Inhibition of myogenesis by ouabain: Effect on protein synthesis

  • Peter G. Pauw
  • Chris R. Kaffer
  • Ryan J. Petersen
  • Sarah A. Semerad
  • Daniel C. Williams
Growth, Differentiation and Senescence

Summary

Ouabain, a specific inhibitor of the sodium- and potassium-activated adenosine triphosphatase, causes reversible inhibition of the fusion of myoblasts to form myotubes. We further examined this observation to investigate whether control of Na/K-ATPase activity may normally contribute to the regulation of myogenesis. In control cultures, fusion was preceded by a small decrease in intracellular sodium concentration, but intracellular sodium and potassium increased significantly during fusion. Levels of ouabain that produce prolonged inhibition of fusion (400 μM) virtually eliminated sodium and potassium gradients. However, lower ouabain levels (10–100 μM) also produced significant changes in intracellular potassium and/or sodium along with little apparent decrease in the eventual extent of fusion. The effect of ouabain on protein synthesis was also examined. Low levels of ouabain (<50 μM) that did not affect myogenesis also did not affect incorporation of radiolabeled amino acids, while higher concentrations produced a decline in protein synthesis that paralleled decreases in the rate of myoblast fusion. Levels of metabolic labeling were reduced 90% in cultures treated with 400 μM ouabain. Inhibition of protein synthesis would prevent membrane remodeling required for fusion and other events in myogenesis. Thus, our results do not support any specific role for the sodium- and potassium-activated adenosine triphosphatase in regulating myogenesis.

