Up-regulation and polarized expression of the sodium-ascorbic acid transporter SVCT1 in post-confluent differentiated CaCo-2 cells
Up-regulation and polarized expression of the sodium-ascorbic acid transporter SVCT1 in post-confluent differentiated CaCo-2 cells
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DOI：
10.1074/jbc.m205119200
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发表时间：
2003-03-14
影响因子：
4.8
通讯作者：
Vera, JC
中科院分区：
文献类型：
--
作者：
Maulén, NP;Henríquez, EA;Vera, JC
人类细胞通过两种不同的转运蛋白系统获得维生素C，即对抗坏血酸具有特异性的钠-抗坏血酸协同转运蛋白和对脱氢抗坏血酸具有特异性的促进性葡萄糖转运蛋白。没有关于维生素C穿过肠道屏障的运输机制的信息，这是决定维生素C在人体中的生物利用度的一个步骤。我们使用结肠癌细胞系CaCo-2作为肠细胞样细胞中维生素C转运的体外模型。运输动力学，钠依赖性，抑制研究和逆转录酶-PCR分析的结果表明，CaCo-2细胞表达钠-抗坏血酸共转运SVCT 1和SVCT 2，脱氢抗坏血酸转运GLUT 1和GLUT 3，和第三脱氢抗坏血酸转运GLUT 2预期的属性。真实的时间定量PCR分析显示，CaCo-2细胞融合分化后SVCT 1 mRNA的稳态水平显著增加（4倍），而SVCT 2 mRNA水平无变化。功能研究表明，分化的细胞只表达一种功能性抗坏血酸转运蛋白，具有SVCT 1的预期特性，并转运抗坏血酸，V（max）与汇合前细胞相比至少增加了2倍。此外，融合后Caco-2细胞在可渗透的过滤器插入物中生长为单层，显示SVCT 1选择性分选至顶端膜室，没有SVCT 2表达的功能证据。SVCT 1作为允许分化的CaCo-2细胞中的抗坏血酸的载体摄取的转运蛋白的鉴定对我们理解维生素C跨肠屏障转运的机制具有直接影响。
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 steadystate 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.