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Am J Physiol Gastrointest Liver Physiol 293: G1166-G1177, 2007. First published September 27, 2007; doi:10.1152/ajpgi.00352.2007
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HORMONES AND SIGNALING

Ca2+ release dynamics in parotid and pancreatic exocrine acinar cells evoked by spatially limited flash photolysis

Jong Hak Won,1,* William J. Cottrell,2,* Thomas H. Foster,2,3 and David I. Yule1

1Departments of Pharmacology and Physiology and 3Imaging Sciences; 2Institute of Optics, University of Rochester, Rochester, New York

Submitted 2 August 2007 ; accepted in final form 25 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intracellular calcium concentration ([Ca2+]i) signals are central to the mechanisms underlying fluid and protein secretion in pancreatic and parotid acinar cells. Calcium release was studied in natively buffered cells following focal laser photolysis of caged molecules. Focal photolysis of caged-inositol 1,4,5 trisphosphate (InsP3) in the apical region resulted in Ca2+ release from the apical trigger zone and, after a latent period, the initiation of an apical-to-basal Ca2+ wave. The latency was longer and the wave speed significantly slower in pancreatic compared with parotid cells. Focal photolysis in basal regions evoked only limited Ca2+ release at the photolysis site and never resulted in a propagating wave. Instead, an apical-to-basal wave was initiated following a latent period. Again, the latent period was significantly longer under all conditions in pancreas than parotid. Although slower in pancreas than parotid, once initiated, the apical-to-basal wave speed was constant in a particular cell type. Photo release of caged-Ca2+ failed to evoke a propagating Ca2+ wave in either cell type. However, the kinetics of the Ca2+ signal evoked following photolysis of caged-InsP3 were significantly dampened by ryanodine in parotid but not pancreas, indicating a more prominent functional role for ryanodine receptor (RyR) following InsP3 receptor (InsP3R) activation. These data suggest that differing expression levels of InsP3R, RyR, and possibly cellular buffering capacity may contribute to the fast kinetics of Ca2+ signals in parotid compared with pancreas. These properties may represent a specialization of the cell type to effectively stimulate Ca2+-dependent effectors important for the differing primary physiological role of each gland.

inositol 1,4,5 trisphosphate receptors; ryanodine receptors; imaging

STIMULATION OF EXOCRINE CELLS derived from the pancreas or salivary glands with G{alpha}q-coupled secretagogues results in an elevation in the cytosolic calcium concentration ([Ca2+]i) (30, 49). This event is a tightly controlled consequence of Ca2+ release from intracellular stores mediated primarily by the action of inositol 1,4,5 trisphosphate (InsP3) on InsP3 receptor (InsP3R), followed by Ca2+ influx from the extracellular space (2, 3, 6, 32, 33, 40, 41, 50). Both phases of the Ca2+ signal are absolutely required for the appropriate activation of effectors important for the primary physiological roles of the tissue, namely, fluid secretion from salivary glands and exocytotic protein secretion from pancreatic acinar cells (29, 46).

The Ca2+ signal stimulated in cells derived from each gland exhibits characteristic temporal and spatial properties. Superficially, these properties and the underlying mechanisms are similar: stimulation of either cell type with secretagogue or global uniform release of InsP3 from a caged precursor results in a signal, which invariably initiates in the apical pole of either cell, and then can spread throughout the cytoplasm to generate a global Ca2+ signal (18, 21, 24, 34, 39, 42, 44, 45). The initiation of the Ca2+ signal is believed to occur largely because of the predominant localization of InsP3Rs, which are tightly apposed to the luminal plasma membrane (23, 24, 34, 43, 52). However, the localization of secretagogue receptors themselves, and thus presumably production of InsP3, has been reported close to the junctional complexes (27, 37), and this may contribute to the initiation of the Ca2+ signal in an agonist-specific manner. Alternatively, it has been suggested that activation of basal secretagogue receptors and subsequent diffusion of InsP3 to apical InsP3R can fully account for the spatial properties of the signal (4). In either case the nonuniform generation of InsP3 acting on a specific distribution of InsP3R is likely to underlie the mechanism, accounting for the Ca2+ signals observed in both cell types.

Although the general characteristics of the Ca2+ signal in each cell type are similar, some important differences have been reported. These characteristics appear generally consistent with the particular specialized exocrine function of each gland. In particular, the machinery in parotid acinar cells appears dedicated to yield rapid, very large, global Ca2+ signals compared with pancreatic acinar cells (17, 23, 50). These Ca2+ signals appear ideally suited to activating the spatially separated ion channel effectors necessary for fluid secretion. For example, a recent study from our laboratory using total internal reflection microscopy to measure membrane-delimited Ca2+ signals has demonstrated that the magnitude of Ca2+ influx is vastly greater in parotid acinar cells compared with cells derived from the exocrine pancreas (50). This difference is most striking at low, presumably physiological, concentrations of secretagogues.

In addition, studies using both confocal and wide-field microscopy have reported that in pancreatic cells standing gradients of Ca2+ can be established locally in the apical domain of the cell (21, 44). These signals occur exclusively following exposure to threshold stimulation. This phenomenon is particularly marked when cells are exposed to a low, spatially uniform InsP3 concentration ([InsP3]), following flash photolysis, or when InsP3 is presented via a whole cell, patch-clamp pipette (17, 20, 39, 44). The majority of studies have, however, not observed local apically limited Ca2+ signals in acinar cells derived from salivary glands (but see Ref. 18 performed in submandibular cells). Important caveats, which apply to this experimental paradigm in general, are that dialysis from the patch pipette will disrupt the native buffering of the cytoplasm and thus may influence the spatial and temporal properties of the signal. In addition, as noted previously, cells are unlikely to experience physiologically a spatially uniform increase in InsP3. Following more intense stimulation, the Ca2+ signals can propagate in a manner typical of a true "wave" toward the basal aspects of the cell; i.e., constant wave speed typical of an "all or nothing" phenomenon dependent on Ca2+-induced Ca2+ release (CICR) (17, 24, 25, 27, 35, 45). CICR can occur through either InsP3R or through the classical CICR channel, the ryanodine receptor (RyR), which is also expressed in exocrine cells (14, 26, 39, 53). In pancreatic acinar cells, two general models have been proposed to account for the propagation of the Ca2+ wave. One model suggests that following initiation in the apical trigger zone, the wave is propagated as a function of the sequential activation of both InsP3R and RyR in the extra apical region (5, 25, 35). The second model does not require a prominent role of RyR but is dependent on the activation of InsP3R present throughout the extra-apical endoplasmic reticulum (ER) at lower density than in the trigger zone. The sequential activation of these InsP3Rs occurs as the wave front progresses as a function of the coagonist activity of Ca2+ at the InsP3R (21). In parotid acinar cells, global, uniform InsP3 exposure results in a signal, which is very rapidly initiated in the apical portion of the cell but, in contrast to pancreas, invariably globalizes to the basal regions, regardless of the [InsP3] (17). At high [InsP3] the kinetics of the globalization are not consistent with properties of a true wave and appear to result in what is termed a "cinematic" Ca2+ wave, initiation in the apical region followed by simultaneous Ca2+ release, taking place essentially autonomously and independently in neighboring subcellular regions as a function of activation of InsP3 and/or RyR.

