0022-3042/78/0601- 1363502.00/0
Journal of Neurochemisrry Vol. 30, pp. 1363-1368 Pergamon Press Ltd. 1978. Printed in Great Britain Q International Society lor Neurochemistry Ltd.
HYPEROSMOLALITY-INDUCED GABA RELEASE FROM RAT BRAIN SLICES: STUDIES OF CALCIUM DEPENDENCY A N D SOURCES OF RELEASE P. H. CHAN,Y. P. WONGand R. A. FISHMAN Department of Neurology, University of California, San Francisco, CA 94143, U.S.A. (Received 20 July 1977. Accepted 17 December 1977)
Abstract-The effects of hyperosmolal superfusion upon the release of preloaded, radio-labeled GABA has been studied, using both first cortical and first pontine brain slices. GABA release was stimulated with either hyperosmolal Na+ or sucrose superfusion in cortical slices. This stimulated release of radio-labeled GABA was partially Ca2+-dependent in cortical slices. When barium ions replaced Ca2 in hyperosmolal medium, a similar effect was seen. High concentration of magnesium in Ca2+-free hyperosmolal medium did not induce stimulation. The increased release of a-aminoisobutyric acid (AIBA), a non-metabolized amino acid induced by hyperosmolality, was not CaZ+-dependent. GABA release was also stimulated with hyperosmolal sucrose superfusion in pontine slices. The effect of pre-treatment of cortical and pontine slices with 8-alanine or L-2,Cdiaminobutyricacid (DABA) was used to study the source of exogenous GABA release induced by hyperosmolality. In cortical slices, 8-alanine blocked the hyperosmolal release of GABA and also slightly inhibited GABA uptake. DABA did not change hyperosmolal GABA release, although it inhibited GABA uptake. In pontine slices, both DABA and 8-alanine inhibited GABA uptake, but were unable to inhibit the hyperosmolal release of GABA. The data suggest that hyperosmolality causes increased release of GABA from neurons, analogous to that seen with K+-depolarization. AIBA, unlike GABA, is released from brain cells as a non-Ca2+dependent response to osmotic equilibration. The observation that pre-treatment with B-alanine inhibits the hyperosmolal release of GABA suggests that hyperosmolality alters glial cell function. +
& RAITERI,1974; SELLSTROMet al., 1976). Release of GABA from nerve terminals and from different brain regions using brain slices has been demonstrated with electrical stimulation, elevated K +-depolarization or Na+-free media (CUTLER& DUDZINSKI,1975; HAMMERSTAD et al., 1971; VARGASet al., 1976). GABA release induced by K+-depolarization is Ca2+-dependent, whereas electrical stimulation of GABA release is not Ca2+-dependent. Early work suggested that DABA and 8-alanine might serve as specific inhibitors of neuronal and glial GABA uptake, respectively & JOHNSTON,1971; SCHON & KELLY,1974; (IVERSEN IVERSEN& KELLY, 1975). GABA release from rat brain synaptosomes induced by elevated K f in the presence of Ca2+, ionophore A23187, ouabain, K + free medium and black widow spider toxin were unaffected with DABA pretreated synaptosomes (LEVI et al., 1976). However, superfusion with DABA nearly abolished K+-stimulated release of C3H]GABA, whereas p-alanine had little effect, leading to the suggestion that the neuronal pool is the source of releasable GABA in rat cortical slices (HAMMERSTAD & LYTLE, 1976). However, further studies failed to This work was presented in part at the March 1977 et al., 1977), support this suggestion (HAMMERSTAD Denver meeting of the American Society of Neurochembecause DABA failed to inhibit the release of istry, supported by NIH Grant NSO 7336. Abbreuiations used: AIBA, a-aminoisobutyric acid; [3H]GABA from K+-depolarized synaptosomes, or AOAA, amino-oxyacetic acid; DABA, ~-2,4-diaminobu- from the protovertrinedepolarized brain slices and tyric acid; HEPES, N-2-hydroxyethylpiperazine-2-ethane synaptosomes. Therefore, DABA is considered as a non-specific inhibitor of GABA transport, and sulfonic acid; mosmol, milliosmoles.
PREVIOUS studies have shown hyperosmolality induces the release of putative neurotransmitter amino acids from cortical slices, using a superfusion method (FISHMANet al., 1977; CHAN & FISHMAN,1977). Hyperosmolality stimulated the release of glycine and taurine, slowly metabolized putative neurotransmitter amino acids as well as a-aminoisobutyric acid (AIBA), a non-metabolized amino acid. However, we failed to demonstrate a significant change in the release of glutamate, aspartate and GABA, more rapidly metabolized neurotransmitter amino acids. Since glycine taurine and GABA are classified as putative inhibitory neurotransmitter amino acids, the release of GABA induced by hyperosmolality required further examination. We have modified our previous experimental techniques to obtain a more sensitive superfusion procedure, similar to that described elsewhere & LYTLE,1976; LEVIet a/., 1976). (HAMMERSTAD A high-affinity uptake process for GABA has been described in rat cerebral cortex and in synaptosomes (IVERSEN & NEAL,1968; BLOOM& IVERSEN, 1971; LEVI
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8-alanine is considered to serve chiefly as a more specific inhibitor of GABA uptake by glia. The present report is an attempt to elucidate the mode of GABA release from first cortical slices induced by hyperosmolality as well as its Ca2+-dependency, and the effects of barium and magnesium. Pretreatment of cortical or pontine slices with DABA or 8-alanine, followed by superfusion with hyperosmolal media has been used to elucidate tile site of GABA release in comparison to the prayiously described effects of K+-depolarization.
1977). The to autoradiographic analysis (CHAN& FISHMAN, per cent GABA released was calculated as described previously.
