Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1978 Jul 1;78(1):274–285. doi: 10.1083/jcb.78.1.274

Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography

PMCID: PMC2110165  PMID: 670295

Abstract

The distribution of acetylcholinesterase (ACHe) in the twitch fibers of the extraocular muscles of the mouse was examined by light and electron microscope autoradiography after labeling with radioactive diisopropyl fluorophosphate (DFP) with, and without, 2-pyridine aldoxime methiodide (2-PAM) reactivation. The values obtained were compared with those previously reported for the diaphragm and sternomastoid muscles. The extraocular muscles were studied because they differ from the other two muscles in that they are among the fastest of the mammalian muscles, yet their endplates have sparse junctional folds. They could thus provide information on the extent to which ACHe concentration is an invariant feature of endplate morphology and what, if any aspects may be related to their fast speed of response. We found, using light microscope autoradiography, that in the twitch fibers of the extraocular muscle, there is n average of 6.4 +/- 2.1 X 10(7) DFP- binding sites per endplate, of which 29% (1.8 X 10(7)) are reactivated by 2-PAM and are thus AChe. The morphology of the extraocular endplates allowed us to conclude, on statistical grounds, that the AChe site are probably localized not only along the surface area of the postjunctional membrane (PJM) but also along the surface of the presynaptic axonal membrane. Based on this localization, we calculate 7,800 DFP sites and 2,500 2-PAM-reactivated sites/micron 2 of surface area of pre-and postjunctional membrane. This stacking density of DFP- binding sites per surface area of membrane ( probably in the overlying sheets of basal lamina) is very similar to that in the diaphragm and sternomastoid muscles.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bach-y-Rita P., Ito F. In vivo studies on fast and slow muscle fibers in cat extraocular muscles. J Gen Physiol. 1966 Jul;49(6):1177–1198. doi: 10.1085/jgp.0491177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Betz W., Sakmann B. "Disjunction" of frog neuromuscular synapses by treatment with proteolytic enzymes. Nat New Biol. 1971 Jul 21;232(29):94–95. doi: 10.1038/newbio232094a0. [DOI] [PubMed] [Google Scholar]
  3. Betz W., Sakmann B. Effects of proteolytic enzymes on function and structure of frog neuromuscular junctions. J Physiol. 1973 May;230(3):673–688. doi: 10.1113/jphysiol.1973.sp010211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown G. L., Harvey A. M. Neuro-muscular transmission in the extrinsic muscles of the eye. J Physiol. 1941 Mar 25;99(3):379–399. doi: 10.1113/jphysiol.1941.sp003910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. COHEN J. A., OOSTERBAAN R. A., JANSZ H. S., BERENDS F. The active site of esterases. J Cell Comp Physiol. 1959 Dec;54:231–244. doi: 10.1002/jcp.1030540419. [DOI] [PubMed] [Google Scholar]
  6. Cooper S. The isometric responses of mammalian muscles. J Physiol. 1930 Jun 27;69(4):377–385. doi: 10.1113/jphysiol.1930.sp002657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coult D. B., Marsh D. J., Read G. Dealkylation studies on inhibited acetylcholinesterase. Biochem J. 1966 Mar;98(3):869–873. doi: 10.1042/bj0980869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DAVIES D. R., GREEN A. L. The kinetics of reactivation, by oximes, of cholinesterase inhibited by organophosphorus compounds. Biochem J. 1956 Aug;63(4):529–535. doi: 10.1042/bj0630529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davis R., Koelle G. B. Electron microscopic localization of acetylcholinesterase and nonspecific cholinesterase at the neuromuscular junction by the gold-thiocholine and gold-thiolacetic acid methods. J Cell Biol. 1967 Jul;34(1):157–171. doi: 10.1083/jcb.34.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fertuck H. C., Salpeter M. M. Localization of acetylcholine receptor by 125I-labeled alpha-bungarotoxin binding at mouse motor endplates. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1376–1378. doi: 10.1073/pnas.71.4.1376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fertuck H. C., Salpeter M. M. Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-alpha-bungarotoxin binding at mouse neuromuscular junctions. J Cell Biol. 1976 Apr;69(1):144–158. doi: 10.1083/jcb.69.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gainer H., Klancher J. E. Neuromuscular junctions in a fast-contracting fish muscle. Comp Biochem Physiol. 1965 Jun;15(2):159–165. doi: 10.1016/0010-406x(65)90343-9. [DOI] [PubMed] [Google Scholar]
