Mucous granule exocytosis and CFTR expression in gallbladder epithelium

Rahul Kuver1,2, Johanne Henriette Klinkspoor4, William R. A. Osborne3 and Sum P. Lee2,4

Departments of 2Medicine and 3Pediatrics, University of Washington School of Medicine and 4Department of Medicine, Veterans Affairs Puget Sound Health Care System, Seattle, WA 98195, USA

Received on May 8, 1999; revised on July 26, 1999; accepted on July 27, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
A mechanistic model of mucous granule exocytosis by columnar epithelial cells must take into account the unique physical-chemical properties of mucin glycoproteins and the resultant mucus gel. In particular, any model must explain the intracellular packaging and the kinetics of release of these large, heavily charged species. We studied mucous granule exocytosis in gallbladder epithelium, a model system for mucus secretion by columnar epithelial cells. Mucous granules released mucus by merocrine exocytosis in mouse gallbladder epithelium when examined by transmission electron microscopy. Spherules of secreted mucus larger than intracellular granules were noted on scanning electron microscopy. Electron probe microanalysis demonstrated increased calcium concentrations within mucous granules. Immunofluorescence microscopic studies revealed intracellular colocalization of mucins and the cystic fibrosis transmembrane conductance regulator (CFTR). Confocal laser immunofluorescence microscopy confirmed colocalization. These observations suggest that calcium in mucous secretory granules provides cationic shielding to keep mucus tightly packed. The data also suggests CFTR chloride channels are present in granule membranes. These observations support a model in which influx of chloride ions into the granule disrupts cationic shielding, leading to rapid swelling, exocytosis and hydration of mucus. Such a model explains the physical-chemical mechanisms involved in mucous granule exocytosis.

Key words: calcium/chloride channel/cystic fibrosis/mucin


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
All columnar epithelial cells are lined by a blanket of mucus. (The term mucus is used to indicate the native, physiological gel-like secretion. Mucins are the main component of mucus and its use implies (biochemically) a species of highly pure glycoprotein and is synonymous with mucous glycoprotein. Mucous is used as an adjective to indicate relatedness to mucus or mucin, such as in mucous secretory granules, mucous (secretory) cells, or mucous glycoprotein. The term mucinous is not used, and its use is discouraged.)

This thin mucus coat separates the host mucosal cells from the exterior milieu. The major constituents of mucus are mucins, glycoproteins with oligosaccharides attached via O-glycosidic bonds to serine or threonine residues on peptide backbones. Mucus is also a highly hydrated gel (95–98% water) and contains a net negative charge due to sialic acid and sulfate residues. In physical-chemical terms, it is a negatively charged hydrogel with the matrix tangled in a randomly woven polyionic network.

There is evidence that mucus is not an inert substance. Physiologically, it may mediate "cytoprotection," as exemplified in the stomach mucosa; regulate the diffusion and direction of ion fluxes across the mucosa; and determine the pathogenicity of microbial organisms. Little is known about the control of mucus secretion. In the intestines, neural and humoral factors, as well as toxins, induce mucus secretion (Forstner et al., 1981Go; Specian and Neutra, 1982Go). In the gallbladder, where mucus hypersecretion is an early and consistent accompaniment in gallstone formation, and where mucus is believed to contribute to stone formation, mucus secretion is hypothesized to be triggered by chemical mediators (LaMorte et al., 1986Go). These studies have focused on mucus secretion at an organ or tissue level. The intracellular events involved in mucus secretion, including the signal transduction pathways involved, have begun to be explored (Forstner, 1995Go).

Regardless of the trigger signal, any model of the molecular mechanism of mucus secretion must account for the observation that within the cell, the mucous granule is extremely tightly packed and condensed, usually contains an excess negative charge, and is relatively underhydrated. Verdugo et al., studying the mucous granule of the giant slug Ariolimax columbianus, observed a marked elevation in calcium concentration within the granule and postulated that calcium acts as a neutralizing cation (Verdugo et al., 1987Go). We hypothesize that the net negative charge in the gallbladder epithelial cell mucous granule is also shielded or neutralized by binding to cations; we further hypothesize that, similar to the giant slug mucous granule, a sudden unshielding of intragranular calcium leads to the expansion of the mucus network. Unshielding may be mediated by calcium channels found on the granule membrane, as originally proposed (Verdugo et al., 1987Go). Alternatively, chloride channels may provide the unshielding mechanism. Whatever the mechanism, the unshielding of cations would drive exocytosis and hydration of the mucous granule to form the mucus gel.

Since secretion of fluid, electrolytes and mucin from biliary epithelial cells can be mediated by a cAMP-dependent pathway (Igimi et al., 1992Go; Lenzen et al., 1992Go; Kuver et al., 1994Go), and since the cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel, we sought to localize CFTR in gallbladder epithelial cells. Indeed, in human gallbladder epithelium, CFTR has been demonstrated in the apical membrane (Dray-Charier et al., 1995Go). CFTR may play a role in the mucin secretory pathway in epithelial cells. Gallbladder epithelial cells that overexpress CFTR via retroviral transduction demonstrated increased mucin secretion (Kuver et al., 1994Go). In rat submandibular acini, CFTR antibodies attenuated ß-adrenergic-stimulated mucin secretion (Mills et al., 1992Go). In human tracheal epithelial cells, protein kinase A–stimulated glycoconjugate release was defective in CF cells, and was corrected with adenoviral-mediated CFTR gene transfer (Mergey et al., 1995Go). ATP-dependent mucin secretion by cystic fibrosis pancreatic epithelial cells is defective (Montserrat et al., 1996Go). Taken together, these studies suggest a role for CFTR in mucin secretion, although none of these studies addressed whether the exo­cytotic process itself is the site of CFTR involvement.

