Regulation of γ-aminobutyric acid synthesis in the vertebrate nervous system

The regulation of glutamic decarboxylase (GAD) activity is undoubtedly the key to the control of the steady-state concentrations of 4-aminobutyric acid (GABA) in the central nervous system. Those factors that might influence GAD activity are reviewed. They include repression and induction of GAD synthesis; the interconversion of the holo- and apo-form of GAD; the availability of substrate and cofactor; the competitive inhibition of GAD by endogenous substances, including GABA; and the involvement of calcium ions in whole-cell preparations. Where possible mechanisms of action are described, and the likelihood that each is of physiological importance is discussed. Experiments are suggested that would help clarify (1) the role of GABA in GAD repression; (2) the possible phosphorylation of GAD; and (3) the existence of multiple forms of the enzyme. In addition, a kinetic mechanism is proposed to explain the possible regulation of GAD by the interconversion of the holo- and apo-forms of the enzyme. It is concluded that the overriding factors responsible for GAD regulation are not yet understood. However, a possible mechanism relying on the direct feedback action of GABA on GAD activity has many attractive features.     

Conversion of 4-aminobutyraldehyde to γ-aminobutyric acid in striatum treated with semicarbazide and kainic acid

4-Aminobutyraldehyde (ABAL) has been shown to cross the blood-brain barrier and to be converted rapidly to -aminobutyric acid (GABA) in various regions of the brain. In this paper, the formation of GABA from ABAL was studied with striatum that had suffered a lesion to GABA synthesis via glutamic acid decarboxylase (GAD). The GABA formation from ABAL was invariably observed in striatum in which GAD was severely inhibited by semicarbazide or kainic acid. Thus, this is another pathway for GABA formation.     

Entropy as a factor in the binding of γ-aminobutyric acid and nipecotic acid to the γ-aminobutyric acid transport system

Nipecotic acid is one of the most potent competitive inhibitors and alternative substrates for the high-affinity -aminobutyric acid transport system in neurons, but the structural basis of this potency is unclear. Because -aminobutyrate is a highly flexible molecule in solution, it would be expected to lose rotational entropy upon binding to the transport system, a change which does not favor binding. Nipecotic acid, in contrast, is a much less flexible molecule, and one would expect the loss of conformational entropy upon binding to be smaller thus favoring the binding of nipecotic acid over -aminobutyric acid. To investigate this possibility, the thermodynamic parameters, G°, H°, and S°, were determined for the binding of -aminobutyrate and nipecotic acid to the high affinity GABA transport system in synaptosomes. In keeping with expectations, the apparent entropy change for nipecotic acid binding (112±13 J·K^–1) was more favorable than the apparent entropy change for -aminobutyric acid binding (61.3±6.6 J·K^–1). The results suggest that restricted conformation per se is an important contributory factor to the affinity of nipecotic acid for the high-affinity transport system for -aminobutyric acid.This work was conducted when both authors were at the Department of Chemistry, University of Maryland, College Park.Special issue dedicated to Dr. Elling Kvamme.     

High-affinity uptake of γ-aminobutyric acid in cultured glial and neuronal cells

Both glial and neuronal cells maintained in primary culture were found to accumulate [³H]GABA by an efficient high-affinity uptake system (apparentK _m=9 M,V _max=0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1 mM) also strongly inhibited uptake of [³H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, -alanine, and 2,4-diaminobutyrate) inhibited uptake of [³H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine,l-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (K _m=92 M,V _max=0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [³H]GABA against a concentration gradient. ApparentK _m of this uptake was relatively high (819 M), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, none of the nontransformed continuous cell lines of either tumoral (glioma, C₆; neuroblastoma, Ml; MINN) or normal (NN; I₆) origin actively accumulated [³H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.     

