INTRODUCTION

Intracellular Ca²⁺ stores play a dominant role in Ca²⁺ signaling in pancreatic acinar cells. Indeed, the Ca²⁺-mobilizing action of the intracellular messenger inositol-1,4,5-trisphosphate (InsP₃)¹ was first demonstrated using a permeabilized pancreatic acinar cell preparation (1). This initial work also identified the endoplasmic reticulum (ER) as the intracellular Ca²⁺ store responsible for agonist-induced increases in [Ca²⁺]_i (1). Recently, however, zymogen granules have been proposed to act as a Ca²⁺ store in pancreatic acinar cells. (2). This hypothesis stemmed initially from the observation that stimulation of pancreatic (and other) acinar cells with acetylcholine results in a polarized rise in cytosolic free [Ca²⁺], with the [Ca²⁺]_i increase being initiated at the apical pole of the cell where zymogen granules are clustered (3-6). Subsequently the rise in [Ca²⁺]_i spreads to the basal pole of the acinar cell (3-6). The role of the proposed zymogen granule Ca²⁺ store would be to act as the releasable Ca²⁺ store responsible for the initiation of the intracellular Ca²⁺ signal at the apical pole (2, 7). Propagation of the increase in [Ca²⁺]_i toward other regions of the cell could then be mediated by the ER Ca²⁺ stores, since the rough endoplasmic reticulum is found throughout the acinar cell (see e.g. Ref. 8). This zymogen granule Ca²⁺ store model is attractive since it can explain the restriction of the [Ca²⁺]_i signal to only the luminal (apical) region when low physiological doses of cholinergic agonists are applied (7, 9). However, there is as yet no conclusive evidence on whether zymogen granules are equipped with intracellular messenger-triggered Ca²⁺ release. Indeed, recent evidence suggests that the report of InsP₃-sensitive Ca²⁺ release from granules (2) may be an artifact produced by the impurity of the zymogen granule preparation employed (10).

A further problem with the report of zymogen granule Ca²⁺ stores (2) is that no evidence was found for a Ca²⁺ uptake mechanism in the proposed zymogen granule Ca²⁺ store. However, an active Ca²⁺-sequestering mechanism is essential to explain the refilling of Ca²⁺ stores and hence the repetitive nature of the agonist-induced [Ca²⁺]_i transients. These conflicting results have prompted us to re-evaluate the suggestion that the endoplasmic reticulum can act as a functional Ca²⁺ store in all subcellular regions of the pancreatic acinar cell. Our results demonstrate that the endoplasmic reticulum can indeed act as a Ca²⁺ store in all subcellular regions, including the apical pole.

EXPERIMENTAL PROCEDURES

Small clusters of acinar cells were prepared from the pancreas of one 200-g male Sprague-Dawley rat by the same enzymatic digestion procedure described in Ref. 11. After isolation, cells were resuspended in a HEPES/Tris-buffered physiological saline containing 133 mM NaCl, 4.2 mM KCl, 1.0 mM CaCl₂, 1.0 MgCl₂, 5.8 mM glucose, 0.2 mg/ml soybean trypsin inhibitor, amino acids as in Eagle's minimal essential medium, 1% bovine serum albumin, and 10 mM HEPES. The pH of this medium was set at 7.4 with Tris. Cells were either used immediately or stored in 1-ml portions on ice until use.

Cells were loaded with 5 µM Magfura-2-AM for 30 min at 37 °C, as described previously (11), and allowed to settle on a poly-L-lysine-coated glass coverslip which formed the bottom of a perfusion chamber. Fluorescence was imaged using a system based on an inverted epifluorescence Nikon Diaphot microscope and a slow scan CCD camera (Digital Pixel Ltd, Brighton, UK). Details of the imaging system were described previously (11). The microscope objective was a Nikon 40 × oil immersion lens (Numerical Aperture 1.3), which allowed images of a field 90 × 135 µm to be captured. For imaging Magfura-2 fluorescence in permeabilized cells, a 3 × 3 binning was applied to the individual pixels on the image sensor to give a theoretical spatial resolution of 0.67 × 0.67 µm/pixel. Background-subtracted 340:380 images were calculated off-line.