The present study was designed to examine intracellular mucous granules in gallbladder epithelium, their cationic content and spatial relationship to CFTR. The objective was to provide evidence for a mechanism of mucous granule exo­cytosis in gallbladder epithelial cells that took into account the unique physical chemical properties of mucus.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Transmission electron microscopy (TEM) studies showed the columnar epithelial cells of the mouse gallbladder contained mucous secretory granules (Figure 1) and these were actively secreted into the lumen of the gallbladder. The intracellular secretory granules were 0.16 ± 0.04 µm in diameter (mean ± SD, n = 20, range 0.1–0.25 µm), and were crowded in the subapical membrane just before secretion. The membrane of the secretory granule then fused with the apical membrane. By way of a breach of the fused membrane, the contents of the mucous granules were released by exocytosis to the exterior of the cell. The phenomenon could be best described as a "merocrine" mechanism of secretion (Lee, 1980Go). Other detailed fine structures of the mouse gallbladder epithelial cell have been described elsewhere (Yamada, 1968Go; Lee and Scott, 1982Go). A continuous mucus blanket was not seen by TEM, most probably because of artifactual disruption of the native mucus layer during fixation and sectioning. However, occasionally cells which had their intracellular mucous granules released extracellularly could be seen with the contents expanded to acquire the form of a spherical mass. We have observed here that not only specialized mucus secreting apparatus such as goblet cells and glands, but also simple columnar epithelial cells of the mouse gallbladder epithelium, can actively secrete mucus.



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Fig. 1. Transmission electron microscopy showing active mucus secretion. (a) Intracellular mucous granules, ranging from 0.1–0.25 µm in diameter are present in the subapical region of the epithelial cell. Some of these granules are immediately adjacent to the apical plasma membrane (arrows). (b) By a breach of the continuity of the fused membrane (secretory granule-apical membrane), the contents of the granule (G) are discharged into the gallbladder lumen. (c) Some of the extracellular discharged contents (M) assume a spherical structure with dimensions many times that of the intracellular granules. All sectionsx13,300.

 
Scanning electron microscopy (SEM) studies with antibody fixation resulted in consistent preservation of the mucus blanket which covered and obscured mucosal details such as the microvillous membrane (Figure 2b). However, the undulations of folds and valleys could still be discerned. If the mucus layer was carefully dissected and lifted under a dissecting microscope before SEM studies, spherical granules were easily observed tangled amongst the microvilli (Figure 2c). These spherules ranged from 0.25 to 0.95 µm (0.48 ± 0.22 µm; mean ± SD; n = 20), and coalesced to form a plaque which annealed with the mucus blanket. In contrast, in mouse gallbladders not fixed with anti-mucus antibodies, the mucus layer and spherules of mucus were not well seen (Figure 2a).



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Fig. 2. Scanning electron microscopy showing the appearance of secreted mucus in the mouse gallbladder. (a) Gallbladder sample prepared in conventional way, showing apical cell membrane, with microvillous projections; 2375x. (b) Gallbladder preparation preincubated with anti-mucus antibody showing preservation of the mucus blanket. This provides a continuous layer covering the apical membrane. The topographic features of an unfilled gallbladder with its "folds and valleys" can still be discerned; 2375x. (c) By lifting the layer of mucus blanket, spheres of mucus can be seen tangled within the microvillous filaments. They range from 0.25–0.95 µm in diameter; 6175x.

 
Electron probe microanalysis of freeze-dried cryosections of the same mouse tissue revealed a striking accumulation of calcium in the intracellular secretory granules (21 ± 13 mmol/kg dry mass; n = 9) compared with the calcium contents in the intracellular cytoplasm (6 ± 2 mmol/kg dry mass; n = 6, p < 0.02, Student’s unpaired t test). Larger perinuclear electron dense mitochondria also had much lower calcium levels (3 ± 3 mmol/kg dry mass, n = 5, p < 0.001 compared with mucous granules, Student’s unpaired t test). Nuclear calcium was 1.0 ± 2.3 mmol/kg dry mass; n = 5, p < 0.01 compared with mucous granules, Student’s unpaired t test.

With the availability of a well-differentiated canine gallbladder epithelial cell culture system (Oda et al., 1991Go), studies were undertaken to determine whether mucin granules colo­calized with an anion channel. CFTR was chosen as a representative anion channel that may play a role in mucous granule exocytosis as suggested by several studies (Mills et al., 1992Go; Kuver et al., 1994Go; Mergey et al., 1995Go; Montserrat et al., 1996Go).

We performed fluorescence immunocytochemistry using a well-characterized CFTR C-terminal murine monoclonal antibody and a dog gallbladder mucin rabbit polyclonal antibody. Negative controls were performed with combinations of primary and secondary antibodies in order to ensure no cross-reactivity existed between primary and/or secondary antibodies (not shown). In addition, negative controls with the filters used (red for TRITC, green for FITC) showed no cross-reactivity.

