GABA-transaminase Inhibitor Research Articles

Electroacupuncture at 2/100 Hz Activates Antinociceptive Spinal Mechanisms Different from Those Activated by Electroacupuncture at 2 and 100 Hz in Responder Rats

We examined the effects of intrathecal injection of desipramine and fluoxetine (selective inhibitors of norepinephrine and 5-HT uptake, resp.), thiorphan and neostigmine (inhibitors of enkephalinase and acetylcholinesterase, resp.), gabapentin (a GABA releaser), and vigabatrin (an inhibitor of GABA-transaminase) on the antinociception induced by 2 Hz, 100 Hz, or 2/100 Hz electroacupuncture (EA) applied bilaterally to the Zusanli (ST36) and Sanyinjiao (SP6) acupoints using the rat tail-flick test. We show that 2 Hz EA antinociception lasts longer after the administration of drugs that increase the spinal availability of norepinephrine, acetylcholine, or GABA; 100 Hz EA antinociception lasts longer after drug that increases the spinal availability of norepinephrine; 2/100 Hz EA antinociception lasts longer after drugs that increase the spinal availability of endogenous opioids or GABA. We conclude that the antinociceptive effect of 2/100 Hz EA is different from the synergistic effect of alternate stimulation at 2 and 100 Hz because the effect of the former is not changed by increasing the spinal availability of serotonin and lasts longer after the administration of vigabatrin. The combination of EA with drugs that increase the availability of spinal neurotransmitters involved in the modulation of nociceptive inputs may result in a synergistic antinociceptive effect in the rat tail-flick test.

Dopamine and GABA Interaction in Basal Ganglia: GABA-A or GABA-B Receptor Stimulation Attenuates L-DOPA-Induced Striatal and Nigral ERK1/2 Signaling in a Rat Model of Parkinson’s Disease

Parkinson’s disease (PD) is characterized by degeneration of nigrostriatal dopamine (DA) neurons. The primary drug used to treat PD symptoms is L-DOPA, but side effects such as dyskinesias limit its use. Previous findings show that L-DOPA treatment induces extracellular signal-regulated kinase (ERK1/2), a MAP-kinase protein. γ-aminobutyric acid (GABA) is intimately involved in basal ganglia function. Our previous study using a unilaterally lesioned rat model of PD indicated that elevating GABA levels by GABA transaminase inhibitor, aminooxyacetic acid significantly attenuated L-DOPA-induced ERK phosphorylation in the striatum and substantia nigra (SN). The aim of the present study was to assess the role of GABA-A and GABA-B receptor by using a selective agonists, muscimol and baclofen respectively, on L-DOPA-induced ERK phosphorylation in the striatum and SN. Unilaterally 6-OHDA-lesioned rats were prescreened by apomorphine induced rotation test for the extent of DA loss. Lesioned rats were treated with L-DOPA alone or after muscimol or baclofen pretreatment. Appropriate control groups were used. Phospho-ERK levels, tyrosine hydroxylase (to ascertain DA loss) and substance P (an indirect marker for DA loss) levels were assessed by immunohistochemistry using coronal slices at the level of striatum and SN. L-DOPA administration induced a robust increase (>300%) in phospho-ERK1/2 levels in the striatum and SN. Muscimol as well as baclofen pretreatment attenuated the L-DOPA-induced increase in phospho-ERK1/2 levels by >60% in the striatum and SN. Muscimol and baclofen pretreatment also greatly reduced the number of L-DOPA induced phospho-ERK1/2 stained cells in the striatum as well as the contralateral rotational behavior. The present data taken together with our previous study indicate that the L-DOPA induced increase in ERK1/2 is attenuated by GABA via a GABA-A and GABA-B receptor linked mechanism. The study provides further insight into a dopamine-GABA-ERK interaction in the therapeutic and/or side effects of L-DOPA in the basal ganglia.

