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Sample records for gaba uptake inhibitors

  1. Do GABA uptake inhibitors herald a new generation of GABA-ergic drugs for trigeminal neuralgia?

    PubMed

    Hon, Ng Chee

    2004-10-01

    Anticonvulsant drugs are the initial treatment of choice in trigeminal neuralgia. Historically, carbamazepine is and remains the gold standard. Many similarities between the pathophysiologic phenomena observed in epilepsy models and neuropathic pain models justify the rationale for the use of anticonvulsants in the management of neuropathic pain disorders. We have evidence that there is a high intensity of Gamma-aminobutyric acid transporters in the trigeminal sensory nuclei, and that Gamma-aminobutyric transporters expression was increased in the spinal trigeminal nucleus of rats after facial carrageenan injections. These are correlated with increased Gamma-aminobutyric acid uptake in the synaptosomal preparation from the spinal trigeminal nucleus of rats that received the injections and can be antagonised with glial uptake inhibitors. We conclude that Gamma-aminobutyric acid uptake inhibitors may prove to be a novel generation of GABAergic drugs for trigeminal neuralgia.

  2. Design, synthesis and SAR studies of GABA uptake inhibitors derived from 2-substituted pyrrolidine-2-yl-acetic acids.

    PubMed

    Steffan, Tobias; Renukappa-Gutke, Thejavathi; Höfner, Georg; Wanner, Klaus T

    2015-03-15

    In this paper, we disclose the design and synthesis of a series of 2-substituted pyrrolidine-2-yl-acetic acid as core structures and the N-arylalkyl derivatives thereof as potential GABA transport inhibitors. The 2-position in the side chain of pyrrolidine-2-yl-acetic acid derivatives was substituted with alkyl, hydroxy and amino groups to modulate the activity and selectivity to mGAT1 and mGAT4 proteins. SAR studies of the compounds performed for the four mouse GABA transporter proteins (mGAT1-mGAT4) implied significant potencies and subtype selectivities for 2-hydroxy-2-pyrrolidine-2-yl-acetic acid derivatives. The racemate rac-(u)-13c exhibited the highest potency (pIC50 5.67) at and selectivity for mGAT1 in GABA uptake assays. In fact, the potency of rac-(u)-13c at hGAT-1 (pIC50 6.14) was even higher than its potency at mGAT1. These uptake results for rac-(u)-13c are in line with the binding affinities to the aforesaid proteins mGAT1 (pKi 6.99) and hGAT-1 (pKi 7.18) determined by MS Binding Assay based on NO711 as marker quantified by LC-ESI-MS-MS analysis. Interestingly, the 2-hydroxy-2-pyrrolidine-2-yl-acetic acid rac-(u)-13d containing 2-{[tris(4-methoxyphenyl)]methoxy} ethyl group at the nitrogen atom of the pyrrolidine ring showed high potency at mGAT4 and a comparatively better selectivity for this protein (>15 against mGAT3) than the well known mGAT4 uptake inhibitor (S)-SNAP-5114.

  3. Perinatal hypoxia: different effects of the inhibitors of GABA transporters GAT1 and GAT3 on the initial velocity of [3H]GABA uptake by cortical, hippocampal, and thalamic nerve terminals.

    PubMed

    Pozdnyakova, Natalia; Dudarenko, Marina; Yatsenko, Ludmila; Himmelreich, Nina; Krupko, Olga; Borisova, Tatiana

    2014-06-01

    AIM. To analyze the effects of highly selective blocker GAT1, NO-711, and substrate inhibitor GAT3, β-alanine, on the initial velocity of [(3)H]GABA uptake by cortical, hippocampal, and thalamic nerve terminals (synaptosomes) after perinatal hypoxia. METHODS. Animals were divided into two groups: control (n=17) and hypoxia (n=12). Rats in the hypoxia group underwent hypoxia and seizures (airtight chamber, 4% O2 and 96% N2) at the age of 10-12 postnatal days and were used in the experiments 8-9 weeks after hypoxia. RESULTS. In cortical synaptosomes, the effects of NO-711 (30 μΜ) and β-alanine (100 μΜ) on [(3)H]GABA uptake were similar in control and hypoxia groups. In hippocampal synaptosomes, NO-711 inhibited 84.3% of the initial velocity of [(3)H]GABA uptake in normal conditions and 80.1% after hypoxia, whereas the effect of β-alanine was increased after hypoxia from 14.4% to 22.1%. In thalamic synaptosomes, the effect of NO-711 was decreased by 79.6% in controls and by 70.9% in hypoxia group, whereas the effect of β-alanine was increased after hypoxia from 20.2% to 30.2%. CONCLUSIONS. The effectiveness of β-alanine to influence GABA uptake was increased in hippocampal and thalamic nerve terminals as a result of perinatal hypoxia and the effectiveness of NO-711 in thalamic nerve terminals was decreased. These results may indicate changes in the ratio of active GAT1/GAT3 expressed in the plasma membrane of nerve terminals after perinatal hypoxia. We showed a possibility to modulate non-GAT1 GABA transporter activity in different brain regions by exogenous and endogenous β-alanine.

  4. Perinatal hypoxia: different effects of the inhibitors of GABA transporters GAT1 and GAT3 on the initial velocity of [3H]GABA uptake by cortical, hippocampal, and thalamic nerve terminals

    PubMed Central

    Pozdnyakova, Natalia; Dudarenko, Marina; Yatsenko, Ludmila; Himmelreich, Nina; Krupko, Olga; Borisova, Tatiana

    2014-01-01

    Aim To analyze the effects of highly selective blocker GAT1, NO-711, and substrate inhibitor GAT3, β-alanine, on the initial velocity of [3H]GABA uptake by cortical, hippocampal, and thalamic nerve terminals (synaptosomes) after perinatal hypoxia. Methods Animals were divided into two groups: control (n = 17) and hypoxia (n = 12). Rats in the hypoxia group underwent hypoxia and seizures (airtight chamber, 4% O2 and 96% N2) at the age of 10-12 postnatal days and were used in the experiments 8-9 weeks after hypoxia. Results In cortical synaptosomes, the effects of NO-711 (30 μΜ) and β-alanine (100 μΜ) on [3H]GABA uptake were similar in control and hypoxia groups. In hippocampal synaptosomes, NO-711 inhibited 84.3% of the initial velocity of [3H]GABA uptake in normal conditions and 80.1% after hypoxia, whereas the effect of β-alanine was increased after hypoxia from 14.4% to 22.1%. In thalamic synaptosomes, the effect of NO-711 was decreased by 79.6% in controls and by 70.9% in hypoxia group, whereas the effect of β-alanine was increased after hypoxia from 20.2% to 30.2%. Conclusions The effectiveness of β-alanine to influence GABA uptake was increased in hippocampal and thalamic nerve terminals as a result of perinatal hypoxia and the effectiveness of NO-711 in thalamic nerve terminals was decreased. These results may indicate changes in the ratio of active GAT1/GAT3 expressed in the plasma membrane of nerve terminals after perinatal hypoxia. We showed a possibility to modulate non-GAT1 GABA transporter activity in different brain regions by exogenous and endogenous β-alanine. PMID:24891283

  5. Effects of NNC 711, a GABA uptake inhibitor, on pentylenetetrazol-induced seizures in developing and adult rats.

    PubMed

    Kubová, H; Haugvicová, R; Mares, P

    1998-09-01

    The anticonvulsant action of NNC 711 [(1-(2-((diphenylmethylene) amino) oxy) ethyl)-1,2,4,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride], an inhibitor of the GABA transporter GAT-1, was studied in a model of pentylenetetrazol-induced motor seizures in rats 7, 12, 18, 25, and 90 days old. NNC 711 at doses of 0.25-20 mg/kg i.p. exhibited two effects in rat pups: a suppression of minimal clonic seizures in age groups in which this type of seizure could be reliably elicited (i.e. in rats aged 18 and 25 days); and a specific suppression or restriction of the tonic phase of generalized tonic-clonic seizures (GTCS) expressed in 18- and especially 12-day-old rats. Effects of NNC 711 on GTCS in 7- and 25-day-old rats were irregular. Adult (i.e. 90-day-old) animals exhibited abolition of generalized tonic-clonic seizures; minimal clonic seizures were suppressed only after substantially higher doses. The abolition of minimal seizures by doses too low to influence generalized tonic-clonic seizures as observed in rat pups is unique among antiepileptic drugs. In addition, an EEG study in rat pups demonstrated dissociation of EEG signs and motor seizures in some animals.

  6. Parawixin2, a novel non-selective GABA uptake inhibitor from Parawixia bistriata spider venom, inhibits pentylenetetrazole-induced chemical kindling in rats.

    PubMed

    Gelfuso, Erica A; Liberato, José L; Cunha, Alexandra O S; Mortari, Márcia R; Beleboni, Renê O; Lopes, Norberto P; Dos Santos, Wagner F

    2013-05-24

    The aims of the present work were to investigate the effects of the repeated administration of Parawixin2 (2-amino-5-ureidopentanamide; formerly FrPbAII), a novel GABA and glycine uptake inhibitor, in rats submitted to PTZ-induced kindling. Wistar rats were randomly divided in groups (n=6-8) for different treatments. Systemic injections of PTZ were administered every 48 h in the dose of 33 mg/kg; i.p., that is sufficient to induce fully kindled seizures in saline i.c.v. treated rats in a short period of time (28 days). Treatments in two types of positive controls (diazepam - DZP and nipecotic acid - NA groups) consisted in daily systemic injections of DZP (2mg/kg; i.p.) or i.c.v. injections of NA (12 μg/μL), while in experimental groups in daily i.c.v. injections of different doses of Parawixin2 (0.15; 0.075; 0.015 μg/μL). Seizures were analyzed using the Lamberty & Klitgaard score and kindling was considered as established after at least three consecutive seizures of score 4 or 5. Cumulative seizure scores for each group were analyzed using repeated measures of ANOVA followed by Tukey test. PTZ induced 4 and 5-score seizures after 12 injections in saline treated rats, whereas daily injection of Parawixin2 inhibited the onset of seizures in a dose dependent manner. Also, the challenging administration of PTZ did not raise seizure score in animals treated with the highest dose of Parawixin2 or those treated with DZP or NA. These findings together with previous data from our laboratory show that Parawixin2 could be a useful probe to design new antiepileptic drugs.

  7. GAT-1 mediated GABA uptake in rat oligodendrocytes.

    PubMed

    Fattorini, Giorgia; Melone, Marcello; Sánchez-Gómez, María Victoria; Arellano, Rogelio O; Bassi, Silvia; Matute, Carlos; Conti, Fiorenzo

    2017-03-01

    Stimulated by the results of a recent paper on the effects of tiagabine, a selective inhibitor of the main GABA transporter GAT-1, on oligodendrogenesis, we verified the possibility that GAT-1 may be expressed in oligodendrocytes using immunocytochemical methods and functional assays. Light microscopic analysis of the subcortical white matter of all animals revealed the presence of numerous GAT-1+ cells of different size (from 3 to 29 µm) and morphology. An electron microscope analysis revealed that, besides fibrous astrocytes and interstitial neurons, GAT-1 immunoreactivity was present in immature and mature oligodendrocytes. Co-localization studies between GAT-1 and markers specific for oligodendrocytes (NG2 and RIP) showed that about 12% of GAT-1 positive cells in the white matter were immature oligodendrocytes, while about 15% were mature oligodendrocytes. In vitro functional assays showed that oligodendrocytes exhibit tiagabine-sensitive Na(+) -dependent GABA uptake. Although relationships between GABA and oligodendrocytes have been known for many years, this is the first demonstration that GAT-1 is expressed in oligodendrocytes. The present results on the one hand definitely closes the era of "neuronal" and "glial" GABA transporters, on the other they suggest that oligodendrocytes may contribute to pathophysiology of the several diseases in which GAT-1 have been implicated to date. GLIA 2017;65:514-522.

  8. Syntheses and GABA uptake properties of 6-ether- and 6-enol ether-substituted nipecotic acids.

    PubMed

    N'Goka, Victor; Bissantz, Caterina; Bisel, Philippe; Stenbøl, Tine B; Krogsgaard-Larsen, Povl; Schlewer, Gilbert

    2004-07-01

    6-aralkylether- and 6-arylenol-ether-substituted nipecotic acids were synthesized. These analogues are poor GABA uptake inhibitors. The electronegative region concept developed in the N-substituted nipecotic acid series cannot be transferred on the side chain of this series of 6-substituted analogues.

  9. Effect of centrally acting drugs on the uptake of γ-aminobutyric acid (GABA) by slices of rat cerebral cortex

    PubMed Central

    Harris, M.; Hopkin, Judy M.; Neal, M. J.

    1973-01-01

    1. The effects of centrally acting drugs on the uptake of 3H-γ-aminobutyric acid (GABA) by slices of rat cerebral cortex have been studied. 2. Many centrally acting drugs at concentrations of 0·1-1·0 mM significantly inhibited the uptake of 3H-GABA by cortical slices, but the only classes of drugs in which all members consistently produced inhibition of uptake were the phenothiazines, tricyclic antidepressants, and butyrophenones. 3. The receptor blocking drugs; phentolamine, propranolol, thymoxamine, mepyramine, and diphenhydramine at concentrations of 0·5-1 mM also significantly reduced the uptake of 3H-GABA. However, atropine, hexamethonium and (+)-tubocurarine had little effect on the uptake of 3H-GABA by cortical slices. 4. Centrally acting drugs, which did not significantly inhibit 3H-GABA uptake, included barbiturates, local anaesthetics, hallucinogens, monoamine oxidase inhibitors, anticonvulsants, and convulsants (except picrotoxin). 5. Chlorpromazine, prochlorperazine, L-2,4,diaminobutyric acid, desmethylimipramine, and iprindole inhibited the uptake of 3H-GABA by 50% (IC50) at concentrations of 30-100 μM. The most potent inhibitor of 3H-GABA uptake was p-chloromercuriphenylsulphonate (IC50 = 18 μM). 6. With the exception of L-2,4,diaminobutyric acid, an outstanding characteristic of these drugs was their complete lack of specificity. Thus at the IC50 for GABA, p-chloromercuriphenylsulphonate, chlorpromazine, prochlorperazine, iprindole, desmethylimipramine, apomorphine and diphenylhydramine also inhibited the uptake of radioactive glycine, alanine, noradrenaline, and 5-hydroxytryptamine. The uptake of the latter two compounds was often inhibited to a greater extent than GABA, glycine and alanine. 7. Kinetic analysis indicated that the inhibition of 3H-GABA by p-chloromercuriphenylsulphonate, chlorpromazine, and desmethylimipramine was noncompetitive. L-2,4,Diaminobutyric acid reduced the uptake of 3H-GABA by a `mixed' type of inhibition. 8. The

  10. Distribution of 3H-GABA uptake sites in the nematode Ascaris

    SciTech Connect

    Guastella, J.; Stretton, A.O. )

    1991-05-22

    The distribution of uptake sites for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the nematode Ascaris suum was examined by autoradiography of 3H-GABA uptake. Single neural processes in both the ventral and dorsal nerve cords were labeled with 3H-GABA. Serial section analysis identified the cells of origin of these processes as the RMEV-like and RMED-like neurons. These cells belong to a set of four neurons in the nerve ring, all of which are labeled by 3H-GABA. 3H-GABA labeling of at least two other sets of cephalic neurons was seen. One of these pairs consists of medium-sized lateral ganglia neurons, located at the level of the amphid commissure bundle. A second pair is located in the lateral ganglia at the level of the deirid commissure bundle. The position and size of these lateral ganglia cells suggest that they are the GABA-immunoreactive lateral ganglia cells frequently seen in whole-mount immunocytochemical preparations. Four neuronal cell bodies located in the retrovesicular ganglion were also labeled with 3H-GABA. These cells, which are probably cholinergic excitatory motor neurons, do not contain detectable GABA-like immunoreactivity. Heavy labeling of muscle cells was also observed. The ventral and dorsal nerve cord inhibitory motor neurons, which are known to contain GABA-like immunoreactivity, were not labeled above background with 3H-GABA. Together with the experiments reported previously, these results define three classes of GABA-associated neurons in Ascaris: (1) neurons that contain endogenous GABA and possess a GABA uptake system; (2) neurons that contain endogenous GABA, but that either lack a GABA uptake system or possess a GABA uptake system of low activity; (3) neurons that possess a GABA uptake system, but that lack endogenous GABA.

  11. Characterization of GABA/sub A/ receptor-mediated /sup 36/chloride uptake in rat brain synaptoneurosomes

    SciTech Connect

    Luu, M.D.; Morrow, A.L.; Paul, S.M.; Schwartz, R.D.

    1987-09-07

    ..gamma..-Aminobutyric acid (GABA) receptor-mediated /sup 36/chloride (/sup 36/Cl/sup -/) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated /sup 36/Cl/sup -/ uptake in a concentration-dependent manner with the following order of potency: Muscimol>GABA>piperidine-4-sulfonic acid (P4S)>4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP)=3-aminopropanesulfonic acid (3APS)>>taurine. Both P4S and 3APS behaved as partial agonists, while the GABA/sub B/ agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated /sup 36/Cl/sup -/ uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated /sup 36/Cl/sup -/ uptake was also dependent on the anion present in the media. The muscinol response varied in media containing the following anions: Br/sup -/>Cl/sup -/greater than or equal toNO/sub 3//sup -/>I/sup -/greater than or equal toSCN/sup -/>>C/sub 3/H/sub 5/OO/sup -/greater than or equal toClO/sub 4//sup -/>F/sup -/, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl/sup -/ channel. 43 references, 4 figures, 3 tables.

  12. [Pharmacological influences on the brain level and transport of GABA. II) Effect of various psychoactive drugs on brain level and uptake of GABA].

    PubMed

    Gabana, M A; Varotto, M; Saladini, M; Zanchin, G; Battistin, L

    1981-04-30

    The effects of some psychoactive drugs on the level and uptake of GABA in the mouse brain was studied using well standardized procedures, mainely the silica-gel cromatography for determining the GABA content and the brain slices for measuring GABA uptake. It was found that levomepromazine, sulpiride, haloperidol and amytryptiline were without effects on the cerebral level of GABA; it was also found that these drugs do not influence the rates of uptake of GABA by mouse brain slices. Such results do indicate that the psychoactive drugs studied are without effects on the level and uptake of GABA in the brain.

  13. Selectivity of the uptake of glutamate and GABA in two morphologically distinct insect neuromuscular synapses.

    PubMed

    van Marle, J; Piek, T; Lammertse, T; Lind, A; Van Weeren-Kramer, J

    1985-11-25

    The common inhibitor (CI) and slow excitor tibiae (SETi) innervated slow muscles 135cd of the locust Schistocerca gregaria were incubated under high-affinity uptake conditions either in [3H]GABA or in [3H]glutamate. [3H]GABA is accumulated in the glia of the nerve endings of the CI as well as the SETi; however, it is accumulated only in the terminal axons of the CI, not in the terminal axons of the SETi. The grain densities above the glia and above the CI terminal axons are approximately 2 grains/micron2. After incubation in [3H]glutamate the grain densities above the CI terminal axons and the SETi terminal axons are approximately 4 grains/micron2; the grain densities above the glia of both types of nerve endings are approximately 17 grains/micron2. The relatively high labeling (3 grains/micron2) of the muscles after incubation in the presence of glutamate is ascribed to the high metabolic requirements of slow muscles. The conclusion is drawn that a high-affinity uptake system for GABA is present in the CI terminal axons and in the glia of both the CI and SETi nerve endings. However, while the glutamate uptake in the CI and SETi nerve endings of the slow 135cd is comparable to the high-affinity uptake of glutamate in the fast excitor tibiae (FETi) nerve endings of the fast retractor unguis muscle, a high-affinity uptake of glutamate was only demonstrated in the glia of both types of nerve endings. A high-affinity uptake in the terminal axons of the CI and SETi may be masked by an extensively low-affinity uptake of glutamate by the muscles.

  14. GABA uptake in embryonic palate mesenchymal cells of two mouse strains.

    PubMed

    Wee, E L; Zimmerman, E F

    1985-12-01

    To obtain further evidence that the inhibitory neurotransmitter GABA functions in palate development, the presence of an active GABA uptake mechanism was sought using primary cultures of embryonic palate mesenchymal cells. Uptake was compared from cells of two inbred mouse strains in which the SWV strain shows greater sensitivity than the AJ strain to effects of GABA on palate morphogenesis and of diazepam in producing cleft palate. Palate cells were capable of accumulating [3H]GABA by saturable uptake mechanisms characteristic of a high and low affinity active transport as indicated by temperature, Na+ ion and carrier dependence as well as Km and Vmax values that were comparable to other biological systems. The Vmax of the high-affinity uptake system from cells of the SWV strain was 1.8 fold higher than that of the AJ. GABA uptake was also observed in fibroblasts from various sources including embryonic mouse limb cells, human skin fibroblasts and 3T3 cells. When active GABA uptake was measured in skin fibroblasts from the mouse SWV and AJ strains, the rate of uptake from SWV cells under high affinity conditions was also 1.8 fold greater than in AJ cells. Thus active GABA uptake appears to be genetically regulated in non-neural cells which may contribute to differential responses to GABA.

  15. 3H-GABA uptake selectively labels identifiable neurons in the leech central nervous system

    SciTech Connect

    Cline, H.T.

    1983-04-10

    Segmental ganglia of the leech ventral nerve cord synthesize the neurotransmitter gamma-aminobutyric acid (GABA) when incubated in the presence of the precursor glutamate, suggesting that there may be GABA-ergic neurons in the leech nerve cord. GABA-accumulating neurons of the two taxonomically distant leech species, Haementeria ghilianii and Hirudo medicinalis, have been labeled by taking advantage of their high-affinity uptake system for the neurotransmitter. Autoradiography of sectioned segmental ganglia previously exposed to 3H-GABA reveals a reproducible pattern of about thirty 3H-GABA-labeled neuronal cell bodies per ganglion. The majority of 3H-GABA-labeled neuronal cell bodies are bilaterally paired, although some apparently unpaired cell bodies also accumulate label. Neuronal processes were reproducibly labeled by GABA uptake and could be traced in the neuropil through commissures and fiber tracts into the segmental nerve roots and interganglionic connectives, respectively.

  16. Hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.

    PubMed

    Yahara, Tohru; Tachikawa, Masanori; Akanuma, Shin-ichi; Hosoya, Ken-ichi

    2010-01-01

    We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [³H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [³H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [³H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and β-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.

  17. The activation of cannabinoid receptors in striatonigral GABAergic neurons inhibited GABA uptake.

    PubMed

    Romero, J; de Miguel, R; Ramos, J A; Fernández-Ruiz, J J

    1998-01-01

    Cannabinoid receptors (CNRs) in basal ganglia are located on striatal efferent neurons which are gamma-aminobutiric acid (GABA)-containing neurons. Recently, we have demonstrated that CN-induced motor inhibition is reversed by GABA-B, but not GABA-A, receptor antagonists, presumably indicating that the activation of CNRs in striatal outflow nuclei, mainly in the substantia nigra, should be followed by an increase of GABA concentrations into the synaptic cleft of GABA-B receptor synapses. The present study was designed to examine whether this was originated by increasing GABA synthesis and/or release or by decreasing GABA uptake. We analyzed: (i) GABA synthesis, by measuring the activity of glutamic acid decarboxylase (GAD) and GABA contents in brain regions that contain striatonigral GABAergic neurons, after in vivo administration of CNs and/or the CNR antagonist SR141716; (ii) [3H]GABA release in vitro in the presence or the absence of a synthetic CN agonist, HU-210, by using perifusion of small fragments of substantia nigra; and (iii) [3H]GABA uptake in vitro in the presence or the absence of WIN-55,212-2, by using synaptosomes obtained from either globus pallidus or substantia nigra. Results were as follows. Delta9-tetrahydrocannabinol (delta9-THC) and HU-210, did not alter neither GAD activity nor GABA contents in both the striatum and the ventral midbrain at any of the two times tested, thus suggesting that CNs apparently failed to change GABA synthesis in striatonigral GABAergic neurons. A similar lack of effect of HU-210 on in vitro [3H]GABA release, both basal and K+-evoked, was seen when this CN was added to perifused substantia nigra fragments, also suggesting no changes at the level of GABA release. However, when synaptosome preparations obtained from the substantia nigra were incubated in the presence of WIN-55,212-2, a decrease in [3H]GABA uptake could be measured. This lowering effect was specific of striatonigral GABAergic neurons since it was not

  18. The effect of polychlorinated biphenyls on the high affinity uptake of the neurotransmitters, dopamine, serotonin, glutamate and GABA, into rat brain synaptosomes.

    PubMed

    Mariussen, E; Fonnum, F

    2001-02-21

    Studies have shown that polychlorinated biphenyls (PCB) may affect cognitive functions both in human and also in experimental animals. We have investigated whether this effect could be caused by an inhibition of the uptake of selected neurotransmitters into rat brain synaptosomes. Ortho-chlorinated biphenyls were found to inhibit transmitter transport into synaptosomes from rat brain. In contrast, several nonortho-chlorinated biphenyls did not inhibit uptake. The uptake of dopamine, glutamate, GABA and serotonin was inhibited by the PCB mixtures, Aroclor 1242 and 1254. Under identical condition, the uptake of dopamine was inhibited more efficient than that of glutamate. The inhibition of neurotransmitter uptake was found to be dependent on the chlorination patterns of the PCB congeners, (i) ortho-chlorinated PCBs with four to five chlorine substituents (with the exception of 2,2',6,6'-TeCB) were the most effective inhibitors; (ii) hexa- or heptachlorinated PCBs were poor inhibitors or partial inhibitors (e.g. 2,2',4,4',5,5'-HCB) of glutamate and GABA uptake. Kinetic studies indicated that Aroclor 1242 inhibited dopamine uptake mainly competitively. The uptake of glutamate and GABA was inhibited in either a mixed competitive or in a non-competitive way, respectively. The neurotoxic consequences of the effect of different PCBs on neurotransmitter uptake on the uptake into synaptosomes are discussed.

  19. Treatment of Huntington disease with gamma-acetylenic GABA an irreversible inhibitor of GABA-transaminase: increased CSF GABA and homocarnosine without clinical amelioration.

    PubMed

    Tell, G; Böhlen, P; Schechter, P J; Koch-Weser, J; Agid, Y; Bonnet, A M; Coquillat, G; Chazot, G; Fischer, C

    1981-02-01

    gamma-Acetylenic GABA (GAG, RMI 71.645), a potent irreversible inhibitor of gamma-aminobutyric acid transaminase, was given orally in various dosage schedules to 14 patients with Huntington disease. The biochemical effects of the drug on cerebrospinal fluid (CSF) concentrations of gamma-aminobutyric acid (GABA) and the GABA-containing dipeptide, homocarnosine, were measured in 10 of 14 patients. Treatment with GAG increased CSF concentrations of GABA and homocarnosine as compared to pretreatment values, suggesting that the drug increased brain GABA concentration. Despite this neurochemical effect, the clinical state was not improved. Except for single seizure episodes in five patients, GAG therapy was well tolerated. These results do not exclude the possibility that agents that augment CNS GABAergic function may prove useful in therapy of Huntington disease.

  20. Inward current caused by sodium-dependent uptake of GABA in the crayfish stretch receptor neurone.

    PubMed Central

    Kaila, K; Rydqvist, B; Pasternack, M; Voipio, J

    1992-01-01

    A two-microelectrode current-voltage clamp and Cl(-)-selective microelectrodes were used to examine the effects of gamma-aminobutyric acid (GABA) on membrane potential, current and intracellular Cl- activity (aiCl) in the crayfish stretch receptor neurone. All experimental solutions were CO2-HCO3- free. 2. GABA (500 microM) produced a mono- or biphasic depolarization (amplitude < or = 10 mV), often with a prominent initial depolarizing component followed by a transient shift to a more negative level. In some neurones, an additional depolarizing phase was seen upon washout of GABA. Receptor desensitization, being absent, played no role in the multiphasic actions of GABA. 3. The pronounced increase in membrane conductance evoked by GABA (500 microM) was associated with an increase in aiCl which indicates that the depolarizing action was not due to a current carried by Cl- ions. 4. The currents activated by GABA under voltage clamp conditions were inwardly directed when recorded at the level of the resting membrane potential, and they often revealed a biphasic character. The reversal potential of peak currents activated by pulses of 500 microM-GABA (EGABA) was 9-12 mV more positive than the reversal potential of the simultaneously measured net Cl- flux (ECl). ECl was 2-7 mV more negative than the resting membrane potential. 5. EGABA (measured using pulses of 500 microM-GABA) was about 10 mV more positive than the reversal potential of the current activated by 500 microM-muscimol, a GABA agonist that is a poor substrate of the Na(+)-dependent GABA uptake system. 6. In the absence of Na+, the depolarization and inward current caused by 500 microM-GABA were converted to a hyperpolarization and to an outward current. Muscimol produced an immediate outward current both in the presence and absence of Na+. 7. Following block of the inhibitory channels by picrotoxin (100-200 microM), the depolarizing effect of 500 microM-GABA was enhanced and the transient hyperpolarizing

  1. Uptake of taurine, GABA, 5-HT, and dopamine by blood platelets in progressive myoclonus epilepsy.

    PubMed

    Airaksinen, E M

    1979-10-01

    The uptakes of four neurotransmitters (taurine, GABA, 5-HT, and dopamine) by blood platelets from patients with degenerative-type progressive myoclonus epilepsy (PME) and from controls were studied using different incubation times and different concentrations. Only the uptakes of taurine differed significantly between patients and controls: patients' uptakes were 70% to 80% of control values at 10, 30, 60, and 120 min of incubation time. Km values were approximately the same, but Vmax values in PME patients were lower, showing quantitative but not qualitative differences in taurine uptake by platelets in PME. These results suggest that a defect or an inhibitory mechanism of some factor needed in the transport or binding of taurine (but not of GABA, 5-HT, and dopamine) is present in PME.

  2. The effect of GABA and the GABA-uptake-blocker NO-711 on the b-wave of the ERG and the responses of horizontal cells to light.

    PubMed

    Hanitzsch, Renate; Küppers, Lea; Flade, Andreas

    2004-09-01

    The effects of GABA in the retina have now become of special interest because the anti-epileptic drug vigabatrin, a GABA analogue, can cause visual field loss in humans. Vigabatrin inhibits the GABA-aminotransferase, which finally results in GABA accumulation in the extracellular space. The b-wave of the electroretinogram (ERG), which originates partly in on-bipolar cells, is influenced by both GABAergic horizontal cells (HCs) and GABAergic amacrine cells (ACs). Their influences, however, are difficult to separate. In an attempt to isolate the effect of GABAergic ACs, use has been made of the specific effect of the GABA-uptake-blocker NO-711, which blocks only the GABA transporter GAT1 of GABAergic ACs. The ERG and the intracellular responses of HCs to light were recorded in the isolated rabbit retina, and the effects of GABA and NO-711, when added separately to the superfusate, were determined. GABA reduced significantly both the light responses of HCs and the b-wave. NO-711 enlarged the b-wave drastically, but did not affect the responses of HCs to light. An increase in the extracellular GABA concentration decreases the b-wave; an impairment of the function of ACs increases the b-wave. These conditions are discussed in the context of the lack of consistent changes to the b-wave during therapy with vigabatrin.

  3. Midbrain dopamine neurons sustain inhibitory transmission using plasma membrane uptake of GABA, not synthesis

    PubMed Central

    Tritsch, Nicolas X; Oh, Won-Jong; Gu, Chenghua; Sabatini, Bernardo L

    2014-01-01

    Synaptic transmission between midbrain dopamine neurons and target neurons in the striatum is essential for the selection and reinforcement of movements. Recent evidence indicates that nigrostriatal dopamine neurons inhibit striatal projection neurons by releasing a neurotransmitter that activates GABAA receptors. Here, we demonstrate that this phenomenon extends to mesolimbic afferents, and confirm that the released neurotransmitter is GABA. However, the GABA synthetic enzymes GAD65 and GAD67 are not detected in midbrain dopamine neurons. Instead, these cells express the membrane GABA transporters mGAT1 (Slc6a1) and mGAT4 (Slc6a11) and inhibition of these transporters prevents GABA co-release. These findings therefore indicate that GABA co-release is a general feature of midbrain dopaminergic neurons that relies on GABA uptake from the extracellular milieu as opposed to de novo synthesis. This atypical mechanism may confer dopaminergic neurons the flexibility to differentially control GABAergic transmission in a target-dependent manner across their extensive axonal arbors. DOI: http://dx.doi.org/10.7554/eLife.01936.001 PMID:24843012

  4. GABA uptake into astrocytes is not associated with significant metabolic cost: implications for brain imaging of inhibitory transmission.

    PubMed

    Chatton, Jean-Yves; Pellerin, Luc; Magistretti, Pierre J

    2003-10-14

    Synaptically released glutamate has been identified as a signal coupling excitatory neuronal activity to increased glucose utilization. The proposed mechanism of this coupling involves glutamate uptake into astrocytes resulting in increased intracellular Na+ (Nai+) and activation of the Na+/K+-ATPase. Increased metabolic demand linked to disruption of Nai+ homeostasis activates glucose uptake and glycolysis in astrocytes. Here, we have examined whether a similar neurometabolic coupling could operate for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), also taken up by Na+-dependent transporters into astrocytes. Thus, we have compared the Nai+ response to GABA and glutamate in mouse astrocytes by microspectrofluorimetry. The Nai+ response to GABA consisted of a rapid rise of 4-6 mM followed by a plateau that did not, however, significantly activate the pump. Indeed, the GABA transporter-evoked Na+ influxes are transient in nature, almost totally shutting off within approximately 30 sec of GABA application. The metabolic consequences of the GABA-induced Nai+ response were evaluated by monitoring cellular ATP changes indirectly in single cells and measuring 2-deoxyglucose uptake in astrocyte populations. Both approaches showed that, whereas glutamate induced a robust metabolic response in astrocytes (decreased ATP levels and glucose uptake stimulation), GABA did not cause any measurable metabolic response, consistent with the Nai+ measurements. Results indicate that GABA does not couple inhibitory neuronal activity with glucose utilization, as does glutamate for excitatory neurotransmission, and suggest that GABA-mediated synaptic transmission does not contribute directly to brain imaging signals based on deoxyglucose.

  5. Receptor regulation of the glutamate, GABA and taurine high-affinity uptake into astrocytes in primary culture.

    PubMed

    Hansson, E; Rönnbäck, L

    1991-05-10

    From experiments using dissociated primary astroglial cultures from newborn rat cerebral cortex, the stimulation of monoamine receptors (alpha, beta and 5HT) was shown to affect the high-affinity uptake kinetics of glutamate, GABA and taurine. In the presence of the alpha 1 agonist phenylephrine, there was an increased uptake (Vmax) of glutamate, while beta adrenoceptor activation slightly inhibited the glutamate uptake and stimulated the GABA and taurine uptakes. 5HT2 receptor stimulation caused a slight inhibition of the taurine uptake. The uptake rate of GABA was not affected by 5HT, alpha 1 or alpha 2 receptor agonists and the glutamate uptake was not affected by 5HT or alpha 2 receptor agonists. Nor was the taurine uptake affected by alpha 1 or alpha 2 receptor agonists. The active uptake of aspartate was unaffected by the presence of any of the monoamine receptor agonists used in this study. When the mechanisms behind these effects were studied, the GABA uptake seemed to be mediated via the G protein-adenylate cyclase complex in the receptor domain. Moreover, the K+ channels seemed to be involved. The taurine uptake, however, did not seem to be regulated by the same mechanism. It seems more probable that there is a direct interaction between the receptor and carrier of taurine at the membrane level. The mechanism underlying the receptor-regulated glutamate uptake is at present unclear, although it does not seem to involve protein kinase C.

  6. GABA (γ-Aminobutyric Acid) Uptake Via the GABA Permease GabP Represses Virulence Gene Expression in Pseudomonas syringae pv. tomato DC3000.

    PubMed

    McCraw, S L; Park, D H; Jones, R; Bentley, M A; Rico, A; Ratcliffe, R G; Kruger, N J; Collmer, A; Preston, G M

    2016-12-01

    The nonprotein amino acid γ-aminobutyric acid (GABA) is the most abundant amino acid in the tomato (Solanum lycopersicum) leaf apoplast and is synthesized by Arabidopsis thaliana in response to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (hereafter called DC3000). High levels of exogenous GABA have previously been shown to repress the expression of the type III secretion system (T3SS) in DC3000, resulting in reduced elicitation of the hypersensitive response (HR) in the nonhost plant tobacco (Nicotiana tabacum). This study demonstrates that the GABA permease GabP provides the primary mechanism for GABA uptake by DC3000 and that the gabP deletion mutant ΔgabP is insensitive to GABA-mediated repression of T3SS expression. ΔgabP displayed an enhanced ability to elicit the HR in young tobacco leaves and in tobacco plants engineered to produce increased levels of GABA, which supports the hypothesis that GABA uptake via GabP acts to regulate T3SS expression in planta. The observation that P. syringae can be rendered insensitive to GABA through loss of gabP but that gabP is retained by this bacterium suggests that GabP is important for DC3000 in a natural setting, either for nutrition or as a mechanism for regulating gene expression. [Formula: see text] Copyright © 2016 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

  7. Inhibition of GABA uptake potentiates the conductance increase produced by GABA-mimetic compounds on single neurones in isolated olfactory cortex slices of the guinea-pig.

    PubMed

    Brown, D A; Scholfield, C N

    1984-09-01

    Membrane potential and input conductance were recorded in single neurones in slices of guinea-pig olfactory cortex in vitro. gamma-Aminobutyric acid (GABA) and GABA-mimetic compounds were applied by bath-perfusion. Potency was measured as the concentration required to double the input conductance. The potency of GABA was increased (i.e. the equi-effective concentrations were reduced) by 15.5 +/- 2.3 times (mean +/- s.e. mean) on reducing external [Na+] from 144 to 20 mmol l-1, by replacement with Mg2+. Corresponding potency changes for other agonists were + 10.8 +/- 2.5 for 3-aminopropanesulphonic acid (3-APS); 3.25 +/- 1.06 for isoguvacine and 2.43 +/- 0.69 for muscimol. Nipecotic acid (0.5 mM) produced the following increases in potency: GABA 2.68 +/- 0.02; 3-aminopropanesulphonic acid, 3.11 +/- 0.07; isoguvacine, 1.92 +/- 0.34; muscimol, 2.24 +/- 0.17. The concentration of GABA in the bathing fluid necessary to double input conductance increased with increasing depth of the recording site from the cut surface. The apparent potency fell 10 times for each 60 micron depth increment up to 150 micron. The recording depth also affected the apparent potency of muscimol and 3-APS but to a lesser extent. Reduction of external [Na+] reduced the depth-dependence of both GABA and 3-APS potency. No clear change in the duration of the recurrent inhibitory postsynaptic conductance could be detected in the presence of 0.5 mmol l-1 nipecotic acid. 6 It is suggested that agonist uptake by a Na+-dependent, nipecotic acid-sensitive mechanism severely attenuates the responses of olfactory neurones to exogenous GABA and to its analogues 3-APS, muscimol and isoguvacine, but has little immediate influence on the duration of the GABA-mediated inhibitory postsynaptic conductance.

  8. (R)-roscovitine, a cyclin-dependent kinase inhibitor, enhances tonic GABA inhibition in rat hippocampus.

    PubMed

    Ivanov, A; Tyzio, R; Zilberter, Y; Ben-Ari, Yehezkel

    2008-10-02

    Pharmacological agents that mediate a persistent GABAergic conductance are of considerable interest for treatment of epilepsy. (R)-roscovitine is a membrane permeable cyclin-dependent kinase inhibitor, designed to block cell division. It is currently undergoing a phase II clinical trial as an anticancer drug. We show that (R)-roscovitine increases a tonic GABA-mediated current in rat hippocampal neurons. This enhanced tonic current appears independent of synaptic GABA release and requires functional transmembrane GABA transport. The effect of (R)-roscovitine is associated with neither modification of GABAA receptors nor protein kinase activity, but is associated with a significant increase in intracellular GABA concentration in hippocampal GABAergic neurons. (R)-roscovitine-induced tonic inhibition significantly suppresses spontaneous spiking activity of hippocampal pyramidal cells. Therefore, (R)-roscovitine is a potent modulator of neuronal activity in rat hippocampus and may provide a tool for preventing paroxysmal activity.

  9. GABA transporters as targets for new drugs.

    PubMed

    Sałat, Kinga; Kulig, Katarzyna

    2011-02-01

    GABA, the principal inhibitory neurotransmitter in the cerebral cortex, maintains the inhibitory tone that counterbalances neuronal excitation. The identification and subsequent development of GABA-transport inhibitors has shown the important role that GABA transporters play in the control of the CNS. To date, four GABA transporters have been cloned (GAT1-4). Compounds that inhibit GABA uptake are targets for epilepsy treatment. Currently, they are also being investigated for other possible indications such as the treatment of psychosis, general anxiety and sleep disorders, drug addiction, acute and chronic pain. These and other issues are discussed in this article.

  10. Distribution and ultrastructure of neurons in opossum piriform cortex displaying immunoreactivity to GABA and GAD and high-affinity tritiated GABA uptake

    SciTech Connect

    Haberly, L.B.; Hansen, D.J.; Feig, S.L.; Presto, S.

    1987-12-08

    GABAergic neurons have been identified in the piriform cortex of the opossum at light and electron microscopic levels by immunocytochemical localization of GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase and by autoradiographic visualization of high-affinity /sup 3/H-GABA uptake. Four major neuron populations have been distinguished on the basis of soma size, shape, and segregation at specific depths and locations: large horizontal cells in layer Ia of the anterior piriform cortex, small globular cells with thin dendrites concentrated in layers Ib and II of the posterior piriform cortex, and multipolar and fusiform cells concentrated in the deep part of layer III in anterior and posterior parts of the piriform cortex and the subjacent endopiriform nucleus. All four populations were well visualized with both antisera, but the large layer Ia horizontal cells displayed only very light /sup 3/H-GABA uptake, thus suggesting a lack of local axon collaterals or lack of high-affinity GABA uptake sites. The large, ultrastructurally distinctive somata of layer Ia horizontal cells receive a very small number of symmetrical synapses; the thin, axonlike dendrites of small globular cells are exclusively postsynaptic and receive large numbers of both symmetrical and asymmetrical synapses, in contrast to somata which receive a small number of both types; and the deep multipolar and fusiform cells receive a highly variable number of symmetrical and asymmetrical synapses on somata and proximal dendrites. Labeled puncta of axon terminal dimensions were found in large numbers in the neuropil surrounding pyramidal cell somata in layer II and in the endopiriform nucleus. Moderately large numbers of labeled puncta were found in layer I at the depth of pyramidal cell apical dendrites with greater numbers in layer Ia at the depth of distal apical segments than in layer Ib.

  11. Impaired GABA synthesis, uptake and release are associated with depression-like behaviors induced by chronic mild stress

    PubMed Central

    Ma, K; Xu, A; Cui, S; Sun, M-R; Xue, Y-C; Wang, J-H

    2016-01-01

    Major depression is a prevalent emotion disorder. Chronic stressful life in genetically susceptible individuals is presumably a major etiology that leads to neuron and synapse atrophy in the limbic system. Molecular mechanisms underlying the pathological changes remain elusive. Mice were treated by chronic unpredictable mild stress (CUMS) until they demonstrated depression-like behavior. GABA release in the medial prefrontal cortex was evaluated by cell electrophysiology and imaging. Molecular profiles related to GABA synthesis and uptake were investigated by the high-throughput sequencings of microRNAs and mRNAs as well as western blot analysis in this cortical area. In CUMS-induced depression mice, there appear the decreases in the innervation and function of GABAergic axons and in the levels of mRNAs and proteins of glutamate decarboxylase-67, vesicular GABA transporter and GABA transporter-3. miRNA-15b-5p, miRNA-144-3p, miRNA-582-5p and miRNA-879-5p that directly downregulate such mRNAs increase in this cortex. Our results suggest that chronic mild stress impairs GABA release and uptake by upregulating miRNAs and downregulating mRNAs and proteins, which may constitute the subcellular and molecular mechanisms for the lowered GABA tone in major depression. PMID:27701406

  12. Enhanced astroglial GABA uptake attenuates tonic GABAA inhibition of the presympathetic hypothalamic paraventricular nucleus neurons in heart failure.

    PubMed

    Pandit, Sudip; Jo, Ji Yoon; Lee, Sang Ung; Lee, Young Jae; Lee, So Yeong; Ryu, Pan Dong; Lee, Jung Un; Kim, Hyun-Woo; Jeon, Byeong Hwa; Park, Jin Bong

    2015-08-01

    γ-Aminobutyric acid (GABA) generates persistent tonic inhibitory currents (Itonic) and conventional inhibitory postsynaptic currents in the hypothalamic paraventricular nucleus (PVN) via activation of GABAA receptors (GABAARs). We investigated the pathophysiological significance of astroglial GABA uptake in the regulation of Itonic in the PVN neurons projecting to the rostral ventrolateral medulla (PVN-RVLM). The Itonic of PVN-RVLM neurons were significantly reduced in heart failure (HF) compared with sham-operated (SHAM) rats. Reduced Itonic sensitivity to THIP argued for the decreased function of GABAAR δ subunits in HF, whereas similar Itonic sensitivity to benzodiazepines argued against the difference of γ2 subunit-containing GABAARs in SHAM and HF rats. HF Itonic attenuation was reversed by a nonselective GABA transporter (GAT) blocker (nipecotic acid, NPA) and a GAT-3 selective blocker, but not by a GAT-1 blocker, suggesting that astroglial GABA clearance increased in HF. Similar and minimal Itonic responses to bestrophin-1 blockade in SHAM and HF neurons further argued against a role for astroglial GABA release in HF Itonic attenuation. Finally, the NPA-induced inhibition of spontaneous firing was greater in HF than in SHAM PVN-RVLM neurons, whereas diazepam induced less inhibition of spontaneous firing in HF than in SHAM neurons. Overall, our results showed that combined with reduced GABAARs function, the enhanced astroglial GABA uptake-induced attenuation of Itonic in HF PVN-RVLM neurons explains the deficit in tonic GABAergic inhibition and increased sympathetic outflow from the PVN during heart failure.

  13. Effects of the GABA-uptake blocker NNC-711 on spontaneous sharp wave-ripple complexes in mouse hippocampal slices.

    PubMed

    Viereckel, Thomas; Kostic, Milos; Bähner, Florian; Draguhn, Andreas; Both, Martin

    2013-05-01

    The precise temporal and spatial activity patterns of neurons in cortical networks are organized by different state-dependent types of network oscillations. GABAergic inhibition plays a key role in the underlying mechanisms of such oscillations and it has been suggested that the duration of widely distributed phasic inhibitory postsynaptic potentials (IPSPs) determines the frequency of the resulting network oscillation. Here, we test this hypothesis in an in vitro model of sharp wave-ripple (SPW-R) complexes, a particularly fast pattern of network oscillations at ∼200 Hz which is involved in memory consolidation. We recorded SPW-R in mouse hippocampal slices in the absence and presence of NCC-711, an inhibitor of GABA uptake. The resulting prolongation of IPSP resulted in reduced occurrence of SPW-R, whereas the superimposed fast oscillations as well as the precision of rhythmic cell synchronization remained stable. Application of Diazepam which is a positive modulator of the GABAA receptor led to consistent results. We conclude that phasic inhibition is a major regulator of network excitability in CA3 (where SPW-Rs are generated), but does not set the frequency of hippocampal ripples. Copyright © 2013 Wiley Periodicals, Inc.

  14. GABA-ergic neurons in the leach central nervous system

    SciTech Connect

    Cline, H.T.

    1985-01-01

    GABA is a candidate for an inhibitory neurotransmitter in the leech central nervous system because of the well-documented inhibitory action of GABA in other invertebrates. To demonstrate that GABA meets the criteria used to identify a substance as a neurotransmitter, the author examined GABA metabolism and synaptic interactions of inhibitory motor neurons in two leech species, Hirudo medicinalis and Haementeria ghilianii. Segmental ganglia of the leech ventral nerve cord and identified inhibitors have the capacity to synthesize GABA when incubated in the presence of the precursor glutamate. Application of GABA to cell bodies of excitatory motor neurons or muscle fibers innervated by the inhibitors hyperpolarizes the membrane potential of the target cell and activates a chloride ion conductance channel, similar to the inhibitory membrane response following intracellular stimulation of the inhibitor. Bicuculline methiodide (5 x 10/sup -5/M), GABA receptor antagonist, blocks reversibly the response to applied GABA and the inhibitory synaptic inputs onto the postsynaptic neurons or muscle fibers without interfering with their excitatory inputs. Furthermore, the inhibitors are included among approximately 25 neurons per segmental ganglion that take up GABA by a high affinity uptake system, as revealed by /sup 3/H-GABA-autoradiography. The development of the capacities to synthesize and to take up GABA were examined in leech embryos. The embryos are able to synthesize GABA at early stages of the development of the nervous system, before any neurons have extended neutrites.

  15. Manganese accumulation in membrane fractions of primary astrocytes is associated with decreased γ-aminobutyric acid (GABA) uptake, and is exacerbated by oleic acid and palmitate.

    PubMed

    Fordahl, Steve C; Erikson, Keith M

    2014-05-01

    Manganese (Mn) exposure interferes with GABA uptake; however, the effects of Mn on GABA transport proteins (GATs) have not been identified. We sought to characterize how Mn impairs GAT function in primary rat astrocytes. Astrocytes exposed to Mn (500 μM) had significantly reduced (3)H-GABA uptake despite no change in membrane or cytosolic GAT3 protein levels. Co-treatment with 100 μM oleic or palmitic acids (both known to be elevated in Mn neurotoxicity), exacerbated the Mn-induced decline in (3)H-GABA uptake. Mn accumulation in the membrane fraction of astrocytes was enhanced with fatty acid administration, and was negatively correlated with (3)H-GABA uptake. Furthermore, control cells exposed to Mn only during the experimental uptake had significantly reduced (3)H-GABA uptake, and the addition of GABA (50 μM) blunted cytosolic Mn accumulation. These data indicate that reduced GAT function in astrocytes is influenced by Mn and fatty acids accumulating at or interacting with the plasma membrane. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Contribution of astrocytic glutamate and GABA uptake to corticostriatal information processing

    PubMed Central

    Goubard, Valérie; Fino, Elodie; Venance, Laurent

    2011-01-01

    Abstract The astrocytes, active elements of the tripartite synapse, remove most of the neurotransmitter that spills over the synaptic cleft. Neurotransmitter uptake operated by astrocytes contributes to the strength and timing of synaptic inputs. The striatum, the main input nucleus of basal ganglia, extracts pertinent cortical signals from the background noise and relays cortical information toward basal ganglia output structures. We investigated the role of striatal astrocytic uptake in the shaping of corticostriatal transmission. We performed dual patch-clamp recordings of striatal output neuron (the medium-sized spiny neurons, MSNs)–astrocyte pairs while stimulating the somatosensory cortex. Cortical activity evoked robust synaptically activated transporter-mediated currents (STCs) in 78% of the recorded astrocytes. STCs originated equally from the activities of glutamate transporters and GABA transporters (GATs). Astrocytic STCs reflected here a presynaptic release of neurotransmitters. STCs displayed a large magnitude associated with fast kinetics, denoting an efficient neurotransmitter clearance at the corticostriatal pathway. Inhibition of glutamate transporters type-1 (GLT-1) and GATs decreased the corticostriatal synaptic transmission, through, respectively, desensitization of AMPA receptors and activation of GABAA receptor. STCs displayed a bidirectional short-term plasticity (facilitation for paired-pulse intervals less than 100 ms and depression up to 1 s). We report a genuine facilitation of STCs for high-frequency cortical activity, which could strengthen the detection properties of cortical activity operated by MSNs. MSN EPSCs showed a triphasic short-term plasticity, which was modified by the blockade of GLT-1 or GATs. We show here that neurotransmitter uptake by astrocytes plays a key role in the corticostriatal information processing. PMID:21486792

  17. Contribution of astrocytic glutamate and GABA uptake to corticostriatal information processing.

    PubMed

    Goubard, Valérie; Fino, Elodie; Venance, Laurent

    2011-05-01

    The astrocytes, active elements of the tripartite synapse, remove most of the neurotransmitter that spills over the synaptic cleft. Neurotransmitter uptake operated by astrocytes contributes to the strength and timing of synaptic inputs. The striatum, the main input nucleus of basal ganglia, extracts pertinent cortical signals from the background noise and relays cortical information toward basal ganglia output structures. We investigated the role of striatal astrocytic uptake in the shaping of corticostriatal transmission.We performed dual patch-clamp recordings of striatal output neuron (the medium-sized spiny neurons, MSNs)–astrocyte pairs while stimulating the somatosensory cortex. Cortical activity evoked robust synaptically activated transporter-mediated currents (STCs) in 78% of the recorded astrocytes. STCs originated equally from the activities of glutamate transporters and GABA transporters (GATs). Astrocytic STCs reflected here a presynaptic release of neurotransmitters. STCs displayed a large magnitude associated with fast kinetics, denoting an efficient neurotransmitter clearance at the corticostriatal pathway. Inhibition of glutamate transporters type-1 (GLT-1) and GATs decreased the corticostriatal synaptic transmission, through, respectively, desensitization of AMPA receptors and activation of GABAA receptor. STCs displayed a bidirectional short-term plasticity (facilitation for paired-pulse intervals less than 100 ms and depression up to 1 s).We report a genuine facilitation of STCs for high-frequency cortical activity, which could strengthen the detection properties of cortical activity operated by MSNs. MSN EPSCs showed a triphasic short-term plasticity, which was modified by the blockade of GLT-1 or GATs. We show here that neurotransmitter uptake by astrocytes plays a key role in the corticostriatal information processing.

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

    SciTech Connect

    Bedwani, J.R.; Mehta, A.

    1987-01-01

    The release of (/sup 3/H)gamma-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 gamma-vinyl GABA. In the experiments carried out without an inhibitor, an ion-exchange column chromatographic technique was used to separate (/sup 3/H)GABA from tritiated metabolites released with it into the superfusate. The presence of gabaculine (5 microM) substantially reduced the Ca2+-dependence of the release of (/sup 3/H)GABA evoked by a 4 min 30 mM K+ pulse, whereas this was not appreciably reduced by the presence of gamma-vinyl GABA (2 mM or 10 mM). Nevertheless, the characteristics of (/sup 3/H)GABA release were not identical in the presence and absence of either inhibitor.

  19. NNC-711, a novel potent and selective gamma-aminobutyric acid uptake inhibitor: pharmacological characterization.

    PubMed

    Suzdak, P D; Frederiksen, K; Andersen, K E; Sørensen, P O; Knutsen, L J; Nielsen, E B

    1992-12-02

    NNC-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3- pyridinecarboxylic acid hydrochloride) is a novel, potent and selective gamma-aminobutyric acid (GABA) uptake inhibitor. NNC-711 inhibited synaptosomal (IC50 = 47 nM), neuronal (IC50 = 1238 nM) and glial (IC50 = 636 nM) GABA uptake in vitro NNC-711 lacked affinity for other neurotransmitter receptor binding sites, uptake sites and ion channels examined in vitro. In vivo, NNC-711 was a potent anticonvulsant compound against rodent seizures induced by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (ED50 (clonic) = 1.2 mg/kg i.p.), pentylenetetrazole (PTZ) (ED50 (tonic) = 0.72 mg/kg i.p., mouse; and ED50 (tonic) = 1.7 mg/kg, rat), or audiogenic (ED50 (clonic and tonic) = 0.23 mg/kg i.p.). At higher doses NNC-711 produced behavioral side effects characterized by inhibition of traction (ED50 = 23 mg/kg i.p.), rotarod (ED50 = 10 mg/kg i.p.) and exploratory locomotor activity (ED50 = 45 mg/kg i.p.) in the mouse. Following acute (3-h) in vivo pretreatment with NNC-711, behavioral tolerance developed to its motor impairing side effects (inhibition of traction, rotarod or exploratory locomotor activity) without corresponding tolerance to the anticonvulsant effects. These data suggest that NNC-711 will be useful for future in vitro and in vivo experiments to elucidate the role of the GABA uptake carrier in the central nervous system.

  20. The taurine uptake inhibitor guanidinoethyl sulphonate is an agonist at gamma-aminobutyric acid(A) receptors in cultured murine cerebellar granule cells.

    PubMed

    Mellor, J R; Gunthorpe, M J; Randall, A D

    2000-05-26

    In patch clamp experiments the beta-amino acid uptake inhibitor guanidinoethyl sulphonate (GES) activated currents in intact cultured murine cerebellar granule neurones. These responses could be attenuated by the gamma-aminobutyric acid(A) (GABA(A)) receptor antagonists bicuculline and picrotoxin. With intracellular chloride concentrations of either 20 or 130 mM, GES-induced current responses reversed polarity near the chloride equilibrium potential. When fast applications of agonist were made to excised granule cell macropatches GES responses were dose-dependent and exhibited significant outward rectification. Like taurine (but unlike GABA and beta-alanine) responses, macroscopic desensitisation of GES-induced currents was slow. Our data indicate that care should be exercised when using GES as a taurine uptake inhibitor in systems that also contain GABA(A) receptors.

  1. GABA uptake blocker NNC-711 exhibits marked anticonvulsant action in two cortical epileptic models in immature rats.

    PubMed

    Bernásková, K; Slamberová, R; Mares, P

    1999-09-01

    To study the anticonvulsant action of a gamma-aminobutyric acid (GABA) uptake inhibitor NNC-711 in two models of cortical epileptogenesis in immature rats. Twelve-, 18-, and 25-day-old rat pups with implanted electrodes were used in this study. Epileptogenic foci were elicited by a local application of bicuculline methiodide (BMI) on the sensorimotor cortical region by means of an implanted cannula, and cortical epileptic afterdischarges (ADs) were induced by low-frequency stimulation (8 Hz) of the same cortical area. Epileptogenic foci were formed after pretreatment with NNC-711 (1 or 10 mg/kg, i.p.), and epileptic ADs were elicited in the second experimental series. Then NNC-711 was administered in the same doses, and stimulation was repeated. NNC-711 did not block the formation of epileptogenic foci, but it significantly suppressed the spontaneous transition of interictal focal into ictal activity in all age groups. The intensity of movements accompanying stimulation of the sensorimotor cortex was less under the influence of NNC-711 in the 18- and 25-day-old rats. The duration of cortical ADs was shortened in all age groups, but transient abolition of ADs was observed only after the higher dose in the 25-day-old rats. In addition, the intensity of clonic seizures appearing during ADs decreased, and the transition of ADs into another type due to an involvement of limbic structures failed to appear in the 18- and 25-day-old rats. Primary epileptogenesis in the cerebral cortex was hardly influenced by NNC-711, but the spread of epileptic activity was markedly suppressed. This effect was better expressed in the 18- and 25-day-old animals than in the youngest group.

  2. Neurochemical characterization of a neuroprotective compound from Parawixia bistriata spider venom that inhibits synaptosomal uptake of GABA and glycine.

    PubMed

    Beleboni, Renê Oliveira; Guizzo, Renato; Fontana, Andréia Cristina Karklin; Pizzo, Andrea Baldocchi; Carolino, Ruither Oliveira Gomes; Gobbo-Neto, Leonardo; Lopes, Norberto Peporine; Coutinho-Netto, Joaquim; Dos Santos, Wagner Ferreira

    2006-06-01

    The major contribution of this work is the isolation of a neuroprotective compound referred to as 2-amino-5-ureidopentanamide (FrPbAII) (M(r) = 174) from Parawixia bistriata spider venom and an investigation of its mode of action. FrPbAII inhibits synaptosomal GABA uptake in a dose-dependent manner and probably does not act on Na(+), K(+), and Ca(2+) channels, GABA(B) receptors, or gamma-aminobutyrate:alpha-ketoglutarate aminotransferase enzyme; therefore, it is not directly dependent on these structures for its action. Direct increase of GABA release and reverse transport are also ruled out as mechanisms of FrPbAII activities as well as unspecific actions on pore membrane formation. Moreover, FrPbAII is selective for GABA and glycine transporters, having slight or no effect on monoamines or glutamate transporters. According to our experimental glaucoma data in rat retina, FrPbAII is able to cross the blood-retina barrier and promote effective protection of retinal layers submitted to ischemic conditions. These studies are of relevance by providing a better understanding of neurochemical mechanisms involved in brain function and for possible development of new neuropharmacological and therapeutic tools.

  3. A novel GABA(A) alpha 5 receptor inhibitor with therapeutic potential.

    PubMed

    Ling, István; Mihalik, Balázs; Etherington, Lori-An; Kapus, Gábor; Pálvölgyi, Adrienn; Gigler, Gábor; Kertész, Szabolcs; Gaál, Attila; Pallagi, Katalin; Kiricsi, Péter; Szabó, Éva; Szénási, Gábor; Papp, Lilla; Hársing, László G; Lévay, György; Spedding, Michael; Lambert, Jeremy J; Belelli, Delia; Barkóczy, József; Volk, Balázs; Simig, Gyula; Gacsályi, István; Antoni, Ferenc A

    2015-10-05

    Novel 2,3-benzodiazepine and related isoquinoline derivatives, substituted at position 1 with a 2-benzothiophenyl moiety, were synthesized to produce compounds that potently inhibited the action of GABA on heterologously expressed GABAA receptors containing the alpha 5 subunit (GABAA α5), with no apparent affinity for the benzodiazepine site. Substitutions of the benzothiophene moiety at position 4 led to compounds with drug-like properties that were putative inhibitors of extra-synaptic GABAA α5 receptors and had substantial blood-brain barrier permeability. Initial characterization in vivo showed that 8-methyl-5-[4-(trifluoromethyl)-1-benzothiophen-2-yl]-1,9-dihydro-2H-[1,3]oxazolo[4,5-h][2,3]benzodiazepin-2-one was devoid of sedative, pro-convulsive or motor side-effects, and enhanced the performance of rats in the object recognition test. In summary, we have discovered a first-in-class GABA-site inhibitor of extra-synaptic GABAA α5 receptors that has promising drug-like properties and warrants further development.

  4. The gamma-aminobutyric acid uptake inhibitor NO-711 potentiates 3-aminopropylphosphinic acid-induced actions in rat neocortical slices.

    PubMed

    Ong, J; Kerr, D I

    1998-04-24

    In rat neocortical slices maintained in Mg2+-free Krebs medium, the GABAB receptor agonists baclofen and 3-aminopropylphosphinic acid dose-dependently reduced the frequency of spontaneous discharges, 3-aminopropylphosphinic acid being 10 times less potent than baclofen. These were sensitive to the antagonist CGP 52432 (3-[[3,4-dichloro-phenyl)methyl]-amino]propyl](-P-diethoxymethyl)- phosphinic acid) (1, 5 and 10 microM). The GABA uptake inhibitor NO-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-+ ++pyridinecarboxylic acid) (5 and 10 microM) produced 2.9 and 9 fold increases in the potency of 3-aminopropylphosphinic acid without affecting baclofen-induced responses. In this study, the low potency of 3-aminopropylphosphinic acid when compared to baclofen, may be attributed to its uptake by NO-711-sensitive GABA transporters.

  5. Combined Oral Administration of GABA and DPP-4 Inhibitor Prevents Beta Cell Damage and Promotes Beta Cell Regeneration in Mice

    PubMed Central

    Liu, Wenjuan; Son, Dong Ok; Lau, Harry K.; Zhou, Yinghui; Prud’homme, Gerald J.; Jin, Tianru; Wang, Qinghua

    2017-01-01

    γ-aminobutyric acid (GABA) or glucagon-like peptide-1 based drugs, such as sitagliptin (a dipeptidyl peptidase-4 inhibitor), were shown to induce beta cell regenerative effects in various diabetic mouse models. We propose that their combined administration can bring forth an additive therapeutic effect. We tested this hypothesis in a multiple low-dose streptozotocin (STZ)-induced beta cell injury mouse model (MDSD). Male C57BL/6J mice were assigned randomly into four groups: non-treatment diabetic control, GABA, sitagliptin, or GABA plus sitagliptin. Oral drug administration was initiated 1 week before STZ injection and maintained for 6 weeks. GABA or sitagliptin administration decreased ambient blood glucose levels and improved the glucose excursion rate. This was associated with elevated plasma insulin and reduced plasma glucagon levels. Importantly, combined use of GABA and sitagliptin significantly enhanced these effects as compared with each of the monotherapies. An additive effect on reducing water consumption was also observed. Immunohistochemical analyses revealed that combined GABA and sitagliptin therapy was superior in increasing beta cell mass, associated with increased small-size islet numbers, Ki67+ and PDX-1+ beta cell counts; and reduced Tunel+ beta cell counts. Thus, beta cell proliferation was increased, whereas apoptosis was reduced. We also noticed a suppressive effect of GABA or sitagliptin on alpha cell mass, which was not significantly altered by combining the two agents. Although either GABA or sitagliptin administration delays the onset of MDSD, our study indicates that combined use of them produces superior therapeutic outcomes. This is likely due to an amelioration of beta cell proliferation and a decrease of beta cell apoptosis. PMID:28676760

  6. Combined Oral Administration of GABA and DPP-4 Inhibitor Prevents Beta Cell Damage and Promotes Beta Cell Regeneration in Mice.

    PubMed

    Liu, Wenjuan; Son, Dong Ok; Lau, Harry K; Zhou, Yinghui; Prud'homme, Gerald J; Jin, Tianru; Wang, Qinghua

    2017-01-01

    γ-aminobutyric acid (GABA) or glucagon-like peptide-1 based drugs, such as sitagliptin (a dipeptidyl peptidase-4 inhibitor), were shown to induce beta cell regenerative effects in various diabetic mouse models. We propose that their combined administration can bring forth an additive therapeutic effect. We tested this hypothesis in a multiple low-dose streptozotocin (STZ)-induced beta cell injury mouse model (MDSD). Male C57BL/6J mice were assigned randomly into four groups: non-treatment diabetic control, GABA, sitagliptin, or GABA plus sitagliptin. Oral drug administration was initiated 1 week before STZ injection and maintained for 6 weeks. GABA or sitagliptin administration decreased ambient blood glucose levels and improved the glucose excursion rate. This was associated with elevated plasma insulin and reduced plasma glucagon levels. Importantly, combined use of GABA and sitagliptin significantly enhanced these effects as compared with each of the monotherapies. An additive effect on reducing water consumption was also observed. Immunohistochemical analyses revealed that combined GABA and sitagliptin therapy was superior in increasing beta cell mass, associated with increased small-size islet numbers, Ki67(+) and PDX-1(+) beta cell counts; and reduced Tunel(+) beta cell counts. Thus, beta cell proliferation was increased, whereas apoptosis was reduced. We also noticed a suppressive effect of GABA or sitagliptin on alpha cell mass, which was not significantly altered by combining the two agents. Although either GABA or sitagliptin administration delays the onset of MDSD, our study indicates that combined use of them produces superior therapeutic outcomes. This is likely due to an amelioration of beta cell proliferation and a decrease of beta cell apoptosis.

  7. Fluvoxamine, a specific 5-hydroxytryptamine uptake inhibitor.

    PubMed

    Claassen, V; Davies, J E; Hertting, G; Placheta, P

    1977-08-01

    1. On the basis of both in vitro and in vivo experiments fluvoxamine has been characterized as a potential anti-depressant drug with almost exclusively 5-hydroxytryptamine (5-HT) uptake inhibiting properties. 2. Fluvoxamine is effective in inhibiting 5-ht uptake by blood platelets and brain synaptosomes. Due to inhibition of the membrane pump the compound prevents 5-HT depletion by the tyramine-derivatives H 75/12 and H 77/77. As a result of the interference with the neuronal re-uptake mechanism for 5-HT, fluvoxamine produces a decreased 5-HT turnover in the brain. Effects of 5-hydroxytryptophan (5-HTP) are potentiated in mice and in combination with pargyline, fluvoxamine induces 5-HT-like behavioural effects. 3. In contrast to tricyclic antidepressants, noradrenaline uptake processes are either unaffected or only slightly inhibited by fluvoxamine. The noradrenaline depleting effects of tyramine derivates are not influenced by fluvoxamine. Reserpine effects, such as ptosis are affected only at very high doses of the test compound. The antagonism by fluvoxamine of the reserpine-induced lowering of the pentamethylenetetrazole convulsive threshold can be regarded as due to an effect upon 5-HT uptake. In contrast to the effects of desmethylimipramine and imipramine, no stimulatory effects are found in rats when rapidly acting reserpine-like compounds are given following a dose of fluvoxamine.

  8. Fluvoxamine, a specific 5-hydroxytryptamine uptake inhibitor.

    PubMed Central

    Claassen, V; Davies, J E; Hertting, G; Placheta, P

    1977-01-01

    1. On the basis of both in vitro and in vivo experiments fluvoxamine has been characterized as a potential anti-depressant drug with almost exclusively 5-hydroxytryptamine (5-HT) uptake inhibiting properties. 2. Fluvoxamine is effective in inhibiting 5-ht uptake by blood platelets and brain synaptosomes. Due to inhibition of the membrane pump the compound prevents 5-HT depletion by the tyramine-derivatives H 75/12 and H 77/77. As a result of the interference with the neuronal re-uptake mechanism for 5-HT, fluvoxamine produces a decreased 5-HT turnover in the brain. Effects of 5-hydroxytryptophan (5-HTP) are potentiated in mice and in combination with pargyline, fluvoxamine induces 5-HT-like behavioural effects. 3. In contrast to tricyclic antidepressants, noradrenaline uptake processes are either unaffected or only slightly inhibited by fluvoxamine. The noradrenaline depleting effects of tyramine derivates are not influenced by fluvoxamine. Reserpine effects, such as ptosis are affected only at very high doses of the test compound. The antagonism by fluvoxamine of the reserpine-induced lowering of the pentamethylenetetrazole convulsive threshold can be regarded as due to an effect upon 5-HT uptake. In contrast to the effects of desmethylimipramine and imipramine, no stimulatory effects are found in rats when rapidly acting reserpine-like compounds are given following a dose of fluvoxamine. PMID:302726

  9. Effect of Uptake-one inhibitors on the uptake of norepinephrine and metaiodobenzylguanidine

    SciTech Connect

    Tobes, M.C.; Jaques, S. Jr.; Wieland, D.M.; Sisson, J.C.

    1985-08-01

    The mechanisms underlying the uptake of the radiopharmaceutical metaiodobenzylguanidine (MIBG) and the catecholamine norepinephrine (NE) were studied using cultured bovine adrenomedullary cells as an in vitro model system. Sodium-dependent and sodium-independent uptake systems have been identified and characterized for both MIBG and NE. The sodium-dependent uptake of Ne and MIBG was inhibited by the selective Uptake-one inhibitors, desmethylimipramine (DMI) and cocaine, whereas the sodium-independent uptake for NE and MIBG was much less sensitive to inhibition by these agents. The sodium-dependent uptake system fulfills the criteria for the neuronal Uptake-one system, and the sodium-independent uptake system fulfills the criteria for a passive diffusion mechanism. Arterial concentrations proximal to the dog adrenal were very small suggesting that the sodium-dependent (Uptake-one) system is predominant in vivo. Consistent with the in vitro observations, the in vivo uptake of MIBG and NE into dog adrenal medullae was effectively blocked by pretreatment with DMI or cocaine. Therefore, iodine-131 MIBG scintigraphy of the adrenal appears to reflect uptake by way of the Uptake-one system.

  10. Cl− uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1

    PubMed Central

    Yamada, Junko; Okabe, Akihito; Toyoda, Hiroki; Kilb, Werner; Luhmann, Heiko J; Fukuda, Atsuo

    2004-01-01

    GABA is the principal inhibitory neurotransmitter in the mature brain, but during early postnatal development the elevated [Cl−]i in immature neocortical neurones causes GABAA receptor activation to be depolarizing. The molecular mechanisms underlying this intracellular Cl− accumulation remain controversial. Therefore, the GABA reversal potential (EGABA) or [Cl−]i in early postnatal rat neocortical neurones was measured by the gramicidin-perforated patch-clamp method, and the relative expression levels of the cation−Cl− cotransporter mRNAs (in the same cells) were examined by semiquantitative single-cell multiplex RT-PCR to look for statistical correlations with [Cl−]i. The mRNA expression levels were positively (the Cl− accumulating Na+,K+−2Cl− cotransporter NKCC1) or negatively (the Cl− extruding K+−Cl− cotransporter KCC2) correlated with [Cl−]i. NKCC1 mRNA expression was high in early postnatal days, but decreased during postnatal development, whereas KCC2 mRNA expression displayed the opposite pattern. [Cl−]i and NKCC1 mRNA expression were each higher in cortical plate (CP) neurones than in the presumably older layer V/VI pyramidal neurones in a given slice. The pharmacological effects of bumetanide on EGABA were consistent with the different expression levels of NKCC1 mRNA. These data suggest that NKCC1 may play a pivotal role in the generation of GABA-mediated depolarization in immature CP cells, while KCC2 promotes the later maturation of GABAergic inhibition in the rat neocortex. PMID:15090604

  11. A functional role for both -aminobutyric acid (GABA) transporter-1 and GABA transporter-3 in the modulation of extracellular GABA and GABAergic tonic conductances in the rat hippocampus.

    PubMed

    Kersanté, Flavie; Rowley, Samuel C S; Pavlov, Ivan; Gutièrrez-Mecinas, María; Semyanov, Alexey; Reul, Johannes M H M; Walker, Matthew C; Linthorst, Astrid C E

    2013-05-15

    Tonic γ-aminobutyric acid (GABA)A receptor-mediated signalling controls neuronal network excitability in the hippocampus. Although the extracellular concentration of GABA (e[GABA]) is critical in determining tonic conductances, knowledge on how e[GABA] is regulated by different GABA transporters (GATs) in vivo is limited. Therefore, we studied the role of GATs in the regulation of hippocampal e[GABA] using in vivo microdialysis in freely moving rats. Here we show that GAT-1, which is predominantly presynaptically located, is the major GABA transporter under baseline, quiescent conditions. Furthermore, a significant contribution of GAT-3 in regulating e[GABA] was revealed by administration of the GAT-3 inhibitor SNAP-5114 during simultaneous blockade of GAT-1 by NNC-711. Thus, the GABA transporting activity of GAT-3 (the expression of which is confined to astrocytes) is apparent under conditions in which GAT-1 is blocked. However, sustained neuronal activation by K(+)-induced depolarization caused a profound spillover of GABA into the extrasynaptic space and this increase in e[GABA] was significantly potentiated by sole blockade of GAT-3 (i.e. even when uptake of GAT-1 is intact). Furthermore, experiments using tetrodotoxin to block action potentials revealed that GAT-3 regulates extrasynaptic GABA levels from action potential-independent sources when GAT-1 is blocked. Importantly, changes in e[GABA] resulting from both GAT-1 and GAT-3 inhibition directly precipitate changes in tonic conductances in dentate granule cells as measured by whole-cell patch-clamp recording. Thus, astrocytic GAT-3 contributes to the regulation of e[GABA] in the hippocampus in vivo and may play an important role in controlling the excitability of hippocampal cells when network activity is increased.

  12. Brofaromine: a monoamine oxidase-A and serotonin uptake inhibitor.

    PubMed

    Waldmeier, P C; Glatt, A; Jaekel, J; Bittiger, H

    1993-01-01

    Brofaromine is a tight-binding, reversible inhibitor of monoamine oxidase-A (MAO-A), with concomitant serotonin (5-HT) uptake-inhibiting properties. In psychopharmacologic investigations, the compound shows the properties expected of an MAO inhibitor, antagonizing the effects of reserpine, tetrabenazine, and 5-hydroxytryptophan in rats and mice, and suppressing rapid eye movement sleep in cats. Brofaromine showed antidepressant-like activity in a rat social conflict test. In radioligand binding assays, brofaromine exhibited weak or no interaction with alpha 1- and alpha 2-noradrenergic, 5-HT1, 5-HT2, 5-HT3, cholinergic, histamine H1 and H2, mu-opiate, GABAA, benzodiazepine, adenosine, neurotensin, and substance P receptors. Comparison of in vitro and in vivo potencies to inhibit 5-HT uptake with those of reference drugs, and direct evidence in patients and volunteers suggest that 5-HT uptake inhibition plays a role in the clinical profile of brofaromine.

  13. Up-regulation of P2X7 receptor-mediated inhibition of GABA uptake by nerve terminals of the human epileptic neocortex.

    PubMed

    Barros-Barbosa, Aurora R; Fonseca, Ana L; Guerra-Gomes, Sónia; Ferreirinha, Fátima; Santos, Agostinho; Rangel, Rui; Lobo, M Graça; Correia-de-Sá, Paulo; Cordeiro, J Miguel

    2016-01-01

    Thirty percent of patients with epilepsy are refractory to medication. The majority of these patients have mesial temporal lobe epilepsy (MTLE). This prompts for new pharmacologic targets, like ATP-mediated signaling pathways, since the extracellular levels of the nucleotide dramatically increase during in vitro epileptic seizures. In this study, we investigated whether sodium-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake by isolated nerve terminals of the human neocortex could be modulated by ATP acting via slow-desensitizing P2X7 receptor (P2X7R). Modulation of [(3) H]GABA and [(14) C]glutamate uptake by ATP, through activation of P2X7R, was investigated in isolated nerve terminals of the neocortex of cadaveric controls and patients with drug-resistant epilepsy (non-MTLE or MTLE) submitted to surgery. Tissue density and distribution of P2X7R in the human neocortex was assessed by Western blot analysis and immunofluorescence confocal microscopy. The P2X7R agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-100 μm) decreased [(3) H]GABA and [(14) C]glutamate uptake by nerve terminals of the neocortex of controls and patients with epilepsy. The inhibitory effect of BzATP (100 μm) was prevented by the selective P2X7R antagonist, A-438079 (3 μm). Down-modulation of [(14) C]glutamate uptake by BzATP (100 μm) was roughly similar in controls and patients with epilepsy, but the P2X7R agonist inhibited more effectively [(3) H]GABA uptake in the epileptic tissue. Neocortical nerve terminals of patients with epilepsy express higher amounts of the P2X7R protein than control samples. High-frequency cortical activity during epileptic seizures releases huge amounts of ATP, which by acting on low-affinity slowly desensitizing ionotropic P2X7R, leads to down-modulation of neuronal GABA and glutamate uptake. Increased P2X7R expression in neocortical nerve terminals of patients with epilepsy may, under high-frequency firing, endure GABA signaling and

  14. The GABA synapse as a target for antiepileptic drugs: a historical overview focused on GABA transporters.

    PubMed

    Schousboe, Arne; Madsen, Karsten K; Barker-Haliski, Melissa L; White, H Steve

    2014-10-01

    It is clear that normal neuronal function relies on a tight balance between excitatory and inhibitory neurotransmission. Inhibitory signaling through the GABAergic system can be tightly regulated at the level of GABA uptake via GABA transporters (GAT). As such, selectively modulating the GABA uptake process through pharmacological agents has been an area of active investigation over several decades. These studies have demonstrated that inhibition of astroglial, but not neuronal, GATs may be preferred for anticonvulsant action. To date, four distinct GAT subtypes have been identified and efforts to selectively target these transporters have led to the proliferation of pharmacological agents aimed at augmenting extrasynaptic GABA levels. These pharmacological tools have provided novel and informative insight into the role of GABA and GABAergic signaling in the brain, but have also provided critical information concerning the regulation of CNS disorders associated with an imbalance in inhibitory tone, such as epilepsy. One such compound with notable inhibitory effects at GATs, tiagabine, has demonstrated clinical anticonvulsant efficacy, and is, to date, the only approved GAT inhibitor for clinical use. Thus, efforts to identify and develop GAT subtype-specific compounds continue to be an area of active investigation for the management of epilepsy and other CNS disorders. Herein, the historical efforts to elucidate the role of GABA in the synapse, as well as the role of GAT inhibitors as anticonvulsants, are described.

