RESUMEN
OBJETIVO: estimar a associação entre fatores socioeconômicos e más oclusões do tipo overbite e overjet em escolares em fase de dentição permanente jovem. MÉTODO: trata-se de um estudo transversal realizado em 251 escolares de sete a 15 anos, de ambos os sexos, regularmente matriculadas no ensino fundamental de escolas públicas e privadas do município de São Luís, Maranhão, Brasil. Todos os sujeitos da amostra realizaram fotografias extrabucais padronizadas de face (frontal em repouso, frontal sorrindo e perfil) e intrabucais (frontal, lateral direita e lateral esquerda em oclusão, oclusal superior e oclusal inferior), para o diagnóstico das alterações. As fotografias foram avaliadas por três examinadores em regime cego e as discordâncias foram discutidas até a obtenção de consenso. Overbite e overjet foram diagnosticados segundo critérios de Angle. RESULTADOS: constatou-se que 35,4% das crianças apresentaram overbite e 18,3%, overjet. Houve associação estatisticamente significante entre a renda familiar com overbite (p=0.003) e com overjet (p=0.029), observando-se que a maioria das crianças que não possuíam overbite (69,4%) era de famílias com baixa renda (até 2 salários mínimos). De modo semelhante, a maioria dos portadores de overjet era de famílias com renda mais elevada, ao passo que a maioria daqueles sem overjet tinha renda familiar baixa. Para as demais variáveis não houve diferenças estatisticamente significante na distribuição de frequência de overjet e overbite. CONCLUSÃO: a prevalência de overbite e overjet nos escolares estudados é alta, especialmente entre aqueles com maior renda familiar.
PURPOSE: to estimate the association between socioeconomic factors and occlusal changes like overbite and overjet in students aged 7 to 15 years. METHOD: this is a cross-sectional study in 251 students of both gender regularly enrolled in the school of public and private schools in São Luís, Maranhão, Brazil. All subjects carried out the sample photos extra-oral standardized face (front resting, smiling front and profile) and intraoral (front, right side and left side in occlusion, occlusal upper and lower occlusal) for the diagnosis of changes. The photographs were evaluated by three observers under blind and disagreements were discussed to achieve consensus. Overbite and overjet were diagnosed according to criteria of Angle. RESULTS: it was found that 35.4% of the children were 18.3% and overbite, overjet. There was a statistically significant association between family income and overbite (p = 0.003) and overjet (p = 0.029), noting that most children who had no overbite (69.4%) were low-income families (up to 2 minimum wage). Similarly, most people with overjet were families with higher incomes, while the majority of those without overjet had low family income. For other variables there was no statistically significant difference in frequency distribution of overjet and overbite. CONCLUSION: the prevalence of overbite and overjet among the studied population is high, especially among those with higher family income.
RESUMEN
Chitin is a representative microbe-associated molecular pattern (MAMP) molecule for various fungi and induces immune responses in many plant species. It has been clarified that the chitin signaling in rice requires a receptor kinase OsCERK1 and a receptor-like protein (Os)CEBiP, which specifically binds chitin oligosaccharides. On the other hand, Arabidopsis requires a receptor kinase (At)CERK1 for chitin signaling but it is not clear whether the plant also requires a CEBiP-like molecule for chitin perception/signaling. To clarify the similarity/difference of the chitin receptor in these two model plants, we first characterized CEBiP homologs in Arabidopsis. Only one of three CEBiP homologs, AtCEBiP (LYM2), showed a high-affinity binding for chitin oligosaccharides similar to rice CEBiP. AtCEBiP also represented the major chitin-binding protein in the Arabidopsis membrane. However, the single/triple knockout (KO) mutants of Arabidopsis CEBiP homologs and the overexpressor of AtCEBiP showed chitin-induced defense responses similar to wild-type Arabidopsis, indicating that AtCEBiP is biochemically functional as a chitin-binding protein but does not contribute to signaling. Studies of the chitin binding properties of the ectodomains of At/OsCERK1 and the chimeric receptors consisting of ecto/cytosolic domains of these molecules indicated that AtCERK1 is sufficient for chitin perception by itself.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Quitina/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Citosol/metabolismo , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Oryza/inmunología , Oryza/microbiología , Plantas Modificadas Genéticamente , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Transducción de Señal , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
