Studies on GABA and protein synthesis

Brain Research, 59 (1973) 339-348

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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

STUDIES ON GABA A N D P R O T E I N SYNTHESIS

S. ROBERT SNODGRASS* Department of Neurology, Children's Hospital Medical Center, Harvard Medical School, Boston, Mass. 02115 (U.S.A.)

(Accepted February 9th, 1973)

SUMMARY The effect of y-aminobutyric acid (GABA) on protein synthesis in vitro by slices and homogenates of guinea pig brain was studied. GABA was shown to significantly increase the incorporation of L-[1-14C]leucine, alanine, and phenylalanine into protein by slices and homogenates of cortex and forebrain. The stimulatory effect of GABA upon protein synthesis was greater in slices than homogenates, and could not be demonstrated in cerebellar slices or homogenates. GABA stimulation of protein synthesis was demonstrable only when additional non-radioactive amino acids were present in the incubation medium. Tissue which had been exposed to G A B A during incubation showed a greater percentage of heavy polysomes, as judged by ultra-violet absorption, than did tissue from control flasks. GABA did not change the incorporation of phenylalanine into polyphenylalanine in response to polyuridylic acid. L-Noradrenaline was shown to stimulate the incorporation of L-[1-14C]leucine into protein, and this increase could be blocked by dichloroisoproterenol.

INTRODUCTION In 1952, Borsook et al. 5 demonstrated that incorporation of 14C radioactive amino acids into protein by rabbit reticulocytes was stimulated by the addition of plasma or additional non-radioactive amino acids to the incubation medium. Amino acids varied in their ability to enhance the incorporation of [14C]glycine, histidine, leucine or lysine into protein, with a mixture of histidine, valine, phenylalanine and * Temporary address: Department of Pharmacology, Cambridge University, Cambridge, Great Britain.

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tyrosine proving as effective as a mixture of 18 amino acids. This same stimulation of incorporation of radioactive amino acid into protein by the addition of other amino acids was also shown for cell-free microsomal systems derived from reticulocytes by Schweet et al. 27, who used the same amino acid mixture as Borsook. Matthei and Nirenberg is found that protein incorporation by E. coli ribosomes was decreased when a mixture of 20 amino acids was omitted. However, this was only true for dialyzed preparations, and subsequent studies failed to demonstrate the stimulatory effect19,2a as had been the case for Borsook in earlier studies on the rat diaphragm 4. Folbergrova s found that a mixture of non-radioactive amino acids inhibited the incorporation of radioactive amino acid into protein by brain slices. She subsequently showed that glutamic and aspartic acids were potent inhibitors of brain protein synthesis in vitro, and that other amino acids were weakly inhibitory. A mixture of amino acids was found to inhibit protein synthesis in a brain microsomal system 34, while Murthy and R a p p o p o r t 20 reported increased incorporation of 14C radioactivity into protein by brain microsomes in the presence of a mixture of 22 amino acids. Orrego and Lipmann 21 showed that electrical stimulation of brain slices, or the addition of any of several acidic amino acids to the incubation medium, substantially decreased incorporation of radioactivity into protein. Neither acidic amino acids nor electrical stimulation altered the synthesis of protein by kidney slices. Mahler and co-workersG, v showed that G A B A stimulated protein synthesis both by mitochondrial and ribosomal systems derived from brain. No other compound stimulated synthesis in both systems. These were the first workers to study the effects of G A B A on brain protein synthesis. Baxter and Tewari 8° also studied the effect of G A B A on protein synthesis by rat brain ribosomes, and found a sodium dependent stimulatory effect, maximal at 5 m M G A B A concentration. Glyeine was also stimulatory, and glutamate inhibitory. The G A B A stimulation could be overcome by adding high concentrations (11 m M ) of glutamate together with GABA. G A B A was still stimulatory when added together with a mixture of 20 other amino acids. This study explored possible mechanisms of the G A B A effect, which was shown to be present in brain slices, and also studied the effects of some other possible neurotransmitters. These studies were done chiefly on brain slices, in which most cellular membranes are intact 17 because of the interest in neurotransmitters, whose first and probably greatest effect should be at cell membranes.

