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D‐Cycloserine transport in human intestinal epithelial (Caco‐2) cells: mediation by a H + ‐coupled amino acid transporter
Author(s) -
Thwaites David T.,
Armstrong Gillian,
Hirst Barry H.,
Simmons Nicholas L.
Publication year - 1995
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1995.tb14998.x
Subject(s) - symporter , alanine , apical membrane , glycine , chemistry , biophysics , amino acid , caco 2 , biochemistry , transporter , biology , cell , membrane , gene
1 The ability of D‐cycloserine to act as a substrate for H + /amino acid symport has been tested in epithelial layers of Caco‐2 human intestinal cells. 2 In Na + ‐free media with the apical bathing media held at pH 6.0, D‐cycloserine (20 mM) is an effective inhibitor of net transepithelial transport (J net ) of L‐alanine (100 μ m ) and its accumulation (across the apical membrane) in a similar manner to amino acid substrates (L‐alanine, β‐alanine, L‐proline and glycine). In contrast L‐valine was ineffective as an inhibitor for H + /amino acid symport. Both inhibition of L‐alanine J net and its accumulation by D‐cycloserine were dose‐dependent, maximal inhibition being achieved by 5–10 mM. 3 Both D‐cycloserine and known substrates for H + /amino acid symport stimulated an inward short circuit current ( I sc ) when voltage‐clamped monolayers of Caco‐2 epitheha, mounted in Ussing chambers, were exposed to apical substrate in Na + ‐free media, with apical pH held at 6.0. The D‐cycloserine dependent increase in I sc was dose‐dependent with an apparent K m = 15.8 + 2.0 (mean ± s.e.mean) mM, and V max = 373±21 nmol cm −2 h −1 . 4 D‐Cycloserine (20 mM) induced a prompt acidification of Caco‐2 cell cytosol when superfused at the apical surface in both Na + and Na + ‐free conditions. Cytosolic acidification in response to D‐cycloserine was dependent upon superfusate pH, being attenuated at pH 8 and enhanced in acidic media. 5 The increment in I sc with 20 mM D‐cycloserine was non‐additive with other amino acid substrates for H + /amino acid symport.