Modulation of Pancreatic Acinar Cell to Cell Coupling during
ACh-evoked Changes in Cytosolic Ca2+*
Marc
Chanson
§¶
,
Patrice
Mollard**,
Paolo
Meda
,
Susanne
Suter§, and
Habo J.
Jongsma
From the
Department of Medical Physiology
and Sport Medicine, Utrecht University, 3508TA Utrecht, The
Netherlands, ** INSERM Unit 469, CCIPE, 34094 Montpellier,
France, and the § Department of Pediatrics and
Department of Morphology, University of Geneva,
1211 Geneva 14, Switzerland
 |
ABSTRACT |
The temporal changes in cytosolic free
Ca2+ ([Ca2+]i),
Ca2+-dependent membrane currents
(Im), and gap junctional current (Ij) elicited by acetylcholine (ACh) were
measured in rat pancreatic acinar cells using digital imaging and dual
perforated patch-clamp recording. ACh (50 nM-5
µM) increased
[Ca2+]i and evoked
Im currents without altering
Ij in 19 of 37 acinar cell pairs. Although
[Ca2+]i rose asynchronously in
cells comprising a cluster, the delay of the
[Ca2+]i responses decreased with
increasing ACh concentrations. Perfusion of inositol
1,4,5-trisphosphate (IP3) into one cell of a cluster
resulted in [Ca2+]i responses in
neighboring cells that were not necessarily in direct contact with the
stimulated one. This suggests that extensive coupling between acinar
cells provides a pathway for cell-to-cell diffusion of
Ca2+-releasing signals. Strikingly, maximal (1-5
µM) ACh concentrations reduced Ij
by 69 ± 15% (n = 9) in 25% of the cell pairs
subjected to dual patch-clamping. This decrease occurred shortly after
the Im peak and was prevented by incubating
acinar cells in a Ca2+-free medium, suggesting that
uncoupling was subsequent to the initiation of the
Ca2+-mobilizing responses. Depletion of
Ca2+-sequestering stores by thapsigargin resulted in a
reduction of intercellular communication similar to that observed with
ACh. In addition, ACh-induced uncoupling was prevented by blocking nitric oxide production with L-nitro-arginine and restored
by exposing acinar cells to dibutyryl cGMP. The results suggest that ACh-induced uncoupling and capacitative Ca2+ entry are
regulated concurrently. Closure of gap junction channels may occur to
functionally isolate nearby cells differing in their intrinsic
sensitivity to ACh and thereby to allow for sustained activity of
groups of secreting cells.
 |
INTRODUCTION |
Gap junctions are intercellular channels formed by twelve subunits
of membrane proteins called connexins
(Cx).1 Six subunits are
contributed by each cell to form hemichannels, the docking of which
provides a low resistance pathway for exchange of ions and small
molecules between cells in contact. Gap junctional coupling was shown
to be involved in the control of embryonic development, cell
proliferation, electrical conduction, and metabolic cooperation (1-3).
Because gap junction channels allow for the potential passage of
molecules of a molecular mass up to 1000 Da, it is conceivable that
second messengers produced in one cell can diffuse between neighboring
cells to coordinate their individual response. In support of this
hypothesis, the passage of Ca2+ waves has been reported in
epithelial cells, glial cells, and various cultured cells (4).
The exocrine pancreas represents a valuable model to search for the
role of gap junctional coupling in signal transduction of nonexcitable
tissues. Acinar cells are extensively electrically and chemically
coupled by Cx32- and Cx26-built gap junction channels (5). A major
group of secretagogues in these cells are the Ca2+-mobilizing agonists, including cholecystokinin and
acetylcholine (ACh). In the highly polarized acinar cells, cytosolic
Ca2+ ([Ca2+]i)
initially rises within the apical secretory region and, when
stimulation is sufficient, subsequently spreads as a wave toward the
basal pole of the cell (6-9). Intercellular propagation of
Ca2+ oscillations and/or Ca2+ waves elicited by
Ca2+-mobilizing secretagogues has been reported to
correlate with gap junctional activity (7, 10-13). Increasing evidence
indicates that open gap junctions coordinate the frequency of
Ca2+ oscillations within individual cells of a same acinus
which, in turn, regulates enzyme secretion. This hypothesis, however, is in apparent contradiction with the observation that these
secretagogues also evoke acinar cell uncoupling, both in
vitro and in vivo, at concentrations that maximally
stimulate enzyme secretion (14-17). Thus, the role of gap junctional
coupling during acute stimulation of pancreatic acinar cells remains tantalizing.