OBJECTIVES: We aimed to clarify the relationships of the different stages of behavior change in dietary habits followed by the mothers of school-age children with the actual breakfast intake of these children and the health-associated behavior of the family. METHODS: We carried out a questionnaire-based survey of 1949 children at 18 elementary schools and of 881 families with children attending seven elementary schools in Kagoshima prefecture. We were supplied with information about children's breakfast intake and content on the day they took the survey and information about mothers' breakfast intake and the stage of behavior change in dietary habits to which they belonged, for which five stages were defined using the stage-of-change model. RESULTS: The collection rates were 83.3% and 83.1% among children and mothers respectively. Of the children, 83.1% ate breakfast every day, while 15.1% were not in the habit of having breakfast. Furthermore, 98.6% children had eaten breakfast on the day of the survey, but 15.1% had eaten only staple foods such as rice or bread; only 34.0% children combined staple foods, a main dish, and vegetables/fruits in their breakfast. Regarding dietary stage, 28.1% of the mothers belonged to the "maintenance" stage; 24.0%, the "action" stage; 6.9%, the "preparation" stage; 9.8%, the "contemplation" stage; and 5.7%, the "precontemplation" stage. Mothers belonging to the first two stages constituted the "action group," because they were already taking care of their dietary habits, and mothers belonging to the latter three stages constituted the "no-action group", because they were not taking care of their dietary habits. The mothers who could provide no answers to the question constituted the "no-answer group" (25.5%). A comparison of the three groups revealed that mothers belonging to the no-answer group had more children who went without breakfast than the action group (P = 0.000). The children of mothers belonging to the no-action group (P = 0.003) and the no-answer group (P = 0.036) were not in general eating vegetables/fruits in their breakfast, in contrast with the action group. Furthermore, in the case of families with mothers belonging to the no-action and no-answer groups, the families did not often talk about diet, and the incidence of smokers among the fathers was high. CONCLUSION: In this study, the breakfast habits of children and the health behavior of families differed by stage of dietary behavior change to which the mother belonged.
Asunto(s)
Familia , Conducta Alimentaria , Conductas Relacionadas con la Salud , Madres , Actitud , Niño , Femenino , Humanos , Masculino , Encuestas y CuestionariosRESUMEN
A novel polymerizable anionic gemini surfactant has been synthesized and the physicochemical properties in aqueous solution have been studied with a combination of various analytical techniques. The surfactant (PA12-2-12) contains two anionic monomeric parts linked with an ethylene spacer and polymerizable methacryloxy groups covalently bound to the terminal of the hydrocarbon chains. The static surface tension data suggest that, when compared with a conventional (non-polymerizable) anionic gemini surfactant (A12-2-12), (i) the interfacial adsorption of PA12-2-12 occurs more effectively from low surfactant concentrations, whereas (ii) a weak interaction of the polymerizable terminal groups with water molecules (and/or the steric hindrance of the polymerizable groups) plays a significant role in the subsequent molecular packing at the air/aqueous solution interface. The latter effect (as well as the electrostatic repulsion between the anionic headgroups) results in a relatively less packed monolayer film, overcoming the strong intermolecular attractive interaction that is frequently seen for gemini surfactant systems. In the region of low added electrolyte concentrations, PA12-2-12 spontaneously forms spherical micelles in aqueous solution, which is confirmed with the Corrin-Harkins analysis (critical micelle concentration (cmc) vs. total counter-ion concentration) and cryogenic transmission electron microscopy (cryo-TEM). The spherical micelles have been polymerized under UV light irradiation in the absence of added electrolytes. Cryo-TEM measurements confirm that no significant change in the original micelle morphology occurs during the polymerization. This offers a possibility that the polymerizable anionic gemini surfactant should be useful as nano-structural organic templates and/or interfacial stabilizers in aqueous solution.
