EAAT1 is involved in transport of L-glutamate during differentiation of the Caco-2 cell line.

Little is known concerning the expression of amino acid transporters during intestinal epithelial cell differentiation. The transport mechanism of L-glutamate and its regulation during the differentiation process were investigated using the human intestinal Caco-2 cell line. Kinetic studies demonstrated the presence of a single, high-affinity, D-aspartate-sensitive L-glutamate transport system in both confluent and fully differentiated Caco-2 cells. This transport was clearly Na(+) dependent, with a Hill coefficient of 2. 9 +/- 0.3, suggesting a 3 Na(+)-to-1 glutamate stoichiometry and corresponding to the well-characterized X(A,G)(-) system. The excitatory amino acid transporter (EAAT)1 transcript was consistently expressed in the Caco-2 cell line, whereas the epithelial and neuronal EAAT3 transporter was barely detected. In contrast with systems B(0) and y(+), which have previously been reported to be downregulated when Caco-2 cells stop proliferating, L-glutamate transport capacity was found to increase steadily between day 8 and day 17. This increase was correlated with the level of EAAT1 mRNA, which might reflect an increase in EAAT1 gene transcription and/or stabilization of the EAAT1 transcript.

Secretion of sparfloxacin from the human intestinal Caco-2 cell line is altered by P-glycoprotein inhibitors.

The mechanism of intestinal secretion of the difluorinated quinolone sparfloxacin was investigated with the epithelial cell line Caco-2 and was compared to that of the P-glycoprotein (P-gp) substrate vinblastine. The P-gp inhibitors verapamil and progesterone significantly increased the epithelial cell accumulation of both vinblastine and sparfloxacin. This increase is likely to result from an inhibition of drug secretion since both vinblastine uptake and sparfloxacin uptake are known to proceed through a passive transmembrane diffusion. The unidirectional fluxes across cell monlayers grown on permeable filters indicated that a net secretion of sparfloxacin and vinblastine occurred across Caco-2 cells. These secretions were significantly inhibited by the MDR-reversing agent verapamil. We conclude that the P-gp is likely to be involved in the intestinal elimination of the difluorinated quinolone sparfloxacin.

Sodium-dependent and -independent transport of L-glutamate in the rat intestinal crypt-like cell line IEC-17.

Mechanisms of L-glutamate transport in intestinal crypts were investigated using the rat intestinal crypt-like cell line IEC-17. Kinetic analysis and competition experiments run in the presence or in the absence of extracellular sodium indicate that L-glutamate uptake occurs through three different transport components: (1) a high affinity Na+-independent component also carrying cystine, similar to system x(c)-; (2) a high affinity Na+-dependent component inhibited by D- and L-aspartate corresponding to the ubiquitous system X(A,G)-; and (3) a low affinity Na+-dependent system resembling the neutral amino acid transport system ASC. The simultaneous presence of these three components suggest that crypt cells are ready to face potential high variations of L-glutamate concentration in the intestinal villus environment.