Upregulation accumulation

of inositol transport mediates inositol in hyperosmolar brain cells

KEVIN STRANGE, REBECCA MORRISON, CHARLES W. HEILIG, SUSAN DIPIETRO, AND STEVEN R. GULLANS Division of Nephrology, Department of Medicine, Children’s Hospital, and Renal Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

STRANGE, KEVIN, REBECCA MORRISON, CHARLES W. HEILIG, SUSAN DIPIETRO, AND STEVEN R. GULLANS. Upregulation of inositol transport mediates inositol accumulation in hyperosmoZar brain ceLZs.Am. J. Physiol. 260 (Cell Physiol. 29): C784C790, 1991.-Attempts to understand brain volume regulation have been greatly hampered by the structural complexity of the mammalian central nervous system, indicating a need for the investigation of cultured brain cell lines whose behavior reflects that observed in situ. We demonstrate here that rat C6 glioma cells exhibit a pattern of hyperosmolar volume regulation qualitatively similar to that of the intact brain. Chronic (2-6 days) acclimation of C6 cells to high NaCl media (440 or 590 mosM) resulted in a 46-133 mM increase in cellular inositol, a known major brain osmolyte. C6 cells exposed acutely to 440 mosM medium shrank abruptly and then underwent a complete regulatory volume increase (RVI) within 4 h. Inositol levels began to increase after 10 h of hyperosmolar stress and reached maximal values by 24 h, suggesting that RVI is initially mediated by inorganic ion uptake. [3H]inositol uptake measurements revealed a sevenfold stimulation of phlorizin-inhibitable inositol transport in hyperosmotic cells. The enhancement of inositol transport paralleled the rise in cellular inositol content. Phlorizin reduced inositol accumulation in hyperosmolar cells by 44%. Our studies provide the first demonstration of RVI and organic osmolyte accumulation in a cultured brain cell line.

volume inositol

regulation;

idiogenic

osmoles; organic

osmolytes;

myo-

OF CELL VOLUME is a physiological process fundamental to all organisms. Cell volume can be altered by changes in intracellular solute content or extracellular osmolality (anisosmotic volume stress). Most mammalian cells are normally protected from anisosmotic volume changes by precise renal regulation of plasma solute and water content. A variety of pathophysiological conditions, however, have significant effects on extracellular osmolality and cell volume. Plasma hyposmolality is a major complication of numerous disease states such as congestive heart failure, syndrome of inappropriate antidiuretic hormone secretion, and Addison’s disease. Increases in plasma osmolality are observed with enteric disorders, renal failure, central and nephrogenic diabetes insipidus, and diabetes mellitus. Some of the most serious clinical consequences of cell volume change are manifested by swelling or shrinking of the brain. Changes in brain cell volume can lead to MAINTENANCE

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$1.50

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severe neurological impairment

and high mortality

(1,

14, 19). Cell volume regulation involves the accumulation or loss of inorganic ions and organic solutes. Extensive studies of osmotically activated ion transport pathways have been carried out in a variety of vertebrate cells and tissues (reviewed in Refs. 4, 8, 9). The utilization of organic osmolytes for volume control has been examined in numerous organisms and cell types spanning the five taxonomic kingdoms (4, 30). Although accumulation or loss of any organic solute would serve to bring a cell to osmotic equilibrium with its environment, only a restricted number of organic osmolytes are used in biological systems. The general classes of organic osmolytes are sugars (e.g., glucose, mannose, trehalose), polyols (e.g., inositol, sorbitol), amino acids (e.g., proline, tauFine, alanine), and methylamines [e.g., betaine, glycerophosphorylcholine (GPC)] . Volume regulatory organic compounds belong to a class of biologically important molecules referred to as “compatible” or “stabilizing” solutes. Compatible solutes can be accumulated by cells to high concentrations without adversely affecting cellular architecture and function (4, 21, 30). Much of our insight into the volume regulatory role of organic osmolytes has come from studies on bacteria and marine invertebrates (4, 30). More recently, however, extensive studies on the mammalian kidney have greatly expanded our understanding of organic compounds and cell volume regulation in mammals (3, 29). In the intact kidney, cultured renal cells and isolated nephron segments, osmoregulatory changes in intracellular sorbitol, inositol, betaine, and GPC have been observed. Alterations in the concentrations of these solutes are brought about by highly specific changes in membrane transport processes and metabolic pathways. In situ investigations of the mammalian brain during osmotic stress have documented acute changes in electrolytes and chronic changes in so-called idiogenic or unidentified organic osmolytes (1,14,19). Many of these organic solutes have now been identified. Intracellular amino acids such as alanine, glycine, taurine, glutamine, and glutamate (5,7,15,26,27), the methylamines betaine (15) and GPC (7, E), and the polyol inositol (7, 15, 17) have all been shown to change in response to osmolality disturbances, suggesting that they play a volume regulatory role in the brain. Despite its physiological and clinical significance, little

