1 Institute of Bioengineering, Miguel Hernandez University, 03550 San Juande Alicante, Spain; 2 Department of Morphology, University of Geneva, 1211 Geneva 4, Switzerland; and 3 Department of Surgery, National University of Singapore, National University Hospital, Singapore 119074
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ABSTRACT |
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Pancreatic -cells constitute a
well-communicating multicellular network that permits a coordinated and
synchronized signal transmission within the islet of Langerhans that is
necessary for proper insulin release. Gap junctions are the molecular
keys that mediate functional cellular connections, which are
responsible for electrical and metabolic coupling in the majority of
cell types. Although the role of gap junctions in
-cell electrical coupling is well documented, metabolic communication is still a matter
of discussion. Here, we have addressed this issue by use of a
fluorescence recovery after photobleaching (FRAP) approach. This
technique has been validated as a reliable and noninvasive approach to
monitor functional gap junctions in real time. We show that control
pancreatic islet cells did not exchange a gap junction-permeant
molecule in either clustered cells or intact islets of Langerhans under
conditions that allowed cell-to-cell exchange of current-carrying ions.
Conversely, we have detected that the same probe was extensively
transferred between islet cells of transgenic mice expressing connexin
32 (Cx32) that have enhanced junctional coupling properties. The
results indicate that the electrical coupling of native islet cells is
more extensive than dye communication. Dye-coupling domains in islet
cells appear more restricted than previously inferred with other methods.
islets of Langerhans; gap junctions; intercellular communication
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INTRODUCTION |
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INSULIN
SECRETION from the islets of Langerhans is a multicellular event
that arises as an emergent property due to -cell intercellular
communication (16). The physiological heterogeneity found
in individual
-cells, when the response to glucose and signaling
patterns are considered (5, 13, 14, 35, 38), is switched
to a coordinated and synchronized behavior when the
-cell population
communicates (31, 39). Such signal coordination yields a
more vigorous secretion and biosynthesis of insulin (5, 35,
38). Furthermore, when cellular connections are severed or
defective, insulin secretion is markedly reduced (23, 50). Thus cell communication within the islets of Langerhans mainly mediates
signal synchronization (39) and leads to a more effective insulin release.
Among the several mechanisms that control cell-to-cell communication
within pancreatic islets (6), the one mediated by gap
junctions is believed to be essential for the recruitment and
synchronization of insulin-secreting cells (50). Gap
junctions, which improve cell contacts, establish transmission
pathways across the islet of Langerhans, allowing cell-to-cell
interaction (50). This physical link is responsible for
electrical and metabolic communication in several types of cells
(19). However, whereas electrical coupling has been well
documented in pancreatic islets (10) and is believed to
account for signal synchronization of islet cells (39),
the extent of metabolic coupling and its influence on islet
synchronization and physiological response are still a matter of
debate. Iontophoretic injection of dye and the evaluation of its
intercellular transfer have been employed as a model for the passage of
small molecules in several types of cells (3, 44). On the
one hand, the iontophoretic injection of dyes (28) and
metabolites (18) into pancreatic -cells has indicated
the existence of limited, spatially restricted territories of dye coupling, in agreement with the observations of intercellular exchanges
of tritiated nucleotides and metabolites that have also suggested the
presence of territories of metabolic cooperation (5, 22).
On the other hand, studies on electrically coupled pairs of islet cells
have failed to provide evidence for a significant dye coupling
(24, 34), leading to controversial conclusions with regard
to the extent of the intraislet communication network.
In this study, we have used the fluorescence recovery after
photobleaching (FRAP) approach, which has been validated as a reliable
technique to analyze functional gap junction-mediated communication,
because of several advantages that it offers over alternative
techniques (9, 51). First, FRAP is a noninvasive technique
and thus is expected to avoid some of the cell injuries that may occur
during cell injection or manipulation. Second, dye passage between
cells through gap junctions takes place as a passive diffusional
process, resembling the one that might be occurring in vivo. Third, the
temporal resolution of the system stands for the on-line monitoring of
gap junction function. We have used this technique to analyze dye
coupling between native cells of control islets and cells from islets
of transgenic mice that specifically overexpressed connexin 32 (Cx32)
(7). These mice are hereafter referred to as rat insulin
promoter (RIP)-Cx32. We show that, although electrical coupling
was present in both control and transgenic cells, it was markedly
enhanced in the latter (7). In contrast, dye transfer was
not observed in native islet cells but was easily detected between
cells of RIP-Cx32 mice. The results suggest that gap junctions of
native -cells ensure electrical coupling but restrict molecular exchanges.
