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首页> 外文期刊>Pflügers Archiv European Journal of Physiology >hPEPT1 is responsible for uptake and transport of Gly-Sar in the human bronchial airway epithelial cell-line Calu-3
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hPEPT1 is responsible for uptake and transport of Gly-Sar in the human bronchial airway epithelial cell-line Calu-3

机译：hPEPT1负责人支气管上皮细胞系Calu-3中Gly-Sar的摄取和运输

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摘要

The purpose of this work was to investigate the apical uptake and transepithelial transport of Gly-Sar along with the expression of the di-/tripeptide transporters hPEPT1 and hPEPT2 in human Calu-3 bronchial epithelial cells. The apical Gly-Sar uptake rate in Calu-3 cells followed Michaelis–Menten kinetics with a K m value of 1.3 ± 0.3 mM and a V max value of 0.60 ± 0.06 nmol ∙ cm−2 ∙ min−1. Transepithelial apical to basolateral transport of 50 μM [3H]-labelled Gly-Sar across the Calu-3 cell monolayer was pH-dependent. The Gly-Sar flux was significantly reduced in the presence of δ-aminolevulinic acid (2.5 mM), cephalexin (25 mM), and captopril (25 mM; p < 0.05, n = 3). Reverse transcriptase polymerase chain reaction (RT-PCR) revealed the presence of both hPEPT1 and hPEPT2 mRNA in the Calu-3 cells. These findings were confirmed in healthy human bronchial cDNA. Restriction-endonuclease analysis identified hPEPT2 in Calu-3 cells to be the hPEPT2*1 haplotype. Western blotting demonstrated expression of the hPEPT1 protein (~80 kDa), and the immunolabel was mainly localized in the apical membrane as judged by immunolocalization studies using confocal laser scanning microscopy (CLSM). This work presents for the first time hPEPT1 and hPEPT2*1 expression in human Calu-3 cells. Surprisingly, the results indicate that Gly-Sar uptake and transport in Calu-3 cells are hPEPT1-mediated rather than hPEPT2-mediated.
机译：这项工作的目的是调查在人Calu-3支气管上皮细胞中Gly-Sar的顶端吸收和经上皮运输以及二肽/三肽转运蛋白hPEPT1和hPEPT2的表达。 Calu-3细胞的顶端Gly-Sar摄取速率遵循Michaelis-Menten动力学，K m 值为1.3±0.3 mM，V max 值为0.60±0.06 nmol∙cm −2 ∙min -1 。跨Calu-3细胞单层的50μM[ 3 H]标记的Gly-Sar跨顶端上皮到基底外侧的转运是pH依赖性的。在δ-氨基乙酰丙酸（2.5 mM），头孢氨苄（25 mM）和卡托普利（25 mM； p <0.05，n = 3）的存在下，Gly-Sar通量显着降低。逆转录聚合酶链反应（RT-PCR）显示，Calu-3细胞中同时存在hPEPT1和hPEPT2 mRNA。这些发现在健康的人支气管cDNA中得到了证实。限制性内切核酸酶分析鉴定出Calu-3细胞中的hPEPT2为hPEPT2 * 1单倍型。 Western blotting证实了hPEPT1蛋白（〜80 kDa）的表达，并且通过使用共聚焦激光扫描显微镜（CLSM）进行的免疫定位研究判断，免疫标记主要位于顶膜中。这项工作首次提出了hPEPT1和hPEPT2 * 1在人Calu-3细胞中的表达。出乎意料的是，结果表明Calu-3细胞中的甘氨酸吸收和转运是hPEPT1介导的，而不是hPEPT2介导的。

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