Ca2+ regulation of gap junctional coupling in
lens epithelial cells
Grant C.
Churchill,
Monica M.
Lurtz, and
Charles F.
Louis
Department of Biochemistry, Molecular Biology and Biophysics,
University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
The quantitative effects of Ca2+
signaling on gap junctional coupling in lens epithelial cells have been
determined using either the spread of Mn2+ that is imaged
by its ability to quench the fluorescence of fura 2 or the spread of
the fluorescent dye Alexa Fluor 594. Gap junctional coupling was
unaffected by a mechanically stimulated cell-to-cell Ca2+
wave. Furthermore, when cytosolic Ca2+ concentration
(Ca
) increased after the addition of the agonist
ATP, coupling was unaffected during the period that
Ca
was maximal. However, coupling decreased
transiently ~5-10 min after agonist addition when
Ca
returned to resting levels, indicating that this
transient decrease in coupling was unlikely due to a direct action of
Ca
on gap junctions. An increase in
Ca
mediated by the ionophore ionomycin that was
sustained for several minutes resulted in a more rapid and sustained
decrease in coupling (IC50 ~300 nM Ca2+, Hill
coefficient of 4), indicating that an increase in Ca
alone could regulate gap junctions. Thus Ca
increases that occurred during agonist stimulation and cell-to-cell Ca2+ waves were too transient to mediate a sustained
uncoupling of lens epithelial cells.
calcium; gap junction; manganese quench; fura 2
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INTRODUCTION |
CELLS CAN COMMUNICATE
DIRECTLY by exchanging small molecules (<1 kDa) through gap
junctional channels that are regulated by several mechanisms including
phosphorylation, cytosolic pH, and cytosolic Ca2+
concentration (Ca
) (10). Although it
has long been known that Ca
can close gap junctions
(20, 23), the Ca2+ reported for this
uncoupling has varied widely from nanomolar (5, 14, 19,
21) to low micromolar (16, 23) to hundreds of
micromolar (1, 8, 25). Moreover, complete
concentration-response relationships have only been generated using
variations of the technique of dual whole cell patch clamp (8,
14, 19), the results of which may not be directly comparable to
the Ca
measured with a Ca2+-sensitive
dye in a monolayer of intact cells, the typical experimental conditions
used for studying Ca2+ signaling. This point is of
particular importance for cell-to-cell Ca2+ waves mediated
by gap junctions (4, 24) in which the possible effect of
Ca2+-mediated uncoupling of gap junctions has not been
examined previously.
It would be desirable to study the effects of Ca
and
gap junctional coupling in the same cells simultaneously. The
techniques currently available for studying Ca
and
coupling in cell monolayers have been limited to ±changes in
Ca
(5, 15). In cell monolayers, several
assays have been used to assess gap junctional coupling (reviewed in
Ref. 17), but none is able to provide rapid quantification
of coupling while simultaneously monitoring Ca2+
concentration. For example, with the dye transfer assay, Lucifer yellow
(442 Da) is the preferred dye because it is fixable and typically
spreads to more cells than other fluorescent tracers (7),
but it cannot be used reliably in cells containing fura 2 due to
overlap of their fluorescence spectra. Other commonly used fluorescent
tracers such as carboxyfluorescein, lissarhodamine, and
4,6-diamidino-2-phenylindole (DAPI) could be imaged concurrently with
fura 2. However, these dyes transfer to fewer cells than Lucifer yellow
(7, 28), making quantification by counting recipient cells
less accurate. Alternatively, the nonfluorescent tracer neurobiotin
(287 Da) (13) could be used because it transfers to more
cells than Lucifer yellow (7). Unlike fluorescent dyes, however, the diffusion of neurobiotin cannot be visualized in real-time
since the cells have to be fixed, permeabilized, and stained to
quantify coupling (13, 17), and, therefore, is not suited
for the rapid quantification of cell coupling.
Recently, Niessen et al. (18) described a technique in
which the gap junction-mediated transfer of the low-molecular-mass (55 Da) Mn2+ can be imaged by its ability to quench the
Ca2+-reporting dye fura 2 (11). We now report
the effects of several different methods that elevate
Ca
on gap junctional coupling in a confluent
monolayer of lens epithelial cells as assessed by the Mn2+
quench of fura 2 fluorescence and Alexa Fluor 594 dye transfer, a
fluorescent dye that can be rapidly and reliably quantified as well as
imaged concurrently with fura 2. The increase in Ca
that occurs during agonist stimulation or a cell-to-cell
Ca2+ wave was of sufficient amplitude but not duration to
decrease gap junctional communication. Agonist stimulation did result
in a delayed, transient decrease in gap junctional coupling that did
not appear to be due to the increase in Ca
per se,
but, rather, it was likely due to a slower covalent modification step
such as the activation of a protein kinase. In conclusion, only a
sustained elevation in Ca
within the physiological
range results in a sustained uncoupling of lens epithelial cells.