One of the first questions to address is whether rises in
[Ca2+]i and changes in junctional
coupling evoked by acinar cell stimulation are parallel events. The
application of the patch-clamp technique to dissociated acinar cells
has revealed that the kinetic of Ca2+-dependent
membrane current activation reflects that of the
[Ca2+]i changes (18-20). However,
accurate monitoring of junctional conductance under dual whole-cell
recording conditions has been limited because spontaneous uncoupling
occurs within seconds, presumably as a result of cytoplasm dialysis
(21-23). To bypass this problem, we applied here a dual perforated
patch-clamp approach, which preserves the integrity of the internal
milieu (24, 25), to monitor pairs of acinar cells stimulated with
increasing ACh concentrations for both
Ca2+-dependent membrane and gap junctional
currents. In parallel experiments, changes in
[Ca2+]i were monitored by digital
imaging of fluo-3-loaded acinar cells. Under these conditions, we
observed that ACh-induced closure of gap junction channels parallels
the phase plateau of the [Ca2+]i response, but
not the initial peak. We further show that acinar cell uncoupling
requires the presence of extracellular Ca2+ and parallels
capacitative Ca2+ entry.
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EXPERIMENTAL PROCEDURES |
Preparation of Acinar Cells--
Acinar cells were prepared as
described previously (26). Briefly, acini were first isolated by
collagenase (CLS 3, Worthington Biochemical Corp.) digestion from the
pancreas of male Wistar or Sprague-Dawley rats (about 200 g),
which were killed by decapitation. Single and paired acinar cells were
prepared by resuspending the intact acini in a Ca2+- and
Mg2+-free Krebs Ringer-bicarbonate medium buffered to pH
7.4 with 12.5 mM Hepes-NaOH and containing 3 mM
EGTA. The resulting cell suspension was repeatedly passed through an
18-gauge needle, centrifuged for 3 min at 100 × g in
Krebs-Ringer bicarbonate medium (KRB) supplemented with 4% bovine
serum albumin. Cells were then resuspended in RPMI 1640 culture medium
(Life Technologies, Inc.) supplemented with 0.1% bovine serum albumin
and 0.01% trypsin inhibitor (Sigma), plated on bacterial Petri dishes
(60 × 15 mm), and kept at 4 °C up to 6 h.
Perforated Patch-Clamp--
Acinar cells were rinsed by
centrifugation and allowed to attach for 15 min at room temperature
onto glass coverslips, previously coated with 0.5 mg/ml
poly-L-lysine (Mr 150,000-300,000,
Sigma) in distilled water. Coverslips with attached acinar cells were transferred to a chamber mounted on the stage of an inverted microscope (TMD-300, Nikon AG). Throughout the experiments, cells were
continuously superfused with a solution containing 136 mM
NaCl, 4 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 2.5 mM glucose, which
was buffered to pH 7.4 with 10 mM Hepes-NaOH (control
solution). In some experiments, CaCl2 was omitted from the
control solution and 1 mM EGTA was added
(Ca2+-free solution). To allow long-term recording with
minimal cell damage, we chose a dual perforated configuration of the
patch-clamp technique (25). To increase the success of seal formation,
the tip of electrodes was first filled with a solution containing 139 mM KCl, 1 mM NaCl, 2 mM
MgCl2, 0.5 mM EGTA, 10 mM Hepes-KOH (pH 7.2) and then back-filled with the same solution supplemented with
120 µg/ml amphotericin B (24). This antibiotic concentration was
prepared from a 60 mg/ml amphotericin B stock solution in dimethyl
sulfoxide (Me2SO) and thoroughly sonicated before use. Under these conditions, the electrode resistance averaged 2.9 ± 0.08 M
(mean ± S.E., n = 56) as measured in
the bathing control solution, and the reversal potential for chloride
and cations was about +3 mV. Once a gigaohm seal was obtained, fast
capacitative current transients were compensated. Under these
conditions, steady series resistances of 10.6 ± 0.5 M
were
reached within 20-30 min (n = 37).