Effects of the inhibitor of the γ-aminobutyrate-transaminase, vinyl-γ-aminobutyrate, on development and nitrogen metabolism in Brassica napus seedlings

γ-aminobutyrate-transaminase (EC 2.6.1.19) catalyzes the first step of the catabolism of γ-aminobutyric acid (GABA), a non-protein amino acid well-known to accumulate in plant in response to environmental stimuli. Recent studies reinforce more and more the role of its metabolism in carbon and/or nitrogen metabolisms and as a signalling molecule in developmental processes. Here we investigated the effects of inhibition of γ-aminobutyrate-transaminase (GABA-T) in seedlings of Brassica napus, using vinyl-GABA (VGB) as a specific inhibitor of GABA-T to prevent enzyme activity. Root growth was reduced by 44% in VGB-treated seedlings but was less inhibited when VGB was associated with exogenous GABA and was not reduced with exogenous GABA alone. Measurements of GABA content in seedlings grown on VGB, GABA or VGB+GABA demonstrated that GABA level in root was not linked with the root length reduction, suggesting that GABA was not the sole component acting in root growth inhibition. Besides, metabolic profiling revealed that in root, VGB-treatment caused a twofold increase in content of almost all amino acids, except for alanine whose content was 19-fold higher than in control. In order to test the involvement of alanine accumulation on growth we studied the effects of exogenous alanine. High alanine content slightly reduced root growth suggesting that VGB-induced alanine accumulation was not responsible for root length reduction. We conclude that root growth inhibition in plants whose GABA catabolism was impaired could result at least partly from the disruption of the primary metabolism as a whole rather than direct effect of GABA on cellular growth process.

Systemic treatment with the inhibitory neurotransmitter gamma-aminobutyric acid aggravates experimental autoimmune encephalomyelitis by affecting proinflammatory immune responses

Transcriptomic and proteomic analyses of multiple sclerosis (MS) lesions indicate alterations in the gamma-aminobutyric acid (GABA) inhibitory system, suggesting its involvement in the disease process. To further elucidate the role of GABA in central nervous system (CNS) inflammation in vivo, the chronic myelin oligodendrocyte glycoprotein (MOG)35–55 experimental autoimmune encephalomyelitis (EAE) model was used. Daily GABA injections (200mg/kg) from day 3 onwards significantly augmented disease severity, which was associated with increased CNS mRNA expression levels of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6. GABA-treated mice showed enhanced MOG-dependent proliferation and were skewed towards a T helper 1 phenotype. Moreover, in vitro, the lipopolysaccharide (LPS)-induced increase in interleukin (IL)-6 production by macrophages was enhanced at low GABA concentrations (0.03–0.3mM). In sharp contrast to exogenous GABA administration, endogenous GABA increment by systemic treatment with the GABA-transaminase inhibitor vigabatrin (250mg/kg) had prophylactic as well as therapeutic potential in EAE. Together, these results indicate an immune amplifying role of GABA in neuroinflammatory diseases like MS.

Cerebellar fastigial nuclear GABAergic projections to the hypothalamus modulate immune function

Our previous work has shown that the cerebellar fastigial nucleus (FN) is involved in modulation of lymphocyte function. Herein, we investigated effect of FN γ-aminobutyric acid (GABA)-ergic projections to the hypothalamus on lymphocytes to understand pathways and mechanisms underlying cerebellar immunomodulation. By injection of Texas red dextran amine (TRDA), an anterograde tracer, into FN, we found that the TRDA-labeled fibers from the FN traveled through the superior cerebellar peduncle (SCP), crossed in decussation of SCP (XSCP), entered the hypothalamus, and primarily terminated in the lateral hypothalamic area (LHA). Further, by injecting Fluoro-Ruby (FR), a retrograde tracer, in LHA, we observed that the FR-stained fibers retrogradely passed through XSCP and reached FN. Among these FR-positive neurons in the FN, there were GABA-immunoreactive cells. We then microinjected vigabatrin, which is an inhibitor of GABA-transaminase (GABA-T) that degrades GABA, bilaterally into FN. The vigabatrin treatment increased both number of GABA-immunoreactive neurons in FN-LHA projections and GABA content in the hypothalamus. Simultaneously, vigabatrin significantly reduced concanavalin A (Con A)-induced lymphocyte proliferation, anti-sheep red blood cell (SRBC) IgM antibody level, and natural killer (NK) cell number and cytotoxicity. In support of these findings, we inhibited GABA synthesis by using 3-mercaptopropionic acid (3-MP), which antagonizes glutamic acid decarboxylase (GAD). We found that the inhibition of GABA synthesis caused changes that were opposite to those when GABA was increased with vigabatrin. These findings show that the cerebellar FN has a direct GABAergic projection to the hypothalamus and that this projection actively participates in modulation of lymphocytes.

