Am J Physiol Gastrointest Liver Physiol 295: G629 –G640, 2008. First published July 31, 2008; doi:10.1152/ajpgi.90217.2008.
Visualizing form and function in organotypic slices of the adult mouse parotid gland Jennifer D. Warner,1 Christian G. Peters,1 Rudel Saunders,1 Jong Hak Won,2 Matthew J. Betzenhauser,2 William T. Gunning III,1 David I. Yule,2 and David R. Giovannucci1 1
Department of Neurosciences, University of Toledo College of Medicine, Health Science Campus, Toledo, Ohio; and 2Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York Submitted 29 February 2008; accepted in final form 24 July 2008
Warner JD, Peters CG, Saunders R, Won JH, Betzenhauser MJ, Gunning WT 3rd, Yule DI, Giovannucci DR. Visualizing form and function in organotypic slices of the adult mouse parotid gland. Am J Physiol Gastrointest Liver Physiol 295: G629 –G640, 2008. First published July 31, 2008; doi:10.1152/ajpgi.90217.2008.—An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function. Ca2⫹ signaling; exocytosis; immunofluorescence; electron microscopy; multiphoton; tissue culture THE SALIVARY GLAND IS AN ESTABLISHED and valued model for studying exocrine gland development (2, 9, 29), protein synthesis and trafficking (5), Ca2⫹ signaling (3, 12, 30), and fluid (16, 18, 21) and protein secretion (6, 10, 20, 28) in health and disease (33). Previous studies on salivary gland function have primarily used whole animal or enzymatically dispersed acini or single cells. However, imaging cellular and subcellular processes such as Ca2⫹ signaling or exocytosis in vivo is difficult to resolve and intravital imaging methods require expensive, specialized instrumentation such as a multiphoton microscope. In contrast, conventional live-cell imaging methods in combination with enzymatically dispersed single acinar cells or small acinar clusters have been used extensively to study exocrine secretion and Ca2⫹ dynamics. However, isolated cells exhibit altered morphology and there is debate as to whether dissociated acinar cells retain normal function (4, 24). In addition, these highly polarized epithelial cells rapidly lose many of their morphological and functional characteristics in primary culture. Other groups have attempted to resolve this by modifying culture conditions or developing cell lines. Al-
Address for reprint requests and other correspondence: D. R. Giovannucci, Dept. of Neurosciences, Health Science Campus, The Univ. of Toledo, 3035 Arlington Ave., Toledo, OH 43614 (e-mail:
[email protected]). http://www.ajpgi.org
though some success has been achieved using immortalized parotid cell lines, no cell line to date recapitulates the naive acinar cell. Recently, a method for primary culture of enzymatically dispersed rat parotid acinar cells that retained the capacity for agonist-induced amylase secretion was reported (11). In these cells, the synthesis and exocytotic release of secretory granules was maintained for up to 48 h. Although a leap forward, dispersed cells rapidly lost polarity and the possibility that ductal cells differentiated into acinar cells could not be excluded. Moreover, these cultures lacked three-dimensional acinar structure and the complexities of salivary gland tissue microenvironment. Therefore, we took a different approach to address this problem. We developed a thin slice preparation of mouse parotid gland fragments and introduced short-term culture methods to advance our understanding of Ca2⫹ and secretory dynamics in the parotid. Freshly prepared slices retained lobular structure including acinar clusters, ducts, vasculature and autonomic nerve fibers. Because no enzymes were used in the preparation, the complement of cell surface receptors was largely unaffected. Moreover, the thin slice was amenable to optical measurements of Ca2⫹ dynamics and exocytotic activity using standard wide-field microscopy. Furthermore, shortterm culture (24 – 48 h) of slices preserved subsets of acinar cell clusters that retained typical polarized morphology, subluminal F-actin distribution, Ca2⫹ signaling, and secretory dynamics largely indistinguishable from freshly prepared slices. In this study we report a significant advance in developing a salivary gland model system that more closely resembles the intact gland and demonstrate the potential of the parotid slice preparation to study integrative exocrine gland function in health and disease. MATERIALS AND METHODS
Preparation of Mouse Parotid Gland Slices Whole parotid glands were quickly dissected from 18- to 25-g male NIH Swiss Webster mice (Charles River) following euthanasia by CO2 asphyxiation and puncture of the heart. Excess fat was removed with fine forceps and the gland was cut with a razor blade into a few large pieces in slice saline solution containing (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 10 Na-HEPES, 10 glucose, 0.8 thiourea, 0.4 ascorbic acid; pH 7. To stabilize and preserve architecture of tissue fragments for slicing, 2 ml of a 3% (wt/vol) low-temperature gelling point agarose solution (Sigma Chemical, type VIIA) in nominal Ca2⫹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.
