©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Imaging of Intracellular Calcium Stores in Individual Permeabilized Pancreatic Acinar Cells
APPARENT HOMOGENEOUS CELLULAR DISTRIBUTION OF INOSITOL 1,4,5-TRISPHOSPHATE-SENSITIVE STORES IN PERMEABILIZED PANCREATIC ACINAR CELLS (*)

(Received for publication, October 30, 1995; and in revised form, December 11, 1995)

Frans H. M. M. van de Put (§) Austin C. Elliott

From the School of Biological Sciences, G. 38 Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Several lines of evidence suggest that the existence of a heterogeneous population of inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3))-sensitive Ca stores underlies the polarized agonist-induced rise in cytosolic Ca concentration ([Ca]) in pancreatic acinar cells (Kasai, H., Li, Y. X., and Miyashita, Y.(1993) Cell 74, 669-677; Thorn, P., Lawrie, A. M., Smith, P. M., Gallacher, D. V., and Petersen, O. H.(1993) Cell 74, 661-668). To investigate whether the apical pole of acinar cells contains Ca stores which are relatively more sensitive to Ins(1,4,5)P(3) than those in basolateral areas, we studied Ca handling by Ca stores in individual streptolysin O (SLO) permeabilized cells using the low affinity Ca indicator Magfura-2 and an in situ imaging technique. The uptake of Ca by intracellular Ca stores was ATP-dependent. A steady-state level was reached within 10 min, and the free Ca concentration inside loaded Ca stores was estimated to be 70 µM. Ins(1,4,5)P(3) induced Ca release in a dose-dependent, ``quantal'' fashion. The kinetics of this release were similar to those reported for suspensions of permeabilized pancreatic acinar cells. Interestingly, the permeabilized acinar cells showed no intercellular variation in Ins(1,4,5)P(3) sensitivity. Although SLO treatment is known to result in a considerable loss of cytosolic factors, permeabilization did not result in a redistribution of zymogen granules, as judged by electron microscope analysis. These results suggest that Ins(1,4,5)P(3)-sensitive Ca stores are unlikely to be redistributed as a result of SLO treatment. The effects of Ins(1,4,5)P(3) were therefore subsequently studied at the subcellular level. Detailed analysis demonstrated that no regional differences in Ins(1,4,5)P(3) sensitivity exist in this permeabilized cell system. Therefore, we propose that additional cytosolic factors and/or the involvement of ryanodine receptors underlie the polarized pattern of agonist-induced Ca signaling in intact pancreatic acinar cells.


INTRODUCTION

In many non-excitable cell types, agonist stimulation results in repetitive oscillations of the free cytosolic Ca concentration ([Ca]) (^1)arising largely from Ca release from intracellular stores(1) . In the polarized exocrine acinar cell of exocrine glands, these agonist-induced intracellular Ca signals are not spatially homogeneous. Thus acetylcholine- or cholecystokinin-octapeptide-induced [Ca] rises are initiated in the luminal pole of the cytosol with the Ca wave subsequently spreading into the basolateral areas of the cell(2, 3, 4) . In some cases little or no [Ca] increase at all is observed in basolateral regions(5, 6) . This spatial pattern of [Ca] signaling has been suggested to be important for both unidirectional fluid secretion (2, 7) and exocytosis (8) .

In permeabilized pancreatic acinar cells, as in many other permeabilized cell systems, the intracellular messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) mobilizes Ca from non-mitochondrial intracellular Ca stores(1, 9) . Recent evidence in both permeabilized (10, 11, 12) and intact cells (5, 6) favors the existence of a heterogeneous population of Ins(1,4,5)P(3)-sensitive Ca stores. In a recent study (12) it was suggested that the Ca imaging data obtained by several laboratories (see above) might be explained by the existence of stores more sensitive to Ins(1,4,5)P(3) localized in the apical pole with stores less sensitive to Ins(1,4,5)P(3) being located in basolateral areas.

To address this question we have now imaged the Ca concentration within Ca stores in pancreatic acinar cells using the in situ imaging technique originally described by Hofer and Machen(13) . The low affinity Ca indicator Magfura-2 was loaded into intracellular stores and the properties of these stores were studied in streptolysin O-permeabilized cells. ATP-driven Ca uptake and Ins(1,4,5)P(3)-dependent Ca release could be clearly demonstrated within individual permeabilized pancreatic acinar cells. However, we were unable to detect any subcellular regional differences in Ins(1,4,5)P(3)-sensitivity. This may indicate that cytosolic modulators of Ins(1,4,5)P(3)-operated Ca channels and/or the involvement of ryanodine receptors, underlie the polarized nature of [Ca] signaling patterns in acinar cells.


