Functional coupling in bovine ciliary epithelial cells is modulated by carbachol

J. W. Stelling and T. J. C. Jacob

Physiology Unit, University of Wales, Cardiff CF1 3US, United Kingdom

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The functional coupling of the ciliary epithelium was studied in isolated pairs (couplets) of pigmented ciliary epithelial (PCE) and nonpigmented ciliary epithelial (NPCE) cells using the whole cell patch clamp and the fluorescent dye lucifer yellow. One cell of the pair (usually the NPCE cell of a NPCE-PCE cell couplet) was accessed with a 2-5 MOmega electrode, containing 1-2 mM lucifer yellow, in the whole cell configuration of the patch clamp. After voltage-clamp experiments were completed, cells were viewed under a fluorescent microscope to confirm that the cells were coupled. The electrical coupling of the cells was also studied by calculating the capacitance (using the time-domain technique), assuming a "supercell" model for coupled cells. The mean capacitance of coupled pairs was 79.8 ± 4.3 (SE) pF (n = 47) compared with single cell capacitances of 36.8 ± 3.4 pF (n = 10) for PCE cells and 38.1 ± 3.1 pF (n = 15) for NPCE cells. Octanol, carbachol (CCh), and raised extracellular Ca2+ concentration ([Ca2+]o) all caused uncoupling in pairs (couplets) of coupled NPCE and PCE cells. At room temperature (22-24°C), the capacitance of the couplets decreased from 70.5 ± 8.0 to 48.0 ± 5.2 pF (n = 5) when exposed to octanol (1 mM), from 73.8 ± 9.2 to 43.2 ± 9.5 pF (n = 4) when exposed to CCh (100 µM), and from 80.5 ± 6.7 to 49.9 ± 7.8 pF (n = 4) when exposed to 10 mM [Ca2+]o. The response to CCh was dose dependent; at higher temperatures of 34-37°C, 10 µM CCh caused a 38% reduction in capacitance, from 53.7 ± 9.7 to 33.5 ± 3.3 pF (n = 7) with a half-time of 249 s, and 100 µM CCh caused a 49% reduction in capacitance, from 51.3 ± 5.6 to 26.0 ± 2.4 pF (n = 7) with a half-time of 124 s. After pairs uncoupled and the uncoupling agent was washed out, the cell pairs often exhibited an increase in capacitance that we interpreted as "recoupling" or a reopening of the gap junctional communication pathway; the half-time for this process was 729 s after uncoupling with 100 µM CCh and 211 s after uncoupling with 10 µM CCh. This interpretation was confirmed optically by the spread of lucifer yellow into both cells of an uncoupled pair with a time course corresponding to the increase in electrical coupling. The controllable coupling of ciliary epithelial cells extends the idea of a functional syncytium involved in active transport. PCE cells take up solute and water from the blood, which then cross to NPCE cells via gap junctions and from there are secreted into the posterior chamber of the eye. Modulation of the coupling between NPCE and PCE cells may provide a mechanism to control secretion.

gap junction; muscarinic; aqueous humor

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE CILIARY EPITHELIUM (CE) of the eye is composed of two distinct cell layers with their apical surfaces facing each other. The basolateral surface of the outer, pigmented layer faces the stroma (and blood vessels), and the basolateral surface of the inner, nonpigmented layer faces the inside of the eye. These cells are responsible for the active secretion of the aqueous humor, and the balance of production and drainage through the trabecular meshwork gives rise to the intraocular pressure (IOP). Obstruction of the outflow leads to increased IOP and glaucoma. The majority of treatments for this disease involve modulation of the production with various drugs, although all produce moderate side effects and there are contradictory reports as to their effects on IOP (13). It is believed that the two layers of CE interactively contribute to the process of aqueous humor secretion, with the pigmented ciliary epithelial (PCE) cells exhibiting solute uptake properties and the nonpigmented ciliary epithelial (NPCE) cells having solute efflux properties (34). Coupling between the cell layers could, therefore, play an important role and might be used to modulate the secretory process, since not only ions but also pharmacological agents or second messengers may pass through the junction (7, 8, 26).

