Glutamic acid decarboxylase activity in striatal slices: Persistent increase following depolarization

When slices prepared from rat corpus striatum were preincubated for 15 min in potassium-enriched Krebs Ringer-Phosphate medium (K⁺-KRP), the activity of glutamic acid decarboxylase measured upon reincubation in normal Krebs-Ringer-Phosphate (KRP) was doubled as compared to GAD activity in slices preincubated in normal KRP. Similarly, when striatal slices were preincubated in KRP containing 100 μM veratridine, GAD activity upon reincubation in normal KRP was increased 66% as compared to activity in slices preincubated in normal KRP. The observed increase in GAD activity was not a function of alterations in glutamate uptake by the slices. These results suggest that GABAergic neurons may regulate transmitter synthesis during the process of depolarization by increasing GAD activity.     

Preincubation stimulates fatty acid synthesis by liver slices and isolated hypatocytes from fasted and diabetic rats.

We have observed that preincubation of 48 hour-fasted or alloxan diabetic rat liver slices, with no exogenous energy supply, for 3 hours resulted in an increased rate of incorporation of [1-¹⁴C] acetate into fatty acids and cholesterol during the following 2 hours. This preincubation effect was enhanced by the presence of glucose (25mM) in or prevented by the addition of dibutyryl cyclic adenosine 3′,5′ monophosphate (10^?4M) to the preincubation medium. Preincubation of normal rat liver slices did not change their rate of incorporation of [1-¹⁴C] acetate into fatty acids or cholesterol. The rate of ¹⁴CO₂ synthesized by normal, fasted or diabetic liver slices was little affected by preincubation. The preincubation effect, i.e. enhanced fatty acid synthesis was also observed in suspensions of hepatocytes from fasted and diabetic rats, preincubated for 2 hours, followed by a 1 hour incubation with either [1-¹⁴C] acetate or [³H] H₂O as precursor. We conclude from these data that there is concurrent and coordinated short- and long-term regulation of fatty acid biosynthesis in fasted and diabetic rat livers. Further, we suggest that the release of inhibition by preincubation of these tissues provides a useful tool for studying the coordinated control     

THE DISTRIBUTION OF GLUTAMATE DECARBOXYLASE IN RAT TISSUES; ISOTOPIC VS FLUORIMETRIC ASSAYS

—The activity of glutamate decarboxylase (GAD, EC 4.1.1.15) in normal and neoplastic rat tissues was determined by two assay methods, one based on the production of ¹⁴CO₂ from [¹⁴C]glutamic acid and the other on the fluorimetric measurement of γ-aminobutyric acid (GABA) formation. Activities obtained with the isotopic assay were high in every tissue (ranging from over 800 in liver and brain to 107nmol CO₂/min/g in lung). They were drastically diminished by Triton X-100, by an oxygen-free atmosphere or by the mitochondrial electron transport inhibitors, rotenone and antimycin A. Activities measured fluorimetrically were significant in only a few tissues and were stimulated by Triton (e.g. from 299 to 569 nmol GABA/min/g brain) but were unaffected by rotenone. For several tissues after Triton treatment the fluorimetric and isotopic assays (in air) gave the same results (i.e. the two end products, CO₂ and GABA were in stoichiometric agreement); however, the fluorimetric assay remains the more reliable measure of GAD activity since Triton may not inhibit completely the non-GAD dependent decarboxylation of glutamate in all types of tissue preparations. The hepatic, renal and mammary tumours tested were devoid of GAD; among non-neural normal tissues, kidney, liver and, possibly, adrenal gland contained significant GAD activity. In kidney and liver the activity was 15 and 10 per cent of that in brain.     

THE METABOLISM OF GABA AND OTHER AMINO ACIDS IN RAT SUBSTANTIA NIGRA SLICES FOLLOWING LESIONS OF THE STRIATO-NIGRAL PATHWAY

