Substrate specificity of the di/tripeptide transporter in human intestinal epithelia (Caco-2): identification of substrates that undergo H(+)-coupled absorption.
1. pH-dependent transepithelial transport and intracellular accumulation of the hydrolysis-resistant dipeptide glycylsarcosine (Gly-Sar) have been demonstrated in the model human intestinal epithelial cell line, Caco-2. 2. Experiments with BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein)-loaded Caco-2 cells demonstrated that dipeptide (Gly-Sar) transport across the apical membrane is coupled to proton flow into the cell. 3. A range of postulated substrates for the intestinal di/tripeptide carrier were tested for their abilities to: (a) inhibit pH-dependent [14C]Gly-Sar apical-to-basal transport and intracellular accumulation and (b) stimulate H+ flow across the apical surface of BCECF-loaded Caco-2 cell monolayers. 4. A range of compounds (including Gly-Gly, Leu-Leu, Gly-Gly-Gly, cefadroxil and cephalexin) caused marked acidification of intracellular pH when perfused at the apical surface of Caco-2 cell monolayers. In contrast leucine and D-Leu-D-Leu failed to induce proton flow. The ability to induce proton-flow across the apical surface by these compounds, in this intestinal epithelium, was directly correlated to the relative inhibitory effects on [14C]-Gly-Sar transport and accumulation. 5. The determination of substrate-induced intracellular pH change in the Caco-2 cell system may provide a useful rapid screen for candidate substrates for absorption via H(+)-coupled transport mechanisms such as the intestinal di/tripeptide carrier in an appropriate physiological context.
Department of Physiological Sciences, University of Newcastle upon Tyne, Medical School.
D T
DT
Thwaites
B H
BH
Hirst
N L
NL
Simmons
D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D002511:Cephalosporins / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism*; D005452:Fluoresceins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007408:Intestinal Absorption; D007413:Intestinal Mucosa / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity
D002511:Cephalosporins; D004151:Dipeptides; D005452:Fluoresceins; D009842:Oligopeptides; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium