The mucus layer continuously covering the gastric mucosa consists of a loosely adherent layer that can be easily removed by suction, leaving a firmly adherent mucus layer attached to the epithelium. These two layers exhibit different gastroprotective roles; therefore, individual regulation of thickness and mucin composition were studied. Mucus thickness was measured in vivo with micropipettes in anesthetized mice [isoflurane; C57BL/6, Muc1−/−, inducible nitric oxide synthase (iNOS)−/−, and neuronal NOS (nNOS)−/−] and rats (inactin) after surgical exposure of the gastric mucosa. The two mucus layers covering the gastric mucosa were differently regulated. Luminal administration of PGE2 increased the thickness of both layers, whereas luminal NO stimulated only firmly adherent mucus accumulation. A new gastroprotective role for iNOS was indicated since iNOS-deficient mice had thinner firmly adherent mucus layers and a lower mucus accumulation rate, whereas nNOS did not appear to be involved in mucus secretion. Downregulation of gastric mucus accumulation was observed in Muc1−/− mice. Both the firmly and loosely adherent mucus layers consisted of Muc5ac mucins. In conclusion, this study showed that, even though both the two mucus layers covering the gastric mucosa consist of Muc5ac, they are differently regulated by luminal PGE2 and NO. A new gastroprotective role for iNOS was indicated since iNOS−/− mice had a thinner firmly adherent mucus layer. In addition, a regulatory role of Muc1 was demonstrated since downregulation of gastric mucus accumulation was observed in Muc1−/− mice.
- nitric oxide
a constantly renewed mucus layer covers the gastrointestinal (GI) tract and functions as a barrier between luminal contents and the mucosa. The thickness of the mucus layer depends on both secretion of mucins and the degree of erosion and proteolytic degradation. An increase in mucus thickness is a normal defensive response to luminal insults, and it is generally believed that, the thicker the mucus, the better the protection (1). Mucus is secreted by epithelial cells, and its main constituents are large glycoproteins, mucins, and water. In the stomach, two cell types secreting different mucins have been identified, surface mucus cells that secrete MUC5AC and mucus neck cells that secrete MUC6 (10). In addition to secreted mucins, the transmembrane mucins MUC1, MUC4, and MUC16 are expressed in the stomach. Transmembrane mucins consist of a large extracellular domain, a transmembrane domain, and a cytoplasmic domain and are suggested to be involved in signal transduction and cell adhesion phenomena (31).
The continuous mucus layer covering the gastric mucosa can be separated into two different layers, in addition to degraded mucus in the lumen (5, 12). The outer of the two layers, the loosely adherent mucus, can be removed by suction or by rubbing with a cotton tip, whereas, the inner layer, the firmly adherent mucus, cannot be removed by mechanical means without destroying the epithelial layer. How the firmly adherent mucus layer attaches or anchors to the mucosa is unknown, as is the contribution of Muc5ac and Muc6 to the different layers.
When pH in the gastric lumen is acidic, the mucus layer is important for establishing and maintaining a pH gradient with a neutral pH in the mucus closest to the epithelium (the juxtamucosal pH). Under normal conditions, the loosely adherent mucus layer of the gastric corpus is not needed for maintaining the neutral juxtamucosal pH (28). This indicates that the firmly adherent mucus is more important in protecting the gastric mucosa from corrosive acid. The loosely adherent mucus has other functions, such as binding luminal noxious agents, binding swallowed nitrite, and continuously releasing nitric oxide (NO) (27). Hence, the two mucus layers covering the gastric mucosa have dissimilar functions, and further understanding of their independent regulation and constituents is required.
The mucus barrier is a highly hydrated extracellular compartment with physical properties that limit the use of conventional in vitro and histological methods. The methodological difficulties in studying mucus in vitro results in a very thin or even discontinuous mucus layer as the mucus becomes dehydrated and eroded (reviewed in Ref. 2). Therefore, in vivo mucus measurements are more reliable for measuring thickness, accumulation, and degradation of the mucus layer in the GI tract. Nevertheless, in vitro studies have reported that prostaglandin E2 (PGE2) and NO stimulate mucus secretion and that the mucus layer can be degraded by proteases originating from enteric parasites (3, 8, 9, 18).
