Swelling-Induced Release of Glutamate, Aspartate, and Taurine from Astrocyte Cultures

The Journal of Neuroscience, May 1990, IO@): 1583-l 591

H. K. Kimelberg,1,2,3 S. K. Goderie,’ S. Higman,’ S. Pang,laa and R. A. Waniewski4 ‘Division of Neurosurgery, *Department of PharmacologyiToxicology, 9nterdepartmental Program in Neuroscience, Albany Medical College, Albany, New York 12208, and 4Wadsworth Center for Laboratories and Research, New York State Department 07 Health, Albany, New York 12201

Swelling of primary astrocyte cultures by exposing them to astrocytes seenin a number of pathological states (Kimelberg hypotonic media caused release of label after the cells had and Ransom, 1986), since swelling of isolated cells and many been allowed to accumulate 3H-L-glutamate, 3H-D-aSpattate, vertebrate and invertebrate tissuesis known to lead to the release or 3H-taurine. Comparable release of endogenous L-gluta- of taurine, glutamate, aspartate,and other amino acids as part mate or taurine, as measured by high-pressure liquid chro- of the processof regulatory volume decrease(RVD) by which matography (HPLC), was also found. Release of label was swollen cells regain their normal volume (Gilles et al., 1987). not affected by treating the cells with cytochalasin B, indi- The pathological conditions in which astroglial swelling is cating that microfilament polymerization was not signifi- observed include experimental (Barron et al., 1988) and human cantly involved. Hypotonic-induced release did not appear (Castejon, 1980) closed head injury, traumatic (stab wound) to principally involve reversal of the Na+-dependent uptake brain edema(Gerschenfeld et al., 1959), ischemia(Garcia et al., system since increasing external K+ to depolarize the cells 1977; Jenkins et al., 1984) hypoglycemia and status epilepticus by replacement of external Na+, thus maintaining isotonic (Siesjo, 1981) prolonged hypoxia (Yu et al., 1972), acute hy- conditions, increased release to a lesser extent. Threo beta- poxia with hypercapnia (Bakay and Lee, 1968) and hepatic hydroxyaspartate, a potent 3H-L-glutamate uptake blocker, encephalopathy(Norenberg, 1981). Astrocytes in vitro have been added externally stimulated efflux of 3H-L-glutamate inde- shown to exhibit RVD after swellingin hypotonic media (Kemp- pendently of the swelling-induced efflux. Upon restoration ski et al., 1983; Kimelberg and Frangakis, 1985; Olson et al., of swollen cells to isotonic medium they showed an unim- 1986) which is associatedwith an increasedpermeability to paired ability to take up 3H-L-glutamate. The swelling-in- mannitol (Kimelberg and Goderie, 1988) reversible membrane duced release of label was inhibited by a number of anion depolarization (Kimelberg and O’Connor, 1988) and releaseof transport inhibitors, one of which has been shown to signif- 3H-taurine (Pasantes-Moralesand Schousboe,1988). icantly improve outcome in an experimental brain trauma/ In this study we show that swelling of primary astrocyte cul- hypoxia model in which astrocyte swelling is an early event. tures in hypotonic media leadsto releaseof label immediately after accumulation of 3H-L-glutamate, H-D-aspartate, and 3H- taurine, and releaseof endogenousglutamate and taurine as Brain damageduring &hernia and other pathological states is measuredby HPLC. Also, this releaseis inhibited by a number now thought to be partly due to the inappropriate releaseof of anion transport blockers, including one compound that we excitatory amino acids (EAAs) such as L-glutamate and L-as- have found significantly improves recovery in an experimental partate, which, through activation of EAA receptors, causes brain trauma/hypoxia model (Cragoeet al., 1986; Kimelberg et death of certain neurons(Olney, 1969; Simon et al., 1984; Wie- al., 1987). In addition, we show that such swelling does not loch, 1985; Choi, 1988; Faden et al., 1989). The origin of the affect reuptake of 3H-L-glutamate upon return of the cells to EAAs has been tacitly assumedto be presynaptic nerve endings. isotonic media, and it is not associatedwith lossof cell viability However, it has long been known that astrocytes avidly take up as measuredby staining with trypan blue, or changesin cell glutamate and aspartateby a Na+-dependentmechanism (Hertz, growth. Also, swelling-induced releaseof 3H-L-glutamate and 1979). The glutamate taken up is then converted to glutamine taurine is not affected by treatment with cytochalasin B. through the action of the astrocyte-specific enzyme, glutamine A preliminary report of this work has been presented(Kim- synthetase(Martinez-Hernandez et al., 1977). This glutamate- elberg et al., 1989b). glutamine pathway constitutes the locus of the small glutamate pool in brain describedmany years ago (Berl et al., 196 1). Re- Materials and Methods leasefrom this pool could occur as a result of the swelling of Cell culture. Primary astrocyte cultures were prepared from the cerebral cortices of 1-d-old rats and grown in 12-well multiwells as previously described (Franaakis and Kimelbera, 1984). Cultures were 34 weeks Received June 26, 1989; revised Oct. 30, 1989; accepted Nov. 20, 1989. old and 295% GFAP positive when’they were used. ThisworkwassupportedbygrantNS23750toH.K.K.andNS21299 toR.A.W. Swelling-induced eflux. For all experiments, cells in 12-well trays We thank E. P. Graham and K. Rebehn for preparing the manuscript. were loaded by removing growth media and then washing 4 times with Correspondence should be addressed to Dr. H. K. Kimelberg, Division of Neu- a HEPES buffered media of the following composition in mmoles/liter; rosurgery A-60, Albany Medical College, Albany, NY 12208. NaCl, 122; KCl, 3.3; CaCl,, 1.2; MgSO,, 0.4; KH,PO,, 1.2; HEPES, 25 = Permanent address: Institute of Biophysics, Academia Sinica, Beijing, China. adjusted with NaOH to obtain a pH of 7.4 and glucose 10. In Na+-free Copyright 0 1990 Society for Neuroscience 0270-6474/90/051583-09$02.00/O medium basic N-methyl-D-glucamine (NMDG’) was added, and the 1584 Kimelberg et al. * Swelling-Induced Release of Amino Acids from Astrocytes

