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Effects of LI+ on the metabolism in brain of glutamate, glutamine, aspartate and gaba from [1-14C]acetate in vitro
BRAIN RESEARCH
203
EFFECTS OF LI + ON T H E METABOLISM IN BRAIN OF G L U T A M A T E , G L U T A M I N E , A S P A R T A T E A N D GABA F R O M [1-14C]ACETATE I N VITRO
S. B E R L AND D . D .
CLARKE
Department of Neurology, Collegeof Physicians and Surgeons, Columbia University, New York, N. Y. 10032 and Chemistry Department, Fordham University, Bronx, N.Y. 10458 (U.S.A.)
(Accepted July 22nd, 1971)
INTRODUCTION An effect of lithium which has been suggested to explain its usefulness in the treatment of mania is that it changes the rate of metabolism of brain neuromediators. Norepinephrine and serotonin have been extensively investigated in this relationship~t,2a, 24. The topic has been reviewed recentlyl°,~L The effect of lithium has also been reviewed~L It has been frequently suggested that certain amino acids may also function as neuromodulators or transmitter substances a. This prompted us to examine the effects of lithium on the metabolism of glutamic acid, glutamine, aspartic acid and GABA in guinea pig brain slices. Previous work had indicated that replacement o f Na + by Li + decreased the incorporation of radioactivity from NaH14COa into citric acid cycle intermediates and associated amino acids in lobster nerve 6. While this work was in progress DeFeudis and Delgado 9 reported that lithium when injected into mice produced a decrease in the level o f brain glutamate. The results reported here indicate that replacement of Na + with Li + does greatly effect the metabolism of the glutamate-glutamine system. METHODS Preparation o f brain slices
Guinea pigs were decapitated, their brains rapidly removed and placed in a moist chamber (Petri dish containing filter paper wet with medium). Slices o f cerebral cortex were cut approximately 0.3 mm thick using a Stadie-Riggs blade and a guide as described by McIlwain and Rodnight 16. The slices were weighed to the nearest 0.1 mg as soon as they were prepared using a Roller-Smith torsion balance. For each experiment approximately 75 mg slices were used. Incubation conditions were as described previously a with the exception that the preincubation period was 15 min and the incubation period was 20 min for all experiments. The medium used was modified Krebs-Ringer phosphate saturated with oxygen and containing 1.55 m M Brain Research, 36 (1972) 203-213
I
b~ PIG BRAIN SLICES, INCUBATION
AND PREINCUBATION
MEDIA
(pmoles/g tissue wet weight -s- S.E.M.)
2.65 ± 0.13 2.54 ± 0.22 1.80 ± 0.33*
Glutamine Control 0.04 M Li" 0.10 M L i ÷ 0.1 M L i ~- N a "
Aspartic acid Control 2.00 ~ 0.18 0.04 M Li + 1.51 • 0.12" 0.10 M Li + 1.13 ± 0.18"* 0.10 M Li + =- Na =
8.68 ± 0.38 8.18 ± 0.t8 6.19 ± 0.07**
Glutamic acM Control 0.04 M Li * 0.10 M Li 0.1 M L i + - - N a +
0.09 i 0.06 0.11 ± 0.06 0.20 + 0.08
0.96 -- 0.07 1.18 ~ 0.04* 1.45 + 0.07**
0.28 ~.~ 0.05 0.82 ± 0.04** 1.67±0.11"*
0.28 £ 0.06 0.41 -i 0.09 0.46 ± 0.12
1.41 ~_0.16 1.63 4- 0.14 2.27 ± 0.20*
1.65 ± 0.15 2.42 ± 0.12"* 2.87 -~- 0.10"*
2.37 0.12 2.02 ± 0.08 1.79 r_ 0.19"
5.01 -- 0.27 5.34 ± 0.22 5.52 - 0.35
10.61 ~ 0.29 11.23 ± 0.21 10.73 ± 0.23
Total
± ± ± ±
0.16 0.41 0.24** 0.34**
2.41 1.93 1.19 1.51
~0.05 :R0.11** -0.05"* -:--0:05"*
1.85 :± 0.12 1.79 ± 0.15 1.71 4 0.17 1.64~0.17
7.85 8.36 6.76 6.75
± ± ± ±
0.03 0.03 0.04 0.13
0.12 0 . 0 1 0 . 0 5 - 0.01" 0.23 : 0.04** 0.21 { 0.01"*
1.18 ± 0.08 1.56 :~ 0.08* 1.64 ~ 0.08** 1.47±0.20
0.24 0.26 0.31 0.35
blcubation medium
Slices
Preincubation medium
Slices
Incubation medium
In 100 % oxygen
In air
± ± ± ±
0.09 0.23 0.15"* 0.14"*
0.18 0 . 0 2 0.26z-0.06 0.52 '--0.06** 0.21 ± 0 . 0 6
1.82 ± 0.10 2.21 ! 0.52 2.73 ± 0.32** 3.20±0.18"*
0.74 1.15 2.01 1.77
Preincubation medium
0.15 0.50* 0.38 0.41
~ 0.25 i 0.46 ± 0.43* ±0.35*
± ± ± ±
2.70-_0.06 2.24 . 0.07 '~* 1.93 ! 0.09** i.93 --0.07"*
4.85 5.55 6.07 6.11
8.87 9.77 9.08 8.86
Total
Guinea pig brain slices (approximately 75 rag) were preincubated for 10 min at 37-C in 2.5 ml Krebs Ringer phosphate m e d i u m containing 55 m M glucose and 1.55 m M Ca 2 . They were then transferred to fresh medium, 2 / t C i of [l-NC]acetate (58/~Ci//maole) added and incubated for 20 min at 37°C. Preincubation and incubation were carried out either in air or 100%/, O2. Where Li ~ was added, an equivalent a m o u n t of NaCI was replaced by LiC1 except in the last set of experiments where 0.1 M LiC1 was added without reduction of Na + (0.1 M Li ~ ~ Na+). N - 4 for all samples except for controls with t00~o O2 (N = 17) and 0.1 M Li + in I00°/~ O2 (N = 7).
LEVELS OF A M I N O A C I D S I N G U I N E A
TABLE I
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GABA
* P < 0.05. ** P < 0.01 by Student 't' test.