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Polímeros/química , Polímeros/síntesis química , Tensoactivos/química , Tensoactivos/síntesis química , Aniones/síntesis química , Aniones/química , Química Física , Microscopía por Crioelectrón , Electrólitos/química , Etilenos/química , Hidrocarburos/química , Metacrilatos/química , Micelas , Estructura Molecular , Tamaño de la Partícula , Soluciones , Electricidad Estática , Propiedades de Superficie , Agua/químicaRESUMEN
PURPOSE: In the present study, we investigate the characteristics of taurine transport in primary cultures of neurons from mouse cerebral cortex to understand the possibility that taurine might attenuate the effects of central nervous system drugs. METHODS: Primary cultured neurons from mouse cerebral cortex were used to determine the transport characteristics of taurine. The expression of taurine transporter (TAUT) in mouse neurons was determined by use of reverse transcriptase-polymerase chain reaction and Western blotting. RESULTS: In vitro transport of taurine in mouse cerebrocortical neurons at day 9 was Na+-dependent and saturable with a Michaelis-Menten constant (Kt) of 10.6 +/- 4.1 microM and a maximum velocity (Vmax) of 6.68 +/- 0.85 nmol/mg protein/10 min. Na+ and Cl- activation kinetics revealed that the Na+-to-Cl(-)-to-taurine stoichiometry was 2:1:1. Na+-dependent [3H]-taurine transport was competitively inhibited by beta-alanine with an inhibitory constant (Ki) of 47.4 +/- 6.5 microM. Gamma-aminobutyric acid also inhibited Na+-dependent [3H]-taurine transport with relatively low affinity (Ki = 273 +/- 71 microM). TAUT mRNA was detected in mouse primary cultured neurons, and TAUT protein was also expressed at approximately 70 kDa. Na+-dependent taurine transport activity was increased with developing neurons and corresponded with the increasing mRNA and protein level of TAUT. CONCLUSIONS: The present study revealed that Na+/Cl(-)-coupled taurine transporter TAUT is responsible for taurine uptake in mouse cerebrocortical neurons, and that the expression of TAUT is increased with developing cerebrocortical neurons.
Asunto(s)
Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Glicoproteínas de Membrana/biosíntesis , Proteínas de Transporte de Membrana/biosíntesis , Neuronas/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Animales , Western Blotting , Células Cultivadas , Corteza Cerebral/crecimiento & desarrollo , Cloruros/metabolismo , Femenino , Ratones , Embarazo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sodio/metabolismo , Especificidad por Sustrato , Taurina/metabolismo , Regulación hacia ArribaRESUMEN
Na+-coupled carboxylate transporters (NaCs) mediate the uptake of tricarboxylic acid cycle intermediates in mammalian tissues. Of these transporters, NaC3 (formerly known as Na+-coupled dicarboxylate transporter 3, NaDC3/SDCT2) and NaC2 (formerly known as Na+-coupled citrate transporter, NaCT) have been shown to be expressed in brain. There is, however, little information available on the precise distribution and function of both transporters in the CNS. In the present study, we investigated the functional characteristics of Na+-dependent succinate and citrate transport in primary cultures of astrocytes and neurons from rat cerebral cortex. Uptake of succinate was Na+ dependent, Li+ sensitive and saturable with a Michaelis constant (Kt) value of 28.4 microM in rat astrocytes. Na+ activation kinetics revealed that the Na+ to succinate stoichiometry was 3:1 and the concentration of Na+ necessary for half-maximal transport was 53 mM. Although uptake of citrate in astrocytes was also Na+ dependent and saturable, its Kt value was significantly higher (approximately 1.2 mM) than that of succinate. Unlabeled succinate (2 mM) inhibited Na+-dependent [14C]succinate (18 microM) and [14C]citrate (4.5 microM) transport completely, whereas unlabeled citrate inhibited Na+-dependent [14C]succinate uptake more weakly. Interestingly, N-acetyl-L-aspartate, which is the second most abundant amino acid in the nervous system, also completely inhibited Na+-dependent succinate transport in rat astrocytes. The inhibition constant (Ki) for the inhibition of [14C]succinate uptake by unlabeled succinate, N-acetyl-L-aspartate and citrate was 15.9, 155 and 764 microM respectively. In primary cultures of neurons, uptake of citrate was also Na+ dependent and saturable with a Kt value of 16.2 microM, which was different from that observed in astrocytes, suggesting that different Na+-dependent citrate transport systems are expressed in neurons and astrocytes. RT-PCR and immunocytochemistry revealed that NaC3 and NaC2 are expressed in cerebrocortical astrocytes and neurons respectively. These results are in good agreement with our previous reports on the brain distribution pattern of NaC2 and NaC3 mRNA using in situ hybridization. This is the first report of the differential expression of different NaCs in astrocytes and neurons. These transporters might play important roles in the trafficking of tricarboxylic acid cycle intermediates and related metabolites between glia and neurons.
