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J. Biol. Chem., Vol. 275, Issue 49, 38680-38686, December 8, 2000

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Agonist-evoked Mitochondrial Ca²⁺ Signals in Mouse Pancreatic Acinar Cells^*

Antonio González, Irene Schulz, and Andreas Schmid^§

From the Department of Physiology, University of the Saarland, D-66421 Homburg/Saar, Germany

Received for publication, June 28, 2000, and in revised form, September 5, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study we have investigated cytosolic and mitochondrial Ca²⁺ signals in isolated mouse pancreatic acinar cells double-loaded with the fluorescent probes fluo-3 and rhod-2. Stimulation of pancreatic acinar cells with 500 nM acetylcholine caused release of Ca²⁺ from intracellular stores and produced cytosolic Ca²⁺ signals in form of Ca²⁺ waves propagating from the luminal to the basal cell pole. The increase in the cytosolic Ca²⁺ concentration was followed by Ca²⁺ uptake into mitochondria. Between onset of cytosolic and mitochondrial Ca²⁺ signals there was a delay of 10.7 ± 0.4 s. Ca²⁺ uptake into mitochondria could be inhibited with Ruthenium Red and carbonyl cyanide m-chlorophenylhydrazone, whereas 2,5-di-tert-butylhydroquinone, which inhibits sarco(endo)plasmic reticulum Ca²⁺ ATPases, did not prevent Ca²⁺ accumulation in mitochondria. Carbonyl cyanide m-chlorophenylhydrazone-induced Ca²⁺ release from mitochondria could only be observed after a preceding stimulation of the cell with a physiological agonist or by treatment with 2,5-di-tert-butylhydroquinone, indicating that under resting conditions mitochondria do not contain releasable Ca²⁺ ions. Analysis of the propagation rate of acetylcholine-induced Ca²⁺ waves revealed that inhibition of mitochondrial Ca²⁺ uptake did not accelerate spreading of cytosolic Ca²⁺ signals. Our experiments indicate that in the early phase of secretagogue-induced Ca²⁺ signals, mitochondria behave as passive Ca²⁺-buffering elements and do not actively suppress spreading of Ca²⁺ signals in pancreatic acinar cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stimulation of pancreatic acinar cells with acetylcholine (ACh),¹ cholecystokinin, or bombesin causes secretion of chloride and enzymes across the apical cell membrane into the acinar lumen. Both chloride and enzyme secretion occurs via Ca²⁺-dependent mechanisms (1). Chloride secretion is accomplished by activation of Ca²⁺-sensitive chloride channels, whereas enzyme secretion involves fusion of enzyme-loaded zymogen granules into the luminal membrane. The Ca²⁺-sensitive step in enzyme secretion probably takes place before the actual fusion triggering event (2-3).

Based on the structural and functional polarization of pancreatic acinar cells, it is not surprising that in this cell type polarized Ca²⁺ signals can be observed. Stimulation of acinar cells with low agonist concentrations causes oscillations in the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt), which are mostly confined to the luminal cell pole (4-5), whereas high agonist concentrations produce intracellular Ca²⁺ waves, which initiate in the secretory region and subsequently travel throughout the cell (6-7). The propagation of Ca²⁺ waves is modulated by secretagogue-dependent activation of protein kinase C (8) and agonist-induced production of arachidonic acid (9). From these data a model was developed which proposes that in pancreatic acinar cells the spatiotemporal characteristics of cytosolic Ca²⁺ signals are controlled by different intracellular Ca²⁺ stores, with different sensitivities to IP₃ and/or Ca²⁺. Since IP₃-evoked Ca²⁺ signals always start at the luminal cell pole, it is assumed that in this cell region the mostly IP₃-sensitive Ca²⁺ stores are located. From the luminal trigger zone, Ca²⁺ signals propagate to the basal cell membrane by some kind of calcium-induced Ca²⁺ release from stores located around the trigger zone and in basal cell regions (4, 8). Spreading of cytosolic Ca²⁺ signals throughout the cell does not only depend on secondary Ca²⁺ release but also on Ca²⁺ uptake into intracellular stores. In pancreatic acinar cells it has been shown that inhibition of endoplasmic reticulum Ca²⁺-ATPases with 2,5-di-tert-butylhydroquinone (tBHQ) causes acceleration of ACh-induced Ca²⁺ waves (8). Furthermore, active Ca²⁺ extrusion via plasma membrane Ca²⁺-ATPases (10) as well as capacitative Ca²⁺ influx triggered by Ca²⁺ store depletion (11) participate in long term regulation of [Ca²⁺]_cyt.

