Mechanism of Ca2’ Wave Propagation in Pancreatic Acinar Cells*

An increase in cytosolic Ca2+ often begins as a Ca2+ wave, and this wave is thought to result from sequential activation of Ca2+-sensitive Ca2+ stores across the cell. We tested that hypothesis in pancreatic acinar cells, and since Ca2+ waves may regulate acinar C1- secretion, we examined whether such waves also are important for amylase secretion. Ca” wave speed and direction was determined in individual cells within rat pancreatic acini using confocal line scanning microscopy. Both acetylcholine (ACh) and cholecystokinin-8 induced rapid Ca2* waves which usually travelled in an apical-to-basal direction. Both caffeine and ryano- dine, at concentrations that inhibit Ca2+-induced Ca” release (CICR), markedly slowed the speed of these waves. Amylase secretion was increased over %fold in response to ACh stimulation, and this increase was preserved in the presence of ryanodine. These results indicate that 1) stimulation of either muscarinic or cholecystokinin-8 receptors induces apical-to-basal Ca2+ waves in pancreatic acinar cells, 2) the speed of such waves is dependent upon mobilization of caffeine- and ryanodine-sensitive Ca2+ stores, and 3) ACh-in-duced amylase secretion is not inhibited by ryanodine. These observations provide direct evidence that Ca2+-induced Ca2+ release is important for propagation of cytosolic Ca2+ waves in pancreatic acinar cells. Agonist-induced Ca2+ waves Leibovitz L-15 medium fetal calf The cells a on the stage of a Zeiss Axiovert microscope, perifused 37 with a HEPES-buffered using a Bio-Rad MRCBOO confocal imaging system. An argon laser was used excite the dye 488 nm, and emission signals above 515 nm were collected. Optical sections between 0.5 and 1.0 pm in thickness were obtained of individual cells within acini; these cells have a diameter of 10-20 pm and are located within a cluster of cells with a typical aggregate diameter of -100 pm. Neither autofluorescence nor other background signals were detectable at the machine settings (i.e. aperture, gain, and black level) that were used. There was also no change in size, shape, or location of cells during the experiments. Acini were stimu- lated under one of the conditions described below, and the resulting Ca'+ signals were detected by confocal line-scanning microscopy (7, 8,13). In this type of confocal microscopy, fluorescence is determined at each point along a single line across the image, rather than at each point across the entire image (Fig. lA), permitting images to be obtained as frequently as every 4 ms without loss of spatial resolution of fluo-3 fluorescence along the line that is scanned. Fluo-3 fluorescence in response to each stimulus was recorded in this way and the resulting line scan was convolved with the following 3 X 3 smoothing filter as follows.

Agonist-induced Ca2+ waves have been observed in the cytosol of cells in animals ranging from echinoderms, sponges, and molluscs to fish, birds, and mammals (1,2). Cytosolic Ca2+ (Ca2+i) waves have also been observed in cells from a number of tissue types, including germinal cells (i.e. oocytes (3,4)) and both excitable (5) and nonexcitable (6, 7) somatic cells (recently reviewed in Ref. 2). There is limited understanding of the significance of this signaling pattern, although oocyte Ca2+i waves initiated by fertilization may induce embryonic development (3,4), while such waves have been proposed to regulate C1-secretion in pancreatic exocrine cells (6) and may be important for intercellular communication in hepatocytes (7)(8)(9). Ca2+i waves propagate across cells in a rapid and nondiminished fashion, so it is hypothesized that Ca2+i wave propagation is mediated by an autocatalytic reaction-diffusion network in which Ca2+-sensitive Ca2+ stores across the cell are sequentially mobilized (1,2,10). The purpose of this investigation was to examine the importance of Ca2+-induced Ca2+ release (CICR)' for the propagation of Ca2+i waves within pancreatic acinar cells.
We examined Ca2+i waves in individual pancreatic cells within clusters of intact acini (containing over 50 cells each), since subcellular Ca2+i signals depend on cell polarity, and since secretion is influenced by intercellular communication, both of which are maintained in this preparation (11,12). Visualization of intracellular Ca2+ waves in this and other cell preparations is often limited for two reasons. Such waves may traverse cells at speeds of over 100 pm/s (5,7,8), necessitating a higher degree of temporal resolution than is available by most imaging methods. In addition, many cell systems (such as mammalian epithelial cell preparations in which polarity has been maintained, or amphibian or fish oocytes) are too thick or dense for detailed subcellular resolution of Ca2+ signals if epifluorescence imaging of Ca2+-sensitive dyes is used. To circumvent these potential problems, subcellular Ca2+i signals in individual cells within acini were observed using confocal line scanning microscopy (7,8,13).

