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J. Biol. Chem., Vol. 276, Issue 47, 44146-44156, November 23, 2001

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Polarized Expression of G Protein-coupled Receptors and an All-or-None Discharge of Ca²⁺ Pools at Initiation Sites of [Ca²⁺]_i Waves in Polarized Exocrine Cells^*

Dong Min Shin, Xiang Luo, Thomas M. Wilkie^§, Laurence J. Miller^¶, Ammon B. Peck, Michael G. Humphreys-Beher^**, and Shmuel Muallem

From the Departments of  Physiology and ^§ Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, the ^¶ Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905, the  Department of Pathology and Laboratory Medicine and the ^** Departments of Oral Biology and Pharmacology and Therapeutics, University of Florida, Gainesville, Florida 32610

Received for publication, June 6, 2001, and in revised form, August 28, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present work we examined localization and behavior of G protein-coupled receptors (GPCR) in polarized exocrine cells to address the questions of how luminal to basal Ca²⁺ waves can be generated in a receptor-specific manner and whether quantal Ca²⁺ release reflects partial release from a continuous pool or an all-or-none release from a compartmentalized pool. Immunolocalization revealed that expression of GPCRs in polarized cells is not uniform, with high levels of GPCR expression at or near the tight junctions. Measurement of phospholipase C activity and receptor-dependent recruitment and trapping of the box domain of RGS4 in GPCRs complexes indicated autonomous functioning of G_q-coupled receptors in acinar cells. These findings explain the generation of receptor-specific Ca²⁺ waves and why the waves are always initiated at the apical pole. The initiation site of Ca²⁺ wave at the apical pole and the pattern of wave propagation were independent of inositol 1,4,5-trisphosphate concentration. Furthermore, a second Ca²⁺ wave with the same initiation site and pattern was launched by inhibition of sarco/endoplasmic reticulum Ca²⁺-ATPase pumps of cells continuously stimulated with sub-maximal agonist concentration. By contrast, rapid sequential application of sub-maximal and maximal agonist concentrations to the same cell triggered Ca²⁺ waves with different initiation sites. These findings indicate that signaling specificity in pancreatic acinar cells is aided by polarized expression and autonomous functioning of GPCRs and that quantal Ca²⁺ release is not due to a partial Ca²⁺ release from a continuous pool, but rather, it is due to an all-or-none Ca²⁺ release from a compartmentalized Ca²⁺ pool.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺ signaling by G protein-coupled receptors (GPCR)¹ involves the generation of inositol 1,4,5-trisphosphate (IP₃) in the cytosol and Ca²⁺ release from the endoplasmic reticulum (1). In polarized exocrine cells, Ca²⁺ release is not uniform but occurs in the form of Ca²⁺ waves. Rooney et al. (2) reported a unique initiation site and propagation pattern of GPCR-evoked Ca²⁺ waves in hepatocytes. Kasai et al. (3) first described the unique feature of initiation of Ca²⁺ waves at the apical pole and their propagation to the basal pole of pancreatic acini. This phenomenon was later confirmed in pancreatic acinar cells (3-6) and was extended to other exocrine cells (7, 8). Subsequent studies showed that expression of high levels of all IP₃ receptor (IP₃Rs) isoforms at the apical pole accounts for the initiation of Ca²⁺ waves at this site (7, 9). Furthermore, the apical pole showed higher sensitivity to Ca²⁺ release by IP₃ than other regions of the cell, including the basal pole (3, 10, 11). However, since the discovery of the polarized Ca²⁺ waves in exocrine cells, it remained a mystery how IP₃ can be generated in the apical pole to initiate the waves. Early functional (12) and radioligand (13) localization of receptors in tissue slices indicated that GPCRs are expressed in the basal membrane. Therefore, it was assumed that during GPCR stimulation IP₃ generated in the basal pole diffuses to the apical pole to initiate Ca²⁺ release and waves (14). This assumption has several difficulties. For example, at maximal stimulus intensity Ca²⁺ release starts within few milliseconds of cell stimulation. The diameter of exocrine acinar cells is about 20 µm. This requires exceptionally high rate of diffusion of IP₃ in the cytosol of this cells. In pancreatic acini, Ca²⁺ release events can remain confined to the apical pole (3, 4). This requires continuous traffic of IP₃ through the cytosol without causing Ca²⁺ release. Another alternative is generation of IP₃ in or close proximity to the apical pole. This requires localization of GPCR at the apical pole. With the development of suitable antibodies for immunolocalization of GPCR and the use of isolated cell clusters that increase the resolution of immunolocalization, it became possible to re-examine GPCR localization in relation to initiation of Ca²⁺ waves.

