RESUMEN
Vigabatrin is an anti-epileptic drug substance. The oral bioavailability of vigabatrin is high (60-70%), however, little is known about the mechanism(s) mediating the intestinal absorption. The aim of the present study was to identify which solute carrier(s) are involved in the absorption of vigabatrin in Caco-2 cells, a cell culture model of the small intestinal epithelium. The uptake and transepithelial flux of vigabatrin was measured using an LC-MS method for quantification. Transepithelial transport of vigabatrin was shown to be proton-dependent and polarized in the apical-to-basolateral (A-B) direction. The A-B flux of vigabatrin had a saturable component and a passive component, indicating the presence of a carrier system in parallel with a passive permeability. The Michaelis constant, Km, of the transepithelial A-B flux of vigabatrin was estimated to be 32.8±7.4 mM (n=3-5), whereas the Km of the apical uptake was found to be 12.7±3.7 mM (n=3). The carrier-mediated transepithelial A-B flux of vigabatrin accounted for 80-95% (50.0-1.0mM) of the total A-B flux. The transepithelial A-B flux (as well as apical uptake) of vigabatrin was significantly decreased upon addition of substrates or inhibitors of the human proton-coupled amino acid transporter (hPAT1) to the apical solution. The present study indicates that the transepithelial A-B flux of vigabatrin is mainly mediated by hPAT1 in Caco-2 cells at dose-relevant concentrations.
Asunto(s)
Transporte Biológico/fisiología , Absorción Intestinal/fisiología , Vigabatrin/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Células CACO-2 , Línea Celular Tumoral , Dipéptidos/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Simportadores/metabolismoRESUMEN
BACKGROUND: We assessed the feasibility of administering a neuroprotective drug, vigabatrin (VGB; gamma-vinyl-gamma-aminobutyric acid) with multimodality monitoring, including cerebral microdialysis, in severe head injury patients, to measure surrogate endpoints and blood-brain barrier (BBB) penetration. METHODS: Patients (n = 20) were randomised to VGB (0.5 g twice-daily, enteric) or control. ICP, ABP, CPP and cerebrovascular pressure reactivity index (PRx) were monitored. Microdialysate glucose, lactate, pyruvate, glutamate, glycerol, amino acids, VGB and GABA were analysed. RESULTS: Preliminary evaluation of results (five VGB-treated patients) showed that VGB levels rose in brain microdialysates, followed by a modest increase in GABA. VGB and GABA increased more in abnormal brain than in sites further from lesions, and were higher after multiple VGB doses. Highest VGB and GABA microdialysate levels were 75 and 4 µmol/L respectively. Microdialysate glucose and glycerol sometimes decreased, and glutamate and tyrosine sometimes increased, following VBG administration; causation unproven. VGB did not overtly affect ICP, ABP, CPP, PRx, or microdialysate lactate, pyruvate and lactate/pyruvate ratio. CONCLUSION: Multimodality monitoring, including cerebral microdialysis, is feasible for studying surrogate endpoints following drug administration. VGB crosses the BBB, leading to modest increases in extracellular GABA. Further analyses are ongoing. Microdialysis may assist the development of neuroprotective agents by determining penetration into extracellular fluid of the brain.