  13. HESS A., PILAR G. SLOW FIBRES IN THE EXTRAOCULAR MUSCLES OF THE CAT. J Physiol. 1963 Dec;169:780–798. doi: 10.1113/jphysiol.1963.sp007296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. HOLMES R., ROBINS E. L. The reversal by oximes of neuromuscular block produced by anticholinesterases. Br J Pharmacol Chemother. 1955 Dec;10(4):490–495. doi: 10.1111/j.1476-5381.1955.tb00110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hall Z. W., Kelly R. B. Enzymatic detachment of endplate acetylcholinesterase from muscle. Nat New Biol. 1971 Jul 14;232(28):62–63. doi: 10.1038/newbio232062a0. [DOI] [PubMed] [Google Scholar]
  16. JANSZ H. S., BRONS D., WARRINGA M. G. Chemical nature of the DFP-binding site of pseudocholinesterase. Biochim Biophys Acta. 1959 Aug;34:573–575. doi: 10.1016/0006-3002(59)90320-8. [DOI] [PubMed] [Google Scholar]
  17. KARNOVSKY M. J., ROOTS L. A "DIRECT-COLORING" THIOCHOLINE METHOD FOR CHOLINESTERASES. J Histochem Cytochem. 1964 Mar;12:219–221. doi: 10.1177/12.3.219. [DOI] [PubMed] [Google Scholar]
  18. Kuffler S. W., Yoshikami D. The number of transmitter molecules in a quantum: an estimate from iontophoretic application of acetylcholine at the neuromuscular synapse. J Physiol. 1975 Oct;251(2):465–482. doi: 10.1113/jphysiol.1975.sp011103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  20. McMahan U. J., Sanes J. R., Marshall L. M. Cholinesterase is associated with the basal lamina at the neuromuscular junction. Nature. 1978 Jan 12;271(5641):172–174. doi: 10.1038/271172a0. [DOI] [PubMed] [Google Scholar]
  21. Porter C. W., Barnard E. A. The density of cholinergic receptors at the endplate postsynaptic membrane: ultrastructural studies in two mammalian species. J Membr Biol. 1975;20(1-2):31–49. doi: 10.1007/BF01870626. [DOI] [PubMed] [Google Scholar]
  22. Rogers A. W., Darzynkiewicz Z., Barnard E. A., Salpeter M. M. Number and location of acetylcholinesterase molecules at motor endplates of the mouse. Nature. 1966 Jun 4;210(5040):1003–1006. doi: 10.1038/2101003a0. [DOI] [PubMed] [Google Scholar]
  23. Rogers A. W., Darzynkiewicz Z., Salpeter M. M., Ostrowski K., Barnard E. A. Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. I. The number of acetylcholinesterase molecules and of other DFP-reactive sites at motor endplates, measured by radioautography. J Cell Biol. 1969 Jun;41(3):665–685. doi: 10.1083/jcb.41.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. SALPETER M. M., BACHMANN L. AUTORADIOGRAPHY WITH THE ELECTRON MICROSCOPE. A PROCEDURE FOR IMPROVING RESOLUTION, SENSITIVITY, AND CONTRAST. J Cell Biol. 1964 Aug;22:469–477. doi: 10.1083/jcb.22.2.469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Salpeter M. M., Elderfrawi M. E. Sizes of end plate compartments, densities of acetylcholine receptor and other quantitative aspects of neuromuscular transmission. J Histochem Cytochem. 1973 Sep;21(9):769–778. doi: 10.1177/21.9.769. [DOI] [PubMed] [Google Scholar]
  26. Salpeter M. M. Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. I. The distribution of acetylcholinesterase at motor end plates of a vertebrate twitch muscle. J Cell Biol. 1967 Feb;32(2):379–389. doi: 10.1083/jcb.32.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Salpeter M. M. Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. II. The distribution of DFP-reactive sties at motor endplates of a vertebrate twitch muscle. J Cell Biol. 1969 Jul;42(1):122–134. doi: 10.1083/jcb.42.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Salpeter M. M., McHenry F. A., Feng H. H. Myoneural junctions in the extraocular muscles of the mouse. Anat Rec. 1974 Jun;179(2):201–224. doi: 10.1002/ar.1091790205. [DOI] [PubMed] [Google Scholar]
  29. Salpeter M. M., Plattner H., Rogers A. W. Quantitative assay of esterases in end plates of mouse diaphragm by electron microscope autoradiography. J Histochem Cytochem. 1972 Dec;20(12):1059–1068. doi: 10.1177/20.12.1059. [DOI] [PubMed] [Google Scholar]
  30. Salpeter M. M., Szabo M. Sensitivity in electron microscope autoradiography. I. The effect of radiation dose. J Histochem Cytochem. 1972 Jun;20(6):425–434. doi: 10.1177/20.6.425. [DOI] [PubMed] [Google Scholar]
  31. WILSON I. B., GINSBURG S., QUAN C. Molecular complementariness as basis for reactivation of alkyl phosphate-inhibited enzyme. Arch Biochem Biophys. 1958 Oct;77(2):286–296. doi: 10.1016/0003-9861(58)90077-8. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES