As motor neurons approach skeletal muscle during development, agrin is released and induces acetylcholine receptor (AChR) clustering. Our laboratory investigates the effect of environmental agents on skeletal muscle development by using C2C12 cell culture. For the current project, we investigated both short-term and long-term exposure to caffeine, nicotine, or both, at physiologically relevant concentrations. Short-term exposure was limited to the last 48 h of myotube formation, whereas a long-term exposure of 2 weeks allowed for several generations of myoblast proliferation followed by myotube formation. Both agrin-induced and spontaneous AChR clustering frequencies were assessed. For agrin-induced AChR clustering, agrin was added for the last 16 h of myotube formation. Caffeine, nicotine, or both significantly decreased agrin-induced AChR clustering during short-term and long-term exposure. Furthermore, caffeine, nicotine, or both significantly decreased spontaneous AChR clustering during long-term, but not short-term exposure. Surprisingly, caffeine and nicotine in combination did not decrease AChR clustering beyond the effect of either treatment alone. We conclude that physiologically relevant concentrations of caffeine or nicotine decrease AChR clustering. Moreover, we predict that fetuses exposed to caffeine or nicotine may be less likely to form appropriate neuromuscular synapses.
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Benowitz NL (1996) Pharmacology of nicotine: addiction and therapeutics. Annu Rev Pharmacol Toxicol 36:597–613
Benowitz NL, Hall SM, Modin G (1989) Persistent increase in caffeine concentrations in people who stop smoking. BMJ 298:1075–1076
Bezakova G, Ruegg MA (2003) New insights into the role of agrin. Nat Rev Mol Cell Biol 4:295–308
Birnbaum M, Reis MA, Shainberg A (1980) Role of calcium in the regulation of acetylcholine receptor synthese in cultured muscle cells. Pflugers Arch 385:37–43
Blau HM, Pavlath GK, Hardeman EC, Chiu CP, Silberstein L, Webster SG, Miller SC, Webster C (1985) Plasticity of the differentiated state. Science 230:758–766
Bloch RJ (1983) Acetylcholine receptor clustering in rat motubes: requirement for Ca2+ and effects of drugs which depolimerize myotubes. J Neurosci 3:2670–2680
Christian MS, Brent RL (2001) Teratogen update: evaluation of the reproductive and developemental risks of caffeine. Teratology 64:51–78
Cook DG, Peacock JL, Feyerabend C, Carey IM, Jarvis MJ, Anderson HR, Bland JM (1996) Relation of caffeine intake and blood caffeine concentrations during pregnancy to fetal growth: prospective population based study. BMJ 313:1358–1362
Ferayorni AJ, Gunville CF, Grow WA (2004) Nicotine decreases agrin signaling and acetylcholine receptor clustering in C2C12 myotube culture. J Neurobiol 60:51–60
Ferns M, Deiner M, Hall Z (1996) Agrin-induced acetylcholine receptor clustering in mammalian muscle requires tyrosine phosphorylation. J Cell Biol 132:937–944
Fortier I, Marcoux S, Beaulac-Baillargeon L (1993) Relation of caffeine intake during pregnancy to intrauterine growth retardation and preterm birth. Am J Epidemiol 137:931–940
Frary CD, Johnson RK, Wang MQ (2005) Food sources and intakes of caffeine in the diets of persons in the United States. J Am Diet Assoc 105:110–113
Fredholm BB, Battig K, Holmen J, Nehlig A, Zvartau EE (1999) Actions of caffeine in the brain with special reference to factors that contribute to its widespread use. Pharmacol Rev 51:83–133
Fuhrer C, Gautam M, Sugiyama JE, Hall ZW (1999) Role of rapsyn and agrin in interaction of postsynaptic proteins with acetylcholine receptors. J Neurosci 19:6405–6416
Gautam M, Noakes PG, Moscoso L, Rupp F, Scheller RH, Merlie JP, Sanes JR (1996) Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Cell 85:525–535
Glass DJ, Bowen DC, Stitt TN, Radziejewski C, Bruno J, Ryan TE, Gies DR, Shah S, Mattsson K, Burden SJ, DiStefano PS, Valenzuela DM, DeChiara TM, Yancopoulos GD (1996) Agrin acts via a MuSK receptor complex. Cell 85:513–523
Godfrey EW, Nitkin RM, Wallace BG, Rubin LL, McMahan UJ (1984) Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells. J Cell Biol 99:615–627
Grow WA, Ferns M, Gordon H (1999) Agrin-independent activation of the agrin signal transduction pathway. J Neurobiol 40:356–365
Grow WA, Gordon H (2000) Acetylcholine receptors are required for postsynaptic aggregation driven by the agrin signaling pathway. Eur J Neurosci 12:467–472