% release ['4C]GABA
=
Total d.p.m. released in 1 0 superfusate x "/, non-metabolized Total d.p.m. in initial uptake of slice' [d.p.m. (slice + superfusate)]
GABA uptake was expressed as tissue/medium ratio after 30 rnin preincubation in control media. First cortical and first pontine slices were stained for myelinated structures and for cytology. No myelinated elements were seen in first cortical slices. An estimated 90-95% of the first MATERIALS AND METHODS pontine slices appeared to consist of myelinated fibers. A [U-'4C]GABA, 49.4 mCi/mmol; y-[2,3-'H(N)]GABA, few pontine nuclei were unavoidably included in the latter 35.07 Ci/mmol and a-[1-'4C]aminoisobutyric acid, 45.25 preparation. The water content of pontine slices was mCi/mmol were obtained from New England Nuclear 77.51% & 0.2 (22) compared to 82.97% & 0.16 (12) in cortiCorporation (Boston, MA); 8-alanine, ~-2,4-diamino- cal slices. butyric acid (DABA), N-2-hydroxyethylpiperazine-N-2RESULTS ethanesulfonic acid (HEPES) were obtained from Sigma Company (St. Louis, MO). Control medium had the folEffects of hyperosmolality on Ci4C]GABA release from lowing composition: (in mM) NaCl, 140; KCI. 3.6; CaCI,, 0.75; KH2P04, 1.4; MgSO,, 0.7; glucose, 10; HEPES, 15 rat brain cortical slices at pH 7.4; and the osmolality was 305 mosmol/l. HyperCortical slices pre-loaded with [I4C]GABA as deosmolal media were obtained by increasing NaCl to scribed were superfused with control medium for 290 mM, or by the addition of 300 mosmol/l sucrose to con- 40 min, followed by the superfusion of control trol medium. In the Ca2+-freebuffer, CaZ+ was omitted medium or hyperosmolal medium (Na+, 290 mM) for from the medium, and 1 mM-EGTA was added. In some another 50 min. The release of GABA was stimulated of the incubation and superfusion media, amino-oxyacetic by hyperosmolal media as shown in Fig. 1. Autoradiacid (AOAA) was added at a concentration of ~ O - , M to prevent GABA metabolism (SCHON& KELLY, 1974; ography using TLC of the pooled superfusate indicated that GABA comprised about 90% of the KENNEDY& VOADEN, 1974). released radioactivity. In some experiments, aminoDetailed descriptions of the methods used including the preparation, incubation and superfusion of rat brain single oxyacetic acid (AOAA) at a final concentration of first cortical slices were reported earlier (FISHMAN er al., M was added to both incubation and superfusion 1977; CHAN& FISHMAN, 1977). Male, adult Spraguemedia to prevent GABA metabolism. Addition of Dawley rats (Simonsen, Gilroy, CA) weighing about lOOg AOAA in this system did not alter GABA metabolism were used. The superfusion procedures have been slightly or release. Thus, AOAA was omitted in all subsequent modified in the present report. Single, first cortical slices experiments. The stimulating effect of hyperosmalality were cut from the surface of each hemisphere, with pia on GABA release was increased slightly when equiintact; weighing 40-50 mg, and 0.35 mm thick. Single, first osmolar sucrose (300mosM) replaced Na+, as indipontine slices were cut from the ventral surface of pons, cated in Fig. 1. 290 mM-sodium chloride is equivalent with pia intact; weighing 10-20 mg, and 0.35 mm thick. in osmolality to 140 mM-sodium chloride plus Individual slices were pre-loaded with ['4C]GABA in a 300 mM-SUCrOse. The 8 rnin detay before the GABA concentration of 16.5 nmole in 5 ml (3.3 phi) medium at 37°C for 50 min. (The final volume of the medium, 5 ml. peak appeared represents the dead space of the tubing was not corrected for the volume of the tissue.) Each 2-min which carried the medium to the superfusion fraction of the superfusate was collected in a vol of 0.8 ml. chamber. 0.1 ml of each fraction was counted with a Packard scintillation counter. Concentrations of Ca2+ ranging from 0.15 Ca'+-Dependency of [14C]GABA release from cortical to 5 mM, as well as Mg2+ IOmM, or Ba2' I mM, were slices stimulated by hyperosmolality used to replace 0.75 mM-Ca2+ in the hyperosmolal media Superfusion of [I4C]GABA pre-loaded cortical for the studies of ionic dependency. In the GABA uptake, slices with Na' or sucrose hyperosmolal medium, in inhibition and release studies, cortical slices or pontine slices were pre-loaded with 0.2 nmol/5 ml (0.4 p ~ ) the absence of calcium plus 1 mM-EGTA, resulted in a significant reduction in the GABA released, as y-[2.3-3H(N)]-GABA plus 16.3nmol/5 ml (3.26 p ~ cold ) GABA in the presence of p-alanine (5 mM) or DABA shown in Fig. 2A. However, the presence of CAZ+ (1 mM). Slices were superfused with control medium for was not absolutely required for the increase in GABA 10 rnin before changing to experimental solutions and release, since Ca2+-free media did not completely superfused for 50 min. The radioactivity released at 2 min abolish the GABA peak. As seen in Fig. 2.4, there intervals was graphed. After b8 min of superfusion was a similar reduction in GABA release using either (0.8 m1/2 rnin) with the experimental medium, there was a Na+ or sucrose hyperosmolal medium in the absence sharp increase in the release of radioactivity, lasting of Ca". The effect of graded changes in calcium conI@ 12 min and then returning to the base level of release. centration upon GABA release was also studied, The fractions that were released were pooled and subjected
Hyperosmolality-induced GABA release
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FIG.1. Stimulated release of [I4C]GABA from brain cortical slices induced by hyperosmolality. --t control medium Naf 140 mM (280 mosM); -0- Na+-hyperosmolal medium, 290 mM-Na+ (580 mom); -Asucrose-hyperosmolal medium, 140 mM-Na+ (280 mow) + 300 mM-sucrose (300 mom). Each point represents the mean of 3-5 experiments. Arrow indicates the time that the hyperosmolal medium was begun. In some of the experiments, amino-oxyacetic acid at a final concentration of 1 0 - 4 ~was added to incubation media as well as control and hyperosmolal superfusion media.
using Ca2+ concentrations of 0.15, 0.5, 3 and 5mM. The stimulated release of GABA was unchanged in 5mM-Ca2+ compared to the response obtained with the usual hyperosmolal medium containing 0.75 mMCaz.+,as shown in Fig. 2A. Similarly, other concentrations (0.15, 0.5, 3mM) of Ca2+ did not affect the release of GABA. A parallel experiment performed with isotonic control media containing different concentrations of Ca2+ also did not affect the release of GABA. The lack of a graded dose-response relationship between Ca2 concentration in hyperosmola1 medium and the amount of GABA released is similar to the pattern seen with the elevated K + depolarization system (VARGASet al., 1977). The dependency upon the presence of CaL+of the +
GABA release stimulated by hyperosmolality was also demonstrated when the brain slices were first incubated in Ca2+-free media, followed by superfusion with hyperosmolal media either with or without the presence of Ca2+.Figure 2B shows that hyperosmolality in the presence of Ca2+ induced a greater stimulating effect on GABA release from cortical slices when the slices were preincubated with Ca2'-free media.