Animals and Materials-Male
Sprague-Dawley rats (80-100 g; Camm Research Lab Animals, Wayne, NJ) were maintained on Purina rodent chow under a constant light cycle and used for all experiments. Acetylcholine (ACh), caffeine, and atropine were obtained from Sigma; fluo-3/AM and ryanodine were obtained from Molecular Probes (Pitchford, OR); and sincalide, a synthetic Cterminal octapeptide of cholecystokinin-8 (CCK), was a gift of Squibb Diagnostics (Princeton, NJ). All other chemicals were of the highest quality commercially available.
Preparation of Pancreatic Acini-Pancreatic acini were prepared from male Sprague-Dawley rats (80-100 g) as described previously (11). Briefly, the pancreas was removed following an overnight fast and placed in a Ca2+-and M%+-free buffer (pH 7.4) containing NaCl (97 mM), KC1 (5 mM), glucose (20 mM), HEPES (20 mM), soy bean trypsin inhibitor (0.1 mg/ml), and bovine serum albumin (0.1%). The pancreas was finely diced, transferred to a siliconized flask in buffer containing CaClZ (2 mM), MgCl2 (1.2 mM), and collagenase (400 units/5 ml), and gently agitated for 5 min at 37 "C. This pancreatic tissue was placed in a Corex tube and shaken by hand for -5-10 min then filtered through 200-pm nylon mesh and rinsed in collagenasefree buffer. This resulted in acini which contained over 50 cells each. In selected experiments, pancreatic acini were dispersed into clusters of only two to four cells each (6,14), by incubating for an additional 10 min in Ca2+-free medium containing EGTA (1 mM) and trypsin (1.5 mg/ml) followed by 5 min in medium containing collagenase (400 units/5 ml). These cells were passed through a second nylon filter and rinsed.
Confocal Microscopic Measurements of Cytosolic Calcium-Isolated rat pancreatic acini were prepared as described above, then loaded with the Ca'+-sensitive fluorescent dye fluo-B/AM (15) (6 p M ) for 20 min at room temperature in Leibovitz L-15 medium (GIBCO) containing 10% fetal calf serum. The cells were transferred to a chamber on the stage of a Zeiss Axiovert microscope, perifused at 37 "C with a HEPES-buffered solution, and observed using a Bio-Rad MRCBOO confocal imaging system. An argon laser was used to excite the dye at 488 nm, and emission signals above 515 nm were collected. Optical sections between 0.5 and 1.0 pm in thickness were obtained of individual cells within acini; these cells have a diameter of 10-20 pm and are located within a cluster of cells with a typical aggregate diameter of -100 pm. Neither autofluorescence nor other background signals were detectable at the machine settings (i.e. aperture, gain, and black level) that were used. There was also no change in size, shape, or location of cells during the experiments. Acini were stimulated under one of the conditions described below, and the resulting Ca'+ signals were detected by confocal line-scanning microscopy (7,8,13). In this type of confocal microscopy, fluorescence is determined a t each point along a single line across the image, rather than at each point across the entire image (Fig. lA), permitting images to be obtained as frequently as every 4 ms without loss of spatial resolution of fluo-3 fluorescence along the line that is scanned. Fluo-3 fluorescence in response to each stimulus was recorded in this way and the resulting line scan was convolved with the following 3 X 3 smoothing filter as follows.
Line scans were displayed as images consisting of 512 X 512 pixels, with a spatial resolution of 0.28 pm/pixel (in the n direction) and a temporal resolution of 4 ms/pixel (in the y direction). Because of this high degree of resolution relative to the typical Ca2+ wave speeds that were detected, the low pass filter served only to reduce noise. The change in fluorescence over time at each point along the scan line was determined from the recorded image using an Itex Series 151 image processor. Velocities of intracellular Ca2+ waves were determined from the rate at which initial increases in fluorescence progressed along the scan line. Specifically, an apical and basal point were identified within the cytosol, and the wave speed between the two points was calculated as (distance between the two points)/(time interval between the initial increase in fluorescence at each of the points). For these measurements, intracellular points not clearly within the cytosol were avoided. The line-scanning approach may overestimate the speed of Ca2+z waves which do not travel along the scan line (by -20% on average) (7), but scan lines were chosen to be oriented along the apical-to-basal axis, which is the direction that Caz+, waves travel in this cell type. Acinar cells stimulated with either ACh or CCK were scanned at a frequency of 250 Hz (every 4 ms) for 512 consecutive scans. Cells stimulated with ACh in the presence of ryanodine or caffeine were instead scanned at a frequency of 10 Hz (every 100 ms), because a frequency of 250 Hz was too rapid to adequately define these slower transcellular Caz+t waves within 512 consecutive scans. For comparison, some Ca'+, waves induced by ACh in the absence of ryanodine or caffeine were also measured at a linescanning frequency of 10 Hz, and their average speed was no different from that measured in ACh-stimulated cells at 250 Hz (Mann-Whitney test). Absolute Ca'+& concentrations were not estimated because fluo-3 cannot be ratio-imaged (15). Since the velocity of a Ca'+ wave is determined independent of Ca2+, concentration, measurements of wave speeds were not limited by use of fluo-3.