EXPERIMENTAL PROCEDURES

Pancreatic Acinar Cells

Acinar cells were prepared from the pancreas of one 200-g male Sprague-Dawley rat. The cell isolation procedure used was the same as described previously for rabbit pancreas (12) . After isolation, acinar cells were resuspended in 6 ml of a HEPES/Tris-buffered (pH 7.4) physiological medium which contained: 133 mM NaCl, 4.2 mM KCl, 1.0 mM CaCl(2), 1.0 mM MgCl(2), 5.8 mM glucose, 0.2 mg/ml soybean trypsin inhibitor, an amino acid mixture according to Eagle(14) , 1% (w/v) bovine serum albumin, and 10 mM HEPES. The pH of the medium was set at 7.4 with Tris. Cells were either used immediately or stored in 1-ml portions on ice until use.

Loading of Pancreatic Acinar Cells with Magfura-2

Pancreatic acinar cells were resuspended in the physiological medium described above with the addition of 5 µM Magfura-2-AM. After a 30-min incubation at 37 °C, cells were washed twice with physiological medium containing 0.1% bovine serum albumin. Cells were allowed to settle on a poly-L-lysine-coated glass coverslip which formed the bottom of a perfusion chamber.

Imaging of Magfura-2-loaded Pancreatic Acinar Cells

The imaging system used was based on an inverted epifluorescence Nikon Diaphot microscope and a times 40 oil immersion lens (numerical aperture: 1.3). A field containing 5-15 cells was selected and the dye-loaded cells were excited alternately with light at 340 and 380 nm using a filterwheel (Lambda 10, Sutter Instruments; 340- and 380-nm band-pass filters were from Ealing Electro-Optics) and a dichroic mirror (400-nm dichroic mirror, 420-nm barrier filter). The emitted fluorescence was captured and digitized at 12-bit resolution by a slow scan CCD camera (Digital Pixel Ltd., Brighton, United Kingdom). An IBM-compatible personal computer and an imaging software package (Kinetic Imaging Ltd., Liverpool, UK) was used to drive the filterwheel and camera and store acquired images. The size of the silicon sensor of the camera and the times 40 objective allowed images of a field 90 times 135 µm to be captured. A 3 times 3 binning was applied to the individual pixels on the image sensor to give a spatial resolution of 0.67 µm/pixel. Since acinar cells have virtually no detectable autofluorescence (results not shown) an empty area of a coverslip was used to determine background levels. All images were background corrected. The ratio of the 340 nm and 380 nm excitations of paired, background-subtracted images were calculated off-line. All experiments were performed at room temperature.

Permeabilization of Pancreatic Acinar Cells

Acinar cells were perfused with Ca uptake medium containing: 135 mM KCl, 1.2 mM KH(2)PO(4), 0.5 mM EGTA, 0.5 mMN-hydroxyethylethylenediaminetriacetic acid, 0.5 mM nitrilotriacetic acid, and 20 mM HEPES/KOH (pH 7.1). The free Mg concentration was 0.9 mM and was adjusted as described by Schoenmakers et al.(15) . SLO (0.4 IU/ml) was used to permeabilize acinar cells.

Since a considerable amount of the total accumulated Magfura-2 was present in the cytosolic compartment, the permeabilization process could be followed on-line (as loss of cytosolic dye) by using the imaging system. At the start of the permeabilization procedure, loaded cells were excited at the isosbestic wavelength for Magfura-2, i.e. 360 nm. Permeabilization was achieved within 10 min and, as a consequence, a significant drop in fluorescence was observed as cytosolic Magfura-2 was lost into the incubation medium (results not shown). Perfusion of the permeabilized cells was subsequently continued with the Ca uptake medium devoid of SLO, as described above.

CaUptake and Release Experiments

Permeabilized cells were continuously perfused throughout the experiments. Ca uptake by intracellular Ca stores was initiated by superfusing cells with a medium containing 1 mM ATP and a free Ca concentration of 0.2 µM; the free Mg concentration remained 0.9 mM (free divalent cation concentrations were calculated again according to Schoenmakers et al.(15) ). Mitochondrial Ca uptake inhibitors were not included in the medium, since mitochondrial Ca uptake has previously been shown not to occur at this ambient free Ca concentration(16) . After a loading period of 15 min, permeabilized cells were stimulated with Ins(1,4,5)P(3), thapsigargin, or cyclic ADP-ribose at various concentrations as described in the text and in the captions of the figures. The Ca concentration in the stores was monitored by determining the ratio of the emitted fluorescence at 340 and 380 nm excitation. Exposure times for individual images were 300 ms, and the interval times between ratio images is indicated in the legend of the figure.

Calibration of Ratio Values

The Ca ionophore 4-Br-A23187 (2 µM) was used to equilibrate extra- and intra-compartment Ca and thus to impose determined free Ca concentrations inside the Magfura-2 containing compartments. The medium used was a 137 mM KCl solution buffered with 20 mM HEPES/KOH to pH 7.1. Media were prepared with various free Ca concentrations (as indicated) in the absence of Mg (since Mg gradients are also dissipated by Ca ionophores and Mg is able to alter the fluorescent properties of Magfura-2). Free Ca concentrations were calculated taking account of the ionic strength of the solution. The activity coefficient for Ca was calculated by using the Guggenheim approximation to the Debye-Hueckel Limiting Law(17, 18) .