  15. A functional role for both γ-aminobutyric acid (GABA) transporter-1 and GABA transporter-3 in the modulation of extracellular GABA and GABAergic tonic conductances in the rat hippocampus

    PubMed Central

    Kersanté, Flavie; Rowley, Samuel C S; Pavlov, Ivan; Gutièrrez-Mecinas, María; Semyanov, Alexey; Reul, Johannes M H M; Walker, Matthew C; Linthorst, Astrid C E

    2013-01-01

    Tonic γ-aminobutyric acid (GABA)A receptor-mediated signalling controls neuronal network excitability in the hippocampus. Although the extracellular concentration of GABA (e[GABA]) is critical in determining tonic conductances, knowledge on how e[GABA] is regulated by different GABA transporters (GATs) in vivo is limited. Therefore, we studied the role of GATs in the regulation of hippocampal e[GABA] using in vivo microdialysis in freely moving rats. Here we show that GAT-1, which is predominantly presynaptically located, is the major GABA transporter under baseline, quiescent conditions. Furthermore, a significant contribution of GAT-3 in regulating e[GABA] was revealed by administration of the GAT-3 inhibitor SNAP-5114 during simultaneous blockade of GAT-1 by NNC-711. Thus, the GABA transporting activity of GAT-3 (the expression of which is confined to astrocytes) is apparent under conditions in which GAT-1 is blocked. However, sustained neuronal activation by K+-induced depolarization caused a profound spillover of GABA into the extrasynaptic space and this increase in e[GABA] was significantly potentiated by sole blockade of GAT-3 (i.e. even when uptake of GAT-1 is intact). Furthermore, experiments using tetrodotoxin to block action potentials revealed that GAT-3 regulates extrasynaptic GABA levels from action potential-independent sources when GAT-1 is blocked. Importantly, changes in e[GABA] resulting from both GAT-1 and GAT-3 inhibition directly precipitate changes in tonic conductances in dentate granule cells as measured by whole-cell patch-clamp recording. Thus, astrocytic GAT-3 contributes to the regulation of e[GABA] in the hippocampus in vivo and may play an important role in controlling the excitability of hippocampal cells when network activity is increased. PMID:23381899

  16. Gamma-aminobutyric acid (GABA) permeates ovine ruminal and jejunal epithelia, mainly by passive diffusion.

    PubMed

    Rackwitz, R; Gäbel, G

    2017-02-01

    Gamma-aminobutyric acid (GABA) represents the most abundant inhibitory neurotransmitter in the mammalian brain. GABA is also produced in plants and/or by the microbial conversion of amino acids. Thus, ruminants may be forced to take up significant amounts of GABA from their diet. However, it is not known whether exogenously acquired GABA might permeate the gastrointestinal barrier in such quantities as to induce systemic alterations. Thus, this study pursues the question of where within the ruminant's GI tract and by which pathways GABA may be taken up from the ingesta. The jejunal and ruminal epithelia of sheep were mounted in Ussing chambers under short-circuit conditions. The flux rates of radiolabelled GABA from the mucosal to the serosal side (Jms ) and vice versa (Jsm ) were measured. GABA was applied in various concentrations with adjustment of the mucosal pH to 6.1 or 7.4. Furthermore, beta-alanine or glycine was used as a competitive inhibitor for GABA transport. In both the jejunal and ruminal epithelium, the Jms of GABA was linearly correlated to the mucosal GABA concentration. However, Jms across the jejunal epithelium was approximately 10-fold higher than Jms across the ruminal epithelium. When 0.5 mmol/l GABA was applied on both sides of the epithelium, no net flux could be observed in the jejunal epithelia. Additionally, there was no effect of decreased mucosal pH or the application of glycine or beta-alanine under these conditions. The Jms and Jsm of GABA were linearly correlated to the transepithelial conductance. Our results suggest that GABA is taken up from the small intestine rather than from the rumen. Due to the lack of influence of pH and competitive inhibitors, this uptake seems to occur primarily via passive diffusion. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

  17. Activity-dependent transport of GABA analogues into specific cell types demonstrated at high resolution using a novel immunocytochemical strategy.

    PubMed

    Pow, D V; Baldridge, W; Crook, D K

    1996-08-01

    We have raised antisera against the GABA analogues gamma-vinyl GABA, diaminobutyric acid and gabaculine. These analogues are thought to be substrates for high-affinity GABA transporters. Retinae were exposed to micromolar concentrations of these analogues in the presence or absence of uptake inhibitors and then fixed and processed for immunocytochemistry at the light and electron microscopic levels. Immunolabelling for gamma-vinyl GABA revealed specific labelling of GABAergic amacrine cells and displaced amacrine cells in retinae of rabbits, cats, chickens, fish and a monkey. GABA-containing horizontal cells of cat and monkey retinae failed to exhibit labelling for gamma-vinyl GABA, suggesting that they lacked an uptake system for this molecule. In light-adapted fish, gamma-vinyl GABA was readily detected in H1 horizontal cells; similar labelling was also observed in light-adapted chicken retinae. The pattern of labelling in the fish and chicken retinae was modified by dark adaptation, when labelling was greatly reduced in the horizontal cells, indicating the activity dependence of GABA (analogue) transport. Intraperitoneal injection of gamma-vinyl GABA into rats resulted in its transport across the blood-brain barrier and subsequent uptake into populations of GABAergic neurons. The other analogues investigated in this study exhibited different patterns of transport; gabaculine was taken up into glial cells, whilst diaminobutyric acid was taken up into neurons, glial cells and retinal pigment epithelia. Thus, these analogues are probably substrates for different GABA transporters. We conclude that immunocytochemical detection of the high-affinity uptake of gamma-vinyl GABA permits the identification of GABAergic neurons which are actively transporting GABA, and suggest that this novel methodology will be a useful tool in rapidly assessing the recent activity of GABAergic neurons at the cellular level.

  18. Attenuation of malonate toxicity in primary mesencephalic cultures using the GABA transport blocker, NO-711.

    PubMed

    Stokes, A H; Bernard, L P; Nicklas, W J; Zeevalk, G D

    2001-04-01

    Cultured rat mesencephalic neurons were used to assess the effects of gamma-aminobutyric acid (GABA) transport blockers on toxicity caused by malonate, a reversible, competitive inhibitor of succinate dehydrogenase. Previous studies utilizing an ex vivo chick retinal preparation have shown that GABA release and cell swelling are early consequences of acute energy impairment and that GABA transport blockers attenuate this toxicity. The present results demonstrate that the nonsubstrate GABA transport blocker, NO-711 (1 nM-1 microM), dose-dependently protected cultured mesencephalic dopamine (DA) and GABA neurons from malonate-induced toxicity. Similar protection was demonstrated with nipecotic acid (1 mM) and SKF89976A (100 nM), substrate and nonsubstrate GABA transport blockers, respectively. These compounds by themselves produced no signs of toxicity, although nipecotic acid caused a long-term decrease in GABA uptake not associated with toxicity. Compounds which decrease intracellular reactive oxygen species (ROS) are protective in this model, but NO-711 did not prevent the rise in intracellular ROS induced by malonate, indicating its protective effects were downstream of ROS production. Supplementation of malonate treated cultures with the GABA(A) agonist, muscimol (10 microM), increased the toxicity toward the DA and GABA neuron populations. Antagonists at the GABA(A) and glycine receptors provided partial protection to both the GABA and DA neurons. These findings suggest that the GABA transporter, GABA(A), and/or glycine channels contribute to cell damage associated with energy impairment in this model. Copyright 2001 Wiley-Liss, Inc.

  19. Significant reductions in human visual gamma frequency by the gaba reuptake inhibitor tiagabine revealed by robust peak frequency estimation

    PubMed Central

    Muthukumaraswamy, Suresh D.; Campbell, Anne E.; Hamandi, Khalid; Lingford‐Hughes, Anne; Myers, Jim F. M.; Nutt, David J.; Sumner, Petroc; Wilson, Sue J.; Singh, Krish D.

    2016-01-01

    Abstract The frequency of visual gamma oscillations is determined by both the neuronal excitation–inhibition balance and the time constants of GABAergic processes. The gamma peak frequency has been linked to sensory processing, cognitive function, cortical structure, and may have a genetic contribution. To disentangle the intricate relationship among these factors, accurate and reliable estimates of peak frequency are required. Here, a bootstrapping approach that provides estimates of peak frequency reliability, thereby increasing the robustness of the inferences made on this parameter was developed. The method using both simulated data and real data from two previous pharmacological MEG studies of visual gamma with alcohol and tiagabine was validated. In particular, the study by Muthukumaraswamy et al. [2013a] (Neuropsychopharmacology 38(6):1105–1112), in which GABAergic enhancement by tiagabine had previously demonstrated a null effect on visual gamma oscillations, contrasting with strong evidence from both animal models and very recent human studies was re‐evaluated. After improved peak frequency estimation and additional exclusion of unreliably measured data, it was found that the GABA reuptake inhibitor tiagabine did produce, as predicted, a marked decrease in visual gamma oscillation frequency. This result demonstrates the potential impact of objective approaches to data quality control, and provides additional translational evidence for the mechanisms of GABAergic transmission generating gamma oscillations in humans. Hum Brain Mapp 37:3882–3896, 2016. © 2016 Wiley Periodicals, Inc. PMID:27273695

  20. Significant reductions in human visual gamma frequency by the gaba reuptake inhibitor tiagabine revealed by robust peak frequency estimation.

    PubMed

    Magazzini, Lorenzo; Muthukumaraswamy, Suresh D; Campbell, Anne E; Hamandi, Khalid; Lingford-Hughes, Anne; Myers, Jim F M; Nutt, David J; Sumner, Petroc; Wilson, Sue J; Singh, Krish D

    2016-11-01

    The frequency of visual gamma oscillations is determined by both the neuronal excitation-inhibition balance and the time constants of GABAergic processes. The gamma peak frequency has been linked to sensory processing, cognitive function, cortical structure, and may have a genetic contribution. To disentangle the intricate relationship among these factors, accurate and reliable estimates of peak frequency are required. Here, a bootstrapping approach that provides estimates of peak frequency reliability, thereby increasing the robustness of the inferences made on this parameter was developed. The method using both simulated data and real data from two previous pharmacological MEG studies of visual gamma with alcohol and tiagabine was validated. In particular, the study by Muthukumaraswamy et al. [] (Neuropsychopharmacology 38(6):1105-1112), in which GABAergic enhancement by tiagabine had previously demonstrated a null effect on visual gamma oscillations, contrasting with strong evidence from both animal models and very recent human studies was re-evaluated. After improved peak frequency estimation and additional exclusion of unreliably measured data, it was found that the GABA reuptake inhibitor tiagabine did produce, as predicted, a marked decrease in visual gamma oscillation frequency. This result demonstrates the potential impact of objective approaches to data quality control, and provides additional translational evidence for the mechanisms of GABAergic transmission generating gamma oscillations in humans. Hum Brain Mapp 37:3882-3896, 2016. © 2016 Wiley Periodicals, Inc. © 2016 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.

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

    PubMed

    Ricci, Lorenzo; Valoti, Massimo; Sgaragli, Giampietro; Frosini, Maria

    2012-06-01

    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 (30-300 μM) and VIGA (30-300 μM) significantly antagonised LDH and glutamate release, as well as tissue water gain caused by oxygen-glucose deprivation and reperfusion. Lower (1-10 μM) or higher concentrations (up to 3,000 μM) were ineffective. ANSA, PSA and ATAHS significantly reduced glutamate and LDH release and tissue water gain in a range of concentrations between 30 and 300 μM. Lower (0-10 μM) or higher (up to 3,000 μM) 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.

  2. The 'specific' tyrosine kinase inhibitor genistein inhibits the enzymic hydrolysis of anandamide: implications for anandamide uptake.

    PubMed

    Thors, L; Alajakku, K; Fowler, C J

    2007-04-01

    The cellular uptake of anandamide is reduced by inhibitors of fatty acid amide hydrolase (FAAH) and by agents disrupting endocytotic mechanisms. However, it is not clear if these events occur over the same time frame and if they occur to the same extent in different cells. We have therefore investigated the effects of such compounds in three cell lines of different origins using different assay incubation times and temperatures. FAAH activity and cellular uptake of anandamide was measured using anandamide, radio-labelled either in the ethanolamine or arachidonoyl part of the molecule. The FAAH inhibitor URB597 inhibited the uptake of anandamide into C6 glioma, RBL2H3 basophilic leukaemia cells and P19 embryonic carcinoma cells at incubation time 4 min. However, a time-dependent and temperature-sensitive residual uptake remained after URB597 treatment. The combination of progesterone and nystatin reduced the uptake, but also decreased the amount of anandamide retained by the wells. Genistein inhibited anandamide uptake in a manner that was not additive to that of URB597. However, genistein was a potent competitive inhibitor of FAAH (K(i) value 8 microM). The reduction of anandamide uptake by genistein can be explained by its ability to inhibit FAAH with a potency which overlaps that for inhibition of tyrosine kinase. The FAAH- resistant but time-dependent uptake of anandamide is seen in all three cell lines studied and is thus presumably a generally occurring process.

  3. Endogenous dopamine increases extracellular concentrations of glutamate and GABA in striatum of the freely moving rat: involvement of D1 and D2 dopamine receptors.

    PubMed

    Expósito, I; Del Arco, A; Segovia, G; Mora, F

    1999-07-01

    Interactions between endogenous dopamine, glutamate, GABA, and taurine were investigated in striatum of the freely moving rat by using microdialysis. Intrastriatal infusions of the selective dopamine uptake inhibitor nomifensine (NMF) were used to increase the endogenous extracellular dopamine. NMF produced a dose-related increase in extracellular dopamine and also increased extracellular concentrations of glutamate, GABA, and taurine. Extracellular increases of dopamine were significantly correlated with extracellular increases of glutamate and GABA, but not taurine. To investigate whether the increased extracellular dopamine produced by NMF was responsible for the concomitant increase of glutamate and GABA, D1, and D2 receptor antagonists were used. Dopamine receptor antagonists D1 (SCH23390) and D2 (sulpiride) significantly attenuated the increases of glutamate and GABA produced by NMF. These data suggest that endogenous dopamine, through both D1 and D2 dopamine receptors, plays a role in releasing glutamate and GABA in striatum of the freely moving rat.

  4. Measuring the serotonin uptake site using (/sup 3/H)paroxetine--a new serotonin uptake inhibitor

    SciTech Connect

    Gleiter, C.H.; Nutt, D.J.

    1988-01-01

    Serotonin is an important neurotransmitter that may be involved in ethanol preference and dependence. It is possible to label the serotonin uptake site in brain using the tricyclic antidepressant imipramine, but this also binds to other sites. We have used the new high-affinity uptake blocker paroxetine to define binding to this site and report it to have advantages over imipramine as a ligand.

  5. On the origin of extracellular GABA collected by brain microdialysis and assayed by a simplified on-line method.

    PubMed

    Westerink, B H; de Vries, J B

    1989-06-01

    The present study describes a simplified on-line system for determination of GABA in brain dialysates. GABA was determined with an isocratic HPLC method after derivatization with o-phthaldialdehyde. One peristaltic pump was sufficient to transport both the perfusion fluid and the derivatizing reagent. The basal release of GABA was stimulated by infusion with either elevated K+ or the GABA uptake inhibitor (-)-nipecotic acid. Basal as well as stimulated GABA release were investigated for possible calcium-dependency by infusing submmolar amounts of the potent calcium antagonist cadmium. Infusion of cadmium did not modify the dialysate concentrations of GABA. In addition basal as well as nipecotic acid enhanced release of GABA dialysate concentrations were investigated for nerve-impulse dependency by infusing mumolar amounts of tetrodotoxin. No change in the GABA output was observed during infusion of TTX. From these results it is concluded that the basal as well as the nipecotic acid induced release of GABA did not fulfill the criteria for classic exocytotic release. Possible explanations for these unexpected findings are discussed.

  6. Enhancement of GABA release through endogenous activation of axonal GABA(A) receptors in juvenile cerebellum.

    PubMed

    Trigo, Federico F; Chat, Mireille; Marty, Alain

    2007-11-14

    Recent evidence indicates the presence of presynaptic GABA(A) receptors (GABA(A)Rs) in the axon domain of several classes of central neurons, including cerebellar basket and stellate cells. Here, we investigate the possibility that these receptors could be activated in the absence of electrical or chemical stimulation. We find that low concentrations of GABA increase the frequency of miniature GABAergic synaptic currents. Submaximal concentrations of a GABA(A)R blocker, gabazine, decrease both the miniature current frequency and the probability of evoked GABA release. Zolpidem, an agonist of the benzodiazepine binding site, and NO-711 (1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride), a blocker of GABA uptake, both increase the frequency of miniature currents. These effects occur up to postnatal day 14, but not later. Immunohistochemistry indicates the presence of alpha1-containing GABA(A)Rs in interneuron presynaptic terminals with a similar age dependence. We conclude that, under resting conditions, axonal GABA(A)Rs are significantly activated, that this activation results in enhanced GABA release, and that it can be augmented by increasing the affinity of GABA(A)Rs or reducing GABA uptake. Our findings suggest the existence of a positive-feedback mechanism involving presynaptic GABA(A)Rs that maintains a high release rate and a high local GABA concentration in the immature cerebellar network.

  7. Effects of polyamine inhibitors on zinc uptake by COMMA-1D mammary epithelial cells

    SciTech Connect

    Allen, J.C.; Haedrich, L.H. )

    1991-03-15

    Zn uptake or transport is stimulated by glucocorticoids in many types of epithelial cells, including the COMMA-1D mouse mammary cell line. The current objective was to determine whether polyamines also mediate glucocorticoid stimulation of Zn-uptake. Initially, cells grown in lactogenic hormone supplemented-media had approximately 65% greater {sup 65}Zn-uptake over 24 h than cells in nonsupplemented growth media (GM). {sup 65}Zn-uptake from HM with 10{sup {minus}5}M methylglyoxal-bis(guanylhydrazone) (MGBG) (s-adenosyl-methionine decarboxylase inhibitor to block polyamine synthesis) added was less than from GM. Exogenous spermidine added to the MGBG-HM media increased {sup 65}Zn-uptake. However, up to 10mM difluoromethylornithine (DFMO), a more specific inhibitor of sperimidine synthesis, had no significant effect on 24-h {sup 65}Zn-uptake by cells in HM. In GM, DFMO caused a slight dose-dependent decrease in {sup 65}Zn-uptake over the range 10{sup {minus}6} to 5 {times} 10{sup 3}M. Also, with 8 h of incubation, DFMO tended to decrease {sup 65}Zn-uptake in HM-stimulated cells. These data cannot yet distinguish between the possibilities that DFMO is inactivated during the 24-h incubation or that the dramatic effects of MGBG on {sup 65}Zn-uptake in these mammary-derived cells is not related to its inhibition of polyamine synthesis. Because COMMA-1D cells alter Zn uptake in response to lactogenic hormones and MGBG, the model system is suitable for further studies of the mechanisms of zinc transport in epithelia.

  8. Iodine status in Turkish populations and exposure to iodide uptake inhibitors.

    PubMed

    Ozpinar, Aysel; Kelestimur, Fahrettin; Songur, Yildiran; Can, Ozge; Valentin, Liza; Caldwell, Kathleen; Arikan, Ender; Unsal, Ibrahim; Serteser, Mustafa; Inal, Tamer; Erdemgil, Yigit; Coskun, Abdurrahman; Bakirci, Nadi; Sezgin, Ozlem; Blount, Ben

    2014-01-01

    Perchlorate, nitrate, and thiocyanate are competitive inhibitors of the sodium iodide symporter of the thyroid membrane. These inhibitors can decrease iodine uptake by the symporter into the thyroid gland and may disrupt thyroid function. This study assesses iodine status and exposure to iodide uptake inhibitors of non-pregnant and non-lactating adult women living in three different cities in Turkey (Istanbul, Isparta and Kayseri). We measured iodine and iodide uptake inhibitors in 24-hr urines collected from study participants (N = 255). All three study populations were mildly iodine deficient, with median urinary iodine (UI) levels of 77.5 µg/L in Istanbul, 58.8 µg/L in Isparta, and 69.8 µg/L in Kayseri. Perchlorate doses were higher in the study population (median 0.13 µg/kg/day), compared with a reference population (median 0.059 µg/kg/day), but lower than the U.S. EPA reference dose (0.7 µg/kg/day). Urinary thiocyanate levels increased with increasing exposure to tobacco smoke, with non-smokers (268 µg/L) significantly lower than light smokers (1110 µg/L), who were significantly lower than heavy smokers (2410 µg/L). This pilot study provides novel data indicating that study participants were moderately iodine deficient and had higher intakes of the iodide uptake inhibitor perchlorate compared with a reference population. Further investigation is needed to characterize the thyroid impact resulting from iodine deficiency coupled with exposure to iodide uptake inhibitors such as perchlorate, thiocyanate and nitrate.

  9. Iodine Status in Turkish Populations and Exposure to Iodide Uptake Inhibitors

    PubMed Central

    Ozpinar, Aysel; Kelestimur, Fahrettin; Songur, Yildiran; Can, Ozge; Valentin, Liza; Caldwell, Kathleen; Arikan, Ender; Unsal, Ibrahim; Serteser, Mustafa; Inal, Tamer; Erdemgil, Yigit; Coskun, Abdurrahman; Bakirci, Nadi; Sezgin, Ozlem; Blount, Ben

    2014-01-01

    Perchlorate, nitrate, and thiocyanate are competitive inhibitors of the sodium iodide symporter of the thyroid membrane. These inhibitors can decrease iodine uptake by the symporter into the thyroid gland and may disrupt thyroid function. This study assesses iodine status and exposure to iodide uptake inhibitors of non-pregnant and non-lactating adult women living in three different cities in Turkey (Istanbul, Isparta and Kayseri). We measured iodine and iodide uptake inhibitors in 24-hr urines collected from study participants (N = 255). All three study populations were mildly iodine deficient, with median urinary iodine (UI) levels of 77.5 µg/L in Istanbul, 58.8 µg/L in Isparta, and 69.8 µg/L in Kayseri. Perchlorate doses were higher in the study population (median 0.13 µg/kg/day), compared with a reference population (median 0.059 µg/kg/day), but lower than the U.S. EPA reference dose (0.7 µg/kg/day). Urinary thiocyanate levels increased with increasing exposure to tobacco smoke, with non-smokers (268 µg/L) significantly lower than light smokers (1110 µg/L), who were significantly lower than heavy smokers (2410 µg/L). This pilot study provides novel data indicating that study participants were moderately iodine deficient and had higher intakes of the iodide uptake inhibitor perchlorate compared with a reference population. Further investigation is needed to characterize the thyroid impact resulting from iodine deficiency coupled with exposure to iodide uptake inhibitors such as perchlorate, thiocyanate and nitrate. PMID:24505430

  10. Extracellular GABA in the ventrolateral thalamus of rats exhibiting spontaneous absence epilepsy: a microdialysis study.

    PubMed

    Richards, D A; Lemos, T; Whitton, P S; Bowery, N G

    1995-10-01

    There is compelling evidence that excessive GABA-mediated inhibition may underlie the abnormal electrical activity, initiated in the thalamus, associated with epileptic absence seizures. In particular, the GABAB receptor subtype seems to play a critical role, because its antagonists are potent inhibitors of absence seizures, whereas its agonists exacerbate seizure activity. Using a validated rat model of absence epilepsy, we have previously found no evidence of abnormal GABAB receptor density or affinity in thalamic tissue. In the present study, we have used in vivo microdialysis to monitor changes in levels of extracellular GABA and other amino acids in this brain region. We have shown that basal extracellular levels of GABA and, to a lesser extent, taurine are increased when compared with values in nonepileptic controls. However, modifying GABAergic transmission with the GABAB agonist (-)-baclofen (2 mg/kg i.p.), the GABAB antagonist CGP-35348 (200 mg/kg i.p.), or the GABA uptake inhibitor tiagabine (100 microM) did not produce any further alteration in extracellular GABA levels, despite the ability of these compounds to increase (baclofen and tiagabine) or decrease (CGP-35348) seizure activity. These findings suggest that the increased basal GABA levels observed in this animal model are not simply a consequence of seizure activity but may contribute to the initiation of absence seizures.

  11. Correlating FAAH and anandamide cellular uptake inhibition using N-alkylcarbamate inhibitors: from ultrapotent to hyperpotent.

    PubMed

    Nicolussi, Simon; Chicca, Andrea; Rau, Mark; Rihs, Sabine; Soeberdt, Michael; Abels, Christoph; Gertsch, Jürg

    2014-12-15

    Besides the suggested role of a putative endocannabinoid membrane transporter mediating the cellular uptake of the endocannabinoid anandamide (AEA), this process is intrinsically coupled to AEA degradation by the fatty acid amide hydrolase (FAAH). Differential blockage of each mechanism is possible using specific small-molecule inhibitors. Starting from the natural product-derived 2E,4E-dodecadiene scaffold previously shown to interact with the endocannabinoid system (ECS), a series of diverse N-alkylcarbamates were prepared with the aim of generating novel ECS modulators. While being inactive at cannabinoid receptors and monoacylglycerol lipase, these N-alkylcarbamates showed potent to ultrapotent picomolar FAAH inhibition in U937 cells. Overall, a highly significant correlation (Spearman's rho=0.91) was found between the inhibition of FAAH and AEA cellular uptake among 54 compounds. Accordingly, in HMC-1 cells lacking FAAH expression the effect on AEA cellular uptake was dramatically reduced. Unexpectedly, 3-(4,5-dihydrothiazol-2-yl)phenyl carbamates and the 3-(1,2,3-thiadiazol-4-yl)phenyl carbamates WOBE490, WOBE491 and WOBE492 showed a potentiation of cellular AEA uptake inhibition in U937 cells, resulting in unprecedented femtomolar (hyperpotent) IC50 values. Potential methodological issues and the role of cellular accumulation of selected probes were investigated. It is shown that albumin impacts the potency of specific N-alkylcarbamates and, more importantly, that accumulation of FAAH inhibitors can significantly increase their effect on cellular AEA uptake. Taken together, this series of N-alkylcarbamates shows a FAAH-dependent inhibition of cellular AEA uptake, which can be strongly potentiated using specific head group modifications. These findings provide a rational basis for the development of hyperpotent AEA uptake inhibitors mediated by ultrapotent FAAH inhibition.

  12. 2,5-Disubstituted tetrahydrofurans as selective serotonin re-uptake inhibitors.

    PubMed

    Voelker, Troy; Xia, Haiji; Fandrick, Keith; Johnson, Robert; Janowsky, Aaron; Cashman, John R

    2009-03-01

    Enhancement of 5-hydroxytryptamine (5-HT, serotonin) neurotransmission is a viable means of treating depression. On the basis of this observation, agents that inhibit re-uptake of 5-HT were prepared based on (-)-cocaine and aryltropanes as lead compounds because they are reasonably potent 5-HT re-uptake inhibitors. Molecular dissection of an aryltropane provided a series of 5- and 6-membered ring compounds. From among this library of compounds a series of disubstituted tetrahydrofurans bearing 2-alkyl aryl and 5-alkyl amino groups were identified as having highly potent and selective 5-HT re-uptake inhibition. The compounds were evaluated for their ability to compete with radiolabeled RTI-55 binding and to inhibit re-uptake of neurotransmitters at the human dopamine, serotonin and norepinephrine transporters. Based on potency (e.g., K(i)=800 pM) and significant functional selectivity (e.g., IC(50) ratios for human dopamine:serotonin or norepinephrine:serotonin, >or=1397) highly potent and selective serotonin re-uptake inhibitors were identified. Optimal features playing a dominant role in binding affinity and re-uptake inhibition included lipophilic substitution on the aromatic moiety, trans relative stereochemistry of the 2,5-disubstituted tetrahydrofuran ring, and a total of four or five methylene groups between the alkyl amine and the alkyl aryl moiety and the tetrahydrofuran group. A number of the most potent serotonin re-uptake inhibitors were tested in Balb/c mice in the forced-swim test (FST), a behavioral test used to measure the effects of antidepressant agents. Acute administration of 32c (10mg/kg), or 32d (10mg/kg) ip tended to decrease the duration of mouse immobility in the FST although the effect was not statistically significant.

  13. An investigation of the origin of extracellular GABA in rat nucleus accumbens measured in vivo by microdialysis.

    PubMed

    Smith, S E; Sharp, T

    1994-01-01

    GABA transmission in the nucleus accumbens is believed to play a central role in motivational processes and the expression of psychostimulant drug action. Here we report measurements of extracellular GABA in nucleus accumbens of the rat and investigate its origin. Extracellular GABA was detected using microdialysis in combination with a novel HPLC-based assay. In the awake rat, GABA in the microdialysates (1) increased 10-fold following perfusion with 0.5 mM nipecotic acid, a GABA releasing agent and uptake blocker, (2) increased 7-fold following local perfusion with 50 mM KCl, (3) decreased 50% following perfusion with tetrodotoxin, (4) decreased 50% following perfusion with a Ca(2+(-free medium and (5) decreased 40% following perfusion with high (12.5 mM) MgCl. Finally, in the anaesthetized rat, GABA in the microdialysates decreased 50% following i.p. injection of 100 mg/kg 3-mercaptoproprionic acid, a GABA synthesis inhibitor. We conclude that GABA in microdialysates from nucleus accumbens of the rat (awake) responds appropriately to selected pharmacological agents and derives at least in part (50%) from neurones.

  14. Triple monoamine uptake inhibitors demonstrate a pharmacologic association between excessive drinking and impulsivity in high alcohol-preferring (HAP) mice

    PubMed Central

    O’Tousa, David S.; Warnock, Kaitlin T.; Matson, Liana M.; Namjoshi, Ojas A.; Halcomb, Meredith E.; Cook, James; Grahame, Nicholas J.; June, Harry L.

    2013-01-01

    Approximately 30% of current drinkers in the US drink excessively, and are referred to as problem/hazardous drinkers. These individuals, who may not meet criteria for alcohol abuse or dependence, comprise binge, heavy drinkers, or both. Given their high prevalence, interventions that reduce the risk of binge and heavy drinking have important public health implications. Impulsivity has been repeatedly associated with excessive drinking in the clinical literature. As impulsivity is correlated with, and may play a critical role in the initiation and maintenance of excessive drinking, this behavior may be an important target for therapeutic intervention. Hence, a better understanding of pharmacological treatments capable of attenuating excessive drinking models and impulsivity may markedly improve clinical outcomes. The high alcohol-preferring (HAP) mice represent an optimal rodent model to study the relationship between impulsivity and excessive alcohol drinking, as recent evidence indicates they consume high levels of alcohol throughout their active cycle and are innately impulsive. Using this model, the present study demonstrates that the triple monoamine uptake inhibitors (TUIs) amitifadine and DOV 102, 677 effectively attenuate binge drinking, heavy drinking assessed via a 24-hour free-choice assay, and impulsivity measured by the delay discounting procedure. In contrast, 3-PBC, a GABA-A α1 preferring ligand, with mixed agonist-antagonist properties attenuates excessive drinking without affecting impulsivity. These findings suggest in the HAP mice, monoamine pathways may predominate as a common mechanism underlying impulsivity and excessive drinking, while the GABAergic system may be more salient in regulating excessive drinking. We further propose that TUIs such as amitifadine and DOV 102, 677 may be used to treat the co-occurrence of impulsivity and excessive drinking. PMID:24118509

  15. Assessment of a glycine uptake inhibitor in animal models of effort-related choice behavior: implications for motivational dysfunctions.

    PubMed

    Yohn, Samantha E; Alberati, Daniela; Correa, Merce; Salamone, John D

    2017-05-01

    Motivated behavior can be characterized by a substantial exertion of effort, and organisms often make effort-related decisions based upon analyses of work-related response costs and reinforcement preference. Moreover, alterations in effort-based choice can be seen in people with major depression and schizophrenia. Effort-related decision making is studied using tasks offering choices between high effort options leading to highly valued reinforces vs low effort/low reward options. Interference with dopamine (DA) transmission by administration of the DA D2 family antagonist haloperidol biases behavior towards the lower effort option that can be obtained with minimal work, and previous research has shown that DA interacts with other transmitters, including adenosine and GABA, to regulate effort-based choice. The present studies focused upon the ability of the glycine transport inhibitor bitopertin to attenuate haloperidol-induced shifts in effort-related choice behavior. Effort-based choice in rats was assessed using the concurrent fixed ratio (FR) 5/chow feeding choice task and the T-maze barrier choice procedure. Haloperidol shifted effort-based choice, biasing animals towards the low effort option in each task. Co-administration of bitopertin (1.0-10.0 mg/kg) significantly attenuated haloperidol-induced shifts in choice behavior, but the same doses of bitopertin had no effect when administered alone. These results indicated that elevation of extracellular glycine via inhibition of glycine uptake was able to reverse the effects of D2 antagonism. Increases in extracellular glycine, possibly through actions on the glycine allosteric site on the NMDA receptor, may be a useful strategy for treating motivational dysfunctions in humans.

  16. The Design, Synthesis and Structure-Activity Relationship of Mixed Serotonin, Norepinephrine and Dopamine Uptake Inhibitors

    NASA Astrophysics Data System (ADS)

    Chen, Zhengming; Yang, Ji; Skolnick, Phil

    The evolution of antidepressants over the past four decades has involved the replacement of drugs with a multiplicity of effects (e.g., TCAs) by those with selective actions (i.e., SSRIs). This strategy was employed to reduce the adverse effects of TCAs, largely by eliminating interactions with certain neurotransmitters or receptors. Although these more selective compounds may be better tolerated by patients, selective drugs, specifically SSRIs, are not superior to older drugs in treating depressed patients as measured by response and remission rates. It may be an advantage to increase synaptic levels of both serotonin and norepinephrine, as in the case of dual uptake inhibitors like duloxetine and venlafaxine. An important recent development has been the emergence of the triple-uptake inhibitors (TUIs/SNDRIs), which inhibit the uptake of the three neurotransmitters most closely linked to depression: serotonin, norepinephrine, and dopamine. Preclinical studies and clinical trials indicate that a drug inhibiting the reuptake of all three of these neurotransmitters could produce more rapid onset of action and greater efficacy than traditional antidepressants. This review will detail the medicinal chemistry involved in the design, synthesis and discovery of mixed serotonin, norepinephrine and dopamine transporter uptake inhibitors.

  17. Fluoxetine at anorectic doses does not have properties of a dopamine uptake inhibitor.

    PubMed

    Fuller, R W; Hemrick-Luecke, S K; Snoddy, H D

    1994-01-01

    Although fluoxetine is a highly selective inhibitor of serotonin uptake in vitro and in vivo, some investigators have suggested that dopamine uptake inhibition may contribute to anorectic actions of fluoxetine. The present experiments were done to determine fluoxetine's effects in some animal protocols in which dopamine uptake inhibitors have characteristic actions. Mazindol prevented the depletion of striatal dopamine and its metabolites by amphetamine in iprindole-pretreated rats, but fluoxetine had no effect. Mazindol prevented the depletion of striatal dopamine and its metabolites by 6-hydroxydopamine injected intracerebroventricularly into rats, but fluoxetine had no effect. Mazindol enhanced the elevation of 3,4-dihydroxyphenylacetic acid concentration in rat brain after spiperone injection, but fluoxetine did not cause that effect. Fluoxetine did not mimic amfonelic acid in antagonizing the retention of alpha-methyl-m-tyramine invant striatum after the injection of alpha-methyl-m-tyrosine. These results show that fluoxetine, at doses that are effective in blocking the serotonin uptake carrier and causing anorexia, does not block the dopamine uptake carrier.

  18. Inhibition of high-affinity gamma-aminobutyric acid uptake in primary astrocyte cultures by phorbol esters and phospholipase C.

    PubMed Central

    Gomeza, J; Casado, M; Gimenez, C; Aragon, C

    1991-01-01

    The effects of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), on high-affinity Na(+)-dependent gamma-aminobutyric acid (GABA) uptake were investigated in primary cultures of neurons and glial cells from rat brain cortex. Incubation of glial cells with PMA led to concentration- and time-dependent decreases in the GABA transport in glial cells. This effect could be completely suppressed by addition of the PKC inhibitor H7. The PMA effects could be mimicked by oleoylacetylglycerol, the diacylglycerol kinase inhibitor R59022 and exogenous phospholipase C. Treatment with PMA did not affect GABA transport in neuronal cells. PMID:1902665

  19. Effects of uptake inhibitors on responses of sheep coronary arteries to catecholamines and sympathetic nerve stimulation.

    PubMed Central

    Brine, F.; Cornish, E. J.; Miller, R. C.

    1979-01-01

    1. Transmural stimulation of intrinsic sympathetic nerves and exogenous catecholamines produce beta 1-adrenoceptor mediated relaxant responses in strips of contracted sheep coronary artery. 2. The neuronal uptake inhibitors, metaraminol, cocaine and desipramine and the extraneuronal uptake inhibitor, cortisol, failed to potentiate responses to noradrenaline or sympathetic stimulation; responses to isoprenaline were enhanced by cortisol. 3. Oxytetracycline, which inhibits binding to connective tissue fibres, did not affect responses to noradrenaline or nerve stimulation. 4. 17 beta-Oestradiol, caffeine and U0521 proved to be unsuitable compounds for studying catecholamine inactivation since they non-selectively potentiated responses to noradrenaline and isoprenaline. 5. It is concluded that catecholamine inactivation processes do not modify transmitter function in sheep coronary arteries. PMID:519107

  20. The presence of a serotonin uptake inhibitor alters pharmacological manipulations of serotonin release.

    PubMed

    Kreiss, D S; Wieland, S; Lucki, I

    1993-01-01

    The present study investigated the effects of the presence of the serotonin uptake inhibitor citalopram in the perfusion medium on pharmacological manipulations which increased and decreased striatal serotonin release using in vivo microdialysis. A high performance liquid chromatography detection system equipped with a microbore column was used which reduced the detection limit to 0.5 fmol serotonin/5 microliters sample and enabled basal striatal serotonin release to be measured without the addition of a serotonin uptake inhibitor to the perfusion medium. Although serotonin uptake inhibitors have frequently been used to enhance the serotonin content of dialysate samples, the effects of the presence of serotonin uptake inhibitors on pharmacological manipulations which increased and decreased the release of serotonin have not yet been characterized. Serotonin release was reduced by the systemic administration of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Although 5-HT release was reduced by 8-OH-DPAT after the addition of citalopram, the 5-HT1A receptor agonist did not reduce absolute levels of extracellular serotonin below basal values of serotonin measured in the absence of citalopram. In addition, citalopram dramatically prevented the four-fold increase in the release of serotonin produced by the systemic administration of the serotonin-releasing agent fenfluramine. The blockade of fenfluramine's effects by citalopram supports the hypothesis that transport of fenfluramine into serotonergic neurons is necessary to increase serotonin release. This study demonstrates that the use of an HPLC detection system equipped with a microbore column can reliably measure basal serotonin release using in vivo microdialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Revisiting serotonin reuptake inhibitors and the therapeutic potential of "uptake-2" in psychiatric disorders.

    PubMed

    Daws, Lynette C; Koek, Wouter; Mitchell, Nathan C

    2013-01-16

    Depression is among the most common psychiatric disorders, and in many patients a disorder for which available medications provide suboptimal or no symptom relief. The most commonly prescribed class of antidepressants, the selective serotonin reuptake inhibitors (SSRIs), are thought to act by increasing extracellular serotonin in brain by blocking its uptake via the high-affinity serotonin transporter (SERT). However, the relative lack of therapeutic efficacy of SSRIs has brought into question the utility of increasing extracellular serotonin for the treatment of depression. In this Viewpoint, we discuss why increasing extracellular serotonin should not be written off as a therapeutic strategy. We describe how "uptake-2" transporters may explain the relative lack of therapeutic efficacy of SSRIs, as well as why "uptake-2" transporters might be useful therapeutic targets.

  2. Uptake inhibitors but not substrates induce protease resistance in extracellular loop two of the dopamine transporter.

    PubMed

    Gaffaney, Jon D; Vaughan, Roxanne A

    2004-03-01

    Changes in protease sensitivity of extracellular loop two (EL2) of the dopamine transporter (DAT) during inhibitor and substrate binding were examined using trypsin proteolysis and epitope-specific immunoblotting. In control rat striatal membranes, proteolysis of DAT in a restricted region of EL2 was produced by 0.001 to 10 microg/ml trypsin. However, in the presence of the dopamine uptake blockers [2-(diphenylmethoxyl) ethyl]-4-(3phenylpropyl) piperazine (GBR 12909), mazindol, 2beta-carbomethoxy-3beta-(4-flourophenyl)tropane (beta-CFT), nomifensine, benztropine, or (-)-cocaine, 100- to 1000-fold higher concentrations of trypsin were required to produce comparable levels of proteolysis. Protease resistance induced by ligands was correlated with their affinity for DAT binding, was not observed with Zn2+, (+)-cocaine, or inhibitors of norepinephrine or serotonin transporters, and was not caused by altered catalytic activity of trypsin. Together, these results support the hypothesis that the interaction of uptake inhibitors with DAT induces a protease-resistant conformation in EL2. In contrast, binding of substrates did not induce protease resistance in EL2, suggesting that substrates and inhibitors interact with DAT differently during binding. To assess the effects of EL2 proteolysis on DAT function, the binding and transport properties of trypsin-digested DAT were assayed with [3H]CFT and [3H]dopamine. Digestion decreased the Bmax for binding and the Vmax for uptake in amounts that were proportional to the extent of proteolysis, indicating that the structural integrity of EL2 is required for maintenance of both DAT binding and transport functions. Together this data provides novel information about inhibitor and substrate interactions at EL2, possibly relating the protease resistant DAT conformation to a mechanism of transport inhibition.

  3. GABA transporter-1 inhibitor NO-711 alters the EEG power spectra and enhances non-rapid eye movement sleep during the active phase in mice.

    PubMed

    Xu, Xin-Hong; Qiu, Mei-Hong; Dong, Hui; Qu, Wei-Min; Urade, Yoshihiro; Huang, Zhi-Li

    2014-04-01

    GABA transporter subtype 1 (GAT1) constructs high affinity reuptake sites for GABA in the CNS and regulates GABAergic transmission. Compounds that inhibit GAT1 are targets often used for the treatment of epilepsy; however sedation has been reported as a side effect of these agents, indicating potential sedative and/or hypnotic uses for these compounds. In the current study, we observed the sleep behaviors of mice treated with NO-711, a selective GAT1 inhibitor, in order to elucidate the role of GAT1 in sleep-wake regulation during the active phase. The data revealed that NO-711 at a high dose of 10 mg/kg caused a marked enhancement of EEG activity in the frequency ranges of 3-25 Hz during wakefulness as well as rapid eye movement (REM) sleep. During the non-REM (NREM) sleep, NO-711 (10 mg/kg) elevated EEG activity in the frequency ranges of 1.5-6.75 Hz. Similar changes were found in mice treated with a low dose of 3 mg/kg. NO-711 administered i.p. at a dose of 1, 3 or 10 mg/kg significantly shortened the sleep latency of NREM sleep, increased the amount of NREM sleep and the number of NREM sleep episodes. NO-711 did not affect the sleep latency and the amount of REM sleep. NO-711 dose-dependently increased c-Fos expression in sleep-promoting nucleus of the ventrolateral preoptic area and median preoptic area. However, c-Fos expression was decreased in the wake-promoting nuclei, tuberomammillary nucleus and lateral hypothalamus. These results indicate that NO-711 can increase NREM sleep in mice. © 2013 Published by Elsevier B.V. and ECNP.

  4. Amino Acid Neurotransmitters; Mechanisms of Their Uptake into Synaptic Vesicles

    DTIC Science & Technology

    1991-08-01

    uptake of the inhibitory neurotransmitters GABA and glycine and the excitatory neurotransmitter glutamate is essential for the understanding of the...1987)i Biochemistry, anatomy, and pharmacology of GABA neurons, In- Meltzer H Y (ed) Psycopharmacology : The third generation of progress, Raven Press...calculated with a linear GABA uptake experiments, regression program (Chou and Chou, 1985). Assay for GABA uptake GABA uptake was determined essentially as

  5. GABA localization in the nematode Ascaris

    SciTech Connect

    Guastella, J.

    1988-01-01

    A histochemical approach was used to examine the distribution of GABA-associated neurons in the nematode Ascaris, an organism whose small number of morphologically simple neurons make it an excellent preparation for analyzing neuronal phenotypes. Two GABAergic markers were examined: GABA-like immunoreactivity (GLIR), a marker for endogenous stores of GABA; and ({sup 3}H)-GABA uptake, a marker for GABA uptake sites. Strong GLIR was present in the cell bodies, neurites and commissures of dorsal and ventral inhibitory motorneurons present in this region. Strong GLIR was also present in the cell bodies and processes of the four RME neurons in the nerve ring and in several other ganglionic neurons. Staining was absent in excitatory motorneurons, in ventral cord interneurons and in muscle cells and hypodermis. GABA uptake sites were found in single neural processes in both the ventral and dorsal nerve cords. ({sup 3}H)-GABA labeling was also observed in the other two RME cells and several other cephalic neurons. Four putative cholinergic excitatory motorneurons in the retrovesicular ganglion (RVG) were heavily labeled. Ventral and dorsal nerve cord inhibitory motorneurons did not take up ({sup 3}H)-GABA. Labeling of the ventral cord excitatory motorneuron somata and cell bodies was at or slightly above background. Heavy labeling of muscle cells was also observed.

  6. Modulation of thyroidal radioiodide uptake by oncological pipeline inhibitors and Apigenin.

    PubMed

    Lakshmanan, Aparna; Scarberry, Daniel; Green, Jill A; Zhang, Xiaoli; Selmi-Ruby, Samia; Jhiang, Sissy M

    2015-10-13

    Targeted radioiodine therapy for thyroid cancer is based on selective stimulation of Na+/I- Symporter (NIS)-mediated radioactive iodide uptake (RAIU) in thyroid cells by thyrotropin. Patients with advanced thyroid cancer do not benefit from radioiodine therapy due to reduced or absent NIS expression. To identify inhibitors that can be readily translated into clinical care, we examined oncological pipeline inhibitors targeting Akt, MEK, PI3K, Hsp90 or BRAF in their ability to increase RAIU in thyroid cells expressing BRAFV600E or RET/PTC3 oncogene. Our data showed that (1) PI3K inhibitor GDC-0941 outperformed other inhibitors in RAIU increase mainly by decreasing iodide efflux rate to a great extent; (2) RAIU increase by all inhibitors was extensively reduced by TGF-β, a cytokine secreted in the invasive fronts of thyroid cancers; (3) RAIU reduction by TGF-β was mainly mediated by NIS reduction and could be reversed by Apigenin, a plant-derived flavonoid; and (4) In the presence of TGF-β, GDC-0941 with Apigenin co-treatment had the highest RAIU level in both BRAFV600E expressing cells and RET/PTC3 expressing cells. Taken together, Apigenin may serve as a dietary supplement along with small molecule inhibitors to improve radioiodine therapeutic efficacy on invasive tumor margins thereby minimizing future metastatic events.

  7. Voltammetric characterization of the effect of monoamine uptake inhibitors and releasers on dopamine and serotonin uptake in mouse caudate-putamen and substantia nigra slices

    PubMed Central

    John, Carrie E.; Jones, Sara R.

    2007-01-01

    Summary Fast scan cyclic voltammetry is an electrochemical technique used to measure dynamics of transporter-mediated monoamine uptake in real time and provides a tool to evaluate the detailed effects of monoamine uptake inhibitors and releasers on dopamine and serotonin transporter function. We measured the effects of cocaine, methylphenidate, 2β-propanoyl–3β-(4tolyl) tropane (PTT), fluoxetine, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), phentermine and fenfluramine on dopamine and serotonin uptake following electrically stimulated release in mouse caudate-putamen and substantia nigra pars reticulata slices. We determined rank orders of uptake inhibition effects based on two variables; increases in apparent Km for dopamine and serotonin uptake and inhibition constant (Ki) values. For example, the rank order of uptake inhibition based on apparent Km values at the dopamine transporter was amphetamine ≥ PTT ≥ methylphenidate ≫ methamphetamine = phentermine = MDMA > cocaine ≫ fluoxetine = fenfluramine, and at the serotonin transporter was fluoxetine = methamphetamine = fenfluramine = MDMA > amphetamine = cocaine = PTT ≥ methylphenidate > phentermine. Additionally, changes in electrically stimulated release were documented. This is the first study using voltammetry to measure the effects of a wide range of monoamine uptake inhibitors and releasers on dopamine and serotonin uptake in mouse brain slices. These studies also highlight methodological considerations for comparison of effects between heterogeneous brain regions. PMID:17459426

  8. Basic aspects of GABA-transmission in alcoholism, with particular reference to GABA-transaminase.

    PubMed

    Sherif, F M; Tawati, A M; Ahmed, S S; Sharif, S I

    1997-02-01

    Neuronal dysfunction is the neurobiological basis for alcoholic behaviour, and ethanol craving seems related to hypofunction of the GABA-ergic activity. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). In several studies, GABA has been shown to be an important target of ethanol in the CNS, partly, as a consequence of damage to membrane-bound enzymes and receptors. GABA is involved in mediating pre- and post-synaptic inhibition of neuronal activity. It is speculated that the initial excitatory effects of ethanol may be due to inhibition of GABA-ergic activity whereas the sedative effects of the higher doses may be mediated by the activation of this inhibitory system. In the CNS, GABA is synthesised from glutamic acid by the enzyme glutamate decarboxylase (GAD) and catabolized into succinic semialdehyde by the enzyme GABA-transaminase (GABA-T), which are pyridoxal phosphate (PLP) dependent enzymes. Platelet GABA-T was characterized as being similar to central GABA-T. Inhibition of GABA-T with certain potent and selective compounds markedly increases the levels of brain GABA. Experimentally, acute ethanol treatment does not alter GABA-T activity whereas chronic treatment produces an increase in the activity, though, with some reservations since a bimodal effect has been found in chronically ethanol-treated rats. Thus, as it will be discussed below, it may be suggested that GABA-T inhibitors (e.g. vigabatrin) could have a potential role in the treatment of alcoholism and in some of the problems of ethanol withdrawal and of other drugs of abuse. Related studies on metabolism and concentrations of GABA are also promising and show a greater increase in our understanding of the aetiology and treatment of ethanol dependence and withdrawal. In general, this article also reviews both the animal and clinical observations in the field of alcoholism with regard to the GABA system.

  9. Modulation of extracellular GABA levels in the retina by activation of glial P2X-purinoceptors.

    PubMed

    Neal, M J; Cunningham, J R; Dent, Z

    1998-05-01

    1. In the rat retina, gamma-aminobutyric acid (GABA) released as a transmitter is inactivated by uptake mainly into glial cells (Müller cells). Activation of P2-purinoceptors in Müller cells increases [Ca2+]i and the present study was undertaken to see whether this action affected the glial release of [3H]-GABA from the superfused rat isolated retina. 2. Adenosine 5'-triphosphate (ATP) and the P2X-purinoceptor agonists, alpha,beta-methylene-ATP (alpha,beta-meATP) and beta,gamma-methyleneATP (beta,gamma-meATP) significantly increased the KCl-evoked release of [3H]-GABA from the retina. 3. Adenosine and the P2Y-purinoceptor agonist, 2-chloroATP, had no effect on the KCl-evoked release of [3H]-GABA from the retina. However, 2-methylthioATP (2-Me-S-ATP) significantly enhanced the evoked release of [3H]-GABA. 4. The effect of ATP on the glial release of [3H]-GABA was abolished by the P2-antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). 5. When the superfused retina was exposed to the GABA uptake inhibitor, SKF89976A, the enhancing effect of alpha,beta-meATP on the KCl-evoked release of GABA was abolished. 6. The KCl-evoked release of [3H]-GABA from the frog retina and rat cerebrocortical slices, which take up GABA mainly into neurones, was not affected by ATP or alpha,beta-meATP. 7. We concluded that the glial Müller cells in the rat retina possess P2-receptors, activation of which increases the 'release' of preloaded [3H]-GABA apparently by reducing uptake. On balance, the results suggest the involvement of P2X-purinoceptors, although we cannot exclude the possibility that P2Y-purinoceptors may be involved. Our results suggest that ATP, as well as being a conventional transmitter in the retina, may be involved in neuronal-glial signalling and modulate the extracellular concentration of GABA.

  10. Proton Pump Inhibitors Inhibit Metformin Uptake by Organic Cation Transporters (OCTs)

    PubMed Central

    Nies, Anne T.; Hofmann, Ute; Resch, Claudia; Schaeffeler, Elke; Rius, Maria; Schwab, Matthias

    2011-01-01

    Metformin, an oral insulin-sensitizing drug, is actively transported into cells by organic cation transporters (OCT) 1, 2, and 3 (encoded by SLC22A1, SLC22A2, or SLC22A3), which are tissue specifically expressed at significant levels in various organs such as liver, muscle, and kidney. Because metformin does not undergo hepatic metabolism, drug-drug interaction by inhibition of OCT transporters may be important. So far, comprehensive data on the interaction of proton pump inhibitors (PPIs) with OCTs are missing although PPIs are frequently used in metformin-treated patients. Using in silico modeling and computational analyses, we derived pharmacophore models indicating that PPIs (i.e. omeprazole, pantoprazole, lansoprazole, rabeprazole, and tenatoprazole) are potent OCT inhibitors. We then established stably transfected cell lines expressing the human uptake transporters OCT1, OCT2, or OCT3 and tested whether these PPIs inhibit OCT-mediated metformin uptake in vitro. All tested PPIs significantly inhibited metformin uptake by OCT1, OCT2, and OCT3 in a concentration-dependent manner. Half-maximal inhibitory concentration values (IC50) were in the low micromolar range (3–36 µM) and thereby in the range of IC50 values of other potent OCT drug inhibitors. Finally, we tested whether the PPIs are also transported by OCTs, but did not identify PPIs as OCT substrates. In conclusion, PPIs are potent inhibitors of the OCT-mediated metformin transport in vitro. Further studies are needed to elucidate the clinical relevance of this drug-drug interaction with potential consequences on metformin disposition and/or efficacy. PMID:21779389

  11. Conjugation of spermine enhances cellular uptake of the stapled peptide-based inhibitors of p53-Mdm2 interaction.

    PubMed

    Muppidi, Avinash; Li, Xiaolong; Chen, Jiandong; Lin, Qing

    2011-12-15

    We report the first synthesis of the C-terminally spermine-conjugated stapled peptide-based inhibitors of the p53-Mdm2 interaction. Subsequent biological, biophysical and cellular uptake assays with the spermine-conjugated stapled peptides revealed that spermine conjugation minimally affects biological activity while significantly increases peptide helicity and cellular uptake without apparent cytotoxicity.

  12. Use of inhibitors for coastal bacteria and phytoplankton: Application to nitrogen uptake measurement

    NASA Astrophysics Data System (ADS)

    Trottet, Aurore; Fouilland, Eric; Leboulanger, Christophe; Lanouguère, Elodie; Bouvy, Marc

    2011-06-01

    For several decades, prokaryotic and eukaryotic inhibitors have been used to exclude bacteria from microalgal cultures and for investigating prey-predator relationships. Recently there has been considerable interest in using specific inhibitors for studying the interactions between bacteria and phytoplankton, by selective repression of either organism's activity. The effectiveness of chemical inhibitors must be tested before applying them to natural communities to partition metabolic activities between functional groups. Six different antibiotics selected from the most commonly reported in the literature were tested, at concentrations varying from 12.5 to 100 mg L -1, for their effect on bacterial growth and functional diversity of natural communities from Mediterranean coastal waters. Penicillin and streptomycin each at a final concentration of 100 mg L -1 significantly reduced bacterial growth within 2 h. There was a greater impact on bacterial functional diversity when both antibiotics were mixed together. This mixture did not have any significant effect on the growth of selected cultured phytoplankton strains, whereas the eukaryote inhibitor cycloheximide at 100 mg L -1 reduced growth within 2 h of incubation. The penicillin-streptomycin mixture and cycloheximide alone successfully partitioned NH 4+ and NO 3- uptake between bacteria and phytoplankton bi-weekly sampled in a coastal lagoon in Autumn, where bacterial contribution to total NH 4+ and NO 3- uptake averaged 46 and 41%, respectively. The use of specific inhibitors may be a valuable method for studying interactions, such as competition and mutualism, or lack of interaction between the different components of microbial communities and could be used to study their relative importance in biogeochemical fluxes.

  13. The four human gamma-aminobutyric acid (GABA) transporters: pharmacological characterization and validation of a highly efficient screening assay.

    PubMed

    Kvist, Trine; Christiansen, Bolette; Jensen, Anders A; Bräuner-Osborne, Hans

    2009-03-01

    The neurotransmission mediated by gamma-aminobutyric acid (GABA) in the mammalian brain is terminated by a family of four GABA transporters (GATs). Inhibition of GATs is currently used in the treatment of epilepsy and these proteins are generally considered as important drug targets. In this study, we perform the first elaborate pharmacological characterization of all four human GAT subtypes. We conduct the experiments in parallel in a [3H]GABA uptake assay using 14 standard GAT substrates and inhibitors. This setup enables direct comparison of the absolute values of inhibitory activities of the compounds between the different GAT subtypes. The results are overall in agreement with data reported by other groups for the orthologous murine GATs. However, there do seem to be some minor variations among species. In contrast to the several subtype selective ligands identified for the GAT-1 subtype, no subtype selective ligands have been reported for the three remaining GATs. Given the potential therapeutic relevance of the individual GAT subtypes, a search for novel structures displaying selectivities for specific GAT subtypes is important. In this study, we validate our [3H]GABA uptake assay for use in high throughput screening. We find that the assay is categorized by high Z'-factors (Z' > 0.5) for all four GAT subtypes, demonstrating that the assay is excellent for a high throughput screen. This [3H]GABA uptake assay therefore enables future high throughput screening of compound libraries at the four human GATs.

  14. Effects of Transport Inhibitors on the Cellular Uptake of Carboxylated Polystyrene Nanoparticles in Different Cell Lines

    PubMed Central

    dos Santos, Tiago; Varela, Juan; Lynch, Iseult; Salvati, Anna; Dawson, Kenneth A.

    2011-01-01

    Nanotechnology is expected to play a vital role in the rapidly developing field of nanomedicine, creating innovative solutions and therapies for currently untreatable diseases, and providing new tools for various biomedical applications, such as drug delivery and gene therapy. In order to optimize the efficacy of nanoparticle (NP) delivery to cells, it is necessary to understand the mechanisms by which NPs are internalized by cells, as this will likely determine their ultimate sub-cellular fate and localisation. Here we have used pharmacological inhibitors of some of the major endocytic pathways to investigate nanoparticle uptake mechanisms in a range of representative human cell lines, including HeLa (cervical cancer), A549 (lung carcinoma) and 1321N1 (brain astrocytoma). Chlorpromazine and genistein were used to inhibit clathrin and caveolin mediated endocytosis, respectively. Cytochalasin A and nocodazole were used to inhibit, respectively, the polymerisation of actin and microtubule cytoskeleton. Uptake experiments were performed systematically across the different cell lines, using carboxylated polystyrene NPs of 40 nm and 200 nm diameters, as model NPs of sizes comparable to typical endocytic cargoes. The results clearly indicated that, in all cases and cell types, NPs entered cells via active energy dependent processes. NP uptake in HeLa and 1321N1 cells was strongly affected by actin depolymerisation, while A549 cells showed a stronger inhibition of NP uptake (in comparison to the other cell types) after microtubule disruption and treatment with genistein. A strong reduction of NP uptake was observed after chlorpromazine treatment only in the case of 1321N1 cells. These outcomes suggested that the same NP might exploit different uptake mechanisms to enter different cell types. PMID:21949717

  15. Design, characterization and cellular uptake studies of fluorescence-labeled prototypic cathepsin inhibitors.

    PubMed

    Kohl, Franziska; Schmitz, Janina; Furtmann, Norbert; Schulz-Fincke, Anna-Christina; Mertens, Matthias D; Küppers, Jim; Benkhoff, Marcel; Tobiasch, Edda; Bartz, Ulrike; Bajorath, Jürgen; Stirnberg, Marit; Gütschow, Michael

    2015-11-07

    Besides their extracellular activity crucial for several pathophysiological conditions, human cysteine cathepsins, in particular cathepsins K and S, represent important intracellular targets for drug development. In the present study, a prototypic dipeptide nitrile inhibitor structure was equipped with a coumarin moiety to function as a fluorescent reporter group. In a second inhibitor, a PEG linker was introduced between the dipeptide nitrile and the fluorophore. These tool compounds 6 and 7 were characterized by kinetic investigations as covalent reversible inhibitors of human cathepsins L, S, K and B. Probe 6 showed a pronounced inhibitory activity against cathepsins K and S, which was corroborated by modeling of inhibition modes. Probe 7 was highly potent (Ki = 93 nM) and selective for cathepsin S. To examine the ability of both probes to enter living cells, human embryonic kidney 293 cells were targeted. At a concentration of 10 μM, cellular uptake of probe 6 was demonstrated by fluorescence measurement after an incubation time of 30 min and 3 h, respectively. The probe's concentration in cell lysates was ascertained on the basis of the emission at 492 nm upon excitation at 450 nm, and the results were expressed as concentrations of probe 6 relative to the protein concentration originating from the lysate. After incubation of 10 μM of probe 6 for 3 h, the cellular uptake was confirmed by fluorescence microscopy. HPLC was used to assess the probes’ lipophilicity, and the obtained

  16. Reviewing the serotonin reuptake inhibitors (SSRIs) footprint in the aquatic biota: uptake, bioaccumulation and ecotoxicology.

    PubMed

    Silva, Liliana J G; Pereira, André M P T; Meisel, Leonor M; Lino, Celeste M; Pena, Angelina

    2015-02-01

    Selective serotonin re-uptake inhibitors (SSRIs) antidepressants are amongst the most prescribed pharmaceutical active substances throughout the world. Their presence, already described in different environmental compartments such as wastewaters, surface, ground and drinking waters, and sediments, and their remarkable effects on non-target organisms justify the growing concern about these emerging environmental pollutants. A comprehensive review of the literature data with focus on their footprint in the aquatic biota, namely their uptake, bioaccumulation and both acute and chronic ecotoxicology is presented. Long-term multigenerational exposure studies, at environmental relevant concentrations and in mixtures of related compounds, such as oestrogenic endocrine disruptors, continue to be sparse and are imperative to better know their environmental impact. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Selective serotonin re-uptake inhibitors (SSRIs) in the aquatic environment: an ecopharmacovigilance approach.

    PubMed

    Silva, Liliana J G; Lino, Celeste M; Meisel, Leonor M; Pena, Angelina

    2012-10-15

    Selective serotonin re-uptake inhibitors (SSRIs) antidepressants are among the most prescribed pharmaceutical active substances throughout the world. The occurrence of these widely used compounds in different environmental compartments (wastewaters, surface, ground and drinking waters, and sediments), justify the growing concern about these emerging environmental pollutants. Viewing an ecopharmacovigilance approach, a comprehensive discussion of the state of the art regarding different contamination sources, fate, degradation and occurrence is presented. Information on the current distribution levels and fate in different environmental matrices continues to be sparse and measures are imperative to improve awareness and encourage precautionary actions to minimize SSRIs' environmental impact.

  18. Modulation of extracellular neurotransmitter levels in the nucleus accumbens by a taurine uptake inhibitor.

    PubMed

    Olive, M F; Mehmert, K K; Hodge, C W

    2000-12-15

    Using in vivo microdialysis, we examined the effect of local perfusion of the taurine uptake inhibitor guanidinoethyl sulfonate on extracellular levels of various neurotransmitters in the rat nucleus accumbens. Guanidinoethyl sulfonate (500 microM-50 mM) produced a concentration-dependent increase in extracellular taurine levels. While 500 microM and 5 mM concentrations of guanidinoethyl sulfonate were largely without effect, 50 mM guanidinoethyl sulfonate produced a significant decrease in extracellular levels of aspartate, glutamate and glycine, with no effect on extracellular dopamine levels. These results indicate that guanidinoethyl sulfonate can modulate extracellular amino acid levels in the nucleus accumbens.

  19. Kinetic studies on the inhibition of GABA-T by gamma-vinyl GABA and taurine.

    PubMed

    Sulaiman, Saba A J; Suliman, Fakhr Eldin O; Barghouthi, Samira

    2003-08-01

    Gamma-aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of gamma-aminobutyric acid. The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: gamma-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid). A kinetic model that describes the transamination process is proposed. GABA-T from Pseudomonas fluorescens is inhibited by gamma-vinyl GABA and taurine at concentrations of 51.0 and 78.5 mM. Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (Ki) values for gamma-vinyl GABA and taurine were found to be 26 +/- 3 mM and 68 +/- 7 mM respectively. The transamination process of alpha-ketoglutarate was not affected by the presence of gamma-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when alpha-ketoglutarate was the substrate. The inhibition dissociation constant (Kii) for this system was found to be 96 +/- 10 mM. The Michaelis-Menten constant (Km) in the absence of inhibition, was found to be 0.79 +/- 0.11 mM, and 0.47 +/- 0.10 mM for GABA and alpha-ketoglutarate respectively.

  20. The amine oxidase inhibitor phenelzine limits lipogenesis in adipocytes without inhibiting insulin action on glucose uptake.

    PubMed

    Carpéné, Christian; Grès, Sandra; Rascalou, Simon

    2013-06-01

    The antidepressant phenelzine is a monoamine oxidase inhibitor known to inhibit various other enzymes, among them semicarbazide-sensitive amine oxidase (currently named primary amine oxidase: SSAO/PrAO), absent from neurones but abundant in adipocytes. It has been reported that phenelzine inhibits adipocyte differentiation of cultured preadipocytes. To further explore the involved mechanisms, our aim was to study in vitro the acute effects of phenelzine on de novo lipogenesis in mature fat cells. Therefore, glucose uptake and incorporation into lipid were measured in mouse adipocytes in response to phenelzine, other hydrazine-based SSAO/PrAO-inhibitors, and reference agents. None of the inhibitors was able to impair the sevenfold activation of 2-deoxyglucose uptake induced by insulin. Phenelzine did not hamper the effect of lower doses of insulin. However, insulin-stimulated glucose incorporation into lipids was dose-dependently inhibited by phenelzine and pentamidine, but not by semicarbazide or BTT2052. In contrast, all these SSAO/PrAO inhibitors abolished the transport and lipogenesis stimulation induced by benzylamine. These data indicate that phenelzine does not inhibit glucose transport, the first step of lipogenesis, but inhibits at 100 μM the intracellular triacylglycerol assembly, consistently with its long-term anti-adipogenic effect and such rapid action was not found with all the hydrazine derivatives tested. Therefore, the alterations of body weight control consecutive to the use of this antidepressant drug might be not only related to central effects on food intake/energy expenditure, but could also depend on its direct action in adipocytes. Nonetheless, phenelzine antilipogenic action is not merely dependent on SSAO/PrAO inhibition.

  1. High-affinity uptake of gamma-aminobutyric acid in cultured glial and neuronal cells.

    PubMed

    Balcar, V J; Mark, J; Borg, J; Mandel, P

    1979-06-01

    Both glial and neuronal cells maintained in primary culture were found to accumulate [3H]GABA by an efficient "high-affinity" uptake system (apparent Km = 9 muM, Vmax = 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 1mM) 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, beta-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 (Km = 92 muM, Vmax = 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. Apparent Km of this uptake was relatively high (819 muM), 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, non of the nontransformed continuous cell lines of either tumoral (glioma, C6; neuroblastoma, M1; M1NN) 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.

  2. Evidence for GABA-Induced Systemic GABA Accumulation in Arabidopsis upon Wounding

    PubMed Central

    Scholz, Sandra S.; Malabarba, Jaiana; Reichelt, Michael; Heyer, Monika; Ludewig, Frank; Mithöfer, Axel

    2017-01-01

    The non-proteinogenic amino acid γ-aminobutyric acid (GABA) is present in all plant species analyzed so far. Its synthesis is stimulated by either acidic conditions occurring after tissue disruption or higher cytosolic calcium level. In mammals, GABA acts as inhibitory neurotransmitter but its function in plants is still not well understood. Besides its involvement in abiotic stress resistance, GABA has a role in the jasmonate-independent defense against invertebrate pests. While the biochemical basis for GABA accumulation in wounded leaves is obvious, the underlying mechanisms for wounding-induced GABA accumulation in systemic leaves remained unclear. Here, the Arabidopsis thaliana knock-out mutant lines pop2-5, unable to degrade GABA, and tpc1-2, lacking a wounding-induced systemic cytosolic calcium elevation, were employed for a comprehensive investigation of systemic GABA accumulation. A wounding-induced systemic GABA accumulation was detected in tpc1-2 plants demonstrating that an increased calcium level was not involved. Similarly, after both mechanical wounding and Spodoptera littoralis feeding, GABA accumulation in pop2-5 plants was significantly higher in local and systemic leaves, compared to wild-type plants. Consequently, larvae feeding on these GABA-enriched mutant plants grew significantly less. Upon exogenous application of a D2-labeled GABA to wounded leaves of pop2-5 plants, its uptake but no translocation to unwounded leaves was detected. In contrast, an accumulation of endogenous GABA was observed in vascular connected systemic leaves. These results suggest that the systemic accumulation of GABA upon wounding does not depend on the translocation of GABA or on an increase in cytosolic calcium. PMID:28382046

  3. Manganese exposure inhibits the clearance of extracellular GABA and influences taurine homeostasis in the striatum of developing rats.

    PubMed

    Fordahl, Steve C; Anderson, Joel G; Cooney, Paula T; Weaver, Tara L; Colyer, Christa L; Erikson, Keith M

    2010-12-01

    Manganese (Mn) accumulation in the brain has been shown to alter the neurochemistry of the basal ganglia. Mn-induced alterations in dopamine biology are fairly well understood, but recently more evidence has emerged characterizing the role of γ-aminobutyric acid (GABA) in this dysfunction. The purpose of this study was to determine if the previously observed Mn-induced increase in extracellular GABA (GABA(EC)) was due to altered GABA transporter (GAT) function, and whether Mn perturbs other amino acid neurotransmitters, namely taurine and glycine (known modulators of GABA). Extracellular GABA, taurine, and glycine concentrations were collected from the striatum of control (CN) or Mn-exposed Sprague-Dawley rats using in vivo microdialysis, and the GAT inhibitor nipecotic acid (NA) was used to probe GAT function. Tissue and extracellular Mn levels were significantly increased, and the Fe:Mn ratio was decreased 36-fold in the extracellular space due to Mn-exposure. NA led to a 2-fold increase in GABA(EC) of CNs, a response that was attenuated by Mn. Taurine responded inversely to GABA, and a novel 10-fold increase in taurine was observed after the removal of NA in CNs. Mn blunted this response and nearly abolished extracellular taurine throughout collection. Striatal taurine transporter (Slc6a6) mRNA levels were significantly increased with Mn-exposure, and Mn significantly increased (3)H-Taurine uptake after 3-min exposure in primary rat astrocytes. These data suggest that Mn increases GABA(EC) by inhibiting the function of GAT, and that perturbed taurine homeostasis potentially impacts neural function by jeopardizing the osmoregulatory and neuromodulatory functions of taurine in the brain.

  4. Blocking the GABA transporter GAT-1 ameliorates spinal GABAergic disinhibition and neuropathic pain induced by paclitaxel

    PubMed Central

    Yadav, Ruchi; Yan, Xisheng; Maixner, Dylan W.; Gao, Mei; Weng, Han-Rong

    2015-01-01

    Paclitaxel is a chemotherapeutic agent widely used for treating carcinomas. Patients receiving paclitaxel often develop neuropathic pain and have a reduced quality of life which hinders the use of this life-saving drug. In this study, we determined the role of GABA transporters in the genesis of paclitaxel-induced neuropathic pain using behavioral tests, electrophysiology, and biochemical techniques. We found that tonic GABA receptor activities in the spinal dorsal horn were reduced in rats with neuropathic pain induced by paclitaxel. In normal controls, tonic GABA receptor activities were mainly controlled by the GABA transporter GAT-1 but not GAT-3. In the spinal dorsal horn, GAT-1 was expressed at presynaptic terminals and astrocytes while GAT-3 was only expressed in astrocytes. In rats with paclitaxel-induced neuropathic pain, the protein expression of GAT-1 was increased while GAT-3 was decreased. This was concurrently associated with an increase of global GABA uptake. The paclitaxel-induced attenuation of GABAergic tonic inhibition was ameliorated by blocking GAT-1 but not GAT-3 transporters. Paclitaxel-induced neuropathic pain was significantly attenuated by the intrathecal injection of a GAT-1 inhibitor. These findings suggest that targeting GAT-1 transporters for reversing disinhibition in the spinal dorsal horn may be a useful approach for treating paclitaxel-induced neuropathic pain. PMID:25827582

  5. GABA and glycine actions on spinal motoneurons.

    PubMed

    Krnjević, K; Puil, E; Werman, R

    1977-06-01

    Applied microiontophoretically in the spinal cord of cats, glycine is consistently more powerful than gamma-aminobutyric acid (GABA) in raising the membrane conductance of lumbosacral motoneurons (mean ratio of equipotent iontophoretic currents tested on same cells is 5.6:1). This is the reverse of the situation in cerebral cortex. The effect of glycine is well maintained during applications lasting about 1 min, but that of GABA, after an early peak, drops to a much lower plateau (mean plateau-over-peak ratio is 0.23). The reversal potentials for the action of GABA and glycine are initially similar but they behave differently during a prolonged application; that for glycine usually remains constant or becomes more negative whereas that for GABA tends to shift in the positive direction. Various explanations of these phenomena are considered. It is suggested that a single process, electrogenic uptake of GABA, may account for both desensitization (by removing GABA from its site of action) and the positive shift in GABA reversal potential (became uptake is probably associated with an influx of Na+).

  6. Interaction between tricyclic and nontricyclic 5-hydroxytryptamine uptake inhibitors and the presynaptic 5-hydroxytryptamine inhibitory autoreceptors in the rat hypothalamus.

    PubMed

    Galzin, A M; Moret, C; Verzier, B; Langer, S Z

    1985-10-01

    In slices of the rat hypothalamus prelabeled with [3H]-5-hydroxytryptamine [( 3H]-5-HT), exposure to lysergic acid diethylamide or 5-methoxytryptamine decreased, in a concentration-dependent manner, the release of 3H-transmitter elicited by electrical stimulation. These inhibitory effects were antagonized by the 5-HT receptor antagonist methiothepin (1 microM). Exposure to methiothepin on its own increased in a concentration-dependent manner the electrically evoked overflow of [3H]-5-HT. Exposure to tricyclic antidepressants, like imipramine and amitriptyline, and to nontricyclic 5-HT uptake inhibitors, like paroxetine and citalopram, did not modify by themselves the electrically evoked overflow of [3H]-5-HT. Yet, the four inhibitors of neuronal uptake of 5-HT, antagonized the inhibition by lysergic acid diethylamide or 5-methoxytryptamine of the electrically induced release of [3H]-5-HT. After depletion of endogenous stores of 5-HT by pretreatment with para-chlorophenylalanine (300 mg/kg i.p.), the inhibitors of 5-HT uptake increased the electrically evoked release of [3H]-5-HT in a concentration-dependent manner. Their order of potency to enhance 5-HT overflow after pretreatment with parachlorophenylalanine paralleled their potency at inhibiting neuronal uptake of 5-HT (paroxetine = citalopram greater than imipramine greater than amitriptyline). In para-chlorophenylalanine-treated rat hypothalamic slices, these inhibitors of 5-HT uptake antagonized the inhibition by 5-HT autoreceptor agonists of the electrically evoked release of [3H]-5-HT to a similar extent than was observed in control rats. It is concluded that inhibition of 5-HT uptake reduces the effectiveness of 5-HT autoreceptor agonists to inhibit the electrically evoked release of [3H]-5-HT, irrespective of the chemical structure of the uptake inhibitor or of the levels of endogenous 5-HT achieved in the synaptic gap.

  7. Effects of phenylethanolamine N-methyltransferase inhibitors on uptake and release of norepinephrine and dopamine from rat brain

    SciTech Connect

    Liang, N.Y.; Hower, J.A.; Borchardt, R.T.

    1985-09-01

    Inhibitors of phenylethanolamine N-methyltransferase (PNMT) and amphetamine were evaluated for their effects on the uptake of (TH)-norepinephrine (TH-NE) and the release of endogenous NE and dopamine (DA) from chopped rat brain tissues. Unlike amphetamine, all of PNMT inhibitors tested produced only slight inhibition of (TH)-NE uptake into chopped cerebral cortex. 2,3-Dichloro-alpha-methylbenzylamine (DCMB) and 7,8-dichloro-1,2,3,4-tetrahydroisoquinoline (SKF64139), but not 2-cyclooctyl-2-hydroxyethylamine (CONH) and 1-aminomethylcycloundecanol (CUNH) produced slight release of endogenous NE and DA from chopped hypothalami, but their effects were less pronounced than those produced by amphetamine.

  8. Uptake of L-tri-iodothyronine by isolated rat liver cells. A process partially inhibited by metabolic inhibitors; attempts to distinguish between uptake and binding to intracellular proteins.

