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Sample records for acid gaba transporter

  1. 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

  2. Efficient production of gamma-aminobutyric acid using Escherichia coli by co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter.

    PubMed

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

    2016-01-01

    Gamma-aminobutyric acid (GABA) is an important bio-product, which is used in pharmaceutical formulations, nutritional supplements, and biopolymer monomer. The traditional GABA process involves the decarboxylation of glutamate. However, the direct production of GABA from glucose is a more efficient process. To construct the recombinant strains of Escherichia coli, a novel synthetic scaffold was introduced. By carrying out the co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter, we redirected the TCA cycle flux to GABA pathway. The genetically engineered E. coli strain produced 1.08 g/L of GABA from 10 g/L of initial glucose. Thus, with the introduction of a synthetic scaffold, we increased GABA production by 2.2-fold. The final GABA concentration was increased by 21.8% by inactivating competing pathways. PMID:26620318

  3. Expression of the γ-Aminobutyric Acid (GABA) Plasma Membrane Transporter-1 in Monkey and Human Retina

    PubMed Central

    Casini, Giovanni; Rickman, Dennis W.; Brecha, Nicholas C.

    2010-01-01

    Purpose To determine the expression pattern of the predominant γ-aminobutyric acid (GABA) plasma membrane transporter GAT-1 in Old World monkey (Macaca mulatta) and human retina. Methods GAT-1 was localized in retinal sections by using immunohistochemical techniques with fluorescence and confocal microscopy. Double-labeling studies were performed with the GAT-1 antibody using antibodies to GABA, vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH), and the bipolar cell marker Mab115A10. Results The pattern of GAT-1 immunostaining was similar in human and monkey retinas. Numerous small immunoreactive somata were in the inner nuclear layer (INL) and were present rarely in the inner plexiform layer (IPL) of all retinal regions. Medium GAT-1 somata were in the ganglion cell layer in the parafoveal and peripheral retinal regions. GAT-1 fibers were densely distributed throughout the IPL. Varicose processes, originating from both the IPL and somata in the INL, arborized in the outer plexiform layer (OPL), forming a sparse network in all retinal regions, except the fovea. Sparsely occurring GAT-1 processes were in the nerve fiber layer in parafoveal regions and near the optic nerve head but not in the optic nerve. In the INL, 99% of the GAT-1 somata contained GABA, and 66% of the GABA immunoreactive somata expressed GAT-1. GAT-1 immunoreactivity was in all VIP-containing cells, but it was absent in TH-immunoreactive amacrine cells and in Mab115A10 immunoreactive bipolar cells. Conclusions GAT-1 in primate retinas is expressed by amacrine and displaced amacrine cells. The predominant expression of GAT-1 in the inner retina is consistent with the idea that GABA transporters influence neurotransmission and thus participate in visual information processing in the retina. PMID:16565409

  4. γ-Amino-butyric acid (GABA) receptor subunit and transporter expression in the gonad and liver of the fathead minnow (Pimephales promelas).

    PubMed

    Biggs, Katie; Seidel, Jason S; Wilson, Alex; Martyniuk, Christopher J

    2013-09-01

    γ-Amino-butyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate central nervous system. GABA receptors and synthesizing enzymes have also been localized to peripheral tissues including the liver, oviduct, uterus and ovary of mammals but the distribution and role of GABA in peripheral tissues of fish has not been fully investigated. The objectives of this study were to (1) determine if mRNA encoding GABA synthesizing enzymes (glutamic acid decarboxylase 65 and 67; gad65 and gad67), GABA transporters, and GABAA receptor subunits are localized to liver and gonad of fathead minnow (Pimephales promelas) (FHM) (2) investigate the effects of GABA on ovarian 17β-estradiol (E2) production, and (3) measure transcript responses in the ovary after in vitro incubation to GABA. Real-time PCR assays were developed for gad65, gad67, vesicular GABA transporter (vgat) and GABA transporter 1 (gat1), and select GABAA receptor subunits (gabra1, gabra5, gabrb1, gabrb2, gabrg1, gabrg2). All transcripts were localized to the brain as expected; however transcripts were also detected in the liver, ovary, and testis of FHMs. In the female liver, gad65 mRNA was significantly higher in expression compared to the male liver. Transcripts for gad67 were the highest in the brain>gonad>liver and in the gonads, gad67 was significantly higher in expression than gad65 mRNA. In the liver and gonad, the relative abundance of the subunits followed a general trend of gabrb1>gabrb2=gabrg1=gabrg2>gabra1=gabra5. To explore the effects of GABA in the ovary, tissue explants from reproductive female FHMs were treated with GABA (10(-10), 10(-8) and 10(-6)M) for 12h. GABA had no significant effect on 17β-estradiol production or on mRNA abundance for genes involved in ovarian steroidogenesis (e.g., 11βhsd, cyp17, cyp19a). There was a significant decrease in estrogen receptor 2a (esr2a) mRNA with 10(-10)M GABA. This study begins to investigate the GABA system in non-neural tissues of

  5. 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

  6. Role for pro-inflammatory cytokines in regulating expression of GABA transporter type 1 and 3 in specific brain regions of kainic acid-induced status epilepticus.

    PubMed

    Su, Jing; Yin, Jian; Qin, Wei; Sha, Suxu; Xu, Jun; Jiang, Changbin

    2015-03-01

    In general, pro-inflammatory cytokines (PICs) contribute to regulation of epilepsy-associated pathophysiological processes in the central nerve system. In this report, we examined the specific activation of PICs, namely IL-1β, IL-6 and TNF-α in rat brain after kainic acid (KA)-induced status epilepticus (SE). Also, we examined the role played by PICs in regulating expression of GABA transporter type 1 and 3 (GAT-1 and GAT-3, respectively), which are the two important subtypes of GATs responsible for the regulation of extracellular GABA levels in the brain. Our results show that IL-1β, IL-6 and TNF-α were significantly increased in the parietal cortex, hippocampus and amygdala of KA-rats as compared with sham control animals (P < 0.05, KA rats vs. control rats). KA-induced SE also significantly increased (P < 0.05 vs. controls) the protein expression of GAT-1 and GAT-3 in those brain regions. In addition, central administration of antagonists to IL-1β and TNF-α receptors significantly attenuated amplified GAT-1 and GAT-3 (P < 0.05 vs. vehicle control for each antagonist group). However, antagonist to IL-6 receptor failed to attenuate enhancement in expression of GAT-1 and GAT-3 induced by KA-induced SE. Overall, our data demonstrate that PIC pathways are activated in the specific brain regions during SE which thereby selectively leads to upregulation of GABA transporters. As a result, it is likely that de-inhibition of GABA system is increased in the brain. This support a role for PICs in engagement of the adaptive mechanisms associated with epileptic activity, and has pharmacological implications to target specific PICs for neuronal dysfunction and vulnerability related to epilepsy. PMID:25708016

  7. 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. PMID:26838342

  8. Library screening by means of mass spectrometry (MS) binding assays-exemplarily demonstrated for a pseudostatic library addressing γ-aminobutyric acid (GABA) transporter 1 (GAT1).

    PubMed

    Sindelar, Miriam; Wanner, Klaus T

    2012-09-01

    In the present study, the application of mass spectrometry (MS) binding assays as a tool for library screening is reported. For library generation, dynamic combinatorial chemistry (DCC) was used. These libraries can be screened by means of MS binding assays when appropriate measures are taken to render the libraries pseudostatic. That way, the efficiency of MS binding assays to determine ligand binding in compound screening with the ease of library generation by DCC is combined. The feasibility of this approach is shown for γ-aminobutyric acid (GABA) transporter 1 (GAT1) as a target, representing the most important subtype of the GABA transporters. For the screening, hydrazone libraries were employed that were generated in the presence of the target by reacting various sets of aldehydes with a hydrazine derivative that is delineated from piperidine-3-carboxylic acid (nipecotic acid), a common fragment of known GAT1 inhibitors. To ensure that the library generated is pseudostatic, a large excess of the nipecotic acid derivative is employed. As the library is generated in a buffer system suitable for binding and the target is already present, the mixtures can be directly analyzed by MS binding assays-the process of library generation and screening thus becoming simple to perform. The binding affinities of the hits identified by deconvolution were confirmed in conventional competitive MS binding assays performed with single compounds obtained by separate synthesis. In this way, two nipecotic acid derivatives exhibiting a biaryl moiety, 1-{2-[2'-(1,1'-biphenyl-2-ylmethylidene)hydrazine]ethyl}piperidine-3-carboxylic acid and 1-(2-{2'-[1-(2-thiophenylphenyl)methylidene]hydrazine}ethyl)piperidine-3-carboxylic acid, were found to be potent GAT1 ligands exhibiting pK(i) values of 6.186 ± 0.028 and 6.229 ± 0.039, respectively. This method enables screening of libraries, whether generated by conventional chemistry or DCC, and is applicable to all kinds of targets including

  9. 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. PMID:24031948

  10. 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

  11. A tonoplast Glu/Asp/GABA exchanger that affects tomato fruit amino acid composition.

    PubMed

    Snowden, Christopher J; Thomas, Benjamin; Baxter, Charles J; Smith, J Andrew C; Sweetlove, Lee J

    2015-03-01

    Vacuolar accumulation of acidic metabolites is an important aspect of tomato fruit flavour and nutritional quality. The amino acids Asp and Glu accumulate to high concentrations during ripening, while γ-aminobutyrate (GABA) shows an approximately stoichiometric decline. Given that GABA can be catabolised to form Glu and subsequently Asp, and the requirement for the fruit to maintain osmotic homeostasis during ripening, we hypothesised the existence of a tonoplast transporter that exports GABA from the vacuole in exchange for import of either Asp or Glu. We show here that the tomato vacuolar membrane possesses such a transport property: transport of Glu across isolated tonoplast vesicle membranes was trans-stimulated in counterexchange mode by GABA, Glu and Asp. We identified SlCAT9 as a candidate protein for this exchanger using quantitative proteomics of a tonoplast-enriched membrane fraction. Transient expression of a SlCAT9-YFP fusion in tobacco confirmed a tonoplast localisation. The function of the protein was examined by overexpression of SlCAT9 in transgenic tomato plants. Tonoplast vesicles isolated from transgenic plants showed higher rates of Glu and GABA transport than wild-type (WT) only when assayed in counterexchange mode with Glu, Asp, or GABA. Moreover, there were substantial increases in the content of all three cognate amino acids in ripe fruit from the transgenic plants. We conclude that SlCAT9 is a tonoplast Glu/Asp/GABA exchanger that strongly influences the accumulation of these amino acids during fruit development. PMID:25602029

  12. GABA transport and calcium dynamics in horizontal cells from the skate retina.

    PubMed Central

    Haugh-Scheidt, L; Malchow, R P; Ripps, H

    1995-01-01

    1. Changes in intracellular calcium concentration [Ca2+]i in response to extracellularly applied gamma-aminobutyric acid (GABA) were studied in isolated horizontal cells from the all-rod skate retina. 2. Calcium measurements were made using fura-2 AM, both with and without whole-cell voltage clamp. Superfusion with GABA, in the absence of voltage clamp, resulted in an increase in [Ca2+]i; the threshold for detection was approximately 50 microM GABA, and a maximal response was elicited by 500 microM GABA. 3. The rise in [Ca2+]i was not mimicked by baclofen nor was it blocked by phaclofen, picrotoxin or bicuculline. However, the GABA-induced [Ca2+]i increase was completely abolished when extracellular sodium was replaced with N-methyl-D-glucamine. 4. With the horizontal cell voltage clamped at -70 mV, GABA evoked a large inward current, but there was no concomitant change in [Ca2+]i. Nifedipine, which blocks L-type voltage-gated Ca2+ channels, suppressed the GABA-induced increase in [Ca2+]i. These findings suggest that the calcium response was initiated by GABA activation of sodium dependent electrogenic transport, and that the resultant depolarization led to the opening of voltage-gated Ca2+ channels, and a rise in [Ca2+]i. 5. The GABA-induced influx of calcium appears not to have been the sole source of the calcium increase. The GABA-induced rise in [Ca2+]i was reduced by dantrolene, indicating that internal Ca2+ stores contributed to the GABA-mediated Ca2+ response. 6. These observations demonstrate that activation of the GABA transporter induces changes in [Ca2+]i which may have important implications for the functional properties of horizontal cells. PMID:8576848

  13. GABA transporters control GABAergic neurotransmission in the mouse subplate.

    PubMed

    Unichenko, P; Kirischuk, S; Luhmann, H J

    2015-09-24

    The subplate is a transient layer between the cortical plate and intermediate zone in the developing cortex. Thalamo-cortical axons form temporary synapses on subplate neurons (SPns) before invading the cortical plate. Neuronal activity within the subplate is of critical importance for the development of neocortical circuits and architecture. Although both glutamatergic and GABAergic inputs on SPns were reported, short-term plasticity of GABAergic transmission has not been investigated yet. GABAergic postsynaptic currents (GPSCs) were recorded from SPns in coronal neocortical slices prepared from postnatal day 3-4 mice using whole-cell patch-clamp technique. Evoked GPSCs (eGPSCs) elicited by electrical paired-pulse stimulation demonstrated paired-pulse depression at all interstimulus intervals tested. Baclofen, a specific GABAB receptor (GABABR) agonist, reduced eGPSC amplitudes and increased paired-pulse ratio (PPR), suggesting presynaptic location of functional GABABRs. Baclofen-induced effects were alleviated by (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid (CGP55845), a selective GABABR blocker. Moreover, CGP55845 increased eGPSC amplitudes and decreased PPR even under control conditions, indicating that GABABRs are tonically activated by ambient GABA. Because extracellular GABA concentration is mainly regulated by GABA transporters (GATs), we asked whether GATs release GABA. 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid (NNC-711) (10μM), a selective GAT-1 blocker, increased eGPSC decay time, decreased eGPSC amplitudes and PPR. The two last effects but not the first one were blocked by CGP55845, indicating that GAT-1 blockade causes an elevation of extracellular GABA concentration and in turn activation of extrasynaptic GABAARs and presynaptic GABABRs. 1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3-piperidinecarboxylic acid (SNAP-5114), a specific GAT-2/3 blocker, failed

  14. GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transporters

    PubMed Central

    Ramesh, Sunita A.; Tyerman, Stephen D.; Xu, Bo; Bose, Jayakumar; Kaur, Satwinder; Conn, Vanessa; Domingos, Patricia; Ullah, Sana; Wege, Stefanie; Shabala, Sergey; Feijó, José A.; Ryan, Peter R.; Gillham, Matthew

    2015-01-01

    The non-protein amino acid, gamma-aminobutyric acid (GABA) rapidly accumulates in plant tissues in response to biotic and abiotic stress, and regulates plant growth. Until now it was not known whether GABA exerts its effects in plants through the regulation of carbon metabolism or via an unidentified signalling pathway. Here, we demonstrate that anion flux through plant aluminium-activated malate transporter (ALMT) proteins is activated by anions and negatively regulated by GABA. Site-directed mutagenesis of selected amino acids within ALMT proteins abolishes GABA efficacy but does not alter other transport properties. GABA modulation of ALMT activity results in altered root growth and altered root tolerance to alkaline pH, acid pH and aluminium ions. We propose that GABA exerts its multiple physiological effects in plants via ALMT, including the regulation of pollen tube and root growth, and that GABA can finally be considered a legitimate signalling molecule in both the plant and animal kingdoms. PMID:26219411

  15. 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

  16. Allosteric modulation of retinal GABA receptors by ascorbic acid

    PubMed Central

    Calero, Cecilia I.; Vickers, Evan; Moraga Cid, Gustavo; Aguayo, Luis G.; von Gersdorff, Henrique; Calvo, Daniel J.

    2011-01-01

    Summary Ionotropic γ-aminobutyric acid receptors (GABAA and GABAC) belong to the cys-loop receptor family of ligand-gated ion channels. GABAC receptors are highly expressed in the retina, mainly localized at the axon terminals of bipolar cells. Ascorbic acid, an endogenous redox agent, modulates the function of diverse proteins, and basal levels of ascorbic acid in the retina are very high. However, the effect of ascorbic acid on retinal GABA receptors has not been studied. Here we show that the function of GABAC and GABAA receptors is regulated by ascorbic acid. Patch-clamp recordings from bipolar cell terminals in goldfish retinal slices revealed that GABAC receptor-mediated currents activated by tonic background levels of extracellular GABA, and GABAC currents elicited by local GABA puffs, are both significantly enhanced by ascorbic acid. In addition, a significant rundown of GABA-puff evoked currents was observed in the absence of ascorbic acid. GABA-evoked Cl- currents mediated by homomeric ρ1 GABAC receptors expressed in Xenopus laevis oocytes were also potentiated by ascorbic acid in a concentration-dependent, stereospecific, reversible, and voltage-independent manner. Studies involving the chemical modification of sulfhydryl groups showed that the two cys-loop cysteines and histidine 141, all located in the ρ1 subunit extracellular domain, each play a key role in the modulation of GABAC receptors by ascorbic acid. Additionally, we show that retinal GABAA IPSCs and heterologously expressed GABAA receptor currents are similarly augmented by ascorbic acid. Our results suggest that ascorbic acid may act as an endogenous agent capable of potentiating GABAergic neurotransmission in the CNS. PMID:21715633

  17. 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.

  18. The transporter GAT1 plays an important role in GABA-mediated carbon-nitrogen interactions in Arabidopsis

    PubMed Central

    Batushansky, Albert; Kirma, Menny; Grillich, Nicole; Pham, Phuong A.; Rentsch, Doris; Galili, Gad; Fernie, Alisdair R.; Fait, Aaron

    2015-01-01

    Glutamate derived γ-aminobutyric acid (GABA) is synthetized in the cytosol prior to delivery to the mitochondria where it is catabolized via the TCA cycle. GABA accumulates under various environmental conditions, but an increasing number of studies show its involvement at the crossroad between C and N metabolism. To assess the role of GABA in modulating cellular metabolism, we exposed seedlings of A. thaliana GABA transporter gat1 mutant to full nutrition medium and media deficient in C and N combined with feeding of different concentrations (0.5 and 1 mM) of exogenous GABA. GC-MS based metabolite profiling showed an expected effect of medium composition on the seedlings metabolism of mutant and wild type alike. That being said, a significant interaction between GAT1 deficiency and medium composition was determined with respect to magnitude of change in relative amino acid levels. The effect of exogenous GABA treatment on metabolism was contingent on both the medium and the genotype, leading for instance to a drop in asparagine under full nutrition and low C conditions and glucose under all tested media, but not to changes in GABA content. We additionally assessed the effect of GAT1 deficiency on the expression of glutamate metabolism related genes and genes involved in abiotic stress responses. These results suggest a role for GAT1 in GABA-mediated metabolic alterations in the context of the C-N equilibrium of plant cells. PMID:26483804

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

    PubMed Central

    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.

    2015-01-01

    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. PMID:25865495

  20. 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

  1. Production of gaba (γ – Aminobutyric acid) by microorganisms: a review

    PubMed Central

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

    2012-01-01

    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. PMID:24031948

  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. 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.

  4. Regulation of Local Ambient GABA Levels via Transporter-Mediated GABA Import and Export for Subliminal Learning.

    PubMed

    Hoshino, Osamu

    2015-06-01

    Perception of supraliminal stimuli might in general be reflected in bursts of action potentials (spikes), and their memory traces could be formed through spike-timing-dependent plasticity (STDP). Memory traces for subliminal stimuli might be formed in a different manner, because subliminal stimulation evokes a fraction (but not a burst) of spikes. Simulations of a cortical neural network model showed that a subliminal stimulus that was too brief (10 msec) to perceive transiently (more than about 500 msec) depolarized stimulus-relevant principal cells and hyperpolarized stimulus-irrelevant principal cells in a subthreshold manner. This led to a small increase or decrease in ongoing-spontaneous spiking activity frequency (less than 1 Hz). Synaptic modification based on STDP during this period effectively enhanced relevant synaptic weights, by which subliminal learning was improved. GABA transporters on GABAergic interneurons modulated local levels of ambient GABA. Ambient GABA molecules acted on extrasynaptic receptors, provided principal cells with tonic inhibitory currents, and contributed to achieving the subthreshold neuronal state. We suggest that ongoing-spontaneous synaptic alteration through STDP following subliminal stimulation may be a possible neuronal mechanism for leaving its memory trace in cortical circuitry. Regulation of local ambient GABA levels by transporter-mediated GABA import and export may be crucial for subliminal learning. PMID:25774546

  5. Synthesis and Proton NMR Spectroscopy of Intra-Vesicular Gamma-Aminobutyric Acid (GABA)*

    PubMed Central

    Wang, Luke Y.-J.; Tong, Rong; Kohane, Daniel S.

    2014-01-01

    We report the synthesis of vesicles containing gamma-aminobutyric acid (GABA), and their proton nuclear magnetic resonance (1H NMR) spectra. These vesicles were constructed to more closely mimic the intracellular environment wherein GABA exists. For this study, these GABA-containing vesicles were examined under 1H NMR as a potential platform for future studies on the differences between aqueous phantoms, ex vivo brain extracts, and in vivo magnetic resonance spectroscopy results. We found that intra-vesicular GABA faithfully yielded the chemical shifts and J-coupling constants of free aqueous GABA, alongside the chemical shift signals of the vesicle wall. PMID:24109882

  6. Up-regulation of GABA transporters and GABA(A) receptor α1 subunit in tremor rat hippocampus.

    PubMed

    Mao, Xiaoyuan; Guo, Feng; Yu, Junling; Min, Dongyu; Wang, Zhanyou; Xie, Ni; Chen, Tianbao; Shaw, Chris; Cai, Jiqun

    2010-12-17

    The loss of GABAergic neurotransmission has been closely linked with epileptogenesis. The modulation of the synaptic activity occurs both via the removal of GABA from the synaptic cleft and by GABA transporters (GATs) and by modulation of GABA receptors. The tremor rat (TRM; tm/tm) is the parent strain of the spontaneously epileptic rat (SER; zi/zi, tm/tm), which exhibits absence-like seizure after 8 weeks of age. However, there are no reports that can elucidate the effects of GATs and GABA(A) receptors (GABARs) on TRMs. The present study was conducted to detect GATs and GABAR α1 subunit in TRMs hippocampus at mRNA and protein levels. In this study, total synaptosomal GABA content was significantly decreased in TRMs hippocampus compared with control Wistar rats by high performance liquid chromatography (HPLC); mRNA and protein expressions of GAT-1, GAT-3 and GABAR α1 subunit were all significantly increased in TRMs hippocampus by real time PCR and Western blot, respectively; GAT-1 and GABAR α1 subunit proteins were localized widely in TRMs and control rats hippocampus including CA1, CA3 and dentate gyrus (DG) regions whereas only a wide distribution of GAT-3 was observed in CA1 region by immunohistochemistry. These data demonstrate that excessive expressions of GAT-1 as well as GAT-3 and GABAR α1 subunit in TRMs hippocampus may provide the potential therapeutic targets for genetic epilepsy. PMID:20851161

  7. Acid Stimulation (Sour Taste) Elicits GABA and Serotonin Release from Mouse Taste Cells

    PubMed Central

    Huang, Yijen A.; Pereira, Elizabeth; Roper, Stephen D.

    2011-01-01

    Several transmitter candidates including serotonin (5-HT), ATP, and norepinephrine (NE) have been identified in taste buds. Recently, γ-aminobutyric acid (GABA) as well as the associated synthetic enzymes and receptors have also been identified in taste cells. GABA reduces taste-evoked ATP secretion from Receptor cells and is considered to be an inhibitory transmitter in taste buds. However, to date, the identity of GABAergic taste cells and the specific stimulus for GABA release are not well understood. In the present study, we used genetically-engineered Chinese hamster ovary (CHO) cells stably co-expressing GABAB receptors and Gαqo5 proteins to measure GABA release from isolated taste buds. We recorded robust responses from GABA biosensors when they were positioned against taste buds isolated from mouse circumvallate papillae and the buds were depolarized with KCl or a stimulated with an acid (sour) taste. In contrast, a mixture of sweet and bitter taste stimuli did not trigger GABA release. KCl- or acid-evoked GABA secretion from taste buds was Ca2+-dependent; removing Ca2+ from the bathing medium eliminated GABA secretion. Finally, we isolated individual taste cells to identify the origin of GABA secretion. GABA was released only from Presynaptic (Type III) cells and not from Receptor (Type II) cells. Previously, we reported that 5-HT released from Presynaptic cells inhibits taste-evoked ATP secretion. Combined with the recent findings that GABA depresses taste-evoked ATP secretion [1], the present results indicate that GABA and 5-HT are inhibitory transmitters in mouse taste buds and both likely play an important role in modulating taste responses. PMID:22028776

  8. Dynamic regulation of glycine–GABA co-transmission at spinal inhibitory synapses by neuronal glutamate transporter

    PubMed Central

    Ishibashi, Hitoshi; Yamaguchi, Junya; Nakahata, Yoshihisa; Nabekura, Junichi

    2013-01-01

    Fast inhibitory neurotransmission in the central nervous system is mediated by γ-aminobutyric acid (GABA) and glycine, which are accumulated into synaptic vesicles by a common vesicular inhibitory amino acid transporter (VIAAT) and are then co-released. However, the mechanisms that control the packaging of GABA + glycine into synaptic vesicles are not fully understood. In this study, we demonstrate the dynamic control of the GABA–glycine co-transmission by the neuronal glutamate transporter, using paired whole-cell patch recording from monosynaptically coupled cultured spinal cord neurons derived from VIAAT-Venus transgenic rats. Short step depolarization of presynaptic neurons evoked unitary (cell-to-cell) inhibitory postsynaptic currents (IPSCs). Under normal conditions, the fractional contribution of postsynaptic GABA or glycine receptors to the unitary IPSCs did not change during a 1 h recording. Intracellular loading of GABA or glycine via a patch pipette enhanced the respective components of inhibitory transmission, indicating the importance of the cytoplasmic concentration of inhibitory transmitters. Raised extracellular glutamate levels increased the amplitude of GABAergic IPSCs but reduced glycine release by enhancing glutamate uptake. Similar effects were observed when presynaptic neurons were intracellularly perfused with glutamate. Interestingly, high-frequency trains of stimulation decreased glycinergic IPSCs more than GABAergic IPSCs, and repetitive stimulation occasionally failed to evoke glycinergic but not GABAergic IPSCs. The present results suggest that the enhancement of GABA release by glutamate uptake may be advantageous for rapid vesicular refilling of the inhibitory transmitter at mixed GABA/glycinergic synapses and thus may help prevent hyperexcitability. PMID:23690564

  9. An analysis of [3H]gamma-aminobutyric acid (GABA) binding in the human brain.

    PubMed

    Lloyd, K G; Dreksler, S

    1979-03-01

    The binding of [3H]GABA to membranes prepared from human brains obtained post morten was examined. This binding was independent of patient sex, age (16--80 years), postmortem interval (4--33 h) or storage time when frozen (0-64 months). In preparations from cerebellar cortex various compounds displaced [3H]GABA binding with the following order of potency: muscimol greater than 3-aminopropanesulfonic acid greater than GABA greater than imidazoleacet acid greater than delta-amino-n-valeric acid greater than 3-hydroxyGABA greater than bicuculline. Other compounds active 'in vitro' included strychnine, homocarnosine and some (e.g. clozapine, thioridazine, pimozide) but not all (chlorpromazine, haloperiodol) neuroleptics. Compounds inactive 'in vitro' included aminooxyacetic acid, baclofen, picrotoxin, anticholinergics, metrazole, anticonvulsants and naloxone. Triton X-100 augmented the [3H]GABA binding (25 nM) by about 10--20-fold in most brain regions. [3H]GABA binding (IC50) was altered in Huntington's chorea and Reye's syndrome, but not in schizophrenics (4-neuroleptic-treated patients) or sudden infant death syndrome. The data presented strongly support the proposal that the measurement of [3H]GABA binding in postmortem human brain offers a reflection of the state of the physiologically relevant GABA receptor. PMID:218679

  10. A fluorescence-coupled assay for gamma aminobutyric acid (GABA) reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

    PubMed

    Ippolito, Joseph E; Piwnica-Worms, David

    2014-01-01

    Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) have been implicated in the pathogenesis of high grade neuroendocrine (NE) neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1), was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC) cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL) activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies. PMID:24551133

  11. Synthesis of 4-substituted nipecotic acid derivatives and their evaluation as potential GABA uptake inhibitors.

    PubMed

    Hellenbrand, Tim; Höfner, Georg; Wein, Thomas; Wanner, Klaus T

    2016-05-01

    In this study, we disclose the design and synthesis of novel 4-susbtituted nipecotic acid derivatives as inhibitors of the GABA transporter mGAT1. Based on molecular modeling studies the compounds are assumed to adopt a binding pose similar to that of the potent mGAT1 inhibitor nipecotic acid. As substitution in 4-position should not cause an energetically unfavorable orientation of nipecotic acid as it is the case for N-substituted derivatives this is expected to lead to highly potent binders. For the synthesis of novel 4-substituted nipecotic acid derivatives a linear synthetic strategy was employed. As a key step, palladium catalyzed cross coupling reactions were used to attach the required biaryl moieties to the ω-position of the alkenyl- or alkynyl spacers of varying length in the 4-position of the nipecotic acid scaffold. The resulting amino acids were characterized with respect to their binding affinities and inhibitory potencies at mGAT1. Though the biological activities found were generally insignificant to poor, two compounds, one of which possesses a reasonable binding affinity for mGAT1, rac-57, the other a notable inhibitory potency at mGAT4, rac-84, both displaying a slight subtype selectivity for the individual transporters, could be identified. PMID:27039250

  12. Contents of Neo-flavored Tea (GABA Kintaro) Containing γ-Aminobutyric Acid

    NASA Astrophysics Data System (ADS)

    Shiraki, Yoshiya

    The contents of γ-aminobutyric acid (GABA), catechins, theaflavins, caffeine and pheophorbide-a in neo-flavored tea (GABA Kintaro tea) were analyzed. 1)The amounts of GABA were increased over 1.5mg/g by means of infrared ray irradiation with agitation treatment. 2)There was a tendency for the amount of catechins to be decreased by this treatment, whereas the amount of theaflavins tended to increase with the same treatment. The composition of these contents in this GABA Kintaro tea was almost the same as that of black tea. 3)There was a tendency for the amount of caffeine to be decreased by this treatment. 4)There was a tendency for the amount of pheophorbide-a to be increased by this treatment. 5)The result of this study showed that the amounts of GABA and theaflavins in this GABA Kintaro tea were higher than ordinary green tea but contained few catechins.It became clear that the amount of pheophorbide-a in this GABA Kintaro tea was less than the standard value established in processed chlorella.

  13. γ-Aminobutyric acid (GABA) homeostasis regulates pollen germination and polarized growth in Picea wilsonii.

    PubMed

    Ling, Yu; Chen, Tong; Jing, Yanping; Fan, Lusheng; Wan, Yinglang; Lin, Jinxing

    2013-11-01

    γ-Aminobutyric acid (GABA) is a four-carbon non-protein amino acid found in a wide range of organisms. Recently, GABA accumulation has been shown to play a role in the stress response and cell growth in angiosperms. However, the effect of GABA deficiency on pollen tube development remains unclear. Here, we demonstrated that specific concentrations of exogenous GABA stimulated pollen tube growth in Picea wilsonii, while an overdose suppressed pollen tube elongation. The germination percentage of pollen grains and morphological variations in pollen tubes responded in a dose-dependent manner to treatment with 3-mercaptopropionic acid (3-MP), a glutamate decarboxylase inhibitor, while the inhibitory effects could be recovered in calcium-containing medium supplemented with GABA. Using immunofluorescence labeling, we found that the actin cables were disorganized in 3-MP treated cells, followed by the transition of endo/exocytosis activating sites from the apex to the whole tube shank. In addition, variations in the deposition of cell wall components were detected upon labeling with JIM5, JIM7, and aniline blue. Our results demonstrated that calcium-dependent GABA signaling regulates pollen germination and polarized tube growth in P. wilsonii by affecting actin filament patterns, vesicle trafficking, and the configuration and distribution of cell wall components. PMID:23900837

  14. Chronic anabolic-androgenic steroid treatment affects brain GABA(A) receptor-gated chloride ion transport.

    PubMed

    Bitran, D; Hilvers, R J; Frye, C A; Erskine, M S

    1996-01-01

    Previous research in this laboratory has shown that chronic treatment of adult male rats with an anabolic-androgenic steroid (AAS) produced anxiolytic behavior and increased the functional response of cortical gamma-aminobutyric acid(A) (GABA(A)) receptors. The experiments reported here were aimed at further characterizing the effect of chronic AAS exposure on cerebral cortical GABA(A) receptors. Adult male rats were injected with dianabol (1,4-androstadien-17alpha-methyl-17beta-ol-3-one; 10 mg/kg/day, SC) for 4 weeks. A significant decrease in ventral prostate gland weight was found after 2 weeks of dianabol, and returned to control levels 3 and 10 days after steroid discontinuation. Testicular weights decreased throughout the treatment period but reached statistical significance only during the withdrawal period. Serum 3alpha-androstanediol level was marginally increased afer 2 weeks of dianabol injection, and was significantly decreased at 3 and 10 days after withdrawal. GABA-stimulated 36chloride (Cl-) influx in cortical synaptoneurosomes was increased in animals treated with dianabol for 2 and 4 weeks, and remained elevated 3 days after dianabol withdrawal, returning to control levels at withdrawal day 10. The increase in receptor efficacy was associated with a transient increase in receptor sensitivity (inverse of EC50), apparent after 2 weeks of AAS treatment and at withdrawal day 3. In a follow-up experiment, metabolites of dianabol were tested for the in vitro efficacy in potentiating GABA-stimulated Cl- transport. Only 3alpha-androstanedial and androsterone were found to have potent stimulatory effects. The 3beta-reduced metabolites were inactive, as were metabolites that contained a methyl group at the 17alpha position. These results point to significant facilitative effects of dianabol treatment on brain GABA(A) receptors via the metabolic formation of neuroactive steroids. PMID:8632710

  15. Effects of traditionally used anxiolytic botanicals on enzymes of the gamma-aminobutyric acid (GABA) system.

    PubMed

    Awad, R; Levac, D; Cybulska, P; Merali, Z; Trudeau, V L; Arnason, J T

    2007-09-01

    In Canada, the use of botanical natural health products (NHPs) for anxiety disorders is on the rise, and a critical evaluation of their safety and efficacy is required. The purpose of this study was to determine whether commercially available botanicals directly affect the primary brain enzymes responsible for gamma-aminobutyric acid (GABA) metabolism. Anxiolytic plants may interact with either glutamic acid decarboxylase (GAD) or GABA transaminase (GABA-T) and ultimately influence brain GABA levels and neurotransmission. Two in vitro rat brain homogenate assays were developed to determine the inhibitory concentrations (IC50) of aqueous and ethanolic plant extracts. Approximately 70% of all extracts that were tested showed little or no inhibitory effect (IC50 values greater than 1 mg/mL) and are therefore unlikely to affect GABA metabolism as tested. The aqueous extract of Melissa officinalis (lemon balm) exhibited the greatest inhibition of GABA-T activity (IC50 = 0.35 mg/mL). Extracts from Centella asiatica (gotu kola) and Valeriana officinalis (valerian) stimulated GAD activity by over 40% at a dose of 1 mg/mL. On the other hand, both Matricaria recutita (German chamomile) and Humulus lupulus (hops) showed significant inhibition of GAD activity (0.11-0.65 mg/mL). Several of these species may therefore warrant further pharmacological investigation. The relation between enzyme activity and possible in vivo mode of action is discussed. PMID:18066140

  16. Metabolic pathways regulated by γ-aminobutyric acid (GABA) contributing to heat tolerance in creeping bentgrass (Agrostis stolonifera)

    PubMed Central

    Li, Zhou; Yu, Jingjin; Peng, Yan; Huang, Bingru

    2016-01-01

    γ-Aminobutyric acid is a non-protein amino acid involved in various metabolic processes. The objectives of this study were to examine whether increased GABA could improve heat tolerance in cool-season creeping bentgrass through physiological analysis, and to determine major metabolic pathways regulated by GABA through metabolic profiling. Plants were pretreated with 0.5 mM GABA or water before exposed to non-stressed condition (21/19 °C) or heat stress (35/30 °C) in controlled growth chambers for 35 d. The growth and physiological analysis demonstrated that exogenous GABA application significantly improved heat tolerance of creeping bentgrass. Metabolic profiling found that exogenous application of GABA led to increases in accumulations of amino acids (glutamic acid, aspartic acid, alanine, threonine, serine, and valine), organic acids (aconitic acid, malic acid, succinic acid, oxalic acid, and threonic acid), sugars (sucrose, fructose, glucose, galactose, and maltose), and sugar alcohols (mannitol and myo-inositol). These findings suggest that GABA-induced heat tolerance in creeping bentgrass could involve the enhancement of photosynthesis and ascorbate-glutathione cycle, the maintenance of osmotic adjustment, and the increase in GABA shunt. The increased GABA shunt could be the supply of intermediates to feed the tricarboxylic acid cycle of respiration metabolism during a long-term heat stress, thereby maintaining metabolic homeostasis. PMID:27455877

  17. Metabolic pathways regulated by γ-aminobutyric acid (GABA) contributing to heat tolerance in creeping bentgrass (Agrostis stolonifera).

    PubMed

    Li, Zhou; Yu, Jingjin; Peng, Yan; Huang, Bingru

    2016-01-01

    γ-Aminobutyric acid is a non-protein amino acid involved in various metabolic processes. The objectives of this study were to examine whether increased GABA could improve heat tolerance in cool-season creeping bentgrass through physiological analysis, and to determine major metabolic pathways regulated by GABA through metabolic profiling. Plants were pretreated with 0.5 mM GABA or water before exposed to non-stressed condition (21/19 °C) or heat stress (35/30 °C) in controlled growth chambers for 35 d. The growth and physiological analysis demonstrated that exogenous GABA application significantly improved heat tolerance of creeping bentgrass. Metabolic profiling found that exogenous application of GABA led to increases in accumulations of amino acids (glutamic acid, aspartic acid, alanine, threonine, serine, and valine), organic acids (aconitic acid, malic acid, succinic acid, oxalic acid, and threonic acid), sugars (sucrose, fructose, glucose, galactose, and maltose), and sugar alcohols (mannitol and myo-inositol). These findings suggest that GABA-induced heat tolerance in creeping bentgrass could involve the enhancement of photosynthesis and ascorbate-glutathione cycle, the maintenance of osmotic adjustment, and the increase in GABA shunt. The increased GABA shunt could be the supply of intermediates to feed the tricarboxylic acid cycle of respiration metabolism during a long-term heat stress, thereby maintaining metabolic homeostasis. PMID:27455877

  18. GABA, 5-HT and amino acids in the rotifers Brachionus plicatilis and Brachionus rotundiformis.

    PubMed

    Gallardo, W G; Hagiwara, A; Hara, K; Soyano, K; Snell, T W

    2000-11-01

    gamma-Aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT) have been shown to increase the reproduction of the Brachionus plicatilis (NH3L strain). In the present study, the endogenous presence of GABA and 5-HT in the rotifers B. plicatilis (NH3L and Kamiura strains) and Brachionus rotundiformis (Langkawi strain) were confirmed by dot blot immunoassay and high-performance liquid chromatography (HPLC). HPLC showed that GABA and 5-HT concentrations in the three rotifer strains range from 71 to 188 pmol/mg and from 12 to 64 pmol/mg, respectively. A total of 33 amino acids were also detected in B. plicatilis and B. rotundiformis, with glutamic acid, serine, glycine, taurine, threonine, alanine, arginine, proline, valine and isoleucine in high concentrations relative to other amino acids. PMID:11118940

  19. The physiological roles of vesicular GABA transporter during embryonic development: a study using knockout mice

    PubMed Central

    2010-01-01

    Background The vesicular GABA transporter (VGAT) loads GABA and glycine from the neuronal cytoplasm into synaptic vesicles. To address functional importance of VGAT during embryonic development, we generated global VGAT knockout mice and analyzed them. Results VGAT knockouts at embryonic day (E) 18.5 exhibited substantial increases in overall GABA and glycine, but not glutamate, contents in the forebrain. Electrophysiological recordings from E17.5-18.5 spinal cord motoneurons demonstrated that VGAT knockouts presented no spontaneous inhibitory postsynaptic currents mediated by GABA and glycine. Histological examination of E18.5 knockout fetuses revealed reductions in the trapezius muscle, hepatic congestion and little alveolar spaces in the lung, indicating that the development of skeletal muscle, liver and lung in these mice was severely affected. Conclusion VGAT is fundamental for the GABA- and/or glycine-mediated transmission that supports embryonic development. VGAT knockout mice will be useful for further investigating the roles of VGAT in normal physiology and pathophysiologic processes. PMID:21190592

  20. An arylaminopyridazine derivative of gamma-aminobutyric acid (GABA) is a selective and competitive antagonist at the GABAA receptor site.

    PubMed Central

    Chambon, J P; Feltz, P; Heaulme, M; Restle, S; Schlichter, R; Biziere, K; Wermuth, C G

    1985-01-01

    In view of finding a new gamma-aminobutyric acid (GABA) receptor ligand we synthesized an arylaminopyridazine derivative of GABA, SR 95103 [2-(carboxy-3'-propyl)-3-amino-4-methyl-6-phenylpyridazinium chloride]. SR 95103 displaced [3H]GABA from rat brain membranes with an apparent Ki of 2.2 microM and a Hill number near 1.0. SR 95103 (1-100 microM) antagonized the GABA-mediated enhancement of [3H]diazepam binding in a concentration-dependent manner without affecting [3H]diazepam binding per se. SR 95103 competitively antagonized GABA-induced membrane depolarization in rat spinal ganglia. In all these experiments, the potency of SR 95103 was close to that of bicuculline. SR 95103 (100 microM) did not interact with a variety of central receptors--in particular the GABAB, the strychnine, and the glutamate receptors--did not inhibit Na+-dependent synaptosomal GABA uptake, and did not affect GABA-transaminase and glutamic acid decarboxylase activities. Intraperitoneally administered SR 95103 elicited clonicotonic seizures in mice (ED50 = 180 mg/kg). On the basis of these results it is postulated that St 95103 is a competitive antagonist of GABA at the GABAA receptor site. In addition to being an interesting lead structure for the search of GABA ligands, SR 95103 could also be a useful tool to investigate GABA receptor subtypes because it is freely soluble in water and chemically stable. Images PMID:2984669

  1. γ-Aminobutyric acid (GABA) concentration inversely correlates with basal perfusion in human occipital lobe.

    PubMed

    Donahue, Manus J; Rane, Swati; Hussey, Erin; Mason, Emily; Pradhan, Subechhya; Waddell, Kevin W; Ally, Brandon A

    2014-03-01

    Commonly used neuroimaging approaches in humans exploit hemodynamic or metabolic indicators of brain function. However, fundamental gaps remain in our ability to relate such hemo-metabolic reactivity to neurotransmission, with recent reports providing paradoxical information regarding the relationship among basal perfusion, functional imaging contrast, and neurotransmission in awake humans. Here, sequential magnetic resonance spectroscopy (MRS) measurements of the primary inhibitory neurotransmitter, γ-aminobutyric acid (GABA+macromolecules normalized by the complex N-acetyl aspartate-N-acetyl aspartyl glutamic acid: [GABA(+)]/[NAA-NAAG]), and magnetic resonance imaging (MRI) measurements of perfusion, fractional gray-matter volume, and arterial arrival time (AAT) are recorded in human visual cortex from a controlled cohort of young adult male volunteers with neurocognitive battery-confirmed comparable cognitive capacity (3 T; n=16; age=23±3 years). Regression analyses reveal an inverse correlation between [GABA(+)]/[NAA-NAAG] and perfusion (R=-0.46; P=0.037), yet no relationship between AAT and [GABA(+)]/[NAA-NAAG] (R=-0.12; P=0.33). Perfusion measurements that do not control for AAT variations reveal reduced correlations between [GABA(+)]/[NAA-NAAG] and perfusion (R=-0.13; P=0.32). These findings largely reconcile contradictory reports between perfusion and inhibitory tone, and underscore the physiologic origins of the growing literature relating functional imaging signals, hemodynamics, and neurotransmission. PMID:24398941

  2. The Uptake of GABA in Trypanosoma cruzi.

    PubMed

    Galvez Rojas, Robert L; Ahn, Il-Young; Suárez Mantilla, Brian; Sant'Anna, Celso; Pral, Elizabeth Mieko Furusho; Silber, Ariel Mariano

    2015-01-01

    Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na(+) , K(+) , and H(+) on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na(+) dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na(+) /GABA symporter energized by Na(+) -exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway. PMID:25851259

  3. 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. PMID:25698617

  4. Cloning of the gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA receptor subunit highly expressed in the retina.

    PubMed Central

    Cutting, G R; Lu, L; O'Hara, B F; Kasch, L M; Montrose-Rafizadeh, C; Donovan, D M; Shimada, S; Antonarakis, S E; Guggino, W B; Uhl, G R

    1991-01-01

    Type A gamma-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. We have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence in 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABAA subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA rho 1, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family. Images PMID:1849271

  5. Localization and Function of GABA Transporters GAT-1 and GAT-3 in the Basal Ganglia

    PubMed Central

    Jin, Xiao-Tao; Galvan, Adriana; Wichmann, Thomas; Smith, Yoland

    2011-01-01

    GABA transporter type 1 and 3 (GAT-1 and GAT-3, respectively) are the two main subtypes of GATs responsible for the regulation of extracellular GABA levels in the central nervous system. These transporters are widely expressed in neuronal (mainly GAT-1) and glial (mainly GAT-3) elements throughout the brain, but most data obtained so far relate to their role in the regulation of GABAA receptor-mediated postsynaptic tonic and phasic inhibition in the hippocampus, cerebral cortex and cerebellum. Taking into consideration the key role of GABAergic transmission within basal ganglia networks, and the importance for these systems to be properly balanced to mediate normal basal ganglia function, we analyzed in detail the localization and function of GAT-1 and GAT-3 in the globus pallidus of normal and Parkinsonian animals, in order to further understand the substrate and possible mechanisms by which GABA transporters may regulate basal ganglia outflow, and may become relevant targets for new therapeutic approaches for the treatment of basal ganglia-related disorders. In this review, we describe the general features of GATs in the basal ganglia, and give a detailed account of recent evidence that GAT-1 and GAT-3 regulation can have a major impact on the firing rate and pattern of basal ganglia neurons through pre- and post-synaptic GABAA- and GABAB-receptor-mediated effects. PMID:21847373

  6. Selective antagonism of the GABA(A) receptor by ciprofloxacin and biphenylacetic acid.

    PubMed

    Green, M A; Halliwell, R F

    1997-10-01

    1. Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit y-aminobutyric acid (GABA)A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels. 2. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique. 3. GABA (50 microM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3-10 microM) and picrotoxin (0.3-10 microM), with IC50 values and 95% confidence intervals (CI) of 1.2 microM (1.1-1.4) and 3.6 microM (3.0-4.3), respectively, and were potentiated by sodium pentobarbitone (30 microM) and diazepam (1 microM) to (mean+/-s.e.mean) 168+/-18% and 117+/-4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 microM)-evoked responses were inhibited by MDL 72222 (1 microM) to 10+/-4% of control; DMPP (10 microM)-evoked responses were inhibited by hexamethonium (100 microM) to 12+/-5% of control, and alphabetaMeATP (30 microM)-evoked responses were inhibited by PPADS (10 microM) to 21+/-5% of control. Together, these data are consistent with activation of GABA(A), 5-HT3, nicotinic ACh and P2X receptors, respectively. 4 Ciprofloxacin (10-3000 microM) inhibited GABA(A)-mediated responses in the vagus nerve with an IC50 (and 95% CI) of 202 microM (148-275). BPAA (1-1000 microM) had little or no effect on the GABA(A)-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times. 5. Responses mediated by 5-HT3, nicotinic ACh and P2X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 microM), BPAA (100 microM) or the combination of these drugs (both at 100 microM). 6. GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC50 of 3.6 microM (2.8-4.5), a value not significantly different

  7. The inhibitory role of γ-aminobutyric acid (GABA) on immunomodulation of Pacific oyster Crassostrea gigas.

    PubMed

    Li, Meijia; Qiu, Limei; Wang, Lingling; Wang, Weilin; Xin, Lusheng; Li, Yiqun; Liu, Zhaoqun; Song, Linsheng

    2016-05-01

    γ-aminobutyric acid (GABA) is an inhibitory neurotransmitter to suppress the immune-mediated pro-inflammatory reactions, and it has been used in the treatment of many inflammation-related diseases in vertebrates, while its immunomodulatory role in invertebrates has never been reported. In the present study, GABA was found to exist in the hemolymph of Pacific oyster Crassostrea gigas, and its concentration decreased slightly from 8.00 ± 0.37 μmol L(-1) at normal condition to 7.73 ± 0.15 μmol L(-1) at 6 h after LPS stimulation, and then increased to 9.34 ± 0.15 μmol L(-1), 8.86 ± 0.68 μmol L(-1) at 12 h and 48 h, respectively. After LPS stimulation, the mRNA expressions of pro-inflammatory cytokines (CgIL-17 and CgTNF) and immune effectors (CgSOD and CgBPI), and the protein expression of NOS increased significantly, and these increased trends were remarkably inhibited by GABA stimulation. At the same time, the phagocytosis rate and apoptosis rate of immunocytes also increased obviously after LPS stimulation, whereas the increase was repressed with the addition of GABA. The results collectively demonstrated that GABA was an indispensable inhibitory agent for both humoral and cellular immune response, which mainly functioned at the late phase of immune response to avoid the excess immune reactions and maintain the immune homeostasis. PMID:26975413

  8. Guanidino acids act as rho1 GABA(C) receptor antagonists.

    PubMed

    Chebib, Mary; Gavande, Navnath; Wong, Kit Yee; Park, Anna; Premoli, Isabella; Mewett, Kenneth N; Allan, Robin D; Duke, Rujee K; Johnston, Graham A R; Hanrahan, Jane R

    2009-10-01

    GABA(C) receptors play a role in myopia, memory-related disorders and circadian rhythms signifying a need to develop potent and selective agents for this class of receptors. Guanidino analogs related to glycine, beta-alanine and taurine were evaluated at human rho(1)GABA(C) receptors expressed in Xenopus oocytes using 2-electrode voltage clamp methods. Of the 12 analogs tested, 8 analogs were active as antagonists and the remaining were inactive. (S)-2-guanidinopropionic acid (IC(50) = 2.2 microM) and guanidinoacetic acid (IC(50) = 5.4 microM; K (B) = 7.75 microM [pK (B) = 5.11 +/- 0.06]) were the most potent being competitive antagonists at this receptor. In contrast, the beta-alanine and GABA guanidino analogs showed reduced activity, indicating the distance between the carboxyl carbon and terminal nitrogen of the guanidino group is critical for activity. Substituting the C2-position of guanidinoacetic acid with various alkyl groups reduced activity indicating that steric effects may impact on activity. The results of this study contribute to the structure-activity-relationship profile required in developing novel therapeutic agents. PMID:19387831

  9. Discovery of a subtype selective inhibitor of the human betaine/GABA transporter 1 (BGT-1) with a non-competitive pharmacological profile.

    PubMed

    Kragholm, Bolette; Kvist, Trine; Madsen, Karsten K; Jørgensen, Lars; Vogensen, Stine B; Schousboe, Arne; Clausen, Rasmus P; Jensen, Anders A; Bräuner-Osborne, Hans

    2013-08-15

    The γ-aminobutyric acid (GABA) transporters (GATs) are essential regulators of the activity in the GABAergic system through their continuous uptake of the neurotransmitter from the synaptic cleft and extrasynaptic space. Four GAT subtypes have been identified to date, each displaying different pharmacological properties and expression patterns. The present study focus on the human betaine/GABA transporter 1 (BGT-1), which has recently emerged as a new target for treatment of epilepsy. However, the lack of selective inhibitors of this transporter has impaired the exploration of this potential considerably. With the objective of identifying novel compounds displaying selectivity for BGT-1, we performed a screening of a small compound library at cells expressing BGT-1 using a [(3)H]GABA uptake assay. The screening resulted in the identification of the compound N-(1-benzyl-4-piperidinyl)-2,4-dichlorobenzamide (BPDBA), a selective inhibitor of the human BGT-1 transporter with a non-competitive profile exhibiting no significant inhibitory activity at the other three human GAT subtypes. The selectivity profile of the compound was subsequently confirmed at cells expressing the four mouse GAT subtypes. Thus, BPDBA constitutes a potential useful pharmacological tool compound for future explorations of the function of the BGT-1 subtype. PMID:23792119

  10. Effect of diphenylhydantoin on gamma aminobutyric acid (GABA) and succinate activity in rat Purkinje cells.

    PubMed Central

    Hitchcock, E; Gabra-Sanders, T

    1977-01-01

    A study has been made of the effect of diphenylhydantoin (DPH) upon the levels of gamma aminobutyric acid (GABA) and succinic dehydrogenase in rat Purkinje cells. DPH was administered over 26 days in chronic experiments using controls receiving the same injection vehicle without DPH. Animals in this group received daily 1.25 mg/kg body weight, 12.5 mg/kg body weight, and 50 mg/kg body weight DPH. Acute experiments were carried out over the course of not more than four days, three groups of animals receiving 75 mg/kg body weight, 87.5 mg/kg body weight, and 100 mg/kg body weight DPH. No effect upon succinic dehydrogenase could be demonstrated at any dose level. There was a significant progressive loss of GABA with increasing dosage of DPH. Images PMID:903771

  11. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability. PMID

  12. Mutations in y-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid '-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome h...

  13. Effects of NaCl Replacement with Gamma-Aminobutyric acid (GABA) on the Quality Characteristics and Sensorial Properties of Model Meat Products

    PubMed Central

    Chun, Ji-Yeon; Cho, Hyung-Yong; Min, Sang-Gi

    2014-01-01

    This study investigated the effects of γ-aminobutylic acid (GABA) on the quality and sensorial properties of both the GABA/NaCl complex and model meat products. GABA/NaCl complex was prepared by spray-drying, and the surface dimensions, morphology, rheology, and saltiness were characterized. For model meat products, pork patties were prepared by replacing NaCl with GABA. For characteristics of the complex, increasing GABA concentration increased the surface dimensions of the complex. However, GABA did not affect the rheological properties of solutions containing the complex. The addition of 2% GABA exhibited significantly higher saltiness than the control (no GABA treatment). In the case of pork patties, sensory testing indicated that the addition of GABA decreased the saltiness intensity. Both the intensity of juiciness and tenderness of patties containing GABA also scored lower than the control, based on the NaCl reduction. These results were consistent with the quality characteristics (cooking loss and texture profile analysis). Nevertheless, overall acceptability of the pork patties showed that up to 1.5%, patties containing GABA did not significantly differ from the control. Consequently, the results indicated that GABA has a potential application in meat products, but also manifested a deterioration of quality by the NaCl reduction, which warrants further exploration. PMID:26761294

  14. Effect of GABA on oxidative stress in the skeletal muscles and plasma free amino acids in mice fed high-fat diet.

    PubMed

    Xie, Z X; Xia, S F; Qiao, Y; Shi, Y H; Le, G W

    2015-06-01

    Increased levels of plasma free amino acids (pFAAs) can disturb the blood glucose levels in patients with obesity, diabetes mellitus and metabolic syndrome (MS) and are associated with enhanced protein oxidation. Oxidation of proteins, especially in the muscles, can promote protein degradation and elevate the levels of pFAAs. Gamma-aminobutyric acid (GABA), a food additive, can reduce high-fat diet (HFD)-induced hyperglycaemia; however, the mechanisms remain unclear. The aim of this study was to evaluate the effects of GABA on protein oxidation and pFAAs changes. One hundred male C57BL/6 mice were randomly divided into five groups that were fed with control diet, HFD and HFD supplied with 0.2%, 0.12% and 0.06% GABA in drinking water for 20 weeks respectively. HFD feeding led to muscular oxidative stress, protein oxidation, pFAA disorders, hyperglycaemia and augmented plasma GABA levels. Treatment with GABA restored normally fasting blood glucose level and dose-dependently inhibited body weight gains, muscular oxidation and protein degradation. While medium and low doses of GABA mitigated HFD-induced pFAA disorders, the high dose of GABA deteriorated the pFAA disorders. Medium dose of GABA increased the levels of GABA, but high dose of GABA reduced the levels of plasma GABA and increased the activity of succinic semialdehyde dehydrogenase in the liver. Therefore, treatment with GABA mitigated HFD-induced hyperglycaemia probably by repairing HFD-induced muscular oxidative stress and pFAA disorders in mice. Our data also suggest that an optimal dose of GABA is crucial for the prevention of excess GABA-related decrease in the levels of pFAA and GABA as well as obesity. PMID:25266692

  15. Identification of amino acids involved in histamine potentiation of GABA A receptors.

    PubMed

    Thiel, Ulrike; Platt, Sarah J; Wolf, Steffen; Hatt, Hanns; Gisselmann, Günter

    2015-01-01

    Histamine is a neurotransmitter involved in a number of physiological and neuronal functions. In mammals, such as humans, and rodents, the histaminergic neurons found in the tuberomamillary nucleus project widely throughout the central nervous system. Histamine acts as positive modulator of GABAA receptors (GABAARs) and, in high concentrations (10 mM), as negative modulator of the strychnine-sensitive glycine receptor. However, the exact molecular mechanisms by which histamine acts on GABAARs are unknown. In our study, we aimed to identify amino acids potentially involved in the modulatory effect of histamine on GABAARs. We expressed GABAARs with 12 different point mutations in Xenopus laevis oocytes and characterized the effect of histamine on GABA-induced currents using the two-electrode voltage clamp technique. Our data demonstrate that the amino acid residues β2(N265) and β2(M286), which are important for modulation by propofol, are not involved in the action of histamine. However, we found that histamine modulation is dependent on the amino acid residues α1(R120), β2(Y157), β2(D163), β3(V175), and β3(Q185). We showed that the amino acid residues β2(Y157) and β3(Q185) mediate the positive modulatory effect of histamine on GABA-induced currents, whereas α1(R120) and β2(D163) form a potential histamine interaction site in GABAARs. PMID:26074818

  16. γ-Aminobutyric acid type A (GABA(A)) receptor subtype inverse agonists as therapeutic agents in cognition.

    PubMed

    Gabriella, Guerrini; Giovanna, Ciciani

    2010-01-01

    The gabaergic system has been identified as a relevant regulator of cognitive and emotional processing. In fact, the discovery that negative allosteric regulators (or inverse agonists) at GABA(A) (γ-aminobutyric acid) α5 subtype receptors improve learning and memory tasks, has further validated this concept. The localization of these extrasynaptic subtype receptors, mainly in the hippocampus, has suggested that they play a key role in the three stages of memory: acquisition, consolidation, and retrieval. The "α5 inverse agonist" binds to an allosteric site at GABA(A) receptor, provoking a reduction of chlorine current, but to elicit this effect, the necessary condition is the binding of agonist neurotransmitter (γ-amino butyric acid) at its orthosteric site. In this case, the GABA(A) receptor is not a "constitutively active receptor" and, however, the presence of spontaneous opening channels for native GABA(A) receptors is rare. Here, we present various classes of nonselective and α5 selective GABA(A) receptor ligands, and the in vitro and in vivo tests to elucidate their affinity and activity. The study of the GABA(A) α5 inverse agonists is one of the important tools, although not the only one, for the development of clinical strategies for treatment of Alzheimer disease and mild cognitive impairment. PMID:21050918

  17. Limitations of Current GABA Agonists in Neonatal Seizures: Toward GABA Modulation Via the Targeting of Neuronal Cl− Transport

    PubMed Central

    Khanna, Arjun; Walcott, Brian Patrick; Kahle, Kristopher T.

    2013-01-01

    Neonatal intensive care has advanced rapidly in the last 40 years, with dramatic decreases in mortality and morbidity; however, for neonatal seizures, neither therapies nor outcomes have changed significantly. Basic and clinical studies indicate that seizures in neonates have long-term neurodevelopmental and psychiatric consequences, highlighting the need for novel pharmacotherapeutics. First-line treatments targeting GABAA receptors, like barbiturates and benzodiazepines, are limited in their efficacy and carry significant risks to the developing brain. Here, we review the use of current GABA agonist therapies for neonatal seizures and suggest other treatment strategies given recent developments in the understanding of disease pathogenesis. One promising avenue is the indirect manipulation of the GABAergic system, via the modulation of neuronal Cl− gradients, by targeting the cation-Cl− cotransporters (NKCC1 and KCC2) or their regulatory signaling molecules. This strategy might yield a novel class of more efficacious anti-epileptics with fewer side effects by specifically addressing disease pathophysiology. Moreover, this strategy may have ramifications for other adult seizure syndromes in which GABA receptor-mediated depolarizations play a pathogenic role, such as temporal lobe epilepsy. PMID:23805124

  18. GABA shunt and polyamine degradation pathway on γ-aminobutyric acid accumulation in germinating fava bean (Vicia faba L.) under hypoxia.

    PubMed

    Yang, Runqiang; Guo, Qianghui; Gu, Zhenxin

    2013-01-01

    GABA shunt and polyamine degradation pathway on γ-aminobutyric acid (GABA) accumulation in germinating fava bean under hypoxia was investigated. GABA content, GAD and DAO activity were significantly increased under hypoxia treatment. Glu and polyamine contents enhanced largely and thus supplied as sufficient substrates for GABA formation. In contrast, GABA content decreased, mainly in the embryo, after removing the hypoxia stress. DAO activity, Glu and polyamines contents decreased, while an increment of GAD activity was observed. This indicated that GAD activity can be not only regulated by hypoxia, but by the rapid growth of embryo after the recovery from hypoxia stress. When treated with AG, DAO activity was almost inhibited completely, and the GABA content decreased by 32.96% and 32.07% after treated for 3 and 5 days, respectively. Hence, it can be inferred that about 30% of GABA formed in germinating fava bean under hypoxia was supplied by polyamine degradation pathway. PMID:23017406

  19. [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. PMID:7272066

  20. SYSTEMIC ADMINISTRATION OF KAINIC ACID INCREASES GABA LEVELS IN PERFUSATE FROM THE HIPPOCAMPUS OF RATS IN VIVO

    EPA Science Inventory

    The ventral hippocampi of male, Fischer-344 rats were implanted with microdialysis probes and the effects of systemically administered kainic acid (KA) (8 mg/kg, s.c.) on the in vivo release of amino acids were measured for four hours after administration. n order to measure GABA...

  1. Exogenous γ-aminobutyric Acid (GABA) Application Improved Early Growth, Net Photosynthesis, and Associated Physio-Biochemical Events in Maize.

    PubMed

    Li, Wu; Liu, Jianhua; Ashraf, Umair; Li, Gaoke; Li, Yuliang; Lu, Wenjia; Gao, Lei; Han, Fuguang; Hu, Jianguang

    2016-01-01

    γ-aminobutyric acid (GABA) is an endogenous signaling molecule and involved in growth regulations and plant development, however, a little information is available on the consequences of exogenous GABA application on growth, development, and associated physio-biochemical processes in maize. The present study examined the GABA-induced regulations in early growth, net photosynthetic rate, gas exchange, osmoregulation, and enzymatic activities in three maize cultivars, i.e., Yuecainuo 6, Zhengtian 68, and Yuecainuo 2. Two levels of GABA, i.e., 0 mg L(-1) and 50 mg L(-1), in solution form, with total application volume of 100 ml per pot containing 15 maize seedlings were exogenously applied. Results revealed that exogenous GABA application improved seedling growth in terms of seedling length and biomass accumulation in all maize cultivars at both 3 and 7 days after treatment (DAT). It also promoted net photosynthesis and variably affected gas exchange attributes, i.e., stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr), as well as leaves SPAD value. Furthermore, lipid peroxidation [in terms of malondialdehyde (MDA)] under GABA treated maize seedlings were also remained variable; however, osmolyte accumulation (protein and proline) and activities of anti-oxidants enzymes, i.e., super-oxide dismutase and peroxidase were also affected differently at both 3 and 7 DAT in all maize cultivars. Furthermore, enzymes involved in nitrogen metabolism, e.g., nitrate reductase and glutamine synthetase were improved. These results suggest the involvement of GABA in various physio-metablical mechanisms which might lead to improvement in morphological growth of maize. In future, research is still needed at molecular and genetic levels to unravel the involvement of GABA-mediated regulations in growth and its associated physio-biochemical mechanisms. PMID:27446149

  2. Exogenous γ-aminobutyric Acid (GABA) Application Improved Early Growth, Net Photosynthesis, and Associated Physio-Biochemical Events in Maize

    PubMed Central

    Li, Wu; Liu, Jianhua; Ashraf, Umair; Li, Gaoke; Li, Yuliang; Lu, Wenjia; Gao, Lei; Han, Fuguang; Hu, Jianguang

    2016-01-01

    γ-aminobutyric acid (GABA) is an endogenous signaling molecule and involved in growth regulations and plant development, however, a little information is available on the consequences of exogenous GABA application on growth, development, and associated physio-biochemical processes in maize. The present study examined the GABA-induced regulations in early growth, net photosynthetic rate, gas exchange, osmoregulation, and enzymatic activities in three maize cultivars, i.e., Yuecainuo 6, Zhengtian 68, and Yuecainuo 2. Two levels of GABA, i.e., 0 mg L-1 and 50 mg L-1, in solution form, with total application volume of 100 ml per pot containing 15 maize seedlings were exogenously applied. Results revealed that exogenous GABA application improved seedling growth in terms of seedling length and biomass accumulation in all maize cultivars at both 3 and 7 days after treatment (DAT). It also promoted net photosynthesis and variably affected gas exchange attributes, i.e., stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr), as well as leaves SPAD value. Furthermore, lipid peroxidation [in terms of malondialdehyde (MDA)] under GABA treated maize seedlings were also remained variable; however, osmolyte accumulation (protein and proline) and activities of anti-oxidants enzymes, i.e., super-oxide dismutase and peroxidase were also affected differently at both 3 and 7 DAT in all maize cultivars. Furthermore, enzymes involved in nitrogen metabolism, e.g., nitrate reductase and glutamine synthetase were improved. These results suggest the involvement of GABA in various physio-metablical mechanisms which might lead to improvement in morphological growth of maize. In future, research is still needed at molecular and genetic levels to unravel the involvement of GABA-mediated regulations in growth and its associated physio-biochemical mechanisms. PMID:27446149

  3. Determination of γ-Aminobutyric Acid (GABA) in Rambutan Fruit cv. Rongrian by HPLC-ELSD and Separation of GABA from Rambutan Fruit Using Dowex 50W-X8 Column.

    PubMed

    Meeploy, Maneerat; Deewatthanawong, Rujira

    2016-03-01

    A high-performance liquid chromatography method coupled with an evaporative light scattering detector (ELSD) was validated for the determination of γ-aminobutyric acid (GABA) in rambutan fruit without any sample pretreatment or derivatization. In the concentration range of 0.05-1.0 mg/mL GABA, the ELSD response was linear with a correlation coefficient (r) >0.999. Limit of detection and limit of quantitation were found to be 0.7 and 2.0 µg/mL, respectively. The method enabled the complete separation of GABA in the aqueous extract of rambutan flesh from the impurity peaks at 45.7 min. The recoveries of sample added GABA were obtained in the range of 92.0-99.3%. Intraday and interday relative standard deviations were <5.3%. Repeatability of the extraction process showed the acceptable precision. From the analysis of GABA content in rambutan flesh, 0.71 ± 0.23 mg of GABA was found in 1 g fresh weight. The recovery of GABA after passing through the Dowex 50W-X8 column was 96.65%. The analytical methodology could be potentially applied to the detection and quantification of GABA in other fruits and complex matrices when a sufficient quantity is available. PMID:26590236

  4. Artificial Autopolyploidization Modifies the Tricarboxylic Acid Cycle and GABA Shunt in Arabidopsis thaliana Col-0

    NASA Astrophysics Data System (ADS)

    Vergara, Fredd; Kikuchi, Jun; Breuer, Christian

    2016-05-01

    Autopolyploidy is a process whereby the chromosome set is multiplied and it is a common phenomenon in angiosperms. Autopolyploidy is thought to be an important evolutionary force that has led to the formation of new plant species. Despite its relevance, the consequences of autopolyploidy in plant metabolism are poorly understood. This study compares the metabolic profiles of natural diploids and artificial autotetraploids of Arabidopsis thaliana Col-0. Different physiological parameters are compared between diploids and autotetraploids using nuclear magnetic resonance (NMR), elemental analysis (carbon:nitrogen balance) and quantitative real-time PCR (qRT-PCR). The main difference between diploid and autotetraploid A. thaliana Col-0 is observed in the concentration of metabolites related to the tricarboxylic acid cycle (TCA) and γ-amino butyric acid (GABA) shunt, as shown by multivariate statistical analysis of NMR spectra. qRT-PCR shows that genes related to the TCA and GABA shunt are also differentially expressed between diploids and autotetraploids following similar trends as their corresponding metabolites. Solid evidence is presented to demonstrate that autopolyploidy influences core plant metabolic processes.

