Glutamate is ubiquitously distributed in brain tissue, where it is present in a higher concentration than any other amino acid. During the last 50 years glutamate in brain has been the subject of numerous studies, and several different functions have been ascribed to it. Early studies by Krebs (1935) suggested that glutamate played a central metabolic role in brain. The complex compartmentation of glutamate metabolism in brain was first noted by Waelsch and coworkers (Berl et al., 1961). These studies were precipitated by the claim that glutamate improved mental behaviour and was beneficial in several neurological disorders including epilepsy and mental retardation. Other scientists pointed out its function in the detoxification of ammonia in brain (Weil-Malherbe, 1950). Glutamate is also an important building block in the synthesis of proteins and peptides, including glutathione (Meister, 1979). The toxic effect of administered glutamate and its analogues kainic acid, ibotenic acid, and N-methyl aspartic acid on CNS neurones has become a large and independent line of research (Lucas and Newhouse, 1957; Olney et al., 1974; Lund-Karlsen and Fonnum, 1976; Coyle, 1983). Attention has also been focused on the role of glutamate as a precursor for the inhibitory neurotransmitter y-aminobutyric acid (GABA) (Roberts and Frankel, 1950). Electrophysiological studies (Curtis and Watkins, 1961) focused early on the powerful and excitatory action of glutamate on spinal cord neurones. Since the action was widespread and effected by both the Dand Lforms, it was at first difficult to believe that glutamate could be a neurotransmitter. During the last 15 years, however, several studies have provided support for the concept that glutamate is a transmitter in brain (for review see Curtis and Johnston, 1974; Fonnum, 1978; 1981; Roberts et al., 1981; DiChiara and Gessa, 1981). Glutamate satisfies today to a large extent the four main criteria for classification as a neurotransmitter: (1) it is presynaptically localized in specific neurones; (2) it is specifically released by physiological stimuli in concentrations high enough to elicit postsynaptic response; (3) it demonstrates identity of action with the naturally occurring transmitter, including response to antagonists; and (4) mechanisms exist that will terminate transmitter action rapidly. The evidence for glutamate as a transmitter at the locust neuromuscular junction has recently been carefully evaluated by Usherwood (1981). In that case the identity of action of glutamate with the naturally occurring transmitter on the neuromuscular receptor, the release from nerve terminals, and its similarity to acetylcholine at the mammalian neuromuscular junction with regard to presynaptic pharmacology and denervation supersensitivity are compelling evidence for glutamate as a neurotransmitter. The main methods used to identify glutamergic pathways in brain will be critically reviewed and discussed. The effect of lesions on high-affinity uptake and release are particularly important, but immunohistochemical methods to study enzymes and glutamate itself are becoming more interesting. The release of glutamate has been demonstrated by several different procedures using both in vivo and in vitro preparations. The synthesis of large groups of specific agonists and antagonists has been important both for identification and characterization of the glutamate receptor by electrophysiological techniques and for the isolation of glutamate receptors. High and perhaps low-affinity uptake into nerve terminals and glial cells is important for the termination of transmitter action. Particular attention is given in this review to the complex compartmentation of glutamate synthesis and the possibility of identifying the transmitter pool of glutamate.

Glutamate: a neurotransmitter in mammalian brain.

2. Evidence for A m i n o Acids as T ransmi t t e r s . . . . . . . . . . . . . . . . . . . . 99 2.1. Synthesis and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 2.2. Synapt ic Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 2.3. Postsynapt ic Act ion . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 2.4. Postsynapt ic An tagon i s t s . . . . . . . . . . . . . . . . . . . . . . . . . 103 2.5. Inac t iva t ion and R e m o v a l . . . . . . . . . . . . . . . . . . . . . . . . . 104

Amino acid transmitters in the mammalian central nervous system

To determine the relationship between cerebral Glc metabolism and glutamatergic neuronal function, we used 13C NMR spectroscopy to measure, simultaneously, the rates of the tricarboxylic acid cycle and Gln synthesis in the rat cortex in vivo. From these measurements, we calculated the rates of oxidative Glc metabolism and glutamate–neurotransmitter cycling between neurons and astrocytes (a quantitative measure of glutamatergic neuronal activity). By measuring the rates of the tricarboxylic acid cycle and Gln synthesis over a range of synaptic activity, we have determined the stoichiometry between oxidative Glc metabolism and glutamate–neurotransmitter cycling in the cortex to be close to 1:1. This finding indicates that the majority of cortical energy production supports functional (synaptic) glutamatergic neuronal activity. Another implication of this result is that brain activation studies, which map cortical oxidative Glc metabolism, provide a quantitative measure of synaptic glutamate release.

https://www.pnas.org/content/pnas/95/1/316.full.pdf

Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity

Metabolism and role of glutamate in mammalian brain.

Functional interactions between neurons and astrocytes I. Turnover and metabolism of putative amino acid transmitters.