0193-1857/08 $8.00 Copyright © 2008 the American Physiological Society
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containing slice saline was prepared and warmed to 90°C until the solution became clear. The agarose solution was then maintained at ⬃35°C, and the pieces were embedded. The tissue-agarose mixture was then gelled at 4 – 8°C and the block trimmed so that minimal agarose (just enough edge to manipulate the slice with fine forceps) remained around the tissue. The block was then glued to the tissue stand of a Vibratome 1000 Classic, and the chamber was filled with ice-cold nominal Ca2⫹ slice saline. Tissue blocks were cut into 50-m-thick sections and slices were kept at 25°C in physiological saline until recording. Experiments were performed 1– 4 h after slice preparation. The remaining slices were maintained in culture at 37°C and 95% O2-5% CO2 for 24 – 48 h in RPMI 1640 medium containing 2-mercaptoenthanol, sodium pyruvate, HEPES, and 10% fetal bovine serum. All experiments using animals were approved by and carried out in strict accordance with the policies of the University of Toledo Institutional Animal Care and Use Committee and the University Committee on Animal Resources at the University of Rochester and conformed to the “Guide for the Care and Use of Laboratory Animals,” National Institutes of Health Publication No. 85-23 (National Research Council, National Academy Press, Washington, DC, 1996). Digital Fluorescence and Time-Differentiated Imaging Fluorescence or transmitted light images were obtained using a Polychrome IV monochromator-based high-speed digital imaging system (TILL Photonics, Gra¨felfing, Germany) ported to a Nikon TE2000 microscope equipped with DIC optics through a fiber optic guide and epifluorescence condenser. For measurement of intracellular Ca2⫹ concentration, slices were alternately illuminated with 340 or 380 nm light focused onto the image plane with a DM400 dichroic mirror and Nikon SuperFluor ⫻40 oil-immersion objective and fluorescence collected through a 525 ⫾ 25 nm band-pass filter (Chroma Technologies, Brattleboro, VT). For experiments in which alternating transmitted light 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 transmitted light and fluorescence images (30- and 50-ms exposure, respectively) were obtained at 2 Hz. Time-differentiated images were generated by subtraction of each transmitted light frame by its preceding frame essentially as previously described. Time-differentiated imaging has been previously validated as a method of visualizing individual zymogen granule fusions (4). Immunofluorescence Organotypic tissue slices were fixed overnight with 4% paraformaldehyde. Fixed tissue was then rinsed three times in PBS and incubated in tissue permeabilization buffer comprised of PBS containing Ca2⫹ and Mg2⫹, 0.3% Triton X-100, and 0.1% bovine serum albumin for 30 min. For immunofluorescent localizations, tissue slices were incubated in blocking buffer (0.2% Triton X-100, 5% goat serum) for 1–2 h to block nonspecific IgG binding sites. Following two washes in PBS, sections were incubated overnight with appropriate polyclonal or monoclonal antisera in PBS-Triton (PBS-Tx) containing 5% goat serum at 4°C. Sections were rinsed in PBS-Tx and incubated for 1 h with Alexa Fluor 488- (or Alexa Fluor 555)conjugated anti-monoclonal/rabbit IgG as needed at room temperature. Anti-tyrosine hydroxylase (TH), anti-vesicular acetylcholine transporter (VAChT) or TUJ, a neuron-specific -tubulin antibody, were used as sympathetic, parasympathetic, or general nerve fiber markers, respectively. F-actin was visualized by staining with Alexa Fluor 546-conjugated phallotoxin (Invitrogen) for 30 min at room temperature. Nuclei were visualized by 4⬘,6-diamidino-2-phenylindole (DAPI) stain. Following several washes, stained sections were mounted on glass slides with Vectashield mounting medium. AJP-Gastrointest Liver Physiol • VOL
Confocal and Two-Photon Microscopy Images were acquired via a Leica TCS SP5 broadband confocal microscope system (Leica, Mannheim, Germany) equipped with argon and diode-pumped solid-state continuous-wave lasers and Coherent Chameleon XR tunable pulsed infrared laser source and coupled to a DMI 6000CS inverted microscope. Optical sections were obtained by use of a ⫻10 objective [0.4 numerical aperture (NA)], ⫻40 immersion oil objective (1.25 NA), or ⫻63.0 (1.40 NA) oil-immersion objective. Alexa-488 and Alexa-546 dyes were excited by using 488- or 561-nm laser lines, respectively. DAPI was excited using multiphoton laser source tuned to 780 nm. Acquisition of emitted light was optimized via a tunable SP detector. A series of optical sections (0.5 m thickness) were collected, rendered as projected images, or processed with Leica LAS, Image J (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/, 1997– 2004) or Photoshop software packages. Real-time imaging of exocytotic activity using fluid phase fluorescent dye and two-photon microscopy was performed similar to that previously described (22). Briefly, slices were placed in 0.5 mM sulforhodamine B (SRB). For SRB excitation, the picosecond pulsed-IR laser was tuned to 830 nm and focused at the focal plane with a ⫻63 water immersion objective (1.2 NA). Confocal and two-photon microscope studies were performed using resources of the Advanced Microscopy and Imaging Center at the University of Toledo Health Science Campus. Electron Microscopy For transmission electron microscopy, parotid slices were fixed with 3% glutaraldehyde for 1 h and postfixed for 2 h with 1% osmium tetroxide followed by treatment with saturated uranyl acetate for 1 h. Dehydration was carried out through a graded series of chilled ethanol solutions, with a final wash with 100% acetone. Cells were embedded in Spurr’s resin (Electron Microscope Sciences, Fort Washington, PA) and sections were collected on copper 300-mesh support grids. Sections were stained with uranyl acetate and lead citrate and examined by use of a Philips CM 10 transmission electron microscope. Cell proliferation and apoptosis. Parotid slices were fixed overnight in 4% paraformaldehyde and incubated for 1 h at room temperature with PBS supplemented with 3% BSA, 2% goat serum, 0.7% cold water fish skin gelatin, and 0.2% Triton-X-100. Slices were then incubated overnight with polyclonal anti-cleaved caspase 3 antibody (1:100) or polyclonal anti-Ki67 (1:100) at 4°C. Slices were rinsed in PBS-Tx and incubated for 1 h with Alexa Fluor 488-conjugated anti-rabbit IgG at room temperature. F-actin was visualized by staining with Alexa Fluor 633-conjugated phallotoxin (Invitrogen, Carlsbad, CA) for 30 min at room temperature. Following several washes, stained slices were mounted on glass slides using Prolong mounting medium and stored at 4°C. The labeling of apoptotic cells was performed using the APObromodeoxyuridine (BrdU) TdT-mediated dUTP nick-end labeling (TUNEL) assay kit as described by the manufacturer (Invitrogen, Carlsbad, CA). Briefly, parotid slices were fixed with 4% paraformaldehyde overnight at 4°C. Slices were added to the DNA-labeling solution consisting of TdT enzyme, BrdUTP, and reaction buffer for 1 h at 37°C. At the end of incubation, slices were rinsed and placed into the antibody staining solution consisting of Alexa Fluor 488labeled anti-BrdU antibody for 30 min, and then 500 l of propidium iodide-RNase A was added to each sample for an additional 30 min before slices were placed on coverslips. All images were captured with a Leica TCS SP5 broadband confocal microscope and visualized as projected images. Field stimulation of parotid slices. Agarose-embedded slices were loaded by incubation with 5 M fura 2-AM. The slice was restrained on a coverslip by use of nylon mesh. The coverslip formed the base of a perfusion chamber. The slice was constantly superfused at a rate of 1 ml/min. Field potential stimulation of endogenous nerves in the slice was accomplished by placing platinum electrodes connected to a 295 • SEPTEMBER 2008 •
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Grass or A-M Systems stimulator (pulse protocol as indicated) into the superfusion chamber. The changes in fluorescence or optical density were recorded via a Till Imaging system as previously described. Viral infection of parotid slices. Agarose-embedded slices were incubated in 35-mm tissue culture dishes in Dulbecco’s modified Eagle’s medium supplemented with 0.5% FBS and penicillin-streptomycin. Recombinant adenovirus encoding green fluorescent proteintagged ribonuclease was added to the dishes at a concentration of 1–2 X 108 plaque-forming units/ml. The infected slices were incubated at 37°C and 5% CO2 for 10 –16 h then washed in HEPES-buffered saline solution prior to confocal imaging. RESULTS
