RESUMEN
In addition to its role as a strong antioxidant, vitamin C regulates the differentiation of several cell lineages. In vertebrate skeletal muscle, the vitamin C transporter SVCT2 is preferentially expressed in slow muscle fibers. To gain insights into the possible involvement of intracellular vitamin C on early myogenesis, we investigated the regulation of SVCT2 expression in cultures of chick fetal myoblasts. SVCT2 expression increases in cultures of both, slow and fast muscle-derived myoblasts, as they fuse to form mainly fast myotubes. Interestingly, we found that SVCT2 could be positively modulated by potassium-induced depolarization of myotubes. These findings suggest that SVCT2-mediated uptake of vitamin C could play diverse roles on skeletal muscle development and physiology.
Asunto(s)
Diferenciación Celular , Fibras Musculares Esqueléticas/citología , Transportadores de Anión Orgánico Sodio-Dependiente/biosíntesis , Simportadores/biosíntesis , Regulación hacia Arriba , Animales , Ácido Ascórbico , Polaridad Celular , Células Cultivadas , Embrión de Pollo , Pollos , Desarrollo de Músculos , Fibras Musculares Esqueléticas/química , Mioblastos , Transportadores de Sodio Acoplados a la Vitamina CRESUMEN
Ascorbic acid (AA) is best known for its role as an essential nutrient in humans and other species. As the brain does not synthesize AA, high levels are achieved in this organ by specific uptake mechanisms, which concentrate AA from the bloodstream to the CSF and from the CSF to the intracellular compartment. Two different isoforms of sodium-vitamin C co-transporters (SVCT1 and SVCT2) have been cloned. Both SVCT proteins mediate high affinity Na(+)-dependent L-AA transport and are necessary for the uptake of vitamin C in many tissues. In the adult brain the expression of SVCT2 was observed in the hippocampus and cortical neurons by in situ hybridization; however, there is no data regarding the expression and distribution of this transporter in the fetal brain. The expression of SVCT2 in embryonal mesencephalic neurons has been shown by RT-PCR suggesting an important role for vitamin C in dopaminergic neuronal differentiation. We analyze SVCT2 expression in human and rat developing brain by RT-PCR. Additionally, we study the normal localization of SVCT2 in rat fetal brain by immunohistochemistry and in situ hybridization demonstrating that SVCT2 is highly expressed in the ventricular and subventricular area of the rat brain. SVCT2 expression and function was also confirmed in neurons isolated from brain cortex and cerebellum. The kinetic parameters associated with the transport of AA in cultured neurons and neuroblastoma cell lines were also studied. We demonstrate two different affinity transport components for AA in these cells. Finally, we show the ability of different flavonoids to inhibit AA uptake in normal or immortalized neurons. Our data demonstrates that brain cortex and cerebellar stem cells, neurons and neuroblastoma cells express SVCT2. Dose-dependent inhibition analysis showed that quercetin inhibited AA transport in cortical neurons and Neuro2a cells.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Tronco Encefálico/metabolismo , Flavonoides/farmacología , Neuroblastoma/metabolismo , Neuronas/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/antagonistas & inhibidores , Transportadores de Anión Orgánico Sodio-Dependiente/biosíntesis , Sodio/fisiología , Simportadores/antagonistas & inhibidores , Simportadores/biosíntesis , Animales , Ácido Ascórbico/metabolismo , Western Blotting , Tronco Encefálico/citología , Línea Celular Tumoral , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , ADN Complementario/biosíntesis , ADN Complementario/genética , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Cinética , Ratones , Neuronas/efectos de los fármacos , Embarazo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transportadores de Sodio Acoplados a la Vitamina CRESUMEN
Human cells acquire vitamin C using two different transporter systems, the sodium-ascorbic acid co-transporters with specificity for ascorbic acid, and the facilitative glucose transporters with specificity for dehydroascorbic acid. There is no information on the mechanism of vitamin C transport across the intestinal barrier, a step that determines the bioavailability of vitamin C in humans. We used the colon carcinoma cell line CaCo-2 as an in vitro model for vitamin C transport in enterocyte-like cells. The results of transport kinetics, sodium dependence, inhibition studies, and reverse transcriptase-PCR analysis indicated that CaCo-2 cells express the sodium-ascorbate co-transporters SVCT1 and SVCT2, the dehydroascorbic acid transporters GLUT1 and GLUT3, and a third dehydroascorbic acid transporter with properties expected for GLUT2. Analysis by real time quantitative PCR revealed that the post-confluent differentiation of CaCo-2 cells was accompanied by a marked increase (4-fold) in the steady-state level of SVCT1 mRNA, without changes in SVCT2 mRNA levels. Functional studies revealed that the differentiated cells expressed only one functional ascorbic acid transporter having properties expected for SVCT1, and transported ascorbic acid with a V(max) that was increased at least 2-fold compared with pre-confluent cells. Moreover, post-confluent Caco-2 cells growing as monolayers in permeable filter inserts showed selective sorting of SVCT1 to the apical membrane compartment, without functional evidence for the expression of SVCT2. The identification of SVCT1 as the transporter that allows vectorial uptake of ascorbic acid in differentiated CaCo-2 cells has a direct impact on our understanding of the mechanism for vitamin C transport across the intestinal barrier.