METHODS

Guinea pigs of either sex, weighing 250-300 g, were decapitated in a cold room. Small slices (0.2 m m × 0.2 m m x 2 mm) of brain tissue were prepared with a McIlwain tissue chopper. The slices were suspended in NaC1 128 m M , KC1 5.5 raM, MgC12 2 mM, CaC12 1 mM, and Hepes (N-2-hydroxyethylpiperazine N-2-ethanesulfonic acid) 20 m M , adjusted to p H 7.4, with 6 m M glucose. Preliminary studies had shown that incorporation of [14C]leucine into protein was greater for Hepes buffered media than for Tris, Krebs phosphate, and Krebs bicarbonate buffers. The

STUDIES O N

GABA

A N D P R O T E I N SYNTHESIS

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slice suspension, equivalent to 25 mg wet weight, was incubated in an atmosphere of 95 % oxygen, 5 % CO2, with a metabolic shaker, usually for 20 min. At the completion of incubation, an aliquot of the suspension was taken for protein determination by the method of Scheuel and Scheue126, 20% trichloroacetic acid containing carrier amino acid was added to the remaining suspension which was then filtered by vacuum filtration, and trapped on a glass fiber filter disc. The precipitate was then rinsed with 10 % trichloroacetic acid, heated to 90 °C, rinsed with ether, methanol and chloroform to extract lipids 14. The filters were placed in vials and counted in a Packard liquid scintillation counter, using a Toluene-Triton X-100 scintillation mixture 22 and internal standardization for quench correction. Incorporation of L-[1-14C]leucine and L-[1-14C]alanine was found to be constant for periods up to 40 rain. In most experiments, brain slices and homogenates were incubated with a mixture of 9 non-radioactive amino acids, the most abundant in rat serum 24. They were L-alanine 0.5 m M concentration, L-threonine 0.5 mM, L-lysine 0.4 mM, glycine 0.3 raM, L-glutamate 0.3 mM, L-glutamine 0.2 mM, L-proline 0.2 mM, and L-valine 0.1 raM. When radioactive alanine was the amino acid whose incorporation was studied, it was omitted from the mixture of non-radioactive amino acids. The concentration of radioactive amino acid was always 0.01 mM. In a few experiments, the non-radioactive amino acids were omitted. In parallel experiments, the uptake of L-[1-~4C]leucine into brain slices was studied, using [3H]mannitol as an indicator for extraeellular space. The trichloroacetic acid soluble leucine radioactivity, after correcting for mannitol space, assuming that leucine and mannitol concentrations were identical in extracellular space and medium, was taken as measuring the intracellular precursor pool available for protein synthesis. Homogenates were prepared by hand homogenization in 10 vol. of 0.32 M sucrose, buffered to pH 7.4 with l0 m M Hepes, and were then suspended in the same medium used for brain slices, without fractionation. Polysome profiles were studied in slices incubated in the presence of 2 m M GABA and incubation medium for 20 rain. Control slices from the same animal were incubated without GABA. The slices were homogenized in 50 m M Tris, 50 m M KCI, 10 m M Mg acetate, pH 7.6 and centrifuged at 15,000 × g for 15 min. The pellet was discarded and the supernatant layered over 2 M sucrose and centrifuged for 2.5 h at 130,000 × g. This pellet was resuspended in 50 m M Tris, 50 m M KC1, 2 m M Mg acetate without sucrose and was then layered on to a continuous linear sucrose density gradient, running from 15 to 40 %. After 4 h centrifugation the gradients were collected by puncturing the bottom of the tube and absorption read in a Beckman spectrophotometer at 254 rim. The area under the curve was determined by cutting out and weighing the paper upon which the curve was plotted. The incorporation of L-phenylalanine into polyphenylalanine was studied with synthetic messenger, poly-uridylic acid, using ribosomes prepared by centrifugation as described, again without inclusion of detergents. The ribosomes were incubated with pH 5 enzyme fraction from brain, NaCI 140 mM, KC1 100 mM, Mg acetate 10 mM, glutathione 6 mM, ATP 5 mM, G T P 1 mM, Hepes 25 mM, sucrose 25 raM, and 10 mg pyruvate kinase, with 1.5 m M phosphoenolpyruvic acid and 100 #g poly-