The metabolism of GABA and other amino acids from various radioactive precursors has been studied in the rat substantia nigra using a sensitive double isotope dansyl derivative assay. Labelled acetate gave greater labelling of glutamate than of glutamine in substantia nigra slices whereas the reverse was the case for cerebral cortex slices. Unilateral transection of the striato-nigral pathway caused a parallel decrease in the GABA and GAD content of the substantia nigra. It also reduced the total synthesis of GABA from all labelled precursors used, namely acetate, glutamate and glucose. After incubation with [1-¹⁴C]acetate the specific activity of glutamate and aspartate, but not that of GABA, increased on the lesioned side compared with the normal side. The specific activity of glutamate, but not that of GABA or aspartate, decreased after incubation with [U-¹⁴C]glucose on the lesioned side compared with the normal side. The results could be explained by the previously proposed hypothesis concerning differential labelling of metabolic pools by the two precursors. [U-¹⁴C]Glutamate lead to increased labelling of GABA on the lesioned side relative to the normal side. Incubation of slices from substantia nigra with β-mercaptopropionic acid caused a decrease of labelling of GABA from glucose and acetate, probably as the result of GAD inhibition. The labelling pattern of the other amino acids, apart from that of glutamate which showed a decrease when synthesised from acetate, did not change appreciably.     

Gamma-Aminobutyric Acid (GABA) Modulates Nitrate Concentrations and Metabolism in the Leaves of Pakchoi (<Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis> Makino) Treated with a Nitrogen-Rich Solution

Pakchoi plants were grown in 32 mM NO₃^? nutrient solution with or without 2.5 mM γ-aminobutyric acid (GABA) to investigate metabolite changes, gene and protein expression levels, and the activities of key enzymes related to nitrate metabolism in the leaves over a period of 0–12 days. High-nitrogen treatment enhanced plant growth and the NO₃^?, NO₂^?, NH₄⁺, Gln, and Glu contents in the leaves; promoted the gene and protein expression of nitrate reductase (NR) and glutamate decarboxylase (GAD); and increased the activities of NR, nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), and GAD. The endogenous GABA concentration in the leaves was enhanced in parallel with the increase in GAD activity. The GABA-treated leaves displayed the greatest increases in the gene and protein expression levels of NR and GAD and in the activities of NR, NiR, GS, GOGAT, and GAD. In addition, accelerated rates of nitrate reduction and assimilation were detected, and these changes occurred concurrently with the observed increases in gene or protein expression and enzyme activity. As a result, the concentrations of NH₄⁺, Gln, Glu, and endogenous GABA were significantly elevated, and the NO₃^? and NO₂^? contents were significantly decreased, in GABA-treated leaves compared with plants exposed to nitrogen-rich conditions. Our results reveal a potential positive that GABA may act as a nitrogen source to improve the plant growth and the most prominent effect of decreasing nitrate contents by accelerating NO₃^? reduction and assimilation. Exogenous GABA plays an important role in reducing the NO₃^? content of leaves, and thereby improves the ability to harvest leafy vegetables containing higher levels of endogenous GABA.     

Bidirectional Movement of γ-Aminobutyric Acid in Rat Spinal Cord Slices

Abstract: The bidirectional movement of GABA (γ-aminobutyric acid) was studied in slices of rat spinal cord which were incubated in small volumes of medium. The appearance in the medium of endogenous GABA and the disappearance from the medium of [¹⁴C]GABA were used to calculate the rates of unidirectional uptake and unidirectional release of GABA. Under these conditions, no net uptake of GABA was observed when slices were incubated in media containing concentrations of GABA as high as 25 μm . Elevated potassium (60 mm ) stimulated the unidirectional release of endogenous GABA from spinal cord slices by a calcium-dependent process. Ouabain (0.1 mm ) more than doubled the unidirectional release of endogenous GABA in a calcium-independent manner, while unidirectional uptake was inhibited by 44%. Nipecotic acid (1.0 mm ) stimulated unidirectional release and inhibited unidirectional uptake of GABA.     

Regional brain GABA metabolism and release during hepatic coma produced in rats chronically treated with carbon tetrachloride

Hepatic coma was induced in rats chronically treated with CCl₄, by means of a single injection of ammonium acetate. The activities of glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), as well as the synaptosomal uptake and release of [³H]GABA, were measured in the following brain areas of the comatose rats: cortex, striatum, hypothalamus, hippocampus, midbrain and cerebellum. Hepatic coma was associated with a general decrease of GAD activity, whereas GABA-T activity was diminished only in the hypothalamus, striatum and midbrain. During hepatic coma, the K⁺-stimulated [³H]GABA release was notably diminished in the striatum and cerebellum, whereas a significant increase was observed in the hippocampus. [³H]GABA uptake increased in most regions after CCl₄ treatment, independently of the presence of coma. The results indicate that GABAergic transmission seems to be decreased in most cerebral regions during hepatic coma.     

