Gamma-aminobutyric Acid Uptake Research Articles

Lipopolysaccharide augments microglial GABA uptake by increasing GABA transporter-1 trafficking and bestrophin-1 expression.

Gamma-aminobutyric acid (GABA), the principal inhibitory neurotransmitter in the brain, affects numerous immune cell functions. Microglia, the brain's resident innate immune cells, regulate GABA signaling through GABA receptors and express the complete GABAergic machinery for GABA synthesis, uptake, and release. Here, the use of primary microglial cell cultures and ex vivo brain tissue sections allowed for demonstrating that treatment with lipopolysaccharide (LPS) increased microglial GABA uptake as well as GABA transporter (GAT)-1 trafficking. This effect was not entirely abolished by treatment with GAT inhibitors (GAT-Is). Notably, LPS also induced microglial upregulation of bestrophin-1 (BEST-1), a Ca2+ -activated Cl- channel permeable to GABA. Combined administration of GAT-Is and a BEST-1 inhibitor completely abolished LPS-induced microglial GABA uptake. Interestingly, increased microglial GAT-1 membrane turnover via syntaxin 1A was detected in LPS-treated cultures after BEST-1 blockade. Altogether, these findings provided evidence for a novel mechanism through which LPS may trigger the inflammatory response by directly altering microglial GABA clearance and identified the GAT-1/BEST-1 interplay as a potential novel mechanism involved in brain inflammation.

GluN2D Subunit in Parvalbumin Interneurons Regulates Prefrontal Cortex Feedforward Inhibitory Circuit and Molecular Networks Relevant to Schizophrenia

BackgroundParvalbumin interneuron (PVI) activity synchronizes the medial prefrontal cortex circuit for normal cognitive function, and its impairment may contribute to schizophrenia (SZ). NMDA receptors in PVIs participate in these activities and form the basis for the NMDA receptor hypofunction hypothesis of SZ. However, the role of the GluN2D subunit, which is enriched in PVIs, in regulating molecular networks relevant to SZ is unknown. MethodsUsing electrophysiology and a mouse model with conditional deletion of GluN2D from PVIs (PV-GluN2D knockout [KO]), we examined the cell excitability and neurotransmission in the medial prefrontal cortex. Histochemical, RNA sequencing analysis and immunoblotting were conducted to understand molecular mechanisms. Behavioral analysis was conducted to test cognitive function. ResultsPVIs in the medial prefrontal cortex were found to express putative GluN1/2B/2D receptors. In a PV-GluN2D KO model, PVIs were hypoexcitable, whereas pyramidal neurons were hyperexcitable. Excitatory neurotransmission was higher in both cell types in PV-GluN2D KO, whereas inhibitory neurotransmission showed contrasting changes, which could be explained by reduced somatostatin interneuron projections and increased PVI projections. Genes associated with GABA (gamma-aminobutyric acid) synthesis, vesicular release, and uptake as well as those involved in formation of inhibitory synapses, specifically GluD1-Cbln4 and Nlgn2, and regulation of dopamine terminals were downregulated in PV-GluN2D KO. SZ susceptibility genes including Disc1, Nrg1, and ErbB4 and their downstream targets were also downregulated. Behaviorally, PV-GluN2D KO mice showed hyperactivity and anxiety behavior and deficits in short-term memory and cognitive flexibility. ConclusionsThese findings demonstrate that GluN2D in PVIs serves as a point of convergence of pathways involved in the regulation of GABAergic synapses relevant to SZ.

Single Cocaine Exposure Inhibits GABA Uptake via Dopamine D1-Like Receptors in Adolescent Mice Frontal Cortex.

Cocaine (COC) is a psychostimulant that acts by increasing catecholaminergic neurotransmission mainly due to its effects on the dopamine transporter (DAT). However, other neurotransmitter systems may also be regulated by COC, including the GABAergic system. Since the effect of COC in modulating gamma-aminobutyric acid (GABA) reuptake is not defined, we investigated the molecular mechanisms related to the increase in GABA uptake induced by acute COC exposure and its effects on locomotor activity in adolescent mice. Behavioral experiments showed that COC increased locomotor activity and decreased immobilization time in mice. A single COC exposure reduced both GABA uptake and GAT-1 protein levels. On the other hand, cyclic adenosine monophosphate (cAMP) levels increased after a COC challenge. The major changes induced by acute COC on behavioral and neurochemical assays were avoided by previous treatment with the selective D1 receptor antagonist SCH-23390 (0.5mg/kg). Our findings suggest that GABA uptake naturally decreases during mice development from preadolescence until adulthood and that dopamine (DA) D1-like receptors are key players in the regulation of GABA uptake levels following a single COC exposure in adolescent mice.