The mechanism(s) which underlie these differences in the Ca2+ signals are not fully understood, but it has been suggested that the number and/or the localization of InsP3R may play a role in defining the specific spatial and temporal characteristics of the signal in each cell. For example, binding studies have reported that parotid acinar cells express approximately threefold more InsP3R compared with pancreas (17). Compared with the structure of the pancreas, the morphology of the salivary gland exhibits a more extensive and elaborate luminal organization. An abundance of InsP3R is associated with these structures and may contribute to the rapid initiation of signals in parotid (23). These data, however, obviously do not address the relative subcellular distribution/abundance of the receptors. Indeed, primarily because of the quantitative limitations of immunofluorescence, there is little information regarding the relative distribution of either InsP3R or RyR on extra-apical ER. Given the limited diffusional mobility of Ca2+, Ca2+ release channel expression would be absolutely required to support the globalization of the signal in pancreas and parotid, regardless of whether the signal occurs by CICR or through autonomous release.

This study was, therefore, designed to compare and probe the mechanism(s) underlying the Ca2+ signal globalization in these individual exocrine cell types. To these ends, we have designed and constructed a relatively simple means of locally releasing chemically caged molecules using high powered, focal laser photolysis. This method, combined with the use of cell permeant forms of caged-InsP3 and caged-Ca2+, has most importantly allowed these data to be obtained in intact and thus more natively buffered acinar cells. In particular, by monitoring the spatial and temporal characteristics of the Ca2+ signals evoked by InsP3, we have functionally defined the relative sensitivities of various subcellular regions of both pancreatic and parotid acinar cells to InsP3 and investigated the role of RyR to the signals observed. The study demonstrates that these exocrine cells are not sensitive to focal InsP3 liberation in any cellular region apart from the extreme apical region. Moreover, localized InsP3 generation in both apical and basal regions of either acinar cells results in an apical-to-basal Ca2+ wave with kinetics consistent with the apical region of parotid cells, exhibiting enhanced sensitivity to InsP3 compared with pancreas. Furthermore, following initiation of an apical-to-basal wave, the cytoplasmic region of parotid acinar cells appears to constitute a more "excitable" medium compared with pancreas. This excitability appears to be attributed in the main to the activity of InsP3R, although RyRs also appear to contribute to the fast kinetics of Ca2+ signals in parotid acinar cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Fluo-4-AM was purchased from Teflabs. O-nitrophenyl ethylenediaminetriacetic acid (NP-EGTA) was from Invitrogen. Caged-isopropylidene inositol 1,4,5 trisphosphate pentoxymethylester (Ci-InsP3/PM) (cell permeable caged-InsP3) was from Axxora. In isopropylidene inositol 1,4,5 trisphosphate (i-InsP3) the 2-3 hydroxyls of the inositol ring are protected by an isopropylidene group (12). This molecule is a full agonist at the InsP3R. Dulbecco's modified Eagle medium (DMEM) was purchased from GIBCO. All other materials were obtained from Sigma.

Preparation of pancreatic acini. These experiments were reviewed, approved by, and carried out in compliance with policies of the University Committee on Animal Resources at the University of Rochester. The investigation conforms to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publ. No. 85-23, revised 1996). Mouse pancreatic acini were prepared essentially as described previously (48). Briefly, following CO2 asphyxiation and cervical dislocation, pancreata were removed from freely fed male NIH-Swiss mice (25 g). The tissue was enzymatically digested with type-II collagenase in DMEM with 0.1% bovine serum albumin (BSA) and 1 mg/ml soybean trypsin inhibitor for 30 min, followed by gentle titration. Acini were then filtered through 100 µm nylon mesh, centrifuged at 75 g through 1% BSA in DMEM, and resuspended in 1% BSA in DMEM.

Preparation of parotid acinar cells. Single cells or small clusters of parotid acinar cells were isolated from freshly dissected parotid glands from NIH-Swiss mice (25 g) by sequential digestion with (single cells) or without (cell clusters) trypsin, followed by collagenase as previously described (13). Following isolation, cells were resuspended in 1% BSA, containing BME supplemented with 2 mM glutamine- penicillin-streptomycin, incubated at 37°C, and gassed with 5% CO2 and 95% O2 until ready for use.

Design and characterization of focal laser photolysis instrumentation. Focal intracellular photolysis was performed using a UV laser (Innova Enterprise; Coherent, Santa Clara, CA), operating at 363.8 nm and coupled into a Nikon TE300 microscope through a dual-port UV flash condenser (Till-photonics, Pleasanton, CA) as shown in Fig. 1A. The laser output was directed through a neutral density filter wheel (FW102; Thorlabs, Newton, NJ) and a shutter before being coupled into a single mode silica fiber (SM400N; Coastal Connections, Anaheim, CA) using a x20 microscope objective. The output of the distal end of the fiber was relayed to an intermediate focus within the condenser system at the focal plane of the primary condenser lens, using a pair of aspheric lenses (C220TM, C280TM, Thorlabs). A dichroic beam splitter allowed the UV beam to enter the condenser while preserving the functionality of a flash-lamp system already in place. The laser was collimated by the condenser lens at a beam diameter of ~18 mm, which slightly overfilled the entrance aperture of the same x40 objective used for imaging. This objective brought the beam to focus in the sample plane.


Figure 1
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  		Fig. 1. Experimental setup for focal laser photolysis and optical characterization. A: a 363.8-nm laser is coupled through a single mode (SM) optical fiber into the illumination port of a Nikon TE300 inverted microscope and brought to a focus in the sample plane using the imaging objective. See MATERIALS AND METHODS for details. ND, neutral density; NA, numerical aperature. B: the focus in the sample plane was characterized using a scanning edge detector to characterize the edge spread function of the system. C: representative edge spread function data, line spread function (LSF), and point spread function (PSF). The full width at half maximum (FWHM) of the PSF at best focus measured 0.65 and 0.74 µm in the x- and y-axes, respectively. See RESULTS for further detail. The modeled FWHM along the z-axis is 2.1 µm.

 
The focus of the beam in the sample plane was characterized by the full width at half maximum (FWHM) of the point spread function (PSF), which was evaluated by directly measuring the edge spread function (ESF) and then taking the first order numerical derivative to generate the line spread function (LSF). Using well known Fourier techniques and assuming cylindrical symmetry of the optical system, the LSF was used to compute the PSF (28). In addition to measurements made at best focus, beam characterization was performed at 2, 5, and 10 µm from best focus. The experimental setup for measuring the ESF is shown in Fig. 1B. The edge of a stainless steel surgical blade was polished to an edge roughness of ~50 nm and mounted ~1 mm from the active region of a plastic encapsulated photodiode (OPT101; Texas Instruments, Dallas, TX), using transparent epoxy. This device was mounted to the electronic translation stage of the microscope and oriented such that the edge of the blade was at a slight angle, {theta}, with respect to the direction of stage translation, Y. Therefore, as shown in Fig. 1B, as the stage was translated along the y-axis, the edge translated through the beam a distance, X, equal to Y times the tangent of {theta}. This technique was used to increase the spatial resolution of the measurement beyond that of the microscope stage. The power incident on the detector was recorded as the blade edge passed through the beam.

A sigmoidal function of the form

Formula 1(1)
was used to fit the ESF data using least squares, where {nu}a, {nu}b, {nu}c, {nu}d, and {nu}e are fit constants, erf is the Gaussian error function, and x is the edge translation through the beam. Representative ESF data and the corresponding sigmoid fit of Eq. 1 are shown in Fig. 1C. The first derivative of the ESF is the LSF, the Fourier transform of which is equivalent to a slice through the two-dimensional Fourier transform of the PSF, whose FWHM defines the maximum spot size of the photolysis system. At best focus, the PSF-FWHM measured 0.65 and 0.74 µm in the x- and y-axes, respectively. Modeling of the optical system in Code V (Optical Research Associates, Pasadena, CA) indicated a best-focus z-axis FWHM of ~2.1 µm. The computed in-plane FWHM was 0.33 µm. The discrepancy between the calculated and measured widths is likely the result of aberrations of the imaging objective at the excitation wavelength and is equivalent to approximately one-quarter wave of spherical aberration. These results suggest that ~70% of the optical power is contained inside a spot with a 1-µm diameter, allowing very targeted molecular uncaging within a well-defined intense focal volume.

