Ashokkumar Balasubramaniem, Vaziri Nosratola D, Said Hamid M
Department of Medicine, University of California, Irvine, CA, USA.
Am J Physiol Renal Physiol. 2006 Oct;291(4):F796-805. doi: 10.1152/ajprenal.00078.2006. Epub 2006 May 16.
Thiamin (vitamin B(1)) is essential for normal cellular functions. The kidneys play a critical role in regulating body thiamin homeostasis, by salvaging the vitamin via reabsorption from the glomerular filtrate, but little is known about the mechanism(s) and regulation of thiamin transport in the human renal epithelia at cellular and molecular levels. Using the human-derived renal epithelial HEK-293 cells as a model, we have addressed these issues. Our results showed [(3)H]thiamin uptake to be 1) temperature and energy dependent but Na(+) independent, 2) pH dependent with higher uptake at alkaline/neutral buffer pH compared with acidic pH, 3) saturable as a function of concentration over the nanomolar (apparent K(m) = 70.0 +/- 18.4 nM) and micromolar (apparent K(m) = 2.66 +/- 0.18 microM) ranges, 4) cis-inhibited by unlabeled thiamin and its structural analogs but not by unrelated organic cations, 5) trans-stimulated by unlabeled thiamin, and 6) competitively inhibited by amiloride with an apparent K(i) of 0.6 mM. Using a gene-specific small-interference RNAs (siRNAs) approach, human thiamin transporters 1 and 2 (hTHTR-1 and hTHTR-2) were both found to be expressed and contributed toward total carrier-mediated thiamin uptake. Maintaining the cells in thiamin-deficient medium led to a significant (P < 0.01) and specific upregulation in [(3)H]thiamin uptake, which was associated with an increase in hTHTR-1 and hTHTR-2 protein and mRNA levels as well as promoter activities. Uptake of thiamin by HEK-293 cells also appeared to be under the regulation of an intracellular Ca(2+)/calmodulin-mediated pathway. These studies demonstrate for the first time that thiamin uptake by HEK-293 cells is mediated via a specific pH-dependent process, which involves both the hTHTR-1 and hTHTR-2. In addition, the uptake process appears to be under the regulation of an intracellular Ca(2+)/CaM-mediated pathway and also adaptively upregulated in thiamin deficiency via transcriptional regulatory mechanism(s) that involves both the hTHTR-1 and hTHTR-2.
硫胺素（维生素B1）对正常细胞功能至关重要。肾脏在调节机体硫胺素稳态方面发挥着关键作用，它通过从肾小球滤液中重吸收来挽救维生素，但在细胞和分子水平上，关于人类肾上皮细胞中硫胺素转运的机制和调节知之甚少。我们以人源肾上皮HEK - 293细胞为模型，解决了这些问题。我们的结果表明，[³H]硫胺素摄取具有以下特点：1）依赖温度和能量，但不依赖Na⁺；2）依赖pH，在碱性/中性缓冲液pH下的摄取高于酸性pH；3）在纳摩尔（表观Kₘ = 70.0 ± 18.4 nM）和微摩尔（表观Kₘ = 2.66 ± 0.18 μM）范围内随浓度呈饱和状态；4）被未标记的硫胺素及其结构类似物顺式抑制，但不被无关的有机阳离子抑制；5）被未标记的硫胺素反式刺激；6）被阿米洛利竞争性抑制，表观Kᵢ为0.6 mM。使用基因特异性小干扰RNA（siRNA）方法，发现人类硫胺素转运蛋白1和2（hTHTR - 1和hTHTR - 2）均有表达，并对载体介导的总硫胺素摄取有贡献。将细胞维持在硫胺素缺乏的培养基中导致[³H]硫胺素摄取显著（P < 0.01）且特异性上调，这与hTHTR - 1和hTHTR - 2蛋白及mRNA水平以及启动子活性的增加有关。HEK - 293细胞对硫胺素的摄取似乎也受细胞内Ca²⁺/钙调蛋白介导的途径调节。这些研究首次证明，HEK - 293细胞对硫胺素的摄取是通过特定的pH依赖过程介导的，该过程涉及hTHTR - 1和hTHTR - 2。此外，摄取过程似乎受细胞内Ca²⁺/钙调蛋白介导的途径调节，并且在硫胺素缺乏时通过涉及hTHTR - 1和hTHTR - 2的转录调节机制适应性上调。
Zotero 插件