  5. Artificial Autopolyploidization Modifies the Tricarboxylic Acid Cycle and GABA Shunt in Arabidopsis thaliana Col-0

    PubMed Central

    Vergara, Fredd; Kikuchi, Jun; Breuer, Christian

    2016-01-01

    Autopolyploidy is a process whereby the chromosome set is multiplied and it is a common phenomenon in angiosperms. Autopolyploidy is thought to be an important evolutionary force that has led to the formation of new plant species. Despite its relevance, the consequences of autopolyploidy in plant metabolism are poorly understood. This study compares the metabolic profiles of natural diploids and artificial autotetraploids of Arabidopsis thaliana Col-0. Different physiological parameters are compared between diploids and autotetraploids using nuclear magnetic resonance (NMR), elemental analysis (carbon:nitrogen balance) and quantitative real-time PCR (qRT-PCR). The main difference between diploid and autotetraploid A. thaliana Col-0 is observed in the concentration of metabolites related to the tricarboxylic acid cycle (TCA) and γ-amino butyric acid (GABA) shunt, as shown by multivariate statistical analysis of NMR spectra. qRT-PCR shows that genes related to the TCA and GABA shunt are also differentially expressed between diploids and autotetraploids following similar trends as their corresponding metabolites. Solid evidence is presented to demonstrate that autopolyploidy influences core plant metabolic processes. PMID:27212081

  6. Artificial Autopolyploidization Modifies the Tricarboxylic Acid Cycle and GABA Shunt in Arabidopsis thaliana Col-0.

    PubMed

    Vergara, Fredd; Kikuchi, Jun; Breuer, Christian

    2016-01-01

    Autopolyploidy is a process whereby the chromosome set is multiplied and it is a common phenomenon in angiosperms. Autopolyploidy is thought to be an important evolutionary force that has led to the formation of new plant species. Despite its relevance, the consequences of autopolyploidy in plant metabolism are poorly understood. This study compares the metabolic profiles of natural diploids and artificial autotetraploids of Arabidopsis thaliana Col-0. Different physiological parameters are compared between diploids and autotetraploids using nuclear magnetic resonance (NMR), elemental analysis (carbon:nitrogen balance) and quantitative real-time PCR (qRT-PCR). The main difference between diploid and autotetraploid A. thaliana Col-0 is observed in the concentration of metabolites related to the tricarboxylic acid cycle (TCA) and γ-amino butyric acid (GABA) shunt, as shown by multivariate statistical analysis of NMR spectra. qRT-PCR shows that genes related to the TCA and GABA shunt are also differentially expressed between diploids and autotetraploids following similar trends as their corresponding metabolites. Solid evidence is presented to demonstrate that autopolyploidy influences core plant metabolic processes. PMID:27212081

  7. Bile acid transporters

    PubMed Central

    Dawson, Paul A.; Lan, Tian; Rao, Anuradha

    2009-01-01

    In liver and intestine, transporters play a critical role in maintaining the enterohepatic circulation and bile acid homeostasis. Over the past two decades, there has been significant progress toward identifying the individual membrane transporters and unraveling their complex regulation. In the liver, bile acids are efficiently transported across the sinusoidal membrane by the Na+ taurocholate cotransporting polypeptide with assistance by members of the organic anion transporting polypeptide family. The bile acids are then secreted in an ATP-dependent fashion across the canalicular membrane by the bile salt export pump. Following their movement with bile into the lumen of the small intestine, bile acids are almost quantitatively reclaimed in the ileum by the apical sodium-dependent bile acid transporter. The bile acids are shuttled across the enterocyte to the basolateral membrane and effluxed into the portal circulation by the recently indentified heteromeric organic solute transporter, OSTα-OSTβ. In addition to the hepatocyte and enterocyte, subgroups of these bile acid transporters are expressed by the biliary, renal, and colonic epithelium where they contribute to maintaining bile acid homeostasis and play important cytoprotective roles. This article will review our current understanding of the physiological role and regulation of these important carriers. PMID:19498215

  8. Lesions of nucleus accumbens affect morphine-induced release of ascorbic acid and GABA but not of glutamate in rats.

    PubMed

    Sun, Ji Y; Yang, Jing Y; Wang, Fang; Wang, Jian Y; Song, Wu; Su, Guang Y; Dong, Ying X; Wu, Chun F

    2011-10-01

    Our previous studies have shown that local perfusion of morphine causes an increase of extracellular ascorbic acid (AA) levels in nucleus accumbens (NAc) of freely moving rats. Lines of evidence showed that glutamatergic and GABAergic were associated with morphine-induced effects on the neurotransmission of the brain, especially on the release of AA. In the present study, the effects of morphine on the release of extracellular AA, γ-aminobutyric acid (GABA) and glutamate (Glu) in the NAc following bilateral NAc lesions induced by kainic acid (KA) were studied by using the microdialysis technique, coupled to high performance liquid chromatography with electrochemical detection (HPLC-ECD) and fluorescent detection (HPLC-FD). The results showed that local perfusion of morphine (100 µM, 1 mM) in NAc dose-dependently increased AA and GABA release, while attenuated Glu release in the NAc. Naloxone (0.4 mM) pretreated by local perfusion to the NAc, significantly blocked the effects of morphine. After NAc lesion by KA (1 µg), morphine-induced increase in AA and GABA were markedly eliminated, while decrease in Glu was not affected. The loss effect of morphine on AA and GABA release after KA lesion could be recovered by GABA agonist, musimol. These results indicate that morphine-induced AA release may be mediated at least by µ-opioid receptor. Moreover, this effect of morphine possibly depend less on the glutamatergic afferents, but more on the GABAergic circuits within this nucleus. Finally, AA release induced by local perfusion of morphine may be GABA-receptor mediated and synaptically localized in the NAc. PMID:20731632

  9. Impact of Precooling and Controlled-Atmosphere Storage on γ-Aminobutyric Acid (GABA) Accumulation in Longan (Dimocarpus longan Lour.) Fruit.

    PubMed

    Zhou, Molin; Ndeurumio, Kessy H; Zhao, Lei; Hu, Zhuoyan

    2016-08-24

    Longan (Dimocarpus longan Lour.) fruit cultivars 'Chuliang' and 'Shixia' were analyzed for γ-aminobutyric acid (GABA) accumulation after precooling and in controlled-atmosphere storage. Fruit were exposed to 5% O2 plus 3%, 5%, or 10% CO2 at 4 °C, and GABA and associated enzymes, aril firmness, and pericarp color were measured. Aril softening and pericarp browning were delayed by 5% CO2 + 5% O2. GABA concentrations and glutamate decarboxylase (GAD; EC 4.1.1.15) activities declined during storage at the higher-CO2 treatments. However, GABA aminotransferase (GABA-T; EC 2.6.1.19) activities in elevated CO2-treated fruit fluctuated during storage. GABA concentrations increased after precooling treatments. GAD activity and GABA-T activity were different between cultivars after precooling. GABA concentrations in fruit increased after 3 days of 10% CO2 + 5% O2 treatment and then declined as storage time increased. GABA accumulation was associated with stimulation of GAD activity rather than inhibition of GABA-T activity. PMID:27412947

  10. GABA as a rising gliotransmitter

    PubMed Central

    Yoon, Bo-Eun; Lee, C. Justin

    2014-01-01

    Gamma-amino butyric acid (GABA) is the major inhibitory neurotransmitter that is known to be synthesized and released from GABAergic neurons in the brain. However, recent studies have shown that not only neurons but also astrocytes contain a considerable amount of GABA that can be released and activate GABA receptors in neighboring neurons. These exciting new findings for glial GABA raise further interesting questions about the source of GABA, its mechanism of release and regulation and the functional role of glial GABA. In this review, we highlight recent studies that identify the presence and release of GABA in glial cells, we show several proposed potential pathways for accumulation and modulation of glial intracellular and extracellular GABA content, and finally we discuss functional roles for glial GABA in the brain. PMID:25565970

  11. Variance analysis of gamma-aminobutyric acid (GABA)-ergic inhibitory postsynaptic currents from melanotropes of Xenopus laevis.

    PubMed Central

    Borst, J G; Kits, K S; Bier, M

    1994-01-01

    We have studied the variance in the decay of large spontaneous gamma-aminobutyric acid (GABA)-ergic inhibitory postsynaptic currents (IPSCs) in melanotropes of Xenopus laevis to obtain information about the number of GABAA receptor channels that bind GABA during the IPSCs. The average decay of the IPSCs is well described by the sum of two exponential functions. This suggests that a three-state Markov model is sufficient to describe the decay phase, with one of the three states being an absorbing state, entered when GABA dissociates from the GABAA receptor. We have compared the variance in the decay of large spontaneous IPSCs with the variance calculated for two different three-state models: a model with one open state, one closed state, and one absorbing state (I), and a model with two open states and one absorbing state (II). The data were better described by the more efficient model II. This suggests that the efficacy of GABA at synaptic GABAA receptor channels is high and that only a small number of channels are involved in generating the GABA-ergic IPSCs. PMID:7918986

  12. The GABA transporter 1 (SLC6A1): a novel candidate gene for anxiety disorders.

    PubMed

    Thoeringer, C K; Ripke, S; Unschuld, P G; Lucae, S; Ising, M; Bettecken, T; Uhr, M; Keck, M E; Mueller-Myhsok, B; Holsboer, F; Binder, E B; Erhardt, A

    2009-06-01

    Recent evidence suggests that the GABA transporter 1 (GAT-1; SLC6A1) plays a role in the pathophysiology and treatment of anxiety disorders. In order to understand the impact of genetic variation within SLC6A1 on pathological anxiety, we performed a case-control association study with anxiety disorder patients with and without syndromal panic attacks. Using the method of sequential addition of cases, we found that polymorphisms in the 5' flanking region of SLC6A1 are highly associated with anxiety disorders when considering the severity of syndromal panic attacks as phenotype covariate. Analysing the effect size of the association, we observed a constant increase in the odds ratio for disease susceptibility with an increase in panic severity (OR approximately 2.5 in severely affected patients). Nominally significant association effects were observed considering the entire patient sample. These data indicate a high load of genetic variance within SLC6A1 on pathological anxiety and highlight GAT-1 as a promising target for treatment of anxiety disorders with panic symptoms. PMID:18607529

  13. Biphenylacetic acid enhances the antagonistic action of fluoroquinolones on the GABA(A)-mediated responses of the isolated guinea-pig ileum.

    PubMed

    Koutsoviti-Papadopoulou, M; Nikolaidis, E; Kounenis, G

    2001-09-01

    This paper examines the effect of biphenylacetic acid on the antagonistic action of norfloxacin and enoxacin on the GABA(A)-mediated responses of the isolated guinea-pig ileum. GABA produced transient contractions followed by relaxation. The contractile effect of exogenously applied GABA was concentration-dependent with EC(50)= 9.8 x 10(-6) M. This contractile effect was not significantly modified by biphenylacetic acid, and the EC(50) value for GABA in the presence of 10(-5) M biphenylacetic acid was 1.15 x 10(-5) M. The GABA contractile effect was inhibited, dose-dependently, by either norfloxacin or enoxacin, but only at concentrations higher than 10(-5) M. The response of the ileum to GABA (at EC(50)) was reduced to 35 and 36% by pretreatment with 10(-5) M norfloxacin or enoxacin, respectively. However, in the presence of 10(-5) M biphenylacetic acid, the response of the ileum to GABA was reduced to 2.2% by pretreatment with 10(-5) M enoxacin, while it was completely abolished by pretreatment with 10(-5) M norfloxacin and the IC(50) values were 5.5 x 10(-7) and 1.5 x 10(-6) M for norfloxacin and enoxacin, respectively. These data show that biphenylacetic acid whilst having no effect at the GABA(A)-mediated contractile response of the guinea-pig ileum, enhances the antagonistic effect of both enoxacin and norfloxacin. This suggests that combined administration of fluoroquinolones and biphenylacetic acid synergistically inhibits GABA(A)-receptors at the intestinal level. PMID:11529690

  14. Modes and nodes explain the mechanism of action of vortioxetine, a multimodal agent (MMA): modifying serotonin's downstream effects on glutamate and GABA (gamma amino butyric acid) release.

    PubMed

    Stahl, Stephen M

    2015-08-01

    Vortioxetine is an antidepressant with multiple pharmacologic modes of action at targets where serotonin neurons connect with other neurons. These actions modify the release of both glutamate and GABA (gamma amino butyric acid) within various brain circuits. PMID:26062900

  15. Valproic acid: brain and plasma levels of the drug and its metabolites, anticonvulsant effects and gamma-aminobutyric acid (GABA) metabolism in the mouse.

    PubMed

    Nau, H; Löscher, W

    1982-03-01

    The slow onset and carry-over effect of valproic acid (VPA) therapy observed in some clinical as well as experimental animal studies have been examined by parallel pharmacokinetic and pharmacological investigations in a mouse model. VPA was rapidly transferred into brain and was cleared from that tissue with rates which exceeded plasma clearance rates. Of several VPA metabolites present in plasma, only one could be found in the brain: 2-propyl-2-pentenoic acid. This metabolite was cleared from plasma and from brain slower than the parent drug. gamma-Aminobutyric acid (GABA) concentrations were increased within 15 min after VPA injection and remained significantly elevated for at least 8 h. A similar time course was found in regard to the increase of the electroconvulsive threshold (maximal seizures) induced by VPA administration. The activity of glutamic acid decarboxylase rose parallel to the elevation of brain GABA levels, whereas the activity of GABA aminotransferase was not affected. Whereas the rapid onset of the effect on electroconvulsive threshold and on GABA metabolism can be explained by the rapid entrance of VPA into brain, the carry-over effects observed correlated with the kinetics of the metabolite 2-propyl-2-pentenoic acid better than with those of VPA due to the persistence of this metabolite in brain. PMID:6801254

  16. The canonical Notch pathway effector RBP-J regulates neuronal plasticity and expression of GABA transporters in hippocampal networks.

    PubMed

    Liu, Shuxi; Wang, Yue; Worley, Paul F; Mattson, Mark P; Gaiano, Nicholas

    2015-05-01

    Activation of the Notch pathway in neurons is essential for learning and memory in various species from invertebrates to mammals. However, it remains unclear how Notch signaling regulates neuronal plasticity, and whether the transcriptional regulator and canonical pathway effector RBP-J plays a role. Here, we report that conditional disruption of RBP-J in the postnatal hippocampus leads to defects in long-term potentiation, long-term depression, and in learning and memory. Using gene expression profiling and chromatin immunoprecipitation, we identified two GABA transporters, GAT2 and BGT1, as putative Notch/RBP-J pathway targets, which may function downstream of RBP-J to limit the accumulation of GABA in the Schaffer collateral pathway. Our results reveal an essential role for canonical Notch/RBP-J signaling in hippocampal synaptic plasticity and suggest that role, at least in part, is mediated by the regulation of GABAergic signaling. PMID:25515406

  17. The canonical Notch pathway effector RBP-J regulates neuronal plasticity and expression of GABA transporters in hippocampal networks

    PubMed Central

    Liu, Shuxi; Wang, Yue; Worley, Paul F.; Mattson, Mark P.; Gaiano, Nicholas

    2014-01-01

    Activation of the Notch pathway in neurons is essential for learning and memory in various species from invertebrates to mammals. However, it remains unclear how Notch signaling regulates neuronal plasticity, and whether the transcriptional regulator and canonical pathway effector RBP-J plays a role. Here we report that conditional disruption of RBP-J in the postnatal hippocampus leads to defects in long-term potentiation (LTP), long-term depression (LTD), and in learning and memory. Using gene expression profiling and chromatin immunoprecipitation, we identified two GABA transporters, GAT2 and BGT1, as putative Notch/RBP-J pathway targets, which may function downstream of RBP-J to limit the accumulation of GABA in the Schaffer collateral pathway. Our results reveal an essential role for canonical Notch/RBP-J signaling in hippocampal synaptic plasticity and suggest that role, at least in part, is mediated by the regulation of GABAergic signaling. PMID:25515406

  18. GABA(B) receptor subunit 1 binds to proteins affected in 22q11 deletion syndrome.

    PubMed

    Zunner, Dagmar; Deschermeier, Christina; Kornau, Hans-Christian

    2010-03-01

    GABA(B) receptors mediate slow inhibitory effects of the neurotransmitter gamma-aminobutyric acid (GABA) on synaptic transmission in the central nervous system. They function as heterodimeric G-protein-coupled receptors composed of the seven-transmembrane domain proteins GABA(B1) and GABA(B2), which are linked through a coiled-coil interaction. The ligand-binding subunit GABA(B1) is at first retained in the endoplasmic reticulum and is transported to the cell surface only upon assembly with GABA(B2). Here, we report that GABA(B1), via the coiled-coil domain, can also bind to soluble proteins of unknown function, that are affected in 22q11 deletion/DiGeorge syndrome and are therefore referred to as DiGeorge critical region 6 (DGCR6). In transfected neurons the GABA(B1)-DGCR6 association resulted in a redistribution of both proteins into intracellular clusters. Furthermore, the C-terminus of GABA(B2) interfered with the novel interaction, consistent with heterodimer formation overriding transient DGCR6-binding to GABA(B1). Thus, sequential coiled-coil interactions may direct GABA(B1) into functional receptors. PMID:20036641

  19. Dual effects of slightly acidic electrolyzed water (SAEW) treatment on the accumulation of γ-aminobutyric acid (GABA) and rutin in germinated buckwheat.

    PubMed

    Hao, Jianxiong; Wu, Tongjiao; Li, Huiying; Wang, Wei; Liu, Haijie

    2016-06-15

    In the present study, the dual effects of slightly acidic electrolyzed water (SAEW) treatment on γ-aminobutyric acid (GABA) and rutin accumulation of germinated buckwheat were evaluated during germination. The results showed that SAEW treatment (pH 5.83, ACC of 20.3 mg/L) could promote the accumulation of GABA and rutin in germinated buckwheat. The GABA and rutin contents of SAEW-germinated buckwheat reached 143.20 and 739.9 mg/100 g respectively, which is significantly higher than those of control (P<0.05). Moreover, SAEW treatment could increase the activity of glutamic acid decarboxylase (GAD) and phenylalanine ammonialyase (PAL) and thus result in the GABA and rutin accumulation of germinated buckwheat. The results suggested that SAEW treatment could promote the rutin accumulation of germinated buckwheat by influencing phenylpropanoid secondary metabolic pathway instead of the inhibition of rutin degrading enzyme (RDE) activity. In addition, SAEW treatment had no adverse impact on the sprouts growth and could reduce the microbial populations of germinated buckwheat during germination. PMID:26868552

  20. A Role for GAT-1 in Presynaptic GABA Homeostasis?

    PubMed Central

    Conti, Fiorenzo; Melone, Marcello; Fattorini, Giorgia; Bragina, Luca; Ciappelloni, Silvia

    2011-01-01

    In monoamine-releasing terminals, neurotransmitter transporters – in addition to terminating synaptic transmission by clearing released transmitters from the extracellular space – are the primary mechanism for replenishing transmitter stores and thus regulate presynaptic homeostasis. Here, we analyze whether GAT-1, the main plasma membrane GABA transporter, plays a similar role in GABAergic terminals. Re-examination of existing literature and recent data gathered in our laboratory show that GABA homeostasis in GABAergic terminals is dominated by the activity of the GABA synthesizing enzyme and that GAT-1-mediated GABA transport contributes to cytosolic GABA levels. However, analysis of GAT-1 KO, besides demonstrating the effects of reduced clearance, reveals the existence of changes compatible with an impaired presynaptic function, as miniature IPSCs frequency is reduced by one-third and glutamic acid decarboxylases and phosphate-activated glutaminase levels are significantly up-regulated. Although the changes observed are less robust than those reported in mice with impaired dopamine, noradrenaline, and serotonin plasma membrane transporters, they suggest that in GABAergic terminals GAT-1 impacts on presynaptic GABA homeostasis, and may contribute to the activity-dependent regulation of inhibitory efficacy. PMID:21503156

  1. A Role for Diminished GABA Transporter Activity in the Cortical Discharge Phenotype of MeCP2-Deficient Mice.

    PubMed

    Zhang, Liang; Wither, Robert G; Lang, Min; Wu, Chiping; Sidorova-Darmos, Elena; Netchev, Hristo; Matolcsy, Catherine B; Snead, Orlando Carter; Eubanks, James H

    2016-05-01

    Cortical network hyper-excitability is a common phenotype in mouse models lacking the transcriptional regulator methyl-CPG-binding protein 2 (MeCP2). Here, we implicate enhanced GABAB receptor activity stemming from diminished cortical expression of the GABA transporter GAT-1 in the genesis of this network hyper-excitability. We found that administering the activity-dependent GABAB receptor allosteric modulator GS-39783 to female Mecp2(+/-) mice at doses producing no effect in wild-type mice strongly potentiated their basal rates of spontaneous cortical discharge activity. Consistently, administering the GABAB receptor antagonist CGP-35348 significantly decreased basal discharge activity in these mice. Expression analysis revealed that while GABAB or extra-synaptic GABAA receptor prevalence is preserved in the MeCP2-deficient cortex, the expression of GAT-1 is significantly reduced from wild-type levels. This decrease in GAT-1 expression is consequential, as low doses of the GAT-1 inhibitor NO-711 that had no effects in wild-type mice strongly exacerbated cortical discharge activity in female Mecp2(+/-) mice. Taken together, these data indicate that the absence of MeCP2 leads to decreased cortical levels of the GAT-1 GABA transporter, which facilitates cortical network hyper-excitability in MeCP2-deficient mice by increasing the activity of cortical GABAB receptors. PMID:26499511

  2. Determination of theanine, GABA, and other amino acids in green, oolong, black, and Pu-erh teas with dabsylation and high-performance liquid chromatography.

    PubMed

    Syu, Kai-Yang; Lin, Chih-Li; Huang, Hsiu-Chen; Lin, Jen-Kun

    2008-09-10

    Dabsyl chloride (dimethylaminoazobenzene sulfonyl chloride), a useful chromophoric labeling reagent for amino acids and amines, was developed in this laboratory in 1975. Although several methods have been developed to determine various types of amino acids, a quick and easy method of determining theanine, GABA, and other amino acids has not been developed in one HPLC system. In this paper are analyzed the free amino acid contents of theanine and GABA in different teas (green tea, black tea, oolong tea, Pu-erh tea, and GABA tea) with a dabsylation and reverse phase high-performance liquid chromatography (HPLC) system coupled with a detector at 425 nm absorbance. Two reverse phase columns, Hypersil GOLD and Zorbax ODS, were used and gave different resolutions of dabsyl amino acids in the gradient elution program. The data suggest that the tea source or the steps of tea-making may contribute to the theanine contents variations. High theanine contents of high-mountain tea were observed in both green tea and oolong tea. Furthermore, the raw (natural fermented) Pu-erh tea contained more theanine than ripe (wet fermented) Pu-erh tea, and the GABA contents in normal teas were generally lower than that in GABA tea. PMID:18652476

  3. Vesicular Glutamate (VGluT), GABA (VGAT), and Acetylcholine (VAChT) Transporters in Basal Forebrain Axon Terminals Innervating the Lateral Hypothalamus

    PubMed Central

    HENNY, PABLO; JONES, BARBARA E.

    2008-01-01

    The basal forebrain (BF) is known to play important roles in cortical activation and sleep, which are likely mediated by chemically differentiated cell groups including cholinergic, γ-aminobutyric acid (GABA)ergic and other unidentified neurons. One important target of these cells is the lateral hypothalamus (LH), which is critical for arousal and the maintenance of wakefulness. To determine whether chemically specific BF neurons provide an innervation to the LH, we employed anterograde transport of 10,000 MW biotinylated dextran amine (BDA) together with immunohistochemical staining of the vesicular transporter proteins (VTPs) for glutamate (VGluT1, -2, and -3), GABA (VGAT), or acetylcholine (ACh, VAChT). In addition, we applied triple staining for the postsynaptic proteins (PSPs), PSD-95 with VGluT or Gephyrin (Geph) with VGAT, to examine whether the BDA-labeled varicosities may form excitatory or inhibitory synapses in the LH. Axons originating from BDA-labeled neurons in the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) descended within the medial forebrain bundle and extended collateral varicose fibers to contact LH neurons. In the LH, the BDA-labeled varicosities were immunopositive (+) for VAChT (~10%), VGluT2 (~25%), or VGAT (~50%), revealing an important influence of newly identified glutamatergic together with GABAergic BF inputs. Moreover, in confocal microscopy, VGluT2+ and VGAT+ terminals were apposed to PSD-95+ and Geph+ profiles respectively, indicating that they formed synaptic contacts with LH neurons. The important inputs from glutamatergic and GABAergic BF cells could thus regulate LH neurons in an opposing manner to stimulate vs. suppress cortical activation and behavioral arousal reciprocally. PMID:16572456

  4. 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.

  5. Targeted Deletion of Vesicular GABA Transporter from Retinal Horizontal Cells Eliminates Feedback Modulation of Photoreceptor Calcium Channels.

    PubMed

    Hirano, Arlene A; Liu, Xue; Boulter, Jim; Grove, James; Pérez de Sevilla Müller, Luis; Barnes, Steven; Brecha, Nicholas C

    2016-01-01

    The cellular mechanisms underlying feedback signaling from horizontal cells to photoreceptors, which are important for the formation of receptive field surrounds of early visual neurons, remain unsettled. Mammalian horizontal cells express a complement of synaptic proteins that are necessary and sufficient for calcium-dependent exocytosis of inhibitory neurotransmitters at their contacts with photoreceptor terminals, suggesting that they are capable of releasing GABA via vesicular release. To test whether horizontal cell vesicular release is involved in feedback signaling, we perturbed inhibitory neurotransmission in these cells by targeted deletion of the vesicular GABA transporter (VGAT), the protein responsible for the uptake of inhibitory transmitter by synaptic vesicles. To manipulate horizontal cells selectively, an iCre mouse line with Cre recombinase expression controlled by connexin57 (Cx57) regulatory elements was generated. In Cx57-iCre mouse retina, only horizontal cells expressed Cre protein, and its expression occurred in all retinal regions. After crossing with a VGAT(flox/flox) mouse line, VGAT was selectively eliminated from horizontal cells, which was confirmed immunohistochemically. Voltage-gated ion channel currents in horizontal cells of Cx57-VGAT(-/-) mice were the same as Cx57-VGAT(+/+) controls, as were the cell responses to the ionotropic glutamate receptor agonist kainate, but the response to the GABAA receptor agonist muscimol in Cx57-VGAT(-/-) mice was larger. In contrast, the feedback inhibition of photoreceptor calcium channels, which in control animals is induced by horizontal cell depolarization, was completely absent in Cx57-VGAT(-/-) mice. The results suggest that vesicular release of GABA from horizontal cells is required for feedback inhibition of photoreceptors. PMID:27022629

  6. Targeted Deletion of Vesicular GABA Transporter from Retinal Horizontal Cells Eliminates Feedback Modulation of Photoreceptor Calcium Channels123

    PubMed Central

    Liu, Xue; Boulter, Jim; Grove, James; Pérez de Sevilla Müller, Luis; Barnes, Steven; Brecha, Nicholas C.

    2016-01-01

    Abstract The cellular mechanisms underlying feedback signaling from horizontal cells to photoreceptors, which are important for the formation of receptive field surrounds of early visual neurons, remain unsettled. Mammalian horizontal cells express a complement of synaptic proteins that are necessary and sufficient for calcium-dependent exocytosis of inhibitory neurotransmitters at their contacts with photoreceptor terminals, suggesting that they are capable of releasing GABA via vesicular release. To test whether horizontal cell vesicular release is involved in feedback signaling, we perturbed inhibitory neurotransmission in these cells by targeted deletion of the vesicular GABA transporter (VGAT), the protein responsible for the uptake of inhibitory transmitter by synaptic vesicles. To manipulate horizontal cells selectively, an iCre mouse line with Cre recombinase expression controlled by connexin57 (Cx57) regulatory elements was generated. In Cx57-iCre mouse retina, only horizontal cells expressed Cre protein, and its expression occurred in all retinal regions. After crossing with a VGATflox/flox mouse line, VGAT was selectively eliminated from horizontal cells, which was confirmed immunohistochemically. Voltage-gated ion channel currents in horizontal cells of Cx57-VGAT−/− mice were the same as Cx57-VGAT+/+ controls, as were the cell responses to the ionotropic glutamate receptor agonist kainate, but the response to the GABAA receptor agonist muscimol in Cx57-VGAT−/− mice was larger. In contrast, the feedback inhibition of photoreceptor calcium channels, which in control animals is induced by horizontal cell depolarization, was completely absent in Cx57-VGAT−/− mice. The results suggest that vesicular release of GABA from horizontal cells is required for feedback inhibition of photoreceptors. PMID:27022629

  7. Stable isotope dilution HILIC-MS/MS method for accurate quantification of glutamic acid, glutamine, pyroglutamic acid, GABA and theanine in mouse brain tissues.

    PubMed

    Inoue, Koichi; Miyazaki, Yasuto; Unno, Keiko; Min, Jun Zhe; Todoroki, Kenichiro; Toyo'oka, Toshimasa

    2016-01-01

    In this study, we developed the stable isotope dilution hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) technique for the accurate, reasonable and simultaneous quantification of glutamic acid (Glu), glutamine (Gln), pyroglutamic acid (pGlu), γ-aminobutyric acid (GABA) and theanine in mouse brain tissues. The quantification of these analytes was accomplished using stable isotope internal standards and the HILIC separating mode to fully correct the intramolecular cyclization during the electrospray ionization. It was shown that linear calibrations were available with high coefficients of correlation (r(2)  > 0.999, range from 10 pmol/mL to 50 mol/mL). For application of the theanine intake, the determination of Glu, Gln, pGlu, GABA and theanine in the hippocampus and central cortex tissues was performed based on our developed method. In the region of the hippocampus, the concentration levels of Glu and pGlu were significantly reduced during reality-based theanine intake. Conversely, the concentration level of GABA increased. This result showed that transited theanine has an effect on the metabolic balance of Glu analogs in the hippocampus. PMID:26033549

  8. Role of a gamma-aminobutryic acid (GABA) receptor mutation in the evolution and spread of Diabrotica virgifera virgifera resistance to cyclodiene insecticides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An alanine to serine amino acid substitution within the Rdl subunit of the gamma-aminobutyric acid (GABA) receptor confers resistance to cyclodiene insecticides in many species. The corn rootworm, Diabrotica virgifera virgifera, is a damaging pest of cultivated corn that was partially controlled by ...

  9. Enhancing Contents of γ-Aminobutyric Acid (GABA) and Other Micronutrients in Dehulled Rice during Germination under Normoxic and Hypoxic Conditions.

    PubMed

    Ding, Junzhou; Yang, Tewu; Feng, Hao; Dong, Mengyi; Slavin, Margaret; Xiong, Shanbai; Zhao, Siming

    2016-02-10

    Biofortification of staple grains with high contents of essential micronutrients is an important strategy to overcome micronutrient malnutrition. However, few attempts have targeted at γ-aminobutyric acid (GABA), a functional nutrient for aging populations. In this study, two rice cultivars, Heinuo and Xianhui 207, were used to investigate changes in GABA and other nutritional compounds of dehulled rice after germination under normoxic and hypoxic conditions. Forty-one metabolites were identified in both cultivars treated by normoxic germination, whereas the germinated dehulled rice of Heinuo and Xianhui 207 under hypoxic treatment had 43 and 41 metabolites identified, respectively. GABA increased in dehulled rice after germination, especially under hypoxia. Meanwhile, a number of other health-beneficial and/or flavor-related compounds such as lysine and d-mannose increased after the hypoxic treatment. The accumulation of GABA exhibited genotype-specific modes in both normoxic and hypoxic treatments. With regard to GABA production, Xianhui 207 was more responsive to the germination process than Heinuo, whereas Heinuo was more responsive to hypoxia than Xianhui 207. This study provides a promising approach to biofortify dehulled rice with increased GABA and other nutrients through metabolomic-based regulation. PMID:26765954

  10. 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. PMID:24829506

  11. Plasmalemmal and Vesicular γ-Aminobutyric Acid Transporter Expression in the Developing Mouse Retina

    PubMed Central

    GUO, CHENYING; STELLA, SALVATORE L.; HIRANO, ARLENE A.; BRECHA, NICHOLAS C.

    2009-01-01

    Plasmalemmal and vesicular γ-aminobutyric acid (GABA) transporters influence neurotransmission by regulating high-affinity GABA uptake and GABA release into the synaptic cleft and extracellular space. Postnatal expression of the plasmalemmal GABA transporter-1 (GAT-1), GAT-3, and the vesicular GABA/glycine transporter (VGAT) were evaluated in the developing mouse retina by using immunohistochemistry with affinity-purified antibodies. Weak transporter immunoreactivity was observed in the inner retina at postnatal day 0 (P0). GAT-1 immunostaining at P0 and at older ages was in amacrine and displaced amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), respectively, and in their processes in the inner plexiform layer (IPL). At P10, weak GAT-1 immunostaining was in Müller cell processes. GAT-3 immunostaining at P0 and older ages was in amacrine cells and their processes, as well as in Müller cells and their processes that extended radially across the retina. At P10, Müller cell somata were observed in the middle of the INL. VGAT immunostaining was present at P0 and older ages in amacrine cells in the INL as well as processes in the IPL. At P5, weak VGAT immunostaining was also observed in horizontal cell somata and processes. By P15, the GAT and VGAT immunostaining patterns appear similar to the adult immunostaining patterns; they reached adult levels by about P20. These findings demonstrate that GABA uptake and release are initially established in the inner retina during the first postnatal week and that these systems subsequently mature in the outer retina during the second postnatal week. PMID:18975268

  12. Up-regulation of gamma-aminobutyric acid transporter I mediates ethanol sensitivity in mice.

    PubMed

    Hu, J-H; Ma, Y-H; Yang, N; Mei, Z-T; Zhang, M-H; Fei, J; Guo, L-H

    2004-01-01

    Ethanol is among the most widely abused drugs in the world. Chronic ethanol consumption leads to ethanol tolerance and addiction, and impairs learning and memory. Na+/Cl- dependent GABA transporters play an important role in controlling the concentration of GABA in the synaptic cleft, and thus they control the intensity and duration of synaptic transmission of GABA. It has been suggested that GABAergic system is involved in ethanol consumption, tolerance and addiction, because chronic ethanol consumption alters the expression of GABAA receptors and drugs on GABA receptors affect ethanol actions. The results of the present study reveal that that activity of GABA transporters in mouse brain after 15-min acute ethanol injection or after chronic ethanol consumption is increased. Moreover, mice pre-injected with a competitive or a noncompetitive antagonist of gamma-aminobutyric acid transporter subtype 1 (GAT1) showed high sensitivity to the sedative/hypnotic effects of ethanol. In contrast, transgenic mice overexpressing GAT1 displayed low sensitivity to ethanol, as shown by the righting reflex test. Mice overexpressing GAT1 survived a lethal dose of ethanol (9 g/kg, i.p.) longer, maintained locomotor activity longer after a sub-lethal dose (1.75 g/kg, i.p.) and exhibited a higher median lethal dose than wild-type littermates. These results suggest that GAT1 plays an important role in sensitivity to ethanol, and might be a therapeutic target for alcoholism prevention and treatment. Acute and chronic ethanol administration resulted in the increase of GABA transporter function. Use of GAT1 selective inhibitors and GAT1 overexpressing mice thus demonstrate that GAT1 should be an important protein mediating sensitivity to ethanol in mice. PMID:14751274

  13. Adenosine-to-inosine RNA editing affects trafficking of the gamma-aminobutyric acid type A (GABA(A)) receptor.

    PubMed

    Daniel, Chammiran; Wahlstedt, Helene; Ohlson, Johan; Björk, Petra; Ohman, Marie

    2011-01-21

    Recoding by adenosine-to-inosine RNA editing plays an important role in diversifying proteins involved in neurotransmission. We have previously shown that the Gabra-3 transcript, coding for the α3 subunit of the GABA(A) receptor is edited in mouse, causing an isoleucine to methionine (I/M) change. Here we show that this editing event is evolutionarily conserved from human to chicken. Analyzing recombinant GABA(A) receptor subunits expressed in HEK293 cells, our results suggest that editing at the I/M site in α3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of α3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for α3 in combination with either the β2 or the β3 subunit. Further, an amino acid substitution at the corresponding I/M site in the α1 subunit has a similar effect on cell surface presentation, indicating the importance of this site for receptor trafficking. We show that the I/M editing during brain development is inversely related to the α3 protein abundance. Our results suggest that editing controls trafficking of α3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of α subunits in the adult brain. PMID:21030585

  14. 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

  15. GABA Signaling and Neuroactive Steroids in Adrenal Medullary Chromaffin Cells.

    PubMed

    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

  16. Promoters inducible by aromatic amino acids and γ-aminobutyrate (GABA) for metabolic engineering applications in Saccharomyces cerevisiae.

    PubMed

    Kim, Sujin; Lee, Kyusung; Bae, Sang-Jeong; Hahn, Ji-Sook

    2015-03-01

    A wide range of promoters with different strengths and regulatory mechanisms are valuable tools in metabolic engineering and synthetic biology. While there are many constitutive promoters available, the number of inducible promoters is still limited for pathway engineering in Saccharomyces cerevisiae. Here, we constructed aromatic amino-acid-inducible promoters based on the binding sites of Aro80 transcription factor, which is involved in the catabolism of aromatic amino acids through transcriptional activation of ARO9 and ARO10 genes in response to aromatic amino acids. A dynamic range of tryptophan-inducible promoter strengths can be obtained by modulating the number of Aro80 binding sites, plasmid copy numbers, and tryptophan concentrations. Using low and high copy number plasmid vectors and different tryptophan concentrations, a 29-fold range of fluorescence intensities of enhanced green fluorescent protein (EGFP) reporter could be achieved from a synthetic U4C ARO9 promoter, which is composed of four repeats of Aro80 binding half site (CCG) and ARO9 core promoter element. The U4C ARO9 promoter was applied to express alsS and alsD genes from Bacillus subtilis for acetoin production in S. cerevisiae, resulting in a gradual increase in acetoin titers depending on tryptophan concentrations. Furthermore, we demonstrated that γ-aminobutyrate (GABA)-inducible UGA4 promoter, regulated by Uga3, can also be used in metabolic engineering as a dose-dependent inducible promoter. The wide range of controllable expression levels provided by these tryptophan- and GABA-inducible promoters might contribute to fine-tuning gene expression levels and timing for the optimization of pathways in metabolic engineering. PMID:25573467

  17. gamma-Aminobutyric acid (GABA)-induced currents of skate Muller (glial) cells are mediated by neuronal-like GABAA receptors.

    PubMed Central

    Malchow, R P; Qian, H H; Ripps, H

    1989-01-01

    Radial glia (Muller cells) of the vertebrate retina appear to be intimately involved in regulating the actions of amino acid neurotransmitters. One of the amino acids thought to be important in mediating retinal information flow is gamma-aminobutyric acid (GABA). The findings of this study indicate that enzymatically isolated skate Muller cells are depolarized by GABA and the GABAA agonist muscimol and that the actions of these agents are reduced by bicuculline and picrotoxin. Membrane currents induced by GABA under voltage clamp were dose dependent, were associated with an increase in membrane conductance, and showed marked desensitization when the concentration of GABA exceeded 2.5 microM. The responses had a reversal potential close to that calculated for chloride, indicating that the currents were generated by ions passing through channels. These data support the view that skate Muller cells possess functional GABAA receptors. The presence of such receptors on retinal glia may have important implications for the role of Muller cells in maintaining the constancy of the extracellular milieu, for neuron-glia interactions within the retina, and for theories concerning the generation of the electroretinogram. Images PMID:2567001

  18. Cellular synthesis and axonal transport of gamma-aminobutyric acid in a photoreceptor cell of the barnacle.

    PubMed Central

    Koike, H; Tsuda, K

    1980-01-01

    1. [3H]glutamate or [3H]gamma-aminobutyric acid (GABA) was injected into the photoreceptor cell of the lateral ocellus of Balanus eburneus, in order to study the transmitter substance of the cell. 2. The photoreceptor cell synthesized [3H]GABA from injected [3H]glutamate. 3. The newly formed [3H]GABA moved inside the photoreceptor axon towards the axon terminal with a velocity of about 0.9 mm/hr. Injected [3H]GABA moved at 0.9 mm/hr and also at 0.4 mm/hr. 4. Axonally transported [3H]GABA reached the axon terminal within several hours following the injection. It did not accumulate at the terminal, but gradually disappeared. 5. Light-microscope and electron-microscope autoradiography following the injection of [3H]GABA revealed that [3H]-reacted silver grains were present in a certain type of axon terminal. The terminal thus identified as that of a photoreceptor cell contains many clear, polymorphic synaptic vesicles about 300-500 A in diameter, some dense-cored vesicles 700-1300 A in diameter, and glycogen granules. The terminal forms many synapses, and each synapse has a synaptic dense body. The terminal always faces two post-synaptic elements at the synapse, forming a triad with a gap distance of about 160-200 A. 6. A GABA analogue, [3H]di-aminobutyric acid, was selectively taken up into the terminals previously identified as those of photoreceptors. 7. These results support the notion that the transmitter substance of the photoreceptor cell of the barnacle is GABA. Images Plate 1 Plate 2 PMID:6160239

  19. [Inherited amino acid transport disorders].

    PubMed

    Igarashi, Y; Tada, K

    1992-07-01

    Disorders due to inherited amino acids transport defect are reviewed. The disorders were categorized into three types of transport defects, namely, brush-border membrane of epithelial cells of small intestine and kidney tubules (Hartnup disease, blue diaper syndrome, cystinuria, iminoglycinuria and lysine malabsorption syndrome), basolateral membrane (lysinuric protein intolerance) and membrane of intracellular organelles (cystinosis and hyperornitinemia-hyperammonemia-homocitrullinuria syndrome). Pathogenesis, clinical feature, laboratory findings, diagnosis, genetics and treatment of these disorders are described, briefly. There is not much data for the transport systems themselves, so that further investigation in molecular and gene levels for transport systems is necessary to clarify the characteristics of the transport and heterogeneity of phenotypes in inherited amino acids transport disorders. PMID:1404888

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

    PubMed Central

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

    2014-01-01

    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. PMID:25485164

  1. Imidase catalyzing desymmetric imide hydrolysis forming optically active 3-substituted glutaric acid monoamides for the synthesis of gamma-aminobutyric acid (GABA) analogs.

    PubMed

    Nojiri, Masutoshi; Hibi, Makoto; Shizawa, Hiroaki; Horinouchi, Nobuyuki; Yasohara, Yoshihiko; Takahashi, Satomi; Ogawa, Jun

    2015-12-01

    The recent use of optically active 3-substituted gamma-aminobutyric acid (GABA) analogs in human therapeutics has identified a need for an efficient, stereoselective method of their synthesis. Here, bacterial strains were screened for enzymes capable of stereospecific hydrolysis of 3-substituted glutarimides to generate (R)-3-substituted glutaric acid monoamides. The bacteria Alcaligenes faecalis NBRC13111 and Burkholderia phytofirmans DSM17436 were discovered to hydrolyze 3-(4-chlorophenyl) glutarimide (CGI) to (R)-3-(4-chlorophenyl) glutaric acid monoamide (CGM) with 98.1% enantiomeric excess (e.e.) and 97.5% e.e., respectively. B. phytofirmans DSM17436 could also hydrolyze 3-isobutyl glutarimide (IBI) to produce (R)-3-isobutyl glutaric acid monoamide (IBM) with 94.9% e.e. BpIH, an imidase, was purified from B. phytofirmans DSM17436 and found to generate (R)-CGM from CGI with specific activity of 0.95 U/mg. The amino acid sequence of BpIH had a 75% sequence identity to that of allantoinase from A. faecalis NBRC13111 (AfIH). The purified recombinant BpIH and AfIH catalyzed (R)-selective hydrolysis of CGI and IBI. In addition, a preliminary investigation of the enzymatic properties of BpIH and AfIH revealed that both enzymes were stable in the range of pH 6-10, with an optimal pH of 9.0, stable at temperatures below 40 °C, and were not metalloproteins. These results indicate that the use of this class of hydrolase to generate optically active 3-substituted glutaric acid monoamide could simplify the production of specific chiral GABA analogs for drug therapeutics. PMID:26205522

  2. Vesicular γ-Aminobutyric Acid Transporter Expression in Amacrine and Horizontal Cells

    PubMed Central

    Cueva, Juan G.; Haverkamp, Silke; Reimer, Richard J.; Edwards, Robert; Wässle, Heinz; Brecha, Nicholas C.

    2010-01-01

    The vesicular γ-aminobutyric acid (GABA) transporter (VGAT), which transports the inhibitory amino acid transmitters GABA and glycine, is localized to synaptic vesicles in axon terminals. The localization of VGAT immunoreactivity to mouse and rat retina was evaluated with light and electron microscopy by using well-characterized VGAT antibodies. Specific VGAT immunoreactivity was localized to numerous varicose processes in all laminae of the inner plexiform layer (IPL) and to the outer plexiform layer (OPL). Amacrine cell somata characterized by weak VGAT immunoreactivity in the cytoplasm were located in the ganglion cell layer and proximal inner nuclear layer (INL) adjacent to the IPL. In rat retina, VGAT-immunoreactive cell bodies also contained GABA, glycine, or parvalbumin (PV) immunoreactivity, suggesting vesicular uptake of GABA or glycine by these cells. A few varicose VGAT-immunoreactive processes entered the OPL from the IPL. VGAT immunoreactivity in the OPL was predominantly localized to horizontal cell processes. VGAT and calcium binding protein-28K immunoreactivities (CaBP; a marker for horizontal cells) were colocalized in processes and terminals distributed to the OPL. Furthermore, VGAT immunoreactivity overlapped or was immediately adjacent to postsynaptic density-95 (PSD-95) immunoreactivity, which is prominent in photoreceptor terminals. Preem-bedding immunoelectron microscopy of mouse and rat retinae showed that VGAT immunoreactivity was localized to horizontal cell processes and their terminals. Immunoreactivity was distributed throughout the cytoplasm of the horizontal cell processes. Taken together, these findings demonstrate VGAT immunoreactivity in both amacrine and horizontal cell processes, suggesting these cells contain vesicles that accumulate GABA and glycine, possibly for vesicular release. PMID:11920703

  3. Hydroxy-1,2,5-oxadiazolyl moiety as bioisoster of the carboxy function. A computational study on gamma-aminobutyric acid (GABA) related compounds.

    PubMed

    Tosco, Paolo; Lolli, Marco L

    2008-04-01

    Recently, our research group has proposed the hydroxyfurazanyl (4-hydroxy-1,2,5-oxadiazole-3-yl) moiety as a new non-classical isoster of the carboxy function in the design of gamma-aminobutyric acid (GABA) analogues. Some compounds showed significant activity at the GABA(A) receptor, representing the only examples of pentatomic heterocycles bearing an omega-aminoalkyl flexible side chain in the position vicinal to the hydroxy group displaying agonist activity at this receptor subtype. In this work, an ab initio analysis of the structural and electronic features of furazan-3-ol is presented, in order to provide a theoretical basis to the claimed bioisosterism with the carboxy function. An ab initio conformational study with the C-PCM implicit solvent model was carried out to elucidate the reasons of the peculiar behaviour of the furazan models. Alongside, another conformational search through molecular dynamics in explicit solvent was accomplished, in order to validate the first method. The electronic features of the 4-hydroxy-1,2,5-oxadiazole-3-yl substructure seem to account for a marked stabilising effect of the putative bioactive conformation at the GABA(A) receptor subtype. The 1,2,5-thiadiazole analogue, which shares the same conformational preference of its oxygenated counterpart, was identified as a potential candidate for synthesis and pharmacological testing. Figure 4-(omega-aminoalkyl)-1,2,5-oxadiazole-3-ol analogues of GABA. PMID:18247067

  4. Retinoic Acid, GABA-ergic, and TGF-β Signaling Systems Are Involved in Human Cleft Palate Fibroblast Phenotype

    PubMed Central

    Baroni, Tiziano; Bellucci, Catia; Lilli, Cinzia; Pezzetti, Furio; Carinci, Francesco; Becchetti, Ennio; Carinci, Paolo; Stabellini, Giordano; Calvitti, Mario; Lumare, Eleonora; Bodo, Maria

    2006-01-01

    During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-β (TGF-β), retinoic acid (RA), and γ-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-β binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA—which, at pharmacologic doses, induces cleft palate in newborns of many species—were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-β3 mRNA expression and TGF-β receptor number were higher and RA receptor-α (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-β3 mRNA expression but reduced the number of TGF-β receptors. TGF-β receptor type I mRNA expression was decreased, TGF-β receptor type II was increased, and TGF-β receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-β signaling systems could be involved in human cleft

  5. 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

  6. Dopaminergic neurons inhibit striatal output via non-canonical release of GABA

    PubMed Central

    Tritsch, Nicolas X.; Ding, Jun B.; Sabatini, Bernardo L.

    2012-01-01

    The substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) contain the two largest populations of dopamine (DA)-releasing neurons in the mammalian brain. These neurons extend elaborate projections in striatum, a large subcortical structure implicated in motor planning and reward-based learning. Phasic activation of dopaminergic neurons in response to salient or reward-predicting stimuli is thought to modulate striatal output via the release of DA to promote and reinforce motor action1–4. Here we show that activation of DA neurons in striatal slices rapidly inhibits action potential firing in both direct-and indirect-pathway striatal projection neurons (SPNs) through vesicular release of the inhibitory transmitter γ-aminobutyric acid (GABA). GABA is released directly from dopaminergic axons but in a manner that is independent of the vesicular GABA transporter VGAT. Instead GABA release requires activity of the vesicular monoamine transporter VMAT2, which is the vesicular transporter for DA. Furthermore, VMAT2 expression in GABAergic neurons lacking VGAT is sufficient to sustain GABA release. Thus, these findings expand the repertoire of synaptic mechanisms employed by DA neurons to influence basal ganglia circuits, reveal a novel substrate whose transport is dependent on VMAT2, and demonstrate that GABA can function as a bona fide co-transmitter in monoaminergic neurons. PMID:23034651

  7. Differential maturation of vesicular glutamate and GABA transporter expression in the mouse auditory forebrain during the first weeks of hearing.

    PubMed

    Hackett, Troy A; Clause, Amanda R; Takahata, Toru; Hackett, Nicholas J; Polley, Daniel B

    2016-06-01

    Vesicular transporter proteins are an essential component of the presynaptic machinery that regulates neurotransmitter storage and release. They also provide a key point of control for homeostatic signaling pathways that maintain balanced excitation and inhibition following changes in activity levels, including the onset of sensory experience. To advance understanding of their roles in the developing auditory forebrain, we tracked the expression of the vesicular transporters of glutamate (VGluT1, VGluT2) and GABA (VGAT) in primary auditory cortex (A1) and medial geniculate body (MGB) of developing mice (P7, P11, P14, P21, adult) before and after ear canal opening (~P11-P13). RNA sequencing, in situ hybridization, and immunohistochemistry were combined to track changes in transporter expression and document regional patterns of transcript and protein localization. Overall, vesicular transporter expression changed the most between P7 and P21. The expression patterns and maturational trajectories of each marker varied by brain region, cortical layer, and MGB subdivision. VGluT1 expression was highest in A1, moderate in MGB, and increased with age in both regions. VGluT2 mRNA levels were low in A1 at all ages, but high in MGB, where adult levels were reached by P14. VGluT2 immunoreactivity was prominent in both regions. VGluT1 (+) and VGluT2 (+) transcripts were co-expressed in MGB and A1 somata, but co-localization of immunoreactive puncta was not detected. In A1, VGAT mRNA levels were relatively stable from P7 to adult, while immunoreactivity increased steadily. VGAT (+) transcripts were rare in MGB neurons, whereas VGAT immunoreactivity was robust at all ages. Morphological changes in immunoreactive puncta were found in two regions after ear canal opening. In the ventral MGB, a decrease in VGluT2 puncta density was accompanied by an increase in puncta size. In A1, perisomatic VGAT and VGluT1 terminals became prominent around the neuronal somata. Overall, the

  8. 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.

  9. Use of sourdough fermentation and pseudo-cereals and leguminous flours for the making of a functional bread enriched of gamma-aminobutyric acid (GABA).

    PubMed

    Coda, Rossana; Rizzello, Carlo Giuseppe; Gobbetti, Marco

    2010-02-28

    Lactobacillus plantarum C48 and Lactococcus lactis subsp. lactis PU1, previously selected for the biosynthesis of gamma-aminobutyric acid (GABA), were used for sourdough fermentation of cereal, pseudo-cereal and leguminous flours. Chickpea, amaranth, quinoa and buckwheat were the flours most suitable to be enriched of GABA. The parameters of sourdough fermentation were optimized. Addition of 0.1mM pyridoxal phosphate, dough yield of 160, inoculum of 5 x 10(7)CFU/g of starter bacteria and fermentation for 24h at 30 degrees C were found to be the optimal conditions. A blend of buckwheat, amaranth, chickpea and quinoa flours (ratio 1:1:5.3:1) was selected and fermented with baker's yeast (non-conventional flour bread, NCB) or with Lb. plantarum C48 sourdough (non-conventional flour sourdough bread, NCSB) and compared to baker's yeast started wheat flour bread (WFB). NCSB had the highest concentration of free amino acids and GABA (ca. 4467 and 504 mg/kg, respectively). The concentration of phenolic compounds and antioxidant activity of NCSB bread was the highest, as well as the rate of in vitro starch hydrolysis was the lowest. Texture analysis showed that sourdough fermentation enhances several characteristics of NCSB with respect to NCB, thus approaching the features of WFB. Sensory analysis showed that sourdough fermentation allowed to get good palatability and overall taste appreciation. PMID:20071045

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

    PubMed

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

    2015-07-01

    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

  11. Gamma-aminobutyric acid (GABA) and neuropeptides in neural areas mediating motion-induced emesis

    NASA Technical Reports Server (NTRS)

    Damelio, F.; Daunton, Nancy G.; Fox, Robert A.

    1991-01-01

    Immunocytochemical methods were employed to localize the neurotransmitter amino acid gamma-aminobutyric acid and the neuropeptides substance P and Met-enkephalin in the area postrema (AP), area subpostrema (ASP), nucleus of the tractus solitarius (NTS), dorsal motor nucleus of the vagus nerve (DMNV), and lateral vestibular nucleus (LVN). Glutamic acid decarboxylase immunoreactive (GAD-IR) terminals and fibers were observed in the AP and particularly in the ASP. A gradual decrease in the density of terminals was seen towards the solitary complex. The DMNV revealed irregularly scattered GAD-IR terminals within the neuropil or closely surrounding neuronal cell bodies. The LVN, particularly the dorsal division, showed numerous axon terminals which were mostly localize around large neurons and their proximal dendrites. Substance P immunoreactive (SP-IR) terminals and fibers showed high density in the solitary complex, in particular within the lateral division. The ASP showed medium to low density of SP-IR fibers and terminals. The AP exhibited a small number of fibers and terminals irregularly distributed. The DMNV revealed a high density of SP-IR terminals and fibers that were mainly concentrated in the periphery. Very few terminals were detected in the LVN. Met-enkephalin immunoreactive (Met-Enk-IR) fibers and terminals showed high density and uniform distribution in the DMNV. Scattered terminals and fibers were observed in the AP, ASP, and NTS (particularly the lateral division). The very few fibers were observed in the LVN surrounded the neuronal cell bodies. The present report is part of a study designed to investigate the interaction between neuropeptides and conventional neurotransmitters under conditions producing motion sickness and in the process of sensory-motor adaptation.

  12. L-DOPA Reverses the Increased Free Amino Acids Tissue Levels Induced by Dopamine Depletion and Rises GABA and Tyrosine in the Striatum.

    PubMed

    Solís, Oscar; García-Sanz, Patricia; Herranz, Antonio S; Asensio, María-José; Moratalla, Rosario

    2016-07-01

    Perturbations in the cerebral levels of various amino acids are associated with neurological disorders, and previous studies have suggested that such alterations have a role in the motor and non-motor symptoms of Parkinson's disease. However, the direct effects of chronic L-DOPA treatment, that produces dyskinesia, on neural tissue amino acid concentrations have not been explored in detail. To evaluate whether striatal amino acid concentrations are altered in peak dose dyskinesia, 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian mice were treated chronically with L-DOPA and tissue amino acid concentrations were assessed by HPLC analysis. These experiments revealed that neither 6-OHDA nor L-DOPA treatment are able to alter glutamate in the striatum. However, glutamine increases after 6-OHDA and returns back to normal levels with L-DOPA treatment, suggesting increased striatal glutamatergic transmission with lack of dopamine. In addition, glycine and taurine levels are increased following dopamine denervation and restored to normal levels by L-DOPA. Interestingly, dyskinetic animals showed increased levels of GABA and tyrosine, while aspartate striatal tissue levels are not altered. Overall, our results indicate that chronic L-DOPA treatment, besides normalizing the altered levels of some amino acids after 6-OHDA, robustly increases striatal GABA and tyrosine levels which may in turn contribute to the development of L-DOPA-induced dyskinesia. PMID:26966009

  13. [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. PMID:9503572

  14. Novel functions of GABA signaling in adult neurogenesis.

    PubMed

    Pontes, Adalto; Zhang, Yonggang; Hu, Wenhui

    2013-10-01

    Neurotransmitter gamma-aminobutiric acid (GABA) through ionotropic GABAA and metabotropic GABAB receptors plays key roles in modulating the development, plasticity and function of neuronal networks. GABA is inhibitory in mature neurons but excitatory in immature neurons, neuroblasts and neural stem/progenitor cells (NSCs/NPCs). The switch from excitatory to inhibitory occurs following the development of glutamatergic synaptic input and results from the dynamic changes in the expression of Na(+)/K(+)/2Cl(-) co-transporter NKCC1 driving Cl(-) influx and neuron-specific K(+)/Cl(-) co-transporter KCC2 driving Cl(-) efflux. The developmental transition of KCC2 expression is regulated by Disrupted-in-Schizophrenia 1 (DISC1) and brain-derived neurotrophic factor (BDNF) signaling. The excitatory GABA signaling during early neurogenesis is important to the activity/experience-induced regulation of NSC quiescence, NPC proliferation, neuroblast migration and newborn neuronal maturation/functional integration. The inhibitory GABA signaling allows for the sparse and static functional networking essential for learning/memory development and maintenance. PMID:24285940

  15. Novel functions of GABA signaling in adult neurogenesis

    PubMed Central

    PONTES, Adalto; ZHANG, Yonggang; HU, Wenhui

    2013-01-01

    Neurotransmitter gamma-aminobutiric acid (GABA) through ionotropic GABAA and metabotropic GABAB receptors plays key roles in modulating the development, plasticity and function of neuronal networks. GABA is inhibitory in mature neurons but excitatory in immature neurons, neuroblasts and neural stem/progenitor cells (NSCs/NPCs). The switch from excitatory to inhibitory occurs following the development of glutamatergic synaptic input and results from the dynamic changes in the expression of Na+/K+/2Cl− co-transporter NKCC1 driving Cl− influx and neuron-specific K+/Cl− co-transporter KCC2 driving Cl− efflux. The developmental transition of KCC2 expression is regulated by Disrupted-in-Schizophrenia 1 (DISC1) and brain-derived neurotrophic factor (BDNF) signaling. The excitatory GABA signaling during early neurogenesis is important to the activity/experience-induced regulation of NSC quiescence, NPC proliferation, neuroblast migration and newborn neuronal maturation/functional integration. The inhibitory GABA signaling allows for the sparse and static functional networking essential for learning/memory development and maintenance. PMID:24285940

  16. GABA induces functionally active low-affinity GABA receptors on cultured cerebellar granule cells.

    PubMed

    Meier, E; Drejer, J; Schousboe, A

    1984-12-01

    The effect of gamma-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo[5-4-c]pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 microM GABA in the culture media led to the induction of low-affinity GABA receptors (KD 546 +/- 117 nM) in addition to the high-affinity receptors (KD 7 +/- 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED50 10 +/- 3 microM) only when the cells had been cultured in the presence of 50 microM GABA, 50 microM muscimol, or 150 microM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 microM bicuculline and mimicked by 50 microM muscimol or 150 microM THIP whereas 150 microM (-)-baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABAA receptors. PMID:6149269

  17. GABA release by hippocampal astrocytes

    PubMed Central

    Le Meur, Karim; Mendizabal-Zubiaga, Juan; Grandes, Pedro; Audinat, Etienne

    2012-01-01

    Astrocytes can directly influence neuronal activity through the release of various transmitters acting on membrane receptors expressed by neurons. However, in contrast to glutamate and ATP for instance, the release of GABA (γ-amino-butyric acid) by astrocytes is still poorly documented. Here, we used whole-cell recordings in rat acute brain slices and electron microscopy to test whether hippocampal astrocytes release the inhibitory transmitter GABA. We observed that slow transient inhibitory currents due to the activation of GABAA receptors occur spontaneously in principal neurons of the three main hippocampal fields (CA1, CA3, and dentate gyrus). These currents share characteristics with the slow NMDA receptor-mediated currents previously shown to result from astrocytic glutamate release: they occur in the absence of synaptic transmission and have variable kinetics and amplitudes as well as low frequencies. Osmotic pressure reduction, known to enhance transmitter release from astrocytes, similarly increased the frequency of non-synaptic GABA and glutamate currents. Simultaneous occurrence of slow inhibitory and excitatory currents was extremely rare. Yet, electron microscopy examination of immunostained hippocampal sections shows that about 80% of hippocampal astrocytes [positive for glial fibrillary acidic protein (GFAP)] were immunostained for GABA. Our results provide quantitative characteristics of the astrocyte-to-neuron GABAergic signaling. They also suggest that all principal neurons of the hippocampal network are under a dual, excitatory and inhibitory, influence of astrocytes. The relevance of the astrocytic release of GABA, and glutamate, on the physiopathology of the hippocampus remains to be established. PMID:22912614

  18. Inherited disorders of GABA metabolism

    PubMed Central

    Pearl, Phillip L; Hartka, Thomas R; Cabalza, Jessica L; Taylor, Jacob; Gibson, Michael K

    2013-01-01

    The inherited disorders of γ-amino butyric acid (GABA) metabolism require an increased index of clinical suspicion. The known genetic disorders are GABA-transaminase deficiency, succinic semialdehyde dehydrogenase (SSADH) deficiency and homocarnosinosis. A recent link has also been made between impaired GABA synthesis and nonsyndromic cleft lip, with or without cleft palate. SSADH deficiency is the most commonly occurring of the inherited disorders of neurotransmitters. The disorder has a nonspecific phenotype with myriad neurological and psychiatric manifestations, and usually has a nonprogressive temporal course. Diagnosis is made by the detection of γ-hydroxybutyrate excretion on urine organic acid testing. The most consistent magnetic resonance imaging abnormality is an increased signal in the globus pallidus. Magnetic resonance spectroscopy has demonstrated the first example of increased endogenous GABA in human brain parenchyma in this disorder. GABA-transaminase deficiency and homocarnosinosis appear to be very rare, but require cerebrospinal fluid for detection, thus allowing for the possibility that these entities, as in the other inherited neurotransmitter disorders, are under-recognized. PMID:23842532

  19. The importance of glutamate, glycine, and {gamma}-aminobutyric acid transport and regulation in manganese, mercury and lead neurotoxicity

    SciTech Connect

    Fitsanakis, Vanessa A.; Aschner, Michael . E-mail: michael.aschner@vanderbilt.edu

    2005-05-01

    Historically, amino acids were studied in the context of their importance in protein synthesis. In the 1950s, the focus of research shifted as amino acids were recognized as putative neurotransmitters. Today, many amino acids are considered important neurochemicals. Although many amino acids play a role in neurotransmission, glutamate (Glu), glycine (Gly), and {gamma}-aminobutyric acid (GABA) are among the more prevalent and better understood. Glu, the major excitatory neurotransmitter, and Gly and GABA, the major inhibitory neurotransmitters, in the central nervous system, are known to be tightly regulated. Prolonged exposure to environmental toxicants, such as manganese (Mn), mercury (Hg), or lead (Pb), however, can lead to dysregulation of these neurochemicals and subsequent neurotoxicity. While the ability of these metals to disrupt the regulation of Glu, Gly and GABA have been studied, few articles have examined the collective role of these amino acids in the respective metal's mechanism of toxicity. For each of the neurotransmitters above, we will provide a brief synopsis of their regulatory function, including the importance of transport and re-uptake in maintaining their optimal function. Additionally, the review will address the hypothesis that aberrant homeostasis of any of these amino acids, or a combination of the three, plays a role in the neurotoxicity of Mn, Hg, or Pb.

  20. Active transport of. gamma. -aminobutyric acid and glycine into synaptic vesicles

    SciTech Connect

    Kish, P.E.; Fischer-Bovenkerk, C.; Ueda, T. )

    1989-05-01

    Although {gamma}-aminobutyric acid (GABA) and glycine are recognized as major amino acid inhibitory neurotransmitters in the central nervous system, their storage is poorly understood. In this study the authors have characterized vesicular GABA and glycine uptakes in the cerebrum and spinal cord, respectively. They present evidence that GABA and glycine are each taken up into isolated synaptic vesicles in an ATP-dependent manner and that the uptake is driven by an electrochemical proton gradient. Uptake for both amino acids exhibited kinetics with low affinity similar to a vesicular glutamate uptake. The ATP-dependent GABA uptake was not inhibited by the putative amino acid neurotransmitters glycine, taurine, glutamate, or aspartate or by GABA analogs, agonists, and antagonists. Similarly, ATP-dependent glycine uptake was hardly affected by GABA, taurine, glutamate, or aspartate or by glycine analogs or antagonists. The GABA uptake was not affected by chloride, which is in contrast to the uptake of the excitatory neurotransmitter glutamate, whereas the glycine uptake was slightly stimulated by low concentrations of chloride. Tissue distribution studies indicate that the vesicular uptake systems for GABA, glycine, and glutamate are distributed in different proportions in the cerebrum and spinal cord. These results suggest that the vesicular uptake systems for GABA, glycine, and glutamate are distinct from each other.

  1. Centrifuge-induced hypergravity: [ 3H]GABA and L-[ 14C]glutamate uptake, exocytosis and efflux mediated by high-affinity, sodium-dependent transporters

    NASA Astrophysics Data System (ADS)

    Borisova, T. A.; Himmelreich, N. H.

    The effects of centrifuge-induced hypergravity on the presynaptic events have been investigated in order to provide further insight into regulation of glutamate and GABA neurotransmission and correlation between excitatory and inhibitory responses under artificial gravity conditions. Exposure of animals to hypergravity (centrifugation of rats at 10 G for 1 h) has been found to cause changes in the synaptic processes of brain, in particular neurotransmitter release and uptake in rat brain synaptosomes. Hypergravity loading resulted in more than two-fold enhancement of GABA transporter activity ( Vmax increased from 1.4 ± 0.3 nmol/min/mg of protein in the control group to 3.3 ± 0.59 nmol/min/mg of protein for the animals exposed to hypergravity ( P ⩽ 0.05)). The maximal velocity of L-[ 14C]glutamate uptake decreased from 12.5 ± 3.2 to 5.6 ± 0.9 nmol/min/mg of protein under artificial gravity conditions. Depolarization-evoked exocytotic release of the neurotransmitters has also changed in response to hypergravity. It increased for GABA (7.2 ± 0.54% and 11.74 ± 1.2% of total accumulated label for control and hypergravity, respectively ( P ⩽ 0.05)), but reduced for glutamate (14.4 ± 0.7% and 6.2 ± 1.9%, for control and hypergravity, respectively). Thus, comparative analysis of the neurotransmitter uptake and release has demonstrated that short-term centrifuge-induced 10 G hypergravity loading intensified inhibitory and attenuated excitatory processes in nerve terminals. The activation or reduction of neurotransmitter uptake appeared to be coupled with similarly directed alterations of the neurotransmitter release.

