Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line.
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.
Nielsen
C U
CU
Department of Pharmaceutics, Royal Danish School of Pharmacy, DK-2100 Copenhagen, Denmark.
Amstrup
J
J
Steffansen
B
B
Frokjaer
S
S
Brodin
B
B
D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D018938:Caco-2 Cells; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D004151:Dipeptides / Q000493:pharmacokinetics*; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D018613:Microscopy, Confocal; D000074011:Peptide Transporter 1; D012333:RNA, Messenger / Q000032:analysis; D027981:Symporters
D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D004151:Dipeptides; D000074011:Peptide Transporter 1; D012333:RNA, Messenger; C082186:SLC15A1 protein, human; D027981:Symporters; C004194:glycylsarcosine; D004815:Epidermal Growth Factor