Before initiating permeabilization acinar cells were perifused with Ca²⁺ uptake medium containing 135 mM KCl, 1.2 mM KH₂PO₄, 0.5 mM EGTA, 0.5 mM HEDTA, 0.5 mM nitrilotriacetic acid, and 20 mM HEPES/KOH, pH 7.1. The free Mg²⁺ concentration was 0.9 mM and was adjusted as described by Schoenmakers et al. (12). SLO (0.4 IU/ml final concentration) was added directly to the perifusion chamber to permeabilize acinar cells as described previously (11). When permeabilization was achieved (as judged by a drop in fluorescence at the dye isosbestic wavelength) cells were re-perifused with the Ca²⁺ uptake medium as described above but devoid of SLO. Perifusion continued for the duration of the experiment. All experiments were performed at room temperature.

Calcium uptake by intracellular Ca²⁺ stores was initiated by superfusing cells with Ca²⁺ uptake medium containing 1 mM ATP, free Ca²⁺ concentration as indicated in the text and figures, and free Mg²⁺ concentration of 0.9 mM (free divalent cation concentrations calculated according to Ref. 12). When free Ca²⁺ concentration was set to 60, 100, or 200 nM, no mitochondrial inhibitors were included in the medium since mitochondrial Ca²⁺ uptake has previously been shown not to occur at this ambient free Ca²⁺ concentration (13). The mitochondrial Ca²⁺ uptake inhibitors oligomycin (5 µM) and antimycin (5 µM) were included when a free Ca²⁺ concentration of 1.0 µM was applied. Calcium uptake was monitored for a period of 20 min with ratio images acquired at 15-s intervals.

Isolated pancreatic acinar cells in suspension were permeabilized by treatment with SLO (0.4 IU/ml) for 10 min as described previously (14), washed twice, resuspended in (Ca²⁺-free) Ca²⁺ uptake medium (see above), and kept at 0 °C until use. Cell density was adjusted to give a protein concentration of around 4 mg of protein/ml. At the beginning of the radiotracer uptake experiment 10 µl of permeabilized cell suspension was added to 87 µl of Ca²⁺ uptake medium which contained 10 mM phosphocreatine, 10 units of creatine kinase/ml, 1 µM thapsigargin or 1% (w/v) Me₂SO, and 5 µCi of ⁴⁵Ca²⁺/ml. The free Mg²⁺ (0.9 mM) and Ca²⁺ (as indicated) concentrations were adjusted as described above. After 3 min, the incubation was started by adding 3 µl of MgATP stock solution to give a final MgATP concentration of 1 mM. The incubation was terminated after 15 min by adding 1.0 ml of ice-cold stop solution containing 150 mM KCl, 5.0 mM MgCl₂, 1.0 mM EGTA, and 20 mM Hepes/KOH, pH 7.1, and the suspension was rapidly filtered (GF/C glass microfiber filters, Whatman, Kent, UK). The filters were washed with 2 × 1.0 ml of ice-cold stop solution, dissolved in scintillation fluid, and counted for radioactivity. Total Ca²⁺ was calculated and expressed as either nmol/mg protein or as mol per liter of endoplasmic reticulum. For the former, cellular protein was determined (after treatment of the cells with 0.1% Triton X-100) with a commercial Coomassie Blue kit (Bio-Rad), using gamma globulin (Bio-Rad) as a standard. For the latter, cell density was determined using a hemocytometer (Weber Scientific International Ltd., Teddington, UK), and the volume of endoplasmic reticulum per incubation was calculated using the number of cells combined with morphological data on pancreatic acinar cells given by Bolender (8). Calcium actively stored by the endoplasmic reticulum was defined as the difference in total Ca²⁺ retained on the filter after incubation in the absence and presence of the endoplasmic reticulum Ca²⁺ uptake inhibitor thapsigargin.