0 1991 the American

Physiological

Society

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is known about the cellular and molecular mechanisms of brain volume maintenance. Attempts to understand osmoregulation in the intact brain have been greatly hampered by the structural complexity of the mammalian central nervous system. The investigations described in this paper were therefore undertaken to develop cell culture models for characterizing brain osmoregulatory processes, particularly the mechanisms of organic osmolyte accumulation and loss. In the present studies, we utilized rat C6 glioma cells. This cell line has characteristics of intact brain glial cells such as expression of S100 protein (25) and glial fibrillary protein (20). Furthermore, C6 cells have been used previously as a model of cytotoxic (12) and postischemic (11) brain cell swelling. Our results demonstrate that C6 cells exposed to acute hyperosmolality (hypernatremia) exhibit a pattern of volume and organic osmolyte regulation qualitatively similar to that observed in the intact mammalian brain. MATERIALS

AND

METHODS

Cell culture. Rat C6 glioma cells were purchased from the American Type Culture Collection (Rockville, MD) and cultured in 25 or 75 cm2 flasks containing Eagle’s minimal essential medium (MEM) with 10% fetal bovine serum and penicillin-streptomycin. Flasks were maintained in a humidified 5% C02-95% air atmosphere at 37”C, and growth media were changed every 2 days. All experiments were initiated after 4-5 days of growth when cultures had reached -7O-90% confluency. Hyperosmotic growth media were made by adding NaCl directly to MEM. Three media, referred to as 290, 440, and 590 mosM MEM, were used in these studies. The measured osmolalities of these solutions were 285295,430-445, and 580-600 mosM, respectively. Organic osmolyte determinations. Cells were harvested by brief trypsinization and then washed three times with saline (PBS) w 2+- and Ca2+-free phosphate-buffered that had the same osmolality as the culture media. Cell pellets were extracted in ice-cold 6% perchloric acid (PCA) for 30 min. PCA supernatants were neutralized with 3 N KOH containing 50 mM K2HP0, and then lyophilized. Precipitated proteins were solubilized in 0.1 N NaOH and quantified using a Lowry assay and bovine serum albumin (Sigma, St. Louis, MO; fraction V powder) as a standard. Cellular organic compounds were initially identified by high-resolution ‘H-nuclear magnetic resonance (NMR) spectroscopy in PCA extracts containing 4-5 mg of cellular protein. Lyophilized PCA extracts were reconstituted in 0.6 ml of D20 (99.7% deuterium) containing 5 mM sodium 3-trimethylsilylpropionate-2,2,3,3-d4 (TSP), which was used to standardize concentration as well as reference chemical shift. NMR spectra were recorded at 23°C using a GN 300 WB NMR spectrometer operating at 300.51 MHz (see Ref. 7). Pulses of 90” were applied at 12-s intervals, and 8,000 data points were collected using a spectral width of +2,500 Hz. Each spectrum was the sum of 128 transients with l-Hz Gaussian line broadening applied prior to Fourier transformation. Cellular inositol content was determined directly by