    PubMed Central

    Eckel, J; Rao, G S; Rao, M L; Breuer, H

    1979-01-01

    1. Rat liver cells obtained by dispersion with collagenase were used to investigate the mode of entry of L-tri-iodothyronine into the cell. 2. The hormone was taken up very rapidly at 23 degrees C; the linear phase of uptake lasted for up to approx. 20 s. 3. A plot of the initial rates of uptake against different concentrations of L-tri-iodothyronine yielded a sigmoidal curve. The Eadie--Hofstee plot (v/[S]2 versus v) yielded two straight lines. The uptake component with an apparent Kt value of 86 +/- 15 pM was designated as system I, and the second uptake component with an apparent Kt of 726 +/- 11 pM as system II. The Hill plot for system I was not linear; the apparent Hill coefficient for system II was calculated to be 2.1.4. Uptake of L-tri-iodothyronine by system I was higher at pH 6.4 than at pH 7.4; system II was relatively insensitive to changes in the pH of the external medium. 5. Both systems exhibited a transition temperature at about 16 degrees C in the Arrhenius plot. The activation energies of the two systems below and above 16 degrees C were 72.8 and 47.7 and 54.4 and 33.1 J/mol respectively. 6. Inhibitors of cellular energy reduced the uptake by system I to a larger extent than that by system II. 7. Replacement of Na+ in the external medium by either K+ or choline led to uptake that followed normal Michaelis--Menten kinetics. 8. Thiol-group-blocking agents reduced the uptake of the hormone by both systems. 9. Treatment of liver cells with beta-glucosidase, Pronase and neuraminidase led to a decrease in the uptake of L-tri-iodothyronine by system I, whereas uptake by system II was decreased after treatment with phospholipase A2, beta-galactosidase. Pronase and neuraminidase. 10. The stereoisomer D-tri-iodothyronine (100--3000 pM) did not affect system I, but uptake by system II decreased with increasing concentration of D-tri-iodothyronine. Reverse L-tri-iodothyronine (2--100 pM) and L-thyroxine (100--3000 pM) did not influence uptake by either

  9. Atypical Dopamine Uptake Inhibitors that Provide Clues About Cocaine's Mechanism at the Dopamine Transporter

    NASA Astrophysics Data System (ADS)

    Hauck Newman, Amy; Katz, Jonathan L.

    The dopamine transporter (DAT) has been a primary target for cocaine abuse/addiction medication discovery. However predicted addiction liability and limited clinical evaluation has provided a formidable challenge for development of these agents for human use. The unique and atypical pharmacological profile of the benztropine (BZT) class of dopamine uptake inhibitors, in preclinical models of cocaine effects and abuse, has encouraged further development of these agents. Moreover, in vivo studies have challenged the original DAT hypothesis and demonstrated that DAT occupancy and subsequent increases in dopamine produced by BZT analogues are significantly delayed and long lasting, as compared to cocaine. These important and distinctive elements are critical to the lack of abuse liability among BZT analogues, and improve their potential for development as treatments for cocaine abuse and possibly other neuropsychiatric disorders.

  10. [Effects of urease/nitrification inhibitors on soil available N and microbial biomass N and on N uptake of wheat].

    PubMed

    Jiao, Xiaoguang; Liang, Wenju; Chen, Lijun; Jiang, Yong; Wen, Dazhong

    2004-10-01

    With an aquic brown earth as test soil, this paper studied the effects of urease inhibitor (NBPT), nitrification inhibitor (DCD) and their combinations on the dynamics of soil available N and microbial biomass N. The results showed that the treatments of inhibitors, especially the combined application of NBPT and DCD, could increase soil NH4+-N by 2%-53%, inhibit NH4+ oxidation, decrease soil NO3(-)-N concentration, increase soil total available N by 34%-44%, and increase wheat N uptake by 0.26%-6.79%. The best treatment was urease inhibitor combined with nitrification inhibitor. The application of inhibitors increased soil microbial biomass N immobilization at the early growth stage of wheat, and promoted soil N mineralization at filling stage.

  11. Protection of malonate-induced GABA but not dopamine loss by GABA transporter blockade in rat striatum.

    PubMed

    Zeevalk, Gail D; Manzino, Lawrence; Sonsalla, Patricia K

    2002-07-01

    Previous work has shown that overstimulation of GABA(A) receptors can potentiate neuronal cell damage during excitotoxic or metabolic stress in vitro and that GABA(A) antagonists or GABA transport blockers are neuroprotective under these situations. Malonate, a reversible succinate dehydrogenase/mitochondrial complex II inhibitor, is frequently used in animals to model cell loss in neurodegenerative diseases such as Parkinson's and Huntington's diseases. To determine if GABA transporter blockade during mitochondrial impairment can protect neurons in vivo as compared with in vitro studies, rats received a stereotaxic infusion of malonate (2 micromol) into the left striatum to induce a metabolic stress. The nonsubstrate GABA transport blocker, NO711 (20 nmol) was infused in some rats 30 min before and 3 h following malonate infusion. After 1 week, dopamine and GABA levels in the striata were measured. Malonate caused a significant loss of striatal dopamine and GABA. Blockade of the GABA transporter significantly attenuated GABA, but not dopamine loss. In contrast with several in vitro reports, GABA(A) receptors were not a downstream mediator of protection by NO711. Intrastriatal infusion of malonate (2 micromol) plus or minus the GABA(A) receptor agonist muscimol (1 micromol), the GABA(A) Cl- binding site antagonist picrotoxin (50 nmol) or the GABA(B) receptor antagonist saclofen (33 nmol) did not modify loss of striatal dopamine or GABA when examined 1 week following infusion. These data show that GABA transporter blockade during mitochondrial impairment in the striatum provides protection to GABAergic neurons. GABA transporter blockade, which is currently a pharmacological strategy for the treatment of epilepsy, may thus also be beneficial in the treatment of acute and chronic conditions involving energy inhibition such as stroke/ischemia or Huntington's disease. These findings also point to fundamental differences between immature and adult neurons in the

  12. R-MODAFINIL (ARMODAFINIL): A UNIQUE DOPAMINE UPTAKE INHIBITOR AND POTENTIAL MEDICATION FOR PSYCHOSTIMULANT ABUSE

    PubMed Central

    Loland, Claus J.; Mereu, Maddalena; Okunola, Oluyomi M.; Cao, J.; Prisinzano, Thomas E.; Mazier, Sonia; Kopajtic, Theresa; Shi, Lei; Katz, Jonathan L.; Tanda, Gianluigi; Newman, Amy Hauck

    2012-01-01

    Background (±)-Modafinil has piqued interest as a treatment for ADHD and stimulant dependence. The R-enantiomer of modafinil may have unique pharmacological properties that should be further investigated. Methods (±)-Modafinil and its R-(−)- and S-(+)-enantiomers were synthesized and tested for inhibition of [3H]DA uptake and [3H]WIN 35,428 binding in hDAT WT and mutants with altered conformational equilibria. Data were compared to cocaine and the atypical dopamine uptake inhibitor, JHW 007. R- and S-modafinil were also evaluated in microdialysis studies in the mouse NAc shell and in a cocaine discrimination procedure. Results (±)-, R- and S-Modafinil bind to the DAT and inhibit dopamine uptake less potently than cocaine, with R-modafinil having ~3-fold higher affinity than its S-enantiomer. Molecular docking studies revealed subtle differences in binding modes for the enantiomers. R-modafinil was significantly less potent in the DAT Y156F mutant compared to wild-type DAT, whereas S-modafinil was affected less. Studies with the Y335A DAT mutant showed that the R- and S-enantiomers tolerated the inward facing conformation better than cocaine, which was further supported by MTSET reactivity on the DAT E2C I159C. Microdialysis studies demonstrated that both R- and S-modafinil produced increases in extracellular DA concentrations in the NAc shell less efficaciously than cocaine, and with a longer duration of action. Both enantiomers fully substituted in mice trained to discriminate cocaine from saline. Conclusions R-modafinil displays an in vitro profile different from cocaine. Future trials with R-modafinil as a substitute therapy with the potential benefit of cognitive enhancement for psychostimulant addiction are warranted. PMID:22537794

  13. Invariance of the density of dopamine uptake sites and dopamine metabolism in the rat brain after a chronic treatment with the dopamine uptake inhibitor GBR 12783.

    PubMed

    Boulay, D; Leroux-Nicollet, I; Duterte-Boucher, D; Naudon, L; Costentin, J

    1994-01-01

    A chronic treatment (10 mg/kg, twice daily during 9 days) with the dopamine uptake inhibitor GBR 12783 was performed in rats at a dose increasing their locomotor activity. Forty-eight hours after the last administration, animals were sacrificed and 3H mazindol binding was performed on brain slices. Autoradiographic analysis revealed no change in this binding relatively to control animals in regions with high dopamine contents: striatum, nucleus accumbens, olfactory tubercle, substantia nigra and ventral tegmentum area. The treatment did not either modify the levels of dopamine (DA) and metabolites (HVA, DOPAC) both in the striatum and the nucleus accumbens. Thus, early after the end of the treatment, the chronic blockade of the dopamine uptake complex regulates neither the dopamine uptake complex nor the dopamine metabolism.

  14. Comparison of desmethylsertraline with sertraline as a monoamine uptake inhibitor in vivo.

    PubMed

    Fuller, R W; Hemrick-Luecke, S K; Littlefield, E S; Audia, J E

    1995-01-01

    1. Desmethylsertraline, a metabolite of the antidepressant drug sertraline, was compared with sertraline for its ability to produce effects characteristic of inhibitors of the serotonin transporter in vivo. Desmethylsertraline antagonized brain serotonin depletion by p-chloroamphetamine, a depletion dependent upon the serotonin transporter, being less potent than sertraline in rats but almost as potent as sertraline in mice. Desmethylsertraline was a weak antagonist of 6-hydroxydopamine-induced depletion of heart norepinephrine in mice; sertraline had no effect at the doses studied. 2. Desmethylsertraline decreased brain concentrations of 5-hydroxyindoleacetic acid (5HIAA) in rats as did sertraline, the duration of the effect after both drugs being at least 24 hrs but less than 48 hrs. 3. After sertraline injection, desmethylsertraline was present in rat brain at higher concentrations than the parent drug at 8 hrs and thereafter. 4. In rats, repeated injections of sertraline, at doses previously shown to diminish beta-adrenergic receptor-mediated responses, led to marked accumulation of desmethylsertraline in brain and to inhibition of the catecholamine transporters. 5. In mice, brain concentrations of desmethylsertraline were higher than those of parent drug within 7 hrs after sertraline injection and probably contributed importantly to the antagonism of p-chloroamphetamine effects. 6. These data show that desmethylsertraline is less potent than sertraline as a serotonin uptake inhibitor in vivo, as the in vitro data would have predicted, but that desmethylsertraline may nonetheless contribute to the prolonged inhibition of the serotonin transporter after sertraline administration, perhaps more in mice than in rats.

  15. A possible role of the non-GAT1 GABA transporters in transfer of GABA from GABAergic to glutamatergic neurons in mouse cerebellar neuronal cultures.

    PubMed

    Suñol, C; Babot, Z; Cristòfol, R; Sonnewald, U; Waagepetersen, H S; Schousboe, A

    2010-09-01

    Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, beta-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC(50) 142 microM), beta-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC(50) 0.8 microM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 microM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of beta-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also

  16. Generation of a 3D model for human GABA transporter hGAT-1 using molecular modeling and investigation of the binding of GABA.

    PubMed

    Wein, Thomas; Wanner, Klaus T

    2010-01-01

    A three-dimensional model of the human Na(+)/Cl(-)-dependent gamma-aminobutyric acid (GABA) transporter hGAT-1 was developed by homology modeling and refined by subsequent molecular modeling using the crystal structure of a bacterial homologue leucine transporter from Aquifex aeolicus (LeuT(Aa)) as the template. Protein structure quality checks show that the resulting structure is particularly suited for the analysis of the substrate binding pocket and virtual screening experiments. Interactions of GABA and the substrate binding pocket were investigated using docking studies. The difference of 6 out of 13 substrate interacting side chains between hGAT-1 and LeuT(Aa) lead to the different substrate preference which can be explained using our three-dimensional model of hGAT-1. In particular the replacement of serine 256 and isoleucine 359 in LeuT(Aa) with glycine and threonine in hGAT-1 seems to facilitate the selection of GABA as the main substrate by changing the hydrogen bonding pattern in the active site to the amino group of the substrate. For a set of 12 compounds flexible docking experiments were performed using LigandFit in combination with the Jain scoring function. With few exceptions the obtained rank order of potency was in line with experimental data. Thus, the method can be assumed to give at least a rough estimate of the potency of the potential of GABA uptake inhibitors.

  17. Not All Antidepressants Are Created Equal: Differential Effects of Monoamine Uptake Inhibitors on Effort-Related Choice Behavior.

    PubMed

    Yohn, Samantha E; Collins, Samantha L; Contreras-Mora, Hector M; Errante, Emily L; Rowland, Margaret A; Correa, Merce; Salamone, John D

    2016-02-01

    Motivated behavior can be characterized by behavioral activation and high work output. Moreover, people with depression and other disorders show effort-related motivational symptoms, such as anergia, psychomotor retardation, and fatigue. Effort-based decision making is studied using tasks offering choices between high effort options leading to highly valued reinforcers vs low effort/low reward options, and such tasks could be useful as animal models of motivational symptoms. In the present studies the effort-related effects of the vesicular monoamine transport (VMAT-2) inhibitor tetrabenazine (TBZ) were investigated. TBZ blocks vesicular storage and also produces depressive symptoms in humans. Moreover, TBZ alters effort-based choice in rats, biasing animals toward low effort alternatives. The present studies investigated the ability of acute administration of various monoamine uptake inhibitors to reverse the effects of TBZ. Effort-related effects of TBZ were attenuated by the catecholamine uptake inhibitor and antidepressant bupropion, and this effect of bupropion was reversed by either D1 or D2 family antagonism. The effort-related effects of TBZ were also attenuated by the selective dopamine uptake blocker GBR12909. The 5-HT uptake inhibitor fluoxetine and the norepinephrine uptake inhibitor desipramine failed to reverse the effects of TBZ, and higher doses of these drugs, given alone or in combination with TBZ, led to further behavioral impairments. These results indicate that drugs acting on dopamine transmission are relatively effective at reversing the effort-related effects of TBZ, and are consistent with the hypothesis that drugs that enhance dopamine transmission may be effective at treating effort-related psychiatric symptoms in humans.

  18. Not All Antidepressants Are Created Equal: Differential Effects of Monoamine Uptake Inhibitors on Effort-Related Choice Behavior

    PubMed Central

    Yohn, Samantha E; Collins, Samantha L; Contreras-Mora, Hector M; Errante, Emily L; Rowland, Margaret A; Correa, Merce; Salamone, John D

    2016-01-01

    Motivated behavior can be characterized by behavioral activation and high work output. Moreover, people with depression and other disorders show effort-related motivational symptoms, such as anergia, psychomotor retardation, and fatigue. Effort-based decision making is studied using tasks offering choices between high effort options leading to highly valued reinforcers vs low effort/low reward options, and such tasks could be useful as animal models of motivational symptoms. In the present studies the effort-related effects of the vesicular monoamine transport (VMAT-2) inhibitor tetrabenazine (TBZ) were investigated. TBZ blocks vesicular storage and also produces depressive symptoms in humans. Moreover, TBZ alters effort-based choice in rats, biasing animals toward low effort alternatives. The present studies investigated the ability of acute administration of various monoamine uptake inhibitors to reverse the effects of TBZ. Effort-related effects of TBZ were attenuated by the catecholamine uptake inhibitor and antidepressant bupropion, and this effect of bupropion was reversed by either D1 or D2 family antagonism. The effort-related effects of TBZ were also attenuated by the selective dopamine uptake blocker GBR12909. The 5-HT uptake inhibitor fluoxetine and the norepinephrine uptake inhibitor desipramine failed to reverse the effects of TBZ, and higher doses of these drugs, given alone or in combination with TBZ, led to further behavioral impairments. These results indicate that drugs acting on dopamine transmission are relatively effective at reversing the effort-related effects of TBZ, and are consistent with the hypothesis that drugs that enhance dopamine transmission may be effective at treating effort-related psychiatric symptoms in humans. PMID:26105139

  19. Revised Ion/Substrate Coupling Stoichiometry of GABA Transporters.

    PubMed

    Eskandari, Sepehr; Willford, Samantha L; Anderson, Cynthia M

    2017-01-01

    The purpose of this review is to highlight recent evidence in support of a 3 Na(+): 1 Cl(-): 1 GABA coupling stoichiometry for plasma membrane GABA transporters (SLC6A1 , SLC6A11 , SLC6A12 , SLC6A13 ) and how the revised stoichiometry impacts our understanding of the contribution of GABA transporters to GABA homeostasis in synaptic and extrasynaptic regions in the brain under physiological and pathophysiological states. Recently, our laboratory probed the GABA transporter stoichiometry by analyzing the results of six independent measurements, which included the shifts in the thermodynamic transporter reversal potential caused by changes in the extracellular Na(+), Cl(-), and GABA concentrations, as well as the ratio of charge flux to substrate flux for Na(+), Cl(-), and GABA under voltage-clamp conditions. The shifts in the transporter reversal potential for a tenfold change in the external concentration of Na(+), Cl(-), and GABA were 84 ± 4, 30 ± 1, and 29 ± 1 mV, respectively. Charge flux to substrate flux ratios were 0.7 ± 0.1 charges/Na(+), 2.0 ± 0.2 charges/Cl(-), and 2.1 ± 0.1 charges/GABA. We then compared these experimental results with the predictions of 150 different transporter stoichiometry models, which included 1-5 Na(+), 0-5 Cl(-), and 1-5 GABA per transport cycle. Only the 3 Na(+): 1 Cl(-): 1 GABA stoichiometry model correctly predicts the results of all six experimental measurements. Using the revised 3 Na(+): 1 Cl(-): 1 GABA stoichiometry, we propose that the GABA transporters mediate GABA uptake under most physiological conditions. Transporter-mediated GABA release likely takes place under pathophysiological or extreme physiological conditions.

  20. Relative efficacies of 1,4-diazepines on GABA-stimulated chloride influx in rat brain vesicles

    SciTech Connect

    Ikeda, Masaaki; Weber, K.H.; Bechtel, W.D.; Malatynska, E.; Yamamura, H.I.

    1989-01-01

    The effects of 1,4-diazepines with two annelated heterocycles (brotizolam (WE 941), ciclotizolam (WE 973) and WE 1008) on gamma-aminobutyric acid (GABA)-stimulated chloride influx into rat brain membrane vesicles were examined. Brotizolam enhanced GABA-stimulated /sup 36/Cl/sup /minus// influx, while ciclotizolam and WE 1008 showed only a small enhancement of GABA-stimulated /sup 36/Cl/sup /minus// uptake. Brotizolam resulted in a left shift of the GABA dose response curve at lower concentrations of GABA, while at higher concentrations of GABA, brotizolam caused a reduction of the maximal response. The enhancement of GABA-stimulated /sup 36/Cl/sup /minus// uptake by brotizolam was antagonized by Ro 15-1788. At higher concentration of GABA (300 /mu/M), brotizolam inhibited GABA-stimulated /sup 36/Cl/sup /minus// uptake in a dose dependent manner and Ro 15-1788 failed to antagonize this effect.

  1. Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells.

    PubMed

    Tomi, Masatoshi; Tajima, Ayumi; Tachikawa, Masanori; Hosoya, Ken-ichi

    2008-10-01

    The purpose of this study was to identify the uptake mechanism of gamma-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na(+)-, Cl(-)-, and concentration-dependent [3H]GABA uptake with a Km of 1.5 mM. Taurine, beta-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [3H]GABA. The uptake of [3H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na(+)-, Cl(-)-, and concentration-dependent with a Km of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and beta-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a Ki of 74 microM. [3H]Taurine also exhibited competitive inhibition with a Ki of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB.

  2. Selective serotonin re-uptake inhibitors (SSRIs) versus placebo for obsessive compulsive disorder (OCD).

    PubMed

    Soomro, G M; Altman, D; Rajagopal, S; Oakley-Browne, M

    2008-01-23

    Obsessive compulsive disorder is a common and disabling disorder. A significant proportion of patients manifest a chronic course. Individual randomised controlled trials (RCTs) have shown that selective serotonin re-uptake inhibitors (SSRIs) are effective in this condition. Previous systematic reviews or meta-analyses summarising the evidence are methodologically problematic or limited in the scope of their analysis. To examine the efficacy and adverse effects of serotonin re-uptake inhibitors (SSRIs) versus placebo for obsessive compulsive disorder (OCD) in adults. CCDANCTR-Studies and CCDANCTR-References were searched on 12/11/2007. Reference lists were checked. Experts in the field were contacted. All RCTs and quasi-RCTs examining the efficacy of SSRIs compared with placebo for OCD in adults were eligible for inclusion. Selection of studies and data extraction were carried out by two review authors independently, and quality assessment of studies was undertaken. Data analysis was conducted using Review Manager software. Summary measures were produced using the weighted mean difference (WMD) for continuous data and relative risk (RR) for dichotomous data, with 95% confidence intervals (CI). SSRIs were examined as an overall group of drugs, and as individual drugs. Seventeen studies were included in the review, involving 3097 participants. Based on all 17 studies, SSRIs as a group were more effective than placebo in reducing the symptoms of OCD between 6 and 13 weeks post-treatment, measured using the Yale-Brown Obsessive Compulsive Scale (YBOCS) (WMD -3.21, 95% CI -3.84 to -2.57). The WMD for individual SSRI drugs were similar and not statistically different. Based on 13 studies (2697 participants), SSRIs were more effective than placebo in achieving clinical response at post-treatment (RR 1.84, 95% CI 1.56 to 2.17). The pooled RR was shown to be similar between individual SSRI drugs. Although reported adverse effects data were more limited, with few exceptions

  3. Synaptic GABA release prevents GABA transporter type-1 reversal during excessive network activity

    PubMed Central

    Savtchenko, Leonid; Megalogeni, Maria; Rusakov, Dmitri A.; Walker, Matthew C.; Pavlov, Ivan

    2015-01-01

    GABA transporters control extracellular GABA, which regulates the key aspects of neuronal and network behaviour. A prevailing view is that modest neuronal depolarization results in GABA transporter type-1 (GAT-1) reversal causing non-vesicular GABA release into the extracellular space during intense network activity. This has important implications for GABA uptake-targeting therapies. Here we combined a realistic kinetic model of GAT-1 with experimental measurements of tonic GABAA receptor currents in ex vivo hippocampal slices to examine GAT-1 operation under varying network conditions. Our simulations predict that synaptic GABA release during network activity robustly prevents GAT-1 reversal. We test this in the 0 Mg2+ model of epileptiform discharges using slices from healthy and chronically epileptic rats and find that epileptiform activity is associated with increased synaptic GABA release and is not accompanied by GAT-1 reversal. We conclude that sustained efflux of GABA through GAT-1 is unlikely to occur during physiological or pathological network activity. PMID:25798861

  4. Neuronal gamma-aminobutyric acid (GABA) type A receptors undergo cognate ligand chaperoning in the endoplasmic reticulum by endogenous GABA

    PubMed Central

    Wang, Ping; Eshaq, Randa S.; Meshul, Charles K.; Moore, Cynthia; Hood, Rebecca L.; Leidenheimer, Nancy J.

    2015-01-01

    GABAA receptors mediate fast inhibitory neurotransmission in the brain. Dysfunction of these receptors is associated with various psychiatric/neurological disorders and drugs targeting this receptor are widely used therapeutic agents. Both the efficacy and plasticity of GABAA receptor-mediated neurotransmission depends on the number of surface GABAA receptors. An understudied aspect of receptor cell surface expression is the post-translational regulation of receptor biogenesis within the endoplasmic reticulum (ER). We have previously shown that exogenous GABA can act as a ligand chaperone of recombinant GABAA receptors in the early secretory pathway leading us to now investigate whether endogenous GABA facilitates the biogenesis of GABAA receptors in primary cerebral cortical cultures. In immunofluorescence labeling experiments, we have determined that neurons expressing surface GABAA receptors contain both GABA and its degradative enzyme GABA transaminase (GABA-T). Treatment of neurons with GABA-T inhibitors, a treatment known to increase intracellular GABA levels, decreases the interaction of the receptor with the ER quality control protein calnexin, concomittantly increasing receptor forward-trafficking and plasma membrane insertion. The effect of GABA-T inhibition on the receptor/calnexin interaction is not due to the activation of surface GABAA or GABAB receptors. Consistent with our hypothesis that GABA acts as a cognate ligand chaperone in the ER, immunogold-labeling of rodent brain slices reveals the presence of GABA within the rough ER. The density of this labeling is similar to that present in mitochondria, the organelle in which GABA is degraded. Lastly, the effect of GABA-T inhibition on the receptor/calnexin interaction was prevented by pretreatment with a GABA transporter inhibitor. Together, these data indicate that endogenous GABA acts in the rough ER as a cognate ligand chaperone to facilitate the biogenesis of neuronal GABAA receptors. PMID

  5. A multivalent inhibitor of the DC-SIGN dependent uptake of HIV-1 and Dengue virus.

    PubMed

    Varga, Norbert; Sutkeviciute, Ieva; Ribeiro-Viana, Renato; Berzi, Angela; Ramdasi, Rasika; Daghetti, Anna; Vettoretti, Gerolamo; Amara, Ali; Clerici, Mario; Rojo, Javier; Fieschi, Franck; Bernardi, Anna

    2014-04-01

    DC-SIGN is a C-type lectin receptor on antigen presenting cells (dendritic cells) which has an important role in some viral infection, notably by HIV and Dengue virus (DV). Multivalent presentation of carbohydrates on dendrimeric scaffolds has been shown to inhibit DC-SIGN binding to HIV envelope glycoprotein gp120, thus blocking viral entry. This approach has interesting potential applications for infection prophylaxis. In an effort to develop high affinity inhibitors of DC-SIGN mediated viral entry, we have synthesized a group of glycodendrimers of different valency that bear different carbohydrates or glycomimetic DC-SIGN ligands and have studied their DC-SIGN binding activity and antiviral properties both in an HIV and a Dengue infection model. Surface Plasmon Resonance (SPR) competition studies have demonstrated that the materials obtained bind efficiently to DC-SIGN with IC50s in the μm range, which depend on the nature of the ligand and on the valency of the scaffold. In particular, a hexavalent presentation of the DC-SIGN selective antagonist 4 displayed high potency, as well as improved accessibility and chemical stability relative to previously reported dendrimers. At low μm concentration the material was shown to block both DC-SIGN mediated uptake of DV by Raji cells and HIV trans-infection of T cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Localization of glycine, GABA and neuropeptide containing neurons in tiger salamander retina

    SciTech Connect

    Yang, C.Y.

    1988-01-01

    Putative glycinergic and GABAergic neurons in the salamander retina were localized by a parallel analysis of high affinity {sup 3}H-glycine uptake and glycine-like immunoreactivity (Gly-IR) and a comparative analysis of high affinity {sup 3}H-GABA uptake, GABA, like immunoreactivity (GABA-IR), and glutamate decarboxylase immunoreactivity (GAD-IR) at the light microscopic level. Good correspondence of labeling of {sup 3}H-glycine uptake and Gly-IR as well as that of {sup 3}H-GABA uptake and GABA-IR were observed. In addition, GAD immunoreactive neurons contained GABA-IR as well. Extensive colocalization of {sup 3}H-glycine uptake and Gly-IR and that of {sup 3}H-GABA uptake, GABA-IR and perhaps GAD-IR were indicated by the similarities in the distribution, morphology and labeling frequency of neurons and lamination in the inner plexiform layer (IPL). However, the Gly-IR and the GABA-IR probes appeared to be more sensitive and can thus be a reliable marker for glycine and GABA containing neurons respectively.

  7. GABA transporter deficiency causes tremor, ataxia, nervousness, and increased GABA-induced tonic conductance in cerebellum.

    PubMed

    Chiu, Chi-Sung; Brickley, Stephen; Jensen, Kimmo; Southwell, Amber; Mckinney, Sheri; Cull-Candy, Stuart; Mody, Istvan; Lester, Henry A

    2005-03-23

    GABA transporter subtype 1 (GAT1) knock-out (KO) mice display normal reproduction and life span but have reduced body weight (female, -10%; male, -20%) and higher body temperature fluctuations in the 0.2-1.5/h frequency range. Mouse GAT1 (mGAT1) KO mice exhibit motor disorders, including gait abnormality, constant 25-32 Hz tremor, which is aggravated by flunitrazepam, reduced rotarod performance, and reduced locomotor activity in the home cage. Open-field tests show delayed exploratory activity, reduced rearing, and reduced visits to the central area, with no change in the total distance traveled. The mGAT1 KO mice display no difference in acoustic startle response but exhibit a deficiency in prepulse inhibition. These open-field and prepulse inhibition results suggest that the mGAT1 KO mice display mild anxiety or nervousness. The compromised GABA uptake in mGAT1 KO mice results in an increased GABA(A) receptor-mediated tonic conductance in both cerebellar granule and Purkinje cells. The reduced rate of GABA clearance from the synaptic cleft is probably responsible for the slower decay of spontaneous IPSCs in cerebellar granule cells. There is little or no compensatory change in other proteins or structures related to GABA transmission in the mGAT1 KO mice, including GAT1-independent GABA uptake, number of GABAergic interneurons, and GABA(A)-, vesicular GABA transporter-, GAD65-, and GAT3-immunoreactive structures in cerebellum or hippocampus. Therefore, the excessive extracellular GABA present in mGAT1 KO mice results in behaviors that partially phenocopy the clinical side effects of tiagabine, suggesting that these side effects are inherent to a therapeutic strategy that targets the widely expressed GAT1 transporter system.

  8. Endogenous glutamate increases extracellular concentrations of dopamine, GABA, and taurine through NMDA and AMPA/kainate receptors in striatum of the freely moving rat: a microdialysis study.

    PubMed

    Segovia, G; Del Arco, A; Mora, F

    1997-10-01

    Interactions between glutamate (Glu), dopamine (DA), GABA, and taurine (Tau) were investigated in striatum of the freely moving rat by using microdialysis. Intrastriatal infusions of the selective Glu uptake inhibitor L-trans-pyrrolidine-3,4-dicarboxylic acid (PDC) were used to increase the endogenous extracellular [Glu]. Correlations between extracellular [Glu] and extracellular [DA], [GABA], and [Tau], and the effects of a selective blockade of ionotropic Glu receptors, were studied. PDC (1, 2, and 4 mM) produced a dose-related increase in extracellular [Glu]. At the highest dose of PDC, [Glu] increased from 1.55 +/- 0.35 to 6.11 +/- 0.88 microM. PDC also increased extracellular [DA], [GABA], and [Tau]. The increasing [Glu] was correlated significantly with increasing [DA], [GABA], and [Tau]. PDC also decreased extracellular concentrations of DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (HVA). Perfusion with the NMDA-receptor antagonist 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (1 mM) or the AMPA/kainate-receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) (1 mM) attenuated the increases produced by PDC (4 mM) on [DA], [GABA], and [Tau], and decreases in [DOPAC] and [HVA]. DNQX also attenuated the increases in [Glu] induced by PDC. These data show that endogenous Glu plays a role in modulating the extracellular concentrations of DA, GABA, and Tau in striatum of the freely moving rat.

  9. Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells

    SciTech Connect

    Saperstein, R.; Vicario, P.P.; Strout, H.V.; Brady, E.; Slater, E.E.; Greenlee, W.J.; Onedyka, D.L.; Patchett, A.A.; Hangauer, D.G. )

    1989-06-27

    An inhibitor of the insulin receptor tyrosine kinase (IRTK), (hydroxy-2-napthalenylmethyl)phosphonic acid, was designed and synthesized and was shown to be an inhibitor of the biological effects of insulin in vitro. With a wheat germ purified human placental insulin receptor preparation, this compound inhibited the insulin-stimulated autophosphorylation of the 95-kDa {beta}-subunit of the insulin receptor. The ability of the kinase to phosphorylate an exogenous peptide substrate, angiotensin II, was also inhibited. Half-maximal inhibition of basal and insulin-stimulated human placental IRTK activity was found at concentrations of 150 and 100 {mu}M, respectively, with 2 mM angiotensin II as the peptide substrate. The inhibitor was found to be specific for tyrosine kinases over serine kinases and noncompetitive with ATP. The inhibitor was converted into various (acyloxy)methyl prodrugs in order to achieve permeability through cell membranes. These prodrugs inhibited insulin-stimulated autophosphorylation of the insulin receptor 95-kDa {beta}-subunit in intact CHO cells transfected with human insulin receptor. Inhibition of insulin-stimulated glucose oxidation in isolated rat adipocytes and 2-deoxyglucose uptake into CHO cells was observed with these prodrugs. The data provide additional evidence for the involvement of the insulin receptor tyrosine kinase in the regulation of glucose uptake and metabolism. These results and additional data reported herein suggest that this class of prodrugs and inhibitors will be useful for modulating the activity of a variety of tyrosine kinases.

  10. Inhibitory Action of Antidepressants on Mouse Betaine/GABA Transporter (BGT1) Heterologously Expressed in Cell Cultures

    PubMed Central

    Gerile; Sogawa, Chiharu; Ohyama, Kazumi; Masuko, Takashi; Kusama, Tadashi; Morita, Katsuya; Sogawa, Norio; Kitayama, Shigeo

    2012-01-01

    Betaine/γ-aminobutyric acid (GABA) transporter (BGT1, SLC6A12) is a member of the Na+- and Cl−-dependent neurotransmitter transporter gene family with a homology to the GABA transporters (GATs), GAT1 (SLC6A1), GAT2 (SLC6A13) and GAT3 (SLC6A11) (HUGO nomenclature). Since antidepressants have been reported to inhibit GABA uptake, we examined those effects on mouse BGT1 (mBGT1) in comparison with other mouse GAT (mGAT) subtypes in the heterologously expressed cell cultures. All antidepressants tested here inhibited the [3H]GABA uptake through mBGT1 and mGATs in a rank order of potency with mBGT1 > mGAT1-3. Kinetic analyses for maprotilline, mianserine and trimipramine revealed that they inhibited mBGT1 and mGAT1 noncompetitively, except that mianserine competitively inhibited mBGT1. These results provided a clue to investigate the structure-function relationship of mBGT1 using antidepressants as a tool, leading to the identification of potential candidates for selective and specific inhibitors of mBGT1. PMID:22489112

  11. Neuronal release and successful astrocyte uptake of aminoacidergic neurotransmitters after spinal cord injury in lampreys.

    PubMed

    Fernández-López, Blanca; Valle-Maroto, Silvia María; Barreiro-Iglesias, Antón; Rodicio, María Celina

    2014-08-01

    In contrast to mammals, the spinal cord of lampreys spontaneously recovers from a complete spinal cord injury (SCI). Understanding the differences between lampreys and mammals in their response to SCI could provide valuable information to propose new therapies. Unique properties of the astrocytes of lampreys probably contribute to the success of spinal cord regeneration. The main aim of our study was to investigate, in the sea lamprey, the release of aminoacidergic neurotransmitters and the subsequent astrocyte uptake of these neurotransmitters during the first week following a complete SCI by detecting glutamate, GABA, glycine, Hu and cytokeratin immunoreactivities. This is the first time that aminoacidergic neurotransmitter release from neurons and the subsequent astrocytic response after SCI are analysed by immunocytochemistry in any vertebrate. Spinal injury caused the immediate loss of glutamate, GABA and glycine immunoreactivities in neurons close to the lesion site (except for the cerebrospinal fluid-contacting GABA cells). Only after SCI, astrocytes showed glutamate, GABA and glycine immunoreactivity. Treatment with an inhibitor of glutamate transporters (DL-TBOA) showed that neuronal glutamate was actively transported into astrocytes after SCI. Moreover, after SCI, a massive accumulation of inhibitory neurotransmitters around some reticulospinal axons was observed. Presence of GABA accumulation significantly correlated with a higher survival ability of these neurons. Our data show that, in contrast to mammals, astrocytes of lampreys have a high capacity to actively uptake glutamate after SCI. GABA may play a protective role that could explain the higher regenerative and survival ability of specific descending neurons of lampreys.

  12. Revisiting Serotonin Reuptake Inhibitors and the Therapeutic Potential of “Uptake-2” in Psychiatric Disorders

    PubMed Central

    2013-01-01

    Depression is among the most common psychiatric disorders, and in many patients a disorder for which available medications provide suboptimal or no symptom relief. The most commonly prescribed class of antidepressants, the selective serotonin reuptake inhibitors (SSRIs), are thought to act by increasing extracellular serotonin in brain by blocking its uptake via the high-affinity serotonin transporter (SERT). However, the relative lack of therapeutic efficacy of SSRIs has brought into question the utility of increasing extracellular serotonin for the treatment of depression. In this Viewpoint, we discuss why increasing extracellular serotonin should not be written off as a therapeutic strategy. We describe how “uptake-2” transporters may explain the relative lack of therapeutic efficacy of SSRIs, as well as why “uptake-2” transporters might be useful therapeutic targets. PMID:23336039

  13. Hazards and uptake of chitin synthesis inhibitors in bumblebees Bombus terrestris.

    PubMed

    Mommaerts, Veerle; Sterk, Guido; Smagghe, Guy

    2006-08-01

    This research project examined the potential hazards of a major class of insect growth regulators (IGRs) to survival, reproduction and larval growth in bumblebees Bombus terrestris L. Eight chitin synthesis inhibitors (CSIs) were tested: buprofezin, cyromazine, diflubenzuron, flucycloxuron, flufenoxuron, lufenuron, novaluron and teflubenzuron. These different IGRs, which are important in the control of pest insects in greenhouses, were applied via three different routes of exposure under laboratory conditions: dermal contact, and orally via the drinking of sugar/water and via pollen. The compounds were tested at their respective maximum field recommended concentrations (MFRC) and also in dose-response assays to calculate LC(50) values. In general, none of the CSIs showed acute worker toxicity. However, there was a dramatic reduction in brood production, especially after oral treatment with pollen and sugar/water. Conspicuously, egg fertility was reduced in all treatments with diflubenzuron and teflubenzuron. In addition to egg mortality, the worker bumblebees removed larvae from the treated nest, and in most cases these individuals were dead first-second instars. Under a binocular microscope, such larvae showed an abnormally formed cuticle leading to mechanical weakness and death. In another series of experiments using (14)C-diflubenzuron and (14)C-flufenoxuron, cuticular penetration in workers was studied for a better understanding of the differences in toxicity. With (14)C-diflubenzuron, transovarial transport and accumulation in the deposited eggs supported the strong reproductive effects. Overall, the present results suggest that CSIs should be applied with caution in combination with bumblebees. The compatibility of each compound to be used in combination with B. terrestris is discussed in relation to calculated LC(50) values, routes of uptake and effects.

  14. Differing effects of transport inhibitor on glutamate uptake by nerve terminals before and after exposure of rats to artificial gravity.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Krisanova, N.; Himmelreich, N.

    Glutamate is the major excitatory neurotransmitter in the brain. Subsequent to its release from glutamatergic neurons and activation of receptors, it is removed from extracellular space by high affinity Na^+-dependent glutamate transporters, which utilize the Na^+/K^+ electrochemical gradient as a driving force and located in nerve terminals and astrocytes. The glutamate transporters may modify the time course of synaptic events. Like glutamate itself, glutamate transporters are somehow involved in almost all aspects of normal and abnormal brain activity (e.g. cerebral ischemia, amyotrophic lateral sclerosis, Alzheimer's disease, traumatic brain injury, epilepsy and schizophrenia). The present study assessed transporter inhibitor for the ability to inhibit glutamate uptake by synaptosomes at the normal and hypergravity conditions (rats were rotated in a long-arm centrifuge at ten-G during one-hour period). DL-threo-beta-benzyloxyaspartate (DL-TBOA) is a newly developed competitive inhibitor of the high-affinity, Na^+-dependent glutamate transporters. As a potent, non- transported inhibitor of glutamate transporters, DL-TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms. We demonstrated that DL-TBOA inhibited glutamate uptake ( 100 μM glutamate, 30 sec incubation period) in dose-dependent manner as in control as in hypergravity. The effect of this transport inhibitor on glutamate uptake by control synaptosomes and synaptosomes prepared of animals exposed to hypergravity was different. IC50 values calculated on the basis of curves of non-linear regression kinetic analysis was 18±2 μM and 11±2 μM ((P≤0,05) before and after exposure to artificial gravity, respectively. Inhibition caused by 10 μM DL-TBOA was significantly increased from 38,0±3,8 % in control group to 51,0±4,1 % in animals, exposed to hypergravity (P≤0,05). Thus, DL-TBOA had complex effect on glutamate uptake process and perhaps, became more potent under

  15. Relative vulnerability of dopamine and GABA neurons in mesencephalic culture to inhibition of succinate dehydrogenase by malonate and 3-nitropropionic acid and protection by NMDA receptor blockade.

    PubMed

    Zeevalk, G D; Derr-Yellin, E; Nicklas, W J

    1995-12-01

    The effects of different severities of metabolic stress on dopamine (DA) and gamma-aminobutyric acid (GABA) cell loss were examined in rat mesencephalic culture. Partial metabolic inhibition was induced in 12-day-old cultures by a 24-hr treatment with various concentrations of 3-nitropropionic acid(3-NPA, 0.1-0.5 mM) or malonate (10-50 mM), irreversible and reversible inhibitors of the Krebs cycle enzyme, succinate dehydrogenase. Cell damage to the DA and GABA populations was assessed after a 48-hr recovery period by simultaneous measurement of high affinity uptake for 3H-DA and 14C-GABA. 3-NPA or malonate caused a dose-dependent loss of DA uptake (EC50 0.21 or 42 mM, respectively). 3-NPA treatment was equally detrimental to the GABA population, whereas malonate exposure did not cause any significant loss of GABA uptake. The presence of the NMDA antagonist, MK-801 (1 microM), during 24 hr of 3-NPA or malonate treatment fully protected against DA and GABA loss with 50 mM malonate or 0.25 mM 3-NPA and partially protected versus 0.5 mM 3-NPA. To determine the degree of metabolic stress imposed by 3-NPA and malonate, 12-day-old cultures were treated with 0.5 mM 3-NPA or 50 mM malonate for 3 hr and the rate of lactate formation was measured. lactate was increased nearly 2-fold at 3 hr of treatment with 3-NPA, but was not significantly elevated above basal with malonate treatment. SDH activity was decreased by 48 or 58% after 3 hr of treatment with 0.25 and 0.5 mM 3-NPA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. A mitochondrial GABA permease connects the GABA shunt and the TCA cycle, and is essential for normal carbon metabolism.

    PubMed

    Michaeli, Simon; Fait, Aaron; Lagor, Kelly; Nunes-Nesi, Adriano; Grillich, Nicole; Yellin, Ayelet; Bar, Dana; Khan, Munziba; Fernie, Alisdair R; Turano, Frank J; Fromm, Hillel

    2011-08-01

    In plants, γ-aminobutyric acid (GABA) accumulates in the cytosol in response to a variety of stresses. GABA is transported into mitochondria, where it is catabolized into TCA cycle or other intermediates. Although there is circumstantial evidence for mitochondrial GABA transporters in eukaryotes, none have yet been identified. Described here is an Arabidopsis protein similar in sequence and topology to unicellular GABA transporters. The expression of this protein complements a GABA-transport-deficient yeast mutant. Thus the protein was termed AtGABP to indicate GABA-permease activity. In vivo localization of GABP fused to GFP and immunobloting of subcellular fractions demonstrate its mitochondrial localization. Direct [(3) H]GABA uptake measurements into isolated mitochondria revealed impaired uptake into mitochondria of a gabp mutant compared with wild-type (WT) mitochondria, implicating AtGABP as a major mitochondrial GABA carrier. Measurements of CO(2) release, derived from radiolabeled substrates in whole seedlings and in isolated mitochondria, demonstrate impaired GABA-derived input into the TCA cycle, and a compensatory increase in TCA cycle activity in gabp mutants. Finally, growth abnormalities of gabp mutants under limited carbon availability on artificial media, and in soil under low light intensity, combined with their metabolite profiles, suggest an important role for AtGABP in primary carbon metabolism and plant growth. Thus, AtGABP-mediated transport of GABA from the cytosol into mitochondria is important to ensure proper GABA-mediated respiration and carbon metabolism. This function is particularly essential for plant growth under conditions of limited carbon.

  17. Neurochemical correlates of. gamma. -aminobutyrate (GABA) inhibition in cat visual cortex

    SciTech Connect

    Balcar, V.J.; Dreher, B. )

    1990-01-01

    High affinity binding of ({sup 3}H){gamma}-aminobutyric acid (GABA) to neuronal membranes from different parts of cat visual cortex was tested for sensitivity to GABA{sub A} agonists isoguvacine and THIP, GABA{sub A} antagonist SR95531 and GABA{sub B} agonist baclofen. Some of the GABA{sub A}-binding sites were found to have a very low affinity for THIP, suggesting the presence and, possibly, uneven distribution of non-synaptic GABA{sub A} receptors in cat visual cortex. There were no differences in K{sub m} and V{sub max} values of high affinity uptake of GABA and in the potency of K{sup +}-stimulated release of GABA, between primary and association cortices. Consequently, the present results indicate that despite the anatomical and physiological differences between the primary and association feline visual cortices the neurochemical characteristics of GABAergic inhibition are very similar in the two regions.

  18. Guineensine is a novel inhibitor of endocannabinoid uptake showing cannabimimetic behavioral effects in BALB/c mice.

    PubMed

    Nicolussi, Simon; Viveros-Paredes, Juan Manuel; Gachet, María Salomé; Rau, Mark; Flores-Soto, Mario Eduardo; Blunder, Martina; Gertsch, Jürg

    2014-02-01

    High-content screening led to the identification of the N-isobutylamide guineensine from Piper nigrum as novel nanomolar inhibitor (EC50=290nM) of cellular uptake of the endocannabinoid anandamide (AEA). Noteworthy, guineensine did not inhibit endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) nor interact with cannabinoid receptors or fatty acid binding protein 5 (FABP5), a major cytoplasmic AEA carrier. Activity-based protein profiling showed no inhibition of serine hydrolases. Guineensine also inhibited the cellular uptake of 2-arachidonoylglycerol (2-AG). Preliminary structure-activity relationships between natural guineensine analogs indicate the importance of the alkyl chain length interconnecting the pharmacophoric isobutylamide and benzodioxol moieties for AEA cellular uptake inhibition. Guineensine dose-dependently induced cannabimimetic effects in BALB/c mice shown by strong catalepsy, hypothermia, reduced locomotion and analgesia. The catalepsy and analgesia were blocked by the CB1 receptor antagonist rimonabant (SR141716A). Guineensine is a novel plant natural product which specifically inhibits endocannabinoid uptake in different cell lines independent of FAAH. Its scaffold may be useful to identify yet unknown targets involved in endocannabinoid transport. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Synthetic Methods for the Preparation of a Functional Analogue of Ru360, a Potent Inhibitor of Mitochondrial Calcium Uptake.

    PubMed

    Nathan, Sarah R; Pino, Nicholas W; Arduino, Daniela M; Perocchi, Fabiana; MacMillan, Samantha N; Wilson, Justin J

    2017-03-20

    The mixed-valent oxo-bridged ruthenium complex [(HCO2)(NH3)4Ru(μ-O)Ru(NH3)4(O2CH)](3+), known as Ru360, is a selective inhibitor of mitochondrial calcium uptake. Although this compound is useful for studying the role of mitochondrial calcium in biological processes, its widespread availability is limited because of challenges in purification and characterization. Here, we describe our investigations of three different synthetic methods for the preparation of a functional analogue of this valuable compound. We demonstrate that this analogue, isolated from our procedures, exhibits potent mitochondrial calcium uptake inhibitory properties in permeabilized HeLa cells and in isolated mitochondria.

  20. Loss of cortical GABA terminals in Unverricht-Lundborg disease.

    PubMed

    Buzzi, Andrea; Chikhladze, Maia; Falcicchia, Chiara; Paradiso, Beatrice; Lanza, Giovanni; Soukupova, Marie; Marti, Matteo; Morari, Michele; Franceschetti, Silvana; Simonato, Michele

    2012-08-01

    Unverricht-Lundborg disease (ULD) is the most common progressive myoclonic epilepsy. Its etiology has been identified in a defect of a protease inhibitor, cystatin B (CSTB), but the mechanism(s) by which this defect translates in the clinical manifestations of the disease are still obscure. We tested the hypothesis that ULD is accompanied by a loss of cortical GABA inhibition in a murine model (the CSTB knockout mouse) and in a human case. Cortical GABA signaling has been investigated measuring VGAT immunohistochemistry (a histological marker of the density of GABA terminals), GABA release from synaptosomes and paired-pulse stimulation. In CSTB knockout mice, a progressive decrease in neocortex thickness was found, associated with a prevalent loss of GABA interneurons. A marked reduction in VGAT labeling was found in the cortex of both CSTB knockout mice and an ULD patient. This implicates a reduction in GABA synaptic transmission, which was confirmed in the mouse model as reduction in GABA release from isolated nerve terminals and as loss of electrophysiologically measured GABA inhibition. The alterations in VGAT immunolabeling progressed in time, paralleling the worsening of myoclonus. These results provide direct evidence that loss of cortical GABA input occurs in a relevant animal model and in a case of human ULD, leading to a condition of latent hyperexcitability that favors myoclonus and seizures. These findings contribute to the understanding of the pathogenic mechanism of ULD and of the neurobiological basis of the effect of currently employed drugs.

  1. Effects of the monoamine uptake inhibitors RTI-112 and RTI-113 on cocaine- and food-maintained responding in rhesus monkeys.

    PubMed

    Negus, S S; Mello, N K; Kimmel, H L; Howell, L L; Carroll, F I

    2009-01-01

    Cocaine blocks uptake of the monoamines dopamine, serotonin and norepinephrine, and monoamine uptake inhibitors constitute one class of drugs under consideration as candidate "agonist" medications for the treatment of cocaine abuse and dependence. The pharmacological selectivity of monoamine uptake inhibitors to block uptake of dopamine, serotonin and norepinephrine is one factor that may influence the efficacy and/or safety of these compounds as drug abuse treatment medications. To address this issue, the present study compared the effects of 7-day treatment with a non-selective monoamine uptake inhibitor (RTI-112) and a dopamine-selective uptake inhibitor (RTI-113) on cocaine- and food-maintained responding in rhesus monkeys. Monkeys (N=3) were trained to respond for cocaine injections (0.01 mg/kg/inj) and food pellets under a second-order schedule [FR2(VR16:S)] during alternating daily components of cocaine and food availability. Both RTI-112 (0.0032-0.01 mg/kg/hr) and RTI-113 (0.01-0.056 mg/kg/h) produced dose-dependent, sustained and nearly complete elimination of cocaine self-administration. However, for both drugs, the potency to reduce cocaine self-administration was similar to the potency to reduce food-maintained responding. These findings do not support the hypothesis that pharmacological selectivity to block dopamine uptake is associated with behavioral selectivity to decrease cocaine- vs. food-maintained responding in rhesus monkeys.

  2. Manganese exposure inhibits the clearance of extracellular GABA and influences taurine homeostasis in the striatum of developing rats

    PubMed Central

    Fordahl, Steve C.; Anderson, Joel G.; Cooney, Paula T.; Weaver, Tara L.; Colyer, Christa L.; Erikson, Keith M.

    2010-01-01

    Manganese (Mn) accumulation in the brain has been shown to alter the neurochemistry of the basal ganglia. Mn-induced alterations in dopamine biology are fairly well understood, but recently more evidence has emerged characterizing the role of γ-aminobutyric acid (GABA) in this dysfunction. The purpose of this study was to determine if the previously observed Mn-induced increase in extracellular GABA (GABAEC) was due to altered GABA transporter (GAT) function, and whether Mn perturbs other amino acid neurotransmitters, namely taurine and glycine (known modulators of GABA). Extracellular GABA, taurine, and glycine concentrations were collected from the striatum of control (CN) or Mn-exposed Sprague-Dawley rats using in vivo microdialysis, and the GAT inhibitor nipecotic acid (NA) was used to probe GAT function. Tissue and extracellular Mn levels were significantly increased, and the Fe:Mn ratio was decreased 36-fold in the extracellular space due to Mn exposure. NA led to a 2-fold increase in GABAEC of CNs, a response that was attenuated by Mn. Taurine responded inversely to GABA, and a novel 10-fold increase in taurine was observed after the removal of NA in CNs. Mn blunted this response and nearly abolished extracellular taurine throughout collection. Striatal taurine transporter (Slc6a6) mRNA levels were significantly increased with Mn exposure, and Mn significantly increased 3H-Taurine uptake after 3-minute exposure in primary rat astrocytes. These data suggest that Mn increases GABAEC by inhibiting the function of GAT, and that perturbed taurine homeostasis potentially impacts neural function by jeopardizing the osmoregulatory and neuromodulatory functions of taurine in the brain. PMID:20832424

  3. GABA predicts visual intelligence.

    PubMed

    Cook, Emily; Hammett, Stephen T; Larsson, Jonas

    2016-10-06

    Early psychological researchers proposed a link between intelligence and low-level perceptual performance. It was recently suggested that this link is driven by individual variations in the ability to suppress irrelevant information, evidenced by the observation of strong correlations between perceptual surround suppression and cognitive performance. However, the neural mechanisms underlying such a link remain unclear. A candidate mechanism is neural inhibition by gamma-aminobutyric acid (GABA), but direct experimental support for GABA-mediated inhibition underlying suppression is inconsistent. Here we report evidence consistent with a global suppressive mechanism involving GABA underlying the link between sensory performance and intelligence. We measured visual cortical GABA concentration, visuo-spatial intelligence and visual surround suppression in a group of healthy adults. Levels of GABA were strongly predictive of both intelligence and surround suppression, with higher levels of intelligence associated with higher levels of GABA and stronger surround suppression. These results indicate that GABA-mediated neural inhibition may be a key factor determining cognitive performance and suggests a physiological mechanism linking surround suppression and intelligence. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  4. Co-Localization of GABA Shunt Enzymes for the Efficient Production of Gamma-Aminobutyric Acid via GABA Shunt Pathway in Escherichia coli.

    PubMed

    Pham, Van Dung; Somasundaram, Sivachandiran; Park, Si Jae; Lee, Seung Hwan; Hong, Soon Ho

    2016-04-28

    Gamma-aminobutyric acid (GABA) is a non-protein amino acid, which is an important inhibitor of neurotransmission in the human brain. GABA is also used as the precursor of biopolymer Nylon-4 production. In this study, the carbon flux from the tricarboxylic acid cycle was directed to the GABA shunt pathway for the production of GABA from glucose. The GABA shunt enzymes succinate-semialdehyde dehydrogenase (GabD) and GABA aminotransferase (GabT) were co-localized along with the GABA transporter (GadC) by using a synthetic scaffold complex. The co-localized enzyme scaffold complex produced 0.71 g/l of GABA from 10 g/l of glucose. Inactivation of competing metabolic pathways in mutant E. coli strains XBM1 and XBM6 increased GABA production 13% to reach 0.80 g/l GABA by the enzymes co-localized and expressed in the mutant strains. The recombinant E. coli system developed in this study demonstrated the possibility of the pathway of the GABA shunt as a novel GABA production pathway.

  5. The Janus kinase 2 inhibitor fedratinib inhibits thiamine uptake: a putative mechanism for the onset of Wernicke's encephalopathy.

    PubMed

    Zhang, Qiang; Zhang, Yan; Diamond, Sharon; Boer, Jason; Harris, Jennifer J; Li, Yu; Rupar, Mark; Behshad, Elham; Gardiner, Christine; Collier, Paul; Liu, Phillip; Burn, Timothy; Wynn, Richard; Hollis, Gregory; Yeleswaram, Swamy

    2014-10-01

    The clinical development of fedratinib, a Janus kinase (JAK2) inhibitor, was terminated after reports of Wernicke's encephalopathy in myelofibrosis patients. Since Wernicke's encephalopathy is induced by thiamine deficiency, investigations were conducted to probe possible mechanisms through which fedratinib may lead to a thiamine-deficient state. In vitro studies indicate that fedratinib potently inhibits the carrier-mediated uptake and transcellular flux of thiamine in Caco-2 cells, suggesting that oral absorption of dietary thiamine is significantly compromised by fedratinib dosing. Transport studies with recombinant human thiamine transporters identified the individual human thiamine transporter (hTHTR2) that is inhibited by fedratinib. Inhibition of thiamine uptake appears unique to fedratinib and is not shared by marketed JAK inhibitors, and this observation is consistent with the known structure-activity relationship for the binding of thiamine to its transporters. The results from these studies provide a molecular basis for the development of Wernicke's encephalopathy upon fedratinib treatment and highlight the need to evaluate interactions of investigational drugs with nutrient transporters in addition to classic xenobiotic transporters.

  6. Selective serotonin re-uptake inhibitors potentiate gene blunting induced by repeated methylphenidate treatment: Zif268 versus Homer1a.

    PubMed

    Van Waes, Vincent; Vandrevala, Malcolm; Beverley, Joel; Steiner, Heinz

    2014-11-01

    There is a growing use of psychostimulants, such as methylphenidate (Ritalin; dopamine re-uptake inhibitor), for medical treatments and as cognitive enhancers in the healthy. Methylphenidate is known to produce some addiction-related gene regulation. Recent findings in animal models show that selective serotonin re-uptake inhibitors (SSRIs), including fluoxetine, can potentiate acute induction of gene expression by methylphenidate, thus indicating an acute facilitatory role for serotonin in dopamine-induced gene regulation. We investigated whether repeated exposure to fluoxetine, in conjunction with methylphenidate, in adolescent rats facilitated a gene regulation effect well established for repeated exposure to illicit psychostimulants such as cocaine-blunting (repression) of gene inducibility. We measured, by in situ hybridization histochemistry, the effects of a 5-day repeated treatment with methylphenidate (5 mg/kg), fluoxetine (5 mg/kg) or a combination on the inducibility (by cocaine) of neuroplasticity-related genes (Zif268, Homer1a) in the striatum. Repeated methylphenidate treatment alone produced minimal gene blunting, while fluoxetine alone had no effect. In contrast, fluoxetine added to methylphenidate robustly potentiated methylphenidate-induced blunting for both genes. This potentiation was widespread throughout the striatum, but was most robust in the lateral, sensorimotor striatum, thus mimicking cocaine effects. For illicit psychostimulants, blunting of gene expression is considered part of the molecular basis of addiction. Our results thus suggest that SSRIs, such as fluoxetine, may increase the addiction liability of methylphenidate.

  7. Localization and expression of GABA transporters in the suprachiasmatic nucleus

    PubMed Central

    Moldavan, Michael; Cravetchi, Olga; Williams, Melissa; Irwin, Robert P.; Aicher, Sue A.; Allen, Charles N.

    2015-01-01

    GABA is a principal neurotransmitter in the suprachiasmatic hypothalamic nucleus (SCN), the master circadian clock. Despite the importance of GABA and GABA uptake for functioning of the circadian pacemaker, the localization and expression of GABA transporters (GATs) in the SCN has not been investigated. The present studies used Western blot analysis, immunohistochemistry, and electron microscopy to demonstrate the presence of GABA transporter 1 (GAT1) and GABA transporter 3 (GAT3) in the SCN. By light microscopy, GAT1 and GAT3 were co-localized throughout the SCN, but were not expressed in the perikarya of arginine vasopressin- or vasoactive intestinal peptide-immunoreactive (−ir) neurons of adult rats, nor in the neuronal processes labeled with the Neurofilament Heavy Chain. By electron microscopy, GAT1- and GAT3-ir was found in glial processes surrounding unlabeled neuronal perikarya, axons, dendrites, and enveloped symmetric and asymmetric axo-dendritic synapses. Glial Fibrillary Acidic Protein-ir astrocytes grown in cell culture were immunopositive for GAT1 and GAT3 – and both GATs could be observed in the same glial cell. These data demonstrate that synapses in the SCN function as “tripartite” synapses consisting of presynaptic axon terminals, postsynaptic membranes, and astrocytes that contain GABA transporters. This model suggests that astrocytes expressing both GATs may regulate the extracellular GABA, and thereby modulate the activity of neuronal networks in the SCN. PMID:26390912

  8. Acute psychological effects of 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") are attenuated by the serotonin uptake inhibitor citalopram.

    PubMed

    Liechti, M E; Baumann, C; Gamma, A; Vollenweider, F X

    2000-05-01

    3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") is a recreational drug that has been shown to release serotonin (5-HT) and dopamine (DA) in animals. The effect of MDMA on 5-HT release can be blocked by 5-HT uptake inhibitors such as citalopram, suggesting that MDMA interacts with the 5-HT uptake site. It is unknown whether this mechanism is also responsible for the psychological effects of MDMA in humans. We investigated the effect of citalopram pretreatment (40 mg iv) on the psychological effects of MDMA (1.5 mg/kg po) in a double-blind placebo-controlled psychometric study in 16 healthy human volunteers. MDMA produced an emotional state with heightened mood, increased self-confidence and extroversion, moderate derealization, and an intensification of sensory perception. Most of these effects were markedly reduced by citalopram. This finding suggests that the psychological effects of MDMA are mediated via action at the 5-HT uptake site to increase 5-HT release through the carrier, as expected from animal studies.

  9. Non-carrier-mediated uptake of the mannosidase I inhibitor 1-deoxymannojirimycin by K562 erythroleukemic cells

    SciTech Connect

    Neefjes, J.J.; Lindhout, J.; Broxterman, H.J.; van der Marel, G.A.; van Boom, J.H.; Ploegh, H.L.

    1989-06-15

    A /sup 3/H label was introduced at the C-1 position of the mannosidase I inhibitor 1-deoxymannojirimycin (dMM) by catalytic hydrogenolysis of benzyl-2,3-O-isopropylidene-5-N-benzyl-6-O-benzyl-alpha-D-mannofurano side with /sup 3/H/sub 2/. 1-(/sup 3/H)dMM as well as its precursor 1-(/sup 3/H)2,3-O-isopropylidene-dMM had identical Rf as the nonradioactive compounds on TLC. Furthermore, alpha 1-antitrypsin secreted by HepG2 cells was modified indistinguishably by treatment of the cells with dMM and 1-(/sup 3/H)dMM. Thus, 1-(/sup 3/H)dMM had chemical and biological properties identical with authentic dMM. Uptake of (/sup 14/C)mannose by K562 cells could be inhibited by glucose but not by the mannose analogue dMM. Thus, dMM does not enter the cell through hexose transporter(s). Uptake of 1-(/sup 3/H)dMM by K562 cells could not be inhibited by increasing concentrations of nonradioactive dMM (from 1-32,000 microM), showing transport of dMM into cells through nonfacilitated diffusion. Furthermore, uptake of 1-(/sup 3/H)dMM by K562 cells was observed at 0/degree/C.

  10. The effect of modifying rooting depths and nitrification inhibitors on nutrient uptake from organic biogas residues in maize

    NASA Astrophysics Data System (ADS)

    Dietrich, Charlotte C.; Koller, Robert; Nagel, Kerstin A.; Schickling, Anke; Schrey, Silvia D.; Jablonowski, Nicolai D.

    2017-04-01

    Optimizing the application of and nutrient uptake from organic nutrient sources, such as the nutrient-rich residues ("digestates") from the biogas industry, is becoming a viable option in remediating fertility on previously unsuitable soils for agricultural utilization. Proposedly, concurrent changes in root system architecture and functioning could also serve as the basis of future phytomining approaches. Herein, we evaluate the effect of spatial nutrient availability and nitrification on maize root architecture and nutrient uptake. We test these effects by applying maize-based digestate at a rate of 170 kg/ha in layers of varying depths (10, 25 and 40 cm) and through either the presence or absence of nitrification inhibitors. In order to regularly monitor above- and below-ground plant biomass production, we used the noninvasive phenotyping platform, GROWSCREEN-Rhizo at the Forschungszentrum Jülich, using rhizotrons (Nagel et al., 2012). Measured parameters included projected plant height and leaf area, as well as root length and spatial distribution. Additionally, root diameters were quantified after the destructive harvest, 21 days after sowing (DAS). Spatial nutrient availability significantly affected root system architecture, as for example root system size -the area occupied by roots- increased alongside nutrient layer depths. Fertilization also positively affected root length density (RLD). Within fertilized layers, the presence of nitrification inhibitors increased RLD by up to 30% and was most pronounced in the fine root biomass fraction (0.1 to 0.5mm). Generally, nitrification inhibitors promoted early plant growth by up to 45% across treatments. However, their effect varied in dependence of layer depths, leading to a time-delayed response in deeper layers, accounting for plants having to grow significantly longer roots in order to reach fertilized substrate. Nitrification inhibitors also initiated the comparatively early on-set of growth differences in

  11. The effect of selective 5-hydroxytryptamine uptake inhibitors on 5-methoxy-N,N-dimethyltryptamine-induced ejaculation in the rat.

    PubMed Central

    Rényi, L.

    1986-01-01

    The ejaculatory response and the 5-hydroxytryptamine (5-HT) behavioural syndrome induced by 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) (3 mg kg-1 i.p.) were studied following acute and repeated treatment of rats with the selective uptake inhibitors of 5-HT, fluoxetine, zimeldine, alaproclate, and citalopram. The oral doses used were based on the respective ED50 values for uptake inhibition. Acute doses of fluoxetine and zimeldine significantly reduced the ejaculatory response when given 48 h before 5-MeODMT. This blockade was prevented by treatment of the rats with the postsynaptic 5-HT receptor antagonist methergoline. An acute dose of fluoxetine given 7 and 14 days before 5-MeODMT significantly enhanced the ejaculatory response. On day 24, the response returned to the control level. Repeated treatment every second day (5 times over 9 days and 10 times over 19 days) with fluoxetine caused a longer blockade of the ejaculatory response and the sensitization of the response came later than after an acute dose. Parallel with the ejaculatory response three other components of the 5-HT behavioural syndrome also decreased significantly. Acute doses of alaproclate and citalopram significantly blocked the ejaculatory response at 1 h, but they failed to affect the response at any other time point after either acute or repeated treatment. Neither did these drugs attentuate the 5-HT syndrome. It is concluded that acute and repeated treatment of rats with different selective 5-HT uptake inhibitors does not produce a common alteration in 5-HT2-receptor functions. PMID:2939912

  12. Interaction between neuronal uptake inhibitors and presynaptic serotonin autoreceptors in rat hypothalamic slices: comparison of K+ and electrical depolarization.

    PubMed

    Passarelli, F; Galzin, A M; Langer, S Z

    1987-09-01

    In rat hypothalamic slices prelabeled with [3H]-5-hydroxytryptamine ([3H]-5-HT), exposure to the 5-HT receptor agonist lysergic acid diethylamide (0.1-1 microM) or 5-methoxytryptamine (0.1-10 microM) decreased in a concentration-dependent manner the release of 3H-transmitter elicited by high K+ or electrical stimulation. Exposure to the 5-HT autoreceptor antagonist methiothepin (0.1-1 microM) increased in a concentration-dependent manner the K+ stimulation-evoked overflow of [3H]-5-HT and a similar increase was observed under conditions of electrical stimulation. In contrast, exposure to the nontricyclic 5-HT uptake inhibitor citalopram (0.1-1 microM) did not modify by itself the electrically evoked overflow of [3H]-5-HT, but increased in a concentration-dependent manner the release of 3H-transmitter elicited by K+ stimulation. This effect of citalopram on transmitter release was potentiated when the endogenous stores of 5-HT were depleted by pretreatment with para-chlorophenylalanine methyl ester (300 mg/kg i.p.). Citalopram was shown previously to antagonize the inhibition by lysergic acid diethylamide of the electrically evoked release of [3H]-5-HT in rat hypothalamic slices. Yet, this inhibitor of neuronal uptake of 5-HT did not antagonize the effects of lysergic acid diethylamide when the release of [3H]-5-HT was evoked by K+ depolarization. Electrical stimulation represents a more physiological experimental model for transmitter release than exposure to high K+, and therefore the interaction between 5-HT uptake blockade and presynaptic inhibitory 5-HT autoreceptors, observed in the hypothalamus with electrical stimulation but not with K+ depolarization, remains of biological relevance.

  13. The Urease Inhibitor NBPT Negatively Affects DUR3-mediated Uptake and Assimilation of Urea in Maize Roots.

    PubMed

    Zanin, Laura; Tomasi, Nicola; Zamboni, Anita; Varanini, Zeno; Pinton, Roberto

    2015-01-01

    Despite the widespread use of urease inhibitors in agriculture, little information is available on their effect on nitrogen (N) uptake and assimilation. Aim of this work was to study, at physiological and transcriptional level, the effects of N-(n-butyl) thiophosphoric triamide (NBPT) on urea nutrition in hydroponically grown maize plants. Presence of NBPT in the nutrient solution limited the capacity of plants to utilize urea as a N-source; this was shown by a decrease in urea uptake rate and (15)N accumulation. Noteworthy, these negative effects were evident only when plants were fed with urea, as NBPT did not alter (15)N accumulation in nitrate-fed plants. NBPT also impaired the growth of Arabidopsis plants when urea was used as N-source, while having no effect on plants grown with nitrate or ammonium. This response was related, at least in part, to a direct effect of NBPT on the high affinity urea transport system. Impact of NBPT on urea uptake was further evaluated using lines of Arabidopsis overexpressing ZmDUR3 and dur3-knockout; results suggest that not only transport but also urea assimilation could be compromised by the inhibitor. This hypothesis was reinforced by an over-accumulation of urea and a decrease in ammonium concentration in NBPT-treated plants. Furthermore, transcriptional analyses showed that in maize roots NBPT treatment severely impaired the expression of genes involved in the cytosolic pathway of ureic-N assimilation and ammonium transport. NBPT also limited the expression of a gene coding for a transcription factor highly induced by urea and possibly playing a crucial role in the regulation of its acquisition. This work provides evidence that NBPT can heavily interfere with urea nutrition in maize plants, limiting influx as well as the following assimilation pathway.

  14. The Urease Inhibitor NBPT Negatively Affects DUR3-mediated Uptake and Assimilation of Urea in Maize Roots

    PubMed Central

    Zanin, Laura; Tomasi, Nicola; Zamboni, Anita; Varanini, Zeno; Pinton, Roberto

    2015-01-01

    Despite the widespread use of urease inhibitors in agriculture, little information is available on their effect on nitrogen (N) uptake and assimilation. Aim of this work was to study, at physiological and transcriptional level, the effects of N-(n-butyl) thiophosphoric triamide (NBPT) on urea nutrition in hydroponically grown maize plants. Presence of NBPT in the nutrient solution limited the capacity of plants to utilize urea as a N-source; this was shown by a decrease in urea uptake rate and 15N accumulation. Noteworthy, these negative effects were evident only when plants were fed with urea, as NBPT did not alter 15N accumulation in nitrate-fed plants. NBPT also impaired the growth of Arabidopsis plants when urea was used as N-source, while having no effect on plants grown with nitrate or ammonium. This response was related, at least in part, to a direct effect of NBPT on the high affinity urea transport system. Impact of NBPT on urea uptake was further evaluated using lines of Arabidopsis overexpressing ZmDUR3 and dur3-knockout; results suggest that not only transport but also urea assimilation could be compromised by the inhibitor. This hypothesis was reinforced by an over-accumulation of urea and a decrease in ammonium concentration in NBPT-treated plants. Furthermore, transcriptional analyses showed that in maize roots NBPT treatment severely impaired the expression of genes involved in the cytosolic pathway of ureic-N assimilation and ammonium transport. NBPT also limited the expression of a gene coding for a transcription factor highly induced by urea and possibly playing a crucial role in the regulation of its acquisition. This work provides evidence that NBPT can heavily interfere with urea nutrition in maize plants, limiting influx as well as the following assimilation pathway. PMID:26635834

  15. Extracellular conversion of guanine-based purines to guanosine specifically enhances astrocyte glutamate uptake.

    PubMed

    Frizzo, Marcos Emílio dos Santos; Antunes Soares, Félix Alexandre; Dall'Onder, Leonara Patrícia; Lara, Diogo Rizzato; Swanson, Raymond A; Souza, Diogo Onofre

    2003-05-16

    Guanosine (GUO) has been shown to stimulate glutamate uptake in primary astrocyte cultures. The purpose of this study was to determine the effect and specificity of guanine- or adenine-based purines on glutamate and GABA uptake in cultured astrocytes. Stimulatory effect on glutamate uptake was observed with GUO, GMP or GTP. Simultaneous exposure with these guanine-based purines did not show an additive effect. We also investigated a possible interconversion of guanine-based purines during incubation time. Action by GTP was excluded since the hydrolysis resistant GTP analog, GMP-PNP did not stimulate glutamate uptake. Addition of an ecto-5'-nucleotidase inhibitor abolished GMP-stimulatory effect on glutamate uptake, without affecting GUO action. Taken together, these results suggest that GUO is the guanine-based purines responsible for glutamate uptake activation. In addition, the stimulatory effect on glutamate uptake was not observed with adenine-based purines. Moreover, GABA uptake was not activated by GUO. These results point to specificity in the interaction between GUO and the astrocyte glutamate uptake system.

  16. Enhancement of muscle glucose uptake by the vasopeptidase inhibitor, omapatrilat, is independent of insulin signaling and the AMP kinase pathway.

    PubMed

    Wong, Victor; Szeto, Linda; Uffelman, Kristine; Fantus, I George; Lewis, Gary F

    2006-08-01

    Omapatrilat (OMA), a vasopeptidase inhibitor (VPI), presently being tested in clinical trials for its antihypertensive properties, inhibits both angiotensin-converting enzyme and neutral endopeptidase, and raises tissue bradykinin levels. Recent studies from our laboratory and those of others have demonstrated that VPIs enhance muscle glucose uptake in animal models, and this effect is mediated by the bradykinin-nitric oxide pathway. The mechanism of the effect of OMA on muscle glucose uptake, however, is presently unknown. To investigate the effect of OMA on insulin signaling, soleus muscle was isolated 2 or 5 min after an i.v. bolus of insulin or saline from male Zucker fatty rats (8-10 weeks of age), following a 5-day treatment period of oral OMA (15 mg/kg per day) or drug vehicle (placebo). OMA resulted in significantly lower systolic blood pressure compared with the placebo-treated group (84.4+/- 7.52 mmHg in OMA vs 112+/-2.18 mmHg in controls, P<0.01). Immunoprecipitation and Western blot analysis of insulin receptor substrate 1 (IRS-1) revealed no changes in protein mass with OMA treatment. OMA did not enhance basal or insulin-stimulated IRS-1 tyrosine phosphorylation or its subsequent association with the p85 regulatory subunit of phosphatidylinositol 3-kinase. Under basal and insulin-stimulated conditions, OMA treatment did not alter the protein mass or the phosphorylation of Akt/protein kinase B, p42/44 extracellular signal-regulated kinase or adenosine monophosphate-activated protein kinase, or GLUT4 protein expression. We conclude that the ability of OMA to enhance whole body and specifically muscle glucose uptake in Zucker fatty rats is not mediated by enhancing insulin or AMPK signaling. Future studies should examine whether hemodynamic effects of the drug, independent of insulin signaling, enhance glucose uptake in insulin-resistant skeletal muscle.

  17. Protease inhibitors to treat hepatitis C in the Swiss HIV Cohort Study: high efficacy but low treatment uptake.

    PubMed

    Schaerer, V; Haubitz, S; Kovari, H; Ledergerber, B; Ambrosioni, J; Cavassini, M; Stoeckle, M; Schmid, P; Decosterd, L; Aouri, M; Böni, J; Günthard, H F; Furrer, H; Metzner, K J; Fehr, J; Rauch, A

    2015-11-01

    Direct-acting antiviral agents (DAAs) have become the standard of care for the treatment of chronic hepatitis C virus (HCV) infection. We aimed to assess treatment uptake and efficacy in routine clinical settings among HIV/HCV coinfected patients after the introduction of the first generation DAAs. Data on all Swiss HIV Cohort Study (SHCS) participants starting HCV protease inhibitor (PI) treatment between September 2011 and August 2013 were collected prospectively. The uptake and efficacy of HCV therapy were compared with those in the time period before the availability of PIs. Upon approval of PI treatment in Switzerland in September 2011, 516 SHCS participants had chronic HCV genotype 1 infection. Of these, 57 (11%) started HCV treatment during the following 2 years with either telaprevir, faldaprevir or boceprevir. Twenty-seven (47%) patients were treatment-naïve, nine (16%) were patients with relapse and 21 (37%) were partial or null responders. Twenty-nine (57%) had advanced fibrosis and 15 (29%) had cirrhosis. End-of-treatment virological response was 84% in treatment-naïve patients, 88% in patients with relapse and 62% in previous nonresponders. Sustained virological response was 78%, 86% and 40% in treatment-naïve patients, patients with relapse and nonresponders, respectively. Treatment uptake was similar before (3.8 per 100 patient-years) and after (6.1 per 100 patient-years) the introduction of PIs, while treatment efficacy increased considerably after the introduction of PIs. The introduction of PI-based HCV treatment in HIV/HCV-coinfected patients improved virological response rates, while treatment uptake remained low. Therefore, the introduction of PIs into the clinical routine was beneficial at the individual level, but had only a modest effect on the burden of HCV infection at the population level. © 2015 British HIV Association.

  18. Different transporter systems regulate extracellular GABA from vesicular and non-vesicular sources

    PubMed Central

    Song, Inseon; Volynski, Kirill; Brenner, Tanja; Ushkaryov, Yuri; Walker, Matthew; Semyanov, Alexey

    2013-01-01

    Tonic GABA type A (GABAA) conductance is a key factor regulating neuronal excitability and computation in neuronal networks. The magnitude of the tonic GABAA conductance depends on the concentration of ambient GABA originating from vesicular and non-vesicular sources and is tightly regulated by GABA uptake. Here we show that the transport system regulating ambient GABA responsible for tonic GABAA conductances in hippocampal CA1 interneurons depends on its source. In mice, GABA from vesicular sources is regulated by mouse GABA transporter 1 (mGAT1), while that from non-vesicular sources by mouse GABA transporters 3/4 (mGAT3/4). This finding suggests that the two transporter systems do not just provide backup for each other, but regulate distinct signaling pathways. This allows individual tuning of the two signaling systems and indicates that drugs designed to act at specific transporters will have distinct therapeutic actions. PMID:23494150

  19. Treating depression in coronary artery disease and chronic heart failure: what's new in using selective serotonin re-uptake inhibitors?

    PubMed

    Paraskevaidis, Ioannis; Palios, John; Parissis, John; Filippatos, Gerasimos; Anastasiou-Nana, Maria

    2012-06-01

    Depression is a common co-morbidity in patients with cardiovascular diseases such as chronic coronary artery disease, acute coronary syndromes, post by-pass surgery and chronic heart failure. The presence of depression is independently associated with a decline in health status and an increase in the risk of hospitalization and death for patients with coronary artery disease or congestive heart failure. Novel treatment modalities such as selective serotonin re-uptake inhibitors (SSRIs) may improve depressive symptoms and prognosis of post-myocardial infarction and heart failure patients interacting with the common pathophysiological mechanisms of depression and cardiovascular disease. This review summarizes current experimental and clinical evidence regarding the effects of SSRIs on platelet functions, immune and neurohormonal activation, and cardiac rhythm disturbances in patients with cardiovascular disease.

  20. Modulation of GABA-stimulated chloride influx into membrane vesicles from rat cerebral cortex by triazolobenzodiazepines

    SciTech Connect

    Obata, T.; Yamamura, H.I.