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uridylic acid. R a d i o a c t i v e L-phenylalanine was the only a m i n o acid present in this system. RESULTS

T a b l e I d e m o n s t r a t e s the rate o f p r o t e i n i n c o r p o r a t i o n f r o m L-[1-14C]leucine by slices o f c e r e b r a l cortex, f o r e b r a i n (defined as t h a t p a r t o f b r a i n r o s t r a l to the superior colliculus) a n d cerebellum. This figure refers to experiments in w h i c h the mixture o f 9 a m i n o acids were present, b u t G A B A was not. Analysis o f variance i n d i c a t e d t h a t t h e differences shown were n o t statistically significant. T h e table also shows the d a t a expressed as relative specific activity, defined as the specific activity o f p r o t e i n derived b y the 14C r a d i o a c t i v i t y in the ' i n t r a c e l l u l a r ' T C A soluble fraction o f the b r a i n slices, w h i c h d a t a derived f r o m p a r a l l e l experiments in the same animals. T h i n - l a y e r c h r o m a t o g r a p h y i n d i c a t e d t h a t 91 ~ 3 ~ o f the r a d i o a c t i v i t y in this f r a c t i o n m i g r a t e d w i t h a n R F identical to t h a t o f s t a n d a r d leucine. Table I b illustrates the effect o f 2 m M G A B A u p o n i n c o r p o r a t i o n o f L-[1-14C]leucine into p r o t e i n b y slices a n d h o m o g e n a t e s

TABLE I (a) INCORPORATIONOF L-[1-14C]LEUCINEINTOPROTEINBY BRAINSLICES Slices or homogenates (25 mg wet wt.) were incubated with L-[1-14C]leucine and a mixture of 9 radioactive amino acids (see text). After 20 min incubation was terminated, the protein was separated by precipitation and its specific activity determined. (See text for definition of relative specific activity.) Tissue

Specific activity (disint./min/mg prot.)

Relative specific activity

Cortex (5) Forebrain (5) Cerebellum (4)

2720 -4- 480 2150 -4- 670 1940 -4- 320

0.24 -4- 0.04 0.20 -4- 0.05 0.17 -4- 0.03

(b) INCREASE IN INCORPORATIONOF L-[1-14C]LEUCINERADIOACTIVITYINTO PROTEIN WHEN GABA ADDEDTO INCUBATIONMIXTURE In (b) slices and homogenates were incubated with radioactive lencine, the 9 additional amino acids and 2 mM GABA. The percentage increase in protein specific activity shown was obtained by comparison of incubations with and without added GABA. Tissue

Percentage increase

Cortex slices (6) Cortex homogenates (5) Forebrain slices (5) Forebrain homogenates (5) Cerebellar slices (4) Cerebellar homogenates (4)

33.1 ± 5.8 14.8 ± 2.8 24.8 -4- 4.2 7.1 -4- 2.5 3.0 -4- 3.4 1.7 -4- 2.3

All figures are means and standard errors, with the number of experiments given in parentheses.

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of cortex, forebrain a n d cerebellum. Baseline protein synthesis was less in h o m o g enates t h a n slices, as was reported by others 31. G A B A had a greater stimulatory effect o n slices t h a n homogenates, a n d had n o significant effect u p o n either slices or h o m o g e n a t e s of cerebellum. Table II shows the effect of G A B A at varying concentrations u p o n the i n c o r p o r a t i o n of L-[1-14C]leucine into forebrain slices. G A B A was s h o w n to stimulate the i n c o r p o r a t i o n of [14C]alanine a n d p h e n y l a l a n i n e into protein, i n a d d i t i o n to leucine. S t i m u l a t i o n by G A B A was d e m o n s t r a b l e with i n c u b a t i o n periods of 5 m i n - 1 h, b u t was n o t evident with i n c u b a t i o n periods less t h a n 5 rain. T h e relative increase in leucine i n c o r p o r a t i o n was 9.8 ~ for 5 m i n i n c u b a t i o n s , 26.4 Yoo for 30 m i n i n c u b a t i o n s a n d 23.7 ~ for 60 m i n incubations. Table I I I indicates that G A B A did n o t increase the uptake of radioactive leucine into the n o n - m a n n i t o l space of b r a i n slices. Slices i n c u b a t e d w i t h o u t the a d d i t i o n of

