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The Endoplasmic Reticulum Can Act as a Functional Ca2+ Store in All Subcellular Regions of the Pancreatic Acinar Cell

1997; Elsevier BV; Volume: 272; Issue: 44 Linguagem: Inglês

10.1074/jbc.272.44.27764

1083-351X

Frans H. M. M. van de Put, A. Elliott,

Endoplasmic Reticulum Stress and Disease

Stimulation of pancreatic acinar cells raises [Ca2+] i via Ca2+ release from inositol-1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores, generally considered to reside within the endoplasmic reticulum (ER). However, with physiological doses of cholinergic agonists, the [Ca2+] i increase is localized to the apical (secretory) pole of the cell, leading to suggestions that zymogen (secretory) granules themselves may constitute an InsP3-sensitive Ca2+ store responsible for localized Ca2+ release. We have therefore re-investigated whether the ER in pancreatic acinar cells is capable of acting as a functional Ca2+ store in all, or only some, cellular regions. In streptolysin O-permeabilized cells, the ER accumulated up to 25 mmol of 45Ca2+ per liter ER volume by an ATP-dependent, thapsigargin-sensitive, process. This tracer Ca2+ uptake was dependent on ambient (loading) [Ca2+], as was the intra-ER free [Ca2+], assessed by imaging the fluorescence of Magfura-2 within the Ca2+ stores. Comparison of free and total intra-ER [Ca2+] indicated that 200–300 Ca2+ions are bound within the ER lumen for every Ca2+ ion remaining free. Subcellular analysis showed that ER stores in all regions of the permeabilized cell took up Ca2+ at loading [Ca2+] between 60 nm and 1 μm. Thapsigargin released Ca2+ from stores in all cellular regions, as did InsP3. Immunofluorescence with antibodies against sarco(endo)plasmic reticulum-2b type Ca2+,Mg2+-ATPase or calreticulin confirmed that ER Ca2+ stores were present throughout the cytoplasm. In summary, these results clearly show that the endoplasmic reticulum can act as a functional Ca2+ store in all regions of the acinar cell, including the apical pole. Stimulation of pancreatic acinar cells raises [Ca2+] i via Ca2+ release from inositol-1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores, generally considered to reside within the endoplasmic reticulum (ER). However, with physiological doses of cholinergic agonists, the [Ca2+] i increase is localized to the apical (secretory) pole of the cell, leading to suggestions that zymogen (secretory) granules themselves may constitute an InsP3-sensitive Ca2+ store responsible for localized Ca2+ release. We have therefore re-investigated whether the ER in pancreatic acinar cells is capable of acting as a functional Ca2+ store in all, or only some, cellular regions. In streptolysin O-permeabilized cells, the ER accumulated up to 25 mmol of 45Ca2+ per liter ER volume by an ATP-dependent, thapsigargin-sensitive, process. This tracer Ca2+ uptake was dependent on ambient (loading) [Ca2+], as was the intra-ER free [Ca2+], assessed by imaging the fluorescence of Magfura-2 within the Ca2+ stores. Comparison of free and total intra-ER [Ca2+] indicated that 200–300 Ca2+ions are bound within the ER lumen for every Ca2+ ion remaining free. Subcellular analysis showed that ER stores in all regions of the permeabilized cell took up Ca2+ at loading [Ca2+] between 60 nm and 1 μm. Thapsigargin released Ca2+ from stores in all cellular regions, as did InsP3. Immunofluorescence with antibodies against sarco(endo)plasmic reticulum-2b type Ca2+,Mg2+-ATPase or calreticulin confirmed that ER Ca2+ stores were present throughout the cytoplasm. In summary, these results clearly show that the endoplasmic reticulum can act as a functional Ca2+ store in all regions of the acinar cell, including the apical pole. 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) 1The abbreviations used are: InsP3,d-myo-inositol 1,4,5-trisphosphate; [Ca2+]lumen, free intraluminal Ca2+ concentration; [Ca2+] i, free cytosolic Ca2+ concentration; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; SERCA-2b, Ca2+-ATPase sarco(endo)plasmic reticulum-2b type Ca2+,Mg2+-ATPase; SLO, streptolysin O; HEDTA,N-hydroxyethylethylenediaminetriacetic acid; ER, endoplasmic reticulum. was first demonstrated using a permeabilized pancreatic acinar cell preparation (1Streb H. Irvine R.F. Berridge M.J. Schulz I. Nature. 1983; 306: 67-69Crossref PubMed Scopus (1797) Google Scholar). This initial work also identified the endoplasmic reticulum (ER) as the intracellular Ca2+ store responsible for agonist-induced increases in [Ca2+] i (1Streb H. Irvine R.F. Berridge M.J. Schulz I. Nature. 1983; 306: 67-69Crossref PubMed Scopus (1797) Google Scholar). Recently, however, zymogen granules have been proposed to act as a Ca2+ store in pancreatic acinar cells. (2Gerasimenko O.V. Gerasimenko J.V. Belan P.V. Petersen O.H. Cell. 1996; 84: 473-480Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). 