Summary
Cell-to-cell junctional permeability in mammalian cell cultures was probed with a series of fluorescent tracers ranging 300 to 800 in molecular weight, during treatment with metabolic inhibitors, Ca-transporting ionophore, and carbon dioxide. Treatment with the combination of cyanide and iodoacetic acid (1–2mm each), but not with either one alone, caused reversible junctional blockade to all tracer molecular species, large and small. (Electrical coupling, however, persisted in a proportion of the junctions tested.) Treatment with the ionophore A23187 (2–10 μm) or with CO2 (an atmosphere of 100% CO2 equilibrated with the medium) produced selective junctional blockade: transmission of a 688 and an 817-dalton tracer was generally blocked, while that of a 376-dalton tracer and, in certain conditions, that of a 559-dalton one, persisted. The junctional effect of the ionophore required the presence of Ca in the external medium; and effective junctional blockade by CO2 required pretreatment in medium with high Ca concentration or, interchangeably, pretreatment in medium with high CO2 concentration. In one cell type, prolonged exposure to medium with high Ca concentration alone sufficed to block transmission of the 688-dalton tracer. These effects are discussed in terms of the Ca hypothesis of junctional permeability regulation. In comparison with mammalian (or other vertebrate and invertebrate) organized tissues or with insect cell cultures, the mammalian cell cultures are more resistant to junctional blockade. This difference in transmission stability is discussed in terms of intracellular Ca-buffering capacities of the junctional locales; in particular, in terms of the electron-microscopic finding in the mammalian cultures of fine, bilateral cell processes connected by gap junctions.
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Åkerman, K.E.O. 1978. Effect of pH and Ca2+ on the retention of Ca2+ by rat liver mitochondria.Arch. Biochem. Biophys. 189:256
Åkerman, K.E.O., Saris, N.-E.L. 1978. Charge and H+/Ca2+ stoichiometries during Ca2+ influx and efflux in rat liver mitochondria.Frontiers Biol. Energetics 2:1187
Azarnia, R., Larsen, W., Loewenstein, W.R. 1974 The membrane junctions in communicating and non-communicating cells, their hybrids and segregants.Proc. Nat. Acad. Sci. 71:880
Azarnia, R., Loewenstein, W.R. 1971. Intercellular communication and tissue growth: V. A cancer cell strain that fails to make permeable membrane junctions with normal cells.J. Membrane Biol. 6:368
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
Baker, P.F. 1972. Transport and metabolism of calcium ions in nerve.Prog. Biophys. Biophys. Chem. 24:177
Baker, P.F., Hodgkin, A.L., Ridgway, E.B. 1971. Depolarization and calcium entry in squid giant axons.J. Physiol. (London) 218:709
Baker, P.F., Honerjäger, P. 1978. Influence of carbon dioxide on level of ionized calcium in squid axons.Nature (London) 273:160
Baker, P.F., Schlaepfer, W. 1975. Calcium uptake by axoplasm extruded from giant axons ofLoligo. J. Physiol. (London) 249:37P
Baux, G., Simonneau, M., Tauc, L., Segundo, J.P. 1978. Uncoupling of electrotonic synapses by calcium.Proc. Nat. Acad. Sci. USA 75:4577
Blaustein, M.P., Ratzlaff, R.W., Kendrick, N.K. 1978. The regulation of intracellular calcium in presynaptic nerve terminals.Ann. N.Y. Acad. Sci. 307:195
Boron, W.F., DeWeer, P. 1976. Intracellular pH transients in squid giant axons caused by CO2, NH3 and metabolic inhibitors.J. Gen. Physiol. 67:91
Brinley, F.J., Tiffert, T., Scarpa, A., Mullins, L. 1977. Intracellular calcium buffering capacity in isolated squid axons.J. Gen. Physiol. 70:355
Bruns, D.E., McDonald, J.M., Jarrett, L. 1976. Energy-dependent calcium transport in endoplasmic reticulum.J. Biol. Chem. 251:7191
Carvalho, A., Sanui, H., Pace, N. 1963. Calcium and magnesium binding properties of cell membrane material.J. Cell Comp. Physiol. 62:311
Chance, B. 1965. The energy linked reaction of calcium with mitochondria.J. Biol. Chem. 240:2728