Another aspect of Ca²⁺ release in terms of initiation and propagation of Ca²⁺ waves is the architecture of the Ca²⁺ pool and the dynamics of Ca²⁺ release. Propagation of Ca²⁺ waves requires either sequential Ca²⁺ release from a compartmentalized pool or release from different sections of a continuous pool along the path of the Ca²⁺ wave. A unique property of Ca²⁺ release from internal stores, the quantal feature of Ca²⁺ release (15), can reflect the spatial organization of the Ca²⁺ pool that is needed for propagation of Ca²⁺ waves. Ca²⁺ release evoked by either stimulation of GPCR (15), activation of the IP₃ receptors (15-17), or activation of the ryanodine receptors (18) has quantal properties, that is at submaximal stimulus intensity only part of the Ca²⁺ pool is released and at increased stimulus an increased fraction of the pool is released. Two main models were proposed to explain quantal release. The first is an all-or-none release of Ca²⁺ from a compartmentalized pool that has a continuum of sensitivity to IP₃ (15, 19, 20). The second model proposes phasic Ca²⁺ release from a homogeneous pool where the phase of release at a given IP₃ concentration is determined by gating of IP₃Rs activity by Ca²⁺ content remaining in the stores (21). The first model fits well with the experimentally observed variable sensitivity of different cellular regions of pancreatic acini to IP₃-mediated Ca²⁺ release (3, 11). On the other hand, recent work (22) in Xenopus oocytes reports that the quantal behavior of Ca²⁺ release stems from rapid adaptation of the IP₃R channels of a continuous Ca²⁺ pool and claimed to refute the compartmentalization model.

The constancy of initiation of Ca²⁺ waves in the apical pole by all GPCR of pancreatic acini and the variable sensitivity of different cellular regions to IP₃-mediated Ca²⁺ release prompted us to determine localization of GPCR in pancreatic acini and to examine their autonomous behavior and what mechanism they use to evoke quantal release. We report that expression of GPCR is highly enriched in the apical pole, at or just underneath the tight junctions. This provides an explanation of how Ca²⁺ waves are initiated at the apical pole. The initiation site and propagation pattern of Ca²⁺ waves are receptor-specific in the same cells. This appears to be the result of autonomous coupling of receptors to G proteins and thus operation of GPCR signaling complexes. Finally and most notably, by using agonist concentration jump protocols and inhibition of the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump of partially stimulated cells, we provide evidence that the quantal properties of Ca²⁺ release are due to an all-or-none Ca²⁺ release from a compartmentalized Ca²⁺ pool.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Thapsigargin was from Alexis. The anti-muscarinic monoclonal antibody (mAb) M35 was from Argene. The hybridoma cells producing the 5H9 mAb against muscarinic type 3 receptor (M3R) and the specificity of the Abs were described elsewhere (23). Two pAbs that recognize different epitopes of the cholecystokinin (CCK) receptor were prepared using peptide antigens, and their specificity was extensively characterized and verified by blocking with peptides that were used to raise the anti-CCKR antibodies (24). A recombinant M2R was a generous gift from Dr. Eliott Ross (University of Texas Southwestern Medical Center, Dallas). Anti-IP₃R2 pAb was a generous gift from Dr. Akihiko Tanimura (University of Hokkaido, Ishikari-Tobetsu, Japan). The box domain of RGS4 (4Box) was prepared as described (25). Anti-IP₃R3 mAb and anti ZO1 pAbs were purchased from ABR and Zymed Laboratories Inc., respectively. Anti-ZO1 mAb was obtained from the Hybridoma Bank at the University of Iowa.

Preparation of Pancreatic Acini and Single Acinar Cells-- Acini were prepared from the pancreas of 100-150-g rats by limited collagenase digestion as described previously (6). After isolation, the acini were resuspended in a standard solution A containing (in mM) 140 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 Hepes (pH 7.4 with NaOH), 10 glucose, and 0.1% bovine serum albumin and kept on ice until use. Doublet or triplet acinar cell clusters were obtained by incubation of a minced pancreas in a 0.025% trypsin, 0.02% EDTA solution for 5 min at 37 °C. After washing with solution A supplemented with 0.02% soybean trypsin inhibitor, doublets and triplets were liberated by a 7-min incubation at 37 °C in the same solution that also contained 160 units/ml pure collagenase. The cells were washed with solution A and kept on ice until use.

[Ca²⁺]_i Imaging-- Pancreatic acinar cells were loaded with Fura 2, and [Ca²⁺]_i was imaged as detailed before (6). Fura 2 fluorescence was measured at a single excitation wavelength of 380 nm, by averaging eight consecutive images for each time point. Under these conditions and using a frame size of 256 × 240 pixels, recording was at a resolution of 90 ms/averaged image. During perfusion with the control solution and just before the first stimulation, the image of resting cells was acquired and was taken as the fluorescence signal at time 0 (F₀). Pixel values of all subsequent images were divided by this image, and the traces and images are the calculated F_t/F₀, where F_t is the fluorescence at time t.

To calculate the distance between initiation sites from which Ca²⁺ waves were triggered by different agonists or sequential application of the same agonist in the same cell, the initiation sites of all waves were marked on the first image, and the image was transferred to Adobe Photoshop. A right triangle was drawn using the ray between the two initiation sites as the hypotenuse. The x and y axes originated from the left and top plane, respectively. The distance between the initiation sites was then calculated by means of the Pythagorean theorem. The distances from multiple experiments were averaged and are given as means ± S.E. Although this procedure allowed quantitative analysis of the distance between initiation sites, such analysis has limitations inherent to the imaging procedure. The most critical limitation is the spatial resolution of cell imaging. The initiation sites and the waves are three-dimensional, whereas imaging at a speed needed to capture Ca²⁺ waves can be done only in two dimensions. Another limitation of the measurement is the temporal resolution of our recording system. This resulted in initiation sites that occupy 1-2 µm². Nevertheless, the differences in Ca²⁺ wave initiation sites and propagation pattern observed when cells were stimulated with two different agonists were sufficiently large to allow quantitative analysis of our measurements.