A small drop of cell suspension in physiological saline (see above) was placed on a silane-coated slide, and the cells were allowed to adhere for 10 min in a moist environment. The cells were then fixed for 15 min in a freshly prepared paraformaldehyde solution (2% w/v in phosphate-buffered saline (PBS)). Cells were washed twice in PBS and were permeabilized using 1% (v/v) Triton dissolved in PBS. The slides were subsequently incubated with primary antibody or preimmune serum in the presence of 0.1% Triton and 1% normal goat serum for 1 h at room temperature. Rabbit polyclonal antiserum against calreticulin (15) was diluted 1:100 and rabbit polyclonal antisera to SERCA-2b Ca²⁺-ATPase (16-18) were used diluted 1:1000. Slides were washed three times with PBS and were then incubated for 1 h at room temperature with FITC-conjugated swine anti-rabbit polyclonal antiserum diluted 1:200 in PBS containing 0.1% (v/v) Triton and 1% (v/v) normal goat serum. Cells were washed three times and mounted in glycerol, which contained SlowFade-Light to prevent photobleaching. Slides were analyzed using the same imaging system employed for Magfura-2 imaging (see above and Ref. 11), except that a FITC dichroic filter set (Chroma Technologies) was used to observe FITC fluorescence. In addition, the resolution of the cooled CCD camera was increased to its maximum by applying a binning of 1 × 1 (i.e. no summation of pixels) to give a theoretical spatial resolution of 0.22 × 0.22 µm per pixel.

Polyclonal antiserum against calreticulin was generously supplied by Drs. D. H. Llewellyn and L. Roderick (UWMC, Cardiff, UK). Two different polyclonal antisera against SERCA-2b Ca²⁺-ATPase were used, kindly supplied by Dr. F. Wuytack (Katholieke Universiteit Leuven, Leuven, Belgium), and by Dr. R. L. Dormer (UWCM, Cardiff, UK). Normal goat serum and FITC-conjugated swine anti-rabbit immunoglobulins were from DAKO, Glostrup, Denmark. Other chemicals were obtained from the following suppliers: SLO from Difco; thapsigargin from Calbiochem; Ins(1, 4, 5)P₃ from Sigma; Magfura-2-AM and SlowFade Light from Molecular Probes; ⁴⁵Ca²⁺ (20 mCi/ml) from NEN Life Science Products. All other chemicals were of analytical grade.

RESULTS

The total exchangeable Ca²⁺ uptake capacity of the thapsigargin-sensitive intracellular Ca²⁺ stores in permeabilized pancreatic acinar cells was determined using the radioactive ⁴⁵Ca²⁺ technique. The effect of the ambient (loading) [Ca²⁺]_i on the total exchangeable uptake capacity of intracellular Ca²⁺ stores was examined over the range 60 nM to 1.0 µM. Total thapsigargin-sensitive Ca²⁺ uptake rose from 1.71 nmol per mg of protein to 6.11 nmol per mg of protein. Fig. 1 shows the uptake data expressed as total thapsigargin-sensitive Ca²⁺ uptake per volume of endoplasmic reticulum (see "Experimental Procedures"). This gives a total exchangeable Ca²⁺ accumulation of 7-25 mmol per liter of endoplasmic reticulum.

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As in our previous paper (11), we used the low affinity Ca²⁺ indicator Magfura-2 to monitor the free Ca²⁺ concentration inside Ca²⁺ storage compartments in real time in individual cells. Calcium uptake was ATP-dependent and reached steady-state levels within 15 min. The Magfura-2 ratio at steady state was dependent on the ambient (loading) [Ca²⁺] and rose from 0.79 to 2.08 as [Ca²⁺]_i was increased from 60 nM to 1 µM (Fig. 2A). The initial rate of Ca²⁺ uptake into the stores was very steeply dependent on ambient [Ca²⁺], increasing more than 6-fold with the change in [Ca²⁺]_i (Fig. 2A). Because of the greatly enhanced initial Ca²⁺ uptake rate, the time taken to reach steady state [Ca²⁺]_lumen was reduced at higher [Ca²⁺]_i.