    1988-01-01

    The effects of triazolobenzodiazepines of GABA-stimulated /sup 36/Cl/sup -/ uptake by membrane vesicles from rat cerebral cortex were examined. Triazolam and alprazolam showed a significant enhancement of GABA-stimulated /sup 36/Cl/sup -/ uptake at 0.01-10 uM. On the other hand, adinazolam showed a small enhancement at 0.1-1 uM followed by a significant inhibition of GABA-stimulated /sup 36/Cl/sup -/ uptake at 100 uM. The enhancement of GABA-stimulated /sup 36/Cl/sup -/ uptake by 1 uM alprazolam was antagonized by Ro15-1788, a benzodiazepine antagonist, but the inhibition of this response by 30 uM adinazolam was not antagonized by Ro15-1788. These results indicate that triazolobenzodiazepines enhanced GABA-stimulated /sup 36/Cl/sup -/ uptake through benzodiazepine receptors. High concentrations of adinazolam inhibit GABA-stimulated /sup 36/Cl/sup -/ uptake which may be due to the direct blockade of GABA-gated chloride channel. 23 references, 4 figures.

  1. GABA predicts time perception.

    PubMed

    Terhune, Devin B; Russo, Sonia; Near, Jamie; Stagg, Charlotte J; Cohen Kadosh, Roi

    2014-03-19

    Our perception of time constrains our experience of the world and exerts a pivotal influence over a myriad array of cognitive and motor functions. There is emerging evidence that the perceived duration of subsecond intervals is driven by sensory-specific neural activity in human and nonhuman animals, but the mechanisms underlying individual differences in time perception remain elusive. We tested the hypothesis that elevated visual cortex GABA impairs the coding of particular visual stimuli, resulting in a dampening of visual processing and concomitant positive time-order error (relative underestimation) in the perceived duration of subsecond visual intervals. Participants completed psychophysical tasks measuring visual interval discrimination and temporal reproduction and we measured in vivo resting state GABA in visual cortex using magnetic resonance spectroscopy. Time-order error selectively correlated with GABA concentrations in visual cortex, with elevated GABA associated with a rightward horizontal shift in psychometric functions, reflecting a positive time-order error (relative underestimation). These results demonstrate anatomical, neurochemical, and task specificity and suggest that visual cortex GABA contributes to individual differences in time perception.

  2. Cerebral uptake of mefloquine enantiomers with and without the P-gp inhibitor elacridar (GF1210918) in mice

    PubMed Central

    de Lagerie, Sylvie Barraud; Comets, Emmanuelle; Gautrand, Céline; Fernandez, Christine; Auchere, Daniel; Singlas, Eric; Mentre, France; Gimenez, François

    2004-01-01

    Mefloquine is a chiral neurotoxic antimalarial agent showing stereoselective brain uptake in humans and rats. It is a substrate and an inhibitor of the efflux protein P-glycoprotein. We investigated the stereoselective uptake and efflux of mefloquine in mice, and the consequences of the combination with an efflux protein inhibitor, elacridar (GF120918) on its brain transport. Racemic mefloquine (25 mg kg−1) was administered intraperitoneally with or without elacridar (10 mg kg−1). Six to seven mice were killed at each of 11 time-points between 30 min and 168 h after administration. Blood and brain concentrations of mefloquine enantiomers were determined using liquid chromatography. A three-compartment model with zero-order absorption from the injection site was found to best represent the pharmacokinetics of both enantiomers in blood and brain. (−)Mefloquine had a lower blood and brain apparent volume of distribution and a lower efflux clearance from the brain, resulting in a larger brain/blood ratio compared to (+)mefloquine. Elacridar did not modify blood concentrations or the elimination rate from blood for either enantiomers. However, cerebral AUCinf of both enantiomers were increased, with a stronger effect on (+)mefloquine. The efflux clearance from the brain decreased for both enantiomers, with a larger decrease for (+)mefloquine. After administration of racemic mefloquine in mice, blood and brain pharmacokinetics are stereoselective, (+)mefloquine being excreted from brain more rapidly than its antipode, showing that mefloquine is a substrate of efflux proteins and that mefloquine enantiomers undergo efflux in a stereoselective manner. Moreover, pretreatment with elacridar reduced the brain efflux clearances with a more pronounced effect on (+)mefloquine. PMID:15023856

  3. A defect in sodium-dependent amino acid uptake in diabetic rabbit peripheral nerve. Correction by an aldose reductase inhibitor or myo-inositol administration.

    PubMed Central

    Greene, D A; Lattimer, S A; Carroll, P B; Fernstrom, J D; Finegold, D N

    1990-01-01

    A myo-inositol-related defect in nerve sodium-potassium ATPase activity in experimental diabetes has been suggested as a possible pathogenetic factor in diabetic neuropathy. Because the sodium-potassium ATPase is essential for other sodium-cotransport systems, and because myo-inositol-derived phosphoinositide metabolites regulate multiple membrane transport processes, sodium gradient-dependent amino acid uptake was examined in vitro in endoneurial preparations derived from nondiabetic and 14-d alloxan diabetic rabbits. Untreated alloxan diabetes reduced endoneurial sodium-gradient dependent uptake of the nonmetabolized amino acid 2-aminoisobutyric acid by greater than 50%. Administration of an aldose reductase inhibitor prevented reductions in both nerve myo-inositol content and endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Myo-inositol supplementation that produced a transient pharmacological elevation in plasma myo-inositol concentration, but did not raise nerve myo-inositol content, reproduced the effect of the aldose reductase inhibitor on endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Phorbol myristate acetate, which acutely normalizes sodium-potassium ATPase activity in diabetic nerve, did not acutely correct 2-aminoisobutyric uptake when added in vitro. These data suggest that depletion of a small myo-inositol pool may be implicated in the pathogenesis of defects in amino acid uptake in diabetic nerve and that rapid correction of sodium-potassium ATPase activity with protein kinase C agonists in vitro does not acutely normalize sodium-dependent 2-aminoisobutyric acid uptake. PMID:2185278

  4. The subcellular localization of GABA transporters and its implication for seizure management.

    PubMed

    Madsen, Karsten K; Hansen, Gert H; Danielsen, E Michael; Schousboe, Arne

    2015-02-01

    The ability to modulate the synaptic GABA levels has been demonstrated by using the clinically effective and selective GAT1 inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid]. N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (EF1502) which not only inhibits GAT1 like tiagabine but also BGT1 has been shown to modulate extrasynaptic GABA levels. The simultaneous inhibition of synaptic and extrasynaptic GABA transporters using tiagabine and EF1502, respectively has been demonstrated to exert a synergistic anticonvulsant effect in several seizure models in mice. The pharmacological profile of these and similar compounds has been thoroughly investigated in in vitro systems, comparing the GAT subtype selectivity with the ability to inhibit GABA uptake in primary cultures of neurons and astrocytes. However, an exact explanation has not yet been found. In the present study, the ability of GATs to form homo and/or heterodimers was investigated as well as to which membrane micro environment the GATs reside. To investigate dimerization of GATs, fusion proteins of GATs tagged with either yellow fluorescent protein or cerulean fluorescent protein were made and fluorescence resonance energy transfer (FRET) was measured. It was found that GATs form both homo- and hetero-dimers in N2A and HEK-293 cells. Microdomain localization of GATs as investigated by detergent resistant membrane fractions after treatment of tissue with Brij-98 or Triton X-100 revealed that BGT1 and GAT1 mostly localize to non-membrane rafts independent of the detergent used. However, GAT3 localizes to membrane rafts when using Brij-98. Taken together, these results suggest that the observed hetero dimerization of GATs in the FRET study is unlikely to have functional implications since the GATs are located to very different cellular compartments and cell types.

  5. Localization and expression of GABA transporters in the suprachiasmatic nucleus.

    PubMed

    Moldavan, Michael; Cravetchi, Olga; Williams, Melissa; Irwin, Robert P; Aicher, Sue A; Allen, Charles N

    2015-12-01

    GABA is a principal neurotransmitter in the suprachiasmatic hypothalamic nucleus (SCN), the master circadian clock. Despite the importance of GABA and GABA uptake for functioning of the circadian pacemaker, the localization and expression of GABA transporters (GATs) in the SCN has not been investigated. The present studies used Western blot analysis, immunohistochemistry and electron microscopy to demonstrate the presence of GABA transporter 1 (GAT1) and GAT3 in the SCN. By using light microscopy, GAT1 and GAT3 were co-localized throughout the SCN, but were not expressed in the perikarya of arginine vasopressin- or vasoactive intestinal peptide-immunoreactive (-ir) neurons of adult rats, nor in the neuronal processes labelled with the neurofilament heavy chain. Using electron microscopy, GAT1- and GAT3-ir was found in glial processes surrounding unlabelled neuronal perikarya, axons, dendrites, and enveloped symmetric and asymmetric axo-dendritic synapses. Glial fibrillary acidic protein-ir astrocytes grown in cell culture were immunopositive for GAT1 and GAT3 and both GATs could be observed in the same glial cell. These data demonstrate that synapses in the SCN function as 'tripartite' synapses consisting of presynaptic axon terminals, postsynaptic membranes and astrocytes that contain GABA transporters. This model suggests that astrocytes expressing both GATs may regulate the extracellular GABA, and thereby modulate the activity of neuronal networks in the SCN. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  6. Selective CNS Uptake of the GCP-II Inhibitor 2-PMPA following Intranasal Administration

    PubMed Central

    Rais, Rana; Wozniak, Krystyna; Wu, Ying; Niwa, Minae; Stathis, Marigo; Alt, Jesse; Giroux, Marc; Sawa, Akira; Rojas, Camilo; Slusher, Barbara S.

    2015-01-01

    Glutamate carboxypeptidase II (GCP-II) is a brain metallopeptidase that hydrolyzes the abundant neuropeptide N-acetyl-aspartyl-glutamate (NAAG) to NAA and glutamate. Small molecule GCP-II inhibitors increase brain NAAG, which activates mGluR3, decreases glutamate, and provide therapeutic utility in a variety of preclinical models of neurodegenerative diseases wherein excess glutamate is presumed pathogenic. Unfortunately no GCP-II inhibitor has advanced clinically, largely due to their highly polar nature resulting in insufficient oral bioavailability and limited brain penetration. Herein we report a non-invasive route for delivery of GCP-II inhibitors to the brain via intranasal (i.n.) administration. Three structurally distinct classes of GCP-II inhibitors were evaluated including DCMC (urea-based), 2-MPPA (thiol-based) and 2-PMPA (phosphonate-based). While all showed some brain penetration following i.n. administration, 2-PMPA exhibited the highest levels and was chosen for further evaluation. Compared to intraperitoneal (i.p.) administration, equivalent doses of i.n. administered 2-PMPA resulted in similar plasma exposures (AUC0-t, i.n./AUC0-t, i.p. = 1.0) but dramatically enhanced brain exposures in the olfactory bulb (AUC0-t, i.n./AUC0-t, i.p. = 67), cortex (AUC0-t, i.n./AUC0-t, i.p. = 46) and cerebellum (AUC0-t, i.n./AUC0-t, i.p. = 6.3). Following i.n. administration, the brain tissue to plasma ratio based on AUC0-t in the olfactory bulb, cortex, and cerebellum were 1.49, 0.71 and 0.10, respectively, compared to an i.p. brain tissue to plasma ratio of less than 0.02 in all areas. Furthermore, i.n. administration of 2-PMPA resulted in complete inhibition of brain GCP-II enzymatic activity ex-vivo confirming target engagement. Lastly, because the rodent nasal system is not similar to humans, we evaluated i.n. 2-PMPA also in a non-human primate. We report that i.n. 2-PMPA provides selective brain delivery with micromolar concentrations. These studies support

  7. Multi-kinase inhibitors induce cutaneous toxicity through OAT6-mediated uptake and MAP3K7-driven cell death

    PubMed Central

    Zimmerman, Eric I.; Gibson, Alice A.; Hu, Shuiying; Vasilyeva, Aksana; Orwick, Shelley J.; Du, Guoqing; Mascara, Gerard P.; Ong, Su Sien; Chen, Taosheng; Vogel, Peter; Inaba, Hiroto; Maitland, Michael L.; Sparreboom, Alex; Baker, Sharyn D.

    2015-01-01

    The use of multi-kinase inhibitors (MKI) in oncology, such as sorafenib, is associated with a cutaneous adverse event called hand-foot skin reaction (HFSR) in which sites of pressure or friction become inflamed and painful, thus significantly impacting quality of life. The pathogenesis of MKI-induced HFSR is unknown, and the only available treatment options involve dose reduction or discontinuation of therapy, which have negative effects on primary disease management. To investigate the underlying mechanisms by which sorafenib promotes keratinocyte cytotoxicity and subsequent HFSR induction, we performed a transporter-directed RNAi screen in human epidermal keratinocytes and identified SLC22A20 (OAT6) as an uptake carrier of sorafenib. Further investigations into the intracellular mechanism of sorafenib activity through in situ kinome profiling identified the mitogen-activated protein kinase MAP3K7 (TAK1) as a target of sorafenib that induces cell death. Finally, we demonstrate that sorafenib induced keratinocyte injury in vivo, and that this effect could be reversed by co-treatment with the OAT6 inhibitor probenecid. Collectively, our findings reveal a novel pathway that regulates the entry of some MKIs into keratinocytes and explains the basis underlying sorafenib-induced skin toxicity, with important implications for the therapeutic management of HFSR. PMID:26677977

  8. Multikinase Inhibitors Induce Cutaneous Toxicity through OAT6-Mediated Uptake and MAP3K7-Driven Cell Death.

    PubMed

    Zimmerman, Eric I; Gibson, Alice A; Hu, Shuiying; Vasilyeva, Aksana; Orwick, Shelley J; Du, Guoqing; Mascara, Gerard P; Ong, Su Sien; Chen, Taosheng; Vogel, Peter; Inaba, Hiroto; Maitland, Michael L; Sparreboom, Alex; Baker, Sharyn D

    2016-01-01

    The use of multikinase inhibitors (MKI) in oncology, such as sorafenib, is associated with a cutaneous adverse event called hand-foot skin reaction (HFSR), in which sites of pressure or friction become inflamed and painful, thus significantly impacting quality of life. The pathogenesis of MKI-induced HFSR is unknown, and the only available treatment options involve dose reduction or discontinuation of therapy, which have negative effects on primary disease management. To investigate the underlying mechanisms by which sorafenib promotes keratinocyte cytotoxicity and subsequent HFSR induction, we performed a transporter-directed RNAi screen in human epidermal keratinocytes and identified SLC22A20 (OAT6) as an uptake carrier of sorafenib. Further investigations into the intracellular mechanism of sorafenib activity through in situ kinome profiling identified the mitogen-activated protein kinase MAP3K7 (TAK1) as a target of sorafenib that induces cell death. Finally, we demonstrate that sorafenib induced keratinocyte injury in vivo and that this effect could be reversed by cotreatment with the OAT6 inhibitor probenecid. Collectively, our findings reveal a novel pathway that regulates the entry of some MKIs into keratinocytes and explains the basis underlying sorafenib-induced skin toxicity, with important implications for the therapeutic management of HFSR.

  9. Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

    PubMed

    Poutiainen, Pekka K; Rönkkö, Teemu; Hinkkanen, Ari E; Palvimo, Jorma J; Närvänen, Ale; Turhanen, Petri; Laatikainen, Reino; Weisell, Janne; Pulkkinen, Juha T

    2014-01-15

    In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 μM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.

  10. Synthesis and evaluation of novel azetidine analogs as potent inhibitors of vesicular [3H]dopamine uptake.

    PubMed

    Ding, Derong; Nickell, Justin R; Deaciuc, Agripina G; Penthala, Narsimha Reddy; Dwoskin, Linda P; Crooks, Peter A

    2013-11-01

    Lobelane analogs that incorporate a central piperidine or pyrrolidine moiety have previously been reported by our group as potent inhibitors of VMAT2 function. Further central ring size reduction of the piperidine moiety in lobelane to a four-membered heterocyclic ring has been carried out in the current study to afford novel cis-and trans-azetidine analogs. These azetidine analogs (15a-15c and 22a-22c) potently inhibited [(3)H]dopamine (DA) uptake into isolated synaptic vesicles (Ki⩽66nM). The cis-4-methoxy analog 22b was the most potent inhibitor (Ki=24nM), and was twofold more potent that either lobelane (2a, Ki=45nM) or norlobelane (2b, Ki=43nM). The trans-methylenedioxy analog, 15c (Ki=31nM), was equipotent with the cis-analog, 22b, in this assay. Thus, cis- and trans-azetidine analogs 22b and 15c represent potential leads in the discovery of new clinical candidates for the treatment of methamphetamine abuse. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Dopamine-dependent hyperactivity in the rat following manipulation of GABA mechanisms in the region of the nucleus accumbens.

    PubMed

    Pycock, C J; Horton, R W

    1979-01-01

    The effect of manipulation of GABA mechanisms in the region of the nucleus accumbens on dopamine-dependent locomotor hyperactivity in the rat has been studied. Two models of hyperactivity were used: (1) the injection of dopamine into the region of the nucleus accumbens in nialamide-pretreated animals and (2) the systemic administration of d-amphetamine. Both GABA and the GABA agonist 3-aminopropane sulphonic acid (3-APS) depressed hyperactivity in a dose-related manner. High concentrations of GABA (greater than 100 micrograms) were required to produce a significant effect and the response was short-lived possibly reflecting the efficient GABA inactivating mechanisms. 3-APS proved to be approximately 10 times more potent as compared to GABA in the dopamine-accumbens hyperactivity model. Conversely GABA receptor antagonism with low doses of either picrotoxin or bicuculline enhanced the mild locomotor response induced by a low dose of dopamine injected into the nucleus accumbens. However such results were difficult to evaluate fairly as higher doses of the GABA antagonists resulted in varying degrees of generalized seizures. Blockade of GABA uptake systems with cis-1, 3-aminocyclohexane carboxylic acid (ACHC), nipecotic acid or beta-alanine within the region of the nucleus accumbens produced dose-related depression of dopamine-dependent hyperactivity in both models. GABA uptake blockade (nipecotic acid) significantly enhanced the GABA-mediated depression of hyperactivity induced by bilateral injection of dopamine into the nucleus accumbens. The results demonstrate an inhibitory action of GABA and drugs facilitating GABA-ergic transmission on dopamine-dependent hyperactivity in the rat. Although open to criticisms of not being able to distinguish between true GABA effects and the results of non-specific neuronal depression the hyperactivity model underlines the potency of the GABA uptake blocking compounds and their possible potential for future clinical use.

  12. Catabolism of GABA, succinic semialdehyde or gamma-hydroxybutyrate through the GABA shunt impair mitochondrial substrate-level phosphorylation.

    PubMed

    Ravasz, Dora; Kacso, Gergely; Fodor, Viktoria; Horvath, Kata; Adam-Vizi, Vera; Chinopoulos, Christos

    2017-03-11

    GABA is catabolized in the mitochondrial matrix through the GABA shunt, encompassing transamination to succinic semialdehyde followed by oxidation to succinate by the concerted actions of GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH), respectively. Gamma-hydroxybutyrate (GHB) is a neurotransmitter and a psychoactive drug that could enter the citric acid cycle through transhydrogenation with α-ketoglutarate to succinic semialdehyde and d-hydroxyglutarate, a reaction catalyzed by hydroxyacid-oxoacid transhydrogenase (HOT). Here, we tested the hypothesis that the elevation in matrix succinate concentration caused by exogenous addition of GABA, succinic semialdehyde or GHB shifts the equilibrium of the reversible reaction catalyzed by succinate-CoA ligase towards ATP (or GTP) hydrolysis, effectively negating substrate-level phosphorylation (SLP). Mitochondrial SLP was addressed by interrogating the directionality of the adenine nucleotide translocase during anoxia in isolated mouse brain and liver mitochondria. GABA eliminated SLP, and this was rescued by the GABA-T inhibitors vigabatrin and aminooxyacetic acid. Succinic semialdehyde was an extremely efficient substrate energizing mitochondria during normoxia but mimicked GABA in abolishing SLP in anoxia, in a manner refractory to vigabatrin and aminooxyacetic acid. GHB could moderately energize liver but not brain mitochondria consistent with the scarcity of HOT expression in the latter. In line with these results, GHB abolished SLP in liver but not brain mitochondria during anoxia and this was unaffected by either vigabatrin or aminooxyacetic acid. It is concluded that when mitochondria catabolize GABA or succinic semialdehyde or GHB through the GABA shunt, their ability to perform SLP is impaired.

  13. Preventative and therapeutic effects of a GABA transporter 1 inhibitor administered systemically in a mouse model of paclitaxel-induced neuropathic pain

    PubMed Central

    Parvathy, Subramanian S.

    2016-01-01

    Background There is a dearth of drugs to manage a dose-limiting painful peripheral neuropathy induced by paclitaxel in some patients during the treatment of cancer. Gamma-aminobutyric acid transporter-1 (GAT-1) whose expression is increased in the brain and spinal cord during paclitaxel-induced neuropathic pain (PINP) might be a potential therapeutic target for managing PINP. Thus, our aim was to evaluate if systemic administration of a GAT-1 inhibitor ameliorates PINP. Methods The reaction latency to thermal stimuli (hot plate test; at 55 °C) and cold stimuli (cold plate test; at 4 °C) of female BALB/c mice was recorded before and after intraperitoneal treatment with paclitaxel, its vehicle, and/or a selective GAT-1 inhibitor NO-711. The effects of NO-711 on motor coordination were evaluated using the rotarod test at a constant speed of 4 rpm or accelerating mode from 4 rpm to 40 rpm over 5 min. Results The coadministration of paclitaxel with NO-711 3 mg/kg prevented the development of paclitaxel-induced thermal hyperalgesia and cold allodynia at day 7 after drug treatment. NO-711 at 3 mg/kg produced antihyperalgesic activity up to 1 h and antiallodynic activity up to 2 h in mice with established paclitaxel-induced thermal hyperalgesia and cold allodynia. No motor deficits were observed with NO-711 at a dose of 3 mg/kg, whereas a higher dose 5 mg/kg caused motor impairment and reduced mean time spent on the rotarod at a constant speed of 4 rpm. However, at a rotarod accelerating mode from 4 rpm to 40 rpm over 5 min, NO-711 3 mg/kg caused motor impairment up to 1 h, but had recovered by 2 h. Conclusions These results show that systemic administration of the GAT-1 inhibitor NO-711 has preventative and therapeutic activity against paclitaxel-induced thermal hyperalgesia and cold allodynia. NO-711’s antiallodynic effects, but not antihyperalgesic effects, were independent of its motor impairment/sedation properties. Thus, low doses of GAT-1 inhibitors

  14. Preventative and therapeutic effects of a GABA transporter 1 inhibitor administered systemically in a mouse model of paclitaxel-induced neuropathic pain.

    PubMed

    Masocha, Willias; Parvathy, Subramanian S

    2016-01-01

    There is a dearth of drugs to manage a dose-limiting painful peripheral neuropathy induced by paclitaxel in some patients during the treatment of cancer. Gamma-aminobutyric acid transporter-1 (GAT-1) whose expression is increased in the brain and spinal cord during paclitaxel-induced neuropathic pain (PINP) might be a potential therapeutic target for managing PINP. Thus, our aim was to evaluate if systemic administration of a GAT-1 inhibitor ameliorates PINP. The reaction latency to thermal stimuli (hot plate test; at 55 °C) and cold stimuli (cold plate test; at 4 °C) of female BALB/c mice was recorded before and after intraperitoneal treatment with paclitaxel, its vehicle, and/or a selective GAT-1 inhibitor NO-711. The effects of NO-711 on motor coordination were evaluated using the rotarod test at a constant speed of 4 rpm or accelerating mode from 4 rpm to 40 rpm over 5 min. The coadministration of paclitaxel with NO-711 3 mg/kg prevented the development of paclitaxel-induced thermal hyperalgesia and cold allodynia at day 7 after drug treatment. NO-711 at 3 mg/kg produced antihyperalgesic activity up to 1 h and antiallodynic activity up to 2 h in mice with established paclitaxel-induced thermal hyperalgesia and cold allodynia. No motor deficits were observed with NO-711 at a dose of 3 mg/kg, whereas a higher dose 5 mg/kg caused motor impairment and reduced mean time spent on the rotarod at a constant speed of 4 rpm. However, at a rotarod accelerating mode from 4 rpm to 40 rpm over 5 min, NO-711 3 mg/kg caused motor impairment up to 1 h, but had recovered by 2 h. These results show that systemic administration of the GAT-1 inhibitor NO-711 has preventative and therapeutic activity against paclitaxel-induced thermal hyperalgesia and cold allodynia. NO-711's antiallodynic effects, but not antihyperalgesic effects, were independent of its motor impairment/sedation properties. Thus, low doses of GAT-1 inhibitors could be useful for the prevention and

  15. GABA receptor agonists: pharmacological spectrum and therapeutic actions.

    PubMed

    Bartholini, G

    1985-01-01

    From the data discussed in this review it appears that GABA receptor agonists exhibit a variety of actions in the central nervous system, some of which are therapeutically useful (Table V). GABA receptor agonists, by changing the firing rate of the corresponding neurons accelerate noradrenaline turnover without changes in postsynaptic receptor density and diminish serotonin liberation with an up-regulation of 5HT2 receptors. These effects differ from those of tricyclic antidepressants which primarily block monoamine re-uptake and cause down-regulation of beta-adrenergic and 5HT2 receptors. The GABA receptor agonist progabide has been shown to exert an antidepressant action which is indistinguishable from that of imipramine in patients with major affective disorders. The fact that: (a) GABA receptor agonists and tricyclic antidepressants affect noradrenergic and serotonergic transmission differently; and (b) tricyclic antidepressants alter GABA-related parameters challenges the classical monoamine hypothesis of depression and suggests that GABA-mediated mechanisms play a role in mood disorders. Decreases in cellular excitability produced by GABAergic stimulation leads to control of seizures in practically all animal models of epilepsy. GABA receptor agonists have a wide spectrum as they antagonize not only seizures which are dependent on decreased GABA synaptic activity but also convulsant states which are apparently independent of alterations in GABA-mediated events. These results in animals are confirmed in a wide range of human epileptic syndromes. GABA receptor agonists decrease dopamine turnover in the basal ganglia and antagonize neuroleptic-induced increase in dopamine release. On repeated treatment, progabide prevents or reverses the neuroleptic-induced up-regulation of dopamine receptors in the rat striatum and antagonizes the concomitant supersensitivity to dopaminomimetics. Behaviorally, GABA receptor agonists diminish the stereotypies induced by

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

    PubMed

    Ikeda, Saori; Tachikawa, Masanori; Akanuma, Shin-ichi; Fujinawa, Jun; Hosoya, Ken-ichi

    2012-08-01

    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.

  17. Release and effect of gamma-aminobutyric acid (GABA) on rat pineal melatonin production in vitro.

    PubMed

    Rosenstein, R E; Chuluyan, H E; Pereyra, E N; Cardinali, D P

    1989-06-01

    1. 3H-gamma-Aminobutyric acid (GABA) release elicited by a depolarizing K+ stimulus or by noradrenergic transmitter was examined in rat pineals in vitro. 2. The release of 3H-GABA was detectable at a 20 mM K+ concentration in medium and increased steadily up to 80 mM K+. 3. In a Ca2+-free medium 3H-GABA release elicited by 30 mM K+, but not that elicited by 50 mM K+, became blunted. 4. Norepinephrine (NE; 10(-6)-10(-4) M) stimulated 3H-GABA release from rat pineal explants in a dose-dependent manner. 5. The activity of 10(-5) M NE on pineal GABA release was suppressed by equimolecular amounts of prazosin or phentolamine (alpha 1- and alpha 1/alpha 2-adrenoceptor blockers, respectively) and was unaffected by propranolol (beta-adrenoceptor blocker). 6. The alpha 1-adrenoceptor agonist phenylephrine (10(-7)-10(-5) M) and the beta-adrenoceptor agonist isoproterenol (10(-5) M) mimicked the GABA releasing activity of NE, while 10(-7) M isoproterenol failed to affect it; the alpha 2-adrenoceptor agonist clonidine (10(-7)-10(-5) M) did not modify 3H-GABA release. 7. The addition of 10(-4) M GABA or of the GABA transaminase inhibitor gamma-acetylenic GABA or aminooxyacetic acid inhibited the melatonin content and/or release to the medium in rat pineal organotypic cultures. 8. GABA at concentrations of 10(-5) M or greater partially inhibited the NE-induced increase in melatonin production by pineal explants. 9. The depressant effect of GABA on melatonin production was inhibited by the GABA type A receptor antagonist bicuculline; bicuculline alone increased the pineal melatonin content. Baclofen, a GABA type B receptor agonist, did not affect the pineal melatonin content or release. 10. The decrease in serotonin (5-HT) content of rat pineal explants brought about by NE was not modified by GABA; GABA by itself increased 5-HT levels. 11. These results indicate that (a) GABA is released from rat pineals by a depolarizing stimulus of K+ through a mechanism which is partially Ca2

  18. An endocannabinoid uptake inhibitor from black pepper exerts pronounced anti-inflammatory effects in mice.

    PubMed

    Reynoso Moreno, Inés; Najar-Guerrero, Israel; Escareno, Noe; Flores, Mario Eduardo; Gertsch, Jürg; Viveros-Paredes, Juan Manuel

    2017-09-24

    Guineensine is a dietary N-isobutylamide widely present in black and long pepper (Piper nigrum and P. longum) previously shown to inhibit cellular endocannabinoid uptake. Given the role of endocannabinoids in inflammation and pain reduction, here we evaluated guineensine in mouse models of acute and inflammatory pain and endotoxemia. Significant dose-dependent anti-inflammatory effects (95.6 ± 3.1 % inhibition of inflammatory pain at 2.5 mg/kg i.p. and 50.0 ± 15.9 % inhibition of edema formation at 5 mg/kg i.p.) and acute analgesia (66.1 ± 28.1 % inhibition at 5.0 mg/kg i.p.) were observed. Moreover, guineensine inhibited pro-inflammatory cytokine production in endotoxemia. Intriguingly, guineensine and LPS independently induced catalepsy but in combination this effect was abolished. Both hypothermia and analgesia were blocked by the CB1 receptor inverse agonist rimonabant but the pronounced hypolocomotion was CB1 receptor-independent. A subsequent screen of 45 CNS-related receptors, ion channels and transporters revealed apparent interactions of guineensine with the dopamine transporter DAT, 5HT2A and sigma receptors, uncovering its prospective polypharmacology. The described potent pharmacological effects of guineensine might relate to the reported anti-inflammatory effects of pepper.

  19. Amitifadine, a triple monoamine re-uptake inhibitor, reduces nicotine self-administration in female rats.

    PubMed

    Levin, Edward D; Wells, Corinne; Johnson, Joshua E; Rezvani, Amir H; Bymaster, Frank P; Rose, Jed E

    2015-10-05

    A wider diversity of drug treatments to aid smoking cessation is needed to help tailor the most efficacious treatment for different types of smokers. This study was conducted to determine whether amitifadine, which inhibits re-uptake of dopamine, norepinephrine and serotonin, would decrease nicotine self-administration at doses that do not cause adverse side effects. Adult female Sprague-Dawley rats were trained to self-administer nicotine intravenous (IV) and were given acute doses of amitifadine in a repeated measures counterbalanced design. Effects of amitifadine on locomotor activity and food motivated responding were also evaluated. Chronic amitifadine effects were also examined. The 30 mg/kg amitifadine dose significantly reduced nicotine self-administration. The 5 and 10 mg/kg doses reduced nicotine self-administration during the first 15 min of the session when the greatest amount of nicotine was self-administered. The 30 mg/kg amitifadine dose, but not the lower doses caused a significant reduction in locomotor activity averaged over the one-hour session and reduced food motivated responding. The 10 mg/kg dose caused hypoactivity at the beginning of the session, but 5 mg/kg did not cause any hypoactivity. The effects of chronic amitifadine treatment (10 mg/kg) over the course of 15 sessions was also determined. Amitifadine caused a significant reduction in nicotine self-administration, which was not seen to diminish over two consecutive weeks of treatment and a week after enforced abstinence. Amitifadine significantly reduced nicotine self-administration. This prompts further research to determine if amitifadine might be an effective treatment for smoking cessation.

  20. Amitifadine, a triple monoamine re-uptake inhibitor, reduces nicotine self-administration in female rats

    PubMed Central

    Levin, Edward D; Wells, Corinne; Johnson, Joshua E; Rezvani, Amir H.; Bymaster, Frank P.; Rose, Jed E.

    2016-01-01

    A wider diversity of drug treatments to aid smoking cessation is needed to help tailor the most efficacious treatment for different types of smokers. This study was conducted to determine whether amitifadine, which inhibits re-uptake of dopamine, norepinephrine and serotonin, would decrease nicotine self-administration at doses that do not cause adverse side effects. Adult female Sprague-Dawley rats were trained to self-administer nicotine intravenous (IV) and were given acute doses of amitifadine in a repeated measures counterbalanced design. Effects of amitifadine on locomotor activity and food motivated responding were also evaluated. Chronic amitifadine effects were also examined. The 30 mg/kg amitifadine dose significantly reduced nicotine self-administration. The 5 and 10 mg/kg doses reduced nicotine self-administration during the first 15 min. of the session when the greatest amount of nicotine was self-administered. The 30 mg/kg amitifadine dose, but not the lower doses caused a significant reduction in locomotor activity averaged over the 1-hour session and reduced food motivated responding. The 10 mg/kg dose caused hypoactivity at the beginning of the session, but 5 mg/kg did not cause any hypoactivity. The effects of chronic amitifadine treatment (10 mg/kg) over the course of 15 sessions was also determined. Amitifadine caused a significant reduction in nicotine self-administration, which was not seen to diminish over two consecutive weeks of treatment and a week after enforced abstinence. Amitifadine significantly reduced nicotine self-administration. This prompts further research to determine if amitifadine might be an effective treatment for smoking cessation. PMID:26101069

  1. Evidence for a Revised Ion/Substrate Coupling Stoichiometry of GABA Transporters.

    PubMed

    Willford, Samantha L; Anderson, Cynthia M; Spencer, Shelly R; Eskandari, Sepehr

    2015-08-01

    Plasma membrane γ-aminobutyric acid (GABA) transporters (GATs) are electrogenic transport proteins that couple the cotranslocation of Na(+), Cl(-), and GABA across the plasma membrane of neurons and glia. A fundamental property of the transporter that determines its ability to concentrate GABA in cells and, hence, regulate synaptic and extra-synaptic GABA concentrations, is the ion/substrate coupling stoichiometry. Here, we scrutinized the currently accepted 2 Na(+):1 Cl(-):1 GABA stoichiometry because it is inconsistent with the measured net charge translocated per co-substrate (Na(+), Cl(-), and GABA). We expressed GAT1 and GAT3 in Xenopus laevis oocytes and utilized thermodynamic and uptake under voltage-clamp measurements to determine the stoichiometry of the GABA transporters. Voltage-clamped GAT1-expressing oocytes were internally loaded with GABA, and the reversal potential (V rev) of the transporter-mediated current was recorded at different external concentrations of Na(+), Cl(-), or GABA. The shifts in V rev for a tenfold change in the external Na(+), Cl(-), and GABA concentration were 84 ± 4, 30 ± 1, and 29 ± 1 mV, respectively. To determine the net charge translocated per Na(+), Cl(-), and GABA, we measured substrate fluxes under voltage clamp in cells expressing GAT1 or GAT3. Charge flux to substrate flux ratios were 0.7 ± 0.1 charge/Na(+), 2.0 ± 0.2 charges/Cl(-), and 2.1 ± 0.1 charges/GABA. Altogether, our results strongly suggest a 3 Na(+):1 Cl(-):1 GABA coupling stoichiometry for the GABA transporters. The revised stoichiometry has important implications for understanding the contribution of GATs to GABAergic signaling in health and disease.

  2. Crucial interactions between selective serotonin uptake inhibitors and sigma-1 receptor in heart failure.

    PubMed

    Bhuiyan, Md Shenuarin; Tagashira, Hideaki; Fukunaga, Kohji

    2013-01-01

    Depression is associated with a substantial increase in the risk of developing heart failure and is independently associated with increased cardiovascular morbidity and mortality. Inversely, cardiovascular disease can lead to severe depression. Thus, therapy with selective serotonin reuptake inhibitors (SSRIs) is strongly recommended to reduce cardiovascular disease-induced morbidity and mortality. However, molecular mechanisms to support evidence-based SSRI treatment of cardiovascular disease have not been elucidated. We recently found very high expression of the sigma-1 receptor, an orphan receptor, in rat heart tissue and defined the cardiac sigma-1 receptor as a direct SSRI target in eliciting cardioprotection in both pressure overload (PO)induced and transverse aortic constriction (TAC)-induced myocardial hypertrophy models in rodents. Our findings suggest that SSRIs such as fluvoxamine protect against PO- and TAC-induced cardiac dysfunction by upregulating sigma-1 receptor expression and stimulating sigma-1 receptor-mediated Akt-eNOS signaling. Here, we discuss the association of depression and cardiovascular diseases, the protective mechanism of SSRIs in heart failure patients, and the pathophysiological relevance of sigma-1 receptors to progression of heart failure. These findings should promote development of clinical therapeutics targeting the sigma-1 receptor in cardiovascular diseases.

  3. Effects of various serotonin agonists, antagonists, and uptake inhibitors on the discriminative stimulus effects of methamphetamine in rats.

    PubMed

    Munzar, P; Laufert, M D; Kutkat, S W; Nováková, J; Goldberg, S R

    1999-10-01

    Neurochemical studies indicate that methamphetamine increases central serotonin (5-HT) levels more markedly than other psychomotor stimulants such as amphetamine or cocaine. In the present study, we investigated 5-HT involvement in the discriminative stimulus effects of methamphetamine. In Sprague-Dawley rats trained to discriminate 1.0 mg/kg methamphetamine i.p. from saline under a fixed-ratio schedule of food presentation, the effects of selected 5-HT agonists, antagonists, and uptake inhibitors were tested. Fluoxetine (1.8-18.0 mg/kg) and clomipramine (3.0-18.0 mg/kg), selective serotonin uptake inhibitors, did not produce any methamphetamine-like discriminative stimulus effects when administered alone, but fluoxetine (5.6 mg/kg), unlike clomipramine (5.6 mg/kg), significantly shifted the methamphetamine dose-response curve to the left. Both 8-hydroxy-2-dipropylaminotetralin (0.03-0.56 mg/kg), a full agonist, and buspirone (1.0-10.0 mg/kg), a partial agonist at 5-HT(1A) receptors, partially generalized to the training dose of methamphetamine but only at high doses that decreased response rate. This generalization was antagonized by the coadministration of the 5-HT(1A) antagonist WAY-100635 (1.0 mg/kg). WAY-100635 (1.0 mg/kg) also partially reversed the leftward shift of the methamphetamine dose-response curve produced by fluoxetine. (+/-)-1-(2, 5-Dimethoxy-4-iodophenyl)-2-aminopropane (0.3 mg/kg), a 5-HT(2A/2C) agonist, shifted the methamphetamine dose-response curve to the left, and this leftward shift was antagonized by the coadministration of ketanserin (3.0 mg/kg), a 5-HT(2A/2C) antagonist. Ketanserin (3.0 mg/kg) also produced a shift to the right in the methamphetamine dose-response curve and completely reversed the leftward shift in the methamphetamine dose-response curve produced by fluoxetine. In contrast, tropisetron (1.0 mg/kg), a 5-HT(3) antagonist, produced a shift to the left of the methamphetamine dose-response curve, and this effect of tropisetron

  4. Vanadium Uptake by Plants: Absorption Kinetics and the Effects of pH, Metabolic Inhibitors, and Other Anions and Cations.

    PubMed

    Welch, R M

    1973-05-01

    The kinetics of vanadium absorption by excised barley (Hordeum vulgare L., cv. Eire) roots were investigated with respect to ionic species of V in solution, time and concentration dependence, Ca sensitivity, and interaction with various anions, cations, and pH levels. The role of metabolism in V absorption was also studied using anaerobic treatment (N(2) gas) and chemical inhibitors (NaN(3), KCN, or 2,4-dinitrophenol). Approximately one-third of the labeled V initially taken up by excised roots was desorbed to a constant level after 45 min in unlabeled V solutions. The rate of absorption of labeled V from 5 mum NH(4)VO(3) solutions containing 0.5 mm CaSO(4) was constant for at least 3 hours. Omission of Ca resulted in a 72% reduction in V uptake when compared to controls with 0.5 mm CaSO(4). The rate of uptake of V was highest at pH 4 but dropped to a very low level at pH 10. It was relatively constant between the pH levels of 5 and 8 at which the VO(3) (-) ion is the predominant ionic species in solution. The rate of absorption of V was followed as a function of concentrations from 0.5 to 100 mum NH(4)VO(3). It was found to be a linear function of concentration and did not follow saturation kinetics. Absorption experiments carried out with labeled V from either N(a)VO(3) or NH(4)VO(3) sources gave similar results. No anion studied (i.e. HPO(4) (2-), HAsO(4) (2-), MoO(4) (2-), SeO(4) (2-), SeO(3) (2-), CrO(4) (2-), BO(3) (3-), No(3) (-), and Cl(-)) interfered appreciably (i.e. less than 30% inhibition) with the absorption of labeled V. Anaerobic treatment of absorption solution with N(2) gas did not inhibit V absorption by excised roots. The results obtained using chemical inhibitors were not consistent. It was concluded that V is not actively absorbed by excised barley roots.

  5. Antagonism of fenfluramine-induced hyperthermia in rats by some, but not all, selective inhibitors of 5-hydroxytryptamine uptake.

    PubMed Central

    Sugrue, M. F.

    1984-01-01

    The injection of fenfluramine (7.5 mg kg-1,i.p.) to rats housed at 27-28 degrees C was associated with an elevation of core body temperature which peaked at approximately 1 h post-injection. One h pretreatment with citalopram (20 mg kg-1, i.p.), chlorimipramine (10 mg kg-1, i.p.), femoxetine (10 mg kg-1, i.p.) and fluoxetine (20 mg kg-1, i.p.) resulted in an attenuated response to fenfluramine. In contrast, Org 6582 (20 mg kg-1) and zimelidine (20 mg kg-1) were devoid of an effect on fenfluramine-induced hyperthermia. The response to fenfluramine was was also blocked by i.p. injections of metergoline (0.2 mg kg-1), methysergide (5 mg kg-1) and mianserin (0.5 mg kg-1). Rectal temperature was unaltered by both the 5-hydroxytryptamine (5-HT) uptake inhibitors and the 5-HT receptor antagonists. The IC50 values (nM) for in vitro inhibition of [3H]-5-HT uptake into rat hypothalamic synaptosomes were for citalopram 2.4, chlorimipramine 8.8, femoxetine 14, fluoxetine 16, Org 6582 75 and zimelidine 250. The injection of all six compounds (20 mg kg-1, i.p.) 1 h before death was associated with an inhibition of [3H]-5-HT uptake into rat hypothalamic synaptosomes which ranged from 47.2% for chlorimipramine to 83.3% for citalopram. Rat hypothalamic 5-HT levels were decreased by approximately 50% 3 h after the injection of fenfluramine (15 mg kg-1, i.p.). This effect was blocked by a 1 h pretreatment with fluoxetine, Org 6582 and zimelidine (all 20 mg kg-1, i.p.). Ki values for displacement of specifically bound [3H]-5-HT (1 nM) to rat hypothalamic membranes were for metergoline 26 nM, methysergide 1.1 microM, mianserin 3.6 microM, chlorimipramine 9.2 microM and fluoxetine 32.7 microM. Values for citalopram, femoxetine, Org 6582 and zimelidine were in excess of 65.4 microM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6722394

  6. Development of Non-GAT1-Selective Inhibitors: Challenges and Achievements.

    PubMed

    Damgaard, Maria; Haugaard, Anne Stæhr; Kickinger, Stefanie; Al-Khawaja, Anas; Lie, Maria E K; Ecker, Gerhard F; Clausen, Rasmus Prætorius; Frølund, Bente

    2017-01-01

    γ-Aminobutyric acid (GABA) neurotransmission is terminated by the GABA transporters (GATs) via uptake of GABA into neurons and surrounding glial cells. Four different transporters have been identified: GAT1, GAT2, GAT3, and the betaine/GABA transporter 1 (BGT1). The GAT1 subtype is the most explored transporter due to its high abundance in the brain and the existence of selective and potent GAT1 inhibitors. Consequently, less is known about the role and therapeutic potential of the non-GAT1 subtypes. Emerging pharmacological evidence suggests that some of these transporters pose interesting targets in several brain disorders. Pharmacological non-GAT1-selective tool compounds are important to further investigate the involvement of GATs in different pathological conditions. Extensive medicinal chemistry efforts have been put into the development of subtype-selective inhibitors, but truly selective and potent inhibitors of non-GAT1 subtypes are still limited. This review covers the advances within the medicinal chemistry area and the structural basis for obtaining non-GAT1-selective inhibitors.

  7. Antidepressants and seizure-interactions at the GABA-receptor chloride-ionophore complex

    SciTech Connect

    Malatynska, E.; Knapp, R.J.; Ikeda, M.; Yamamura, H.I.

    1988-01-01

    Convulsive seizures are a potential side effect of antidepressant drug treatment and can be produced by all classes of antidepressants. It is also know that some convulsant and anticonvulsant drug actions are mediated by the GABA-receptor chloride-ionophore complex. Drugs acting at this complex appear to induce convulsions by inhibiting chloride conductance through the associated chloride channel. Using the method of GABA-stimulated /sup 36/Cl-uptake by rat cerebral cortical vesicles, we show that some antidepressant drugs can inhibit the GABA-receptor chloride uptake, and that the degree of chloride channel inhibition by these drugs correlates with the frequency of convulsive seizures induced by them.

  8. The serotonin uptake inhibitor citalopram reduces acute cardiovascular and vegetative effects of 3,4-methylenedioxymethamphetamine ('Ecstasy') in healthy volunteers.

    PubMed

    Liechti, M E; Vollenweider, F X

    2000-01-01

    MDMA (3,4-methylenedioxymethamphetamine) or 'Ecstasy' is a widely used recreational drug that produces a state of heightened mood but also cardiovascular and vegetative side-effects. In animals, MDMA releases serotonin and, to a lesser extent, dopamine and norepinephrine. The release of serotonin can be blocked by serotonin uptake inhibitors such as citalopram. It is unknown to what extent this mechanism is also responsible for the physiological side-effects of MDMA seen in humans. We investigated the effect of citalopram pretreatment (40 mg i.v.) on vegetative and cardiovascular effects of MDMA (1.5 mg/kg p.o.) in a double-blind placebo-controlled study in 16 healthy volunteers. MDMA moderately increased blood pressure and heart rate, slightly elevated body temperature and produced a broad range of acute and short-term side-effects. Citalopram reduced all these MDMA-induced physiological changes except for body temperature. These findings suggest that physiological effects of MDMA in humans are partially due to an interaction of MDMA with the serotonin carrier and a subsequent release of serotonin.

  9. Characteristics of the ambulation-increasing effect of GBR-12909, a selective dopamine uptake inhibitor, in mice.

    PubMed

    Hirate, K; Kuribara, H

    1991-04-01

    Behavioral effects of a dopamine uptake inhibitor, GBR-12909 (GBR), were evaluated by ambulatory activity in mice. The single administration of over 10 mg/kg of GBR, i.p. and p.o., significantly increased the ambulatory activity. The repeated administration of GBR, at only 10 mg/kg, produced a reverse tolerance to its ambulation-increasing effect. However, a cross-reverse tolerance was induced between GBR (10 and 20 mg/kg) and methamphetamine (2 mg/kg) in both directions. Furthermore, 5 mg/kg of GBR significantly enhanced the effects of methamphetamine, cocaine, imipramine, morphine, scopolamine and caffeine. R-THBP, a coenzyme of tyrosine hydroxylase, also enhanced the effect of GBR. In contrast, the ambulation-increasing effect of 10 mg/kg of GBR was markedly reduced by haloperidol, chlorpromazine, tetrabenazine, oxypertine, reserpine and alpha-methyl-p-tyrosine. On the other hand, the effect of GBR was only slightly and/or scarcely modified by apomorphine, caerulein, physostigmine, pilocarpine, N6-(L-2-phenylisopropyl)-adenosine and naloxone. The neurochemical experiment in rats, not in mice, revealed that GBR possessed more dominant action on dopaminergic systems than noradrenergic or serotonergic systems. However, the behavioral characteristics of GBR are similar to those of methamphetamine and cocaine, which possess less selective action than GBR on dopaminergic and noradrenergic systems.

  10. Transport and equilibrium uptake of a peptide inhibitor of PACE4 into articular cartilage is dominated by electrostatic interactions.

    PubMed

    Byun, Sangwon; Tortorella, Micky D; Malfait, Anne-Marie; Fok, Kam; Frank, Eliot H; Grodzinsky, Alan J

    2010-07-01

    The availability of therapeutic molecules to targets within cartilage depends on transport through the avascular matrix. We studied equilibrium partitioning and non-equilibrium transport into cartilage of Pf-pep, a 760 Da positively charged peptide inhibitor of the proprotein convertase PACE4. Competitive binding measurements revealed negligible binding of Pf-pep to sites within cartilage. Uptake of Pf-pep depended on glycosaminoglycan charge density, and was consistent with predictions of Donnan equilibrium given the known charge of Pf-pep. In separate transport experiments, the diffusivity of Pf-pep in cartilage was measured to be approximately 1 x 10(-6) cm(2)/s, close to other similarly-sized non-binding solutes. These results suggest that small positively charged therapeutics will have a higher concentration within cartilage than in the surrounding synovial fluid, a desired property for local delivery; however, such therapeutics may rapidly diffuse out of cartilage unless there is additional specific binding to intra-tissue substrates that can maintain enhanced intra-tissue concentration for local delivery.

  11. Disorders of GABA metabolism: SSADH and GABA-transaminase deficiencies.

    PubMed

    Parviz, Mahsa; Vogel, Kara; Gibson, K Michael; Pearl, Phillip L

    2014-11-25

    Clinical disorders known to affect inherited gamma-amino butyric acid (GABA) metabolism are autosomal recessively inherited succinic semialdehyde dehydrogenase and GABA-transaminase deficiency. The clinical presentation of succinic semialdehyde dehydrogenase deficiency includes intellectual disability, ataxia, obsessive-compulsive disorder and epilepsy with a nonprogressive course in typical cases, although a progressive form in early childhood as well as deterioration in adulthood with worsening epilepsy are reported. GABA-transaminase deficiency is associated with a severe neonatal-infantile epileptic encephalopathy.

  12. Electronic and structural features of γ-aminobutyric acid (GABA) and four of its direct agonists

    NASA Astrophysics Data System (ADS)

    Lipkowitz, Kenny B.; Gilardi, Richard D.; Aprison, M. H.

    1989-04-01

    To understand better how the inhibitory neurotransmitter γ-aminobutyric acid (GABA) functions at its postsynaptic receptor site, electronic and structural features of the natural inhibitor were compared with four direct GABA agonists: muscimol, trans-3-amino-1-cyclopentane carboxylic acid ( trans-3 ACPC), isoguvacine and 4,5,6,7-tetrahydroisoxazolo [5,4-c]-pyridin-3-ol (THIP). The structures of isoguvacine and THIP were determined by X-ray crystallography. The structures of GABA and muscimol were retrieved from the literature and that of trans-3 ACPC was computed with AM1. A relationship was found between published IC50 values obtained from ( 3H)-GABA binding data and the per cent polar surface area scaled by molecular ionization potential. The structural features of GABA and its agonists were compared and a hypothesis for GABA agonist activity based upon position of the ammonium ion with respect to the carboxylate is presented.

  13. Ethanol, not metabolized in brain, significantly reduces brain metabolism, probably via specific GABA(A) receptors

    PubMed Central

    Rae, Caroline D.; Davidson, Joanne E.; Maher, Anthony D.; Rowlands, Benjamin D.; Kashem, Mohammed A.; Nasrallah, Fatima A.; Rallapalli, Sundari K.; Cook, James M; Balcar, Vladimir J.

    2014-01-01

    Ethanol is a known neuromodulatory agent with reported actions at a range of neurotransmitter receptors. Here, we used an indirect approach, measuring the effect of alcohol on metabolism of [3-13C]pyruvate in the adult Guinea pig brain cortical tissue slice and comparing the outcomes to those from a library of ligands active in the GABAergic system as well as studying the metabolic fate of [1,2-13C]ethanol. Ethanol (10, 30 and 60 mM) significantly reduced metabolic flux into all measured isotopomers and reduced all metabolic pool sizes. The metabolic profiles of these three concentrations of ethanol were similar and clustered with that of the α4β3δ positive allosteric modulator DS2 (4-Chloro-N-[2-(2-thienyl)imidazo[1,2a]-pyridin-3-yl]benzamide). Ethanol at a very low concentration (0.1 mM) produced a metabolic profile which clustered with those from inhibitors of GABA uptake, and ligands showing affinity for α5, and to a lesser extent, α1-containing GABA(A)R. There was no measureable metabolism of [1,2-13C]ethanol with no significant incorporation of 13C from [1,2-13C]ethanol into any measured metabolite above natural abundance, although there were measurable effects on total metabolite sizes similar to those seen with unlabeled ethanol. The reduction in metabolism seen in the presence of ethanol is therefore likely to be due to its actions at neurotransmitter receptors, particularly α4β3δ receptors, and not because ethanol is substituting as a substrate or because of the effects of ethanol catabolites acetaldehyde or acetate. We suggest that the stimulatory effects of very low concentrations of ethanol are due to release of GABA via GAT1 and the subsequent interaction of this GABA with local α5-containing, and to a lesser extent, α1-containing GABA(A)R. PMID:24313287

  14. Inhibition of recombinant N-type and native high voltage-gated neuronal Ca{sup 2+} channels by AdGABA: Mechanism of action studies

    SciTech Connect

    Martinez-Hernandez, Elizabeth; Sandoval, Alejandro; Gonzalez-Ramirez, Ricardo; Zoidis, Grigoris; Felix, Ricardo

    2011-02-01

    High-voltage activated Ca{sup 2+} (Ca{sub V}) channels play a key role in the regulation of numerous physiological events by causing transient changes in the intracellular Ca{sup 2+} concentration. These channels consist of a pore-forming Ca{sub V}{alpha}{sub 1} protein and three auxiliary subunits (Ca{sub V}{beta}, Ca{sub V}{alpha}{sub 2}{delta} and Ca{sub V}{gamma}). Ca{sub V}{alpha}{sub 2}{delta} is an important component of Ca{sub V} channels in many tissues and of great interest as a drug target. It is well known that anticonvulsant agent gabapentin (GBP) binds to Ca{sub V}{alpha}{sub 2}{delta} and reduces Ca{sup 2+} currents by modulating the expression and/or function of the Ca{sub V}{alpha}{sub 1} subunit. Recently, we showed that an adamantane derivative of GABA, AdGABA, has also inhibitory effects on Ca{sub V} channels. However, the importance of the interaction of AdGABA with the Ca{sub V}{alpha}{sub 2}{delta} subunit has not been conclusively demonstrated and the mechanism of action of the drug has yet to be elucidated. Here, we describe studies on the mechanism of action of AdGABA. Using a combined approach of patch-clamp recordings and molecular biology we show that AdGABA inhibits Ca{sup 2+} currents acting on Ca{sub V}{alpha}{sub 2}{delta} only when applied chronically, both in a heterologous expression system and in dorsal root-ganglion neurons. AdGABA seems to require uptake and be acting intracellularly given that its effects are prevented by an inhibitor of the L-amino acid transport system. Interestingly, a mutation in the Ca{sub V}{alpha}{sub 2}{delta} that abolishes GBP binding did not affect AdGABA actions, revealing that its mechanism of action is similar but not identical to that of GBP. These results indicate that AdGABA is an important Ca{sub V}{alpha}{sub 2}{delta} ligand that regulates Ca{sub V} channels.

  15. Health resource use and costs of vilazodone and other selective serotonin re-uptake inhibitors in treating major depressive disorder.

    PubMed

    Zhou, Zheng-Yi; Sun, Shawn; Chopra, Pooja; Zhong, Yichen; Totev, Todor; Signorovitch, James

    2015-01-01

    Selective serotonin re-uptake inhibitors (SSRIs) are widely prescribed antidepressants. This claims database study compared healthcare resource use and costs among patients with major depressive disorder (MDD) treated with vilazodone vs other SSRIs. Adults with an MDD diagnosis and ≥ 1 prescription fill for vilazodone, citalopram, escitalopram, fluoxetine, paroxetine, or sertraline were identified from administrative claims data (2010-2012). Patients who concomitantly used adjunctive medication, either a second-generation antidepressant or antipsychotic, were excluded. All-cause and MDD-related healthcare resource use and costs (in 2012 USD) were compared between patients treated with vilazodone vs other SSRIs over a 6-month follow-up period using unadjusted and multivariable analyses. The study cohort included 49 861 patients (mean age = 44.0 years; 70% female). Compared with the vilazodone cohort (n = 3527), patients in the citalopram (n = 12 187), escitalopram (n = 8275), fluoxetine (n = 10 142), paroxetine (n = 3146), and sertraline (n = 12 584) cohorts had significantly more all-cause inpatient hospital visits, longer hospital stays and more frequent emergency department visits, following the index date, after adjusting for baseline characteristics. All-cause medical service costs (inpatient + outpatient + emergency department visits) were significantly higher across all other SSRI cohorts vs vilazodone by $758-$1165 (p < 0.05). Similarly, all-cause total costs, were significantly or numerically (non-significantly) higher across all SSRI cohorts vs vilazodone by $351-$780. The was no clinical measurement of disease severity, partial coverage of the Medicare-eligible population, and short follow-up. MDD treatment with vilazodone was associated with significantly lower rates of inpatient and emergency services, and with significantly lower all-cause medical service costs and numerically (non-significantly) lower total costs to payers than with the other SSRIs

  16. Increased uptake of [123I]-meta-iodobenzylguanidine and [18F]-dopamine in mouse pheochromocytoma cells and tumors after treatment with the histone deacetylase inhibitors romidepsin and trichostatin A

    PubMed Central

    Martiniova, Lucia; Perera, Shiromi M.; Brouwers, Frederieke M.; Alesci, Salvatore; Abu-Asab, Mones; Marvelle, Amanda F.; Kiesewetter, Dale O.; Thomasson, David; Morris, John C.; Kvetnansky, Richard; Tischler, Arthur S.; Reynolds, James C; Fojo, A. Tito; Pacak, Karel

    2014-01-01

    Purpose [131I]-meta-iodobenzylguanidine ([131I]-MIBG) is the most commonly employed treatment for metastatic pheochromocytoma and paraganglioma; however, its success is limited. Its efficacy depends on the [131I]-MIBG concentration reached within the tumor through its uptake via the norepinephrine transporter and retention in neurosecretory granules. Purpose is to enhance [123I]-MIBG uptake in cells and liver pheochromocytoma tumors. Experimental Design We report the in vitro effects of two histone deacetylase (HDAC) inhibitors, romidepsin and trichostatin A, on increased uptake of [3H]-norepinephrine and [123I]-MIBG in mouse pheochromocytoma (MPC) cells, and the effect of romidepsin on [18F]-fluorodopamine and [123I]-MIBG uptake in a mouse model of metastatic pheochromocytoma. The effects of both inhibitors on norepinephrine transporter activity were assessed in MPC cells by [123I]-MIBG uptake studies with and without the transporter blocking agent desipramine and the vesicular blocking agent reserpine. Results Both HDAC inhibitors increased [3H]-norepinephrine, [123I]-MIBG, and [18F]-fluorodopamine uptake through the norepinephrine transporter in MPC cells. In vivo, inhibitor treatment resulted in increased uptake of [18F]-fluorodopamine and in pheochromocytoma liver metastases as measured by maximal standardized uptake values on PET imaging (p < 0.001). Analysis of biodistribution after inhibitor treatment confirmed the PET results in that uptake of [123I]-MIBG was significantly increased in liver metastases (p < 0.05). Therefore, HDAC inhibitor treatment increased radioisotope uptake in MPC cells in vitro and in liver metastases in vivo, through increased norepinephrine transporter activity. Conclusion These results suggest that HDAC inhibitors could enhance the therapeutic efficacy of [131I]-MIBG treatment in patients with malignant pheochromocytoma. PMID:21098082

  17. Identification and functional characterization of a dual GABA/taurine transporter in the bullfrog retinal pigment epithelium

    PubMed Central

    1995-01-01

    Intracellular microelectrodes, fluorescence imaging, and radiotracer flux techniques were used to investigate the physiological response of the retinal pigment epithelium (RPE) to the major retinal inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). GABA is released tonically in the dark by amphibian horizontal cells, but is not taken up by the nearby Muller cells. Addition of GABA to the apical bath produced voltage responses in the bullfrog RPE that were not blocked nor mimicked by any of the major GABA-receptor antagonists or agonists. Nipecotic acid, a substrate for GABA transport, inhibited the voltage effects of GABA. GABA and nipecotic acid also inhibited the voltage effects of taurine, suggesting that the previously characterized beta- alanine sensitive taurine carrier also takes up GABA. The voltage responses of GABA, taurine, nipecotic acid, and beta-alanine all showed first-order saturable kinetics with the following Km's: GABA (Km = 160 microM), beta-alanine (Km = 250 microM), nipecotic acid (Km = 420 microM), and taurine (Km = 850 microM). This low affinity GABA transporter is dependent on external Na, partially dependent on external Cl, and is stimulated in low [K]o, which approximates subretinal space [K]o during light onset. Apical GABA also produced a significant conductance increase at the basolateral membrane. These GABA-induced conductance changes were blocked by basal Ba2+, suggesting that GABA decreased basolateral membrane K conductance. In addition, the apical membrane Na/K ATPase was stimulated in the presence of GABA. A model for the interaction between the GABA transporter, the Na/K ATPase, and the basolateral membrane K conductance accounts for the electrical effects of GABA. Net apical-to-basal flux of [3H]-GABA was also observed in radioactive flux experiments. The present study shows that a high capacity GABA uptake mechanism with unique pharmacological properties is located at the RPE apical membrane and could play an

  18. Isolation-induced aggression in mice: effects of 5-hydroxytryptamine uptake inhibitors and involvement of postsynaptic 5-HT1A receptors.

    PubMed

    Sánchez, C; Hyttel, J

    1994-11-03

    The inhibitory potencies of selective serotonin (5-hydroxytryptamine, 5-HT) uptake inhibitors on isolation-induced aggressive behaviour in male mice were studied. Furthermore, the role of postsynaptic 5-HT1A receptors in the mediation of aggressive behaviour was studied. The selective 5-HT uptake inhibitors, sertraline, floxetine, femoxetine and fluvoxamine, showed weak antiaggressive effects, and citalopram and paroxetine were ineffective. This rank of potencies corresponded with neither uptake inhibitory potencies in vitro nor potentiation of 1-5-hydroxytryptophan (1,5-HTP)-induced motor effects in vivo, as citalopram and paroxetine were among the most potent compounds in these tests. A subeffective dose of 1,5-HTP (110 mumol/kg = 25 mg/kg, s.c.) potentiated the antiaggressive effect of citalopram and paroxetine more than 110 and 1600 times, respectively. The effects of sertraline, fluvoxamine, fluoxetine and femoxetine were only potentiated 3, 36, 4 and 16 times, respectively. The 5-HT releasing compound fenfluramine inhibited the aggressive behaviour dose dependently, and depletion of 5-HT by treatment with p-chloro-phenylalanine methyl ester attenuated this effect significantly. p-Chloro-phenylalanine methyl ester was ineffective itself, but potentiated the antiaggressive effect of the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamin)tetralin (8-OH-DPAT). The beta-adrenoceptor/5-HT1A receptor antagonist, (-)-penbutolol, reversed the antiaggressive effects of 8-OHDPAT. In conclusion, selective 5-HT uptake inhibitors act in different ways on isolation-induced aggressive behaviour, and postsynaptic 5-HT1A receptors are involved in mediating the aggressive behaviour.

  19. T-1032, a cyclic GMP phosphodiesterase-5 inhibitor, acutely blocks physiologic insulin-mediated muscle haemodynamic effects and glucose uptake in vivo.

    PubMed

    Mahajan, Hema; Richards, Stephen M; Rattigan, Stephen; Clark, Michael G

    2003-12-01

    1. Cyclic GMP phosphodiesterase-5 inhibitors have been shown to alter blood flow in specific tissues by potentiating local NO-dependent vasodilatory mechanisms. Since the haemodynamic effects of physiologic insulin, particularly capillary recruitment, may be critical for muscle glucose uptake in vivo and are blocked by inhibitors of nitric oxide synthase, we have explored the acute effects of the specific cGMP phosphodiesterase-5 inhibitor T-1032 on physiologic insulin action in anaesthetized healthy rats in vivo. 2. Whole-body glucose infusion (GIR), femoral blood flow (FBF), hind leg vascular resistance (VR), hind leg glucose uptake (HGU), 2-deoxyglucose uptake into muscles of the lower leg (R'g), hind leg metabolism of infused 1-methylxanthine (1-MX), a measure of capillary recruitment, and muscle cGMP were determined. The experimental groups were T-1032 (10 microg min-1 kg-1) infused for 1 h before and during a euglycaemic insulin clamp (3 mU min-1 kg-1 x 2 h), T-1032 infused for 3 h with saline, T-1032 during a 2 h clamp, T-1032 with saline for 2 h, and a 2 h saline control. 3. Insulin increased GIR from zero to 13 mg min-1 kg-1, HGU from 0.1+/-0.01 to 0.43+/-0.05 micromol min-1, R'g and 1-MX, marginally increased FBF, and had no effect on blood pressure or heart rate. T-1032 alone had no effect on blood pressure, heart rate, FBF, VR, HGU, R'g or 1-MX, but increased muscle cGMP. T-1032 1 h before and during insulin completely blocked GIR (1 h), HGU (2 h), R'g (2 h), and 1-MX (2 h). T-1032 commenced with insulin had only partial blocking activity against insulin. 4. We conclude that T-1032 is a potent acutely acting inhibitor of the muscle effects of physiologic insulin on capillary recruitment and glucose uptake in vivo. These, together with inhibition of whole-body glucose infusion during insulin, may caution against the use of isoenzyme-5-specific cyclic GMP phosphodiesterase inhibitors as therapeutic agents.

  20. GABA deficiency in NF1

    PubMed Central

    Patricio, Miguel; Bernardino, Inês; Rebola, José; Abrunhosa, Antero J.; Ferreira, Nuno; Castelo-Branco, Miguel

    2016-01-01

    Objective: To provide a comprehensive investigation of the γ-aminobutyric acid (GABA) system in patients with neurofibromatosis type 1 (NF1) that allows understanding the nature of the GABA imbalance in humans at pre- and postsynaptic levels. Methods: In this cross-sectional study, we employed multimodal imaging and spectroscopy measures to investigate GABA type A (GABAA) receptor binding, using [11C]-flumazenil PET, and GABA concentration, using magnetic resonance spectroscopy (MRS). Fourteen adult patients with NF1 and 13 matched controls were included in the study. MRS was performed in the occipital cortex and in a frontal region centered in the functionally localized frontal eye fields. PET and MRS acquisitions were performed in the same day. Results: Patients with NF1 have reduced concentration of GABA+ in the occipital cortex (p = 0.004) and frontal eye fields (p = 0.026). PET results showed decreased binding of GABAA receptors in patients in the parieto-occipital cortex, midbrain, and thalamus, which are not explained by decreased gray matter levels. Conclusions: Abnormalities in the GABA system in NF1 involve both GABA concentration and GABAA receptor density suggestive of neurodevelopmental synaptopathy with both pre- and postsynaptic involvement. PMID:27473134

  1. Microperfusion of 3-MPA into the brain augments GABA

    PubMed Central

    Mayer, Andrew P.; Osorio, Ivan; Lunte, Craig E.

    2014-01-01

    In vivo effects of microperfusion of a GABA synthesis inhibitor (3-MPA) into the striatum and hippocampus on amino acid concentrations and electrical neuronal activity were investigated. Paradoxical elevations in GABA in the striatum (5-fold in anesthetized and 50-fold in awake rats) and hippocampus (2-fold in anesthetized and 15-fold in awake rats) were documented under steady-state concentrations of 3-MPA along with expected increases in glutamate (a 15-fold increase and a 250-fold increase in the striatum of anesthetized and awake rats, respectively; a 7-fold increase and a 25-fold increase in the hippocampus of anesthetized and awake rats, respectively). There was no clear epileptiform or seizure activity. Explanations for the paradoxical increase in GABA are offered, and emphasis is placed on the dependency of disinhibition on the model in which its effects are studied as well as on the prevailing level of activation of the probed network. PMID:24094842

  2. Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin-like activity.

    PubMed

    De Petrocellis, L; Bisogno, T; Davis, J B; Pertwee, R G; Di Marzo, V

    2000-10-13

    Some synthetic agonists of the VR1 vanilloid (capsaicin) receptor also inhibit the facilitated transport into cells of the endogenous cannabinoid anandamide (arachidonoylethanolamide, AEA). Here we tested several AEA derivatives containing various derivatized phenyl groups or different alkyl chains as either inhibitors of the AEA membrane transporter (AMT) in intact cells or functional agonists of the VR1 vanilloid receptor in HEK cells transfected with the human VR1. We found that four known AMT inhibitors, AM404, arvanil, olvanil and linvanil, activate VR1 receptors at concentrations 400-10000-fold lower than those necessary to inhibit the AMT. However, we also found three novel AEA derivatives, named VDM11, VDM12 and VDM13, which inhibit the AMT as potently as AM404 but exhibit little or no agonist activity at hVR1. These compounds are weak inhibitors of AEA enzymatic hydrolysis and poor CB(1)/CB(2) receptor ligands. We show for the first time that, despite the overlap between the chemical moieties of AMT inhibitors and VR1 agonists, selective inhibitors of AEA uptake that do not activate VR1 (e.g. VDM11) can be developed.

  3. R59949, a diacylglycerol kinase inhibitor, inhibits inducible nitric oxide production through decreasing transplasmalemmal L-arginine uptake in vascular smooth muscle cells.

    PubMed

    Shimomura, Tomoko; Nakano, Tomoyuki; Goto, Kaoru; Wakabayashi, Ichiro

    2017-02-01

    Although diacylglycerol kinase (DGK) is known to be expressed in vascular smooth muscle cell, its functional significance remains to be clarified. We hypothesized that DGK is involved in the pathway of cytokine-induced nitric oxide (NO) production in vascular smooth muscle cells. The purpose of this study was to investigate the effects of R59949, a diacylglycerol kinase inhibitor, on inducible nitric oxide production in vascular smooth muscle cell. Cultured rat aortic smooth muscle cells (RASMCs) were used to elucidate the effects of R59949 on basal and interleukin-1β (IL-1β)-induced NO production. The effects of R59949 on protein and mRNA expression of induced nitric oxide synthase (iNOS) and on transplasmalemmal L-arginine uptake were also evaluated using RASMCs. Treatment of RASMCs with R59949 (10 μM) inhibited IL-1β (10 ng/ml)-induced NO production but not basal NO production. Neither protein nor mRNA expression level of iNOS after stimulation with IL-1β was significantly affected by R59949. Estimated enzymatic activities of iNOS in RASMCs were comparable in the absence and presence of R59949. Stimulation of RASMCs with IL-1β caused a marked increase in transplasmalemmal L-arginine uptake into RASMCs. L-Arginine uptake in the presence of IL-1β was markedly inhibited by R59949, while basal L-arginine uptake was not significantly affected by R59949. Both IL-1β-induced NO production and L-arginine uptake were abolished in the presence of cycloheximide (1 μM). The results indicate that R59949 inhibits inducible NO production through decreasing transplasmalemmal L-arginine uptake. DGK is suggested to be involved in cytokine-stimulated L-arginine transport and regulate its intracellular concentration in vascular smooth muscle cell.

  4. Influence of proton-pump inhibitors on stomach wall uptake of 99mTc-tetrofosmin in cadmium-zinc-telluride SPECT myocardial perfusion imaging.

    PubMed

    Mouden, Mohamed; Rijkee, Karlijn S; Schreuder, Nanno; Timmer, Jorik R; Jager, Pieter L

    2015-02-01

    Proton-pump inhibitors (PPIs) induce potentially interfering stomach wall activity in single-photon emission computed tomography myocardial perfusion imaging (SPECT-MPI) with technetium-99m ((99m)Tc)-sestamibi. However, no data are available for (99m)Tc-tetrofosmin. We assessed the influence of prolonged (>2 weeks) PPI use on the stomach wall uptake of (99m)Tc-tetrofosmin in patients referred for stress MPI with a cadmium-zinc-telluride-based SPECT camera and its relation with dyspepsia symptoms. Consecutive patients (n=127) underwent a 1-day adenosine stress-first SPECT-MPI with (99m)Tc-tetrofosmin, of whom 54 (43%) patients had been on PPIs for more than 2 weeks. Stomach wall activity was identified on stress SPECT using computed tomographic attenuation maps and was scored using a four-point grading scale into clinically relevant (scores 2 or 3) or nonrelevant (scores 0 or 1).Patients on PPIs had stomach wall uptake more frequently as compared with patients not using PPIs (22 vs. 7%, P=0.017). Dyspepsia was similar in both groups. Prolonged use of PPIs is associated with stomach wall uptake of (99m)Tc-tetrofosmin in stress cadmium-zinc-telluride-SPECT images. Gastric symptoms were not associated with stomach wall uptake.

  5. Actions of insecticides on the insect GABA receptor complex

    SciTech Connect

    Bermudez, I.; Hawkins, C.A.; Taylor, A.M.; Beadle, D.J. )

    1991-01-01

    The actions of insecticides on the insect gamma-aminobutyric acid (GABA) receptor were investigated using (35S)t-butylbicyclophosphorothionate (( 35S)TBPS) binding and voltage-clamp techniques. Specific binding of (35S)TBPS to a membrane homogenate derived from the brain of Locusta migratoria locusts is characterised by a Kd value of 79.3 {plus minus} 2.9 nM and a Bmax value of 1770 {plus minus} 40 fmol/mg protein. (35S)TBPS binding is inhibited by mM concentrations of barbiturates and benzodiazepines. In contrast dieldrin, ivermectin, lindane, picrotoxin and TBPS are inhibitors of (35S)TBPS binding at the nanomolar range. Bicuculline, baclofen and pyrethroid insecticides have no effect on (35S)TBPS binding. These results are similar to those obtained in electrophysiological studies of the current elicited by GABA in both Locusta and Periplaneta americana central neurones. Noise analysis of the effects of lindane, TBPS, dieldrin and picrotoxin on the cockroach GABA responses reveals that these compounds decrease the variance of the GABA-induced current but have no effect on its mean open time. All these compounds, with the exception of dieldrin, significantly decrease the conductance of GABA-evoked single current.

  6. Aldose reductase inhibitor improves insulin-mediated glucose uptake and prevents migration of human coronary artery smooth muscle cells induced by high glucose.

    PubMed

    Yasunari, K; Kohno, M; Kano, H; Minami, M; Yoshikawa, J

    2000-05-01

    We examined involvement of the polyol pathway in high glucose-induced human coronary artery smooth muscle cell (SMC) migration using Boyden's chamber method. Chronic glucose treatment for 72 hours potentiated, in a concentration-dependent manner (5.6 to 22.2 mol/L), platelet-derived growth factor (PDGF) BB-mediated SMC migration. This potentiation was accompanied by an increase in PDGF BB binding, because of an increased number of PDGF-beta receptors, and this potentiation was blocked by the aldose reductase inhibitor epalrestat. Epalrestat at concentrations of 10 and 100 nmol/L inhibited high glucose-potentiated (22.2 mmol/L), PDGF BB-mediated migration. Epalrestat at 100 nmol/L inhibited a high glucose-induced increase in the reduced/oxidized nicotinamide adenine dinucleotide ratio and membrane-bound protein kinase C (PKC) activity in SMCs. PKC inhibitors calphostin C (100 nmol/L) and chelerythrine (1 micromol/L) each inhibited high glucose-induced, PDGF BB-mediated SMC migration. High glucose-induced suppression of insulin-mediated [(3)H]-deoxyglucose uptake, which was blocked by both calphostin C (100 nmol/L) and chelerythrine (1 micromol/L), was decreased by epalrestat (100 nmol/L). Chronic high glucose treatment for 72 hours increased intracellular oxidative stress, which was directly measured by flow cytometry using carboxydichlorofluorescein diacetate bis-acetoxymethyl ester, and this increase was significantly suppressed by epalrestat (100 nmol/L). Antisense oligonucleotide to PKC-beta isoform inhibited high glucose-mediated changes in SMC migration, insulin-mediated [(3)H]-deoxyglucose uptake, and oxidative stress. These findings suggest that high glucose concentrations potentiate SMC migration in coronary artery and that the aldose reductase inhibitor epalrestat inhibits high glucose-potentiated, PDGF BB-induced SMC migration, possibly through suppression of PKC (PKC-beta), impaired insulin-mediated glucose uptake, and oxidative stress.

  7. Frequency-dependent depression of inhibition in guinea-pig neocortex in vitro by GABAB receptor feed-back on GABA release.

    PubMed Central

    Deisz, R A; Prince, D A

    1989-01-01

    induced constant responses; at higher frequencies, the responses sometimes declined. The current-voltage relationships of the two GABA responses were similar to each other and to the early IPSP. An apparently fivefold higher conductance was estimated at lower Ems, suggesting that the GABA response had a voltage sensitivity. The slope conductance of IPSPs was decreased by up to 50% for tens of seconds after postsynaptically detectable effects of GABA had dissipated. 6. Application of the GABA uptake inhibitor nipecotic acid (50-500 microM) reduced the conductance of both components of orthodromically evoked inhibition and shortened the IPSP at low frequencies, but had no additional effects at higher stimulation rates.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2557431

  8. Combinational Spinal GAD65 Gene Delivery and Systemic GABA-Mimetic Treatment for Modulation of Spasticity

    PubMed Central

    Kakinohana, Osamu; Hefferan, Michael P.; Miyanohara, Atsushi; Nejime, Tetsuya; Marsala, Silvia; Juhas, Stefan; Juhasova, Jana; Motlik, Jan; Kucharova, Karolina; Strnadel, Jan; Platoshyn, Oleksandr; Lazar, Peter; Galik, Jan; Vinay, Laurent; Marsala, Martin

    2012-01-01

    Background Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABAB receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments. Methods/Principal Findings Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2–3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene. Conclusions/Significance These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel

  9. Neuronal calcium channel inhibitors. Synthesis of omega-conotoxin GVIA and effects on 45Ca uptake by synaptosomes

    SciTech Connect

    Rivier, J.; Galyean, R.; Gray, W.R.; Azimi-Zonooz, A.; McIntosh, J.M.; Cruz, L.J.; Olivera, B.M.

