RESUMEN
The constant transport of ions across the alveolar epithelial barrier regulates alveolar fluid homeostasis. Dysregulation or inhibition of Na+ transport causes fluid accumulation in the distal airspaces resulting in impaired gas exchange and respiratory failure. Previous studies have primarily focused on the critical role of amiloride-sensitive epithelial sodium channel (ENaC) in alveolar fluid clearance (AFC), yet activation of ENaC failed to attenuate pulmonary edema in clinical trials. Since 40% of AFC is amiloride-insensitive, Na+ channels/transporters other than ENaC such as Na+-coupled neutral amino acid transporters (SNATs) may provide novel therapeutic targets. Here, we identified a key role for SNAT2 (SLC38A2) in AFC and pulmonary edema resolution. In isolated perfused mouse and rat lungs, pharmacological inhibition of SNATs by HgCl2 and α-methylaminoisobutyric acid (MeAIB) impaired AFC. Quantitative RT-PCR identified SNAT2 as the highest expressed System A transporter in pulmonary epithelial cells. Pharmacological inhibition or siRNA-mediated knockdown of SNAT2 reduced transport of l-alanine across pulmonary epithelial cells. Homozygous Slc38a2-/- mice were subviable and died shortly after birth with severe cyanosis. Isolated lungs of Slc38a2+/- mice developed higher wet-to-dry weight ratios (W/D) as compared to wild type (WT) in response to hydrostatic stress. Similarly, W/D ratios were increased in Slc38a2+/- mice as compared to controls in an acid-induced lung injury model. Our results identify SNAT2 as a functional transporter for Na+ and neutral amino acids in pulmonary epithelial cells with a relevant role in AFC and the resolution of lung edema. Activation of SNAT2 may provide a new therapeutic strategy to counteract and/or reverse pulmonary edema.
Asunto(s)
Lesión Pulmonar Aguda/prevención & control , Sistema de Transporte de Aminoácidos A/fisiología , Agua Pulmonar Extravascular/metabolismo , Alveolos Pulmonares/metabolismo , Edema Pulmonar/prevención & control , Sodio/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Ácido Clorhídrico/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Edema Pulmonar/metabolismo , Edema Pulmonar/patología , Ratas , Ratas Sprague-DawleyRESUMEN
The green tea amino acid theanine is abundant in green tea rather than black and oolong teas, which are all made of the identical tea plant "Chanoki" (Camellia sinensis). Theanine has a molecular structure close to glutamine (GLN) compared to glutamic acid (Glu), in terms of the absence of a free carboxylic acid moiety from the gamma carbon position. Theanine efficiently inhibits [3H]GLN uptake without affecting [3H]Glu uptake in rat brain synaptosomes. In contrast to GLN, however, theanine markedly stimulates the abilities to replicate and to commit to a neuronal lineage following prolonged exposure in cultured neural progenitor cells (NPCs) prepared from embryonic and adult rodent brains. Upregulation of transcript expression is found for one of the GLN transporter isoforms, Slc38a1, besides the promotion of both proliferation and neuronal commitment along with acceleration of the phosphorylation of mechanistic target of rapamycin (mTOR) and relevant downstream proteins, in murine NPCs cultured with theanine. Stable overexpression of Slc38a1 similarly facilitates both cellular replication and neuronal commitment in pluripotent embryonic carcinoma P19â¯cells. In P19â¯cells with stable overexpression of Slc38a1, marked phosphorylation is seen for mTOR and downstream proteins in a manner insensitive to further additional phosphorylation by theanine. Taken together, theanine would exhibit a novel pharmacological property to up-regulate Slc38a1 expression for activation of the intracellular mTOR signaling pathway required for neurogenesis after sustained exposure in undifferentiated NPCs in the brain. In this review, a novel neurogenic property of the green tea amino acid theanine is summarized for embryonic and adult neurogenesis with a focus on the endogenous amino acid GLN on the basis of our accumulating evidence to date.
Asunto(s)
Encéfalo/efectos de los fármacos , Glutamatos/farmacología , Glutamina/fisiología , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Sistema de Transporte de Aminoácidos A/fisiología , Animales , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/prevención & control , Método Doble Ciego , Predicción , Glutamatos/química , Glutamatos/uso terapéutico , Glutamina/química , Hipocampo/efectos de los fármacos , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Transgénicos , Trastornos del Humor/tratamiento farmacológico , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Fosforilación/efectos de los fármacos , Fitoterapia , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ensayos Clínicos Controlados Aleatorios como Asunto , Ratas , Trastornos del Sueño-Vigilia/tratamiento farmacológico , Serina-Treonina Quinasas TOR/metabolismo , Té/químicaRESUMEN
OBJECTIVE: To validate the gene expression profile obtained from the previous microarray analysis and to further study the biological functions of these genes in endometrial cancer. From our previous study, we identified 621 differentially expressed genes in laser-captured microdissected endometrioid endometrial cancer as compared to normal endometrial cells. Among these genes, 146 were significantly up-regulated in endometrial cancer. MATERIALS AND METHODS: A total of 20 genes were selected from the list of up-regulated genes for the validation assay. The qPCR confirmed that 19 out of the 20 genes were up-regulated in endometrial cancer compared with normal endometrium. RNA interference (RNAi) was used to knockdown the expression of the upregulated genes in ECC-1 and HEC-1A endometrial cancer cell lines and its effect on proliferation, migration and invasion were examined. RESULTS: Knockdown of MIF, SOD2, HIF1A and SLC7A5 by RNAi significantly decreased the proliferation of ECC-1 cells (p < 0.05). Our results also showed that the knockdown of MIF, SOD2 and SLC7A5 by RNAi significantly decreased the proliferation and migration abilities of HEC-1A cells (p < 0.05). Moreover, the knockdown of SLC38A1 and HIF1A by RNAi resulted in a significant decrease in the proliferation of HEC1A cells (p < 0.05). CONCLUSION: We have identified the biological roles of SLC38A1, MIF, SOD2, HIF1A and SLC7A5 in endometrial cancer, which opens up the possibility of using the RNAi silencing approach to design therapeutic strategies for treatment of endometrial cancer.
