The regulation of L-proline transport by insulin-like growth factor-I in human osteoblast-like SaOS-2 cells.
The effect of insulin-like growth factor-I on amino acid transport was studied by measuring the uptake of tritiated L-proline in the cultured human osteoblast-like SaOS-2 cells. The uptake of L-proline was supported by both transport system A, ASC and Gly and by Na+-dependent amino acid transport system A, and by Na+-independent system L. The initial rate of total L-proline uptake as a function of concentration showed saturation and obeyed Michaelis-Menten kinetics with Michaelis constant (Km) and maximum velocity (Vmax) values of 1.87 mM and 8.89 nmol x (mg protein)-1 x (3 min)-1, respectively. Na+-dependent L-proline uptake was significantly stimulated by insulin-like growth factor-I in a time- and concentration-dependent manner. Kinetic analysis showed that insulin-like growth factor-I enhanced transport activity by increasing the Vmax of transport without significant changes in the affinity (Km) of the carrier for the substrate. The increase in transport activity was significantly reduced by cycloheximide. The stimulated increment above basal L-proline uptake was completely inhibited by alpha-(methylamino) isobutyric acid, suggesting that only system A was affected by insulin-like growth factor-I. Na+-dependent L-proline uptake was also stimulated by insulin-like growth factor-II and insulin-like growth factor-I analogues. The insulin-like growth factor-I-stimulated L-proline uptake was inhibited by one of its binding protein, insulin-like growth factor binding protein-4, in a concentration-dependent manner.
Kudo
Y
Y
Department of Obstetrics and Gynecology, Tokyo Women's Medical College, 8-1 Kawada, Shinjuku, Tokyo 162, Japan.
Iwashita
M
M
Iguchi
T
T
Takeda
Y
Y
D000596:Amino Acids / Q000494:pharmacology; D000598:Amino Acids, Cyclic; D001693:Biological Transport, Active / Q000187:drug effects; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000494:pharmacology
D000596:Amino Acids; D000598:Amino Acids, Cyclic; D018974:Insulin-Like Growth Factor Binding Protein 4; D011500:Protein Synthesis Inhibitors; D015091:beta-Alanine; C012924:2,2-dimethyl-beta-alanine; C006504:2-aminobicyclo(2,2,1)heptane-2-carboxylic acid; D007334:Insulin-Like Growth Factor I; D003513:Cycloheximide; D011392:Proline; D007930:Leucine