithelial cells (361, 1134), MDCK cells (48, 1029, 1334), lymphocytes (421), Ehrlich cells (188), and chromaffin cells (287). Yet, as shown in Ehrlich ascites tumor cells, Ca²⁺ triggers the formation of leukotrienes, which do activate the channels (539). In kidney cortex, Ca²⁺-sensitive Cl channels have been found in endosomes and proposed to serve cell volume regulation (991). Calcium is also thought to trigger the fusion of vesicles, allowing the release of sorbitol (629), whereas it is apparently not required for release of GPC (629) or taurine (1051).

Calmodulin antagonists and inhibitory peptides against Ca²⁺/calmodulin-dependent kinase (116) have been shown to inhibit RVD or activation of cell volume regulatory ion channels (71, 263, 421, 424, 543, 546, 815), and it has been concluded that calmodulin/Ca²⁺ complexes are important for activation of RVD. In other cells, however, Ca²⁺-sensitive K⁺ channels involved in cell volume regulation did not require calmodulin and were actually activated by calmodulin antagonists (950).

Beyond its putative role during RVD, calmodulin has been implicated in activation of the Na⁺-K⁺-2Cl cotransport in RVI (583).

Inhibitors of G proteins such as pertussis toxin or cholera toxin have been shown to blunt the RVD (539) as well as swelling-induced osmolyte efflux (1037), increases of intracellular Ca²⁺ concentration (30), mitogen-activated protein kinase (MAPK) activity (895, 1072), vesicular acidification (1088), and swelling-induced stimulation of taurocholate excretion (895), suggesting that G proteins do mediate some effects of cell swelling. Activation of the Na⁺/H⁺ exchanger during cell shrinkage has similarly been claimed to involve G proteins (108, 249).

In addition to heterotrimeric G proteins, small G proteins have been implicated in cell volume regulation. Clostridium botulinus C₃ exoenzyme, which depolymerizes the actin filament network by ADP-ribosylation of rho (8), blunts the volume regulatory anion efflux (1209). In neurons, osmotic cell shrinkage stimulates the expression of ₁-chimerin (286), a GTPase-activating protein that inactivates the small G protein Rac. The impact of this effect on cell volume regulation is not explored.

Decreased intracellular Cl activity is apparently required for full activation of Na⁺-K⁺-2Cl cotransport during cell shrinkage (539, 548, 1245, 1370). Beyond its influence on one of the driving forces of Na⁺-K⁺-2Cl cotransport, a decreased intracellular Cl concentration is thought to play a permissive role for the activation of both Na⁺-K⁺-2Cl cotransport (119, 734, 735, 1009) and Na⁺/H⁺ exchange (108, 250, 933, 934, 1009). If extracellular osmolarity is made hypertonic by increased extracellular NaCl concentration, the increase of intracellular Cl activity could thus impede RVI (539). Accordingly, some cells are unable to regulate their volume during exposure to hypertonic extracellular fluid (308, 437, 522, 539, 544, 545, 634). If intracellular Cl is lowered by prior RVD (308, 420, 522, 634) or by activation of Cl channels with cAMP (437, 1177) or vasopressin (351, 519, 1177), the same cells do accomplish RVI. Moreover, if cells are exposed to short-chain fatty acids, they swell by accumulation of the acids along with Na⁺ and are forced to release Cl for RVD (see Table 2). In the presence of these acids, they display RVI after exposure to hypertonic extracellular fluid (1016).

Intracellular Cl inhibits the glycogen synthase phosphatase (408), and the decrease of intracellular Cl activity participates in the stimulation of glycogen synthesis during osmotic or glutamine-induced cell swelling (555, 819).

The dilution and concentration of intracellular solutes during cell swelling or shrinkage lead to the respective alterations of Mg²⁺ concentration. Magnesium has been described to inhibit volume regulatory KCl cotransport (78, 295). During cell swelling, the decrease of intracellular Mg²⁺ concentration may partially account for the activation of KCl cotransport (78, 295). Conversely, an increase of intracellular Mg²⁺ activity stimulates Na⁺/H⁺ exchange (944) and Na⁺-K⁺-2Cl cotransport (794) and may thus participate in regulatory cell volume increase.

Cell swelling has been shown to activate phospholipase A₂ (542, 800), possibly in part through decrease of macromolecular crowding (539). Metabolites of arachidonic acid include the products of cyclooxygenase (e.g., prostaglandins), 15-lipoxygenase (such as hepoxilin A₃), 5-lipoxygenase [e.g., leukotriene (LT) D₄], and epoxygenase (epoxyeicosatrienoic acids) (675). On the other hand, as shown in Ehrlich ascites tumor cells, swelling stimulates the formation of the leukotrienes, namely, LTD₄ , at the expense of prostaglandins such as prostaglandin (PG) E₂ (675). Both phospholipase A₂ and 5-lipoxygenase are activated by Ca²⁺ (542, 675). Thus an increase of intracellular Ca²⁺ concentration during cell swelling could participate in the activation of these two enzymes during cell swelling. Along these lines, LTD₄ may overcome the inhibitory effect of the calmodulin antagonist pimozide on cell volume regulation, suggesting that LTD₄ is a signal downstream of Ca²⁺ (673).

Arachidonic acid has been shown to inhibit glial cell volume regulation (1052) and to inhibit volume regulatory Cl channels (403, 673, 679, 1047). On the other hand, it may increase Ca²⁺ concentration in renal collecting duct and activate K⁺ channels in neurons (622, 1213). Moreover, the 15-lipoxygenase product of arachidonic acid hepoxilin A₃ activates volume regulatory K⁺ channels in platelets (798-801), and the 5-lipoxygenase product LTD₄ activates volume regulatory K⁺ and Cl channels (547, 592, 674, 675, 679) and volume regulatory taurine release (592, 678) in Ehrlich ascites tumor cells, as well as taurine release in fish erythrocytes (1207). 5-Lipoxygenase products similarly appear to mediate regulatory cell volume decrease in colonic epithelium (277, 281, 282), chromaffin cells (287), MDCK cells (947), and human fibroblasts (808). However, in most of these cell types, the evidence comes largely from effects of 5-lipoxygenase inhibitors such as nordihydroguaiaretic acid (NDGA). In Ehrlich ascites tumor cells (679) and proximal renal tubules (Völkl and Lang, unpublished observations), on the other hand, the inhibitory effect of NDGA was not overcome by the addition of LTD₄ , pointing to an additional effect of the drug not related to 5-lipoxygenase inhibition. It may more directly inhibit the volume regulatory Cl channels (438) or be effective by increasing arachidonic acid concentration (1052).

The enhanced formation of leukotrienes in swollen Ehrlich ascites tumor cells parallels a decreased formation of PGE₂ , an effect possibly accounting for the inhibition of Na⁺ channels (679). Those channels are thought to be stimulated by PGE₂ (679). On the other hand, in ciliary epithelial cells, PGE₂ was thought to mediate the activation of volume regulatory K⁺ channels during cell swelling (191). Prostaglandin E₂ similarly activates K⁺ channels in MDCK cells (1153) and erythrocytes (743).

In collecting duct principal cells, inhibition of phospholipase A₂ with quinacrine blunted the activation of volume regulatory Ca²⁺-sensitive K⁺ channels, which, on the other hand, were activated by arachidonic acid (751). In LLC-PK₁ cells, however, volume regulatory rubidium flux was not modified by arachidonic acid, even though it was inhibited by an arachidonic acid antagonist (262).

Ketoconazole, an inhibitor of epoxygenase (cytochrome P-450), impedes volume regulatory efflux of osmolytes, such as sorbitol, betaine, myo-inositol, or amino acids from renal papillary cells (354), MDCK cells (38), and C6 glioma cells (818, 1171). However, the inhibitory effect is not reversed by addition of hydroxyeicosatetraenoic acids (818), indicating that the inhibitory effect on osmolyte flux is not due to inhibition of epoxygenase. Similarly, in Necturus gallbladder, ketoconazole does not prevent regulatory KCl efflux but stimulates NaCl entry, leading to cell swelling (615).

In addition to their influence on volume-sensitive ion channels, phospholipase A₂ and a cytochrome P-450 product of arachidonic acid have been invoked in mediating the swelling-induced cellular release of sorbitol (354).

The fatty acid composition of the cell membrane can be modulated by dietary polyunsaturated fatty acids, which lead to enhanced formation of leukotrienes and thus to acceleration of RVD in Ehrlich ascites tumor cells (712).

