RAPID COMMUNICATION
Effects of arginine vasopressin on cell volume regulation in brain astrocyte in culture

Darya Sarfaraz and Cosmo L. Fraser

Department of Medicine, Division of Gerontology, University of California at San Francisco, and Veterans Affairs Medical Center, San Francisco, California 94121


    ABSTRACT
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

Astrocytes initially swell when exposed to hypotonic medium but rapidly return to normal volume by the process of regulatory volume decrease (RVD). The role that arginine vasopressin (AVP) plays in hypotonically mediated RVD in astrocytes is unknown. This study was therefore designed to determine whether AVP might play a role in astrocyte RVD. With the use of 3-O-[3H]methyl-D-glucose to determine water space, AVP treatment resulted in significantly increased 3-O-methyl-D-glucose water space within 30 s of hypotonic exposure (P = 0.0001) and remained significantly elevated above baseline (1.75 µl/mg protein) at 5 min (P < 0.021). In contrast, in untreated cells, complete RVD was achieved by 5 min. At 30 s, cell volume with AVP treatment was 37% greater than in cells that received no treatment (2.9 vs. 2.26 µl/mg protein, respectively; P < 0.006). The rate of cell volume increase (dV/dt) over 30 s was highly significant (0.038 vs. 0.019 µl · mg protein-1 · s-1 in the AVP-treated vs. untreated group; P = 0.0004 by regression analysis). Additionally, the rate of cell volume decrease over the next 4.5 min was also significantly greater with vasopressin treatment (-dV/dt = 0.0027 vs. 0.0013 µl · mg protein-1 · s-1; P = 0.0306). The effect of AVP was concentration dependent with EC50 = 3.5 nM. To determine whether AVP action was receptor mediated, we performed RVD studies in the presence of the V1-receptor antagonists benzamil and ethylisopropryl amiloride and the V2-receptor agonist 1-desamino-8-D-arginine vasopressin (DDAVP). Both V1-receptor antagonists significantly inhibited AVP-mediated volume increase by 40-47% (P < 0.005), whereas DDAVP had no stimulatory effects above control. Taken together, these data suggest that AVP treatment of brain astrocytes in culture appears to increase 3-O-methyl-D-glucose water space during RVD through V1 receptor-mediated mechanisms. The significance of these findings is presently unclear.

regulatory volume decrease; hypotonicity; V1-receptor agonist; V2-receptor agonist


    INTRODUCTION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

ASTROCYTES are important regulators of brain water, and they make up ~20% of brain intracellular space (19, 45). They contain high Na-K-ATPase activity (46) and express sex hormone receptors (11, 17). Like most cells, astrocytes swell when exposed to hypotonic conditions (12, 20, 21, 37, 42) but subsequently return to near-normal volume by the process of regulatory volume decrease (RVD). This process is accomplished by the extrusion of osmotically active solutes from within cells (21, 22, 23, 36, 37, 41, 42). This volume-regulatory response of cells to hypotonicity is a cell survival mechanism that prevents uncontrolled volume increases that could result in cell lysis and cell death. In addition to hypotonicity, other factors can cause cell swelling and prevent RVD as well (36). It has been shown that when astrocytes were exposed to hypotonic medium without calcium, RVD was abolished. In the absence of calcium, cells would swell normally when exposed to hypotonicity but were unable to decrease their volume to achieve effective RVD. However, with the addition of calcium to swollen cells containing calcium-free medium, RVD proceeds rapidly. Calcium has also been shown to play a critical role in RVD in many types of cells, including cardiac (8, 39, 52), renal (41), platelet (43, 48, 50), and muscle cells (2, 26, 31). Glutamate and ouabain have also been shown to cause cell swelling and prevent RVD (12, 18, 25, 30). In a more recent study (12), our laboratory has shown that the female sex hormones estrogen and progesterone prevented appropriate RVD in brain astrocytes in culture during hypotonic challenge.

The hormone arginine vasopressin (AVP) induces pressor and antidiuretic responses by the activation of two distinct types of vasopressin receptors. The V1 and V2 receptors are coupled to two different intracellular second messengers that differ in their sizes and sites of action (55). Whereas V1-receptor activation in hepatocytes and platelets is associated with phosphoinositol turnover, V2-receptor activation in renal tubular cells is said to result in increased cAMP production (43). More recently, it has been shown that AVP may also stimulate phosphoinositol hydrolysis via V2 receptors in rat inner medullary collecting tubular cells as well (47).