Asunto(s)
Neoplasias Endometriales/genética , Silenciador del Gen , Sistema de Transporte de Aminoácidos A/genética , Sistema de Transporte de Aminoácidos A/fisiología , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Endometriales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/fisiología , Transportador de Aminoácidos Neutros Grandes 1/genética , Transportador de Aminoácidos Neutros Grandes 1/fisiología , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/fisiología , Interferencia de ARN , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Superóxido Dismutasa/genética , Superóxido Dismutasa/fisiología , Regulación hacia ArribaRESUMEN
Many cancer cells depend on glutamine as they use the glutaminolysis pathway to generate building blocks and energy for anabolic purposes. As a result, glutamine transporters are essential for cancer growth and are potential targets for cancer chemotherapy with ASCT2 (SLC1A5) being investigated most intensively. Here we show that HeLa epithelial cervical cancer cells and 143B osteosarcoma cells express a set of glutamine transporters including SNAT1 (SLC38A1), SNAT2 (SLC38A2), SNAT4 (SLC38A4), LAT1 (SLC7A5), and ASCT2 (SLC1A5). Net glutamine uptake did not depend on ASCT2 but required expression of SNAT1 and SNAT2. Deletion of ASCT2 did not reduce cell growth but caused an amino acid starvation response and up-regulation of SNAT1 to replace ASCT2 functionally. Silencing of GCN2 in the ASCT2(-/-) background reduced cell growth, showing that a combined targeted approach would inhibit growth of glutamine-dependent cancer cells.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/genética , Sistema de Transporte de Aminoácidos A/fisiología , Proliferación Celular , Eliminación de Gen , Expresión Génica , Glutamina/metabolismo , Células HeLa , Homeostasis , Humanos , Transporte Iónico , Redes y Vías Metabólicas , Antígenos de Histocompatibilidad MenorRESUMEN
RATIONALE: There is evidence that impairments in nitric oxide (NO) signaling contribute to chronic hypoxia-induced pulmonary hypertension. The L-arginine-NO precursor, L-citrulline, has been shown to ameliorate pulmonary hypertension. Sodium-coupled neutral amino acid transporters (SNATs) are involved in the transport of L-citrulline into pulmonary arterial endothelial cells (PAECs). The functional link between the SNATs, L-citrulline, and NO signaling has not yet been explored. OBJECTIVE: We tested the hypothesis that changes in SNAT1 expression and transport function regulate NO production by modulating eNOS coupling in newborn piglet PAECs. METHODS AND RESULTS: A silencing RNA (siRNA) technique was used to assess the contribution of SNAT1 to NO production and eNOS coupling (eNOS dimer-to-monomer ratios) in PAECs from newborn piglets cultured under normoxic and hypoxic conditions in the presence and absence of L-citrulline. SNAT1 siRNA reduced basal NO production in normoxic PAECs and prevented L-citrulline-induced elevations in NO production in both normoxic and hypoxic PAECs. SNAT1 siRNA reduced basal eNOS dimer-to-monomer ratios in normoxic PAECs and prevented L-citrulline-induced increases in eNOS dimer-to-monomer ratios in hypoxic PAECs. CONCLUSIONS: SNAT1 mediated L-citrulline transport modulates eNOS coupling and thus regulates NO production in hypoxic PAECs from newborn piglets. Strategies that increase SNAT1-mediated transport and supply of L-citrulline may serve as novel therapeutic approaches to enhance NO production in patients with pulmonary vascular disease.
