title: A fluorescent hPept1 transporter substrate for uptake screening.
abstract: OBJECTIVE
  To synthesize fluorescent analogues of hPept1 substrates, FITC-Val-OCH3, Lys-FITC-OH, and Lys-FITC-OCH3, and to characterize their hPept1 transporter-mediated uptake.
  
  
  METHODS
  FITC analogues of amino acids were synthesized using established synthetic procedures, and the extent of their [3H]Gly-Sar uptake inhibition in HeLa/hPept1 cells was determined. The uptake of Lys-FITC-OCH3 was evaluated in HeLa, HeLa/hPept1, and Caco-2 cells in the presence and absence of Gly-Sar using a fluorescence microscopy-based assay. The uptake and transport of the Lys-FITC analogues were also determined in Caco-2 cells using HPLC assays.
  
  
  RESULTS
  In HeLa/hPept1 cells, [3H]Gly-Sar uptake was significantly inhibited by Lys-FITC-OCH3 (74%) but not by FITC-Val-OCH3 (22%). The uptake of Lys-FITC-OCH3 (100 microM) was approximately 10-fold higher in HeLa/hPept1 cells. Also, Lys-FITC-OCH3 (100 microM) uptake in HeLa/hPept1 and Caco-2 cells was reduced by 77% and 80%, respectively, in the presence of 1 mM Gly-Sar. Dipeptides and cephalexin significantly reduced Lys-FITC-OCH3 uptake in Caco-2 cells. The apical permeability of Lys-FITC-OCH3 (1.5 x 10(6) cm/s) in Caco-2 cells was significantly lowered in the presence of Gly-Sar. Fluorescence micrographs revealed that this analogue was localized in the cytoplasm and in the nucleus.
  
  
  CONCLUSIONS
  The combined results indicate that Lys-FITC-OCH3 is recognized and transported by hPept1 in HeLa/hPept1 and by peptide transporters in Caco-2 cells. The results also suggest that Lys-FITC-OCH3 might be a useful fluorescent substrate for rapid assessment of peptide transporter activity in cells of interest.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Landowski
authors.forename: Hyo-kyung
authors.initials: HK
authors.lastname: Han
authors.forename: Kyung-Dall
authors.initials: KD
authors.lastname: Lee
authors.forename: Gordon L
authors.initials: GL
authors.lastname: Amidon
mesh_terms: D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry / Q000493:pharmacokinetics*; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry / Q000493:pharmacokinetics*; D006367:HeLa Cells; D006801:Humans; D000074011:Peptide Transporter 1; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D027981:Symporters
chemical_list: D002352:Carrier Proteins; D005456:Fluorescent Dyes; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters; D016650:Fluorescein-5-isothiocyanate