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Fig. 2B shows the relationship between steady-state free [Ca²⁺]_lumen (as indicated by the steady-state Magfura-2 ratio from Fig. 2A) and total thapsigargin-sensitive exchangeable stored Ca²⁺ (taken from Fig. 1) in pancreatic acinar cells. The two parameters change in parallel over the entire range of loading [Ca²⁺] studied, showing that increased Ca²⁺ uptake at the higher loading [Ca²⁺]_i values does not saturate the intra-store Ca²⁺ buffering system. In addition, the parallelism of total and free [Ca²⁺] within the stores implies that the free [Ca²⁺]_lumen in any given part of the cell can be used to infer the total exchangeable Ca²⁺ in the same compartment.

We proceeded to study Ca²⁺ uptake in more detail by comparing uptake in apical and basal areas of acinar cells. This was achieved by applying the same method of subcellular regional analysis used in our previous study (11), i.e. selecting regions of interest in the apical area and in the basal area of the same cell(s). At 60 and 100 nM [Ca²⁺]_i, both initial uptake rates and steady-state ratio values were almost identical between apical and basal regions (Fig. 3, A and B). At higher values of [Ca²⁺]_i, namely 200 nM and 1 µM, initial uptake rates were also similar for the two regions. Interestingly, however, the [Ca²⁺]_lumen reached at steady state at these higher values of [Ca²⁺]_i was noticeably greater in the basal area of the cell (Fig. 3, C and D).

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Thapsigargin has been widely used in intact acinar cells to induce "global" [Ca²⁺]_i release (see e.g. Ref. 4). In contrast to the apical pole [Ca²⁺]_i signals observed with acetylcholine and thapsigargin (and other organellar Ca²⁺-ATPase inhibitors) evoke a homogeneous elevation in cytosolic [Ca²⁺]_i (4) or, in some cases, a [Ca²⁺]_i rise which is largest in the basolateral pole (6). In permeabilized cells, thapsigargin completely prevents ATP-driven ⁴⁵Ca²⁺ accumulation (13). We tested whether thapsigargin could deplete previously loaded Ca²⁺ stores in permeabilized pancreatic acinar cells. Fig. 4A shows that, as expected, thapsigargin caused a slow depletion of Ca²⁺ stores which was similar in both the apical and basolateral poles. Prevention of Ca²⁺-ATPase activity by removal of ATP had essentially similar results (data not shown). The kinetics of thapsigargin-induced depletion were examined by normalizing the data shown in Fig. 4A to the total size of the ATP-sensitive Ca²⁺ pool (i.e. taking the ratio before loading the stores in each experiment as 0% and the steady-state ratio following loading as 100%). This gave depletion curves for apical and basolateral stores which were not significantly different (data not shown), again indicating that thapsigargin has equal actions on Ca²⁺ stores in the two cellular regions.

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We have previously shown that thapsigargin enhances the rate of InsP₃-evoked Ca²⁺ release from loaded intracellular Ca²⁺ stores and also converts apparent "quantal" release of Ca²⁺ from stores into an essentially monophasic release process (11). Fig. 4B presents subcellular regional analysis of the effects of sequential addition of thapsigargin and InsP₃. To facilitate comparison between the apical and basolateral poles, data were normalized as described above. It is again clear that the kinetics of Ca²⁺ store depletion by thapsigargin and InsP₃ are identical for Ca²⁺ stores in the apical and basolateral poles.

We used immunohistochemistry to study the distribution of SERCA-2b Ca²⁺-ATPase and of calreticulin in pancreatic acinar cells. Both SERCA-2b Ca²⁺-ATPase (Fig. 5A) and calreticulin (Fig. 5B) appeared to be present in all regions of the cell. Immunostaining for the SERCA-2b Ca²⁺-ATPase was slightly weaker in the apical area than in other regions of the cell (Fig. 5A). Weak decoration with the Ca²⁺-ATPase antibody in the central portion of the basal areas of the cells indicated the presence of the nucleus, which was verified by staining DNA with 4,6-diamidino-2-phenylindole (results not shown). The polyclonal antibody against calreticulin decorated acinar cells in essentially the same pattern as observed with Ca²⁺-ATPase antiserum. The only major difference was that the anti-calreticulin staining was slightly more punctate. As with the SERCA-2b antibody, both the apical region and the nucleus appeared less decorated than the basolateral cytoplasm. Since SERCA-2B Ca²⁺-ATPase and calreticulin are both markers of the endoplasmic reticulum in pancreatic acinar cells, the less intense decoration of the apical cytoplasm probably reflects the fact that the apical region contains relatively less endoplasmic reticulum compared with other areas of the cell. This is well known from numerous morphological studies (see e.g. Ref. 8) and arises from the fact that a large part of the apical cytoplasm is occupied by zymogen granules.