Asunto(s)
Sistema de Transporte de Aminoácidos A/fisiología , Citrulina/metabolismo , Células Endoteliales/metabolismo , Óxido Nítrico/metabolismo , Arteria Pulmonar/patología , Transducción de Señal , Animales , Transporte Biológico , Hipoxia de la Célula , Células Cultivadas , Endotelio Vascular/patología , Hipertensión Pulmonar/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Superóxidos/metabolismo , Sus scrofaRESUMEN
Growth hormone (GH) is important in maternal adaptation to pregnancy, and maternal circulating GH concentrations are reduced in human growth-restricted pregnancies. In the pig, maternal GH treatment throughout early to mid pregnancy increases fetal growth, despite constraining effects of adolescent and primiparous pregnancy, high litter size, and restricted maternal nutrition. Because GH cannot cross the placenta and does not increase placental weight, we hypothesized that its effects on fetal growth might be via improved placental structure or function. We therefore investigated effects of maternal GH treatment in pigs on structural correlates of placental function and placental expression of nutrient transporters important to fetal growth. Multiparous (sows) and primiparous pregnant pigs (gilts) were treated with GH (~15 µg kg(-1) day(-1)) or vehicle from Days 25-50 of gestation (n = 7-8 per group, term ~115 days). Placentas were collected at Day 50 of gestation, and we measured structural correlates of function and expression of SLC2A1 (previously known as GLUT1) and SLC38A2 (previously known as SNAT2) nutrient transporters. Maternal GH treatment did not alter placental size or structure, increased protein expression of SLC2A1 in trophoblast (+35%; P = 0.037) and on its basal membrane (+44%; P = 0.011), and increased SLC38A2 protein expression in the basal (+44%; P = 0.001) but not the apical cytoplasm of trophoblast. Our findings suggest that maternal GH treatment increases fetal growth, in part, by enhancing placental nutrient transporter protein expression and hence fetal nutrient supply as well as trophoblast proliferation and differentiation and may have the potential to ameliorate intrauterine growth restriction.
Asunto(s)
Sistema de Transporte de Aminoácidos A/análisis , Desarrollo Fetal/efectos de los fármacos , Transportador de Glucosa de Tipo 1/análisis , Hormona del Crecimiento/administración & dosificación , Placenta/fisiología , Sus scrofa , Sistema de Transporte de Aminoácidos A/fisiología , Animales , Femenino , Desarrollo Fetal/fisiología , Peso Fetal/efectos de los fármacos , Edad Gestacional , Transportador de Glucosa de Tipo 1/fisiología , Inmunohistoquímica , Tamaño de los Órganos , Placenta/química , Placenta/efectos de los fármacos , Embarazo , Receptor IGF Tipo 1/análisis , Trofoblastos/químicaRESUMEN
OBJECTIVES: Deletion of the placental-specific P0 transcript of the insulin-like growth factor gene (Igf2) reduces placental growth from early pregnancy onwards. In Igf2 P0 knockout fetuses (P0), maternofetal flux of (14)C-methylaminoisobutyric acid ((14)C-MeAIB) mediated by system A amino acid transporter activity is increased at embryonic day 16 (E16), but this stimulation is not sustained, and by E19, fetal growth restriction (FGR) ensues. Here, we investigated whether upregulated (14)C-MeAIB transfer does occur concomitantly with a change in System A amino acid transporter activity and whether altered uteroplacental vascular function contributes to the FGR. We tested the hypothesis that FGR in P0 mice is attributable to altered nutrient transport rather than aberrant uteroplacental vascular function. METHODS: Plasma membrane vesicles were isolated from placentas of P0 and wild-type (WT) fetuses at E16 and E19. System A amino acid transporter activity was measured as sodium-dependent (14)C-MeAIB uptake over 60s. Wire myography was performed on uterine artery branches supplying P0 or WT implantation sites and agonist-induced constriction and dilation measured. RESULTS: Sodium-dependent uptake of (14)C-MeAIB (at 60s) was significantly (P < 0.05) higher in P0 compared to WT vesicles at E16; at E19 (14)C-MeAIB uptake was similar between P0 and WT. Uterine artery branch vascular reactivity was comparable between groups. CONCLUSIONS: System A activity in the maternal-facing plasma membrane of syncytiotrophoblast layer II underpins the adaptations observed in the transplacental MeAIB flux of P0 mice. Unaltered uterine artery vascular function suggests that the FGR phenotype of P0 fetuses is primarily due to deficient placental nutrient exchange capacity.
Asunto(s)
Sistema de Transporte de Aminoácidos A/metabolismo , Vasos Sanguíneos/fisiología , Factor II del Crecimiento Similar a la Insulina/genética , Placenta/metabolismo , Sistema de Transporte de Aminoácidos A/fisiología , Animales , Transporte Biológico , Vasos Sanguíneos/metabolismo , Fraccionamiento Celular , Femenino , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos/genética , Placenta/irrigación sanguínea , Circulación Placentaria/genética , Circulación Placentaria/fisiología , Embarazo , Arteria Uterina/metabolismo , Arteria Uterina/fisiología , beta-Alanina/análogos & derivados , beta-Alanina/farmacocinéticaRESUMEN
The inhibitory neurotransmitter glycine is known to enhance microglial nitric oxide production. However, up to now, the mechanism is undocumented. Since calcium is an important second messenger in both immune and glial cells, we studied the effects of glycine on intracellular calcium signaling. We found that millimolar concentrations of glycine enhance microglial intracellular calcium transients induced by 100 µM ATP or by 500 nM thapsigargin. This modulation was unaffected by the glycine receptor antagonist strychnine and could not be mimicked by glycine receptor agonists such as taurine or ß-alanine, indicating glycine receptor independency. The modulation of calcium responses could be mimicked by several structurally related amino acids (e.g., serine, alanine, or glutamine) and was inhibited in the presence of the neutral amino acid transporter substrate α-aminoisobutyric acid (AIB). We correlated these findings to immunofluorescence glycine uptake experiments which showed a clear glycine uptake which was inhibited by AIB. Furthermore, all amino acids that were shown to modulate calcium responses also evoked AIB-sensitive inward currents, mainly carried by sodium, as demonstrated by patch clamp experiments. Based on these findings, we propose that sodium-coupled neutral amino acid transporters are responsible for the observed glycine modulation of intracellular calcium responses.
