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31P NMR analysis of intracellular pH of Swiss mouse 3T3 cells: Effects of extracellular Na+ and K+ and mitogenic stimulation

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Summary

Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional31P and19F probes of intracellular pH (pH c ) were found to be impracticable. Cells were therefore superfused with 1 to 4mm 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na+ with choline or K+ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pH c on external Na+ concentration (c oNa ). pH c also depended on intracellular Na+ concentration (c oNa ). Increasingc cNa by withdrawing external K+ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducingc oNa produced a larger acid shift in pH c than with external K+ present. Comparison of separate preparations indicated that pH c was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pH c of Swiss mouse 3T3 cells using31P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event.

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References

  1. Ackerman, J.J.H., Gadian, D.G., Radda, G.K., Wong, G.G. 1981. Observations of1H NMR signals with receiver coils tuned for other nuclides.J. Magn. Reson. 42:498–500

    Google Scholar 

  2. Bailey, I.A., Williams, S.R., Radda, G.K., Gadian, D.G. 1981. Activity of phosphorylase in total global ischaemia in the rat heart: A phosphorus-31 nuclear-magnetic-resonance study.Biochem. J. 196:171–178

    PubMed  Google Scholar 

  3. Burns, C.P., Rozengurt, E. 1983. Serum, platelet-derived growth factor, vasopressin and phorbol esters increase intracellular pH in Swiss mouse 3T3 cells.Biochem. Biophys. Res. Commun. 116:931–938

    PubMed  Google Scholar 

  4. Burns, C.P., Rozengurt, E. 1984. Extracellular Na+ and initiation of DNA synthesis: Role of intracellular pH and K+.J. Cell. Biol. 98:1082–1089

    Article  PubMed  Google Scholar 

  5. Cassel, D., Rothenberg, P., Zhuang, Y.X., Deuel, T.F., Glaser, L. 1983. Platelet-derived growth factor stimulates Na+/H+ exchange and induces cytoplasmic alkalinization in NR6 cells.Proc. Natl. Acad. Sci. USA 80:6224–6228

    PubMed  Google Scholar 

  6. Civan, M.M., Lin, L.E., Peterson-Yantorno, K., Taylor, J., Deutsch, C. 1984. Intracellular pH of perfused single frog skin: Combined19F- and31P-NMR analysis.Am. J. Physiol. 16:C506-C510

    Google Scholar 

  7. DeLong, J., Civan, M.M. 1984. Apical sodium entry in split frog skin: Current-voltage relationship.J. Membrane Biol. 82:25–40

    Article  Google Scholar 

  8. Dicker, P., Rozengurt, E. 1980. Phorbol esters and vasopressin stimulate DNA synthesis by a common mechanism.Nature (London) 287:607–612

    Article  Google Scholar 

  9. Felber, S.M., Brand, M.D. 1983. Concanavalin A causes an increase in sodium permeability and intracellular sodium content of pig lymphocytes.Biochem. J. 210:893–897

    PubMed  Google Scholar 

  10. Gadian, D.G. 1982. Nuclear Magnetic Resonance and its Applications to Living Systems. Clarendon, Oxford

    Google Scholar 

  11. Gadian, D.G., Robinson, F.N.H. 1979. Radiofrequency losses in NMR experiments on electrically conducting samples.J. Magn. Reson. 34:449–455

    Google Scholar 

  12. Graber, M.L., DiLillo, D.C., Friedman, B.L., Pastoriza-Munoz, E. 1986. Characteristics of fluoroprobes for measuring intracellular pH.Anal. Biochem. 156:202–212

    Article  PubMed  Google Scholar 

  13. Gupta, R.K., Gupta, P. 1982. Direct observation of resolved resonances from intra- and extracellular sodium-23 ions in NMR studies of intact cells and tissues using dysprosium (ITT) tripolyphosphate as paramagnetic shift reagent.J. Magn. Reson. 47:344–350

    Google Scholar 

  14. Hoult, D.I., Lauterbur, P.C. 1979. The sensitivity of the zeugmatographic experiment involving human samples.J. Magn. Reson. 34:425–433

    Google Scholar 

  15. Hoult, D.I., Richards, R.E. 1976. The signal-to-noise ratio of the nuclear magnetic resonance equipment.J. Magn. Reson. 24:71–85

    Google Scholar 

  16. Kimhi, Y., Palfrey, C., Spector, I., Barak, Y., Littauer, U.Z. 1976. Maturation of neuroblastoma cells in the presence of dimethylsulfoxide.Proc. Natl. Acad. Sci. USA 73:462–466

    PubMed  Google Scholar 

  17. Koch, K.S., Leffert, H.L. 1979. Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation.Cell 18:155–163

    Article  Google Scholar 

  18. Lichtshtein, D., Kaback, H.R., Blume, A.J. 1979. Use of a lipophilic cation for determination of membrane potential in neuroblastoma-glioma hybrid cell suspensions.Proc. Natl. Acad. Sci. USA 76:650–654

