Journal of Neurochemistry. 1975. Vol. 25, pp. 393-398. Pergamon Press. Printed in Great Britain

NEURONAL AND GLIAL SYSTEMS FOR y-AMINOBUTYRIC ACID METABOLISM A. SELLSIROM,L.-B. SJOSERG and A. HAMBERGER Institute of Neurobiology, University of Goteborg, Goteberg, Sweden, and Astra Nutrition, Molndal, Sweden (Rrwiued 31 July 1974. Accrpird 7 Fihxary 1975) Abstract-Bulk prepared neuronal perikarya, nerve endings and glial cells have been used to study amino acid concentrations and GABA metabolism in uitro. All amino acids were more conccntrated in synaptosomes and glial cells than in neuronal perikarya. Cell specificity was found with respect to the relative distribution of some amino acids. Glutamate decarboxylase activity was considerably higher in synaptosomes than in glial cells. The inhibitory effect of amino-oxyacetic acid on glutamate decarboxylase activity differed between synaptosomes and glial cells. y-Aminobutyric acid-a-ketoglutarate transaminase had the highest activity in the glial cell fraction; the inhibition of amino-oxyacetic acid differed between glial and neuronal material. The metabolism of exogenous GABA just accumulated by a cell showed similar time characteristics in neuronal and glial material.

A CENTRAL theme in the field of metabolic compart- GABA system. The occurrence of a high affinity mentation in the brain concerns glutamic acid and uptake system of GABA both in nerve endings and its metabolites, particularly glutamine, aspartic acid in glial cells suggests different functions and rate of and y-aminobutyric acid (GABA) (BALAZS& CREMER, metabolism for the amino acid in the two compart1973). The outlined compartmentation is based on ments. The steady state levels of a 4 n o acids in differresults obtained from whole brain and could have ent cellular compartments, the activity of enzymes inthe morphological counterpart in inter- and/or intra- volved in GABA formation and destruction and the cellular specialization. As glutamic acid, GABA and in v i m metabolism of GABA into neuronal cell body, aspartic acid are considered to play a role as neuro- glial cell and synaptosome preparations have theretransmitters (WATKINS,1973), regulation of synaptic fore been compared. function could be one aspect of metabolic compartmentation. The research on these amino acid transMATERIALS AND METHOD mitters has been focussed on their production, release Materials. Ficoll@ was obtained from Pharmacia Fine and inactivation. GABA is released from the pre- Chemicals. y-[2,3-3H]Aminobutyric acid, y-[l-' 4C]aminosynaptic site where it is produced by glutamate decar- butyric acid, and [1-'4C]glutamic acid was purchased from boxylase (GAD; EC 4.1.15; FONNU\I, 1973). Inactiva- NEN Chemicals GmbH, amino-oxyacetic acid (AOAA) tion of amino acid transmitters seems to occur by and y-aminobutyric acid from Sigma Chemicals, St. Louis, rapid reuptake in analogy to what has been shown and bicuculline from K. & K. Laboratories, N.Y. Preparation of cells enriched fractions and synaptosornes. for biogenic amines (AXELROD,1965). Following its withdrawal from the receptive region of the neuronal Fractions enriched in neuronal cell bodies and in glial membrane, the transmitter is either stored or broken cells were prepared as previously describcd (BLOMSTRAND & HAMBERGER, 1969, 1970). Cerebral cortices or whole down. brains from 6-8 white rabbits were sliced with a mechaniHigh affinity uptake systems for GABA in cerebral cal chopper and incubated for 45min at 37°C. The suscortex (IVERSEN & NEAL,1968) have been suggested pended tissue was further disrupted by passage through to be presynaptic (BLOOM& IVERSEN, 1971). This was nylon mesh attached to the end of a plastic syringe. The supported by results obtained with synaptosomal resulting suspension was filtered through a series of nylon preparations (KURIYAMA et al., 1969; MARTIN& meshes with pore sizes down to 50 pm; the final filtration SMITH, 1972). However, a high affinity uptake system included passage through a double layer of 50pm nylon localized to glial cells (HOKFLLT & LJUNGDAHL, 1970; mesh. The filtrate was centrifuged for 5min at 150g and HFNN& HAMBERGEK, 1971 ; HABEK e t al., 1973) shows the resulting pellet was mixed with Ficoll. T 4 neuronal speclfic characteristics (KELLYet a/., 1973; SELLSTROMand glial fractions were prepared by centrifugation on a & HAMBERGER, 1975). The old hypothesis (KOELLE, discontinuous sucrose-Ficall. gradieht at 81,500 g for llOmin in a SW 27 rotor in a Beckman Spinco L2 65B 1955) that glial cells remove and inactivate synaptic ulttacentrifuge. The fractions were aspirated with Pasteur transmitters may thus receive new evidence from the pipettes, diluted with 3-4 vol of 0.32 M sucrose and pelleted by centrifugation at 2000 g. The synaptosomal fraction was & MATTHEWS (1971). The Abbreviations used: AOAA, amino-oxyacetic acid; GAD, prepared according to COTMAN glutamate decarboxylase; GABA-T, y-aminobutyrate-a- chopped rabbit brains were homogenized in 0 . 3 2 ~sucrose containing 10 mM Tris-HC1 (pH 7.4). The homogenate ketoglutarate transaminase. 393