    1987-01-25

    We previously described a 27-amino acid peptide neurotoxin from the venom of Conus geographus, omega-conotoxin GVIA, which inhibits neuronal voltage-activated calcium channels. In this paper we describe the total synthesis of omega-conotoxin GVIA and demonstrate that it efficiently blocks voltage-activated uptake of /sup 45/Ca by standard synaptosomal preparations from chick brain. Dihydropyridines do not block /sup 45/Ca uptake under these conditions. Thus, the omega-conotoxin-sensitive, but dihydropyridine-insensitive uptake of /sup 45/Ca/sup 2 +/ by chick brain synaptosomes serves as a functional assay for a Ca channel target of omega-conotoxin. The use of synthetic GVIA should rapidly accelerate our understanding of the molecular biology of Ca/sup 2 +/ channels and their role in neuronal function.

  10. Evidence That GABA Mediates Dopaminergic and Serotonergic Pathways Associated with Locomotor Activity in Juvenile Chinook Salmon (Oncorhynchus tshawytscha)

    USGS Publications Warehouse

    Clements, S.; Schreck, C.B.

    2004-01-01

    The authors examined the control of locomotor activity in juvenile salmon (Oncorhynchus tshawytscha) by manipulating 3 neurotransmitter systems-gamma-amino-n-butyric acid (GABA), dopamine, and serotonin-as well as the neuropeptide corticotropin releasing hormone (CRH). Intracerebroventricular (ICV) injections of CRH and the GABAAagonist muscimol stimulated locomotor activity. The effect of muscimol was attenuated by administration of a dopamine receptor antagonist, haloperidol. Conversely, the administration of a dopamine uptake inhibitor (4???,4??? -difluoro-3-alpha-[diphenylmethoxy] tropane hydrochloride [DUI]) potentiated the effect of muscimol. They found no evidence that CRH-induced hyperactivity is mediated by dopaminergic systems following concurrent injections of haloperidol or DUI with CRH. Administration of muscimol either had no effect or attenuated the locomotor response to concurrent injections of CRH and fluoxetine, whereas the GABAA antagonist bicuculline methiodide potentiated the effect of CRH and fluoxetine.

  11. Does extracellular calcium determine what pool of GABA is the target for alpha-latrotoxin?

    PubMed

    Storchak, L G; Linetska, M V; Himmelreich, N H

    2002-04-01

    Presynaptic neurotoxin alpha-latrotoxin, from the venom of Latrodectus mactans tredecimguttatus, causes massive [(3)H]GABA release from rat brain synaptosomes, irrespective of calcium presence in the extracellular medium. Whether the binding of alpha-latrotoxin to Ca(2+)-dependent (neurexin 1 alpha) or to Ca(2+)-independent (latrophilin) receptor triggers [(3)H]GABA release by the same mechanisms or different ones, inducing either exocytotic process or outflow by mobile membrane GABA transporter, is unknown. We examined alpha-latrotoxin-evoked [(3)H]GABA release from synaptosomes which cytosolic [(3)H]GABA pool was depleted either by applying competitive inhibitors of the GABA transporter, nipecotic acid and 2,4-diaminobutyric acid, or by permeation with digitonin. We also compared the effect of the GABA transporter inhibitors on depolarisation-evoked and alpha-latrotoxin-evoked [(3)H]GABA release using as depolarising agents 4-aminopyridine and high KCl in the Ca(2+)-containing and in Ca(2+)-free medium, respectively. Incubation of synaptosomes with nipecotic acid induced the essential acceleration of unstimulated [(3)H]GABA release and deep inhibition of high KCl-evoked Ca(2+)-independent [(3)H]GABA release. In contrast, at the similar conditions the effect of alpha-latrotoxin was greatly augmented with respect to the control response. Another way to assay what GABA pool was involved in alpha-latrotoxin-induced release lays in an analysis of the effects of depolarisation and alpha-latrotoxin in consecutive order. The preliminary 4-aminopyridine-stimulated [(3)H]GABA release attenuated the toxin effect. But when depolarisation occurred in Ca(2+)-free medium, no influence on alpha-latrotoxin effect was revealed. Employing digitonin-permeated synaptosomes, we have shown that alpha-latrotoxin could stimulate [3H]GABA release in the medium with 1mM EGTA, this effect of the toxin was blocked by concanavalin A and was ATP-dependent. The latter suggests that alpha

  12. Participation of a GABA-ergic system in the processes of neuroimmunomodulation.

    PubMed

    Devoino, L; Idova, G; Beletskaya, I

    1992-01-01

    Participation of a GABA-ergic system in neuroimmunomodulation was established through the use of a large number of chemical compounds which selectively modulate the activity of the GABA-BD-receptor-ionophore complex. Activation of the GABA-receptors with muscimol or activation of the BD-receptors with diazepam or tazepam had stimulatory effects upon immunogenesis. A decrease in the GABA-BD-receptor-ionophore complex activity led to a suppression of the immune response. The effect was achieved with: a blockade of the complex with bicuculline--a competitive inhibitor of the GABA-receptors: administration of a specific antagonist of the BD-receptors flumazenil or Ro 15-3505: or with blockade of chloride channels with picrotoxin. Activation of the GABA-ergic system causes an increase in bone marrow content of T-helper cells marked by L3T4. The immunomodulatory action of the GABA-ergic system is of central origin and can occur only when the hypothalamo-pituitary system is intact. Section of the pituitary stalk prevents accumulation of the T-helper cells in the bone marrow. The result show that the influence of GABA-ergic system on immunogenesis requires participation of both dopaminergic and serotoninergic systems.

  13. Increased uptake of [¹²³I]meta-iodobenzylguanidine, [¹⁸F]fluorodopamine, and [³H]norepinephrine in mouse pheochromocytoma cells and tumors after treatment with the histone deacetylase inhibitors.

    PubMed

    Martiniova, Lucia; Perera, Shiromi M; Brouwers, Frederieke M; Alesci, Salvatore; Abu-Asab, Mones; Marvelle, Amanda F; Kiesewetter, Dale O; Thomasson, David; Morris, John C; Kvetnansky, Richard; Tischler, Arthur S; Reynolds, James C; Fojo, Antonio Tito; Pacak, Karel

    2011-02-01

    [¹³¹I]meta-iodobenzylguanidine ([¹³¹I]MIBG) is the most commonly used treatment for metastatic pheochromocytoma and paraganglioma. It enters the chromaffin cells via the membrane norepinephrine transporter; however, its success has been modest. We studied the ability of histone deacetylase (HDAC) inhibitors to enhance [¹²³I]MIBG uptake by tumors in a mouse metastatic pheochromocytoma model. HDAC inhibitors are known to arrest growth, induce differentiation and apoptosis in various cancer cells, and further inhibit tumor growth. We report the in vitro and in vivo effects of two HDAC inhibitors, romidepsin and trichostatin A, on the uptake of [(3)H]norepinephrine, [¹²³I]MIBG, and [(18)F]fluorodopamine in a mouse model of metastatic pheochromocytoma. The effects of both inhibitors on norepinephrine transporter activity were assessed in mouse pheochromocytoma (MPC) cells by using the transporter-blocking agent desipramine and the vesicular-blocking agent reserpine. HDAC inhibitors increased [(3)H]norepinephrine, [¹²³I]MIBG, and [(18)F]fluorodopamine uptake through the norepinephrine transporter in MPC cells. In vivo, inhibitor treatment resulted in significantly increased uptake of [(18)F]fluorodopamine positron emission tomography (PET) in pheochromocytoma liver metastases (19.1 ± 3.2% injected dose per gram of tumor (%ID/g) compared to liver metastases in pretreatment scans 5.9 ± 0.6%; P<0.001). Biodistribution analysis after inhibitors treatment confirmed the PET results. The uptake of [(123)I]MIBG was significantly increased in liver metastases 9.5 ± 1.1% compared to 3.19 ± 0.4% in untreated control liver metastases (P<0.05). We found that HDAC inhibitors caused an increase in the amount of norepinephrine transporter expressed in tumors. HDAC inhibitors may enhance the therapeutic efficacy of [(131)I]MIBG treatment in patients with advanced malignant pheochromocytoma and paraganglioma.

  14. Quinolyl analogues of norlobelane: novel potent inhibitors of [(3)H]dihydrotetrabenazine binding and [(3)H]dopamine uptake at the vesicular monoamine transporter-2.

    PubMed

    Ding, Derong; Nickell, Justin R; Dwoskin, Linda P; Crooks, Peter A

    2015-07-01

    We have previously shown that quinolyl moieties are attractive structural replacements for the phenyl groups in lobelane. These quinolyl analogues had improved water-solubility over lobelane and retained the potent vesicular monoamine transporter-2 (VMAT-2) inhibitory properties of the parent compound, with quinlobelane (4) exhibiting potent inhibition of uptake at VMAT-2 (Ki=51nM). However, the VMAT-2 inhibitory properties of quinolyl analogues of norlobelane, which is equipotent with lobeline as an inhibitor of [(3)H]dopamine (DA) uptake at VMAT-2, have not been reported. In the current communication, we describe the synthesis of some novel des-methyl quinolyl analogues of lobelane that exhibit greater affinity (Ki=178-647nM) for the dihydrotetrabenazine binding site located on VMAT-2 compared with lobelane (Ki=970nM), norlobelane (Ki=2310nM) and quinlobelane (Ki=2640nM). The most potent compounds, 14 and 15, also exhibited inhibition of [(3)H]DA uptake at VMAT-2 (Ki=42nM) which was comparable to both lobelane (Ki=45nM) and norlobelane (Ki=43nM). Results reveal that binding affinity at VMAT-2 serves as an accurate predictor of inhibition of the function of VMAT-2 for the majority of these analogues. These novel analogues are under consideration for further development as treatments for methamphetamine abuse. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Differential effects of GABA in modulating nociceptive vs. non-nociceptive synapses.

    PubMed

    Wang, Y; Summers, T; Peterson, W; Miiller, E; Burrell, B D

    2015-07-09

    GABA (γ-amino-butyric acid) -mediated signaling is normally associated with synaptic inhibition due to ionotropic GABA receptors that gate an inward Cl(-) current, hyperpolarizing the membrane potential. However, there are also situations where ionotropic GABA receptors trigger a Cl(-) efflux that results in depolarization. The well-characterized central nervous system of the medicinal leech was used to study the functional significance of opposing effects of GABA at the synaptic circuit level. Specifically, we focused on synapses made by the nociceptive N cell and the non-nociceptive P (pressure) cell that converge onto a common postsynaptic target. It is already known that GABA hyperpolarizes the P cell, but depolarizes the N cell and that inhibition of ionotropic GABA receptors by bicuculline (BIC) has opposing effects on the synapses made by these two inputs; enhancing P cell synaptic transmission, but depressing N cell synapses. The goal of the present study was to determine whether the opposing effects of GABA were due to differences in Cl(-) homeostasis between the two presynaptic neurons. VU 0240551 (VU), an inhibitor of the Cl(-) exporter K-Cl co-transporter isoform 2 (KCC2), attenuated GABA-mediated hyperpolarization of the non-nociceptive afferent while bumetanide (BUM), an inhibitor of the Cl(-) importer Na-K-Cl co-transporter isoform 1 (NKCC1), reduced GABA-mediated depolarization of the nociceptive neuron. VU treatment also enhanced P cell synaptic signaling, similar to the previously observed effects of BIC and consistent with the idea that GABA inhibits synaptic signaling at the presynaptic level. BUM treatment depressed N cell synapses, again similar to what is observed following BIC treatment and suggests that GABA has an excitatory effect on these synapses. The opposing effects of GABA could also be observed at the behavioral level with BIC and VU increasing responsiveness to non-nociceptive stimulation while BIC and BUM decreased responsiveness

  16. Selaginellin and biflavonoids as protein tyrosine phosphatase 1B inhibitors from Selaginella tamariscina and their glucose uptake stimulatory effects.

    PubMed

    Nguyen, Phi-Hung; Ji, Da-Jung; Han, Yu-Ran; Choi, Jae-Sue; Rhyu, Dong-Young; Min, Byung-Sun; Woo, Mi-Hee

    2015-07-01

    As part of an ongoing search for new antidiabetic agents from medicinal plants, the methanol extract of the aerial parts of Selaginella tamariscina was found to possess stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. Thus, bioassay-guided isolation of this active extract yielded two new compounds (1 and 2) along with five known biflavonoids (3-7). Their structures were elucidated by extensive analysis of spectroscopic and physicochemical data. The absolute configuration of compound 2 was determined by specific rotation and CD data analysis. All isolates exhibited potent inhibitory effects on PTP1B enzyme with IC50 values ranging from 4.5±0.1 to 13.2±0.8μM. Furthermore, the isolates (1-7) showed significant stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. Of these, compounds (1, 6, and 7) which exhibited mixed-competitive inhibition modes against PTP1B, showed potent stimulatory effects on 2-NBDG uptake. This result indicated the potential of these biflavonoids as lead molecules for development of antidiabetic agents and the beneficial use of S. tamariscina against hyperglycemia.

  17. GABA protects pancreatic beta cells against apoptosis by increasing SIRT1 expression and activity.

    PubMed

    Prud'homme, Gérald J; Glinka, Yelena; Udovyk, Oleksandr; Hasilo, Craig; Paraskevas, Steven; Wang, Qinghua

    2014-09-26

    We have previously shown that GABA protects pancreatic islet cells against apoptosis and exerts anti-inflammatory effects. Notably, GABA inhibited the activation of NF-κB in both islet cells and lymphocytes. NF-κB activation is detrimental to beta cells by promoting apoptosis. However, the mechanisms by which GABA mediates these effects are unknown. Because the above-mentioned effects mimic the activity of sirtuin 1 (SIRT1) in beta cells, we investigated whether it is involved. SIRT1 is an NAD(+)-dependent deacetylase that enhances insulin secretion, and counteracts inflammatory signals in beta cells. We found that the incubation of a clonal beta-cell line (rat INS-1) with GABA increased the expression of SIRT1, as did GABA receptor agonists acting on either type A or B receptors. NAD(+) (an essential cofactor of SIRT1) was also increased. GABA augmented SIRT1 enzymatic activity, which resulted in deacetylation of the p65 component of NF-κB, and this is known to interfere with the activation this pathway. GABA increased insulin production and reduced drug-induced apoptosis, and these actions were reversed by SIRT1 inhibitors. We examined whether SIRT1 is similarly induced in newly isolated human islet cells. Indeed, GABA increased both NAD(+) and SIRT1 (but not sirtuins 2, 3 and 6). It protected human islet cells against spontaneous apoptosis in culture, and this was negated by a SIRT1 inhibitor. Thus, our findings suggest that major beneficial effects of GABA on beta cells are due to increased SIRT1 and NAD(+), and point to a new pathway for diabetes therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Regulation of GABA and benzodiazepine receptors following neurotoxin-induced striatal and medial forebrain bundle lesions

    SciTech Connect

    Pan, H.S.I.

    1985-01-01

    GABA, a major inhibitory transmitter, is used by many projection neurons of the striatum. To investigate the role of GABA in striatal function, the GABA receptor complex was studied after lesions of the striatum or the nigrostriatal neurons. Quantitative receptor autoradiography using thaw-mounted tissue slices was developed for the study of GABA and benzodiazepine (BDZ) receptors. With the technique established, binding to GABA and BDZ receptors after unilateral striatal kainate lesions was examined. Subsequently, changes in GABA and BDZ receptors were studied following the destruction of dopaminergic nigrostriatal cells by unilateral 6-hydroxydopamine lesion of the medial forebrain bundle. In summary, quantitative receptor autoradiography allowed the detection of GABA and BDZ receptor changes in multiple small areas in each lesioned brain. This technique made it feasible to carry out kinetic saturation, and competition studies using less than 1 mg of tissue. The data suggest that dopamine is functionally inhibitory on striatopallidal neurons but is functionally excitatory on striatoentopeduncular and striatonigral cells which in turn inhibit the thalamus. This quantitative autoradiographic technique can be generalized to study other transmitter receptors and can be combined with 2-deoxyglucose uptake studies.

  19. GAD67-mediated GABA Synthesis and Signaling Regulate Inhibitory Synaptic Innervation in the Visual Cortex

    PubMed Central

    Chattopadhyaya, Bidisha; Di Cristo, Graziella; Wu, Cai Zhi; Knott, Graham; Kuhlman, Sandra; Fu, Yu; Palmiter, Richard D.; Huang, Z. Josh

    2007-01-01

    The development of GABAergic inhibitory circuits is shaped by neural activity, but the underlying mechanisms are unclear. we demonstrate a novel function of GABA in regulating GABAergic innervation in the adolescent brain, when GABA is mainly known as an inhibitory transmitter. Conditional knockdown of the rate-limiting synthetic enzyme GAD67 in basket interneurons in adolescent visual cortex resulted in cell autonomous deficits in axon branching, perisomatic synapse formation around pyramidal neurons, and complexity of the innervation fields; the same manipulation had little influence on the subsequent maintenance of perisomatic synapses. These effects of GABA deficiency were rescued by suppressing GABA re-uptake and by GABA receptor agonists. Germ-line knockdown of GAD67 but not GAD65 showed similar deficits, suggesting a specific role of GAD67 in the maturation of perisomatic innervation. Since intracellular GABA levels are modulated by neuronal activity, our results implicate GAD67-mediated GABA synthesis in activity-dependent regulation of inhibitory innervation patterns. PMID:17582330

  20. GABA transporters in the mammalian cerebral cortex: localization, development and pathological implications.

    PubMed

    Conti, Fiorenzo; Minelli, Andrea; Melone, Marcello

    2004-07-01

    The extracellular levels of gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the mammalian cerebral cortex, are regulated by specific high-affinity, Na+/Cl- dependent transporters. Four distinct genes encoding GABA transporters (GATs), named GAT-1, GAT-2, GAT-3, and BGT-1 have been identified using molecular cloning. Of these, GAT-1 and -3 are expressed in the cerebral cortex. Studies of the cortical distribution, cellular localization, ontogeny and relationships of GATs with GABA-releasing elements using a variety of light and electron microscopic immunocytochemical techniques have shown that: (i) a fraction of GATs is strategically placed to mediate GABA uptake at fast inhibitory synapses, terminating GABA's action and shaping inhibitory postsynaptic responses; (ii) another fraction may participate in functions such as the regulation of GABA's diffusion to neighboring synapses and of GABA levels in cerebrospinal fluid; (iii) GATs may play a role in the complex processes regulating cortical maturation; and (iv) GATs may contribute to the dysregulation of neuronal excitability that accompanies at least two major human diseases: epilepsy and ischemia.

  1. A study on the involvement of GABA-transaminase in MCT induced pulmonary hypertension.

    PubMed

    Lingeshwar, Poorella; Kaur, Gurpreet; Singh, Neetu; Singh, Seema; Mishra, Akanksha; Shukla, Shubha; Ramakrishna, Rachumallu; Laxman, Tulsankar Sachin; Bhatta, Rabi Sankar; Siddiqui, Hefazat H; Hanif, Kashif

    2016-02-01

    Increased sympathetic nervous system (SNS) activity is associated with cardiovascular diseases but its role has not been completely explored in pulmonary hypertension (PH). Increased SNS activity is distinguished by elevated level of norepinephrine (NE) and activity of γ-Amino butyric acid Transminase (GABA-T) which degrades GABA, an inhibitory neurotransmitter within the central and peripheral nervous system. Therefore, we hypothesized that GABA-T may contribute in pathophysiology of PH by modulating level of GABA and NE. The effect of daily oral administration of GABA-T inhibitor, Vigabatrin (GVG, 50 and 75 mg/kg/day, 35 days) was studied following a single subcutaneous administration of monocrotaline (MCT, 60 mg/kg) in male SD rats. The pressure and hypertrophy of right ventricle (RV), oxidative stress, inflammation, pulmonary vascular remodelling were assessed after 35 days in MCT treated rats. The expression of GABA-T and HIF-1α was studied in lung tissue. The levels of plasma NE (by High performance liquid chromatography coupled with electrochemical detector; HPLC-ECD) and lung GABA (by liquid chromatography-mass spectrometry) were also estimated. GVG at both doses significantly attenuated increased in pressure (35.82 ± 4.80 mm Hg, p < 0.001; 28.37 ± 3.32 mm Hg, p < 0.001 respectively) and hypertrophy of RV, pulmonary vascular remodelling, oxidative stress and inflammation in lungs of MCT exposed rats. GVG also reduced the expression of GABA-T and HIF-1α in MCT treated rats. Increased NE level and decreased GABA level was also reversed by GVG in MCT exposed rats. GABA-T plays an important role in PH by modulating SNS activity and may be considered as a therapeutic target in PH.

  2. Effects of 5-HT uptake inhibitors, agonists and antagonists on the burying of harmless objects by mice; a putative test for anxiolytic agents.

    PubMed

    Njung'e, K; Handley, S L

    1991-09-01

    1. The effects of 5-hydroxytryptamine (5-HT) uptake inhibitors, agonists and antagonists have been evaluated on mouse marble-burying behaviour, a putative test for anxiolytic agents. The high levels of locomotor activity occurring on first exposure to a circular runway (runway were used as a separate test of non-specific drug effects. 2. Fluvoxamine, zimeldine, indalpine and citalopram dose-dependently inhibited burying without affecting runway activity. 5-Hydroxytryptophan (5-HTP, with carbidopa), 5-methoxy-N,N-dimethyltryptamine, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OHDPAT), buspirione, gepirone and ipsapirone reduced burying only at doses reducing runway activity. RU 24969 increased runway activity at all effective doses. 1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI), 1,-(3-trifluoromethylphenyl) piperazine (TFMPP) and 1-(3-chlorophenyl)-piperazine (mCPP) potently and differentially reduced burying at doses below those affecting runway activity. 3. 5-HT antagonists only reduced burying at high doses which also reduced runway activity. Burying inhibition by DOI was antagonized by ritanserin, ICI 169,369 and cyproheptadine but not by pindolol or a low (0.25 mg kg-1) dose of metergoline. Burying inhibition by mCPP was not altered by any of these agents except that it was potentiated by pindolol 5 mg kg-1. 4. Zimeldine burying inhibition was potentiated by ritanserine, ICI 169,369, ICS 205-930, cyproheptadine and pindolol. Runway activity was not affected by these drug combinations. 5. Zimeldine was administered in drinking water at a dose of 10 mg kg-1 daily for 21 days. Burying inhibition had disappeared by day 14 and did not recur 24 or 48h after withdrawal at which times responses to DOI were at control levels.6. Selective inhibition of marble burying was not found to be a property of 5-HT-related putative and actual anxiolytics such as buspirone, gepirone, ipsapirone, ritanserin and ondansetron. Nevertheless it was a general property of both 5-HT

  3. Gestational changes of GABA levels and GABA binding in the human uterus

    SciTech Connect

    Erdoe, S.L.; Villanyi, P.; Laszlo, A.

    1989-01-01

    The concentrations of gamma-aminobutyric acid (GABA), the activities of L-glutamate decarboxylase and GABA-transaminase, and the nature of the sodium-independent binding of GABA were examined in uterine tissue pieces obtained surgically from pregnant and non-pregnant women. GABA concentrations were reduced, while the activity of GABA-transaminase and the specific binding of (/sup 3/H)GABA significantly increased in specimens from pregnant subjects. These findings suggest some gestation-related functional role for the GABA system in the human uterus.

  4. Effects of nitrification inhibitors (DCD and DMPP) on nitrous oxide emission, crop yield and nitrogen uptake in a wheat-maize cropping system

    NASA Astrophysics Data System (ADS)

    Liu, C.; Wang, K.; Zheng, X.

    2013-01-01

    The application of nitrification inhibitors together with ammonium-based fertilizers is proposed as a potent method to decrease nitrous oxide (N2O) emission while promoting yield and nitrogen use efficiency in fertilized agricultural fields. To evaluate the effects of nitrification inhibitors, we conducted year-round measurements of N2O fluxes, yield, aboveground biomass, plant carbon and nitrogen contents, soil inorganic nitrogen and dissolved organic carbon contents and the main environmental factors for urea (U), urea + dicyandiamide (DCD) and urea + 3,4-dimethylpyrazol-phosphate (DMPP) treatments in a wheat-maize rotation field. The cumulative N2O emissions were calculated to be 4.49 ± 0.21, 2.93 ± 0.06 and 2.78 ± 0.16 kg N ha-1 yr-1 for the U, DCD and DMPP treatments, respectively. Therefore, the DCD and DMPP treatments decreased the annual emissions by 35% and 38%, respectively. The variations of soil temperature, moisture and inorganic nitrogen content regulated the seasonal fluctuation of N2O emissions. When the emissions presented clearly temporal variations, year-round and high-frequency measurements should be adopted to estimate annual cumulative emissions and treatment effects. The application of nitrification inhibitors increased the soil inorganic nitrogen and dissolved organic carbon availability and shifted the main soil inorganic nitrogen form from nitrate to ammonium. The annual yield, aboveground biomass and nitrogen uptake by aboveground plants increased by 8.5-9.1%, 8.6-9.7% and 10.9-13.2%, respectively, for the DCD and DMPP treatments compared with the U treatment. The results demonstrate the roles the nitrification inhibitors play in enhancing yield and nitrogen use efficiency and reducing N2O emission from the wheat-maize cropping system.

  5. Effects of nitrification inhibitors (DCD and DMPP) on nitrous oxide emission, crop yield and nitrogen uptake in a wheat-maize cropping system

    NASA Astrophysics Data System (ADS)

    Liu, C.; Wang, K.; Zheng, X.

    2013-04-01

    The application of nitrification inhibitors together with ammonium-based fertilizers is proposed as a potent method to decrease nitrous oxide (N2O) emission while promoting crop yield and nitrogen use efficiency in fertilized agricultural fields. To evaluate the effects of nitrification inhibitors, we conducted year-round measurements of N2O fluxes, yield, aboveground biomass, plant carbon and nitrogen contents, soil inorganic nitrogen and dissolved organic carbon contents and the main environmental factors for urea (U), urea + dicyandiamide (DCD) and urea + 3,4-dimethylpyrazol phosphate (DMPP) treatments in a wheat-maize rotation field. The cumulative N2O emissions were calculated to be 4.49 ± 0.21, 2.93 ± 0.06 and 2.78 ± 0.16 kg N ha-1 yr-1 for the U, DCD and DMPP treatments, respectively. Therefore, the DCD and DMPP treatments significantly decreased the annual emissions by 35% and 38%, respectively (p < 0.01). The variations of soil temperature, moisture and inorganic nitrogen content regulated the seasonal fluctuation of N2O emissions. When the emissions presented clearly temporal variations, high-frequency measurements or optimized sampling schedule for intermittent measurements would likely provide more accurate estimations of annual cumulative emission and treatment effect. The application of nitrification inhibitors significantly increased the soil inorganic nitrogen content (p < 0.01); shifted the main soil inorganic nitrogen form from nitrate to ammonium; and tended to increase the dissolved organic carbon content, crop yield, aboveground biomass and nitrogen uptake by aboveground plant. The results demonstrate the roles the nitrification inhibitors play in enhancing yield and nitrogen use efficiency and reducing N2O emission from the wheat-maize cropping system.

  6. Selective serotonin re-uptake inhibitors for the treatment of depression in coronary artery disease and chronic heart failure: evidence for pleiotropic effects.

    PubMed

    Paraskevaidis, Ioannis; Parissis, John T; Fountoulaki, Katerina; Filippatos, Gerasimos; Kremastinos, Dimitrios

    2006-10-01

    Depression is a common co-morbidity in patients with cardiovascular diseases such as chronic coronary artery disease, acute coronary syndromes, post by-pass surgery and chronic heart failure. There is a significant body of evidence suggesting that the presence of depression is independently associated with a decline in health status and an increase in the risk of hospitalization and death for patients with coronary artery disease or congestive heart failure. Novel treatment modalities such as selective serotonin re-uptake inhibitors (SSRIs) may improve depressive symptoms and prognosis of post-myocardial infarction and heart failure patients interacting with the common pathophysiologic mechanisms of depression and cardiovascular disease. This review summarizes current experimental and clinical evidence regarding the pleiotropic effects of SSRIs on platelet functions, immune and neurohormonal activation, and cardiac rhythm disturbances in patients with cardiovascular disease. These bio-modulatory properties of SSRIs may be translated into improvement of patient clinical outcomes beyond their anti-depressant action.

  7. Mutations in the GABA Transporter SLC6A1 Cause Epilepsy with Myoclonic-Atonic Seizures.

    PubMed

    Carvill, Gemma L; McMahon, Jacinta M; Schneider, Amy; Zemel, Matthew; Myers, Candace T; Saykally, Julia; Nguyen, John; Robbiano, Angela; Zara, Federico; Specchio, Nicola; Mecarelli, Oriano; Smith, Robert L; Leventer, Richard J; Møller, Rikke S; Nikanorova, Marina; Dimova, Petia; Jordanova, Albena; Petrou, Steven; Helbig, Ingo; Striano, Pasquale; Weckhuysen, Sarah; Berkovic, Samuel F; Scheffer, Ingrid E; Mefford, Heather C

    2015-05-07

    GAT-1, encoded by SLC6A1, is one of the major gamma-aminobutyric acid (GABA) transporters in the brain and is responsible for re-uptake of GABA from the synapse. In this study, targeted resequencing of 644 individuals with epileptic encephalopathies led to the identification of six SLC6A1 mutations in seven individuals, all of whom have epilepsy with myoclonic-atonic seizures (MAE). We describe two truncations and four missense alterations, all of which most likely lead to loss of function of GAT-1 and thus reduced GABA re-uptake from the synapse. These individuals share many of the electrophysiological properties of Gat1-deficient mice, including spontaneous spike-wave discharges. Overall, pathogenic mutations occurred in 6/160 individuals with MAE, accounting for ~4% of unsolved MAE cases.

  8. Free radical modification of high-affinity GABA transport in the central nervous system

    SciTech Connect

    Debler, E.A.

    1985-01-01

    Presynaptic nerve terminals of DNS neurons can be characterized as dynamic self-contained subunits of the overall neuron. These neuron subunits together with the adjacent post-synaptic terminals constitute the functional unit of neurotransmission. One major function of the presynaptic nerve terminal in neurotransmission is its role in the release and subsequent reuptake of neurotransmitters. Neurochemical and morphological analysis of this high-affinity uptake process, for the amino acid neurotransmitter gamma-aminobutyric acid (GABA), revealed that free radical modification (lipid peroxidation) of presynaptic nerve terminal membrane structures resulted in a decrease in high-affinity (/sup 14/C)GABA uptake. Depolarized nerve terminals exposed to horseradish peroxidase (HRP)-generated superoxide radicals (.O/sub 2//sup -/) exhibited an alteration of synaptic vesicle membrane structure and a reduction of high-affinity (/sup 14/C)GABA uptake. In the kinetic analysis of high affinity (/sup 14/C)GABA uptake, hypoxanthine/xanthine oxidase (HPX/XOD)-treated synaptosomes exhibited a significant reduction in V/sub max/ but no significant alteration of K/sub m/. This HPX/XOD treatment also resulted in significant structural alterations of both the synaptic vesicle membrane and the synaptosomal plasma membrane. Structural and functional correlates appear to exist between synaptic vesicle and plasma membrane systems and high-affinity (/sup 14/C)GABA uptake. Together, the selectivity of the .O/sub 2//sup -/ toxicity and that of the high-K/sup +//Na/sup +/-free effect support the idea of two separate and different amino acid uptake systems in CNS tissue.

  9. Selective serotonin re-uptake inhibitors potentiate gene blunting induced by repeated methylphenidate treatment: Zif268 vs. Homer1a

    PubMed Central

    Van Waes, Vincent; Vandrevala, Malcolm; Beverley, Joel; Steiner, Heinz

    2015-01-01

    There is a growing use of psychostimulants such as methylphenidate (Ritalin; dopamine reuptake inhibitor) for medical treatments and as cognitive enhancers in the healthy. Methylphenidate is known to produce some addiction-related gene regulation. Recent findings in animal models show that selective serotonin reuptake inhibitors (SSRIs) including fluoxetine can potentiate acute induction of gene expression by methylphenidate, thus indicating an acute facilitatory role for serotonin in dopamine-induced gene regulation. We investigated whether repeated exposure to fluoxetine in conjunction with methylphenidate in adolescent rats facilitated a gene regulation effect well-established for repeated exposure to illicit psychostimulants such as cocaine - blunting (repression) of gene inducibility. We measured, by in situ hybridization histochemistry, the effects of a 5-day repeated treatment with methylphenidate (5 mg/kg), fluoxetine (5 mg/kg) or a combination on the inducibility (by cocaine) of neuroplasticity-related genes (Zif268, Homer1a) in the striatum. Repeated methylphenidate treatment alone produced minimal gene blunting, while fluoxetine alone had no effect. In contrast, fluoxetine added to methylphenidate robustly potentiated methylphenidate-induced blunting for both genes. This potentiation was widespread throughout the striatum, but was most robust in the lateral, sensorimotor striatum, thus mimicking cocaine effects. For illicit psychostimulants, blunting of gene expression is considered part of the molecular basis of addiction. Our results thus suggest that SSRIs such as fluoxetine may increase the addiction liability of methylphenidate. PMID:23763573

  10. Effect of GABA agonists and GABA-A receptor modulators on cocaine- and food-maintained responding and cocaine discrimination in rats.

    PubMed

    Barrett, Andrew C; Negus, S Stevens; Mello, Nancy K; Caine, S Barak

    2005-11-01

    Recent studies indicate that GABAergic ligands modulate abuse-related effects of cocaine. The goal of this study was to evaluate the effects of a mechanistically diverse group of GABAergic ligands on the discriminative stimulus and reinforcing effects of cocaine in rats. One group of rats was trained to discriminate 5.6 mg/kg cocaine from saline in a two-lever, food-reinforced, drug discrimination procedure. In two other groups, responding was maintained by cocaine (0-3.2 mg/kg/injection) or liquid food (0-100%) under a fixed ratio 5 schedule. Six GABA agonists were tested: the GABA-A receptor agonist muscimol, the GABA-B receptor agonist baclofen, the GABA transaminase inhibitor gamma-vinyl-GABA (GVG), and three GABA-A receptor modulators (the barbiturate pentobarbital, the high-efficacy benzodiazepine midazolam, and the low-efficacy benzodiazepine enazenil). When tested alone, none of the compounds substituted fully for the discriminative stimulus effects of cocaine. As acute pretreatments, select doses of midazolam and pentobarbital produced 2.2- to 3.6-fold rightward shifts in the cocaine dose-effect function. In contrast, muscimol, baclofen, GVG, and enazenil failed to alter the discriminative stimulus effects of cocaine. In assays of cocaine- and food-maintained responding, midazolam and pentobarbital decreased cocaine self-administration at doses 9.6- and 3.3-fold lower, respectively, than those that decreased food-maintained responding. In contrast, muscimol, baclofen, and GVG decreased cocaine self-administration at doses that also decreased food-maintained responding. Enazenil failed to alter cocaine self-administration. Together with previous studies, these data suggest that among mechanistically diverse GABA agonists, high-efficacy GABA-A modulators may be the most effective for modifying the abuse-related effects of cocaine.

  11. Estimation of ambient GABA levels in layer I of the mouse neonatal cortex in brain slices

    PubMed Central

    Dvorzhak, Anton; Myakhar, Olga; Unichenko, Petr; Kirmse, Knut; Kirischuk, Sergei

    2010-01-01

    GABAergic synapses on Cajal–Retzius neurons in layer I of the murine neocortex experience GABAB receptor (GABABR)-mediated tonic inhibition. Extracellular GABA concentration ([GABA]o) that determines the strength of GABABR-mediated inhibition is controlled by GABA transporters (GATs). In this study, we hypothesized that the strength ofpresynaptic GABABR activation reflects [GABA]o in the vicinity of synaptic contacts. Slices obtained from two age groups were used, namely postnatal days (P)2–3 and P5–7. GABAergic postsynaptic currents (IPSCs) were recorded using the whole-cell patch-clamp technique. Minimal electrical stimulation in layer I was applied to elicit evoked IPSCs (eIPSCs) using a paired-pulse protocol. Three parameters were selected for comparison: the mean eIPSC amplitude, paired-pulse ratio, and failure rate. When GAT-1 and GAT-2/3 were blocked by NO-711 (10 μm) and SNAP-5114 (40 μm), respectively, no tonic GABABR-mediated inhibition was observed. In order to restore the control levels of GABABR-mediated inhibition, 250 and 125 nm exogenous GABA was required at P2–3 and P5–7, respectively. Addition of 3-mercaptopropionic acid, a glutamate decarboxylase inhibitor, did not significantly change the obtained values arguing against the suggestion that a mechanism different from GATs contributes to [GABA]o control. We conclude that juxtasynaptic [GABA]o is higher (about 250 nm) at P2–3 than at P5–7 (about 125 nm). As both radial cell migration and corticogenesis in general are strongly dependent on [GABA]o and the formation of the last layer 2/3 is finished by P4 in rodents, the observed [GABA]o reduction in layer I might reflect this crucial event in the cortical development. PMID:20421290

  12. Striatal cholinergic interneurons Drive GABA release from dopamine terminals.

    PubMed

    Nelson, Alexandra B; Hammack, Nora; Yang, Cindy F; Shah, Nirao M; Seal, Rebecca P; Kreitzer, Anatol C

    2014-04-02

    Striatal cholinergic interneurons are implicated in motor control, associative plasticity, and reward-dependent learning. Synchronous activation of cholinergic interneurons triggers large inhibitory synaptic currents in dorsal striatal projection neurons, providing one potential substrate for control of striatal output, but the mechanism for these GABAergic currents is not fully understood. Using optogenetics and whole-cell recordings in brain slices, we find that a large component of these inhibitory responses derive from action-potential-independent disynaptic neurotransmission mediated by nicotinic receptors. Cholinergically driven IPSCs were not affected by ablation of striatal fast-spiking interneurons but were greatly reduced after acute treatment with vesicular monoamine transport inhibitors or selective destruction of dopamine terminals with 6-hydroxydopamine, indicating that GABA release originated from dopamine terminals. These results delineate a mechanism in which striatal cholinergic interneurons can co-opt dopamine terminals to drive GABA release and rapidly inhibit striatal output neurons. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Striatal cholinergic interneurons drive GABA release from dopamine terminals

    PubMed Central

    Nelson, Alexandra B.; Hammack, Nora; Yang, Cindy F.; Shah, Nirao M.; Seal, Rebecca P.; Kreitzer, Anatol C.

    2014-01-01

    Summary Striatal cholinergic interneurons are implicated in motor control, associative plasticity, and reward-dependent learning. Synchronous activation of cholinergic interneurons triggers large inhibitory synaptic currents in dorsal striatal projection neurons, providing one potential substrate for control of striatal output, but the mechanism for these GABAergic currents is not fully understood. Using optogenetics and whole-cell recordings in brain slices, we find that a large component of these inhibitory responses derive from action-potential-independent disynaptic neurotransmission mediated by nicotinic receptors. Cholinergically-driven IPSCs were not affected by ablation of striatal fast-spiking interneurons, but were greatly reduced after acute treatment with vesicular monoamine transport inhibitors or selective destruction of dopamine terminals with 6-hydroxydopamine, indicating that GABA release originated from dopamine terminals. These results delineate a mechanism in which striatal cholinergic interneurons can co-opt dopamine terminals to drive GABA release and rapidly inhibit striatal output neurons. PMID:24613418

  14. Perinatal hypoxia induces a long-lasting increase in unstimulated gaba release in rat brain cortex and hippocampus. The protective effect of pyruvate.

    PubMed

    Pozdnyakova, N; Yatsenko, L; Parkhomenko, N; Himmelreich, N

    2011-01-01

    Hypoxia and seizures early in life can cause multiple neurological deficits and even chronic epilepsy. Here, we report the data obtained in rats exposed to hypoxia and seizures at age 10-12 postnatal days and taken in experiments 8-9 weeks after hypoxia treatment. A level of the extracellular GABA and the initial velocity of GABA uptake were measured in the brain cortex, hippocampus and thalamus using isolated nerve terminals (synaptosomes). It has been revealed that the extracellular [(3)H]GABA level maintained by cortical and hippocampal synaptosomes in standard conditions (with glucose as an energy substrate) was significantly higher in adult rats exposed to hypoxia/seizures at P10-12 than in the control ones, and, moreover, became unstable with tendency to increase. Pyruvate as a single energy substrate was shown to be a highly effective for lowering and stabilizing the extracellular [(3)H]GABA level. This effect of pyruvate was tightly correlated with increase in GABA uptake and GATs affinity to GABA. Thalamus was insensible to the action of perinatal hypoxia/seizures, and thalamic GATs, in contrast to cortical and hippocampal ones, had a lower affinity to GABA (the apparent Km is 39.2±3.1 μM GABA vs 8.9±1.8 μM GABA in the hippocampus). A selective vulnerability of brain regions to hypoxia is suggested to be attributed to distinct terms of their maturation at the postnatal period. Thus, perinatal hypoxia/seizures evoke a long-lasting increase in the extracellular GABA level that could be attenuated by pyruvate treatment. This effect of pyruvate is likely due to a significant increase in GATs-mediated GABA uptake and modulation of GATs kinetic properties. Copyright © 2010 Elsevier Ltd. All rights reserved.

  15. Novel dose-dependent alterations in excitatory GABA during embryonic development associated with lead (Pb) neurotoxicity

    PubMed Central

    Wirbisky, Sara E.; Weber, Gregory J.; Lee, Jang-Won; Cannon, Jason R.; Freeman, Jennifer L.

    2014-01-01

    Lead (Pb) is a heavy metal that is toxic to numerous physiological processes. Its use in industrial applications is widespread and results in an increased risk of human environmental exposure. The central nervous system (CNS) is most sensitive to Pb exposure during early development due to rapid cell proliferation and migration, axonal growth, and synaptogenesis. One of the key components of CNS development is the Gamma-aminobutyric acid (GABA)ergic system. GABA is the primary inhibitory neurotransmitter in the adult brain. However, during development GABA acts as an excitatory neurotrophic factor which contributes to these cellular processes. Multiple studies report effects of Pb on GABA in the mature brain; however, little is known regarding the adverse effects of Pb exposure on the GABAergic system during embryonic development. To characterize the effects of Pb on the GABAergic system during development, zebrafish embryos were exposed to 10, 50, or 100 ppb Pb or a control treatment. Tissue up-take, gross morphological alterations, gene expression, and neurotransmitter levels were analyzed. Analysis revealed that alterations in gene expression throughout the GABAergic system and GABA levels were dose and developmental time point specific. These data provide a framework for further analysis of the effects of Pb on the GABAergic system during the excitatory phase and as GABA transitions to an inhibitory neurotransmitter during development. PMID:24875535

  16. Novel dose-dependent alterations in excitatory GABA during embryonic development associated with lead (Pb) neurotoxicity.

    PubMed

    Wirbisky, Sara E; Weber, Gregory J; Lee, Jang-Won; Cannon, Jason R; Freeman, Jennifer L

    2014-08-17

    Lead (Pb) is a heavy metal that is toxic to numerous physiological processes. Its use in industrial applications is widespread and results in an increased risk of human environmental exposure. The central nervous system (CNS) is most sensitive to Pb exposure during early development due to rapid cell proliferation and migration, axonal growth, and synaptogenesis. One of the key components of CNS development is the Gamma-aminobutyric acid (GABA)-ergic system. GABA is the primary inhibitory neurotransmitter in the adult brain. However, during development GABA acts as an excitatory neurotrophic factor which contributes to these cellular processes. Multiple studies report effects of Pb on GABA in the mature brain; however, little is known regarding the adverse effects of Pb exposure on the GABAergic system during embryonic development. To characterize the effects of Pb on the GABAergic system during development, zebrafish embryos were exposed to 10, 50, or 100 ppb Pb or a control treatment. Tissue up-take, gross morphological alterations, gene expression, and neurotransmitter levels were analyzed. Analysis revealed that alterations in gene expression throughout the GABAergic system and GABA levels were dose and developmental time point specific. These data provide a framework for further analysis of the effects of Pb on the GABAergic system during the excitatory phase and as GABA transitions to an inhibitory neurotransmitter during development.

  17. Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate.

    PubMed

    Vandevoorde, Séverine; Fowler, Christopher J

    2005-08-01

    There is some dispute concerning the extent to which the uptake inhibitor VDM11 (N-(4-hydroxy-2-methylphenyl) arachidonoyl amide) is capable of inhibiting the metabolism of the endocannabinoid anandamide (AEA) by fatty acid amide hydrolase (FAAH). In view of a recent study demonstrating that the closely related compound AM404 (N-(4-hydroxyphenyl)arachidonylamide) is a substrate for FAAH, we re-examined the interaction of VDM11 with FAAH. In the presence of fatty acid-free bovine serum albumin (BSA, 0.125% w v(-1)), both AM404 and VDM11 inhibited the metabolism of AEA by rat brain FAAH with similar potencies (IC(50) values of 2.1 and 2.6 microM, respectively). The compounds were about 10-fold less potent as inhibitors of the metabolism of 2-oleoylglycerol (2-OG) by cytosolic monoacylglycerol lipase (MAGL). The potency of VDM11 towards FAAH was dependent upon the assay concentration of fatty acid-free bovine serum albumin (BSA). Thus, in the absence of fatty acid-free BSA, the IC(50) value for inhibition of FAAH was reduced by a factor of about two (from 2.9 to 1.6 microM). A similar reduction in the IC(50) value for the inhibition of membrane bound MAGL by both this compound (from 14 to 6 microM) and by arachidonoyl serinol (from 24 to 13 microM) was seen. An HPLC assay was set up to measure 4-amino-m-cresol, the hypothesised product of FAAH-catalysed VDM11 hydrolysis. 4-Amino-m-cresol was eluted with a retention time of approximately 2.4 min, but showed a time-dependent degradation to compounds eluting at peaks of approximately 5.6 and approximately 8 min. Peaks with the same retention times were also found following incubation of the membranes with VDM11, but were not seen when the membranes were preincubated with the FAAH inhibitors URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) and CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1-one) prior to addition of VDM11. The rate of metabolism of VDM11 was estimated to be roughly 15-20% of that for

  18. Methamphetamine-evoked depression of GABA(B) receptor signaling in GABA neurons of the VTA.

    PubMed

    Padgett, Claire L; Lalive, Arnaud L; Tan, Kelly R; Terunuma, Miho; Munoz, Michaelanne B; Pangalos, Menelas N; Martínez-Hernández, José; Watanabe, Masahiko; Moss, Stephen J; Luján, Rafael; Lüscher, Christian; Slesinger, Paul A

    2012-03-08

    Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Suicide trends in discharged patients with mood disorders: associations with selective serotonin uptake inhibitors and comorbid substance misuse.

    PubMed

    Fazel, Seena; Grann, Martin; Goodwin, Guy M

    2006-03-01

    We determined the rates of suicide in two consecutive time intervals of low and high selective serotonin reuptake inhibitor (SSRI) prescribing for men and women with mood disorders, who were at relatively high risk of suicide, in a discharged hospitalized sample. High quality Swedish national registers were cross-linked to estimate rates of suicide. The results were stratified by three age-bands, and rates of comorbid substance misuse were explored. These were compared with general population suicide rates. There were 1606 suicides in discharged patients with mood disorders. Suicide rates decreased for all women during 1989-94 and 1995-2000 [odds ratio (OR) = 0.74, 95% confidence interval (CI) = 0.64-0.85] and a trend was found towards lower rates in men (OR = 0.89, 95% CI = 0.78-1.02). Approximately 90% of suicides occurred in those aged over 40 years; thus, the inpatient sample lacked the power to discriminate clear effects in the younger age groups. There was no increase or decrease in the rates of substance use disorders in patients who committed suicide. There were 20 851 suicides in the general population during 1989-2000. Suicide rates were lower at all ages for men and women in the general population in the interval of high SSRI prescribing. We conclude that there is no evidence from this study of an increase in suicide rates following the introduction of SSRIs in the general population or in a high-risk inpatient sample.

  20. Anion transport and GABA signaling

    PubMed Central

    Hübner, Christian A.; Holthoff, Knut

    2013-01-01

    Whereas activation of GABAA receptors by GABA usually results in a hyperpolarizing influx of chloride into the neuron, the reversed chloride driving force in the immature nervous system results in a depolarizing efflux of chloride. This GABAergic depolarization is deemed to be important for the maturation of the neuronal network. The concept of a developmental GABA switch has mainly been derived from in vitro experiments and reliable in vivo evidence is still missing. As GABAA receptors are permeable for both chloride and bicarbonate, the net effect of GABA also critically depends on the distribution of bicarbonate. Whereas chloride can either mediate depolarizing or hyperpolarizing currents, bicarbonate invariably mediates a depolarizing current under physiological conditions. Intracellular bicarbonate is quickly replenished by cytosolic carbonic anhydrases. Intracellular bicarbonate levels also depend on different bicarbonate transporters expressed by neurons. The expression of these proteins is not only developmentally regulated but also differs between cell types and even subcellular regions. In this review we will summarize current knowledge about the role of some of these transporters for brain development and brain function. PMID:24187533

  1. GABA and Central Neuropathic Pain following Spinal Cord Injury

    PubMed Central

    Gwak, Young S.; Hulsebosch, Claire E.

    2012-01-01

    Spinal cord injury induces maladaptive synaptic transmission in the somatosensory system that results in chronic central neuropathic pain. Recent literature suggests that glial-neuronal interactions are important modulators in synaptic transmission following spinal cord injury. Neuronal hyperexcitability is one of the predominant phenomenon caused by maladaptive synaptic transmission via altered glial-neuronal interactions after spinal cord injury. In the somatosensory system, spinal inhibitory neurons counter balance the enhanced synaptic transmission from peripheral input. For a decade, the literature suggests that hypofunction of GABAergic inhibitory tone is an important factor in the enhanced synaptic transmission that often results in neuronal hyperexcitability in dorsal horn neurons following spinal cord injury. Neurons and glial cells synergistically control intracellular chloride ion gradients via modulation of chloride transporters, extracellular glutamate and GABA concentrations via uptake mechanisms. Thus, the intracellular “GABA-glutamate-glutamine cycle” is maintained for normal physiological homeostasis. However, hyperexcitable neurons and glial activation after spinal cord injury disrupts the balance of chloride ions, glutamate and GABA distribution in the spinal dorsal horn and results in chronic neuropathic pain. In this review, we address spinal cord injury induced mechanisms in hypofunction of GABAergic tone that results in chronic central neuropathic pain. PMID:21216257

  2. Stimulation of [3H] GABA and beta-[3H] alanine release from rat brain slices by cis-4-aminocrotonic acid.

    PubMed

    Chebib, M; Johnston, G A

    1997-02-01

    cis-4-Aminocrotonic acid (CACA; 100 microM), an analogue of GABA in a folded conformation, stimulated the passive release of [3H] GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of beta-[3H]alanine from slices of cerebellum and spinal cord without influencing potassium-evoked release. In contrast, CACA (100 microM) did not stimulate the passive release of [3H]taurine from slices of cerebellum and spinal cord or of D-[3H]aspartate from slices of cerebellum and did not influence potassium-evoked release of [3H]-taurine from the cerebellum and spinal cord and D-[3H]-aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and beta-alanine release are due to CACA acting as a substrate for a beta-alanine-sensitive GABA transport system, consistent with CACA inhibiting the uptake of beta-[3H]alanine into slices of rat cerebellum and cerebral cortex. The observed Ki for CACA against beta-[3H]alanine uptake in the cerebellum was 750 +/- 60 microM. CACA appears to be 10-fold weaker as a substrate for the transporter system than as an agonist for the GABAc receptor. The effects of CACA on GABA and beta-alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, beta-alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT-3, cloned from rat CNS. The structural similarities of GABA, beta-alanine, CACA, and nipecotic acid are demonstrated by computer-aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.

  3. Short- and Long-Term Effects of Interleukin-2 Treatment on the Sensitivity of Periadolescent Female Mice to Interleukin-2 and Dopamine Uptake Inhibitor

    PubMed Central

    Rankin, James S.; Zalcman, Steven S.; Zhu, Youhua; Siegel, Allan

    2013-01-01

    Interleukin (IL)-2, a T-helper 1 (Th1) cell-derived cytokine, which potently modulates dopamine activity and neuronal excitability in mesolimbic structures, is linked with pathological outcomes (e.g., schizophrenia, depression, etc.) that at least partly reflect alterations in central dopaminergic processes. It has been suggested that dopamine neurons undergo pruning during adolescence and abnormalities in pruning predispose individuals to behavioral disorders. Since IL-2 is known as a neurodevelopmental factor affecting associated behavioral processes, the present study tested whether IL-2 can modulate stereotypic behaviors in both the periadolescent and adult periods. This study determined whether IL-2 treatment would produce long-lasting changes in sensitivity to a later challenge with IL-2 or GBR 12909, a highly selective dopamine uptake inhibitor. Four experiments were conducted. Firstly, a decrease in novelty-induced stereotypic behavior was observed in BALB/c periadolescent mice (38 days of age) following IL-2 administration (0.4 µg/2 ml) relative to vehicle control. In the second experiment, an initial dose of IL-2 was given in the periadolescent period, but did not affect rearing responses. A second dose of IL-2 given to the animals 30 days later as adults, resulted in a significant increase in rearing behaviors relative to control animals. In the third experiment, separate groups of experimental and control mice were administered GBR 12909, a highly selective dopamine reuptake inhibitor, 30 days following treatment with either IL-2 or vehicle. It was noted that this experimental group, which initially received IL-2, exhibited stereotypy, as evidenced by increased sniffing behavior. A fourth experiment revealed that IL-2 administered in periadolesecence and adulthood had no effect on other motor responses, indicating that IL-2 selectively modulates selective stereotypic behaviors. The results provide evidence, for the first time, that long-term changes in

  4. Unique pH dynamics in GABAergic synaptic vesicles illuminates the mechanism and kinetics of GABA loading

    PubMed Central

    Egashira, Yoshihiro; Takase, Miki; Watanabe, Shoji; Ishida, Junji; Fukamizu, Akiyoshi; Kaneko, Ryosuke; Yanagawa, Yuchio; Takamori, Shigeo

    2016-01-01

    GABA acts as the major inhibitory neurotransmitter in the mammalian brain, shaping neuronal and circuit activity. For sustained synaptic transmission, synaptic vesicles (SVs) are required to be recycled and refilled with neurotransmitters using an H+ electrochemical gradient. However, neither the mechanism underlying vesicular GABA uptake nor the kinetics of GABA loading in living neurons have been fully elucidated. To characterize the process of GABA uptake into SVs in functional synapses, we monitored luminal pH of GABAergic SVs separately from that of excitatory glutamatergic SVs in cultured hippocampal neurons. By using a pH sensor optimal for the SV lumen, we found that GABAergic SVs exhibited an unexpectedly higher resting pH (∼6.4) than glutamatergic SVs (pH ∼5.8). Moreover, unlike glutamatergic SVs, GABAergic SVs displayed unique pH dynamics after endocytosis that involved initial overacidification and subsequent alkalization that restored their resting pH. GABAergic SVs that lacked the vesicular GABA transporter (VGAT) did not show the pH overshoot and acidified further to ∼6.0. Comparison of luminal pH dynamics in the presence or absence of VGAT showed that VGAT operates as a GABA/H+ exchanger, which is continuously required to offset GABA leakage. Furthermore, the kinetics of GABA transport was slower (τ > 20 s at physiological temperature) than that of glutamate uptake and may exceed the time required for reuse of exocytosed SVs, allowing reuse of incompletely filled vesicles in the presence of high demand for inhibitory transmission. PMID:27601664

  5. GABA interaction with lipids in organic medium

    SciTech Connect

    Beltramo, D.; Kivatinitz, S.; Lassaga, E.; Arce, A.

    1987-08-10

    The interaction of TH-GABA and UC-glutamate with lipids in an aqueous organic partition system was studied. With this partition system TH-GABA and UC-glutamate were able to interact with sphingomyelin, sulfatide, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidic acid but not with cholesterol or ceramide. In an homogeneous aqueous medium the authors could not demonstrate any interaction between TH-GABA-lipids. The apparent dissociation constants (K/sub d/) for TH-GABA-lipids or UC-glutamate-lipids interactions inorganic medium were in the millimolar range and maximal charge between 3 and 7 moles of GABA or glutamate by mole of lipid. Amino acids such as glutamic acid, US -alanine and glycine displaced TH-GABA with the same potency as GABA itself; thus these results show that the interaction lacks pharmacological specificity. To detect this interaction lipid concentrations higher than 2 M were required and in the partition system TH-GABA and lipid phosphorus were both concentrated at the interface. Therefore, lipids tested with a biphasic partition system do not fulfill the classical criteria for a neurotransmitter receptor at least not for GABA and glutamate. 15 references, 1 figure, 3 tables.

  6. GABA pharmacology: the search for analgesics.

    PubMed

    McCarson, Kenneth E; Enna, S J

    2014-10-01

    Decades of research have been devoted to defining the role of GABAergic transmission in nociceptive processing. Much of this work was performed using rigid, orthosteric GABA analogs created by Povl Krogsgaard-Larsen and his associates. A relationship between GABA and pain is suggested by the anatomical distribution of GABA receptors and the ability of some GABA agonists to alter nociceptive responsiveness. Outlined in this report are data supporting this proposition, with particular emphasis on the anatomical localization and function of GABA-containing neurons and the molecular and pharmacological properties of GABAA and GABAB receptor subtypes. Reference is made to changes in overall GABAergic tone, GABA receptor expression and activity as a function of the duration and intensity of a painful stimulus or exposure to GABAergic agents. Evidence is presented that the plasticity of this receptor system may be responsible for the variability in the antinociceptive effectiveness of compounds that influence GABA transmission. These findings demonstrate that at least some types of persistent pain are associated with a regionally selective decline in GABAergic tone, highlighting the need for agents that enhance GABA activity in the affected regions without compromising GABA function over the long-term. As subtype selective positive allosteric modulators may accomplish these goals, such compounds might represent a new class of analgesic drugs.

  7. Correlation between the enhancement of flunitrazepam binding by GABA and seizure susceptibility in mice

    SciTech Connect

    Marley, R.J.; Wehner, J.M.

    1987-06-08

    Various populations of mice exhibit differential sensitivity to seizure-inducing agents. The relationship of seizure susceptibility to alterations in the GABA receptor complex was investigated in six different populations of mice consisting of four inbred strains (C57BL, DBA, C3H, and BALB) and two selected lines (long sleep and short sleep). Seizure activity was induced by intraperitoneal administration of the GAD inhibitor, 3-mercaptopropionic acid, and latencies to seizure onset and tonus were measured. In naive mice of the same populations, GABA enhancement of TH-flunitrazepam binding was measured in extensively washed whole brain membranes at several GABA concentrations. Both differential seizure sensitivity to 3-mercaptopropionic acid and differential enhancement of TH-flunitrazepam binding by GABA were observed in these six populations of mice. Correlational analyses indicated a positive correlation between the degree of GABA enhancement of TH-flunitrazepam binding and resistance to the seizure-inducing properties of 3-mercaptopropionic acid. These data suggest that genetic differences in sensitivity to seizure-inducing agents that disrupt the GABAergic system may be related to differences in coupling between the various receptors associated with the GABA receptor complex.

  8. Fluoxetine, a selective inhibitor of serotonin uptake, potentiates morphine analgesia without altering its discriminative stimulus properties or affinity for opioid receptors

    SciTech Connect

    Hynes, M.D.; Lochner, M.A.; Bemis, K.G.; Hymson, D.L.

    1985-06-17

    The analgesic effect of morphine in the rat tail jerk assay was enhanced by the serotonin uptake inhibitor, fluoxetine. Tail jerk latency was not affected by fluoxetine alone. Morphine's affinity for opioid receptors labeled in vitro with /sup 3/H-naloxone or /sup 3/H-D-Ala/sup 2/-D-Leu/sup 5/-enkephalin was not altered by fluoxetine, which has no affinity for these sites at concentrations as high as 1000 nM. In rats trained to discriminate morphine from saline, fluoxetine at doses of 5 or 10 mg/kg were recognized as saline. Increasing the fluoxetine dose to 20 mg/kg did not result in generalization to either saline or morphine. The dose response curve for morphine generalization was not significantly altered by fluoxetine doses of 5 or 10 mg/kg. Those rats treated with the combination of morphine and 20 mg/kg of fluoxetine did not exhibit saline or morphine appropriate responding. Fluoxetine potentiates the analgesic properties of morphine without enhancing its affinity for opioid receptors or its discriminative stimulus properties. 30 references, 2 figures, 2 tables.

  9. A neurobiological perspective on attachment problems in sexual offenders and the role of selective serotonin re-uptake inhibitors in the treatment of such problems.

    PubMed

    Beech, Anthony R; Mitchell, Ian J

    2005-02-01

    This paper describes what is currently known about attachment from the development, social-cognitive and biological literatures and outlines the impact on organisms given adverse development experiences that can have an effect upon attachment formation in childhood across these three literatures. We then describe the effects that 'insecure' attachment styles arising in childhood can affect brain chemistry and brain function and subsequently adult social/romantic relationships. In the paper, we note that a number of sexual offenders report adverse childhood experiences and that they possess attachment styles that, taken together, make it likely that they will either seek out intimate attachments in ways where they will have sex with children, perhaps confusing sex with intimacy or in aggressive ways as particularly happens with men who sexually assault adult women. The last section of the paper describes chemical treatment for sexual offenders, focusing on the use of selective serotonin re-uptake inhibitors (SSRIs). We note evidence for the role of SSRIs in promoting more social/affiliative behaviors and speculate on the effects that SSRIs have in the treatment of sexual offenders by targeting areas of the social brain. Here, we would argue that it would be useful to carry out treatment where there is a combination of SSRI treatment (to promote more prosocial feelings and behaviors) in conjunction with therapy that typically addresses thoughts and behaviors, i.e., cognitive-behavioral therapy/schema-focused therapy.

  10. Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction.

    PubMed

    Malomouzh, Artem I; Petrov, Konstantin A; Nurullin, Leniz F; Nikolsky, Evgeny E

    2015-12-01

    Gamma-aminobutyric acid (GABA) is an amino acid which acts as a neurotransmitter in the central nervous system. Here, we studied the effects of GABA on non-quantal, spontaneous, and evoked quantal acetylcholine (ACh) release from motor nerve endings. We found that while the application of 10 μM of GABA had no effect on spontaneous quantal ACh release, as detected by the frequency of miniature endplate potentials, GABA reduced the non-quantal ACh release by 57%, as determined by the H-effect value. Finally, the evoked quantal ACh release, estimated by calculating the quantal content of full-sized endplate potentials (EPPs), was reduced by 34%. GABA's inhibitory effect remained unchanged after pre-incubation with picrotoxin, an ionotropic GABAA receptor blocker, but was attenuated following application of the GABAB receptor blocker CGP 55845, which itself had no effect on ACh release. An inhibitor of phospholipase C, U73122, completely prevented the GABA-induced decrease in ACh release. Immunofluorescence demonstrated the presence of both subunits of the GABAB receptor (GABAB R1 and GABAB R2) in the neuromuscular junction. These findings suggest that metabotropic GABAB receptors are expressed in the mammalian neuromuscular synapse and their activation results in a phospholipase C-mediated reduction in the intensity of non-quantal and evoked quantal ACh release. We investigated the effect of gamma-aminobutyric acid (GABA) on neuromuscular transmission. GABA reduced the non-quantal and evoked quantal release of acetylcholine. These effects are mediated by GABAB receptors and are implemented via phospholipase C (PLC) activation. Our findings suggest that in the mammalian neuromuscular synapse, metabotropic GABAB receptors are expressed and their activation results in a reduction in the intensity of acetylcholine release. © 2015 International Society for Neurochemistry.

  11. Taurine is a potent activator of extrasynaptic GABA(A) receptors in the thalamus.

    PubMed

    Jia, Fan; Yue, Minerva; Chandra, Dev; Keramidas, Angelo; Goldstein, Peter A; Homanics, Gregg E; Harrison, Neil L

    2008-01-02

    Taurine is one of the most abundant free amino acids in the brain. In a number of studies, taurine has been reported to activate glycine receptors (Gly-Rs) at moderate concentrations (> or = 100 microM), and to be a weak agonist at GABA(A) receptors (GABA(A)-Rs), which are usually activated at high concentrations (> or = 1 mM). In this study, we show that taurine reduced the excitability of thalamocortical relay neurons and activated both extrasynaptic GABA(A)-Rs and Gly-Rs in neurons in the mouse ventrobasal (VB) thalamus. Low concentrations of taurine (10-100 microM) decreased neuronal input resistance and firing frequency, and elicited a steady outward current under voltage clamp, but had no effects on fast inhibitory synaptic currents. Currents elicited by 50 microM taurine were abolished by gabazine, insensitive to midazolam, and partially blocked by 20 microM Zn2+, consistent with the pharmacological properties of extrasynaptic GABA(A)-Rs (alpha4beta2delta subtype) involved in tonic inhibition in the thalamus. Tonic inhibition was enhanced by an inhibitor of taurine transport, suggesting that taurine can act as an endogenous activator of these receptors. Taurine-evoked currents were absent in relay neurons from GABA(A)-R alpha4 subunit knock-out mice. The amplitude of the taurine current was larger in neurons from adult mice than juvenile mice. Taurine was a more potent agonist at recombinant alpha4beta2delta GABA(A)-Rs than at alpha1beta2gamma2 GABA(A)-Rs. We conclude that physiological concentrations of taurine can inhibit VB neurons via activation of extrasynaptic GABA(A)-Rs and that taurine may function as an endogenous regulator of excitability and network activity in the thalamus.

  12. Homocysteine alters cerebral microvascular integrity and causes remodeling by antagonizing GABA-A receptor.

    PubMed

    Lominadze, David; Tyagi, Neetu; Sen, Utpal; Ovechkin, Alexander; Tyagi, Suresh C

    2012-12-01

    High levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), are associated with cerebrovascular diseases, such as vascular dementia, stroke, and Alzheimer's disease. The γ-amino butyric acid (GABA) is an inhibitory neurotransmitter and a ligand of GABA-A receptor. By inhibiting excitatory response, it may decrease complications associated with vascular dementia and stroke. Hcy specifically competes with the GABA-A receptors and acts as an excitotoxic neurotransmitter. Previously, we have shown that Hcy increases levels of NADPH oxidase and reactive oxygen species (ROS), and decreases levels of thioredoxin and peroxiredoxin by antagonizing the GABA-A receptor. Hcy treatment leads to activation of matrix metalloproteinases (MMPs) in cerebral circulation by inducing redox stress and ROS. The hypothesis is that Hcy induces MMPs and suppresses tissue inhibitors of metalloproteinase (TIMPs), in part, by inhibiting the GABA-A receptor. This leads to degradation of the matrix and disruption of the blood brain barrier. The brain cortex of transgenic mouse model of HHcy (cystathionine β-synthase, CBS-/+) and GABA-A receptor null mice treated with and without muscimol (GABA-A receptor agonist) was analysed. The mRNA levels were measured by Q-RT-PCR. Levels of MMP-2, -9, -13, and TIMP-1, -2, -3, and -4 were evaluated by in situ labeling and PCR-gene arrays. Pial venular permeability to fluorescence-labeled albumin was assessed with intravital fluorescence microscopy. We found that Hcy increases metalloproteinase activity and decreases TIMP-4 by antagonizing the GABA-A receptor. The results demonstrate a novel mechanism in which brain microvascular permeability changes during HHcy and vascular dementias, and have therapeutic ramifications for microvascular disease in Alzheimer's patients.

  13. The GABA transaminase, ABAT, is essential for mitochondrial nucleoside metabolism

    PubMed Central

    Besse, Arnaud; Wu, Ping; Bruni, Francesco; Donti, Taraka; Graham, Brett H.; Craigen, William J.; McFarland, Robert; Moretti, Paolo; Lalani, Seema; Scott, Kenneth L.; Taylor, Robert W.; Bonnen, Penelope E.

    2015-01-01

    Summary ABAT is a key enzyme responsible for catabolism of principal inhibitory neurotransmitter gamma-aminobutyric acid (GABA). We report an essential role for ABAT in a seemingly unrelated pathway, mitochondrial nucleoside salvage, and demonstrate that mutations in this enzyme cause an autosomal recessive neurometabolic disorder and mtDNA depletion syndrome (MDS). We describe a family with encephalomyopathic MDS caused by a homozygous missense mutation in ABAT that results in elevated GABA in subjects’ brains as well as decreased mtDNA levels in subjects’ fibroblasts. Nucleoside rescue and co-IP experiments pinpoint that ABAT functions in the mitochondrial nucleoside salvage pathway to facilitate conversion of dNDPs to dNTPs. Pharmacological inhibition of ABAT through the irreversible inhibitor Vigabatrin caused depletion of mtDNA in photoreceptor cells that was prevented through addition of dNTPs in cell culture media. This work reveals ABAT as a connection between GABA metabolism and nucleoside metabolism and defines a neurometabolic disorder that includes MDS. PMID:25738457

  14. PKCβ Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux.

    PubMed

    Zestos, Alexander G; Mikelman, Sarah R; Kennedy, Robert T; Gnegy, Margaret E

    2016-06-15

    Amphetamine abuse afflicts over 13 million people, and there is currently no universally accepted treatment for amphetamine addiction. Amphetamine serves as a substrate for the dopamine transporter and reverses the transporter to cause an increase in extracellular dopamine. Activation of the beta subunit of protein kinase C (PKCβ) enhances extracellular dopamine in the presence of amphetamine by facilitating the reverse transport of dopamine and internalizing the D2 autoreceptor. We previously demonstrated that PKCβ inhibitors block amphetamine-stimulated dopamine efflux in synaptosomes from rat striatum in vitro. In this study, we utilized in vivo microdialysis in live, behaving rats to assess the effect of the PKCβ inhibitors, enzastaurin and ruboxistaurin, on amphetamine-stimulated locomotion and increases in monoamines and their metabolites. A 30 min perfusion of the nucleus accumbens core with 1 μM enzastaurin or 1 μM ruboxistaurin reduced efflux of dopamine and its metabolite 3-methoxytyramine induced by amphetamine by approximately 50%. The inhibitors also significantly reduced amphetamine-stimulated extracellular levels of norepinephrine. The stimulation of locomotor behavior by amphetamine, measured simultaneously with the analytes, was comparably reduced by the PKCβ inhibitors. Using a stable isotope label retrodialysis procedure, we determined that ruboxistaurin had no effect on basal levels of dopamine, norepinephrine, glutamate, or GABA. In addition, normal uptake function through the dopamine transporter was unaltered by the PKCβ inhibitors, as measured in rat synaptosomes. Our results support the utility of using PKCβ inhibitors to reduce the effects of amphetamine.

  15. Altered GABA Signaling in Early Life Epilepsies

    PubMed Central

    Briggs, Stephen W.; Galanopoulou, Aristea S.

    2011-01-01

    The incidence of seizures is particularly high in the early ages of life. The immaturity of inhibitory systems, such as GABA, during normal brain development and its further dysregulation under pathological conditions that predispose to seizures have been speculated to play a major role in facilitating seizures. Seizures can further impair or disrupt GABAA signaling by reshuffling the subunit composition of its receptors or causing aberrant reappearance of depolarizing or hyperpolarizing GABAA receptor currents. Such effects may not result in epileptogenesis as frequently as they do in adults. Given the central role of GABAA signaling in brain function and development, perturbation of its physiological role may interfere with neuronal morphology, differentiation, and connectivity, manifesting as cognitive or neurodevelopmental deficits. The current GABAergic antiepileptic drugs, while often effective for adults, are not always capable of stopping seizures and preventing their sequelae in neonates. Recent studies have explored the therapeutic potential of chloride cotransporter inhibitors, such as bumetanide, as adjunctive therapies of neonatal seizures. However, more needs to be known so as to develop therapies capable of stopping seizures while preserving the age- and sex-appropriate development of the brain. PMID:21826277

  16. Closing the loop on the GABA shunt in plants: are GABA metabolism and signaling entwined?

    PubMed

    Michaeli, Simon; Fromm, Hillel

    2015-01-01

    γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that is found in uni- and multi-cellular organisms and is involved in many aspects of plant life cycle. GABA metabolism occurs by the action of evolutionary conserved enzymes that constitute the GABA shunt, bypassing two steps of the TCA cycle. The central position of GABA in the interface between plant carbon and nitrogen metabolism is well established. In parallel, there is evidence to support a role for GABA as a signaling molecule in plants. Here we cover some of the recent findings on GABA metabolism and signaling in plants and further suggest that the metabolic and signaling aspects of GABA may actually be inseparable.

  17. Closing the loop on the GABA shunt in plants: are GABA metabolism and signaling entwined?

    PubMed Central

    Michaeli, Simon; Fromm, Hillel

    2015-01-01

    γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that is found in uni- and multi-cellular organisms and is involved in many aspects of plant life cycle. GABA metabolism occurs by the action of evolutionary conserved enzymes that constitute the GABA shunt, bypassing two steps of the TCA cycle. The central position of GABA in the interface between plant carbon and nitrogen metabolism is well established. In parallel, there is evidence to support a role for GABA as a signaling molecule in plants. Here we cover some of the recent findings on GABA metabolism and signaling in plants and further suggest that the metabolic and signaling aspects of GABA may actually be inseparable. PMID:26106401

  18. The effect of anaesthetics on the uptake and release of gamma-aminobutyrate and D-aspartate in rat brain slices.

    PubMed Central

    Minchin, M. C.

    1981-01-01

    1 The effect of various concentrations of thiopentone, pentobarbitone, methohexitone, hydroxydione, alphaxalone/alphadolone, ketamine, alpha-chloralose, and urethane on the transport of radiolabelled gamma-aminobutyric acid (GABA) and D-aspartate was investigated. 2 Uptake of the amino acids was weakly inhibited, if at all, by the anaesthetics and it is unlikely that such effects contribute significantly to their physiological function. 3 The spontaneous efflux of GABA and D-aspartate was not detectably altered by any of the drugs tested. 4 Thiopentone, pentobarbitone, methohexitone and hydroxydione inhibited K+-stimulated GABA and D-aspartate release. The other anaesthetics had no effect on K+-stimulated amino acid release. 5 The rank order of potency of the inhibitors of K+-stimulated amino acid release did not correlate with their anaesthetic potency. Furthermore not all inhibitors appeared to be very effective at anaesthetic concentrations. 6 It is concluded that although it is possible that inhibition of excitatory transmitter release may be involved in the anaesthetic action of some anaesthetics, for many of the substances tested in this study such as mechanism does not appear to be implicated. PMID:6265017

  19. Structure, function, and plasticity of GABA transporters

    PubMed Central

    Scimemi, Annalisa

    2014-01-01

    GABA transporters belong to a large family of neurotransmitter:sodium symporters. They are widely expressed throughout the brain, with different levels of expression in different brain regions. GABA transporters are present in neurons and in astrocytes and their activity is crucial to regulate the extracellular concentration of GABA under basal conditions and during ongoing synaptic events. Numerous efforts have been devoted to determine the structural and functional properties of GABA transporters. There is also evidence that the expression of GABA transporters on the cell membrane and their lateral mobility can be modulated by different intracellular signaling cascades. The strength of individual synaptic contacts and the activity of entire neuronal networks may be finely tuned by altering the density, distribution and diffusion rate of GABA transporters within the cell membrane. These findings are intriguing because they suggest the existence of complex regulatory systems that control the plasticity of GABAergic transmission in the brain. Here we review the current knowledge on the structural and functional properties of GABA transporters and highlight the molecular mechanisms that alter the expression and mobility of GABA transporters at central synapses. PMID:24987330

  20. GABA maintains the proliferation of progenitors in the developing chick ciliary marginal zone and non-pigmented ciliary epithelium.

    PubMed

    Ring, Henrik; Mendu, Suresh Kumar; Shirazi-Fard, Shahrzad; Birnir, Bryndis; Hallböök, Finn

    2012-01-01

    GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABA(A) receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABA(A) receptor system. To quantify the effects on proliferation by GABA(A) receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABA(A) receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABA(A) receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl-transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABA(A) receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABA(A) receptors. This supported the depolarising role for the GABA(A) receptors. Inhibition of L-type voltage-gated Ca(2+) channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABA(A) receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27(KIP1), along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27(KIP1) after inhibition of either the GABA(A) receptors or the L-type VGCCs suggests a link between the GABA(A) receptors, membrane potential, and intracellular Ca

  1. Cortical GABA Levels in Primary Insomnia

    PubMed Central

    Morgan, Peter T.; Pace-Schott, Edward F.; Mason, Graeme F.; Forselius, Erica; Fasula, Madonna; Valentine, Gerald W.; Sanacora, Gerard

    2012-01-01

    Study Objectives: GABA is increasingly recognized as an important neurotransmitter for the initiation and maintenance of sleep. We sought to measure cortical GABA content through proton magnetic resonance spectroscopy (MRS) in persons with and without primary insomnia, and relate brain GABA levels to polysomnographic sleep measures. Design: Two-group comparison study. Setting: Outpatient study at a university research clinic. Participants: Non-medicated persons with primary insomnia (N = 16) and no sleep complaints (N = 17). Interventions: Participants kept sleep diaries and a regular time-in-bed schedule for 9 days, culminating in 2 consecutive nights of ambulatory polysomnography and a single proton MRS session. The main outcome measure was occipital GABA/creatine ratios; secondary measures included sleep measurements and relationship between polysomnographically measured time awake after sleep onset and occipital GABA content. Measurements and Results: The primary insomnia group was distinguished from persons with no sleep complaints on self-reported and polysomnographically measured sleep. The two groups did not differ in age, sex, body mass index, habitual bed- and wake-times, napping, use of caffeine, or use of cigarettes. Mean occipital GABA level was 12% higher in persons with insomnia than in persons without sleep complaints (P < 0.05). In both groups, GABA levels correlated negatively with polysomnographically measured time awake after sleep onset (P < 0.05). Conclusions: Increased GABA levels in persons with insomnia may reflect an allostatic response to chronic hyperarousal. The preserved, negative relationship between GABA and time awake after sleep onset supports this notion, indicating that the possible allostatic response is adaptive. Citation: Morgan PT; Pace-Schott EF; Mason GF; Forselius E; Fasula M; Valentine GW; Sanacora G. Cortical GABA levels in primary insomnia. SLEEP 2012;35(6):807-814. PMID:22654200

  2. Developmental expression of GABA transporter-1 and 3 during formation of the GABAergic synapses in the mouse cerebellar cortex.