Figure 1. Hypoosmotic-media-induced efflux of label for several labeled compounds from primary astrocyte cultures. The cells were loaded with the appropriate compounds for different times and under varying conditions (see Materials and Methods). Release is expressed as a percentage of the total label accumulated and represents the release in hypoosmotic minus release in isoosmotic media. The key for the labeled compounds and the osmolarity of the media is as follows. Data represents mean ? SEM, n = 4 wells. This is the same for the data in all the figures unless stated otherwise. Minus 100 mM NaCl (dotted lines): (t - -0) ‘H-taurine, (m - q) 3H-L-glutamate, (V- - -V) 86Rb+. Minus 50 mM NaCl (continuous lines); (0-O) 3H-D-aSpartate, (04) ‘H-L-glutamate, (O----O) 3H-taurine, (v-v) *6Rb+, (A-A) I I I I I ‘H-5-HT. Na+-free medium (-50 mM 0 10 20 30 40 N-methyl-D-ghicamineC1 (NMDG.Cl) (H) 3H-L-glutamate. Time (min)

pH was then adjusted to 7.4 with HCl. The cells were loaded for 30 or to the media samples and the neutralized cell extracts. These samples 60 min by adding 0.5 ml of warmed (37°C) Na+-containing buffer, plus were then stored at -70°C until analyzed by HPLC. Samples were the different labeled compounds as follows: 0.4 PCi L-[3,4,-‘H] glutamic automatically derivatized with orthophthalaldehyde (OPA) and 2-mer- acid (S.A., 69.7 Ci/mmol) brought to a final concentration of 100 FM captoethanol in a borate buffer (pH 10.5). After derivatization, the L-glutamic acid by addition of nonradioactive L-glutamate plus 10 mM sample was injected onto a reverse-phase Cl8 column (Waters Assoc.). L-methionine sulfoximine to inhibit glutamine synthetase; 0.4 &i D-[2,3,- The column eluate was passed through a flow cell in a fluorescence 3H] aspartic acid (S.A., 26 Ci/mmol) plus unlabeled n-aspartate for a spectrophotometer with excitation wavelengths set at 340 nm and emis- final concentration of 100 PM aspartate; 0.4 &i [ 1,2,-)H] taurine (S.A., sion set at 450 nm. The fluorescent signal was then transmitted to a 35 Ci/mmol) plus unlabeled taurine for a final concentration of 50 PM computerized integrator and the areas under the peaks calculated and taurine; 0.62 &i 5-hydroxy [6-3H] tryptamine creatinine sulfate sero- stored. These HPLC chromatographic conditions are modifications of tonin (5-HT) (S.A., 12 Ci/mmol) at a final concentration of 0.1 PM plus those published by Hill et al. (1979) as described by Spink et al. (1986), 10m4M pargyline to inhibit monoamine oxidase and 10m5 M ascorbate; with the elution modified to optimize the separation of aspartate, glu- 2.5 PCi rubidium-86 (S.A., l-8 mCi/mg) used as a tracer for K+. All tamate, SCMC, asparagine, glutamine, alanine, and taurine. The limit radioisotopes were from Amersham, except ~-[3,4,-~H] glutamic acid, of detection was 2 pmol in a 10-J sample. which was from New England Nuclear. Amino acids were quantified with respect to standard solutions con- After the loading medium was removed, the cells were rapidly washed taining the amino acids ofinterest and SCMC, all at a final concentration 34 times with warmed (37°C) isotonic buffered media. Efflux into the of 10 PM and prepared identically to samples and run in quadruplicate final 1 ml of isotonic or hypotonic media was measured by removing with each daily analysis of samples. 20-~1 aliquots of the media, and the radioactivity was measured by liquid scintillation counting. In some experiments (Figs. 1, 3, 5, 7) the effluxed radioactivity and the final cell content were added back to obtain Results the initial cell content and the efflux at increasing times expressed as a Swelling-inducede@ux of labeledamino acids percentage of this. In the other experiments the amount of label left in the cells after the efflux period and the cell protein was measured and In Figure 1 we show the effect of reducing medium osmolarity compared to cells in isotonic media only. After the final sampling the by 100 or 200 mOsm on the release of label after uptake of a cells were washed 3 times in ice-cold mannitol-wash medium (0.3 M number of labeled compounds. The cultures were loaded in mannitol, 10 mM Tris Cl, 0.5 mM Ca(NO,),‘4 H,O, final pH 7.4). The normal media and then exposed to normal media or media made cells in each well were then solubilized in 1 N NaOH and aliquots taken for scintillation counting and protein determination using the Pierce hypoosmotic by removal of NaCl or NMDG in Na+-free media bicinchoninic acid (BCA) reagent as modified for use in monolayer cell in which Na+ was replaced with NMDG. The results show the cultures (Goldschmidt and Kimelberg, 1989). accumulative hypotonic-medium-induced efflux as a percentage Uptake studies. These experiments were done in the same manner as of the initial total label present at the end of the loading period. the loading part of the efflux experiments described above. The cells were rapidly washed at the desired times after radioisotope was added Generally, the amount of label released depended on the os- with the mannitol wash solution, and the cellular content of isotope molarity of the medium. For example, for 3H-taurine and 3H- and cell protein determined exactly as described above for the efflux glutamate release was much greater (75-80%) when 100 mM experiments. NaCl was removed than when 50 mM NaCl was removed (25- High-pressure liquid chromatography (HPLC). The cellular contents 30%), all measured at 40 min. There were also marked differ- of endogenous amino acids were determined by adding perchloric acid (PCA) to the cell monolayers immediately after removing the media ences between the different compounds. Release of label was and washing the cells; 0.3 ml PCA (0.4 M) was left in contact with the similar in the case of 3H-L-glutamate, 3H-D-aspartate, and 3H- cells for 2-3 min and the 12-well tray was then sonicated with the bottom taurine. However, hypotonic-medium-induced efflux of 86Rb+ of the tray immersed in a bath-type sonicator. The PCA cell extracts was considerably lower, and there was no hypotonic-medium- were centrifuged and the supernatant neutralized with 1 M KOHKHCO,. The amino acid contents of the media were determined by taking 0.2- induced efflux of labeled 3H-5-HT. ml aliquots. An internal standard, s-carboxymethylcysteine (SCMC; 10 We found that the presence or absence of Na+ in the medium PM) and 0.0 1% sodium azide to prevent bacterial growth was then added had no significant effect on the hypotonic-medium-induced ef- The Journal of Neuroscience, May 1990, 70(5) 1585