The use of deuterium-labeled gamma-aminobutyric (D6-GABA) to study uptake, translocation, and metabolism of exogenous GABA in plants

BackgroundExogenous application of gamma-aminobutyric acid (GABA) could relieve stress symptoms caused by abiotic stresses including anoxia, heat and cold shock, drought, and salt. However, studying translocation and metabolism of exogenous GABA is challenged by the presence of endogenous GABA.ResultsHerein, we used D6-GABA in order to investigate the uptake, translocation, and the metabolism of exogenous GABA in Mexican lime (Citrus aurantifolia) seedlings using gas chromatography-mass spectrometry (GC–MS). The GC–MS analysis showed that D6-GABA could be easily distinguished from the non-labeled GABA after methyl chloroformate (MCF) derivatization. The D6-GABA was detected in the cortex (phloem), inner stem (xylem), and leaves after root drench. Girdling did not affect the translocation of D6-GABA, indicating that it is mainly translocated via the xylem. In addition, D4-labled succinic acid was detected in D6-GABA-treated plants, indicating that exogenous GABA was metabolized to succinic acid. The half-life of D6-GABA in citrus was about 1.3 h, indicating a quick conversion to succinic acid.ConclusionThe use of D6-GABA offers a valuable tool to study the translocation and metabolism of GABA in plants. D6-GABA and its metabolite (D4-succinic acid) can be easily distinguished from the endogenous GABA and succinic acid using GC–MS.

Efficacy of steroids in childhood drug-resistant epilepsy

Background Epilepsy is considered the most frequent chronic neurologic condition in childhood. In childhood epilepsy, the incidence rates range from 0.5 to 8 per 1000 children per year. Drug-resistant epilepsy (DRE) is defined according to the International League against Epilepsy as a failure of adequate trials of greater than or equal to 2 appropriately used, tolerated, and appropriately chosen regimens of antiepileptic drugs to achieve seizure freedom. It has been described that steroids and adrenocorticotropic hormone are effective treatment modalities in DRE children. They may have anticonvulsive properties through their endocrine criteria and their effect on neuronal excitability and stabilization of membrane. They are believed to induce changes in the transmission of neurochemicals due to changes in gamma-aminobutyric acid uptake or serotonin turnover mediated through the mineralocorticoid and glucocorticoid receptors.Objectives The aim of this study was to evaluate the efficacy of oral prednisolone regimen and hybrid corticosteroid regimens in the treatment of DRE and to assess the possible side effects of both regimens.Patients and methods This study was a prospective randomized clinical trial which was conducted at the neurology outpatient clinic and department of the Mansoura University Children Hospital. It included 49 pediatric patients diagnosed as DRE according to the International League against Epilepsy 2010 definition as failure of adequate trials of two or more drugs to achieve freedom from seizures. They were divided into two groups, the first group (25 patients) received oral prednisolone and the second (24 patients) received intravenous methylprednisolone followed by oral prednisolone. Both regimens were applied in addition to conventional antiepileptic drugs. The response to the treatment is graded as: ‘free from seizures,’ ‘response is good’ (reduction of seizures more than 50% including seizure free), or ‘response is poor’ (<50% reduction, considered as therapy failure).Results Our study showed that of the 25 patients enrolled in group 1 and received high-dose oral prednisolone (14/25) 56% showed good response (decrease in seizure frequency by >50%), of whom seven (28%) became seizure free, while (11/25) 44% showed poor response (decrease of seizure frequency by <50%) and of the 24 patients enrolled in group 2 and received hybrid regimen consists of methylprednisolone and low-dose prednisolone (13/24) 54% showed good response, six (25%) of them became free from seizures, while (11/24) 46% showed poor response. And there is no significant difference as regards the efficacy of the steroid regimens used in the two groups (P=0.97) but the regimen used in group 2 showed lesser adverse effects.Conclusion The use of hybrid corticosteroid regimen composed of initial pulses of methylprednisolone followed by low-dose oral prednisolone in the treatment of DRE is as effective as the use of high-dose oral prednisolone from the start but with less adverse-effect profile.

