The Journal of Neuroscience, May 1990, IO@): 1583-l 591 Swelling-Induced Release of Glutamate, Aspartate, and Taurine from Astrocyte Cultures 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 increased permeability 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 leads to 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 described many 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-in- duced 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 ex- pressed as a percentage of the total label accumulated and represents the release in hypoosmotic minus release in isoos- motic 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-induced e@ux of labeled amino acids percentage of this.