Asunto(s)
Astrocitos/metabolismo , Corteza Cerebral/metabolismo , Transportadores de Ácidos Dicarboxílicos/metabolismo , Neuronas/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Sodio/metabolismo , Simportadores/metabolismo , Animales , Animales Recién Nacidos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacología , Astrocitos/efectos de los fármacos , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/fisiología , Células Cultivadas , Ácido Cítrico/metabolismo , Ciclo del Ácido Cítrico/efectos de los fármacos , Ciclo del Ácido Cítrico/fisiología , Transportadores de Ácidos Dicarboxílicos/efectos de los fármacos , Litio/farmacología , Neuronas/efectos de los fármacos , Transportadores de Anión Orgánico Sodio-Dependiente/efectos de los fármacos , Ratas , Ratas Wistar , Sodio/farmacología , Ácido Succínico/metabolismo , Simportadores/efectos de los fármacosRESUMEN
Neurons are known to express a high-affinity Na+ -coupled dicarboxylate transporter(s) for uptake of tricarboxylic acid cycle intermediates, such as alpha-ketoglutarate and malate, which are precursors for neurotransmitters including glutamate and gamma-aminobutyric acid. There is, however, little information available on the molecular identity of the transporters responsible for this uptake process in neurons. In the present study, we investigated the characteristics of Na+ -dependent citrate transport in primary cultures of neurons from mouse cerebral cortex and established the molecular identity of this transport system as the Na+ -coupled citrate transporter (NaC2/NaCT). Reverse transcriptase (RT)-PCR and immunocytochemical analyses revealed that only NaC2/NaCT was expressed in mouse cerebrocortical neurons but not in astrocytes. Uptake of citrate in neurons was Na+ -dependent, Li+ -sensitive, and saturable with the Kt value of 12.3 microM. This Kt value was comparable with that in the case of Na+ -dependent succinate transport (Kt = 9.2 microM). Na+ -activation kinetics revealed that the Na+ -to-citrate stoichiometry was 3.4:1 and concentration of Na+ necessary for half-maximal activation (K0.5(Na)) was 45.7 mM. Na+ -dependent uptake of [14C]citrate (18 microM) was significantly inhibited by unlabeled citrate as well as dicarboxylates such as succinate, malate, fumarate, and alpha-ketoglutarate. This is the first report demonstrating the molecular identity of the Na+ -coupled di/tricarboxylate transport system expressed in neurons as NaC2/NaCT, which can transport the tricarboxylate citrate as well as dicarboxylates such as succinate, alpha-ketoglutarate, and malate.
Asunto(s)
Proteínas Portadoras/metabolismo , Corteza Cerebral/citología , Expresión Génica/fisiología , Neuronas/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Animales , Animales Recién Nacidos , Transporte Biológico/efectos de los fármacos , Isótopos de Carbono/farmacocinética , Proteínas Portadoras/genética , Células Cultivadas , Ácido Cítrico/farmacocinética , Relación Dosis-Respuesta a Droga , Litio/farmacología , Malatos/farmacología , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/efectos de los fármacos , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Formación de Roseta/métodos , Sodio/farmacología , Ácido Succínico/farmacología , Factores de TiempoRESUMEN
The extracellular levels of gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the mammalian cerebral cortex, are regulated by specific high-affinity Na(+)/Cl(-) dependent transporters (GATs). GAT1 mainly expressed in cerebrocortical neurons is thought to play an important role for clearance of GABA in the extracellular fluid, whereas there is a little information available for pharmacological importance for astrocytic GABA transporters. In the present study, we therefore described the functional characterization of GABA transport in primary cultures of astrocytes from rat cerebral cortex and the identification of GABA transporter subtype(s). GABA transport was Na(+) and Cl(-) dependent and saturable with a Michaelis constant (K(t)) of 9.3+/-2.8 microM. Na(+)- and Cl(-)- activation kinetics revealed that the Na(+)-Cl(-)-to-GABA stoichiometry was 2:1:1 and concentrations of Na(+) and Cl(-) necessary for half-maximal transport (K(0.5)(Na) and K(0.5)(Cl)) were 78+/-28 mM and 9.6+/-2.6 mM, respectively. Na(+)-dependent GABA transport was competitively inhibited by various GABA transport inhibitors, especially GAT2- or GAT3-selective inhibitor. In addition, Zn(2+), which has been reported to be a potent inhibitor of GAT3, was found to have a significantly but partially inhibitory effect on the Na(+)-dependent GABA transport in a concentration-dependent manner. Furthermore, reverse transcription-PCR and Western blot analyses revealed that GAT2 and GAT3 are expressed in primary cultures of astrocytes. These results clearly showed that zinc is a useful reagent for separating GAT3 activity from GAT1- and GAT2-activities in CNS. To our knowledge, the present study represents the first report on the inhibitory effect of zinc on the Na(+)-dependent GABA transport in rat cerebrocortical astrocytes.