Release of Endogenous Glutamate,Aspartate, GABA,and Taurine from Hippocampal Slices from Adult and Developing Mice under Cell-Damaging Conditions

The releases of endogenous glutamate, aspartate, GABA and taurine from hippocampal slices from 7-day-, 3-, 12-, and 18-month-old mice were investigated under cell-damaging conditions using a superfusion system. The slices were superfused under hypoxic conditions in the presence and absence of glucose and exposed to hydrogen peroxide. In the adult hippocampus under normal conditions the basal release of taurine was highest, with a response only about 2-fold to potassium stimulation (50 mM). The low basal releases of glutamate, aspartate, and GABA were markedly potentiated by K⁺ ions. In general, the release of the four amino acids was enhanced under all above cell-damaging conditions. In hypoxia and ischemia (i.e., hypoxia in the absence of glucose) the release of glutamate, aspartate and GABA increased relatively more than that of taurine, and membrane depolarization by K⁺ markedly potentiated the release processes. Taurine release was doubled in hypoxia and tripled in ischemia but K⁺ stimulation was abolished. In both the mature and immature hippocampus the release of glutamate and aspartate was greatly enhanced in the presence of H₂O₂, that of aspartate particularly in developing mice. In the immature hippocampus the increase in taurine release was 10-fold in hypoxia and 30-fold in ischemia, and potassium stimulation was partly preserved. The release processes of the four amino acids in ischemia were all partially Ca²⁺-dependent. High concentrations of excitatory amino acids released under cell-damaging conditions are neurotoxic and contribute to neuronal death during ischemia. The substantial amounts of the inhibitory amino acids GABA and taurine released simultaneously may constitute an important protective mechanism against excitatory amino acids in excess, counteracting their harmful effects. In the immature hippocampus in particular, the massive release of taurine under cell-damaging conditions may have a significant function in protecting neural cells and aiding in preserving their viability.     

Turnover and release of GABA in rat cortical slices: Effect of a GABA-T inhibitor,gabaculine

The turnover and release of endogenous and labeled GABA were followed in rat cortical slices after incubation with [³H]GABA. High performance liquid chromatography was used to measure endogenous GABA and to separate [³H]GABA from its metabolites. During superfusion with 3 mM K⁺ the slices rapidly lost their [³H]GABA content while maintaining constant GABA levels. Exposure to 50 mM K⁺ for 25 min caused an initial rapid rise in the release of both endogenous and [³H]GABA followed by a more rapid decline in the release of the latter. The specific activity of released GABA was two to four times higher than that in the slices. Depolarization lead to a net synthesis of GABA. The GABA-T inhibitor, gabaculine, (5 M) in vitro arrested the metabolism of [³H]GABA and rapidly doubled the GABA content but did not significantly increase the high K⁺ evoked release of endogenous GABA. In vivo pretreatment with 0.5 mM/kg gabaculine quadrupled GABA content and increased both the spontaneous and evoked release of endogenous GABA but while its Ca²⁺-dependent release increased by 50%, the Ca²⁺-independent release was enhanced sevenfold. This large Ca²⁺-independent release of GABA is likely to have different functional significance from the normal Ca²⁺-dependent release.     

EFFECTS OF TETRODOTOXIN, CALCIUM AND MAGNESIUM ON THE RELEASE OF AMINO ACIDS FROM SLICES OF GUINEA-PIG CEREBRAL CORTEX

Abstract— Tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ were used in an effort to identify the portion of the evoked release of endogenous amino acids, labelled via metabolism of [¹⁴C]-glucose, and several exogenous labelled amino acids, that came from nerve terminals when slices of guinea pig cerebral cortex were superfused with glucose-free solutions and stimulated electrically. With some exceptions, spontaneous release of labelled amino acids was decreased by 2 μm -tetrodotoxin but increased in Ca²⁺-free medium and in solutions containing an extra 24 mm -MgCl₂. Tetrodotoxin suppressed 85–90% of the stimulated release of almost all labelled amino acids, but had a smaller effect on the release of endogenous ¹⁴C-labelled threonine-serine-glutamine (unseparated). In Ca²⁺-free solution, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 80–90%, but that of endogenous ¹⁴C-labelled threonine-serine-glutamine was unaffected as was most of the release of the other labelled amino acids. In medium containing an extra 24mM-MgCl₂, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 75-85%, that of exogenous labelled aspartate and GABA by 50–65%, but the release of the other labelled amino acids was unaffected. The control stimulated releases of endogenous ¹⁴C-labelled glutamate, aspartate and GABA were much larger than those of other labelled amino acids but were reduced by tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ to a level similar to that of the control stimulated releases of the other labelled amino acids. These results suggest that almost all of the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA came from nerve terminals while those of the other labelled amino acids came from other tissue elements. In addition, they are in accord with a transmitter role for glutamate, aspartate and GABA in cerebral cortex.     