0 [3H]Taurin. 9QU c3H] Glutamate . 100 .5 I [3H] Aspartata E 0 80 K c= 80 iz 2 40 8 aki 20 25 -a u B 0 olii I 122 112 102 0 10 20 30 40 50 (293) (273) (253) (2%) $3) (1%:) (8, mM NMDG.CI TIME (min) (total m0smales) Figure3. Hypotonic-media-induced efflux of endogenous glutamate, Figure 2. Dose-response curve of the effect of varying media osmo- glutamine, ahd taurine as determined by HPLC. Primary astrocyte cul- larity on efflux of label for the amino acids shown. Cells were preloaded tures were first washed 3 times in normal HEPES buffered media (see in normal Na+-containing medium (see Materials and Methods). The Materials and Methods) and then exposed to isotonic Na+-free (Na+ cells were then exposed to Na+-free medium in which the 122 mM NaCl replaced with NMDG) or hypotonic (- 50 mM NMDG’Cl) media. Levels was replaced by the different concentrations of N-methyl-D-glucamine. of the endogenous amino acids in the media were determined at the Cl (NMDG.Cl) shown. Efflux was then measured for a lo-min period times indicated as described in Materials and Methods. These levels by measuring the cell content at the end of this period. were then expressed as an accumulative mean percentage of the level of amino acids present in the same cultures immediately after washing, by determining the content of the amino acids left in the cells after the final sample was removed and adding back this amount to the amounts flux, even though it is essentialfor uptake. We show this for effluxed (see Materials and Methods). one case in Figure 1; efflux of 3H-L-glutamateinto hypotonic Na+-free medium was only slightly lower than in Na+-containing medium. The lack of effect of extracellular Na+ in promoting Methods. 3H-taurine is unlikely to be metabolized by thesecells reuptake is probably due to the large dilution of the effluxed (Shain and Martin, 1984). However, in order to confirm that material. Basalefflux for 3H-L-glutamateand 3H-taurine in iso- authentic glutamate and taurine is releasedwhen the cells swell tonic medium averagedaround 20% (seedata in Figs. 2, 5, 6, in hypotonic medium, we also measuredefflux of endogenous 7). Basal efflux of 3H-D-aspartatewas similar, while for 86Rb+ taurine and glutamate by HPLC, in the absenceof L-methionine and 3H-5-HT, efflux into isotonic medium was 46 and 62%, sulfoximine. These resultsare shown in Figure 3, where it can respectively, over a 20-min period (data not shown). be seen that exposure to Na+-free medium from which 100 In Figure 2 we show a dose-responserelationship for hypo- mOsm of NMDG.Cl had been removed led to increasedefflux. tonic-media-induced efflux of label for 3H-taurine, 3H-L-gluta- At 30 min the efflux into isotonic Na+-free medium was 20% mate, and 3H-D-aspartateinto Na+-free media where all the for both taurine and L-glutamate, and increasedto 89 and 63%, NaCl wasreplaced by varying concentrations of NMDG.Cl. As respectively, in hypotonic medium (- 50 mM NMDG. Cl). This can be seen,the efflux of label for all 3 amino acids was similar gives a hypotonic-medium-induced efflux of 69% for taurine and increasedwith decreasingosmolarity. and 43% for L-glutamate. These values correspond reasonably well to the values for the newly accumulated labeled amino Swelling-inducedeflux of endogenousamino acids acids, except the efflux of endogenoustaurine is greater than The identical behavior of the efflux of 3H from D-aspartateor efflux of endogenousL-glutamate, unlike the results from ex- L-glutamate suggeststhat we are indeed studying efflux of 3H- periments with the labeledcompounds, where the efflux of label labeledL-glutamate, since D-aspartate is nonmetabolizable.Also, is essentially the same. Efflux of endogenousglutamine in hy- uptake of 3H-L-glutamate was only for 30 min, there was no potonic medium was the sameas for taurine, but efflux in iso- exogenoussource of NH, (Waniewski and Martin, 1986), and tonic medium was greater, so the hypotonic-medium-induced we added an inhibitor of glutamine synthetase,L-methionine efflux of glutamine was lower. sulfoximine (Nicklas et al., 1987), as describedin Materials and In Table 1 we show the endogenouslevels in the cells at 45

Table 1. Amino acid levels in cells incubated for 45 min in isotonic and hypotonic medium as a percent of initial levels

Aspartate Asparagine Alanine Glutamate Glutamine Taurine Isotonic 113.0 95.0 58.0 83.4 28.7 17.6 Hypotonic 65.4 46.9 32.9 36.1 7.1 8.6 A % change -41.6 -48.1 -25.1 -47.3 -21.0 -69.0 Intracellular levels were determined by HPLC and are from the experiment shown in Figure 3. A % change = % hypotonic minus % isotonic levels. Hypotonic medium was -50 rnM NMDG’CI. The zero time values in nmol/mg protein for the different amino acids were: Asp, 4.31 ? 0.33; Asn, 2.89 t- 0.98; Ala, 51.1 + 5.9; Glu, 55.4 + 5.4; Gln, 85.3 + 17.4; Tau, 398.0 k 28.9 (mean f SD, n = 3). 1588 Kimelberg et al. . Swelling-Induced Release of Amino Acids from Astrocytes

100 0 I50 T A [3H] L-Glutamate - HYPO

200- / f .E 60 . E 3 0 \ 60

2 loo- E 40 1 150- 0 / O-O Preincubatian in s . 122mM NaCl HC03- c 8 20 0 - l Preincubation in E 50- / 22mM NaCl HC03- 0 0, Control 5mM 5mM 1mM 1mM 1mM 0 5 10 15 20 25 30 Furu SITS L-844,711 Bumat TWA Time (min.) 100 0 Is0 B [‘HI Tautinn Figure 4. Uptake of 3H-L-glutamate by cells preincubated in isotonic - HYPO (122 mM NaCl) or hypotonic (22 mM NaCl) media for 20 min, as indicated. After this exposure both media were replaced with isotonic media and a time course for the uptake of 3H-L-glutamate (see Materials and Methods) was done. In thisexperiment 25 mM HCO,m replaced HEPES as the buffer. The exneriment was done in a 5%X0,/95% air atmosphere using a CO, incubator.