As evidenced from acridine orange and fluorescein isothiocyanate-dextran fluorescence, hepatocyte swelling leads to alkalinization of acidic cellular compartments, whereas cell shrinkage enhances the acidity in those compartments (156, 683, 1088, 1089, 1279, 1280).

The lysosomal proteases are known to have their pH optimum in the acidic range, and alkalinization of the lysosomes is well known to inhibit hepatic proteolysis (859, 860). Thus the alkalinizing effect on acidic cellular compartments could at least in theory contribute to the antiproteolytic action of cell swelling. Along these lines, alkalinization of acidic cellular compartments parallels the cell swelling and antiproteolytic effect of transforming growth factor-1 on LLC-PK₁ cells (752). However, the contribution of vesicular alkalinization to the antiproteolytic effect of cell swelling remains to be proven. At least in liver cells, swelling alkalinizes prelysosomal rather than lysosomal compartments (766, 1090). Moreover, inhibition of tyrosine kinase by erbstatin interferes with prelysosomal alkalinization but not with inhibition of proteolysis (S. vom Dahl and D. Häussinger, unpublished observations), pointing to some antiproteolytic mechanisms independent of lysosomal pH.

The alkalinization of acidic cellular compartments in hepatocytes occurs not only if cell swelling is due to decrease of extracellular osmolarity but also if cell swelling is caused by inhibition of K⁺ channels and by concentrative uptake of amino acids (1279).

It appears that the influence of cell volume on pH of acidic cellular compartments is not confined to prelysosomes in hepatocytes but involves a number of distinct compartments in a great variety of cells, such as pancreatic -cells, glial cells, neurons, vascular smooth muscle cells, proximal renal tubules, MDCK cells, alveolar cells, macrophages, and fibroblasts (153-157, 684, 1090, 1283). Accordingly, the functions of these compartments may be modified by alterations of cell volume.

Both cell swelling and cell shrinkage markedly influence the expression of a wide variety of genes (see Table 1).

As indicated in section IIC, exposure of cells to enhanced extracellular osmolarity or ionic strength stimulates the expression of aldose reductase and the Na⁺-coupled transport systems for inositol, betaine, taurine, and amino acids. The function of these proteins serves cellular accumulation of osmolytes and thus reestablishes osmotic equilibrium and cell volume constancy (144). Moreover, cell shrinkage stimulates the expression of heat shock proteins that serve to stabilize the proteins and thus to counteract the detrimental effects of increased salt concentrations. The cell volume regulated kinase h-sgk (see sect. IIIH) is a putative element of the signaling cascade triggering cell volume regulation. Furthermore, cell shrinkage stimulates the expression of proteins with diverse functions not obviously related to RVI, such as P-glycoprotein, ClC-K1, and Na⁺-K⁺-ATPase ₁-subunit, cyclooxygenase-2, the GTPase-activating protein for Rac ₁-chimerin, the immediate early gene transcription factors Egr1-1 and c-Fos, vasopressin, phosphoenolpyruvate carboxykinase, tyrosine aminotransferase, tyrosine hydroxylase, dopamine -hydroxylase, matrix metalloproteinase 9, and several matrix proteins (see Table 1).

Cell swelling similarly stimulates the expression of a variety of proteins including -actin, tubulin, cyclooxygenase-2, extracellular signal-regulated kinases ERK-1 and ERK-2, JNK, the transcription factors c-Jun and c-Fos, ornithine decarboxylase, and tissue plasminogen activator (see Table 1).

Information on the mechanisms triggering altered gene expression remains scanty (1254). Some evidence points to involvement of the cytoskeleton (76). The expression of aldose reductase is regulated by a distinct osmolarity-responsive element (320, 321, 1036). The stimulation of c-fos expression by swelling of cardiac myocytes is apparently secondary to tyrosine phosphorylation (1044); the c-jun transcription after swelling of hepatocytes is at least partially the result of MAPK activation, followed by phosphorylation of c-Jun (895, 1072, 1211). Cell volume changes modify phosphorylation of a histonelike nuclear protein (1057), and hypertonicity has been shown to alter the karyotype (1237).

In mice, a gene (rol) has been identified that renders erythrocytes resistant to osmotic lysis. The product of this gene is likely to be involved in the stimulation of volume regulatory K⁺ fluxes. However, the precise function of this gene remains elusive (318).

	IV. CHALLENGES OF CELL VOLUME CONSTANCY

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A multitude of mechanisms alter cell volume. They may do so by overriding the volume regulatory mechanims, by knocking them out, or by shifting their volume regulatory set point.

In mammalian tissues, most cells are exposed to extracellular fluid with well-controlled osmolarity. A notable exception is the kidney medulla, where extracellular osmolarity may approach values exceeding isotonicity by a factor of >4 (1033). Any blood cell passing the kidney medulla experiences exposure to this high ambient osmolarity and subsequent return to isosmolarity within seconds. Medullary cells have not only to cope with this excessive extracellular osmolarity for prolonged periods but encounter rapid changes of osmolarity during transition from antidiuresis to diuresis, when medullary osmolarity rapidly decreases toward isosmolarity (63).

Less dramatic alterations of extracellular osmolarity occur during intestinal absorption, which exposes intestinal cells to anisosmotic luminal fluid and may modify portal blood osmolarity and liver cell volume (460).

Other tissues are exposed to altered extracellular osmolarity during a variety of disorders. Although moderate, these alterations are still highly relevant challenges to cell volume control.

Because Na⁺ salts (mainly NaCl) contribute >90% to extracellular osmolarity, a significant decrease of extracellular osmolarity is necessarily paralleled by hyponatremia. A variety of clinical conditions can lead to hyponatremia (20, 21, 90, 816, 905, 1265). Hyponatremia may reflect an excess of water, either due to excessive oral load or due to impaired renal elimination, or a deficit of Na⁺ due to renal or extrarenal loss (90, 606, 1264). In both cases, the hyponatremia reflects a decreased extracellular osmolarity, leading to cell swelling. Excessive water intake is seen in psychiatric disorders (22). Causes for impaired renal water elimination include inappropriate antidiuretic hormone (ADH) secretion, glucocorticoid deficiency, hypothyroidism, and renal and hepatic failure. Renal and/or extrarenal loss of Na⁺ may result from mineralocorticoid deficiency, salt losing kidney, nephrotic syndrome, osmotic diuresis, vomiting, and diarrhea (90). Moreover, a wide variety of drugs including diuretics, cyclooxygenase inhibitors, and certain central nervous system active drugs may lead to hyponatremia due to loss of Na⁺ and/or to retention of water (90). Hyposmolar hyponatremia is further observed after burns, pancreatitis, and crush syndrome (90).

Hyponatremia does not necessarily indicate hyposmolarity but may occur in isosmolar or even hyperosmolar states (90). Extracellular osmolarity may be enhanced despite normal or even decreased extracellular Na⁺ concentration during hyperglycemia in uncontrolled diabetes mellitus (27) and ethanol poisoning (1010). Moreover, hyponatremia cannot be equated with cell swelling. As detailed in section IVF, cell swelling or cell shrinkage may prevail in diabetes mellitus. Burns, pancreatitis, and severe trauma, all conditions associated with hyponatremia (see above), may actually lead to muscle cell shrinkage rather than cell swelling (507).

Extracellular osmolarity is increased in hypernatremia, due to excessive oral intake and/or renal retention of Na⁺ and/or renal and extrarenal loss of water (325, 553, 858, 1142). Causes include excessive sweating, osmotic diuresis, lack of ADH or defective renal response to ADH, and drinking of seawater (553).

Even though extracellular osmolarity increases due to accumulation of urea in uremia (803), urea easily passes cell membranes and does thus not usually cause osmotic gradients across the cell membrane. Nevertheless, as shown in several cell types, high extracellular urea concentrations may trigger cell shrinkage by modifying the set point for volume regulatory mechanisms (see sect. IIIA). Cell shrinkage may be the signal for increase of osmolyte concentration in the brain, which has been observed to parallel enhanced urea concentration in uremia (1223).

Rapid correction of chronically enhanced osmolarity may lead to cell swelling, namely, to cerebral edema (28, 1157). Chronic increases of extracellular osmolarity are compensated by cells through accumulation of osmolytes, which may not be rapidly readjusted. Cerebral betaine, inositol, and glycerophosphorylcholine, for instance, may remain enhanced for days after correction of extracellular hypertonicity (746, 1208). Conversely, rapid correction of hyponatremia may prove similarly harmful (1141, 1156).