Imaging. Before experimentation, aliquots of cell suspensions from pancreas or parotid were loaded by incubation with 4 µM Fluo-4-AM and either Ci-InsP3/PM (cell permeable InsP3) or NP-EGTA-AM (cell permeable caged-Ca2+), as indicated in a HEPES-buffered physiological saline solution (HEPES-PSS) containing in mM 5.5 glucose, 137 NaCl, 0.56 MgCl2, 4.7 KCl, 1 Na2HPO4, 10 HEPES (pH 7.4), and 1.2 CaCl2, after which they were resuspended in HEPES-PSS and kept at 4°C until ready for use. Before experiments, acini were seeded on coverslips, which formed the base of a superfusion chamber, and mounted on the stage of a Nikon TE300 inverted microscope. Solution changes were accomplished by selecting flow from a multichambered, valve-controlled, gravity-fed reservoir. [Ca2+]i imaging was performed essentially as described previously (39, 47, 50) by using an inverted epifluorescence Nikon microscope with a x40 oil immersion objective lens (numerical aperture, 1.3). Cells were excited at 488 ± 10 nm using a monochrometer (TILL Photonics). Fluorescence images were captured and digitized with a digital camera driven by TILL Photonics Vision software. Images were captured every ~40–80 ms with an exposure of 10 ms and 2 x 2 binning. Changes in Fluo-4 fluorescence are expressed as {Delta}F/F0, where F is the fluorescence captured at a particular time, and F0 is the mean of the initial 10 fluorescence images captured.

Laser photolysis. Before initiating experiments each day, the x-y position of the laser spot in relation to the field of view was assessed by illumination of a drop of fura-2 placed on the coverslip as shown in (Fig. 2A and graphical representation in 2B). Similarly, the beam was focused in the z plane to attain the brightest spot, and this position was fixed by means of a motorized z drive. The camera chip coordinates of the brightest pixel were recorded and regarded as the center of the spot illumination. Small acini were positioned using a motorized stage such that the laser beam illuminated the desired region of the cell. Photolysis of caged compounds was accomplished following a brief (50 ms) shutter opening. In initial experiments it was determined empirically that a power density of 16 µW·µm–2 was sufficient to effectively uncage both NP-EGTA and Ci-InsP3. The energy resulting from this laser exposure did not have any obvious deleterious effects on [Ca2+]i in cells not incubated with cage but loaded with dye and was therefore used throughout the study.


Figure 2
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  		Fig. 2. Visualization of the laser spot size. A: the system was used to excite a field containing 20 µM fura-2. The spot size is illustrated in relation to a 15-µm polystyrene bead fixed on the cover-slip. B: 3D graphical representation of the intensity of the laser illumination.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Focal uncaging of Ci-InsP3 in pancreatic acinar cells. The apical pole of acinar cells has been documented to be preferentially sensitive to the effects of InsP3 compared with other regions of exocrine cells. Consistent with these earlier observations, as shown in the images in Fig. 3A and the corresponding kinetic in Fig. 3a, brief focal release of i-InsP3 close to the luminal plasma membrane in pancreatic acini incubated with 5 µM Ci-InsP3/PM resulted in a transient increase in [Ca2+]i at the photolysis site. This initial Ca2+ signal was followed after a marked latency by initiation of a Ca2+ wave in the extreme apical pole of the cell. Of note, despite spatially limited photolysis, the Ca2+ signal always subsequently propagated toward the basal aspects of the cell. On average the latency between photolysis and initiation of the propagating wave was 0.83 ± 0.2 s (defined as time between the laser exposure and an increase in fluorescence at the apical pole of 5% above baseline). The latency was not significantly shortened by increasing the laser power to maximum values (24 µW·µm–2) or by increasing the incubated Ci-InsP3/PM concentration ([Ci-InsP3/PM]) to 10 µM. Thus this time appears to approach an absolute latency for initiation of the Ca2+ signal. In contrast, the latency increased significantly following photolysis in cells incubated with lower concentrations of Ci-InsP3/PM. For example, in cells incubated with 1 µM Ci-InsP3/PM the latency increased dramatically to 7.04 ± 0.51 s (see pooled kinetic data in GoFig. 5, A and E). Once initiated, the Ca2+ wave invariably propagated throughout the cell with a constant velocity of 10.90 ± 0.54 µm/s (5 µM Ci-InsP3; n = 21 cells). Despite drastically lengthening the latency, the Ca2+ wave velocity was not significantly different in cells incubated with 1 µM Ci-IP3/PM (12.17 ± 0.48 µm/s; n = 11), consistent with initiation of an "all or nothing" process.


Figure 3
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  		Fig. 3. Photolysis of caged-isopropylidene inositol 1,4,5 trisphosphate (Ci-InsP3) in pancreatic acinar cells. A: a series of images at the times indicated from a small pancreatic acini shown in brightfield (top, left) incubated with 5 µM Ci-InsP3 and the Ca2+ indicator Fluo-4. The laser was positioned in the extreme apical region of one cell (as indicated by the lightning bolt) and exposed to laser illumination to release Ci-InsP3. The images show the initiation of the Ca2+ signal in the apical domain followed by propagation to the basal region. The corresponding kinetic recorded from an apical (blue box) or basal (red box) are shown in a. B (images) and b (kinetic): a typical experiment is shown following photolysis toward the center of a doublet of acinar cells. A limited increase in intracellular calcium concentration ([Ca2+]i) is observed at the photolysis site, which is then followed after a significant latency by initiation and propagation of an apical-to-basal Ca2+ wave. C (images) and c (kinetic): a typical experiment where photolysis was performed in the extreme basal aspects of a small pancreatic acinus. A limited Ca2+ signal is observed at the photolysis site, which is followed by an extended latency before initiation of an apical-to-basal wave. Local apically delimited signals were never observed under these conditions. Changes in Fluo-4 fluorescence are expressed as {Delta}F/F0, where F is the fluorescence captured at a particular time, and F0 is the mean of the initial 10 fluorescence images captured.

 

Figure 4
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  		Fig. 4. Photolysis of Ci-InsP3 in parotid acinar cells. A: a series of images at the times indicated from a small parotid acini shown in brightfield (top, left) incubated with 5 µM Ci-InsP3 and the Ca2+ indicator Fluo-4. The laser was positioned in the extreme apical region of one cell (as indicated by the lightning bolt) and exposed to laser illumination to release Ci-InsP3. The images show the initiation of the Ca2+ signal in the apical domain followed by propagation to the basal region. The corresponding kinetic recorded from an apical (blue box) or basal (red box) are shown in a. Apical photolysis results in the rapid initiation of an apical-to-basal wave. B (images) and b (kinetics): photolysis is performed in a more central location in a small parotid acinus. A limited increase in [Ca2+]i is observed at the photolysis site, which is then followed after short latency by initiation and propagation of an apical-to-basal Ca2+ wave. C (images) and c (kinetic): a typical experiment where photolysis was performed in the extreme basal aspects of a small pancreatic acinus. A limited Ca2+ signal is observed at the photolysis site, which is followed by a latent period before initiation of an apical-to-basal wave. Note the much shorter latencies in parotid vs. pancreas.