Additional controls were performed to ensure lack of cross-reactivity between the mucin and CFTR antibodies. Mucins, purified from dog gallbladder bile and culture media of the dog gallbladder epithelial cells, were subjected to SDS–PAGE and Western blotting. Whereas the mucin polyclonal antibody showed a specific staining reaction with the purified mucins, no staining was observed with the CFTR monoclonal antibody (data not shown). Specifically, staining of high molecular weight proteins, which migrated in the stacking and upper part of the separating gel, was observed, consistent with immunostaining for mucin. However, no staining was noted in the molecular weight range expected for CFTR (140–180 kDa). No staining of mucin was seen with the preimmune serum. Also, western blotting of purified dog gallbladder mucin using the anti-mucin antibody did not show low-molecular weight bands, indicating specificity of this antibody for high mole­cular weight mucins. Finally, the mucin antibody showed reactivity for mucins after periodate oxidation using an ELISA assay, indicating that reactivity was not lost following cleavage of carbohydrate groups (data not shown).

Dog gallbladder epithelial cells stained extensively for intracellular mucin using the specific dog gallbladder mucin antibody (Figure 3, red). Intracellular CFTR staining was seen with the murine monoclonal CFTR antibody (Fig 3, green). Computer-assisted digitization of the immunofluorescence images was performed in order to demonstrate colocalization with fusion of pseudocolors. The merged images on the same fields (with the areas of colocalization shown in yellow) indicated intracellular granule colocalization of mucin and CFTR in these cells.



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Fig. 3. Digitized images of immunofluorescence cytochemical studies on subconfluent dog gallbladder epithelial cells grown on glass coverslips showing intracellular colocalization of CFTR and mucin. Staining with the rabbit polyclonal anti-mucin antibody and the mouse monoclonal anti-CFTR antibody followed by incubation with the two secondary antibodies performed sequentially on the same slide showed specific staining for mucin granules in the majority of cells, as well as staining with the CFTR antibody. Shown are representative slides. CFTR signal (green pseudocolor) corresponding to FITC filter. Mucin signal (red pseudocolor) corresponding to TRITC filter. Composite image analysis (yellow pseudocolor) showing areas of colocalization (87x).

 
We also performed fluorescence immunocytochemistry on dog gallbladder epithelial cells transduced with the retroviral vector LCFSN. These cells overexpress CFTR, and demonstrate increased constitutive mucin secretion (Kuver et al., 1994Go). In addition, cells transduced with the control retroviral vector LNL6 which contains the neomycin phosphotransferase gene (Kuver et al., 1994Go) did not show increased CFTR signal on immunofluorescence studies (not shown). As shown in Figure 4, cells overexpressing CFTR showed a corresponding increase in the CFTR signal (compare Figure 3 and 4, green pseudocolor images). Furthermore, colocalization of mucin and CFTR in LCFSN-transduced cells is clearly demonstrated using digitized images and fusion of pseudocolors (Figure 4, yellow). In these images, colocalization appears nearly complete for CFTR staining granules, although the mucin signal was more pronounced in the cells.



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Fig. 4. Digitized images of immunofluorescence cytochemical studies on subconfluent dog gallbladder epithelial cells overexpressing CFTR grown on glass coverslips. Staining and digitized pseudocolor analysis were performed as described for Figure 3. CFTR signal (green pseudocolor). Mucin signal (red pseudocolor). Composite signal (yellow pseudocolor; 870x).

 
In order to increase the resolution of these images and thereby more convincingly demonstrate colocalization, CFTR-overexpressing dog gallbladder epithelial cells were stained with CFTR and mucin antibodies, then scanned using fluorescence filters with laser confocal microscopy at 0.2–0.5 µm intervals. As shown in Figure 5, the fusion of the red (mucin) and green (CFTR) signals to give the yellow color is clearly demonstrated in intracellular granular structures. A strong mucin signal is noted on the plasma membrane and intra­cellular regions. Similar images were obtained with untransduced dog gallbladder epithelial cells double-labeled with mucin and CFTR antibodies. Within cells overexpressing CFTR, more granules stained with the mucin antibody than with the CFTR antibody.



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Fig. 5. Confocal laser scanning immunofluorescence microscopy of subconfluent dog gallbladder epithelial cells overexpressing CFTR. Shown is a representative sample with CFTR signal in green, mucin signal in red and composite signal in yellow (1920x).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The molecular mechanisms that allow mucus to be densely packaged, stored and released from cells are not well known. How, once outside the cell, the granular contents rapidly become a hydrated gel with many times its intracellular water content and volume is equally unclear. Mucous granules are discharged into the mouse gallbladder lumen (Lee, 1980Go; Axelsson et al., 1979Go). We have extended this observation by showing the granules were discharged as a "merocrine" secretion, existing as spherules many times larger than their intra­cellular dimensions. Nevertheless, such morphologic observations were made at only one point in time of a complex dynamic process. Verdugo used video enhanced microscopy to examine exocytosis of rabbit tracheal mucous cells and recorded the dimension of the mucous spherules at millisecond intervals (Verdugo, 1984Go). A dramatic rapid expansion in the radius of these spherules was observed. The swelling behavior followed first order kinetics conforming to a Donnan equilibrium process. That is, the rate of swelling was dependent on the pH and ionic concentration in the extracellular gel medium (Verdugo, 1984Go).

