Abstract
Electrical coupling between cells is usually measured using the double patch-clamp technique with cell pairs. Here, a single patch-clamp technique that is not limited to cell pairs is described to determine electrical coupling between cells. Capacitance measurements in clusters of normal rat kidney (NRK) fibroblasts were used to study intercellular communication. In the whole-cell patch-clamp configuration capacitive transients were evoked by applying small voltage pulses. Total membrane capacitance was calculated from these capacitive transients after determination of access resistance, membrane conductance, and the decay constant of the transients, or alternatively by integrating the current transient. We found that in clusters of one to ten cells, membrane capacitance increased linearly with cell number, showing that the cells are electrically coupled. Membrane conductance of the cluster of cells also increased, as expected for cells that are well coupled. In subconfluent and confluent cultures, high membrane conductances together with large capacitive transients were observed, indicative of electrical coupling. Capacitance could only be determined qualitatively under these conditions, due to space clamp problems. In the presence of the gap junctional inhibitors halothane, heptanol or octanol, capacitance of all clusters of cells fell to single-cell levels, showing a complete uncoupling of the cells. The tumour promoter 12-O-tetradecanoylphor-bol-13-acetate (TPA) also uncoupled the cells completely, within 10 min. We conclude that capacitance measurements can provide a useful tool to study changes in intercellular communication in clusters of cells.
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References
Armstrong CM, Gilly WF (1992) Access resistance and space clamp problems associated with whole-cell patch clamping. Methods Enzymol 207:100–122
Brissette JL, Kumar NM, Gilula NB, Hall JE, Dotto GP (1994) Switch in gap junction protein expression is associated with selective changes in junctional permeability during keratinocyte differentiation. Proc Natl Acad Sci USA 91:6453–6457
Burt JM, Spray DC (1989) Volatile anesthetics block intercellular communication between neonatal rat myocardial cells. Circ Res 65:829–837
Daut J, Mehrke G, Nees S, Newman WH (1988) Passive electrical properties and electrogenic sodium transport of cultured guinea-pig coronary endothelial cells. J Physiol (Lond) 402: 237–254
El-Fouley MH, Trosko JE, Chang CC (1987) A rapid and simple technique to study gap junctional intercellular communication. Exp Cell Res 168:422–430
Goldberg GS, Bechberger JF, Naus CCG (1995) Scrape loading and dye transfer. A pre-loading method of evaluating gap junctional communication by fluorescent dye transfer. Biotechniques 18:490–497
Holder JW, Elmore E, Barrett JC (1993) Gap junction function and cancer. Cancer Res 53:3475–3485
Hooper ML (1982) Metabolic co-operation between mammalian cells in culture. Biochim Biophys Acta 651:85–103
Kado RT (1993) Membrane area and electrical capacitance. Methods Enzymol 221:273–299
Kalimi GH, Lo CW (1988) Communicating compartments in the gastrulating mouse embryo. J Cell Biol 107:241–255
Kiang DT, Kollander R, Lin HH, LaVilla S, Atkinson MM (1994) Measurement of gap junctional communication by fluorescence activated cell sorting. In Vitro Cell Dev Biol 30A:796–802
Lindau M, Neher E (1988) Patch-clamp techniques for timeresolved capacitance measurements in single cells. Pflügers Arch 411:137–146
Loewenstein WR (1981) Junctional intercellular communication: the cell-to-cell membrane channel. Physiol Rev 61:829–913
Maldonado PE, Rose B, Loewenstein WR (1988) Growth factors modulate junctional cell-to-cell communication. J Membr Biol 106:203–210 ((203-210))
Murray AW, Fitzgerald DJ (1979) Tumor promoters inhibit metabolic cooperation in cocultures of epidermal and 3T3 cells. Biochem Biophys Res Commun 91:395–401
Neyton J, Trautmann A (1985) Single-channel currents of an intercellular junction. Nature 317:331–335
Paulson AF, Johnson RG, Atkinson MM (1994) Intercellular communication is reduced by TPA and Ki-ras p21 in quiescent, but not proliferating, NRK cells. Exp Cell Res 213:64–70
Santos RM, Rosario LM, Nadal A, Garcia-Sancho J, Soria B, Valdeolmillos M (1991) Widespread synchronous [Ca2−]i, oscillations due to bursting electrical activity in single pancreatic islets. Pflügers Arch 418:417–422
Simpson I, Rose B, Loewenstein WR (1977) Size limit of molecules permeating the junctional membrane channels. Science 195:294–296
Spray DC, Bennett MVL (1985) Physiology and pharmacology of gap junctions. Annu Rev Physiol 47:281–303
Spray DC, Harris AL, Bennett MVL (1981) Equilibrium properties of a voltage-dependent junctional conductance. J Gen Physiol 77:77–93
Steinberg TH, Civitelli R, Geist ST, Robertson AJ, Hick E, Veenstra RD, Wang H-Z, Warlow PM, Westphale EM, Laing JG, Beyer EC (1994) Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells. EMBO J 13:744–750
Stewart WW (1978) Gap junctional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell 14:741–759
Takens-Kwak BR, Jongsma HJ, Rook MB, van Ginneken ACG (1992) Mechanisms of heptanol induced uncoupling of cardiac junctions: a perforated patch clamp study. Am J Physiol 262:C1531-C1538
Traub O, Eckert R, Lichtenberg-Fraté H, Elfgang C, Bastide B, Scheidtmann KH, Hülser DF, Willecke K (1994) Immunochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells. Eur J Cell Biol 64:101–112
Trosko JE, Chang CC, Madhakar BV, Klaunig JE (1990) Chemical, oncogene and growth factor inhibition of gap functional intercellular communication: an integrative hypothesis of carcinogenesis. Pathobiology 58:265–278
Veenstra RD, Wang H-Z, Beyer EC, Ramanan SV, Brink PR (1994) Connexin 37 forms high conductance gap junction channels with subconductance state activity and selective dye and ionic permeabilities. Biophys J 66:1915–1928
Wade MH, Trosko JE, Schindler MA (1986) A fluorescent photobleaching assay of gap junction mediated communication between human cells. Science 232:525–528
von der Weid PY, Bény J-L (1993) Simultaneous oscillations in the membrane potential of pig coronary artery endothelial and smooth muscle cells. J Physiol (Lond) 471:13–24
Yotti LP, Chang CC, Trosko JE (1979) Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter. Science 206:1089–1091
van Zoelen EJJ, Tertoolen LGJ (1991) Transforming growth factor-β enhances the extent of intercellular communication between normal rat kidney cells. J Biol Chem 266:12075–12081
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de Roos, A.D.G., van Zoelen, E.J.J. & Theuvenet, A.P.R. Determination of gap junctional intercellular communication by capacitance measurements. Pflugers Arch. 431, 556–563 (1996). https://doi.org/10.1007/BF02191903
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DOI: https://doi.org/10.1007/BF02191903