Key words

cardiac glycosides sodium gradient Na/K-pump myoblast fusion 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Antin, P. B.; Ordahl, C. P.. Isolation and characterization of an avian myogenic cell line. Dev. Biol. 143:111–121; 1991.PubMedCrossRefGoogle Scholar
  2. Antolovic, R.; Kost, H.; Mohadjerani, M.; Linder, D.; Linder, M.; Schoner, W. A specific binding protein for cardiac glycosides exists in bovine serum. J. Biol. Chem. 273:16259–16264; 1998.PubMedCrossRefGoogle Scholar
  3. Ash, J. F.; Fineman, R. M.; Kalka, T.; Morgan, M.; Wire, B.. Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection. J. Cell Biol. 99:971–983; 1984.PubMedCrossRefGoogle Scholar
  4. Baghian, A.; Kousoulas, K. G.. Role of the Na+, K+ pump in Herpes Simplex Type 1-induced cell fusion: melittin causes specific reversion of syncytial mutants with the Syn1 mutation to Syn + (wild-type) phenotype. Virology 196:548–556; 1993.PubMedCrossRefGoogle Scholar
  5. Blanco, G.; Mercer, R. W.. Isozymes of the Na−K-ATPase: heterogeneity in structure, diversity in function. Am. J. Phys. 275:F633-F650; 1998.Google Scholar
  6. Christensen, H. N.. Role of amino acid transport and countertrasport in nutrition and metabolism. Physiol. Rev. 70:43–77; 1990.PubMedGoogle Scholar
  7. DeRosier, S. M.; Mosey, T. R.; Piper, M. T.; Pauw, P. G.. Changes in ouabain affinity during development of MDCK and a ouabain-resistant mutant. J. Cell. Biol. 115:401a; 1991.Google Scholar
  8. Dornand, J.; Kaplan, J. G.. Persistent effects of ouabain treatment on human lymphocytes: synthesis of DNA, RNA and protein in stimulated and unstimulated cells. Can. J. Biochem. 54:280–286; 1976.PubMedCrossRefGoogle Scholar
  9. Elliott, M. E.; Hadjokas, N. E.; Goodfriend, T. L.. Effects of ouabain and potassium on protein synthesis and angiotensin-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. Endocrinology 118:1469–1475; 1986.PubMedCrossRefGoogle Scholar
  10. Entwistle, A.; Zalin, R. J.; Bevan, S.; Warner, A. E.. The control of chick myoblast fusion by ion channels operated by prostaglandins and acetylcholine. J. Cell Biol. 106:1693–1702; 1988.PubMedCrossRefGoogle Scholar
  11. Frantz, C. N.; Stiles, C. D.; Pledger, W. J.; Scher, C. D.. Effect of ouabain on growth regulation by serum components in Balb/c-3T3 cells: inhibition of entry into S phase by decreased protein synthesis. J. Cell. Physiol. 105:439–448; 1980.PubMedCrossRefGoogle Scholar
  12. Guidotti, G. G.; Borghetti, A. F.; Gazzola, G. C.. The regulation of amino acid transport in animal cells. Biochim. Biophys. Acta 515:329–366; 1978.PubMedGoogle Scholar
  13. Hamlyn, J. M.; Blaustein, M. P.; Bova, S.; DuCharme, D. W.; Harris, D. W.; Mandel, F.; Mathews, W. R.; Ludens, J. H.. Identification and characterization of a ouabain-like compound from human plasma. Proc. Natl. Acad. Sci. USA 88:6259–6263; 1991.PubMedCrossRefGoogle Scholar
  14. Higham, S. C.; Melikian, J.; Karin, N. J.; Ismail-Beigi, F.; Pressley, T. A. Na,K-ATPase expression in C2C12 cells during myogenesis: minimal contribution of α2 isoform to Na,K transport. J. Membrane Biol. 131:129–136; 1993.Google Scholar
  15. Ichikawa, K.; Mimura, N.; Asano, A.. Brefeldin A inhibits muscle-specific gene expression during differentiation in C2C12 myoblasts. Exp. Cell Res. 209:333–341; 1993.PubMedCrossRefGoogle Scholar
  16. Kaufman, S. J.; Foster, R. F.. Remodeling of the myoblast membrane accompanies development. Dev. Biol. 110:1–14; 1985.PubMedCrossRefGoogle Scholar
  17. Kent, C.. Inhibition of myoblast fusion by lysosomotropic amines. Dev. Biol. 90:91–98; 1982.PubMedCrossRefGoogle Scholar
  18. Laemmli, U. K.. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond), 227:680–685; 1970.CrossRefGoogle Scholar
  19. Lerner, J.. Effectors of amino acid transport processes in animal cell membranes. Comp. Biochem. Physiol. A. 81:713–739; 1985.PubMedCrossRefGoogle Scholar
  20. Linask, K. K.; Gui, Y.-H.. Inhibitory effects of ouabain on early heart development and cardiomyogenesis in the chick embryo. Dev. Dyn. 203:93–105; 1995.PubMedGoogle Scholar
  21. Mark, R. J.; Hensley, K.; Butterfield, D. A.. Amyloid beta-peptide impairs ion-motive ATPase activities: evidence for a role in loss of neuronal Ca2+ homeostasis and cell death. J. Neurosci. 15:6239–6249; 1995.PubMedGoogle Scholar
  22. Morrow, J. S.; Cianci, C. D.; Ardito, T.; Mann, A. S.; Kashgarian, M.. Ankyrin links fodrin to the alpha subunit of Na,K-ATPase in Madin-Darby canine kidney cells and in intact renal tubule cells. J. Cell Biol. 108:455–465; 1989.PubMedCrossRefGoogle Scholar
  23. Nelson, W. J.; Hammerton, R. W.. A membrane-cytoskeleton complex containing Na+, K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells; implications for the biogenesis of epithelial cell polarity. J. Cell Biol. 108:893–902; 1989.PubMedCrossRefGoogle Scholar
  24. Olej, B.; dos Santos, N. F.; Leal, L.; Rumjanek, V. M.. Ouabain induces apoptosis on PHA- activated lymphocytes. Biosci. Rep. 18:1–7; 1998.PubMedCrossRefGoogle Scholar
  25. Orlowski, J.; Lingrel, J. B.. Differential expression of the Na,K-ATPase α1 and α2 subunit genes in a murine myogenic cell line: induction of the α2 isozyme during myocyte differentiation. J. Biol. Chem. 263:17817–17821; 1988.PubMedGoogle Scholar
  26. Pauw, P. G.; Ash, J. F.. Graded amplification of the Na,K-ATPase across a subclonal series: effects on membrane physiology. J. Cell. Physiol. 130:199–206; 1987.PubMedCrossRefGoogle Scholar
  27. Pauw, P. G.; David, J. D.. Alterations in surface proteins during myogenesis of a rat myoblast cell line. Dev. Biol. 70:27–38; 1979.PubMedCrossRefGoogle Scholar
  28. Pauw, P. G.; David, J. D.. A unique subset of developmentally regulated surface proteins turns over rapidly during fusion of the L6 rat myoblast cell line. Exp. Cell Res. 186:74–82; 1990.PubMedCrossRefGoogle Scholar
  29. Pauw, P. G.; Hermann, G. J.. Ouabain is a reversible inhibitor of myogenic fusion. In Vitro Cell. Dev. Biol. 30A:9–11; 1994.Google Scholar
  30. Pauw, P. G.; Sheck, R. N.; Ash, J. F.. Steady-state physiological variations across a graded series of Na,K-ATPase-amplified cells. Mol. Cell. Biol. 9:116–123; 1989.PubMedGoogle Scholar
  31. Rodriguez, H. J.; Yates, J. T.. Studies on amino acid incorporation in isolated toad bladder epithelial cells. Seasonal changes in protein synthesis. Biochim. Biophys. Acta 596:64–80; 1980.PubMedCrossRefGoogle Scholar
  32. Sweadner, K. J.. Isozymes of the Na+/K+ ATPase. Biochim. Biophys. Acta 988:185–220; 1989.PubMedGoogle Scholar
  33. Tang, M. J.; Cheng, Y. R.; Lin, H. H.. Role of apoptosis in growth and differentiation of proximal tubule cells in primary cultures. Biochem. Biophys. Res. Commun. 218:658–664; 1996.PubMedCrossRefGoogle Scholar
  34. Vandenburgh, H. H.. Relationship of muscle growth in vitro to sodium pump activity and transmembrane potential. J. Cell. Physiol. 119:283–295; 1984.PubMedCrossRefGoogle Scholar
  35. Vigne, P.; Frelin, C.; Lazdunski, M.. Ontogeny of the (Na+, K+)-ATPase during chick skeletal myogenesis. J. Biol. Chem. 257:5380–5384; 1982.PubMedGoogle Scholar
  36. Yaffe, D.; Saxel, O.. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature (Lond) 270:725–727; 1977.CrossRefGoogle Scholar
  37. Zhao, N.; Lo, L. C.; Berova, N.; Nakanishi, K.; Tymiak, A. A.; Ludens, J. H.; Haupert, G. T., Jr. Na,K-ATPase inhibitors from bovine hypothalamus and human plasma are different from ouabain: nanogram scale CD structural analysis. Biochemistry 34:9893–9896; 1995.PubMedCrossRefGoogle Scholar

Copyright information

© Society for In Vitro Biology 2000

Authors and Affiliations

  • Peter G. Pauw
    • 1
  • Chris R. Kaffer
    • 1
  • Ryan J. Petersen
    • 1
  • Sarah A. Semerad
    • 1
  • Daniel C. Williams
    • 1
  1. 1.Gonzaga UniversitySpokane

Personalised recommendations