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enzyme assay (28). Neutralized PCA extracts containing 0.5-1.5 mg of cellular protein were lyophilized and then reconstituted in 1.0 ml of H,O. Inositol was determined in duplicate aliquots of 400-450 ~1. Measurement of cell volume. Cell volume was determined using video- and computer-enhanced differential interference contrast (DIC) optical sectioning microscopy (24). Briefly, C6 cells were cultured in 20-mmdiameter culture dishes with glass cover slip bottoms. Cultures were maintained in 290 mosM MEM or transferred abruptly to 440 mosM MEM. At various times, dishes were removed from the incubator and placed on the stage of an inverted microscope equipped with DIC optics. Single cells were imaged using a Zeiss Neofluar x63 objective lens, a Leitz x32 objective-condenser lens, and a Dage model 65 television camera. The objective lens was step focused through the cell under computer control. At the bottom of the cell and at each 1.3 ,urn of focal displacement, a video image was recorded on video disk. These images were digitized at a later time, and the cross-sectional area of each optical section was determined by image processing techniques. With the use of the cross-sectional area and known focal displacement between optical sections, cell volume was calculated as described previously (23, 24). [3H]inositol uptake measurements. C6 cells were grown in 6-well culture dishes (Costar, Cambridge, MA; 3.5-cm diameter) and exposed to control or 440 mosM MEM for 0 to 96 h. At selected time points, control or hyperosmotic cells were washed twice with 3 ml of isosmotic or 440 mosM PBS as appropriate. The cells were then exposed to 2 ml of 290 or 440 mosM MEM containing 1.0 &I/ ml of [3H] inositol (New England Nuclear, Billerica, MA). After 1 min, the uptake medium was removed, and the cells were washed three times with 3 ml of ice-cold stop solution containing either 100 mM (isosmotic) or 150 mM (hyperosmotic) MgCl, plus 1.0 mM phlorizin. Cells were extracted in 0.5 ml of 0.25 M PCA and then scraped into a centrifuge tube and spun at 5,000 g for 10 min. [3H]inositol in the supernatants was determined by liquid scintillation counting, and protein was quantified in the cell pellet as described above. For each experiment a parallel series of uptake measurements was performed in the presence of 2.0 mM phlorizin (Sigma), a known inhibitor of Na+-inositol cotransport (11). Pilot studies indicated that phlorizin-inhibitable [3H] inositol uptake was linear between 1 and 30 min. RESULTS

Cellular organic osmolytes. Hypernatremia is a common cause of plasma hyperosmolality (1, 14, 19). The present studies were therefore aimed at determining whether C6 cells could survive in high-NaCl culture media. To examine this, cells were gradually acclimated to hyperosmotic MEM. This was carried out by changing the growth media every 2 days. At each change, the medium osmolality was increased by adding 25 mM NaCl until a final osmolality of 440 or 590 mosM was achieved. Cell survival was assessed as the ability of the cells to remain attached to the culture flasks and continue growing. C6 cells showed normal attachment to the growth

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surface, had normal morphology, and continued to grow, albeit more slowly, in both 440 and 590 mosM media. To determine whether organic osmolytes changed in response to medium osmolality, high-resolution ‘HNMR spectroscopy was conducted on PCA extracts of C6 cells. Cultures were either grown in isosmotic MEM (290 mosM) or gradually acclimated to 440 and 590 mosM MEM and then maintained under these hyperosmotic conditions for an additional 2-6 days. Figure 1 shows typical ‘H-NMR spectra of C6 cell extracts. These spectra are qualitatively similar to those observed in the intact rat brain (7). Under control conditions the major easily resolved organic solutes are lactate, phosphocreatine and creatine, inositol, acetate, and various amino acids including alanine, glutamate, and glutamine. Dramatic increases in cellular inositol levels were observed in cells cultured in 440 and 590 mosM MEM. An apparent decrease in lactate levels accompanied the rise in inositol (Fig. 1). Smaller increases in GPC, choline, and some amino acids were also apparent in the spectra. Further measurements are needed to provide additional LAC

Control Medium (290 mOsm)

PCr

High NaCl Medium (440 mOsm)

Amino Acids

High NaCl Medium (590 mOsm)