  2. 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…

  3. Targeting γ-aminobutyric acid (GABA) carriers to the brain: potential relevance as antiepileptic pro-drugs.

    PubMed

    Semreen, Mohammad H; El-Shorbagi, Abdel-Nasser; Al-Tel, Taleb H; Alsalahat, Izzeddin M M

    2010-05-01

    The search for antiepileptic compounds with more selective activity continues to be an area of intensive investigation in medicinal chemistry. 3,5-Disubstituted tetrahydro-2H-1,3,5-thiadiazine-2-thione (THTT) derivatives, 3a-g, potential prodrugs incorporating the neurotransmitter GABA were synthesized and studied for crossing the blood-brain barrier (BBB). Compounds were prepared from primary amines and carbon disulfide to give dithiocarbamates 2a-g which upon reaction in situ with formaldehyde provided the intermediates Ia-g. Addition of Ia-g onto GABA furnished the title compounds 3a-g. The structures were verified by spectral data and the amounts of the compounds in the brain were investigated by using HPLC. The concentration profiles of the tested compounds in mice brain were determined and the in vivo anticonvulsant activity was measured. PMID:20632978

  4. Role of a γ-aminobutryic acid (GABA) receptor mutation in the evolution and spread of Diabrotica virgifera virgifera resistance to cyclodiene insecticides.

    PubMed

    Wang, H; Coates, B S; Chen, H; Sappington, T W; Guillemaud, T; Siegfried, B D

    2013-10-01

    The western corn rootworm, Diabrotica virgifera virgifera, is a damaging pest of cultivated corn that was controlled by applications of cyclodiene insecticides from the late 1940s until resistance evolved ∼10 years later. Range expansion from the western plains into eastern USA coincides with resistance development. An alanine to serine amino acid substitution within the Rdl subunit of the gamma-aminobutyric acid (GABA) receptor confers resistance to cyclodiene insecticides in many species. We found that the non-synonymous single nucleotide polymorphism (SNP) G/T at the GABA receptor cDNA position 838 (G/T(838)) of D. v. virgifera resulted in the alanine to serine change, and the codominant SNP allele T(838) was genetically linked to survival of beetles in aldrin bioassays. A phenotypic gradient of decreasing susceptibility from west to east was correlated with higher frequencies of the resistance-conferring T(838) allele in the eastern-most populations. This pattern exists in opposition to perceived selective pressures since the more eastern and most resistant populations probably experienced reduced exposure. The reasons for the observed distribution are uncertain, but historical records of the range expansion combined with the distribution of susceptible and resistant phenotypes and genotypes provide an opportunity to better understand factors affecting the species' range expansion. PMID:23841833

  5. Straightforward and effective synthesis of γ-aminobutyric acid transporter subtype 2-selective acyl-substituted azaspiro[4.5]decanes.

    PubMed

    Ma, Xiaofeng; Lubin, Hodney; Ioja, Enikő; Kékesi, Orsolya; Simon, Ágnes; Apáti, Ágota; Orbán, Tamás I; Héja, László; Kardos, Julianna; Markó, István E

    2016-01-15

    Supply of major metabolites such as γ-aminobutyric acid (GABA), β-alanine and taurine is an essential instrument that shapes signalling, proper cell functioning and survival in the brain and peripheral organs. This background motivates the synthesis of novel classes of compounds regulating their selective transport through various fluid-organ barriers via the low-affinity γ-aminobutyric acid (GABA) transporter subtype 2 (GAT2). Natural and synthetic spirocyclic compounds or therapeutics with a range of structures and biological activity are increasingly recognised in this regard. Based on pre-validated GABA transport activity, straightforward and efficient synthesis method was developed to provide an azaspiro[4.5]decane scaffold, holding a variety of charge, substituent and 3D constrain of spirocyclic amine. Investigation of the azaspiro[4.5]decane scaffold in cell lines expressing the four GABA transporter subtypes led to the discovery of a subclass of a GAT2-selective compounds with acyl-substituted azaspiro[4.5]decane core. PMID:26706177

  6. Functional reconstitution of the. gamma. -aminobutyric acid transporter from synaptic vesicles using artificial ion gradients

    SciTech Connect

    Hell, J.W.; Edelmann, L.; Hartinger, J.; Jahn, R. )

    1991-12-24

    The {gamma}-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3{prime}-diisopropylthiodicarbocyanine iodide, and changes of the H{sup +} gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K{sup +} gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of ({sup 3}H)GABA which was saturable. Similarly, ({sup 3}H)glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K{sup +}-loaded proteoliposomes in a buffer free of K{sup +} or Na{sup +} ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H{sup +} ATPase by incubation of K{sup +}-loaded proteoliposomes in equimolar K{sup +} buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and {beta}-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, these data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.

  7. Pharmacology of GABA.

    PubMed

    Meldrum, B

    1982-01-01

    GABA-ergic systems are involved in all the main functions of the brain. In most brain regions impairment of this system produces epileptic activity. GABA-mediated inhibitory function can be enhanced by drugs of at least seven different types. They act on the metabolism or synaptic release of GABA, or its reuptake into neurones of glia, or on various components of the GABA receptor complex (GABA recognition site, "benzodiazepine" receptor or chloride ionophore). Among such compounds, those which act most specifically and potently on GABA receptors remain primarily research tools. Among compounds in clinical use, valproate, benzodiazepines, and anticonvulsant barbiturates al enhance GABA-mediated inhibition. In the future, new inhibitors of GABA uptake, new GABA agonists and potent inhibitors of GABA-transaminase are likely to become available. Trials of drugs enhancing GABA-ergic function have been made in a wide variety of neurological disorders. In most forms of epilepsy a therapeutic effect is evident. Real benefit from GABA therapies has not been demonstrated in the principal disorders of movement (Huntington's chorea, Parkinson's disease, dystonias), except in so far as they have a myoclonic or paroxysmal component. Among psychiatric disorders the acute symptoms of schizophrenia are exacerbated by enhanced GABA-ergic function. Abstinence syndromes (alcohol, barbiturate or narcotic withdrawal) are ameliorated by drugs enhancing GABA-ergic function, and there is some evidence for a beneficial action in anxiety states and mania. Attempts to relate the molecular neurobiology of GABA with clinical pharmacology are of very recent origin. Improved understanding of the variety of GABA receptor mechanisms will provide the key to the more selective pharmacological manipulations that are required for therapeutic success. PMID:6214305

  8. Inhibition of the betaine-GABA transporter (mGAT2/BGT-1) modulates spontaneous electrographic bursting in the medial entorhinal cortex (mEC).

    PubMed

    Smith, Misty D; Saunders, Gerald W; Clausen, Rasmus P; Frølund, Bente; Krogsgaard-Larsen, Povl; Larsson, Orla M; Schousboe, Arne; Wilcox, Karen S; White, H Steve

    2008-03-01

    Disruptions in GABAergic neurotransmission have been implicated in numerous CNS disorders, including epilepsy and neuropathic pain. Selective inhibition of neuronal and glial GABA transporter subtypes may offer unique therapeutic options for regaining balance between inhibitory and excitatory systems. The ability of two GABA transport inhibitors to modulate inhibitory tone via inhibition of mGAT1 (tiagabine) or mGAT2/BGT-1 (N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-4-(methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol), also known as EF1502) was evaluated using an in vitro model of spontaneous interictal-like bursting (SB). SBs were recorded extracellularly in combined mEC-HC horizontal brain slices (400 microm; 31+/-1 degrees C) obtained from KA-treated rats. Slice recordings demonstrated that EF1502 exhibited a concentration-dependent reduction in SB frequency. EF1502 significantly reduced SB rate to 32% of control at the 30 microM concentration, while reducing the area and duration of SB activity to 60% and 46% of control, respectively, at the 10 microM concentration. In contrast, the GAT1 selective inhibitor tiagabine (3, 10, and 30 microM) was unable to significantly reduce the frequency of SB activity in the mEC, despite significantly reducing both the duration (51% of control) and area (58% of control) of the SB at concentrations as low as 3 microM. The ability of EF1502, but not tiagabine, to inhibit SBs in the mEC suggests that this in vitro model of pharmacoresistant SB activity is useful to differentiate between novel anticonvulsants with similar mechanisms of action and suggests a therapeutic potential for non-GAT1 transport inhibitors. PMID:18262393

  9. Amino Acid Transport in Pseudomonas aeruginosa

    PubMed Central

    Kay, W. W.; Gronlund, Audrey F.

    1969-01-01

    Properties of the transport systems for amino acids in Pseudomonas aeruginosa were investigated. Exogenous 14C-labeled amino acids were shown to equilibrate with the internal native amino acid pool prior to incorporation into protein. When added at low external concentrations, the majority of the amino acids examined entered the protein of the cell unaltered. The rates of amino acid transport, established at low concentrations with 18 commonly occurring amino acids, varied as much as 40-fold. The transport process became saturated at high external amino acid concentrations, was temperature-sensitive, and was inhibited by sodium azide and iodoacetamide. Intracellular to extracellular amino acid ratios of 100- to 300-fold were maintained during exponential growth of the population in a glucose minimal medium. When the medium became depleted of glucose, neither extracellular nor intracellular amino acids could be detected. PMID:4974392

  10. Linking Metabolism to Membrane Signaling: The GABA-Malate Connection.

    PubMed

    Gilliham, Matthew; Tyerman, Stephen D

    2016-04-01

    γ-Aminobutyric acid (GABA) concentration increases rapidly in tissues when plants encounter abiotic or biotic stress, and GABA manipulation affects growth. This, coupled to GABA's well-described role as a neurotransmitter in mammals, led to over a decade of speculation that GABA is a signal in plants. The discovery of GABA-regulated anion channels in plants provides compelling mechanistic proof that GABA is a legitimate plant-signaling molecule. Here we examine research avenues unlocked by this finding and propose that these plant 'GABA receptors' possess novel properties ideally suited to translating changes in metabolic status into physiological responses. Specifically, we suggest they have a role in signaling altered cycling of tricarboxylic acid (TCA) intermediates during stress via eliciting changes in electrical potential differences across membranes. PMID:26723562

  11. 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. PMID:24844747

  12. Brain regional distribution of GABA(A) receptors exhibiting atypical GABA agonism: roles of receptor subunits.

    PubMed

    Halonen, Lauri M; Sinkkonen, Saku T; Chandra, Dev; Homanics, Gregg E; Korpi, Esa R

    2009-11-01

    The major inhibitory neurotransmitter in the brain, gamma-aminobutyric acid (GABA), has only partial efficacy at certain subtypes of GABA(A) receptors. To characterize these minor receptor populations in rat and mouse brains, we used autoradiographic imaging of t-butylbicyclophosphoro[(35)S]thionate ([(35)S]TBPS) binding to GABA(A) receptors in brain sections and compared the displacing capacities of 10mM GABA and 1mM 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a competitive GABA-site agonist. Brains from GABA(A) receptor alpha1, alpha4, delta, and alpha4+delta subunit knockout (KO) mouse lines were used to understand the contribution of these particular receptor subunits to "GABA-insensitive" (GIS) [(35)S]TBPS binding. THIP displaced more [(35)S]TBPS binding than GABA in several brain regions, indicating that THIP also inhibited GIS-binding. In these regions, GABA prevented the effect of THIP on GIS-binding. GIS-binding was increased in the cerebellar granule cell layer of delta KO and alpha4+delta KO mice, being only slightly diminished in that of alpha1 KO mice. In the thalamus and some other forebrain regions of wild-type mice, a significant amount of GIS-binding was detected. This GIS-binding was higher in alpha4 KO mice. However, it was fully abolished in alpha1 KO mice, indicating that the alpha1 subunit was obligatory for the GIS-binding in the forebrain. Our results suggest that native GABA(A) receptors in brain sections showing reduced displacing capacity of [(35)S]TBPS binding by GABA (partial agonism) minimally require the assembly of alpha1 and beta subunits in the forebrain and of alpha6 and beta subunits in the cerebellar granule cell layer. These receptors may function as extrasynaptic GABA(A) receptors. PMID:19397945

  13. Brain γ-aminobutyric acid (GABA) detection in vivo with the J-editing (1) H MRS technique: a comprehensive methodological evaluation of sensitivity enhancement, macromolecule contamination and test-retest reliability.

    PubMed

    Shungu, Dikoma C; Mao, Xiangling; Gonzales, Robyn; Soones, Tacara N; Dyke, Jonathan P; van der Veen, Jan Willem; Kegeles, Lawrence S

    2016-07-01

    Abnormalities in brain γ-aminobutyric acid (GABA) have been implicated in various neuropsychiatric and neurological disorders. However, in vivo GABA detection by (1) H MRS presents significant challenges arising from the low brain concentration, overlap by much stronger resonances and contamination by mobile macromolecule (MM) signals. This study addresses these impediments to reliable brain GABA detection with the J-editing difference technique on a 3-T MR system in healthy human subjects by: (i) assessing the sensitivity gains attainable with an eight-channel phased-array head coil; (ii) determining the magnitude and anatomic variation of the contamination of GABA by MM; and (iii) estimating the test-retest reliability of the measurement of GABA with this method. Sensitivity gains and test-retest reliability were examined in the dorsolateral prefrontal cortex (DLPFC), whereas MM levels were compared across three cortical regions: DLPFC, the medial prefrontal cortex (MPFC) and the occipital cortex (OCC). A three-fold higher GABA detection sensitivity was attained with the eight-channel head coil compared with the standard single-channel head coil in DLPFC. Despite significant anatomical variation in GABA + MM and MM across the three brain regions (p < 0.05), the contribution of MM to GABA + MM was relatively stable across the three voxels, ranging from 41% to 49%, a non-significant regional variation (p = 0.58). The test-retest reliability of GABA measurement, expressed as either the ratio to voxel tissue water (W) or to total creatine, was found to be very high for both the single-channel coil and the eight-channel phased-array coil. For the eight-channel coil, for example, Pearson's correlation coefficient of test vs. retest for GABA/W was 0.98 (R(2)  = 0.96, p = 0.0007), the percentage coefficient of variation (CV) was 1.25% and the intraclass correlation coefficient (ICC) was 0.98. Similar reliability was also found for the co-edited resonance

  14. Effect of γ-Aminobutyric Acid (GABA) Producing Bacteria on In vitro Rumen Fermentation, Biogenic Amine Production and Anti-oxidation Using Corn Meal as Substrate

    PubMed Central

    Ku, Bum Seung; Mamuad, Lovelia L.; Kim, Seon-Ho; Jeong, Chang Dae; Soriano, Alvin P.; Lee, Ho-Il; Nam, Ki-Chang; Ha, Jong K.; Lee, Sang Suk

    2013-01-01

    The effects and significance of γ-amino butyric acid (GABA) producing bacteria (GPB) on in vitro rumen fermentation and reduction of biogenic amines (histamine, methylamine, ethylamine, and tyramine) using corn meal as a substrate were determined. Ruminal samples collected from ruminally fistulated Holstein cows served as inoculum and corn was used as substrate at 2% dry matter (DM). Different inclusion rates of GPB and GABA were evaluated. After incubation, addition of GPB had no significant effect on in vitro fermentation pH and total gas production, but significantly increased the ammonia nitrogen (NH3-N) concentration and reduced the total biogenic amines production (p<0.05). Furthermore, antioxidation activity was improved as indicated by the significantly higher concentration of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) among treated samples when compared to the control (p<0.05). Additionally, 0.2% GPB was established as the optimum inclusion level. Taken together, these results suggest the potential of utilizing GPB as feed additives to improve growth performance in ruminants by reducing biogenic amines and increasing anti-oxidation. PMID:25049853

  15. 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

  16. 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. PMID:27135813

  17. [Autoantibodies to glutamate and GABA in opiate addiction].

    PubMed

    Vetrile, L A; Fomina, V G; Nevidimova, T I; Vetlugina, T P; Batukhtina, E I; Savochkina, D N; Zakharova, I A; Davydova, T V

    2015-01-01

    Blood serum from 129 patients with opium addiction at different stages of the disease and 63 donors (control group) was examined for the presence of autoantibodies to the exciting and inhibitory amino acids glutamate and GABA. It was shown enhanced production of autoantibodies to glutamate and GABA. Dependence of the level and frequency of detec- tion of autoantibodies to glutamate and GABA on the stage of the disease was revealed. PMID:26852594

  18. Effects of prenatal exposure to 2,4-D/2,4,5-T mixture on postnatal changes in rat brain glutamate, GABA protein, and nucleic acid levels

    SciTech Connect

    Mohammad, F.K.; Omer, V.E.V.

    1988-02-01

    The opportunity of maternal exposure to various chemicals in the work place and the general environments have increased, and the fetus and neonate may be at greater risk than the adult. However, the embryotoxic and teratogenic effects of the chlorinated phenoxy herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), the main chemicals in Agent Orange, are well documented only in laboratory animals. The brain of the developing fetus is vulnerable to the toxic effects of the phenoxy herbicides which readily cross the placental barrier and distribute into fetal tissues, including brain. Although the neurochemical basis for the behavioral teratogenicity of the phenoxy herbicides is not know, it was recently reported that non-teratogenic doses of a 1:1 mixture of 2,4-D and 2,4,5-T delayed the ontogeny of dopamine and serotonin in the brain of the developing rate. This communication provides further descriptive information about the ontogeny of rat brain nucleic acid, protein, glutamate and ..gamma..-aminobutyrate (GABA) following in utero exposure to non-teratogenic levels of a 1:1 mixture of 2,4-D/2,4,5-T.

  19. Quantitative autoradiographic characterization of GA-BA sub B receptors in mammalian central nervous system

    SciTech Connect

    Chu, D.Chin-Mei.

    1989-01-01

    The inhibitory effects of the amino acid neurotransmitter {gamma}-aminobutyric acid (GABA) within the nervous system appear to be mediated through two distinct classes of receptors: GABA{sub A} and GABA{sub B} receptors. A quantitative autoradiographic method with {sup 3}H-GABA was developed to examine the hypotheses that GABA{sub A} and GABA{sub B} sites have distinct anatomical distributions, pharmacologic properties, and synaptic localizations within the rodent nervous system. The method was also applied to a comparative study of these receptors in postmortem human brain from individuals afflicted with Alzheimer's disease and those without neurologic disease. The results indicated that GABA{sub B} receptors occur in fewer numbers and have a lower affinity for GABA than GABA{sub A} receptors in both rodent and human brain. Within rodent brain, the distribution of these two receptor populations were clearly distinct. GABA{sub B} receptors were enriched in the medial habenula, interpeduncular nucleus, cerebellar molecular layer and olfactory glomerular layer. After selective lesions of postsynaptic neurons of the corticostriatal and perforant pathway, both GABA{sub B} and GABA{sub A} receptors were significantly decreased in number. Lesions of the presynaptic limbs of the perforant but not the corticostriatal pathway resulted in upregulation of both GABA receptors in the area of innervation. GABA{sub B} receptors were also upregulated in CA3 dendritic regions after destruction of dentate granule neurons.

  20. MaxiK channel interactome reveals its interaction with GABA transporter 3 and heat shock protein 60 in the mammalian brain.

    PubMed

    Singh, H; Li, M; Hall, L; Chen, S; Sukur, S; Lu, R; Caputo, A; Meredith, A L; Stefani, E; Toro, L

    2016-03-11

    Large conductance voltage and calcium-activated potassium (MaxiK) channels are activated by membrane depolarization and elevated cytosolic Ca(2+). In the brain, they localize to neurons and astrocytes, where they play roles such as resetting the membrane potential during an action potential, neurotransmitter release, and neurovascular coupling. MaxiK channels are known to associate with several modulatory proteins and accessory subunits, and each of these interactions can have distinct physiological consequences. To uncover new players in MaxiK channel brain physiology, we applied a directed proteomic approach and obtained MaxiK channel pore-forming α subunit brain interactome using specific antibodies. Controls included immunoprecipitations with rabbit immunoglobulin G (IgG) and with anti-MaxiK antibodies in wild type and MaxiK channel knockout mice (Kcnma1(-/-)), respectively. We have found known and unreported interactive partners that localize to the plasma membrane, extracellular space, cytosol and intracellular organelles including mitochondria, nucleus, endoplasmic reticulum and Golgi apparatus. Localization of MaxiK channel to mitochondria was further confirmed using purified brain mitochondria colabeled with MitoTracker. Independent proof of MaxiK channel interaction with previously unidentified partners is given for GABA transporter 3 (GAT3) and heat shock protein 60 (HSP60). In human embryonic kidney 293 cells containing SV40 T-antigen (HEK293T) cells, both GAT3 and HSP60 coimmunoprecipitated and colocalized with MaxiK channel; colabeling was observed mainly at the cell periphery with GAT3 and intracellularly with HSP60 with protein proximity indices of ∼ 0.6 and ∼ 0.4, respectively. In rat primary hippocampal neurons, colocalization index was identical for GAT3 (∼ 0.6) and slightly higher for HSP60 (∼ 0.5) association with MaxiK channel. The results of this study provide a complete interactome of MaxiK channel the mouse brain, further establish

  1. γ-Aminobutyric Acid Transporter 2 Mediates the Hepatic Uptake of Guanidinoacetate, the Creatine Biosynthetic Precursor, in Rats

    PubMed Central

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

    2012-01-01

    Guanidinoacetic acid (GAA) is the biosynthetic precursor of creatine which is involved in storage and transmission of phosphate-bound energy. Hepatocytes readily convert GAA to creatine, raising the possibility that the active uptake of GAA by hepatocytes is a regulatory factor. The purpose of this study is to investigate and identify the transporter responsible for GAA uptake by hepatocytes. The characteristics of [14C]GAA uptake by hepatocytes were elucidated using the in vivo liver uptake method, freshly isolated rat hepatocytes, an expression system of Xenopus laevis oocytes, gene knockdown, and an immunohistochemical technique. In vivo injection of [14C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [14C]GAA by the liver. The uptake was markedly inhibited by γ-aminobutyric acid (GABA) and nipecotinic acid, an inhibitor of GABA transporters (GATs). The characteristics of Na+- and Cl−-dependent [14C]GAA uptake by freshly isolated rat hepatocytes were consistent with those of GAT2. The Km value of the GAA uptake (134 µM) was close to that of GAT2-mediated GAA transport (78.9 µM). GABA caused a marked inhibition with an IC50 value of 8.81 µM. The [14C]GAA uptake exhibited a significant reduction corresponding to the reduction in GAT2 protein expression. GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. This distribution pattern was consistent with that of the creatine biosynthetic enzyme, S-adenosylmethionine∶guanidinoacetate N-methyltransferase. GAT2 makes a major contribution to the sinusoidal GAA uptake by periportal hepatocytes, thus regulating creatine biosynthesis in the liver. PMID:22384273

  2. γ-Aminobutyric acid transporter 2 mediates the hepatic uptake of guanidinoacetate, the creatine biosynthetic precursor, in rats.

    PubMed

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

    2012-01-01

    Guanidinoacetic acid (GAA) is the biosynthetic precursor of creatine which is involved in storage and transmission of phosphate-bound energy. Hepatocytes readily convert GAA to creatine, raising the possibility that the active uptake of GAA by hepatocytes is a regulatory factor. The purpose of this study is to investigate and identify the transporter responsible for GAA uptake by hepatocytes. The characteristics of [(14)C]GAA uptake by hepatocytes were elucidated using the in vivo liver uptake method, freshly isolated rat hepatocytes, an expression system of Xenopus laevis oocytes, gene knockdown, and an immunohistochemical technique. In vivo injection of [(14)C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [(14)C]GAA by the liver. The uptake was markedly inhibited by γ-aminobutyric acid (GABA) and nipecotinic acid, an inhibitor of GABA transporters (GATs). The characteristics of Na(+)- and Cl(-)-dependent [(14)C]GAA uptake by freshly isolated rat hepatocytes were consistent with those of GAT2. The Km value of the GAA uptake (134 µM) was close to that of GAT2-mediated GAA transport (78.9 µM). GABA caused a marked inhibition with an IC(50) value of 8.81 µM. The [(14)C]GAA uptake exhibited a significant reduction corresponding to the reduction in GAT2 protein expression. GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. This distribution pattern was consistent with that of the creatine biosynthetic enzyme, S-adenosylmethionine:guanidinoacetate N-methyltransferase. GAT2 makes a major contribution to the sinusoidal GAA uptake by periportal hepatocytes, thus regulating creatine biosynthesis in the liver. PMID:22384273

  3. Intestinal transport and metabolism of bile acids

    PubMed Central

    Dawson, Paul A.; Karpen, Saul J.

    2015-01-01

    In addition to their classical roles as detergents to aid in the process of digestion, bile acids have been identified as important signaling molecules that function through various nuclear and G protein-coupled receptors to regulate a myriad of cellular and molecular functions across both metabolic and nonmetabolic pathways. Signaling via these pathways will vary depending on the tissue and the concentration and chemical structure of the bile acid species. Important determinants of the size and composition of the bile acid pool are their efficient enterohepatic recirculation, their host and microbial metabolism, and the homeostatic feedback mechanisms connecting hepatocytes, enterocytes, and the luminal microbiota. This review focuses on the mammalian intestine, discussing the physiology of bile acid transport, the metabolism of bile acids in the gut, and new developments in our understanding of how intestinal metabolism, particularly by the gut microbiota, affects bile acid signaling. PMID:25210150

  4. Transcriptional Dysregulation of γ-Aminobutyric Acid Transporter in Parvalbumin-Containing Inhibitory Neurons in the Prefrontal Cortex in Schizophrenia

    PubMed Central

    Bitanihirwe, Byron K. Y.; Woo, Tsung-Ung W.

    2015-01-01

    Parvalbumin (PV)-containing neurons are functionally compromised in schizophrenia. Using double in situ hybridization in postmortem human prefrontal cortex, we found that the messenger RNA (mRNA) for the γ-aminobutyric acid transporter GAT-1 was undetectable in 22-41% of PV neurons in layers 3-4 in schizophrenia. In the remaining PV neurons with detectable GAT-1 mRNA, transcript expression was decreased by 26% in layer 3. Hence, the dysfunction of PV neurons involves the molecular dysregulation of presynaptic GABA reuptake. PMID:25312391

  5. Isoflurane modulates excitability in the mouse thalamus via GABA-dependent and GABA-independent mechanisms.

    PubMed

    Ying, Shui-Wang; Werner, David F; Homanics, Gregg E; Harrison, Neil L; Goldstein, Peter A

    2009-02-01

    GABAergic neurons in the reticular thalamic nucleus (RTN) synapse onto thalamocortical neurons in the ventrobasal (VB) thalamus, and this reticulo-thalamocortical pathway is considered an anatomic target for general anesthetic-induced unconsciousness. A mutant mouse was engineered to harbor two amino acid substitutions (S270H, L277A) in the GABA(A) receptor (GABA(A)-R) alpha1 subunit; this mutation abolished sensitivity to the volatile anesthetic isoflurane in recombinant GABA(A)-Rs, and reduced in vivo sensitivity to isoflurane in the loss-of-righting-reflex assay. We examined the effects of the double mutation on GABA(A)-R-mediated synaptic currents and isoflurane sensitivity by recording from thalamic neurons in brain slices. The double mutation accelerated the decay, and decreased the (1/2) width of, evoked inhibitory postsynaptic currents (eIPSCs) in VB neurons and attenuated isoflurane-induced prolongation of the eIPSC. The hypnotic zolpidem, a selective modulator of GABA(A)-Rs containing the alpha1 subunit, prolonged eIPSC duration regardless of genotype, indicating that mutant mice incorporate alpha1 subunit-containing GABA(A)-Rs into synapses. In RTN neurons, which lack the alpha1 subunit, eIPSC duration was longer than in VB, regardless of genotype. Isoflurane reduced the efficacy of GABAergic transmission from RTN to VB, independent of genotype, suggesting a presynaptic action in RTN neurons. Consistent with this observation, isoflurane inhibited both tonic action potential and rebound burst firing in the presence of GABA(A)-R blockade. The suppressed excitability in RTN neurons is likely mediated by isoflurane-enhanced Ba(2+)-sensitive, but 4-aminopyridine-insenstive, potassium conductances. We conclude that isoflurane enhances inhibition of thalamic neurons in VB via GABA(A)-R-dependent, but in RTN via GABA(A)-R-independent, mechanisms. PMID:18948126

  6. Cocaine disinhibits dopamine neurons in the ventral tegmental area via use-dependent blockade of GABA neuron voltage-sensitive sodium channels

    PubMed Central

    Steffensen, Scott C.; Taylor, Seth R.; Horton, Malia L.; Barber, Elise N.; Lyle, Laura T.; Stobbs, Sarah H.; Allison, David W.

    2010-01-01

    The aim of this study was to evaluate the effects of cocaine on γ-aminobutyric acid (GABA) and dopamine (DA) neurons in the ventral tegmental area (VTA). Utilizing single-unit recordings in vivo, microelectrophoretic administration of DA enhanced the firing rate of VTA GABA neurons via D2/D3 DA receptor activation. Lower doses of intravenous cocaine (0.25–0.5 mg/kg), or the DA transporter (DAT) blocker methamphetamine, enhanced VTA GABA neuron firing rate via D2/D3 receptor activation. Higher doses of cocaine (1.0–2.0 mg/kg) inhibited their firing rate, which was not sensitive to the D2/D3 antagonist eticlopride. The voltage-sensitive sodium channel (VSSC) blocker lidocaine inhibited the firing rate of VTA GABA neurons at all doses tested (0.25–2.0 mg/kg). Cocaine or lidocaine reduced VTA GABA neuron spike discharges induced by stimulation of the internal capsule (ICPSDs) at dose levels 0.25–2 mg/kg (IC50 1.2 mg/kg). There was no effect of DA or methamphetamine on ICPSDs, or of DA antagonists on cocaine inhibition of ICPSDs. In VTA GABA neurons in vitro, cocaine reduced (IC50 13 μm) current-evoked spikes and TTX-sensitive sodium currents in a use-dependent manner. In VTA DA neurons, cocaine reduced IPSCs (IC50 13 μm), increased IPSC paired-pulse facilitation and decreased spontaneous IPSC frequency, without affecting miniature IPSC frequency or amplitude. These findings suggest that cocaine acts on GABA neurons to reduce activity-dependent GABA release on DA neurons in the VTA, and that cocaine's use-dependent blockade of VTA GABA neuron VSSCs may synergize with its DAT inhibiting properties to enhance mesolimbic DA transmission implicated in cocaine reinforcement. PMID:19046384

  7. 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

  8. Accumulation of GABAergic Neurons, Causing a Focal Ambient GABA Gradient, and Downregulation of KCC2 Are Induced During Microgyrus Formation in a Mouse Model of Polymicrogyria

    PubMed Central

    Wang, Tianying; Kumada, Tatsuro; Morishima, Toshitaka; Iwata, Satomi; Kaneko, Takeshi; Yanagawa, Yuchio; Yoshida, Sachiko; Fukuda, Atsuo

    2014-01-01

    Although focal cortical malformations are considered neuronal migration disorders, their formation mechanisms remain unknown. We addressed how the γ-aminobutyric acid (GABA)ergic system affects the GABAergic and glutamatergic neuronal migration underlying such malformations. A focal freeze-lesion (FFL) of the postnatal day zero (P0) glutamic acid decarboxylase–green fluorescent protein knock-in mouse neocortex produced a 3- or 4-layered microgyrus at P7. GABAergic interneurons accumulated around the necrosis including the superficial region during microgyrus formation at P4, whereas E17.5-born, Cux1-positive pyramidal neurons outlined the GABAergic neurons and were absent from the superficial layer, forming cell-dense areas in layer 2 of the P7 microgyrus. GABA imaging showed that an extracellular GABA level temporally increased in the GABAergic neuron-positive area, including the necrotic center, at P4. The expression of the Cl– transporter KCC2 was downregulated in the microgyrus-forming GABAergic and E17.5-born glutamatergic neurons at P4; these cells may need a high intracellular Cl– concentration to induce depolarizing GABA effects. Bicuculline decreased the frequency of spontaneous Ca2+ oscillations in these microgyrus-forming cells. Thus, neonatal FFL causes specific neuronal accumulation, preceded by an increase in ambient GABA during microgyrus formation. This GABA increase induces GABAA receptor-mediated Ca2+ oscillation in KCC2-downregulated microgyrus-forming cells, as seen in migrating cells during early neocortical development. PMID:23246779

  9. 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.

  10. Glial GABA, synthesized by monoamine oxidase B, mediates tonic inhibition.

    PubMed

    Yoon, Bo-Eun; Woo, Junsung; Chun, Ye-Eun; Chun, Heejung; Jo, Seonmi; Bae, Jin Young; An, Heeyoung; Min, Joo Ok; Oh, Soo-Jin; Han, Kyung-Seok; Kim, Hye Yun; Kim, Taekeun; Kim, Young Soo; Bae, Yong Chul; Lee, C Justin

    2014-11-15

    GABA is the major inhibitory transmitter in the brain and is released not only from a subset of neurons but also from glia. Although neuronal GABA is well known to be synthesized by glutamic acid decarboxylase (GAD), the source of glial GABA is unknown. After estimating the concentration of GABA in Bergmann glia to be around 5-10 mM by immunogold electron microscopy, we demonstrate that GABA production in glia requires MAOB, a key enzyme in the putrescine degradation pathway. In cultured cerebellar glia, both Ca(2+)-induced and tonic GABA release are significantly reduced by both gene silencing of MAOB and the MAOB inhibitor selegiline. In the cerebellum and striatum of adult mice, general gene silencing, knock out of MAOB or selegiline treatment resulted in elimination of tonic GABA currents recorded from granule neurons and medium spiny neurons. Glial-specific rescue of MAOB resulted in complete rescue of tonic GABA currents. Our results identify MAOB as a key synthesizing enzyme of glial GABA, which is released via bestrophin 1 (Best1) channel to mediate tonic inhibition in the brain. PMID:25239459

  11. Modeling Electrical Transport through Nucleic Acids

    NASA Astrophysics Data System (ADS)

    Qi, Jianqing

    Nucleic acids play a vital role in many biological systems and activities. In recent years, engineers and scientists have been interested in studying their electrical properties. The motivation for these studies stems from the following facts: (1) the bases, which form the building blocks of nucleic acids, have unique ionization potentials. Further, nucleic acids are one of the few nanomaterials that can be reproducibly manufactured with a high degree of accuracy (though admittedly their placement at desired locations remains a challenge). As a result, designed strands with specific sequences may offer unique device properties; (2) electrical methods offer potential for sequencing nucleic acids based on a single molecule; (3) electrical methods for disease detection based on the current flowing through nucleic acids are beginning to be demonstrated. While experiments in the above mentioned areas is promising, a deeper understanding of the electrical current flow through the nucleic acids needs to be developed. The modeling of current flowing in these molecules is complex because: (1) they are based on atomic scale contacts between nucleic acids and metal, which cannot be reproducibly built; (2) the conductivity of nucleic acids is easily influenced by the environment, which is constantly changing; and (3) the nucleic acids by themselves are floppy. This thesis focuses on the modeling of electrical transport through nucleic acids that are connected to two metal electrodes at nanoscale. We first develop a decoherent transport model for the double-stranded helix based on the Landauer-Buttiker framework. This model is rationalized by comparison with an experiment that measured the conductance of four different DNA strands. The developed model is then used to study the: (1) potential to make barriers and wells for quantum transport using specifically engineered sequences; (2) change in the electrical properties of a specific DNA strand with and without methylation; (3

  12. Biphasic effects of direct, but not indirect, GABA mimetics and antagonists on haloperidol-induced catalepsy.

    PubMed

    Worms, P; Lloyd, K G

    1980-03-01

    At very low doses the GABA agonists SL 76002 and muscimol diminish haloperidol-induced catalepsy. At somewhat higher doses these compounds potentiate catalepsy. Biphasic effects on DA-receptor mediated functions have previously been noted with bicuculline and picrotoxinin. In contrast, manipulation of GABA levels by enzyme inhibition induced only a monophasic effect on dopamine-mediated behaviour. The potentiation of GABA levels by enzyme inhibition induced only a monophasic effect on dopamine-mediated behaviour. The potentiation of haloperidol-induced catalepsy by GABA mimetics is also observed with dipropylacetate, delta-aminovaleric acid and gamma-acetylenic GABA. This GABA-mimetic potentiation of catakepsy was blocked by the coadministration of bicuculline. These results confirm and extend the hypothesis that GABA-neurons influence DA neuron function. Furthermore they suggest that more than one group of GABA receptors influence directly and/or indirectly DA neuronal function, with different resultant effects. PMID:7189827

  13. 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

  14. Reactive solute transport in acidic streams

    USGS Publications Warehouse

    Broshears, R.E.

    1996-01-01

    Spatial and temporal profiles of Ph and concentrations of toxic metals in streams affected by acid mine drainage are the result of the interplay of physical and biogeochemical processes. This paper describes a reactive solute transport model that provides a physically and thermodynamically quantitative interpretation of these profiles. The model combines a transport module that includes advection-dispersion and transient storage with a geochemical speciation module based on MINTEQA2. Input to the model includes stream hydrologic properties derived from tracer-dilution experiments, headwater and lateral inflow concentrations analyzed in field samples, and a thermodynamic database. Simulations reproduced the general features of steady-state patterns of observed pH and concentrations of aluminum and sulfate in St. Kevin Gulch, an acid mine drainage stream near Leadville, Colorado. These patterns were altered temporarily by injection of sodium carbonate into the stream. A transient simulation reproduced the observed effects of the base injection.

  15. Abscisic Acid Transport in Human Erythrocytes*

    PubMed Central

    Vigliarolo, Tiziana; Guida, Lucrezia; Millo, Enrico; Fresia, Chiara; Turco, Emilia; De Flora, Antonio; Zocchi, Elena

    2015-01-01

    Abscisic acid (ABA) is a plant hormone involved in the response to environmental stress. Recently, ABA has been shown to be present and active also in mammals, where it stimulates the functional activity of innate immune cells, of mesenchymal and hemopoietic stem cells, and insulin-releasing pancreatic β-cells. LANCL2, the ABA receptor in mammalian cells, is a peripheral membrane protein that localizes at the intracellular side of the plasma membrane. Here we investigated the mechanism enabling ABA transport across the plasmamembrane of human red blood cells (RBC). Both influx and efflux of [3H]ABA occur across intact RBC, as detected by radiometric and chromatographic methods. ABA binds specifically to Band 3 (the RBC anion transporter), as determined by labeling of RBC membranes with biotinylated ABA. Proteoliposomes reconstituted with human purified Band 3 transport [3H]ABA and [35S]sulfate, and ABA transport is sensitive to the specific Band 3 inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. Once inside RBC, ABA stimulates ATP release through the LANCL2-mediated activation of adenylate cyclase. As ATP released from RBC is known to exert a vasodilator response, these results suggest a role for plasma ABA in the regulation of vascular tone. PMID:25847240

  16. First photoswitchable neurotransmitter transporter inhibitor: light-induced control of γ-aminobutyric acid transporter 1 (GAT1) activity in mouse brain.

    PubMed

    Quandt, Gabriele; Höfner, Georg; Pabel, Jörg; Dine, Julien; Eder, Matthias; Wanner, Klaus T

    2014-08-14

    Inhibition of mGAT1, the most abundant GABA transporter in the brain, enhances GABA signaling and alleviates symptoms of CNS disorders such as epilepsy assumed to be associated with low GABA levels. We have now developed a potent and subtype selective photoswitchable inhibitor of this transporter, which for the first time extends the photoswitch concept for the light-induced control of ligand affinity to active membrane transporters. The new inhibitor exhibited reduced activity upon irradiation with light, as demonstrated in GABA uptake assays and electrophysiological experiments with brain slices, and might be used as a tool compound for deepening the understanding of mGAT1 function in brain. PMID:25025595

  17. Chronic benzodiazepine treatment and cortical responses to adenosine and GABA.

    PubMed

    Mally, J; Connick, J H; Stone, T W

    1990-10-22

    The effects of chronic treatment of mice with clonazepam have been examined on the responses of neocortical slices to adenosine, 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA). Responses to these agonists were measured as changes in the depolarisation induced by N-methyl-D-aspartate (NMDA). Added to the superfusion medium diazepam blocked responses to adenosine but not 5-HT; this effect was not observed with 2-chloroadenosine or in the presence of 2-hydroxynitrobenzylthioguanosine. GABA was inactive in control slices but chronic treatment with clonazepam induced responses to GABA and enhanced responses to adenosine but not 5-HT. It is suggested that the induction of GABA responses may reflect the up-regulation of GABA receptors, but the increase of adenosine responses by clonazepam implies that there is no simple relationship between adenosine receptor binding and functional responses. PMID:1979931

  18. Neurotoxins from snake venoms and α-conotoxin ImI inhibit functionally active ionotropic γ-aminobutyric acid (GABA) receptors.

    PubMed

    Kudryavtsev, Denis S; Shelukhina, Irina V; Son, Lina V; Ojomoko, Lucy O; Kryukova, Elena V; Lyukmanova, Ekaterina N; Zhmak, Maxim N; Dolgikh, Dmitry A; Ivanov, Igor A; Kasheverov, Igor E; Starkov, Vladislav G; Ramerstorfer, Joachim; Sieghart, Werner; Tsetlin, Victor I; Utkin, Yuri N

    2015-09-11

    Ionotropic receptors of γ-aminobutyric acid (GABAAR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of α-cobratoxin (α-Ctx), belonging to the family of three-finger toxins from snake venoms, specifically stained the α1β3γ2 receptor; and at 10 μm α-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nm) and less potently inhibited α1β2γ2 ≈ α2β2γ2 > α5β2γ2 > α2β3γ2 and α1β3δ GABAARs. The α1β3γ2 receptor was also inhibited by some other three-finger toxins, long α-neurotoxin Ls III and nonconventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and noncompetitive α-Ctx action. Fluorescent α-Ctx, however, could be displaced by muscimol indicating that most of the α-Ctx-binding sites overlap with the orthosteric sites at the β/α subunit interface. Modeling and molecular dynamic studies indicated that α-Ctx or α-bungarotoxin seem to interact with GABAAR in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with α-conotoxin ImI and a chimeric Naja oxiana α-neurotoxin indicating that the major role in α-Ctx binding to GABAAR is played by the tip of its central loop II accommodating under loop C of the receptors. PMID:26221036

  19. Fine Tuning of Synaptic Plasticity and Filtering by GABA Released from Hippocampal Autaptic Granule Cells.

    PubMed

    Valente, Pierluigi; Orlando, Marta; Raimondi, Andrea; Benfenati, Fabio; Baldelli, Pietro

    2016-03-01

    The functional consequence of γ-aminobutyric acid (GABA) release at mossy fiber terminals is still a debated topic. Here, we provide multiple evidence of GABA release in cultured autaptic hippocampal granule cells. In ∼50% of the excitatory autaptic neurons, GABA, VGAT, or GAD67 colocalized with vesicular glutamate transporter 1-positive puncta, where both GABAB and GABAA receptors (Rs) were present. Patch-clamp recordings showed a clear enhancement of autaptic excitatory postsynaptic currents in response to the application of the GABABR antagonist CGP58845 only in neurons positive to the selective granule cell marker Prox1, and expressing low levels of GAD67. Indeed, GCP non-responsive excitatory autaptic neurons were both Prox1- and GAD67-negative. Although the amount of released GABA was not sufficient to activate functional postsynaptic GABAARs, it effectively activated presynaptic GABABRs that maintain a tonic "brake" on the probability of release and on the size of the readily releasable pool and contributed to resting potential hyperpolarization possibly through extrasynaptic GABAAR activation. The autocrine inhibition exerted by GABABRs on glutamate release enhanced both paired-pulse facilitation and post-tetanic potentiation. Such GABABR-mediated changes in short-term plasticity confer to immature granule cells the capability to modulate their filtering properties in an activity-dependent fashion, with remarkable consequences on the dynamic behavior of neural circuits. PMID:25576534

  20. 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. PMID:22507692

  1. 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. PMID:26471792

  2. First molecular genotyping of A302S mutation in the gamma aminobutyric acid (GABA) receptor in Aedes albopictus from Malaysia.

    PubMed

    Low, V L; Vinnie-Siow, W Y; Lim Y, A L; Tan, T K; Leong, C S; Chen, C D; Azidah, A A; Sofian-Azirun, M

    2015-09-01

    Given the lack of molecular evidence in altered target-site insecticide resistance mechanism in Aedes albopictus (Skuse) worldwide, the present study aims to detect the presence of A302S mutation in the gene encoding the gamma aminobutyric acid receptor resistant to dieldrin (Rdl) in Ae. albopictus for the first time from its native range of South East Asia, namely Malaysia. World Health Organization (WHO) adult susceptibility bioassay indicated a relatively low level of dieldrin resistance (two-fold) in Ae. albopictus from Petaling Jaya, Selangor. However, PCR-RFLP and direct sequencing methods revealed the presence of the A302S mutation with the predomination of heterozygous genotype (40 out of 82 individuals), followed by the resistant genotype with 11 individuals. This study represents the first field evolved instance of A302S mutation in Malaysian insect species. PMID:26695218

  3. Pharmacodynamic effects and possible therapeutic uses of THIP, a specific GABA-agonist.

    PubMed

    Christensen, A V; Svendsen, O; Krogsgaard-Larsen, P

    1982-10-22

    THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a potent and specific GABA receptor agonist which does not influence the GABA uptake system or GABA metabolizing enzymes. The specificity for the GABA receptor is also demonstrated by lack of action on monoaminergic, cholinergic, histaminergic or opiate receptors. Since in recent years GABA receptor stimulants-among others THIP--have become available many have speculated as to what clinical indication GABA-ergic stimulation might be an important element. The first suggestion was that GABA-ergic drugs by an inhibitory effect on the dopamine neurons would improve the antischizophrenic effect of neuroleptics and improve tardive dyskinesia. Furthermore, studies on brains of deceased Parkinson and Huntington's chorea patients have demonstrated a low level of GABA and its synthesizing enzyme glutamic acid decarboxylase (GAD) in the basal ganglia. Also in epilepsy and diseases with dementia a deficit in the GABA system has been proposed. Therefore a therapeutic strategy for these diseases may be supplementary treatment with drugs which increase GABA receptor activity. Furthermore, recent results in humans have shown that GABA agonists perhaps also could be of benefit in mania and depressions. When considering the neurophysiological elements of nociception and muscle tone it is also reasonable to suggest that GABA-ergic stimulation may reduce pain perception and muscle tone. PMID:6292818

  4. 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. PMID:19476215

  5. An evolutionarily conserved switch in response to GABA affects development and behavior of the locomotor circuit of Caenorhabditis elegans.

    PubMed

    Han, Bingjie; Bellemer, Andrew; Koelle, Michael R

    2015-04-01

    The neurotransmitter gamma-aminobutyric acid (GABA) is depolarizing in the developing vertebrate brain, but in older animals switches to hyperpolarizing and becomes the major inhibitory neurotransmitter in adults. We discovered a similar developmental switch in GABA response in Caenorhabditis elegans and have genetically analyzed its mechanism and function in a well-defined circuit. Worm GABA neurons innervate body wall muscles to control locomotion. Activation of GABAA receptors with their agonist muscimol in newly hatched first larval (L1) stage animals excites muscle contraction and thus is depolarizing. At the mid-L1 stage, as the GABAergic neurons rewire onto their mature muscle targets, muscimol shifts to relaxing muscles and thus has switched to hyperpolarizing. This muscimol response switch depends on chloride transporters in the muscles analogous to those that control GABA response in mammalian neurons: the chloride accumulator sodium-potassium-chloride-cotransporter-1 (NKCC-1) is required for the early depolarizing muscimol response, while the two chloride extruders potassium-chloride-cotransporter-2 (KCC-2) and anion-bicarbonate-transporter-1 (ABTS-1) are required for the later hyperpolarizing response. Using mutations that disrupt GABA signaling, we found that neural circuit development still proceeds to completion but with an ∼6-hr delay. Using optogenetic activation of GABAergic neurons, we found that endogenous GABAA signaling in early L1 animals, although presumably depolarizing, does not cause an excitatory response. Thus a developmental depolarizing-to-hyperpolarizing shift is an ancient conserved feature of GABA signaling, but existing theories for why this shift occurs appear inadequate to explain its function upon rigorous genetic analysis of a well-defined neural circuit. PMID:25644702

  6. An Evolutionarily Conserved Switch in Response to GABA Affects Development and Behavior of the Locomotor Circuit of Caenorhabditis elegans

    PubMed Central

    Han, Bingjie; Bellemer, Andrew; Koelle, Michael R.

    2015-01-01

    The neurotransmitter gamma-aminobutyric acid (GABA) is depolarizing in the developing vertebrate brain, but in older animals switches to hyperpolarizing and becomes the major inhibitory neurotransmitter in adults. We discovered a similar developmental switch in GABA response in Caenorhabditis elegans and have genetically analyzed its mechanism and function in a well-defined circuit. Worm GABA neurons innervate body wall muscles to control locomotion. Activation of GABAA receptors with their agonist muscimol in newly hatched first larval (L1) stage animals excites muscle contraction and thus is depolarizing. At the mid-L1 stage, as the GABAergic neurons rewire onto their mature muscle targets, muscimol shifts to relaxing muscles and thus has switched to hyperpolarizing. This muscimol response switch depends on chloride transporters in the muscles analogous to those that control GABA response in mammalian neurons: the chloride accumulator sodium-potassium-chloride-cotransporter-1 (NKCC-1) is required for the early depolarizing muscimol response, while the two chloride extruders potassium-chloride-cotransporter-2 (KCC-2) and anion-bicarbonate-transporter-1 (ABTS-1) are required for the later hyperpolarizing response. Using mutations that disrupt GABA signaling, we found that neural circuit development still proceeds to completion but with an ∼6-hr delay. Using optogenetic activation of GABAergic neurons, we found that endogenous GABAA signaling in early L1 animals, although presumably depolarizing, does not cause an excitatory response. Thus a developmental depolarizing-to-hyperpolarizing shift is an ancient conserved feature of GABA signaling, but existing theories for why this shift occurs appear inadequate to explain its function upon rigorous genetic analysis of a well-defined neural circuit. PMID:25644702

  7. Carnitine transport and fatty acid oxidation.

    PubMed

    Longo, Nicola; Frigeni, Marta; Pasquali, Marzia

    2016-10-01

    Carnitine is essential for the transfer of long-chain fatty acids across the inner mitochondrial membrane for subsequent β-oxidation. It can be synthesized by the body or assumed with the diet from meat and dairy products. Defects in carnitine biosynthesis do not routinely result in low plasma carnitine levels. Carnitine is accumulated by the cells and retained by kidneys using OCTN2, a high affinity organic cation transporter specific for carnitine. Defects in the OCTN2 carnitine transporter results in autosomal recessive primary carnitine deficiency characterized by decreased intracellular carnitine accumulation, increased losses of carnitine in the urine, and low serum carnitine levels. Patients can present early in life with hypoketotic hypoglycemia and hepatic encephalopathy, or later in life with skeletal and cardiac myopathy or sudden death from cardiac arrhythmia, usually triggered by fasting or catabolic state. This disease responds to oral carnitine that, in pharmacological doses, enters cells using the amino acid transporter B(0,+). Primary carnitine deficiency can be suspected from the clinical presentation or identified by low levels of free carnitine (C0) in the newborn screening. Some adult patients have been diagnosed following the birth of an unaffected child with very low carnitine levels in the newborn screening. The diagnosis is confirmed by measuring low carnitine uptake in the patients' fibroblasts or by DNA sequencing of the SLC22A5 gene encoding the OCTN2 carnitine transporter. Some mutations are specific for certain ethnic backgrounds, but the majority are private and identified only in individual families. Although the genotype usually does not correlate with metabolic or cardiac involvement in primary carnitine deficiency, patients presenting as adults tend to have at least one missense mutation retaining residual activity. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler

  8. Boramino acid as a marker for amino acid transporters

    PubMed Central

    Liu, Zhibo; Chen, Haojun; Chen, Kai; Shao, Yihan; Kiesewetter, Dale O.; Niu, Gang; Chen, Xiaoyuan

    2015-01-01

    Amino acid transporters (AATs) are a series of integral channels for uphill cellular uptake of nutrients and neurotransmitters. Abnormal expression of AATs is often associated with cancer, addiction, and multiple mental diseases. Although methods to evaluate in vivo expression of AATs would be highly useful, efforts to develop them have been hampered by a lack of appropriate tracers. We describe a new class of AA mimics—boramino acids (BAAs)—that can serve as general imaging probes for AATs. The structure of a BAA is identical to that of the corresponding natural AA, except for an exotic replacement of the carboxylate with -BF3−. Cellular studies demonstrate strong AAT-mediated cell uptake, and animal studies show high tumor-specific accumulation, suggesting that BAAs hold great promise for the development of new imaging probes and smart AAT-targeting drugs. PMID:26601275

  9. High Selectivity of the γ-Aminobutyric Acid Transporter 2 (GAT-2, SLC6A13) Revealed by Structure-based Approach*

    PubMed Central

    Schlessinger, Avner; Wittwer, Matthias B.; Dahlin, Amber; Khuri, Natalia; Bonomi, Massimiliano; Fan, Hao; Giacomini, Kathleen M.; Sali, Andrej

    2012-01-01

    The solute carrier 6 (SLC6) is a family of ion-dependent transporters that mediate uptake into the cell of osmolytes such as neurotransmitters and amino acids. Four SLC6 members transport GABA, a key neurotransmitter that triggers inhibitory signaling pathways via various receptors (e.g., GABAA). The GABA transporters (GATs) regulate the concentration of GABA available for signaling and are thus targeted by a variety of anticonvulsant and relaxant drugs. Here, we characterize GAT-2, a transporter that plays a role in peripheral GABAergic mechanisms, by constructing comparative structural models based on crystallographic structures of the leucine transporter LeuT. Models of GAT-2 in two different conformations were constructed and experimentally validated, using site-directed mutagenesis. Computational screening of 594,166 compounds including drugs, metabolites, and fragment-like molecules from the ZINC database revealed distinct ligands for the two GAT-2 models. 31 small molecules, including high scoring compounds and molecules chemically related to known and predicted GAT-2 ligands, were experimentally tested in inhibition assays. Twelve ligands were found, six of which were chemically novel (e.g., homotaurine). Our results suggest that GAT-2 is a high selectivity/low affinity transporter that is resistant to inhibition by typical GABAergic inhibitors. Finally, we compared the binding site of GAT-2 with those of other SLC6 members, including the norepinephrine transporter and other GATs, to identify ligand specificity determinants for this family. Our combined approach may be useful for characterizing interactions between small molecules and other membrane proteins, as well as for describing substrate specificities in other protein families. PMID:22932902

  10. Local GABA Concentration Predicts Perceptual Improvements After Repetitive Sensory Stimulation in Humans

    PubMed Central

    Heba, Stefanie; Puts, Nicolaas A. J.; Kalisch, Tobias; Glaubitz, Benjamin; Haag, Lauren M.; Lenz, Melanie; Dinse, Hubert R.; Edden, Richard A. E.; Tegenthoff, Martin; Schmidt-Wilcke, Tobias

    2016-01-01

    Learning mechanisms are based on synaptic plasticity processes. Numerous studies on synaptic plasticity suggest that the regulation of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) plays a central role maintaining the delicate balance of inhibition and excitation. However, in humans, a link between learning outcome and GABA levels has not been shown so far. Using magnetic resonance spectroscopy of GABA prior to and after repetitive tactile stimulation, we show here that baseline GABA+ levels predict changes in perceptual outcome. Although no net changes in GABA+ are observed, the GABA+ concentration prior to intervention explains almost 60% of the variance in learning outcome. Our data suggest that behavioral effects can be predicted by baseline GABA+ levels, which provide new insights into the role of inhibitory mechanisms during perceptual learning. PMID:26637451

  11. The role of GABA in the regulation of GnRH neurons

    PubMed Central

    Watanabe, Miho; Fukuda, Atsuo; Nabekura, Junichi

    2014-01-01

    Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction. Gamma-amino butyric acid (GABA) has long been implicated as one of the major players in the regulation of GnRH neurons. Although GABA is typically an inhibitory neurotransmitter in the mature adult central nervous system, most mature GnRH neurons show the unusual characteristic of being excited by GABA. While many reports have provided much insight into the contribution of GABA to the activity of GnRH neurons, the precise physiological role of the excitatory action of GABA on GnRH neurons remains elusive. This brief review presents the current knowledge of the role of GABA signaling in GnRH neuronal activity. We also discuss the modulation of GABA signaling by neurotransmitters and neuromodulators and the functional consequence of GABAergic inputs to GnRH neurons in both the physiology and pathology of reproduction. PMID:25506316

  12. The importance of the excitatory amino acid transporter 3 (EAAT3).

    PubMed

    Bjørn-Yoshimoto, Walden E; Underhill, Suzanne M

    2016-09-01

    The neuronal excitatory amino acid transporter 3 (EAAT3) is fairly ubiquitously expressed in the brain, though it does not necessarily maintain the same function everywhere. It is important in maintaining low local concentrations of glutamate, where its predominant post-synaptic localization can buffer nearby glutamate receptors and modulate excitatory neurotransmission and synaptic plasticity. It is also the main neuronal cysteine uptake system acting as the rate-limiting factor for the synthesis of glutathione, a potent antioxidant, in EAAT3 expressing neurons, while on GABAergic neurons, it is important in supplying glutamate as a precursor for GABA synthesis. Several diseases implicate EAAT3, and modulation of this transporter could prove a useful therapeutic approach. Regulation of EAAT3 could be targeted at several points for functional modulation, including the level of transcription, trafficking and direct pharmacological modulation, and indeed, compounds and experimental treatments have been identified that regulate EAAT3 function at different stages, which together with observations of EAAT3 regulation in patients is giving us insight into the endogenous function of this transporter, as well as the consequences of altered function. This review summarizes work done on elucidating the role and regulation of EAAT3. PMID:27233497

  13. Apical transporters for neutral amino acids: physiology and pathophysiology.

    PubMed

    Bröer, Stefan

    2008-04-01

    Absorption of amino acids in kidney and intestine involves a variety of transporters for different groups of amino acids. This is illustrated by inherited disorders of amino acid absorption, such as Hartnup disorder, cystinuria, iminoglycinuria, dicarboxylic aminoaciduria, and lysinuric protein intolerance, affecting separate groups of amino acids. Recent advances in the molecular identification of apical neutral amino acid transporters has shed a light on the molecular basis of Hartnup disorder and iminoglycinuria. PMID:18400692

  14. Electrogenicity of Na(+)-coupled bile acid transporters.

    PubMed Central

    Weinman, S. A.

    1997-01-01

    The Na(+)-bile acid cotransporters NTCP and ASBT are largely responsible for the Na(+)-dependent bile acid uptake in hepatocytes and intestinal epithelial cells, respectively. This review discusses the experimental methods available for demonstrating electrogenicity and examines the accumulating evidence that coupled transport by each of these bile acid transporters is electrogenic. The evidence includes measurements of transport-associated currents by patch clamp electrophysiological techniques, as well as direct measurement of fluorescent bile acid transport rates in whole cell patch clamped, voltage clamped cells. The results support a Na+:bile acid coupling stoichiometry of 2:1. PMID:9626753

  15. Fast detection of extrasynaptic GABA with a whole-cell sniffer

    PubMed Central

    Christensen, Rasmus K.; Petersen, Anders V.; Schmitt, Nicole; Perrier, Jean-François

    2014-01-01

    Gamma-amino-butyric acid (GABA) is the main inhibitory transmitter of the brain. It operates by binding to specific receptors located both inside and outside synapses. The extrasynaptic receptors are activated by spillover from GABAergic synapses and by ambient GABA in the extracellular space. Ambient GABA is essential for adjusting the excitability of neurons. However, due to the lack of suitable methods, little is known about its dynamics. Here we describe a new technique that allows detection of GABA transients and measurement of the steady state GABA concentration with high spatial and temporal resolution. We used a human embryonic kidney (HEK) cell line that stably expresses GABAA receptors composed of α1, β2, and γ2 subunits. We recorded from such a HEK cell with the whole-cell patch-clamp technique. The presence of GABA near the HEK cell generated a measurable electric current whose magnitude increased with concentration. A fraction of the current did not inactivate during prolonged exposition to GABA. This technique, which we refer to as a “sniffer” allows the measurement of ambient GABA concentration inside nervous tissue with a resolution of few tens of nanomolars. In addition, the sniffer detects variations in the extrasynaptic GABA concentration with millisecond time resolution. Pilot experiments demonstrate that the sniffer is able to report spillover of GABA induced by synaptic activation in real time. This is the first report on a GABA sensor that combines the ability to detect fast transients and to measure steady concentrations. PMID:24860433

  16. Dopamine-related drugs act presynaptically to potentiate GABA(A) receptor currents in VTA dopamine neurons.

    PubMed

    Michaeli, Avner; Yaka, Rami

    2011-01-01

    Electrical activity of ventral tegmental area (VTA) dopamine (DA) neurons is immediately inhibited following in vivo administration of cocaine and other DA-related drugs. While various forms of synaptic modulation were demonstrated in the VTA following exposure to DA-related drugs, comprehensive understanding of their ability to inhibit the activity of DA neurons, however, is still lacking. In this study, using whole-cell patch-clamp recordings from rat brain slices, a novel form of synaptic modulation induced by DA-related drugs was isolated. DA exposure was shown to cause potentiation of γ-amino-butyric acid (GABA) receptor type A (GABA(A)R)-mediated evoked inhibitory postsynaptic currents (eIPSCs), recorded from VTA DA neurons, under conditions of potassium channels blockade. The potentiation of these eIPSCs lasted for more than twenty minutes, could be mimicked by activation of D2-like but not D1-like DA receptors, and was accompanied by an increase in the frequency of GABA(A)R-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Furthermore, exposure to inhibitors of DA transporter (DAT) led to potentiation of GABA(A) currents in a manner similar to the DA-mediated potentiation. Finally, a prolonged presence of l-NAME, an inhibitor of nitric-oxide (NO) signaling was found to conceal the potentiation of GABA(A) currents induced by the DA-related drugs. Taken together, this study demonstrates a new modulatory form of VTA GABA(A) neurotransmission mediated by DA-related drugs. These results also suggest better understanding of the initial inhibitory action of DA-related drugs on the activity of DA neurons in the VTA. PMID:21527263

  17. Bioactivity-guided isolation of GABA(A) receptor modulating constituents from the rhizomes of Actaea racemosa.

    PubMed

    Cicek, Serhat S; Khom, Sophia; Taferner, Barbara; Hering, Steffen; Stuppner, Hermann

    2010-12-27

    Black cohosh (Actaea racemosa) is a frequently used herbal remedy for the treatment of mild climacteric symptoms. In the present study, the modulation of γ-aminobutryic acid (GABA)-induced chloride currents (I(GABA)) through GABA type A (GABA(A)) receptors by black cohosh extracts and isolated compounds was investigated. GABA(A) receptors, consisting of α(1), β(2), and γ(2S) subunits, were expressed in Xenopus laevis oocytes, and potentiation of I(GABA) was measured using the two-microelectrode voltage clamp technique. In a bioactivity-guided isolation procedure the positive modulation of I(GABA) could be restricted to the plant terpenoid fractions, resulting in the isolation of 11 cycloartane glycosides, of which four significantly (p < 0.05) enhanced I(GABA). The most efficient effect was observed for 23-O-acetylshengmanol 3-O-β-d-xylopyranoside (4, 100 μM), enhancing I(GABA) by 1692 ± 201%, while actein (1), cimigenol 3-O-β-d-xylopyranoside (6), and 25-O-acetylcimigenol 3-O-α-l-arabinopyranoside (8) were significantly less active. In the absence of GABA, only 4 induced small (not exceeding 1% of I(GABA-max)) chloride inward currents through GABA(A) receptors. It is hypothesized that the established positive allosteric modulation of GABA(A) receptors may contribute to beneficial effects of black cohosh extracts in the treatment of climacteric symptoms. PMID:21082802

  18. 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

  19. An Electrostatic Funnel in the GABA-Binding Pathway.

    PubMed

    Carpenter, Timothy S; Lightstone, Felice C

    2016-04-01

    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

  20. GABA and GABA receptors in the gastrointestinal tract: from motility to inflammation.

    PubMed

    Auteri, Michelangelo; Zizzo, Maria Grazia; Serio, Rosa

    2015-03-01

    Although an extensive body of literature confirmed γ-aminobutyric acid (GABA) as mediator within the enteric nervous system (ENS) controlling gastrointestinal (GI) function, the true significance of GABAergic signalling in the gut is still a matter of debate. GABAergic cells in the bowel include neuronal and endocrine-like cells, suggesting GABA as modulator of both motor and secretory GI activity. GABA effects in the GI tract depend on the activation of ionotropic GABAA and GABAC receptors and metabotropic GABAB receptors, resulting in a potential noteworthy regulation of both the excitatory and inhibitory signalling in the ENS. However, the preservation of GABAergic signalling in the gut could not be limited to the maintenance of physiologic intestinal activity. Indeed, a series of interesting studies have suggested a potential key role of GABA in the promising field of neuroimmune interaction, being involved in the modulation of immune cell activity associated with different systemic and enteric inflammatory conditions. Given the urgency of novel therapeutic strategies against chronic immunity-related pathologies, i.e. multiple sclerosis and Inflammatory Bowel Disease, an in-depth comprehension of the enteric GABAergic system in health and disease could provide the basis for new clinical application of nerve-driven immunity. Hence, in the attempt to drive novel researches addressing both the physiological and pathological importance of the GABAergic signalling in the gut, we summarized current evidence on GABA and GABA receptor function in the different parts of the GI tract, with particular focus on the potential involvement in the modulation of GI motility and inflammation. PMID:25526825

  1. Influence of cold stress on contents of soluble sugars, vitamin C and free amino acids including gamma-aminobutyric acid (GABA) in spinach (Spinacia oleracea).

    PubMed

    Yoon, Young-Eun; Kuppusamy, Saranya; Cho, Kye Man; Kim, Pil Joo; Kwack, Yong-Bum; Lee, Yong Bok

    2017-01-15

    The contents of soluble sugars (sucrose, fructose, glucose, maltose and raffinose), vitamin C and free amino acids (34 compounds, essential and non-essential) were quantified in open-field and greenhouse-grown spinaches in response to cold stress using liquid chromatography. In general, greenhouse cultivation produced nutritionally high value spinach in a shorter growing period, where the soluble sugars, vitamin C and total amino acids concentrations, including essential were in larger amounts compared to those grown in open-field scenarios. Further, low temperature exposure of spinach during a shorter growth period resulted in the production of spinach with high sucrose, ascorbate, proline, gamma-aminobutyric acid, valine and leucine content, and these constitute the most important energy/nutrient sources. In conclusion, cultivation of spinach in greenhouse at a low temperature (4-7°C) and exposure for a shorter period (7-21days) before harvest is recommended. This strategy will produce a high quality product that people can eat. PMID:27542466

  2. The betaine-GABA transporter (BGT1, slc6a12) is predominantly expressed in the liver and at lower levels in the kidneys and at the brain surface.

