Selective secretion and replenishment of discrete mucin glycoforms from intestinal goblet cells

C. Michael Stanley and Thomas E. Phillips

Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211-7400


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies against MUC2, MUC3, and MUC5AC peptide epitopes stained the secretory contents of all goblet cells in the human colon-derived HT29-18N2 cell line. In contrast, four carbohydrate-specific monoclonal antibodies stained mucin glycoforms in consistent subsets of goblet cells. Cholinergic agonist-evoked decreases in total mucin stores were not always mirrored by proportional changes in mucin glycoforms in the same monolayers. Selective secretion of mucin glycoforms did not result from differences in receptor distribution, since cholinergic stimulation was found to increase intracellular free calcium in all cells and selective secretion was also observed when the cells were directly stimulated with the protein kinase C activator phorbol myristate acetate. The results demonstrate that goblet cells cycle through transient periods in which their exocytotic response is unresponsive to cholinergic or protein kinase C-mediated stimuli. Goblet cells replenished intracellular mucin stores to control levels within 1 h, but the relative proportion of mucin glycoforms was not always restored until 24 h after stimulation.

mucus; HT-29 cells; cholinergic; protein kinase C; exocytosis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MUCINS ARE A HETEROGENEOUS group of high-molecular-weight, O-linked glycoproteins. Part of the heterogeneity of mucins results from the existence of at least nine "MUC" genes in humans (9, 22). Additional heterogeneity can result from allelic length variations in the mucin RNAs, which reflect variable numbers of tandem-repeat polymorphisms present at the DNA level (6). Finally, another level of heterogeneity results from diversity in the length, composition, branching, and degree of sulfation and acetylation of the oligosaccharide chains that account for up to 85% of the weight of mucin (12).

The expression of different mucins, defined by differences either in their protein backbones or in their glycosylation patterns, has been shown to vary both between tissues and within tissues (9, 12). For example, in the normal human colon, MUC2 expression is present along the entire length of the crypt, whereas MUC3 labeling is limited to the cells along the surface or upper crypt (4). Similarly, lectins and monoclonal antibodies that recognize defined carbohydrate sequences have been used to demonstrate heterogeneous expression of mucin glycoforms in distinctive populations of intestinal goblet cells along the crypt-villus axis (7), between intestinal regions (8), and during fetal and neonatal development (5).

Intestinal goblet cells secrete the contents of individual mucous secretory granules by simple exocytosis at a slow, variable rate (13). Cholinergic stimulation causes rapid acceleration of mucin discharge via compound exocytosis from crypt cells, but, until recently, goblet cells located on villi or colonic luminal surfaces were thought to be unresponsive (16, 23). Computerized morphometric analysis demonstrated, however, that cholinergic stimulation accelerates mucin discharge from these cells via a mechanism that does not result in the deep apical membrane cavitation associated with recent compound exocytotic activity (14, 18). Earlier secretory studies have mostly considered mucin as a single entity and generally not examined the possibility of selective secretion of discrete mucin types. It may be overly simplistic to assume that all goblet cells express the same types of functional secretagogue receptors. It was clear in our earlier in vivo morphometric studies that cholinergic stimulation failed to accelerate mucin secretion from some intestinal goblet cells, even though adjacent cells had clearly discharged their mucin stores (14, 18). Furthermore, whether goblet cells that have undergone an accelerated discharge of their mucin stores refill with the same types of mucin they had been synthesizing before secretion has never been investigated. The human colonic adenocarcinoma-derived HT29-18N2 cell line, which grows as a monolayer of mucin-secreting goblet cells (15, 17, 19), allowed these issues to be addressed. Computer-assisted morphometric analysis was used to quantify the relative proportions of different mucins stained by a panel of peptide- and carbohydrate-specific antibodies in monolayers before and immediately after stimulation, as well as following replenishment of secreted stores.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. The HT29-18N2 cell line was generously provided by Dr. Daniel Louvard (Pasteur Institute, Paris, France) and grown as previously described (17). For routine propagation, cells were grown in 75-cm2 flasks in DMEgal [glucose-free, glutamine-free DMEM (Sigma, St. Louis, MO) supplemented with 5 mM galactose plus 10% dialyzed fetal bovine serum (JRH Biosciences, Lenexa, KS)]. Cells were seeded onto 12-mm-diameter glass coverslips in 16-mm-diameter plastic wells of 24-well trays at a density of 600,000-800,000 cells per well in DMEgal. After 24 h, the coverslips were transferred to 22-mm-diameter wells of a 12-well tray containing 1 ml of protein-free, hybridoma medium (PFHM-II; GIBCO, Grand Island, NY). The cells were typically confluent within 2-4 days after seeding. Cells were fed with 1 ml of fresh medium every other day.

