Dynamic regulation of mucus gel thickness in rat duodenum

Yasutada Akiba1,2, Paul H. Guth3, Eli Engel4, Igor Nastaskin5, and Jonathan D. Kaunitz1,2,3

3 Greater Los Angeles Veterans Affairs Healthcare System, 1 CURE: Digestive Diseases Research Center, and 2 Department of Medicine, School of Medicine, and 4 Department of Biomathematics, 5 College of Letters and Science, University of California, Los Angeles, California 90073


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the dynamic regulation of mucus gel thickness (MGT) in vivo in rat duodenum in response to luminal acid, cyclooxygenase (COX) inhibition, and exogenous PGE2. An in vivo microscopic technique was used to measure MGT with fluorescent microspheres in urethan-anesthetized rats. Duodenal mucosa was topically superfused with pH 7.0 or pH 2.2 solutions with or without PGE2 and indomethacin treatments. Glycoprotein concentration of duodenal loop perfusates was measured with periodic acid/Schiff (PAS) or Alcian blue (AB) staining. MGT and perfusate glycoprotein concentration were stable during a 35-min perfusion with pH 7.0 solution. Acid exposure increased MGT and PAS- and AB-positive perfusate glycoprotein concentrations. Indomethacin pretreatment increased both PAS- and AB-positive perfusate glycoprotein at baseline; subsequent acid superfusion decreased perfusate glycoproteins and gel thickness. PGE2 (1 mg/kg iv) simultaneously increased MGT and PAS-positive perfusate glycoprotein concentrations followed by a transient increase in AB-positive glycoprotein concentration, suggesting contributions from goblet cells and Brunner's glands. Parallel changes in MGT and perfusate glycoprotein concentration in response to luminal acid and PGE2 suggest that rapid MGT variations reflect alterations in the balance between mucus secretion and exudation, which in turn are regulated by a COX-related pathway. Luminal acid and PGE2 augment mucus secretion from goblet cells and Brunner's glands.

fluorescent microspheres; perfusate glycoprotein concentration; indomethacin; periodic acid/Schiff staining; Alcian blue staining; Brunner's glands


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PROXIMAL DUODENAL MUCOSA is exposed to frequent pulses of gastric acid. Unlike other acid-exposed organs such as the stomach or esophagus, the duodenum is functionally "leaky" in that the epithelial cell layer is an incomplete barrier to solute diffusion, increasing the importance of nonstructural defense mechanisms such as the mucus gel layer, bicarbonate secretion, and blood flow. The viscoelastic mucus gel layer is the first line of defense against luminal contents and is thought to play an important role in decreasing mucosal injury due to acid. Mucus secretion is increased by luminal acid (19, 33) and the neuro- and inflammatory mediators acetylcholine and interleukin-1 (8), vasoactive intestinal polypeptide (15), secretin (16, 19), and guanylin (10) in duodenum. In the stomach and intestine, mucus secretion and gel thickness are increased by prostaglandins (5, 20, 24, 36), pentagastrin (26, 45), carbachol (30, 31), nitric oxide (7, 30), bradykinin (41) and phorbol esters (29). Of these factors, prostaglandins, products of the cyclooxygenase (COX) pathway, are of paramount clinical importance, because COX inhibition is an important cause of clinical duodenal ulceration (38).

In the gastrointestinal tract, mucus secretion is generally inferred from examination of histological sections stained by the periodic acid/Schiff (PAS) or Alcian blue (AB) techniques (25, 28). Secretion has also been extrapolated from measurements of mucus glycoprotein of luminal perfusates by PAS staining (24, 36) or hexosamine measurement (19); mucus gel thickness (MGT) has been measured in tissue sections by the method developed by Kerss et al (14). In the duodenum, Sababi et al. (33) performed the only study in which MGT was continuously measured in vivo. In that study, the duodenal mucus gel was shown to form a spontaneously secreted, continuous layer. Furthermore, recovery of MGT after mechanical removal was accelerated by luminal acid but inhibited by indomethacin (Indo) and a nitric oxide synthase inhibitor (33). No study, however, has addressed the regulation of MGT in a superfused system in which glycoprotein sloughing (exudation) was measured in the basal steady state or in response to secretory stimuli. Furthermore, because the duodenum secretes at least two mucin subtypes, muc2, which is synthesized in intestinal goblet cells (48), and muc6 from Brunner's glands (12), it is unknown which mucin subtype contributes to the regulation of MGT.

In this study, we describe a novel optical, noninvasive technique for measuring duodenal MGT and compare MGT with perfusate glycoprotein concentration measured by PAS and AB staining. We demonstrate that MGT is governed by the balance between mucus secretion and release into the superfusate, that spontaneous and stimulated mucus secretion and luminal exudation are regulated by luminal pH and the COX pathway, and that both goblet cells and Brunner's glands contribute to the changes in MGT and exuded luminal mucus.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and Chemicals

Male Sprague-Dawley rats weighing ~225-275 g (Harlan Laboratories, San Diego, CA) were fasted overnight but had free access to water. All studies were approved by the Animal Use Committee of the Greater Los Angeles Veterans Affairs Healthcare System.

2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM (BCECF-AM) was obtained from Molecular Probes (Eugene, OR). Pink-fluorescent microspheres (2 µm; excitation, 575 nm; emission, 600 nm) were obtained from Bangs Laboratories (Fishers, IN). PGE2 was obtained from Oxford Biochemical (Oxford, MI). SDS and polyacrylamide/bis-polyacrylamide solution were from Bio-Rad (Richmond, CA). Indo, HEPES, 2-mercaptoethanol, acridine orange, AB 8 GX, and all other chemicals were obtained from Sigma Chemical (St. Louis, MO). Krebs solution contained (in mM) 136 NaCl, 2.6 KCl, 1.8 CaCl2, and 10 HEPES at pH 7.0. For acid superfusion, Krebs solution was titrated to pH 2.2 with 1 N HCl and adjusted to isotonicity (300 mM). Each solution was prewarmed to 37°C using a water bath, and temperature was maintained with a heating pad during the experiment. PGE2 was dissolved with absolute ethanol to make a concentrated stock solution.

