Methods
Caco-2 cells where grown on polycarbonate filters in media supplemented with serum and antibiotics for 26-28 days in the absence or presence of EGF. Cell morphology and hPepT1 localisation were studied using confocal laser scanning microscopy. Integrity of the monolayer was verified using measurements of transepithelial electrical resistance and fluxes of radiolabelled mannitol. Transepithelial peptide transport and apical peptide uptake were measured in flux studies, using radiolabelled glycylsarcosine (Gly-Sar). hPepT1 protein was immunoprecipitated using specific antibodies and subjected to Western blotting. hPepT1 mRNA-levels were studied using quantitaive RT-PCR, using 6-phosphate dehydrogenase mRNA as a control.

Results
Long-term treatment with EGF caused a dose dependent decrease in transepithelial peptide transport with an ED50 value of 1.21±0.33 ng/ml and a maximal inhibiton of 50% of the control flux. Kinetic analysis revealed that this was caused by a decrease in the carrier-mediated component of the fluxes. Apical peptide uptake displayed a similar pattern, with an ED50 value of 0.36 ng/ml and a maximal inhibition of 40 % of the control uptake. Immunoprecipitation of total cell protein using a hPepT1-antibody, followed by western blotting, revealed a decrease in hPepT1 protein, while quantitative RT-PCR showed a decrease in hPepT1 mRNA. CLSM-studies showed hPepT1 localisation to be primarily in the apical membrane.

Conclusions
Long-term treatment with EGF decreases transepithelial peptide transport in Caco-2 cell monolayers by decreasing the expresion of hPepT1 in the apical membrane. This correlates with other studies showing that EGF inhibits expression of brush-border specific proteins (for refs. see Delie and Rubas, 1997).

Acknowledgements: B. Brodin and C. U. Nielsen were funded by the "Center for Drug Delivery and Transport", a project grant from the Danish Medical Research Council.