The present in vivo study investigated whether the membrane-bound mucin Muc1 was involved in anchoring the firmly adherent mucus layer to the mucosa. The effect of PGE2 and NO from different NO synthases (NOS) in regulating the thickness of the two different mucus layers was also studied. In addition, the contribution of different mucins to the two mucus layers was determined by proteomics.
MATERIALS AND METHODS
All animals were kept under standardized conditions of temperature (21–22°C) and illumination (12 h light/12 h dark). They were kept in cages with mesh bottoms and had free access to tap water and pelleted food (Ewos, Södertälje, Sweden; Lactamin, Kinstad, Sweden).
Male F1 hybrids of Lewis and Dark Agouti rats (Animal Department at the Biomedical Centre, Uppsala, Sweden) weighing between 172–270 g were used. The rats were deprived of food but not water for 17–20 h before they were anesthetized by an intraperitoneal injection of thiobutabarbital sodium (Inactin; Research Biochemicals International, Natick, MA; 120 mg/kg body wt). Spontaneous breathing was facilitated by a short PE-200 cannula placed in the trachea, and body temperature was maintained at 37.5 ± 0.5°C by means of a heating pad controlled by a rectal thermistor. A PE-50 cannula containing heparin (KabiVitrum, Stockholm, Sweden; Leo Pharma, Malmö, Sweden; 12.5 IU/ml) dissolved in isotonic saline was placed in the right femoral artery to monitor blood pressure. The right femoral vein was catheterized for administration of Ringer's solution at an infusion rate of 1.0 ml/h (120 mM NaCl, 2.5 mM KCl, 0.75 mM CaCl2, and 25 mM NaHCO3).
Mice weighing between 22–43 g were used. Breeding pairs of heterozygous neuronal NOS (nNOS)-deficient mice were kept under standardized conditions as mentioned above. The mice (background C57BL/6 × 129Sv) were generated by gene targeting in embryonic stem cells (16). Both female and male mice were used for the experiments. The genotype of each nNOS mouse was determined by PCR analysis of DNA isolated from tail tissue. Mice deficient in the inducible NOS (iNOS) gene (background C57BL/6 × 129SvEv) were generated by gene targeting in embryonic stem cells as previously described (21). Homozygous iNOS mutants were maintained by interbreeding the F2 generation, and only males were used in the experiments. For wild-type controls, male C57BL/6 × 129Sv were used (Taconic Farms, Germantown, NY) and pooled with the littermates (nNOS+/+) from the nNOS breeding. Homozygous Muc1-deficient mice (both sexes, background C57BL/6, 32) were compared with wild-type C57BL/6 mice (males; B & K Universal, Stockholm, Sweden).
The mice were anesthetized with spontaneous inhalation of isoflurane (Forene; Abbott Scandinavia, Kista, Sweden). The inhalation gas (≈2.2% isoflurane) was continuously administered through a breathing mask (Simtec Engineering, Askim, Sweden) in a mixture of 40% oxygen and 60% nitrogen. Body temperature was maintained at 37°C by means of a heating pad controlled by a rectal thermistor probe. A catheter containing heparin dissolved in isotonic saline was placed in the left carotid artery to monitor blood pressure. The jugular vein was cannulated for continuous infusion of Ringer's solution at a rate of 0.35 ml/h.
All experimental procedures in this study have been approved by the Swedish Laboratory Animal Ethical Committee in Uppsala and were conducted in accordance with guidelines of the Swedish National Board for Laboratory Animals.