TABLE II Results given are the mean and standard errors of 3 or 4 separate experiments, with L-[14C]leucine as substrate unless otherwise indicated. GABA concentration (mM)

% increase in specific activity of protein

0.01 0.4 2 6 10 2, with [14C]alanine as substrate 2, with [14C]phenylalanine as substrate

3.9 4- 3.1 24.5 4- 4.4 34.1 4- 5.6 22.5 4- 5.0 5.7 4- 3.5 17.3 4- 3.0 11.5 4- 3.3

TABLE III 14C ]RADIOACTIVITY IN THE ~NON-MANNITOL SPACE'

Slices (25 mg wet wt.) were suspended in incubation medium and the mixture of 9 amino acids, with or without GABA (conc. 2 raM). Flasks contained 0.8 #Ci L-[1-14C]leucineand 2 #Ci D-[aH]mannitol. After incubation, slices were collected by centrifugation, homogenized in 10 ~ trichloroacetic acid containing carrier amino acid, and aliquots of supernatant counted in a scintillation counter. The 14C radioactivity due to trapped extracellular fluid was calculated as described in the text and subtracted from total radioactivity to obtain the values shown. Disint./min Cortex Controls Controls* GABA GABA*

9,600 45,500 410,300 i 5,000 4-

800 600 1000 500

Forebrain

Cerebellum

10,900 4- 1000

13,000 4- 1200

10,700 4- 900

11,600 4- 700

* Incubated for 10 rain instead of 20.

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the 9 non-radioactive amino acids showed less incorporation of radioactive leucine into protein than the slices incubated with the amino acid mixture. I n c o r p o r a t i o n was 1 9 ~ less for 10 min incubation periods, and 29~o less for 20 min incubation periods. These differences were statistically significant at the 0.05 level. Under these circumstances, the addition o f G A B A , at concentrations of either 2 or 5 m M , produced only a small increase in incorporation (10 ~ , 6 ~ ) not statistically significant. Polysome profiles were c o m p a r e d and a small increase in the relative n u m b e r of heavy polysomes found in gradients made from slices which had been incubated with 2 m M G A B A (Table IV). The differences found were small, but were present in all 6 animals so studied, and were statistically significant, whether one c o m p a r e d m o n o mers against the heavier portion of the gradient, or b o t h m o n o m e r s and dimers against the heavier polysomes. I n c o r p o r a t i o n o f radioactive phenylalanine into polyphenylalanine did not differ between ribosomes prepared f r o m slices incubated with G A B A , and those prepared f r o m slices not exposed to G A B A . The results were: G A B A ribosomes, 8850 ± 900 disint./min; control ribosomes, 7940 ± 1100 disint./min; based u p o n 3 experiments. TABLE IV ANALYSIS OF POLYSOME PROFILES

Monomers alone Monomers and dinaers Trimers and larger polysomes

% of gradient corresponding to control slices

GABA

0.181 ~ 0.013 0.443 2- 0.015 0.557 z~ 0.029

0.155 ± 0.010' 0.385 q- 0.012" 0.611 -4- 0.030*

* Difference significant from control value at P ~< 0.001 by Student's 2-tailed t-test. TABLE V EFFECTS OF POSSIBLE NEUROTRANSMITTERS ON INCORPORATION OF L [14C]LEUC1NE INTO PROTEIN BY GUINEA P IG FOREBRAIN SLICES

Abbreviations: NA, noradrenaline, DCI, dichloroisoproterenol. Number of experiments in parentheses. Compound

% change in incorporation compared with control

Serotonin, 1 mM Dopamine, 2 mM NA, 0.5 mM NA, 2 mM NA, 0.5 mM ÷ DCI 0.01 rnM NA, 0.5 m M ÷ phentolamine 1 mM NA, 0.5 m M + theophylline, 2 mM Glycine, 2 mM

-- 18.2 ~ 4.7 (4)

2.4 d= 3.9 (3) 60.3 ~ 5.8 (4) 21.5 z~ 4.3 (4) 11.5 :~ 2.6 (4) 45.7 :~ 7.2 (3) 55.7 ± 8.4 (3) 16.9 ± 4.6 (3)

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I+~+T

~'~A~A

A~+T1F~.

