A Fluorescent hPept1 Transporter Substrate for Uptake Screening

Christopher P. Landowski, Hyo Kyung Han, Kyung Dall Lee, Gordon L. Amidon

Research output: Contribution to journalArticleScientificpeer-review

10 Citations (Scopus)

Abstract

Purpose. 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 μM) was approximately 10-fold higher in HeLa/hPept1 cells. Also, LyS-FITC-OCH3 (100 μM) 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 × 106 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.

Original languageEnglish
Pages (from-to)1738-1745
Number of pages8
JournalPharmaceutical Research
Volume20
Issue number11
DOIs
Publication statusPublished - 1 Nov 2003
MoE publication typeA1 Journal article-refereed

Fingerprint

Fluorescein-5-isothiocyanate
Screening
Substrates
Caco-2 Cells
HeLa Cells
Assays
Cephalexin
Dipeptides
Fluorescence microscopy
Fluorescence Microscopy
Permeability
Cytoplasm

Keywords

  • Caco-2
  • Fluorescent substrate
  • HeLa
  • hPept1
  • Oligopeptide transporter
  • Permeability

Cite this

Landowski, Christopher P. ; Han, Hyo Kyung ; Lee, Kyung Dall ; Amidon, Gordon L. / A Fluorescent hPept1 Transporter Substrate for Uptake Screening. In: Pharmaceutical Research. 2003 ; Vol. 20, No. 11. pp. 1738-1745.
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title = "A Fluorescent hPept1 Transporter Substrate for Uptake Screening",
abstract = "Purpose. 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 μM) was approximately 10-fold higher in HeLa/hPept1 cells. Also, LyS-FITC-OCH3 (100 μM) 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 × 106 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.",
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A Fluorescent hPept1 Transporter Substrate for Uptake Screening. / Landowski, Christopher P.; Han, Hyo Kyung; Lee, Kyung Dall; Amidon, Gordon L.

In: Pharmaceutical Research, Vol. 20, No. 11, 01.11.2003, p. 1738-1745.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - A Fluorescent hPept1 Transporter Substrate for Uptake Screening

AU - Landowski, Christopher P.

AU - Han, Hyo Kyung

AU - Lee, Kyung Dall

AU - Amidon, Gordon L.

PY - 2003/11/1

Y1 - 2003/11/1

N2 - Purpose. 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 μM) was approximately 10-fold higher in HeLa/hPept1 cells. Also, LyS-FITC-OCH3 (100 μM) 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 × 106 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.

AB - Purpose. 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 μM) was approximately 10-fold higher in HeLa/hPept1 cells. Also, LyS-FITC-OCH3 (100 μM) 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 × 106 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.

KW - Caco-2

KW - Fluorescent substrate

KW - HeLa

KW - hPept1

KW - Oligopeptide transporter

KW - Permeability

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U2 - 10.1023/B:PHAM.0000003369.64891.51

DO - 10.1023/B:PHAM.0000003369.64891.51

M3 - Article

C2 - 14661916

AN - SCOPUS:0344084057

VL - 20

SP - 1738

EP - 1745

JO - Pharmaceutical Research

JF - Pharmaceutical Research

SN - 0724-8741

IS - 11

ER -