The Journal of Membrane Biology

, Volume 63, Issue 1–2, pp 133–146 | Cite as

Cell junction and cyclic AMP: III. Promotion of junctional membrane permeability and junctional membrane particles in a junction-deficient cell type

  • R. Azarnia
  • G. Dahl
  • W. R. Loewenstein


The cyclic nucleotide effect on junction was studied in C1-1D cells, a mouse cancer cell type that fails to make permeable junctions in ordinary confluent culture. Upon administration of cyclic AMP, dibutyryl cyclic AMP, dibutyryl cyclic AMP plus caffeine (db-cAMP-caffeine), or cholera toxin (an adenylate cyclase activator), the cells acquired permeable junctions; they became electrically coupled and transferred fluorescent tracer molecules among each other—a transfer exhibiting the molecular size limit of permeation of normal cell-to-cell channels. The effect took several hours to develop. With the db-cAMP-caffeine treatment, junctional permeability emerged within two hours in one-fifth of the cell opopulation, and within the next few hours in the entire population. This development was not prevented by the cytokinesis inhibitor cytochalasin B. Permeable junctions formed also in two other conditions where the cell-endogenous cyclic AMP level may be expected to increase: serum starvation and low cell density. After three weeks of starving the cells of serum, a junctional permeability arose in confluent cultures, which on feeding with serum disappeared within two to three days. At low cell density, namely below confluency, the cells made permeable junctions, unstarved. In cultures of rather uniform density, the frequency of permeable junctions was inversely related to the average density, over the subconfluent range; at densities of about 1×104 cells/cm2, where the cells had few mutual contacts, 80% of the pairs presumed to be in contact were electrically coupled. In cultures with adjoining territories of high (confluent) and low cell density, there was coupling only in the last, and in this low-density state the cells were also capable of coupling with other mammalian cell types (mouse 3T3-BalbC and human Lesch-Nyhan cells).

Correlated electron microscopy of freeze-fractured cell junctions showed no membrane differentiation in confluent C1-1D cultures. The junctions acquired differentiations, namely particle clusters of gap junction and strands of tight junction, upon cyclic nucleotide application or serum starvation and in the lowdensity condition. With db-cAMP-caffeine, these differentiations appeared within 4 hr of the treatment (confluent cultures), growing in size over the next hours. Treatment with cycloheximide, but not with cytochalasin B, prevented the development of recognizable gap junction and tight junction in cultures supplied with db-cAMP-caffeine.

