Skip to main content
Log in

Endplates after esterase inactivationin vivo: correlation between esterase concentration, functional response and fine structure

  • Published:
Journal of Neurocytology

Summary

Mouse sternomastoid muscles were incubated with diisopropylfluorophosphate (DFP)in vivo, and the time course of recovery was studied using histochemistry, EM autoradiography and physiology. We found that: (1) the ability of the muscle to sustain tetanus in response to nerve stimulation is eliminated when the esterases at the neuromuscular junctions are saturated with DFP. This ability is regained partially when <10% of the DFP-binding sites have recovered. (2) There is a positive correlation between the frequency of stimulation at which the tetanic response can be maintained and the extent of acetylcholinesterase (AChE) recovery. (3) Tetanic responses at fusion frequency (about 100 Hz) appear indistinguishable from controls with only about 25% of normal AChE. (4) Butyrylcholinesterase (BuChE) possibly of Schwann cell origin recovers more rapidly than does AChE. (5) The muscle shows fine structural changes involving Z band dissolution and the breakdown of sarcoplasmic reticulum within hours after esterase inactivation. (6) This myopathy reaches a peak at three days after esterase inactivation and is almost fully recovered by two weeks. (7) It can be eliminated if, at the time of esterase inactivation, the nerve is cut or the acetylcholine receptors at the endplate are inactivated by α-bungarotoxin.

We suggest that the myopathy, seen after DFP, is mediated by Ca2+ fluxes due to prolonged action of acetylcholine (ACh) in the absence of esterases.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Ahmad, K. andLewis, J. J. (1962) The influence of drugs which stimulate skeletal muscle and their antagonists on flux of calcium, potassium and sodium ions.Journal of Pharmacology and Experimental Therapeutics 136, 298–304.

    PubMed  Google Scholar 

  • Ariens, A. Th., Meeter, E., Woltheus, O. L. andVan Benthan, R. M. J. (1969) Reversible necrosis at the end-plate region in striated muscles of the rat poisoned with cholinesterase inhibitors.Experiencia 25, 57–9.

    Google Scholar 

  • Austin, L. andBerry, W. K. (1953). Two selective inhibitors of cholinesterase.Biochemical Journal 54, 695–700.

    PubMed  Google Scholar 

  • Barnard, E. A., Rymaszewska, T. andWieckowski, J. (1971) Cholinesterases at individual neuromuscular junctions. InCholinergic Ligand Interactions (edited byTriggle, D. J., Moran, J. F. andBarnard, E. A.) pp. 175–197. New York: Academic Press.

    Google Scholar 

  • Barnes, J. M. andDuff, J. I. (1953) The role of cholinesterases at the myoneural junction.British Journal of Pharmacology 8, 334–9.

    PubMed  Google Scholar 

  • Barstad, J. A. B. (1960) Cholinesterase inhibition and the effect of anticholinesterases on indirectly evoked single and tetanic muscle contractions in the phrenic nerve-diaphragm preparation from the rat.Archives Internationales de Pharmacodynamie et de Therapie 128, 143–68.

    PubMed  Google Scholar 

  • Beaty, G. N. andStefani, E. (1976) Calcium dependent electrical activity in twitch muscle fibres of the frog.Proceedings of the Royal Society of London B194, 141–50.

    Google Scholar 

  • Betz, W. andSakmann, B. (1971) ‘Disjunction’ of frog neuromuscular synapses by treatment with proteolytic enzymes.Nature New Biology 232, 94–5.

    PubMed  Google Scholar 

  • Betz, W. andSakmann, B. (1973) Effects of proteolytic enzymes on function and structure of frog neuromuscular junctions.Journal of Physiology (London)230, 673–88.

    Google Scholar 

  • Busch, W. A., Stromer, M. H., Goll, D. E. andSuzuki, A. (1972) Ca2+ — specific removal of Z lines from rabbit skeletal muscle.Journal of Cell Biology 52, 367–81.

    PubMed  Google Scholar 

  • Chang, C. C. andLee, C. Y. (1963) Isolation of neurotoxins from the venom ofBungaris multicinctus and their modes of neuromuscular blocking action.Archives Internationales de Pharmacodynamie et de Therapie 144, 241–57.

    PubMed  Google Scholar 

  • Chang, H. W. andNeumann, E. (1976) Dynamic properties of isolated acetylcholine receptor proteins: Release of calcium ions caused by acetylcholine binding.Proceedings of the National Academy of Sciences, U.S.A. 73, 3364–8.

    Google Scholar 

  • Clouet, D. H. andWaelsch, H. (1961) Amino acid and protein metabolism of the brain — VIII. The recovery of cholinesterase in the nervous system of the frog after inhibition.Journal of Neurochemistry 8, 201–15.

