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 (Ca-in(2+)) concentrations by Ca2+ channel blockers or by Ca2+ ionophores affect [C-14]cefixime absorption. Reduction of Ca-in(2+) levels by Ca2+ channel blockers (nifedipine, verapamil, diltiazem, or bepridil) at concentrations of 100 muM led to 35 to 50% increases in the cellular uptake of 1 mM [C-14] cefixime. Increases in Ca-in(2+) levels by Ca2+ ionophores, on the other hand, led to 40% reductions in [C-14] 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 Ca 2(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 Ca-in(2+) 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.