Asunto(s)
Anticonvulsivantes/administración & dosificación , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/metabolismo , Microdiálisis , Vigabatrin/administración & dosificación , Administración Oral , Adolescente , Adulto , Anciano , Aminoácidos/metabolismo , Presión Sanguínea/efectos de los fármacos , Química Encefálica , Cromatografía Líquida de Alta Presión , Femenino , Glucosa/metabolismo , Ácido Glutámico/metabolismo , Humanos , Presión Intracraneal/efectos de los fármacos , Presión Intracraneal/fisiología , Ácido Láctico/metabolismo , Masculino , Persona de Mediana Edad , Monitoreo Fisiológico , Factores de Tiempo , Vigabatrin/metabolismo , Adulto Joven , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The enzyme methylenetetrahydrofolate reductase (MTHFR) is a part of the homocysteine and folate metabolic pathways, affecting the methylations of DNA, RNA, and proteins. Mthfr deficiency was reported as a risk factor for neurodevelopmental disorders such as autism spectrum disorder and schizophrenia. Neonatal disruption of the GABAergic system is also associated with behavioral outcomes. The interaction between the epigenetic influence of Mthfr deficiency and neonatal exposure to the GABA potentiating drug vigabatrin (GVG) in mice has been shown to have gender-dependent effects on mice anxiety and to have memory impairment effects in a gender-independent manner. Here we show that Mthfr deficiency interacts with neonatal GABA potentiation to alter social behavior in female, but not male, mice. This impairment was associated with a gender-dependent enhancement of proteins implicated in excitatory synapse plasticity in the female cortex. Reelin and fragile X mental retardation 1 protein (FMRP) levels and membrane GluR1/GluR2 ratios were elevated in wild-type mice treated neonatally with GVG and in Mthfr+/- mice treated with saline, but not in Mthfr+/- mice treated with GVG, compared with control groups (wild type treated with saline). A minor influence on the levels of these proteins was observed in male mice cortices, possibly due to high basal protein levels. Interaction between gender, genotype, and treatment was also observed in the GABA pathway. In female mice, GABA Aα2/gephyrin ratios were suppressed in all test groups; in male mice, a genotype-specific enhancement of GABA Aα2/gephyrin was observed. The lack of an effect on either reln or Fmr1 transcription suggests post-transcriptional regulation of these genes. Taken together, these findings suggest that Mthfr deficiency may interact with neonatal GABA potentiation in a gender-dependent manner to interrupt synaptic function. This may illustrate a possible mechanism for the epigenetic involvement of Mthfr deficiency in neurodevelopmental disorders.
Asunto(s)
Corteza Cerebral/metabolismo , Metilenotetrahidrofolato Reductasa (NADPH2)/deficiencia , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Proteínas del Tejido Nervioso/metabolismo , Caracteres Sexuales , Vigabatrin/farmacología , Animales , Corteza Cerebral/efectos de los fármacos , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal/fisiología , Unión Proteica/fisiología , Distribución Aleatoria , Proteína Reelina , Membranas Sinápticas/genética , Membranas Sinápticas/metabolismo , Vigabatrin/metabolismoRESUMEN
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 Ca(2+) 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 GABA(A) receptors, occluded SOCs, whereas the benzodiazepine site inverse agonist ß-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 GABA(A) 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.
Asunto(s)
Receptores de GABA-A/metabolismo , Transducción de Señal , Tálamo/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Femenino , GABAérgicos/metabolismo , Masculino , Ratones , Ratones Noqueados , Neuronas/citología , Neuronas/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptores de GABA-A/genética , Tálamo/citología , Vigabatrin/metabolismoRESUMEN
γ-Aminobutyric acid (GABA) synthesis from glutamate is catalyzed by glutamate decarboxylase (GAD) of which two isoforms, GAD65 and GAD67, have been identified. The GAD65 has repeatedly been shown to be important during intensified synaptic activity. To specifically elucidate the significance of GAD65 for maintenance of the highly compartmentalized intracellular and intercellular GABA homeostasis, GAD65 knockout and corresponding wild-type mice were injected with [1-(13)C]glucose and the astrocyte-specific substrate [1,2-(13)C]acetate. Synthesis of GABA from glutamine in the GABAergic synapses was further investigated in GAD65 knockout and wild-type mice using [1,2-(13)C]acetate and in some cases γ-vinylGABA (GVG, Vigabatrin), an inhibitor of GABA degradation. A detailed metabolic mapping was obtained by nuclear magnetic resonance (NMR) spectroscopic analysis of tissue extracts of cerebral cortex and hippocampus. The GABA content in both brain regions was reduced by â¼20%. Moreover, it was revealed that GAD65 is crucial for maintenance of biosynthesis of synaptic GABA particularly by direct synthesis from astrocytic glutamine via glutamate. The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism. Furthermore, a severe neuronal hypometabolism, involving glycolysis and tricarboxylic acid (TCA) cycle activity, was observed in cerebral cortex of GAD65 knockout mice.