    PubMed

    Takayama, Chitoshi; Inoue, Yoshiro

    2005-08-08

    In the brain, gamma-amino butyric acid (GABA), released extrasynaptically and synaptically from GABAergic neurons, plays important roles in morphogenesis, expression of higher functions and so on. In the GABAergic transmission system, plasma membrane GABA transporters (GATs) mediate GABA-uptake from the synaptic cleft in the mature brain and are thought to mediate diacrine of cytosolic GABA in the immature brain. In the present study, we focused on two GATs (GAT-1 and GAT-3) in the mouse cerebellar cortex, which are widely localized in neural and glial cells. Firstly, we examined the localization of GATs in the dendrites and cell bodies of developing GABAergic neurons, where GABA is extrasynaptically distributed, to clarify the GABA-diacrine before synaptogenesis. Secondly, we examined the developmental changes in the localization of GATs to reveal the development of the GABA-uptake system. Neither transporter was detected within the dendrites and cell bodies of GABAergic neurons, including Purkinje, stellate, basket and Golgi cells, in the immature cerebellar cortex. GAT-1 was observed within the Golgi cell axon terminals after postnatal day 5 (P5) and presynaptic axons of stellate and basket cells after P7. GAT-3 was localized within the astrocyte processes, sealing the GABAergic synapses in the Purkinje cell and granular layers after P10. These results indicated that GABA-diacrine did not work in the mouse cerebellar cortex. The onset of GAT-1-expression was prior to that of GAT-3. GAT-1 started to be localized within the GABAergic axon terminals during synapse formation. GAT-3 started to be localized within astrocyte processes when they sealed the synapses.

  3. Effects of the novel endocannabinoid uptake inhibitor, LY2183240, on fear-potentiated startle and alcohol-seeking behaviors in mice selectively bred for high alcohol preference.

    PubMed

    Powers, Matthew S; Barrenha, Gustavo D; Mlinac, Nate S; Barker, Eric L; Chester, Julia A

    2010-12-01

    Alcohol-use disorders often occur together with anxiety disorders in humans which may be partly due to common inherited genetic factors. Evidence suggests that the endocannabinoid system (ECS) is a promising therapeutic target for the treatment of individuals with anxiety and/or alcohol-use disorders. The present study assessed the effects of a novel endocannabinoid uptake inhibitor, LY2183240, on anxiety- and alcohol-seeking behaviors in a unique animal model that may represent increased genetic risk to develop co-morbid anxiety and alcohol-use disorders in humans. Mice selectively bred for high alcohol preference (HAP) show greater fear-potentiated startle (FPS) than mice selectively bred for low alcohol preference (LAP). We examined the effects of LY2183240 on the expression of FPS in HAP and LAP mice and on alcohol-induced conditioned place preference (CPP) and limited-access alcohol drinking behavior in HAP mice. Repeated administration of LY2183240 (30 mg/kg) reduced the expression of FPS in HAP but not LAP mice when given prior to a second FPS test 48 h after fear conditioning. Both the 10 and 30 mg/kg doses of LY2183240 enhanced the expression of alcohol-induced CPP and this effect persisted in the absence of the drug. LY2183240 did not alter limited-access alcohol drinking behavior, unconditioned startle responding, or locomotor activity. These findings suggest that ECS modulation influences both conditioned fear and conditioned alcohol reward behavior. LY2183240 may be an effective pharmacotherapy for individuals with anxiety disorders, such as post-traumatic stress disorder, but may not be appropriate for individuals with co-morbid anxiety and alcohol-use disorders.

  4. Effects of the novel endocannabinoid uptake inhibitor, LY2183240, on fear-potentiated startle and alcohol-seeking behaviors in mice selectively bred for high alcohol preference

    PubMed Central

    Powers, Matthew S.; Barrenha, Gustavo D.; Mlinac, Nate S.; Barker, Eric L.; Chester, Julia A.

    2010-01-01

    Rationale Alcohol-use disorders often occur together with anxiety disorders in humans which may be partly due to common inherited genetic factors. Evidence suggests that the endocannabinoid system (ECS) is a promising therapeutic target for the treatment of individuals with anxiety and/or alcohol-use disorders. Objectives The present study assessed the effects of a novel endocannabinoid uptake inhibitor, LY2183240, on anxiety- and alcohol-seeking behaviors in a unique animal model that may represent increased genetic risk to develop comorbid anxiety and alcohol-use disorders in humans. Mice selectively bred for high alcohol preference (HAP) show greater fear-potentiated startle (FPS) than mice selectively bred for low alcohol preference (LAP). We examined the effects of LY2183240 on the expression of FPS in HAP and LAP mice and on alcohol-induced conditioned place preference (CPP) and limited-access alcohol drinking behavior in HAP mice. Results Repeated administration of LY2183240 (30 mg/kg) reduced the expression of FPS in HAP but not LAP mice when given prior to a second FPS test 48 h after fear conditioning. Both the 10 and 30 mg/kg doses of LY2183240 enhanced the expression of alcohol-induced CPP and this effect persisted in the absence of the drug. LY2183240 did not alter limited-access alcohol drinking behavior, unconditioned startle responding, or locomotor activity. Conclusions These findings suggest that ECS modulation influences both conditioned fear and conditioned alcohol reward behavior. LY2183240 may be an effective pharmacotherapy for individuals with anxiety disorders, such as post-traumatic stress disorder, but may not be appropriate for individuals with co-morbid anxiety and alcohol-use disorders. PMID:20838777

  5. Amoxapine inhibition of GABA-stimulated chloride conductance: Investigations of potential sites of activity

    SciTech Connect

    Ikeda, M.; Knapp, R.J.; Yamamura, H.I. ); Malatynska, E. )

    1989-01-01

    Amoxapine inhibits GABA-stimulated chloride conductance by acting on the GABA{sub A}-receptor chloride-ionophore complex which can be studied using membrane vesicles prepared from rat cerebral cortex. Amoxapine produces a right shift in the GABA concentration-response curve for the stimulation of {sup 36}Cl{sup {minus}} uptake into these vesicles with no apparent change in the maximum response. Schild analysis of these data gave a pA{sub 2} value of 5.52 with a slope of 0.79. Amoxapine inhibits the binding of the GABA{sub A} receptor selective antagonist ({sup 3}H)SR 95531 with an IC{sub 50} value of 3.45 {mu}M and a pseudo Hill coefficient of 0.83. In contrast, 10 {mu}M amoxapine inhibits ({sup 3}H) flunitrazepam binding by less than 25% while the benzodiazepine antagonist Ro 15-1788 reduces the amoxapine inhibition of GABA-stimulated chloride conductance only at high concentrations.

  6. Effects of glutamate decarboxylase and gamma-aminobutyric acid (GABA) transporter on the bioconversion of GABA in engineered Escherichia coli.

    PubMed

    Le Vo, Tam Dinh; Kim, Tae Wan; Hong, Soon Ho

    2012-05-01

    Gamma-aminobutyric acid (GABA) is a non-essential amino acid and a precursor of pyrrolidone, a monomer of nylon 4. GABA can be biosynthesized through the decarboxylation of L: -glutamate by glutamate decarboxylase. In this study, the effects of glutamate decarboxylase (gadA, gadB), glutamate/GABA antiporter (gadC) and GABA aminotransferase (gabT) on GABA production were investigated in Escherichia coli. Glutamate decarboxylase was overexpressed alone or with the glutamate/GABA antiporter to enhance GABA synthesis. GABA aminotransferase, which redirects GABA into the TCA cycle, was knock-out mutated. When gadB and gadC were co-overexpressed in the gabT mutant strain, a final GABA concentration of 5.46 g/l was obtained from 10 g/l of monosodium glutamate (MSG), which corresponded to a GABA yield of 89.5%.

  7. Neuronal GABA release and GABA inhibition of ACh release in guinea pig urinary bladder.

    PubMed

    Kusunoki, M; Taniyama, K; Tanaka, C

    1984-04-01

    gamma-Aminobutyric acid (GABA) and glutamate decarboxylase (GAD) are present in the urinary bladder of guinea pigs, and the possible correlation in regional distribution between GABA, GAD, and the number of vesical ganglion cells was studied. Electrical stimulation of the bladder strips produced an increase in the calcium-dependent and tetrodotoxin-sensitive [3H]GABA release and contractions in the strips preloaded with [3H]GABA. Nicotine, acetylcholine chloride (ACh), and hexamethonium did not significantly alter the release of [3H]GABA. Bicuculline significantly enhanced [3H]ACh release and cholinergic components of contractions evoked by electrical stimulation of the bladder strips preloaded with [3H]choline, thereby suggesting that this compound antagonizes the effect of endogenous GABA released during stimulation. GABA and muscimol but not baclofen reduced both the [3H]ACh release and contractions evoked by nicotine. These effects of GABA were antagonized by bicuculline and furosemide but not by alpha- and beta-adrenergic blockers. These findings suggest that GABA may be a noncholinergic nonadrenergic inhibitory neurotransmitter in the urinary bladder. The motility of the urinary bladder is thus inhibited by reducing the release of ACh from the postganglionic cholinergic neurons through bicuculline-sensitive GABA receptors probably associated with the chloride ion channel.

  8. The putative P-gp inhibitor telmisartan does not affect the transcellular permeability and cellular uptake of the calcium channel antagonist verapamil in the P-glycoprotein expressing cell line MDCK II MDR1

    PubMed Central

    Saaby, Lasse; Tfelt-Hansen, Peer; Brodin, Birger

    2015-01-01

    Verapamil is used in high doses for the treatment of cluster headache. Verapamil has been described as a P-glycoprotein (P-gp, ABCB1) substrate. We wished to evaluate in vitro whether co administration of a P-gp inhibitor with verapamil could be a feasible strategy for increasing CNS uptake of verapamil. Fluxes of radiolabelled verapamil across MDCK II MDR1 monolayers were measured in the absence and presence of the putative P-gp inhibitor telmisartan (a clinically approved drug compound). Verapamil displayed a vectorial basolateral-to-apical transepithelial efflux across the MDCK II MDR1 monolayers with a permeability of 5.7 × 10−5 cm sec−1 compared to an apical to basolateral permeability of 1.3 × 10−5 cm sec-1. The efflux could be inhibited with the P-gp inhibitor zosuquidar. Zosuquidar (0.4 μmol/L) reduced the efflux ratio (PB-A/PA-B) for verapamil 4.6–1.6. The presence of telmisartan, however, only caused a slight reduction in P-gp-mediated verapamil transport to an efflux ratio of 3.4. Overall, the results of the present in vitro approach indicate, that clinical use of telmisartan as a P-gp inhibitor may not be an effective strategy for increasing brain uptake of verapamil by co-administration with telmisartan. PMID:26171231

  9. A1R-A2AR heteromers coupled to Gs and G i/0 proteins modulate GABA transport into astrocytes.

    PubMed

    Cristóvão-Ferreira, Sofia; Navarro, Gemma; Brugarolas, Marc; Pérez-Capote, Kamil; Vaz, Sandra H; Fattorini, Giorgia; Conti, Fiorenzo; Lluis, Carmen; Ribeiro, Joaquim A; McCormick, Peter J; Casadó, Vicent; Franco, Rafael; Sebastião, Ana M

    2013-09-01

    Astrocytes play a key role in modulating synaptic transmission by controlling extracellular gamma-aminobutyric acid (GABA) levels via GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that a further level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A1 (A1R) and A2A (A2AR) receptors. This regulation occurs through A1R-A2AR heteromers that signal via two different G proteins, Gs and Gi/0, and either enhances (A2AR) or inhibits (A1R) GABA uptake. These results provide novel mechanistic insight into how GPCR heteromers signal. Furthermore, we uncover a previously unknown mechanism where adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glia-neuron) synapse.

  10. [GABA: a functional duality? Transition during neurodevelopment].

    PubMed

    Cortes-Romero, C; Galindo, F; Galicia-Isasmendi, S; Flores, A

    2011-06-01

    Gamma-aminobutyric acid (GABA) is the most important inhibitory-type neurotransmitter and its actions are mediated by ionotropic (GABA(A)) and metabotropic (GABA(B)) type receptors, which are widely distributed throughout the tissue of the central nervous system. To review the structure of GABA receptors and their involvement in physiological processes in the central nervous system. The study addresses the structure and diversity of the GABA receptors, especially during neurodevelopment, and reference is made to the excitatory and inhibitory nature of GABAergic transmission, where the participation of the cotransporters NKCC1 and KCC2 plays a key role in this functional duality in the transition from an embryonic to a post-natal state. Likewise, the interest in GABA receptors as a pharmacological target for clinical use is also discussed. This is manifested by the presence of under-explored allosteric modulation sites in the aforementioned complex-receptor. The physiological and pharmacological knowledge of the great diversity of subunits that make up a particular subtype of GABA receptor, as well as the correct expression in time and space in order to ensure the viability of the organism, promise to be the answer to long-time severe disorders like epilepsy or drug addiction, and such complex ones as neurodevelopment.

  11. Intracellular trafficking of GABA(A) receptors.

    PubMed

    Barnes, E M

    2000-02-11

    Some of the mechanisms that control the intracellular trafficking of GABA(A) receptors have recently been described. Following the synthesis of alpha, beta, and gamma subunits in the endoplasmic reticulum, ternary receptor complexes assemble slowly and are inefficiently inserted into surface membranes of heterologous cells. While beta3, beta4, and gamma2S subunits appear to contain polypeptide sequences that alone are sufficient for surface targeting, these sequences are neither conserved nor essential for surface expression of heteromeric GABA(A) receptors formed from alpha1beta or alpha1betagamma subunits. At the neuronal surface, native GABA(A) receptor clustering and synaptic targeting require a gamma2 subunit and the participation of gephyrin, a clustering protein for glycine receptors. A linker protein, such as the GABA(A) receptor associated protein (GABARAP), may be necessary for the formation of GABA(A) receptor aggregates containing gephyrin. A substantial fraction of surface receptors are sequestered by endocytosis, another process which apparently requires a GABA(A) receptor gamma2 subunit. In heterologous cells, constitutive endocytosis seems to predominate while, in cortical neurons, internalization is evoked when receptors are occupied by GABA(A) agonists. After constitutive endocytosis, receptors are relatively stable and can be rapidly recycled to the cell surface, a process that may be regulated by protein kinase C. On the other hand, a portion of the intracellular GABA(A) receptors derived from ligand-dependent endocytosis is apparently degraded. The clustering of GABA(A) receptors at synapses and at coated pits are two mechanisms that may compete for a pool of diffusable receptors, providing a model for plasticity at inhibitory synapses.

  12. Low nanomolar GABA effects at extrasynaptic α4β1/β3δ GABA(A) receptor subtypes indicate a different binding mode for GABA at these receptors.

    PubMed

    Karim, Nasiara; Wellendorph, Petrine; Absalom, Nathan; Bang, Line Haunstrup; Jensen, Marianne Lerbech; Hansen, Maja Michelle; Lee, Ho Joon; Johnston, Graham A R; Hanrahan, Jane R; Chebib, Mary

    2012-08-15

    Ionotropic GABA(A) receptors are a highly heterogenous population of receptors assembled from a combination of multiple subunits. The aims of this study were to characterize the potency of GABA at human recombinant δ-containing extrasynaptic GABA(A) receptors expressed in Xenopus oocytes using the two-electrode voltage clamp technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at α4β3δ GABA(A) receptors. α4/δ-Containing GABA(A) receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating α4β1δ (EC₅₀=24 nM) and α4β3δ (EC₅₀=12 nM) receptors. In the majority of oocytes expressing α4β3δ subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC₅₀(1)=16 nM; EC₅₀(2)=1.2 μM). At α4β2δ, GABA had low micromolar activity (EC₅₀=1 μM). An analysis of 10 N-terminal singly mutated α4β3δ receptors shows that GABA interacts with amino acids different to those reported for α1β2γ2 GABA(A) receptors. Residues Y205 and R207 of the β3-subunit significantly affected GABA potency, while the residue F71 of the α4- and the residue Y97 of the β3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the δ-subunit, equivalent to the GABA binding residue R207 of the β2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the δ-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic δ-containing GABA(A) receptors compared to their synaptic counterparts.

  13. Muscimol as an ionotropic GABA receptor agonist.

    PubMed

    Johnston, Graham A R

    2014-10-01

    Muscimol, a psychoactive isoxazole from Amanita muscaria and related mushrooms, has proved to be a remarkably selective agonist at ionotropic receptors for the inhibitory neurotransmitter GABA. This historic overview highlights the discovery and development of muscimol and related compounds as a GABA agonist by Danish and Australian neurochemists. Muscimol is widely used as a ligand to probe GABA receptors and was the lead compound in the development of a range of GABAergic agents including nipecotic acid, tiagabine, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, (Gaboxadol(®)) and 4-PIOL.

  14. Synthesis and evaluation of novel heteroaromatic substrates of GABA aminotransferase

    PubMed Central

    Hawker, Dustin D.; Silverman, Richard B.

    2012-01-01

    Two principal neurotransmitters are involved in the regulation of mammalian neuronal activity, namely, γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, and L-glutamic acid, an excitatory neurotransmitter. Low GABA levels in the brain have been implicated in epilepsy and several other neurological diseases. Because of GABA’s poor ability to cross the blood-brain barrier (BBB), a successful strategy to raise brain GABA concentrations is the use of a compound that does cross the BBB and inhibits or inactivates GABA aminotransferase (GABA-AT), the enzyme responsible for GABA catabolism. Vigabatrin, a mechanism-based inactivator of GABA-AT, is currently a successful therapeutic for epilepsy, but has harmful side effects, leaving a need for improved GABA-AT inactivators. Here, we report the synthesis and evaluation of a series of heteroaromatic GABA analogues as substrates of GABA-AT, which will be used as the basis for the design of novel enzyme inactivators. PMID:22944334

  15. PR-independent neurosteroid regulation of α2-GABA-A receptors in the hippocampus subfields.

    PubMed

    Reddy, Doodipala Samba; Gangisetty, Omkaram; Wu, Xin

    2017-03-15

    Progesterone (P) binding to the intracellular progesterone receptors (PRs) plays a key role in epilepsy via modulation of GABA-A receptor plasticity in the brain. This is thought to occur via conversion of P to neurosteroids such as allopregnanolone, an allosteric modulator of GABA-A receptors. In the female brain, the composition of GABA-A receptors is not static and undergoes dynamic spatial changes in response to fluctuations in P and neurosteroid levels. Synaptic α2-containing GABA-A receptors contribute to phasic neuronal excitability and seizure susceptibility. However, the mechanisms underlying α2-subunit plasticity remain unclear. Here, we utilized the neurosteroid synthesis inhibitor finasteride and PR knockout mice to investigate the role of PRs in α2-subunit in the hippocampus. α2-Subunit expression was significantly upregulated during the high-P state of diestrous stage and with P treatment in wildtype and PR knockout mice. In contrast, there was no change in α2-subunit expression when metabolism of P into neurosteroids was blocked by finasteride in both genotypes. These findings suggest that ovarian cycle-related P and neurosteroids regulate α2-GABA-A receptor expression in the hippocampus via a non-PR pathway, which may be relevant to menstrual-cycle related brain conditions.

  16. Interactions between dopamine and GABA in the control of ambulatory activity.

    PubMed

    Agmo, A; Belzung, C; Giordano, M

    1996-01-01

    Ambulatory activity of male rats was quantified in an open field. The subjects were treated with DL-amphetamine and amfonelic acid alone or combined with the GABA transaminase inhibitors gamma-acetylen GABA (GAG) and sodium valproate as well as with the GABAA agonist THIP and the GABAB agonist baclofen. Subeffective doses of the GABAergic drugs did not modify the effects of moderate doses of the dopaminergic stimulants whereas effective doses continued to reduce ambulatory activity just as in the absence of dopaminergic activation. When DL-amphetamine or amfonelic acid were administered in doses that strongly enhanced ambulatory activity, doses of the GABAergic drugs that were inhibitory in the absence of dopaminergic stimulation were no longer effective. The mixed D1/D2 dopamine antagonist pimozide, the D1 antagonist SCH 23390 and the D2 antagonist sulpiride were then combined with subeffective doses of the GABA agonists. GAG, sodium valproate and baclofen were potentiated by pimozide and SCH 23390 but not by sulpiride. THIP was ineffective. These data show that GABAergic drugs had a reduced effect after stimulation of dopaminergic neurotransmission. On the other hand, when dopamine D1 receptors were blocked, nonselective GABA agonists and the GABAB agonist baclofen were potentiated. This was not the case for the GABAA agonist THIP, suggesting that the GABAA receptor is of slight importance for the interactions between GABA and dopamine in the control of ambulatory activity. No potentiation of GABAergic agonists was obtained after treatment with a dopamine D2 antagonist.

  17. Acute increases in synaptic GABA detectable in the living human brain: a [¹¹C]Ro15-4513 PET study.

    PubMed

    Stokes, Paul R A; Myers, Jim F; Kalk, Nicola J; Watson, Ben J; Erritzoe, David; Wilson, Sue J; Cunningham, Vincent J; Riano Barros, Daniela; Hammers, Alexander; Turkheimer, Federico E; Nutt, David J; Lingford-Hughes, Anne R

    2014-10-01

    The inhibitory γ-aminobutyric acid (GABA) neurotransmitter system is associated with the regulation of normal cognitive functions and dysregulation has been reported in a number of neuropsychiatric disorders including anxiety disorders, schizophrenia and addictions. Investigating the role of GABA in both health and disease has been constrained by difficulties in measuring acute changes in synaptic GABA using neurochemical imaging. The aim of this study was to investigate whether acute increases in synaptic GABA are detectable in the living human brain using the inverse agonist GABA-benzodiazepine receptor (GABA-BZR) positron emission tomography (PET) tracer, [(11)C]Ro15-4513. We examined the effect of 15 mg oral tiagabine, which increases synaptic GABA by inhibiting the GAT1 GABA uptake transporter, on [(11)C]Ro15-4513 binding in 12 male participants using a paired, double blind, placebo-controlled protocol. Spectral analysis was used to examine synaptic α1 and extrasynaptic α5 GABA-BZR subtype availability in brain regions with high levels of [(11)C]Ro15-4513 binding. We also examined the test-retest reliability of α1 and a5-specific [(11)C]Ro15-4513 binding in a separate cohort of 4 participants using the same spectral analysis protocol. Tiagabine administration produced significant reductions in hippocampal, parahippocampal, amygdala and anterior cingulate synaptic α1 [(11)C]Ro15-4513 binding, and a trend significance reduction in the nucleus accumbens. These reductions were greater than test-retest reliability, indicating that they are not the result of chance observations. Our results suggest that acute increases in endogenous synaptic GABA are detectable in the living human brain using [(11)C]Ro15-4513 PET. These findings have potentially major implications for the investigation of GABA function in brain disorders and in the development of new treatments targeting this neurotransmitter system. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Guinea Pig Horizontal Cells Express GABA, the GABA-Synthesizing Enzyme GAD65, and the GABA Vesicular Transporter

    PubMed Central

    Guo, Chenying; Hirano, Arlene A.; Stella, Salvatore L.; Bitzer, Michaela; Brecha, Nicholas C.

    2013-01-01

    γ-Aminobutyric acid (GABA) is likely expressed in horizontal cells of all species, although conflicting physiological findings have led to considerable controversy regarding its role as a transmitter in the outer retina. This study has evaluated key components of the GABA system in the outer retina of guinea pig, an emerging retinal model system. The presence of GABA, its rate-limiting synthetic enzyme glutamic acid decarboxylase (GAD65 and GAD67 isoforms), the plasma membrane GABA transporters (GAT-1 and GAT-3), and the vesicular GABA transporter (VGAT) was evaluated by using immunohistochemistry with well-characterized antibodies. The presence of GAD65 mRNA was also evaluated by using laser capture microdissection and reverse transcriptase-polymerase chain reaction. Specific GABA, GAD65, and VGAT immunostaining was localized to horizontal cell bodies, as well as to their processes and tips in the outer plexiform layer. Furthermore, immunostaining of retinal whole mounts and acutely dissociated retinas showed GAD65 and VGAT immunoreactivity in both A-type and B-type horizontal cells. However, these cells did not contain GAD67, GAT-1, or GAT-3 immunoreactivity. GAD65 mRNA was detected in horizontal cells, and sequencing of the amplified GAD65 fragment showed approximately 85% identity with other mammalian GAD65 mRNAs. These studies demonstrate the presence of GABA, GAD65, and VGAT in horizontal cells of the guinea pig retina, and support the idea that GABA is synthesized from GAD65, taken up into synaptic vesicles by VGAT, and likely released by a vesicular mechanism from horizontal cells. PMID:20235161

  19. Cannabinoids: influence on neurotransmitter uptake in rat brain synaptosomes.

    PubMed

    Banerjee, S P; Snyder, S H; Mechoulam, R

    1975-07-01

    We have examined the effect of Delta1-tetrahydrocannabinol (delat1-THC) and 12 of its derivatives on the uptake of 3H-labeled norepinephrine (NE), dopamine (DA), serotonin (5-HT) gamma-aminobutyric acid (GABA) into synaptosomes in homogenates of various regions of rat brain. Delta1-THC inhibits the accumulation of NE and 5-HT into hypothalamic preparations and DA into the corpus striatum with Ki values of about 12 to 25 muM while GABA uptake into cerebral cortical preparations is inhibited less (Ki = 140 muM). The affinities of delta6-THC, 7-hydroxy-delta1-THC, 7-hydroxy-delta6-THC and cannabidiol for 5-HT, NE and GABA transports are similar to values for delta1-THC, while cannabigerol, cannabinol and delta6-THC-7-oic acid have substantially less affinity. Thus, hydroxylation of C-7 in delta6-THC does not alter inhibitory potency, but its oxidation to an acid and aromatization of ring A greatly reduce affinity. The hydroxyl at C-3(1) of ring C is critical for inhibition of NE, 5-HT and GABA uptake, since its acetylation or methylation abolishes activity. Inhibition of NE, DA, 5-HT and GABA uptake by all cannabinoids examined is noncompetitive. Only about 1% of delta1-THC and delta6-THC and 5% of cannabidiol are fully soluble under our experimental conditions.

  20. GABA Australis, some reflections on the history of GABA receptor research in Australia.

    PubMed

    Johnston, Graham A R

    2017-02-01

    Research on GABA receptors has a long history in Australia dating from 1958 with David Curtis and his colleagues in Canberra. This review traces many of the advances made in Australia guided by highly cited publications and some obscure ones. It covers the discovery of key chemicals with which to investigate GABA receptor function including bicuculline, muscimol, phaclofen, THIP and (+)-CAMP. Also described are findings relevant to the involvement of mutant GABA receptors in inherited epilepsy. The modulation of GABA receptors by a bewildering range of chemicals, especially by flavonoids and terpenoids, is discussed.

  1. Inhibition of GABA release by presynaptic ionotropic GABA receptors in hippocampal CA3.

    PubMed

    Axmacher, Nikolai; Draguhn, Andreas

    2004-02-09

    Vesicular transmitter release can be regulated by transmitter-gated ion channels at presynaptic axon terminals. The central inhibitory transmitter GABA acts on such presynaptic ionotropic receptors in various cells, including inhibitory interneurons. Here we report that GABA-mediated postsynaptic inhibitory currents in CA3 pyramidal cells of rat hippocampal slices are suppressed by agonists of GABAA receptors. The effect is present for both stimulus-induced and miniature IPSCs, indicating a reduction in the probability of vesicular release by presynaptic, action-potential-independent mechanisms. We conclude that the release of GABA from hippocampal CA3 interneurons is regulated by a negative feedback via presynaptic ionotropic GABA autoreceptors.

  2. Presynaptic Na+-dependent transport and exocytose of GABA and glutamate in brain in hypergravity.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Pozdnyakova, N.; Krisanova, N.; Himmelreich, N.

    γ-Aminobutyric acid (GABA) and L-glutamate are the most widespread neurotransmitter amino acids in the mammalian central nervous system. GABA is now widely recognized as the major inhibitory neurotransmitter. L-glutamate mediates the most of excitatory synaptic neurotransmission in the brain. They involved in the main aspects of normal brain function. The nerve terminals (synaptosomes) offer several advantages as a model system for the study of general mechanisms of neurosecretion. Our data allowed to conclude that exposure of animals to hypergravity (centrifugation of rats at 10G for 1 hour) had a profound effect on synaptic processes in brain. Comparative analysis of uptake and release of GABA and glutamate have demonstrated that hypergravity loading evokes oppositely directed alterations in inhibitory and excitatory signal transmission. We studied the maximal velocities of [^3H]GABA reuptake and revealed more than twofold enhancement of GABA transporter activity (Vmax rises from 1.4 |pm 0.3 nmol/min/mg of protein in the control group to 3.3 ± 0.59 nmol/min/mg of protein for animals exposed to hypergravity (P ≤ 0.05)). Recently we have also demonstrated the significant lowering of glutamate transporter activity (Vmax of glutamate reuptake decreased from 12.5 ± 3.2 nmol/min/mg of protein in the control group to 5.6 ± 0.9 nmol/min/mg of protein in the group of animals, exposed to the hypergravity stress (P ≤ 0.05)). Significant changes occurred in release of neurotransmitters induced by stimulating exocytosis with the agents, which depolarized nerve terminal plasma membrane. Depolarization-evoked Ca2+-stimulated release was more abundant for GABA (7.2 ± 0.54% and 11,74 ±1,2 % of total accumulated label for control and hypergravity, respectively (P≤0.05)) and was essentially less for glutamate (14.4 ± 0.7% and 6.2 ± 1.9%) after exposure of animals to centrifuge induced artificial gravity. Changes observed in depolarization-evoked exocytotic release

  3. Effects of Tityus serrulatus scorpion venom and its toxin TsTX-V on neurotransmitter uptake in vitro.

    PubMed

    Cecchini, Alessandra L; Vasconcelos, Flávio; Amara, Susan G; Giglio, José Roberto; Arantes, Eliane Candiani

    2006-12-01

    Scorpion neurotoxins targeting the Na(v) channel can be classified into two classes: alpha- and beta-neurotoxins and are reported as highly active in mammalian brain. In this work, we evaluate the effects of Tityus serrulatus venom (Ts venom) and its alpha-neurotoxin TsTX-V on gamma-aminobutyric acid (GABA), dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes. TsTX-V was isolated from Ts venom by ion exchange chromatography followed by reverse-phase (C18) high-performance liquid chromatography. Neither Ts venom nor TsTX-V was able to affect (3)H-Glu uptake. On the other hand, Ts venom (0.13 microg/mg) significantly inhibited both (3)H-GABA and (3)H-DA uptake ( approximately 50%). TsTX-V showed IC(50) values of 9.37 microM and 22.2 microM for the inhibition of (3)H-GABA and (3)H-DA uptake, respectively. These effects were abolished by pre-treatment with tetrodotoxin (TTX, 1 microM), indicating the involvement of voltage-gated Na(+) channels in this process. In the absence of Ca(2+), and at low Ts venom concentrations, the reduction of (3)H-GABA uptake was not as marked as in the presence of Ca(2+). TsTX-V did not reduce (3)H-GABA uptake in COS-7 cells expressing the GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced (3)H-GABA uptake by synaptosomes might be due to rapid cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes a reduction of (3)H-GABA and (3)H-DA uptake in a Ca(2+)-dependent manner, not directly affecting GABA transporters, but, in consequence of depolarization, involving voltage-gated Na(+) channels.

  4. New GABA amides activating GABAA-receptors.

    PubMed

    Raster, Peter; Späth, Andreas; Bultakova, Svetlana; Gorostiza, Pau; König, Burkhard; Bregestovski, Piotr

    2013-01-01

    We have prepared a series of new and some literature-reported GABA-amides and determined their effect on the activation of GABAA-receptors expressed in CHO cells. Special attention was paid to the purification of the target compounds to remove even traces of GABA contaminations, which may arise from deprotection steps in the synthesis. GABA-amides were previously reported to be partial, full or superagonists. In our hands these compounds were not able to activate GABAA-receptor channels in whole-cell patch-clamp recordings. New GABA-amides, however, gave moderate activation responses with a clear structure-activity relationship suggesting some of these compounds as promising molecular tools for the functional analysis of GABAA-receptors.

  5. New GABA amides activating GABAA-receptors

    PubMed Central

    Raster, Peter; Späth, Andreas; Bultakova, Svetlana; Gorostiza, Pau

    2013-01-01

    Summary We have prepared a series of new and some literature-reported GABA-amides and determined their effect on the activation of GABAA-receptors expressed in CHO cells. Special attention was paid to the purification of the target compounds to remove even traces of GABA contaminations, which may arise from deprotection steps in the synthesis. GABA-amides were previously reported to be partial, full or superagonists. In our hands these compounds were not able to activate GABAA-receptor channels in whole-cell patch-clamp recordings. New GABA-amides, however, gave moderate activation responses with a clear structure–activity relationship suggesting some of these compounds as promising molecular tools for the functional analysis of GABAA-receptors. PMID:23503884

  6. Novel GABA receptor pesticide targets.

    PubMed

    Casida, John E; Durkin, Kathleen A

    2015-06-01

    The γ-aminobutyric acid (GABA) receptor has four distinct but overlapping and coupled targets of pesticide action importantly associated with little or no cross-resistance. The target sites are differentiated by binding assays with specific radioligands, resistant strains, site-directed mutagenesis and molecular modeling. Three of the targets are for non-competitive antagonists (NCAs) or channel blockers of widely varied chemotypes. The target of the first generation (20th century) NCAs differs between the larger or elongated compounds (NCA-IA) including many important insecticides of the past (cyclodienes and polychlorocycloalkanes) or present (fiproles) and the smaller or compact compounds (NCA-IB) highly toxic to mammals and known as cage convulsants, rodenticides or chemical threat agents. The target of greatest current interest is designated NCA-II for the second generation (21st century) of NCAs consisting for now of isoxazolines and meta-diamides. This new and uniquely different NCA-II site apparently differs enough between insects and mammals to confer selective toxicity. The fourth target is the avermectin site (AVE) for allosteric modulators of the chloride channel. NCA pesticides vary in molecular surface area and solvent accessible volume relative to avermectin with NCA-IBs at 20-22%, NCA-IAs at 40-45% and NCA-IIs at 57-60%. The same type of relationship relative to ligand-docked length is 27-43% for NCA-IBs, 63-71% for NCA-IAs and 85-105% for NCA-IIs. The four targets are compared by molecular modeling for the Drosophila melanogaster GABA-R. The principal sites of interaction are proposed to be: pore V1' and A2' for NCA-IB compounds; pore A2', L6' and T9' for NCA-IA compounds; pore T9' to S15' in proximity to M1/M3 subunit interface (or alternatively an interstitial site) for NCA-II compounds; and M1/M3, M2 interfaces for AVE. Understanding the relationships of these four binding sites is important in resistance management and in the discovery and use

  7. Development of an (S)-1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}piperidine-3-carboxylic acid [(S)-SNAP-5114] carba analogue inhibitor for murine γ-aminobutyric acid transporter type 4.

    PubMed

    Pabel, Jörg; Faust, Mark; Prehn, Cornelia; Wörlein, Babette; Allmendinger, Lars; Höfner, Georg; Wanner, Klaus T

    2012-07-01

    A series of GABA uptake inhibitors related to (S)-1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}piperidine-3-carboxylic acid [(S)-SNAP-5114], the most potent mGAT4 inhibitor known so far, were synthesized and biologically evaluated for their inhibitory potency at the four GABA uptake transporters mGAT1-4 stably expressed in HEK-293 cell lines. New analogues were developed with potencies that are similar to or slightly higher than those of current mGAT4 inhibitors, but with distinctly improved chemical stability. (S)-Nipecotic acid derivatives possessing a 2-[1-(4-methoxy-2-methylphenyl)-1,1-bis(4-methoxyphenyl)methoxy]ethyl (DDPM-859) or a 4,4,4-tris(4-methoxyphenyl)but-2-en-1-yl moiety (DDPM-1457) were found to exhibit pIC(50) values of 5.78 and 5.87, respectively. Thus, as mGAT4 inhibitors, these compounds compare well with (S)-SNAP-5114 (pIC(50) =5.71), but are far more stable than the latter. Moreover, DDPM-859 displays a more favorable subtype selectivity for mGAT4 versus mGAT3 than does (S)-SNAP-5114. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Differential effects of phosphonic analogues of GABA on GABA(B) autoreceptors in rat neocortical slices.

    PubMed

    Ong, J; Marino, V; Parker, D A; Kerr, D I

    1998-04-01

    The effects of five phosphonic derivatives of GABA on the release of [3H]-GABA from rat neocortical slices, preloaded with [3H]-GABA, were investigated. Phaclofen and 4-aminobutylphosphonic acid (4-ABPA) increased the overflow of [3H] evoked by electrical stimulation (2 Hz) in a concentration-dependent manner, with similar potencies (phaclofen EC50=0.3 mmol/l, 4-ABPA EC50=0.4 mmol/l). At 3 mmol/l, phaclofen increased the release of [3H]-GABA by 82.6+/-8.6%, and 4-ABPA increased the release by 81.3+/-9.0%. 2-Amino-ethylphosphonic acid (2-AEPA) increased the overflow of [3H] by 46.8+/-10.9% at the highest concentration tested (3 mmol/l). In contrast, the lower phosphonic homologue 3-aminopropylphosphonic acid (3-APPA), and 2-amino-2-(p-chlorophenyl)-ethylphosphonic acid (2-CPEPA), a baclofen analogue, did not modify the stimulated overflow. These results suggest that phaclofen, 4-ABPA and 2-AEPA are antagonists at GABA(B) autoreceptors, the latter being the weakest antagonist, whilst neither 3-APPA nor 2-CPEPA are active at these receptors. Since phaclofen, 4-ABPA and 2-CPEPA are antagonists and 3-APPA a partial agonist/antagonist on GABA(B) heteroreceptors, the lack of effect of 3-APPA and 2-CPEPA on [3H]-GABA release in this study suggests that GABA(B) autoreceptors may be pharmacologically distinct from the heteroreceptors.

  9. Excitatory effects of GABA on procerebrum neurons in a slug.

    PubMed

    Kobayashi, Suguru; Matsuo, Ryota; Sadamoto, Hisayo; Watanabe, Satoshi; Ito, Etsuro

    2012-08-01

    Classical neurotransmitters, such as glutamate and γ-aminobutyric acid (GABA), often have different actions on invertebrate neurons from those reported for vertebrate neurons. In the terrestrial mollusk Limax, glutamate was found to function as an inhibitory transmitter in the procerebrum (PC), but it has not yet been clarified how GABA acts in the PC. We thus examined what effects GABA exerts on PC neurons in the present study. For this purpose, we first applied GABA to isolated PC preparations and recorded postsynaptic currents and potentials in PC neurons. The GABA application reduced the amplitude of inhibitory postsynaptic currents and depolarization-induced outward currents recorded in nonbursting neurons and increased the number of spontaneous spikes of nonbursting neurons. However, direct GABA-induced currents were not observed in either bursting or nonbursting neurons. These results suggest a potential direct effect of GABA on outward currents resulting in enhanced excitability of PC neurons. Next, we measured the change in [Ca(2+)](i) in cultured PC neurons by application of GABA. The GABA application increased spontaneous Ca(2+) events in cultured neurons. These Ca(2+) events were ascribable to the influx of extracellular Ca(2+). We then confirmed the presence of GABA and GABA receptors in the PC. The GABA-like immunoreactivity was observed in the neuropil layers of the PC, and the mRNAs for both GABA(A) and GABA(B) receptors were expressed in the PC. In particular, GABA(B) receptor mRNA, rather than GABA(A), was found to be more abundantly expressed in the PC. These results suggest that GABA functions as an excitatory modulator for PC neurons via mainly GABA(B) receptors.

  10. micro-Opioid receptor endocytosis prevents adaptations in ventral tegmental area GABA transmission induced during naloxone-precipitated morphine withdrawal.

    PubMed

    Madhavan, Anuradha; He, Li; Stuber, Garret D; Bonci, Antonello; Whistler, Jennifer L

    2010-03-03

    Chronic morphine drives adaptations in synaptic transmission thought to underlie opiate dependence. Here we examine the role of micro-opioid receptor (MOR) trafficking in one of these adaptations, specifically, changes in GABA transmission in the ventral tegmental area (VTA). To address this question, we used a knock-in mouse, RMOR (for recycling MOR), in which genetic change in the MOR promotes morphine-induced receptor desensitization and endocytosis in GABA interneurons of the VTA. In wild-type mice (postnatal days 23-28) chronic morphine (10 mg/kg, s.c., twice daily for 5 d), induced a cAMP-dependent increase in the probability of GABA release onto VTA dopamine neurons. The increased GABA release frequency correlated with physical dependence on morphine measured by counting somatic signs of morphine withdrawal, such as, tremors, jumps, rears, wet-dog shakes, and grooming behavior precipitated by subcutaneous administration of naloxone (NLX) (2 mg/kg). This adaptation in GABA release was prevented in RMOR mice given the same morphine treatment, implicating MOR trafficking in this morphine-induced change in plasticity. Importantly, treatment with the cAMP activity inhibitor rp-cAMPS [(R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium] (50 ng/0.5 microl), directly to the VTA, attenuated somatic withdrawal signs to systemic morphine produced by intra-VTA NLX (500 ng/0.5 microl), directly tying enhanced cAMP-driven GABA release to naloxone-precipitated morphine withdrawal in the VTA.

  11. GABA(B2) is essential for g-protein coupling of the GABA(B) receptor heterodimer.

    PubMed

    Robbins, M J; Calver, A R; Filippov, A K; Hirst, W D; Russell, R B; Wood, M D; Nasir, S; Couve, A; Brown, D A; Moss, S J; Pangalos, M N

    2001-10-15

    GABA(B) receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA(B1) and GABA(B2), for functional expression. Whereas GABA(B1) is capable of binding receptor agonists and antagonists, the role of each GABA(B) subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA(B2) that are critical for the coupling of GABA(B) receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA(B2) subunit in G-protein coupling of the GABA(B) receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.

  12. Basic mechanism leading to stimulation of glycogenolysis by isoproterenol, EGF, elevated extracellular K+ concentrations, or GABA.

    PubMed

    Xu, Junnan; Song, Dan; Bai, Qiufang; Cai, Liping; Hertz, Leif; Peng, Liang

    2014-04-01

    Glycogenolysis, in brain parenchyma an astrocyte-specific process, has changed from being envisaged as an emergency procedure to playing central roles during brain response to whisker stimulation, memory formation, astrocytic K(+) uptake and stimulated release of ATP. It is activated by several transmitters and by even very small increases in extracellular K(+) concentration, and to be critically dependent upon an increase in free cytosolic Ca(2+) concentration ([Ca(2+)]i), whereas cAMP plays only a facilitatory role together with increased [Ca(2+)]i. Detailed knowledge about the signaling pathways eliciting glycogenolysis is therefore of interest and was investigated in the present study in well differentiated cultures of mouse astrocytes. The β-adrenergic agonist isoproterenol stimulated glycogenolysis by a β1-adrenergic effect, which initiated a pathway in which cAMP/protein kinase A activated a Gi/Gs shift, leading to Ca(2+)-activated glycogenolysis. Inhibition of this pathway downstream of cAMP but upstream of the Gi/Gs shift abolished the glycogenolysis. However, inhibitors operating downstream of the Ca(2+)-sensitive step, but preventing transactivation-mediated epidermal growth factor (EGF) receptor stimulation, a later step in the activated pathway, also caused inhibition of glycogenolysis. For this reason the effect of EGF was investigated and it was found to be glycogenolytic. Large increases in extracellular K(+) activated glycogenolysis by a nifedipine-inhibited L-channel opening allowing influx of Ca(2+), known to be glycogenolysis-dependent. Small increases (addition of 5 mM KCl) caused a smaller effect by a similarly glycogenolysis-reliant opening of an IP3 receptor-dependent ouabain signaling pathway. The same pathway could be activated by GABA (also in brain slices) due to its depolarizing effect in astrocytes.

  13. GABA and Glutamate Pathways Are Spatially and Developmentally Affected in the Brain of Mecp2-Deficient Mice

    PubMed Central

    Matagne, Valérie; Ghata, Adeline; Villard, Laurent; Roux, Jean-Christophe

    2014-01-01

    Proper brain functioning requires a fine-tuning between excitatory and inhibitory neurotransmission, a balance maintained through the regulation and release of glutamate and GABA. Rett syndrome (RTT) is a rare genetic disorder caused by mutations in the methyl-CpG binding protein 2 (MECP2) gene affecting the postnatal brain development. Dysfunctions in the GABAergic and glutamatergic systems have been implicated in the neuropathology of RTT and a disruption of the balance between excitation and inhibition, together with a perturbation of the electrophysiological properties of GABA and glutamate neurons, were reported in the brain of the Mecp2-deficient mouse. However, to date, the extent and the nature of the GABA/glutamate deficit affecting the Mecp2-deficient mouse brain are unclear. In order to better characterize these deficits, we simultaneously analyzed the GABA and glutamate levels in Mecp2-deficient mice at 2 different ages (P35 and P55) and in several brain areas. We used a multilevel approach including the quantification of GABA and glutamate levels, as well as the quantification of the mRNA and protein expression levels of key genes involved in the GABAergic and glutamatergic pathways. Our results show that Mecp2-deficient mice displayed regional- and age-dependent variations in the GABA pathway and, to a lesser extent, in the glutamate pathway. The implication of the GABA pathway in the RTT neuropathology was further confirmed using an in vivo treatment with a GABA reuptake inhibitor that significantly improved the lifespan of Mecp2-deficient mice. Our results confirm that RTT mouse present a deficit in the GABAergic pathway and suggest that GABAergic modulators could be interesting therapeutic agents for this severe neurological disorder. PMID:24667344

  14. Chronic lamotrigine treatment increases rat hippocampal GABA shunt activity and elevates cerebral taurine levels.

    PubMed

    Hassel, B; Taubøll, E; Gjerstad, L

    2001-02-01

    The mechanism of action of the antiepileptic drug lamotrigine has previously been investigated only in acute experiments and is thought to involve inhibition of voltage-dependent sodium channels. However, lamotrigine is effective against more forms of epilepsies than other antiepileptic drugs that also inhibit sodium channels. We investigated whether chronic lamotrigine treatment may affect cerebral amino acid levels. Rats received lamotrigine, 10 mg/kg/day, for 90 days. The hippocampal level of GABA increased 25%, and the activities of glutamate decarboxylase and succinic semialdehyde/GABA transaminase increased 12 and 21% (p< 0.05), respectively, indicating increased GABA turnover. The uptake of GABA and glutamate into proteoliposomes remained unaltered. The level of taurine increased 27% in the hippocampus and 16% in the frontal and parietal cortices. The activities of hexokinase and alpha-ketoglutarate dehydrogenase, remained at control values. Serum lamotrigine was 41.7+/-1.5 microM (mean+/-S.E.M.), which is within the range seen in epileptic patients. Acute experiments with 5, 20 or 100 mg lamotrigine/kg, caused no changes in brain amino acid levels. The results suggest that chronic lamotrigine treatment increases GABAergic activity in the hippocampus. The cerebral increase in taurine, which has neuromodulatory properties, may contribute to the antiepileptic effect of lamotrigine.

  15. Production of gaba (γ - Aminobutyric acid) by microorganisms: a review.

    PubMed

    Dhakal, Radhika; Bajpai, Vivek K; Baek, Kwang-Hyun

    2012-10-01

    GABA (γ-aminobutyric acid) is a four carbon non-protein amino acid that is widely distributed in plants, animals and microorganisms. As a metabolic product of plants and microorganisms produced by the decarboxylation of glutamic acid, GABA functions as an inhibitory neurotransmitter in the brain that directly affects the personality and the stress management. A wide range of traditional foods produced by microbial fermentation contain GABA, in which GABA is safe and eco-friendly, and also has the possibility of providing new health-benefited products enriched with GABA. Synthesis of GABA is catalyzed by glutamate decarboxylase, therefore, the optimal fermentation condition is mainly based on the biochemical properties of the enzyme. Major GABA producing microorganisms are lactic acid bacteria (LAB), which make food spoilage pathogens unable to grow and act as probiotics in the gastrointestinal tract. The major factors affecting the production of GABA by microbial fermentation are temperature, pH, fermentation time and different media additives, therefore, these factors are summarized to provide the most up-dated information for effective GABA synthesis. There has been a huge accumulation of knowledge on GABA application for human health accompanying with a demand on natural GABA supply. Only the GABA production by microorganisms can fulfill the demand with GABA-enriched health beneficial foods.

  16. My close encounter with GABA(B) receptors.

    PubMed

    Nicoll, Roger A

    2004-10-15

    In this review, I summarize the sequence of events involved in characterizing the functional role of GABA(B) receptors in the CNS and their involvement in synaptic transmission. The story was launched with the realization that baclofen was a selective agonist of GABA(B) receptors. This lead to the discovery in the CNS that GABA(B) receptor activation could result in a presynaptic inhibition of transmitter release as well as a postsynaptic increase in potassium conductance. Based on this information, it was found that GABA also activated a potassium conductance. A role for GABA(B) receptors in synaptic transmission was suggested by the fact that activation of GABAergic interneurons could generate a slow IPSP mediated by an increase in potassium conductance. To link this slow IPSP to GABA(B) receptors required a selective GABA(B) antagonist. Phaclofen was the first antagonist developed and was found to antagonize the action of baclofen and the GABA(A) independent action of GABA. Most importantly, it blocked the slow IPSP. The properties of GABA(A) and GABA(B) IPSPs are remarkably different. GABA(A) IPSPs powerfully inhibit neurons and rapidly curtail excitatory inputs. This greatly enhances the precision of excitatory synaptic transmission. GABA(B) IPSPs are recruited with repetitive and synchronous activity and are postulated to modulate the rhythmic network activity of cortical tissue.

  17. Treatment of chronic pruritus with the selective serotonin re-uptake inhibitors paroxetine and fluvoxamine: results of an open-labelled, two-arm proof-of-concept study.

    PubMed

    Ständer, Sonja; Böckenholt, Barbara; Schürmeyer-Horst, Funda; Weishaupt, Carsten; Heuft, Gereon; Luger, Thomas A; Schneider, Gudrun

    2009-01-01

    Chronic pruritus is difficult to treat and requires the evaluation of new therapeutic modalities. We initiated an open-labelled, two-arm prospective, proof-of-concept study applying two selective serotonin re-uptake inhibitors on a long-term basis. Paroxetine and fluvoxamine were tested in a total of 72 pruritic patients (27 men, 45 women, age range 28-88 years, mean age 59.2 years). The reduction in pruritus was evaluated by analysis of visual analogue scores and determination of the maximal antipruritic effect (maximal percentual reduction in pruritus). Forty-nine of 72 patients (68.0%) experienced a weak (n=9), good (n=16) or very good (n=24) antipruritic effect. Statistical analysis proved the efficacy of paroxetine and fluvoxamine with no significant difference. The best response was observed in patients with pruritus due to atopic dermatitis, systemic lymphoma and solid carcinoma. Chronic scratch lesions healed completely in 14/31 patients and partially in 17/31 patients. Adverse drug effects were observed in 70.8% of patients, resulting in discontinuation of treatment in 18 patients. These results support previous reports of high antipruritic potency of selective serotonin re-uptake inhibitors, which are a good alternative treatment modality in chronic pruritus. This should be confirmed in future double-blind studies.

  18. Flexible and biomimetic analogs of triple uptake inhibitor 4-((((3S,6S)-6-benzhydryltetrahydro-2H-pyran-3-yl)amino)methyl)phenol: Synthesis, biological characterization, and development of a pharmacophore model.

    PubMed

    Sharma, Horrick; Santra, Soumava; Debnath, Joy; Antonio, Tamara; Reith, Maarten; Dutta, Aloke

    2014-01-01

    In this study we have generated a pharmacophore model of triple uptake inhibitor compounds based on novel asymmetric pyran derivatives and the newly developed asymmetric furan derivatives. The model revealed features important for inhibitors to exhibit a balanced activity against dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). In particular, a 'folded' conformation was found common to the active pyran compounds in the training set and was crucial to triple uptake inhibitory activity. Furthermore, the distances between the benzhydryl moiety and the N-benzyl group as well as the orientation of the secondary nitrogen were also important for TUI activity. We have validated our findings by synthesizing and testing novel asymmetric pyran analogs. The present work has also resulted in the discovery of a new series of asymmetric tetrahydrofuran derivatives as novel TUIs. Lead compounds 41 and 42 exhibited moderate TUI activity. Interestingly, the highest TUI activity by lead tetrahydrofuran compounds for example, 41 and 42, was exhibited in a stereochemical preference similar to pyran TUI for example, D-161. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Flexible and biomimetic analogs of triple uptake inhibitor 4-((((3S,6S)-6-benzhydryltetrahydro-2H-pyran-3-yl)amino)methyl)phenol : Synthesis, biological characterization, and development of a pharmacophore model

    PubMed Central

    Sharma, Horrick; Santra, Soumava; Debnath, Joy; Antonio, Tamara; Reith, Maarten; Dutta, Aloke

    2014-01-01

    In this study we have generated a pharmacophore model of triple uptake inhibitor compounds based on novel asymmetric pyran derivatives and the newly developed asymmetric furan derivatives. The model revealed features important for inhibitors to exhibit a balanced activity against dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). In particular, a ‘folded’ conformation was found common to the active pyran compounds in the training set and was crucial to triple uptake inhibitory activity. Furthermore, the distances between the benzhydryl moiety and the N-benzyl group as well as the orientation of the secondary nitrogen were also important for TUI activity. We have validated our findings by synthesizing and testing novel asymmetric pyran analogs. The present work has also resulted in the discovery of a new series of asymmetric tetrahydrofuran derivatives as novel TUIs. Lead compounds 41 and 42 exhibited moderate TUI activity. Interestingly, the highest TUI activity by lead tetrahydrofuran compounds e.g. 41 and 42, was exhibited in a stereochemical preference similar to pyran TUI e.g. D-161. PMID:24315194

  20. Taurine activates GABA(A) but not GABA(B) receptors in rat hippocampal CA1 area.

    PubMed

    del Olmo, N; Bustamante, J; del Río, R M; Solís, J M

    2000-05-12

    We investigated if taurine, an endogenous GABA analog, could mimic both hyperpolarizing and depolarizing GABA(A)-mediated responses as well as pre- and postsynaptic GABA(B)-mediated actions in the CA1 region of rat hippocampal slices. Taurine (10 mM) perfusion induced changes in membrane potential and input resistance that are compatible with GABA(A) receptor activation. Local pressure application of taurine and GABA from a double barrel pipette positioned along the dendritic shaft of pyramidal cells revealed that taurine evoked a very small change of membrane potential and resistance compared with the large changes induced by GABA in these parameters. Moreover, in the presence of GABA(A) antagonists, local application of GABA on the dendrites evoked a GABA(B)-mediated hyperpolarization while taurine did not induce any change. Taurine neither mimicked baclofen inhibitory actions on presynaptic release of glutamate and GABA as judging by the lack of taurine effect on paired-pulse facilitation ratio and slow inhibitory postsynaptic potentials, respectively. These results show that taurine mainly activates GABA(A) receptors located on the cell body, indicating therefore that if taurine has any action on the dendrites it will not be mediated by either GABA(A) or GABA(B) receptors activation.

  1. Trophic actions of GABA on neuronal development.

    PubMed

    Represa, Alfonso; Ben-Ari, Yehezkel

    2005-06-01

    During brain development, transmitter-gated receptors are operative before synapse formation, suggesting that their action is not restricted to synaptic transmission. GABA, which is the principal excitatory transmitter in the developing brain, acts as an epigenetic factor to control processes including cell proliferation, neuroblast migration and dendritic maturation. These effects appear to be mediated through a paracrine, diffuse, non-synaptic mode of action that precedes the more focused, rapid mode of operation characteristic of synaptic connections. This sequential operation implies that GABA is used as an informative agent but in a unique context at an early developmental stage. This sequence also implies that by altering these effects, drugs acting on the GABA system could be pathogenic during pregnancy.

  2. An excitatory GABA loop operating in vivo

    PubMed Central

    Astorga, Guadalupe; Bao, Jin; Marty, Alain; Augustine, George J.; Franconville, Romain; Jalil, Abdelali; Bradley, Jonathan; Llano, Isabel

    2015-01-01

    While it has been proposed that the conventional inhibitory neurotransmitter GABA can be excitatory in the mammalian brain, much remains to be learned concerning the circumstances and the cellular mechanisms governing potential excitatory GABA action. Using a combination of optogenetics and two-photon calcium imaging in vivo, we find that activation of chloride-permeable GABAA receptors in parallel fibers (PFs) of the cerebellar molecular layer of adult mice causes parallel fiber excitation. Stimulation of PFs at submaximal stimulus intensities leads to GABA release from molecular layer interneurons (MLIs), thus creating a positive feedback loop that enhances excitation near the center of an activated PF bundle. Our results imply that elevated chloride concentration can occur in specific intracellular compartments of mature mammalian neurons and suggest an excitatory role for GABAA receptors in the cerebellar cortex of adult mice. PMID:26236197

  3. Advantages of an antagonist: bicuculline and other GABA antagonists

    PubMed Central

    Johnston, Graham AR

    2013-01-01

    The convulsant alkaloid bicuculline continues to be investigated more than 40 years after the first publication of its action as an antagonist of receptors for the inhibitory neurotransmitter GABA. This historical perspective highlights key aspects of the discovery of bicuculline as a GABA antagonist and the sustained interest in this and other GABA antagonists. The exciting advances in the molecular biology, pharmacology and physiology of GABA receptors provide a continuing stimulus for the discovery of new antagonists with increasing selectivity for the myriad of GABA receptor subclasses. Interesting GABA antagonists not structurally related to bicuculline include gabazine, salicylidene salicylhydrazide, RU5135 and 4-(3-biphenyl-5-(4-piperidyl)-3-isoxazole. Bicuculline became the benchmark antagonist for what became known as GABAA receptors, but not all ionotropic GABA receptors are susceptible to bicuculline. In addition, not all GABAA receptor antagonists are convulsants. Thus there are still surprises in store as the study of GABA receptors evolves. PMID:23425285

  4. Inositol uptake in rat aorta

    SciTech Connect

    Rapoport, R.M.; Van Gorp, C.; Chang, Ki-Churl )

    1990-01-01

    {sup 3}H-inositol uptake into deendothelialized aorta was linear for at least 2 h and was composed of both a saturable, Na{sup +}-dependent, and a nonsaturable, Na{sup +}-independent component. The Na{sup +}-dependent component of inositol uptake had a K{sub m} of 50 {mu}M and a V{sub max} of 289 pmol/mg prot/h. Exposure to LiCl, ouabain, or Ca{sup 2+} - free Krebs-Ringer bicarbonate solution inhibited uptake. Metabolic poisoning with dinitrophenol, as well as incubation with phloretin, an inhibitor of carrier-mediated hexose transport, also inhibited uptake. Exposure to norepinephrine decreased inositol uptake, while phorbol myristate acetate was without effect. Isobutylmethylxanthine significantly increased inositol uptake, while the increased uptake due to dibutyryl cyclic AMP and forskolin were not statistically significant. Sodium nitroprusside, and activator of guanylate cyclase, and 8-bromo cyclic GMP, were without effect on uptake, as was methylene blue, an inhibitor of guanylate cyclase. Inositol uptake into the aorta was increased when the endothelium was allowed to remain intact, although this effect was likely due to uptake in both the endothelial and smooth muscle cells.

  5. Glutamate and GABA in Appetite Regulation

    PubMed Central

    Delgado, Teresa C.

    2013-01-01

    Appetite is regulated by a coordinated interplay between gut, adipose tissue, and brain. A primary site for the regulation of appetite is the hypothalamus where interaction between orexigenic neurons, expressing Neuropeptide Y/Agouti-related protein, and anorexigenic neurons, expressing Pro-opiomelanocortin cocaine/Amphetamine-related transcript, controls energy homeostasis. Within the hypothalamus, several peripheral signals have been shown to modulate the activity of these neurons, including the orexigenic peptide ghrelin and the anorexigenic hormones insulin and leptin. In addition to the accumulated knowledge on neuropeptide signaling, presence and function of amino acid neurotransmitters in key hypothalamic neurons brought a new light into appetite regulation. Therefore, the principal aim of this review will be to describe the current knowledge of the role of amino acid neurotransmitters in the mechanism of neuronal activation during appetite regulation and the associated neuronal-astrocytic metabolic coupling mechanisms. Glutamate and GABA dominate synaptic transmission in the hypothalamus and administration of their receptors agonists into hypothalamic nuclei stimulates feeding. By using 13C High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance spectroscopy based analysis, the Cerdán group has shown that increased neuronal firing in mice hypothalamus, as triggered by appetite during the feeding-fasting paradigm, may stimulate the use of lactate as neuronal fuel leading to increased astrocytic glucose consumption and glycolysis. Moreover, fasted mice showed increased hypothalamic [2-13C]GABA content, which may be explained by the existence of GABAergic neurons in key appetite regulation hypothalamic nuclei. Interestingly, increased [2-13C]GABA concentration in the hypothalamus of fasted animals appears to result mainly from reduction in GABA metabolizing pathways, rather than increased GABA synthesis by augmented activity of the glutamate-glutamine-GABA

  6. Glutamate and GABA in Appetite Regulation.

    PubMed

    Delgado, Teresa C

    2013-01-01

    Appetite is regulated by a coordinated interplay between gut, adipose tissue, and brain. A primary site for the regulation of appetite is the hypothalamus where interaction between orexigenic neurons, expressing Neuropeptide Y/Agouti-related protein, and anorexigenic neurons, expressing Pro-opiomelanocortin cocaine/Amphetamine-related transcript, controls energy homeostasis. Within the hypothalamus, several peripheral signals have been shown to modulate the activity of these neurons, including the orexigenic peptide ghrelin and the anorexigenic hormones insulin and leptin. In addition to the accumulated knowledge on neuropeptide signaling, presence and function of amino acid neurotransmitters in key hypothalamic neurons brought a new light into appetite regulation. Therefore, the principal aim of this review will be to describe the current knowledge of the role of amino acid neurotransmitters in the mechanism of neuronal activation during appetite regulation and the associated neuronal-astrocytic metabolic coupling mechanisms. Glutamate and GABA dominate synaptic transmission in the hypothalamus and administration of their receptors agonists into hypothalamic nuclei stimulates feeding. By using (13)C High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance spectroscopy based analysis, the Cerdán group has shown that increased neuronal firing in mice hypothalamus, as triggered by appetite during the feeding-fasting paradigm, may stimulate the use of lactate as neuronal fuel leading to increased astrocytic glucose consumption and glycolysis. Moreover, fasted mice showed increased hypothalamic [2-(13)C]GABA content, which may be explained by the existence of GABAergic neurons in key appetite regulation hypothalamic nuclei. Interestingly, increased [2-(13)C]GABA concentration in the hypothalamus of fasted animals appears to result mainly from reduction in GABA metabolizing pathways, rather than increased GABA synthesis by augmented activity of the glutamate-glutamine-GABA

  7. [GABA-ergic system in defense against excitatory kynurenines].

    PubMed

    Lapin, I P

    1997-01-01

    Protection against the excitatory action of L-kynurenine and quinolinic acid in mice is related to the activation of GABA-B and dopamine receptors of the brain and to much lesser degree to the activation of GABA-A receptors. It is hardly believable that the anticonvulsant effect of phenibut (beta-phenyl-GABA), baclofen (CL-phenibut), sodium hydroxybutyrate and taurine against seizures induced by these two kynurenines is determined by alterations in metabolism of GABA.

  8. Synergistic GABA-Enhancing Therapy against Seizures in a Mouse Model of Dravet Syndrome

    PubMed Central

    Oakley, John C.; Cho, Alvin R.; Cheah, Christine S.; Scheuer, Todd

    2013-01-01

    Seizures remain uncontrolled in 30% of patients with epilepsy, even with concurrent use of multiple drugs, and uncontrolled seizures result in increased morbidity and mortality. An extreme example is Dravet syndrome (DS), an infantile-onset severe epilepsy caused by heterozygous loss of function mutations in SCN1A, the gene encoding the brain type-I voltage-gated sodium channel NaV1.1. Studies in Scn1a heterozygous knockout mice demonstrate reduced excitability of GABAergic interneurons, suggesting that enhancement of GABA signaling may improve seizure control and comorbidities. We studied the efficacy of two GABA-enhancing drugs, clonazepam and tiagabine, alone and in combination, against thermally evoked myoclonic and generalized tonic-clonic seizures. Clonazepam, a positive allosteric modulator of GABA-A receptors, protected against myoclonic and generalized tonic-clonic seizures. Tiagabine, a presynaptic GABA reuptake inhibitor, was protective against generalized tonic-clonic seizures but only minimally protective against myoclonic seizures and enhanced myoclonic seizure susceptibility at high doses. Combined therapy with clonazepam and tiagabine was synergistic against generalized tonic-clonic seizures but was additive against myoclonic seizures. Toxicity determined by rotorod testing was additive for combination therapy. The synergistic actions of clonazepam and tiagabine gave enhanced seizure protection and reduced toxicity, suggesting that combination therapy may be well tolerated and effective for seizures in DS. PMID:23424217

  9. Oxytocin regulates changes of extracellular glutamate and GABA levels induced by methamphetamine in the mouse brain.

    PubMed

    Qi, Jia; Han, Wen-Yan; Yang, Jing-Yu; Wang, Li-Hui; Dong, Ying-Xu; Wang, Fang; Song, Ming; Wu, Chun-Fu

    2012-07-01

    Oxytocin (OT), a neurohypophyseal neuropeptide, affects adaptive processes of the central nervous system. In the present study, we investigated the effects of OT on extracellular levels of glutamate (Glu) and γ-aminobutyric acid (GABA) induced by methamphetamine (MAP) in the medial prefrontal cortex (mPFC) and dorsal hippocampus (DHC) of freely moving mice, using in vivo microdialysis coupled to high-performance liquid chromatography and fluorescence detection. The results showed that OT had no effect on basal Glu levels, but attenuated MAP-induced Glu increase in the mPFC and decrease in the DHC. OT increased the basal levels of extracellular GABA in mPFC and DHC of mice, and inhibited the MAP-induced GABA decrease in DHC. Western blot results indicated that OT significantly inhibited the increased glutamatergic receptor (NR1 subunit) levels in the PFC after acute MAP administration, whereas OT further enhanced the elevated levels of glutamatergic transporter (GLT1) induced by MAP in the hippocampus of mice. Atosiban, a selective inhibitor of OT receptor, antagonized the effects of OT. The results provided the first neurochemical evidence that OT, which exerted its action via its receptor, decreased Glu release induced by MAP, and attenuated the changes in glutamatergic neurotransmission partially via regulation of NR1 and GLT1 expression. OT-induced extracellular GABA increase also suggests that OT acts potentially as an inhibitory neuromodulator in mPFC and DHC of mice.

  10. GABA selectively controls the secretory activity of oxytocin neurons in the rat supraoptic nucleus.

    PubMed

    Engelmann, Mario; Bull, Philip M; Brown, Colin H; Landgraf, Rainer; Horn, Thomas F W; Singewald, Nicolas; Ludwig, Mike; Wotjak, Carsten T

    2004-02-01

    Recently we reported that a single social defeat experience triggers the release of oxytocin (OXT) from somata and dendrites, but not axon terminals, of neurons of the hypothalamic-neurohypophysial system. To further investigate the regulatory mechanisms underlying this dissociated release, we exposed male Wistar rats to a 30-min social defeat and monitored release of the inhibitory amino acids gamma amino butyric acid (GABA) and taurine within the hypothalamic supraoptic nucleus (SON) using microdialysis. Social defeat caused a significant increase in the release of both GABA and taurine within the SON (up to 480%; P < 0.01 vs. prestress release). To reveal the physiological significance of centrally released GABA, the specific GABAA-receptor antagonist bicuculline (0.02 mm) was administered into the SON via retrodialysis. This approach caused a significant increase in the release of OXT both within the SON and into the blood under basal conditions and during stress (up to 300 and 200%, respectively; P < 0.05 vs. basal values), without affecting plasma vasopressin. Electrophysiological studies confirmed the selective action of bicuculline on the firing activity of OXT neurons in the SON. Taken together, our data demonstrate that GABA is released within the SON during emotional stress to act as a selective inhibitor of both central and peripheral OXT secretion.

  11. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and gamma-vinyl-gamma-aminobutyric acid (gamma-vinyl GABA) alter neurotransmitter concentrations in the nervous tissue of the goldfish (Carassius auratus) but not the cockroach (Periplaneta americana).

    PubMed

    Sloley, B D; McKenna, K F

    1993-02-01

    1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium iodide (MPP+) and gamma-vinyl-gamma-aminobutyric acid (gamma-vinyl GABA) are drugs demonstrated to alter catecholamine or gamma-aminobutyric acid (GABA) concentrations in vertebrate nervous tissue. MPTP and MPP+, which are potent and selective vertebrate neurotoxins, are effective in depleting noradrenaline and dopamine concentrations in goldfish. However, only MPP+ depletes dopamine in the central nervous tissues of the cockroach, and only when injected directly into the nervous tissue. Systemic injection of gamma-vinyl GABA, a selective GABA transaminase inhibitor in vertebrates, increases GABA concentrations in goldfish but not cockroach nervous tissue. Incubations of both goldfish hypothalamus and cockroach nervous tissue demonstrated the presence of GABA transaminase activity in vitro. However, the GABA transaminase activity obtained from goldfish tissues was much more sensitive to inhibition by gamma-vinyl GABA than that obtained from cockroach nervous tissue. These results demonstrate that MPTP, MPP+ and gamma-vinyl GABA are useful pharmacological tools which can alter neurotransmitter concentrations in a lower vertebrate. Unfortunately, they possess limited effectiveness in the cockroach.

  12. Assignment of the human GABA transporter gene (GABATHG) locus to chromosome 3p24-p25

    SciTech Connect

    Huang, Fang; Fei, Jian; Guo, Li-He

    1995-09-01

    An essential regulatory process of synaptic transmission is the inactivation of released neurotransmitters by the transmitter-specific uptake mechanism, {gamma}-Aminobutyric acid (GABA) is an inhibitory transmitter in the vertebrate central nervous system; its activity is terminated by a high-affinity Na{sup +} and Cl{sup -} -dependent specific GABA transporter (GAT), which carries the neurotransmitter to the presynaptic neuron and/or glial elements surrounding the synaptic cleft. Deficiency of the transporter may cause epilepsy and some other nervous diseases. The human GAT gene (GABATHG), approximately 25 kb in length, has been cloned and sequenced by our colleagues (7). Here the results of the in situ hybridization mapping with the gene are presented. 10 refs., 1 fig.

  13. Frontal GABA levels change during working memory.

    PubMed

    Michels, Lars; Martin, Ernst; Klaver, Peter; Edden, Richard; Zelaya, Fernando; Lythgoe, David J; Lüchinger, Rafael; Brandeis, Daniel; O'Gorman, Ruth L

    2012-01-01

    Functional neuroimaging metrics are thought to reflect changes in neurotransmitter flux, but changes in neurotransmitter levels have not been demonstrated in humans during a cognitive task, and the relationship between neurotransmitter dynamics and hemodynamic activity during cognition has not yet been established. We evaluate the concentration of the major inhibitory (GABA) and excitatory (glutamate + glutamine: Glx) neurotransmitters and the cerebral perfusion at rest and during a prolonged delayed match-to-sample working memory task. Resting GABA levels in the dorsolateral prefrontal cortex correlated positively with the resting perfusion and inversely with the change in perfusion during the task. Further, only GABA increased significantly during the first working memory run and then decreased continuously across subsequent task runs. The decrease of GABA over time was paralleled by a trend towards decreased reaction times and higher task accuracy. These results demonstrate a link between neurotransmitter dynamics and hemodynamic activity during working memory, indicating that functional neuroimaging metrics depend on the balance of excitation and inhibition required for cognitive processing.

  14. Plant reproduction: GABA gradient, guidance and growth.

    PubMed

    Ma, Hong

    2003-10-28

    How a pollen tube manages to navigate through the female tissues during plant reproduction has been a mystery. A new analysis of an Arabidopsis mutant has provided the strongest evidence yet that a GABA gradient may be a critical signal for correct targeting of the pollen tube.

  15. Drug interactions at GABA(A) receptors.

    PubMed

    Korpi, Esa R; Gründer, Gerhard; Lüddens, Hartmut

    2002-06-01

    Neurotransmitter receptor systems have been the focus of intensive pharmacological research for more than 20 years for basic and applied scientific reasons, but only recently has there been a better understanding of their key features. One of these systems includes the type A receptor for the gamma-aminobutyric acid (GABA), which forms an integral anion channel from a pentameric subunit assembly and mediates most of the fast inhibitory neurotransmission in the adult vertebrate central nervous system. Up to now, depending on the definition, 16-19 mammalian subunits have been cloned and localized on different genes. Their assembly into proteins in a poorly defined stoichiometry forms the basis of functional and pharmacological GABA(A) receptor diversity, i.e. the receptor subtypes. The latter has been well documented in autoradiographic studies using ligands that label some of the receptors' various binding sites, corroborated by recombinant expression studies using the same tools. Significantly less heterogeneity has been found at the physiological level in native receptors, where the subunit combinations have been difficult to dissect. This review focuses on the characteristics, use and usefulness of various ligands and their binding sites to probe GABA(A) receptor properties and to gain insight into the biological function from fish to man and into evolutionary conserved GABA(A) receptor heterogeneity. We also summarize the properties of the novel mouse models created for the study of various brain functions and review the state-of-the-art imaging of brain GABA(A) receptors in various human neuropsychiatric conditions. The data indicate that the present ligands are only partly satisfactory tools and further ligands with subtype-selective properties are needed for imaging purposes and for confirming the behavioral and functional results of the studies presently carried out in gene-targeted mice with other species, including man.

  16. Comparative immunohistochemical localisation of GABA(B1a), GABA(B1b) and GABA(B2) subunits in rat brain, spinal cord and dorsal root ganglion.

    PubMed

    Charles, K J; Evans, M L; Robbins, M J; Calver, A R; Leslie, R A; Pangalos, M N

    2001-01-01

    GABA(B) receptors are G-protein-coupled receptors mediating the slow onset and prolonged synaptic actions of GABA in the CNS. The recent cloning of two genes, GABA(B1) and GABA(B2), has revealed a novel requirement for GABA(B) receptor signalling. Studies have demonstrated that the two receptor subunits associate as a GABA(B1)/GABA(B2) heterodimer to form a functional GABA(B) receptor. In this study we have developed polyclonal antisera specific to two splice variants of the GABA(B1) subunit, GABA(B1a) and GABA(B1b), as well as an antiserum to the GABA(B2) subunit. Using affinity-purified antibodies derived from these antisera we have mapped out the distribution profile of each subunit in rat brain, spinal cord and dorsal root ganglion. In brain the highest areas of GABA(B1a), GABA(B1b) and GABA(B2) subunit expression were found in neocortex, hippocampus, thalamus, cerebellum and habenula. In spinal cord, GABA(B1) and GABA(B2) subunits were expressed in the superficial layers of the dorsal horn, as well as in motor neurones in the deeper layers of the ventral horn. GABA(B) receptor subunit immunoreactivity in dorsal root ganglion suggested that expression of GABA(B1b) was restricted to the large diameter neurones, in contrast to GABA(B1a) and GABA(B2) subunits which were expressed in both large and small diameter neurones. Although expression levels of GABA(B1) and GABA(B2) subunits varied we found no areas in which GABA(B1) was expressed in the absence of GABA(B2). This suggests that most, if not all, GABA(B1) immunoreactivity may represent functional GABA(B) receptors. Although our data are in general agreement with functional studies, some discrepancies in GABA(B1) subunit expression occurred with respect to other immunohistochemical studies. Overall our data suggest that GABA(B) receptors are widely expressed throughout the brain and spinal cord, and that GABA(B1a) and GABA(B1b) subunits can associate with GABA(B2) to form both pre- and post-synaptic receptors.

  17. Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs cycle in rat pancreatic islets.

    PubMed

    Hernández-Fisac, Inés; Fernández-Pascual, Sergio; Ortsäter, Henrik; Pizarro-Delgado, Javier; Martín del Río, Rafael; Bergsten, Peter; Tamarit-Rodriguez, Jorge

    2006-11-15

    OMP (oxo-4-methylpentanoic acid) stimulates by itself a biphasic secretion of insulin whereas L-leucine requires the presence of L-glutamine. L-Glutamine is predominantly converted into GABA (gamma-aminobutyric acid) in rat islets and L-leucine seems to promote its metabolism in the 'GABA shunt' [Fernández-Pascual, Mukala-Nsengu-Tshibangu, Martín del Río and Tamarit-Rodríguez (2004) Biochem. J. 379, 721-729]. In the present study, we have investigated how 10 mM OMP affects L-glutamine metabolism to uncover possible differences with L-leucine that might help to elucidate whether they share a common mechanism of stimulation of insulin secretion. In contrast with L-leucine, OMP alone stimulated a biphasic insulin secretion in rat perifused islets and decreased the islet content of GABA without modifying its extracellular release irrespective of the concentration of L-glutamine in the medium. GABA was transaminated to L-leucine whose intracellular concentration did not change because it was efficiently transported out of the islet cells. The L-[U-14C]-Glutamine (at 0.5 and 10.0 mM) conversion to 14CO2 was enhanced by 10 mM OMP within 30% and 70% respectively. Gabaculine (250 microM), a GABA transaminase inhibitor, suppressed OMP-induced oxygen consumption but not L-leucine- or glucose-stimulated respiration. It also suppressed the OMP-induced decrease in islet GABA content and the OMP-induced increase in insulin release. These results support the view that OMP promotes islet metabolism in the 'GABA shunt' generating 2-oxo-glutarate, in the branched-chain alpha-amino acid transaminase reaction, which would in turn trigger GABA deamination by GABA transaminase. OMP, but not L-leucine, suppressed islet semialdehyde succinic acid reductase activity and this might shift the metabolic flux of the 'GABA shunt' from gamma-hydroxybutyrate to succinic acid production.

  18. Potentiation by WIN 55,212-2 of GABA-activated currents in rat trigeminal ganglion neurones.

    PubMed

    Li, Zhi-Wang; Zhang, Jian; Ouyang, Chang-han; Li, Chao-Ying; Zhao, Feng-Bo; Liu, Yu-Wei; Ai, Yong-Xun; Hu, Wang-Ping

    2009-12-01

    Although both natural and synthetic cannabinoid compounds have been shown to exert an antinociceptive effect on acute and persistent pain, the anatomical locus of the target of cannabinoid-induced analgesia has not been fully elucidated. Here, we investigated the effects of the cannabinoid agonist WIN 55,212-2 on GABA-activated currents (I(GABA)) in rat primary sensory neurones. In the present study, experiments were performed on neurones freshly isolated from rat trigeminal ganglion (TG) by using whole-cell patch clamp and repatch techniques. GABA-evoked inward currents were potentiated by pretreatment with WIN 55,212-2 in a concentration-dependent manner (10(-10)-10(-8) M). WIN 55,212-2 shifted the GABA concentration-response curve upwards, with an increase of 30.3 +/- 3.7% in the maximal current response but with no significant change in the EC(50) (agonist concentration producing a half-maximal response) value. WIN 55,212-2 potentiated the responses to GABA in a manner independent of holding potential and in the absence of any change in the reversal potential of the current. This potentiation of I(GABA) induced by WIN 55,212-2 was almost completely blocked by AM 251 (3 x 10(-8) M), a CB(1) receptor antagonist, and, using the repatch technique, was found to be abolished after intracellular dialysis with the protein kinase A (PKA) activator cAMP or the PKA inhibitor H89. The potentiation by WIN 55,212-2 of I(GABA) in primary sensory neurones may help to elucidate the mechanism underlying the modulation of analgesia by cannabinoids in the spinal dorsal horn.