MGT In Vivo

In vivo microscopic preparation. An in vivo microscopic technique was used to visualize epithelial cells of rat duodenal villus tips as previously described (3). Briefly, after urethan (1.25 g/kg) anesthesia, a tracheal cannula was inserted and warmed saline was continuously infused through the left femoral vein at a rate of 1.08 ml/h using a Harvard infusion pump. Body temperature was maintained at 36-37°C by a heating pad, with rectal temperature monitored throughout the experiment by a rectal thermistor (Yellow Springs Instruments, Yellow Springs, OH). Arterial blood pressure was monitored via a catheter placed in the left femoral artery. The abdomen was opened, and the duodenum was exposed. The pylorus was tightly ligated to prevent gastric juice from entering into the proximal duodenum, and the duodenum was temporarily closed with a nylon suture proximal to the ligament of Treitz, before filling the duodenal loop with 0.5 ml saline prewarmed at 37°C. The anterior wall of the duodenum was incised distal to the pylorus and just proximal to the common entrance of the bile and pancreatic ducts using a miniature electrocautery to prevent bile-pancreatic juice from contaminating the observed duodenal mucosa. A concave stainless steel disk (16-mm diameter and 1-2 mm deep) with 3-mm central aperture was fixed watertight on the mucosal surface with a silicone plastic adherent (Silly Putty, Binney & Smith, Easton, PA). The serosal surface of the duodenum just below the chambered mucosa was supported with a right-angle probe (R type, Transonic, Ithaca, NY) surrounded with a silicone plastic adherent as previously described (2). A thin plastic coverslip was fixed to the disk with the silicone adherent to permit closed superfusion with solutions (total vol, 50 µl; rate, 0.25 ml/min) using a Harvard infusion pump. Two PE-50 polyethylene perfusion lines were inserted into the chamber to enable rapid changes of superfusate. The exposed mucosa was incubated with 50 µl Krebs solution (pH 7.0) containing 10 µM BCECF-AM for 15 min to load the duodenal epithelial cells before starting the experiment. BCECF localization in the epithelial cells of villus tips has previously been confirmed by examination of cryostat sections (3).

Measurement of MGT. Duodenal MGT measurement was adapted from a technique used to measure MGT in rat stomach (13). After BCECF loading, fluorescent microspheres diluted to 0.05% wt/vol with prewarmed Krebs buffer (pH 7.0) were placed over the mucosa to delineate the luminal surface of the gel layer. After a 5-s incubation, the microsphere-containing solution was removed from the chamber and superfusion with Krebs buffer commenced. Fluorescence of the microscopically observed chambered segment of duodenal mucosa was visualized with a charge-coupled device color video camera (Optronics Engineering, Goleta, CA) and was captured and stored using an Intel Pentium-based IBM-compatible microcomputer with a FlashPoint framegrabbing videographic card (Integral Technologies, Silver Spring, MD) and image-processing software (Image-Pro Plus v. 1.3, Media Cybernetics, Silver Spring, MD). Optical mucus thickness measurements were made by alternately focusing on the fluorescent cell surface, using a 495-nm excitation and a 515-nm emission filter (green filter set, Chroma, Battleboro, VT), and then focusing on the microspheres layer, visualized with a 575-nm excitation and a 600-nm emission filter (red filter set). The vertical travel of the microscope objective from the plane of the fluorescent cell surface to the plane of the microspheres was measured by using a digital z-axis measuring device (Quick-Check, Metronics, Bedford, NH) connected to the microscope, providing a measure of gel thickness. Gel thickness was measured every 5 min, after capture of the BCECF images of the mucosa. Paired images of mucosa and microspheres were taken a maximum of 10 s apart.

Histological sections were examined to localize the microspheres to the gel surface. After microsphere loading and a 30-min pH 7.0 Krebs superfusion, the observed area of duodenum was excised and mounted in OCT compound (Miles, Elkhart, IN) at -20°C. Frozen cryostat sections (10 µm) were mounted on glass slides (Fisher Scientific, Willard, OH) and counterstained with 10 µM acridine orange. Sections were coverslipped using glycerol and observed by a Zeiss MPS microscope with a dual-band filter (Chroma) that enabled simultaneous visualization of the green acridine orange and red microsphere fluorescence.

Effect of luminal acid on MGT. After stabilization of the preparation with a 30-min superfusion of pH 7.0 Krebs buffer, the time was set as time 0. The duodenal mucosa was superfused with pH 7.0 Krebs buffer from time 0 to time 10 (baseline period), pH 7.0 or 2.2 buffer from time 10 to time 20 (challenge period), and pH 7.0 solution from time 20 to time 35 (recovery period).

Experimental design. The following four groups were studied: control (no pretreatment and challenge with pH 7.0); acid (no pretreatment and challenge with pH 2.2); Indo (5 mg/kg ip pretreatment 1 h before anesthesia and challenge with pH 2.2); and PGE2 (1 mg/kg iv infused at time 10 during continuous pH 7.0 buffer superfusion and challenge with pH 7.0). MGT and perfusate mucus concentration were measured in each group and condition in separate animals.

