PEPT1-mediated cefixime uptake into human intestinal epithelial cells is increased by Ca2+ channel blockers.
Ca2+ channel blockers like nifedipine have been shown to increase the oral bioavailability of beta-lactam antibiotics, such as cefixime, in humans. The molecular mode of action of Ca2+ channel blockers on beta-lactam absorption, however, has not yet been defined. Using the Caco-2 human intestinal epithelial cell line, we assessed whether alterations in intracellular free Ca2+ ion (Ca2+in) concentrations by Ca2+ channel blockers or by Ca2+ ionophores affect [14C]cefixime absorption. Reduction of Ca2+in levels by Ca2+ channel blockers (nifedipine, verapamil, diltiazem, or bepridil) at concentrations of 100 microM led to 35 to 50% increases in the cellular uptake of 1 mM [14C]cefixime. Increases in Ca2+in levels by Ca2+ ionophores, on the other hand, led to 40% reductions in [14C]cefixime absorption. Nifedipine increased the V(max) of cefixime transport by 67%, whereas the K(m) of cefixime transport remained unaffected. By measuring the pH in Caco-2 cells loaded with the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein, we show that cefixime transport mediated by the intestinal H+-coupled peptide transporter PEPT1 leads to intracellular acidification. This acid load was reduced by nifedipine, although the Ca2+ channel blocker increased the level of H+ and cefixime cotransport. Increases in Ca2+in levels by ionomycin enhanced the decline in intracellular pH induced by cefixime alone, although ionomycin reduced the level of H+ and cefixime cotransport. In conclusion, our studies demonstrate that alterations of Ca2+in levels, e.g., by Ca2+ channel blockers, affect pH regulatory systems, such as apical Na+ and H+ exchange, and thereby alter the H+ gradient that serves as the driving force for uptake of beta-lactams into intestinal epithelial cells.
Wenzel
Uwe
U
Department of Food and Nutrition, Molecular Nutrition Unit, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany.
Kuntz
Sabine
S
Diestel
Simone
S
Daniel
Hannelore
H
D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000378:metabolism / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism / Q000494:pharmacology*; D020682:Cefixime / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D007476:Ionophores / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D000074011:Peptide Transporter 1; D027981:Symporters
D002121:Calcium Channel Blockers; D002352:Carrier Proteins; D007476:Ionophores; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters; D020682:Cefixime; D002118:Calcium