Effects of barium and magnesium on the GABA release from cortical slices induced by hyperosmolality Figure 3 shows that Ba2+ ions (1 mM), when used to replace Ca2+ in the hyperosmolal medium, also could induce increased GABA release from cortical
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FIG.2. Ionic effects on the stimulated release of [I4C]-GABA from cortical slices induced by hyperosmolality. Each point is the mean of three experimental values. Figure 2A shows the Ca2+-dependency of GABA release obtained with 0 and 5 mM-Ca2+ concentration in hyperosmolal superfusion media. -(+ Na+ 290 mM (Caz+-free + 1 mM-EGTA); -ANa+ 140 mM + sucrose 300 mM (Ca2+free + 1 mM-EGTA); --t Na+ 2 9 0 m ~+ 5rnM-Ca". Figure 2B: The stimulated release of ["CI-GABA from cortical slices pre-incubated in Cat +-free medium (plus 1 mM-EGTA) induced by hyperosmolality. -ANa+ 140 mM-(Ca2+-free, + 1 mM-EGTA); -+ Na+ 290 mM-(CaZ+-free+ 1 mM-EGTA); --t Na+ 290 mM.
P. H. CHAN,Y. P. WONGand R. A. FISHMAN
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FIG. 3. Effects of barium and magnesium ions on the stimulated release of ["CI-GABA from cortical slices induced by hyperosmolal superfusion. Na+ 290 mM, Ba2+ 1 mM; -tNa+ 1 4 0 m ~ sucrose , 3 0 0 m ~ Ba2+ , l m ~ -A; Na+ 2 9 0 m ~ ,Mg*+ 10mM; -0Na+ 290 mM, CaZ 0.75 mM.
-*
+
slices. Hypertonic sucrose medium containing Ba2 had a slightly greater effect than hypertonic Na+, results that were similar to that obtained with hyperosmolal sucrose medium in the presence of CaZ+. However, both the Na+ and sucrose hyperosmolal media in the presence of BaZ+induced a second small peak of GABA release whereas the hyperosmolal media with Ca2+only induced a single peak, suggesting there might be more than single compartment involved in Ba2+-induced GABA release. Figure 3 also shows that high concentrations of Mgz+ ( l o r n ) in the CaZ+-free hyperosmolal medium failed to stimulate GABA release in cortical slices. Thus, the data suggest that Mgz+ cannot be substituted for Ca2+, or the chemically related divalent cation Ba2+ for the stimulation of GABA release from cortical slices induced by hyperosmolality. +
The absence of Ca2+ dependency of [14C]AIBA release from cortical slices stimulated by hyperosmolality The pattern of release of a non-metabolized amino acid, AIBA, from the cortical slice induced by Ca2 containing or CaZ -free hyperosmolal superfusion is shown in Fig. 4. Unlike the partial dependency upon the presence of calcium for the stimulation of GABA release, the release of AIBA from cortical slices irr duced by hypertonic medium was similar, regardless of the presence or absence of Ca2+ in the medium. Two distinct peaks were observed in both AIBA superfusates, suggesting that AIBA is released from two distinct pools within the cortical slice. +-
+
Efjcts of hyperosmolality on the release of GABA from cortical and pontine slices pre-treated with DABA or p-alanine First cortical slices which are largely grey matter and first pontine slices which are largely white matter in composition, have been used to study whether
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FIG.4. Ca2+-dependencyof [I4C]GABA release from cortical slices induced by hyperosmohlity. -0- Na+ 1 4 0 m ~-A; Na+ 2 9 0 m ~ -A; Na' 290mM-(Ca2+free + 1 mM-EGTA).Arrow indicates the time the hyperosmolal medium was begun. Each point is the mean of three experimental values.
GABA release is regionally or structurally specific. Brain tissues were pre-incubated with DABA or p-alanine in the presence of C3H]GABA for 30min, followed by superfusion with hyperosmolal media. The inhibition of uptake and release of C3H]GABA by DABA or p-alanine using first cortical and first pontine slices is summarized in Table 1. The release of C3H]GABA from both cortical and pontine slices was stimulated significantly with hypertonic sucrose superfusion. The stimulated increase was similar in both cortical and pontine slices, although the uptake of GABA was much less in pontine slices. Pre-incubation with 5 mM-p-alanine or 1 mM-DABA did not alter the release of GABA significantly in cortical slices when control buffer was used for superfusion. Although 1 mM-DABA inhibited GABA uptake in cortical slices, the release of GABA was not affected by hyperosmolal sucrose superfusion. However, palanine pre-treatment of cortical slices blocked the stimulated release of GABA induced by hyperosmolal medium. In pontine slices, both 8-alanine and DABA inhibited the GABA uptake, but there were no significant changes in GABA release induced by hyperosmolal sucrose superfusion. Thus, the two amino acid inhibitors reduced the uptake (T/M ratio) of C3H]GABA in both grey and white matter. However, the stimulation in release of C3H]GABA induced by hyperosmolal superfusion was not affected by either inhibitor in pontine white matter, but in the cortical grey matter, only 8-alanine blocked the stimulated release induced by hyperosmolality. DISCUSSION
The present studies have demonstrated that increased release of GABA from brain slices occurs as
Hyperosmolality-induced GABA release TABLE1. EFFECTSOF
HYPEROSMOLALITY ON THE RELEASE OF C3H]GABA FROM CORTICAL OR PONTINE SLICES PRE-TREATED WITH DABA OR
p-ALANINE
Preincubation
Superfusion
Tissue
medium
T/M Ratio
medium
23.42 f 0.77 (12) 23.42 f 0.77(12)
+sucrose
Cortical slices
Control Control Control +B-alanine + DABA Control +B-alanine + DABA Control Control Control + /3-alanine + DABA Control + p-alanine + DABA
5.26 f 0.34 (10) 5.26 f 0.34 (1 0)
Pontine slices
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23.42 f 0.77(12) 19.49 f 1.3 (7)t 7.92 f 0.26 (7)*
5.26 f 0.34(10) 3.66 k 0.20 (6)f 2.45 f 0.10(6)*
Control Control Control Control +sucrose +sucrose +sucrose
% Release 11.75 f 1.33 (6) 16.27 f 0.57 (6)t 11.75 f 1.33 (6) 11.09 k 1.3 (2) 13.49 If: 0.05 (2) 16.27 0.57 (6) 12.38 f 1.41 (5)t 16.30 f 0.48 (5)
Control +sucrose Control Control Control
17.06 f 0.38 (5) 21.34 i-0.89 (5)
+sucrose
21.34 f 0.89 (5) 19.38 k 1.57(4) 19.25 f 1.76(4)
+sucrose +sucrose
17.06 f 0.38 (5) 16.37 0.79 (2) 16.10 f 0.23 (2)
+
The concentrations used in this study were 1 mM and 5 mM respectively for DABA and p-alanine. Control medium, 140mM-Na'; sucrose medium, 1 4 0 m ~ - N a + 300rnM-sucrose. Mean values f S.E.M. are given. (n) = number of slices. * P < 0.001. t P < 0.02. 1P < 0.05. AOAA was omitted in both preincubation and superfusion media.