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METHODS |
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Islet cell isolation.
All experiments were conducted according to regulations approved by our
institution. Seven Swiss albino OF1 and five homozygotic transgenic
(RIP-Cx32 strain) mice (7) were killed by cervical dislocation. The design of the RIP-Cx32 construct and the junctional and physiological characteristics of transgenic mice expressing Cx32
have been shown elsewhere (7). Pancreatic islets were isolated by collagenase digestion as previously described (31, 36) and were dispersed into single cells and clusters by
enzymatic digestion in the presence of 0.05% trypsin and 0.02% EDTA
for 2 min. Cells and clusters were plated onto coverslips and cultured for 24 h in RPMI 1640 supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 5.6 mmol/l glucose. Intact islets were used 2 h after isolation. No significant
difference in cellular coupling was found between normal OF1 and C57B16
mice, which were used to control the experiments on transgenic mice (not shown) (7). We conclude that the majority of
clustered cells that we used in FRAP experiments were pancreatic
-cells, because ~85% of cultured islet cells were identified as
-cells by means of immunofluorescence labeling of insulin content,
as described elsewhere (36). In addition,
-cells can be
easily distinguished by their large size and low nuclear-to-cytoplasmic ratio compared with other islet cell types (4, 36).
Astrocyte isolation. Astrocytes were isolated from cerebral cortices of 1- to 3-day-old rats and cultured for 2-4 wk, as previously described (30, 32). At least 98% of the cells expressed glial fibrillary acidic protein, a glial-specific marker, and had the characteristic flat, type 1-like astrocyte morphology. Astrocytes were plated onto coverslips 2-4 days before use. Coverslips were transferred from the culture medium (DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin) to Hanks' solution (140 mM NaCl, 5.4 KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Na-HEPES, pH 7.35) for photobleaching experiments.
FRAP.
Cell clusters and intact islets were loaded with 5 µmol/l of the
lypophyllic dye 5-(and-6)-carboxyfluorescein diacetate
[5(6)-CFDA; Molecular Probes, Madrid, Spain] for 5 min
at room temperature and then washed with the perfusion medium (see
below). Once inside the cell, the dye is retained due to a conversion
by cytoplasmic esterases into a nonpermeant and fluorescent
carboxyfluorescein (CF) molecule. This Ca2+-independent
fluorescent dye of small size (molecular mass of the hydrolyzed
derivative = 376 Da) has been shown to permeate gap junction
channels of different kinds of cells (9, 30, 51) and of
islet cells when iontophoretic techniques are used (18,
25). Cell clusters or islets were perfused with a modified Krebs-Ringer buffer containing (in mmol/l): 119 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 2.5 CaCl2, which was constantly bubbled with a mixture of 95% O2-5% CO2,
giving a final pH of 7.4. Glucose concentration was 16 mmol/l except
when otherwise indicated. All experiments were performed at 37°C.
Fluorescence was monitored under a Zeiss LSM 510 confocal microscope
(Zeiss, Jena, Germany) by use of a ×40 oil immersion lens (numerical
aperture 1.3) or a ×63 oil immersion lens (numerical aperture 1.25)
and fluorescein filters. Photobleaching was performed by concentrating the confocal laser beam onto a selected cell and transiently increasing its intensity to the maximum (25 mW) for 30 or 60 s for each cell. After bleaching, recovery of fluorescence in this cell was monitored by
recording an image every 4-6 s. Fluorescence was expressed in
arbitrary units (from 0 to 255) as a function of time. The laser
intensity used during image records did not produce photobleaching in
any case. Results were plotted using commercial software (Sigmaplot; Jandel Scientific, Erkrath, Germany). Fluorescence recovery takes place
as a result of dye transfer through gap junctions from surrounding nonphotobleached cells (30, 51). To control the
specificity of the observed fluorescent changes, we first studied
single cells. We observed no recovery of CF fluorescence after these
cells were bleached, indicating that the recovery observed in pairs,
clusters, and islets could not be accounted for by the incorporation of dye present in the medium. Fluorescence recovery was also reversibly blocked by 20 µmol/l 18-glycyrrhetinic acid, a gap junction
blocker, providing further evidence that connexin channels mediated the diffusion of CF from nonbleached into photobleached cells (17, 30).
Calcium measurements. Calcium measurements after bleaching were performed by loading cells with 4 µmol/l fluo 3-AM (Molecular Probes, Madrid, Spain) for 20 min at 37°C and by monitoring fluorescence changes under the aforementioned confocal microscope (31, 36).