  19. Activation of VTA GABA neurons disrupts reward consumption

    PubMed Central

    van Zessen, Ruud; Phillips, Jana L.; Budygin, Evgeny A.; Stuber, Garret D.

    2012-01-01

    The activity of Ventral Tegmental Area (VTA) dopamine (DA) neurons promotes behavioral responses to rewards and environmental stimuli that predict them. VTA GABA inputs synapse directly onto DA neurons and may regulate DA neuronal activity to alter reward-related behaviors, however, the functional consequences of selective activation of VTA GABA neurons remains unknown. Here, we show that in vivo optogenetic activation of VTA GABA neurons disrupts reward consummatory behavior, but not conditioned anticipatory behavior in response to reward-predictive cues. In addition, direct activation of VTA GABA projections to the nucleus accumbens (NAc) resulted in detectable GABA release, but did not alter reward consumption. Furthermore, optogenetic stimulation of VTA GABA neurons directly suppressed the activity and excitability of neighboring DA neurons, as well as the release of DA in the NAc, suggesting that the dynamic interplay between VTA DA and GABA neurons can control the initiation and termination of reward-related behaviors. PMID:22445345

  20. Activation of GABA-A Receptor Ameliorates Homocysteine-Induced MMP-9 Activation by ERK Pathway

    PubMed Central

    TYAGI, NEETU; GILLESPIE, WILLIAM; VACEK, JONATHAN C.; SEN, UTPAL; TYAGI, SURESH C.; LOMINADZE, DAVID

    2010-01-01

    Hyperhomocysteinemia (HHcy) is a risk factor for neuroinflammatory and neurodegenerative diseases. Homocysteine (Hcy) induces redox stress, in part, by activating matrix metalloproteinase-9 (MMP-9), which degrades the matrix and leads to blood–brain barrier dysfunction. Hcy competitively binds to γ-aminbutyric acid (GABA) receptors, which are excitatory neurotransmitter receptors. However, the role of GABA-A receptor in Hcy-induced cerebrovascular remodeling is not clear. We hypothesized that Hcy causes cerebrovascular remodeling by increasing redox stress and MMP-9 activity via the extracellular signal-regulated kinase (ERK) signaling pathway and by inhibition of GABA-A receptors, thus behaving as an inhibitory neurotransmitter. Hcy-induced reactive oxygen species production was detected using the fluorescent probe, 2′–7′-dichlorodihydrofluorescein diacetate. Hcy increased nicotinamide adenine dinucleotide phosphate-oxidase-4 concomitantly suppressing thioredoxin. Hcy caused activation of MMP-9, measured by gelatin zymography. The GABA-A receptor agonist, muscimol ameliorated the Hcy-mediated MMP-9 activation. In parallel, Hcy caused phosphorylation of ERK and selectively decreased levels of tissue inhibitors of metalloproteinase-4 (TIMP-4). Treatment of the endothelial cell with muscimol restored the levels of TIMP-4 to the levels in control group. Hcy induced expression of iNOS and decreased eNOS expression, which lead to a decreased NO bioavailability. Furthermore muscimol attenuated Hcy-induced MMP-9 via ERK signaling pathway. These results suggest that Hcy competes with GABA-A receptors, inducing the oxidative stress transduction pathway and leading to ERK activation. PMID:19308943

  1. Excitatory GABA in rodent developing neocortex in vitro.

    PubMed

    Rheims, Sylvain; Minlebaev, Marat; Ivanov, Anton; Represa, Alfonso; Khazipov, Rustem; Holmes, Gregory L; Ben-Ari, Yehezkel; Zilberter, Yuri

    2008-08-01

    GABA depolarizes immature cortical neurons. However, whether GABA excites immature neocortical neurons and drives network oscillations as in other brain structures remains controversial. Excitatory actions of GABA depend on three fundamental parameters: the resting membrane potential (Em), reversal potential of GABA (E(GABA)), and threshold of action potential generation (Vthr). We have shown recently that conventional invasive recording techniques provide an erroneous estimation of these parameters in immature neurons. In this study, we used noninvasive single N-methyl-d-aspartate and GABA channel recordings in rodent brain slices to measure both Em and E(GABA) in the same neuron. We show that GABA strongly depolarizes pyramidal neurons and interneurons in both deep and superficial layers of the immature neocortex (P2-P10). However, GABA generates action potentials in layer 5/6 (L5/6) but not L2/3 pyramidal cells, since L5/6 pyramidal cells have more depolarized resting potentials and more hyperpolarized Vthr. The excitatory GABA transiently drives oscillations generated by L5/6 pyramidal cells and interneurons during development (P5-P12). The NKCC1 co-transporter antagonist bumetanide strongly reduces [Cl(-)]i, GABA-induced depolarization, and network oscillations, confirming the importance of GABA signaling. Thus a strong GABA excitatory drive coupled with high intrinsic excitability of L5/6 pyramidal neurons and interneurons provide a powerful mechanism of synapse-driven oscillatory activity in the rodent neocortex in vitro. In the companion paper, we show that the excitatory GABA drives layer-specific seizures in the immature neocortex.

  2. MET-88, a gamma-butyrobetaine hydroxylase inhibitor, improves cardiac SR Ca2+ uptake activity in rats with congestive heart failure following myocardial infarction.

    PubMed

    Hayashi, Y; Ishida, H; Hoshiai, M; Hoshiai, K; Kirimoto, T; Kanno, T; Nakano, M; Tajima, K; Miyake, H; Matsuura, N; Nakazawa, H

    2000-06-01

    We previously reported that MET-88, 3-(2,2,2-trimethylhydrazinium) propionate, improved left ventricular diastolic dysfunction induced by congestive heart failure (CHF) in rats. The present study was designed to investigate the mechanism by which MET-88 improved the cardiac relaxation impaired in CHF rats. The left coronary artery of the animals was ligated, and the rats were then orally administered vehicle (control), MET-88 at 50 or 100 mg/kg or captopril at 20 mg/kg for 20 days. Myocytes were isolated from the non-infarcted region in the left ventricle, and cell shortening and [Ca2+]i transients were measured with a video-edge detector and by fluorescence analysis, respectively. In CHF control rats, the diastolic phase of cell shortening was prolonged compared with that of the sham-operated (sham) rats. This prolongation was prevented by treatment with MET-88 at 100 mg/kg or captopril at 20 mg/kg. CHF control rats also showed an increase in the decay time of [Ca2+]i transients compared with sham rats. MET-88 at 100 mg/kg and captopril at 20 mg/kg attenuated the increase in decay time of [Ca2+]i transients. Ca2+ uptake activity of the sarcoplasmic reticulum (SR) isolated from the non-infarcted region in the left ventricle was measured, and Lineweaver-Burk plot analysis of the activity was performed. CHF control rats revealed a decrease in the Vmax for SR Ca2+ uptake activity without alteration in Kd. MET-88 at 100 mg/kg significantly prevented the decrease in Vmax, but had no effect on Kd. Also, treatment with MET-88 at 100 mg/kg improved myocardial high-energy phosphate levels impaired in CHF rats. These results suggest that one of the mechanisms by which MET-88 improved cardiac relaxation in CHF rats is based on the amelioration of [Ca2+]i transients through increase of SR Ca2+ uptake activity.

  3. Effects of ABA and CaCl₂ on GABA accumulation in fava bean germinating under hypoxia-NaCl stress.

    PubMed

    Yang, Runqiang; Hui, Qianru; Gu, Zhenxin

    2016-01-01

    Effects of exogenous abscisic acid (ABA) and CaCl2 on γ-aminobutyric acid (GABA) accumulation of germinated fava bean under hypoxia-NaCl stress were investigated. Exogenous ABA resulted in the enhancement of glutamate decarboxylase (GAD) and diamine oxidase (DAO) activity as well as GABA content in cotyledon and shoot. CaCl2 increased both enzyme activities in shoot and GABA content in cotyledon and shoot. ABA downregulated GAD expression in cotyledon and radicle, while upregulated that in shoot; it also upregulated DAO expression in each organ. CaCl2 upregulated GAD expression in cotyledon, while downregulated that in radicle. However, it upregulated DAO expression in shoot, downregulated that in radicle. ABA inhibitor fluridon and ethylenediaminetetraacetic acid inhibited GAD and DAO activities significantly so that inhibited GABA accumulation through reducing ABA biosynthesis and chelating Ca(2+), respectively. However, they upregulated GAD and DAO expression in varying degrees. These results indicate that ABA and Ca(2+) participate in GABA biosynthesis in fava bean during germination under hypoxia-NaCl stress.

  4. [Effects of agonists and antagonists of benzodiazepine, GABA and NMDA receptors, on caffeine-induced seizures in mice].

    PubMed

    Inano, S

    1992-08-01

    In mice, tonic convulsive seizure induced by intravenous administration of caffeine (adenosine A1, A2 receptors antagonist) was significantly potentiated by any one of L-PIA (adenosine A1 receptor agonist), NECA (adenosine A2 receptor agonist) and 2-ClAd (adenosine A1, A2 receptors agonist). The caffeine-induced seizure was unaffected by diazepam (benzodiazepine receptor agonist), but was inhibited by Ro 15-1788 (antagonist or partial agonist). beta-DMCM (antagonist or inverse agonist) increased the seizure. Muscimol (GABA-a receptor agonist), baclofen (GABA-b receptor agonist) and AOAA (GABA transaminase inhibitor) did not show significant effect on caffeine-induced convulsion. Bicuculline (GABA-a receptor antagonist) and picrotoxin (chloride channel blocker) significantly potentiated the convulsion at the doses which did not induce it. Caffeine-induced convulsion was potentiated by NMDA with its non-convulsive dose. CPP (competitive NMDA receptor antagonist) and MK-801 (non-competitive NMDA receptor antagonist) significantly inhibited the seizures. These results suggest that caffeine-induced seizure is not caused by blockade of adenosine receptors. Caffeine may act to beta-carboline sensitive benzodiazepine receptor (Type 1) which has no linkage with GABA-a receptor. Furthermore, it is implied that caffeine plays some role at NMDA receptor calcium ion channel complex.

  5. Impact of exogenous GABA treatments on endogenous GABA metabolism in anthurium cut flowers in response to postharvest chilling temperature.

    PubMed

    Aghdam, Morteza Soleimani; Naderi, Roohangiz; Jannatizadeh, Abbasali; Babalar, Mesbah; Sarcheshmeh, Mohammad Ali Askari; Faradonbe, Mojtaba Zamani

    2016-09-01

    Anthurium flowers are susceptible to chilling injury, and the optimum storage temperature is 12.5-20 °C. The γ-aminobutyric acid (GABA) shunt pathway may alleviate chilling stress in horticultural commodities by providing energy (ATP), reducing molecules (NADH), and minimizing accumulation of reactive oxygen species (ROS). In this experiment, the impact of a preharvest spray treatment with 1 mM GABA and postharvest treatment of 5 mM GABA stem-end dipping on GABA shunt pathway activity of anthurium cut flowers (cv. Sirion) in response to cold storage (4 °C for 21 days) was investigated. GABA treatments resulted in lower glutamate decarboxylase (GAD) and higher GABA transaminase (GABA-T) activities in flowers during cold storage, which was associated with lower GABA content and coincided with higher ATP content. GABA treatments also enhanced accumulation of endogenous glycine betaine (GB) in flowers during cold storage, as well as higher spathe relative water content (RWC). These findings suggest that GABA treatments may alleviate chilling injury of anthurium cut flowers by enhancing GABA shunt pathway activity leading to provide sufficient ATP and promoting endogenous GB accumulation. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  6. Medicinal and aromatic plant materials as nitrification inhibitors for augmenting yield and nitrogen uptake of Japanese mint (Mentha arvensis L. Var. Piperascens).

    PubMed

    Kiran, Usha; Patra, D D

    2003-02-01

    Pot experiments were conducted to evaluate the relative performance of medicinal and aromatic plant materials and dicyandiamide (DCD) as nitrification inhibitors to regulate transformation of N from urea. Their effect on the efficiencies of use of N by Japanese mint (Mentha arvensis cv. Hy 77) was tested. Urea was coated with these materials viz., Mentha spicata, Artemisia annua or DCD at the rate of 5% (w/w) of fertilizer urea using an appropriate coating technique. Nimin (tetranortriterpenoids, an ethanol extract of neem (Azadirachta indica Juss) coating was done at the rate of 1% w/w of urea. Fertilizer nitrogen was applied at 100 and 200 mg kg(-1) soil. These natural coating materials significantly increased the herb and essential oil yields of the crop at both rates of fertilizer nitrogen compared to urea alone and were found to be as effective as DCD in retarding NO3- formation in soil. Herb yield increased by 6-81% when compared to uncoated urea. The increase in essential oil yield ranged between 3% and 68% due to coating. The effectiveness of the nitrification-inhibitor--coated urea, however, varied with the soils used and the rate of fertilizer nitrogen applied. The results suggest that the natural products could be potential nitrification inhibitors for increasing fertilizer N use efficiency.

  7. Effects of inhibitors of key enzymes of sphingolipid metabolism on insulin-induced glucose uptake and glycogen synthesis in liver cells of old rats.

    PubMed

    Babenko, N A; Kharchenko, V S

    2015-01-01

    Sphingolipids play an important role in the development of insulin resistance. Ceramides are the most potent inhibitors of insulin signal transduction. Ceramides are generated in response to stress stimuli and in old age. In this work, we studied the possible contribution of different pathways of sphingolipid metabolism in age-dependent insulin resistance development in liver cells. Inhibition of key enzymes of sphingolipid synthesis (serine palmitoyl transferase, ceramide synthase) and degradation (neutral and acidic SMases) by means of specific inhibitors (myriocin, fumonisin B1, imipramine, and GW4869) was followed with the reduction of ceramide level and partly improved insulin regulation of glucose metabolism in "old" hepatocytes. Imipramine and GW4869 decreased significantly the acidic and neutral SMase activities, respectively. Treatment of "old" cells with myriocin or fumonisin B1 reduced the elevated in old age ceramide and SM synthesis. Ceramide and SM levels and glucose metabolism regulation by insulin could be improved with concerted action of all tested inhibitors of sphingolipid turnover on hepatocytes. The data demonstrate that not only newly synthesized ceramide and SM but also neutral and acidic SMase-dependent ceramide accumulation plays an important role in development of age-dependent insulin resistance.

  8. The influence of proteasome inhibitor MG132, external radiation, and unlabeled antibody on the tumor uptake and biodistribution of (188)re-labeled anti-E6 C1P5 antibody in cervical cancer in mice.

    PubMed

    Phaeton, Rébécca; Wang, Xing Guo; Einstein, Mark H; Goldberg, Gary L; Casadevall, Arturo; Dadachova, Ekaterina

    2010-02-15

    Human papillomavirus (HPV) infection is considered a necessary step for the development of cervical cancer, and >95% of all cervical cancers have detectable HPV sequences. The authors of this report recently demonstrated the efficacy of radioimmunotherapy (RIT) targeting viral oncoprotein E6 in the treatment of experimental cervical cancer. They hypothesized that the pretreatment of tumor cells with various agents that cause cell death and/or elevation of E6 levels would increase the accumulation of radiolabeled antibodies to E6 in cervical tumors. HPV type 16 (HPV-16)-positive CasKi cells were treated in vitro with up to 6 grays of external radiation, or with the proteasome inhibitor MG-132, or with unlabeled anti-E6 antibody C1P5; and cell death was assessed. The biodistribution of (188)Re-labeled C1P5 antibody was determined in both control and radiation MG-132-treated CasKi tumor-bearing nude mice. (188)Re-C1P5 antibody demonstrated tumor specificity, very low uptake, and fast clearance from the major organs. The amount of tumor uptake was enhanced by MG-132 but was unaffected by pretreatment with radiation. In addition, in vitro studies demonstrated an unanticipated effect of unlabeled antibody on the amount of cell death, a finding that was suggested by the authors' previous in vivo studies in a CasKi tumor model. The current results indicated that pretreatment of cervical tumors with the proteasome inhibitor MG-132 and with unlabeled antibody to E6 can serve as a means to generate nonviable cancer cells and to elevate the levels of target oncoproteins in the cells for increasing the accumulation of targeted radiolabeled antibodies in tumors. These results favor the further development of RIT for cervical cancers targeting viral antigens. (c) 2010 American Cancer Society.

  9. The influence of proteasome inhibitor MG132, external radiation and unlabeled antibody on the tumor uptake and biodistribution of 188Re-labeled anti-E6 C1P5 antibody in cervical cancer in mice

    PubMed Central

    Phaeton, Rébécca; Wang, Xing Guo; Einstein, Mark H.; Goldberg, Gary L.; Casadevall, Arturo; Dadachova, Ekaterina

    2009-01-01

    Background Human Papillomavirus (HPV) infection is considered a necessary step for the development of cervical cancer and >95% of all cervical cancers have detectable HPV sequences. We have recently demonstrated the efficacy of radioimmunotherapy (RIT) which targeted viral oncoprotein E6 in treatment of experimental cervical cancer We hypothesized that pre-treatment of tumor cells with various agents which cause cell death and/or elevation of E6 levels would increase the accumulation of radiolabeled antibodies to E6 in cervical tumors. Methods HPV-16 positive CasKi cells were treated in vitro with up to 6 Gy of external radiation, or proteasome inhibitor MG-132 or unlabeled anti-E6 antibody C1P5 and cell death was assessed. Biodistribution of 188Rhenium (188Re)-labeled C1P5 antibody was performed in both control and radiation MG-132 treated CasKi tumor-bearing nude mice. Results . 188Re-C1P5 antibody demonstrated tumor specificity and very low uptake and fast clearance from the major organs. The amount of tumor uptake was enhanced by MG-132 but was unaffected by pre-treatment with radiation. In addition, in vitro studies demonstrated an unanticipated effect of unlabeled antibody on the amount of cell death, a finding that was suggested by our previous in vivo studies in CasKi tumor model. Conclusion We demonstrated that pre-treatment of cervical tumors with proteasome inhibitor MG-132 and with unlabeled antibody to E6 can serve as a means to generate non-viable cancer cells and to elevate the levels of target oncoproteins in the cells for increasing the accumulation of targeted radiolabeled antibodies in tumors. These results favor further development of RIT of cervical cancers targeting viral antigens. PMID:20127955

  10. (±)-(1S,2R,5S)-5-Amino-2-fluorocyclohex-3-ene Carboxylic Acid. A Potent GABA Aminotransferase Inactivator that Irreversibly Inhibits through an Elimination-Aromatization Pathway†

    PubMed Central

    Wang, Zhiyong; Yuan, Hai; Nikolic, Dejan; Van Breemen, Richard B.; Silverman, Richard B.

    2008-01-01

    Inhibition of γ-aminobutyric acid aminotransferase (GABA-AT) raises the concentration of GABA, an inhibitory neurotransmitter in human brain, which could have therapeutic applications for a variety of neurological diseases including epilepsy. Based on studies of several previously synthesized conformationally-restricted GABA-AT inhibitors, (±)- (1S,2R,5S)-5-amino-2-fluorocyclohex-3-ene carboxylic acid (12) was designed as a mechanismbased inactivator. This compound was shown to irreversibly inhibit GABA-AT; substrate protects the enzyme from inactivation. Mechanistic experiments demonstrated the loss of one fluoride ion per active site during inactivation and the formation of N-m-carboxyphenylpyridoxamine 5′-phosphate (26), the same product generated by inactivation of GABA-AT by gabaculine (8). An elimination-aromatization mechanism is proposed to account for these results. PMID:17128990

  11. A neuronal lactate uptake inhibitor slows recovery of extracellular ion concentration changes in the hippocampal CA3 region by affecting energy metabolism.

    PubMed

    Angamo, Eskedar Ayele; Rösner, Joerg; Liotta, Agustin; Kovács, Richard; Heinemann, Uwe

    2016-11-01

    Astrocyte-derived lactate supports pathologically enhanced neuronal metabolism, but its role under physiological conditions is still a matter of debate. Here, we determined the contribution of astrocytic neuronal lactate shuttle for maintenance of ion homeostasis and energy metabolism. We tested for the effects of α-cyano-4-hydroxycinnamic acid (4-CIN), which could interfere with energy metabolism by blocking monocarboxylate-transporter 2 (MCT2)-mediated neuronal lactate uptake, on evoked potentials, stimulus-induced changes in K(+), Na(+), Ca(2+), and oxygen concentrations as well as on changes in flavin adenine dinucleotide (FAD) autofluorescence in the hippocampal area CA3. MCT2 blockade by 4-CIN reduced synaptically evoked but not antidromic population spikes. This effect was dependent on the activation of KATP channels indicating reduced neuronal ATP synthesis. By contrast, lactate receptor activation by 3,5-dihydroxybenzoic acid (3,5-DHBA) resulted in increased antidromic and orthodromic population spikes suggesting that 4-CIN effects are not mediated by lactate accumulation and subsequent activation of lactate receptors. Recovery kinetics of all ion transients were prolonged and baseline K(+) concentration became elevated by blockade of lactate uptake. Lactate contributed to oxidative metabolism as both baseline respiration and stimulus-induced changes in Po2 were decreased, while FAD fluorescence increased likely due to a reduced conversion of FAD into FADH2 These data suggest that lactate shuttle contributes to regulation of ion homeostatsis and synaptic signaling even in the presence of ample glucose. Copyright © 2016 the American Physiological Society.

  12. [ERK activation effects on GABA secretion inhibition induced by SDF-1 in hippocampal neurons of rats].

    PubMed

    Zhang, Zi-juan; Guo, Mei-xia; Xing, Ying

    2015-09-01

    To investigate the effect of extracellular regulating kinase (ERK) signaling pathway on the secretion of gamma-aminobutyric acid (GABA) in cultured rat hippocampal neurons induced by stromal cell derived factor-1 (SDF-1). The hippocampal neurons of newborn SD rats were cultured and identified in vitro; the phosphorylation level of ERK1/2 was examined by Western blot; ELISA was used to detect the effect of PD98059, a ERK1/2 specific blocker on GABA secretion of cultured hippocampal neurons and Western blot were adopted to measure the protein expression levels of glutamate decarboxylase (GAD65/67) and gamma aminobutyric acid transporter (GAT); after blocking ERK1/2 signaling pathway with PD98059; RT-PCR was used to detect the mRNA expression levels of GAT-1 and GAD65 after treated with PD98059. The levels of ERKl/2 phosphorylation were increased significantly by SDF1 acting on hippocampal neurons, and CX-CR4 receptor blocker AMD3100, could inhibit SDF-1 induced ERK1/2 activation; SDF-1 could inhibit the secretion of GABA in cultured hippocampal neurons, and ERK1/2 specific inhibitor PD98059, could partly reverse the inhibition of GABA secretion by SDF-1. The effects of SDF-1 on cultured hippocampal neurons was to decrease the mRNA genesis of glutamic acid decarboxylase GAD65 and GABA transporter GAT-1, besides, ERK inhibitor PD98059 could effectively flip the effect of SDF-1. The results of Western blot showed that SDF-1 could inhibit the protein expression of GAT-1 and GAD65/67 in hippocampal neurons and the inhibition of GAT-1 and GAD65/67 protein expression could be partially restored by ERK1/2 blocker. SDF-1 acts on the CXCR4 of hippocampal neurons in vitro, and inhibits the expression of GAD by activating the ERK1/2 signaling pathway, and this may represent one possible pathway of GABA secretion inhibition.

  13. Excitatory actions of GABA in developing rat hypothalamic neurones.

    PubMed Central

    Chen, G; Trombley, P Q; van den Pol, A N

    1996-01-01

    1. Gramicidin-perforated patch clamp recording was employed to study GABA-mediated responses in rat hypothalamic neurones (n = 102) with an intracellular Cl- concentration unaltered by the pipette solution. 2. In young cultures after 1-7 days in vitro (DIV), GABA induced depolarizing membrane potentials (+16.5 +/- 1.3 mV) that often surpassed the threshold for the firing of action potentials (-42 +/- 1 mV) and resulted in an increase in neuronal activity. The depolarizing responses to GABA in young cultures were dose dependent. The concentration of GABA necessary to evoke the half-maximal depolarization (EC50) was 2.8 microM. In contrast, GABA induced hyperpolarizing membrane potentials (-12.0 +/- 1.4 mV) and a decrease in neuronal activity in older neurones (20-33 DIV). Both the depolarization and the hyperpolarization induced by GABA were blocked by bicuculline, indicating a mediation by GABAA receptors. 3. The reversal potentials of the GABA-evoked currents were between -40 to -50 mV during the first week of culture, and shifted to below -70 mV after 3 weeks of culture. In parallel, neurones that were dissociated from older animals (postnatal day 5) had a more negative reversal potential for the GABA-evoked currents than cells from younger animals (embryonic day 15), suggesting that the negative shift of the reversal potential occurs both in vitro and in vivo. Our data suggest that the mechanism for GABA-induced depolarization is the depolarized Cl- reversal potential found in young but not older neurones. 4. Consistent with the depolarizing response to exogenous application of GABA, some spontaneous depolarizing postsynaptic potentials in young cultures were insensitive to AP5-CNQX, but were eliminated by bicuculline, indicating that synaptically released GABA mediated excitatory synaptic transmission in early development. 5. By combining a rapid computer-controlled delivery of GABA with subthreshold positive current injections into recorded neurones, we found

  14. Evidence for glutamate, GABA and NO in coordinating behaviour in the sponge, Ephydatia muelleri (Demospongiae, Spongillidae).

    PubMed

    Elliott, Glen R D; Leys, Sally P

    2010-07-01

    The view that sponges lack tissue level organisation, epithelia, sensory cells and coordinated behaviour is challenged by recent molecular studies showing the existence in Porifera of molecules and proteins that define cell signalling systems in higher order metazoans. Demonstration that freshwater sponges can contract their canals in an organised manner in response to both external and endogenous stimuli prompted us to examine the physiology of the contraction behaviour. Using a combination of digital time-lapse microscopy, high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis, immunocytochemistry and pharmacological manipulations, we tested the role of the diffusible amino acids glutamate and gamma-aminobutyric acid (GABA) and a short-lived diffusible gas, nitric oxide (NO), in triggering or modulating contractions in Ephydatia muelleri. We identified pools of glutamate, glutamine and GABA used to maintain a metabotropic glutamate and GABA receptor signalling system. Glutamate induced contractions and propagation of a stereotypical behaviour inflating and deflating the canal system, acting in a dose-dependent manner. Glutamate-triggered contractions were blocked by the metabatropic glutamate receptor inhibitor AP3 and by incubation of the sponge in an allosteric competitive inhibitor of glutamate, Kynurenic acid. Incubation in GABA inhibited glutamate-triggered contractions of the sponge. Nitric oxide synthase, involved in the formation of the diffusible gas NO, was localised using NADPH-diaphorase to mesenchyme cells in the osculum and pinacoderm. A cGMP assay showed the same cells were labelled suggesting that the NO system is functional. Our findings suggest sponges coordinate behaviour using chemical messenger systems common to other animals.

  15. Interaction of GABA-mimetics with the taurine transporter (TauT, Slc6a6) in hyperosmotic treated Caco-2, LLC-PK1 and rat renal SKPT cells.

    PubMed

    Rasmussen, Rune Nørgaard; Lagunas, Candela; Plum, Jakob; Holm, René; Nielsen, Carsten Uhd

    2016-01-20

    The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells, porcine LLC-PK1 cells, and rat SKPT cells using radiolabelled taurine. Hyperosmotic conditions were obtained by incubation with raffinose (final osmolality of 500mOsm) for 24h prior to the uptake experiments. Expression of the taurine transporter, TauT, was investigated at the mRNA level by real-time PCR. Uptake of the GABA-mimetics gaboxadol and vigabatrin was investigated in SKPT cells, and quantified by liquid scintillation or HPLC-MS/MS analysis, respectively. The uptake rate of [(3)H]-taurine was Na(+) and Cl(-) and concentration dependent with taurine with an apparent Vmax of 6.3±1.6pmolcm(-2)min(-1) and a Km of 24.9±15.0μM. β-alanine, nipecotic acid, gaboxadol, GABA, vigabatrin, δ-ALA and guvacine inhibited the taurine uptake rate in a concentration dependent manner. The order of affinity for TauT was β-alanine>GABA>nipecotic acid>guvacine>δ-ALA>vigabatrin>gaboxadol with IC50-values of 0.04, 1.07, 2.02, 4.19, 4.94, 31.4 and 39.9mM, respectively. In conclusion, GABA mimetics inhibited taurine uptake in hyperosmotic rat renal SKPT cells. SKPT cells, which seem to be a useful model for investigating taurine transport in the short-term presence of high concentrations of osmolytes. Furthermore, analogues of β-alanine appear to have higher affinities for TauT than GABA-analogues.

  16. Design and Mechanism of Tetrahydrothiophene-based GABA Aminotransferase Inactivators

    PubMed Central

    Le, Hoang V.; Hawker, Dustin D.; Wu, Rui; Doud, Emma; Widom, Julia; Sanishvili, Ruslan; Liu, Dali; Kelleher, Neil L.; Silverman, Richard B.

    2015-01-01

    Low levels of γ-aminobutyric acid (GABA), one of two major neurotransmitters that regulate brain neuronal activity, are associated with many neurological disorders, such as epilepsy, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and cocaine addiction. One of the main methods to raise the GABA level in human brain is to use small molecules that cross the blood-brain barrier and inhibit the activity of γ-aminobutyric acid aminotransferase (GABA-AT), the enzyme that degrades GABA. We have designed a series of conformationally-restricted, tetrahydrothiophene-based GABA analogs with a properly-positioned leaving group that could facilitate a ring-opening mechanism, leading to inactivation of GABA-AT. One compound in the series is eight times more efficient an inactivator of GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. Our mechanistic studies show that the compound inactivates GABA-AT by a new mechanism. The metabolite resulting from inactivation does not covalently bind to amino acid residues of GABA-AT but stays in the active site via H-bond interactions with Arg-192, a π-π interaction with Phe-189, and a weak nonbonded S···O=C interaction with Glu-270, thereby inactivating the enzyme. PMID:25781189

  17. Pharmacologically novel GABA receptor in human dorsal root ganglion neurons.

    PubMed

    Valeyev, A Y; Hackman, J C; Wood, P M; Davidoff, R A

    1996-11-01

    1. Whole cell voltage-clamp studies of gamma-aminobutyric acid (GABA) receptors were performed on large (> 80 microns) cultured human dorsal root ganglion (DRG) neurons. 2. GABA and pentobarbital sodium when applied in micromolar concentrations evoked inward Cl- currents in DRG neurons voltage clamped at negative membrane potentials. 3. Diazepam (10 microM) and pentobarbital (10 microM) upmodulated the GABA current by approximately 149 and 168%, respectively. 4. The GABA currents in human DRG cells were unaffected by the classical GABA antagonists picrotoxin and bicuclline (100 microM). In contrast, the GABA responses evoked in adult rat DRG cells cultured in an identical manner were inhibited by both antagonists. The glycine receptor antagonist strychnine (100 microM) did not alter GABA currents in human DRG cells. 5. Human DRG cells did not respond to glycine (10-100 microM) or taurine (10-100 microM). The GABAB agonist baclofen had no effect on the holding current when patch pipettes were filled with 130 mM KCl. The GABAB antagonists saclofen applied either alone or with GABA was without effect. 6. The differences between the GABA receptors described here and GABA receptors in other species may reflect the presence of receptor subunits unique to human DRG cells.

  18. Design and Mechanism of Tetrahydrothiophene-based GABA Aminotransferase Inactivators

    SciTech Connect

    Le, Hoang V.; Hawker, Dustin D.; Wu, Rui; Doud, Emma; Widom, Julia; Sanishvili, Ruslan; Liu, Dali; Kelleher, Neil L.; Silverman, Richard B

    2015-04-08

    Low levels of gamma-aminobutyric acid (GABA), one of two major neurotransmitters that regulate brain neuronal activity, are associated with many neurological disorders, such as epilepsy, Parkinsons disease, Alzheimers disease, Huntingtons disease, and cocaine addiction. One of the main methods to raise the GABA level in human brain is to use small molecules that cross the bloodbrain barrier and inhibit the activity of gamma-aminobutyric acid aminotransferase (GABA-AT), the enzyme that degrades GABA. We have designed a series of conformationally restricted tetrahydrothiophene-based GABA analogues with a properly positioned leaving group that could facilitate a ring-opening mechanism, leading to inactivation of GABA-AT. One compound in the series is 8 times more efficient an inactivator of GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. Our mechanistic studies show that the compound inactivates GABA-AT by a new mechanism. The metabolite resulting from inactivation does not covalently bind to amino acid residues of GABA-AT but stays in the active site via H-bonding interactions with Arg-192, a pi-pi interaction with Phe-189, and a weak nonbonded (SO)-O-...=C interaction with Glu-270, thereby inactivating the enzyme.

  19. Astrocytic GABA transporter activity modulates excitatory neurotransmission

    PubMed Central

    Boddum, Kim; Jensen, Thomas P.; Magloire, Vincent; Kristiansen, Uffe; Rusakov, Dmitri A.; Pavlov, Ivan; Walker, Matthew C.

    2016-01-01

    Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na+ concentrations and a consequent increase in astrocytic Ca2+ through Na+/Ca2+ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus. PMID:27886179

  20. Development of potent dopamine-norepinephrine uptake inhibitors (DNRIs) based on a (2S,4R,5R)-2-benzhydryl-5-((4-methoxybenzyl)amino)tetrahydro-2H-pyran-4-ol molecular template.

    PubMed

    Santra, Soumava; Sharma, Horrick; Vedachalam, Seenuvasan; Antonio, Tamara; Reith, Maarten; Dutta, Aloke

    2015-02-15

    Current therapy of depression is less than ideal with remission rates of only 25-35% and response rates of 45-60%. It has been hypothesized that a dysfunctional dopaminergic system in the mesocorticolimbic pathway in depressive disorder may lead to development of anhedonia associated with loss of pleasure and interest along with loss of motivation. The current antidepressants do not address dopamine dysfunction which might explain their low efficacy. In this report, we have described an SAR study on our pyran-based triple reuptake inhibitors (TRIs) which are being investigated as the next-generation antidepressants. In the present work we demonstrate that our lead TRIs can be modified with appropriate aromatic substitutions to display a highly potent SSRI profile for compounds 2a and 4a (Ki (SERT); 0.71 and 2.68nM, respectively) or a potent DNRI profile for compounds 6b and 6h (Ki (DAT/NET); 8.94/4.76 and 13/7.37nM, respectively). Compounds 4g-4i exhibited potencies at all three monoamine transporters. The results provide insights into the structural requirements for developing selective dual- and triple-uptake inhibitors from a unique pyran molecular template for an effective management of depression and related disorders.

  1. Mutation of a single threonine in the cytoplasmic NH2 terminus disrupts trafficking of renal betaine-GABA transporter 1 during hypertonic stress.

    PubMed

    Schweikhard, Eva S; Kempson, Stephen A; Ziegler, Christine; Burckhardt, Birgitta C

    2014-07-01

    Betaine is an important osmolyte and is, compared with other organs, much more abundant in the kidneys, where it enters cells in the medulla by betaine-GABA transporter 1 (BGT1) to balance osmoregulation in the countercurrent system. In wild-type (wt-)BGT1-expressing oocytes, GABA-mediated currents were diminished by preincubation of oocytes with 100 nM PMA or 5 μM dioctanoyl-sn-glycerol, activators of PKC, whereas the application of staurosporine before the application of dioctanoyl-sn-glycerol restored the response to GABA. Four potential phosphorylation sites on BGT1 were mutated to alanine by site-directed mutagenesis. Three mutants (T235A, S428A, and S564A) evoked GABA currents comparable in magnitude to currents observed in wt-BGT1-expressing oocytes, whereas GABA currents in T40A were barely detectable. Uptake of [(3)H]GABA was also determined in human embryonic kidney-293 cells expressing enhanced green fluorescent protein (EGFP)-tagged BGT1 with the same mutations. T235A, S428A, and S564A showed upregulation of GABA uptake after hypertonic stress and downregulation by PMA similar to EGFP-wt-BGT1. In contrast, T40A did not respond to either hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in Madin-Darby canine kidney cells revealed that T40A was present in the cytoplasm after 24 h of hypertonic stress. whereas the other mutants and EGFP-wt-BGT1 were in the plasma membrane. All mutants, including T40A, comigrated with wt-BGT1 on Western blots, suggesting that they are full-length proteins. T40A, however, cannot be phosphorylated, as revealed using a specific anti-phosphoantibody, and, therefore, T40 may be important for the trafficking and insertion of BGT1 in the plasma membrane. Copyright © 2014 the American Physiological Society.

  2. Effect of sodium lithium and proton concentrations on the electrophysiological properties of the four mouse GABA transporters expressed in Xenopus oocytes.

    PubMed

    Grossman, Tamar R; Nelson, Nathan

    2003-01-01

    Mouse GABA transporters belong to the family of Na(+) and Cl(-) dependent neurotransmitter transporter. GABA transport, by these family members, was shown to be electrogenic and driven by sodium ions. It was demonstrated that, as in several other transporters, sodium binding and release by GAT1, GAT3 and BGT-1, the canine homolog of GAT2, resulted in the appearance of presteady-state currents. In this work we show that each of the four GABA transporters exhibit unique presteady-state currents when expressed in Xenopus oocytes. The properties of the presteady-state currents correspond to the transporters affinities to Na(+). At 100 mM GAT1 exhibited symmetric presteady-state currents at all imposed potentials, whereas GAT2 exhibited asymmetric presteady-state currents exclusively at negative imposed potentials, GAT3 or GAT4 exhibited presteady-state currents predominantly at positive imposed potentials. GABA uptake by GAT2 and GAT4 was much more sensitive to external pH than GAT1 and GAT3. Reducing the external Na(+) concentration rendered the GABA uptake activity by GAT1 and GAT3 to be sensitive to pH. Lowering the external pH reduced the Na(+) affinity of GAT1. Substitution of the external Na(+) to Li(+) resulted in the appearance of leak currents exclusively at negative potentials in Xenopus oocyte expressing GAT1 and GAT3. Low Na(+) concentrations inhibited the leak currents of GAT1 but Na(+) had little effect on the leak currents of GAT3. Washing of occluded Na(+) in GAT1 enhanced the leak currents. Similarly addition of GABA in the presence of 80 mM Li(+), that presumably accelerated the release of the bound Na(+), also induced the leak currents. Conversely, addition of GABA to GAT3 expressing oocytes, in the presence of 80 mM Li(+), inhibited the leak currents.

  3. Effects of adrenoceptor antagonists and neuronal uptake inhibitors on dimethylphenylpiperazinium-induced release of catecholamines from the rabbit isolated adrenal gland and guinea-pig atria

    SciTech Connect

    Collett, A.R.; Story, D.F.

    1984-11-01

    Isolated rabbit adrenal glands were perfused with Krebs-Henseleit solution at 37 degrees C and the catecholamine storage sites were labeled with (/sup 3/H)epinephrine. Release of radioactivity was evoked by 2-min periods of perfusion with dimethylphenylpiperazinium (DMPP, 100 microM). DMPP-induced efflux of radioactivity was decreased by desipramine (1 microM), cocaine (30 microM), phenoxybenzamine (1 and 10 microM), phentolamine (1, 3 and 10 microM) and propranolol (1 microM). The reduction in DMPP-induced efflux cannot be accounted for by interactions with alpha adrenoceptors, as prazosin (1 microM) and yohimbine (1 microM) were without effect. There also was no correlation between inhibition of DMPP-induced efflux and ability of the drugs to inhibit catecholamine uptake as phentolamine (1 microM) and propranolol (1 microM) did not affect the incorporation of (/sup 3/H)epinephrine by the gland. In guinea-pig atria, in which the catecholamine storage sites had been labeled with (/sup 3/H)norepinephrine, efflux of radioactivity was elicited by 1-min periods of contact with DMPP. DMPP-induced efflux of radioactivity from atria was decreased by desipramine (1 microM), cocaine (30 microM), phenoxybenzamine (1 and 10 microM), phentolamine (1 and 10 microM) and propranolol (1 microM) but not by prazosin (1 microM) or yohimbine (1 microM). The inhibition of DMPP-induced efflux in guinea-pig atria could not be correlated with alpha adrenoceptor antagonism or blockade of neuronal uptake. There were differences between the two preparations in the degree of inhibition of DMPP-induced release produced by the above drugs.

  4. Changes in antiviral susceptibility to entry inhibitors and endocytic uptake of dengue-2 virus serially passaged in Vero or C6/36 cells.

    PubMed

    Acosta, Eliana G; Piccini, Luana E; Talarico, Laura B; Castilla, Viviana; Damonte, Elsa B

    2014-05-12

    The aim of the present study was to analyze the influence of virus origin, mammalian or mosquito cell-derived, on antiviral susceptibility of DENV-2 to entry inhibitors and the association of this effect with any alteration in the mode of entry into the cell. To this end, ten serial passages of DENV-2 were performed in mosquito C6/36 cells or monkey Vero cells and the antiviral susceptibility of each virus passage to sulfated polysaccharides (SPs), like heparin and carrageenans, was evaluated by a virus plaque reduction assay. After serial passaging in Vero cells, DENV-2 became increasingly resistant to SP inhibition whereas the antiviral susceptibility was not altered in virus propagated in C6/36 cells. The change in antiviral susceptibility was associated to a differential mode of entry into the host cell. The route of endocytic entry for productive Vero cell infection was altered from a non-classical clathrin independent pathway for C6/36-grown virus to a clathrin-mediated endocytosis when the virus was serially propagated in Vero cells. Our results show the impact of the cellular system used for successive propagation of DENV on the initial interaction between the host cell and the virion in the next round of infection and the relevant consequences it might have during the in vitro evaluation of entry inhibitors.

  5. GABA and GABA receptors alterations in the primary visual cortex of concave lens-induced myopic model.

    PubMed

    Zhao, Wen; Bi, Ai-Ling; Xu, Chao-Li; Ye, Xiang; Chen, Mei-Qing; Wang, Xin-Ting; Zhang, Xiao-Yan; Guo, Jun-Guo; Jiang, Wen-Jun; Zhang, Jin; Bi, Hong-Sheng

    2017-02-02

    Until recently most researches on myopia mechanisms have mainly been focused on the eye ball and few investigations were explored on the upper visual pathway, such as the visual cortex. The roles of gamma-aminobutyric acid (GABA) in the retinal and in the upper visual pathway are inter-correlated. As the retinal glutamate decarboxylase (GAD), GABA, and the mRNA levels of GABA receptors increased during the concave lens induced myopia formation, however, whether GABA alterations also occurred in the visual cortex during the concave lens induction is still unknown. In the present study, using HPLC, Enzyme-Linked Immunosorbent Assay (ELISA) and Real-Time Quantitative-PCR (RT-PCR) methods, we observed the changing trends of GABA, glutamate decarboxylase (GAD), and GABA receptors in the visual cortex of concave lens-induced myopic guinea pigs. Similar to the changing patterns of retinal GABA, the concentrations of GAD, GABA and the mRNA levels of GABA receptors in the visual cortex also increased. These results indicate that the exploration on myopia mechanisms should possibly be investigated on the whole visual pathway and the detailed significance of cortical GABA alterations needs further investigation.

  6. GABA and Neuroactive Steroid Interactions in Glia: New Roles for Old Players?

    PubMed Central

    Magnaghi, Valerio

    2007-01-01

    In recent years it has becoming clear that glial cells of the central and peripheral nervous system play a crucial role from the earliest stages of development throughout adult life. Glial cells are important for neuronal plasticity, axonal conduction and synaptic transmission. In this respect, glial cells are able to produce, uptake and metabolize many factors that are essential for neuronal physiology, including classic neurotransmitters and neuroactive steroids. In particular, neuroactive steroids, which are mainly synthesized by glial cells, are able to modulate some neurotransmitter receptors affecting both glia and neurons. Among the signaling systems that are specialized for neuron-glial communication, we can include neurotransmitter GABA. The main focus of this review is to illustrate the cross-talk between neurons and glial cells in terms of GABA neurotransmission and actions of neuroactive steroids. To this purpose, we will review the presence of the different GABA receptors in the glial cells of the central and peripheral nervous system. Then, we will discuss their modulation by some neuroactive steroids. PMID:18615153

  7. [Effect of synaptosomal cytosolic [3H]GABA pool depletion on secretory ability of alpha-latrotoxin].

    PubMed

    Linets'ka, M V; Storchak, L H; Himmelreĭch, N H

    2002-01-01

    alpha-Latrotoxin, a presynaptic neurotoxin from the venom of Latrodectus mactans tredecimguttatus, induces massive [3H]GABA release from rat brain synaptosomes as a result of interaction with either Ca(2+)-dependent (neurexin 1 alpha or Ca(2+)-independent (latrophilin) membrane receptor. The main aim of the study was to elucidate whether the binding of alpha-latrotoxin to different types of receptors led to [3H]GABA secretion from one pool or in each case the source of neurotransmitter differs: in the presence of Ca2+ exocytosis is induced, while in the absence of Ca(2+)--outflow by mobile membrane GABA transporter from cytoplasm. We examined the effect of the depletion of cytosolic [3H]GABA pool by competitive inhibitors of the GABA transporter (nipecotic acid and 2,4-diaminobutyric acid) on the alpha-latrotoxin-stimulated neurotransmitter release. We also compared the influence of these agents on neurosecretion, evoked by depolarization with that evoked by alpha-latrotoxin. Depolarization was stimulated by 4-aminopyridine in the Ca(2+)-containing saline and high KCl in Ca(2+)-free medium. In synaptosomes treated with nipecotic acid unstimulated [3H]GABA release was significantly augmented and high KCl-evoked Ca(2+)-independent [3H]GABA release was essentially inhibited. But under the same conditions neurosecretion stimulated by alpha-latrotoxin greatly raised with respect to the control response. The similar results were obtained with the synaptosomes treated with 2,4-diaminobutyric acid. Another way to determine which of GABA pool is the target of alpha-latrotoxin action lay in analysis of the toxin effects on the preliminary depolarized synaptosomes. alpha-Latrotoxin influence was diminished by the preceding depolarization by 4-aminopyridine in Ca2+ presence. But after the high KCl stimulation effect of alpha-latrotoxin didn't change. These data suggest that alpha-latrotoxin triggers neurotransmitter release from synaptic vesicles via exocytosis. We suppose

  8. Comparative metabolic profiling revealed limitations in xylose-fermenting yeast during co-fermentation of glucose and xylose in the presence of inhibitors.

    PubMed

    Wang, Xin; Jin, Mingjie; Balan, Venkatesh; Jones, A Daniel; Li, Xia; Li, Bing-Zhi; Dale, Bruce E; Yuan, Ying-Jin

    2014-01-01

    During lignocellulosic ethanol fermentation, yeasts are exposed to various lignocellulose-derived inhibitors, which disrupt the efficiency of hexose and pentose co-fermentation. To understand the metabolic response of fermentation microbes to these inhibitors, a comparative metabolomic investigation was performed on a xylose-fermenting Saccharomyces cerevisiae 424A (LNH-ST) and its parental strain 4124 with and without three typical inhibitors (furfural, acetic acid, and phenol). Three traits were uncovered according to fermentation results. First, the growth of strain 424A (LNH-ST) was more sensitive to inhibitors than strain 4124. Through metabolomic analysis, the variance of trehalose, cadaverine, glutamate and g-aminobutyric acid (GABA) suggested that strain 424A (LNH-ST) had a lower capability to buffer redox changes caused by inhibitors. Second, lower ethanol yield in glucose and xylose co-fermentation than glucose fermentation was observed in strain 424A (LNH-ST), which was considered to be correlated with the generation of xylitol, as well as the reduced levels of lysine, glutamate, glycine and isoleucine in strain 424A (LNH-ST). Accumulation of glycerol, galactinol and mannitol was also observed in strain 424A (LNH-ST) during xylose fermentation. Third, xylose utilization of strain 424A (LNH-ST) was more significantly disturbed by inhibitors than glucose utilization. Through the analysis of fermentation and metabolomic results, it was suggested that xylose catabolism and energy supply, rather than xylose uptake, were the limiting steps in xylose utilization in the presence of inhibitors.

  9. Suppression of γ-aminobutyric acid (GABA) transaminases induces prominent GABA accumulation, dwarfism and infertility in the tomato (Solanum lycopersicum L.).

    PubMed

    Koike, Satoshi; Matsukura, Chiaki; Takayama, Mariko; Asamizu, Erika; Ezura, Hiroshi

    2013-05-01

    Tomatoes accumulate γ-aminobutyric acid (GABA) at high levels in the immature fruits. GABA is rapidly converted to succinate during fruit ripening through the activities of GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH). Although three genes encoding GABA-T and both pyruvate- and α-ketoglutarate-dependent GABA-T activities have been detected in tomato fruits, the mechanism underlying the GABA-T-mediated conversion of GABA has not been fully understood. In this work, we conducted loss-of-function analyses utilizing RNA interference (RNAi) transgenic plants with suppressed pyruvate- and glyoxylate-dependent GABA-T gene expression to clarify which GABA-T isoforms are essential for its function. The RNAi plants with suppressed SlGABA-T gene expression, particularly SlGABA-T1, showed severe dwarfism and infertility. SlGABA-T1 expression was inversely associated with GABA levels in the fruit at the red ripe stage. The GABA contents in 35S::SlGABA-T1(RNAi) lines were 1.3-2.0 times and 6.8-9.2 times higher in mature green and red ripe fruits, respectively, than the contents in wild-type fruits. In addition, SlGABA-T1 expression was strongly suppressed in the GABA-accumulating lines. These results indicate that pyruvate- and glyoxylate-dependent GABA-T is the essential isoform for GABA metabolism in tomato plants and that GABA-T1 primarily contributes to GABA reduction in the ripening fruits.

  10. Effects of RHC 80267, a diglyceride lipase inhibitor, on prolactin secretion and calcium uptake in GH/sub 3/ pituitary cells

    SciTech Connect

    Camoratto, A.M.; Grandison, L.

    1987-01-19

    The effect of the diglyceride lipase inhibitor RHC 80267 on the prolactin secretory process was examined in clonal anterior pituitary GH/sub 3/ cells. This compound reduced basal prolactin secretion as well as secretion induced by TRH and phospholipase C but not that induced by phorbol myristate acetate. Although exogenous phospholipase C increased diglyceride, no increase in the products of diglyceride lipase was detected. Moreover, low doses of RHC 80267 were observed to effectively block potassium-stimulated /sup 45/calcium influx. It is unlikely that RHC 80267 inhibits prolactin release solely by inhibiting diglyceride lipase. These data suggest blockage of plasma membrane calcium channels as an alternate mechanism for the inhibitory actions of RHC 80267 on intact GH/sub 3/ cells. These observations may have implications for RHC 80267 action in other cell types.

  11. Effects of nitrogen application rate and a nitrification inhibitor dicyandiamide on methanotroph abundance and methane uptake in a grazed pasture soil.

    PubMed

    Dai, Yu; Di, Hong J; Cameron, Keith C; He, Ji-Zheng

    2013-12-01

    Methane-oxidizing bacteria (methanotrophs) in the soil are a unique group of methylotrophic bacteria that utilize methane (CH4) as their sole source of carbon and energy which limit the flux of methane to the atmosphere from soils and consume atmospheric methane. A field experiment was conducted to determine the effect of nitrogen application rates and the nitrification inhibitor dicyandiamide (DCD) on the abundance of methanotrophs and on methane flux in a grazed pasture soil. Nitrogen (N) was applied at four different rates, with urea applied at 50 and 100 kg N ha(-1) and animal urine at 300 and 600 kg N ha(-1). DCD was applied at 10 kg ha(-1). The results showed that both the DNA and selected mRNA copy numbers of the methanotroph pmoA gene were not affected by the application of urea, urine or DCD. The methanotroph DNA and mRNA pmoA gene copy numbers were low in this soil, below 7.13 × 10(3) g(-1) soil and 3.75 × 10(3) μg(-1) RNA, respectively. Daily CH4 flux varied slightly among different treatments during the experimental period, ranging from -12.89 g CH4 ha(-1) day(-1) to -0.83 g CH4 ha(-1) day(-1), but no significant treatment effect was found. This study suggests that the application of urea fertilizer, animal urine returns and the use of the nitrification inhibitor DCD do not significantly affect soil methanotroph abundance or daily CH4 fluxes in grazed grassland soils.

  12. WAY-318068: a novel, potent and selective noradrenaline re-uptake inhibitor with activity in rodent models of pain and depression

    PubMed Central

    Whiteside, GT; Dwyer, JM; Harrison, JE; Beyer, CE; Cummons, T; Manzino, L; Mark, L; Johnston, GH; Strassle, BW; Adedoyin, A; Lu, P; Piesla, MJ; Pulicicchio, CM; Erve, JCL; Platt, BJ; Hughes, ZA; Rogers, KE; Deecher, DC; Trybulski, EJ; Kennedy, JD; Zhang, P; Leventhal, L

    2010-01-01

    Background and purpose: Antidepressants, which raise the CNS concentrations of 5-HT and noradrenaline, are frequently used in the treatment of chronic pain; however, it is not known if increasing CNS noradrenaline levels alone is sufficient for efficacy, in part resulting from a lack of small molecules with sufficient selectivity. Experimental approach: In this report, we present the in vitro pharmacological and in vivo pharmacokinetic and pharmacological properties of the novel, orally available and CNS penetrant inhibitor of the noradrenaline transporter (NET), WAY-318068 (1-[(1S,2R)-1-(3,5-difluorophenyl)-2-hydroxy-3-(methylamino)propyl]-7-fluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-one). Key results: WAY-318068 is a potent and effective inhibitor of the NET with a Ki of 8.7 nM in a binding assay, and an IC50 of 6.8 nM in an assay of transporter function, without significant binding to the dopamine transporter. Furthermore, the compound has only weak activity at the 5-HT transporter, leading to a functional selectivity of greater than 2500-fold. It is orally bioavailable with substantial quantities of the compound found in the CNS after oral dosing. As measured by microdialysis in rats, the compound causes a robust and significant increase in cortical noradrenaline levels without affecting 5-HT. WAY-318068 was effective in models of acute, visceral, inflammatory, osteoarthritic, neuropathic, diabetic and bone cancer pain, as well as in traditional models of depression at doses that do not cause motor deficits. Conclusions and implications: Collectively, the present results support the conclusion that selectively increasing CNS levels of noradrenaline is sufficient for efficacy in models of depression and pain. PMID:20590604

  13. GABA-BZD Receptor Modulating Mechanism of Panax quinquefolius against 72-h Sleep Deprivation Induced Anxiety like Behavior: Possible Roles of Oxidative Stress, Mitochondrial Dysfunction and Neuroinflammation

    PubMed Central

    Chanana, Priyanka; Kumar, Anil

    2016-01-01

    Rationale: Panax quinquefolius (American Ginseng) is known for its therapeutic potential against various neurological disorders, but its plausible mechanism of action still remains undeciphered. GABA (Gamma Amino Butyric Acid) plays an important role in sleep wake cycle homeostasis. Thus, there exists rationale in exploring the GABA-ergic potential of Panax quinquefolius as neuroprotective strategy in sleep deprivation induced secondary neurological problems. Objective: The present study was designed to explore the possible GABA-ergic mechanism in the neuro-protective effect of Panax quinquefolius against 72-h sleep deprivation induced anxiety like behavior, oxidative stress, mitochondrial dysfunction, HPA-axis activation and neuroinflammation. Materials and Methods: Male laca mice were sleep deprived for 72-h by using Grid suspended over water method. Panax quinquefolius (American Ginseng 50, 100, and 200 mg/kg) was administered alone and in combination with GABA modulators (GABA Cl− channel inhibitor, GABA-benzodiazepine receptor inhibitor and GABAA agonist) for 8 days, starting 5 days prior to 72-h sleep deprivation period. Various behavioral (locomotor activity, mirror chamber test), biochemical (lipid peroxidation, reduced glutathione, catalase, nitrite levels), mitochondrial complexes, neuroinflammation marker (Tumor Necrosis Factor, TNF-alpha), serum corticosterone, and histopathological sections of brains were assessed. Results: Seventy two hours sleep deprivation significantly impaired locomotor activity, caused anxiety-like behavior, conditions of oxidative stress, alterations in mitochondrial enzyme complex activities, raised serum corticosterone levels, brain TNFα levels and led to neuroinflammation like signs in discrete brain areas as compared to naive group. Panax quinquefolius (100 and 200 mg/kg) treatment restored the behavioral, biochemical, mitochondrial, molecular and histopathological alterations. Pre-treatment of GABA Cl− channel

  14. GABA neurons of the VTA drive conditioned place aversion.

    PubMed

    Tan, Kelly R; Yvon, Cédric; Turiault, Marc; Mirzabekov, Julie J; Doehner, Jana; Labouèbe, Gwenaël; Deisseroth, Karl; Tye, Kay M; Lüscher, Christian

    2012-03-22

    Salient but aversive stimuli inhibit the majority of dopamine (DA) neurons in the ventral tegmental area (VTA) and cause conditioned place aversion (CPA). The cellular mechanism underlying DA neuron inhibition has not been investigated and the causal link to behavior remains elusive. Here, we show that GABA neurons of the VTA inhibit DA neurons through neurotransmission at GABA(A) receptors. We also observe that GABA neurons increase their firing in response to a footshock and provide evidence that driving GABA neurons with optogenetic effectors is sufficient to affect behavior. Taken together, our data demonstrate that synaptic inhibition of DA neurons drives place aversion. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. GABA modulates baroreflex in the ventral tegmental area in rat.

    PubMed

    Hatam, Masoumeh; Rasoulpanah, Minoo; Nasimi, Ali

    2015-12-01

    There are some reports demonstrating the cardiovascular functions of the ventral tegmental area (VTA). About 20-30% of the VTA neurons are GABAergic, which might play a role in baroreflex modulation. This study was performed to find the effects of GABA(A), GABA(B) receptors and reversible synaptic blockade of the VTA on baroreflex. Drugs were microinjected into the VTA of urethane anesthetized rats, and the maximum change of blood pressure and the gain of the reflex bradycardia in response to intravenous phenylephrine (Phe) injection were compared with the preinjection and the control values. Microinjection of bicuculline methiodide (BMI, 100 pmol/100 nl), a GABA(A) antagonist, into the VTA strongly decreased the Phe-induced hypertension, indicating that GABA itself attenuated the baroreflex. Muscimol, a GABA(A) agonist (30 mM, 100 nl), produced no significant changes. Baclofen, a GABA(B) receptor agonist (1000 pmole/100 nl), moderately attenuated the baroreflex, however phaclofen, a GABA(B) receptor antagonist (1000 pmole/100 nl), had no significant effect. In conclusion, for the first time, we demonstrated that GABA(A) receptors of the VTA strongly attenuate and GABA(B) receptors of the VTA moderately attenuate baroreflex in rat. © 2015 Wiley Periodicals, Inc.

  16. Noisy Galvanic Vestibular Stimulation Promotes GABA Release in the Substantia Nigra and Improves Locomotion in Hemiparkinsonian Rats

    PubMed Central

    Samoudi, Ghazaleh; Nissbrandt, Hans; Dutia, Mayank B.; Bergquist, Filip

    2012-01-01

    Background The vestibular system is connected to spinal, cerebellar and cerebral motor control structures and can be selectively activated with external electrodes. The resulting sensation of disturbed balance can be avoided by using stochastic stimulation patterns. Adding noise to the nervous system sometimes improves function. Small clinical trials suggest that stochastic vestibular stimulation (SVS) may improve symptoms in Parkinson's disease. We have investigated this claim and possible mechanisms using the 6-hydroxydopamine (6-OHDA) hemilesion model of Parkinson's disease. Methodology/Principal Findings Animals were tested in the accelerating rod test and the Montoya staircase test of skilled forelimb use. In 6-OHDA hemilesioned animals, SVS improved rod performance by 56±11 s. At group level L-DOPA treatment had no effect, but positive responders improved time on rod by 60±19 s. Skilled forelimb use was not altered by SVS. To investigate how SVS may influence basal ganglia network activity, intracerebral microdialysis was employed in four regions of interest during and after SVS. In presence of the γ-amino buturic acid (GABA) transporter inhibitor NNC 711, SVS induced an increase in GABA to 150±15% of baseline in the substantia nigra (SN) of unlesioned animals, but had no effect in the pedunculopontine nucleus (PPN), the striatum or the ventromedial thalamus (VM). Dopamine release remained stable in all areas, as did GABA and amine concentrations in the SN of unstimulated controls. Following SVS, a sustained increase in GABA concentrations was observed in the ipsilesional, but not in the contralesional SN of 6-OHDA hemilesioned rats. In contrast, L-DOPA treatment produced a similar increase of GABA in the ipsi- and contra-lesional SN. Conclusions/Significance SVS improves rod performance in a rat model of Parkinson's disease, possibly by increasing nigral GABA release in a dopamine independent way. We propose that SVS could be useful for treating symptoms

  17. Neuroprotection by estradiol: a role of aromatase against spine synapse loss after blockade of GABA(A) receptors.

    PubMed

    Zhou, Lepu; Lehan, Nadine; Wehrenberg, Uwe; Disteldorf, Erik; von Lossow, Richard; Mares, Ute; Jarry, Hubertus; Rune, Gabriele M

    2007-01-01

    Estrogen has been suggested to be pro-epileptic by reducing GABA synthesis, resulting in increased spine density and a decreased threshold for seizures in the hippocampus, which, once they occur, are characterized by a dramatic spine loss in the affected brain areas. As considerable amounts of estradiol are synthesized in the hippocampus, in this study we focused on aromatase, the rate-limiting enzyme in estrogen synthesis in order to examine the role of locally synthesized estrogens in epilepsy. To this end, we first examined the effects of letrozole, a potent aromatase inhibitor, on GABA metabolism in single interneurons of hippocampal dispersion cultures. Letrozole downregulated estradiol release into the medium, as well as glutamate decarboxylase (GAD) expression and GABA synthesis, and decreased the number of GAD positive cells in the cultures. Next, we counted spine synapses and measured estradiol release of hippocampal slice cultures, in which GABA(A) receptors had been blocked by bicuculline, in order to mimic epileptic activity. Treatment of slice cultures with bicuculline resulted in a dramatic decrease in the number of spine synapses and in a significant suppression of estrogen synthesis. The decrease in synapse number in response to bicuculline was restored by combined application of estradiol and bicuculline. Surprisingly, estradiol alone had no effect on either spine synapse number or on GAD expression and GABA synthesis. "Rescue" of synapse number in "epileptic slices" by estradiol and maintenance of GABA metabolism by hippocampus-derived estradiol points to a neuroprotective role of aromatase in epilepsy. Re-filling of estradiol stores after their depletion due to overexcitation may therefore add to therapeutical strategies in epilepsy.

  18. The selective dopamine uptake inhibitor, D-84, suppresses cocaine self-administration, but does not occasion cocaine-like levels of generalization

    PubMed Central

    Batman, Angela M.; Dutta, Aloke K.; Reith, Maarten E. A.; Beardsley, Patrick M.

    2010-01-01

    A successful replacement pharmacotherapy for treating cocaine dependency would likely reduce cocaine's abuse, support a low abuse liability, overlap cocaine's subjective effects, and have a long duration of action. Inhibitors with varying selectivity at the dopamine transporter (DAT) have approximated these properties. The objective of the present study was to characterize the behavioural effects of an extremely selective DAT inhibitor, (+) trans-4-(2-Benzhydryloxyethyl)-1-(4-fluorobenzyl) piperadin-3-ol (D-84), a 3-hydroxy substituted piperidine derivative of GBR-12935, for its cocaine-like discriminative stimulus effects, its effects on cocaine self-administration, and for its own self-administration. During cocaine discrimination tests, cocaine occasioned the 10 mg/kg cocaine training stimulus with an ED50 value of 3.13 (1.54-6.34) mg/kg, and reduced response rates with an ED50 value of 20.39 (7.24-57.44) mg/kg. D-84 incompletely generalized to the cocaine stimulus occasioning a maximal 76% cocaine lever responding, while reducing response rates with lower potency than cocaine (ED50=30.94 (12.34-77.60) mg/kg). Pretreatment with D-84 (9.6-30.4 mg/kg) significantly (P<0.05) reduced cocaine intake at 17.1 mg/kg D-84 when cocaine was self-administered at 0.5 mg/kg/infusion, and at 30.4 mg/kg D-84 when cocaine was self-administered at 0.1, 0.5 .and 1.0 mg/kg/infusion. During self-administration tests with D-84 (0.1-1 mg/kg/infusion), numbers of infusions significantly exceeded vehicle levels at 0.3 mg/kg/infusion. These results show that D-84 pre-treatment can decrease cocaine intake especially when high doses of cocaine are being self-administered. This observation, combined with its incomplete generalization to the cocaine discriminative stimulus and its reported long duration of action, provides a profile consistent with a potential replacement therapy for treating cocaine abusing patients. PMID:20840845

  19. The selective dopamine uptake inhibitor, D-84, suppresses cocaine self-administration, but does not occasion cocaine-like levels of generalization.

    PubMed

    Batman, Angela M; Dutta, Aloke K; Reith, Maarten E A; Beardsley, Patrick M

    2010-12-01

    A successful replacement pharmacotherapy for treating cocaine dependency would likely reduce cocaine's abuse, support a low abuse liability, overlap cocaine's subjective effects, and have a long duration of action. Inhibitors with varying selectivity at the dopamine transporter (DAT) have approximated these properties. The objective of the present study was to characterize the behavioural effects of an extremely selective DAT inhibitor, (+) trans-4-(2-Benzhydryloxyethyl)-1-(4-fluorobenzyl) piperadin-3-ol (D-84), a 3-hydroxy substituted piperidine derivative of GBR-12935, for its cocaine-like discriminative stimulus effects, its effects on cocaine self-administration, and for its own self-administration. During cocaine discrimination tests, cocaine occasioned the 10 mg/kg cocaine training stimulus with an ED(50) value of 3.13 (1.54-6.34) mg/kg, and reduced response rates with an ED(50) value of 20.39 (7.24-57.44) mg/kg. D-84 incompletely generalized to the cocaine stimulus occasioning a maximal 76% cocaine-lever responding, while reducing response rates with lower potency than cocaine (ED(50)=30.94 (12.34-77.60) mg/kg). Pretreatment with D-84 (9.6-30.4 mg/kg) significantly (P<0.05) reduced cocaine intake at 17.1 mg/kg D-84 when cocaine was self-administered at 0.5 mg/kg/infusion, and at 30.4 mg/kg D-84 when cocaine was self-administered at 0.1, 0.5 .and 1.0 mg/kg/infusion. During self-administration tests with D-84 (0.1-1 mg/kg/infusion), numbers of infusions significantly exceeded vehicle levels at 0.3 mg/kg/infusion. These results show that D-84 pretreatment can decrease cocaine intake especially when high doses of cocaine are being self-administered. This observation, combined with its incomplete generalization to the cocaine discriminative stimulus and its reported long duration of action, provides a profile consistent with a potential replacement therapy for treating cocaine-abusing patients. Copyright © 2010 Elsevier B.V. All rights reserved.

  20. Target-specific suppression of GABA release from parvalbumin interneurons in the basolateral amygdala by dopamine.

    PubMed

    Chu, Hong-Yuan; Ito, Wataru; Li, Jiayang; Morozov, Alexei

    2012-10-17

    Dopamine (DA) in the basolateral amygdala (BLA) promotes fear learning by disinhibiting principal neurons (PNs) and enabling synaptic plasticity in their sensory inputs. While BLA interneurons (INs) are heterogeneous, it is unclear which interneuron subtypes decrease GABAergic input to PNs in the presence of DA. Here, using cell type-selective photostimulation by channelrhodopsin 2 in BLA slices from mouse brain, we examined the role of parvalbumin-positive INs (PV-INs), the major interneuronal subpopulation in BLA, in the disinhibitory effect of DA. We found that DA selectively suppressed GABAergic transmission from PV-INs to PNs by acting on presynaptic D(2) receptors, and this effect was mimicked by Rp-cAMP, an inhibitor of cAMP-dependent signaling. In contrast, DA did not alter GABA release from PV-INs to INs. Furthermore, neither suppressing cAMP-dependent signaling by Rp-cAMP nor enhancing it by forskolin altered GABA release from PV-INs to BLA INs. Overall, DA disinhibits BLA, at least in part, by suppressing GABA release from PV-INs in the target cell-specific manner that results from differential control of this release by cAMP-dependent signaling.

  1. Long-Lasting Effects of GABA Infusion Into the Cerebral Cortex of the Rat

    PubMed Central

    Montiel, Teresa; Almeida, Daniel; Arango, Iván; Calixto, Eduardo; Casasola, César; Brailowsky, Simón

    2000-01-01

    In electrophysiological terms, experimental models of durable information storage in the brain include long-term potentiation (LTP), long-term depression, and kindling. Protein synthesis correlates with these enduring processes. We propose a fourth example of long-lasting information storage in the brain, which we call the GABA-withdrawal syndrome (GWS). In rats, withdrawal of a chronic intracortical infusion of GABA, a ubiquitous inhibitory neurotransmitter, induced epileptogenesis at the infusion site. This overt GWS lasted for days. Anisomycin, a protein synthesis inhibitor, prevented the appearance of GWS in vivo. Hippocampal and neocortical slices showed a similar post-GABA hyperexcitability in vitro and an enhanced susceptibility to LTP induction. One to four months after the epileptic behavior disappeared, systemic administration of a subconvulsant dose of pentylenetetrazol produced the reappearance of paroxysmal activity. The long-lasting effects of tonic GABAA receptor stimulation may be involved in long-term information storage processes at the cortical level, whereas the cessation of GABAA receptor stimulation may be involved in chronic pathological conditions, such as epilepsy. Furthermore, we propose that GWS may represent a common key factor in the addiction to GABAergic agents (for example, barbiturates, benzodiazepines, and ethanol). GWS represents a novel form of neurono-glial plasticity. The mechanisms of this phenomenon remain to be understood. PMID:10709209

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

    PubMed

    Hu, Jing; Fei, Jian; Reutter, Werner; Fan, Hua

    2011-03-01

    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.

  3. Synthesis, cytotoxicity, cellular uptake and influence on eicosanoid metabolism of cobalt-alkyne modified fructoses in comparison to auranofin and the cytotoxic COX inhibitor Co-ASS.

    PubMed

    Ott, Ingo; Koch, Thao; Shorafa, Hashem; Bai, Zhenlin; Poeckel, Daniel; Steinhilber, Dieter; Gust, Ronald

    2005-06-21

    Propargylhexacarbonyldicobalt complexes with fructopyranose ligands were prepared and investigated for cytotoxicity in the MCF-7 human breast cancer cell line. The antiproliferative effects depended on the presence of isopropylidene protecting groups in the carbohydrate ligand and correlated with the cellular concentration of the complexes. IC(50) values of > 20 microM demonstrated that the fructose derivatives were only moderately active compared to the references auranofin and the aspirin (ASS) derivative [2-acetoxy(2-propynyl)benzoate]hexacarbonyldicobalt (Co-ASS). In continuation of our studies on the mode of action of cobalt-alkyne complexes we studied the influence of the compounds on the formation of 12-HHT (COX-1 product) and 12-HETE (12-LOX product) by human platelets as an indication of the interference in the eicosanoid metabolism, which is discussed as a target system of cytostatics. Co-ASS was an efficient COX-1 inhibitor without LOX inhibitory activity and auranofin inhibited both COX-1 and 12-LOX eicosanoid production. The missing activity of the fructopyranose complexes at the 12-LOX and the only moderate effects at COX-1 indicate that COX/LOX inhibition may be in part responsible for the pharmacological effects of auranofin and Co-ASS but not for those of the fructopyranose complexes.

  4. Signaling Cascades for δ-Opioid Receptor-Mediated Inhibition of GABA Synaptic Transmission and Behavioral Antinociception

    PubMed Central

    Zhang, Zhi

    2012-01-01

    Membrane trafficking of the δ-opioid receptor (DOR) from intracellular compartments to plasma membrane in central neurons, induced by various pathological conditions such as long-term opioid exposure, represents unique receptor plasticity involved in the mechanisms of long-term opioid effects in opioid addiction and opioid treatment of chronic pain. However, the signaling pathways coupled to the newly emerged functional DOR in central neurons are largely unknown at present. In this study, we investigated the signaling cascades of long-term morphine-induced DOR for its cellular and behavioral effects in neurons of the rat brainstem nucleus raphe magnus (NRM), a key supraspinal site for opioid analgesia. We found that, among the three phospholipase A2 (PLA2)-regulated arachidonic acid (AA) metabolic pathways of lipoxygenase, cyclooxygenase, and epoxygenase, 12-lipoxygenase of the lipoxygenase pathway primarily mediated DOR inhibition of GABA synaptic transmission, because inhibitors of 12-lipoxygenase as well as lipoxygenases and PLA2 largely blocked the DOR- or AA-induced GABA inhibition in NRM neurons in brainstem slices in vitro. Blockade of the epoxygenase pathway was ineffective, whereas blocking either 5-lipoxygenase of the lipoxygenase pathway or the cyclooxygenase pathway enhanced the DOR-mediated GABA inhibition. Behaviorally in rats in vivo, NRM infusion of 12-lipoxygenase inhibitors significantly reduced DOR-induced antinociceptive effect whereas inhibitors of 5-lipoxygenase and cyclooxygenase augmented the DOR antinociception. These findings suggest the PLA2-AA-12-lipoxygenase pathway as a primary signaling cascade for DOR-mediated analgesia through inhibition of GABA neurotransmission and indicate potential therapeutic benefits of combining 5-lipoxygenase and cyclooxygenase inhibitors for maximal pain inhibition. PMID:22144670

  5. Expression of spinal cord GABA transporter 1 in morphine-tolerant male Wistar rats.

    PubMed

    Shokoofeh, Siroosi; Homa, Manaheji; Leila, Dargahi; Samira, Daniali

    2015-11-15

    Chronic morphine exposure produces morphine tolerance. One of the mechanisms of morphine tolerance involves γ-aminobutric acid (GABA), whose level is regulated by GABA transporter 1 (GAT-1). The aim of this study was to investigate the expression of GAT-1 in the spinal cord during morphine treatment. Morphine was administrated to rats via drinking water for 21 days. On day 21, a single dose of morphine (10mg/kg) was injected, followed by the administration of 5% formalin after 30 min. Expression of GAT-1 in the lumbar spinal cord during morphine treatment was analyzed by Western blotting and immunohistochemistry assay. In another set of experiments, a morphine-tolerant group was treated with a GAT-1 inhibitor, ethyl nipecotate (60 mg/kg), 5 min prior to the formalin test. To assess a possible analgesic effect of the GAT-1 inhibitor, a non-tolerant group was injected only with ethyl nipecotate 5 min prior to the formalin test. Our results indicated that a chronic consumption of morphine led to morphine tolerance. Morphine tolerance was also concomitant with GAT-1 up-regulation in the lumbar spinal cord. The GAT-1 inhibitor ethyl nipecotate improved the antinociceptive effect of morphine in the morphine-tolerant group. Ethyl nipecotate also had an antinociceptive effect on the non-tolerant group. Thus, our data suggest that GAT-1 overexpression in the spinal cord plays an important role in morphine tolerance.

  6. Studies, using in vivo microdialysis, on the effect of the dopamine uptake inhibitor GBR 12909 on 3,4-methylenedioxymethamphetamine ('ecstasy')-induced dopamine release and free radical formation in the mouse striatum.

    PubMed

    Camarero, Jorge; Sanchez, Veronica; O'Shea, Esther; Green, A Richard; Colado, M Isabel

    2002-06-01

    The present study examined the mechanisms by which 3,4-methylenedioxymethamphetamine (MDMA) produces long-term neurotoxicity of striatal dopamine neurones in mice and the protective action of the dopamine uptake inhibitor GBR 12909. MDMA (30 mg/kg, i.p.), given three times at 3-h intervals, produced a rapid increase in striatal dopamine release measured by in vivo microdialysis (maximum increase to 380 +/- 64% of baseline). This increase was enhanced to 576 +/- 109% of baseline by GBR 12909 (10 mg/kg, i.p.) administered 30 min before each dose of MDMA, supporting the contention that MDMA enters the terminal by diffusion and not via the dopamine uptake site. This, in addition to the fact that perfusion of the probe with a low Ca(2+) medium inhibited the MDMA-induced increase in extracellular dopamine, indicates that the neurotransmitter may be released by a Ca(2+) -dependent mechanism not related to the dopamine transporter. MDMA (30 mg/kg x 3) increased the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) from salicylic acid perfused through a probe implanted in the striatum, indicating that MDMA increased free radical formation. GBR 12909 pre-treatment attenuated the MDMA-induced increase in 2,3-DHBA formation by approximately 50%, but had no significant intrinsic radical trapping activity. MDMA administration increased lipid peroxidation in striatal synaptosomes, an effect reduced by approximately 60% by GBR 12909 pre-treatment. GBR 12909 did not modify the MDMA-induced changes in body temperature. These data suggest that MDMA-induced toxicity of dopamine neurones in mice results from free radical formation which in turn induces an oxidative stress process. The data also indicate that the free radical formation is probably not associated with the MDMA-induced dopamine release and that MDMA does not induce dopamine release via an action at the dopamine transporter.

  7. The expression of GABA(B1) and GABA(B2) receptor subunits in the cNS differs from that in peripheral tissues.

    PubMed

    Calver, A R; Medhurst, A D; Robbins, M J; Charles, K J; Evans, M L; Harrison, D C; Stammers, M; Hughes, S A; Hervieu, G; Couve, A; Moss, S J; Middlemiss, D N; Pangalos, M N

    2000-01-01

    GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.

  8. Norepinephrine turnover in the goldfish brain is modulated by sex steroids and GABA.

    PubMed

    Trudeau, V L; Sloley, B D; Peter, R E

    1993-10-08

    It is known that norepinephrine (NE) is important in the neuroendocrine control of pituitary gonadotropin II (GTH-II) and growth hormone (GH) release but very little is known about the factors regulating NE neurons in the goldfish brain. Female gonad-intact goldfish were implanted intraperitoneally (100 micrograms/g) with testosterone (T) or estradiol (E2) to elevate serum steroid levels. High-performance liquid chromatography measurements showed that steroid implantation had no effect on NE content in the telencephalon, including preoptic area (TEL-POA), or the hypothalamus (HYP). The turnover rate of NE was estimated from the rate of depletion of NE content from tissues following inhibition of tyrosine hydroxylase by alpha-methyl-p-tyrosine (240 micrograms/g). The present study demonstrates that E2 can decrease NE turnover rates in TEL-POA and HYP of sexually regressed goldfish (August). The results in recrudescent fish (November), however, indicate a more complex interaction of E2 with NE neurons since E2 increased NE turnover in TEL-POA and HYP in these animals. Testosterone (T) has less prominent effects on NE turnover rates in TEL-POA and HYP; the only significant effect of T-implantation was a small reduction of NE turnover in the TEL-POA of sexually recrudescent fish. Elevation of endogenous brain GABA concentrations by injection of the GABA transaminase inhibitor, gamma-vinyl-GABA (300 micrograms/g), significantly reduced NE turnover in TEL-POA. These data demonstrate that goldfish NE neurons in the TEL-POA are sensitive to regulation by changes in circulating sex steroids and by increases in brain GABA.