Electrophysiology-- The whole cell configuration of the patch clamp technique was used for measurement of Ca²⁺-activated Cl current, which correlates with changes in [Ca²⁺]_i near the plasma membrane (26). The experiments were performed with single acinar cells perfused with solution A. The standard pipette solution contained the following (in mM): 140 KCl, 1 MgCl₂, 0.5 EGTA, 5 ATP, 10 Hepes (pH 7.3 with KOH) with or without 100 nM 4Box, as described in previous studies (27). The 4Box was dialyzed against an ATP-free pipette solution and concentrated to about 5 µM with a centricone system. Seals of 6-10 gigohms were produced on the cell membrane, and the whole cell configuration was obtained by gentle suction or voltage pulses of 0.5 V for 0.3-1 ms. The patch clamp output (Axopatch-1B, Axon Instruments) was filtered at 20 Hz. Recording was performed with patch clamp 6 and a Digi-Data 1200 interface (Axon Instruments). All traces shown were recorded at a holding potential of 40 mV.

Measurement of IP₃-- IP₃ levels were measured by a radioligand assay as described elsewhere (28). Acini in solution A were stimulated with the indicated combination of agonists for 10 s at 37 °C. The reactions were stopped by addition of 20 µl of ice-cold 20% perchloric acid to 200 µl of samples, vigorous mixing, and incubation on ice for at least 10 min to allow precipitation of proteins. The supernatants were collected and transferred to clean tubes. Standards of IP₃ were prepared in the same manner. The perchloric acid was removed and IP₃ extracted by the addition of 0.15 ml of Freon and 0.15 ml of tri-n-octylamine. IP₃ content in the aqueous phase was measured by displacement of [³H]IP₃ using microsomes prepared from bovine brain cerebella (28).

Immunocytochemistry-- The immunostaining procedure was described previously (29). In brief, cells attached to glass coverslips were fixed and permeabilized with cold methanol. After removal of methanol, nonspecific sites were blocked by a 1-h incubation in blocking medium prior to incubation with 50 µl of blocking medium containing control serum (controls) or the following antibodies: 1:500 dilution of the M35 mAb that recognizes all muscarinic receptors; the same mAbs that were reabsorbed with recombinant M2R; 1:250 dilution of the 5H9 mAb recognizing the M3R; 1:200 dilution of pAb specific for the M3R; 1:50 dilution of pAb1 and a 1:100 dilution of pAb2 that recognize the CCK receptor; 1:200 dilution of mAb against IP₃R3; 1:100 dilution of pAb against IP₃R2; 1:20 dilution of mAb against ZO1; and 1:100 dilution of pAb recognizing ZO1. After incubation with the various primary antibodies overnight at 4 °C, the cells were washed three times with the incubation buffer. For the experiment with the 5H9 mAbs in Fig. 4A, two different protocols were used. The first was a standard protocol using permeabilized cells. Under these conditions, all pools of M3R were detected. Because this antibody recognizes an extracellular epitope of the M3R diagnostic of Sjögren syndrome (23), it was used to label the receptors in the plasma membrane by incubating a 0.5-ml suspension of intact cells with 50 µl of 5H9 mAb for 30 min at 37 °C. The cells were then washed 3 times with phosphate-buffered saline to remove excess antibodies before fixation with 0.5 ml of cold methanol. All steps post-fixation were the same as with permeabilized cells. The primary Abs were detected with goat anti-rabbit or anti-mouse IgG tagged with fluorescein or rhodamine. Images were collected with a Bio-Rad MRC 1024 confocal microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Receptor-specific Ca²⁺ Waves-- Repetitive stimulation of pancreatic acinar cells with the same high concentration of agonist evokes Ca²⁺ waves with the same initiation site and propagation pattern (6). The quantal properties of agonist-mediated Ca²⁺ release (15) raised the question of whether partial and maximal discharge of the pool generates the same or different Ca²⁺ waves. In particular, it was of interest to determine whether the wave initiates from the same site at low and high IP₃ concentrations generated by weak and intense agonist stimulation. The properties of Ca²⁺ waves evoked by repetitive, brief stimulation with increasing concentration of agonist (5, 50, and 500 µM carbachol) are depicted in Fig. 1. Similar results were obtained in seven different cell preparations. All experiments were performed with clusters of doublet or triplet cells to ensure polarized expression of signaling protein was maintained. The arrowheads in the first image of each series show that the initiation site is constant and is independent of the IP₃ concentration used to initiate the wave. The calculated distance between initiation sites (see "Experimental Procedures") averaged 0.19 ± 0.05 µm (n = 7). The diameter of an acinar cell was about 20 µm. In addition, the pattern of wave propagation remained constant at all agonist concentrations. Increased stimulation intensity only increased the rate of Ca²⁺ wave propagation.