Perfusate Glycoprotein Concentration Measurement

Preparation and protocol. A duodenal loop was prepared and perfused as modified from previously described methods (43, 44). In urethan-anesthetized rats, the abdomen was opened and the forestomach wall was incised 0.5 cm using a miniature electrocautery. A polyethylene tube (diameter, 5 mm) was inserted through the incision until it was 0.5 cm caudal from the pyloric ring, where it was secured with a nylon ligature. The distal duodenum was ligated proximal to the ligament of Treitz, before the duodenal loop was filled with 1 ml saline prewarmed at 37°C. The distal duodenum was then incised, through which another polyethylene tube was inserted and sutured into place. To prevent the contamination of the perfusate with bile-pancreatic juice, the pancreaticobiliary duct was ligated just proximal to its insertion into the duodenal wall. The resultant closed proximal duodenal loop (perfused length, 2 cm) was perfused with prewarmed saline using a Harvard infusion pump at 1 ml/min. A similar method was used to isolate and perfuse a 2-cm segment of distal duodenum, which extended distally from the common entrance of the bile and pancreatic ducts, a segment that in the rat is devoid of Brunner's glands (1). After a 30-min stabilization with pH 7.0 saline (time -40 to time -10), the perfusate was collected from time -10 to time 0 and for each subsequent 5-min period of perfusion. Solutions were perfused identically to the protocol described previously for MGT measurement; pH 7.0 saline was perfused for 10 min (time 0 to time 10; baseline), followed by either pH 7.0 or pH 2.2 saline for 10 min (time 10 to time 20; challenge period), and pH 7.0 saline for 15 min (time 20 to time 35; recovery period). The duodenum solution was gently flushed with 5 ml of perfusate to rapidly change the perfusate composition at time 10 and time 20. A 1-ml aliquot of sample was lyophilized with a Speed Vac (SC110, Savant Instruments, Holbrook, NY) and resuspended with 50 µl of distilled water (resuspended sample solution). The perfusate was frozen at less than -20°C before measurement of mucus glycoprotein.

Detection of glycoprotein in sample solutions. Mucus concentration in the perfusates was measured with PAS staining or pH 2.5 AB staining of polyvinylidene difluoride (PVDF) membranes as modified from previously described methods (11, 42). Five microliters of the resuspended sample solution were blotted on PVDF membrane (Hybond-P, Amersham International, Little Chalfont, UK) set on wet filter paper. For PAS staining, after a brief distilled water rinse, the membrane was exposed to 0.5% periodic acid solution for 15 min, rinsed with distilled water, and exposed to Schiff's reagent for 30 min followed by two 3-min exposures to 0.6% sodium metabisulfite. For AB staining, after a 15-min incubation with 3% BSA to reduce background, the membrane was stained with 1% AB in 3% acetic acid for 30 min followed by rinsing with 3% acetic acid and distilled water. After a final distilled water rinse, the membrane was dried, and density was digitized by a scanner (ScanJet 6100C, Hewlett Packard, Boise, ID). Image analysis was performed by measurement of dot-blot density as an eight-bit monochrome image (256 shades of gray) with the image-processing software described in Measurement of MGT.

To confirm the molecular mass of the PAS-positive protein in resuspended samples, SDS-PAGE was performed as previously described (47). Samples were boiled for 5 min in sample buffer containing 2.5% (vol/vol) 2-mercaptoethanol and 1% (wt/vol) SDS. Gels were composed of 4% stacking gel/7.5% running gel or 3% stacking gel/4% running gel. High and low molecular markers from Pharmacia (Piscataway, NJ) were used. Gels were stained with Coomassie brilliant blue R-250 (CBB; Bio-Rad) or PAS, as described previously (17, 35, 37).

Statistics

All data are means ± SE. Comparisons between groups were made by one-way ANOVA followed by Fisher's least significant difference test. P < 0.05 was taken as significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Imaging of Loaded Mucosa In Vivo and in Microsphere-Loaded Frozen Sections

Examination of mucosa loaded with BCECF and fluorescent microspheres in vivo produced images in which the focal plane included the fluorescent cell surface (Fig. 1A) or the red microspheres adherent to the luminal gel surface (Fig. 1C). Figure 1B depicts the red fluorescence at the tissue plane, in which out-of-focus microspheres are visible, and Fig. 1D depicts green fluorescence at the plane of the gel surface, in which out-of-focus tissue fluorescence is visible. z-Axis travel between these planes is also shown in Fig. 1, A-D (at lower right). The microspheres appear to lie on a plane parallel to that of the mucosal surface. Figure 1F depicts microspheres placed on a glass slide, with a similar appearance to those in Fig. 1D. This further confirms the quasiplanar nature of the microsphere distribution in the z-axis. Before the initial superfusion, the thickness of the gel overlying the duodenal mucosa was 136 ± 4 µm (n = 63, range = 92-236 µm) and stabilized to 90 ± 1 µm (n = 360, range = 54-139 µm) during a 1-h superfusion with Krebs buffer (pH 7.0). During superfusion, the microspheres remained visible in a planar configuration at the luminal gel surface, but the number of visible microspheres slowly decreased over time, indicating gradual removal of the surface layers of the mucus. Figure 2 depicts the temporal spatial distribution of microspheres during neutral and acid superfusions. Although some microspheres disappeared during the course of the experiment, the overall spatial distribution remained constant across time and changes in superfusate composition and MGT, confirming the firm adherence of the microspheres to the gel layer. Microsphere localization was confirmed with frozen sections of tissue loaded in vivo. In the sections, the microspheres were almost exclusively located on the gel surface (Fig. 1E). Interestingly, as shown previously (3), the mucus gel layer was clearly delineated by acridine orange staining. Furthermore, the gel spans the villi, indicating that the mucus gel layer is continuous over the mucosa.


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Fig. 1.   Imaging of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and microsphere-loaded mucosa in vivo and in frozen section. A and B: focal plane of mucosa. C and D: focal plane of luminal gel surface. Mucosal images under BCECF fluorescence are in focus under green fluorescence in A but out of focus in C. Conversely, the microspheres were out of focus in B but in focus in D. A-D: z-axis travel between focal plane is depicted at the lower right, with the mucosal plane defined as 0 µm. E: frozen section of microsphere-loaded mucosa counterstained with acridine orange reveals that red microspheres localized on the surface of the adherent mucus gel layer overlying the duodenal mucosa. F: fluorescent microspheres placed on a glass slide, demonstrating a similar distribution pattern to those overlying the mucus gel in D. Bars, 100 µm.



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Fig. 2.   Temporal distribution of microspheres in vivo. The microscope was focused on the focal plane of the microspheres. Superfusate was changed from pH 7.0 to pH 2.2 solution at 10 min and from pH 2.2 to pH 7.0 at 20 min. Yellow outline, corresponding microscopic fields. Sequential images of the same field show that the distribution of the microspheres remained constant despite changes of superfusate composition and an increase of mucus gel thickness (MGT) at time 15. Elapsed time is shown (each time is given as h:min:s:<FR><NU>1</NU><DE>10</DE></FR>-<FR><NU>1</NU><DE>100</DE></FR> s) with time 0 defined as the start of the baseline superfusion period.

