Pflügers Archiv

, Volume 422, Issue 6, pp 591–598 | Cite as

Activation of the nicotinic acetylcholine receptor mobilizes calcium from caffeine-insensitive stores in C2C12 mouse myotubes

  • F. Grassi
  • A. Giovannelli
  • S. Fucile
  • F. Eusebi
Excitable Tissues and Central Nervous Physiology


In cultured mouse C2C12 myotubes, digital Ca2+ imaging fluorescence microscopy using the acetoxymethyl ester of Fura-2, Fura-2-AM, showed that, in the absence of extracellular Ca2+, acetylcholine (ACh) and nicotine, but not muscarine, raised the intracellular concentration of Ca2+ ([Ca2+]i) by about tenfold. AChinduced Ca2+ mobilization was prevented by thapsigargin, a drug known to deplete inositol 1,4,5-trisphosphate (InsP3)-sensitive stores, and was concomitant with InsP3 accumulation. Caffeine, which releases Ca2+ from the ryanodine-sensitive stores of the sarcoplasmic reticulum, did not interfere with the ACh-induced [Ca2+]i increase. Ca2+ mobilization was also inhibited when myotubes were depolarized by high K+, or when extracellular Na+ was omitted. Nicotinic ACh receptor (nAChR) stimulation lowered intracellular pH with a time course slower than the [Ca2+]i increase. Possible mechanisms linking the current flowing through the nAChR pore to [Ca2+]i increase are discussed.