  9. Sleep and GABA levels in the oral part of rat pontine reticular formation are decreased by local and systemic administration of morphine.

    PubMed

    Watson, C J; Lydic, R; Baghdoyan, H A

    2007-01-05

    Morphine, a mu-opioid receptor agonist, is a commonly prescribed treatment for pain. Although highly efficacious, morphine has many unwanted side effects including disruption of sleep and obtundation of wakefulness. One mechanism by which morphine alters sleep and wakefulness may be by modulating GABAergic signaling in brain regions regulating arousal, including the pontine reticular nucleus, oral part (PnO). This study used in vivo microdialysis in unanesthetized Sprague-Dawley rat to test the hypothesis that mu-opioid receptors modulate PnO GABA levels. Validation of the high performance liquid chromatographic technique used to quantify GABA was obtained by dialyzing the PnO (n=4 rats) with the GABA reuptake inhibitor nipecotic acid (500 microM). Nipecotic acid caused a 185+/-20% increase in PnO GABA levels, confirming chromatographic detection of GABA and demonstrating the existence of functional GABA transporters in rat PnO. Morphine caused a concentration-dependent decrease in PnO GABA levels (n=25 rats). Coadministration of morphine (100 microM) with naloxone (1 microM), a mu-opioid receptor antagonist, blocked the morphine-induced decrease in PnO GABA levels (n=5 rats). These results show for the first time that mu-opioid receptors in rat PnO modulate GABA levels. A second group of rats (n=6) was used to test the hypothesis that systemically administered morphine also decreases PnO GABA levels. I.v. morphine caused a significant (P<0.05) decrease (19%) in PnO GABA levels relative to control i.v. infusions of saline. Finally, microinjections followed by 2 h recordings of electroencephalogram and electromyogram tested the hypothesis that PnO morphine administration disrupts sleep (n=8 rats). Morphine significantly (P<0.05) increased the percent of time spent in wakefulness (65%) and significantly (P<0.05) decreased the percent of rapid eye movement (REM) sleep (-53%) and non-REM sleep (-69%). The neurochemical and behavioral data suggest that morphine may

  10. The VMAT-2 inhibitor tetrabenazine alters effort-related decision making as measured by the T-maze barrier choice task: reversal with the adenosine A2A antagonist MSX-3 and the catecholamine uptake blocker bupropion.

    PubMed

    Yohn, Samantha E; Thompson, Christian; Randall, Patrick A; Lee, Christie A; Müller, Christa E; Baqi, Younis; Correa, Mercè; Salamone, John D

    2015-04-01

    Depressed people show effort-related motivational symptoms, such as anergia, retardation, lassitude, and fatigue. Animal tests can model these motivational symptoms, and the present studies characterized the effort-related effects of the vesicular monoamine transport (VMAT-2) inhibitor tetrabenazine. Tetrabenazine produces depressive symptoms in humans and, at low doses, preferentially depletes dopamine. The current studies investigated the effects of tetrabenazine on effort-based decision making using the T-maze barrier task. Rats were tested in a T-maze in which the choice arms of the maze contain different reinforcement densities, and under some conditions, a vertical barrier was placed in the high-density arm to provide an effort-related challenge. The first experiment assessed the effects of tetrabenazine under different maze conditions: a barrier in the arm with 4 food pellets and 2 pellets in the no barrier arm (4-2 barrier), 4 pellets in one arm and 2 pellets in the other with no barrier in either arm (no barrier), and 4 pellets in the barrier arm with no pellets in the other (4-0 barrier). Tetrabenazine (0.25-0.75 mg/kg IP) decreased selection of the high cost/high reward arm when the barrier was present, but had no effect on choice under the no barrier and 4-0 barrier conditions. The effects of tetrabenazine on barrier climbing in the 4-2 condition were reversed by the adenosine A2A antagonist MSX-3 and the catecholamine uptake inhibitor and antidepressant bupropion. These studies have implications for the development of animal models of the motivational symptoms of depression and other disorders.

  11. GABA blocks pathological but not acute TRPV1 pain signals.

    PubMed

    Hanack, Christina; Moroni, Mirko; Lima, Wanessa C; Wende, Hagen; Kirchner, Marieluise; Adelfinger, Lisa; Schrenk-Siemens, Katrin; Tappe-Theodor, Anke; Wetzel, Christiane; Kuich, P Henning; Gassmann, Martin; Roggenkamp, Dennis; Bettler, Bernhard; Lewin, Gary R; Selbach, Matthias; Siemens, Jan

    2015-02-12

    Sensitization of the capsaicin receptor TRPV1 is central to the initiation of pathological forms of pain, and multiple signaling cascades are known to enhance TRPV1 activity under inflammatory conditions. How might detrimental escalation of TRPV1 activity be counteracted? Using a genetic-proteomic approach, we identify the GABAB1 receptor subunit as bona fide inhibitor of TRPV1 sensitization in the context of diverse inflammatory settings. We find that the endogenous GABAB agonist, GABA, is released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism limiting TRPV1 sensitization. The effect of GABAB on TRPV1 is independent of canonical G protein signaling and rather relies on close juxtaposition of the GABAB1 receptor subunit and TRPV1. Activating the GABAB1 receptor subunit does not attenuate normal functioning of the capsaicin receptor but exclusively reverts its sensitized state. Thus, harnessing this mechanism for anti-pain therapy may prevent adverse effects associated with currently available TRPV1 blockers. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. A new role for GABA: inhibition of tumor cell migration.

    PubMed

    Ortega, Arturo

    2003-04-01

    GABA, the main inhibitory neurotransmitter in the vertebrate brain, participates outside the CNS in diverse functions such as platelet aggregation and the acrosomal reaction in spermatozoa. A recent study now demonstrates that GABA inhibits the migration of colon carcinoma cells, paving the way to the development of specific pharmacological agents that delay or inhibit invasion and metastasis of various cancer types.

  13. γ-Aminobutyric acid (GABA) signalling in plants.

    PubMed

    Ramesh, Sunita A; Tyerman, Stephen D; Gilliham, Matthew; Xu, Bo

    2017-05-01

    The role of γ-aminobutyric acid (GABA) as a signal in animals has been documented for over 60 years. In contrast, evidence that GABA is a signal in plants has only emerged in the last 15 years, and it was not until last year that a mechanism by which this could occur was identified-a plant 'GABA receptor' that inhibits anion passage through the aluminium-activated malate transporter family of proteins (ALMTs). ALMTs are multigenic, expressed in different organs and present on different membranes. We propose GABA regulation of ALMT activity could function as a signal that modulates plant growth, development, and stress response. In this review, we compare and contrast the plant 'GABA receptor' with mammalian GABAA receptors in terms of their molecular identity, predicted topology, mode of action, and signalling roles. We also explore the implications of the discovery that GABA modulates anion flux in plants, its role in signal transduction for the regulation of plant physiology, and predict the possibility that there are other GABA interaction sites in the N termini of ALMT proteins through in silico evolutionary coupling analysis; we also explore the potential interactions between GABA and other signalling molecules.

  14. GABA transaminases from Saccharomyces cerevisiae and Arabidopsis thaliana complement function in cytosol and mitochondria.

    PubMed

    Cao, Juxiang; Barbosa, Jose M; Singh, Narendra; Locy, Robert D

    2013-07-01

    GABA transaminase (GABA-T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA-T from Saccharomyces cerevisiae (ScGABA-TKG) is an α-ketoglutarate-dependent enzyme encoded by the UGA1 gene, while higher plant GABA-T is a pyruvate/glyoxylate-dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA-T). The GABA-T from A. thaliana is localized in mitochondria and mediated by an 18-amino acid N-terminal mitochondrial targeting peptide predicated by both web-based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA-TKG and AtGABA-T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA-TKG and AtGABA-T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA-TKG deletion mutant Δuga1 with AtGABA-T or ScGABA-TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat-induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA-T is functionally interchangeable with ScGABA-TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA-T is about half as efficient in doing so as ScGABA-TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate-limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces. Copyright © 2013 John Wiley & Sons, Ltd.

  15. Cloning and expression of a rat brain GABA transporter

    SciTech Connect

    Guastella, J.; Czyzyk, L.; Davidson, N.; Lester, H.A. ); Nelson, N.; Nelson, H.; Miedel, M.C. ); Keynan, S.; Kanner, B.I. )

    1990-09-14

    A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter {gamma}-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated ({sup 3}H)GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium- and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.

  16. A noncanonical release of GABA and glutamate modulates neuronal migration.

    PubMed

    Manent, Jean-Bernard; Demarque, Michaël; Jorquera, Isabel; Pellegrino, Christophe; Ben-Ari, Yehezkel; Aniksztejn, Laurent; Represa, Alfonso

    2005-05-11

    Immature neurons express GABA and glutamate receptors before synapse formation, and both transmitters are released at an early developmental stage. We have now tested the hypothesis that the ongoing release of GABA and glutamate modulates neuronal migration. Using 5-bromo-2'-deoxyuridine labeling and cocultures of hippocampal slices obtained from naive and green fluorescent protein-transgenic mice, we report that migration is severely affected by GABA(A) or NMDA receptor antagonist treatments. These effects were also present in munc18-1 knock-out slices in which soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicular secretion of transmitters has been deleted. GABA(A) antagonists were more efficient than NMDA antagonists to reduce cell migration, in keeping with the earlier maturation of GABAergic mechanisms. We conclude that GABA and, to a lesser degree, glutamate released in a SNARE-independent mechanism exert a paracrine action on neuronal migration.

  17. Diversity of inhibitory neurotransmission through GABA(A) receptors.

    PubMed

    Mody, Istvan; Pearce, Robert A

    2004-09-01

    In the brain, highly connected and heterogeneous GABAergic cells are crucial in controling the activity of neuronal networks. They accomplish this task by communicating through remarkably diverse sets of inhibitory processes, the complexity of which is reflected by the variety of interneuron classification schemes proposed in recent years. It is now becoming clear that the subcellular localization and intrinsic properties of heteropentameric GABA(A) receptors themselves also constitute major sources of diversity in GABA-mediated signaling. This review summarizes some of the factors underlying this diversity, including GABA(A) receptor subunit composition, localization, activation, number and phosphorylation states, variance of GABA concentration in the synaptic cleft, and some of the presynaptic factors regulating GABA release.

  18. Neurosteroids and GABA-A Receptor Function

    PubMed Central

    Wang, Mingde

    2011-01-01

    Neurosteroids represent a class of endogenous steroids that are synthesized in the brain, the adrenals, and the gonads and have potent and selective effects on the GABAA-receptor. 3α-hydroxy A-ring reduced metabolites of progesterone, deoxycorticosterone, and testosterone are positive modulators of GABAA-receptor in a non-genomic manner. Allopregnanolone (3α-OH-5α-pregnan-20-one), 5α-androstane-3α, 17α-diol (Adiol), and 3α5α-tetrahydrodeoxycorticosterone (3α5α-THDOC) enhance the GABA-mediated Cl- currents acting on a site (or sites) distinct from the GABA, benzodiazepine, barbiturate, and picrotoxin binding sites. 3α5α-P and 3α5α-THDOC potentiate synaptic GABAA-receptor function and activate δ-subunit containing extrasynaptic receptors that mediate tonic currents. On the contrary, 3β-OH pregnane steroids and pregnenolone sulfate (PS) are GABAA-receptor antagonists and induce activation-dependent inhibition of the receptor. The activities of neurosteroid are dependent on brain regions and types of neurons. In addition to the slow genomic action of the parent steroids, the non-genomic, and rapid actions of neurosteroids play a significant role in the GABAA-receptor function and shift in mood and memory function. This review describes molecular mechanisms underlying neurosteroid action on the GABAA-receptor, mood changes, and cognitive functions. PMID:22654809

  19. Bicyclic γ-amino acids as inhibitors of γ-aminobutyrate aminotransferase.

    PubMed

    Pinto, Andrea; Tamborini, Lucia; Pennacchietti, Eugenia; Coluccia, Antonio; Silvestri, Romano; Cullia, Gregorio; De Micheli, Carlo; Conti, Paola; De Biase, Daniela

    2016-01-01

    The γ-aminobutyrate (GABA)-degradative enzyme GABA aminotransferase (GABA-AT) is regarded as an attractive target to control GABA levels in the central nervous system: this has important implications in the treatment of several neurological disorders and drug dependencies. We have investigated the ability of newly synthesized compounds to act as GABA-AT inhibitors. These compounds have a unique bicyclic structure: the carbocyclic ring bears the GABA skeleton, while the fused 3-Br-isoxazoline ring contains an electrophilic warhead susceptible of nucleophilic attack by an active site residue of the target enzyme. Out of the four compounds tested, only the one named (+)-3 was found to significantly inhibit mammalian GABA-AT in vitro. Docking studies, performed on the available structures of GABA-AT, support the experimental findings: out of the four tested compounds, only (+)-3 suitably orients the electrophilic 3-Br-isoxazoline warhead towards the active site nucleophilic residue Lys329, thereby explaining the irreversible inhibition of GABA-AT observed experimentally.

  20. Laser photolysis of DPNI-GABA, a tool for investigating the properties and distribution of GABA receptors and for silencing neurons in situ.

    PubMed

    Trigo, Federico F; Papageorgiou, George; Corrie, John E T; Ogden, David

    2009-07-30

    Laser photolysis to release GABA at precisely defined times and locations permits investigation of the distribution of functional GABA(A) receptors in neuronal compartments, the activation kinetics and pharmacology of GABA(A) receptors in situ, and the role of individual neurons in neural circuits by selective silencing with low GABA concentrations. We describe the experimental evaluation and applications of a new nitroindoline-caged GABA, DPNI-GABA, modified to minimize the pharmacological interference commonly found with caged GABA reagents, but retaining the advantages of nitroindoline cages. Unlike the 5-methoxycarbonylmethyl-7-nitroindolinyl-GABA tested previously, DPNI-GABA inhibited GABA(A) receptors with much lower affinity, reducing peak GABA-evoked responses with an IC(50) of approximately 0.5 mM. Most importantly, the kinetics of receptor activation, determined as 10-90% rise-times, were comparable to synaptic events and were little affected by DPNI-GABA present at 1mM concentration, permitting photolysis of DPNI-GABA to mimic synaptic activation of GABA(A) receptors. With a laser spot of 1 microm applied to cerebellar molecular layer interneurons, the spatial resolution of uncaging DPNI-GABA in dendrites was estimated as 2 microm laterally and 7.5 microm focally. Finally, at low DPNI-GABA concentration, photorelease restricted to the area of the soma suppressed spiking in single Purkinje neurons or molecular layer interneurons for periods controlled by the flash intensity and duration. DPNI-GABA has properties better adapted for fast kinetic studies with laser photolysis at GABA(A) receptors than previously reported caged GABA reagents, and can be used in experiments where spatial resolution is determined by the dimensions of the laser light spot.

  1. Effects of Tityus serrulatus scorpion venom and its toxin TsTX-V on neurotransmitter uptake in vitro

    SciTech Connect

    Cecchini, Alessandra L.; Vasconcelos, Flavio; Giglio, Jose Roberto; Arantes, Eliane Candiani . E-mail: ecabraga@fcfrp.usp.br

    2006-12-01

    Scorpion neurotoxins targeting the Na{sub v} channel can be classified into two classes: {alpha}- and {beta}-neurotoxins and are reported as highly active in mammalian brain. In this work, we evaluate the effects of Tityus serrulatus venom (Ts venom) and its {alpha}-neurotoxin TsTX-V on {gamma}-aminobutyric acid (GABA), dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes. TsTX-V was isolated from Ts venom by ion exchange chromatography followed by reverse-phase (C18) high-performance liquid chromatography. Neither Ts venom nor TsTX-V was able to affect {sup 3}H-Glu uptake. On the other hand, Ts venom (0.13 {mu}g/mg) significantly inhibited both {sup 3}H-GABA and {sup 3}H-DA uptake ({approx} 50%). TsTX-V showed IC{sub 5} values of 9.37 {mu}M and 22.2 {mu}M for the inhibition of {sup 3}H-GABA and {sup 3}H-DA uptake, respectively. These effects were abolished by pre-treatment with tetrodotoxin (TTX, 1 {mu}M), indicating the involvement of voltage-gated Na{sup +} channels in this process. In the absence of Ca{sup 2+}, and at low Ts venom concentrations, the reduction of {sup 3}H-GABA uptake was not as marked as in the presence of Ca{sup 2+}. TsTX-V did not reduce {sup 3}H-GABA uptake in COS-7 cells expressing the GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced {sup 3}H-GABA uptake by synaptosomes might be due to rapid cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes a reduction of {sup 3}H-GABA and {sup 3}H-DA uptake in a Ca{sup 2+}-dependent manner, not directly affecting GABA transporters, but, in consequence of depolarization, involving voltage-gated Na{sup +} channels.

  2. Neurotransmitter GABA activates muscle but not α7 nicotinic receptors.

    PubMed

    Dionisio, Leonardo; Bergé, Ignacio; Bravo, Matías; Esandi, María Del Carmen; Bouzat, Cecilia

    2015-01-01

    Cys-loop receptors are neurotransmitter-activated ion channels involved in synaptic and extrasynaptic transmission in the brain and are also present in non-neuronal cells. As GABAA and nicotinic receptors (nAChR) belong to this family, we explored by macroscopic and single-channel recordings whether the inhibitory neurotransmitter GABA has the ability to activate excitatory nAChRs. GABA differentially activates nAChR subtypes. It activates muscle nAChRs, with maximal peak currents of about 10% of those elicited by acetylcholine (ACh) and 15-fold higher EC50 with respect to ACh. At the single-channel level, the weak agonism is revealed by the requirement of 20-fold higher concentration of GABA for detectable channel openings, a major population of brief openings, and absence of clusters of openings when compared with ACh. Mutations at key residues of the principal binding-site face of muscle nAChRs (αY190 and αG153) affect GABA activation similarly as ACh activation, whereas a mutation at the complementary face (εG57) shows a selective effect for GABA. Studies with subunit-lacking receptors show that GABA can activate muscle nAChRs through the α/δ interface. Interestingly, single-channel activity elicited by GABA is similar to that elicited by ACh in gain-of-function nAChR mutants associated to congenital myasthenic syndromes, which could be important in the progression of the disorders due to steady exposure to serum GABA. In contrast, GABA cannot elicit single-channel or macroscopic currents of α7 or the chimeric α7-serotonin-type 3 receptor, a feature important for preserving an adequate excitatory/inhibitory balance in the brain as well as for avoiding activation of non-neuronal receptors by serum GABA. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  3. Study of GABA in Healthy Volunteers: Pharmacokinetics and Pharmacodynamics

    PubMed Central

    Li, Junfeng; Zhang, Zhaoyun; Liu, Xiaoxia; Wang, Yi; Mao, Fei; Mao, Junjun; Lu, Xiaolan; Jiang, Dongdong; Wan, Yun; Lv, Jia-Ying; Cao, Guoying; Zhang, Jing; Zhao, Naiqing; Atkinson, Mark; Greiner, Dale L.; Prud'homme, Gerald J.; Jiao, Zheng; Li, Yiming; Wang, Qinghua

    2015-01-01

    Preclinical studies show that GABA exerts anti-diabetic effects in rodent models of type 1 diabetes. Because little is known about its absorption and effects in humans, we investigated the pharmacokinetics and pharmacodynamics of GABA in healthy volunteers. Twelve subjects were subjected to an open-labeled, three-period trial involving sequential oral administration of placebo, 2 g GABA once, and 2 g GABA three times/day for 7 days, with a 7-day washout between each period. GABA was rapidly absorbed (Tmax: 0.5 ~ 1 h) with the half-life (t1/2) of 5 h. No accumulation was observed after repeated oral GABA administration for 7 days. Remarkably, GABA significantly increased circulating insulin levels in the subjects under either fasting (1.6-fold, single dose; 2.0-fold, repeated dose; p < 0.01) or fed conditions (1.4-fold, single dose; 1.6-fold, repeated dose; p < 0.01). GABA also increased glucagon levels only under fasting conditions (1.3-fold, single dose, p < 0.05; 1.5-fold, repeated dose, p < 0.01). However, there were no significant differences in the insulin-to-glucagon ratio and no significant change in glucose levels in these healthy subjects during the study period. Importantly, GABA significantly decreased glycated albumin levels in the repeated dosing period. Subjects with repeated dosing showed an elevated incidence of minor adverse events in comparison to placebo or the single dosing period, most notably transient discomforts such as dizziness and sore throat. However, there were no serious adverse events observed throughout the study. Our data show that GABA is rapidly absorbed and tolerated in human beings; its endocrine effects, exemplified by increasing islet hormonal secretion, suggest potential therapeutic benefits for diabetes. PMID:26617516

  4. GABA selectively increases mucin-1 expression in isolated pig jejunum.

    PubMed

    Braun, Hannah-Sophie; Sponder, Gerhard; Pieper, Robert; Aschenbach, Jörg R; Deiner, Carolin

    2015-11-01

    The inhibitory neurotransmitter GABA (γ-aminobutyric acid) is synthesized by glutamic acid decarboxylase, which is expressed in the central nervous system and in various other tissues including the intestine. Moreover, GABA can be ingested in vegetarian diets or produced by bacterial commensals in the gastrointestinal tract. As previous studies in lung have suggested a link between locally increased GABA availability and mucin 5AC production, the present study sought to test whether the presence or lack of GABA (and its precursor glutamine) has an effect on intestinal mucin expression. Porcine jejunum epithelial preparations were incubated with two different amounts of GABA or glutamine on the mucosal side for 4 h, and changes in the relative gene expression of seven different mucins, enzymes involved in mucin shedding, GABA B receptor, enzymes involved in glutamine/GABA metabolism, glutathione peroxidase 2, and interleukin 10 were examined by quantitative PCR (TaqMan(®) assays). Protein expression of mucin-1 (MUC1) was analyzed by Western blot. On the RNA level, only MUC1 was significantly up-regulated by both GABA concentrations compared with the control. Glutamine-treated groups showed the same trend. On the protein level, all treatment groups showed a significantly higher MUC1 expression than the control group. We conclude that GABA selectively increases the expression of MUC1, a cell surface mucin that prevents the adhesion of microorganisms, because of its size and negative charge, and therefore propose that the well-described positive effects of glutamine on enterocytes and intestinal integrity are partly attributable to effects of its metabolite GABA.

  5. Study of GABA in Healthy Volunteers: Pharmacokinetics and Pharmacodynamics.

    PubMed

    Li, Junfeng; Zhang, Zhaoyun; Liu, Xiaoxia; Wang, Yi; Mao, Fei; Mao, Junjun; Lu, Xiaolan; Jiang, Dongdong; Wan, Yun; Lv, Jia-Ying; Cao, Guoying; Zhang, Jing; Zhao, Naiqing; Atkinson, Mark; Greiner, Dale L; Prud'homme, Gerald J; Jiao, Zheng; Li, Yiming; Wang, Qinghua

    2015-01-01

    Preclinical studies show that GABA exerts anti-diabetic effects in rodent models of type 1 diabetes. Because little is known about its absorption and effects in humans, we investigated the pharmacokinetics and pharmacodynamics of GABA in healthy volunteers. Twelve subjects were subjected to an open-labeled, three-period trial involving sequential oral administration of placebo, 2 g GABA once, and 2 g GABA three times/day for 7 days, with a 7-day washout between each period. GABA was rapidly absorbed (Tmax: 0.5 ~ 1 h) with the half-life (t1/2) of 5 h. No accumulation was observed after repeated oral GABA administration for 7 days. Remarkably, GABA significantly increased circulating insulin levels in the subjects under either fasting (1.6-fold, single dose; 2.0-fold, repeated dose; p < 0.01) or fed conditions (1.4-fold, single dose; 1.6-fold, repeated dose; p < 0.01). GABA also increased glucagon levels only under fasting conditions (1.3-fold, single dose, p < 0.05; 1.5-fold, repeated dose, p < 0.01). However, there were no significant differences in the insulin-to-glucagon ratio and no significant change in glucose levels in these healthy subjects during the study period. Importantly, GABA significantly decreased glycated albumin levels in the repeated dosing period. Subjects with repeated dosing showed an elevated incidence of minor adverse events in comparison to placebo or the single dosing period, most notably transient discomforts such as dizziness and sore throat. However, there were no serious adverse events observed throughout the study. Our data show that GABA is rapidly absorbed and tolerated in human beings; its endocrine effects, exemplified by increasing islet hormonal secretion, suggest potential therapeutic benefits for diabetes.

  6. The effects of agonists of ionotropic GABA(A) and metabotropic GABA(B) receptors on learning.

    PubMed

    Zyablitseva, Evgeniya A; Kositsyn, Nikolay S; Shul'gina, Galina I

    2009-05-01

    The research described here investigates the role played by inhibitory processes in the discriminations made by the nervous system of humans and animals between familiar and unfamiliar and significant and nonsignificant events. This research compared the effects of two inhibitory mediators of gamma-aminobutyric acid (GABA): 1) phenibut, a nonselective agonist of ionotropic GABA(A) and metabotropic GABA(B) receptors and 2) gaboxadol a selective agonist of ionotropic GABA(A) receptors on the process of developing active defensive and inhibitory conditioned reflexes in alert non-immobilized rabbits. It was found that phenibut, but not gaboxadol, accelerates the development of defensive reflexes at an early stage of conditioning. Both phenibut and gaboxadol facilitate the development of conditioned inhibition, but the effect of gaboxadol occurs at later stages of conditioning and is less stable than that of phenibut. The earlier and more stable effects of phenibut, as compared to gaboxadol, on storage in memory of the inhibitory significance of a stimulus may occur because GABA(B) receptors play the dominant role in the development of internal inhibition during an early stage of conditioning. On the other hand this may occur because the participation of both GABA(A) and GABA(B) receptors are essential to the process. We discuss the polyfunctionality of GABA receptors as a function of their structure and the positions of the relevant neurons in the brain as this factor can affect regulation of various types of psychological processes.

  7. GABA-A and GABA-B receptors in the cuneate nucleus of the rat in vivo.

    PubMed

    Orviz, P; Cecchini, B G; Andrés-Trelles, F

    1986-09-01

    Electric stimulation of the rat forepaw evokes a negative potential (N-wave) at the ipsilateral cuneate nucleus. The responses of the N-wave to microiontophoretically applied GABA agonists and antagonists have been studied. Applications of GABA-A agonists (3-amino-propanesulfonic acid and muscimol) reduce the amplitude of the N-wave. This effect decreases during prolonged application, suggesting a desensitization of GABA-A receptors. In addition the effect of muscimol is reduced by (-)-bicuculline methiodide. Baclofen (a GABA-B agonist) also depresses the N-wave but its action lasts longer, is less reversible, shows no desensitization and is not blocked by (-)-bicuculline methiodide. The different responses of the N-wave to GABA-A and GABA-B agonists are compatible with the existence of different types of functional receptors for them in the cuneate nucleus of the rat. The receptors activated by muscimol (GABA-A) are clearly not the same as the ones activated by baclofen (conceivably GABA-B).

  8. Glia of the cholinergic electromotor nucleus of Torpedo are the source of the cDNA encoding a GAT-1-like GABA transporter.

    PubMed

    Swanson, G T; Umbach, J A; Gundersen, C B

    1994-07-01

    A PCR-based strategy was used to clone DNAs encoding Na(+)- and Cl(-)-dependent cotransport proteins using DNA from the cholinergic electromotor nucleus of Torpedo californica. This cloning strategy resulted in the isolation of a cDNA clone that shows strong nucleotide sequence homology to the GABA transporter-1 (GAT-1) types of rat and human brain. When expressed in frog oocytes, this transporter mediates the uptake of GABA. Moreover, physiologically and pharmacologically, the Torpedo protein behaves very similarly to the rat and human GAT-1 proteins. However, in contrast to the predominantly neuronal localization of the mammalian GAT-1 proteins, the mRNA for the fish protein is found almost exclusively in glial elements of the electromotor nucleus. This unexpected discovery of a GABA transporter cDNA in a nucleus that has no previously characterized GABAergic innervation raises questions about the role of GABA and this transporter in the electromotor system. Several speculative models for GABA function are proposed.

  9. Nickel uptake in Bradyrhizobium japonicum

    SciTech Connect

    Stults, L.W.; Mallick, S.; Maier, R.J.

    1987-04-01

    Free-living Bradyrhizobium japonicum grown heterotrophically with 1 ..mu..M /sup 63/Ni/sup 2 +/ accumulated label. Strain SR470, a Hup/sup c/ mutant, accumulated almost 10-fold more /sup 63/Ni/sup 2 +/ on a per-cell basis than did strain SR, the wild type. Nongrowing cells were also able to accumulate nickel over a 2-h period, with the Hup/sup c/ mutant strain SR470 again accumulating significantly more /sup 63/Ni/sup 2 +/ than strain SR. These results suggest that this mutant is constitutive for nickel uptake as well as for hydrogenase expression. The uptake process was relatively specific for nickel; only Cu/sup 2 +/ and Zn/sup 2 +/ (10 ..mu..M) were found to appreciably inhibit the uptake of 1 ..mu..M Ni, while a 10-fold excess of Mg/sup 2 +/, Co/sup 2 +/, Fe/sup 3 +/, or Mn/sup 2 +/ did not affect Ni/sup 2 +/ uptake. The lack of inhibition by Mg/sup 2 +/ indicates that nickel is not transported by a magnesium uptake system. Nickel uptake was also inhibited by cold and slightly by the ionophores nigericin and carbonyl cyanide m-chlorophenylhydrazone. The cytochrome c oxidase inhibitors azide, cyanide, and hydroxylamine did not inhibit Ni/sup 2 +/ uptake, even at concentrations (of cyanide and hydroxylamine) that inhibited O/sub 2/ uptake. The addition of oxidizable substrates such as succinate or gluconate did not increase nickel uptake, even though they increased respiratory activity. Nickel uptake showed a pH dependence with an optimum at 6.0. Most (approximately 85%) of the /sup 63/Ni/sup 2 +/ taken up in 1 min by strain SR470 was not exchangeable with cold nickel.

  10. Modulation of GABA transport by adenosine A1R-A2AR heteromers, which are coupled to both Gs- and G(i/o)-proteins.

    PubMed

    Cristóvão-Ferreira, Sofia; Navarro, Gemma; Brugarolas, Marc; Pérez-Capote, Kamil; Vaz, Sandra H; Fattorini, Giorgia; Conti, Fiorenzo; Lluis, Carmen; Ribeiro, Joaquim A; McCormick, Peter J; Casadó, Vicent; Franco, Rafael; Sebastião, Ana M

    2011-11-02

    Astrocytes play a key role in modulating synaptic transmission by controlling the available extracellular GABA via the GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that an additional level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A(1) (A(1)R) and A(2A) (A(2A)R) receptors. This regulation occurs through a complex of heterotetramers (two interacting homodimers) of A(1)R-A(2A)R that signal via two different G-proteins, G(s) and G(i/o), and either enhances (A(2A)R) or inhibits (A(1)R) GABA uptake. These results provide novel mechanistic insight into how G-protein-coupled receptor heteromers signal. Furthermore, we uncover a previously unknown mechanism in which adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glia-neuron) synapse.

  11. Immunocytochemical Evidence that Monkey Rod Bipolar Cells Use GABA

    PubMed Central

    Lassová, Luisa; Fina, Marie; Sulaiman, Pyroja; Vard, Noga

    2010-01-01

    Certain bipolar cells in most species immunostain for GABA or its synthesizing enzyme, GAD. However it is unknown whether they actually release GABA, and if so, from which cellular compartment, and by what release mechanism. We investigated these questions in monkey retina where rod bipolar cells immunostain for GABA. We found that rod bipolar cells immunostain for one isoform of GAD, GAD65, in their somas, dendrites, and axon terminals. Near the fovea, the somatic stain of rod bipolar cells is weaker than that of horizontal cells, but at the periphery, it is stronger. Staining for the vesicular GABA transporter in monkey rod bipolar cells is negative. On the other hand, staining for the GABA transporter, GAT3, is positive in the soma and primary dendrites (but not in the axon terminals). Staining for GAT3 is also positive in horizontal cells. Double staining of rod bipolar cells and the alpha subunit of the GABAA receptor, reveals scarce GABAA puncta that appose rod bipolar dendrites. We conclude that monkey rod bipolar cells use GABA, and discuss the possibility that they tonically release GABA from their dendrites using a reverse action of GAT3. PMID:20384812

  12. Corelease of acetylcholine and GABA from cholinergic forebrain neurons

    PubMed Central

    Saunders, Arpiar; Granger, Adam J; Sabatini, Bernardo L

    2015-01-01

    Neurotransmitter corelease is emerging as a common theme of central neuromodulatory systems. Though corelease of glutamate or GABA with acetylcholine has been reported within the cholinergic system, the full extent is unknown. To explore synaptic signaling of cholinergic forebrain neurons, we activated choline acetyltransferase expressing neurons using channelrhodopsin while recording post-synaptic currents (PSCs) in layer 1 interneurons. Surprisingly, we observed PSCs mediated by GABAA receptors in addition to nicotinic acetylcholine receptors. Based on PSC latency and pharmacological sensitivity, our results suggest monosynaptic release of both GABA and ACh. Anatomical analysis showed that forebrain cholinergic neurons express the GABA synthetic enzyme Gad2 and the vesicular GABA transporter (Slc32a1). We confirmed the direct release of GABA by knocking out Slc32a1 from cholinergic neurons. Our results identify GABA as an overlooked fast neurotransmitter utilized throughout the forebrain cholinergic system. GABA/ACh corelease may have major implications for modulation of cortical function by cholinergic neurons. DOI: http://dx.doi.org/10.7554/eLife.06412.001 PMID:25723967

  13. Identification and Characterization of γ-Aminobutyric Acid Uptake System GabPCg (NCgl0464) in Corynebacterium glutamicum

    PubMed Central

    Zhao, Zhi; Ma, Wen-hua; Zhou, Ning-Yi

    2012-01-01

    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 (GabPCg) encoded by ncgl0464 was identified and characterized. GabPCg plays a major role in GABA uptake and is essential to C. glutamicum growing on GABA. GABA uptake by GabPCg was weakly competed by l-Asn and l-Gln and stimulated by sodium ion (Na+). The Km and Vmax 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. GabPCg 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 GabPCg deletion showed 12.5% higher productivity of GABA than RES167/pGXKZ9. It is concluded that GabPCg represents a new type of GABA transporter and is potentially important for engineering GABA-producing C. glutamicum strains. PMID:22307305

  14. gamma-Hydroxybutyrate (GHB) induces GABA(B) receptor independent intracellular Ca2+ transients in astrocytes, but has no effect on GHB or GABA(B) receptors of medium spiny neurons in the nucleus accumbens.

    PubMed

    Molnár, T; Antal, K; Nyitrai, G; Emri, Z

    2009-08-18

    We report on cellular actions of the illicit recreational drug gamma-hydroxybutyrate (GHB) in the brain reward area nucleus accumbens. First, we compared the effects of GHB and the GABA(B) receptor agonist baclofen. Neither of them affected the membrane currents of medium spiny neurons in rat nucleus accumbens slices. GABAergic and glutamatergic synaptic potentials of medium spiny neurons, however, were reduced by baclofen but not GHB. These results indicate the lack of GHB as well as postsynaptic GABA(B) receptors, and the presence of GHB insensitive presynaptic GABA(B) receptors in medium spiny neurons. In astrocytes GHB induced intracellular Ca(2+) transients, preserved in slices from GABA(B) receptor type 1 subunit knockout mice. The effects of tetrodotoxin, zero added Ca(2+) with/without intracellular Ca(2+) store depletor cyclopiazonic acid or vacuolar H-ATPase inhibitor bafilomycin A1 indicate that GHB-evoked Ca(2+) transients depend on external Ca(2+) and intracellular Ca(2+) stores, but not on vesicular transmitter release. GHB-induced astrocytic Ca(2+) transients were not affected by the GHB receptor-specific antagonist NCS-382, suggesting the presence of a novel NCS-382-insensitive target for GHB in astrocytes. The activation of astrocytes by GHB implies their involvement in physiological actions of GHB. Our findings disclose a novel profile of GHB action in the nucleus accumbens. Here, unlike in other brain areas, GHB does not act on GABA(B) receptors, but activates an NCS-382 insensitive GHB-specific target in a subpopulation of astrocytes. The lack of either post- or presynaptic effects on medium spiny neurons in the nucleus accumbens distinguishes GHB from many drugs and natural rewards with addictive properties and might explain why GHB has only a weak reinforcing capacity.

  15. Interaction of pyracetam with specific /sup 3/H-imipramine binding sites and GABA-benzodiazepine receptor complex of brain membranes

    SciTech Connect

    Rozhanets, V.V.; Chakhbra, K.K.; Danchev, N.D.; Malin, K.M.; Rusakov, D.Yu.; Val'dman, A.V.

    1986-06-01

    This paper studies the effect of pyracetam on parameters of specific binding of tritium-imipramine and GABA-activated binding of tritium-flunitrazepam with rat brain membranes. The experimental method is described and it is shown that pyracetam and mebicar in experiments in vivo on normal animals can exert their anxiolytic action without the participation of bensodiazepine receptors. Either the interaction of pyracetam and mebicar with benzodiazeprine receptors has a different interpretation than competition of these compounds with specific binding sites of tritium-flunitrazepam, or in experiments on normal animals in vivo GABA-benzodiazepine receptor complex does not accept pyracetam and mebicar, for it contains endogenous inhibitors of GABA-modulating action.

  16. Presynaptic modulation by L-glutamate and GABA of sympathetic co-transmission in rat isolated vas deferens.

    PubMed

    Kwan, Y W; Ngan, M P; Tsang, K Y; Lee, H M; Chu, L A

    1996-06-01

    1. The modulatory effects of L-glutamate and its structural analogues, and of gamma-aminobutyric acid (GABA), on sympathetic co-transmission were studied in the rat isolated vas deferens exposed to electrical field stimulation (EFS). 2. Application of exogenous L-glutamate caused a concentration-dependent (1 microM-3 mM) inhibition of the rapid twitch component of the biphasic EFS contraction. However, L-glutamate (1 microM-3 mM) had a minimal effect on the phasic contraction induced by exogenous adenosine 5'-triphosphate (ATP, 150 microM) and noradrenaline (50 microM). Unlike L-glutamate, D-glutamate had no effect on the EFS contraction. 3. The L-glutamate-induced inhibition of the EFS contractions was significantly attenuated by the glutamate decarboxylase (GAD) inhibitor 3-mercapto-propionic acid (150 microM) and was abolished in the presence of the GABA transaminase (GABA-T) inhibitor, 2-aminoethyl hydrogen sulphate (500 microM). 4. The L-glutamate-induced inhibition of the electrically evoked contraction was not affected by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)(30 nM), reactive blue 2 (30 microM) or the GABAA receptor antagonist bicuculline (50 microM). However, the GABAB receptor antagonist 2-hydroxysaclofen (50 microM) significantly inhibited the L-glutamate effect. 5. Similar to L-glutamate, GABA also caused a concentration-dependent (0.1-100 microM) inhibition of the EFS contractions. This GABA-induced inhibition was not affected by either the GABAA receptor antagonist bicuculline (50 microM) or reactive blue 2 (30 microM). However, a significant attenuation of the GABA-mediated effect was recorded with the GABAB receptor antagonist 2-hydroxysaclofen (50 microM). Contractions of the vas deferens induced by exogenous ATP and noradrenaline were not affected by GABA (0.1-100 microM). 6. The L-glutamate analogues, N-methyl-D-aspartate (NMDA) (1 microM-1 mM) and quisqualate (Quis 0.1 microM-0.3 mM) had no effect

  17. GABA(B) receptors mediate motility signals for migrating embryonic cortical cells.

    PubMed

    Behar, T N; Smith, S V; Kennedy, R T; McKenzie, J M; Maric, I; Barker, J L

    2001-08-01

    During development, postmitotic neurons migrate from germinal regions into the cortical plate (cp), where lamination occurs. In rats, GABA is transiently expressed in the cp, near target destinations for migrating neurons. In vitro GABA stimulates neuronal motility, suggesting cp cells release GABA, which acts as a chemoattractant during corticogenesis. Pharmacological studies indicate GABA stimulates migration via GABA(B)-receptor (GABA(B)-R) activation. Using immunohistochemistry, RT-PCR and Western blotting, we examined embryonic cortical cell expression of GABA(B)-Rs in vivo. At E17, GABA(B)-R1(+) cells were identified in the ventricular zone (vz) and cp. RT-PCR and Western blotting demonstrated the presence of GABA(B)-R1a and GABA(B)-R1b mRNA and proteins. Using immuno- cytochemistry, GABA(B)-R expression was examined in vz and cp cell dissociates before and after migration to GABA in an in vitro chemotaxis assay. GABA-induced migration resulted in an increase of GABA(B)-R(+) cells in the migrated population. While <20% of each starting dissociate was GABA(B)-R(+), >70% of migrated cells were immunopositive. We used a microchemotaxis assay to analyze cp cell release of diffusible chemotropic factor(s). In vitro, cp dissociates induced vz cell migration in a cell density-dependent manner that was blocked by micromolar saclofen (a GABA(B)-R antagonist). HPLC demonstrated cp cells release micromolar levels of GABA and taurine in several hours. Micromolar levels of both molecules stimulated cell migration that was blocked by micromolar saclofen. Thus, migratory cortical cells express GABA(B)-Rs, cp cells release GABA and taurine, and both molecules stimulate cortical cell movement. Together these findings suggest GABA and/or taurine act as chemoattractants for neurons during rat cortical histogenesis via mechanisms involving GABA(B)-Rs.

  18. Regulation of GABA-modulin phosphorylation and GABA receptor binding by excitatory amino acids

    SciTech Connect

    Vaccarino, F.; Guidotti, A.

    1987-05-01

    Primary cultures of cerebellar granule cells phosphorylate numerous proteins including GABA-modulin (GM), which is a putative allosteric modulator of GABA receptors. Cell depolarization and treatment with dicarboxylic excitatory amino acids, which activate PI turnover, Ca/sup 2 +/ influx and guanylate cyclase in granule cells increase the phosphorylation of specific proteins. To determine GM phosphorylation by endogenous protein kinases in living granule cell cultures, GM was isolated by immunoprecipitation and reverse-phase HPLC. High K/sup +/, veratridine, glutamate and NMDA treatment stimulated GM phosphorylation over 2-fold. This increase was abolished by the absence of extracellular Ca/sup 2 +/ and was antagonized by Mg/sup 2 +/ ions and by AVP. The excitatory amino acid action was mimicked by phorbol esters but not by forskolin or by cGMP, and thus may be mediated by an activation of protein kinase C (PKC). Moreover, excitatory amino acids increase /sup 3/H-labelled phorbol ester binding sites in granule cell membrane. The same cultures, treated with glutamate or kainate, showed a 50-fold greater efficacy of muscimol for the stimulation of benzodiazepine (BZ) binding. These data-suggest that excitatory amino acid stimulation of neurons triggers PKC translocation and the activated enzyme phosphorylates GM. The extent of GM phosphorylation may regulate the coupling between GABA and BZ binding sites.

  19. GABA[subscript A] Receptor Downregulation in Brains of Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Thuras, Paul D.

    2009-01-01

    Gamma-aminobutyric acid A (GABA[subscript A]) receptors are ligand-gated ion channels responsible for mediation of fast inhibitory action of GABA in the brain. Preliminary reports have demonstrated altered expression of GABA receptors in the brains of subjects with autism suggesting GABA/glutamate system dysregulation. We investigated the…

  20. GABA[subscript A] Receptor Downregulation in Brains of Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Thuras, Paul D.

    2009-01-01

    Gamma-aminobutyric acid A (GABA[subscript A]) receptors are ligand-gated ion channels responsible for mediation of fast inhibitory action of GABA in the brain. Preliminary reports have demonstrated altered expression of GABA receptors in the brains of subjects with autism suggesting GABA/glutamate system dysregulation. We investigated the…

  1. A Gut Feeling about GABA: Focus on GABAB Receptors

    PubMed Central

    Hyland, Niall P.; Cryan, John F.

    2010-01-01

    γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the body and hence GABA-mediated neurotransmission regulates many physiological functions, including those in the gastrointestinal (GI) tract. GABA is located throughout the GI tract and is found in enteric nerves as well as in endocrine-like cells, implicating GABA as both a neurotransmitter and an endocrine mediator influencing GI function. GABA mediates its effects via GABA receptors which are either ionotropic GABAA or metabotropic GABAB. The latter which respond to the agonist baclofen have been least characterized, however accumulating data suggest that they play a key role in GI function in health and disease. Like GABA, GABAB receptors have been detected throughout the gut of several species in the enteric nervous system, muscle, epithelial layers as well as on endocrine-like cells. Such widespread distribution of this metabotropic GABA receptor is consistent with its significant modulatory role over intestinal motility, gastric emptying, gastric acid secretion, transient lower esophageal sphincter relaxation and visceral sensation of painful colonic stimuli. More intriguing findings, the mechanisms underlying which have yet to be determined, suggest GABAB receptors inhibit GI carcinogenesis and tumor growth. Therefore, the diversity of GI functions regulated by GABAB receptors makes it a potentially useful target in the treatment of several GI disorders. In light of the development of novel compounds such as peripherally acting GABAB receptor agonists, positive allosteric modulators of the GABAB receptor and GABA producing enteric bacteria, we review and summarize current knowledge on the function of GABAB receptors within the GI tract. PMID:21833169

  2. An Electrostatic Funnel in the GABA-Binding Pathway

    PubMed Central

    Lightstone, Felice C.

    2016-01-01

    The γ-aminobutyric acid type A receptor (GABAA-R) is a major inhibitory neuroreceptor that is activated by the binding of GABA. The structure of the GABAA-R is well characterized, and many of the binding site residues have been identified. However, most of these residues are obscured behind the C-loop that acts as a cover to the binding site. Thus, the mechanism by which the GABA molecule recognizes the binding site, and the pathway it takes to enter the binding site are both unclear. Through the completion and detailed analysis of 100 short, unbiased, independent molecular dynamics simulations, we have investigated this phenomenon of GABA entering the binding site. In each system, GABA was placed quasi-randomly near the binding site of a GABAA-R homology model, and atomistic simulations were carried out to observe the behavior of the GABA molecules. GABA fully entered the binding site in 19 of the 100 simulations. The pathway taken by these molecules was consistent and non-random; the GABA molecules approach the binding site from below, before passing up behind the C-loop and into the binding site. This binding pathway is driven by long-range electrostatic interactions, whereby the electrostatic field acts as a ‘funnel’ that sweeps the GABA molecules towards the binding site, at which point more specific atomic interactions take over. These findings define a nuanced mechanism whereby the GABAA-R uses the general zwitterionic features of the GABA molecule to identify a potential ligand some 2 nm away from the binding site. PMID:27119953

  3. Mechanisms of carbacholine and GABA action on resting membrane potential and Na+/K+-ATPase of Lumbricus terrestris body wall muscles.

    PubMed

    Volkov, Eugeny M; Nurullin, Leniz F; Volkov, Michael E; Nikolsky, Eugeny E; Vyskočil, Frantisek

    2011-04-01

    This work was aimed to identify the action of several ion channel and pump inhibitors as well as nicotinic, GABAergic, purinergic and serotoninergic drugs on the resting membrane potential (RMP) and assess the role of cholinergic and GABAergic sensitivity in earthworm muscle electrogenesis. The nicotinic agonists acetylcholine (ACh), carbacholine (CCh) and nicotine depolarize the RMP at concentrations of 5 μM and higher. The nicotinic antagonists (+)tubocurarine, α-bungarotoxin, muscarinic antagonists atropine and hexamethonium do not remove or prevent the CCh-induced depolarization. Verapamil, tetrodotoxin, removal of Cl(-) and Ca(2+) from the solution also cannot prevent the depolarization by CCh. In a Na(+)-free medium, however, CCh lost this depolarization ability and this indicates that the drug opens the sodium permeable pathway. Serotonin, glutamate, glycine, adenosine triphosphate (ATP) and cis-4-aminocrotonic acid (GABA(C) receptor antagonist) had no effect on the RMP. On the other hand, isoguvacin, γ-aminobutyric acid (GABA) and baclofen (GABA(B) receptor agonist) hyperpolarized the RMP. Ouabain, bicucullin (GABA(A) antagonist) and phaclofen (GABA(B) antagonist), as well as the removal of Cl(-), suppressed the effect of GABA and baclofen. CCh did not enhance the depolarization generated by ouabain but, on the other hand, hindered the hyperpolarizing activity of baclofen both in the absence and presence of atropine and (+)tubocurarine. The long-term application of CCh depolarizes the RMP primarily by inhibiting the Na(+)/K(+)-ATPase. The muscle membrane also contains A and B type GABA binding sites, the activation of which increases the RMP at the expense of increasing the action of ouabain- and Cl(-) -sensitive electrogenic pumps. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Agonist pharmacology of two Drosophila GABA receptor splice variants.

    PubMed Central

    Hosie, A. M.; Sattelle, D. B.

    1996-01-01

    1. The Drosophila melanogaster gamma-aminobutyric acid (GABA) receptor subunits, RDLac and DRC 17-1-2, form functional homo-oligomeric receptors when heterologously expressed in Xenopus laevis oocytes. The subunits differ in only 17 amino acids, principally in regions of the N-terminal domain which determine agonist pharmacology in vertebrate ionotropic neurotransmitter receptors. A range of conformationally restricted GABA analogues were tested on the two homo-oligomers and their agonists pharmacology compared with that of insect and vertebrate iontropic GABA receptors. 2. The actions of GABA, isoguvacine and isonipecotic acid on RDLac and DRC 17-1-2 homo-oligomers were compared, by use of two-electrode voltage-clamp. All three compounds were full agonists of both receptors, but were 4-6 fold less potent agonists of DRC 17-1-2 homo-oligomers than of RDLac. However, the relative potencies of these agonists on each receptor were very similar. 3. A more complete agonist profile was established for RDLac homo-oligomers. The most potent agonists of these receptors were GABA, muscimol and trans-aminocrotonic acid (TACA), which were approximately equipotent. RDLac homo-oligomers were fully activated by a range of GABA analogues, with the order of potency: GABA > ZAPA ((Z)-3-[(aminoiminomethyl)thio]prop-2-enoic acid) > isoguvacine > imidazole-4-acetic acid > or = isonipecotic acid > or = cis-aminocrotonic acid (CACA) > beta-alanine. 3-Aminopropane sulphonic acid (3-APS), a partial agonist of RDLac homo-oligomers, was the weakest agonist tested and 100 fold less potent than GABA. 4. SR95531, an antagonist of vertebrate GABAA receptors, competitively inhibited the GABA responses of RDLac homo-oligomers, which have previously been found to insensitive to bicuculline. However, its potency (IC50 500 microM) was much reduced when compared to GABAA receptors. 5. The agonist pharmacology of Drosophila RDLac homo-oligomers exhibits aspects of the characteristic pharmacology of

  5. Distribution of GABA-like immunoreactivity in the octopus brain.

    PubMed

    Cornwell, C J; Messenger, J B; Williamson, R

    1993-09-10

    This paper presents the first evidence that some neurons in the octopus CNS contain delta-amino butyric acid (GABA). Using conventional immunohistochemical methods with appropriate controls, we obtained positive staining with an antibody to GABA in fibres in the neuropil of many lobes of the brain of the northern octopus Eledone cirrhosa. In several lobes cell bodies were also stained. Staining was not uniformly distributed in the brain nor within a particular lobe: some regions stained strongly, others not at all. These findings suggest that GABA should be added to the already long list of putative neurotransmitters in the cephalopod CNS.

  6. Dopaminergic neurons modulate GABA neuron migration in the embryonic midbrain

    PubMed Central

    Vasudevan, Anju; Won, Chungkil; Li, Suyan; Erdélyi, Ferenc; Szabó, Gábor; Kim, Kwang-Soo

    2012-01-01

    Neuronal migration, a key event during brain development, remains largely unexplored in the mesencephalon, where dopaminergic (DA) and GABA neurons constitute two major neuronal populations. Here we study the migrational trajectories of DA and GABA neurons and show that they occupy ventral mesencephalic territory in a temporally and spatially specific manner. Our results from the Pitx3-deficient aphakia mouse suggest that pre-existing DA neurons modulate GABA neuronal migration to their final destination, providing novel insights and fresh perspectives concerning neuronal migration and connectivity in the mesencephalon in normal as well as diseased brains. PMID:22872083

  7. Dopaminergic neurons modulate GABA neuron migration in the embryonic midbrain.

    PubMed

    Vasudevan, Anju; Won, Chungkil; Li, Suyan; Erdélyi, Ferenc; Szabó, Gábor; Kim, Kwang-Soo

    2012-09-01

    Neuronal migration, a key event during brain development, remains largely unexplored in the mesencephalon, where dopaminergic (DA) and GABA neurons constitute two major neuronal populations. Here we study the migrational trajectories of DA and GABA neurons and show that they occupy ventral mesencephalic territory in a temporally and spatially specific manner. Our results from the Pitx3-deficient aphakia mouse suggest that pre-existing DA neurons modulate GABA neuronal migration to their final destination, providing novel insights and fresh perspectives concerning neuronal migration and connectivity in the mesencephalon in normal as well as diseased brains.

  8. Trans-synaptic (GABA-dopamine) modulation of cocaine induced dopamine release: A potential therapeutic strategy for cocaine abuse

    SciTech Connect

    Dewey, S.L.; Straughter-Moore, R.; Chen, R.

    1995-05-01

    We recently developed a new experimental strategy for measuring interactions between functionally-linked neurotransmitter systems in the primate and human brain with PET. As part of this research, we demonstrated that increases in endogenous GABA concentrations significantly reduced striatal dopamine concentrations in the primate brain. We report here the application of the neurotransmitter interaction paradigm with PET and with microdialysis to the investigation of a novel therapeutic strategy for treating cocaine abuse based on the ability of GABA to inhibit cocaine induced increases in striatal dopamine. Using gamma-vinyl GABA (GVG, a suicide inhibitor of GABA transaminase), we performed a series of PET studies where animals received a baseline PET scan with labeled raclopride injection, animals received cocaine (2.0 mg/kg). Normally, a cocaine challenge significantly reduces the striatal binding of {sup 11}C-raclopride. However, in animals pretreated with GVG, {sup 11}C-raclopride binding was less affected by a cocaine challenge compared to control studies. Furthermore, microdialysis studies in freely moving rats demonstrate that GVG (300 mg/kg) significantly inhibited cocaine-induced increases in extracellular dopamine release. GVG also attenuated cocaine-induced increases in locomotor activity. However, at a dose of 100 mg/kg, GVG had no effect. Similar findings were obtained with alcohol. Alcohol pretreatment dose dependantly (1-4 g/kg) inhibited cocaine-induced increases in extracellular dopamine concentrations in freely moving rats. Taken together, these studies suggest that therapeutic strategies targeted at increasing central GABA concentrations may be beneficial for the treatment of cocaine abuse.

  9. [Axolemmal transporters for neurotransmitter uptake].

    PubMed

    García-López, M

    Neurotransmission is a fundamental process in interneuronal communication. It starts with the release of the neurotransmitter following a nerve impulse and ends either by uptake by specific specific transporters or by metabolization to an inactive compound. In this review we will consider the molecular, ion dependence and electrogenic properties of the axolemal transporters for neurotransmitters and also the pathological consequences of their impairment as well as the drugs that can interact with them. Most axolemmal transporters have been cloned and grouped into two large families according to their molecular characteristics and electrogenic properties: 1. Those dependent on Na+/Cl- include transporters of GABA, noradrenaline, dopamine, serotonin, choline, proline, betaine, glycine and taurine, and 2. Those dependent on Na+/K+, which include the transporters of glutamate, alanine, serine and cysteine. The clonation of transporters has permitted (and will continue to permit) the correlation of molecular alterations of transporters with different neuro-degenerative disorders (e.g. multiple sclerosis, Parkinson's disease, Alzheimer's disease), with brain lesions (e.g. cerebral ischemia, status epilepticus) and with psychiatric alterations (e.g. schizophrenia, depression). In this respect, chemical synthesis of new selective drugs which interact with the different systems for uptake of neurotransmitters will offer new approaches to the treatment of many disorders of the central nervous system which still have no satisfactory drug treatment.

  10. Saturable binding of /sup 35/S-t-butylbicyclophosphorothionate to the sites linked to the GABA receptor and the interaction with gabaergic agents

    SciTech Connect

    Wong, D.T.; Threlkeld, P.G.; Bymaster, F.P.; Squires, R.F.

    1984-02-27

    /sup 35/-S-t-Butylbicyclophosphorothionate (/sup 35/S-TBPS) binds in a concentration-saturable manner to specific sites on membranes from rat cerebral cortex. Using a filtration assay at 25/sup 0/C, in 250 mM NaCl, specific binding of /sup 35/S-TBPS constitutes about 84 to 94 percent of total binding, depending on radioligand concentrations. /sup 35/S-TBPS binding is optimal in the presence of NaCl or NaBr and substantially less in the presence of NaI or NaF. It is sensitive to the treatment with 0.05 percent Triton X-100 but not to repeated freezing and thawing, procedures which increase /sup 3/H-GABA binding. Pharmacological studies show that /sup 35/S-TBPS binding is strongly inhibited by GABA-A receptor agonists (e.g., GABA and muscimol) and by the noncompetitive antagonist, picrotoxin, but not the competitive antagonist, bicuculline. Compounds which enhance binding of radioactive GABA and benzodiazepines, such as the pyrazolopyridines, cartazolate and trazolate, and a diaryl-triazine, LY81067, are also potent inhibitors of /sup 35/S-TBPS binding, with LY81067 being the most effective. The effects of GABA, picrotoxin

  11. The effects of endomorphins on striatal [3H]GABA release induced by electrical stimulation: an in vitro superfusion study in rats.

    PubMed

    Bagosi, Zsolt; Jászberényi, Miklós; Telegdy, Gyula

    2009-05-01

    The endomorphins (EM1 and EM2) are selective endogenous ligands for mu-opioid receptors (MOR1 and MOR2) with neurotransmitter and neuromodulator roles in mammals. In the present study we investigated the potential actions of EMs on striatal GABA release and the implication of different MORs in these processes. Rat striatal slices were preincubated with tritium-labelled GABA ([(3)H]GABA), pretreated with selective MOR1 and MOR2 antagonist beta-funaltrexamine and selective MOR1 antagonist naloxonazine and then superfused with the selective MOR agonists, EM1 and EM2. EM1 significantly decreased the striatal [(3)H]GABA release induced by electrical stimulation. Beta-funaltrexamine antagonized the inhibitory action of EM1, but naloxonazine did not affect it considerably. EM2 was ineffective, even in case of specific enzyme inhibitor diprotin A pretreatment. The results demonstrate that EM1 decreases GABA release in the basal ganglia through MOR2, while EM2 does not influence it.

  12. Effects of graded oral doses of a new 5-hydroxytryptamine/noradrenaline uptake inhibitor (Ro 15-8081) in comparison with 60 mg codeine and placebo on experimentally induced pain and side effect profile in healthy men.

    PubMed Central

    Stacher, G; Steinringer, H; Schneider, S; Mittelbach, G; Gaupmann, G; Abatzi, T A; Stacher-Janotta, G

    1987-01-01

    1. Ro 15-8081 (Hoffmann-La Roche, Basle, Switzerland) is a novel mixed 5-HT/noradrenaline uptake inhibitor producing potent antinociceptive effects in animal pain models. 2. In healthy man, two models with electrically and thermally induced pain, respectively, have been shown to reliably discriminate between the effects of opioid as well as of antipyretic analgesics and placebo. 3. This study investigated the effects of single oral doses of 10, 25, and 50 mg Ro 15-8081 in comparison with 60 mg codeine and placebo on threshold and tolerance to electrically induced pain and on threshold to thermally induced pain. Furthermore, the effects on psychomotor function, self-rated subjective feelings, and side effect profile were studied. 4. Twenty healthy males participated each in five experiments in which they received, in random double-blind fashion, each of the treatments. Every experiment comprised two series of measurements before and twelve after drug administration, carried out at 30 min intervals. 5. Ro 15-8081 produced marked elevations of threshold and tolerance to electrically and of threshold to thermally induced pain. The effects of all doses of Ro 15-8081 were significantly superior to those of placebo. Threshold and tolerance to electrically induced pain were not affected differently by the three doses of Ro 15-8081, whereas the threshold to thermally induced pain was elevated significantly more by 50 mg than by 10 and 25 mg Ro 15-8081. 6. Codeine 60 mg had a more rapid onset of action and greater maximal effects than Ro 15-8081.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3501728

  13. GABA sub A (gamma-aminobutyric acid) type binding sites on membranes of spermatozoa

    SciTech Connect

    Erdoe, S.L. ); Wekerle, L. )

    1990-01-01

    The binding of ({sup 3}H) gamma-aminobutyric acid (GABA) to seminal membranes of swines and rams was examined. Specific, GABA binding was demonstrated in both species, which showed the features of GABA{sub A} type receptors. The affinity of binding was similar in both species, whereas the density of seminal GABA binding sites was 5 times higher in swine. Our findings suggest that GABA may have a direct effect on spermatozoa.

  14. Bioassay-guided fractionation of lemon balm (Melissa officinalis L.) using an in vitro measure of GABA transaminase activity.

    PubMed

    Awad, Rosalie; Muhammad, Asim; Durst, Tony; Trudeau, Vance L; Arnason, John T

    2009-08-01

    A novel pharmacological mechanism of action for the anxiolytic botanical Melissa officinalis L. (lemon balm) is reported. The methanol extract was identified as a potent in vitro inhibitor of rat brain GABA transaminase (GABA-T), an enzyme target in the therapy of anxiety, epilepsy and related neurological disorders. Bioassay-guided fractionation led to the identification and isolation of rosmarinic acid (RA) and the triterpenoids, ursolic acid (UA) and oleanolic acid (OA) as active principles. Phytochemical characterization of the crude extract determined RA as the major compound responsible for activity (40% inhibition at 100 microg/mL) since it represented approximately 1.5% of the dry mass of the leaves. Synergistic effects may also play a role.

  15. GABA transporter 1 tunes GABAergic synaptic transmission at output neurons of the mouse neostriatum

    PubMed Central

    Kirmse, Knut; Dvorzhak, Anton; Kirischuk, Sergei; Grantyn, Rosemarie

    2008-01-01

    GABAergic medium-sized striatal output neurons (SONs) provide the principal output for the neostriatum. In vitro and in vivo data indicate that spike discharge of SONs is tightly controlled by effective synaptic inhibition. Although phasic GABAergic transmission critically depends on ambient GABA levels, the role of GABA transporters (GATs) in neostriatal GABAergic synaptic transmission is largely unknown. In the present study we aimed at elucidating the role of GAT-1 in the developing mouse neostriatum (postnatal day (P) 7–34). We recorded GABAergic postsynaptic currents (PSCs) using the whole-cell patch-clamp technique. Based on the effects of NO-711, a specific GAT-1 blocker, we demonstrate that GAT-1 is operative at this age and influences GABAergic synaptic transmission by presynaptic and postsynaptic mechanisms. Presynaptic GABABR-mediated suppression of GABA release was found to be functional at all ages tested; however, there was no evidence for persistent GABABR activity under control conditions, unless GAT-1 was blocked (P12–34). In addition, whereas no tonic GABAAR-mediated conductances were detected in SONs until P14, application of a specific GABAAR antagonist caused distinct tonic outward currents later in development (P19–34). In the presence of NO-711, tonic GABAAR-mediated currents were also observed at P7–14 and were dramatically increased at more mature stages. Furthermore, GAT-1 block reduced the median amplitude of GABAergic miniature PSCs indicating a decrease in quantal size. We conclude that in the murine neostriatum GAT-1 operates in a net uptake mode. It prevents the persistent activation of presynaptic GABABRs (P12–34) and prevents (P7–14) or reduces (P19–34) tonic postsynaptic GABAAR activity. PMID:18832421

  16. The betaine/GABA transporter and betaine: roles in brain, kidney, and liver

    PubMed Central

    Kempson, Stephen A.; Zhou, Yun; Danbolt, Niels C.

    2014-01-01

    The physiological roles of the betaine/GABA transporter (BGT1; slc6a12) are still being debated. BGT1 is a member of the solute carrier family 6 (the neurotransmitter, sodium symporter transporter family) and mediates cellular uptake of betaine and GABA in a sodium- and chloride-dependent process. Most of the studies of BGT1 concern its function and regulation in the kidney medulla where its role is best understood. The conditions here are hostile due to hyperosmolarity and significant concentrations of NH4Cl and urea. To withstand the hyperosmolarity, cells trigger osmotic adaptation, involving concentration of a transcriptional factor TonEBP/NFAT5 in the nucleus, and accumulate betaine and other osmolytes. Data from renal cells in culture, primarily MDCK, revealed that transcriptional regulation of BGT1 by TonEBP/NFAT5 is relatively slow. To allow more acute control of the abundance of BGT1 protein in the plasma membrane, there is also post-translation regulation of BGT1 protein trafficking which is dependent on intracellular calcium and ATP. Further, betaine may be important in liver metabolism as a methyl donor. In fact, in the mouse the liver is the organ with the highest content of BGT1. Hepatocytes express high levels of both BGT1 and the only enzyme that can metabolize betaine, namely betaine:homocysteine –S-methyltransferase (BHMT1). The BHMT1 enzyme removes a methyl group from betaine and transfers it to homocysteine, a potential risk factor for cardiovascular disease. Finally, BGT1 has been proposed to play a role in controlling brain excitability and thereby represents a target for anticonvulsive drug development. The latter hypothesis is controversial due to very low expression levels of BGT1 relative to other GABA transporters in brain, and also the primary location of BGT1 at the surface of the brain in the leptomeninges. These issues are discussed in detail. PMID:24795654

  17. How and why does tomato accumulate a large amount of GABA in the fruit?

    PubMed Central

    Takayama, Mariko; Ezura, Hiroshi

    2015-01-01

    Gamma-aminobutyric acid (GABA) has received much attention as a health-promoting functional compound, and several GABA-enriched foods have been commercialized. In higher plants, GABA is primarily metabolized via a short pathway called the GABA shunt. The GABA shunt bypasses two steps (the oxidation of α-ketoglutarate to succinate) of the tricarboxylic acid (TCA) cycle via reactions catalyzed by three enzymes: glutamate decarboxylase, GABA transaminase, and succinic semialdehyde dehydrogenase. The GABA shunt plays a major role in primary carbon and nitrogen metabolism and is an integral part of the TCA cycle under stress and non-stress conditions. Tomato is one of the major crops that accumulate a relatively high level of GABA in its fruits. The GABA levels in tomato fruits dramatically change during fruit development; the GABA levels increase from flowering to the mature green stage and then rapidly decrease during the ripening stage. Although GABA constitutes up to 50% of the free amino acids at the mature green stage, the molecular mechanism of GABA accumulation and the physiological function of GABA during tomato fruit development remain unclear. In this review, we summarize recent studies of GABA accumulation in tomato fruits and discuss the potential biological roles of GABA in tomato fruit development. PMID:26322056

  18. [GABA-Receptors in Modulation of Fear Memory Extinction].

    PubMed

    Dubrovina, N I

    2016-01-01

    GABA is the major inhibitory neurotransmitter in the central nervous system determining the efficacy of neuronal interaction. GABA-receptors play a key role in different aspects of fear memory--acquisition and consolidation, retention, reconsolidation and extinction. Extinction is an important behavioural phenomenon which allows organism to adapt its behavior to a changing environment. Extinction of fear memory is a form of new inhibitory learning which interferes with expression of the initial acquired fear conditioning. Resistance to extinction is symptom of depression and posttraumatic stress disorder. The aim of the present review was to summarize own and literary data about GABAergic modulation of fear extinction and pharmacological correction of extinction impairment at influences on GABA(A)- and GABA(B)- receptors.

  19. GABA Metabolism and Transport: Effects on Synaptic Efficacy

    PubMed Central

    Roth, Fabian C.; Draguhn, Andreas

    2012-01-01

    GABAergic inhibition is an important regulator of excitability in neuronal networks. In addition, inhibitory synaptic signals contribute crucially to the organization of spatiotemporal patterns of network activity, especially during coherent oscillations. In order to maintain stable network states, the release of GABA by interneurons must be plastic in timing and amount. This homeostatic regulation is achieved by several pre- and postsynaptic mechanisms and is triggered by various activity-dependent local signals such as excitatory input or ambient levels of neurotransmitters. Here, we review findings on the availability of GABA for release at presynaptic terminals of interneurons. Presynaptic GABA content seems to be an important determinant of inhibitory efficacy and can be differentially regulated by changing synthesis, transport, and degradation of GABA or related molecules. We will discuss the functional impact of such regulations on neuronal network patterns and, finally, point towards pharmacological approaches targeting these processes. PMID:22530158

  20. Dual role of GABA in the neonatal rat hippocampus.

    PubMed

    Khalilov, I; Dzhala, V; Ben-Ari, Y; Khazipov, R

    1999-11-01

    The effects of modulators of GABA-A receptors on neuronal network activity were studied in the neonatal (postnatal days 0-5) rat hippocampus in vitro. Under control conditions, the physiological pattern of activity of the neonatal hippocampal network was characterized by spontaneous network-driven giant depolarizing potentials (GDPs). The GABA-A receptor agonist isoguvacine (1-2 microM) and the allosteric modulator diazepam (2 microM) induced biphasic responses: initially the frequency of GDPs increased 3 to 4 fold followed by blockade of GDPs and desynchronization of the network activity. The GABA-A receptor antagonists bicuculline (10 microM) and picrotoxin (100 microM) blocked GDPs and induced glutamate (AMPA and NMDA)-receptor-mediated interictal- and ictal-like activities in the hippocampal slices and the intact hippocampus. These data suggest that at early postnatal ages GABA can exert a dual - both excitatory and inhibitory - action on the network activity.

  1. GABA-Activated Chloride Channels in Secretory Nerve Endings

    NASA Astrophysics Data System (ADS)

    Zhang, Shuanglin J.; Jackson, Meyer B.

    1993-01-01

    Neurotransmitters acting on presynaptic terminals regulate synaptic transmission and plasticity. Because of the difficulty of direct electrophysiological recording from small presynaptic terminals, little is known about the ion channels that mediate these actions or about the mechanisms by which transmitter secretion is altered. The patch-clamp technique is used to show that the predominant inhibitory presynaptic neurotransmitter, γ-aminobutyric acid (GABA), activates a GABA_A receptor and gates a chloride channel in the membranes of peptidergic nerve terminals of the posterior pituitary. The opening of a chloride channel by GABA weakly depolarizes the nerve terminal membrane and blocks action potentials. In this way, GABA limits secretion by retarding the spread of excitation into the terminal arborization.

  2. Endogenous synthesis of taurine and GABA in rat ocular tissues.

    PubMed

    Heinämäki, A A

    1988-01-01

    The endogenous production of taurine and gamma-aminobutyric acid (GABA) in rat ocular tissues was investigated. The activities of taurine-producing enzyme, cysteine sulfinic acid decarboxylase (CSAD), and GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), were observed in the retina, lens, iris-ciliary body and cornea. The highest specific activity of CSAD was in the cornea and that of GAD in the retina. The discrepancy between CSAD activity and taurine content within the ocular tissues indicates that intra- or extraocular transport processes may regulate the concentration of taurine in the rat eye. The GAD activity and the content of GABA were distributed in parallel within the rat ocular tissues. The quantitative results suggest that the GAD/GABA system has functional significance only in the retina of the rat eye.

  3. GABA level, gamma oscillation, and working memory performance in schizophrenia

    PubMed Central

    Chen, Chi-Ming A.; Stanford, Arielle D.; Mao, Xiangling; Abi-Dargham, Anissa; Shungu, Dikoma C.; Lisanby, Sarah H.; Schroeder, Charles E.; Kegeles, Lawrence S.

    2014-01-01

    A relationship between working memory impairment, disordered neuronal oscillations, and abnormal prefrontal GABA function has been hypothesized in schizophrenia; however, in vivo GABA measurements and gamma band neural synchrony have not yet been compared in schizophrenia. This case–control pilot study (N = 24) compared baseline and working memory task-induced neuronal oscillations acquired with high-density electroencephalograms (EEGs) to GABA levels measured in vivo with magnetic resonance spectroscopy. Working memory performance, baseline GABA level in the left dorsolateral prefrontal cortex (DLPFC), and measures of gamma oscillations from EEGs at baseline and during a working memory task were obtained. A major limitation of this study is a relatively small sample size for several analyses due to the integration of diverse methodologies and participant compliance. Working memory performance was significantly lower for patients than for controls. During the working memory task, patients (n = 7) had significantly lower amplitudes in gamma oscillations than controls (n = 9). However, both at rest and across working memory stages, there were significant correlations between gamma oscillation amplitude and left DLPFC GABA level. Peak gamma frequency during the encoding stage of the working memory task (n = 16) significantly correlated with GABA level and working memory performance. Despite gamma band amplitude deficits in patients across working memory stages, both baseline and working memory-induced gamma oscillations showed strong dependence on baseline GABA levels in patients and controls. These findings suggest a critical role for GABA function in gamma band oscillations, even under conditions of system and cognitive impairments as seen in schizophrenia. PMID:24749063

  4. GABA Signaling and Neuroactive Steroids in Adrenal Medullary Chromaffin Cells

    PubMed Central

    Harada, Keita; Matsuoka, Hidetada; Fujihara, Hiroaki; Ueta, Yoichi; Yanagawa, Yuchio; Inoue, Masumi

    2016-01-01

    Gamma-aminobutyric acid (GABA) is produced not only in the brain, but also in endocrine cells by the two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67. In rat adrenal medullary chromaffin cells only GAD67 is expressed, and GABA is stored in large dense core vesicles (LDCVs), but not synaptic-like microvesicles (SLMVs). The α3β2/3γ2 complex represents the majority of GABAA receptors expressed in rat and guinea pig chromaffin cells, whereas PC12 cells, an immortalized rat chromaffin cell line, express the α1 subunit as well as the α3. The expression of α3, but not α1, in PC12 cells is enhanced by glucocorticoid activity, which may be mediated by both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). GABA has two actions mediated by GABAA receptors in chromaffin cells: it induces catecholamine secretion by itself and produces an inhibition of synaptically evoked secretion by a shunt effect. Allopregnanolone, a neuroactive steroid which is secreted from the adrenal cortex, produces a marked facilitation of GABAA receptor channel activity. Since there are no GABAergic nerve fibers in the adrenal medulla, GABA may function as a para/autocrine factor in the chromaffin cells. This function of GABA may be facilitated by expression of the immature isoforms of GAD and GABAA receptors and the lack of expression of plasma membrane GABA transporters (GATs). In this review, we will consider how the para/autocrine function of GABA is achieved, focusing on the structural and molecular mechanisms for GABA signaling. PMID:27147972

  5. Benzodiazepines do not potentiate GABA responses in neonatal hippocampal neurons.

    PubMed

    Rovira, C; Ben-Ari, Y

    1991-09-16

    Benzodiazepines (midazolam; flunitrazepam) and pentobarbital increase the response to exogenous gamma-aminobutyric acid (GABA) in adult hippocampal cells. We report in this paper that in contrast pentobarbital but not benzodiazepine potentiate the effects of exogenous (GABA) in neurons recorded from slices of less than two weeks old. This finding suggests that the functional association of benzodiazepine and GABAA receptors is changed during early postnatal life.

  6. Parkinson's Disease and Neurodegeneration: GABA-Collapse Hypothesis

    PubMed Central

    Błaszczyk, Janusz W.

    2016-01-01

    Neurodegenerative diseases constitute a heterogeneous group of age-related disorders that are characterized by a slow but irreversible deterioration of brain functions. Evidence accumulated over more than two decades has implicated calcium-related homeostatic mechanisms, giving rise to the Ca2+ hypothesis of brain aging and, ultimately, cell death. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter within the central (CNS), peripheral and enteric nervous systems. It appears to be involved in a wide variety of physiological functions within and outside the nervous system, that are maintained through a complex interaction between GABA and calcium-dependent neurotransmission and cellular metabolic functions. Within CNS the Ca2+/GABA mechanism stabilizes neuronal activity both at cellular and systemic levels. Decline in the Ca2+/GABA control initiates several cascading processes leading to both weakened protective barriers (in particular the blood-brain barrier) and accumulations of intracellular deposits of calcium and Lewy bodies. Linking such a vital mechanism of synaptic transmission with metabolism (both at cellular and tissue level) by means of a common reciprocal Ca2+/GABA inhibition results in a fragile balance, which is prone to destabilization and auto-destruction. The GABA decline etiology proposed here appears to apply to all human neurodegenerative processes initiated by abnormal intracellular calcium levels. Therefore, the original description of Parkinson's disease (PD) as due to the selective damage of dopaminergic neurons in the mesencephalon should be updated into the concept of a severe multisystemic neurodegenerative disorder of the nervous system, whose clinical symptoms reflect the localization and progression of the most advanced GABA pathology. A future and more complete therapeutic approach to PD should be aimed first at slowing (or stopping) the progression of Ca2+/GABA functional decline. PMID:27375426

  7. Modulation of horizontal cell function by GABA(A) and GABA(C) receptors in dark- and light-adapted tiger salamander retina.

    PubMed

    Yang, X L; Gao, F; Wu, S M

    1999-01-01

    The physiological function of GABA transporters and GABA receptors in retinal horizontal cells (HCs) under dark-and light-adapted conditions were studied by whole-cell voltage clamp and intracellular recording techniques in retinal slices and whole-mounted isolated retinas of the larval tiger salamander. Puff application of GABA in picrotoxin elicited a NO-711 (a potent GABA transporter blocker)-sensitive inward current that did not exhibit a reversal potential in the physiological range, consistent with the idea that these HCs contain electrogenic GABA transporters. Application of GABA in NO-711 elicited a chloride current in HCs; about half of the current was suppressed by bicuculline or I4AA (a GABA(C) receptor antagonist), and the remaining half was suppressed by bicuculline + I4AA or picrotoxin. In whole-mount retinas, NO-711, bicuculline, I4AA, or picrotoxin hyperpolarized the HCs and enhanced the light responses under dark-adapted conditions, and blocked the time-dependent recovery of HC membrane potential and light responses during background illumination. Based on the parallel conductance model, GABA released in darkness mediates a chloride conductance about three times greater than the leak conductance or the glutamate-gated cation conductance. About half of this chloride conductance is mediated by GABA(A) receptors, and the other half is mediated by GABA