    PubMed

    Zhou, Y; Holmseth, S; Hua, R; Lehre, A C; Olofsson, A M; Poblete-Naredo, I; Kempson, S A; Danbolt, N C

    2012-02-01

    The Na(+)- and Cl(-)-dependent GABA-betaine transporter (BGT1) has received attention mostly as a protector against osmolarity changes in the kidney and as a potential controller of the neurotransmitter GABA in the brain. Nevertheless, the cellular distribution of BGT1, and its physiological importance, is not fully understood. Here we have quantified mRNA levels using TaqMan real-time PCR, produced a number of BGT1 antibodies, and used these to study BGT1 distribution in mice. BGT1 (protein and mRNA) is predominantly expressed in the liver (sinusoidal hepatocyte plasma membranes) and not in the endothelium. BGT1 is also present in the renal medulla, where it localizes to the basolateral membranes of collecting ducts (particularly at the papilla tip) and the thick ascending limbs of Henle. There is some BGT1 in the leptomeninges, but brain parenchyma, brain blood vessels, ependymal cells, the renal cortex, and the intestine are virtually BGT1 deficient in 1- to 3-mo-old mice. Labeling specificity was assured by processing tissue from BGT1-deficient littermates in parallel as negative controls. Addition of 2.5% sodium chloride to the drinking water for 48 h induced a two- to threefold upregulation of BGT1, tonicity-responsive enhancer binding protein, and sodium-myo-inositol cotransporter 1 (slc5a3) in the renal medulla, but not in the brain and barely in the liver. BGT1-deficient and wild-type mice appeared to tolerate the salt treatment equally well, possibly because betaine is one of several osmolytes. In conclusion, this study suggests that BGT1 plays its main role in the liver, thereby complementing other betaine-transporting carrier proteins (e.g., slc6a20) that are predominantly expressed in the small intestine or kidney rather than the liver. PMID:22071246

  3. Novel Lactate Transporters from Carboxylic Acid-Producing Rhizopus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fungus Rhizopus is frequently used for fermentative production of lactic acid, but little is known about the mechanisms or proteins for transporting this carboxylic acid. Since transport of the lactate anion across the plasma membrane is critical to prevent acidification of the cytoplasm, we ev...

  4. Neocortical GABA release at high intracellular sodium and low extracellular calcium: an anti-seizure mechanism.

    PubMed

    Rassner, Michael P; Moser, Andreas; Follo, Marie; Joseph, Kevin; van Velthoven-Wurster, Vera; Feuerstein, Thomas J

    2016-04-01

    In epilepsy, the GABA and glutamate balance may be disrupted and a transient decrease in extracellular calcium occurs before and during a seizure. Flow Cytometry based fluorescence activated particle sorting experiments quantified synaptosomes from human neocortical tissue, from both epileptic and non-epileptic patients (27.7% vs. 36.9% GABAergic synaptosomes, respectively). Transporter-mediated release of GABA in human and rat neocortical synaptosomes was measured using the superfusion technique for the measurement of endogenous GABA. GABA release was evoked by either a sodium channel activator or a sodium/potassium-ATPase inhibitor when exocytosis was possible or prevented, and when the sodium/calcium exchanger was active or inhibited. The transporter-mediated release of GABA is because of elevated intracellular sodium. A reduction in the extracellular calcium increased this release (in both non-epileptic and epileptic, except Rasmussen encephalitis, synaptosomes). The inverse was seen during calcium doubling. In humans, GABA release was not affected by exocytosis inhibition, that is, it was solely transporter-mediated. However, in rat synaptosomes, an increase in GABA release at zero calcium was only exhibited when the exocytosis was prevented. The absence of calcium amplified the sodium/calcium exchanger activity, leading to elevated intracellular sodium, which, together with the stimulation-evoked intracellular sodium increment, enhanced GABA transporter reversal. Sodium/calcium exchange inhibitors diminished GABA release. Thus, an important seizure-induced extracellular calcium reduction might trigger a transporter- and sodium/calcium exchanger-related anti-seizure mechanism by augmenting transporter-mediated GABA release, a mechanism absent in rats. Uniquely, the additional increase in GABA release because of calcium-withdrawal dwindled during the course of illness in Rasmussen encephalitis. Seizures cause high Na(+) influx through action potentials. A

  5. Structural Determinants for Transport Across the Intestinal Bile Acid Transporter Using C-24 Bile Acid Conjugates

    PubMed Central

    Rais, Rana; Acharya, Chayan; MacKerell, Alexander D.; Polli, James E.

    2010-01-01

    The human apical sodium dependent bile acid transporter (hASBT) re-absorbs gram quantities of bile acid daily and is a potential prodrug target to increase oral drug absorption. In the absence of a high resolution hASBT crystal structure, 3D-QSAR modeling may prove beneficial in designing prodrug targets to hASBT. The objective was to derive a conformationally sampled pharmacophore 3D–QSAR (CSP-SAR) model for the uptake of bile acid conjugates by hASBT. A series of bile acid conjugates of glutamyl chenodeoxycholate were evaluated in terms of Km and normalized Vmax(normVmax) using hASBT-MDCK cells. All mono-anionic conjugates were potent substrates. Dianions, cations and zwitterions, which bound with a high affinity, were not substrates. CSP-SAR models were derived using structural and physicochemical descriptors, and evaluated via cross-validation. The best CSP-SAR model for Km included two structural and two physiochemical descriptors, where substrate hydrophobicity enhanced affinity. A best CSP-SAR model for Km/normVmax employed one structural and three physicochemical descriptors, also indicating hydrophobicity enhanced efficiency. Overall, the bile acid C-24 region accommodated a range of substituted anilines, provided a single negative charge was present near C-24. In comparing uptake findings to prior inhibition results, increased hydrophobicity enhanced activity, with dianions and zwitterions hindering activity. PMID:20939504

  6. A GABA-activated chloride-conductance not blocked by picrotoxin on spiny lobster neuromuscular preparations.

    PubMed Central

    Albert, J.; Lingle, C. J.; Marder, E.; O'Neil, M. B.

    1986-01-01

    Conductance increases to gamma-aminobutyric acid (GABA) were recorded in the gm6b and opener muscle of the spiny lobsters, Panulirus interruptus and P. argus. GABA-evoked responses were insensitive to picrotoxin at concentrations as high as 5 X 10(-5) M. Some blockade by picrotoxin was observed at higher concentrations. In normal physiological saline, the reversal potential of the Panulirus GABA-induced response was near the resting potential. The reversal potential was unaffected by reductions in sodium and calcium. Reduction of chloride by 50% resulted in a greater than 10 mV shift in the reversal potential of the GABA-induced response. Muscimol was able to mimic the action of GABA while baclofen was without effect. Bicuculline was a weak blocker. Avermectin B1a irreversibly increased the chloride permeability of the gm6b membrane. This conductance increase was blocked by picrotoxin over a range of concentrations similar to those required for blockade of the GABA-induced response. GABA-induced responses of the gm6b muscle of Homarus americanus were blocked almost completely by picrotoxin 10(-6) M. Sensitivity to picrotoxin is not invariably associated with GABA-activated chloride-mediated conductance increases. It is suggested that alteration in the binding-site for picrotoxin on the GABA-activated chloride-ion channel does not change other functional characteristics of the GABA-induced response. PMID:3708210

  7. Extent of colocalization of serotonin and GABA in neurons of the ventral medulla oblongata in rat.

    PubMed

    Millhorn, D E; Hökfelt, T; Seroogy, K; Verhofstad, A A

    1988-09-27

    The colocalization of serotonin (5-hydroxytryptamine; 5-HT) and gamma-aminobutyric acid (GABA) in the ventral aspect of the rat medulla oblongata was studied using antibodies directed against 5-HT and GABA. Although 5-HT- and GABA-immunoreactive cell bodies were observed over the entire rostral-caudal extent of the ventral medulla, the colocalization of these two classical neurotransmitters in single cells was, for the most part, limited to a region that corresponds anatomically to nucleus raphe magnus/nucleus paragigantocellularis. Schematic drawings showing the distribution of 5-HT/GABA cell bodies in the ventral medulla are provided. PMID:3066433

  8. Ionic Mechanisms of Neuronal Excitation by Inhibitory GABA_A Receptors

    NASA Astrophysics Data System (ADS)

    Staley, Kevin J.; Soldo, Brandi L.; Proctor, William R.

    1995-08-01

    Gamma-aminobutyric acid A (GABA_A) receptors are the principal mediators of synaptic inhibition, and yet when intensely activated, dendritic GABA_A receptors excite rather than inhibit neurons. The membrane depolarization mediated by GABA_A receptors is a result of the differential, activity-dependent collapse of the opposing concentration gradients of chloride and bicarbonate, the anions that permeate the GABA_A ionophore. Because this depolarization diminishes the voltage-dependent block of the N-methyl-D-aspartate (NMDA) receptor by magnesium, the activity-dependent depolarization mediated by GABA is sufficient to account for frequency modulation of synaptic NMDA receptor activation. Anionic gradient shifts may represent a mechanism whereby the rate and coherence of synaptic activity determine whether dendritic GABA_A receptor activation is excitatory or inhibitory.

  9. Aldehyde Dehydrogenase 1a1 Mediates a GABA Synthesis Pathway in Midbrain Dopaminergic Neurons

    PubMed Central

    Kim, Jae-Ick; Ganesan, Subhashree; Luo, Sarah X.; Wu, Yu-Wei; Park, Esther; Huang, Eric J.; Chen, Lu; Ding, Jun B.

    2016-01-01

    Midbrain dopamine neurons are an essential component of the basal ganglia circuitry, playing key roles in the control of fine movement and reward. Recently, it has been demonstrated that γ-aminobutyric acid (GABA), the chief inhibitory neurotransmitter, is co-released by dopamine neurons. Here we show that GABA corelease in dopamine neurons does not utilize the conventional GABA synthesizing enzymes, glutamate decarboxylases GAD65 and GAD67. Our experiments reveal an evolutionarily conserved GABA synthesis pathway mediated by aldehyde dehydrogenase 1a1 (ALDH1a1). Moreover, GABA co-release is modulated by ethanol at binge drinking blood alcohol concentrations and diminished ALDH1a1 leads to enhanced alcohol consumption and preference. These findings provide insights into the functional role of GABA co-release in midbrain dopamine neurons, which may be essential for reward-based behavior and addiction. PMID:26430123

  10. In vivo measurements of glutamate, GABA, and NAAG in schizophrenia.

    PubMed

    Rowland, Laura M; Kontson, Kimberly; West, Jeffrey; Edden, Richard A; Zhu, He; Wijtenburg, S Andrea; Holcomb, Henry H; Barker, Peter B

    2013-09-01

    The major excitatory and inhibitory neurotransmitters, glutamate (Glu) and gamma-aminobutyric acid (GABA), respectively, are implicated in the pathophysiology of schizophrenia. N-acetyl-aspartyl-glutamate (NAAG), a neuropeptide that modulates the Glu system, may also be altered in schizophrenia. This study investigated GABA, Glu + glutamine (Glx), and NAAG levels in younger and older subjects with schizophrenia. Forty-one subjects, 21 with chronic schizophrenia and 20 healthy controls, participated in this study. Proton magnetic resonance spectroscopy ((1)H-MRS) was used to measure GABA, Glx, and NAAG levels in the anterior cingulate (AC) and centrum semiovale (CSO) regions. NAAG in the CSO was higher in younger schizophrenia subjects compared with younger control subjects. The opposite pattern was observed in the older groups. Glx was reduced in the schizophrenia group irrespective of age group and brain region. There was a trend for reduced AC GABA in older schizophrenia subjects compared with older control subjects. Poor attention performance was correlated to lower AC GABA levels in both groups. Higher levels of CSO NAAG were associated with greater negative symptom severity in schizophrenia. These results provide support for altered glutamatergic and GABAergic function associated with illness course and cognitive and negative symptoms in schizophrenia. The study also highlights the importance of studies that combine MRS measurements of NAAG, GABA, and Glu for a more comprehensive neurochemical characterization of schizophrenia. PMID:23081992

  11. 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

  12. Parkinson's Disease and Neurodegeneration: GABA-Collapse Hypothesis.

    PubMed

    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 Ca(2+) 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 Ca(2+)/GABA mechanism stabilizes neuronal activity both at cellular and systemic levels. Decline in the Ca(2+)/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 Ca(2+)/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 Ca(2+)/GABA functional decline. PMID:27375426

  13. Loss of function mutation of the Slc38a3 glutamine transporter reveals its critical role for amino acid metabolism in the liver, brain, and kidney.

    PubMed

    Chan, Kessara; Busque, Stephanie M; Sailer, Manuela; Stoeger, Claudia; Bröer, Stefan; Daniel, Hannelore; Rubio-Aliaga, Isabel; Wagner, Carsten A

    2016-02-01

    Glutamine, the most abundant amino acid in mammals, is critical for cell and organ functions. Its metabolism depends on the ability of cells to take up or release glutamine by transporters located in the plasma membrane. Several solute carrier (SLC) families transport glutamine, but the SLC38 family has been thought to be mostly responsible for glutamine transport. We demonstrate that despite the large number of glutamine transporters, the loss of Snat3/Slc38a3 glutamine transporter has a major impact on the function of organs expressing it. Snat3 mutant mice were generated by N-ethyl-N-nitrosurea (ENU) mutagenesis and showed stunted growth, altered amino acid levels, hypoglycemia, and died around 20 days after birth. Hepatic concentrations of glutamine, glutamate, leucine, phenylalanine, and tryptophan were highly reduced paralleled by downregulation of the mTOR pathway possibly linking reduced amino acid availability to impaired growth and glucose homeostasis. Snat3-deficient mice had altered urea levels paralleled by dysregulation of the urea cycle, gluconeogenesis, and glutamine synthesis. Mice were ataxic with higher glutamine but reduced glutamate and gamma-aminobutyric acid (GABA) levels in brain consistent with a major role of Snat3 in the glutamine-glutamate cycle. Renal ammonium excretion was lower, and the expression of enzymes and amino acid transporters involved in ammoniagenesis were altered. Thus, SNAT3 is a glutamine transporter required for amino acid homeostasis and determines critical functions in various organs. Despite the large number of glutamine transporters, loss of Snat3 cannot be compensated, suggesting that this transporter is a major route of glutamine transport in the liver, brain, and kidney. PMID:26490457

  14. Amino acid transporters: roles in amino acid sensing and signalling in animal cells.

    PubMed Central

    Hyde, Russell; Taylor, Peter M; Hundal, Harinder S

    2003-01-01

    Amino acid availability regulates cellular physiology by modulating gene expression and signal transduction pathways. However, although the signalling intermediates between nutrient availability and altered gene expression have become increasingly well documented, how eukaryotic cells sense the presence of either a nutritionally rich or deprived medium is still uncertain. From recent studies it appears that the intracellular amino acid pool size is particularly important in regulating translational effectors, thus, regulated transport of amino acids across the plasma membrane represents a means by which the cellular response to amino acids could be controlled. Furthermore, evidence from studies with transportable amino acid analogues has demonstrated that flux through amino acid transporters may act as an initiator of nutritional signalling. This evidence, coupled with the substrate selectivity and sensitivity to nutrient availability classically associated with amino acid transporters, plus the recent discovery of transporter-associated signalling proteins, demonstrates a potential role for nutrient transporters as initiators of cellular nutrient signalling. Here, we review the evidence supporting the idea that distinct amino acid "receptors" function to detect and transmit certain nutrient stimuli in higher eukaryotes. In particular, we focus on the role that amino acid transporters may play in the sensing of amino acid levels, both directly as initiators of nutrient signalling and indirectly as regulators of external amino acid access to intracellular receptor/signalling mechanisms. PMID:12879880

  15. GABA shapes the dynamics of bistable perception.

    PubMed

    van Loon, Anouk M; Knapen, Tomas; Scholte, H Steven; St John-Saaltink, Elexa; Donner, Tobias H; Lamme, Victor A F

    2013-05-01

    Sometimes, perception fluctuates spontaneously between two distinct interpretations of a constant sensory input. These bistable perceptual phenomena provide a unique window into the neural mechanisms that create the contents of conscious perception. Models of bistable perception posit that mutual inhibition between stimulus-selective neural populations in visual cortex plays a key role in these spontaneous perceptual fluctuations. However, a direct link between neural inhibition and bistable perception has not yet been established experimentally. Here, we link perceptual dynamics in three distinct bistable visual illusions (binocular rivalry, motion-induced blindness, and structure from motion) to measurements of gamma-aminobutyric acid (GABA) concentrations in human visual cortex (as measured with magnetic resonance spectroscopy) and to pharmacological stimulation of the GABAA receptor by means of lorazepam. As predicted by a model of neural interactions underlying bistability, both higher GABA concentrations in visual cortex and lorazepam administration induced slower perceptual dynamics, as reflected in a reduced number of perceptual switches and a lengthening of percept durations. Thus, we show that GABA, the main inhibitory neurotransmitter, shapes the dynamics of bistable perception. These results pave the way for future studies into the competitive neural interactions across the visual cortical hierarchy that elicit conscious perception. PMID:23602476

  16. Downregulation of GABA[Subscript A] Receptor Protein Subunits a6, ß2, d, e, ?2, ?, and ?2 in Superior Frontal Cortex of Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rustan, Oyvind G.; Rooney, Robert J.; Thuras, Paul D.

    2014-01-01

    We measured protein and mRNA levels for nine gamma-aminobutyric acid A (GABA[subscript A]) receptor subunits in three brain regions (cerebellum, superior frontal cortex, and parietal cortex) in subjects with autism versus matched controls. We observed changes in mRNA for a number of GABA[subscript A] and GABA[subscript B] subunits and overall…

  17. Molecular cloning of mouse amino acid transport system B0, a neutral amino acid transporter related to Hartnup disorder.

    PubMed

    Bröer, Angelika; Klingel, Karin; Kowalczuk, Sonja; Rasko, John E J; Cavanaugh, Juleen; Bröer, Stefan

    2004-06-01

    Resorption of amino acids in kidney and intestine is mediated by transporters, which prefer groups of amino acids with similar physico-chemical properties. It is generally assumed that most neutral amino acids are transported across the apical membrane of epithelial cells by system B(0). Here we have characterized a novel member of the Na(+)-dependent neurotransmitter transporter family (B(0)AT1) isolated from mouse kidney, which shows all properties of system B(0). Flux experiments showed that the transporter is Na(+)-dependent, electrogenic, and actively transports most neutral amino acids but not anionic or cationic amino acids. Superfusion of mB(0)AT1-expressing oocytes with neutral amino acids generated inward currents, which were proportional to the fluxes observed with labeled amino acids. In situ hybridization showed strong expression in intestinal microvilli and in the proximal tubule of the kidney. Expression of mouse B(0)AT1 was restricted to kidney, intestine, and skin. It is generally assumed that mutations of the system B(0) transporter underlie autosomal recessive Hartnup disorder. In support of this notion mB(0)AT1 is located on mouse chromosome 13 in a region syntenic to human chromosome 5p15, the locus of Hartnup disorder. Thus, the human homologue of this transporter is an excellent functional and positional candidate for Hartnup disorder. PMID:15044460

  18. Identification and application of keto acids transporters in Yarrowia lipolytica.

    PubMed

    Guo, Hongwei; Liu, Peiran; Madzak, Catherine; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2015-01-01

    Production of organic acids by microorganisms is of great importance for obtaining building-block chemicals from sustainable biomass. Extracellular accumulation of organic acids involved a series of transporters, which play important roles in the accumulation of specific organic acid while lack of systematic demonstration in eukaryotic microorganisms. To circumvent accumulation of by-product, efforts have being orchestrated to carboxylate transport mechanism for potential clue in Yarrowia lipolytica WSH-Z06. Six endogenous putative transporter genes, YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D24607g, YALI0D20108g and YALI0E32901g, were identified. Transport characteristics and substrate specificities were further investigated using a carboxylate-transport-deficient Saccharomyces cerevisiae strain. These transporters were expressed in Y. lipolytica WSH-Z06 to assess their roles in regulating extracellular keto acids accumulation. In a Y. lipolytica T1 line over expressing YALI0B19470g, α-ketoglutarate accumulated to 46.7 g·L(-1), whereas the concentration of pyruvate decreased to 12.3 g·L(-1). Systematic identification of these keto acids transporters would provide clues to further improve the accumulation of specific organic acids with higher efficiency in eukaryotic microorganisms. PMID:25633653

  19. Identification and application of keto acids transporters in Yarrowia lipolytica

    PubMed Central

    Guo, Hongwei; Liu, Peiran; Madzak, Catherine; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2015-01-01

    Production of organic acids by microorganisms is of great importance for obtaining building-block chemicals from sustainable biomass. Extracellular accumulation of organic acids involved a series of transporters, which play important roles in the accumulation of specific organic acid while lack of systematic demonstration in eukaryotic microorganisms. To circumvent accumulation of by-product, efforts have being orchestrated to carboxylate transport mechanism for potential clue in Yarrowia lipolytica WSH-Z06. Six endogenous putative transporter genes, YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D24607g, YALI0D20108g and YALI0E32901g, were identified. Transport characteristics and substrate specificities were further investigated using a carboxylate-transport-deficient Saccharomyces cerevisiae strain. These transporters were expressed in Y. lipolytica WSH-Z06 to assess their roles in regulating extracellular keto acids accumulation. In a Y. lipolytica T1 line over expressing YALI0B19470g, α-ketoglutarate accumulated to 46.7 g·L−1, whereas the concentration of pyruvate decreased to 12.3 g·L−1. Systematic identification of these keto acids transporters would provide clues to further improve the accumulation of specific organic acids with higher efficiency in eukaryotic microorganisms. PMID:25633653

  20. Transport, metabolism, and effect of chronic feeding of lagodeoxycholic acid. A new, natural bile acid.

    PubMed

    Schmassmann, A; Angellotti, M A; Clerici, C; Hofmann, A F; Ton-Nu, H T; Schteingart, C D; Marcus, S N; Hagey, L R; Rossi, S S; Aigner, A

    1990-10-01

    Ursodeoxycholic acid, the 7 beta-hydroxy epimer of chenodeoxycholic acid, is more hydrophilic and less hepatotoxic than chenodeoxycholic acid. Because "lagodeoxycholic acid," the 12 beta-hydroxy epimer of deoxycholic acid, is also more hydrophilic than deoxycholic acid, it was hypothesized that it should also be less hepatotoxic than deoxycholic acid. To test this, lagodeoxycholic acid was synthesized, and its transport and metabolism were examined in the rat, rabbit, and hamster. The taurine conjugate of lagodeoxycholic acid was moderately well transported by the perfused rat ileum (Tmax = 2 mumol/min.kg). In rats and hamsters with biliary fistulas, the taurine conjugate of lagodeoxycholic acid was well transported by the liver with a Tmax greater than 20 mumol/min.kg; for the taurine conjugate of deoxycholic acid, doses infused at a rate greater than 2.5 mumol/min.kg are known to cause cholestasis and death. Hepatic biotransformation of lagodeoxycholic acid in the rabbit was limited to conjugation with glycine; in the hamster, lagodeoxycholic acid was conjugated with glycine or taurine; in addition, 7-hydroxylation occurred to a slight extent (approximately 10%). When lagodeoxycholic acid was instilled in the rabbit colon, it was absorbed as such although within hours it was progressively epimerized by bacteria to deoxycholic acid. When injected intravenously and allowed to circulate enterohepatically, lagodeoxycholic acid was largely epimerized to deoxycholic acid in 24 hours. Surgical creation of a distal ileostomy abolished epimerization in the rabbit, indicating that exposure to colonic bacterial enzymes was required for the epimerization. Lagodeoxycholic acid was administered for 3 weeks at a dose of 180 mumol/day (0.1% by weight of a chow diet; 2-4 times the endogenous bile acid synthesis rate); other groups received identical doses of deoxycholic acid (hamster) or cholyltaurine, a known precursor of deoxycholic acid (rabbit). After 3 weeks of

  1. Carboxylic Acids Plasma Membrane Transporters in Saccharomyces cerevisiae.

    PubMed

    Casal, Margarida; Queirós, Odília; Talaia, Gabriel; Ribas, David; Paiva, Sandra

    2016-01-01

    This chapter covers the functionally characterized plasma membrane carboxylic acids transporters Jen1, Ady2, Fps1 and Pdr12 in the yeast Saccharomyces cerevisiae, addressing also their homologues in other microorganisms, as filamentous fungi and bacteria. Carboxylic acids can either be transported into the cells, to be used as nutrients, or extruded in response to acid stress conditions. The secondary active transporters Jen1 and Ady2 can mediate the uptake of the anionic form of these substrates by a H(+)-symport mechanism. The undissociated form of carboxylic acids is lipid-soluble, crossing the plasma membrane by simple diffusion. Furthermore, acetic acid can also be transported by facilitated diffusion via Fps1 channel. At the cytoplasmic physiological pH, the anionic form of the acid prevails and it can be exported by the Pdr12 pump. This review will highlight the mechanisms involving carboxylic acids transporters, and the way they operate according to the yeast cell response to environmental changes, as carbon source availability, extracellular pH and acid stress conditions. PMID:26721276

  2. Cortical Gene Expression After a Conditional Knockout of 67 kDa Glutamic Acid Decarboxylase in Parvalbumin Neurons.

    PubMed

    Georgiev, Danko; Yoshihara, Toru; Kawabata, Rika; Matsubara, Takurou; Tsubomoto, Makoto; Minabe, Yoshio; Lewis, David A; Hashimoto, Takanori

    2016-07-01

    In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis. PMID:26980143

  3. Endogenous gamma-aminobutyric acid (GABA)(A) receptor active neurosteroids and the sedative/hypnotic action of gamma-hydroxybutyric acid (GHB): a study in GHB-S (sensitive) and GHB-R (resistant) rat lines.

    PubMed

    Barbaccia, Maria Luisa; Carai, Mauro A M; Colombo, Giancarlo; Lobina, Carla; Purdy, Robert H; Gessa, Gian Luigi

    2005-07-01

    In the rat brain, gamma-hydroxybutyric-acid (GHB) increases the concentrations of 3alpha-hydroxy,5alpha-pregnan-20-one (allopregnanolone, 3alpha,5alpha-THP) and 3alpha,21-dihydroxy,5alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone/3alpha,5alphaTHDOC), two neurosteroids acting as positive allosteric modulators of gamma-aminobutyric acid (GABA)(A) receptors. This study was aimed at assessing whether neurosteroids play a role in GHB-induced loss of righting reflex (LORR). Basal and GHB-stimulated brain concentrations of endogenous 3alpha,5alpha-THP and 3alpha,5alpha-THDOC were analyzed in two rat lines, GHB-sensitive (GHB-S) and GHB-resistant (GHB-R), selectively bred for opposite sensitivity to GHB-induced sedation/hypnosis. Basal neurosteroid concentrations were similar in brain cortex of the two rat lines. However, in male GHB-S rats, administration of GHB (1000 mg/kg, i.p., 30 min) increased brain cortical concentrations of 3alpha,5alpha-THP and 3alpha,5alpha-THDOC 7- and 2.5-fold, respectively, whilst male GHB-R animals displayed only a 4- and 2-fold increase, respectively. In GHB-S rats this increase lasted up to 90 min and declined 180 min following GHB administration, a time course that matches LORR onset and duration. In contrast, in GHB-R rats, which failed to show GHB-induced LORR, brain cortical 3alpha,5alpha-THP and 3alpha,5alpha-THDOC had returned to control values within 90 min. At onset of LORR, a similar increase in brain cortical levels of 3alpha,5alpha-THP and 3alpha,5alpha-THDOC (2-3-fold) was observed in GHB-S female rats and in the few female GHB-R rats that lost the righting reflex after GHB administration, but not in female GHB-R rats failing to show LORR. Sub-hypnotic doses (7.5 and 12.5 mg/kg, i.p.) of pregnanolone, administered 10 min before GHB, dose-dependently facilitated the expression of GHB-induced LORR in GHB-R male rats. These results suggest that the GHB-induced increases of brain 3alpha,5alpha-THP and 3alpha,5alpha

  4. The plant GABA signaling downregulates horizontal transfer of the Agrobacterium tumefaciens virulence plasmid.

    PubMed

    Lang, Julien; Gonzalez-Mula, Almudena; Taconnat, Ludivine; Clement, Gilles; Faure, Denis

    2016-05-01

    In the tumor-inducing (Ti) Agrobacterium tumefaciens, quorum sensing activates the horizontal transfer of the virulent Ti plasmid. In pure culture, this process can be impaired by the A. tumefaciens BlcC lactonase, whose expression is induced by gamma-aminobutyrate (GABA). It was therefore hypothesized that host GABA content might modulate quorum sensing and virulence gene dissemination during A. tumefaciens infection. We examined GABA metabolism and transport in Arabidopsis thaliana tumors combining transcriptomic, metabolomic and histological approaches. In addition, using genetically modified plants and bacteria, we evaluated the impact of plant host GABA content on Ti plasmid dissemination. The results showed that GABA and free proline, which acts as an antagonist of GABA uptake in A. tumefaciens, accumulated in wild-type tumors relative to uninfected plant tissues. Moreover, comparisons of tumors induced on Col-0 and her1 plants showed that the increase in the plant GABA : proline ratio was associated with both the upregulated expression of the blcC gene and the decreased dissemination of Ti plasmid in tumor-colonizing A. tumefaciens populations. This work demonstrates experimentally that the variation in the GABA content in plant tumors can interfere with the dissemination of A. tumefaciens Ti plasmids, and therefore highlights plant GABA content as an important trait in the struggle against pathogenic bacteria. PMID:26714842

  5. Sialic acid acquisition in bacteria-one substrate, many transporters.

    PubMed

    Thomas, Gavin H

    2016-06-15

    The sialic acids are a family of 9-carbon sugar acids found predominantly on the cell-surface glycans of humans and other animals within the Deuterostomes and are also used in the biology of a wide range of bacteria that often live in association with these animals. For many bacteria sialic acids are simply a convenient source of food, whereas for some pathogens they are also used in immune evasion strategies. Many bacteria that use sialic acids derive them from the environment and so are dependent on sialic acid uptake. In this mini-review I will describe the discovery and characterization of bacterial sialic acids transporters, revealing that they have evolved multiple times across multiple diverse families of transporters, including the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic (TRAP), major facilitator superfamily (MFS) and sodium solute symporter (SSS) transporter families. In addition there is evidence for protein-mediated transport of sialic acids across the outer membrane of Gram negative bacteria, which can be coupled to periplasmic processing of different sialic acids to the most common form, β-D-N-acetylneuraminic acid (Neu5Ac) that is most frequently taken up into the cell. PMID:27284039

  6. Sialic Acid Transport Contributes to Pneumococcal Colonization ▿

    PubMed Central

    Marion, Carolyn; Burnaugh, Amanda M.; Woodiga, Shireen A.; King, Samantha J.

    2011-01-01

    Streptococcus pneumoniae is a major cause of pneumonia and meningitis. Airway colonization is a necessary precursor to disease, but little is known about how the bacteria establish and maintain colonization. Carbohydrates are required as a carbon source for pneumococcal growth and, therefore, for colonization. Free carbohydrates are not readily available in the naso-oropharynx; however, N- and O-linked glycans are common in the airway. Sialic acid is the most common terminal modification on N- and O-linked glycans and is likely encountered frequently by S. pneumoniae in the airway. Here we demonstrate that sialic acid supports pneumococcal growth when provided as a sole carbon source. Growth on sialic acid requires import into the bacterium. Three genetic regions have been proposed to encode pneumococcal sialic acid transporters: one sodium solute symporter and two ATP binding cassette (ABC) transporters. Data demonstrate that one of these, satABC, is required for transport of sialic acid. A satABC mutant displayed significantly reduced growth on both sialic acid and the human glycoprotein alpha-1. The importance of satABC for growth on human glycoprotein suggests that sialic acid transport may be important in vivo. Indeed, the satABC mutant was significantly reduced in colonization of the murine upper respiratory tract. This work demonstrates that S. pneumoniae is able to use sialic acid as a sole carbon source and that utilization of sialic acid is likely important during pneumococcal colonization. PMID:21189320

  7. Regulation of renal amino acid transporters during metabolic acidosis.

    PubMed

    Moret, Caroline; Dave, Mital H; Schulz, Nicole; Jiang, Jean X; Verrey, Francois; Wagner, Carsten A

    2007-02-01

    The kidney plays a major role in acid-base homeostasis by adapting the excretion of acid equivalents to dietary intake and metabolism. Urinary acid excretion is mediated by the secretion of protons and titratable acids, particularly ammonia. NH(3) is synthesized in proximal tubule cells from glutamine taken up via specific amino acid transporters. We tested whether kidney amino acid transporters are regulated in mice in which metabolic acidosis was induced with NH(4)Cl. Blood gas and urine analysis confirmed metabolic acidosis. Real-time RT-PCR was performed to quantify the mRNAs of 16 amino acid transporters. The mRNA of phosphoenolpyruvate carboxykinase (PEPCK) was quantified as positive control for the regulation and that of GAPDH, as internal standard. In acidosis, the mRNA of kidney system N amino acid transporter SNAT3 (SLC38A3/SN1) showed a strong induction similar to that of PEPCK, whereas all other tested mRNAs encoding glutamine or glutamate transporters were unchanged or reduced in abundance. At the protein level, Western blotting and immunohistochemistry demonstrated an increased abundance of SNAT3 and reduced expression of the basolateral cationic amino acid/neutral amino acid exchanger subunit y(+)-LAT1 (SLC7A7). SNAT3 was localized to the basolateral membrane of the late proximal tubule S3 segment in control animals, whereas its expression was extended to the earlier S2 segment of the proximal tubule during acidosis. Our results suggest that the selective regulation of SNAT3 and y(+)LAT1 expression may serve a major role in the renal adaptation to acid secretion and thus for systemic acid-base balance. PMID:17003226

  8. Neurotransmitters as food supplements: the effects of GABA on brain and behavior

    PubMed Central

    Boonstra, Evert; de Kleijn, Roy; Colzato, Lorenza S.; Alkemade, Anneke; Forstmann, Birte U.; Nieuwenhuis, Sander

    2015-01-01

    Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the human cortex. The food supplement version of GABA is widely available online. Although many consumers claim that they experience benefits from the use of these products, it is unclear whether these supplements confer benefits beyond a placebo effect. Currently, the mechanism of action behind these products is unknown. It has long been thought that GABA is unable to cross the blood–brain barrier (BBB), but the studies that have assessed this issue are often contradictory and range widely in their employed methods. Accordingly, future research needs to establish the effects of oral GABA administration on GABA levels in the human brain, for example using magnetic resonance spectroscopy. There is some evidence in favor of a calming effect of GABA food supplements, but most of this evidence was reported by researchers with a potential conflict of interest. We suggest that any veridical effects of GABA food supplements on brain and cognition might be exerted through BBB passage or, more indirectly, via an effect on the enteric nervous system. We conclude that the mechanism of action of GABA food supplements is far from clear, and that further work is needed to establish the behavioral effects of GABA. PMID:26500584

  9. GABA-mediated regulation of the activity-dependent olfactory bulb dopaminergic phenotype

    PubMed Central

    Akiba, Yosuke; Sasaki, Hayato; Huerta, Patricio T.; Estevez, Alvaro G.; Baker, Harriet; Cave, John W.

    2009-01-01

    Gamma-amino-butyric acid (GABA) regulates the proliferation and migration of olfactory bulb (OB) interneuron progenitors derived from the subventricular zone (SVZ), but the role of GABA in the differentiation of these progenitors has been largely unexplored. This study examined the role of GABA in the differentiation of OB dopaminergic interneurons using neonatal forebrain organotypic slice cultures prepared from transgenic mice expressing GFP under the control of the tyrosine hydroxylase (Th) gene promoter (ThGFP). KCl-mediated depolarization of the slices induced ThGFP expression. The addition of GABA to the depolarized slices further increased GFP fluorescence by inducing ThGFP expression in an additional set of periglomerular cells. These findings showed that GABA promoted differentiation of SVZ-derived OB dopaminergic interneurons and suggested that GABA indirectly regulated Th expression and OB dopaminergic neuron differentiation through an acceleration of the maturation rate for the dopaminergic progenitors. Additional studies revealed that the effect of GABA on ThGFP expression required activation of L- and P/Q-type Ca+2 channels as well as GABAA and GABAB receptors. These voltage-gated Ca+2 channels and GABA receptors have previously been shown to be required for the co-expressed GABAergic phenotype in the OB interneurons. Together, these findings suggest that Th expression and the differentiation of OB dopaminergic interneurons are coupled to the co-expressed GABAergic phenotype, and demonstrate a novel role for GABA in neurogenesis. PMID:19301430

  10. Neurotransmitters as food supplements: the effects of GABA on brain and behavior.

    PubMed

    Boonstra, Evert; de Kleijn, Roy; Colzato, Lorenza S; Alkemade, Anneke; Forstmann, Birte U; Nieuwenhuis, Sander

    2015-01-01

    Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the human cortex. The food supplement version of GABA is widely available online. Although many consumers claim that they experience benefits from the use of these products, it is unclear whether these supplements confer benefits beyond a placebo effect. Currently, the mechanism of action behind these products is unknown. It has long been thought that GABA is unable to cross the blood-brain barrier (BBB), but the studies that have assessed this issue are often contradictory and range widely in their employed methods. Accordingly, future research needs to establish the effects of oral GABA administration on GABA levels in the human brain, for example using magnetic resonance spectroscopy. There is some evidence in favor of a calming effect of GABA food supplements, but most of this evidence was reported by researchers with a potential conflict of interest. We suggest that any veridical effects of GABA food supplements on brain and cognition might be exerted through BBB passage or, more indirectly, via an effect on the enteric nervous system. We conclude that the mechanism of action of GABA food supplements is far from clear, and that further work is needed to establish the behavioral effects of GABA. PMID:26500584

  11. Comparative Mapping of GABA-Immunoreactive Neurons in the Buccal Ganglia of Nudipleura Molluscs.

    PubMed

    Gunaratne, Charuni A; Katz, Paul S

    2016-04-15

    Phylogenetic comparisons of neurotransmitter distribution are important for understanding the ground plan organization of nervous systems. This study describes the γ-aminobutyric acid (GABA)-immunoreactive (GABA-ir) neurons in the buccal ganglia of six sea slug species (Mollusca, Gastropoda, Euthyneura, Nudipleura). In the nudibranch species, Hermissenda crassicornis, Tritonia diomedea, Tochuina tetraquetra, and Dendronotus iris, the number of GABA-ir neurons was highly consistent. Another nudibranch, Melibe leonina, however, contained approximately half the number of GABA-ir neurons. This may relate to its loss of a radula and its unique feeding behavior. The GABA immunoreactivity in a sister group to the nudibranchs, Pleurobranchaea californica, differed drastically from that of the nudibranchs. Not only did it have significantly more GABA-ir neurons but it also had a unique GABA distribution pattern. Furthermore, unlike the nudibranchs, the Pleurobranchaea GABA distribution was also different from that of other, more distantly related, euopisthobranch and panpulmonate snails and slugs. This suggests that the Pleurobranchaea GABA distribution may be a derived feature, unique to this lineage. The majority of GABA-ir axons and neuropil in the Nudipleura were restricted to the buccal ganglia, commissures, and connectives. However, in Tritonia and Pleurobranchaea, we detected a few GABA-ir fibers in buccal nerves that innervate feeding muscles. Although the specific functions of the GABA-ir neurons in the species in this study are not known, the innervation pattern suggests these neurons may play an integrative or regulatory role in bilaterally coordinated behaviors in the Nudipleura. PMID:26355705

  12. Stimulation of TM3 Leydig cell proliferation via GABAA receptors: A new role for testicular GABA

    PubMed Central

    Geigerseder, Christof; Doepner, Richard FG; Thalhammer, Andrea; Krieger, Annette; Mayerhofer, Artur

    2004-01-01

    The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABAA and GABAB receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABAA receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABAA receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABAA receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABAA agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABAA antagonist bicuculline, implying a role for GABAA receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABAA receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment. PMID:15040802

  13. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site

    PubMed Central

    Snell, Heather D.

    2015-01-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride’s positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound’s potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ. PMID:25829529

  14. Conditional Knock-Out of Vesicular GABA Transporter Gene from Starburst Amacrine Cells Reveals the Contributions of Multiple Synaptic Mechanisms Underlying Direction Selectivity in the Retina

    PubMed Central

    Pei, Zhe; Chen, Qiang; Koren, David; Giammarinaro, Benno; Acaron Ledesma, Hector

    2015-01-01

    Direction selectivity of direction-selective ganglion cells (DSGCs) in the retina results from patterned excitatory and inhibitory inputs onto DSGCs during motion stimuli. The inhibitory inputs onto DSGCs are directionally tuned to the antipreferred (null) direction and therefore potently suppress spiking during motion in the null direction. However, whether direction-selective inhibition is indispensable for direction selectivity is unclear. Here, we selectively eliminated the directional tuning of inhibitory inputs onto DSGCs by disrupting GABA release from the presynaptic interneuron starburst amacrine cell in the mouse retina. We found that, even without directionally tuned inhibition, direction selectivity can still be implemented in a subset of On-Off DSGCs by direction-selective excitation and a temporal offset between excitation and isotropic inhibition. Our results therefore demonstrate the concerted action of multiple synaptic mechanisms for robust direction selectivity in the retina. SIGNIFICANCE STATEMENT The direction-selective circuit in the retina has been a classic model to study neural computations by the brain. An important but unresolved question is how direction selectivity is implemented by directionally tuned excitatory and inhibitory mechanisms. Here we specifically removed the direction tuning of inhibition from the circuit. We found that direction tuning of inhibition is important but not indispensable for direction selectivity of DSGCs' spiking activity, and that the residual direction selectivity is implemented by direction-selective excitation and temporal offset between excitation and inhibition. Our results highlight the concerted actions of synaptic excitation and inhibition required for robust direction selectivity in the retina and provide critical insights into how patterned excitation and inhibition collectively implement sensory processing. PMID:26400950

  15. Acid aerosol transport episodes in Toronto, Ontario

    SciTech Connect

    Thurston, G.D. . Inst. of Environmental Medicine); Waldman, J. )

    1987-01-01

    In this paper, the authors examine the pollution data collected during a 1986 field study in order to assess the nature and sources of acidic aerosols in the Toronto metropolitan area during this period. Through the examination of the continuous and filter aerosol data, isobaric back-trajectories of air masses, weather maps, and available trace element data, assessment are made of the character and possible sources of acid aerosols in this Southern Ontario city.

  16. Physiological Adaptation to the Loss of Amino Acid Transport Ability

    PubMed Central

    DeBusk, Ruth M.; Ogilvie-Villa, Susan

    1982-01-01

    A strain of Neurospora crassa devoid of constitutive amino acid transport ability can utilize arginine as the sole nitrogen source. Nitrogen starvation, presence of arginine, and mutational inactivation of the general permease are key factors in signaling production of an extracellular enzyme which removes the alpha-amino group from the amino acid. PMID:6214547

  17. Nucleic acids encoding metal uptake transporters and their uses

    DOEpatents

    Schroeder, Julian I.; Antosiewicz, Danuta M.; Schachtman, Daniel P.; Clemens, Stephan

    1999-01-01

    The invention provides LCT1 nucleic acids which encode metal ion uptake transporters. The invention also provides methods of modulating heavy metal and alkali metal uptake in plants. The methods involve producing transgenic plants comprising a recombinant expression cassette containing an LCT1 nucleic acid linked to a plant promoter.

  18. Potentiation of the ionotropic GABA receptor response by whiskey fragrance.

    PubMed

    Hossain, Sheikh Julfikar; Aoshima, Hitoshi; Koda, Hirofumi; Kiso, Yoshinobu

    2002-11-01

    It is well-known that the target of most mood-defining compounds is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activity in the human brain. To study the effects of whiskey fragrance on the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting rat whole brain mRNA or cRNA prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors. Most whiskey components such as phenol, ethoxy, and lactone derivatives potentiated the electrical responses of GABA(A) receptors, especially ethyl phenylpropanoate (EPP), which strongly potentiated the response. When this compound was applied to mice through respiration, the convulsions induced by pentetrazole were delayed, suggesting that EPP was absorbed by the brain, where it could potentiate the GABA(A) receptor responses. The extract of other alcoholic drinks such as wine, sake, brandy, and shochu also potentiated the responses to varying degrees. Although these fragrant components are present in alcoholic drinks at low concentrations (extremely small quantities compared with ethanol), they may also modulate the mood or consciousness of the human through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic fragrant compounds are easily absorbed into the brain through the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response. PMID:12405783

  19. Molecular Evolution of Plant AAP and LHT Amino Acid Transporters.

    PubMed

    Tegeder, Mechthild; Ward, John M

    2012-01-01

    Nitrogen is an essential mineral nutrient and it is often transported within living organisms in its reduced form, as amino acids. Transport of amino acids across cellular membranes requires proteins, and here we report the phylogenetic analysis across taxa of two amino acid transporter families, the amino acid permeases (AAPs) and the lysine-histidine-like transporters (LHTs). We found that the two transporter families form two distinct groups in plants supporting the concept that both are essential. AAP transporters seem to be restricted to land plants. They were found in Selaginella moellendorffii and Physcomitrella patens but not in Chlorophyte, Charophyte, or Rhodophyte algae. AAPs were strongly represented in vascular plants, consistent with their major function in phloem (vascular tissue) loading of amino acids for sink nitrogen supply. LHTs on the other hand appeared prior to land plants. LHTs were not found in chlorophyte algae Chlamydomonas reinhardtii and Volvox carterii. However, the characean alga Klebsormidium flaccidum encodes KfLHT13 and phylogenetic analysis indicates that it is basal to land plant LHTs. This is consistent with the hypothesis that characean algae are ancestral to land plants. LHTs were also found in both S. moellendorffii and P. patens as well as in monocots and eudicots. To date, AAPs and LHTs have mainly been characterized in Arabidopsis (eudicots) and these studies provide clues to the functions of the newly identified homologs. PMID:22645574

  20. Molecular Evolution of Plant AAP and LHT Amino Acid Transporters

    PubMed Central

    Tegeder, Mechthild; Ward, John M.

    2012-01-01

    Nitrogen is an essential mineral nutrient and it is often transported within living organisms in its reduced form, as amino acids. Transport of amino acids across cellular membranes requires proteins, and here we report the phylogenetic analysis across taxa of two amino acid transporter families, the amino acid permeases (AAPs) and the lysine–histidine-like transporters (LHTs). We found that the two transporter families form two distinct groups in plants supporting the concept that both are essential. AAP transporters seem to be restricted to land plants. They were found in Selaginella moellendorffii and Physcomitrella patens but not in Chlorophyte, Charophyte, or Rhodophyte algae. AAPs were strongly represented in vascular plants, consistent with their major function in phloem (vascular tissue) loading of amino acids for sink nitrogen supply. LHTs on the other hand appeared prior to land plants. LHTs were not found in chlorophyte algae Chlamydomonas reinhardtii and Volvox carterii. However, the characean alga Klebsormidium flaccidum encodes KfLHT13 and phylogenetic analysis indicates that it is basal to land plant LHTs. This is consistent with the hypothesis that characean algae are ancestral to land plants. LHTs were also found in both S. moellendorffii and P. patens as well as in monocots and eudicots. To date, AAPs and LHTs have mainly been characterized in Arabidopsis (eudicots) and these studies provide clues to the functions of the newly identified homologs. PMID:22645574

  1. Acid aerosol transport episodes in Toronto, Ontario

    SciTech Connect

    Thurston, G.D.; Waldman, J.M.

    1987-07-01

    Authors used recently developed equipment to continuously monitor levels of H/sub 2/SO/sub 4/, NH/sub 4/HSO/sub 4/ and (NH/sub 4/)/sub 2/SO/sub 4/ concentrations in the ambient air outside Toronto, Ontario. These data were combined with 48-hour isobaric air mass back-trajectories ending in Toronto on each of the four days with highest acid (and sulfate) aerosol levels. The air masses with highest acid levels were found to have first passed over the SO/sub 2/ source region of the U.S. and then across the Great Lakes to Toronto. The role of ammonia as a modulator of aerosol acidity for eastern U.S. cities but not for Toronto (where the Great Lakes serve as ammonia sinks) is also discussed.

  2. Attenuated inhibition by levofloxacin, l-isomer of ofloxacin, on GABA response in the dissociated rat hippocampal neurons.

    PubMed

    Imanishi, T; Akahane, K; Akaike, N

    1995-06-30

    The effects of ofloxacin (OFLX) and its isomers, levofloxacin (LVFX) and DR-3354, on the gamma-aminobutyric acid (GABA)-induced Cl- current in acutely dissociated rat hippocampal CA1 neurons were investigated using nystatin perforated patch recording configuration under voltage-clamp conditions. At 10(-5) M these 3 compounds themselves did not affect the GABA response. Biphenylacetic acid (BPA) at 10(-5) M also had no effect on the GABA response, but BPA greatly suppressed the GABA response in combination with these 3 compounds without affecting the reversal potential of GABA response. The inhibitory effects of OFLX and DR-3354 on the GABA response were stronger than that of LVFX. LVFX inhibited the response in a competitive and voltage-independent manner. The results suggest that LVFX has lower CNS adverse effects, such as convulsions, compared to OFLX. PMID:7478164

  3. Role of proline and GABA in sexual reproduction of angiosperms.

    PubMed

    Biancucci, Marco; Mattioli, Roberto; Forlani, Giuseppe; Funck, Dietmar; Costantino, Paolo; Trovato, Maurizio

    2015-01-01

    Two glutamate derivatives, proline and γ-aminobutyric acid (GABA), appear to play pivotal roles in different aspects of sexual reproduction in angiosperms, although their precise function in plant reproduction and the molecular basis of their action are not yet fully understood. Proline and GABA have long been regarded as pivotal amino acids in pollen vitality and fertility. Proline may constitute up to 70% of the free amino acid pool in pollen grains and it has been recently shown that Arabidopsis mutants affected in the first and rate-limiting step in proline synthesis produce aberrant and infertile pollen grains, indicating that proline synthesis is required for pollen development and fertility. Concerning GABA, a large body of evidence points to this glutamate derivative as a key determinant of post-pollination fertilization. Intriguingly, proline has also been associated with pollination, another aspect of sexual reproduction, since honeybees were reported to show a strong preference for proline-enriched nectars. In this review, we survey current knowledge on the roles of proline and GABA in plant fertility, and discuss future perspectives potentially capable to improve our understanding on the functions of these amino acids in pollen development, pollination, and pollen tube guidance. PMID:26388884

  4. Role of proline and GABA in sexual reproduction of angiosperms

    PubMed Central

    Biancucci, Marco; Mattioli, Roberto; Forlani, Giuseppe; Funck, Dietmar; Costantino, Paolo; Trovato, Maurizio

    2015-01-01

    Two glutamate derivatives, proline and γ-aminobutyric acid (GABA), appear to play pivotal roles in different aspects of sexual reproduction in angiosperms, although their precise function in plant reproduction and the molecular basis of their action are not yet fully understood. Proline and GABA have long been regarded as pivotal amino acids in pollen vitality and fertility. Proline may constitute up to 70% of the free amino acid pool in pollen grains and it has been recently shown that Arabidopsis mutants affected in the first and rate-limiting step in proline synthesis produce aberrant and infertile pollen grains, indicating that proline synthesis is required for pollen development and fertility. Concerning GABA, a large body of evidence points to this glutamate derivative as a key determinant of post-pollination fertilization. Intriguingly, proline has also been associated with pollination, another aspect of sexual reproduction, since honeybees were reported to show a strong preference for proline-enriched nectars. In this review, we survey current knowledge on the roles of proline and GABA in plant fertility, and discuss future perspectives potentially capable to improve our understanding on the functions of these amino acids in pollen development, pollination, and pollen tube guidance. PMID:26388884

  5. Transport of D-serine via the amino acid transporter ATB(0,+) expressed in the colon.

    PubMed

    Hatanaka, Takahiro; Huang, Wei; Nakanishi, Takeo; Bridges, Christy C; Smith, Sylvia B; Prasad, Puttur D; Ganapathy, Malliga E; Ganapathy, Vadivel

    2002-02-22

    D-Serine, synthesized endogenously in the brain, is an important modulator of glutamatergic neurotransmission. Since colonic bacteria produce D-serine, we asked the question whether there are transport mechanisms in the colon that might make this exogenously produced D-serine available to the host. Here we identify for the first time an amino acid transporter in the intestine for high-affinity active transport of D-serine. This transporter, called ATB(0,+), is a Na(+)- and Cl(-)-coupled transporter for L-enantiomers of neutral and cationic amino acids. Here we demonstrate that ATB(0,+) is also capable of mediating the Na(+)- and Cl(-)-coupled transport of D-serine. The affinity of ATB(0,+) for L-serine and D-serine is similar, the K(t) value for the two enantiomers being approximately 150 microM. In addition to D-serine, ATB(0,+) transports D-alanine, D-methionine, D-leucine, and D-tryptophan. However, several other neutral and cationic amino acids that are transportable substrates for ATB(0,+) as L-enantiomers are not transported when presented as D-enantiomers. ATB(0,+) is expressed in the intestinal tract, interestingly not in the proximal intestine but in the distal intestine. Expression is most predominant in the colon where the transporter is localized to the luminal membrane of colonocytes, making this transporter uniquely suitable for absorption of bacteria-derived D-serine. PMID:11846403

  6. 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.

  7. GABA accumulation causes cell elongation defects and a decrease in expression of genes encoding secreted and cell wall-related proteins in Arabidopsis thaliana.

    PubMed

    Renault, Hugues; El Amrani, Abdelhak; Palanivelu, Ravishankar; Updegraff, Emily P; Yu, Agnès; Renou, Jean-Pierre; Preuss, Daphne; Bouchereau, Alain; Deleu, Carole

    2011-05-01

    GABA (γ-aminobutyric acid), a non-protein amino acid, is a signaling factor in many organisms. In plants, GABA is known to accumulate under a variety of stresses. However, the consequence of GABA accumulation, especially in vegetative tissues, remains poorly understood. Moreover, gene expression changes as a consequence of GABA accumulation in plants are largely unknown. The pop2 mutant, which is defective in GABA catabolism and accumulates GABA, is a good model to examine the effects of GABA accumulation on plant development. Here, we show that the pop2 mutants have pollen tube elongation defects in the transmitting tract of pistils. Additionally, we observed growth inhibition of primary root and dark-grown hypocotyl, at least in part due to cell elongation defects, upon exposure to exogenous GABA. Microarray analysis of pop2-1 seedlings grown in GABA-supplemented medium revealed that 60% of genes whose expression decreased encode secreted proteins. Besides, functional classification of genes with decreased expression in the pop2-1 mutant showed that cell wall-related genes were significantly enriched in the microarray data set, consistent with the cell elongation defects observed in pop2 mutants. Our study identifies cell elongation defects caused by GABA accumulation in both reproductive and vegetative tissues. Additionally, our results show that genes that encode secreted and cell wall-related proteins may mediate some of the effects of GABA accumulation. The potential function of GABA as a growth control factor under stressful conditions is discussed. PMID:21471118

  8. Insect Herbivory-Elicited GABA Accumulation in Plants is a Wound-Induced, Direct, Systemic, and Jasmonate-Independent Defense Response

    PubMed Central

    Scholz, Sandra S.; Reichelt, Michael; Mekonnen, Dereje W.; Ludewig, Frank; Mithöfer, Axel

    2015-01-01

    The non-proteinogenic amino acid γ-aminobutyric acid (GABA) is present in all organisms analyzed so far. In invertebrates GABA acts as a neurotransmitter; in plants different functions are under discussion. Among others, its involvement in abiotic stress reactions and as a defensive compound against feeding insects is suggested. GABA is synthesized from glutamate by glutamate decarboxylases and degraded by GABA-transaminases. Here, in Arabidopsis thaliana, gad1/2 double mutants showing reduced GABA concentrations as well as GABA-enriched triple mutants (gad1/2 x pop2-5) were generated and employed for a systematic study of GABA induction, accumulation and related effects in Arabidopsis leaves upon herbivory. The results demonstrate that GABA accumulation is stimulated by insect feeding-like wounding by a robotic caterpillar, MecWorm, as well as by real insect (Spodoptera littoralis) herbivory. Higher GABA levels in both plant tissue and artificial dietary supplements in turn affect the performance of feeding larvae. GABA enrichment occurs not only in the challenged but also in adjacent leaf. This induced response is neither dependent on herbivore defense-related phytohormones, jasmonates, nor is jasmonate induction dependent on the presence of GABA. Thus, in Arabidopsis the rapid accumulation of GABA very likely represents a general, direct and systemic defense reaction against insect herbivores. PMID:26734035

  9. Alternate mechanism for amino acid entry into Neurospora crassa: extracellular deamination and subsequent keto acid transport.

    PubMed Central

    DeBusk, R M; Brown, D T; DeBusk, A G; Penderghast, R D

    1981-01-01

    The growth of the pm nbg mutant strain of Neurospora crassa was inhibited by the amino acid analog para-fluorophenylalanine despite the fact that none of the three constitutive amino acid permeases is functional in this strain. This observation led to the detection of both a deaminase which was released into the growth medium in response to para-fluorophenylalanine and a keto acid transport system which allowed entry of the resulting keto acid into the cell. The transported keto acid was recovered in cellular protein, suggesting its regeneration as the amino acid. The cooperative activity of these two systems represents an additional mechanism for the intracellular accumulation of amino acids, which is distinct from the known amino acid permeases. Images PMID:6452443

  10. Transport Function of Rice Amino Acid Permeases (AAPs).

    PubMed

    Taylor, Margaret R; Reinders, Anke; Ward, John M

    2015-07-01

    The transport function of four rice (Oryza sativa) amino acid permeases (AAPs), OsAAP1 (Os07g04180), OsAAP3 (Os06g36180), OsAAP7 (Os05g34980) and OsAAP16 (Os12g08090), was analyzed by expression in Xenopus laevis oocytes and electrophysiology. OsAAP1, OsAAP7 and OsAAP16 functioned, similarly to Arabidopsis AAPs, as general amino acid permeases. OsAAP3 had a distinct substrate specificity compared with other rice or Arabidopsis AAPs. OsAAP3 transported the basic amino acids lysine and arginine well but selected against aromatic amino acids. The transport of basic amino acids was further analyzed for OsAAP1 and OsAAP3, and the results support the transport of both neutral and positively charged forms of basic amino acids by the rice AAPs. Cellular localization using the tandem enhanced green fluorescent protein (EGFP)-red fluorescent protein (RFP) reporter pHusion showed that OsAAP1 and OsAAP3 localized to the plasma membrane after transient expression in onion epidermal cells or stable expression in Arabidopsis. PMID:25907566

  11. Inhibition of 5-methyltetrahydrofolic acid transport by amphipathic drugs.

    PubMed

    Branda, R F; Nelson, N L

    1981-01-01

    Numerous chemically unrelated drugs after the membrane transport of folate compounds. To investigate drug structure-activity relationships, we measured the effect of amphipathic drugs (that is, compounds with polar-apolar character) on 5-methyltetrahydrofolic acid permeability of human erythrocytes. All drugs tested were inhibitory, but only compounds that exist at least partially in the anionic form were highly active. Ethacrynic acid, sulfinpyrazone, phenylbutazone, sulfasalazine, and furosemide were effective transport inhibitors in micromolar concentrations. In contrast, compounds that are capable of forming cations at physiologic pH, such as chlorpromazine, procaine, tetracaine, and papaverine, were inhibitory only in millimolar concentrations or caused hemolysis before major inhibition was seen. Inhibitory activity correlated with drug dissociation constant (r = 0.87). A double-reciprocal plot analysis of drug effect on 5-methyltetrahydrofolic acid transport showed changes in both Km and Vmax (indicating a mixture of competitive and noncompetitive inhibition) by ethacrynic acid, sulfasalazine, and phlorizin. Inhibitory activity of a series of eight phenoxyacetic derivatives, including ethacrynic acid, correlated highly with measurements of liposolubility (r = 0.87) but only weakly with the Hammet substituent constant (r = 0.56). These results suggest that the effect of amphipathic drugs on 5-methyltetrahydrofolic acid transport is influenced by drug pKa and by the presence of hydrophobic substituents, but is relatively independent of electron-attracting groups. PMID:6926815

  12. Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

    PubMed Central

    Aleksunes, Lauren M.

    2010-01-01

    Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting β polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) α and β] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of regulatory

  13. Pharmacological modulation of brain levels of glutamate and GABA in rats exposed to total sleep deprivation

    PubMed Central

    Kamal, Sahar Mohamed

    2010-01-01

    Modulation of gamma-aminobutyric acid (GABA) and glutamate by selected antidepressants and anticonvulsants could play a beneficial role in total sleep deprivation (TSD) caused by depressed mood. In the present study, albino rats were exposed to TSD for five days. On the sixth day, the brains were removed, and GABA and glutamate levels were measured in the prefrontal cortex and thalamus to identify TSD-induced changes in untreated rats and in rats treated with carbamazepine 40 mg/kg intraperitoneally (IP), fluoxetine 20 mg/kg IP, or desipramine 10 mg/kg IP. Carbamazepine and fluoxetine significantly increased GABA and reduced glutamate levels in both brain areas. Desipramine administration did not affect GABA or glutamate concentrations in the tested brain areas; levels were comparable with those induced by TSD without treatment. These results suggest that administration of carbamazepine or fluoxetine could have a beneficial effect by increasing GABA levels during TSD.

  14. Transported acid aerosols measured in southern Ontario

    NASA Astrophysics Data System (ADS)

    Keeler, Gerald J.; Spengler, John D.; Koutrakis, Petros; Allen, George A.; Raizenne, Mark; Stern, Bonnie

    During the period 29 June 1986-9 August 1986, a field health study assessing the acute health effects of air pollutants on children was conducted at a summer girls' camp on the northern shore of Lake Erie in SW Ontario. Continuous air pollution measurements of SO 2, O 3, NO x, particulate sulfates, light scattering, and meteorological measurements including temperature, dew point, and wind speed and direction were made. Twelve-hour integrated samples of size fractioned particles were also obtained using dichotomous samplers and Harvard impactors equipped with an ammonia denuder for subsequent hydrogen ion determination. Particulate samples were analyzed for trace elements by X-ray fluorescence and Neutron Activation, and for organic and elemental carbon by a thermal/optical technique. The measured aerosol was periodically very acidic with observed 12-h averaged H + concentrations in the range < 10-560 nmoles m -3. The aerosol H + appeared to represent the net strong acidity after H 2SO 4 reaction with NH 3(g). Average daytime concentrations were higher than night-time for aerosol H +, sulfate, fine mass and ozone. Prolonged episodes of atmospheric acidity, sulfate, and ozone were associated with air masses arriving at the measurement site from the west and from the southwest over Lake Erie. Sulfate concentrations measured at the lakeshore camp were more than twice those measured at inland sites during extreme pollution episodes. The concentration gradient observed with onshore flow was potentially due to enhanced deposition near the lakeshore caused by discontinuities in the meteorological fields in this region.

  15. 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.

  16. Reduction of Phosphorylated Synapsin I (Ser-553) Leads to Spatial Memory Impairment by Attenuating GABA Release after Microwave Exposure in Wistar Rats

    PubMed Central

    Qiao, Simo; Peng, Ruiyun; Yan, Haitao; Gao, Yabing; Wang, Changzhen; Wang, Shuiming; Zou, Yong; Xu, Xinping; Zhao, Li; Dong, Ji; Su, Zhentao; Feng, Xinxin; Wang, Lifeng; Hu, Xiangjun

    2014-01-01

    Background Abnormal release of neurotransmitters after microwave exposure can cause learning and memory deficits. This study investigated the mechanism of this effect by exploring the potential role of phosphorylated synapsin I (p-Syn I). Methods Wistar rats, rat hippocampal synaptosomes, and differentiated (neuronal) PC12 cells were exposed to microwave radiation for 5 min at a mean power density of 30 mW/cm2. Sham group rats, synaptosomes, and cells were otherwise identically treated and acted as controls for all of the following post-exposure analyses. Spatial learning and memory in rats was assessed using the Morris Water Maze (MWM) navigation task. The protein expression and presynaptic distribution of p-Syn I and neurotransmitter transporters were examined via western blotting and immunoelectron microscopy, respectively. Levels amino acid neurotransmitter release from rat hippocampal synaptosomes and PC12 cells were measured using high performance liquid chromatograph (HPLC) at 6 hours after exposure, with or without synapsin I silencing via shRNA transfection. Results In the rat experiments, there was a decrease in spatial memory performance after microwave exposure. The expression of p-Syn I (ser-553) was decreased at 3 days post-exposure and elevated at later time points. Vesicular GABA transporter (VGAT) was significantly elevated after exposure. The GABA release from synaptosomes was attenuated and p-Syn I (ser-553) and VGAT were both enriched in small clear synaptic vesicles, which abnormally assembled in the presynaptic terminal after exposure. In the PC12 cell experiments, the expression of p-Syn I (ser-553) and GABA release were both attenuated at 6 hours after exposure. Both microwave exposure and p-Syn I silencing reduced GABA release and maximal reduction was found for the combination of the two, indicating a synergetic effect. Conclusion p-Syn I (ser-553) was found to play a key role in the impaired GABA release and cognitive dysfunction that was

  17. Inflammatory bowel disease alters intestinal bile acid transporter expression.

    PubMed

    Jahnel, Jörg; Fickert, Peter; Hauer, Almuthe C; Högenauer, Christoph; Avian, Alexander; Trauner, Michael

    2014-09-01

    The enterohepatic circulation of bile acids (BAs) critically depends on absorption of BA in the terminal ileum and colon, which can be affected by inflammatory bowel disease (IBD). Diarrhea in IBD is believed to result in part from BA malabsorption (BAM). We explored whether IBD alters mRNA expression of key intestinal BA transporters, BA detoxifying systems, and nuclear receptors that regulate BA transport and detoxification. Using real-time polymerase chain reaction, mucosal biopsy specimens from the terminal ileum in Crohn's disease (CD) patients and from the descending colon in ulcerative colitis (UC) patients were assessed for mRNA expression. Levels were compared with healthy controls. The main ileal BA uptake transporter, the apical sodium dependent bile acid transporter, was downregulated in active CD and UC and in CD in remission. Other significant changes such as repression of breast cancer-related protein and sulphotransferase 2A1 were seen only during active disease. In UC, pancolitis (but not exclusively left-sided colitis) was associated with altered expression of major BA transporters [multidrug resistance-associated protein 3 (MRP3), MRP4, multidrug resistance gene 1, organic solute transporter α/β] and nuclear receptors (pregnane X receptor, vitamin D receptor) in the descending colon. UC pancolitis leads to broad changes and CD ileitis to selective changes in intestinal BA transporter expression. Early medical manipulation of intestinal BA transporters may help prevent BAM. PMID:24965812

  18. Segregation of Acetylcholine and GABA in the Rat Superior Cervical Ganglia: Functional Correlation.

    PubMed

    Elinos, Diana; Rodríguez, Raúl; Martínez, Luis Andres; Zetina, María Elena; Cifuentes, Fredy; Morales, Miguel Angel

    2016-01-01

    Sympathetic neurons have the capability to segregate their neurotransmitters (NTs) and co-transmitters to separate varicosities of single axons; furthermore, in culture, these neurons can even segregate classical transmitters. In vivo sympathetic neurons employ acetylcholine (ACh) and other classical NTs such as gamma aminobutyric acid (GABA). Herein, we explore whether these neurons in vivo segregate these classical NTs in the superior cervical ganglia of the rat. We determined the topographical distribution of GABAergic varicosities, somatic GABAA receptor, as well as the regional distribution of the segregation of ACh and GABA. We evaluated possible regional differences in efficacy of ganglionic synaptic transmission, in the sensitivity of GABAA receptor to GABA and to the competitive antagonist picrotoxin (PTX). We found that sympathetic preganglionic neurons in vivo do segregate ACh and GABA. GABAergic varicosities and GABAA receptor expression showed a rostro-caudal gradient along ganglia; in contrast, segregation exhibited a caudo-rostral gradient. These uneven regional distributions in expression of GABA, GABAA receptors, and level of segregation correlate with stronger synaptic transmission found in the caudal region. Accordingly, GABAA receptors of rostral region showed larger sensitivity to GABA and PTX. These results suggest the presence of different types of GABAA receptors in each region that result in a different regional levels of endogenous GABA inhibition. Finally, we discuss a possible correlation of these different levels of GABA modulation and the function of the target organs innervated by rostral and caudal ganglionic neurons. PMID:27092054

  19. Effect of GABA, a bacterial metabolite, on Pseudomonas fluorescens surface properties and cytotoxicity.

    PubMed

    Dagorn, Audrey; Chapalain, Annelise; Mijouin, Lily; Hillion, Mélanie; Duclairoir-Poc, Cécile; Chevalier, Sylvie; Taupin, Laure; Orange, Nicole; Feuilloley, Marc G J

    2013-01-01

    Different bacterial species and, particularly Pseudomonas fluorescens, can produce gamma-aminobutyric acid (GABA) and express GABA-binding proteins. In this study, we investigated the effect of GABA on the virulence and biofilm formation activity of different strains of P. fluorescens. Exposure of a psychotropic strain of P. fluorescens (MF37) to GABA (10-5 M) increased its necrotic-like activity on eukaryotic (glial) cells, but reduced its apoptotic effect. Conversely, muscimol and bicuculline, the selective agonist and antagonist of eukaryote GABAA receptors, respectively, were ineffective. P. fluorescens MF37 did not produce biosurfactants, and its caseinase, esterase, amylase, hemolytic activity or pyoverdine productions were unchanged. In contrast, the effect of GABA was associated to rearrangements of the lipopolysaccharide (LPS) structure, particularly in the lipid A region. The surface hydrophobicity of MF37 was marginally modified, and GABA reduced its biofilm formation activity on PVC, but not on glass, although the initial adhesion was increased. Five other P. fluorescens strains were studied, and only one, MFP05, a strain isolated from human skin, showed structural differences of biofilm maturation after exposure to GABA. These results reveal that GABA can regulate the LPS structure and cytotoxicity of P. fluorescens, but that this property is specific to some strains. PMID:23743829

  20. Effect of GABA, a Bacterial Metabolite, on Pseudomonas fluorescens Surface Properties and Cytotoxicity

    PubMed Central

    Dagorn, Audrey; Chapalain, Annelise; Mijouin, Lily; Hillion, Mélanie; Duclairoir-Poc, Cécile; Chevalier, Sylvie; Taupin, Laure; Orange, Nicole; Feuilloley, Marc G. J.