Control 17.34 4- 1.04 0.04 M Li ÷ 15.83 4- 0.73 0.10MLi + 12.134-0.73"* 0.10 M Li + ÷ N a +
Sum o f the amino acids
Control 4.01 4- 0.35 0.04 M Li + 3.60 i 0.27 0.10 M Li + 3.09 4- 0.15" 0.10 M Li + + N a + 1.73 4- 0.23 2.57 4- 0.19" 4.194-0.35"*
0.40 4- 0.05 0.46 ± 0.05 0.87 4- 0.09**
3.76 4- 0.43 5.01 ± 0.43* 6.544-0.47**
0.42 4- 0.06 0.55 :t: 0.08 0.94 4- 0.05**
22.81 4- 1.01 23.20 4- 0.83 22.934-0.96
4.82 4- 0.33 4.61 ± 0.32 4.89 4- 0.19
14.53 14.88 11.51 13.38
2.42 2.80 1.85 3.48 4- 0.50 4- 0.81 4- 0.80 4- 0.83
1.93 2.40 2.72 2.69
4444-
0.15 0.20 0.22 0.39
4- 0.17 0.39 4- 0.03 4- 0.14 0.53 4- 0.08 4- 0.34 0.54 4- 0.06* 4- 0.27** 0.66 4- 0.05** 3.40 4.38 6.34 6.43
0.66 0.76 1.08 1.25 ± 444-
0.27 0.96 0.73** 0.57**
4- 0.06 4- 0.15 ± 0.19"* ± 0.19"* 19.89 21.64 20.55 22.30
3.47 4.08 3.47 5.40 i 444-
± 4441.32 1.30 1.35 1.26
0.86 0.27 0.45 0.43**
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S. B E R L A N D 1). ['). C L A R K [
Ca 2+ and 55 mMglucose. In the experiments labeled Li ~-, LiCI replaced an equal molar concentration of NaCI. In the experiments labeled Li ~ + N a ' the LiCI was added to the Krebs-Ringer medium without decreasing the NaCI concentration. In the experiments labeled '100 ~o oxygen', this gas was constantly bubbled through the medium during both the preincubation and incubation periods. In the experiments labeled 'in air' the tubes were merely exposed to the air during the preincubation and incubation periods. Zero time was taken as the point at which 2 ,uCi of [l-14C]acetate (58 #Ci/#mole) were added to the incubation medium. The slices were prepared at room temperature and incubated at 37°C. At the end of the preincubation period and at the end of the experiments 2 ml of medium was rapidly pipetted into 0.2 ml of 50",~i (w/v) TCA and the supernatant analyzed as previously described 1. Glutamic acid, glutamine, aspartic acid and GABA were isolated from slices and media, determined and counted 3. RESULTS
The effect of added Li + on the levels of the amino acids in the slices and media is described in Table I. The presence of this ion causes the levels of each of the 4 amino acids to decrease in the slices and to increase in the incubation and preincubation media. This leakage of amino acids from the slices is most pronounced in the preincubation media. In the presence of 100~ oxygen the leakage is less than in air in both the preincubation and incubation media. Even in the controls glutamine leaks out to a much greater extent than do the other amino acids and it is increased by Li +. However, the greatest effect of this cation appears to be on the leakage of glutamate from slices incubated in air. O2 counteracts this leakage of glutamate; in contrast 02 does not appear to affect the leakage of the other amino acids. 'In air' the total quantity of each amino acid in the slice, preincubation plus incubation media, is unaffected by Li ÷ and, except for GABA, resembles the levels observed in vivo. 'In oxygen' the total glutamate level is unaffected while glutamine increases and aspartate decreases. The sum of the amino acids do not differ significantly under the various conditions. The presence of LiC1 plus NaCI, however, greatly elevates the level o f GABA in the slices. In 100 ~o 02 the slices take up almost twice as much radioactive acetate as in air (Table II). Li ÷ decreases the amount of radioactivity taken up by the slices. In air this is evident even at 0.04 M Li ÷ while in 100~ oxygen the decrease is significant only at 0.1 M Li ~-. A corresponding decrease in the conversion of [l-14C]acetate to the amino acids is observed. Examination of the distribution of radioactivity in the individual amino acids shows that the labeling of glutamine is severely reduced in the presence of Li ÷ (Table III). This effect is greater in air than in 100~ oxygen. While the per cent of radioactivity in the glutamine is decreased that in the glutamic acid and aspartic acid is increased. In air this increase of radioactivity is most evident in the incubation medium into which the amino acids have leaked. The percentage of radioactivity in GABA is relatively unaffected by Li ÷. Brain Research, 36 (1972) 203-213
I,o
I
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~D ,-o
2.73 4, 0.24 1.59 ± 0.10"* 1.37 4- 0.20**
35.8 4- 1.9 35.0 4- 2.0 39.3 -t- 1.1
* P < 0.05 by S t u d e n t ' t ' test. ** P < 0.01 by S t u d e n t ' t ' test.
TOTAL RADIOACTIVITY/G TISSUE IN AMINO ACIDS ( c o u n t s / m i n 4. S.E.M.) x 10 -6 Control 1.66 -4- 0.16 0.38 4. 0.05 0.04 M Li + 0.74 =/= 0.03** 0.20 4- 0.01 * 0.10 M Li + 0.48 4. 0.14"* 0.15 4- 0.01"* 0.1 M Li ÷ + N a +
Control 0.04 M Li + 0.10 M Li + 0.1 M Li + -t- N a +
4 4 4
4 4 4
3.70 3.66 1.67 1.14
4.76 4.42 2.43 2.09 4- 0.25 4- 0.69 -t- 0.05** 4. 0.22**
4- 0.19 4" 0.47 4- 0.06** 4- 0.24**
Slices
N
Slices
Incubation medium
In 100 % 02
In air
See Table I for i n c u b a t i o n conditions.
TOTAL RADIOACTIVITY/G TISSUE IN TRICHLOROACETIC ACID EXTRACT ( c o u n t s / m i n 4- S.E.M.) × 10 6
T A B L E II
0.09 0.18 0.02** 0.09**
0.90 2.2 2.1 2.6 4444-
444. 4, 1.04 1.25 0.46 0.33
33.4 33.2 33.9 37.9
Incubation medium
17 4 7 4
17 4 7 4
N
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* Calculations percentages are ** P < 0,005 *** P < 0.001
61.1 4. 3.9 46.9 4. 3.0** 40.5 4. 3.0** -75.9 75.6 68.1 53.4
1.1 0.9 0.9 1.4
3.7 4.5 6.3 5.6
45.7 37.2 21.9 15.7
25.4 33.0 39.0 30.7
4. 4. 4. 4.
4. i 4. ~
i 4. 4. ~
4. 4. ± 4.
4. ± 4. 4.
3.6 7.0 4.6 5.4**
0.1 0.3 0.1 0.1"*
0.2 0.3** 0.5*** 0.8***
2.1 3.8 2.1"** 0.8***
1.2 2.6** 0.9*** 3.7
15 2 5 4
17 4 7 4
17 4 7 4
17 4 7 4
0.4 ± 0.11 0.6 4. 0.08 1.1 ± 0.27 --
0.4 4. 0.07 1.7 -3=0.04*** 4.7 4. 0.32*** --
83.4 4. 3.9 67.4 4. 2.4** 40.9 4. 3.0*** --
16.0 4. 3.7 32.5 4. 2.4*** 54.5 4. 3.0*** --
4 4 4
2 2 3
4 4 4
4 4 4
N
0.5 0.6 0.5 0.5
0.4 0.7 1.6 1.5
95.5 94.5 86.2 84.5
3.6 4.5 11.6 13.5
%
0.3 0.5 1.7"** 3.3***
4. 4. ± 4.
0.11 0.15 0.08 0.05
4.4-0.05 ± 0.19 4. 0.26*** 4- 0.22***
44. 4. 4.
4. 0.3 4. 0.5 -+- 1.4"** ± 3.1"*
In 100 % 02
17 4 4 4
17 4 7 4
17 4 7 4
17 4 7 4
N
for the slices are based on the total radioactivity in the trichloroacetic acid extract of the slices (see Table ll); for the media the based on the total radioactivity in the amino acids (see Table II). by Student ' t ' test. by Student ' t ' test.
