Aspartic acid as a precursor for glutamic acid and glycine

Brain Research, 67 i 19,'~} 358- 362

358

:l! Elsevier Scientilic Publishing Compally, Amsterdam

Printed in The Netherlands

Aspartic acid as a precursor for glutamic acid and glycine

J E F F E R Y LEE JOHNSON

Department of Physiology and Pharmacology, School of Medicine, University of South Dakota, Vermillion, S. D. 57069 (U.S.A.) (Accepted November 14th, 1973)

Glutamic acid and glycine have been put forth as synaptic transmitter candidates for the dorsal sensory neurona, 4 and inhibitory interneurons in the spinal grey 1° respectively. Aspartic acid can lead to glutamate and gtutamine production in cortical or brain tissues1,2, 9 and serine has been shown to be an important precursor of glycine 7. The importance of aspartate in providing a supply of carbon atoms for the synthesis of glutamate and glycine of a magnitude great enough to be a significant factor has never been weighed in the dorsal sensory neuron and spinal grey. The purpose of this analysis, therefore, is to determine if aspartic acid is an effective precursor of amino acids of key interest in the dorsal sensory neuron where glutamate is the best transmitter candidate. Analysis of spinal grey and ventral root tissues will provide useful data for comparison purposes, since the spinal grey is an area where both glutamate and glycine are transmitter candidates, and the ventral root contains axons where neither amino acid is presently entertained as a transmitter. Adult cats anesthetized with sodium pentobarbital were used throughout. Methods of surgical exposure and direct labeled precursor injection were carried out as outlined previously ~,6. The amino acids were isolated and determined according to the method of Shank and Aprison 8. After analysis of variance, individual values were compared with each other via the Newman-Keuls method of individual comparisons (a -= 0.05). For the first set of experiments, [U-14C]aspartic acid was injected into the dorsal root ganglion, and at 15 min (Table I, part A) and 60 min (Table I, part B) the dorsal root ganglion and dorsal root were taken for analysis. The dorsal root was divided into two portions of equal length designated as dorsal root proximal (DRP) and dorsal root distal (DRD), these being the segment nearest and furthest away from the ganglion respectively. The glutamine/glutamate relative specific radioactivity ratio showed a progressive increase from ganglion to proximal and distal root at 60 rain but not at 15 rain, with glutamine accounting for a significantly greater percentage o f the injected aspartate label at 60 min compared to 15 rain in the two root tissues. In contrast, the aspartate label accounted for a smaller percentage of the total radioactivity in the two root tissues at 60 rain compared to 15 min.Glycine showed anunexpectedly high labeling from [U-14C]aspartate, with a significant proximo-distal increase in the

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relative specific r a d i o a c t i v i t y r a t i o at 15 min, which was m u c h less in evidence at 60 min. This suggests a very early labeling o f glycine b y aspartate. A t the left h a n d side o f T a b l e I, the specific r a d i o a c t i v i t y o f g l u t a m a t e is given for the 3 tissues o f interest. In passing f r o m the injection site at the g a n g l i o n to the p r o x i m a l a n d distal d o r s a l r o o t there is a m a r k e d decline in g l u t a m a t e specific r a d i o a c t i v i t y in the 15- a n d 60-min periods, with this decline being less at 60 min. This is also true for the respective radioactivities o f glutamine, glycine a n d aspartate. In a similar set o f experiments, [U-14C]aspartate was injected into the ventral spinal grey, a n d 60 min later the ventral spinal grey a n d ventral r o o t tissues were t a k e n for analysis. T h e ventral r o o t was divided into two p o r t i o n s o f equal length designated as ventral r o o t p r o x i m a l (nearest the cord) a n d ventral r o o t distal (furthest a w a y f r o m the cord). The d a t a are shown in T a b l e II. The g l u t a m i n e / g l u t a m a t e relative specific r a d i o a c t i v i t y r a t i o showed no consistent p r o x i m o - d i s t a l gradient. Since the g l u t a m i n e label a c c o u n t e d for m o r e t h a n 50 ~ o f the t o t a l label in all 5 substances m e a s u r e d in the r o o t tissues, a s p a r t a t e w o u l d a p p e a r to be a g o o d p r e c u r s o r for g l u t a m i n e in these tissues. T h e a s p a r t a t e / g l u t a m a t e relative specific r a d i o a c t i v i t y r a t i o was highest in the distal ventral root, next followed b y the p r o x i n m l ventral root, with the spinal grey showing the lowest values. The glycine/glutamate relative specific