To monitor ACh-evoked Ca2+-dependent membrane
and gap junctional currents, voltage sweeps of 1 s were repeatedly
applied at 3-s intervals in both cells of a pair using an EPC-9 (HEKA
elektronik) and a PC-501A (Warner Instrument Corp.) patch-clamp
amplifier. First, both cells (cell 1 being connected to the EPC-9, and
cell 2 connected to the PC501A) were hyperpolarized for 200 ms from a
holding potential of +3 mV to
30 mV. This voltage step allowed for
the monitoring of Ca2+-dependent chloride and
cation membrane currents (Im1 and
Im2) evoked during ACh stimulation. After
returning to the holding potential for 100 msec, a negative pulse of 10 mV and 300 ms was applied to cell 2 to elicit a transjunctional
potential that allowed for the measurement of junctional current
(Ij') in cell 1. Currents were acquired at a
sampling rate of 1 kHz. Series resistance (Rs1 and Rs2) was compensated in both cells. Off-line
analysis of the electrophysiological data was performed using the Pulse
software (HEKA elektronik). For each sweep, average values of membrane currents recorded at +3 mV and
30 mV were calculated in cells 1 and
2, and amplitude of Ij' was determined in cell
1. These values were then expressed as a function of time. Final
display of the traces as well as plots were generated using the IGOR
software (WaveMetrics Inc.). Thus, Im1 and
Im2 were defined as the difference between the
current measured at
30 mV and +3 mV. True junctional current
(Ij) was calculated by
Ij = Ij'
[1+(Rs1/r1)], where
r1 is the input resistance of cell 1 evaluated
at +3 mV, and Ij' and Rs1
are as described above (21).
Measurements of [Ca2+]i--
Acinar
cells were incubated in the presence of 10 µM fluo-3
acetoxymethylester (fluo-3/AM, Molecular Probes) and allowed to adhere
for 15 min at room temperature to coverslips coated with poly-L-lysine. Coverslips were then transferred to the
stage of an upright microscope fitted with differential interference
contrast optics (Axioskop FS, Zeiss). A peristaltic pump was used to
continuously superfuse the cells with KRB. Global stimulation of acinar
cells was achieved by superfusing KRB supplemented with various
concentrations of ACh (Sigma), as indicated under "Results."
Stimulation of individual cells was achieved by injecting 10-40
µM inositol 1,4,5-trisphosphate (IP3) into
one cell within a cluster using a patch electrode in the whole-cell
configuration of the patch-clamp technique. In this case, the
electrode-filling solution was supplemented with 10 mM
Na2-phosphocreatine, 25 units/ml creatine phosphokinase, and 1 mM MgATP to minimize the rapid and spontaneous
uncoupling of acinar cells (26). ACh-evoked mobilization of
[Ca2+]i was measured using a real
time (30 images/s with averaging 16 frames) confocal laser scanning
microscope equipped with an Ar/Kr laser (Odyssey XL with InterVision,
Ver. 1.4.1 software, Noran Instruments Inc.). Cells were viewed with a
63 × 0.9 numerical aperture achroplan water immersion objective
lens (Zeiss). A 100 µm slit was used for
[Ca2+]i signals, giving bright
images with a 3.1 µm axial resolution. Fluo-3 was excited through a
488-nm band pass filter, and the emitted fluorescence was collected
through a 515 nm barrier filter. To follow the time course of fluo-3
emission changes, the "bright over time" tool of the InterVision
software package was applied to areas that surrounded cells on live
images using an Indy R4600SC/133 MHz Silicon Graphics station. Because
fluo-3 is a single-wavelength dye, its emission is a function of both intracellular Ca2+ and dye concentration.