Semicarbazone – a versatile therapeutic pharmacophore for fragment based anticonvulsant drug design

During the last fifteen years, semicarbazones have been extensively investigated for their anticonvulsant properties. 4-(4-Flurophenoxy) benzaldehyde semicarbazone (C0102862, V102862) was discovered as a lead molecule and is being developed as a potent antiepileptic drug, with maximal electroshock (MES) ED₅₀ of i.p. 12.9 mg kg⁻¹. In MES (oral screen), this compound has a protective index (PI = TD₅₀/ED₅₀ > 315) higher than carbamazepine (PI 101), phenytoin (PI > 21.6) and valproate (PI 2.17). The compound is a potent sodium channel blocker. Other semicarbazones have demonstrated activity in various chemoshock screens, like subcutaneous pentylenetetrazole, subcutaneous strychnine, subcutaneous picrotoxin and subcutaneous bicculine. Semicarbazones are also GABA-transaminase inhibitors. Extensive structure- -activity relationship has demonstrated that F, Cl, Br and NO₂ substituents in the arylhydrophobic pocket and a hydrogen bonding domain (HBD) are generally found in active anticonvulsant agents.

An overview of third-generation antiseizure drugs: Clobazam, lacosamide, rufinamide, and vigabatrin.

Four antiseizure drugs have been approved in the United States since 2008. Clobazam, a 1,5-benzodiazepine, was approved in October 2011 as an adjunctive therapy for Lennox-Gastaut syndrome (LGS) in patients 2 years and older. Lacosamide, an amino acid that selectively enhances the slow inactivation of voltage-gated sodium channels, was approved in October 2008 as an add-on therapy for partial onset seizures in patients 17 years and older. Rufinamide, a triazole derivative, was approved in November 2008 as an adjunctive therapy for LGS in patients 4 years and older. Vigabatrin, an irreversible inhibitor of GABA transaminase, was approved in August 2009 for the treatment of infantile spasms in children ages 1 month to 2 years and intractable complex partial seizures in adults.

Involvement of GABAergic and glutamatergic systems in the anticonvulsant activity of 3-alkynyl selenophene in 21 day-old rats

In this study, we investigated the role of GABAergic and glutamatergic systems in the anticonvulsant action of 3-alkynyl selenophene (3-ASP) in a pilocarpine (PC) model of seizures. To this purpose, 21 day-old rats were administered with an anticonvulsant dose of 3-ASP (50 mg/kg, per oral, p.o.), and [(3)H]γ-aminobutyric acid (GABA) and [(3)H]glutamate uptakes were carried out in slices of cerebral cortex and hippocampus. [(3)H]GABA uptake was decreased in cerebral cortex (64%) and hippocampus (58%) slices of 21 day-old rats treated with 3-ASP. In contrast, no alteration was observed in [(3)H]glutamate uptake in cerebral cortex and hippocampus slices of 21 day-old rats that received 3-ASP. Considering the drugs that increase synaptic GABA levels, by inhibiting its uptake or catabolism, are effective anticonvulsants, we further investigated the possible interaction between sub-effective doses of 3-ASP and GABA uptake or GABA transaminase (GABA-T) inhibitors in PC-induced seizures in 21 day-old rats. For this end, sub-effective doses of 3-ASP (10 mg/kg, p.o.) and DL-2,4-diamino-n-butyric acid hydrochloride (DABA, an inhibitor of GABA uptake--2 mg/kg, intraperitoneally; i.p.) or aminooxyacetic acid hemihydrochloride (AOAA; a GABA-T inhibitor--10 mg/kg, i.p.) were co-administrated to 21 day-old rats before PC (400 mg/kg; i.p.) treatment, and the appearance of seizures was recorded. Results demonstrated that treatment with AOAA and 3-ASP or DABA and 3-ASP significantly abolished the number of convulsing animals induced by PC. The present study indicates that 3-ASP reduced [(3)H]GABA uptake, suggesting that its anticonvulsant action is related to an increase in inhibitory tonus.

