Journal of Muscle Research & Cell Motility

, Volume 27, Issue 8, pp 577–584 | Cite as

Arginine supplementation induces myoblast fusion via augmentation of nitric oxide production

  • Jodi H. D. Long
  • Vitor A. Lira
  • Quinlyn A. Soltow
  • Jenna L. Betters
  • Jeff E. Sellman
  • David S. Criswell
Original Paper


The semi-essential amino acid, l-arginine (l-Arg), is the substrate for endogenous synthesis of nitric oxide, a molecule that is involved in myoblast proliferation and fusion. Since l-Arg supply may limit nitric oxide synthase (NOS) activity in endothelial cells, we examined l-Arg supplementation in differentiating mouse myoblasts and tested the hypothesis that l-Arg exerts direct effects on myoblast fusion via augmentation of endogenous nitric oxide production. C2C12 myoblasts in differentiation media received one of␣the␣following treatments for 120 h: 1 mM l-Arg, 0.1 mM N-nitro-l-arginine methyl ester (l-NAME), l-Arg  + l-NAME, 10 mM l-Lysine, or no supplement (Control). Cultures were fixed and stained with hematoxylin and eosin for microphotometric image analysis of myotube density, nuclear density, and fusion index (% of total nuclei in myotubes). Endogenous production of nitric oxide during the treatment period peaked between 24 and 48 h. l-Arg amplified nitric oxide production between 0 and 24 h and increased myotube density, total nuclei number, and nuclear fusion index. These l-Arg effects were prevented by the NOS inhibitor, l-NAME. Further, l-Lysine, a competitive inhibitor of l-Arg uptake, repressed nitric oxide production and reduced myotube density and fusion index. In summary, l-Arg augments myotube formation and increases nitric oxide production in a process limited by cellular l-Arg uptake.


C2C12 myotubes L-NAME Skeletal muscle Differentiation 


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Funded by the University of Florida Research Opportunity Fund (DSC).


