1Department of Neurosciences, Medical College of Ohio, Toledo, Ohio; and 2Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York
Submitted 5 April 2005 ; accepted in final form 29 June 2005
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ABSTRACT |
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membrane capacitance; differential imaging; zymogen; gland slice; exocrine cells
In the present study we sought to address this gap in our understanding by using time-differential imaging analysis in combination with highly time-resolved membrane capacitance (Cm) measurements to image the secretory activity of single mouse parotid acinar cells in real time after agonist-induced or direct photolytic elevation of Ca2+. Moreover, optical methods were successfully applied to a parotid gland organotypic slice preparation. Our observations revealed that there are two apparent Ca2+-dependent vesicle pools. After moderate elevation of Ca2+, one pool was rapidly induced to fuse with the plasma membrane. Although imaged with time-resolved Cm methods, single exocytotic events could not be resolved either electrically or optically. The other vesicle pool underwent exocytosis after a delay, was resolvable by both electrophysiological and optical methods, and most likely corresponded to the individual fusion events of dense core zymogen-containing secretory granules (ZSGs). ZSG fusion events produced persistent postfusion structures, were often found to coincide at similar subcellular sites or "hot spots," and produced a spatial pattern of exocytotic activity that progressed outward from apical or lateral aspects of the cell toward the cell interior when robustly stimulated. These observations suggest that there are multiple classes of secretory vesicles in parotid acinar cells that differ in their size, Ca2+ sensitivity, kinetics of exocytosis, and membrane retrieval mechanisms. Thus exocytotic activity in parotid acinar cells exhibits characteristics both similar to and divergent from those of the better-studied pancreatic acini.
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MATERIALS AND METHODS |
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For slice preparation, 2 ml of a 3% (wt/vol) low-temperature gelling point agarose solution (Sigma type VIIA) was prepared in physiological salt solution (PSS) that contained (in mM) 140 NaCl, 10 HEPES-NaOH, 4.7 KCl, 1.13 MgCl2, 1 CaCl2, and 10 D-glucose, pH 7.3, and warmed to 90°C. The agarose solution was then maintained at 35°C, and isolated lobes, or in some cases the entire parotid glands, were embedded. The tissue-agarose mixture was then gelled at 48°C, and the block was trimmed so that minimal agarose remained around the tissue. The block was then glued to the tissue stand of a vibratome, and the chamber was filled with ice-cold nominal-Ca2+ PSS. Parotid gland tissue prepared by this method was cut into 40- to 50-µm-thick sections. The slices were collected and kept at 25°C until recording. Experiments monitoring Ca2+ or secretory dynamics in isolated cells or slices were performed with PSS at
25°C 14 h after slice preparation.
Time-resolved Cm measurements. Cm measurements were performed with an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster, CA), an ITC-16 digital interface (Instrutech, Port Washington, NY), and an Apple G4 computer running IGOR Pro (Wavemetrics, Lake Oswego, OR) and a software-based phase-sensitive detector (Pulse Control software, Dr. Richard Bookman, University of Miami Medical School, Coral Gables, FL) as previously described (8). The standard intracellular recording/caged-Ca2+ solution contained (in mM) 110 CsMeSO4, 20 CsCl, 10 HEPES-Tris, 10 NP-EGTA, 5 CaCl2, 3.2 MgCl2, 2 Tris-ATP, 0.2 GTP, and 0.075 Oregon Green BAPTA-5N, pH 7.2.
Flash photolysis, digital imaging, and time-differential imaging analysis.
