Pflügers Arch – Eur J Physiol (1996) 431 : 556–563 © Springer-Verlag 1996

ORIGINAL ARTICLE

Albert D. G. de Roos · Everardus J. J. van ZoelenAlexander P. R. Theuvenet

Determination of gap junctional intercellular communicationby capacitance measurements

Received: 27 July 1995/Received after revison: 2 October 1995/Accepted: 3 October 1995

Abstract Electrical coupling between cells is usuallymeasured using the double patch-clamp technique withcell pairs. Here, a single patch-clamp technique that isnot limited to cell pairs is described to determine elec-trical coupling between cells. Capacitance measure-ments in clusters of normal rat kidney (NRK)fibroblasts were used to study intercellular communi-cation. In the whole-cell patch-clamp configurationcapacitive transients were evoked by applying smallvoltage pulses. Total membrane capacitance was cal-culated from these capacitive transients after determi-nation of access resistance, membrane conductance,and the decay constant of the transients, or alterna-tively by integrating the current transient. We foundthat in clusters of one to ten cells, membrane capaci-tance increased linearly with cell number, showing thatthe cells are electrically coupled. Membrane conduc-tance of the cluster of cells also increased, as expectedfor cells that are well coupled. In subconfluent andconfluent cultures, high membrane conductancestogether with large capacitive transients were observed,indicative of electrical coupling. Capacitance couldonly be determined qualitatively under these condi-tions, due to space clamp problems. In the presence ofthe gap junctional inhibitors halothane, heptanol oroctanol, capacitance of all clusters of cells fell to sin-gle-cell levels, showing a complete uncoupling of thecells. The tumour promoter 12-O-tetradecanoylphor-bol-13-acetate (TPA) also uncoupled the cells com-pletely, within 10 min. We conclude that capacitancemeasurements can provide a useful tool to studychanges in intercellular communication in clusters ofcells.

Key words Intercellular communication ·Gap junctions · Electrical coupling · Capacitance ·Patch clamp · Whole cell

Introduction

Gap junctional intercellular communication plays animportant role in many biological processes. Couplingbetween cells has, for instance, been implicated in thepropagation of cardiac action potentials and contrac-tion of smooth muscle [20], embryonic development[10], synchronization of hormone secretion in the pan-creas [18], regulation of blood flow [4], and in the regu-lation of cell proliferation [13].

Intercellular communication through gap junctionsis mediated by connexin proteins and can be modu-lated by several mechanisms. Elevation of the intracel-lular calcium concentration, intracellular acidification,and lipophilic substances such as heptanol, octanol,and halothane have been shown to reduce intercellu-lar communication. Phosphorylation, stimulated by adenosine 3',5'-cyclic monophosphate (cAMP) or 12-O-tetradecanoylphorbol-13-acetate (TPA),also decreases coupling between cells (reviewed in [7]).

Several methods exist to study intercellular com-munication. Various biochemical techniques have beendeveloped in which the transfer of a tracer molecule isfollowed. These techniques include dye transfer afterscrape loading [5] or microinjection [23], fluorescencerecovery after photobleaching [28], metabolic co-oper-ation [30], and radioactive metabolite transfer [8].Electrical communication in cell pairs can be measuredusing the double voltage-clamp technique with micro-electrodes [21] or by determining the gap junctionalconductance using the double whole-cell patch-clamptechnique [16].

Although gap junctions allow transfer of moleculesup to 1 kDa [19], molecules smaller than 1 kDa are

A. D. G. de Roos (*) · E. J. J. van Zoelen · A. P. R. TheuvenetDepartment of Cell Biology, University of Nijmegen,Toernooiveld 1, 6525 ED Nijmegen, The Netherlands

not always transferred effectively and even transferof molecules of the same size can be different [2, 27, 29].Moreover, cells that are not dye coupled can stillbe electrically coupled [22, 25]. Differences in dye and electrical coupling may depend on the physical and chemical properties of the dye, and the typeof connexin involved [2, 22, 25, 27]. Therefore, inter-cellular communication studied using biochemicaltechniques only shows whether the cells are coupledfor the dye used and will not always give informationabout electrical coupling. On the other hand, mea-surement of electrical coupling using double voltage-clamp techniques is technically difficult because it requires the use of at least two electrodes.Theoretically, the double patch- clamp techniquecan be applied to multicellular preparations but mathematical analysis of the data obtained from those experiments is complicated. Therefore, this technique is mostly used to determine electrical coup-ling of isolated cell pairs only.