    PubMed  Google Scholar 

  19. Lin, L.E., Shporer, M., Civan, M.M. 1985.31P-nuclear magnetic resonance analysis of perfused single frog skins.Am. J. Phsyiol. 248:C177-C180

    Google Scholar 

  20. Lopez-Rivas, A., Adelberg, E.A., Rozengurt, E. 1982. Intracellular K+ and the mitogenic response of 3T3 cells to peptide factors in serum-free medium.Proc. Natl. Acad. Sci. USA 79:6275–6279

    PubMed  Google Scholar 

  21. Mendoza, S.A., Wigglesworth, N.M., Pohjanpelto, P., Rozengurt, E. 1980. Na entry and Na−K pump activity in murine, hamster, and human cells. Effect of monensin, serum, platelet extract and viral transformation.J. Cell. Physiol. 103:17–27

    PubMed  Google Scholar 

  22. Mendoza, S.A., Wigglesworth, N.M., Rozengurt, E. 1980. Vasopressin rapidly stimulates Na+ entry and Na−K pump activity in quiescent cultures of mouse 3T3 cells.J. Cell. Physiol. 105:153–162

    Article  PubMed  Google Scholar 

  23. Mierjewski, K., Rozengurt, E. 1977. Vitamin B12 enhances the stimulation of DNA synthesis by serum in resting cultures of 3T6 cells.Exp. Cell Res. 106:394–396

    Article  PubMed  Google Scholar 

  24. Moolenaar, W.H., Boonstra, J., Saag, P.T., van der, Laat, S.W. de. 1981. Sodium/proton exchange in mouse neuroblastoma cells.J. Biol. Chem. 256:12883–12887

    Google Scholar 

  25. Moolenaar, W.H., Tertoolen, L.G.J., Laat, S.W. de. 1984. The regulation of cytoplasmic pH in human fibroblasts.J. Biol. Chem. 259:7563–7569

    PubMed  Google Scholar 

  26. Moolenaar, W.H., Yarden, Y., Laat, S.W. de, Schlessinger, J. 1982. Epidermal growth factor induces electrically silent Na+ influx in human fibroblasts.J. Biol. Chem. 257:8502–8506

    PubMed  Google Scholar 

  27. Moon, R.B., Richards, J.H. 1973. Determination of intracellular pH by31P magnetic resonance.J. Biol. Chem. 248:7276–7278

    PubMed  Google Scholar 

  28. Navon, G., Ogawa, S., Shulman, R.G., Yamane, T. 1977.31P Nuclear magnetic resonance studies of Ehrlich ascites tumor cells.Proc. Natl. Acad. Sci. USA 74:87–91

    PubMed  Google Scholar 

  29. Ogino, T., Shulman, G.I., Avison, M.J., Gullans, S.R., Hollander, J.A. den, Shulman, R.G. 1985.23Na and39K NMR studies of ion transport in human erythrocytes.Proc. Natl. Acad. Sci. USA 82:1099–1103

    PubMed  Google Scholar 

  30. Owen, N.E., Villereal, M.L. 1983. Lys-bradykinin stimulates Na+ influx and DNA synthesis in cultured human fibroblasts.Cell 32:979–985

    Article  PubMed  Google Scholar 

  31. Paris, S., Pouysségur, J. 1983. Biochemical characterization of the amiloride-sensitive Na+/H+ antiport in Chinese hamster lung fibroblasts.J. Biol. Chem. 258:3503–3508

    PubMed  Google Scholar 

  32. Paris, S., Pouysségur, J. 1984. Growth factors activate the Na+/H+ antiporter in quiescent fibroblasts by increasing its affinity for intracellular H+.J. Biol. Chem. 259:10989–10994

    PubMed  Google Scholar 

  33. Philo, R.D., Eddy, A.A. 1978. The membrane potential of mouse ascites-tumor cells studied with the fluorescent probe 3,3′-dipropyloxadicarbocyanine: Amplitude of the depolarization caused by amino acids.Biochem. J. 174:801–810

    PubMed  Google Scholar 

  34. Pike, M.M., Springer, C.S., Jr. 1982. Aqueous shift reagents for high-resolution cationic nuclear magnetic resonance.J. Magn. Reson. 46:348–353

    Google Scholar 

  35. Pouysségur, J., Sardet, C., Franchi, A., L'Allemain, G., Paris, S. 1984. A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH.Proc. Natl. Acad. Sci. USA 81:4833–4837

    PubMed  Google Scholar 

  36. Rothenberg, P., Glaser, L., Schlesinger, P., Cassel, D. 1983. Epidermal growth factor stimulates amiloride-sensitive22Na+ uptake in A431 cells: Evidence for Na+/H+ exchange.J. Biol. Chem. 258:4883–4889

