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.     

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.     

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.     

Effects of γ-aminobutyric acid on 33Pi incorporation into rat brain phospholipids in vivo

The effects of γ-aminobutyric acid (GABA), bicuculline and strychnine on the incorporation $\text{in vivo}$ of ³³Pi into phospholipids of rat brain were studied at 10 and 30 minutes after intracisternal injection of the radionuclide. GABA inhibited labeling of phospholipids in the three brain regions studied at both times. Bicuculline by itself had no significant effect on ³³Pi incorporation, but totally blocked the inhibitory effect of GABA in all three brain regions. Strychnine by itself inhibited phospholipid labeling in the brain stem and forebrain, had no significant effect on GABA inhibition of ³³Pi incorporation in the cerebellum and forebrain, and partially blocked the GABA effect in the brain stem. GABA inhibited ³³Pi incorporation into phosphatidic acid, phosphatidylinositol, phosphatidyl choline and phosphatidyl ethanolamine but had no effect on phosphatidyl serine. The data suggest that the inhibitory effects of GABA on CNS phospholipid labeling are mediated specifically through GABA receptor sites.     

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.     

Impact of postharvest exogenous γ-aminobutyric acid treatment on cucumber fruit in response to chilling tolerance

Low-temperature storage is generally used to extend postharvest lifetime and to inhibit decay of cucumber fruit, but it also enhances the intensity of chilling injury. The capability of γ-aminobutyric acid to enhance antioxidant enzyme activities and reduce chilling injury was studied in cucumber (Cucumis sativus L.) fruit stored at 1 °C for 5 weeks. The purpose of this study was to define if the GABA-induced modification in antioxidant system and phospholipase activity is linked to the reduced chilling injury in cold-stored cucumber fruit. Alleviation of chilling injury by GABA treatment was related to increased content of proline, endogenous GABA and enhanced activities of CAT and SOD, together with reduced activities of PLC, PLD and LOX. We suggest that PLC, LOX and PLD are associated with chilling injury initiation by involvement in a signaling pathway and membrane deterioration. Therefore the results obtained in this study suggest GABA’s potential for postharvest applications for reducing chilling injury symptom in cucumber fruit.     

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.     

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.     

Effects of α-bungarotoxin on acetylcholine-induced response at the Helix neuronal membrane

The effects of -bungarotoxin (-BT) on two patterns of acetylcholine (ACh)-induced response differing in desensitization rate were investigated in isolated mollusk neurons using intracellular dialysis and concentration clamping techniques. It was found that -BT depressed both types of ACh-induced response — a reversible action in the majority of experiments performed. It also exerted a blocking effect on ACh-induced currents dependent on the presence of albumin, although albumin itself produced no noticeable change in ACh-induced response. Concentration dependence of -BT-induced blockade on both types of currents evoked by 1 and 10 µM ACh was investigated. The -BT concentrations producing a 50% suppression of the current evoked by 1 µM ACh were calculated by approximating concentration plots as (13.85±1.25)×10^–9 and (5.56±1.0)×10^–8 g/ml for type A and B cells respectively.Institute of Experimental Biology. Academy of Sciences of the Armenian SSR, Erevan. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 729–735, November–December, 1989.     

Effects of γ-rays on Poly-L-glutamic acid and Poly-D,L-glutamic acid in the solid state

Summary Poly-L-glutamic acid and poly-D,L-glutamic acid, as models of proteins, were irradiated with⁶⁰Co--radiation in air and under vacuo to examine whether or not the changes caused by the exposure to ionizing radiation depend on the conformations of polypeptides.It was found that theG- values (yield of main-chain scissions per 100 eV of energy absorbed) of both polypeptides are approximately equal for the irradiation in air, while under vacuo theG- value of poly-D,L-glutamic acid is larger than that of poly-L-glutamic acid. This observation for irradiation under vacuo was ascribed to the stabilizing effect of intramolecular hydrogen bond bridges in poly-L-glutamic acid. It was also found that the-helical structure of poly-L-glutamic acid is destroyed by the exposure to ionizing radiation.     

Synthesis of γ-aminobutyric acid analogs based on carbohydrate scaffolds

γ-Aminobutyric acid analogs based on sugar scaffolds were prepared in six to nine steps starting from d-glucal and d-galactal. The key step in the synthesis is the Vilsmeier-Haack reaction that affords the corresponding 2-C-formyl glycal on treatment with DMF and POCl₃. Oxidation of the aldehyde and reduction of the 4-azido group provided the corresponding GABA analog. Acylamide and tetrazole analogs were also prepared as the bioisosteres of the carboxylic acid.     