394

A. SELLSTROM, L.-B. S J ~ B E Rand G A. HAMBERGER

was centrifuged for 10 min at 900 g. The supernatant fluid was centrifuged for 20min at 10,aoOg to sediment the crude mitochondria1 fraction, which was resuspended in the sucrose solution and placed over layers of 7.5 and 13% Ficoll made up in the sucrose solution, and centrifuged for 45 min at 65,000 g. The band at the interphas between 7.5 and 13% Ficoll was collected, diluted with 0-32 M sucrose and pelleted by centrifugation for 30 min at 10,OOO8. Incubations. Samples of the fractions were incubated in a medium containing: 35 mM Tris-HCI (pH 7.4), 120mM NaCl, 5 n i ~KCI, 25mM MgCI,, 2 0 m ~glucose and 15mM CaCI, and radioactive GABA as indicated in the Results section. Incubations were made in a shaking water bath at 37°C in 15 or 50 ml open test tubes, and terminated by centrifugation of the suspensions through a layer of 032 M sucrose. In the experimentswhere ninhydrin reactive material was determined, the suspension was simply pelleted. The amino acid analyses were done on sulphosalicylic acid extracts (final concentration of sulphosalicyclic acid, 3%). Thin layer chromatography (TLC) was performed on samples which had accumulated C3H]GABA for 10min. Samples incubated with 5 x M AOAA were preincubated for 5min. The cells were pelleted through sucrose, and then resuspended and incubated in fresh non-radioactive medium at 37°C. After varying time periods equal volumes of ice-cold 10% TCA were added. The TCA supernatants were extracted twice with equal volumes of ether before TLC. Determination of total ninhydrin-reacting material. The colorimetric method for the determination of total amino acids has been used, based on the reaction with ninhydrin (YEMM& COCKING,1955). The reaction was carried out on the sulphosalicylic acid (3%) supernatant fluids in a citric acid buffer, and the results expressed as O.D. per mg wet weight.