Measurement of Perfusate Glycoprotein Concentration With Dot Blot Using PAS and AB Staining

Figure 3A depicts the dot blot images of solutions of partially purified pig gastric mucus (0.001-10 µg/µl) blotted on PVDF membrane and stained with PAS. The calibration curve depicted in Fig. 3B is the density of each dot calculated with image analysis as a function of the mucus concentration. An abbreviated calibration curve was included on each dot blot of experimental samples to control for development time and other variables. Because the calibration curve indicates a usable range of 0.03-1 µg/µl, resuspended lyophilized samples were used to increase sensitivity. A similar calibration curve was constructed by AB staining of blotted pig gastric mucus (data not shown).


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Fig. 3.   Detection of glycoprotein on polyvinylidene difluoride (PVDF) membrane with periodic acid/Schiff (PAS) staining. A: dot blot images of standard solutions of partially purified pig gastric mucus (0.001-10 µg/µl) blotted on PVDF membrane and stained with PAS. Lane 1, 10 µg/µl; lane 2, 5 µg/µl; lane 3, 1 µg/µl; lane 4, 0.5 µg/µl; lane 5, 0.33 µg/µl; lane 6, 0.2 µg/µl; lane 7, 0.1 µg/µl; lane 8, 0.05 µg/µl; lane 9, 0.033 µg/µl; lane 10, 0.02 µg/µl; lane 11, 0.01 µg/µl; lane 12, 0.005 µg/µl; lane 13, 0.001 µg/µl. B: calibration curve of dot-blot density vs. pig gastric mucus concentration.

Figure 4 depicts PAS (Fig. 4A) and AB staining (Fig. 4B) of concentrated perfusates obtained from the four experimental groups. Note that the density in the samples obtained from the control group was stable with minimum density fluctuation, whereas the density of the samples obtained from the acid group increased suddenly at time 15, gradually declining over the remaining time toward baseline. In samples obtained from the Indo group, baseline density (time 0 to time 10) was high, with density decreasing during and after acid exposure. In samples obtained from the PGE2 group, density progressively increased at time 15 and beyond for both stains.


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Fig. 4.   Dot-blot images of PAS- (A) and Alcian blue (AB)-positive (B) perfusate glycoproteins in control, acid, indomethacin (Indo), and PGE2 groups. Similar results in each group are obtained from 6 rats in a group. Note that pH 2.2 was perfused from time 10 to time 20 in the acid and Indo groups. PGE2 was injected intravenously at time 10.

Figure 5 depicts SDS-PAGE stained with CBB (Fig. 5, A and C) and PAS (Fig. 5, B and D) of 100 µg of partially purified gastric mucus and 10 µl of resuspended samples from the control (time 15), acid (time 15), and PGE2 groups (time 30). Fig. 5, A and B (7.5% running gel), and Fig. 5, C and D (4.0% running gel), were cut from the same gel, but stained differently. In Fig. 5A, although CBB stained several proteins with molecular masses of 40-100 kDa, the only PAS-positive material was of extremely high molecular mass, having barely entered the stacking gel. In Fig. 5D, these extreme high-molecular-weight bands are visible in addition to bands near the bottom of the gel, which are low-molecular-weight materials not visible in the corresponding location of the 7.5% gel. It is likely that the very high-mass PAS-positive material is mucus glycoprotein, which has a molecular mass of >600 kDa, consistent with the polymeric form of gastric mucin [molecular mass, 2 × 106 Da (20)], and the low density material is degraded glycoprotein, which is inferred from its molecular mass of <60 kDa and its presence in the sample of partially purified mucin.


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Fig. 5.   Detection of glycoproteins in SDS gels. Gels (A and B: 7.5%; C and D: 4%) were stained with Coomassie brilliant blue (CBB) (A and C) and PAS (B and D), respectively. Molecular markers (M) are indicated at left of each gel. Partially purified pig gastric mucus (G; 100 µg) and resuspended perfusate samples in control (c; time 15), acid (a; time 15), and PGE2 (PG; time 30) groups (10 µl each) were loaded.

Effect of Luminal Acid and Indo Pretreatment on MGT and Perfusate Glycoprotein Concentration

Figure 6 depicts MGT (Fig. 6A) and perfusate glycoprotein concentration measured by PAS (Fig. 6B) and AB staining (Fig. 6C) for the control, acid, and Indo groups. In the control group, MGT and PAS- and AB-positive perfusate glycoprotein concentrations were stable, not affected by solution changes at time 10 and time 20. In the acid group, MGT rapidly increased after mucosal exposure to pH 2.2, with rapid restoration of MGT toward baseline during the recovery period. Acid exposure also increased PAS-positive perfusate glycoprotein concentration from time 15 until time 22, followed by recovery to the baseline, whereas AB-positive glycoprotein transiently increased at time 15 only. Rapid parallel increases of MGT and perfusate glycoprotein concentration are consistent with simultaneous augmentation of mucus secretion and exudation rates, with the rate of increase of secretion exceeding the rate of exudation until a new steady state is reached. The rapid decrease of MGT corresponds to a decrease of secretion rate while the rate of exudation remains elevated, followed by a decrease in the exudation rate until a new steady state is reached. Rapid parallel increases of PAS- and AB-positive glycoprotein concentration are consistent with an increase of goblet cell-type mucus release, whereas sustained increase of only PAS-positive glycoprotein concentration may correspond to Brunner's gland-type mucus exudation. In the Indo group, baseline MGT was the same as in the control group whereas PAS-positive perfusate glycoprotein concentration was over fourfold higher and AB-positive was sixfold higher in the Indo group than in the control group. During the acid-challenge period, MGT decreased and remained below baseline during the recovery period. No further increase in perfusate glycoprotein concentration was observed during acid exposure, with PAS-positive perfusate glycoprotein concentration remaining higher than in the control group until time 20, whereas AB-positive glycoprotein decreased rapidly during acid exposure. Decreased MGT coupled with increased PAS-positive and decreased AB-positive perfusate glycoprotein concentration is consistent with Indo pretreatment destabilizing the gel, increasing exudation while inhibiting secretion from goblet cells during acid exposure.