Electron Microscopy on Intact and Permeabilized Pancreatic Acinar Cells

Intact or SLO-permeabilized acinar cells (permeabilization procedure, see above) were diluted 1:1 into fixation buffer (0.1 M sodium cacodylite, 5% glutaraldehyde). Preparations were washed twice, postfixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4), dehydrated, and embedded in resin. A Reichert Ultracut E microtome was used to cut 60-nm sections. Sections were stained using uranyl acetate and lead citrate, and a Philips transmission electron microscope (200) was used to visualize the samples. The magnifications used are indicated in the figures.

Analysis of Data

The mean ratio values mentioned under ``Results'' were calculated by determining ratio values for each of the individual cells in a field. From these values, an average value for a particular experiment was determined. These values were in turn averaged between preparations to give the values presented in the text. The value of n given thus refers to the number of cell preparations on which a given experiment was performed, although the total number of individual cells analyzed was between 19 and 180 for different experimental procedures.

Materials

Chemicals were obtained from the following suppliers: SLO from Difco; thapsigargin from Calbiochem; Ins(1,4,5)P(3) and 4-Br-A23187 from Sigma; Magfura-2-AM from Molecular Probes, Eugene, OR; cyclic ADP-ribose from Amersham International plc, Buckinghamshire, UK. All other chemicals were of analytical grade.


RESULTS

Effect of Streptolysin O Treatment on Pancreatic Acinar Cells

The morphology of pancreatic acinar cells was altered after permeabilization. Acinar cells had a more swollen appearance in the light microscope, and the cytosol became paler, reflecting loss of proteins. Permeabilized cells coupled to each other in doublets or triplets clearly retained their polarized morphology, whereas the polarity in individual cell was, just as in intact cells, largely lost (see e.g.Fig. 5B for an example). To study the effect of SLO treatment on the cellular structure in more detail, electron microscopy was performed on both intact and permeabilized cells. Fig. 1A shows a representative triplet of intact acinar cells. Cells retained a polarized morphology, since the large densely stained secretory granules were restricted to the apical pole. The endoplasmic reticulum retained its typical tubular arrangement and was present in all parts of the cell, although it was more abundant in basolateral regions. A representative example of permeabilized pancreatic acinar cells is shown in Fig. 1B. The cells had a much brighter appearance, reflecting loss of the majority of the cytosolic content after permeabilization. The picture shows two coupled cells which were, similar to intact cells, polarized. The endoplasmic reticulum was rearranged but appeared to be continuous. The changes in endoplasmic reticulum morphology may be due to the swelling of the cells after permeabilization.


Figure 5: Effect of stepwise increases in Ins(1,4,5)P(3) concentration on loaded intracellular Ca stores. Intracellular stores of permeabilized pancreatic acinar cells loaded Ca in an ATP-dependent fashion for 15 min. The experiment was started and image pairs were collected at 15 s intervals. After 1.5 min 0.1 µM Ins(1,4,5)P(3) was included for 5 min in the medium and its concentration was increased at 5-min intervals to 0.3, 1.0, and 10 µM. Finally, permeabilized cells were reperfused with control medium until the end of the experiment. A, individual responses of seven acinar cells plotted in a similar format to that of Fig. 2. B and C show, respectively, a brightfield image and a fluorescence image (340 nm excitation). Six ratio images are presented in D. The ratio values were converted to pseudocolor to clarify the ratio levels measured. The pseudocolor scale used is shown at the side. The images shown are taken at time points indicated with the corresponding numbers in A. The calibration squares in B and C, and the sizes of the boxes used to number the images in D, represent 25 µm. The results shown are representative for three independent experiments.




Figure 1: Electron micrographs on intact and permeabilized pancreatic acinar cells. Intact pancreatic acinar cells or cells permeabilized by steptolysin O treatment were fixed and processed as described under ``Experimental Procedures.'' A and B show the morphology of a triplet of intact cells and two coupled permeabilized cells, respectively. The magnification was in both cases 3000 times and the calibration bar represents 5 µM.




Figure 2: Effect of ATP and Ca on the Ca content of intracellular Ca stores in permeabilized pancreatic acinar cells. Magfura-2-loaded pancreatic acinar cells were permeabilized by SLO treatment, and the Magfura-2 remaining inside the intracellular compartments was used to monitor store lumen Ca changes. A field of nine permeabilized cells was excited alternately at 340 and 380 nm and the emitted fluorescence captured by a digital camera as described under ``Experimental Procedures.'' Pairs of images were acquired at 1 min intervals. The off-line calculated 340/380 nm ratio of each individual cell is depicted. After assessment of permeabilization, cells were initially perfused with Ca uptake medium in the absence of Ca or ATP, but with a free Mg concentration of 0.9 mM. At the beginning of the experiment, ATP was added to this medium. After 19 min, Ca was introduced to the Mg- and ATP-containing medium at a free concentration of 0.2 µM. The observation is representative of two independent experiments.