    2013-01-01

    Different bacterial species and, particularly Pseudomonas fluorescens, can produce gamma-aminobutyric acid (GABA) and express GABA-binding proteins. In this study, we investigated the effect of GABA on the virulence and biofilm formation activity of different strains of P. fluorescens. Exposure of a psychotropic strain of P. fluorescens (MF37) to GABA (10−5 M) increased its necrotic-like activity on eukaryotic (glial) cells, but reduced its apoptotic effect. Conversely, muscimol and bicuculline, the selective agonist and antagonist of eukaryote GABAA receptors, respectively, were ineffective. P. fluorescens MF37 did not produce biosurfactants, and its caseinase, esterase, amylase, hemolytic activity or pyoverdine productions were unchanged. In contrast, the effect of GABA was associated to rearrangements of the lipopolysaccharide (LPS) structure, particularly in the lipid A region. The surface hydrophobicity of MF37 was marginally modified, and GABA reduced its biofilm formation activity on PVC, but not on glass, although the initial adhesion was increased. Five other P. fluorescens strains were studied, and only one, MFP05, a strain isolated from human skin, showed structural differences of biofilm maturation after exposure to GABA. These results reveal that GABA can regulate the LPS structure and cytotoxicity of P. fluorescens, but that this property is specific to some strains. PMID:23743829

  1. Immunocytochemistry of GABA in the brain and suboesophageal ganglion of Manduca sexta.

    PubMed

    Homberg, U; Kingan, T G; Hildebrand, J G

    1987-04-01

    We have used specific antisera against protein-conjugated gamma-aminobutyric acid (GABA) in immunocytochemical preparations to investigate the distribution of putatively GABAergic neurons in the brain and suboesophageal ganglion of the sphinx moth Manduca sexta. About 20,000 neurons per brain hemisphere exhibit GABA-immunoreactivity. Most of these are optic-lobe interneurons, especially morphologically centrifugal neurons of the lamina and tangential neurons that innervate the medulla or the lobula complex. Many GABA-immunoreactive neurons, among them giant fibers of the lobula plate, project into the median protocerebrum. Among prominent GABA-immunoreactive neurons of the median protocerebrum are about 150 putatively negative-feedback fibers of the mushroom body, innervating both the calyces and lobes, and a group of large, fan-shaped neurons of the lower division of the central body. Several commissures in the supra- and suboesophageal ganglion exhibit GABA-like immunoreactivity. In the suboesophageal ganglion, a group of contralaterally descending neurons shows GABA-like immunoreactivity. The frontal ganglion is innervated by immunoreactive processes from the tritocerebrum but does not contain GABA-immunoreactive somata. With few exceptions the brain nerves do not contain GABA-immunoreactive fibers. PMID:3552234

  2. Segregation of Acetylcholine and GABA in the Rat Superior Cervical Ganglia: Functional Correlation

    PubMed Central

    Elinos, Diana; Rodríguez, Raúl; Martínez, Luis Andres; Zetina, María Elena; Cifuentes, Fredy; Morales, Miguel Angel

    2016-01-01

    Sympathetic neurons have the capability to segregate their neurotransmitters (NTs) and co-transmitters to separate varicosities of single axons; furthermore, in culture, these neurons can even segregate classical transmitters. In vivo sympathetic neurons employ acetylcholine (ACh) and other classical NTs such as gamma aminobutyric acid (GABA). Herein, we explore whether these neurons in vivo segregate these classical NTs in the superior cervical ganglia of the rat. We determined the topographical distribution of GABAergic varicosities, somatic GABAA receptor, as well as the regional distribution of the segregation of ACh and GABA. We evaluated possible regional differences in efficacy of ganglionic synaptic transmission, in the sensitivity of GABAA receptor to GABA and to the competitive antagonist picrotoxin (PTX). We found that sympathetic preganglionic neurons in vivo do segregate ACh and GABA. GABAergic varicosities and GABAA receptor expression showed a rostro-caudal gradient along ganglia; in contrast, segregation exhibited a caudo-rostral gradient. These uneven regional distributions in expression of GABA, GABAA receptors, and level of segregation correlate with stronger synaptic transmission found in the caudal region. Accordingly, GABAA receptors of rostral region showed larger sensitivity to GABA and PTX. These results suggest the presence of different types of GABAA receptors in each region that result in a different regional levels of endogenous GABA inhibition. Finally, we discuss a possible correlation of these different levels of GABA modulation and the function of the target organs innervated by rostral and caudal ganglionic neurons. PMID:27092054

  3. Temperature dependence and GABA modulation of (TH)triazolam binding in the rat brain

    SciTech Connect

    Earle, M.E.; Concas, A.; Wamsley, J.K.; Yamamura, H.I.

    1987-07-27

    The hypnotic triazolam (TZ), a triazolobenzodiazepine displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. The authors major objectives were the direct measurement of the temperature dependence and the gamma-aminobutyric acid (GABA) effect of (TH)TZ binding in the rat brain. Saturation studies showed a shift to lower affinity with increasing temperatures (K/sub d/ = 0.27 +/- 08 nM at 0C; K/sub d/ = 1.96 +/- 0.85 nM at 37C) while the B/sub max/ values remained unchanged (1220 +/- 176 fmoles/mg protein at 0C and 1160 +/- 383 fmoles/mg protein at 37C). Saturation studies of (TH)TZ binding in the presence or absence of GABA (100 M) showed a GABA-shift. At 0C the K/sub d/ values were (K/sub d/ = 0.24 +/- 0.03 nM/-GABA; K/sub d/ = 0.16 +/- 0.04/+GABA) and at 37C the K/sub d/ values were (K/sub d/ = 1.84 +/- 0.44 nM/-GABA; K/sub d/ = 0.95 +/- 0.29 nM/+GABA). In contrast to reported literature, the authors findings show that TZ interacts with benzodiazepine receptors with a temperature dependence and GABA-shift consistent with predicted behavior for benzodiazepine agonists. 20 references, 3 tables.

  4. GABA-shunt enzymes activity in GH3 cells with reduced level of PMCA2 or PMCA3 isoform

    SciTech Connect

    Kowalski, Antoni

    2011-08-12

    Highlights: {yields} Suppression of PMCA2 or PMCA3 slows down proliferation of GH3 cells. {yields} PMCA2 suppression lowers the activity of GABA-shunt enzymes. {yields} PMCA3 suppression increases the expression of glutamate decarboxylase 65. {yields} PMCA2 and PMCA3 function appears to be linked to regulation of GABA metabolism. -- Abstract: GABA ({gamma}-aminobutyric acid) is important neurotransmitter and regulator of endocrine functions. Its metabolism involves three enzymes: glutamate decarboxylase (GAD65 and GAD67), GABA aminotransferase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). As many cellular processes GABA turnover can depend on calcium homeostasis, which is maintained by plasma membrane calcium ATPases (PMCAs). In excitable cells PMCA2 and PMCA3 isoforms are particularly important. In this study we focused on GABA-metabolizing enzymes expression and activity in rat anterior pituitary GH3 cells with suppressed expression of PMCA2 or PMCA3. We observed that PMCA3-reduced cells have increased GAD65 expression. Suppression of PMCA2 caused a decrease in total GAD and GABA-T activity. These results indicate that PMCA2 and PMCA3 presence may be an important regulatory factor in GABA metabolism. Results suggest that PMCA2 and PMCA3 function is rather related to regulation of GABA synthesis and degradation than supplying cells with metabolites, which can be potentially energetic source.

  5. Development of a high-affinity GABA uptake system in embryonic amphibian spinal neurons.

    PubMed

    Lamborghini, J E; Iles, A

    1985-11-01

    High-affinity uptake systems for amino acid neurotransmitter precursors have been highly correlated with the use of the particular amino acid or its derivative as a transmitter. We have found interneurons in the Xenopus embryo spinal cord which accumulate GABA by a high-affinity uptake system. They originate near the end of gastrulation and their ability to accumulate GABA first appears at the early tail bud stage. By position and appearance they are comparable to some of the embryonic interneurons described by A. Roberts and J. D. W. Clarke (1982, Phil. Trans. R. Soc. London Ser. B 296, 195-212). GABA-accumulating neurons also develop in dissociated cell cultures made from the presumptive spinal cord of neural plate stage Xenopus embryos. GABA accumulation in cultured neurons, as in cells in vivo, occurs via a high-affinity uptake system; GABA-accumulating cells have the same time of origin as the cells in vivo, and the ability to accumulate GABA in the population of cultured neurons appears at a time equivalent to that observed in intact sibling embryos. Thus it seems likely that the population of GABA-accumulating neurons developing in cell culture corresponds to the GABA-accumulating interneurons in vivo. The development of these neurons in dissociated cell cultures permits perturbation experiments that would be difficult to perform in vivo. We have examined the development of high-affinity GABA uptake in conditions that permit no electrical impulse activity in the cultures. The onset and extent of development of GABA accumulation in the neuronal population are normal under these conditions. PMID:3932109

  6. Na+ Interactions with the Neutral Amino Acid Transporter ASCT1*

    PubMed Central

    Scopelliti, Amanda J.; Heinzelmann, Germano; Kuyucak, Serdar; Ryan, Renae M.; Vandenberg, Robert J.

    2014-01-01

    The alanine, serine, cysteine transporters (ASCTs) belong to the solute carrier family 1A (SLC1A), which also includes the excitatory amino acid transporters (EAATs) and the prokaryotic aspartate transporter GltPh. Acidic amino acid transport by the EAATs is coupled to the co-transport of three Na+ ions and one proton, and the counter-transport of one K+ ion. In contrast, neutral amino acid exchange by the ASCTs does not require protons or the counter-transport of K+ ions and the number of Na+ ions required is not well established. One property common to SLC1A family members is a substrate-activated anion conductance. We have investigated the number and location of Na+ ions required by ASCT1 by mutating residues in ASCT1 that correspond to residues in the EAATs and GltPh that are involved in Na+ binding. Mutations to all three proposed Na+ sites influence the binding of substrate and/or Na+, or the rate of substrate exchange. A G422S mutation near the Na2 site reduced Na+ affinity, without affecting the rate of exchange. D467T and D467A mutations in the Na1 site reduce Na+ and substrate affinity and also the rate of substrate exchange. T124A and D380A mutations in the Na3 site selectively reduce the affinity for Na+ and the rate of substrate exchange without affecting substrate affinity. In many of the mutants that reduce the rate of substrate transport the amplitudes of the substrate-activated anion conductances are not substantially affected indicating altered ion dependence for channel activation compared with substrate exchange. PMID:24808181

  7. Primordial transport of sugars and amino acids via Schiff bases

    NASA Astrophysics Data System (ADS)

    Stillwell, William; Rau, Aruna

    1981-09-01

    Experimental support is given for a model concerning the origin of a primordial transport system. The model is based on the facilitated diffusion of amino acids stimulated by aliphatic aldehyde carriers and sugars stimulated by aliphatic amine carriers. The lipid-soluble diffusing species is the Schiff base. The possible role of this simple transport system in the origin of an early protocell is discussed.

  8. Renal Transport of Uric Acid: Evolving Concepts and Uncertainties

    PubMed Central

    Bobulescu, Ion Alexandru; Moe, Orson W.

    2013-01-01

    In addition to its role as a metabolic waste product, uric acid has been proposed to be an important molecule with multiple functions in human physiology and pathophysiology and may be linked to human diseases beyond nephrolithiasis and gout. Uric acid homeostasis is determined by the balance between production, intestinal secretion, and renal excretion. The kidney is an important regulator of circulating uric acid levels, by reabsorbing around 90% of filtered urate, while being responsible for 60–70% of total body uric acid excretion. Defective renal handling of urate is a frequent pathophysiologic factor underpinning hyperuricemia and gout. In spite of tremendous advances over the past decade, the molecular mechanisms of renal urate transport are still incompletely understood. Many transport proteins are candidate participants in urate handling, with URAT1 and GLUT9 being the best characterized to date. Understanding these transporters is increasingly important for the practicing clinician as new research unveils their physiology, importance in drug action, and genetic association with uric acid levels in human populations. The future may see the introduction of new drugs that specifically act on individual renal urate transporters for the treatment of hyperuricemia and gout. PMID:23089270

  9. A molecular characterization of the agonist binding site of a nematode cys-loop GABA receptor

    PubMed Central

    Kaji, Mark D; Kwaka, Ariel; Callanan, Micah K; Nusrat, Humza; Desaulniers, Jean-Paul; Forrester, Sean G

    2015-01-01

    Background and Purpose Cys-loop GABA receptors represent important targets for human chemotherapeutics and insecticides and are potential targets for novel anthelmintics (nematicides). However, compared with insect and mammalian receptors, little is known regarding the pharmacological characteristics of nematode Cys-loop GABA receptors. Here we have investigated the agonist binding site of the Cys-loop GABA receptor UNC-49 (Hco-UNC-49) from the parasitic nematode Haemonchus contortus. Experimental Approach We used two-electrode voltage-clamp electrophysiology to measure channel activation by classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus laevis oocytes, along with site-directed mutagenesis and in silico homology modelling. Key Results The sulphonated molecules P4S and taurine had no effect on Hco-UNC-49. Other classical Cys-loop GABAA receptor agonists tested on the Hco-UNC-49B/C heteromeric channel had a rank order efficacy of GABA > trans-4-aminocrotonic acid > isoguvacine > imidazole-4-acetic acid (IMA) > (R)-(−)-4-amino-3-hydroxybutyric acid [R(−)-GABOB] > (S)-(+)-4-amino-3-hydroxybutyric acid [S(+)-GABOB] > guanidinoacetic acid > isonipecotic acid > 5-aminovaleric acid (DAVA) (partial agonist) > β-alanine (partial agonist). In silico ligand docking revealed some variation in binding between agonists. Mutagenesis of a key serine residue in binding loop C to threonine had minimal effects on GABA and IMA but significantly increased the maximal response to DAVA and decreased twofold the EC50 for R(−)- and S(+)-GABOB. Conclusions and Implications The pharmacological profile of Hco-UNC-49 differed from that of vertebrate Cys-loop GABA receptors and insect resistance to dieldrin receptors, suggesting differences in the agonist binding pocket. These findings could be exploited to develop new drugs that specifically target GABA receptors of parasitic nematodes. PMID:25850584

  10. A comparative density functional theory study of electronic structure and optical properties of γ-aminobutyric acid and its cocrystals with oxalic and benzoic acid

    NASA Astrophysics Data System (ADS)

    da Silva Filho, J. G.; Freire, V. N.; Caetano, E. W. S.; Ladeira, L. O.; Fulco, U. L.; Albuquerque, E. L.

    2013-11-01

    In this letter, we study the electronic structure and optical properties of the active medicinal component γ-aminobutyric acid (GABA) and its cocrystals with oxalic (OXA) and benzoic (BZA) acid by means of the density functional theory formalism. It is shown that the cocrystallization strongly weakens the zwitterionic character of the GABA molecule leading to striking differences among the electronic band structures and optical absorption spectra of the GABA crystal and GABA:OXA, GABA:BZA cocrystals, originating from distinct sets of hydrogen bonds. Calculated band widths and Δ-sol band gap estimates indicate that both GABA and GABA:OXA, GABA:BZA cocrystals are indirect gap insulators.

  11. Single rodent mesohabenular axons release glutamate and GABA

    PubMed Central

    Root, David H.; Mejias-Aponte, Carlos; Zhang, Shiliang; Wang, Huiling; Hoffman, Alexander F.; Lupica, Carl R.; Morales, Marisela

    2016-01-01

    The lateral habenula (LHb) is involved in reward, aversion, addiction, and depression, through descending interactions with several brain structures, including the ventral tegmental area (VTA). VTA provides reciprocal inputs to LHb, but their actions are unclear. Here we show that the majority of rat and mouse VTA neurons innervating LHb co-express markers for both glutamate-signaling (vesicular glutamate transporter 2, VGluT2) and GABA-signaling (glutamate decarboxylase, GAD; and vesicular GABA transporter, VGaT). A single axon from these mesohabenular neurons co-expresses VGluT2-protein and VGaT-protein, and surprisingly establishes symmetric and asymmetric synapses on LHb neurons. In LHb slices, light activation of mesohabenular fibers expressing channelrhodopsin-2 (ChR2) driven by VGluT2 or VGaT promoters elicits release of both glutamate and GABA onto single LHb neurons. In vivo light-activation of mesohabenular terminals inhibits or excites LHb neurons. Our findings reveal an unanticipated type of VTA neuron that co-transmits glutamate and GABA, and provides the majority of mesohabenular inputs. PMID:25242304

  12. Fabrication of the Optical Fiber GABA Sensor Based on the NADP+ -Functionalized Quantum Dots.

    PubMed

    Zhao, Fei; Yoo, Jeongha; Kim, Jongsung

    2016-02-01

    A novel quantum dots (QDs)-based optical fiber biosensor has been developed to detect gamma-amino butyric acid (GABA) directly, via QD fluorescence quenching and recovery. QDs were immobilized on the surface of an optical-fiber through the EDC/Sulfo-NHS coupling reaction. The QDs were functionalized by 3-aminophenyl boronic acid and then by NADP+. The fluorescence of the NADP+ -functionalized QDs was quenched by electron transfer from QDs to NADP+. However, by the metabolic conversion of GABA to succinic acid by GABase, NADP+ was reduced to NADPH, which hindered the electron transfer. As a result, the fluorescence of the QDs could recover. The recovery rate of the fluorescence intensity of QDs depended on the concentration of GABA. This shows the possibility of detection of low concentrations of GABA via measurement of the fluorescence intensity. PMID:27433599

  13. Neutral amino acid transport in bovine articular chondrocytes.

    PubMed

    Barker, G A; Wilkins, R J; Golding, S; Ellory, J C

    1999-02-01

    1. The sodium-dependent amino acid transport systems responsible for proline, glycine and glutamine transport, together with the sodium-independent systems for leucine and tryptophan, have been investigated in isolated bovine chondrocytes by inhibition studies and ion replacement. Each system was characterized kinetically. 2. Transport via system A was identified using the system-specific analogue alpha-methylaminoisobutyric acid (MeAIB) as an inhibitor of proline, glycine and glutamine transport. 3. Uptake of proline, glycine and glutamine via system ASC was identified by inhibition with alanine or serine. 4. System Gly was identified by the inhibition of glycine transport with excess sarcosine (a substrate for system Gly) whilst systems A and ASC were inhibited. This system, having a very limited substrate specificity and tissue distribution, was also shown to be Na+ and Cl- dependent. Evidence for expression of the system Gly component GLYT-1 was obtained using the reverse transcriptase-polymerase chain reaction (RT-PCR). 5. System N, also of narrow substrate specificity and tissue distribution, was shown to be present in chondrocytes. Na+-dependent glutamine uptake was inhibited by high concentrations of histidine (a substrate of system N) in the presence of excess MeAIB and serine. 6. System L was identified using the system specific analogue 2-aminobicyclo(2,2, 1)heptane-2-carboxylic acid (BCH) and D-leucine as inhibitors of leucine and tryptophan transport. 7. The presence of system T was tested by using leucine, tryptophan and tyrosine inhibition. It was concluded that this system was absent in the chondrocyte. 8. Kinetic analysis showed the Na+-independent chondrocyte L system to have apparent affinities for leucine and tryptophan of 125 +/- 27 and 36 +/- 11 microM, respectively. 9. Transport of the essential amino acids leucine and tryptophan into bovine chondrocytes occurs only by the Na+-independent system L, but with a higher affinity than the

  14. GABA Production in Lactococcus lactis Is Enhanced by Arginine and Co-addition of Malate.

    PubMed

    Laroute, Valérie; Yasaro, Chonthicha; Narin, Waranya; Mazzoli, Roberto; Pessione, Enrica; Cocaign-Bousquet, Muriel; Loubière, Pascal

    2016-01-01

    Lactococcus lactis NCDO 2118 was previously selected for its ability to decarboxylate glutamate to γ-aminobutyric acid (GABA), an interesting nutritional supplement able to improve mood and relaxation. Amino acid decarboxylation is generally considered as among the biochemical systems allowing lactic acid bacteria to counteracting acidic stress and obtaining metabolic energy. These strategies also include arginine deiminase pathway and malolactic fermentation but little is known about their possible interactions of with GABA production. In the present study, the effects of glutamate, arginine, and malate (i.e., the substrates of these acid-resistance pathways) on L. lactis NCDO 2118 growth and GABA production performances were analyzed. Both malate and arginine supplementation resulted in an efficient reduction of acidity and improvement of bacterial biomass compared to glutamate supplementation. Glutamate decarboxylation was limited to narrow environmental conditions (pH < 5.1) and physiological state (stationary phase). However, some conditions were able to improve GABA production or activate glutamate decarboxylation system even outside of this compass. Arginine clearly stimulated glutamate decarboxylation: the highest GABA production (8.6 mM) was observed in cultures supplemented with both arginine and glutamate. The simultaneous addition of arginine, malate, and glutamate enabled earlier GABA production (i.e., during exponential growth) at relatively high pH (6.5). As far as we know, no previous study has reported GABA production in such conditions. Although further studies are needed to understand the molecular basis of these phenomena, these results represent important keys suitable of application in GABA production processes. PMID:27458444

  15. GABA Production in Lactococcus lactis Is Enhanced by Arginine and Co-addition of Malate

    PubMed Central

    Laroute, Valérie; Yasaro, Chonthicha; Narin, Waranya; Mazzoli, Roberto; Pessione, Enrica; Cocaign-Bousquet, Muriel; Loubière, Pascal

    2016-01-01

    Lactococcus lactis NCDO 2118 was previously selected for its ability to decarboxylate glutamate to γ-aminobutyric acid (GABA), an interesting nutritional supplement able to improve mood and relaxation. Amino acid decarboxylation is generally considered as among the biochemical systems allowing lactic acid bacteria to counteracting acidic stress and obtaining metabolic energy. These strategies also include arginine deiminase pathway and malolactic fermentation but little is known about their possible interactions of with GABA production. In the present study, the effects of glutamate, arginine, and malate (i.e., the substrates of these acid-resistance pathways) on L. lactis NCDO 2118 growth and GABA production performances were analyzed. Both malate and arginine supplementation resulted in an efficient reduction of acidity and improvement of bacterial biomass compared to glutamate supplementation. Glutamate decarboxylation was limited to narrow environmental conditions (pH < 5.1) and physiological state (stationary phase). However, some conditions were able to improve GABA production or activate glutamate decarboxylation system even outside of this compass. Arginine clearly stimulated glutamate decarboxylation: the highest GABA production (8.6 mM) was observed in cultures supplemented with both arginine and glutamate. The simultaneous addition of arginine, malate, and glutamate enabled earlier GABA production (i.e., during exponential growth) at relatively high pH (6.5). As far as we know, no previous study has reported GABA production in such conditions. Although further studies are needed to understand the molecular basis of these phenomena, these results represent important keys suitable of application in GABA production processes. PMID:27458444

  16. Acid-base transport in pancreas—new challenges

    PubMed Central

    Novak, Ivana; Haanes, Kristian A.; Wang, Jing

    2013-01-01

    Along the gastrointestinal tract a number of epithelia contribute with acid or basic secretions in order to aid digestive processes. The stomach and pancreas are the most extreme examples of acid (H+) and base (HCO−3) transporters, respectively. Nevertheless, they share the same challenges of transporting acid and bases across epithelia and effectively regulating their intracellular pH. In this review, we will make use of comparative physiology to enlighten the cellular mechanisms of pancreatic HCO−3 and fluid secretion, which is still challenging physiologists. Some of the novel transporters to consider in pancreas are the proton pumps (H+-K+-ATPases), as well as the calcium-activated K+ and Cl− channels, such as KCa3.1 and TMEM16A/ANO1. Local regulators, such as purinergic signaling, fine-tune, and coordinate pancreatic secretion. Lastly, we speculate whether dys-regulation of acid-base transport contributes to pancreatic diseases including cystic fibrosis, pancreatitis, and cancer. PMID:24391597

  17. Energetics of low affinity amino acid transport into brain slices.

    PubMed

    Banay-Schwartz, M; Teller, D N; Lajtha, A

    1976-01-01

    It appears possible to dissect and study some of the potential energy sources for amino acid transport in brain slices despite the apparent complexity of the tissue in comparison to that of isolated bacterial vesicles23. The uptake capability of the tissue may be inadvertently damaged in some experimental protocols so that very special controls must be used to ensure that the treatment did not somehow inactivate the very mechanism that thereafter will be tested. We have presented some evidence that brain slice amino acid transport may not be obligatorily linked to glycolysis, ATP levels, Na+, K+-ATPase activity, K+ levels or direction of flux, or to Na+ flux. However, the energy source linkage for different amino acids appears to be rather specific, so that further generalizations are difficult to sustain. For instance, the incubation media and conditions we describe here were experimentally adjusted to maximize uptake of D-glu or alpha-AIB in the absence of glucose, or in lowered K+ or Na+. Therefore, these procedures, the results of which directly challenge some common assumptions regarding the energy basis for active transport in brain slices, probably will not be universally extensible to all other actively transported amino acids. PMID:782193

  18. Transport of amino acids and nucleic acid precursors in malarial parasites

    PubMed Central

    Sherman, I. W.

    1977-01-01

    In vitro studies have shown that exogenously supplied amino acids are transferred into the malaria-infected cell, where they are incorporated into proteins. Most amino acids appear to enter the cell by facilitated or simple diffusion; however, the high distribution ratios seen in Plasmodium knowlesi-infected cells are difficult to explain on this basis. The changes (leakiness) observed in amino acid transport in P. lophurae infected cells are probably the result of ATP depletion in the host cell as well as the elaboration of plasmodial substances. Depletion of isoleucine, methionine, and cysteine from the medium strikingly depresses the in vitro growth of P. knowlesi. The degree of amino acid incorporation into the malaria-infected cell is not correlated with the amount of a particular amino acid in the host cell haemoglobin, the decline of that amino acid in the plasma of infected animals, or the ratio of free amino acids of the erythrocyte to those of the plasma. In erythrocyte-“free” P. lophurae, carrier-mediated transport is apparently limited to a small number of amino acids; all others seem to enter by simple diffusion. Malaria-infected erythrocytes transport exogenously supplied purines at substantially higher rates than uninfected red cells. The preferred purines are adenosine, hypoxanthine, and inosine. The only pyrimidine incorporated is orotic acid. Thymidine, cytidine, and uridine do not readily enter the red cell, and incorporation does not take place because the parasites lack the appropriate enzyme for conversion to nucleotides. Erythrocyte-“free” P. berghei and P. lophurae take up purines and orotic acid. It has been suggested that in vivo the preferred purines are hypoxanthine and inosine, and that the transport locus for erythrocytes is specific for 6-oxopurines. Similar results of purine incorporation are reported for the insect stages of P. cynomolgi and P. berghei, although transport studies have not been carried out. PMID:338180

  19. Sodium-coupled sugar and amino acid transport in an acidic microenvironment.

    PubMed

    Ahearn, G A; Clay, L P

    1988-01-01

    1. Nutrient transport mechanisms of lobster hepatopancreatic epithelial brush border membrane vesicles (BBMV) are strongly influenced by the acidic nature of the tubular lumen. 2. Sodium-dependent glucose uptake by BBMV was electrogenic and was stimulated at low pH by reducing sugar transport Ki, without affecting JM. 3. Glutamate was largely transported in zwitterionic form at pH 4.0 by an electrically silent cotransport mechanism with both Na and Cl. 4. Increased H+ concentration tripled the apparent membrane permeability to glutamate as well as the amino acid transport JM. 5. At pH 4.0 leucine was transported as a cation by two dissimilar carrier systems: a Na-independent process shared by polar amino acids, and an electroneutral Na-2Cl-dependent mechanism shared with non-polar amino acids. 6. A model is proposed for hepatopancreatic BBMV at acidic pH which employs ionic chemical gradients and membrane potential as nutrient transport driving forces. PMID:2902970

  20. Focal Uncaging of GABA Reveals a Temporally Defined Role for GABAergic Inhibition during Appetitive Associative Olfactory Conditioning in Honeybees

    ERIC Educational Resources Information Center

    Raccuglia, Davide; Mueller, Uli

    2013-01-01

    Throughout the animal kingdom, the inhibitory neurotransmitter ?-aminobutyric acid (GABA) is a key modulator of physiological processes including learning. With respect to associative learning, the exact time in which GABA interferes with the molecular events of learning has not yet been clearly defined. To address this issue, we used two…

  1. The Memory-Impairing Effects of Septal GABA Receptor Activation Involve GABAergic Septo-Hippocampal Projection Neurons

    ERIC Educational Resources Information Center

    Krebs-Kraft, Desiree L.; Wheeler, Marina G.; Parent, Marise B.

    2007-01-01

    Septal infusions of the [gamma]-aminobutyric acid (GABA)[subscript A] agonist muscimol impair memory, and the effect likely involves the hippocampus. GABA[subscript A] receptors are present on the perikarya of cholinergic and GABAergic septo-hippocampal (SH) projections. The current experiments determined whether GABAergic SH projections are…

  2. GABA System in Schizophrenia and Mood Disorders: A Mini Review on Third-Generation Imaging Studies.

    PubMed

    Chiapponi, Chiara; Piras, Federica; Piras, Fabrizio; Caltagirone, Carlo; Spalletta, Gianfranco

    2016-01-01

    Third-generation neuroimaging research has been enriched by advances in magnetic resonance spectroscopy (MRS) measuring the concentration of important neurotrasmitters, such as the inhibitory amino acid GABA. Here, we performed a systematic mini-review on brain MRS studies measuring GABA concentration in patients affected by schizophrenia (SZ), bipolar disorder (BD), and major depressive disorder (MDD). We wondered whether multimodal investigations could overcome intrinsic technical limits of MRS giving a broader view of mental disorders pathogenesis. In SZ, unimodal studies gave mixed results, as increased, decreased, or unaltered GABA levels were reported depending on region, disease phase, and treatment. Conversely, multimodal results showed reduced level of glutamate, but not of GABA, in patients mirrored by in vitro biochemical findings revealing hippocampal reduction in glutamate signaling in SZ, and no deficits in GABA synthesis. Moreover, a mouse model confirmed the unique pathological characteristic of glutamate function in SZ. Unimodal studies in BD revealed again, inconsistent results, while no multimodal investigations including MRS on GABA exist. In MDD, unimodal studies could not differentiate patients from controls nor characterize high-risk subjects and remitted patients. However, a multimodal study combining functional magnetic resonance imaging and MRS revealed that cingulate cortex activity is related to glutamate, N-acetylaspartate levels and anhedonia in patients, and to GABA concentration in healthy subjects, improving the distinction between MDD and physiology. Overall, our results show that unimodal studies do not indicate GABA as a biomarker for the psychiatric disorders considered. Conversely, multimodal studies can widen the understanding of the link between psychopathology, genetics, neuroanatomy, and functional-biochemical brain activity in mental disorders. Although scarce, multimodal approaches seem promising for moving from GABA MRS

  3. GABA System in Schizophrenia and Mood Disorders: A Mini Review on Third-Generation Imaging Studies

    PubMed Central

    Chiapponi, Chiara; Piras, Federica; Piras, Fabrizio; Caltagirone, Carlo; Spalletta, Gianfranco

    2016-01-01

    Third-generation neuroimaging research has been enriched by advances in magnetic resonance spectroscopy (MRS) measuring the concentration of important neurotrasmitters, such as the inhibitory amino acid GABA. Here, we performed a systematic mini-review on brain MRS studies measuring GABA concentration in patients affected by schizophrenia (SZ), bipolar disorder (BD), and major depressive disorder (MDD). We wondered whether multimodal investigations could overcome intrinsic technical limits of MRS giving a broader view of mental disorders pathogenesis. In SZ, unimodal studies gave mixed results, as increased, decreased, or unaltered GABA levels were reported depending on region, disease phase, and treatment. Conversely, multimodal results showed reduced level of glutamate, but not of GABA, in patients mirrored by in vitro biochemical findings revealing hippocampal reduction in glutamate signaling in SZ, and no deficits in GABA synthesis. Moreover, a mouse model confirmed the unique pathological characteristic of glutamate function in SZ. Unimodal studies in BD revealed again, inconsistent results, while no multimodal investigations including MRS on GABA exist. In MDD, unimodal studies could not differentiate patients from controls nor characterize high-risk subjects and remitted patients. However, a multimodal study combining functional magnetic resonance imaging and MRS revealed that cingulate cortex activity is related to glutamate, N-acetylaspartate levels and anhedonia in patients, and to GABA concentration in healthy subjects, improving the distinction between MDD and physiology. Overall, our results show that unimodal studies do not indicate GABA as a biomarker for the psychiatric disorders considered. Conversely, multimodal studies can widen the understanding of the link between psychopathology, genetics, neuroanatomy, and functional–biochemical brain activity in mental disorders. Although scarce, multimodal approaches seem promising for moving from GABA

  4. 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. PMID:26608704

  5. GABA's Control of Stem and Cancer Cell Proliferation in Adult Neural and Peripheral Niches

    PubMed Central

    Young, Stephanie Z.; Bordey, Angélique

    2010-01-01

    Aside from traditional neurotransmission and regulation of secretion, γ-amino butyric acid (GABA) through GABAA receptors negatively regulates proliferation of pluripotent and neural stem cells in embryonic and adult tissue. There has also been evidence that GABAergic signaling and its control over proliferation is not only limited to the nervous system, but is widespread through peripheral organs containing adult stem cells. GABA has emerged as a tumor signaling molecule in the periphery that controls the proliferation of tumor cells and perhaps tumor stem cells. Here, we will discuss GABA's presence as a near-universal signal that may be altered in tumor cells resulting in modified mitotic activity. PMID:19509127

  6. Non-neuronal, slow GABA signalling in the ventrobasal thalamus targets δ-subunit-containing GABAA receptors

    PubMed Central

    Jiménez-González, Cristina; Pirttimaki, Tiina; Cope, David W; Parri, H R

    2011-01-01

    The rodent ventrobasal (VB) thalamus contains a relatively uniform population of thalamocortical (TC) neurons that receive glutamatergic input from the vibrissae and the somatosensory cortex, and inhibitory input from the nucleus reticularis thalami (nRT). In this study we describe γ-aminobutyric acid (GABA)A receptor-dependent slow outward currents (SOCs) in TC neurons that are distinct from fast inhibitory postsynaptic currents (IPSCs) and tonic currents. SOCs occurred spontaneously or could be evoked by hypo-osmotic stimulus, and were not blocked by tetrodotoxin, removal of extracellular Ca2+ or bafilomycin A1, indicating a non-synaptic, non-vesicular GABA origin. SOCs were more common in TC neurons of the VB compared with the dorsal lateral geniculate nucleus, and were rarely observed in nRT neurons, whilst SOC frequency in the VB increased with age. Application of THIP, a selective agonist at δ-subunit-containing GABAA receptors, occluded SOCs, whereas the benzodiazepine site inverse agonist β-CCB had no effect, but did inhibit spontaneous and evoked IPSCs. In addition, the occurrence of SOCs was reduced in mice lacking the δ-subunit, and their kinetics were also altered. The anti-epileptic drug vigabatrin increased SOC frequency in a time-dependent manner, but this effect was not due to reversal of GABA transporters. Together, these data indicate that SOCs in TC neurons arise from astrocytic GABA release, and are mediated by δ-subunit-containing GABAA receptors. Furthermore, these findings suggest that the therapeutic action of vigabatrin may occur through the augmentation of this astrocyte–neuron interaction, and highlight the importance of glial cells in CNS (patho) physiology. PMID:21395866

  7. Fatty Acid and Lipid Transport in Plant Cells.

    PubMed

    Li, Nannan; Xu, Changcheng; Li-Beisson, Yonghua; Philippar, Katrin

    2016-02-01

    Fatty acids (FAs) and lipids are essential - not only as membrane constituents but also for growth and development. In plants and algae, FAs are synthesized in plastids and to a large extent transported to the endoplasmic reticulum for modification and lipid assembly. Subsequently, lipophilic compounds are distributed within the cell, and thus are transported across most membrane systems. Membrane-intrinsic transporters and proteins for cellular FA/lipid transfer therefore represent key components for delivery and dissemination. In addition to highlighting their role in lipid homeostasis and plant performance, different transport mechanisms for land plants and green algae - in the model systems Arabidopsis thaliana, Chlamydomonas reinhardtii - are compared, thereby providing a current perspective on protein-mediated FA and lipid trafficking in photosynthetic cells. PMID:26616197

  8. Cation-halide transport through peptide pores containing aminopicolinic acid.

    PubMed

    Basak, Debajyoti; Sridhar, Sucheta; Bera, Amal K; Madhavan, Nandita

    2016-05-18

    Synthetic pores that selectively transport ions of biological significance through membranes could be potentially used in medical diagnostics or therapeutics. Herein, we report cation-selective octapeptide pores derived from alanine and aminopicolinic acid. The ion transport mechanism through the pores has been established to be a cation-chloride symport. The cation-chloride co-transport is biologically essential for the efficient functioning of the central nervous system and has been implicated in diseases such as epilepsy. The pores formed in synthetic lipid bilayers do not exhibit any closing events. The ease of synthesis as well as infinite lifetimes of these pores provides scope for modifying their transport behaviour to develop sensors. PMID:27137995

  9. [Enhancers on the transmembrane transport of chlorogenic acid].

    PubMed

    Ren, Jing; Deng, Sheng-Qi; Jiang, Xue-Hua; Wang, Ling-Ling; Xiao, Yu

    2014-02-01

    To investigate the influence of the difference enhancers on the transport mechanism of chlorogenic acid (CGA) across Caco-2 cells model, a RP-HPLC method was adopted to detect the concentrations of CGA. At the concentrations of 20 to 80 microg x mL(-1), the difference of absorption rate constants (K(a)) was not statistically significant. At the concentrations of 40 and 20 microg x mL(-1), the ratios of apparent permeability coefficients (P(app)) of the apical to basolateral and the basolateral to apical were 1.14 and 1.18, respectively. With the effect of enhancers K(a) and P(app) increased, the absorption half-life (T1/2) decreased. CGA passed through the Caco-2 cell membrane mainly by passive transport. It showed that monocarboxylic acid transporter (MCT) could be involved in the across membrane transport process of CGA. Borneol had no effect on the cell membrane transport processes. The order of increasing absorption of CGA caused by the enhancers was sodium lauryl sulphate > sodium taurocholate > carbomer. PMID:24761618

  10. GABA metabolism pathway genes, UGA1 and GAD1, regulate replicative lifespan in Saccharomycescerevisiae

    SciTech Connect

    Kamei, Yuka; Tamura, Takayuki; Yoshida, Ryo; Ohta, Shinji; Fukusaki, Eiichiro; Mukai, Yukio

    2011-04-01

    Highlights: {yields}We demonstrate that two genes in the yeast GABA metabolism pathway affect aging. {yields} Deletion of the UGA1 or GAD1 genes extends replicative lifespan. {yields} Addition of GABA to wild-type cultures has no effect on lifespan. {yields} Intracellular GABA levels do not differ in longevity mutants and wild-type cells. {yields} Levels of tricarboxylic acid cycle intermediates positively correlate with lifespan. -- Abstract: Many of the genes involved in aging have been identified in organisms ranging from yeast to human. Our previous study showed that deletion of the UGA3 gene-which encodes a zinc-finger transcription factor necessary for {gamma}-aminobutyric acid (GABA)-dependent induction of the UGA1 (GABA aminotransferase), UGA2 (succinate semialdehyde dehydrogenase), and UGA4 (GABA permease) genes-extends replicative lifespan in the budding yeast Saccharomycescerevisiae. Here, we found that deletion of UGA1 lengthened the lifespan, as did deletion of UGA3; in contrast, strains with UGA2 or UGA4 deletions exhibited no lifespan extension. The {Delta}uga1 strain cannot deaminate GABA to succinate semialdehyde. Deletion of GAD1, which encodes the glutamate decarboxylase that converts glutamate into GABA, also increased lifespan. Therefore, two genes in the GABA metabolism pathway, UGA1 and GAD1, were identified as aging genes. Unexpectedly, intracellular GABA levels in mutant cells (except for {Delta}uga2 cells) did not differ from those in wild-type cells. Addition of GABA to culture media, which induces transcription of the UGA structural genes, had no effect on replicative lifespan of wild-type cells. Multivariate analysis of {sup 1}H nuclear magnetic resonance spectra for the whole-cell metabolite levels demonstrated a separation between long-lived and normal-lived strains. Gas chromatography-mass spectrometry analysis of identified metabolites showed that levels of tricarboxylic acid cycle intermediates positively correlated with lifespan

  11. Action of tremorgenic mycotoxins on GABA/sub A/ receptor

    SciTech Connect

    Gant, D.B.; Cole, R.J.; Valdes, J.J.; Eldefrawi, M.E.; Eldefrawi, A.T.

    1987-11-09

    The effects of four tremorgenic and one nontremorgenic mycotoxins were studied on ..gamma..-aminobutyric acid (GABA/sub A/) receptor binding and function in rat brain and on binding of a voltage-operated Cl/sup -/ channel in Torpedo electric organ. None of the mycotoxins had significant effect on (/sup 3/H)muscimol or (/sup 3/H)flunitrazepam binding to the GAMA/sup A/ receptor. However, only the four tremorgenic mycotoxins inhibited GABA-induced /sup 36/Cl/sup -/ influx and (/sup 35/S)t-butylbicyclophosphorothionate ((/sup 35/S)TBPS) binding in rate brain membranes, while the nontremorgenic verruculotoxin had no effect. Inhibition of (/sup 35/S)TBPS binding by paspalinine was non-competitive. This suggests that tremorgenic mycotoxins inhibit GABA/sub A/ receptor function by binding close to the receptor's Cl/sup -/ channel. On the voltage-operated Cl/sup -/ channel, only high concentrations of verruculogen and verruculotoxin caused significant inhibition of the channel's binding of (/sup 35/S)TBPS. The data suggest that the tremorgenic action of these mycotoxins may be due in part to their inhibition of GABA/sub A/ receptor function. 21 references, 4 figures, 2 tables.

  12. Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport.

    PubMed

    Kobayashi, T; Beuchat, M H; Lindsay, M; Frias, S; Palmiter, R D; Sakuraba, H; Parton, R G; Gruenberg, J

    1999-06-01

    The fate of free cholesterol released after endocytosis of low-density lipoproteins remains obscure. Here we report that late endosomes have a pivotal role in intracellular cholesterol transport. We find that in the genetic disease Niemann-Pick type C (NPC), and in drug-treated cells that mimic NPC, cholesterol accumulates in late endosomes and sorting of the lysosomal enzyme receptor is impaired. Our results show that the characteristic network of lysobisphosphatidic acid-rich membranes contained within multivesicular late endosomes regulates cholesterol transport, presumably by acting as a collection and distribution device. The results also suggest that similar endosomal defects accompany the anti-phospholipid syndrome and NPC. PMID:10559883

  13. Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking.

    PubMed

    Sandoval, Angel; Fraisl, Peter; Arias-Barrau, Elsa; Dirusso, Concetta C; Singer, Diane; Sealls, Whitney; Black, Paul N

    2008-09-15

    These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis-Menten kinetics; the apparent efficiency (k(cat)/K(T)) of this process increases over 2-fold (2.1 x 10(6)-4.5 x 10(6)s(-1)M(-1)) upon adipocyte differentiation. The V(max) values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 x 10(6)s(-1)M(-1) versus 1.5 x 10(6)s(-1)M(-1)). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V(max) values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The

  14. [The radioprotective effect of GABA-tropic substances, gamma-hydroxybutyrate and piracetam].

    PubMed

    Kulinskiĭ, V I; Klimova, A D

    1993-01-01

    From experiments in mice, it is shown that with a radiation dose of 8 Gy (LD96) the radioprotective effect was exerted by gamma-aminobutyric acid (GABA), substances that increase its concentration in tissues (progabide and valproate), and synthetic agonists of both receptor types, particularly baclofen, a GABA-receptor agonist. The radioprotective effect is also exerted by gamma-hydroxybutyrate, not piracetam. PMID:8469734

  15. GABA-mediated inhibition of the anaphylactic response in the guinea-pig trachea.

    PubMed Central

    Gentilini, G.; Franchi-Micheli, S.; Mugnai, S.; Bindi, D.; Zilletti, L.

    1995-01-01

    1. In sensitized guinea-pigs, the effects of gamma-aminobutyric acid (GABA) and GABAmimetic drugs have been investigated on tracheal segments contracted by cumulative application of an allergen (ovoalbumin, OA) and on serosal mast cells. The same drugs have also been tested on activation of alveolar macrophages isolated from unsensitized guinea-pigs. 2. Superfusion with GABA (1-1000 microM) reduced the contraction intensity of tracheal strips. The effect of GABA (100 microM) was not affected by the carrier blockers, nipecotic acid and beta-alanine (300 microM each). It was mimicked by the GABAB agonist (-)-baclofen (100 microM) but not 3-aminopropanephosphinic acid (100 microM, 3-APA). The GABAA agonist, isoguvacine (100 microM) did not exert any effect. GABA (10 microM)-induced inhibition of tracheal contractions was reduced by the GABAB antagonist, 2-hydroxysaclofen (100 microM, 2-HS), but not by the GABAA antagonist, bicuculline (30 microM). 3. The reduction in contraction intensity induced by GABA (100 microM) was prevented by a 40 min preincubation of tracheal strips with capsaicin (10 microM), but not tetrodotoxin (TTX, 0.3 microM). The effect of GABA (1000 microM) was absent after preincubation with indomethacin (2.8 microM) but unmodified when nordihydroguaiaretic acid (NDGA, 3.3 microM) was used. Finally, removal of the epithelium prevented the GABA effect. 4. Anaphylactic histamine release from serosal mast cells isolated from sensitized animals was not affected either by GABA (10-1000 microM) or the selective receptor agonists (-)-baclofen (0.1-1000 microM) and isoguvacine (10-1000 microM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582447

  16. Uptake of aristolochic acid I into Caco-2 cells by monocarboxylic acid transporters.

    PubMed

    Kimura, Osamu; Haraguchi, Koichi; Ohta, Chiho; Koga, Nobuyuki; Kato, Yoshihisa; Endo, Tetsuya

    2014-01-01

    The uptake mechanism of aristolochic acid I (AAI) was investigated using Caco-2 cells cultured on dishes and permeable membranes. The uptake of AAI from the apical membrane of Caco-2 cells cultured on a dish was rapid, and a decrease in the pH of the incubation medium significantly increased uptake. Incubation at low temperature (4°C) and treatment with sodium azide (a metabolic inhibitor) or carbonylcyanide p-trifluoromethoxyphenylhydrazone (a protonophore) significantly inhibited the AAI uptake. Coincubation with L-lactic acid or benzoic acid, typical substrates for the proton-linked monocarboxylic acid transporters (MCTs), significantly decreased the AAI uptake, as did coincubation with α-cyano-4-hydroxycinnamate (an inhibitor of MCTs). Dixon plotting revealed the competitive inhibition of benzoic acid on the AAI uptake. To confirm the AAI uptake via MCTs, the apical-to-basolateral transport of AAI was investigated using the Caco-2 cells cultured on the permeable membranes. The transport of AAI at pH 6.0 was markedly higher than that at pH 7.4, and was significantly decreased by coincubation with benzoic acid. These results suggest that the uptake of AAI from the apical membrane of Caco-2 cells is mediated mainly by MCTs along with benzoic acid. PMID:25177030

  17. Sodium-dependent GABA-induced currents in GAT1-transfected HeLa cells.

    PubMed Central

    Risso, S; DeFelice, L J; Blakely, R D

    1996-01-01

    1. HeLa cells were infected with recombinant vaccinia virus containing the T7 RNA polymerase gene and transfected with the cDNA for a rat GABA transporter, GAT1, cloned downstream of a T7 RNA polymerase promoter. Six to sixteen hours after transfection, whole-cell recording with a voltage ramp in the range -90 to 50 mV revealed GABA-induced currents (approximately -100 pA at -60 mV in 100 microM GABA, 16 h after transfection at room temperature). No GABA-induced currents were observed in parental HeLa cells or in mock-transfected cells. 2. GABA-induced currents were suppressed by extracellular perfusion with GABA-free solutions or addition of GAT1 inhibitors SKF89976-A or SKF100330-A. At fixed voltage the GABA dependence of the inward current fitted the Michaelis-Menten equation with a Hill coefficient, n, near unity and an equilibrium constant, K(m), near 3 microM. The Na+ dependence of the inward currents fitted the Michaelis-Menten equation with n approximately equal to 2 and K(m) approximately equal to 10 mM. The constants n and K(m) for GABA and Na+ were independent of voltage in the range -90 to -30 mV. 3. GABA-induced currents reverse direction in the range 5-10 mV. The implication of this result is that GAT1 can mediate electrogenic (electrophoretic) influx or efflux of GABA depending on the membrane voltage. The presence of an outward current in our experiments is consistent with radioactive-labelled flux data from resealed vesicle studies. However, it is inconsistent with frog oocyte expression experiments using the sample clone. In oocytes, GAT1 generates no outward current in a similar voltage range. Smaller intracellular volume or higher turnover rates in the mammalian expression system may explain the outward currents. 4. External GABA induces inward current, and internal GABA induces outward current. However, in cells initially devoid of internal GABA, external GABA can also facilitate an outward current. This GAT1-mediated outward current occurs

  18. GABA(A) receptor downregulation in brains of subjects with autism.

    PubMed

    Fatemi, S Hossein; Reutiman, Teri J; Folsom, Timothy D; Thuras, Paul D

    2009-02-01

    Gamma-aminobutyric acid A (GABA(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 expression of four GABA(A) receptor subunits and observed significant reductions in GABRA1, GABRA2, GABRA3, and GABRB3 in parietal cortex (Brodmann's Area 40 (BA40)), while GABRA1 and GABRB3 were significantly altered in cerebellum, and GABRA1 was significantly altered in superior frontal cortex (BA9). The presence of seizure disorder did not have a significant impact on GABA(A) receptor subunit expression in the three brain areas. Our results demonstrate that GABA(A) receptors are reduced in three brain regions that have previously been implicated in the pathogenesis of autism, suggesting widespread GABAergic dysfunction in the brains of subjects with autism. PMID:18821008

  19. Imbalance between Glutamate and GABA in Fmr1 Knockout Astrocytes Influences Neuronal Development

    PubMed Central

    Wang, Lu; Wang, Yan; Zhou, Shimeng; Yang, Liukun; Shi, Qixin; Li, Yujiao; Zhang, Kun; Yang, Le; Zhao, Minggao; Yang, Qi

    2016-01-01

    Fragile X syndrome (FXS) is a form of inherited mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), the product of the Fmr1 gene. Numerous studies have shown that FMRP expression in astrocytes is important in the development of FXS. Although astrocytes affect neuronal dendrite development in Fmr1 knockout (KO) mice, the factors released by astrocytes are still unclear. We cultured wild type (WT) cortical neurons in astrocyte-conditioned medium (ACM) from WT or Fmr1 KO mice. Immunocytochemistry and Western blotting were performed to detect the dendritic growth of both WT and KO neurons. We determined glutamate and γ-aminobutyric acid (GABA) levels using high-performance liquid chromatography (HPLC). The total neuronal dendritic length was reduced when cultured in the Fmr1 KO ACM. This neurotoxicity was triggered by an imbalanced release of glutamate and GABA from Fmr1 KO astrocytes. We found increased glutaminase and GABA transaminase (GABA-T) expression and decreased monoamine oxidase B expression in Fmr1 KO astrocytes. The elevated levels of glutamate contributed to oxidative stress in the cultured neurons. Vigabatrin (VGB), a GABA-T inhibitor, reversed the changes caused by glutamate and GABA release in Fmr1 KO astrocytes and the abnormal behaviors in Fmr1 KO mice. Our results indicate that the imbalance in the astrocytic glutamate and GABA release may be involved in the neuropathology and the underlying symptoms of FXS, and provides a therapeutic target for treatment. PMID:27517961

  20. The involvement of L-type amino acid transporters in theanine transport.

    PubMed

    Yamamoto, Sachiko; Kimura, Toru; Tachiki, Takashi; Anzai, Naohiko; Sakurai, Takuya; Ushimaru, Makoto

    2012-01-01

    L-Theanine has favorable physiological effects in terms of human health, but the mechanisms that transport it to its target organs or cells are not completely defined. To identify the major transport mechanisms of L-theanine, we screened for candidate transporters of L-3H-theanine in several mammal cell lines that intrinsically express multiple transporters with various specificities. All of the cells tested, T24, HepG2, COS1, 293A, Neuro2a, and HuH7, absorbed L-3H-theanine. Uptake was significantly inhibited by the addition of L-leucine and by a specific inhibitor of the system L transport system, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). L-3H-Theanine uptake occurred mostly independently of Na+. These results indicate that L-theanine was taken up via a system L like transport system in all of the cells tested. Additionally, in experiments using cells stably expressing two system L isoforms, LAT1 and LAT2, we found that the two isoforms mediated L-theanine transport to similar extents. Taken together, our results indicate that L-theanine is transported mostly via the system L transport pathway and its isoforms. PMID:23221699

  1. Comparison of taurine, GABA, Glu, and Asp as scavengers of malondialdehyde in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Deng, Yan; Wang, Wei; Yu, Pingfeng; Xi, Zhijiang; Xu, Lijian; Li, Xiaolong; He, Nongyue

    2013-04-01

    The purpose of this study is to determine if amino acid neurotransmitters such as gamma-aminobutyric acid (GABA), taurine, glutamate (Glu), and aspartate (Asp) can scavenge activated carbonyl toxicants. In vitro, direct reaction between malondialdehyde (MDA) and amino acids was researched using different analytical methods. The results indicated that scavenging activated carbonyl function of taurine and GABA is very strong and that of Glu and Asp is very weak in pathophysiological situations. The results provided perspective into the reaction mechanism of taurine and GABA as targets of activated carbonyl such as MDA in protecting nerve terminals. In vivo, we studied the effect of taurine and GABA as antioxidants by detecting MDA concentration and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. It was shown that MDA concentration was decreased significantly, and the activities of SOD and GSH-Px were increased significantly in the cerebral cortex and hippocampus of acute epileptic state rats, after the administration of taurine and GABA. The results indicated that the peripherally administered taurine and GABA can scavenge free radicals and protect the tissue against activated carbonyl in vivo and in vitro.

  2. 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.

  3. Sleep-promoting effects of the GABA/5-HTP mixture in vertebrate models.

    PubMed

    Hong, Ki-Bae; Park, Yooheon; Suh, Hyung Joo

    2016-09-01

    The aim of this study was to investigate the sleep-promoting effect of combined γ-aminobutyric acid (GABA) and 5-hydroxytryptophan (5-HTP) on sleep quality and quantity in vertebrate models. Pentobarbital-induced sleep test and electroencephalogram (EEG) analysis were applied to investigate sleep latency, duration, total sleeping time and sleep quality of two amino acids and GABA/5-HTP mixture. In addition, real-time PCR and HPLC analysis were applied to analyze the signaling pathway. The GABA/5-HTP mixture significantly regulated the sleep latency, duration (p<0.005), and also increased the sleep quality than single administration of the amino acids (p<0.000). Long-term administration increased the transcript levels of GABAA receptor (1.37-fold, p<0.000) and also increased the GABA content compared with the control group 12h after administration (1.43-fold, p<0.000). Our available evidence suggests that the GABA/5-HTP mixture modulates both GABAergic and serotonergic signaling. Moreover, the sleep architecture can be controlled by the regulation of GABAA receptor and GABA content with 5-HTP. PMID:27150227

  4. Structural insights into thyroid hormone transport mechanisms of the L-type amino acid transporter 2.

    PubMed

    Hinz, Katrin M; Meyer, Katja; Kinne, Anita; Schülein, Ralf; Köhrle, Josef; Krause, Gerd

    2015-06-01

    Thyroid hormones (THs) are transported across cell membranes by different transmembrane transporter proteins. In previous studies, we showed marked 3,3'-diiodothyronine (3,3'-T2) but moderate T3 uptake by the L-type amino acid transporter 2 (Lat2). We have now studied the structure-function relationships of this transporter and TH-like molecules. Our Lat2 homology model is based on 2 crystal structures of the homologous 12-transmembrane helix transporters arginine/agmatine antiporter and amino acid/polyamine/organocation transporter. Model-driven mutagenesis of residues lining an extracellular recognition site and a TH-traversing channel identified 9 sensitive residues. Using Xenopus laevis oocytes as expression system, we found that side chain shortening (N51S, N133S, N248S, and Y130A) expanded the channel and increased 3,3'-T2 transport. Side chain enlargements (T140F, Y130R, and I137M) decreased 3,3'-T2 uptake, indicating channel obstructions. The opposite results with mutations maintaining (F242W) or impairing (F242V) uptake suggest that F242 may have a gating function. Competitive inhibition studies of 14 TH-like compounds revealed that recognition by Lat2 requires amino and carboxylic acid groups. The size of the adjacent hydrophobic group is restricted. Bulky substituents in positions 3 and 5 of the tyrosine ring are allowed. The phenolic ring may be enlarged, provided that the whole molecule is flexible enough to fit into the distinctly shaped TH-traversing channel of Lat2. Taken together, the next Lat2 features were identified 1) TH recognition site; 2) TH-traversing channel in the center of Lat2; and 3) switch site that potentially facilitates intracellular substrate release. Together with identified substrate features, these data help to elucidate the molecular mechanisms and role of Lat2 in T2 transport. PMID:25945809

  5. Mitochondrial transporters for ornithine and related amino acids: a review.

    PubMed

    Monné, Magnus; Miniero, Daniela Valeria; Daddabbo, Lucia; Palmieri, Luigi; Porcelli, Vito; Palmieri, Ferdinando

    2015-09-01

    Among the members of the mitochondrial carrier family, there are transporters that catalyze the translocation of ornithine and related substrates, such as arginine, homoarginine, lysine, histidine, and citrulline, across the inner mitochondrial membrane. The mitochondrial carriers ORC1, ORC2, and SLC25A29 from Homo sapiens, BAC1 and BAC2 from Arabidopsis thaliana, and Ort1p from Saccharomyces cerevisiae have been biochemically characterized by transport assays in liposomes. All of them transport ornithine and amino acids with side chains terminating at least with one amine. There are, however, marked differences in their substrate specificities including their affinity for ornithine (KM values in the mM to μM range). These differences are most likely reflected by minor differences in the substrate binding sites of these carriers. The physiological role of the above-mentioned mitochondrial carriers is to link several metabolic pathways that take place partly in the cytosol and partly in the mitochondrial matrix and to provide basic amino acids for mitochondrial translation. In the liver, human ORC1 catalyzes the citrulline/ornithine exchange across the mitochondrial inner membrane, which is required for the urea cycle. Human ORC1, ORC2, and SLC25A29 are likely to be involved in the biosynthesis and transport of arginine, which can be used as a precursor for the synthesis of NO, agmatine, polyamines, creatine, glutamine, glutamate, and proline, as well as in the degradation of basic amino acids. BAC1 and BAC2 are implicated in some processes similar to those of their human counterparts and in nitrogen and amino acid metabolism linked to stress conditions and the development of plants. Ort1p is involved in the biosynthesis of arginine and polyamines in yeast. PMID:26002808

  6. Acute Immobilization Stress Modulate GABA Release from Rat Olfactory Bulb: Involvement of Endocannabinoids—Cannabinoids and Acute Stress Modulate GABA Release

    PubMed Central

    Delgado, Alejandra; Jaffé, Erica H.

    2011-01-01

    We studied the effects of cannabinoids and acute immobilization stress on the regulation of GABA release in the olfactory bulb. Glutamate-stimulated 3H-GABA release was measured in superfused slices. We report that cannabinoids as WIN55, 212-2, methanandamide, and 2-arachidonoylglycerol were able to inhibit glutamate- and KCl-stimulated 3H-GABA release. This effect was blocked by the CB1 antagonist AM281. On the other hand, acute stress was able per se to increase endocannabinoid activity. This effect was evident since the inhibition of stimulated GABA release by acute stress was reversed with AM281 and tetrahydrolipstatin. Inhibition of the endocannabinoid transport or its catabolism showed reduction of GABA release, antagonized by AM281 in control and stressed animals. These results point to endocannabinoids as inhibitory modulators of GABA release in the olfactory bulb acting through an autocrine mechanism. Apparently, stress increases the endocannabinoid system, modulating GABAergic synaptic function in a primary sensory organ. PMID:21785597

  7. Abscisic acid transporters cooperate to control seed germination

    PubMed Central

    Kang, Joohyun; Yim, Sojeong; Choi, Hyunju; Kim, Areum; Lee, Keun Pyo; Lopez-Molina, Luis; Martinoia, Enrico; Lee, Youngsook

    2015-01-01

    Seed germination is a key developmental process that has to be tightly controlled to avoid germination under unfavourable conditions. Abscisic acid (ABA) is an essential repressor of seed germination. In Arabidopsis, it has been shown that the endosperm, a single cell layer surrounding the embryo, synthesizes and continuously releases ABA towards the embryo. The mechanism of ABA transport from the endosperm to the embryo was hitherto unknown. Here we show that four AtABCG transporters act in concert to deliver ABA from the endosperm to the embryo: AtABCG25 and AtABCG31 export ABA from the endosperm, whereas AtABCG30 and AtABCG40 import ABA into the embryo. Thus, this work establishes that radicle extension and subsequent embryonic growth are suppressed by the coordinated activity of multiple ABA transporters expressed in different tissues. PMID:26334616

  8. Synthesis of new fluorinated analogs of GABA, Pregabalin bioisosteres, and their effects on [(3)H]GABA uptake by rat brain nerve terminals.

    PubMed

    Borisova, T; Pozdnyakova, N; Shaitanova, E; Gerus, I; Dudarenko, M; Mironets, R; Haufe, G; Kukhar, V

    2015-08-01

    Fluorinated analogs of natural substances take an essential place in the design of new biologically active compounds. New fluorinated analogs of γ-aminobutyric acid, that is, β-polyfluoroalkyl-GABAs (FGABAs), were synthesized with substituents: β-CF3-β-OH (1), β-CF3 (2); β-CF2CF2H (3). FGABAs are bioisosteres of Pregabalin (Lyrica®, Pfizer's blockbuster drug, β-i-Bu-GABA), and have lipophilicity close to this medicine. The effects of synthesized FGABAs on [(3)H]GABA uptake by isolated rat brain nerve terminals (synaptosomes) were assessed and compared with those of Pregabalin. FGABAs 1-3 (100μM) did not influence the initial velocity of [(3)H]GABA uptake when applied acutely, whereas an increase in this parameter was found after preliminary incubation of FGABAs with synaptosomes. Pregabalin after preliminary incubation with synaptosomes caused unidirectional changes in the initial velocity of [(3)H]GABA uptake. Using specific inhibitors of GAT1 and GAT3, NO-711 and SNAP5114, respectively, the ability of FGABAs 1-3 to influence non-GAT1 and non-GAT3 uptake activity of nerve terminals was analyzed, but no specificity was found. Therefore, new synthesized FGABAs are structural but not functional analogs of GABA (because they did not inhibit synaptosomal [(3)H]GABA uptake). Moreover, FGABAs are able to increase the initial velocity of [(3)H]GABA uptake by synaptosomes, and this effect is higher than that of Pregabalin. PMID:26138193

  9. Microtransplantation of cellular membranes from squid stellate ganglion reveals ionotropic GABA receptors.

    PubMed

    Conti, Luca; Limon, Agenor; Palma, Eleonora; Miledi, Ricardo

    2013-02-01

    The squid has been the most studied cephalopod, and it has served as a very useful model for investigating the events associated with nerve impulse generation and synaptic transmission. While the physiology of squid giant axons has been extensively studied, very little is known about the distribution and function of the neurotransmitters and receptors that mediate inhibitory transmission at the synapses. In this study we investigated whether γ-aminobutyric acid (GABA) activates neurotransmitter receptors in stellate ganglia membranes. To overcome the low abundance of GABA-like mRNAs in invertebrates and the low expression of GABA in cephalopods, we used a two-electrode voltage clamp technique to determine if Xenopus laevis oocytes injected with cell membranes from squid stellate ganglia responded to GABA. Using this method, membrane patches containing proteins and ion channels from the squid's stellate ganglion were incorporated into the surface of oocytes. We demonstrated that GABA activates membrane receptors in cellular membranes isolated from squid stellate ganglia. Using the same approach, we were able to record native glutamate-evoked currents. The squid's GABA receptors showed an EC(50) of 98 μmol l(-1) to GABA and were inhibited by zinc (IC(50) = 356 μmol l(-1)). Interestingly, GABA receptors from the squid were only partially blocked by bicuculline. These results indicate that the microtransplantation of native cell membranes is useful to identify and characterize scarce membrane proteins. Moreover, our data also support the role of GABA as an ionotropic neurotransmitter in cephalopods, acting through chloride-permeable membrane receptors. PMID:23493508

  10. Aging of whiskey increases the potentiation of GABA(A) receptor response.

    PubMed

    Koda, Hirofumi; Hossain, Sheikh Julfikar; Kiso, Yoshinobu; Aoshima, Hitoshi

    2003-08-27

    It is known that the target of most mood-defining compounds such as ethanol is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activities in the human brain. Because both extracts of whiskey by pentane and fragrant components in whiskey potentiate the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting cRNAs prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors in order to study the effects of whiskey itself on the GABA(A) receptor-mediated response. Whiskey itself also potentiated the electrical responses of GABA(A) receptors generally more than ethanol at the same concentration as that of the whiskey. The potentiation of the GABA(A) receptor-mediated response increased with the aging period of the whiskey. Inhalation of whiskey to mice increased the sleeping time induced by pentobarbital more than that of the same concentration of ethanol as the whiskey. These results suggest that not only ethanol but also minor components in whiskey play an important role in the potentiation of GABA(A) receptor-mediated response and possibly the sedative effect of whiskey. Although the minor components are present in extremely small quantities compared with ethanol in alcoholic beverages, they may modulate the mood or consciousness of humans through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic compounds are easily absorbed into the brain across the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response. PMID:12926865

  11. GABA(B) receptors and synaptic modulation.

    PubMed

    Kornau, Hans-Christian

    2006-11-01

    GABA(B) receptors modulate transmitter release and postsynaptic membrane potential at various types of central synapses. They function as heterodimers of two related seven-transmembrane domain receptor subunits. Trafficking, activation and signalling of GABA(B) receptors are regulated both by allosteric interactions between the subunits and by the binding of additional proteins. Recent studies have shed light on the roles of GABA(B) receptors in plasticity processes at excitatory synapses. This review summarizes our knowledge of the localization, structure and function of GABA(B) receptors in the central nervous system and their use as drug targets for neurological and psychiatric disorders. PMID:16932937

  12. GABA and GAD expression in the X-organ sinus gland system of the Procambarus clarkii crayfish: inhibition mediated by GABA between X-organ neurons.