Drugs. A 1-mg/ml stock of carbachol (carbamylcholine; Sigma) in culture medium was prepared fresh for each experiment. A stock solution of phorbol 12-myristate 13-acetate (PMA, 1 mg/ml; Sigma) was prepared in ethanol and stored at 4°C.

Morphological assessment. Monolayers, in triplicate, were immersed in cold paraformaldehyde fixative (2% freshly depolymerized paraformaldehyde in 70 mM NaCl, 30 mM HEPES, and 2 mM CaCl2, pH 7.4) for 1 h at 4°C. After three rinses in HEPES-wash buffer (HWB: 70 mM NaCl, and 30 mM HEPES, pH 7.4), the monolayers were dehydrated with an ethanol series and embedded in JB-4 resin (Polysciences, Warrington, PA).

Rabbit polyclonal antisera against synthetic MUC2 and MUC3 tandem-repeat consensus sequences were obtained from Biomeda (Foster City, CA). Monoclonal antibody 45M1 against a MUC5AC protein epitope and monoclonal GP1.4 against the Asp-Thr-Arg-Pro epitope present in the tandem repeats of MUC1 were obtained from LabVision (Union City, CA). The protein-epitope-specific antibodies were tested on both untreated and neuraminidase pretreated sections. Untreated semithin (0.9 µm) sections were incubated overnight in a dilution (typically 1:100 in HWB + 10% normal goat serum) of the antibodies, rinsed thoroughly, and stained with a fluorescein-tagged goat secondary antibody (Jackson ImmunoResearch, West Grove, PA) diluted in the same buffer for 4 h. In some experiments, the sections were first incubated overnight with 100 µl of a 50 U/ml solution of neuraminidase (type V, from Clostridium perfringens; Sigma) in buffer (50 mM sodium acetate, 110 mM NaCl, and 4 mM CaCl2, pH 5.0) at 37°C. After they were washed in HWB, sections were then stained using the same protocol as untreated sections.

To evaluate the relative proportions of mucins with different glycosylation patterns, four carbohydrate-specific monoclonal antibodies (generously provided by Dr. Daniel K. Podolsky, Massachusetts General Hospital, Boston, MA) were used. The epitopes recognized by these antibodies are shown in Table 1 (20). Sections were incubated overnight in undiluted hybridoma supernatant. On the following day, the incubation fluid was replaced with a fresh aliquot of the monoclonal antibody containing a 1:100 dilution of a rabbit polyclonal antisera specific for unfractionated high-molecular-weight HT29-18N2 mucin glycoproteins (21). This second incubation was again allowed to proceed overnight at room temperature, and then the slides were washed carefully and incubated overnight in a FITC-conjugated donkey antiserum against mouse immunoglobulins absorbed against rabbit immunoglobulins (Jackson ImmunoResearch) and a lissamine rhodamine-conjugated donkey antiserum against rabbit immunoglobulins absorbed against mouse immunoglobulins (Jackson ImmunoResearch). After they were rinsed, the sections were coverslipped and stored at 4°C until examination.

                              
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Table 1.   Binding patterns and specificities of monoclonal antibodies

A Bio-Rad MRC-600 confocal scanning fluorescence microscope coupled to a Nikon Diaphot microscope equipped with a ×60 (numerical aperture 1.4) objective was used to capture the fluorescent images from randomly selected fields. Although the optical sectioning characteristics of the confocal microscope were not needed for these semithin resin sections, we found that the signal-to-noise ratio of the confocal system was superior to that of signal-intensified video microscopy alternatives. Digital images were printed using a Fuji Pictrography 3000 color printer at a 200 dpi resolution.