Functional coupling between the layers has been suggested previously on the basis of electron microscopy (24) and dye-coupling experiments (7, 21, 26). Electrical coupling of the layers has been demonstrated in rabbit CE (10). The gap junctions therefore allow the cells to function as a syncytium, allowing ions, water, and intracellular messengers to pass between the cell layers.

We have previously demonstrated that the effects of the muscarinic agonist carbachol (CCh) on the whole cell conductance of PCE cells caused a transient increase in the K+ conductances acting via Ca2+ (33). It is known that several types of gap junction are also modulated by intracellular Ca2+ concentration ([Ca2+]i) (17, 22, 30). The link between secretomotor agonists (such as acetylcholine) and gap junction closure has been demonstrated in a variety of tissues, such as pancreas, salivary, and lacrimal glands (see Ref. 23 for a review). It was therefore of interest to continue the previous study (33) and examine the effects of the muscarinic agonist CCh and modulators of gap junction conductance on pairs of coupled cells.

The present study uses electrical and dye-coupling techniques to investigate the functional properties of the intercellular junction in CE in response to CCh. We demonstrate the coupling, uncoupling, and recoupling of pairs of NPCE and PCE cells and show that this process is modulated by CCh in a dose-dependent manner.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

The tips of the ciliary body were dissected from bovine eyes within 3-5 h postmortem. Cells were isolated using 0.25% trypsin and plated on coverslips overnight in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered culture medium E199 (Sigma, Poole, UK) containing 10% fetal calf serum (Sigma). Single and coupled cells were investigated using both the whole cell patch-clamp technique and intracellular electrodes. Coverslips were mounted in a recording chamber and perfused with a HEPES-buffered physiological saline solution. This solution contained (in mM) 125 NaCl, 5 KCl, 10 NaHCO3, 10 HEPES, 0.5 MgCl2, 2 CaCl2, 3.2 glucose, and 20 sucrose; the pH was adjusted to 7.4 with 1 M NaOH.

Pipettes contained a standard intracellular buffer for electrophysiological recording containing (in mM) 56 KCl, 84 potassium gluconate, 10 HEPES, 2 MgCl2, 20 sucrose, 1.1 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 0.1 CaCl2 (buffered Ca2+ was 10-8 M unless indicated). The pH was adjusted to 7.25 with 1 M NaOH. In dye-coupling experiments, 1-2 mM lucifer yellow was added to the pipette solution. The relatively low-impedance electrodes (4-8 MOmega ) allowed the dye to enter the cell with the application of hyperpolarizing voltage steps or by diffusion (fully loaded within 2 min).

In electrical coupling experiments, the cell pairs were studied electrophysiologically using a single patch-clamp technique (4) and the dye coupling was examined afterward with a fluorescence microscope. One cell of the pair (usually the NPCE cell of a NPCE-PCE cell couplet) was accessed using a 2-5 MOmega electrode in the whole cell configuration of the patch clamp. After voltage-clamp experiments were completed, the cells were viewed under a fluorescence microscope with an excitation wavelength of 450-488 nm and an emission wavelength >510 nm to confirm that the cells were coupled. The membrane capacitance of cell(s) was calculated off-line with the time-domain analysis technique (15, 16), assuming a "supercell" model for coupled cells. The access resistance (RA) and capacitance can be calculated from the following equations (15)
<IT>R</IT><SUB>A</SUB> = <IT>V ⋅ R</IT><SUB>S</SUB>/(<IT>I</IT><SUB>o</SUB> ⋅ <IT>R</IT><SUB>S</SUB> − <IT>V</IT>) (1)
where V is the step potential, RS is the seal resistance, and Io is the peak capacitative current at time zero
<IT>G</IT><SUB>M</SUB> = 1/[<IT>V</IT> ⋅ <IT>R</IT><SUB>S</SUB>/(<IT>I</IT><SUB>ss</SUB> ⋅ <IT>R</IT><SUB>S</SUB> − <IT>V</IT>) − <IT>R</IT><SUB>A</SUB>] (2)
where GM is the membrane conductance and Iss is the steady-state current
<IT>C</IT><SUB>M</SUB> = &tgr; ⋅ (1/<IT>R</IT><SUB>A</SUB> + <IT>G</IT><SUB>M</SUB>) (3)
where CM is the membrane capacitance and tau  is the time constant.