Here, we describe a simple single patch-clamptechnique that is not limited to cell pairs to assay electrical intercellular communication. With increasingnumbers of cells in a cluster, the total membranesurface, and thus the capacitance, will increasewhen the cells are electrically coupled. We usedthis principle to determine intercellular coupling inclusters of normal rat kidney (NRK) fibroblasts. NRKcells have been shown to be coupled biochemically [6, 11, 14] as well as electrically [14]. Using capacitancemeasurements, we show that clusters of NRKfibroblasts are electrically well coupled and mayprovide a good cell system to study electrical intercel-lular communication.

Materials and methods

Chemicals

Octanol, heptanol and halothane were from Aldrich (Steinheim,Germany), TPA was from Sigma (St. Louis, Mo., USA).

Cell culture

NRK fibroblasts (clone 49F) were seeded on plastic tissue culturedishes at a density of 103 to 104 cells per cm2 in bicarbonate-bufferedDulbecco’s modified Eagle’s medium, supplemented with 10%bovine calf serum (HyClone Laboratories, Logan, VT, USA). Cellswere cultured for 1–5 days at 37° C in 5% CO2. Single cells, smallclusters of cells (two to ten cells), subconfluent and confluent cellcultures were used for patch-clamp studies. Cultures were calledsubconfluent when 30–50% of the culture dish was covered withcells and the patched cell was part of a cluster of at least 100 cells.At 10–15 min before using the cells for patch-clamp measurements,medium was replaced with a physiological salt solution, containing(in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 Tris, 10 glucose,pH 7.4 at room temperature. In experiments in which the mediumwas changed, the cells were perfused at a rate of 1 ml/min in anincubation chamber with a volume of 0.6–0.8 ml.

Whole-cell patch-clamp measurements

Conventional whole-cell patch-clamp methods were used. Patchpipettes were made from thin-walled glass (SG150T, ClarkeElectromedical Instruments, Pangbourne, UK) using a two-stagepipette puller (L/M-3P-A, List Electronic, Darmstadt, Germany).Pipettes were carefully coated with Sylgard 184 (Dow Corning,Midland, Mich., USA). Pipettes were filled with a pipette solution[in mM: 25 NaCl, 120 KCl, 1 CaCl2, 1 MgCl2, 10 Tris, 3.5 ethyl-ene glycol bis(aminoethylether)tetraacetic acid (EGTA), pH 7.4]and had resistances of 4–6 megaohms. Membrane currents weredetected with an EPC-7 patch- clamp amplifier (List Electronic). Inthe cell-attached configuration, capacitance of the pipette was com-pensated. In the whole-cell configuration, however, capacative tran-sients of the cell membrane were not cancelled. In some experimentsmembrane potential was determined by switching to the current-clamp mode. Voltage-clamp protocols and data acquisition wereperformed using CED software in conjunction with a CED 1401interface (Cambridge Electronic Design, Cambridge, UK). Datawere filtered at 5 kHz, sampled at 12.5 kHz, and stored on harddisk for subsequent analysis.

Theoretical background

Determination of capacitance and membrane conductance were per-formed according to the method described by Lindau and Neher[12]. Figure 1A shows the minimal equivalent circuit of the whole-cell patch-clamp configuration. The cell membrane is characterizedby the parallel combination of the membrane capacitance, Cm and

