Increased L-arginine transport in a nitric oxide-producing metastatic colon cancer cell line

Background: Little is known about amino acid transport in human neoplastic cells. We previously characterized L-arginine transport in the primary human colon cancer cell line, SW480, and found it is principally mediated by the sodium-independent system y(+). In this study, we characterized L-arginine transport in the metastatic cell line, SW620, and compared it with that in the primary cell line, SW480. Methods: Transport of H-3-L-arginine in cell monolayers was analyzed in the presence and absence of sodium. Kinetic studies were performed over a range of L-arginine concentrations to determine transporter affinity (K-m) and maximal transport velocity (V-max). Transport was further characterized through blockade with known amino acids. In addition, the effect of cell age (i.e., time in culture) on arginine transport was examined at 2 and 9 days after seeding. Cellular proliferation was asssessed by using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Results: L-Arginine uptake was primarily sodium independent in the SW620 cell line. Kinetic and amino acid-inhibition studies revealed a single high-affinity, sodium-independent L-arginine transporter (V-max = 1286.3 +/- 158.3 pmol/mg protein/30 s; K-m = 46.8 +/- 4.2 mu M). Sodium-independent transport was blocked by system y(+) substrates L-homoarginine, L-ornithine and L-lysine, Sodium-dependent uptake occurs through a single transporter with system B-O,B-+ characteristics (K-m = 16.15 +/- 2.1 mu M; V-max = 329.94 +/- 29.7 pmol/mg protein/30 s). Arginine transport increased with time in culture with day 2 cells transport velocity = 241.7 +/- 33.6 pmol/mg protein/30s, whereas day 9 cells transport velocity = 377 +/- 15.4 pmol/mg protein/30 s (p < 0.01). Cellular-proliferation studies revealed a doubling time of 3.2 days for SW620 and 5.4 days for SW480 (p < 0.05). Conclusions: L-Arginine transport in these neoplastic cell lines occurs primarily through sodium-independent, high-affinity system y(+), V-max was increased 180% in the metastatic variant (SW620), suggesting upregulation of the y(+) transporter. The increased y(+) activity may be a mechanism to provide continuous substrate for tumor growth.