CaUptake by Permeabilized Pancreatic Acinar Cells

After permeabilization, cells were exposed to an ``intracellular'' medium with an ambient free Ca concentration of 0.2 µM Ca and 1 mM ATP. This resulted in an increase in the Magfura ratio, presumably reflecting an increase in the free Ca concentration in intracellular Ca stores (Fig. 2). To rule out the possibility that changes in the Magfura ratio were due to changes in intra-organelle [Mg], rather than [Ca], the experimental protocol was adapted by perfusing cells initially with Ca uptake medium devoid of Ca but containing ATP and Mg. Fig. 2shows that the ratio remained unaltered and could only be increased by including Ca in the medium. The combined addition of Ca and ATP increased the Magfura ratio from 0.53 (S.E. = 0.04; n = 14 cell preparations and 137 individual cells analyzed) to 1.44 (S.E. = 0.04; n = 16 cell preparations and 178 individual cells analyzed) within 10 min. This increased level remained constant for a considerable period thereafter (at least 10 min).

To demonstrate that the Magfura signal was not saturated in loaded Ca stores under these conditions, the Ca ionophore 4-Br-A23187 (2 µM) was included and the ambient free Ca concentration in the medium was increased to 1 mM (for details of the medium used, see ``Experimental Procedures''). This treatment resulted in a further increase in the ratio to 3.43 (S.E. 0.02; n = 3 cell preparations and 19 individual cells analyzed; results not shown, but see also Fig. 3). This indicates that the free Ca concentration of steady-state loaded intracellular Ca stores of pancreatic acinar cells is not in the millimolar range. A tentative calibration was made by perfusing permeabilized cells with Ca ionophore and a medium containing different ambient free Ca concentrations. Fig. 3shows the result of this calibration. The average Magfura ratio in stores loaded at steady-state was 1.44 (see above), and therefore the free Ca concentration in loaded intracellular Ca stores is estimated to be 70 µM. The minimum ratio, obtained with permeabilized cells in a Ca free medium, including ionophore, was 0.37 (S.E. = 0.02; n = 4 cell preparations and 37 individual cells analyzed). The average ratio in unloaded stores without ionophore in the same set of experiments was 0.47 (S.E. = 0.03; n = 4 cell preparations and 37 cells analyzed). This shows that, before initiation of Ca uptake, the Ca stores in the permeabilized cells were virtually depleted of their Ca content.


Figure 3: Calibration of the free Ca levels inside intracellular Ca stores of permeabilized pancreatic acinar cells. Permeabilized pancreatic acinar cells were allowed to load Ca in the presence of ATP. After 15 min, Mg and ATP were removed from the medium and various Ca concentrations were imposed by using the non-fluorescent Ca ionophore 4-Br-A23187 (2 µM). Image pairs were acquired every 3.5 min. Details of the various Ca conditions are described under ``Experimental Procedures.'' The observation is representative of two independent experiments.



Effects of Ins(1,4,5)P(3)on Loaded Intracellular CaStores

Perfusion of permeabilized pancreatic acinar cells with an Ins(1,4,5)P(3) containing medium resulted in a release of Ca, indicated by a fall in the Magfura ratio. Fig. 4, A and B, show the effect of 0.3 and 1.0 µM Ins(1,4,5)P(3), respectively, on loaded stores in permeabilized cells. Ins(1,4,5)P(3) induced a release of Ca in both cases. The rate of release, and the new reduced intravesicular [Ca] achieved, both varied with the concentration of Ins(1,4,5)P(3) applied. The intravesicular Ca levels could be further reduced by increasing the Ins(1,4,5)P(3) concentration, which also indicated that the release process was not desensitized despite the 12 min presence of submaximal doses of Ins(1,4,5)P(3). At 10 µM Ins(1,4,5)P(3), a maximal Ca releasing effect was obtained and the ratio was reduced to 0.62 (S.E. = 0.02, n = 5 cell preparations and 52 individual cells analyzed). This level was close to the ratio in unloaded stores, indicating that the Ca stores were virtually emptied by a maximally effective dose of Ins(1,4,5)P(3) (10 µM was shown to be a maximally effective dose, since 30 µM did not induce any further Ca release). The half-maximal effect of Ins(1,4,5)P(3) occurred at 0.3 µM, a value slightly lower than, but in a range similar to that reported by others using permeabilized pancreatic acinar cell preparations (e.g. Refs. 9, 10, 19, and 20).


Figure 4: Effects of Ins(1,4,5)P(3) on loaded intracellular Ca stores in permeabilized pancreatic acinar cells. Permeabilized pancreatic acinar cells were perfused during a 15-min period, allowing intracellular Ca stores to accumulate Ca to a steady-state level. The experiment was started after the loading period and a pair of images was acquired each 15 s as described under ``Experimental Procedures'' and the legend of Fig. 2. After 1.5 min, Ins(1,4,5)P(3) was added with a concentration of 0.3 or 1.0 µM in A and B, respectively. After a period of 12 min, the Ins(1,4,5)P(3) concentration was increased to 10 µM until the experiment was terminated. For reasons of clarity, the averaged responses of five and seven cells are presented in A and B, respectively. For each situation, all permeabilized cells responded in a similar manner. The results represent a single observation; the effects of 0.3 and 1.0 µM Ins(1,4,5)P(3) applied for a period of 5 min were tested in four independent experiments and gave results similar to those presented.