Control 0.04 M Li + 0.10 M Li + 0 . 1 0 M L i + + Na +
Total
Control 1.4 ± 0.3 0.04 M Li ÷ 1.0 i 0.1 0.10 M Li + 0.5 4 . 0 . 1 " * 0.10 M Li + + N a + - -
GABA
Control 3.4 4. 0.2 0.04 M Li + 3.3 ± 0.1 0.10 M Li + 3.0 4. 0.5 0.10MLi ++ Na + --
Aspartic acid
Control 30.1 4- 2.1 0.04 M Li + 14.3 4. 0.8*** 0.10 M L i 3.5 4. 0.4*** 0 . 1 0 M L i + + Na + - -
Glutamine
Control 26.2 4. 1.3 0.04 M Li + 28.3 4. 2.0 0.10 M Li + 33.5 ± 2.0** 0.10 M Li + -- Na +
Glutamic acid
%
%
% N
In air
In 100 % 02
In air N
Me~um
Slices
PER CENT DISTRIBUTION OF RADIOACTIVITY IN AMINO ACIDS*
TABLE I I I
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t.o to
--,.I
2"
GABA
ISOLATED FROM GUINEA P IG
9.7 ± 2.0*
15.5 -4- 0.9 17.84-1.8 14.1 4- 0.9
8.3 -4- 1.0 5.44-0.3* 6.1 4- 1.9
2.5
=9 0.2**
7.9 ± 0.3 5.4 ,4,1.1"* 2.3 4- 0.3**
3.8 =k 0.2 1.7 4-0.1"* 0.52 4- 0.04**
* P < 0.05 by Student ' t ' test. ** P < 0.01 by Student ' t ' test.
0.10 M L i + ÷ Na +
0.10 M Li +
In oxygen Control O.IM M Li +
In air Control 0.04 M Li + O.IOMLi + 0.10 -4- 0.02 0.07 4- 0.01 0.04 4- 0.01 *
0.83 ± 0 . 0 6 * *
0.09 ± 0 . 0 1 "
0.47 -4- 0.02 0.14 4- 0.01 0.58 4- 0.03* 0.08 4- 0.02** 0.91 4- 0.04** 0.10 4- 0.02**
0.58 4- 0.01 0.65 4- 0.05 0.66 4- 0.07
13.9 ± 1.4
17.1 ± 1.7 21.7 ± 2.2 17.34-0.7
13.1 -4- 3.0 6.5 -4- 0.6* 5.7 4- 0.5*
1.3 ± 0.2**
5.7 4- 0.6 3.6 4- 0.7 1.5 4-0.1"*
2.9 -4- 0.6 1.8 4- 0.1 0.78 4- 0.09*
0.18 4- 0.06
0.22 -4- 0.08 0.26 ± 0.13 0.20-4-0.04
0.04 ± 0.03 0.13 4- 0.08 0.43 4- 0.18
Specific activity Relative specific activity glutamic acid x 10 -4 Glutamine Aspartic acid
Specific activity Relative specific activity glutamic acid x 10 -4 Glutamine Aspartic acid GABA
Incubation medium
Slices
0.03 4- 0.01
0.09 -k 0.02 0.07 ± 0.02 0.02 :[: 0.01"
0.03 :t: 0.01 0.04 4- 0.01 0.04 4- 0.01
GABA
4
17 4 7
4 4 4
N
See Table I for incubation conditions. Specific activity is expressed as counts/min/ttmole -4- S.E.M.; relative specific activity is defined as the ratio o f the specific activity of the amino acid to that of glutamic acid. N = number of samples of tissue slices,
SPECIFIC RADIOACTIVITY OF G L U T A M I C A C I D AND RELATIVE SPECIFIC ACTIVITY OF G L U T A M I N E , ASPARTIC ACID AND BRAIN SLICES AND INCUBATION MEDIA
TABLE IV
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210
S, BERL A N D D. 12}. C L A R K E
The specific activity of glutamic acid and glutamine in the slices tends to be similar to that in the corresponding media (Table IV). However, the specific activity of aspartic acid and GABA relative to glutamic acid tends to be lower in the media than in the slices. The specific activity of glutamic acid is greater in oxygen than in air (Table IV). This parallels the increased entry of acetate in the slices in oxygen (Table II). The relative specific activity of glutamine (glutamic acid - 1) is markedly and consistently decreased by Li ~ both in oxygen and in air. In oxygen the relative specific activity of aspartate in the slice is increased by Li ~ while that of G A B A tends to be decreased. DISCUSSION
It has been shown that drugs such as reserpine which release catecholamines and serotonin from their stores in the central nervous system also alter the metabolism of glutamic acid, glutamine, aspartic acid and GABA, amino acids which are associated with the tricarboxylic acid cycle '~. Several studies have suggested that the metabolism of the norepinephrine system in brain is influenced by treatment with lithium saltsZ1,23, 24. Since catecholamines as well as glutamic acid and GABA have been discussed as possible transmitter agents it was decided to study the affect of Li + on the metabolism of these amino acids. This interest was enhanced by the report of DeFeudis and Delgado 9 that the levels of glutamate in mouse brain were decreased by the in rivo administration of LiCI ; the levels of other amino acids were not affected. It was decided to examine the effects of Li + on brain slices since the results would not be influenced by metabolism in other organs. While the leakage of amino acids into the medium usually observed upon incubation of brain slices was stimulated by Li ~ that of glutamate was most affected (Table I). The leakage of glutamate could be counteracted by the use of 100% Oz rather than air as the gas phase. While the latter condition is closer to that which prevails in vivo, the former indicates that the leakage is dependent upon the energy state of the tissue. The sum of the levels of the amino acids in the slices plus media were relatively constant and approximated the values observed in fresh tissue (except for GABA) particularly when incubated in air. In 100~o 02 there was a distinct tendency for the levels of aspartate to decrease and a corresponding increase of glutamate or glutamine. These observations are in keeping with those of Krebs and Bellamy 14 that the oxidation of glucose stimulates the conversion of aspartate to glutamate. While at first glance it would seem that the unaffected levels of total glutamate are in contradiction to the results of DeFeudis and Delgado 9 this is not necessarily so. It is probable that Li ÷ stimulates the leakage of glutamate from brain into the cerebrospinal fluid or blood. This is suggested by observation that while the levels of amino acids in the media increase, those in the slices decrease. We also find a decrease in the slice level of glutamine but we cannot compare these values with those of DeFeudis and Delgado 9 since their glutamine fraction contained other amino acids. We also find a decrease in the slice level of aspartate which they did not observe. While DeFeudis and Delgado 9 reported a 1 0 - 2 0 ~ decrease in the entrance of Brain Research, 36 (1972) 203-213
EFFECTS OF LI + ON METABOLISM OF BRAIN NEUROMEDIATORS
211