1. Cerebral-cortex slices prelabelled with gamma-amino[1-(14)C]butyrate (GABA) were incubated in a glucose-saline medium. After the initial rapid uptake there was no appreciable re-entry of (14)C into the GABA pool, either from the medium or from labelled metabolites formed in the tissue. The kinetic constants of GABA metabolism were determined by computer simulation of the experimental results by using mathematical procedures. The GABA flux was estimated to be 0.03mumol per min/g, or about 8% of the total flux through the tricarboxylic acid cycle. It was found that the assumption of compartmentation did not greatly affect the estimates of the GABA flux. 2. The time-course of incorporation of (14)C into amino acids associated with the tricarboxylic acid cycle was followed with [1-(14)C]GABA and [U-(14)C]-glucose as labelled substrates. The results were consistent with the utilization of GABA via succinate. This was confirmed by determining the position of (14)C in the carbon skeletons of aspartate and glutamate formed after the oxidation of [1-(14)C]GABA. These results also indicated that under the experimental conditions the reversal of reactions catalysed by alpha-oxoglutarate dehydrogenase and glutamate decarboxylase respectively was negligible. The conversion of [(14)C]GABA into gamma-hydroxybutyrate was probably also of minor importance, but decarboxylation of oxaloacetate did occur at a relatively slow rate. 3. When [1-(14)C]GABA was the labelled substrate there was evidence of a metabolic compartmentation of glutamate since, even before the peak of the incorporation of (14)C into glutamate had been reached, the glutamine/glutamate specific-radioactivity ratio was greater than unity. When [U-(14)C]glucose was oxidized this ratio was less than unity. The heterogeneity of the glutamate pool was indicated also by the relatively high specific radioactivity of GABA, which was comparable with that of aspartate during the whole incubation time (40min). The rates of equilibration of labelled amino acids between slice and medium gave evidence that the permeability properties of the glutamate compartments labelled as a result of oxidation of [1-(14)C]GABA were different from those labelled by the metabolism of [(14)C]glucose. The results showed therefore that in brain tissue incubated under the conditions used, the organization underlying metabolic compartmentation was preserved. The observed concentration ratios of amino acids between tissue and medium were also similar to those obtaining in vivo. These ratios decreased in the order: GABA>acidic acids>neutral amino acids>glutamine. 4. The approximate pool sizes of the amino acids in the different metabolic compartments were calculated. The glutamate content of the pool responsible for most of the labelling of glutamine during oxidation of [1-(14)C]GABA was estimated to be not more than 30% of the total tissue glutamate. The GABA content of the ;transmitter pool' was estimated to be 25-30% of the total GABA in the tissue. The structural correlates of metabolic compartmentation were considered.

The operation of the γ-aminobutyrate bypath of the tricarboxylic acid cycle in brain tissue in vitro

THE METABOLISM of the brain is characterized by a high rate of incorporation of glucose carbon into the free amino acids. The brain differs in this respect from other organs examined (ROBERTS, FLEXNER and FLEXNER, 1959; BUSCH, FUJIWARA and KEER, 1960; VRBA, GAITONDE and RICHTER, 1962; GAITONDE, DAHL and ELLIOTT, 1965). At 22 min after subcutaneous injection of uniformly labelled [14C]glucose in the cat, more than 70 per cent of the total 14C in the brain was found in the amino acids, whereas only 8-16 per cent of the 14C had entered the amino acids in liver, kidney, lung, muscle or spleen (GAITONDE, MARCHI and RICHTER, 1964). The work hitherto reported on the entry of glucose carbon into amino acids was carried out on adult animals or on animals in which the brain was functionally mature. This raises the question: does the rapid incorporation of glucose carbon into amino acids occur also in the immature brain, or at what age does this characteristic of brain metabolism appear ? The following experiments were undertaken to answer this question.

Changes with age in the utilization of glucose carbon in liver and brain.

WHEN [14C] glucose is administered in oiuo, part is oxidized to CO,, part is stored as glycogen and part is used for the synthesis of amino acids and other compounds in the tissues (WINZLER et al., 1952; PROCHOROVA, 1954; ALLWEIS and MAGNES, 1958a,b; GEIGER, 1958; BUSCH et al., 1960). ROBERTS, FLEXNER and FLEXNER (1959) reported a relatively high rate of conversion of glucose carbon into protein in the liver and brain in the mouse. They found that the distribution of I4C in the different amino acids of the proteins was different in the two organs : there was a greater incorporation of I4C into alanine than into glutamic acid in the liver, whereas in brain tissue the converse was true. The object of the present work was to study the conversion of glucose-carbon into protein in the rat brain. Orientating experiments were first carried out to find out to what extent carbon from [14C]glucose is incorporated in the free amino acids and proteins of other organs besides the brain. The incorporation was studied for a period of up to 12 hr and the distribution of I4C was examined in the dicarboxylic, neutral and basic amino acid fractions.

