Modulation of Pancreatic Acinar Cell to Cell Coupling during ACh-evoked Changes in Cytosolic Ca2+*

Marc ChansonDagger §parallel , Patrice Mollard**, Paolo MedaDagger , Susanne Suter§, and Habo J. JongsmaDagger

From the Dagger  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 parallel  Department of Morphology, University of Geneva, 1211 Geneva 14, Switzerland

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
REFERENCES

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.

    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 MOmega (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 MOmega 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 MOmega ) 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).


View larger version (17K):
[in this window]
[in a new window]
 
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.

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).


View larger version (39K):
[in this window]
[in a new window]
 
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.

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).


View larger version (19K):
[in this window]
[in a new window]
 
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.

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.


View larger version (37K):
[in this window]
[in a new window]
 
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.


View larger version (29K):
[in this window]
[in a new window]
 
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.


    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; Omega , ohm..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
REFERENCES
  1. Bennett, M. V. L., Barrio, L. C, Bargiello, T. A., Spray, D. C., Hertzberg, E., and Sàez, J. C. (1991) Neuron 6, 305-320[Medline] [Order article via Infotrieve]
  2. Kumar, N. M., and Gilula, N. B. (1996) Cell 84, 381-388[Medline] [Order article via Infotrieve]
  3. Goodenough, D. A., Goliger, J. A., and Paul, D. L. (1996) Annu. Rev. Biochem. 65, 475-502[CrossRef][Medline] [Order article via Infotrieve]
  4. Sanderson, M. J. (1996) News Physiol. Sci. 11, 262-269[Abstract/Free Full Text]
  5. Meda, P., Bruzzone, R., Chanson, M., and Bosco, D. (1988) in Modern Cell Biology (Hertzberg, E. L., and Johnson, R. G., eds), Vol. 7, pp. 353-364, Alan R. Liss, Inc., New York
  6. Kasai, H., and Agustine, G. J. (1990) Nature 348, 735-738[CrossRef][Medline] [Order article via Infotrieve]
  7. Nathanson, M. H., Padfield, P. J., O'Sullivan, A. J., Burghstahler, A. D., and Jamieson, J. D. (1992) J. Biol. Chem. 267, 18118-18121[Abstract/Free Full Text]
  8. Toescu, E. C., Lawrie, A. M., Petersen, O. H., and Gallacher, D. V. (1992) EMBO J. 11, 1623-1629[Abstract]
  9. Kasai, H., Li, Y. X., and Miyashita, Y. (1993) Cell 74, 669-677[Medline] [Order article via Infotrieve]
  10. Petersen, C. C. H., and Petersen, O. H. (1991) FEBS Lett. 284, 113-116[CrossRef][Medline] [Order article via Infotrieve]
  11. Stauffer, P. L., Zhao, H., Luby-Phelps, K., Moss, R. L., Star, R. A., and Muallem, S. (1993) J. Biol. Chem. 268, 19769-19775[Abstract/Free Full Text]
  12. Ngezahayo, A., and Kolb, H.-A. (1993) Pfluegers Arch. 422, 413-415[Medline] [Order article via Infotrieve]
  13. Yule, D. I., Stuenkel, E., and Williams, J. A. (1996) Am. J. Physiol. 271, C1285-C1294[Abstract/Free Full Text]
  14. Petersen, O. H., and Ueda, N. (1975) J. Physiol. (Lond.) 247, 461-471[Abstract]
  15. Iwatsuki, N., and Petersen, O. H. (1978) J. Physiol. (Lond.) 274, 81-96[Medline] [Order article via Infotrieve]
  16. Meda, P., Bruzzone, R., Chanson, M., Bosco, D., and Orci, L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4901-4904[Abstract]
  17. Chanson, M., Orci, L., and Meda, P. (1991) Am. J. Physiol. 261, G28-G36[Abstract/Free Full Text]
  18. Osipchuk, Y. V., Wakui, M., Yule, D. I., Gallacher, D. V., and Petersen, O. H. (1990) EMBO J. 9, 697-704[Abstract]
