H~+-coupled α-methylaminoisobutyric acid transport in human intestinal Caco-2 cells

摘要

Transepithelial apical-to-basal transport and cellular uptake of the non-metabolisable amino acid α-methylaminoisobutyric acid (MeAIB) across confluent monolayers of the human intestinal epithelial cell line Caco-2 are enhanced by a transepithelial pH gradient (apical pH 6.0, basolateral pH 7.4). In Na+-free conditions (apical pH 7.4, basolateral pH 7.4), net absorption (120 ± 58 pmol/cm2per h, N = 13) and uptake across the apical membrane (cell/medium ratio 0.56 ± 0.06, N = 13) are low. However, in Na+-free conditions with apical pH 6.0, net absorption (685 ± 95 pmol/cm2 per h, N = 15) and intracellular accumulation (cell/medium ratio 3.63 ± 0.29, N = 14) were marked. Continuous monitoring of intracellular pH (pHi) in BCECF (2′,7′-bis(2-carbozyethyl)-5(6)-carboxyfluorescein)-loaded Caco-2 cell monolayers indicated that apical addition of MeAIB (20 mM) was associated with H+-flow across the apical membrane in both Na+ and Na+-free conditions. This transport process is rheogenic in Na+-free media, stimulating an inward short-circuit current in voltage-clamped Caco-2 cell monolayers. On the basis of competition for MeAIB accumulation and pHi experiments, -proline, glycine, -alanine and β-alanine are also substrates for H+-linked transport at the apical membrane of Caco-2 cells but -valine, -leucine and -phenylalanine are not. These data are consistent with the expression, in the apical brush-border membrane of Caco-2 cells, of a H+-coupled, Na+-independent MeAIB carrier.