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Fig. 6.   Effects of luminal acid and Indo pretreatment on MGT and perfusate glycoprotein concentration. In the control group (pH 7.0 only), MGT (A) and PAS- (B) and AB-positive (C) perfusate glycoprotein concentration were stabilized during the entire experimental period. During the challenge period, pH 2.2 rapidly increased MGT and perfusate glycoprotein concentration. In the Indo group, perfusate glycoprotein concentration was markedly elevated during the baseline period and decreased during the challenge period. * P < 0.05 vs. control group; dagger  P < 0.05 vs. acid group. Values are expressed as means ± SE from 6 rats.

To study the effect of Brunner's gland secretion on the composition of exuded mucus, parallel studies were done using an isolated segment of duodenum caudal to the common entrance of the bile and pancreatic ducts in untreated (control) rats. At time 0 and time 35, PAS-positive perfusate glycoproteins measured 0.79 ± 0.15 and 0.76 ± 0.14 µg/ml, respectively. AB-positive perfusate glycoproteins measured 0.80 ± 0.03 and 0.70 ± 0.14 µg/ml, respectively.

Effect of PGE2 on MGT and Perfusate Glycoprotein Concentration

An intravenous PGE2 bolus injection significantly increased PAS- and AB-positive perfusate glycoprotein concentration and MGT, which remained higher than in the untreated rats until time 35 (Fig. 7, A-C). The parallel increase of MGT and PAS-positive perfusate glycoprotein concentration induced by PGE2 is consistent with a simultaneous increase in mucus secretion rate and exudation, the latter occurring in the absence of perfused acid. Delayed increase of AB-positive glycoprotein with rapid increase of PAS-positive glycoprotein suggests that PGE2-stimulated mucus secretion augments Brunner's gland secretion before that of goblet cells.


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Fig. 7.   Effects of PGE2 on MGT and perfusate glycoprotein concentration. PGE2 (1 mg/kg iv) progressively increased MGT (A) and PAS- (B) and AB-positive (C) perfusate glycoprotein concentration. * P < 0.05 vs. control group. Values are expressed as means ± SE from 6 rats.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Described herein is a new method for the study of the dynamic regulation of rat duodenal MGT in response to luminal acid, PGE2, and COX inhibition in the duodenum. This is the first study in which steady-state duodenal MGT was measured in vivo in parallel to measurement of perfusate mucus concentration, the first to demonstrate a rapid response of duodenal MGT to exogenous prostaglandins, and the first to document a spontaneous, rapid decrease of MGT. Furthermore, this is the first study in which the relative contributions of Brunner's glands and goblet cells to the regulation of MGT were examined. With this system, we were able to formulate a preliminary understanding of how duodenal MGT is regulated.

The dynamic regulation of MGT in the gastrointestinal tract is one of the least-studied aspects of mucosal barrier function, perhaps due to the difficulties inherent in obtaining this measure in a living preparation. To measure MGT, we initially attempted to delineate the luminal gel surface with several different types of particles, including carbon. We found that polystyrene microspheres reliably and faithfully marked the luminal surface of the adherent gel. The spheres were chosen to be of a size large enough to be visualized but small enough not to interfere with the underlying cellular fluorescence; to have emission at a longer wavelength than BCECF fluorescence, which enabled microsphere identification on the basis of color; and to have density and surface characteristics that enable the microspheres to adhere to the luminal gel surface. MGT presumably represents only the contribution of the "firmly adherent gel," e.g., the component that resists hydromechanical shear forces, as opposed to the "loosely adherent" overlying layer, as originally described by Sababi et al. (33) and Sellers et al. (36). Shear forces were provided by continuous superfusion at a rate chosen to mimic physiological transit of luminal contents. Our studies are strongly consistent with removal of the loosely adherent layer during the stabilization period in which the superfusion rate provides the mechanical forces necessary to remove this layer, exposing the stable, adherent layer. MGT measured by this method (presuperfusion, 136 µm; steady state, 90 µm) is similar to that measured by other methods, i.e., 82 ± 7 µm in rats (24), 162 ± 45 µm in humans (34), and 236 ± 56 µm (before mechanical removal) and 74 ± 6 µm (recovery period after mechanical removal) in rats in vivo (33), but smaller than ~450 µm or 833 ± 72 µm in rats in vivo (9, 23), the latter reflecting the contribution of the unstirred layer (4).

Physiological secretion of mucus is controlled in three distinct phases. In the spontaneous or basal state (phase I), mucus is slowly secreted by exocytosis, with the rate of synthesis presumably matching the secretory rate (40). In phase I, goblet cells are loaded with apical mucus granules. In the steady state, MGT is governed by the opposing forces of mucus secretion and exudation. In response to secretory stimuli such as cholinergic agonists, prostaglandins, or guanylin, a rapid burst of secretion occurs by compound exocytosis within 2 min of the inciting signal, depleting the cells of preformed granules [phase II (10, 28, 39)] accompanied by membrane cavitation. In our studies, this presumed phase II secretory burst after acid exposure or PGE2 was accompanied by a rapid increase of MGT, followed by stabilization and a gradual decline toward baseline. Increased mucus secretion relative to exudation increases MGT whereas increased exudation relative to secretion decreases net MGT. The rapid increase of MGT in response to acid or PGE2 must hence reflect a marked increase of secretion relative to a smaller increase in rate of release into the perfusate. De novo mucus synthesis, occurring over hours, is far too slow to produce these changes (39). Enhanced exudation accompanied the sudden increase of MGT in response to acid or PGE2, as has been observed previously in the stomach (36), suggesting that a sudden increase of gel thickness per se, rather than acid exposure, increased mucus exudation. As an alternative possibility, we had considered that mucosal bicarbonate secretion, which is also enhanced by luminal acid and prostaglandins (43), may expand the gel layer by rapid generation of intragel CO2 produced in response to an acid-bicarbonate mixture. Increased mucus exudation in response to PGE2 and Indo in the absence of acid does not support this possibility. On the basis of our observations, the most likely explanation for the rapid shifts of MGT is that neurochemical stimuli such as luminal acid or prostaglandins induce phase II mucus secretion, which rapidly increases MGT. This suddenly thick gel layer is somewhat less stable than normal, raising its susceptibility to shear forces and thus increasing the rate of mucus release into the superfusate. These forces decrease MGT, which then reaches a new steady state as the increased rates of secretion and exudation come into balance. As the rate of secretion diminishes, MGT decreases toward baseline. To restore the mucus granule population, the synthesis rate increases in the ensuing hours (phase III).