V1 receptors are the predominant type of AVP receptors in brain and are located in a number of sites including astrocytes (9, 38). Although other studies (6, 7, 27, 40) showed that vasopressin can cause astrocyte swelling, we are unaware of other studies that have reported a relationship between AVP and hypotonically mediated cell volume regulation in brain astrocytes in culture. A recent study (27) utilizing the 3-O-methyl-D-glucose method has shown that AVP causes cell swelling in the absence of hypotonicity. However, the question of whether AVP action was affected by hypotonicity was not addressed. Our study was therefore designed to investigate whether AVP further affected astrocyte cell volume during hypotonically mediated RVD.


    MATERIALS AND METHODS
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

Astrocyte culture. Primary cortical astrocyte cultures were prepared from newborn female rats (Simonsen, Gilroy, CA) with the method of Hertz et al. (14). Dissociated cortices were suspended in Eagle's minimal essential medium with 10% fetal bovine serum (Hyclone, Ogden, UT) and 2 mM glutamine, plated onto Falcon 24-well tissue culture plates (Becton-Dickinson, Oxnard, CA) and incubated at 37°C in a 5% CO2 atmosphere. At confluency (12-15 days in vitro), 10 µM cytosine arabinoside were added to inhibit growth of other cell types. After 48 h, this medium was replaced with 0.5 ml of serum-free medium consisting of a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) plus 100 mg/ml transferrin, 5 mg/ml insulin, 0.1 mM putrescine, 30 nM sodium selenate, 0.5 mg/ml albumin, and 2 mM glutamine. After 7 days, an additional 0.25 ml of the same medium was added to each well. Cells were used to determine RVD at 24-34 days in vitro.

Volume regulation. Astrocyte volume regulation was measured by the method of Kletzien et al. (24) as modified by Bender et al. (1) with room air and temperature. Cells were equilibrated for 1 h in 0.5 ml/well of Hanks' balanced salt solution (HBSS; 137 mM NaCl, 5 mM KCl, 0.44 mM KOH2PO4, 4 mM NaHCO3, 1.3 mM CaCl2, 0.8 mM MgSO4, and 0.5 mM MgCl2 · 6H2O) with 10 mM glucose. At the end of this time period, the medium was replaced with 0.5 ml/well of glucose-free HBSS containing 1 mM 3-O-methyl-D-glucose and 1 mCi/ml 3-O-[3H]methyl-D-glucose for 15 min. A hypotonic challenge was presented by exchanging this isotonic medium (osmolality = 300 mosmol/kgH2O) with a medium of identical composition except NaCl was reduced to between 75 and 100 mM (osmolality = 150-195 mosmol/kgH2O by vapor pressure; Fisket Associates, Needham Heights, MA). Control well media were exchanged with isotonic medium. In some experiments, 1-100 µM benzamil, 1-100 µM ethylisopropyl amiloride (EIPA), 1 µM DDAVP, and various concentrations of AVP were added during the hypotonic challenge as the experiments required. Incubation was terminated at the appropriate times by four washes with ice-cold 290 mM sucrose solution containing 1 mM phloretin, 0.5 mM calcium dinitrate, and 10 mM Tris-nitrate, pH 7.4. The cells were digested in 1 M NaOH, and aliquots were taken for counting on a Packard liquid scintillation counter (model 2000CA) (10). Protein determinations were made as described previously (29). Extracellular water space was determined with either [methoxy-3H]inulin or [methoxy-14C]inulin, which were added during the final hypotonic challenge. Intracellular water space was determined by subtracting the external water space from the total 3-O-methyl-D-glucose water space (12).

Data analysis. All data are expressed as means ± SD. Significance was determined by either two-way ANOVA with Fisher's post hoc comparisons or by linear regression analysis where indicated. A probability of P < 0.05 was considered to be significant. Analysis was performed with StatView version 4.5 software package.


