The objective of this study was to characterize the expression profile, transport kinetics, and regulation of peptide transporters in bovine mammary epithelial cells (BMECs). Quantitative reverse-transcription real-time PCR, Western blotting, and immunofluorescence staining were used to investigate the expression of peptide transporters in bovine mammary tissues. The effects of time, pH, concentration, and specific inhibitors on β-alanyl-l-lysyl-Nε-7-amino-4-methyl-coumarin-3-acetic acid (β-Ala-Lys-AMCA) uptake in BMECs were also studied. The results showed that the peptide transporters PepT2 and PhT1 are both expressed in bovine mammary glands. The optimal pH for the uptake of β-Ala-Lys-AMCA in BMECs was 6.5. The transport-kinetics study suggested that the uptake of β-Ala-Lys-AMCA in BMECs is saturable over the tested concentration, with a K_m value of 82 ± 18 μM and a V_max of 124 ± 11 pmol/min per milligram of protein. Other dipeptides, including Gly-Sar, Met-Gly, and Met-Met, competitively inhibited β-Ala-Lys-AMCA uptake in BMECs. However, histidine had no effect on β-Ala-Lys-AMCA uptake. Furthermore, knocking down PepT2 could significantly reduce β-Ala-Lys-AMCA uptake, but PhT1 interference had no effect on peptide uptake in BMECs. The inhibition of PI3K and Akt decreased the uptake of β-Ala-Lys-AMCA. The above results revealed functional characteristics of peptide transporters and demonstrated that PepT2 may play a major role in β-Ala-Lys-AMCA uptake in BMECs. Moreover, the PI3K–Akt signaling pathway may regulate the uptake of β-Ala-Lys-AMCA in BMECs.

中文翻译：

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牛乳腺上皮细胞中肽转运蛋白的功能表征

这项研究的目的是表征牛乳腺上皮细胞（BMEC）中的表达谱，转运动力学和肽转运蛋白的调控。定量逆转录实时荧光定量PCR，Western印迹和免疫荧光染色用于研究牛乳腺组织中肽转运蛋白的表达。时间，pH，浓度和特异性抑制剂对β-丙氨酰-1-赖氨酰-N的影响还研究了BMEC中ε-7-氨基-4-甲基香豆素-3-乙酸（β-Ala-Lys-AMCA）的吸收。结果表明，肽转运蛋白PepT2和PhT1均在牛乳腺中表达。在BMEC中摄取β-Ala-Lys-AMCA的最佳pH为6.5。运输动力学研究表明，BMEC中β-Ala-Lys-AMCA的吸收在测试浓度范围内是饱和的，K _m值为82± 18μM，V _max每毫克蛋白质124±11 pmol / min。其他二肽，包括Gly-Sar，Met-Gly和Met-Met，竞争性抑制BMEC中β-Ala-Lys-AMCA的摄取。但是，组氨酸对β-Ala-Lys-AMCA的摄取没有影响。此外，敲除PepT2可以显着降低β-Ala-Lys-AMCA的摄取，但是PhT1干扰对BMECs的肽摄取没有影响。PI3K和Akt的抑制降低了β-Ala-Lys-AMCA的摄取。以上结果揭示了肽转运蛋白的功能特征，并证明PepT2可能在BMEC中β-Ala-Lys-AMCA摄取中起主要作用。此外，PI3K–Akt信号通路可能调节BMEC中β-Ala-Lys-AMCA的摄取。