Asunto(s)
Astrocitos/metabolismo , Corteza Cerebral/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Zinc/farmacología , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Transporte Biológico , Células Cultivadas , Cartilla de ADN , Proteínas Transportadoras de GABA en la Membrana Plasmática/efectos de los fármacos , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Inmunohistoquímica , Ratas , Ácido gamma-Aminobutírico/metabolismoRESUMEN
GABA(B) receptor subunits are widely expressed on neurons throughout the central nervous system (CNS), at both pre- and postsynaptic sites, where they mediate the late and slow component of the inhibitory response to the major inhibitory neurotransmitter GABA. Recently, GABA(B) receptors have been reported to be expressed in astrocytes and microglia in the rat CNS by immunocytochemistry. However, there are few reports available for the functional characterization of GABA(B) receptors on astrocytes. In the present study, we therefore investigated the functional expression and characteristics of GABA(B) receptors in primary cultures of astrocytes from rat cerebral cortex. In the presence of 10 microM GTP, forskolin concentration-dependently increased adenylylcyclase (AC) activity in membranes prepared from rat astrocytes. The selective GABA(B) agonist (R)-baclofen concentration-dependently reduced forskolin-stimulated AC activity in the presence of 10 microM GTP. This effect was reversed by the selective GABA(B) antagonists, CGP-55845 and CGP-54626, and was completely abolished by treatment of astrocytic membranes with pertussis toxin. In addition, RT-PCR, Western blotting, and immunocytochemistry clearly showed that metabotropic GABA(B) receptor isoforms (GABA(B)R1 and GABA(B)R2) are expressed in rat cerebrocortical astrocytes. Taken collectively, these results demonstrate that functionally active metabotropic GABA(B) receptors are expressed in rat cerebrocortical astrocytes.
Asunto(s)
Astrocitos/metabolismo , Corteza Cerebral/citología , Receptores de GABA-B/metabolismo , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Astrocitos/efectos de los fármacos , Baclofeno/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Colforsina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Guanosina Trifosfato/farmacología , ARN Mensajero/genética , RatasRESUMEN
In our previous studies, we demonstrated that the high-affinity type peptide transporter PEPT2 is expressed in rat cerebral cortex using synaptosomal membrane study and that the uptake of dipeptide [14C]glycylsarcosine into synaptosomes was stimulated by an inwardly directed H+ gradient (Fujita et al., Brain Res. 972, 52-61, 2004). However, there is no information available for the driving force of PEPT2 function in the nervous system. In the present study, we investigated functional characteristics of PEPT2 mediated transport of Gly-Sar in primary cultured astrocytes from mouse cerebral cortex and examined the effects of Na+/H+ exchanger (NHE) inhibitor on Gly-Sar uptake in mouse astrocytes. In mouse astrocytes, extracellular H+ influenced only the maximal velocity (Vmax) of Gly-Sar uptake without affecting the apparent affinity (Kt). Interestingly, removal of Na+ from uptake buffer significantly reduced Gly-Sar uptake and Gly-Sar uptake was modulated by NHE inhibitors. The treatment of EIPA, an NHE inhibitor, altered the Vmax value of Gly-Sar uptake but had no effect on its Kt value. RT-PCR revealed that NHE1 and NHE2 mRNA are expressed in mouse cerebrocortical astrocytes. These results demonstrated that NHE activity is required to allow optimal uptake of dipeptides mediated by PEPT2 into the astrocytes. This study represents the first description of the functional co-operation of PEPT2 and NHE1 and/or NHE2 in cerebrocortical astrocytes.