Even at constant extracellular osmolarity, cell volume constancy may be challenged by altered extracellular ion composition (see Table 2).

Most importantly, an increase of extracellular K⁺ concentration depolarizes the cell membrane and eventually leads to cellular uptake of K⁺ with accompanying anions (mainly Cl and HCO₃) and subsequent cell swelling. Conversely, a decrease of extracellular K⁺ could result in cell shrinkage due to cellular loss of KCl (see Table 2).

An increase of extracellular HCO₃ concentration could swell cells by electrogenic entry, hyperpolarization, reduced driving force for K⁺ exit, and subsequent accumulation of KHCO₃ (976). During correction of extracellular acidosis in the course of the treatment of diabetic ketoacidosis, increasing extracellular pH allows the cells to extrude H⁺ through the Na⁺/H⁺ exchanger, similarly leading to cell swelling (1255).

Several organic anions such as acetate, lactate, and proprionate swell cells by entry of the unionized acid, intracellular dissociation, stimulation of Na⁺/H⁺ exchange by cytosolic acidosis, and subsequent accumulation of Na⁺ and organic anions (see Table 2). A similar effect is exerted by CO₂ . In general, acidosis favors cell swelling, whereas cellular alkalosis has the opposite effect (see Table 2). Along these lines, the cellular accumulation of lactate in muscle exercise triggers volume regulatory mechanisms (1048).

Isotonic replacement of Cl with gluconate leads to cell shrinkage due to cellular loss of Cl (and K⁺) (see Table 2).

As pointed out in section IIB, the maintenance of a constant cell volume requires the expenditure of energy to fuel the Na⁺-K⁺-ATPase, which is required to establish the ionic gradients across the cell membrane. Inhibition of Na⁺-K⁺-ATPase by ouabain or during ischemia eventually leads to cell swelling. In cardiac myocytes, the swelling is preceded by transient cell shrinkage due to increase of intracellular Ca²⁺ and hypercontraction (31, 174, 1131).

As would be expected, ischemia leads to swelling of the brain (347, 1229) by impairment of Na⁺-K⁺-ATPase and subsequent accumulation of NaCl (347, 593). However, according to in vitro studies on glial and neuronal cells, energy depletion alone does not result in cell swelling (35, 613, 775, 1396). Rather, additional factors such as the intracellular acidosis (91, 578, 611, 1299) may account for cell swelling observed during ischemia (625). Furthermore, cell swelling in cerebral ischemia is favored by an increase of extracellular K⁺ concentration (612) and by extracellular accumulation of glutamate (42, 1396), which stimulates cationic channels through N-methyl-D-aspartate (NMDA) receptors and leads to subsequent accumulation of Na⁺, depolarization, and uptake of Cl (185, 186). In the heart, recovery from ischemia is facilitated in the presence of the Na⁺/H⁺ exchange inhibitor 3-methylsulfonyl-4-piperidinobenzoyl-guanidine-mesilate (HOE-694) (1085).

Alterations of cell volume are encountered during cryopreservation of organs (366, 394, 681). Low temperatures inhibit the Na⁺-K⁺-ATPase and may thus be expected to eventually result in cell swelling.

As listed in Table 2, a wide variety of hormones has been shown to alter cell volume.

Most importantly, insulin swells hepatocytes by activation of both Na⁺/H⁺ exchange and Na⁺-K⁺-2Cl cotransport (4, 472, 953), and glucagon shrinks hepatocytes, presumably by activation of ion channels (473, 1286, 1287). The effect of these hormones on cell volume accounts for several of the effects on hepatocyte metabolism (504, 506, 1286, 1287). It should be pointed out that insulin and glucagon modify cell volume at hormone concentrations well encountered under physiological conditions. This is not necessarily true for all hormones listed in Table 2. Similar to insulin, several growth factors increase cell volume in a variety of cells by stimulation of Na⁺/H⁺ exchange and in some cases of Na⁺-K⁺-2Cl cotransport (see Table 2). An increase of cell volume appears to be required for cell proliferation (see sect. VI).

Activation of Na⁺ channels or nonselective cation channels by excitatory neurotransmitters such as glutamate tends to swell neurons, whereas activation of K⁺ channels or anion channels by inhibitory neurotransmitters such as GABA tends to shrink neurons (see Table 2).

Secretagogues may stimulate transepithelial transport by enhancing cellular ion entry, ion exit, or both. If stimulation of ion entry prevails, the cells swell, whereas if ion exit is preferentially activated, the cells shrink. Thus secretagogues may either swell (e.g., epinephrine) or shrink (e.g., acetylcholine) the cells (see Table 2).

In kidney medulla, ADH enhances extracellular osmolarity and thus forces the medullary cells to accumulate osmolytes (1033, 1075). Furthermore, ADH may more directly trigger the formation or accumulation of osmolytes (880).

The transport and cellular accumulation of amino acids lead to cell swelling (Table 2). Especially Na⁺-coupled transport processes can generate large chemical gradients across the cell membrane, due to the steep electrochemical gradient for Na⁺. For instance, cellular glutamine has been observed to increase by >30 mM after the addition of 3 mM glutamine to portal blood (502).

As listed in Table 2, transport of several other substrates such as glucose, taurine, and taurocholeate similarly increases cell volume.

Exercising muscle may lead to cellular accumulation of lactate and thus may increase cell volume (1048). The developing intracellular acidosis may compound cell swelling by activation of the Na⁺/H⁺ exchanger.

Diabetic ketoacidosis may cause cell swelling (125, 197, 646, 1388) due to cellular uptake of acids and enhanced Na⁺/H⁺ exchange activity in compensation for cellular H⁺ generation (1255). More importantly, high glucose concentrations favor cellular formation and accumulation of sorbitol through aldose reductase (143, 180, 559, 748, 990, 1196). As a consequence, cells decrease other osmolytes such as myo-inositol (143, 411, 1079, 1158, 1222, 1386, 1387), an effect which can be reversed by inhibition of aldose reductase with sorbinil (303, 326, 1218). On the other hand, hyperglycemia is paralleled by hyperosmolarity, which shrinks cells. In fact, some evidence points to shrinkage of polymorphonuclear lymphocytes in hyperosmolar diabetes mellitus (269).

In addition to creating osmotically active substances, metabolic pathways may alter cell volume indirectly through modification of transport processes across the cell membrane. For instance, a decrease of cellular ATP could activate ATP-sensitive K⁺ channels and thus shrink susceptible cells, a possibility which has, however, not yet been explored. On the other hand, the cellular formation of peroxides has been shown to shrink hepatocytes due to activation of K⁺ channels at the cell membrane (1045). Peroxides similarly activate K⁺ channels in pancreatic -cells (652) and vascular smooth muscle cells (651) but inhibit N-type K⁺ channels in lymphocytes (1183) and minK channels (152), which are expressed in a variety of cells (150). In endothelial cells, peroxides inhibit Na⁺-K⁺-2Cl cotransport (305). However, the consequences on cell volume have not been tested in any of those cells.

In addition to hormones, a great number of drugs and toxins lead to cell swelling or cell shrinkage (Table 2). For most substances, the functional significance of the effect on cell volume has not been explored.

In several stress situations, such as surgical intervention (306), acute pancreatitis (1027), severe injury, burns, and sepsis (79), a decrease of muscle intracellular space has been observed, leading to disinhibition of proteolysis and thus to hypercatabolism (507). However, the mechanisms underlying muscle cell shrinkage have not yet been elucidated.

	V. ROLE OF CELL VOLUME REGULATORY MECHANISMS IN CELL FUNCTIONS

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Erythrocyte volume and shape are important determinants of blood viscosity. Cell volume regulatory mechanisms are specifically important in limiting alterations of cell volume during their passage through the hypertonic kidney medulla and during HCO₃ transport in the lung and the periphery. One disorder exacerbated by altered erythrocyte cell volume regulatory mechanisms is sickle cell anemia, where mutations of the hemoglobin chain (HbS) favor the polymerization of deoxygenated hemoglobin, leading to characteristic changes of cell shape (sickling) and impaired deformability of the erythrocytes (591, 737); the consequence is a severe increase of blood viscosity (591). The polymerization of hemoglobin is highly dependent on protein concentration and thus on cell volume (297, 298). In HbS erythrocytes, volume regulatory KCl cotransport (133, 163, 164, 343, 1276) is enhanced, partially due to direct interaction with the mutated hemoglobin (914). Furthermore, cell shrinkage is presumably favored by enhanced activity of Ca²⁺-sensitive K⁺ channels (105, 134, 343) due to increase of intracellular Ca²⁺ concentration. The ensuing cell shrinkage further favors the polymerization of hemoglobin (591). The expression of the Na⁺/H⁺ exchanger is enhanced, possibly in compensation for cell shrinkage (165). Similarly, cell volume is decreased in homozygous hemoglobin C disease (135).