    PubMed

    Pérez-Polanco, Paola; Garduño, Julieta; Cebada, Jorge; Zarco, Natanael; Segovia, José; Lamas, Mónica; García, Ubaldo

    2011-09-01

    In crustaceans, the X-organ-sinus gland (XO-SG) neurosecretory system is formed of distinct populations of neurons that produce two families of neuropeptides: crustacean hyperglycemic hormone and adipokinetic hormone/red pigment-concentrating hormone. On the basis of electrophysiological evidence, it has been proposed that γ-aminobutyric acid (GABA) regulates both electrical and secretory activity of the XO-SG system. In this work we observed that depolarizing current pulses to neurons located in the external rim of the X-organ induced repetitive firing that suppressed the spontaneous firing of previously active X-organ neurons. Picrotoxin reversibly blocked this inhibitory effect suggesting that the GABA released from the stimulated neuron inhibited neighboring cells. Immunoperoxidase in X-organ serial sections showed co-localization of GABA and glutamic acid decarboxylase (GAD) including the aforementioned neurons. Immunofluorescence in whole mount preparations showed that two subpopulations of crustacean hyperglycemic hormone-containing neurons colocalized with GABA. The expression of GAD mRNA was determined in crayfish tissue and X-organ single cells by RT-PCR. Bioinformatics analysis shows, within the amplified region, 90.4% consensus and 41.9% identity at the amino acid level compared with Drosophila melanogaster and Caenorhabditis elegans. We suggest that crustacean hyperglycemic hormone-GABA-containing neurons can regulate the excitability of other X-organ neurons that produce different neurohormones. PMID:21626307

  13. Molecular basis of essential amino acid transport from studies of insect nutrient amino acid transporters of the SLC6 family (NAT-SLC6)

    PubMed Central

    Boudko, Dmitri Y.

    2012-01-01

    Two protein families that represent major components of essential amino acid transport in insects have been identified. They are annotated as the SLC6 and SLC7 families of transporters according to phylogenetic proximity to characterized amino acid transporters (HUGO nomenclature). Members of these families have been identified as important apical and basolateral parts of transepithelial essential amino acid absorption in the metazoan alimentary canal. Synergistically, they play critical physiological roles as essential substrate providers to diverse metabolic processes, including generic protein synthesis. This review briefly clarifies the requirements for amino acid transport and a variety of amino acid transport mechanisms, including the aforementioned families. Further it focuses on the large group of Nutrient Amino acid Transporters (NATs), which comprise a recently identified subfamily of the Neurotransmitter Sodium Symporter family (NSS or SLC6). The first insect NAT, cloned from the caterpillar gut, has a broad substrate spectrum similar to mammalian B0 transporters. Several new NAT-SLC6 members have been characterized in an effort to explore mechanisms for the essential amino acid absorption in model dipteran insects. The identification and functional characterization of new B0-like and narrow specificity transporters of essential amino acids in fruit fly and mosquitoes leads to a fundamentally important insight: that NATs evolved and act together as the integrated active core of a transport network that mediates active alimentary absorption and systemic distribution of essential amino acids. This role of NATs is projected from the most primitive prokaryotes to the most complex metazoan organisms, and represents an interesting platform for unraveling the molecular evolution of amino acid transport and modeling amino acid transport disorders. The comparative study of NATs elucidates important adaptive differences between essential amino acid transportomes

  14. Transport in Halobacterium Halobium: Light-Induced Cation-Gradients, Amino Acid Transport Kinetics, and Properties of Transport Carriers

    NASA Technical Reports Server (NTRS)

    Lanyi, Janos K.

    1977-01-01

    Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na(+). Measurements of Na-22 flux, exterior pH change, and membrane potential, Delta(psi) (with the dye 3,3'-dipentyloxadicarbocyanine) indicate that the means of Na(+) transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H(+)/Na(++ greater than 1). The resulting large chemical gradient for Na(+) (outside much greater than inside), as well as the membrane potential, will drive the transport of 18 amino acids. The I9th, glutamate, is unique in that its accumulation is indifferent to Delta(psi): this amino acid is transported only when a chemical gradient for Na(+) is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H+() collapses within 1 min, while the large Na(+) gradient and glutamate transporting activity persists for 10- 15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na(+), arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V(sub max) and K(sub m) comparable to light-induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na(+), in an electrically neutral fashion, so that only the chemical component of the Na(+) gradient is a driving force.

  15. Decreased Phosphorylated Protein Kinase B (Akt) in Individuals with Autism Associated with High Epidermal Growth Factor Receptor (EGFR) and Low Gamma-Aminobutyric Acid (GABA)

    PubMed Central

    Russo, Anthony J

    2015-01-01

    Dysregulation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway could contribute to the pathogenesis of autism spectrum disorders. In this study, phosphorylated Akt concentration was measured in 37 autistic children and 12, gender and age similar neurotypical, controls using an enzyme-linked immunosorbent assay. Akt levels were compared to biomarkers known to be associated with epidermal growth factor receptor (EGFR) and c-Met (hepatocyte growth factor (HGF) receptor) pathways and severity levels of 19 autism-related symptoms. We found phosphorylated Akt levels significantly lower in autistic children and low Akt levels correlated with high EGFR and HGF and low gamma-aminobutyric acid, but not other biomarkers. Low Akt levels also correlated significantly with increased severity of receptive language, conversational language, hypotonia, rocking and pacing, and stimming, These results suggest a relationship between decreased phosphorylated Akt and selected symptom severity in autistic children and support the suggestion that the AKT pathways may be associated with the etiology of autism. PMID:26508828

  16. The Arabidopsis her1 mutant implicates GABA in E-2-hexenal responsiveness.

    PubMed

    Mirabella, Rossana; Rauwerda, Han; Struys, Eduard A; Jakobs, Cornelis; Triantaphylidès, Christian; Haring, Michel A; Schuurink, Robert C

    2008-01-01

    When wounded or attacked by herbivores or pathogens, plants produce a blend of six-carbon alcohols, aldehydes and esters, known as C6-volatiles. Undamaged plants, when exposed to C6-volatiles, respond by inducing defense-related genes and secondary metabolites, suggesting that C6-volatiles can act as signaling molecules regulating plant defense responses. However, to date, the molecular mechanisms by which plants perceive and respond to these volatiles are unknown. To elucidate such mechanisms, we decided to isolate Arabidopsis thaliana mutants in which responses to C6-volatiles were altered. We observed that treatment of Arabidopsis seedlings with the C6-volatile E-2-hexenal inhibits root elongation. Among C6-volatiles this response is specific to E-2-hexenal, and is not dependent on ethylene, jasmonic and salicylic acid. Using this bioassay, we isolated 18 E-2-hexenal-response (her) mutants that showed sustained root growth after E-2-hexenal treatment. Here, we focused on the molecular characterization of one of these mutants, her1. Microarray and map-based cloning revealed that her1 encodes a gamma-amino butyric acid transaminase (GABA-TP), an enzyme that degrades GABA. As a consequence of the mutation, her1 plants accumulate high GABA levels in all their organs. Based on the observation that E-2-hexenal treatment induces GABA accumulation, and that high GABA levels confer resistance to E-2-hexenal, we propose a role for GABA in mediating E-2-hexenal responses. PMID:17971036

  17. Aluminum in acidic surface waters: chemistry, transport, and effects.

    PubMed Central

    Driscoll, C T

    1985-01-01

    Ecologically significant concentrations of Al have been reported in surface waters draining "acid-sensitive" watersheds that are receiving elevated inputs of acidic deposition. It has been hypothesized that mineral acids from atmospheric deposition have remobilized Al previously precipitated within the soil during soil development. This Al is then thought to be transported to adjacent surface waters. Dissolved mononuclear Al occurs as aquo Al, as well as OH-, F-, SO4(2-), and organic complexes. Although past investigations have often ignored non-hydroxide complexes of Al, it appears that organic and F complexes are the predominant forms of Al in dilute (low ionic strength) acidic surface waters. The concentration of inorganic forms of Al increases exponentially with decreases in solution pH. This response is similar to the theoretical pH dependent solubility of Al mineral phases. The concentration of organic forms of Al, however, is strongly correlated with variations in organic carbon concentration of surface waters rather than pH. Elevated concentrations of Al in dilute acidic waters are of interest because: Al is an important pH buffer; Al may influence the cycling of important elements like P, organic carbon, and trace metals; and Al is potentially toxic to aquatic organisms. An understanding of the aqueous speciation of Al is essential for an evaluation of these processes. PMID:3935428

  18. Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid.

    PubMed

    Nguyen, Long N; Ma, Dongliang; Shui, Guanghou; Wong, Peiyan; Cazenave-Gassiot, Amaury; Zhang, Xiaodong; Wenk, Markus R; Goh, Eyleen L K; Silver, David L

    2014-05-22

    Docosahexaenoic acid (DHA) is an omega-3 fatty acid that is essential for normal brain growth and cognitive function. Consistent with its importance in the brain, DHA is highly enriched in brain phospholipids. Despite being an abundant fatty acid in brain phospholipids, DHA cannot be de novo synthesized in brain and must be imported across the blood-brain barrier, but mechanisms for DHA uptake in brain have remained enigmatic. Here we identify a member of the major facilitator superfamily--Mfsd2a (previously an orphan transporter)--as the major transporter for DHA uptake into brain. Mfsd2a is found to be expressed exclusively in endothelium of the blood-brain barrier of micro-vessels. Lipidomic analysis indicates that Mfsd2a-deficient (Mfsd2a-knockout) mice show markedly reduced levels of DHA in brain accompanied by neuronal cell loss in hippocampus and cerebellum, as well as cognitive deficits and severe anxiety, and microcephaly. Unexpectedly, cell-based studies indicate that Mfsd2a transports DHA in the form of lysophosphatidylcholine (LPC), but not unesterified fatty acid, in a sodium-dependent manner. Notably, Mfsd2a transports common plasma LPCs carrying long-chain fatty acids such LPC oleate and LPC palmitate, but not LPCs with less than a 14-carbon acyl chain. Moreover, we determine that the phosphor-zwitterionic headgroup of LPC is critical for transport. Importantly, Mfsd2a-knockout mice have markedly reduced uptake of labelled LPC DHA, and other LPCs, from plasma into brain, demonstrating that Mfsd2a is required for brain uptake of DHA. Our findings reveal an unexpected essential physiological role of plasma-derived LPCs in brain growth and function. PMID:24828044

  19. Regulation of hepatic bile acid transporters Ntcp and Bsep expression.

    PubMed

    Cheng, Xingguo; Buckley, David; Klaassen, Curtis D

    2007-12-01

    Sodium-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) are two key transporters for hepatic bile acid uptake and excretion. Alterations in Ntcp and Bsep expression have been reported in pathophysiological conditions. In the present study, the effects of age, gender, and various chemicals on the regulation of these two transporters were characterized in mice. Ntcp and Bsep mRNA levels in mouse liver were low in the fetus, but increased to its highest expression at parturition. After birth, mouse Ntcp and Bsep mRNA decreased by more than 50%, and then gradually increased to adult levels by day 30. Expression of mouse Ntcp mRNA and protein exhibit higher levels in female than male livers. No gender difference exists in BSEP/Bsep expression in human and mouse livers. Hormone replacements conducted in gonadectomized, hypophysectomized, and lit/lit mice indicate that female-predominant Ntcp expression in mouse liver is due to the inhibitory effect of male-pattern GH secretion, but not sex hormones. Ntcp and Bsep expression are in general resistant to induction by a large battery of microsomal enzyme inducers. Administration of cholestyramine increased Ntcp, whereas chenodeoxycholic acid (CDCA) increased Bsep mRNA expression. In conclusion, mouse Ntcp and Bsep are regulated by age, gender, cholestyramine, and bile acid, but resistant to induction by most microsomal enzyme inducers. PMID:17897632

  20. MATE Transporter-Dependent Export of Hydroxycinnamic Acid Amides.

    PubMed

    Dobritzsch, Melanie; Lübken, Tilo; Eschen-Lippold, Lennart; Gorzolka, Karin; Blum, Elke; Matern, Andreas; Marillonnet, Sylvestre; Böttcher, Christoph; Dräger, Birgit; Rosahl, Sabine

    2016-02-01

    The ability of Arabidopsis thaliana to successfully prevent colonization by Phytophthora infestans, the causal agent of late blight disease of potato (Solanum tuberosum), depends on multilayered defense responses. To address the role of surface-localized secondary metabolites for entry control, droplets of a P. infestans zoospore suspension, incubated on Arabidopsis leaves, were subjected to untargeted metabolite profiling. The hydroxycinnamic acid amide coumaroylagmatine was among the metabolites secreted into the inoculum. In vitro assays revealed an inhibitory activity of coumaroylagmatine on P. infestans spore germination. Mutant analyses suggested a requirement of the p-coumaroyl-CoA:agmatine N4-p-coumaroyl transferase ACT for the biosynthesis and of the MATE transporter DTX18 for the extracellular accumulation of coumaroylagmatine. The host plant potato is not able to efficiently secrete coumaroylagmatine. This inability is overcome in transgenic potato plants expressing the two Arabidopsis genes ACT and DTX18. These plants secrete agmatine and putrescine conjugates to high levels, indicating that DTX18 is a hydroxycinnamic acid amide transporter with a distinct specificity. The export of hydroxycinnamic acid amides correlates with a decreased ability of P. infestans spores to germinate, suggesting a contribution of secreted antimicrobial compounds to pathogen defense at the leaf surface. PMID:26744218

  1. mRNA and Protein Levels for GABA[subscript A][alpha]4, [alpha]5, [beta]1 and GABA[subscript B]R1 Receptors are Altered in Brains from Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rooney, Robert J.; Patel, Diven H.; Thuras, Paul D.

    2010-01-01

    We have shown altered expression of gamma-aminobutyric acid A (GABA[subscript A]) and gamma-aminobutyric acid B (GABA[subscript B]) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3…

  2. GABA Not Only a Neurotransmitter: Osmotic Regulation by GABAAR Signaling

    PubMed Central

    Cesetti, Tiziana; Ciccolini, Francesca; Li, Yuting

    2012-01-01

    Mature macroglia and almost all neural progenitor types express γ-aminobutyric (GABA) A receptors (GABAARs), whose activation by ambient or synaptic GABA, leads to influx or efflux of chloride (Cl−) depending on its electro-chemical gradient (ECl). Since the flux of Cl− is indissolubly associated to that of osmotically obliged water, GABAARs regulate water movements by modulating ion gradients. In addition, since water movements also occur through specialized water channels and transporters, GABAAR signaling could affect the movement of water by regulating the function of the channels and transporters involved, thereby affecting not only the direction of the water fluxes but also their dynamics. We will here review recent observations indicating that in neural cells GABAAR-mediated osmotic regulation affects the cellular volume thereby activating multiple intracellular signaling mechanisms important for cell proliferation, maturation, and survival. In addition, we will discuss evidence that the osmotic regulation exerted by GABA may contribute to brain water homeostasis in physiological and in pathological conditions causing brain edema, in which the GABAergic transmission is often altered. PMID:22319472

  3. 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

  4. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms

    PubMed Central

    Widdows, Kate L.; Panitchob, Nuttanont; Crocker, Ian P.; Please, Colin P.; Hanson, Mark A.; Sibley, Colin P.; Johnstone, Edward D.; Sengers, Bram G.; Lewis, Rohan M.; Glazier, Jocelyn D.

    2015-01-01

    Uptake of system L amino acid substrates into isolated placental plasma membrane vesicles in the absence of opposing side amino acid (zero-trans uptake) is incompatible with the concept of obligatory exchange, where influx of amino acid is coupled to efflux. We therefore hypothesized that system L amino acid exchange transporters are not fully obligatory and/or that amino acids are initially present inside the vesicles. To address this, we combined computational modeling with vesicle transport assays and transporter localization studies to investigate the mechanisms mediating [14C]l-serine (a system L substrate) transport into human placental microvillous plasma membrane (MVM) vesicles. The carrier model provided a quantitative framework to test the 2 hypotheses that l-serine transport occurs by either obligate exchange or nonobligate exchange coupled with facilitated transport (mixed transport model). The computational model could only account for experimental [14C]l-serine uptake data when the transporter was not exclusively in exchange mode, best described by the mixed transport model. MVM vesicle isolates contained endogenous amino acids allowing for potential contribution to zero-trans uptake. Both L-type amino acid transporter (LAT)1 and LAT2 subtypes of system L were distributed to MVM, with l-serine transport attributed to LAT2. These findings suggest that exchange transporters do not function exclusively as obligate exchangers.—Widdows, K. L., Panitchob, N., Crocker, I. P., Please, C. P., Hanson, M. A., Sibley, C. P., Johnstone, E. D., Sengers, B. G., Lewis, R. M., Glazier, J. D. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms. PMID:25761365

  5. Choline inhibition of amino acid transport in preimplantation mouse blastocysts

    SciTech Connect

    Campione, A.L.; Haghighat, N.; Gorman, J.; Van Winkle, L.J.

    1987-05-01

    Addition of 70 mM choline chloride to Brinster's medium (140 mM Na/sup +/) inhibited uptake of approx. 1 ..mu..M (/sup 3/H)glycine, leucine, lysine and alanine in blastocysts by about 50% each during a five-minute incubation period at 37/sup 0/C, whereas 70 mM LiCl, sodium acetate and NaCl or 140 mM mannitol had no effect. They attribute the apparent linear relationship between Gly transport in blastocysts and the square of the (Na/sup +/), observed when choline was substituted for Na/sup +/ in Brinster's medium, to concomitant, concentration-dependent enhancement and inhibition of transport by Na/sup +/ and choline, respectively. As expected, Gly uptake and the (Na/sup +/) were linearly related up to 116 mM Na/sup +/, when Na/sup +/ was replaced with Li/sup +/. The rates of Na/sup +/-independent Gly and Ala uptake were <5% and <2% of the total, respectively, and similar when either Li/sup +/ or choline replaced Na/sup +/. Therefore, neither Li/sup +/ nor choline appears to substitute for Na/sup +/ in supporting Na/sup +/-dependent transport in blastocysts. Na/sup +/-independent Leu uptake was 20 times faster than Gly or Ala uptake and appeared to be inhibited by choline in blastocysts since it was about 37% slower when choline instead of Li/sup +/ was substituted for Na/sup +/. In contrast to blastocysts, choline had no effect on amino acid transport in cleavage-stage mouse embryos. The unexpected sensitivity of transport to choline in blastocysts underscores the importance of testing the effects of this substance when it is used to replace Na/sup +/ in new transport studies.

  6. Transport and metabolism of glycolic acid by Chlamydomonas reinhardtii

    SciTech Connect

    Wilson, B.J.

    1987-01-01

    In order to understand the excretion of glycolate from Chlamydomonas reinhardtii, the conditions affecting glycolate synthesis and metabolism were investigated. Although glycolate is synthesized only in the light, the metabolism occurs in the light and dark with greater metabolism in the light due to refixation of photorespiratory CO/sub 2/. The amount of internal glycolate will affect the metabolism of externally added glycolate. When glycolate synthesis exceeds the metabolic capacity, glycolate is excreted from the cell. The transport of glycolate into the cells occurs very rapidly. Equilibrium is achieved at 4/sup 0/C within the time cells are pelleted by the silicone oil centrifugation technique through a layer of (/sup 14/C) glycolate. Glycolate uptake does not show the same time, temperature and pH dependencies as diffusion of benzoate. Uptake can be inhibited by treatment of cells with N-ethylmaleimide and stimulated in the presence of valino-mycin/KCl. Acetate and lactate are taken up as quickly as glycolate. The hypothesis was made that glycolate is transported by a protein carrier that transports monocarboxylic acids. The equilibrium concentration of glycolate is dependent on the cell density, implying that there may be a large number of transporter sites and that uptake is limited by substrate availability.

  7. Perfluorocarboxylic acid (PFCA) atmospheric formation and transport to the Arctic.

    NASA Astrophysics Data System (ADS)

    Pike-thackray, C.; Selin, N. E.

    2015-12-01

    Perfluorocarboxylic acids (PFCAs) are highly persistent and toxic environmental contaminants that have been found in remote locations such as the Arctic, far from emission sources. These persistent organic pollutants are emitted directly to the atmosphere as well as being produced by the degradation of precursor compounds in the atmosphere, but recent trends towards increasing precursor emissions and decreasing direct emissions raise the importance of production in the atmosphere. Our work aims to improve understanding of the atmospheric degradation of fluorotelomer precursor compounds to form the long-chain PFCAs PFOA (C8) and PFNA (C9).Using the atmospheric chemical transport model GEOS-Chem, which uses assimilated meteorology to simulate the atmospheric transport of trace gas species, we investigate the interaction of the atmospheric formation of PFCAs and the atmospheric transport of their precursor species. Our simulations are a first application of the GEOS-Chem framework to PFCA chemistry. We highlight the importance of the spatial and temporal variability of background atmospheric chemical conditions experienced during transport. We find that yields and formation times of PFOA and PFNA respond differently and strongly to the photochemical conditions of the atmosphere, such as the abundance of NO, HO2, and other photochemical species.

  8. Inhibition of GABA release from slices prepared from several brain regions of rats at various times following a convulsion.

    PubMed Central

    Green, A. R.; Minchin, M. C.; Vincent, N. D.

    1987-01-01

    1 A method is described for the measurement of the K+-evoked release of endogenous gamma-aminobutyric acid (GABA) from slices of rat cortex, hippocampus and striatum. 2 In tissue prepared 30 min following an electroconvulsive shock, K+-evoked GABA release (above basal release) was inhibited by 45% in cortex, 50% in hippocampus and 75% in striatum. A similar inhibition of release was observed with slices prepared from rats in which a convulsion had been induced by flurothyl. There was no change in spontaneous (basal) release following either procedure. 3 An inhibition of K+-evoked endogenous GABA release was also seen in tissue prepared 4 min postictally but not 2 h after the seizure. 4 No difference was observed in the release of [3H]-GABA from preloaded cortical slices prepared from rats given a single electroconvulsive shock. 5 It is proposed that a convulsion results in an inhibition of GABA release and that this inhibition may in turn inhibit GABA synthesis as described in the preceding paper. 6 It is also proposed that changes in the endogenous releasable pool of GABA may not be detected by preloading slices with [3H]-GABA. PMID:3664084

  9. 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

  10. Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica.

    PubMed

    Dulermo, Rémi; Gamboa-Meléndez, Heber; Ledesma-Amaro, Rodrigo; Thévenieau, France; Nicaud, Jean-Marc

    2015-09-01

    Fatty acid (FA) transport and activation have been extensively studied in the model yeast species Saccharomyces cerevisiae but have rarely been examined in oleaginous yeasts, such as Yarrowia lipolytica. Because the latter begins to be used in biodiesel production, understanding its FA transport and activation mechanisms is essential. We found that Y. lipolytica has FA transport and activation proteins similar to those of S. cerevisiae (Faa1p, Pxa1p, Pxa2p, Ant1p) but mechanism of FA peroxisomal transport and activation differs greatly with that of S. cerevisiae. While the ScPxa1p/ScPxa2p heterodimer is essential for growth on long-chain FAs, ΔYlpxa1 ΔYlpxa2 is not impaired for growth on FAs. Meanwhile, ScAnt1p and YlAnt1p are both essential for yeast growth on medium-chain FAs, suggesting they function similarly. Interestingly, we found that the ΔYlpxa1 ΔYlpxa2 ΔYlant1 mutant was unable to grow on short-, medium-, or long-chain FAs, suggesting that YlPxa1p, YlPxa2p, and YlAnt1p belong to two different FA degradation pathways. We also found that YlFaa1p is involved in FA storage in lipid bodies and that FA remobilization largely depended on YlFat1p, YlPxa1p and YlPxa2p. This study is the first to comprehensively examine FA intracellular transport and activation in oleaginous yeast. PMID:25887939

  11. Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition.

    PubMed

    Lakhani, Ronak; Vogel, Kara R; Till, Andreas; Liu, Jingjing; Burnett, Sarah F; Gibson, K Michael; Subramani, Suresh

    2014-04-01

    In addition to key roles in embryonic neurogenesis and myelinogenesis, γ-aminobutyric acid (GABA) serves as the primary inhibitory mammalian neurotransmitter. In yeast, we have identified a new role for GABA that augments activity of the pivotal kinase, Tor1. GABA inhibits the selective autophagy pathways, mitophagy and pexophagy, through Sch9, the homolog of the mammalian kinase, S6K1, leading to oxidative stress, all of which can be mitigated by the Tor1 inhibitor, rapamycin. To confirm these processes in mammals, we examined the succinic semialdehyde dehydrogenase (SSADH)-deficient mouse model that accumulates supraphysiological GABA in the central nervous system and other tissues. Mutant mice displayed increased mitochondrial numbers in the brain and liver, expected with a defect in mitophagy, and morphologically abnormal mitochondria. Administration of rapamycin to these mice reduced mTOR activity, reduced the elevated mitochondrial numbers, and normalized aberrant antioxidant levels. These results confirm a novel role for GABA in cell signaling and highlight potential pathomechanisms and treatments in various human pathologies, including SSADH deficiency, as well as other diseases characterized by elevated levels of GABA. PMID:24578415

  12. Treatment of GABA from Fermented Rice Germ Ameliorates Caffeine-Induced Sleep Disturbance in Mice

    PubMed Central

    Mabunga, Darine Froy N.; Gonzales, Edson Luck T.; Kim, Hee Jin; Choung, Se Young

    2015-01-01

    γ-Aminobutyric acid (GABA), a major inhibitory neurotransmitter in the mammalian central nervous system, is involved in sleep physiology. Caffeine is widely used psychoactive substance known to induce wakefulness and insomnia to its consumers. This study was performed to examine whether GABA extracts from fermented rice germ ameliorates caffeine-induced sleep disturbance in mice, without affecting spontaneous locomotor activity and motor coordination. Indeed, caffeine (10 mg/kg, i.p.) delayed sleep onset and reduced sleep duration of mice. Conversely, rice germ ferment extracts-GABA treatment (10, 30, or 100 mg/kg, p.o.), especially at 100 mg/kg, normalized the sleep disturbance induced by caffeine. In locomotor tests, rice germ ferment extracts-GABA slightly but not significantly reduced the caffeine-induced increase in locomotor activity without affecting motor coordination. Additionally, rice germ ferment extracts-GABA per se did not affect the spontaneous locomotor activity and motor coordination of mice. In conclusion, rice germ ferment extracts-GABA supplementation can counter the sleep disturbance induced by caffeine, without affecting the general locomotor activities of mice. PMID:25995826

  13. Treatment of GABA from Fermented Rice Germ Ameliorates Caffeine-Induced Sleep Disturbance in Mice.

    PubMed

    Mabunga, Darine Froy N; Gonzales, Edson Luck T; Kim, Hee Jin; Choung, Se Young

    2015-05-01

    γ-Aminobutyric acid (GABA), a major inhibitory neurotransmitter in the mammalian central nervous system, is involved in sleep physiology. Caffeine is widely used psychoactive substance known to induce wakefulness and insomnia to its consumers. This study was performed to examine whether GABA extracts from fermented rice germ ameliorates caffeine-induced sleep disturbance in mice, without affecting spontaneous locomotor activity and motor coordination. Indeed, caffeine (10 mg/kg, i.p.) delayed sleep onset and reduced sleep duration of mice. Conversely, rice germ ferment extracts-GABA treatment (10, 30, or 100 mg/kg, p.o.), especially at 100 mg/kg, normalized the sleep disturbance induced by caffeine. In locomotor tests, rice germ ferment extracts-GABA slightly but not significantly reduced the caffeine-induced increase in locomotor activity without affecting motor coordination. Additionally, rice germ ferment extracts-GABA per se did not affect the spontaneous locomotor activity and motor coordination of mice. In conclusion, rice germ ferment extracts-GABA supplementation can counter the sleep disturbance induced by caffeine, without affecting the general locomotor activities of mice. PMID:25995826

  14. GABA regulates synaptic integration of newly generated neurons in the adult brain

    NASA Astrophysics Data System (ADS)

    Ge, Shaoyu; Goh, Eyleen L. K.; Sailor, Kurt A.; Kitabatake, Yasuji; Ming, Guo-Li; Song, Hongjun

    2006-02-01

    Adult neurogenesis, the birth and integration of new neurons from adult neural stem cells, is a striking form of structural plasticity and highlights the regenerative capacity of the adult mammalian brain. Accumulating evidence suggests that neuronal activity regulates adult neurogenesis and that new neurons contribute to specific brain functions. The mechanism that regulates the integration of newly generated neurons into the pre-existing functional circuitry in the adult brain is unknown. Here we show that newborn granule cells in the dentate gyrus of the adult hippocampus are tonically activated by ambient GABA (γ-aminobutyric acid) before being sequentially innervated by GABA- and glutamate-mediated synaptic inputs. GABA, the major inhibitory neurotransmitter in the adult brain, initially exerts an excitatory action on newborn neurons owing to their high cytoplasmic chloride ion content. Conversion of GABA-induced depolarization (excitation) into hyperpolarization (inhibition) in newborn neurons leads to marked defects in their synapse formation and dendritic development in vivo. Our study identifies an essential role for GABA in the synaptic integration of newly generated neurons in the adult brain, and suggests an unexpected mechanism for activity-dependent regulation of adult neurogenesis, in which newborn neurons may sense neuronal network activity through tonic and phasic GABA activation.

  15. Wavelength-Selective One- and Two-Photon Uncaging of GABA

    PubMed Central

    2013-01-01

    We have synthesized photolabile 7-diethylamino coumarin (DEAC) derivatives of γ-aminobutyric acid (GABA). These caged neurotransmitters efficiently release GABA using linear or nonlinear excitation. We used a new DEAC-based caging chromophore that has a vinyl acrylate substituent at the 3-position that shifts the absorption maximum of DEAC to about 450 nm and thus is named “DEAC450”. DEAC450-caged GABA is photolyzed with a quantum yield of 0.39 and is highly soluble and stable in physiological buffer. We found that DEAC450-caged GABA is relatively inactive toward two-photon excitation at 720 nm, so when paired with a nitroaromatic caged glutamate that is efficiently excited at such wavelengths, we could photorelease glutamate and GABA around single spine heads on neurons in brain slices with excellent wavelength selectivity using two- and one-photon photolysis, respectively. Furthermore, we found that DEAC450-caged GABA could be effectively released using two-photon excitation at 900 nm with spatial resolution of about 3 μm. Taken together, our experiments show that the DEAC450 caging chromophore holds great promise for the development of new caged compounds that will enable wavelength-selective, two-color interrogation of neuronal signaling with excellent subcellular resolution. PMID:24304264

  16. Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition

    PubMed Central

    Lakhani, Ronak; Vogel, Kara R; Till, Andreas; Liu, Jingjing; Burnett, Sarah F; Gibson, K Michael; Subramani, Suresh

    2014-01-01

    In addition to key roles in embryonic neurogenesis and myelinogenesis, γ-aminobutyric acid (GABA) serves as the primary inhibitory mammalian neurotransmitter. In yeast, we have identified a new role for GABA that augments activity of the pivotal kinase, Tor1. GABA inhibits the selective autophagy pathways, mitophagy and pexophagy, through Sch9, the homolog of the mammalian kinase, S6K1, leading to oxidative stress, all of which can be mitigated by the Tor1 inhibitor, rapamycin. To confirm these processes in mammals, we examined the succinic semialdehyde dehydrogenase (SSADH)-deficient mouse model that accumulates supraphysiological GABA in the central nervous system and other tissues. Mutant mice displayed increased mitochondrial numbers in the brain and liver, expected with a defect in mitophagy, and morphologically abnormal mitochondria. Administration of rapamycin to these mice reduced mTOR activity, reduced the elevated mitochondrial numbers, and normalized aberrant antioxidant levels. These results confirm a novel role for GABA in cell signaling and highlight potential pathomechanisms and treatments in various human pathologies, including SSADH deficiency, as well as other diseases characterized by elevated levels of GABA. PMID:24578415

  17. Increased Rat Placental Fatty Acid, but Decreased Amino Acid and Glucose Transporters Potentially Modify Intrauterine Programming.

    PubMed

    Nüsken, Eva; Gellhaus, Alexandra; Kühnel, Elisabeth; Swoboda, Isabelle; Wohlfarth, Maria; Vohlen, Christina; Schneider, Holm; Dötsch, Jörg; Nüsken, Kai-Dietrich

    2016-07-01

    Regulation of placental nutrient transport significantly affects fetal development and may modify intrauterine growth restriction (IUGR) and fetal programming. We hypothesized that placental nutrient transporters are differentially affected both by utero-placental insufficiency and prenatal surgical stress. Pregnant rats underwent bilateral uterine artery and vein ligation (LIG), sham operation (SOP) or no operation (controls, C) on gestational day E19. Placentas were obtained by caesarean section 4 h (LIG, n=20 placentas; SOP, n=24; C, n=12), 24 h (LIG, n=28; SOP, n=20; C, n=12) and 72 h (LIG, n=20; SOP, n=20; C, n=24) after surgery. Gene and protein expression of placental nutrient transporters for fatty acids (h-FABP, CD36), amino acids (SNAT1, SNAT2) and glucose (GLUT-1, Connexin 26) were examined by qRT-PCR, western blot and immunohistochemistry. Interestingly, the mean protein expression of h-FABP was doubled in placentas of LIG and SOP animals 4, 24 (SOP significant) and 72 h (SOP significant) after surgery. CD36 protein was significantly increased in LIG after 72 h. SNAT1 and SNAT2 protein and gene expressions were significantly reduced in LIG and SOP after 24 h. Further significantly reduced proteins were GLUT-1 in LIG (4 h, 72 h) and SOP (24 h), and Connexin 26 in LIG (72 h). In conclusion, placental nutrient transporters are differentially affected both by reduced blood flow and stress, probably modifying the already disturbed intrauterine milieu and contributing to IUGR and fetal programming. Increased fatty acid transport capacity may affect energy metabolism and could be a compensatory reaction with positive effects on brain development. J. Cell. Biochem. 117: 1594-1603, 2016. © 2015 Wiley Periodicals, Inc. PMID:26590355

  18. Regulation of hepatic bile acid transporters Ntcp and Bsep expression

    PubMed Central

    Cheng, Xingguo; Buckley, David; Klaassen, Curtis D.

    2009-01-01

    Sodium-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) are two key transporters for hepatic bile acid uptake and excretion. Alterations in Ntcp and Bsep expression have been reported in pathophysiological conditions. In the present study, the effects of age, gender, and various chemicals on the regulation of these two transporters were characterized in mice. Ntcp and Bsep mRNA levels in mouse liver were low in the fetus, but increased to its highest expression at parturition. After birth, mouse Ntcp and Bsep mRNA decreased by more than 50%, and then gradually increased to adult levels by day 30. Expression of mouse Ntcp mRNA and protein exhibit higher levels in female than male livers, which is consistent with the trend of human NTCP mRNA expression between men and women. No gender difference exists in BSEP/Bsep expression in human and mouse livers. Hormone replacements conducted in gonadectomized, hypophysectomized, and lit/lit mice indicate that female-predominant Ntcp expression in mouse liver is due to the inhibitory effect of male-pattern GH secretion, but not sex hormones. Ntcp and Bsep expression are in general resistant to induction by a large battery of microsomal enzyme inducers. Administration of cholestyramine increased Ntcp, whereas chenodeoxycholic acid increased Bsep mRNA expression. In silico analysis indicates that female-predominant mouse and human Ntcp/NTCP expression may be due to GH. In conclusion, mouse Ntcp and Bsep are regulated by age, gender, cholestyramine, and bile acid, but resistant to induction by most microsomal enzyme inducers. PMID:17897632

  19. Transport of the two natural auxins, indole-3-butyric acid and indole-3-acetic acid, in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Rashotte, Aaron M.; Poupart, Julie; Waddell, Candace S.; Muday, Gloria K.; Brown, C. S. (Principal Investigator)

    2003-01-01

    Polar transport of the natural auxin indole-3-acetic acid (IAA) is important in a number of plant developmental processes. However, few studies have investigated the polar transport of other endogenous auxins, such as indole-3-butyric acid (IBA), in Arabidopsis. This study details the similarities and differences between IBA and IAA transport in several tissues of Arabidopsis. In the inflorescence axis, no significant IBA movement was detected, whereas IAA is transported in a basipetal direction from the meristem tip. In young seedlings, both IBA and IAA were transported only in a basipetal direction in the hypocotyl. In roots, both auxins moved in two distinct polarities and in specific tissues. The kinetics of IBA and IAA transport appear similar, with transport rates of 8 to 10 mm per hour. In addition, IBA transport, like IAA transport, is saturable at high concentrations of auxin, suggesting that IBA transport is protein mediated. Interestingly, IAA efflux inhibitors and mutations in genes encoding putative IAA transport proteins reduce IAA transport but do not alter IBA movement, suggesting that different auxin transport protein complexes are likely to mediate IBA and IAA transport. Finally, the physiological effects of IBA and IAA on hypocotyl elongation under several light conditions were examined and analyzed in the context of the differences in IBA and IAA transport. Together, these results present a detailed picture of IBA transport and provide the basis for a better understanding of the transport of these two endogenous auxins.

  20. A Plasma Membrane Association Module in Yeast Amino Acid Transporters.

    PubMed

    Popov-Čeleketić, Dušan; Bianchi, Frans; Ruiz, Stephanie J; Meutiawati, Febrina; Poolman, Bert

    2016-07-29

    Amino acid permeases (AAPs) in the plasma membrane (PM) of Saccharomyces cerevisiae are responsible for the uptake of amino acids and involved in regulation of their cellular levels. Here, we report on a strong and complex module for PM association found in the C-terminal tail of AAPs. Using in silico analyses and mutational studies we found that the C-terminal sequences of Gap1, Bap2, Hip1, Tat1, Tat2, Mmp1, Sam3, Agp1, and Gnp1 are about 50 residues long, associate with the PM, and have features that discriminate them from the termini of organellar amino acid transporters. We show that this sequence (named PMasseq) contains an amphipathic α-helix and the FWC signature, which is palmitoylated by palmitoyltransferase Pfa4. Variations of PMasseq, found in different AAPs, lead to different mobilities and localization patterns, whereas the disruption of the sequence has an adverse effect on cell viability. We propose that PMasseq modulates the function and localization of AAPs along the PM. PMasseq is one of the most complex protein signals for plasma membrane association across species and can be used as a delivery vehicle for the PM. PMID:27226538

  1. Amino Acid Transporters and Release of Hydrophobic Amino Acids in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

    PubMed Central

    Pernil, Rafael; Picossi, Silvia; Herrero, Antonia; Flores, Enrique; Mariscal, Vicente

    2015-01-01

    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can use inorganic compounds such as nitrate or ammonium as nitrogen sources. In the absence of combined nitrogen, it can fix N2 in differentiated cells called heterocysts. Anabaena also shows substantial activities of amino acid uptake, and three ABC-type transporters for amino acids have been previously characterized. Seven new loci encoding predicted amino acid transporters were identified in the Anabaena genomic sequence and inactivated. Two of them were involved in amino acid uptake. Locus alr2535-alr2541 encodes the elements of a hydrophobic amino acid ABC-type transporter that is mainly involved in the uptake of glycine. ORF all0342 encodes a putative transporter from the dicarboxylate/amino acid:cation symporter (DAACS) family whose inactivation resulted in an increased uptake of a broad range of amino acids. An assay to study amino acid release from Anabaena filaments to the external medium was set up. Net release of the alanine analogue α-aminoisobutyric acid (AIB) was observed when transport system N-I (a hydrophobic amino acid ABC-type transporter) was engaged in the uptake of a specific substrate. The rate of AIB release was directly proportional to the intracellular AIB concentration, suggesting leakage from the cells by diffusion. PMID:25915115

  2. Amino Acid Transporters and Release of Hydrophobic Amino Acids in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

    PubMed

    Pernil, Rafael; Picossi, Silvia; Herrero, Antonia; Flores, Enrique; Mariscal, Vicente

    2015-01-01

    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can use inorganic compounds such as nitrate or ammonium as nitrogen sources. In the absence of combined nitrogen, it can fix N2 in differentiated cells called heterocysts. Anabaena also shows substantial activities of amino acid uptake, and three ABC-type transporters for amino acids have been previously characterized. Seven new loci encoding predicted amino acid transporters were identified in the Anabaena genomic sequence and inactivated. Two of them were involved in amino acid uptake. Locus alr2535-alr2541 encodes the elements of a hydrophobic amino acid ABC-type transporter that is mainly involved in the uptake of glycine. ORF all0342 encodes a putative transporter from the dicarboxylate/amino acid:cation symporter (DAACS) family whose inactivation resulted in an increased uptake of a broad range of amino acids. An assay to study amino acid release from Anabaena filaments to the external medium was set up. Net release of the alanine analogue α-aminoisobutyric acid (AIB) was observed when transport system N-I (a hydrophobic amino acid ABC-type transporter) was engaged in the uptake of a specific substrate. The rate of AIB release was directly proportional to the intracellular AIB concentration, suggesting leakage from the cells by diffusion. PMID:25915115

  3. Heteromeric amino acid transporters. In search of the molecular bases of transport cycle mechanisms.

    PubMed

    Palacín, Manuel; Errasti-Murugarren, Ekaitz; Rosell, Albert

    2016-06-15

    Heteromeric amino acid transporters (HATs) are relevant targets for structural studies. On the one hand, HATs are involved in inherited and acquired human pathologies. On the other hand, these molecules are the only known examples of solute transporters composed of two subunits (heavy and light) linked by a disulfide bridge. Unfortunately, structural knowledge of HATs is scarce and limited to the atomic structure of the ectodomain of a heavy subunit (human 4F2hc-ED) and distant prokaryotic homologues of the light subunits that share a LeuT-fold. Recent data on human 4F2hc/LAT2 at nanometer resolution revealed 4F2hc-ED positioned on top of the external loops of the light subunit LAT2. Improved resolution of the structure of HATs, combined with conformational studies, is essential to establish the structural bases for light subunit recognition and to evaluate the functional relevance of heavy and light subunit interactions for the amino acid transport cycle. PMID:27284037

  4. {gamma}-aminobutyric acid{sub A} (GABA{sub A}) receptor regulates ERK1/2 phosphorylation in rat hippocampus in high doses of Methyl Tert-Butyl Ether (MTBE)-induced impairment of spatial memory

    SciTech Connect

    Zheng Gang; Zhang Wenbin; Zhang Yun; Chen Yaoming; Liu Mingchao; Yao Ting; Yang Yanxia; Zhao Fang; Li Jingxia; Huang Chuanshu; Luo Wenjing Chen Jingyuan

    2009-04-15

    Experimental and occupational exposure to Methyl Tert-Butyl Ether (MTBE) has been reported to induce neurotoxicological and neurobehavioral effects, such as headache, nausea, dizziness, and disorientation, etc. However, the molecular mechanisms involved in MTBE-induced neurotoxicity are still not well understood. In the present study, we investigated the effects of MTBE on spatial memory and the expression and function of GABA{sub A} receptor in the hippocampus. Our results demonstrated that intraventricular injection of MTBE impaired the performance of the rats in a Morris water maze task, and significantly increased the expression of GABA{sub A} receptor {alpha}1 subunit in the hippocampus. The phosphorylation of ERK1/2 decreased after the MTBE injection. Furthermore, the decreased ability of learning and the reduction of phosphorylated ERK1/2 level of the MTBE-treated rats was partly reversed by bicuculline injected 30 min before the training. These results suggested that MTBE exposure could result in impaired spatial memory. GABA{sub A} receptor may play an important role in the MTBE-induced impairment of learning and memory by regulating the phosphorylation of ERK in the hippocampus.

  5. Bipotential precursors of putative fibrous astrocytes and oligodendrocytes in rat cerebellar cultures express distinct surface features and neuron-like. gamma. -aminobutyric acid transport

    SciTech Connect

    Levi, G.; Gallo, V.; Ciotti, T.

    1986-03-01

    When postnatal rat cerebellar cells were cultured in a chemically defined, serum-free medium, the only type of astrocyte present was unable to accumulate ..gamma..-(/sup 3/H)aminobutyric acid (GABA), did not express surface antigens recognized by two monoclonal antibodies, A2B5 and LB1, and showed minimal proliferation. In these cultures, nonneuronal A2B5/sup +/, LB1/sup +/ stellate cells exhibiting neuron-like (/sup 3/H)GABA uptake formed cell colonies of increasing size and were GFAP/sup -/. After about one week of culturing, the A2B5/sup +/, LB1/sup +/, GABA-uptake positive cell groups became galactocerebroside (GalCer) positive. Immunocytolysis of the A2B5/sup +/ cells at 3 and 4 days in vitro prevented the appearance of the A2B5/sup +/, LB1/sup +/, GABA-uptake positive cell colonies, and also of the GalCer/sup +/ cell groups. If 10% (vol/vol) fetal calf serum was added to 6-day cultures, the A2B5/sup +/, LB1/sup +/, GABA-uptake positive cell groups expressed GFAP and not GalCer. If the serum was added to the cultures 2 days after lysing the A2B5/sup +/ cells, only A2B5/sup -/, LB1/sup -/, GABA-uptake negative astrocytes proliferated. It is concluded that the putative fibrous astrocytes previously described in serum-containing cultures derive from bipotential precursors that differentiate into oligodendrocytes (GalCer/sup +/) in serum-free medium or into astrocytes (GFAP/sup +/) in the presence of serum, while the epithelioid A2B5/sup -/, LB1/sup -/, GABA-uptake negative astrocytes originate from a different precursor not yet identified.

  6. Comparison between the modes of action of novel meta-diamide and macrocyclic lactone insecticides on the RDL GABA receptor.

    PubMed

    Nakao, Toshifumi; Banba, Shinichi; Hirase, Kangetsu

    2015-05-01

    Macrocyclic lactones, avermectins, and milbemycins are widely used to control arthropods, nematodes, and endo- and ectoparasites in livestock and pets. Their main targets are glutamate-gated chloride channels. Furthermore, macrocyclic lactones reportedly interact with insect RDL γ-aminobutyric acid (GABA) receptors, but their modes of action on insect RDL GABA receptors remain unknown. In this study, we attempted to better understand the modes of action of macrocyclic lactones on RDL GABA receptors. We observed that ivermectin and milbemectin behaved as allosteric agonists of the Drosophila RDL GABA receptor. G336A, G336S, and G336T mutations had profound effects on the activities of ivermectin and milbemectin, and a G336M mutation abolished the allosteric agonist and antagonist activities of these macrocyclic lactones. These results suggest that G336 in TM3 of the Drosophila RDL GABA receptor is important for the binding of macrocyclic lactones. Recently, it has been suggested that a novel RDL GABA receptor antagonist, 3-benzamido-N-(2-bromo-4-perfluoroisopropyl-6-(trifluoromethyl)phenyl)-2-fluorobenzamide (meta-diamide 7), binds to the transmembrane intersubunit pocket near G336 in the Drosophila RDL GABA receptor. Thus, we compared the effects of mutations around G336 and A302 mutations in TM2 on the activities of macrocyclic lactone and meta-diamide 7. The effects of L281C, V340Q, V340N, A302S, and A302N mutations on the activity of meta-diamide 7 differed from those on ivermectin and milbemectin. Molecular modeling studies showed that macrocyclic lactones docked in the intersubunit pocket near G336 in the Drosophila RDL GABA receptor in the open state. In contrast, meta-diamide 7 docked into the Drosophila RDL GABA receptor in the closed state. This suggests that the modes of action of macrocyclic lactone binding to the wild-type Drosophila RDL GABA receptor differ from those of meta-diamide binding. PMID:25987227

  7. 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

  8. Phenotyping GABA transaminase deficiency: a case description and literature review.

    PubMed

    Louro, Pedro; Ramos, Lina; Robalo, Conceição; Cancelinha, Cândida; Dinis, Alexandra; Veiga, Ricardo; Pina, Raquel; Rebelo, Olinda; Pop, Ana; Diogo, Luísa; Salomons, Gajja S; Garcia, Paula

    2016-09-01

    Gamma-aminobutyric acid transaminase (GABA-T) deficiency is an autosomal recessive disorder reported in only three unrelated families. It is caused by mutations in the ABAT gene, which encodes 4-aminobutyrate transaminase, an enzyme of GABA catabolism and mitochondrial nucleoside salvage. We report the case of a boy, deceased at 12 months of age, with early-onset epileptic encephalopathy, severe psychomotor retardation, hypotonia, lower-limb hyporeflexia, central hypoventilation, and rapid increase in weight and, to a lesser rate, length and head circumference. He presented signs of premature pubarche, thermal instability, and water-electrolyte imbalance. Serum total testosterone was elevated (43.3 ng/dl; normal range <16), as well as serum growth hormone (7.7 ng/ml; normal range <1). Brain magnetic resonance imaging (MRI) showed decreased myelination and generalized brain atrophy, later confirmed by post-mortem examination. ABAT gene sequencing was performed post-mortem, identifying a homozygous variant c.888G > T (p.Gln296His),not previously described. In vitro analysis concluded that this variant is pathogenic. The clinical features of this patient are similar to those reported so far in GABA-T deficiency. However, distinct mutations may have a different effect on enzymatic activity, which potentially could lead to a variable clinical outcome. Clinical investigation aiming for a diagnosis should not end with the patient's death, as it may allow a more precise genetic counselling for the family. PMID:27376954

  9. Phenibut (beta-phenyl-GABA): a tranquilizer and nootropic drug.

    PubMed

    Lapin, I

    2001-01-01

    Phenibut (beta-phenyl-gamma-aminobutyric acid HCl) is a neuropsychotropic drug that was discovered and introduced into clinical practice in Russia in the 1960s. It has anxiolytic and nootropic (cognition enhancing) effects. It acts as a GABA-mimetic, primarily at GABA(B) and, to some extent, at GABA(A) receptors. It also stimulates dopamine receptors and antagonizes beta-phenethylamine (PEA), a putative endogenous anxiogenic. The psychopharmacological activity of phenibut is similar to that of baclofen, a p-Cl-derivative of phenibut. This article reviews the structure-activity relationship of phenibut and its derivatives. Emphasis is placed on the importance of the position of the phenyl ring, the role of the carboxyl group, and the activity of optical isomers. Comparison of phenibut with piracetam and diazepam reveals similarities and differences in their pharmacological and clinical effects. Phenibut is widely used in Russia to relieve tension, anxiety, and fear, to improve sleep in psychosomatic or neurotic patients; as well as a pre- or post-operative medication. It is also used in the therapy of disorders characterized by asthenia and depression, as well as in post-traumatic stress, stuttering and vestibular disorders. PMID:11830761

  10. Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases

    PubMed Central

    Yuan, Hongjie; Low, Chian-Ming; Moody, Olivia A.; Jenkins, Andrew

    2015-01-01

    The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-d-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases. PMID:25904555

  11. Induction of amino acid transporters expression by endurance exercise in rat skeletal muscle

    SciTech Connect

    Murakami, Taro Yoshinaga, Mariko

    2013-10-04

    Highlights: •Regulation of amino acid transporter expression in working muscle remains unclear. •Expression of amino acid transporters for leucine were induced by a bout of exercise. •Requirement of leucine in muscle cells might regulate expression of its transporters. •This information is beneficial for understanding the muscle remodeling by exercise. -- Abstract: We here investigated whether an acute bout of endurance exercise would induce the expression of amino acid transporters that regulate leucine transport across plasma and lysosomal membranes in rat skeletal muscle. Rats ran on a motor-driven treadmill at a speed of 28 m/min for 90 min. Immediately after the exercise, we observed that expression of mRNAs encoding L-type amino acid transporter 1 (LAT1) and CD98 was induced in the gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. Sodium-coupled neutral amino acid transporter 2 (SNAT2) mRNA was also induced by the exercise in those three muscles. Expression of proton-assisted amino acid transporter 1 (PAT1) mRNA was slightly but not significantly induced by a single bout of exercise in soleus and EDL muscles. Exercise-induced mRNA expression of these amino acid transporters appeared to be attenuated by repeated bouts of the exercise. These results suggested that the expression of amino acid transporters for leucine may be induced in response to an increase in the requirement for this amino acid in the cells of working skeletal muscles.

  12. ABC transporter AtABCG25 is involved in abscisic acid transport and responses

    PubMed Central

    Kuromori, Takashi; Miyaji, Takaaki; Yabuuchi, Hikaru; Shimizu, Hidetada; Sugimoto, Eriko; Kamiya, Asako; Moriyama, Yoshinori; Shinozaki, Kazuo

    2010-01-01

    Abscisic acid (ABA) is one of the most important phytohormones involved in abiotic stress responses, seed maturation, germination, and senescence. ABA is predominantly produced in vascular tissues and exerts hormonal responses in various cells, including guard cells. Although ABA responses require extrusion of ABA from ABA-producing cells in an intercellular ABA signaling pathway, the transport mechanisms of ABA through the plasma membrane remain unknown. Here we isolated an ATP-binding cassette (ABC) transporter gene, AtABCG25, from Arabidopsis by genetically screening for ABA sensitivity. AtABCG25 was expressed mainly in vascular tissues. The fluorescent protein-fused AtABCG25 was localized at the plasma membrane in plant cells. In membrane vesicles derived from AtABCG25-expressing insect cells, AtABCG25 exhibited ATP-dependent ABA transport. The AtABCG25-overexpressing plants showed higher leaf temperatures, implying an influence on stomatal regulation. These results strongly suggest that AtABCG25 is an exporter of ABA and is involved in the intercellular ABA signaling pathway. The presence of the ABA transport mechanism sheds light on the active control of multicellular ABA responses to environmental stresses among plant cells. PMID:20133881

  13. The impact of GABA in harpin-elicited biotic stress responses in Nicotiana tabaccum.

    PubMed

    Dimlioğlu, Gizem; Daş, Zeycan Akcan; Bor, Melike; Özdemir, Filiz; Türkan, İsmail

    2015-09-01

    Harpin is a bacterial elicitor protein that was first isolated from Erwinia amylovora. Infiltration of this elicitor into the leaves of plants activates systemic acquired resistance against a variety of plant pathogens via the salicyclic acid defense pathway. The non-protein amino acid, neurotransmission inhibitor molecule of mammals-GABA- is found in all organisms and is known to be an important component of stress responses in plants. We hypothesized a possible interaction between harpin-induced defense responses and GABA shunt. Therefore, we conducted experiments on harpin-infiltrated tobacco and analyzed the components of GABA shunt in relation to growth, photosynthesis and H2O2 levels. RGR, RWC and photosynthetic efficiency were all affected in harpin-infiltrated tobacco leaves, but the rate of decline was more remarkable on RGR. H2O2 levels showed significant difference on 7 days after harpin infiltration when the necrotic lesions were also visible. GABA accumulation was increased and glutamate levels were decreased parallel to the differences in GDH and GAD enzyme activities, especially on days 5 and 7 of harpin infiltration. Transcript abundance of GDH and GAD encoding genes were differentially regulated in harpin-infiltrated leaves as compared to that of control and mock groups. In the present study, for the first time we showed a relationship between harpin-elicited responses and GABA in tobacco that was not mediated by H2O2 accumulation. Harpin infiltration significantly induced the first components of the GABA shunt such as GDH, GAD, glutamate and GABA in tobacco. PMID:26432406

  14. Impact of Microbial Growth on Subsurface Perfluoroalkyl Acid Transport

    NASA Astrophysics Data System (ADS)

    Weathers, T. S.; Higgins, C. P.; Sharp, J.

    2014-12-01

    The fate and transport of poly and perfluoroalkyl substances (PFASs) in the presence of active microbial communities has not been widely investigated. These emerging contaminants are commonly utilized in aqueous film-forming foams (AFFF) and have often been detected in groundwater. This study explores the transport of a suite of perfluorocarboxylic acids and perfluoroalkylsulfonates, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), in microbially active settings. Single point organic carbon normalized sorption coefficients derived by exposing inactive cellular material to PFASs result in more than an order of magnitude increase in sorption compared to soil organic carbon sorption coefficients found in literature. For example, the sorption coefficients for PFOS are 4.05±0.07 L/kg and 2.80±0.08 L/kg for cellular organic carbon and soil organic carbon respectively. This increase in sorption, coupled with enhanced extracellular polymeric substance production observed during growth of a common hydrocarbon degrading soil microbe exposed to source-level concentrations of PFASs (10 mg/L of 11 analytes, 110 mg/L total) may result in PFAS retardation in situ. To address the upscaling of this phenomenon, flow-through columns packed with low-organic carbon sediment and biostimulated with 10 mg/L glucose were exposed to PFAS concentrations from 15 μg/L to 10 mg/L of each 11 analytes. Breakthrough and tailing of each analyte was measured and modeled with Hydrus-1D to explore sorption coefficients over time for microbially active columns.

  15. Acid-base transport by the renal proximal tubule

    PubMed Central

    Skelton, Lara A.; Boron, Walter F.; Zhou, Yuehan

    2015-01-01

    Each day, the kidneys filter 180 L of blood plasma, equating to some 4,300 mmol of the major blood buffer, bicarbonate (HCO3−). The glomerular filtrate enters the lumen of the proximal tubule (PT), and the majority of filtered HCO3− is reclaimed along the early (S1) and convoluted (S2) portions of the PT in a manner coupled to the secretion of H+ into the lumen. The PT also uses the secreted H+ to titrate non-HCO3− buffers in the lumen, in the process creating “new HCO3−” for transport into the blood. Thus, the PT – along with more distal renal segments – is largely responsible for regulating plasma [HCO3−]. In this review we first focus on the milestone discoveries over the past 50+ years that define the mechanism and regulation of acid-base transport by the proximal tubule. Further on in the review, we will summarize research still in progress from our laboratory, work that addresses the problem of how the PT is able to finely adapt to acid–base disturbances by rapidly sensing changes in basolateral levels of HCO3− and CO2 (but not pH), and thereby to exert tight control over the acid–base composition of the blood plasma. PMID:21170887

  16. Butyric acid increases transepithelial transport of ferulic acid through upregulation of the monocarboxylate transporters SLC16A1 (MCT1) and SLC16A3 (MCT4).

    PubMed

    Ziegler, Kerstin; Kerimi, Asimina; Poquet, Laure; Williamson, Gary

    2016-06-01

    Ferulic acid is released by microbial hydrolysis in the colon, where butyric acid, a major by-product of fermentation, constitutes the main energy source for colonic enterocytes. We investigated how varying concentrations of this short chain fatty acid may influence the absorption of the phenolic acid. Chronic treatment of Caco-2 cells with butyric acid resulted in increased mRNA and protein abundance of the monocarboxylate transporters SLC16A1 (MCT1) and SLC16A3 (MCT4), previously proposed to facilitate ferulic acid absorption in addition to passive diffusion. Short term incubation with butyric acid only led to upregulation of MCT4 while both conditions increased transepithelial transport of ferulic acid in the apical to basolateral, but not basolateral to apical, direction. Chronic treatment also elevated intracellular concentrations of ferulic acid, which in turn gave rise to increased concentrations of ferulic acid metabolites. Immunofluorescence staining of cells revealed uniform distribution of MCT1 protein in the cell membrane, whereas MCT4 was only detected in the lateral plasma membrane sections of Caco-2 cells. We therefore propose that MCT1 may be acting as an uptake transporter and MCT4 as an efflux system across the basolateral membrane for ferulic acid, and that this process is stimulated by butyric acid. PMID:26854723

  17. GABA and glycine in the developing brain.

    PubMed

    Ito, Susumu

    2016-09-01

    GABA and glycine are major inhibitory neurotransmitters in the CNS and act on receptors coupled to chloride channels. During early developmental periods, both GABA and glycine depolarize membrane potentials due to the relatively high intracellular Cl(-) concentration. Therefore, they can act as excitatory neurotransmitters. GABA and glycine are involved in spontaneous neural network activities in the immature CNS such as giant depolarizing potentials (GDPs) in neonatal hippocampal neurons, which are generated by the synchronous activity of GABAergic interneurons and glutamatergic principal neurons. GDPs and GDP-like activities in the developing brains are thought to be important for the activity-dependent functiogenesis through Ca(2+) influx and/or other intracellular signaling pathways activated by depolarization or stimulation of metabotropic receptors. However, if GABA and glycine do not shift from excitatory to inhibitory neurotransmitters at the birth and in maturation, it may result in neural disorders including autism spectrum disorders. PMID:26951057

  18. Possible involvement of lipoic acid in binding protein-dependent transport systems in Escherichia coli.

    PubMed

    Richarme, G

    1985-04-01

    We describe the properties of the binding protein dependent-transport of ribose, galactose, and maltose and of the lactose permease, and the phosphoenolpyruvate-glucose phosphotransferase transport systems in a strain of Escherichia coli which is deficient in the synthesis of lipoic acid, a cofactor involved in alpha-keto acid dehydrogenation. Such a strain can grow in the absence of lipoic acid in minimal medium supplemented with acetate and succinate. Although the lactose permease and the phosphoenolypyruvate-glucose phosphotransferase are not affected by lipoic acid deprivation, the binding protein-dependent transports are reduced by 70% in conditions of lipoic acid deprivation when compared with their activity in conditions of lipoic acid supply. The remaining transport is not affected by arsenate but is inhibited by the uncoupler carbonylcyanide-m-chlorophenylhydrazone; however the lipoic acid-dependent transport is completely inhibited by arsenate and only weakly inhibited by carbonylcyanide-m-chlorophenylhydrazone. The known inhibitor of alpha-keto acid dehydrogenases, 5-methoxyindole-2-carboxylic acid, completely inhibits all binding protein-dependent transports whether in conditions of lipoic supply or deprivation; the results suggest a possible relation between binding protein-dependent transport and alpha-keto acid dehydrogenases and shed light on the inhibition of these transports by arsenicals and uncouplers. PMID:3920206

  19. Transport and signaling via the amino acid binding site of the yeast Gap1 amino acid transceptor.

    PubMed

    Van Zeebroeck, Griet; Bonini, Beatriz Monge; Versele, Matthias; Thevelein, Johan M

    2009-01-01

    Transporter-related nutrient sensors, called transceptors, mediate nutrient activation of signaling pathways through the plasma membrane. The mechanism of action of transporting and nontransporting transceptors is unknown. We have screened 319 amino acid analogs to identify compounds that act on Gap1, a transporting amino acid transceptor in yeast that triggers activation of the protein kinase A pathway. We identified competitive and noncompetitive inhibitors of transport, either with or without agonist action for signaling, including nontransported agonists. Using substituted cysteine accessibility method (SCAM) analysis, we identified Ser388 and Val389 as being exposed into the amino acid binding site, and we show that agonist action for signaling uses the same binding site as used for transport. Our results provide the first insight, to our knowledge, into the mechanism of action of transceptors. They indicate that signaling requires a ligand-induced specific conformational change that may be part of but does not require the complete transport cycle. PMID:19060912

  20. Deletion of the γ-aminobutyric acid transporter 2 (GAT2 and SLC6A13) gene in mice leads to changes in liver and brain taurine contents.

    PubMed

    Zhou, Yun; Holmseth, Silvia; Guo, Caiying; Hassel, Bjørnar; Höfner, Georg; Huitfeldt, Henrik S; Wanner, Klaus T; Danbolt, Niels C

    2012-10-12

    The GABA transporters (GAT1, GAT2, GAT3, and BGT1) have mostly been discussed in relation to their potential roles in controlling the action of transmitter GABA in the nervous system. We have generated the first mice lacking the GAT2 (slc6a13) gene. Deletion of GAT2 (both mRNA and protein) neither affected growth, fertility, nor life span under nonchallenging rearing conditions. Immunocytochemistry showed that the GAT2 protein was predominantly expressed in the plasma membranes of periportal hepatocytes and in the basolateral membranes of proximal tubules in the renal cortex. This was validated by processing tissue from wild-type and knockout mice in parallel. Deletion of GAT2 reduced liver taurine levels by 50%, without affecting the expression of the taurine transporter TAUT. These results suggest an important role for GAT2 in taurine uptake from portal blood into liver. In support of this notion, GAT2-transfected HEK293 cells transported [(3)H]taurine. Furthermore, most of the uptake of [(3)H]GABA by cultured rat hepatocytes was due to GAT2, and this uptake was inhibited by taurine. GAT2 was not detected in brain parenchyma proper, excluding a role in GABA inactivation. It was, however, expressed in the leptomeninges and in a subpopulation of brain blood vessels. Deletion of GAT2 increased brain taurine levels by 20%, suggesting a taurine-exporting role for GAT2 in the brain. PMID:22896705

  1. Unexpected Photo-instability of 2,6-Sulfonamide-Substituted BODIPYs and Its Application to Caged GABA.

    PubMed

    Takeda, Aoi; Komatsu, Toru; Nomura, Hiroshi; Naka, Masamitsu; Matsuki, Norio; Ikegaya, Yuji; Terai, Takuya; Ueno, Tasuku; Hanaoka, Kenjiro; Nagano, Tetsuo; Urano, Yasuteru

    2016-07-01

    Investigation of the unexpected photo-instability of 2,6-sulfonamide-substituted derivatives of the boron dipyrromethene (BODIPY) fluorophore led to the discovery of a photoreaction accompanied by multiple bond scissions. We characterized the photoproducts and utilized the photoreaction to design a caged γ-aminobutyric acid (GABA) derivative that can release GABA upon irradiation in the visible range (>450 nm). This allowed us to stimulate neural cells in mouse brain slices. PMID:27038199

  2. The glutamine-glutamate/GABA cycle: function, regional differences in glutamate and GABA production and effects of interference with GABA metabolism.