Cell viability test. Viability of cells was tested after photobleaching by loading cells with 1 µmol/l ethidium homodimer-1 (Molecular Probes, Madrid, Spain).
Electrophysiological recordings.
Microdissected islets were attached in a 50-µl chamber and perfused
at 37°C with 0.8 ml/min modified Krebs medium supplemented with 11 mM
glucose. Individual cells were impaled using borosilicate glass
microelectrodes of 80-120 M. Cell membrane potentials were recorded using an Axoclamp 2B amplifier acquired using Axoscope 8.2 (Axon Instruments, Foster City, CA) and analyzed using Origin 5.0 (Microcal Software, Northampton, MA). To evaluate electrical synchronization and coupling, two islet cells were simultaneously recorded within a single islet by use of electrodes whose tips were
apart from each other 85 ± 15 µm (1, 2).
Synchronization was evaluated by measuring the interval between
corresponding spikes on the recordings of the two cells.
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RESULTS |
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Electrical coupling was assessed using two microelectrodes that
simultaneously recorded events within two cells of a single islet. We
observed that the glucose-induced typical bursts of electrical
activity, which characterize the insulin-producing -cells, occurred
concomitantly in the cells monitored in both control (Fig.
1A) and transgenic islets
(Fig. 1B). However, whereas small temporal delays (
1 s)
between spikes occurred in cells from control islets (Fig. 1,
A and C),
-cells of RIP-Cx32 transgenic mice
were fully synchronous (Fig. 1, B and D).
Although the duration of the active and silent phases was longer in
transgenic than control cells, the degree of electrical activity, which
is measured by the ratio of active phase to total period, was the same
in both cells (7). Some differences in the electrical
activity may arise because of the existence of two different sets of
connexins mediating intercellular connections, mainly Cx36 in native
islets (41) and Cx32 in transgenic cells (7),
which may provide different junctional conductances (43,
45). Actually, variations of the gap-junctional conductance can
modify synchrony, duration of bursts, and electrical pattern of the
islet network (42). When the cable properties of the
system were evaluated by measuring the propagation of pulses between
cells separated by ~85 µm, we noted that currents injected into
-cells of RIP-Cx32 mice were able to propagate voltage deflections
(Fig. 1D), whereas control cells were not at a detectable
level (Fig. 1C). Note in Fig. 1D that islet cells
from transgenic mice could transmit voltage deflections even during the
silent phase. In this situation, coupling conductance may be smaller
than during the active period (2). These results are
representative of eight experiments from three different preparations for native islet cells and three experiments from three preparations for transgenic islet cells. Thus, although electrical coupling is
widespread in the islet of Langerhans (2, 10, 31, 39), transgenic islets exhibited enhanced electrical synchronization and
coupling properties.
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Dye coupling was tested by monitoring the intercellular transfer of CF
by use of a FRAP approach. To validate the technique, we first set up
the experimental conditions using astrocytes (Fig. 2), since these cells are coupled by
large junctional conductances (11) and show a rapid
recovery of fluorescence after bleaching (30). In these
experiments, we observed that the conditions that we used were adequate
to rapidly assess junctional coupling of astrocytes. FRAP had an
exponential time course (Fig. 2B) with a time constant of
59.21 ± 13.45 s (n = 8 cells from 8 clusters from
2 different preparations). An exponential fluorescence decay was often
recorded from the adjacent nonbleached cells, which were coupled to the
photobleached one (Fig. 2B) and which functioned as donors
of the fluorescent probe. Thus astrocytes provided a reliable model and
could be used as positive controls of FRAP experiments in native islet
cells.
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To investigate whether the laser may have damaged islet cells, we
tested cell viability with ethidium homodimer-1 staining. We observed
that, even under conditions more severe than those employed during the
experiments, the impermeant DNA-binding probe did not stain the nuclei
of photobleached cells (n = 7 from 3 different
preparations), suggesting that these cells were still viable after
laser bleaching (Fig. 3A). In
contrast, nuclei stained red by ethidium homodimer-1 were
occasionally observed in cells that were also unable to retain CF
fluorescence after loading with 5(6)-CFDA (Fig.
3A). Additional CFDA incorporated into the medium resulted
in reaccumulation of the dye in the photobleached cells. Measuring the
glucose-induced Ca2+ changes, after cells were loaded with
the Ca2+-sensitive fluorescent probe fluo 3, provided
another control for cell viability. We observed that cells that had
been submitted to the FRAP protocol (n = 6 cells from 6 clusters from 2 different preparations) showed a typical response
pattern to glucose (48) (Fig. 3B).