Asunto(s)
Sistema de Transporte de Aminoácidos A/fisiología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Glicina/farmacología , Microglía/efectos de los fármacos , Microglía/fisiología , Ácidos Aminoisobutíricos/farmacología , Animales , Línea Celular , Glicina/metabolismo , Glicinérgicos/farmacología , Ratones , Modelos Animales , Técnicas de Placa-Clamp , Receptores de Glicina/agonistas , Receptores de Glicina/antagonistas & inhibidores , Receptores de Glicina/fisiología , Estricnina/farmacología , Taurina/farmacología , beta-Alanina/farmacologíaRESUMEN
The marked increase in skeletal muscle mass during the neonatal period is largely due to a high rate of postprandial protein synthesis that is modulated by an enhanced sensitivity to insulin and amino acids. The amino acid signaling pathway leading to the stimulation of protein synthesis has not been fully elucidated. Among the amino acids, leucine is considered to be a principal anabolic agent that regulates protein synthesis. mTORC1, which controls protein synthesis, has been implicated as a target for leucine. Until recently, there have been few studies exploring the role of amino acids in enhancing muscle protein synthesis in vivo. In this review, we discuss amino acid-induced protein synthesis in muscle in the neonate, focusing on current knowledge of the role of amino acids in the activation of mTORC1 leading to mRNA translation. The role of the amino acid transporters, SNAT2, LAT1, and PAT, in the modulation of mTORC1 activation and the role of amino acids in the activation of putative regulators of mTORC1, i.e., raptor, Rheb, MAP4K3, Vps34, and Rag GTPases, are discussed.
Asunto(s)
Aminoácidos/fisiología , Proteínas Musculares/biosíntesis , Músculo Esquelético/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Sistema de Transporte de Aminoácidos A/fisiología , Sistemas de Transporte de Aminoácidos/fisiología , Animales , Animales Recién Nacidos , Fosfatidilinositol 3-Quinasas Clase III/fisiología , Humanos , Recién Nacido , Transportador de Aminoácidos Neutros Grandes 1/fisiología , Leucina/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas de Unión al GTP Monoméricas/fisiología , Complejos Multiproteicos , Músculo Esquelético/efectos de los fármacos , Neuropéptidos/fisiología , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas/fisiología , ARN Mensajero/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro , Proteína Reguladora Asociada a mTOR , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Porcinos , Simportadores/fisiología , Serina-Treonina Quinasas TOR , Factores de Intercambio de Guanina Nucleótido ras/fisiologíaRESUMEN
Elevated maternal plasma concentrations of homocysteine (Hcy) are associated with pregnancy complications and adverse neonatal outcomes. The postulate that we wish to advance here is that placental transport of Hcy, by competing with endogenous amino acids for transporter activity, may account for some of the damaging impacts of Hcy on placental metabolism and function as well as fetal development. In this article, we provide an overview of some recent studies characterising the transport mechanisms for Hcy across the microvillous plasma membrane (MVM) of the syncytiotrophoblast, the transporting epithelium of human placenta. Three Hcy transport systems have been identified, systems L, A and y(+)L. This was accomplished using a strategy of competitive inhibition to investigate the effects of Hcy on the uptake of well-characterised radiolabelled substrates for each transport system into isolated MVM vesicles. The reverse experiments were also performed, examining the effects of model substrates on [³5S]L-Hcy uptake. This article describes the evidence for systems L, A and y(+)L involvement in placental Hcy transport and discusses the physiological implications of these findings with respect to placental function and fetal development.