Organotypic slices provide tractable model preparations to study complex physiological processes. For example, brain slices have been extensively used to image Ca2⫹ signaling and morphological dynamics in delicate structures such as dendrites (27, 34). Slice preparations have also been used successfully to monitor Ca2⫹ signaling in lung (26), exocytotic and ion channel activity in adrenal gland (7), and perinatal development and function of the pancreas (19). Recently, we developed a mouse parotid gland slice preparation. By this procedure, slices can be imaged both by transmitted light and by fluorescence following incubation with indicator using a standard inverted fluorescence or confocal/ multiphoton microscope. This preparation has practical advantages over studying isolated cells. For example, no enzymes were used to dissociate the tissue into isolated cells and, thus, the complement of cell surface receptors remained largely unaffected. Additionally, the structural integrity of the tissue was maintained such that cells retained normal cell-cell associations. A parotid slice shown in Fig. 1 demonstrates that the relationships between acinar clusters, ductal structures, and innervating nerve terminals are maintained. Maintenance of associations between acinar clusters may be of particular importance because loss of junctional complexes following dissociation may alter the distribution of membrane proteins, such as receptors in individual cells. Consistent with this notion, we found that acinar cells in slices, in contrast to enzymatically dispersed acini, retained polarized morphology over hours to days. Transmitted light images of whole lobules as well as individual acinar units using DIC optics are shown in Fig. 2, a–d. In freshly prepared or 24-h cultured slices (Fig. 2, c and d, respectively), zymogencontaining secretory granules were found mainly at apical and basolateral regions of cells in tightly coupled acinar clusters. Although on average there was some expansion of the area occupied by secretory granules in slices maintained in culture for 24 h, subsets of acini were visually indistinguishable from acini of freshly prepared slices. The maintenance of polarized distribution of granules was also observed at the ultrastructural level. Figure 2, e–h compares electron micrographs of acinar clusters from freshly prepared slices and slices cultured for 24 or 48 h (f). Luminal structure was also largely preserved at 24 h, as shown by presence of tight junctions. Another feature indicative of acinar cell polarity is an elaborate subluminal F-actin network. This network has been previously shown to be critical for maintaining secretory function and signaling in exocrine tissue. To test whether normal F-actin distribution was preserved, we treated slices with AJP-Gastrointest Liver Physiol • VOL
Fig. 1. Confocal image of a lobule from a mouse parotid salivary gland slice preparation. F-actin (green signal) was visualized by treatment with Alexa Fluor 488-conjugated phallotoxin to distinguish ductal and acinar structures. Nuclei were identified by 4⬘,6-diamidino-2-phenylindole (blue signal) and sympathetic nerve elements were localized by using tyrosine hydroxylase antisera in combination with Alexa Fluor 546-conjugated secondary antibody (red signal).
fluorescently labeled phallotoxin, a fungal toxin that selectivity binds to F-actin, and used confocal microscopy to localize F-actin distribution. As shown in Fig. 2, the F-actin signal following 24 h in culture (j) was similar to that for freshly prepared slices (i) with only minor expansion of the lumen and a slightly elevated background fluorescence compared with fresh slices. However, we observed a time-dependent reorganization of this signal. Thus, although a subluminal F-actin signal was still evident, significant luminal dilation and redistribution of F-actin to basal regions of acinar cells was evident in slices cultured for greater than 48 h (data not shown). Because of the loss of normal morphology of slices over an extended period in culture, we evaluated tissue viability at 0-, 24- and 48-h time points using markers of cell proliferation and apoptosis. To assess whether there were short-term changes in the proliferative capacity of organotypic slices, we used immunofluorescence localization with antibody to Ki67, a nuclear antigen present in all non-G0 cell cycle phases. As shown in Fig. 3, a–c, proliferating cells were rarely observed in freshly isolated or 24-h cultured slices. In contrast, there were many more proliferating cells observed in cultured slices at the 48-h time point. In a parallel set of experiments, TUNEL assays were performed to determine the numbers of apoptotic cells in slices prior to and following culturing. As shown in Fig. 3, d–f, there was a time-dependent increase in the numbers of apoptotic cells compared with total cell number indicated by propidium iodide staining. Similar data were obtained by use of anti-cleaved-caspase 3 antibody (Fig. 3, g–i). These data suggest that, although there is substantial increase in both apoptotic cells and in the number of unidentified 295 • SEPTEMBER 2008 •
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Fig. 2. Comparison of the acinar structure of freshly prepared and short-term cultured parotid slices. Differential interference contrast (DIC) images of freshly prepared 50 m thick slice at ⫻40 (a) and ⫻400 (b) magnifications demonstrated the usefulness of lowgelling-point agarose to maintain integrity of tissue structure. Comparison of individual acini from a freshly prepared slice (c) and a slice maintained in culture for 24 h (d). Electron micrographs comparing ultrastructural features of polarized acini from slices maintained in culture for 0 (e) or 24 h (f). Corresponding images at higher power magnification (g and h, respectively). Visualization of subluminal F-actin by confocal microscopy in freshly prepared (i) and 24-h cultured slices (j).
proliferative cells in tissue with longer-term culture, there are only modest differences in the turnover of cells within the first 24 h of culture. Short-Term Culture of Parotid Gland Slices Preserves Physiological Function Having demonstrated that short-term culture of parotid gland organotypic slices retained markers of normal acinar cell morphology, we determined whether function was also preserved. The primary function of this exocrine gland is to secrete fluid and protein in response to autonomic neural input. Secretory activity is controlled by parasympathetic and sympathetic neuron-mediated increases in cytosolic levels of Ca2⫹ and cAMP. Therefore, to assay functional integrity, we tested the effectiveness of cholinergic and/or adrenergic agonists to induce Ca2⫹ signals or exocytotic secretory activity using freshly prepared or cultured slices. Carbacholamine-Evoked Calcium Dynamics To assess Ca2⫹ signaling, slices were loaded with fura 2-AM and transferred onto glass bottom dishes. The tissue slices were perifused by local application of physiological saline with or without agonist using a small glass tube positioned above the slice and an air pressure driven reservoir system. Fluorescence intensity changes were monitored by live-cell digital fluorescence imaging methods. Although attempts were made to place regions of interest on each cell in a field of view, it was often difficult to accurately distinguish individual cells because they were not always at the same focal AJP-Gastrointest Liver Physiol • VOL
plane. In addition, other than avoiding obvious ductal structures, no specific effort was made to conclusively identify specific fura 2-loaded cell types. However, the majority of cells in the slices are serous acinar cells. It should be noted that for the present study we used an inverted microscope to record signals from cells adjacent to the coverslip while superfusing from the top or side of the slice. This configuration was the same for fresh or cultured slices. Because slices were maintained in minimal volume, bath solutions could be readily exchanged. To address concerns regarding accessibility of agonists, we generally focused on groups of acini that were near the periphery of the slice. To load slices with a sufficient amount of indicator we found it necessary to adjust our standard loading protocol and treat with higher levels of fura 2-AM (2–5 M) and for longer time periods (1 h) than that previously demonstrated to effectively load enzymatically dispersed acini (1 M, 30 min). Despite these adjustments and a generally lower level of absolute fluorescence compared with isolated acini, agonist-induced Ca2⫹ signals were found to be qualitatively similar to signals evoked in enzymatically dispersed acini. To assess whether Ca2⫹ signaling was preserved in slices maintained in shortterm culture, we compared the resting levels and agonistevoked changes in Ca2⫹ following maintenance in culture for 24 h. On average, the resting (prestimulus) ratio values of individual cells for freshly prepared and 24-h cultured cells were 0.387 ⫾ 0.006 ratio units (n ⫽ 238) and 0.414 ⫾ 0.005 ratio units (n ⫽ 346), respectively. These values indicated that on average there was a significant elevation of resting Ca2⫹ 295 • SEPTEMBER 2008 •
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Fig. 3. Assessment of proliferation and apoptosis in slices in freshly prepared slices (a, d, g) and slices maintained in culture for 24 h (b, e, h) or 48 h (c, f, i). In a–c, acinar cell structure was visualized by phalloidin (blue signal) and proliferating cells were identified by anti-Ki67 (green signal). In d–f, apoptotic cells were stained with an APObromodeoxyuridine (BrdU) TdT-mediated dUTP nick-end labeling (TUNEL) assay kit. Cell nuclei were visualized by propidium iodide (PI; red signal) and apoptotic cells were identified by anti-BrdU (green signal). g–i: Similar data were obtained by using anti-cleaved-caspase 3 antibody (green signal) and phalloidin (red signal).
