LAUSR

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 Km value of 82 ± 18 μM and a Vmax 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.