The gastric preparation for in vivo microscopy has previously been described in detail (12, 15). Briefly, exteriorization of the stomach through a midline abdominal incision was followed by an incision along the greater curvature in the forestomach. The animal was placed on its left side on a Lucite table. Part of the corpus of the stomach was loosely draped over a truncated cone in the center of the table with the mucosal surface facing upwards. A mucosal chamber, with a hole in the bottom corresponding to the position of the cone, was placed over the mucosa, exposing ∼1.2 cm2 of the rat gastric mucosa and 0.3 cm2 of the mouse gastric mucosa through the hole. The mucosal chamber did not touch the mucosa, to not impair blood flow, and the edges of the hole were sealed with silicon grease (Dow Corning High Vacuum Grease; Dow Corning, Wiesbaden, Germany). The mucosal chamber was filled with 5 ml (rat) or 3 ml (mouse) of 0.9% NaCl maintained at 37°C by circulating warm water in a jacket in the bottom of the chamber. The gastric mucosa was observed in a stereomicroscope (Leica MZ12; Leica, Heerbrugg, Switzerland), and the gastric mucosa was transilluminated with light from a 150 W light source guided by fiber optics.
Measurements of Mucus Gel Thickness
Mucus thickness was measured with micropipettes connected to a micromanipulator (Leitz, Wetzlar, Germany) with a digimatic indicator (IDC Series 543; Mitutoyo, Tokyo, Japan). Glass tubing (borosilicate tubing with 1.2 mm OD and 0.6 mm ID; Frederick Haer, Brunswick, ME) was pulled with a pipette puller (pp-83; Narishige Scientific Instrument Laboratories, Tokyo, Japan) to a tip diameter of 1–3 μm. To prevent mucus adhering to glass, the pipettes were siliconized by dipping the tip of the micropipette into a silicon solution (MS1107, 25% acetone), followed by drying at 100°C for 30 min. The luminal surface of the mucus gel was visualized by placing graphite particles (activated charcoal, extra pure; Merck, Darmstadt, Germany) on the gel, and the gastric epithelial cell surface was visible through the microscope. The micropipette was inserted into the mucus gel at an angle of ∼30 degrees to the surface. The angle was measured with a protractor, and the same angle was maintained throughout an experiment. The distances travelled by the micropipette from the luminal surface of the mucus gel to the epithelial cell surface were measured, and a mean value was calculated. The mucus thickness, which is the vertical distance between the cell surface and the luminal mucus surface, was then calculated. The mean value of four to five different measurements was taken as one thickness value.
The loosely adherent mucus layer was removed by gentle suction with a thin polyethylene cannula connected either to a syringe or to a vacuum pump. This procedure was conducted under supervision through a stereomicroscope to avoid contact with the epithelium.
When the blood pressure had stabilized after surgery (30–60-min rest period), experiments were commenced. Total mucus thickness was measured before removal of the loosely adherent layer, which was immediately followed by a measurement of the firmly adherent mucus layer. Mucus accumulation was then studied by measuring the mucus thickness in rats every 10 min for 20 min and, in mice, every 15 min for 30 min. The loosely adherent mucus layer was then removed again, and the thickness of the firmly adherent mucus layer was measured a second time. In some instances in control rats and mice, slight decreases of the firmly adherent layer after the second removal compared with the first removal were noted, most probably due to remaining loose mucus after the first removal. The values of the firmly adherent mucus thickness obtained after the second mucus removal is therefore presented in the graphs.
To study the effect of prostaglandins on the accumulation of the mucus layers in rats, PGE2 (1 or 10 μg/ml; Sigma Aldrich, Stockholm, Sweden) was applied luminally for two consecutive 10-min periods after the first mucus removal. To inhibit endogenous prostaglandin production, one group of rats was pretreated with the cyclooxygenase inhibitor indomethacin (indo) (3 mg/kg iv, diluted in saline; Confortid; Dumex, Copenhagen, Denmark) 1 h before mucus measurements commenced. Control mice (C57BL/6) were pretreated with diclofenac (5 mg/kg iv, diluted in saline; Voltaren; Novartis, Täby, Sweden) 60 min before mucus measurements.