levels after 24 h in culture, perhaps suggesting a diminishment of slice health. An alternative explanation was that the elevated averaged resting Ca2⫹ level reflected the contribution of a subpopulation of acini that were damaged by slicing and developed altered Ca2⫹ homeostasis over a period of many hours. We therefore examined the distribution of ratio values by plotting the data in 0.01-unit bins and fit the resulting histograms using a peak detection algorithm (IgorPro, WaveMetrics, Lake Oswego, OR). As shown in the histogram in Fig. 4a, the majority of cells in freshly prepared and in cultured slices exhibited resting ratio values less than 0.4 ratio units and were distributed with Gaussian peak values of 0.330 and 0.350 ratio units, respectively. This indicated that in both fresh and cultured slices the majority of the cells retained the ability to maintain Ca2⫹ homeostasis. However, following 24 h in culture, a substantial additional number of cells showed elevated resting ratio values distributed around 0.540 ratio units, indicating the emergence of a population of cells that exhibited compromised Ca2⫹ homeostasis following short-term culture. It was unlikely that time-dependent changes in autofluorescence accounted for the elevated resting ratios. Although there was minimal autofluorescence in both fresh and unloaded slices, we observed on average about a 25% increase in F380 gray value levels at 24 h that remained stable out to 48 h, without significant changes in F340 values. This increase would have little substantive effect to account for the increased resting ratio values because the contribution to the fura signal AJP-Gastrointest Liver Physiol • VOL
was minimal and an increase in autofluorescent signal induced by F380 would actually diminish the ratio value. Next, we characterized the Ca2⫹ responses induced in slices by the application of the acetylcholine receptor agonist, carbacholamine (CCh). Although there was found to be a greater number of unresponsive cells, or cells that exhibited altered signaling in 24-h cultured slices compared with fresh slices, many of the cells showed functional indexes that were nearly indistinguishable from those of freshly prepared slices. Treatment with acetylcholine or CCh is known to evoke rapid apical-to-basal Ca2⫹ responses in parotid acinar cells that typically consist of rapid sinusoidal oscillations on an elevated steady-state or “plateau” level. Similarly, in both freshly prepared and cultured slices, the increase in cytosolic Ca2⫹ was generally found to initiate in a subluminal “trigger” zone that has previously been shown to contain the majority of InsP3sensitive intracellular Ca2⫹ channels (15). Moreover, in an initial set of experiments, the agonist-induced increase in cytosolic Ca2⫹ was demonstrated to be concentration dependent. As shown in Fig. 4b, randomly selected acini from freshly prepared slices responded to application of 0.1, 0.3, and 1 M with sustained increases in intracellular Ca2⫹ concentration of 0.058 ⫾ 0.009, 0.128 ⫾ 0.014, and 0.162 ⫾ 0.016 ratio units, respectively (12 ⱕ n ⱕ 18). Corresponding responses for slices maintained for 24 h in culture were 0.083 ⫾ 0.012, 0.117 ⫾ 0.010, and 0.191 ⫾ 0.016 ratio units, respectively (15 ⱕ n ⱕ 18). 295 • SEPTEMBER 2008 •
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Fig. 4. Comparison of Ca2⫹ signals measured by fura 2 in freshly prepared slices (black) and slices maintained in culture for 24 h (gray). a: Histograms of resting ratio values in freshly prepared slices and slices cultured for 24 h. b: Concentration-dependent steady-state ratio increases in freshly prepared slices (solid symbols) and cultured slices (shaded symbols) in response to application of CCh (0.1–1 M). Brackets denote concentration. c: Examples of cytosolic Ca2⫹ changes evoked by 1 M CCh in single acinar cells from freshly prepared or 24-h cultured slices. au, Arbitrary units. d and e: Distributions of steady-state ratio changes and frequencies of oscillations (osc.) evoked by 1 M CCh. Inset demonstrates a lack of clear relationship between resting level and the plateau amplitude for either fresh or 24-h cultured slices. f: Agonist-induced decreases in acinus area in freshly prepared slices (control) and slices cultured for 24 h. IsoCh, cotreatment with isoproterenol and carbachol.