In permeabilized gastric epithelial cells, mitochondrial uptake of Ca contributed substantially to the ATP- and Ca-dependent increase in the signal from compartmentalized Magfura-2(21) . Thus a considerable part of the ATP-dependent Ca pool in this cell type was Ins(1,4,5)P(3)-insensitive but sensitive to mitochondrial Ca uptake inhibitors. In pancreatic acinar cells, however, a maximal dose of Ins(1,4,5)P(3) released virtually all the Ca that had been taken up in an ATP-dependent manner, indicating that mitochondria were not active under the conditions used (see above). In addition, when Ca uptake was performed in the presence of the mitochondrial inhibitors antimycin and oligomycin, no change in uptake characteristics was observed (results not shown). These observations confirm previous studies with radiotracer techniques on permeabilized pancreatic acinar cells, in which it was shown that mitochondrial Ca uptake was inactive at an ambient free Ca concentration identical to that used in the present study(16) .

Effects of Ins(1,4,5)P(3)on Cellular and Subcellular Level

We went on to study the kinetics and sensitivity of Ins(1,4,5)P(3)-induced Ca release in individual cells in more detail. Fig. 5A shows the effect of sequential additions of increasing Ins(1,4,5)P concentrations. The addition of 0.1 µM Ins(1,4,5)P resulted in minor decrease in the Ca levels in the intracellular Ca stores. Subsequent addition of 0.3 µM induced a more pronounced release, which leveled off within 5 min of application (see also Fig. 4A). The addition of 1.0 µM Ins(1,4,5)P nearly completed the Ca release process, since the maximal effective dose of 10.0 µM Ins(1,4,5)P induced only a small further reduction of the Magfura ratio. The ``steps'' in store Ca content on increasing Ins(1,4,5)P resemble the so-called ``quantal'' Ca release previously observed in these and other cells(10, 12, 22) . The effect of Ins(1,4,5)P was reversible, since reperfusion with control medium resulted in a reuptake of Ca (this reversibility was tested in four experiments and was observed in all cases). Fig. 5A also shows that the effects of Ins(1,4,5)P were observed in all seven cells analyzed from this field. Ins(1,4,5)P evoked a simultaneous and equal response in all cells, indicating that there were no intercellular differences in Ins(1,4,5)P sensitivity.

The action of Ins(1,4,5)P(3) was studied in more detail by analyzing the ratio images of the experiment. Fig. 5, B and C show a brightfield and a fluorescence image respectively of the selected field of cells. The brightfield image shows again that permeabilized cells organized in triplets clearly maintained their polarized morphology. The first ratio image (Fig. 5D, image 1) shows Ca stores in permeabilized cells loaded to a steady-state level. The ratio intensity throughout the different regions of the cells was not homogeneous, although virtually all regions had a ratio value of about 1 or higher (i.e. the free [Ca] was 40 µM or higher). Stimulation with a low dose of Ins(1,4,5)P(3) (0.3 µM) resulted in a simultaneous decrease of Ca levels in all regions of the permeabilized cells (Fig. 5D, images 2 and 3). Elevation of the dose to 1.0 µM (Fig. 5D, image 4) and then to 10.0 µM (Fig. 5D, image 5) resulted again in a simultaneous reaction in all subcellular regions in all permeabilized acinar cells. Subsequent removal of Ins(1,4,5)P(3) resulted in Ca reuptake in all regions of the permeabilized cells (Fig. 5D, image 6). (The regional analysis, as presented in Fig. 5D, was performed in four additional experiments; in two experiments sequential Ins(1,4,5)P(3) additions were made as presented in Fig. 5, and in the other two experiments, 0.3 µM Ins(1,4,5)P(3) added to loaded stores of permeabilized cells. All experiments analyzed gave similar results to those presented in Fig. 5). To further demonstrate the uniform Ins(1,4,5)P(3) sensitivity, the kinetics of Ins(1,4,5)P(3)-induced Ca release were compared between selected areas of interest in apical and basolateral regions in the same field of cells. Fig. 6A shows the selected areas and Fig. 6B shows the averaged and normalized kinetics of Ca release induced by Ins(1,4,5)P(3) in apical and basolateral regions. Again, the results demonstrate that both regions were equally sensitive to Ins(1,4,5)P(3). Taken together, the results demonstrate that Ca stores in SLO-permeabilized acinar cells display neither regional nor intercellular differences in their sensitivity toward Ins(1,4,5)P(3).