label from D-[6-14C]glucose into the amino acids there was no selective effect on the labeling of any o f the amino acids. We decided to use [1-14C]acetate since previous experience had shown that this precursor preferentially labels a pool of glutamate active in glutamine formation 4. The decrease in the amount of labeled acetate which entered the slices in the presence of lithium (Table II) indicates that this ion interferes with the active transport of acetate presumably by affecting the N a + - K + pump mechanism4,10. Further support for the idea that entry of acetate is energy dependent is shown by the results obtained using 100 ~ 02 where there is an approximately 2-fold increase in the amount of radioactivity taken up by slices as compared with those incubated in air. The general pattern for the radioactivity incorporated into amino acids parallels the entry of acetate (Table II), however, the distribution of radioactivity in the individual amino acids showed specific effects (Table III). The Li + decreased the percentage of radioactivity in the sum of the 4 amino acids particularly in the presence of air. Most striking was the decrease in the amount of radioactivity in glutamine while that in glutamic acid tended to increase (Table III). This inhibition of the labeling of glutamine coald be due to inhibition of glutamine synthetase. However, it has been shown that the addition of ouabain to, or the omission of Ca 2+ from, the medium I or the addition of fluoroacetate or fluorocitrate to the medium 7 all have a similar pattern of affects to those described here, e.g., a decrease in the entry of acetate into the slices and the formation of labeled glutamine. In addition, a specific inhibition of glutamine synthetase by ouabain 11 or by fluorocitrate 17 has been ruled out. A second hypothesis is that the pool of ATP required for the operation of the N a + - K + pump is either the same or closely related to the pool of ATP required for glutamine synthesis. Presently available experimental evidence does not allow further evaluation of this idea. A third hypothesis which would not necessarily exclude the above would be that a small pool of glutamate (transmitter glutamate?) formed at one site must be transported to another site for conversion to glutamine. The observations reported here indicate an effect on the active transport system similar to that of ouabain. In addition, Ca 2+, a cation essential for synaptic transmission 5,11,13, is also required for glutamine formation in brain tissue. Although metabolic inactivation of a transmitter is not essential, it can be postulated for glutamate. If we were to suppose that glutamate released by synaptic transmission entered the postsynaptic membrane with the influx of Na ÷ it would encounter glutamine synthetase concentrated in the endoplasmic reticulum and be converted to glutamine, a relatively inactive pharmacological agent. A number of investigators have demonstrated a relationship between the cellular influx of glutamate and Na+lS,ls, 19. Another possible buffer zone for the inactivation of released glutamate would be the glia. Trachtenberg and Pollen 26 have suggested that the glial cells may act as a buffer of the immediate extraneural space at areas of synaptic contact against increases in external K + concentration that accompany postsynaptic and spike activity. In line with this suggestion is our report that increasing the concentration of K + in the medium markedly stimulates the labeling of glutamine from [1-14C]acetate as well as from other labeled precursors3, 4. Brain Research, 36 (1972) 203-213
212
S. BERL AND l). D. CLARK[:
In addition, it has been reported that brain slices bathed in medium in which Li ' is substituted for Na*, lose K ÷ to the same extent as that found in brain slices under anaerobic conditions 2°. Li ÷ also inhibits the uptake of K + by brain slices previously depleted of K ÷ by anaerobisislL If Li ÷ were to inhibit the reuptake of glutamate it presumably would be free to diffuse out into the medium as reported here or out into the cerebrospinal fluid in vivo. This would be consistent with the decreased levels of mouse brain glutamate reported by DeFeudis and Delgado 9. It is tempting to suggest that this depletion of 'transmitter glutamate' is associated with the therapeutic effects of Li + treatment. SUMMARY
Guinea pig brain slices were incubated in media in which Li ÷ replaced part of the Na ÷. Leakage of glutamic acid, glutamine, aspartic acid and GABA into the media were increased markedly; glutamic acid was most affected. The uptake of [l-14C]acetate was decreased in the presence of Li ÷. O f the amino acids related to the citric acid cycle the labeling of glutamine was most affected. The presence of 100 ~ O~ counteracts in part the Li + effects. The results were interpretable on the basis of the compartmentation of citric acid cycle metabolism in brain. It is suggested that the pool of 'transmitter' glutamate is related to the small pool of glutamate which actively labels glutamine. ACKNOWLEDGEMENTS
This work was supported in part by Grants NS-04064, NS-07890 and Research Career Award 5K 3-NS-5177 (S.B.) from the National Institute of Neurological Diseases and Stroke and by the Clinical Center for Research in Parkinson's and Allied Diseases, NS-05184. We thank Iren Tar and Yolanda Veliz for their technical assistance.
REFERENCES 1 BERL,S., CLARKE,D. D., ANDNICKLAS,W. J., Compartmentation of citric acid cyclemetabolism in brain slices, J. Neurochem., 17 (1970) 999-1007. 2 BERL, S., AND FRIGYESI,T. L., Effect of reserpine on the turnover of glutamate, glutamine, aspartate and GABA labeled with [1-14C]acetatein caudate nucleus, thalamus and sensorimotor cortex (cat), Brain Research, 14 (1969) 683-695. 3 BERL,S., NICKLAS,W. J., AND CLARKE,D. O., Compartmentation of glutamic acid metabolism in brain slices, J. Neurochem., 15 (1968) 131-140. 4 BERL,S., NICKLAS,W. J., AND CLARKE, D. O., Compartmentation of citric acid cycle metabolism in brain: Labelling of glutamate, glutamine, aspartate and GABA by several radioactive tracer metabolites, J. Neurochem., 17 (1970) 1009-1015. 5 BLAUSTEIN,M. P., Preganglionic stimulation increases calcium uptake by sympathetic ganglia, Science, 172 (1971) 391-393. 6 CHENG, S.-C., AND MELA, P., CO2 fixation in the nervous system. II. Environmental effects of COs fixation in lobster nerve, J. Neuroehem., 13 (1966) 281-287. 7 CLARKE, D. D., NICKLAS,W. J., AND BERL, S., Tricarboxylic acid cycle metabolism in brain. Brain Research, 36 (1972) 203-213
EFFECTS OF LI + ON METABOLISM OF BRAIN NEUROMEDIATORS
8 9 10 11 12 13 14 15 16 17 18 19 20
21 22 23 24 25 26
213