View larger version (97K): [in this window] [in a new window]   Fig. 1.   Ca2+ wave initiation site and pattern are independent of [IP3]. Fura 2-loaded doublet or triplet cell clusters were stimulated with 5, 50, and 500 µM carbachol. The cells were perfused with solution A for 3 min between the stimulations. The top left bright field image is × 600 magnification and the middle image is × 1000 magnification of the cell marked by an asterisk. The top right image shows the Fura 2 ratio under resting conditions. The first image in each row shows the first detected [Ca2+]i increase by the respective carbachol concentration. The arrowheads mark the exact same spot in each image, demonstrating the same initiation site at all carbachol concentrations. The sequential images show the identical Ca2+ wave patterns at all carbachol concentrations. The bottom panel show the changes in Ft/F0 ratios during the [Ca2+]i rise phase for the three carbachol concentrations. Traces 1-5 show the [Ca2+]i changes in the areas marked 1-5 in the bright-field image. Please note the different times at top left of each sequence of images and the different time scales for the traces at each agonist concentration, demonstrating the effect of increased agonist concentration on the speed of the wave. AM, apical membrane; BM, basal membrane; SG, secretory granule; N, nucleus.

Constant initiation site and propagation patterns of Ca²⁺ waves were also observed with repetitive stimulation of another GPCR, the CCK receptor (see below). However, in previous work (6) we reported that stimulations of multiple GPCRs in the same rat pancreatic acinar cell evoked receptor-specific Ca²⁺ waves in terms of initiation sites and propagation patterns. Cancela et al. (30) used mouse pancreatic acinar cells to suggest that Ca²⁺ waves are stochastic and are similar for all GPCRs in a given cell. Therefore, we examined Ca²⁺ waves evoked by carbachol and CCK stimulation in the same cell of small acinar clusters. Fig. 2 shows three examples of five experiments with similar results. The cells were stimulated with submaximal agonist concentrations to increase the temporal resolution of the waves. The white (carbachol) and magenta (CCK) arrowheads in the first image of each experiment show that, without exception, the initiation site of Ca²⁺ waves was different between the two agonists. The calculated distance between the carbachol and CCK initiation averaged 4.5 ± 1.5 µm (range 1.2-9.1 µm, n = 5). Although the waves initiated by stimulation of the two receptors propagated along the cell periphery, differences in wave patterns were noticeable in most experiments. Close examination of the results in Fig. 5A of Cancela et al. (30) also show that in a single isolated mouse pancreatic acinar cell activation of the muscarinic and CCK receptors resulted in different initiation sites of Ca²⁺ waves. This is particularly evident when the third image of the acetylcholine series is compared with the second image of the CCK series. The Ca²⁺ waves evoked by the two GPCRs had different spatial propagation pattern.

View larger version (103K): [in this window] [in a new window]   Fig. 2.   Receptor-specific initiation site and propagation pattern of Ca2+ waves. Cells were sequentially stimulated with 5 µM carbachol and 0.25 nM CCK, as indicated in the traces in the upper panel. A shows the initiation site and pattern of the Ca2+ wave evoked by activation of the two receptors in the same cell. B and C show additional examples of the different Ca2+ wave initiation sites evoked by carbachol (white arrowhead) and CCK (magenta arrowhead) stimulation in the same cells. AM, apical membrane; BM, basal membrane.

Autonomous Behavior of Signaling Complexes-- The constancy of Ca²⁺ waves evoked by repetitive stimulation of the same GPCR, their independence of stimulus intensity, and the different Ca²⁺ wave evoked by activation of different GPCRs in the same cell suggest autonomous functioning of GPCRs. We used two experimental protocols to test this prediction. In the first set of experiments we examined IP₃ production by maximal agonist concentrations added individually or in combination. Although maximal stimulation of each of the GPCRs can mobilize the entire Ca²⁺ pool (see below), Fig. 3A shows that all combinations of agonists produced a nearly additive increase in IP₃ concentration. Hence, each GPCR can activate different pools of cellular PLC or distinct portions of a single pool that is in excess to the total number of GPCRs.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Autonomous behavior of GPCR complexes. A shows stimulation of IP3 production by 1 mM carbachol (Car), 10 nM CCK8b, and 100 nM BS alone (open column) and combinations of two (gray columns) or the three agonists (filled column). B-E show the Ca2+-activated Cl current recorded from control cells or cells dialyzed with 100 nM 4Box for 7 min before the first stimulation. The bars indicate where the cells were stimulated with the indicated agonist concentration. Note that CCK8 and BS had no effect on [Ca2+]i in carbachol-stimulated cells under control conditions but evoked a maximal response in cells dialyzed with 4Box.

In the second protocol we used the RGS domain of RGS4 (4Box) to demonstrate autonomous functioning of the GPCRs in the same cell. The 4Box lacks receptor recognition domain and is a poor inhibitor of signaling in resting cells (31). The 4Box binds to activated -subunits of G proteins (25, 32) and is nearly as effective as full-length RGS4 in stimulating GTPase activity of G_q (31). Consequently, upon cell stimulation the 4Box is recruited to signaling complexes to inhibit their activity and remains trapped in the complexes for as long as the cells are stimulated (see Ref. 27 for further details on the mode of action of the 4Box). We took advantage of the mode of interaction of the 4Box with GPCR signaling complexes to examine whether maximal activation of one GPCR type is sufficient to recruit 4Box to all GPCRs complexes, or stimulation of each GPCR complex is needed to inhibit signaling of the stimulated complex. Fig. 3, B and C, shows that stimulation of cells with 1 mM carbachol resulted in complete mobilization of the Ca²⁺ pool, as evident from the failure of 10 nM CCK8 and 100 nM bombesin (BS) to increase [Ca²⁺]_i in cells continuously stimulated with carbachol. When the cells were infused with 100 nM 4Box, stimulation with 1 mM carbachol resulted in a maximal initial signal that terminated rapidly and returned to base line at continuous stimulation with carbachol, due to receptor-dependent recruitment and trapping of 4Box (27). Fig. 3, D and E, shows that at continuous stimulation with carbachol the cells responded with a maximal signal to CCK8 (n = 15) and BS stimulation (n = 4). The response to CCK8 and BS had the same kinetics as that to carbachol, with a rapid onset and inhibition, and it remained inhibited for the duration of cell stimulation. This behavior is best explained if the various receptors are coupled to separate pools of cellular G_q and PLC.