557

Fig. 1A Minimal equivalent circuit of the whole-cell patch-clamptechnique. The cell membrane is represented by a capacitance Cm

and a resistance Rm. The resistance between the pipette and the cellis indicated as the access resistance Ra. Seal resistance is assumedto be perfect and capacitance of the pipette assumed to be can-celled by the patch-clamp amplifier. B Current transient of a modelcell as a result of a short voltage pulse (Vp) delivered through thepipette. I0 is peak current at t = 0. Current will decay exponentially,and Iss is the steady-state current after capacitor (i.e. membrane)has been charged. The area under the transient (Q) represents thecharge accumulated on the membrane

the membrane conductance, Gm. The access resistance, Ra, of thepipette tip is in series with Cm and Gm. Figure 1B shows the cur-rent response to a voltage step delivered through the pipette (Vp).From this current response, values for the current at t = 0 (I0), thesteady-state current (Iss), and the decay constant of transient (τ) canbe obtained. The circuit parameters Ra, Gm and Cm can be relatedto the these parameters through the following equations [12].

Ra = Vp/I0 (1)Gm = Iss/ (Vp[Ra·Iss) (2)Cm = τ·[(1/Ra) + Gm] (3)

In this minimal equivalent circuit of the whole-cell patch-clampconfiguration, Gm is considered to be of pure ohmic behaviour. Sinceseal resistances were at least 20 GΩ, capacitance error will be lessthan 0.1% [12]. The area under the transient represents the totalcharge accumulated on the membrane [9]. Capacitance can be cal-culated by dividing the total charge (Q) by the applied voltage.

Data analysis

Capacitive current transients were obtained by applying a 10-mVvoltage pulse. Capacitive transients were fit to a single exponentialfunction using Bio-Patch software (Bio-Logic, France). The firstthree or four data points (about 0.3 ms) were neglected becausethey represent filter artefacts. When clusters of less than ten cellswere used, all transients could be fit to a single exponential func-tion, and I0, Iss and τ were obtained from this fit. By insertion inEqs. 1–3 Ra, Gm, and Cm were calculated. By integrating the cur-rent transient using Bio-Patch software, the accumulated chargeQ (Fig. 1B) could be determined and Cm was obtained by dividingQ by Vp. In subconfluent and confluent cultures, data could no longer be fit to a single exponential. In these cases, I0 wasdetermined from a fit from the first section of the data, Iss was measured from the raw data, and only Ra and Gm were calculated.

Results

NRK fibroblasts were patched in the whole-cell patch-clamp configuration and small (10 mV) voltage pulseswere applied. In Fig. 2, the resulting current transientis shown in small clusters of cells (up to ten cells), andin subconfluent and confluent cell cultures. In patchedsingle cells, the current transient was characterized bya small decay constant and a low steady-state current.With increasing cluster size, decay constants becamelarger and also steady-state current increased. Averagedecay constants were 0.51 ± 0.36 ms for single cells,and 5.31 ± 1.44 ms for clusters of ten cells. The aver-age steady-state current values ranged from 1.05 ± 0.36pA in single cells to 11.39 ± 4.82 pA in clusters of tencells. Peak currents represent the access resistance ofthe pipette tip (Eq. 1) and are not related to clustersize. When subconfluent and confluent cultures wereused, decay constants became even larger and very largesteady-state currents became apparent. Increasingsteady-state currents, at equal access resistances, indi-cate increasing membrane conductance, while increas-ing decay constants suggest an increasing capacitance.Thus, with increasing cluster size, the current transientsindicated that membrane conductance and capacitanceof the cluster of cells increased. This implied that thecells were electrically coupled.

Since decay constants are related to membranecapacitance, membrane capacitance was calculatedfrom the evoked capacitive transients in clusters of upto ten cells. The capacitance was calculated after deter-mination of access resistance, membrane conductanceand decay constant (Fig. 3A), and alternatively throughintegration of the current transient (Fig. 3B), asdescribed in Materials and methods. Single-cell capac-itance was about 30 pS, while capacitance of a clusterof ten cells was about 300 pS. Figure 3A, B shows thatcapacitance increased linearly with cell number indi-cating electrical coupling. Figure 3C shows that bothmethods to determine capacitance yielded the sameresults, indicating that the variation in membranecapacitance at a specific cell number is not the resultof variation in the method used to determine mem-brane capacitance, but represents an actual variationin the capacitance of the clusters of cells. This is mostlikely due to a variation in cell size.