min of all the amino acids we measured.These values are ex- o- pressedas a percentageof the levels seenat 0 time. The relative Control 5mY 5mM 1mM 1mY lossesin isotonic and hypotonic media for glutamate, glutamine, FWOB sit5 L-644.71 1 BUfMt and taurine mirror the amounts found in the media (seeFig. 3). Aspartate and asparaginelevels were very low, but hypo- Figure 5. Effect of different anion transport inhibitors on hypotonic (50 mM NMDG.Cl) media-induced efflux of 3H-L-glutamate (A) and tonic-media-induced efflux could be detected. Taurine is clearly ‘H-taurine (B) into Na+-free medium. SITS, 4-acetamido-4’-isothio- the endogenousamino acid present in the highest amounts (see cyanostilbene-2,2’-disulfonic acid; Furos.,furosemide; Bumet.,bume- legend to Table 1) and also showed the greatest percentageof tanide; L-644,7 11, (R(+) [(5,6-dichloro 9a-propyl 2,3,9,9a-tetrahydro hypotonic-media-induced efflux. Basedon an intracellular vol- 3-0x0-1H fluoren-7-yl)oxy] acetic acid (Cragoe et al., 1986; Kimelberg et al., 1987); TBHA, threo beta-hydroxyaspartate. Total efluxed radio- ume of 3.5 &mg protein (Kimelberg and Frangakis, 1985; Ki- activity was expressed as a percent of the total initial radioactivity melberg and Goderie, 1988) the endogenousL-glutamate and present. Inhibitors were added at 0 time when medium was changed taurine contentscorrespond to 15.8 and 113.7 mM, respectively, from normal isotonic to either Na+-free isotonic or hypotonic medium if both these compounds are freely distributed. The value of (Na+ replaced with NMDG’). Cells were loaded as described in Figure 55.4 nmol/mg protein for glutamate correspondswell to the 1. TBHA (see text) is an inhibitor of Na+-dependent L-glutamate uptake in astrocytes(Kimelberg et al., 1989a).All hypotonic-inducedeffluxes mean value for glutamate of 67.9 nmol/mg protein for 3 different were different from the efflux in isotonic medium plus inhibitors at astrocytic clones (Cambier and Pessac,1987). levels of significance ofp = 0.000 1 or less by 2-tailed t test. For L-644,7 11 in A and SITS in B the p levels were 0.020 and 0.024, respectively. Efect of swelling on cell viability One question that arises about this hypotonic-media-induced efflux is whether it is a result of nonspecific leakage.One ar- Inhibition by anion transport inhibitors gument against this is the selectivity shown in Figure 1 and The swelling-inducedefflux of 3H-L-glutamateand 3H-taurine Table 1. Also, the cells exclude trypan blue during the peak of was inhibited by several anion transport blockers (Fig. 5). All the swelling (1 min) and continue to exclude trypan blue during the effective inhibitors are inhibitors of the Cl-/HCO,- anion the entire 30-min exposureto hypotonic media (data not shown). exchange system, as shown in red blood cells for SITS (Ca- We also found that the processwas reversible and the cells did bantchik et al., 1978), furosemide (Brazy and Gunn, 1976), and not lose important functions. Thus, after exposure to hypotonic L-644,7 11 (Garay et al., 1986), a fluorenyl derivative of etha- medium, cultures replaced in isotonic media were able to take crynic acid (Cragoeet al., 1986). In contrast, bumetanide,a spe- up 3H-L-glutamateas well as cultures exposedto isotonic media cific inhibitor of the K+ + Na+ + 2Cll cotransport system(War- for the same period of time (Fig. 4). In this one experiment neck et al., 1983), was relatively ineffective at the highest HCO,- replaced HEPES as the buffer (seelegend). These data concentration we could obtain in aqueoussolution. None of the also suggestthat glutamate homo-exchange is not a significant inhibitors had any effect on the initial hypotonic-media-induced component of the uptake of 3H-L-glutamate, as also suggested swelling, as this involves only inward movement of water. by Hertz et al. (1978) since after swelling almost 90% of the endogenousglutamate should be lost (seeFig. 2). In addition, Swelling-inducede&x appearsnot to be due to reversal of the cells continued to divide and grow the sameafter exposure Na+-dependentuptake to hypotonic medium as they did without any such prior ex- In the caseof 3H-L-glutamate,we were also interested in deter- posure (data not shown). mining whether part of the efflux involved a reversal of the Na+- The Journal of Neuroscience, May 1990, 70(5) 1587

iso + 1mMTBHA iso + 1mM Glut

-5OmM

-5OmM +TBHA Figure 6. Efflux of ‘H-L-glutamatein isotonicand hypotonic mediaand the effectof externallyadded 1 mM TBHA and L-glutamateat 0 time asindicated. Aliquots of the mediawere taken at the time indicatedand addedback to the 4 lb iii io ' final cellcontent and expressed as a percent of the initial level (seeMaterials Time (min) and Methods).