+
a rapid response to hyperosmolality. This stimulated Whether the release of other putative neurotransrelease was dependent in part upon the presence of mitter amino acids such as taurine, glycine, glutamate Ca2+ in the superfusion medium, because in its and aspartate induced by hyperosmolality is Ca2+absence stimulated GABA release was reduced more dependent requires further elucidation. Despite the than 50%. It has been reported recently that the lack of Ca'+-dependency, a second AIBA peak was stimulated release of neurotransmitter quanta in frog detected in its hyperosmolal release pattern. This neuromuscular junctions induced by hyperosmolality phenomenon was also found with glutamine and tauris dependent upon intracellular Ca2 concentration ine release induced by hyperosmolality (Chan & Fish(SHIMONI et al., 1977). The calcium dependency was man, unpublished data). The fact that two peaks were graphically illustrated by the even greater response seen with AIBA, a non-metabolized amino acid, in GABA release obtained when calcium was omitted favors the existence of two or more compartments. The present observations are analogous to other from the pre-incubation medium, and then replaced with the hyperosmolal medium. However, there was studies of the release of pre-loaded exogenous radionot a graded dose-response relationship between labeled GABA from various brain tissues, spinal cord Ca2+ concentration and the magnitude of GABA slices and synaptosomes upon electrical stimulation & release. GABA release was stimulated also by hyper- or depolarization with potassium (DAVIDOFF & VOADEN,1974; CUTLER& osmolality when Ba2+ was added to the superfusion ADAIR,1976; KENNEDY 1975; HAMMERSTAD et al., 1971; VARGAS medium to replace Ca2+. It has been shown that DUDZINSKI, Ba2+, when used to replace Ca2+ in the 'perfusion et al., 1977). VARGAS and colleagues (1977) have sugmedium, had a similar stimulating effect on neuro- gested that K+-depolarization results in an exit of transmitter release in the adrenal medulla (DOUGLASGABA from a cytoplasmic compartment, rather than & RUBIN, 1964). However, there was no stimulation from nerve endings. It is not clear whether the GABA of GABA release with a high concentration of Mg2+ release with K+-depolarization is derived from (10 mM) indicating that specific divalent ions are neurons or glia. required for stimulated GABA release from cortical Early studies suggested that L-2,Cdiaminqbutyric acid (DABA) and /3-alanine are specific inhibitors of slices. We have previously demonstrated that the release GABA uptake by neuronal and glial 'structures of [14C]AIBA was stimulated by hyperosmolal super- (SCHON& KELLY,1974; IVERSEN19 KELLY,1975; fusion (CHAN& FISHMAN, 1977). The present studies LODGEet al., 1976). The release of C3H]GABA with have confirmed this finding by using a slightly modi- K+-depolarization is inhibited by the superfusion of fied superfusion technique. The release of AIBA in- DABA (HAMMERSTAD & LYTLE,1976). It has been duced by hypertonicity was not Ca2+-dependent. demonstrated that GABA release obtained from +
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P. H. CHAN,Y. P. WONGand R. A. FISHMAN
DABA pre-treated synaptosomes was affected by both unlabeled GABA and DABA, but was unaffected by elevated K t . This suggests that K + induces GABA release in synaptosomes by a non-DABA carriermediated system (LEVIet al., 1976). However, further confirmation is required because of uncertainty regarding the specificity of DABA inhibition of neuronal GABA uptake and release induced by K + (HAMMERSTAD et al., 1977; BOWERY et al., 1976). The results we have obtained with hyperosmolality-induced GABA release are similar to that demonstrated with the elevated K + system. First, the percentage of release of ['HIGABA obtained from DABA pretreated cortical slices induced by hyperosmolality was similar, suggesting the existence of a transport system for GABA not inhibited by DABA. Secondly, the release of C3H]GABA from B-alanine pre-treated cortical slices induced by hyperosmolality was largely blocked. This suggests the glial elements in cortical slices might be involved in GABA release processes. It has been suggested that glial structures are the main compartments for the re-uptake of neurotransmitters located in synaptic clefts, in order to terminate the action of released neurotransmitters (HE" & HAMBERGER, 1971; IVERSEN& NEAL,1968). The reuptake of GABA by synaptosomes might serve as a direct source for the GABA released with subsequent nerve volleys (RYAN& ROSKOSKI,1975). The mechanism of re-uptake and release of GABA by glial elements related to hyperosmolal modulation requires further elucidation. Our data further demonstrated that GABA uptake was inhibited significantly with both DABA and j-alanine pre-treated pontine slices, but there were no significant differences in GABA release. These data may indicate the presence of cellular compartments in pontine slices that respond with a non-specific increase in GABA release as the tissue adapts to hyperosmolality. The use of glial cells and neuroblastoma cells in culture might elucidate the differences in the responses of glial and neuronal elements to hyperosmolality.