Although agonist-evoked Ca²⁺ signaling in pancreatic acinar cells was the subject of numerous studies during the last years, there is only little information about the role of mitochondria in Ca²⁺ signaling. It is well known that mitochondria can sequester a considerable amount of Ca²⁺ (12). Ca²⁺ uptake into mitochondria is mediated by a high capacity uniporter in the inner membrane that permits transport of Ca²⁺ down its electrochemical gradient. Ca²⁺ efflux occurs via Na⁺-dependent and Na⁺-independent mechanisms (12). Furthermore, a Ca²⁺-induced Ca²⁺ release mechanism via opening of permeability transition pores in the inner mitochondrial membrane has been identified (12-13). Ca²⁺ fluxes across the mitochondrial membranes have two fundamental physiological consequences: (i) the mitochondrial metabolism can be adapted to the actual energy demand of the cell (14-15), and (ii) mitochondria can modulate cytosolic Ca²⁺ signals (15-23). It has been shown that both rapid accumulation of Ca²⁺ via the high capacity, low affinity uniporter as well as subsequent release of Ca²⁺ sequestered in mitochondria determine amplitude and shape of cytosolic Ca²⁺ signals (24). Furthermore, mitochondria participate in the regulation of capacitative Ca²⁺ influx by spatial redistribution of Ca²⁺ that entered the cell via store-operated Ca²⁺ channels (25). Amplification of IP₃-induced Ca²⁺ mobilization by Ca²⁺-induced Ca²⁺ release from mitochondria has been suggested by Ichas et al. (13).

In a recent study on mouse pancreatic acinar cells it has been shown that in this cell type mitochondria are preferentially localized in a belt surrounding the apical zymogen granule-rich cell pole (26). Since by treatment of the cells with the mitochondrial inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and antimycin, IP₃-induced local Ca²⁺ signals in the secretory cell pole were transformed into global Ca²⁺ signals, which travel throughout the cell, it was suggested that in pancreatic acinar cells mitochondria act as a diffusion barrier for Ca²⁺ and thereby confine cytosolic Ca²⁺ signals evoked by low agonist concentrations to the secretory cell pole.

To address the question of whether in pancreatic acinar cells mitochondria are active elements in modulation of agonist-evoked cytosolic Ca²⁺ signals more directly, we double-loaded pancreatic acinar cells with the two fluorescent Ca²⁺ indicators fluo-3 and rhod-2. Selective accumulation of rhod-2 in mitochondria was confirmed by co-staining of rhod-2 with MitoTracker^TM Green FM. Mitochondrial potentials (_m) were monitored with the ratiometric dye JC-1. In most cells mitochondria could be found close to the secretory pole but also within the apical cell pole as well as beneath the plasma membrane at the basolateral side. Stimulation of the cells with ACh led first to an increase in [Ca²⁺]_cyt and with some latency in the mitochondrial Ca²⁺ concentration ([Ca²⁺]_m). Mitochondrial Ca²⁺ uptake could be inhibited with Ruthenium Red and CCCP. Inhibition of mitochondrial Ca²⁺ uptake did not accelerate propagation of ACh-induced Ca²⁺ waves. From these data we conclude that in pancreatic acinar cells, mitochondria are able to participate in long term regulation of cytosolic Ca²⁺ signals but are not actively involved in formation of the early agonist-evoked cytosolic Ca²⁺ signals. Transfer of cytosolic Ca²⁺ signals into mitochondria may adapt mitochondrial metabolism to the increased energy demand of the cell during enzyme and electrolyte secretion.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials and Statistical Analysis-- fluo-3/AM, rhod-2/AM, MitoTracker^TM Green FM, and JC-1 were obtained from Molecular Probes (Europe), tBHQ was from Aldrich, and Ruthenium Red was from Merck. CCCP and all other reagents were obtained from Sigma.

Experiments were performed at room temperature (21-24 °C). Mean values are expressed as mean ± S.E. Statistical analysis was performed by Student's t test, and only P values < 0.05 were considered as significant.

Cell Preparation-- Adult male CD-1 mice (35-40 g) were sacrificed by cervical dislocation. The pancreas was removed and placed into a preparation buffer with the following composition: 130 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl₂, 1 mM MgCl₂, 1.2 mM KH₂PO₄, 10 mM glucose, 0.2% (w/v) albumin, 0.01% (w/v) trypsin inhibitor, 10 Hepes (pH 7.4 adjusted with NaOH). The pancreas was injected with 1 ml of preparation buffer supplemented with collagenase type V (30 units/ml) and incubated in 2 ml of the same buffer in a shaking water bath at 37 °C for 10 min. Enzymatic digestion was followed by mechanical dissociation of cells by gentle pipetting. The resulting cell suspension was centrifuged, and the cells were washed twice in preparation buffer without collagenase. With this isolation procedure we obtained single acinar cells as well as small cell clusters consisting of 2 up to 5 cells.

Dye Loading-- For quantitative measurement of mitochondrial Ca²⁺ signals, freshly isolated cells were loaded at 4 °C for 15 min with 8 µM rhod-2/AM in preparation buffer. Low temperature was chosen to diminish enzymatic cleavage of rhod-2 acetoxymethylesters in the cytosol (23). After incubation with rhod-2/AM, cells were centrifuged for 2 min at 30 × g, resuspended in preparation buffer, and incubated at room temperature (21-24 °C) for 30 min to allow hydrolysis of rhod-2/AM trapped in mitochondria. After rhod-2 loading, cells were kept at 4 °C, and experiments were performed within the next 3 h.

To confirm that rhod-2 fluorescence signals originate from mitochondria, pancreatic acinar cells were double-loaded with rhod-2/AM and the fluorescent mitochondrial marker MitoTracker^TM Green FM (25, 27). After loading of rhod-2 at 4 °C, cells were centrifuged, resuspended in preparation buffer, and then incubated in presence of 100 nM MitoTracker^TM Green FM for 30 min at room temperature.

