Cell volume regulation in renal cortical cells

Both proximal renal tubule cells and cultured Madin-Darby canine kidney (MDCK) cells are capable of regulating their volume in hypotonic media. Regulatory cell volume decrease in proximal straight tubules is impaired by barium, amiloride and acetazolamide and depends on the presence of bicarbonate and of sodium, whereas it is unaffected by complete removal of extracellular chloride. The observations may point to parallel loss of potassium through potassium channels as well as of bicarbonate and sodium via a bicarbonate-sodium cotransport. Alternatively, potassium/hydrogen ion exchange or potassium bicarbonate cotransport could be involved. In MDCK cells, exposure to hypotonic media apparently leads to the activation of an anion channel, while potassium conductance is rather decreased. In both proximal tubules and MDCK cells, volume regulatory decrease is possibly triggered by leucotrienes, which may be released during cell swelling. Cell volume is altered in a variety of conditions even at isotonic extracellular fluid and cell volume-regulatory mechanisms are likely to participate in regulation of renal transepithelial transport.     

Volume regulation of Chinese hamster ovary cells in anisoosmotic media

Chinese hamster ovary (CHO) cells when suspended in anisoosmotic media regulate their volumes by the activation of specific ion transport pathways. In hypoosmotic media the cells first swell and then return to their isoosmotic volumes by the loss of cellular KCl and osmotically obliged water. This regulatory volume decrease (RVD) is insensitive to ouabain or bumetanide but is blocked by quinine, cetiedil and oligomycin C. Based on cell volume and membrane potential measurements under various experimental conditions, we conclude that hypoosmotic shock activates independent, conductive transport pathways for K+ and for Cl-, respectively. The anion pathway can also transport NO3- and SCN- but not gluconate- anions. Osmotic shrinkage of CHO cells does not produce a regulatory volume increase (RVI) unless the cells have previously undergone a cycle of RVD. RVI is a Na+-dependent, amiloride-sensitive, but ouabain- and oligomycin-insensitive process, probably involving a Na+-H+ exchange system. Internal acidification of isoosmotic cells by addition of a permeable weak acid also activates an amiloride-sensitive Na+-H+ exchange, producing a volume increase. Both RVD and RVI in CHO cells seem to involve molecular mechanisms similar to those described for the volume regulation of lymphocytes, indicating the prevalence of these phenomena in nucleated mammalian cells. Cultured CHO cell lines may provide a basis for a genetic characterization of the volume-regulatory transport pathways.     

Effect of exogenous ATP on the volume of TA3 ascites tumor cells

When exogenous ATP is added to suspensions of TA₃ ascites tumor cells suspended in Ca⁺⁺ and Mg⁺⁺ free media, a significant increase in cell volume can be measured. This increase is reversible upon addition of Ca⁺⁺ and/or Mg⁺⁺ back to the media. The enlargement of these cells is temperature sensitive and specific for ATP; no other nucleotides, EDTA or ouabain were effective. The evidence suggest that this phenomena may be due to an alteration in membrane permeability and that the regulation of membrane permeability is an energy dependent process.     

Relations between cell volume control, microfilaments and microtubules networks in T2 and PC12 cultured cells

The possible relations between cell volume, microfilaments and microtubules networks have been studied in cultured mice fibrosarcoma cells of line T2 and rat pheochromocytoma cells of line PC12. The obtained results show that: 1. Changes in volume induced by application of hypo-osmotic medium are concomitant with a modification in the organization of the microfilaments network as visualized by immunocytochemistry. The microtubules lattice is not affected in these conditions. 2. Disruption of the microfilaments network by cytochalasin B causes a significant decrease in cell volume in isosmotic conditions. It also deeply affects the volume regulation response of cells swollen in hypo-osmotic media. 3. Disruption of the microtubules lattice by colchicine has no effect on volume in isosmotic conditions nor on the volume regulation that follows application of hypo-osmotic shock. The possible role of microfilaments in cell volume control is discussed.     