Effect of gabaculine on metabolism and release of γ-aminobutyric acid in synaptosomes

Synaptosomes isolated from mouse brain were incubated with [¹⁴C]glutamate and [³H]-aminobutyric acid ([³H]GABA), and then [¹⁴C]GABA (newly synthesized GABA) and [³H]GABA (newly captured GABA) in the synaptosomes were analysed. (1) the [³H]GABA was rapidly degraded in the synaptosomes, (2) when the synaptosomes were treated with gabaculine (a potent inhibitor of GABA aminotransferase), the degradation of [³H]GABA was strongly inhibited, (3) the gabaculine treatment brough about a significant increase in Ca²⁺-independent release of [³H]GABA with no effect on Ca²⁺-dependent release, (4) no effects of gabaculine on degradation and release of [¹⁴C]GABA were observed. The results indicate that there are at least two pools of GABA in synaptosomes and support the possibilities that GABA taken up into a pool which is under the influence of GABA aminotransferase is released Ca²⁺-independently and that GABA synthesized in another pool which is not under the influence of GABA aminotransferase is released Ca²⁺-dependently.     

Light- and electron-microscopic visualization of γ-aminobutyric acid and GABA-transaminase in the oviduct of rats

Summary The distribution in the rat oviduct of -aminobutyric acid and its catabolic enzyme GABA-transaminase was studied by the use of immunocytochemical and enzymehistochemical techniques. At the light-microscopic level, both GABA immunoreactivity and GABA-transaminase enzyme reactivity were found primarily in the tubal epithelium while in the muscle layers of the organ only a faint GABA and GABA-transaminase positive staining could be detected. Electron-microscopic evaluation of the GABA immunoreactivity revealed a heavy labelling of the basal bodies (kinetosomes) and a moderate staining of the cilia. These findings indicate that the role of GABA in the oviduct is not related to neurotransmission but may be related to ciliary functions.     

Involvement of sialic acid in the regulation of γ--aminobutyric acid uptake activity of γ-aminobutyric acid transporter 1

The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.     

Fluorescence ‘turn-off–on’ assays for neomycin sulphate and K+ ions with orange-red fluorescent molybdenum nanoclusters

Fluorescent metal nanoclusters (MNCs) have found extensive application in recognizing molecular species. Here, orange-red fluorescent Arg–A. paniculata–MoNCs were synthesized using Andrographis paniculata leaf extract, arginine as a ligand, and MoCl₅ as a metal precursor. The Arg–A. paniculata–MoNCs complex exhibited a quantum yield (QY) of 16.91% and excitation/emission wavelengths of 400/665 nm. The synthesized Arg–A. paniculata–MoNCs successfully acted as a probe for assaying neomycin sulphate (NS) via fluorescence turn-off and K⁺ ions via fluorescence turn-on mechanisms, respectively. Moreover, the developed probe was effectively used to develop a cellulose paper strip-based sensor for detection of NS and K⁺ ions. Arg–A. paniculata–MoNCs demonstrated great potential for sensing NS and K⁺ ions, with concentration ranges of 0.1–80 and 0.25–110 μM for NS and K⁺ ions, respectively. The as-synthesized Arg–A. paniculata–MoNCs efficiently detected NS and K⁺ ions in food and biofluid samples, respectively.     

Synergistic effect of phosphatidylserine with γ-aminobutyric acid in antagonizing the isoniazid-induced convulsions in mice

The influence of phosphatidylserine (PS) on the isoniazid-induced convulsions has been studied in mice. Sonicated dispersions of this phospholipid given intravenously do not show anticonvulsant activity but they do so when -aminobutyric acid (GABA) is simultaneously injected. GABA alone is inactive. The synergism between PS and GABA is influenced by the structure of the phospholipid liposomes. In contrast to multilamellar vesicles, oligolamellar vesicles are active. Under these conditions the effect shows head group specificity, in that the neutral phosphatidylcholine (PC) or the acidic phosphatidylinositol (PI) are inactive, either in the presence or in the absence of GABA. Lysophosphatidylserine (lysoPS), the deacylated PS derivative, shows increased efficacy as an isoniazid antagonist in the presence of GABA, and has anticonvulsant activity also in the absence of GABA. Other lysophospholipids are inactive. It is suggested that PS, after its metabolic conversion to lysoPS, enhances the anticonvulsant effect of GABA.     