Evidence for GABA as an Inhibitory Neurotransmitter in the Neural Circuit Regulating Metamorphosis in the Marine Mollusc Ilyanassa obsoleta

Event Abstract Back to Event Evidence for GABA as an Inhibitory Neurotransmitter in the Neural Circuit Regulating Metamorphosis in the Marine Mollusc Ilyanassa obsoleta Dhani Biscocho1 and Esther M. Leise1* 1 University of North Carolina Greensboro, Biology, United States The marine mud snail, Ilyanassa obsoleta, displays a biphasic life cycle. The initial phase consists of a larval stage in which the animals have ciliated feeding structures on the head (velar lobes) that allow them to swim weakly in the water column. The second phase consists of an adult stage during which the animals reside on intertidal mudflats as obligate omnivores. The event that links these life history stages is metamorphosis. Our interest lies in this transition from larva to non-reproductive adult, the neural circuit that controls metamorphosis, and the cues which are responsible for initiating this process. Molluscan metamorphosis requires multiple changes in cellular action, morphology, physiology and behavioral interactions. I. obsoleta undergoes transformations that include loss of one brain ganglion (the apical ganglion), reorganization of other brain regions, and disappearance of the velar lobes. The neurotransmitter γ-aminobutyric acid (GABA) can induce metamorphosis in a related mollusc, the abalone Haliotis rufescens (Morse, et al. 1979, Science 204, 407-410), but results of our experiments with I. obsoleta have revealed an inhibitory action for GABA. Bath application of GABA to metamorphically competent larvae of I. obsoleta does not elicit metamorphosis. However, exposure of larvae to seawater with reduced chloride concentrations induced metamorphosis in a dose-dependent fashion. Earlier work on I. obsoleta has demonstrated that the neurotransmitters serotonin (5-HT) and nitric oxide function as a promoter and repressor of metamorphosis, respectively. GABA can inhibit serotonergically induced metamorphosis, suggesting that GABA acts either downstream of, or directly on serotonergic neurons. We are currently exploiting reagents that modify the function of GABA transport, synthesis and metabolism to verify that GABA has a role in this system. In bath application, muscimol, a GABA-A agonist, inhibits spontaneous larval metamorphosis for 48 hours. Bath application of isoguvacine, another GABA-A agonist or aminooxyacetic acid (AOA), a major inhibitor of GABA-Transaminase can also repress metamorphosis in 5-HT-induced larvae. In current experiments, we are injecting reagents into competent larvae to decrease chances of interactions between neuroactive compounds and epidermal sensory receptors. These experiments should further support the idea that GABAergic activity occurs internally within the larval central nervous system, perhaps in the apical ganglion. Together, our data will help to elucidate the role of GABA in this molluscan metamorphic pathway. Acknowledgements Funded in part by UNCG Department of Biology Keywords: GABA, gastropod, Larva, metamorphosis, mollusc, Nitric Oxide, Serotonin, Snail Conference: Tenth International Congress of Neuroethology, College Park. Maryland USA, United States, 5 Aug - 10 Aug, 2012. Presentation Type: Poster Presentation (see alternatives below as well) Topic: Development Citation: Biscocho D and Leise EM (2012). Evidence for GABA as an Inhibitory Neurotransmitter in the Neural Circuit Regulating Metamorphosis in the Marine Mollusc Ilyanassa obsoleta. Conference Abstract: Tenth International Congress of Neuroethology. doi: 10.3389/conf.fnbeh.2012.27.00131 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Apr 2012; Published Online: 07 Jul 2012. * Correspondence: Dr. Esther M Leise, University of North Carolina Greensboro, Biology, Greensboro, NC, 27412, United States, esther_leise@uncg.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. 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Enhanced GABAergic tone in the ventral pallidum: memory of unpleasant experiences?