DDE't~I'~|~T ~ V ~ T ~ I ~ I J ~ I e

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generally a;;ear-to have-a-high;; ;;rceniag;- ;f-m;n;m;rsa-nd-dimers than those in this study. The mixture of non-radioactive amino acids was selected on the premise that

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s.R. SNODGRASS

amino acids reach brain cells primarily from the blood, and that those present in greatest amount are the most important. Because tryptophan, present in low amounts in serum, is quite important in hepatic protein synthesis 2s, this approach is an oversimplification. It was felt more desirable to reflect the amino acid content of blood rather than that of cerebral tissue, as some investigators have done. Other investigations with different systems have sometimes found inhibition rather than stimulation when additional non-radioactive amino acids are added to the incubation medium 4. Although stimulation of protein synthesis in brain subcellular fractions has been reported 6,7,3°, Goldberg 1~ found that 1 m M GABA had no effect on protein synthesis by synaptosomal and mitochondrial fractions of rat brain and that noradrenaline was inhibitory. The difference between the present results and Goldberg's may be partly due to the different preparations used: brain slices as opposed to purified subcellular fractions. Small changes in incubation media and other technical matters may account for much of the variability reported in the literature on protein synthesis and its modification by amino acids and other small molecules. GABA did not alter the response of brain ribosomes to artificial messenger, and it seems unlikely that the stimulatory effect of GABA requires increased synthesis of messenger RNA. It seems that GABA brings about a general change in the protein synthesizing apparatus, which is accompanied by increased aggregation of brain polysomes, and increased incorporation of amino acids into protein. GABA has a positive stimulatory effect even if the inhibitory amino acid glutamate is removed from the group of supplementary amino acids. The nature of the noradrenaline stimulatory effect remains unexplained, but the fact that it is blocked by small concentrations of dichloroisoproterenol suggests that it is a specific effect. Evidence was presented that the noradrenaline stimulatory effect could not be reduced to a change in the amino acid precursor pool. In terms of synaptic function, there would be greater value in the demonstration of increases in the synthesis of specific proteins, rather than the generalized increase demonstrated here. The effect of these substances (GABA, noradrenaline, and other neurotransmitters) on the synthesis of specific proteins remains an important subject for future investigation. ACKNOWLEDGEMENTS

This study is supported in part by Grant NS 05172 of the National Institute of Neurological Diseases and Stroke, and the C.H.M.C. Mental Retardation and Human Development Research Program Grant HD03773. S.R.S. is a recipient of Teacher-Investigator Award 5 F l l N S l l , 010 02NSRB.