Dietary γ-aminobutyric acid affects the brain protein synthesis rate in young rats

Summary. The purpose of this study was to determine whether the γ-aminobutyric acid (GABA) affects the rate of brain protein synthesis in male rats. Two experiments were done on five or three groups of young rats (5 wk) given the diets containing 20% casein administrated 0 mg, 25 mg, 50 mg, 100 mg or 200 mg/100 g body weight GABA dissolved in saline by oral gavage for 1 day (d) (Experiment 1), and given the diets contained 0%, 0.25% or 0.5% GABA added to the 20% casein diet (Experiment 2) for 10 d. The plasma concentration of growth hormone (GH) was the highest in rats administrated 50 mg and 100 mg/100 g body weight GABA. The concentration of serum GABA increased significantly with the supplementation groups. The fractional (Ks) rates of protein synthesis in brain regions, liver and gastrocnemius muscle increased significantly with the 20% casein + 0.25% GABA diet and still more 20% casein + 0.5% GABA compared with the 20% casein diet. In brain regions, liver and gastrocnemius muscle, the RNA activity [g protein synthesized/(g RNA·d)] significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. Our results suggest that the treatment of GABA to young male rats are likely to increase the concentrations of plasma GH and the rate of protein synthesis in the brain, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.     

Monitoring γ-aminobutyric acid in human brain and plasma microdialysates using micellar electrokinetic chromatography and laser-induced fluorescence detection

Due to its low electrophoretic mobility, few studies have been able to measure gamma aminobutyric acid (GABA) in biological samples by means of capillary zone electrophoresis. Nevertheless, in micellar electrokinetic chromatography (MEKC) by adding a surfactant to the mobile phase separation can be carried out on the basis of the partition coefficient of the molecules rather than their electrophoretic mobility. In the present study microdialysis coupled to MEKC with laser induced fluorescence detection was used to successfully monitor GABA from cerebrospinal fluid and plasma dialysates. Moreover, we monitored changes in extracellular GABA from a human brain. Microdialysis samples were collected from a Parkinson’s disease patient undergoing a thallamotomy as part of her treatment. Significant decreases in extracellular GABA were detected during high frequency electrical stimulation and following a thermolesion of the thalamus. These results demonstrate the feasibility of MEKC coupled to laser-induced fluorescence detection in resolving neutral amino acids, specifically GABA, from different human body fluids.     

Metabolic engineering of microorganisms for the production of multifunctional non-protein amino acids: γ-aminobutyric acid and δ-aminolevulinic acid

Gamma-aminobutyric acid (GABA) and delta-aminolevulinic acid (ALA), playing important roles in agriculture, medicine and other fields, are multifunctional non-protein amino acids with similar and comparable properties and biosynthesis pathways. Recently, microbial synthesis has become an inevitable trend to produce GABA and ALA due to its green and sustainable characteristics. In addition, the development of metabolic engineering and synthetic biology has continuously accelerated and increased the GABA and ALA yield in microorganisms. Here, focusing on the current trends in metabolic engineering strategies for microbial synthesis of GABA and ALA, we analysed and compared the efficiency of various metabolic strategies in detail. Moreover, we provide the insights to meet challenges of realizing industrially competitive strains and highlight the future perspectives of GABA and ALA production.     

The role of γ-aminobutyric acid and its receptors in the nucleus of basal optic root in pigeons

The effects of γ-aminobutyric acid (GABA) and its antagonists bicuculline and 2-hydroxysaclofen on neuronal firings in the nucleus of basal optic root (nBOR) in pigeons were studied by using extracellular recording and microiontophoretic techniques. The results suggest that GABA may be an inhibitory neurotransmitter or modulator within nBOR, functioning by means of main mediation of GABAA receptors and of minor mediation of GABAB receptors. Furthermore, GABA and its GABAA receptors are involved in the modulation of directional selectivity in part of nBOR neurons.     

