CHARACTERIZATION AND GROWTH-FACTOR STIMULATION OF L-ARGININE TRANSPORT IN A HUMAN COLON-CANCER CELL-LINE

Background: Epidermal growth factor (EGF) and transforming growth fact or alpha (TGF alpha) are potent mitogens that contribute to abnormal g rowth regulation in colon cancer. Growth factors have been shown to re gulate transmembrane nutrient uptake as an adaptive response to suppor t cellular proliferation. Methods: The transport of L-arginine by the SW480 primary human colon adenocarcinoma cell line was characterized b y assaying the uptake of [H-3]L-arginine in the presence and absence o f sodium, Kinetic studies were performed over a range of L-arginine co ncentrations to determine transport affinity (Km) and maximal transpor t velocity (Vmax). To further characterize the specific transporters, [H-3]L-arginine uptake was measured in the presence of selected amino acids, hormones, and under conditions of varying external pH. To inves tigate the effects of EGF and TGF alpha, cells were incubated with inc reasing doses of growth factors (1, 10, 50 ng/ml) and L-arginine trans port was measured at various time intervals (8, 12, 24 h). Proliferati on was assessed by the colorimetric (4,5-dimethylthiazol-2-yl)-2,5-dip henyltetrazolium bromide (MTT) assay 3 days after growth factor stimul ation, Results: The majority of carrier-mediated L-arginine transport was via a sodium-independent process (65-70%), whereas the remainder w as sodium-dependent (28-30%). Diffusion contributed a small amount to total L-arginine uptake (2%). Kinetic studies of arginine transport re vealed a single high-affinity Na+-independent transporter with a Km = 55.8 +/- 5.8 mu M and a Vmax = 710.6 +/- 87.3 pM/mg protein/30 s. Na+- independent arginine uptake was pH-insensitive and markedly inhibited by system y(+) substrates L-homoarginine, L-lysine, and L-ornithine. A single Na+-dependent transporter with a Km = 19.8 +/- 2.3 mu M and a Vmax = 159.1 +/- 8.9 pM/mg protein/30 s was identified. Na+-dependent arginine uptake was inhibited by system B-O,B-+ substrates L-lysine, L -ornithine, L-leucine, L-cysteine, and L-glutamine, but not by 2-methy laminoisobutyric acid. In addition, Na+-dependent arginine uptake was pH- and hormone-insensitive. Incubation with EGF or TGF alpha had no e ffect on Na+-independent L-arginine uptake; however, Na+-dependent upt ake was enhanced 60% by EGF (10 ng/ml, p < 0.05) and 100% by TGF alpha (10 ng/ml, p < 0.05), whereas cellular proliferation was increased 27 % by EGF (10 ng/ml, p < 0.05) and 37% by TGF alpha (10 ng/ml, p < 0.01 ). Conclusions: L-arginine transport in the SW480 colon cancer cell li ne is principally mediated by the Na+-independent system y(+) and to a lesser extent by the Na+-dependent system B-O,B-+. Furthermore, EGF a nd TGF alpha preferentially stimulate L-arginine uptake via the Na+-de pendent transporter, ostensibly to accommodate for the mitogenic stimu lus.