The Journal of Membrane Biology

, Volume 93, Issue 1, pp 11–21 | Cite as

Quantitative gap junction alterations in mammalian heart cells quickly frozen or chemically fixed after electrical uncoupling

  • J. Délèze
  • J. C. Hervé


The gap junction morphology was quantified in freeze-fracture replicas prepared from rat auricles that had been either quickly frozen at 6 K or chemically fixed by glutaraldehyde, in a state of normal cell-to-cell conduction or in a state of electrical uncoupling. The general appearance of the gap junctions was similar after both preparative procedures. A quantitative analysis of three gap junctional dimensions provided the following measurements in the quickly frozen conducting auricles (mean±sd): (a) P-face particles' diameter 8.27±0.74 nm (n =5709), (b) P-face particles' center-to-center distance 10.78±2.12 nm (n=4800), and (c) E-face pits' distance 9.99±2.19 nm (n=1600). Corresponding values obtained from chemically fixed tissues were decreased by about 3% for the particle's diameter and about 5% for the particles' and pits' distances. Electrical uncoupling by the action of either 1 mM 2–4-dinitrophenol (DNP), or 3.5 mMn-Heptan-1-ol (heptanol), induced a decrease of the particle's diameter, which amounted to −0.69±0.01 nm (mean ±se) in the quickly frozen preparations and −0.71±0.01 nm in the chemically fixed ones. The particles' distance was decreased by −0.96±0.04 nm in the quickly frozen samples and by −0.90 ±0.03 nm in the chemically fixed ones and the E-face pits' distance was similarly reduced. All differences were statistically significant (P<0.001 for all dimensions). Electrical recoupling after the heptanol effect promoted a return of these gap junctional dimensions towards normal values, which was about 50% complete within 20 min. It is concluded that very similar morphological alterations of the gap junctional structure are induced in the mammalian heart by different treatments promoting electrical uncoupling and that these conformational changes appear independently of the preparative procedure. The suggestion that the observed decrease of the particles' diameter is genuinely related to the closing mechanism of the unit cell-to-cell channel set in thei centers is thus confirmed.

Key Words

mammalian heart cell-to-cell conduction cell-to-cell conduction block electrical uncoupling gap junction quantitative gap junction electron microscopy quick-freezing at 6K 