TABLE I THE LABELING OF SEVERAL KEY AMINO ACIDS IN THE DORSAL SENSORY SYSTEM FROM [U-14CIASPARTATE 15 AND 6 0

min AFTER INJECTION

INTO THE DORSAL ROOT GANGLION

The relative specific radioactivity ratio (glutamate = 1) is shown for glutamine, aspartate, alanine, and glycine :k the standard deviation, from data in disint./min/#mole of substance. For the 15-min data (section A) N -- 5, and for the 60-min data (section B) N ~ 4. The number in parentheses for the glutamate values is the disint./min/#mole in each tissue to be used as a standard reference point. From the data in disint./min/g tissue, the percentage distribution of labeling is given for each of the 5 substances in the indicated tissues. DRG -- dorsal root ganglion, DRP = dorsal root proximal, and DRD -- dorsal root distal. Both for the dorsal root ganglion (Table I) and the spinal grey (Table II) injection experiments 10-12 #Ci of [U-14C]aspartate were applied. This amount was parcelled out into the two L7 and two SI ganglia, or into the ventral spinal grey of L7 and S1 segments bilaterally. Glutamate

Glutamine

Aspartate

Alanine

Glycine

1.0 (820,000) 23.7 ~ 1.0 (40,000) 34.6 ~ 1.0 (2400) 22%

1.59 ± 0.10 40.4~ 1.19 ± 0.22 27.0 ~ 2.28 4- 0.34 34.2%

1.96 ± 0.73 28.0~ 2.99 ± 0.63 28.8 % 5.27 ± 1.5 32.7 %

0.380 3- 0.1 3.9 ~ 0.71l ± 0.24 1.6 % ---

0.708 ~: 0.063 4.4~ 2.08 ± 0.49 7.9 % 6.46 ± 0.58 11.2%

1.0 (440,000) 20.4 ~ 1.0 (80,000) 28.5 ~ 1.0 (18,000) 22.8~

2.04 ± 0.18 45.4~ 2.62 ± 0.30 49.8 ~ 3.83 ± 0.43 58~

2.19 ± 0.45 26.8 % 1.82 ± 0.22 14.2 ~ 2.35 ± 0.47 13~

0.202 ± 0.04 1.8 ~ 0.652 ± 0.15 1.2 ~ 1.32 ~= 0.32 1.4~

1.08 ~: 0.28 5.7 2.07 ~_ 0.22 6.3 2.78 ~ 0.40 4.7~

(A) 15 min

DRG DRP DRD ( B) 60 min

DRG DRP DRD

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TABLE II THE LABELING OF SEVERAL KEY AMINO ACIDS IN THE VENTRAL SPINAL GREY AND VENTRAL ROOT FROM

[U-14C]ASPARTATE60 min A F T E R

INJECTION INTO THE VENTRAL SPINAL GREY

The relative specific radioactivity ratio (glutamate -- 1) is shown for glutamine, aspartate, alanine, and glycine ± the standard deviation of 4 animals, from data in disint./min/#mole of substance. The number in parentheses for glutamate is the disint./min//~mole in each tissue to be used as a standard reference point. From the data in disint./min/g tissue, the percentage distribution of labeling is given for each of the 5 substances in the indicated tissues. SCG -- spinal cord grey, VRP = ventral root proximal, and VRD = ventral root distal.