The nucleus accumbens (NAc) has emerged as an important part of the neural circuitry regulating depressive-like behaviors. Given that the NAc GABAergic medium spiny neurons project to the ventral pallidum (VP), it is reasonable to suggest that the VP may also be involved in these behaviors. Consequently, we explored the role of the VP GABAergic terminals during depressive-like behaviors in rats using the forced swim test (FST) and the sucrose preference test (SPT). Microdialysis coupled with micellar electrokinetic chromatography was used to monitor in vivo changes of GABA in the VP during the FST. GABA levels significantly increased during day-1 and day-2 during swimming, returning to the pre-swimming levels after the test. Basal concentrations of GABA on day-2 of the FST significantly increased with respect to day-1. In another set of experiments, intra-VP injections of vigabatrin (a GABA transaminase inhibitor) increased extracellular GABA and immobility behaviors in the FST while the direct GABAA receptor antagonist bicuculline reduced immobility behaviors. In the SPT, intra-VP vigabatrin injection significantly reduced preference for sucrose while bicuculline did not produce any change. At the postsynaptic side, we used semiquantitative RT-PCR to measure mRNA expression of 17 GABAA receptor subunits (α1-α6, β1-β3, γ2, δ, ε, θ, π, and ρ1-ρ3) in rats subjected to the FST. We found a significant reduction of α3 and γ2 subunit expression and an increase of δ subunit expression after day-2 in rats subject to the FST which might enhance tonic inhibition of the VP. Furthermore, immunoblot experiments revealed that protein expression of γ2 and δ subunits changed 6 days after FST in a way similar to mRNA expression. These results suggest that the enhanced VP-GABAergic tone might trigger a low motivational state, anhedonia and a possible memory mechanism for unpleasant experiences.

Mechanisms of GABA release from human astrocytes

We have previously demonstrated that human astrocytes are GABAergic cells. Throughout the adult human brain, they express the GABA synthesizing enzyme GAD 67, the GABA metabolizing enzyme GABA-T, and the GABA(A) and GABA(B) receptors. GABA modulates the actions of microglia, indicating an important role for astrocytes beyond that of influencing neurotransmitter function. Here we report on the mechanisms by which astrocytes release GABA. Astrocytes were found to express the mRNA and protein for multiple GABA transporters, and multiple receptors for glutamate, GABA, and glycine. In culture, untreated human astrocytes maintained an intracellular GABA level of 2.32 mM. They exported GABA into the culture medium so that an intracellular-extracellular gradient of 3.64 fold was reached. Inhibitors of the GABA transporters GAT1, GAT2, and GAT3, significantly reduced this export in a Ca(2+)-independent fashion. Intracellular GABA levels were enhanced by treatment with the GABA-T inhibitors gabaculine or vigabatrin. Treatment with glutamate increased GABA release in a concentration-dependent fashion. This was partially inhibited by blockers of N-methyl-D-aspartate and kainate receptors. Conversely, glycine and D-serine, co-agonists of NMDA receptors, enhanced the GABA release. GABA release was accompanied by an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and was reduced by adding the Ca(2+) chelator, BAPTA-AM to the medium. These data indicate that astrocytes continuously synthesize GABA and that there are multiple mechanisms which can mediate its release. Each of these may play a role in the physiological functioning of astrocytes.