Cytosolic Ca²⁺ signals were monitored using the Ca²⁺-sensitive dye fluo-3. Cells were loaded with fluo-3/AM at a concentration of 4 µM for 30 min at room temperature (8). For simultaneous registration of cytosolic and mitochondrial Ca²⁺ signals, cells were double-loaded with rhod-2 and fluo-3. Rhod-2 loading was performed at 4 °C for 15 min with 8 µM rhod-2/AM. Then, cells were washed and incubated with 4 µM fluo-3/AM for 30 min at room temperature.

Changes in the mitochondrial membrane potential were registered using the cationic dye JC-1 (28). Freshly isolated mouse pancreatic acinar cells were loaded with 10 µg/ml JC-1 at 37 °C for 10 min. After loading, cells were kept on ice until use.

Confocal Microscopy-- Dye-loaded cells were transferred to a perfusion chamber, mounted on the stage of an inverse microscope (Zeiss, Axiovert 100 TV) equipped with a confocal laser-scanning system from Bio-Rad (MRC-1024). Cells were allowed to adhere to the glass coverslip for several minutes and then were continuously superfused with a bath solution consisting of 140 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, 10 mM Hepes (pH 7.4 adjusted with NaOH). Confocal fluorescence images of 128 × 128 pixels with a resolution of 0.334 µm/pixel were recorded every 2 s. Cells double-loaded with fluo-3 and rhod-2 were simultaneously excited at 488 and 568 nm with a 15-mW krypton/argon laser. Laser intensity was reduced to 1-3% with neutral density filters to reduce photobleaching. The emitted light was filtered with band pass filters and detected with two photomultipliers at 522/35 nm and 605/32 nm, respectively. The same protocol was used for cells double-loaded with rhod-2 and MitoTracker^TM Green FM.

The propagation rate of ACh-induced Ca²⁺ waves was determined as described previously (8, 29). Cells loaded solely with fluo-3 were excited at 488 nm, and fluorescence images (emission > 515 nm) of 128 × 128 pixels were taken every 0.25 s. In the luminal and basal cell pole, small rectangular areas were selected, and the speed of ACh-induced Ca²⁺ waves was calculated from the distance between these areas and the time lag between the increase in [Ca²⁺]_cyt at the luminal and basal cell region.

Changes in _m were monitored by use of the ratiometric dye JC-1. This dye accumulates in mitochondria, where it forms red fluorescent aggregates at high mitochondrial membrane potentials. At low membrane potential JC-1 exists mainly in the green fluorescent monomeric form. JC-1-loaded cells were excited at 488 nm, and emission light was captured at 605/32 nm (JC-1 aggregates) and 522/35 nm (JC-1 monomers) with two separate photomultipliers. Data are presented as emission ratios (605 nm/522 nm).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Simultaneous Registration of Cytosolic and Mitochondrial Ca²⁺ Signals-- Pancreatic acinar cells loaded with rhod-2/AM displayed an inhomogeneous fluorescence pattern with bright structures of globular and filamentous shape within a diffusely and very faintly labeled cytosol. Accumulation of rhod-2 in the nucleus, probably in nucleoli, could sometimes also be noticed (23). Bright fluorescent spots were in principle spread all through the cytosolic area but often concentrated near the zymogen granule area and beneath the basolateral plasma membrane. When images were taken every five seconds for several minutes, changes in the position of the fluorescent structures could be noticed, indicating that rhod-2 accumulated within movable organelles. To confirm that rhod-2 fluorescence did indeed originate from mitochondria, pancreatic acinar cells were double-loaded with rhod-2/AM and MitoTracker^TM Green FM (n = 6), an indicator that specifically labels mitochondria regardless of mitochondrial membrane potentials (25). Coincidence of rhod-2 and MitoTracker^TM Green FM fluorescence in almost all bright spots (>50 cells) proved that rhod-2 indeed accumulated in mitochondria. In most cells, clusters of mitochondria were localized in the lateral cell regions near the secretory granules between the nucleus and zymogen granules and beneath the basal plasma membrane. Single mitochondria could also be found in between the zymogen granules in the luminal cell pole as well as in the endoplasmic reticulum-containing basal cell region. An arrangement of mitochondria in the form of a continuous belt between secretory and basal cell pole, as described by Tinel et al. (26), could sometimes be observed. However, scanning of these cells in z axis revealed that a three-dimensional network of mitochondria that completely covers the secretory cell pole and forms a thorough barrier between luminal and basal cell pole only exceptionally exists. Accumulation of mitochondria in the region between zymogen granules and nucleus seems to be the casual result of displacement of mitochondria by those organelles. No differences in the distribution of mitochondria could be found when rhod-2 staining at low temperature was omitted and the cells were loaded immediately after cell preparation with MitoTracker^TM Green FM at room temperature (n = 5; >500 cells), indicating that temporary exposure of acinar cells to 4 °C does not alter mitochondrial localization.