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IN

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quantification of cellular lactate, GPC, choline, and amino acid levels. Enzymatic assays confirmed the presence of inositol in cell extracts (Fig. 2). In isosmotic MEM cellular inositol content was 28 t 1 nmol/mg protein (n = 4). This value increased to 133 t 4 (n = 4) and 334 t 20 nmol/mg protein (n = 5) in 440 and 590 mosM MEM, respectively. Similar changes in cell inositol levels were observed when medium osmolality was increased to 440 mosM by addition of raffinose (Strange and Morrison, unpublished observations). With the use of previous estimates of the relationship between cell volume and cell protein content (11) and with the assumption that 79% of the cell volume is osmotically active under both control and hyperosmotic conditions (see below), cytoplasmic inositol concentrations of 12, 58, and 145 mM can be calculated. External inositol concentration in MEM with 10% serum is -16 PM, indicating that this polyol was accumulated against a 750- to 9,100-fold chemical gradient. In 440 and 590 mosM MEM, inositol accounts for 38 and 48%, respectively, of the increased cellular osmolality. Cell volume regulation and inositol accumulation. C6 cells could also be transferred abruptly to 440 mosM MEM with no apparent loss of viability. DIC microscopy was used to examine volume regulation in these cells. Figure 3 shows a typical optical section scan of a single C6 cell grown in 290 mosM MEM. Preliminary studies indicated that optical sectioning could detect minimum cell volume changes of 5%. C6 cells transferred to 440 mosM MEM shrank 27% (Fig. 4), a volume change expected for a cell in which 21% of its volume is osmotically inactive. Such a value is well within the range observed for many other cells types (8). Four hours after exposure to 440 mosM MEM, cell volume had returned to a value that was not significantly different (P > 0.6) from control and remained constant for an additional 6 h (Fig. 4). Figure 5 shows the time course of cellular inositol accumulation following abrupt exposure to 440 mosM MEM. Four hours after the exposure cell inositol concentration had not changed significantly. After 10 h in hyperosmotic MEM, however, cell inositol had increased threefold and bv 24 h had reached maximal levels. p50 .Q) %oo k ,,, 4 200 E 5150

l”“l”“l”“l”“l”“~““l”“~“‘~~ 4.5 4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

PPM FIG. 1. High resolution ‘H-nuclear magnetic resonance (NMR) spectra of extracts of C6 cells grown in isosmotic or high NaCl media. Each spectrum was scaled with respect to sample protein content so that peak heights reflect relative changes in intracellular levels of various organic compounds. I, inositol; LAC, lactate; ALA, alanine; PCr, phosphocreatine; Cr, creatine; AC, acetate. Peaks labeled 1 and 2 are glycerophosphorylcholine (GPC) and choline, respectively.

&o !s z50

290

mOsm MEM

440

mOsm MEM

590

mOsm MEM

FIG. 2. Enzymatic quantification of inositol content in grown in isosmotic or high NaCl media. *Value is significantly than control (P < 0.0003); n = 4-6.

C6 cells greater

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FIG. 3. Representative differential interference contrast (DIC) microscopy optical section scan of a single C6 cell grown on a glass cover slip in 290 mosM MEM. Optical sectioning begins at side of cell closest to growth surface (A) and proceeds to the top of cell (H). Focal displacement between successive images is 1.3 pm.

Mechanism of inositol accumulation. Cellular inositol levels can be increased during hyperosmotic stress by alterations in metabolism and/or changes in membrane transport. To assess the role of inositol transport in volume regulation, the effect of 2.0 mM phlorizin, a wellknown inhibitor of Na+-inositol cotransport (lo), was examined. Table 1 demonstrates that phlorizin reduced inositol concentrations by 44-68% in both control cells

and cells acclimated to 440 mosM MEM for 24 h. Inositol transport was further examined by conducting [3H]inositol uptake measurements. The initial rate of inositol uptake in control cells was 65 + 11 pmol . min-’ . mg protein-’ (n = 38) and was inhibited 77 & 2% (n = 38) by 2.0 mM phlorizin. As shown in Fig. 6, 4 h of exposure to 440 mosM MEM had no effect on inositol transport. After 24 h of exposure, however, there was a

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t

* l-

w =16003 B Ihoo

290

8

mOsm

MEM

1 12ooJ

r( 0

440 I

2

mOsm

MEM I

4

TIME

I

I

8

10

(ho~rs)~

*

-

*

mOsm

MEM

290

mOsm

MEM

T

FIG.

tained mosM

0

8I

16I

24I

TIb%1

(hou:os)I

-

4t

48I

56I

1 72

I

64

5. Time course of changes in inositol levels in C6 cells mainin isosmotic medium or in cells transferred abruptly to 440 MEM. * Value is significantly greater than control (P < 0.004);

n = 4-6.

