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.     

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.     

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.     

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.     

Identification and characterization of γ-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum

Corynebacterium glutamicum is widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as γ-aminobutyric acid (GABA). In this study, a C. glutamicum GABA-specific transporter (GabP(Cg)) encoded by ncgl0464 was identified and characterized. GabP(Cg) plays a major role in GABA uptake and is essential to C. glutamicum growing on GABA. GABA uptake by GabP(Cg) was weakly competed by l-Asn and l-Gln and stimulated by sodium ion (Na(+)). The K(m) and V(max) values were determined to be 41.1 ± 4.5 μM and 36.8 ± 2.6 nmol min(-1) (mg dry weight [DW])(-1), respectively, at pH 6.5 and 34.2 ± 1.1 μM and 67.3 ± 1.0 nmol min(-1) (mg DW)(-1), respectively, at pH 7.5. GabP(Cg) has 29% amino acid sequence identity to a previously and functionally identified aromatic amino acid transporter (TyrP) of Escherichia coli but low identities to the currently known GABA transporters (17% and 15% to E. coli GabP and Bacillus subtilis GabP, respectively). The mutant RES167 Δncgl0464/pGXKZ9 with the GabP(Cg) deletion showed 12.5% higher productivity of GABA than RES167/pGXKZ9. It is concluded that GabP(Cg) represents a new type of GABA transporter and is potentially important for engineering GABA-producing C. glutamicum strains.     

Nitrate uptake and utilization is modulated by exogenous γ-aminobutyric acid in Arabidopsis thaliana seedlings

Exogenously applied GABA modulates root growth by inhibition of root elongation when seedlings were grown in vitro on full-strength Murashige and Skoog (MS) salts, but root elongation was stimulated when seedlings were grown on 1/8 strength MS salts. When the concentration of single ions in MS salts was individually varied, the control of growth between inhibition and stimulation was found to be related to the level of nitrate (NO₃^?) in the growth medium. At NO₃^? concentrations below 40 mM (full-strength MS salts level), root growth was stimulated by the addition of GABA to the growth medium; whereas at concentrations above 40 mM NO₃^?, the addition of GABA to the growth medium inhibited root elongation. GABA promoted NO₃^? uptake at low NO₃^?, while GABA inhibited NO₃^? uptake at high NO₃^?. Activities of several enzymes involved in nitrogen and carbon metabolism including nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (NADH-GOGAT), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and phosphoenol pyruvate carboxylase (PEPCase) were regulated by GABA in the growth medium. Supplementing 1/8 strength MS medium with 50 mM GABA enhanced the activities of all of the above enzymes except ICDH activities in root tissues. However, at full-strength MS, GABA showed no inhibitory effect on the activities of these enzymes, except on GS in both root and shoot tissues, and PEPCase activity in shoot tissues. Exogenous GABA increased the amount of NR protein rather than its activation status in the tissues. This study shows that GABA affects the growth of Arabidopsis, possibly by acting as a signaling molecule, modulating the activity of enzymes involved in primary nitrogen metabolism and nitrate uptake.     

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.     

Distribution and uptake of glycine,glutamate and γ-aminobutyric acid in the vagal nuclei and eight other regions of the rat medulla oblongata

In order to study the central neurochemical control of the vagus nerve, the contents of glycine, GABA, glutamate and five other amino acids have been measured in ten anatomically distinct regions of the rat medulla oblongata. Additionally, the high affinity uptake of glycine, GABA, glutamate, and leucine were measured in the same ten medullary regions. The data support published evidence for glutamatergic and GABAergic transmission in the nucleus of the tractus solitarius (NTS), and glycinergic inhibition in the hypoglossal nucleus. The data also lead to the suggestion that GABA and glutamate may be taken up into glial cells which exist along fiber tracts.     