Interactions of sodium transport, cell volume, and calcium in frog urinary bladder

The volume of individual cells in intact frog urinary bladders was determined by quantitative microscopy and changes in volume were used to monitor the movement of solute across the basolateral membrane. When exposed to a serosal hyposmotic solution, the cells swell as expected for an osmometer, but then regulate their volume back to near control in a process that involves the loss of KCl. We show here that volume regulation is abolished by Ba++, which suggests that KCl movements are mediated by conductive channels for both ions. Volume regulation is also inhibited by removing Ca++ from the serosal perfusate, which suggests that the channels are activated by this cation. Previously, amiloride was observed to inhibit volume regulation: in this study, amiloride-inhibited, hyposmotically swollen cells lost volume when the Ca++ ionophore A23187 was added to Ca++-replete media. We attempted to effect volume changes under isosmotic conditions by suddenly inhibiting Na+ entry across the apical membrane with amiloride, or Na+ exit across the basolateral membrane with ouabain. Neither of these Na+ transport inhibitors produced the expected results. Amiloride, instead of causing a decrease in cell volume, had no effect, and ouabain, instead of causing cell swelling, caused cell shrinkage. However, increasing cell Ca++ with A23187, in both the absence and presence of amiloride, caused cells to lose volume, and Ca++-free Ringer's solution (serosal perfusate only) caused ouabain-blocked cells to swell. Finally, again under isosmotic conditions, removal of Na+ from the serosal perfusate caused a loss of volume from cells exposed to amiloride. These results strongly suggest that intracellular Ca++ mediates cell volume regulation by exerting a negative control on apical membrane Na+ permeability and a positive control on basolateral membrane K+ permeability. They also are compatible with the existence of a basolateral Na+/Ca++ exchanger.     

Hyper-osmotic cell volume regulation in vivo and in vitro in flounder (Platichthys flesus) heart ventricles

Summary Cell volume regulation in heart ventricles of the flounderPlatichthys flesus has been studied under hyper-osmotic conditions in vivo and in vitro.During reacclimation of flounders from fresh water to sea water the osmolality of blood plasma increased to the sea water level in 7 days. The water content of the heart ventricle cells remained constant after reacclimation while the intracellular concentration of K⁺ and taurine increased to values found in sea water flounders.Hearts taken from fresh water flounders were mounted in vitro and perfused with hyper-osmotic ringer solution. Upon perfusion the cellular water content of the ventricle decreased initially but then steadily increased. After 6 h the cellular water content was still below the control values reflecting that the ventricle cells have an incomplete volume regulation in a hyper-osmotic medium. The cellular amount of K⁺ and taurine increased during the volume regulation. The perfusion media did not contain taurine or amino acids and the gain in cellular taurine must be due to intracellular production.     

Characterization of low potassium-resistant mutants of the Madin-Darby canine kidney cell line with defects in NaCl/KCl symport

Three independent mutants of the Madin-Darby canine kidney cell line (MDCK) have been isolated which were capable of growth in media containing low concentrations of potassium. All three mutants were deficient to varying extents in furosemide- and bumetanide-sensitive 22Na+, 86+b+, and 36Cl- uptake. The two mutants most resistant to low K+ media had lost essentially all of the 22Na+, 86Rb+, and 36Cl- uptake activities of this system. The third mutant was partially resistant to low K+ media and had reduced levels of bumetanide-sensitive uptake for all three ions. Extrapolated initial uptake rates for 22Na+, 86Rb+, and 36Cl- revealed that the partial mutant exhibited approximately 50% of the parental uptake rates for all three ions. The stoichiometries of bumetanide-sensitive uptake in both the parental cell line and the partial mutant approximated 1 Rb+:1 Na+:2 Cl-. The results of this study provide genetic evidence for a single tightly-coupled NaCl/KCl symporter in MDCK cells. The correlation between the ability to grow in low K+ media and decreased activity of the bumetanide-sensitive co-transport system suggests that the bumetanide-sensitive transport system catalyzes net K+ efflux from cells in low K+ media. The results of 86Rb+ efflux studies conducted on ouabain-pretreated mutant and parental cells are consistent with this interpretation. Cell volume measurements made on cells at different densities in media containing normal K+ concentrations showed that none of the mutants differed significantly in volume from the parental strain at a similar cell density. Furthermore, all three mutants were able to readjust their volume after suspension in hypotonic media. These results suggest that in the MDCK cell line, the bumetanide-sensitive NaCl/KCl symport system does not function in the regulation of cell volume under the conditions employed.     