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Astrocitos/fisiología , Corteza Cerebral/citología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Simportadores/metabolismo , Animales , Astrocitos/efectos de los fármacos , Northern Blotting/métodos , Western Blotting/métodos , Células Cultivadas , Dipéptidos/farmacocinética , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Concentración de Iones de Hidrógeno , Inmunohistoquímica/métodos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Intercambiadores de Sodio-Hidrógeno/clasificación , Intercambiadores de Sodio-Hidrógeno/genética , Especificidad por Sustrato/efectos de los fármacos , Simportadores/antagonistas & inhibidores , Simportadores/genética , Proteína 25 Asociada a Sinaptosomas , Tritio/farmacocinéticaRESUMEN
We describe the regulation mechanisms of the Na(+)-dependent neutral amino acid transporter ASCT2 via nitric oxide (NO) in the human intestinal cell line, Caco-2. Exposure of Caco-2 cells to S-nitrosothiol, such as S-nitroso-N-acetyl-DL-penicillamine (SNAP) and S-nitrosoglutathione, and the NO-donor, NOC12, concentration- and time-dependently increased Na(+)-dependent alanine uptake. Kinetic analyses indicated that SNAP increases the maximal velocity (V(max)) of Na(+)-dependent alanine uptake in Caco-2 cells without affecting the Michaelis-Menten constant (K(t)). The stimulatory effect was partially eliminated by actinomycin D and cycloheximide. Increased Na(+)-dependent alanine uptake by SNAP was partially abolished by the NO scavengers, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide sodium salt (carboxy-PTIO) and N-(dithiocarboxy)sarcosine disodium salts (DTCS), as well as the NADPH oxidase inhibitor, diphenyleneiodonium. RT-PCR revealed that Caco-2 cells expressed the Na(+)-dependent neutral amino acid transporter ASCT2, but not the other Na(+)-dependent neutral amino acid transporters ATB(0,+) and B(0)AT1. These results suggested that functional up-regulation of ASCT2 by SNAP might be partially associated with an increase in the density of transporter protein via de novo synthesis.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/metabolismo , S-Nitrosotioles/farmacología , Sodio/farmacología , Alanina/metabolismo , Transporte Biológico/efectos de los fármacos , Células CACO-2 , Cicloheximida/farmacología , Dactinomicina/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Humanos , Cinética , Antígenos de Histocompatibilidad Menor , Donantes de Óxido Nítrico/metabolismo , S-Nitroso-N-Acetilpenicilamina/farmacología , S-Nitrosotioles/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
We investigated in the present study the transport characteristics of N-acetyl-L-aspartate in primary cultures of astrocytes from rat cerebral cortex and the involvement of NA+-coupled high-affinity carboxylate transporter NaC3 (formerly known as NaDC3) responsible for N-acetyl-L-aspartate transport. N-acetyl-L-aspartate transport was NA+-dependent and saturable with a Michaelis-Menten constant (Km) of approximately 110 microm. NA+-activation kinetics revealed that the NA+ to-N-acetyl-L-aspartate stoichiometry was 3 : 1 and concentration of Na+ necessary for half-maximal transport (KNA m) was 70 mm. NA+-dependent N-acetyl-L-aspartate transport was competitively inhibited by succinate with an inhibitory constant (Ki) of 14.7 microm, which was comparable to the Km value of NA+-dependent succinate transport (29.4 microm). L-aspartate also inhibited NA+-dependent [14C]N-acetyl-L-aspartate transport with relatively low affinity (Ki = 2.2 mm), whereas N-acetyl-L-aspartate was not able to inhibit NA+-dependent aspartate transport in astrocytes. In addition, Li+ was found to have a significant inhibitory effect on the NA+-dependent N-acetyl-L-aspartate transport in a concentration-dependent manner. Furthermore, RT-PCR and western blot analyses revealed that NaC3 is expressed in primary cultures of astrocytes. Taken collectively, these results indicate that NaC3 expressed in rat cerebrocortical astrocytes is responsible for NA+-dependent N-acetyl-L-aspartate transport. This transporter is likely to be an essential prerequisite for the metabolic role of N-acetyl-L-aspartate in the process of myelination.