Chiu, R.S., Black, L.M. 1967. Monolayer cultures of insect cell lines and their inoculation with a plant virus.Nature (London) 215:1076
Dahl, G., Schudt, C., Gratzl, M. 1978. Fusion of isolated myoblast plasma membranes. An approach to the mechanism.Biochim. Biophys. Acta 514:105
Délèze, J., Loewenstein, W.R. 1976. Permeability of a cell junction, during intracellular injection of divalent cations.J. Membrane Biol. 28:71
DeMello, W.C. 1975. Effect of intracellular injection of calcium and strontium on cell communication in heart.J. Physiol. (London) 250:231
Epstein, M.L., Gilula, N.B. 1977. A study of communication specificity between cells in culture.J. Cell Biol. 75:769
Epstein, M.L., Sheridan, J.D., Johnson, R.G. 1977. Formation of low-resistance junctions in vitro in the absence of protein synthesis and ATP production.Exp. Cell Res. 104:25
Farquhar, M.G., Palade, G.E. 1963. Junctional complexes in various epithelia.J. Cell Biol. 17:375
Fentiman, I.S., Taylor-Papadimitriou, J., Stoker, M. 1976. Selective contact-dependent cell communication.Nature (London) 264:760
Flagg-Newton, J.L., Loewenstein, W.R. 1979. Modulation of junctional mammalian permeability by cell-intrinsic factors.J. Membrane Biol. (in press)
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
Furshpan, E.J., Potter, D.D. 1968. Low resistance junctions between cells in embryos and tissue culture.Curr. Top. Dev. Biol. 3:95
Gilula, N.B., Epstein, M.J. 1976. Cell-to-cell communication gap junctions and calcium.1975 Symp. Soc. Exp. Biol. 30:257
Hales, C.N., Luzio, J.P., Chandler, J.A., Herman, L. 1974. Localization of calcium in the smooth endoplasmic reticulum of rat isolated fat cells.J. Cell Sci. 15:1
Hasselbach, W. 1974. Sarcoplasmic membrane ATPases.In: The Enzymes. Vol. 10, p. 431. Academic Press, New York
Ito, S., Sato, E., Loewenstein, W.R. 1974. Studies on the formation of a permeable cell membrane junction. I. Coupling under various conditions of membrane contact. Colchicine, cytochalasin B, dinitrophenol.J. Membrane Biol. 19:305
Jacobs, M.H. 1920. The production of intracellular acidity by neutral and alkaline solutions containing carbon dioxide.Am. J. Physiol. 53:457
Katchalsky, A. 1964.In: Connective Tissue, Intercellular Macromolecules. New York Heart Association, editors. p. 9. Little Brown, Boston
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
Lawrence, T.S., Ginzberg, R.D., Gilula, N.B., Beers, W.H. 1979. Hormonally induced shape changes in cultured rat ovaries granulosc cells.J. Cell Biol. 80:21
Lea, T.J., Ashley, C.C. 1978. Increase in free Ca2+ in muscle after exposure to CO2.Nature (London) 275:236
Lehninger, A.L. 1970. Mitochondria and calcium transport.Biochem. J. 119:129
Lehninger, A.L., Carafoli, E., Rossi, C.J. 1967. Energy-linked ion movements in mitochondrial systems.Adv. Enzymol. 29:259
Loewenstein, W.R. 1966. Permeability of membrane junctions.Ann. N.Y. Acad. Sci. 137:441
Loewenstein, W.R. 1967. On the genesis of cellular communication.Dev. Biol. 15:503
Loewenstein, W.R. 1975. Permeable junctions.Cold Spring Harbor Symp. Quant. Biol. 40:49
Loewenstein, W.R. 1979a. Junctional intercellular communication and the control of growth.Biochim. Biophys. Acta Cancer Rev. 560:1
Loewenstein, W.R. 1979b. Intercellular communication. The cell-to-cell membrane channel.Physiol. Rev. (in press)
Loewenstein, W.R., Kanno, Y., Socolar, S.J. 1978a. Quantum jumps of conductance during formation of membrane channels at cell-cell junction.Nature (London) 274:133
Loewenstein, W.R., Kanno, Y., Socolar, S.J. 1978b. The cell-to-cell channel.Fed. Proc. Symp. 37:89
Loewenstein, W.R., Nakas, M., Socolar, S.J. 1967. Junctional membrane uncoupling. Permeability transformations at a cell membrane junction.J. Gen. Physiol. 50:1865
Loewenstein, W.R., Rose, B. 1978. Calcium in (junctional) intercellular communication and a thought on its behavior in intracellular communication.Ann. N.Y. Acad. Sci. 307:285
Maroudas, N.G. 1974. Short range diffusion gradients. Appendix.Cell 3:217
