The transport of acidic amino acids and their analogues across monolayers of human intestinal absorptive (Caco-2) cells in vitro

AbstractThe X−AG system, a sodium-dependent, acidic amino-acid transport system has been implicated in the transport of l-aspartate and l-glutamate across monolayers of human Caco-2 cells, an in vitro model of intestinal absorption. This system, which shares many properties with the l-glutamate carrier present in the human jejunum, is highly saturable (> 95% at 50 μM), vectorial (apical-to-basolateral ⪢ basolateral-to-apical) and sodium-, pH- and temperature-dependent. l-Aspartate was also transported against a 10-fold reverse concentration gradient. These data are consistent with a major (saturable) carrier-mediated pathway superimposed onto a minor non-saturable (diffusional) pathway. The carrier has an absolute sodium-dependence and the Michaelis constants for the sodium-dependent transport component (Km) for l-aspartate and l-glutamate were 56 ± 3 μM and 65 ± μM, respectively. Cross-inhibition studies showed that strong interaction with the carrier was limited to close analogues of the natural substrates. Potent inhibitors included l-aspartate, d-aspartate (Ki, 70 μM), l-glutamate (Ki, 180 μM) and threo-ß-hydroxy-dl-aspartate (Ki, 55 μM), while partial inhibitors included α-methyl-dl-aspartate, d-glutamate, l-asparagine, l-proline and l-alanine. Replacement of the side-chain -COO− group (aspartate) with -SO−3 (l-cysteate, Ki, 65 μM) or -(H)P(O)O− (dl-3-(hydroxyphosphoryl)alanine, Ki, 0 60 μM) maintained strong interaction with the carrier while -As(O)(OH)O− (dl-3-arsonoalanie, Ki, 1100 μM) and -P(O)(OH)O− (dl-3-phosphonoalanine, Ki, 3270 μM) were much more weakly bound, with the larger, but probably less ionised, arsono analogue being more tightly bound than the phosphoro compound. The corresponding analogues of glutamate (homologous extension of the methylene chain) showed negligible interaction. We conclude that Caco-2 monolayers are a relevant experimental model for the study of the transport of acidic amino acids and their analogues in man