To assess more thoroughly the consequences on Ca2⫹ dynamics of culturing slices, we placed a region of interest on each cell observable in the fields of view to monitor their Ca2⫹ responses. We then compared both the steady-state (plateau) amplitude of the changes in Ca2⫹ as well as the Ca2⫹ oscillation frequencies, in response to application of 1 M CCh for freshly prepared and 24-h cultured slices. Representative fura 2 traces from individual acinar cells in freshly prepared slices or 24-h cultured slices following continuous bath application of CCh are shown in Fig. 4c. The average steady-state amplitude evoked in cells of freshly prepared slices was 0.129 ⫾ 0.005 ratio units (n ⫽ 179). In comparison, the average amplitude evoked in cultured slices was 0.162 ⫾ 0.004 ratio units (n ⫽ 204). On average, there was a significant elevation in the evoked change in Ca2⫹ in slices maintained in culture. As outlined above, the data were plotted as histograms to determine the distribution of responses. The plateau values for fresh and cultured slices were distributed with peaks at 0.110 and 0.130 ratio units, respectively, as shown in Fig. 4d. This analysis revealed that many of the cells in cultured slices retained the plateau characteristics we observed in freshly AJP-Gastrointest Liver Physiol • VOL
prepared slices. Thus, despite a broadening in the distribution profile for 24-h cultured slices, there was substantial overlap of the range of plateau values between freshly prepared and cultured slices. As shown in the inset, there was no obvious relationship between resting level and the plateau amplitude for either fresh or 24-h cultured slices. The frequency of oscillations, which has previously been shown in pancreatic acinar cells to be highly dependent on integrity of the acinar cell cytoskeleton and gap junction communication, was also used as an index of acinar cell function (32). The oscillation frequency in response to application of 1 M CCh was compared between freshly prepared and 24-h cultures slices. The average frequencies of oscillations were 10 ⫾ 0.5 oscillations/min (n ⫽ 179) and 7 ⫾ 0.4 oscillations/min (n ⫽ 204), respectively. These data indicated a significant reduction in the frequency of oscillations in slices maintained in short-term culture. When the responses from individual cells were plotted as histograms it was revealed that a greater number of cells in cultured slices failed to exhibit oscillations in response to agonist treatment (Fig. 2e). For cells that responded, however, the distribution of oscillation fre295 • SEPTEMBER 2008 •
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quencies for fresh and cultured slices generally overlapped. The overlap indicated that a substantial portion of acinar cells responded with oscillatory signatures that were similar to those induced in freshly prepared slices. Our data indicated that a substantial number of acinar cells in slices could retain functionally intact Ca2⫹ signaling machinery for nearly 2 days. Consistent with the principle that Ca2⫹ release is the dominant signal for fluid secretion, we also observed robust apparent volume changes in acini in freshly prepared and cultured slices. Relative changes in acinar area were estimated directly from the transmitted light images. This was achieved by circumscribing the basal border of an acinus and integrating area prior to and at 30 s following agonist application. As shown in Fig. 4f, the maximal decreases in area in response to 1 M CCh application for freshly prepared and cultured slices were 23.6 ⫾ 4.6% (n ⫽ 7) and 14.1 ⫾ 6.4%, respectively (n ⫽ 3). The changes were transient and the area typically returned to prestimulus levels in concert with recovery of the Ca2⫹ signal. Cotreatment with 1 M isoproterenol (Iso) produced similar maximal decreases in acini area in freshly prepared and cultured slices [24.5 ⫾ 4.4% (n ⫽ 8) and 10.7 ⫾ 2.6% (n ⫽ 6), respectively] whereas application of 1 M Iso alone did not evoke decreases in area. Agonist-Evoked Zymogen Granule Exocytosis The exocytotic fusion of zymogen granules and the release of protein and other cargo molecules into the lumen are critical for the production of saliva. To further test the utility of the slice preparation as a model to study parotid gland function, we evoked and characterized exocytotic secretion in slices. We previously demonstrated that CCh and isoproterenol are potent secretagogues for protein secretion in freshly prepared parotid acinar cell slices. As an additional testament to the viability of the slice preparation, stimulation of exocytosis was significantly more sensitive than in isolated parotid acinar cells. In the present study we used treatment with Ca2⫹ and cyclic AMP-raising agonists in combination with time-differentiated imaging analysis to track and assess the spatiotemporal properties of exocytotic activity evoked in slices that were freshly prepared or maintained in short-term culture. Individual acinar cell clusters, typically comprised of five to six cells, were imaged and exocytotic activity was monitored over a 6- to 8-min time period. The total numbers of exocytotic events were counted frame by frame prior to and during continuous application of cholinergic and/or adrenergic agonist. An example of an acinar cluster imaged in this manner is shown in Fig. 5a. In general, there was little exocytotic activity in slices that were not stimulated. The rate of exocytosis prior to application of agonist for freshly prepared and cultured slices was 0.74 ⫾ 0.13 events 䡠 cell⫺1 䡠 min⫺1 (n ⫽ 30) and 0.42 ⫾ 0.17 events䡠cell⫺1 䡠min⫺1 (n ⫽ 34), respectively. The data indicated that the resting rates of exocytosis were not significantly different between freshly prepared and cultured slices. Acini with a high basal rate of activity (⬎3 events䡠cell⫺1 䡠min⫺1) were assumed to be damaged and were excluded from subsequent analysis. Application of CCh, Iso, or CCh/Iso induced robust exocytotic responses in freshly prepared slices. As shown by plot in Fig. 5b, the average rates of exocytosis evoked by these challenges were 3.25 ⫾ 0.78, 2.49 ⫾ 0.52, and 5.62 ⫾ 1.20 events䡠cell⫺1 䡠min⫺1, respectively (6 ⱕ n ⱕ 9). When tested AJP-Gastrointest Liver Physiol • VOL
Fig. 5. Time-differentiated imaging was used to quantify the exocytotic activity of acinar clusters in slices. Transmitted light image (a) and corresponding time-differentiated images (b) depicting a small acinus. In this example, the consecutive images shown were generated by subtracting each frame by an average of the 5 preceding frames to visualize the spatial and temporal characteristics of the exocytotic events (circles) in response to continuous application of 1 M CCh. c: Bar graph comparing the rates of exocytosis in freshly prepared slices (control) and slices cultured for ⬃24 h. Exocytotic rates of individual acini were estimated by time-differentiated imaging following activation of exocytotic activity using 1 M CCh, 1 M isoproterenol (Iso), or coapplication of CCh and Iso. For estimation of rates, exocytotic activity was followed for 8.3 min. The same data was also replotted (symbols) to show the scatter of the rate values of the individual acini.
on slices that were cultured for 24 h, agonist treatment revealed that exocytotic function was largely retained but that activity was on average diminished. The rates of exocytosis evoked by CCh, Iso, or CCh/Iso challenges were 1.38 ⫾ 0.39, 1.58 ⫾ 0.22, and 3.50 ⫾ 0.69 events䡠cell⫺1 䡠min⫺1, respectively (5 ⱕ n ⱕ 9). However, the scatter of the data indicated that despite the reduced rate of exocytotic activity on average, a subpopulation of individual acini or acinar cells in cultured slices responded to agonist treatment with exocytotic rates and profiles that were indistinguishable from those of freshly prepared slices. As example, Fig. 6 shows that analysis of the spatial and kinetic profiles of fusion events in representative acini following cotreatment with 1 M CCh and Iso yielded similar results. Typically, the majority of the exocytotic fusion events mapped to granule-rich apical and lateral regions of individual acinar cells in both fresh and cultured slices. This is shown in Fig. 6, a and b, where masks indicating the sites of fusion events were 295 • SEPTEMBER 2008 •
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Fig. 6. Spatial and temporal dynamics of exocytosis using transmitted light microscopy and time-differentiated imaging analysis. DIC images of acini from freshly prepared (a) or 24-h cultured slices (b) were collected at 2 Hz. c: Corresponding maps and histograms of fusion events evoked by continuous application of 1 M CCh coapplied with 1 M Iso at 50 image frames are shown. d: Example of the kinetic profile of an individual cell (marked by white asterisk) of the acinus shown in b. This cell displayed the typical kinetic profile observed in fresh slices. Events per 20 image frames were binned and plotted as histograms.
