D-CYCLOSERINE TRANSPORT IN HUMAN INTESTINAL EPITHELIAL (CACO-2) CELLS - MEDIATION BY A H+-COUPLED AMINO-ACID TRANSPORTER

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 gn effective inhibitor of net transepithelial transport (J(net)) of L-alanine (100 mu M) and its accumulation (across the apical membrane) in a similar manner to amino acid substrates (L-alanine, beta-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 epithelia, 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 Km = 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.