 

Figure 5
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  		Fig. 5. Summary of Ci-InsP3 photolysis experiments. A: pooled data for all experiments performed in pancreatic acini following apical photolysis of Ci-InsP3. Each individual experiment was normalized to its maximal amplitude. The blue trace represents data from apical regions of interest, whereas the red trace is from basal regions of interest at the [Ci-InsP3] indicated. Note the significant latency following the flash. The wave speed is represented by the time difference between red and blue traces and is relatively constant at different [Ci-InsP3]. B: shows pooled data following basal uncaging. Note the extended latent period at each [Ci-InsP3]. C: pooled data is shown for apical photolysis in parotid acinar cells. D: pooled data is shown for basal photolysis. In C and D for comparison the scales are identical to A and B, respectively, and illustrate that the kinetic characteristics of parotid acinar cells are much faster compared with pancreatic cells. Insets: show the same data on an expanded scale. E: pooled data showing differences in latency in parotid vs. pancreas at the indicated uncaging sites. F: pooled data showing the Ca2+ wave speeds in parotid vs. pancreas at the indicated uncaging sites.

 
To probe the InsP3 sensitivity of regions outside the apical pole of the cell, the effects of photo release of i-InsP3 toward the middle and basal aspects of the acinar cells were next investigated. Figure 3, B (images) and b (kinetic), shows a representative experiment in which photolysis of Ci-InsP3 was achieved toward the middle of a small acinus. Again, a limited increase in fluorescence was generated at the photolysis site. Notably, this initial increase in [Ca2+]i never resulted in a propagating Ca2+ signal. Instead, following a marked latency of, on average, 3.53 ± 0.39 s (5 µM Ci-InsP3; n = 20), a Ca2+ wave was initiated from the trigger zone Ca2+ release sites. Once initiated, the Ca2+ wave propagated at a velocity not significantly different from that following apical photolysis of Ci-InsP3 (11.60 ± 0.90 µm/s; n = 21 cells; P = 0.538). In a similar fashion, photolysis of Ci-InsP3 close to the basal plasma membrane never resulted in a Ca2+ signal propagating from the photolysis site as seen in Fig. 3, C (images) and c (kinetic), and pooled data in Fig. 5B. This observation held even when cells were incubated with higher [Ci-InsP3/PM] or when the laser power was increased to 24 µW·µm–2. Again, following an extended latency, which was on average 4.87 ± 0.91 s (5 µM; Ci-InsP3/PM n = 9) and even longer, averaging 16.91 ± 0.89s at 1 µM Ci-InsP3/PM (pooled data, Fig. 5E), a Ca2+ wave was initiated from the trigger zone and then subsequently propagated toward the basal region of the cell at a velocity of 9.40 ± 1.60 µm/s (n = 4). This wave speed was not significantly different from that initiated by apical focal photolysis (P = 0.181). It should also be noted that local Ca2+ release limited to the apical region following photolysis in any cellular region was never observed; if a Ca2+ signal was initiated in the apical region, it invariably propagated to the basal region. In total, these data confirm that the apical domain of pancreatic acinar cells is most sensitive to InsP3 but indicate, somewhat surprisingly, that other regions of the cell are practically so insensitive to InsP3 that they are unable to support sufficient Ca2+ release to initiate a Ca2+ wave from the photolysis site. Instead, propagation always occurs from the apical trigger zone to the basal aspects of the cell following a variable latency. These data, however, do not exclude the possibility that following initiation, the propagation of the Ca2+ signal is largely independent of the InsP3R and instead relies on CICR through RyR.

Focal uncaging in parotid acinar cells. Previous reports have shown that the apical region of salivary gland acinar cells also constitutes a trigger zone where Ca2+ signals are initiated following exposure to InsP3 or agonist stimulation. In contrast to pancreatic acinar cells, where signals at threshold stimulation can be localized, in parotid or submandibular salivary glands the Ca2+ signals invariably globalize in a manner that is consistent, especially at higher [InsP3], with largely autonomous Ca2+ release from extra-apical sites and not a true Ca2+ wave (17, 38). A prediction from these data is that the extra-apical regions of parotid acinar cells are more sensitive to InsP3 and may support at least limited, regenerative Ca2+ release. This idea was tested by focal photolysis at sites throughout small parotid acini. When the laser was positioned at the apical pole of the cell, photolysis of Ci-InsP3 resulted in Ca2+ release at the spot site followed rapidly by extensive Ca2+ release from the trigger zone, as shown in Fig. 4, A (images) and a (kinetic), and pooled data (Fig. 5C). On average the latency in cells incubated with 5 µM Ci-InsP3/PM was 0.15 ± 0.02 s, (n = 21) and longer at 1 µM Ci-InsP3/PM (0.37 ± 0.03 s; n = 10) and was not significantly shortened by increasing the laser power to 24 µW·µm–2. The latency was significantly shorter than that observed under similar conditions in pancreatic acinar cells and consistent with findings reported following global InsP3 photolysis (17). In a similar fashion to pancreatic acinar cells, following initiation the Ca2+ wave propagated throughout the parotid acinar cell in a manner consistent with a true Ca2+ wave. Notably, the wave proceeded with a faster velocity of 27.81 ± 2.62 µm/s (5 µM Ci-InsP3/PM) than observed in pancreatic acinar cells but again was not altered significantly as a function of [Ci-InsP3/PM] (Ca2+ wave velocity 26.29 ± 3.01 µm/s; n = 17; 1 µM Ci-InsP3/PM). Next, photolysis of Ci-InsP3 was performed in the mid and basal regions of parotid cells. As shown in Fig. 4, B (images) and b (kinetics), photo release in the mid region resulted in modest Ca2+ release at the site of laser exposure but did not result in a propagating wave from the photolysis site. Increasing laser power or [Ci-InsP3/PM] similarly did not result in a wave propagating from the photolysis spot. These data suggest that this cytoplasmic region of the parotid acinar cell does not express sufficient density of InsP3R to support the initiation of a propagating wave. Again, following a latency of 0.21 ± 0.02 s, a Ca2+ wave was initiated at the trigger zone which propagated at a velocity of 24.20 ± 1.57 µm/s toward the basal pole of the cell. This wave speed was significantly faster than observed in pancreas, although not different from that initiated following photolysis in other regions of parotid cells. No regenerative Ca2+ wave occurred at the initial photolysis site when laser exposure was targeted in the extreme basal pole as shown in Fig. 4, C (images) and c (kinetics), and pooled data in Fig. 5D. Instead, an apical-basal Ca2+ wave propagating at a velocity of 21.91 ± 1.62 µm/s was initiated after a latency of 0.38 ± 0.05 s (pooled data in Fig. 5).

In summary, focal production of InsP3 resulted in Ca2+ signals in either cell type with similar spatial characteristics; production of InsP3 in any region of the cell resulted only in a modest transient Ca2+ signal at the photolysis site followed by initiation of a Ca2+ wave from the extreme luminal pole of the cell, traveling at a fixed velocity in each particular cell type. The latent period was closely related to the distance between the photolysis site and the trigger zone. Despite these similarities, the Ca2+ signals in each cell type exhibited very different kinetic profiles, including markedly shorter latent periods prior to wave initiation and faster wave speeds when initiated in parotid acinar cells.