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DISCUSSION

This study describes the Ca²⁺ sequestering properties of intracellular Ca²⁺ stores in pancreatic acinar cells at the subcellular level. In situ imaging of intracellular Ca²⁺ stores revealed that Ca²⁺ can be accumulated in all regions of this polarized cell type. The [Ca²⁺]_lumen levels reached at steady state were dependent on the ambient [Ca²⁺]_i used to load the stores. The total Ca²⁺ taken up by the Ca²⁺ stores also rose with increasing [Ca²⁺]_i. These observations show that increased Ca²⁺ uptake does not result in saturation of the intravesicular Ca²⁺-buffering system, since both total Ca²⁺ and [Ca²⁺]_lumen increased upon elevation of [Ca²⁺]_i. In addition, these data tend to suggest that a single type of intra-store Ca²⁺ buffer with a single class of binding site is highly unlikely to account for all intra-luminal Ca²⁺ buffering.

Detailed subcellular analysis of the Ca²⁺ uptake process showed that ATP-driven Ca²⁺ sequestration occurred in both apical and basal regions of the cell. The two subcellular regions did not differ in their capacity to accumulate Ca²⁺ at lower values of [Ca²⁺]_i. At 0.2 and 1.0 µM [Ca²⁺]_i, however, stores in the basal area were able to accumulate significantly more Ca²⁺ than stores in the apical region, as judged by a higher steady-state Magfura ratio. Immunohistochemistry revealed a higher density of SERCA-2b Ca²⁺-ATPases in the basal area. However, this most probably reflects the simple fact that the basal area contains relatively more endoplasmic reticulum (8). We conclude that intracellular endoplasmic reticulum stores in the apical and basal area are able to accumulate Ca²⁺ but that stores in the basal area have an increased capacity for storing Ca²⁺.

In our previous study we showed that, at an ambient [Ca²⁺]_i of 0.2 µM, the steady-state intravesicular [Ca²⁺] was 70 µM (11). This figure can be compared with the measured total thapsigargin-sensitive Ca²⁺ uptake under identical conditions, which was 19 mmol of Ca²⁺ per liter of endoplasmic reticulum (Fig. 1B). From this comparison we conclude that around 270 Ca²⁺ ions are bound for every free Ca²⁺ ion. This calculation confirms that most stored Ca²⁺ is buffered within intracellular Ca²⁺ stores. Despite this heavy Ca²⁺ buffering, large amounts of Ca²⁺ are immediately accessible for rapid mobilization from these stores in both intact and permeabilized cell systems. This indicates that stored Ca²⁺ is not tightly bound or trapped once accumulated.

Calreticulin is proposed to act as a major Ca²⁺ buffer within Ca²⁺ stores, as well as having an important role as an intra-ER molecular chaperone (19). The calreticulin molecule has the ability to bind up to 25 mol of Ca²⁺ per mol with a low millimolar affinity. Pancreatic tissue is the richest known source of calreticulin (calreticulin content of the pancreas is 540 µg/g of tissue (20)), presumably because the high protein synthesis rate of acinar cells imposes a requirements for molecular chaperones. Simple calculation reveals that the calreticulin concentration within the pancreatic endoplasmic reticulum is about 52 µmol/liter ER.² Our estimate of [Ca²⁺]_lumen indicates that calreticulin will not be saturated under these conditions. Even if one assumes a [Ca²⁺]_lumen of 1 mM (instead of our 70 µM estimate), as has been suggested for other cell types on the basis of work with ER-targeted aequorin (21), calreticulin can only account for the binding of 3-4% of the total amount of stored Ca²⁺. This calculation shows that calreticulin may not be as important in organellar Ca²⁺ buffering as originally proposed. This is in agreement with recent work on fibroblasts from calreticulin null mice, which showed no differences in ER Ca²⁺ storage from wild-type cells (22). In fact, recent studies have provided evidence that calreticulin may have other functions in Ca²⁺ signaling than organellar Ca²⁺ buffering. For instance, although calreticulin overexpression inhibited Ca²⁺ waves in Xenopus oocytes, this action was found to be associated with the high affinity, low capacity, Ca²⁺-binding site, rather than with the low affinity, high capacity binding site which confers Ca²⁺ buffering properties (23). In addition, recent overexpression studies in epithelial and fibroblast cell lines revealed that calreticulin may play an important role in the regulation of Ca²⁺ influx (24, 25). Finally, the knockout studies indicate that calreticulin plays a critical role in transmitting signals from integrin extracellular matrix receptors to the cell interior (22).