Such a rapid explosive swelling cannot be due to simple diffusion of water into the condensed mass of mucin molecules, and dilution to form a gel. The huge molecular weight of mucins (2–10 x 106) would mean hours before any passive diffusion would reach equilibrium. The sudden expansion of the intracellular mucous granule after its release from the cell must mean that there is intrinsic energy stored within the granule, with exocytosis releasing this endogenously stored force (Verdugo et al., 1987Go). This concept, and the subsequent swelling characteristics, can be explained by the observation that mucins contain net negative charges which repulse and cause the gel to swell as predicted by the Donnan equilibrium (Verdugo, 1984Go).

Two other findings in the present study are relevant in considering mucin exocytosis. One is the selective entrapment of calcium within the secretory granules; the other is the intracellular colocalization of CFTR chloride channels with mucous granules. To maintain electrical neutrality, the negative charges of mucins inside the granule must be neutralized or shielded. Our observation of elevated calcium in the mucous secretory granules is consistent with the idea that calcium provides the cationic shielding mechanism (Nicaise et al., 1992Go). The mucous granule in the gallbladder epithelium is not the only intracellular product that uses cationic shielding. The shielding species can be a protein, such as the spermine in DNA which neutralizes negative charges on phosphate groups (Marquet et al., 1985Go). Elevated calcium has been recorded in secretory granules of cells in the salivary duct, labial glands and the pancreas (Roomans et al., 1982Go; Sasake et al., 1983Go; Izutsu et al., 1985Go). In mucous secretory granules of the terrestrial slug Ariolimax columbianus, an even more marked concentration of calcium exists (Verdugo et al., 1987Go). There is a sudden release of calcium from the mucous secretory granules which occurs immediately preceding the release of granule contents, a step which is often associated with fusion of the membrane of the secretory granule with the apical plasma membrane (Zimmemberg and Whitaker, 1985Go). In some tissues such as the chicken trachea, calcium is co-secreted with mucus (Kent and Mian, 1987Go).

A malfunction of CFTR (cystic fibrosis) will result in a relative deficiency of fluid and electrolytes and thereby not allow proper hydration of the exocytosed mucus. This effect may be enhanced by the sodium hyperabsorption characteristic of CF cells resulting from abnormal regulation of an epithelial sodium channel by CFTR (Stutts et al., 1995Go). Alternatively, if CFTR functions on the mucous granule membrane as the trigger for mucus release, then absent or defective CFTR may lead to unregulated mucus release, contributing to the mucus hypersecretion characteristic of CF. In CF, the secreted mucus remains as a highly condensed tangled polyionic network and manifests itself as highly viscous "inspissated" mucus. This speculation can be examined by concomitantly testing CFTR function with respect to Cl, fluid, and mucus secretion.

For immunofluorescence studies, dog gallbladder epithelial cells were grown on glass coverslips. No significant plasma membrane CFTR staining was noted. This finding may have several explanations. As culture conditions did not allow differentiation into apical and basolateral membrane domains, membrane localization of CFTR may have been affected. Targeting of CFTR to the apical plasma membrane is linked to polarization of cultured human pancreatic duct cells, and a similar phenomenon may occur with cultured gallbladder epithelial cells (Hollande et al., 1998Go). Alternatively, in these cells under these culture conditions, CFTR may be present in low copy number in unstimulated cells on the plasma membrane. Stimulation with agonists that elevate intracellular cAMP may be required to allow trafficking of CFTR from intracellular vesicles. The combination of lack of polarization and lack of CFTR stimulation may therefore account for the absence of plasma membrane CFTR staining, as reported for other cultured epithelial cells (Morris et al., 1994Go).

The question of cross-reactivity between the CFTR and mucin antibodies was rigorously addressed in light of the colocalization seen with immunofluorescence studies. Several controls were used to ensure lack of cross-reactivity. First, no cross-reactivity between the primary and secondary antibodies was noted, nor was there signal crossover between the two filters used. Second, Western blotting of purified dog gallbladder mucin with the mucin antibody showed the expected high molecular weight bands, which were clearly distinguished from the molecular weight regions expected for CFTR. Finally, the CFTR-overexpressing cells showed an increase in staining for CFTR, but no increase in intracellular mucin staining. While the latter finding supports the lack of cross-reactivity between the two antibodies, this seems to contradict our earlier observation that CFTR-overexpressing cells showed increased mucin secretion (Kuver et al., 1994Go). One explanation for this discrepancy is that while the rate of mucin secretion was increased in CFTR-overexpressing cells, the sum of intracellular mucin stores was not significantly different. These findings, however, deserve further investigation.

Chloride channels are functionally involved in the regulation of exocytosis (Gasser et al., 1988Go) and chloride channels and G proteins have been found in the membrane of secretory granules (Gasser et al., 1988Go; Watson et al., 1992Go). In intestinal, salivary and biliary epithelium, both Cl (encompassing Na+ and fluid) secretion as well as mucus secretion can be concomitantly driven by a cAMP-dependent pathway (Igimi et al., 1992Go; Fitz et al., 1993Go; Kuver et al., 1994Go). This simultaneous response to a common biochemical intracellular signal transduction pathway is appropriate physical-chemically since abundant fluid must accompany mucus exocytosis so that extracellular mucus can be hydrated. Cholera toxin, for example, stimulates copious intestinal secretion of fluid and chloride ions, as well as mucin (Forstner et al., 1981Go).