Focal release of Ca2+ in exocrine acinar cells. Next, experiments were performed to explore the possible role of classical CICR in the propagation of acinar cell Ca2+ signals. Acinar cells were incubated with caged-Ca2+ (25 µM NP-EGTA for 30 min) during the loading with Fluo-4. Photolysis was accomplished in either the apical or basal region of each acinar cell type. Fig. 6, A (images) and a (kinetic), shows a representative example of uncaging Ca2+ in the apical region of a small pancreatic acinus and illustrates that a limited Ca2+ signal could be observed, which decayed within ~400 ms and never expanded significantly from the initial photolysis site (n = 10). Similar spatial characteristics were observed when Ca2+ was released by photolysis in the basal pole of pancreatic acinar cells; specifically no significant propagation of the signal away from the photolysis site occurred (n = 12), although the magnitude of the Ca2+ signals realized were significantly larger [1.01 ± 0.22 vs. 3.60 ± 0.50 {Delta}F/F0 units (apical vs. basal), respectively] as seen in Fig. 6C. As shown in Fig. 6, B (images) b (kinetics), qualitatively and quantitatively similar data was obtained following photo release of Ca2+ in parotid acinar cells; photolysis of NP-EGTA resulted in a limited Ca2+ rise, which failed to propagate. In addition, the magnitude of the Ca2+ signals were not significantly different when parotid and pancreas were compared, as shown in Fig. 6C. These data indicate that under these conditions, raising Ca2+ can itself not support significant CICR in exocrine cells and further suggests that RyR activation, at least in isolation, plays a limited role in Ca2+ wave propagation in exocrine cells.


Figure 6
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  		Fig. 6. Photolysis of O-nitrophenyl ethylenediaminetriacetic acid (NP-EGTA). A: a typical experiment in which a small pancreatic acinus shown in brightfield (left) was loaded with 25 µM NP-EGTA-AM (cell permeant caged-Ca2+) and Fluo-4. A shows images and a the kinetic follow photolysis in the apical region. A limited rise in [Ca2+]i was observed at the photolysis site which never propagated away. B: a similar representative experiment is shown performed in parotid cells; B shows images, and b shows the kinetics. Again, a limited, nonpropagating increase in [Ca2+]i was observed at the photolysis site. C: shows the pooled data showing the maximum amplitude of the [Ca2+]i signal attained from both cell types following photolysis in either the apical or basal region. Qualitatively similar spatial data was observed following basal photolysis; the Ca2+ signal did not propagate. Significantly larger limited Ca2+ signals were observed following basal photolysis, which were not significantly different when comparing pancreas and parotid.

 
To further investigate the role of RyR in Ca2+ signal initiation and propagation, we next compared the spatial and temporal characteristics of signals generated by focal uncaging in the presence or absence of a blocking concentration of ryanodine (25, 35, 39). Small acini were incubated in 200 µM ryanodine for 10 min prior to photolysis of Ci-InsP3. As shown in Fig. 7A, blockade of RyR failed to markedly alter the kinetic characteristics of Ca2+ signals evoked by apical uncaging of Ci-InsP3 in pancreatic acini. For example, following photo release in cells incubated with 5 µM Ci-InsP3/PM (n = 17), the latency was 1.08 ± 0.15 s vs. 0.825 ± 0.21 s (no ryanodine), wave speed was 10.43 ± 0.75 µm/s vs. 10.97 ± 0.53 µm/s (no ryanodine), and time to half peak (t1/2) for the rise to peak in the apical region was 2.45 ± 0.27 s vs. 2.26 ± 0.25 s (no ryanodine). These parameters were not significantly different from those obtained in the absence of ryanodine. In contrast, in parotid acini (n = 16), ryanodine incubation increased the latency for the initiation of a Ca2+ signal following laser exposure from 0.15 ± 0.1 s to 0.3 ± 0.1 s (P = 0.001), significantly slowed the wave speed to 20.5 ± 2.0 from 27.7 ± 2.59 (P = 0.001), and increased the t1/2 to peak from 0.75 ± 0.02 to 1.08 ± 0.11. Pooled data is shown in Fig. 7B. These data suggest that in parotid acinar cells, in contrast to pancreatic cells, RyRs play a more prominent role in Ca2+ wave propagation following activation of InsP3R.


Figure 7
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  		Fig. 7. Ca2+ wave propagation in the presence of ryanodine. A: pooled, normalized data from pancreatic cells incubated with 200 µM ryanodine before uncaging of 5 µM Ci-InsP3. The red traces represent control traces from the apical (squares) or basal (circles) region. The blue traces are experiments in which cells were incubated with ryanodine. No significant effect was observed of ryanodine on latency, wave velocity of time-to-half peak in pancreatic acinar cells. B: in contrast, ryanodine incubation significantly lengthened the latency and slowed the wave speed and time to half maximum in parotid acinar cells.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
We have designed and developed a system to realize a spatially limited spot of intense laser illumination through a high numerical aperture objective on the stage of an inverted microscope. The equipment facilitated experiments to locally photo release cell permeant caged second messengers, allowing a comparison of the spatial and temporal characteristics of Ca2+-signaling events in two distinct but related polarized exocrine cells. The design of the apparatus is optically straightforward and was relatively inexpensive to construct and could be duplicated by other investigators with similar experimental needs. The experimental paradigm was designed primarily to investigate signaling events in natively buffered intact cells and secondarily to more closely approximate local production of messengers likely to occur under physiological stimulation of the gland in situ. These studies revealed that the individual cell types exhibit both similar and divergent characteristics, which appear to occur largely as a consequence of utilizing common pathways.

Role of classical CICR. Photo-production of Ca2+ never resulted in a propagating wave in either cell type, indicating that classical CICR through RyR in isolation does not appear to be a dominant mechanism in these exocrine cells. These data are consistent with a recent study showing that photo release of Ca2+ in the absence of a global increase in [InsP3] failed to evoke regenerative release (5). In pancreatic acinar cells, little effect was observed of blocking RyR, even on Ca2+ signals initiated by Ci-InsP3. These data contrast to some earlier reports that RyRs contribute to the propagation of Ca2+ waves in pancreatic acinar cells (25, 35, 39) (but see Ref. 15) and are thus most consistent with Ca2+ wave propagation involving sequential activation of InsP3R by InsP3 and Ca2+. A caveat that applies to this experimental paradigm is that these data may reflect the fact that blockade of RyR is generally thought to be use dependent and often requires repetitive stimulation in the presence of ryanodine to observe functional effects. Thus, in the present experiments, which were designed to monitor signals following acute exposure to relatively low InsP3, some effects previously attributed to RyR may be underestimated. Notwithstanding this idea, significant effects of RyR blockade were observed in parotid acinar cells, including slowing latency, rise time, and wave propagation velocity. These data could indicate a comparably more significant role of RyR in parotid acinar cells and is consistent with the higher functional sensitivity of parotid to RyR agonists (24). Following RyR blockade, the Ca2+ signaling characteristics in parotid and pancreas were, however, still significantly different, indicating that increased utilization of RyR by parotid acinar cells cannot totally account for the differences observed between pancreas and parotid acinar cells.