This model assumes infinite conductance1 between the cells and has been used successfully to measure exocytosis in whole cell clamped tissue (20) with a frequency domain technique. Similarly, if the series resistance compensation accelerates charging, the capacitance can be estimated using a two-compartment model (see appendix of Ref. 16). We have taken the approach of curve fitting the uncompensated capacitative transient produced by a low test pulse (10-20 mV) with a single exponential, using an iterative least-squares method, with RA and tau  as the variable parameters, until the variance was <10-12, to obtain measurements of cell capacitance. The RA values for cell pairs were 66.2 ± 13.0 MOmega (n = 5) in control solution and 35.8 ± 11.2 MOmega in 100 µM CCh, and this recovered to 63.4 ± 11.9 MOmega following washout. Similar results were obtained with the lower dose of CCh: 46.9 ± 4.5 MOmega (n = 5) in control, 32.6 ± 3.0 MOmega in 10 µM CCh, and 46.2 ± 7.4 MOmega following washout. This reduction after exposure to CCh and uncoupling may reflect the removal of the contribution of the pigmented cell to RA following uncoupling and its reappearance following recoupling.

One can estimate cell capacitance from the surface area of the cell. The diameter of NPCE cells is (in µm) 15.76 ± 0.54 (12) and 15.43 ± 0.40 (5) at 22 and 35°C, respectively, whereas that of the PCE cells is (in µm) 16.14 ± 0.28 (20) and 15.17 ± 0.30 (19) at 22 and 35°C, respectively. With measurements of 2.6 µF/cm2 for membrane capacitance (12) and a temperature dependence of 1.5%/°C (1), these values equate with calculated capacitance values of 20.3 and 20.9 pF at 22 and 35°C, respectively, for the NPCE cells and 21.3 and 20.2 pF at 22 and 35°C, respectively, for PCE cells. These values are likely to be underestimates, since they are based on the cell diameter and thus make no allowances for cell membrane folds and microvilli with which these cells are well endowed (14).

All recordings were performed with a List L/M EPC-7 (List Medical, Darmstadt, Germany) patch-clamp amplifier in voltage-clamp mode with low-pass filtering at 10 kHz. Recordings were made both with electrode capacitance compensated and uncompensated by the List amplifier and were stored after digitization at 5 kHz through a CED1401 interface (CED, Cambridge, UK) on a computer (DCS 486DX-66 PC, Edinburgh, UK). Pulse generation, data capture, analysis, and curve fitting were performed with the vCLAMP and PATCH series of programs (CED).

The effects of uncoupling agents on the capacitance were also tested, with measurements taken before and after bath application of CCh, 1-octanol, and 10 mM Ca2+.

In dye-coupling experiments, the cell pairs were injected with 1-2 mM lucifer yellow while being viewed on an optical fluorescence (Leica DM-1L) or confocal (Noran Odyssey) microscope, using intracellular electrodes of ~10-30 MOmega impedance. In these experiments, the bath Ca2+ was lowered to 0.5 mM. Experiments were performed at room temperature (20-22°C) and at body temperatures of 34-37°C, as indicated.

Data are presented as means ± SE.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

One cell of the NPCE-PCE cell pair was whole cell patch clamped with a pipette containing lucifer yellow. In all cases in which dye perfused both cells of the NPCE-PCE pair, a process that was complete in ~2 min, the cell membrane capacitance displayed a component with a long time constant (3-10 ms). In cell pairs that did not display the long time constant (electrical coupling), the dye perfused only the cell to which the patch pipette was attached. With the assumption that the cell pairs function as a two-compartment syncytium or supercell connected by a junction of infinite conductance, we have calculated the capacitance of single cells and electrically coupled cell pairs. The average cell capacitance of single cells was approximately one-half that of coupled cell pairs in which the cells were of equivalent size (Fig. 1). The mean capacitance of coupled pairs was 79.8 ± 4.3 pF (n = 47) compared with single cell capacitances of 36.8 ± 3.4 pF (n = 10) for PCE cells and 38.1 ± 3.1 pF (n = 15) for NPCE cells.


