Abstract
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.
Similar content being viewed by others
References
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):26–33
Anderson JE (2000) A role for nitric oxide in muscle repair: nitric oxide-mediated activation of muscle satellite cells. Mol Biol Cell 11:1859–1874
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):751–760
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 344:265–272
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):143–150
Bustin SA (2002) Quantification of mRNA using rea l-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39
Closs EI, Simon A, Vékony N, Rotmann A (2004) Plasma membrane transporters for arginine. J Nutr 134:2752S–2759S
Chromiak JA, Antonio J (2002) Use of amino acids as growth hormone-releasing agents by athletes. Nutrition 18(7–8): 657–661
Durante W (2001) Regulation of l-arginine transport and metabolism in vascular smooth muscle cells. Cell Biochem Biophys 35(1):19–34
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):534–539
Gokce N (2004) l-arginine and hypertension. J Nutr 134(10suppl):2807S–2811S
Herrera M, Garvin JL (2005) Recent advances in the regulation of nitric oxide in the kidney. Hypertension 45(6):1062–1067
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): 19951–19957
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):515–528
Kakuda DK, Finley KD, Maruyama M, MacLeod CL (1998) Stress differentially induces cationic amino acid transporter gene expression. Biochem Biophys Acta 1414(1–2):75–84
Koh TJ, Tidball JG (1999) Nitric oxide synthase inhibitors reduce sarcomere addition in rat skeletal muscle. J Phys 519:189–196
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):14371–14374
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):237–242
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):992–999
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):11–16
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):291–298
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):233–44
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 122:784–789
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):403–407
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):258–265
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):1352–1356
Smith LW, Smith JD, Criswell DS (2002) Involvement of nitric oxide synthase in skeletal muscle adaptation to chronic overload. J Appl Physiol 92(5):2005–11
Stamler JS, Meissner G (2001) Physiology of nitric oxide in skeletal muscle. Physiol Rev 81(1):209–237
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): 261–266
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):215–218
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):213–219
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–I207
Wu G, Morris SM (1998) Arginine maetabolism: nitric oxide and beyond. Biochm J 336:1–17
Wyatt AW, Steinert JR, Mann GE (2004) Modulation of the l-arginine/nitric oxide signaling pathway in vascular endothelial cells. Biochem Soc Symp 71:143–156
Acknowledgments
Funded by the University of Florida Research Opportunity Fund (DSC).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Long, J.H.D., Lira, V.A., Soltow, Q.A. et al. Arginine supplementation induces myoblast fusion via augmentation of nitric oxide production. J Muscle Res Cell Motil 27, 577–584 (2006). https://doi.org/10.1007/s10974-006-9078-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10974-006-9078-1