With larger clusters (more than ten cells), decay con-stants could not be determined anymore, because thetransients could not be fit with a single exponential,probably due to space clamp problems. These spaceclamp problems arise from the fact that the cytoplasmas well as the gap junctions have a certain resistance.

558

Fig. 2 Current response to a voltage pulse in normal rat kidney(NRK) cells. One cell of a cluster of cells was patched in the whole-cell configuration and a voltage pulse of 10 mV was given. Pipettecapacitance was cancelled and transients represent capacitive tran-sients of the cell membrane

These resistances cause the voltage to drop and aftera certain distance, voltage will be zero [1]. Because ofthese space clamp limitations, voltage will no longer beuniformly distributed and the monolayer can only bepartly voltage-clamped.

In electrically coupled cells, it is expected that totalmembrane conductance will also increase with cellnumber. The steady state currents in Fig. 2 already sug-gested that membrane conductance increased withincreasing cell numbers. Figure 4 shows the membraneconductance as a function of cell number. Membraneconductance was calculated from the access resistanceand the steady-state currents (Eq. 2). From one to tencells, membrane conductance increased, but variationin membrane conductance also increased (Fig. 4A).Membrane resistance of single cells was in the gigaohmrange, but resistance of the membrane was only a fewmegaohms in confluent cells. This also showed thatcells were electrically coupled. From the average num-

bers in Fig 4A no clear linear relation could be detectedbetween membrane conductance and cell number, incontrast to membrane capacitance and cell number. Alinear relation could be obscured by a large variationin membrane conductance in single cells, which couldresult from a variation in channel density. It is alsopossible that channel activity, and thereby membraneconductance, is a function of cell number and in thatcase, there does not have to be a linear relation. Whenwe compared larger clusters of cells (10–30 cells)with subconfluent and confluent cells, membrane conductance increased dramatically (Fig. 4B). Thisincrease suggests that coupling is maintained at highcell densities. Despite space clamp problems thatprobably occurred, the difference between subconfluentand confluent cells could be clearly seen. However, theobserved membrane conductance will not be the con-ductance of the whole monolayer, because only part ofthe monolayer will be voltage-clamped.

We also tested the known gap junctional blockershalothane, heptanol and octanol on the ability to blockgap junctional communication in NRK cells by deter-mining their effect on evoked capacitive transients.Figure 5 shows the effect of exposure of confluent,subconfluent and single cells to halothane (1.5 mM).Within seconds after addition of halothane to the bath,

559

Fig. 3A–C Capacitance as a function of cell number. A Capacitancewas determined after calculation of Ra, Gm and decay constant ofcapacitive current transients. B The integral of current transient wasused to determine capacitance. C Capacitance calculated via theintegral plotted against capacitance calculated via decay constant(τ), Ra and Gm. Each data point represents the value of an individ-ual measurement at the indicated cell number

Fig. 4A, B Gm as a function ofcell number. A Gm of clustersof one to ten cells. Data pointsrepresent individualmeasurements. B. Membraneconductance in clusters of10–30, subconfluent andconfluent cells. Errors barsrepresent SEM values. Gm isthe reciprocal of Rm

the decay constant and steady-state current of thecapacitive transient fell to levels similar to that of sin-gle cells. This shows that halothane treatment resultsin complete functional uncoupling of the cells. Figure5 also shows that after evaporation of halothane, cou-pling was re-established. Similar results (not shown)were obtained with heptanol (1.5 mM) and octanol(1.5 mM). These results show that the electrical com-munication in NRK cells is mediated through gap junc-tions and that capacitance measurements can be usedto study modulation of this communication.