REFERENCES BLOOMF. E. & IVERSEN L. L. (1971) Nature, Lond. 229, 628-630. BOWERYN. G., JONES G. P. & NEALM. J. (1976) Nature, Lond. 264, 281-284. CHANP. H. & FISHMANR. A. (1977) J . Neurochem. 29, 179-18 1. CUTLERR . W. P. & DUDZINSKI D. S. (1975) Brain Res. 88, 41S423. DAVIDOFF R. A. & ADAIR R. (1976) Brain Res. 118, 403-415. DOUGLAS W. W. & RUBINR. P. (1964) Nature, Lond. 203, 305-307. FISHMAN R. A., REINERM. & CHANP. H. (1977) J . Neurochern. 28, 1061-1067. HAMMERSTAD J. P., CAWTHON M. & LYTLEC. R. (1977) Trans. Am. SOC.Neurochem. 8, 250. HAMMERSTAD J. P., MURRAYJ. E. & CUTLERR. W. P. (1971) Brain Res. 35, 357-367. HAMMERSTAD J. P. & LYTLEC. R. (1976) J . Neurochem. 21, 399-403. HENNF. A. & HAMBERGER A. (1971) Proc. natn. Acad. Sci. V.S.A. 68, 2686-2690. IVERSEN L. L. & JOHNSTON G. A. R. (1971) J . Neurochern. 18, 1939-1950. IVERSENL. L. & KELLYJ. S. (1975) Biochem. Pharmac. 24, 933-9323, IVERSENL. L. & NEALM. J. (1968) J . Neurochem. 15, 1141-1 149. KENNEDY A. J. & VOADENM. J. (1974) J . Neurochem. 22, 63-7 1. LEVIG., ROBERTS P. J. & RAITERII. M. (1976) Neurochem. Res. 1, 409416. LEVIG. & RAITERI M. (1974) Nature, Lond. 250, 735-737. LODGED., JOHNSON G. A. R. & STEPHENSON A. L. (1976) J . Neurochem. 27, 1569-1570. RYAN L. D. & ROSKOSKIR. Jr. (1975) Nature, Lond. 258, 254-256. SCHONF. & KELLYJ. S . (1974) Brain Res. 66, 289-300. SELLSTROM A., VENEMA R. & HENNF. (1976) Nature, Lond. 264, 652-653. SHIMONI Y., ALNAESE. & RAHAMINOFFR. (1977) Nature, Lond. 267, 17C-172. VARGASO., DELORENZO D. E. D., SALDATEM. C. & ORREGO F. (1977) J . Neurochem. 28, 165-170. VARGASO., MIRANDA R. & ORREGO F. (1976) Neuroscience 1, 137-145.
Journal of Neurochemisrry Vol. 30, pp. 1363-1368 Pergamon Press Ltd. 1978. Printed in Great Britain Q International Society lor Neurochemistry Ltd.
HYPEROSMOLALITY-INDUCED GABA RELEASE FROM RAT BRAIN SLICES: STUDIES OF CALCIUM DEPENDENCY A N D SOURCES OF RELEASE P. H. CHAN,Y. P. WONGand R. A. FISHMAN Department of Neurology, University of California, San Francisco, CA 94143, U.S.A. (Received 20 July 1977. Accepted 17 December 1977)
Abstract-The effects of hyperosmolal superfusion upon the release of preloaded, radio-labeled GABA has been studied, using both first cortical and first pontine brain slices. GABA release was stimulated with either hyperosmolal Na+ or sucrose superfusion in cortical slices. This stimulated release of radio-labeled GABA was partially Ca2+-dependent in cortical slices. When barium ions replaced Ca2 in hyperosmolal medium, a similar effect was seen. High concentration of magnesium in Ca2+-free hyperosmolal medium did not induce stimulation. The increased release of a-aminoisobutyric acid (AIBA), a non-metabolized amino acid induced by hyperosmolality, was not CaZ+-dependent. GABA release was also stimulated with hyperosmolal sucrose superfusion in pontine slices. The effect of pre-treatment of cortical and pontine slices with 8-alanine or L-2,Cdiaminobutyricacid (DABA) was used to study the source of exogenous GABA release induced by hyperosmolality. In cortical slices, 8-alanine blocked the hyperosmolal release of GABA and also slightly inhibited GABA uptake. DABA did not change hyperosmolal GABA release, although it inhibited GABA uptake. In pontine slices, both DABA and 8-alanine inhibited GABA uptake, but were unable to inhibit the hyperosmolal release of GABA. The data suggest that hyperosmolality causes increased release of GABA from neurons, analogous to that seen with K+-depolarization. AIBA, unlike GABA, is released from brain cells as a non-Ca2+dependent response to osmotic equilibration. The observation that pre-treatment with B-alanine inhibits the hyperosmolal release of GABA suggests that hyperosmolality alters glial cell function. +
& RAITERI,1974; SELLSTROMet al., 1976). Release of GABA from nerve terminals and from different brain regions using brain slices has been demonstrated with electrical stimulation, elevated K +-depolarization or Na+-free media (CUTLER& DUDZINSKI,1975; HAMMERSTAD et al., 1971; VARGASet al., 1976). GABA release induced by K+-depolarization is Ca2+-dependent, whereas electrical stimulation of GABA release is not Ca2+-dependent. Early work suggested that DABA and 8-alanine might serve as specific inhibitors of neuronal and glial GABA uptake, respectively & JOHNSTON,1971; SCHON & KELLY,1974; (IVERSEN IVERSEN& KELLY, 1975). GABA release from rat brain synaptosomes induced by elevated K f in the presence of Ca2+, ionophore A23187, ouabain, K + free medium and black widow spider toxin were unaffected with DABA pretreated synaptosomes (LEVI et al., 1976). However, superfusion with DABA nearly abolished K+-stimulated release of C3H]GABA, whereas p-alanine had little effect, leading to the suggestion that the neuronal pool is the source of releasable GABA in rat cortical slices (HAMMERSTAD & LYTLE, 1976). However, further studies failed to This work was presented in part at the March 1977 et al., 1977), support this suggestion (HAMMERSTAD Denver meeting of the American Society of Neurochembecause DABA failed to inhibit the release of istry, supported by NIH Grant NSO 7336. Abbreuiations used: AIBA, a-aminoisobutyric acid; [3H]GABA from K+-depolarized synaptosomes, or AOAA, amino-oxyacetic acid; DABA, ~-2,4-diaminobu- from the protovertrinedepolarized brain slices and tyric acid; HEPES, N-2-hydroxyethylpiperazine-2-ethane synaptosomes. Therefore, DABA is considered as a non-specific inhibitor of GABA transport, and sulfonic acid; mosmol, milliosmoles.
PREVIOUS studies have shown hyperosmolality induces the release of putative neurotransmitter amino acids from cortical slices, using a superfusion method (FISHMANet al., 1977; CHAN & FISHMAN,1977). Hyperosmolality stimulated the release of glycine and taurine, slowly metabolized putative neurotransmitter amino acids as well as a-aminoisobutyric acid (AIBA), a non-metabolized amino acid. However, we failed to demonstrate a significant change in the release of glutamate, aspartate and GABA, more rapidly metabolized neurotransmitter amino acids. Since glycine taurine and GABA are classified as putative inhibitory neurotransmitter amino acids, the release of GABA induced by hyperosmolality required further examination. We have modified our previous experimental techniques to obtain a more sensitive superfusion procedure, similar to that described elsewhere & LYTLE,1976; LEVIet a/., 1976). (HAMMERSTAD A high-affinity uptake process for GABA has been described in rat cerebral cortex and in synaptosomes (IVERSEN & NEAL,1968; BLOOM& IVERSEN, 1971; LEVI
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8-alanine is considered to serve chiefly as a more specific inhibitor of GABA uptake by glia. The present report is an attempt to elucidate the mode of GABA release from first cortical slices induced by hyperosmolality as well as its Ca2+-dependency, and the effects of barium and magnesium. Pretreatment of cortical or pontine slices with DABA or 8-alanine, followed by superfusion with hyperosmolal media has been used to elucidate tile site of GABA release in comparison to the prayiously described effects of K+-depolarization.