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In islet cell clusters cultured for 24 h, no recovery of CF
fluorescence was recorded after photobleaching (n = 16 cells from 10 clusters from 4 different preparations), whatever the
number of clustered cells (Fig. 4, A and
B). To evaluate whether lack of intercellular exchange of CF reflected alterations of gap junctions resulting from the cell isolation procedure, identical experiments were
performed in freshly isolated islets of Langerhans (Fig. 4C). No case of fluorescence recovery was observed
irrespective of whether islets were freshly isolated (n = 30 cells from 17 islets) or cultured for 24 h (n = 12 from 6 islets; not shown) or whether glucose was switched to
different concentrations during fluorescence monitoring
(n = 17 from 7 islets; not shown). Results were
obtained from at least three different preparations.
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Conversely, a consistent fluorescence recovery was recorded when the
FRAP procedure was used to test cells in clusters and in intact islets
of Langerhans obtained from transgenic mice that were forced to
overexpress Cx32 (Fig. 5). Dye passage
may take place between -cells, given that RIP-Cx32 is specifically
expressed in this islet cell population (7, 15), and
recent studies indicate an absence of coupling in non-
-cells
(12, 31, 36). In these experiments, fluorescence recovery
followed an exponential time course with a time constant of 225.9 ± 42.6 s in cell clusters (Fig. 5A; n = 8 cells from 8 clusters) and 182.3 ± 52.6 s in isolated islets of Langerhans (Fig. 5B; n = 3 cells
from 3 islets). Results were obtained from three different
preparations. The donor, nonphotobleached cells exhibited a parallel
fluorescence decay, due to probe transfer to the coupled photobleached
cell receiving this dye (Fig. 5, A and B). The
intensity of this change, which was also observed in astrocytes,
decreased with the increase in the number of clustered cells,
presumably because the source of dye was then enlarged by the presence
of more donor cells. The fluorescence values of recovery varied among
different experiments. It might be attributable to divergences in the
number of donor cells that were coupled in each case or to differences
in tracer dilution due to diffusion as observed in other cell types
(8). At any rate, we demonstrate that, under the same
experimental conditions, native islet cells did not exchange CF,
whereas transgenic cells with enhanced junctional properties allowed
for the passage of the dye.
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DISCUSSION |
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Gap junctions offer permeability to ions and metabolites,
providing a mechanism for intercellular communication in multiple cellular systems (19). The system permits electrical and
metabolic coupling of cells, affecting several cell functions including secretion (19, 52). Iontophoretic injection of dyes and
monitoring of their transfer into neighboring cells have traditionally
been utilized to trace molecular exchanges (3, 44).
However, when applied to pancreatic -cells, this technique has led
to different conclusions. On the one hand, passage of dyes has been
observed between
-cells of monolayer cultures (18, 25)
and whole islets of Langerhans (7, 28), in agreement with
the intercellular exchange of endogenous tritiated nucleotides and
amino acids, which was monitored between the same cells without the use
of cell microinjection (5, 22). On the other hand, the
technique has failed to provide direct evidence for dye coupling in
pairs of electrically coupled islet cells (24,
34). Furthermore, microinjection of dyes into islet
cells has supported the idea of dye coupling between different types of
endocrine islet cells (25, 28). Although the occurrence of
such a coupling is consistent with the observation of typical gap
junction plaques (33) between different types of islet
cells, it does contrast with recent investigations indicating the
absence of calcium signal synchronization and electrical coupling
between
- and non-
-cells and between
- or
-cells (12,
31, 36). Thus it is possible that the microinjection approach
led to overestimated domains of dye coupling made of pancreatic
-cells, possibly because of secondary effects of cell injection
and/or electrophoresis (20).