Taurine-like GABA aminotransferase inhibitors prevent rabbit brain slices against oxygen–glucose deprivation-induced damage

The activation of the GABAergic system has been shown to protect brain tissues against the damage that occurs after cerebral ischaemia. On the other hand, the taurine analogues (±)Piperidine-3-sulphonic- (PSA), 2-aminoethane phosphonic- (AEP), 2-(N-acetylamino) cyclohexane sulfonic-acids (ATAHS) and 2-aminobenzene sulfonate-acids (ANSA) have been reported to block GABA metabolism by inhibiting rabbit brain GABA aminotransferase and to increase GABA content in rabbit brain slices. The present investigation explored the neuroprotection provided by GABA, Vigabatrin (VIGA) and taurine analogues in the course of oxygen-glucose deprivation and reperfusion induced damage of rabbit brain slices. Tissue damage was assessed by measuring the release of glutamate and lactate dehydrogenase (LDH) during reperfusion and by determining final tissue water gain, measured as the index of cell swelling. GABA (30-300μM) and VIGA (30-300μM) significantly antagonised LDH and glutamate release, as well as tissue water gain caused by oxygen-glucose deprivation and reperfusion. Lower (1-10μM) or higher concentrations (up to 3,000μM) were ineffective. ANSA, PSA and ATAHS significantly reduced glutamate and LDH release and tissue water gain in a range of concentrations between 30 and 300μM. Lower (0-10μM) or higher (up to 3,000μM) concentrations were ineffective. Both mechanisms suggest hormetic ("U-shaped") effects. These results indicate that the GABAergic system activation performed directly by GABA or indirectly through GABA aminotransferase inhibition is a promising approach for protecting the brain against ischemia and reperfusion-induced damage.

Muscimol and related GABA receptor agonists: the potency of GABAergic drugs in vivo determined after intranigral injection.

Contralateral turning behaviour following unilateral intranigral injection of a large series of GABA analogues was investigated. The results indicated that the turning behaviour was induced stereospecifically and was selectively antagonized by the GABA antagonist bicuculline methochloride. The comparative potencies of a series of GABA agonists related to muscimol in general corresponded well to the affinity for 3H-GABA receptor sites and to the depressant action on single neurons using microelectrophoretic administration. However, the GABA agonists trans-aminocrotonic acid and 3-aminopropanesulphonic acid were much weaker than expected from in vitro studies. The GABA-uptake inhibitors nipecotic acid and guvacine showed only weak and short-lasting effects. The GABA-transaminase inhibitor gamma-acetylenic GABA showed delayed effects compared with the agonists which acted immediately. It is proposed that this behavioural effect may be a sensitive and quantitative method for evaluation of GABA agonists in vivo.

May GABA transaminase inhibitors improve stereotyped behaviors in Rett syndrome?

May GABA transaminase inhibitors improve stereotyped behaviors in Rett syndrome?

An Historical Perspective on Gabaergic Drugs

In 1950, γ-aminobutyric acid (GABA) was discovered in the brain and in 1967 it was recognized as an inhibitory neurotransmitter. The discovery of the benzodiazepines Librium® (launched in 1960) and Valium® by Sternbach initiated huge research activities resulting in 50 marketed drugs. In 1975, Haefely found that GABA is involved in the actions of benzodiazepines. The baclofen-sensitive, bicuculline-insensitive GABA(B) receptor was discovered by Bowery in 1980, and the baclofen-insensitive, bicuculline-insensitive GABA(C) receptor by Johnston in 1984. Barnard & Seeburg reported the cloning of the GABA(A) receptor in 1987, Cutting the GABA(C) receptor in 1991 and Bettler the GABA(B1a) and GABA(B1b) receptors in 1997. Six groups cloned the GABA(B2) receptor in 1998/1999 showing that the GABA(B) receptor functions as a heterodimer with GABA(B1b)/GABA(B2) mediating postsynaptic inhibition and GABA(B1a)/GABA(B2) mediating presynaptic inhibition. Möhler and McKernan dissected the pharmacology of the benzodiazepine-receptor subtypes. Antagonists and positive allosteric modulators of GABA(B) receptors were discovered in 1987 and 2001, respectively. GABA transporter inhibitor, tiagabine, was launched in 1996, a GABA aminotransferase inhibitor, vigabatrin, in 1998 and a glutamic acid decarboxylase activator, pregabalin, in 2004. Most recently, brain-penetrating GABA(C)-receptor antagonists were reported in 2009.