Microglial expression of GAT-1 in the cerebral cortex.

Microglial cells are the immune cells of the brain that, by sensing the microenvironment, permit a correct brain development and function. They communicate with other glial cells and with neurons, releasing and responding to a number of molecules that exert effects on surrounding cells. Among these, neurotransmitters and, in particular, gamma-aminobutyric acid (GABA) has recently gained interest in this context. We demonstrated the expression of GABA transporter 1 (GAT-1) in microglial cells both in soma and cell processes. We show that microglial cell treatment with 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid hydrochloride (NNC-711), a potent and selective GAT-1 inhibitor, significantly reduced Na+ -dependent GABA uptake. On the other hand, GABA uptake was significantly increased by cell treatment with (S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-3-piperidinecarboxylic acid (SNAP-5114), a GAT-2/3 inhibitor, and this effect was completely blocked by the botulinum toxin BoNT/C1, that specifically cleaves and inactives syntaxin 1A (STX1A). Overall, these findings show that microglial cells express GAT-1 and indicate that STX1A plays an important role in the regulation of GAT-1-dependent GABA uptake in microglia.

Altered release and uptake of gamma-aminobutyric acid in the cerebellum of dystrophin-deficient mice

Dystrophin deficiency caused by mutations of the related gene leads to muscle wasting in Duchenne muscular dystrophy (DMD). Some patients with DMD also present with intellectual disability and various degrees of neurological disorders, which have been related to a decreased number of postsynaptic gamma-aminobutyric acid type A receptors (GABAARs) in the hippocampus (HPC) and cerebellum (CBL). The aim of this study was to examine the relevance of dystrophin in the presynaptic GABAergic function in brain regions in which this protein is normally abundant. [3H]-GABA release, induced by nicotinic receptor (nAChR) activation or K+ depolarization, and [3H]-GABA uptake were determined using synaptosomes extracted from the cortex (CTX), HPC, and CBL of littermate control and mdx mice. Superfusion of the synaptosomes with nicotine or high K+ solutions led to a concentration-dependent and Ca2+-dependent [3H]-GABA release in control and mdx synaptosomes. [3H]-GABA release induced by 10 μM nicotine in mdx CBL synaptosomes was 47% less than that in control mice. K+-induced [3H]-GABA release did not differ between control and mdx synaptosomes. α7-containing and β2-containing nAChRs were involved in nicotine-induced [3H]-GABA release in control and mdx synaptosomes. Kinetic analysis of [3H]-GABA uptake in mdx CBL synaptosomes showed a reduced (50%) half-maximal uptake time (t1/2) and increased (44%) rate of [3H]-GABA uptake (Vmax) compared to controls. The apparent transporter affinity (Km) for GABA was not altered. Our findings show that dystrophin deficiency in mdx mice is associated with significant changes in the release and uptake of GABA in the CBL. These presynaptic alterations may be related to the reported decrease in postsynaptic GABAAR in the same brain region. The results indicate possible dysfunction of GABAergic synapses associated with dystrophin deficiency in the CBL, which may contribute to the cognitive and neurobehavioral disorders in mdx mice and patients with DMD.

7-Fluoro-1,3-diphenylisoquinoline-1-amine reverses the reduction in self-care behavior induced by maternal separation stress in rats by modulating glutamatergic/GABAergic systems