  1. Adriao M, Chrisman CJ, Bielavsky M, Olinto SC, Shiraishi EM, Nunes MT (2004) Arginine increases growth hormone gene expression in rat pituitary and GH3 cells. Neuroendocrinology 79(1):26–33PubMedCrossRefGoogle Scholar
  2. Anderson JE (2000) A role for nitric oxide in muscle repair: nitric oxide-mediated activation of muscle satellite cells. Mol Biol Cell 11:1859–1874PubMedGoogle Scholar
  3. Barton ER, Morris L, Kawana M, Bish LT, Toursel T (2005) Systemic administration of l-arginine benefits mdx skeletal muscle function. Muscle Nerve 32(6):751–760PubMedCrossRefGoogle Scholar
  4. Baydoun AR, Wileman SM, Wheeler-Jones CPD, Marber MS, Mann GE, Pearson JD, Closs EI (1999) Transmembrane signaling mechanisms regulating expression of cationic amino acid transporters and inducible nitric oxide synthase in rat vascular smooth muscle cells. Biochem J 344:265–272PubMedCrossRefGoogle Scholar
  5. Bode-Boger SM, Boger RH, Galland A, Frolich JC (1998) Differential inhibition of human platelet aggregation and thromboxane A2 formation by l-arginine in vivo and in vitro. Naunyn Schmiedebergs Arch Pharmacol 357(2):143–150PubMedCrossRefGoogle Scholar
  6. Bustin SA (2002) Quantification of mRNA using rea l-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39PubMedCrossRefGoogle Scholar
  7. Closs EI, Simon A, Vékony N, Rotmann A (2004) Plasma membrane transporters for arginine. J Nutr 134:2752S–2759SPubMedGoogle Scholar
  8. Chromiak JA, Antonio J (2002) Use of amino acids as growth hormone-releasing agents by athletes. Nutrition 18(7–8): 657–661PubMedCrossRefGoogle Scholar
  9. Durante W (2001) Regulation of l-arginine transport and metabolism in vascular smooth muscle cells. Cell Biochem Biophys 35(1):19–34PubMedCrossRefGoogle Scholar
  10. Evans RW, Fernstrom JD, Thompson J, Morris SM, Jr, Kuller LH (2004) Biochemical responses of healthy subjects during dietary supplementation with l-arginine. J Nutr Biochem 15(9):534–539PubMedCrossRefGoogle Scholar
  11. Gokce N (2004) l-arginine and hypertension. J Nutr 134(10suppl):2807S–2811SPubMedGoogle Scholar
  12. Herrera M, Garvin JL (2005) Recent advances in the regulation of nitric oxide in the kidney. Hypertension 45(6):1062–1067PubMedCrossRefGoogle Scholar
  13. Hyatt SL, Aulak KS, Malandro M, Kilberg MS, Hatzoglou M (1997) Adaptive regulation of the cationic amino acid transporter–1 (Cat-1) in Fao cells. J Bio Chem 272(32): 19951–19957CrossRefGoogle Scholar
  14. Ito K, Chen J, Seshan SV, Khodadadian JJ, Gallagher R, Chaar ME, Vaughan ED, Jr, Poppas DP, Felsen D (2005) Dietary arginine supplementation attenuates renal damage after relief of unilateral ureteral obstruction in rats. Kidney Int 68(2):515–528PubMedCrossRefGoogle Scholar
  15. Kakuda DK, Finley KD, Maruyama M, MacLeod CL (1998) Stress differentially induces cationic amino acid transporter gene expression. Biochem Biophys Acta 1414(1–2):75–84PubMedGoogle Scholar
  16. Koh TJ, Tidball JG (1999) Nitric oxide synthase inhibitors reduce sarcomere addition in rat skeletal muscle. J Phys 519:189–196CrossRefGoogle Scholar
  17. Lee KH, Baek MY, Moon KY, Song WK, Chung CH, Ha DB, Kang MS (1994) Nitric oxide as a messenger molecule for myoblast fusion. J Biol Chem 269(20):14371–14374PubMedGoogle Scholar
  18. Lee KH, Kim DG, Shin NY, Song WK, Kwon H, Chung CH, Kang MS (1997) NF-kappaB-dependent expression of nitric oxide synthase is required for membrane fusion of chick embryonic myoblasts. Biochem J 324(1):237–242PubMedGoogle Scholar
  19. Lin WT, Yang SC, Chen KT, Huang CC, Lee NY (2005) Protective effects of l-arginine on pulmonary oxidative stress and antioxidant defenses during exhaustive exercise in rats. Acta Pharmacol Sin 26(8):992–999PubMedCrossRefGoogle Scholar
  20. Misko TP, Schilling RJ, Salvemini D, Moore WM, Currie MG (1993) A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem 214(1):11–16PubMedCrossRefGoogle Scholar
  21. Nakai Y, Voisine P, Bianchi C, Xu SH, Feng J, Malik T, Rosinberg A, Sellke FW (2005) Effects of l-arginine on the endogenous angiogenic response in a model of hypercholesterolemia. Surgery 138(2):291–298PubMedCrossRefGoogle Scholar
  22. Pisconti A, Brunelli S, Di Padova M, De Palma C, Deponti D, Baesso S, Sartorelli V, Cossu G, Clementi E (2006) Follistatin induction by nitric oxide through cyclic GMP: a tightly regulated signaling pathway that controls myoblast fusion. J Cell Biol 172(2):233–44PubMedCrossRefGoogle Scholar
  23. Saito H, Trocki O, Wang SL, Gonce SJ, Joffe SN, Alexander JW (1987) Metabolic and immune effects of dietary arginine supplementation after burn. Arch Surg 122:784–789PubMedGoogle Scholar
  24. Schaefer A, Piquard F, Geny B, Doutreleau S, Lampert E, Mettauer B, Lonsdorfer J (2002) l-arginine reduces exercise-induced increase in plasma lactate and ammonia. Int J Sports Med 23(6):403–407PubMedCrossRefGoogle Scholar
  25. Sellman JE, Deruisseau KC, Betters JL, Lira VA, Soltow QA, Selsby JT, Criswell DS (2006) In vivo inhibition of nitric oxide synthase impairs up-regulation of contractile protein mRNA in overloaded plantaris muscle. J Appl Physiol 100(1):258–265PubMedCrossRefGoogle Scholar
  26. Shima Y, Maeda T, Aizawa S, Tsuboi I, Kobayashi D, Kato R, Tamai I (2006) l-Arginine import via cationic amino acid transporter CAT1 is essential for both differentiation and proliferation of erythrocytes. Blood 107(4):1352–1356PubMedCrossRefGoogle Scholar
  27. Smith LW, Smith JD, Criswell DS (2002) Involvement of nitric oxide synthase in skeletal muscle adaptation to chronic overload. J Appl Physiol 92(5):2005–11PubMedGoogle Scholar
  28. Stamler JS, Meissner G (2001) Physiology of nitric oxide in skeletal muscle. Physiol Rev 81(1):209–237PubMedGoogle Scholar
  29. Tangphao O, Grossmann M, Chalon S, Hoffman BB, Blaschke TF (1999) Pharmacokinetics of intravenous and oral l-arginine in normal volunteers. Br J Clin Pharmacol 47(3): 261–266PubMedCrossRefGoogle Scholar
  30. Ulibarri JA, Mozdziak PE, Schultz E, Cook C, Best TM (1999) Nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetylpencillamine, stimulate myoblast proliferation in vitro. In Vitro Cell Dev Biol Anim 35(4):215–218PubMedGoogle Scholar
  31. Valverde I, Penalva A, Ghigo E, Casanueva FF, Dieguez C (2001) Involvement of nitric oxide in the regulation of growth hormone secretion in dogs. Neuroendocrinology 74(4):213–219PubMedCrossRefGoogle Scholar
  32. Voisine P, Li J, Bianchi C, Khan TA, Ruel M, Xu SH, Feng J, Rosinberg A, Malik T, Nakai Y, Sellke FW (2005) Effects of l-arginine on fibroblast growth factor 2-induced angiogenesis in a model of endothelial dysfunction. Circulation 112(9 Suppl):I202–I207PubMedGoogle Scholar
  33. Wu G, Morris SM (1998) Arginine maetabolism: nitric oxide and beyond. Biochm J 336:1–17Google Scholar
  34. Wyatt AW, Steinert JR, Mann GE (2004) Modulation of the l-arginine/nitric oxide signaling pathway in vascular endothelial cells. Biochem Soc Symp 71:143–156PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Jodi H. D. Long
    • 1
  • Vitor A. Lira
    • 1
  • Quinlyn A. Soltow
    • 1
  • Jenna L. Betters
    • 1
  • Jeff E. Sellman
    • 1
  • David S. Criswell
    • 1
  1. 1.Center for Exercise Science, Department of Applied Physiology & KinesiologyUniversity of FloridaGainesvilleUSA

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