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Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Astrocitos/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Sodio/metabolismo , Simportadores/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Células Cultivadas , Transportadores de Ácidos Dicarboxílicos/metabolismo , Relación Dosis-Respuesta a Droga , Cinética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Ratas Wistar , Sodio/farmacologíaRESUMEN
We report the functional characterization of constitutive nitric oxide synthase(s) (NOS) such as neuronal and endothelial NOS in cultured human astrocytes. Exposure of cultured human astrocytes to 1 microM veratridine or 50 mM KCl produced a pronounced increase in a calmodulin-dependent NOS activity estimated from cGMP formation. The functional expression of voltage-gated Na(+) channel, which is estimated by the response to veratridine, appeared to be earlier (at second day in culture) than that of voltage-gated Ca(2+) channels, which are estimated by the response to the KCl stimulation (at fourth day in culture). The KCl-evoked NO synthesis was totally reversed by L-type Ca(2+) channel blockers such as nifedipine and verapamil, but not by omega-conotoxin GVIA, an N-type Ca(2+) channel blocker, or omega-agatoxin IVA, a P/Q-type Ca(2+) channel blocker. In addition, verapamil abolished the KCl-induced increase in the intracellular free Ca(2+) concentration. RT-PCR analysis revealed that mRNA for neuronal and endothelial NOS was expressed in human astrocytes. In addition, Western blot analysis and double labeling of NOS and glial fibrillary acidic protein (GFAP) showed that cultured human astrocytes expressed neuronal NOS and endothelial NOS as well as the alpha(1) subunit of Ca(2+) channel. These results suggest that human astrocytes express constitutive NOS that are regulated by voltage-gated L-type Ca(2+) channel as well as Na(+) channel.
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Astrocitos/enzimología , Canales de Calcio/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/fisiología , Óxido Nítrico Sintasa/biosíntesis , Canales de Sodio/fisiología , Astrocitos/efectos de los fármacos , Células Cultivadas , GMP Cíclico/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas del Tejido Nervioso/biosíntesis , Óxido Nítrico Sintasa/fisiología , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo III , Cloruro de Potasio/farmacología , Veratridina/farmacologíaRESUMEN
In this report, we studied the functional characteristics of a brain peptide transporter using synaptosomes prepared from rat cerebral cortex. Crude synaptosomes (P(2) fraction) were prepared from cerebral cortices in male Wistar rats. Uptake of [14C]glycylsarcosine (Gly-Sar), a substrate for H(+)/oligopeptide transporters PEPT1 and PEPT2, and [3H]histidine, a substrate for peptide/histidine transporters PHT1 and PHT2, was measured at 37 degrees C by a rapid filtration technique. The uptake of [14C]Gly-Sar into synaptosomes was stimulated by an inwardly directed H(+)-gradient. The uptake system exhibited a Michaelis-Menten constant (K(t)) of 110+/-20 microM for Gly-Sar. This value is comparable to the K(t) value for Gly-Sar uptake via the high-affinity H(+)/peptide transporter PEPT2. The H(+)-dependent uptake of [14C]Gly-Sar into synaptosomes was inhibited by di- and tripeptides and beta-lactam antibiotics, but was unaffected by amino acids glycine and histidine. In particular, kyotorphin (Tyr-Arg) completely inhibited Gly-Sar uptake with the K(i) value of 29+/-14 microM. These uptake properties of the brain peptide transporter (i.e., the K(t) value for Gly-Sar uptake and the K(i) value of kyotorphin for Gly-Sar uptake) are very similar to those of PEPT2. RT-PCR and Western blotting analyses revealed that PEPT2 is actually expressed in the cerebral cortex in rat. These results indicate that a H(+)-coupled high affinity peptide transport system is functionally expressed in the cerebral cortex and that this transport system is identical to PEPT2.