To follow changes in both [Ca²⁺]_cyt and [Ca²⁺]_m, we double-loaded pancreatic acinar cells with rhod-2/AM and fluo-3/AM (n > 20). In contrast to rhod-2, fluo-3 remains in the cytosol and, therefore, reports changes in [Ca²⁺]_cyt (30). Due to differences in the excitation and emission spectra, changes in the fluorescence of rhod-2 and fluo-3 can be registered with only little interference. Fig. 1A shows a representative fluorescence image of a pancreatic acinar cells stained with fluo-3 and rhod-2. The cell was simultaneously excited at 488 and 568 nm, and fluo-3 and rhod-2 fluorescence was captured with two separate photomultipliers at 522/35 nm and 605/32 nm, respectively. The two resulting images were superimposed, presenting the fluo-3 image in green and the rhod-2 image in red. The image shows the typical punctuated pattern for rhod-2 staining of mitochondria and the more or less homogeneous distribution of fluo-3 in the cytosol. Yellow regions ,which would indicate co-localization of both dyes at similar concentrations, could not be recognized. To test cross-reactivity of rhod-2 and fluo-3 fluorescence signals, we stimulated the cell with 1 µM ACh and analyzed the fluorescence signals originating from several small areas within the cell. When solely green fluorescent areas were analyzed, the typical rapid time course of an agonist-evoked cytosolic Ca²⁺ signal could be registered in the green detection channel, whereas the fluorescence signal captured in the red detection channel remained more or less constant (Fig. 1B). On the other hand, when a brightly red fluorescent area was selected, there was an increase in the red fluorescence signal, whereas the green fluorescence signal was almost unchanged (Fig. 1C). When larger regions enclosing both red and green fluorescence were selected, changes in the fluorescence signals could be observed in both detection channels (Fig. 1D). The time course of the fluorescence signal registered in the green detection channel was identical to the time course of the fluorescence signal of a purely green fluorescent region, whereas the time course of the fluorescence signal registered in the red detection channel corresponded to the much slower time course of the signal from a purely red fluorescent spot. The data indicate that there is only little interference of the two signals and thereby confirm that the bulk of fluo-3 and rhod-2 accumulated in distinct intracellular compartments. Since accumulation of rhod-2 in mitochondria could be demonstrated by double-staining with rhod-2 and MitoTracker^TM Green FM, we can conclude that our loading procedure for staining pancreatic acinar cells with rhod-2 and fluo-3 is suitable for monitoring changes in both [Ca²⁺]_cyt and [Ca²⁺]_m at the same time.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Double-loading of pancreatic acinar cells with rhod-2/AM and fluo-3/AM allows simultaneous measurement of changes in [Ca2+]cyt and [Ca2+]m. Panel A shows a superimposed image with the rhod-2 fluorescence signal in red and the fluo-3 fluorescence in green. Stimulation of the cell with 1 µM ACh produced a rapid rise in [Ca2+]cyt reflected by a fast increase in fluo-3 fluorescence (B). The increase in [Ca2+]m, indicated by a rise in rhod-2 fluorescence, followed with some delay (C). Analysis of larger areas, which showed both rhod-2 and fluo-3 fluorescence (D), indicates that changes in [Ca2+]cyt and [Ca2+]m can be investigated without major interference.

Time Course of ACh-evoked Cytosolic and Mitochondrial Ca²⁺ Signals-- Stimulation of pancreatic acinar cells with the secretagogues ACh, cholecystokinin, or bombesin at supramaximal concentrations induces cytosolic Ca²⁺ signals that start in the luminal cell pole and subsequently spread to the basolateral cell membrane (6-7). The propagation rate of Ca²⁺ waves depends on sequential release of Ca²⁺ from stores in series involving Ca²⁺-induced Ca²⁺ release. Furthermore, it has been shown that thapsigargin-sensitive Ca²⁺ re-uptake into Ca²⁺ stores can slow down the propagation of cytosolic Ca²⁺ waves (8). Intracellular organelles, which are also known to take up Ca²⁺ in response to elevated cytosolic Ca²⁺ levels, are mitochondria (12). To investigate whether in pancreatic acinar cells spreading of agonist-evoked Ca²⁺ signals is also modulated by mitochondrial Ca²⁺ uptake, we measured [Ca²⁺]_cyt and [Ca²⁺]_m in cells double-loaded with fluo-3 and rhod-2. Fig. 2 shows a representative experiment in which fluo-3 fluorescence was measured in small areas in the cytosol near the luminal and basal cell membrane, respectively, whereas rhod-2 fluorescence was captured from a red spot located in the middle of the cell near the zymogen granules. After stimulation of the cell with 500 nM ACh, [Ca²⁺]_cyt first rose in the luminal (lum) and then in the basal (bl) cell region, indicating propagation of the cytosolic Ca²⁺ signal from the luminal to the basal cell pole. During spreading of the cytosolic Ca²⁺ signal, there was no significant increase in the rhod-2 fluorescence. An increase in rhod-2 fluorescence could only be recognized when [Ca²⁺]_cyt had already reached its maximal value. After removal of ACh, [Ca²⁺]_cyt slowly declined to prestimulation level, whereas rhod-2 fluorescence still increased for several seconds and then also slowly declined to prestimulation level. Statistical analysis of the time courses of cytosolic and mitochondrial Ca²⁺ signals revealed that the rhod-2 fluorescence started to rise 10.7 ± 0.4 s (n = 36; 333 mitochondria) later than the fluo-3 fluorescence in the surrounding area of the respective mitochondrion. The fluo-3 fluorescence reached its maximum 8.4 ± 0.4 s after [Ca²⁺]_cyt started to rise. This means that in most experiments an increase in rhod-2 fluorescence could be observed only when [Ca²⁺]_cyt had already reached its maximal value. A similar delay between cytosolic and mitochondrial Ca²⁺ signals could be observed when cells were stimulated with ACh at low concentration (20 nM), which causes oscillation in [Ca²⁺]_cyt (n = 7; 63 mitochondria). Since during prolonged Ca²⁺ spiking successive Ca²⁺ spikes in the cytosol occurred before [Ca²⁺]_m returned to resting level, long term accumulation of Ca²⁺ in the mitochondrial matrix could be observed under such conditions. These data argue against an active role of mitochondria in short term regulation of Ca²⁺ signals in pancreatic acinar cells but suggest a regulatory function in long term regulation of [Ca²⁺]_cyt during sustained hormonal stimulation.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Time course of cytosolic and mitochondrial Ca2+ signals. Stimulation of pancreatic acinar cells with ACh produces a rise in the fluo-3 fluorescence first at the luminal (lum) and then at the basal (bl) cell pole. Simultaneous measurement of fluo-3 and rhod-2 signals show that ACh-induced Ca2+ waves had already traveled throughout the cell before the rhod-2 fluorescence from a mitochondrion near the zymogen granule area started to rise.