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3 BANKER,G., AND COTMAN,C. W., Characteristics of different amino acids as protein precursors in mouse brain: advantages of certain carboxyl labelled amino acids, Arch. Biochem. Biophys. 142 (1971) 565-573. 4 BORSOOK,H., DEASEY,C. L., HAAGEN-SMIT,A. J., KEIGHLY,G., ANDLOWRY,P. H., Incorporation in vitro of labelled amino acid into rat diaphragm proteins, J. bioL Chem., 186 (1950) 309-315. 5 BORSOOK,H., DEASEY,C. L., HAAGEN-SMIT,G. J., KEIGHLY,G., AND LOWRY,P. H., Incorporation in vitro of labelled amino acids into proteins of rabbit reticulocytes, J. biol. Chem., 196 (1952) 669-694. 6 CAMPAGNONI,A. T., AND MAnLER,H. R., Isolation and properties of polyribosomes from cerebral cortex, Biochemistry, 6 (1967) 956-967. 7 CAMPBELL,M. K., MAHLER,H. R., MOORE,W. J., ANDTEWARI,S., Protein synthesis systems from rat brain, Biochemistry, 5 (1966) 1174-1184. 8 FOLBERGROVA,J., Incorporation of labelled amino acid into the proteins of brain cortex slices in vitro in the presence of other non-radioactive amino acid, J. Neurochem., 13 (1966) 553-562. 9 GEIGER,A., HOVARTH,N., AND KAWAKITA,J., Incorporation of 14C derived from glucose into the proteins of the brain cortex, J. Neurochem., 5 (1960) 311-322. 10 GIORGI,P. P., Polyamines and amino acid incorporation in vitro, Biochem. J., 120 (1970) 643-651. 11 GIORGI,P. P., Polyamines and regulation of protein synthesis in brain, Biochem. J., 127 (1972) 6P. 12 GOLDBERG,M. A., Inhibition of synaptosomal protein synthesis by neurotransmitter substances, Brain Research, 39 (1972) 171-179. 13 JONES,D. A., AND MCILWAIN,H., Amino acid distribution and incorporation into proteins in isolated electrically stimulated cerebral tissues, J. Neurochem., 18 (1971) 41-58. 14 KENNELL,D., Use of filters to separate radioactivity in RNA, DNA and protein. In N. O. KAPLAN AND S. P. COLOWICK(Eds.), Methods ofEnzymology, Vol. Xlla, Academic Press, New York, 1967, pp. 686-693. 15 LE CocQ, R. E., CANTRAINE,F., KEYHANI,E., CLAUDE,A., DELACROIX,C., AND DUMONT,J. E., Quantitative evaluation of polysomes and ribosomes by density gradient centrifugation and electron microscopy, Analyt. Biochem., 43 (1971) 71-79. 16 LEANER,M. P., AND JOHNSON,T. C., Regulation of protein synthesis in developing mouse brain, J. biol. Chem., 245 (1970) 1388-1393. 17 M C1LWA1N,H., ANDRODNIGHT,R., Practical Neurochemistry, Little, Brown, Boston, Mass., 1962, 296 pp. 18 MATTHEI,H. J., ANDNIRENBERG,M. W., Characterization and stabilization of DNA-ase sensitive protein synthesis in E. coli extracts, Proc. nat. Acad. Sci. (Wash.), 47 (1961) 1580-1588. 19 MAXWELL,E. S., Study of amino acid incorporation into protein by natural and synthetic polyribonucleotides, Proc. nat. Acad. Sci. (Wash.), 48 (1962) 1639-1643. 20 MURTHY,M. R., AND RAPPOPORT,D. A., Developing rat brain. VI. Preparation and properties of ribosomes, Biochim. biophys. Acta (Amst.), 95 (1965) 132-145. 21 ORREGO,F., AND LIPMANN,F., Protein synthesis in brain slices, J. biol. Chem., 242 (1967) 665-671. 22 PATTERSON,M. S., AND GREENE, R. C., Measurement of low energy beta-emitters in aqueous solution by liquid scintillation counting of emulsion, Anal. Chem., 37 (1965) 854-861. 23 REGIER,J. C., AND KAFATOS,F. C., Microtechnique for determining specific activity of radioactive intracellular leucine and application to in vivo studies of protein synthesis, J. biol. Chem., 246 (1971) 6480-6488. 24 ROGERS,Q. R., SPOLTER,P. D., AND HARPER,A. E., Effect of leucine-isoleucine antagonism on plasma amino acid pattern in rats, Arch. Biochem. Biophys., 97 (1962) 497-504. 25 SATAKE,M., MASE,K., TAKAHASI4I,Y., ANDOGATA,K., Incorporation of leucine into microsomal protein by a cell free system of guinea pig brain, Biochim. biophys. Acta (Amst.), 41 (1960) 366367. 26 SCHEUEL,H., AND SCHEUEL,R., Automated determination of protein in the presence of sucrose, Analyt. Biochem., 20 (1967) 86-93. 27 SCHWEET,R., LAMFROM,H., AND ALLEN, E., The synthesis of hemoglobin in a cell free system, Proc. nat. Acad. Sci. (Wash.), 44 0958) 1029-1035. 28 SIDRANSKY,H., SARMA,D., BONGIORUO,M., AND VERNEY,E. E., Effect of dietary tryptophan on hepatic polyribosomes and protein synthesis in fasted mice, J. bioL Chem., 243 (1968) 1123-1132. 29 SIEGEL,F. L., AOKI, A., AND COLWELL,R. E., Polyribosome disaggregation and cell free protein synthesis in preparations from the cerebral cortex of hyperphenylalaninemic rats, J. Neurochem., 18 (1971) 537-547. 30 TEWARI, S., AND BAXTER, C. F., Stimulatory effect of ?-aminobutyric acid upon amino acid

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