Figure 6: Comparison of Ins(1,4,5)P(3) sensitivity in apical and basolateral regions of permeabilized pancreatic acinar cells. A, in the same field of seven selected cells shown in Fig. 5, a region of interest was selected in the apical (black square symbols) and basolateral part of each cell (white square symbols). The selected regions of interest are shown on the corresponding brightfield image. B shows the effect of stepwise increases in Ins(1,4,5)P(3) on the averaged and normalized size of the Ins(1,4,5)P(3)-sensitive store in the apical regions (solid line) and in the basolateral (dashed line) regions. Maximal store size was defined as the difference in ratio between unstimulated and maximally Ins((1,4,5)P(3)-stimulated Ca stores and the averaged results were normalized to this value; this is directly analogous to the definition of ``Ins(1,4,5)P(3)-sensitive Ca pools'' commonly employed in radiotracer experiments. The results presented are from the same experiment as shown in Fig. 5and are typical for three independent experiments.



Effect of Thapsigargin on Ins(1,4,5)P(3)-induced CaRelease

To study the effect of Ca pump activity on Ins(1,4,5)P(3)-induced Ca release, thapsigargin was used to completely block all Ca pumping into the intracellular Ca stores(12, 16) . Addition of thapsigargin (1 µM) resulted in a slow but sustained efflux of Ca from the stores with kinetics similar to that observed in permeabilized acinar cell suspensions (Fig. 7). Addition of 0.1 µM Ins(1,4,5)P(3) resulted in an increased efflux rate, indicating that Ins(1,4,5)P(3)-operated Ca channels were indeed activated at this low dose. After 5 min the Ins(1,4,5)P(3) concentration was increased to 0.3 µM; the efflux rate increased again and remained elevated. Under these conditions suboptimal doses of Ins(1,4,5)P(3) were indeed more effective in depleting stores compared with the situation where Ca pumps remained active. The Ins(1,4,5)P(3)-sensitive Ca stores in the acinar cells were virtually depleted after prolonged treatment with 0.3 µM Ins(1,4,5)P(3), since a further increase of the Ins(1,4,5)P(3) concentration to 1.0 µM evoked only a minor further release of Ca.


Figure 7: Effect of stepwise increases in Ins(1,4,5)P(3) concentration on loaded intracellular Ca stores in the absence of intracellular Ca-ATPase activity. Intracellular Ca stores of permeabilized pancreatic acinar cells were loaded with Ca to a steady-state level for 15 min as described under ``Experimental Procedures.'' At the start of the experiment, Ca pumps of the loaded intracellular stores were inhibited by including 1 µM thapsigargin in the medium. After 5 min the Ins(1,4,5)P(3) concentration was increased from 0.1 to 0.3 µM and finally to 1.0 µM at 5-min intervals. The Magfura ratio was determined each 15 s and the average value obtained from the eight cells in the field is shown. All permeabilized cells responded to the applications in a similar fashion and the result shown is representative of three independent experiments.




DISCUSSION

The major aim of the present study was to characterize the spatial organization of intracellular Ca stores in pancreatic acinar cells. To address this question we imaged Ca stores in individual permeabilized pancreatic acinar cells using a Ca-sensitive dye compartmentalized in organelles. Our main finding is that Ins(1,4,5)P(3)-sensitive Ca stores are located throughout the acinar cell cytoplasm and that no regional differences in Ins(1,4,5)P(3) sensitivity exist, at least in the absence of cytosolic modulatory factors.

During the cell permeabilization process, cytosolic factors are lost via the pores created by SLO in the plasma membrane(23) . One of our concerns was that SLO treatment affected intracellular structures. Both conventional and electron microscopy demonstrated that SLO permeabilized pancreatic acinar cells had a more swollen appearance. However, the cell architecture remained polarized, since the localization of zymogen granules remained restricted to the apical pole of the cells. Other studies on SLO-permeabilized pancreatic acinar cells have also shown that they retain their polarity and remain functionally active, both in terms of agonist- or Ins(1,4,5)P(3)-stimulated Ca release from intracellular stores and in terms of agonist- or Ins(1,4,5)P(3)- or Ca-stimulated enzyme secretion(24, 25) . Electron microscopy revealed that the endoplasmic reticulum was less strictly arranged compared with intact cells, an effect that was most likely caused by the swelling. We therefore cannot rule out the possibility that some rearrangement of the endoplasmic reticulum Ca stores may have occurred. However, our hypothesis is that the relative position of the components of the Ca stores is most likely not altered given that the permeabilized cells clearly retained their polarized morphology.

