We determined the extent of Na(+)-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na(+)-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na(+)-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, beta-alanine, alpha-aminoisobutyric acid (AIB), alpha-methylaminoisobutyric acid (MeAIB), tau-amino-n-butyric acid and L-alanine are likely substrates for pH-dependent transport in the brush border of Caco-2 cells. Both D-serine and D-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and D-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caco-2 cell monolayers loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (Isc) in voltage-clamped Caco-2 cell monolayers in Na(+)-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, serine, glutamine, asparagine, D-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pHi and inward Isc. In conclusion, Caco-2 cells express a Na(+)-independent, H(+)-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial transport of a range of amino acids across this human intestinal epithelium.

In human Caco-2 intestinal epithelial layers, L-alanine absorption can be energized by a proton gradient across the brush-border membrane. Acidification of the apical medium, even in Na(+)-free media, is associated with a saturable net transepithelial absorption of L-alanine. L-Alanine transport causes cytosolic acidification consistent with proton/amino acid symport. L-Alanine transport in Na(+)-free media is rheogenic, stimulating an inward short-circuit current in voltage-clamped epithelial monolayers. By measurement of rapid L-alanine influx across the apical membrane, L-alanine-stimulated inward short-circuit current and intracellular acidification in the same cell batch, we estimate L-alanine/proton stoichiometry to be 1:0.62 +/- 0.25 (SD) (short-circuit current) or 1:0.73 +/- 0.19 (intracellular acidification). From competition studies, it is likely that L-proline, alpha-aminoisobutyric acid, and beta-alanine, but not L-valine and L-serine, are substrates for proton-linked, substrate transport in the brush border of Caco-2 cells.

Previously, absorption of l‐proline across the apical membrane of the intestinal enterocyte has been attributed to transport via the Na+‐dependent Imino system. However, net (absorptive) transport of proline across intact Caco‐2 cell monolayers was enhanced by acidification of the apical environment, under both Na+‐containing and Na+‐free conditions. This Na+‐independent pH‐dependent proline flux was associated with H+ flow across the apical membrane as determined by continuous measurement of intracellular pH. H+/proline symport was associated with an inward I sc in voltage‐clamped Caco‐2 epithelial layers demonstrating the electrogenic nature of this transport process. In conclusion Caco‐2 cells possess an apically‐localised, Na+‐independent, electrogenic H+/imino acid transporter which may play an important role in intestinal proline absorption.

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