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. 1989 May 1;108(5):1823–1832. doi: 10.1083/jcb.108.5.1823

Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization

PMCID: PMC2115558  PMID: 2715181

Abstract

Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.

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Selected References

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  1. Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
  2. Baldwin T. J., Yoshihara C. M., Blackmer K., Kintner C. R., Burden S. J. Regulation of acetylcholine receptor transcript expression during development in Xenopus laevis. J Cell Biol. 1988 Feb;106(2):469–478. doi: 10.1083/jcb.106.2.469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boulter J., Evans K., Martin G., Mason P., Stengelin S., Goldman D., Heinemann S., Patrick J. Isolation and sequence of cDNA clones coding for the precursor to the gamma subunit of mouse muscle nicotinic acetylcholine receptor. J Neurosci Res. 1986;16(1):37–49. doi: 10.1002/jnr.490160106. [DOI] [PubMed] [Google Scholar]
  4. Boulter J., Luyten W., Evans K., Mason P., Ballivet M., Goldman D., Stengelin S., Martin G., Heinemann S., Patrick J. Isolation of a clone coding for the alpha-subunit of a mouse acetylcholine receptor. J Neurosci. 1985 Sep;5(9):2545–2552. doi: 10.1523/JNEUROSCI.05-09-02545.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buc-Caron M. H., Nystrom P., Fischbach G. D. Induction of acetylcholine receptor synthesis and aggregation: partial purification of low-molecular-weight activity. Dev Biol. 1983 Feb;95(2):378–386. doi: 10.1016/0012-1606(83)90039-8. [DOI] [PubMed] [Google Scholar]
  6. Buonanno A., Merlie J. P. Transcriptional regulation of nicotinic acetylcholine receptor genes during muscle development. J Biol Chem. 1986 Sep 5;261(25):11452–11455. [PubMed] [Google Scholar]
  7. Carlin B. E., Lawrence J. C., Jr, Lindstrom J. M., Merlie J. P. Inhibition of acetylcholine receptor assembly by activity in primary cultures of embryonic rat muscle cells. J Biol Chem. 1986 Apr 15;261(11):5180–5186. [PubMed] [Google Scholar]
  8. Catterall W. A. Activation and inhibition of the action potential Na+ ionophore of cultured rat muscle cells by neurotoxins. Biochem Biophys Res Commun. 1976 Jan 12;68(1):136–142. doi: 10.1016/0006-291x(76)90020-6. [DOI] [PubMed] [Google Scholar]
  9. Connolly J. A., St John P. A., Fischbach G. D. Extracts of electric lobe and electric organ from Torpedo californica increase the total number as well as the number of aggregates of chick myotube acetylcholine receptors. J Neurosci. 1982 Sep;2(9):1207–1213. doi: 10.1523/JNEUROSCI.02-09-01207.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cox K. H., DeLeon D. V., Angerer L. M., Angerer R. C. Detection of mrnas in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol. 1984 Feb;101(2):485–502. doi: 10.1016/0012-1606(84)90162-3. [DOI] [PubMed] [Google Scholar]
  11. Evans S., Goldman D., Heinemann S., Patrick J. Muscle acetylcholine receptor biosynthesis. Regulation by transcript availability. J Biol Chem. 1987 Apr 5;262(10):4911–4916. [PMC free article] [PubMed] [Google Scholar]
  12. Fambrough D. M. Control of acetylcholine receptors in skeletal muscle. Physiol Rev. 1979 Jan;59(1):165–227. doi: 10.1152/physrev.1979.59.1.165. [DOI] [PubMed] [Google Scholar]
  13. Fontaine B., Klarsfeld A., Changeux J. P. Calcitonin gene-related peptide and muscle activity regulate acetylcholine receptor alpha-subunit mRNA levels by distinct intracellular pathways. J Cell Biol. 1987 Sep;105(3):1337–1342. doi: 10.1083/jcb.105.3.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fontaine B., Klarsfeld A., Hökfelt T., Changeux J. P. Calcitonin gene-related peptide, a peptide present in spinal cord motoneurons, increases the number of acetylcholine receptors in primary cultures of chick embryo myotubes. Neurosci Lett. 1986 Oct 30;71(1):59–65. doi: 10.1016/0304-3940(86)90257-0. [DOI] [PubMed] [Google Scholar]
  15. Fontaine B., Sassoon D., Buckingham M., Changeux J. P. Detection of the nicotinic acetylcholine receptor alpha-subunit mRNA by in situ hybridization at neuromuscular junctions of 15-day-old chick striated muscles. EMBO J. 1988 Mar;7(3):603–609. doi: 10.1002/j.1460-2075.1988.tb02853.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goldman D., Brenner H. R., Heinemann S. Acetylcholine receptor alpha-, beta-, gamma-, and delta-subunit mRNA levels are regulated by muscle activity. Neuron. 1988 Jun;1(4):329–333. doi: 10.1016/0896-6273(88)90081-5. [DOI] [PubMed] [Google Scholar]