Inhibition of γ-aminobutyric acid release from rat cerebral cortex slices by barbiturate anesthesia

Amobarbital and pentobarbital anesthesia inhibited the potassium-stimulated, Ca-dependent release of -aminobutyric acid (GABA) from rat cerebral cortex slices during incubation in vitro. Inhibition of GABA release was not found when slices were prepared from rats shortly after they awakened from amobarbital anesthesia. Phenobarbital anesthesia did not affect the release of GABA.     

Effects of γ-aminobutyric acid on feed intake,growth performance and expression of related genes in growing lambs

This study was conducted to investigate the effects of rumen-protected γ-aminobutyric acid (GABA) on feed intake, growth performance and expression of related genes in growing lambs. A total of 24 lambs weaned at age of 50 days were divided into four block of six based on their BW, six lambs within a block were allocated to three pairs, which were then assigned randomly to three treatments with addition of rumen-protected GABA at levels of 0, 70 or 140 mg/day for 6 weeks. Dry matter intake was recorded weekly in three consecutive days, and BW was recorded every two weeks. At the end of the trial, four lambs from each group were slaughtered, and duodenum and ileum mucosa were obtained for measurement of mRNA abundance of GABA receptor and cholecystokinin receptor. Dry matter intake was higher (P<0.01) in the lambs fed 140 mg/day GABA than that in the control or 70 mg GABA-fed lambs. Average daily gain and nutrients digestibility were not different (P>0.05) among treatments. Lambs fed 140 mg/day GABA had higher mRNA abundance of GABA-B receptor (P<0.01) and lower mRNA abundance of cholecystokinin-2 receptor (P<0.01) in duodenum mucosa. Serum CCK content was lower (P<0.01) in lambs fed 140 mg/day GABA than that in control. It is indicated that GABA may enhance feed intake by regulating GABA- and cholecystokinin-related genes.     

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.     

Different effects of α- and γ-glycine on the aberrant activity of pyramidal neurons of hippocampal slices

Experiments in vitro on hippocampal slices of mouse have shown that solutions prepared from polymorphic modifications α- and γ-glycine have different effect on the aberrant activity of neurons. In the presence of α-glycine the excitability of these neurons decreased more slowly, prolonging its modulating effect on NMDA type glutamate receptors. This effect agrees with higher biological activity of α-polymorphic modifications (as compared with that of the α-form) that previously observed with respect to behavior of mice from the line with genetic diathesis to catalepsy, which were used as a biological model for investigation of some pathological behavior forms.     

Autoradiographic studies of the γ-aminobutyric acid (GABA) system in the rat pancreas

Summary The -cells of the pancreatic islets have been shown to contain -aminobutyric acid (GABA) together with insulin. Autoradiographic analysis indicated that high affinity GABA binding sites (GABA receptors) are not present in the pancreas. High affinity GABA uptake sites are present, not in -cells, but in a few cells on the periphery of the islets. These observations cast doubt on the suggestion that GABA has a paracrine role in the pancreas.     

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.     

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.     

Primary afferent depolarization distribution of the γ-aminobutyric acid system in frog spinal cord

In the frog spinal cord primary afferent depolarization (PAD) constitutes a powerful inhibitory control mechanism. It has been suggested that -aminobutyric acid (GABA) is the transmitter substance involved in the genesis of PAD. In these studies we show that maximal glutamic acid decarboxylase activity is localized roughly 400–600 m from the dorsal surface, and that correlates well with the intraspinal distribution of field potentials associated with PAD. Measurement of GABA in serial spinal cord sections cut in a dorsal-ventral direction shows that high levels of GABA are seen at 400–600 m, with a peak at 800 m from the dorsal surface. Stimulation at frequencies shown to produce PAD augments the release of endogenous GABA from a superfused frog hemicord preparation.     

Is the concentration of γ-aminobutyric acid in the nerve terminal regulated via product inhibition of glutamic acid decarboxylase?

Fractions of synaptosomes were used to study the regulation of -aminobutyric acid (GABA) synthesis. The isolated synaptosomes were superfused in media of various compositions. [³H]GABA and GABA released into the medium or remaining in the synaptosomes were analyzed by liquid scintillation and HPLC techniques. Different conditions, designed to increase the GABA efflux rate were used: the rate of superfusion was varied and the concentrations of K⁺ and Ca²⁺ were altered. Stimulation of GABA efflux was paralleled with an increased synthesis of GABA, since, in spite of the increased GABA efflux, a relatively constant intraterminal level was found. The findings suggest that the intraterminal concentration of GABA and thus also its synthesis is regulated via product inhibition. In addition, [³H]GABA, exogenous, and GABA, endogenous, responded to external stimulae (Ca²⁺, veretradine, various GABA concentrations and the glutaminase inhibitor diazo-nor-leucine) in a way which was compatible with them being localized in and/or released from different compartments.