To study the effect of NO, one group of rats received the NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 0.3 mM; Sigma Aldrich) topically for two consecutive 10-min periods after the first mucus removal. To inhibit endogenous NO production, the nonspecific NOS inhibitor Nω-nitro-l-arginine (l-NNA, Sigma Aldrich) or the iNOS-specific inhibitor l-N6-(1-aminoethyl)-lysine (l-NIL; Alexis, Läufelfingen, Switzerland) was given in a dose of 10 mg/kg iv bolus followed by 3 mg/kg per h continuous intravenous infusion throughout the experiment. Mucus measurements were then performed when blood pressure stabilized and at least 30 min after the bolus dose.
Indo and l-NIL treatments were combined to investigate whether mucus secretion could be further decreased. In these experiments, two sets of mucus measurements were performed per rat, with a 40-min resting period in between. This resulted in longer experiments: a stabilization period of 60 min followed by a mucus measurement period as described above, followed by a 40-min stabilization period, and then the last mucus measurement set. The following combinations were investigated: untreated + untreated, indo + indo, l-NIL + l-NIL, and indo + l-NIL.
To investigate whether NO had the same effect in mice as in rats, 0.3 mM SNAP was applied topically for 30 min after the first mucus removal. The involvement of the neuronal and inducible isoforms of NO synthesizing enzymes (nNOS and iNOS) in mucus accumulation of the different mucus layers was studied in genetically modified animals. To examine whether prostaglandin inhibition could further decrease the firmly adherent mucus layer in iNOS-deficient mice, diclofenac (5 mg/kg iv, diluted in saline) was given after the first measurement of the firmly adherent mucus, and the firmly adherent mucus was measured again 60 min later.
The involvement of the membrane-bound mucin Muc1 in mucus layer thickness and adhesion to the mucosa was investigated in mice deficient in Muc1.
Mucus accumulation rate.
Mucus accumulation rate (μm/min) was calculated as the difference between the 20-min value of mucus thickness (30-min value in mice) and the thickness of the mucus after the first mucus removal divided with the time (min) between these mucus measurements.
The loosely adherent mucus thickness.
The loosely adherent mucus thickness (μm) was calculated by subtracting the 20-min value of mucus thickness (30-min value in mice) with the thickness of the mucus after the second mucus removal. This value gave the thickness of the loosely adherent mucus that was removed at 20 min (30 min in mice).
Analysis of mucin contents in the two different mucus layers.
The two mucus layers were collected from C57BL/6 mice by suction (loosely adherent), followed by gentle scraping (firmly adherent). Mucus sampled from the two layers was analyzed by electrophoresis on a 4–12% acrylamide gel. Separated proteins were stained with Imperial stain (Pierce, Rockford, IL). Stained high molecular mass bands were selected and excised and then were destained for 3 × 45 min (500 μl, 50% acetonitrile, 25 mM NH4HCO3). The gel pieces were dried in vacuum, and then 12 μl trypsin (10 μg/ml in 25 mM NH4HCO3; Promega, Madison, WI) was added; they were incubated overnight at 37°C. Peptides were extracted in 15 μl of stop solution (50% acetonitrile, 2% trifluoracetic acid) for 30 min and transferred to 0.6-ml tubes (AxyGen, Union City, CA). The extraction was repeated in 20 μl (50% acetonitrile, 0.2% trifluoracetic acid) and the extracts pooled. The extracts were lyophilized and resuspended in 0.1% acetic acid and stored in −20°C until analyzed. The samples were centrifuged at 16,000 g for 15 min, and the supernatant was used for the analysis.
Samples were separated by nano-LC coupled to a hybrid linear ion trap-FT-ICR mass spectrometry (MS) equipped with a 7T ICR magnet (LTQ-FT; Thermo Finnegan, Bremen, Germany). The peptide analysis has previously been described in detail (4).