    PubMed

    Walls, Anne B; Waagepetersen, Helle S; Bak, Lasse K; Schousboe, Arne; Sonnewald, Ursula

    2015-02-01

    The operation of a glutamine-glutamate/GABA cycle in the brain consisting of the transfer of glutamine from astrocytes to neurons and neurotransmitter glutamate or GABA from neurons to astrocytes is a well-known concept. In neurons, glutamine is not only used for energy production and protein synthesis, as in other cells, but is also an essential precursor for biosynthesis of amino acid neurotransmitters. An excellent tool for the study of glutamine transfer from astrocytes to neurons is [(14)C]acetate or [(13)C]acetate and the glial specific enzyme inhibitors, i.e. the glutamine synthetase inhibitor methionine sulfoximine and the tricarboxylic acid cycle (aconitase) inhibitors fluoro-acetate and -citrate. Acetate is metabolized exclusively by glial cells, and [(13)C]acetate is thus capable when used in combination with magnetic resonance spectroscopy or mass spectrometry, to provide information about glutamine transfer. The present review will give information about glutamine trafficking and the tools used to map it as exemplified by discussions of published work employing brain cell cultures as well as intact animals. It will be documented that considerably more glutamine is transferred from astrocytes to glutamatergic than to GABAergic neurons. However, glutamine does have an important role in GABAergic neurons despite their capability of re-utilizing their neurotransmitter by re-uptake. PMID:25380696

  3. The D-amino acid transport by the invertebrate SLC6 transporters KAAT1 and CAATCH1 from Manduca sexta.

    PubMed

    Vollero, Alessandra; Imperiali, Francesca G; Cinquetti, Raffaella; Margheritis, Eleonora; Peres, Antonio; Bossi, Elena

    2016-02-01

    The ability of the SLC6 family members, the insect neutral amino acid cotransporter KAAT1(K(+)-coupled amino acid transporter 1) and its homologous CAATCH1(cation anion activated amino acid transporter/channel), to transport D-amino acids has been investigated through heterologous expression in Xenopus laevis oocytes and electrophysiological techniques. In the presence of D-isomers of leucine, serine, and proline, the msKAAT1 generates inward, transport-associated, currents with variable relative potencies, depending on the driving ion Na(+) or K(+). Higher concentrations of D-leucine (≥1 mmol/L) give rise to an anomalous response that suggests the existence of a second binding site with inhibitory action on the transport process. msCAATCH1 is also able to transport the D-amino acids tested, including D-leucine, whereas L-leucine acts as a blocker. A similar behavior is exhibited by the KAAT1 mutant S308T, confirming the relevance of the residue in this position in L-leucine binding and the different interaction of D-leucine with residues involved in transport mechanism. D-leucine and D-serine on various vertebrate orthologs B(0)AT1 (SLC6A19) elicited only a very small current and singular behavior was not observed, indicating that it is specific of the insect neutral amino acid transporters. These findings highlight the relevance of D-amino acid absorption in the insect nutrition and metabolism and may provide new evidences in the molecular transport mechanism of SLC6 family. PMID:26884475

  4. Fatty acid transport protein 1 can compensate for fatty acid transport protein 4 in the developing mouse epidermis.

    PubMed

    Lin, Meei-Hua; Miner, Jeffrey H

    2015-02-01

    Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very-long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective barrier; they die neonatally because of dehydration and restricted movements. Mutations in SLC27A4, the gene encoding FATP4, cause ichthyosis prematurity syndrome (IPS), characterized by premature birth, respiratory distress, and edematous skin with severe ichthyotic scaling. Symptoms of surviving patients become mild, although atopic manifestations are common. We previously showed that suprabasal keratinocyte expression of a Fatp4 transgene in Fatp4 mutant skin rescues the lethality and ameliorates the skin phenotype. Here we tested the hypothesis that FATP1, the closest FATP4 homolog, can compensate for the lack of FATP4 in our mouse model of IPS, as it might do postnatally in IPS patients. Transgenic expression of FATP1 in suprabasal keratinocytes rescued the phenotype of Fatp4 mutants, and FATP1 sorted to the same intracellular organelles as endogenous FATP4. Thus, FATP1 and FATP4 likely have overlapping substrate specificities, enzymatic activities, and biological functions. These results suggest that increasing expression of FATP1 in suprabasal keratinocytes could normalize the skin of IPS patients and perhaps prevent the atopic manifestations. PMID:25184958

  5. Comparative mapping of GABA-immunoreactive neurons in the central nervous systems of nudibranch molluscs.

    PubMed

    Gunaratne, Charuni A; Sakurai, Akira; Katz, Paul S

    2014-03-01

    The relative simplicity of certain invertebrate nervous systems, such as those of gastropod molluscs, allows behaviors to be dissected at the level of small neural circuits composed of individually identifiable neurons. Elucidating the neurotransmitter phenotype of neurons in neural circuits is important for understanding how those neural circuits function. In this study, we examined the distribution of γ-aminobutyric-acid;-immunoreactive (GABA-ir) neurons in four species of sea slugs (Mollusca, Gastropoda, Opisthobranchia, Nudibranchia): Tritonia diomedea, Melibe leonina, Dendronotus iris, and Hermissenda crassicornis. We found consistent patterns of GABA immunoreactivity in the pedal and cerebral-pleural ganglia across species. In particular, there were bilateral clusters in the lateral and medial regions of the dorsal surface of the cerebral ganglia as well as a cluster on the ventral surface of the pedal ganglia. There were also individual GABA-ir neurons that were recognizable across species. The invariant presence of these individual neurons and clusters suggests that they are homologous, although there were interspecies differences in the numbers of neurons in the clusters. The GABAergic system was largely restricted to the central nervous system, with the majority of axons confined to ganglionic connectives and commissures, suggesting a central, integrative role for GABA. GABA was a candidate inhibitory neurotransmitter for neurons in central pattern generator (CPG) circuits underlying swimming behaviors in these species, however none of the known swim CPG neurons were GABA-ir. Although the functions of these GABA-ir neurons are not known, it is clear that their presence has been strongly conserved across nudibranchs. PMID:24638845

  6. Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels

    PubMed Central

    Ihara, Makoto; Ling, Yun; Yang, Xinling; Kai, Kenji; Hayashi, Hideo; Matsuda, Kazuhiko

    2015-01-01

    Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs. PMID:25902139

  7. Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels.

    PubMed

    Xu, Yan; Furutani, Shogo; Ihara, Makoto; Ling, Yun; Yang, Xinling; Kai, Kenji; Hayashi, Hideo; Matsuda, Kazuhiko

    2015-01-01

    Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to 10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs. PMID:25902139

  8. Brain regional distribution of GABAA receptors exhibiting atypical GABA agonism: roles of receptor subunits

    PubMed Central

    Halonen, Lauri M.; Sinkkonen, Saku T.; Chandra, Dev; Homanics, Gregg E.; Korpi, Esa R.

    2009-01-01

    The major inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), has only partial efficacy at certain subtypes of GABAA receptors. To characterize these minor receptor populations in rat and mouse brains, we used autoradiographic imaging of t-butylbicyclophosphoro[35S]thionate ([35S]TBPS) binding to GABAA receptors in brain sections and compared the displacing capacities of 10 mM GABA and 1 mM 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a competitive GABA-site agonist. Brains from GABAA receptor α1, α4, δ, and α4 + δ subunit knockout (KO) mouse lines were used to understand the contribution of these particular receptor subunits to “GABA-insensitive” (GIS) [35S]TBPS binding. THIP displaced more [35S]TBPS binding than GABA in several brain regions, indicating that THIP also inhibited GIS-binding. In these regions, GABA prevented the effect of THIP on GIS-binding. GIS-binding was increased in the cerebellar granule cell layer of δ KO and α4 + δ KO mice, being only slightly diminished in that of α1 KO mice. In the thalamus and some other forebrain regions of wild-type mice, a significant amount of GIS-binding was detected. This GIS-binding was higher in α4 KO mice. However, it was fully abolished in α1 KO mice, indicating that the α1 subunit was obligatory for the GIS-binding in the forebrain. Our results suggest that native GABAA receptors in brain sections showing reduced displacing capacity of [35S]TBPS binding by GABA (partial agonism) minimally require the assembly of α1 and β subunits in the forebrain and of α6 and β subunits in the cerebellar granule cell layer. These receptors may function as extrasynaptic GABAA receptors. PMID:19397945

  9. Characterization of an N-system amino acid transporter expressed in retina and its involvement in glutamine transport.

    PubMed

    Gu, S; Roderick, H L; Camacho, P; Jiang, J X

    2001-06-29

    We report here on the characterization of a mouse N-system amino acid transporter protein, which is involved in the transport of glutamine. This protein of 485 amino acids shares 52% sequence homology with an N-system amino acid transporter, mouse N-system amino acid transporter (mNAT) and its orthologs. Because this protein shares a high degree of sequence homology and functional similarity to mNAT, we named it mNAT2. mNAT2 is predominately expressed in the retina and to a slightly lesser extent in the brain. In the retina, it is located in the axons of ganglion cells in the nerve fiber layer and in the bundles of the optic nerve. Functional analysis of mNAT2 expressed in Xenopus oocytes revealed that the strongest transport activities were specific for l-glutamine. In addition, mNAT2 is a Na(+)- and pH-dependent, high affinity transporter and partially tolerates substitution of Na(+) by Li(+). Additionally, mNAT2 functions as a carrier-mediated transporter that facilitates efflux. The unique expression pattern and selective glutamine transport properties of mNAT2 suggest that it plays a specific role in the uptake of glutamine involved in the generation of the neurotransmitter glutamate in retina. PMID:11325958

  10. Enterobacteria modulate intestinal bile acid transport and homeostasis through apical sodium-dependent bile acid transporter (SLC10A2) expression.

    PubMed

    Miyata, Masaaki; Yamakawa, Hiroki; Hamatsu, Mayumi; Kuribayashi, Hideaki; Takamatsu, Yuki; Yamazoe, Yasushi

    2011-01-01

    In our study, ampicillin (AMP)-mediated decrease of enterobacteria caused increases in hepatic bile acid concentration through (at least in part) elevation of bile acid synthesis in C57BL/6N mice. We investigated the involvement of enterobacteria on intestinal bile acid absorption in AMP-treated mice in the present study. Fecal enterobacterial levels and fecal bile acid excretion rates were markedly decreased in mice treated with AMP (100 mg/kg) for 3 days, whereas bile acid concentrations in portal blood were significantly increased compared with those in mice treated with a vehicle. Ileal apical sodium-dependent bile acid transporter (SLC10A2) mRNA levels and ileal SLC10A2 protein levels in brush-border membranes were significantly increased compared with those in mice treated with the vehicle. In AMP-treated mice, total bile acid levels were increased, whereas levels of enterobacteria-biotransformed bile acid, taurodeoxycholic acid, and cholic acid were decreased in intestinal lumen. These phenomena were also observed in farnesoid X receptor-null mice treated with AMP for 3 days. Discontinuation of AMP administration after 3 days (vehicle administration for 4 days) increased levels of fecal enterobacteria, fecal bile acid excretion, and taurodeoxycholic acid and cholic acid in the intestinal lumen, whereas the discontinuation decreased ileal SLC10A2 expression and bile acid concentrations in the portal blood. Coadministration of taurodeoxycholic acid or cholic acid decreased ileal SLC10A2 expression in mice treated with AMP. These results suggest that enterobacteria-mediated bile acid biotransformation modulates intestinal bile acid transport and homeostasis through down-regulation of ileal SLC10A2 expression. PMID:20884752

  11. Reactive Transport Modeling of Acid Gas Generation and Condensation

    SciTech Connect

    G. Zhahg; N. Spycher; E. Sonnenthal; C. Steefel

    2005-01-25

    Pulvirenti et al. (2004) recently conducted a laboratory evaporation/condensation experiment on a synthetic solution of primarily calcium chloride. This solution represents one potential type of evaporated pore water at Yucca Mountain, Nevada, a site proposed for geologic storage of high-level nuclear waste. These authors reported that boiling this solution to near dryness (a concentration factor >75,000 relative to actual pore waters) leads to the generation of acid condensate (pH 4.5) presumably due to volatilization of HCl (and minor HF and/or HNO{sub 3}). To investigate the various processes taking place, including boiling, gas transport, and condensation, their experiment was simulated by modifying an existing multicomponent and multiphase reactive transport code (TOUGHREACT). This code was extended with a Pitzer ion-interaction model to deal with high ionic strength. The model of the experiment was set-up to capture the observed increase in boiling temperature (143 C at {approx}1 bar) resulting from high concentrations of dissolved salts (up to 8 m CaCl{sub 2}). The computed HCI fugacity ({approx} 10{sup -4} bars) generated by boiling under these conditions is not sufficient to lower the pH of the condensate (cooled to 80 and 25 C) down to observed values unless the H{sub 2}O mass fraction in gas is reduced below {approx}10%. This is because the condensate becomes progressively diluted by H{sub 2}O gas condensation. However, when the system is modeled to remove water vapor, the computed pH of instantaneous condensates decreases to {approx}1.7, consistent with the experiment (Figure 1). The results also show that the HCl fugacity increases, and calcite, gypsum, sylvite, halite, MgCl{sub 2}4H{sub 2}O and CaCl{sub 2} precipitate sequentially with increasing concentration factors.

  12. L-aspartic acid transport by cat erythrocytes

    SciTech Connect

    Chen, C.W.; Preston, R.L.

    1986-03-01

    Cat and dog red cells are unusual in that they have no Na/K ATPase and contain low K and high Na intracellularly. They also show significant Na dependent L-aspartate (L-asp) transport. The authors have characterized this system in cat RBCs. The influx of /sup 3/H-L-asp (typically 2..mu..M) was measured in washed RBCs incubated for 60 s at 37/sup 0/C in medium containing 140 mM NaCl, 5 mM Kcl, 2 mM CaCl/sub 2/, 15 mM MOPS pH 7.4, 5 mM glucose, and /sup 14/C-PEG as a space marker. The cells were washed 3 times in the medium immediately before incubation which was terminated by centrifuging the RBCs through a layer of dibutylphthalate. Over an L-asp concentration range of 0.5-1000..mu..M, influx obeyed Michaelis-Menten kinetics with a small added linear diffusion component. The Kt and Jmax of the saturable component were 5.40 +/- 0.34 ..mu..M and 148.8 +/- 7.2 ..mu..mol 1. cell/sup -1/h/sup -1/ respectively. Replacement of Na with Li, K, Rb, Cs or choline reduce influx to diffusion. With the addition of asp analogues (4/sup +/M L-asp, 40/sup +/M inhibitor), the following sequence of inhibition was observed (range 80% to 40% inhib.): L-glutamate > L-cysteine sulfonate > D-asp > L-cysteic acid > D-glutamate. Other amino acids such as L-alanine, L-proline, L-lysine, L-cysteine, and taurine showed no inhibition (<5%). These data suggest that cat red cells contain a high-affinity Na dependent transport system for L-asp, glutamate, and closely related analogues which resembles that found in the RBCs of other carnivores and in neural tissues.

  13. Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid.

    PubMed

    Pan, Yijun; Scanlon, Martin J; Owada, Yuji; Yamamoto, Yui; Porter, Christopher J H; Nicolazzo, Joseph A

    2015-12-01

    The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of (14)C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (Kin) of (14)C-DHA between FABP5-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in (14)C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of (14)C-DHA (1 min)), the Kin of (14)C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5(+/+) mice, the Kin of (14)C-DHA decreased 36.7 ± 12.4% in FABP5(-/-) mice

  14. Report membrane transport of lactic acid in the filamentous fungus Rhizopus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fungus Rhizopus is frequently used for fermentative production of lactic acid, but little is known about the mechanisms or proteins for transporting this carboxylic acid. Since transport of the lactate anion across the plasma membrane is critical to prevent acidification of the cytoplasm, we ev...

  15. Distinct modes of dopamine and GABA release in a dual transmitter neuron

    PubMed Central

    Borisovska, Maria; Bensen, AeSoon; Chong, Gene; Westbrook, Gary L.

    2013-01-01

    We now know of a surprising number of cases where single neurons contain multiple neurotransmitters. Neurons that contain a fast-acting neurotransmitter such as glutamate or GABA, and a modulatory transmitter such as dopamine are a particularly interesting case because they presumably serve dual signaling functions. The olfactory bulb contains a large population of GABA and dopamine-containing neurons, which have been implicated in normal olfaction as well as in Parkinson’s disease. Yet, they have been classified as non-exocytotic catecholamine neurons because of the apparent lack of vesicular monoamine transporters. Thus we examined how dopamine is stored and released from tyrosine-hydroxylase-positive-GFP (TH+-GFP) mouse periglomerular neurons in vitro. TH+ cells expressed both VMAT2 and VGAT, consistent with vesicular storage of both dopamine and GABA. Carbon fiber amperometry revealed that release of dopamine was quantal and calcium-dependent, but quantal size was much less than expected for large dense core vesicles, suggesting that release originated from EM-identified small clear vesicles. A single action potential in a TH+ neuron evoked a brief GABA synaptic current whereas evoked dopamine release was asynchronous, lasting for tens of seconds. Our data suggests that dopamine and GABA serve temporally distinct roles in these dual transmitter neurons. PMID:23365218

  16. Regulatory signals for intestinal amino acid transporters and peptidases

    SciTech Connect

    Ferraris, R.P.; Kwan, W.W.; Diamond, J. )

    1988-08-01

    Dietary protein ultimately regulates many processes involved in protein digestion, but it is often unclear whether proteins themselves, peptides, or amino acids (AAs) are the proximate regulatory signal. Hence the authors compared several processes involved in protein digestion in mice adapted to one of three rations, identical except for containing 54% of either casein, a partial hydrolysate of casein, or a free AA mixture simulating a complete hydrolysate of casein. The authors measured brush-border uptakes of seven AAs that variously serve as substrates for four AA transporters, and brush-border and cytosolic activities of four peptidases. The three rations yielded essentially the same AA uptake rates. Peptidase activities tended to be lower on the AA ration than on the protein ration. In other studies, all three rations yielded the same rates of brush-border peptide uptake; protein is only modestly more effective than AAs at inducing synthesis of pancreatic proteases; and, depending on the animal species, protein is either much less or much more effective than AAs at stimulating release of cholecystokinin and hence of pancreatic enzymes. Thus the regulators of each process involved in protein digestion are not necessarily that process's substrate.

  17. Hypoxia and GABA shunt activation in the pathogenesis of Alzheimer's disease.

    PubMed

    Salminen, Antero; Jouhten, Paula; Sarajärvi, Timo; Haapasalo, Annakaisa; Hiltunen, Mikko

    2016-01-01

    We have previously observed that the conversion of mild cognitive impairment to definitive Alzheimer's disease (AD) is associated with a significant increase in the serum level of 2,4-dihydroxybutyrate (2,4-DHBA). The metabolic generation of 2,4-DHBA is linked to the activation of the γ-aminobutyric acid (GABA) shunt, an alternative energy production pathway activated during cellular stress, when the function of Krebs cycle is compromised. The GABA shunt can be triggered by local hypoperfusion and subsequent hypoxia in AD brains caused by cerebral amyloid angiopathy. Succinic semialdehyde dehydrogenase (SSADH) is a key enzyme in the GABA shunt, converting succinic semialdehyde (SSA) into succinate, a Krebs cycle intermediate. A deficiency of SSADH activity stimulates the conversion of SSA into γ-hydroxybutyrate (GHB), an alternative route from the GABA shunt. GHB can exert not only acute neuroprotective activities but unfortunately also chronic detrimental effects which may lead to cognitive impairment. Subsequently, GHB can be metabolized to 2,4-DHBA and secreted from the brain. Thus, the activation of the GABA shunt and the generation of GHB and 2,4-DHBA can have an important role in the early phase of AD pathogenesis. PMID:26617286

  18. The role of the GABA system in amphetamine-type stimulant use disorders

    PubMed Central

    Jiao, Dongliang; liu, Yao; Li, Xiaohong; liu, Jinggen; Zhao, Min

    2015-01-01

    Abuse of amphetamine-type stimulants (ATS) has become a global public health problem. ATS causes severe neurotoxicity, which could lead to addiction and could induce psychotic disorders or cognitive dysfunctions. However, until now, there has been a lack of effective medicines for treating ATS-related problems. Findings from recent studies indicate that in addition to the traditional dopamine-ergic system, the GABA (gamma-aminobutyric acid)-ergic system plays an important role in ATS abuse. However, the exact mechanisms of the GABA-ergic system in amphetamine-type stimulant use disorders are not fully understood. This review discusses the role of the GABA-ergic system in ATS use disorders, including ATS induced psychotic disorders and cognitive dysfunctions. We conclude that the GABA-ergic system are importantly involved in the development of ATS use disorders through multiple pathways, and that therapies or medicines that target specific members of the GABA-ergic system may be novel effective interventions for the treatment of ATS use disorders. PMID:25999814

  19. Effect of pressure on (/sup 3/H)GABA release by synaptosomes isolated from cerebral cortex

    SciTech Connect

    Gilman, S.C.; Colton, J.S.; Hallenbeck, J.M.

    1986-12-01

    High hydrostatic pressure has been shown to produce neurological changes in humans which manifest, in part, as tremor, myoclonic jerks, electroencephalographic changes, and convulsions. This clinical pattern has been termed high-pressure nervous syndrome (HPNS). These symptoms may represent an alteration in synaptic transmission in the central nervous system with the inhibitory neural pathways being affected in particular. Since gamma-aminobutyric acid (GABA) transmission has been implicated in other seizure disorders, it was of interest to study GABAergic function at high pressure. Isolated synaptosomes were used to follow GABA release at 67.7 ATA of pressure. The major observation was a 33% depression in total (/sup 3/H)GABA efflux from depolarized cerebrocortical synaptosomes at 67.7 ATA. The Ca2+-dependent component of release was found to be completely blocked during the 1st min of (/sup 3/H)GABA efflux with a slow rise over the subsequent 3 min. These findings lead us to conclude that high pressure interferes with the intraterminal cascade for Ca2+-dependent release of GABA.

  20. GABA/benzodiazepine receptor complex in long-sleep and short-sleep mice

    SciTech Connect

    Marley, R.J.

    1987-01-01

    LS mice are more sensitive to benzodiazepine-induced anesthesia; however, the two lines do not differ in their hypothermic response to flurazepam. SS mice are more resistant to 3-mercaptopropionic acid-induced seizures and more sensitive to the anticonvulsant effects of benzodiazepines. The various correlates of GABA and benzodiazepine actions probably are the results of different mechanisms of action and/or differential regional control. Bicuculline competition for /sup 3/H-GABA binding sites is greater in SS cerebellar tissue and /sup 3/H-flunitrazepam binding is greater in the mid-brain region of LS mice. GABA enhancement of /sup 3/H-flunitrazepma binding is greater in SS mice. Ethanol also enhances /sup 3/H-flunitrazepam binding and increases the levels of /sup 3/H-flunitrazepam binding above those observed for GABA. Using correlational techniques on data from LS and SS mice and several inbred mouse strains, it was demonstrated that a positive relationship exists between the degree of receptor coupling within the GABA receptor complex and the degree of resistance to seizures.

  1. “Brain MR spectroscopy in autism spectrum disorder—the GABA excitatory/inhibitory imbalance theory revisited”

    PubMed Central

    Brix, Maiken K.; Ersland, Lars; Hugdahl, Kenneth; Grüner, Renate; Posserud, Maj-Britt; Hammar, Åsa; Craven, Alexander R.; Noeske, Ralph; Evans, C. John; Walker, Hanne B.; Midtvedt, Tore; Beyer, Mona K.

    2015-01-01

    Magnetic resonance spectroscopy (MRS) from voxels placed in the left anterior cingulate cortex (ACC) was measured from 14 boys with Autism Spectrum Disorder (ASD) and 24 gender and age-matched typically developing (TD) control group. Our main aims were to compare the concentration of γ-aminobutyric acid (GABA) between the two groups, and to investigate the relationship between brain metabolites and autism symptom severity in the ASD group. We did find a significant negative correlation in the ASD group between Autism Spectrum Screening Questionnaire (ASSQ) and GABA+/Cr, which may imply that severity of symptoms in ASD is associated with differences in the level of GABA in the brain, supporting the excitatory/inhibitory (E/I) imbalance theory. However we did not find a significant difference between the two groups in GABA levels. PMID:26157380

  2. Contribution of polyamines metabolism and GABA shunt to chilling tolerance induced by nitric oxide in cold-stored banana fruit.

    PubMed

    Wang, Yansheng; Luo, Zisheng; Mao, Linchun; Ying, Tiejin

    2016-04-15

    Effect of exogenous nitric oxide (NO) on polyamines (PAs) catabolism, γ-aminobutyric acid (GABA) shunt, proline accumulation and chilling injury of banana fruit under cold storage was investigated. Banana fruit treated with NO sustained lower chilling injury index than the control. Notably elevated nitric oxide synthetase activity and endogenous NO level were observed in NO-treated banana fruit. PAs contents in treated fruit were significantly higher than control fruit, due to the elevated activities of arginine decarboxylase and ornithine decarboxylase. NO treatment increased the activities of diamine oxidase, polyamine oxidase and glutamate decarboxylase, while reduced GABA transaminase activity to lower levels compared with control fruit, which resulted the accumulation of GABA. Besides, NO treatment upregulated proline content and significantly enhanced the ornithine aminotransferase activity. These results indicated that the chilling tolerance induced by NO treatment might be ascribed to the enhanced catabolism of PAs, GABA and proline. PMID:26616957

  3. Ornithine aminotransferase vs. GABA aminotransferase. Implications for the design of new anticancer drugs

    PubMed Central

    Lee, Hyunbeom; Juncosa, Jose I.; Silverman, Richard B.

    2015-01-01

    Ornithine aminotransferase (OAT) and γ-aminobutyric acid aminotransferase (GABA-AT) are classified under the same evolutionary subgroup and share a large portion of structural, functional, and mechanistic features. Therefore, it is not surprising that many molecules that bind to GABA-AT also bind well to OAT. Unlike GABA-AT, OAT had not been viewed as a potential therapeutic target until recently; consequently, the number of therapeutically viable molecules that target OAT is very limited. In this review the two enzymes are compared with respect to their active site structures, catalytic and inactivation mechanisms, and selective inhibitors. Insight is offered that could aid in the design and development of new selective inhibitors of OAT for the treatment of cancer. PMID:25145640

  4. Genetic differences in the ethanol sensitivity of GABA sub A receptors expressed in Xenopus oocytes

    SciTech Connect

    Wafford, K.A.; Burnett, D.M.; Dunwiddie, T.V.; Harris, R.A. )

    1990-07-20

    Animal lines selected for differences in drug sensitivity can be used to help determine the molecular basis of drug action. Long-sleep (LS) and short-sleep (SS) mice differ markedly in their genetic sensitivity to ethanol. To investigate the molecular basis for this difference, mRNA from brains of LS and SS mice was expressed in Xenopus oocytes and the ethanol sensitivity of gamma-aminobutyric acid A (GABA{sub A})- and N-methyl D-aspartate (NMDA) - activated ion channels was tested. Ethanol facilitated GABA responses in oocytes injected with mRNA from LS mice but antagonized responses in oocytes injected with mRNA from SS animals. Ethanol inhibited NMDA responses equally in the two lines. Thus, genes coding for the GABA{sub A} receptor or associated proteins may be critical determinants of individual differences in ethanol sensitivity.

  5. Designing Novel Nanoformulations Targeting Glutamate Transporter Excitatory Amino Acid Transporter 2: Implications in Treating Drug Addiction

    PubMed Central

    Rao, PSS; Yallapu, Murali M.; Sari, Youssef; Fisher, Paul B.; Kumar, Santosh

    2015-01-01

    Chronic drug abuse is associated with elevated extracellular glutamate concentration in the brain reward regions. Deficit of glutamate clearance has been identified as a contributing factor that leads to enhanced glutamate concentration following extended drug abuse. Importantly, normalization of glutamate level through induction of glutamate transporter 1 (GLT1)/ excitatory amino acid transporter 2 (EAAT2) expression has been described in several in vivo studies. GLT1 upregulators including ceftriaxone, a beta-lactam antibiotic, have been effective in attenuating drug-seeking and drug-consumption behavior in rodent models. However, potential obstacles toward clinical translation of GLT1 (EAAT2) upregulators as treatment for drug addiction might include poor gastrointestinal absorption, serious peripheral adverse effects, and/or suboptimal CNS concentrations. Given the growing success of nanotechnology in targeting CNS ailments, nanoformulating known GLT1 (EAAT2) upregulators for selective uptake across the blood brain barrier presents an ideal therapeutic approach for treating drug addiction. In this review, we summarize the results obtained with promising GLT1 (EAAT2) inducing compounds in animal models recapitulating drug addiction. Additionally, the various nanoformulations that can be employed for selectively increasing the CNS bioavailability of GLT1 (EAAT2) upregulators are discussed. Finally, the applicability of GLT1 (EAAT2) induction via central delivery of drug-loaded nanoformulations is described. PMID:26635971

  6. Influence of GABA and GABA-producing Lactobacillus brevis DPC 6108 on the development of diabetes in a streptozotocin rat model.

    PubMed

    Marques, T M; Patterson, E; Wall, R; O'Sullivan, O; Fitzgerald, G F; Cotter, P D; Dinan, T G; Cryan, J F; Ross, R P; Stanton, C

    2016-06-01

    The aim of this study was to investigate if dietary administration of γ-aminobutyric acid (GABA)-producing Lactobacillus brevis DPC 6108 and pure GABA exert protective effects against the development of diabetes in streptozotocin (STZ)-induced diabetic Sprague Dawley rats. In a first experiment, healthy rats were divided in 3 groups (n=10/group) receiving placebo, 2.6 mg/kg body weight (bw) pure GABA or L. brevis DPC 6108 (~10(9)microorganisms). In a second experiment, rats (n=15/group) were randomised to five groups and four of these received an injection of STZ to induce type 1 diabetes. Diabetic and non-diabetic controls received placebo [4% (w/v) yeast extract in dH2O], while the other three diabetic groups received one of the following dietary supplements: 2.6 mg/kg bw GABA (low GABA), 200 mg/kg bw GABA (high GABA) or ~10(9) L. brevis DPC 6108. L. brevis DPC 6108 supplementation was associated with increased serum insulin levels (P<0.05), but did not alter other metabolic markers in healthy rats. Diabetes induced by STZ injection decreased body weight (P<0.05), increased intestinal length (P<0.05) and stimulated water and food intake. Insulin was decreased (P<0.05), whereas glucose was increased (P<0.001) in all diabetic groups, compared with non-diabetic controls. A decrease (P<0.01) in glucose levels was observed in diabetic rats receiving L. brevis DPC 6108, compared with diabetic-controls. Both the composition and diversity of the intestinal microbiota were affected by diabetes. Microbial diversity in diabetic rats supplemented with low GABA was not reduced (P>0.05), compared with non-diabetic controls while all other diabetic groups displayed reduced diversity (P<0.05). L. brevis DPC 6108 attenuated hyperglycaemia induced by diabetes but additional studies are needed to understand the mechanisms involved in this reduction. PMID:27013462

  7. gamma-Glutamyl amino acids. Transport and conversion to 5-oxoproline in the kidney

    SciTech Connect

    Bridges, R.J.; Meister, A.

    1985-06-25

    Transport of gamma-glutamyl amino acids, a step in the proposed glutathione-gamma-glutamyl transpeptidase-mediated amino acid transport pathway, was examined in mouse kidney. The transport of gamma-glutamyl amino acids was demonstrated in vitro in studies on kidney slices. Transport was followed by measuring uptake of /sup 35/S after incubation of the slices in media containing gamma-glutamyl methionine (/sup 35/S)sulfone. The experimental complication associated with extracellular conversion of the gamma-glutamyl amino acid to amino acid and uptake of the latter by slices was overcome by using 5-oxoproline formation (catalyzed by intracellular gamma-glutamyl-cyclotransferase) as an indicator of gamma-glutamyl amino acid transport. This method was also successfully applied to studies on transport of gamma-glutamyl amino acids in vivo. Transport of gamma-glutamyl amino acids in vitro and in vivo is inhibited by several inhibitors of gamma-glutamyl transpeptidase and also by high extracellular levels of glutathione. This seems to explain urinary excretion of gamma-glutamylcystine by humans with gamma-glutamyl transpeptidase deficiency and by mice treated with inhibitors of this enzyme. Mice depleted of glutathione by treatment with buthionine sulfoximine (which inhibits glutathione synthesis) or by treatment with 2,6-dimethyl-2,5-heptadiene-4-one (which effectively interacts with tissue glutathione) exhibited significantly less transport of gamma-glutamyl amino acids than did untreated controls. The findings suggest that intracellular glutathione functions in transport of gamma-glutamyl amino acids. Evidence was also obtained for transport of gamma-glutamyl gamma-glutamylphenylalanine into kidney slices.

  8. The Relation Between GABA and L-Arginine Levels With Some Stroke Risk Factors in Acute Ischemic Stroke Patients

    PubMed Central

    Hosinian, Mohsen; Qujeq, Durdi; Ahmadi Ahangar, Alijan

    2016-01-01

    Changes in extra and intracellular neurotransmitter amino acids concentration in the early stage of acute cerebral ischemia have been reported. In this the study, serum level of gamma aminobutyric acid (GABA) and L-Arginine in acute ischemic stroke patients was assessed. 60 patients with acute ischemic stroke and sixthy healthy volunteers as a control group were assessed. Serum GABA was measured with modified enzymatic method and serum L- Arginine was measured by modified Sakaguchi method. Serum GABA level in stroke cases was lower than that of the control group. There was no relationship between GABA level and age or gender. Also, no significant correlation was observed between GABA levels with ischemic stroke risk factors such as smoking, diabetes mellitus, and hypertension. Serum L- Arginine level in patients was slightly increased in comparison with control group. There was a positive relationship between serum L- Arginine level and acute ischemic stroke risk factors. Serum GABA level was reduced in patients and had no correlation with acute ischemic stroke risk factors. PMID:27478806

  9. The Relation Between GABA and L-Arginine Levels With Some Stroke Risk Factors in Acute Ischemic Stroke Patients.

    PubMed

    Hosinian, Mohsen; Qujeq, Durdi; Ahmadi Ahangar, Alijan

    2016-01-01

    Changes in extra and intracellular neurotransmitter amino acids concentration in the early stage of acute cerebral ischemia have been reported. In this the study, serum level of gamma aminobutyric acid (GABA) and L-Arginine in acute ischemic stroke patients was assessed. 60 patients with acute ischemic stroke and sixthy healthy volunteers as a control group were assessed. Serum GABA was measured with modified enzymatic method and serum L- Arginine was measured by modified Sakaguchi method. Serum GABA level in stroke cases was lower than that of the control group. There was no relationship between GABA level and age or gender. Also, no significant correlation was observed between GABA levels with ischemic stroke risk factors such as smoking, diabetes mellitus, and hypertension. Serum L- Arginine level in patients was slightly increased in comparison with control group. There was a positive relationship between serum L- Arginine level and acute ischemic stroke risk factors. Serum GABA level was reduced in patients and had no correlation with acute ischemic stroke risk factors. PMID:27478806

  10. Homomeric RDL and heteromeric RDL/LCCH3 GABA receptors in the honeybee antennal lobes: two candidates for inhibitory transmission in olfactory processing.

    PubMed

    Dupuis, Julien Pierre; Bazelot, Michaël; Barbara, Guillaume Stéphane; Paute, Sandrine; Gauthier, Monique; Raymond-Delpech, Valérie

    2010-01-01

    gamma-Aminobutyric acid (GABA)-gated chloride channel receptors are abundant in the CNS, where their physiological role is to mediate fast inhibitory neurotransmission. In insects, this inhibitory transmission plays a crucial role in olfactory information processing. In an effort to understand the nature and properties of the ionotropic receptors involved in these processes in the honeybee Apis mellifera, we performed a pharmacological and molecular characterization of GABA-gated channels in the primary olfactory neuropile of the honeybee brain-the antennal lobe (AL)-using whole cell patch-clamp recordings coupled with single-cell RT-PCR. Application of GABA onto AL cells at -110 mV elicited fast inward currents, demonstrating the existence of ionotropic GABA-gated chloride channels. Molecular analysis of the GABA-responding cells revealed that both subunits RDL and LCCH3 were expressed out of the three orthologs of Drosophila melanogaster GABA-receptor subunits encoded within the honeybee genome (RDL, resistant to dieldrin; GRD, GABA/glycine-like receptor of Drosophila; LCCH3, ligand-gated chloride channel homologue 3), opening the door to possible homo- and/or heteromeric associations. The resulting receptors were activated by insect GABA-receptor agonists muscimol and CACA and blocked by antagonists fipronil, dieldrin, and picrotoxin, but not bicuculline, displaying a typical RDL-like pharmacology. Interestingly, increasing the intracellular calcium concentration potentiated GABA-elicited currents, suggesting a modulating effect of calcium on GABA receptors possibly through phosphorylation processes that remain to be determined. These results indicate that adult honeybee AL cells express typical RDL-like GABA receptors whose properties support a major role in synaptic inhibitory transmission during olfactory information processing. PMID:19906878

  11. Effects of benzodiazepines and non-benzodiazepine compounds on the GABA-induced response in frog isolated sensory neurones.

    PubMed

    Yakushiji, T; Fukuda, T; Oyama, Y; Akaike, N

    1989-11-01

    1. The effects of benzodiazepines and non-benzodiazepine compounds on the gamma-aminobutyric acid (GABA)-induced chloride current (ICl) were studied in frog isolated sensory neurones by use of a concentration-jump (termed 'concentration-clamp') technique, under single-electrode voltage-clamp conditions. The drugs used were classified into four categories as follows: full benzodiazepine receptor agonists (diazepam, clonazepam, nitrazepam, midazolam, clotiazepam and etizolam), partial agonists (CL 218,872, Ro 16-6028, Ro 17-1812 and Ro 23-0364), inverse agonists (Ro 15-3505, FG 7142 and beta-CCE) and a benzodiazepine receptor antagonist, Ro 15-1788 (flumazenil). 2. All full agonists at concentrations of 3 x 10(-6) M or less increased dose-dependently the peak amplitude of ICl elicited by 3 x 10(-6) M GABA to twice to three times larger than the control. However, no further augmentation of the GABA response was observed at concentrations of 1 x 10(-5) M or higher. Partial agonists also showed a dose-dependent augmentation of the GABA response at concentrations ranging from 3 x 10(-8) M to 3 x 10(-5) M, but their efficacies of augmentation of the GABA response were only about half or less of those of full agonists. Of the inverse agonists, beta-CCE had a unique dose-dependent effect on the GABA response. Beta-CCE reduced dose-dependently the GABA response at concentrations of less than 3 x 10(-6) M, but augmented it at concentrations of 3 x 10(-5) M and 6 x 10(-5) M. The inverse agonists reduced dose-dependently the GABA response. The benzodiazepine antagonist, flumazenil, slightly augmented the GABA response at concentrations between 3 x 10 7M and 3 x 10 5 M. 3. These results show clear differences in the effects on the GABA response between these four categories of compounds known to affect the benzodiazepine recognition site of the GABA/ benzodiazepine receptor-chloride channel complex. Our experimental system of frog isolated sensory neurones and a 'concentration

  12. Adsorption and transport of polymaleic acid on Callovo-Oxfordian clay stone: Batch and transport experiments

    NASA Astrophysics Data System (ADS)

    Durce, Delphine; Landesman, Catherine; Grambow, Bernd; Ribet, Solange; Giffaut, Eric

    2014-08-01

    Dissolved Organic Matter (DOM) can affect the mobility of radionuclides in pore water of clay-rich geological formations, such as those intended to be used for nuclear waste disposal. The present work studies the adsorption and transport properties of a polycarboxylic acid, polymaleic acid (PMA, Mw = 1.9 kDa), on Callovo-Oxfordian argillite samples (COx). Even though this molecule is rather different from the natural organic matter found in clay rock, the study of its retention properties on both dispersed and intact samples allows assessing to which extent organic acids may undergo sorption under natural conditions (pH 7) and what could be the impact on their mobility. PMA sorption and desorption were investigated in dispersed systems. The degree of sorption was measured after 1, 8 and 21 days and for a range of PMA initial concentrations from 4.5 × 10- 7 to 1.4 × 10- 3 mol.L- 1. The reversibility of the sorption process was estimated by desorption experiments performed after the sorption experiments. At the sorption steady state, the sorption was described by a two-site Langmuir model. A total sorption capacity of COx for PMA was found to be 1.01×10- 2 mol.kg- 1 distributed on two sorption sites, one weak and one strong. The desorption of PMA was incomplete, independently of the duration of the sorption phase. The amount of desorbable PMA even appeared to decrease for sorption phases from 1 to 21 days. To describe the apparent desorption hysteresis, two conceptual models were applied. The two-box diffusion model accounted for intraparticle diffusion and more generally for nonequilibrium processes. The two-box first-order non-reversible model accounted for a first-order non-reversible sorption and more generally for kinetically-controlled irreversible sorption processes. The use of the two models revealed that desorption hysteresis was not the result of nonequilibrium processes but was due to irreversible sorption. Irreversible sorption on the strong site was

  13. Characterizing MttA as a mitochondrial cis-aconitic acid transporter by metabolic engineering.

    PubMed

    Steiger, Matthias G; Punt, Peter J; Ram, Arthur F J; Mattanovich, Diethard; Sauer, Michael

    2016-05-01

    The mitochondrial carrier protein MttA is involved in the biosynthesis of itaconic acid in Aspergillus terreus. In this paper, the transport specificity of MttA is analyzed making use of different metabolically engineered Aspergillus niger strains. Furthermore, the mitochondrial localization of this protein is confirmed using fluorescence microscopy. It was found that MttA preferentially transports cis-aconitic acid over citric acid and does not transport itaconic acid. The expression of MttA in selected A. niger strains results in secretion of aconitic acid. MttA can be used in further strain engineering strategies to transport cis-aconitic acid to the cytosol to produce itaconic acid or related metabolites. The microbial production of aconitic acid (9g/L) is achieved in strains expressing this transport protein. Thus, metabolic engineering can be used for both the in vivo characterization of transport protein function like MttA and to make use of this protein by creating aconitic acid producing strains. PMID:26875555

  14. Excitatory actions of GABA in developing chick vestibular afferents: effects on resting electrical activity.

    PubMed

    Cortes, Celso; Galindo, Fabian; Galicia, Salvador; Cebada, Jorge; Flores, Amira

    2013-07-01

    The aim of this study was to characterize the effect of γ-aminobutyric acid (GABA) in the resting multiunit activity of the vestibular afferents during development using the isolated inner ear of embryonic and postnatal chickens (E15-E21 and P5). GABA (10(-3) to 10(-5) M; n = 133) and muscimol (10(-3) M) elicited an increase in the frequency of the basal discharge of the vestibular afferents. We found that GABA action was dose-dependent and inversely related to animal age. Thus, the largest effect was observed in embryonic ages such as E15 and E17 and decreases in E21 and P5. The GABAA receptor antagonists, bicuculline (10(-5) M; n = 10) and picrotoxin (10(-4) M; n = 10), significantly decreased the excitatory action of GABA and muscimol (10(-3) M). Additionally, CNQX 10(-6) M, MCPG 10(-5) M and 7ClKyn 10(-5) M (n = 5) were co-applied by bath substitution (n = 5). Both the basal discharge and the GABA action significantly decreased in these experimental conditions. The chloride channel blocker 9-AC 0.5 mM produced an important reduction in the effect of GABA 10(-3) (n = 5) and 10(-4) M (n = 5). Thus, our results suggest an excitatory role of GABA in the resting activity of the vestibular afferents that can be explained by changes in the gradient of concentration of Cl(-) during development. We show for the first time that the magnitude of this GABA effect decreases at later stages of embryonic and early postnatal development. Taking into account the results with glutamatergic antagonists, we conclude that GABA has a presynaptic action but is not the neurotransmitter in the vestibular afferent synapses, although it could act as a facilitator of the spontaneous activity and may regulate glutamate release. PMID:23401185

  15. Skeletal muscle amino acid transporter expression is increased in young and older adults following resistance exercise

    PubMed Central

    Fry, Christopher S.; Glynn, Erin L.; Timmerman, Kyle L.; Dickinson, Jared M.; Walker, Dillon K.; Gundermann, David M.; Volpi, Elena; Rasmussen, Blake B.

    2011-01-01

    Amino acid transporters and mammalian target of rapamycin complex 1 (mTORC1) signaling are important contributors to muscle protein anabolism. Aging is associated with reduced mTORC1 signaling following resistance exercise, but the role of amino acid transporters is unknown. Young (n = 13; 28 ± 2 yr) and older (n = 13; 68 ± 2 yr) subjects performed a bout of resistance exercise. Skeletal muscle biopsies (vastus lateralis) were obtained at basal and 3, 6, and 24 h postexercise and were analyzed for amino acid transporter mRNA and protein expression and regulators of amino acid transporter transcription utilizing real-time PCR and Western blotting. We found that basal amino acid transporter expression was similar in young and older adults (P > 0.05). Exercise increased L-type amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, sodium-coupled neutral amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, and cationic amino acid transporter 1/SLC7A1 mRNA expression in both young and older adults (P < 0.05). L-type amino acid transporter 1 and CD98 protein increased only in younger adults (P < 0.05). eukaryotic initiation factor 2 α-subunit (S52) increased similarly in young and older adults postexercise (P < 0.05). Ribosomal protein S6 (S240/244) and activating transcription factor 4 nuclear protein expression tended to be higher in the young, while nuclear signal transducer and activator of transcription 3 (STAT3) (Y705) was higher in the older subjects postexercise (P < 0.05). These results suggest that the rapid upregulation of amino acid transporter expression following resistance exercise may be regulated differently between the age groups, but involves a combination of mTORC1, activating transcription factor 4, eukaryotic initiation factor 2 α-subunit, and STAT3. We propose an increase in amino acid transporter expression may contribute to enhanced amino acid sensitivity following exercise in young and older

  16. Transporter-targeted cholic acid-cytarabine conjugates for improved oral absorption.

    PubMed

    Zhang, Dong; Li, Dongpo; Shang, Lei; He, Zhonggui; Sun, Jin

    2016-09-10

    Cytarabine has a poor oral absorption due to its rapid deamination and poor membrane permeability. Bile acid transporters are highly expressed both in enterocytes and hepatocytes and to increase the oral bioavailability and investigate the potential application of cytarabine for liver cancers, a transporter- recognizing prodrug strategy was applied to design and synthesize four conjugates of cytarabine with cholic acid (CA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA) and ursodeoxycholic acid (UDCA). The anticancer activities against HepG2 cells were evaluated by MTT assay and the role of bile acid transporters during cellular transport was investigated in a competitive inhibition experiment. The in vitro and in vivo metabolic stabilities of these conjugates were studied in rat plasma and liver homogenates. Finally, an oral bioavailability study was conducted in rats. All the cholic acid-cytarabine conjugates (40μM) showed potent antiproliferative activities (up to 70%) against HepG2 cells after incubation for 48h. The addition of bile acids could markedly reduce the antitumor activities of these conjugates. The N(4)-ursodeoxycholic acid conjugate of cytarabine (compound 5) exhibited optimal stability (t1/2=90min) in vitro and a 3.9-fold prolonged half-life of cytarabine in vivo. More importantly, compound 5 increased the oral bioavailability 2-fold compared with cytarabine. The results of the present study suggest that the prodrug strategy based on the bile acid transporters is suitable for improving the oral absorption and the clinical application of cytarabine. PMID:27377011

  17. Identification and characterization of an amino acid transporter expressed differentially in liver

    PubMed Central

    Gu, Sumin; Roderick, Hywel Llewelyn; Camacho, Patricia; Jiang, Jean X.

    2000-01-01

    Cellular metabolic needs are fulfilled by transport of amino acids across the plasma membrane by means of specialized transporter proteins. Although many of the classical amino acid transporters have been characterized functionally, less than half of these proteins have been cloned. In this report, we identify and characterize a cDNA encoding a plasma membrane amino acid transporter. The deduced amino acid sequence is 505 residues and is highly hydrophobic with the likely predicted structure of 9 transmembrane domains, which putatively place the amino terminus in the cytoplasm and the carboxy terminus on the cell surface. Expression of the cRNA in Xenopus laevis oocytes revealed strong transport activities specific for histidine and glutamine. This protein is a Na+- and pH-dependent transporter and tolerates substitution of Na+ by Li+. Furthermore, this transporter is not an obligatory exchanger because efflux occurs in the absence of influx. This transporter is expressed predominantly in the liver, although it is also present in the kidney, brain, and heart. In the liver, it is located in the plasma membrane of hepatocytes, and the strongest expression was detected in those adjacent to the central vein, gradually decreasing towards the portal tract. Because this protein displays functional similarities to the N-system amino acid transport, we have termed it mNAT, for murine N-system amino acid transporter. This is the first transporter gene identified within the N-system, one of the major amino acid transport systems in the body. The expression pattern displayed by mNAT suggests a potential role in hepatocyte physiology. PMID:10716701

  18. 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

  19. Potency of individual bile acids to regulate bile acid synthesis and transport genes in primary human hepatocyte cultures.

    PubMed

    Liu, Jie; Lu, Hong; Lu, Yuan-Fu; Lei, Xiaohong; Cui, Julia Yue; Ellis, Ewa; Strom, Stephen C; Klaassen, Curtis D

    2014-10-01

    Bile acids (BAs) are known to regulate their own homeostasis, but the potency of individual bile acids is not known. This study examined the effects of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) on expression of BA synthesis and transport genes in human primary hepatocyte cultures. Hepatocytes were treated with the individual BAs at 10, 30, and 100μM for 48 h, and RNA was extracted for real-time PCR analysis. For the classic pathway of BA synthesis, BAs except for UDCA markedly suppressed CYP7A1 (70-95%), the rate-limiting enzyme of bile acid synthesis, but only moderately (35%) down-regulated CYP8B1 at a high concentration of 100μM. BAs had minimal effects on mRNA of two enzymes of the alternative pathway of BA synthesis, namely CYP27A1 and CYP7B1. BAs increased the two major target genes of the farnesoid X receptor (FXR), namely the small heterodimer partner (SHP) by fourfold, and markedly induced fibroblast growth factor 19 (FGF19) over 100-fold. The BA uptake transporter Na(+)-taurocholate co-transporting polypeptide was unaffected, whereas the efflux transporter bile salt export pump was increased 15-fold and OSTα/β were increased 10-100-fold by BAs. The expression of the organic anion transporting polypeptide 1B3 (OATP1B3; sixfold), ATP-binding cassette (ABC) transporter G5 (ABCG5; sixfold), multidrug associated protein-2 (MRP2; twofold), and MRP3 (threefold) were also increased, albeit to lesser degrees. In general, CDCA was the most potent and effective BA in regulating these genes important for BA homeostasis, whereas DCA and CA were intermediate, LCA the least, and UDCA ineffective. PMID:25055961

  20. Glutamate and GABA in Vestibulo-Sympathetic Pathway Neurons

    PubMed Central

    Holstein, Gay R.; Friedrich, Victor L. Jr.; Martinelli, Giorgio P.

    2016-01-01

    The vestibulo-sympathetic reflex (VSR) actively modulates blood pressure during changes in posture. This reflex allows humans to stand up and quadrupeds to rear or climb without a precipitous decline in cerebral perfusion. The VSR pathway conveys signals from the vestibular end organs to the caudal vestibular nuclei. These cells, in turn, project to pre-sympathetic neurons in the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively). The present study assessed glutamate- and GABA-related immunofluorescence associated with central vestibular neurons of the VSR pathway in rats. Retrograde FluoroGold tract tracing was used to label vestibular neurons with projections to RVLM or CVLM, and sinusoidal galvanic vestibular stimulation (GVS) was employed to activate these pathways. Central vestibular neurons of the VSR were identified by co-localization of FluoroGold and cFos protein, which accumulates in some vestibular neurons following galvanic stimulation. Triple-label immunofluorescence was used to co-localize glutamate- or GABA- labeling in the identified VSR pathway neurons. Most activated projection neurons displayed intense glutamate immunofluorescence, suggestive of glutamatergic neurotransmission. To support this, anterograde tracer was injected into the caudal vestibular nuclei. Vestibular axons and terminals in RVLM and CVLM co-localized the anterograde tracer and vesicular glutamate transporter-2 signals. Other retrogradely-labeled cFos-positive neurons displayed intense GABA immunofluorescence. VSR pathway neurons of both phenotypes were present in the caudal medial and spinal vestibular nuclei, and projected to both RVLM and CVLM. As a group, however, triple-labeled vestibular cells with intense glutamate immunofluorescence were located more rostrally in the vestibular nuclei than the GABAergic neurons. Only the GABAergic VSR pathway neurons showed a target preference, projecting predominantly to CVLM. These data provide the first

  1. Glutamate and GABA in Vestibulo-Sympathetic Pathway Neurons.

    PubMed

    Holstein, Gay R; Friedrich, Victor L; Martinelli, Giorgio P

    2016-01-01

    The vestibulo-sympathetic reflex (VSR) actively modulates blood pressure during changes in posture. This reflex allows humans to stand up and quadrupeds to rear or climb without a precipitous decline in cerebral perfusion. The VSR pathway conveys signals from the vestibular end organs to the caudal vestibular nuclei. These cells, in turn, project to pre-sympathetic neurons in the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively). The present study assessed glutamate- and GABA-related immunofluorescence associated with central vestibular neurons of the VSR pathway in rats. Retrograde FluoroGold tract tracing was used to label vestibular neurons with projections to RVLM or CVLM, and sinusoidal galvanic vestibular stimulation (GVS) was employed to activate these pathways. Central vestibular neurons of the VSR were identified by co-localization of FluoroGold and cFos protein, which accumulates in some vestibular neurons following galvanic stimulation. Triple-label immunofluorescence was used to co-localize glutamate- or GABA- labeling in the identified VSR pathway neurons. Most activated projection neurons displayed intense glutamate immunofluorescence, suggestive of glutamatergic neurotransmission. To support this, anterograde tracer was injected into the caudal vestibular nuclei. Vestibular axons and terminals in RVLM and CVLM co-localized the anterograde tracer and vesicular glutamate transporter-2 signals. Other retrogradely-labeled cFos-positive neurons displayed intense GABA immunofluorescence. VSR pathway neurons of both phenotypes were present in the caudal medial and spinal vestibular nuclei, and projected to both RVLM and CVLM. As a group, however, triple-labeled vestibular cells with intense glutamate immunofluorescence were located more rostrally in the vestibular nuclei than the GABAergic neurons. Only the GABAergic VSR pathway neurons showed a target preference, projecting predominantly to CVLM. These data provide the first

  2. β-Hydroxybutyrate is the preferred substrate for GABA and glutamate synthesis while glucose is indispensable during depolarization in cultured GABAergic neurons.

    PubMed

    Lund, Trine M; Obel, Linea F; Risa, Øystein; Sonnewald, Ursula

    2011-08-01

    The ketogenic diet has multiple beneficial effects not only in treatment of epilepsy, but also in that of glucose transporter 1 deficiency, cancer, Parkinson's disease, obesity and pain. Thus, there is an increasing interest in understanding the mechanism behind this metabolic therapy. Patients on a ketogenic diet reach high plasma levels of ketone bodies, which are used by the brain as energy substrates. The interaction between glucose and ketone bodies is complex and there is still controversy as to what extent it affects the homeostasis of the neurotransmitters glutamate, aspartate and GABA. The present study was conducted to study this metabolic interaction in cultured GABAergic neurons exposed to different combinations of (13)C-labeled and unlabeled glucose and β-hydroxybutyrate. Depolarization was induced and the incorporation of (13)C into glutamate, GABA and aspartate was analyzed. The presence of β-hydroxybutyrate together with glucose did not affect the total GABA content but did, however, decrease the aspartate content to a lower value than when either glucose or β-hydroxybutyrate was employed alone. When combinations of the two substrates were used (13)C-atoms from β-hydroxybutyrate were found in all three amino acids to a greater extent than (13)C-atoms from glucose, but only the (13)C contribution from [1,6-(13)C]glucose increased upon depolarization. In conclusion, β-hydroxybutyrate was preferred over glucose as substrate for amino acid synthesis but the total content of aspartate decreased when both substrates were present. Furthermore only the use of glucose increased upon depolarization. PMID:21684314

  3. Concentration-dependent effects of GABA on insensitivity to fipronil in the A2'S mutant RDL GABA receptor from fipronil-resistant Oulema oryzae (Coleoptera: Chrysomelidae).

    PubMed

    Nakao, Toshifumi; Naoi, Atsuko; Hama, Masako; Kawahara, Nobuyuki; Hirase, Kangetsu

    2012-10-01

    The beetle Oulema oryzae Kuwayama (Coleoptera: Chrysomelidae), an important pest of rice, has developed fipronil resistance in Japan. Molecular cloning and sequence analysis of O. oryzae RDL gamma-aminobutyric acid (GABA) receptor subunit (OO-RDL) genes from fipronil-susceptible and -resistant O. oryzae identified the A2'S mutation (index number for the M2 membrane-spanning region). To investigate the effect of the A2'S mutation on fipronil resistance, we stably expressed the wild-type and mutant OO-RDL homomers in Drosophila Mel-2 cells. A membrane potential assay exhibited that the IC50 values of fipronil for inhibition of the response to EC80 GABA of the wild-type and A2'S mutant OO-RDL homomers were 0.09 microM and 0.11 microM, respectively. However, the IC50 values of fipronil for inhibition of the response to EC95 GABA of the wild-type and A2'S mutant OO-RDL homomers were 0.11 microM and approximately equal to 5 microM, respectively. These results suggest that the GABA concentration is an important factor affecting fipronil resistance in O. oryzae carrying the A2'S mutation in OO-RDL. PMID:23156177

  4. A novel action of highly specific acaricide; bifenazate as a synergist for a GABA-gated chloride channel of Tetranychus urticae [Acari: Tetranychidae].

    PubMed

    Hiragaki, Susumu; Kobayashi, Takeru; Ochiai, Noriaki; Toshima, Kayoko; Dekeyser, Mark A; Matsuda, Kazuhiko; Takeda, Makio

    2012-06-01

    Bifenazate is a very selective acaricide that controls the spider mite, Tetranychus urticae. Bifenazate is the first example of a carbazate acaricide. Its mode of action remains unclear. Bifenazate and its active metabolite diazene induce paralysis in spider mites, suggesting that they may act on the nervous system. Here we have employed a homologue (TuGABAR) of RDL (Resistance to dieldrin), a subunit of ionotropic γ-aminobutyric acid (GABA) receptor, from T. urticae to investigate the action of bifenazate and its active metabolite diazene on this receptor function. Although neither acaricide showed a GABA agonist action, 30 μM of bifenazate or diazene significantly enhanced the GABA-induced response of TuGABAR in a dose-dependent manner, shifting the EC(50) of GABA from 24.8 μM to 4.83 μM and 10.8 μM, respectively. This action demonstrates a positive allosteric modulator effect of bifenazate on T. urticae GABA receptors. This synergistic action is likely the result of bifenazate binding to a site distinct from that of the GABA binding site causing a conformational change that affects the magnitude of the GABA response. Precisely how the observed GABA synergist action correlates with the acaricidal activity of bifenazate, if at all, has yet to be determined. PMID:22330756

  5. The putative Cationic Amino Acid Transporter 9 is targeted to vesicles and may be involved in plant amino acid homeostasis

    PubMed Central

    Yang, Huaiyu; Stierhof, York-Dieter; Ludewig, Uwe

    2015-01-01

    Amino acids are major primary metabolites. Their uptake, translocation, compartmentation, and re-mobilization require a diverse set of cellular transporters. Here, the broadly expressed gene product of CATIONIC AMINO ACID TRANSPORTER 9 (CAT9) was identified as mainly localized to vesicular membranes that are involved in vacuolar trafficking, including those of the trans-Golgi network. In order to probe whether and how these compartments are involved in amino acid homeostasis, a loss-of-function cat9-1 mutant and ectopic over-expressor plants were isolated. Under restricted nitrogen supply in soil, cat9-1 showed a chlorotic phenotype, which was reversed in the over-expressors. The total soluble amino acid pools were affected in the mutants, but this was only significant under poor nitrogen supply. Upon nitrogen starvation, the soluble amino acid leaf pools were lower in the over-expressor, compared with cat9-1. Over-expression generally affected total soluble amino acid concentrations, slightly delayed development, and finally improved the survival upon severe nitrogen starvation. The results potentially identify a novel function of vesicular amino acid transport mediated by CAT9 in the cellular nitrogen-dependent amino acid homeostasis. PMID:25883600

  6. The putative Cationic Amino Acid Transporter 9 is targeted to vesicles and may be involved in plant amino acid homeostasis.

    PubMed

    Yang, Huaiyu; Stierhof, York-Dieter; Ludewig, Uwe

    2015-01-01

    Amino acids are major primary metabolites. Their uptake, translocation, compartmentation, and re-mobilization require a diverse set of cellular transporters. Here, the broadly expressed gene product of CATIONIC AMINO ACID TRANSPORTER 9 (CAT9) was identified as mainly localized to vesicular membranes that are involved in vacuolar trafficking, including those of the trans-Golgi network. In order to probe whether and how these compartments are involved in amino acid homeostasis, a loss-of-function cat9-1 mutant and ectopic over-expressor plants were isolated. Under restricted nitrogen supply in soil, cat9-1 showed a chlorotic phenotype, which was reversed in the over-expressors. The total soluble amino acid pools were affected in the mutants, but this was only significant under poor nitrogen supply. Upon nitrogen starvation, the soluble amino acid leaf pools were lower in the over-expressor, compared with cat9-1. Over-expression generally affected total soluble amino acid concentrations, slightly delayed development, and finally improved the survival upon severe nitrogen starvation. The results potentially identify a novel function of vesicular amino acid transport mediated by CAT9 in the cellular nitrogen-dependent amino acid homeostasis. PMID:25883600

  7. Auxin Transport in Zea mays Coleoptiles II. Influence of Light on the Transport of Indoleacetic Acid-2-C.

    PubMed

    Naqvi, S M; Gordon, S A

    1967-01-01

    The effect of bilateral irradiation with white light (1000 Meter Candle Sec) on the basipetal transport of auxin has been investigated. Illumination of either the intact shoot or the excised coleoptile tip of the Zea seedling, decreased the amount of diffusible auxin obtained from the tip, and decreased Avena curvature response to unilaterally applied indoleacetic acid. Irradiation of the intact Zea seedling did not affect the absorption of (14)C-labeled indoleacetic acid from an agar block subsequently placed on the decapitated coleoptile. However, light caused a significant decrease in the amount of labeled auxin basipetally transported, without affecting materially the velocity of that transport. These and other observations are interpreted as support for the hypothesis that the primary hormonal phenomenon in first-positive phototropism is a light-induced impairment in the basipetal transport of auxin. PMID:16656477

  8. Structural basis of the alternating-access mechanism in a bile acid transporter

    NASA Astrophysics Data System (ADS)

    Zhou, Xiaoming; Levin, Elena J.; Pan, Yaping; McCoy, Jason G.; Sharma, Ruchika; Kloss, Brian; Bruni, Renato; Quick, Matthias; Zhou, Ming

    2014-01-01

    Bile acids are synthesized from cholesterol in hepatocytes and secreted through the biliary tract into the small intestine, where they aid in absorption of lipids and fat-soluble vitamins. Through a process known as enterohepatic recirculation, more than 90% of secreted bile acids are then retrieved from the intestine and returned to the liver for resecretion. In humans, there are two Na+-dependent bile acid transporters involved in enterohepatic recirculation, the Na+-taurocholate co-transporting polypeptide (NTCP; also known as SLC10A1) expressed in hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT; also known as SLC10A2) expressed on enterocytes in the terminal ileum. In recent years, ASBT has attracted much interest as a potential drug target for treatment of hypercholesterolaemia, because inhibition of ASBT reduces reabsorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption. However, a lack of three-dimensional structures of bile acid transporters hampers our ability to understand the molecular mechanisms of substrate selectivity and transport, and to interpret the wealth of existing functional data. The crystal structure of an ASBT homologue from Neisseria meningitidis (ASBTNM) in detergent was reported recently, showing the protein in an inward-open conformation bound to two Na+ and a taurocholic acid. However, the structural changes that bring bile acid and Na+ across the membrane are difficult to infer from a single structure. To understand the structural changes associated with the coupled transport of Na+ and bile acids, here we solved two structures of an ASBT homologue from Yersinia frederiksenii (ASBTYf) in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives the conserved `crossover' region, where two discontinuous helices cross each other, alternating accessibility from either side of the cell membrane. This result has implications

  9. Amino acid absorption and homeostasis in mice lacking the intestinal peptide transporter PEPT1.

    PubMed

    Nässl, Anna-Maria; Rubio-Aliaga, Isabel; Fenselau, Henning; Marth, Mena Katharina; Kottra, Gabor; Daniel, Hannelore

    2011-07-01

    The intestinal peptide transporter PEPT1 mediates the uptake of di- and tripeptides derived from dietary protein breakdown into epithelial cells. Whereas the transporter appears to be essential to compensate for the reduced amino acid delivery in patients with mutations in amino acid transporter genes, such as in cystinuria or Hartnup disease, its physiological role in overall amino acid absorption is still not known. To assess the quantitative importance of PEPT1 in overall amino acid absorption and metabolism, PEPT1-deficient mice were studied by using brush border membrane vesicles, everted gut sacs, and Ussing chambers, as well as by transcriptome and proteome analysis of intestinal tissue samples. Neither gene expression nor proteome profiling nor functional analysis revealed evidence for any compensatory changes in the levels and/or function of transporters for free amino acids in the intestine. However, most plasma amino acid levels were increased in Pept1(-/-) compared with Pept1(+/+) animals, suggesting that amino acid handling is altered. Plasma appearance rates of (15)N-labeled amino acids determined after intragastric administration of a low dose of protein remained unchanged, whereas administration of a large protein load via gavage revealed marked differences in plasma appearance of selected amino acids. PEPT1 seems, therefore, important for overall amino acid absorption only after high dietary protein intake when amino acid transport processes are saturated and PEPT1 can provide additional absorption capacity. Since renal amino acid excretion remained unchanged, elevated basal concentrations of plasma amino acids in PEPT1-deficient animals seem to arise mainly from alterations in hepatic amino acid metabolism. PMID:21350187

  10. Decreased auditory GABA+ concentrations in presbycusis demonstrated by edited magnetic resonance spectroscopy

    PubMed Central

    Gao, Fei; Wang, Guangbin; Ma, Wen; Ren, Fuxin; Li, Muwei; Dong, Yuling; Liu, Cheng; Liu, Bo; Bai, Xue; Zhao, Bin; Edden, Richard A.E.

    2014-01-01

    Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central auditory system. Altered GABAergic neurotransmission has been found in both the inferior colliculus and the auditory cortex in animal models of presbycusis. Edited magnetic resonance spectroscopy (MRS), using the MEGA-PRESS sequence, is the most widely used technique for detecting GABA in the human brain. However, to date there has been a paucity of studies exploring changes to the GABA concentrations in the auditory region of patients with presbycusis. In this study, sixteen patients with presbycusis (5 males/11 females, mean age 63.1 ± 2.6 years) and twenty healthy controls (6 males/14 females, mean age 62.5 ± 2.3 years) underwent audiological and MRS examinations. Pure tone audiometry from 0.125 to 8 KHz and tympanometry were used to assess the hearing abilities of all subjects. The pure tone average (PTA; the average of hearing thresholds at 0.5, 1, 2, and 4 kHz) was calculated. The MEGA-PRESS sequence was used to measure GABA+ concentrations in 4 × 3 × 3 cm3 volumes centered on the left and right Heschl’s gyri. GABA+ concentrations were significantly lower in the presbycusis group compared to the control group (left auditory regions: p = 0.002, right auditory regions: p = 0.008). Significant negative correlations were observed between PTA and GABA+ concentrations in the presbycusis group (r = −0.57, p = 0.02), while a similar trend was found in the control group (r = −0.40, p = 0.08). These results are consistent with a hypothesis of dysfunctional GABAergic neurotransmission in the central auditory system in presbycusis, and suggest a potential treatment target for presbycusis. PMID:25463460

  11. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A) receptor-mediated signaling.

    PubMed

    Kakehashi, Anna; Kato, Ayumi; Ishii, Naomi; Wei, Min; Morimura, Keiichirou; Fukushima, Shoji; Wanibuchi, Hideki

    2014-01-01

    Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P(+)) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+) foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1), p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P(+) foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+) foci by activating GABA(A)R-mediated signaling. PMID:25419570

  12. GABA content within the ventromedial prefrontal cortex is related to trait anxiety.

    PubMed

    Delli Pizzi, Stefano; Padulo, Caterina; Brancucci, Alfredo; Bubbico, Giovanna; Edden, Richard A; Ferretti, Antonio; Franciotti, Raffaella; Manippa, Valerio; Marzoli, Daniele; Onofrj, Marco; Sepede, Gianna; Tartaro, Armando; Tommasi, Luca; Puglisi-Allegra, Stefano; Bonanni, Laura

    2016-05-01

    The ventromedial prefrontal cortex (vmPFC) plays a key role in emotion processing and regulation. vmPFC dysfunction may lead to disinhibition of amygdala causing high anxiety levels. γ-Aminobutyric acid (GABA) inter-neurons within vmPFC shape the information flow to amygdala. Thus, we hypothesize that GABA content within vmPFC could be relevant to trait anxiety. Forty-three healthy volunteers aged between 20 and 88 years were assessed for trait anxiety with the Subscale-2 of the State-Trait-Anxiety Inventory (STAI-Y2) and were studied with proton magnetic resonance spectroscopy to investigate GABA and Glx (glutamate+glutamine) contents within vmPFC. Total creatine (tCr) was used as internal reference. Partial correlations assessed the association between metabolite levels and STAI-Y2 scores, removing the effect of possible nuisance factors including age, educational level, volumes of gray matter and white matter within magnetic resonance spectroscopy voxel. We observed a positive relationship between GABA/tCr and STAI-Y2 scores. No significant relationships were found between Glx/tCr and STAI-Y2 and between tCr/water and STAI-Y2. No differences were found between males and females as regards to age, STAI-Y2, GABA/tCr, Glx/tCr, tCr/water, gray matter and white matter volumes. We suggest a close relationship between GABA content within vmPFC and trait anxiety providing new insights in the physiology of emotional brain. PMID:26722018

  13. Valerian Inhibits Rat Hepatocarcinogenesis by Activating GABA(A) Receptor-Mediated Signaling

    PubMed Central

    Kakehashi, Anna; Kato, Ayumi; Ishii, Naomi; Wei, Min; Morimura, Keiichirou; Fukushima, Shoji; Wanibuchi, Hideki

    2014-01-01

    Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P+) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2′-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P+ foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21Waf1/Cip1, p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P+ foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P+ foci by activating GABA(A)R-mediated signaling. PMID:25419570

  14. Effects of benzodiazepines and non-benzodiazepine compounds on the GABA-induced response in frog isolated sensory neurones.