The phorbol ester TPA was used to follow a morephysiological approach to modulate gap junctions.Activation of protein kinase C by TPA [7, 15] has beenshown to block gap junctions by a phosphorylation ofconnexin. Figure 6A shows the capacitive transients ofa cluster of 11 cells after treatment with 100 ng/mlTPA. After 5 min of TPA treatment the transient wasstill the same as in the control, but after 10 min theshape of the transient changed. First, the area underthe curve became smaller, indicating a reduced capaci-tance. Second, the decay constant became larger. After13 min of TPA treatment, both the area under the tran-sient and the decay constant reached single-cell levels.We interpret these data as follows. When gap junctionsare partially blocked, resistance between cells becomeslarger. Therefore, the decay constant, which is equal tothe product of the total resistance (Ra and 1/Gm) andthe capacitance, (from Eq. 3) becomes larger. Thereduced area under the curve, i.e. the accumulatedcharge, decreases because the higher resistance between

cells prevents charging of all cells in the cluster. Whenthe gap junctions are not totally blocked by TPA, thecurrent transient represents the fast charging of thepatched cell, and the slow charging of the rest of thecluster with much larger decay constants. The smalldecay constant at partial TPA block (0.10 ms) wassimilar to the decay constant at maximal TPA block(0.11 ms). Moreover, the capacitance calculated froman exponential fit of this fast part of the transientyielded a value comparable to those measured in sin-gle cells. Finally, when the gap junctions are totallyblocked, the resistance between cells is very high, andonly the capacitance of a single cell can be measured.

Figure 6B shows the calculated capacitance of thecluster of 11 cells in response to TPA treatment.Capacitance fell from a value of 300 pF, characteristicfor a cluster of 11 cells, to a capacitance of 20 pF, char-acteristic for a single cell. No stepwise decreases incapacitance were observed. The figure shows the capac-

560

Fig. 6A, B The effect of 12-O-tetradecanoylphorbol-13-acetate(TPA) on coupling and membrane potential. A Capacitive currenttransients are shown in a cluster of 11 cells at different times afterperfusion of 100 ng/ml TPA. B Corresponding calculated capaci-tance and membrane potential. Capacitance was calculated via inte-gration of the capacitive current transients. Membrane potentialwas determined in the current-clamp mode

Fig. 5A–C The effect of the uncoupler halothane on capacitivecurrent transient in NRK cells. A Single, B subconfluent and C confluent cells were treated with 1.5 mM halothane. Left: con-trol current transients, middle: after treatment with halothane, right:after evaporation of halothane (about 5 min after addition)

itance calculated from the integrated transient, becausethe data could not be fit with a single exponential whenTPA block was not complete. Control values and val-ues at complete block calculated through the decay con-stant yielded similar results to those calculated via theintegral of the current. From the decreased membranecapacitance values, one cannot deduce the number ofcoupled cells, but the values will only give an estimateas to what extent coupling is reduced. Membranepotential also dropped (Fig. 6B) after addition of TPAfrom an initial value of [70 mV to [5 mV. It has beenreported that transmembrane potential can affect inter-cellular coupling [16]. Clearly, membrane depolariza-tion preceded the decrease in capacitance, and is,therefore, not directly related to the change in capaci-tance. We cannot exclude the possibility, however, thatmembrane depolarization is a prerequisite for uncou-pling.

Discussion

Several methods have been described to measure gapjunctional communication. These include techniques toassay biochemical communication, in which thediffusion of tracer molecules was followed, and elec-trical communication, using voltage-clamp techniques.Biochemical communication may not be a good meas-ure for electrical coupling. In several instances electri-cal coupling of cells has been demonstrated in theabsence of dye coupling [22, 25, 29]. Also, a reductionin gap junctional coupling may abolish transfer of, forinstance, fluorescent dyes, but remaining electrical cou-pling may be sufficient to transduce electrical signals[2, 27]. Measurements of electrical communicationbetween cells are usually performed using double volt-age-clamp techniques on cell pairs, but these measure-ments have been hampered by the fact that they aretechnically difficult and require two patch-clamp set-ups. The mathematical analysis when clusters of cellsare used is complex and requires cable analysis [1].