Peak lists were extracted from raw data with the program ‘extract_ msn.exe’ (Thermo Finnegan). No smoothing was applied. Searches were with the search program MASCOT (Version 2.1.0; Matrix Science, London, UK). The search parameters were set to MS accuracy 5 ppm, MS/MS accuracy 0.5 Da, one missed cleavage allowed, fixed propionamide modification of cysteine, and variable modification of oxidized methionine. The three upper bands from two gels were merged in the search. The tandem mass spectra from the LC-FT MS/MS experiments were searched against an in-house mucin database (www.medkem.gu.se/mucinbiology/databases) containing the assembled mouse mucin sequences version 1. The database contains the Muc5ac and Muc6 sequences with sequence gaps in the mucin domain.
The results are expressed as means ± SE. For statistical evaluations of differences between data within a group, ANOVA for repeated measures was used; ANOVA for multiple comparisons compared data between groups. ANOVA was followed by Fisher's protected least-significant difference test. To compare single values, Student's t-test for paired or unpaired data was used. All statistical calculations were performed with StatView II SE Graphics (Abacus Concepts, Berkeley, CA). The differences were regarded as significant at P < 0.05.
The transmembrane mucin Muc1 regulates mucus accumulation of both firmly and loosely adherent mucus layers.
To test the hypothesis that Muc1 was involved in anchoring the firmly adherent mucus to the mucosa, the mucus was measured in mice deficient in Muc1. The inner firmly adherent mucus layer was still present and could not be removed by suction in Muc1−/− mice, demonstrating that Muc1 does not contribute to the attachment of the mucus layer to the mucosa (Fig. 1). The mucus measurements in Muc1−/− mice revealed that Muc1 was involved in regulating the thickness of the mucus layers. The total mucus accumulation rate decreased, and the inner firmly adherent mucus layer was thinner in Muc1−/− mice than in wild-type mice (Fig. 1).
Luminal NO donor increases mucus accumulation of firmly but not loosely adherent mucus layer.
To investigate whether luminal NO stimulated mucus secretion, the NO donor SNAP, 0.3 mM, was applied on the rat gastric mucosa for 20 min. This resulted in an amplified total mucus accumulation rate compared with untreated rats and was due to increased growth of the firmly adherent mucus layer but not an increase in the loosely adherent mucus layer (Fig. 2).
When the NO donor SNAP was applied (0.3 mM) topically on the gastric mucosa in C57BL/6 mice, the thickness of the firmly adherent mucus layer increased (Fig. 1), but the accumulation of the loosely adherent mucus layer remained unchanged. Total mucus accumulation tended to increase compared with controls even though this did not attain statistical significance (P = 0.10; SNAP, 0.89 ± 0.14 μm/min; control, 0.58 ± 0.11 μm/min). These results indicated that luminal NO increased the firmly but not the loosely adherent mucus layer.
iNOS-derived, not nNOS-derived, NO increases mucus accumulation.
The role of endogenously produced NO in mucus secretion was then studied. Rats were pretreated with the nonspecific NOS inhibitor l-NNA (10 mg/kg iv bolus followed by 3 mg/kg per h continuous intravenous infusion), but this did not significantly decrease total mucus accumulation even though there was a tendency toward a reduction in the firmly adherent layer after the second mucus removal (Fig. 2).
Constitutively expressed iNOS in the gastric epithelium has been identified (29), and, in the present study, its role in mucus secretion was investigated by pretreating rats with the iNOS-specific inhibitor l-NIL (10 mg/kg iv bolus followed by 3 mg/kg per h continuous intravenous infusion). After 40 min with l-NIL and the first mucus removal, the firmly adherent mucus layer decreased (49 ± 1 μm) compared with untreated (72 ± 4 μm) and l-NNA-treated (64 ± 6 μm) rats.
To clarify whether iNOS regulated mucus secretion in mice as well as rats, and to avoid problems related to pharmacological inhibition of the enzyme, mice with an inactivated gene for iNOS were used. Total mucus accumulation rate in iNOS−/− mice was about 25% of that observed in wild-type mice (C57BL/6 × 129Sv, Fig. 1). iNOS−/− mice had a thinner firmly adherent mucus layer compared with wild-type mice after the second mucus removal (Fig. 1). In mice with inactivated gene for nNOS, total mucus accumulation did not differ compared with wild-type controls (C57BL/6 × 129Sv, Fig. 1).