[Ca2+]i changes were therefore
expressed as the F1/F0 ratio where
F0 was the initial fluorescence intensity measured during the recording (27, 28). Acquired data were then processed for analysis
using either the Indy station or a PowerPC 8100/100 MHz (NIH Image,
Ver. 1.6.0; Adobe Photoshop, Ver. 3.0.5; or Igor Pro, Ver. 2.03).
Dye Coupling--
For dye coupling studies, intact acini
attached to plastic dishes coated with poly-L-lysine were
used. Acini were incubated in KRB supplemented with either 1 µM ACh, 0.5 µM thapsigargin, or 1 mM dibutyryl cGMP (Sigma) for up to 30 min. Specificity of these agents was tested by depleting acinar cells for their internal Ca2+. To deplete internal Ca2+ stores, acini
were incubated for 15 min in a Ca2+-free KRB supplemented
with 1 mM EGTA and 500 nM ACh, and rinsed for
an additional 15 min in the Ca2+-free solution (29). To
assess coupling, one cell per acinus was impaled with a glass
microelectrode (150-200 M
) filled with 4% Lucifer Yellow in 150 mM LiCl and buffered to pH 7.2 with 10 mM
Hepes. The microelectrode was connected to a pulse generator for
passing current and recording membrane potential (30). After successful
cell impalement, 0.1 nA negative square pulses of 900 ms duration and
0.5 Hz frequency were applied to the electrode for 3 min to inject the
dye. At the end of the injection period, acini were photographed under
fluorescence and phase-contrast illuminations. To quantitate the extent
of dye coupling, color slides of the microinjected acini were projected
on a graphic tablet connected to a personal computer to measure the
surfaces of the whole acinus and the Lucifer Yellow-labeled cells. Cell coupling was then expressed as percentage of the acinus area. All data
are expressed as mean ± S.E. and compared with controls using an
unpaired t test. Values were not corrected for the slight overestimation of the Lucifer Yellow stained areas which resulted from
their fluorescent labeling (30).
 |
RESULTS |
Temporal Relationship between ACh-induced Uncoupling and
[Ca2+]i Changes--
The dual perforated
patch-clamp approach was applied to pairs of acinar cells to
simultaneously monitor Ca2+-dependent membrane
(Im) and gap junctional currents
(Ij). All cell pairs exhibited a large and
stable initial junctional conductance, which averaged 42 ± 4 ns
(mean ± S.E., n = 37) over a 30-90 min period of
recording. Nine of the 37 cell pairs studied did not respond to ACh at
concentrations ranging from 100 nM to 5 µM. In 19 cell pairs, ACh triggered Im currents
without affecting the extensive electrical coupling
(Ij traces) of the cell pairs (Fig.
1A). In the remainder nine
pairs, a similar paradigm was observed with ACh concentrations
1 µM. However, ACh induced a marked decrease in
junctional coupling, averaging 69 ± 15% (n = 9)
of the initial conductance, at concentrations of 1-5 µM. This uncoupling was reversible when the secretagogue was washed out and
was inducible again during a second exposure to ACh (Fig. 1B). The decrease in junctional conductance was low, if any,
at the Im peaks. In contrast, uncoupling was
maximal 1-2 min after the Im peaks, taking
place during the sustained phase of the Im response (Figs. 1B and 3). Close inspection of the
recordings showed that Ca2+-dependent currents
(Im1 and Im2) were
synchronized in both cells of a pair when junctional coupling was high.
In contrast, the pattern of Im1 and
Im2 currents was different when junctional coupling was low (Fig. 1B, arrows). In the
example shown in Fig. 1B, while
Ca2+-dependent membrane currents were still
observed in one cell (Im1 trace),
they were readily discontinued in the other (Im2
trace). Similar observations were made in stimulated pairs
that spontaneously uncoupled during recording (n = 4)
or which were experimentally uncoupled by superfusing 3.5 mM heptanol (n = 2).

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Fig. 1.