  17. Harris D. A., Falls D. L., Dill-Devor R. M., Fischbach G. D. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor alpha subunit. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1983–1987. doi: 10.1073/pnas.85.6.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Harris D. A., Falls D. L., Fischbach G. D. Differential activation of myotube nuclei following exposure to an acetylcholine receptor-inducing factor. Nature. 1989 Jan 12;337(6203):173–176. doi: 10.1038/337173a0. [DOI] [PubMed] [Google Scholar]
  19. Hartzell H. C., Fambrough D. M. Acetycholine receptor production and incorporation into membranes of developing muscle fibers. Dev Biol. 1973 Jan;30(1):153–165. doi: 10.1016/0012-1606(73)90054-7. [DOI] [PubMed] [Google Scholar]
  20. Horovitz O., Spitsberg V., Salpeter M. M. Regulation of acetylcholine receptor synthesis at the level of translation in rat primary muscle cells. J Cell Biol. 1989 May;108(5):1817–1822. doi: 10.1083/jcb.108.5.1817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jessell T. M., Siegel R. E., Fischbach G. D. Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5397–5401. doi: 10.1073/pnas.76.10.5397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Klarsfeld A., Changeux J. P. Activity regulates the levels of acetylcholine receptor alpha-subunit mRNA in cultured chicken myotubes. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4558–4562. doi: 10.1073/pnas.82.13.4558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Klier F. G., Schubert D., Heinemann S. The ultrastructural differentiation of the clonal myogenic cell line L6 in normal and high K+ medium. Dev Biol. 1977 Jun;57(2):440–449. doi: 10.1016/0012-1606(77)90228-7. [DOI] [PubMed] [Google Scholar]
  24. Knaack D., Podleski T. R., Salpeter M. M. Ascorbic acid and acetylcholine receptor expression. Ann N Y Acad Sci. 1987;498:77–89. doi: 10.1111/j.1749-6632.1987.tb23752.x. [DOI] [PubMed] [Google Scholar]
  25. Knaack D., Podleski T. Ascorbic acid mediates acetylcholine receptor increase induced by brain extract on myogenic cells. Proc Natl Acad Sci U S A. 1985 Jan;82(2):575–579. doi: 10.1073/pnas.82.2.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Knaack D., Shen I., Salpeter M. M., Podleski T. R. Selective effects of ascorbic acid on acetylcholine receptor number and distribution. J Cell Biol. 1986 Mar;102(3):795–802. doi: 10.1083/jcb.102.3.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. LaPolla R. J., Mayne K. M., Davidson N. Isolation and characterization of a cDNA clone for the complete protein coding region of the delta subunit of the mouse acetylcholine receptor. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7970–7974. doi: 10.1073/pnas.81.24.7970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Land B. R., Podleski T. R., Salpeter E. E., Salpeter M. M. Acetylcholine receptor distribution on myotubes in culture correlated to acetylcholine sensitivity. J Physiol. 1977 Jul;269(1):155–176. doi: 10.1113/jphysiol.1977.sp011897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mason R. T., Peterfreund R. A., Sawchenko P. E., Corrigan A. Z., Rivier J. E., Vale W. W. Release of the predicted calcitonin gene-related peptide from cultured rat trigeminal ganglion cells. Nature. 1984 Apr 12;308(5960):653–655. doi: 10.1038/308653a0. [DOI] [PubMed] [Google Scholar]
  30. Merlie J. P., Sanes J. R. Concentration of acetylcholine receptor mRNA in synaptic regions of adult muscle fibres. Nature. 1985 Sep 5;317(6032):66–68. doi: 10.1038/317066a0. [DOI] [PubMed] [Google Scholar]
  31. Merlie J. P., Sebbane R., Gardner S., Lindstrom J. cDNA clone for the alpha subunit of the acetylcholine receptor from the mouse muscle cell line BC3H-1. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3845–3849. doi: 10.1073/pnas.80.12.3845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moss S. J., Beeson D. M., Jackson J. F., Darlison M. G., Barnard E. A. Differential expression of nicotinic acetylcholine receptor genes in innervated and denervated chicken muscle. EMBO J. 1987 Dec 20;6(13):3917–3921. doi: 10.1002/j.1460-2075.1987.tb02732.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Neugebauer K., Salpeter M. M., Podleski T. R. Differential responses of L5 and rat primary muscle cells to factors in rat brain extract. Brain Res. 1985 Oct 28;346(1):58–69. doi: 10.1016/0006-8993(85)91094-7. [DOI] [PubMed] [Google Scholar]