A computer-assisted image analysis system (Image 1-AT, Universal Imaging, West Chester, PA) was used to calculate the surface area of intracellular mucous granule stores per length of monolayer analyzed (17, 19). The "paint" function of the software was used to mask the fluorescence of extracellular mucus and background fluorescence outside the thecal stores of secretory granules. Each image of a pair was then thresholded to show only the fluorescently stained portions of the images and area of the resulting binary image quantified. Total mucin stores were defined as the area resulting from the addition of the binary monoclonal and polyclonal images. Repeated measurements (n = 5) of a single representative image by each of three separate investigators had coefficients of variation of <0.05. The coefficient of variation for the measurement of the same sample between investigators was 0.04.

Several safeguards were employed to prevent biased evaluation. Coded sections from three identically treated monolayers were analyzed for each treatment. The analysis of six distinct regions of each monolayer averaging 105 µm in length resulted in 1,890 µm or more of monolayer length being evaluated for each condition. Statistical analysis of the data was performed using a two-group unpaired Mann-Whitney U test.

To detect changes in intracellular free calcium concentration, partially confluent monolayers of cells growing on glass coverslips were rinsed several times in Dulbecco's PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 0.5 mM Mg2Cl, and 0.9 mM CaCl2) and 5 mM glucose and then incubated in 7.64 µM fura red-AM (Molecular Probes, Eugene, OR) in the same buffer for 45 min. After they were rinsed in fresh buffer, the coverslips were mounted in a perfusion chamber and imaged using the 488-nm excitation line on the confocal microscope. The fluorescence intensity of each cell was quantified using the "average gray value" tool in the Image 1-AT image analysis software.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies against peptide epitopes present on specific mucins were used to stain semithin cross sections of HT29-18N2 cells. On sections that had not been pretreated in any manner, anti-MUC2 and anti-MUC3 polyclonal antisera stained mature mucins in secretory granules in only a small proportion of the goblet cells. The MUC2 and MUC3 antibodies did consistently stain most cells in a punctate or crescent-shaped perinuclear pattern, which was consistent with the antibodies staining apomucin molecules in the Golgi region (data not shown). The heavy carbohydrate coat on mature mucins in secretory granules apparently prevented the antibodies from reaching their binding sites on the protein backbone. When the sections were pretreated with neuraminidase, the MUC2 and MUC3 antibodies stained the intracellular mucin stores in the secretory granules of all HT29-18N2 goblet cells (Fig. 1, A and B). Antibody 45M1 is a member of a panel of monoclonal antibodies that recognize a class of mucin antigens, called M1, that are common to gastric mucosa and ovarian mucinous cysts (3). The binding pattern of 45M1 to mucins expressed mainly in the surface epithelium of normal gastric mucosa and in fetal and precancerous colon coincides with the known distribution of MUC5AC (2, 3). Furthermore, gastric M1 mucin was recently shown to be encoded by the MUC5AC gene (1). The binding site for 45M1 is presumed to be a protein epitope, since it can react with deglycosylated gastric mucin, whereas its binding to native mucins is eliminated or reduced following their treatment with beta -mercaptoethanol or protease (1, 3). In our hands, antibody 45M1 stained the secretory contents of all HT29-18N2 goblet cells in untreated JB-4 sections and this staining pattern was not altered by neuraminidase pretreatment (Fig. 1C). MUC1 is an integral membrane mucin glycoprotein. A monoclonal antibody specific for a MUC1 peptide epitope caused staining predominately along the apical membrane along with diffuse cytoplasmic staining and occasional punctate intracellular staining (Fig. 1D). Although the intensity of the apical membrane staining varied along the length of the monolayer, most cells showed at least a weak signal. There was no sign of MUC1 expression within mucous secretory granules.


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Fig. 1.   Immunocytochemical staining of HT29-18N2 goblet cells by antibodies specific for peptide epitopes on mucin proteins. MUC2 (A)- and MUC3 (B)-specific antisera stain the intracellular mucin stores of all goblet cells in sections that have been pretreated with neuraminidase to increase access to the mucin protein backbone. C: monoclonal antibody 45M1, a MUC5AC-specific antibody, resulted in a similar staining of intracellular mucin stores in all goblet cells even in the absence of neuraminidase pretreatment. D: monoclonal antibody GP1.4, which recognizes a MUC1 peptide epitope, stained the apical membrane of the cells (arrows) and caused some punctate and weak diffuse cytoplasmic staining. Intracellular mucous secretory granules were unlabeled by the anti-MUC1 antibodies. MUC1 and MUC5AC staining patterns were not altered by neuraminidase pretreatment. Bar = 20 µm.