Polarized Expression of Ca²⁺ Signaling Proteins and Receptors-- Polarized and restricted expression of Ca²⁺ channels and Ca²⁺ pumps in the luminal pole of epithelial cells appears to be important for initiation and propagation of polarized Ca²⁺ waves (7, 33). Co-immunoprecipitation of various Ca²⁺ signaling proteins indicates organization of the proteins into complexes in cellular microdomains (29). GPCRs are believed to be expressed mostly in the basal membrane. However, a recent report (34) suggested the possible expression of selective GPCRs next the luminal pole in rat goblet cells. With the development of selective antibodies that recognize several GPCRs, it was of interest to re-examine localization of GPCRs in pancreatic acini. Fig. 4 demonstrates the co-localization of the type 3 muscarinic (M3Rs) and CCK receptors (CCKRs) with Ca²⁺-signaling proteins. Both receptors are expressed at high levels at the apical pole of pancreatic acini. Several antibodies used to localize the M3Rs revealed two cellular pools of these receptors, a plasma membrane and a Golgi-like pools. Double staining with pAb recognizing M3Rs and ZO1, a specific tight-junction protein (Fig. 4A, top panels), or IP₃R2 (not shown) showed that M3Rs localized in a non-uniform fashion with high levels at the lateral border, in close proximity or at the tight junctions, and low levels at the basal region (n = 10). However, the M35 and the 5H9 mAbs showed different patterns of localization. In permeabilized cells, the two mAbs showed strong labeling of an intracellular compartment and lower staining of the plasma membrane (n = 9 for M35, n = 6 for 5H9). This is particularly evident with the M35 mAb (Fig. 4, lower middle panel). The staining with this mAb was eliminated by adsorbing the mAb with recombinant M2Rs (Fig. 4, lower left panel), demonstrating specificity of the Abs. Because the 5H9 mAbs recognize an extracellular epitope evident of Sjögren syndrome (23), the plasma membrane staining was isolated by incubating intact cells with the Ab prior to cell fixation and permeabilization. Fig. 4A, lower right panel, shows that the M3Rs receptors are highly localized to the lateral membrane next to tight junctions (n = 4).

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Polarized expression of GPCRs, ZO1, and IP3Rs. Isolated pancreatic acini were fixed and stained with the following Abs: A, upper panels, double staining with anti-ZO1 and anti-M3Rs; middle panels, double staining with anti-ZO1 and the anti-M3R mAb clone 5H9 after cell permeabilization; lower left panel, staining with the anti-muscarinic receptors mAb M35 pre-adsorbed with recombinant M2 receptors; middle panels, double staining with anti-M3Rs clone M35 and anti-ZO1; lower right panel, staining with anti-M3Rs mAb clone 5H9 in intact cells before permeabilization (see "Experimental Procedures"). B, main panels, double staining with anti-CCKRs raised against amino acids 411-429 of the CCKR and anti-ZO1 or anti-IP3R2, as indicated; right panel, staining with an Ab that was raised against amino acids 30-42 of the CCKR.

Selective localization was also observed with the CCKRs using two different pAbs recognizing different domains of the receptor, a pAb raised against amino acids 30-42 and a pAb raised against amino acids 411-429 of the type A CCKR (24). The two Abs specifically recognized recombinant and native CCKRs. Localization of the CCKRs at the apical pole of the lateral membrane (Fig. 4B) was similar to that of the M3Rs at the plasma membrane. The CCKRs at the plasma membrane were concentrated very near the IP₃R3 at the apical pole (n = 10). The anti-CCKRs Abs did not recognize a Golgi pool of this receptor.

Quantal Behavior and an All-or-None Ca²⁺ Release-- The independence of the Ca²⁺ waves initiation site and propagation pattern of IP₃ concentration (Fig. 1) offered us the opportunity to examine whether local Ca²⁺ release is due to an all-or-none release from a compartmentalized pool (Fig. 5, model A) or a partial release from a continuous pool (Fig. 5, model B). The experiments used to distinguish between the two models and their predicted outcome according to each model are depicted in Fig. 5. Resting cells are in state a. Shortly after cell stimulation with submaximal agonist concentration (state b), all the Ca²⁺ is released from a sub-compartment of the pool (model A) or part of the Ca²⁺ is released from the entire pool (model B). At continuous stimulation (state c), high [Ca²⁺]_i at the mouth of the IP₃Rs reduces channel activity to allow partial reloading of the just discharged sub-pool with Ca²⁺ by the activated SERCA pumps (35) (model A). In this scenario, Ca²⁺ permeability of the sub-pool remains elevated, whereas Ca²⁺ permeability of the remaining pool is similar to that in resting cells. By contrast, model B assumes that at continuous stimulation, the activity of all the IP₃Rs channels is adapted to the lower Ca²⁺ content in the entire pool to terminate Ca²⁺ release so that Ca²⁺ permeability of the pool is similar to that of resting cells.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Models of quantal Ca2+ release. Model A illustrates quantal Ca2+ release by an all-or-none mechanism and model B Ca2+ release by IP3Rs adaptation mechanism. The text depicts the predicted outcomes of the agonist concentration jump and SERCA pump inhibition protocols by each model.