placed on tracings of acinar clusters to produce exocytotic maps. Typically, agonist application induced an initial exocytotic “burst” followed by a phase of persistent activity. This burst was not universally evident, since individual cells of acini did not always activate in a synchronous fashion or display this characteristic. Figure 6d gives an example of the kinetic profile of an individual cell (marked by asterisk) of the acinus shown in b. This cell displayed the typical kinetic profile. This is demonstrated in the corresponding histograms plotted in Fig. 6, c and d. Two-Photon Excitation Imaging of Exocytosis in Parotid Gland Slices Sequential compound exocytosis has been visualized in real-time in rat pancreas (22) and guinea pig nasal gland acini (23) by two-photon microscopy. The deeper penetration and reduced scatter associated with two-photon microscopy has an
advantage over conventional confocal microscopy in visualizing apical membrane structure in acini. Therefore, we used two-photon microscopy as an alternative method to monitor individual exocytotic events in 200- to 400-m-thick parotid slices. Following immersion in physiological saline containing 0.5 mM SRB, a fluorescent fluid phase marker, detailed images of acinar structure in slices were readily made apparent. As shown in Fig. 7a, two-photon excitation at 830 nm revealed SRB fluorescence labeling of intercellular spaces. In most slices the fluorescent image remained stable over a time period of 10 –20 min. Following stimulation by addition of 10 M CCh to the bath solution, the formation of primary, secondary, and tertiary exocytotic fusions was visualized. This was achieved by rapid diffusional equilibrium of SRB with the lumen of secretory granules that fused to the plasma membrane or the membrane of previously fused granules. Granule fusions produced stable, long-lived structures that decorated the apical
Fig. 7. Spatial and temporal dynamics of exocytotic activity were visualized by 2-photon microscopy. Exocytotic events in parotid acini imaged by 2-photon excitation of the fluid phase dye marker, sulforhodamine B (SRB), added to the bath solution. SRB fluorescence was evident at intercellular spaces and solution surrounding acinar clusters. Granule fusions were visualized as bright long-lived fluorescent spots primarily at apical and lateral borders of acinar cells. Images revealed exocytotic activity at a single image plane of a 400 M thick slice.
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and lateral borders of acinar cells, consistent with that observed in pancreatic acini (31) and our previous data using timedifferentiated imaging analysis (8). Example of these structures are shown in Fig. 7b (arrows). Field Stimulation-Evoked Ca2⫹ Signals in Slices An advantage of the organotypic slice preparation is preservation of diversity and integration of signaling and function of the parotid gland at the supracellular level. Maintenance of the integrative relationships between specific tissue types in the organ allowed us to the study secretory function in a situation that more completely resembled the gland in vivo. For example, secretory activity in the parotid gland is largely regulated by autonomic nervous system control. Sympathetic and parasympathetic innervations via superior cervical and otic ganglia, respectively, terminate on the basal regions of the acinar cells. Although cholinergic stimulation is the primary signal for fluid secretion, a variety of studies have demonstrated that both sympathetic and parasympathetic stimulation can induce substantial protein secretion. We presumed that these connections were preserved in the slice. To demonstrate this we visualized the nerve fibers in the slice and gauged their spatial relationship to acini and ducts using confocal microscopy. As shown in Fig. 8, a–c, in a slice costained with TUJ, an antibody against neuron specific -tubulin (red signal) and Alexa Fluor-488 phallotoxin to visualize F-actin (green signal), there is extensive neural input within the slice preparation. The overlay of fluorescent images revealed that autonomic innervations essentially encapsulate each individual acinus. Innervation of the slice was comprised of sympathetic (Fig. 8d) and parasympathetic (Fig. 8e) input in roughly equal proportions as indicated by labeling with specific antisera for VAChT or TH as markers, respectively.
The preservation of the neural structures suggested that it would be possible to induce neurotransmitter release and subsequent activation of acinar and ductal cell receptor signaling. Field stimulation of slices (stimulus application at 20 Hz unless otherwise indicated) was used to test whether the remnant fibers were functional. As illustrated in Fig. 9, field stimulation of endogenous nerves with platinum electrodes placed in a perfusion chamber evoked a robust Ca2⫹ signal in the acinar cells of the slice. The temporal trace in Fig. 9d shows the profile of the Ca2⫹ signal from an individual acinar cell. These signals were largely inhibited by pretreatment with 10 mM atropine, indicating that they occurred as a result of muscarinic receptor stimulation (data not shown). These data are consistent with activation of parasympathetic input to the gland. We also demonstrated that field stimulation could be used to induce exocytotic activity in slices. An example is shown in Fig. 10. Adenovirus Infection in Slices Genetic studies to probe salivary gland function have largely been advanced through the use of transgenic animals or injection of adenovirus vectors into the parotid duct to introduce genes of interest. However, these experiments are relatively labor, time, and cost intensive. Overexpression, knockdown, or knockin studies are potentially useful approaches, but their use has been limited because exocrine acinar cells rapidly lose function when isolated in primary culture. Thus an in vitro system in which one could utilize optical and electrophysiological approaches in combination with genetic manipulation would provide a valuable tool for investigating salivary gland function. Thus these approaches are not suitable to maintain acinar cells in culture long enough for expression of potential genes of interest.
Fig. 8. Autonomic innervation in parotid slices visualized using confocal imaging. a: Lobule structure was visualized by actin labeled with Alexa Fluor 488-conjugated phallotoxin (green). b: Nerve fibers were labeled with TUJ antibody against a neuronspecific tubulin protein (red). c: Overlay indicates that individual acinar clusters appear to be encapsulated by autonomic nerve fibers. d and e: Labeling of sympathetic and parasympathetic innervation, respectively, using anti-tyrosine hydroxylase (red) or antivesicular acetylcholine transporter antisera (green). f: Sympathetic and parasympathetic nerve terminals appear to innervate the exocrine and vascular units of parotid lobules labeled by Alexa Fluor 633-conjugated phallotoxin (blue).
