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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>i</SUB><SUP>2+</SUP></UP>) increased after the addition of the agonist ATP, coupling was unaffected during the period that Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> was maximal. However, coupling decreased transiently ~5-10 min after agonist addition when Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> returned to resting levels, indicating that this transient decrease in coupling was unlikely due to a direct action of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> on gap junctions. An increase in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> alone could regulate gap junctions. Thus Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>i</SUB><SUP>2+</SUP></UP>) (10). Although it has long been known that Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> and gap junctional coupling in the same cells simultaneously. The techniques currently available for studying Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> and coupling in cell monolayers have been limited to ±changes in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> (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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> within the physiological range results in a sustained uncoupling of lens epithelial cells.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>i</SUB><SUP>2<UP>+</UP></SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP>. 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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 MOmega 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> and gap junctional coupling was curve fit to a four-parameter logistic (Hill) equation (12) using SigmaPlot (SPSS, Chicago, IL).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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<UP><SUB>i</SUB><SUP>2<UP>+</UP></SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP>) (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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> (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.

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<UP><SUB>i</SUB><SUP>2+</SUP></UP> and the surrounding cells, and 2) whether an increase in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> experienced by both sides of the gap junctional channel reduced the rate of fura 2 fluorescence quenching. Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> (cells 3 and 4) with the rate of quenching in the cell that maintained its Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> (B) and total fura 2 fluorescence (C) during 2 cell-to-cell Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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.

Effect of an agonist-mediated Ca<UP><SUB>i</SUB><SUP>2<UP>+</UP></SUP></UP> increase on gap junctional coupling. In other cell types, agonist-mediated increases in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> was either at its peak concentration or still elevated relative to the prestimulation Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> in this cell (Fig. 4). When Mn2+ was injected 30 s after the peak increase in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> while Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> was not elevated sufficiently to close the gap junctions, or that agonist-induced elevation of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> increase of an agonist-mediated Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> transient. A: effect of extracellular ATP (100 µM) on Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> in 5 selected cells. B: effect of the agonist-mediated Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> when it is saturated with Mn2+, only the Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> from cell 5 is shown for the whole time course. The numbered traces correspond to the numbered cells (inset). The average maximal Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> was 515 ± 29 nM (n = 11 experiments; Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> for each experiment was calculated from 25 cells). Data are representative of those obtained in 11 similar experiments.

To determine whether uncoupling occurs at some time after the initial ATP-mediated Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> (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<UP><SUB>i</SUB><SUP>2+</SUP></UP> increase on gap junctional coupling between lens epithelial cells. A: effect of addition of extracellular ATP (100 µM) on Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> over time. Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> was calculated as the average of the Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP>. 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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.

Effect of ionomycin-mediated Ca<UP><SUB>i</SUB><SUP>2<UP>+</UP></SUP></UP> increase on gap junction-mediated cell-to-cell coupling. To determine the extent of uncoupling that was due to an increase in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> per se, the Ca2+ ionophore ionomycin was used to increase Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> (Fig. 6A). The final equilibrium Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> and time. For example, in one experiment (Fig. 6, A and B, squares), Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> reached an equilibrium concentration of ~900 nM, and uncoupling was complete within 30 s of the peak increase in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>. In contrast, in another experiment where the cells maintained a lower Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> (~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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> due to an intervening step(s), after 3-5 min the critical factor affecting the extent of uncoupling was Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>. Similar results were obtained in experiments using Alexa Fluor 594 (Fig. 6E). Control cells had an average Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> on gap junctional coupling between lens epithelial cells. A: average Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> response to ionomycin (iono) addition in the presence of "high" extracellular Ca2+ concentration [either 5 mM Ca2+ ( and diamond ) or 10 mM Ca2+ (open circle , , and triangle )]. 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> assessed with Alexa Fluor 594 dye transfer. Average Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> was 112 ± 9 nM in the presence of 1.8 mM extracellular Ca2+ (ionomycin, low Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>) and 1,198 ± 138 nM in the presence of 11.8 mM extracellular Ca2+ (ionomycin, high Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>). 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.