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 (3Kasai H. Augustine G.J. Nature. 1990; 348: 735-738Crossref PubMed Scopus (314) Google Scholar, 4Toescu E.C. Lawrie A.M. Gallacher D.V. Petersen O.H. EMBO J. 1992; 11: 1623-1629Crossref PubMed Scopus (124) Google Scholar, 5Tan Y.P. Marty A. Trautmann A.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11229-11233Crossref PubMed Scopus (42) Google Scholar, 6Elliott A.C. Cairns S.P. Allen D.G. Eur. J. Physiol. 1992; 422: 245-252Crossref PubMed Scopus (27) Google Scholar). Subsequently the rise in [Ca2+] i spreads to the basal pole of the acinar cell (3Kasai H. Augustine G.J. Nature. 1990; 348: 735-738Crossref PubMed Scopus (314) Google Scholar, 4Toescu E.C. Lawrie A.M. Gallacher D.V. Petersen O.H. EMBO J. 1992; 11: 1623-1629Crossref PubMed Scopus (124) Google Scholar, 5Tan Y.P. Marty A. Trautmann A.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11229-11233Crossref PubMed Scopus (42) Google Scholar, 6Elliott A.C. Cairns S.P. Allen D.G. Eur. J. Physiol. 1992; 422: 245-252Crossref PubMed Scopus (27) Google Scholar). 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 (2Gerasimenko O.V. Gerasimenko J.V. Belan P.V. Petersen O.H. Cell. 1996; 84: 473-480Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 7Kasai Li Y.X. Miyashita Y. Cell. 1993; 74: 669-677Abstract Full Text PDF PubMed Scopus (317) Google Scholar). 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. 8Bolender R.P. J. Cell Biol. 1974; 61: 269-287Crossref PubMed Scopus (206) Google Scholar). 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 (7Kasai Li Y.X. Miyashita Y. Cell. 1993; 74: 669-677Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 9Thorn P. Lawrie A.M. Smith P.M. Gallacher D.V. Petersen O.H. Cell. 1993; 74: 661-668Abstract Full Text PDF PubMed Scopus (430) Google Scholar). 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 (2Gerasimenko O.V. Gerasimenko J.V. Belan P.V. Petersen O.H. Cell. 1996; 84: 473-480Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) may be an artifact produced by the impurity of the zymogen granule preparation employed (10Yule D.I. Ernst S.A. Ohnishi H. Wojcikiewicz R.J.H. J. Biol. Chem. 1997; 272: 9093-9098Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). A further problem with the report of zymogen granule Ca2+stores (2Gerasimenko O.V. Gerasimenko J.V. Belan P.V. Petersen O.H. Cell. 1996; 84: 473-480Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) 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. 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. 11Van de Put F.H.M.M. Elliott A.C. J. Biol. Chem. 1996; 271: 4999-5006Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar. 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 (11Van de Put F.H.M.M. Elliott A.C. J. Biol. Chem. 1996; 271: 4999-5006Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), 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 (11Van de Put F.H.M.M. Elliott A.C. J. Biol. Chem. 1996; 271: 4999-5006Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). 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 mmnitrilotriacetic 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. (12Schoenmakers T.J.M. Visser G.J. Flik G. Theuvenet A.P.R. BioTechniques. 1992; 12: 870-879PubMed Google Scholar). SLO (0.4 IU/ml final concentration) was added directly to the perifusion chamber to permeabilize acinar cells as described previously (11Van de Put F.H.M.M. Elliott A.C. J. Biol. Chem. 1996; 271: 4999-5006Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). 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. 12Schoenmakers T.J.M. Visser G.J. Flik G. Theuvenet A.P.R. BioTechniques. 1992; 12: 870-879PubMed Google Scholar). 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 (13Van de Put F.H.M.M. Hoenderop J.G.J. De Pont J.J.H.H.M. Willems P.H.G.M. J. Membr. Biol. 1993; 135: 153-163Crossref PubMed Scopus (12) Google Scholar). 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 (14Van de Put F.H.M.M. De Pont J.J.H.H.M. Willems P.H.G.M. J. Biol. Chem. 1994; 269: 12438-12443Abstract Full Text PDF PubMed Google Scholar), 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 (8Bolender R.P. J. Cell Biol. 1974; 61: 269-287Crossref PubMed Scopus (206) Google Scholar). 