1. Effects of 2.0 mM phlorizin on intracellular inositol content of C6 cells TABLE

Medium

290 mosM 290 mosM 440 mosM 440 mosM

MEM MEM MEM MEM

+ phlorizin + phlorizin

24I

36I

I

TIME &urs)

60I

I

1

72

84

1 96

[3H]inositol uptake in growth medium to 440 than control (P < 0.005);

* 440

4P

0

12I

FIG. 6. Relative change in phlorizin-sensitive C6 cells transferred abruptly from isosmotic mosM MEM. * Value is significantly greater n = 4-12.

FIG. 4. Time course of volume changes in C6 cells abruptly transferred to 440 mosM culture medium (MEM). * Value is significantly less than control (P < 0.005); n = 12-19.

350 .Q) z k125

OJ 0f

Inositol, nmol/mg protein

n

83.7t4.9 26.8t2.1* 176.7t3.7 99.0t2.1*

6 4 3 4

Values are means t SE; n, no. of observations. C6 cells were grown to 70-90% confluency in isosmotic MEM and then exposed to either isosmotic or hyperosmotic growth medium with or without 2.0 mM phlorizin for 24 h. * Values are significantly less than control (P < 0.0001).

200% increase in inositol uptake. Continued hyperosmotic stress further augmented inositol transport to a maximal rate sevenfold greater than control. In cells exposed to 440 mosM MEM for 24,48, and 96 h, phlorizin inhibited inositol uptake by 88 t 1 (n = 12), 94 t 1 (n = II), and 88 t 2% (n = 7)) respectively, indicating that there was a selective upregulation of the phlorizin-inhibitable inositol transport pathway. DISCUSSIBN

Studies on whole animals have shown that brain water content is regulated in the continued presence of hyper-

osmotic disturbances (1, 2, 14, 19). The cellular and molecular mechanisms by which this occurs, however, are largely unknown. Attempts to understand brain volume regulation have been greatly hampered by the structural complexity of the mammalian central nervous system, which is composed of multiple cell types and multiple physiologically distinct regions and compartments. Such complexity argues strongly for the need to develop and investigate cultured cell models exhibiting volume regulatory behavior similar to that observed in situ. The present studies demonstrate that rat C6 glioma cells provide such a model system. We have observed a pattern of volume and organic osmolyte regulation in this cell line that is qualitatively similar to that observed in the intact brain. NMR and enzyme analyses demonstrated that large quantities of inositol were accumulated by C6 cells during chronic acclimation to high NaCl media (Figs. 1 and 2). Similar findings have been made in situ. Lohr et al. (17) recently demonstrated that chronic hypernatremia in rats (3 days) caused a 60% increase in brain inositol levels. NMR studies by Heilig et al. (7) in salt-loaded rats demonstrated a 36% increase in brain inositol levels and revealed that inositol is one of the major organic solutes present in rat brain. Lien et al. (15) have recently shown that chronic hypernatremia in rats induces a 65% elevation of brain inositol. Whereas further studies are needed, our NMR analyses also suggested that cellular GPC and choline levels are increased and lactate levels are decreased by exposure of C6 cells to high NaCl media. Again, similar findings have been made in the intact brain. Heilig et al. (7) have observed significant increases in GPC and choline concentrations in brains of hypernatremic rats. Increases in rat brain GPC levels during hypernatremia have also been observed by Lien et al. (15). In mice, hypernatremia has been shown to reduce brain lactate levels, possibly reflecting an effect of hypertonicity on brain energy metabolism (16, 26). Acute exposure to 440 mosM culture medium caused C6 cells to shrink 27% and then activate regulatory volume increase (RVI) mechanisms that restored cell

