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Am J Physiol Gastrointest Liver Physiol 281: G191-G199, 2001;
0193-1857/01 $5.00
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Vol. 281, Issue 1, G191-G199, July 2001

Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line

Carsten Uhd Nielsen1, Jan Amstrup2, Bente Steffansen1, Sven Frokjaer1, and Birger Brodin1

1 Department of Pharmaceutics, Royal Danish School of Pharmacy; and 2 Department of Zoophysiology, August Krogh Institute, DK-2100 Copenhagen, Denmark

The human intestinal cell line Caco-2 was used as a model system to study the effects of epidermal growth factor (EGF) on peptide transport. EGF decreased apical-to-basolateral fluxes of [14C]glycylsarcosine ([14C]Gly-Sar) up to 50.2 ± 3.6% (n = 6) of control values. Kinetic analysis of the fluxes showed that maximal flux (Vmax) of transepithelial transport decreased from 3.00 ± 0.17 nmol · cm-2 · min-1 in control cells to 0.50 ± 0.07 nmol · cm-2 · min-1 in cells treated with 5 ng/ml EGF (n = 6, P < 0.01). The apparent Michaelis-Menten constant (Km) was 2.71 ± 0.31 mM (n = 6) in control cells and 1.89 ± 0.28 mM (n = 6, not significantly different from control) in EGF-treated cells. Similarly, apical uptake of [14C]Gly-Sar decreased in cells treated with EGF, with an ED50 value of 0.36 ± 0.06 ng/ml (n = 6) EGF and a maximal inhibition of 80 ± 0.02% (n = 6). Vmax decreased from 2.61 ± 0.4 to 1.06 ± 0.1 nmol · cm-2 · min-1 (n = 3, P < 0.05), whereas Km remained constant. Basolateral Gly-Sar uptake showed no changes in Vmax or Km after EGF treatment (n = 3). RT-PCR showed a decrease in hPepT1 mRNA (using glucose-6-phosphate dehydrogenase mRNA as control) in cells treated with EGF. Western blotting indicated a decrease in hPepT1 protein in cell lysates. We conclude that EGF treatment decreases Gly-Sar transport in Caco-2 cells by decreasing the number of peptide transporter molecules in the apical membrane.

intestinal oligopeptide transporter; growth factor; immunocytochemistry; laser scanning confocal microscopy


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