Effect of fluoroacetate and fluorocitrate on the labelling of glutamate, aspartate~ glutamine and 7-aminobutyrate, Biochem. J., 120 (1970) 345-351. CURTIS, D. R., ANDJOHNSTON,G. A. R., Amino acid transmitters. In A. LAJTHA(Ed.), Handbook of Neurochemistry, Vol. 4, Plenum Press, New York, 1970, pp. 115-134. DEFEUDIS, E. V., AND DELGADO, J. M. R., Effects of lithium on amino-acids in mouse brain in vivo, Nature (Lond.), 225 (1970) 749-750. GERSrlON, S., Lithium in mania, Clin. Pharmacol. exp. Ther., 11 (1970) 168-187. GONDA, O., AND QUASTEL, J. H., Effects of ouabain on cerebral metabolism and transport mechanisms in vitro, Biochem. J., 84 (1962) 394-406. ISRAEL,Y., KALANT,H., ANDLEBLANC,A. E., Effects of lower alcohols on potassium transport and microsomal adenosine triphosphatase activity of rat cerebral cortex, Biochem. J., 100 (1966) 27-33. KATZ, B., AND MILEDI, R., The release of acetylcholine from nerve endings by graded electric pulses, Proc. roy. Sac. B, 167 (1967) 23-38. KREBS, H. H., AND BELLAMY,D., Interconversion of glutamic acid and aspartic acid in respiring tissues, Biochem. J., 75 (1960) 523-529. MCILWAIN, H., HORVEY,J. A., AND RODRIGUEZ,G., Tetrodotoxin on the sodium and other ions of cerebral tissues, excited electrically and with glutamate, J. Neurochem., 16 (1969) 363-370. MCILWAIN, H., AND RODNIGHT, R., Practical Neurochemistry, Little, Brown, Boston, Mass., 1962, p. 109. NICKLAS, W. J., Compartmentation of Amino Acid Metabolism in Brain, Ph. D. Thesis, Fordham University, New York, 1969, p. 61. OKAMOTO,t . , AND QUASTEL,J. H., Water uptake and energy metabolism in brain slices from the rat, Biochem. J., 120 (1970) 25-36. ORAMOTO,K., AND QUASTEL,J. H., Tetrodotoxin-sensitive uptake of ions and water by slices of rat brain in vitro, Biochem. J., 120 (1970) 37-47. PAPPIUS, H. M., ROSENFELD,M., JOHNSON,D. M., AND ELLIOTT, K. A. C., Effects of sodium-free media upon the metabolism and the potassium and water contents of brain slices, Canad. J. Biochern. Physiol., 36 (1958) 217-226. SCHANBERG,S. M., SCHILDKRAUT,J. J., AND KOPIN, I. J., Effects of psychoactive drugs on [aH]norepinephrine metabolism in brain, Biochem. Pharmacol., 16 (1967) 393-399. SCHILDKRAUT,J. J., AND GERSnON, E. S., Antidepressants and related drugs. In A. LAJTHA(Ed.), Handbook of Neurochemistr.v, VoL 6, Plenum Press, New York, 1971, pp. 357-386. SCHILDKRAUT,J. J., LOGUE, M. A., AND DODGE, G. A., The effects of lithium salts on the turnover and metabolism of norepinephrine in rat brain, Psychopharmacologia (BerL), 14 (1969) 135-141. SCHILDKRAUT,J. J., SCHANBERG,S. M., AND KOPIN, L J., The effect of lithium on [aH]norepinephrine metabolism in brain, Life Sci., 5 (1966) 1479-1483. Scnou, M., Lithium. In A. LAJTHA (Ed.), Handbook of Neurochemistry, Vol. 6, Plenum Press, New York, 1971, pp. 387-393. TRACHTENBERG,M. C., AND POLLEN, O. A., Neuroglia: Biophysical properties and physiologic function, Science, 167 (1970) 1248-1252.
Brain Research, 36 (1972) 203-213
203
EFFECTS OF LI + ON T H E METABOLISM IN BRAIN OF G L U T A M A T E , G L U T A M I N E , A S P A R T A T E A N D GABA F R O M [1-14C]ACETATE I N VITRO
S. B E R L AND D . D .
CLARKE
Department of Neurology, Collegeof Physicians and Surgeons, Columbia University, New York, N. Y. 10032 and Chemistry Department, Fordham University, Bronx, N.Y. 10458 (U.S.A.)
(Accepted July 22nd, 1971)
INTRODUCTION An effect of lithium which has been suggested to explain its usefulness in the treatment of mania is that it changes the rate of metabolism of brain neuromediators. Norepinephrine and serotonin have been extensively investigated in this relationship~t,2a, 24. The topic has been reviewed recentlyl°,~L The effect of lithium has also been reviewed~L It has been frequently suggested that certain amino acids may also function as neuromodulators or transmitter substances a. This prompted us to examine the effects of lithium on the metabolism of glutamic acid, glutamine, aspartic acid and GABA in guinea pig brain slices. Previous work had indicated that replacement o f Na + by Li + decreased the incorporation of radioactivity from NaH14COa into citric acid cycle intermediates and associated amino acids in lobster nerve 6. While this work was in progress DeFeudis and Delgado 9 reported that lithium when injected into mice produced a decrease in the level o f brain glutamate. The results reported here indicate that replacement of Na + with Li + does greatly effect the metabolism of the glutamate-glutamine system. METHODS Preparation o f brain slices
Guinea pigs were decapitated, their brains rapidly removed and placed in a moist chamber (Petri dish containing filter paper wet with medium). Slices o f cerebral cortex were cut approximately 0.3 mm thick using a Stadie-Riggs blade and a guide as described by McIlwain and Rodnight 16. The slices were weighed to the nearest 0.1 mg as soon as they were prepared using a Roller-Smith torsion balance. For each experiment approximately 75 mg slices were used. Incubation conditions were as described previously a with the exception that the preincubation period was 15 min and the incubation period was 20 min for all experiments. The medium used was modified Krebs-Ringer phosphate saturated with oxygen and containing 1.55 m M Brain Research, 36 (1972) 203-213
I
b~ PIG BRAIN SLICES, INCUBATION
AND PREINCUBATION
MEDIA
(pmoles/g tissue wet weight -s- S.E.M.)
2.65 ± 0.13 2.54 ± 0.22 1.80 ± 0.33*
Glutamine Control 0.04 M Li" 0.10 M L i ÷ 0.1 M L i ~- N a "
Aspartic acid Control 2.00 ~ 0.18 0.04 M Li + 1.51 • 0.12" 0.10 M Li + 1.13 ± 0.18"* 0.10 M Li + =- Na =
8.68 ± 0.38 8.18 ± 0.t8 6.19 ± 0.07**
Glutamic acM Control 0.04 M Li * 0.10 M Li 0.1 M L i + - - N a +
0.09 i 0.06 0.11 ± 0.06 0.20 + 0.08
0.96 -- 0.07 1.18 ~ 0.04* 1.45 + 0.07**
0.28 ~.~ 0.05 0.82 ± 0.04** 1.67±0.11"*
0.28 £ 0.06 0.41 -i 0.09 0.46 ± 0.12
1.41 ~_0.16 1.63 4- 0.14 2.27 ± 0.20*
1.65 ± 0.15 2.42 ± 0.12"* 2.87 -~- 0.10"*
2.37 0.12 2.02 ± 0.08 1.79 r_ 0.19"
5.01 -- 0.27 5.34 ± 0.22 5.52 - 0.35
10.61 ~ 0.29 11.23 ± 0.21 10.73 ± 0.23
Total
± ± ± ±
0.16 0.41 0.24** 0.34**
2.41 1.93 1.19 1.51
~0.05 :R0.11** -0.05"* -:--0:05"*
1.85 :± 0.12 1.79 ± 0.15 1.71 4 0.17 1.64~0.17
7.85 8.36 6.76 6.75
± ± ± ±
0.03 0.03 0.04 0.13
0.12 0 . 0 1 0 . 0 5 - 0.01" 0.23 : 0.04** 0.21 { 0.01"*
1.18 ± 0.08 1.56 :~ 0.08* 1.64 ~ 0.08** 1.47±0.20
0.24 0.26 0.31 0.35
blcubation medium
Slices
Preincubation medium
Slices
Incubation medium
In 100 % oxygen
In air
± ± ± ±
0.09 0.23 0.15"* 0.14"*
0.18 0 . 0 2 0.26z-0.06 0.52 '--0.06** 0.21 ± 0 . 0 6
1.82 ± 0.10 2.21 ! 0.52 2.73 ± 0.32** 3.20±0.18"*
0.74 1.15 2.01 1.77
Preincubation medium
0.15 0.50* 0.38 0.41
~ 0.25 i 0.46 ± 0.43* ±0.35*
± ± ± ±
2.70-_0.06 2.24 . 0.07 '~* 1.93 ! 0.09** i.93 --0.07"*
4.85 5.55 6.07 6.11
8.87 9.77 9.08 8.86
Total
Guinea pig brain slices (approximately 75 rag) were preincubated for 10 min at 37-C in 2.5 ml Krebs Ringer phosphate m e d i u m containing 55 m M glucose and 1.55 m M Ca 2 . They were then transferred to fresh medium, 2 / t C i of [l-NC]acetate (58/~Ci//maole) added and incubated for 20 min at 37°C. Preincubation and incubation were carried out either in air or 100%/, O2. Where Li ~ was added, an equivalent a m o u n t of NaCI was replaced by LiC1 except in the last set of experiments where 0.1 M LiC1 was added without reduction of Na + (0.1 M Li ~ ~ Na+). N - 4 for all samples except for controls with t00~o O2 (N = 17) and 0.1 M Li + in I00°/~ O2 (N = 7).