Volume regulation in cortical collecting duct cells: role of AQP2

BACKGROUND INFORMATION: The renal CCD (cortical collecting duct) plays a role in final volume and concentration of urine by a process that is regulated by the antidiuretic hormone, [arginine]vasopressin. This hormone induces an increase in water permeability due to the translocation of AQP2 (aquaporin 2) from the intracellular vesicles to the apical membrane of principal cells. During the transition from antidiuresis to diuresis, CCD cells are exposed to changes in environmental osmolality, and cell-volume regulation may be especially important for the maintenance of intracellular homoeostasis. Despite its importance, cell-volume regulation in CCD cells has not been widely investigated. Moreover, no studies have been carried out till date to evaluate the putative role of AQPs during this process in renal cells. RESULTS: In the present study, we have studied the regulatory cell-volume responses to hypo-osmotic or hyperosmotic challenges in two CCD cell lines: one not expressing AQPs and the other stably transfected with AQP2. We have used a fluorescent probe technique in which the acquisition of single-cell kinetic data can be simultaneously recorded with the intracellular pH. Experiments with hyperosmotic mannitol media demonstrated that, independent of AQP2 expression, CCD cells shrink but fail to show regulatory volume increase, at least under the studied conditions. In contrast, under hypo-osmotic shocks, regulatory volume decrease occurs and the activation of these mechanisms is more rapid in AQP2 transfected cells. This regulatory response takes place in parallel with intracellular acidification, which is faster in cells expressing AQP2. The acidification and the initial regulatory volume decrease response were inhibited by glibenclamide and BaCl2 only in AQP2 cells. CONCLUSIONS: These results suggest that increases in the osmotic water permeability due to the expression of AQP2 are critical for a rapid activation of regulatory volume decrease mechanisms, which would be linked to cystic fibrosis transmembrane conductance regulator and to barium-sensitive potassium channels.     

Osmotic Balance in a Marine Pulmonate,Amphibola crenata

The pulmonate mud‐snail Amphibola crenata is an osmoconformer in 25%‐125% sea water, its haemolymph being slightly hyperosmotic and hyperionic to the medium. It maintains its haemolymph markedly hyperosmotic in media below 25% sea water and in freshwater, in which it survives for a week or longer. On exposure to dilute or concentrated media, water which enters or leaves the tissues preferentially enters or leaves the cells. Regulation of cell volume towards initial values is accomplished within 5 days. Measurements of changes in cell volume, and of distribution of tissue water between the intracellular and extracellular compartments, in snails moved from 100% sea water into dilute or concentrated media, and in snails moved back to 100% sea water, indicate that mechanisms of cell volume regulation are minimal in ca 60–65% sea water. Changes in the concentration of tissue free amino acids are in accord with current theories of mechanisms of isosmotic intracellular regulation. The results are discussed in the light of the taxonomic, evolutionary and ecological status of Amphibola as a shore‐living, primitive, marine representative of a predominantly terrestrial and freshwater group of gastropod molluscs.     

Physiological significance of volume-regulatory transporters

Research over thepast 25 years has identified specific ion transporters and channelsthat are activated by acute changes in cell volume and that serve torestore steady-state volume. The mechanism by which cells sense changesin cell volume and activate the appropriate transporters remains amystery, but recent studies are providing important clues. A curiousaspect of volume regulation in mammalian cells is that it is oftenabsent or incomplete in anisosmotic media, whereas complete volumeregulation is observed with isosmotic shrinkage and swelling. The basisfor this may lie in an important role of intracellularCl in controllingvolume-regulatory transporters. This is physiologically relevant, sincethe principal threat to cell volume in vivo is not changes inextracellular osmolarity but rather changes in the cellular content ofosmotically active molecules. Volume-regulatory transporters are alsoclosely linked to cell growth and metabolism, producing requisitechanges in cell volume that may also signal subsequent growth andmetabolic events. Thus, despite the relatively constant osmolarity inmammals, volume-regulatory transporters have important roles inmammalian physiology.     

Regulation of sheep erythrocyte volume in anisotonic media.

Sheep erythrocytes of high and low potassium types were incubated in non-haemolytic hypotonic and hypertonic media for 4-5 h at 30 degrees. After initial swelling or shrinking, they readjusted their volume toward their initial isotonic volume. The volume regulation was associated with specific changes in cation fluxes. In the swollen cells, efflux of both sodium and potassium was increased and influx of both cations was slightly decreased; the converse was true for the shrunken cells. All four fluxes were changed in a direction that led to return to normal volume. The difference in the response of the two types of sheep erythrocytes to changes of extracellular fluid osmolality resided in the different activity of their cation transport systems. It is concluded that sheep erythrocytes possess some means of regulating their volume in vitro which is linked to cation permeability. The exact nature of the physical mechanisms by which they accomplish this remains to be elucidated.     