  34. New H. V., Mudge A. W. Calcitonin gene-related peptide regulates muscle acetylcholine receptor synthesis. 1986 Oct 30-Nov 5Nature. 323(6091):809–811. doi: 10.1038/323809a0. [DOI] [PubMed] [Google Scholar]
  35. Patrick J., Boulter J., Goldman D., Gardner P., Heinemann S. Molecular biology of nicotinic acetylcholine receptors. Ann N Y Acad Sci. 1987;505:194–207. doi: 10.1111/j.1749-6632.1987.tb51292.x. [DOI] [PubMed] [Google Scholar]
  36. Podleski T. R., Axelrod D., Ravdin P., Greenberg I., Johnson M. M., Salpeter M. M. Nerve extract induces increase and redistribution of acetylcholine receptors on cloned muscle cells. Proc Natl Acad Sci U S A. 1978 Apr;75(4):2035–2039. doi: 10.1073/pnas.75.4.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Podleski T. R., Nichols S., Ravdin P., Salpeter M. M. Cloned myogenic cells during differentiation: membrane biochemistry and fine structural observations. Dev Biol. 1979 Jan;68(1):239–258. doi: 10.1016/0012-1606(79)90256-2. [DOI] [PubMed] [Google Scholar]
  38. Rosenfeld M. G., Mermod J. J., Amara S. G., Swanson L. W., Sawchenko P. E., Rivier J., Vale W. W., Evans R. M. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature. 1983 Jul 14;304(5922):129–135. doi: 10.1038/304129a0. [DOI] [PubMed] [Google Scholar]
  39. Salpeter M. M., Loring R. H. Nicotinic acetylcholine receptors in vertebrate muscle: properties, distribution and neural control. Prog Neurobiol. 1985;25(4):297–325. doi: 10.1016/0301-0082(85)90018-8. [DOI] [PubMed] [Google Scholar]
  40. Salpeter M. M., Marchaterre M., Harris R. Distribution of extrajunctional acetylcholine receptors on a vertebrate muscle: evaluated by using a scanning electron microscope autoradiographic procedure. J Cell Biol. 1988 Jun;106(6):2087–2093. doi: 10.1083/jcb.106.6.2087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Salpeter M. M., Spanton S., Holley K., Podleski T. R. Brain extract causes acetylcholine receptor redistribution which mimics some early events at developing neuromuscular junctions. J Cell Biol. 1982 May;93(2):417–425. doi: 10.1083/jcb.93.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Takami K., Kawai Y., Shiosaka S., Lee Y., Girgis S., Hillyard C. J., MacIntyre I., Emson P. C., Tohyama M. Immunohistochemical evidence for the coexistence of calcitonin gene-related peptide- and choline acetyltransferase-like immunoreactivity in neurons of the rat hypoglossal, facial and ambiguus nuclei. Brain Res. 1985 Mar 4;328(2):386–389. doi: 10.1016/0006-8993(85)91055-8. [DOI] [PubMed] [Google Scholar]
  43. Takami K., Kawai Y., Uchida S., Tohyama M., Shiotani Y., Yoshida H., Emson P. C., Girgis S., Hillyard C. J., MacIntyre I. Effect of calcitonin gene-related peptide on contraction of striated muscle in the mouse. Neurosci Lett. 1985 Sep 30;60(2):227–230. doi: 10.1016/0304-3940(85)90248-4. [DOI] [PubMed] [Google Scholar]
  44. Usdin T. B., Fischbach G. D. Purification and characterization of a polypeptide from chick brain that promotes the accumulation of acetylcholine receptors in chick myotubes. J Cell Biol. 1986 Aug;103(2):493–507. doi: 10.1083/jcb.103.2.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Witzemann V., Barg B., Nishikawa Y., Sakmann B., Numa S. Differential regulation of muscle acetylcholine receptor gamma- and epsilon-subunit mRNAs. FEBS Lett. 1987 Oct 19;223(1):104–112. doi: 10.1016/0014-5793(87)80518-5. [DOI] [PubMed] [Google Scholar]
  46. YAFFE D., FELDMAN M. THE EFFECT OF ACTINOMYCIN D ON HEART AND THIGH MUSCLE CELLS GROWN IN VITRO. Dev Biol. 1964 Jun;9:347–366. doi: 10.1016/0012-1606(64)90030-2. [DOI] [PubMed] [Google Scholar]
  47. Yaffe D. Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc Natl Acad Sci U S A. 1968 Oct;61(2):477–483. doi: 10.1073/pnas.61.2.477. [DOI] [PMC free article] [PubMed] [Google Scholar]

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