In intestine, gallbladder, and renal proximal tubules (see Fig. 1A), the luminal uptake of substrates for Na⁺-coupled transport, such as glucose or amino acids, tends to swell the cells, leading to volume regulatory activation of K⁺ channels in the basolateral cell membrane (67, 68, 122, 173, 355, 493, 687, 692, 706, 782, 995, 1092-1096, 1230). The activation of these channels not only limits cell swelling but maintains the electrical driving force for continued transport.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Three examples illustrating role of cell volume in coupling of apical to basolateral cell membranes in epithelia. A: Na+-coupled transport across apical cell membrane of proximal renal tubules leads to accumulation of Na+ and substrate [e.g., amino acids (AA)] and thus to cell swelling, which activates basolateral K+ channels. B: electrolyte uptake by Na+-K+-2Cl cotransport across basolateral cell membrane in dark vestibular cells leads to cell swelling and subsequent activation of luminal K+ channels. C: stimulation of apical Cl channels in Cl-secreting cells leads to loss of Cl and, because of depolarization, of K+. Cell shrinkage and decrease of intracellular Cl activity in turn stimulate basolateral Na+-K+-2Cl cotransport.

In the NaCl-reabsorbing thick ascending limb of Henle's loop and diluting segment of the amphibian kidney, NaCl entry is accomplished by luminal Na⁺-K⁺-2Cl cotransport, basolateral Cl channels, and Na⁺-K⁺-ATPase as well as apical and basolateral K⁺ channels (416, 900, 1178). Inhibition of Na⁺-K⁺-ATPase leads to rapid cell swelling, which is prevented by inhibition of luminal Na⁺-K⁺-2Cl cotransport (444, 520, 1178). On the other hand, stimulation of transport by ADH involves a well-coordinated increase of transport rate across both luminal and basolateral cell membranes (521) without any appreciable increase of cell volume (519). If the cells are shrunk by increased extracellular osmolarity, they do not display RVI unless they are stimulated with ADH or cAMP (520, 522, 1177). The hormone not only activates luminal Na⁺-K⁺-2Cl cotransport but also a Na⁺/H⁺ exchanger in the basolateral cell membrane, which contributes to ion accumulation during RVI (520, 522, 1177).

Potassium secretion in dark vestibular cells (see Fig. 1B) and stria vascularis marginal cells of the inner ear is accomplished by basolateral Na⁺-K⁺-2Cl cotransport, Na⁺-K⁺-ATPase, and Cl channels as well as apical minK channels (1311). The K⁺ channels are activated by cell swelling (1120, 1312), and the basolateral Na⁺-K⁺-2Cl cotransport by cell shrinkage (1315). Cell volume couples the two cell membranes, since excess Na⁺-K⁺-2Cl cotransport would swell the cells and thus activate the apical K⁺ channels, and excess K⁺ channel activity would shrink the cell and thus turn on Na⁺-K⁺-2Cl cotransport. Moreover, exposure of the basolateral side to a hypotonic medium stimulates transepithelial transport (1312), possibly by decreasing intracellular Cl and subsequent activation of Na⁺-K⁺-2Cl cotransport.

In tight epithelia reabsorbing Na⁺, such as urinary bladder, Na⁺ entry through luminal Na⁺ channels is similarly coordinated with Na⁺-K⁺-ATPase and K⁺ channels at the basolateral cell membrane (179, 329, 742, 789, 995, 1232, 1233, 1303, 1349). Accordingly, inhibition of the Na⁺-K⁺-ATPase in toad urinary bladder leads to parallel inhibition of luminal Na⁺ channels, preventing luminal Na⁺ entry and cell swelling (252).

In several tight epithelia, insertion of Na⁺ channels was stimulated by decrease of extracellular osmolarity (1231, 1347), a function obviously serving Na⁺ homeostasis rather than cell volume regulation.

Activation of K⁺ and Cl channels during stimulation of secretion in several epithelia (Fig. 1C) may lead to cell shrinkage due to cellular KCl loss (229, 338, 339, 873, 1363). Shrinkage then turns on volume regulatory Na⁺/H⁺ exchange and/or Na⁺-K⁺-2Cl cotransport, which may partially recover cell volume and at the same time supply the cell with further Cl for secretion (341, 549, 796, 797).

Thus, during both reabsorption of Na⁺ and substrates and secretion of Cl, cell volume participates in the coupling of the basolateral and luminal cell membrane, the so-called cross-talk between the opposing cell membranes (995). It should be kept in mind, however, that cell volume participates in, but does not fully account for, the coupling of the cell membranes (687). Accordingly, even though osmotic cell swelling mimics many effects of swelling induced by substrate transport, the underlying mechanisms are not necessarily identical. For instance, the depolarization resulting from Na⁺-coupled transport alkalinizes the cytosol by impairment of HCO₃ exit (687), whereas osmotic cell swelling leads to cytosolic acidification (see sect. IIIE). Because K⁺ channels are highly sensitive to cytosolic pH (692), their activation is expected to be different between osmotic and substrate-induced cell swelling. In enterocytes, RVD apparently depends on Ca²⁺ from outside and calmodulin-sensitive K⁺ channels, whereas substrate-induced cell swelling appears to be counterregulated by cellular Ca²⁺ release and subsequent activation of Cl channels by protein kinase C (782, 788).

Volume-mediated activation of transporters not only involves ion channels and ion transporters. As pointed out above, cell shrinkage stimulates the expression and/or activity of Na⁺-coupled transporters for inositol (877), betaine (1242, 1372), taurine (1238), and neutral amino acids (182, 380, 1135, 1373). Cell swelling, on the other hand, stimulates cellular release of the above osmolytes and of glutathione (503) and cellular uptake of alanine and glutamine (502, 1335) and of taurocholate (469, 497). Stimulation of osmolyte flux during alterations of cell volume serves to regulate cell volume rather than transepithelial transport, but entry and/or exit may be polarized (377, 480, 630, 1372), and in the liver, cell swelling indeed stimulates transepithelial transport of taurocholate (469, 508) and leukotrienes (1340).

Hyperosmolarity stimulates urea transport (392, 393) but inhibits transport of NaCl in inner medullary collecting duct (410) and salivary gland (874).

Stimulation of transport during cell swelling may be the result of insertion of the carriers and/or channels into the cell membrane by exocytosis (114, 132, 462, 497, 912, 969, 1260, 1354). Exocytosis may be stimulated by an increase of intracellular Ca²⁺ activity. In lung alveolar type II cells, the increase of intracellular Ca²⁺ concentration due to mechanical stress not only accounts for exocytosis but also for stimulation of surfactant secretion (1354). Protein secretion in seminal vesicles, on the other hand, is inhibited by both hyper- and hypotonic extracellular fluid (554). Exposure of the apical side of the pulmonary epithelium to hypotonic fluid is thought to stimulate the secretion of a humoral factor, leading to bronchodilation (367).

Whether the polarized trafficking of vesicles in epithelia is influenced by cell volume has not yet been explored. The polarized distribution of secretory proteins is modified by an alkalinization of vesicular pH (167, 931) and could thus theoretically be sensitive to cell volume. However, the alkalinization of vesicles during cell swelling may be too small to significantly interfere with trafficking.

In addition to its effect on transcellular transport, cell volume has been demonstrated to modify the permeability of tight junctions and thus paracellular transport. However, the reported effects are not consistent (29, 111, 402, 930, 962, 1348, 1384).

As listed in Table 3, cell volume changes modify a wide variety of metabolic functions. Most importantly, cell swelling favors the synthesis and inhibits the degradation of proteins, glycogen, and to a lesser extent lipids (504, 506). Cell shrinkage has the opposite effect. Thus cell swelling can be considered as an anabolic signal, whereas cell shrinkage favors cell catabolism.

