Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
Life Sci. 2013 Oct 17;93(17):630-6. doi: 10.1016/j.lfs.2013.08.008. Epub 2013 Aug 27.
The purpose of this study was to clarify the expression and function of peptide transporter 2 (PEPT2) in primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells.
Real-time PCR analysis, uptake study of [(3)H]Gly-Sar, and immunostaining were performed in alveolar epithelial cells.
The expression of PEPT2 mRNA in type II cells isolated from rat lungs was highest at day 0, and decreased rapidly during culture of the cells. In accordance with this change, PEPT2 activity estimated as cefadroxil-sensitive [(3)H]Gly-Sar uptake also decreased along with transdifferentiation. The expression of PEPT2 protein in type II cells was confirmed by immunostaining and Western blot analysis. The uptake of [(3)H]Gly-Sar in type II cells was time- and pH-dependent. In contrast, minimal time-dependence and no pH-dependence of [(3)H]Gly-Sar uptake were observed in type I-like cells. The maximal [(3)H]Gly-Sar uptake was observed at pH6.0, and the uptake decreased at higher pHs in type II cells. The uptake of [(3)H]Gly-Sar in type II cells was inhibited by cefadroxil in a concentration-dependent manner, the IC50 value being 4.3 μM. On the other hand, no significant inhibition by cefadroxil was observed in type I-like cells. In addition, [(3)H]Gly-Sar uptake in type II cells was saturable, the Km value being 72.0 μM.
PEPT2 is functionally expressed in alveolar type II epithelial cells, but the expression decreases along with transdifferentiation, and PEPT2 would be almost completely lost in type I cells.
本研究旨在阐明肽转运蛋白 2（PEPT2）在原代培养的肺泡 II 型上皮细胞和转分化的 I 型样细胞中的表达和功能。
采用实时 PCR 分析、[(3)H]甘氨酰-丝氨酸摄取研究和免疫染色法在肺泡上皮细胞中进行。
从大鼠肺中分离的 II 型细胞中，PEPT2 mRNA 的表达在第 0 天最高，在细胞培养过程中迅速下降。与此变化一致，作为头孢羟氨苄敏感的 [(3)H]甘氨酰-丝氨酸摄取的 PEPT2 活性也随着转分化而降低。通过免疫染色和 Western blot 分析证实了 II 型细胞中 PEPT2 蛋白的表达。[(3)H]甘氨酰-丝氨酸在 II 型细胞中的摄取具有时间和 pH 依赖性。相比之下，在 I 型样细胞中，[(3)H]甘氨酰-丝氨酸的摄取观察到最小的时间依赖性和无 pH 依赖性。在 pH6.0 时观察到最大的 [(3)H]甘氨酰-丝氨酸摄取，在 II 型细胞中摄取在较高 pH 值时降低。头孢羟氨苄以浓度依赖的方式抑制 [(3)H]甘氨酰-丝氨酸在 II 型细胞中的摄取，IC50 值为 4.3 μM。另一方面，在 I 型样细胞中未观察到头孢羟氨苄的显著抑制。此外，[(3)H]甘氨酰-丝氨酸在 II 型细胞中的摄取是饱和的，Km 值为 72.0 μM。
PEPT2 在肺泡 II 型上皮细胞中功能性表达，但表达随转分化而下降，PEPT2 在 I 型细胞中几乎完全丢失。