The first protocol we used is an agonist concentration jump after state c, long after return of [Ca²⁺]_i to near resting level. Model A predicts that the agonist (or IP₃) concentration jump will generate a Ca²⁺ wave with the same initiation site and pattern. Model B predicts generation of Ca²⁺ waves from random initiation sites and propagation pattern, because of the adaptation of the IP₃Rs that generated the first wave. Fig. 6 shows the results of such experiments with carbachol and CCK. It is clear that the Ca²⁺ waves initiated by low agonist and the concentration jump have the same initiation site and propagation pattern. In five experiments with carbachol and four experiments with CCK, the average distances between the two initiation sites were 0.16 ± 0.03 and 0.10 ± 0.03 µm, respectively. Localized generation of IP₃ due to the polarized expression of the muscarinic receptors is likely to contribute to the constancy of the wave. Hence, in addition to testing the first prediction in Fig. 5, the constancy of the wave observed with the protocol of Fig. 6 lend further support for the autonomous behavior of signaling complexes

View larger version (105K): [in this window] [in a new window]   Fig. 6.   Successive stimulation by a delayed agonist concentration jump. A, the cell was stimulated with 5 µM carbachol, and after return of Ca2+ to basal levels carbachol concentration was rapidly increased to 0.5 mM. B, the cell was stimulated with 0.25 nM CCK, and after return of Ca2+ to resting levels CCK concentration was increased to 10 nM. The white arrowheads show the Ca2+ wave initiation site for each stimulation period. Note the same initiation site by the low and high agonist concentrations in all experiments.

In the second protocol, the second Ca²⁺ wave was initiated by applying the concentration jump at the end of Ca²⁺ release evoked by the low agonist and before any reduction in [Ca²⁺]_i due to IP₃Rs channel adaptation. Model A predicts a second Ca²⁺ wave with different initiation site, due to discharge of all the Ca²⁺ from the pool mobilized by low agonist. Model B predicts a second Ca²⁺ wave from the same initiation site because Ca²⁺ is released prior to adaptation of the IP₃Rs activated by the first stimulus. The trace in Fig. 7 shows that this particular cell was exposed to the supermaximal concentration of 5 mM carbachol at 1.63 s after simulation with 5 µM carbachol. The concentration of 5 mM was used for the second stimulus, which was applied by rapid injection from a needle adjacent to the cell, to minimize the delay between agonist application and cell stimulation. The images in Fig. 7 show the results of four of five independent experiments with similar results. In contrast with the prediction of model B, but as predicted by model A, in all experiments the second Ca²⁺ wave evoked by the agonist concentration jump was initiated at a different site than the first Ca²⁺ wave. The calculated distance between the initiation sites averaged 7.3 ± 1.3 µm (range 3.3-10.9 µm, n = 5), which is statistically different (p < 0.01) from the 0.19 ± 0.05 µm measured after delayed application of carbachol.

View larger version (97K): [in this window] [in a new window]   Fig. 7.   Successive stimulation by a rapid agonist concentration jump. The trace shows the experimental protocol and the Ft/F0 ratio of a typical experiment. The cells were stimulated with 5 mM carbachol at the peak of the [Ca2+]i increase evoked by 5 µM carbachol. The images in A-D show the initiation site of the Ca2+ waves evoked by rapid, consecutive application of 5 µM (white arrowhead) and 5 mM carbachol (magenta arrowhead) to the same cells. Note the different initiation sites by the low and high agonist concentrations in all experiments.

A shift in the initiation site of Ca²⁺ waves when the second wave was initiated at the end of the first Ca²⁺ release (Fig. 7) can be either because the initiation site still contained Ca²⁺ but the IP₃Rs at this site became refractory to a jump in IP₃ concentration, or because all the Ca²⁺ was released from this site by the first stimulus, and Ca²⁺ re-uptake is needed to initiate a second Ca²⁺ wave from the same site. The results in Fig. 8 provide direct evidence that Ca²⁺ release is an all-or-none process and that Ca²⁺ uptake must occur to initiate multiple Ca²⁺ waves from the same site. In these experiments we blocked the SERCA pumps with 10 µM thapsigargin (Tg) at different times after initiation of a Ca²⁺ wave. Model A predicts that inhibition SERCA pumps of cells continuously stimulated with low agonist at any time will trigger a Ca²⁺ wave with the same initiation site and pattern. Model B predicts that inhibition of SERCA pumps will not trigger a wave but rather will cause slow Ca²⁺ release because of adaptation of all IP₃Rs. Fig. 8A shows that in the absence of agonist stimulation, Tg caused a slow, peripheral to central Ca²⁺ release with no apparent Ca²⁺ wave or preferential release from any cellular region. As shown by the trace in Fig. 8A, [Ca²⁺]_i continued to increase for at least 30 s after exposure to Tg. Hence, in the absence of agonist stimulation Tg never initiated a Ca²⁺ wave.