PGE2 increases mucus accumulation of both firmly and loosely adherent mucus layers.
Application of luminal PGE2 for 20 min in a concentration of 10 μg/ml increased total mucus accumulation rate more than four times compared with control rats (Fig. 2). This was due to increased accumulation of both the firmly adherent and the loosely adherent mucus layers. A lower concentration of PGE2 (1 μg/ml) did not cause an increase in total mucus accumulation (data not shown). Indomethacin (3 mg/kg iv 60 min before mucus measurements) caused a thinner firmly adherent mucus layer than in untreated control rats (Fig. 2). In agreement to this, diclofenac-inhibited prostaglandin synthesis in mice resulted in a significantly decreased firmly adherent mucus layer (Fig. 3B).
Prostaglandin inhibition does not further decrease mucus accumulation during iNOS inhibition.
To investigate whether inhibition of both prostaglandins and NO would result in a further decreased firmly adherent mucus thickness, iNOS was inhibited by l-NIL during prostaglandin synthesis inhibition with indomethacin. Mucus thickness measurements revealed that l-NIL treatment resulted in a thinner firmly adherent mucus layer than in untreated and indomethacin-treated rats (Fig. 3A). However, the firmly adherent mucus layer did not become any thinner whether indomethacin was given in combination with l-NIL.
Similarly, the firmly adherent mucus layer in iNOS-deficient mice was not significantly thinner after 60 min of prostaglandin inhibition (diclofenac) compared with the measurement made before prostaglandin inhibition (Fig. 3B). Even less difference was observed if compared with the firmly adherent mucus layer measurement after the second mucus removal in iNOS−/− mice (Fig. 1).
Proteomics analysis reveals Muc5ac as a main mucin component in the loosely and firmly adherent mucus layers.
The molecular nature of the mucins in the different mucus layers was analyzed by proteomics adapted for the identification of mucins core protein. Muc5ac mucin was a main component in both layers with a for mucin good peptide coverage (Fig. 4; a complete peptide list is given in Supplementary Table 1; supplemental information for this article is provided online at the American Journal of Physiology Gastrointestinal and Liver Physiology website). Slightly fewer tryptic peptides covering the Muc5ac protein core were found in the loosely adherent mucus layer compared with the firm mucus layer (Fig. 4). This indicates that the loose layer Muc5ac mucin has been further processed and degraded. Because of the heavy glycosylation of mucins, most peptides are modified and are thus not possible to identify with the proteomics approach used. Only a few peptides from Muc2 and Muc5b were identified in the mucus, but no peptides corresponding to the Muc6 mucin were detected (data not shown). Thus the Muc5ac mucin was the most readily detected mucin, suggesting that the Muc5ac mucin was a major mucin of both the gastric mucus layers.
In this study, an in vivo model was used where the thickness and accumulation of the two mucus layers covering the gastric mucosa could be measured and collected separately. Muc1-deficient mice had thinner mucus layers and decreased accumulation rate, indicating that this membrane-bound mucin played a regulatory role in secretion of the gel-forming mucins. In addition, PGE2- and iNOS-derived NO, but not nNOS-derived NO, were important in stimulating growth of the protective firmly and loosely adherent mucus layers. By using proteomics, Muc5ac was identified as a major mucin in both the loosely and firmly adherent mucus layers.