In hepatocytes, the influence of cell volume on metabolism is one way that insulin and glucagon exert their metabolic effects. Insulin increases liver cell volume by activation of Na⁺/H⁺ exchange and Na⁺-K⁺-2Cl cotransport and thus triggers a variety of metabolic functions, including protein and glycogen synthesis and inhibition of protein and glycogen degradation (4, 504, 506). The effect of insulin on proteolysis is fully accounted for by the cell swelling effect of the hormone. Conversely, glucagon and cAMP stimulate proteolysis and glycogenolysis and inhibit protein synthesis in part by cell shrinkage due to activation of ion channels and subsequent release of KCl (504). Although cell shrinkage correlates well with the inhibitory effect of cAMP and ADH on protein synthesis, this is not true for the effects of insulin and phenylephrine (1159). Thus cell volume may play a less prominent role in hormonal regulation of protein synthesis than in proteolysis. The same probably holds true for glycogen metabolism and lipogenesis.

The antiproteolytic effect of cell swelling depends on an intact microtubule network (156, 1284) and could thus not be reproduced in freshly isolated hepatocytes (820), which suffer from a disintegrated microtubule network (511, 1284).

The set points of volume regulatory mechanisms and thus cell volume can be altered by a wide variety of other hormones and transmitters, which thus trigger the metabolic pattern typical for swollen or shrunken cells (Table 1). By this means, the mediators exploit volume regulatory mechanisms to exert their effects on cellular metabolism.

In addition to hormones, nutrients may modify protein, glycogen, and lipid metabolism in part through their influence on cell volume. In fact, the antiproteolytic effect of glutamine and glycine in liver has been shown to be completely accounted for by their influence on cell volume (471). The antiproteolytic and swelling effect of glycine is potentiated after starvation (471, 1285), which upregulates the glycine transporting system A (515). However, the antiproteolytic action of other amino acids such as phenylalanine, serine, alanine, and proline cannot be fully explained by their effects on cell volume. Thus mechanisms other than cell swelling contribute to the antiproteolytic action of some amino acids.

The influence of cell volume on metabolism is not restricted to macromolecular synthesis and breakdown. Swelling of hepatocytes apparently interferes with the transfer of reducing equivalents through the mitochondrial malate/aspartate shuttle (503). The lack of aspartate impedes the formation of urea from NH₃ , an effect that is overcome by addition of lactate and pyruvate, allowing the mitochondrial regeneration of oxaloacetate (503). The formation of urea from glutamine is enhanced after cell swelling (500).

Some effects of cell swelling caused by a decrease of extracellular osmolarity may actually be due to concomitant mitochondrial swelling (969) because of decreasing ambient osmolarity. Glutamine breakdown (502) and glycine oxidation (510), for instance, are stimulated not only by decrease of extracellular osmolarity but also by glucagon, cAMP, and several Ca²⁺-mobilizing hormones that swell mitochondria (465-467), but at the same time shrink hepatocytes (see Table 2). Similarly, the swelling-induced decrease of the -hydroxybutyrate-to-acetoacetate ratio (510) is probably due to stimulation of the respiratory chain due to concomitant mitochondrial swelling (274, 466).

Peroxides may modify cell volume by activation of ion channels (see Table 2). They shrink hepatocytes by activation of K⁺ channels (470). On the other hand, superoxide has been shown to swell erythrocytes (1247). Cell volume in turn influences the peroxide metabolism; cell swelling stimulates and cell shrinkage inhibits flux through the pentose phosphate pathway and NADPH generation (1046). Thus cell swelling provides NADPH for glutathione reductase to produce reduced glutathione (GSH) and strengthens the protective mechanisms against peroxides (1046). On the other hand, cell swelling should favor the formation of reactive oxygen species by NADPH oxidase, which is inhibited by high osmolarity (1186). Furthermore, components of the cytosolic burst oxidase have been found to be dissociated by high osmolarity (573). Moreover, cell swelling stimulates formation of arachidonic acid (see sect. IIIK), which is required for activation of NADPH oxidase (526). Accordingly, osmotic cell swelling stimulates (602) and osmotic cell shrinkage inhibits formation of peroxides in neutrophils (602, 659, 795, 810).

The enhanced formation of sorbitol in diabetes mellitus (see sect. IVF) has been implicated in the generation of several diabetic complications such as neuropathy, retinopathy, microangiopathy, and cataracts (143). Similarly, the cellular accumulation of galactitol with subsequent decrease of other osmolytes has been implicated in the pathophysiology of galactosemia (80, 302). However, the mechanisms linking cell swelling with defined sequelae of diabetes mellitus or galactosemia are far from understood.

Information on metabolic effects of cell volume in mammalian cells other than hepatocytes is still scarce (see Table 3), even though it appears highly unlikely that the influence of altered cell volume on metabolism is restricted to hepatocytes. For instance, mechanical or osmotic deformation of chondrocytes or osteoblasts, respectively, may stimulate the synthesis of proteoglycans and proteins (123, 623, 1244) and thus foster cartilage and bone growth, which indeed correlated with chondrocyte volume (661). Moreover, a decrease of muscle cell volume was correlated with hypercatabolism in several clinical conditions (507).

Certainly, more experimental information is needed on the interaction of cell volume and cell metabolism in other cells, such as glial cells and adipocytes.

The formation of coated pits and internalization of low-density lipoproteins (LDL) and transferrin receptors are inhibited by both increase of extracellular osmolarity (246, 485, 531, 644, 909) and K⁺ depletion (485, 700, 701), both maneuvers expected to shrink cells. Conversely, internalization of LDL or ferritin is increased by hypotonic extracellular fluid (644, 707). However, because almost identical effects were exerted by KCl and NaCl but not by glucose or urea, the altered internalization appeared to be due to the ionic strength rather than cell volume (644). Increased ionic strength may impede internalization by interference with the formation of coated pits (531). On the other hand, cell swelling has been shown to inhibit endocytosis (1316).

Cell swelling leads rather to cytosolic acidification (see sect. IIIE), which has been shown to interfere with endocytosis from coated pits (485, 530, 1056). Moreover, cell swelling reverses lysosomal acidification, which is a prerequisite for normal recycling of LDL (57) and transferrin receptors (248). Accordingly, cell swelling would have been expected to impede receptor recycling. The vesicular alkalinization and cytosolic acidification after cell swelling may not be sufficient to significantly interfere with receptor recycling, and the effects of ionic strength exceed the weak effects of cell volume. The reason for the interference of K⁺ depletion with the formation of coated pits (701) remains, however, unexplained.

In glial cells, NH₃ , which swells the cells (10, 897, 898) and alkalinizes their lysosomes (153), leads to upregulation of peripheral type benzodiazepine receptors (571, 572). Moreover, binding of benzodiazepine to glial cells (570), muscarinic drugs to peritoneal cells (537), atrial natriuretic peptide to collecting duct cells (561), and endothelin to renal cells (1268) increases after hypotonic exposure. Excessive osmotic shrinkage, on the other hand, has been shown to induce clustering and internalization of cytokine receptors and thus to mimic effects of the ligands (1020).

Taken together, these data do not suggest a uniform influence of cell volume as such on the regulation of cell membrane receptors.

An increase in cell membrane tension, as occurs during cell swelling, has been described to trigger fusion of endocytotic vesicles with the plasma membrane, leading to release of vesicle contents and insertion of ion channels in the cell membrane (132, 417, 462, 508, 969, 1260). The mechanism requires Ca²⁺ and an intact actin filament network. If the same is true for secretory vesicles, osmotic cell swelling should stimulate hormone release (Fig. 2).

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Mechanism of stimulated hormone release and of contraction after swelling of endocrine and vascular smooth muscle cells, respectively. Swelling leads to activation of anion channels, and exit of Cl depolarizes cell membrane. Subsequent activation of voltage-sensitive Ca2+ channels stimulates Ca2+ entry. Ca2+ then triggers hormone release from endocrine cells or contraction of smooth muscle cells, respectively.

Cell swelling has indeed been shown to trigger the release of insulin (95, 768), prolactin (414, 1063, 1066, 1070, 1304, 1306, 1307, 1309), gonadotropin-releasing hormone (563), luteinizing hormone (414, 415), thyrotropin (414, 1065, 1306), aldosterone (514, 1081-1083, 1301), and renin (345, 582, 1128, 1129). Where tested, the hormone-releasing effect was correlated with an increase of intracellular Ca²⁺ activity (514, 983, 1062, 1064-1069, 1080, 1308). In insulin-secreting -cells, Ca²⁺ entry during cell swelling is partially due to activation of Cl channels (82), with subsequent depolarization of the cell membrane and opening of voltage-sensitive Ca²⁺ channels (124).