Here, we describe a simple single patch-clamp tech-nique, that is not limited to cell pairs, to study electri-cal intercellular communication. The advantage of thetechnique presented here, is the fact that only onepatch-clamp electrode is needed to assay electrical com-munication, in contrast to the double voltage-clamptechniques. The capacitance of clusters of cells wasmeasured to determine gap junctional coupling. Sincethe membrane acts as a capacitor, capacitance isdirectly related to membrane area and therefore cellsize. The specific membrane capacitance is about1 µF/cm2 [9]. In NRK cells, capacitance increased lin-early with cell number, showing that capacitance meas-urements can be used to assess electrical coupling inthese cells. When large cells or clusters of cells are volt-age-clamped, measurements will be limited by the space

clamp phenomenon: applied voltage via the pipette willgradually decrease in space, due to the resistance of thecytosol and the gap junctions [1]. The fact that in smallclusters of cells, capacitance increased linearly with cellnumber, indicates that gap junctional resistance is solow that in these small clusters of cells no space clampproblems occur. Space clamp problems will result in anon-linear relation between capacitance and membranearea or capacitance. Since NRK cells in culture tendto move and spread out, it is hard to find nicely aggre-gated clusters of more than 15 cells. Therefore, we couldnot determine precisely at what cluster size the linearrelation between cell number and capacitance was lost.However, current transients could be fit by a singleexponential in clusters of 30 cells, indicating nosignificant space clamp problems.

In subconfluent and confluent cell cultures, currenttransients could no longer be fit by a single exponen-tial. In these cases, we used membrane conductance asan indication for coupling. In electrically coupled cellsmembrane conductance of neighbouring cells will bemeasured. We observed an increase in total membraneconductance as a function of cell number in NRK cells,indicating electrical coupling of the cells. Here, a clearlinear relation could not be found, in contrast with thecapacitance measurements. Although capacitance is adirect measure of membrane area, membrane conduc-tance is determined by the number and the open prob-ability of ion channels in the membrane. Theseparameters can be influenced by coupling or cell den-sity and therefore, membrane conductance does nothave to be directly related to cell number in coupledcells.

We also showed that capacitance measurements can be used to measure a reduction in gap junctionalintercellular communication. Gap junctions can beblocked by lipophilic agents such as heptanol, octanolor halothane [3, 24], and by activation of proteinkinase C using TPA [15, 26, 30]. Here, we showedthat lipophilic agents and TPA inhibit intercellularcommunication in NRK cells, which demonstrates that electrical coupling is mediated through gap junctions.

Capacitance measurements can show uncoupling ina cluster of cells, although no absolute values of coup-ling can be obtained. Since NRK cells are well coup-led, a complete uncoupling can be easily shown by acapacitance value that is similar to single-cell values.Partial uncoupling of a cluster of cells can mean thatsome of the cells uncouple from the other cells thatremain coupled. In these cases, a decreased capacitancewill be measured, namely the capacitance value of thecells that remain electrically coupled to the patchedcell. In these cases a stepwise decrease in capacitancecan be expected. Since differences in capacitance ofclusters of cells of different cell number could be determined (Fig. 3), it should be possible to measurea stepwise decrease in capacitance resulting from the

561

uncoupling of certain cells in a cluster of cells. However,in our experiments stepwise decreases were never seen.

A partial uncoupling will also be seen, when the gapjunctional conductances of all the cells is reduced. Adecrease in gap junctional conductance will results ina larger decay constant (see Eq. 3). This will lead to alower measured membrane capacitance, except whenthis conductance decrease does not prevent completecharging of the cluster of cells. However, also a decreasein membrane conductance will cause a larger decayconstant (Eq. 3), but in these cases capacitance willalways stay the same. In our experiments, partialuncoupling by TPA resulted in a larger decay constant(Fig. 6), together with a decrease in capacitance.Calculated capacitance would not have been decreasedif TPA decreased membrane conductance, but we can-not exclude the possibility that the increased decayconstant is partly due to a decreased membrane con-ductance. In conclusion, capacitance measurementscan give qualitative information about modulation ofcoupling, but no absolute values of uncoupling can beobtained.

NRK cells have been shown to be coupled electri-cally using microelectrodes and biochemically using dyemicroinjection [14], dye transfer with a preloadingtechnique [6] or fluorescence-activated cell sorting [11].Measurement of biochemical coupling appears todepend on the dye used, because in some experimentsonly a limited coupling was observed in NRK cells [31].However, these conflicting data on gap junctional cou-pling of NRK cells might be related to possibledifferences in growth state of the cells used in thesestudies. Recently, namely, a cell-state-dependent modu-lation of gap junctional communication in NRK cellshas been reported [17].