As already discussed above, we can use morphological data to express our permeabilized cell Ca²⁺ uptake data in terms of Ca²⁺ uptake per unit volume of rough endoplasmic reticulum. It is interesting to compare these data with Ca²⁺ fluxes observed in intact acinar cell systems. In isolated intact pancreas, the neurotransmitter acetylcholine can mobilize 0.5 µmol of Ca²⁺ per g of tissue.³ (26). Assuming the endoplasmic reticulum is the agonist-sensitive intracellular Ca²⁺ storage site, this value suggests the InsP₃-sensitive store holds approximately 2.6 mmol of Ca²⁺ per liter of endoplasmic reticulum. Dormer et al. (27) reported that agonists mobilized around 3 nmol of Ca²⁺ per mg of protein from intact acini, equal to approximately 1.8 mmol of Ca²⁺ per liter rough endoplasmic reticulum.⁴ In permeabilized pancreatic acinar cells, 60% of the thapsigargin-sensitive Ca²⁺ store is InsP₃-sensitive (13, 14). Although the corresponding value for the thapsigargin-sensitive store in intact pancreas is not known, the thapsigargin-sensitive Ca²⁺ store is known to be bigger than the agonist-sensitive intracellular store (29). If we assume that the InsP₃-sensitive store in intact tissues accounts for 60% of the thapsigargin-sensitive store, then the thapsigargin-sensitive intracellular Ca²⁺ stores of intact cells would contain about 4 mmol of Ca²⁺ per liter endoplasmic reticulum. These calculations show that intracellular Ca²⁺ stores in permeabilized cell systems are not by any means reduced compared with those in intact cells. In fact, under the standard conditions used in our study (0.2 µM free Ca²⁺) intracellular Ca²⁺ stores in permeabilized cells accumulate considerably more Ca²⁺ than in intact cells. The most likely explanation for this difference is that the ambient free [Ca²⁺] which we have used to load the stores is higher than the free [Ca²⁺]_i in unstimulated intact cells. At the lowest ambient (loading) [Ca²⁺] used in this study, 60 nM, the stores accumulated around 6 mmol of Ca²⁺ per liter endoplasmic reticulum. This suggests, albeit indirectly, that the cytosolic [Ca²⁺]_i under resting conditions in intact cells is below 60 nM.

It well established that the endoplasmic reticulum can be found in all regions of the pancreatic acinar cell, including the apical pole (e.g. Refs. 8 and 11). The ER is, therefore, a plausible candidate for an apical pole Ca²⁺ store. The same is true for other acinar cell types, for instance lacrimal cells (see Ref. 6 for references). In our previous studies we have shown that all ATP-dependent Ca²⁺ uptake in permeabilized acinar cells can be inhibited by the SERCA Ca²⁺-ATPase inhibitor thapsigargin, indicating that the endoplasmic reticulum is wholly responsible for ATP-driven Ca²⁺ sequestration (13). The in situ imaging experiments in the present study confirm that ATP-driven Ca²⁺ uptake can indeed be observed in all regions of the acinar cell. To seek further evidence that the endoplasmic reticulum can act as Ca²⁺ stores in all cellular regions, we mapped the distribution of the SERCA-2B isoform of the microsomal Ca²⁺ pump, known to be present in acinar cells (18), at low (non-confocal) resolution. Immunofluorescence demonstrated that SERCA-2B Ca²⁺-ATPases are present in all regions of the pancreatic acinar cell. In keeping with this result, addition of thapsigargin caused depletion of Ca²⁺ stores in all cellular regions. We can therefore conclude that Ca²⁺ can be actively reaccumulated by the endoplasmic reticulum in any area of the acinar cell. Calcium accumulation will occur during rises in [Ca²⁺]_i, regardless of whether the increase in [Ca²⁺]_i derives from intracellular stores or from influx from the extracellular environment.