1977). The to autoradiographic analysis (CHAN& FISHMAN, per cent GABA released was calculated as described previously.
% release ['4C]GABA
=
Total d.p.m. released in 1 0 superfusate x "/, non-metabolized Total d.p.m. in initial uptake of slice' [d.p.m. (slice + superfusate)]
GABA uptake was expressed as tissue/medium ratio after 30 rnin preincubation in control media. First cortical and first pontine slices were stained for myelinated structures and for cytology. No myelinated elements were seen in first cortical slices. An estimated 90-95% of the first MATERIALS AND METHODS pontine slices appeared to consist of myelinated fibers. A [U-'4C]GABA, 49.4 mCi/mmol; y-[2,3-'H(N)]GABA, few pontine nuclei were unavoidably included in the latter 35.07 Ci/mmol and a-[1-'4C]aminoisobutyric acid, 45.25 preparation. The water content of pontine slices was mCi/mmol were obtained from New England Nuclear 77.51% & 0.2 (22) compared to 82.97% & 0.16 (12) in cortiCorporation (Boston, MA); 8-alanine, ~-2,4-diamino- cal slices. butyric acid (DABA), N-2-hydroxyethylpiperazine-N-2RESULTS ethanesulfonic acid (HEPES) were obtained from Sigma Company (St. Louis, MO). Control medium had the folEffects of hyperosmolality on Ci4C]GABA release from lowing composition: (in mM) NaCl, 140; KCI. 3.6; CaCI,, 0.75; KH2P04, 1.4; MgSO,, 0.7; glucose, 10; HEPES, 15 rat brain cortical slices at pH 7.4; and the osmolality was 305 mosmol/l. HyperCortical slices pre-loaded with [I4C]GABA as deosmolal media were obtained by increasing NaCl to scribed were superfused with control medium for 290 mM, or by the addition of 300 mosmol/l sucrose to con- 40 min, followed by the superfusion of control trol medium. In the Ca2+-freebuffer, CaZ+ was omitted medium or hyperosmolal medium (Na+, 290 mM) for from the medium, and 1 mM-EGTA was added. In some another 50 min. The release of GABA was stimulated of the incubation and superfusion media, amino-oxyacetic by hyperosmolal media as shown in Fig. 1. Autoradiacid (AOAA) was added at a concentration of ~ O - , M to prevent GABA metabolism (SCHON& KELLY, 1974; ography using TLC of the pooled superfusate indicated that GABA comprised about 90% of the KENNEDY& VOADEN, 1974). released radioactivity. In some experiments, aminoDetailed descriptions of the methods used including the preparation, incubation and superfusion of rat brain single oxyacetic acid (AOAA) at a final concentration of first cortical slices were reported earlier (FISHMAN er al., M was added to both incubation and superfusion 1977; CHAN& FISHMAN, 1977). Male, adult Spraguemedia to prevent GABA metabolism. Addition of Dawley rats (Simonsen, Gilroy, CA) weighing about lOOg AOAA in this system did not alter GABA metabolism were used. The superfusion procedures have been slightly or release. Thus, AOAA was omitted in all subsequent modified in the present report. Single, first cortical slices experiments. The stimulating effect of hyperosmalality were cut from the surface of each hemisphere, with pia on GABA release was increased slightly when equiintact; weighing 40-50 mg, and 0.35 mm thick. Single, first osmolar sucrose (300mosM) replaced Na+, as indipontine slices were cut from the ventral surface of pons, cated in Fig. 1. 290 mM-sodium chloride is equivalent with pia intact; weighing 10-20 mg, and 0.35 mm thick. in osmolality to 140 mM-sodium chloride plus Individual slices were pre-loaded with ['4C]GABA in a 300 mM-SUCrOse. The 8 rnin detay before the GABA concentration of 16.5 nmole in 5 ml (3.3 phi) medium at 37°C for 50 min. (The final volume of the medium, 5 ml. peak appeared represents the dead space of the tubing was not corrected for the volume of the tissue.) Each 2-min which carried the medium to the superfusion fraction of the superfusate was collected in a vol of 0.8 ml. chamber. 0.1 ml of each fraction was counted with a Packard scintillation counter. Concentrations of Ca2+ ranging from 0.15 Ca'+-Dependency of [14C]GABA release from cortical to 5 mM, as well as Mg2+ IOmM, or Ba2' I mM, were slices stimulated by hyperosmolality used to replace 0.75 mM-Ca2+ in the hyperosmolal media Superfusion of [I4C]GABA pre-loaded cortical for the studies of ionic dependency. In the GABA uptake, slices with Na' or sucrose hyperosmolal medium, in inhibition and release studies, cortical slices or pontine slices were pre-loaded with 0.2 nmol/5 ml (0.4 p ~ ) the absence of calcium plus 1 mM-EGTA, resulted in a significant reduction in the GABA released, as y-[2.3-3H(N)]-GABA plus 16.3nmol/5 ml (3.26 p ~ cold ) GABA in the presence of p-alanine (5 mM) or DABA shown in Fig. 2A. However, the presence of CAZ+ (1 mM). Slices were superfused with control medium for was not absolutely required for the increase in GABA 10 rnin before changing to experimental solutions and release, since Ca2+-free media did not completely superfused for 50 min. The radioactivity released at 2 min abolish the GABA peak. As seen in Fig. 2.4, there intervals was graphed. After b8 min of superfusion was a similar reduction in GABA release using either (0.8 m1/2 rnin) with the experimental medium, there was a Na+ or sucrose hyperosmolal medium in the absence sharp increase in the release of radioactivity, lasting of Ca". The effect of graded changes in calcium conI@ 12 min and then returning to the base level of release. centration upon GABA release was also studied, The fractions that were released were pooled and subjected
Hyperosmolality-induced GABA release
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FIG.1. Stimulated release of [I4C]GABA from brain cortical slices induced by hyperosmolality. --t control medium Naf 140 mM (280 mosM); -0- Na+-hyperosmolal medium, 290 mM-Na+ (580 mom); -Asucrose-hyperosmolal medium, 140 mM-Na+ (280 mow) + 300 mM-sucrose (300 mom). Each point represents the mean of 3-5 experiments. Arrow indicates the time that the hyperosmolal medium was begun. In some of the experiments, amino-oxyacetic acid at a final concentration of 1 0 - 4 ~was added to incubation media as well as control and hyperosmolal superfusion media.
