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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Feb;86(3):783–786. doi: 10.1073/pnas.86.3.783

Altered sensitivity of system A amino acid transport to ouabain in normal and transformed C3H-10T1/2 cells during the cell cycle.

K J Leister 1, M A Schenerman 1, E Racker 1
PMCID: PMC286561  PMID: 2536932

Abstract

Quiescent C3H-10T1/2 mouse fibroblasts that have not undergone any type of stress have a relatively low rate of 2-aminoisobutyrate (Aib) uptake by means of system A, which is primarily energized by the transmembrane Na+ chemical gradient potential. System A activity in these cells is not sensitive to ouabain or proton ionophores. In contrast, methylcholanthrene-transformed and confluent C3H-10T1/2 cells treated with 0.4 mM ouabain for 16-20 hr utilize the membrane potential generated by the Na+, K+-ATPase pump to drive Aib transport by means of system A as shown by the sensitivity of transport activity to ouabain and proton ionophores. Since glucose is present during the assay, the proton ionophores do not affect the availability of ATP, as indicated by the undiminished uptake of 86Rb+ by the Na+, K+-ATPase pump. As cells progress through the G1 phase of the cell cycle, they show an increased system A activity prior to entry into the S phase, which is also dependent on the electrogenicity of the Na+, K+-ATPase pump. There appears to be in all these cases a qualitative shift in the bioenergetic mechanism for the uptake of Aib as well as a marked quantitative increase in Aib uptake. The high activity after ouabain treatment was sustained in the transformed cells after removal of the ouabain, whereas in the confluent 10T1/2 cells the rate of uptake decayed rapidly, suggesting a difference in the mode of regulation. We conclude that transformed cells and normal cells in late G1 or under stress make use of the membrane potential generated by the Na+, K+-ATPase pump to drive amino acid uptake by means of system A.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Colombini M., Johnstone R. M. Na+-gradient-stimulated AIB transport in membrane vesicles from Ehrlich ascites cells. J Membr Biol. 1974;18(3-4):315–334. doi: 10.1007/BF01870120. [DOI] [PubMed] [Google Scholar]
  2. EAGLE H., PIEZ K. A., LEVY M. The intracellular amino acid concentrations required for protein synthesis in cultured human cells. J Biol Chem. 1961 Jul;236:2039–2042. [PubMed] [Google Scholar]
  3. Foster D. O., Pardee A. B. Transport of amino acids by confluent and nonconfluent 3T3 and polyoma virus-transformed 3T3 cells growing on glass cover slips. J Biol Chem. 1969 May 25;244(10):2675–2681. [PubMed] [Google Scholar]
  4. Graves J. S., Wheeler D. D. Increase in K+ and alpha-AIB active transport in CHO cells after low [K+] treatment. Am J Physiol. 1982 Sep;243(3):C124–C132. doi: 10.1152/ajpcell.1982.243.3.C124. [DOI] [PubMed] [Google Scholar]
  5. Guidotti G. G., Borghetti A. F., Gazzola G. C. The regulation of amino acid transport in animal cells. Biochim Biophys Acta. 1978 Dec 15;515(4):329–366. doi: 10.1016/0304-4157(78)90009-6. [DOI] [PubMed] [Google Scholar]
  6. Heinz A., Jackson J. W., Richey B. E., Sachs G., Schafer J. A. Amino Acid Transport and stimulation by substrates in the absence of a Na2+ electrochemical potential gradient. J Membr Biol. 1981;62(1-2):149–160. doi: 10.1007/BF01870207. [DOI] [PubMed] [Google Scholar]
  7. Heytler P. G. Uncouplers of oxidative phosphorylation. Methods Enzymol. 1979;55:462–442. doi: 10.1016/0076-6879(79)55060-5. [DOI] [PubMed] [Google Scholar]
  8. Isselbacher K. J. Increased uptake of amino acids and 2-deoxy-D-glucose by virus-transformed cells in culture. Proc Natl Acad Sci U S A. 1972 Mar;69(3):585–589. doi: 10.1073/pnas.69.3.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Johnson E., Eddy A. A. Effect of ouabain on amino acid uptake by mouse ascites-tumour cells in the presence of nigericin. Biochem J. 1985 Mar 15;226(3):773–779. doi: 10.1042/bj2260773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Leister K. J., Schenerman M. A., Racker E. Energetic mechanism of system A amino acid transport in normal and transformed mouse fibroblasts. J Cell Physiol. 1988 May;135(2):163–168. doi: 10.1002/jcp.1041350203. [DOI] [PubMed] [Google Scholar]
  12. Leister K. J., Wenner C. E., Tomei L. D. Correlation of ouabain-sensitive ion movements with cell-cycle activation. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1599–1603. doi: 10.1073/pnas.82.6.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lever J. E. Membrane potential and neutral amino acid transport in plasma membrane vesicles from Simian virus 40 transformed mouse fibroblasts. Biochemistry. 1977 Sep 20;16(19):4328–4334. doi: 10.1021/bi00638a031. [DOI] [PubMed] [Google Scholar]
  14. Mills B., Tupper J. T. Cell cycle dependent changes in potassium transport. J Cell Physiol. 1976 Sep;89(1):123–132. doi: 10.1002/jcp.1040890112. [DOI] [PubMed] [Google Scholar]
  15. OXENDER D. L., CHRISTENSEN H. N. DISTINCT MEDIATING SYSTEMS FOR THE TRANSPORT OF NEUTRAL AMINO ACIDS BY THE EHRLICH CELL. J Biol Chem. 1963 Nov;238:3686–3699. [PubMed] [Google Scholar]
  16. Pardee A. B. A restriction point for control of normal animal cell proliferation. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1286–1290. doi: 10.1073/pnas.71.4.1286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pietrzyk C., Geck P., Heinz E. Regulation of the electrogenic (Na+ + K+)-pump of Ehrlich cells by intracellular cation levels. Biochim Biophys Acta. 1978 Oct 19;513(1):89–98. doi: 10.1016/0005-2736(78)90114-1. [DOI] [PubMed] [Google Scholar]
  18. Sander G., Pardee A. B. Transport changes in synchronously growing CHO and L cells. J Cell Physiol. 1972 Oct;80(2):267–271. doi: 10.1002/jcp.1040800214. [DOI] [PubMed] [Google Scholar]
  19. Schenerman M. A., Leister K. J., Trachtenberg D. K., Racker E. Induction of system A amino acid transport through long-term treatment with ouabain: correlation with increased (Na+/K+)-ATPase activity. J Cell Physiol. 1988 May;135(2):157–162. doi: 10.1002/jcp.1041350202. [DOI] [PubMed] [Google Scholar]
  20. Tupper J. T., Mills B., Zorgniotti F. Membrane transport in synchronized Ehrlich ascites tumor cells: uptake of amino acids by the A and L system during the cell cycle. J Cell Physiol. 1976 May;88(1):77–87. doi: 10.1002/jcp.1040880110. [DOI] [PubMed] [Google Scholar]
  21. Vaughan G. L., Cook J. S. Regeneration of cation-transport capacity in HeLa cell membranes after specific blockade by ouabain. Proc Natl Acad Sci U S A. 1972 Sep;69(9):2627–2631. doi: 10.1073/pnas.69.9.2627. [DOI] [PMC free article] [PubMed] [Google Scholar]

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