A functional agonist-sensitive Ca²⁺ store minimally requires an active Ca²⁺ uptake mechanism, an intra-store Ca²⁺ buffering system, and a Ca²⁺ release mechanism. The present work clearly shows that the first two are ubiquitous in the acinar cell cytoplasm. In our previous work we demonstrated that the InsP₃-sensitive release mechanism is also present in all subcellular regions of the acinar cell (11). This latter result is re-emphasized in Fig. 4B, which shows that thapsigargin alters the kinetics of InsP₃-evoked Ca²⁺ release in both apical and basolateral poles (contrast Fig. 4B with Fig. 6B of Ref. 11). This shows that the Ca²⁺ store compartment(s) where the InsP₃-evoked Ca²⁺ release mechanism is located are loaded by a thapsigargin-sensitive uptake mechanism. It is thus clear that endoplasmic reticulum stores able to take up, store, and release Ca²⁺ are present in the apical pole of pancreatic acinar cells.

The present study and the recent work of Mogami et al. (30) show unequivocally that the endoplasmic reticulum in the apical pole can act as a functional Ca²⁺ store. Zymogen granules have also been suggested to be an InsP₃-sensitive Ca²⁺ storage compartment in this cellular region, a view based largely on work on an isolated pancreatic zymogen granule preparation (2). However, a very recent cell fractionation study concluded that zymogen granules did not show InsP₃-sensitive Ca²⁺ release (10). In fact, Yule et al. (10) showed that the zymogen granule preparation used by Gerasimenko et al. (2) was contaminated with mitochondria, nuclei, and endoplasmic reticulum. This "mixed" preparation showed Ca²⁺ release on treatment with InsP₃, although Ca²⁺ release was not evoked by thapsigargin (2, 10).⁵ Further purification resulted in a homogenous zymogen granule preparation that had lost its InsP₃ sensitivity, although the granules did contain Ca²⁺, which could be released using Ca²⁺ ionophores (10). Immunolocalization studies in intact cells also failed to detect any InsP₃ receptors in the granule region (32), and it has been suggested very recently that some zymogen granules may "acquire" InsP₃ sensitivity during isolation by fusion with nearby membrane domains containing high concentrations of InsP₃ receptors (33).

In addition to the recent work of Yule et al. (10) discounting the role of zymogen granules in [Ca²⁺]_i signaling, several older studies employing radioactive ⁴⁵Ca²⁺ in isolated intact pancreas also specifically ruled out zymogen granules as an agonist-mobilizable Ca²⁺ store (34-36). In particular, zymogen granules were unable to exchange Ca²⁺ for radioactive ⁴⁵Ca²⁺ despite prolonged labeling protocols (34-36). The authors of these studies concluded that during protein processing the divalent cations Ca²⁺ and Mg²⁺ become trapped inside zymogen granules (34-36). This is consistent with the classical observation that, during agonist stimulation, pancreatic enzyme secretion is tightly correlated with the secretion of Ca²⁺ and Mg²⁺ into the luminal space (where divalent cations are believed to be necessary for the activation of digestive enzymes) (37, 38).

In conclusion, InsP₃-sensitive Ca²⁺ stores can only act as fully functional Ca²⁺ stores when they are equipped with a Ca²⁺-pumping mechanism. This is mandatory in order for the stores to refill following depletion. The combination of techniques applied in this study to investigate organellar Ca²⁺ uptake provides clear evidence that the endoplasmic reticulum can act as a functional Ca²⁺ store in all subcellular regions of the pancreatic acinar cell.