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MATERIALS AND METHODS |
Materials.
Sheep eyes were obtained from John Morrell (Sioux Falls, SD) and Iowa
Lamb (Hawarden, IA). Fetal calf serum was obtained from Hyclone (Logan,
UT). Fura 2, fura 2-AM, and Alexa Fluor 594 were obtained from
Molecular Probes (Eugene, OR). Medium 199, Hanks' balanced salt
solution (HBSS), MnCl2, and all other chemicals were
obtained from Sigma Chemical (St. Louis, MO).
Cell culture.
Primary cultures of cells isolated from the equatorial region of fresh
ovine lenses were prepared as described previously (26).
Briefly, eyeballs were removed from freshly slaughtered lambs and
maintained on ice until removal of the lenses 3-7 h later. Six
lenses were placed in 6 ml of HBSS without added Ca2+ and
Mg2+ (HBSS-CMF) and digested with 2.5 mg/ml of trypsin for
15 min. Forty milliliters of ice-cold HBSS-CMF was added to slow the
reaction, and the solution was triturated 20 times. Cells were
centrifuged (230 g for 4 min) and resuspended at a density
of 5 × 105 cells/ml in medium 199, which included
10% fetal calf serum, 100 U/ml penicillin, and 100 U/ml streptomycin.
Two milliliters of this cell suspension was placed into a 35-mm plastic
petri plate containing a 25-mm-diameter glass coverslip coated with poly-L-ornithine (100 µg/ml). Cells were grown in
medium 199 at 37°C in a humidified atmosphere containing 5%
CO2. The ovine lens epithelial cells used in this study
were grown in culture for 5-28 days. Although some cells
differentiated into fiberlike lentoid cells in these cultures
(26), only regions of epithelial-like cells, which we have
shown previously to express both connexin 43 and connexin 49 (27), were used in this study.
Ca
determination and image
analysis.
Ca2+ imaging was performed as described previously
(4). Briefly, cells were loaded with fura 2 by incubation
in 1 µM fura 2-AM in HBSS-H (HBSS supplemented with 10 mM HEPES, pH
7.2) at 22°C in the dark for 20-40 min and were then rinsed
three times with HBSS-H. The glass coverslip with attached cells formed
the bottom of a microincubation culture chamber (MS 200D; Harvard Apparatus, Holliston, MA), which was maintained at 22°C. The
chamber was mounted on the stage of an inverted epifluorescence
microscope (IM 35, Zeiss or TE 300, Nikon) supported on a
vibration-isolated table (Technical Manufacturing, Peabody, MA). Cells
were viewed through a ×40, 1.3 numerical aperture, oil-immersion
objective lens (Fluor 40, Nikon). Ca
was determined by the ratio method and based on an in vitro calibration
(11). Fura 2 was excited alternately at 340 and 380 nm,
and fluorescence at 510 nm was detected with a SIT camera (VE-1000,
Dage-MTI). Images were stored digitally with an optical memory disk
recorder (TQ-3031F; Panasonic, Secaucus, NJ) and processed with the
software Image-1/Fluorescence (Universal Imaging). Alternatively, data collected using the Nikon TE 300 were imaged with a digital
charge-coupled device camera (Hamamatsu Photonics) and processed with
MetaFluor imaging software (Universal Imaging). A background
subtraction and a shading correction were applied before calculating
the ratio image.
Assessment of gap junctional coupling using
Mn2+ quench of fura 2 fluorescence.
The ability of Mn2+ to quench fura 2 fluorescence was used
to assess gap junctional coupling. With a molecular mass of 55 Da, Mn2+ would be predicted to readily pass through gap
junctional channels (cutoff of >1 kDa) (10). Upon
injecting Mn2+ into a single cell in a monolayer of fura
2-loaded cells, the Mn2+ quenched the fura 2 fluorescence
in the injected cell, and this was followed by the spread of this
Mn2+ quenching to the surrounding cells.
Mn2+-mediated quenching of fura 2 fluorescence was assessed
either by directly counting quenched cells 5 min after the injection (cells in which fluorescence was <50% of its initial intensity) or by
continuously monitoring Ca2+-insensitive fura 2 fluorescence (Ftotal). Ftotal was monitored in
the injected and four tiers of surrounding cells to obtain a plot of
quenching over time in the injected and surrounding tiers of cells. As
described previously (9), Ftotal was
calculated with the equation Ftotal = F340 + (xF380), where
F340 is the fluorescence at 340 nm excitation,
F380 is the fluorescence at 380 nm excitation, and
x is a scaling coefficient. The value of x
(typically 0.6-1) was determined empirically by plotting
Ca
and Ftotal over time with
x initially set to 1 and then altering x until
the Ftotal did not change during an increase in
Ca
. In experiments where cell-to-cell transfer of
Alexa Fluor 594 was determined, the number of cells receiving dye was
counted 5 min after the injection of fluorescent dye.