7-Fluoro-1,3-diphenylisoquinoline-1-amine (FDPI) is a promising isoquinoline that elicits an antidepressant-like action in rodents. In this study, an animal model of stress induced by maternal separation was used to investigate the effects of FDPI in Wistar rats of 30 and 90 days of age. It was investigated the effects of maternal separation in the self-care behavior and the contribution of glutamatergic and gamma-aminobutyric acid (GABA)ergic systems in the FDPI action. Male Wistar rats were separated from their mothers for 3 h/day from postnatal day (PND) 1–10. The rats were treated at different ages (PND-30 and PND-90) with FDPI (5 mg/kg, intragastrically/7 days) and performed the splash test. Maternal separation reduced total grooming time in the splash test, an index of motivational and self-care behavior, and FDPI treatment was effective in reversing this behavior in rats at both ages. The neurochemical parameters were differently affected, dependent on the age of rats, by maternal separation and FDPI. Maternal separation increased the GABA uptake and the excitatory amino acid transporter 1 levels in the prefrontal cortices of rats at PND-30 and FDPI was effective against these alterations. At PND-90, maternal separation decreased the glutamate uptake and increased the GABA uptake and the N-methyl-D-aspartate (NMDA) receptor 2B levels in the prefrontal cortices of rats. FDPI reversed the neurochemical alterations caused by maternal separation in the prefrontal cortices of rats at PND-90. The results of this study demonstrated that FDPI reversed the reduction in self-care behavior induced by maternal separation stress in rats by modulating the glutamatergic/GABAergic systems in rats.

Central GABAA Receptors Provoke Inflammatory and Cardiovascular Consequences of Endotoxemia in Conscious Rats

Gamma‐aminobutyric acid (GABA), the principal brain inhibitory neurotransmitter, plays a key role in inflammatory and neurodegenerative disease. In this study, we tested the hypothesis that central GABAergic neurotransmission mediates the detrimental inflammatory, hemodynamic, and cardiac autonomic actions of endotoxemia. The effects of drugs that block GABA receptors or interfere with GABA uptake or degradation on blood pressure (BP), heart rate (HR), and HR variability (HRV) responses elicited by i.v. lipopolysaccharide (LPS) were assessed in conscious rats. The BP lowering effect of LPS (10 mg/kg) was blunted after intracisternal (i.c.) administration of bicuculline (GABAA receptor antagonist, 3 μg/rat) or saclofen (selective GABAB receptor antagonist, 50 μg/rat). By contrast, the concomitant LPS‐evoked tachycardia and decreases in time‐ and frequency domain indices of HRV, measures of cardiac autonomic control, were abolished upon treatment with bicuculline but not saclofen. The increases in serum tumor necrosis factor‐α and interleukin‐6 caused by LPS disappeared in rats treated concurrently with bicuculline or saclofen, whereas the LPS‐evoked increases in serum nitric oxide metabolites was reversed by bicuculline only. We also report that none of the effects of endotoxemia was altered in rats treated with i.c. tiagabine (GABA reuptake inhibitor, 100 μg/rat) or vigabatrin (GABA transaminase inhibitor, 200 μg//rat). These data suggest a major role for central GABAA receptors in the inflammatory and cardiovascular effects of endotoxemia.Support or Funding InformationSupported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895).

The Uptake of GABA in Trypanosoma cruzi.

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.

Characteristics of endogenous γ-aminobutyric acid (GABA) in human platelets: functional studies of a novel collagen glycoprotein VI inhibitor

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system, and it also appears in peripheral tissues. Platelets are anuclear blood cells that play a central role in hemostatic processes. Although platelets possess a GABA uptake system, the functional activity of GABA in platelets has remained unclear. We determined that GABA is abundantly distributed in the platelets at a concentration of approximately 1.03 ng/10(6) cells. GABA (0.5 μM) specifically inhibited collagen-induced platelet activation accompanied by [Ca(2+)]i mobilization, phospholipase Cγ2, protein kinase C, Akt phosphorylation, and hydroxyl radical formation. In addition, GABA interfered with fluorescein isothiocyanate-collagen binding to platelet membranes and produced a concentration-dependent shift in the collagen concentration-response curve and a Schild plot slope of -0.96 ± 0.11, indicating competitive inhibition. Platelet activation induced by convulxin, a glycoprotein VI agonist, was inhibited by GABA, whereas activation induced by the integrin α(2)β(1) agonist, aggretin, was not. Immunoprecipitation and surface plasmon resonance revealed that GABA binds directly to glycoprotein VI in human platelets with equilibrium dissociation (binding) constant (K(D)) of 41.4 nM. The closure time of whole blood and the occlusion time of platelet plug formation were significantly prolonged by GABA in vivo. In this study, GABA is a specific inhibitor of collagen glycoprotein VI and may be involved in an endogenous negative feedback mechanism for platelet activation. Thus, GABA may represent a potential target for the development of novel interventions for the treatment of cardiovascular diseases associated with platelet activation, such as stroke and myocardial infarction. GABA is abundantly distributed in the platelets. GABA inhibited platelet activation stimulated by convulxin. GABA directly associated with glycoprotein VI in platelet membrane. GABA prolonged the closure time of whole blood and the occlusion time of platelet plug formation in vivo.

