Function and immunolocalization of overexpressed human intestinal H+/peptide cotransporter in adenovirus-transduced Caco-2 cells.
OBJECTIVE
To determine the localization of the human intestinal H+/peptide cotransporter (hPepT1) and its function in intestinal epithelial cells after adenoviral transduction.


METHODS
Caco-2 cells grown on Transwell membrane filters were transduced with a recombinant replication-deficient adenovirus carrying the hPepT1 gene. The transport of Gly-Sar across both apical and basolateral membranes was measured after adenoviral transduction as a function of pH, temperature, inhibitors, and substrate concentration. The localization of hPepT1 was examined by immunocytochemistry using confocal laser scanning microscopy.


RESULTS
The apical-to-basolateral and basolateral-to-apical transport of Gly-Sar in Caco-2 cells after viral transduction was increased 3.3 and 3.5-fold, respectively. The similar magnitude of Gly-Sar permeability from either direction indicates involvement of identical transport pathways in both membranes. This was further confirmed by immunocytochemistry showing that hPepT1 was localized in the apical and basolateral membrane of Caco-2 cells after adenoviral transduction. In both directions, Gly-Sar transport was enhanced in the presence of a pH gradient. In addition, the basolateral-to-apical Gly-Sar transport was dependent on temperature, multiplicity of infection (MOI), and Gly-Sar concentration. It was inhibited in the presence of excess Gly-Pro and cephalexin.


CONCLUSIONS
Caco-2 cell monolayers represent an appropriate model to study gene expression in intestinal epithelial cells. Transport characteristics of Gly-Sar from the basolateral to the apical side in adenovirus-transduced Caco-2 cells are in agreement with those from the apical to the basolateral side, indicating that hPepT1 is also expressed in the basolateral membrane and displays a similar level of transport enhancement after adenovirus mediated hPepT1 gene expression.
Hsu
C P
CP
Johnson & Johnson, Drug Metabolism, Route 202, P.O. Box 300, Raritan, NJ 08869, USA. chsu1@prius.jnj.com
Walter
E
E
Merkle
H P
HP
Rothen-Rutishauser
B
B
Wunderli-Allenspach
H
H
Hilfinger
J M
JM
Amidon
G L
GL
D000256:Adenoviridae / Q000235:genetics*; D001692:Biological Transport; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism*; D020895:Enterocytes / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000378:metabolism / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D018613:Microscopy, Confocal; D008871:Microvilli / Q000378:metabolism; D000074011:Peptide Transporter 1; D010539:Permeability; D027981:Symporters; D013696:Temperature; D014161:Transduction, Genetic
D002352:Carrier Proteins; D004151:Dipeptides; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters; C004194:glycylsarcosine