Measurement of Amylase Secretion-Isolated rat pancreatic acini were prepared as described above, then 50-pl aliquots of the acini were preincubated with 50 pl of buffer (kryanodine) for 5 min. An additional 100 pl of buffer k agonist was added to the cells, and acini were incubated under these conditions for 25 min. All steps were carried out at 37 "C. Cells were then centrifuged for 2 min at 2,000 x g, and the Bernfeld assay (16) was used to measure amylase in each supernatant and lysed cell pellet as described previously (17). Amylase release was calculated from these measurements and expressed as a fraction of the total amylase present. Amylase release was measured in acinar cells prepared from three rats. For each of these three preparations measurements were made in quadruplicate.
Statistical Analysis-Outliers were identified a priori using an F test based on the Mahalanobis distance (18). Comparisons between groups were then made using a one-tailed two-sample t test for Ca2+z wave speeds, and using a paired t test for amylase secretion. Caz+, wave speeds and measures of amylase secretion are expressed as mean k S.E.

RESULTS AND DISCUSSION
Ca2+ Waves Induced by ACh and CCK-Ca2+, waves were induced in pancreatic acinar cells by stimulation with either ACh or CCK; ACh-induced Ca2+i waves observed in a representative acinus are shown in Fig. 1 4). CCK (500 p~, n = 14) also induced rapid (60 2 11 pm/s) Ca2+i waves, and most waves induced by either ACh (73%) or CCK (77%) traveled in an apical-tobasal direction. These observations demonstrate that stimulation of either muscarinic or CCK receptors induces rapid apical-to-basal Ca2+i waves in pancreatic acinar cells. The wave speed appears to be dose-dependent, since submaximal stimulation with ACh (0.1 p~) induced slower waves.
It has previously been reported that ACh (10 pM)-induced Ca2+: waves travel across pancreatic exocrine cells a t -15 pm/s (6), but these measurements were made in cells from acini that had been further digested and dispersed into groups of two to four cells. For comparison, we also examined Ca2+i waves in pancreatic acinar cells prepared and stimulated in this way (Fig. 2). We observed that stimulation with 10 p~ ACh induced apical-to-basal CaZci waves in 100% of these cells (n = 15), but at a speed of only 37 f 4 pm/s ( p < 0.0005 relative to those cells within intact acini stimulated with 10 p M ACh). Our observations thus indicate that the manner in which cells are prepared may influence Ca2+i wave speed, since waves were markedly slower in cells from acini that were dispersed to a greater extent, even though these cells were maximally stimulated as well. Possible explanations for this behavior of such cells most likely relate to the way in which the cells are prepared, rather than to the fact that the cells were dispersed per se. First, prolonged incubation of cells in Ca2+-free medium may partially deplete their Ca2+-induced Ca2+ release (CICR) stores. Alternatively, fewer muscarinic receptors may be present on the basal membrane of such cells (eo that the cells respond as if stimulated with a lower dose of ACh), possibly because of digestion of receptors by the collagenase and trypsin preparation, or because receptors may have relocalized across loosened tight junctions to the apical membrane (19). Ca2+ wave speeds measured in dispersed acinar cells in our study were nonetheless faster than previously reported (6), but this may relate to the temperature at which our study was performed (2) (37 "C, as opposed to room temperature in a previous study (6)). Together, our findings indicate that stimulation of pancreatic acinar cells results in a rapid apical-to-basal Ca2+, wave, regardless of the type of receptor that is stimulated, and the wave travels at a speed that is dependent upon the magnitude of the stimulus and the Effects of Caffeine and Ryanodine on Ca2+ Wave Speed-To assess the importance of CICR for Ca2+i wave propagation, pancreatic cells within acini were stimulated with 1 p~ ACh in the presence of 20 mM caffeine or 10 or 50 pM ryanodine (n = 16,15, and 35, respectively). We chose this concentration of caffeine because others had shown it to block CICR in this cell type (20). Caffeine affects ryanodine-sensitive rather than inositol 1,4,5-trisphosphate (IP&sensitive stores (21)(22)(23)(24), although there is conflicting evidence as to whether this action of caffeine is due to discharge of CICR stores (21)(22)  These findings demonstrate that mobilization of caffeine-and ryanodine-sensitive Ca2+ stores are necessary for rapid propagation of Ca2+ waves in pancreatic acinar cells. Estimates of the diffusion constant for Ca2+ in cytosol have ranged from -10 to 400 pm2/s (10,25,26), suggesting that agonist-induced Ca" waves travel much faster than can be explained by diffusion. These observations are consistent with the hypoth-esis that Ca2+; wave propagation in the acinar cell is mediated by sequential release of spatially distributed CICR stores, serving as a positive feedback mechanism to accelerate wave speed (1,2).
