The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers.
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.
Thwaites
D T
DT
Department of Physiological Sciences, University of Newcastle upon Tyne, Medical School, United Kingdom.
McEwan
G T
GT
Simmons
N L
NL
D000596:Amino Acids / Q000378:metabolism*; D001693:Biological Transport, Active; D002460:Cell Line; D003470:Culture Media; D004563:Electrochemistry; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007408:Intestinal Absorption / Q000502:physiology*; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism; D011522:Protons; D012964:Sodium / Q000378:metabolism
D000596:Amino Acids; D003470:Culture Media; D011522:Protons; D012964:Sodium