View larger version (82K): [in this window] [in a new window]   Fig. 8.   Ca2+ waves are evoked by inhibition of SERCA pumps in cells continuously stimulated with low agonist concentration. A, maximal inhibition of SERCA pumps by 10 µM thapsigargin (Tg) in resting cells causes a slow peripheral to central Ca2+ release. B, maximal inhibition of SERCA pumps by Tg in cells continuously treated with 5 µM carbachol after return of [Ca2+]i to basal level launch a Ca2+ wave with the same initiation site (white arrowheads) and propagation pattern as the initial wave evoked by carbachol. C, cells were stimulated with 5 µM carbachol (a) or 0.25 nM CCK (b). At different times during the reduction of [Ca2+]i to basal levels, SERCA pumps were inhibited with 10 µM Tg. In all cases Tg evoked a rapid increase in [Ca2+]i (in the form of a luminal-to-basal Ca2+ wave) with a subsequent slow reduction in [Ca2+]i.

Very different results were obtained when the cells were stimulated with submaximal agonist concentration and allowed to reduce [Ca²⁺]_i back to near basal level. As shown in Fig. 8B, under these conditions Tg evoked rapid, luminal to basal Ca²⁺ waves. Remarkably, in all experiments the Tg-evoked Ca²⁺ waves had the same initiation site and propagation pattern as the first Ca²⁺ wave evoked by the agonists. The distance between the initiation sites of the first waves evoked by carbachol and the second waves evoked by Tg in the same cells averaged 0.25 ± 0.1 µm (n = 5), which is not different from the 0.16 ± 0.03 µm measured with delayed concentration jump. The traces in C show that the magnitude of Ca²⁺ release by Tg was dependent on the incubation time with agonist. Thus, simultaneous stimulation of the cells with 5 µM carbachol and 10 µM Tg resulted in a rapid Ca²⁺ release but slow return of [Ca²⁺]_i to basal levels, much slower than the rate observed with agonist alone (compare red to all other traces in C). Application of Tg at different times after cell stimulation caused a rapid increase in [Ca²⁺]_i, to approximately the same level as that caused by the agonist and a slow reduction toward basal levels. Hence, Tg rapidly discharged only the Ca²⁺ that was incorporated into the initiation site during continuous stimulation and never to a level significantly higher than the initial level caused by the agonists. This behavior indicates that Ca²⁺ release from the initiation site must be an all-or-none process and that the initiation site reloads with Ca²⁺ during continuous stimulation with submaximal agonist concentration (model A). Furthermore, this behavior is incompatible with model B.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present work we addressed two important topics in Ca²⁺ signaling: how luminal to basal Ca²⁺ waves can be generated in a receptor-specific manner and whether quantal Ca²⁺ release reflects partial release from a continuous pool or an all-or-none release from a compartmentalized pool. Polarized exocrine cells are a good model system to address these questions because of the polarized nature of their Ca²⁺ signals (3), the polarized expression of high levels of Ca²⁺ transport proteins in the apical pole (7, 9, 29, 33, 36), and the generation of receptor-specific Ca²⁺ signals in these cells (6, 37). Our results indicate that signaling specificity in pancreatic acinar cells is aided by polarized expression and autonomous functioning of GPCRs and that quantal Ca²⁺ release is due to an all-or-none Ca²⁺ release from a compartmentalized Ca²⁺ pool.

Signaling specificity is a central topic in cell signaling (38). Several mechanisms have been shown to contribute to generation of specific GPCR-dependent Ca²⁺ signals, which include selective phosphorylation of IP₃Rs (39), differential couplings of Ca²⁺ release to Ca²⁺ entry (40), and different involvement of cADP-ribose and NADP-mediated Ca²⁺ release in the response (30). However, ultimately signaling specificity must reside in the communication between the receptor and downstream effectors. Indeed, RGS proteins function in a receptor-specific manner to confer receptor-specific signaling (41). Interaction of receptors with effectors requires their co-localization in cellular microdomains at sites of Ca²⁺ release. In polarized exocrine cells Ca²⁺ waves are always initiated at the apical pole. Whereas expression of all known IP₃R isoforms (7, 9), specific isoforms of SERCA pumps (33), and plasma membrane Ca²⁺ ATPase pumps (36) has been demonstrated, it remains a puzzle as to how IP₃ can be generated at this cellular microdomain because GPCRs are believed to reside largely at the basal pole. The localization of the M3Rs and CCKRs found at the present work clarifies this issue. The use of three anti-M3Rs and two anti-CCKRs, which recognize different epitopes, showed that both GPCR types are expressed at high level at close proximity or at the tight junctions. This localization removes the need to assume long range diffusion of IP₃ from the basal to the apical pole to initiate Ca²⁺ waves. Moreover, such localization of GPCRs is likely to contribute to the constancy of the Ca²⁺ waves generated by repetitive stimulation of the same GPCR complex.

