Function and immunolocalization of overexpressed human intestinal H+/peptide cotransporter in adenovirus-transduced Caco-2 cells
Journal Title: The AAPS Journal - Year 1999, Vol 1, Issue 3
Abstract
Purpose. 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.
Authors and Affiliations
Cheng-Pang Hsu, Elke Walter, Hans P. Merkle, Barbara Rothen-Rutishauser, Heidi Wunderli-Allenspach, John M. Hilfinger, Gordon L. Amidon
Hidalgo, I. J., Raub, T. J., and Borchardt, R. T.: Characterization of the Human Colon Carcinoma Cell Line (Caco-2) as a Model System for Intestinal Epithelial Permeability, Gastroenterology, 96, 736–749, 1989—The Backstory
This manuscript was written with the knowledge and input of Ismael J. Hidalgo and Thomas J. Raub, who were coauthors of the 1989 Gastroenterology paper describing for the first time Caco-2 cell monolayers as a cell cultu...
Solvation and hydration characteristics of ibuprofen and acetylsalicylic acid
Ibuprofen and acetylsalicylic acid were studied by thermoanalytical methods: sublimation calorimetry, solution calorimetry, and with respect to solubility. Upon measuring the temperature dependences of the saturated vapo...
Food Effect in Humans: Predicting the Risk Through In Vitro Dissolution and In Vivo Pharmacokinetic Models
In vitro and in vivo experimental models are frequently used to assess a new chemical entity’s (NCE) biopharmaceutical performance risk for food effect (FE) in humans. Their ability to predict human FE hinges on...
Rationalizing Structure and Target Relationships between Current Drugs
A recent analysis of structure and target relationships between current drugs and bioactive compounds has revealed that only a small fraction of drugs that are active against the same or overlapping targets are involved...
Bladder Tissue Pharmacokinetics of Intravesical Mitomycin C and Suramin in Dogs
Suramin, at non-cytotoxic doses, reverses chemoresistance and enhances the activity of mitomycin C (MMC) in mice bearing human bladder xenograft tumors. The present study evaluated the pharmacokinetics of the intravesica...