Asunto(s)
Astrocitos/metabolismo , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/fisiología , Glutamina/metabolismo , Sinapsis/metabolismo , Ácido gamma-Aminobutírico/biosíntesis , Ácido gamma-Aminobutírico/genética , Acetatos/metabolismo , Animales , Western Blotting , Corteza Cerebral/metabolismo , Ciclo del Ácido Cítrico/fisiología , GABAérgicos/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Glucosa/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Vigabatrin/metabolismoRESUMEN
PURPOSE: In 2002, we reported our preliminary experience using the ketogenic diet (KD) for predominantly intractable infantile spasms (IS) in 23 infants. Since that time, we have increased our use of the KD for this condition including those with new-onset IS. METHODS: Infants were referred and prospectively started on the traditional KD from 1996 to 2009 at our institution. Included subjects had documented clinical IS, hypsarrhythmia on electroencephalography (EEG), and parental consent to start the KD. Efficacy was assessed through phone communication, clinic visits, and EEG every 3 months. RESULTS: One hundred four infants, mean age 1.2 years, were started on the KD for IS, of which 74 (71%) had a symptomatic etiology. Previous therapy for this patients included a mean of 3.6 anticonvulsants; 71% including corticosteroids or vigabatrin. Using an intent-to-treat analysis, > 50% spasm improvement occurred in 64% at 6 months and 77% after 1-2 years. Thirty-eight (37%) became spasm-free for at least a 6-month period within a median 2.4 months of starting the KD. In addition, 62% reported improvement in development, 35% had EEG improvement, and 29% were able to reduce concurrent anticonvulsants. Adverse effects were noted in 33%, of which 6% had diminished linear growth. Older age at onset of IS and fewer prior anticonvulsants were more likely to be associated with > 90% spasm improvement at 6 months. DISCUSSION: The KD is an efficacious therapy for IS in approximately two-thirds of patients treated, and it should be considered strongly after failure of corticosteroids and vigabatrin.
Asunto(s)
Dieta Cetogénica/métodos , Espasmos Infantiles/dietoterapia , Corticoesteroides/metabolismo , Electroencefalografía/métodos , Femenino , Humanos , Lactante , Masculino , Valor Predictivo de las Pruebas , Estudios Prospectivos , Estudios Retrospectivos , Factores de Tiempo , Resultado del Tratamiento , Vigabatrin/metabolismoRESUMEN
In this study, the relationship between endogenous brain GABA concentration and glutamate-glutamine cycling flux (V (cyc)) was investigated using in vivo (1)H and (1)H{(13)C} magnetic resonance spectroscopy techniques. Graded elevations of brain GABA levels were induced in rat brain after administration of the highly specific GABA-transaminase inhibitor vigabatrin (gamma-vinyl-GABA). The glial-specific substrate [2-(13)C]acetate and (1)H{(13)C} magnetic resonance spectroscopy were used to measure V (cyc) at different GABA levels. Significantly reduced V (cyc) was found in rats pretreated with vigabatrin. The reduction in group mean V (cyc) over the range of GABA concentrations investigated in this study (1.0 +/- 0.3-5.1 +/- 0.5 micromol/g) was found to be nonlinear: Delta V (cyc)/V (cyc) = [GABA (micromol/g)](-0.35 )- 1.0 (r (2) = 0.98). The results demonstrate that V (cyc) is modulated by endogenous GABA levels, and that glutamatergic and GABAergic interactions can be studied in vivo using noninvasive magnetic resonance spectroscopy techniques.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Neuronas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , 4-Aminobutirato Transaminasa/antagonistas & inhibidores , Acetatos/administración & dosificación , Acetatos/metabolismo , Análisis de Varianza , Animales , Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , Isótopos de Carbono/metabolismo , Radioisótopos de Carbono/metabolismo , Inyecciones Intravenosas , Espectroscopía de Resonancia Magnética/métodos , Masculino , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Vigabatrin/administración & dosificación , Vigabatrin/metabolismoRESUMEN
Changed neuropeptide Y (NPY) system in the hippocampus has been reported in various experimental epileptic models. However, there have been little data concerning the alteration in the NPY system in the epileptic hippocampus following treatment of anti-epileptic drugs (AEDs). In the present study, therefore, we performed analyses of effects of vigabatrin (VGB) and zonisamide (ZNS) treatment on the NPY system in the hippocampus of the seizure sensitive (SS) gerbils. In SS gerbil, NPY immunoreactivity in the hippocampus was lower than that in seizure resistant gerbil. Following VGB treatment, the number of NPY immunoreactive neurons and NPY mRNA expression were increased in the hilus and the hippocampus proper. In contrast, ZNS treatment markedly elevated only the density of NPY immunoreactive fibers in the dentate gyrus, not in the hippocampus proper, as compared with saline-treated animals. These patterns were observed in the dose-dependent manners. These findings suggest that AEDs treatments may distinctly affect the NPY system in the SS gerbil hippocampus.