The validity of using fura 2 to simultaneously monitor
Ca
and the spread of Mn2+ was determined
in a spectrofluorimeter (LS-50B Fluorimeter; Perkin-Elmer). A complete
Ca2+ calibration with fura 2 (5 µM) in the presence and
absence of Mn2+ was performed by reciprocal dilutions of
solutions containing 10 mM Ca2+-EGTA and 10 mM EGTA
(Calcium Calibration Buffer Kit C-3721; Molecular Probes). Because
Mn2+ is bound by both EGTA and fura 2, 2 mM
MnCl2 was required to quench fura 2 to ~20% of its
Mn2+-free total fluorescence. Total fura 2 fluorescence was
calculated as described above.
Microinjection.
Micropipettes were pulled from 2-mm outside diameter borosilicate
capillary tubing (WPI, Sarasota, FL) on a Flaming/Brown-type pipette
puller (P-87; Sutter Instruments, Novato, CA). Micropipettes had tip
diameters of <1 µm and resistances of ~60-200 M
when filled with 10 mM MnCl2. MnCl2 was dissolved in
distilled deionized water, and micropipettes were loaded by backfilling
with 10-60 µl of injection solution using a 1-ml syringe and a
28-gauge, plastic-coated glass needle (WPI). Current was delivered
through a chlorodized Ag wire in contact with the micropipette
solution, which contained 10 mM KCl to provide Cl
for the
Ag-AgCl half-cell. The micropipette was positioned with a low-drift
hydraulic micromanipulator (MW-3; Narishige, Greenvale, NY), and
Mn2+ was microinjected with a micropipette lowered 1-3
µm beyond apparent contact with the cell's plasma membrane, which
either impaled or dimpled the cell. Then the "tickle" button was
depressed while delivering current pulses of
30 to
60 nA, applied
for 5 ms every 100 ms for 40-120 s. In the experiments using Alexa
Fluor 594, pipettes contained a 100 µM solution of Alexa Fluor 594 dissolved in distilled deionized water. Alexa Fluor 594 was injected
with a train of 5-ms current pulses applied every 100 ms for 60 s. If the micropipette became plugged, it was replaced with a new micropipette, and the data from such a partial injection were excluded
from the analysis. Current was generated with an electrometer (Intra
767 or Duo 773; WPI). Current duration, magnitude, and polarity were
controlled with a pulse generator (A310 Accupulser; WPI), and current
passage through the micropipette was monitored with an oscilloscope and
verified to be of the expected magnitude during each injection.
Manipulation of
Cai 2+
with ionomycin.
To control Ca
in an agonist-independent manner,
ionomycin (2 µM) was added to the cells, and extracellular Ca2+ concentration was varied from 100 µM to 20 mM, as
reported previously (5, 15), by adding CaCl2
to HBSS without compensating for the increase in ionic strength of the
medium. This enabled Ca
to be maintained at specific
concentrations encompassing the entire physiological range.
Data analysis.
Data are presented as either representative single experiments or the
mean ± SE of the mean based on pooled data from several experiments. All data are presented as the raw number of cells showing
communication for clarity and ease of comparison among figures and with
data from other published reports. Where appropriate, differences among
treatments were determined by analysis of variance with means separated
by Fisher's protected least-significant differences test or the
Student's t-test, with P = 0.01. The concentration-inhibition relationship between Ca
and gap junctional coupling was curve fit to a four-parameter logistic
(Hill) equation (12) using SigmaPlot (SPSS, Chicago, IL).
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RESULTS |
Mn2+ quenching of fura 2 fluorescence
can be used to assess gap junctional coupling.
To determine whether gap junctional coupling between lens epithelial
cells could be assessed by monitoring the passage of Mn2+
through junctional channels, cells were loaded with fura 2, and a
single cell was injected with Mn2+ as described by Niessen
et al. (18). When injected, Mn2+ immediately
quenched the fura 2 fluorescence in the injected cell, and within 5 min
quenched the fura 2 fluorescence in all cells out to at least five
tiers (Fig. 1, A and
B). Five minutes after the Mn2+
injection, the fluorescence in 36 ± 4 (n = 23)
cells was quenched. When the cells were incubated in
CO2-saturated medium, which acidifies their cytosols and
closes gap junctions (25), the quenching was limited to
the injected cell (Fig. 1C). Thus the spread of the fura 2 fluorescence quenching between cells is likely due to the passage of
Mn2+ from cell to cell through gap junctions.

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Fig. 1.
