H(+)-coupled alpha-methylaminoisobutyric acid transport in human intestinal Caco-2 cells

D T Thwaites, G T McEwan, B H Hirst, N L Simmons

Research output: Contribution to journalArticle

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Abstract

Transepithelial apical-to-basal transport and cellular uptake of the non-metabolisable amino acid alpha-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/cm2 per 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-carboxyethyl)-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, L-proline, glycine, L-alanine and beta-alanine are also substrates for H(+)-linked transport at the apical membrane of Caco-2 cells but L-valine, L-leucine and L-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.
Original languageEnglish
Pages (from-to)111-8
Number of pages8
JournalBiochimica et Biophysica Acta
Volume1234
Issue number1
Publication statusPublished - 8 Mar 1995

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Caco-2 Cells
Acids
Membranes
alanylglycine
beta-Alanine
Proton-Motive Force
Valine
Microvilli
Phenylalanine
Proline
Leucine
Epithelial Cells
Cell Membrane
Amino Acids
Cell Line

Keywords

  • Biological Transport
  • Cell Line
  • Cell Polarity
  • Cytoplasm
  • Epithelium
  • Humans
  • Hydrogen-Ion Concentration
  • Intestinal Absorption
  • Intestinal Mucosa
  • Intestines
  • Patch-Clamp Techniques
  • Sodium
  • beta-Alanine

Cite this

H(+)-coupled alpha-methylaminoisobutyric acid transport in human intestinal Caco-2 cells. / Thwaites, D T; McEwan, G T; Hirst, B H; Simmons, N L.

In: Biochimica et Biophysica Acta, Vol. 1234, No. 1, 08.03.1995, p. 111-8.

Research output: Contribution to journalArticle

Thwaites, D T ; McEwan, G T ; Hirst, B H ; Simmons, N L. / H(+)-coupled alpha-methylaminoisobutyric acid transport in human intestinal Caco-2 cells. In: Biochimica et Biophysica Acta. 1995 ; Vol. 1234, No. 1. pp. 111-8.
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T1 - H(+)-coupled alpha-methylaminoisobutyric acid transport in human intestinal Caco-2 cells

AU - Thwaites, D T

AU - McEwan, G T

AU - Hirst, B H

AU - Simmons, N L

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N2 - Transepithelial apical-to-basal transport and cellular uptake of the non-metabolisable amino acid alpha-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/cm2 per 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-carboxyethyl)-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, L-proline, glycine, L-alanine and beta-alanine are also substrates for H(+)-linked transport at the apical membrane of Caco-2 cells but L-valine, L-leucine and L-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.

AB - Transepithelial apical-to-basal transport and cellular uptake of the non-metabolisable amino acid alpha-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/cm2 per 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-carboxyethyl)-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, L-proline, glycine, L-alanine and beta-alanine are also substrates for H(+)-linked transport at the apical membrane of Caco-2 cells but L-valine, L-leucine and L-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.

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KW - Cell Line

KW - Cell Polarity

KW - Cytoplasm

KW - Epithelium

KW - Humans

KW - Hydrogen-Ion Concentration

KW - Intestinal Absorption

KW - Intestinal Mucosa

KW - Intestines

KW - Patch-Clamp Techniques

KW - Sodium

KW - beta-Alanine

M3 - Article

C2 - 7880851

VL - 1234

SP - 111

EP - 118

JO - Biochimica et Biophysica Acta

JF - Biochimica et Biophysica Acta

SN - 0006-3002

IS - 1

ER -