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 an effective inhibitor of net transepithelial transport (Jnet) of L-alanine (100 microM) 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 Jnet 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 (Isc) 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 Isc was dose-dependent with an apparent Km = 15.8 +/- 2.0 (mean +/- s.e. mean) mM, and Vmax = 373 +/- 21 nmol cm-2h-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 Isc with 20 mM D-cycloserine was non-additive with other amino acid substrates for H+/amino acid symport.
Department of Physiological Sciences, University of Newcastle upon Tyne, Medical School.
D T
DT
Thwaites
G
G
Armstrong
B H
BH
Hirst
N L
NL
Simmons
D026905:Amino Acid Transport Systems; D000596:Amino Acids / Q000378:metabolism; D000963:Antimetabolites / Q000378:metabolism*; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000378:metabolism*; D003470:Culture Media; D003523:Cycloserine / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004594:Electrophysiology; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D012964:Sodium / Q000502:physiology
D026905:Amino Acid Transport Systems; D000596:Amino Acids; D000963:Antimetabolites; D002352:Carrier Proteins; D003470:Culture Media; D003523:Cycloserine; D012964:Sodium