Proton-coupled oligopeptide transporter (POT) family expression in human nasal epithelium and their drug transport potential

Mol Pharm. 2011 Jun 6;8(3):664-72. doi: 10.1021/mp100234z. Epub 2011 Mar 24.

Abstract

The molecular and functional expression of peptide transporters (PEPT1 and PEPT2, PHT1, PHT2) in human nasal epithelium was investigated. Quantitative/reverse transcriptase polymerase chain reaction (qPCR/RT-PCR), Western blotting and indirect immuno-histochemistry were used to investigate the functional gene and protein expression for the transporters. Uptake and transport studies were performed using metabolically stable peptides [β-alanyl-L-lysyl-Nε-7-amino-4-methyl-coumarin-3-acetic acid (β-Ala-Lys-AMCA) and β-alanyl-L-histidine (carnosine)]. The effects of concentration, temperature, polarity, competing peptides, and inhibitors on peptide uptake and transport were investigated. PCR products corresponding to PEPT1 (150 bp), PEPT2 (127 bp), PHT1 (110 bp) and PHT2 (198 bp) were detected. Immunohistochemistry and Western blotting confirmed the functional expression of PEPT1 and PEPT2 genes. The uptake of β-Ala-Lys-AMCA was concentration-dependent and saturable (Vmax =4.1 ( 0.07 μmol/min/mg protein, Km = 0.6 ( 0.07 μM). The optimal pH for intracellular accumulation of β-Ala-Lys-AMCA was 6.5. Whereas dipeptides and carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly inhibited peptide uptake and transport, L-Phe had no effect on peptide transport. The permeation of β-alanyl-L-histidine was concentration-, direction-, and temperature-dependent. The uptake, permeation, qPCR/RT-PCR and protein expression data showed that the human nasal epithelium functionally expresses proton-coupled oligopeptide transporters.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Transport / genetics
  • Biological Transport / physiology
  • Blotting, Western
  • Carnosine / metabolism
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Coumarins / metabolism
  • Humans
  • Immunohistochemistry
  • In Vitro Techniques
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Nasal Mucosa
  • Oligopeptides / metabolism
  • Peptide Transporter 1
  • Reverse Transcriptase Polymerase Chain Reaction
  • Symporters / genetics
  • Symporters / metabolism*
  • Temperature

Substances

  • Coumarins
  • Membrane Transport Proteins
  • Oligopeptides
  • Peptide Transporter 1
  • SLC15A1 protein, human
  • Symporters
  • alanyl-lysyl-amca
  • hydrogen-coupled oligopeptide transporter PepT2
  • Carnosine