Functional Characterization of Peptide Transporters in Bovine Mammary Epithelial Cells

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 beta-alanyl-L-lysyl-N epsilon-7-amino-4-methyl-coumarin-3-acetic acid (beta-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 beta-Ala-Lys-AMCA in BMECs was 6.5. The transport-kinetics study suggested that the uptake of beta-Ala-Lys-AMCA in BMECs is saturable over the tested concentration, with a K-m value of 82 +/- 18 mu M 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 beta-Ala-Lys-AMCA uptake in BMECs. However, histidine had no effect on beta-Ala-Lys-AMCA uptake. Furthermore, knocking down PepT2 could significantly reduce beta-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 beta-Ala-LysAMCA. The above results revealed functional characteristics of peptide transporters and demonstrated that PepT2 may play a major role in beta-Ala-Lys-AMCA uptake in BMECs. Moreover, the PI3K Akt signaling pathway may regulate the uptake of beta-Ala-Lys-AMCA in BMECs.