Asunto(s)
Membrana Celular/metabolismo , Homocisteína/metabolismo , Microvellosidades/metabolismo , Placenta/metabolismo , Sistema de Transporte de Aminoácidos A/metabolismo , Sistema de Transporte de Aminoácidos A/fisiología , Sistema de Transporte de Aminoácidos L/metabolismo , Sistema de Transporte de Aminoácidos L/fisiología , Sistema de Transporte de Aminoácidos y+L/metabolismo , Sistema de Transporte de Aminoácidos y+L/fisiología , Transporte Biológico , Femenino , Homocisteína/farmacocinética , Humanos , Microvellosidades/ultraestructura , Modelos Biológicos , Placenta/ultraestructura , Embarazo , Radioisótopos de Azufre/farmacocinéticaRESUMEN
The purpose of this study was to clarify the cytoprotective mechanism(s) induced in a conditionally immortalized syncytiotrophoblast cell line (TR-TBT 18d-1) exposed to hypertonic conditions. Hypertonicity-induced apoptosis of TR-TBT 18d-1 cells, but this was blocked by addition of 1 mM taurine to the culture medium. TauT-knockdown using siRNA revealed that TauT is a major contributor to taurine uptake by TR-TBT 18d-1 cells, at least under normal conditions. Cellular uptake of [(3)H]taurine and [(14)C]betaine by TR-TBT 18d-1 cells cultured under hypertonic conditions was increased compared to that under normal conditions. TauT, BGT-1, ATA2 and HSP70 mRNAs were upregulated by hypertonicity, while OCTN2, ENT1 and CNT1 mRNAs were downregulated. [(3)H]Taurine uptake was strongly inhibited by TauT inhibitors such as hypotaurine and ß-alanine. MeAIB, a system A specific substrate, inhibited hypertonic stress-induced [(14)C]betaine uptake. These results suggest that TauT and system A play cytoprotective roles in syncytiotrophoblasts exposed to hypertonic stress.
Asunto(s)
Sistema de Transporte de Aminoácidos A/fisiología , Citoprotección , Glicoproteínas de Membrana/fisiología , Proteínas de Transporte de Membrana/fisiología , Estrés Fisiológico , Taurina/metabolismo , Trofoblastos/patología , Sistema de Transporte de Aminoácidos A/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos A/genética , Animales , Apoptosis , Betaína/metabolismo , Transporte Biológico/efectos de los fármacos , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Soluciones Hipertónicas , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Moduladores del Transporte de Membrana/farmacología , Proteínas de Transporte de Membrana/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Ratas , Taurina/análogos & derivados , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismo , beta-Alanina/análogos & derivados , beta-Alanina/metabolismo , beta-Alanina/farmacologíaRESUMEN
GABAergic neurotransmission adapts to maintain normal brain function in a wide range of activity states through multiple mechanisms; pre-synaptic control of quantal size has only recently gained recognition as one of those mechanisms. GABA synthesis from glutamate is coupled with vesicular packaging, and therefore the supply of glutamate can affect inhibitory synaptic strength. Because System A transporters supply glutamine to neurons, where it is converted to glutamate, we hypothesized that regulation of the activity of these transporters could alter glutamine uptake and provide a mechanism to link supply to demand for neurotransmitter GABA. In immature and mature rat hippocampus, after a period of hyperexcitability, we observed a System A-dependent enhancement of inhibitory synaptic strength along with an increase in System A activity in synaptosomes under the same conditions. Under resting conditions, System A's contribution of glutamine to synaptic GABA diminished with age, correlating with reduced SNAT1/SAT1 expression and, even more so, with its activity on synaptic membranes. We conclude that System A activity is highly regulated, by depolarization and developmental cues, to dynamically modulate GABAergic transmission. Our evidence suggests that SNAT1/SAT1 is the transporter that plays a critical role in dynamically modulating inhibition in response to metabolic demands.
Asunto(s)
Envejecimiento/fisiología , Sistema de Transporte de Aminoácidos A/fisiología , Proteínas Portadoras/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/fisiología , Neurotransmisores/fisiología , Ácido gamma-Aminobutírico/fisiología , Envejecimiento/metabolismo , Sistema de Transporte de Aminoácidos A/metabolismo , Animales , Hipocampo/crecimiento & desarrollo , Inhibición Neural/fisiología , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/biosíntesisRESUMEN
Changes in placental nutrient transport are closely associated with abnormal fetal growth. However, the molecular mechanisms underlying the regulation of placental amino acid transporters are unknown. We demonstrate that physiological concentrations of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha stimulate the activity of amino acid transporter system A, but not system L, in cultured human primary trophoblast cells. Both cytokines increased the gene and protein expression of the Na(+)-coupled neutral amino acid transporter (SNAT)2 isoform and upregulated SNAT1 protein expression. IL-6 increased Tyr705 phosphorylation of signal transducer and activator of transcription 3 (STAT3). In cells transfected with small interfering RNA (siRNA) targeting STAT3, the RNA and protein expression of SNAT2, but not SNAT1, was reduced and the stimulating effect of IL-6 on system A activity was abolished. Despite eliciting similar responses in amino acid transport activity and transporter expression, TNF-alpha effects on system A activity were not mediated through the JAK/STAT pathway. In conclusion, we have identified a novel regulatory pathway involving increased gene expression of the SNAT2 isoform mediated by a STAT-dependent pathway, which links IL-6 to increased activity of system A, a ubiquitously expressed transporter of neutral amino acids. From these new findings, we propose that upregulation of amino acid transporters by cytokines may contribute to increased placental nutrient transport and fetal overgrowth, which are commonly found in pregnancies complicated by maternal diabetes and obesity.