Caged compounds were photoconverted with a pulsed xenon arc flash lamp system (TILL Photonics, Eugene, OR) ported to a Nikon TE2000 microscope equipped with differential interference contrast (DIC) optics through a fiber-optic guide and epifluorescence condenser. High-intensity UV light flashes (0.11 ms) were focused onto the image plane with a DM400 dichroic mirror and a Nikon SuperFluor x40 oil-immersion objective. Fluorescence or transmitted light images were obtained with a TILL Photonics monochrometer-based high-speed digital imaging system. For measurement of [Ca2+]i cells were illuminated with 340-, 380-, or 488-nm light and fluorescence was collected through a 525 ± 25-nm band-pass filter (Chroma Technologies, Brattleboro, VT). The numerical apertures of the objective and condenser for time-differential imaging analysis were 1.3 and 0.52, respectively. Transmitted light optical resolution was estimated to be 350 nm. For experiments in which alternating brightfield and fluorescent images were collected, a high-speed Uniblitz VS35 optical shutter (Vincent Associates, Rochester, NY) was placed in the tungsten lamp illumination path. Pairs of brightfield and fluorescence images (30- and 50-ms exposure, respectively) were obtained at 5 Hz. Time-differential images were generated by subtraction of each brightfield frame by its preceding frame. Time-differential imaging has been used and validated previously in pancreatic acinar cells and other cell types as a method of visualizing individual secretory granule fusions (2). Parotid acinar cells were amenable for this approach, likely because of the large size and phase-dark core of their ZSGs. Exocytotic fusion created a change in optical density that was detected as a spot in the difference image. Average spot diameter after calibration with 1-µm beads (Molecular Probes) as standards was estimated to be 890 nm. Only those spots that appeared and disappeared within one or two frames were considered to be exocytotic events.
Data analysis. Fluorescence and time-differentiated images were generated, analyzed, and represented with TillVisION (TILL Photonics) and ImageJ (W. S. Rasband, National Institutes of Health, Bethesda, MD) software packages. For statistical analysis, all numerical values of the data are expressed as means ± SE, where n is the number of cells examined. Statistical significance was determined where appropriate by Student's t-test with Prism3 software.
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RESULTS |
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Optical measurements of secretory dynamics.
The observations with time-resolved Cm measurements suggested that distinct vesicle populations of differing sizes might underlie the two apparent phases of activity induced by Ca2+ elevation. To test whether ZSG fusion was linked to one or both of these phases of exocytosis, we applied time-differential imaging methods to DIC images of patch-clamped acinar cells to visualize exocytotic fusion dynamics at the single-cell level. These events are detected as changes in optical density as phase-dark core material is extruded from the ZSG on exocytotic fusion. This method has been previously applied to image single exocytotic events in pancreatic acinar cells (1, 2, 11) and submandibular salivary gland cells (26). Initially, to determine applicability of time-differential imaging in the parotid, we used 13 µM Iso treatment for 3 min before high-intensity flash photorelease of Ca2+ to activate robust exocytotic activity in acinar cells. As shown in Fig. 2, application of a high-intensity flash to an acinar cell loaded via the patch pipette with caged Ca2+ evoked a burst of ZSG fusions after a delay of 400 ms. On average the latency between the flash and the first exocytotic event was 600 ± 144 ms (n = 6). This delay was significantly longer than the rapid activation of increases in membrane surface area measured by the Cm method. The highest rates of exocytosis occurred within the first 1020 s after flash but persisted, generally at a lower rate for much longer periods (see Fig. 4C). An advantage of the time-differential imaging analysis method over the Cm method is the ability to create a spatial map of exocytotic activity. For example, as shown in Fig. 3B, ZSG fusions induced by flash photolysis of caged Ca2+ began at the apical border of the cell, and subsequent events occurred in progressively deeper regions of the cell. Thus the burst of ZSG fusions generally initiated in the apical region and proceeded sequentially into deeper regions of the cell interior. These events appeared and disappeared within 12 frames and had an average spot diameter of 1.2 ± 0.03 µm (estimated from pixel numbers and before calibration with bead standards; n = 160). In addition, larger events with a more complex profile were occasionally observed. These events appeared to reflect the multivesicular fusion of two to four ZSGs. As shown in Fig. 3C, a Gaussian fit to a histogram describing the distributions of diameter sizes of events that could be resolved as discrete spots indicated that events were distributed around 1.14 ± 0.04 µm. Spot diameter after correction by calibration with bead standards was estimated to be 890 nm, comparable to the average size of a single parotid ZSG determined from electron micrographs. Moreover, ZSG fusions often occurred in clusters or exocytotic hot spots that were largely confined to the apical or lateral granule-containing regions of the cells (1). In many cases where exocytotic activity was robust, ZSG fusions at these hot spots occurred over several successive image frames. This sequential activity suggested that the fusion of one ZSG can recruit or coordinate the subsequent fusion of other granules. An example of this coordinated behavior is shown in Fig. 4B.
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Estimates of exocytotic rates with time-differential imaging analysis.