    RESULTS
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

When brain astrocytes in culture in isotonic medium (osmolality of 300 mosmol/kgH2O) were exposed to hypotonic medium (osmolality of 194 mosmol/kgH2O), cell volume rapidly increased from 1.75 to 2.26 µl/mg protein within 30 s of exposure to the hypotonic medium. Cell volume subsequently decreased toward its initial level to a point where it was not significantly different (1.91 µl/mg protein) from baseline at 5 min. This indicates an increase in cell volume from baseline of 29% (P = 0.001) at 30 s and 9% at 5 min (Fig. 1). However, when cells were treated with 1 µM AVP, the maximum volume reached at 30 s was 2.90 µl/mg protein, which is 66% (P < 0.0001) above the initial cell volume of 1.75 µl/mg protein and 37% greater (P = 0.006) than the volume of 2.26 µl/mg protein reached by the untreated cells at the same time point. Cell volume in the AVP-treated group subsequently decreased to 2.17 µl/mg protein at 5 min, which is 15% above that of control cells and 24% above (P < 0.021) the initial cell volume. Although there were no significant differences between the end volumes in both the treated and untreated groups, only with AVP treatment was the value at 5 min significantly greater than the initial cell volume of 1.75 µl/mg protein (P < 0.021).


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Fig. 1.   Effect of 1 µM arginine vasopressin (AVP) on brain astrocyte regulatory volume decrease (RVD). Compared with control cells that show normal cell volume regulation, AVP treatment resulted in significantly greater volume increase over time. Data are means ± SD of 6 experiments, and each time point was done in triplicate. NS, not significant. AVP volume effects were significantly greater than in control cells at 0.5 min (P < 0.006), 2 min (P < 0.041), and 3 min (P < 0.002). Both groups had initial cell volume of 1.75 µl/mg protein as determined by 3-O-methyl-D-glucose. There was no significant difference between groups at 5 min, although with AVP treatment, end volume at 5 min was significantly greater than initial volume at time 0 (P < 0.021). By linear regression analysis, rates of cellular volume increase and decrease were significantly different between treated and untreated groups (see Table 1).

The rate of cell volume increase (dV/dt) in the AVP treatment group within the first 30 s was 0.038 µl · mg protein-1 · s-1 compared with only 0.019 µl · mg protein-1 · s-1 in the control group (Table 1), a difference that was highly significant (P = 0.0004) between the groups. At the same time, the rate of cell volume decrease in the AVP treatment group over the final 4.5 min of observation was also significantly greater than that in the untreated group (-dV/dt = 0.0027 µl · mg protein-1 · s-1 compared with 0.0013 µl · mg protein-1 · s-1, respectively; P = 0.0306). Thus both the rate of cell volume increase and the rate of cell volume decrease were at least twice as great with AVP treatment as with no treatment. Thus it appears that AVP treatment is able to stimulate transmembrane water flux in these cells, although the mechanism by which this occurs is presently unknown.

                              
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Table 1.   Rate of cell volume increase and decrease during RVD of brain astrocyte in culture

Although the AVP-treated cells were able to volume-regulate at a faster rate than control cells, complete RVD was not achieved with AVP after 5 min of hypotonic exposure (Fig. 1). To determine whether the action of AVP on cell volume regulation was concentration dependent, we first performed studies with a high and a low concentration of AVP (100 µM and 1 nM) and compared the results to that of control cells that were not treated with AVP. The result indicates that with either AVP concentration, cell volume was significantly greater than that of the untreated cells (P < 0.05). The increased volume was approximately twice as large with 100 µM AVP compared with the lower 1 nM AVP concentration. As a result of this observation, we investigated the effect of various concentrations of AVP on cell volume regulation, the results of which are shown by the AVP concentration curve in Fig. 2. As shown in Fig. 2, at 2 min of hypotonic exposure, cell volume progressively increased with increasing concentrations of AVP, reaching a maximum volume at AVP concentrations >1 µM and with EC50 = 3.5 nM. The increase in 3-O-methyl-D-glucose space was significantly greater than that of control cells at AVP concentrations >1 nM (P < 0.002). No increase in cell volume above control values was observed at an AVP concentration <0.1 nM.


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Fig. 2.   Concentration effect of AVP on astrocyte during cell RVD after 2 min of exposure to hypotonic medium (osmolality 194 mosmol/kgH2O). 3-O-methyl-D-glucose water space increased with increasing AVP concentration ([AVP]) and reached a plateau at concentrations >= 1 µM. Data are means ± SD of 4 experiments. Each time point was done in triplicate. Cell volume was significantly greater than that of control cells (no added AVP) at [AVP] >=  1 nM (P < 0.002). EC50 = 3.5 nM AVP.