Stimulation of Synaptosomal γ-Aminobutyric Acid Synthesis by Glutamate and Glutamine

gamma-Aminobutyric acid (GABA) synthesis was studied in rat brain synaptosomes by measuring the increase of GABA level in the presence of the GABA-transaminase inhibitor gabaculine. The basal rate of synaptosomal GABA synthesis in glucose-containing medium (25.9 nmol/h/mg of protein) was only 3% of the maximal activity of glutamate decarboxylase (GAD; 804 +/- 83 nmol/h/mg of protein), a result indicating that synaptosomal GAD operates at only a small fraction of its catalytic capacity. Synaptosomal GABA synthesis was stimulated more than threefold by adding 500 microM glutamine. Glutamate also stimulated GABA synthesis, but the effect was smaller (1.5-fold). These results indicate that synaptosomal GAD is not saturated by endogenous levels of its substrate, glutamate, and account for part of the unused catalytic capacity. The greater stimulation of GABA synthesis by glutamine indicates that the GAD-containing compartment is more accessible to extrasynaptosomal glutamine than glutamate. The strong stimulation by glutamine also shows that the rates of uptake of glutamine and its conversion to glutamate can be sufficiently rapid to support GABA synthesis in nerve terminals. Synaptosomes carried out a slow net synthesis of aspartate in glucose-containing medium (7.7 nmol/h/mg of protein). Aspartate synthesis was strongly stimulated by glutamate and glutamine, but in this case the stimulation by glutamate was greater. Thus, the larger part of synaptosomal aspartate synthesis occurs in a different compartment than does GABA synthesis.     

POSTNATAL ALTERATIONS IN EFFECTS OF POTASSIUM ON UPTAKE AND RELEASE OF GLUTAMATE AND GABA IN RAT BRAIN CORTEX SLICES

The uptake and release of glutamate and of GABA, as well as the effect of high potassium concentrations (35 or 80 mM) hereupon, were studied by aid of ¹⁴C-labelled amino acids in brain cortex slices from rats of different ages between birth and adulthood. Both the extent of the uptake (i.e. the tissue/medium ratio of ¹⁴C at, or close to, equilibrium) and the rate of uptake (i.e. the tissue/ medium ratio of ¹⁴C after short (5 min) incubation periods) increased with age. Differences were, however, found between glutamate and GABA, and the extent of the GABA uptake had a distinct maximum during the second postnatal week. At all ages, high concentrations of potassium caused a decrease in the rate of GABA uptake but were without effect on the rate with which glutamate was taken up. The release of the two amino acids occurred with approximately the same half-time (50 min) in slices from animals of at least 14 days of age. Before that time the release of glutamate was somewhat faster, whereas that of GABA was much slower, especially during the first postnatal week (half-time 90 min). The ontogenetic alterations in the effect of excess potassium were complex and varied both between the two potassium concentrations used and between the two amino acids. The results are thus compatible with the existence of different transport systems for the two amino acids, They also suggest that glutamate may exert other functions in addition to its role as a putative transmitter.     

POTASSIUM-INDUCED RELEASE OF [3H]GABA AND OF [3H]NORADRENALINE FROM NORMAL AND RESERPINIZED RAT BRAIN CORTEX SLICES. DIFFERENCES IN CALCIUMDEPENDENCY, AND IN SENSITIVITY TO POTASSIUM IONS

The release of [³H]GABA induced by elevated extracellular potassium (K)_o, from thin rat brain cortex slices, has been compared with that of [³H]noradrenaline ([³H]NA), released by the same procedures, both from normal slices, and from slices pre-treated with reserpine and nialamide, [³H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM-(K)_o released considerably more [³H]NA from normal than from drug-treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [³H]NA from drug-treated slices increased in proportion to (K)_o, up to 36-46 mM and then declined considerably with higher (K)_o. The dependency of potassium-induced release on the concentration of calcium in the medium, indicated that release of [³H]NA from normal slices was proportional to calcium up to 1.5-2 mM, while that of [³H]NA from drug-treated slices increased up to 0.5 mM-Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM-calcium, but no further changes were observed at higher concentrations. The calcium antagonist D-600 inhibited high (K)_o-induced release of [³H]NA from normal slices to a greater extent than that of [³H]GABA or of [³H]NA from drug-treated slices. These results, in which elevated (K)_o-induced release of [³H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [³H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)_o as a stimulus for inducing transmitter secretions.     