After cholinergic stimulation for either 5 or 60 min, the total intracellular mucin stores were decreased (described below), but the intracellular mucin stores in all cells in neuraminidase-treated sections could still be stained by the MUC2 and MUC3 antibodies and in untreated sections by antibody 45M1. Similarly, goblet cells in monolayers allowed to recover for 1, 4, or 24 h after a 1-h cholinergic stimulation were still all stained by the panel of three secretory mucin peptide-specific antibodies. At all time points during the secretion and replenishment cycle, the MUC2 and MUC3 antibodies continued to stain the contents of mucous secretory granules in only a small proportion of cells in sections that had not been pretreated with neuraminidase. This indicates that there was not an influx of newly synthesized partially glycosylated mucins or apomucins into the secretory granules during replenishment. The expression of the nonsecretory, transmembrane MUC1 protein was not studied in stimulated monolayers.

Each of the four carbohydrate-specific monoclonal antibodies stained mucin glycoforms in specific subsets of cells, whereas the polyclonal antisera against unfractionated HT29-18N2 mucin stained all goblet cell mucin stores (Fig. 2). To determine whether the carbohydrate-specific monoclonal antibodies stained reproducible fractions of the intracellular mucin stores, computer-assisted morphometric analysis was used to quantify intracellular mucin stores in several seedings of cells. The digital fluorescent images were thresholded and converted to binary images (e.g., Fig. 2, m-o). Although the polyclonal serum against unfractionated mucin should have recognized all intracellular mucin stores, it was conceivable that heavy staining of the monoclonal antibody could have sterically hindered binding of the polyclonal serum. Total mucin stores were, therefore, based on a composite image obtained by using the logic "or" function to add the binary images from each pair. In practice, the difference in area between mucin stores in the composite images and the polyclonal antisera-only images averaged <10%. With the use of three different seedings of HT29-18N2 cells, it was found that the proportion of monoclonal antibody staining to total intracellular mucin stores was remarkably constant (Table 1).


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Fig. 2.   Dual-label immunocytochemical staining of HT29-18N2 cells by a polyclonal antisera against unfractionated mucins and carbohydrate-specific monoclonal antibodies. Left column illustrates how monoclonal antibodies (a: M5, d: Q5, g: WE9, j: 2D55) stain mucin glycoforms in only a subset of the cells, whereas polyclonal antisera [middle column (b, e, h, k)] recognize the mucin secretory product in all the goblet cells in the same sections. All of these digital images were collected by confocal microscopy and pseudo-colored using Adobe Photoshop. Right column (c, f, i, l) shows a superimposition of the monoclonal and polyclonal antibody staining patterns. Yellow areas indicate regions of coexpression, whereas red regions show goblet cells that do not express the mucin glycoform stained by the monoclonal antibody. Bottom row (m, n, o) shows images j, k, l that have been thresholded and converted to binary images to illustrate how the mucin stores were measured using computer-assisted morphometrics. Extracellular mucin and background signal outside the thecal stores of mucous secretory granules are removed using the "paint" function in the image analysis software. Total mucin stores were defined as the area formed by superimposing the binary pair of images (e.g., m + n = o). The few purple pixels present in the binary images result from a slight mismatch in the digital resolution of the confocal microscope (72 dpi) and the color printer (200 dpi). These output errors would not have resulted in an error in measurement. Bar = 20 µm.

When it was determined that the carbohydrate-specific monoclonal antibodies stained reproducible fractions of the total mucin pool, the next step was to determine if the relative proportions of mucin glycoforms were altered immediately after accelerated secretion or during the subsequent replenishment phase. Four sets of cross sections from a single seeding (seeding 1) of monolayers, fixed at various times before, during and after cholinergic stimulation by 100 µM carbachol, were prepared. Each of the monoclonal antibodies was used in combination with the polyclonal antisera against unfractionated mucin to stain one of the sets of cross sections. On the basis of the average response for the four sets of monoclonal and polyclonal composite images, carbachol decreased total intracellular mucin stores by 31 ± 3% (SE; P < 0.01) within 5 min and 33 ± 3% (P < 0.01) at 60 min. When monolayers were allowed to recover for 1, 4, or 24 h after an initial 60-min exposure to carbachol, total intracellular mucin stores were replenished to control levels (88 ± 4%, 93 ± 3%, and 86 ± 3%, respectively; P > 0.1). There were no significant differences (P > 0.1) in the measurements of total mucin between the four sets of slides stained with each of the monoclonal and polyclonal antibody combinations when similar treatment conditions were compared (e.g., control set 1 vs. control sets 2, 3, or 4).

