Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line
Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line
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DOI：
10.1152/ajpgi.2001.281.1.g191
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发表时间：
2001-07-01
影响因子：
4.5
通讯作者：
Brodin, B
中科院分区：
文献类型：
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作者：
Nielsen, CU;Amstrup, J;Brodin, B
以人肠上皮细胞系Caco-2为模型系统，研究表皮生长因子（EGF）对肽转运的影响。EGF使[C-14]甘氨酰肌氨酸（[C-14]Gly-Sar）的顶侧至基底侧通量降低至对照值的50.2 +/- 3.6%（n = 6）。流量的动力学分析显示，跨上皮转运的最大流量（V-max）从对照细胞的3.00 +/- 0.17 nmol.cm（-2）.min（-1）降低到用5 ng/ ml EGF处理的细胞的0.50 +/- 0.07 nmol.cm（-2）.min（-1）（n = 6，P < 0.01）。表观Michaelis-Menten常数（Km）在对照细胞中为2.71 +/- 0.31 mM（n = 6），在EGF处理的细胞中为1.89 +/- 0.28 mM（n = 5 6，与对照无显著差异）。类似地，在用EGF处理的细胞中，[C-14]Gly-Sar的顶端摄取减少，EGF的ED 50值为0.36 +/- 0.06 ng/ ml（n = 6），最大抑制为80 +/- 0.02%（n = 6）。V-max从2.61 ± 0.4降至1.06 ± nmol.cm（-2）.min（-1）（n = 3，P < 0.05），而K-m保持不变。基底外侧Gly-Sar摄取显示EGF治疗后V-max或K-m无变化（n 5 - 3）。RT-PCR显示EGF处理的细胞中hPepT 1 mRNA（使用葡萄糖-6-磷酸脱氢酶mRNA作为对照）减少。蛋白质印迹法表明细胞裂解物中hPepT 1蛋白质减少。我们的结论是，EGF治疗减少Gly-Sar的运输在Caco-2细胞的顶端膜中的肽转运分子的数量减少。
The human intestinal cell line Caco-2 was used as a model system to study the effects of epidermal growth factor (EGF) on peptide transport. EGF decreased apical-to-basolateral fluxes of [C-14]glycylsarcosine ([C-14]Gly-Sar) up to 50.2 +/- 3.6% (n = 6) of control values. Kinetic analysis of the fluxes showed that maximal flux (V-max) of transepithelial transport decreased from 3.00 +/- 0.17 nmol.cm(-2).min(-1) in control cells to 0.50 +/- 0.07 nmol.cm(-2).min(-1) in cells treated with 5 ng/ ml EGF (n = 6, P < 0.01). The apparent Michaelis-Menten constant (K-m) was 2.71 +/- 0.31 mM (n = 6) in control cells and 1.89 +/- 0.28 mM (n 5 6, not significantly different from control) in EGF-treated cells. Similarly, apical uptake of [C-14]Gly-Sar decreased in cells treated with EGF, with an ED50 value of 0.36 +/- 0.06 ng/ ml (n = 6) EGF and a maximal inhibition of 80 +/- 0.02% (n = 6). V-max decreased from 2.61 +/- 0.4 to 1.06 +/- 0.1 nmol.cm(-2).min(-1) (n = 3, P < 0.05), whereas K-m remained constant. Basolateral Gly-Sar uptake showed no changes in V-max or K-m after EGF treatment (n 5 3). RT-PCR showed a decrease in hPepT1 mRNA (using glucose-6-phosphate dehydrogenase mRNA as control) in cells treated with EGF. Western blotting indicated a decrease in hPepT1 protein in cell lysates. We conclude that EGF treatment decreases Gly-Sar transport in Caco-2 cells by decreasing the number of peptide transporter molecules in the apical membrane.