ACh-evoked
Ca2+-dependent membrane currents in acinar cell
pairs. A, example of a pair comprising coupled acinar
cells that showed changes in Ca2+-dependent
membrane currents (Im1 and
Im2) during ACh stimulation. As shown, low
concentrations of ACh evoked transient changes in
Im, suggesting release of
[Ca2+]i from internal stores.
These changes were, however, without effect on the junctional current
(Ij). B, in contrast, 1 µM ACh markedly reduced Ij in some
cell pairs, reflecting closure of gap junction channels. Uncoupling was
also observed during a second exposure of the cell pair to ACh. The
maximal reduction of Ij was delayed 1-2 min
after the peaks of Im currents. During
uncoupling, the individual response of the two cells was revealed, cell
1 showing larger changes in Im1 compared with
cell 2 which exhibited only a basal level of activity.
Arrows indicate time at which uncoupling was maximal during
a second exposure of the pair to ACh.
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One possible explanation for the synchronized Im
responses observed in highly coupled pairs is that cells mobilized
[Ca2+]i simultaneously.
Alternatively, junctional coupling could be so high that the patch
pipettes only recorded the average current of the whole cell pair. To
address this question, [Ca2+]i
changes were monitored in multiple acinar cells by digital imaging. As
shown in Fig. 2A, superfusion
of an acinar cell pair with 100 nM ACh induced first a rise
in [Ca2+]i in one cell followed by
a delayed Ca2+ response in the second cell. An increase in
the concentration of ACh was associated with shortening of the delay
between the onset of responses, an event which was observed in all 13 cell pairs tested. At higher ACh concentrations, the Ca2+
responses exhibited a typical biphasic "peak-and-plateau" profile (Fig. 2A). Two mechanisms, not mutually exclusive, could be
involved in the lack of synchronization of the Ca2+
response between coupled acinar cells. The long lasting intervals between individual responses at low ACh concentrations could be because
of heterogeneity in ACh responsiveness (11, 13, 31). Furthermore,
distinct IP3 receptor sensitivity may exist in acinar cells
as illustrated in Fig. 2B. Dialysis of a cell using a patch pipette containing 10-40 µM IP3 triggered
transient rises in [Ca2+]i, which
was followed by delayed Ca2+ responses in either
neighboring or distant cells (n = 5).

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Fig. 2.
Ca2+ mobilization in acinar cells
comprising a cluster in response to global and focal stimulation.
A, global stimulation was achieved by superfusing cells with
various ACh concentrations as indicated. Between stimulations, cells
were washed with KRB for several minutes. Interruptions in
the traces indicates periods in which no data acquisition was
performed. In the cell pair illustrated, 100 nM ACh evoked
Ca2+ responses in both cells (traces 1 and
2) which had, however, different time courses. Increasing
the ACh concentration to 300 nM resulted in shortening the
delay of Ca2+ responses in both cells, which still showed
different patterns. At 1 µM ACh, cells raised
[Ca2+]i synchronously in a typical
peak-and-plateau fashion. B, focal stimulation was achieved
by applying intracellularly 10 µM IP3 to one
cell of an acinus using a patch electrode in the whole-cell
configuration. IP3 first increased
[Ca2+]i in the perfused cell (cell
number 1) and in the neighboring cells after a delay
(left panel). These secondary changes in
[Ca2+]i were also detected in
cells (numbers 2, 3, and 4) that were not in
direct contact with the stimulated one. Note that the superfusion flux
was orientated against the propagation of Ca2+ waves. The
fluorescent image of the acinus captured before the experiment and the
location of the cells monitored for
[Ca2+]i are shown in the
right panel.
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Together, these data indicate that acinar cells can exhibit
asynchronous rises in cytosolic Ca2+ in response to
Ca2+-mobilizing mediators. In dual-patched acinar cells,
the asynchrony of Ca2+-dependent membrane
currents was only unmasked when junctional coupling was strongly
reduced (Figs. 1B and 3).

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Fig. 3.