dependent L-glutamate uptake system, since we make the so- nor, 1988) in conjunction with a reversal of the Na+ gradient. lution hypotonic by reducing medium NaCl, or we measure In Table 2 we showthat the efflux of 3H-taurine, 3H-L-glutamate, efflux into hypotonic Na+-free medium. An effective inhibitor and 3H-D-aspartatewas unaffected or affected to a much lesser of L-glutamate uptake in primary astrocyte cultures, threo beta- extent when external K+ was increasedfrom 4.5 to 100 mM by hydroxyaspartate (TBHA; Kimelberg et al., 1989a),had no effect replacement of Na+, which was thus reduced from 122 to 22 on efflux of 3H label into hypoosmotic Na+-free medium after mM, thus the isotonicity of the medium was maintained than the cells were loaded with 3H-L-glutamate(Fig. 5A). However, when the solution was made hypotonic by removal of 100 mM this is a competitive blocker of 3H-L-glutamateuptake in astro- NaCl (compare Table 2 with Figs. 1, 2, 5, 7). cytes and we do not know if TBHA has freely entered the cells. Indeed, one interesting feature of this compound is that it stim- Effects of cytochalasin B on swelling-inducede&x ulated 3H-L-glutamateefflux in isotonic media (as shown in Fig. It hasbeen suggested(Sachs, 1987, 1988)that the microfilament SA). This is shown in more detail in Figure 6, where it can be componentsof the cytoskeleton may mediate the activation of seenthat efflux into isotonic media was stimulated by the pres- stretch-activated channels(SACS), and by analogy it is possible ence of 1 mM TBHA. This appearsto be by exchange since 1 that the swelling-induced amino acid transport changescould mM L-glutamate did the same thing, as originally reported by be mediatedin the sameway. In Figure 7 we showthat treatment Hertz et al. (1978). TBHA also acceleratedthe efflux in hypo- of the cells with 1, 10, or 30 PM cytochalasin B did not affect tonic media, suggestingthat the exchange system operatesin- the efflux of label for 3H-taurine or 3H-L-glutamatein isotonic dependently of the swelling-activated systemand the 2 processes medium and also did not alter the hypotonic-medium-induced are additive. release.The cells were pretreated with cytochalasin B to obtain Exposure of the cells to hypotonic media could causereversal maximum morphological changes.Shown in Figure 8B are the of the Na+-dependent L-glutamate uptake system due to de- marked morphological changescaused by 10 WM cytochalasin polarization of the membranepotential (Kimelberg and O’Con- B (compare with the normal cultures shown in Fig. 8A).

Table 2. Effect of increased K+ and decreased Na+ under isotonic conditions on ‘H-L-glutamate, ‘H-L+ aspartate, and “H-taurine efflux

Compositionof media Time (min) (changedat 0 time) 2 5 10 20 4.5 mM K+ 122mM Na+ 3H-L-glutamate 8.8 k 1.72 9.4 III 2.18 16.7 k 2.04 18.2 + 1.41 ‘H-D-aspartate 8.9 k 1.15 11.0 * 1.73 10.6? 0.78 8.1 + 0.94 3H-taurine 6.9 IL 0.73 7.4 * 1.54 8.2 k 1.32 7.8 + 1.10 100mM K+ 22 mM Na+ 3H-L-glutamate 6.9 + 1.28 14.6 + 2.60 17.2 k 0.96 24.2 + 1.50 ‘H-D-aspartate 4.8 k 1.19 7.4 + 0.50 7.6 k 0.70 9.8 k 0.81 )H-taurine 19.9 2 1.17 15.7 * 3.17 23.5 + 2.31 28.5 + 2.71 Values representefflux at timesindicated as a percentage of content at 0 time. Cells were loaded with )H-L-glutamate, ‘H-D-aspartate, or ‘H-taurine (see Materials and Methods). 100 rn~ Na+ was replaced with 100 rn~ K’ where indicated. Values are means + SEM. n was 4 wells in all cases. The 100 rn~ K’, 20-min time for glutamate, and the IO-min time for aspartate were significantly different from 4.5 rnM K+ values at p = 0.01-0.05 level. For taurine all values were significantly different at the p i 0.001 level or less, except at the 5-min time (p = 0.057). 1588 Kimelberg et al. l Swelling-Induced Release of Amino Acids from Astrocytes

looI A [3H] Taurine

100 B [3H] L-Glutamate

CONTROL DMSO 1UM 10uM 30uM Cytochalaain B

Figure 7. Effect of increasing concentrations of cytochalasin B on the amount of radioactivity left in cells after exnosure to Na+-free medium of varying osmolarity; -15 ~1 dimethyl sulfoxide (DMSO) alone or different concentrations of cytochalasin B in DMSO up to this maximum volume were first added for a 1-hr preincubation period with 3H-taurine or a 30-min preincubation with 3H-glutamate in Na+-containing medium. Cytochalasin up to 30 PM had no effect on these uptakes. The cultures were then washed in isotonic media (see Materials and Meth- ods) and then exposed to isotonic or hypotonic Na+-free media (NMDG+ replacing Na+) also containing DMSO or the different concentrations of cytochalasin B shown in DMSO. Open bars, Isotonic control; stippled bars, - 50mM NMDG.Cl$lZed bars, - 100 mM NMDG. Cl. The percent label remaining associated with the cells was measured after 10 min and compared to the initial level in the cells after loading.