The characteristics of store loading, and the ratio values in unloaded and loaded stores of permeabilized pancreatic acinar cells, were similar to those observed in permeabilized gastric epithelial cells by Hofer and Machen(13) . Our estimate of the free intra-organellar Ca concentration in steady-state loaded intracellular Ca stores as 70 µM is also in broad agreement with the value of 127 µM found in gastric epithelial cells. It might be argued that Mg interferes to some extent with the Ca signals reported by Magfura-2. However, this seems unlikely, since (i) Mg is not transported in an ATP-dependent manner and (ii) the resting Magfura ratio in unloaded stores was very low, i.e. nearly equivalent to the minimum ratio for the dye in the total absence of all divalent cations. In addition, if Mg was present inside stores its free concentration would need to exceed a value of 150 µM to give a significant contribution to the Magfura-2 signal, since the apparent affinity of the dye for Mg is very low, i.e. 1.5 mM(26) . The Magfura-2 signal in organelles was clearly not saturated with Ca under normal conditions, since the ratio was increased markedly by exposure of Ca stores to the Ca ionophore 4-Br-A23187 in the presence of 1 mM ambient Ca.

Recent developments have allowed measurements of Ca levels inside the endoplasmic reticulum by targeting the Ca-sensitive bioluminescent protein aequorin to this organelle(27, 28, 29) . In the first work of this type, Kendall et al. (27, 28) have reported that in COS-7 cells free Ca inside the endoplasmic reticulum was around 1-5 µM, approximately 5-20 times the free cytosolic Ca concentration. Very recently, however, it has been reported by Montero et al.(29) that Ca concentrations inside the endoplasmic reticulum of HeLa cells exceeded 100 µM. By using the Ca surrogate Sr, these workers concluded that even millimolar free concentrations of divalent cations could occur within the endoplasmic reticulum and they argued from this that Ca levels might reach similar values. Taken at face value, however, the widely differing estimates reported using the aequorin technique suggest that a ubiquitous conclusion about Ca levels inside Ca stores cannot be reached. Although, as discussed above, we cannot exclude the possibility that properties of the endoplasmic reticulum are altered during permeabilization, we suggest that our estimate of a free Ca concentration within the endoplasmic reticulum of 70 µM might well be applicable to loaded Ca stores in intact pancreatic acinar cells.

The second messenger Ins(1,4,5)P(3) released Ca from intracellular Ca stores in permeabilized pancreatic acinar cells. The characteristics of this release were similar to the fluxes observed in suspensions of permeabilized acinar cells using the radioactive tracer Ca(10, 12) . Ca release was of a quantal nature, apparently due to the compensatory action of the organelle Ca pump during suboptimal stimulation. Thus, suboptimal concentrations of Ins(1,4,5)P(3) were much more efficient in releasing Ca in the absence of Ca pumping activity. An interesting observation was that all permeabilized cells showed similar sensitivities to Ins(1,4,5)P(3). This observation rules out the often raised possibility that intercellular differences in Ins(1,4,5)P(3) sensitivity determine the quantal nature of Ins(1,4,5)P(3)-induced Ca release.

Compartmentalized dye techniques similar to those applied here have been employed in a number of cell types, including hepatocytes, gastric epithelial cells, AR4-2J pancreatoma cells, and smooth muscle cells (13, 21, 30, 31, 32) . In both hepatocytes (31) and DDT(1)MF-2 smooth muscle cells (32) the intracellular Ca stores function as a single homogeneous pool, and electron microscopy has shown that the endoplasmic reticulum, which presumably acts as the intracellular Ca storage compartment, is a continuous compartment. However, the properties of Ins(1,4,5)P(3)-induced Ca release from intracellular Ca stores clearly differed between the two cell types. In permeabilized hepatocytes attached to coverslips, the Ins(1,4,5)P(3)-induced Ca release was non-quantal, in that, while the kinetics of Ca release depended on the concentration of Ins(1,4,5)P(3) used, all doses of Ins(1,4,5)P(3) eventually induced total depletion of the Ca stores. In smooth muscle cells, in contrast, Ca release induced by Ins(1,4,5)P(3) was of a quantal nature, similar to what we have observed for pancreatic acinar cells.