Monoclonal antibody 2D55 stained 49% of the total intracellular mucin stores in monolayers from seeding 1. The decrease in mucin stores stained by 2D55 after cholinergic stimulation was not significantly different from that predicted from the decrease in total mucin stores in the same set of sections (Fig. 3A). The mucin area/length (µm2/µm) stained by 2D55 decreased by 39% after a 5-min and 47% after a 60-min exposure to carbachol. These decreases were 108% (P > 0.1) and 115% (P > 0.1) of the decreases in total mucin stores in the same sections at 5 and 60 min, respectively. Like the total mucin pool, 2D55-stained mucins returned to control levels when the monolayers were allowed to recover for 1, 4, or 24 h after cholinergic stimulation.


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Fig. 3.   Intracellular mucin stores after either a 5- or 60-min stimulation with 100 µM carbachol and following a 1-, 4-, or 24-h recovery from the 60-min stimulation. Sections were double labeled with a polyclonal antisera against unfractionated mucin and 1 of 4 monoclonal antibodies against a carbohydrate-specific epitope (A: 2D55, B: M5, C: Q5, D: WE9). Total mucin stores (solid bars) and mucin stained by the carbohydrate-specific monoclonal antibody (gray bars) were measured and expressed as a percentage of their levels in mock-stimulated control monolayers. * Volume of mucin was significantly less (P < 0.01) than volume in the corresponding control. Numerical P values above each pair of bars indicate the statistical significance of the difference in the change in the mucin glycoform compared with the change in the total mucin pool in the same sections. Each bar represents the average response of 3 monolayers (means ± SE).

Monoclonal antibody M5 stained 35% of the total intracellular mucin stores in monolayers from seeding 1. The decrease in mucin stores stained by M5 after cholinergic stimulation was significantly greater than that predicted from the decrease in total mucin stores (Fig. 3B). The mucin area/length stained by M5 decreased by 45% after a 5-min and 51% after a 60-min exposure to carbachol. These decreases were 185% (P < 0.01) and 176% (P < 0.01) of the decreases in total mucin stores in the same sections at 5 and 60 min, respectively. M5-stained mucins returned to control levels within 1 h after stimulation.

Monoclonal antibody Q5 stained 40% of the total intracellular mucin stores in monolayers from seeding 1. The decrease in mucin stores stained by Q5 after cholinergic stimulation was significantly greater than that predicted from the decrease in total mucin stores (Fig. 3C). The mucin area/length stained by Q5 decreased by 46% after a 5-min and 42% after a 60-min exposure to carbachol. These decreases were 133% (P < 0.05) and 162% (P < 0.01) of the decreases in total mucin stores in the same sections at 5 and 60 min, respectively. The mucin area/length stained by Q5 was indistinguishable from control levels (101%, P > 0.1) at 24 h after the initial 1-h stimulation. At the 1-h and 4-h poststimulation time points, however, a statistically significant increase (115%, P < 0.01 at 1 h and 139%, P < 0.01 at 4 h) in the mucin area/length stained by Q5 was observed.

Monoclonal antibody WE9 stained 30% of the total intracellular mucin stores in this set of monolayers. The decrease in mucin stores stained by WE9 after cholinergic stimulation was not significantly different from that predicted from the decrease in total mucin stores (Fig. 3D). The mucin area/length stained by WE9 decreased by 25% after a 5-min and 30% after a 60-min exposure to carbachol. These decreases were 86% (P > 0.1) and 87% (P > 0.1) of the decreases in total mucin stores in the same sections at 5 and 60 min, respectively. WE9-stained mucins returned to control levels within 1 h of stimulation.

To determine if the selective secretion of mucin glycoforms in response to cholinergic stimulation was consistent, the response to a 1-h stimulation with 100 µM carbachol was examined in a second seeding of monolayers. Differences between the secretory responses of cells within a population could result from either variations in their expression of receptors or postreceptor events involving the signal transduction pathway. To distinguish between these two possibilities, additional monolayers from this seeding were stimulated for 1 h with 2 µM PMA, a direct activator of protein kinase C.