Osmotic cell shrinkage has been shown to inhibit prolactin release, presumably by inhibiting Ca²⁺ influx (1065, 1305). Furthermore, increase of extracellular osmolarity decreases the formation and release of endothelin-1 (640).

For atrial natriuretic factor (ANF), the position is less clear. It is released from cardiac myocytes in response to mechanical stretch (198, 1071) and osmotic cell swelling (412). Cell volume may be part of a negative-feedback loop limiting ANF release. Atrial natriuretic factor inhibits the cardiac Na⁺/K⁺/2Cl exchanger via guanosine 3',5'-cyclic monophosphate (cGMP), with the resulting cell shrinkage then inhibiting ANF release (199, 201). On the other hand, ANF release has been postulated to be stimulated by cell shrinkage (1399, 1400).

In contrast to the above hormones, vasopressin is released during cell shrinkage, which apparently leads to disinhibition of a stretch-inactivated cation channel. The activation of this channel leads to depolarization and accelerated action potentials (913). Interestingly, ethanol, which triggers hormone release from other cells (1063, 1068), is known to inhibit vasopressin release (90). In addition to ADH, the release of nitric oxide (1152) and of the putative hormones ouabain or ouabainlike factors (98, 744) may be stimulated by an increase in plasma osmolarity. Furthermore, hyperosmolarity stimulates the release of histamine from basophil granulocytes (892) and of substance P from C-fiber neurons (375). Alterations of NaCl concentrations have further been shown to modify transmitter release from various regions of the brain (551).

Renin secretion is inhibited by an increase of intracellular Ca²⁺ activity, the so-called Ca²⁺ paradox of renin secretion (461, 923). It has been suggested that an increase of intracellular Ca²⁺ activity activates Ca²⁺-sensitive Cl channels, thus leading to cellular loss of KCl and cell shinkage, which in turn would inhibit renin release (665).

Cell volume may further modify hormone and transmitter release through pH changes in secretory vesicles. In pancreatic -cells, for instance, acidic proteases within the acidic secretory granules cleave proinsulin to yield insulin, a function probably compromised by cell swelling and fostered by cell shrinkage. Furthermore, the release of insulin may be modified by the luminal pH of the secretory granules and thus be sensitive to alterations of cell volume. In neurons, metabolism, uptake, and release of neurotransmitters may be modified by the luminal pH of synaptic vesicles. For instance, the uptake of neurotransmitters such as catecholamines, glutamate, GABA, and acetylcholine into small synaptic vesicles is driven by a proton gradient between the cytosol and the acid lumen (254). Uptake of glutamate and aspartate into glial cells has indeed been found to be impaired by osmotic cell swelling (628).

Transmitter metabolism may be further influenced by cell volume through effects on the expression of enzymes. At enhanced extracellular K⁺, an increase of extracellular osmolarity inhibits the expression of tyrosine hydroxylase and dopamine -hydroxylase in PC12 cells (621). Increasing extracellular osmolarity at low extracellular K⁺ was, however, without effect on the expression of these enzymes (621).

Some of the osmolytes released during cell swelling of neurons such as glutamate (148, 304, 523, 550, 865, 1339), aspartate (550, 891), GABA (774, 891), glycine (891), and taurine (184, 560) function as neurotransmitters in the brain. Hyperosmolar glucose or sorbitol concentrations inhibited K⁺-induced GABA release and inhibited K⁺-induced release of norepinephrine and serotonin (327).

After swelling of cardiac cells, volume-sensitive Cl currents have been shown to depolarize the cell membrane (1253, 1394), enhance excitability, and reduce the duration of the action potential (1139). On the other hand, moderate osmotic shrinkage exerts a positive inotropic effect in the heart (74, 432). This latter effect may be due to a direct influence on the contractile elements. As shown in skinned muscle fibers, increased ionic strength destabilizes the actinomyosin complex, thus interfering with the Ca²⁺-activated force (41, 398). To the extent that cell shrinkage leads to increase of intracellular ionic strength, this effect could modify muscular contraction. Osmotic swelling of smooth muscle cells (see Fig. 2) activates a depolarizing anion conductance, leading to depolarization, opening of voltage-gated Ca²⁺ channels, increase of intracellular Ca²⁺ activity, and contraction (685). Conversely, osmolar cell shrinkage leads to vasodilation (1112, 1152).

An enhanced activity of Na⁺/H⁺ exchanger with resulting cell swelling has been implicated in the generation of one type of essential hypertension (271, 314, 348, 558, 664, 793, 887-889, 1022-1024, 1026, 1109, 1124, 1330, 1390). On the one hand, cell swelling should increase contractility of smooth muscle cells, and on the other hand, enhanced Na⁺/H⁺ exchange activity should favor cell proliferation and thus hypertrophy of vascular smooth muscle cells. Proliferation of vascular smooth muscle cells has been shown to be stimulated by mechanical stretch (770, 979).

Neuronal excitability could be affected in several ways by cell volume. Any release of K⁺ for cell volume regulation should enhance extracellular K⁺, decrease the K⁺ equilibrium potential, and thus depolarize the cell membrane. Possibly, however, swollen neurons do not volume regulate by rapid release of electrolytes (23).

As discussed in section VE, glutamate, aspartate, GABA, glycine, and taurine serve both as osmolytes and as neurotransmitters. When released from swollen cells, they may modify the function of neighboring cells (628, 1059). Conversely, the osmosensitive betaine transporter transports GABA at higher affinity than betaine (1374). Moreover, sensitivity of neurons to neurotransmitters may be modified by cell volume. For instance, NMDA receptors have been found to be mechanosensitive (929). Cell volume changes may further interfere with neuronal excitability by modifying the pH and trafficking of secretory vesicles (see above). Increases of osmolarity have been shown to increase the percentage of slow miniature end-plate potentials (1262).

Chloride concentration in neurons is regulated by furosemide-sensitive Na⁺-K⁺-2Cl cotransport (45, 839). Activation of K⁺ and Cl channels shrinks the cells by KCl loss, decreases intracellular Cl concentration, and thus dissipates the Cl gradient. The cell shrinkage activates the Na⁺-K⁺-2Cl cotransport, which not only restores cell volume but also maintains intracellular Cl activity. Accordingly, GABA-induced depolarization was rendered transient by addition of furosemide (45). Moreover, furosemide has been shown to block synchronized burst discharges in hippocampal slices (538).

Excitability of neurons critically depends on glial cell function (612). One of the major tasks of glial cells is to maintain constancy of extracellular K⁺. Because of the minimal extracellular space, any release of K⁺ from neurons during depolarization would lead to rapid increase of extracellular K⁺ concentration, a dissipation of the chemical K⁺ gradient, and thus to impairment of repolarization. Glial cells accumulate K⁺ and thus blunt the increase of extracellular K⁺ concentration in part by uptake of K⁺ through Na⁺-K⁺-2Cl cotransport in parallel with Na⁺-K⁺-ATPase (1298). This function is expected to be compromised during glial cell swelling. Glial cell swelling has indeed been implicated in a wide variety of disorders affecting the brain (505, 624, 649, 650, 896, 898, 1220). In hepatic encephalopathy, for instance, the enhanced NH₃ concentration forces the formation and cellular accumulation of glutamine, leading to glial cell swelling (218, 896, 898). One of the consequences is cellular loss of inositol (218, 649, 1021). Accordingly, inhibition of glutamine synthetase has been found to be protective against hepatic encephalopathy (512). A striking increase of brain inositol is observed in Alzheimer's disease (1122). It is not clear, though, whether this change relates to altered cell volume regulation and contributes to the pathophysiology of this disease.

In part as a result of the above interactions, increased plasma osmolarity decreases and reduced plasma osmolarity increases the susceptibility to epileptic seizures (22, 921). It must be kept in mind, though, that alterations of plasma osmolarity primarily create an osmotic gradient across the blood-brain barrier, leading to respective changes of extracellular space (235, 1125) and movements of electrolytes from or to brain tissue (822). The decrease of extracellular space during osmotic cell swelling may be a major cause for altered excitability (921, 1224).

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Polarized cell volume regulatory ion transport in migrating cells. Cell migrates from left to right. At rear end, oscillations of intracellular Ca2+ activity (Cai) lead to activation of Ca2+-sensitive K+ channels, favoring regulatory cell volume decrease (RVD). At leading edge, Na+/H+ exchange and Na+-K+-2Cl cotransport favor regulatory cell volume increase (RVI). Ca2+ oscillations further trigger depolymerization of actin filaments at rear end. Fragments are transported to leading edge, where polymerization of actin filaments prevails.

