Uptake of 4-chloro-2-methylphenoxyacetic acid (MCPA) from the apical membrane of Caco-2 cells by the monocarboxylic acid transporter

Abstract

The cellular uptake mechanism of 4-chloro-2-methylphenoxyacetic acid (MCPA), a phenoxyacetic acid derivative, was investigated using Caco-2 epithelial cells. The cells were incubated with 50 μM MCPA at pH 6.0 and 37 °C, and the uptake of MCPA from the apical membranes was measured. The uptake of MCPA was significantly decreased by incubation at low temperature (4 °C) and markedly increased by lowering the extracellular pH. Pretreatment with a protonophore, carbonylcyanide- p-(trifluoromethoxy)phenylhydrazone (25 μM), or metabolic inhibitors, 2,4-dinitrophenol (1 mM) and sodium azide (10 mM), significantly decreased the uptake of MCPA by 53%, 45% and 48%, respectively. Coincubation of MCPA with 10 mM l-lactic acid or α-cyano-4-hydroxycinnamate, which is a substrate or an inhibitor of the monocarboxylic acid transporters (MCTs), significantly decreased the uptake of MCPA by 31% and 20%, respectively, and coincubation with benzoic acid profoundly decreased the uptake by 68%. In contrast, coincubation with succinic acid (a dicarboxylic acid) did not affect the uptake. Kinetic analysis of initial MCPA uptake suggested that MCPA is taken up via a carrier-mediated process [ K m = 1.37 ± 0.15 mM, V max = 115 ± 6 nmol (mg protein) − 1 (3 min) − 1 ]. Lineweaver–Burk plots show that benzoic acid competitively inhibits the uptake of MCPA with a K i value of 4.68 ± 1.76 mM. A trans-stimulation effect on MCPA uptake was found in cells preloaded with benzoic acid. These results suggest that the uptake of MCPA from the apical membrane of Caco-2 cells is mainly mediated by common MCTs along with benzoic acid but also in part by l-lactic acid.