Amylase Secretion-Since apical-to-basal Ca2+; waves are thought to regulate lumenal C1-secretion in pancreatic acinar cells (6), we wished to determine whether such waves also affect amylase secretion, an indicator of apical exocytosis. Stimulation of acini with 1 ~L M ACh induced a greater than 3fold rise in amylase secretion relative to unstimulated controls ( p < 0.025, paired t test; Fig. 3). Amylase release in the presence of 50 p~ ryanodine alone was no different than in unstimulated controls (Fig. 3). Secretion of amylase was increased almost 3-fold in acini incubated with both ryanodine and ACh ( p < 0.005), identical to the effect observed with ACh alone (Fig. 3). These findings suggest that, unlike C1secretion, secretion of amylase by pancreatic acinar cells is not dependent on Ca2+; wave speed. However, these findings merely relate the initial -200 ms of ACh-induced Ca2+; signals to the amylase secretion that follows over the next -20 min. There is considerable evidence that other features of AChinduced Ca2+ signals are causally related to exocytosis (27).
It has been hypothesized that CICR provides a unifying mechanism for spatial and temporal organization of Ca2+ signals within the cytosol (1, 2, 10, 28). Documented effects of CICR on Ca2+; signals include increasing the magnitude of Ca2+ elevations (29) and establishing the threshold for (20) and maintaining (28, 30) Ca2+ oscillations. By demonstrating that this positive-feedback mechanism also greatly enhances Ca", wave speed independent of the receptor type stimulated, we provide experimental evidence that CICR is the common subcellular mechanism by which intracellular Ca2+ signals are coordinated.
These findings are in agreement with the previous observation that ACh-induced Ca2+ waves begin apically and spread to the basal pole in pancreatic acinar cells (6). In addition, we report that this subcellular pattern of Ca2+ release is elicited by CCK, which suggests that apical-to-basal Ca2+ waves occur independent of the type of receptor that is stimulated. Since both ACh-and CCK-induced increases in Ca2+; are initiated by release of Ca2+ from IP3-sensitive stores (31), these findings would suggest that such stores are located in the apical region (6). Since ryanodine-sensitive (ie. CICR) Ca2+ stores are thought to be distributed across the cell (l), our findings would also suggest that apical release of Ca2+ (by IPS) leads to sequential release of additional Ca2+ from apically to basally distributed ryanodine-sensitive stores. This Increase relative to unstimulated controls is significant ( p < 0.025) by paired t test.
pattern of Ca2+ release would result in apical-to-basal Ca2+ waves, as observed. Selective inhibition of CICR would again result in apical release of Ca2+ from IP3-sensitive stores, but followed by a slower apical-to-basal Ca2+ wave, as we also observed. The subcellular distribution of receptors for IPB and ryanodine in exocrine pancreas has not been reported, but IP3 receptors in the hepatocyte are located apically while hepatocyte ryanodine receptors are located elsewhere (23, 32). Ca2+L wave speeds of -100 pm/s, as reported here in pancreatic acinar cells, have also been observed in excitable cells (e.g. myocytes (5)) and nonexcitable cells (hepatocytes (7,8)). The significance of such waves for acinar cell function is not fully understood. The importance of Ca2+; waves for lumenal C1-secretion has been suggested (6), but the present work suggests that amylase secretion is not controlled in a similar fashion. In hepatocyte couplets and triplets, agonist-induced Ca2+; signals travel across gap junctions in a rapid, synchronized, and wave-like fashion (7,8), and cells within pancreatic acini also communicate across gap junctions (12,33). Disruption of conductance across gap junctions influences exocrine pancreatic secretion (12), and agonist-induced Ca2+; oscillations are synchronized among cells within a single acinus (34). These considerations raise the question of whether CICRinduced Ca2+& waves in this cell type play a role in establishing intercellular communication and coordination. Further work will be needed to determine whether CICR indeed leads to synchronized and integrative behavior among cells comprising the pancreatic secretory epithelium.