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Fig. 1.   Cell capacitance in single cells and coupled pairs. A comparison of cell membrane capacitance of single ciliary epithelial cells and coupled pairs of pigmented ciliary epithelial (PCE)-nonpigmented ciliary epithelial (NPCE) cells. Mean capacitance of coupled pairs was 79.8 ± 4.3 (SE) pF (n = 47) compared with a single cell capacitance of 37.6 ± 2.2 pF (n = 25) for all ciliary epithelial cells. Different cell types had capacitances of 36.8 ± 3.4 pF (n = 10) for PCE cells and 38.1 ± 3.1 pF (n = 15) for NPCE cells. Experiments were performed at room temperature (20-22°C).

Effects of uncoupling agents. In experiments with known gap junction uncoupling agents, we determined the cell capacitance in cell pairs before and after the bath application of the agent. Octanol (1 mM), CCh (100 µM, a muscarinic agonist that can raise cell Ca2+), and raised bath Ca2+ (10 mM) caused an uncoupling, as measured by a decrease in capacitance. The results indicate that the average capacitance of cell pairs decreased by about one-third after application of uncoupling agents (Fig. 2). The time constants of the capacitance transient induced by a 20-mV step (Fig. 3) decreased in the presence of these uncoupling agents, allowing a calculation of change in capacitance. The mean capacitance of cells at 20-22°C decreased ("uncoupled") from 70.5 ± 8.0 to 48.0 ± 5.2 pF (n = 5) when exposed to 1 mM octanol (5/6 cells), from 73.8 ± 9.2 to 43.16 ± 9.5 pF (n = 4) with 100 µM CCh (4/6 cells), and from 80.5 ± 6.7 to 49.9 ± 7.8 pF (n = 4) when the bath Ca2+ was raised to 10 mM (4/6 cells).


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Fig. 2.   Capacitance in cell pairs before and after application of uncoupling agents. A: with application of 1 mM octanol, mean capacitance dropped from 70.5 ± 8.0 to 48.0 ± 5.2 pF (n = 5); P = 0.056. B: mean capacitance drops from 80.5 ± 6.7 to 50.0 ± 7.8 pF (n = 4) when the bath Ca2+ (Ca2+e) is raised to 10 mM; P = 0.0002. C: bath application of 100 µM carbachol (CCh) reduces mean capacitance from 73.8 ± 9.2 to 43.2 ± 9.5 pF (n = 4); P = 0.02. All experiments were performed at room temperature (20-22°C). All values shown are means ± SE. Significance was determined using a paired t-test.


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Fig. 3.   Capacitance changes of cell pairs with CCh. A voltage-clamp current transient recorded from an NPCE cell of a PCE-NPCE cell pair. Waveform of the capacitative transient changes when the cell pair is exposed to CCh. Decay of the capacitative transient is longer in control (coupled, broken trace) compared with 100 µM CCh (solid trace), indicating a decrease in capacitance or an uncoupling of the cells. A single exponential was fitted to the capacitative transient by a least-squares algorithm until the variance was <10-12, with access resistance (RA) and time constant (tau ) as the variable parameters. From tau  and RA, the cell capacitance can be determined (see METHODS). Values are as follows: for control, RA = 123.1 MOmega , tau  = 5.6 ms; in 100 µM CCh, RA = 90.8 MOmega , tau  = 1.7 ms. Experiment was performed at 35°C. i, Current; V, voltage.

Dose dependence of CCh-induced uncoupling. The response was concentration dependent, with 100 µM CCh (n = 11) acting more quickly and producing a larger reduction in the capacitance than 10 µM CCh (n = 5). Experiments were performed at the higher temperature of 34-37°C. At this temperature, a slightly lower value for capacitance was measured compared with room temperature. Of 15 cell pairs exposed to 100 µM CCh at 34-37°C, 11 exhibited a drop in capacitance, with no visible change in either cell size, from 51.3 ± 5.6 (n = 7) to 26.0 ± 2.4 pF (n = 7, e.g., Fig. 4). After ~3 min, the bath solution was changed to wash off the CCh. Of the 11 cells that exhibited a reduction in capacitance, 5 responded to washout with a recovery of their capacitance. During this recoupling phase, which took between 4 and 10 min, the capacitance increased from 25.1 ± 1.7 to 41.0 ± 3.3 pF (n = 5) (Fig. 4). In comparison, the capacitance of coupled cells exposed to 10 µM CCh decreased from 53.7 ± 9.7 to 33.5 ± 3.3 pF (n = 7), of which 5 recoupled once CCh was washed off and their capacitance increased to 48.6 ± 5.5 pF after 6-10 min. The half-time of uncoupling was 124.3 ± 12.6 s for 100 µM CCh compared with 248.6 ± 42.0 s for 10 µM CCh, and the half-time of recoupling was 728.6 ± 308.5 s (n = 5) for 100 µM CCh compared with 211.4 ± 41.2 s (n = 5) for 10 µM CCh. Both uncoupling and recoupling with different doses of CCh (Fig. 4) were significantly different (two-tail t-test, P = 0.025).