using Ca2+ concentrations of 0.15, 0.5, 3 and 5mM. The stimulated release of GABA was unchanged in 5mM-Ca2+ compared to the response obtained with the usual hyperosmolal medium containing 0.75 mMCaz.+,as shown in Fig. 2A. Similarly, other concentrations (0.15, 0.5, 3mM) of Ca2+ did not affect the release of GABA. A parallel experiment performed with isotonic control media containing different concentrations of Ca2+ also did not affect the release of GABA. The lack of a graded dose-response relationship between Ca2 concentration in hyperosmola1 medium and the amount of GABA released is similar to the pattern seen with the elevated K + depolarization system (VARGASet al., 1977). The dependency upon the presence of CaL+of the +
GABA release stimulated by hyperosmolality was also demonstrated when the brain slices were first incubated in Ca2+-free media, followed by superfusion with hyperosmolal media either with or without the presence of Ca2+.Figure 2B shows that hyperosmolality in the presence of Ca2+ induced a greater stimulating effect on GABA release from cortical slices when the slices were preincubated with Ca2'-free media.
Effects of barium and magnesium on the GABA release from cortical slices induced by hyperosmolality Figure 3 shows that Ba2+ ions (1 mM), when used to replace Ca2+ in the hyperosmolal medium, also could induce increased GABA release from cortical
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FIG.2. Ionic effects on the stimulated release of [I4C]-GABA from cortical slices induced by hyperosmolality. Each point is the mean of three experimental values. Figure 2A shows the Ca2+-dependency of GABA release obtained with 0 and 5 mM-Ca2+ concentration in hyperosmolal superfusion media. -(+ Na+ 290 mM (Caz+-free + 1 mM-EGTA); -ANa+ 140 mM + sucrose 300 mM (Ca2+free + 1 mM-EGTA); --t Na+ 2 9 0 m ~+ 5rnM-Ca". Figure 2B: The stimulated release of ["CI-GABA from cortical slices pre-incubated in Cat +-free medium (plus 1 mM-EGTA) induced by hyperosmolality. -ANa+ 140 mM-(Ca2+-free, + 1 mM-EGTA); -+ Na+ 290 mM-(CaZ+-free+ 1 mM-EGTA); --t Na+ 290 mM.
P. H. CHAN,Y. P. WONGand R. A. FISHMAN
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FIG. 3. Effects of barium and magnesium ions on the stimulated release of ["CI-GABA from cortical slices induced by hyperosmolal superfusion. Na+ 290 mM, Ba2+ 1 mM; -tNa+ 1 4 0 m ~ sucrose , 3 0 0 m ~ Ba2+ , l m ~ -A; Na+ 2 9 0 m ~ ,Mg*+ 10mM; -0Na+ 290 mM, CaZ 0.75 mM.
-*
+
slices. Hypertonic sucrose medium containing Ba2 had a slightly greater effect than hypertonic Na+, results that were similar to that obtained with hyperosmolal sucrose medium in the presence of CaZ+. However, both the Na+ and sucrose hyperosmolal media in the presence of BaZ+induced a second small peak of GABA release whereas the hyperosmolal media with Ca2+only induced a single peak, suggesting there might be more than single compartment involved in Ba2+-induced GABA release. Figure 3 also shows that high concentrations of Mgz+ ( l o r n ) in the CaZ+-free hyperosmolal medium failed to stimulate GABA release in cortical slices. Thus, the data suggest that Mgz+ cannot be substituted for Ca2+, or the chemically related divalent cation Ba2+ for the stimulation of GABA release from cortical slices induced by hyperosmolality. +
The absence of Ca2+ dependency of [14C]AIBA release from cortical slices stimulated by hyperosmolality The pattern of release of a non-metabolized amino acid, AIBA, from the cortical slice induced by Ca2 containing or CaZ -free hyperosmolal superfusion is shown in Fig. 4. Unlike the partial dependency upon the presence of calcium for the stimulation of GABA release, the release of AIBA from cortical slices irr duced by hypertonic medium was similar, regardless of the presence or absence of Ca2+ in the medium. Two distinct peaks were observed in both AIBA superfusates, suggesting that AIBA is released from two distinct pools within the cortical slice. +-
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Efjcts of hyperosmolality on the release of GABA from cortical and pontine slices pre-treated with DABA or p-alanine First cortical slices which are largely grey matter and first pontine slices which are largely white matter in composition, have been used to study whether
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FIG.4. Ca2+-dependencyof [I4C]GABA release from cortical slices induced by hyperosmohlity. -0- Na+ 1 4 0 m ~-A; Na+ 2 9 0 m ~ -A; Na' 290mM-(Ca2+free + 1 mM-EGTA).Arrow indicates the time the hyperosmolal medium was begun. Each point is the mean of three experimental values.