Capacitance measurements may provide a usefultool to study modulation of electrical intercellular com-munication in NRK cells. It has been shown thatgrowth factors like epidermal growth factor andplatelet-derived growth factor inhibited gap junctionalcommunication in NRK cells [14]. Intracellular pH,phosphorylation and the intracellular calcium concen-tration can affect intercellular coupling in cells [7]. Weare currently investigating how these components ofmitogenic/transforming pathways may modulate gapjunctional communication in NRK fibroblasts.

Our observation that capacitance of small clustersof NRK cells increased linearly with cell numbershowed that, electrically, small clusters of cells behavelike a functional syncytium. In larger clusters andsubconfluent and confluent cells, capacitance could notbe determined quantitatively anymore, but large capa-citive transients together with high membrane con-ductances suggested that many, if not all, cells in amonolayer were coupled. Strong electrical couplingimplicates that the membrane of the whole monolayerof cells is isopotential. This has also been shown in cul-tured guinea-pig coronary endothelial cells [4]. Like

NRK cells, monolayers of these cells were also char-acterized by low input resistances. In strongly coupledcells, measurement of membrane potential in mono-layers will actually represent the average membranepotential of many cells. In coupled cells, de- or hyper-polarizations of the membrane will be also uniformlydistributed. We recently showed that agonists, such asbradykinin and prostaglandin F2α depolarize NRKcells (A.D.G. de Roos et al., submitted). A function ofelectrical coupling between NRK cells may be thetransduction of de- or hyperpolarizations and changesin gap junctional coupling may affect this transduction.Moreover, membrane potential is much more stablein subconfluent and confluent cells than in small clus-ters of cells (A. D. G. de Roos et al., unpublished).Therefore, another function of electrical couplingmight be stabilization of the membrane potential.

In conclusion, capacitance measurements using thesingle whole-cell patch-clamp technique provide a use-ful tool to determine intercellular electrical coupling inclusters of cells. Since NRK cells are electrically wellcoupled, they provide an excellent model system tostudy control of intercellular communication.

Acknowledgements We would like to thank Dr. Maarten Kansen,department of Biochemistry, University of Nijmegen, for criticallyreading the manuscript and Dr. Martin Rook, department ofMedical Physiology, University of Utrecht, for helpful discussions.This research was funded by the Netherlands foundation for lifesciences.

References

1. Armstrong CM, Gilly WF (1992) Access resistance and spaceclamp problems associated with whole-cell patch clamping.Methods Enzymol 207:100–122

2. Brissette JL, Kumar NM, Gilula NB, Hall JE, Dotto GP (1994) Switch in gap junction protein expression is associatedwith selective changes in junctional permeability during keratinocyte differentiation. Proc Natl Acad Sci USA91:6453–6457

3. Burt JM, Spray DC (1989) Volatile anesthetics block intercel-lular communication between neonatal rat myocardial cells.Circ Res 65:829–837

4. Daut J, Mehrke G, Nees S, Newman WH (1988) Passive elec-trical properties and electrogenic sodium transport of culturedguinea-pig coronary endothelial cells. J Physiol (Lond) 402:237–254

5. El-Fouley MH, Trosko JE, Chang CC (1987) A rapid and sim-ple technique to study gap junctional intercellular communica-tion. Exp Cell Res 168:422–430

6. Goldberg GS, Bechberger JF, Naus CCG (1995) Scrape load-ing and dye transfer. A pre-loading method of evaluating gapjunctional communication by fluorescent dye transfer. Bio-techniques 18:490–497

7. Holder JW, Elmore E, Barrett JC (1993) Gap junction func-tion and cancer. Cancer Res 53:3475–3485

8. Hooper ML (1982) Metabolic co-operation between mam-malian cells in culture. Biochim Biophys Acta 651:85–103

9. Kado RT (1993) Membrane area and electrical capacitance.Methods Enzymol 221:273–299

10. Kalimi GH, Lo CW (1988) Communicating compartments inthe gastrulating mouse embryo. J Cell Biol 107:241–255

562

11. Kiang DT, Kollander R, Lin HH, LaVilla S, Atkinson MM(1994) Measurement of gap junctional communication byfluorescence activated cell sorting. In Vitro Cell Dev Biol 30A:796–802