    PubMed  Google Scholar 

  • Cohen, J. A., Oosterbaan, R. A., Jansz, H. S. andBerendes, F. (1959) The active sites of estenses.Journal of Cellular and Comparative Physiology 54, 231–44.

    Google Scholar 

  • Couteaux, R. (1972) Structure and cytochemical characteristics of the neuromuscular junction. InInternational Encyclopedia of Pharmacology and Therapeutics (edited byChaymol, J.) pp. 7–56. New York: Pergamon Press.

    Google Scholar 

  • Csillik, B. (1965)Functional structure of the post-synaptic membrane in the myoneural junction. Budapest: Akademiai Kiado.

    Google Scholar 

  • Davison, A. N. (1953) Return of cholinesterase activity in the rat after inhibition by organophosphorus compounds 1. Diethylp-nitrophenyl phosphate (E 600, Paraoxan).Biochemical Journal 54, 583–90.

    PubMed  Google Scholar 

  • Dayton, W. R., Reville, W. J., Goll, D. E. andStromer, M. H. (1976) A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme.Biochemistry 15, 1259–2166.

    Google Scholar 

  • Eccles, J. C. andMacFarlane, W. V. (1949) Action of anticholinesterases on endplate potential of frog muscle.Journal of Neurophysiology 12, 59–80.

    Google Scholar 

  • Evans, R. H. (1974) The entry of labelled calcium into the innervated region of the mouse diaphragm muscle.Journal of Physiology (London) 240, 517–33.

    Google Scholar 

  • Feng, H., Rogers, A. W. andSalpeter, M. M. (1973) Recovery of esterases at muscle endplates inactivated by DFP.Journal of Cell Biology 59, 98a.

    Google Scholar 

  • Fenichel, G. M., Dettbarn, W. D. andNewman, T. M. (1974) An experimental myopathy secondary to excessive acetylcholine release.Neurology 24, 41–5.

    PubMed  Google Scholar 

  • Fenichel, G. M., Kibler, W. B., Olson, W. H. andDettbarn, W. D. (1972) Chronic inhibition of cholinesterase as a cause of myopathy.Neurology 22, 1026–33.

    PubMed  Google Scholar 

  • Fertuck, H. C., Woodward, W. andSalpeter, M. M. (1975)In vivo recovery of muscle contraction after α-bungarotoxin binding.Journal of Cell Biology 66, 209–13.

    PubMed  Google Scholar 

  • Fertuck, H. C. andSalpeter, M. M. (1976) Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after125I-α-bungarotoxin binding at mouse neuromuscular junctions.Journal of Cell Biology 69, 144–58.

    Google Scholar 

  • Filogamo, G. andGabella, G. (1966) Cholinesterase behaviour in the denervated and reinnervated muscles.Acta Anatomica 63, 199–214.

    PubMed  Google Scholar 

  • Fischer, G. (1968) Inhibierung und Restitution der Azetylcholinesterase an der motorischen Endplatte im Zerchfell der Ratte nach Intoxikation mit Soman.Histochemie 16, 144–9.

    PubMed  Google Scholar 

  • Gage, P. W. andMcBurney, R. N. (1975) Effects of membrane potential, temperature and neostigmine on the conductance change caused by a quantum of acetylcholine at the toad neuromuscular junction.Journal of Physiology (London) 244, 385–407.

    Google Scholar 

  • Hall, Z. andKelly, R. B. (1971) Enzymatic detachment of endplate acetylcholinesterase from muscle.Nature New Biology 232, 62–3.

    PubMed  Google Scholar 

  • Hartzell, H. C., Kuffler, S. W. andYoshikami, D. (1975) Post-synaptic potentation: Interaction between quanta of acetylcholine at the skeletal neuromuscular synapse.Journal of Physiology (London) 251, 427–63.

    Google Scholar 

  • Jenkinson, D. H. andNicholls, J. G. (1961) Contractures and permeability changes produced by acetylcholine in depolarized denervated muscle.Journal of Physiology (London) 159, 111–27.

    Google Scholar 

  • Karnovsky, M. J. andRoots, L. (1964). A ‘direct-coloring’ thiocholine method for cholinesterase.Journal of Histochemistry and Cytochemistry 12, 219–21.

    PubMed  Google Scholar 

  • Katz, B. andMiledi, R. (1973) The binding of acetylcholine to receptors and its removal from the synaptic cleft.Journal of Physiology (London) 231, 549–74.

    Google Scholar 

  • Koelle, G. B. (1962). A new general concept of the neurohumoral functions of acetylcholine and acetylcholinesterase.Journal of Pharmacy and Pharmacology 14, 65–90.