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Fig. 4.   Electrical uncoupling and recoupling by CCh. Membrane capacitance (in pF) of an NPCE-PCE cell pair is plotted as a function of time during application of CCh. Examples of uncoupling and recoupling by 10 µM CCh (bullet ) and 100 µM CCh (black-square) are illustrated. Capacitance values are normalized to control value. Experiments were performed at 35°C.

Dye spread following uncoupling. The recoupling of NPCE-PCE cell pairs following CCh exposure and washout was confirmed with the spread (by diffusion) of lucifer yellow and confocal microscopy at room temperature. First, cell pairs were uncoupled by 100 µM CCh in the bath solution. They were then impaled with intracellular electrodes containing lucifer yellow. The extracellular Ca2+ concentration was reduced to 0.5 mM to reduce influx around the electrode. In control solutions, lucifer yellow spread to both impaled and unimpaled cells of a coupled pair within 2-4 min by diffusion (n = 3). In the presence of 100 µM CCh, the dye was confined to the impaled cell of the pair. The cell pairs remained uncoupled until washout of CCh, after which the dye spread to the unimpaled cell with a time course similar to that of the observed increase in capacitance (Fig. 5, see also Fig. 4).


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Fig. 5.   Recoupling in NPCE-PCE cell pair shown by dye spread. A: phase contrast image of a pair of NPCE-PCE cells uncoupled by CCh (100 µM). B: confocal fluorescence image of same pair of cells 2 min after uncoupling by CCh. C-F: fluorescence images taken at the intervals indicated, illustrating dye (2 mM lucifer yellow) spread as the cells recouple. Digital images were taken with a confocal microscope (Noran Odyssey), with ×20 water immersion phase-contrast objective. Experiments were performed at room temperature (20-22°C).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

In this study, we have demonstrated the functional coupling of NPCE-PCE cell pairs using both electrical capacitance measurements and dye diffusion. The junction between the cell pair is modulated by several agents such as octanol and high external Ca2+, as has been shown in the intact rabbit CE (21). The loss of cell-to-cell coupling in the presence of the muscarinic agonist CCh has been demonstrated both electrically and with dye spread. The observation that high external Ca2+ causes junctional closure is consistent with a role for internal Ca2+ in this process.

Gap junction coupling is thought to contribute to the ability of many fluid-secreting and -absorbing epithelia to function as a syncytium, allowing cells to coordinate and synchronize their behavior (25, 31). However, the CE presents a different aspect of functional coupling, since the fluid secretion is across the two cell types and the junctions are in series (as well as parallel). In other tissues where secretomotor agonists affect gap junctions, such as pancreatic, salivary, or lacrimal glands (23), the gap junctions are lateral connections between the cells, thus coordinating their responses and allowing passage of ions and second messengers. Any effect on the gap junctions in the ciliary bilayer will either allow or prevent the flow of ions, water, and second messengers between the two cell types and will therefore modulate fluid secretion.