GABA release is regionally or structurally specific. Brain tissues were pre-incubated with DABA or p-alanine in the presence of C3H]GABA for 30min, followed by superfusion with hyperosmolal media. The inhibition of uptake and release of C3H]GABA by DABA or p-alanine using first cortical and first pontine slices is summarized in Table 1. The release of C3H]GABA from both cortical and pontine slices was stimulated significantly with hypertonic sucrose superfusion. The stimulated increase was similar in both cortical and pontine slices, although the uptake of GABA was much less in pontine slices. Pre-incubation with 5 mM-p-alanine or 1 mM-DABA did not alter the release of GABA significantly in cortical slices when control buffer was used for superfusion. Although 1 mM-DABA inhibited GABA uptake in cortical slices, the release of GABA was not affected by hyperosmolal sucrose superfusion. However, palanine pre-treatment of cortical slices blocked the stimulated release of GABA induced by hyperosmolal medium. In pontine slices, both 8-alanine and DABA inhibited the GABA uptake, but there were no significant changes in GABA release induced by hyperosmolal sucrose superfusion. Thus, the two amino acid inhibitors reduced the uptake (T/M ratio) of C3H]GABA in both grey and white matter. However, the stimulation in release of C3H]GABA induced by hyperosmolal superfusion was not affected by either inhibitor in pontine white matter, but in the cortical grey matter, only 8-alanine blocked the stimulated release induced by hyperosmolality. DISCUSSION
The present studies have demonstrated that increased release of GABA from brain slices occurs as
Hyperosmolality-induced GABA release TABLE1. EFFECTSOF
HYPEROSMOLALITY ON THE RELEASE OF C3H]GABA FROM CORTICAL OR PONTINE SLICES PRE-TREATED WITH DABA OR
p-ALANINE
Preincubation
Superfusion
Tissue
medium
T/M Ratio
medium
23.42 f 0.77 (12) 23.42 f 0.77(12)
+sucrose
Cortical slices
Control Control Control +B-alanine + DABA Control +B-alanine + DABA Control Control Control + /3-alanine + DABA Control + p-alanine + DABA
5.26 f 0.34 (10) 5.26 f 0.34 (1 0)
Pontine slices
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23.42 f 0.77(12) 19.49 f 1.3 (7)t 7.92 f 0.26 (7)*
5.26 f 0.34(10) 3.66 k 0.20 (6)f 2.45 f 0.10(6)*
Control Control Control Control +sucrose +sucrose +sucrose
% Release 11.75 f 1.33 (6) 16.27 f 0.57 (6)t 11.75 f 1.33 (6) 11.09 k 1.3 (2) 13.49 If: 0.05 (2) 16.27 0.57 (6) 12.38 f 1.41 (5)t 16.30 f 0.48 (5)
Control +sucrose Control Control Control
17.06 f 0.38 (5) 21.34 i-0.89 (5)
+sucrose
21.34 f 0.89 (5) 19.38 k 1.57(4) 19.25 f 1.76(4)
+sucrose +sucrose
17.06 f 0.38 (5) 16.37 0.79 (2) 16.10 f 0.23 (2)
+
The concentrations used in this study were 1 mM and 5 mM respectively for DABA and p-alanine. Control medium, 140mM-Na'; sucrose medium, 1 4 0 m ~ - N a + 300rnM-sucrose. Mean values f S.E.M. are given. (n) = number of slices. * P < 0.001. t P < 0.02. 1P < 0.05. AOAA was omitted in both preincubation and superfusion media.
+
a rapid response to hyperosmolality. This stimulated Whether the release of other putative neurotransrelease was dependent in part upon the presence of mitter amino acids such as taurine, glycine, glutamate Ca2+ in the superfusion medium, because in its and aspartate induced by hyperosmolality is Ca2+absence stimulated GABA release was reduced more dependent requires further elucidation. Despite the than 50%. It has been reported recently that the lack of Ca'+-dependency, a second AIBA peak was stimulated release of neurotransmitter quanta in frog detected in its hyperosmolal release pattern. This neuromuscular junctions induced by hyperosmolality phenomenon was also found with glutamine and tauris dependent upon intracellular Ca2 concentration ine release induced by hyperosmolality (Chan & Fish(SHIMONI et al., 1977). The calcium dependency was man, unpublished data). The fact that two peaks were graphically illustrated by the even greater response seen with AIBA, a non-metabolized amino acid, in GABA release obtained when calcium was omitted favors the existence of two or more compartments. The present observations are analogous to other from the pre-incubation medium, and then replaced with the hyperosmolal medium. However, there was studies of the release of pre-loaded exogenous radionot a graded dose-response relationship between labeled GABA from various brain tissues, spinal cord Ca2+ concentration and the magnitude of GABA slices and synaptosomes upon electrical stimulation & release. GABA release was stimulated also by hyper- or depolarization with potassium (DAVIDOFF & VOADEN,1974; CUTLER& osmolality when Ba2+ was added to the superfusion ADAIR,1976; KENNEDY 1975; HAMMERSTAD et al., 1971; VARGAS medium to replace Ca2+. It has been shown that DUDZINSKI, Ba2+, when used to replace Ca2+ in the 'perfusion et al., 1977). VARGAS and colleagues (1977) have sugmedium, had a similar stimulating effect on neuro- gested that K+-depolarization results in an exit of transmitter release in the adrenal medulla (DOUGLASGABA from a cytoplasmic compartment, rather than & RUBIN, 1964). However, there was no stimulation from nerve endings. It is not clear whether the GABA of GABA release with a high concentration of Mg2+ release with K+-depolarization is derived from (10 mM) indicating that specific divalent ions are neurons or glia. required for stimulated GABA release from cortical Early studies suggested that L-2,Cdiaminqbutyric acid (DABA) and /3-alanine are specific inhibitors of slices. We have previously demonstrated that the release GABA uptake by neuronal and glial 'structures of [14C]AIBA was stimulated by hyperosmolal super- (SCHON& KELLY,1974; IVERSEN19 KELLY,1975; fusion (CHAN& FISHMAN, 1977). The present studies LODGEet al., 1976). The release of C3H]GABA with have confirmed this finding by using a slightly modi- K+-depolarization is inhibited by the superfusion of fied superfusion technique. The release of AIBA in- DABA (HAMMERSTAD & LYTLE,1976). It has been duced by hypertonicity was not Ca2+-dependent. demonstrated that GABA release obtained from +
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DABA pre-treated synaptosomes was affected by both unlabeled GABA and DABA, but was unaffected by elevated K t . This suggests that K + induces GABA release in synaptosomes by a non-DABA carriermediated system (LEVIet al., 1976). However, further confirmation is required because of uncertainty regarding the specificity of DABA inhibition of neuronal GABA uptake and release induced by K + (HAMMERSTAD et al., 1977; BOWERY et al., 1976). The results we have obtained with hyperosmolality-induced GABA release are similar to that demonstrated with the elevated K + system. First, the percentage of release of ['HIGABA obtained from DABA pretreated cortical slices induced by hyperosmolality was similar, suggesting the existence of a transport system for GABA not inhibited by DABA. Secondly, the release of C3H]GABA from B-alanine pre-treated cortical slices induced by hyperosmolality was largely blocked. This suggests the glial elements in cortical slices might be involved in GABA release processes. It has been suggested that glial structures are the main compartments for the re-uptake of neurotransmitters located in synaptic clefts, in order to terminate the action of released neurotransmitters (HE" & HAMBERGER, 1971; IVERSEN& NEAL,1968). The reuptake of GABA by synaptosomes might serve as a direct source for the GABA released with subsequent nerve volleys (RYAN& ROSKOSKI,1975). The mechanism of re-uptake and release of GABA by glial elements related to hyperosmolal modulation requires further elucidation. Our data further demonstrated that GABA uptake was inhibited significantly with both DABA and j-alanine pre-treated pontine slices, but there were no significant differences in GABA release. These data may indicate the presence of cellular compartments in pontine slices that respond with a non-specific increase in GABA release as the tissue adapts to hyperosmolality. The use of glial cells and neuroblastoma cells in culture might elucidate the differences in the responses of glial and neuronal elements to hyperosmolality.
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