Spatial characteristics of the Ca2+ wave. Following focal generation of i-InsP3, the spatial aspects of the Ca2+ signal in pancreas and parotid were remarkably similar. Local generation of i-InsP3 in any region other than in the extreme apical domain also invariably resulted in only limited Ca2+ release at the photolysis site. Propagation away from sites outside the apical domain was never observed. Photolysis, regardless of the cellular location, always resulted in initiation of substantial Ca2+ release from the apical trigger zone and subsequent entrainment of an apical-to-basal Ca2+ wave. In an earlier study by Fogarty and colleagues (15), in which caged-InsP3 was introduced by microinjection, generally similar conclusions were made regarding the enhanced sensitivity of the apical domain of pancreatic acinar cells over other regions of the cell. However, in a minority of cells in this study, photolysis in some more basally localized regions resulted in a Ca2+ wave propagating away from the photolysis site. Although the reasons for these seemingly disparate results are presently unclear, a possible contributing factor is that the spot size achieved in the previous study was approximately threefold larger than in the present study (FWHM ~>2 µm vs. 0.65 µm this study), giving an effective photolysis spot of ~4 µm. This less spatially limited spot would be predicted to activate many more InsP3R during laser exposure and possibly, in turn, generate sufficient Ca2+ release to facilitate initiation of a wave. This might occur especially if extra-apical photolysis engaged InsP3R, known to be in relative abundance close to lateral plasma membranes at cell-to-cell borders (23, 24, 52).

Although qualitatively similar in both cell types and at all [Ci-InsP3] tested, the exclusive activation of the apical-to-basal wave was most striking following photolysis of lower [Ci-InsP3] limited to the extreme basal region. Under these conditions, a particularly extended latent period (~10 s; Fig. 5B) was observed before initiation of the Ca2+ wave in pancreatic acinar cells. In parotid acinar, although this latent period was markedly shorter (~350 ms, Fig. 5, C–E), a clear refractory period following the initial Ca2+ release was always observed before initiation of a wave. A conclusion from these experiments is that the density of InsP3R throughout the extra-apical ER in either pancreas or parotid is not sufficient to initiate and then support regenerative CICR. This is consistent with numerous immunocytochemical studies, which show a low abundance of InsP3R in all regions of acinar cells compared with the extreme apical domain (24, 34, 43, 52). Presumably the lack of responsiveness of extra-apical InsP3R occurs because the receptor is not exposed to sufficiently elevated Ca2+ required for coactivation of InsP3-induced Ca2+ release until the apical release sites are engaged (8, 16). Physiological stimulation is unlikely to result in uniform InsP3 generation and would be predicted to result in locally high [InsP3], which subsequently dissipates by diffusion and metabolism. This might occur, for example, at sites of synapsing nerves. The inability of InsP3 in isolation to activate extra-apical Ca2+ release may ensure the consistent initiation of an apical-to-basal wave, an event thought to be fundamentally important for the appropriate activation of effectors that underlie the physiological activation of secretion in exocrine cells. In addition, the exclusive apical initiation would also be rendered independent of the localization of secretagogue receptors and primary sites of InsP3 production.

Ca2+ wave initiation. The initiation of a Ca2+ wave is proposed to rely on the progressive recruitment of elemental Ca2+ release events through clusters of InsP3R, which sum to reach a critical level and thus provide sufficient diffusing Ca2+ for activation of neighboring Ca2+ release sites (9, 10). According to this generally accepted model, the latent period following i-InsP3 equilibration would represent the time required to engage sufficient InsP3 in the apical region to provide the trigger for Ca2+ wave propagation. In parotid acinar cells, this time approximates the sum of the time for InsP3 to diffuse [estimate of the diffusion rate: 200 µm2/s (1)] and the shortest latent period observed following apical uncaging. In contrast, in pancreas under identical conditions, markedly extended latent periods were observed. These times were significantly greater than required for diffusion and may reflect the increased time to recruit a productive Ca2+ release event from lower densities or less sensitive InsP3R in the apical region of pancreas compared with parotid. These ideas are consistent with the known sensitivities of the predominant InsP3R expressed and the relative abundance of total InsP3R, given the assumption that the increase in InsP3R number previously noted is predominately expressed in the apical region of each tissue (19, 24, 52).

Ca2+ wave propagation. Following initiation of the apical Ca2+ signal, the Ca2+ wave velocity was remarkably constant under the various conditions in a particular cell type; for example, the wave velocity was not influenced either by [Ci-InsP3/PM] or the site of i-InsP3 generation in either cell type. These observations are consistent with activation of an "all or none" process, a primary characteristic of a true propagating wave, which relies on the sequential recruitment of release sites throughout the cytoplasm. In pancreatic acinar cells, we have previously reported similar conclusions based on observations under conditions that resulted in a global and uniform photolysis of InsP3 (17). In this study, over a wide range of [InsP3], a relatively constant wave speed was generated. In contrast, in parotid acinar cells, wave speed was observed to increase dramatically with [InsP3] and be more consistent with global Ca2+ signals underpinned by largely autonomous Ca2+ release from areas of ER outside the trigger zone, rather than a true propagating wave (17). It should be noted, however, that in the present study, it is unlikely that the high global [InsP3] achieved following uniform photolysis is ever achieved following focal uncaging. Based on an estimate of the diffusion rate of InsP3, the latency observed in each cell type would be sufficient for [Ci-InsP3] to equilibrate before entrainment of the apical-to-basal wave (cell diameter ~15 µm) (1). This would, however, be predicted to result in a relatively low global [i-InsP3]. These present experiments, therefore, more closely approximate the situation following uniform photolysis of low [InsP3] and perhaps physiological activation of cells. Under these conditions, in the earlier study, Ca2+ signals with properties more consistent with a propagating wave were also elicited in parotid acinar cells following uniform photolysis. Interestingly, the wave velocity of secretagogue-induced Ca2+ waves in pancreatic acinar cells has been reported to be dependent on agonist concentration (37). Thus although the present data provide information indicative of an InsP3-induced wave produced in isolation, secretagogue stimulation results in a more complex series of signal transduction events, which are capable of modulating the properties of the wave. These events could potentially include agonist-stimulated production of other calcium-mobilizing second messengers (51) or activation of kinases that regulate the Ca2+-handling machinery (47).

Although the apical-to-basal Ca2+ wave velocity was constant in each cell type under various [Ci-InsP3] and following photolysis at various sites, the absolute velocity in parotid acinar cells was ~2.5-fold faster when compared with pancreatic acinar cells. Although it cannot be excluded that these data could simply be explained by a difference in the rate of InsP3 diffusion in the two cell types, the observation is again superficially consistent with higher expression/greater sensitivity of InsP3R in parotid acinar cells compared with pancreas (17). To underlie the increase in wave speed, the increased abundance/sensitivity of InsP3R would have to be reflected in channels throughout the ER of parotid acinar cells. It should be noted, however, that, while possible, the present study provides little functional evidence, and immunocytochemical studies present little structural evidence to support this idea.

A further consideration is that the spatial and temporal properties of Ca2+ signals can be markedly influenced by mobile and immobile cellular Ca2+ buffers. For example, local buffering markedly affects the properties of the InsP3R by altering both Ca2+ activation and inhibition of the receptor (11, 22). The Ca2+-binding capacity is reported to be 1,500 in pancreatic acinar cells (31) (essentially the ratio of total Ca2+ to free Ca2+). Although this value has not been reported for parotid acinar cells, the buffering capacity of pancreatic acinar cells is considered high compared with other cell types including chromaffin, cardiac myocytes, and neurons (7, 31, 36) (values reported between 10–100). The high buffer capacity has been suggested as important for the generation of locally limited Ca2+ signals that are observed at threshold levels of stimulation in pancreatic acinar cells (31). It should, however, be stressed that in this study, under close to native buffering conditions, locally delimited Ca2+ signals were not observed. Additionally, this profile of Ca2+ signal is not observed in parotid acinar cells. Differing intrinsic buffering in pancreas vs. parotid could conceivably contribute to the distinct temporal characteristics of the signals observed in each cell type. In a majority of previous studies by our group and others, buffering was achieved through dialysis of solutions from a whole cell, patch-clamp pipette. It is probable that imposition of buffering in this manner could markedly influence the characteristics of the Ca2+ signals observed. For example, in contrast to the present study, wave speeds at comparable stimulation intensity were essentially identical in both cell types under patch-clamp conditions (~25 µm/s) (17). This comparison reinforces the importance of measuring Ca2+ signaling characteristics under conditions as close to native buffering as possible.