In this second group, carbachol caused a 21 ± 1% decrease (P < 0.01) and PMA caused a 27 ± 4% decrease (P < 0.01) in total intracellular mucin stores, based on the average for the four sets of monoclonal and polyclonal composite images. The reproducibility of the morphometric technique was demonstrated by the finding that, once again, there was no significant difference (P > 0.1) in the measurements of total mucin between the four sets of slides stained with each of the monoclonal and polyclonal antibody combinations when similar treatment conditions were compared. The amount of 2D55-stained mucin following carbachol stimulation was 178% of the change predicted by the drop in total mucin stores, but the difference was only marginally significant (P = 0.09; Fig. 4A). The decrease in 2D55-stained mucin following PMA stimulation, on the other hand, was 160% of the predicted decline, and this difference had a high degree of statistical significance (P < 0.01). There was no significant decrease in M5-stained mucin after stimulation by either carbachol (10% decrease; P > 0.1) or PMA (10% decrease; P > 0.1) despite the fact that total mucin stores in the same sections dropped by 19% (P < 0.01) and 16% (P < 0.01) after carbachol and PMA treatment, respectively (Fig. 4B). Significantly more Q5-stained mucin was released following stimulation by carbachol (168%; P < 0.05) and PMA (130%; P < 0.05) than was predicted by the change in total mucin stores in the same sections (Fig. 4C). Mucins stained by WE9 dropped by 92% (carbachol) and only 40% (PMA) of the amount predicted by the decrease in total mucin stores in the same sections, but neither difference was statistically significant (P > 0.1; Fig. 4D).


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Fig. 4.   Effect of a 1-h stimulation with either 100 µM carbachol or 2 µM phorbol 12-myristate 13-acetate (PMA) on intracellular mucin stores. Sections were double labeled with a polyclonal antisera against unfractionated mucin and 1 of 4 monoclonal antibodies against a carbohydrate-specific epitope (A: 2D55, B: M5, C: Q5, D: WE9). Total mucin stores (solid bars) and mucin stained by the carbohydrate-specific monoclonal antibody (gray bars) were measured and expressed as a percentage of their levels in mock-stimulated control monolayers. * Volume of mucin was significantly less (P < 0.01) than the corresponding volume in controls. Numerical P values above each pair of bars indicate the statistical significance of the difference in the change in the mucin glycoform compared with the change in the total mucin pool in the same sections. Each bar represents the average response of 3 monolayers (means ± SE).

The inability of PMA to accelerate mucin secretion from some goblet cells (e.g., those making M5-stained mucins in seeding 2) suggested that these cells might have active cholinergic receptors still linked to the phosphoinositol pathway but that the calcium and protein kinase C signal transduction pathways had been uncoupled from the exocytotic process. To give further insight into whether all HT29-18N2 cells, including those that were not accelerating their release of mucin, were responding to cholinergic stimulation, cells were loaded with fura red, a fluorometric indicator whose fluorescence is inversely proportional to the concentration of calcium. In cells in which the cytosolic calcium level is increased by cholinergic stimulation, a decrease in fluorescence would result. It was not possible to effectively load fura red-AM into confluent monolayers of HT29-18N2 cells. The efficiency of loading acetoxymethyl esters of indicator dyes is well known to vary between different cell and tissue types, and an interaction between these highly hydrophobic compounds and the extracellular mucin coat present on confluent monolayers would not be unexpected. Reasonable loading of the fura red-AM into partially confluent cell cultures was achieved. When a cluster of 16 cells in a partially confluent monolayer was scanned every 10 s during a 30-s control perfusion period, the average pixel brightness for 16 individual cells at the 30-s time point was not significantly different (P > 0.1) from the matched 0-s time point (Fig. 5A). On average, there was less than a 1% change in the average pixel brightness during the 30-s control period. Within 10 s of exposure to 100 µM carbachol, 15 of the 16 cells showed a significant drop in brightness averaging 15 ± 3% (mean ± SE; P < 0.01). By 20 s of carbachol perfusion, all 16 cells were responding to carbachol. After a 180-s exposure, the average decrease was 17 ± 2% (P < 0.01; Fig. 5B). Within 10 s of washing out the carbachol, all 16 cells were fluorescing significantly brighter (9 ± 1% higher than the carbachol response at 180 s; P < 0.01). An additional 20 s of washing did not result in a further recovery (7 ± 1% higher than the carbachol response at 180 s). When the calcium ionophore ionomycin was added to the perfusate, there was little effect for the first 10 s, but, by 20 s, 12 of the 16 cells showed decreased fluorescence (6 ± 2% less than the value after the 30-s wash period; P < 0.02), and, by 180 s of ionomycin exposure, all 16 cells were responding (19 ± 2% less than the value after the 30-s wash period; P < 0.01). Other clusters of cells showed similar responses.