Migration involves substantial reorganization of the cytoskeleton both at the leading edge and the rear of the cell (202, 215, 1162) (see Fig. 3). At the leading edge, polymerization of actin filaments prevails, whereas at the rear of the cells, actin filaments are depolymerized (1162). The depolymerization is mediated by capping or escort proteins, such as gelsolin, which are stimulated by Ca²⁺ (237, 492, 1162, 1355). In fact, after chemotactic stimuli, Ca²⁺ concentration increases primarily at the rear of the cell (136, 463, 756). Bound to the capping or escort proteins, the actin fragments travel toward the leading edge, where they are reutilized for elongation of the actin filaments. The elongation is stimulated by polyphosphoinositides, which bind and thus neutralize the capping proteins (1162). The addition of new elements to the actin filament is thought to be facilitated by protrusion of the cell membrane, which may be caused by local osmotic swelling (1162).

Migration of leukocytes can be stimulated by chemoattractants such as formylpeptides (727). N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulates Na⁺/H⁺ cotransport in neutrophils, leading to cell swelling (842, 870, 871, 1003, 1019, 1365). Activation of Na⁺/H⁺ exchange and cell swelling are required for migration, which is impeded by inhibitors of the carrier and osmotic cell shrinkage (1003, 1019). Similarly, inhibition of Na⁺-K⁺-2Cl cotransport with bumetanide inhibited migration of transformed MDCK cells (1101). In those cells, migration further requires the operation of Ca²⁺-sensitive K⁺ channels, which are activated by oscillating intracellular Ca²⁺ activity (240, 1101). Inhibition of these channels similarly prevented migration. It is attractive to postulate that migration involves RVD with Ca²⁺ oscillations and activation of K⁺ channels at the rear and RVI with activation of Na⁺/H⁺ exchange and/or Na⁺-K⁺-2Cl cotransport at the leading edge of a migrating cell. The migration would require asymmetrical distribution and/or activation of channels and carriers. As a matter of fact, the Na⁺/H⁺ exchanger is concentrated at the leading edge (431) and K⁺ channel activity at the rear of the cell (1099, 1100), and a Ca²⁺ gradient has been observed within a migrating cell with highest values at the rear (136, 401, 463, 1098).

The elongation of the neuritic cylinder, but not the extension of the growth cone of neurons, has been observed to be stimulated by a decrease of extracellular osmolarity (117). Accordingly, the role of osmotic gradients in the protrusion of the leading edge is still a matter of debate (117, 965, 1162). Obviously, variation of extracellular osmolarity does more than modify the detachment of the cell membrane from the cytoskeleton. In any case, more experimentation is needed to elucidate the role of cell volume regulatory mechanisms in the machinery of migration.

Reports on the interplay of cell volume and infection are scarce. It is well known that cholera toxin activates both Cl and K⁺ channels via cAMP (337), an effect expected to result in cell shrinkage. The heat-stable toxin from Escherichia coli has been shown to activate Cl channels via cGMP (749), and erythrocytes infected with malaria display new ion permeation pathways (633). Moreover, several bacteria produce porins, which may be inserted into the host cell membrane (97, 771, 1328). Because porins may function as Cl channels (1035, 1338), it is tempting to speculate that they serve to alter host cell volume. Exposure of erythrocytes to Schistosoma mansoni membrane fractions has indeed been shown to produce marked cell shrinkage (1201). However, whether the metabolic alterations triggered by cell swelling or shrinkage are favorable for the pathogen is similarly unknown. As outlined above, cell shrinkage inhibits O₂ formation in neutrophils, an effect possibly contributing to the inhibitory effect on bacterial killing in a hypertonic environment (481). On the other hand, a hypertonic environment increases adherence and invasion of Salmonella typhi (1195).

Similar scanty information is available on the role of cell volume in viral infection. The M₂ protein of influenza virus serves as an ion channel (974). Whether the insertion of this channel into the host membrane alters cell volume is not known. Tacaribe virus infection was shown to inhibit Na⁺-K⁺-ATPase (996), and vaccinia virus infection of HeLa cells led to increases of Na⁺ at the expense of K⁺ (899). Infection of fibroblasts with herpes simplex virus leads to profound cell swelling (441). Cell swelling was enhanced in the presence of the antiviral drug 3'-azido-3'-deoxythymidine, which inhibits I_Cln , the putative volume regulatory Cl channel (441). Infection of renal tubule cells with simian virus 40 results in the appearance of K⁺ channels (1199). Changes in cell volume, on the other hand, may influence viral replication. For instance, a decrease of extracellular NaCl has been shown to reduce replication of reticuloendotheliosis (93) and Sindbis virus (1290) as well as maturation of poliovirus (5). Furthermore, infection of MDCK cells with vesicular stomatitis virus is impaired by increasing extracellular K⁺ at the expense of Na⁺ (7), a maneuver known to induce cell swelling (see Table 2). The effect could not be explained by altered intracellular Ca²⁺ activity but was assumed to be secondary to a depolarization of the cell membrane (7). In duck hepatocytes, increase of extracellular osmolarity markedly reduced duck hepatitis B virus DNA, mRNA, and protein (904). Because shrinkage of hepatocytes leads to depolarization (406), the effect could have been due to either depolarization or cell shrinkage.

In tracheal epithelium, sulfation and sialation of membrane proteins has been shown to be sensitive to luminal pH of acidic cellular compartments, and it has been argued that defective acidification of these compartments in cystic fibrosis leads to altered sulfation and sialation and thus to enhanced adhesion of Pseudomonas aeruginosa (54). Because the primary defect of cystic fibrosis leads to impaired Cl channel activity (92, 170, 171, 350, 1118, 1336, 1337) and impairment of cell volume regulation (1252), it may be paralleled by cell swelling, which increases lysosomal pH (see sect. IIIL). Thus cell swelling could enhance adhesion of P. aeruginosa and infection, a possibility, however, not yet explored.

Clearly, the role of cell volume in the interaction of host and pathogen is far from understood. The data available thus far, however, are sufficiently intriguing to justify additional experimental effort.

A wide variety of mitogenic factors (for review, see Ref. 1000) activate the Na⁺/H⁺ exchanger, and many factors stimulate Na⁺-K⁺-2Cl cotransport (85, 428, 826, 844, 928, 1000, 1274, 1275, 1291, 1292, 1293). One expected consequence of the activation of these transport systems is an increase of cell volume.

Cell proliferation has indeed been shown to correlate with increases of cell volume in fibroblasts (694, 695, 824, 960), mesangial cells (1371), lymphocytes (284, 285, 454, 1114, 1117), HL-60 cells (120, 146), GAP A3 hybridoma cells (884), smooth muscle cells (317, 881), and HeLa cells (1185). As shown for fibroblasts, the cell volume increase parallels the entry of fibroblasts from the G₁ into the S phase (960), which is accompanied by inhibition of K⁺ channels (253). Moreover, large variations of volume regulatory Cl channels have been observed in ascidian embryos (1273).

Osmotic alterations of cell volume indeed modify cell proliferation. Hypertonic shrinkage inhibits (9, 533, 840, 967, 1008, 1380) and slight osmotic cell swelling has been shown to accelerate (18) cell proliferation. When exposed to enhanced extracellular ionic strength, the cells may overcome cell shrinkage by cellular accumulation of osmolytes which then allows them to proliferate normally (720, 1379, 1380).

As illustrated in Figure 4, Ras oncogene expression in fibroblasts is paralleled by enhanced Na⁺/H⁺ exchange and Na⁺-K⁺-2Cl cotransport activity, leading to an increase of cell volume (694, 695, 824). The increase of cell volume is related to oscillations of intracellular Ca²⁺ activity (242), which can be triggered by bradykinin, bombesin, or serum in those cells (686, 694, 697, 1296, 1359, 1360). The Ca²⁺ oscillations cause rapid transient cell shrinkage due to activation of Ca²⁺-sensitive K⁺ channels (1005, 1358), followed by a sustained increase of cell volume presumably due to a depolymerization of the actin filaments (243, 1004) and subsequent shift of the set point for cell volume regulation (242). The Ca²⁺ oscillations are in turn favored by cell shrinkage (1006), pointing to a negative-feedback loop (Fig. 4).