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

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

Human neuromelanin: an endogenous microglial activator for dopaminergic neuron death.

Substantial evidence indicates that neuroinflammation caused by microglial activation in substantia nigra is critical in the pathogenesis of dopaminergic neurodegeneration in Parkinson's disease (PD). Increasing data demonstrates that environmental factors such as rotenone, paraquat play pivotal roles in dopaminergic neuron death. Here, potential role and mechanism of neuromelanin (NM), a major endogenous component in dopaminergic neurons of substantia nigra, on microglial activation and associated dopaminergic neurotoxicity were investigated. Using multiple primary mesencephalic cultures, we found that HNM caused dopaminergic neurodegeneration characterized by the decreased dopamine uptake and reduced numbers and shorted dendrites. HNM was selectively toxic to dopaminergic neurons since the other types of neurons determined by either gamma-aminobutyric acid uptake and total neuronal numbers showed smaller decrease. HNM produced modest dopaminergic neurotoxicity in neuron-enriched cultures; in contrast, much greater neurotoxicity was observed in the presence of microglia. HNM morphologically activated microglia and produced proinflammatory and neurotoxic factors. Thus, HNM can be a potent endogenous activator of microglial reactivation, mediating PD progression. Hence, inhibition of microglial reactivation can be a useful strategy for PD therapy.

Platelet uptake of GABA and glutamate in patients with bipolar disorder

Gamma aminobutyric acid (GABA) and glutamate (Glu) are the major neurotransmitters of the human central nervous system, and their actions are determined by specific transporters. Several studies suggest that GABA- and Glu-uptake mechanisms are modified in patients with bipolar disorder (BD). We explored the functionality of the GABA and Glu transporters in three groups of patients with BD, each with a different polarity of index episode (manic, depressive, or euthymic) at the time of blood draw. Forty patients with a diagnosis of BD, according to DSM-IV-TR criteria, and 15 healthy subjects were enrolled in the study. GABA and Glu uptake were evaluated in freshly prepared platelets using [(3) H]GABA or [(3) H]glutamate. Compared to controls, GABA uptake was significantly increased in patients with depressive episodes and significantly decreased in subjects with manic episodes. Glu uptake was significantly increased in patients with index manic episodes and in euthymic patients compared to healthy controls. Moreover, a positive correlation was found between GABA platelet uptake and Hamilton Depression Rating Scale scores and between Glu platelet uptake and Young Mania Rating Scale scores in patients with manic episodes. We found a relationship between GABA- and Glu-uptake levels and the polarity of episodes in patients with BD. Our data suggest that the functionality of both GABA and Glu transporters could represent a useful neurobiological marker to characterize the real polarity of an index episode of illness in patients with BD.

Low concentrations of methamidophos do not alter AChE activity but modulate neurotransmitters uptake in hippocampus and striatum in vitro

AimsMethamidophos (Meth) is a toxic organophosphorus compound (OP) that inhibits acetylcholinesterase enzyme (AChE) and induces neurotoxicity. As the mechanism of its neurotoxic effects is not well understood, the aim of the present study was to evaluate the effects of Meth on glutamate and gamma aminobutyric acid (GABA) uptake and correlate with cell viability and AChE and Na+/K+-ATPase enzyme activities in striatum and hippocampus slices exposed to low concentrations (0.05 to 1.0μM) of Meth. Main methodsHippocampal and striatal slices of rat brain were exposed to Meth for 5min ([3H]Glutamate uptake) or 15min ([3H]GABA uptake) for assays. The enzyme activities and cell viability were also accessed at both times in hippocampal and striatal slices and homogenates. Key findingsAt concentrations that did not inhibit AChE, Meth caused changes in glutamate uptake in striatal (0.05 and 1.0μM Meth) and hippocampal (1.0μM Meth) slices. GABA uptake was increased by the pesticide in striatum at 0.5 and 1.0μM and in hippocampus at 0.05μM. After 3.5h of Meth exposure, striatal and hippocampal cells showed no changes in viability as well as no inhibition of Na+/K+-ATPase were observed after 5 or 15min exposure to Meth in the same brain structures. SignificanceResults suggest that Meth, even without changing the AChE activity can modify somehow the neurotransmitters uptake. However, further studies are necessary to clarify if this modulation in glutamate or GABA uptake may be responsible to cause some disturbance in behavior or in other neurochemical parameters following low Meth exposure in vivo.