Regulation of intracellular osmotic pressure during the initial stages of salt stress in a salt-tolerant yeast, Zygosaccharomyces rouxii.

The accumulation of glycerol and inorganic ions as it related to osmotic pressure, and the regulation of intracellular osmotic pressure in a salt-tolerant yeast, Zygosaccharomyces rouxii, were examined for several hours after salt stress. Intracellular contents of glycerol increased for up to 6 h in media supplemented with 1 M and 2 M NaCl and did not increase in medium containing 3 M NaCl. Intracellular contents of Na+ and Cl- reached a maximum value within 1 and 3 h, respectively, in all NaCl-containing media and increases were proportional to the concentration of NaCl in the medium. As glycerol was accumulated in cells, the intracellular contents of Na+ and Cl- gradually decreased in media containing 1 M and 2 M NaCl. After salt stress, cell volume decreased within 1 h and the original volume was re-established for 3 to 6 h in media with 1 M and 2 M NaCl but not in medium with 3 M NaCl. Intracellular concentrations of solutes, which were calculated from the total contents of glycerol and inorganic ions and the cell volume, became almost equivalent to the external osmotic pressure within 1 h after salt stress. Experiments using various inhibitors showed that a large amount of ATP was required not only for the synthesis and accumulation of glycerol but also for the exclusion of Na+ and Cl- from cells under salt-stressed conditions.     

Contribution of organic and inorganic osmolytes to volume regulation in rat brain cells in culture

In this work we examined the time course and the amount released, by hyposmolarity, for the most abundant free amino acids (FAA) in rat brain cortex astrocytes and neurons in culture. The aim was to evaluate their contribution to the process of cell volume regulation. Taurine, glutamate, andd-aspartate in the two types of cells, -alanine in astrocytes and GABA in neurons were promptly released by hyposmolarity, reaching a maximum within 1–2 min. after an osmolarity change. A substantial amount of the intracellular pool of these amino acids was mobilized in response to hyposmolarity. The amount released in media with osmolarity reduced from 300 mOsm to 150 mOsm or 210 mOsm, represented 50%–65% and 13%–31%, respectively, of the total amino acid content in cells. In both astrocytes and neurons, the efflux of glutamine and alanine was higher under isosmotic conditions and increased only marginally during hyposmotic conditions.⁸⁶Rb⁺, used as tracer for K⁺, was released from astrocytes, 30% and 11%, respectively, in hyposmotic media of 150 mOsm or 210 mOsm but was not transported in neurons. From these results it was calculated that FAA contribute 54% and inorganic ions 46% to the process of volume regulation in astrocytes exposed to a 150 mOsm hyposmotic medium. This contribution was 55% for FAA and 45% for K⁺ and Cl^– in cells exposed to 210 mOsm hyposmotic solutions. These results indicate that the contribution of FAA to the process of cell volume regulation is higher in astrocytes than in other cell types including renal and blood cells.Special issue dedicated to Dr. Claude Baxter.     

An electrophysiological technique to measure change in hepatocyte water volume

We have applied an electrophysiologic technique (Reuss, L. (1985) Proc. Natl. Acad. Sci. USA 82, 6014) to measure changes in steady-state hepatocyte volume during osmotic stress. Hepatocytes in mouse liver slices were loaded with tetramethylammonium ion (TMA+) during transient exposure of cells to nystatin. Intracellular TMA+ activity (alpha 1TMA) was measured with TMA(+)-sensitive, double-barrelled microelectrodes. Loading hepatocytes with TMA+ did not change their membrane potential (Vm), and under steady-state conditions alpha iTMA remained constant over 4 min in a single impalement. Hyperosmotic solutions (50, 100 and 150 mM sucrose added to media) and hyposmotic solutions (sucrose in media reduced by 50 and 100 mM) increased and decreased alpha iTMA, respectively, which demonstrated transmembrane water movements. The slope of the plot of change in steady-state cell water volume, [(alpha iTMA)0/(alpha iTMA)4min] -1, on the relative osmolality of media, (experimental mosmol/control mosmol) -1, was less predicted for a perfect osmometer. Corresponding measurements of Vm showed that its magnitude increased with hyposmolality and decreased with hyperosmolality. When Ba2+ (2 mM) was present during hyposmotic stress of 0.66 X 286 mosmol (control), cell water volume increased by a factor of 1.44 +/- 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.12 +/- 0.02, P less than 0.001. Ba2+ also decreased the hyperpolarization of hyposmotic stress from a factor of 1.62 +/- 0.04 to 1.24 +/- 0.09, P less than 0.01. We conclude that hepatocytes partially regulate their steady-state volume during hypo- and hyperosmotic stress. However, volume regulation during hyposmotic stress diminished along with hyperpolarization of Vm in the presence of K(+)-channel blocker, Ba2+. This shows that variation in Vm during osmotic stress provides an intercurrent, electromotive force for hepatocyte volume regulation.     