Asunto(s)
Sistema de Transporte de Aminoácidos A/metabolismo , Regulación de la Expresión Génica , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/fisiología , Trofoblastos/metabolismo , Sistema de Transporte de Aminoácidos A/genética , Sistema de Transporte de Aminoácidos A/fisiología , Western Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Expresión Génica , Humanos , Interleucina-6 , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
The System L transporter facilitates cellular import of large neutral amino acids (AAs) such as Leu, a potent activator of the intracellular target of rapamycin (TOR) pathway, which signals for cell growth. System L is an AA exchanger, proposed to accumulate certain AAs by coupling to dissipation of concentration gradient(s) of exchange substrates generated by secondary active AA transporters such as System A (SNAT2). We addressed the hypothesis that this type of coupling (termed tertiary active transport) acts as an indirect mechanism to extend the range of AA stimulating TOR to those transported by both Systems A and L (e.g., Gln) through downstream enhancement of Leu accumulation. System A overexpression enabled Xenopus oocytes to accumulate substrate AAs (notably Ser, Gln, Ala, Pro, Met; totaling 2.6 nmol/oocyte) from medium containing a physiological AA mixture at plasma concentrations. Net accumulation of System L (4F2hc-xLAT1) substrates from this medium by System L-overexpressing oocytes was increased by 90% (from 0.7 to 1.35 nmol/oocyte; mainly Leu, Ile) when Systems A and L were coexpressed, coincident with a decline in accumulation of specific System A substrates (Gln, Ser, Met), as expected if the latter were also System L substrates and functional coupling of the transport Systems occurred. AA flux coupling was confirmed as trans-stimulation of Leu influx in System L-expressing oocytes by Gln injection (0.5 nmol/oocyte). The observed changes in Leu accumulation are sufficient to activate the TOR pathway in oocytes, although intracellular AA metabolism limits the potential for AA accumulation by tertiary active transport in this system.
Asunto(s)
Sistema de Transporte de Aminoácidos A/genética , Sistema de Transporte de Aminoácidos L/genética , Aminoácidos/metabolismo , Expresión Génica , Oocitos/metabolismo , Xenopus/genética , Sistema de Transporte de Aminoácidos A/metabolismo , Sistema de Transporte de Aminoácidos A/fisiología , Sistema de Transporte de Aminoácidos L/metabolismo , Sistema de Transporte de Aminoácidos L/fisiología , Animales , Transporte Biológico/genética , Femenino , Expresión Génica/fisiología , Modelos Biológicos , Factores de Tiempo , Transfección , Xenopus/metabolismo , beta-Alanina/análogos & derivados , beta-Alanina/farmacocinéticaRESUMEN
Mechanisms by which gut luminal content regulates secretion and motility are ill understood. We evaluated whether neuroendocrine enterochromaffin (EC) cells act as luminal sensors for a wide variety of nutrients and defined the secretory mechanisms of this process. Pure (98-99%) FACS-sorted human EC cells and neoplastic EC cells (KRJ-I) were studied. RT-PCR identified transcripts for T2R1 (bitter), OR1G1 (class II olfactory) and trace amine (TAR1) G protein-coupled receptors (GPCRs) and transporters for glutamine (SNAT1/2), glucose (GLUT1/3/SGLT1), and bile salts (ABST). Glutamine and sodium deoxycholate stimulated 5-HT release (EC(50) = 0.002-0.2 microM; 2-fold release) but were 10-100 times more potent in neoplastic EC cells, which also secreted 6-13 times more 5-HT. Tastants (caffeine, tyramine, octopamine) and olfactants (thymol and eugenol) also stimulated normal and neoplastic EC cell 5-HT secretion (EC(50) = 1.2 nM to 2.1 microM and 0.05 nM to 0.1 microM release, respectively); 2-deoxyglucose and the artificial sweetener sucralose also stimulated (EC(50) = 9.2 and 0.38 nM). 5-HT release was associated with ERK phosphorylation (1.5-fold, P < 0.02) and could be inhibited by a somatostatin analog (IC(50) = 1 pM). Eleven secretory associated genes including the vesicle docking inhibitor STXBP3 were upregulated in response to glutamine and bile salt stimulation in neoplastic EC cells. Targeting STXBP3 expression by use of antisense knockdown significantly (P < 0.05) reduced 5-HT secretion. In conclusion, EC cells express GPCRs and transporters for luminal tastants, olfactants, glutamine, glucose, and bile salts. Activation includes a panel of secretory genes, ERK phosphorylation, and 5-HT secretion. Luminal EC cell regulation is likely to be as important as G cell regulation in gastric acid secretion; development of agents to target EC cell function is therefore a critical therapeutic goal.
