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
Keywords
Related Articles
- EP ID EP682118
- DOI 10.1208/ps010312
- Views 90
- Downloads 0
Reaction Phenotyping: Current Industry Efforts to Identify Enzymes Responsible for Metabolizing Drug Candidates
Reaction phenotyping studies to identify specific enzymes involved in the metabolism of drug candidates are increasingly important in drug discovery efforts. Experimental approaches used for CYP reaction phenotyping incl...
Macromolecular Modelling and Docking Simulations for the Discovery of Selective GPER Ligands
Estrogens influence multiple physiological processes and are implicated in many diseases as well. Cellular responses to estrogens are mainly mediated by the estrogen receptors (ER)α and ERβ, which act as...
Activation of G-proteins in brain by endogenous and exogenous cannabinoids
The biological response to cannabinoid agonist begins when the agonist-bound receptor activates G-protein Gα subunits, thus initiating a cascade of signal transduction pathways. For this reason, information about...
Pharmacodynamic Model of Sodium–Glucose Transporter 2 (SGLT2) Inhibition: Implications for Quantitative Translational Pharmacology
Sodium–glucose co-transporter-2 (SGLT2) inhibitors are an emerging class of agents for use in the treatment of type 2 diabetes mellitus (T2DM). Inhibition of SGLT2 leads to improved glycemic control through incre...
Cloning and characterization of the rat multidrug resistance-associated protein 1
Multidrug resistance-associated protein 1 (MRP1) was originally shown to confer resistance of human tumor cells to a broad range of natural product anticancer drugs. MRP1 has also been shown to mediate efflux transport o...