Circular Swimming by the Medaka, Oryzias latipes, Induced by Microinjection of GABA-ergic Agonists and Antagonists into the Posterior Thalamus

The thalamic mechanism involved in locomotor control, particularly in steering control, was studied in a teleost, Oryzias latipes. Ipsilateral circular swimming was induced by injection of bicuculline, a selective gamma-aminobutyric acid (GABA)-A antagonist, into the unilateral posterior thalamus. δ-Amino-valeric acid, phaclofen and 2-hydroxysaclofen (weak GABA-B antagonists) had weak, but similar effects. In contrast to antagonists, injection of GABA, muscimol (GABA-A agonist), baclofen (GABA-B agonist), nipecotic acid (blocker of Na + -coupled GABA uptake carrier), and γ-hydroxybutyric acid (possible GABA-A and -B agonist) into the unilateral posterior thalamus induced contralateral circular swimming

The Response to Postnatal Stress: Amino Acids Transporters and PKC Activity

It is well known that animals exposed to stressful stimuli during their early life develop different neurological disorders when they become adults. In this study, we evaluated the effect of acute cold stress on gamma-aminobutyric acid (GABA) and L-Serine (L-Ser) transporters in vitro, using the uptake of [(3)H]-GABA and [(3)H]L-Ser by synaptosomes-enriched fractions isolated from rat cerebral cortex during postnatal development. GABA and L-Ser uptake studies in vitro will be used in this investigation as a colateral evidence of changes in the expression of transporters of GABA and L-Ser. We observed that the maximum velocity (V (max)) in L-Ser and GABA uptake after stress session increased in all stages studied. In contrast, K (m) values of L-Ser uptake enhancent in almost age calculated, excluding at PD21 after cold stress during development, at the same time as K (m) (uptake affinity) values of GABA increased in just about age considered but not at PD5 compared with the control group. Finally we investigated the mechanism by which cells regulate the substrate affinity of L-Ser and GABA transporters. We demonstrated a significantly increase in total PKC activity to PD5 from PD21. Pretreatment with PKC inhibitor: staurosporine (SP) led to a restoration of control uptake in several postnatal-days suggesting a relationship between amino acids system and PKC activation. These findings suggest that a single exposure to postnatal cold stress at different periods after birth modifies both GABA and L-Ser transporters and the related increase in total PKC activity could be intracellular events that participate in neuronal plasticity by early life stress, which could be relevant to function of transporters in the adult rat brain.

A convergent approach to (R)-Tiagabine by a regio- and stereocontrolled hydroiodination of alkynes

The occurrence of unsaturated systems in natural products combined with the mildness and the wide range of applicability of CeCl(3) promoted methodologies suggest several potential future synthetic applications within the field of total synthesis of biologically active molecules. On this concept, the use of CeCl(3).7H(2)O-NaI system as an efficient heterogeneous promoter has been highlighted in the iodofunctionalization of carbon-carbon triple bonds. The study has shown that this method would be particularly interesting for the stereoselective formation of trisubstituted (Z)- or (E)-iodoalkenes by simply changing the nature of the solvent. The methodology has been successfully applied to the synthesis of (R)-1-[4,4-bis-(3-methyl-2-thienyl)-3-butenyl]-3-piperidinecarboxylic acid , named (R)-Tiagabine, which is a potent and selective gamma-aminobutyric acid (GABA) uptake inhibitor with proven anticonvulsant efficacy in humans.

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

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