    PubMed  Google Scholar 

  37. Rothenberg, P., Reuss, L., Glaser, L. 1982. Serum and epidermal growth factor transiently depolarize quiescent BSC-1 epithelial cells.Proc. Natl. Acad. Sci. USA 79:7783–7787

    PubMed  Google Scholar 

  38. Rozengurt, E. 1980. Stimulation of DNA synthesis in quiescent cultured cells: Exogenous agents, internal signals and early events.Curr. Top. Cell. Regul. 17:59–88

    PubMed  Google Scholar 

  39. Rozengurt, E. 1981. Stimulation of Na influx, Na−K pump activity and DNA synthesis in quiescent cultured cells.Adv. Enzyme Regul. 19:61–85

    Article  Google Scholar 

  40. Rozengurt, E. 1981. Na influx as a mitogenic signal for cultured fibroblastic cells.In: Cellular Responses to Molecular Modulators. L.W. Mozes, J. Schultz, W.A. Scott, and R. Werner, editors. pp. 149–168. Academic Press, New York

    Google Scholar 

  41. Rozengurt, E., Gelehrter, T.D., Legg, A., Pettican, P. 1981. Mellitin stimulates Na entry, Na−K pump activity and DNA synthesis in quiescent cultures of mouse cells.Cell 23:781–788

    Article  PubMed  Google Scholar 

  42. Rozengurt, E., Heppel, L.A. 1975. Serum rapidly stimulates ouabain-sensitive86Rb+ influx in quiescent 3T3 cells.Proc. Natl. Acad. Sci. USA 72:4492–4495

    PubMed  Google Scholar 

  43. Rozengurt, E., Mendoza, S.A. Synergistic signals in mitogenesis: Role of ion fluxes, cyclic nucleotides and protein kinase C in Swiss mouse 3T3 cells.J. Cell Sci. (in press)

  44. Rozengurt, E., Mendoza, S.A. 1986. Early stimulation of Na+/K+ pump activity and Ca2+ fluxes in fibroblast mitogenesis.Curr. Top. Membr. Transp. (in press)

  45. Schuldiner, S., Rozengurt, E. 1982. Na+/H+ antiport in Swiss 3T3 cells: Mitogenic stimulation leads to cytoplasmic alkalinizationProc. Natl. Acad. Sci. USA 79:7778–7782

    PubMed  Google Scholar 

  46. Smith, J.B., Rozengurt, E. 1978. Serum stimulates the Na+,K+ pump in quiescent fibroblasts by increasing Na+ entry.Proc. Natl. Acad. Sci. USA 75:5560–5564

    PubMed  Google Scholar 

  47. Taylor, J., Deutsch, C. 1983. Fluorinated α-methylamino acids as19F indicators of intracellular pH.Biophys. J. 43:261–267

    PubMed  Google Scholar 

  48. Todaro, G.J., Green, H. 1963. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines.J. Cell Biol. 17:299–313

    Article  PubMed  Google Scholar 

  49. Tupper, J.T., Zorgniotti, F., Mills, B. 1977. Potassium transport and content during G1 and S phase following serum stimulation of 3T3 cells.J. Cell. Physiol. 91:429–440

    PubMed  Google Scholar 

  50. Ugurbil, K., Guernsey, D.L., Brown, T.R., Glynn, P., Tobkes, N., Edelman, I.S. 1981.31P NMR studies of intact anchorage-dependent mouse embryo fibroblasts.Proc. Natl. Acad. Sci. USA 78:4843–4847

    PubMed  Google Scholar 

  51. Villereal, M.L. 1981. Sodium fluxes in human fibroblasts: Kinetics of serum-dependent and serum-independent pathways.J. Cell. Physiol. 108:251–259

    PubMed  Google Scholar 

  52. Villereal, M.L., Cook, J.S. 1978. Regulation of active amino acid transport by growth-related changes in membrane potential in a human fibroblast.J. Biol. Chem. 253:8257–8262

    PubMed  Google Scholar 

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Authors and Affiliations

  1. Imperial Cancer Research Fund Laboratories, Lincoln's Inn Fields, WC2A, London, England

    Mortimer M. Civan & Enrique Rozengurt

  2. Royal College of Surgeons, Lincoln's Inn Fields, WC2A, London, England

    Stephen R. Williams & David G. Gadian

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  1. Mortimer M. Civan
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Civan, M.M., Williams, S.R., Gadian, D.G. et al. 31P NMR analysis of intracellular pH of Swiss mouse 3T3 cells: Effects of extracellular Na+ and K+ and mitogenic stimulation. J. Membrain Biol. 94, 55–64 (1986). https://doi.org/10.1007/BF01901013

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  • DOI: https://doi.org/10.1007/BF01901013

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