To determine whether the observed action of AVP on astrocyte RVD was receptor mediated, we performed studies to determine the effect that V1-receptor antagonists and V2-receptor agonist may have on AVP-associated RVD (Fig. 3). When cells were treated with 1 µM AVP in the presence of the V1-receptor antagonists benzamil and EIPA (49), no increase in cell volume was observed, unlike that which occurred with AVP alone. Both the amiloride analogs benzamil and EIPA at concentrations of either 1, 10, or 100 µM significantly inhibited the AVP-induced volume increase seen with AVP alone (P < 0.001), with a maximum effect observed at 100 µM AVP (Fig. 3). Cell volume in the presence of AVP alone was decreased by >40% with either benzamil or EIPA (Fig. 3). Thus it appears that the V1-receptor antagonists completely inhibited the stimulatory action of AVP in our cell preparation. In contrast, the V2-receptor agonist DDAVP had no apparent stimulatory effect on cell volume and did not affect control RVD. Neither benzamil nor EIPA alone had any effect on hypotonically mediated cell volume regulation in either the treated or untreated cells.


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Fig. 3.   Effects of AVP agonist 1-desamino-8-D-arginine vasopressin (DDAVP) and antagonists on AVP-mediated 3-O-methyl-D-glucose water space. Data are means ± SD; 4 experiments were performed. In presence of 1 µM AVP, V1-receptor antagonists [100 µM benzamil and 100 µM ethylisopropyl amiloride (EIPA)] significantly inhibited effect of AVP (P < 0.005). In contrast, amiloride antagonist of Na/H antiports had no significant effect on AVP action (data not shown). Similarly, V2-receptor agonist DDAVP had no effect on cell volume as is manifested by AVP. cpm, Counts/min.


    DISCUSSION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

The results of this study provide evidence to support the idea that AVP acting through V1 receptor-mediated mechanisms causes an increase in 3-O-methyl-D-glucose water space in brain astrocyte in culture during RVD. Although other studies (4, 6) have shown that AVP can increase 3-O-methyl-D-glucose water space in astroglia, to our knowledge the present study is the first to show such an action of AVP during hypotonically mediated cell volume regulation. Not only did AVP increase hypotonically mediated cell swelling during the first 30 s of exposure but the rate at which the cell volume returned toward the initial baseline value was increased as well (Table 1). The rate of cell volume increase in the AVP treatment group within the first 30 s (dV/dt) was 0.038 µl · mg protein-1 · s-1 compared with only 0.019 µl · mg protein-1 · s-1 in the control group. At the same time, the rate at which cell volume decreased in the AVP treatment group over the final 4.5 min (-dV/dt) was 0.0027 µl · mg protein-1 · s-1 compared with only 0.0013 µl · mg protein-1 · s-1 in the untreated group. Thus both the initial rates of cell volume increase and decrease are at least twice as rapid with AVP treatment and were significantly greater than the control rate (Table 1). This increased rate of cell volume increase with AVP treatment occurred when both groups had identical initial starting volumes and osmolality (Fig. 1). Thus the difference in the rate of cell volume increase between the two groups cannot be readily explained on the basis of differences in driving forces due to either tonicity or initial cell size. However, in contrast, because after 30 s the cell volume in AVP-treated cells was greater than in control cells, the increased rate of cell volume decrease with AVP could partially be explained by the differences in cell volume at 30 s (Fig. 1).

The response of AVP-treated cells to hypotonic challenge (Fig. 1) indicates either that AVP may have resulted in failure of important adaptive mechanisms during cell volume regulation or that it promotes transmembrane water transport during hypotonic challenge through water channels. One possible pathway is the aquaporin (AQP) channels, which are novel types of water channels described in both brain and kidney (35). Studies have indicated that these distinctive channels are important mediators of water transport. The only AQP known to be regulated by AVP is AQP channel AQP-2, which may be important in AVP-mediated urinary concentrating ability in humans. AQP-2 is also felt to be a site of mutation in some forms of nephrogenic diabetes insipidus (5). AQP-2 has not been found in astrocytes, so it is unlikely that the AVP effects we observed involve this channel. In addition to its role in human urinary concentration, AQP-2 is felt to be also involved in water retention in pathological states, such as the syndrome of inappropriate secretion of antidiuretic hormone, and in alcoholic cirrhosis (5, 13). Another type of water channel that could play a role in these phenomena is AQP-4, which is found in the central nervous system. It appears to be the osmoreceptor that regulates body water and mediates water flow within the central nervous system (16). AQP-4 is expressed in a number of sites in brain, including the astrocytes (33, 34). At the present time, we are not aware of any evidence that suggests that AQP-4 is capable of interacting with AVP (3).