A Substituted Anilino Enaminone Acts as a Novel Positive Allosteric Modulator of GABAA Receptors in the Mouse Brain

A small library of anilino enaminones was analyzed for potential anticonvulsant agents. We examined the effects of three anilino enaminones on neuronal activity of output neurons, mitral cells (MC), in an olfactory bulb brain slice preparation using whole-cell patch-clamp recording. These compounds are known to be effective in attenuating pentylenetetrazol-induced convulsions. Among the three compounds tested, 5-methyl-3-(4-trifluoromethoxy-phenylamino)-cyclohex-2-enone (KRS-5Me-4-OCF₃) showed potent inhibition of MC activity with an EC₅₀ of 24.5 μM. It hyperpolarized the membrane potential of MCs accompanied by suppression of spontaneous firing. Neither ionotropic glutamate receptor blockers nor a GABA(B) receptor blocker prevented the KRS-5Me-4-OCF(3)-evoked inhibitory effects. In the presence of GABA(A) receptor antagonists, KRS-5Me-4-OCF(3) completely failed to evoke inhibition of MC spiking activity, suggesting that KRS-5Me-4-OCF₃-induced inhibition may be mediated by direct action on GABA(A) receptors or indirect action through the elevation of tissue GABA levels. Neither vigabatrin (a selective GABA-T inhibitor) nor 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid hydrochloride (NNC-711) (a selective inhibitor of GABA uptake by GABA transporter 1) eliminated the effect of KRS-5ME-4-OCF₃ on neuronal excitability, indicating that the inhibitory effect of the enaminone resulted from direct activation of GABA(A) receptors. The concentration-response curves for GABA are left-shifted by KRS-5Me-4-OCF₃, demonstrating that KRS-5Me-4-OCF₃ enhanced GABA affinity and acted as a positive allosteric modulator of GABA(A) receptors. The effect of KRS-5Me-4-OCF₃ was blocked by applying a benzodiazepine site antagonist, suggesting that KRS-5Me-4-OCF₃ binds at the classic benzodiazepine site to exert its pharmacological action. The results suggest clinical use of enaminones as anticonvulsants in seizures and as a potential anxiolytic in mental disorders.

A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells

The GABase assay is widely used to rapidly and accurately quantify levels of extracellular γ-aminobutyric acid (GABA). Here we demonstrate a modification of this assay that enables quantification of intracellular GABA in bacterial cells. Cells are lysed by boiling and ethanolamine-O-sulphate, a GABA transaminase inhibitor is used to distinguish between GABA and succinate semialdehyde.

Glucose promotion of GABA metabolism contributes to the stimulation of insulin secretion in β-cells