Conclusions. In conclusion, although acinar cells derived from pancreas and parotid share many morphological and functional features, parotid acinar cells exhibit Ca2+ signaling kinetics, which are more rapid than pancreatic cells. This appears to occur in the main by using generally similar pathways. The enhanced kinetics are consistent with the differing primary effectors for the Ca2+ signals in the individual cells. In pancreas, Ca2+ is required primarily in the apical domain to satisfy the requirement for the relatively slow process of exocytosis. In contrast, the rapid kinetics in parotid acinar cells appear to reflect fine coordination for the appropriate activation of spatially separated ion channels necessary for the initiation of fluid secretion.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The work was supported by National Institutes of Health Grants R01-DK-54568, R01/R56-DE-14756, R01-DE-16999 (D. I. Yule), and RO1-CA-68409 (T. H. Foster). W. J. Cottrell was supported by an NIH training Grant T32-HL-66988.


   ACKNOWLEDGMENTS
 
The authors thank Trevor Shuttleworth, Mathew Betzenhauser, David Brown, and Larry Wagner for helpful discussion throughout the study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. I. Yule, Univ. of Rochester, 601 Elmwood Ave., Rochester, NY 14642 (e-mail: David_Yule{at}URMC.Rochester.edu)

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.

* J. H. Won and W. J. Cottrell contributed equally to this work. Back


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TOP
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 REFERENCES
 