Discussion Swelling-inducede&x of amino acids in astrocytes Figure 8. A, Representative phase-contrast micrograph of living cultures exposed to isotonic medium + 15 ~1 DMSO in 1 ml HEPES There is a considerablebody of information that amino acids buffered media (see Materials and Methods) for 30 min. B, The same such as taurine, alanine, glutamate, and aspartateare involved conditions except 10 WM cytochalasin B was also present. in osmoregulatjonin euryhaline vertebrates and invertebrates (Gilles, 1987). The amino acidsinvolved can vary from organism ta organism and even in different tissueswithin the same The basicobservation stimulating our work is that astrocytes animal. Thus in the common skate(Baja erinacea),taurine and are commonly, and usually specifically, swollenin a number of beta alanine are major amino acids in the nucleated red blood pathological states (seeintroductory remarks). We are using cells of this animal, while in its skeletal muscle sarcosineand hypotonic-media-induced swellingof primary astrocyte cultures beta alanine predominate. These amino acids are those which as a convenient model systemfor exploring the functional con- undergo major decreaseswhen the cells or tissue are exposed sequencesof such swelling.The reasonswhy astrocytes swell so to hypotonic solutions(Forster and Goldstein, 1979).The trans- readily under pathological conditions are still unclear (seeKi- port changesinduced by exposing tissue to hypotonic media melbergand Ransom, 1986, for a recent discussionof this ques- underlie the processesof RVD, and the needfor theseprocesses tion). In viva, significant hypoosmolarity is almost always due in euryhaline animals is clear. However, volume regulation is to lowered plasma Na+ concentrations, i.e., hyponatremia, as also seenin mammalian cells and the substancesreleased in- encountered in electrolyte imbalance in hospitalized patients. clude the major intracellular cations K+ and Cl-, aswell asamino Experimentally, hypoosmolarity leadsto lossof tam-me,as well acids such as taurine and alanine (Grinstein et al., 1984; Hoff- as aspartic and glutamic acids, from the brain (Thurston et al., mann, 1987). 1975, 1987; Wade et al., 1988; Law, 1989). In a rat model of The Journal of Neuroscience, May 1990, IO@) 1589