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Fig. 9. Ca2⫹ dynamics evoked by field stimulation of a mouse parotid slice. a: Fluorescence image of a parotid lobule following loading with 4 M fura 2-AM. b: Transmitted light image of the same slice. (Note shadow grid bars from nylon mesh used to hold slice in place during bath perifusion). c: Fluorescence image of an acinus from the depicted slice and corresponding pseudocolored images demonstrating Ca2⫹ signals evoked by application of field stimulation (FS; 9 V, 5-ms pulse train). d: Line trace representing the Ca2⫹ response recorded from an individual acinar cell marked by the red box in c.
The use of parotid slices, which can be cultured for up to 48 h and retain viability, potentially provides a system where exogenous genes of interest can be introduced and expressed to probe the molecular underpinnings of salivary gland physiology. To test this possibility, we used adenovirus infection to introduce a green fluorescent protein (GFP)-ribonuclease fusion protein construct known to target expression of fluorescent cargo to secretory granules. As shown in Fig. 11, slices can be infected with adenovirus to produce robust expression of exogenous protein within 16 h. As indicated by the fluorescent signal (b) and the overlay with the transmitted light image (c), the fusion protein appeared localized to secretory granules, indicating that synthesis and sorting of granule protein is maintained in cultured organotypic slices. The bright fluorescence indicated that the GFP signal was not appreciably quenched and suggested that the labeled intracellular compartments were not strongly acidic. (The fluorescence of GFP is stable from pH 6 to 10 but decreases at pH ⬍ 6; Ref. 25). The moderate acidity of zymogen secretory granules is not unexpected. Whereas the secretory granules of neuroendocrine cells such as chromaffin cells or pancreatic beta cells have an intragranule acidity ranging from pH 5 to 5.5 (13, 14), the intragranule acidity of zymogen granules, in contrast, is estimated at pH 6.5 (1). Thus we are confident that the observation of GFP fluorescence in zymogen granules in situ is not an indication of loss of intragranule pH homeostasis or unhealthy slices. These data underscore the potential usefulness of this preparation to study salivary gland function by overexpression methods. AJP-Gastrointest Liver Physiol • VOL
DISCUSSION
In this study, we demonstrated that the organotypic slice of the adult mouse parotid gland is a useful preparation to study acinar cell morphology and secretory function using a variety of optical approaches. There are several advantages of this approach over the enzymatic digestion of tissue and preparation of isolated cells. First, tissue slicing is relatively rapid, taking only tens of minutes following gland isolation, and obviates the need for time- and labor-intensive treatment with enzymes such as collagenase (with activity that can vary depending on lot) and mechanical dissociation of the tissue. Second, slicing not only preserves much of the normal three-dimensional associations between acinar, ductal, and myoepithelial cells that comprise the exocrine tissue, but associated connective tissue, autonomic nerve fibers, and microvasculature as well. In addition, 40- to 100-m-thick slices are well suited for imaging with standard bench-top microscopy techniques because they are typically one to four cell layers thick. Furthermore, the semi-intact nature of the organotypic slice allows access to study supracellular signaling and complex integrative functions over a variety of cell types in a more physiological context. For example, in slices acinar tissue is exposed as the connective tissue capsule that normally sheaths the tissue is removed en face. This enables efficient loading of cell-permeable dyes and access for fluid phase markers, drugs, or agonists. Importantly, slicing allows for the free exchange of gases and nutrients. Consistent with this notion, we observed that the polarized cell mor295 • SEPTEMBER 2008 •
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Fig. 10. Exocytotic secretory dynamics evoked by field stimulation of parotid slice. a: Transmitted light images demonstrating rapid and transient volume changes of an acinus in a parotid slice induced by field stimulus application (10 V, 5 ms, 20 Hz) as indicated by arrows. b: Examples of individual exocytotic events (circles) captured by time-differentiated imaging. c: Individual events were counted, binned, and plotted as a histogram.
phology of thin slices was maintained in short-term culture better than thick slices (⬎200 m). We reasoned that acinar cell function in thin slices in short-term culture might also be preserved. A significant advantage and step forward through the use of the slice preparation was the ability to maintain polarized morphology and secretory function of acinar cells out to 24 to 48 h in short-term organotypic culture. Modifications of the slice culture methods will likely extend slice viability and functionality. Although we did not optimize cell viability, there are a variety of strategies that can potentially improve long-term slice viability. Necrosis might be reduced through the use of a vibratome equipped with diamond knife rather than the featherweight razor blades used
for the present study. This would likely reduce tearing or mechanical damage of the tissue and improve cell viability. In some cases, transient heat shock of the tissue following slicing has also been shown to help maintain slice health. Culture conditions may be modified to reduce apoptosis (17). For example, toxins that promote activation of apoptosis such as ammonia or oxidants can be reduced by placing slices on substrate such as a permeable filter membrane, agitating medium, and addition of antioxidants to the medium. Similarly, addition of growth factors or low level, periodic electrical stimulation or addition of factors mimicking neural maintenance of the tissue could be included. We are currently testing these possibilities, as well as the application of this technique to other exocrine tissues. For
Fig. 11. Adenoviral infection of a parotid slice. Transmitted light (a) and fluorescence images (b) of a slice 16 h postinfection with GFP-ribonuclease fusion I protein construct that localizes to zymogen secretory granules. c: Overlay of fluorescence image with transmitted light image demonstrating that a punctate fluorescent signal is primarily seen in apical regions of acini. AJP-Gastrointest Liver Physiol • VOL