  19. Maruyama, Y., Inooka, G., Li, Y. X., Miyashita, Y., and Kasai, H. (1993) EMBO J. 8, 3017-3022
  20. Thorn, P., Lawrie, A. M., Smith, P., Gallacher, D. V., and Petersen, O. H. (1993) Cell 74, 661-668[Medline] [Order article via Infotrieve]
  21. Neyton, J., and Trautmann, A. (1985) Nature 317, 331-335[Medline] [Order article via Infotrieve]
  22. Chanson, M., Bruzzone, R., Spray, D. C., Regazzi, R., and Meda, P. (1988) Am. J. Physiol. 255, C699-C704[Abstract/Free Full Text]
  23. Somogyi, R., and Kolb, H.-A. (1988) Pfluegers Arch. 412, 54-65[Medline] [Order article via Infotrieve]
  24. Horn, R., and Marty, A. (1988) J. Gen. Physiol. 92, 145-159[Abstract]
  25. Takens-Kwak, B. R., and Jongsma, H. J. (1992) Pfluegers Arch. 422, 198-200[Medline] [Order article via Infotrieve]
  26. Chanson, M., Bruzzone, R., Bosco, D., and Meda, P. (1989) J. Cell. Physiol. 139, 147-156[Medline] [Order article via Infotrieve]
  27. Mollard, P., Seward, E. P., and Nowycky, M. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3065-3069[Abstract]
  28. Guérineau, N. C., Bonnefont, X., Stoeckel, L., and Mollard, P. (1998) J. Biol. Chem. 273, 10389-10395[Abstract/Free Full Text]
  29. Xu, X., Star, R. A., Tortorici, G., and Muallem, S. (1994) J. Biol. Chem. 269, 12645-12653[Abstract/Free Full Text]
  30. Meda, P., Bruzzone, R., Knodel, S., and Orci, L. (1986) J. Cell Biol. 103, 475-483[Abstract]
  31. Willems, P. H. G. M., Van Emst-De Vries, S. E., Van Os, C. H., and De Pont, J. J. H. H. M. (1993) Cell Calcium 14, 145-159[Medline] [Order article via Infotrieve]
  32. Berridge, M. J. (1993) Nature 361, 315-325[CrossRef][Medline] [Order article via Infotrieve]
  33. Putney, J. W., Bird, G., and St, J. (1993) Cell 75, 199-201[Medline] [Order article via Infotrieve]
  34. Yule, D. I., and Williams, J. A. (1994) in Physiology of the GI Tract (Johnson, L. R., ed), pp. 1447-1472, Raven Press, New York
  35. Thastrup, O., Cullen, P. J., Drobak, B. J., Hanley, M. R., and Dawson, A. P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2466-2470[Abstract]
  36. Bahnson, T. D., Pandol, S. J., and Dionne, V. E. (1993) J. Biol. Chem. 268, 10808-10812[Abstract/Free Full Text]
  37. Gukovskaya, A., and Pandol, S. (1994) Am. J. Physiol. 266, G350-G356[Abstract/Free Full Text]
  38. Chanson, M., and Meda, P. (1993) in Progress in Cell Research (Hall, J. E., Zampighi, G. A., and Davis, R. M., eds), Vol. 3, pp. 199-205, Elsevier Science Publishers B.V., Amsterdam
  39. Tsien, R. W., and Tsien, R. Y. (1990) Annu. Rev. Cell Biol. 6, 715-760[CrossRef]
  40. Parekh, A. B., and Penner, R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7907-7911[Abstract]
  41. Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. (1995) J. Biol. Chem. 270, 1149-1155[Abstract/Free Full Text]
  42. Pandol, S. J., and Schoeffield-Payne, M. S. (1990) J. Biol. Chem. 265, 12846-12853[Abstract/Free Full Text]
  43. Xu, X., Kitamura, K., Lau, K. S., Muallem, S., and Miller, R. T. (1995) J. Biol. Chem. 270, 29169-29175[Abstract/Free Full Text]
  44. Berlin, R. D., and Preston, S. F. (1993) Cell Calcium 14, 379-386[Medline] [Order article via Infotrieve]
  45. Parekh, A. B., Terlau, H., and Stühmer, W. (1993) Nature 364, 814-818[CrossRef][Medline] [Order article via Infotrieve]
  46. Lu, C., and McMahon, D. G. (1997) J. Physiol. (Lond.) 499, 689-699[Abstract]
  47. Chanson, M., Fanjul, M., Bosco, D., Nelles, E., Suter, S., Willecke, K., and Meda, P. (1998) J. Cell Biol. 141, 1267-1275[Abstract/Free Full Text]
  48. Bosco, D., Soriano, J. V., Chanson, M., and Meda, P. (1994) J. Cell. Physiol. 160, 378-388[Medline] [Order article via Infotrieve]
  49. Bosco, D., Chanson, M., Bruzzone, R., and Meda, P. (1988) Am. J. Physiol. 254, G664-G670[Abstract/Free Full Text]
  50. Yule, D. I., Lawrie, A. M., and Petersen, O. H. (1991) Cell Calcium 12, 145-151[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.