incubated with 50pmol of Tris-HCI, pH 8.9, 1Opmol pmercaptoethanol, 3 pmol NAD, 1.8 pmol succinate, 0.3 pmol pyridoxal phosphate, 20 pmol a-ketoglutarate and 0.22 pmol [I-14C]GABA in a total volume of 2.2 ml. Inhibitors were added to the reaction mixture as indicated in the Results section. Incubation was carried out for 30 min at 37°C. The reaction was linear with time for 60 min with the amounts of enzyme used. The reaction was stopped by adding 1 0 0 4 45% TCA, containing 115 mM unlabelled GABA. After centrifugation, the supernatant fluid was applied to a 0.7 x 5cm column of Dowex-50-H’. The radioactivity in the effluent solution, mainly succinate, was measured. Thin layer chromatography ( T L C ) . The TLC was performed as described by MARTIN& SMITH(1972). Carriers of different metabolites were added. Samples of 10-20 pl were spotted on a 250pm silica gel G thin layer plate and run for 240min in a mixture of butanol-acetic acidwater (60:20:20, by vol.). The plate was dried in an oven for 30min at 100°C. The citric acid cycle intermediates were visualized by spraying with 0 1% (w/v) bromcresol purple in ethanol and the amino acids by spraying with 01% ninhydrin. The regions of interest were scraped off the plate and extracted in water for 1 h. Samples were taken for radioactivity measurements in a Beckman TriCarb liquid scintillation spectrometer. The fractions separated by TLC could be collected in three groups, the fast migrating group, containing succinate (RF0.53), fumarate (0.47), oxaloacetate (0.32), cis-aconitate (0.31) and malate (0.30). In the second group, the GABA spot (026) could not be separated from a-ketoglutarate (0.27). The slowly migrating group consisted of glutamate (0.19), glycine (0.15), glutamine (015), asparate (0-13), citrate (0.12) and isocitrate (0.12). Amino acid analysis by ion-exchange chromatography. The automatic amino acid analyser Jeol JLC-5AH was used. Both the columns were loaded with the resin Jeol RC-2. The basic compounds were eluted from a 26 cm column as follows: 0.13 M Na-citrate buffer + 5.5% methanol MeOH, pH 4.23, at 36°C for 150 min, and 0 . 1 2 ~ Na-citrate buffer, pH 5.25, at 55°C till the end, total 230 min. The acidic compounds were eluted from a 46 cm column as follows: 0 . 1 0 ~ Li-citrate buffer + 8% MeOH, pH 280, at 36°C for 105min; 0 . 1 0 ~Li-citrate buffer + 10.5% MeOH. pH MX). at 36°C for 30min: 0 . 1 0 ~Licitrate buffer + IO.So/, MeOH, pH 3.00, at 5 5 T for 25 min; 0 . 1 0 ~ Li-citrate buffer + 10.5% Na, pH 3.00, at 36°C for 80min; and finally 0 . 1 0 ~Li-citrate buffer, pH 4.21, at 36°C for 165 min.

Determination of enzyme activities Glutamate decarboxyiase (GAD EC 4.1.1.15). The activity was determined by measuring 14C02 liberated from [l‘4C]glutamic acid, using the procedure of ROBERTS& SIMONSEN (1963) as described by WOOD& PEESKER (1972): 0.1 ml buffer substrate was re-placed in a 25 ml glass vial with a screw cap, where the top was placed by a rubber membrane. A polyethylene liquid scintillation flask, containing 0.4 ml 1.0 M hyaminc hydroxide (in methanol), was connected through a side arm on the glass vial. Two hypodermic needles were pushed through the rubber membrane to gas the flask with nitrogen for 2 min. A 10%(w/v) homogenate in l 0 0 m ~K-phosphate buffer, pH 6.5, containing 0.25% Triton X-100 to ensure maximal activity, and 0.02 M mercaptoethanol was prepared in a Teflon-glass homoRESULTS genizer. A portion of the homogenate (0.4ml) was injected Amino acid content. Table 1 illustrates the content into the reaction flask which was then shaken a t 37°C for IOmin, homogenates were not preincubated. To stop of free amino acids per mg protein in rabbit brain the reaction, 0.1 ml of 4~ H2S04 was injected, and the preparations. The amino acid concentration in brain flask shaken at 37°C for a further 30min. Next, 15ml tissue decreased to approx. So”/, when brain slices toluene containing 03% PPO and 01% POPOP was were incubated for 30min at 37°C. After disruption added to the hyamine hydroxide. The radioactivity was of the incubated slices, a crude cell suspension was measured in a Packard liquid scintillation spectrometer collected by centrifugation. The amino acid conequipped with an absolute activity-analyser. centration in this suspension was 3540% of that oriGABA-a-krtoglutarate transaminase (GABA-T EC ginally present in brain tissue. Nerve cell and glial 2.6.1.19). The radiochemical method of HALL& KRAVITZ (1967) was used. The fractions were homogenized in 10 m~ cell fractions were isolated from the crude cell suspenpyridoxal phosphate, pH 8.9, for maximal activity and sion by gradient centrifugation. The neurons had 4% enzyme stability according to SHERIDAN et al. (1967). Six and the glial cells 20% of the amino acid confreezings and thawings of the homogenate were required centration present in fresh brain. The synaptosomal for maximal activity. One millilitre of the homogenate was preparation which was obtained after homogenization