Comparison of release of endogenous dopamine and γ-aminobutyric acid from rat caudate synaptosomes

Release of endogenous dopamine (DA) and -aminobutyric acid (GABA) from superfused rat caudate synaptosomes was monitored with liquid chromatography with electrochemical detection. Dopamine was analyzed by oxidative detection following alumina extraction while GABA was analyzed with reductive detection following pre-column derivatization with trinitrobenzenesulfonic acid and extraction. Both spontaneous and K⁺-stimulated (40 mM) release were examined as well as the effect of several possible neuromodulatory agents (DA, GABA, muscimol, ascorbic acid, acetylcholine). The content of GABA in the sample and the amount released by K⁺ were approximately fifty times those of DA although the relative amounts released by repetitive K⁺ stimulations were similar. Muscimol and DA significantly attenuated both the spontaneous and stimulated release of GABA while ascorbate and acetylcholine had no effect. Acetylcholine significantly increased both the stimulated and spontaneous release of DA while the other agents had no effect. Dopamine showed an absolute dependence on calcium for stimulated release while GABA exhibited a significant calcium-independent release. These results indicate that profound differences exist in the factors which modulate the release of endogenous DA and GABA.     

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.     

Autoradiographic localization of 3H-γ-aminobutyric acid uptake in the lamina ganglionaris of Musca and Drosophila

Summary The uptake of ³H-GABA in the visual system of half-head preparations of Musca and Drosophila was studied by means of light and electron microscope autoradiography. Of all three ganglia, only the first synaptic region, the lamina ganglionaris, showed accumulation of radioactive grains, and there a preferential glial uptake could be found. Under normal light conditions at incubation (constant light flux of 100 Lux) the maximum of radio-activity was found in the marginal glia cells. Increasing the time of incubation produced also an increase in the number of grains per surface unit in the marginal glia cells. After changing the light intensity during incubation, quantitative modifications of the distribution of radio-activity were observed: incubating with stroboscopic illumination, the number of grains diminished in the marginal glia cells and remained constant in the epithelial cells; incubated in darkness, the epithelial cells became more intensely labelled whilst the number of grains decreased in the marginal cells.The possibility is discussed that the receptor axons 1–6 are the neurological elements of the lamina which use GABA as a transmitter. This hypothesis is lent some support from results of similar experiments with neurological mutants of Drosophila. In opm 18 there was a delayed uptake of ³H-GABA whereas in opm 3 and ebony the results were comparable to those found in Musca incubated in darkness. Behavioral studies on these mutants have demonstrated a defect, most probably related to the receptor system 1–6.     

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.     

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.     

Electrophysiological characterization of ivermectin triple actions on Musca chloride channels gated by l-glutamic acid and γ-aminobutyric acid

Ivermectin (IVM) is a macrocyclic lactone that exerts antifilarial, antiparasitic, and insecticidal effects on nematodes and insects by acting on l-glutamic acid-gated chloride channels (GluCls). IVM also acts as an allosteric modulator of various other ion channels. Although the IVM binding site in the Caenorhabditis elegans GluCl was identified by X-ray crystallographic analysis, the mechanism of action of IVM in insects is not well defined. We therefore examined the action of IVM on the housefly (Musca domestica) GluCl and γ-aminobutyric acid (GABA)-gated ion channel (GABACl). For both channels, IVM induced currents by itself, potentiated currents induced by low concentrations of agonists, and inhibited currents induced by high concentrations of agonists. Despite exerting common actions on both types of channels, GluCls were more susceptible to IVM actions than GABACls, indicating that GluCls are the primary target of IVM. Substitution of an amino acid residue in the third transmembrane segment (G312M in GluCls, and G333A and G333M in GABACls) resulted in significantly reduced levels or loss of activation, potentiation, and antagonism of the channels, indicating that these three actions result from the interaction of IVM with amino acid residues in the transmembrane intersubunit crevice.     

Effects of the γ-aminobutyrate transaminase inhibitors gabaculine and γ-vinyl GABA on γ-aminobutyric acid release from slices of rat cerebral cortex

The release of [³H]-aminobutyric acid (GABA) from pre-loaded slices of rat cerebral cortex was investigated in the presence and absence of the GABA-transaminase inhibitors gabaculine and -vinyl GABA. In the experiments carried out without an inhibitor, an ion-exchange column chromatographic technique was used to separate [³H]GABA from tritiated metabolites released with it into the superfusate. The presence of gabaculine (5 M) substantially reduced the Ca²⁺-dependence of the release of [³H]GABA evoked by a 4 min 30 mM K⁺ pulse, whereas this was not appreciably reduced by the presence of -vinyl GABA (2 mM or 10 mM). Nevertheless, the characteristics of [³H]GABA release were not identical in the presence and absence of either inhibitor.     

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