To determine whether the gap junctional uncoupling observed after the addition of ionomycin required a sustained increase in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>, the effect of a transient increase in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> on gap junctional coupling was investigated. When extracellular Ca2+ concentration was 1.8 mM, ionomycin addition resulted in a brief transient increase in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> (Fig. 6C). The magnitude of the transient Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> increase (Fig. 6C) was similar to the magnitude of the sustained Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> increases achieved by either mechanical activation (Fig. 2) or ionomycin plus elevated extracellular Ca2+ (Fig. 6A), yet the transient Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> increase did not affect the extent of gap junctional coupling measured using either Mn2+ (Fig. 6D) or Alexa Fluor 594 (Fig. 6E). The Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> increase was transient likely because of the endoplasmic reticulum and plasma membrane Ca2+ pumps of lens epithelial cells that can restore Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2<UP>+</UP></SUP></UP> dependence of gap junction-mediated cell-to-cell coupling. To better define the relationship between Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> and gap junctional coupling, the extent of junctional coupling was assessed over a range of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> concentrations. Figure 7 shows the results from one of these experiments in which Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> (Fig. 7, pseudocolor images). This illustrates a major advantage of measuring Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> in the very same cells in which gap junctional coupling is assessed. In the presence of 2 µM ionomycin, when Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> (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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> on gap junctional coupling between lens epithelial cells. Each row of images shows Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> before Mn2+ injection (left) and fura 2 fluorescence intensity at 340-nm excitation before (middle) and 5 min after (right) injection of Mn2+. Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> on gap junctional coupling between lens epithelial cells. Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> and activating protein kinase C (10). These two pathways can be delineated by comparing the agonist-mediated increases in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> with the ionomycin-mediated increases in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>. Agonist-mediated uncoupling of lens cells occurred between 6 and 15 min after the maximal increase in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> (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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP>, 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> elicited by the Ca2+ wave. Indeed, when ionomycin was used to increase Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>, 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> with an IC50 of ~300 nM, which is well within the physiological range (0.1-1 µM). However, increases in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arellano, RO, Rivera A, and Ramon F. Protein phosphorylation and hydrogen ions modulate calcium-induced closure of gap junction channels. Biophys J 57: 363-367, 1990[Abstract].

2.   Berridge, MJ. Inositol trisphosphate and calcium signalling. Nature 361: 315-325, 1993[ISI][Medline].

3.   Cao, F, Eckert R, Elfgang C, Nitsche JM, Snyder SA, Hulser DF, Willecke K, and Nicholson BJ. A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes. J Cell Sci 111: 31-43, 1998[Abstract/Free Full Text].

4.   Churchill, GC, Atkinson MM, and Louis CF. Mechanical stimulation initiates cell-to-cell calcium signaling in ovine lens epithelial cells. J Cell Sci 109: 355-365, 1996[Abstract/Free Full Text].

5.   Crow, JM, Atkinson MM, and Johnson RG. Micromolar levels of intracellular calcium reduce gap junctional permeability in lens cultures. Invest Ophthalmol Vis Sci 35: 3332-3341, 1994[Abstract].

6.   Duncan, G, Webb SF, Dawson AP, Bootman MD, and Elliott AJ. Calcium regulation in tissue-cultured human and bovine lens epithelial cells. Invest Ophthalmol Vis Sci 34: 2835-2842, 1993[Abstract].

7.   Elfgang, C, Eckert R, Lichtenberg-Frate H, Butterweck A, Traub O, Klein RA, Hulser DF, and Willecke K. Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J Cell Biol 129: 805-817, 1995[Abstract].

8.   Firek, L, and Weingart R. Modification of gap junction conductance by divalent cations and protons in neonatal rat heart cells. J Mol Cell Cardiol 27: 1633-1643, 1995[ISI][Medline].