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 (15Roderick H.L. Campbell A.K. Llewellyn D.H. FEBS Lett. 1997; 405: 181-185Crossref PubMed Scopus (82) Google Scholar) was diluted 1:100 and rabbit polyclonal antisera to SERCA-2b Ca2+-ATPase (16Wuytack F. Eggermont J. Raeymaekers L. Plessers L. Casteels R. Biochem. J. 1989; 264: 765-769Crossref PubMed Scopus (80) Google Scholar, 17Eggermont J.A. Wuytack F. Verbist J. Casteels R. Biochem. J. 1990; 271: 649-653Crossref PubMed Scopus (121) Google Scholar, 18Dormer R.L. Capurro D.E. Morris R. Webb R. Biochim. Biophys. Acta. 1993; 1152: 225-230Crossref PubMed Scopus (14) Google Scholar) 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. 11Van de Put F.H.M.M. Elliott A.C. J. Biol. Chem. 1996; 271: 4999-5006Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), 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. The total exchangeable Ca2+ uptake capacity of the thapsigargin-sensitive intracellular Ca2+ stores in permeabilized pancreatic acinar cells was determined using the radioactive45Ca2+ 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. As in our previous paper (11Van de Put F.H.M.M. Elliott A.C. J. Biol. Chem. 1996; 271: 4999-5006Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), 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.2 A). 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. 2 A). Because of the greatly enhanced initial Ca2+ uptake rate, the time taken to reach steady state [Ca2+]lumen was reduced at higher [Ca2+] i . Fig. 2 B shows the relationship between steady-state free [Ca2+]lumen (as indicated by the steady-state Magfura-2 ratio from Fig. 2 A) 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 (11Van de Put F.H.M.M. Elliott A.C. J. Biol. Chem. 1996; 271: 4999-5006Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), 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 andD). Thapsigargin has been widely used in intact acinar cells to induce “global” [Ca2+] i release (see e.g.Ref. 4Toescu E.C. Lawrie A.M. Gallacher D.V. Petersen O.H. EMBO J. 1992; 11: 1623-1629Crossref PubMed Scopus (124) Google Scholar). 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 (6Elliott A.C. Cairns S.P. Allen D.G. Eur. J. Physiol. 1992; 422: 245-252Crossref PubMed Scopus (27) Google Scholar). In permeabilized cells, thapsigargin completely prevents ATP-driven 45Ca2+accumulation (13Van de Put F.H.M.M. Hoenderop J.G.J. De Pont J.J.H.H.M. Willems P.H.G.M. J. Membr. Biol. 1993; 135: 153-163Crossref PubMed Scopus (12) Google Scholar). We tested whether thapsigargin could deplete previously loaded Ca2+ stores in permeabilized pancreatic acinar cells. Fig. 4 A 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. 4 A 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. 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 (11Van de Put F.H.M.M. Elliott A.C. J. Biol. Chem. 1996; 271: 4999-5006Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Fig. 4 Bpresents 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 InsP3are 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.5 A) and calreticulin (Fig.5 B) 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.5 A). 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 (seee.g. Ref. 8Bolender R.P. J. Cell Biol. 1974; 61: 269-287Crossref PubMed Scopus (206) Google Scholar) and arises from the fact that a large part of the apical cytoplasm is occupied by zymogen granules. 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 (8Bolender R.P. J. Cell Biol. 1974; 61: 269-287Crossref PubMed Scopus (206) Google Scholar). 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 (11Van de Put F.H.M.M. Elliott A.C. J. Biol. Chem. 1996; 271: 4999-5006Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). 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.1 B). 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 (19Michalak M. Milner R.E. Burns K. Opas M. Biochem. J. 1992; 285: 681-692Crossref PubMed Scopus (411) Google Scholar). 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 (20Khanna N.C. Waisman D.M. Biochemistry. 1986; 25: 1078-1082Crossref PubMed Scopus (23) Google Scholar)), 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. 2Eighty-two percent of pancreatic volume consists of acinar cells (8Bolender R.P. J. Cell Biol. 1974; 61: 269-287Crossref PubMed Scopus (206) Google Scholar). To simplify the calculation we have assumed that all calreticulin in the pancreas is contained in acinar cells. 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 (21Monteru M. Brini M. Marsault R. Sitia R. Pozzan T. Rizzuto R. EMBO J. 1995; 14: 5467-5475Crossref PubMed Scopus (265) Google Scholar), 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 (22Coppolino M.G. Woodside M.J. Demaurex N. Grinstein S. St-Arnaud R. Dedhar S. Nature. 1997; 386: 843-847Crossref PubMed Scopus (348) Google Scholar). 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 Xenopusoocytes, this action was found to be associated with the high affinity, low capacity, Ca2+-binding site, rather than with the low affinity, high capacity b