The methods used to measure fluid mucus concentration have not been standardized. We chose the PAS and AB blotting methods for their sensitivity and simplicity (11, 22), bearing in mind that a reliable and reproducible means of quantitating fluid mucin content was more desirable than an exhaustive reckoning of all glycoprotein species. To minimize contributions from free carbohydrates, several modifications to the staining techniques were made, such as the use of PVDF membranes, which only bind to glycoproteins (42). The dot-blot system hence only detected PAS- and AB-positive glycoproteins. Furthermore, PAS staining of SDS gels showed that PAS-positive material in the perfusate had either a molecular mass of >600 kDa or <60 kDa, consistent with previous studies (35, 37) in which PAS-stained PAGE of crude or gel-purified mucins revealed bands that barely penetrated the gel. Mucins are the only very high-molecular-mass (>600 kDa) glycoproteins that are likely to be present in an intestinal perfusate. The lack of PAS-positive material with molecular mass between the very high and low molecular mass materials is consistent with a lack of contamination by nonmucin glycoproteins. The low molecular mass materials are likely to be degraded mucin glycoproteins that are compressed into a narrow band as an artifact of running the 4% gel. Our data are also supported by a previous study (19), in which perfusate hexosamine concentration was increased in perfused rabbit duodenal loops in response to acid exposure.

Histological staining of duodenum indicates that the two major mucus subtypes are identified by PAS and AB staining: PAS-positive Brunner's gland mucus and mixed AB- and PAS-positive goblet cell mucus. The correspondence between histological staining and core mucin polypeptide subtype (e.g., muc2, muc3, and muc6) has not yet been firmly established (12, 25). Acid-induced augmentation of mixed PAS- and AB-positive glycoproteins suggests that increased mucus secretion and exudation originates mainly from goblet cells. PGE2-associated increases in PAS-positive glycoproteins followed by increases in AB-positive glycoproteins suggest that PGE2 increases mucus secretion and exudation from both Brunner's glands and goblet cells. The short duration of increased AB-positive perfusate glycoprotein in response to acid and PGE2 suggests that goblet cells are rapidly depleted of mucus granules (10, 39). These studies indicate that proximal duodenal mucus secretion, and by implication the adherent mucus gel, is composed of mixed Brunner's gland and goblet cell secretions. A similar paradigm is likely present with gastric mucus, the adherent gel being composed of a laminar array of foveolar (MUC5AC) and deep gland (MUC6)-derived mucus (27). The limitation of the staining technique is that only relative changes can be measured because measurement of absolute amounts of mucus secretion requires comparison against purified rat goblet cell or Brunner's gland-derived mucus, which would be further complicated by the heterogeneous nature of goblet cell mucins and mucus (12). Nevertheless, the relative changes in perfusate glycoprotein concentration provide valuable data regarding the interplay between MGT, mucus secretion, and mucus exudation in the presence of acid, COX inhibition, and PGE2. Furthermore, the similar relative amounts of PAS- and AB-positive mucus derived from distal duodenum provide strong evidence in favor of goblet cell-derived mucus being an important component of exuded mucus from proximal and distal duodenum.

Perhaps the most difficult data to interpret were those obtained from the Indo group. At baseline, MGT was normal, although perfusate glycoprotein concentration was markedly elevated. To explain these data, we initially hypothesized that Indo pretreatment increased exudation of nonmucin glycoproteins into the perfusate, a contention not supported by our data, in which the only visible glycoproteins by PAGE were typical of mucins. The few studies of the effects of COX inhibition and exogenous prostaglandins on duodenal mucus secretion, MGT, and exudation also shed little light on this observation. For example, neither intragastric 16,16-dimethyl PGE2 nor Indo changed rat duodenal MGT, as measured by examination of thick sections (24). In dogs, intravenous Indo did not increase baseline duodenal perfusate glycoprotein release, although Indo inhibited a marked increase of mucin release by the prostaglandin precursor arachidonic acid (18). Indo reduced surface hydrophobicity of rat duodena, which may reflect altered mucus composition (21). Furthermore, Indo decreased mucin synthesis in gastric explants (41). Nevertheless, acute Indo administration does not alter MGT in stomach (6, 24, 46). Our observations are in substantial agreement with the findings of Sababi et al. (33), in which Indo inhibited the rate of MGT restoration after mechanical removal (33), which would correspond to the blunted increase of MGT during acid superfusion. Taking these data into account, it is possible to integrate our observations and those of others under a unifying hypothesis. In the steady state, Indo accelerates spontaneous phase I mucus secretory rate while inhibiting basal synthesis, gradually depleting the cells of granules. The mucus secreted in this fashion, perhaps due to altered composition, is less stable than normal and thus sheds into the lumen more readily. Combined increased exudation and secretion did not alter steady-state phase I MGT, at least within 1 h of Indo administration. With acid perfusion, there is no further secretion due to depletion of the remaining mucus granules, inhibiting the usual increase of MGT. In the recovery period, MGT stabilizes at a lower level as the mucus stores are exhausted. Over time, MGT presumably will decrease below baseline, because phase III synthesis is presumably inhibited. Another explanation is the effect of Indo on motility, since Indo increases gastroduodenal motility (32), which may affect the exudation rate.

In summary, the thickness of the duodenal mucus gel layer represents the balance between the opposing forces of mucus secretion and the sloughing of the luminal portion of the gel surface by mechanical forces. The gel in its steady state is intrinsically stable relative to a gel that has suddenly increased thickness or one that has formed after exposure to systemic Indo. The significant role played by prostaglandins and COX inhibition on mucus dynamics may explain how COX inhibitors weaken the preepithelial component of the duodenal mucosal barrier to acid, increasing susceptibility to injury.