sustained hypoosmolarity (Verbalis and Drutarosky, 1988), the to 10 PM cytochalasin B for 12 hr changed their morphology brain water content was kept constant by loss of K+, Na+, and from a myotube to a myoball type and also resulted in a large Cl-. However, half the anion loss was due to unidentified com- increase in the sensitivity to the applied pressureof the prob- pounds which could have been organic acids. These in vivo ability of channel opening, but channel activity was not abol- results thus correspond to our results for cultured astrocytes ished. In our studiescytochalasin B had no effect on efflux of exposed to hypoosmolar solutions. Release of 3H-taurine has 3H-taurine or 3H-L-glutamateunder either hypotonic or isotonic also recently been reported by Pasantes-Morales and Schousboe conditions (Fig. 7). However, cytochalasin B did causemarked (1988) for primary astrocyte cultures swollen in hypotonic me- morphological changesin our cells (Fig. 8) confirming previous dium. However, these workers found a much smaller release of work by Ciesielski-Treskaet al. (1982). Thus these data also 3H-L-glutamate and H-D-aspartate, with which the cells had show that marked cellular and membrane shapechanges per se previously been loaded. The reasons for this discrepancy are do not affect the swelling-inducedefflux. not clear. Also, since high extracellular K+ has been shown to We found that hypotonic-media-induced swelling of astro- cause swelling of primary astrocyte cultures and is thought to cytes causesincreased permeability to relatively large com- be an important cause of pathological astrocytic swelling in vivo pounds such as aspartate and glutamate, although the cells did (see Kimelberg and Ransom, 1986) it is of interest that exposure not take up trypan blue (data not shown), which is a 960-mo- of astrocytes and other glial cultures to medium containing raised lecular-weight anion. The channelsopened in E. coli by applying [K+] leads to release of taurine (Martin et al., 1988; Philibert et pressureto patchescan be permeableto ions as large as gluta- al., 1988; and see Table 2) under isotonic conditions. mate with a large single-channelconductance of 970 pS (Mar- tinac et al., 1987) but the channels opened by this technique Mechanisms of swelling-induced amino acid release in animal cells are generally only permeant to monovalent cat- The swelling-induced release processes show some degree of ions such as K+ and divalent cations such as Ca2+(Guhary and selectivity since all the amino acids were released to a greater Sachs,1984; Kullberg, 1987; Lansmanet al., 1987; Sachs,1987, extent than preloaded 86Rb+ (an analog for K+), while preloaded 1988). However, earlier studies on the cell currents produced 3H-5-HT was not released at all (Fig. 1). The process was clearly in crayfish stretch receptors by stretching the muscle showed proportional to the degree of swelling (Fig. 2), is not associated that large cations such as Tris, tetraethylammonium, and cho- with entry of trypan blue, and there was no loss of cell viability line could pass(Brown et al., 1978). (data not shown). Hypotonic-media-induced swelling is also to- SACS are thought to respond to membrane tension, rather tally reversible in terms of both subsequent uptake of 3H-~- than directly to pressure,and a threshold tension of 5 rnN.rnml glutamate in isoosmotic media (Fig. 4) and swelling-induced was needed to activate these channels in yeast (Gustin et al., membrane potential depolarization (Kimelberg and O’Connor, 1988). We have calculated the tensionsgenerated in astrocytes 1988). These data suggesttransient changesin specifictransport under the pressuresgenerated by osmotic differencesof lOO- processes,rather than nonspecific “prelytic” opening of non- 200 mOsm assuminga spherewith a smooth surfacesuch that selective “holes” in the membrane. In further support of a se- volume increasescan only be accommodated by stretching the lective process,we have found that a group of compounds that surface membrane. On this basisthe membrane tensionsgen- have in