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volume to control values within 4 h (Fig. 4).l This initial RVI is most likely mediated by inorganic ion transport pathways because it was complete several hours before inositol accumulation was observed (cf. Figs. 4 and 5). Inorganic ion transport pathways play an important volume regulatory role in numerous cell types (4, 8, 9). For example, RVI in the unicellular alga, Platymonas subcordiformis, is mediated by a rapid, transitory increase in intracellular NaCl and a slower permanent accumulation of mannitol. In C6 cells, part of the inorganic ions accumulated during the initial RVI are presumably exchanged for inositol as the cellular machinery required for accumulation of this polyol is activated. Measurements of intracellular ions and inorganic ion transport in C6 cells undergoing acclimation to high-NaCl media are required to test this hypothesis. The time course of both RVI and inositol accumulation in C6 cells reflects that observed in the intact mammalian brain. For example, Cserr and co-workers (6) demonstrated that exposure of rats to acute hypernatremia for 15-120 min resulted in brain shrinkage. The shrinkage, however, was much less than that predicted for an ideal osmometer, indicating that RVI mechanisms had been activated. This rapid volume regulatory behavior was accounted for by the uptake of inorganic ions (6). In contrast, the accumulation of organic solutes in response to hyperosmotic stress requires chronic exposure to hyperosmolality for several hours to several days (1, 2, 14, 19) Membrane transport has been shown previously to play an important role in the regulation of brain cell inositol levels (22). The accumulation of inositol by C6 cells undergoing acclimation to 440 mosM medium is mediated, at least in part, by increased membrane transport. Cellular levels of inositol in both control and high NaCl cells were reduced 44-68% by exposure of C6 cultures to phlorizin (Table I), a potent inhibitor of Na+inositol cotransport (10). [3H]inositol uptake measurements demonstrated a dramatic sevenfold stimulation of inositol transport in C6 cells acclimated to 440 mosM MEM (Fig. 6). This enhanced transport was not observed until lo-24 h after exposure to high NaCl medium and coincided with the first observed increases in cell inositol content (cf. Figs. 5 and 6). Phlorizin inhibited [3H] inositol uptake by 80-90% in both control and high NaCl cells, indicating that there was a selective upregulation of a phlorizin-inhibitable inositol transport pathway. The mechanisms of inositol transport in both control and hyperosmotic C6 cells, as well as the mechanism by which high NaCl increases inositol transport, remain to be determined. Upregulation of phlorizin-inhibitable inositol transport has also recently been observed in cultured kidney cells exposed to hyperosmotic media and is thought to ’ Technical considerations limited the temporal resolution of optical sectioning measurements of cell volume regulation in C6 cells. It is possible, indeed likely, that RVI is extremely rapid in this cell type. Recent studies in this laboratory have utilized a laser light-scattering method for quantification of volume changes in cells cultured on glass cover slips. Preliminary results (K. Strange and J. Fischbarg, unpublished observations) have demonstrated that C6 cells undergo RVI that is complete within 2-3 min after exposure to a 60 mosM hyperosmotic stress.

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play an important role in cell volume maintenance. As demonstrated by Nakanishi et al. (18), exposure of Madin-Darby canine kidney (MDCK) cells to a growth medium made hypertonic (700 mosM) by the addition of NaCl and urea resulted in a SOO-400% increase in inositol levels. This increase occurs much more slowly (i.e., 2-4 days) than that observed in C6 cells but is mediated by phlorizin-sensitive transport from the external medium. Hypertonicity enhanced inositol uptake -400% by increasing the maximum velocity ( Vmax)of the transporter two- to threefold. Because the K, of the uptake mechanism was unchanged by hypertonicity, these investigators suggested that volume regulation in MDCK cells was mediated by increasing the number of functional inositol transporters. In summary, our investigations are the first demonstration of RVI and organic osmolyte accumulation in a cultured brain cell line. Furthermore, our studies have provided the first evidence as to how the accumulation of inositol and possibly other organic osmolytes may be enhanced during exposure of the brain to hyperosmolar stress. The pattern of hyperosmolar volume regulation we observed in C6 cells is qualitatively similar to that observed in situ, indicating that this cell line is an excellent model system for the characterization of brain osmoregulatory mechanisms. Results described in this paper provide an essential foundation for elucidating the cellular and molecular basis of hyperosmolar adaptation in the brain. A detailed understanding of brain volume homeostasis is of fundamental importance for the proper treatment of numerous debilitating and fatal human diseases. We thank Dr. Robert Grubbs for use of his tissue culture facilities and Patty Beltz for extensive advice on tissue culture methods. This study was supported by a grant from the American Diabetes Association and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-36031. Address for reprint requests: K. Strange, Div. of Nephrology, Dept. of Medicine, The Children’s Hospital, Harvard Medical School, Boston, MA 02115. Received

27 August

1990; accepted

in final

form

19 November

1990.