ACh-evoked uncoupling is prevented in the
absence of extracellular Ca2+. A first stimulation
with 1 µM ACh resulted in activation of
Ca2+-dependent membrane currents
(Im1 and Im2) associated
with a modest reduction in junctional current
(Ij) in a cell pair superfused with a
Ca2+-free medium. In contrast, a second stimulation did not
evoke marked changes in either Im or
Ij, suggesting that the internal
Ca2+ stores were not refilled between the two stimulations.
Superfusing the cells with an extracellular solution containing
Ca2+ restored both ACh-induced Im
and Ij changes.
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ACh-induced Uncoupling Is Dependent on Extracellular
Ca2+--
In acinar and other cell types, it has been
shown that the rapid phase of
[Ca2+]i changes reflects
Ca2+ release from internal stores, whereas the later
sustained phase depends on capacitative Ca2+ entry into the
cells (32-34). We therefore investigated whether removal of external
Ca2+ could affect ACh-induced changes in junctional
coupling. As shown in Fig. 3, a first exposure of acinar cells to ACh
induced Im currents and a delayed reduction of
Ij. During a second stimulation, the amplitude
of the Im currents was markedly decreased and
junctional coupling was not affected. Larger ACh-induced
Im and Ij responses were
readily restored after reintroduction of Ca2+ in the
superfusing solution. The ACh-induced uncoupling of acinar cells
observed in cells incubated in the absence of external Ca2+
was typically reduced by 49 ± 8% (n = 4) as
compared with that measured in pairs exposed to the control solution.
To investigate a possible relationship between Ca2+ entry
and cell uncoupling, the extent of intercellular communication was studied by injecting Lucifer Yellow in isolated acini exposed to
various conditions known to deplete internal Ca2+ stores
and/or to activate capacitative Ca2+ entry. Thapsigargin is
a specific inhibitor of the endoplasmic reticulum
Ca2+-ATPase (35), which induces a slow depletion of
Ca2+ stores and, hence, capacitative entry of
Ca2+. As shown in Figs. 4 and
5, a 15-20-min exposure to 0.5 µM thapsigargin resulted in a marked reduction of
intercellular communication. Although Lucifer yellow rapidly spread
from the injected cell into all its neighbors in control acini (Fig.
4A), the diffusion of the tracer was restricted to the site
of injection in the presence of thapsigargin (Fig. 4B).
Quantitative analysis revealed that the surface labeled by Lucifer
yellow represented 51 ± 6% (n = 17) of the
acinus profile, a value that is markedly reduced (p < 0.001) as compared with that measured under control conditions (Fig.
5A). A similar blockade of acinar cell coupling
(p < 0.001) was observed with 1 mM
dibutyryl cGMP (Figs. 4C and 5A), an agent known
to activate Ca2+ entry (36). The effects of both agents
were inhibited when internal Ca2+ stores were previously
depleted and acinar cells incubated in the absence of extracellular
Ca2+ (Fig. 5A). We therefore studied whether
Ca2+-mobilizing secretagogues could cause parallel
activation of capacitative Ca2+ entry and cell uncoupling.
ACh has been shown previously to stimulate capacitative
Ca2+ entry by activation of nitric-oxide synthase, leading
to generation of nitric oxide (NO) which, in turn, increases
intracellular cGMP concentration (29, 37). When cells were incubated in
the presence of 1 mM L-nitro-arginine, an
inhibitor of nitric-oxide synthase, the ACh-induced uncoupling was
fully prevented (Fig. 5B). In contrast, application of
dibutyryl cGMP to acini pre-treated with L-nitro-arginine fully reduced (p < 0.001) acinar cell coupling by an
extent similar to that observed in the presence of ACh alone (Figs.
4D and 5B). These results suggest that closure of
gap junctions by ACh is closely associated with the activation of
capacitative Ca2+ entry.

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Fig. 4.
Depletion of internal Ca2+ stores
and/or activation of capacitative Ca2+ entry reduced dye
coupling in dispersed acini. Fluorescence views of dispersed acini
injected 3 min with Lucifer Yellow. Under control conditions, all cells
of the acinus were found to be coupled to each other (A). In contrast,
the diffusion of Lucifer Yellow was markedly reduced in acini exposed
to thapsigargin (B), dibutyryl cGMP (C), and ACh
(D). Note the tip of the Lucifer Yellow-filled
microelectrode in panel B. Bar = 30 µm.