Use of Mn2+ quenching of fura 2 fluorescence
to assess gap junctional coupling among lens epithelial cells.
A: fluorescence images of lens cells containing fura 2 before and at various times after injection of a single cell (labeled
1) with Mn2+. Images reflect 510-nm fluorescence
intensity (340-nm excitation). Scale bar, 20 µm. B: time
course of Mn2+ quenching of fura 2 fluorescence in 5 selected cells (outlined cells). C: effect of
CO2-mediated cytosolic acidification on the time course of
Mn2+ quenching of fura 2 fluorescence in selected cells
(outlined cells). D: effect of Ca2+
concentration on total fura 2 fluorescence in the presence or absence
of partial Mn2+ quench. Fluorescence is expressed as the
percentage of the total Ca2+-insensitive fura 2 fluorescence (Ftotal) and was calculated by summing the
fluorescence intensities at 340-nm excitation with that obtained at
380-nm excitation as described in MATERIALS AND METHODS.
Data are representative of those obtained in 183 (A and
B) and 7 (C) similar experiments.
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One concern with the Mn2+ quench technique is that as the
concentration of Ca2+ is increased, more Mn2+
would be required to obtain the same degree of total quenching of fura
2 fluorescence. Increasing the concentration of one cation will
displace the other because both are in reversible equilibrium with fura
2. High concentrations of Ca2+ could protect fura 2 from
Mn2+ quench, resulting in an underestimate of the spread of
Mn2+. To address this concern, the effect of
Mn2+ on total fura 2 fluorescence was quantified over the
range of Ca2+ concentrations used in this study. Although
Mn2+ quenched fura 2 fluorescence to ~20% of its
control value, Ftotal was not affected by Ca2+
concentration (Fig. 1D). The lack of effect is
likely due to the 50-fold higher affinity of Mn2+ for fura
2 relative to Ca2+ (11).
Effect of a mechanically stimulated
Ca
increase on gap junctional
coupling.
As reported previously, mechanically stimulating a single cell with a
micropipette initiated a cell-to-cell Ca2+ wave (Fig.
2) (4). After ~1 min, the
same micropipette, which contained 10 mM MnCl2, was used to
inject the mechanically stimulated cell. The injected Mn2+
quenched the fura 2 fluorescence of the injected cell and subsequently spread into and quenched the surrounding cells (Fig. 2). These results
demonstrate that a mechanically initiated cell-to-cell Ca2+
wave did not have a detectable effect on gap junctional coupling 1 min
after a cell-to-cell Ca2+ wave. Similar experiments
conducted with Alexa Fluor 594 injected at 1, 5, 10, and 25 min
postmechanical stimulation showed no change in gap junctional coupling
(analysis of variance, P = 0.377; data not shown).

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Fig. 2.
Effect of a cell-to-cell
Ca2+ wave on gap junctional coupling between lens
epithelial cells. A: images showing cytosolic
Ca2+ concentration (Ca ) (ratio) and fura
2 fluorescence intensity (340 nm) during a cell-to-cell
Ca2+ wave and subsequent Mn2+ injection. Images
are from times indicated, which correspond to the x-axis of
the bottom traces in B and C. Fura 2 does not provide an accurate measurement of Ca after
being quenched; therefore, in the last ratio image (293 s), all pixels
in which the fura 2 fluorescence was quenched beyond 60% of its
initial value were excluded from the ratio calculation and appear
black. Time course of Ca (B) and total
fura 2 fluorescence (C) during a cell-to-cell
Ca2+ wave and subsequent Mn2+ injection. The
Ca2+ wave was initiated by mechanically stimulating a
single cell, which was then injected with Mn2+. Data are
representative of those obtained in 18 similar
experiments.
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It was reported previously that hemichannels on the two sides of a gap
junction may be modulated independently (22).
Hemichannel-hemichannel interactions between cells may prevent a
channel from closing when only one hemichannel is receiving a signal to
close. Therefore, it was important to determine 1) whether
the rate of fura 2 fluorescence quenching was reduced between a cell
with elevated Ca
and the surrounding cells, and
2) whether an increase in Ca
experienced
by both sides of the gap junctional channel reduced the rate of fura 2 fluorescence quenching. Ca
in two contacting cells
was increased by mechanically stimulating two cells in quick succession
(Fig. 3). From the comparison of the rate
of quenching in the adjacent cells that experienced a smaller,
transient increase in Ca
(cells 3 and 4) with the
rate of quenching in the cell that maintained its Ca
above the resting level (cell 2), it can be seen that the rate of
quenching was not slowed when the mechanically induced elevation of
Ca
was maintained for up to 2 min.

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Fig. 3.