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Corteza Cerebral/metabolismo , Simportadores/metabolismo , Animales , Antibacterianos/farmacología , Transporte Biológico , Northern Blotting , Western Blotting , Isótopos de Carbono/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Dipéptidos/metabolismo , Relación Dosis-Respuesta a Droga , Histidina/metabolismo , Cinética , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Transportador de Péptidos 1 , Protones , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Especificidad por Sustrato , Simportadores/genética , Simportadores/aislamiento & purificación , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Factores de Tiempo , Tritio/metabolismoRESUMEN
PURPOSE: The effect of gabapentin [1-(aminomethyl)cyclohexane acetic acid] on Ca2+ channels involving the activation of nitric oxide synthase (NOS) was investigated in primary neuronal culture of mouse cerebral cortex. METHODS: The expression of alpha2delta subunits of Ca2+ channels was investigated by RT-PCR using specific primer sets. The K(+)-evoked NOS activity was estimated by guanosine 3'5' cyclic monophosphate (cGMP) formation. RESULTS: mRNA for alpha2delta subunits of Ca2+ channels is found in these cells. Gabapentin blocked the K(+)-evoked NOS activity estimated from cGMP formation in a concentration dependent manner. The increase in NOS activity by the K(+)-stimulation was almost completely reversed by the combination of nifedipine, an L-type Ca2+ channel blocker, and omega-agatoxin VIA, a P/Q-type Ca2+ channel blocker. On the other hand, omega-conotoxin GVIA, an N-type Ca2+ channel blocker, was failed to reverse the increase in NOS activity by the K(+)-stimulation, indicating that the activation of NOS by the depolarizing stimulation might be not mediated by N-type Ca2+ channel. Under the presence of nifedipine or omega-agatoxin IVA, gabapentin inhibited the increase in NOS activity concentration-dependently. CONCLUSIONS: These results suggest that gabapentin inhibits depolarization-induced NOS activation in murine cortical neuronal culture via blockade of both P/Q-type and L-type Ca2+ channels.
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Acetatos/farmacología , Aminas , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo P/efectos de los fármacos , Canales de Calcio Tipo Q/efectos de los fármacos , Corteza Cerebral/citología , Ácidos Ciclohexanocarboxílicos , Neuronas/enzimología , Óxido Nítrico Sintasa/biosíntesis , Ácido gamma-Aminobutírico , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/enzimología , GMP Cíclico/biosíntesis , Femenino , Gabapentina , Ratones , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Nifedipino/farmacología , Óxido Nítrico Sintasa de Tipo I , Cloruro de Potasio/farmacología , Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , omega-Conotoxina GVIA/farmacologíaRESUMEN
1 The mode of Ca(2+) channel blocking by gabapentin [1-(aminomethyl)cyclohexane acetic acid] was compared to those of other Ca(2+) channel blockers, and the potential role of Ca(2+) channel antagonists in providing protection against hypoxic injury was subsequently investigated in rat cerebrocortical slices. 2 mRNA for the alpha(2)delta subunits of Ca(2+) channels was found in rat cerebral cortex. 3 Nitric oxide (NO) synthesis estimated from cGMP formation was enhanced by KCl stimulation, which was mediated primarily by the activation of N- and P/Q-type Ca(2+) channels. Gabapentin blocked both types of Ca(2+) channels, and preferentially reversed the response to 30 mM K(+) stimulation compared with 50 mM K(+) stimulation. In contrast, verapamil preferentially inhibited the response to depolarization by the higher concentration (50 mM) of K(+). 4 Gabapentin inhibited KCl-induced elevation of intracellular Ca(2+) in primary neuronal culture. 5 Hypoxic injury was induced in cerebrocortical slices by oxygen deprivation in the absence (severe injury) or presence of 3 mM glucose (mild injury). Gabapentin preferentially inhibited mild injury, while verapamil suppressed only severe injury. omega-Conotoxin GVIA (omega-CTX) and omega-agatoxin IVA (omega-Aga) were effective in both models. 6 NO synthesis was enhanced in a manner dependent on the severity of hypoxic insults. Gabapentin reversed the NO synthesis induced by mild insults, while verapamil inhibited that elicited by severe insults. omega-CTX and omega-Aga were effective in both the cases. 7 Therefore, the data suggest that gabapentin and verapamil cause activity-dependent Ca(2+) channel blocking by different mechanisms, which are associated with their cerebroprotective actions and are dependent on the severity of hypoxic insults.