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  1. Abramowitz, M., Stegun, I.A. 1964. Handbook of Mathematical Functions, Applied Mathematics Series. Vol. 55 U.S. Department of Commerce, National Bureau of StandardsGoogle Scholar
  2. Baldwin, K.M. 1979. Cardiac gap junction configuration after an uncoupling treatment as a function of time.J. Cell Biol. 82:66–75PubMedGoogle Scholar
  3. Bernardini, G., Peracchia, C., Peracchia, L.L. 1982. Reversible gap junction crystallization and electrical uncoupling by heptanol.Biophys. J. 37:285aGoogle Scholar
  4. Bernardini, G., Peracchia, C., Peracchia, L.L. 1984. Reversible effects of heptanol on gap junction structure and cell-to-cell electrical coupling.Eur. J. Cell Biol. 34:307–312PubMedGoogle Scholar
  5. Brink, P., Barr, L. 1977. The resistance of the septum of the median giant axon of the earthworm.J. Gen. Physiol. 69:517–536PubMedGoogle Scholar
  6. Caspar, D.L.D., Goodenough, D.A., Makowski, L., Phillips, W.C. 1977. Gap junction structures. I. Correlated electron microscopy and X-ray diffraction.J. Cell Biol. 74:605–628PubMedGoogle Scholar
  7. Dahl, G., Isenberg, G. 1980. Decoupling of heart muscle cells: Correlation with increased cytoplasmic calcium activity and with changes of nexus ultrastructure.J. Membrane Biol. 53:63–75Google Scholar
  8. Délèze, J., Hervé, J.C. 1983. Effect of several uncouplers of cell-to-cell communication on gap junction morphology in mammalian heart.J. Membrane Biol. 74:203–215Google Scholar
  9. Délèze, J., Hervé, J.C. 1984. Effects of electrical uncoupling on the size and spacing of the gap junction particles in rat auricles examined after quick freezing at 6oK.J. Physiol. (London) 351:41PGoogle Scholar
  10. Délèze, J., Loewenstein, W.R. 1976. Permeability of a cell junction during intracellular injection of divalent cations.J. Membrane Biol. 28:71–86Google Scholar
  11. De Mello, W.C. 1979. Effect of 2–4-dinitrophenol on intracellular communication in mammalian cardiac fibres.Pfluegers Arch. 380:267–276Google Scholar
  12. Dewey, M.M., Barr, L. 1962. Intercellular connections between smooth muscle cells: The nexus.Science 137:670–672Google Scholar
  13. Escaig, J. 1982. New instruments which facilitate rapid freezing at 83 K and 6 K.J. Microsc. (Oxford) 126:221–229Google Scholar
  14. Green, C.R., Severs, N.J. 1984. Gap junction connexon configuration in rapidly frozen myocardium and isolated intercalated disks.J. Cell Biol. 99:453–463PubMedGoogle Scholar
  15. Hanna, R.B., Pappas, G.D., Bennett, M.V.L. 1984. The fine structure of identified electrotonic synapses following increased coupling resistance.Cell Tissue Res. 235:243–249PubMedGoogle Scholar
  16. Harreveld, A. van, Crowell, J. 1964. Electron microscopy after rapid freezing on a metal surface and substitution fixation.Anat. Rec. 149:381–386PubMedGoogle Scholar
  17. Heuser, J.E., Reese, T.S., Landis, D.M.D. 1976. Preservation of synaptic structure by rapid freezing.Cold Spring Harbor Symp. Quant. Biol. 40:17–24PubMedGoogle Scholar
  18. Hopwood, D. 1972. Theoretical and practical aspects of glutaraldehyde fixation.Histochem. J. 4:267–303PubMedGoogle Scholar
  19. Johnston, M.F., Simon, S.A., Ramón, F. 1980. Interaction of anaesthetics with electrical synapses.Nature (London) 286:498–500Google Scholar
  20. Jongsma, H.J., Rijn, H.E van 1972. Electrotonic spread of current in monolayer cultures of neonatal rat heart cells.J. Membrane Biol. 9:341–360Google Scholar
  21. Loewenstein, W.R. 1981. Junctional intercellular communication: The cell-to-cell membrane channel.Physiol. Rev. 61:829–913PubMedGoogle Scholar
  22. Makowski, L., Caspar, D.L.D., Phillips, W.C., Goodenough, D.A. 1977. Gap junction structures. II. Analysis of the X-ray diffraction data.J. Cell Biol. 74:629–645PubMedGoogle Scholar
  23. McNutt, N.S., Weinstein, R.S. 1970. The ultrastructure of the nexus. A correlated thin-section and freeze-cleave study.J. Cell Biol. 47:666–688PubMedGoogle Scholar
  24. Miller, T.M., Goodenough, D.A. 1985. Gap junction structures after experimental alteration of junctional channel conductance.J. Cell Biol. 101:1741–1748PubMedGoogle Scholar
  25. Noble, D. 1962. The voltage dependence of the cardiac membrane conductance.Biophys. J. 2:381–393PubMedGoogle Scholar
  26. Peracchia, C. 1977. Gap junctions: Structural changes after uncoupling procedures.J. Cell Biol. 72:628–641PubMedGoogle Scholar
  27. Peracchia, C., Dulhunty, A.F. 1976. Low resistance junctions in crayfish. Structural changes with functional uncoupling.J. Cell Biol. 70:419–439PubMedGoogle Scholar
  28. Politoff, A.L., Pappas, G.D. 1972. Mechanisms of increase in coupling resistance at electrotonic synapses of the crayfish septate axon.Anat. Rec. 172:384–385Google Scholar
  29. Politoff, A.L., Socolar, S.J., Loewenstein, W.R. 1969. Permeability of a cell membrane junction. Dependence on energy metabolism.J. Gen. Physiol. 53:498–515PubMedGoogle Scholar
  30. Raviola, E., Goodenough, D.A., Raviola, G. 1980. Structure of rapidly frozen gap junctions.J. Cell Biol. 87:273–279PubMedGoogle Scholar
  31. Rose, B., Loewenstein, W.R. 1976. Permeability of a cell junction and the local cytoplasmic free ionized calcium concentration: A study with aequorin.J. Membrane Biol. 28:87–119Google Scholar
  32. Schwabe, K.G., Terracio, L. 1980. Ultrastructural and thermocouple evaluation of rapid freezing techniques.Cryobiology 17:571–584PubMedGoogle Scholar
  33. Schwarzmann, G., Wiegandt, H., Rose, B., Zimmermann, A., Ben-Haim, D., Loewenstein, W.R. 1981. Diameter of the cell-to-cell junctional membrane channels as probed with neutral molecules.Science 213:551–553PubMedGoogle Scholar
  34. Shibata, Y., Page, E. 1981. Gap junctional structure in intact and cut sheep cardiac Purkinje fibers: A freeze-fracture study of Ca2+-induced resealing.J. Ultrastruct. Res. 75:195–204PubMedGoogle Scholar
  35. Socolar, S.J. 1977. Appendix: The coupling coefficient as an index of junctional conductance.J. Membrane Biol. 34:29–37Google Scholar
  36. Socolar, S.J., Loewenstein, W.R. 1979. Methods for studying tranmission through permeable cell-to-cell junctions.In: Methods in Membrane Biology. E. Korn, editor. Vol. 10, pp 123–179. Plenum, New YorkGoogle Scholar
  37. Tanaka, I., Sasaki, Y. 1966. On the electrotonic spread in cardiac muscle of the mouse.J. Gen. Physiol. 49:1089–1110PubMedGoogle Scholar
  38. Unwin, P.N.T., Ennis, P.D. 1983. Calcium-mediated changes in gap junction structure: Evidence from the low angle X-ray pattern.J. Cell Biol. 97:1459–1466PubMedGoogle Scholar
  39. Unwin, P.N.T., Ennis, P.D. 1984. Two configurations of a channel-forming membrane protein.Nature (London) 307:609–613CrossRefGoogle Scholar
  40. Unwin, P.N.T., Zampighi, G. 1980. Structure of the junction between communicating cells.Nature (London) 283:545–549Google Scholar
  41. Vassort, G., Whittembury, J., Mullins, L.J. 1986. Increases in internal Ca2+ and decreases in internal H+ are induced by general anesthetics in squid axons.Biophys. J. 50:11–19PubMedGoogle Scholar
  42. Weidmann, S. 1952. The electrical constants of Purkinje fibres.J. Physiol. (London) 118:348–360Google Scholar
  43. Woodbury, J.W., Crill, W.E. 1961. On the problem of impulse conduction in the atrium.In: Nervous Inhibition. E. Florey, editor. pp. 124–135. Pergamon, New YorkGoogle Scholar

Copyright information

© Springer-Verlag New York Inc. 1986

Authors and Affiliations

  • J. Délèze
    • 1
  • J. C. Hervé
    • 1
  1. 1.Physiologie Cellulaire, Unité Associée au CNRS no. 290Université de PoitiersPoitiersFrance

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