    ACKNOWLEDGEMENTS

We thank Dipty Shah and William Matterer for technical assistance.


    FOOTNOTES

This study was supported by Veterans Affairs Merit Review funding and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54221.

Address for reprint requests and other correspondence: J. D. Kaunitz, Bldg. 114, Suite 217, West Los Angeles VA Medical Center, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail: jake{at}ucla.edu).

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.

Received 17 September 1999; accepted in final form 7 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ainsworth, MA, Koss MA, Hogan DL, and Isenberg JI. Higher proximal duodenal mucosal bicarbonate secretion is independent of Brunner's glands in rats and rabbits. Gastroenterology 109: 1160-1166, 1997[ISI][Medline].

2.   Akiba, Y, Guth PH, Engel E, Nastaskin I, and Kaunitz JD. Acid-sensing pathways of rat duodenum. Am J Physiol Gastrointest Liver Physiol 277: G268-G274, 1999[Abstract/Free Full Text].

3.   Akiba, Y, and Kaunitz JD. Regulation of intracellular pH and blood flow in rat duodenal epithelium in vivo. Am J Physiol Gastrointest Liver Physiol 276: G293-G302, 1999[Abstract/Free Full Text].

4.   Allen, A, and Carroll NJ. Adherent and soluble mucus in the stomach and duodenum. Dig Dis Sci 30, Suppl: 55S-62S, 1985[Medline].

5.   Bickel, M. Effect of 16,16-dimethyl prostaglandin E2 on gastric mucus gel thickness. Prostaglandins 21, Suppl: 63-65, 1981[Medline].

6.   Bickel, M, and Kauffman J. Gastric gel mucus thickness: effect of distention, 16,16-dimethyl prostaglandin E2 and carbenoxolone. Gastroenterology 80: 770-775, 1981[ISI][Medline].

7.   Brown, JF, Hanson PJ, and Whittle BJ. Nitric oxide donors increase mucus gel thickness in rat stomach. Eur J Pharmacol 223: 103-104, 1992[ISI][Medline].

8.   Cohan, VL, Scott AL, Dinarello CA, and Prendergast RA. Interleukin-1 is a mucus secretagogue. Cell Immunol 136: 425-434, 1991[ISI][Medline].

9.   Flemström, G, and Kivilaakso E. Demonstration of a pH gradient at the luminal surface of rat duodenum in vivo and its dependence on mucosal alkaline secretion. Gastroenterology 84: 787-794, 1983[ISI][Medline].

10.   Furuya, S, Naruse S, and Hayakawa T. Intravenous injection of guanylin induces mucus secretion from goblet cells in rat duodenal crypts. Anat Embryol (Berl) 197: 359-367, 1998[ISI][Medline].

11.   Goso, Y, and Hotta K. Dot blot analysis of rat gastric mucin using histochemical staining methods. Anal Biochem 223: 274-279, 1994[ISI][Medline].

12.   Ho, SB, Robertson AM, Shekels LL, Lyftogt CT, Niehans GA, and Toribara NW. Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression. Gastroenterology 109: 735-747, 1995[ISI][Medline].

13.   Kaunitz, JD, Nishizaki Y, Kaneko K, and Guth PH. Effect of orogastric nicotine on rat gastric mucosal gel thickness, surface cell viability, and intracellular pH. J Pharmacol Exp Ther 265: 948-954, 1993[Abstract].

14.   Kerss, S, Allen A, and Garner A. A simple method for measuring thickness of the mucus gel layer adherent to rat, frog and human gastric mucosa: influence to feeding, prostaglandin, N-acetylcysteine and other agents. Clin Sci (Colch) 63: 187-195, 1982[ISI][Medline].

15.   Kirkegaard, P, Lundberg JM, Poulsen SS, Olsen PS, Fahrenkrug J, Hokfelt T, and Christiansen J. Vasoactive intestinal polypeptidergic nerves and Brunner's gland secretion in the rat. Gastroenterology 81: 872-878, 1981[ISI][Medline].

16.   Kirkegaard, P, Skov O, Seier P, Holst JJ, Schaffalitzky de Muckadell OB, and Christiansen J. Effect of secretin and glucagon on Brunner's gland secretion in the rat. Gut 25: 264-268, 1984[Abstract].

17.   Konat, G, Offner H, and Mellah J. Improved sensitivity for detection and quantitation of glycoproteins on polyacrylamide gels. Experientia 40: 303-304, 1984[ISI].

18.   Kosmala, M, Carter SR, Konturek SJ, Slomiany A, and Slomiany BL. Mucus glycoprotein secretion by duodenal mucosa in response to luminal arachidonic acid. Biochim Biophys Acta 884: 419-428, 1986[ISI][Medline].

19.   Lang, IM, and Tansy MF. Mechanisms of the secretory and motor responses of the Brunner's gland region of the intestines to duodenal acidification. Pflügers Arch 396: 115-120, 1983[ISI][Medline].

20.   Lee, SP, Nicholls JF, and Robertson AM. Effects of trimoprostil, a prostaglandin E2 analog, on human gastric acid secretion and soluble mucin output. Eur J Clin Invest 17: 1-6, 1987[ISI][Medline].

21.   Lugea, A, Antolin M, Mourelle M, Guarner F, and Malagelada JR. Deranged hydrophobic barrier of the rat gastroduodenal mucosa after parenteral nonsteroidal anti-inflammatory drugs. Gastroenterology 112: 1931-1939, 1997[ISI][Medline].

22.   Mantle, M, and Allen A. A colorimetric assay for glycoproteins based on the periodic acid/Schiff stain. Biochem Soc Trans 6: 607-609, 1978[Medline].

23.   Matsueda, K, Muraoka A, Umeda N, Misaki N, Uchida M, and Kawano O. In vitro measurement of the pH gradient and thickness of the duodenal mucus gel layer in rats. Scand J Gastroenterol 24, Suppl162: 31-34, 1989[ISI].

