Characterization and growth factor stimulation of L-arginine transport in a human colon cancer cell line.
BACKGROUND
Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are potent mitogens that contribute to abnormal growth regulation in colon cancer. Growth factors have been shown to regulate transmembrane nutrient uptake as an adaptive response to support cellular proliferation.


METHODS
The transport of L-arginine by the SW480 primary human colon adenocarcinoma cell line was characterized by assaying the uptake of [3H]L-arginine in the presence and absence of sodium. Kinetic studies were performed over a range of L-arginine concentrations to determine transport affinity (Km) and maximal transport velocity (Vmax). To further characterize the specific transporters, [3H]L-arginine uptake was measured in the presence of selected amino acids, hormones, and under conditions of varying external pH. To investigate the effects of EGF and TGF alpha, cells were incubated with increasing doses of growth factors (1, 10, 50 ng/ml) and L-arginine transport was measured at various time intervals (8, 12, 24 h). Proliferation was assessed by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 3 days after growth factor stimulation.


RESULTS
The majority of carrier-mediated L-arginine transport was via a sodium-independent process (65-70%), whereas the remainder was sodium-dependent (28-30%). Diffusion contributed a small amount to total L-arginine uptake (2%). Kinetic studies of arginine transport revealed a single high-affinity Na(+)-independent transporter with a Km = 55.8 +/- 5.8 microM 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 microM and a Vmax = 159.1 +/- 8.9 pM/mg protein/30 s was identified. Na(+)-dependent arginine uptake was inhibited by system BO,+ substrates L-lysine, L-ornithine, L-leucine, L-cysteine, and L-glutamine, but not by 2-methylaminoisobutyric acid. In addition, Na(+)-dependent arginine uptake was pH- and hormone-insensitive. Incubation with EGF or TGF alpha had no effect on Na(+)-independent L-arginine uptake; however, Na(+)-dependent uptake 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 line is principally mediated by the Na(+)-independent system y+ and to a lesser extent by the Na(+)-dependent system BO,+. Furthermore, EGF and TGF alpha preferentially stimulate L-arginine uptake via the Na(+)-dependent transporter, ostensibly to accommodate for the mitogenic stimulus.
Cendan
J C
JC
Department of General Surgery, University of Florida College of Medicine, Gainesville 32610, USA.
Souba
W W
WW
Copeland
E M
EM
Lind
D S
DS
D000230:Adenocarcinoma / Q000378:metabolism*; D000596:Amino Acids / Q000494:pharmacology; D001120:Arginine / Q000493:pharmacokinetics*; D002352:Carrier Proteins; D002455:Cell Division / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D003110:Colonic Neoplasms / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects; D009569:Nitric Oxide / Q000096:biosynthesis; D011868:Radioisotopes; D013997:Time Factors; D016211:Transforming Growth Factor alpha / Q000737:chemistry / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000378:metabolism
D000596:Amino Acids; D002352:Carrier Proteins; D011868:Radioisotopes; D016211:Transforming Growth Factor alpha; D009569:Nitric Oxide; D004815:Epidermal Growth Factor; D001120:Arginine