Key words

Intercellular communication cell junction gap junction junctional permeability cell-to-cell membrane channels promotion of cell-to-cell membrane channels membrane permeability cyclic AMP cancer cell 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Azarnia, R., Larsen, W., Loewenstein, W.R. 1974. The membrane junctions in communicating and non-communicating cells, their hybrids and segregants.Proc. Natl. Acad. Sci. USA 71:880–884PubMedGoogle Scholar
  2. Azarnia, R., Loewenstein, W. R. 1977. Intercellular communication and tissue growth. VIII. A genetic analysis of junctional communication and cancerous growth.J. Membrane Biol. 34:1–37CrossRefGoogle Scholar
  3. Brunton, L. L., Guerrant, R. L. 1974.V. cholerae andE. coli enterotoxins and cyclic AMP (cAMP) metabolism in cultured cells.Fed. Proc. 33:507Google Scholar
  4. Caspar, D. L. C., 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
  5. Chlapowski, F. J., Kelly, L. A., Butcher, R. W. 1975. Cyclic nucleotides in cultured cells.In: Advances in Cyclic Nucleotide Research. P. Greengard and G. A. Robison, editors. pp. 245–338. Raven Press, New YorkGoogle Scholar
  6. Dahl, G., Azarnia, R., Werner, R. 1981. Induction of cell-cell channel formation by mRNA.Nature (London) 289:683–685CrossRefGoogle Scholar
  7. Dubbs, D. R., Kit, S. 1964. Effect of halogenated pyrimidines and thymidine on growth of L-cells and a subline lacking thymidine kinase.Exp. Cell Res. 33:19–28PubMedGoogle Scholar
  8. Flagg-Newton, J. L., Dahl, G., Loewenstein, W. R. 1981. Cell junction and cyclic AMP. I. Upregulation of junctional membrane permeability and junctional membrane particles by administration of cyclic nucleotide or phosphodiesterase inhibitor.J. Membrane Biol. 63:105–121Google Scholar
  9. Flagg-Newton, J. L., Loewenstein, W. R. 1981. Cell junction and cyclic AMP. II. Modulations of junctional membrane permeability, dependent on serum and cell density.J. Membrane-Biol. 63:123–131Google Scholar
  10. Flagg-Newton, J. L., Simpson, I., Loewenstein, W. R. 1979. Permeability of the cell-to-cell membrane channels in mammalian cell junction.Science 205:404–407PubMedGoogle Scholar
  11. Fujimoto, W. Y., Seegmüller, J. E. 1970. Hypoxanthine-quanine phosphoribosyltransferase deficiency: Activity in normal, mutant and heterozygote-cultured human skin fibroblasts.Proc. Natl. Acad. Sci. USA 65:557–559PubMedGoogle Scholar
  12. Furshpan, E. J., Potter, D. D. 1968. Low resistance junctions between cells in embryos and tissue culture.Curr. Topics Devel. Biol. 3:95–127Google Scholar
  13. Grinnell, F., Milan, M., Srere, P. A. 1973. Cyclic AMP does not affect the rate at which cells attach to a substratum.Nature New Biol. 241:82–83PubMedGoogle Scholar
  14. Holley, R. W., Armour, R., Baldwin, J. H. 1977. Density-dependent regulation of growth of BSC-1 cells in cell culture: Control of growth by low molecular weight nutrients.Proc. Natl. Acad. Sci. USA 75:339–341Google Scholar
  15. Holley, R. W., Kiernan, J. A. 1971.In: CIBA Foundation Symposium on Growth Control in Cell Cultures. G. E. W. Wolstenholme and J. Knight, editors. pp. 3–15. J. A. Churchill, LondonGoogle Scholar
  16. Johson, G. S., Pastan, I. 1972. Cyclic AMP increases the adhesion of fibroblasts to substratum.Nature New Biol. 236:247–249Google Scholar
  17. Kram, R., Mamont, P., Tomkins, G. 1973: Pleiotypic control by adenosine 3′∶5′-cyclic monophosphate: A model for growth control in animal cells.Proc. Natl. Acad. Sci. USA 70:1432–1436PubMedGoogle Scholar
  18. Larsen, W. J., Azarnia, R., Loewenstein, W. R. 1977. Intercellular communication and tissue growth. IX. Junctional membrane structure of hybrids between communication-competent and communication-incompetent cells.J. Membrane Biol. 34:39–54CrossRefGoogle Scholar
  19. Loewenstein, W. R. 1979. Junctional intercellular communication and the control of growth.Biochim. Biophys. Acta Cancer Rev. 560:1–65CrossRefGoogle Scholar
  20. Loewenstein, W. R. 1981. Junctional intercellular communication. The cell-to-cell membrane channel.Physiol. Rev. 6/(4):Google Scholar
  21. Michalke, W., Loewenstein, W. R. 1971. Communication between cells of different types.Nature (London) 232:121–122Google Scholar
  22. O'Keefe, E., Cuatrecasas, P. 1974. Cholera toxin mimics melanocyte stimulating hormone in inducing differentiation in melanoma cells.Proc. Natl. Acad. Sci. USA 71:2500–2504PubMedGoogle Scholar
  23. Otten, J., Johnson, G. S., Pastan, I. 1971. Cyclic AMP levels in fibroblasts: Relationship to growth rate and contact inhibition of growth.Biochem. Biophys. Res. Commun. 44:1192–1198PubMedGoogle Scholar
  24. Revel, J. P., Karnovsky, M. J. 1967. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver.J. Cell Biol. 33:C7–18PubMedGoogle Scholar
  25. Rozengurt, E., Pardee, A. B. 1972. Opposite effects of dibutyryl adenosine 3′,5′-cyclic monophosphate and serum on growth of Chinese hamster cells.J. Cell. Physiol. 80:273–292CrossRefPubMedGoogle Scholar
  26. Ryan, W. L., Curtis, G. L. 1973. Chemical carcinogenesis and cyclic AMP.In: Role of Cyclic Nucleotides in Carcinogenesis. H. Gratzner and J. Schultz, editors. Academic Press, New YorkGoogle Scholar
  27. Schimmer, B. P. 1981. The adrenocortical tumor cell line Yl.In: Functionally Differentiated Cell Lines. G. H. Sato, editor. Ar-Liss, New York (in press)Google Scholar
  28. Schwartzmann, G., Wiegandt, H., Rose, B., Zimmermann, A., Ben-Haim, D., Loewenstein, W. R. 1981. The diameter of the cell-to-cell channel as probed with neutral molecules.Science 213:551–553PubMedGoogle Scholar
  29. Sheppard, J. R. 1972. Difference in the cyclic adenosine 3′-5′-monophosphate levels in normal and transformed cells.Nature New Biol. 236:14–16PubMedGoogle Scholar
  30. Simpson, I., Rose, B., Loewenstein, W. R. 1977 Size limit of molecules permeating the junctional membrane channels.Science 195:294–296PubMedGoogle Scholar
  31. Socolar, S. J., Loewenstein, W. R. 1979. Methods for studying transmission through permeable cell-to-cell junctions.In: Methods in Membrane Biology. E. Korn, editor. Vol. 10, pp. 123–179. Plenum, New YorkGoogle Scholar
  32. Staehelin, L. A. 1974. The structure and function of intercellular junctions.Int. Rev. Cytol. 39:191–283PubMedGoogle Scholar
  33. Temin, H. M. 1967. Control of factors in serum of multiplication of uninfected cells and cells infected and converted by avian sarcoma viruses.In: Growth Regulatory Substances for Animal Cells in Culture. V. Defendi, editor. Wistar Institute-Symp. Monogr.7:103–116. Wistar Inst. Press, PhiladelphiaGoogle Scholar
  34. Todaro, G. J., Lazar, G. K., Green, H. 1965. The initiation of cell division in a contact-inhibited mammalian cell line.J. Cell. Comp. Physiol. 66:325–342CrossRefGoogle Scholar
  35. Vogt, M., Dulbecco, R. 1960. Virus-cell interaction with a tumorproducing virus.Proc. Natl. Acad. Sci. USA 46:365Google Scholar
  36. Willingham, M. C. 1976. Cyclic AMP and cell behaviour in cultured cells.Int. Rev. Cytol. 44:319–363PubMedGoogle Scholar
  37. Wolff, J., Temple, R., Cook, G. H. 1973. Stimulation of steroid secretion in adrenal tumor cells by choleragen.Proc. Natl. Acad. Sci. USA 70:2741–2744PubMedGoogle Scholar

Copyright information

© Springer-Verlag New York Inc. 1981

Authors and Affiliations

  • R. Azarnia
    • 1
  • G. Dahl
    • 1
  • W. R. Loewenstein
    • 1
  1. 1.Department of Physiology and BiophysicsUniversity of Miami, School of MedicineMiami

Personalised recommendations