View larger version (41K): [in this window] [in a new window]   FIG. 4.   Cell volume in proliferating cells. Cellular mechanisms triggered by expression of ras oncogene leading to activation or inhibition of cell volume regulatory mechanisms. Expression of Ras oncogene (RAS) sensitizes phospholipase C (PLC) for growth promotors such as bradykinin, bombesin, or serum. As a result, bradykinin-induced formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) is enhanced. Instead of a single release of cellular Ca2+, bradykinin induces sustained oscillations of intracellular Ca2+ concentration by triggering both Ca2+ entry through Ca2+ channels and Ca2+ release from cellular stores. On one hand, Ca2+ oscillations cause repetitive activation of Ca2+-sensitive K+ channels, leading to oscillations of cell membrane potential and transient decrease of cell volume. On the other hand, Ca2+ oscillations lead to depolymerization of actin filaments, which presumably facilitates activation of Na+/H+ exchanger and Na+-K+-2Cl cotransport. Activation of these carriers results in uptake of KCl and NaCl. Because Na+ is replaced by K+ by action of Na+-K+-ATPase, cells accumulate mainly KCl. Ion uptake increases cell volume, which is one prerequisite for stimulation of cell proliferation. Increase of cell volume is limited by inhibition of Na+/H+ exchanger and Na+-K+-2Cl cotransport by cell swelling (ICS and ECS stand for intracellular and extracellular space, respectively).

Apparently, the activation of Na⁺/H⁺ exchange and Na⁺-K⁺-2Cl cotransport is required for stimulation of cell proliferation by ras oncogene (694, 697, 824, 1000). In several cell types, cell proliferation is similarly correlated with enhanced Ca²⁺ and K⁺ channel activity (for review, see Ref. 1000) and is impeded by respective channel inhibitors (17, 726, 739, 893, 981, 1091, 1123, 1199, 1256, 1369). However, the activity of certain ion channels and transporters may not always be required for cell proliferation to occur. Inhibition of the Na⁺/H⁺ exchanger, for instance, is not always found to interfere with cell proliferation (83, 189, 598, 827, 1025).

How alterations of cell volume interact with cell cycle control is not known. As outlined above, cell swelling stimulates the protein kinases ERK-1 and ERK-2 (1072), proteins probably involved in regulation of cell cycle (113).

In proliferating cells, the role of increased cell volume in altering further volume-sensitive cellular functions such as alkalinization of lysosomal vesicles (588), decreased proteolysis (501), and increased LDL receptor expression at the cell surface (1136) has not yet been explored.

In contrast to cell proliferation, cell differentiation is accompanied by cell shrinkage in erythroleukemia cells (264, 458, 607, 725), HL-60 leukemic cells (311, 475), and EMT6/ro mouse mammary sarcoma cells (118). HL-60 cells shrink despite enhanced expression of Na⁺/H⁺ exchanger (986), which displays altered kinetic properties (227, 228). On the other hand, senescent fibroblasts have been shown to gain cell volume (960, 961).

Apoptotic cell death (617, 1367) is triggered by a wide variety of factors including stimulation of specific receptors at the cell membrane, such as the receptor for Fas (CD95) (1074) or tumor necrosis factor- (655).

One of the hallmarks of apoptotic cell death is cell shrinkage (1, 207). Shrinkage may in some cells be secondary to increased intracellular Ca²⁺ activity (922, 1226) and subsequent activation of Ca²⁺-sensitive K⁺ and/or Cl channels. Accordingly, it could be inhibited by K⁺ channel blockers or increased extracellular K⁺ concentration (55, 61). Stimulation of the Fas-receptor leads to rapid activation of cell volume regulatory Cl channels (I. Szabo, E. Gulbins, and F. Lang, unpublished observations) as well as delayed taurine release, the latter effect paralleling cell shrinkage (686a). The loss of cell volume may be functionally important, since a doubling of extracellular osmolarity has been shown to trigger apoptosis (109, 811). Moreover, the ability of cells to resist osmotic shrinkage by cell volume regulation paralleled their resistance to apoptosis after an osmotic shock (109). As discussed in section IIIH, osmotic stress triggers the MAPK pathway, leading to activation of JNK via the MAPK kinase (MKK4) (809, 853, 1020, 1216). Both JNK and p38 kinase, another target of MKK4, have been invoked in the triggering of apoptosis (1368). On the other hand, MKK4 has been postulated to protect from apoptosis (894). Thus the precise function of these kinases in apoptosis remains elusive. Whether the proteolytic effect of cell shrinkage (see Table 1) contributes to apoptosis remains to be tested.

Surprisingly, Fas-induced cell death is inhibited during exposure of the cells to moderately hypertonic extracellular fluid (451). Moreover, in lymphocytes, stimulation of the Fas receptor leads to inhibition of the n-type K⁺ channel or Kv1.3 via tyrosine phosphorylation (449, 1183), which is the volume regulatory K⁺ channel in lymphocytes (273). It appears that cell shrinkage interferes at some point with the signaling cascade of apoptotic cell death. Fas-induced cell death is triggered by activation of caspases with subsequent stimulation of sphingomyelinases, ceramide formation, activation of Ras, PI 3-kinase, Rac, MKK4, and JNK or p38 kinase (446, 448, 1368). In addition, activation of Rac triggers formation of active oxygen species (AOS), presumably by the NADPH oxidase (447). Cell shrinkage apparently does not interfere with the cascade leading to Ras activation but prevents the formation of AOS, since the decrease of GSH is blunted in shrunken lymphocytes (451).

Several mechanisms could mediate the inhibition of the signaling cascade: cell shrinkage stimulates the expression of ₁-chimerin (see Table 1), a GTPase activating protein for Rac. If a similar protein is expressed in lymphocytes as a function of cell volume, it could account for some inhibition of NADP oxidase activity. Moreover, cell shrinkage inhibits glucose flux through the pentose phosphate pathway, decreasing the availability of NADPH (see sect. VC). Furthermore, hyperosmolarity interferes with the respiratory burst oxidase (573). Along these lines, hyperosmotic cell shrinkage blunts agonist-triggered AOS formation (602, 659, 795, 810), and hyposmotic cell swelling stimulates AOS formation (602) in polymorphonuclear leukocytes. Leukocyte AOS production through NADPH oxidase is further inhibited by high concentrations of urea (1187), which at least in some cells leads to cell shrinkage (see sect. IIIA) and similar to osmotic shrinkage inhibits Fas-induced cell death (E. Gulbins and F. Lang, unpublished observations).

Although the decreased NADPH availability in shrunken cells may interfere with endogenous formation of AOS and thus protects from Fas-induced cell death, it renders the cells more vulnerable to exogenous oxidative stress, whereas cell swelling appears to protect from exogenous oxidative stress (804, 1046).

Beyond the role of AOS, cell shrinkage is expected to turn on Na⁺/H⁺ exchange, leading to alkalinization (see sect. IIB). Intracellular acidosis, on the other hand, has been considered a prerequisite for apoptosis (405).

Because cell shrinkage apparently interferes with the Fas signaling pathway, the inhibition of K⁺ channels after activation of the Fas receptor could serve to prevent premature inhibition of the signaling cascade by cell shrinkage. In analogy with Fas-induced cell death, methylprednisolone-induced apoptosis of thymocytes was observed to be inhibited by low doses of the K⁺ ionophore valinomycin (255). Furthermore, tumor necrosis factor- has been shown to stimulate Na⁺/H⁺ exchange (1248).

Clearly, the role of cell volume regulatory mechanisms in apoptotic cell death is still ill-defined, and at this point, their functional significance remains a matter of speculation.

Phagocytosis, for instance, could be expected to increase cell volume. Indeed, phagocytotic Kupffer cells do contain betaine, which is released during phagocytosis (1319, 1393). Similarly, the ion channels activated during phagocytosis (641) may at least in part serve RVD. Moreover, phagocytosis has been shown to be sensitive to ambient osmolarity (1321).

Activation of thrombocytes is paralleled by excessive cell shrinkage, which participates in the metamorphosis of the cells (59).

Furthermore, some evidence points to the involvement of cell volume regulatory mechanisms during lymphocyte adhesion, which may play a permissive role in the activation of Na⁺/H⁺ exchange (562, 1103-1106). Binding to L-selectins, with the receptors mediating the first contact of lymphocytes with the endothelial cell surface (1018), triggers an intracellular cascade involving Ras activation (121), ultimately leading to an increase of cell volume (E. Gulbins, B. Brenner, and F. Lang, unpublished observations). The functional significance of these events is still elusive but may relate to the considerable mechanical forces operative during adhesion of lymphocytes to the endothelial surface (1395).

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