GABA metabolism in neurons and glia

TABLE 1. AMINVACID COiiTENT IN

DIFFERENT PREPARATIONS OF RABBIT BRAIN EXPRESSED AS mg a.a./g PROTEIN

395

TABLE 2. 7-AYINOBLTYRIC ACID: Z-OXOGLUTARATE TRANSAMINASE (GABA-T) ACTIVITY IN NEURONAL, GLlAL AKD SYNAPTOSOMAL FRACTIONS

Taurine Phosphoethanolamine Aspdrtic acid Tbreonine Serine Glutamine Glutamic acid Proline Glycinc Citrulline Aliiiiiiic cy,t'IIIl1OIIIIlc y-Aminohu lyric acid Omithine Lysine Histidine Arginine

Brain

Slice (incubated 30 min)

2.06

1.37

0.61

011

075

2.86 3.73 014 0.62 995 1933 017 077 0-36 0.40 1.51

1.78 2.66 023 0.67 2.41 11.52 0.14

1.18

017 013

090 1.33 0-17

Synaptosomes Neurons

.

050 0.16 0.30 059

3.47 011 018 0.58 4.35 0.07 016 003 0. I 7 0.25

0.05 018 0.16 032 ~

008 001

0.06 0.14

Clial cells

035 0.89 271 0.04 036 014 0.27 0.36

Homogenate Neurons Glia Synaptosomes

Control

#mole/h/g Protein 5 x M AOAA

53.6 f 5.7 (4) 20.8? 125(15) 58.3 f 25-1(16) 41.4 f 120(7)

3.2' i 3.0(8) 462t f 34.1 (4) 2-8* k 0.8 (4)

lo-'

M

Ricucolline

-

21-5 f 17.3(8) 292 15.9(5) 39 9 f 200 (7)

Values are mean S.D. Number of experiments within brackets. * P 0.01 vs control. t P 0.8 vs control.

The distribution of amino acids in the neuronal fraction corresponds fairly well to that of an incubated 0.26 slice. Some differences may, however, be pointed out: 0.18 0.26 the relative amounts of serine and cystathionine were increased, and the glutamic acid/glutamine ratio was Total 4426 23.93 12.12 1.73 9.84 decreased in the neuronal fraction. No specific Each value represents the mean of 6 experiments, with changes could be noticed in the glial fraction. The a S.D. Of 51.5%. synaptosomal preparation showed two characteristic alterations in the distribution pattern of amino acids: and centrifugation had an amino acid concentration a prominent increase in the relative level of aspartic which was approx. 27% of that in fresh tissue. The acid and an increase in the glutamic acid/glutamine loss of amino acids from brain slices during incuba- ratio. The changes in ninhydrin reactive material durtion was due to a fairly even loss of all amino acids, ing incubation is shown in Fig. 1. The loss of amino since the distribution of amino acids was approxi- acids from brain slices during incubation (see also mately similar in fresh brain tissue and in brain slices Table 1) occurred during the first 5 min of incubation. which had been incubated. One exception was gluta- Crude cell suspensions were incubated in media with mine which showed a further substantial decrease. and without Ca2+ ions, and in both cases a fairly stable level of ninhydrin reactive material was found. Also the level in synaptosomes was fairly stable durlo+ ing a 30 min incubation. Neuronal and glial cell fractions showed an initial decrease in O.D./mg wet wt., whereafter a plateau or a return to initial O.D. was obtained. GABA-T and GAD activity. The specific activity of GABA-T (see Table 2) of the glia cell fraction was approx. 3 times that of the neuronal fraction and 50% d higher than the activity of the synaptosomes. The GABA-T activity in the glial cells was only slightly E" \ inhibited by M AOAA, but inhibited to 50% by 0 0 M bicuculline. The neuronal and synaptosomal GABA-T activity was strongly inhibited with the same concentration of AOAA but hardly affected by bicuculline. The GAD activity (Table 3) in the synaptosome preparation from rabbit brain was approx. 1.51