Recent evidence from Ussing chamber studies of the short-circuit current across the rabbit iris-ciliary body suggests a Ca2+-mediated cholinergic inhibition of the NPCE-PCE junctional path (29). Our present study therefore confirms this finding. Schütte and Wolosin (27) suggest that the gap junctions connecting these two cell types may rectify with respect to Ca2+ mobilization, allowing Ca2+ or a "mediator" that causes Ca2+ release in the PCE cells to pass from the NPCE to the PCE only. The data we have presented do not contradict this idea. We found that 100 µM CCh caused uncoupling with a half-time of 124 s; the peak of the Ca2+ mobilization response to epinephrine and CCh in Schütte and Wolosin's study occurred at 1 min, well before the half-time of the uncoupling we observed. In their study, the authors (27) pointed out that a very large local messenger gradient may overcome a reduction in junctional permeability, which would result from the elevated Ca2+ concentration, and allow the Ca2+-mobilizing signal to pass from the NPCE to the PCE cells. Together, the results of Shi et al. (29), Schutte and Wolosin (27), and our present study suggest that the NPCE-PCE junction is closed by increases in [Ca2+]i, as in other tissues (22).

We have also demonstrated for the first time the ability of the cells to recouple after uncoupling by CCh, both electrically and with dye spread. The NPCE-PCE cell pairs will become fully functionally coupled ~7 min (using dye spread at 20-22°C) to 18 min (using capacitance calculation at 32-34°C) after the muscarinic agonist CCh is washed off. This time course is relatively slow and may reflect that of cellular uptake and homeostatic mechanisms for Ca2+ or other second messengers. We have previously demonstrated that the response to CCh of single PCE cells at room temperature is fully repeatable after this time (33).

The important question is whether modulation of junctional conductance affects secretion. Current models of aqueous humor secretion postulate a vectorial flow of ions across the ciliary bilayer (3, 32-34), with the two cell types acting together. The effect of closing the gap junctions between homotypic cells (NPCE-NPCE cells and PCE-PCE cells) could increase secretion by reducing damping and synchronization, thereby increasing the current amplitude as in other secretory tissues (28). However, what is the effect of closing the heterotypic junction between the NPCE and PCE cells? This would allow control over secretion. The CE may be described by a two-compartment model. The first stage would be the loading of ions and solute into the first compartment, the PCE cells, with subsequent water loading. If the junction was open, ions, solute, and water would flow into the second compartment, the NPCE cells. It has been suggested (35) that this would cause the NPCE cells to swell, activating volume-sensitive ion channels. Ions, solute, and water would then efflux into the eye. We have demonstrated that coupling between the two compartments is under muscarinic control, and this would allow a regulation of transfer of solute and water and would thus modulate secretion. The effects of muscarinic stimulation on aqueous secretion and IOP are unclear. Previous reports have found an increase (18), a decrease (9), or no change (11) in the production of aqueous humor. Once the uncoupling has occurred with muscarinic elevation of [Ca2+]i, we have shown that the junctions will reopen as, presumably, normal Ca2+ levels are restored.

We have previously shown the transient activation of K+ channels in the PCE by CCh (33). Activation of K+ channels in other secretory epithelia has been associated both with K+ efflux and with providing the hyperpolarizing drive for Cl- efflux (5, 19), as first proposed in the models of Cartwright and co-workers (2) and Dharmsathaphorn and Pandol (6). Thus muscarinic stimulation could control aqueous humor secretion at two levels: first, the efflux of K+ and/or Cl- and, second, the transfer of solute across the tissue.

This study, by demonstrating that the junctional coupling between the two cell layers of the CE (the NPCE and PCE cells), opens up a novel target for pharmaceutical intervention in glaucoma therapy. The control of aqueous secretion by selectively targeting the junctional conductance needs to be investigated.

    ACKNOWLEDGEMENTS

This study was supported by the Medical Research Council, the Royal Society, and the Wellcome Trust.

    FOOTNOTES

J. W. Stelling is a Wellcome Trust Vision Research Fellow.

1 The gap junctional conductance is clearly not infinite, but the consequences of a finite gap junctional resistance (Rj) do not seriously affect our conclusions. When Rj is zero, we have a supercell and the capacitance of the cell pair is 2× that of a single cell. When Rj is infinite, then we only measure the capacitance of cell 1, i.e., a single cell capacitance. For any value of Rj between these two extremes, we will underestimate the capacitance associated with cell 2.

Address for reprint requests: T. J. C. Jacob, Physiology Unit, PO Box 911, MOMED, Univ. of Wales, Cardiff CF1 3US, UK.

Received 8 January 1997; accepted in final form 26 August 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Cell Physiol 273(6):C1876-C1881
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