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Fig. 5.   Partially confluent monolayers of HT29-18N2 cells were loaded with the calcium indicator dye fura red-AM. Fluorescence intensity (average pixel gray-scale values on an arbitrary 0-255 scale) of each cell was measured and digitally stamped on top of each cell in the image. A: unstimulated cells (30-s control time point). B: same cells as A at 180 s after exposure to 100 µM carbachol. In each cell, a rise in cytosolic free calcium is indicated by a decrease in the fura red fluorescence. Bar = 20 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mucus is a complex mixture of high-molecular-weight mucin glycoproteins, water, electrolytes, serum- and cell-derived macromolecules, bacteria, and sloughed epithelial cells. Intestinal mucus acts as a lubricant and an anchoring matrix for secretory IgA and digestive enzymes and participates in the delicate process of maintaining a favorable environment for normal enteric flora while preventing the establishment of pathogenic microorganisms (12). There is also evidence that intestinal mucus acts to limit damage to the mucosal surface from exogenous or endogenous luminal irritants such as laxatives (28) or bile salts (24). Most of the physical properties of mucus are derived from the mucin glycoproteins. Consistent with the multiple functions of mucus, it is well established that goblet cells produce a heterogenous range of mucins that can differ in both their protein backbone (4, 9, 22) and glycosylation pattern (5, 7, 8). It is also widely accepted that there are shifts in the relative proportions of mucin protein types and glycoforms, which occur in several common intestinal diseases (4, 9, 12). Differences in the relative proportions of individual mucin types secreted might have profound effects on any of the multiple functions of extracellular mucus.

In the present study, each of the carbohydrate-specific monoclonal antibodies stained a proportion of the total mucin pool that was essentially identical in monolayers from different experiments. Acceleration of mucin secretion by the cholinergic agonist carbachol resulted in a nonuniform release of mucin glycoforms. Because the particular glycoforms secreted varied widely between experiments, it is clear that cholinergic responsiveness is not permanently linked to cells expressing a fixed subset of mucin glycoforms. The cholinergic sensitivity of a given cell cannot be random, however, because nonproportional release of mucin glycoforms was consistently observed. The fraction of cholinergically responsive cells must somehow cycle through the monolayer in a manner that allows it to partially or wholly overlap, at times, the subsets of cells synthesizing particular glycoforms. The factors that control which glycoforms a goblet cell synthesizes are not known, but, if any of these factors also partially contributed to the cholinergic sensitivity of a cell, one would predict a variable overlap of the two responses. For example, if the cholinergic sensitivity and type of glycoform being synthesized were both dependent on temporal cycles of different duration, the degree of overlap between the two responses would also be cycling.

Receptor occupation of muscarinic M3 receptors, such as those found on HT29-18N2 cells, normally results in an elevation of intracellular calcium and activation of protein kinase C (11). Two lines of evidence suggest that the selective secretion of carbohydrate-defined mucin glycoforms was a result of the exocytotic steps becoming uncoupled from the signal transduction pathway. First, cholinergic stimulation increased intracellular calcium in all HT29-18N2 cells in semiconfluent monolayers. This clearly demonstrates that, at least in this stage of cell growth, all HT29-18N2 cells have functional cholinergic receptors coupled to the phosphoinositiol pathway. Second, the direct activation of protein kinase C with PMA consistently resulted in a decrease in total mucin stores but did not always cause a similar decrease in mucin types stained by monoclonal antibodies in the same sections. These results imply that the differences in mucin discharge from goblet cells within monolayers are more likely a result of the secretory response becoming uncoupled from the signal transduction pathway rather than variations in cholinergic receptor distribution or an uncoupling of the receptor from the signal transduction pathway. This conclusion is consistent with earlier studies that have also shown that some goblet cells in vivo appear to be transiently insensitive to stimulation by cholinomimetics. It is not uncommon to observe goblet cells that appear maximally filled with secretory granules next to cells that have lost their entire mucin stores after cholinergic stimulation in vivo (14, 18). Additional evidence that the coupling of the cholinergic signal transduction pathway to the exocytotic process undergoes changes as the goblet cell matures in vivo comes from the observation that more recently differentiated goblet cells in the crypt region of the small and large intestines respond to cholinergic stimulation by compound exocytotic discharge of mucin whereas more mature, villus goblet cells in the small intestine and upper crypt and surface goblet cells in the colon respond by accelerating their rate of simple exocytosis without ever undergoing the deep cavitation associated with compound exocytotic activity (14, 18). Earlier studies have shown that PMA-mediated activation of protein kinase C in HT29-18N2 goblet cells results in an acceleration of mucin secretion by compound exocytotic discharge similar to the response of crypt goblet cells (19). Stimulation of HT29-18N2 cells with carbachol or a calcium ionophore, on the other hand, results in an acceleration of simple exocytosis such as seen in villus or colonic surface goblet cells (19). The present study demonstrates that both types of secretory responses can be transiently inactive with populations of HT29-18N2 cells.