The mucus layer covering the gastric (and colonic) mucosa can be divided into two different layers since the most luminal of the two layers, the loosely adherent mucus, can easily be removed by suction, whereas, the inner layer, the firmly adherent mucus, cannot (5). Therefore, the loosely adherent mucus layer is most likely rubbed off and mixed with the gastric contents after a meal; meanwhile, the firmly adherent mucus layer provides the physical barrier protecting the epithelium. The firmly adherent mucus layer retards hydrogen ion back diffusion but most importantly creates an unstirred environment where neutralization of hydrogen ions by secreted bicarbonate from the epithelia occurs, maintaining a pH gradient that protects the gastric mucosa from luminal acid. The firmly, but not the loosely, adherent mucus layer in the stomach is important for the maintenance of the pH gradient (28). It is plausible to conclude that an unstirred water layer within the mucus is a prerequisite for formation of a pH gradient, and, if this mucus layer is disrupted or decreased below a certain thickness, the pH gradient will be distorted and the epithelium thereby exposed to corrosive acid. Therefore, studies on mucus thickness and accumulation of the firmly adherent mucus layer are important for the understanding, development, and prevention of injuries of the gastric mucosa.
Prostaglandins are important in gastric mucosal protection and the severe gastrointestinal side effects of NSAIDs have to some extent been attributed to the inhibition of the prostaglandin-synthesizing enzymes and concomitant reduced gastric mucus thickness (11, 14). In addition, several in vitro studies have determined increased mucus secretion upon prostaglandin stimulation (6, 17, 23, 33). In the present in vivo study, prostaglandins stimulated mucus secretion, resulting in increased thickness of both the firmly and the loosely adherent mucus layers. In concordance with this, pretreatment with the NSAID indomethacin decreased mucus secretion and thickness of the firmly adherent mucus layer, confirming the important role of prostaglandins in mucosal defense. Prostaglandins have earlier been shown to increase mucus secretion in an in vivo model, but the distinction between the different mucus layers has not previously been studied (25).
NO present in the gastric mucosa can be either NOS-derived or generated nonenzymatically in the gastric lumen (19, 20). To investigate the effect of NO generated luminally, the NO donor SNAP was applied to the gastric mucosa. SNAP has earlier been shown to increase mucus gel thickness by in vitro phase contrast microscopy of rat stomach (8). In the study presented here, SNAP increased the thickness of the firmly adherent mucus layer both in rats and mice and total mucus accumulation rates were increased. However, the accumulation of the loosely adherent mucus layer did not increase after SNAP addition. These results indicated that the two mucus layers covering the gastric mucosa are differently regulated since luminal PGE2 increased the thickness of both layers, whereas a luminal NO donor increased the thickness of only the inner firmly adherent mucus layer. The thickness of the firmly adherent, but not the loosely adherent, mucus layer also increased in a previous study (7) when acidified nitrite was applied topically on the gastric mucosa, which results in high luminal concentrations of NO through conversion of nitrite. Recently, cysteine proteases secreted by the parasite Entamoeba histolytica were reported to degrade the Muc2 mucus gel as part of its mechanism for invasion (18). However, it is also possible that endogenously produced proteases can degrade the mucus gel. A possible explanation for the observation that NO and PGE2 affects the thickness of two mucus layers differently could be that the secretion or activity of proteases was influenced by these agents. Only the firmly adherent mucus layer was increased by luminal NO, and, assuming that the loosely adherent layer is generated from the firm adherent mucus, this may suggest inhibited protease activity or decreased protease secretion. The importance of endogenous proteases on the mucus gel formation and transition is not fully understood.
To study the influence of NOS-derived NO on mucus secretion, nNOS- and iNOS-deficient mice were used. nNOS is found in the gastric surface mucus cells and has previously been suggested to be involved in mucus secretion (8, 9, 30). However, when mucus thickness and accumulation was measured in the nNOS-deficient mice (Fig. 2), no difference was detected compared with the wild-type controls.
Constitutively expressed iNOS is present in the surface epithelial cells in the mouse corpus (29). Thus the involvement of iNOS in mucus secretion was studied, and a thinner mucus layer with a lower rate of accumulation in iNOS-deficient mice was determined. This observation supported the gastroprotective role of epithelial iNOS that has been previously suggested (29). These results also explained why the mucus was not affected by l-NNA pretreatment (in a concentration efficient in increasing blood pressure, Refs. 27 and 29) because this unspecific inhibitor of NOS is reported to be more potent in inhibiting eNOS and nNOS than iNOS (24). iNOS-derived NO appeared to be most efficient in regulating accumulation of the firmly adherent mucus layer because specific iNOS inhibition or depletion caused thinner mucus layers compared with prostaglandin inhibition. No further decrease was detected after inhibition or inactivation of iNOS in combination with inhibited prostaglandin synthesis.