  1. Allbritton NL, Meyer T, Stryer L. Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science 258: 1812–1815, 1992.[Abstract/Free Full Text]
  2. Ambudkar IS, Hiramatsu Y, Lockwich T, Baum BJ. Activation and regulation of calcium entry in rat parotid gland acinar cells. Crit Rev Oral Biol Med 4: 421–425, 1993.[Free Full Text]
  3. Ambudkar IS, Lockwich T, Hiramatsu Y, Baum BJ. Calcium entry in rat parotid acinar cells. Mol Cell Biochem 114: 73–77, 1992.[Web of Science][Medline]
  4. Ashby MC, Camello-Almaraz C, Gerasimenko OV, Petersen OH, Tepikin AV. Long distance communication between muscarinic receptors and Ca2+ release channels revealed by carbachol uncaging in cell-attached patch pipette. J Biol Chem 278: 20860–20864, 2003.[Abstract/Free Full Text]
  5. Ashby MC, Craske M, Park MK, Gerasimenko OV, Burgoyne RD, Petersen OH, Tepikin AV. Localized Ca2+ uncaging reveals polarized distribution of Ca2+-sensitive Ca2+ release sites: mechanism of unidirectional Ca2+ waves. J Cell Biol 158: 283–292, 2002.[Abstract/Free Full Text]
  6. Ashby MC, Tepikin AV. Polarized calcium and calmodulin signaling in secretory epithelia. Physiol Rev 82: 701–734, 2002.[Abstract/Free Full Text]
  7. Berlin JR, Bassani JW, Bers DM. Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes. Biophys J 67: 1775–1787, 1994.[Web of Science][Medline]
  8. Bezprozvanny I, Watras J, Ehrlich BE. Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 351: 751–754, 1991.[CrossRef][Medline]
  9. Bootman MD, Berridge MJ. The elemental principles of calcium signaling. Cell 83: 675–678, 1995.[CrossRef][Web of Science][Medline]
  10. Bootman MD, Berridge MJ, Lipp P. Cooking with calcium: the recipes for composing global signals from elementary events. Cell 91: 367–373, 1997.[CrossRef][Web of Science][Medline]
  11. Callamaras N, Parker I. Inositol 1,4,5-trisphosphate receptors in Xenopus laevis oocytes: localization and modulation by Ca2+. Cell Calcium 15: 66–78, 1994.[CrossRef][Web of Science][Medline]
  12. Dakin K, Li WH. Cell membrane permeable esters of D-myo-inositol 1,4,5-trisphosphate. Cell Calcium 42: 291–301, 2007.[CrossRef][Web of Science][Medline]
  13. Evans RL, Bell SM, Schultheis PJ, Shull GE, Melvin JE. Targeted disruption of the Nhe1 gene prevents muscarinic agonist-induced up-regulation of Na(+)/H(+) exchange in mouse parotid acinar cells. J Biol Chem 274: 29025–29030, 1999.[Abstract/Free Full Text]
  14. Fitzsimmons TJ, Gukovsky I, McRoberts JA, Rodriguez E, Lai FA, Pandol SJ. Multiple isoforms of the ryanodine receptor are expressed in rat pancreatic acinar cells. Biochem J 351: 265–271, 2000.[CrossRef][Web of Science][Medline]
  15. Fogarty KE, Kidd JF, Tuft DA, Thorn P. Mechanisms underlying InsP3-evoked global Ca2+ signals in mouse pancreatic acinar cells. J Physiol 3: 515–526, 2000.
  16. Foskett JK, White C, Cheung KH, Mak DO. Inositol trisphosphate receptor Ca2+ release channels. Physiol Rev 87: 593–658, 2007.[Abstract/Free Full Text]
  17. Giovannucci DR, Bruce JI, Straub SV, Arreola J, Sneyd J, Shuttleworth TJ, Yule DI. Cytosolic Ca(2+) and Ca(2+)-activated Cl(-) current dynamics: insights from two functionally distinct mouse exocrine cells. J Physiol 540: 469–484, 2002.[Abstract/Free Full Text]
  18. Harmer AR, Smith PM, Gallacher DV. Local and global calcium signals and fluid and electrolyte secretion in mouse submandibular acinar cells. Am J Physiol Gastrointest Liver Physiol 288: G118–G124, 2005.[Abstract/Free Full Text]
  19. Iwai M, Michikawa T, Bosanac I, Ikura M, Mikoshiba K. Molecular basis of the isoform-specific ligand-binding affinity of inositol 1,4,5-trisphosphate receptors. J Biol Chem 282: 12755–12764, 2007.[Abstract/Free Full Text]
  20. Kasai H, Augustine GJ. Cytosolic Ca2+ gradients triggering unidirectional fluid secretion from exocrine pancreas. Nature 348: 735–738, 1990.[CrossRef][Medline]
  21. Kasai H, Li YX, Miyashita Y. Subcellular distribution of Ca2+ release channels underlying Ca2+ waves and oscillations in exocrine pancreas. Cell 74: 669–677, 1993.[CrossRef][Web of Science][Medline]
  22. Kidd JF, Fogarty KE, Tuft RA, Thorn P. The role of Ca2+ feedback in shaping InsP3-evoked Ca2+ signals in mouse pancreatic acinar cells. J Physiol 1: 187–201, 1999.
  23. Larina O, Thorn P. Ca2+ dynamics in salivary acinar cells: distinct morphology of the acinar lumen underlies near-synchronous global Ca2+ responses. J Cell Sci 118: 4131–4139, 2005.[Abstract/Free Full Text]
  24. Lee MG, Xu X, Zeng W, Diaz J, Wojcikiewicz RJ, Kuo TH, Wuytack F, Racymaekers L, Muallem S. Polarized expression of Ca2+ channels in pancreatic and salivary gland cells. Correlation with initiation and propagation of [Ca2+]i waves. J Biol Chem 272: 15765–15770, 1997.[Abstract/Free Full Text]
  25. Leite MF, Burgstahler AD, Nathanson MH. Ca2+ waves require sequential activation of inositol trisphosphate receptors and ryanodine receptors in pancreatic acini. Gastroenterology 122: 415–427, 2002.[CrossRef][Web of Science][Medline]
  26. Leite MF, Dranoff JA, Gao L, Nathanson MH. Expression and subcellular localization of the ryanodine receptor in rat pancreatic acinar cells. Biochem J 337: 305–309, 1999.[CrossRef][Web of Science][Medline]
  27. Li Q, Luo X, Muallem S. Functional mapping of Ca2+ signaling complexes in plasma membrane microdomains of polarized cells. J Biol Chem 279: 27837–27840, 2004.[Abstract/Free Full Text]
  28. Marchand EW. Derivation of the point spread function from the line spread function. J Opt Soc Am A 54: 915–919, 1964.
  29. Melvin JE, Koek L, Zhang GH. A capacitative Ca2+ influx is required for sustained fluid secretion in sublingual mucous acini. Am J Physiol Gastrointest Liver Physiol 261: G1043–G1050, 1991.[Abstract/Free Full Text]
  30. Melvin JE, Yule D, Shuttleworth T, Begenisich T. Regulation of fluid and electrolyte secretion in salivary gland acinar cells. Annu Rev Physiol 67: 445–469, 2005.[CrossRef][Web of Science][Medline]
  31. Mogami H, Gardner J, Gerasimenko OV, Camello P, Petersen OH, Tepikin AV. Calcium binding capacity of the cytosol and endoplasmic reticulum of mouse pancreatic acinar cells. J Physiol 518: 463–467, 1999.[Abstract/Free Full Text]
  32. Muallem S, Pandol SJ, Beeker TG. Two components of hormone-evoked calcium release from intracellular stores of pancreatic acinar cells. Biochem J 255: 301–307, 1988.[Web of Science][Medline]
  33. Muallem S, Schoeffield M, Pandol S, Sachs G. Inositol trisphosphate modification of ion transport in rough endoplasmic reticulum. Proc Natl Acad Sci USA 82: 4433–4437, 1985.[Abstract/Free Full Text]
  34. Nathanson MH, Fallon MB, Padfield PJ, Maranto AR. Localization of the type 3 inositol 1,4,5-trisphosphate receptor in the Ca2+ wave trigger zone of pancreatic acinar cells. J Biol Chem 269: 4693–4696, 1994.[Abstract/Free Full Text]
  35. Nathanson MH, Padfield PJ, O'Sullivan AJ, Burgstahler AD, Jamieson JD. Mechanism of Ca2+ wave propagation in pancreatic acinar cells. J Biol Chem 267: 18118–18121, 1992.[Abstract/Free Full Text]
  36. Rose HJ, Dargan S, Shuai J, Parker I. ‘Trigger’ events precede calcium puffs in Xenopus oocytes. Biophys J 91: 4024–4032, 2006.[CrossRef][Web of Science][Medline]
  37. Shin DM, Luo X, Wilkie TM, Miller LJ, Peck AB, Humphreys-Beher MG, Muallem S. 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. J Biol Chem 276: 44146–44156, 2001.[Abstract/Free Full Text]
  38. Sneyd J, Tsaneva-Atanasova K, Bruce JI, Straub SV, Giovannucci DR, Yule DI. A model of calcium waves in pancreatic and parotid acinar cells. Biophys J 85: 1392–1405, 2003.[Web of Science][Medline]
  39. Straub SV, Giovannucci DR, Yule DI. Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria. J Gen Physiol 116: 547–560, 2000.[Abstract/Free Full Text]
  40. Streb H, Irvine RF, Berridge MJ, Schulz I. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature 306: 67–69, 1983.[CrossRef][Medline]
  41. Takemura H, Putney JW Jr. Capacitative calcium entry in parotid acinar cells. Biochem J 258: 409–412, 1989.[Web of Science][Medline]
  42. Tanimura A, Matsumoto Y, Tojyo Y. Polarized Ca2+ release in saponin-permeabilized parotid acinar cells evoked by flash photolysis of 'caged' inositol 1,4,5-trisphosphate. Biochem J 332: 769–772, 1998.[Web of Science][Medline]
  43. Tanimura A, Tojyo Y, Turner RJ. Evidence that type I, II, and III inositol 1,4,5-trisphosphate receptors can occur as integral plasma membrane proteins. J Biol Chem 275: 27488–27493, 2000.[Abstract/Free Full Text]
  44. Thorn P, Lawrie AM, Smith PM, Gallacher DV, Petersen OH. Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate. Cell 74: 661–668, 1993.[CrossRef][Web of Science][Medline]
  45. Tojyo Y, Tanimura A, Matsumoto Y. Imaging of intracellular Ca2+ waves induced by muscarinic receptor stimulation in rat parotid acinar cells. Cell Calcium 22: 455–462, 1997.[CrossRef][Web of Science][Medline]
  46. Tsunoda Y, Stuenkel EL, Williams JA. Characterization of sustained [Ca2+]i increase in pancreatic acinar cells and its relation to amylase secretion. Am J Physiol Gastrointest Liver Physiol 259: G792–G801, 1990.[Abstract/Free Full Text]
  47. Wagner LE, 2nd Li WH, Joseph SK, Yule DI. Functional consequences of phosphomimetic mutations at key cAMP-dependent protein kinase phosphorylation sites in the type 1 inositol 1,4,5-trisphosphate receptor. J Biol Chem 279: 46242–46252, 2004.[Abstract/Free Full Text]
  48. Williams JA, Korc M, Dormer RL. Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini. Am J Physiol 235: 517–524, 1978.[Medline]
  49. Williams JA, Yule DI. Stimulus secretion coupling in pancreatic acinar cells. In: Physiology of the Gastrointestinal Tract (4th ed.), edited by Johnson LR. San Diego, CA: Academic, 2006, p. 1337–1369.
  50. Won JH, Yule DI. Measurement of Ca2+ signaling dynamics in exocrine cells with total internal reflection microscopy. Am J Physiol Gastrointest Liver Physiol 291: G146–G155, 2006.[Abstract/Free Full Text]
  51. Yamasaki M, Thomas JM, Churchill GC, Garnham C, Lewis AM, Cancela JM, Patel S, Galione A. Role of NAADP and cADPR in the induction and maintenance of agonist-evoked Ca2+ spiking in mouse pancreatic acinar cells. Curr Biol 15: 874–878, 2005.[CrossRef][Web of Science][Medline]
  52. Yule DI, Ernst SA, Ohnishi H, Wojcikiewicz RJ. Evidence that zymogen granules are not a physiologically relevant calcium pool. Defining the distribution of inositol 1,4,5-trisphosphate receptors in pancreatic acinar cells. J Biol Chem 272: 9093–9098, 1997.[Abstract/Free Full Text]
  53. Zhang X, Wen J, Bidasee KR, Besch HR Jr, Wojcikiewicz RJ, Lee B, Rubin RP. Ryanodine and inositol trisphosphate receptors are differentially distributed and expressed in rat parotid gland. Biochem J 340: 519–527, 1999.[CrossRef][Web of Science][Medline]




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