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example, we recently prepared viable thin slices of mouse submandibular gland and pancreas with only minimal modifications of the slice preparation methods. The ability to combine optical and electrophysiological methods with genetic manipulation in an in vitro model system should greatly facilitate investigations of fluid and protein secretion in the parotid and other exocrine glands. ACKNOWLEDGMENTS The authors thank Dr. Corey Smith for valuable advice in establishing the slice preparation, Dr. Peter Thorn for suggestions regarding the multiphoton studies, and Stefanie Brown for assistance with some of the experiments. We are grateful to Dr. Marthe Howard for providing the TUJ and TH antisera and Drs. John A. Williams and Xuequn Chen for the GFP-ribonuclease I fusion protein plasmid. Confocal, multiphoton, and electron microscope images were acquired by use of the resources of the Advanced Microscopy and Imaging Center at the University of Toledo Health Science Campus. GRANTS This work was supported by the National Institute of Dental and Craniofacial Research grant DE-014756 to D. I. Yule and D. R. Giovannucci. REFERENCES 1. Anderie I, Thevenod F. Evidence for involvement of a zymogen granule Na⫹/H⫹ exchanger in enzyme secretion from rat pancreatic acinar cells. J Membr Biol 152: 195–205, 1996. 2. Ann DK, Lin HH, Kousvelari E. Regulation of salivary-gland-specific gene expression. Crit Rev Oral Biol Med 8: 244 –252, 1997. 3. Bruce JI, Shuttleworth TJ, Giovannucci DR, Yule DI. Phosphorylation of inositol 1,4,5-trisphosphate receptors in parotid acinar cells. A mechanism for the synergistic effects of cAMP on Ca2⫹ signaling. J Biol Chem 277: 1340 –1348, 2002. 4. Campos-Toimil M, Edwardson JM, Thomas P. Acetylcholine-induced zymogen granule exocytosis: comparison between acini and single pancreatic acinar cells. Pancreas 24: 179 –183, 2002. 5. Castle D, Castle A. Intracellular transport and secretion of salivary proteins. Crit Rev Oral Biol Med 9: 4 –22, 1998. 6. Castle JD. Protein secretion by rat parotid acinar cells. Pathways and regulation. Ann NY Acad Sci 842: 115–124, 1998. 7. Chan SA, Polo-Parada L, Landmesser LT, Smith C. Adrenal chromaffin cells exhibit impaired granule trafficking in NCAM knockout mice. J Neurophysiol 94: 1037–1047, 2005. 8. Chen Y, Warner JD, Yule DI, Giovannucci DR. Spatiotemporal analysis of exocytosis in mouse parotid acinar cells. Am J Physiol Cell Physiol 289: C1209 –C1219, 2005. 9. Denny PC, Ball WD, Redman RS. Salivary glands: a paradigm for diversity of gland development. Crit Rev Oral Biol Med 8: 51–75, 1997. 10. Fujita-Yoshigaki J. Divergence and convergence in regulated exocytosis: the characteristics of cAMP-dependent enzyme secretion of parotid salivary acinar cells. Cell Signal 10: 371–375, 1998. 11. Fujita-Yoshigaki J, Tagashira A, Yoshigaki T, Furuyama S, Sugiya H. A primary culture of parotid acinar cells retaining capacity for agonistsinduced amylase secretion and generation of new secretory granules. Cell Tissue Res 320: 455– 464, 2005. 12. Giovannucci DR, Bruce JI, Straub SV, Arreola J, Sneyd J, Shuttleworth TJ, Yule DI. Cytosolic Ca2⫹ and Ca2⫹-activated Cl⫺ current dynamics: insights from two functionally distinct mouse exocrine cells. J Physiol 540: 469 – 484, 2002.
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13. Hutton JC. The internal pH and membrane potential of the insulinsecretory granule. Biochem J 204: 171–178, 1982. 14. Johnson RG, Scarpa A. Ion permeability of isolated chromaffin granules. J Gen Physiol 68: 601– 631, 1976. 15. 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. 16. Kidd JF, Thorn P. Intracellular Ca2⫹ and Cl⫺ channel activation in secretory cells. Annu Rev Physiol 62: 493–513, 2000. 17. Laken HA, Leonard MW. Understanding and modulating apoptosis in industrial cell culture. Curr Opin Biotechnol 12: 175–179, 2001. 18. Maruyama Y, Gallacher DV, Petersen OH. Voltage and Ca2⫹-activated K⫹ channel in baso-lateral acinar cell membranes of mammalian salivary glands. Nature 302: 827– 829, 1983. 19. Meneghel-Rozzo T, Rozzo A, Poppi L, Rupnik M. In vivo and in vitro development of mouse pancreatic beta-cells in organotypic slices. Cell Tissue Res 316: 295–303, 2004. 20. Murakami M, Yoshimura K, Segawa A, Loffredo F, Riva A. Relationship between amylase and fluid secretion in the isolated perfused whole parotid gland of the rat. Eur J Morphol 38: 243–247, 2000. 21. Nauntofte B. Regulation of electrolyte and fluid secretion in salivary acinar cells. Am J Physiol Gastrointest Liver Physiol 263: G823–G837, 1992. 22. Nemoto T, Kimura R, Ito K, Tachikawa A, Miyashita Y, Iino M, Kasai H. Sequential-replenishment mechanism of exocytosis in pancreatic acini. Nat Cell Biol 3: 253–258, 2001. 23. Oshima A, Kojima T, Dejima K, Hisa Y, Kasai H, Nemoto T. Two-photon microscopic analysis of acetylcholine-induced mucus secretion in guinea pig nasal glands. Cell Calcium 37: 349 –357, 2005. 24. Park MK, Lee M, Petersen OH. Morphological and functional changes of dissociated single pancreatic acinar cells: testing the suitability of the single cell as a model for exocytosis and calcium signaling. Cell Calcium 35: 367–379, 2004. 25. Patterson GH, Knobel SM, Sharif WD, Kain SR, Piston DW. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J 73: 2782–2790, 1997. 26. Pintelon I, De Proost I, Brouns I, Van Herck H, Van Genechten J, Van Meir F, Timmermans JP, Adriaensen D. Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di-2-ASP in various animal species. Cell Tissue Res 321: 21–33, 2005. 27. Sabatini BL, Regehr WG. Detecting changes in calcium influx which contribute to synaptic modulation in mammalian brain slice. Neuropharmacology 34: 1453–1467, 1995. 28. Segawa A, Loffredo F, Puxeddu R, Yamashina S, Testa Riva F, Riva A. Cell biology of human salivary secretion. Eur J Morphol 38: 237–241, 2000. 29. Soltoff SP, Grubman SA, Jefferson DM. Development of salivary gland cell lines for studies of signaling and physiology. Ann NY Acad Sci 842: 100 –107, 1998. 30. Takemura H, Yamashina S, Segawa A. Millisecond analyses of Ca2⫹ initiation sites evoked by muscarinic receptor stimulation in exocrine acinar cells. Biochem Biophys Res Commun 259: 656 – 660, 1999. 31. Thorn P, Fogarty KE, Parker I. Zymogen granule exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity. Proc Natl Acad Sci USA 101: 6774 – 6779, 2004. 32. Turvey MR, Fogarty KE, Thorn P. Inositol (1,4,5)-trisphosphate receptor links to filamentous actin are important for generating local Ca2⫹ signals in pancreatic acinar cells. J Cell Sci 118: 971–980, 2005. 33. Wiedemann HR. Salivary gland disorders and heredity. Am J Med Genet 68: 222–224, 1997. 34. Yasuda R, Nimchinsky EA, Scheuss V, Pologruto TA, Oertner TG, Sabatini BL, Svoboda K. Imaging calcium concentration dynamics in small neuronal compartments. Sci STKE 2004: pl5, 2004.
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