SCG VRP VRD

Glutamate

Glutamine

Aspartate

Alanine

Glycine

(850,000) 25.9~ (25,000) 15.7~ (6,000) 14.6 ~

2.26 ± 0.40 34.1 ~ 1.17 ± 0.15 54~ 3.02 ;- 0.13 55 }/o

2.48 ± 0.24 24.6~ 3.14 ± 0.64 16.5~ 3.74 ± 0.20 23.5 ~

0.146 ± 0.04 0.24~ 1.44 ± 0.21 1.6~ 1.61 ± 0.12 1.1 /o°/

0.632 ± 0.03 15~ 1.28 ± 0.42 11.8~ 3.24 ± 0.74 5.6

radioactivity ratio also showed a proximo-distal increase, with the glycine label in the ventral grey, where it is a transmitter suspect 10, accounting for a considerable percentage of the total radioactivity. A most unexpected finding in this study was the effectiveness with which aspartic acid was able to act as a precursor for glycine. It has already been revealed that glucose is a relatively poor precursor of glycine and that serine is an effective glycine precursor in the nervous systemV. The pathway which is envisioned for the production of glycine from aspartate is shown in Fig. 1. Aspartate is first converted to oxaloacetate via transamination. A portion of this oxaloacetate will, through tricarboxylic acid cycle function 9, result in a-ketoglutarate production which via the action of glutamic dehydrogenase can be converted to glutamate. The oxaloacetate may also result in glycine production by the steps indicated, with the final intermediate leading to glycine being serine. Since this pathway is only proposed as a working framework to be subject to further testing, Table Ill shows one simple test of this proposed mechanism. One of the intermediates, 3-phospho-I>[U-14C]glycerate was injected into the dorsal root ganglion and the amino acid labeling analyzed. All substances were labeled poorly by 3-phospho-D-[U-14C]glycerate, with glutamate showing the highest specific radioactivity. Aspartate, alanine, and serine showed a similar degree of labeling. Glycine showed the lowest labeling of all substances measured. When the glycine labeling from the labeled aspartate and 3-phospho-D-glycerate precursors are compared, aspartate stands out as a very effective supply of carbon atoms leading to glycine, with the fact that 3-phospho-D-glycerate did label serine and glycine suggesting that it may be an intermediate in this conversion. 3-Phospho-D-glycerate in the tissues in general, however, does not seem to be an effective carbon source leading to glycine, most of it entering into normal glycolytic pathways for energy production. Several observations suggest that we are dealing with uptake of the injected labeled aspartate with subsequent proximo-distal axoplasmic transport of the aspar-

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ASPARTATE

GLYCINE

transa,minase OXALOACETATE l carboxykinase PHOSPHOENOL PYRUIVATE l eno,lase 2-PHOSPHOGLYCERATE

T

se,6nerran's'hydroxyme,thylase

SERINE l

p~ho:sp~ham'se

3-PHOSPHOSERINE

T tra'nsa,m,i'nase 3-PHOS'PHOHYDROXYPYRUVATE dehydrogenase OSPHOGL~YCERATE

~~tSf:H

Fig. 1. A possible pathway for the production of glycine from aspartate.

tate precursor, associated enzyme systems, a n d p r o d u c t s d o w n the d o r s a l r o o t a x o n s : (1) the p r o x i m o - d i s t a l decrease in the glutamate, glutamine, aspartate, a n d glycine specific radioactivities were each, respectively, m u c h steeper at 15 min t h a n at 60 m i n ; (2) the a s p a r t a t e label a c c o u n t e d for a smaller percentage o f the t o t a l label in all 5 m e a s u r e d substances in the d o r s a l r o o t tissues at 60 m i n c o m p a r e d to 15 m i n after injection into the ganglion; a n d (3) the g l u t a m i n e label a c c o u n t e d for a greater percentage o f the label in the r o o t tissues at 60 min c o m p a r e d to 15 min. Glycine w o u l d a p p e a r to be f o r m e d quite r a p i d l y f r o m a s p a r t a t e as suggested b y its high TABLE III THE LABELING OF SEVERAL KEY AMINO ACIDS INTO THE DORSAL ROOT GANGLION

60 rain AFTER INJECTION OF 3-PHOSPHO-D-[U-14C]GLYCERATE

An injection of 8-10/zCi of the labeled precursor was made into the dorsal root ganglia of L7 and S1 on the left and right sides, this being the total amount which was parcelled out into these 4 ganglia. The data shown are for the dorsal root ganglion (DRG), the counts in the dorsal root tissue being too low to record effectively.