    PubMed Central

    Yakushiji, T.; Fukuda, T.; Oyama, Y.; Akaike, N.

    1989-01-01

    1. The effects of benzodiazepines and non-benzodiazepine compounds on the gamma-aminobutyric acid (GABA)-induced chloride current (ICl) were studied in frog isolated sensory neurones by use of a concentration-jump (termed 'concentration-clamp') technique, under single-electrode voltage-clamp conditions. The drugs used were classified into four categories as follows: full benzodiazepine receptor agonists (diazepam, clonazepam, nitrazepam, midazolam, clotiazepam and etizolam), partial agonists (CL 218,872, Ro 16-6028, Ro 17-1812 and Ro 23-0364), inverse agonists (Ro 15-3505, FG 7142 and beta-CCE) and a benzodiazepine receptor antagonist, Ro 15-1788 (flumazenil). 2. All full agonists at concentrations of 3 x 10(-6) M or less increased dose-dependently the peak amplitude of ICl elicited by 3 x 10(-6) M GABA to twice to three times larger than the control. However, no further augmentation of the GABA response was observed at concentrations of 1 x 10(-5) M or higher. Partial agonists also showed a dose-dependent augmentation of the GABA response at concentrations ranging from 3 x 10(-8) M to 3 x 10(-5) M, but their efficacies of augmentation of the GABA response were only about half or less of those of full agonists. Of the inverse agonists, beta-CCE had a unique dose-dependent effect on the GABA response. Beta-CCE reduced dose-dependently the GABA response at concentrations of less than 3 x 10(-6) M, but augmented it at concentrations of 3 x 10(-5) M and 6 x 10(-5) M. The inverse agonists reduced dose-dependently the GABA response.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2574062

  15. GABA(A) receptors in visual and auditory cortex and neural activity changes during basic visual stimulation.

    PubMed

    Qin, Pengmin; Duncan, Niall W; Wiebking, Christine; Gravel, Paul; Lyttelton, Oliver; Hayes, Dave J; Verhaeghe, Jeroen; Kostikov, Alexey; Schirrmacher, Ralf; Reader, Andrew J; Northoff, Georg

    2012-01-01

    Recent imaging studies have demonstrated that levels of resting γ-aminobutyric acid (GABA) in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABA(A) receptors, in the changes in brain activity between the eyes closed (EC) and eyes open (EO) state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: an EO and EC block design, allowing the modeling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [(18)F]Flumazenil PET to measure GABA(A) receptor binding potentials. It was demonstrated that the local-to-global ratio of GABA(A) receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABA(A) receptor binding potential in the visual cortex also predicted the change in functional connectivity between the visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABA(A) receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity. PMID:23293594

  16. Plasmodium falciparum Malaria Elicits Inflammatory Responses that Dysregulate Placental Amino Acid Transport

    PubMed Central

    Boeuf, Philippe; Aitken, Elizabeth H.; Chandrasiri, Upeksha; Chua, Caroline Lin Lin; McInerney, Bernie; McQuade, Leon; Duffy, Michael; Molyneux, Malcolm; Brown, Graham; Glazier, Jocelyn; Rogerson, Stephen J.

    2013-01-01

    Placental malaria (PM) can lead to poor neonatal outcomes, including low birthweight due to fetal growth restriction (FGR), especially when associated with local inflammation (intervillositis or IV). The pathogenesis of PM-associated FGR is largely unknown, but in idiopathic FGR, impaired transplacental amino acid transport, especially through the system A group of amino acid transporters, has been implicated. We hypothesized that PM-associated FGR could result from impairment of transplacental amino acid transport triggered by IV. In a cohort of Malawian women and their infants, the expression and activity of system A (measured by Na+-dependent 14C-MeAIB uptake) were reduced in PM, especially when associated with IV, compared to uninfected placentas. In an in vitro model of PM with IV, placental cells exposed to monocyte/infected erythrocytes conditioned medium showed decreased system A activity. Amino acid concentrations analyzed by reversed phase ultra performance liquid chromatography in paired maternal and cord plasmas revealed specific alterations of amino acid transport by PM, especially with IV. Overall, our data suggest that the fetoplacental unit responds to PM by altering its placental amino acid transport to maintain adequate fetal growth. However, IV more profoundly compromises placental amino acid transport function, leading to FGR. Our study offers the first pathogenetic explanation for FGR in PM. PMID:23408887

  17. Antigen-specific stimulation of amino acid transport in bovine lymphocytes

    SciTech Connect

    Tate, E.H.

    1982-01-01

    Treatment of bovine lymphocytes isolated from animals which were either infected with Mycobacterium bovis or sensitized to a purified protein derivative (PPD-B) from this organism induced an increase in the transport of a-aminoisobutyric acid (AIB) and a-methylaminoisobutyric acid (MeAIB). PPD-B did not stimulate these transport activities in lymphocytes from nonsensitized animals. The transport stimulation was first measurable after about 7 hours of treatment, reached about a two-fold enhancement after 20 hours, and continued to increase to 30- to 40-fold after 6 days. The stimulation of AIB transport was inhibited by both ouabain and cycloheximide. Experiments to determine transport system specificities in nonstimulated lymphocytes showed that MeAIB transport was primarily by the Na/sup +/-dependent, A-system,and leucine transport was mostly by Na/sup +/-independent system(s). In contrast, AIB transport was about 25% by the A-system, 25% by at least one Na/sup +/-dependent, non-A-system, and 50% by one or more Na/sup +/-independent system(s). Analysis of the three components of AIB transport after treatment with PPD-B showed that: 1) transport by both the A-system and the Na/sup +/-independent system(s) was stimulated; 2) A-system transport was stimulated to a larger extent than Na/sup +/-independent transport; and 3) Na/sup +/-dependent, non-A-system transport was not stimulated significantly.

  18. Synthesis and Pharmacological Properties of Novel Esters Based on Monocyclic Terpenes and GABA

    PubMed Central

    Nesterkina, Mariia; Kravchenko, Iryna

    2016-01-01

    Novel esters of γ-aminobutyric acid (GABA) with monocyclic terpenes were synthesized via Steglich esterification and characterized by 1H-NMR, IR and mass spectral studies. Their anticonvulsant, analgesic and anti-inflammatory activities were evaluated by a PTZ-induced convulsion model, AITC-induced hyperalgesia and AITC-induced paw edema, respectively. All studied esters, as well as their parent terpenes, were found to produce antinociceptive effects in the AITC-induced model and attenuate acute pain more than the reference drug benzocaine after their topical application. GABA esters of l-menthol and thymol were also shown to exceed the reference drug ibuprofen in their ability to decrease the inflammatory state induced by intraplantar injection of the TRPA1 activator AITC. The present findings indicate that GABA esters of carvacrol and guaiacol are not a classical prodrug and possess their own pharmacological activity. Prolonged antiseizure action of the ester based on the amino acid and guaiacol (200 mg/kg) was revealed at 24 h after oral administration. Furthermore, orally co-administered gidazepam (1 mg/kg) and GABA esters of l-menthol, thymol and carvacrol produce synergistic seizure prevention effects. PMID:27304960

  19. Synthesis and Pharmacological Properties of Novel Esters Based on Monocyclic Terpenes and GABA.

    PubMed

    Nesterkina, Mariia; Kravchenko, Iryna

    2016-01-01

    Novel esters of γ-aminobutyric acid (GABA) with monocyclic terpenes were synthesized via Steglich esterification and characterized by ¹H-NMR, IR and mass spectral studies. Their anticonvulsant, analgesic and anti-inflammatory activities were evaluated by a PTZ-induced convulsion model, AITC-induced hyperalgesia and AITC-induced paw edema, respectively. All studied esters, as well as their parent terpenes, were found to produce antinociceptive effects in the AITC-induced model and attenuate acute pain more than the reference drug benzocaine after their topical application. GABA esters of l-menthol and thymol were also shown to exceed the reference drug ibuprofen in their ability to decrease the inflammatory state induced by intraplantar injection of the TRPA1 activator AITC. The present findings indicate that GABA esters of carvacrol and guaiacol are not a classical prodrug and possess their own pharmacological activity. Prolonged antiseizure action of the ester based on the amino acid and guaiacol (200 mg/kg) was revealed at 24 h after oral administration. Furthermore, orally co-administered gidazepam (1 mg/kg) and GABA esters of l-menthol, thymol and carvacrol produce synergistic seizure prevention effects. PMID:27304960

  20. [Anti-arrhythmic properties of GABA and GABA-ergic system activators].

    PubMed

    Tiurenkov, I N; Perfilova, V N

    2002-01-01

    Clinical and experimental data available in the literature are summarized, which are indicative of the antiarrhythmogenic properties of GABA and substances possessing GABA-positive activity (phenibut, piracetam, sodium hydroxybutyrate, lithium hydroxybutyrate, etc.). The antiarrhythmic effects are manifested in various cases of the heart rhythm violation. The mechanism of this action is related to activation of the central and peripheral retarding GABAergic system, as well as to antihypoxant, antioxidant, and antistressor effects. PMID:12025796

  1. Colocalization of synaptic GABA(C)-receptors with GABA (A)-receptors and glycine-receptors in the rodent central nervous system.

    PubMed

    Frazao, Renata; Nogueira, Maria Ines; Wässle, Heinz

    2007-10-01

    Fast inhibition in the nervous system is preferentially mediated by GABA- and glycine-receptors. Two types of ionotropic GABA-receptor, the GABA(A)-receptor and GABA(C)-receptor, have been identified; they have specific molecular compositions, different sensitivities to GABA, different kinetics, and distinct pharmacological profiles. We have studied, by immunocytochemistry, the synaptic localization of glycine-, GABA(A)-, and GABA(C)-receptors in rodent retina, spinal cord, midbrain, and brain-stem. Antibodies specific for the alpha1 subunit of the glycine-receptor, the gamma2 subunit of the GABA(A)-receptor, and the rho subunits of the GABA(C)-receptor have been applied. Using double-immunolabeling, we have determined whether these receptors are expressed at the same postsynaptic sites. In the retina, no such colocalization was observed. However, in the spinal cord, we found the colocalization of glycine-receptors with GABA(A)- or GABA(C)-receptors and the colocalization of GABA(A)- and GABA(C)-receptors in approximately 25% of the synapses. In the midbrain and brain-stem, GABA(A)- and GABA(C)-receptors were colocalized in 10%-15% of the postsynaptic sites. We discuss the possible expression of heteromeric (hybrid) receptors assembled from GABA(A)- and GABA(C)-receptor subunits. Our results suggest that GABA(A)- and GABA(C)-receptors are colocalized in a minority of synapses of the central nervous system. PMID:17610086

  2. 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.

  3. Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii.

    PubMed

    Jiménez-Vázquez, Eric N; Díaz-Velásquez, Clara E; Uribe, R M; Arias, Juan M; García, Ubaldo

    2016-02-01

    Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA β subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates. PMID:26577600

  4. 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. PMID:26644273

  5. GABA binding to an insect GABA receptor: a molecular dynamics and mutagenesis study.

    PubMed

    Ashby, Jamie A; McGonigle, Ian V; Price, Kerry L; Cohen, Netta; Comitani, Federico; Dougherty, Dennis A; Molteni, Carla; Lummis, Sarah C R

    2012-11-21

    RDL receptors are GABA-activated inhibitory Cys-loop receptors found throughout the insect CNS. They are a key target for insecticides. Here, we characterize the GABA binding site in RDL receptors using computational and electrophysiological techniques. A homology model of the extracellular domain of RDL was generated and GABA docked into the binding site. Molecular dynamics simulations predicted critical GABA binding interactions with aromatic residues F206, Y254, and Y109 and hydrophilic residues E204, S176, R111, R166, S176, and T251. These residues were mutated, expressed in Xenopus oocytes, and their functions assessed using electrophysiology. The data support the binding mechanism provided by the simulations, which predict that GABA forms many interactions with binding site residues, the most significant of which are cation-π interactions with F206 and Y254, H-bonds with E204, S205, R111, S176, T251, and ionic interactions with R111 and E204. These findings clarify the roles of a range of residues in binding GABA in the RDL receptor, and also show that molecular dynamics simulations are a useful tool to identify specific interactions in Cys-loop receptors. PMID:23200041

  6. Characteristics of the transport of ascorbic acid into leucocytes

    SciTech Connect

    Raghoebar, M.; Huisman, J.A.M.; van den Berg, W.B.; van Ginneken, C.A.M.

    1987-02-02

    The degree and the mode of association of (/sup 14/C)-ascorbic acid with leucocytes are examined. The degree of association of ascorbic acid with polymorphonuclear leucocytes (1-3 %) is dependent on cell type, extracellular concentration of ascorbic acid, incubation temperature, intactness of the cells and the extracellular pH. All experiments are performed according to strict protocols as these compounds are labile in aqueous solutions. Further it is noticed that in all experiments an outward gradient of leucocyte endogenic ascorbic acid exists. The results suggest that the association process comprises at least one saturable pathway. The activation of polymorphonuclear leucocytes by phorbol myristate acetate increases the accumulation of ascorbic acid threefold. 30 references, 7 figures, 3 tables.

  7. Regulation of amino acid transporters in pluripotent cell populations in the embryo and in culture; novel roles for sodium-coupled neutral amino acid transporters.

    PubMed

    Tan, Boon Siang Nicholas; Rathjen, Peter D; Harvey, Alexandra J; Gardner, David K; Rathjen, Joy

    2016-08-01

    The developmental outcomes of preimplantation mammalian embryos are regulated directly by the surrounding microenvironment, and inappropriate concentrations of amino acids, or the loss of amino acid-sensing mechanisms, can be detrimental and impact further development. A specific role for l-proline in the differentiation of embryonic stem (ES) cells, a cell population derived from the blastocyst, has been shown in culture. l-proline acts as a signalling molecule, exerting its effects through cell uptake and subsequent metabolism. Uptake in ES cells occurs predominantly through the sodium-coupled neutral amino acid transporter 2, Slc38a2 (SNAT2). Dynamic expression of amino acid transporters has been shown in the early mammalian embryo, reflecting functional roles for amino acids in embryogenesis. The expression of SNAT2 and family member Slc38a1 (SNAT1) was determined in mouse embryos from the 2-cell stage through to the early post-implantation pre-gastrulation embryo. Key changes in expression were validated in cell culture models of development. Both transporters showed temporal dynamic expression patterns and changes in intracellular localisation as differentiation progressed. Changes in transporter expression likely reflect different amino acid requirements during development. Findings include the differential expression of SNAT1 in the inner and outer cells of the compacted morula and nuclear localisation of SNAT2 in the trophectoderm and placental lineages. Furthermore, SNAT2 expression was up-regulated in the epiblast prior to primitive ectoderm formation, an expression pattern consistent with a role for the transporter in later developmental decisions within the pluripotent lineage. We propose that the differential expression of SNAT2 in the epiblast provides evidence for an l-proline-mediated mechanism contributing to the regulation of embryonic development. PMID:27373508

  8. Double isotopic method using dansyl chloride for the determination of GABA in rat C6 astrocytoma cell cultures

    SciTech Connect

    Kohl, R.L.; Quay, W.B.; Perez-Polo, J.R.

    1986-01-01

    Methods are described for the quantitative measurement of GABA in culture. The method can be adapted to any amino acid or dansyl-chloride-reactive species. The sensitivity and selectivity of the procedure result from the double isotopic design in which (/sup 14/C)-labeled internal standard was added to the samples before reaction with (3M)-labeled dansyl chloride. Values obtained by ion-exchange amino acid analysis of cultures agree closely with the values obtained by the double isotopic method. This method is sensitive enough to measure GABA intracellularly and the condition medium.

  9. GABA{sub A} receptor open-state conformation determines non-competitive antagonist binding

    SciTech Connect

    Chen Ligong; Xue Ling; Giacomini, Kathleen M.; Casida, John E.

    2011-02-01

    The {gamma}-aminobutyric acid (GABA) type A receptor (GABA{sub A}R) is one of the most important targets for insecticide action. The human recombinant {beta}3 homomer is the best available model for this binding site and 4-n-[{sup 3}H]propyl-4'-ethynylbicycloorthobenzoate ([{sup 3}H]EBOB) is the preferred non-competitive antagonist (NCA) radioligand. The uniquely high sensitivity of the {beta}3 homomer relative to the much-less-active but structurally very-similar {beta}1 homomer provides an ideal comparison to elucidate structural and functional features important for NCA binding. The {beta}1 and {beta}3 subunits were compared using chimeragenesis and mutagenesis and various combinations with the {alpha}1 subunit and modulators. Chimera {beta}3/{beta}1 with the {beta}3 subunit extracellular domain and the {beta}1 subunit transmembrane helices retained the high [{sup 3}H]EBOB binding level of the {beta}3 homomer while chimera {beta}1/{beta}3 with the {beta}1 subunit extracellular domain and the {beta}3 subunit transmembrane helices had low binding activity similar to the {beta}1 homomer. GABA at 3 {mu}M stimulated heteromers {alpha}1{beta}1 and {alpha}1{beta}3 binding levels more than 2-fold by increasing the open probability of the channel. Addition of the {alpha}1 subunit rescued the inactive {beta}1/{beta}3 chimera close to wildtype {alpha}1{beta}1 activity. EBOB binding was significantly altered by mutations {beta}1S15'N and {beta}3N15'S compared with wildtype {beta}1 and {beta}3, respectively. However, the binding activity of {alpha}1{beta}1S15'N was insensitive to GABA and {alpha}1{beta}3N15'S was stimulated much less than wildtype {alpha}1{beta}3 by GABA. The inhibitory effect of etomidate on NCA binding was reduced more than 5-fold by the mutation {beta}3N15'S. Therefore, the NCA binding site is tightly regulated by the open-state conformation that largely determines GABA{sub A} receptor sensitivity. - Graphical Abstract: Display Omitted Research Highlights

  10. Transport of hop bitter acids across intestinal Caco-2 cell monolayers.

    PubMed

    Cattoor, Ko; Bracke, Marc; Deforce, Dieter; De Keukeleire, Denis; Heyerick, Arne

    2010-04-14

    Several health-beneficial properties of hop bitter acids have been reported (inhibition of bone resorption and anticarcinogenic and anti-inflammatory activities); however, scientific data on the bioavailability of these compounds are lacking. As a first approach to study the bioavailability, the epithelial transport of hop alpha- and beta-acids across Caco-2 monolayers was investigated. Hop acids were added either to the apical or to the basolateral chamber and, at various time points, amounts transported to the receiving compartment were determined. The monolayer integrity control was performed by using marker compounds (atenolol and propranolol), transepithelial electrical resistance (TEER) measurement, and determination of the fluorescein efflux. The TEER and fluorescein efflux confirmed the preservation of the monolayer integrity. The membrane permeability of the alpha-acids (apparent permeability coefficients for apical to basolateral transport (P(appAB)) ranged from 14 x 10(-6) to 41 x 10(-6) cm/s) was determined to be substantially higher than that of the beta-acids (P(appAB) values ranging from 0.9 x 10(-6) to 2.1 x 10(-6) cm/s). Notably, the beta-acids exhibited significantly different bidirectional P(app) values with efflux ratios around 10. The involvement of carrier-mediated transport for beta-acids (active efflux pathway by P-gp, BCRP, and/or MRP-2 type efflux pumps) could be confirmed by transport experiments with specific inhibitors (verapamil and indomethacin). It appears that alpha-acids are efficiently absorbed, whereas the permeability of beta-acids is low. Limiting factors in the absorption of beta-acids could involve P-gp and MRP-2 type efflux transporters and phase II metabolism. PMID:20329731

  11. Aphid amino acid transporter regulates glutamine supply to intracellular bacterial symbionts.

    PubMed

    Price, Daniel R G; Feng, Honglin; Baker, James D; Bavan, Selvan; Luetje, Charles W; Wilson, Alex C C

    2014-01-01

    Endosymbiotic associations have played a major role in evolution. However, the molecular basis for the biochemical interdependence of these associations remains poorly understood. The aphid-Buchnera endosymbiosis provides a powerful system to elucidate how these symbioses are regulated. In aphids, the supply of essential amino acids depends on an ancient nutritional symbiotic association with the gamma-proteobacterium Buchnera aphidicola. Buchnera cells are densely packed in specialized aphid bacteriocyte cells. Here we confirm that five putative amino acid transporters are highly expressed and/or highly enriched in Acyrthosiphon pisum bacteriocyte tissues. When expressed in Xenopus laevis oocytes, two bacteriocyte amino acid transporters displayed significant levels of glutamine uptake, with transporter ACYPI001018, LOC100159667 (named here as Acyrthosiphon pisum glutamine transporter 1, ApGLNT1) functioning as the most active glutamine transporter. Transporter ApGLNT1 has narrow substrate selectivity, with high glutamine and low arginine transport capacity. Notably, ApGLNT1 has high binding affinity for arginine, and arginine acts as a competitive inhibitor for glutamine transport. Using immunocytochemistry, we show that ApGLNT1 is localized predominantly to the bacteriocyte plasma membrane, a location consistent with the transport of glutamine from A. pisum hemolymph to the bacteriocyte cytoplasm. On the basis of functional transport data and localization, we propose a substrate feedback inhibition model in which the accumulation of the essential amino acid arginine in A. pisum hemolymph reduces the transport of the precursor glutamine into bacteriocytes, thereby regulating amino acid biosynthesis in the bacteriocyte. Structural similarities in the arrangement of hosts and symbionts across endosymbiotic systems suggest that substrate feedback inhibition may be mechanistically important in other endosymbioses. PMID:24367072

  12. The orally active antihyperglycemic drug beta-guanidinopropionic acid is transported by the human proton-coupled amino acid transporter hPAT1.

    PubMed

    Metzner, Linda; Dorn, Madlen; Markwardt, Fritz; Brandsch, Matthias

    2009-01-01

    The orally administered creatine analogue beta-guanidinopropionic acid (beta-GPA) decreases plasma glucose levels by increasing the sensitivity to insulin. This effect is based on a beta-GPA induced expression of mRNA and total protein content of the insulin-responsive glucose transporter GLUT4. Although the oral availability of beta-GPA is well established, the underlying uptake mechanism has not yet been studied. We investigated whether the H(+)-coupled amino acid transporter PAT1, which is expressed in the apical membrane of intestinal cells, accepts guanidine derivatives as substrates. Uptake of l-[(3)H]proline into Caco-2 cells expressing hPAT1 constitutively was strongly inhibited by beta-GPA and its derivatives guanidinoacetic acid (GAA) and 4-guanidinobutyric acid (4-GBA). Competition assays revealed apparent affinity constants of about 1.5 mM. Electrophysiological measurements at hPAT1-expressing Xenopus laevis oocytes unequivocally demonstrated that beta-GPA, GAA and 4-GBA are effectively transported by this transport system in an electrogenic manner. We conclude that hPAT1 might be responsible for the intestinal absorption of beta-GPA thereby allowing its oral administration. Moreover, with beta-GPA we identified a new high affinity hPAT1 substrate that might be an interesting starting point for future drug design-drug delivery strategies. PMID:19358571

  13. A binding mode hypothesis of tiagabine confirms liothyronine effect on γ-aminobutyric acid transporter 1 (GAT1).

    PubMed

    Jurik, Andreas; Zdrazil, Barbara; Holy, Marion; Stockner, Thomas; Sitte, Harald H; Ecker, Gerhard F

    2015-03-12

    Elevating GABA levels in the synaptic cleft by inhibiting its reuptake carrier GAT1 is an established approach for the treatment of CNS disorders like epilepsy. With the increasing availability of crystal structures of transmembrane transporters, structure-based approaches to elucidate the molecular basis of ligand-transporter interaction also become feasible. Experimental data guided docking of derivatives of the GAT1 inhibitor tiagabine into a protein homology model of GAT1 allowed derivation of a common binding mode for this class of inhibitors that is able to account for the distinct structure-activity relationship pattern of the data set. Translating essential binding features into a pharmacophore model followed by in silico screening of the DrugBank identified liothyronine as a drug potentially exerting a similar effect on GAT1. Experimental testing further confirmed the GAT1 inhibiting properties of this thyroid hormone. PMID:25679268

  14. A Binding Mode Hypothesis of Tiagabine Confirms Liothyronine Effect on γ-Aminobutyric Acid Transporter 1 (GAT1)

    PubMed Central

    2015-01-01

    Elevating GABA levels in the synaptic cleft by inhibiting its reuptake carrier GAT1 is an established approach for the treatment of CNS disorders like epilepsy. With the increasing availability of crystal structures of transmembrane transporters, structure-based approaches to elucidate the molecular basis of ligand–transporter interaction also become feasible. Experimental data guided docking of derivatives of the GAT1 inhibitor tiagabine into a protein homology model of GAT1 allowed derivation of a common binding mode for this class of inhibitors that is able to account for the distinct structure–activity relationship pattern of the data set. Translating essential binding features into a pharmacophore model followed by in silico screening of the DrugBank identified liothyronine as a drug potentially exerting a similar effect on GAT1. Experimental testing further confirmed the GAT1 inhibiting properties of this thyroid hormone. PMID:25679268

  15. Utilization of Lactic Acid by Fusarium oxysporum var. lini: Regulation of Transport and Metabolism

    PubMed Central

    Castro, Ieso M.; Loureiro-Dias, Maria C.

    1994-01-01

    Lactic acid was transported in Fusarium oxysporum var. lini ATCC 10960 by a saturable transport system that had a half-saturation constant of 56.6 ± 7.5 μM and a maximum velocity of 0.61 ± 0.10 mmol h-1 g-1 (dry weight) at 26°C and pH 5.0. This transport system was inducible and was not expressed in the presence of a repressing substrate. Evidence is presented that the anionic form lactate- was taken up by the cells. Propionic, acetic, pyruvic, and bromoacetic acids but not succinic acid competitively inhibited the transport of lactic acid. Bromoacetic acid, which was not metabolized, was taken up to a steady-state level when intracellular and extracellular concentrations were identical, indicating that the transport system was not accumulative. The enzymatic activity that was physiologically more relevant in the metabolism of lactic acid was lactate: ferricytochrome c oxidase. This enzyme did not exhibit stereospecifity and was induced by lactic acid. PMID:16349143

  16. Role of organic acids in promoting colloidal transport of mercury from mine tailings

    USGS Publications Warehouse

    Slowey, A.J.; Johnson, S.B.; Rytuba, J.J.; Brown, Gordon E., Jr.

    2005-01-01

    A number of factors affect the transport of dissolved and paniculate mercury (Hg) from inoperative Hg mines, including the presence of organic acids in the rooting zone of vegetated mine waste. We examined the role of the two most common organic acids in soils (oxalic and citric acid) on Hg transport from such waste by pumping a mixed organic acid solution (pH 5.7) at 1 mL/min through Hg mine tailings columns. For the two total organic acid concentrations investigated (20 ??M and 1 mM), particle-associated Hg was mobilized, with the onset of paniculate Hg transport occurring later for the lower organic acid concentration. Chemical analyses of column effluent indicate that 98 wt % of Hg mobilized from the column was paniculate. Hg speciation was determined using extended X-ray absorption fine structure spectroscopy and transmission electron microscopy, showing that HgS minerals are dominant in the mobilized particles. Hg adsorbed to colloids is another likely mode of transport due to the abundance of Fe-(oxyhydr)oxides, Fe-sulfides, alunite, and jarosite in the tailings to which Hg(II) adsorbs. Organic acids produced by plants are likely to enhance the transport of colloid-associated Hg from vegetated Hg mine tailings by dissolving cements to enable colloid release. ?? 2005 American Chemical Society.

  17. Characterization of methylaminoisobutyric acid transport by system A in rat mammary gland.

    PubMed

    Tovar, A R; Avila, E; DeSantiago, S; Torres, N

    2000-07-01

    During lactation, the mammary gland has a large demand for amino acids for the synthesis of milk proteins and fatty acids. Arteriovenous differences in amino acids across the mammary gland show an elevated uptake of small neutral amino acids that are mainly transported via system A. The purpose of this study was to characterize the transport of methylaminoisobutyric acid (MeAIB), an amino acid analog used to model transport by system A in lactating rat mammary gland explants. MeAIB accumulation in mammary gland cells increased steadily, and after 3 hours of incubation, the intracellular concentration of the analog was 8-fold higher than the concentration in the medium. MeAIB transport into mammary gland explants showed a Km of 3.3 +/- 0.4 mmol/L and a maximal velocity (Vmax) of 555 +/- 23 pmol/microL intracellular fluid (ICF) x min, indicating a system with high capacity but low affinity for its substrate. MeAIB transport into mammary tissue depended highly on Na+, and the uptake was inhibited by addition of natural and analog small neutral amino acids. Cationic, anionic, and large neutral amino acids did not reduce MeAIB transport into mammary gland explants. Preincubation of mammary gland explants in an amino acid-free medium stimulated MeAIB transport, suggesting an adaptive regulation. The addition of an equimolar mixture of alanine, glycine, and serine to the preincubation medium inhibited stimulation of MeAIB transport. Furthermore, stimulation of MeAIB uptake by amino acid starvation was also prevented by the addition of actinomycin D, cycloheximide, tunicamycin, and colchicine. Dibutyryl cyclic adenosine monophosphate (cAMP) increased MeAIB uptake, whereas phorbol 12-myristate 13-acetate (PMA) did not stimulate MeAIB transport. During the first postweaning days, kinetic analyses showed a decrease of 27% in the Vmax. Injection of rat lactating mammary gland mRNA into Xenopus laevis oocytes induced expression of the MeAIB transport system; however, the

  18. LHT1, a lysine- and histidine-specific amino acid transporter in arabidopsis.

    PubMed Central

    Chen, L; Bush, D R

    1997-01-01

    We have identified a new amino acid transporter from the Arabidopsis thaliana expressed sequence tag cDNA collection by functional complementation of a yeast amino acid transport mutant. Transport analysis of the expressed protein in yeast shows that it is a high-affinity transporter for both lysine (Lys) and histidine with Michaelis constant values of 175 and 400 microM, respectively. This transporter (LHT1, lysine histidine transporter) has little affinity for arginine when measured directly in uptake experiments or indirectly with substrate competition. The cDNA is 1.7 kb with an open reading frame that codes for a protein with 446 amino acids and a calculated molecular mass of 50.5 kD. Hydropathy analysis shows that LHT1 is an integral membrane protein with 9 to 10 putative membrane-spanning domains. Southern-blot analysis suggests that LHT1 is a single-copy gene in the Arabidopsis genome. RNA gel-blot analysis shows that this transporter is present in all tissues, with the strongest expression in young leaves, flowers, and siliques. Wholemount, in situ hybridization revealed that expression is further localized on the surface of roots in young seedlings and in pollen. Overall, LHT1 belongs to a new class of amino acid transporter that is specific for Lys and histidine, and, given its substrate specificity, it has significant promise as a tool for improving the Lys content of Lys-deficient grains. PMID:9390441

  19. Regulation of amino acid transport in Escherichia coli by transcription termination factor rho.

    PubMed Central

    Quay, S C; Oxender, D L

    1977-01-01

    Amino acid transport rates and amino acid binding proteins were examined in a strain containing the rho-120 mutation (formerly SuA), which has been shown to lower the rho-dependent, ribonucleic acid-activated adenosine triphosphatase activity to 9% of the rho activity in the isogenic wild-type strain. Tryptophan and proline transport, which occur by membrane-bound systems, were not altered. On the other hand, arginine, histidine, leucine, isoleucine, and valine transport were variably increased by a factor of 1.4 to 5.0. Kinetics of leucine transport showed that the LIV (leucine, isoleucine, and valine)-I (binding protein-associated) transport system is increased 8.5-fold, whereas the LIV-II (membrane-bound) system is increased 1.5-fold in the rho mutant under leucine-limited growth conditions. The leucine binding protein is increased fourfold under the same growth conditions. The difference in leucine transport in these strains was greatest during leucine-limited growth; growth on complex media repressed both strains to the same transport activity. We propose that rho-dependent transcriptional termination is important for leucine-specific repression of branched-chain amino acid transport, although rho-independent regulation, presumably by a corepressor-aporepressor-type mechanism, must also occur. PMID:324970

  20. Maternal bile acid transporter deficiency promotes neonatal demise

    PubMed Central

    Zhang, Yuanyuan; Li, Fei; Wang, Yao; Pitre, Aaron; Fang, Zhong-ze; Frank, Matthew W.; Calabrese, Christopher; Krausz, Kristopher W.; Neale, Geoffrey; Frase, Sharon; Vogel, Peter; Rock, Charles O.; Gonzalez, Frank J.; Schuetz, John D.

    2015-01-01

    Intrahepatic cholestasis of pregnancy (ICP) is associated with adverse neonatal survival and is estimated to impact between 0.4 and 5% of pregnancies worldwide. Here we show that maternal cholestasis (due to Abcb11 deficiency) produces neonatal death among all offspring within 24 h of birth due to atelectasis-producing pulmonary hypoxia, which recapitulates the neonatal respiratory distress of human ICP. Neonates of Abcb11-deficient mothers have elevated pulmonary bile acids and altered pulmonary surfactant structure. Maternal absence of Nr1i2 superimposed on Abcb11 deficiency strongly reduces maternal serum bile acid concentrations and increases neonatal survival. We identify pulmonary bile acids as a key factor in the disruption of the structure of pulmonary surfactant in neonates of ICP. These findings have important implications for neonatal respiratory failure, especially when maternal bile acids are elevated during pregnancy, and highlight potential pathways and targets amenable to therapeutic intervention to ameliorate this condition. PMID:26416771

  1. Transportation impact analysis for the shipment of low specific activity nitric acid. Revisison 1

    SciTech Connect

    Green, J.R.

    1995-05-16

    This is in support of the Plutonium-Uranium Extraction (PUREX) Facility Low Specific Activity (LSA) Nitric Acid Shipment Environmental Assessment. It analyzes potential toxicological and radiological risks associated with transportation of PUREX Facility LSA Nitric Acid from the Hanford Site to Portsmouth VA, Baltimore MD, and Port Elizabeth NJ.

  2. Chronic stress shifts the GABA reversal potential in the hippocampus and increases seizure susceptibility.

    PubMed

    MacKenzie, Georgina; Maguire, Jamie

    2015-01-01

    The most commonly reported precipitating factor for seizures is stress. However, the underlying mechanisms whereby stress triggers seizures are not yet fully understood. Here we demonstrate a potential mechanism underlying changes in neuronal excitability in the hippocampus following chronic stress, involving a shift in the reversal potential for GABA (EGABA) associated with a dephosphorylation of the potassium chloride co-transporter, KCC2. Mice subjected to chronic restraint stress (30min/day for 14 consecutive days) exhibit an increase in serum corticosterone levels which is associated with increased susceptibility to seizures induced with kainic acid (20mg/kg). Following chronic stress, but not acute stress, we observe a dephosphorylation of KCC2 residue S940, which regulates KCC2 cell surface expression and function, in the hippocampus. To determine the impact of alterations in KCC2 expression following chronic stress, we performed gramicidin perforated patch recordings to measure changes in EGABA and neuronal excitability of principal hippocampal neurons. We observe a depolarizing shift in EGABA in hippocampal CA1 pyramidal neurons after chronic stress. In addition, there is an increase in the intrinsic excitability of CA1 pyramidal neurons, evident by a shift in the input-output curve which could be reversed with the NKCC1 inhibitor, bumetanide. These data uncover a potential mechanism involving chronic stress-induced plasticity in chloride homeostasis which may contribute to stress-induced seizure susceptibility. PMID:25524838

  3. Chronic stress shifts the GABA reversal potential in the hippocampus and increases seizure susceptibility

    PubMed Central

    MacKenzie, Georgina; Maguire, Jamie

    2014-01-01

    The most commonly reported precipitating factor for seizures is stress. However, the underlying mechanisms whereby stress triggers seizures are not yet fully understood. Here we demonstrate a potential mechanism underlying changes in neuronal excitability in the hippocampus following chronic stress, involving a shift in the reversal potential for GABA (EGABA) associated with a dephosphorylation of the potassium chloride co-transporter, KCC2. Mice subjected to chronic restraint stress (30 mins/day for 14 consecutive days) exhibit an increase in serum corticosterone levels which is associated with increased susceptibility to seizures induced with kainic acid (20 mg/kg). Following chronic stress, but not acute stress, we observe a dephosphorylation of KCC2 residue S940, which regulates KCC2 cell surface expression and function, in the hippocampus. To determine the impact of alterations in KCC2 expression following chronic stress, we performed gramicidin perforated patch recordings to measure changes in EGABA and neuronal excitability of principal hippocampal neurons. We observe a depolarizing shift in EGABA in hippocampal CA1 pyramidal neurons after chronic stress. In addition, there is an increase in the intrinsic excitability of CA1 pyramidal neurons, evident by a shift in the input-output curve which could be reversed with the NKCC1 inhibitor, bumetanide. These data uncover a potential mechanism involving chronic stress-induced plasticity in chloride homeostasis which may contribute to stress-induced seizure susceptibility. PMID:25524838

  4. Ion Transport Dynamics in Acid Variable Charge Subsoils

    SciTech Connect

    Qafoku, Nik; Sumner, Malcolm E.; Toma, Mitsuru

    2005-06-06

    This is a mini-review of the research work conducted by the authors with the objective of studying ion transport in variable charge subsoils collected from different areas around the world. An attempt is made in these studies to relate the unique behavior manifested during ionic transport in these subsoils with their mineralogical, physical and chemical properties, which are markedly different from those in soils from temperate regions. The variable charge subsoils have a relatively high salt sorption capacity and anion exchange capacity (AEC) that retards anions downward movement. The AEC correlates closely with the anion retardation coefficients. Ca2+ applied with gypsum in topsoil may be transported to the subsoil and may improve the subsoil chemical properties. These results may help in developing appropriate management strategies under a range of mineralogical, physical, and chemical conditions.

  5. Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot ( Scophthalmus maximus L.)

    NASA Astrophysics Data System (ADS)

    Xu, Dandan; He, Gen; Mai, Kangsen; Zhou, Huihui; Xu, Wei; Song, Fei

    2016-04-01

    Turbot ( Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B0-type amino acid transporter 1 (B0AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y+L-type amino acid transporter 1 (y+LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y+L-type amino acid transporter 2 (y+LAT2) abundantly expressed in stomach. In addition, system b0,+ transporters (rBAT and b0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

  6. Redirection of Metabolic Flux into Novel Gamma-Aminobutyric Acid Production Pathway by Introduction of Synthetic Scaffolds Strategy in Escherichia Coli.

    PubMed

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

    2016-04-01

    In general, gamma-aminobutyric acid (GABA) pathway involves the decarboxylation of glutamate, which is produced from sugar by Corynebacterium fermentation. GABA can be used for the production of pharmaceuticals and functional foods. Due to the increasing demand of GABA, it is essential to create an effective alternative pathway for the GABA production. In this study, Escherichia coli were engineered to produce GABA from glucose via GABA shunt, which consists of succinate dehydrogenase, succinate-semialdehyde dehydrogenase, and GABA aminotransferase. The three enzymes were physically attached to each other through a synthetic scaffold, and the Krebs cycle flux was redirected to the GABA pathway. By introduction of synthetic scaffold, 0.75 g/l of GABA was produced from 10 g/l of glucose at 30 °C and pH 6.5. The inactivation of competing metabolic pathways provided 15.4 % increase in the final GABA concentration. PMID:26667817

  7. Shifted pallidal co-release of GABA and glutamate in habenula drives cocaine withdrawal and relapse.

    PubMed

    Meye, Frank J; Soiza-Reilly, Mariano; Smit, Tamar; Diana, Marco A; Schwarz, Martin K; Mameli, Manuel

    2016-08-01

    Cocaine withdrawal produces aversive states and vulnerability to relapse, hallmarks of addiction. The lateral habenula (LHb) encodes negative stimuli and contributes to aversive withdrawal symptoms. However, it remains unclear which inputs to LHb promote this and what the consequences are for relapse susceptibility. We report, using rabies-based retrolabeling and optogenetic mapping, that the entopeduncular nucleus (EPN, the mouse equivalent of the globus pallidus interna) projects to an LHb neuronal subset innervating aversion-encoding midbrain GABA neurons. EPN-to-LHb excitatory signaling is limited by GABAergic cotransmission. This inhibitory component decreases during cocaine withdrawal as a result of reduced presynaptic vesicular GABA transporter (VGAT). This shifts the EPN-to-LHb GABA/glutamate balance, disinhibiting EPN-driven LHb activity. Selective virally mediated VGAT overexpression at EPN-to-LHb terminals during withdrawal normalizes GABAergic neurotransmission. This intervention rescues cocaine-evoked aversive states and prevents stress-induced reinstatement, used to model relapse. This identifies diminished inhibitory transmission at EPN-to-LHb GABA/glutamate synapses as a mechanism contributing to the relapsing feature of addictive behavior. PMID:27348214

  8. Isothiouronium compounds as gamma-aminobutyric acid agonists.

    PubMed Central

    Allan, R. D.; Dickenson, H. W.; Hiern, B. P.; Johnston, G. A.; Kazlauskas, R.

    1986-01-01

    Analogues of gamma-aminobutyric acid (GABA) incorporating an isothiouronium salt as a replacement for a protonated amino functional group have been investigated for activity on: GABA receptors in the guinea-pig ileum; [3H]-GABA and [3H]-diazepam binding to rat brain membranes; and GABA uptake and transamination. For the homologous series of omega-isothiouronium alkanoic acids, maximum GABA-mimetic activity was found at 3-[(aminoiminomethyl)thio]propanoic acid. Introduction of unsaturation into this compound gave two isomeric conformationally restricted analogues. The trans isomer was inactive at GABA receptors while the cis compound ((Z)-3-[(aminoiminomethyl)thio]prop-2-enoic acid (ZAPA)) was more potent than muscimol and GABA as a GABA agonist with respect to low affinity GABA receptor sites. Both isomers were moderately potent at inhibiting the uptake of [3H]-GABA into rat brain slices. Comparison of possible conformations of the two unsaturated isomers by interactive computer graphics modelling and comparison with muscimol has led to a plausible active conformation of ZAPA, which may be a selective and potent agonist for low affinity GABA binding sites. PMID:3015310

  9. The SLC36 family of proton-coupled amino acid transporters and their potential role in drug transport

    PubMed Central

    Thwaites, David T; Anderson, Catriona MH

    2011-01-01

    Members of the solute carrier (SLC) 36 family are involved in transmembrane movement of amino acids and derivatives. SLC36 consists of four members. SLC36A1 and SLC36A2 both function as H+-coupled amino acid symporters. SLC36A1 is expressed at the luminal surface of the small intestine but is also commonly found in lysosomes in many cell types (including neurones), suggesting that it is a multipurpose carrier with distinct roles in different cells including absorption in the small intestine and as an efflux pathway following intralysosomal protein breakdown. SLC36A1 has a relatively low affinity (Km 1–10 mM) for its substrates, which include zwitterionic amino and imino acids, heterocyclic amino acids and amino acid-based drugs and derivatives used experimentally and/or clinically to treat epilepsy, schizophrenia, bacterial infections, hyperglycaemia and cancer. SLC36A2 is expressed at the apical surface of the human renal proximal tubule where it functions in the reabsorption of glycine, proline and hydroxyproline. SLC36A2 also transports amino acid derivatives but has a narrower substrate selectivity and higher affinity (Km 0.1–0.7 mM) than SLC36A1. Mutations in SLC36A2 lead to hyperglycinuria and iminoglycinuria. SLC36A3 is expressed only in testes and is an orphan transporter with no known function. SLC36A4 is widely distributed at the mRNA level and is a high-affinity (Km 2–3 µM) transporter for proline and tryptophan. We have much to learn about this family of transporters, but from current knowledge, it seems likely that their function will influence the pharmacokinetic profiles of amino acid-based drugs by mediating transport in both the small intestine and kidney. LINKED ARTICLES This article is part of a themed section on Transporters. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2011.164.issue-7 PMID:21501141

  10. Characterization of the role of ABCG2 as a bile acid transporter in liver and placenta.

    PubMed

    Blazquez, Alba G; Briz, Oscar; Romero, Marta R; Rosales, Ruben; Monte, Maria J; Vaquero, Javier; Macias, Rocio I R; Cassio, Doris; Marin, Jose J G

    2012-02-01

    ABCG2 is involved in epithelial transport/barrier functions. Here, we have investigated its ability to transport bile acids in liver and placenta. Cholylglycylamido fluorescein (CGamF) was exported by WIF-B9/R cells, which do not express the bile salt export pump (BSEP). Sensitivity to typical inhibitors suggested that CGamF export was mainly mediated by ABCG2. In Chinese hamster ovary (CHO cells), coexpression of rat Oatp1a1 and human ABCG2 enhanced the uptake and efflux, respectively, of CGamF, cholic acid (CA), glycoCA (GCA), tauroCA, and taurolithocholic acid-3-sulfate. The ability of ABCG2 to export these bile acids was confirmed by microinjecting them together with inulin in Xenopus laevis oocytes expressing this pump. ABCG2-mediated bile acid transport was inhibited by estradiol 17β-d-glucuronide and fumitremorgin C. Placental barrier for bile acids accounted for <2-fold increase in fetal cholanemia despite >14-fold increased maternal cholanemia induced by obstructive cholestasis in pregnant rats. In rat placenta, the expression of Abcg2, which was much higher than that of Bsep, was not affected by short-term cholestasis. In pregnant rats, fumitremorgin C did not affect uptake/secretion of GCA by the liver but inhibited its fetal-maternal transfer. Compared with wild-type mice, obstructive cholestasis in pregnant Abcg2(-/-) knockout mice induced similar bile acid accumulation in maternal serum but higher accumulation in placenta, fetal serum, and liver. In conclusion, ABCG2 is able to transport bile acids. The importance of this function depends on the relative expression in the same epithelium of other bile acid exporters. Thus, ABCG2 may play a key role in bile acid transport in placenta, as BSEP does in liver. PMID:22096226

  11. Substrate specificity and transport mechanism of amino-acid transceptor Slimfast from Aedes aegypti.

    PubMed

    Boudko, Dmitri Y; Tsujimoto, Hitoshi; Rodriguez, Stacy D; Meleshkevitch, Ella A; Price, David P; Drake, Lisa L; Hansen, Immo A

    2015-01-01

    Anautogenous mosquitoes depend on vertebrate blood as nutrient source for their eggs. A highly efficient set of membrane transporters mediates the massive movement of nutrient amino acids between mosquito tissues after a blood meal. Here we report the characterization of the amino-acid transporter Slimfast (Slif) from the yellow-fever mosquito Aedes aegypti using codon-optimized heterologous expression. Slif is a well-known component of the target-of-rapamycin signalling pathway and fat body nutrient sensor, but its substrate specificity and transport mechanism were unknown. We found that Slif transports essential cationic and neutral amino acids with preference for arginine. It has an unusual dual-affinity mechanism with only the high affinity being Na(+) dependent. Tissue-specific expression and blood meal-dependent regulation of Slif are consistent with conveyance of essential amino acids from gut to fat body. Slif represents a novel transport system and type of transceptor for sensing and transporting essential amino acids during mosquito reproduction. PMID:26449545

  12. Substrate specificity and transport mechanism of amino-acid transceptor Slimfast from Aedes aegypti

    PubMed Central

    Boudko, Dmitri Y.; Tsujimoto, Hitoshi; Rodriguez, Stacy D.; Meleshkevitch, Ella A.; Price, David P.; Drake, Lisa L.; Hansen, Immo A.

    2015-01-01

    Anautogenous mosquitoes depend on vertebrate blood as nutrient source for their eggs. A highly efficient set of membrane transporters mediates the massive movement of nutrient amino acids between mosquito tissues after a blood meal. Here we report the characterization of the amino-acid transporter Slimfast (Slif) from the yellow-fever mosquito Aedes aegypti using codon-optimized heterologous expression. Slif is a well-known component of the target-of-rapamycin signalling pathway and fat body nutrient sensor, but its substrate specificity and transport mechanism were unknown. We found that Slif transports essential cationic and neutral amino acids with preference for arginine. It has an unusual dual-affinity mechanism with only the high affinity being Na+ dependent. Tissue-specific expression and blood meal-dependent regulation of Slif are consistent with conveyance of essential amino acids from gut to fat body. Slif represents a novel transport system and type of transceptor for sensing and transporting essential amino acids during mosquito reproduction. PMID:26449545

  13. Role for Ion Transport in Porcine Vocal Fold Epithelial Defense to Acid Challenge

    PubMed Central

    Erickson-Levendoski, Elizabeth; Sivasankar, M. Preeti

    2012-01-01

    Objective The vocal fold epithelium is routinely exposed to gastric contents, including acid and pepsin, during laryngopharyngeal reflux events. The epithelium may possess intrinsic defenses to reflux. The first objective of the current study was to examine whether vocal fold epithelial ion transport is one potential mechanism of defense to gastric contents. The second objective was to determine whether ion transport in response to gastric contents is associated with the secretion of bicarbonate. Study Design Prospective design in excised porcine larynges. Setting Laboratory. Subjects and Methods Porcine vocal folds (N = 56) were exposed on the luminal surface to acid, pepsin, or sham challenges. Ion transport at baseline and following challenge exposure was measured using electrophysiological techniques. To examine specific ion transport mechanisms, vocal folds were pretreated with either a sodium channel blocker or bicarbonate channel blocker. Results Within 60 seconds of acid but not pepsin exposure, there was a significant increase in ion transport. This rapid increase in ion transport was transient and related to bicarbonate secretion. Conclusion The current data suggest that porcine vocal folds immediately increase bicarbonate secretion following exposure to acid. Bicarbonate secretion may act to neutralize acid. These findings contribute to the identification of the mechanisms underlying vocal fold defense to reflux and offer implications for the development of treatments for reflux-induced vocal fold injury. PMID:22086905

  14. Inhibitory actions of GABA on rabbit urinary bladder muscle strips: mediation by potassium channels.

    PubMed

    Ferguson, D R; Marchant, J S

    1995-05-01

    1. The actions of gamma-aminobutyric acid (GABA) upon rabbit urinary bladder muscle were investigated to determine whether they were mediated through potassium channels. 2. In vitro experiments were undertaken in which bladder muscle strips were caused to contract with carbachol. Addition of GABA or baclofen reduced the size of such evoked contractions in the case of GABA by 20.7 +/- 3.2%, in the case of baclofen by 22.4 +/- 2.2%. 3. Electrical stimulation of autonomic nerves in bladder wall strips also evoked contractions which were significantly smaller in potassium-free Krebs solution. The size of contractions produced by carbachol on the other hand were unaffected by the absence of potassium in the Krebs solution. 4. The inhibitory actions of GABA and baclofen on carbachol-induced contractions of bladder muscle were detected at much lower concentrations in potassium-free compared with potassium containing solutions. 5. The inhibitory effects of baclofen were completely reversed by tetraethyl ammonium chloride between 1 and 5 mM, caesium chloride between 0.5 and 3 mM and barium chloride between 0.5 and 2.5 mM. The actions of baclofen were only partially reversed by 4-amino-pyridine between 1 and 5 mM. 6. It was concluded that the GABAB receptor-mediated inhibitory actions on rabbit urinary bladder smooth muscle cells were produced by activation of potassium channels. PMID:7647988

  15. A novel GABA-mediated corticotropin-releasing hormone secretory mechanism in the median eminence

    PubMed Central

    Kakizawa, Keisuke; Watanabe, Miho; Mutoh, Hiroki; Okawa, Yuta; Yamashita, Miho; Yanagawa, Yuchio; Itoi, Keiichi; Suda, Takafumi; Oki, Yutaka; Fukuda, Atsuo

    2016-01-01

    Corticotropin-releasing hormone (CRH), which is synthesized in the paraventricular nucleus (PVN) of the hypothalamus, plays an important role in the endocrine stress response. The excitability of CRH neurons is regulated by γ-aminobutyric acid (GABA)–containing neurons projecting to the PVN. We investigated the role of GABA in the regulation of CRH release. The release of CRH was impaired, accumulating in the cell bodies of CRH neurons in heterozygous GAD67-GFP (green fluorescent protein) knock-in mice (GAD67+/GFP), which exhibited decreased GABA content. The GABAA receptor (GABAAR) and the Na+-K+-2Cl− cotransporter (NKCC1), but not the K+-Cl− cotransporter (KCC2), were expressed in the terminals of the CRH neurons at the median eminence (ME). In contrast, CRH neuronal somata were enriched with KCC2 but not with NKCC1. Thus, intracellular Cl− concentrations ([Cl−]i) may be increased at the terminals of CRH neurons compared with concentrations in the cell body. Moreover, GABAergic terminals projecting from the arcuate nucleus were present in close proximity to CRH-positive nerve terminals. Furthermore, a GABAAR agonist increased the intracellular calcium (Ca2+) levels in the CRH neuron terminals but decreased the Ca2+ levels in their somata. In addition, the increases in Ca2+ concentrations were prevented by an NKCC1 inhibitor. We propose a novel mechanism by which the excitatory action of GABA maintains a steady-state CRH release from axon terminals in the ME. PMID:27540587

  16. Contributions of GABA to alcohol responsivity during adolescence: Insights from preclinical and clinical studies

    PubMed Central

    Silveri, Marisa M.

    2015-01-01

    There is a considerable body of literature demonstrating that adolescence is a unique age period, which includes rapid and dramatic maturation of behavioral, cognitive, hormonal and neurobiological systems. Most notably, adolescence is also a period of unique responsiveness to alcohol effects, with both hyposensitivity and hypersensitivity observed to the various effects of alcohol. Multiple neurotransmitter systems are undergoing fine-tuning during this critical period of brain development, including those that contribute to the rewarding effects of drugs of abuse. The role of developmental maturation of the γ-amino-butyric acid (GABA) system, however, has received less attention in contributing to age-specific alcohol sensitivities. This review integrates GABA findings from human magnetic resonance spectroscopy studies as they may translate to understanding adolescent-specific responsiveness to alcohol effects. Better understanding of the vulnerability of the GABA system both during adolescent development, and in psychiatric conditions that include alcohol dependence, could point to a putative mechanism, boosting brain GABA, that may have increased effectiveness for treating alcohol abuse disorders. PMID:24631274

  17. The role of vasopressin, somatostatin and GABA in febrile convulsion in rat pups.

    PubMed

    Nagaki, S; Nagaki, S; Minatogawa, Y; Sadamatsu, M; Kato, N; Osawa, M; Fukuyama, Y

    1996-01-01

    In order to further elucidate a possible role of neuropeptides and GABA in the pathogenesis of febrile convulsions, we studied changes of immunoreactive-arginine vasopressin (IR-AVP), IR-somatostatin (IR-SRIF) and gamma-aminobutyric acid (GABA) in the rat brain after febrile convulsions induced by ultra-red light (UR). Male Wistar rats at 16 days of age irradiated with UR developed generalized convulsions after 4.9 +/- 0.5 min irradiation. Six rats were killed by microwave irradiation 3 min after UR irradiation prior to convulsion development, and 29 rats were killed either 0 min, 2 h, 6 h, 24 h or 48 h after febrile convulsions. Non-irradiated rats served as controls. The rat brain was dissected into 4 regions; amygdala, hypothalamus, cortex and hippocampus, and subjected to radioimmunoassays. IR-AVP levels in hypothalamus were increased 3 min after UR and decreased at 2 h and 6 h after the convulsions. IR-SRIF levels were increased in cortex and hippocampus at 3 min after UR and 0 min after the convulsions. The GABA content increased in all regions tested at 2 h and 6 h after the convulsions. These results suggest that AVP, SRIF and GABA may be involved in the pathogenesis of febrile convulsions in different ways. PMID:8649210

  18. A novel GABA-mediated corticotropin-releasing hormone secretory mechanism in the median eminence.

    PubMed

    Kakizawa, Keisuke; Watanabe, Miho; Mutoh, Hiroki; Okawa, Yuta; Yamashita, Miho; Yanagawa, Yuchio; Itoi, Keiichi; Suda, Takafumi; Oki, Yutaka; Fukuda, Atsuo

    2016-08-01

    Corticotropin-releasing hormone (CRH), which is synthesized in the paraventricular nucleus (PVN) of the hypothalamus, plays an important role in the endocrine stress response. The excitability of CRH neurons is regulated by γ-aminobutyric acid (GABA)-containing neurons projecting to the PVN. We investigated the role of GABA in the regulation of CRH release. The release of CRH was impaired, accumulating in the cell bodies of CRH neurons in heterozygous GAD67-GFP (green fluorescent protein) knock-in mice (GAD67(+/GFP)), which exhibited decreased GABA content. The GABAA receptor (GABAAR) and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), but not the K(+)-Cl(-) cotransporter (KCC2), were expressed in the terminals of the CRH neurons at the median eminence (ME). In contrast, CRH neuronal somata were enriched with KCC2 but not with NKCC1. Thus, intracellular Cl(-) concentrations ([Cl(-)]i) may be increased at the terminals of CRH neurons compared with concentrations in the cell body. Moreover, GABAergic terminals projecting from the arcuate nucleus were present in close proximity to CRH-positive nerve terminals. Furthermore, a GABAAR agonist increased the intracellular calcium (Ca(2+)) levels in the CRH neuron terminals but decreased the Ca(2+) levels in their somata. In addition, the increases in Ca(2+) concentrations were prevented by an NKCC1 inhibitor. We propose a novel mechanism by which the excitatory action of GABA maintains a steady-state CRH release from axon terminals in the ME. PMID:27540587

  19. Involvement of GABA in environmental temperature-induced change in body temperature.

    PubMed

    Biswas, S; Poddar, M K

    1988-12-01

    Acute exposure of adult male albino rats (110-120 g) to higher environmental temperature (40 +/- 1 degrees C) increased body temperature (BT). This increase of BT was also dependent on the duration of exposure. Treatment with muscimol (1 mg/kg, i.p.), a GABA agonist, produced hypothermia at room temperature (28 +/- 1 degree C) and resistance to increase the body temperature when exposed to higher temperature (40 +/- 1 degree C). Administration of bicuculline (1 mg/kg, i.p.), a GABA antagonist, on the other hand, enhanced BT more than that observed in control (normal) rat exposed to higher temperature (40 +/- 1 degree C), although at room temperature bicuculline treatment did not show any effect on BT. Pretreatment with ethanolamine-O-sulfate (EOS) (2 g/kg, s.c.), a GABA transaminase inhibitor, to rats exposed to higher temperature increased BT as in control (normal) rat. Inhibition of central GAD activity with mercaptopropionic acid (MPA) (70 mg/kg, i.p.) produced resistance to increase BT during its period of action when rats were exposed to higher environmental temperature (28 +/- 1 degree C). These results thus suggest that central inhibitory neuron, GABA, plays a regulatory role in thermoregulation. PMID:3236943

  20. Lamina-specific alterations in cortical GABA(A) receptor subunit expression in schizophrenia.

    PubMed

    Beneyto, Monica; Abbott, Andrew; Hashimoto, Takanori; Lewis, David A

    2011-05-01

    Dysfunction of the dorsolateral prefrontal cortex (DLPFC) in schizophrenia is associated with lamina-specific alterations in particular subpopulations of interneurons. In pyramidal cells, postsynaptic γ-aminobutyric acid (GABA(A)) receptors containing different α subunits are inserted preferentially in distinct subcellular locations targeted by inputs from specific interneuron subpopulations. We used in situ hybridization to quantify the laminar expression of α1, α2, α3, and α5 subunit, and of β1-3 subunit, mRNAs in the DLFPC of schizophrenia, and matched normal comparison subjects. In subjects with schizophrenia, mean GABA(A) α1 mRNA expression was 17% lower in layers 3 and 4, α2 expression was 14% higher in layer 2, α5 expression was 15% lower in layer 4, and α3 expression did not differ relative to comparison subjects. The mRNA expression of β2, which preferentially assembles with α1 subunits, was also 20% lower in layers 3 and 4, whereas β1 and β3 mRNA levels were not altered in schizophrenia. These expression differences were not attributable to medication effects or other potential confounds. These findings suggest that GABA neurotransmission in the DLPFC is altered at the postsynaptic level in a receptor subunit- and layer-specific manner in subjects with schizophrenia and support the hypothesis that GABA neurotransmission in this illness is predominantly impaired in certain cortical microcircuits. PMID:20843900

  1. Increased GABA concentrations in type 2 diabetes mellitus are related to lower cognitive functioning.

    PubMed

    van Bussel, Frank C G; Backes, Walter H; Hofman, Paul A M; Puts, Nicolaas A J; Edden, Richard A E; van Boxtel, Martin P J; Schram, Miranda T; Stehouwer, Coen D A; Wildberger, Joachim E; Jansen, Jacobus F A

    2016-09-01

    Type 2 diabetes mellitus is associated with accelerated cognitive decline. The underlying pathophysiological mechanisms still remain to be elucidated although it is known that insulin signaling modulates neurotransmitter activity, including inhibitory γ-aminobutyric acid (GABA) and excitatory glutamate (Glu) receptors. Therefore, we examined whether levels of GABA and Glu are related to diabetes status and cognitive performance.Forty-one participants with type 2 diabetes and 39 participants without type 2 diabetes underwent detailed cognitive assessments and 3-Tesla proton MR spectroscopy. The associations of neurotransmitters with type 2 diabetes and cognitive performance were examined using multivariate regression analyses controlling for age, sex, education, BMI, and percentage gray/white matter ratio in spectroscopic voxel.Analysis revealed higher GABA+ levels in participants with type 2 diabetes, in participants with higher fasting blood glucose levels and in participants with higher HbA1c levels, and higher GABA+ levels in participants with both high HbA1c levels and less cognitive performance.To conclude, participants with type 2 diabetes have alterations in the GABAergic neurotransmitter system, which are related to lower cognitive functioning, and hint at the involvement of an underlying metabolic mechanism. PMID:27603392

  2. Differential effects of cyclosporin A on transport of bile acids by rat hepatocytes: relationship to individual serum bile acid levels.

    PubMed

    Azer, S A; Stacey, N H

    1994-02-01

    Cyclosporin A treatment has been reported to induce hepatotoxicity marked by a rise in total serum bile acid and total bilirubin. The mechanism of cyclosporin A-induced hepatotoxicity seems to be related to interference with hepatocellular transport of these substrates although this remains to be fully substantiated. The purpose of this study was to investigate whether the hepatocellular uptake of the different bile acids, in the presence of cyclosporin A, is consistent with the changes in their respective individual serum bile acid concentrations. High-performance liquid chromatography has been used to assay individual serum bile acids in cyclosporin A-treated rats at doses of 0.1, 1, and 10 mg/kg/day for 4 days. Control rats were treated with Cremophor (1 ml/kg/day). At the higher doses, cyclosporin A produced a significant increase in levels of cholic acid, taurocholic acid, chenodeoxycholic acid, and deoxycholic acid compared with controls. Serum glycocholate was unaffected even at the highest dose. Inhibition of initial rate of uptake and accumulation of [14C]cholic acid, [14C]chenodeoxycholic acid, and [14C]deoxycholic acid by isolated rat hepatocytes was consistent with the changes in their respective serum bile acids. Coincubation of rat hepatocytes with unlabeled cholic acid (100 microM), the major serum bile acid in cyclosporin A-treated rats, showed a further inhibitory effect on [14C]cholic acid and [14C]deoxycholic acid accumulation. The initial rate of uptake of [14C]glycocholate was also inhibited. However, accumulation of glycocholic acid did not show significant changes at the longer incubation times (2-30 min). In addition, coincubation of rat hepatocytes with unlabeled cholic acid (100 microM) plus cyclosporin A did not induce any inhibition of glycocholate accumulation. Together, these differences provide an explanation for the unchanged serum levels of glycocholate. In conclusion, the changes in individual serum bile acids in cyclosporin A

  3. Light-activated amino acid transport in Halobacterium halobium envelope vesicles

    NASA Technical Reports Server (NTRS)

    Macdonald, R. E.; Lanyi, J. K.

    1977-01-01

    Vesicles prepared from Halobacterium halobium cell envelopes accumulate amino acids in response to light-induced electrical and chemical gradients. Nineteen of 20 commonly occurring amino acids have been shown to be actively accumulated by these vesicles in response to illumination or in response to an artificially created Na+ gradient. On the basis of shared common carriers the transport systems can be divided into eight classes, each responsible for the transport of one or several amino acids: arginine, lysine, histidine; asparagine, glutamine; alanine, glycine, threonine, serine; leucine, valine, isoleucine, methionine; phenylalanine, tyrosine, tryptophan; aspartate; glutamate; proline. Available evidence suggests that these carriers are symmetrical in that amino acids can be transported equally well in both directions across the vesicle membranes. A tentative working model to account for these observations is presented.

  4. Myosin 1b Regulates Amino Acid Transport by Associating Transporters with the Apical Plasma Membrane of Kidney Cells

    PubMed Central

    Komaba, Shigeru; Coluccio, Lynne M.

    2015-01-01

    Amino acid transporters (AATers) in the brush border of the apical plasma membrane (APM) of renal proximal tubule (PT) cells mediate amino acid transport (AAT). We found that the membrane-associated class I myosin myosin 1b (Myo1b) localized at the apical brush border membrane of PTs. In opossum kidney (OK) 3B/2 epithelial cells, which are derived from PTs, expressed rat Myo1b-GFP colocalized in patched microvilli with expressed mouse V5-tagged SIT1 (SIT1-V5), which mediates neutral amino acid transport in OK cells. Lentivirus-mediated delivery of opossum Myo1b-specific shRNA resulted in knockdown (kd) of Myo1b expression, less SIT1-V5 at the APM as determined by localization studies, and a decrease in neutral AAT as determined by radioactive uptake assays. Myo1b kd had no effect on Pi transport or noticeable change in microvilli structure as determined by rhodamine phalloidin staining. The studies are the first to define a physiological role for Myo1b, that of regulating renal AAT by modulating the association of AATers with the APM. PMID:26361046

  5. Bibliography for acid-rock drainage and selected acid-mine drainage issues related to acid-rock drainage from transportation activities

    USGS Publications Warehouse

    Bradley, Michael W.; Worland, Scott C.

    2015-01-01

    Acid-rock drainage occurs through the interaction of rainfall on pyrite-bearing formations. When pyrite (FeS2) is exposed to oxygen and water in mine workings or roadcuts, the mineral decomposes and sulfur may react to form sulfuric acid, which often results in environmental problems and potential damage to the transportation infrastructure. The accelerated oxidation of pyrite and other sulfidic minerals generates low pH water with potentially high concentrations of trace metals. Much attention has been given to contamination arising from acid mine drainage, but studies related to acid-rock drainage from road construction are relatively limited. The U.S. Geological Survey, in cooperation with the Tennessee Department of Transportation, is conducting an investigation to evaluate the occurrence and processes controlling acid-rock drainage and contaminant transport from roadcuts in Tennessee. The basic components of acid-rock drainage resulting from transportation activities are described and a bibliography, organized by relevant categories (remediation, geochemical, microbial, biological impact, and secondary mineralization) is presented.

  6. Taxol induced apoptosis regulates amino acid transport in breast cancer cells.

    PubMed

    Wu, Yanyuan; Shen, Dejun; Chen, Zujian; Clayton, Sheila; Vadgama, Jaydutt V

    2007-03-01

    A major outcome from Taxol treatment is induction of tumor cell apoptosis. However, metabolic responses to Taxol-induced apoptosis are poorly understood. In this study, we hypothesize that alterations in specific amino acid transporters may affect the Taxol-induced apoptosis in breast cancer cells. In this case, the activity of the given transporter may serve as a biomarker that could provide a biological assessment of response to drug treatment. We have examined the mechanisms responsible for Taxol-induced neutral amino acid uptake by breast cancer cells, such as MCF-7, BT474, MDAMB231 and T47D. The biochemical and molecular studies include: (1) growth-inhibition (MTT); (2) transport kinetics: (3) substrate-specific inhibition; (4) effect of thiol-modifying agents NEM and NPM; (5) gene expression of amino acid transporters; and (6) apoptotic assays. Our data show that Taxol treatment of MCF-7 cells induced a transient increase in Na(+)-dependent transport of the neutral amino acid transporter B0 at both gene and protein level. This increase was attenuated by blocking the transporter in the