Angiotensin II AT2 receptor localization in cardiovascular tissues by its antibody developed in AT2 gene-deleted mice

In contrast to well-established physiological roles of the angiotensin II type 1 receptor (AT1), the significance of the type 2 receptor (AT2) remains largely unclear. AT2-knockout (AT2KO) mice have a phenotype associated with mild hypertension. This implies that AT2 has a role for the regulation of blood pressure. To gain insight into the mechanism by which AT2 regulates systemic blood pressure, we have investigated the expression of the AT2 receptor protein in adult rat cardiovascular tissues, using a newly developed polyclonal anti-AT2 antiserum that was successfully obtained in the AT2KO mice by immunizing with a peptide fragment of the receptor protein. In blood vessels, a stronger immunoreactivity was observed in endothelial cells than in the muscular media of resistant arteries. In the thoracic aorta, AT2 was observed only in muscular media. Abundant AT2 immunoreactivity was detected in perivascular nerve fibers. In the heart, positive immunostaining for AT2 was restricted to the coronary blood vessels. These data suggest that AT2 expressed in the vascular endothelial cells and muscular media in resistant arteries may play a pivotal role in systemic blood pressure regulation. AT2 was observed for the first time in the perivascular nerve fibers and may also play a role in neuronal blood pressure regulation.     

Volume regulation by Amphiuma red blood cells: strategies for identifying alkali metal/H+ transport

The regulation of cell volume in response to anisotonic media, and in a broader perspective electroneutral alkali/metal H+ exchange transport, are currently areas of general interest to transport physiologists. In this paper I outline the basic features of volume-sensitive ion fluxes as studied with Amphiuma red blood cells. As has been shown in previous studies the alkali metal ion fluxes that are responsible for volume regulation by these cells are electroneutral by virtue of obligatory counter coupling with H+. The criteria for establishing the existence of electroneutral alkali metal/H+ exchange in these cells will be reviewed and expanded on. In the process, behavior and phenomena consistent with, as well as those unique to, electroneutral alkali metal/H+ exchange will be introduced, illustrated with experimental data, and discussed. Finally, based on thermodynamic considerations, kinetic behavior will be evaluated in terms of electroneutral alkali metal/H+ transport.     

Ion channels and transporters involved in cell volume regulation and sensor mechanisms

All animal cell types have an appropriate volume. Even under physiological conditions of constant extracellular osmolarity, cells must regulate their volume. Cell volume is subjected to alterations because of persistent physicochemical osmotic load resulting from Donnan-type colloid osmotic pressure and of cell activity-associated changes in intracellular osmolarity resulting from osmolyte transport and metabolism. The strategy adopted by animal cells for coping with volume regulation on osmotic perturbation is to activate transport pathways, including channels and transporters, mainly for inorganic osmolytes to drive water flow. Under normotonic conditions, cells undergo volume regulation by pump-mediated mechanisms. Under anisotonic conditions, volume regulation occurs by additional channel/transporter-mediated mechanisms. Cell volume regulation is also attained through adjustment of intracellular levels not only of inorganic but also of organic osmolytes with changing the expression of their transporters or regulation of metabolism. In cell volume regulation mechanism, several "volume sensors" are thought to be involved. A volume-sensitive Cl- channel has lately attracted considerable attention in this regard.     