One hypothesis to explain these observations is that in mucus exocytosis, the initial event is Cl moving into the granule from the cytoplasmic compartment. This event may coincide with changes mediated by the family of proteins which controls vesicular docking and membrane fusion (Naren et al., 1997Go; Low et al., 1998Go; Schimmoller et al., 1998Go). Swelling due to this ionic movement leads to the expulsion of the granule. Cationic shielding by calcium explains how mucus can be condensed and stored in an intracellular granule. This hypothesis also describes a common intracellular pathway in the control of mucus secretion, whether this is initiated by chemical mediators (LaMorte et al., 1986Go), by adrenergic, peptidergic (Fleming et al., 1987Go), or cholinergic (Forstner et al., 1981Go; Specian and Neutra, 1982Go) mechanisms. That the mucous granule, in the form of a tightly packed and condensed network of polyions, once released of its cationic shielding, will rapidly expand according to a Donnan equilibrium process (Verdugo et al., 1987Go; Nanavati and Fernandez, 1993Go; Dray-Charier et al., 1995Go) and can be immediately hydrated with the optimum amount of extracellular fluid is also predicted by this hypothesis.

Functional data for chloride channel involvement in mucous granule exocytosis is beyond the scope of this report. Others have shown the presence of functional ionic channels on zymogen granules from pancreas and salivary gland (Gasser et al., 1988Go; Gasser and Hopfer, 1990Go). Characterization of these channels on granule membranes has been carried out (Thevenod et al., 1990Go). In order to perform similar studies on mucous granules, methods for isolating intact and stable mucous granules will need to be developed. Ongoing studies address the feasibility of isolating such intact mucous granules from columnar epithelial cells. The ultimate proof of a role for CFTR or another anion channel in mucous granule exocytosis will come from such functional studies.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
All procedures involving animals were approved by the institutional animal use committee, and received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health.

Transmission electron microscopy (TEM)
Male inbred pathogen-free albino mice weighing 20–30 g were used. They were housed four to a stainless steel cage with wire mesh bottom to prevent coprophagia. The cages were kept in a laboratory equipped with an automatic diurnal lighting system and at a temperature of 21 ± 2.0°C. Animals were fed a regular Purina Chow diet and were euthanized at 9:00–10:00 a.m. without prior fasting by cervical dislocation. Mouse gallbladders (n = 20) were immediately removed, opened and carefully washed with 0.15 N saline, cut into 2 mm sections and rapidly transferred to 2.5% glutaraldehyde at 4°C for fixation. These were post-fixed in osmic acid and dehydrated in graded ethanol. They were oriented and embedded in Epon 812, sectioned on a Reichert ultratome, stained with 50% methanolic uranyl acetate and Reynolds lead acetate, and studied with a Philips EM 300 electron microscope as previously described (Lee and Scott, 1982Go).

Scanning electron microscopy (SEM)
The relationship between columnar epithelial cells and the adjacent mucus coat has not been studied adequately by SEM because routine preparative techniques including fixation and dehydration leave the mucus as condensed and disrupted strands. In the rat colon, we have found that pretreatment of specimens with anti-mucus antibodies reliably preserved the mucus in relation to the epithelium (Bollard et al., 1986Go).

Cholecystectomies were performed on 35 mice and gallbladder mucus was collected by scraping away the native mucus gel with a glass slide. Pooled mucus was homo­genized in nine volumes of phosphate-buffered saline and stored at –70°C until used in the immunization schedule in rabbits. Phosphate-buffered saline (0.5 ml) containing 100 µg of protein was emulsified with 1.5 ml of Freund’s complete adjuvant using two syringes and a double hub connector. This emulsion was injected subcutaneously at eight sites on both sides of the rabbit’s spine.

Four weeks thereafter rabbits were given a booster injection (0.5 ml) by the same technique. The animals were bled (10–20 ml) at 7 days after the booster injection and the serum separated by centrifugation. Antibody activity was confirmed using the tube precipitin test, double diffusion in agarose, and binding to immobilized mucus on cellulose nitrate membranes. The serum of the highest titer (1:100) was used throughout. Complement activity in the sera was removed either by adding solid Na2EDTA to a final concentration of 10 mM or by incubation at 56°C for 30 min.

Laparotomies were performed on mice (n = 20) and cholecystectomies performed after in vivo perfusion–fixation using 4% glutaraldehyde (Bollard et al., 1986Go). Mouse gallbladders were opened, washed and oriented on a wax impregnated mesh that was then pinned onto a wax petri dish. The mucus antibody was added to cover the specimen and incubated at 4°C for 30 min to allow antibody–mucus complexing. Samples were immersed in 4% glutaraldehyde for 16 h and subjected to critical point drying. Control samples (n = 20) without antibody pretreatment were also used. The dried tissue specimens, mounted on aluminum stubs with copper conducting paint, were coated with carbon, then gold by vacuum evaporation before examination in an ISI DS-130 research scanning electron microscope fitted with a Robinson back scattered electron detector (Bollard et al., 1986Go).

X-Ray elemental microanalysis
Immediately after removal, gallbladders (n = 4) were opened and cut into 2 mm squares, then plunged rapidly into freon slush at –160 to –180°C. We obtained cryosections at –110°C by cutting the tissue, glued with toluene to metal chucks, using a Sorvall MT2B ultramicrotome with FS 1000 cryokit. Thin cryosections were then transferred dry to carbon-coated Formvar support films on folding grids and freeze-dried in an oil-free vacuum system.