Involvement of γ-aminobutyric acid transporter 2 in the hepatic uptake of taurine in rats

Taurine is essential for the hepatic synthesis of bile salts and, although taurine is synthesized mainly in pericentral hepatocytes, taurine and taurine-conjugated bile acids are abundant in periportal hepatocytes. One possible explanation for this discrepancy is that the active supply of taurine to hepatocytes from the blood stream is a key regulatory factor. The purpose of the present study is to investigate and identify the transporter responsible for taurine uptake by periportal hepatocytes. An in vivo bolus injection of [(3)H]taurine into the rat portal vein demonstrated that 25% of the injected [(3)H]taurine was taken up by the liver on a single pass. The in vivo uptake was significantly inhibited by GABA, taurine, β-alanine, and nipecotic acid, a GABA transporter (GAT) inhibitor, each at a concentration of 10 mM. The characteristics of Na(+)- and Cl(-)-dependent [(3)H]taurine uptake by freshly isolated rat hepatocytes were consistent with those of GAT2 (solute carrier SLC6A13). Indeed, the K(m) value of the saturable uptake (594 μM) was close to that of mouse SLC6A13-mediated taurine transport. Although GABA, taurine, and β-alanine inhibited the [(3)H]taurine uptake by > 50%, each at a concentration of 10 mM, GABA caused a marked inhibition with an IC(50) value of 95 μM. The [(3)H]taurine uptake exhibited a significant reduction when the GAT2 gene was silenced. Immunohistochemical analysis showed that GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. These results suggest that GAT2 is responsible for taurine transport from the circulating blood to hepatocytes predominantly in the periportal region.     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

Cycling treatment of anaerobic and aerobic incubation increases the content of γ-aminobutyric acid in tea shoots

Summary. γ-Aminobutyric acid (GABA), a hypotensive compound, is formed from glutamic acid under anaerobic condition in tea shoots. Glutamic acid was exhausted in the first three hours of anaerobic incubation and the increase of GABA stopped. After that, when tea shoots were released under aerobic condition, glutamic acid reproduced rapidly. After one hour of aerobic incubation, tea shoots were given three hours of anaerobic incubation again and then accumulated glutamic acid changed to GABA. The content of GABA increased much more than usual anaerobic incubation. GABA was more in the tea stem than in the leaf. Received January 4, 2000 Accepted March 1, 2000     

Is hypoxia a stimulus for synthesis of oxidative enzymes and myoglobin?

To compare two situations with similar magnitudes of mitochondrial substrate flux but different blood oxygen contents, one-legged training was employed. Ten healthy subjects trained one leg under normobaric conditions and the other under hypobaric conditions. At each session the subjects trained each leg for 30 min. The absolute work intensity was the same for both legs and was chosen to correspond to 65% of the average (right and left) pretraining one-legged maximal work capacity. There were three to four training sessions per week for 4 wk. Muscle biopsies from each leg were taken before and after training and analyzed for fiber types, capillaries, myoglobin, and oxidative and glycolytic enzymes. The most striking finding was a greater increase of citrate synthase activity under hypobaric conditions than under normobaric conditions. In addition, the myoglobin content increased in the leg trained under hypobaric conditions, whereas it tended to decrease in the normobarically trained leg. Because both legs were trained at the same intensity, the oxygen turnover and the substrate flux through the carboxylic acid cycle and the respiratory chain must have been of similar magnitude. Thus a difference in substrate flux is less likely to have caused the differences in enzyme activities and myoglobin content between training under normobaric and hypobaric conditions. Instead, the stimulus seems to be related to the blood oxygen content or tension.