When cells are exposed to hypotonic medium, a number of volume-regulatory mechanisms come into play to help maintain constant cell volume. These regulatory mechanisms depend on the type of cells involved and on the rate and extent to which swelling has occurred. The main goal of RVD is to decrease intracellular solutes to lower intracellular osmolality and prevent excessive water influx that could produce hypotonic lysis and cell death. Some of these compensatory mechanisms include the loss of sodium by the Na-K-ATPase pump (12, 28, 51, 53), loss of potassium and chloride (36, 42), and the gain of calcium that facilitates potassium efflux through stretch-activated potassium channels and also sodium efflux via the Na/Ca exchanger (32, 36). In the present study, no assumptions are being made regarding the mechanism of action of AVP. It is not known if AVP is activating water channels or if it is in any way interacting with other established mechanisms that are responsible for successful RVD.

3-O-methyl-D-glucose was utilized in this study to estimate cell volume because it is a well established technique that has been used to study cell volume regulation in monolayer culture by numerous other investigators (1, 12, 21, 22, 24). The utility of 3-O-methyl-D-glucose to determine intracellular water space is based on the fact that intracellular water space can be determined in cells while they are attached to a culture dish (12). This property allows cell volume to be determined in attached cells while they maintain their normal morphology and membrane properties. This method also takes advantage of the transport of the nonmetabolizable hexose 3-O-methyl-D-glucose to an intracellular concentration equal to that of its extracellular concentration, and the inhibition of its efflux by phloretin, a potent inhibitor of sugar transport (24). Thus it requires only a determination of the nanomolar amount of the hexose taken up at equilibrium and the protein concentration of the cells (24). Although there are other techniques available to assess transmembrane water transport, we decided to utilize the well-established 3-O-methyl-D-glucose method to assess cell volume because our laboratory has had much experience with this technique (12).

Because AVP appears to increase both the rate of cell volume increase in the first 30 s after hypotonic exposure and the rate of cell volume decrease over the next 4.5 min, it is possible that AVP is capable of producing multiple effects during RVD in our cell population. It could be that the initial action of AVP under these conditions is to stimulate transmembrane water transport from the hypotonic extracellular environment to the relatively hypertonic intracellular space, resulting in increased cell volume in the AVP treatment group. This increased cell volume would then serve as a driving force, on the basis of cell size alone, to stimulate RVD through stretch-activated receptors (15, 32, 54). The AVP-treated cells would therefore be stimulated to extrude osmotically active solutes at a more rapid rate than control cells. Another scenario is that if AVP prevented cells from effectively extruding osmotically active solutes during hypotonic exposure (6, 27), then cell volume in the AVP group would be larger than that of control cells on the basis of a higher transmembrane osmolar gradient acting as a driving force for water influx. As before, but for different reasons, RVD may then be enhanced as secondary adaptive mechanisms come into play to defend against the rapid rise in cell volume that was driven by osmolality. Thus the increased rate of cell volume decrease after the initial cell volume increase observed with AVP treatment might indeed be dependent on a number of physiological factors that are yet to be determined.


    ACKNOWLEDGEMENTS

We thank Dr. Allen I. Arieff of the Dept. of Medicine at the Univ. of California at San Francisco for reviewing the manuscript and providing helpful comments and suggestions. We are grateful to Dr. Raymond Swanson of the Neurology Dept. for providing us with the astrocytes in culture.


    FOOTNOTES

This work was supported in part by National Institute on Aging Grant AG-08575-02S1.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: C. L. Fraser, Veterans Affairs Medical Center, Dept. of Medicine, Gerontology Section (NH), 4150 Clement St., San Francisco, CA 94121.

Received 22 September 1998; accepted in final form 16 November 1998.


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Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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