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Fig. 5.
Role of capacitative Ca2+ influx
on acinar cell uncoupling. A, the extent of dye coupling
between acinar cells incubated in the presence of 0.5 µM
thapsigargin (Tg) or 1 mM dibutyryl cGMP
(dB-cGMP) was markedly reduced (p < 0.001) as
compared with that observed under control conditions (CONT
or Me2SO (DMSO) see "Experimental
Procedures"). The uncoupling effect induced by both Tg and
dB-cGMP was prevented when Ca2+-sequestering stores were
depleted and acini incubated in a Ca2+-free medium
(Ca2+-depleted). B, the uncoupling evoked by 1 µM ACh could be prevented by a 10-min preincubation of
acini with 1 mM L-nitro-Arginine
(L-NA). Addition of dB-cGMP to acini exposed to
L-NA and ACh restored the uncoupling effect
(p < 0.001). Stars indicate differences at
p < 0.001 levels compared with controls.
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 |
DISCUSSION |
Our results describe the temporal relationship between changes in
[Ca2+]i and gap junctional
conductance within pairs of pancreatic acinar cells exposed to ACh.
Uncoupling of pancreatic acinar cells by concentrations of
Ca2+-mobilizing secretagogues that maximally stimulate
exocrine secretion is well documented (14-17). However, the
intracellular mechanism that mediates this effect is not known (38).
Using a dual perforated patch-clamp approach and confocal digital
imaging, we report here that ACh-induced uncoupling and
[Ca2+]i changes had distinct
kinetics. Thus, decrease in junctional conductance of acinar cells
consistently develops after the initial [Ca2+]i peak and is maximal during
the [Ca2+]i plateau. Furthermore,
ACh-induced uncoupling was no longer detected when reloading of
internal Ca2+ stores was prevented by incubation of the
cells in a Ca2+-free medium. These results suggest that
depletion-activated Ca2+ entry was a key determinant for
the ACh-induced uncoupling of acinar cells.
In many nonexcitable cells, depletion of intracellular Ca2+
stores by IP3 is the primary mechanism by which cell
surface receptors activate Ca2+ influx. This phenomenon,
which is termed capacitative Ca2+ entry (33), has been
involved in the control of Ca2+ oscillations (32, 39),
secretion (40), and enzymatic regulation (41). The signal that couples
store depletion to Ca2+ entry has not yet been identified
(33). In pancreatic acinar cells, however, there is definite evidence
that NO produced by nitric-oxide synthase mediates the stimulation of
cGMP formation by cholinergic agonists. cGMP, in turn, is known to
modulate Ca2+ entry (29, 36, 37, 42, 43). Consistent with
these data, we observed that activation of capacitative
Ca2+ entry by either depletion of internal Ca2+
stores with thapsigargin or by exposure of acinar cells to dibutyryl cGMP decreased intercellular communication to an extent similar to that
observed with ACh alone. Side effects of these agents on intercellular
communication appear unlikely because uncoupling was abolished in acini
that were depleted for their internal Ca2+ and incubated in
a Ca2+-free medium. In addition, cell uncoupling evoked by
ACh was prevented in the presence of a nitric-oxide synthase inhibitor,
supporting the view that gap junctional conductance and capacitative
Ca2+ entry are concurrently regulated. These results,
however, do not rule out the possibility that capacitative
Ca2+ entry may activate another intracellular pathway
leading to modulation of junctional conductance. Support for this idea
is provided by the earlier observation that okadaic acid, a phosphatase
inhibitor that modulates capacitative Ca2+ entry (44, 45),
also prevents ACh-induced uncoupling (38). Although several studies
have shown that gap junction channels are blocked by cGMP (25) and
nitric oxide (46), our data provide the first observation that
ACh-induced uncoupling is linked to capacitative Ca2+ entry.