    PubMed  Google Scholar 

  • Koelle, G. B., Koelle, W. A. andSmyrl, E. G. (1977) Effects of inactivation of butyrylcholinesterase on steady state and regenerating levels of ganglionic acetylcholinesterase.Journal of Neuro chemistry 28, 313–9.

    Google Scholar 

  • Koelle, W. A., Smyrl, E. G., Ruch, G. A., Siddons, V. E. andKoelle, G. B. (1977) Effects of protection of butyrylcholinesterase on regeneration of ganglionic acetylcholinesterase.Journal of Neurochemistry 28, 307–11.

    PubMed  Google Scholar 

  • Koenig, E. (1965) Synthetic mechanisms in the axon. I: Local axonal synthesis of acetylcholinesterase.Journal of Neurochemistry 12, 343–55.

    PubMed  Google Scholar 

  • Koenig, E. andKoelle, G. B. (1961) Mode of regeneration of acetylcholinesterase in cholinergic neurons following irreversible inactivation.Journal of Neurochemistry 8, 169–88.

    PubMed  Google Scholar 

  • Koenig, J. andVigny, M. (1978) Neural induction of the 16S acetylcholinesterase in muscle cell cultures.Nature 271, 75–7.

    PubMed  Google Scholar 

  • Kuba, K. andTomita, T. (1971) Effects of prostigmine on the time course of the endplate potential in the rat diaphragm.Journal of Physiology (London) 213, 533–44.

    Google Scholar 

  • Laskowski, M. B., Olson, W. H. andDettbarn, W. D. (1975) Ultrastructural changes at the motor end-plate produced by an irreversible cholinesterase inhibitor.Experimental Neurology 47, 290–306.

    PubMed  Google Scholar 

  • Laskowski, M. B., Olson, W. H. andDettbarn, W. D. (1977) Initial ultrastructural abnormalities at the motor endplate produced by a cholinesterase inhibitor.Experimental Neurology 57, 13–33.

    PubMed  Google Scholar 

  • Lowndes, H. E., Baker, T. andRiker, W. F. (1974) Motor nerve dysfunction in delayed DFP neuropathy.European Journal of Pharmacology 29, 66–73.

    PubMed  Google Scholar 

  • Lubinska, L. (1966) Influence of denervatipn on acetylcholinesterase in developing fast and slow muscles of the rat. InExploratory Concepts in Muscular Dystrophy (edited byMilhorat, A. T.) pp. 168–175. Amsterdam: Exerpta Medica.

    Google Scholar 

  • Luft, J. H. (1961) Improvements in epoxy resin embedding methods.Journal of Biophysical and Biochemical Cytology 9, 409–14.

    PubMed  Google Scholar 

  • Martonosi, A. (1972) Biochemical and clinical aspects of sarcoplasmic reticulum function. InCurrent Topics in Membranes and Transport (edited byBronner, F. andKleinzeller, A.) pp. 83–197. New York: Academic Press.

    Google Scholar 

  • Matthews-Bellinger, J. andSalpeter, M. M. (1978) Distribution of acetylcholine receptors at frog neuromuscular junctions with a discussion of some physiological implications.Journal of Physiology (London) 279, 197–213.

    Google Scholar 

  • McMahan, U. J., Sanes, J. R. andMarshall, L. M. (1978) Cholinesterase is associated with the basal lamina at the neuromuscular junction.Nature 271, 172–4.

    PubMed  Google Scholar 

  • Miledi, R., Parker, I. andSchalow, G. (1977) Calcium entry across the post-junctional membrane during transmitter action.Journal of Physiology 268, 32-3 P.

    Google Scholar 

  • Preusser, H. J. (1967) Die Ultrastruktur der motorischen Endplatte in Zwerchfell der Ratte und Veranderungen nach Inhibierung der Acetylcholinesterase.Zeitschrift für Zellforschung und mikroskopische Anatomie 80, 436–45.

    Google Scholar 

  • Ranish, N. andOchs, S. (1972) Fast axoplasmic transport of acetylcholinesterase in mammalian nerve fibers.Journal of Neurochemistry 19, 2641–9.

    PubMed  Google Scholar 

  • Reddy, M. K., Etlinger, J. D., Fischman, D. A., Rabinowitz, M. andZak, R. (1975) Removal of Z lines and α-actinin from isolated myofibrils by a calcium activated neutral protease.Journal of Biological Chemistry 250, 4278–84.

    PubMed  Google Scholar 

  • Reville, W. J., Goll, D. E., Stromer, M. H., Robson, R. M. andDayton, W. R. (1976) A Ca2+-activated protease possibly involved in myofibrillar protein turnover: subcellular localization of the protease in porcine skeletal muscle.Journal of Cell Biology 70, 1–8.