Key words

Calcium Acetylcholine Nicotine Nicotinic acetylcholine receptor C2C12 myotubes Inositol 1,4,5-trisphosphate 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Airey JA, Baring MD, Sutko JL (1991) Ryanodine receptor protein is expressed during differentiation in the muscle cell lines BC3H1 and C2C12. Dev Biol 148:365–374Google Scholar
  2. 2.
    Ambroz C, Fein HG, Smallridge RC (1990) Na+-ionophore, monensin-induced rise in cytoplasmic free calcium depends on the presence of extracellular calcium in FRTL-5 rat thyroid cells. Biochim Biophys Acta 1028:229–235Google Scholar
  3. 3.
    Asotra K, Lagos N, Vergara J (1991) Synthesis of polyphosphoinositides in transverse tubule and sarcoplasmic reticulum membranes of frog skeletal muscle. Biochim Biophys Acta 1081:229–237Google Scholar
  4. 4.
    Berridge MJ (1987) Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 56:159–193Google Scholar
  5. 5.
    Bolosover S, Silver RA (1991) Artifacts in calcium measurements: recognition and remedies. Trends Cell Biol 1:71–74Google Scholar
  6. 6.
    Bright GR, Fisher GW, Rogowska J, Taylor DL (1987) Fluorescence ratio imaging microscopy: temporal and spatial measurements of cytoplasmic pH. J Cell Biol 104:1019–1033Google Scholar
  7. 7.
    Cossu G, Eusebi F, Grassi F, Wanke E (1987) Acetylcholine receptor channels are present in undifferentiated satellite cells but not in embryonic myoblasts in culture. Dev Biol 123:43–50Google Scholar
  8. 8.
    Doroshenko P (1991) Second messengers mediating activation of chloride current by intracellular GTPγS in bovine chromaffin cells. J Physiol (Lond) 436:725–738Google Scholar
  9. 9.
    Drummond AH (1989) Inositol lipid signalling in excitable cells. In: Mitchell RH, Drummond AH, Downes PC (eds) Inositol lipids in cell signalling. Academic Press, New York, pp 305–309Google Scholar
  10. 10.
    Elliott AC, Lau KR, Brown PD (1991) The effects of Na+ replacement on intracellular pH and [Ca2+] in rabbit salivary gland acinar cells. J Physiol (Lond) 444:419–439Google Scholar
  11. 11.
    Eusebi F, Molinaro M, Zani BM (1985) Agents that activate protein kinase C reduce acetylcholine sensitivity during myogenesis. J Cell Biol 100:1339–1342Google Scholar
  12. 12.
    Eusebi F, Grassi F, Molinaro M, Zani BM (1987) Acetylcholine regulation of nicotinic receptor channels through a putative G protein in chick myotubes. J Physiol (Lond) 393:635–645Google Scholar
  13. 13.
    Eusebi F, Grassi F, Nervi C, Caporale C, Adamo S, Zani BM, Molinaro M (1987) Acetylcholine may regulate its own nicotinic receptor-channel through the C-kinase system. Proc R Soc Lond [Biol] 230:355–365Google Scholar
  14. 14.
    Giovannelli A, Grassi F, Mattel E, Mileo AM, Eusebi F (1991) Acetylcholine induces voltage-independent increase of cytosolic calcium in mouse myotubes. Proc Natl Acad Sci USA 88:10069–101073Google Scholar
  15. 15.
    Grassi F, Polenzani L, Mileo AM, Caratsch CG, Eusebi F, Miledi R (1993) 5-Hydroxytryptamine alters the function of muscular nicotinic acetylcholine receptor (in press)Google Scholar
  16. 16.
    Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450Google Scholar
  17. 17.
    Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85–100Google Scholar
  18. 18.
    Mason WT, Hoyland J, Rawlings SR, Relf GT (1990) Techniques and technology for dynamic video imaging of cellular fluorescence. Methods Neurosci 3:109–135Google Scholar
  19. 19.
    Meldolesi J, Clementi E, Fasolato C, Zacchetti D, Pozzan T (1991) Ca2+ influx following receptor activation. Trends Pharmacol Sci 12:289–292Google Scholar
  20. 20.
    Miledi R (1980) Intracellular calcium and desensitization of acetylcholine receptors. Proc R Soc Lond [Biol] 209:447–452Google Scholar
  21. 21.
    Miledi R, Parker I, Schalow G (1980) Transmitter-induced calcium entry across the post-synaptic membrane at frog endplates measured using Arsenazo III. J Physiol (Lond) 300:197–212Google Scholar
  22. 22.
    Nishizuka Y (1984) Turnover of inositol phospholipids and signal transduction. Science 225:1365–1370Google Scholar
  23. 23.
    Parker I, Miledi R (1986) Changes in intracellular calcium and in membrane currents evoked by injection of inositol trisphosphate into Xenopus oocytes. Proc R Soc Lond [Biol] 228:307–315Google Scholar
  24. 24.
    Prentki M, Deeney JT, Matschinsky FM, Suresh KJ (1986) Neomycin: a specific drug to study the inositol-phospholipid signalling system? FEBS Lett 197:285–288Google Scholar
  25. 25.
    Ríos E, Pizarro G (1991) Voltage sensor of excitation-contraction coupling in skeletal muscle. Physiol Rev 71:849–908Google Scholar
  26. 26.
    Ross A, Rapuano M, Prives J (1988) Induction of phosphorylation and cell surface redistribution of acetylcholine receptors by phorbol ester and carbamylcholine in cultured chick muscle cells. J Cell Biol 107:1139–1145Google Scholar
  27. 27.
    Simpson CMF, Batty IH, Hawthorne JN (1987) Physiological responses to receptor activation: phosphoinositide turnover. In: Turner AJ, Bachelard HS (eds) Neurochemistry: a practical approach. IRL Press, Oxford, pp 193–223Google Scholar
  28. 28.
    Sugiyama H, Ito I, Hirono C (1987) A new type of glutamate receptor linked to inositol phospholipid metabolism. Nature 325:531–533Google Scholar
  29. 29.
    Thastrup O, Cullen PJ, Drøbak, BK, Hanley MR, Dawson AP (1990) Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci USA 87:2466–2470Google Scholar
  30. 30.
    Tilly BC, Lambrechts AC, Tertoolen LGJ, Laat SW de, Moolenaar WH (1990) Regulation of phosphoinositide hydrolysis induced by histamine and guanine nucleotides in human HeLa carcinoma cells. FEBS Lett 265:80–84Google Scholar
  31. 31.
    Tsien RY (1988) Fluorescence measurements and photochemical manipulation of cytosolic free calcium. Trends Neurosci 11:419–424Google Scholar
  32. 32.
    Tsien RY (1989) Fluorescent probes of cell signalling. Annu Rev Neuroscie 12:227–253Google Scholar
  33. 33.
    Vergara J, Tsien RY, Delay M (1985) Inositol 1,4,5-trisphosphate: a possible chemical link in excitation-contraction coupling in muscle. Proc Natl Acad Sci USA 82:6352–6356Google Scholar
  34. 34.
    Volpe P, Di Virgilio F, Bruschi G, Regolisti G, Pozzan T (1989) Phosphoinositide metabolism and excitation-contraction coupling in smooth, cardiac and skeletal muscles. In: Michell RH, Drummond AH, Downes CD (eds) Inositol lipids in cell signalling. Academic Press, London, pp 377–404Google Scholar
  35. 35.
    Yaffe D, Saxel O (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270:725–727Google Scholar
  36. 36.
    Zarain-Herzberg A, MacLennan DH, Periasamy M (1990) Characterization of rabbit cardiac sarco(endo)plasmic reticulum Ca2+-ATPase gene. J Biol Chem 265:4670–4677Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • F. Grassi
    • 1
  • A. Giovannelli
    • 2
  • S. Fucile
    • 1
  • F. Eusebi
    • 1
  1. 1.Department of Experimental MedicineUniversity of RomeRomeItaly
  2. 2.Department of Experimental MedicineUniversity of L'AquilaL'AquilaItaly

Personalised recommendations