015

0-95 0.16 0.18 0.10 0.21

0.45 007 0.18 010 016

0.17 005 0.07 0.03 -

13.74 019 0.28 023 013

TABLE 3. GLUTAMIC ACID 0

5

I0

20

30

60

DECARBOXYLASE (GAD) ACTIVITY IN HOMOGENATE AND FRACTIONS OF SYNAPTOSOMES, NFURONAL AND GLlAL CELLS

min

FIG.1. Extinction (O.D.)per mg wet wt of ninhydrin-reactive material as a function of increasing time of incubation. Incubation in a glucose-saline medium containing calcium (see Methods): brain slice: -@-; crude cell suspension: ,+-; synaptosomes: --4--; gial cells: -4-; neurons: Incubation medium not containing calcium: crude cellsuspensions A; synaptosomes: V; glial cells: 0; neurons: 0. The values are means from at least three experiments with S.D. of about 10%.

G A D Activity (pmole/h/g protein) Control 5 x 1 0 . ' ~ AOAA Homogena te Synaptosomes Neurons Glial cells

3462 k 3.71 ( 5 ) 77.52 f 1 I-49(5) 11.82 f 246 (6) 7.39 f 3 31 (6)

688* k 0 08 (4) 15.74* f 0 82 (4) 3.27. ir 0.36 (4) 6.43 k 0.56(4)

Values are mean f S.D. Number of experiments within brackets. * Significant for P < 0,001. t Significant for P < 0.2.

A. SFLLSTR~YL.-B. SJ~BERG and A. HAMBERGER

396

TABLE 4. RELATIVEDISTRIBUTION OF

INTRACELLULAR RADIOACTIVITY AND SPECIFIC RADIOACTIVITIES OF AMINO ACIDS RECOVERED FROM THE AMINO ACID ANALYSER IN NEURONAL. GLlAL AND SYNAPTOSOMAL FRACTIONS AFTER INCUBATION WITH

5 x Neuronal (%)

GABA Aspartic acid Glutarnic acid (glutamine) Alanine Total recovered radioactivity (d.o.m.1

98 2

0.I

-

h57.Xon

lo-'

M (2,3-3H]GABA OK

2x

[2,3-'H]GABA, 15 min incubation Glial Synaptosomal S.A. (%) S.A. (%I -

97

-

2

-

02

-

0.04 1.424.350

Ill 1.3 04 -

93 6 04

0.1

4XI.Xl(l

M

S.A.

[l-'4C]GABA

Neuronal (%)

[I-"C]GABA. 30 min incubation Glial Synaptosomal S.A. (%) S.A. (%)

S.A.

122

150

92

8-8

1.6

6

1-7

87 10

02

I 0.1

05

3

145

-

0.4

2.5 0.6 -

5 0.4

-

-

y?.xni

78 17

1.6

I(W).IIW)

hVi.(HN)

y!, = ':b of recovered radioactivity. S.A. = specific radioactivity. Ci/mole. Each value represents the mean from 2 experiments except the value for neurons after I5 min incubation (one expt.). twice that in a homogenate. The activity in the homogenate and the synaptosomes was strongly inhibited by M AOAA. The enzyme activity in the neuronal and glial cells was approx. one-tenth of that of the synaptosomes. The neuronal activity was inhibited to 3077 of control by 10- M AOAA, while the glial enzyme retained 80% of its initial activity at this concentration of inhibitor. GABA metabolism. In order to determine the metabolites of labelled GABA recovered intracellularly after incubation, TCA-soluble extracts were analysed by TLC and sulphosalicylic acid extracts by ion exchange chromatography of amino acids. The TLC experiments showed that approx. 70% of the intracellular radioactivity was recovered as GABA after incubation in 5 x lo-' M [2,3-3H]GABA for 10 min. There was no appreciable difference between the fractions in this respect, and the percentage recovered as GABA showed little change (- 5%) during 30 min accumulation at 37°C. This result was not unexpected as long as the extracellular radiolabelled GABA was in excess. On analysis of the intracellular free amino acid metabolites by TLC, amino acid fractions other than GABA and a carboxylic acid fraction were obtained, each of which contained 2-5% of the total radioactivity. The change with time @ (3 0min) was small also in this case. The remaining 20-25% was in a volatile fraction, mainly as tritiated water. Four separate amino acid analyses by ion exchange chromatography were also done on the intracellular content after incubation of fractions containing neuronal cells, glial cells and synaptosomes for 15min with C3H]GABA, and 2 after 30min with [I4C]GABA. The results show that the main metabolites of GABA, aspartic acid and glutamic acid, were labelled in similar proportions for the different fractions (Table 4). Although special precautions were taken to isolate radiolabelled glutamine, none or only very little labelled glutamine could be found. This may be due to a rapid transport of newly-synthesized glutamine to the incubation medium. Such a transfer has been observed by BALAZSet al. (1970), working with GABA metabolism in brain slices. In both the experiments utilizing C3H]GABA and [14C]GABA, there was a big peak of radioactivity close to the taurine fraction, the amount of radioacti-

