PEPT1-Mediated Cefixime Uptake into Human Intestinal Epithelial Cells Is Increased by Ca 2+ Channel Blockers

Abstract

ABSTRACT Ca 2+ channel blockers like nifedipine have been shown to increase the oral bioavailability of β-lactam antibiotics, such as cefixime, in humans. The molecular mode of action of Ca 2+ channel blockers on β-lactam absorption, however, has not yet been defined. Using the Caco-2 human intestinal epithelial cell line, we assessed whether alterations in intracellular free Ca 2+ ion (Ca 2+ in ) concentrations by Ca 2+ channel blockers or by Ca 2+ ionophores affect [ 14 C]cefixime absorption. Reduction of Ca 2+ in levels by Ca 2+ channel blockers (nifedipine, verapamil, diltiazem, or bepridil) at concentrations of 100 μM led to 35 to 50% increases in the cellular uptake of 1 mM [ 14 C]cefixime. Increases in Ca 2+ in levels by Ca 2+ ionophores, on the other hand, led to 40% reductions in [ 14 C]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 Ca 2+ 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 2+ in levels, e.g., by Ca 2+ 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 β-lactams into intestinal epithelial cells.