Effect of Ruthenium Red and CCCP on Ca²⁺ Wave Propagation-- There are in principle two ways to explain the delay of the rhod-2 fluorescence signals in comparison to the fluo-3 signals. One possible explanation is that under resting conditions the mitochondrial Ca²⁺ uniporter is maintained in an inactive mode and requires activation before Ca²⁺ can be taken up efficiently into the mitochondrial matrix (12). The other possibility is that under in vitro conditions in the mitochondrial matrix rhod-2 could have a much lower affinity to Ca²⁺ than in the cell-free systems used for determination of K_d (K_d 570 nM). Under resting conditions [Ca²⁺]_m is similar to or only slightly higher than [Ca²⁺]_cyt (24, 31). If in the mitochondrial matrix the K_d of rhod-2 would be in the 10 µM range, it is imaginable that rhod-2 fluorescence increases only when a considerable amount of Ca²⁺ has already been taken up into the mitochondrial matrix. Since the K_d for Ca²⁺ binding of rhod-2 in intact mitochondria within intact cells is difficult to determine, we decided to investigate the possibility that mitochondrial Ca²⁺ uptake already occurs during spreading of agonist-evoked Ca²⁺ waves in a more indirect way. If significant Ca²⁺ uptake into mitochondria takes place during Ca²⁺ wave propagation, inhibition of mitochondrial Ca²⁺ uptake should lead to an acceleration of Ca²⁺ waves traveling through the cell, similar as shown before for inhibition of Ca²⁺ re-uptake into intracellular Ca²⁺ stores with thapsigargin or tBHQ (8).

The propagation rate of ACh-evoked Ca²⁺ waves was measured in cells loaded with the cytosolic Ca²⁺ indicator fluo-3. Small areas in the luminal and basal cell pole were selected, and the fluorescence changes within these areas were measured simultaneously. From the distance between the areas and the delay in the fluorescence increase, we determined the speed of Ca²⁺ waves. In one set of experiments (n = 13; 38 cells) the mean propagation rate of Ca²⁺ signals in response to 500 nM ACh was 16.5 ± 0.9 µm/s, which is close to the values found in previous studies (8, 29). When cells from the same preparation were preincubated for 5 min with 100 µM Ruthenium Red, which is an inhibitor of the mitochondrial Ca²⁺ uniporter (12, 24), not a faster but a slower spreading of Ca²⁺ waves (12.7 ± 0.6 µm/s, n = 12; 28 cells) could be observed. However, it is known that Ruthenium Red not only inhibits mitochondrial Ca²⁺ uptake but also Ca²⁺-induced Ca²⁺ release from non-mitochondrial stores (32), which could explain the slower spreading of cytosolic Ca²⁺ waves (8). Therefore, the experiments with Ruthenium Red alone cannot definitely rule out a role of mitochondria in regulation of Ca²⁺ wave spreading. Another way to inhibit mitochondrial Ca²⁺ uptake is treatment of cells with the protonophore CCCP, which dissipates the proton gradient across the inner mitochondrial membrane and therefore causes breakdown of the mitochondrial membrane potential (_m). This results in a loss of the inward driving force for mitochondrial Ca²⁺ uptake. When in a second set of experiments cells were stimulated with ACh in the presence of 100 nM CCCP (30-s preincubation), also a slower propagation rate of Ca²⁺ waves was observed in comparison with cells from the same cell preparations stimulated with ACh alone (10.2 ± 0.7 µm/s; n = 22; 60 cells versus 13.9 ± 1.0 µm/s; n = 17; 45 cells). The slower spreading of Ca²⁺ waves in the presence of CCCP cannot be explained by rapid ATP depletion of the cell. Impairment of ATP-dependent Ca²⁺ uptake into intracellular Ca²⁺ stores should lead to an acceleration of cytosolic Ca²⁺ waves similar to that shown in a former study by use of inhibitors of the endoplasmic reticulum Ca²⁺ ATPase (8). Therefore, the experiments with Ruthenium Red and CCCP argue against a negative feedback exerted by mitochondrial Ca²⁺ uptake on Ca²⁺ wave spreading. The mean values of the propagation rate of Ca²⁺ signals in response to ACh alone and after preincubation with either Ruthenium Red or CCCP for both sets of experiments are summarized in Fig. 3.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Propagation rate of cytosolic Ca2+ waves in the presence of mitochondrial inhibitors. Inhibition of Ca2+ uptake into mitochondria by preincubation of cells with Ruthenium Red (R.R., 100 µM) did not accelerate but slowed down ACh-evoked Ca2+ waves (left panel). Furthermore, treatment of cells with the protonophore CCCP (100 nM), which dissipates the proton gradient across the mitochondrial membrane and therefore inhibits mitochondrial Ca2+ uptake in an indirect way, also did not produce a faster spreading of ACh-induced Ca2+ waves. The data indicate that spreading of agonist-evoked cytosolic Ca2+ waves is not directly controlled by mitochondrial Ca2+ uptake.