In the present work we confirmed and extended the finding of receptor-specific Ca²⁺ waves by various GPCRs expressed in the same cell. By using the unique property of recruitment and trapping of the 4Box within signaling complexes (27), we demonstrated autonomous functioning of GPCRs. Furthermore, production of IP₃ by multiple GPCRs was nearly additive. The combined findings indicate that different GPCRs communicate with separate portions of the cellular pool of G_q and PLC to generate IP₃ at separate cellular microdomains. The finding that the initiation site of Ca²⁺ waves was independent of IP₃ concentration, remaining constant when initiated by low (weak stimulation) or high IP₃ concentration (intense agonist stimulation), indicates that each GPCR communicates with a separate portion of the Ca²⁺ pool at the apical pole to generate Ca²⁺ waves with distinct initiation sites. Communication with a distinct portion of the Ca²⁺ pool may also reflect localized generation of IP₃ at the locus from which Ca²⁺ waves are initiated. Measurement of IP₃ in single pancreatic acinar cells is needed to test this point. At present, this is not feasible.

Since the discovery of the quantal behavior of Ca²⁺ release (15), two models were proposed as possible mechanisms of this phenomenon (see Fig. 5). The first model proposes that the intracellular Ca²⁺ pool is compartmentalized with respect to Ca²⁺ release, and the IP₃R Ca²⁺ release channels in the different compartments have variable affinity to IP₃ and Ca²⁺ release from individual compartments in an all-or-none process (15, 20). The second model proposed that the intracellular Ca²⁺ pool is continuous, and the affinity of all IP₃Rs to IP₃ is the same but is sensitive to Ca²⁺ content in the stores, and Ca²⁺ release is incremental due to rapid inactivating adaptation of the IP₃Rs (21). The evidence in favor of the adaptation model is the finding that stored Ca²⁺ regulates the affinity of the IP₃Rs for IP₃ in virtually all cell types examined (42), and a recent report (22) indicates that two rapid successive increases in IP₃ can liberate Ca²⁺ from the same site. The first evidence (42) is not mutually exclusive with the all-or-none model, and in the second case (22) rapid Ca²⁺ re-uptake into the stores was not prevented by inhibition of the SERCA pumps which, as shown in the present work, can explain the second Ca²⁺ release event.

Previous evidence in support of the all-or-none model was developed by measurement of Ca²⁺ permeability of the stores at increasing stimulus intensity (15). Increased agonist concentration mobilized a larger fraction of the Ca²⁺ pool. At persistent stimulation with submaximal agonist concentrations, Ca²⁺ permeability of the mobilized fraction of the pool remained very high, whereas Ca²⁺ permeability of the immobilized fraction remained very low, identical to that measured in resting cells (15). Another important finding in support of the all-or-none model is the variable sensitivity of IP₃ to Ca²⁺ release from different regions of pancreatic acinar cells (3, 11). The adaptation model predicts uniform IP₃ sensitivity throughout the cell.

In the present work we provide what we believe is strong evidence in support of the all-or-none model by first showing that the initiation site of Ca²⁺ waves is independent of IP₃ concentration. The implication of this finding is that there must exist a sub-pool that is more sensitive to IP₃ than the remaining cellular Ca²⁺ pool, and this pool is always discharged first upon cell stimulation. This provides additional evidence that the cellular Ca²⁺ pool is not uniform with respect to sensitivity to IP₃. Next, we used an agonist concentration jump to apply two consecutive rapid or delayed applications of IP₃. This protocol is equivalent to the two consecutive IP₃ application protocols used to conclude multiple Ca²⁺ release events from the same site (22). Two consecutive applications of IP₃ in pancreatic acini released Ca²⁺ from two separate sites when the second application was applied immediately at the end of Ca²⁺ release by the first application (Fig. 7). The difference between our results and those obtained in Xenopus oocytes (22) can be due to species differences or partial reloading of the just released pool with Ca²⁺.

The adaptation model requires that at continued exposure to the same IP₃ concentration, the IP₃R channels adapt, becoming refractory to IP₃, and Ca²⁺ permeability of the pool is reduced back to resting level to terminate Ca²⁺ release. We used Tg to provide compelling evidence that this is not the case, at least in pancreatic acini. In fact, the Ca²⁺ pool liberated by submaximal receptor stimulation reloads with Ca²⁺ with continuous exposure to a constant agonist concentration. Furthermore, and most important, the Ca²⁺ permeability of the released and reloaded pool remains high, resulting in a Tg-evoked Ca²⁺ wave. The Tg-evoked Ca²⁺ wave had the same initiation site and propagation pattern as the Ca²⁺ wave evoked by the agonist (Fig. 8). Hence, the maintained high Ca²⁺ permeability of the liberated sub-pool, reloading of the sub-pool at continued stimulation, and the shift in the initiation site of Ca²⁺ wave observed at rapid agonist concentration jump are all compatible only with an all-or-non model of Ca²⁺ release. Therefore, we conclude that the principal mechanism behind the quantal behavior of Ca²⁺ release is an all-or-none Ca²⁺ release from highly compartmentalized intracellular Ca²⁺ pool.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: the University of Texas Southwestern Medical Center, Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9040. Tel.: 214-648-2593; Fax: 214-648-8879; E-mail: SHMUEL.MUALLEM@utsouthwestern.edu.

Published, JBC Papers in Press, September 11, 2001, DOI 10.1074/jbc.M105203200

	ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptors; IP₃, inositol 1,4,5-trisphosphate; IP₃R, IP₃ receptor(s); SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase; PLC, phospholipase C; M3R, muscarinic type 3 receptor; Ab, antibody; mAb, monoclonal antibody; pAb, polyclonal antibody; CCK, cholecystokinin; BS, bombesin; CCK, CCK receptor(s); Tg, thapsigargin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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