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

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.

Altered Diurnal Rhythm of Intestinal Peptide Transporter by Fasting and Its Effects on the Pharmacokinetics of Ceftibuten

“…Furthermore, molecular identification of PEPT1 and PEPT2 provided a novel opportunity to determine the mechanisms of their regulation. For example, it was reported that the intestinal PEPT1 is regulated by various factors, including dietary conditions (Ogihara et al, 1999;Shiraga et al, 1999;Naruhashi et al, 2002), hormones such as insulin, leptin, and thyroid hormone (Buyse et al, 2001;Ashida et al, 2002;Gangopadhyay et al, 2002), epidermal growth factor (Nielsen et al, 2001), development (Shen et al, 2001), and some pharmacological agents (Fujita et al, 1999;Berlioz et al, 2000).…”

Peptide absorption after massive proximal small bowel resection: Mechanisms of ileal adaptation

“…Moreover, although hyperplasia and increased uptake were evident even at 1 wk post-resection, cellular mRNA levels in the ileal enterocyte decreased 3-fold at 1 wk post-resection, while cellular protein levels remained unchanged. This decrease in gene expression of PepT1 despite the strong catabolic stress with a 70% proximal small bowel resection might be related to some counterregulatory hormones (such as epidermal growth factor and triiodothyronine) or other negative regulators of PepT1 gene expression as has been suggested by other in vitro studies (in cell cultures) [23][24]; however, we have no objective data to support this statement. Similar findings of a decrease in jejunal mRNA for PepT1 were reported after enterectomy in rabbits, but no protein or uptake studies were carried out [25].…”

Changes of biological functions of dipeptide transporter (PepT1) and hormonal regulation in severe scald rats

“…However, few reports have dealt with the hormonal regulation of PepT1. Insulin could stimulate dipeptide transport by increasing membrane insertion of PepT1 from a preformed cytoplasmic pool [9] , and choleratoxin could decrease dipeptide transport by inhibiting the activity of PepT1 through an increase in intracellular concentration of adenosine 3', 5'-cyclic monophosphate [24] . Strong evidences have demonstrated that growth hormone (GH) was an important growth factor for intestine [25] .…”

“…Studies showed that some hormones metabolically regulated the expression of intestinal dipeptide transporter [8,9] . For example, insulin could increase the membrane population of PepT 1 by increasing its translocation from a preformed cytoplasmic pool [9] .…”

“…For example, insulin could increase the membrane population of PepT 1 by increasing its translocation from a preformed cytoplasmic pool [9] . Our previous study [10] showed that rhGH markedly stimulated the uptake and transport of cephalexin in Caco-2 cells with normal or anoxia/reoxygenation management.…”