Effect of mechanically induced Ca2+
elevation in 2 adjacent cells on gap junctional coupling between lens
epithelial cells. A: diagram of the experimental protocol
and spatial relationship of the cells to each other. Time course of
Ca (B) and total fura 2 fluorescence
(C) during 2 cell-to-cell Ca waves
initiated by mechanically stimulating 2 different cells (cell
1 and cell 2) followed by Mn2+ injection
into cell 1. Data are representative of those obtained in 12 similar experiments.
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Effect of an agonist-mediated
Ca
increase on gap junctional
coupling.
In other cell types, agonist-mediated increases in
Ca
have been reported to result in the uncoupling of
gap junctional communication when supramaximal concentrations of
agonist are applied (28). To determine whether
agonist-mediated Ca2+ signaling closed gap junctions in
lens epithelial cell cultures, Mn2+ was injected soon after
the application of the agonist ATP such that Ca
was
either at its peak concentration or still elevated relative to the
prestimulation Ca
in this cell (Fig.
4). When Mn2+ was injected
30 s after the peak increase in Ca
while
Ca
was ~550 nM, there was an extensive
cell-to-cell spread of the Mn2+ quenching of fura 2 fluorescence comparable to that seen before addition of the agonist
(Fig. 4). This result could mean that the Ca2+-dependent
uncoupling is time dependent, that Ca
was not
elevated sufficiently to close the gap junctions, or that agonist-induced elevation of Ca
does not affect gap
junctional coupling between lens epithelial cells.

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Fig. 4.
Gap junctional coupling between lens epithelial cells
during the peak Ca increase of an agonist-mediated
Ca transient. A: effect of extracellular
ATP (100 µM) on Ca in 5 selected cells.
B: effect of the agonist-mediated Ca
increase on the cell-to-cell spread of Mn2+. Although the
experiment used a confluent monolayer of cells, only the 5 cells shown
schematically (inset) were monitored. Note that because fura
2 does not accurately report Ca when it is saturated
with Mn2+, only the Ca from cell
5 is shown for the whole time course. The numbered traces
correspond to the numbered cells (inset). The average
maximal Ca was 515 ± 29 nM (n = 11 experiments; Ca for each experiment was
calculated from 25 cells). Data are representative of those obtained in
11 similar experiments.
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To determine whether uncoupling occurs at some time after the initial
ATP-mediated Ca
increase, Mn2+
injections were performed at ~5-min intervals after the addition of
ATP. Gap junctional coupling decreased within 5 to 10 min after ATP
addition and then recovered to prestimulation levels ~25 min after
the initial elevation of Ca
(Fig.
5, A and
B). This trend is also evident in the data pooled from
several experiments in which the number of coupled cells declined
transiently by approximately twofold (Fig. 5C). To confirm that the decreased cell-to-cell coupling was indeed due to gap junction
closure, cell-to-cell coupling following ATP addition was also
determined using the fluorescent dye Alexa Fluor 594 (Fig.
5D), which also can be used to assess ovine lens cell
coupling (15 ± 0.7 cells coupled under control conditions with
Alexa Fluor 594). The results obtained using Alexa Fluor 594 were very
similar to those obtained using the Mn2+ quench technique
in that there was a 5-9 min delay in the decrease in cell coupling
following addition of ATP to the cell culture and a return to control
levels of coupling within 25 min post-ATP addition (Fig.
5D).

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Fig. 5.
Time-dependent effect of an agonist-mediated
Ca increase on gap junctional coupling between lens
epithelial cells. A: effect of addition of extracellular ATP
(100 µM) on Ca over time. Ca
was calculated as the average of the Ca of the cell
subsequently injected with Mn2+ and the immediately
adjacent cells (typically 6). B: number of cells coupled at
the indicated times before and after the ATP-mediated increase in
Ca . The number of coupled cells was determined by
injecting a single cell with Mn2+ and then monitoring the
cell-to-cell spread of Mn2+ by the quenching of fura 2 fluorescence. C: summary data of gap junctional permeability
assessed with Mn2+ before and after an ATP-mediated
Ca increase. The decrease in gap junctional
permeability is somewhat masked by pooling the raw data because the
time of the decrease varied from 6 to 17 min (13 ± 1 min) and
variation in the absolute number of cells coupled in control conditions
among different experiments, 21 to 50 cells (33 ± 3.1).
D: summary data of gap junctional permeability assessed with
Alexa Fluor 594 dye transfer before and after an ATP-mediated
Ca increase. Data in A and B
are from 1 experiment representative of 11 similar experiments using 7 coverslips of cells, and data in C are results from all 11 experiments.
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Effect of ionomycin-mediated
Ca
increase on gap
junction-mediated cell-to-cell coupling.
To determine the extent of uncoupling that was due to an increase in
Ca
per se, the Ca2+ ionophore ionomycin
was used to increase Ca
in an agonist-independent
manner. When extracellular Ca2+ concentration was
maintained at either 5 or 10 mM (as indicated in Fig.