Effects of spinal transection on presynaptic markers for glutamatergic neurons in the rat

To evaluate the hypothesis that glutamic acid may be the neurotransmitter of descending, excitatory supraspinal pathways, the uptake and release ofl-[³H] glutamate and the levels of endogenous glutamate were measured in preparations from rat lumbar spinal cord following complete mid-thoracic transection. Following transection, the activity of the synaptosomal high-affinty glutamate uptake process was increased in both dorsal and ventral halves of lumbar cord between 1 and 14 days after transection and returned to control levels by 21 days posttransection. At 7 days, the increased activity of the uptake process forl-[³H] glutamate resulted in elevation ofV _max with no significant alteration inK _t as compared to age-matched controls. Depolarization-induced release ofl-[³H]glutamate from prelabeled slices did not differ significantly from control in the lesioned rat except at 21 days after lesion when the amount of tritium release was significantly greater in the transected preparations than in control. Amino acid analysis of the lumbar cord from control and transected rats indicated only a 10% decrease in the level of endogenous glutamate and no alterations in the concentration of GABA and glycine 7 days after lesion. These findings do not support the hypothesis that glutamate serves as a major excitatory neurotransmitter in supraspinal pathways innervating the lumbar cord of the rat.     

The rate of turnover of cortical GABA from [1-13C]glucose is reduced in rats treated with the GABA-transaminase inhibitor vigabatrin (γ-vinyl GABA)

Brain GABA levels rise and plateau following prolonged administration of the irreversible GABA-transaminase inhibitor vigabatrin (γ-vinylGABA). Recently it has been shown that increased GABA levels reduces GAD₆₇ protein, one of two major isoforms of glutamic acid decarboxylase (GAD). The effects of GABA elevation on GABA synthesis were assessed in vivo using¹H and¹³C-edited NMR spectroscopy. Rates of turnover of cortical glutamate and GABA from intravenously administered [1-¹³C]glucose were measured in α-chloralose anesthetized rats 24 hours after receiving vigabatrin (500 mg/kg, i.p.) and in non-treated controls. GABA concentration was increased 2-fold at 24 hours (from 1.3±0.4 to 2.7±0.9 μmol/g) and GABA-T activity was inhibited by 60%. Tricarboxylic acid cycle flux was not affected by vigabatrin treatment compared to non-treated rats (0.47±0.19 versus 0.52±0.18 μmol/g, respectively). GABA-C2 fractional enrichment (FE) measured in acid extracts rose more slowly in vigabatrin-treated compared to nontreated rats, reaching >90% of the glutamate FE after 3 hours. In contrast, GABA FE≥glutamate FE in non-treated rats. A metabolic model consisting of a single glutamate pool failed to account for the rapid labeling of GABA from glutamate. Metabolic modelling analysis based on two (non-communicating) glutamate pools revealed a ∼70% decrease in the rate of GABA synthesis following vigabatrin-treatment, from 0.14 (non-treated) to 0.04 μmol/g/min (vigabatrin-treated). These findings, in conjunction with the previously reported differential effects of elevated GABA on the GAD isoforms, suggests that GAD₆₇ may account for a major fraction of cortical GABA synthesis in the α-chloralose anesthetized rat brain in vivo. Special issue dedicated to Dr. Herman Bachelard.     