Differential imaging analysis was used to investigate the Ca2+ dependence of ZSG exocytosis. Events evoked after activation of exocytosis were counted and expressed as the rate of ZSG fusion (events/min). As shown in the histogram in Fig. 4C, the delay following flash photorelease of Ca2+ for the first 10 fusion events/cell was generally 14 s. These data demonstrated that Ca2+-activated ZSG fusion follows a biphasic relationship in which the majority of events are evoked in the first 20 s after Ca2+ elevation. Average rates of ZSG fusions under different experimental conditions are shown in Fig. 4D. The rates of exocytotic activity in single patch-clamped cells after attainment of whole cell configuration with pipettes containing no added Ca2+ and BAPTA, 60 nM Ca2+, or 600 nM Ca2+ were 0.25 ± 0.25, 2.1 ± 1.5 and 4 events/min (1
n
4), respectively. In contrast, the instantaneous elevation of Ca2+ to micromolar levels by UV flash photorelease evoked an average response of 63 events/min (n = 2). To assess the magnitude of the Ca2+-induced exocytotic response in the presence of elevated cAMP, acinar cells were treated with 3 µM Iso for
3 min before flash photolysis. The average response to the photolytic release of Ca2+ after Iso treatment was 165 ± 13 events/min (n = 6). The rate of exocytotic events was augmented nearly threefold compared with control cells. Furthermore, in contrast to the enhancement estimated by Cm measurements, the total number of ZSG fusions determined by imaging over a 30-s interval after flash photolysis was nearly fivefold that of control. These data indicate that elevated cAMP levels greatly enhanced the number of ZSGs available for Ca2+-evoked exocytotic release. The mismatch between estimates of the cAMP-mediated enhancement obtained with Cm measurements and optical methods suggests that Cm measurements may not properly track changes in surface area due to sequential compound exocytosis, perhaps because of space clamp errors.
Exocytotic rates in parotid slices.
Enzymatically dispersed single acinar cells or small clusters of three to eight acinar cells (small acini) have been used extensively as models to study exocrine secretion and calcium dynamics, and their study has resulted in a wealth of information regarding these processes. However, some reports have debated whether dissociated acinar cells can exhibit altered morphology and function (1, 20). To address this possibility, we developed an organotypic slice preparation of the mouse parotid gland, using a vibratome that circumvented the need to use enzymes. As shown in Fig. 5A, this preparation largely preserved the normal three-dimensional arrangements of cell types in this tissue as indicated by F-actin localization. Moreover, as shown in Fig. 5, BE, parotid slices were readily imaged with conventional digital brightfield and fluorescence microscopy. For example, an intact acinus from a slice (Fig. 5B) was treated with 1 µM Iso and imaged with transmitted light and time-differential analysis. A projection of nearly 1,000 frames of the exocytotic events (image inverted for clarity) induced over 8 min of continuous Iso application is shown in Fig. 5C. In addition, as shown in Fig. 5, D and E, spatial and temporal aspects of Ca2+ dynamics could also be visualized in slices. Slices were loaded for 1 h at 25°C with fura-2 AM, placed onto glass-bottomed dishes, and held in place with a small metal washer. The majority of the cells in slices responded to CCh treatment with increases in cytosolic Ca2+. For example, the Ca2+ response of a small three- to four-cell acinus of a slice to application of 300 nM CCh is shown in the montage in Fig. 5D. Line traces obtained from ROIs placed in apical and basal regions of one acinar cell from this cluster demonstrate that CCh application could induce Ca2+ oscillations that were qualitatively similar to those characterized in isolated acini.
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DISCUSSION |
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Comparison of time-resolved Cm measurements and time-differential imaging analysis of membrane dynamics.