24.   McQueen, S, Hutton D, Allen A, and Garner A. Gastric and duodenal surface mucus gel thickness in rat: effects of prostaglandins and damaging agents. Am J Physiol Gastrointest Liver Physiol 245: G388-G393, 1983[Free Full Text].

25.   Morrissey, SM, Ward PM, Jayaraj AP, Tovey FI, and Clark CG. Histochemical changes in mucus in duodenal ulceration. Gut 24: 909-913, 1983[Abstract].

26.   Nishizaki, Y, Guth PH, Kim G, Wayland H, and Kaunitz JD. Pentagastrin enhances gastric mucosal defenses in vivo: luminal acid-dependent and independent effects. Am J Physiol Gastrointest Liver Physiol 267: G94-G104, 1994[Abstract/Free Full Text].

27.   Ota, H, and Katsuyama T. Alternating laminated array of two types of mucin in the human gastric surface mucous layer. Histochem J 24: 86-92, 1992[ISI][Medline].

28.   Phillips, TE. Both crypt and villus intestinal goblet cells secrete mucin in response to cholinergic stimulation. Am J Physiol Gastrointest Liver Physiol 262: G327-G331, 1992[Abstract/Free Full Text].

29.   Phillips, TE, and Wilson J. Signal transduction pathways mediating mucin secretion from intestinal goblet cells. Dig Dis Sci 38: 1046-1054, 1993[ISI][Medline].

30.   Price, KJ, Hanson PJ, and Whittle BJ. Stimulation by carbachol of mucus gel thickness in rat stomach involves nitric oxide. Eur J Pharmacol 263: 199-202, 1994[ISI][Medline].

31.   Rubinstein, A, and Tirosh B. Mucus gel thickness and turnover in the gastrointestinal tract of the rat: response to cholinergic stimulus and implication for mucoadhesion. Pharm Res 11: 794-799, 1994[ISI][Medline].

32.   Sababi, M, Hällgren A, and Nylander O. Interaction between prostanoids, NO, and VIP in modulation of duodenal alkaline secretion and motility. Am J Physiol Gastrointest Liver Physiol 271: G582-G590, 1996[Abstract/Free Full Text].

33.   Sababi, M, Nilsson E, and Holm L. Mucus and alkali secretion in the rat duodenum: effects of indomethacin, Nomega -nitro-L-arginine, and luminal acid. Gastroenterology 109: 1526-1534, 1995[ISI][Medline].

34.   Sarosiek, J, Marshall BJ, Puera DA, Hoffman S, Feng T, and McCallum RW. Gastroduodenal mucus gel thickness in patients with Helicobacter pylori: a method for assessment of biopsy specimens. Am J Gastroenterol 86: 729-734, 1991[ISI][Medline].

35.   Schumacher, U, and Krause WJ. Molecular anatomy of an endodermal gland: investigations on mucus glycoproteins and cell turnover in Brunner's glands of Didelphis virginiana using lectins and PCNA immunoreactivity. J Cell Biochem 58: 56-64, 1995[ISI][Medline].

36.   Sellers, LA, Carroll NJH, and Allen A. Misoprostol-induced increases in adherent gastric mucus thickness and luminal mucus output. Dig Dis Sci 31, Suppl: 91S-95S, 1986[Medline].

37.   Smits, HL, van Kerkhof PJ, and Kramer MF. Isolation and partial characterization of rat duodenal-gland (Brunner's-gland) mucus glycoprotein. Biochem J 203: 779-785, 1982[ISI][Medline].

38.   Soll, A. Pathogenesis of nonsteroidal anti-inflammatory drug-related upper gastrointestinal toxicity. Am J Med 105, Suppl: 10S-16S, 1998[Medline].

39.   Specian, RD, and Neutra MR. Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine. J Cell Biol 85: 626-640, 1980[Abstract].

40.   Specian, RD, and Neutra MR. Regulation of intestinal goblet cell secretion. I. Role of parasympathetic stimulation. Am J Physiol Gastrointest Liver Physiol 242: G370-G379, 1982[Abstract/Free Full Text].

41.   Stanley, CM, and Phillips TE. Bradykinin modulates mucin secretion but not synthesis from an intestinal goblet cell line. Agents Actions 42: 141-145, 1994[ISI][Medline].

42.   Stromqvist, M, and Gruffman H. Periodic acid/Schiff staining of glycoproteins immobilized on a blotting matrix. Biotechniques 13: 744-746, 1992[ISI][Medline].

43.   Takeuchi, K, Matsumoto J, Ueshima K, and Okabe S. Role of capsaicin-sensitive afferent neurons in alkaline secretory response to luminal acid in the rat duodenum. Gastroenterology 101: 954-961, 1991[ISI][Medline].

44.   Takeuchi, K, Tanaka H, Furukawa O, and Okabe S. Gastroduodenal HCO3- secretion in anesthetized rats: effects of 16,16-dimethyl PGE2, topical acid, and acetazolamide. Jpn J Pharmacol 41: 87-99, 1986[ISI][Medline].

45.   Tanaka, S, Akiba Y, and Kaunitz JD. Pentagastrin gastroprotection against acid is related to H2 receptor activation but not acid secretion. Gut 43: 334-341, 1998[Abstract/Free Full Text].

46.   Tanaka, S, and Kaunitz JD. Indomethacin does not alter the effect of pentagastrin on rat gastric defense mechanisms. Peptides 17: 155-159, 1996[ISI][Medline].

47.   Tytgat, KM, Bovelander FJ, Opdam FJ, Einerhand AW, Buller HA, and Dekker J. Biosynthesis of rat MUC2 in colon and its analogy with human MUC2. Biochem J 309: 221-229, 1995[ISI][Medline].

48.   van Klinken, BJ, Einerhand AW, Duits LA, Makkink MK, Tytgat KM, Renes IB, Verburg M, Buller HA, and Dekker J. Gastrointestinal expression and partial cDNA cloning of murine Muc2. Am J Physiol Gastrointest Liver Physiol 276: G115-G124, 1999[Abstract/Free Full Text].


Am J Physiol Gastrointest Liver Physiol 279(2):G437-G447