Previous in vitro and in vivo studies have
documented a relationship between intercellular communication and the
secretory activity of pancreatic acinar cells (17, 30, 47). In this context, gap junctional coupling is thought to coordinate the Ca2+ response of individual acinar cells within an acinus
and thereby to regulate exocytosis (11, 48). This idea is supported by our present finding that the large and stable junctional conductance observed in most acinar cell pairs was not altered during changes in
[Ca2+]i evoked by ACh. In
agreement with a previous study using cholecystokinin (13), we observed
that rises in [Ca2+]i were
asynchronous in acinar cells stimulated with low concentrations of ACh.
Increasing the agonist concentration was associated with shortening of
the delay between the onset of the Ca2+ responses,
suggesting that these cells were coupled in terms of Ca2+
mobilization. Also, perfusion of IP3 into one cell evoked a
rise in [Ca2+]i in neighboring
cells even though not necessarily in those that directly contacted the
stimulated one. This observation suggests that rat pancreatic acinar
cells differ in their ability to mobilize Ca2+ from
internal stores, as indicated by previous studies reporting similar
heterogeneity in [Ca2+]i
mobilization (31) and amylase secretion (48, 49). This differential
responsiveness may be essential to provide a properly modulable
response to agonist-specific stimulation (11, 50).
These results, however, are not immediately reconcilable with the
observation that ACh decreases gap junctional coupling while maximally
stimulating the secretory activity of acinar cells (30, 47, 48). The
blockade of acinar cell-to-cell communication is known to enhance the
basal release of amylase in vitro and in vivo
(16, 26, 30, 47). Under conditions of gap junction blockade, the
potency of several agonists in stimulating exocytosis has also been
found to be reduced when the effect of acinar cell uncoupling on basal
secretion was taken into account (11, 16, 30). Therefore, uncoupling
may provide acinar cells with a mechanism to sustain enzyme release
during acute stimulation by increasing their rate of basal secretion.
In this context, the delayed uncoupling evoked by ACh may
compartmentalize cells that are highly sensitive to ACh from cells that
are less sensitive to the secretagogue, therefore decreasing the
effective volume of cytoplasm of interconnected cells. This regulation
may be essential to ensure that the intracellular levels of critical
factor(s) are maintained to allow for sustained activity of groups of
actively secreting cells. Future studies should determine whether
uncoupling induced by Ca2+ store depletion is a common
mechanism to control junctional communication in other types of
nonexcitable cells.
 |
ACKNOWLEDGEMENTS |
We thank D. Bosco, N. Guérineau,
M. B. Rook, and S. Verheule for continuous support during the
preparation of this work, and Luc Analbers and Isabelle Duperrut for
excellent technical help.
 |
FOOTNOTES |
*
This work was supported by European Union Grant
SC1-CT91-0644 (to H. J. J.); by the University of Utrecht, the Swiss
National Science Foundation Grants 95-34090.92, and the Assocation
Française de Lutte contre la Mucoviscidose (to M.C.); by the
INSERM, European Union (Biotech-2 PL970517), the Association Pour La
Recherche Contre Le Cancer, Région Languedoc-Roussillon, MENSER
(ACC-SV11) and the Fondation Pour La Recherche Médicale (to P. Mollard); and by the Swiss National Science Foundation Grant
31-53720.98 (to P. Meda). The European Science Foundation is gratefully
acknowledged for a travel grant (to M. C., P. Mollard, and H. J. J.).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.
¶
To whom correspondence should be addressed: Laboratory of
Clinical Investigation III, Dept. of Pediatrics, HUG - PO BOX 14, Micheli-du-Crest, 24, 1211 Geneva 14, Switzerland. Tel.:
41-22-37-24-609; Fax.: 41-22-37-24-088; E-mail:
Marc.Chanson{at}hcuge.ch.
 |
ABBREVIATIONS |
The abbreviations used are:
Cx, connexin;
ACh, acetylcholine;
[Ca2+]i, intracellular free calcium concentration;
KRB, Krebs-Ringer
bicarbonate;
fluo-3, fluo-3-acetoxymethyl ester;
DMSO, dimethyl
sulfoxide;
IP3, inositol 1,4,5-trisphosphate;
NO, nitric
oxide;
, ohm..
 |
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