The human placental trophoblast tumour cell line BeWo can be induced to syncytialise following forskolin. During a microarray expression study of forskolin-regulated genes in these cells (Kudo et al. 2002) we noted striking and reciprocal changes (up-regulation, down-regulation respectively) in the levels of mRNA encoding the amino acid transporters y⁺LAT-2 and y⁺LAT-1. These membrane proteins are the two isoforms of the catalytic light chains of the y⁺L amino acid transport system. We have therefore studied transport through system y⁺L in these cells with and without 36 h pre-treatment with forskolin to see whether there are alterations in transport function associated with the altered gene expression.

Because we were interested in determining both affinity and velocity for different known system y⁺L substrates we used a mixture of two cationic amino acids (lysine, arginine) as substrates, each at low concentration (1 µM) with a modified protocol for determining amino acid influx (Kudo & Boyd, 2002) under initial rate conditions (3 min, 37 °C) using double label (L-[¹⁴C]arginine, L-[³H]lysine). We observe high affinity inhibition of more than 95 % of total cationic amino acid influx by 10 mM glutamine in both control and forskolin (100 µM) treated cells (K_i is 90 µM in control and 30 µM in treated cells). Since in the absence of sodium (choline substituting), total cationic amino acid transport fluxes are substantially unaltered whereas the affinity for inhibition by glutamine is very markedly reduced, we conclude that at the low substrate concentration used in this study virtually all of the flux of cationic amino acids into these cells is through system y⁺L (in contrast to the conclusion of Way et al. 1998). We find (Tables 1 and 2) that cells treated with forskolin have a higher affinity for inhibition (V_i) of system y⁺L by either amino acid than do control (vehicle-only) cells. Taken together with the array data this indicates that, in mammalian cells at 37 °C, y⁺LAT1 has a lower affinity than does y⁺LAT2 for cationic amino acids (compatible with findings made in the Xenopus oocyte expression system at room temperature; Pfeiffer et al. 1999; Broer et al. 2000). A novel behaviour of system y⁺L in these cells is the finding that arginine is a better inhibitor of arginine flux than of lysine flux. Lysine on the other hand appears to have the same effect on both fluxes. This may be explained if there are different transport activities for each amino acid.

We are grateful to the Wellcome Trust for financial support.