vity was in the same range as for aspartate. This radioactivity was probably due to citric acid intermediates. The hlgher percentage of label in the metabolites in the [14C]GABA experiments is partly an effect of the longer incubation time, but mainly due to the dehydrogenation of the GABA skeleton which lowers the specific activity of the [2,3-3H]GABA metabolites. The differences obtained with respect to radioactivity recovered as GABA do not necessarily reflect differences in GABA metabolism. It could also be a different substrate to cell material ratio, since the amount of protein varied, with the highest concentration for synaptosomes. The specific radioactivity for GABA did not differ largely between the fractions and was in decreasing order: synaptosomes, glia, neurons. Aspartic acid and glutamic acid had specific radioactivities which for all fractions agreed with values reported for brain slices (BALAZs et al., 1970).

loor

-

I

0

10

Time,

1

1

20

30

min

FIG.2. Percentage of recovered C3H]GABA during varying incubation periods following a 10 rnin preincubation with [2,3-3H]GABA. Normal glucose-saline: neurons -0-; glial cells -V: synaptosomes -0--. Normal glucosesaline + 5 x M-AOAA: neurons -4-; glial cells --V--; synaptosomes Values are means from two or three experiments, some S.D. values are indicated.

GABA metabolism in neurons and glia

397

be made, if it is assumed that amino acids leak to the same extent. The amino acid pattern for fresh brain corresponds well with that obtained by LEV^ e t a / . (1967).Thc amino acids with proposed transmitter function had the highest concentration in whole brain, and this was reflected in all fractions. The relatively specific decrease in the glutamine level of all fractions is in agreement with the specific efflux of glutamine to the incubation medium from brain slices found by BALAZSet al. (1970). The concentrations of amino acids in the synaptosomes correspond well with those reported by BRADFORD (1970).The distribution suggests a large aspartic acid pool and a small pool of glutamine within the presynaptic region. Serine was relatively highly concentrated in the neuronal perikarya, which is interesting in connection with the DISCUSSION active serine-phosphoethanolamine base exchange Recovery and steady state levels of amino acids. Re- system for phospholipid biosynthesis present in gional variation in the distribution of free amino acids neurons (G~RACCI et al., 1973). The high glutamine/ in brain (UNDERA et al., 1968) may reflect alterations glutamic acid ratio in the neurons compared to glia in the cell composition, since the amino acid pool and synaptosomes suggests that the efflux of glutacould be different in neurons and glia (LAJTHA, 1974). mine from brain slices could appear due to leakage However: any quantitative differences between the cell preferentially from glia and synaptosomes. The stable types are difficult to evaluate when the cells have been level of ninhydrin reactive material during incubation isolated in aqueous media. Leakage of small mole- in glucose-saline media favours the use of cell prepcules, like amino acids, inevitably occurs during frac- arations for metabolic experiments. Similarly, stable tionation of any organ. Incubation of brain slices in levels have been observed for phospholipid precursors standard glucose-saline media causes a rapid loss of in incubated ncuronal and glial cells (POKCELLATI, amino acids (LEVI et a/., 1965; (SHERAYILet al., 1967). personal communication). Amino acids are probably concentrated in the cytoGABA metabolism. The attempt to evaluate plasm of a cell. Consequently, the high nuclear/cyto- whether GABA uptake is specifically correlated to plasm ratio in the isolated neuronal perikarya, and to transmitter inactivation in all GABA systcms has a some extent in the astrocytes (HAMBERGEK et al., definite drawback in the present approach using 1974), may contribute to the lower value for amino material from whole brain. Autoradiographic studies acids per protein. It may still be an open question generally demonstrate GABA accumulation in occawhether the finding of a very low level of free amino sional neurons while the greater part of neuronal peracids in the neuronal perikarya, i.e. only 20% of that ikarya is conspicuously free of granules (HOSLIet al., found in glia and synaptosomes, simply rcflects a 