Effect of Ruthenium Red and CCCP on [Ca²⁺]_cyt and [Ca²⁺]_m-- To verify that, under the experimental conditions used for determination of Ca²⁺ wave propagation, Ruthenium Red and CCCP indeed inhibited mitochondrial Ca²⁺ uptake, we performed experiments in which [Ca²⁺]_cyt and [Ca²⁺]_m was measured during application of these substances. Preincubation of pancreatic acinar cells with 100 µM Ruthenium Red for 5 min neither changed resting [Ca²⁺]_cyt nor resting [Ca²⁺]_m (n = 11). When cells were stimulated with 1 µM ACh in the presence of Ruthenium Red, [Ca²⁺]_cyt rapidly increased, whereas in most experiments (8 out of 11 experiments, 160 mitochondria) rhod-2 fluorescence remained unchanged (Fig. 4A). Only in 3 out of 11 experiments, an elevation in [Ca²⁺]_m could be observed (100 mitochondria). In those experiments in which [Ca²⁺]_m increased, the time until rhod-2 fluorescence reached a maximum was longer (t_max = 43.7 ± 1.6 s; n = 3; 40 mitochondria) than in control experiments without Ruthenium Red (t_max = 34.9 ± 0.9 s; n = 36; 333 mitochondria; p < 0.001), indicating that Ruthenium Red had at least partially blocked mitochondrial Ca²⁺ uptake. When cells loaded with the mitochondria-specific voltage-sensitive dye JC-1 were perfused with a bath solution containing 100 µM Ruthenium Red, no changes in the JC-1 fluorescence ratio could be observed (n = 6; 40 mitochondria). Incubation of cells with Ruthenium Red had therefore no direct effect on the mitochondrial membrane potential (_m) in resting cells.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effect of Ruthenium Red and CCCP on [Ca2+]m. Preincubation of pancreatic acinar cells with 100 µM Ruthenium Red inhibits mitochondrial Ca2+ uptake after stimulation of the cell with ACh (A). When cells were treated with CCCP at low concentration (100 nM), often a spontaneous increase in [Ca2+]cyt could be observed (B), whereas [Ca2+]m remained constant. Rapid breakdown of m by 100 nM CCCP could be demonstrated in parallel experiments with JC-1 (B). CCCP releases Ca2+ from mitochondria only when accumulation of Ca2+ in mitochondria was produced by a preceding stimulation of cells with ACh (C).

In contrast, application of the protonophore CCCP (100 nM) to resting pancreatic acinar cells produced a rapid breakdown of _m that could be demonstrated with JC-1 (Fig. 4B). A few seconds after application of CCCP, the JC-1 fluorescence ratio started to decline. Within 30 s the normalized fluorescence ratio dropped from 1.00 to 0.48 ± 0.01, and a mean steady state of about 0.34 ± 0.01 was reached after about 60 s (n = 15; 112 mitochondria). During the first minute after application of CCCP a decrease in rhod-2 fluorescence could never be observed (n = 7; 14 cells; 64 mitochondria), indicating that under resting conditions mitochondria do not retain a significant amount of releasable Ca²⁺ due to the negative _m. Nevertheless, in three out of seven experiments, an increase in [Ca²⁺]_cyt could be noticed 40-60 s after application of CCCP. This rise in [Ca²⁺]_cyt is probably due to CCCP-induced Ca²⁺ release from agonist-controlled non-mitochondrial Ca²⁺ stores (33) or a decrease in H⁺ gradient-driven Ca²⁺ uptake into the endoplasmic reticulum by a Ca²⁺/H⁺ exchange (34).

CCCP-induced Ca²⁺ release from mitochondria could be demonstrated when accumulation of Ca²⁺ in mitochondria was allowed by a preceding stimulation of cells, e.g. with ACh. Fig. 4C shows the typical time course of cytosolic and mitochondrial Ca²⁺ signals when a cell was first stimulated with 500 nM ACh for 2 min and then challenged with 100 nM CCCP. In the presence of Ach, [Ca²⁺]_cyt rapidly peaked and then slowly declined to a plateau level. The delayed accumulation of Ca²⁺ in mitochondria is reflected by the slow rise in rhod-2 fluorescence. When ACh was removed after 2 min and CCCP was added instead, a decrease in [Ca²⁺]_m could be observed, indicating that accumulation of Ca²⁺ in mitochondria during stimulation of the cell with ACh was driven by the mitochondrial membrane potential _m. The CCCP-induced Ca²⁺ release from mitochondria caused an elevation of [Ca²⁺]_cyt, as indicated by a renewed increase in fluo-3 fluorescence. However, since CCCP, as shown before, can also induce Ca²⁺ release from non-mitochondrial stores, part of the cytosolic Ca²⁺ signals might reflect Ca²⁺ release from other stores.