LEVELS OF A M I N O A C I D S I N G U I N E A
TABLE I
7:
r>
r>
Lo
I Fo
to
~O --d
.q'
GABA
* P < 0.05. ** P < 0.01 by Student 't' test.
Control 17.34 4- 1.04 0.04 M Li ÷ 15.83 4- 0.73 0.10MLi + 12.134-0.73"* 0.10 M Li + ÷ N a +
Sum o f the amino acids
Control 4.01 4- 0.35 0.04 M Li + 3.60 i 0.27 0.10 M Li + 3.09 4- 0.15" 0.10 M Li + + N a + 1.73 4- 0.23 2.57 4- 0.19" 4.194-0.35"*
0.40 4- 0.05 0.46 ± 0.05 0.87 4- 0.09**
3.76 4- 0.43 5.01 ± 0.43* 6.544-0.47**
0.42 4- 0.06 0.55 :t: 0.08 0.94 4- 0.05**
22.81 4- 1.01 23.20 4- 0.83 22.934-0.96
4.82 4- 0.33 4.61 ± 0.32 4.89 4- 0.19
14.53 14.88 11.51 13.38
2.42 2.80 1.85 3.48 4- 0.50 4- 0.81 4- 0.80 4- 0.83
1.93 2.40 2.72 2.69
4444-
0.15 0.20 0.22 0.39
4- 0.17 0.39 4- 0.03 4- 0.14 0.53 4- 0.08 4- 0.34 0.54 4- 0.06* 4- 0.27** 0.66 4- 0.05** 3.40 4.38 6.34 6.43
0.66 0.76 1.08 1.25 ± 444-
0.27 0.96 0.73** 0.57**
4- 0.06 4- 0.15 ± 0.19"* ± 0.19"* 19.89 21.64 20.55 22.30
3.47 4.08 3.47 5.40 i 444-
± 4441.32 1.30 1.35 1.26
0.86 0.27 0.45 0.43**
bo O
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S. B E R L A N D 1). ['). C L A R K [
Ca 2+ and 55 mMglucose. In the experiments labeled Li ~-, LiCI replaced an equal molar concentration of NaCI. In the experiments labeled Li ~ + N a ' the LiCI was added to the Krebs-Ringer medium without decreasing the NaCI concentration. In the experiments labeled '100 ~o oxygen', this gas was constantly bubbled through the medium during both the preincubation and incubation periods. In the experiments labeled 'in air' the tubes were merely exposed to the air during the preincubation and incubation periods. Zero time was taken as the point at which 2 ,uCi of [l-14C]acetate (58 #Ci/#mole) were added to the incubation medium. The slices were prepared at room temperature and incubated at 37°C. At the end of the preincubation period and at the end of the experiments 2 ml of medium was rapidly pipetted into 0.2 ml of 50",~i (w/v) TCA and the supernatant analyzed as previously described 1. Glutamic acid, glutamine, aspartic acid and GABA were isolated from slices and media, determined and counted 3. RESULTS
The effect of added Li + on the levels of the amino acids in the slices and media is described in Table I. The presence of this ion causes the levels of each of the 4 amino acids to decrease in the slices and to increase in the incubation and preincubation media. This leakage of amino acids from the slices is most pronounced in the preincubation media. In the presence of 100~ oxygen the leakage is less than in air in both the preincubation and incubation media. Even in the controls glutamine leaks out to a much greater extent than do the other amino acids and it is increased by Li +. However, the greatest effect of this cation appears to be on the leakage of glutamate from slices incubated in air. O2 counteracts this leakage of glutamate; in contrast 02 does not appear to affect the leakage of the other amino acids. 'In air' the total quantity of each amino acid in the slice, preincubation plus incubation media, is unaffected by Li ÷ and, except for GABA, resembles the levels observed in vivo. 'In oxygen' the total glutamate level is unaffected while glutamine increases and aspartate decreases. The sum of the amino acids do not differ significantly under the various conditions. The presence of LiC1 plus NaCI, however, greatly elevates the level o f GABA in the slices. In 100 ~o 02 the slices take up almost twice as much radioactive acetate as in air (Table II). Li ÷ decreases the amount of radioactivity taken up by the slices. In air this is evident even at 0.04 M Li ÷ while in 100~ oxygen the decrease is significant only at 0.1 M Li ~-. A corresponding decrease in the conversion of [l-14C]acetate to the amino acids is observed. Examination of the distribution of radioactivity in the individual amino acids shows that the labeling of glutamine is severely reduced in the presence of Li ÷ (Table III). This effect is greater in air than in 100~ oxygen. While the per cent of radioactivity in the glutamine is decreased that in the glutamic acid and aspartic acid is increased. In air this increase of radioactivity is most evident in the incubation medium into which the amino acids have leaked. The percentage of radioactivity in GABA is relatively unaffected by Li ÷. Brain Research, 36 (1972) 203-213
I,o
I
IxJ
~D ,-o
2.73 4, 0.24 1.59 ± 0.10"* 1.37 4- 0.20**
35.8 4- 1.9 35.0 4- 2.0 39.3 -t- 1.1
* P < 0.05 by S t u d e n t ' t ' test. ** P < 0.01 by S t u d e n t ' t ' test.
TOTAL RADIOACTIVITY/G TISSUE IN AMINO ACIDS ( c o u n t s / m i n 4. S.E.M.) x 10 -6 Control 1.66 -4- 0.16 0.38 4. 0.05 0.04 M Li + 0.74 =/= 0.03** 0.20 4- 0.01 * 0.10 M Li + 0.48 4. 0.14"* 0.15 4- 0.01"* 0.1 M Li ÷ + N a +
Control 0.04 M Li + 0.10 M Li + 0.1 M Li + -t- N a +
4 4 4
4 4 4
3.70 3.66 1.67 1.14
4.76 4.42 2.43 2.09 4- 0.25 4- 0.69 -t- 0.05** 4. 0.22**
4- 0.19 4" 0.47 4- 0.06** 4- 0.24**
Slices
N
Slices
Incubation medium
In 100 % 02
In air
See Table I for i n c u b a t i o n conditions.