The conversion of glucose carbon into protein in the brain and other organs of the rat

1. The metabolism of γ-aminobutyrate (GABA) was investigated in cerebral-cortex slices incubated in glucose–saline medium with [1-14C]GABA and [U-14C]-glucose as labelled substrates. 2. A rapid release of GABA from the tissue, amounting to 25–30% of the total, was observed on addition of 66m-equiv. of K+/1 to the medium; the liberation of other amino acids was relatively small. The effect was apparently specific for K+; GABA was not released on addition of equivalent amounts of Na+ or on increasing the respiration rate with 10mm-ammonium chloride. The results show that GABA behaves like the transmitter compounds (acetylcholine, catecholamines) on K+ stimulation, and therefore now satisfies certain of the criteria required for a transmitter in mammalian brain. 3. The release of GABA from the tissue on addition of K+ was followed by a slow re-uptake. The rate of uptake of GABA in a medium containing 5.9m-equiv. of K+/1 was more than four times that in a medium containing 66m-equiv. of K+/1. 4. The concentration of GABA in brain tissue incubated for 1h in a medium containing 66m-equiv. of K+/1 was about 50% higher than that observed under normal conditions. 5. There was evidence that exogenous [14C]GABA mixed with the endogenous pool(s), since the proportion of the total GABA released on K+ stimulation was the same, and the specific radioactivity of the liberated GABA was close to that remaining in the tissue, whether the GABA was labelled by [1-14C]GABA from the medium or generated in the tissue from [14C]glucose. 6. On the basis of these findings and the observations outlined in the preceding papers it was possible to calculate the kinetic constants of GABA metabolism by computer simulation of the results. K+ stimulation led to a 2.5-fold increase in the flux through the tricarboxylic acid cycle, whereas the flux in the GABA bypath was little affected; as a result the flux through the GABA bypath, which under normal conditions was 8% of that through the tricarboxylic acid cycle, decreased to 3–5%. 7. The metabolism of glutamine was greatly affected by K+-stimulation. The ratio of the concentration of glutamine in the slices to that in the medium, which under normal conditions was the smallest among the amino acids investigated, increased from about 17 to 63 in 1h. This effect was attributable partly to an uptake of glutamine from the medium (1.8μmol/h per g) and partly to a net increase in the total amount of glutamine (2.6μmol/h per g). At 1h after the addition of K+ the net gain of glutamine could be accounted for by the decrease of glutamate. 8. Metabolic compartmentation was evident when brain-cortex slices were incubated in glucose–saline medium and the labelled substrate was [14C]GABA, since the specific radioactivity of glutamine exceeded that of glutamate. On addition of K+ the signs of metabolic compartmentation promptly disappeared: this effect was apparently associated with an increase in the permeability of the compartments containing labelled metabolites derived from [14C]GABA. The change in the permeability, however, did not affect all the compartments; when the labelled substrate was [14C]glucose the equilibration of labelled amino acids between tissue and medium was similar under normal conditions and in the presence of high concentrations of K+. 9. The metabolism of [14C]glucose was followed by measuring oxygen uptake, respiratory 14CO2, and incorporation of 14C into amino acids. The results showed that K+ stimulation increased the flux of glucose carbon, both in the glycolytic pathway and in the tricarboxylic acid cycle.

/pdf/the-metabolism-of-g-aminobutyrate-and-glucose-in-potassium-mmhtprck68.pdf

The metabolism of γ-aminobutyrate and glucose in potassium ion-stimulated brain tissue in vitro

THE ROLE of amino acids in the functional activity of the brain has been studied from many angles. It is known that 14C from [14C]glucose is incorporated into amino acids, and especially into glutamate, at a relatively high rate in the brain (cf. VRBA, GAITONDE and RICHTER, 1962). Furthermore it has been found that certain amino acids, including GABA, glutamate and aspartate, affect the rate of firing of nerve cells; and it has been suggested that they may be involved in determining the normal excitability of neuronal membranes in vivo (CURTIS and WATKINS, 1960). There is evidence suggesting that the metabolism of brain cortex slices stimulated by a high K+ ion concentration, or by electrical pulses, approximates more nearly to that obtaining in duo (QUASTEL and QUASTEL, 1961 ; MCILWAIN, 1966). The experiments of KINI and QUASTEL (1959) indicated that the metabolism of GABA in brain slices is influenced by the K+ ion concentration. Furthermore the binding of GABA to subcellular particles seems to be affected by monovalent ions (ELLIOTT, 1965; VARON, WEINSTEIN, BAXTER and ROBERTS, 1965). In view of these considerations a study was made of the effects of high concentrations of K+ ions on the metabolism and distribution of GABA in brain slices in vitro.

D. Richter

Papers

The operation of the γ-aminobutyrate bypath of the tricarboxylic acid cycle in brain tissue in vitro

Changes with age in the utilization of glucose carbon in liver and brain.

The conversion of glucose carbon into protein in the brain and other organs of the rat

The metabolism of γ-aminobutyrate and glucose in potassium ion-stimulated brain tissue in vitro

Effect of K+-stimulation on GABA metabolism in brain slices in vitro.