Effect of Ca²⁺ Pump Inhibition on [Ca²⁺]_cyt and [Ca²⁺]_m-- In RBL cells it has been shown that local coupling of Ca²⁺ uptake sites in mitochondria to Ca²⁺ release sites in endoplasmic reticulum membranes allows rapid transmission of Ca²⁺ signals from the endoplasmic reticulum into the mitochondrial matrix (35). To test whether in pancreatic acinar cells local transmission of Ca²⁺ signals is also a central event in regulation of [Ca²⁺]_m, we performed experiments in which [Ca²⁺]_cyt was elevated not by activation of the IP₃ signal cascade but by inhibition of Ca²⁺ re-uptake into intracellular stores with the Ca²⁺ ATPase inhibitor tBHQ. About 10 to 20 s after application of 10 µM tBHQ to pancreatic acinar cells, [Ca²⁺]_cyt started to increase, first slowly and then 10-20 s later, with a much faster rate. A maximum in [Ca²⁺]_cyt was reached about 60 s after drug application and was followed by a plateau phase similar to the presence of the physiological agonist ACh. A look at the rhod-2 fluorescence signal revealed that already in the slow-rising phase of [Ca²⁺]_cyt, about 10 s after the start of the cytopsolic Ca²⁺ signal, a weak increase in [Ca²⁺]_m could be observed. The mean delay between an initial rise in [Ca²⁺]_cyt and [Ca²⁺]_m after application of tBHQ was 10.2 ± 0.9 s (n = 8, 14 cells, 40 mitochondria), which is close to the value found in the experiments with ACh as stimulus. The time course of the mitochondrial Ca²⁺ signal evoked by application of tBHQ was similar to that observed in the presence of ACh, indicating that mitochondrial Ca²⁺ uptake is not impaired by the Ca²⁺ ATPase inhibitor tBHQ. Mitochondrial Ca²⁺ uptake is part of a counterbalancing system that avoids an excessive increase in [Ca²⁺]_cyt in the presence of tBHQ. This can be demonstrated by dissipation of _m with the protonophore CCCP, which causes release of Ca²⁺ buffered in the mitochondrial matrix followed by an increase in [Ca²⁺]_cyt (Fig. 5B).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Effect of tBHQ on [Ca2+]m. Elevation of [Ca2+]cyt by depletion of cytosolic Ca2+ stores with the Ca2+ ATPase inhibitor tBHQ (10 µM) produced a rise in [Ca2+]m (A) with a similar delay as in the presence of ACh. The data indicate that in pancreatic acinar cells, Ca2+ signals from the endoplasmic reticulum to the mitochondrial matrix are not transmitted by co-localization of Ca2+ release sites in endoplasmic reticulum membranes and mitochondrial Ca2+ uptake sites. Ca2+ taken up by mitochondria in the presence of tBHQ could be released by the addition of CCCP (B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The spatiotemporal pattern of agonist-evoked cytosolic Ca²⁺ signals is controlled by a complex network of Ca²⁺ release from intracellular stores, Ca²⁺ re-uptake into these stores, as well as Ca²⁺ influx and extrusion across the plasma membrane. Although it is well documented that the mitochondrial membrane also possesses Ca²⁺ uptake and release mechanisms, it had been assumed for a long time that mitochondrial Ca²⁺ uptake only occurs at non-physiologically high [Ca²⁺]_cyt, and a significant contribution of mitochondria to the regulation of cytosolic Ca²⁺ signals was therefore neglected. However, recent studies on several cell types using mitochondria-specific Ca²⁺ indicators revealed that already under physiological conditions [Ca²⁺]_m responds to agonist-evoked changes in [Ca²⁺]_cyt (15-23). Furthermore, it has been shown that mitochondrial Ca²⁺ uptake and release not only influences the time course of cytosolic Ca²⁺ signals but also causes spatial redistribution of cytosolic Ca²⁺ and, therefore, is capable to regulate local Ca²⁺-dependent processes (25, 36).