12. Lindau M, Neher E (1988) Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pflügers Arch411:137–146

13. Loewenstein WR (1981) Junctional intercellular communica-tion: the cell-to-cell membrane channel. Physiol Rev 61:829–913

14. Maldonado PE, Rose B, Loewenstein WR (1988) Growth fac-tors modulate junctional cell-to-cell communication. J MembrBiol 106:203–210 ((203–210))

15. Murray AW, Fitzgerald DJ (1979) Tumor promoters inhibitmetabolic cooperation in cocultures of epidermal and 3T3 cells.Biochem Biophys Res Commun 91:395–401

16. Neyton J, Trautmann A (1985) Single-channel currents of anintercellular junction. Nature 317:331–335

17. Paulson AF, Johnson RG, Atkinson MM (1994) Intercellularcommunication is reduced by TPA and Ki-ras p21 in quiescent,but not proliferating, NRK cells. Exp Cell Res 213:64–70

18. Santos RM, Rosario LM, Nadal A, Garcia-Sancho J, Soria B,Valdeolmillos M (1991) Widespread synchronous [Ca2+]i oscil-lations due to bursting electrical activity in single pancreaticislets. Pflügers Arch 418:417–422

19. Simpson I, Rose B, Loewenstein WR (1977) Size limit of mol-ecules permeating the junctional membrane channels. Science195:294–296

20. Spray DC, Bennett MVL (1985) Physiology and pharmacologyof gap junctions. Annu Rev Physiol 47:281–303

21. Spray DC, Harris AL, Bennett MVL (1981) Equilibrium prop-erties of a voltage-dependent junctional conductance. J GenPhysiol 77:77–93

22. 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 gapjunctions with different molecular permeabilities in osteoblas-tic cells. EMBO J 13:744–750

23. Stewart WW (1978) Gap junctional connections between cellsas revealed by dye-coupling with a highly fluorescent naph-thalimide tracer. Cell 14:741–759

24. Takens-Kwak BR, Jongsma HJ, Rook MB, van GinnekenACG (1992) Mechanisms of heptanol induced uncoupling ofcardiac junctions: a perforated patch clamp study. Am J Physiol262:C1531-C1538

25. Traub O, Eckert R, Lichtenberg-Fraté H, Elfgang C, BastideB, Scheidtmann KH, Hülser DF, Willecke K (1994)Immunochemical and electrophysiological characterization ofmurine connexin40 and [43 in mouse tissues and transfectedhuman cells. Eur J Cell Biol 64:101–112

26. Trosko JE, Chang CC, Madhakar BV, Klaunig JE (1990)Chemical, oncogene and growth factor inhibition of gap junc-tional intercellular communication: an integrative hypothesisof carcinogenesis. Pathobiology 58:265–278

27. Veenstra RD, Wang H-Z, Beyer EC, Ramanan SV, Brink PR(1994) Connexin 37 forms high conductance gap junction chan-nels with subconductance state activity and selective dye andionic permeabilities. Biophys J 66:1915–1928

28. Wade MH, Trosko JE, Schindler MA (1986) A fluorescent pho-tobleaching assay of gap junction mediated communicationbetween human cells. Science 232:525–528

29. von der Weid PY, Bény J-L (1993) Simultaneous oscillationsin the membrane potential of pig coronary artery endothelialand smooth muscle cells. J Physiol (Lond) 471:13–24

30. Yotti LP, Chang CC, Trosko JE (1979) Elimination of meta-bolic cooperation in Chinese hamster cells by a tumor pro-moter. Science 206:1089–1091

31. van Zoelen EJJ, Tertoolen LGJ (1991) Transforming growthfactor-β enhances the extent of intercellular communicationbetween normal rat kidney cells. J Biol Chem 266:12075–12081

563