6A), addition of ionomycin (2 µM) resulted in a sustained increase in Ca
(Fig.
6A). The final equilibrium Ca
varied with
both the concentration of extracellular Ca2+ and the sets
of cells studied (Fig. 6A). The effect on gap junctional coupling was dependent on both Ca
and time. For
example, in one experiment (Fig. 6, A and B,
squares), Ca
reached an equilibrium concentration of
~900 nM, and uncoupling was complete within 30 s of the peak increase in Ca
. In contrast, in another experiment
where the cells maintained a lower Ca
(~500 nM;
Fig. 6, A and B, diamonds), coupling was reduced
to ~10 cells within 3 min following the addition of ionomycin and remained at this level for the next 20 min until Ca
increased to 750 nM (Fig. 6, A and B). This
indicates that although there may be a time requirement for achieving
gap junctional uncoupling in response to an increase in
Ca
due to an intervening step(s), after 3-5 min
the critical factor affecting the extent of uncoupling was
Ca
. Similar results were obtained in experiments
using Alexa Fluor 594 (Fig. 6E). Control cells had an
average Ca
of 112 ± 9 nM and dye transferred
to 16.1 ± 0.5 cells. With addition of ionomycin in the presence
of low (1.8 mM) external Ca2+, the average
Ca
was 158 ± 22 nM, and the degree of dye
transfer (13.0 ± 2 cells) did not decrease significantly.
However, in the presence of both 2 µM ionomycin and elevated
extracellular Ca2+ (11.8 mM), the average
Ca
was 1,198 ± 138 nM, and dye transfer
decreased significantly (5.0 ± 0.7 cells; P < 0.001, t-test).

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Fig. 6.
Effect of a sustained increase in Ca
on gap junctional coupling between lens epithelial cells. A:
average Ca response to ionomycin (iono) addition in
the presence of "high" extracellular Ca2+ concentration
[either 5 mM Ca2+ ( and )
or 10 mM Ca2+ ( , , and
)]. Data from 5 sets of cells (coverslips) with each
represented by a different symbol. B: gap junctional
coupling of cells in A expressed as the number of cells in
which the fura 2 fluorescence was quenched 5 min after a single cell
was injected with Mn2+. C: average
Ca response after ionomycin addition in the presence
of "low" extracellular Ca2+ (1.8 mM). D: gap
junctional coupling of cells in C expressed as the number of
cells in which fura 2 fluorescence was quenched 5 min after a single
cell was injected with Mn2+. E: gap junctional
coupling of cells exhibiting a sustained increase in
Ca assessed with Alexa Fluor 594 dye transfer.
Average Ca was 112 ± 9 nM in the presence of
1.8 mM extracellular Ca2+ (ionomycin, low
Ca ) and 1,198 ± 138 nM in the presence of 11.8 mM extracellular Ca2+ (ionomycin, high
Ca ). Data are from 5 (A and B)
or 4 (C and D) experiments. Experiments in
A-D were conducted by using 1 set (coverslip) of cells
and performing Mn2+ injections at various times in
different regions of the monolayer. Average Ca2+ was
determined immediately before Mn2+ injection, and the
number of cells coupled was determined 5 min after Mn2+
injection. A new region of cells on the same coverslip was then
selected, and the process was repeated. Thus each data point is from a
separate field of view and a different group of cells, but the group of
cells is on the same coverslip.
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To determine whether the gap junctional uncoupling observed after the
addition of ionomycin required a sustained increase in
Ca
, the effect of a transient increase in
Ca
on gap junctional coupling was investigated. When
extracellular Ca2+ concentration was 1.8 mM, ionomycin
addition resulted in a brief transient increase in
Ca
(Fig. 6C). The magnitude of the
transient Ca
increase (Fig. 6C) was
similar to the magnitude of the sustained Ca
increases achieved by either mechanical activation (Fig. 2) or
ionomycin plus elevated extracellular Ca2+ (Fig.
6A), yet the transient Ca
increase did
not affect the extent of gap junctional coupling measured using either
Mn2+ (Fig. 6D) or Alexa Fluor 594 (Fig.
6E). The Ca
increase was transient likely
because of the endoplasmic reticulum and plasma membrane
Ca2+ pumps of lens epithelial cells that can restore
Ca
to resting levels (6). For
example, during calibration of fura 2 in human lens cells,
saturation of the dye was not achieved with ionomycin and elevated
extracellular Ca2+ alone but only when the plasma membrane
Ca2+ pump was inhibited (6).
Ca
dependence of gap
junction-mediated cell-to-cell coupling.