Effects of alterations in endothelial cell volume on transendothelial albumin permeability

We examined the effects of alterations in endothelial cell volume on transendothelial albumin permeability. Studies were done using a confluent monolayer of bovine pulmonary artery endothelial cells grown on gelatinized microporous filters. When endothelial cells were exposed to media made hypertonic with 200 mM mannitol, the intracellular volume (measured with 14C-urea) decreased twofold and remained decreased over a 30-minute time-span, thus showing no significant regulatory volume increase (RVI) within this time period. When endothelial cells were exposed to hypotonic media, intracellular volume rapidly doubled within 2 minutes, and then decreased to baseline values within 10 minutes in spite of the sustained hypotonic environment, a process known as regulatory volume decrease (RVD). We also measured the transendothelial flux of 125I-albumin with the cells exposed to the same osmotic changes. We observed that only under hypertonic conditions was there a significant change in the 125I-albumin permeability. These results indicate that the pulmonary artery endothelial cells in culture alter their cell volume when exposed to variations in the osmotic environment, and also show RVD in response to hypotonic conditions but no RVI within 40 minutes after exposure to hypertonic conditions. The transendothelial albumin permeability did not change under hypotonic conditions but increased under hypertonic conditions. Thus, endothelial cells shrinkage may be an important mechanism of increased endothelial macromolecule permeability. These volume changes may occur in endothelial cells in situ and have a role in inducing alterations in the transendothelial permeability to proteins.     

Volume regulation in lens epithelial cells and differentiating lens fiber cells

Previous studies from our laboratory have led us to conclude that lens cell elongation is caused by an increase in cell volume. This volume increase results from an increase in the potassium content of the cells due to decreased potassium efflux. In contrast, an increase in the volume of most cells triggers a regulatory volume decrease (RVD) that is usually mediated by increased potassium efflux. For this reason, chicken embryo lens epithelial cells were tested to see whether they were capable of typical cell volume regulation. Changes in cell volume during lens fiber differentiation were first estimated by 3H2O water uptake. Cell water increased in proportion to cell length in elongating lens cells. Treatment of epithelial cells cultured in basal medium with dilute or concentrated medium, or with medium containing 50 mM sucrose, resulted in typical volume regulatory responses. Cells lost or gained volume in response to osmotic stress, then returned to their previous volume. In addition, the elongation and increase in cell volume that accompanies lens fiber cell differentiation occurred normally in either hypo- or hypertonic media. This observation showed that the activation of mechanisms to compensate for osmotic stress did not interfere with the increase in volume that accompanies elongation. The ability of elongating cells to volume regulate was also tested. Lens epithelial cells were stimulated to elongate by exposure to embryonic vitreous humor, then challenged with hypotonic medium. These elongating cells regulated their volume as effectively as unstimulated cells. Therefore, cells that were increasing their volume due to reduced potassium efflux could adjust their volume in response to osmotic stress, presumably by increasing potassium efflux. This suggests that the changes in potassium efflux that occur during differentiation and RVD are regulated by distinct mechanisms.     

The Na+/K+/Cl- cotransport in C6 glioma cells. Properties and role in volume regulation

The role of the Na+/K+/Cl- cotransporter in the regulation of the volume of C6 astrocytoma cells was analyzed using isotopic fluxes and cell cytometry measurements of the cell volume. The system was inhibited by 'loop diuretics' with the following order of potency: benzmetanide greater than bumetanide greater than piretanide greater than furosemide. Under physiological conditions of osmolarity of the incubation media, equal rates of bumetanide-sensitive inward and outward K+ fluxes were observed. Blockade of the Na+/K+/Cl- cotransporter with bumetanide did not lead to a modification in the mean cell volume. When C6 cells were incubated in an hyperosmotic solution, a cell shrinkage was observed. It was accompanied by a twofold increase in the activity of the Na+/K+/Cl- cotransport, which then catalyzed the net influx of K+. In spite of this increased activity, no cell swelling could be measured. Incubation of the cells in an iso-osmotic medium deprived of either Na+, K+ or Cl- also produced cell shrinkage. Large activations (up to tenfold) of the Na+/K+/Cl- cotransport together with a cell swelling back to the normal volume were observed upon returning ion-deprived C6 cells to a physiological solution. This cell swelling was completely prevented in the presence of bumetanide. It is concluded that the Na+/K+/Cl- cotransport system is one of the transport systems involved in volume regulation of glial cells. The system can either be physiologically quiescent or active depending on the conditions used. A distinct volume regulating mechanism is the Na+/H+ exchange system.