Relative Specific Radioactivity Glutamine/ Glutamate

DRG

Aspartate/ Glutamate

Alanine/ Glutamate

Glycine! Glutamate

Serine/ Glutamate

0.653~0.082 0.743~-0.061 0.608±0.042 0.256~-0.10

0.5892c0.10

Glutamate (disint./min/ pmole) 15,000

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labeling in the distal dorsal r o o t as early as 15 rain after labeled a s p a r t a t e injection. The labeled a s p a r t a t e injected was shown to be an effective precursor o f glutamate in the g l u t a m i n e p r o d u c i n g c o m p a r t m e n t a n d o f glycine b o t h in the dorsal and ventral r o o t systems, as well as in the spinal grey. This suggests t h a t one role o f the free a s p a r t a t e pool is to p a r t i c i p a t e in the r e g u l a t i o n o f the g l u t a m a t e g t u t a m i n e system a n d o f glycine. Thus, in the d o r s a l sensory system where g l u t a m a t e is presently e n t e r t a i n e d as a synaptic t r a n s m i t t e r suspect a,4` and in the spinal i n h i b i t o r y intern e u r o n s where glycine is a t r a n s m i t t e r suspect x°, free a s p a r t a t e p o o l s could be o f great i m p o r t a n c e in t r a n s m i t t e r maintenance. Since these two a m i n o acids glycine a n d g l u t a m a t e have roles in a d d i t i o n to that o f synaptic t r a n s m i s s i o n , and a s p a r t a t e can lead to g l u t a m a t e and glycine p r o d u c t i o n in tissues where neither substance is entertained as a synaptic t r a n s m i t t e r as shown in the p r e s e n t study, the difficult t a s k remains o f discerning the role o f a s p a r t a t e in the m a i n t e n a n c e o f t r a n s m i t t e r a n d n o n - t r a n s m i t t e r pools o f these a m i n o acids. S u p p o r t e d by research g r a n t U.S. Public H e a l t h Service G r a n t 1-R01-NS-10508 to the a u t h o r .

1 BERL, S., AND CLARKE, D. D., Compartmentation of amino acid metabolism. In A. LAJTHA(Ed.), Handbook of Neurochemistry, Vol. lI, Plenum Press. New York, 1969, pp. 447-472. 2 BERL, S., CLARKE, D. D., AND NICKLAS, W. J., Compartmentation of citric acid cycle metabolism

in brain: effect of aminooxyacetic acid, ouabain, and Ca +~ on the labeling of glutamate, glutamine, aspartate, and GABA by [l-14C]acetate, [U-14C]glutamate, and [U-14C]aspartate, J. Neurochem., 17 (1970) 999-1007. 3 HAMMERSCHLAG,R., AND WEINREICH.D., Glutamic acid and primary afferent transmission In E. COSTA, L. L. lVERSEN AND R. PAOLETTIrEds.), Advances in Biochemical Psychopharmacology, Vol. 6, Raven Press, New York, 1972. pp. 165-180. 4 JOHNSON, J. L., Glutamic acid as a synaptic transmitter in the nervous system. A review, Brain Research, 37 0972) 1-19. 50CHS, S., JOHNSON, J. L., AND NG, M. H., Protein incorporation and axoplasmic flow in motoneuron fibers following intra-cord injection of labeled leucine, J. Neurochem., 14 (1967) 307-31 l. 60CHS, S., SABRI, M. I., AND JOHNSON. J. L . Fast transport system of materials in mammalian nerve fibers, Science, 163 0969) 686--687. 7 SHANK, R. P., AND APRISON, M. H., The metabolism in vivo of glycine and serine m eight areas of the rat central nervous system, J. Neurochem., 17 (1970) 1461-1475. 8 SHANK, R. P., AND APRISON, M. H., Method for multiple analysis of concentration and specific radioactivity of individual amino acids in nervous tissue extracts. Analyt. Biochem., 35 (1970)

136-145. 9 SIMON, G., DROR1, J. B., AND COHEN. M. H., Mechanism of conversion of aspartate into glutamate in cerebral cortex slices, Biochem. J.. 102 (1967) 153-I62. 10 WERMAN, R., AND APRISON, M. H., The search for a spinal cord inhibitory transmitter. In Proceedings of the Fourth International Meeting of Neurobiologists held in Stockholm, Structure and Function of Inhibitory Neuronal Mechanisms, Pergamon Press, New York. 1968, pp. 473-486.