1972; LASHER,1974). In contrast to this, autorahigher degree of neuronal cell damage. Both ROSE diographic studies of GABA accumulation in glia T LJUNG(1968) and NAGATA et al. (1974) reported considerably show a more general distribution ( H O ~ E L& higher levels of free amino acids in the neurons, while DAHL, 1970; HOSLIet a/., 1972). The data on GAD and GABA-T activities support the levels in the glial fractions prepared by these authors correspond well with the present data. This previous work, in that GABA is formed mainly in & DE ROBEKTIS, discrepancy with respect to neuronal amino acid level the nerve endings (SALGANICOFF 1968). The low levels of GAD in glia could be due to differences in the purity of the frac- 1965; FONNUM, tions or in the disruption procedures. The authors and neuronal perikarya could represent an admixture cited utilized a similar cell separation technique, in of nerve endings and/or a true localization of the which the tissue is dissociated in the cold in the pres- enzyme in these compartments. The low degree of ence of high potassium concentrations. The technique inhibition obtained with the glial homogenate upon employed in this study involves slice incubation at addition of AOAA may favour the latter possibility. 37°C in salt solution with a similar composition to GABA-T is distributed differently from GAD. It has extracellular fluid. Although it is at present impossible been observed in studies with subcellular fractions to conclude which result most closely reflects the in from whole brain that GABA-T is localized mainly vivo level of amino acids, it is not unlikely that iso- in the fraction containing nonsynaptosomal mito& DE ROBEKTIS,1965; VAN lated glial cells retain higher levels of amino acids, chondria (SALGANICOFF et a/., 1965). The glial enzymes had the highsince their surface area/volume ratio is considerably KEMPEN larger (HAMBERGER et al., 1974) and their transport est activity and were distributed differently from the ATPase activity higher (HE” et al., 1972; MEDZIH- neuronal and synaptosomal enzymes. This may indicate different types of GABA-T (Ho, 1973) although RADSKY et a[., 1971, 1972). An extrapolation back to the in vivo pattern of it is premature to draw definite conclusions from the amino acids in different brain compartments could present data. In the experiments employing TLC to follow the radioactivity remaining as GABA, each fraction had been loaded with C3H]GABA, as described in the Methods section. The experiments (Fig. 2) were started by the resuspension of the loaded pellets in a final volume of approx. 200~1.Since a new tissue medium distribution was then established, a strict comparison of C3H]GABA turnover rates is difficult, because it is dependent on both the relative volume of resuspending medium and the GABA accumulating ability. The amount of resuspending medium was kept minimal. Figure 2 shows that no appreciable difference between the fractions could be found in GABA breakdown with this system, and that the inhibition by AOAA was close to 100% for all fractions.

398

A. SELLSTROWL.-B. WBERG and A. HAMBERGER

If the ratio of GAD/GABA-T could be used to obtain information on the roles of the different compartments, it would indicate GABA production in the presynaptic site and GABA destruction in the glial cell. However, this evidence had little support in the results obtained by TLC or ionexchange chromatography analysis of amino acids. In the study of GABA metabolism by isolated fractions, no significant differences were observed in metabolite pattern or turnover rate. The previously observed difference in AOAA sensitivity was also absent from these systems, but the conditions, substrate-inhibitor ratio, etc.. differed considerably. Under the experimental conditions used for turnover studies, i.e. preloading followed by recording of the breakdown of the accumulated GABA, it seemed that the cells metabolize the substrate at a rate proportional to their uptake ability. With the exception of GABA formation which clearly is a presynaptic event, no cell specitic characteristics were found. It seems reasonable to question the direct relationship between high affinity uptake and transmitter inactivation unless GABA is used also by glial cells to influence neuronal activity.

HABER B., WERRBACH K., VANCE C. & HUTCHEON H. T.

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