Freeze-dried cryosections were viewed and analyzed in STEM mode in a JEOL 1200 EX electron microscope. X-Ray spectra were collected and processed using a Link AN 10,000 and 30 mm2 detector. Quantitation procedures which have been described elsewhere (Izutsu et al., 1985Go) make use of protein and salt standards (Shuman et al., 1976Go). Square rasters were placed over regions of granules (200 x 200 nm), cytosol (600 x 600 nm) and nuclei (1 µm x 1 µm). Electron dense structures assumed to be secretory granules were identified on the basis of their size and location (apical to nucleus) in the cells. Larger perinuclear electron dense mitochondria were also studied. Spectra were collected from samples at room temperature and corrections were made for beam-induced mass loss (Cantino et al., 1986Go).

Fluorescence immunocytochemistry
Mucin purification.
Gallbladder bile from a mongrel dog was obtained during surgery by aspiration of the gallbladder by needle puncture and frozen at –20°C. Purification of mucin from the gallbladder bile was performed as described previously (Smith, 1987Go), with slight modifications. Briefly, 5 ml of bile was saturated with ammonium sulfate. After incubation at 4°C for 16 h, a floating layer of lipids and mucin was removed and dissolved in PBS, containing 20 mM sodium cholate and 0.02% sodium azide, pH 7.4. Mucin was purified by repeated preparative cesium chloride (CsCl) equilibrium-density-gradient ultracentrifugation. After Amicon filtration and lyophilization, the purified dog gallbladder mucin was dissolved in 0.5 ml of PBS. The purified mucin was subjected to SDS–polyacrylamide gel electrophoresis. Staining with Coomassie brilliant blue for total protein and periodic acid–Schiff for sugar residues demonstrated that the preparation was free from low-molecular weight contaminants and contained only high-molecular weight glycoproteins.

Preparation of polyclonal antibody to dog gallbladder mucin.
A New Zealand White rabbit was injected subcutaneously with 100 µg of mucin purified from dog gallbladder bile in complete Freund’s adjuvant. At 15 and 36 days, this was repeated with injection of 50 µg of mucin in Freund’s incomplete adjuvant. On day 68 a final injection of 50 µg of mucin in incomplete adjuvant was given and after 10 days 20 ml of serum was collected and stored at –20°C. Preimmune serum was obtained before the first immunization. The reactivity of the polyclonal antibody with the purified dog gallbladder mucin was tested after SDS–PAGE and Western blotting. The antibody was shown to give a specific staining reaction with the purified mucin, whereas no staining was seen with the preimmune serum.

Immunofluorescence studies.
Normal dog gallbladder epithelial cells and dog gallbladder epithelial cells overexpressing CFTR were cultured on glass coverslips coated with 2.5 µg/cm2 rat tail collagen type I to approximately 50% confluence. CFTR-overexpression was achieved via retroviral transduction with the vector LCFSN encoding the human CFTR cDNA driven by the retroviral LTR promoter, as described previously (Kuver et al., 1994Go). Cells were washed once with PBS, and fixed in cold methanol (-20°C) for 5 min, followed by cold acetone for 5 min (-20°C). The cells were washed with 70% ETOH in PBS/Tween-20 (0.05%, v/v) then incubated with mouse CFTR C-terminal monoclonal antibody (Genzyme) at 1:50 dilution in PBS/Tween-20/10% normal goat serum (NGS) for 60 min. The rabbit mucin antibody was applied at 1:50 dilution in PBS/Tween-20/NGS for 60 min. Secondary antibodies were goat-anti-rabbit-tetramethylrhodamine B isothiocyanate (TRITC) and goat anti-mouse-fluorescein isothiocyanate (FITC) (Molecular Probes, Eugene, OR), each applied at 1:50 dilution for 60 min. The slides were washed three times with PBS/Tween-20 and sealed in mounting media containing p-phenylenediamine.

The cells were examined using epifluorescence microscopy, utilizing standard filter sets to visualize rhodamine and fluorescein fluorochromes. Photographs were taken on Kodak Ektachrome PRD200X film, using a Zeiss Axiovert 135 fluorescence microscope (Oberkochen, Germany), and a Contax 167 MT camera (Tokyo, Japan). The cells were also examined using a Zeiss Axiophot fluorescence microscope. Images were captured using a Hamamatsu C4880 fast-cooled integrating CCD camera and MCID M2 software (Imaging Research, St. Catherines, Ontario, Canada). Images from the same group of cells, but using different excitation filters were digitized into separate channels, and assigned either a red (mucin) or a green (CFTR) pseudocolor. The channels were subsequently merged into a single image, using the software’s image fusion function, resulting in the simultaneous visualization of two signals in the same group of cells. The same fields were fused resulting in visualization of areas of colocalization.

Confocal laser scanning immunofluorescence microscopy
Samples were prepared as described above. Confocal images were acquired on a Bio-Rad MRC-1024 fitted to a Nikon Diaphot 200 fluorescence microscope. Samples were illuminated with all lines of a krypton/argon laser and scanned at intervals of 0.2–0.5 µm using the 60x or 100x oil immersion objective.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Su Wan Chen, Manuel Villalon, Marie Cantino, Marilyn Skelly and Andrew Shirk for valuable technical assistance; Dr. Andy McShea for confocal laser scanning fluorescence microscopy assistance; John Breiniger for fluorescence immunocytochemistry assistance; and Drs. Kenneth Izutsu and Pedro Verdugo for helpful discussions. NIH RO1 DK 50246, Medical Research Service of the Department of Veterans Affairs, NIH CF Core Grant DK 47754, and Cystic Fibrosis Foundation Post-doctoral Fellowship.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CFTR, cystic fibrosis transmembrane conductance regulator; FITC, fluorescein isothiocyanate; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TRITC, tetramethylrhodamine B isothiocyanate.