REFERENCES 1. ARIEFF, A. I., AND R. GUISADO. Effects on the central nervous system of hypernatremic and hyponatremic states. Kidney Int. 10: 104-116, 1976. 2. ARIEFF, A. I., R. GUISADO, AND V. C. LAZAROWITZ. Pathophysiology of hyperosmolar states. In: Disturbances in Body Fluid Osmolality, edited by T. E. Andreoli, J. J. Grantham, and F. C. Rector, Jr. Bethesda, MD: Am. Physiol. Sot., 1977, p. 227-250. 3. BURG, M. B. Role of aldose reductase and sorbitol in maintaining the medullary intracellular milieu. Kidney Int. 33: 635-641, 1988. 4. CHAMBERLIN, M. E., AND K. STRANGE. Anisosmotic volume regulation: a comparative view. Am. J. Physiol. 257 (Cell Physiol. 26): c159-C173,1989. 5. CHAN, P. H., AND R. A. FISHMAN. Elevation of rat brain amino acids, ammonia and idiogenic osmoles induced by hyperosmolality. Bruin Res. 161: 293-301, 1979. 6. CSERR, H. F., M. DEPASQUALE, AND C. S. PATLAK. Regulation of brain water and electrolytes during acute hyperosmolality in rats. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F522F529,1987. 7. HEILIG, C. W., M. E. STROMSKI, J. D. BLUMENFELD, J. P. LEE, AND S. R. GULLANS. Characterization of the major brain osmolytes that accumulate in salt-loaded rats. Am. J. Phvsiol. 257 (Renal

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Fluid Electrolyte Physiol. 26): F1108-F1116, 1989. 8. HOFFMANN, E. K. Control of cell volume. In: Transport of Ions and Water in Animals, edited by B. L. Gupta, R. B. Moreno, J. L. Aschman, and B. J. Wall. London: Academic, 1977, p. 285-332. 9. HOFFMANN, E. K., AND L. 0. SIMONSEN. Membrane mechanisms in volume and pH regulation in vertebrate cells. Physiol. Reu. 69: 315-382,1989. 10. HOPPER, U. Membrane transport mechanisms for hexoses and amino acids in the small intestine. In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by L. R. Johnson. New York: Raven, 1987, p. 1499-1526. 11. JACUBOVICZ, D. S., S. GRINSTEIN, AND A. KLIP. Cell swelling following recovery from acidification in C6 glioma cells: an in vitro model of postischemic brain edema. Brain Res. 435: 138-146,1987. 12. KEMPSKI, 0. L., L. CHAUSSY, U. GROSS, M. ZIMMER, AND A. BAETH-MANN. Volume regulation and metabolism of suspended C6 glioma cells: an in vitro model to study cytotoxic brain edema. Brain Res. 279: 217-228, 1983. 13. KIRST, G. 0. Coordination of ionic relations and mannitol concentrations in the euryhaline unicellular alga, Platymonas subcordiformis (Hazen) after osmotic shocks. Planta Berl. 135: 69-75, 1977. 14. KLEEMAN, C. R. Metabolic coma. Kidney Int. 36: 1142-1158,1989. 15. LIEN, Y.-H. H., J. L. SHAPIRO, AND L. CHAN. Effects of hypernatremia on organic brain osmoles. J. Clin. Invest. 85: 1427-1435, 1990. 16. LOCKWOOD, A. H. Acute and chronic hyperosmolality. Arch. Neurol. 32: 62-64, 1975. 17. LOHR, J. W., J. MCREYNOLDS, T. GRIMALDI, AND M. ACARA. Effect of acute and chronic hypernatremia on myoinositol and sorbitol concentration in rat brain and kidney. Life Sci. 43: 271276,1988. 18. NAKANISHI, T., R. J. TURNER, AND M. B. BURG. Osmoregulatory changes in myo-inositol transport by renal cells. Proc. Natl. Acad. Sci. USA 86: 6002-6006,1989. 19. POLLOCK, A. S., AND A. I. ARIEFF. Abnormalities of cell volume regulation and their functional consequences. Am. J. Physiol. 239

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Upregulation of inositol transport mediates inositol accumulation in hyperosmolar brain cells.

Attempts to understand brain volume regulation have been greatly hampered by the structural complexity of the mammalian central nervous system, indica...
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