One way to bypass these problems is to use an alternative, noninvasive methodology such as the FRAP approach, which allows for the on-line monitoring of gap junction-mediated dye transfer between cells (9, 51). In contrast to microinjection (44), cells are not physically invaded during FRAP, with the exception of the exposure to a laser beam, which, however, can be performed under conditions that prevent major cell injury (9, 51). Instead, dyes available in a membrane-permeant form are allowed to diffuse into cells, where they are retained after enzymatic conversion into an impermeant, fluorescent derivative. Furthermore, and contrasting with microinjection procedures that were often used in combination with fixation of the injected cells for subsequent analysis (7, 28), FRAP combined with confocal sectioning allows for a direct measurement of dye passage in freshly isolated islets or living cultures, providing a real-time estimate of junctional permeability. Eventually, compared with microinjection conditions, which impose the prolonged injection of charged currents with various effects on cells (20, 27, 43), FRAP allows for a simple, diffusion-driven passage of dyes from one cell to another along concentration gradients, permitting experimental conditions that may be closer to those that islet cells face under physiological situations (9). In contrast, the electrical gradients imposed during iontophoresis (20) may affect the diffusion of charged fluorescent dyes. In this study, we have taken advantage of the potential benefits of the FRAP approach to revisit the existence of dye coupling between islet cells. Although earlier studies have sustained the idea of domains of metabolic cooperation in islet cells according to the intercellular transfer of several tracers, we show here that molecular exchange is much more constrained than previously reported. Our results suggest that electrical coupling, which is a widespread event in the islet of Langerhans (2, 10, 12, 31, 39), might have a major role in islet synchronization rather than metabolic communication.
We have first shown that FRAP did not affect the viability of islet
cells, as judged by retention after photobleaching of both membrane
impermeability to a DNA-binding dye and glucose-induced increase in
intracellular calcium. We have then observed that the approach allowed
for the detection of transfer of the small dye CF between astrocytes,
as well as between the islet cells of transgenic RIP-Cx32 mice that
show enhanced junctional properties (7). In the latter
model, the observations made with the FRAP approach are fully
consistent with those previously made using dye microinjection
(7), indicating that the latter approach does not, per se,
abolish molecular exchanges between islet cells. In contrast, similar
experimental conditions did not provide evidence for a molecular
exchange between control islet cells that were studied within either
monolayer clusters or isolated islets of Langerhans. Electrical
coupling in the absence of detectable dye passage has been observed in
pairs of pancreatic -cells (24, 34) and several other
systems (29, 37, 40, 47, 49), possibly depending on the
connexin isoform linking the cells under study (21). Islet
cells express several connexin isoforms, among which Cx36 predominates
between
-cells (41). Interestingly, the channels formed
by this protein have a smaller unitary conductance than those formed by
other mammalian connexins (43, 45). This property is well
in agreement with the low junctional conductance recorded from
-cells in pairs (
215 pS) (24, 34) and in whole islet
of Langerhans (
0.5-1 nS) (1, 2, 12). Conceivably, this small unitary conductance may also restrict the intercellular exchange of some dyes between cells coupled by Cx36 channels. Thus
conflicting results about whether Cx36 channels allow dye coupling have
been reported in monolayer cultures of HeLa cells transfected to
express this protein (43, 47). Here, we provide novel
evidence that molecules of the size, electrical charge, and hydrated
volume similar to those of CF cannot permeate the channels, most likely
made by Cx36 (41), that electrically couple mouse islet
cells. The use of other gap junction tracers differing in sizes and
electrical charges (9) is now required to assess whether
this defect is absolute or provides for the cell-to-cell exchange of
only selected types of molecules.
At any rate, the present results suggest that, although electrical
coupling is widespread in the islets of Langerhans, dye coupling is
more restricted, and possibly much more limited for selected molecules,
than what has been previously proposed. The characteristically small
conductance of Cx36 channels and the whole conductance provided by gap
junctions at - to
-cell interfaces ensure electrical
communication but, at least when studied for short-term periods
(15-25 min), restrict the exchange of molecules similar to CF.
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ACKNOWLEDGEMENTS |
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We thank A. Charollais, E. Pérez, N. Illera, and A. Pérez Vergara for technical assistance, and P. L. Herrera for helpful discussion of the manuscript.
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FOOTNOTES |
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Our work was supported in part by grants from Secretaria de Estado de Universidades e Investigacion (PM99-0142), Fundació Marató TV3 (99-1210), Fundación Salud 2000, Juvenile Diabetes Foundation (1-2000-575 to B. Soria and 1-2001-622 to P. Meda), Generalitat Valenciana (GV99-139-1-04), Swiss National Science Foundation (31-67788.02 to P. Meda), European Union (QLG1-CT-1999-00516 to P. Meda), and National Institute of Health (DK-63443 to P. Meda).
Present address of I. Quesada: Department of Bioengineering, University of Washington, Seattle, WA 98195
Address for reprint requests and other correspondence: Bernat Soria MD, Institute of Bioengineering, Miguel Hernandez Univ., San Juan Campus, 03550 San Juan de Alicante, Spain (E-mail: bernat.soria{at}umh.es).
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.
First published January 7, 2003;10.1152/ajpendo.00473.2002
Received 31 October 2002; accepted in final form 29 December 2002.
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