    Footnotes
 
1 To whom correspondence should be addressed at: Division of Gastroenterology, Box 356424, University of Washington, Seattle, WA 98195 Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Axelsson,H., Danielsson,R., Henriksson,T. and Wahlin,T. (1979) Secretory behavior and ultrastructural changes in mouse gallbladder principal cells after stimulation with cholinergic drugs. Gastroenterology, 76, 335–340.[Medline]

Bollard,J., Vanderwee,M., Smith,G., Tasman-Jones,C., Gavin,J. and Lee,S. (1986) Preservation of mucus in situ in rat colon. Dig. Dis. Sci., 31, 1338–1344.[ISI][Medline]

Cantino,M., Wilkinson,L., Goddard,M. and Johnson,D. (1986) Beam induced mass loss in high resolution biological microanalysis. J. Microsc., 144, 317–327.[ISI][Medline]

Dray-Charier,N., Paul,A., Veissiere,D., Mergey,M., Scoazec,J.-Y., Capeau,J., Brahimi-Horn,C. and Housset,C. (1995) Expression of cystic fibrosis transmembrane conductance regulator in human gallbladder epithelial cells. Lab. Invest., 73, 828–836.[ISI][Medline]

Fitz,J., Basavappa,S., McGill,J., Melhus,O. and Cohn,J. (1993) Regulation of membrane chloride currents in rat bile duct epithelial cells. J. Clin. Invest., 91, 319–328.[ISI][Medline]

Fleming,N., Bilan,P., Slivinski-Lis,E. and Carvalho,V. (1987) Muscarinic, 1-{alpha} adrenergic and peptidergic agonists stimulate phosphoinositide hydrolysis and regulate mucin secretion in rat submandibular gland cells. Eur. J. Physiol., 409, 416–421.[ISI][Medline]

Forstner,G. (1995) Signal transduction, packaging and secretion of mucins. Annu. Rev. Physiol., 57, 585–605.[ISI][Medline]

Forstner,J., Roomi,N., Fahim,R. and Forstner,G. (1981) Cholera toxin stimulates secretion of immunoreactive intestinal mucin. Am. J. Physiol., 240, G10–G16.[Abstract/Free Full Text]

Gasser,K. and Hopfer,U. (1990) Chloride transport across the membrane of parotid secretory granules. Am. J. Physiol., 259, C413–C420[Abstract/Free Full Text]

Gasser,K., DiDomenico,J. and Hopfer,U. (1988) Secretogogues activate chloride transport pathways in pancreatic zymogen granules. Am. J. Physiol., 254, G93–G99.[Abstract/Free Full Text]

Hollande,E., Fanjul,M., Chemin-Thomas,C., Devaux,C., Demolombe,S., Van Rietschoten,J., Guy-Crotte,O. and Figarella,C. (1998) Targeting of CFTR protein is linked to the polarization of human pancreatic duct cells in culture. Eur. J. Cell Biol., 76, 220–227.[ISI][Medline]

Igimi,H., Yamamoto,F. and Lee,S. (1992) Gallbladder mucosal function: studies in absorption and secretion in humans and in dog gallbladder epithelium. Am. J. Physiol., 263, G69–G74.[ISI]

Izutsu,K., Johnson,D., Schubert,M., Wang,E., Ramsey,B., Tamarin,A., Truelove,G., Ensign,W. and Young,M. (1985) Electron microprobe analysis of human labial gland secretory granules in cystic fibrosis. J. Clin. Invest., 75, 1951–1956.[ISI][Medline]

Kent,P. and Mian,N. (1987) Transmural calcium fluxes and role of mucins as cellular calcium transport vehicles in chicken trachea in vitro. J. Physiol. (Lond.), 388, 121–140.[Abstract]

Kuver,R., Savard,C., Oda,D. and Lee,S. (1994) PGE generates intracellular cAMP and accelerates mucin secretion by cultured dog gallbladder epithelial cells. Am. J. Physiol., 267, G998–G1003.[Abstract/Free Full Text]

Kuver,R., Ramesh,N., Lau,S., Savard,C., Lee,S. and Osborne,W. (1994) Constitutive mucin secretion linked to CFTR expression. Biochem. Biophys. Res. Commun., 203, 1457–1462.[ISI][Medline]

LaMorte,W., LaMont,J., Hale,W., Booker,M., Scott,T. and Turner,B. (1986) Gallbladder prostaglandins and lysophospholipids as mediators of mucin secretion during cholelithiasis. Am. J. Physiol., 251, G701–G709.[ISI]

Lee,S. (1980) The mechanism of mucus secretion by the gallbladder epithelium. Br. J. Exp. Pathol., 131, 117–119.