TOTAL RADIOACTIVITY/G TISSUE IN TRICHLOROACETIC ACID EXTRACT ( c o u n t s / m i n 4- S.E.M.) × 10 6
T A B L E II
0.09 0.18 0.02** 0.09**
0.90 2.2 2.1 2.6 4444-
444. 4, 1.04 1.25 0.46 0.33
33.4 33.2 33.9 37.9
Incubation medium
17 4 7 4
17 4 7 4
N
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t,~
* Calculations percentages are ** P < 0,005 *** P < 0.001
61.1 4. 3.9 46.9 4. 3.0** 40.5 4. 3.0** -75.9 75.6 68.1 53.4
1.1 0.9 0.9 1.4
3.7 4.5 6.3 5.6
45.7 37.2 21.9 15.7
25.4 33.0 39.0 30.7
4. 4. 4. 4.
4. i 4. ~
i 4. 4. ~
4. 4. ± 4.
4. ± 4. 4.
3.6 7.0 4.6 5.4**
0.1 0.3 0.1 0.1"*
0.2 0.3** 0.5*** 0.8***
2.1 3.8 2.1"** 0.8***
1.2 2.6** 0.9*** 3.7
15 2 5 4
17 4 7 4
17 4 7 4
17 4 7 4
0.4 ± 0.11 0.6 4. 0.08 1.1 ± 0.27 --
0.4 4. 0.07 1.7 -3=0.04*** 4.7 4. 0.32*** --
83.4 4. 3.9 67.4 4. 2.4** 40.9 4. 3.0*** --
16.0 4. 3.7 32.5 4. 2.4*** 54.5 4. 3.0*** --
4 4 4
2 2 3
4 4 4
4 4 4
N
0.5 0.6 0.5 0.5
0.4 0.7 1.6 1.5
95.5 94.5 86.2 84.5
3.6 4.5 11.6 13.5
%
0.3 0.5 1.7"** 3.3***
4. 4. ± 4.
0.11 0.15 0.08 0.05
4.4-0.05 ± 0.19 4. 0.26*** 4- 0.22***
44. 4. 4.
4. 0.3 4. 0.5 -+- 1.4"** ± 3.1"*
In 100 % 02
17 4 4 4
17 4 7 4
17 4 7 4
17 4 7 4
N
for the slices are based on the total radioactivity in the trichloroacetic acid extract of the slices (see Table ll); for the media the based on the total radioactivity in the amino acids (see Table II). by Student ' t ' test. by Student ' t ' test.
Control 0.04 M Li + 0.10 M Li + 0 . 1 0 M L i + + Na +
Total
Control 1.4 ± 0.3 0.04 M Li ÷ 1.0 i 0.1 0.10 M Li + 0.5 4 . 0 . 1 " * 0.10 M Li + + N a + - -
GABA
Control 3.4 4. 0.2 0.04 M Li + 3.3 ± 0.1 0.10 M Li + 3.0 4. 0.5 0.10MLi ++ Na + --
Aspartic acid
Control 30.1 4- 2.1 0.04 M Li + 14.3 4. 0.8*** 0.10 M L i 3.5 4. 0.4*** 0 . 1 0 M L i + + Na + - -
Glutamine
Control 26.2 4. 1.3 0.04 M Li + 28.3 4. 2.0 0.10 M Li + 33.5 ± 2.0** 0.10 M Li + -- Na +
Glutamic acid
%
%
% N
In air
In 100 % 02
In air N
Me~um
Slices
PER CENT DISTRIBUTION OF RADIOACTIVITY IN AMINO ACIDS*
TABLE I I I
Oo
t.o to
--,.I
2"
GABA
ISOLATED FROM GUINEA P IG
9.7 ± 2.0*
15.5 -4- 0.9 17.84-1.8 14.1 4- 0.9
8.3 -4- 1.0 5.44-0.3* 6.1 4- 1.9
2.5
=9 0.2**
7.9 ± 0.3 5.4 ,4,1.1"* 2.3 4- 0.3**
3.8 =k 0.2 1.7 4-0.1"* 0.52 4- 0.04**
* P < 0.05 by Student ' t ' test. ** P < 0.01 by Student ' t ' test.
0.10 M L i + ÷ Na +
0.10 M Li +
In oxygen Control O.IM M Li +
In air Control 0.04 M Li + O.IOMLi + 0.10 -4- 0.02 0.07 4- 0.01 0.04 4- 0.01 *
0.83 ± 0 . 0 6 * *
0.09 ± 0 . 0 1 "
0.47 -4- 0.02 0.14 4- 0.01 0.58 4- 0.03* 0.08 4- 0.02** 0.91 4- 0.04** 0.10 4- 0.02**
0.58 4- 0.01 0.65 4- 0.05 0.66 4- 0.07
13.9 ± 1.4
17.1 ± 1.7 21.7 ± 2.2 17.34-0.7
13.1 -4- 3.0 6.5 -4- 0.6* 5.7 4- 0.5*
1.3 ± 0.2**
5.7 4- 0.6 3.6 4- 0.7 1.5 4-0.1"*
2.9 -4- 0.6 1.8 4- 0.1 0.78 4- 0.09*
0.18 4- 0.06
0.22 -4- 0.08 0.26 ± 0.13 0.20-4-0.04
0.04 ± 0.03 0.13 4- 0.08 0.43 4- 0.18
Specific activity Relative specific activity glutamic acid x 10 -4 Glutamine Aspartic acid
Specific activity Relative specific activity glutamic acid x 10 -4 Glutamine Aspartic acid GABA
Incubation medium
Slices
0.03 4- 0.01
0.09 -k 0.02 0.07 ± 0.02 0.02 :[: 0.01"
0.03 :t: 0.01 0.04 4- 0.01 0.04 4- 0.01
GABA
4
17 4 7
4 4 4
N
See Table I for incubation conditions. Specific activity is expressed as counts/min/ttmole -4- S.E.M.; relative specific activity is defined as the ratio o f the specific activity of the amino acid to that of glutamic acid. N = number of samples of tissue slices,
SPECIFIC RADIOACTIVITY OF G L U T A M I C A C I D AND RELATIVE SPECIFIC ACTIVITY OF G L U T A M I N E , ASPARTIC ACID AND BRAIN SLICES AND INCUBATION MEDIA
TABLE IV
o
t-,J
0
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o
+
0
210
S, BERL A N D D. 12}. C L A R K E
The specific activity of glutamic acid and glutamine in the slices tends to be similar to that in the corresponding media (Table IV). However, the specific activity of aspartic acid and GABA relative to glutamic acid tends to be lower in the media than in the slices. The specific activity of glutamic acid is greater in oxygen than in air (Table IV). This parallels the increased entry of acetate in the slices in oxygen (Table II). The relative specific activity of glutamine (glutamic acid - 1) is markedly and consistently decreased by Li ~ both in oxygen and in air. In oxygen the relative specific activity of aspartate in the slice is increased by Li ~ while that of G A B A tends to be decreased. DISCUSSION
It has been shown that drugs such as reserpine which release catecholamines and serotonin from their stores in the central nervous system also alter the metabolism of glutamic acid, glutamine, aspartic acid and GABA, amino acids which are associated with the tricarboxylic acid cycle '~. Several studies have suggested that the metabolism of the norepinephrine system in brain is influenced by treatment with lithium saltsZ1,23, 24. Since catecholamines as well as glutamic acid and GABA have been discussed as possible transmitter agents it was decided to study the affect of Li + on the metabolism of these amino acids. This interest was enhanced by the report of DeFeudis and Delgado 9 that the levels of glutamate in mouse brain were decreased by the in rivo administration of LiCI ; the levels of other amino acids were not affected. It was decided to examine the effects of Li + on brain slices since the results would not be influenced by metabolism in other organs. While the leakage of amino acids into the medium usually observed upon incubation of brain slices was stimulated by Li ~ that of glutamate was most affected (Table I). The leakage of glutamate could be counteracted by the use of 100% Oz rather than air as the gas phase. While the latter condition is closer to that which prevails in vivo, the former indicates that the leakage is dependent upon the energy state of the tissue. The sum of the levels of the amino acids in the slices plus media were relatively constant and approximated the values observed in fresh tissue (except for GABA) particularly when incubated in air. In 100~o 02 there was a distinct tendency for the levels of aspartate to decrease and a corresponding increase of glutamate or glutamine. These observations are in keeping with those of Krebs and Bellamy 14 that the oxidation of glucose stimulates the conversion of aspartate to glutamate. While at first glance it would seem that the unaffected levels of total glutamate are in contradiction to the results of DeFeudis and Delgado 9 this is not necessarily so. It is probable that Li ÷ stimulates the leakage of glutamate from brain into the cerebrospinal fluid or blood. This is suggested by observation that while the levels of amino acids in the media increase, those in the slices decrease. We also find a decrease in the slice level of glutamine but we cannot compare these values with those of DeFeudis and Delgado 9 since their glutamine fraction contained other amino acids. We also find a decrease in the slice level of aspartate which they did not observe. While DeFeudis and Delgado 9 reported a 1 0 - 2 0 ~ decrease in the entrance of Brain Research, 36 (1972) 203-213