The rate of exocytosis induced by continuous low-level photolysis and determined by counting stepwise events from Cm records roughly matched that of measured by differential imaging analysis (4 events/min). However, when absolute rises in Cm rather than steps were counted, the estimate of exocytotic activity was two- or threefold greater. This apparent discrepancy could largely be accounted for if a significant number of the stepwise increases observed reported compound exocytotic events. Consistent with this idea, many of the Cm steps were indeed multiples of 20 fF (the amount of membrane estimated for 1 ZSG fusion). Similarly, with the understanding that the time resolution was significantly slower than Cm measurements (100 ms vs. 10 ms), examination of time-differentiated images often indicated what appeared to be multivesicular fusion events. On the other hand, high-intensity flash photorelease of Ca2+ produced an exponential rise in Cm that rapidly reached a new steady state and suggested the nearly simultaneous, unresolved fusion of
30 ZSGs. These estimates indicated a greater number of individual fusion events than that observed by direct visualization of fusion events by optical methods. Moreover, the increase in Cm initiated within a few milliseconds after cessation of the flash, in contrast to the delay (600 ms on average) between the flash and the initial fusion event detected by differential imaging. Indeed, the shortest delay observed with optical methods was at least 200 ms. This observation suggested that, in addition to ZSG fusion, Cm measurements also detected the rapid fusion of another vesicle type. The idea that there are multiple vesicle types that differ in size, Ca2+ sensitivity, and kinetics of exocytosis is not without precedence. Moreover, multiple pathways of amylase release have been identified in parotid acinar cells (4). Constitutive and minor regulated pathways account for the majority of release of
-amylase under basal conditions. The majority of evoked release is via regulated fusion of ZSGs in response to adrenoceptor and muscarinic cholinoceptor receptor activation. However, recent studies have demonstrated that the minor regulated pathway is functionally linked to granule exocytosis. Exocytotic fusion of the minor regulated pathway is apically directed, precedes granule exocytosis, and can be selectively discharged under low-level stimulus conditions (i.e., 1 µM Iso) (3, 5). It has been proposed that the minor pathway can recruit syntaxin 3 to the apical membrane (or remove via endocytosis) and modulate the number of docking sites for exocytotic fusion of ZSGs (3). The initial phase of Cm increase we observed exhibits features that are qualitatively similar to the minor regulated pathway in the parotid. The rise in Cm was continuous, suggesting fusion of a population of vesicles that were too small to be resolved by either Cm or optical methods. The initial, sometimes biphasic Cm change was induced by low-level stimulation and was abolished by strong intracellular Ca2+ buffering with BAPTA. In addition, BAPTA treatment and block of the initial phase of exocytosis abolished the secondary stepwise increases in exocytosis. Conversely, pretreatment with low concentrations of Iso enhanced the secondary phase of ZSG fusion evoked by low-level photolysis of caged Ca2+. Thus it is tempting to speculate that the initial phase of exocytosis induced by Ca2+ ramps identified in the present study reflects recruitment of the minor pathway. Indeed, ultrastructural studies of parotid and submandibular salivary glands have demonstrated the presence of small-diameter vesicles (<150 nm) at the subluminal face of apical membrane or on or around ZSGs (23, 27). However, it is also possible that transient fusion of ZSGs, delayed fusion pore expansion, or retention of the phase-dense material underlies the initial phase reported by Cm measurements. Accordingly, as differential imaging detects the loss of phase-dark material, we would not be able to visualize ZSG fusions faster than the time required to extrude core material. Indeed, we did occasionally detect a fusion event that projected over two image frames (100 ms/frame), indicating that this process may require 0.10.3 s to complete. Therefore, additional study specifically addressing this will be needed for a complete understanding of the relationship between these two phases of exocytosis.
Comparison of ZSG exocytosis in parotid and pancreatic acinar cells.
In part because Cm measurements distinguish neither the specific site nor the identity of a vesicle that fuses with the cell surface, we used time-differential imaging methods pioneered by others to optically assess parotid ZSG fusion dynamics in real time. We found that parotid cells had a relatively low rate of basal exocytosis but that high rates of ZSG fusion could be evoked after the controlled photorelease of caged Ca2+ with a minimum delay of 200 ms. In fact, the average delay between the flash application and the first ZSG fusion (600 ms) was more than an order of magnitude shorter in parotid cells than the delay reported for photolysis-evoked fusion in pancreatic cells (10 s). In general the kinetics of the responses were similar to those reported with the time-differential imaging analysis of pancreatic acinar cells. We found that increases in cytosolic Ca2+ by direct elevation or CCh application produced a biphasic response characterized by an initial burst of exocytosis with rates that decreased over the subsequent 2530 s and then persisted at a lower rate over minutes. (Interestingly, Iso application produced a largely monotonic, but persistent, level of fusion events that lasted throughout the stimulus application.) However, whereas the overall rates of exocytosis and total numbers of fusion events induced by agonist treatment were comparable between parotid cells and those reported for pancreatic cells, the rate and number of events evoked by flash photolysis were much greater. Furthermore, under strong stimulation, ZSG fusion proceeded from apical or sometimes lateral borders toward the cell interior. We interpreted this activity as the sequential compound fusion of ZSGs that proceeded from the primary sites of fusion at the cell surface into successively deeper regions of the cell interior (see below). Consistent with this idea, ZSG fusions were shown coordinate subsequent fusions at so-called hot spots. Indeed, our observations and interpretations match well with those of Campos-Toimil et al. (2), with the exception that sequential fusion at hot spots was much more rapid in parotid than in pancreatic acinar cells. For example in pancreatic cells, the delay between events at defined hot spots was
50 s. In the parotid, frame-to-frame analysis showed that multiple events at hot spots could occur over several successive frames (
100 ms of each other). Moreover, unlike the pancreatic cells, a number of events appeared to be comprised of multivesicular fusions.
