研究叶酸转运蛋白在 Staten's Seruminstitut 兔角膜上皮细胞系中的功能特征。
Functional characterization of folate transport proteins in Staten's Seruminstitut rabbit corneal epithelial cell line.
机构信息
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108-2718, USA.
出版信息
Curr Eye Res. 2011 May;36(5):404-16. doi: 10.3109/02713683.2011.566411.
PURPOSE
The overall objective of this study was to investigate and characterize the expression of folate transport proteins in Staten's Seruminstitut rabbit corneal (SIRC) epithelial cell line.
METHODS
[(3)H]Folic acid uptake was studied with respect to time, pH, temperature, sodium, and chloride ion dependency. Inhibition studies were conducted with structural analogs, vitamins, and metabolic inhibitors. [(3)H]Folic acid uptake was also determined with varying concentrations of cold folic acid. Uptake kinetics was studied in the presence of various modulators of intracellular regulatory pathways, protein kinases A and C (PKA and PKC), protein tyrosine kinase (PTK), and calcium-calmodulin modulators. Ex vivo corneal permeability studies were carried out with [(3)H]folic acid in the presence and absence of 1 mM cold folic acid.
RESULTS
Linear increase in [(3)H]folic acid uptake was observed over 30 min. The process followed saturation kinetics with apparent K(m) of 14.2 ± 0.2 nM, V(max) of (1.5 ± 0.1)*10(-5) micro.moles/min/mg protein, and K(d) of (2.1 ± 0.2)*10(-6) min(-1). The uptake process was found to be dependent on pH, sodium ions, chloride ions, temperature, and energy. Uptake was inhibited in the presence of structural analogs (cold folic acid, methyltetrahydro folate, and methotrexate), but structurally unrelated vitamins did not show any effect. Membrane transport inhibitors SITS, DIDS, probenecid and endocytic inhibitor, colchicine significantly inhibited the [(3)H]folic acid uptake indicating the involvement of receptor/transporter mediated process. PKA, PTK, and Ca(2+)/calmodulin pathways significantly regulate the process. RT-PCR and Western blot analysis confirmed the presence of folate receptor-α (FR-alpha) and proton-coupled folate transporter (PCFT). Permeability of [(3)H]folic acid across the rabbit cornea was (1.48 ± 0.13)*10(-05) cm/sec, and in the presence of cold folic acid it was (1.08 ± 0.10)*10(-05) cm/sec.
CONCLUSIONS
This work demonstrated the functional and molecular presence of FR-alpha and PCFT in SIRC epithelial cell line.
目的
本研究的总体目标是研究并描述叶酸转运蛋白在 Staten 血清研究所兔角膜（SIRC）上皮细胞系中的表达。
方法
研究了[(3)H]叶酸摄取与时间、pH 值、温度、钠离子和氯离子依赖性的关系。进行了结构类似物、维生素和代谢抑制剂的抑制研究。还测定了不同浓度冷叶酸对[(3)H]叶酸摄取的影响。在各种细胞内调节途径的调节剂、蛋白激酶 A 和 C（PKA 和 PKC）、蛋白酪氨酸激酶（PTK）和钙钙调蛋白调节剂的存在下研究了 [(3)H]叶酸摄取的动力学。在用和不用 1mM 冷叶酸的情况下，进行了 [(3)H]叶酸的离体角膜通透性研究。
结果
在 30 分钟内观察到 [(3)H]叶酸摄取的线性增加。该过程遵循饱和动力学，表观 K(m)为 14.2±0.2nM，V(max)为(1.5±0.1)*10(-5)微摩尔/分钟/毫克蛋白，K(d)为(2.1±0.2)*10(-6)分钟(-1)。发现摄取过程依赖于 pH 值、钠离子、氯离子、温度和能量。在结构类似物（冷叶酸、甲基四氢叶酸和甲氨蝶呤）存在下，摄取受到抑制，但结构上不相关的维生素没有显示出任何效果。膜转运抑制剂 SITS、DIDS、丙磺舒和内吞抑制剂长春花碱显著抑制 [(3)H]叶酸摄取，表明涉及受体/转运蛋白介导的过程。PKA、PTK 和 Ca(2+)/钙调蛋白途径显著调节该过程。RT-PCR 和 Western blot 分析证实了叶酸受体-α（FR-α）和质子偶联叶酸转运蛋白（PCFT）的存在。[(3)H]叶酸在兔角膜中的通透性为(1.48±0.13)*10(-05)cm/sec，在冷叶酸存在下为(1.08±0.10)*10(-05)cm/sec。
结论
本工作证明了 FR-α和 PCFT 在 SIRC 上皮细胞系中的功能和分子存在。
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