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

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.