Related Experiment Videos
Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line
C U Nielsen1, J Amstrup, B Steffansen
1Department of Pharmaceutics, Royal Danish School of Pharmacy, DK-2100 Copenhagen, Denmark.
Abstract:
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 [(14)C]glycylsarcosine ([(14)C]Gly-Sar) up to 50.2 +/- 3.6% (n = 6) of control values. Kinetic analysis of the fluxes showed that maximal flux (V(max)) of transepithelial transport decreased from 3.00 +/- 0.17 nmol x cm(-2) x min(-1) in control cells to 0.50 +/- 0.07 nmol x cm(-2) x min(-1) in cells treated with 5 ng/ml EGF (n = 6, P < 0.01). The apparent Michaelis-Menten constant (K(m)) 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 [(14)C]Gly-Sar decreased in cells treated with EGF, with an ED(50) value of 0.36 +/- 0.06 ng/ml (n = 6) EGF and a maximal inhibition of 80 +/- 0.02% (n = 6). V(max) decreased from 2.61 +/- 0.4 to 1.06 +/- 0.1 nmol x cm(-2) x min(-1) (n = 3, P < 0.05), whereas K(m) remained constant. Basolateral Gly-Sar uptake showed no changes in V(max) or K(m) 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.
Insights
Epidermal growth factor (EGF) significantly reduces peptide transport in Caco-2 intestinal cells by decreasing the number of peptide transporter 1 (hPepT1) molecules. This impacts nutrient absorption and drug delivery research.
Area of Science:
- Gastroenterology and Nutrition
- Molecular Biology
- Cell Biology
Background:
- The Caco-2 cell line serves as a validated model for studying intestinal epithelial transport.
- Epidermal Growth Factor (EGF) is a key regulator of cell growth and differentiation, with potential roles in intestinal transport.
- Peptide transporters, like hPepT1, are crucial for absorbing dietary peptides and certain drugs in the intestine.
Purpose of the Study:
- To investigate the effect of EGF on the transport of the dipeptide glycylsarcosine (Gly-Sar) in Caco-2 cells.
- To elucidate the molecular mechanisms underlying EGF-mediated changes in peptide transport.
- To assess the impact of EGF on the expression and function of the human peptide transporter 1 (hPepT1).
Main Methods:
- Utilized Caco-2 cell monolayers to measure apical-to-basolateral fluxes of radiolabeled glycylsarcosine ([14C]Gly-Sar).
- Performed kinetic analyses to determine maximal transport velocity (Vmax) and Michaelis-Menten constant (Km).
- Employed RT-PCR and Western blotting to quantify hPepT1 mRNA and protein levels, respectively.
Main Results:
- EGF treatment significantly reduced Gly-Sar transepithelial transport by up to 50.2%, primarily by decreasing Vmax.
- Apical uptake of Gly-Sar was inhibited by EGF, with an ED50 of 0.36 ng/ml, while Km remained unchanged.
- EGF decreased both hPepT1 mRNA and protein expression in Caco-2 cells, indicating reduced transporter availability.
Conclusions:
- EGF diminishes peptide transport in Caco-2 cells through a reduction in the number of apical hPepT1 transporters.
- This downregulation of hPepT1 by EGF has implications for understanding nutrient absorption and the efficacy of peptide-based drugs.
- The findings highlight EGF's role in regulating intestinal peptide transport at the molecular level.