We are unaware of any other reports of unstimulated "resting" exocrine secretory cells going through transient periods in which their exocytotic machinery is uncoupled from the second messengers that normally evoke a secretory response. It must be stressed, however, that most biochemical, immunochemical, or even morphological assays of secretion would not be expected to be able to detect such a population of uncoupled cells. There is precedent for physiologically or pharmacologically stimulated cells to become transiently uncoupled from the exocytotic process at points distal to second messenger generation. For example, epinephrine is a physiological inhibitor of insulin secretion from beta -cells. It exerts this inhibition, via a heterotrimeric G protein, at a step distal to the generation of cAMP and the rise in cytosolic free calcium that normally stimulate insulin secretion (26). Overexpression of monomeric G proteins in chromaffin cells inhibits exocytotic secretion in response to elevated cytosolic free calcium (10). In the related HT29-19A colonic enterocyte cell line, activation of G proteins by somatostatin or clonidine inhibits chloride secretion in response to rises in either cAMP or cytosolic free calcium (27). Whether similar G proteins can inhibit mucin secretion at a distal point in the exocytotic pathway is unknown, but the present results raise the exciting possibility that G proteins may cycle through active and inactive states even in unstimulated, resting cells. The present results identify a cell model in which this hypothesis could be tested.

When goblet cells replenished their intracellular stores following an acute challenge, they generally refilled with the same relative proportions of carbohydrate-defined mucin glycoforms as found in the prestimulated monolayers. One explanation of the increased proportion of M5-stained mucin found at 1 and 4 h poststimulation would be that some cells normally further glycosylate, sulfate, or acetylate the oligosaccharide chain to a form no longer recognized by the antibody. The heavy load of nascent mucin molecules passing through the Golgi following discharge may temporarily overwhelm the enzymatic machinery. Because the normal, unstimulated baseline secretion of mucin results in a turnover of intracellular stores every 24 h in HT29-18N2 goblet cells (15), it is not surprising that all cells have returned to their prestimulated proportions of mucin glycoforms by 24 h poststimulation. It is surprising that the relative proportions of carbohydrate-defined mucin glycoforms is so consistent between different seedings of HT29-18N2 cells. The factors that regulate the relative proportions are not yet understood, but the clonal nature of the HT29-18N2 line eliminates the possibility that these differences reflect a mixture of goblet cell phenotypes with fixed synthetic pathways. Identification of the factors regulating synthesis of particular mucin glycoforms could provide important insights into diseases such as ulcerative colitis in which a selective reduction in one mucin glycoforms precedes the onset of clinical symptoms (25).


    ACKNOWLEDGEMENTS

We thank Bryan MacDonald for excellent technical assistance in working out the neuraminidase pretreatment procedure.


    FOOTNOTES

This work was supported by a grant from the Crohn's and Colitis Foundation and a gift from the Mid-Missouri Inflammatory Bowel Disease Support Group. Dr. Daniel K. Podolsky generously provided the panel of carbohydrate-specific monoclonal antibodies.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. E. Phillips, Division of Biological Sciences, Tucker Hall, Univ. of Missouri, Columbia, MO 65211-7400 (E-mail: phillipst{at}missouri.edu).

Received 23 October 1998; accepted in final form 23 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 277(1):G191-G200
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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