Henningfield JE, Stapleton JM, Benowitz NL, Grayson RF, London ED (1993) Higher levels of nicotine in arterial than in venous blood after cigarette smoking. Drug Alcohol Depend 33:23–29
Hinds TS, West WL, Knight EM, Harland BF (1996) The effect of caffeine on pregnancy outcome variables. Nutr Rev 54:203–207
Horta BL, Victora CG, Menezes AM, Halpern R, Barros FC (1997) Low birthweight, preterm births and intrauterine growth retardation in relation to maternal smoking. Paediatr Perinat Epidemiol 11:140–151
Jacobson C, Cote PD, Rossi SG, Rotundo RL, Carbonetto S (2001) The dystroglycan complex is necessary for stabilization of acetylcholine receptor clusters at neuromuscular junctions and formation of the synaptic basement membrane. J Cell Biol 152:435–450
Kaiser L, Allen LH (2008) Position of the American Dietetic Association: nutrition and lifestyle for a healthy pregnancy outcome. J Am Diet Assoc 108:553–561
Luo ZG, Wang Q, Zhou JZ, Wang J, Luo Z, Liu M, He X, Wynshaw-Boris A, Xiong WC, Lu B, Mei L (2002) Regulation of AChR clustering by disheveled interacting with MuSK and PAK1. Neuron 35:489–505
Martin TR, Bracken MB (1987) The association between low birth weight and caffeine consumption during pregnancy. Am J Epidemiol 126:813–821
McCusker RR, Goldberger BA, Cone EJ (2003) Caffeine content of specialty coffees. J Anal Toxicol 27:520–522
McCusker RR, Goldberger BA, Cone EJ (2006) Caffeine content of energy drinks, carbonated sodas, and other beverages. J Anal Toxicol 30:112–114
McMahan UJ (1990) The agrin hypothesis. Cold Spring Harbor Symp Quant Biol 55:407–418
Megeath LJ, Fallon JR (1998) Intracellular calcium regulates agrin-induced acetylcholine receptor clustering. J Neurosci 18:672–678
Nastuk MA, Lieth E, Ma JY, Cardasis CA, Moynihan EB, McKechnie BA, Fallon JR (1991) The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering. Neuron 7:807–818
Nitkin RM, Smith MA, Magill C, Fallon JR, Yao YM, Wallace BG, McMahan UJ (1987) Identification of agrin, a synaptic organizing protein from Torpedo electric organ. J Cell Biol 105:2471–2478
Ravdin P, Axelrod D (1977) Fluorescent tetramethyl rhodamine derivatives of alpha-bungarotoxin: preparation, separation, and characterization. Anal Biochem 80:585–592
Shainberg A, Freud-Silverberg M, Brik H (1987) Changes in the levels of acetylcholine receptors mediated by calcium concentration in the sarcoplasmic reticulum. Prog Clin Biol Res 253:303–314
Signorello LB, McLaughlin JK (2004) Maternal caffeine consumption and spontaneous abortion: a review of the epidemiologic evidence. Epidemiology 15:229–239
Soyka LF (1981) Caffeine ingestion during pregnancy: in utero exposure and possible effects. Semin Perinatol 5:305–309
St. John PA, Gordon H (2001) Agonists cause endocytosis of nicotinic acetylcholine receptors on cultured myotubes. J Neurobiol 49:212–223
Swanson JA, Lee JW, Hopp JW (1994) Caffeine and nicotine: a review of their joint use and possible interactive effects in tobacco withdrawal. Addict Behav 19:229–256
Weng X, Odouli R, Li DK (2008) Maternal caffeine consumption during pregnancy and the risk of miscarriage: a prospective cohort study. Am J Obstet Gynecol 198:279.e1–279.e8
Williams MA, Mittendorf R, Stubblefield PG, Lieberman E, Schoenbaum SC, Monson RR (1992) Cigarettes, coffee, and preterm premature rupture of the membranes. Am J Epidemiol 135:895–903
Wisborg K, Kesmodel U, Bech BH, Hedegaard M, Henriksen TB (2003) Maternal consumption of coffee during pregnancy and stillbirth and infant death in the first year of life: prospective study. BMJ 326:420–423
Yaffe D, Saxel O (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270:725–727
Zhou H, Glass DJ, Yancopoulos GD, Sanes JR (1999) Distinct domains of Musk mediate its abilities to induce and to associate with postsynaptic specializations. J Cell Biol 146:1133–1146
We thank Dr. James Stoehr and Dr. Carleton Jones for their insightful commentary in the creating and editing of this manuscript.
This work was supported in part by the Office of Research and Sponsored Programs at Midwestern University, which provided intramural funding, and by the Physician Assistant Program, which provided additional funding.
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Kordosky-Herrera, K., Grow, W.A. Caffeine and nicotine decrease acetylcholine receptor clustering in C2C12 myotube culture. Cell Tissue Res 335, 341–348 (2009). https://doi.org/10.1007/s00441-008-0691-8
- Acetylcholine receptor
- C2C12 cell culture (mouse)