Asunto(s)
Ácidos y Sales Biliares/fisiología , Tumor Carcinoide/metabolismo , Células Enterocromafines/metabolismo , Neoplasias Intestinales/metabolismo , Serotonina/metabolismo , Sistema de Transporte de Aminoácidos A/fisiología , Cafeína/farmacología , Tumor Carcinoide/genética , Línea Celular Tumoral , Ácido Desoxicólico/farmacología , Desoxiglucosa/farmacología , Células Enterocromafines/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Proteínas Facilitadoras del Transporte de la Glucosa/fisiología , Glutamina/farmacología , Humanos , Neoplasias Intestinales/genética , Intestino Delgado , Transportadores de Anión Orgánico Sodio-Dependiente/fisiología , Receptores Acoplados a Proteínas G/fisiología , Receptores de Glutamato Metabotrópico/fisiología , Receptores Odorantes/fisiología , Transportador 1 de Sodio-Glucosa/fisiología , Somatostatina/farmacología , Sacarosa/análogos & derivados , Sacarosa/farmacología , Simportadores/fisiología , Tirosina/farmacologíaRESUMEN
L-carnitine is absorbed in the intestinal tract via the carnitine transporter OCTN2 and the amino acid transporter ATB(0,+). Loss-of-function mutations in OCTN2 may be associated with inflammatory bowel disease (IBD), suggesting a role for carnitine in intestinal/colonic health. In contrast, ATB(0,+) is upregulated in bowel inflammation. Butyrate, a bacterial fermentation product, is beneficial for prevention/treatment of ulcerative colitis. Butyryl-L-carnitine (BC), a butyrate ester of carnitine, may have potential for treatment of gut inflammation, since BC would supply both butyrate and carnitine. We examined the transport of BC via ATB(0,+) to determine if this transporter could serve as a delivery system for BC. We also examined the transport of BC via OCTN2. Studies were done with cloned ATB(0,+) and OCTN2 in heterologous expression systems. BC inhibited ATB(0,+)-mediated glycine transport in mammalian cells (IC(50), 4.6 +/- 0.7 mM). In Xenopus laevis oocytes expressing human ATB(0,+), BC induced Na(+) -dependent inward currents under voltage-clamp conditions. The currents were saturable with a K(0.5) of 1.4 +/- 0.1 mM. Na(+) activation kinetics of BC-induced currents suggested involvement of two Na(+) per transport cycle. BC also inhibited OCTN2-mediated carnitine uptake (IC(50), 1.5 +/- 0.3 microM). Transport of BC via OCTN2 is electrogenic, as evidenced from BC-induced inward currents. These currents were Na(+) dependent and saturable (K(0.5), 0.40 +/- 0.02 microM). We conclude that ATB(0,+) is a low-affinity/high-capacity transporter for BC, whereas OCTN2 is a high-affinity/low-capacity transporter. ATB(0,+) may mediate intestinal absorption of BC when OCTN2 is defective.
Asunto(s)
Sistemas de Transporte de Aminoácidos/fisiología , Carnitina/análogos & derivados , Proteínas de Transporte de Catión Orgánico/fisiología , Sistema de Transporte de Aminoácidos A/fisiología , Transporte Biológico/efectos de los fármacos , Carnitina/farmacocinética , Carnitina/farmacología , Línea Celular , Clonación Molecular , Femenino , Glicina/metabolismo , Humanos , Cinética , Oocitos/efectos de los fármacos , Oocitos/fisiología , Placenta , Embarazo , Profármacos/farmacocinéticaRESUMEN
Recently, we cloned the ATA/SNAT transporters responsible for amino acid transport system A. System A is one of the major transport systems for small neutral and glucogenic amino acids represented by alanine and is involved in the metabolism of glucose and fat. Here, we describe the cellular mechanisms that participate in the acute translocation of ATA2 by insulin stimulus in 3T3-L1 adipocytes. We monitored this insulin-stimulated translocation of ATA2 using an expression system of enhanced green fluorescent protein-tagged ATA2. Studies in living cells revealed that ATA2 is stored in a discrete perinuclear site and that the transporter is released in vesicles from this site toward the plasma membrane. In immunofluorescent analysis, the storage site of ATA2 overlapped with the location of syntaxin 6, a marker of the trans-Golgi network (TGN), but not with that of EEA1, a marker of the early endosomes. The ATA2-containing vesicles on or near the plasma membrane were distinct from GLUT4-containing vesicles. Brefeldin A, an inhibitor of vesicular exit from the TGN, caused morphological changes in the ATA2 storage site along with the similar changes in the TGN. In non-transfected adipocytes, brefeldin A inhibited insulin-stimulated uptake of alpha-(methylamino)isobutyric acid more profoundly than insulin-stimulated uptake of 2-deoxy-d-glucose. These data demonstrate that the ATA2 storage site is specifically associated with the TGN and not with the general endosomal recycling system. Thus, the insulin-stimulated translocation pathways for ATA2 and GLUT4 in adipocytes are distinct, involving different storage sites.