Lee,S. and Scott,A. (1982) The evolution of morphologic changes in the gallbladder before stone formation in mice fed a cholesterol cholic acid diet. Am. J. Pathol., 108, 1–8.[Abstract]

Lenzen,R., Alpini,G. and Tavaloni,N. (1992) Secretin stimulates bile ductular secretory activity through the cAMP system. Am. J. Physiol., 263, G527–G532.[ISI]

Low,S., Chapin,S., Wimmer,C., Whiteheart,S., Komuves,L., Mostov,K. and Weimbs,T. (1998) The SNARE machinery is involved in apical plasma membrane trafficking in MDCK cells. J. Cell Biol., 141, 1503–1513.[Abstract/Free Full Text]

Marquet,R., Houssier,C. and Fredericz,E. (1985) An electro-optical study of the mechanism of DNA condensation induced by spermine. Biochim. Biophys. Acta, 825, 365–374.[ISI][Medline]

Mergey,M., Lemnaouar,M., Veissiere,D., Perricaudet,M., Gruenert,D., Picard,J., Capeau,J., Brahimi-Horn,M.-C. and Paul,A. (1995) CFTR gene transfer corrects defective glycoconjugate secretion in human CF tracheal epithelial cells. Am. J. Physiol., 269, L855–L864.[Abstract/Free Full Text]

Mills,C., Pereira,M., Dormer,R. and McPherson,M. (1992) An antibody against a CFTR-derived synthetic peptide, incorporated into living submandibular cells, inhibits beta-adrenergic stimulation of mucin secretion. Biochem. Biophys. Res. Commun., 188, 1146–1152.[ISI][Medline]

Montserrat,C., Merten,M. and Figarella,C. (1996) Defective ATP-dependent mucin secretion by cystic fibrosis pancreatic epithelial cells. FEBS Lett., 393, 264–268.[ISI][Medline]

Morris,A., Cunningham,S., Tousson,A., Benos,D. and Frizzell,R. (1994) Polarization-dependent apical membrane CFTR targeting underlies cAMP-stimulated Cl- secretion in epithelial cells. Am. J. Physiol., 266, C254–C268.[Abstract/Free Full Text]

Nanavati,C. and Fernandez,J. (1993) The secretory granule matrix: a fast-acting smart polymer. Science, 259, 96–98.

Naren,A., Nelson,D., Xie,W., Jovov,B., Pevsner,J., Bennett,M., Benos,D., Quick,M. and Kirk,K. (1997) Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms. Nature, 390, 302–305.[ISI][Medline]

Nicaise,G., Maggio,K., Thirion,S., Horoyan,M. and Keicher,E. (1992) The calcium loading of secretory granules. A possible key event in stimulus-secretion coupling. Biol. Cell., 75, 89–99.[ISI][Medline]

Oda,D., Lee,S. and Hayashi,A. (1991) Long term culture and partial characterization of dog gallbladder epithelial cells. Lab. Invest., 64, 682–692.[ISI][Medline]

Roomans,G., Wei,X., Ceder,O. and Kollberg,H. (1982) The reserpinized rat in the study of cystic fibrosis–X-ray microanalysis of submandibular gland and pancreas. Ultrastruct. Pathol., 3, 285–293.[ISI][Medline]

Sasake,S., Nakagaki,I., Mori,H. and Imai,Y. (1983) Intracellular calcium stores and transport of elements in acinar cells of the salivary gland determined by electron probe x-ray microanalysis. Jpn. J. Physiol., 33, 69–83.

Schimmoller,F., Simon,I. and Pfeffer,S. (1998) Rab GTPases, directors of vesicle docking. J. Biol. Chem., 273, 22161–22164.[Free Full Text]

Shuman,H., Somlyo,A. and Somlyo,A. (1976) Quantitative electron probe microanalysis of biological thin sections: methods and validity. Ultramicroscopy, 1, 317–339.[ISI][Medline]

Smith,B. (1987) Human gallbladder mucin binds biliary lipids and promotes crystal nucleation in model bile. J. Lipid Res., 28, 1088–1097[Abstract]

Specian,R. and Neutra,M. (1982) Regulation of intestinal mucus secretion: role of parasympathetic stimulation. Am. J. Physiol., 242, G370–G379.[ISI]

Stutts,M., Canessa,C., Olsen,J., Hamrick,M., Cohn,J., Rossier,B. and Boucher,R. (1995) CFTR as a cAMP-dependent regulator of sodium channels. Science, 269, 847–850.[ISI][Medline]

Thevenod,F., Gasser,K. and Hopfer,U. (1990) Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules. Biochem. J., 272, 119–126.[ISI][Medline]

Verdugo,P. (1984) Hydration kinetics of exocytosed mucins in cultured secretory cells of the rabbit trachea: a new model. Mucus and Mucosa. Ciba Foundation Symposium 109. Pitman, London, pp. 212–234.

Verdugo,P., Deyrup-Olsen,I., Aitken,M., Villalon,M. and Johnson,D. (1987) Molecular mechanism of mucin secretion. I. The role of intragranular charge shielding. J. Dent. Res., 66, 506–508.[Abstract]

Watson,E., DiJulio,D., Kauffman,D., Iversen,J., Robinovitch,M. and Izutsu,K. (1992) Evidence for G proteins in rat parotid plasma membranes and secretory granule membranes. Biochem. J., 285, 441–449.[ISI][Medline]

Yamada,K. (1968) Some aspects of the fine structure of the gallbladder epithelium of the mouse. Folia Anat. Jpn., 45, 11–21.

Zimmemberg,J. and Whitaker,M. (1985) Irreversible swelling of secretory granules during exocytosis caused by calcium. Nature, 315, 581–584.[ISI][Medline]