9.   Garcia-Sancho, J, Alonso MT, and Sanchez A. Receptor-operated calcium channels in human platelets. Biochem Soc Trans 17: 980-982, 1989[ISI][Medline].

10.   Goodenough, DA, Goliger JA, and Paul DL. Connexins, connexons, and intercellular communication. Annu Rev Biochem 65: 475-502, 1996[ISI][Medline].

11.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

12.   Hill, AV. The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J Physiol 40: iv-vii, 1910.

13.   Kita, A, and Armstrong W. A biotin-containing compound N-(2-aminoethyl)biotinamide for intracellular labeling and neuraonal tracing studies: comparison with biocytin. J Neurosci Methods 37: 141-150, 1991[ISI][Medline].

14.   Lazrak, A, and Peracchia C. Gap junction gating sensitivity to physiological internal calcium regardless of pH in Novikoff hepatoma cells. Biophys J 65: 2002-2012, 1993[Abstract].

15.   Massas, R, and Benayahu D. Parathyroid hormone effect on cell-to-cell communication in stromal and osteoblastic cells. J Cell Biochem 69: 81-86, 1998[ISI][Medline].

16.   Maurer, P, and Weingart R. Cell pairs isolated from adult guinea pig and rat hearts: effects of [Ca2+]i on nexal membrane resistance. Pflügers Arch 409: 394-402, 1987[ISI][Medline].

17.   Mobbs, P, Becker D, Williamson R, Bate M, and Warner A. Techniques for dye injection and cell labelling. In: Microelectrode Techniques (2nd ed.), edited by Ogden D.. Cambridge, UK: Company of Biologists Limited, 1994.

18.   Niessen, H, Harz H, Bedner P, Kramer K, and Willecke K. Selective permeability of different connexin channels to the second messenger inositol 1,4,5-trisphosphate. J Cell Sci 113: 1365-1372, 2000[Abstract/Free Full Text].

19.   Noma, A, and Tsuboi N. Dependence of junctional conductance on proton, calcium and magnesium ions in cardiac paired cells of guinea-pig. J Physiol 382: 193-211, 1987[Abstract].

20.   Peracchia, C. Calcium effects on gap junction structure and cell coupling. Nature 271: 669-671, 1978[ISI][Medline].

21.   Peracchia, C. Increase in gap junction resistance with acidification in crayfish septate axons is closely related to changes in intracellular calcium but not hydrogen ion concentration. J Membr Biol 113: 75-92, 1990[ISI][Medline].

22.   Pereda, AE, and Faber DS. Activity-dependent short-term enhancement of intercellular coupling. J Neurosci 16: 983-992, 1996[Abstract].

23.   Rose, B, and Loewenstein WR. Permeability of cell junction depends on local cytoplasmic calcium activity. Nature 254: 250-252, 1975[ISI][Medline].

24.   Sanderson, MJ, Charles AC, Boitano S, and Dirksen ER. Mechanisms and function of intercellular calcium signaling. Mol Cell Endocrinol 98: 173-187, 1994[ISI][Medline].

25.   Spray, DC, Stern AL, Harris AL, and Bennett MV. Gap junctional conductance: comparison of sensitivities to H and Ca ions. Proc Natl Acad Sci USA 79: 441-445, 1982[Abstract].

26.   TenBroek, EM, Johnson RG, and Louis CF. Cell-to-cell communication in a differentiating ovine lens culture system. Invest Ophthalmol Vis Sci 35: 215-228, 1994[Abstract].

27.   Yang, DI, and Louis CF. Molecular cloning of ovine lens connexin44 and temporal expression patterns of gap junction proteins in lens primary cell culture. Invest Ophthalmol Vis Sci 41: 2558-2564, 2000[Abstract/Free Full Text].

28.   Yule, DI, Stuenkel E, and Williams JA. Intercellular calcium waves in rat pancreatic acini: mechanism of transmission. Am J Physiol Cell Physiol 271: C1285-C1294, 1996[Abstract/Free Full Text].


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