    PubMed  Google Scholar 

  • Rogers, A. W., Darzynkiewicz, Z., Barnard, E. A. andSalpeter, M. M. (1966) Number and location of acetylcholinesterase molecules at motor endplates of the mouse.Nature (London) 210, 1003–6.

    Google Scholar 

  • Rogers, A. W., Darzynkiewicz, A., Ostrowski, K., Salpeter, M. M. andBarnard, E. A. (1969) 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.Journal of Cell Biology 41, 665–85.

    PubMed  Google Scholar 

  • Rose, S. andGlow, P. H. (1967) Denervation effects on the presumedde novo synthesis of muscle cholinesterase and the effects of acetylcholine availability on retinal cholinesterase.Experimental Neurology 18, 267–75.

    PubMed  Google Scholar 

  • Rubsamen, H., Hess, G. P., Eldefrawi, A. T. andEldefrawi, M. E. (1976) Interaction between calcium and ligand-binding sites of the purified acetylcholine receptor studies by use of a fluorescent lanthanide.Biochemical and Biophysical Research Communications 68, 56–63.

    PubMed  Google Scholar 

  • Salpeter, M. M. (1967) Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. I. The distribution of acetylcholinesterase at motor endplates of a vertebrate twitch muscle.Journal of Cell Biology 32, 379–89.

    PubMed  Google Scholar 

  • Salpeter, M. M. (1969) Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. II. The distribution of DFP-reactive sites at motor endplates of a vertebrate twitch muscle.Journal of Cell Biology 42, 122–34.

    PubMed  Google Scholar 

  • Salpeter, M. M. andBachmann, L. (1964) Autoradiography with the electron microscope.Journal of Cell Biology 22, 469–77.

    PubMed  Google Scholar 

  • Salpeter, M. M. andBachmann, L. (1972) Electron microscope autoradiography InPrinciples and Techniques of Electron Microscopy, Biological Applications (edited byHayat, M. A.) Vol. 2, pp. 221–78. New York: Van Nostrand Reinhold.

    Google Scholar 

  • Salpeter, M. M., Bachmann, L. andSalpeter, E. E. (1969). Resolution in electron microscope radioautography.Journal of Cell Biology 41, 1–20.

    Google Scholar 

  • Salpeter, M. M., Plattner, H. andRogers, A. W. (1972) Quantitative assay of esterases in endplates of mouse diaphragm by electron microscope autoradiography.Journal of Histochemistry and Cytochemistry 20, 1059–68.

    PubMed  Google Scholar 

  • Salpeter, M. M., Rogers, A. W., Kasprzak, H. andMcHenry, F. A. (1978) Acetylcholinesterase in the fast extraocular muscle of the mouse.Journal of Cell Biology 78, 274–85.

    PubMed  Google Scholar 

  • Salpeter, M. M. andSzabo, M. (1972). Sensitivity in electron microscope autoradiography. I. The effect of radiation dose.Journal of Histochemistry and Cytochemistry 20, 425–34.

    PubMed  Google Scholar 

  • Silver, A. (1974).The Biology of Cholinesterases. Amsterdam: North Holland.

    Google Scholar 

  • Tennyson, V. M., Brzin, M. andKremzner, L. T. (1973) Acetylcholinesterase activity in the myotubes and muscle satellite cell of the fetal rabbit. An electron microscopic-cytochemical and biochemical study.Journal of Histochemistry and Cytochemistry 21, 634–52.

    PubMed  Google Scholar 

  • Wecker, L. andDettbarn, W. D. (1976) Paraoxon-induced myopathy: Muscle specificity and acetylcholine involvement.Experimental Neurology 51, 281–91.

    PubMed  Google Scholar 

  • Welsch, F. andDettbarn, W. D. (1972) Inhibition of cholinesterases of rat diaphragm muscle by organophosphates and spontaneous recovery of enzyme activityin vitro.Biochemical Pharmacology 21, 1039–49.

    PubMed  Google Scholar 

  • Wilson, I. B. andFroede, H. C. (1971) The design of reactivators for irreversibly blocked acetylcholinesterase. InDrug Design (edited byAriens, E. J.) Vol. 2, pp. 213–29. New York: Academic Press.

    Google Scholar 

  • Wilson, I. B., Ginsburg, S. andQuan, C. (1958) Molecular complimentariness as basis for reactivation of alklyl phosphate inhibited enzyme.Archive of Biochemistry and Biophysics 77, 286–96.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Salpeter, M.M., Kasprzak, H., Feng, H. et al. Endplates after esterase inactivationin vivo: correlation between esterase concentration, functional response and fine structure. J Neurocytol 8, 95–115 (1979). https://doi.org/10.1007/BF01206461

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01206461

Keywords

Navigation