Asunto(s)
Sistema de Transporte de Aminoácidos A/fisiología , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/metabolismo , Red trans-Golgi/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Sistema de Transporte de Aminoácidos A/metabolismo , Animales , Transporte Biológico , Brefeldino A/farmacología , Células CHO , Membrana Celular/metabolismo , Cricetinae , Desoxiglucosa/farmacología , Ratones , Inhibidores de la Síntesis de la Proteína/farmacologíaRESUMEN
Sodium-dependent neutral amino acid transporter-2 (SNAT2), the ubiquitous member of SLC38 family, accounts for the activity of transport system A for neutral amino acids in most mammalian tissues. As the transport process performed by SNAT2 is highly energized, system A substrates, such as glutamine, glycine, proline and alanine, reach high transmembrane gradients and constitute major components of the intracellular amino acid pool. Moreover, through a complex array of exchange fluxes, involving other amino acid transporters, and of metabolic reactions, such as the synthesis of glutamate from glutamine, SNAT2 activity influences the cell content of most amino acids, thus determining the overall size and the composition of the intracellular amino acid pool. As amino acids represent a large fraction of cell organic osmolytes, changes of SNAT2 activity are followed by modifications in both cell amino acids and cell volume. This mechanism is utilized by many cell types to perform an effective regulatory volume increase (RVI) upon hypertonic exposure. Under these conditions, the expression of SNAT2 gene is induced and newly synthesized SNAT2 proteins are preferentially targeted to the cell membrane, leading to a significant increase of system A transport Vmax. In cultured human fibroblasts incubated under hypertonic conditions, the specific silencing of SNAT2 expression, obtained with anti-SNAT2 siRNAs, prevents the increase in system A transport activity, hinders the expansion of intracellular amino acid pool, and significantly delays cell volume recovery. These results demonstrate the pivotal role played by SNAT2 induction in the short-term hypertonic RVI and suggest that neutral amino acids behave as compatible osmolytes in hypertonically stressed cells.
Asunto(s)
Sistema de Transporte de Aminoácidos A/fisiología , Aminoácidos Neutros/metabolismo , Adaptación Fisiológica , Transporte Biológico , Membrana Celular/metabolismo , Tamaño de la Célula , Células Cultivadas , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Soluciones Hipertónicas , Interferencia de ARN , ARN Interferente Pequeño , Equilibrio HidroelectrolíticoRESUMEN
Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Transport of L-homocysteine into and out of the human vascular endothelium is poorly understood. We hypothesized that cultured human aortic endothelial cells (HAEC) would import L-homocysteine on one or more of the L-cysteine transport systems. Inhibitors of the transporters were used to characterize the uptake of [35S]L-homocysteine, [35S]L-homocystine, and [35S]L-cysteine. We found that L-homocysteine uptake is mediated by the sodium-dependent cysteine transport systems X(AG), ASC, and A, and the sodium-independent transport system L. Thus, HAEC utilize multiple cysteine transporters (X(AG) > or = L > ASC > A) to import L-homocysteine. Kinetic analysis supported the uptake results. Michaelis-Menten constants (Km) for the four systems yielded values of 19.0, 27.1, 112, and 1000 microM for systems L, X(AG), ASC, and A, respectively. The binding and uptake of [35S]L-homocystine, the disulfide homodimer of L-homocysteine, was mediated by systems X(AG), L, and ASC but not by system A. In contrast to [35S]L-homocysteine, system x(c) was active for [35S]L-homocystine uptake. A similar pattern was observed for [35S]L-cysteine. Thus, L-homocysteine and L-homocystine found in hyperhomocysteinemic subjects can gain entry into the vascular endothelium by way of multiple L-cysteine transporters.
Asunto(s)
Células Endoteliales/metabolismo , Homocisteína/metabolismo , Sistema de Transporte de Aminoácidos A/fisiología , Sistema de Transporte de Aminoácidos ASC/fisiología , Sistema de Transporte de Aminoácidos L/fisiología , Sistema de Transporte de Aminoácidos X-AG/fisiología , Aorta Torácica/citología , Transporte Biológico Activo , Células Cultivadas , Cisteína/metabolismo , Cisteína/farmacocinética , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Homocisteína/farmacocinética , Humanos , Unión Proteica , Radioisótopos de AzufreRESUMEN
Although the sulfate/anion transporter (sat-1; SLC26A1) was isolated from a rat liver cDNA library by expression cloning, localization of sat-1 within the liver and its contribution to the transport of sulfate and organo sulfates have remained unresolved. In situ hybridization and immunohistochemical studies were undertaken to demonstrate the localization of sat-1 in liver tissue. RT-PCR studies on isolated hepatocytes and liver endothelial and stellate cells in culture were performed to test for the presence of sat-1 in these cells. In sulfate uptake and efflux experiments, the substrate specificity of sat-1 was evaluated. Sat-1 mRNA was found in hepatocytes and endothelial cells. Sat-1 protein was localized in sinusoidal membranes and along the borders of hepatocytes. The canalicular region and bile capillaries were not stained. Sulfate uptake was only slightly affected by sulfamoyl diuretics or organo sulfates. Sulfate efflux from sat-1-expressing oocytes was enhanced in the presence of bicarbonate, indicating sulfate/bicarbonate exchange. Estrone sulfate was not transported by sat-1. Sat-1 may be responsible for the uptake of inorganic sulfate from the blood into hepatocytes to enable sulfation reactions. In hepatocytes and endothelial cells, sat-1 may also supply sulfate for proteoglycan synthesis.