Although we concede the possibility that under strong stimulus conditions primary fusions might be induced at distal sites on the cell surface, our interpretations are largely consistent with recent observations on the exocytotic process in pancreatic acinar cells. Recently, several groups have applied multiphoton and confocal methods to directly visualize exocytotic activity in pancreatic acinar cells. An emerging consensus regarding the process of ZSG fusion in pancreatic acinar cells is that after activation by Ca2+ levels >1 µM, a subpopulation of ZSGs fuse initially at the apical membrane, and additional granule-granule fusions proceed in a sequential fashion as they "daisy-chain" on each other (18). It is believed that sequential compound exocytosis is an adaptation to increase the efficiency of protein secretion where access to the subluminal apical domain is limited. Importantly, after primary fusion events, additional ZSG fusions appear to proceed independently of receptor activity or Ca2+ gradients (19). Therefore, it is hypothesized that sequential compound exocytosis requires the redistribution of a surface membrane protein to sites on the newly fused granules that in turn act as nuclei for subsequent granule-granule fusions (18, 21). Thus the recruitment of secondary or tertiary granules is believed to be limited by the diffusion of protein factor(s) from the plasma membrane to the granular membranes without the mixing of plasma membrane and granule membrane lipids (18, 28). Consistent with this idea, ZSG fusions in parotid acinar cells were shown to produce persistent, phase-clear structures that generally lasted for 220 s. The lifetimes of these events were similar to the F-actin-stabilized postfusion structures, or secretory granule "ghosts," produced in pancreatic acinar cells shown by multiphoton microscopy (19, 29, 31).
Monitoring exocytosis in parotid gland slice.
In contrast to those reported for pancreatic acinar cells, many of the exocytotic events in single parotid cells appeared to comprise multivesicular ZSG fusions. To ascertain whether this was a relevant characteristic of parotid cells or a consequence of patch-clamping isolated cells and artificial stimulus conditions, we treated parotid organotypic slices with agonist and optically monitored ZSG fusions. Numerous multivesicular fusions were also observed in response to Iso or CCh treatment in slices, and thus this mode of secretory activity likely represents a real physiological property of these cells. In contrast, we found that isolated cells did not respond to low levels of Iso or CCh with ZSG fusions, whereas cells in acini in semi-intact slices responded to 1 µM Iso with robust secretory activity. Therefore, although isolated cells were a good model for studying mechanistic aspects of Ca2+ and secretory activity, enzymatic treatment may alter the amount or integrity of surface receptors and reduce the exocytotic response. Thus this indicates that some caution in using isolated cells is warranted and that the parotid slice preparation may provide a useful system to address questions regarding these processes in a less invasive and more physiologically relevant context. It is also important to note that the overall rate of secretion, as measured by time-differential image analysis, in parotid slices is lower than that reported for pancreatic acini. However, the total mass of zymogen release may be similar because the percentage of multivesicular fusions appears greater in parotid. Whether the lower rate of exocytosis reflects a true difference in the magnitude of secretory activity remains to be determined.
In conclusion, despite remarkable similarity in morphology and general function between these two cell types, parotid cells demonstrate secretory kinetics that are more rapid and perhaps subject to more dynamic regulation than pancreatic cells. These differences may reflect the multifunctional role for and the need to rapidly and dynamically adjust saliva output in processes such as mastication, digestion, oral health, and speech.
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GRANTS |
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FOOTNOTES |
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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.
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