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.     

Properties of γ-aminobutyric acid synthesis by rat renal cortex

Substantial synthesis of γ-aminobutyric acid occurs in rat renal cortex. Renal glutamate decarboxylase activity (24.3±2.9 (S.E.) nmols/mg protein per h) is 15% of that in brain; renal γ-aminobutyric acid content (39.5±5.3 (S.E.) nmols/g wet wt.) is 5% of the whole brain concentration. Properties of glutamate decarboxylase were studied in homogenates of rat renal cortex and rat brain under conditions for which γ-aminobutyric acid formation from [2,3-³H]glutamate and CO₂ release from [1-¹⁴C]glutamate were equal. Several properties of renal glutamate decarboxylase distinguish it from the corresponding brain enzyme: (1) renal glutamate decarboxylase is selectively inhibited by cysteine sulfinic acid (K_i = 5·10^?5 M) ; (20 renal glutamate decarboxylase is less sensitive (K_i = 3–5·10^?5 M)_to inhibition by aminooxyacetic acid than is the brain enzyme (K_i = 1·10^?6 M); (3) brain but not renal glutamate decarboxylase activity can be substantially stimulated in vitro by the addition of exogenous pyridoxal 5′-phosphate; (4) renal glutamate decarboxylase is significantly decreased in renal cortex from rats on a low-salt diet. Proximal tubules are enriched in glutamate decarboxylase compared to the activity in whole renal cortex or glomeruli (42, 22 and 14 nmols/mg protein per h, respectively). We speculate that renal γ-aminobutyric acid synthesis does not reflect the presence of GABAergic renal nerves, but may serve a function in proximal tubular cells.     

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.     

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.     

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.     

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.     

Microwave irradiation and brain γ-aminobutyric acid levels in mice

Microwave irradiation sufficient to raise the core brain temperatures of mice to >60°C is essential to prevent post mortem increases in γ-aminobutyric acid levels. Physiological or drug-induced changes in the body temperatures of mice profoundly effect the final temperatures of the brains after microwave treatment and may lead to spurious values for putative neurotransmitter concentration estimates. This paper points out the importance of certain data each laboratory $\text{must}$ establish for itself when applying microwave technique.     

Rapid and sensitive ion-exchange fluorometric measurement of γ-aminobutyric acid in physiological fluids

Improvements of the ion-exchange/fluorometric method for measuring γ-aminobutyric acid (GABA) have resulted in an automatic three-column analyzer which produces an analysis every 15 min using 200-μl samples to a sensitivity of 1 pmol. This high-performance liquid chromatographic procedure utilizes three stainless-steel microbore columns containing cation-exchange resin. GABA is eluted using a lithium citrate buffer and then detected in the flow stream after reaction with the fluorogenic reagent orthophthalaldehyde. Each column utilizes a timer-controlled, pneumatically actuated two-way 10-port valve containing a 200-μl loop for sample injection and a 1-ml loop for regeneration. Similar 4-port valves sequentially direct the output from one of the columns to the reaction manifold. Measurement of GABA levels in sequential aliquets of the first 20 ml of human lumbar cerebrospinal fluid revealed an increasing gradient of GABA level in cerebrospinal fluid from six patients and no gradient in a seventh patient, the mean rate of increase being 2% per milliliter.     

Biosynthesis of γ-aminobutyric acid from spermine in maize seedlings

The administration of labelled spermine [tetramethylene-1,4-¹⁴C] to Zea mays shoots resulted in the formation of radioactive γ-aminobutyric acid (GABA). A chemical degradation of radioactive GABA suggested that its radioactivity was located on C-1 and C-4, indicating that GABA is a product of spermine metabolism in maize seedlings.     

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.     

Efflux of γ-aminobutyric acid from and appearance of free arachidonic acid inside synaptosomes

When synaptosomes were depolarized in the presence of Ca²⁺, or when Ca²⁺ was added to synaptosomes pretreated with Ca²⁺ ionophore (A23187), free arachidonic acid was clearly increased within synaptosomes, and at the same time an efflux of γ-aminobutyric acid from synaptosomes was observed. Moreover, when synaptosomes labelled with [¹⁴C]arachidonic acid were depolarized in the presence of Ca²⁺, there was a significant decrease in the radioactivity of the fatty acid of phosphatidylinositol and phosphatidylcholine. Exogenously added arachidonic acid, but not other fatty acids, stimulated the efflux of γ-aminobutyric acid in the absence of Ca²⁺. These observations suggest that the release of arachidonic acid from phospholipids is an intrinsic part of the biochemical mechanism that modulates the γ-aminobutyric acid efflux.     

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.     

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.