THE SPONTANEOUS AND ELECTRICALLY EVOKED RELEASE, FROM SLICES OF GUINEA-PIG CEREBRAL CORTEX, OF ENDOGENOUS AMINO ACIDS LABELLED VIA METABOLISM OF d-[U-14C]GLUCOSE

Abstract— In an effort to identify neurotransmitters in slices of guinea-pig cerebral cortex, a study was made of the release of endogenous amino acids which had become labelled via metabolism of d -[U-¹⁴C]glucose. While incorporation of ¹⁴C into endogenous glutamate, aspartate, GABA, alanine and threonine-serine-glutamine (unseparated) was large enough to permit measurement of their release, that into other amino acids was not. In parallel experiments, the release of exogeneous labelled glutamate, aspartate, GABA and α-aminoisobutyrate was examined. Electrical field stimulation evoked a transient increase in the release of all the adequately labelled endogenous amino acids and all the exogenous amino acids. The stimulated ‘increase’ in the release of each of the endogenous ¹⁴C-labelled transmitter candidates (glutamate, aspartate and GABA) was larger than that of any other amino acid (except that of exogenous GABA). When the experiments were performed without the glucose (5 mm ) usually present in the medium bathing the slices, larger amounts of each labelled amino acid were released from the slices than in the presence of glucose. Moreover, the pattern of selective release of the endogenous labelled transmitter candidates was much more pronounced in the absence of glucose. It is likely that in the absence of glucose, release from the tissue was larger because cells in the slice were relatively depolarized and uptake of amino acids into cells was impaired. Because previous evidence suggests that over 90% of glucose consumption occurs in the ‘large metabolic compartment’ which is thought to be composed of neuronal elements, neurons were probably the main site from which the larger release of endogenous ¹⁴C-labelled transmitter candidates was evoked. The exogenous amino acids were probably released from several cellular elements in the slices. It was concluded that the pattern of a selective release of the endogenous labelled transmitter candidates may have been indicative of a transmitter releasing mechanism in nerve terminals.     

Distribution and tissue specificity of glutamate decarboxylase (EC 4.1.1.15).

Abstract— The activity of L–glutamate decarboxylase (EC 4.1.1.15) (GAD) in various mouse tissues was determined by five different methods, namely, the radiometric CO₂ method, column separation, electro–phoretic separation, the filtration method, and amino acid analysis. Results from the latter four methods agreed well, showing that brain had the highest activity, 4.27 nmol/min/mg protein (100%), followed by heart (7.4%), kidney (6.3%) and liver (1.5%). Measurement of brain GAD using the radiometric CO₂ assay method agreed with the other techniques. However, in heart, kidney, and liver, the GAD activities measured by the CO₂ method were about 3–4 times higher than those obtained by the GABA method, suggesting that the CO₂ method does not give a valid measurement of GAD activity in a crude non–neural tissue preparation. GAD activity also was detected in adrenal gland but not in pituitary, stomach, testis, muscle, uterus, lung, salivary gland, or spleen. GAD from brain, spinal cord, heart, kidney and liver were further compared by double immunodiffusion, enzyme inhibition by antibody, and microcomplement fixation using antibody against GAD purified from mouse brain. GAD from brain and spinal cord appear to be identical as judged from the following results: the immunoprecipitin bands fused together without a spur; the enzyme activity was inhibited by anti–GAD to the same extent; and the microcomplement fixation curves were similar in both the shape of the curve and the extent of fixation. No crossreactivity was observed between GAD from heart, kidney or liver and antibody against brain GAD in all the immunochemical tests described above, suggesting that GAD in non–neural tissues is different from that in brain and spinal cord.     

A Two-Translocator Model for the Transport of 2-Oxoglutarate and Glutamate in Chloroplasts during Ammonia Assimilation in the Light

This study examines the transport of 2-oxoglutarate (2-OG) and other dicarboxylates during ammonia assimilation in illuminated spinach chloroplasts. The transport of all dicarboxylates examined was strongly inhibited by NH₄Cl preincubation in the light. Treatment with NH₄Cl caused a rapid depletion of the endogenous glutamate pool and a corresponding increase in endogenous glutamine content. The inhibition of transport activity by NH₄Cl was apparently linked to its metabolism in the light because inhibition of glutamine synthetase activity by the addition of l-methionine sulfoximine or carbonylcyanide-m-chlorophenylhydrazone abolished this affect. Measurements of endogenous metabolite pools showed that malate was most rapidly exchanged during the uptake of all exogenous dicarboxylates examined. Depending on the exogenous substrates used, the apparent half-times of efflux measured for endogenous malate, aspartate and glutamate were 10, 10 to 30, and 15 to 240 seconds, respectively. The transport of 2-OG was also inhibited by malate. But chloroplasts preincubated with malate in the presence or absence of NH₄Cl were found to have high transport activity similar to untreated chloroplasts. A two-translocator model is proposed to explain the stimulation of 2-OG transport as well as the stimulation of (NH₃, 2-OG)-dependent O₂ evolution by malate (KC Woo, CB Osmond 1982 Plant Physiol 69: 591-596) in isolated chloroplasts. In this model the transport of 2-OG on the 2-OG translocator and glutamate on the dicarboxylate translocator is coupled to malate counter-exchange in a cascade-like manner. This results in a net 2-OG/glutamate exchange with no net malate transport. Thus, during NH₃ assimilation the transport of 2-OG into and the export of glutamate out of the chloroplast occurs via the 2-OG and the dicarboxylate translocators, respectively.     