Asunto(s)
Anticonvulsivantes/farmacología , Hipocampo/efectos de los fármacos , Isoxazoles/farmacología , Neuropéptido Y/metabolismo , Vigabatrin/farmacología , Animales , Anticonvulsivantes/metabolismo , Gerbillinae , Hipocampo/citología , Hibridación in Situ , Isoxazoles/metabolismo , Neuropéptido Y/genética , Convulsiones/fisiopatología , Vigabatrin/metabolismo , ZonisamidaRESUMEN
Nitric oxide (NO) modulates the uptake and/or release of neurotransmitters through a variety of cellular mechanisms. However, the pharmacological and biochemical processes underlying these neurochemical effects of NO often remain unclear. In our study, we used immunocytochemical methods to study the effects of NO, cyclic guanosine monophosphate (cGMP), and peroxynitrite on the uptake and release of gamma-aminobutyric acid (GABA) and glycine in the turtle retina. In addition, we examined the involvement of glutamate receptors, calcium, and the GABA transporter in this GABA uptake and release. We also tested for interactions between the GABAergic and glycinergic systems. In general, we show that NO stimulated GABA release and inhibited glycine release. The NO-stimulated GABA release involved calcium-dependent or calcium-independent synaptic release or reversal of the GABA transporter. Some effects of NO on GABA release involved glutamate, cGMP, or peroxynitrite. NO promoted glycine uptake and inhibited its release, and this inhibition of glycine release was influenced by GABAergic modulation. These findings indicate that NO modulates the levels of the inhibitory transmitters GABA and glycine through several specific biochemical mechanisms in different retinal cell types and layers. Thus it appears that some of the previously described reciprocal interactions between GABA and glycine in the retina function through specific NO signaling pathways.
Asunto(s)
Citrulina/análogos & derivados , GMP Cíclico/análogos & derivados , Depuradores de Radicales Libres/farmacología , Glicina/metabolismo , Molsidomina/análogos & derivados , Inhibición Neural/efectos de los fármacos , Óxido Nítrico/farmacología , Retina/efectos de los fármacos , Tiourea/análogos & derivados , Ácido gamma-Aminobutírico/metabolismo , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Bicuculina/farmacología , Cadmio/farmacología , Citrulina/farmacología , GMP Cíclico/farmacología , DEET/farmacología , Maleato de Dizocilpina/farmacología , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Inmunohistoquímica/métodos , Técnicas In Vitro , Molsidomina/farmacología , Ácidos Nipecóticos/farmacología , Potasio/farmacología , Retina/metabolismo , Tinción con Nitrato de Plata/métodos , Tiourea/farmacología , Tortugas , Vigabatrin/metabolismoRESUMEN
The X-ray crystal structures of Escherichia coli gamma-aminobutyrate aminotransferase unbound and bound to the inhibitor aminooxyacetate are reported. The enzyme crystallizes from ammonium sulfate solutions in the P3(2)21 space group with a tetramer in the asymmetric unit. Diffraction data were collected to 2.4 A resolution for the unliganded enzyme and 1.9 A resolution for the aminooxyacetate complex. The overall structure of the enzyme is similar to those of other aminotransferase subgroup II enzymes. The ability of gamma-aminobutyrate aminotransferase to act on primary amine substrates (gamma-aminobutyrate) in the first half-reaction and alpha-amino acids in the second is proposed to be enabled by the presence of Glu211, whose side chain carboxylate alternates between interactions with Arg398 in the primary amine half-reaction and an alternative binding site in the alpha-amino acid half-reaction, in which Arg398 binds the substrate alpha-carboxylate. The specificity for a carboxylate group on the substrate side chain is due primarily to the presence of Arg141, but also requires substantial local main chain rearrangements relative to the structurally homologous enzyme dialkylglycine decarboxylase, which is specific for small alkyl side chains. No iron-sulfur cluster is found in the bacterial enzyme as was found in the pig enzyme [Storici, P., De Biase, D., Bossa, F., Bruno, S., Mozzarelli, A., Peneff, C., Silverman, R. B., and Schirmer, T. (2004) J. Biol. Chem. 279, 363-73.]. The binding of aminooxyacetate causes remarkably small changes in the active site structure, and no large domain movements are observed. Active site structure comparisons with pig gamma-aminobutyrate aminotransferase and dialkylglycine decarboxylase are discussed.