We have demonstrated recently that branched-chain α-keto acid stimulation of insulin secretion is dependent on islet GABA (γ-aminobutyric acid) metabolism: GABA transamination to succinic semialdehyde is increased by 2-oxoglutarate, generated in α-keto acid transamination to its corresponding α-amino acid. The present work was aimed at investigating whether glucose also promotes islet GABA metabolism and whether the latter contributes to the stimulation of insulin secretion. Glucose (20 mM) decreased both the content and release of islet GABA. Gabaculine (1 mM), a GABA transaminase inhibitor, partially suppressed the secretory response of rat perifused islets to 20 mM glucose at different L-glutamine concentrations (0, 1 and 10 mM), as well as the glucose-induced decrease in islet GABA. The drug also reduced islet ATP content and the ATP/ADP ratio at 20 mM glucose. Exogenous succinic semialdehyde induced a dose-dependent increase in islet GABA content by reversal of GABA transamination and a biphasic insulin secretion in the absence of glucose. It depolarized isolated β-cells and triggered action potential firing, accompanied by a reduction of membrane currents through ATP-sensitive K(+) channels. The gene expression and enzyme activity of GABA transaminase were severalfold higher than that of 2-oxoglutarate dehydrogenase in islet homogenates. We conclude that, at high glucose concentrations, there is an increased diversion of glucose metabolism from the citric acid cycle into the 'GABA shunt'. Semialdehyde succinic acid is a cell-permeant 'GABA-shunt' metabolite that increases ATP and the ATP/ADP ratio, depolarizes β-cells and stimulates insulin secretion. In summary, an increased islet GABA metabolism may trigger insulin secretion.

Docking and QSAR studies of β-phenylethylidenehydrazine derivatives as a Gamma-aminobutyric acid aminotransferase inhibitor

β-Phenylethylidenehydrazine (PEH) derivatives have been recognized as Gamma-aminobutyric acid aminotransferase (GABA-AT) inhibitors. In this research a group of newly synthesized of PEH analogs, possessing a variety of substituents (Me, OMe, Cl, and CF3) at the 2-, 3-, and 4-position of the phenyl ring, were subjected to docking study and quantitative structure activity relationship (QSAR) analysis. PEH analogs were built by HYPERCHEM program, and conformational studies were performed through semi-empirical method followed by PM3 method. QSAR descriptors were obtained from the EDRAGON and HYPERCHEM, and equations were derived from multilinear regression (MLR) method. The sums of the JGI2, H6m, and E2s were identified as the most significant descriptors. This simple equation can be used to estimate the GABA-AT inhibitory activity for new derivatives of this series of compounds. Docking study was performed by using AutoDock4 program on the all compounds. The obtained results show that the phenyl ring is inserted into the lipophilic pocket and that the NHNH2 moiety is situated in a mainly polar region of the enzyme. These computational studies can offer some useful references for understanding the action mechanism and performing the molecular design or modification of this series of GABA-AT inhibitors.

γ‐Hydroxybutyrate and the GABAergic footprint: a metabolomic approach to unpicking the actions of GHB

Gamma-hydroxybutyrate is found both naturally in the brain and self-administered as a drug of abuse. It has been reported to act at endogenous γ-hydroxybutyrate (GHB) receptors and GABA(B) receptors [GABA(B)R], and may also be metabolized to GABA. Here, the metabolic fingerprints of a range of concentrations of GHB were measured in brain cortical tissue slices and compared with those of ligands active at GHB and GABA-R using principal components analysis (PCA) to identify sites of GHB activity. Low concentrations of GHB (1.0 μM) produced fingerprints similar to those of ligands active at GHB receptors and α4-containing GABA(A)R. A total of 10 μM GHB clustered proximate to mainstream GABAergic synapse ligands, such as 1.0 μM baclofen, a GABA(B)R agonist. Higher concentrations of GHB (30 μM) clustered with GABA(C)R agonists and the metabolic responses induced by blockade of the GABA transporter-1 (GAT1). The metabolic responses induced by 60 and 100 μM GHB were mimicked by simultaneous blockade of GAT1 and GAT3, addition of low concentrations of GABA(C)R antagonists, or increasing cytoplasmic GABA concentrations by incubation with the GABA transaminase inhibitor vigabatrin. These data suggest that at concentrations > 30 μM, GHB may be active via metabolism to GABA, which is then acting upon an unidentified GABAergic master switch receptor (possibly a high-affinity extrasynaptic receptor), or GHB may itself be acting directly on an extrasynaptic GABA-R, capable of turning off large numbers of cells. These results offer an explanation for the steep dose-response curve of GHB seen in vivo, and suggest potential target receptors for further investigation.