Regulation of γ-Aminobutyric Acid Synthesis in the Brain

Abstract: γ-Aminobutyric acid (GABA) is synthesized in brain in at least two compartments, commonly called the transmitter and metabolic compartments, and because reglatory processes must serve the physiologic function of each compartment, the regulation of GABA synthesis presents a complex problem. Brain contains at least two molecular forms of glutamate decarboxylase (GAD), the principal synthetic enzyme for GABA. Two forms, termed GAD₆₅ and GAD₆₇, are the products of two genes and differ in sequence, molecular weight, interaction with the cofactor, pyridoxal 5′-phosphate (pyridoxal-P), and level of expression among brain regions. GAD₆₅ appears to be localized in nerve terminals to a greater degree than GAD₆₇, which appears to be more uniformly distributed throughout the cell. The interaction of GAD with pyridoxal-P is a major factor in the short-term regulation of GAD activity. At least 50% of GAD is present in brain as apoenzyme (GAD without bound cofactor; apoGAD), which serves as a reservoir of inactive GAD that can be drawn on when additional GABA synthesis is needed. A substantial majority of apoGAD in brain is accounted for by GAD₆₅, but GAD₆₇ also contributes to the pool of apoGAD. The apparent localization of GAD₆₅ in nerve terminals and the large reserve of apo-GAD₆₅ suggest that GAD₆₅ is specialized to respond to short-term changes in demand for transmitter GABA. The levels of apoGAD and the holoenzyme of GAD (holoGAD) are controlled by a cycle of reactions that is regulated by physiologically relevant concentrations of ATP and other polyanions and by inorganic phosphate, and it appears possible that GAD activity is linked to neuronal activity through energy metabolism. GAD is not saturated by glutamate in synaptosomes or cortical slices, but there is no evidence that GABA synthesis in vivo is regulated physiologically by the availability of glutamate. GABA competitively inhibits GAD and converts holo- to apoGAD, but it is not clear if intracellular GABA levels are high enough to regulate GAD. There is no evidence of short-term regulation by second messengers. The syntheses of GAD₆₅ and GAD₆₇ proteins are regulated separately. GAD₆₇ regulation is complex; it not only is present as apoGAD₆₇, but the expression of GAD₆₇ protein is regulated by two mechanisms: (a) by control of mRNA levels and (b) at the level of translation or protein stability. The latter mechanism appears to be mediated by intracellular GABA levels.     

Glyoxylate decreases the oxygen sensitivity of nitrogenase activity and photosynthesis in the cyanobacterium Anabaena cylindrica

Raising the pO₂ reduced nitrogenase activity (C₂H₂ reduction) of Anabaena cylindrica for both glyoxylate-treated (5 mM) and untreated cells. The stimulation caused by glyoxylate, however, increased with increases of pO₂ from 2 to 99 kPa. As the pO₂ increased the net CO₂ fixation was lowered (Warburg effect) while the CO₂ compensation point increased. Glyoxylate partly relieved this sensitivity of net photosynthesis to oxygen and reduced the compensation point considerably. The cells used were preincubated in the dark to exhaust photosynthetic pools. A more pronounced reduction in sensitivity of nitrogenase to oxygen for glyoxylate-treated cells was evident when a preincubation in air with reduced pCO₂ (13 l l^-1) was used. This was, however, not evident until after a 10-h incubation in air. Before this point 2 kPa O₂ sustained the highest nitrogenase activity. Addition of 0.5 and 5 mM of HCO ₃ ^- to Anabaena cultures preincubated at low CO₂ levels (29 l l^-1) abolished the stimulatory effect of glyoxylate on the nitrogenase. Thus, the results sustain the suggestion that glyoxylate may act as an inhibitor of photorespiratory activities in cyanobacteria and can be used as a means of increasing their nitrogen and CO₂ fixation capacities.Abbreviation RuBP ribulose 1,5-bisphosphate