The transport of selected neutral and cationic amino acids has been studied in Balb/c 3T3, SV3T3, and SV3T3 revertant cell lines. After properly timed preincubations to control the size of internal amino acid pools, the activity of systems A, ASC, L, and Ly+ has been discriminated by measurements of amino acid uptake (initial entry rate) in the presence and absence of sodium and of transport-specific model substrates. L-Proline, 2-aminoisobutyric acid, and glycine were primarily taken up by system A; L-alanine and L-serine by system ASC; L-phenylalanine by system L; and L-lysine by system Ly+ in SV3T3 cells. L-Proline and L-serine were also preferential substrates of systems A and ASC, respectively, in 3T3 and SV3T3 revertant cells. Transport activity of the Na+-dependent systems A and ASC decreased markedly with the increase of cell density, whereas the activity of the Na+-independent systems L and Ly+ remained substantially unchanged. The density-dependent change in activity of system A occurred through a mechanism affecting transport maximum (Vmax) rather than substrate concentration for half-maximal velocity (Km). Transport activity of systems A and ASC was several-fold higher in transformed SV3T3 cells than in 3T3 parental cells at all the culture densities that could be compared. In SV3T3 revertant cells, transport activity by these systems remained substantially similar to that observed in transformed SV3T3 cells. The results presented here add cell density as a regulatory factor of the activity of systems A and ASC, and show that this control mechanism of amino acid transport is maintained in SV40 virus-transformed 3T3 cells that have lost density-dependent inhibition of growth, as well as in SV3T3 revertant cells that have resumed it.
When chicken serum was added to serum-deprived quiescent cultures of chick embryo fibroblasts the activity of amino acid transport by means of the A system, as measured by a-aminoisobutyric acid and L-proline uptake after discrimination of the contribution of interacting systems, increased with time of exposure to serum between 30 and 120 minutes (remaining constantly high thereafter). Under the same conditions, DNA synthesis, as measured by thymidine incorporation, increased abruptly six to eight hours after the addition of serum. Serum-mediated increases of transport activity by the A system have also been detected with glycine, L-alanine and L-serine. Transport activities of systems ASC, L and Ly+ did not change appreciably (or decreased slightly) after the addition of serum. The stimulation of amino acid transport was apparently proportional to the length of exposure to serum; its rate declined progressively with time after withdrawal of serum from the culture medium. Kinetic analysis indicated that stimulation of the activity of transport system A by serum occurred through a mechanism affecting V, , , rather than K,; stimulation was prevented by inhibitors of protein synthesis. Our results indicate that the A transport system is the only system which is regulated by serum in cultured avian fibroblasts.Remarkably, the A transport system appears to be the target on which widely different factors and conditions converge to regulate amino acid transport in eukaryotic cells. Each system acts on a discrete group of amino acids with rather extensive overlap of substrate reactivity (Christensen, '69, '73). Therefore, the characterization of a specific regulatory effect on the transport process re-J. CELL. PHYSIOL., 93: 425-434.quires the formal identification of the system(s) involved (Christensen, '76).The purpose of the present study was to characterize the serum-dependent control mechanism of amino acid transport in cultured avian fibroblasts. The results to be presented indicate that changes in transport activity for amino acids occur earlier than the onset of DNA synthesis in cells committed by serum to enter into the S phase of the cell cycle. These changes consist of increases in activity of only one discrete transport system for amino acids; the A system. They require continuous exposure to serum, are likely to reflect an acceleration of the maximal transport velocity and are abolished by inhibitors of protein synthesis.
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