The physical differences responsible for the dissimilar behavior of the two mucus layers have not yet been described. However, apart from water, the mucus gel consists mainly of secreted mucins (1), and there are two types of gel-forming mucins in the stomach, which are secreted by cell types located in different regions in the gastric mucosa. MUC5AC is expressed in the surface mucus cells and MUC6 by the mucus neck cells in the gastric glands (26, 34). Immunohistochemistry of human biopsies have revealed that the adherent mucus layer contains segregated MUC5AC and MUC6 mucins, which are arranged in a laminated linear way, with a continuous sheet of MUC5AC mucins immediately adjacent to the surface epithelium (13). MUC6 mucins are found in thinner, more truncated layers interspersed in the MUC5AC mucus layer. Proteomics were used to determine the protein cores of the mucins in the different mucus layers in the mouse, and Muc5ac was found to be a main component of both mucus layers; however, no detectable levels of Muc6 were found with this method. The differences in results between the present study and Ho et al. (13) might be due to species differences or the methodology used since antibodies against mucins are difficult to use because of the heavy and variable glycosylation of the protein core. From our data, it was concluded that the contribution of Muc6 to the mucus layers was negligible but could not be excluded. However, the reasons for the different physicochemical properties and behavior of the different mucus layers are still unclear, as is the cause of the transition from a firmly to a loosely adherent mucus layer. The latter question is intriguing because the thickness of the firmly adherent mucus layer appears preset and variations between individuals are minimal. What determines this thickness is unknown. Fewer tryptic peptides of Muc5ac at the proteomics analysis were detected in the loosely adherent mucus layer, suggesting that processing or degradation of the firmly adherent mucus layer might cause formation of the loosely adherent mucus layer. Proteases have been suggested to dissolve mucus in the colon, but whether this also occurs in the stomach remains to be determined (18).
In addition to the gel-forming mucins, Muc1, a transmembrane mucin that cannot form a mucus gel, is found at the apical membrane of epithelial cells (31). In addition to being part of the mucus gel after its secretion, it is also possible that transmembrane mucins can act as sensors and transmit outside-in signaling from the exterior to the interior of epithelial cells, providing a second line of defense. Muc1 is more prominently expressed in the stomach compared with the colon and thus less likely to have a structural role for the formation of the colon mucus than for the stomach. Still it was found that Muc1−/− mice have a thinner firmly adherent mucus layer in the colon (22). These colon observations suggest a regulatory role rather than a structural role of Muc1 in the formation of the colonic mucus composed of the gel-forming Muc2 mucin. In the present study, Muc1−/− were shown to have a thinner firmly adherent mucus also in the stomach, and a similar role as described in the colon is suggested.
In conclusion, this study suggests a regulatory role of Muc1 since downregulation of gastric mucus accumulation is observed in Muc1−/− mice. In addition, the study shows that, even though the firmly and loosely adherent mucus layers covering the gastric mucosa consist of Muc5ac, they are differently regulated. Luminal administration of PGE2 results in increases of both layers, whereas luminal NO stimulates only firmly adherent mucus accumulation. A new gastroprotective role for iNOS is indicated because iNOS-deficient mice have a thinner firmly adherent mucus layer and a lower mucus accumulation rate.
The study was supported by grants from the Swedish Research Council (57P-20680-01-4, 57x-20675-01-4, 07491 and 08646), The Wallenberg Foundation, Ingabritt and Arne Lundberg Foundation, Swedish Foundation for Strategic Research, Mucosal Immunobiology and Vaccine Center (MIVAC), and Swedish Society for Medical Research (SSMF).
The authors thank Annika Jägare for excellent technical assistance. We also thank Johan Sällström for genotyping the nNOS mice.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2008 the American Physiological Society