To better define the relationship between Ca
and gap junctional coupling, the extent of junctional coupling was
assessed over a range of Ca
concentrations. Figure
7 shows the results from one of these
experiments in which Ca
was controlled by incubating the cells with ionomycin (2 µM) and varying the concentration of
extracellular Ca2+ from 1.8 to 20 mM to achieve
Ca
values that ranged from 50 to 1,000 nM, the
approximate physiological range in most cells (2),
including lens epithelial cells (4). Note that even when
exposed to the same concentrations of ionomycin and extracellular
Ca2+, different cells maintained different
Ca
(Fig. 7, pseudocolor images). This illustrates a
major advantage of measuring Ca
in the very same
cells in which gap junctional coupling is assessed. In the presence of
2 µM ionomycin, when Ca
was ~100 nM, cells were
well coupled (Fig. 7, top). As the extracellular concentration of Ca2+ was increased, there was an
accompanying increase in Ca
(Fig. 7, pseudocolor
images) that correlated with a decrease in the number of fura
2-containing cells that were quenched after the injection of
Mn2+ (Fig. 7, 340-nm images). When the average
Ca
of the injected and immediately adjacent cells
was 950 nM, Mn2+ quenching of the fura 2 fluorescence was
limited entirely to the injected cell (Fig. 7, bottom).

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Fig. 7.
Effect of Ca
on gap junctional coupling between lens epithelial cells. Each row of
images shows Ca before Mn2+ injection
(left) and fura 2 fluorescence intensity at 340-nm
excitation before (middle) and 5 min after
(right) injection of Mn2+. Ca
was varied by adding ionomycin (2 µM) to the cells while they were
bathed in medium with ~40 nM free Ca2+ and then
increasing Ca2+ concentration in the medium by adding
1-µl aliquots of a 1 M CaCl2 solution. The average
Ca for a region comprising the injected and
immediately adjacent cells was 150, 340, 650, and 920 nM
Ca2+, respectively, from top to
bottom. Data are representative of 8 similar
experiments.
|
|
The results from eight experiments similar to the one shown in Fig. 7
are summarized in a plot of Ca
vs. the number of
cells coupled (Fig. 8). This
concentration-inhibition relationship was well fit
(r2 of 0.83) by a four-parameter logistic
equation (12) that yielded an IC50 of 286 ± 21 nM, a Hill coefficient of
4.1 ± 1.0, a
y-maximum response of 37 ± 3.4, and a
y-minimum response of 1.0 ± 2.1.

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Fig. 8.
Effect of ionomycin-adjusted Ca on gap
junctional coupling between lens epithelial cells.
Ca was adjusted by incubating the cells with
ionomycin and varying extracellular Ca2+ concentrations.
Gap junctional coupling was assessed by monitoring the number of
cells in which fura 2 was quenched 5 min after a single cell was
injected with Mn2+, as described in MATERIALS AND
METHODS. Data were fitted with a four-parameter logistic equation
(Hill equation), which yielded an r2 of 0.83, an
IC50 of 286 ± 21 nM, a Hill coefficient
(nH) of 4.1 ± 1.0, a maximum response of
37 ± 3.4, and a minimum response of 1.0 ± 2.1. Data are
from 44 injections of Mn2+ using 8 cultures.
|
|
 |
DISCUSSION |
In this report, we describe an assay in which the gap
junction-mediated transfer of the low-molecular-mass (55 Da)
Mn2+ is imaged by its ability to quench the fluorescence of
the Ca2+-reporting dye fura 2, reflecting the direct
imaging of electrical coupling among cells in a monolayer. Advantages
of this technique include the use of cells in a confluent monolayer,
the measurement of both Ca
and coupling in the same cells in real time, and that a single culture dish of cells can be used
for multiple timed injections. The small size of Mn2+ (55 Da), which is smaller than any of the fluorescent dyes currently used
(287-1,000 Da) (17), should detect levels of
cell-to-cell coupling that, in contrast to previously used fluorescent
dyes, is closer to those achievable by measuring electrical coupling (7, 17), yet Mn2+ can be imaged with commonly
available fura 2 imaging systems. In contrast to certain fluorescent
tracers such as DAPI (279 Da) and lissarhodamine (540 Da) that label
primarily first-tier cells (3, 28) due to either
hydrophobicity or DNA binding limiting their diffusion,
Mn2+ diffuses rapidly through 4 to 7 tiers of cells. We
confirmed results obtained using the Mn2+ quench technique
with the relatively new dye Alexa Fluor 594 (759 Da) that, although it
transfers to fewer cells than Mn2+, can be used
simultaneously with fura 2 because their spectra do not overlap.