Meech, R.W., Thomas, R.C. 1977. The effect of calcium injection on the intracellular sodium and pH of snail neurones.J. Physiol. (London) 265:867
Moore, L., Fitzpatrick, D.F., Chen, T.S., Landon, E.J. 1974. Calcium pump activity of the renal plasma membrane and renal microsomes.Biochim. Biophys. Acta 345:405
Moore, L., Pastan, I. 1977. Energy-dependent calcium uptake. Activity in cultured fibroblast microsomes. Regulation of the uptake system by cell density.J. Biol. Chem. 252:6304
Moore, L., Pastan, I. 1978. Energy dependent calcium uptake by fibroblast microsomes.Ann. N.Y. Acad. Sci. 307:177
Oliveira-Castro, G.M., Loewenstein, W.R. 1971. Junctional membrane permeability: Effects of divalent cations.J. Membrane Biol. 5:51
Pauli, B.V., Weinstein, R., Soble, L.W., Alroy, J. 1977. Freeze-fracture of monolayer cultures.J. Cell Biol. 72:763
Peracchia, C., Dulhunty, A. 1976. Low resistance junctions in crayfish. Structural changes with functional uncoupling.J. Cell Biol. 70:419
Pitts, J.D. 1977. Direct communication between animal cells.In: International Cell Biology 1976–1977. B.R. Brinkley and K.R. Porter, editors. p. 43. Rockefeller University Press, New York
Poisner, A.M., Hava, M. 1970. The role of adenosine triphosphate and adenosine triphosphatase in the release of catecholamines from the adrenal medulla. IV. Adenosine triphosphate-activated uptake of calcium by microsomes and mitochondria.Mol. Pharmacol. 6:407
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
Requena, J., DiPolo, R., Brinley, F.J., Jr., Mullins, L.J. 1977. The control of ionized calcium in squid axons.J. Gen. Physiol. 70:329
Rose, B., Loewenstein, W.R. 1971. Junctional membrane permeability. Depression by substitution of Li for extracellular Na, and by long-term lack of Ca and Mg; restoration by cell repolarization.J. Membrane Biol. 5:20
Rose, B., Loewenstein, W.R. 1975a. Calcium ion distribution in cytoplasm visualized by aequorin: Diffusion in the cytosol is restricted due to energized sequestering.Science 190:1204
Rose, B., Loewenstein, W.R. 1975b. Permeability of cell junction depends on local cytoplasmic calcium activity.Nature (London) 254:250
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
Rose, B., Rick, R. 1978. Intracellular pH, intracellular free Ca, and junctional cell-cell coupling.J. Membrane Biol. 44:377
Rose, B., Simpson, I., Loewenstein, W.R. 1977. Calcium ion produces graded changes in permeability of membrane channels in cell junction.Nature (London) 267:625
Simpson, I., Rose, B., Loewenstein, W.R. 1977. Size limit of molecules permeating the junctional membrane channels.Science 195:294
Socolar, S.J. 1977. Appendix: The coupling coefficient as an index of junctional conductance.J. Membrane Biol. 34:29
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, p. 123. Plenum, New York
Spray, D.C., Harris, A.L., Bennett, M.V.L. 1979. Intracellular pH controls conductance of embryonic gap junctions.Biophys. J. 25:81a
Staehelin, L.A. 1974. Structure and functions of intercellular junctions.Int. Rev. Cytol. 39:191
Stoker, M.G.P. 1973. Role of diffusion boundary layer in contact inhibition of growth.Nature (London) 246:200
Thomas, R. 1976. The effect of carbon dioxide on the intracellular pH and buffering power of snail neurons.J. Physiol. (London) 255:715
Turin, L., Warner, A. 1977. Carbon dioxide reversibly abolishes ionic communication between cells of early amphibian embryo.Nature (London) 270:56
Vogt, M., Dulbecco, R. 1960. Virus-cell interaction with a tumor-producing virus.Proc. Nat. Acad. Sci. USA 46:365
Wasserman, R.H., Corradino, R.A. 1973. Vitamin D, calcium, and protein synthesis.Vitam. Horm. 32:43
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Flagg-Newton, J., Loewenstein, W.R. Experimental depression of junctional membrane permeability in mammalian cell culture. A study with tracer molecules in the 300 to 800 dalton range. J. Membrain Biol. 50, 65–100 (1979). https://doi.org/10.1007/BF01868788
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DOI: https://doi.org/10.1007/BF01868788