Agonist stimulation of intact acinar cells initiates a rise of cytosolic Ca in the apical pole of the cell, with the increase in [Ca](i) subsequently spreading into basolateral areas of the cell(2, 3, 4, 5, 6, 7) . It is notable, however, that some reports indicate that [Ca](i) rises uniformly throughout acinar cells upon agonist stimulation(33, 34) . In recent studies on intact acinar cells, employing the combination of imaging techniques and patch-clamp recording, evidence was obtained for a heterogeneous distribution of Ca stores(5, 6) . By infusing acinar cells with a low dose of Ins(1,4,5)P(3), or its non-metabolizable analogue inositol 1,4,5-trisphosphorothioate, it was shown that Ca spikes could be generated exclusively in the apical pole. These results therefore strongly support the idea that a heterogeneous population and distribution of Ins(1,4,5)P(3)-sensitive Ca pools do exist in individual pancreatic acinar cells. Biochemical evidence suggested that this store heterogeneity might be explained by differences in numbers of Ins(1,4,5)P(3)-operated Ca channels and/or by differences in sensitivity to Ins(1,4,5)P(3)(12) . In particular, it was suggested that, during suboptimal stimulation, the most sensitive stores were completely depleted, whereas less sensitive stores remained partially filled due to a compensatory pumping mechanism. This model (12) could explain a number of observations in intact acinar cells in which the apical pole Ca stores display higher apparent Ins(1,4,5)P(3) and Ca sensitivity(5, 6) . In the present study we have examined directly whether Ca stores were heterogeneously distributed in individual permeabilized cells. However, no subcellular differences in Ins(1,4,5)P(3) sensitivity could be detected. One possible interpretation of this result is that subcellular regional differences in Ins(1,4,5)P(3) sensitivity depend critically on some aspects of cellular or cytoskeletal or endoplasmic reticulum architecture, which is disrupted by permeabilization. However, as discussed above, we feel it is unlikely that cell permeabilization results in a major redistribution of Ca stores. If our results can be extrapolated to the intact acinar cell, an alternative explanation of why Ca starts to rise in the apical region on agonist stimulation may be the selective presence of an additional Ca release mechanisms in this area of the cell(5, 35) . Several studies suggest the existence of cyclic ADP-ribose-induced Ca release which might be mediated by ryanodine receptors (35, 36) . The experimental evidence in those studies has been interpreted to suggest that the combined activation of Ins(1,4,5)P(3)-sensitive and cyclic ADP-ribose-sensitive mechanisms is required to explain polarized Ca spike generation. However, 5 µM cyclic ADP-ribose failed to change the Magfura signal in permeabilized pancreatic acinar cells, whereas in the same cells Ins(1,4,5)P(3) induced a normal response. (^2)It is possible that cytosolic factors, which are lost during permeabilization and the subsequent extensive perfusion, may be required for the cyclic ADP-ribose response.

Interestingly, other lines of evidence argue against our finding that no regional differences in Ins(1,4,5)P(3) sensitivity exist. Several isoforms of the Ins(1,4,5)P(3) receptor are known to be expressed in pancreatic acinar cells(37, 38) . Therefore, multiple isoforms are likely to be translated into functionally operating receptors in pancreatic acinar cells, possibly with non-homogeneous distributions within the cell. So far, immunocytochemistry with antibodies directed against Ins(1,4,5)P(3) receptors has shown that these receptors are present in the apical pole of pancreatic and airway gland acinar cells(24, 39) . In pancreatic acinar cells only type 3 Ins(1,4,5)P(3) receptors were detected, with no evidence being found for the presence of type 1 Ins(1,4,5)P(3) receptors(24) . This observation is surprisingly for two reasons: (i) Ins(1,4,5)P(3) sensitivity in basolateral areas has been demonstrated many times in intact cells (5, 6) and in permeabilized cells (this study) and (ii) more than half of the Ins(1,4,5)P(3) receptor mRNA expressed in the whole pancreas was mRNA of type 1 receptors(37, 38) .

The present study is, however, consistent with Ins(1,4,5)P(3) binding studies in a pancreatic microsomal fraction, which revealed the presence of a single class of binding sites(20) . In addition, the dose-response curve for Ins(1,4,5)P(3)-induced Ca release gave no indications of the presence of multiple binding sites (e.g.(19) and (20) ). Multiple Ins(1,4,5)P(3) binding sites and a broad dose-response relationship for Ins(1,4,5)P(3)-induced Ca release would be expected if the intrinsic properties of Ins(1,4,5)P(3) receptor subtypes included different Ins(1,4,5)P(3) sensitivities. If the spatial pattern of Ca signaling observed in pancreatic acinar cells is not a result of the intrinsic properties of Ins(1,4,5)P(3) receptors, it must involve additional cytosolic factors regulating the opening of these receptor-operated ion channels. Multiple kinases and cytosolic factors like Ca are known to be involved in the complex regulation of Ins(1,4,5)P(3) receptors(1, 40, 41) . In peripherial tissues, cytosolic Ca levels and/or the phosphorylation status of Ins(1,4,5)P(3) receptors have been shown to play an important role in controlling Ca release mechanisms(42, 43) .

In conclusion, imaging of Ca within intracellular stores revealed that Ins(1,4,5)P(3)-sensitive Ca stores are found in all regions of the polarized pancreatic acinar cell. Furthermore, the stores did not display a heterogeneous sensitivity toward Ins(1,4,5)P(3). The polarized Ca signaling observed in intact acinar cells is therefore likely to be controlled by additional cytosolic factors and/or ryanodine receptors possibly present in the apical pole of acinar cells.


FOOTNOTES

*
This work was supported by a grant from the Wellcome Trust (to A. C. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: School of Biological Sciences, G. 38 Stopford Bldg., Oxford Rd., Manchester M13 9PT, United Kingdom. Tel.: 44-161-275-5452; Fax: 44-161-275-5600; vandeput{at}fs4.scg.man.ac.uk.

(^1)
The abbreviations used are: [Ca], free cytosolic Ca concentration; Ins(1,4,5)P(3), D-myo-inositol 1,4,5-trisphosphate; SLO, streptolysin O.

(^2)
F. H. M. M. Van de Put and A. C. Elliott, unpublished observations.


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