Nutritional challenge induces the adaptive regulation of several nutrient transporters including the amino acid transport system A. Depriving the human choriocarcinoma BeWo cell line of system A substrates causes up-regulation of both system A transport and gene expression. This response is believed to be biphasic, consisting of an acute response involving the trafficking of pre-made transporters from intracellular stores to the plasma membrane similar to the process seen in insulin-stimulated translocation of the glucose transporter GLUT4 (Bose et al.2002). The chronic response produces up-regulation of gene expression and increased transporter protein synthesis. In order to study this biphasic response we used immunocytochemistry to track changes in the localisation of the system A transporter ATA2 in the BeWo cell line.

Cells were maintained in DMEM supplemented with 10 % fetal calf serum and 2 % penicillin/ streptomycin. Cells were seeded onto glass coverslips and grown overnight to allow them to achieve a flattened morphology. Amino acid deprivation was induced by culturing cells in Deprivation Media (DM, Earles Balanced Salt Solution, pH7.4 supplemented with 1 X MEM essential amino acid solution without non-essential amino acids). Cells exposed to either control or DM media were fixed for immunocytochemistry at 0, 0.5, 1, 2, 4, 6 and 8 h (3 coverslips/treatment at each time point). ATA2 was detected using a human-specific rabbit anti-ATA2 antibody.

Increased staining of the plasma membrane was seen after just 30 min of exposure of the cells to DM accompanied by depletion in intracellular ATA2 stores compared to control. Intracellular stores started to be replenished after 4 h of exposure to DM. These data differ slightly from our Western blot data (Jones HN et al. unpublished), where there is no apparent change at 6 h deprivation. This discrepancy is currently being investigated. Staining intensity of the intracellular stores and the plasma membrane increased further with exposure times to DM of 6 and 8 hours.

These results support the hypothesis that adaptive regulation of system A in the BeWo choriocarcinoma cell line is a biphasic process involving the trafficking of ATA2 between cellular compartments.

We thank Dr A. L. Schwartz (St. Louis Children’s Hospital, St. Louis, Missouri, USA) for the supply of the BeWo cells and Dr P. Sarmientos (PRIMM, Milan, Italy) for production of the ATA2 antibody. This work is funded by SEERAD flexible fund and Framework V QLK-1999-003377.