common the fact that they will block anion transport erated are almost 2 orders of magnitude higher than the above processesinhibited the releaseof the amino acids. Recently, value. However, it is likely that the volume increasesare initially furosemide was reported to block hypotonic-media-induced re- accommodated by an unfolding of the surfacemembrane (Sar- leaseof taurine using in vivo microdialysis (Solis et al., 1988). kadi et al., 1984; Parsons et al., 1989). It is possiblethat only Reversal of the uptake system appears unlikely to be the when the volume of astrocytes increases2- to 3-fold is a thresh- dominant mechanism in swelling-induced amino acid efflux, old tension for SACS generatedin the membrane, as proposed since reduction of external Na+ by replacementwith K+, main- for the biconcave to sphericaltransition for erythrocytes (Dav- taining isotonic conditions, causedmuch smallereffluxes of the son, 1960), and may accountfor the lag seenin the dose-response labeled amino acids than did removal of comparable amounts curve shown in Figure 2. of NaCl, resulting in a hypotonic medium (seeTable 2). The swelling-induced transport systemsfor the amino acids Pathological consequencesof astrocytic swelling could be mechanistically related to the SACS that have recently Our studiesin vitro suggestthat swollen astrocytes may be an been discovered using patch-clamp, single-channelanalysis (re- additional sourceof releasefor L-glutamate and L-aspartate,and viewed in Sachs,1987, 1988; and Kullberg, 1987). SACS have perhapsother neurotoxins such as quinolinic acid that are syn- now been found in a wide variety of cell membranesfrom bac- thetized in astrocytes (Whetsell et al., 1988) which could con- teria to mammalian cells, including rat primary astrocyte cul- tribute to the excitotoxin-induced neuronal injury (seeintro- tures prepared identically to those used in this study (Ding et ductory remarks). In pathological statesrelease of amino acids al., 1989). They are generally activated in either cell-attached may be massive and general, and indeed the failure of volume or excised membrane patches when negative or positive pres- regulation, as indicated by the persistenceof astrocytic swelling sures are applied through the patch pipette. SACS have also under theseconditions in situ (seeKimelberg and Ransom,1986), been reported to be activated in cell-attached patcheswhen the is perhapsdue to the releasefrom intracellular compartments cells were swollen in hypotonic medium (Christensen, 1987; of much of these volume-regulatory substances.Decreases of Sachs, 1988; Falke and Misler, 1989). plasma osmolality of 10 mOsm are seenin normal human preg- It is not clear how mechanicaldeformations of the membrane nancy (Law, 1989) and even larger decreasesin hyponatremia are transduced to effect channel openings, but discussionsof are encountered in kidney dialysis (Thurston et al., 1975, 1987) this have centered on activation of the cytoskeleton, especially and other hospitalized patients (Verbalis and Drutarosky, 1988). actin microfilaments (Sachs, 1987, 1988). Guhary and Sachs However, whether the changeswe have observed with the swell- (1984) showed that exposure of cultured chick pectoral muscle ing induced by the larger decreasesin osmolarity correspond to 1590 Kimelberg et al. * Swelling-Induced Release of Amino Acids from Astrocytes those seen when astrocytes swell for other reasons, even though Forster, R. P., and L. Goldstein (1979) Amino acids and cell volume the magnitude of the swelling is comparable (Kimelberg and regulation. Yale J. Biol. Med. 52: 497-5 15. Ransom, 1986), will need further investigation. In spite of such Frangakis, M. V., and H. K. Kimelberg (1984) Dissociation ofneonatal rat brain by dispase for preparation of primary astrocyte cultures. uncertainties, treatment of excitotoxin-mediated CNS injury with Neurochem. Res. 9: 1689-1698. agents such as L644,7 11 might provide a potentially useful ad- Garay, R. P., P. A. Hannaert, C. Nazaret, and E. J. 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