A central role of mitochondrial Ca²⁺ uptake in the regulation of agonist-evoked Ca²⁺ signals was also suggested for pancreatic acinar cells (26). However, in this study changes in [Ca²⁺]_m were not measured, and therefore, only indirect evidence was provided for an active role of mitochondria in the early phase of agonist-evoked cytosolic Ca²⁺ signals. To get a more direct insight into agonist-induced mitochondrial Ca²⁺ signals, we double-loaded pancreatic acinar cells with the fluorescent probes fluo-3 and rhod-2 and employed confocal laser-scanning microscopy to monitor simultaneously changes in both [Ca²⁺]_cyt and [Ca²⁺]_m. After stimulation with ACh, [Ca²⁺] first rose in the cytosol and then in the mitochondrial matrix. The delay between the rise in fluo-3 and rhod-2 fluorescence was surprisingly long. [Ca²⁺]_m started to increase only when [Ca²⁺]_cyt in the region surrounding the respective mitochondrion had almost reached its maximal value. This is in contrast to studies in hepatocytes, which showed an almost parallel rise in [Ca²⁺]_cyt and [Ca²⁺]_m (15, 37). However, a delay of 10-20 s between peaks in [Ca²⁺]_cyt and [Ca²⁺]_m was also observed, e.g. in studies on the epithelium-derived cell line HT29 (21, 38). The delay between cytosolic and mitochondrial Ca²⁺ signals might be explained in several ways. First, there is a threshold for efficient Ca²⁺ uptake into mitochondria that is in the range of 500 nM to several µM (12, 39-41), and second, the mitochondrial uniporter might, under resting conditions, be maintained in an inactive mode and requires activation before Ca²⁺ can be taken up efficiently (12, 39, 41). After removal of the agonist, in our experiments recovery of resting [Ca²⁺]_m also lagged behind that of [Ca²⁺]_cyt, which is in accordance to many studies on other cell types (17-18, 21, 24, 37, 42-43). Differences in the response of individual mitochondria in dependence on the location in the cell, as shown in aortic myocytes (43) or oligodendrocytes (22), could not be observed in pancreatic acinar cells.

It has been suggested that mitochondria might be actively involved in conveying cytosolic Ca²⁺ signals by Ca²⁺-induced breakdown of mitochondrial membrane potential (13). Agonist-evoked Ca²⁺ release from mitochondria has been reported for oligodendrocytes (22). However, in this study only a small fraction of the mitochondria present in a single cell showed agonist-dependent Ca²⁺ release, whereas [Ca²⁺]_m in all other mitochondria was constant or even increased. The regulatory mechanism underlying the heterogeneity in mitochondrial responses to agonist treatment is not clear at the moment. In our experiments on pancreatic acinar cells we could never observe agonist-evoked mitochondrial Ca²⁺ release, indicating that in this cell type, mitochondrial Ca²⁺ release does not contribute to propagation of cytosolic Ca²⁺ signals. Actually, under resting conditions, mitochondria in pancreatic acinar cells do not seem to contain any readily releasable Ca²⁺, since treatment of cells with CCCP did not produce any decrease in the rhod-2 fluorescence, despite the fact that CCCP caused a rapid breakdown of mitochondrial membrane potential (Fig. 4B). CCCP-induced Ca²⁺ release from mitochondria could only be observed when mitochondria were allowed to accumulate Ca²⁺ in the presence of ACh or tBHQ before application of CCCP.

A recent study describes that in pancreatic acinar cells mitochondria are often located near to the luminal cell pole and form a belt surrounding the granule region (26). Furthermore, treatment of the cells with CCCP or antimycin transformed IP₃-evoked local Ca²⁺ signals to global Ca²⁺ signals. It was therefore suggested that in pancreatic acinar cells mitochondria might act as a diffusion barrier that confines cytosolic Ca²⁺ signals, evoked by low agonist concentrations, to the luminal cell pole (26). In the present study we demonstrate that during spreading of ACh-evoked Ca²⁺ waves, no significant increase in [Ca²⁺]_m could be detected. Inhibition of mitochondrial Ca²⁺ uptake with CCCP and Ruthenium Red caused slow-down of ACh-induced Ca²⁺ waves instead of acceleration, which should be expected if mitochondria acted as diffusion barrier (26) or as a suppressing system of local feedback activation of inositol 1,4,5-trisphosphate receptors by Ca²⁺ (36). Furthermore, in our hands, staining of cells with MitoTracker^TM Green revealed that in acinar cells mitochondria only rarely formed a close-meshed network around the granule area and, therefore, probably are not suitable to prevent Ca²⁺ signal spreading efficiently. Therefore, our data argue against a significant role of mitochondria in the regulation of the very early phase of agonist-evoked Ca²⁺ signals in pancreatic acinar cells. However, the delay in mitochondrial Ca²⁺ signals in comparison with cytosolic Ca²⁺ signals indicates that mitochondria are involved in long term regulation of [Ca²⁺]_cyt.

	ACKNOWLEDGEMENTS

We thank S. Plant and P. Hammes for excellent technical assistance.

	FOOTNOTES

^* This work was supported by a Deutsche Forschungsgemeinschaft Grant (Schm 876/2-1).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.

Present address: Dept. of Physiology, University of Extremadura, Faculty of Veterinary Sciences, Avenida University, P. O. Box 643, 10071 Cáceres, Spain

^§ To whom correspondence should be addressed. Tel.: 0049-6841-166454; Fax.: 0049-6841-166655; E-mail: andreas.schmid@med-rz.uni-saarland.de.

Published, JBC Papers in Press, September 19, 2000, DOI 10.1074/jbc.M005667200

	ABBREVIATIONS

The abbreviations used are: ACh, acetylcholine; CCCP, carbonyl cyanide m-chlorophenylhydrazone; tBHQ, 2,5-di-tert-butylhydroquinone; [Ca²⁺]_cyt, cytosolic Ca²⁺ concentration; _m, mitochondrial membrane potential; [Ca²⁺]_m, mitochondrial Ca²⁺ concentration; IP₃, inositol 1,4,5-trisphosphate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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