Agonists can close gap junctions by both increasing
Ca
and activating protein kinase C
(10). These two pathways can be delineated by comparing
the agonist-mediated increases in Ca
with the
ionomycin-mediated increases in Ca
. Agonist-mediated uncoupling of lens cells occurred between 6 and 15 min after the maximal increase in Ca
(Fig. 5), which is comparable
to the time course reported for direct activation of protein kinase C
with phorbol esters in these cultures (26). In contrast,
uncoupling by an ionomycin-mediated sustained increase in
Ca
was detected after ~30-90 s and was
maximal within 3-5 min (Fig. 6). The slower time course of the
agonist-mediated uncoupling of gap junctions suggests that this
involves a covalent modification step such as the phosphorylation of a
junctional protein. The relative effect of either uncoupling pathway
may depend, however, on both the particular agonist and the cell type.
For example, Yule et al. (28) reported that there was
significant uncoupling of gap junctions during the plateau phase of an
agonist-mediated increase in Ca
in pancreatic acinar cells.
In a confluent monolayer of lens epithelial cells treated with
ionomycin and various concentrations of extracellular Ca2+,
IC50 for the uncoupling of gap junctions was 286 ± 21 nM. This IC50 is consistent with previous reports showing
that nanomolar Ca
can close gap junctions in both monolayers (5) and in isolated cell pairs (14, 16,
19). It should be noted, however, that in contrast to these
previous studies, our IC50 value is based on the
Ca2+-reporting dye fura 2 and an in vitro calibration. It
is widely acknowledged that dyes can underestimate the true
Ca2+ concentration by as much as threefold due to
differences between the cytosolic environment (e.g., pH, free
Mg2+, binding to proteins, viscosity) and the calibration
solutions (11). Therefore, the true IC50 might
be as high as 1 µM. Regardless of what the true IC50 for
Ca
was, the same calibration method was used for
calculating the concentration from fura 2 fluorescence in all the
experiments, so the values obtained are internally consistent. The
point to note is that in lens cells, a transient increase in
Ca
, such as occurs during cell-to-cell
Ca2+ waves, does not result in the uncoupling of gap
junctions. In contrast, agonists such as ATP can produce a transient
and delayed uncoupling that may result from the activation of a protein
kinase. However, the presence and extent of uncoupling is likely
dependent on both the cell type and agonist. A more prolonged plateau
phase of the increase in Ca
would be predicted to
result in significant Ca2+-dependent uncoupling. Indeed,
Yule et al. (28) reported complete gap junctional
uncoupling during the plateau phase in pancreatic acinar cells
responding to cholecystokinin or carbachol.
Although cell-to-cell Ca2+ waves and
Ca2+-dependent uncoupling of gap junctions are inextricably
linked, all previous reports have focused on the effect of modulating
gap junction coupling on the spread of the Ca2+ wave. This
is the first report to examine the reciprocal effect, namely, the
effect of cell-to-cell Ca2+ waves on gap junctional
coupling. During the cell-to-cell Ca2+ wave, gap junctional
coupling was not detectably reduced even though Ca
increased two- to threefold above the IC50 (~300 nM) for
Ca2+-dependent gap junctional uncoupling in these lens
epithelial cells. This lack of uncoupling is likely attributable to the
brief, transient nature of the increase in Ca
elicited by the Ca2+ wave. Indeed, when ionomycin was used
to increase Ca
, gap junctional coupling was only
reduced detectably if the Ca2+ increase was sustained,
suggesting a mechanism of gap junctional closure distinct from the
delayed, transient decrease in gap junction permeability after addition
of agonist.
In summary, we have used Mn2+ quenching and Alexa Fluor 594 dye transfer to investigate the effects of Ca2+ signaling
on gap junctional coupling. Gap junctions are closed by a sustained
elevation of Ca
with an IC50 of ~300
nM, which is well within the physiological range (0.1-1 µM).
However, increases in Ca
mediated by either agonist
or mechanical stimulation were not of sufficient duration to produce a
sustained, Ca2+-dependent uncoupling of lens cells. Rather,
agonists such as ATP that have been shown to activate phospholipase C
and produce a transient but delayed decrease in lens cell-to-cell
coupling are more likely, acting via the diacylglycerol/protein kinase C branch of this bifurcating signaling pathway. Understanding the
molecular basis by which these different protocols affect gap junction
coupling will provide important insights into our understanding of
intracellular Ca2+ signaling in vivo.
 |
ACKNOWLEDGEMENTS |
We thank S. Patel for helpful review and comment.
 |
FOOTNOTES |
This research was supported by National Eye Institute Grant EY-05684.
Present address of G. C. Churchill: Dept. of Pharmacology,
University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.
Address for reprint requests and other correspondence: C. F. Louis, Georgia State Univ., 30 Courtland St., Rm. 326, University Plaza, Atlanta, GA 30303-3083 (E-mail: clouis{at}gsu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 December 2000; accepted in final form 3 May 2001.
 |
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