EFFECTS OF LI + ON METABOLISM OF BRAIN NEUROMEDIATORS
211
label from D-[6-14C]glucose into the amino acids there was no selective effect on the labeling of any o f the amino acids. We decided to use [1-14C]acetate since previous experience had shown that this precursor preferentially labels a pool of glutamate active in glutamine formation 4. The decrease in the amount of labeled acetate which entered the slices in the presence of lithium (Table II) indicates that this ion interferes with the active transport of acetate presumably by affecting the N a + - K + pump mechanism4,10. Further support for the idea that entry of acetate is energy dependent is shown by the results obtained using 100 ~ 02 where there is an approximately 2-fold increase in the amount of radioactivity taken up by slices as compared with those incubated in air. The general pattern for the radioactivity incorporated into amino acids parallels the entry of acetate (Table II), however, the distribution of radioactivity in the individual amino acids showed specific effects (Table III). The Li + decreased the percentage of radioactivity in the sum of the 4 amino acids particularly in the presence of air. Most striking was the decrease in the amount of radioactivity in glutamine while that in glutamic acid tended to increase (Table III). This inhibition of the labeling of glutamine coald be due to inhibition of glutamine synthetase. However, it has been shown that the addition of ouabain to, or the omission of Ca 2+ from, the medium I or the addition of fluoroacetate or fluorocitrate to the medium 7 all have a similar pattern of affects to those described here, e.g., a decrease in the entry of acetate into the slices and the formation of labeled glutamine. In addition, a specific inhibition of glutamine synthetase by ouabain 11 or by fluorocitrate 17 has been ruled out. A second hypothesis is that the pool of ATP required for the operation of the N a + - K + pump is either the same or closely related to the pool of ATP required for glutamine synthesis. Presently available experimental evidence does not allow further evaluation of this idea. A third hypothesis which would not necessarily exclude the above would be that a small pool of glutamate (transmitter glutamate?) formed at one site must be transported to another site for conversion to glutamine. The observations reported here indicate an effect on the active transport system similar to that of ouabain. In addition, Ca 2+, a cation essential for synaptic transmission 5,11,13, is also required for glutamine formation in brain tissue. Although metabolic inactivation of a transmitter is not essential, it can be postulated for glutamate. If we were to suppose that glutamate released by synaptic transmission entered the postsynaptic membrane with the influx of Na ÷ it would encounter glutamine synthetase concentrated in the endoplasmic reticulum and be converted to glutamine, a relatively inactive pharmacological agent. A number of investigators have demonstrated a relationship between the cellular influx of glutamate and Na+lS,ls, 19. Another possible buffer zone for the inactivation of released glutamate would be the glia. Trachtenberg and Pollen 26 have suggested that the glial cells may act as a buffer of the immediate extraneural space at areas of synaptic contact against increases in external K + concentration that accompany postsynaptic and spike activity. In line with this suggestion is our report that increasing the concentration of K + in the medium markedly stimulates the labeling of glutamine from [1-14C]acetate as well as from other labeled precursors3, 4. Brain Research, 36 (1972) 203-213
212
S. BERL AND l). D. CLARK[:
In addition, it has been reported that brain slices bathed in medium in which Li ' is substituted for Na*, lose K ÷ to the same extent as that found in brain slices under anaerobic conditions 2°. Li ÷ also inhibits the uptake of K + by brain slices previously depleted of K ÷ by anaerobisislL If Li ÷ were to inhibit the reuptake of glutamate it presumably would be free to diffuse out into the medium as reported here or out into the cerebrospinal fluid in vivo. This would be consistent with the decreased levels of mouse brain glutamate reported by DeFeudis and Delgado 9. It is tempting to suggest that this depletion of 'transmitter glutamate' is associated with the therapeutic effects of Li + treatment. SUMMARY
Guinea pig brain slices were incubated in media in which Li ÷ replaced part of the Na ÷. Leakage of glutamic acid, glutamine, aspartic acid and GABA into the media were increased markedly; glutamic acid was most affected. The uptake of [l-14C]acetate was decreased in the presence of Li ÷. O f the amino acids related to the citric acid cycle the labeling of glutamine was most affected. The presence of 100 ~ O~ counteracts in part the Li + effects. The results were interpretable on the basis of the compartmentation of citric acid cycle metabolism in brain. It is suggested that the pool of 'transmitter' glutamate is related to the small pool of glutamate which actively labels glutamine. ACKNOWLEDGEMENTS
This work was supported in part by Grants NS-04064, NS-07890 and Research Career Award 5K 3-NS-5177 (S.B.) from the National Institute of Neurological Diseases and Stroke and by the Clinical Center for Research in Parkinson's and Allied Diseases, NS-05184. We thank Iren Tar and Yolanda Veliz for their technical assistance.
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